Potentiation of thermoregulatory to isoproterenol by ,8-adrenergic HARRY
J. CARLISLE
AND MICHAEL
responses antagonists
J. STOCK
Department of Psychology, University of California, Santa Barbara, California 93106; and Department of Physiology, St George’s Hospital Medical School, London S W17 ORE, United Kingdom Carlisle, Harry J., and Michael J. Stock. Potentiation of thermoregulatory responsesto isoproterenol by ,&adrenergic antagonists.Am. J. Physiol. 263 (Regulatory Integrative Comp. Physiol. 32): R915R923, 1992.-The thermoregulatory effects of isothermogenicdosesof isoproterenol (Iso) and a novel ,&agonist (BRL 35135) were tested in rats at 22°C and in rats trained to bar pressfor radiant heat at -8°C. BRL 35135produced hyperthermia at 22°C and reduced operant responding for heat at -8’C, whereasIso reduced body temperature and increasedoperant responding.In both situations, the negative effects of Iso on thermal balancewere abolishedby propranolol at dosesthat did not inhibit heat production. In anesthetized rats, propranolol potentiated the Iso-induced rise in brown adipose tissue and colonic temperature. The potentiation was more marked with the &-selective antagonist ICI 118,551, whereastreatment with the &-selective antagonist atenolol resulted in a profound Iso-induced reduction in temperature. The two selective antagonists also produced divergent responsesin operant behavior in Iso-treated rats at -8°C. These experiments demonstrate the extent to which responsesto a nonselective agonist can be manipulated using appropriately low dosesof selectiveantagonistsand indicate that the effects of Iso on thermal balance are due to its ,& activity. propranolol; atenolol; ICI 118,551; hypothermia; thermogenesis; operant responding OR FACULTATIVE FORMS of thermogenesis, such as nonshivering and diet-induced thermogenesis, are due mainly to increased sympathetic activity, with brown adipose tissue (BAT) acting as the main effector tissue (see Refs. 8, 15 for reviews). Activation of BAT thermogenesis by synaptic release of norepinephrine is mediated by ,&adrenergic receptors (I), and for many experimental studies peripheral administration of ,& adrenergic agonists, such as isoproterenol, have been used. Isoproterenol is a full agonist of thermogenesis, but because it activates &- and ,&-adrenoceptors in other tissues, it affects many other functions. However, these other effects of P-adrenergic activation can be avoided by using some of the new selective thermogenic agonists, such as BRL 35135, Ro 40-2148, and ICI D7114. The selectivity of these ,&adrenergic agonists is believed to be due to the atypical nature of the BAT ,&adrenoceptor (2), which is now thought to correspond to or resemble the ,&-adrenoceptor subtype identified in the human genome (5); these novel thermogenic drugs are therefore often referred to as ,&,-agonists. In a previous study (4), the nonselective P-adrenoceptor agonist isoproterenol was found to cause a decrease in colonic temperature in rats housed at 22°C and increased operant responding for exogenous heat in rats tested at -8OC. These paradoxical hypothermic responses to this thermogenic agonist differed markedly from the responses seen when &-selective agonists (Ro 40-2148 and ICI D7114) were used. As might be predicted, these two thermogenic drugs produced hyperthermia in rats kept at room temperature and decreased ADAPTIVE
behavioral responding for heat in the cold. The conclusion from this study was that the thermogenic (i.e., ,&) actions of isoproterenol were negated by its other (i.e., ,& or &) effects and resulted in heat loss exceeding heat production. It would seem reasonable to assume that this increase in heat loss was due to ,&-mediated effects on peripheral blood flow, particularly to important areas of heat loss, such as the tail (12, 14). However, norepinephrine (which causes vasoconstriction) also produces increased operant responding for heat in a cold environment (21)) and Fregly (7) has reported that the effect of isoproterenol on rat tail temperature is mediated by ,&adrenoceptors and not by &-adrenoceptors. The principal aim of the current study was to attempt a pharmacological “dissection” of the ,&adrenoceptor subtypes to determine which was responsible for the temperature lowering effects of isoproterenol. In the absence of a selective P3-adrenoceptor antagonist, the approach adopted took advantage of the low affinity of the &-adrenoceptor for conventional P-adrenergic antagonists. For example, the concentrations of nonselective and selective (& or &) antagonists required to inhibit BAT adipocyte thermogenesis are two to three orders of magnitude greater than those required to inhibit typical &- or &-mediated responses (1, 18). This explains why previous workers have had to use exceptionally large doses (20-30 mg/kg body wt) of propranolol to block nonshivering and diet-induced thermogenesis (1) or to block the thermogenic response to &-agonists (13). Thus it should be possible to use a low dose of a conventional antagonist, such as propranolol, which would be sufficient to inhibit &- and ,&-mediated responses but would not be high enough to inhibit & activation of thermogenesis. This approach appeared to be successful, and further studies were undertaken using low doses of a selective &-antagonist (atenolol) and a selective &-antagonist (ICI 118,551). For these antagonist studies, a third experimental paradigm (temperature and heart rate responses in anesthetized rats) was added to the two behavioral tests (thermoregulatory responses at 22 and -8OC) used in the earlier study. In addition, the thermogenic (i.e., heat production) and behavioral responses to a different ,&-agonist (BRL 35135) from those used in the previous study were compared with the responses obtained with isoproterenol. A preliminary summary of the results has been reported previously (17). METHODS Animals. For the operant thermoregulatory tests, lean (+/?) 8-mo-old femalerats of “fatty” Zucker strain wereobtained from the colony at the University of California, Santa Barbara. The animalswere fed a standard stock diet (Purina 5001)ad libitum and housedin groups of two to four in a room maintained at 22°C with 50% relative humidity and a 12:12-hlight-dark cycle R915
0363-6119/92 $2.00 Copyright 0 1992 the American Physiological Society Downloaded from www.physiology.org/journal/ajpregu by ${individualUser.givenNames} ${individualUser.surname} (132.174.254.157) on January 17, 2019.
R916
ADRENERGIC
RECEPTORS
(lights on at 0700 h); all testing was carried out during the light phase of the cycle. Training for the operant tests had taken place at regular intervals (twice a week) over the previous month. The other experiments did not require any prior training and were carried out using 3mo-old male Sprague-Dawley rats purchased from Bantin & Kingman (Fremont, CA) and housed two per cage under identical conditions to the above. No other animals were housed in the room, and access was restricted to the researchers on those days when thermoregulatory responses at 22°C were measured to ensure the animals were not disturbed. Different groups of Sprague-Dawley rats were used for the temperature and calorimetry studies, and at a later stage were used for the terminal experiments carried out under anesthesia. Within each experiment, care was taken to ensure that the groups were matched for age and weight, and often the animals acted as their own controls. Body temperature. For the behavioral tests at 22 and -8”C, the animal was removed from the home or experimental cage and colonic temperature was taken within 2 min using a Sensortek (Clifton, NJ) BAT-12 meter and thermocouple probe insertedvia the rectum at least 6 cm until peak temperature was indicated. Temperature recordings were made before and at 30-min intervals after drug injections with the animal sitting unrestrained on a bench. Measurementsof colonic, interscapular BAT and tail temperatures in anesthetized rats were made on two to four animals at a time using a combination of Sensortek thermocouples and YSI-400 (Yellow Springs Instruments) thermistor probes (all calibrated to give identical readings). The lead of the rectal probe was taped to the tail probe, which was positioned on the dorsal surface 1 cm distal to the base of the tail. The fur over the scapulaewas shaved, and a small skin incision madeto allow the interscapular BAT probe to be inserted beneath the brown fat pad, so that the tip of the probe sat in the interscapular cleft closeto the large vein (Sulzer’svein) that drains the fat pad. The BAT thermocoupleprobe was made in the laboratory from fine (36-gauge)copper-constantan wire sheathed in PE-50 tubing. Temperature readings were taken at 15min intervals for 45 min before and 120 min after drug injections. Heart rate. Electrocardiographic (ECG) recordings from anesthetizedrats were madeat 15-min intervals using fine needle recording electrodesin the skin and connected to a Gould recorder (Gould 2400 series). Anesthesia. The rats were anesthetizedwith urethan (1.2 g/kg ip) before the temperature probes and ECG electrodes were attached. Measurementswere madewith the animal in a prone position in a room maintained at 25-26°C. No additional heating was provided, and the animal was killed at the end of the experiment. Operant responding. Shaved rats were placedin a cageinside a freezer maintained at -8”C, having previously beentrained to pressa lever that activated two infrared lamps that together provided 300 W (equivalent to an irradiance of 180 mW/cm2). The experimental test sessionslasted90 min, and the number of bar pressesand duration of heat reinforcements (secondsof heat) were recorded continuously; at least 3 days intervened betweentests on each animal. The bar-pressapparatusand test procedurehave been describedin detail previously (4). Calorimetry. Measurements of heat production by indirect calorimetry were madeusing a system describedin detail previously (10). In outline, the procedure involved placing the animal in a temperature-controlled cabinet through which dry air wasdrawn. A samplewaspassedthrough a Beckman 755 paramagnetic oxygen analyzer (Beckman, Fullerton, CA) before recombining with remaining effluent air and passingthrough a dry-gas meter. Oxygen consumption was recorded for 30 min before and 60 min after drug injection using an on-line minicomputer, and the rate of heat production (W/kg) was calculated and printed every 2 min.
AND
BODY
TEMPERATURE
Drugs. (-) -1soproterenol HCl (Iso), (+) -propranolol HCl (Prop), and (A)-atenolol (Aten) were obtained from Sigma (St. Louis, MO), BRL 35135 (BRL) from SmithKline Beecham (Epsom,UK), and ICI 118,551(ICI) from ICI Pharmaceuticals (Macclesfield, UK). All drugs were prepared for injection by dissolvingin a minimum volume of dimethyl sulfoxide (DMSO; Sigma), making up to volume with 5% gum arabic in normal saline (0.9% NaCl) and sonicating before use. The control vehicle was10% DMSO in 5% gum arabic-saline.All injections (1 ml/kg) were subcutaneous. Data analysis. Differences betweentreatments were assessed from changesin body temperatures (colonic, BAT, tail), heat production, and heart rate as appropriate. The bar-pressexperiments were more difficult to analyze becauseof the problemof trying to compare simultaneously changesin heat influx (HI, mW/cm2) with changesin colonic temperature (“C). This problem was resolvedby adopting the analysisdescribedin the previous study (4), where the data were transformed to common units of thermal balance (kJ) basedon changesin heat storage (dS) and HI from the infrared lamps. These parameterswere then usedto calculate net thermal balance(NTB, kJ = HI - dS) and net thermal efficiency (NTE, % = dS/HI x 100). The significanceof these derived measuresis that a high NTB or a low NTE indicates a greater dependenceon exogenousheat to maintain thermal balance(4). All resultshave beenexpressedas mean values t SE, and differences between vehicle (Con) and drug-treated meansassessed using Student’s t test for matched or unmatched data, asappropriate; quoted probabilities are two tailed. EXPERIMENTSAND
RESULTS
There were nine separate experiments that followed a chronological and logical sequence. For this reason and for clarity, the experimental design, results, and reasoning that led to the next experiment will be described here before discussing their significance in DISCUSSION. Experiment 1. This experiment involved comparing operant responding for heat at -8°C in rats receiving Iso and one of the novel ,&-agonists (BRL) that had not been tested in the previous study (4). Trained lean Zucker rats (300-340 g; n = 6) each received on separate occasions Iso (75 pg/kg), BRL (40 pg/kg), or vehicle (Con); the treatments were randomized. The dose of 75 pg Isa/kg is a maximal thermogenic dose (Stock, unpublished data), whereas the 40-pg BRL/kg dose was chosen to be supramaximal [maximal effective dose (EDloo) - 10 pg/kg]. Supramaximal doses of Iso were avoided because they can be lethal in cold-exposed rats, whereas the high dose of BRL was chosen deliberately to test for any loss of p3 selectivity. Figure 1 shows that all rats increased colonic temperature during the first 30 min in the bar-press apparatus. However, the rise in Iso rats was very much less (OBOC) than in the other groups (1.7-1.8”C) despite bar pressing for three times more heat than the BRL or Con rats over the same period (Fig. 1). Rats receiving BRL responded much as did Con rats, although the 90-min summary of results (Table 1) shows that BRL produced a significant improvement in NTB. Judged from Fig. 1, this was probably due to a slightly lower level of heat reinforcements over the last 60 min of the 90-min test, and calculation of NTB relative to control values over the last hour shows that it was decreased by 20% (P < 0.05); this implies a greater contribution to thermal balance from endogenous heat production in the BRL-treated rats. By contrast, the
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ADRENERGIC
RECEPTORS
I
I
I
1
0
30
60
90
Time
(mid
Fig. 1. Ex@ 1: colonic temperature (A) and heat reinforcements (B) in lean Zucker rats after vehicle (o), 75 pg isoproterenol (Iso)/kg (o), and 40 pg BRL 35135 (BRL)/kg (A). Mean values & SE; n = 6. Compared with vehicle (by paired t test): * P < 0.05; ** P < 0.01; *** P < 0.001.
negative effects of Iso on thermal balance were clearly obvious from the doubling in operant responding for HI, the twofold increase in NTB, and the 75% decrease in NTE (Table 1). The next experiment was designed to see whether these diffe rences were still apparent when animals were in their normal home-cage environment . To conserve the trained lean Zucker rats for operant studies, these experiments were carried out on Sprague-Dawley rats. Experiment 2. Measurements of colonic temperature were made in three groups (n = 8/group) of SpragueDawley rats (320-340 g) housed in their home cages at 22”C, with each group- receiving one of the three treatments described above. There were no significant differences in colonic temperature (mean = 37.1 t O.l”C) before injection, but the changes over the 2.5 h after injections (Fig. 2) revealed a marked decrease in temperature in rats receiving Iso. The intermittent handling and disturbance produced a small 0.6-0.7OC sustained increase in controls, and the mean difference over 2.5 h between Iso and Con rats amounts to >l.O”C (P < 0.001). This contrasts with the BRL-treated rats, which exhibited a larger mean rise than vehicle-treated rats (BRL = 1.14 t 0.08, Con = 0.68 t 0.14; P < 0.05), with the differences in response being particularly noticeable over the first 90 min. The contrast between the effects of BRL and Iso on colonic temperature raises questions about their relative thermogenic potency, and although maximal thermogenic doses of both agonists were used in these experiments, it was decided to see whether these were isothermogenic. Experiment 3. Heat production (W) was estimated
AND
BODY
TEMPERATURE
R917
from measurements of oxygen consumption in SpragueDawley rats (480-500 g; n = 5) before and after receiving vehicle, I so and B RL (doses as before) in a randomized sequence at three env ironmental temperatures. Two of these temperatures (20 and 25°C) fell-either side of the room temperature used for experiment 2, and the third (29OC) corresponds to the thermoneutral temperature of the rat. Each rat was tested at each environmental temperature, and at least 3 days elapsed between treatments. As explained for experiment 1, the dose of Iso (75 pg/ kg) was intended to be maximal and the BRL dose (40 pg/kg) was chosen to be supramaximal. Dependent on environmental temperature, both agonists produced 4070% increases in heat production (Fig. 3) with no significant differences between the two at any of the three environmental temperatures; i.e., the doses were isothermogenic. After it had been established that the differences between the effects of Iso and BRL on thermoregulation could not be due to differences in the activation of thermogenesis, the emphasis switched to trying to determine the role of the ,&- and &-adrenoceptor subtypes in mediating the effects of Iso on thermal balance seen in the previous experiments. Experiment 4. The object of this experiment was to see whether low doses of a nonselective @-adrenergic antagonist (Prop) could be used to inhibit the hypothermic effects of Iso without inhibiting its thermogenic actions. As explained in the introduction, high doses (120 mg/kg) of Prop are required to block thermogenesis, and a dose of 100 pg/kg was eventually selected after preliminary barpress trials with lean Zucker rats using doses that decreased progressively from 5 to 2 to 1 to 0.1 mg/kg. After these preliminary experiments, operant responses for exogenous heat at -8°C were recorded in three groups of lean Zucker rats (330-350 g; n = G/group) after receiving vehicle, Iso or BRL alone, and the same treatment in combination with 100 pg Prop/kg (injected 5 min earlier). The test sequence (agonist alone followed by agonist + Prop) was reversed for half the rats in each group, with at least a 3-day interval between tests. The results (Table 2) show that this low dose of Prop had no discernible effect on any of the parameters of behavioral thermoregulation (colonic temperature, barpress rate, or duration) or thermal balance (dS, HI, NTB, or NTE) in control and BRL-treated rats. However, Prop had a dramatic effect in Iso-treated rats, and completely reversed the responses seen with the agonist alone, so that all parameters of behavior and thermal balance were similar to those in the control and BRL groups. In the absence of Prop, thermal balance (e.g., dS, HI, and NTB) in the Iso rats was significantly (P < 0.001, Sheffe’s test) impaired in comparison to all the other treatment groups. Inspection of’30-min data records (data not shown) indicated that the ability of Prop to reverse the effects of Iso on thermal balance were more apparent during the first 60 min of the 90-min test. Over this period, there was a significant (P < 0.001) 60% decrease in heat reinforcements and NTB and a eightfold increase in NTE in Iso plus Prop rats compared with the same rats receiving Iso alone. Experiment 5. After observation of the potent effects of low-level nonselective ,&adrenergic block on responses at -8”C, exactly the same protocol was adopted to see
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R918
ADRENERGIC
Table 1. Effect of maximal thermogenic on operant
responding T*in9 “C
Con Is0 BRL
AND
doses of isoproterenol
BODY
TEMPERATURE
and BRL 35135
for heat (expt 1) T out.7 “C
36.77 to.26 36.35 to.19 36.73 to.16
RECEPTORS
dT, “C
38.43 to.10 37.15* to.21 38.72 to.15
R s/min
R/BP, s/press
ds, kJ
HI, kJ
NTB, kJ
NTE, %
11.94 to.64 22.97* to.59 10.42 to.57
4.27 k0.42 11.04* to.81 4.43 zkO.85
1.67 to.27 0.79 to.18 1.93 to.27
21.15 tl.14 40.59* tl.78 18.08 to.89
19.48 a.19 39.80* tl.85 16.15$ to.79
8.07 tl.54 2.oq to.47 10.65 kl.26
BP, presses/min
1.67 to.28 0.80-f to.19 1.98 to.28
2.87 to.19 2.15t kO.19 2.64 to.33
Values are means t SE; n = 6. Tin, Tout, colonic temperature before and after 90-min test; dT, change in colonic temperature; BP, bar presses; R, heat reinforcements; dS, change in body heat content; HI, heat influx; NTB, net thermal balance; NTE, net thermal efficiency. Con, control (vehicle); Iso, isoproterenol; BRL, BRL 35135. Significantly different vs. Con (paired t test): * P c 0.001; t P c 0.05; $ P < 0.01. 1.5
--------I 6 -0.5
-1 .o
I
I
0
30
I
I
I
I
60 90 120 150 Time (mins) Fig. 2. Expt 2: changes in colonic temperature in Sprague-Dawley rats at 22°C relative to preinjection values after vehicle (o), 75 rug Isa/kg (e), and 40 pg BRL/kg (A). Mean values t SE; n = 8. Initial colonic temperatures were 37.1 t 0.2, 37.0 & 0.2, and 36.9 t 0.2OC for vehicle, Iso, and BRL groups, respectively. Compared with vehicle (by unpaired t test): * f < 0.05; ** P < 0.01; *** P < 0.0-01.
Ambient
25’C Temperature
Fig. 3. Expt 3: heat production in Sprague-Dawley rats at 3 environmental temperatures after vehicle (open bars), 75 pg Isa/kg (filled bars), and 40 pg BRL/kg (crosshatched bars). Mean values t SE; n = 5. Compared with vehicle (by paired t test): ** P < 0.01; *** P < 0.001.
whether this could be repeated at normal room temperature (i.e., 22°C) using three groups of Sprague-Dawley rats (330-350 g; n = 8/group). The results in Fig. 4 show that, as in the previous experiment, Prop had no effect on vehicle and BRL-treated rats but completely reversed the decrease in colonic temperature produced by Iso alone. The fact that such low doses of Prop could produce the effects seen in the last two experiments prompted the introduction of experiments on anesthetized rats to determine the effect of Prop on another ,&adrenoceptor response (heart rate), as well as recording temperatures at more sites (BAT and tail) than colonic. To concentrate
on the mechanisms responsible for the paradoxical effects of Iso on thermal balance, BRL was dropped from the study at this point. Experiment 6. After preparation under anesthesia and stable baseline values (see METHODS), two = 10) of Sprague-Dawley rats (360-380 g) received either Iso or Iso plus Prop (doses as above), and recordings continued at 15min intervals for the next 2 h. Additional heating was deliberately avoided, and the animals became slightly hypothermic during induction of anesthesia and preparation; colonic and tail temperatures at the end of the baseline period were 35.0-35.5 and 27.0-29.0°C, respectively. If the animal was left or injetted with vehicle, there was little change or only a small slow decline in temperature over the 2-h postinjection period. For this reason and to conserve animals, no further tests with vehicle were carried out. The time course of the changes in heart rate and BAT, colonic, and tail temperatures are shown in Fig. 5, where it will be noticed that, apart from a transient dip in coionic temperature, Iso produced a steady rise in both BAT and colonic temperatures. The initial BAT temperature (recorded before injection) actually started below colonic temperature but rose more rapidly and exceeded colonic temperature after -45 min. When combined with Prop, the BAT response was larger and faster but started to decline toward the end of the 2-h test, when the Iso response was still rising slowly. The mean response over 2 h was significantly (P < 0.01) greater with Iso plus Prop (1.54 t 0.09OC) than with Iso alone (1.09 t O.ll”C). Colonic temperature showed a similar pattern of response to BAT but was less pronounced and lagged behind the changes in BAT temperature. Records of tail temperature gave no sign of an early vasodilator response to Iso, and after a slow decline, it rose above preinjection values only in the last 30 min, when colonic temperature had risen by -1.5”C, and was approaching 37OC. The effect of Prop on the Iso response was to cause an earlier and sustained rise in tail temperature. As would be predicted for a ,&adrenergic agonist, Iso caused a large increase in heart rate (mean increase = 117 t 12 beats/min) over baseline rate (260 beats/min). The effect of Prop was to cause a significant (P < 0.05) reduction in the response (mean increase = 79 t 12 beats/ min), indicating that, despite the low dose used in this and the previous two experiments, it was sufficient to cause at least a partial ,& block. Experiment 7. The inhibition of the negative effects of Iso on thermal balance in rats at 22 and -8°C (expts 4 and
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ADRENERGIC
RECEPTORS
AND
BODY
R919
TEMPERATURE
Table 2. Effect of propranolol on operant responding for heat (expt 4) T- in? “C
Con Con
37.18 to.16 36.90 kO.19 36.98 k0.27 36.68 kO.19 36.83 t0.18 36.60 to.20
+ Prop
Is0 Is0 + Prop BRL BRL
T out? “C
+ Prop
38.48 zto.12 38.50 to.12 37.57 to.24 38.50* to.11 38.40 t0.18 38.52 to.10
Values are means k SE; n = 6. Prop, * P < 0.01; t P c 0.05; $ P c 0.001. 39.0 38.0
dT “C
1.30 to.22 1.60 to.20 0.58 to.34 1.82.t to.20 1.57 to.20 1.92 kO.18
propranolol;
BP, presses/min
2.32 to.25 1.98 to.06 1.82 to.13 2.18 to.20 1.85 t0.18 1.91 kO.18 see Table 1
A
1
39.0 1c
36.0’
I
I
I
0
30
60
Time
I
90
I
120
R/BP, s/press
ds, kJ
HI, kJ
NTB, kJ
NTE, %
13.08 t2.16 10.64 21.29 28.78 k2.90 12.37$ tl.29 7.27 kO.99 9.45 kl.28
5.67 to.73 5.46 kO.77 16.25 t2.04 6.06* to.99 3.98 to.46 4.93 kO.59
1.52 t0.28 1.96 k0.26 0.55 to.32 1.98* to.26 1.87 to.39 2.30 to.29
25.60 k3.95 21.94 t3.77 50.88 t4.51 23.095 k2.87 13.99 k2.14 18.58 k2.74
24.08 t3.81 19.98 t3.60 50.33 24.69 21.12$ t2.71 12.12 tl.86 16.28 t2.73
6.35 tl.27 9.77 kl.70 1.30 t0.81 9.00$ 21.32 13.64 a.90 15.05 A4.15
for other abbreviations. Significantly
l-; 36.0 1 0 0 0
R, s/min
I
150
(mins)
Fig. 4. Ex@ 5: colonic temperature in Sprague-Dawley rats at 22°C receiving vehicle (A), 40 hg BRL/kg (B) and 75 pugIsa/kg (C) with (solid lines) and without (dotted lines) 100 pg propranolol (Prop)/kg. Mean values & SE; n = 8. Compared with agonist alone (paired t test): * P < 0.05; ** P < 0.01; *** P c 0.001.
5), and the potentiation of thermal responses in anesthetized rats (expt 6) by Prop suggested that this ,&adrenergic antagonist could be potentiating the thermogenic effects of Iso. Although unlikely, this possibility was tested by measuring heat production in two groups of SpragueDawley rats (480-500 g; n = 5) after receiving vehicle or Iso with and without Prop (doses as before) at the three environmental temperatures used in experiment 3. The results (Fig. 6) show that Prop had no effect on heat production in either vehicle- or Iso-treated rats. Dependent on ambient temperature, the increases in heat production due to Iso ranged from 45 to 67% of control values regardless of whether the animals had received Prop. Experiment 8. Because it had been established that a low dose of a nonselective ,&adrenergic antagonist could block the effects of Iso on thermal balance without affecting its thermogenic actions, the next experiment was designed using anesthetized rats to see whether the same approach could be used with selective &- (Aten) and ,&(ICI) antagonists. Preliminary tests showed that the same dose of ICI as used for Prop (i.e., 100 hg/kg) was
different vs. agonist alone (paired t test):
effective, but a lower dose of Aten (10 pg/kg) was selected because it appeared to give more pronounced effects at this level. The protocol was the same as for experiment 6 but involved three groups (n = 6) of Sprague-Dawley rats (350-380 g) receiving 75 pg Isa/kg, Iso plus 10 pg Aten/ kg, or Iso plus 100 pg ICI/kg. Preinjection temperatures (BAT = 34.4-34.7”C; coionic = 35.2-35.5”C; tail = 27.3-27.6”C) were similar in all groups but, as seen previously (expt 6), Iso caused a more marked and rapid rise in BAT than in colonic temperature (Fig. 7); there was a late slow rise in tail temperature that barely differed from baseline values. The effects of the two selective antagonists were very different, producing divergent responses in BAT, colonic, and tail temperatures. Aten reversed the rise in BAT and colonic temperature and caused a marked fall in both temperatures. Tail temperatures in Aten-treated rats were no different from those with Iso alone, whereas the ,&-selective antagonist reduced the heart rate response to Iso by >50% (mean increase over 2 h: Iso = 116 t 4, Iso/Aten = 55 t 1 beats/min; P < 0.001). Compared with Iso alone, selective & block with ICI caused BAT temperature to rise very rapidly, with the peak increase (2.62 t 0.2O”C) occurring at 90 min and exceeding (1.41 t 0.3O”C; P < 0.01) and preceding (120 min) that with Iso. BAT temperature started to fall in the last 30 min, but the mean rise over 2 h was nearly double that of Iso alone (Iso = 1.0 & 0.14, ISOICI = 1.92 t 0.2O”C; P < 0.001). Colonic temperature also increased much more than with Iso alone, although the rise lagged behind that of BAT; there was also a delayed but obvious elevation in tail temperature. Unlike the effects of Aten, the heart rate response to Iso was not affected significantly by ICI, although there was an indication that the time course was slightly different. Experiment 9. Because of the divergent effects of selective p block on Iso responses in anesthetized rats, the final experiment in this series was designed to test the effects of these antagonists on behavioral responses in the bar-press apparatus. Two groups of lean Zucker rats (320-350g; n = 8) were tested, with one group receiving on separate occasions Iso and Iso plus Aten and the other receiving Iso and Iso plus ICI (doses as above). The test sequence (agonist alone followed by agonist plus antagonist) was reversed for half the rats, with at least 3 days between tests.
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R920
ADRENERGIC
RECEPTORS
= .E u) w
160
2 -
120
AND BODY TEMPERATURE
Fig. 5. Expt 6: changes in brown adipose tissue (BAT), colonic, and tail temperatures and heart rate in anesthetized Sprague-Dawley rats receiving 75 pg Isa/kg (0) or Iso + 100 pg Prop/kg (0). Mean values t SE; n = 10. Compared with Iso alone (by unpaired t test): * P < 0.05; ** P c 0.01; *** P c 0.001.
al E 80 G s .-c
40
& z I 30
1 90
sb
Time
I 120
6
0
I
1
1
I
0
30
60
90
(mind
The benavioral responses ana cnanges in thermal balance are shown in Table 3, where it will be seen that the effect of Aten on heat reinforcements in Iso-treated rats was neglible. However, the rise in colonic temperature was reduced significantly, resulting in reductions in dS and NTE. In contrast, the effect of ICI on Iso responses was to reduce heat reinforcements and HI by 40%, resulting in a corresponding improvement in therma1 balance and a doubling in NTE.
Time
I
120
(mins)
x*
1
DISCUSSION
For practical and ethical reasons (see METHODS), female lean Zucker rats were used for the operant tests and male Sprague-Dawley rats for the other protocols and ultimately for the experiments carried out under terminal anesthesia. The switch between strain and sex may have introduced some confounding variables, but the fact that the results obtained with the various agonist-antagonist combinations in Sprague-Dawley rats were checked and found consistent with the behavioral responses in Zucker rats suggests that this should strengthen rather than vitiate the overall significance of the results obtained. Perhaps the most intriguing outcome of this series of experiments was the realization that one could use an antagonist to potentiate the effects of an agonist. Moreover, by using a mixed adrenoceptor agonist (Iso) in combination with selective adrenoceptor antagonists (Aten and ICI 118,551), one could manipulate and potentiate the opposing actions of the agonist on body temperature; i.e., Iso could be converted into a selective agonist with positive or negative effects on thermal balance. These effects and the similarities between its thermogenic actions and those of the selective &-agonist (BRL 35135) indicate that Iso is truly a mixed agonist, capable of activating p1-, pZ-, and &-adrenoceptors, The effect of BRL on body temperature and operant responding for heat was similar to that observed previously (4) with two other selective thermogenic agonists (Ro 40-2148 and ICI D7114) and provides additional
12
L
J. CARLISLE
AND MICHAEL
responses antagonists
J. STOCK
Department of Psychology, University of California, Santa Barbara, California 93106; and Department of Physiology, St George’s Hospital Medical School, London S W17 ORE, United Kingdom Carlisle, Harry J., and Michael J. Stock. Potentiation of thermoregulatory responsesto isoproterenol by ,&adrenergic antagonists.Am. J. Physiol. 263 (Regulatory Integrative Comp. Physiol. 32): R915R923, 1992.-The thermoregulatory effects of isothermogenicdosesof isoproterenol (Iso) and a novel ,&agonist (BRL 35135) were tested in rats at 22°C and in rats trained to bar pressfor radiant heat at -8°C. BRL 35135produced hyperthermia at 22°C and reduced operant responding for heat at -8’C, whereasIso reduced body temperature and increasedoperant responding.In both situations, the negative effects of Iso on thermal balancewere abolishedby propranolol at dosesthat did not inhibit heat production. In anesthetized rats, propranolol potentiated the Iso-induced rise in brown adipose tissue and colonic temperature. The potentiation was more marked with the &-selective antagonist ICI 118,551, whereastreatment with the &-selective antagonist atenolol resulted in a profound Iso-induced reduction in temperature. The two selective antagonists also produced divergent responsesin operant behavior in Iso-treated rats at -8°C. These experiments demonstrate the extent to which responsesto a nonselective agonist can be manipulated using appropriately low dosesof selectiveantagonistsand indicate that the effects of Iso on thermal balance are due to its ,& activity. propranolol; atenolol; ICI 118,551; hypothermia; thermogenesis; operant responding OR FACULTATIVE FORMS of thermogenesis, such as nonshivering and diet-induced thermogenesis, are due mainly to increased sympathetic activity, with brown adipose tissue (BAT) acting as the main effector tissue (see Refs. 8, 15 for reviews). Activation of BAT thermogenesis by synaptic release of norepinephrine is mediated by ,&adrenergic receptors (I), and for many experimental studies peripheral administration of ,& adrenergic agonists, such as isoproterenol, have been used. Isoproterenol is a full agonist of thermogenesis, but because it activates &- and ,&-adrenoceptors in other tissues, it affects many other functions. However, these other effects of P-adrenergic activation can be avoided by using some of the new selective thermogenic agonists, such as BRL 35135, Ro 40-2148, and ICI D7114. The selectivity of these ,&adrenergic agonists is believed to be due to the atypical nature of the BAT ,&adrenoceptor (2), which is now thought to correspond to or resemble the ,&-adrenoceptor subtype identified in the human genome (5); these novel thermogenic drugs are therefore often referred to as ,&,-agonists. In a previous study (4), the nonselective P-adrenoceptor agonist isoproterenol was found to cause a decrease in colonic temperature in rats housed at 22°C and increased operant responding for exogenous heat in rats tested at -8OC. These paradoxical hypothermic responses to this thermogenic agonist differed markedly from the responses seen when &-selective agonists (Ro 40-2148 and ICI D7114) were used. As might be predicted, these two thermogenic drugs produced hyperthermia in rats kept at room temperature and decreased ADAPTIVE
behavioral responding for heat in the cold. The conclusion from this study was that the thermogenic (i.e., ,&) actions of isoproterenol were negated by its other (i.e., ,& or &) effects and resulted in heat loss exceeding heat production. It would seem reasonable to assume that this increase in heat loss was due to ,&-mediated effects on peripheral blood flow, particularly to important areas of heat loss, such as the tail (12, 14). However, norepinephrine (which causes vasoconstriction) also produces increased operant responding for heat in a cold environment (21)) and Fregly (7) has reported that the effect of isoproterenol on rat tail temperature is mediated by ,&adrenoceptors and not by &-adrenoceptors. The principal aim of the current study was to attempt a pharmacological “dissection” of the ,&adrenoceptor subtypes to determine which was responsible for the temperature lowering effects of isoproterenol. In the absence of a selective P3-adrenoceptor antagonist, the approach adopted took advantage of the low affinity of the &-adrenoceptor for conventional P-adrenergic antagonists. For example, the concentrations of nonselective and selective (& or &) antagonists required to inhibit BAT adipocyte thermogenesis are two to three orders of magnitude greater than those required to inhibit typical &- or &-mediated responses (1, 18). This explains why previous workers have had to use exceptionally large doses (20-30 mg/kg body wt) of propranolol to block nonshivering and diet-induced thermogenesis (1) or to block the thermogenic response to &-agonists (13). Thus it should be possible to use a low dose of a conventional antagonist, such as propranolol, which would be sufficient to inhibit &- and ,&-mediated responses but would not be high enough to inhibit & activation of thermogenesis. This approach appeared to be successful, and further studies were undertaken using low doses of a selective &-antagonist (atenolol) and a selective &-antagonist (ICI 118,551). For these antagonist studies, a third experimental paradigm (temperature and heart rate responses in anesthetized rats) was added to the two behavioral tests (thermoregulatory responses at 22 and -8OC) used in the earlier study. In addition, the thermogenic (i.e., heat production) and behavioral responses to a different ,&-agonist (BRL 35135) from those used in the previous study were compared with the responses obtained with isoproterenol. A preliminary summary of the results has been reported previously (17). METHODS Animals. For the operant thermoregulatory tests, lean (+/?) 8-mo-old femalerats of “fatty” Zucker strain wereobtained from the colony at the University of California, Santa Barbara. The animalswere fed a standard stock diet (Purina 5001)ad libitum and housedin groups of two to four in a room maintained at 22°C with 50% relative humidity and a 12:12-hlight-dark cycle R915
0363-6119/92 $2.00 Copyright 0 1992 the American Physiological Society Downloaded from www.physiology.org/journal/ajpregu by ${individualUser.givenNames} ${individualUser.surname} (132.174.254.157) on January 17, 2019.
R916
ADRENERGIC
RECEPTORS
(lights on at 0700 h); all testing was carried out during the light phase of the cycle. Training for the operant tests had taken place at regular intervals (twice a week) over the previous month. The other experiments did not require any prior training and were carried out using 3mo-old male Sprague-Dawley rats purchased from Bantin & Kingman (Fremont, CA) and housed two per cage under identical conditions to the above. No other animals were housed in the room, and access was restricted to the researchers on those days when thermoregulatory responses at 22°C were measured to ensure the animals were not disturbed. Different groups of Sprague-Dawley rats were used for the temperature and calorimetry studies, and at a later stage were used for the terminal experiments carried out under anesthesia. Within each experiment, care was taken to ensure that the groups were matched for age and weight, and often the animals acted as their own controls. Body temperature. For the behavioral tests at 22 and -8”C, the animal was removed from the home or experimental cage and colonic temperature was taken within 2 min using a Sensortek (Clifton, NJ) BAT-12 meter and thermocouple probe insertedvia the rectum at least 6 cm until peak temperature was indicated. Temperature recordings were made before and at 30-min intervals after drug injections with the animal sitting unrestrained on a bench. Measurementsof colonic, interscapular BAT and tail temperatures in anesthetized rats were made on two to four animals at a time using a combination of Sensortek thermocouples and YSI-400 (Yellow Springs Instruments) thermistor probes (all calibrated to give identical readings). The lead of the rectal probe was taped to the tail probe, which was positioned on the dorsal surface 1 cm distal to the base of the tail. The fur over the scapulaewas shaved, and a small skin incision madeto allow the interscapular BAT probe to be inserted beneath the brown fat pad, so that the tip of the probe sat in the interscapular cleft closeto the large vein (Sulzer’svein) that drains the fat pad. The BAT thermocoupleprobe was made in the laboratory from fine (36-gauge)copper-constantan wire sheathed in PE-50 tubing. Temperature readings were taken at 15min intervals for 45 min before and 120 min after drug injections. Heart rate. Electrocardiographic (ECG) recordings from anesthetizedrats were madeat 15-min intervals using fine needle recording electrodesin the skin and connected to a Gould recorder (Gould 2400 series). Anesthesia. The rats were anesthetizedwith urethan (1.2 g/kg ip) before the temperature probes and ECG electrodes were attached. Measurementswere madewith the animal in a prone position in a room maintained at 25-26°C. No additional heating was provided, and the animal was killed at the end of the experiment. Operant responding. Shaved rats were placedin a cageinside a freezer maintained at -8”C, having previously beentrained to pressa lever that activated two infrared lamps that together provided 300 W (equivalent to an irradiance of 180 mW/cm2). The experimental test sessionslasted90 min, and the number of bar pressesand duration of heat reinforcements (secondsof heat) were recorded continuously; at least 3 days intervened betweentests on each animal. The bar-pressapparatusand test procedurehave been describedin detail previously (4). Calorimetry. Measurements of heat production by indirect calorimetry were madeusing a system describedin detail previously (10). In outline, the procedure involved placing the animal in a temperature-controlled cabinet through which dry air wasdrawn. A samplewaspassedthrough a Beckman 755 paramagnetic oxygen analyzer (Beckman, Fullerton, CA) before recombining with remaining effluent air and passingthrough a dry-gas meter. Oxygen consumption was recorded for 30 min before and 60 min after drug injection using an on-line minicomputer, and the rate of heat production (W/kg) was calculated and printed every 2 min.
AND
BODY
TEMPERATURE
Drugs. (-) -1soproterenol HCl (Iso), (+) -propranolol HCl (Prop), and (A)-atenolol (Aten) were obtained from Sigma (St. Louis, MO), BRL 35135 (BRL) from SmithKline Beecham (Epsom,UK), and ICI 118,551(ICI) from ICI Pharmaceuticals (Macclesfield, UK). All drugs were prepared for injection by dissolvingin a minimum volume of dimethyl sulfoxide (DMSO; Sigma), making up to volume with 5% gum arabic in normal saline (0.9% NaCl) and sonicating before use. The control vehicle was10% DMSO in 5% gum arabic-saline.All injections (1 ml/kg) were subcutaneous. Data analysis. Differences betweentreatments were assessed from changesin body temperatures (colonic, BAT, tail), heat production, and heart rate as appropriate. The bar-pressexperiments were more difficult to analyze becauseof the problemof trying to compare simultaneously changesin heat influx (HI, mW/cm2) with changesin colonic temperature (“C). This problem was resolvedby adopting the analysisdescribedin the previous study (4), where the data were transformed to common units of thermal balance (kJ) basedon changesin heat storage (dS) and HI from the infrared lamps. These parameterswere then usedto calculate net thermal balance(NTB, kJ = HI - dS) and net thermal efficiency (NTE, % = dS/HI x 100). The significanceof these derived measuresis that a high NTB or a low NTE indicates a greater dependenceon exogenousheat to maintain thermal balance(4). All resultshave beenexpressedas mean values t SE, and differences between vehicle (Con) and drug-treated meansassessed using Student’s t test for matched or unmatched data, asappropriate; quoted probabilities are two tailed. EXPERIMENTSAND
RESULTS
There were nine separate experiments that followed a chronological and logical sequence. For this reason and for clarity, the experimental design, results, and reasoning that led to the next experiment will be described here before discussing their significance in DISCUSSION. Experiment 1. This experiment involved comparing operant responding for heat at -8°C in rats receiving Iso and one of the novel ,&-agonists (BRL) that had not been tested in the previous study (4). Trained lean Zucker rats (300-340 g; n = 6) each received on separate occasions Iso (75 pg/kg), BRL (40 pg/kg), or vehicle (Con); the treatments were randomized. The dose of 75 pg Isa/kg is a maximal thermogenic dose (Stock, unpublished data), whereas the 40-pg BRL/kg dose was chosen to be supramaximal [maximal effective dose (EDloo) - 10 pg/kg]. Supramaximal doses of Iso were avoided because they can be lethal in cold-exposed rats, whereas the high dose of BRL was chosen deliberately to test for any loss of p3 selectivity. Figure 1 shows that all rats increased colonic temperature during the first 30 min in the bar-press apparatus. However, the rise in Iso rats was very much less (OBOC) than in the other groups (1.7-1.8”C) despite bar pressing for three times more heat than the BRL or Con rats over the same period (Fig. 1). Rats receiving BRL responded much as did Con rats, although the 90-min summary of results (Table 1) shows that BRL produced a significant improvement in NTB. Judged from Fig. 1, this was probably due to a slightly lower level of heat reinforcements over the last 60 min of the 90-min test, and calculation of NTB relative to control values over the last hour shows that it was decreased by 20% (P < 0.05); this implies a greater contribution to thermal balance from endogenous heat production in the BRL-treated rats. By contrast, the
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ADRENERGIC
RECEPTORS
I
I
I
1
0
30
60
90
Time
(mid
Fig. 1. Ex@ 1: colonic temperature (A) and heat reinforcements (B) in lean Zucker rats after vehicle (o), 75 pg isoproterenol (Iso)/kg (o), and 40 pg BRL 35135 (BRL)/kg (A). Mean values & SE; n = 6. Compared with vehicle (by paired t test): * P < 0.05; ** P < 0.01; *** P < 0.001.
negative effects of Iso on thermal balance were clearly obvious from the doubling in operant responding for HI, the twofold increase in NTB, and the 75% decrease in NTE (Table 1). The next experiment was designed to see whether these diffe rences were still apparent when animals were in their normal home-cage environment . To conserve the trained lean Zucker rats for operant studies, these experiments were carried out on Sprague-Dawley rats. Experiment 2. Measurements of colonic temperature were made in three groups (n = 8/group) of SpragueDawley rats (320-340 g) housed in their home cages at 22”C, with each group- receiving one of the three treatments described above. There were no significant differences in colonic temperature (mean = 37.1 t O.l”C) before injection, but the changes over the 2.5 h after injections (Fig. 2) revealed a marked decrease in temperature in rats receiving Iso. The intermittent handling and disturbance produced a small 0.6-0.7OC sustained increase in controls, and the mean difference over 2.5 h between Iso and Con rats amounts to >l.O”C (P < 0.001). This contrasts with the BRL-treated rats, which exhibited a larger mean rise than vehicle-treated rats (BRL = 1.14 t 0.08, Con = 0.68 t 0.14; P < 0.05), with the differences in response being particularly noticeable over the first 90 min. The contrast between the effects of BRL and Iso on colonic temperature raises questions about their relative thermogenic potency, and although maximal thermogenic doses of both agonists were used in these experiments, it was decided to see whether these were isothermogenic. Experiment 3. Heat production (W) was estimated
AND
BODY
TEMPERATURE
R917
from measurements of oxygen consumption in SpragueDawley rats (480-500 g; n = 5) before and after receiving vehicle, I so and B RL (doses as before) in a randomized sequence at three env ironmental temperatures. Two of these temperatures (20 and 25°C) fell-either side of the room temperature used for experiment 2, and the third (29OC) corresponds to the thermoneutral temperature of the rat. Each rat was tested at each environmental temperature, and at least 3 days elapsed between treatments. As explained for experiment 1, the dose of Iso (75 pg/ kg) was intended to be maximal and the BRL dose (40 pg/kg) was chosen to be supramaximal. Dependent on environmental temperature, both agonists produced 4070% increases in heat production (Fig. 3) with no significant differences between the two at any of the three environmental temperatures; i.e., the doses were isothermogenic. After it had been established that the differences between the effects of Iso and BRL on thermoregulation could not be due to differences in the activation of thermogenesis, the emphasis switched to trying to determine the role of the ,&- and &-adrenoceptor subtypes in mediating the effects of Iso on thermal balance seen in the previous experiments. Experiment 4. The object of this experiment was to see whether low doses of a nonselective @-adrenergic antagonist (Prop) could be used to inhibit the hypothermic effects of Iso without inhibiting its thermogenic actions. As explained in the introduction, high doses (120 mg/kg) of Prop are required to block thermogenesis, and a dose of 100 pg/kg was eventually selected after preliminary barpress trials with lean Zucker rats using doses that decreased progressively from 5 to 2 to 1 to 0.1 mg/kg. After these preliminary experiments, operant responses for exogenous heat at -8°C were recorded in three groups of lean Zucker rats (330-350 g; n = G/group) after receiving vehicle, Iso or BRL alone, and the same treatment in combination with 100 pg Prop/kg (injected 5 min earlier). The test sequence (agonist alone followed by agonist + Prop) was reversed for half the rats in each group, with at least a 3-day interval between tests. The results (Table 2) show that this low dose of Prop had no discernible effect on any of the parameters of behavioral thermoregulation (colonic temperature, barpress rate, or duration) or thermal balance (dS, HI, NTB, or NTE) in control and BRL-treated rats. However, Prop had a dramatic effect in Iso-treated rats, and completely reversed the responses seen with the agonist alone, so that all parameters of behavior and thermal balance were similar to those in the control and BRL groups. In the absence of Prop, thermal balance (e.g., dS, HI, and NTB) in the Iso rats was significantly (P < 0.001, Sheffe’s test) impaired in comparison to all the other treatment groups. Inspection of’30-min data records (data not shown) indicated that the ability of Prop to reverse the effects of Iso on thermal balance were more apparent during the first 60 min of the 90-min test. Over this period, there was a significant (P < 0.001) 60% decrease in heat reinforcements and NTB and a eightfold increase in NTE in Iso plus Prop rats compared with the same rats receiving Iso alone. Experiment 5. After observation of the potent effects of low-level nonselective ,&adrenergic block on responses at -8”C, exactly the same protocol was adopted to see
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R918
ADRENERGIC
Table 1. Effect of maximal thermogenic on operant
responding T*in9 “C
Con Is0 BRL
AND
doses of isoproterenol
BODY
TEMPERATURE
and BRL 35135
for heat (expt 1) T out.7 “C
36.77 to.26 36.35 to.19 36.73 to.16
RECEPTORS
dT, “C
38.43 to.10 37.15* to.21 38.72 to.15
R s/min
R/BP, s/press
ds, kJ
HI, kJ
NTB, kJ
NTE, %
11.94 to.64 22.97* to.59 10.42 to.57
4.27 k0.42 11.04* to.81 4.43 zkO.85
1.67 to.27 0.79 to.18 1.93 to.27
21.15 tl.14 40.59* tl.78 18.08 to.89
19.48 a.19 39.80* tl.85 16.15$ to.79
8.07 tl.54 2.oq to.47 10.65 kl.26
BP, presses/min
1.67 to.28 0.80-f to.19 1.98 to.28
2.87 to.19 2.15t kO.19 2.64 to.33
Values are means t SE; n = 6. Tin, Tout, colonic temperature before and after 90-min test; dT, change in colonic temperature; BP, bar presses; R, heat reinforcements; dS, change in body heat content; HI, heat influx; NTB, net thermal balance; NTE, net thermal efficiency. Con, control (vehicle); Iso, isoproterenol; BRL, BRL 35135. Significantly different vs. Con (paired t test): * P c 0.001; t P c 0.05; $ P < 0.01. 1.5
--------I 6 -0.5
-1 .o
I
I
0
30
I
I
I
I
60 90 120 150 Time (mins) Fig. 2. Expt 2: changes in colonic temperature in Sprague-Dawley rats at 22°C relative to preinjection values after vehicle (o), 75 rug Isa/kg (e), and 40 pg BRL/kg (A). Mean values t SE; n = 8. Initial colonic temperatures were 37.1 t 0.2, 37.0 & 0.2, and 36.9 t 0.2OC for vehicle, Iso, and BRL groups, respectively. Compared with vehicle (by unpaired t test): * f < 0.05; ** P < 0.01; *** P < 0.0-01.
Ambient
25’C Temperature
Fig. 3. Expt 3: heat production in Sprague-Dawley rats at 3 environmental temperatures after vehicle (open bars), 75 pg Isa/kg (filled bars), and 40 pg BRL/kg (crosshatched bars). Mean values t SE; n = 5. Compared with vehicle (by paired t test): ** P < 0.01; *** P < 0.001.
whether this could be repeated at normal room temperature (i.e., 22°C) using three groups of Sprague-Dawley rats (330-350 g; n = 8/group). The results in Fig. 4 show that, as in the previous experiment, Prop had no effect on vehicle and BRL-treated rats but completely reversed the decrease in colonic temperature produced by Iso alone. The fact that such low doses of Prop could produce the effects seen in the last two experiments prompted the introduction of experiments on anesthetized rats to determine the effect of Prop on another ,&adrenoceptor response (heart rate), as well as recording temperatures at more sites (BAT and tail) than colonic. To concentrate
on the mechanisms responsible for the paradoxical effects of Iso on thermal balance, BRL was dropped from the study at this point. Experiment 6. After preparation under anesthesia and stable baseline values (see METHODS), two = 10) of Sprague-Dawley rats (360-380 g) received either Iso or Iso plus Prop (doses as above), and recordings continued at 15min intervals for the next 2 h. Additional heating was deliberately avoided, and the animals became slightly hypothermic during induction of anesthesia and preparation; colonic and tail temperatures at the end of the baseline period were 35.0-35.5 and 27.0-29.0°C, respectively. If the animal was left or injetted with vehicle, there was little change or only a small slow decline in temperature over the 2-h postinjection period. For this reason and to conserve animals, no further tests with vehicle were carried out. The time course of the changes in heart rate and BAT, colonic, and tail temperatures are shown in Fig. 5, where it will be noticed that, apart from a transient dip in coionic temperature, Iso produced a steady rise in both BAT and colonic temperatures. The initial BAT temperature (recorded before injection) actually started below colonic temperature but rose more rapidly and exceeded colonic temperature after -45 min. When combined with Prop, the BAT response was larger and faster but started to decline toward the end of the 2-h test, when the Iso response was still rising slowly. The mean response over 2 h was significantly (P < 0.01) greater with Iso plus Prop (1.54 t 0.09OC) than with Iso alone (1.09 t O.ll”C). Colonic temperature showed a similar pattern of response to BAT but was less pronounced and lagged behind the changes in BAT temperature. Records of tail temperature gave no sign of an early vasodilator response to Iso, and after a slow decline, it rose above preinjection values only in the last 30 min, when colonic temperature had risen by -1.5”C, and was approaching 37OC. The effect of Prop on the Iso response was to cause an earlier and sustained rise in tail temperature. As would be predicted for a ,&adrenergic agonist, Iso caused a large increase in heart rate (mean increase = 117 t 12 beats/min) over baseline rate (260 beats/min). The effect of Prop was to cause a significant (P < 0.05) reduction in the response (mean increase = 79 t 12 beats/ min), indicating that, despite the low dose used in this and the previous two experiments, it was sufficient to cause at least a partial ,& block. Experiment 7. The inhibition of the negative effects of Iso on thermal balance in rats at 22 and -8°C (expts 4 and
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ADRENERGIC
RECEPTORS
AND
BODY
R919
TEMPERATURE
Table 2. Effect of propranolol on operant responding for heat (expt 4) T- in? “C
Con Con
37.18 to.16 36.90 kO.19 36.98 k0.27 36.68 kO.19 36.83 t0.18 36.60 to.20
+ Prop
Is0 Is0 + Prop BRL BRL
T out? “C
+ Prop
38.48 zto.12 38.50 to.12 37.57 to.24 38.50* to.11 38.40 t0.18 38.52 to.10
Values are means k SE; n = 6. Prop, * P < 0.01; t P c 0.05; $ P c 0.001. 39.0 38.0
dT “C
1.30 to.22 1.60 to.20 0.58 to.34 1.82.t to.20 1.57 to.20 1.92 kO.18
propranolol;
BP, presses/min
2.32 to.25 1.98 to.06 1.82 to.13 2.18 to.20 1.85 t0.18 1.91 kO.18 see Table 1
A
1
39.0 1c
36.0’
I
I
I
0
30
60
Time
I
90
I
120
R/BP, s/press
ds, kJ
HI, kJ
NTB, kJ
NTE, %
13.08 t2.16 10.64 21.29 28.78 k2.90 12.37$ tl.29 7.27 kO.99 9.45 kl.28
5.67 to.73 5.46 kO.77 16.25 t2.04 6.06* to.99 3.98 to.46 4.93 kO.59
1.52 t0.28 1.96 k0.26 0.55 to.32 1.98* to.26 1.87 to.39 2.30 to.29
25.60 k3.95 21.94 t3.77 50.88 t4.51 23.095 k2.87 13.99 k2.14 18.58 k2.74
24.08 t3.81 19.98 t3.60 50.33 24.69 21.12$ t2.71 12.12 tl.86 16.28 t2.73
6.35 tl.27 9.77 kl.70 1.30 t0.81 9.00$ 21.32 13.64 a.90 15.05 A4.15
for other abbreviations. Significantly
l-; 36.0 1 0 0 0
R, s/min
I
150
(mins)
Fig. 4. Ex@ 5: colonic temperature in Sprague-Dawley rats at 22°C receiving vehicle (A), 40 hg BRL/kg (B) and 75 pugIsa/kg (C) with (solid lines) and without (dotted lines) 100 pg propranolol (Prop)/kg. Mean values & SE; n = 8. Compared with agonist alone (paired t test): * P < 0.05; ** P < 0.01; *** P c 0.001.
5), and the potentiation of thermal responses in anesthetized rats (expt 6) by Prop suggested that this ,&adrenergic antagonist could be potentiating the thermogenic effects of Iso. Although unlikely, this possibility was tested by measuring heat production in two groups of SpragueDawley rats (480-500 g; n = 5) after receiving vehicle or Iso with and without Prop (doses as before) at the three environmental temperatures used in experiment 3. The results (Fig. 6) show that Prop had no effect on heat production in either vehicle- or Iso-treated rats. Dependent on ambient temperature, the increases in heat production due to Iso ranged from 45 to 67% of control values regardless of whether the animals had received Prop. Experiment 8. Because it had been established that a low dose of a nonselective ,&adrenergic antagonist could block the effects of Iso on thermal balance without affecting its thermogenic actions, the next experiment was designed using anesthetized rats to see whether the same approach could be used with selective &- (Aten) and ,&(ICI) antagonists. Preliminary tests showed that the same dose of ICI as used for Prop (i.e., 100 hg/kg) was
different vs. agonist alone (paired t test):
effective, but a lower dose of Aten (10 pg/kg) was selected because it appeared to give more pronounced effects at this level. The protocol was the same as for experiment 6 but involved three groups (n = 6) of Sprague-Dawley rats (350-380 g) receiving 75 pg Isa/kg, Iso plus 10 pg Aten/ kg, or Iso plus 100 pg ICI/kg. Preinjection temperatures (BAT = 34.4-34.7”C; coionic = 35.2-35.5”C; tail = 27.3-27.6”C) were similar in all groups but, as seen previously (expt 6), Iso caused a more marked and rapid rise in BAT than in colonic temperature (Fig. 7); there was a late slow rise in tail temperature that barely differed from baseline values. The effects of the two selective antagonists were very different, producing divergent responses in BAT, colonic, and tail temperatures. Aten reversed the rise in BAT and colonic temperature and caused a marked fall in both temperatures. Tail temperatures in Aten-treated rats were no different from those with Iso alone, whereas the ,&-selective antagonist reduced the heart rate response to Iso by >50% (mean increase over 2 h: Iso = 116 t 4, Iso/Aten = 55 t 1 beats/min; P < 0.001). Compared with Iso alone, selective & block with ICI caused BAT temperature to rise very rapidly, with the peak increase (2.62 t 0.2O”C) occurring at 90 min and exceeding (1.41 t 0.3O”C; P < 0.01) and preceding (120 min) that with Iso. BAT temperature started to fall in the last 30 min, but the mean rise over 2 h was nearly double that of Iso alone (Iso = 1.0 & 0.14, ISOICI = 1.92 t 0.2O”C; P < 0.001). Colonic temperature also increased much more than with Iso alone, although the rise lagged behind that of BAT; there was also a delayed but obvious elevation in tail temperature. Unlike the effects of Aten, the heart rate response to Iso was not affected significantly by ICI, although there was an indication that the time course was slightly different. Experiment 9. Because of the divergent effects of selective p block on Iso responses in anesthetized rats, the final experiment in this series was designed to test the effects of these antagonists on behavioral responses in the bar-press apparatus. Two groups of lean Zucker rats (320-350g; n = 8) were tested, with one group receiving on separate occasions Iso and Iso plus Aten and the other receiving Iso and Iso plus ICI (doses as above). The test sequence (agonist alone followed by agonist plus antagonist) was reversed for half the rats, with at least 3 days between tests.
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Fig. 5. Expt 6: changes in brown adipose tissue (BAT), colonic, and tail temperatures and heart rate in anesthetized Sprague-Dawley rats receiving 75 pg Isa/kg (0) or Iso + 100 pg Prop/kg (0). Mean values t SE; n = 10. Compared with Iso alone (by unpaired t test): * P < 0.05; ** P c 0.01; *** P c 0.001.
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The benavioral responses ana cnanges in thermal balance are shown in Table 3, where it will be seen that the effect of Aten on heat reinforcements in Iso-treated rats was neglible. However, the rise in colonic temperature was reduced significantly, resulting in reductions in dS and NTE. In contrast, the effect of ICI on Iso responses was to reduce heat reinforcements and HI by 40%, resulting in a corresponding improvement in therma1 balance and a doubling in NTE.
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DISCUSSION
For practical and ethical reasons (see METHODS), female lean Zucker rats were used for the operant tests and male Sprague-Dawley rats for the other protocols and ultimately for the experiments carried out under terminal anesthesia. The switch between strain and sex may have introduced some confounding variables, but the fact that the results obtained with the various agonist-antagonist combinations in Sprague-Dawley rats were checked and found consistent with the behavioral responses in Zucker rats suggests that this should strengthen rather than vitiate the overall significance of the results obtained. Perhaps the most intriguing outcome of this series of experiments was the realization that one could use an antagonist to potentiate the effects of an agonist. Moreover, by using a mixed adrenoceptor agonist (Iso) in combination with selective adrenoceptor antagonists (Aten and ICI 118,551), one could manipulate and potentiate the opposing actions of the agonist on body temperature; i.e., Iso could be converted into a selective agonist with positive or negative effects on thermal balance. These effects and the similarities between its thermogenic actions and those of the selective &-agonist (BRL 35135) indicate that Iso is truly a mixed agonist, capable of activating p1-, pZ-, and &-adrenoceptors, The effect of BRL on body temperature and operant responding for heat was similar to that observed previously (4) with two other selective thermogenic agonists (Ro 40-2148 and ICI D7114) and provides additional
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