European Journal of Pharmacology, 52 (1978) 85--92

85

© Elsevier/North-Holland Biomedical Press

EFFECT OF GLUCAGON ON RENIN SECRETION IN THE DOG JURO

UEDA, HITOSHI NAKANISHI

* and Y O U I C H I

A B E **

Department of Pharmacology, Teikyo UniversitySchool of Medicine, Tokyo, Japan, and • * Department of Pharmacology, Osaka City UniversityMedical School, Osaka, Japan Received 14 December 1978, revised MS received 10 May 1978, accepted 6 July 1978 J. UEDA, H. NAKANISHI and Y. ABE, Effect ofglucagon on renin secretion in the dog, European J. Pharmacol. 52 (1978) 85---92. The effects of glucagon alone or in combination with theophylline on renin section were studied in relation to renal hemodynamic responses in anesthetized dogs. The intrarenal infusion of glucagon (0.5 pg/kg/min) increased heart rate, renal blood flow, glomerular filtration rate and urine flow without any effect on renin secretion, but at a higher dose (1.0/~g/kg/min) it increased renin secretion significantly. Theophylline (0.1 mg/kg/min) did not affect renal hemodynamics but caused a slight increase in renin secretion after 30--60 rain infusion. The combined infusion of glucagon (0.5 pg/kg/min) with theophy|line (0.1 mg/kg/min) increased renin secretion markedly, although it produced renal hemodynamic changes similar to those induced by glucagon alone. These effects were not suppressed by d,l-propranolol (1.0 pg/kg/min). It is suggested that the increase in renin secretion caused by the combined infusion of glucagon and theophylline resulted mainly from an increase in cyclic AMP in the juxtaglomerular cells, and not from stimulation of ~-adrenoceptors. Glucagon

Theophylline

Renin secretion

1. Introduction Glucagon exerts glycogenolytic, lipolytic and cardiac effects, which, like the effects produced by catecholamines, are mediated through increases in cyclic AMP levels in target organs (Sutherland et al., 1968; ~ye and Langslet, 1972; Sobel and Mayer, 1973; Lucchesi, 1977). Recently, we have found that the intrarenal infusion of glucagon caused renal hemodynamic and urine flow responses similar to those produced by dibutyryl cyclic AMP (Okahara, 1974; Miyazaki et al., 1975; Nakanishi et al., 1977; Ueda et al., 1977). Broadus et al. (1970) showed increases in the cyclic AMP levels of human plasma and urine during an infusion of glucagon, and Mulvehill et al. (1976) demonstrated that glucagon stimulated adenyl cyclase in human renal medullary tis-

Cyclic AMP

sue preparation in vitro. Fernandez-Cruz et al. (1975} and Olsen (1977) have found that glucagon increased plasma renin activity in humans and inconsistently in dogs, and Vandongen et al. (1973) reported that glucagon at a high dose stimulated renin secretion in the isolated rat kidney. Furthermore, both dibutyryl cyclic AMP and theophyUine increased renin release in vivo and in vitro (Reid et al., 1972; Okahara et al., 1977). Thus, there are close relations between renin secretion and cyclic AMP production. In view of these findings, we attempted to clarify the effect of glucagon and theophylline, alone and in combination, on renin secretion in relation to renal hemodynamic responses in anesthetized dogs. 2. Materials and methods 2.1. General

* T o w h o m correspondence should be sent at the above address.

Mongrel dogs of either sex weighing between 12--20 kg were anesthetized with intravenous

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pentobarbital sodium (30 mg/kg), followed b y a maintenance dose as necessary. The left kidney was exposed through a retroperitoneal flank incision and was denervated b y dissecting all visible nerve fibers and the tissue connecting to the renal hilum cephalic to the renal artery. Renal blood flow (RBF) was measured b y an electromagnetic flowmeter (Nihonkoden MF-26), and renal arterial pressure (RAP) was considered equal to aortic pressure measured at the level of the renal artery through a left femoral arterial cannula. Heart rate (HR) was monitored from the pulse b y a biotachometer (Nihonkoden RF5). All of the parameters (RAP, HR and RBF) were recorded on a multipurpose polygraph (Nihondoden RM-45). A No. 25 gauge h o o k e d needle attached to a polyethylene catheter was introduced into the left renal artery proximal to the flow probe and used for the intrarenal infusion of saline and drug solutions at a rate of 0.5 ml/ min. Another catheter was inserted into the right brachial vein to infuse saline at a rate of 1.0--1.5 ml/min.. For the measurement of glomerular filtration rate (GFR) a priming dose of creatinine (100 mg/kg) was given into the right brachial vein, followed b y a continuous infusion at a rate of 50 mg/kg/h to maintain a constant blood level of creatinine. A polyethylene catheter was inserted into the left ureter and urine was collected. After completion of surgery, the dog was left for 90 min to allow RBF, H R and urine flow (UF) stabilize. When these parameters had stabilized, urine was collected during 3 consecutive 10-min control renal clearance periods. At the midpoint of each period, systemic arterial and renal venous blood was collected from the right brachial artery and from the renal vein via a catheter introduced through the left spermatic or ovarian vein. These b l o o d and mine samples were used for renin, creatinine and electrolyte estimation. After control periods, test drugs were infused into the left renal artery for 30--60 min and urine and blood samples were collected in the same manner as in control periods.

J. U E D A ET AL.

G F R was calculated as follows: G F R = (systemic arterial concentration of creatinine-renal venous concentration of creatinine) X renal plasma flow/systemic arterial concentration of creatinine. Sodium in plasma and urine was analyzed with a flame p h o t o m e t e r (Coleman, Model 51). Plasma renin activity (PRA) was measured b y radioimmunoassay according to the technique described b y Stockigt et al. (1971) and expressed as ng angiotension I formed/ml of plasma during a 3 h incubation. The renin secretion rate (RSR) was calculated as the product of renal plasma flow and of the difference b e t w e e n renal venous PRA (VPRA) and arterial PRA (A-PRA) and was expressed as ng angiotensin I per g kidney/ min.

2.2. Experimental design The experiments were carried o u t according to the following protocols.

2.2.1. Glucagon When H R , R B F and UF were stabilized, three 10-min urine samples were collected along with arterial and renal venous blood samples. After the third control period, glucagon was infused into the renal artery for 30 min at a rate of 0.5 or 1.0 pg/kg/min. During the glucagon infusion, urine was collected for three consecutive 10-min clearance periods and at the midpoint of each period blood samples were withdrawn for plasma analysis.

2.2.2. Theophylline After three 10-min control periods, theophylline ethylenediamine was infused into the renal artery at a rate of 0.1 mg/kg/min for 60 min. Urine and blood samples were taken in the same w a y as described for glucagon alone.

2.2.3. Theophylline and glucagon To test the combined effect o f t h e o p h y l l i n e and glucagon, theophylline was infused into the renal artery at a rate of 0.1 mg/kg/min for

E F F E C T O F G L U C A G O N ON R E N I N S E C R E T I O N

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30 min and then glucagon (0.5 pg/kg/min) plus theophylline were infused for 30 min. Urine and blood samples were obtained by the same procedure as described for glucagon alone.

2.2.4. Theophylline, glucagon

d,l-propranolol

and

An infusion of theophylline (0.1 mg/kg/ min) with propranolol (1.0 pg/kg/min) was made into the renal artery for 60 min. At 30 min after the treatment with theophylline and propranolol, glucagon was infused at a rate of 0.5 pg/kg/min for 30 min.

2.3. Statistical evaluation The values presented in this paper are means + S.E. Statistical significance was evaluated by means of Student's paired t-test.

2.4. Drugs used in the experiment Glucagon hydrochloride (Novo), theophylline ethylenediamine (Hoeiyakko) and d,1propranolol hydrochloride (Sumitomokagaku). All drugs were dissolved in saline.

3. Results All the data in tables 1--5 are means of the three consecutive 10-rain periods.

3.1. Effect of glucagon infusion Tables 1 and 2 show the effects of intrarenal infusion of glucagon on renal hemodynamics, UF, PRA and RSR. Glucagon infusion at a rate of 0.5/~g/kg/min resulted in a significant increase in HR, RBF, GFR and UF without any change in RAP. These responses reached their maximum about 10 min after the start of infusion and remained at a high level for consecutive periods. UF and urinary excretion of sodium (UNaV) increased approximately three-fold. Although there were significant changes in renal hemodynamics and UF, PRA and RSR remained unchanged. The infusion of glucagon at a rate of 1.0/~g/ kg/min produced renal hemodynamic and diuretic responses qualitatively similar to those observed with 0.5/~g/kg/min, with maximal responses after 10 min. A-PRA, VPRA and RSR rose during the first 10 rain of the infusion and were maintained at high levels in consecutive periods.

TABLE 1 Effects o f glucagon o n r e n a l h e m o d y n a m i c s , p l a s m a r e n i n activity a n d r e n i n s e c r e t i o n r a t e (n = 8). All values are m e a n s +_ S.E. n = n u m b e r o f dogs t e s t e d , R A P = renal arterial pressure, H R = h e a r t rate, R B F = renal b l o o d flow, G F R = g l o m e r u l a r f i l t r a t i o n r a t e , U F = u r i n e flow, UNaV = u r i n a r y s o d i u m e x c r e t i o n , P R A = p l a s m a r e n i n activity, A = arterial b l o o d , V = renal v e n o u s b l o o d , R S R = r e n i n s e c r e t i o n rate. Time min

RAP HR m m Hg b e a t s / min

RBF ml/g/min

GFR ml/g/min

UF }ll/g/min

UNaV pEq/g/min

PRA

135 -+5

2.79 -+0.10

0.56 -+0.04

9.82 -+0.74

1.06 -+0.21

8.70 -+1.97

10.30 -+2.31

3.23 -+1.48

3.66 1 -+0.15

0.73 1 -+0.04

26.82 1 -+4.22

3.26 1 -+0.33

7.44 +1.80

10.47 -+2.26

8.53 -+4.10

A V ang.I-eq, n g / m l

RSR ang. I-eq. ng/g/min

Saline --30 0

127 -+8

Glucagon (0.5 pg/kg/min) 0 30

127 -+5

180 I -+15

I I n d i c a t e s significance o f the difference b e t w e e n c o n t r o l a n d e x p e r i m e n t a l p e r i o d s (P < 0.01).

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J. UEDA ET AL.

TABLE 2 Effects of glucagon on renal hemodynamics, plasma renin activity and renin secretion rate (n = 4 ). All values are means _+ S.E. See legend to table 1 for abbreviations. Time min

GFR ml/g/min

UF pl/g/min

UNaV

RAP HR mm Hg beats/ min

RBF ml/g/min

PRA

128 _+3

3.81 _+0.34

0.57 _+0.05

11.69 _+1.69

1.76 _+0.33

8.15 _+1.49

9.20 _+1.84

2.59 _+1.13

4.05 _+0.33

0.68 _+0.07

23.54 1 _+3.69

3.71 _+1.01

17.66 i ±2.63

28.74 1 _+5.32

28.87 l _+8.60

pEq/g/min A V ang.I-eq, ng/ml

RSR ang. I-eq. ng/g/min

Saline --30 0

133 _+5

Glucagon (1.0 tlg/kg/min) 0 30

123 _+4

205 +_12

1 Indicates significance of the difference between control and experimental periods (P < 0.05).

3.2. Effect o f theophylline infusion Data obtained with dogs infused with theop h y l l i n e a t a r a t e o f 0.1 m g / k g / m i n f o r 6 0 m i n a r e s u m m a r i z e d i n t a b l e 3. F o l l o w i n g the infusion of theophylline, there were no

c h a n g e s in H R a n d r e n a l h e m o d y n a m i c s , b u t U F a n d UNaV i n c r e a s e d s o o n a f t e r t h e s t a r t o f i n f u s i o n , r e a c h i n g 2.4- a n d 2 . 0 - f o l d i n c r e a s e s r e s p e c t i v e l y at t h e s e c o n d 30 m i n o f infusion. A-PRA, V-PRA and RSR increased gradually during the infusion, showing significant

TABLE 3 Effects of theophylline on renal hemodynamics, plasma renin activity and renin secretion rate (n = 6). All values are means _+ S.E. See legend to table 1 for abbreviations. Time min

RAP HR mm Hg beats/ rain

RBF ml/g/min

125 _+5

3.48 +-0.22

GFR ml/g/min

UF pl/g/min

UNaV

PRA

}aEq/g/min A V ang.I-eq, ng/ml

RSR ang. I-eq. ng/g/min

Saline --30 0

135 _+9

0.64 +_0.03

7.24 _+0.64

1.70 -+0.28

4.10 _+0.68

4.65 _+0.82

1.56 +_0.74

0.65 _+0.05

13.65 2 +_0.95

2.96 +_0.48

6.25 +_1.46

8.92 _+2.86

5.93 _+2.42

0.65 _+0.06

17.26 2 +_2.23

3.44 _+0.73

7.89 1 _+1.51

10.12 I +_1.89

6.46 l +_1.86

Theophylline (0.1 mg/kg/min) 0 30

125 +_5

127 _+7

3.36 +_0.15

Theophylline (0.i mg/kg/min) 30 60

126 +_5

131 +_9

3.38 _+0.29

1 and 2 indicate significance of the difference between control and experimental periods (l p < 0.05 and 2 p < 0.01).

E F F E C T O F G L U C A G O N ON R E N I N S E C R E T I O N

89

increases at 30--60 min; there was no significant difference between the first and the second 30 min of infusion.

3.3. Effect o f glucagon infusion in combination with theophylline Following the control period, theophylline (0.1 mg/kg/min) was infused for 30 min, and a glucagon infusion (0.5 ~g/kg/min) was then superimposed on the theophylline infusion for a further 30 min. The results are shown in table 4. The combined infusion produced significant increases in HR, RBF and GFR similar to those seen with glucagon alone. UF and UNaV alSO increased 3.6- and 3.2-fold over the control levels. During the first 10 rain of the infusion, A-PRA and V-PRA increased from 11.4+- 1.0 and 14.1+ 1.5 to 20.8-+ 4.1 ( P < 0.01) and 29.6-+ 5.0 ng/ ml (P < 0.01), respectively. RSR also increased from 4.5 +- 1.0 to 17.6 +- 6.2 ng/g/min (P < 0.05). These responses were followed by further increases during the two successive

infusion periods. There were no significant differences between the magnitude of the renal hemodynamic responses to glucagon with or without theophylline, but both PRA and RSR were significantly increased by the combined infusion.

3.4. Effect o f propranolol on the responses to glucagon and theophylline Table 5 shows the effects of d,l-propranolol (1.0 pg/kg/min) on the renal hemodynamic and renin responses of 5 dogs to glucagon plus theophylline. When theophylline (0.1 mg/kg/ min) was infused into the renal artery in combination with propranolol, there were no significant changes in HR, RBF and RSR. The superimposition of glucagon on theophylline plus propranolol produced a significant rise in A-PRA, V-PRA and RSR, associated with the increases in HR, RBF, UF and UN,V. The responses to glucagon plus theophylline were not suppressed by d,l-propranolol.

TABLE 4 Effects o f t h e o p h y l l i n e a n d glucagon in c o m b i n a t i o n o n r e n a l h e m o d y n a m i c s , p l a s m a r e n i n activity a n d r e n i n s e c r e t i o n r a t e (n = 6). All values are m e a n s -+ S.E. See legend t o t a b l e 1 for a b b r e v i a t i o n s . Time min

RAP HR m m Hg b e a t s / rain

RBF ml/g/min

131 +-4

2.55 -+0.10

GFR ml/g/min

UF lal/g/min

UNaV

PRA

pEq/g/min A V ang.I-eq, n g / m l

RSR ang. I-eq. ng/g/min

Saline --30 0

117 +-7

0.50 -+0.04

13.79 +-2.91

1.93 +-0.41

10.22 +-1.63

10.73 +-1.13

2.01 -+0.54

0.49 +_0.02

25.37 +_4.91

3.45 -+0.77

11.36 +-1.03

14.12 -+1.52

4.47 -+0.96

6.33 1 +1.35

25.57 2 +3.69

40.07 2 +5.09

27.30 2 +-5.78

Theophylline (0.1 mg/kg/min) 0 30

132 +-4

118 +-7

2.62 -+0.13

TheophyUine (0.1 mg/kg/min) + glucagon (0.5 pg/hg/min) 30 60

125 +-4

168 1 +-13

3.25 1 +-0.24

0.65 1 +0.04

49.41 I -+11.72

1 a n d 2 i n d i c a t e significance o f t h e d i f f e r e n c e b e t w e e n c o n t r o l a n d e x p e r i m e n t a l p e r i o d s (1 p < 0.05 a n d 2 p