Effect of adrenoceptor-blocking agents on the renin response to isoproterenol in dogs

EUROPEAN JOURNAL OF PHARMACOLOGY 26 (1974) 285-297. NORTH-HOLLAND PUBLISHING COMPANY

EFFECT OF ADRENOCEPTOR-BLOCKING AGENTS ON THE RENIN RESPONSE TO ISOPROTERENOL IN DOGS Tatiana A. ASSAYKEEN, Hiromi TANIGAWA* and Donna J. ALLISON Departments o f Surgery (Urology) and Pharmacology, Stanford University School o f Medicine, Stanford, California 94305, U.S.A.

Received 27 February 1973

Accepted 14 February 1974

T.A. ASSAYKEEN, H. TANIGAWA and D.J. ALLISON, Effect o f adrenoceptor-blocking agents on the renin response to isoproterenol in dogs, European J. Pharmacol. 26 (1974) 285-297. Isoproterenol, infused into the kidney of unilaterally nephrectomized dogs at doses of 0.26-0.01 gg/kg/min, was tested for its effect on plasma renin activity in the absence and presence of peripheral infusions of adrenoceptor-blocking agents. Neither phenoxybenzamine (4 mg/kg) nor phentolamine (4.4 and 8.8 gg/kg/min) caused a significant reduction in the renin response during isoproterenol infusion. Likewise D-propranolol, at doses of 2.2 and 22 ~tg/kg/min, also was ineffective in preventing significant increases in plasma renin activity during isoproterenol administration. However, both L- and D,L-propranolol forms of the drug possessing significant ~-adrenoceptor activity, consistently prevented isoproterenol-induced changes in plasma renin levels at doses as low as 2.2 and 5.0 #g/kg/min. The data strongly support the theory that isoproterenol-induced renin release is mediated by ~3-adrenoceptors. No evidence was obtained to suggest the involvement of c~-adrenoceptors in the renin release which occurs during isoproterenol infusion. Adrenergic receptors Phenoxybenzamine

Isoproterenol Phentolamine

1. Introduction Previous studies from our laboratory have shown that in dogs activation of the sympathetic nervous system by insulin-induced hypoglycemia or stimulation o f the medulla oblongata increases plasma renin activity through a mechanism that is blocked by propranolol but not by phenoxybenzamine (Assaykeen et al., 1970; Passo et al., 1971). Additional studies showed that the effect on renin of the peripheral infusion of epinephrine (Assaykeen et al., 1970) is also blocked by propranolol but not by phenoxybenzamine. Work from other laboratories supports the concept that sympathetic mediation of renin release involves /~- but not a-adrenoceptors (Alexandre et al., 1970; Meurer, 1971 ; Michelakis and McAllister, 1972). However, interpretation of these and our stud* Bay Area Heart Research Committee Fellow.

D,L-Propranolol D-Propranolol

L-Propranolol Renin

ies are complicated by the fact that stimulation of the sympathetic nervous system in the presence of ablockade often results in a decrease in mean blood pressure which, of itself, could be responsible for the renin rise seen (Skinner et al., 1964). Winer and his co-workers (Winer et al., 1969; Wirter et al., 1971), using the a-antagonist, phentolamine, have been able to demonstrate a role o f both a- and/3-adrenoceptors in mediating renin release in a variety of experimental conditions, and these experiments do not appear to be complicated by systemic blood pressure changes. Their work receives support from the findings of Hauger-Klevene et al. (1972) that both propranolol and phenoxybenzamine lower renin in hyperthyroid patients and normal volunteers. Administration o f isoproterenol, a ~-adrenoceptor agonist, has been reported to increase plasma renin activity in dogs with chronic renal hypertension (Ayers et al., 1969), in normal dogs (Ueda et al.,

286

T.A. Assaykeen et al., Isoproterenol, adrenergic receptors and renin

1970; Winer et al., 1971; Reid et al., 1972) and in rats (Peskar et al., 1970; Meyer et al., 1971). The work of Ueda et al. (1970) and Meyer et al. (1971) suggests involvement of/3-adrenergic receptors, whereas Winer et al. (1971) show evidence of an action on a-receptors as well. Because of the controversy over the importance of a-adrenoceptors in mediating renin release, particularly in response to the ~-agonist, isoproterenol, the studies to be presented were undertaken. The effect of isoproterenol, administered into the kidney of unilaterally nephrectomized dogs was studied alone and in the presence of either phenoxybenzamine, phentolamine or propranolol. Since Winer et al. (1971) also reported that D-propranolol is as effective as L-propranolol in blocking renin release, we utilized both forms of the drug in our experiments. If renin release following isoproterenol involves a specific/3adrenoceptor phenomenon, only the L-form of propranolol should be effective; the D-form is thought.to be essentially devoid of/3.blocking activity in the dose range used (Howe and Shanks, 1966). Preliminary reports of the data to be presented have been published (Allison et al., 1970; Assaykeen and Tanigawa, 1972; Assaykeen, 1973).

2. Materials and methods 2.1. General

Male mongrel dogs weighing 10-21 kg were anesthetized with pentobarbital sodium (30 mg/kg) with supplements administered as required. The animals had been fasted for 24 hr but were allowed free access to water. An indwelling catheter was inserted into a foreleg vein and patency was maintained by a saline infusion at 0.23 ml/min, except in Groups I and II in which a 5% mannitol solution was infused at 2.4 ml/min. A femoral artery was cannulated for withdrawal of blood samples and monitoring of blood pressure via a Statham strain gauge connected to a Model 7 Grass polygraph. A femoral vein was also cannulated. Through a right flank incision, the left kidney was removed and the right ureter was cannulated. A fish-hook-shaped modified scalp vein needle (23 gauge) was then inserted into the right renal artery. Patency of the needle was maintained by

infusion of a heparinized solution of 5% dextrose in water at a rate of 0.23 ml/min (0.47 ml/min in Groups I and II). These solutions were also used for subsequent intrarenal administration of isoproterenol. Handling of the kidney and dissection of the renal pedicle were minimal. 1.5 hr after completion of surgery, an arterial plasma sample was withdrawn as a blank for subsequent PAH and inulin determinations. Priming doses of PAH (sodium aminohippurate, Merck, Sharpe and Dohme) and inulin (Amar-Stone Labs., Inc.) were then administered, followed by a continuous infusion of both drugs via the foreleg indwelling catheter. The priming and maintenance doses were calculated for each dog and designed to maintain arterial concentrations of PAH and inulin at 2 and 20 mg/100 ml blood, respectively. 30 min later, in Groups I and II, 2 control 10 min clearance periods were performed 20 rain apart. At the midpoint of these and subsequent clearance periods a 10 ml sample of arterial blood was collected in heparin for measurement of Na ÷, K +, PAH and inulin. A 30 ml sample of femoral arterial blood was also collected into ethylenediamine tetra-acetic acid (1 ml of a 3.8% solution per 9 ml of blood) for measurement of plasma renin activity by bioassay. In the remaining groups (III-VI) only a single 15 min control period was taken. At the midpoint of this and all subsequent clearance periods, a 10 ml sample of arterial blood was also collected in heparin for measurement of electrolytes, PAH and inulin. In addition, a 10 ml blood sample was collected into ethylenediamine tetra-acetic acid (1 ml of a 0.3 M solution, pH 7.4, per 9 ml of blood) for measurement of plasma renin activity by radioimmunoassay. In all groups blood withdrawn throughout the experiments was replaced with blood from a nephrectomized donor dog. The following adrenoceptor agonists and antagonists were used in the experimental protocols to be described: isoproterenol hydrochloride (Mann Research Laboratories); phenoxybenzamine hydrochloride (Dibenzyline®, Smith, Kline and French); phentolamine mesylate (Regitine ®, Ciba); D- and L-propranolol (Ayerst Laboratories) and D,L-propranolol hydrochloride (Inderal ®, Ayerst Laboratories). Drug doses in the text for isoproterenol and D- and L-propranolol refer to the free base; doses for all other drugs refer to the salt.

T.A. Assaykeen et al., Isoproterenol, adrenergic receptors and renin 2.2. Group I

In 6 dogs following control clearance periods, isoproterenol was infused into the right renal artery at a dose of 0.26/ag/kg/min. Blood samples were collected at 5, 15, 30 and 60 min after the start of the infusion, each time being the midpoint of a 10 min clearance period. The isoproterenol was stopped and a priming dose of D,L-propranolol, 0.6 mg/kg, was administered. This was followed by an i.v. infusion of D,L-propranolol (0.3 mg/kg/hr) which was continued throughout the remainder of the experiment. 25 min after propranolol administration was initiated, a control clearance period was performed. The infusion of isoproterenol was then reinstituted and additional blood samples were collected 30 and 60 min after the start of the second isoproterenol infusion. 2.3. Group H

The 8 dogs in this group received an intrarenal infusion of isoproterenol at a lower dose of 0.09 /ag/kg/min. Blood samples were collected 15 and 30 min after the start of the infusion. The isoproterenol infusion was then discontinued and over the next hr, 4 mg/kg phenoxybenzamine dissolved in normal saline was infused i.v. at a rate of 0.24 ml/min. A waiting period of 1 additional hr was allowed for the drug to exert its full effect. After a control clearance period isoproterenol was again administered and blood samples taken 15 and 30 min after the start of the infusion. Isoproterenol administration was then stopped and a priming dose of D,L-propranolol (0.6 mg/kg) was administered followed by a continuous i.v. infusion of the drug at a dose of 0.3 mg/kg/hr. 25 min later, a control clearance period was begun. The infusion of isoproterenol was again instituted at the conclusion of this clearance period and blood samples were collected 15 and 30 min after the start of the isoproterenol infusion. 2. 4. Group III

The 6 dogs in this group received an intrarenal infusion of isoproterenol at a rate of 0.06/ag/kg/min for 30 min. This infusion was then repeated 3 additional times along with a simultaneous infusion of phentolamine (8.8 tag/kg/min), D-propranolol (2.2

287

/ag/kg/min) or L-propranolol (2.2 /~g/kg/min). The latter were made up in saline and administered via the foreleg indwelling catheter. Blood samples were taken before and at 15 and 30 min after the start of each isoproterenol infusion. 30 min were allowed to elapse between the end of one infusion and the next control (0 min) sample. Renal function measurements with clearance periods of 15 min duration were made in this and all subsequent groups. 2.5. Group I V

The protocol for the 6 animals in this group was identical to that of Group III except that a still lower dose of isoproterenol was used (0.01 /ag/kg/min). However, in this series the order of drug administration was varied in 3 of the animals. 2 dogs received phentolamine first, then D-propranolol, L-propranolol and finally isoproterenol alone. The remaining dog received phentolamine followed by isoproterenol alone, D- and L-propranolol. 2. 6. Group V

The 7 dogs in this group received isoproterenol at 0.06 /~g/kg/min for 30 min. After a 30 min control period the animals then received isoproterenol again along with a peripheral infusion of phentolamine at 4.4/ag/kg/min for 60 min. During the last 30 min of this second isoproterenol infusion, D,L-propranolol (5 pg/kg/min) was added to the phentolamine infusion. 2. 7. Group VI

In this series 6 dogs received isoproterenol alone (0.06/~g/kg/min) and during the simultaneous peripheral infusion of D-propranolol (22 #g/kg/min) and D,L-propranolol (5/~g/kg/min; n = 4). 1 hr was allowed to elapse between the end of each drug infusion and the subsequent 0 min control sample. Otherwise the protocol was identical to those of Groups III and IV. Standard clearance techniques were used to determine the glomerular f'dtration rate (GFR, inulin clearance) and effective renal plasma flow (RPF, PAH clearance). Inulin concentration in plasma and urine samples was measured by the method of Higashi and

288

T.A. Assaykeen et al., IsoproterenoL adrenergic receptors and renin

Peters (1950) as modified by Fjeldbo and Stamey (1968). PAH concentration in plasma and urine was determined as described by Harvey and Brothers (1962). The products of both reactions were measured spectrophotometrically on a Technicon Autoanalyzer. In Groups I and II, plasma renin activity, expressed as m/ag of angiotensin II per ml of plasma per 3 hr of incubation, was measured according to the method of Boucher et al. (1964) with slight modifications that have been detailed elsewhere (Assaykeen et al., 1968). In the remaining groups plasma renin activity, expressed as mug of angiotensin I formed per ml of plasma per 3 hr of incubation, was measured by radioimmunoassay as described by Stockigt et al. (1971, 1972). The between-assay variation of this method in our laboratory, based on 48 replicate samples at 2 different renin levels, was 13% (coefficient of variation). Plasma and urinary electrolytes were determined by flame photometry. The values presented in this paper are means + 1 standard error. Statistical analysis of the difference between control and experimental periods was determined by use of the t-test on paired observations (Goldstein, 1964).

3. R e s u l t s

3.1. Group I (table 1) Isoproterenol infusion int o the renal artery at a dose of 0.26 tag/kg/min was associated with a rise in plasma renin activity that wa~ statistically significant (p < 0.01) at the 30 and 6~ min sampling periods. However, in spite of the intrarenal route of administration, this dose of isoprotercnol also caused significant decreases in mean blood pressure and plasma [K÷] at the 30 and 6Omin sampling periods. There was no effect on plasma [Na+]. Although 2 sets of control values were determined in this group and in Group II (below), only the values for the period closest to the isoproterenol infusion are shown in tables 1 and 2. In addition, renal function data for Group I has been omitted since technical difficulties made the number of values too small to be meaningful. In general, renal function was depressed with this dose of isoproterenol. In the presence of D,L-propranolol, isoproterenol no longer caused a significant rise in plasma rertin activity and the effects on blood pressure and plasma [K÷] were either blocked or reversed.

Table 1 Effects of isoproterenol infusion into the renal artery (0.26/2g/kg/min) in dogs (Group I; n = 6). Isoproterenol 0 rain

Isoproterenol + (0.3 mg/kg/hO +5 rain

+15 rain

+30 min

+60 min

0 mill

D, L-propranolol

+30 min

+60 rain

Plasma renin activity (m/~g/ml/3 hr) 10.0 ±

1.1"

11.6 ±

1.3

21.0 ± 6.0

27.3 ± 5.1 b

26.2 ± 2.2 b 1 2 , 7 ± 1

15.0 ± 3.3

11.6 ± 1.8

Mean blood pressure

(mmHg) Plasma potassium (mEq/L)

136 ±7

136 ±3

4.4 ± 0.2

4.9 ± 0.5 a

122 ±8 a 4.2 ± 0.4

114 ±7 b 3.9 ± 0.2b

* Data expressed as means ± S.E.

a p < 0.05 when compared to the respective 0 min control value. bp< 0.01 when compared to the respective 0 min control value.

119 ±7 b 3.4 ± 0.2h

127 ±4 3.6 ± 0.2

133 ±3 b 4.1 ± 0.3 a

132 ±5 4.4 ± 0.5

61 42

± 18 ±6

66 45

± 17 +-9

42 47

± 15 ± 12

2.0±0.3

2.4±0.3

± 14 ±8

4.8±0.5

6.2±0.5

58 39

1.6~0.2

±5

1.8±0.2

126

4.9±0.5

±3

11.8±2.9

3.7±0.2 b

128

21.9±4.3 a

* Data expressed as means ± S.E. a p < 0.05 when compared to the respective 0 min control value. b p < 0.01 when compared to the respective 0 min control value.

Sodium Potassium

(.Eq/min):

Plasma renin activity (m~g/ml/ 3 hr) 13.3±3.4" 18.9±3.6 b Mean blood pressure (ram Hg) 127 ± 4 127 ± 3 Plasma potassium (mEq]l) 4.120.2 4.0±0.2 GFR (ml/min/kg) 1.7±0.2 1.820.2 RPF (ml/min/kg) 6.6~0.7 6.5±0.4 Urine flow (ml/min) 2.3±0.2 2.4±0.3 Urinary electrolytes ±5 b

51 42

± 18 ±7

2.0±0.3

4.9±0.5

1.6±0.1

4.4±0.4 a

112

23.9±5.3 a

+15 min

0 min

+30 min

0 min

+15 min

Isoproterenol + phenoxybenzamine (4 mg/kg)

Isoproterenol

±6 b

43 43

± 15 ±8

1.8±0.3

5.320.4

1.6±0.1

4.1±0.3 a

111

24.2±6.6 a

+30 min

±6

31 26

± 10 ±4

1.7±0.2

4.3±0.2

1.4±0.1

4.0±0.2

121

13.522.6

0 min

±7

42 34

± 12 a ±7

1.820.3

4.7±0.4

1.520.1

4.1±0.2

123

12.8±3.0

+15 min

±9

48 41

± 12 ±6 b

1.8±0.2

4.3±0.5

1.4±0.2

4.1±0.2

124

12.0±2.6

+30 min

Isoproterenol + phenoxybenzamine (4 mg/kg) and D, L-propranolol (0.3 mg]kg/hr)

Table 2 Effects of isoproterenol infusion into the renal artery (0.09 t~g/kg/min) in dogs (Group II; n = 8).

~D

t~

q~

N

290

T.A. Assaykeen et al., lsoproterenol, adrenergicreceptorsand renin

3.2. Group H (table 2) Lowering the dose of isoproterenol to 0.09 /ag/kg/min caused a smaller rise in plasma renin activity but the changes were significant at both the 15 and 30 min sampling periods (p < 0.01 and 0.05, respectively). This dose of isoproterenol did not cause a statistically significant change in systemic blood pressure or plasma [Na+]. Plasma [K÷] was unchanged at 15 min but had decreased significantly ( p < 0.01)by the 30 min sampling period. No significant changes were noted in GFR, RPF, urine flow or Na ÷ and K÷ excretion. In the presence of a-blockade with phenoxybenzamine, isoproterenol again caused a significant increase in plasma renin activity at both the 15 and 30 min sampling periods. The mean change from control was greater in the presence of phenoxybenzamine than with isoproterenol alone. With t~-adrenoceptor blockade in effect, mean blood pressure and plasma [K+] were significantly decreased during infusion of isoproterenol, as seen in the experiments in Group I with isoproterenol alone. Again, no significant changes in renal function or plasma [Na+] were noted. Isoproterenol infusion in the presence of D,L-propranolol no longer was associated with a rise in plasma renin activity. Systemic blood pressure and plasma [Na+] and [K÷] were also unchanged. No measureable changes in GFR, RPF, or urine flow were noted but Na+ and K+ excretion increased significantly.

3.3. Group III (table 3) Intrarenal administration of isoproterenol at a dose of 0.06/ag/kg/min caused a significant increase in peripheral plasma renin activity. Repeated administration of the amine in the presence of either phentolamine or D-propranolol was also accompanied by significant increases in plasma renin activity. This response (in terms of either absolute or percentage change) to isoproterenol in the presence of either drug was not statistically significantly different from that seen with isoproterenol alone. L-propranolol completely blocked the renin response to isoproterenol infusion. Mean blood pressure did not change significantly in the presence of isoproterenol except

for a slight fall during the infusion of L-propranolol. Diastolic blood pressure did decrease significantly, however, when isoproterenol was infused in the presence of phentolamine (128 -+ 6 to 106 -+ 5 mm Hg,p < 0.05). Again there was a tendency for plasma [K+] to fall during isoproterenol administration except in the presence of L-propranolol. No significant changes in plasma [Na+], urine volume or electrolyte excretion were noted. There was a slight but significant fall in GFR 30 min after the infusion of isoproterenol plus phentolamine, but similar significant decreases in both GFR and RPF also occurred during L-propranolol administration.

3.4. Group IV(table 4) When the dose of isoproterenol was lowered to 0.01 ttg/kg/min, a significant rise in plasma renin activity was no longer observed, except at the 30 min sampling period in the presence of phentolamine. It may be noteworthy that at this time period other ~effects of isoproterenol appeared to be enhanced. Diastolic blood pressure decreased from 115 -+ 4 to 89 -+ 9 mm Hg (p < 0.05) and there was a significant decrease in plasma [K+]. Again D-propranolol was without effect (not shown in table 4) but L-propranolol caused significant decreases in plasma renin activity compared to control levels. The latter changes were accompanied by significant elevations in plasma [K+].

3.5. Group V (table 5) Because of the tendency of phentolamine to cause a decrease in mean blood pressure during the infusion of isoproterenol, the 0.06 tag/kg/min dose was repeated in the presence of a lower dose of phentolamine (4.4 /gg/kg/min). Such decreases in blood pressure could have caused the renin rises seen, thus masking a blocking effect of phentolamine on renin secretion. It was reasoned, therefore, that lowering the dose of phentolamine might obliterate the blood pressure changes and, if phentolamine has any effect on renin secretion, at least a partial block could then be observed. The degree of t~-adrenoceptor blockade obtained with a 4.4 /ag/kg/min dose of phentolamine was determined in 3 dogs against a test dose of epinephrine (2 tag/kg). The mean systolic and dias-

291

I:A. Assaykeen et al., Isoproterenol, adrenergic receptors and renin Table 3 Effects of isoproterenol infusion into the renal artery (0.06/~g/kg/min) in dogs (Group III; n = 6). Isoproterenol

0 min Plasma renin activity (mtag/ml/3 hr) Mean blood pressure (mmHg) Plasma potassium (mEq/1) GFR (ml/min/kg) RPF (ml/min/kg) Urine flow (ml/min) Urinary electrolytes (uEq/min): Sodium Potassium

+15 min

12.5 ± 3.2*

140

Isoproterenol + phentolamine (8.8 ~tg/kg/min)

±6

25.1 ± 5.4 b

139

±5

139

± 4

3.4 ± 0.2

3.2-+

1.5 ± 0.2

1.6 ± 0.2

3.7 ± 0.4 0.3 ± 0.0

27 25

-+6 -+5

22.1 ± 4.5

139

± 4

0 min

27,2 ± 6.7 a

3.5 ± 0.2

19.6 ± 4.2

143

± 7

31.1 ± 4.5 b

132

± 5

+30 min

35,3 ± 5.2 b

125

+-6

3.4 ± 0.2

3.1 ± 0.2

1.6 ± 0.2

1.9 ± 0.3

1.2 ± 0.3

1.5 ± 0.3 a

3.5 ± 0.4

3.6 ± 0.4

3.6 ± 0.5

2.4 ± 0.5

3.4 ± 0.6

0.3 ~ 0.1

0.3 ± 0.1

0.4 ± 0.2

0.3 ± 0.1

0.2 +- 0.1

23 21

±9 ±5

40.1 +- 5.9 b

139

54

25 22

0.1 b

+15 min

3.5 ± 0.3 c

Isoproterenol + D-propranolol (2.2 ug/kg/min) 0 min +15 min Plasma renin activity (m~g/ml/3 hr) Menn blood pressure (mmHg) Plasma potassium (mEq/l) GFR (ml/min/kg) RPF (ml]min/kg) Urine flow (ml/min) Urinary electrolytes (~Eq/min): Sodium Potassium

+30 min

± 10 ± 5

+30 min

37.0 ± 4.8 b

142

54

28 28

-+ 15 ± 9

23 21

± 10 ± 7

Isoproterenol + L-propranolol (2.2/~g/kg/min) 0 min +15 min

25.0 ± 4.0

144

± 4

24.0 ± 4.0

141

± 4

20 22

± 8 ±5

+30 min

24.1 +- 6.4

138

± 4a

3.3 ± 0.2 c

3.3 ± 0.1

3.1 ± 0.1

3.5 ± 0.1

3.5 ± 0.1

3.5 ± 0.1

1.5 + 0.3

1.5 ± 0.2

1.5 ± 0.1

1.5 ± 0.2

1.3 ± 0.2

1.2 ± 0.2 b

2.8 ± 0.5

2.9 ± 0.3

2.9 ± 0.2

2.6 ± 0.3

2.4 ± 0.3

2.3 ± 0.4 a

0.2 ± 0.1

0.2 ± 0.0

0.2 ± 0.0

0.2 ± 0.1

0.2 ± 0.0

0.2 +- 0.1

28 26

+- 10 ± 7

24 26

±6 ±6

27 26

+-7 ±5

* Data expressed as means ± S.E. a p < 0.05 when compared to the respective 0 rain control value. b p < 0.01 when compared to the respective 0 rain control value. c The entire group is calculated on the values from only 5 dogs.

30 27

± 11 ± 6

30 28

± 10 ± 7

31 30

± 12 ± 8

4 15

128

±4

0.1 + 0.0

-+ 0.0

-+1 ±4

3.4 -+ 0.5 a

2 0.6 c

3 14

1.2 2 0.3 a

2 0.2 c

21 -+3

4.2 -+ 0.4

-+ 0.6

24 a

25.1 ± 4.8

± 5.0*

+6

4 16

±1 -+6

0.2 ± 0.1

3.2 ± 0.5 a

1.1 2 0.2 b

4.3 -+ 0.5

125

25.9 ± 3.6

* Data expressed as m e a n s ± S.E. a p < 0.05 w h e n compared to t h e respective 0 m i n c o n t r o l value. b p < 0.01 w h e n compared to t h e respective 0 rain control value. c T h e enti~e group is calculated o n t h e values f r o m o n l y 5 dogs. d T h e entire group is calculated o n t h e values f r o m only 4 dogs.

Sodium Potassium

O,~/min):

electrolytes

Urinan,

Plasma renin activity (ml~g/ml/ 3 hx) 27.5 Mean blood pressure (mmHg) 125 Plasma potassium (mEq]D 4.3 GFR (ml/min/kg) 1.5 RPF (ml/min/kg) 4.6 Urine flow (ml/min) 0.2 -+3

6 14

±3 ±2

0.2 2 0.0

3.5 2 0.3 e

1.4 2 0.4 d

4.1 -+ 0.3

129

21.1 ± 3.4

5 15

0.0

-+ 2 2 4

0.2 2

2.8 + 0.6

1.0 ± 0"4 a

0.3

±11

3.9 2

109

28.8 + 4.2

+15 m i n

0 min

+30 m i n

0 min

+15 rain

Isoproterenol + p h e n t o l a m i n e (8.8 ~g/kg]min)

lsoproterenol

-+11 a

0.6

3 6

0.0 a

2 2a -+ 4 b

0.1 2

1.4 -+ 0.9

0.7 2

3.7 ± 0.3 a

98

38.9 + 8.4 a

+30 m i n

26

9 26

+2 c -+7

0.2 2 0.1

2.3 ± 0.3

0.9 ± 0.2

4.4 2 0.5

130

25.6 ± 7.9

0 min

28

8 23

22 -+8

0.2 -+ 0.0

2.1 2 0.3

0.8 ± 0.2

4.7 -+0 . 5 b

127

19.4 + 5.8 a

+15 m i n

Isoproterenol + L-propranolol (2.2 ~g/kg/min)

Table 4 Effects o f isoproterenol infusion into t h e renal artery (0.01 ~tg/kg/min) in dogs ( G r o u p IV; n = 6).

±8

9 26

+3 ±8

0.2 ± 0.1

1.8 2 0.4

0.7 ± 0.2

4.7 ± 0.5 b

126

17.9 + 5.3 a

+30 m i n

e~

tO

bO

24 28

±9 ±6

22 30

±9 ±9

0.2 ± 0.0

4.4 ± 0.6 a

4.8 ± 0.7

0.2 ± 0.0

1.9 ± 0.3

±8

1.9 ± 0.3

136

3.5 ± 0.1

±7

16.9 ± 5.7

3.6 ± 0.1

144

8.6 ± 1.8"

+-7

19 26

±8 ±5

0.2 ± 0.0

4.6 ± 0.5

1.8 -+ 0.2

3.3 ± 0.1 a

141

17.2 ± 4.6

* Data expressed as means ± S.E. a p < 0.05 when compared to the respective 0 rain control value. b p < 0.01 when compared to the respective 0 rain control value.

Sodium Potassium

(uEq/min):

(ml/min) Urinary electrolytes

Urine flow

Plasma renin activity (mug]ml/3 hr) Mean b l o o d pressure (mmHg) Plasma potassium (mF_xl]l) GFR (ml/min/kg) RPF (ml/min/kg) ±8

18 27

±7 ±8

0.2 ± 0.0

3.9 ± 0.6

1.7 ± 0.3

3.7 ± 0.2

148

11.4 ± 2.9

±8

22 27

±8 ±6

0.2 ± 0.0

3.2 ± 0.5 a

1.4 -+ 0.2 a

3.5 ± 0.2

139

18.7 ± 6.1

+15 rain

0 min

+30 rain

0 rain

+15 rain

Isoproterenol + phentolamine (4.4 gg/kg]min)

Isoproterenol

±7

18 26

±6 ±4

0.2 ± 0.0

3.7 ± 0.6

1.6 ± 0.2

3.2 ± 0.1 a

138

18.9 ± 3.4 b

+30 rain

Table 5 Effects o f isoproterenol infusion into the renal artery (0.06/~g/kg/min) in dogs (Group V; n = 7).

±8 a

16 23

±4 ±4

0.2 ± 0.0 a

3.3 ± 0.6

1.4 ± 0.2

3.6 ± 0.2 a

140

15.0 ± 3.0 b

+45 min

+-9 a

20 21

±6 ±5

0.2 ± 0.0

3.2 ± 0.6

1.5 ± 0.2

3.5 ± 0.2

129

12.6 ± 2.9 a

+60 min

Isoproteren~! + phentolamine (4.4 t~g/kg/min) + D, L-propranolol (5/~g/kg/min)

tO ~D

t~

± 7

3.9 ± 0.2

126

20.2 ± 5.0*

~6

3.7±0.3

129

30.8±6.2 a

±7

3.6±0.3 a

126

32.6±6.4 a

* D a t a expressed as means + S . E . a/7 < 0.05 when compared to the respective 0 rain control value. b p < 0.01 when compared to the respective 0 rain control value. e The entire group is calculated on the values from only 5 dogs.

~hr) Mean blood pressure (mmHg) Plasma potassium (mEq]L)

Plasma renin activity

±5

4.0±0.1 c

130

16.8±4.7

±4

3.9±0.2

131

26.8±7.7 a

+15 min

0 min

+30 min

0 rain

+15 min

Isoproterenol + D-propanolol (22 t~g/kg/min; n = 6)

Isoproterenol (n = 6)

±5

3.8±0.2

132

29.3±9.0 a

+30 rain

±3

4.2±0.4

142

21.8±6.0

0 rain

±6

4.2±0.4

140

21.6±4.9

+15 min

Isoproterenol + D, L-propranolol (5 ~tg/kg/min; n = 4)

Table 6 Effects of isoproterenol infusion into the renal artery (0.06 t~g/kg/min) in dogs (Group VI).

±6

4.2±0.4

142

21.5±5.4

+30 min

~a

4~

T.A. Assaykeen et al., lsoproterenol, adrenergic receptors and renin

tolic blood pressure rises following epinephrine alone were 101 -+ 1.5 and 61 +- 7 mm Hg, respectively. After 15-30 min of phentolamine infusion the same dose of epinephrine caused only a 63 --- 20 and 30 -+ 11 mm Hg rise in systolic and diastolic blood pressure. Both changes were statistically significant (p < 0.05) and represent an approximately 50% decrease in the response to epinephrine at the dose used. When isoproterenol alone was infused into the animals of this series, plasma renin activity rose in each of 7 dogs but, presumably due to high values in 1 dog, the rise for the group as a whole was not statistically significant. However, when the amine was administered along with phentolamine, a significant rise in renin was noted at the 30 min sampling period, which was not accompanied by significant changes in either diastolic or mean blood pressure. Statistical analysis of the actual or percentage change in plasma renin activity again showed that the renin response to isoproterenol was not significantly decreased in the presence of phentolamine. When propranolol was then administered along with the isoproterenol and phentolamine, plasma renin activity decreased toward control levels. The mean renin value at the 60 min time period, though still elevated when compared to the 0 min sample, had significantly decreased if compared to the value before propranolol administration (12.6-+ 2.9 verses 18.9 -+ 3.4; p < 0.01). Thus propranolol could block the renin rise following isoproterenol plus phentolamine and there was again no evidence for the involvement of a-receptors in mediating isoproterenol-induced renin release.

3.6. Group VI (table 6)

In this series the effect of a very large dose of D-propranolol was tested. The level chosen (22 /ag/kg/min) was 10 times the dose of L-propranolol which had been sl-,own previously to effectively block the renin response to 0.06 /ag/kg/min isoproteren01 (see table 3). In the 6 animals studied there was again a significant rise in plasma renin activity whether isoproterenol was given alone or in the presence of a large dose of D-propranolol. D,L-propranolot completely blocked the renin response to isoproterenol in the 4 animals studied. These experirnents were not complicated by any changes in blood pressure or

295

renal function, but predictable changes in plasma [K÷] were observed.

4. Discussion The results presented indicate that, in dogs, intrarenal administration of isoproterenol at doses of 0.26-0.06 /~g/kg/min causes a significant increase in plasma renin activity within 15-30 min. Previous studies in other laboratories in dogs (Ayers et al., 1969; Ueda et al., 1970; Winer et al., 1971; Reid et al., 1972) and in rats (Peskar et at., 1970; Meyer et al., 1971) have also shown that administration of isoproterenol is associated with an increase in plasma renin activity. In addition, in agreement with earlier reports (Ueda et al., 1970; Meyer et al., 1971; Winer et al., 1971), the data presented indicate quite clearly that the rise in plasma renin activity during isoproterenol infusion can be blocked by the /3-adrenoceptor antagonist, propranolol. That this is a result of specific ~adrenoceptor-blocking activity was demonstrated in our experiments by the lack of effect of even high doses of the D-isomer. In anesthetized dogs, D-propranolol has been reported to possess only 1/60-1/100th of the /3-blocking activity of the D,L-form (Howe and Shanks, 1966). Our results, therefore, do not support earlier reports which indicate that identical doses of either the D- or L-forms of/3-adrenoceptor-blocking agents equally affect renin secretion (Winer et al., 1971; Distler et al., 1971). They are confirmed, however, by a recent publication (Meyer et al., 1973) which demonstrated that in rats L-propranolol but not an equal dose of D-propranolol blocked the renin response to isoproterenol. We have no explanation for these discrepancies, but the lack of effect in our experiments of a dose of D-propranolel, 10 times the dose of L-propranolol which completely blocks the renin response to isoproterenot, adds strength to our conclusion. The participation of a-adrenoceptors in the renin response to isoproterenol is not suggested by the data presented. Similarly, Meurer (1971) has reported that in man the renin stimulating effect of the/3-agonist, orciprenalin, is blocked by propranolol but not by phentolamine. The data of Winer et al. (1971), on the other hand, disagree with both the present study and

296

T.A. Assaykeen et al., Isoproterenol, adrenergic receptors and renin

that of Meurer (1971), and show evidence for a blocking effect of phentolamine on isoproterenolinduced renin secretion. It is difficult to explain such a major discrepancy between our data and that of Winer et al. (1971) but some differences in the protocol and results of both studies should be pointed out. The experiments shown in table 4 reproduce to the best of our knowledge the protocol and drug doses published by Winer et al. (197.1) with the exception that our animals were uninephrectomized. The left kidney was removed in order that peripheral plasma renin levels could be used as an indicator of renin secretion from the infused right kidney. In a dog removal of the left kidney through a right flank incision is a very simple and quick procedure and is unlikely to add significantly to the surgical stress incurred by the exposure and cannulation of the right kidney. With the lowest dose of isoproterenol used (0.01 tag/kg/min; table4) we, unlike Winer et al. (1971), were not able to show any significant change in plasma renin activity. This may have been due to the higher than normal control renin levels observed in our animals in this series. However, in spite of the lack of effect of isoproterenol alone at 0.01 /ag/kg/ min, there was a significant effect of the amine on renin in the presence of phentolamine, but this increase in renin was accompanied by significant decreases in both mean blood pressure and plasma [K+]. Such systemic changes were not reported by Winer's group in spite of the similar protocol and identical doses of the two drugs used. It could be argued that the difference between our results and those of Winer et al. (1971) can be explained on the basis of the blood pressure changes that take place during phentolamine or phenoxybenzamine administration in our hands. Thus, the ,v-adrenoceptor antagonists may have blocked the renin rise due to isoproterenol, but this effect was masked by the renin changes that resulted from the decrease in mean blood pressure. However, several lines of evidence from our data would seem to negate this argument. Table 3 shows quite clearly that isoproterenol can raise plasma renin activity significantly in the presence of phentolamine without a concomitant statistically significant decrease in mean blood pressure. Secondly, 2 out of 6 dogs receiving either 0.06 or 0.01/ag/kg/min isoproterenol showed rises in plasma renin activity in the presence of phentolamine

without a simultaneous decrease in blood pressure. And lastly, during the first 30 min of phentolamine infusion at 4.4 gg/kg/min there was no significant change in blood pressure while plasma renin activity rose significantly, in spite of at least partial a-adrenoceptor blockade. By the end of the 60 min infusion, mean blood pressure had fallen approximately 20 mm Hg (p < 0.05) yet propranolol was able to significantly lower renin levels. Also of interest is the report by Loeffler et al. (1972) that rapid administration of phenoxybenzamine alone to dogs results in a decline in systemic blood pressure which is accompanied by a marked rise in plasma renin activity. On the other hand, the administration of phenoxybenzamine combined with propranolol causes a similar decrease in blood pressure but is not accompanied by any significant change in plasma renin activity. These results could be interpreted to mean that o~-adrenoceptors are inhibitory to renin secretion. Such a concept is supported by the observation that the a.agonist, methoxamine, inhibits the renin response to isoproterenol in the isolated rat kidney (R. Vandongen, Dept. of Med., Univ. of Queensland, personal communications). Furthermore, since our experiments were completed two papers have appeared in the literature which add support to our conclusion that o~-adrenoceptors do not participate in the renin rise that occurs in response to catecholamine administration. Using isolated perfused rat kidneys, Vandongen et al. (1973) have reported that the increased renin secretion seen following the infusion of either norepinephrine or isoproterenol is unaffected by phenoxybenzamine but is markedly suppressed by D,L-propranolol. Similarly, Nelly et al. (1973), using rat kidney slices, have reported that the renin response to norepinephrine is enhanced by both phenoxybenzamine and phentolamine but is blocked by either L- or D,L-propranolol. Thus, on the basis of these and our own results we conclude that isoproterenol increases plasma renin activity through a/3- and not an ~,-adrenoceptor.

Acknowledgements

The authors are indebted to Dr. Alex Sahigian-Edwardsof Ayerst Laboratories for generous supplies of D-, L- and D,L-propranolol (Inderal®). The technical assistance of Miss

T.A. Assaykeen et al., lsoproterenol, adrenergic receptors and renin V.M. Brorsson, Miss J. Jackson and Miss J. Bragonje is also gratefully acknowledged. This work was supported by USPHS research grant AM 13548 and training grant GH 0322.

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