Group Title: Influence of preconditioning temperatures and neuropeptides on metabolic and endocrine responses of rats in the heat
Title: Influence of preconditioning temperatures and neuropeptides on metabolic and endocrine responses of rats in the heat /
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Title: Influence of preconditioning temperatures and neuropeptides on metabolic and endocrine responses of rats in the heat /
Physical Description: xii, 151 leaves : ill. ; 28 cm.
Language: English
Creator: Gwosdow, Andrea Rose, 1954-
Publication Date: 1984
Copyright Date: 1984
 Subjects
Subject: Temperature -- Physiological effect   ( lcsh )
Heat -- Physiological effect   ( lcsh )
Acclimatization   ( lcsh )
Rats -- Physiology   ( lcsh )
Animal Science thesis Ph. D
Dissertations, Academic -- Animal Science -- UF
Genre: bibliography   ( marcgt )
non-fiction   ( marcgt )
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Thesis: Thesis (Ph. D.)--University of Florida, 1984.
Bibliography: Bibliography: leaves 137-150.
General Note: Typescript.
General Note: Vita.
Statement of Responsibility: by Andrea Rose Gwosdow.
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Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
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Resource Identifier: alephbibnum - 000450188
oclc - 11427644
notis - ACL1856

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INFLUENCE OF PRECONDITIONING TEMPERATURES AND
NEUROPEPTIDES ON METABOLIC AND ENDOCRINE
RESPONSES OF RATS IN THE HEAT
















BY

ANDREA ROSE GWOSDOW

















A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR
THE DEGREE OF DOCTOR OF PHILOSOPHY



UNIVERSITY OF FLORIDA


































Copyright 1984

by

Andrea Rose Gwosdow















ACKNOWLEDGEMENTS


Andrea wishes to express her sincere appreciation to her mentor,

Emerson L. Besch, for his patience, support and valuable advice in the

planning and execution of this significant, original and independent

research project. The guidance, cooperation and assistance of members

of her committee, Drs. C. L. Chen, M. J. Fregly, F. B. Mather and W. P.

Palmore, are greatly appreciated.

The author acknowledges the generous donations of naloxone by

Dr. M. S. A. Kumar, Department of Anatomy and Cell Biology, Tufts

University; beta-endorphin anuiserum by Dr. Gregory P. Mueller,

Department of Physiology, Uniformed Armed Services University of the

Health Sciences, and phentolamine by CIBA-GEIGY Pharmaceuticals.

Thanks are extended to Drs. M. S. A. Kumar and J. M. Liu for

teaching the author stereotaxic and surgical techniques; to Robin

Brigmon and Dr. 0. W. I. Li for technical assistance; Tim Ganey and

Dr. P. W. Poulos for cerebral ventriculography and radiographs. The

patience and cooperation of Janet Eldred for typing this dissertation

is equally appreciated.
















TABLE OF CONTENTS


Page

ACKNOWLEDGEMENTS ................................................ iii

LIST OF TABLES ........................ ........................ vi

LIST OF FIGURES .................. . ....................... ... .ix

ABSTRACT ........................................................ xi

INTRODUCTION .................................................... 1

CHAPTER I ....................................................... 16

Introduction ............................................... 16
Materials & Methods ........................................ 17
Results .................................................... 32
Discussion ................................................. 44
Summary .................................................... 48

CHAPTER II ...................................................... 50

Introduction ............................................... 50
Materials & Methods ........................................ 51
Results .................................................... 57
Discussion ................................................. 72
Summary .................................................. . 76

CHAPTER III ..................................................... 77

Introduction ............................................... 77
Materials & Methods ........................................ 78
Results .................................................... 80
Discussion ................................................. 85

SUMMARY OF RESULTS .............................................. 90

CONCLUSIONS ..................................................... 93

APPENDIX A: TABLES OF RESEARCH DATA ............................ 103

APPENDIX B: RADIOIMMUNOASSAY (RIA) OF SERUM
CORTICOSTERONE IN RATS ............................. 112



iv









Page

APPENDIX C: THE USE OF CEREBRAL VENTRICULOGRAPHY FOR
VERIFICATION OF INTRACEREBROVENTRICULAR
(IVT) CANNULA PLACEMENT IN HIE LIVE RAT ............ 131

REFERENCES .................................................... 137

BIOGRAPHICAL SKETCH ............................................. 151















LIST OF TABLES


Table Page

1 Factors contributing to heat balance in
homeothermic animals .................................... 7

2 Heat production and evaporative heat loss as a
function of environmental temperature ................... 42

3 Relative food and water intake and body mass of
rats preconditioned to different environmental
temperatures ............................................ 43

4 Rectal temperature differential in rats at
24.50 C 60 min post-intracerebroventricular
injection of angiotensin II and leucine-
enkephalin .............................................. 60

5 Corticosterone and rectal temperature differentials
of intact rats 60 min post-intracerebroventricular
injection of beta-endorphin ............................. 68

6 Thyroxine (T4), triiodothyronine (T3) and rectal
temperature differentials of intact rats 60 min
post-intracerebroventricular injection of beta-
endorphin ............................................... 69

7 Differential effects of metabolic rate,
evaporative water loss and rectal and tail skin
temperature of rats preconditioned to different
environmental temperatures and exposed to
32.50 C for 1 hr ........................................ 81

8 Blood levels of thyroid hormones, corticosterone
and beta-endorphin-like-immunoreactivity in rats
preconditioned to different environmental tempera-
tures and exposed to 32.50 C for 1 hr ................... 83

9 Pituitary beta-endorphin-like-immunoreactivity in
rats preconditioned to different environmental
temperatures and acutely exposed to 32.5 C
for 1 hr ........................................... .... 84








Table Page

A-1 Metabolic rate (oxygen consumption) of rats
preconditioned to different environmental
temperatures (24.5 or 29.20 C) and exposed to
experimental temperatures between 18.0 and 34.50 C
for 3 hr ................................................ 103

A-2 Rectal temperature differential of rats precondi-
tioned to different environmental temperatures
(24.5 or 29.20 C) and exposed to experimental
temperatures between 18.0 and 34.5C C for 3 hr .......... 104

A-3 Tail skin temperature differential of rats
preconditioned to different environmental
temperatures (24.5 or 29.2 C) and exposed to
experimental temperatures between 18.0 and
32.50 C for 3 hr ........................................ 105

A-4 Evaporative water loss differential of rats
preconditioned to different environmental
temperatures (24.5 or 29.20 C) and exposed to
experimental temperatures between 18.0 and
34.50 C for 1 hr ........................................ 106

A-5 Comparison of intravenous (IV) and intracerebro-
ventricular (IVT) injections of varying doses of
beta-endorphin on body temperature changes 60 min
post-injection .......................................... 107

A-6 Time course effects on rectal temperature of beta-
endorphin followed by saline, beta-endorphin
followed by naloxone and saline followed by
naloxone in rats at 24.50 C. Beta-endorphin
(10 pg) or saline (5 pl) were injected intra-
cerebroventricularly (IVT). Naloxone (1 mg/kg)
or saline (0.3 ml) were injected intraperitoneally
at the arrow ............................................ 108

A-7 Rectal temperature differentials obtained from
intact, adrenalectomized and hypophysectomized rats
receiving post-intracerebroventricular injections
of varying doses of beta-endorphin ...................... 109

A-8 Rectal temperature differential of rats admin-
istered saline, propranolol (6 mg/kg), phentolamine
(6 mg/kg) or propranolol and phentolamine 30 min
prior to intracerebroventricular injections of
beta-endorphin .......................................... 110

B-1 The cross-reaction of anti-corticosterone-BSA-3
antiserum to corticosterone and a variety of
steroids ................................................ 120









Table Page

B-2 Characteristics of regression equations obtained
from serum samples for adrenalectomized (A) and
intact male (Im) rats assayed in serial dilution ........ 123

B-3 Comparison of corticosterone values obtained from
ethanol extraction alone, methanol extraction
alone and form chromatographic purification ............ 125















LIST OF FIGURES


Figure Page

1 Schematic of the events involved in the process
of physiological adaptation ............................. 3

2 A detailed diagram of the containers used for
evaporative water loss and metabolic rate
measurements ............................................ 22

3 Schematic of the apparatus used to measure
evaporative water loss .................................. 24

4 Schematic of the apparatus used to measure
metabolic rate .......................................... 29

5 Typical record of oxygen consumption for rats ........... 31

6 Metabolic rate (oxygen consumption) of rats pre-
conditioned to different environmental temperatures
(24.5 or 29.20 C) and exposed to experimental
temperatures between 18.0 and 34.50 C for 3 hr .......... 34

7 Rectal temperature differential of rats precondi-
tioned to different environmental temperatures
(24.5 or 29.2 C) and exposed to experimental
temperatures between 18.0 and 24.50 C for 3 hr .......... 36

8 Tail skin temperature differential of rats
preconditioned to different environmental
temperatures (24.5 or 29.20 C) and exposed to
experimental temperatures between 18.0 and
32.50 C for 3 hr ........................................ 39

9 Evaporative water loss differential of rats
preconditioned to different environmental
temperatures (24.5 or 29.20 C) and exposed to
experimental temperatures between 18.0 and
34.50 C for 1 hr ........................................ 41

10 Diagrammatic sketch of the sampling box used for
intravenous injections and blood sampling ............... 55











11 Comparison of intravenous (IV) and intracerebro-
ventricular (IVT) injections of varying doses of
beta-endorphin on body temperature changes 60 min
post-injection .......................................... 59

12 The relationship between dose of beta-endorphin
and change in rectal temperature in the rat ............. 62

13 Time course effects of beta-endorphin followed by
saline (.), beta-endorphin followed by naloxone (o)
and saline followed by naloxone (i) on rectal
temperature differential for rats at 24.50 C ............ 65

14 Rectal temperature differential for intact (o),
adrenalectomized (e) and hypophysectomized (E)
rats receiving intracerebroventricular
injections of varying doses of beta-endorphin ........... 67

15 Time course effects of rats pretreated with
saline (N), propranolol (o), phentolamine (*)
or propranolol and phentolamine (A) 30 min prior
to intracerebroventricular administration of beta-
endorphin (10 Pg) at the arrow .......................... 71

16 Schematic of body temperature regulation ................ 95

17 Proposed diagram of the events involved in
thermoregulation in the heat ............................ 97

B-1 Standard curve for corticosterone ....................... 118

B-2 Values for recovered amounts of corticosterone
added to adrenalectomized rat sera ...................... 122

C-1 Specimen radiographs showing placement of
intracerebroventricular cannula before (A)
and after (B) injection of fluorescent dye .............. 135


Figure


Page















Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment of the Requirements
for the Degree of Doctor of Philosophy


INFLUENCE OF PRECONDITIONING TEMPERATURES AND
NEUROPEPTIDES ON METABOLIC AND ENDOCRINE
RESPONSES OF RATS IN THE HEAT

By

Andrea Rose Gwosdow

April, 1984

Chairman: Emerson L. Besch
Major Department: Animal Science

Increased environmental temperature produces elevated rectal

temperatures (Tr) in rats. Some of the factors contributing to the

elevated Tr include increased heat production, stimulation of adrenal

and thyroid glands, and naturally occurring endogenous substances, such

as beta-endorphin (B-END). The experiments reported herein were

conducted to evaluate the effects of preconditioning temperatures and

neuropeptides on the metabolic and endocrine responses of rats to

varying thermal environments.

After preconditioning individually housed male Sprague-Dawley rats

to temperatures of 24.50 C or 29.20 C for 14 days, animals were either

exposed to experimental temperatures between 18.00 C and 34.5 C or

administered B-END intracerebroventricularly (IVT) at 24.50 C in a

controlled environment room. Relative humidity of 50 0.3% and a

12L:12D photoperiod (L = 0900 to 2100 hr) were maintained. Metabolic

rate (MR) and evaporative water loss (EWL) were measured using an









open-flow system; rectal and tail skin (Tts) temperatures using

thermistors; serum corticosterone, B-END-like-immunoreactivity

(B-END-LI) and thyroid hormones by radioimmunoassay. Food and water

were available ad libitum.

Compared to 24.50 C adapted rats, rats adapted to 29.20 C main-

tained elevated absolute Tr and Tts, serum and pituitary gland

B-END-LI, reduced serum thyroid hormones and comparable serum

corticosterone levels. The thermoneutral zone of these two groups of

rats was different. At temperatures above 29.20 C, changes in Tts and

EWL were of a greater magnitude for the 24.50 C adapted rats compared

to the 29.20 C group. The ATr of rats preconditioned to 24.50 C and

exposed to 32.50 C were similar to those produced by IVT injections of

B-END at 24.50 C; the concentration of B-END-LI in the bloodstream of

these two groups of rats was similar. Beta-endorphin injected IVT

produced a dose-related hyperthermic response that was antagonized by

naloxone but not adrenergic antagonists.

These data indicate that the response of rats to changing

environmental conditions is influenced by their thermal history.

Preconditioning temperatures may alter the reference point around which

Tr is regulated. Beta-endorphin appeared to elevate Tr centrally,

though p opiate receptors. Additionally, stimuli which released

B-END-LI into the bloodstream may also increase the synthesis or

delivery of B-END-LI to the pituitary gland. Beta-endorphin appears

to be involved in physiological adaptation of rats to heat.


xii
















INTRODUCTION


When an animal is exposed to an extreme change in environment, a

process of adjustment or physiological adaptation results (Prosser,

1964). Although the term "adaptation" includes genetic heritage and

natural selection, "physiological adaptation" refers to the readjust-

ment of the animal to itself, other organisms and/or to its surrounding

environment. Another term, acclimatization, is used to describe the

long term physiological adjustments induced by complex environmental

factors such as seasonal and climatic changes; acclimation or

conditioning refers to changes in response to a single variable, as in

a controlled experiment (Hart, 1957).

Adolph (1964) proposed a generalized scheme to describe the

adaptation process (Figure 1). A change in the animal's thermal

environment is sensed by temperature receptors (located on the skin,

etc.) which transmit this information to a transmitter which relays

this message to an effector organ (vasomotor, metabolic, sudomotor).

Through hormonal or neural mechanisms, the effector organ responds with

an appropriate output (vasodilation, sweating, etc.). This response is

coordinated by the central nervous system regulator (hypothalamus) that

maintains body temperature at a predetermined ("set-point") level and

is influenced by feedforward and feedbackward information. As a

result, the organism is able to respond appropriately to the "new"

environment. Physiological adaptation develops slowly on exposure to



















Figure 1. Schematic of the events involved in the process of physiological
adaptation. (See text for details.)

SOURCE: Adolph (1964).





























f FEEDBACK
LOOP


1^1








the new environment and disappears slowly when the stress is removed.

These processes are called adaptation and deadaptation, respectively.

The center of the thermoregulatory system is believed to be the

preoptic area of the anterior hypothalamus (POAH). Thermosensitive

neurons in this area receive afferent input from cutaneous thermo-

receptors (Pierau and Wurster, 1981) and from deep body thermosensitive

neurons in the spinal cord (Boulant and Hardy, 1974; Guieu and Hardy,

1970), posterior hypothalamus (Nutik, 1973; Wunnerberg and Hardy, 1972)

or other sites within the central nervous system (Mercer and Jessen,

1978). Afferent information from peripheral thermoreceptors is

relayed through unmyelineated C fibers and thinly myelinated A delta

fibers which are sensitive to heat and cold, respectively. This skin

temperature information travels upward with pain information through

the lateral spinothalamic tract (Brodal et al., 1967). This pathway

projects to the ventrobasal thalamus with collateral projections to

neurons in the reticular formation (Nakayama and Hardy, 1969), midbrain

and raphe nuclei (Dickenson, 1976) which relay this thermosensory

information to areas that include the POAH (Fuxe, 1965). Other extra

hypothalamic areas also may be involved in temperature regulation

(Boulant, 1981).

The thermosensitive neurons in the POAH integrate hypothalamic

thermal information with thermal information from peripheral and deep

body structures. This includes information from thermally active

endogenous substances as well as local neuronal input. The sensitivity

of the body to heat may be reflected by the 3:1 ratio of warm to cold

sensitive neurons (Boulant, 1981). As a result of this integration,

appropriate thermoregulatory responses are evoked through efferent









pathways to lower regions of the brain stem and spinal cord (Saper

et al., 1976) for maintenance of body temperature.

Body heat loss and heat production -esponses depend on the degree

of neuronal stimulation or temperature change of the POAH. The magni-

tude of this response is proportional to the change sensed by the POAH

(Boulant, 1981). Feldberg and Myers (1963) reported the balance of

norepinephrine and serotonin in the POAH may determine "set-point"

temperature. This is supported by Hellon (1981) because the raphe

nuclei is the only source of serotonin containing neurons in the brain.

Fibers in this area may relay thermal information to the POAH using

serotonin as a neurotransmitter. Direct heating of the POAH may

inhibit the release of thyroid releasing and stimulating hormones and,

consequently, the release of thyroid hormones (Andersson et al., 1962).

Lomax and Green (1981) found biochemical evidence that histamine may be

a neurotransmitter in the POAH. Opioid peptides may act on the thermo-

regulatory system through serotonin (Lin et al., 1979; Martin and

Bacino, 1979), dopamine (Lal et al., 1976; Martin et al., 1980),

acetylcholine (Yehuda and Kastin, 1980) or prostaglandin E2 (Martin

et al., 1977). These and other hormones or endogenous substances may

alter body temperature by acting on any part of the thermoregulatory

system together or alone.

Thermoregulation enables homeotherms to maintain constant body

temperatures within a range of environmental temperatures. The thermal

state of the environment stimulates physiological, behavioral and

morphological responses and determines heat transfer between the

environment and the animal. Important elements in this transfer are

environmental dry bulb temperature, air humidity, radiant temperatures








(infrared and solar radiation) and air movement (Ingram and Mount,

1975).

Between the extremes of hot and cold environments lies a range of

temperature in which homeotherms maintain a relatively constant body

temperature; this is the zone of homeothermy. Within this zone is a

smaller range of environmental temperatures in which an animal main-

tains a relatively constant body temperature with minimal adjustments

in metabolic rate; this is the zone of thermal neutrality. The latter

zone is bounded by the upper and lower critical temperatures--for heat

and cold, respectively (Hart, 1957), and varies with the age, sex and

species of the animal (Mitchell and Carman, 1926; Benedict and MacLeod,

1929; Gelineo, 1964; Folk, 1974). Above and below the critical

temperatures an animal increases metabolic rate to prevent a change in

body temperature.

Maintenance of a relatively constant body temperature is a balance

between heat loss and heat producing mechanisms (Table 1). The balance

between these two factors may be altered by changes within the body or

by the external environment. The resultant response is controlled by

the central thermoregulatory system which involves endogenous sub-

stances at each step in the process described above. Substances that

have been shown to alter the thermoregulatory processes include

acetylcholine, serotonin, dopamine, histamine, norepinephrine,

adrenocorticotropic hormone (ACTH), beta-endorphin (B-END), bombesin,

arginine vasopressin, neurotensin, glutamate, aspartate and taurine

(Blatteis, 1981).

Heat may be lost to the environment through conduction, convec-

tion, radiation or evaporation. However, the amount of heat lost by
















Table 1. Factors contributing to heat balance in homeothermic animals.


FACTORS INCREASING HEAT PRODUCTION
(over basal metabolic rate)

1. Exercise or shivering
2. Imperceptible tensing of muscles
3. Chemical increase of metabolic rate
(thyroid hormones, catecholamines)
4. Specific dynamic action of food
(hunger)
5. Disease (fever)
6. Nonshivering thermogenesis


FACTORS DECREASING HEAT LOSS

1. Shift in blood distribution
(vasoconstriction)
2. Decrease in tissue conductance
3. Counter-current heat exchange
4. Piloerection
5. Behavioral adjustment (curling up)


FACTORS DECREASING HEAT PRODUCTION

1. Chemical decrease of metabolic rate
(thyroid hormones, catecholamines)
2. Decreased activity
3. Decreased food intake


FACTORS ENHANCING HEAT LOSS

1. Sweating
2. Panting
3. Cooler environment
4. Increased skin circulation
(vasodilation)
5. Decreased clothing or shorter fur
insulation
6. Increased insensible water loss
7. Increased radiating surface
8. Increased air movement
(convection)
9. Behavioral adjustment
(laying "spread-eagle")


SOURCES: Folk (1974); Ganong (1977).









each of these means varies with ambient environment conditions. Heat

is lost by conduction through physical contact of the animal with air

and objects at a lower temperature. Loss of heat by conduction is

minimized by insulation with fur and clothing. Convection is similar

to conduction and is sometimes referred to as air conduction.

Radiation is the loss (or gain) of heat in the form of electromagnetic

infrared heat waves. These avenues all contribute to sensible or non-

evaporative heat loss (Folk, 1974).

On the other hand, evaporative heat loss--also called latent

loss--involves a change of phase of water from the liquid to the

gaseous state. Latent loss usually involves perspiration (sweating) or

increased evaporation from the lungs (panting). In the latter, shallow

breathing increases the amount of water vaporized in the mouth and

respiratory passages and is an effective heat loss mechanism. So

called "insensible heat loss" is a form of latent heat loss and results

from continual diffusion of water molecules through the skin and

respiratory tract. Other losses of heat through the skin depend on the

skin temperature, which is regulated by tissue conductance, blood flow

(vasoconstriction and vasodilation) and evaporation (Yoshimura, 1964).

Evaporative heat loss is an effective thermoregulatory mechanism

because 0.58 cal of heat is lost for each gram of water evaporated from

the body surface (Gelineo, 1964; Folk, 1974).

Under elevated temperatures, an animal may gain heat from the

environment but generally loses heat via physiological and behavioral

mechanisms (e.g., panting, burrowing and vasodilation). The ability

of animals to survive in the heat depends upon their ability to both

dissipate heat and maintain water balances within their bodies.








Some animals such as the kangaroo rat have the ability to utilize

metabolic water and excrete a more concentrated urine (Schmidt-Nielsen,

1979).

Morphological changes during prolonged exposure to heat include

thinning of the fur (Gelineo, 1949) and increase in the relative length

of the tail (Sumner, 1909; Sundstroem, 1922; Heroux, 1961). Behavioral

adaptations include laying "spread-eagle" to increase surface area in

addition to burrowing and estivating, as seen in some desert species

(Schmidt-Nielsen, 1964).

Many mammals, such as the rat, do not sweat or pant in the heat

but increase heat loss by regulating the flow of blood to the tail

(Rand et al., 1965), through evaporation of water from their

respiratory tract, and by spreading saliva on their body surfaces

(Hainsworth, 1968). The tail of the rat may account for as much as

10% of the cardiac output or for dissipating from 5-25% of the rat's

metabolic heat production (Rand et al., 1965). Heat also may be lost

through the ears and feet (Dawson and Keber, 1975), although Grant

(1963) and Gemmell and Hales (1977) demonstrated that the ears of the

rat do not function in response to thermal stimuli as do the ears of

other animals such as the rabbit. Moreover, heat loss from nude areas

of the rat other than the tail is insufficient alone to support

thermoregulation and is accompanied by increased evaporative water loss

(EWL) in the heat (Stricker and Hainsworth, 1970). Evaporation

accounts for more than 49.2% of the heat lost by rats at 330 C (Swift

and Forbes, 1939; Hainsworth and Stricker, 1970). Male rats also groom

saliva on the testes and scrotum which increases the surface area for

evaporation (Herrington, 1940; Hainsworth, 1967), survival time in the








heat (Stricker et al., 1968), and may account for the ability of males

to dissipate heat more effectively than females (Hainsworth, 1968).

The mechanisms of heat loss through the tail and by evaporation are

interdependent: removal of either impairs the rat's ability to regu-

late body temperature, while removal of both leaves the rat vulnerable

to heat stress (Stricker and Hainsworth, 1971).

Yousef and Johnson (1967) reported initial exposure of rats to

heat is marked by an increased rectal temperature (Tr) and metabolic

rate (MR). Elevated MR is commonly seen when animals are exposed to

conditions for which they are unaccustomed or nonadapted (Herrington,

1940; Yousef and Johnson, 1967) and signals the start of physiological

adjustment to the new environment (Prosser, 1964). When Tr reaches

38.50 C saliva secretion begins (Stricker and Hainsworth, 1970).

Circulating catecholamines from the adrenal medulla also may stimulate

salivary secretion as intravenous (IV) administration of epinephrine

elicited submaxillary saliva secretion in rats (Elmer and Ohlin, 1971).

The saliva is groomed onto body surfaces and through evaporation helps

prevent further increases in Tr (Hainsworth, 1968). Malan and Hildwein

(1969) observed increased EWL at a Tr of 39.50 C. At a Tr of 39.20 C

(Little and Stoner, 1968) and hypothalamic temperature (Thy) between

37.50 C and 39.00 C (Young and Dawson, 1982) vasodilation of the rat

tail occurs. This response continues until Thy falls by 0.2-0.40 C, at

which time vasoconstriction occurs until Thy rises again (Young and

Dawson, 1982).

After several hours (6-18 hr) in the heat, Tr reaches maximal

level plateaus and is maintained at a new elevated level 0.5-2.0 C

above control values (Yousef and Johnson, 1967; Horowitz, 1976).








Tail skin temperature (Tts) increases to 0.5-3.0 C below Tr

(Johanssen, 1962). The temperatures of the fore and hind limbs act in

a similar manner (Gemmell and Hales, 1977). Metabolic rate remains

elevated from 48 hr (Yousef and Johnson, 1967) to several weeks

(Gelineo, 1934; Beattie and Chambers, 1953) after which time MR

declines to control values.

The elevated MR observed on initial exposure to heat may be

attributed to increased secretions from the adrenal and thyroid glands

or increased muscular exertion of respiration (Yousef and Johnson,

1967). Glucocorticoids have been shown to exert a significant

calorigenic effect in rats (Evans et al., 1957). Kotby and Johnson

(1967) found elevated plasma corticosterone levels in rats after 1 hr

in the heat and maximum levels of this hormone were reached after

24 hr at 340 C. Similar findings were observed in this laboratory

(unpublished observations).

All of these hormones are among those that regulate energy

metabolism (Collins and Weiner, 1968). Jones et al. (1976) reported

decreased utilization of norepinephrine by heat adapted animals,

suggesting decreased sympathetic activity accompanies heat adaptation.

Adrenocortical hormones regulate glucose-6-phosphatase activity

(Collins and Weiner, 1968). Chayoth and Cassuto (1971a, b) reported

reduced activity of this enzyme in heat acclimated hamsters. Other

biochemical adaptations include reduced rates of mitochondrial

respiration and alterations in carbohydrate and fat metabolism in heat

producing organs (liver, kidney and brown adipose tissue) of heat

acclimated hamsters (Cassuto, 1968, 1970; Cassuto et al., 1970; Chayoth

and Cassuto, 1971a, b; Inbar et al., 1975; Rabi and Cassuto, 1976).








Such biochemical adaptations are directed toward lowering the activity

of energy producing metabolic pathways (Cassuto, 1968) and may be

reversed by administration of triiodothyronine to heat-acclimated

animals for at least 3 days (Cassuto et al., 1970).

Adrenal and thyroid hormones may act at regulatory site(s) of

these metabolic pathways to aid in chemical thermosuppression (Cassuto,

1968) and decrease metabolic heat production in rats in the heat.

Physiological adaptation to heat is accompanied by reduced thyroid

function (Dempsey and Astwood, 1943) and adrenal cortical activity

(Kotby and Johnson, 1967). Yousef and Johnson (1968) found shifts in

the amounts of iodine compounds in the plasma of heat acclimated rats:

the relative amount of thyroxine decreased, while that of free iodide

and monoiodotransferase increased during heat acclimation. These

hormonal and biochemical responses occur jointly with the lowered MR

observed after exposure to heat for 10 days.

Other physiological adjustments on exposure to heat include a rise

in total daily water consumption (Hainsworth, 1968; Attah and Besch,

1977; Gwosdow-Cohen, 1980) which may replace water lost by evaporative

cooling (Hainsworth, 1968; Attah and Besch, 1977; Gwosdow-Cohen, 1980).

Total daily food consumption is decreased (Brobeck, 1960; Hamilton,

1963; Attah and Besch, 1977; Gwosdow-Cohen, 1980) and may be a factor

in reducing thyroid activity in the heat (Yousef and Johnson, 1968;

Valtorta et al., 1982). Fluid shifts between compartments (Jones

et al., 1976; Horowitz, 1976) and an increased body water turnover

(Attah and Besch, 1977; Gwosdow-Cohen, 1980) are found in these

animals. Jansky and Hart (1968), Jones et al. (1976) and Bobeck et al.









(1980) reported a redistribution of blood flow away from heat producing

organs, including the thyroid gland.

Exposure to heat also causes reduced growth rates of rats (Ray

et al., 1968). Body weight initially decreases during the first 3 days

of heat exposure but increases after this time (Horowitz and Soskoline,

1978). The heat decreases the weights of most organs (Ray et al.,

1968; Jones et al., 1976) with the exception of the salivary glands

which begin to enlarge within 48-96 hr after heat exposure (Elmer and

Ohlin, 1969, 1970; Horowitz, 1976). Salivary gland enlargement serves

as a mechanism to increase saliva production for EWL in the heat

(Horowitz and Soskoline, 1978). These manifestations of heat adapta-

tion are completed in 7 to 10 days (Yousef and Johnson, 1967; Horowitz,

1976) and within 2 to 4 weeks return to original levels (Gelineo, 1934;

Sellers et al., 1951; Folk, 1974).

Gelineo (1934) and Schwabe et al. (1938) reported that MR

(measured as oxygen consumption) of rats was influenced by the

temperature at which animals were physiologically adapted. Rats

adapted to heat (30-320 C) consumed less oxygen at 5 C than rats

adapted to cold (0-20 C). These cold adapted rats regulated Tr at

-10 C while heat adapted animals became hypothermic at temperatures

below 250 C. Cold acclimated rats also survived longer at -100 C

compared to warm acclimated animals (Hart, 1957). In contrast, Cottle

and Carlson (1954) found no effect of thermal history on MR of 5 C

and 250 C adapted rats.

Rand et al. (1965) showed that thermal history influenced the

critical temperature for vasodilation of the rat tail. Lewis et al.

(1960) were unable to dehydrate the same rat at different ambient









temperatures due to physiological adaptation. Gwosdow-Cohen (1980)

reported that preconditioning temperature (24.50 C or 29.20 C) and

route of exposure (direct or stepwise) influenced the rat's EWL, body

water turnover, food intake and water intake responses to prolonged

(2 weeks) heat exposure. Previous studies of thermal history

emphasized individual thermoregulatory responses, and although these

responses are important, interactions of these and other adjustments

ultimately determine the rat's response to thermal stress. However, no

studies have been conducted to measure the influence of thermal history

on MR, EWL, Tr and Tts in the same rat.

Changes in the endocrine state of rats preconditioned to different

environmental temperatures may be responsible for thermoregulatory

alterations which could provide information on possible mechanisms of

action. Since ACTH and B-END are derived from the common precursor,

preopiomelanocortin (Blatteis, 1981), and released from the pituitary

gland together (Guilleman et al., 1977; Csontos et al., 1979; Mueller,

1980) on exposure to heat (Deeter and Mueller, 1981), and in response

to stressful stimuli (Millan et al., 1981; Mueller, 1981), it seems

likely that both of these hormones may be involved in the physiological

response to heat. The amount of B-END released is directly propor-

tional to ACTH levels in blood (Kreiger et al., 1979). In addition,

physiological levels of B-END produce hyperthermia (Blasig et al.,

1979; Thornhill and Wilfong, 1982) and appear to increase the "set-

point" around which body temperature is regulated, regardless of

ambient temperature (Bloom and Tseng, 1981; Clark, 1981).

Lin et al. (1979) suggest that at 220 C or below, B-END increases

sensible heat loss and decreases MR. Alternatively, naloxone was





15


reported to increase body temperature in rats exposed to the heat

(Holaday et al., 1978b; Thornhill et al., 1980) which infers that B-END

may prevent changes in body temperature on exposure to heat. The

ability of B-END to alter body temperature suggests involvement in the

process of physiological adaptation. Mobilization of B-END stores may

be influenced by environmental temperature which directly or indirectly

produce body temperature changes seen on exposure to different environ-

mental temperatures. These considerations led to the experiments

reported here.















CHAPTER 1
THERMOREGULATORY CHANGES IN RATS PRECONDITIONED
TO DIFFERENT ENVIRONMENTAL TEMPERATURES


Introduction


The influence of thermal history on the physiological adaptation

of rats in the cold has received more attention than rats in the heat

(Hart, 1957). Rand et al. (1965) and Thompson and Stevenson (1966)

showed that acclimatization temperature influenced the temperature for

vasodilation of the rat tail. In addition, Lewis et al. (1960) were

unable to dehydrate the same rat at different ambient temperatures due

to physiological adaptation.

For rats adapted to environmental temperatures between 0-25 C,

the upper critical temperature remains relatively constant at 29-30 C

(Swift and Forbes, 1939; Herrington, 1940) but increases to 34.50 C for

rats adapted to 30-320 C (Gelineo, 1934). Poole and Stephenson (1977)

suggested 18-280 C as a thermoneutral zone and this commonly is used as

the range of animal room temperatures for the housing of rats. On the

other hand, reproductive and growth differences in rats housed within

this temperature range have been reported (Yamauchi et al., 1981), and

20-26' C was suggested as the optimal temperature range for the housing

of rats. Benedict and MacLeod (1929) reported metabolic rate (MR)

differences in rats housed at environmental temperatures as little as

3 C apart.









Significant increases in water intake and body water turnover have

been reported (Attah and Besch, 1977) in rats physiologically adapted

to 29.20 C and 34.00 C compared to 24.50 C. Yet, for rats precondi-

tioned to room temperature (25.00 C), evaporative water loss (EWL) does

not increase until the ambient temperature exceeds 33.0 C to 34.0 C

(Hainsworth, 1968). Other evidence (Hainsworth and Stricker, 1970)

suggests that increased salivary secretions in rats at 320 C are the

primary contributors to EWL between 29-360 C.

What is not clear from prior research is the role of precondition-

ing environmental temperature on the relationship between the rat's

MR, shifts in upper critical temperature, and EWL. Previous reports

emphasized responses to changes in single variables and, although

these responses are important, interactions of multiple adjustments

ultimately determine the response of the animal to thermal stress.

Hence, the present study was conducted to determine the influence of

preconditioning temperatures on the rat's responses (MR, EWL, and

rectal and tail skin temperature) to elevated temperatures. The

results are reported here.


Materials and Methods


Animals


Sixty adult (3 months old), male Sprague-Dawley rats [Caw:

CFE (SD)] were used in these experiments. These rats (416.3 8.6 g,

mean S.E.) were housed individually in metabolic cages (Model

4-641-000, Acme) and were fed pulverized rat food (Laboratory Rodent

Chow, Ralston Purina) from feed cups with perforated inserts to









minimize spillage. Water bottles were equipped with sipper tubes and

attached to each cage. Food and water were available ad libitum.

Use of duplicate feed cups and water bottles allowed for rapid daily

interchange, caused minimal disturbance to the rats, and provided an

efficient procedure for quantifying food and water intake (Besch,

1970).


Environmental Room


Two environmental chambers (Model C7-88, Forma Scientific) were

used in this study: one for preconditioning and one for thermal

exposures. Dry-bulb temperatures of 24.5 0.10 C or 29.2 0.1 C

were utilized as preconditioning temperatures. Relative humidity of

50 0.3% and a 12L:12D photoperiod (L = 0900 to 2100 hr) were

maintained. Continuous monitoring of the ambient temperature in the

environmental room was accomplished through thermistors (Model 43TD,

Yellow Springs Instruments) interfaced with a data acquisition system

(Model PD2064, Esterline Angus).


Experimental Design and Procedure


Rats were exposed to either 24.5 or 29.20 C for at least 2 weeks

before beginning each experiment. Two weeks were allowed for physio-

logical adaptation because of previous reports (Gelineo, 1934; Cassuto

and Chaffee, 1963; Yousef and Johnson, 1967; Folk, 1974; Horowitz,

1976; Horowitz and Soskoline, 1978) that primary changes associated

with long term physiological adaptation are completed in 10 days.

Furthermore, when the experiments described herein were replicated with









rats preconditioned for 6 weeks, quantitatively similar results to

those reported were obtained.

After this adjustment period, the rats were randomly selected and

paired for acute (3 hr) exposure to one of the experimental tempera-

tures ranging between 18.0 and 34.5 C. The order of exposure of each

rat to each temperature remained constant throughout the experiment and

was separated by 3-5 days. Measurements of rectal (Tr) and tail skin

(Tts) temperatures and body mass were made at the beginning and end of

each 3 hr experimental session. Evaporative water loss and MR for each

rat were measured at each temperature.


Measurements


Measurements were performed between 0900 and 1200 hr.


Food and water intake. Food and water intake and body mass were

measured using a top loading balance (Model P1200N, Mettler). Food and

water intake measurements were calculated as the difference in weight

of the respective containers between the beginning and end of each 24

hr period.


Body temperature. Rectal temperature was measured using a

thermistor (Model 402, Yellow Springs Instruments) inserted 5 cm into

the rectum. Tail skin temperature was measured with a thermistor

(Model 409, Yellow Springs Instruments) positioned 2.5 cm from the base

on the ventral surface of the tail (Young and Dawson, 1982). Both

thermistors were held in place with gauze tape wrapped around the tail.

Each thermistor was connected to a telethermometer (Model 44TD, Yellow

Springs Instruments) for visual display of the temperature. Rectal and









tail skin temperature differentials were computed as the difference in

Tr and Tts, respectively, at the beginning and end of each experimental

session.


Evaporative water loss. The open-flow system of Hainsworth

(1968), as modified by Gwosdow-Cohen (1980), was used for determination

of EWL in rats. Simultaneous measurements were made on two rats one in

each of two cylindrical (height 26 cm; diameter 22 cm), 8-liter poly-

propylene containers (Model 5304, Nalgene) with slip-on lids (Model

5308, Nalgene) as illustrated in Figure 2. To complete the assembly,

the containers were placed in a plywood frame that was secured with an

adjustable strap and attached to the system detailed in Figure 3.

To assure an air-tight container, a 2 cm wide strip of foam

insulation tape was glued to the lid to provide a seal between the lid

and the walls of the container. A wire mesh grid was attached to each

lid to prevent the rats from chewing the tape. The lid of each

container was equipped with two sealed tube connector fittings (Model

4-HC-A-401, Cajon) which served as air inlet and outlet openings.

A 12.5 cm galvanized steel pipe extension was attached to each

connector fitting to deliver air into the middle of each container

through plastic (Tygon) tubing. A third opening in the lid was

equipped with a quick-disconnect fitting (Model QC4-B-4PM, Swagelok)

which allowed for monitoring of container temperature and pressure.

No temperature or pressure changes between container and outside were

detected in any of the experiments. Each container was equipped with a

wire mesh grid placed 5 cm above the container floor. A 2.5 cm layer




















Figure 2. A detailed diagram of the containers used for evaporative water loss
and metabolic rate measurements.

(See text for details.)








AIR INLET AND OUTLET


WIRE MES-

MINERAL
OIL


ADJUSTABLE
STRAP


WOOD FRAME




















Figure 3. Schematic of the apparatus used to measure evaporative water loss.

For sake of simplicity, the manometer and thermometer are shown
attached to only one container although pressure and temperature
measurements were made in each container. (See text for details.)











































VENTED TO
SURROUNDING AIR









of mineral oil (Mallinkrodt) on the floor of each container prevented

urinary and fecal moisture from contributing to EWL measurements.

Before EWL data were collected, a period of 25 min was allowed for

the development of the saliva spreading and Tr responses in each rat

(Hainsworth, 1968). Tanked dry air was passed through a tube con-

taining loosely packed anhydrous calcium sulfate (Drierite, Hammond

Drierite Co.) and a flowmeter (Model 100H, Lab-Crest Century) at a

constant rate of 8 liter/min before entering the sealed containers

(i.e., 4 liter/min through each container). Air exited from each

container through three drying tubes (12.7 cm each) containing loosely

packed anhydrous calcium sulfate to absorb the water vapor for a 15 min

collection period. The tubes then were removed from the environmental

chamber and allowed to equilibrate with room temperature (24.50 C) for

10 min before weighing to the nearest 0.01 mg on an analytical balance

(Model 2474, Sartorius). The drying tubes were handled with gloves at

all times. Evaporative water loss was determined by the change in

weight of the tube between the beginning and end of the 15 min

collecting period and calculated as water loss per unit of body mass

per hour (mg/g- hr- ). Data from this collection period represent EWL

of rats in the heat because EWL as a function of time indicates a

relatively constant rate of evaporation in the heat (Hainsworth and

Stricker, 1970).

Following each experiment, the drying tubes were washed (Alconox,

Alconox, Inc.), rinsed with distilled water, dried at 75.00 C (Stabil-

Therm gravity oven, Blue M Electric Co.) and filled with fresh

anhydrous calcium sulfate. Glass wool plugs were placed in the ends

of each tube to prevent loss of calcium sulfate. Tubes were stored in








an air-tight vacuum dessicator until use, usually within 24 hr. The

ends of each drying tube were wrapped with parafilm when not in use and

no changes in tube weight were detected as the result of storage.

To measure the repeatability and effectiveness of the water vapor

recovery technique, the rat was replaced in the container by its heat

equivalent in the form of an electrical resistor (resistance = 100.5

Ohms). The resistor, in a beaker containing 100 ml of water, was

connected to a power supply and, by applying 1.54 watts of power, the

resultant water vapor released to the container simulated the EWL of a

rat (Yuhaski, 1979). Evaporative water loss was measured, as described

above, and compared to the volume of water lost from the beaker. From

this a percent water recovery was calculated. This value, 0.961

0.004 (mean S.E.), was obtained daily, before and after each experi-

mental session. The daily average recovery error was used to correct

all experimentally obtained EWL values.

The rate of airflow through the system was measured by flowmeters

before and after each experiment (Figure 3). For each chamber,

(1) airflow at the inlet was compared to airflow at the outlet and

(2) airflow at the inlet was compared to airflow exiting from the

complete system at the last calcium sulfate tube. No differences in

inlet or outlet airflow rates were detected.


Metabolic rate. After the EWL measurements were completed,

usually about 45 min following exposure to the experimental

temperature, the rats remained in their respective containers and

dry air passed through a tube containing anhydrous calcium sulfate

and a flowmeter, at a constant rate of 500 ml/min, before entering









each container. Metabolic rate was measured according to the oxygen

consumption method of Depocas and Hart (1957) as modified by

Gwosdow-Cohen (1980). During the MR measurements, one container was

vented to the room while the other was connected to the oxygen analyzer

(Model 755, Beckman). From the exit port of one container the air

passed through soda lime (Fisher Scientific) and calcium sulfate tubes

to remove carbon dioxide and water vapor, respectively, before entering

the oxygen analyzer (Figure 4). The latter was attached to a strip

chart recorder (Model 110, Gould) and a graphic recording of oxygen

consumption measurements over a 60 min period was obtained (Figure 5).

The vented container was then attached to the oxygen analyzer for

measurement of MR (ml oxygen consumed/min kg0 75) in the second rat.

Oxygen consumption (V02) was calculated from the tracings (Figure

5) according to the method of Kelleher (1978):


0.75 air FO2in 2out
VO2 (ml/min- kg ) = 0.75 x
BW0 100


where, Vair is air flow rate in ml per min; BW is body mass in kilo-

grams; F02 is the fraction of oxygen in the air entering (in) and

exiting (out) the container; and 100 converts this fraction to a

decimal. All volumes of consumed oxygen were corrected to standard

conditions of temperature and pressure.


Statistics


Differences between treatments were determined by analysis of

variance. Duncan's multiple range test was used to compare the means.

Significance was assumed when P < 0.05.




















Figure 4. Schematic of the apparatus used to measure metabolic rate.

Everything to the left of the dashed line was in the environmental room.
For the sake of clarity, the manometer and thermometer are shown attached
to only one container although pressure and temperature measurements were
made in each container. (See text for details.)























THERMOMETER


SURROUNDING
AIR


CHART
RECORDER
ENTED TO


ANALYZER



















Figure 5. Typical record of oxygen consumption for rats.

In the usual case, F02 is measured at the beginning and end of the data
collection period. The average of these numerical values is used in the 0.75
equation for measurement of metabolic rate as ml oxygen consumed/min kg

V FO FO
air FO2in FO2ut
VO2 x 2t
2 BW075 100

where Vair is air flow in milliliters per minute; BW is body mass in
kilograms; FO2 is the fraction of oxygen in the air entering (in) and exiting
(out) the container and 100 converts this fraction to a decimal. In this
figure, the FO2 values (3.11 and 3.35) are averaged and the AFO2 is 3.23.
The result of this sample calculation (16.72) has not been corrected for STPD.
























vo2 250mi/ 323= 16.72 mr
03789 kg75 00 min-kg75

0 t-/- Period for Instrument Calibration | Period of Data Collection -/




0
z 2
z 3 ------- 3.11

< 3.35

RAT NO 38
-, MALE
S15 JUNE 83

5-

0 10 15 20 25 30 55 60

TIME (min)









Results


For rats adapted to the preconditioning temperature of 24.50 C,

MR was relatively constant between experimental temperatures of 22.2

to 27.00 C, but significantly (P < 0.05) elevated at experimental

temperatures of 20.0 and 29.20 C (Figure 6). Exposure to lower

(18.00 C) and higher experimental temperatures (above 29.20 C) produced

additional increases in MR. The MR of rats physiologically adapted to

29.20 C was relatively constant between experimental temperatures of

20.0 to 29.20 C (Figure 6) but significantly elevated following acute

exposure to experimental temperatures above and below this range.

Rectal and tail skin temperatures varied with preconditioning

temperature. Rats adapted to 29.2 C maintained highly significant

(P < 0.01) elevations in Tr (37.0 0.05, mean S.E., n = 190)

compared to rats (36.5 0.07, n = 117) adapted to 24.50 C. Tail skin

temperatures produced similar results: 29.20 C adapted rats maintained

highly significant elevations in Tts (32.1 0.10, n = 50) compared to

24.5 C adapted rats (28.5 0.13, n = 50).

Minimal rectal temperature differentials (ATr) for rats adapted to

24.50 C were observed between 20 to 24.50 C (Figure 7), a range that is

similar to that for relatively constant MR (Figure 6). These rats had

significantly reduced ATr at an experimental temperature of 18.00 C and

a significantly increased ATr at temperatures above 29.20 C. Compared

to 24.50 C adapted rats, the 29.20 C adapted group displayed a reduced

but relatively constant ATr between 20.0 and 27.00 C, a range that is

quantitatively similar to that for relatively constant MR (Figure 6).



















Figure 6. Metabolic rate (oxygen consumption) of rats preconditioned to different
environmental temperatures (24.5 or 29.20 C) and exposed to experimental
temperatures between 18.0 and 34.50 C for 3 hr.

Each point represents the mean value and the vertical bars the standard
errors. Asterisks indicate significant (P < 0.05) differences between
groups and values in parentheses indicate the number of rats used.
















24.50C ADAPTED
25- o 29.20C ADAPTED


d (10)
S8(10) ( 20)
20-

S18 (20) ( 18)


15- (24) ( (28)
(28)
((16) (28)
- (0)
o

S10-


5-




10 15 20 25 30 35
ENVIRONMENTAL TEMPERATURE (C)
































Figure 7. Rectal temperature differential of rats
preconditioned to different environmental
temperatures (24.5 or 29.20 C) and exposed
to experimental temperatures between 18.0
and 34.5 C for 3 hr.

Each point represents the mean value and
the vertical bars the standard errors.
Asterisks indicate significant (P < 0.05)
differences between groups and the values
in parentheses indicate the number of rats
used.





















* 24.50C ADAPTED
o 29.20C ADAPTED


o---------


10 15 20 25 30 35

ENVIRONMENTAL TEMPERATURE (-C)








At 32.50 C, the ATr of the 29.20 C adapted animals was significantly

increased, but no further elevation was observed at 34.50 C.

Changes in Tts were more variable than in ATr. Rats adapted to

24.50 C displayed significantly decreased tail skin temperature

differentials (ATts) when acutely exposed to 20.00 C and 18.00 C

compared to 24.50 C (Figure 8). On the other hand, a significantly

elevated ATts occurred at 29.20 C and 32.50 C. Although ATts followed

a similar pattern for 29.2 C adapted rats, these changes were greater

in magnitude than for the 24.50 C adapted rats. The 29.20 C adapted

group maintained significantly lower ATts than 24.50 C adapted rats at

all experimental temperatures.

Evaporative water loss differentials (AEWL) for both groups of

rats were unchanged between 18.0 and 27.00 C (Figure 9). However, the

24.5 C adapted group maintained a consistently elevated AEWL (P <

0.05) compared to the 29.20 C adapted group. Rats adapted to 24.50 C

displayed significantly elevated AEWL at temperatures of 29.20 C or

higher whereas the AEWL for the 29.20 C adapted group was significantly

elevated at temperatures of 32.50 C or above.

Estimates of heat loss through evaporation indicate that evapora-

tion accounts for a great amount of the metabolic heat production as

environmental temperatures are raised (Table 2). Rats adapted to

29.20 C dissipated more heat at experimental temperatures above 32.50 C

compared to 24.50 C adapted rats; at 34.50 C these rats lost 20% more

heat by evaporation than their 24.50 C adapted counterparts.

The relative food intake of 24.50 C adapted rats were greater (P <

0.05) than that of the 29.20 C group (Table 3). The latter group
































Figure 8. Tail skin temperature differential of rats
preconditioned to different environmental
temperatures (24.5 or 29.20 C) and exposed
to experimental temperatures between 18.0
and 32.50 C for 3 hr.

Each point represents the mean value and the
vertical bars the standard errors for 10 rats.
Asterisks indicate significant (P < 0.05)
differences between groups.



















4- /
24.50C ADAPTED
*/
o 29.20C ADAPTED

Y2
_J

z
w0- -_ --- - -

LL

n-

S-2
n
ENVIRONMENTAL TEMPERATURE (C)


2 -4
Q- *
z







-8





10 15 20 25 30 35
ENVIRONMENTAL TEMPERATURE (C)



















Figure 9. Evaporative water loss differential of rats preconditioned to different
environmental temperatures (24.5 or 29.20 C) and exposed to experimental
temperatures between 18.0 and 34.50 C for 1 hr.

Each point represents the mean value and the vertical bars the standard
errors. Asterisks indicate significant (P < 0.05) differences between
groups and the values in parentheses indicate the number of rats used.



















* 24.50C ADAPTED
o 29.2*C ADAPTED


2o 25
ENVIRONMENTAL TEMPERATURE (*C)


I 2
E

-J



uJ
it


u- I
u-


U-
3 0.

w
I -
: -1.0.

w
9.-
' -2
0
a-
4
W --.


15

















Table 2. Heat production and evaporative heat loss as a function of environmental temperature.


Temperature (O C)


Precondition


24.5

24.5

24.5

29.2

29.2

29.2


Experimental


24.5

32.5

34.5

29.2

32.5

34.5


Metabolic rate*

ml/g -1 cal/g hr
ml/g hr cal/g hr


1.07

1.42

1.62

1.11

1.20

1.26


5.16

6.84

7.82

5,35

5.78

6.07


Evaporative water loss


mg/ hr al/g hr
mg/g -hr cal/g hr


1.72

2.86

4.60

1.97

2.97

5.61


1.00

1.66

2.67

1.14

1.72

3.25


% heat
produced
lost by
evaporation


19.38

24.27

34.14

21.31

29.76

53.54


SThe caloric value = 4,82 calories per ml of oxygen consumed (Ganong, 1977).

















Table 3. Relative food and water intake and body mass of rats preconditioned to different
environmental temperatures.


Group Daily food intake* Daily water intake',* Body mass**
(g/100 g body wt) (g/100 g body wt) (g)



24.50 C adapted 6.6 0.07a*** 11.2 0.38a 385.48 i 6.24a

29.20 C adapted 5.5 0.10b 12.7 0.40b 447.66 10.90b



All values are expressed as mean S.E. for 26 rats adapted to 24.50 C and 34 rats
preconditioned to 29.2 C for 24 days.

Initial body mass for 24.50 C adapted and 29.20 C adapted rats after the two week
preconditioning period.

** Different superscripts (a-b) within each column differ significantly (P < 0.05) as
determined by analysis of variance.









consumed (P < 0.05) more water than 24.50 C rats. In general, rats

adapted to 29.20 C were larger than 24.50 C adapted rats.


Discussion


The present research extends previous findings on thermoneutral

zones (TNZ) for rats and suggests that these zones depend upon the

thermal history of the rat. For example, rats adapted to 24.50 C

displayed a TNZ of about 22.20 C to 27.00 C, while their 29.20 C

counterparts had a TNZ of about 20.00 C to 29.20 C (Figure 6). Within

these temperature ranges the rats maintained a relatively constant ATr

and MR.

The increased metabolic heat production of 24.5 C adapted rats

resulted in relatively constant ATr when exposed to cool (20.00 C)

temperatures and contributed to an increased ATr at 29.20 C or above.

At these higher temperatures the elevated MR of 24.50 C adapted rats

were parallel to increases in ATr (Figure 7). Rats adapted to 29.20 C

maintained a tTr below their preconditioning temperature and this may

be due to decreased thyroid or adrenal gland activity or thinned fur

coat. Such physiological adaptations ensure survival at elevated

temperatures by reducing metabolic heat production and facilitating

heat loss (Collins and Weiner, 1968).

At their respective preconditioned temperatures, the absolute Tr

and Tts of 29.2 C adapted rats were maintained at an elevated level

compared to 24.5 C adapted rats. The resultant increased temperature

gradient between the rat and the environment may have facilitated heat

loss when the animals were exposed to the various experimental

temperatures. Maintenance of Tr at a constant new level 0.5-1.00 C









above control values indicates physiological adaptation to the heat

(Horowitz, 1976) and suggests an increased reference point for

regulation of body temperature.

By regulating the blood flow to the tail, rats may conserve or

dissipate heat (Grant, 1963; Rand et al., 1965; Stricker and

Hainsworth, 1971; Hellstrom, 1975; Young and Dawson, 1982). In the

present experiment, Tts is used as an index of heat conservation

(vasoconstriction) or heat dissipation (vasodilation). Generally,

tail vasoconstriction was evident at the lower temperatures (18.0 to

20.00 C) and tail vasodilation at elevated environmental temperatures

(32.50 C or above) for both groups of rats (Figure 8). Both the

magnitude of ATts and the experimental temperatures at which

vasoconstriction and vasodilation occurred varied with preconditioning

temperature. Rand et al. (1965) and Thompson and Stevenson (1966) also

found the ambient temperature causing vasodilation of the rat tail

varied with acclimatization temperature. These changes may be due to

a modified sensitivity of peripheral (Rand et al., 1965) or central

(Dawson and Keeber, 1979) thermal receptors.

As environmental temperatures increase, the temperature gradient

between the rat and the environment decreases and EWL becomes an

important heat loss mechanism (Hainsworth, 1968). By increasing Tr in

the heat, the rats widen the temperature gradient between themselves

and the environment which facilitates heat loss by conduction, convec-

tion and radiation. Because heat loss by these means is not sufficient

for body temperature regulation in the heat (Stricker and Hainsworth,

1970), rats must cool themselves by evaporating saliva which is groomed

onto their body surfaces (Hainsworth et al., 1968). Both groups of









rats in the current experiment increased ATr to similar values and the

ATts of 24.50 C adapted rats was higher (P < 0.05) than the 29.20 C

adapted group, indicating a greater loss of heat through the tail for

the former group. At 32.50 C, the 24.5' C adapted rats also maintained

an elevated EWL compared to the 29.20 C adapted rats.

Swift and Forbes (1939) have shown that 250 C adapted rats lost

49.2% of their body heat by evaporation at 330 C. Studies by Hainsworth

and Stricker (1971) produced similar results. In the experiment

reported herein, rats adapted to 24.50 C lost 24.3% while 29.20 C

adapted rats lost 29.8% of their body heat by evaporation in the heat

(32.50 C). At 34.50 C these values are 34.1% and 53.5% for 24.50 C and

29.20 C adapted rats, respectively (Table 2).

Because water is the main source of cooling for the body under

elevated temperatures, physiological mechanisms operate to replace

water lost by evaporative cooling (Hainsworth, 1968). In this study,

water was available ad libitum and significant changes in water

intake (Table 3) were observed on exposure to heat. The increased

water intake may have compensated for the water lost by evaporation.

Although increased drinking by rats in the heat has been shown to be

secondary to dehydration (Hainsworth et al., 1968), water intake

also may be triggered by the stimulation of peripheral or central

thermoreceptors (Andersson and Larson, 1961) and drying of the

oropharyngeal mucous membranes (Gregerson and Cannon, 1932; Lund

et al., 1969).

As environmental temperatures increased, the appearances and

behavior of the rats were indicative of the rat's ability to

thermoregulate as reported by Stricker et al. (1968). All rats









appeared to have similar behavioral adjustments after placement in

the EWL chamber for about 10 min. The rats settled into a prone

resting position and, except for periodic adjustments and grooming

behavior, remained quiet for the duration of the experimental session.

Initially the rats groomed the face and neck, but as ambient tempera-

tures increased, the scrotum and tail were emphasized. Eventually, the

entire ventral surface was covered with saliva and the animals were

"soaking wet."

The decreased food intake observed in rats preconditioned to

29.20 C for at least 2 weeks agrees with previous reports by Attah and

Besch (1977) and Gwosdow-Cohen (1980). Brobeck (1960) and Hamilton

(1963) also have reported decreased food intake at high ambient

temperatures. The reduced food intake may indicate a lower energy

requirement which, in turn, may reflect changes in metabolic activity

in the heat (Cassuto, 1968). Efficiency of food conversion is

increased in the heat, so the decreased food intake may be utilized

more efficiently (Pennycuik, 1964). This may account for some of the

body mass differences between 24.50 C and 29.20 C adapted rats. Attah

and Besch (1977) reported that rats gained weight in the heat despite

significantly reduced food intake. Also, rats at high environmental

temperatures are less active (Howard et al., 1959). These changes

contribute to the decreased internal heat production of heat adapted

rats (Brobeck, 1960). Such control of food intake has been reported by

Collins and Weiner (1968) to be crucial in counteracting hyperthermia.

At their respective preconditioned temperatures, the absolute

rectal and tail skin temperatures of 29.20 C adapted rats were main-

tained at an elevated level compared to 24.50 C adapted rats.









At temperatures above 29.2 C changes in MR, Tts and EIL all were of a

greater magnitude for the 24.50 C adapted rats compared to the 29.20 C

rats but similar ATr were observed in both groups. Because exposure of

29.20 C adapted rats to temperatures below 29.20 C produced greater

changes than for 24.50 C adapted rats, it seems likely that these two

groups were regulating their thermal responses around different

reference points. Moreover, the different upper critical temperatures

of the thermoneutral zones of both groups also indicate a changed

sensitivity to heat. Thermoregulatory responses are regulated around a

"set-point" temperature (Adolph, 1964; Folk, 1974), and the altered

"heat sense" of these two groups of rats could indicate that the

preconditioning temperatures in this study shifted this reference point

for regulation of body temperature.

Based on these results, future studies on physiological responses

of rats to heat should take into consideration the rat's thermal

history. This would enable a comparison of data from animals exposed

to temperatures within the range of 22.2 to 27.00 C (control

temperature). Moreover, it is necessary that control temperatures be

precisely reported so that the responses of rats may be compared to

similar experiments from other laboratories.


Summary


After preconditioning individually housed male Sprague-Dawley

rats to temperatures of either 24.5 C or 29.20 C for 14 days,

randomly paired animals from each group were acutely exposed (3 hr),

in series, to experimental temperatures between 18.00 C and 34.50 C

in a controlled environment room. Relative humidity of 50 0.3% and









a 12L:12D photoperiod (L = 0900 to 2100 hr) were maintained for

all experiments. Metabolic rate (MR) and evaporative water loss (EWL)

were measured using an openflow system; rectal (Tr) and tail skin (Tts)

temperatures using thermistors. For rats physiologically adapted to

24.50 C, MR was relatively constant over a temperature range of 22.2 to

27.00 C; for 29.20 C rats, over a range of 20.0 to 29.20 C. Above and

below these ranges, MR for both groups was significantly (P < 0.05)

elevated. Between 20.0 to 29.20 C, Tr changes for 24.50 C adapted rats

were significantly (P < 0.05) different from 29.20 C rats; similar

changes were observed in Tts. Although EWL for both groups was rela-

tively constant between 18.0 to 27.00 C, 24.50 C adapted rats displayed

consistently higher EWL changes at all environmental temperatures above

27.00 C. These data suggest that preconditioning temperatures may

alter the reference point around which body temperature is regulated.















CHAPTER 2
BODY TEMPERATURE RESPONSES INDUCED BY
BETA-ENDORPHIN IN RATS AT 24.50 C


Introduction


The data presented in Chapter 1 suggest that preconditioning

temperatures alter thermoregulatory responses in the rat which, in

turn, change the reference point around which body temperature is

regulated. It was previously reported that "set-point" temperatures

also may be modified by opioids (Holaday et al., 1980; Clark, 1981);

these body temperature alterations appear to be influenced by dose and

route of opioid injection (Yehuda and Kastin, 1980).

It has been reported that both physiological levels and low

doses of beta-endorphin (B-END) produce hyperthermia (Blasig et al.,

1979), but high doses of B-END produce either hypothermia or

hyperthermia followed by a hypothermic (biphasic) response (Clark,

1981). Although B-END does not readily cross the blood-brain barrier

(Reilly et al., 1980), intravenous (IV) administration produces a

slight hyperthermia in rats at room temperature (Yehuda et al., 1980).

Intracerebroventricular (IVT) injections of B-END cause both thermal

and analgesic responses which are dependent on the administered dose

(Yehuda and Kastin, 1980).

Naloxone has been reported to block (Holaday et al., 1978a; Clark,

1981), or antagonize (Brown et al., 1977; Blasig et al., 1978) the

body temperature response to B-END. In some experiments, naloxone









has had no effect on body temperature (Bloom and Tseng, 1979). Bloom

and Tseng (1981) reported naloxone antagonism of opioid-induced

hypothermia but not hyperthermia.

Although other substances (e.g., prostaglandin E2) may produce

body temperature changes similar to those previously observed for B-END

(Yehuda and Kastin, 1980), no studies dealing with alterations in body

temperature by leucine-enkephalin and angiotensin II in rats have been

reported. Leucine-enkephalin is similar in structure to B-END while

angiotensin II possesses a molecular weight similar to B-END.

The present study was conducted to clarify the dose-response rela-

tionship between B-END and body temperature in the rat and to compare

the specificity of action of B-END, leucine-enkephalin (opioid) and

angiotensin II (non-opioid) on body temperature in rats preconditioned

to a thermoneutral temperature of 24.50 C. The role of calorigenic

hormones from the adrenal and thyroid glands also was examined. The

results are reported here.


Materials and Methods


Animals


Adult (2 months old), male Sprague-Dawley rats [Caw: CFE (SD)]

were used in these experiments. The rats (350.5 6.2 g, mean S.E.)

were housed individually in metabolic cages (Model 4-641-000, Acme) in

a controlled environment room (Model C7-88, Forma Scientific) at 24.5

0.10 C, 50 0.3% relative humidity and a 12L:12D photoperiod (L = 0900

to 2100 hr) for at least 2 weeks before experiments began. Water

bottles equipped with sipper tubes were attached to each cage.









Food (Laboratory Rodent Chow, Ralston Purina Company) and water were

available ad libitum.


Environmental Room


The ambient temperature in the environmental room was monitored as

described in Chapter 1.


Drugs


Varying doses of B-END (0.05-50 pg), leucine-enkephalin (1-50 pg)

and angiotensin II (0.01-1 pg) were prepared in sterile physiological

saline. The injection volumes for all substances (Beckman) were 5 p1

and 0.3 ml for IVT and IV routes of administration, respectively.

Naloxone (Endo Laboratories), propranolol (Sigma Chemicals, Inc.) and

phentolamine (Ciba-Geigy Pharmaceuticals) were injected intraperi-

toneally (IP). Ampicillin (Bristol Laboratories) and procaine

penicillin-G (Pfizer) were used post-operatively to prevent infection.

Antibiotics were discontinued 48 hr before beginning experiments and

caused no observed rectal temperature (Tr) changes. A series of

control rats received similar treatment.


Experimental Procedures


General. Experiments were conducted on unrestrained rats placed

in standard rodent shoebox cages in a controlled environment room.

After a 30 min control period, rats were injected IVT and briefly

removed from the cages, at 0.5 hr intervals, for measurement of rectal

temperature over 3 hr. An additional Tr measurement was recorded 75

min post-injection for rats injected with naloxone. The Tr of rats








pretreated with propranolol and phentolamine were measured every 15 min

for the first hour followed by 30 min measurements for 2 additional hr.


Intracerebroventricular cannulae. Intracerebroventricular

injections were through stainless steel cannulae implanted into the

right lateral ventricle while each rat was under ether anesthesia.

Cannula placement was determined by the de Groot system as modified by

Pellegrino et al. (1979) at least 72 hr before experiments began.

Correct positioning of each cannula was verified initially by the back-

ward flow of cerebrospinal fluid. Further verification was obtained at

necropsy and by cerebral ventriculography (Appendix C).


Intravenous cannulae. Under ether anesthesia, a permanent

intravenous (IV) cannula was implanted into the right jugular vein of

each rat, as detailed by Popovic et al. (1963). A 24 hr recovery

period was allowed before beginning experiments. Patency of the

cannula was maintained by flushing daily with 0.1 ml of heparinized

(Lypo-Med, Inc.) saline solution (1000 units/ml).

Experiments requiring IV injections or blood sampling were

conducted in an acrylic plastic (Plexiglas) sampling box 7 cm wide,

17.5 cm long and 6 cm high (Figure 10). The top of the sampling box

was equipped with a 10 cm long and 1 cm wide opening for easy access to

the IV cannula. A sliding door with an opening for the rat's tail

allowed easy access but assured containment of the rat. The box

allowed free movement of the rat.


Adrenalectomized rats. Adrenalectomy was performed under

ether anesthesia through the bilateral paralumbar approach (Zarrow



















Figure 10. Diagrammatic sketch of the sampling box used for intravenous injections
and blood sampling.

(See text for details.)














:NING FOR CANNULA


HIDINGG DOOR


IING FOR RAT'S TAIL









et al., 1964). Following surgery, rats were maintained on a 0.9% NaCI

drinking solution, available ad libitum. Completeness of adrena-

lectomy was verified at necropsy and by serum corticosterone levels

(Gwosdow-Cohen et al., 1982, Appendix B). A 1 week recovery period was

allowed before beginning experiments.


Hypophysectomized rats. Hypophysectomized rats (Charles River

Breeding Laboratories) were maintained on a 0.5% dextrose drinking

solution, available ad libitum. Hypophysectomy was verified by

lack of change in body mass. A 2 week recovery period was allowed

before experiments began.


Radioimmunoassays (RIA). Serum corticosterone levels were

measured according to the procedure (Appendix B) detailed by Gwosdow-

Cohen et al. (1982). Measurements of serum thyroid hormones were made

using commercially available RIA kits which appear to be appropriate

for determining these hormones in rat serum (Clinical Assays).


Measurements


All measurements or sample collection were performed between 1500

and 1800 hr.


Body temperature. The procedure for measurement of rectal

temperature was described in Chapter 1.


Statistical Analysis


Differences between treatments were determined by analysis of

variance. Duncan's multiple range test was used to compare the means.









Analysis of variance, log transformation and linear regression were

used to determine dose-response relationships. Significance was

assumed when P < 0.05.


Results


Intracerebroventricular injections of varying doses (5-50 ug) of

B-END resulted in increased rectal temperature differentials (ATr)

within 30 min post-injection. Rectal temperature remained elevated

for at least 90 min, then declined and returned to control values of

saline-treated rats within 180 min post-injection. Because the maximal

Tr response occurred 60 min post-injection, this time interval was

used in all subsequent measurements.

Following IV administration of B-END in doses between 0.05 and

50.0 g, no significant differences in ATr were observed (Figure 11).

However, for rats injected IVT with B-END, the ATr was highly

significantly (P < 0.01) elevated above the ATr of IV administered rats

at all doses between 5.0 and 50 pg. On the other hand, IVT injections

of varying doses of leucine-enkephalin (1-50 pg) and angiotensin II

(0.01-1.0 ug) did not alter ATr (Table 4).

A significant relationship between ATr and doses of B-END between

0.05 and 50 pg was observed during the 60 min post-IVT injection time

interval (Figure 12). The dose-response curve (Figure 12) was plotted

as ATr vs. the log dose of B-END and calculated using analysis of

variance and linear regression. Verification of the results were

obtained using the log transformation (Alder and Roessler, 1964).

Correctness of fit for both statistical models was highly significant



















Figure 11. Comparison of intravenous (IV) and intracerebroventricular (IVT)
injections of varying doses of beta-endorphin on body temperature
changes 60 min post-injection.

Each point represents the mean value and the vertical bars the standard
errors. Asterisks indicate significant (P < 0.05) differences between
groups and the values in parentheses indicate the number of rats used.













o IVT INJECTIONS
* IV INJECTIONS


0 1.0

LOG /3-ENDORPHIN DOSE (p.g)


* *


Z)
0



z
[i

Lu 1.0
LL
U-

LE
D

[L


a-
I


bii


rr












Table 4. Rectal temperature differentials in rats at 24.50 C 60 min
post-intracerebroventricular injection of angiotensin II
and leucine-enkephalin.


Rectal temperature differential* (0 C)
Dose
(Fg)
n** Angiotensin II n Leucine-Enkephalin



0.01 7 -0.1 0.21

0.10 6 0.2 0.19

1.0 7 0.1 0.36 6 0.2 0.21

10.0 6 0.7 0.27

50.0 6 0.0 0.26


60 min post-
for saline


** Number of rats.


* Rectal temperature differential = rectal temperature
injection minus rectal temperature 0 time, corrected
value and expressed as mean S.E.

































Figure 12. The relationship between dose of beta-
endorphin and change in rectal temperature
in the rat.

Each point represents the mean value and the
vertical bars the standard errors. Values
in parentheses indicate the number of rats
used.

















I(19) (8)

(7)



(12)










(7) y= 0.47x+ 0.51
n= 60
r = 0.97
P<0.OI


LOG /3-ENDORPHIN DOSE (pg)


1.20


0
j 0.98

I--
z

- 0.77-
LL
IL

W
c 0.55-


--
w
S0.33-


- -
-J

0.12-
w
0'









(P < 0.01). The data (Figure 12) is represented by the simpler

statistical model.

The hyperthermia produced by IVT injections of B-END was

antagonized by naloxone injected IP (Figure 13). This response was

rapid. and LTr returned to control levels within 15 min following

naloxone injection. Administration of saline did not alter the

hyperthermia resulting from IVT injected B-END. Rats receiving IVT

saline followed by naloxone did not display significant changes in ATr.

Adrenalectomized and hypophysectomized rats receiving varying

doses (between 0.05 and 50.0 pg) of IVT injected B-END generally

displayed an increased ATr, but not to the same level as intact rats

(Figure 14). Beta-endorphin doses between 10 and 50 ug induced highly

significant decreases in ATr of adrenalectomized compared to intact

rats. For hypophysectomized rats, lowered (P < 0.01) LTr were observed

only at doses of 10 and 50 vg of B-END. Moreover, a dose-response

relationship similar to that demonstrated with intact rats (Figure 12)

could not be obtained with either adrenalectomized or hypophysectomized

rats. Dose-related changes in serum corticosterone (Table 5) or

thyroid hormone differentials (Table 6) could not be detected in rats

receiving IVT injections of varying doses of B-END. However, serum

levels (absolute and differential values) were significantly reduced

below saline (zero baseline) levels at all doses of B-END administered.

Intraperitoneal injections of propranolol (6 mg/kg IP) lowered,

although not significantly, ATr compared to saline controls (Figure

15). Rats administered phentolamine (6 mg/kg IP) significantly

decreased ATr compared to saline-treated animals. The ATr effect



















Figure 13. Time course
saline (e),
by naloxone


effects on rectal temperature of beta-endorphin followed by
beta-endorphin followed by naloxone (o) and saline followed
(a) on rectal temperature differential for rats at 24.50 C.


Intracerebroventricular injection of beta-endorphin (10 ug) or saline
was made at 0 time. The arrow represents intraperitoneal administration
of naloxone (1 mg/kg) or saline. Each point represents the mean and the
vertical bars the standard errors for 10 rats.











o /3-ENDORPHIN/NALOXONE

* 3-ENDORPHIN/SALINE

m SALINE/NALOXONE


TIME (MINUTES)


2.0-

-j

z 1.5.
Lu
It
IL
0 1.0
Lu
It
~0.5
Lu

0~
L:
I-

~0.5



















Figure 14. Rectal temperature differential of intact (o) adrenalectomized (e) and
hypophysectomized (U) rats receiving intracerebroventricular injections
of varying doses of beta-endorphin.

Each point represents the mean value and the vertical bars the standard
errors. Values in parentheses indicate the number of rats used.











2.0



1.5-
0
-I

z 1.0
br
IL-
LL
o 0.5
n

I--





'- -0.5
-ij
I.-


o INTACT

* ADRENALECTOMIZED

* HYPOPHYSECTOMIZED











(11)


1.5 -1.0 10 2.0

LOG -O-ENDORPHIN DOSE (/pg)












Table 5. Corticosterone and rectal temperature differentials of
intact rats 60 min post-intracerebroventricular injection
of beta-endorphin.


Differential*
Beta-endorphin
Dose (pg)
n** Corticosterone n Rectal temperature
(vg/dl) (0 C)



0.05 5 11.8 3.46a*** 5 0.0 0.36a

1.0 6 5.1 3.81 a 6 0.3 0.43a

5.0 6 10.7 3.03a 6 0.8 0.42ab

10.0 4 6.7 0.94a 4 1.5 0.15b

25.0 5 6.6 1.15a 6 1.3 0.26b

50.0 7 15.1 3.95a 6 1.3 0.50b



Corticosterone and rectal temperature differential = 60 min post-
injection value minus 0 time value, corrected for saline value and
expressed as mean S.E.

** Number of rats.

*** Values with different superscripts (a-b) differ significantly (P <
0.05) within each column as determined by analysis of variance and
Duncan's multiple range test.















Table 6. Thyroxine (T4), triiodothyronine (T3) and rectal temperature differentials of intact
rats 60 min post-intracerebroventricular injection of beta-endorphin.


Differential*
Beta-endorphin
dose
(pg) n** T4 n T3 n Tr
(pg/dl) (ng/dl) ( C)



1.0 6 0.05 0.12a*** 6 -7.93 + 1.98 a,b 7 0.4 0.0 a

10.0 6 -0.32 0.22a 6 -10.48 1.92 a 19 1.1 0.13 b

25.0 6 -0.23 0.20 a 6 -4.25 1.51 b 7 1.2 0.30 b



Thyroxine, triiodothyronine and rectal temperature differential = 60 min post-injection
value minus 0 time measurement, corrected for saline value and expressed as mean S.E.

** Number of rats.

*** Values with different superscripts (a-b) differ significantly (P < 0.05) within each
column as determined by analysis of variance and Duncan's multiple range test.





















Figure 15. Time course effects of rats pretreated with saline (M), propranolol (o),
phentolamine (*) or propranolol and phentolamine (A) 30 min prior to
intracerebroventricular administration of beta-endorphin (10 Pg) at the
arrow.

Each point represents the mean value and the vertical bars the standard errors
for six rats.











m SALINE
o PROPRANOLOL
* PHENTOLAMINE
a PROPRANOLOL AND
PHENTOLAMINE


0 15 30 45 60
TIME (MINUTES)


90 120









resulting from simultaneous administration of propranolol and

phentolamine was greater than individual injections of each drug.

Intracerebroventricular administration of B-END to rats pretreated

with propranolol for 0.5 hr increased 6Tr after 30 min (Figure 15).

Administration of B-END to rats pretreated with phentolamine caused the

ATr to increase but not to the 60 min level of either the propranolol

or saline groups. The ATr increased following IVT administration of

B-END in the propranolol/phentolamine group.


Discussion


In previous reports on the effects of opioids on body temperature

in rats, three major patterns appear: hyperthermia (Blasig et al.,

1979), hypothermia, or a biphasic response (Clark, 1981). These

different results may be explained by considering the range (0.6 pg -

0.5 mg) of B-END doses and the routes (IP, IVT, IV) of administration

(Yehuda and Kastin, 1980) used in those studies. On the other hand,

the doses of B-END (5 to 50 pg) used in the present study produced

hyperthermic responses only when injected IVT; IV administration of the

same doses did not elevate ATr. Although only a small percent (0.3%)

of IV injected B-END crosses the blood-brain barrier (Reilly et al.,

1980), the current findings strongly suggest that Tr responses induced

by B-END are mediated through a central (IVT) mechanism of action.

Beta-endorphin may mediate changes in body temperature either

through central thermoreceptors or through calorigenic or vasoactive

hormones. This action may involve B-END alone or in combination with

one or more neurotransmitters. The response obtained may depend on the

dose of B-END administered. Low doses of B-END may produce opiate








effects while administration of higher doses also may induce non-opiate

effects which cannot be antagonized by naloxone (Yehuda and Kastin,

1980).

No linear dose-response curve for ATr and B-END previously has

been reported (Yehuda and Kastin, 1980). However, doses reported

herein resulted in a hyperthermic response that correlated linearly

with the log dose of B-END using the statistical models described

above (Figure 12). This hyperthermia appears to be specific for B-END

as varying doses of leucine-enkephalin and angiotensin II did not

alter ATr.

In general, the doses (0.05-50 ug) of IVT injected B-END did not

appear to change the behavior of the rats. Occasionally, high doses

(25, 50 pg) of B-END resulted in quiescent rats while lower doses (5,

10 yg) excited some rats.

Antagonism of the hyperthermic effects of B-END by naloxone

previously has been reported (Rudy and Yaksh, 1977; Blasig et al.,

1978; Huidobro-Toro and Way, 1979) but others have found B-END-induced

hyperthermia to be resistant (Cox et al., 1976) or partially resistant

(Martin and Bacino, 1979) to naloxone antagonism. The data presented

here (Figure 13) demonstrate the antagonistic effect of naloxone on the

B-END caused hyperthermia. This suggests that naloxone-antagonized

opioid receptors are involved. It is known that naloxone strongly

antagonizes 1 receptors but is less dominant at the other opioid

(K,6,o) receptors (Martin, 1981). When injected alone, naloxone had

little effect on body temperature. This is in agreement with Ferri

et al. (1978), Holaday et al. (1978), Thornhill et al. (1978),

Blasig et al. (1979), Cox et al. (1979) and Wong and Bentley (1979).









All reported that naloxone, given IP or subcutaneously, had no effect

on body temperature in doses ranging from 3-40 mg/kg at room

temperature.

Neither adrenalectomized or hypophysectomized rats demonstrated a

dose-response relationship (Figure 14) comparable to that produced by

intact rats (Figure 12) at any time interval studied. Holaday et al.

(1977) found no alteration in opiate potency following IVT administra-

tion in adrenalectomized animals. Hypophysectomized rats had reduced

plasma (Mueller, 1980) and brain (Kerdelhue et al., 1982) B-END-like-

immunoreactivity which may alter the sensitivity of opiate receptors.

Adrenal atrophy, a consequence of hypophysectomy, may alter the hormone

balance necessary to produce the thermal effects of B-END.

Involvement of the hypothalamo-pituitary-adrenal axis implicates

adrenal cortical hormones in mediating the Tr responses reported in

this study. In addition, the findings of Herrmann (1942), Numan and

Lal (1981) and Thornhill and Wilfong (1982) suggest adrenocortical

involvement in opiate-induced hyperthermia. However, in the study

reported herein, serum corticosterone differentials were not altered by

the dose of B-END administered. Moreover, serum corticosterone

differentials were not related to the Tr changes. Risch et al. (1983)

reported positive correlations between plasma levels of B-END and ACTH,

but not cortisol, in humans. These authors attributed this response to

different adrenal receptor sensitivities to ACTH or differences in

metabolic clearance rates of plasma ACTH and cortisol. These data may

reflect concomitant releases of ACTH and B-END from the pituitary

gland. Alternatively, B-END and ACTH may act in opposition to maintain

a relatively constant body temperature (Thornhill and Wilfong, 1982).








Although serum corticosterone does not appear to be directly involved

in B-END-induced hyperthermia, this hormone may have a permissive role

(Gale, 1973).

Modification of endogenous levels of B-END in brain tissues by

thyroid hormones has been reported by Gambert et al., 1980. The

present findings suggest thyroid hormone involvement because the

doses of B-END administered inhibit serum T3 levels. In contrast,

thyrotropin releasing hormone reversed B-END-induced hyperthermia in

rats (Holaday et al., 1978a).

Body temperature also may be influenced by the catecholamines,

epinephrine and norepinephrine (Fregly et al., 1979) which stimulate

a- and 8-adrenergic receptors. Blockage of these receptors with

propranolol, phentolamine or both drugs in combination decreased ATr.

Body temperature was lowered,because of tail vasodilation due to loss

of vasconstrictor tone (a-receptor mediated) or decreased MR

(predominantly B-receptor mediated). Nevertheless, B-END administered

IVT to all of these groups increased Tr (Figure 14), suggesting that

B-END-induced hyperthermia is not mediated through a or B receptors.

The data presented herein suggest a central (IVT) role for B-END-

induced hyperthermia. The doses (0.05-50 yg) presented produced a

linear log B-END dose-rectal temperature response relationship,

specific for B-END. The actions of B-END involve V opiate receptors.

Adrenergic receptors do not appear to be involved in the hyperthermic

response produced by B-END. Beta-endorphin administration had no

effect on serum corticosterone; however, serum T3 was inhibited. More

detailed studies will be required to understand the interactions of

these hormones.








Summary


The effects of beta-endorphin (B-END) on body temperature and the

role of the adrenal and thyroid glands were examined in male, Sprague-

Dawley rats in a controlled environment room at 24.50 C. Relative

humidity of 50 0.3% and a 12L:12D photoperiod (L = 0900 to 2100 hr)

were maintained. Rectal temperature (Tr) was measured using

thermistors, corticosterone and thyroid hormones by radioimmunoassay.

Intracerebroventricular (IVT) administration of varying doses 0.05-50

pg of B-END resulted in a dose-related hyperthermia that began 30 min

post-IVT injection and continued for an additional hr. Intravenous

injections of the same doses of B-END resulted in little or no Tr

response. The B-END-induced hyperthermia was antagonized by naloxone.

Pretreatment with propranolol, phentolamine or both drugs in combina-

tion did not block the hyperthermia caused by B-END. Adrenalectomized

and hypophysectomized rats injected IVT with B-END did not consistently

increase Tr. Beta-endorphin administration had no effect on serum

corticosterone; however, serum triiodothyronine was inhibited. These

data suggest the hyperthermic action of B-END is mediated centrally

through i opiate receptors. Adrenergic receptors do not appear to be

involved in the hyperthermic response produced by B-END.















CHAPTER 3
THE EFFECTS OF PRECONDITIONING TEMPERATURE ON PITUITARY
AND BLOOD LEVELS OF BETA-ENDORPHIN IN RATS AT 32.50 C


Introduction


The data presented in Chapter 2 demonstrate that rectal

temperature differentials (ATr) induced by intracerebroventricular

(IVT) injections of beta-endorphin (B-END) were similar to those of

rats preconditioned to 24.50 C and acutely (3 hr) exposed to 32.50 C

(Chapter 1). The ability of B-END to alter body temperature suggests

involvement in the process of physiological adaptation. Plasma levels

of B-END-like-immunoreactivity (B-END-LI) increase during exposure to

heat (Deeter and Mueller, 1981) and physiological stress (Millan

et al., 1981; Mueller, 1981). Thornhill et al. (1980) suggest that

blockade of brain and pituitary endorphins by opiate antagonists alters

the rat's ability to physiologically adapt to severe changes in

environmental temperature.

Using a purified preparation of corticotropin releasing factor,

Vale et al. (1978) directly stimulated the in vitro release of

B-END and adrenocorticotropic hormone (ACTH) by rat pituitary tissue.

These findings suggest a common mechanism for release of B-END and ACTH

from the rat pituitary gland. Co-release of ACTH and B-END from the

anterior pituitary gland was observed by Guilleman et al. (1977),

Mains et al. (1977), Pellefier et al. (1977) and Rossier et al. (1977).

Increased glucocorticoids have been observed in rats injected with ACTH









(Evans et al., 1957) and rats exposed to environmental temperatures of

29.20 C (Attah and Besch, 1977) and 32.50 C (Gwosdow-Cohen et al.,

1982, Appendix B). Like ACTH, the secretion of B-END is inhibited by

glucocorticoids (Guilleman et al., 1977; Hollt et al., 1979; Wardlaw

and Frantz, 1979; Wiedemann et al., 1979; Mueller, 1980).

These commonalities suggest that B-END may contribute to the

rectal temperature changes observed in rats preconditioned to different

environmental temperatures. Hence, the present study was conducted to

evaluate the influence of preconditioning temperatures on pituitary and

blood serum levels of B-END-LI in rats acutely exposed to the heat

(32.50 C). Also measured were the blood serum levels of thyroid

hormones and corticosterone. The results are reported here.


Materials and Methods


Animals


Sixty-four adult (2 months old), male Sprague-Dawley rats [Caw:

CFE (SD)] were used in these experiments. These rats (351.3 4.1 g,

mean S.E.) were housed individually in metabolic cages (Model

4-641-000, Acme) for at least 2 weeks before experiments began. Water

bottles equipped with sipper tubes were attached to each cage. Food

(Laboratory Rodent Chow, Ralston Purina Company) and water were

available ad libitum.


Environmental Room


Two environmental chambers (Model C7-88, Forma Scientific)

were used in this study: one for preconditioning and one for








thermal exposures. Dry-bulb temperatures of 24.5 0.10 C or 29.2

0.10 C were utilized as preconditioning temperatures. Relative

humidity of 50 0.3% and a 12L:12D photoperiod (L = 0900 to 2100 hr)

were maintained. The ambient temperature in the environmental room was

monitored as described in Chapter 1.


Experimental Procedures


General. All rats were preconditioned to either 24.50 C or

29.20 C (control temperatures) for at least 2 weeks before beginning

each experiment. The rats then were randomly selected and divided into

equal groups for exposure to the respective control temperature or

32.50 C in standard rodent shoebox cages for 1 hr because Deeter and

Mueller (1981) reported plasma levels of B-END-LI are maximal 60 min

after exposure to heat. Measurements of rectal (Tr) and tail skin

(Tts) temperature and body mass were made at the beginning and end of

each 1 hr experimental session. Evaporative water loss and metabolic

rate were measured 1 hr after temperature exposure. Immediately

following these measurements, the rats were decapitated and blood

was collected in plastic test tubes containing 0.5 ml of 10%

ethylenediaminetetraacetic acid (EDTA) and chilled on ice. Plasma was

separated by centrifugation and stored at -70 C until hormone assays

were conducted. The pituitary neurointermediate lobe (NIL) was

dissected from the anterior lobe (AL) and both tissues were homogenized

in 0.5 ml of 1 N acetic acid. Aliquots of the homogenates were removed

for protein determination (Protein Assay Kit, BioRad Laboratories) and

all samples were stored at -700 C until tissue and protein assays were

conducted.









Radioimmunoassays (RIA). Serum B-END-LI was measured according

to the procedure of Mueller (1980). The RIA procedures used to

determine serum corticosterone and thyroid hormones were described in

Chapter 2.


Measurements


All measurements were performed between 0900 and 1200 hr.


Body temperature. The procedures for measurement of Tr and Tts

were described in Chapter 1.


Evaporative water loss (EWL). The open-flow system used for

determination of EWL in rats was detailed in Chapter 1.


Metabolic rate (MR). The oxygen consumption method for measure-

ment of metabolic rate was described earlier (Chapter 1). Oxygen con-

sumption was measured in one rat during each 1 hr experimental session.


Statistical Analysis


Differences between treatments were determined by analysis of

variance. Duncan's multiple range test was used to compare the means.

Significance was assumed when P < 0.05.


Results


Rats adapted to either 24.50 C or 29.20 C displayed elevated

evaporative water loss (AEWL) and tail skin temperature (ATts)

differentials following exposure to 32.50 C for 1 hr (Table 7); the

AEWL and ATts of 24.50 C adapted rats were significantly (P < 0.05)















Table 7. Differential effects of metabolic rate, evaporative water loss and rectal and tail
skin temperature of rats preconditioned to different environmental temperatures and
exposed to 32.50 C for 1 hr.


Differential* n** 24.50 C adapted n 29.20 C adapted P**


Metabolic rate 6 1.62 0.37 6 1.20 0.31 NS
(ml/min kg. 75)

Evaporative water 6 3.19 0.36 6 0.71 0.19 < 0.01
loss (mg/g. hr-)

Rectal temperature 10 0.7 0.18 10 0.7 0.19 NS
(0 C)

Tail skin temperature 10 4.6 0.43 10 1.7 0.43 < 0.01
(o C)


Differential = final measurement 1 hr post-exposure to 32.50 C minus initial measurement
at preconditioned temperature (0 time), expressed as mean S.E.

** Number of rats.

*~* P value denotes significant differences as determined by analysis of variance.








higher than 29.20 C adapted animals. On the other hand, the rectal

temperature (ATr) and metabolic rate (AMR) differentials of both groups

of rats were elevated to comparable levels following acute (1 hr)

exposure to the heat (32.50 C).

The influence of preconditioning temperature on blood levels of

thyroid hormones, corticosterone, and B-END-LI are shown in Table 8.

The thyroid hormones, T3 and T4, were significantly reduced in the

control group of rats adapted to 29.20 C compared to those adapted to

24.50 C. For both groups of rats acute exposure to the heat (32.50 C)

did not change T3 levels. On the other hand, 24.50 C adapted rats

significantly increased T4 levels following acute exposure to 32.50 C;

T4 levels of rats adapted to 29.20 C were not altered on exposure to

32.50 C. Serum corticosterone levels of both groups of rats were

quantitatively similar at control temperatures but were significantly

elevated following 1 hr in the heat (32.50 C).

Control rats adapted to 29.20 C displayed highly significant (P <

0.01) elevations in B-END-LI compared to 24.50 C adapted rats (Table

8). Following exposure to 32.50 C, B-END-LI of 24.5 C adapted rats

was significantly elevated and was similar to that of the 29.20 C

adapted control rats. Rats physiologically adapted to 29.20 C

displayed a slight, but not significant, elevation in B-END-LI

following acute exposure to 32.50 C.

The content of B-END-LI in the NIL of the pituitary gland of

29.20 C adapted control rats was significantly elevated compared to

24.50 C controls, but no differences were detected in the AL (Table 9).

On exposure to 32.50 C, the B-END-LI of the NIL and AL was signifi-

cantly elevated for both experimental groups of rats. Exposure to the











Table 8. Blood levels of thyroid hormones, corticosterone and beta-endorphin-like-
immunoreactivity in rats preconditioned to different environmental temperatures and
exposed to 32.50 C for 1 hr.


24.5 C adapted 29.20 C adapted
Hormone

Control Experimental Control Experimental
(24.50 C) (32.50 C) (29.20 C) (32.50 C)



Triiodothyronine 52.71 2.00** 56.88 1.70a 41.60 0.96b 39.17 1.47b
(ng/dl)

Thyroxine 3.83 0.12a 4.40 0.06b 2.99 0.10' 3.00 0.07c
(pg/dl)

Corticosterone 17.96 0.89a 23.72 1.02b 17.00 0.67a 22.03 1.06b
(pg/dl)

Beta-endorphin-like a b b
immunoreactivity 0.24 0.03 0.56 0.04 0.67 0.09c 0.85 0.07
(ng/ml)



Mean S.E.; values are based on duplicate determinations of samples from 10 rats.

** Values with different superscripts (a-c) differ significantly (P < 0.05) between columns as
determined by analysis of variance and Duncan's multiple range test.


















Table 9. Pituitary beta-endorphin-like immunoreactivity in rats preconditioned to different
environmental temperatures and acutely exposed to 32.50 C for 1 hr.


Temperature ( C) Neurointcrmediate lobe* Anterior lobe


Precondition Experimental ug/gland pg/mg protein pg/gland ug/mg protein


24.5 24.5 5.47 + 0.27a** 32.18 0.24d 0.28 0.01h 0.22 0.01j

24.5 32.5 7.60 0.39b 40.00 0.42e 0.32 0.011 0.27 0.02k

29.2 29.2 8.20 0.34b 51.25 0.37f 0.27 + 0.01h 0.19 + 0.03j

29.2 32.5 9.72 0.53c 54.18 0.418 0.35 + 0.011 0.29 0.02k


Mean S.E.; values are based on duplicate determinations for 10 rats.

Values with different superscripts (a-k) differ significantly (P < 0.05) within each column as
determined by analysis of variance and Duncan's multiple range test.









heat resulted in elevation of the B-END-LI to similar levels in the AL

and NIL for both groups of rats.

Exposure to 32.50 C for 1 hr increased ATr of both groups of rats

by 0.7 C (Table 7). However, the B-END-LI differentials of 24.50 C

and 29.2' C adapted rats were 0.32 and 0.81 ng/ml, respectively (Table

8). These LTr results are similar to those obtained 1 hr post-IVT

injection of varying doses (5-50 pg) of B-END (Figure 11). Moreover,

serum concentration of B-END for preconditioned rats (0.003-0.011 ug)

was qualitatively similar to that of IVT injected rats (0.067-0.007

ug).


Discussion


Elevated MR and Tr commonly occur when animals are exposed to

environmental conditions for which they are unaccustomed or nonadapted

(Herrington, 1940; Yousef and Johnson, 1967) and signals the start of

physiological adjustment to the new environment (Prosser, 1964).

However, in the study reported here, rats adapted to 24.50 C appeared

to dissipate greater amounts of heat from the tail and by evaporation

of water compared to 29.2' C adapted rats. These findings agree with

those of Chapter 1 which suggest that the thermoneutral zone for

24.5' C adapted rats is different from the thermoneutral zone of

29.20 C rats. The different upper critical temperatures of the thermo-

neutral zones of both groups of rats (as described in Chapter 1) may

indicate a changed sensitivity to heat of the sensory component of the

rat's thermoregulatory mechanism. This altered "heat sense" could

account for the different heat dissipation responses of 24.5' C and

29.2' C adapted rats following acute exposure to 32.50 C.








The increased MR observed on exposure to heat may be the result

of increased adrenal (Yousef and Johnson, 1967; Kotby et al., 1967)

or thyroid (Yousef and Johnson, 1968) activity. The secretions from

these glands which include thyroid hormones, catecholamines and

corticosterone, all are calorigenic hormones (Evans et al., 1957;

Collins and Weiner, 1968). In the present experiments (Table 8),

control rats adapted to 24.50 C and 29.2* C had similar levels of serum

corticosterone which indicate physiological adaptation to heat.

Corticosterone increased similarly in both groups after 1 hr exposure

to 32.5 C. The elevated serum corticosterone levels observed in the

present study (Table 8) at 32.50 C probably contributed to the

increased MR (Table 7; Kotby and Johnson, 1967; Kotby et al., 1967).

Increased corticosterone secretion has been observed in rats adapted to

24.50 C and exposed to experimental temperatures of 29.20 C (Attah and

Besch, 1977) and 32.50 C (Gwosdow-Cohen et al., 1982, Appendix B). It

has been reported (Yousef and Johnson, 1967; Gwosdow-Cohen et al.,

1982, Appendix B) that corticosterone levels peak 24 hr after exposure

to heat before returning to control levels.

On physiological adaptation to heat, thyroid activity decreases

(Dempsey and Astwood, 1943; Chaffee and Roberts, 1971) and MR returns

to control levels (Gelineo, 1934). Yousef and Johnson (1968) attribute

part of the decline in MR to the concomitant decreased thyroid

activity. Similar findings have been reported by Kotby and Johnson

(1967) and Kotby et al. (1967). In the present study, 29.20 C adapted

rats maintained significantly reduced levels of T3 and T4 compared to

their 24.50 C counterparts but on acute exposure to heat, the 24.5' C

animals had elevated T4 levels only. The reduced thyroid hormone








levels of 29.20 C adapted rats, compared to 24.50 C adapted rats, and

the maintenance of this lowered level following exposure to 32.50 C are

additional indicators of the rat's physiological adaptation to heat.

The elevated MR displayed by 29.20 C and 24.50 C adapted rats

after 1 hr in the heat (Table 7) may be due to the actions of

corticosterone or may represent the initial response of these rats to

heat. Three hr after exposure to 32.50 C (Chapter 1), the 29.20 C

adapted rats had significantly lowered MR compared to 24.50 C adapted

rats. This reduction in MR in 29.20 C rats (Figure 6) may be due

partially to the lower thyroid hormone levels maintained by these rats

(Table 8). The resultant lower thyroid activity nay be responsible for

the decreased MR and heat output observed in the heat adapted animals

(Cassuto and Chaffee, 1966).

Circulating levels of B-END-LI increase in the heat (Mueller,

1980; Deeter and Mueller, 1981) and in response to stressful stimuli

(Mueller, 1981). Mueller (1981) suggests that the increase in B-END-LI

in the blood is directly related to the intensity of the stimulus. In

the experiment reported here, 29.20 C adapted rats had elevated

B-END-LI compared to 24.50 C adapted animals. On exposure to heat, the

levels of B-END-LI significantly increased in 24.50 C animals, but not

in the 29.20 C group. Moreover, at 32.50 C both groups of rats

maintained comparable levels of B-END-LI. These data suggest that

32.5 C was not a "stressful" environment for the 29.20 C adapted rats

compared to the 24.50 C adapted animals.

Mueller (1980) found that approximately 95% of the rat's B-END-LI

is located in the NIL. The data presented in Table 9 support these

findings. The B-END-LI content of this tissue was increased in 29.20 C









adapted control rats compared to the 24.50 C controls and was elevated

in both groups of rats in the heat. While Guilleman et al. (1977)

suggest that changes in B-END-LI reflect changes in pituitary secre-

tion, the data reported herein suggest that pituitary content of

B-END-LI is elevated concomitantly with increases in circulating

levels. This may indicate that stimuli which release B-END-LI into the

bloodstream also increase the synthesis or delivery of B-END-LI to the

pituitary gland (Bergland and Page, 1979).

The thyroid and corticosterone responses reported in this chapter

are similar to those reported for heat adapted rats (Dempsey and

Astwood, 1943; Kotby and Johnson, 1967). Preconditioning and heat

exposure appear to have altered the blood and pituitary levels of

B-END-LI, and these hormonal and rectal temperature responses mimic

those induced by IVT injections of B-END (Chapter 2). Therefore,

B-END may be responsible for the Tr increases observed in the

experiments reported herein and may play a role in the process of

physiological adaptation to heat.

In order to compare the response of IVT injected B-END with

B-END-LJ resulting from heat exposure, the metabolic clearance rate and

half-life of B-END need to be estimated. Little is known about the

metabolic clearance rate of B-END, but Houghten et al. (1980) reported

the plasma half-life of B-END was 45 min. Skeleton (1927) estimated

that a 300 g rat has 195.9 ml of water of which 13.3 ml (6.9%) is

blood. Reilly et al. (1980) demonstrated that 0.3% of the B-END

injected IV crosses the blood-brain barrier. From these data, it is

possible to calculate the total concentration of serum B-END-LI in

preconditioned and IVT injected rats. The similarity of obtained




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