Effect of chronic exposure to cold on some responses to catecholamines M. J. FREGLY, F. P. FIELD, E. L. NELSON, JR., P. E. TYLER, AND R. DASLER Department of Physiology, Colleges of Medicine and Pharmacy, University of Florida, Gainesville, Florida 32610
FREGLY, M.J.,F. P. FIELD, E.L. NELSON, JR., P.E. TYLER, AND R. DASLER. Effect of chronic exposure to cold on some responses to catecholamines. J. Appl. Physiol.: Respirat. Environ. Exercise Physiol. 42(3): 349-354, 1977. -An objective of these studies was to test the responsiveness of cold-adapted (8 wk, 5°C) rats to a specific P-adrenergic agonist. Twenty-four hours after removal from cold, increases in tail skin temperature (?I,,,) and colonic temperature (T,.,,) were measured for 2 h in air at 25°C following subcutaneous (SC) administration of 28, 70 or 136 pg d,b-isoproterenol sulfate dihydrate/kg body wt to restrained male rats. Cold-adapted rats responded to each dose of isoproterenol with greater increases in Tsr( than controls. T(.(, of both groups increased at the two highest doses, but coldadapted rats showed a greater rise. Thus, cold-adapted rats showed greater metabolic (T,$,,) and vascular (?I,,> responsiveness to the P-adrenergic agonist, isoproterenol, than nonadapted controls. No effect of the a-adrenergic agonist, phenylephrine (50 or 100 ,ug/kg body weight, SC), was observed on TcBo or Tsl,. A second objective was to study the tension developed by aortic smooth muscle rings of cold-adapted and control rats both during stimulation of cr-adrenergic receptors by norepinephrine and membrane depolarization by KCl. Adaptation to cold air appeared to suppress cu-adrenergic responsiveness in aortic segments but did not alter responsiveness to KCl. This suggests an unchanged contractile mechanism in aortic rings of cold-adapted rats and a reduced responsiveness either at the level of the a-receptor or at a site immediately beyond. cold adaptation; isoproterenol; perature; vascular reactivity;
norepinephrine; aortic rings
tail skin tem-
RATS have an enhanced metabolic responsiveness to both norepinephrine (NE) and epinephrine (E) (3, 4, 8, 9, 16). Th is increased responsiveness has been termed “nonshivering thermogenesis” since it appears to replace the metabolic effects of shivering during the process of adaptation to cold in rats. The increased responsiveness to both NE and E is accompanied by an increase in their rate of production during both acute and chronic exposure to cold (11, 12, 15). These catecholamines may play an important role in mobilization of substrates for energy production, including carbohydrate and lipid, and in this way may participate in nonshivering thermogenesis. The relative importance of endogenous NE, as compared with endogenous E, for nonshivering thermogenesis in cold-adapted rats is not yet settled. Equally unsettled is the importance of cy- versus p-adrenergic COLD-ADAPTED
stimulation for maintenance of nonshivering thermogenesis in cold-adapted rats. Both NE and E have the potential for stimulating cy- and p-adrenergic receptors although the dominant effect of each catecholamine varies considerably from organ to organ. Studies have shown that pharmacological blockade of the P-adrenergic receptors reduced survival of mice exposed acutely to cold (51, and increased both shivering and fall in colonic temperature of cold-adapted rats that were anesthetized and exposed acutely to cold air (14). Although evidence is gradually accumulating that the P-adrenergic receptors are primarily important for the maintenance of nonshivering thermogenesis in coldadapted rats, the issue is not completely settled. The experiments described here were carried out to test the metabolic responsiveness of cold-adapted rats to a P-adrenergic agonist, isoproterenol. Other studies were carried out to test the vascular reactivity of coldadapted rats to the cx-adrenergic effects of norepinephrine. METHODS
Experiment 1. Effect of isoproterenol on tail skin and colonic temperatures of cold-adapted and control rats. Six male rats of the Blue-Spruce Farms (Sprague Dawley) strain were kept in individual wire cages in a room maintained at 5 t -1°C for 8 weeks. Six additional male rats served as controls and were maintained in individual wire cages in air at 25 t 1°C. Illumination in both the cold and warm rooms was from 8 A.M. to 6 P.M. All rats received tap water and Purina laboratory chow ad libitum. At the end of the 8-wk period, each rat was restrained in a tunnel-type cage containing a galvanized, mesh wire cover and a wooden floor (1). The cage was large enough to hold a rat comfortably but prevented movement of the rat from head to tail. Each rat had a copperconstantan thermocouple inserted 5 cm beyond its anal orifice and held in place by a small piece of adhesive tape. A second thermocouple was woven into one layer of a gauze sponge, 2.5 cm wide. The junction of this thermocouple contacted the skin at the base of the tail. A piece of adhesive tape was placed at the front and rear of the gauze sponge to hold it in place. The thermocouples were led off to a potentiometer that recorded the temperatures at 1-min intervals.
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350 An initial series of experiments was carried out when it was observed that rats removed from air at 5°C to air at 25°C for 1 h maintained a fully dilated tail with increased tail skin temperature (T,& Three days later, the same group of cold-treated rats was returned to air at 25°C for 5 h prior to measurement of tail skin and colonic temperatures (T,.,,). At the end of a 30-min control period, isotonic saline (0.2 ml) was injected subcutaneously through a slot in the cage. Tsk and Tco were then measured for an additional 2 h. Rats that had been maintained in air at 25OC served as controls. Because Tsk of cold-treated rats was still elevated even at 5 h after removal from cold, the cold-adapted group was removed from cold for 24 h before temperature measurements began. At the end of the 30-min control period, all rats were administered isotonic saline subcutaneously (SC). Temperatures were measured for an additional 2 h. Rats adapted to 25OC served as controls. Since a 24-h sojourn in air at 25°C was required for stability of Tsk in cold-adapted rats, all subsequent experiments followed this procedure. Three additional identical studies separated by 3-4 days from each other were performed on the same rats. The experiments were the same in detail as those described above except that d,Z-isoproterenol sulfate dihydrate (28, 70, and 139 ,ug/kg body wt) was administered SC at the end of the control period. The dose was calculated as the salt. Tsk and Tco were measured for an additional 2 h after treatment. Rats adapted to 25°C served as controls. Experiment 2. Effect of phenylephrine on tail skin and colonic temperatures of cold-adapted and control rats. These studies were carried out in the same fashion as those in experiment 1 except that the a-adrenergic agonist, phenylephrine, at doses of 50 and 100 r_Lg/kg body wt, was administered SCat the end of the control period. Experiment 3. Vascular reactivity of cold-adapted rats to norepinephrine and potassium chloride. Wistar (CFN) male rats weighing 150-180 g were divided into two groups. Twelve rats were maintained in air at 5 t 1°C for 6 wk while 12 control rats were maintained at 26 t 1°C. Both rooms were illuminated from 8 A.M. to 6 P.M. All rats received tap water and Purina laboratory chow ad libitum. During the 7th through the 11th wk of the study, one rat from each group was anesthetized with ether and killed by cervical dislocation. At death, 3-cm segments of aorta (taken 1 cm from the aortic arch) were excised and immediately placed in a modified Krebs solution. After adherent fat and loose connective tissue were removed, one or two 4-mm rings were cut from each segment by the use of two surgical blades mounted on a plastic block. The segments were suspended between two stainless steel hooks inserted into the lumen to record circular muscle contraction. The isolated tissue was mounted in a 20-ml water-jacketed muscle bath and equilibrated for 3 h under 2 g of tension which was maintained. Contractions were recorded isometrically, employing a Narco-Bio microdisplacement transducer (F-50) with a Narco-Bio Physiograph (DMP4B) recorder.
FREGLY
ET
AL.
The modified Krebs solution containing dextrose (11 mM) and disodium EDTA (0.01 mM) was maintained at 37°C and aerated with an O,-CO, mixture (95:5%) which maintained the pH at 7.30. The bath solutions were changed once every 15 min during equilibration and between dose-response determinations. After the 3-h equilibration period, an accumulative norepinephrine dose-response curve was determined on each segment. About 90 min after the norepinephrine was washed from the bath, KC1 was added accumulatively. Statistical analysis of all data was made by means of the Student t-test for the 95% confidence limit (12). RESULTS
Experiment 1. One hour after removal from cold air Tsk was approximately 30°C. During the 30-min control period and the 2 h following SCadministration of saline, Tsk remained high and varied from 27.2 to 30.2”C. In contrast, Tsk of the warm-adapted group during the control period was approximately 24OC and increased 0.8OC after saline injection but fell within 20 min to preinjection level. Tsk of the cold-adapted group was significantly (P < 0.05-0.01) higher than that of the control group throughout the experiment while T,() of the two groups were not significantly different with both declining about 0.6OCduring the treatment period. Five hours after removal from cold air, Tsk of the coldadapted rats varied from 28.4 to 3O.O”C. Tslcdecreased about 2°C following administration of saline and reached control level by the end of the 2-h experimental period. Tsk of the control group declined about 1°C during the experimental period. T 0.05) different from the control group and by 24 h, both groups had attained nearly identical Tsk values (Fig. lA >, Tco of the two groups did not differ significantly in any of the three studies (Fig. W Administration of the lowest dose of d,Z-isoproterenol (28 r-cglkg) to the control group induced an average rise in Tsk of about 1.5”C and an average rise in T(.() of approximately 0.3”C (Fig. 2A). Although the coldadapted group appeared to have a greater mean increase in Tsk than the control group, the difference
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COLD-TREATED
RATS
AND
351
CATECHOLAMINES
A
CONTROL
30
GROUP
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1. Mean tail skin (A) and colonic (B) temperatures of coldadapted and control rats, measured 30 min after SC administration of saline, are shown at 1, 5, and 24 h after removal from cold air. One standard error is set off at the means.
60 MINUTES
FIG.
between groups was not significant at any time during the experiment (Fig. 2B). TcOof the cold-adapted group remained stable throughout the experiment and did not differ significantly from that of the control group at any time. Administration of 70 pg d,Z-isoproterenol/kg of body wt to the control group increased Tsk 54°C (Fig. 3A). An early rise (0.3”C) in TcOmay have preceded slightly the rise in Tsk. Cold-adapted rats appeared to have a somewhat greater rise in both Tsk (7.1"C) and TcO(0.9OC)than the control group (Fig. 3B). Tsk of the cold-adapted group exceeded significantly (P < 0.05) that of the control group during the first 20-min period following administration of isoproterenol. No differences between mean Tsk of the two groups were observed thereafter. Mean T.. of the two groups differed from each other (P < 0.05) only at 120min after administration of isoproterenol. When 139 rugd,l-isoproterenol/kg body wt was administered to the control group, a striking increase in Tsk (75°C) occurred (Fig. 4A). A slight (0.3”C) but not significant rise in TcOoccurred followed by a decline. In the case of the cold-adapted group a greater elevation of Tsk (9.l”C) occurred than was observed in the control group (Fig. 4B). Mean Tsk of the cold-adapted group did not differ significantly from the control group prior to administration of isoproterenol. Following administration of isoproterenol, mean Tsk of the cold-adapted group differed significantly (P < 0.05) from the control group at 30, 40, 50, 60, and 70 min. Thereafter, Tsk of the two groups no longer differed significantly. TcO of the two groups also differed significantly (P < 0.05-0.01) at 20, 30,40, 50, and 60 min after administration of isoproterenol, with the cold-adanted g-roun being the higher.
80
FIG. 2. Tail skin (Left ordinate) and colonic (r&ht ordinate) temperatures of control (A) and cold-treated (B) groups following SC administration of d,L-isoproterenol (28 pg/kg body wt) at 0. One standard error is set off at the means. CONTROL d,~ I
GR3UP ISOPR0TERENOL (70 pg/kg B.W. ‘3 C.!
TAIL SKIN TEMP
1
I 0
I 20
1
I I 1 40 60 MINUTES
1
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TAIL SKIN TEMC
LCOLONIC I 1 ITEMP 1 L1 I 1 1 11 FIG.
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2 except at 0.
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d,Z-isoproterenol
(70
pg/kg
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FREGLY
352
DISCUSSION
of these experiments
was
to test the COLD-TREATED
GROUP TAIL SKIN TEMI?
35CONTROL
33A 34
GROUP
349
t
d.1 ISOPROTERENOL (139 yg/kg B.W. S.C.)
AL.
responsiveness of cold-adapted rats to cy- and P-adrenergic agonists. The increase in Tsk of rats in response to administration of isoproterenol has been studied under other conditions in this laboratory (2, 7). It appears to arise as a result of specific p-adrenergic stimulation and can be blocked by the P-adrenergic antagonist, propran0101 (7). In addition, an increase in Tsk could not be elicited in either warm- or cold-adapted rats by administration of the a-adrenergic agonist, phenylephrine (7) (Fig. 5). Using the increase in Tsk as a criterion, the results of these studies show that adaptation to cold air is accompanied by an increased responsiveness to p-adrenergic stimulation in the rat (Figs. 2-4). LeBlanc et al. (10) have arrived at a similar conclusion using as criteria both the heart rate and rate of increase in oxygen consumption in response to an acute infusion of isoprotere-
Experiment 2. The adrenergic agonist, phenylephrine, at either 50 pg/kg or 100 pg/kg body wt had no significant effect on either Tsk or TccJof control and coldadapted rats. Since this was the case the results shown in Fig. 5 are for the higher dose only. Experiment 3. The tension developed by aortic rings of control and cold-adapted rats following the addition of norepinephrine to the test bath is shown in Fig. 6A. The aortic vascular smooth muscle reactivity to norepinephrine was reduced significantly in cold-adapted rats compared with controls. In contrast, the response to KC1 depolarization was not significantly altered by prior cold exposure (Fig. W).
The objective
ET
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COLON/C TEMP. I I IL I loo 120
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z
-
FIG. 4. Same d,l-isoproterenol was administered
as Fig. 2 except that (139 pg/kg body wt) at 0.
8 41 r 8 0 4o+ 2 392 c 3e; cg 375
MINUTES
MINUTES
CONTROL GROUP (5) A. PHENYLEPHRINE (100 pg/kg. SC. )
27~
COLONIC
TEMP
FIG. 5. Same as Fig. 2 except that phenylephrine (100 pg/kg, body wt) was administered at 0. Number of rats per group in parentheses.
MINUTES
.
COLD - TREATED 0.
GROUP (5)
.
COLONIC
TEMR
. PHENYLEPHRINE (I00 yg/kg. S.C.)
MINUTES
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COLD-TREATED
RATS
AND
353
CATECHOLAMINES
COLD
0 ,I
d 10 -10
lo-9
’ ’ IO -8 10-7
NOREPINEPHRINE f
’
’
’
to-6 IO -5 IO -4 (Moles/Liter 1
O.Sr
CONTROL
I
I
d 1 IO
I 20
1 I 30 40 KCL (mM/Li
1 50 ter)
I 60
I 70
I 80
FIG. 6. A: tension (g/mg wet wt) developed by aortic rings of control (0) and cold-adapted (a) rats when graded concentrations of norepinephrine were placed accumulatively in bathing medium. One standard error is set off at the means. Significance of the difference in tension developed between control and cold-treated rats is shown in the figure. B: tension developed by aortic rings of control and coldadapted rats when graded concentrations of KC1 were added accumulatively to the bathing medium. One standard error is set off at the means.
nol. Schonbaum et al. (14) observed that administration of the P-adrenergic antagonist, propranolol, to coldadapted rats altered heat production from nonshivering to shivering thermogenesis. Estler and Ammon (5) administered several different P-adrenergic antagonists to mice and showed that survival rate after a 4-h exposure to 0°C was sharply reduced compared with untreated controls. It appears likely that the P-adrenergic system is of primary importance for the metabolic responses of rats to cold and for adaptation. When rats were removed from cold for 1 h, their tail blood vessels were dilated and tail skin temperatures remained elevated for over 2 h. At 5 h after removal from cold, tail blood vessels were still dilated. A 24-h sojourn at 25°C was necessary for stability of tail skin temperature comparable to that of warm-adapted controls (Fig. 1). Since the tail of the rat acts as an important avenue of heat loss, the elevated tail skin temperature suggests that during the first 24 h after removal from cold, the cold-adapted rat produced heat in excess of its requirement for maintenance of body temperature (13). Hence tail skin temperature increased in an effort to dissipate the excess heat produced. Thus a possibility production inexists tha .t the increased catecholamine duced bY expos ure to cold decreased to that of warmadapted controls within 24 h after removal from cold. The increased responsiveness to exogenously adminis-
tered, or endogenously produced, catecholamines may persist for a longer period of time. Leduc (11) has shown that the increased catecholamine excretion by coldtreated rats returned to control levels within 2 days after return to air at 22OC. The mechanism by which isoproterenol increases tail skin temperature is not known with certainty. The response could be the result of either a direct action of isoproterenol on tail blood vessels or an indirect effect occurring secondary to an increase in heat production stimulated by isoproterenol. Studies are in progress to clarify the mechanism. In addition, the mechanism by which the increased responsiveness to P-adrenergic stimulation occurs in cold-treated rats cannot be stated at present. P-adrenergic agents are known to act by way of the cyclic AMP system. Whether cold exposure induces changes in receptor sensitivity or in sites beyond, e.g., at the level of the kinase enzymes, is unknown. It seems likely that the increased metabolic responsiveness of cold-adapted rats to administration of norepinephrine reported by many investigators is due to its padrenergic component (3, 4, 9, 11). Administration of the a-adrenergic agonist, phenylephrine, had no effect on the tail skin temperature of either cold-treated or control rats at two separate doses. The results of the aortic ring studies offer evidence of diminished cu-adrenergic sensitivity in cold-adapted rats. The possibility that these results occurred’ because of increased P-receptor activity seems untenable since previous experiments with P-receptor blocking agents indicated the presence of only cu-adrenergic receptors in aortic smooth muscle of the adult rat (6). Further, attempts to demonstrate P-adrenergic activity in these tissues Wi .th isoproterenol treatment proved fruitless (unpublished). The decreased sensitivity of the a-receptor system in aortic smooth muscle of cold-adapted rats is relatively specific for the receptor mechanism since KC1 depolarization elicited the same development of tension from both groups. At the present time it is impossible to state where in the activation system the diminished pharmacomechanical coupling occurs. It is also possible that the high levels of circulating catecholamines present during cold stress directly decreases the sensitivity of the cyreceptors. Studies designed to clarify the mechanism by which reduced cw-adrenergic responsiveness occurs are now in progress. The value of an increase in P-adrenergic responsiveness to cold-adapted rats may be in facilitating an increase in metabolic rate to offset the increased heat loss induced by cold exposure. The increased rate of secretion of P-adrenergic catecholamines, in conjunction with an increased responsiveness to them, would appear to make the P-adrenergic system the one most likely responsible for nonshivering thermogenesis in the coldadapted rat. The value of the reduced a-adrenergic responsiveness in cold-adapted rats, if it occurs uniformly for all cw-adrenergic-mediated responses, is more difficult to rationalize. It is unknown at present whether, during cold exposure, the increased rate of secretion of catecholamines with ar-adrenergic potential for stimul .a-
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354
FREGLY
tion is offset by the reduction in responsiveness Such a comparison remains for further study.
to them.
This Contract Medicine
Research Bureau of
research was supported N00014-75C-0199, with and Surgery.
by the Office funds provided
of Naval by the
ET AL.
Present addresses: E. L. Nelson, Jr., Dept. of Physiology, Oklahoma College of Osteopathic Medicine and Surgery, Tulsa, Okla. 74119; P. E. Tyler and R. Dasler, EMR Project Office, Naval Medical Research and Development Command, National Naval Medical Center, Bethesda, Md. 20014. Received
for publication
26 July
1976.
REFERENCES 1. ADOLPH, E. F., J. P. BARKER, AND P. A. HOY. Multiple factors in thirst. Am. J. Physiol. 178: 538-562, 1954. 2. BLACK, D. J., M. J. FREGLY, T. N. THRASHER, AND A. H. MORELAND. Reduced P-adrenergic responsiveness in rats treated with estrogenic agents. J. Pharmacol. Exptl. Therap. 197: 362-370, 1976. 3 COTTLE, W., AND L. D. CARLSON. Regulation of heat production in cold-adapted rats. Proc. Sot. Exptl. Biol. Med. 92: 845-848, 1956. 4 DEPOCAS, F. The caloric response of cold-acclimated white rats to infused noradrenaline. Can. J. B&hem. PhysioZ. 38: 107-114, 1960. 5 ESTLER, C. J., AND H. P. T. AMMON. The importance of the adrenergic beta-receptors for thermogenesis and survival of acutely cold-exposed mice. Can. J. Ph.ysiol. PharmacoZ. 47: 427434, 1969. 6 FIELD, F. P., R. A. JANIS, AND D. J. TRIGGLE. Aortic reactivity of rats with genetic and experimental renal hypertension. Can. J. Physiol. Pharmacol. 50: 1072-1079, 1972. 7. FREGLY, M. J., E. L. NELSON, JR., G. E. RESCH, F. P. FIELD, AND L. 0. LUTHERER. Reduced P-adrenergic responsiveness in hypothyroid rats. Am. J. Physiol. 229: 916-924, 1975. 8. HART, J. S., 0. HEROUX, AND F. DEPOCAS. Cold acclimation and the electromyogram of unanesthetized rats. J. Appl. Physiol. 9: 404-408, 1956. 9. HSIEH, A. C. L., AND L. D. CARL~ON. Role of adrenaline and
10.
11. 12.
13.
14.
15.
16.
17.
noradrenaline in chemical regulation of heat production. Am. J. Physiol. 190: 243-246, 1957. LEBLANC, J., J. VALIERES, AND C. VACHON. Beta-receptor sensitization by repeated injections of isoproterenol and by-cold adaptation. Am. J. Physiol. 222: 1043-1046, 1972. LEDUC, J. Effect of acclimation to cold on the production and release of catecholamines. Acta PhysioL. &and. Suppl. 53: 1961. LUTHERER, L. O., M. J. FREGLY, AND A. ANTON. An interrelationship between theophylline and catecholamines in the hypothyroid rat acutely exposed to cold. Federation Proc. 28: 12381242, 1969. RAND, R. P., A. C. BURTON, AND T. ING. The tail of the rat in temperature regulation and acclimatization. Can. J. Physiol. Pharmacol. 43: 257-267, 1965. SCH~NBAUM, E., G. E. JOHNSON, E. A. SELLERS AND M. J. GILL. Adrenergic P-receptors and non-shivering thermogenesis. Nature 210: 426, 1966. SELLERS, E. A., K. V. FLATTERY, A. SHUM, AND G. E. JOHNSON. Thyroid status in relation to catecholamines in cold and warm environments. Can. J. Physiol. Pharmacol. 49: 268-275, 1971. SELLERS, E. A., J. W. SCOTT, AND N. THOMAS. Electrical activity of skeletal muscle of normal and acclimatized rats on exposure to cold. Am. J. PhysioZ. 177: 372-376, 1954. SNEDECOR, G. W., AND W. G. COCHRAN. Statistical Methods (5th ed.). Ames, Iowa: Iowa State Univ. Press, 1956, p. 35-65.
Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (150.135.135.069) on July 31, 2018. Copyright © 1977 American Physiological Society. All rights reserved.
FREGLY, M.J.,F. P. FIELD, E.L. NELSON, JR., P.E. TYLER, AND R. DASLER. Effect of chronic exposure to cold on some responses to catecholamines. J. Appl. Physiol.: Respirat. Environ. Exercise Physiol. 42(3): 349-354, 1977. -An objective of these studies was to test the responsiveness of cold-adapted (8 wk, 5°C) rats to a specific P-adrenergic agonist. Twenty-four hours after removal from cold, increases in tail skin temperature (?I,,,) and colonic temperature (T,.,,) were measured for 2 h in air at 25°C following subcutaneous (SC) administration of 28, 70 or 136 pg d,b-isoproterenol sulfate dihydrate/kg body wt to restrained male rats. Cold-adapted rats responded to each dose of isoproterenol with greater increases in Tsr( than controls. T(.(, of both groups increased at the two highest doses, but coldadapted rats showed a greater rise. Thus, cold-adapted rats showed greater metabolic (T,$,,) and vascular (?I,,> responsiveness to the P-adrenergic agonist, isoproterenol, than nonadapted controls. No effect of the a-adrenergic agonist, phenylephrine (50 or 100 ,ug/kg body weight, SC), was observed on TcBo or Tsl,. A second objective was to study the tension developed by aortic smooth muscle rings of cold-adapted and control rats both during stimulation of cr-adrenergic receptors by norepinephrine and membrane depolarization by KCl. Adaptation to cold air appeared to suppress cu-adrenergic responsiveness in aortic segments but did not alter responsiveness to KCl. This suggests an unchanged contractile mechanism in aortic rings of cold-adapted rats and a reduced responsiveness either at the level of the a-receptor or at a site immediately beyond. cold adaptation; isoproterenol; perature; vascular reactivity;
norepinephrine; aortic rings
tail skin tem-
RATS have an enhanced metabolic responsiveness to both norepinephrine (NE) and epinephrine (E) (3, 4, 8, 9, 16). Th is increased responsiveness has been termed “nonshivering thermogenesis” since it appears to replace the metabolic effects of shivering during the process of adaptation to cold in rats. The increased responsiveness to both NE and E is accompanied by an increase in their rate of production during both acute and chronic exposure to cold (11, 12, 15). These catecholamines may play an important role in mobilization of substrates for energy production, including carbohydrate and lipid, and in this way may participate in nonshivering thermogenesis. The relative importance of endogenous NE, as compared with endogenous E, for nonshivering thermogenesis in cold-adapted rats is not yet settled. Equally unsettled is the importance of cy- versus p-adrenergic COLD-ADAPTED
stimulation for maintenance of nonshivering thermogenesis in cold-adapted rats. Both NE and E have the potential for stimulating cy- and p-adrenergic receptors although the dominant effect of each catecholamine varies considerably from organ to organ. Studies have shown that pharmacological blockade of the P-adrenergic receptors reduced survival of mice exposed acutely to cold (51, and increased both shivering and fall in colonic temperature of cold-adapted rats that were anesthetized and exposed acutely to cold air (14). Although evidence is gradually accumulating that the P-adrenergic receptors are primarily important for the maintenance of nonshivering thermogenesis in coldadapted rats, the issue is not completely settled. The experiments described here were carried out to test the metabolic responsiveness of cold-adapted rats to a P-adrenergic agonist, isoproterenol. Other studies were carried out to test the vascular reactivity of coldadapted rats to the cx-adrenergic effects of norepinephrine. METHODS
Experiment 1. Effect of isoproterenol on tail skin and colonic temperatures of cold-adapted and control rats. Six male rats of the Blue-Spruce Farms (Sprague Dawley) strain were kept in individual wire cages in a room maintained at 5 t -1°C for 8 weeks. Six additional male rats served as controls and were maintained in individual wire cages in air at 25 t 1°C. Illumination in both the cold and warm rooms was from 8 A.M. to 6 P.M. All rats received tap water and Purina laboratory chow ad libitum. At the end of the 8-wk period, each rat was restrained in a tunnel-type cage containing a galvanized, mesh wire cover and a wooden floor (1). The cage was large enough to hold a rat comfortably but prevented movement of the rat from head to tail. Each rat had a copperconstantan thermocouple inserted 5 cm beyond its anal orifice and held in place by a small piece of adhesive tape. A second thermocouple was woven into one layer of a gauze sponge, 2.5 cm wide. The junction of this thermocouple contacted the skin at the base of the tail. A piece of adhesive tape was placed at the front and rear of the gauze sponge to hold it in place. The thermocouples were led off to a potentiometer that recorded the temperatures at 1-min intervals.
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350 An initial series of experiments was carried out when it was observed that rats removed from air at 5°C to air at 25°C for 1 h maintained a fully dilated tail with increased tail skin temperature (T,& Three days later, the same group of cold-treated rats was returned to air at 25°C for 5 h prior to measurement of tail skin and colonic temperatures (T,.,,). At the end of a 30-min control period, isotonic saline (0.2 ml) was injected subcutaneously through a slot in the cage. Tsk and Tco were then measured for an additional 2 h. Rats that had been maintained in air at 25OC served as controls. Because Tsk of cold-treated rats was still elevated even at 5 h after removal from cold, the cold-adapted group was removed from cold for 24 h before temperature measurements began. At the end of the 30-min control period, all rats were administered isotonic saline subcutaneously (SC). Temperatures were measured for an additional 2 h. Rats adapted to 25OC served as controls. Since a 24-h sojourn in air at 25°C was required for stability of Tsk in cold-adapted rats, all subsequent experiments followed this procedure. Three additional identical studies separated by 3-4 days from each other were performed on the same rats. The experiments were the same in detail as those described above except that d,Z-isoproterenol sulfate dihydrate (28, 70, and 139 ,ug/kg body wt) was administered SC at the end of the control period. The dose was calculated as the salt. Tsk and Tco were measured for an additional 2 h after treatment. Rats adapted to 25°C served as controls. Experiment 2. Effect of phenylephrine on tail skin and colonic temperatures of cold-adapted and control rats. These studies were carried out in the same fashion as those in experiment 1 except that the a-adrenergic agonist, phenylephrine, at doses of 50 and 100 r_Lg/kg body wt, was administered SCat the end of the control period. Experiment 3. Vascular reactivity of cold-adapted rats to norepinephrine and potassium chloride. Wistar (CFN) male rats weighing 150-180 g were divided into two groups. Twelve rats were maintained in air at 5 t 1°C for 6 wk while 12 control rats were maintained at 26 t 1°C. Both rooms were illuminated from 8 A.M. to 6 P.M. All rats received tap water and Purina laboratory chow ad libitum. During the 7th through the 11th wk of the study, one rat from each group was anesthetized with ether and killed by cervical dislocation. At death, 3-cm segments of aorta (taken 1 cm from the aortic arch) were excised and immediately placed in a modified Krebs solution. After adherent fat and loose connective tissue were removed, one or two 4-mm rings were cut from each segment by the use of two surgical blades mounted on a plastic block. The segments were suspended between two stainless steel hooks inserted into the lumen to record circular muscle contraction. The isolated tissue was mounted in a 20-ml water-jacketed muscle bath and equilibrated for 3 h under 2 g of tension which was maintained. Contractions were recorded isometrically, employing a Narco-Bio microdisplacement transducer (F-50) with a Narco-Bio Physiograph (DMP4B) recorder.
FREGLY
ET
AL.
The modified Krebs solution containing dextrose (11 mM) and disodium EDTA (0.01 mM) was maintained at 37°C and aerated with an O,-CO, mixture (95:5%) which maintained the pH at 7.30. The bath solutions were changed once every 15 min during equilibration and between dose-response determinations. After the 3-h equilibration period, an accumulative norepinephrine dose-response curve was determined on each segment. About 90 min after the norepinephrine was washed from the bath, KC1 was added accumulatively. Statistical analysis of all data was made by means of the Student t-test for the 95% confidence limit (12). RESULTS
Experiment 1. One hour after removal from cold air Tsk was approximately 30°C. During the 30-min control period and the 2 h following SCadministration of saline, Tsk remained high and varied from 27.2 to 30.2”C. In contrast, Tsk of the warm-adapted group during the control period was approximately 24OC and increased 0.8OC after saline injection but fell within 20 min to preinjection level. Tsk of the cold-adapted group was significantly (P < 0.05-0.01) higher than that of the control group throughout the experiment while T,() of the two groups were not significantly different with both declining about 0.6OCduring the treatment period. Five hours after removal from cold air, Tsk of the coldadapted rats varied from 28.4 to 3O.O”C. Tslcdecreased about 2°C following administration of saline and reached control level by the end of the 2-h experimental period. Tsk of the control group declined about 1°C during the experimental period. T 0.05) different from the control group and by 24 h, both groups had attained nearly identical Tsk values (Fig. lA >, Tco of the two groups did not differ significantly in any of the three studies (Fig. W Administration of the lowest dose of d,Z-isoproterenol (28 r-cglkg) to the control group induced an average rise in Tsk of about 1.5”C and an average rise in T(.() of approximately 0.3”C (Fig. 2A). Although the coldadapted group appeared to have a greater mean increase in Tsk than the control group, the difference
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COLD-TREATED
RATS
AND
351
CATECHOLAMINES
A
CONTROL
30
GROUP
A I
d, I ISOPROTERENOL yg/kg 6.b’. s.c.1
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SKIN
TEMl?
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COLD-TREATED
I
GROUP
d, I ISOPROTERENOL (28 pg/kg B.W. SC.)
B
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COLD-ADAPTED
37b
CONTROL
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HOURS AFTER
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1. Mean tail skin (A) and colonic (B) temperatures of coldadapted and control rats, measured 30 min after SC administration of saline, are shown at 1, 5, and 24 h after removal from cold air. One standard error is set off at the means.
60 MINUTES
FIG.
between groups was not significant at any time during the experiment (Fig. 2B). TcOof the cold-adapted group remained stable throughout the experiment and did not differ significantly from that of the control group at any time. Administration of 70 pg d,Z-isoproterenol/kg of body wt to the control group increased Tsk 54°C (Fig. 3A). An early rise (0.3”C) in TcOmay have preceded slightly the rise in Tsk. Cold-adapted rats appeared to have a somewhat greater rise in both Tsk (7.1"C) and TcO(0.9OC)than the control group (Fig. 3B). Tsk of the cold-adapted group exceeded significantly (P < 0.05) that of the control group during the first 20-min period following administration of isoproterenol. No differences between mean Tsk of the two groups were observed thereafter. Mean T.. of the two groups differed from each other (P < 0.05) only at 120min after administration of isoproterenol. When 139 rugd,l-isoproterenol/kg body wt was administered to the control group, a striking increase in Tsk (75°C) occurred (Fig. 4A). A slight (0.3”C) but not significant rise in TcOoccurred followed by a decline. In the case of the cold-adapted group a greater elevation of Tsk (9.l”C) occurred than was observed in the control group (Fig. 4B). Mean Tsk of the cold-adapted group did not differ significantly from the control group prior to administration of isoproterenol. Following administration of isoproterenol, mean Tsk of the cold-adapted group differed significantly (P < 0.05) from the control group at 30, 40, 50, 60, and 70 min. Thereafter, Tsk of the two groups no longer differed significantly. TcO of the two groups also differed significantly (P < 0.05-0.01) at 20, 30,40, 50, and 60 min after administration of isoproterenol, with the cold-adanted g-roun being the higher.
80
FIG. 2. Tail skin (Left ordinate) and colonic (r&ht ordinate) temperatures of control (A) and cold-treated (B) groups following SC administration of d,L-isoproterenol (28 pg/kg body wt) at 0. One standard error is set off at the means. CONTROL d,~ I
GR3UP ISOPR0TERENOL (70 pg/kg B.W. ‘3 C.!
TAIL SKIN TEMP
1
I 0
I 20
1
I I 1 40 60 MINUTES
1
’ 80
’
’ ’ ’ 100 120
37=
COLD-TREATED GROUP d , I ISOPPOTEHENOL
TAIL SKIN TEMC
LCOLONIC I 1 ITEMP 1 L1 I 1 1 11 FIG.
bodv
wt)
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2 except at 0.
that
d,Z-isoproterenol
(70
pg/kg
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FREGLY
352
DISCUSSION
of these experiments
was
to test the COLD-TREATED
GROUP TAIL SKIN TEMI?
35CONTROL
33A 34
GROUP
349
t
d.1 ISOPROTERENOL (139 yg/kg B.W. S.C.)
AL.
responsiveness of cold-adapted rats to cy- and P-adrenergic agonists. The increase in Tsk of rats in response to administration of isoproterenol has been studied under other conditions in this laboratory (2, 7). It appears to arise as a result of specific p-adrenergic stimulation and can be blocked by the P-adrenergic antagonist, propran0101 (7). In addition, an increase in Tsk could not be elicited in either warm- or cold-adapted rats by administration of the a-adrenergic agonist, phenylephrine (7) (Fig. 5). Using the increase in Tsk as a criterion, the results of these studies show that adaptation to cold air is accompanied by an increased responsiveness to p-adrenergic stimulation in the rat (Figs. 2-4). LeBlanc et al. (10) have arrived at a similar conclusion using as criteria both the heart rate and rate of increase in oxygen consumption in response to an acute infusion of isoprotere-
Experiment 2. The adrenergic agonist, phenylephrine, at either 50 pg/kg or 100 pg/kg body wt had no significant effect on either Tsk or TccJof control and coldadapted rats. Since this was the case the results shown in Fig. 5 are for the higher dose only. Experiment 3. The tension developed by aortic rings of control and cold-adapted rats following the addition of norepinephrine to the test bath is shown in Fig. 6A. The aortic vascular smooth muscle reactivity to norepinephrine was reduced significantly in cold-adapted rats compared with controls. In contrast, the response to KC1 depolarization was not significantly altered by prior cold exposure (Fig. W).
The objective
ET
B
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32
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I,
COLON/C TEMP. I I IL I loo 120
d, I ISOPROTERENOL (I39 yg/kg B.W. S.C.)
z
-
FIG. 4. Same d,l-isoproterenol was administered
as Fig. 2 except that (139 pg/kg body wt) at 0.
8 41 r 8 0 4o+ 2 392 c 3e; cg 375
MINUTES
MINUTES
CONTROL GROUP (5) A. PHENYLEPHRINE (100 pg/kg. SC. )
27~
COLONIC
TEMP
FIG. 5. Same as Fig. 2 except that phenylephrine (100 pg/kg, body wt) was administered at 0. Number of rats per group in parentheses.
MINUTES
.
COLD - TREATED 0.
GROUP (5)
.
COLONIC
TEMR
. PHENYLEPHRINE (I00 yg/kg. S.C.)
MINUTES
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COLD-TREATED
RATS
AND
353
CATECHOLAMINES
COLD
0 ,I
d 10 -10
lo-9
’ ’ IO -8 10-7
NOREPINEPHRINE f
’
’
’
to-6 IO -5 IO -4 (Moles/Liter 1
O.Sr
CONTROL
I
I
d 1 IO
I 20
1 I 30 40 KCL (mM/Li
1 50 ter)
I 60
I 70
I 80
FIG. 6. A: tension (g/mg wet wt) developed by aortic rings of control (0) and cold-adapted (a) rats when graded concentrations of norepinephrine were placed accumulatively in bathing medium. One standard error is set off at the means. Significance of the difference in tension developed between control and cold-treated rats is shown in the figure. B: tension developed by aortic rings of control and coldadapted rats when graded concentrations of KC1 were added accumulatively to the bathing medium. One standard error is set off at the means.
nol. Schonbaum et al. (14) observed that administration of the P-adrenergic antagonist, propranolol, to coldadapted rats altered heat production from nonshivering to shivering thermogenesis. Estler and Ammon (5) administered several different P-adrenergic antagonists to mice and showed that survival rate after a 4-h exposure to 0°C was sharply reduced compared with untreated controls. It appears likely that the P-adrenergic system is of primary importance for the metabolic responses of rats to cold and for adaptation. When rats were removed from cold for 1 h, their tail blood vessels were dilated and tail skin temperatures remained elevated for over 2 h. At 5 h after removal from cold, tail blood vessels were still dilated. A 24-h sojourn at 25°C was necessary for stability of tail skin temperature comparable to that of warm-adapted controls (Fig. 1). Since the tail of the rat acts as an important avenue of heat loss, the elevated tail skin temperature suggests that during the first 24 h after removal from cold, the cold-adapted rat produced heat in excess of its requirement for maintenance of body temperature (13). Hence tail skin temperature increased in an effort to dissipate the excess heat produced. Thus a possibility production inexists tha .t the increased catecholamine duced bY expos ure to cold decreased to that of warmadapted controls within 24 h after removal from cold. The increased responsiveness to exogenously adminis-
tered, or endogenously produced, catecholamines may persist for a longer period of time. Leduc (11) has shown that the increased catecholamine excretion by coldtreated rats returned to control levels within 2 days after return to air at 22OC. The mechanism by which isoproterenol increases tail skin temperature is not known with certainty. The response could be the result of either a direct action of isoproterenol on tail blood vessels or an indirect effect occurring secondary to an increase in heat production stimulated by isoproterenol. Studies are in progress to clarify the mechanism. In addition, the mechanism by which the increased responsiveness to P-adrenergic stimulation occurs in cold-treated rats cannot be stated at present. P-adrenergic agents are known to act by way of the cyclic AMP system. Whether cold exposure induces changes in receptor sensitivity or in sites beyond, e.g., at the level of the kinase enzymes, is unknown. It seems likely that the increased metabolic responsiveness of cold-adapted rats to administration of norepinephrine reported by many investigators is due to its padrenergic component (3, 4, 9, 11). Administration of the a-adrenergic agonist, phenylephrine, had no effect on the tail skin temperature of either cold-treated or control rats at two separate doses. The results of the aortic ring studies offer evidence of diminished cu-adrenergic sensitivity in cold-adapted rats. The possibility that these results occurred’ because of increased P-receptor activity seems untenable since previous experiments with P-receptor blocking agents indicated the presence of only cu-adrenergic receptors in aortic smooth muscle of the adult rat (6). Further, attempts to demonstrate P-adrenergic activity in these tissues Wi .th isoproterenol treatment proved fruitless (unpublished). The decreased sensitivity of the a-receptor system in aortic smooth muscle of cold-adapted rats is relatively specific for the receptor mechanism since KC1 depolarization elicited the same development of tension from both groups. At the present time it is impossible to state where in the activation system the diminished pharmacomechanical coupling occurs. It is also possible that the high levels of circulating catecholamines present during cold stress directly decreases the sensitivity of the cyreceptors. Studies designed to clarify the mechanism by which reduced cw-adrenergic responsiveness occurs are now in progress. The value of an increase in P-adrenergic responsiveness to cold-adapted rats may be in facilitating an increase in metabolic rate to offset the increased heat loss induced by cold exposure. The increased rate of secretion of P-adrenergic catecholamines, in conjunction with an increased responsiveness to them, would appear to make the P-adrenergic system the one most likely responsible for nonshivering thermogenesis in the coldadapted rat. The value of the reduced a-adrenergic responsiveness in cold-adapted rats, if it occurs uniformly for all cw-adrenergic-mediated responses, is more difficult to rationalize. It is unknown at present whether, during cold exposure, the increased rate of secretion of catecholamines with ar-adrenergic potential for stimul .a-
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354
FREGLY
tion is offset by the reduction in responsiveness Such a comparison remains for further study.
to them.
This Contract Medicine
Research Bureau of
research was supported N00014-75C-0199, with and Surgery.
by the Office funds provided
of Naval by the
ET AL.
Present addresses: E. L. Nelson, Jr., Dept. of Physiology, Oklahoma College of Osteopathic Medicine and Surgery, Tulsa, Okla. 74119; P. E. Tyler and R. Dasler, EMR Project Office, Naval Medical Research and Development Command, National Naval Medical Center, Bethesda, Md. 20014. Received
for publication
26 July
1976.
REFERENCES 1. ADOLPH, E. F., J. P. BARKER, AND P. A. HOY. Multiple factors in thirst. Am. J. Physiol. 178: 538-562, 1954. 2. BLACK, D. J., M. J. FREGLY, T. N. THRASHER, AND A. H. MORELAND. Reduced P-adrenergic responsiveness in rats treated with estrogenic agents. J. Pharmacol. Exptl. Therap. 197: 362-370, 1976. 3 COTTLE, W., AND L. D. CARLSON. Regulation of heat production in cold-adapted rats. Proc. Sot. Exptl. Biol. Med. 92: 845-848, 1956. 4 DEPOCAS, F. The caloric response of cold-acclimated white rats to infused noradrenaline. Can. J. B&hem. PhysioZ. 38: 107-114, 1960. 5 ESTLER, C. J., AND H. P. T. AMMON. The importance of the adrenergic beta-receptors for thermogenesis and survival of acutely cold-exposed mice. Can. J. Ph.ysiol. PharmacoZ. 47: 427434, 1969. 6 FIELD, F. P., R. A. JANIS, AND D. J. TRIGGLE. Aortic reactivity of rats with genetic and experimental renal hypertension. Can. J. Physiol. Pharmacol. 50: 1072-1079, 1972. 7. FREGLY, M. J., E. L. NELSON, JR., G. E. RESCH, F. P. FIELD, AND L. 0. LUTHERER. Reduced P-adrenergic responsiveness in hypothyroid rats. Am. J. Physiol. 229: 916-924, 1975. 8. HART, J. S., 0. HEROUX, AND F. DEPOCAS. Cold acclimation and the electromyogram of unanesthetized rats. J. Appl. Physiol. 9: 404-408, 1956. 9. HSIEH, A. C. L., AND L. D. CARL~ON. Role of adrenaline and
10.
11. 12.
13.
14.
15.
16.
17.
noradrenaline in chemical regulation of heat production. Am. J. Physiol. 190: 243-246, 1957. LEBLANC, J., J. VALIERES, AND C. VACHON. Beta-receptor sensitization by repeated injections of isoproterenol and by-cold adaptation. Am. J. Physiol. 222: 1043-1046, 1972. LEDUC, J. Effect of acclimation to cold on the production and release of catecholamines. Acta PhysioL. &and. Suppl. 53: 1961. LUTHERER, L. O., M. J. FREGLY, AND A. ANTON. An interrelationship between theophylline and catecholamines in the hypothyroid rat acutely exposed to cold. Federation Proc. 28: 12381242, 1969. RAND, R. P., A. C. BURTON, AND T. ING. The tail of the rat in temperature regulation and acclimatization. Can. J. Physiol. Pharmacol. 43: 257-267, 1965. SCH~NBAUM, E., G. E. JOHNSON, E. A. SELLERS AND M. J. GILL. Adrenergic P-receptors and non-shivering thermogenesis. Nature 210: 426, 1966. SELLERS, E. A., K. V. FLATTERY, A. SHUM, AND G. E. JOHNSON. Thyroid status in relation to catecholamines in cold and warm environments. Can. J. Physiol. Pharmacol. 49: 268-275, 1971. SELLERS, E. A., J. W. SCOTT, AND N. THOMAS. Electrical activity of skeletal muscle of normal and acclimatized rats on exposure to cold. Am. J. PhysioZ. 177: 372-376, 1954. SNEDECOR, G. W., AND W. G. COCHRAN. Statistical Methods (5th ed.). Ames, Iowa: Iowa State Univ. Press, 1956, p. 35-65.
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