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Lack of effect of verapamil on urinary prostaglandin E2 excretion in the rabbit
European Journal of Pharmacolog)', 132 (1986) 57-59
57
Elsevier EJP 498SC
Short communication
Lack of effect of verapamil on urinary prostaglandin E 2 excretion in the rabbit A h m a d A. A t t a l l a h Department of Pllvsiologv, College of Medicine and AIlied Sciences, P.O. Box 12653, Jeddah 21483, Saudi Arabia Received 2 October 1986, accepted 14 October 1986
The effects of verapamil on renal function, the renin-angiotensin-aldosterone system and renal prostaglandin E 2 were investigated in the rabbit. Verapamil, administered with rate-controlled osmotic pumps, showed no significant effects on plasma renin activity, plasma aldosterone and urinary prostaglandin E 2 excretion. There were marked rises in the glomerular filtration rate and calcium excretion but no significant change in slat and water excretion was observed with verapamil. It is concluded that the hypotensive response to verapamil is unrelated to the renin-angiotensin-aldosterone system and to renal prostaglandin E 2. Verapamil; Prostaglandin E2; Calcium blockade; Plasma renin
1. Introduction Calcium entry blockers (CEB) are becoming widely used in the treatment of cardiac failure and human hypertension (Fleckenstein, 1984). Their pharmacological effects are assumed to be secondary to inhibition of calcium influx into vascular smooth muscle cells, with resultant vasodilatation that might underlie their antihypertensive actions. Calmoduline (BostriSm et al., 1981) and interference with az-adrenergic-induced vasoconstriction (Van Zwieten et al., 1986) have in addition been suggested as important in the vascular relaxation caused by CEB. Although the systemic vasoconstrictor action of exogenously administered angiotensin II is significantly suppressed with CEB (Magometchnigg et al., 1984), no consistent effect on plasma renin activity and aldosterone concentration has been observed with CEB (Bauer et al., 1985). In fact, there is a poor response to CEB in patients with high renin activity (Mtiller et al., 1986) and no significant alterations in salt and water excretion are produced (Bauer et al., 1985), suggesting lack 0014-2999/86/$03.50 ~? 1986 Elsevier Science Publishers B.V.
of involvement of the renin-angiotensin-aldosterone system in the hypotensive response to CEB in man. Since calcium is a well-documented stimulator of renal PG synthesis and is thought to be required for the PG secretion induced by vasopressin (Ausiello and Zusman, 1984) and furosemide (Craven and Derubertis, 1982), and since PGs are thought to be involved in the vasomotor tone of precapillary arterioles, we investigated the effect of calcium entry blockade with verapamil on the renal excretion of prostaglandin E z in the normal rabbit.
2. Materials and methods Sixteen New Zealand white male rabbits (2.5-3.0 kg) were fed regular rabbit chow (Ralston Purina, St. Louis) and permitted free access to tap water. After a steady state of sodium excretion was reached (2 weeks), 24 h urine specimens and plasma samples were collected at mid-day over a 2 week period. Verapamil was administered during
58
the second week via rate-controlled osmotic pumps (Model 2 MLI, Alza Corporation, USA). The pump was implanted subcutaneously under the scapular region with light local anaesthesia (Nembutal, Abbott, 2 mg/kg). The release of verapamil was estimated to be 7 m g / k g per day. During the control and verapamil period, days 4 and 11 respectively, four animals were anaesthetized with sodium pentobarbitone (60 m g / k g i.p.) and a carotid artery was cannulated for blood pressure measurements (Statham P23 Db pressure transducer linked to a Grass model 7 polygraph). Urinary sodium, potassium and chloride were measured with a Beckman autoanalyser (Model E4A). Plasma and urinary creatinine were determined with a CobasBio autoanalyser. Urinary calcium was measured with a Clinical analyser (Beckman Model 42). Urinary PGE 2 was measured using a modification of the radioimmunoassay described previously (Kayama et al., 1984). PGE 2 antiserum was obtained from the Institut Pasteur (Paris, France) and 125I-PGE2 from New England Nuclear (Boston, MA). The antiserum was found to cross-react 11.5% with 15-keto-PGE 2, 5.5% with PGE 1 but less than 0.1% with PGA 2, PGB 2, PGD2, PG~,~ 6-keto-PGFt,, and TXB 2. The detection limit, defined as the apparent concentration two standard deviations below the counts at maximum binding, was found to be 0.5 pg/ml. A 50% displacement of 125I-PGE2 was observed at 4 pg/ml. In initial studies, PGE 2 was measured in urine directly and following extraction and chromatographic purifi-
cation. Linear regression analysis of the results from 50 urinary samples showed close agreement between the two methods (y = 0.954x + 0.043: r = 0.965; P ~<0.001), The chromatographic step was thus omitted and the results for urinary PGE 2 reported in this study were obtained from direct measurements following appropriate dilution with 0.1 M sodium phosphate buffer, pH 7.4, containing 0.3% y globulin. Plasma renin activity was determined using a radioimmunoassay for angiotensin I (Fyrquist et al., 1976). The plasma aldosterone concentration was measured by radioimmunoassay, utilizing commercially available kits (Diagnostic Products Corp., Los Angeles, CA). The results are expressed as mean values + S.E.M. Means were compared by Student's t-test, employing two-tailed probabilities, with values less than 0.05 being considered significant. These and all the other analyses of the data were done on our central computer using a statistical package (SPSS).
3. Results
Table 1 presents the effects of verapamil, administered for 7 days, on urinary flow (UV), sodium (UNaV), potassium (UKV), chloride (UC1V) and calcium (UCaV) excretions, glomerular filtration rate, measured as creatinine clearance (Ccr), plasma renin activity (PRA), plasma aldosterone concentration (PAC) and urinary PGE 2 excretion (UPGE2V). Verapamil caused no signifi-
TABLE 1 Effect of verapamil on urinary_ flow (UV), urinary, sodium (UNaV), potassium (UKV), chloride (UC1V) and calcium (UCaV) excretion, creatinine clearance (Ccr/, plasma renin activity (PRA), plasma aldosterone concentration (PAC) and urinary prostaglandin E 2 excretion (UPGE2V). Each value represents the mean _+S.E.M., n = 70 observations from 12 rabbits. UV (ml/day)
UNaV (mEq/day)
UKV (mEq/day)
UC1V (mEq/day)
UCaV (mg/day)
Ccr (L/day)
PRA (ng/ml per h)
PAC (ng/ml)
UP(jE:V (ng/day)
14.7 +0.33
29.8 +0.61
21.1 ±0.46
54.2_+3.2
5.3±0.59
2.35 :~-0.4
2.04±0.2
632_+39
16.0_+0.63
29.7_+1.1
21.8_+0.87
79.1 +7.1
6.2_+0.71
2.63±0.83
1.78k0.2
643_+45
NS
NS
NS
~< 0.001
~< 0.001
NS
NS
NS
Control 165.g+ 5.2
Verapamil 159.6+8.5 P NS
59 cant effects on UV, U N a V , U K V and U C I V but m a r k e d l y increased U C a V and Ccr. In addition, P R A and P A C were essentially u n c h a n g e d by verapamil. T h e r e was no significant alteration in ur i n ar y P G E 2 excretion. T h e m e a n arterial blood pressure was 85 + 5 m m H g in the control group a n d it was significantly (P ~< 0.01) reduced to 77 + 4 m m H g with verapamil.
4. Discussion Th es e studies d e m o n s t r a t e d that verapamil, at doses which caused a significant r e d u c t i o n in arterial b l o o d pressure, had no effect on urinary salt and wat er excretion, the renin-angiotensin-ald o s t e r o n e axis or the renal synthesis of prostaglandin E 2 in the rabbit, Verapamil, however, caused a m a r k e d increase in urinary calcium and in the rate of g l o m e r u l a r filtration, O u r findings for the renal excretory function are in a g r e e m e n t with previous results o b t a i n e d in m a n (Bauer et al., 1985), c o n f i r m i n g the ability of C E B to lower b l o o d pressure while preserving renal function. T h e lack of effect of v e r a p a m i l on p l a s m a renin activity an d a l d o s t e r o n e c o n c e n t r a t i o n further supports the a s s u m p t i o n that the C E B - i n d u c e d re d u ct i o n in arterial b l o o d pressure is unrelated to i n h i b i t i o n of the r e n i n - a n g i o t e n s i n - a l d o s t e r o n e system. A l t h o u g h cal c i u m is t h o u g h t to be essential in the release of renal PG, the findings from this study exclude an i n t e r m e d i a r y role for renal P G E 2 in the h y p o t e n s i v e response to verapamil.
Acknowledgements This study was supported by a grant from the Saudi Arabian National Center for Science and Technology
(SANCST). The author is grateful to Ms. Kathy Ellen Franccs and Ms. Jackie Marriott for technical assistance and to Mr. Anthony Silva for secretarial help.
References Ausiello, D. and R. Zusman, 1984, The role of calcium in the stimulation of prostaglandin synthesis by vasopressin in rabbit renal medullary interstitial cells in tissue culture, Biochem. J. 220, 139. Bauer, J., S. Sunderrajan and G. Reams, 1985, Effects of calcium entry blockers on renin-angiotensin-aldosterone system, renal function and haemodynamics, salt and water excretion and body fluid composition, Am. I. Cardiol. 56, 624. Bostr~m, S.L., B. Ljung, S. Mards, S. Forsen and E. Thulin, 1981, Interaction of the antihypertensive drug felodipine with calmodulin, Nature 292, 777. Craven, P. and F. Derubertis, 1982, Calcium-dependent stimulation of renal medullary prostaglandin synthesis by furosemide, J. Pharmacol. Exp. Ther. 222, 306. Flcckenstein, A., 1984, Calcium antagonism: history and prospects for a muhifaceted pharmacodynamic principle, in: Calcium Antagonists and Cardiovascular Disease, ed. L.H. Opie (Raven Press. New York) p. 9. Fyrqvist, P., P. Soveri, L. Putula and V. Sterman, 1976, Radioimmunoassay of plasma renin activity, Clin. Chem. 22, 250. Kayama, S., A. Attallah, R. Stahl, D. Block and J. Lee, 1984, Mechanism of furosemide-induced natriuresis by direct stimulation of renal prostaglandin E 2, Am. J. Physiol. 247, F555. Magometschnigg, D., H. Hormagl and H. Ramels, 1984, Diltiazem and verapamil: functional antagonism of exogenous norepinephrine and angiotensin II in man, European J. Clin. Pharmacol. 26, 303. Miiller, F., P. Bolli, P. Evne, W. Kiuwski and F. Btihler, 1986, Calcium antagonism - A new concept for treating essential hypertension, Am. J. Cardiol. 56, 50 D. Van Zwieten, P.A., P. Timmermans, M. Thoolen, B. Wilffert and A. Dc Jonge, 1986, Inhibitory effect of calcium antagonist drugs on vasoconstriction induced by vascular alpha-adrenoceptor stimulation, Am. J. Cardiol. 57, 11 D.
57
Elsevier EJP 498SC
Short communication
Lack of effect of verapamil on urinary prostaglandin E 2 excretion in the rabbit A h m a d A. A t t a l l a h Department of Pllvsiologv, College of Medicine and AIlied Sciences, P.O. Box 12653, Jeddah 21483, Saudi Arabia Received 2 October 1986, accepted 14 October 1986
The effects of verapamil on renal function, the renin-angiotensin-aldosterone system and renal prostaglandin E 2 were investigated in the rabbit. Verapamil, administered with rate-controlled osmotic pumps, showed no significant effects on plasma renin activity, plasma aldosterone and urinary prostaglandin E 2 excretion. There were marked rises in the glomerular filtration rate and calcium excretion but no significant change in slat and water excretion was observed with verapamil. It is concluded that the hypotensive response to verapamil is unrelated to the renin-angiotensin-aldosterone system and to renal prostaglandin E 2. Verapamil; Prostaglandin E2; Calcium blockade; Plasma renin
1. Introduction Calcium entry blockers (CEB) are becoming widely used in the treatment of cardiac failure and human hypertension (Fleckenstein, 1984). Their pharmacological effects are assumed to be secondary to inhibition of calcium influx into vascular smooth muscle cells, with resultant vasodilatation that might underlie their antihypertensive actions. Calmoduline (BostriSm et al., 1981) and interference with az-adrenergic-induced vasoconstriction (Van Zwieten et al., 1986) have in addition been suggested as important in the vascular relaxation caused by CEB. Although the systemic vasoconstrictor action of exogenously administered angiotensin II is significantly suppressed with CEB (Magometchnigg et al., 1984), no consistent effect on plasma renin activity and aldosterone concentration has been observed with CEB (Bauer et al., 1985). In fact, there is a poor response to CEB in patients with high renin activity (Mtiller et al., 1986) and no significant alterations in salt and water excretion are produced (Bauer et al., 1985), suggesting lack 0014-2999/86/$03.50 ~? 1986 Elsevier Science Publishers B.V.
of involvement of the renin-angiotensin-aldosterone system in the hypotensive response to CEB in man. Since calcium is a well-documented stimulator of renal PG synthesis and is thought to be required for the PG secretion induced by vasopressin (Ausiello and Zusman, 1984) and furosemide (Craven and Derubertis, 1982), and since PGs are thought to be involved in the vasomotor tone of precapillary arterioles, we investigated the effect of calcium entry blockade with verapamil on the renal excretion of prostaglandin E z in the normal rabbit.
2. Materials and methods Sixteen New Zealand white male rabbits (2.5-3.0 kg) were fed regular rabbit chow (Ralston Purina, St. Louis) and permitted free access to tap water. After a steady state of sodium excretion was reached (2 weeks), 24 h urine specimens and plasma samples were collected at mid-day over a 2 week period. Verapamil was administered during
58
the second week via rate-controlled osmotic pumps (Model 2 MLI, Alza Corporation, USA). The pump was implanted subcutaneously under the scapular region with light local anaesthesia (Nembutal, Abbott, 2 mg/kg). The release of verapamil was estimated to be 7 m g / k g per day. During the control and verapamil period, days 4 and 11 respectively, four animals were anaesthetized with sodium pentobarbitone (60 m g / k g i.p.) and a carotid artery was cannulated for blood pressure measurements (Statham P23 Db pressure transducer linked to a Grass model 7 polygraph). Urinary sodium, potassium and chloride were measured with a Beckman autoanalyser (Model E4A). Plasma and urinary creatinine were determined with a CobasBio autoanalyser. Urinary calcium was measured with a Clinical analyser (Beckman Model 42). Urinary PGE 2 was measured using a modification of the radioimmunoassay described previously (Kayama et al., 1984). PGE 2 antiserum was obtained from the Institut Pasteur (Paris, France) and 125I-PGE2 from New England Nuclear (Boston, MA). The antiserum was found to cross-react 11.5% with 15-keto-PGE 2, 5.5% with PGE 1 but less than 0.1% with PGA 2, PGB 2, PGD2, PG~,~ 6-keto-PGFt,, and TXB 2. The detection limit, defined as the apparent concentration two standard deviations below the counts at maximum binding, was found to be 0.5 pg/ml. A 50% displacement of 125I-PGE2 was observed at 4 pg/ml. In initial studies, PGE 2 was measured in urine directly and following extraction and chromatographic purifi-
cation. Linear regression analysis of the results from 50 urinary samples showed close agreement between the two methods (y = 0.954x + 0.043: r = 0.965; P ~<0.001), The chromatographic step was thus omitted and the results for urinary PGE 2 reported in this study were obtained from direct measurements following appropriate dilution with 0.1 M sodium phosphate buffer, pH 7.4, containing 0.3% y globulin. Plasma renin activity was determined using a radioimmunoassay for angiotensin I (Fyrquist et al., 1976). The plasma aldosterone concentration was measured by radioimmunoassay, utilizing commercially available kits (Diagnostic Products Corp., Los Angeles, CA). The results are expressed as mean values + S.E.M. Means were compared by Student's t-test, employing two-tailed probabilities, with values less than 0.05 being considered significant. These and all the other analyses of the data were done on our central computer using a statistical package (SPSS).
3. Results
Table 1 presents the effects of verapamil, administered for 7 days, on urinary flow (UV), sodium (UNaV), potassium (UKV), chloride (UC1V) and calcium (UCaV) excretions, glomerular filtration rate, measured as creatinine clearance (Ccr), plasma renin activity (PRA), plasma aldosterone concentration (PAC) and urinary PGE 2 excretion (UPGE2V). Verapamil caused no signifi-
TABLE 1 Effect of verapamil on urinary_ flow (UV), urinary, sodium (UNaV), potassium (UKV), chloride (UC1V) and calcium (UCaV) excretion, creatinine clearance (Ccr/, plasma renin activity (PRA), plasma aldosterone concentration (PAC) and urinary prostaglandin E 2 excretion (UPGE2V). Each value represents the mean _+S.E.M., n = 70 observations from 12 rabbits. UV (ml/day)
UNaV (mEq/day)
UKV (mEq/day)
UC1V (mEq/day)
UCaV (mg/day)
Ccr (L/day)
PRA (ng/ml per h)
PAC (ng/ml)
UP(jE:V (ng/day)
14.7 +0.33
29.8 +0.61
21.1 ±0.46
54.2_+3.2
5.3±0.59
2.35 :~-0.4
2.04±0.2
632_+39
16.0_+0.63
29.7_+1.1
21.8_+0.87
79.1 +7.1
6.2_+0.71
2.63±0.83
1.78k0.2
643_+45
NS
NS
NS
~< 0.001
~< 0.001
NS
NS
NS
Control 165.g+ 5.2
Verapamil 159.6+8.5 P NS
59 cant effects on UV, U N a V , U K V and U C I V but m a r k e d l y increased U C a V and Ccr. In addition, P R A and P A C were essentially u n c h a n g e d by verapamil. T h e r e was no significant alteration in ur i n ar y P G E 2 excretion. T h e m e a n arterial blood pressure was 85 + 5 m m H g in the control group a n d it was significantly (P ~< 0.01) reduced to 77 + 4 m m H g with verapamil.
4. Discussion Th es e studies d e m o n s t r a t e d that verapamil, at doses which caused a significant r e d u c t i o n in arterial b l o o d pressure, had no effect on urinary salt and wat er excretion, the renin-angiotensin-ald o s t e r o n e axis or the renal synthesis of prostaglandin E 2 in the rabbit, Verapamil, however, caused a m a r k e d increase in urinary calcium and in the rate of g l o m e r u l a r filtration, O u r findings for the renal excretory function are in a g r e e m e n t with previous results o b t a i n e d in m a n (Bauer et al., 1985), c o n f i r m i n g the ability of C E B to lower b l o o d pressure while preserving renal function. T h e lack of effect of v e r a p a m i l on p l a s m a renin activity an d a l d o s t e r o n e c o n c e n t r a t i o n further supports the a s s u m p t i o n that the C E B - i n d u c e d re d u ct i o n in arterial b l o o d pressure is unrelated to i n h i b i t i o n of the r e n i n - a n g i o t e n s i n - a l d o s t e r o n e system. A l t h o u g h cal c i u m is t h o u g h t to be essential in the release of renal PG, the findings from this study exclude an i n t e r m e d i a r y role for renal P G E 2 in the h y p o t e n s i v e response to verapamil.
Acknowledgements This study was supported by a grant from the Saudi Arabian National Center for Science and Technology
(SANCST). The author is grateful to Ms. Kathy Ellen Franccs and Ms. Jackie Marriott for technical assistance and to Mr. Anthony Silva for secretarial help.
References Ausiello, D. and R. Zusman, 1984, The role of calcium in the stimulation of prostaglandin synthesis by vasopressin in rabbit renal medullary interstitial cells in tissue culture, Biochem. J. 220, 139. Bauer, J., S. Sunderrajan and G. Reams, 1985, Effects of calcium entry blockers on renin-angiotensin-aldosterone system, renal function and haemodynamics, salt and water excretion and body fluid composition, Am. I. Cardiol. 56, 624. Bostr~m, S.L., B. Ljung, S. Mards, S. Forsen and E. Thulin, 1981, Interaction of the antihypertensive drug felodipine with calmodulin, Nature 292, 777. Craven, P. and F. Derubertis, 1982, Calcium-dependent stimulation of renal medullary prostaglandin synthesis by furosemide, J. Pharmacol. Exp. Ther. 222, 306. Flcckenstein, A., 1984, Calcium antagonism: history and prospects for a muhifaceted pharmacodynamic principle, in: Calcium Antagonists and Cardiovascular Disease, ed. L.H. Opie (Raven Press. New York) p. 9. Fyrqvist, P., P. Soveri, L. Putula and V. Sterman, 1976, Radioimmunoassay of plasma renin activity, Clin. Chem. 22, 250. Kayama, S., A. Attallah, R. Stahl, D. Block and J. Lee, 1984, Mechanism of furosemide-induced natriuresis by direct stimulation of renal prostaglandin E 2, Am. J. Physiol. 247, F555. Magometschnigg, D., H. Hormagl and H. Ramels, 1984, Diltiazem and verapamil: functional antagonism of exogenous norepinephrine and angiotensin II in man, European J. Clin. Pharmacol. 26, 303. Miiller, F., P. Bolli, P. Evne, W. Kiuwski and F. Btihler, 1986, Calcium antagonism - A new concept for treating essential hypertension, Am. J. Cardiol. 56, 50 D. Van Zwieten, P.A., P. Timmermans, M. Thoolen, B. Wilffert and A. Dc Jonge, 1986, Inhibitory effect of calcium antagonist drugs on vasoconstriction induced by vascular alpha-adrenoceptor stimulation, Am. J. Cardiol. 57, 11 D.