111

Tica JOURNAL o PHARMACOLOGY AND EXPZRIMENTAL THERAPEUTICSCopyright 1967 by The Williams & WilkinsCo.

Vol. 157, No. 1Printed in U.S.A.

THE SYMPATHETIC NERVOUS SYSTEM AS A HOMEOSTATICMECHANISM. II. EFFECT OF ADRENOCORTICAL HORMONES

ON BODY TEMPERATURE MAINTENANCE OFCOLD-EXPOSED ADRENALECTOMIZED RATS

R. P. MAICKEL, D. N. STERN, E. TAKABATAKE4 AND B. B. BRODIE

Laboratory of Chemical Pharmacology, National Heart Institute, Bethesda, Marylandand Departments of Pharmacology and Psychology, Indiana University,

Bloomington, Indiana

Accepted for publication February 16, 1967

ABSTRACT

MAICKEL, R. P., D. N. STERN, E. TAKABATAKE AND B. B. BRODIE : The sympathetic nervoussystem as a homeostatic mechanism. II. Effect of adrenocortical hormones on body tempera-ture maintenance of cold-exposed adrenalectomized rats. J. Pharmacol. Exp. Therap. 157:111-116, 1967. Intact rats exposed to low environmental temperature (4#{176}C)maintain bodytemperature by piloerection, vasoconstriction, shivering and mobilization of glucose andfree fatty acids. In contrast, totally adrenalectomized rats are unable to activate any ofthese systems ; they lose body heat rapidly and die in a few hours when exposed to lowtemperature. The sugar- and lipid-mobilizing actions of exogenous catecholamines are alsomarkedly reduced by adrenalectomy. The inability of adrenalectomized rats to survive inthe cold can be reversed by acute treatment with glucocorticoids such as cortisone, dexa-methasone or prednisolone or by chronic treatment with aldosterone. This treatment alsorestores responsiveness to catecholamines, suggesting that the adrenalectomized rat may beconsidered as a functionally sympathectomized animal because of impaired responsivenessof effector systems which normally are activated by sympathetic function.

The preceding paper (Maickel et at., 1967)has described the similarity of responses inadrenalectomized and chemically sympathecto-mized rats exposed to cold. The evidence sug-gested that the failure of adrenalectomized ani-mals to maintain normal body temperature ina cold enviroment was due to a breakdown ofperipheral sympathetic functions. Since theeffects of adrenalectomy were reversed by ad-ministration of cortisone, but not by admin-istration of catecholamines, it seemed rea-sonable that the defect in adrenalectomy wasdue to the lack of adrenal cortical hormones.

A number of reports in the literature mdi-cate that adrenalectomized animals are poorly

Received for publication October 21, 1966.1 Supported in part by U.S. Public Health

Service Grant MH-06997.2 Present address : Departments of Pharmacology

and Psychology, Indiana University, Bloomington,Ind. 47401.

5 Present address: Department of Psychiatry,New York State Psychiatric Institute, New York,N.Y.

4 Present address : Pharmaceutical Faculty, Na-gasaki University, Nagasaki, Japan.

responsive to administration of catecholamines(Shafrir and Steinberg, 1960; Shafrir et at.,1960 ; Maickel and Brodie, 1963) . Similarly,the increased sensitivity of adrenalectomizedanimals to cold-exposure is also well known(Secker, 1939 ; Sawyer and Schlossberg, 1933).

In both of these situations, the responses ofadrenalectomized animals can be restored byadministration of cortisone or hydrocortisone.Thus, the breakdown of temperature regulationin cold-exposed, adrenalectomized rats wouldbe due to the lack of adrenocortical function.

The present investigation of the impairedresponse of energy-producing mechanisms incold-exposed, adrenalectomized rats was de-signed to test the ability of various steroidsto reverse the effects of adrenalectomy. Sinceadrenalectomized animals also show decreasedresponses to the sugar- and lipid-mobilizingeffects of catecholamines, these responses wereused as a comparison. The results are con-sistent with the hypothesis that the adreno-cortical hormones maintain the functionalresponsiveness of peripheral sympathetic ef-

112 MAICK.EL ET AL. Vol. 157

fectors. Thus, the adrenalectomized animalmay also be considered as a functionallysympathectomized animal.

METHODS. Adult, male, Sprague-Dawley rats(170-200 g) were used in all experiments. Adrenal-ectomized rats were obtained from the HormoneAssay Laboratories, Chicago, Ill., and were main-tamed on normal laboratory chow (containing0.5% NaCl) and drinking water containing 1%NaCl-5% glucose until used 4 to 10 days aftersurgery. Measurement of plasma levels of corti-costerone by the method of Guillemin et at.(1959) indicated no measurable steroid content.

Animals were exposed to cold (4#{176}C)in in-dividual screen wire cages. The steroids used werecommercially obtainable materials, administeredas described in the tables.

Rectal temperatures were measured with asmall animal probe and a Yellow Springs Tele-Thermometer. Animals were stunned, decapitatedand blood was collected into heparin-treatedbeakers. After transfer to tubes, the blood wascentrifuged immediately and aliquots of plasmawere used for the determination of free fattyacids (FFA), by the method of Dole (1956).Blood glucose levels were determined with theGlucostat (Worthington Biochemical Corpora-tion) reagent, and liver glycogen was determinedby the method of Carroll et at. (1956).

Calculations of estimated mean survival time(M.S.T.) and rate of change of heat content(-dQ/dt) were made as described in the precedingpaper (Maickel et al., 1967).

Materials. Cortisone acetate, aldosterone, dex-amethasone, prednisolone, d,t-norepinephrine hy-drochioride and t-epinephrine bitartrate werecommercially available compounds.

RESULTS. Effect of NaC1 deprivation on body

temperature of cold-exposed adrenalectomizedrats. Since the adrenalectomized animal hasan impaired ability to retain Nat, a studywas made of the effects of a decreased Na in-take on the ability of adrenalectomized ani-mals to maintain their body temperature whenexposed to cold. The data in table 1 show theeffects of drinking tap water for 1, 2 or 3 dayson the ability of adrenalectomized rats tomaintain body temperature in the cold. Witheach succeeding day of decreased Na intake,the body temperature fell at a more rapid rate.After 3 days of drinking tap water, the rateof heat loss was more than twice the rate ofadrenalectomized rats drinking 1% NaC1-5%glucose. Apparently, the greater Na imbalancereduced the ability of the animals to main-tam their internal temperature when exposedto cold.

Further evidence for this conclusion isgiven in table 2, a comparison of the abilityof adrenalectomized rats drinking 1% NaCl or5% glucose to maintain body temperature inthe cold. It is evident that glucose alone didnot prevent the rapid loss in body temperature,while NaCL alone was as effective as the NaC1-glucose mixture usually provided to the ani-mals. It is interesting to note that the animalsgiven only glucose did not lose much heat inthe first 2 hr of exposure, presumably owingto the higher level of blood glucose. The energysupplied by this source, however, was rapidlyused up-by 4 hr of exposure the body tem-perature of these animals was falling rapidly.

Reversal of effects of adrenalectomy byacute treatment with steroids. Since Na dep-

TABLE 1Eff!ts of partial salt deprivation on body temperature of adrenalectomized rats exposed to cold

Days on H,OObr lhr 2hr 3hr 4hr

M.S.T. .-dQ/dt

0123

oc

36.7 0.8 34.4 2.2 31.7 2.9 28.3 3.4 24.5 2.336.6 0.5 33.9 0.5 32.2 0.6 28.9 1.2 23.9 2.035.0 0.5 31.1 1.6 27.8 2.5 21.1 3.2 18.9 3.435.6 0.6 30.0 1.3 25.6 2.5 20.6 2.3 12.8 1.4

hr

7.97.25.23.8

cal/kr

546603765

1083

Each value is the mean S.E. of 14 animals. Animals were maintained on 1% NaCl-5% glucose indrinking water for 4 days after surgery, then tap water was substituted for 1, 2 or 3 days prior to cold-exposure. The fall in body temperature of animals maintained on NaC1-glucose did not vary signifi-cantly when exposed to cold on days 4 through 10 after surgery.

Rectal Temperature after Cold-Exposure for

1967 HOMEOSTATIC MECHANISMS. II 113

Effect of steroid hormones on lipolytic and

TABLE 2

Differential effect of drinking water containing NaCI or glucose on ability of adrenalectomized rats tomaintain body temperature when exposed to cold

TreatmentRectal Temperature after Cold-Exposure for

M.S.T. -dQ/dtOhr lhr 2hz 3hz 4hz

1% NaCI 5% glu-cose

1% NaCl (1 clay)1% NaCl (3 days)5% Glucose (1 day)5% Glucose (3 days)

.c

36.7 0.8 34.4 E 2.1 31.7 2.9 28.3 3.4 24.5 2.3

36.7 0.4 32.8 0.6 30.6 1.4 28.9 1.4 27.2 1.536.9 1.2 33.4 0.4 32.2 0.6 30.0 0.3 27.7 1.136.1 0.7 32.2 0.5 31.1 1.0 25.6 1.8 18.3 2.036.6 0.6 31.7 1.1 28.4 1.9 27.2 1.8 18.9 2.6

hr

7.9

8.48.66.06.1

cal/hr

546

461437846841

Each value is the mean S.D. of six animals except that NaCl-glucose controls are 14 animals. Ani-mals were maintained on 1% NaCl-5% glucose for 4 days after surgery, then groups were treated asdescribed below.

TABLE 3

Protective effect of acute administration of various steroids on adrenalectomized, salt-deprived ratsexposed to cold

Steroid TreatmentRectal Temperature after Cold-Exposure for

M.S.T. -.dQ/d:Ohr lhr 2hr 3hr 4hr

ControlAidosteroneCortisoneDexamethasonePrednisolone

oc

35.0 0.5 31.1 1.6 27.8 2.5 21.1 3.2 18.9 3.436.1 0.4 31.7 1.3 28.9 2.4 23.9 3.4 21.7 4.935.6 0.6 84.5 0.4 83.4 0.5 32.8 0.8 81.7 1.136.0 0.6 82.8 0.4 8L2 0.8 SI. 0.5 -35.6 0.8 32.2 0.5 81.7 1.0 30.0 1.8 30.8 1.8

hr

5.26.0

21.718.218.3

cal/kr

765684186209238

Each value is the mean S.D. of nine animals, except that H20 controls are 13 animals. Animalswere maintained on tap water for 2 days, then given cortisone acetate (26 mg/kg i.p., as an aqueoussuspension), prednisolone (5 mg/kg s.c., in oil), dexamethasone (0.126 mg/kg i.m., as an aqueous solu-tion) or aldosterone (1.25 mg/kg s.c., in oil), 4 hr before exposure to cold. Values in italics are signifi-cantly different from untreated controls at each time point (P < .05).

rivation seemed to be damaging to the tem-perature-maintaining mechanisms, the pro-tective effect of various steroids administeredprior to cold-exposure was examined. The testsystem chosen was adrenalectomized rats thathad been drinking tap water instead of 1%NaC1-5% glucose for 2 days. A single doseof steroid was administered 4 hr prior to cold-exposure. The rectal temperature changes dur-ing subsequent cold-exposure (table 3) demon-strate that all the steroids except aldosteronewere effective in reversing the loss of bodyheat of the adrenalectomized rats. Thus, itwould seem that aldosterone was unable to re-verse the effects of adrenalectomy and salinedeprivation in a 4-hr period of treatment.

Reversal of effects of adrenalectomy bychronic administration of steroids. Furtherstudies were carried out in which adrenalecto-mized animals were given daily doses of thesteroids during the 2-day period while theywere drinking tap water instead of 1% NaC1-5% glucose. Under these conditions, all of thesteroids tested were able to reverse the effectsof adrenalectomy (table 4). When comparedwith table 3, these results suggest that the ac-tion of aldosterone in reversing the effects ofadrenalectomy is a slower process than that ofcortisone, dexamethasone or prednisolone, sug-gesting a different locus or mechanism of ac-tion.

MAICKEL ET AL. Vol. 157

TABLE 4Protective effect of chronic administration of various steroids on adrenalectomized, salt-deprived rats

exposed to cold

Steroid Treatment

Rectal Temperature after Cold-Exposure forM.S.T. -dQ/di

Ohr lhr 2hr 3hr 4hr

ControlAldosteroneCortisoneDexamethasonePrednisolone

oc

35.0 0.5 31.1 1.6 27.8 2.5 21.1 3.2 18.9 3.436.1 0.6 35.0 0.8 33.9 1.4 31.7 5.4 82.2 1.136.2 0.4 86.1 0.9 35.0 1.5 33.9 1.4 31.1 1.436.7 0.4 37.2 0.4 35.6 0.4 53.9 0.8 -36.1 0.6 35.0 0.9 53.4 1.1 32.5 1.3 30.6 2.1

hr

5.223.2

19.320.519.5

cal/hr

765185

242177262

Each value is the mean S.D. of seven animals, except that H,O controls are 13 animals. Animalswere maintained on tap water for 2 days, and steroids were given once daily at the doses described intable 4. The exposure to cold was made approximately 16 hr after the last dose of steroid. Values initalics are significantly different from controls at each time point (P < .05).

TABLE 5

Effect of steroid pretreatment on glycolytic and lipolytic response to catecholamines in adrenalectomized rats

Animals Treatment

Plasma FFA Plasma GlucoseUver

Glycogen(Control)Control Norepinepbrine Control Epinephrine

pEq/ml mg/I 00 ml mg/g

Intact control 0.35 0.05 0.84 0.07 112 3 52 14 31 6Adrenalectomy + 0.37 0.03 0.49 0. 10 87 5 ii 16 io 2

1%Adrenalectomy + 0.45 0.08 0.41 0.04 59 5 78 15 7 2

tap water (2 days)Acute

AldosteroneCortisoneDexamethasonePrednisolone

ChronicAldosteroneCortisoneDexamethasonePrednisolone

0.27 0.010.33 0.060.37 0.06

-

0.34 0.030.40 0.030.36 0.06

-

0.31 0.040.71 0.110.57 0.15

-

0.64 d 0.080.80 0.060.87 0.16

-

81 886 7

-

95 9

77 16113 5

-

107 11

89 10117 11

-

138 ii

136 1217 15

-

195 10

12 423 6

-

-

17 729 3

-

-

Each value is the mean S.D. of six animals. Steroid pretreatments were as described in table 3(acute) and table 4 (chronic). Plasma FFA elevations were induced by norepinephrine (0.1 mg/kg,aqueous, i.m.) and blood glucose by epinephrine (0.2 mg/kg, in oil, i.m.), 30 mm before measurement ofblood levels. Values in italics indicate significant response to catecholamines (P < .05).

glycolytic actions of catecholamines in adrenal- exposure and to the administration of cate-ectomized animals. The increased levels of cholamine seemed more than coincidental. Theplasma FFA and blood glucose evoked by ex- results of studies of the effects of steroid pre-ogenous catecholamines were markedly reduced treatment on the lipolytic and glycolytic ac-in adrenalectomized animals (Maickel et at., tions of catecholamines in adrenalectomized1967) . This similarity between the reduced rats are shown in table 5. The response ofresponses of adrenalectomized animals to cold- plasma FFA levels to norepinephrine mirrored

1967 HOMEOSTATIC MECHANISMS. II 115

the results obtained with cold-exposure ; acuteadministration of cortisone or dexamethasonerestored the lipolytic action of the norepi-nephrine, while aldosterone was ineffective.In contrast, chronic administration of all threesteroids restored the FFA response to norepi-nephrine.

The results for blood glucose levels weresomewhat more complicated, although the samegeneral picture was obtained. Treatment witha single dose of cortisone or prednisoloneshortly before the catecholamine test broughtthe glucose response back to the level of the ad-renalectomized animal maintained on saline,while aldosterone had no effect. The lowerresponse of the steroid-treated animals waspresumably due to a lower initial level of liverglycogen. This possibility was strengthened bythe results obtained after treatment with ster-oids for 2 days. With the longer period oftreatment, liver glycogen stores were returnedto levels near normal and the epinephrine-induced glycosuria was close to that observedin normal animals. Again, the animals treatedwith aldosterone had a poor response, pre-sumably reflecting the lower glucocorticoidactivity of this steroid.

DISCUssIoN. The decreased ability ofadrenalectomized animals to withstand ab-normal environmental conditions is wellknown. A general opinion has held that thelack of adrenal corticoids causes an increasedsensitivity to stress, but that the adrenalec-tomized animal can survive in a careful con-trolled environment. As recently as 1957,Ramey and Goldstein (1957) considered theprimary physicochemical site of action ofthe adrenal hormones to be obscure. Thedata presented in the present paper are consis-tent with the hypothesis that the adrenocorticalsteroids are necessary to maintain the func-tional responsiveness of the effector systems,enabling the animal to respond to changes inenvironmental conditions which require adap-tation of such systems.

The supportive action of the adrenal cor-tical steroids is replaceable to some extent byordinary saline. In fact, adrenalectomizedrats can be kept alive for some time on anadequate intake of saline provided they arenot subjected to situations requiring extra

metabolic energy. This was demonstrated byexposing rats to cold after maintaining themon tap water instead of saline. The longer theanimals were deprived of saline, the morerapid was the decline in body temperature.Rats maintained on saline until exposure tocold survived almost 8 hr ; with each succes-sive day of salt deprivation, cold-exposureproduced a more rapid decline in tempera-ture until finally the animals deprived ofsaline for 3 days and placed in the cold sur-vived less than 4 hr. These animals were un-able to mobilize extra energy substrates and toconserve body heat. The rate of heat loss wasmore than 1000 calories/hr (cal/br) , a ratesimilar to that seen after chemical sympathec-tomy. The results show that the lack of salineexacerbates the effects of corticoid insuffi-ciency and suggest that imbalance of electro-lvtes may be responsible in some way for theinability of adrenalectomized rats to respondto situations requiring an increased energyexpenditure. It seems particularly pertinentthat adrenalectomized animals, exposed to coldafter 2 days without saline, display no signs ofpiloerection, vasoconstriction or shivering.Furthermore, even exogenous catecholaminesdo not increase levels of fatty acids or glucose,suggesting that effector systems are no longerresponsive to catecholamines. That ionicchanges are probably involved in this break-down of the responsiveness to catecholaminesis further suggested by the studies which showthat adrenalectomized rats maintained onglucose are unable to maintain their bodytemperature in the cold.

Evidence that the effects of adrenalectomyare due to the lack of steroids comes from thereplacement experiments described in tables 3and 4. For example, pretreatment of adrenalec-tomized rats with cortisone, dexamethasoneor prednisolone 4 hr prior to cold-exposureenables the animals to maintain their bodytemperature. The steroid-pretreated animalshad -dQ/dt of approximately 200 cal/hr, lessthan % that of untreated, adrenalectomizedrats. In contrast, pretreatment with a singledose of aldosterone had virtually no protec-tive action under these conditions ; such ani-mals lost heat almost as fast as untreated rats.

However, if the animals were given daily

116 MAICKEL ET AL. Vol. 157

cose and FFA.

doses of steroids, aldosterone was just as ef-fective as the other steroids (table 4) in en-abling the adrenalectomized animals to main-tam their body temperature in the cold. Onemight speculate that the glucocorticoids areeffective in some acute manner, while al-dosterone is effective through its renal actionon sodium, thus requiring a longer time. Adifference in actions of gluco- and mineralo-corticoids has been reported by Swingle etat. (1960), who found that prednisolone main-tains vigor and normal activity in adrenalec-tomized dogs despite a low level of plasmasodium and chloride. In contrast, deoxycor-ticosterone acetate in doses that increased thelevels of plasma sodium and chloride did notcounteract the fall in plasma volume and bloodpressure.

In summary, it is not unreasonable to pos-tulate that the lethal effect of cold on adrenalec-tomized rats may be attributed to the poorresponsiveness of the end-organs to cate-cholamines. This concept is strengthenedby the data presented in tables 3 and 4.Adrenalectomized animals, exposed to cold, areunable to mobilize energy substrates such asglucose and FFA to meet the needs of theirbodies for additional metabolic fuel. They arealso unable to conserve body heat and theirtemperature drops rapidly until death occurs.Pretreatment of the animals with steroids re-stores the ability of the animals to vasocon-strict, pioerect, shiver and to mobilize glu-

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