Effect of vasopressin V1 (OPC-21268) and V2 (OPC-31260) antagonists on renal hemodynamics and excretory function

Life Sciences,Vol. 55, No. 4, pp. PL 67-72, 1994

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0024-3205(94)00123-5 PHARMACOLOGY LETTERS Accelerated Communication

EFFECT OF VASOPRESSIN V, (OPC-21268) and V~ (OPC-31260) ANTAGONISTS ON RENAL HEMODY'NAMICS AND EXCRETORY FUNCTION Testuya Nakamura ~, Tetsuo Sakamaki, Toshiaki Kurashina, Jin Hoshino, Kunio Sato, Zenpei Ono and Kazuhiko Murata The Second Department of Internal Medicine, Gunma University School of Medicine, Maebashi, 371 Japan (Submitted March 29, 1994; accepted April 18, 1994; received in final form May 13, 1994)

Abstract: Our objective was to assess the effect of endogenous AVP on renal hemodynamics and excretory function. We measured mean arterial pressure (MAP), renal blood flow (RBF), glomerular filtration rate (GFR) and urine osmolality before and after the intravenous infusion of a V 1 antagonist (OPC-21268), a V, antagonist (OPC-31260) and their vehicle (saline) in anesthetized male Wistar rats. The iiafusion of the VA antagonist increased the urine flow rate and reduced the urine osmolality significantly (p < 6.05). The infusion of saline and the V. antagonist did not change the urine flow rate or the urine osmolality. The infusion of saline, th~ V~ antagonist and the V. antagonist had no effect on MAP, RBF or GFR. These results suggest that endogenous AVP plZays a critical role in the regulation of renal water reabsorption mediated through the V 2 receptor. Key Words: vasopressin antagonist, renal blood flow, glomerular filtration rate

Introduction Two subtypes of peripheral arginine vasopressin (AVP) receptors have been functionally and pharmacologically distinguished from one another. In the kidney, AVP exerts an antidiuretic effect through V~ vasopressin receptors via an adenosine 3', 5'-monophosphate (cAMP)dependent mechariism (1). In vascular smooth muscle, AVP elicits vasoconstriction through the Vlreceptors by a cAMP-independent mechanism that is coupled with phosphoinositide turnover (2). Although many AVP antagonists have been developed (3, 4), these antagonists are all peptide analogues and do not have adequate oral bioavailability. Recently, nonpeptide vasopressin V. and V. receptor antagonists which have adequate oral bioavailability, have been developed for possible human use (5, 6). 1

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While renal hemodynamic and excretory changes may occur after V.- and V.-vasopressinergic antagonism, the effect of specific nonpeptide antagonists of AVP oh these p~arameters has not been determined in vivo. The purpose of the present study was to assess the roles of endogenous AVP on renal hemodynamics and excretory function, and to distinguish between responses caused by V. and V. receptors. The effects of the nonpeptide V. receptor-selective antagonist, 1-{ 1-[4-(3-acetylammopropoxy) benzoyl]-4-prperidyl}-3,4-dthydro-2(1H)-quinohnone (OPC21268) were c o m p a r e d with those of the V~ r e c e p t o r - s e l e c t i v e a n t a g o n i s t , 5-dimethylamino- 1- {4-(2-methylbenzoylamino) benzoy-I }-2,3,4,5-tetrahydro- 1H-benzazepine 1

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lTestuya Nakamura, The Second Department of Internal Medicine, Gunma University School of Medicine, Maebashi, 371 Japan

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(OPC-31260)(5, 6). The concentration of OPC-21268 that displaced 50% of the specific AVP binding is 4 x 10-7 M for V, receptors and > 10 -4 M for V~ receptors (5). The concentration of OPC-31260 that displaced 5~0% of the specific AVP bindin~ is 1.4 x 10.8 M for V z receptors and 1.2 x 10 -6 M for V 1 receptors (6). Thus, OPC-21268 and OPC-31260 are selective nonpeptide V1 and V 2 antagonists, respectively. Methods

Surgical Preparation: Male Wistar rats (Imai Rats, Fukaya, Japan), age 8-10 weeks, were anesthetized with pentobarbital (40 mg/kg i.p.). After making an incision in the right flank, the right kidney was removed. Then, prophylactic antibiotic (carumonam 30 mg/kg, i.m.) was injected. The rats were individually caged, fed rat-chow (Oriental Yeast Co., Tokyo, Japan) and water ad libitum, and maintained on a 12-hour light/dark cycle until the experiments began 2 weeks later. Overnight, prior to the acute experimental studies, all the rats were fasted with free access to water. The rats were anesthetized intraperitoneally with urethane 80 mg/kg and ct-chloralose 8 mg/kg and placed on a thermostaticaUy-controlled warming table to maintain a body temperature of 37 °C. A tracheotomy was performed, and a PE-200 tube (3 cm long) was inserted into the trachea. Polyethylene catheters (PE-50) were placed in the left jugular vein (for maintenance infusion) and the left carotid artery (for blood sampling). The right femoral artery was cannulated using PE-10 tubing for continuous measurement of the arterial pressure. The femoral arterial catheters were connected to a pressure transducer (Model SPB-108, Biokit, NEC San-ei, Tokyo, Japan), and the arterial pressure was recorded on a polygraph (Model 363 and 8M14, Omnicorder, NEC San-ei). An anterior midline incision was made and the bladder was cannulated using a flare-tipped PE-50 tube (3 cm long) for urine collection. An electromagnetic flow probe (Model MFV-3100, Nihon Koden, Tokyo, Japan) was placed around the left renal artery.

Experimental Protocol: After surgery, all rats were given a bolus injection of polyfructosan (Inutest, Laevosan-Gesellschaft m.b.H, Linz, Germany) 360 mg/kg followed by a maintenance infusion of isotonic saline containing 14.3 mg/ml polyfructosan into the jugular vein at a rate of 7 ml/hr. The isotonic saline solution was infused for 60 min, and urine samples were collected during a 15-min control period. Then, the rats were divided into three groups. The V 1 antagonist 5 mg/kg (OPC-21268; n = 6 rats) or the V~ antagonist 2 mg/kg (OPC-31260; n = 7) at a volume of 0.5 ml or their vehicle (saline 0.5 ml; n-= 8) was given as a bolus injection through a jugular venous catheter. Then, the V 1 or V9 antagonist was included in the maintenance saline solution and infused at the rate of 100 or 4fflxg/kg/min, respectively. Preliminarily, we confirmed that the intravenous injection of OPC-21268 5 mg/kg followed by the continuous infusion of OPC21268 100 Ixg/kg/min completely abolished the pressor response induced by the bolus injection of AVP 30 mU/kg. It is also reported (5, 6) that these doses could effectively and maximally block V. and V~ receptors. Urine samples were collected twice (at 15 and 45 min after the control unne collection) during the 15-min period. A 15-min interval was allowed between the two collections. Blood samples (0.8 ml) were drawn from the carotid artery in the middle of each 15-min clearance period to determine serum osmolality and serum concentrations of sodium and polyfructosan. 1

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Sodium and potassium concentrations were measured using flame photometry (HITACHI 73660E, Hitachi Medical, Tokyo, Japan). Serum and urine osmolality was measured using freezingpoint depression osmometry (Digital Micro-osmometer, Vogel, West Germany). cAMP in urine was measured by r a d i o i m m u n o a s s a y (Cyclic AMP assay kit, Yamasa, Tokyo, Japan). Polyfructosan in serum and urine was measured using anthrone methods. OPC-21268 and OPC31260 were kindly donated by Otsuka Pharmaceutical (Tokyo, Japan).

Statistical Analyses: Values are given as mean + SEM. Multiple data were analyzed by analysis of variance, followed by multiple comparisons made with Scheffe F-test. Statistical significance was considered to be p<0.05.

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Table I Changes in Renal Hemodynamics in Response to Infusion of Saline or Vasopressin V~ or V z Antagonist. Saline (n = 8) BW (g) MAP (mmHg) Cont 1st 2nd R B F (ml/min/g) Cont 1st 2nd GFR (ml/min/g) Cont 1st 2nd

V, Antagonist (n = 6)

V~ Antagonist (n = 7)

389 + 29

396 + 16

401 + 25

108 + 5 109 + 6 113 + 7

105 + 6 104 + 3 109 + 4

113 + 3 110+ 3 112 + 4

4.4 + 0.4 4.8 + 0.5 5.2 + 0.6

4.2 + 0.6 4.1 + 0.6 4.6 + 0.7

4.8 + 0.4 5.0 + 0.5 5.1 + 0.6

0.9+0.1 1.0+ 0.1 1.2 + 0.1

1.0+0.2 1.1 + 0 . 2 1.2 + 0.2

1.1 +0.1 1.3 +0.1 1.3 + 0.1

Values are mean + SEM. BW: body weight; MAP: mean arterial pressure; RBF: renal blood flow; GFR: glomerular filtration rate; Cont: urine collection period during the control period; I st: the first urine collection period after starting the infusion o f antagonists or vehicle; 2nd: the second urine collection period after starting the infusion of antagonists or vehicle. Table II Changes in Free Water Clearance and Urinary Excretion of Sodium, Potassium and c A M P in Response to Infusion of Saline or Vasopressin V1 or V 2 Antagonist. Saline (n = 8) F W C (~l/min/g) Cont -11.2+ 1.9 1st -16.5 + 3.0 2nd -23.2 + 3.3* UNaV (~tEq/min/g) Cont 0.19 + 0.03 1st 0.23+0.05 2nd 0.49+0.12" FENa (%) Cont 0.16 + 0.03 1st 0.16 + 0.03 2nd 0.27 + 0.05* UKV (I.tEq/min/g) Cont 0.79+0.14 1st 1.02 + 0.21 2nd 1.30 + 0.22 c A M P / G F R (ixmol/ml) Cont 84.2 + 7.4 1st 84.2 + 7.6 2nd 75.4 + 4.9

V. Antagonist '(n = 6) -15.4+3.1 -13.5 + 3.3 -16.0 + 3.5

V z Antagonist (n = 7) -12.4+2.0 -19.1 + 2.6 -9.3 + 4.2+

0.24 + 0.09 0.26+0.16 0.42+0.18"

0.24 + 0.06 0.37+0.10 0.69+0.15"+

0.17 + 0.06 0.16 + 0.08 0.27 + 0.12"

0.17 + 0.06 0.21 + 0.05 0.42 + 0.10"+

0.84+0.18 0.72 + 0.22 0.79 + 0.25

0.92+0.15 0.97 + 0.20 1.03 + 0.23

72.3 + 10.2 72.1 + 12.1 59.9 + 10.9

78.8 + 8.3 73.0 + 3.9 79.0 + 5.8

Values are m e a n + SEM. F W C : free water clearance; UNaV: urinary e x c r e t i o n o f sodium; FENa: fractional e x c r e t i o n o f sodium; UKV: urinary e x c r e t i o n o f potassium; c A M P : cyclic adenosine monophosphate. Other abbreviations are defined in those in Table I. *p < 0.05 vs. Cont, +p < 0.05 vs. Saline and V 1 antagonist.

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40 SALINE V1 ANTAGONIST

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Time (min) Fig. 1. Changes in urine flow rate and urine osmolality in response to infusion of saline or vasopressin V 1 or V 2 antagonist. Results There was no significant difference in body weight among the 3 groups (Table 1). The infusion of saline, V. antagonist and V~ antagonist had no effect on mean arterial pressure, renal blood flow or glomerular fdtrat~on rate (Table 1). There was no significant difference in these measurements among the 3 groups. 1

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The infusion of V 2 antagonist increased the urine flow rate significantly (p < 0.05) from 4.5 + 0.3

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Ixl/min during the control period to 25.4 +_ 4.9 ~tl/min during the third urine collection period (Fig. 1). The infusion of saline or V 1 antagonist did not change the urine flow rate significantly (Fig.l). The infusion of V, antagomst decreased urine osmolality significantly from 1084 _+94 mOsm kg 1 during the con{rol period to 509 +_ 86 mOsm kg -I during the third urine collection period (Fig.l). Urine osmolality was significantly lower in the V~ antagonist group than in the other groups during the third urine collection period (saline:'1396 _+ 102 m O s m k g l ; V 1 antagonist: 1160 + 120 mOsm kgl)(Fig.1). The free w a t e r c l e a r a n c e was s i g n i f i c a n t l y h i g h e r in the V. a n t a g o n i s t g r o u p than in the other groups during the thtrd urine collection period (Table 2). The unnary excretion of sodium and the fractional excretion of sodium increased slightly (p < 0.05) in all groups, and was significantly (p < 0•05) higher in the V,antagonist group than in the other groups during the third urine collection period (Table 2). The urinary excretion of potassium did not differ significantly among the 3 groups (Table 2). The infusion of saline, V. antagonist or V. antagonist had no effect on urinary excretion of cAMP corrected by glomerular filtration rate; these parameters did not differ among the 3 groups (Table 2). .

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Discussion Although AVP is one of the strongest vasoconstrictors, it remains controversial whether endogenous AVP contributes to the maintenance of blood pressure and regional blood flow in vivo. In studies of water-deprived and anesthetized rats (7), administration of a peptidergic V~ receptor antagonist decreased arterial pressure because water deprivation increased the release of AVP. Other experiments on conscious rats have shown no effect on blood pressure after peptidergic V j-receptor antagonism either during water diuresis or antidiuresis (8, 9); however, all these studies were conducted using a peptidergic structural analogue of AVP. The present study has shown that the administration of a new nonpeptide specific V 1 antagonist did not influence systemic arterial pressure or renal blood flow in anesthetized hydrated rats. It has been postulated that AVP is both antidiuretic and antinatriuretic on kidney• AVP infused at a low rate (10) exerted sodium-retaining effects on conscious rats in vivo and increased sodium reabsorption in vitro, as in the isolated perfused rat kidney (11, 12) and isolated nephron segments (13). However, the effect of a specific V9 receptor antagonist should be determined to clarify the role of AVP at physiologically relevant levels and unmask the endogenous activity of AVP in the kidney• Earlier studies found that the intravenous injection of peptidergic V^ antagonist was associated with an increase in sodium excretion (14). Oral OPC-31260 has been reported to increase sodium and water excretion (2). In the present study, we confirmed that OPC-31260 increased the urinary excretion of sodium, although the change in urinary sodium excretion was small compared with the marked increase in urine volume and the decrease in urine osmolality. These results imply that OPC-31260 may be useful for the treatment of diseases associated with excessive AVP and/or excessive body water, such as the syndrome of inappropriate secretion of AVE hyponatremia or congestive heart failure. •

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Administration of exogenous AVP could influence the urinary excretion of cAMP because AVE like parathyroid hormone, activates adenylate cyclase in specific regions of the nephron; however, approximately 50% of urinary cAMP excretion is normally accounted for by glomerular filtration of cAMP from plasma, and the remainder is synthesized and excreted by the kidney under direct control of the circulating parathyroid hormone (15). The intravenous infusion of AVP reportedly causes only minor changes in the excretion of cAMP (16). The present study showed that endogenous AVP has a minor influence on urinary excretion of cAMP. In conclusion, the V~ vasopressin antagonist increased urine flow rate and free water clearance without renal vasodOation in rats. A specific V 1 antagonist had no significant effect on mean arterial pressure or renal hemodynamics. These results suggest that endogenous AVP plays a critical role in the regulation of renal water reabsorption mediated through the V. receptor. OPC-31260, a specific nonpeptide V^ receptor antagonist, may be useful for the trratment of diseases associated with excessive AVI5 and body water.

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Acknowledgements The authors appreciate the excellent technical assistance of Mrs. Mari Kurosawa and Mrs. Shizuko Saiki. References 1. J. BOCKAERT, C. ROY, R. RAJERISON and S. JARD, J. Biol. Chem. 24859225931 (1973) 2. M.E.M. TOLBERT, A.C. WHITE, K. ASPRY, J. CUTTS and J.N. FAIN, J. Biol. Chem. 2551938-1944 (1980) 3. W.H. SAWYER, P.K.T. PANG, J. SETO, M. McENROE, B. LAMMEK and M. MANNING, Science. 21249-51 (1981) 4. M. MANNING and W.H. SAWYER, J. Lab. Clin. Med. 114617-632 (1989) 5. Y. YAMAMURA, H. OGAWA, T CHIHARA, K. KONDO, T. ONOGAWA, S. NAKAMURA, 1 MORI, M. TOMINAGA and Y. YABUUCHI, Science. 252572-574 (1991). 6. Y. YAMAMURA, H. OGAWA, H. YAMASHITA, T. CHIHARA, H. MIYAMOTO, S. NAKAMURA, T. ONOGAWA, T. YAMASHITA, T. HOSOKAWA, T. MORI, M. TOMINAGA and Y. YABUUCHI, Br. J. Pharmacol. 105787-791 (1992) 7. C.E. ANDREWS, Jr. and B.M. BRENNER, Circ. Res. 4._.88254-258 (1981) 8. B.L. BRIZZEE, L. HARRISON-BERNARD, H.A. PRETUS, G.G. CLIFTON and B.R. WALKER, Am. J. Physiol. 255 R46-R51 (1988) 9. R.W. ROCKHOLD, L. SHARE, J.T. CROFTON and D.P. BROOKS, Neuroendocrinology. 38 139-144 (1984) 10. M. GELLAI, J.H. SILVERSTEIN, J. HWANG, F.T. LaROCHELLE, Jr., and H. VALTIN, Am. J. Physiol. 246 F819-F827, (1984) 11. W. LIEBERTHAL, M.L. VASILEVSKY, C.R. VALERI and N.G. LEVINSKY, Am. J. Physiol. 252 F331-F337 (1987) 12. D.D. SMYTH, S. UMEMURA and W.A. PETTINGER, Am. J. Physiol. 248 F767F772, (1985) 13. M.C. REIF, S.L. TROUTMAN and A. SCHAFER, J. Clin. Invest. 7__271291-1298, (1986) 14. D.E. BLANDFORD and D.D. SMYTH, J. Pharmacol. Exp. Ther. 255264-270, (1990) 15. G.D. AURBACH, S.J. MARX and A.M. SPIEGEL, Parathyroid Hormone. Calcitonin. and the Calciferols, in Williams Textbook of Endocrinology, J.D. Wilson and D.W. Foster (Eds), 1397-1517, W.B. Saunders, Philadelphia (1992) 16. K. RAIJ, J. PERHEENTUPA and M. H ~ R K ~ E N , Scand. J. clin. Lab. Invest. 3__44 177-184 (1974)