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MK 421 and prevention of genetic hypertension development in young spontaneously hypertensive rats
European Journal of Pharmacology, 79 (1982) 23-29 Elsevier Biomedical Press
23
M K 421 AND P R E V E N T I O N OF G E N E T I C H Y P E R T E N S I O N D E V E L O P M E N T IN Y O U N G S P O N T A N E O U S L Y H Y P E R T E N S I V E RATS CHRISTINE RICHER, MARIE-PASCALE DOUSSAU and JEAN-FRANCOIS GIUDICELLI
*
Dbpartement de Pharmacologie, Facultb de Mbdeeine Paris-Sud, 63 rue Gabriel P&i, 942 70-Le Kremlin-Bicbtre, France Received 8 September 1981, revised MS received 28 October 1981, accepted 14 December 1981
C. RICHER, M.P. DOUSSAU and J.F. GIUDICELLI, MK 421 and prevention of genetic hypertension development in young spontaneously hypertensive rats, European J. Pharmacol. 79 (1982) 23-29. MK 421, at the dose of 25 mg/kg, administered daily by gavage to spontaneously hypertensive rats (SHRs) from their 4th to 15th weeks of age almost completely inhibited development of genetic hypertension. Since heart rate and cardiac and systolic indexes were not affected by the drug, prevention of genetic hypertension development was solely related to an early, potent and long-lasting reduction of the progressive increase of the peripheral resistance which generally develops in SHRs during ageing. MK 421 reduced body growth but did not modify fluid intake, plasma Na + and urine volume, thus water and salt retention did not develop. MK 421 enhanced vascular responsiveness to norepinephrine and angiotensin II and reduced myocardial hypertrophy. Plasma renin concentration was increased and urinary antidiuretic hormone did not change. Finally, MK 421's preventive effects against genetic hypertension development persisted up to 10 weeks after discontinuation of treatment. Spontaneously hypertensive rats Converting enzyme inhibitors Genetic hypertension development
1. Introduction
Angiotensin I converting enzyme inhibitors (CEI) are a new class of potent antihypertensive drugs. Captopril, the first orally active drug in the group is an efficient drug in human arterial hypertension (Gavras et al., 1978; Brunner et al., 1979) but also in different types of experimental hypertension in animals, including low renin models. Thus, captopril not only lowers blood pressure in adult SHRs with established hypertension (Laffan et al., 1978; "Muirhead et al., 1978) but also prevents genetic hypertension development in the chronically-treated young SHR (Ferrone and Antonaccio, 1979; Giudicelli et al., 1980). N-[(S)1-(ethoxycarbonyl)-3-phenylpropyl]-L-Ala-L-Pro or M K 421 is a new potent and orally active CEI which has a pharmacological profile qualitatively similar to captopril (Patchett et al., 1980; Sweet et
* To whom all correspondence should be addressed. 0014-2999/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press
MK 421
al., 1981). The present study was designed in order to investigate whether M K 421 also prevents genetic hypertension development in the young S H R and if so, to determine the underlying mechanism(s).
2. Materials and methods 2.1. Animals and treatments
Forty 4-week old male SHRs (Okamoto strain, Charles River, St. Aubin-les-Elbeuf, France) were randomly divided into two groups of 20 animals each. Rats from each group were housed together in subgroups of 6 to 8 animals, fed ad libitum with standard diet (0.130 m E q / g Na + and 0.205 m E q / g K +) and had free access to tap water. All animals were maintained under identical conditions of temperature (21-+ l°C), humidity and lighting. Starting from the 28th day of life and up to the 15th week of age, the two groups received by
24 gavage every day at the same time a single dose of M K 421 (25 mg/kg, l m l / 1 0 0 g of an aqueous solution) or distilled water (1 ml / 100 g). The dose of M K 421 used was selected as follows: captopril prevents genetic hypertension development in SHRs at an oral daily dose of 100 m g / k g (Giudicelli et al., 1980) and M K 421 is 4.3-fold more potent than captopril as a CEI in the anesthetized rat (Gross et al., 1981).
2.2. Pardmeters investigated Body weight (BW) and systolic blood pressure (SBP), recorded (Physiograph DMP, Narcobiosystems Inc.) in the conscious animals using the indirect tail cuff method (Byrom and Wilson, 1938), were measured in the two groups before the onset of treatment and then every week during the treatment period. H R was obtained from the SBP tracings. Furthermore, systolic blood pressure (SBP), heart rate (HR), cardiac index (CI), systolic index (SI), renal blood flow (RBF), peripheral resistance (PR), kidney and heart weights were measured or calculated at 14 weeks of age in 6 anesthetized (sodium pentobarbital, 50 m g / k g i.p.) animals randomly selected in each group 24 h after SBP and H R had been determined in the conscious state. SBP and H R were monitored using a P 50 Statham blood pressure transducer, a Gould Brush computer model 13421404 and a Gould Brush recorder model 6610-06. Cardiac output and RBF were measured with radioactive microspheres according to the technique described by McDevitt and Nies (1976). After the animals had been sacrificed, hearts and kidneys were immediately removed, carefully cleaned, blotted for 3 min and weighed. Responses to exogenous norepinephrine (0.25, 0.5, 1 and 2/~g/kg) and angiotensin II (10 n g / k g ) were determined at 14 weeks of age in 8 anesthetized animals (sodium pentobarbital, 50 mg/kg, i.p.) randomly selected from each group. Following anesthesia, the trachea was cannulated but the animals breathed spontaneously. Two catheters (PE 10 and PE 50) previously filled with heparinized saline (heparine 50 U / m l ) were introduced in the femoral vein for injections of drugs
and in the carotid artery, respectively. The arterial catheter was connected to a P 50 Statham blood pressure transducer. Blood pressure, recorded on a Gould Brush recorder model 6610-06, was allowed to stabilize for 15 min before starting the experiments and to return to baseline values between the different injections. Body temperature was maintained at 37°C. At the 13th week of age 6 treated and 6 control SHRs randomly selected were housed individually in metabolism cages. Fluid intake and urine volume were measured daily for 5 days and averaged. An aliquot of each urine sample was used for measurement of urinary A D H according to the method of Fressinaud et al. (1974). Immediately after urine collection, two blood samples (1 ml each) were taken from the jugular vein under light ether anesthesia. The first, collected in a tube rinsed with 15% ammonium EDTA solution, pH 6.5 was used to measure plasma renin concentration (PRC) according to the method of Mrnard and Catt (1972). The second, collected in a tube rinsed with heparin was used for determination of plasma Na ÷, K + (flame photometry), urea nitrogen, creatinin and uric acid (Technicon Autoanalyzer). Finally, after discontinuation of treatment (15th week of age), SBP was measured at weekly intervals in the 6 surviving animals of each group up to the age of 25 weeks. All parameters investigated were determined 20 h after the last drug administration.
2.3. Drugs The drugs used were MK 421 (Merck Sharp and Dohme), norepinephrine bitartrate (Levophed R, Winthrop) and angiotensin II (Hypertensin R, Ciba). Doses are expressed in terms of the ester (MK 421), the base (norepinephrine) and the peptide (angiotensin II).
2.4. Statistics Means -+S.E.M. are reported and statistical analysis of the results (treated vs controls) was performed using Student's t-test.
25 2 2C
3. Results
!
3.1. Cardiovascular parameters
2OO
Chronic oral administration of single daily doses of M K 421 at 25 m g / k g to the young SHR markedly inhibited genetic hypertension development (fig. 1). Thus, at the end of the treatment period, SBP measured in the unanesthetized animal was 142.0±2.5 mmHg in the M K 421-treated animals vs. 197.0 ± 3.7 mmHg in the controls. H R was slightly but significantly reduced by M K 421 between the 9th (449.9 ± 8.9 in the treated animals vs. 470.7 ± 10.7 in the controls, P < 0.05) and the 12th week of age (451.4 ± 6.9 in the treated animals vs. 470.3±9.1 in the controls, P < 0 . 0 5 ) but this effect was no longer observed later. In the 14-week old anesthetized SHRs, HR, CI and SI were not treatment-affected (table 1) but SBP was strongly reduced due to the MK 421induced inhibition of PR increase with age. Moreover, RBF was significantly increased in M K 421treated SHRs. Heart weight (HW) was significantly reduced in the treated group at the 14th week of age (table 1). H W / B W ratio also decreased but because of the M K 421-induced lesser age-related BW increase (see below), this decrease did not reach statistical significance. Finally, kidney weight (KW) was not altered by M K 421 (table 1). Ten weeks after discontinuation of treat-
~ 180 a o
q 16o m L)
AGE
(weeks)
Fig. 1. Effect of age on systolic blood pressure of unanesthetized MK 421-treated (25 m g / k g , p.o., daily) ( A ) or control ( 0 ) SHRs. Values depicted are means -+ S.E.M. Values significantly different from respective control values: *** P<0.001.
ment, SBP in the conscious, previously MK 421treated SHRs plateaued at 172.0 ± 2.6 mmHg, a value significantly lower than that observed in controls (216.2 ± 5.5 mmHg, P < 0.001). Fig. 2 depicts the dose-response curves to norepinephrine in both groups. M K 421 produces a shift to the left, significantly so for the two highest doses, indicating an enhanced sensitivity to norepinephrine induced by the chronic converting enzyme blockade. A similar trend, although not
TABL E 1 Effects of MK 421 chronic oral treatment (25 m g / k g per day) on various hemodynamic and biological parameters in the young SHR.
SBPC(mmHg) HR c (beats/mitl) CI c ( m l / m i n per kg) SI~ ( m l / b e a t per kg) P R c ( m m H g / m l per min per kg) RBFC ( m l / m i n per g) H W c (mg) KW ~ (mg) PRC d (rig Ang I / m l per 2 h) ADH d (pg/h)
Control SHRs
MK 421-treated SHRs
165.0 -+ 5.3 428.7 + 9.7 129.1 -+ 11.3 0.30-+ 0.03 1.14-+ 0.11 5.5 + 0.8 1056.4 +26.8 951.2 -+16.4 104.7 ± 9.5 197.9 --+36.3
135.3 --+ 7.0 a 456.4 + 8.8 162.1 -+ 14.5 0.36-+ 0.03 0 . 7 2+- 0.10 a 10.4 -- 1.8 a 937.3 ± 2 5 . 2 b 900.7 -+19.2 220.8 -+32.5 a 282.7 -+40.9
a P < 0 . 0 5 ; b P<0 .0 1 from controls. c Measured in the anesthetized animal at 14 weeks of age (10 weeks of treatment). d Measured on blood or urine samples taken at 13 weeks of age (9 weeks of treatment).
26
302.0 ± 4.0 g (controls), P < 0.05. Furthermore, total fluid intake and urine volume were not drugaffected. Finally, urinary A D H values were not significantly modified (table 1).
A
gE ~4c
4. Discussion UJ < 25
o.'25 . 0.5 NOREPINEPHRINE
~ (~g/kg)
Fig. 2. Mean arterial pressure increases in response to intravenous norepinephrine in anesthetized M K 421-treated (25 m g / k g p.o., daily) ( A ) and control ( 0 ) 14-week old SHRs. Values depicted are m e a n s -4-S.E.M. Values significantly different from respective control values: * P<0.05.
significant, to hyperresponsiveness was also observed with angiotensin II, 10 ng/kg: blood pressure increases were 45.0 - 5.0 in the treated animals and 33.3 ± 5.4 m m H g in the controls, respectively.
3.2. Plasma renin concentration (PRC) and other plasma parameters PRC was significantly augmented in the M K 421-treated SHRs showing a two-fold increase at the 13th week of age (table 1). Plasma Na + (136.6 ± 0.7 in treated vs. 137.4 ± 0.7 mEq/1 in controls), K +, urea nitrogen and creatinine values were not affected by the treatment but plasma uric acid was significantly reduced (39.5 ± 8.8 in treated vs. 73.2 ± 5.7/~mol/1 in controls, P < 0.01).
3.3. Body weight, fluid intake, urinary volume and ADH The age-related increase of the BW was significantly reduced in M K 421-treated SHRs, starting from the 4th week of treatment until its discontinuation. Thus, at 8, 10 and 15 weeks of age (4, 6 and 11 weeks of treatment), mean BW were 157.5 - 2 . 6 g (treated) and 165.7 ± 1.9 (controls), P < 0.05; 212.9 ± 3.1 g (treated) and 226.0 ± 2.4g (controis), P < 0.01 and 269.0-4- 12.0 g (treated) and
M K 421 is a potent and long-lasting oral inhibitor of angiotensin converting enzyme (Patchett et al., 1980; Gross et al., 1981). Its antihypertensive efficacy upon acute administration has been demonstrated in several models of renal hypertension (Sweet et al., 1981), but also in the adult spontaneously hypertensive rat in which a significant decrease of BP has been observed as early as 60 min after a single oral dose of M K 421, with full recovery of initial blood pressure value within 4 h (Sweet et al., 1981). Our experiments demonstrate that M K 421, 25 m g / k g per day chronically administered orally to young SHRs, from their 4th week of age, is also able to prevent genetic hypertension development. Since similar results have already been reported under analogous experimental conditions with a four-fold higher daily oral dose of captopril (Ferrone and Antonaccio, 1979; Giudicelli et al., 1980), our data indicate that M K 421 is at least 4-times more potent on a weight basis than captopril in preventing BP rise in this model. Since the cardiac index is not significantly affected by the drug, it appears that the preventive effect of M K 421 against genetic hypertension development is solely due to the inhibition of the age-related increase in PR which generally develops in SHRs. As previously shown with captopril (Koike et al., 1980; Giudicelli et al., 1981a), early blockade of angiotensin II formation by M K 421 is directly involved and mainly responsible for this inhibition of PR increase, specially in the renal vascular bed which is particularly sensitive to this hormone (Heyndrickx et al., 1976). However, the reduction of norepinephrine release resulting from the blockade of angiotensin II formation (Zimmerman and Gisslen, 1968; Malik and Nasjletti, 1976) may also contribute to the prevention of genetic hypertension development (and perhaps also to the transient bradycardia observed during the first weeks of treatment), specially since sympathetic
27 system hyperactivity plays a dominant role in the first stages of genetic hypertension development (Yamori, 1976; Cutilletta et al., 1977; Richer et al., 1980; Collis et al., 1980). There are contradictory data in the literature regarding the vasopressin secretion in the young SHR as compared to the young Wistar-Kyoto rats. Crofton et al. (1979) have provided evidence for an increased release of vasopressin from the neurohypophysis in the young SHR while Rascher et al. (1981) reported a reduced vasopressin plasma concentration in the young and adolescent stroke-prone SHR. Strain as well as methodology differences may be responsible for this discrepancy but whether vasopressin is involved or not in the early stages of genetic hypertension development in SHRs has not yet been established. However, the fact that in our experiments urinary ADH was not reduced by MK 421 after 9 weeks of treatment, suggests that this mechanism does not participate in the MK 421-induced prevention of genetic hypertension development. Chronic converting enzyme blockade by MK 421 resulted in our experiments in a tendency to an increase in the pressor responses of SHRs to exogenous norepinephrine and angiotensin II. Spertini et al. (1981) also found an enhanced response to angiotensin II but a reduced response to norepinephrine in normotensive rats chronically treated with captopril while Antonaccio and Kerwin (1981) reported a decreased response to both norepinephrine and angiotensin II in SHRs chronically treated with captopril. These data are difficult to reconcile. Increased sensitivity to exogenous angiotensin II, already previously reported with teprotide (Thurston and Laragh, 1975), has been thought to be related to the CEI-induced decrease in circulating angiotensin II which may result in greater availability of vascular receptor sites to exogenous angiotensin II (Thurston and Laragh, 1975; Spertini et al., 1981). Regarding the increased sensitivity to norepinephrine, the same hypothesis could be put forward if one assumes that inhibition of angiotensin II formation leads to a reduction in endogenous norepinephrine release via a presynaptic mechanism (Antonaccio and Kerwin, 1981) and hence possibly to an increased receptor responsiveness to norepinephrine. Fur-
thermore, the fact that the BP was lower in MK 421-treated SHRs than in controls may also have contributed to an enhanced response of the treated rats to the two hormones. However, the data by Antonaccio and Kerwin (1981) do not support this hypothesis and therefore the mechanism of the slight increase in pressor responsiveness to norepinephrine and angiotensin II observed in our experiments remains unclear. Because of the reduction in afterload, the decrease in sympathetic tone and the suppression of the trophic role of angiotensin II, the HW of MK 421-treated SHRs is, as expected, significantly reduced, indicating less myocardial hypertrophy. Thus, like captopril (Giudicelli et al., 1980; Sen et al., 1980; Koike et al., 1980), MK 421 is able to simultaneously prevent the rise in PR and reduce the HW and this parallel effect of the two drugs on the development of functional a n d / o r structural vascular and cardiac abnormalities in the young SHR may account, as previously postulated for captopril (Giudicelli et al., 1981b), for the long-lasting protection which MK-421 and captopril afford against genetic hypertension development, up to 10 weeks or more after discontinuation of treatment. A dissociation between the kinetics of MK 421induced fall in blood pressure and plasma converting enzyme inhibition has been reported by Sweet et al. (1981) in acute experiments performed in the adult SHR. This suggests that the two phenomena might not be related or that the antihypertensive effect could result from blockade of converting enzyme at other sites, e.g. within the vascular wall or the brain. Our experiments, in which MK 421 has been administered chronically to young SHRs only indicate that after 9 weeks of treatment, the drug induces plasma converting enzyme inhibition, as shown by the rise in PRC, and prevention of genetic hypertension development simultaneously. However, one cannot exclude that MK 421 might oppose genetic hypertension development by inhibition of brain converting enzyme, like captopril upon chronic treatment (Mann et al., 1981). The MK 421-induced reduction of BW growth with age and the lack of change in plasma Na + , fluid intake and urine volume after 9weeks of treatment, clearly indicate that MK 421 does not
28
induce sodium and water retention, a property shared by captopril and YS 980, another CEI (Crofton et al., 1979; Unger et al., 1981). In summary, chronic blockade of converting enzyme by MK 421 in the young SHR prevents genetic hypertension development by reducing the age-related increase in PR, vasodilating the renal vascular bed and reducing the cardiac hypertrophy without simultaneously inducing tachycardia and body fluid volume expansion.
Acknowledgements The authors want to express their gratitude to Dr. C. Sweet of Merck Institute for Therapeutic Research, West Point (Pa), U.S.A., for a generous supply of MK 421. They also wish to thank Pr. J. M+nard and Mrs. A. Cottard for ADH measurements.
References Antonaccio, M.J. and L. Kerwin, 1981, Pre- and postjunctional inhibition of vascular sympathetic function by captopril in SHR, Hypertension 3, 154. Brunner, H.R., H. Gavras, B. Waeber, G.R. Kershaw, G.A. Turini, R.A. Vukovich, D.N. McKinstry and I. Gavras, 1979, Oral angiotensin converting enzyme inhibitor in longterm treatment of hypertensive patients, Ann. Int. Med. 90, 19. Byrom, F.B. and C.A. Wilson, 1938, A plethysmographic method for measuring blood pressure in the rat, J. Physiol. (London) 93, 301. Collis, M.G., C. Demey and P.M. Vanhoutte, 1980, Renal vascular reactivity in the young spontaneously hypertensive rat, Hypertension 2, 45. Crofton, J.T., L. Share and Z. Horovitz, 1979, The effect of SQ 14,225 on systolic blood pressure and urinary excretion of vasopressin in the developing spontaneously hypertensive rat, Hypertension 1,462. Cutilletta, A., L. Erinoff, A. Heller, J. Low and S. Oparil, 1977, Development of left ventricular hypertrophy in young spontaneously hypertensive rats after peripheral sympathectomy, Circ. Res. 40, 428. Ferrone, R.A. and M.J. Antonaccio, 1979, Prevention of the development of spontaneous hypertension in rats by captopril (SQ 14,225), European J. Pharmacol. 60, 131. Fressinaud, P., P. Corvol and J. Menard, 1974, Radioimmunoassay of urinary antidiuretic hormone in man. Stimulation suppression tests, Kidney Int. 6, 184. Gavras, H., H.R. Brunner, G.A. Turini, G.B. Kershaw, C.P. Tifft, S. Cuttelod, I. Gavras, R.A. Vukovich and D.N. McKinstry, 1978, Antihypertensive effect of the oral con-
verting enzyme inhibitor SQ 14,225 in man, New Engl. J. Med. 298, 991. Giudicelli, J.F., J.L. Freslon, S. Glasson and C. Richer, 1980, Captopril and hypertension development in the SHR. Clin. Exp. Hypertension 2, 1083. Giudicelli, J.F., J.L. Freslon and C. Richer, 1981a, Captopril et prrvention du d~veloppement de l'hypertension grnetique du rat SHR, Nouv. Presse Med. 10, 1547. Giudicelli, J.F., C. Richer and J.L. Freslon, 1981b, Antihypertensive drugs and prevention of genetic hypertension development in SHRs, in: New Trends in Arterial Hypertension, M. Worcel et al., eds., Elsevier, Amsterdam, p. 45. Gross, D.M., C.S. Sweet, E.H. Ulm, E.P. Backlund, A.A. Morris, D. Weitz, D.L. Bohn, H.C. Henger, T.C. Vassil and C.A. Stone, 1981, Effect of N - [ ( S ) - l - c a r b o x y - 3 phenylpropyl]-L-Ala-L-Pro and its ethyl ester (MK 421) on angiotensin converting enzyme in vitro and angiotensin I pressor responses in vivo, J. Pharmacol. Exp. Ther. 216, 552. Heyndrickx, G.R., D.H. Boettcher and S.F. Vatner, 1976, Effects of angiotensin, vasopressin, and methoxamine on cardiac function and blood flow distribution on conscious dogs, Am. J. Physiol. 231, 1579. Koike, H., I. Katsuaki, M. Miyamoto and N. Nishino, 1980, Effects of long-term blockade of angiotensin converting enzyme with captopril (SQ 14,225) on hemodynamics and circulating blood volume in SHR, Hypertension 2, 299. Laffan, R.J., M.E. Goldberg, J.P. High, T.R. Schaeffer, M.H. Waugh and B. Rubin, 1978, Antihypertensive activity in rats of SQ 14,225, an orally active inhibitor of angiotensin I converting enzyme, J. Pharmacol. Exp. Ther. 204, 281. Malik, K.V. and A. Nasjletti, 1976, Facilitation of adrenergic transmission by locally generated angiotensin II in rat mesenteric arteries, Circ. Res. 38, 26. Mann, J.F.E., J. Genest, Th. Unger, B. Scholkens, W. Rascher, A. Schomig and D. Ganten, 1981, A role for the reninangiotensin system in central mechanisms of blood pressure control, in: New Trends in Arterial Hypertension, M. Worcel et al., eds., Elsevier, Amsterdam, p. 101. McDevitt, D.G. and A.S. Nies, 1976, Simultaneous measurement of cardiac output and its distribution with microspheres in the rat, Cardiovasc. Res. 10, 494. M~nard, J. and K.J. Catt, 1972, Measurement of renin activity, concentration and substrate in rat plasma by radioimmunoassay of angiotensin I, Endocrinology 90, 422. Muirhead, E.E., R.L. Prewitt, B. Brooks and W.L. Brosius, 1978, Antihypertensive action of the orally active converting enzyme inhibitor (SQ 14,225) in spontaneously hypertensive rats, Circ. Res. 43 (Suppl. I), 53. Patchett, A., E. Harris, E. Tristram, M. Wyvratt, M. Wu, D. Taub, E. Paterson, T. Ikeler, J. Ten Broeke, L. Payne, T. Ondeyka, E. Thorsett, W. Greenlee, N. Lohr, R. Hoffsommer, H. Joshua, W. Ruyle and C.A. Stone, 1980, A new class of converting enzyme inhibitors, Nature 288, 280. Rascher, W., E. Weidmann and F. Gross, 1981, Vasopressin in the plasma of stroke-prone spontaneously hypertensive rats, Clin. Sci. 61,295.
29 Richer, C., N. Venturini-Souto, J.R. Boissier and J.F. Giudicelli, 1980, fl-adrenoceptor blockade and genetic hypertension development in rats, Clin. Exp. Hypertension 2, 99. Sen, S., R. Tarazi and F.M. Bumpus, 1980, Effect of converting enzyme inhibitor (SQ 14,225) on myocardial hypertrophy in spontaneously hypertensive rats, Hypertension 2, 169. Spertini, F., H.R. Brunner, B. Waeber and H. Gavras, 1981, The opposing effects of chronic angiotensin-converting enzyme blockade by captopril on the responses to exogenous angiotensin II and vasopressin vs norepinephrine in rats, Circ. Res. 48, 612. Sweet, C.S., D.M. Gross, P.T. Arbegast, P.M. Britt, C.T. Ludden, D. Weitz and C.A. Stone, 1981, Antihypertensive activity of N-[(S)-l-(ethoxycarbonyl)-3-phenylpropyl]-LAla-L-Pro (MK 421), an orally active converting enzyme inhibitor, J. Pharmacol. Exp. Ther. 216, 558.
Thurston, H. and J.H. Laragh, 1975, Prior receptor occupancy as a determinant of the pressor activity of infused A II in the rat, Circ. Res. 36, 113. Unger, Th., R.W. Rockhold, G. Brnner, W. Rascher, K. Schaz, G. Speck, A. Schrmig and D. Ganten, 1981, Antihypertensive effects of the novel converting-enzyme inhibitor YS 980 in spontaneously hypertensive rats, Clin. Exp. Hypertension 3, 121. Yamori, Y., 1976, Regulation of blood pressure by the central nervous system, G. Onesti et al. eds., Grunne and Stratton, New York, p. 65. Zimmerman, B.G. and J. Gisslen, 1968, Pattern of renal vasoconstriction and transmitter release during sympathetic stimulation in presence of angiotensin and cocaine, J. Pharmacol. Exp. Ther. 163,320.
23
M K 421 AND P R E V E N T I O N OF G E N E T I C H Y P E R T E N S I O N D E V E L O P M E N T IN Y O U N G S P O N T A N E O U S L Y H Y P E R T E N S I V E RATS CHRISTINE RICHER, MARIE-PASCALE DOUSSAU and JEAN-FRANCOIS GIUDICELLI
*
Dbpartement de Pharmacologie, Facultb de Mbdeeine Paris-Sud, 63 rue Gabriel P&i, 942 70-Le Kremlin-Bicbtre, France Received 8 September 1981, revised MS received 28 October 1981, accepted 14 December 1981
C. RICHER, M.P. DOUSSAU and J.F. GIUDICELLI, MK 421 and prevention of genetic hypertension development in young spontaneously hypertensive rats, European J. Pharmacol. 79 (1982) 23-29. MK 421, at the dose of 25 mg/kg, administered daily by gavage to spontaneously hypertensive rats (SHRs) from their 4th to 15th weeks of age almost completely inhibited development of genetic hypertension. Since heart rate and cardiac and systolic indexes were not affected by the drug, prevention of genetic hypertension development was solely related to an early, potent and long-lasting reduction of the progressive increase of the peripheral resistance which generally develops in SHRs during ageing. MK 421 reduced body growth but did not modify fluid intake, plasma Na + and urine volume, thus water and salt retention did not develop. MK 421 enhanced vascular responsiveness to norepinephrine and angiotensin II and reduced myocardial hypertrophy. Plasma renin concentration was increased and urinary antidiuretic hormone did not change. Finally, MK 421's preventive effects against genetic hypertension development persisted up to 10 weeks after discontinuation of treatment. Spontaneously hypertensive rats Converting enzyme inhibitors Genetic hypertension development
1. Introduction
Angiotensin I converting enzyme inhibitors (CEI) are a new class of potent antihypertensive drugs. Captopril, the first orally active drug in the group is an efficient drug in human arterial hypertension (Gavras et al., 1978; Brunner et al., 1979) but also in different types of experimental hypertension in animals, including low renin models. Thus, captopril not only lowers blood pressure in adult SHRs with established hypertension (Laffan et al., 1978; "Muirhead et al., 1978) but also prevents genetic hypertension development in the chronically-treated young SHR (Ferrone and Antonaccio, 1979; Giudicelli et al., 1980). N-[(S)1-(ethoxycarbonyl)-3-phenylpropyl]-L-Ala-L-Pro or M K 421 is a new potent and orally active CEI which has a pharmacological profile qualitatively similar to captopril (Patchett et al., 1980; Sweet et
* To whom all correspondence should be addressed. 0014-2999/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press
MK 421
al., 1981). The present study was designed in order to investigate whether M K 421 also prevents genetic hypertension development in the young S H R and if so, to determine the underlying mechanism(s).
2. Materials and methods 2.1. Animals and treatments
Forty 4-week old male SHRs (Okamoto strain, Charles River, St. Aubin-les-Elbeuf, France) were randomly divided into two groups of 20 animals each. Rats from each group were housed together in subgroups of 6 to 8 animals, fed ad libitum with standard diet (0.130 m E q / g Na + and 0.205 m E q / g K +) and had free access to tap water. All animals were maintained under identical conditions of temperature (21-+ l°C), humidity and lighting. Starting from the 28th day of life and up to the 15th week of age, the two groups received by
24 gavage every day at the same time a single dose of M K 421 (25 mg/kg, l m l / 1 0 0 g of an aqueous solution) or distilled water (1 ml / 100 g). The dose of M K 421 used was selected as follows: captopril prevents genetic hypertension development in SHRs at an oral daily dose of 100 m g / k g (Giudicelli et al., 1980) and M K 421 is 4.3-fold more potent than captopril as a CEI in the anesthetized rat (Gross et al., 1981).
2.2. Pardmeters investigated Body weight (BW) and systolic blood pressure (SBP), recorded (Physiograph DMP, Narcobiosystems Inc.) in the conscious animals using the indirect tail cuff method (Byrom and Wilson, 1938), were measured in the two groups before the onset of treatment and then every week during the treatment period. H R was obtained from the SBP tracings. Furthermore, systolic blood pressure (SBP), heart rate (HR), cardiac index (CI), systolic index (SI), renal blood flow (RBF), peripheral resistance (PR), kidney and heart weights were measured or calculated at 14 weeks of age in 6 anesthetized (sodium pentobarbital, 50 m g / k g i.p.) animals randomly selected in each group 24 h after SBP and H R had been determined in the conscious state. SBP and H R were monitored using a P 50 Statham blood pressure transducer, a Gould Brush computer model 13421404 and a Gould Brush recorder model 6610-06. Cardiac output and RBF were measured with radioactive microspheres according to the technique described by McDevitt and Nies (1976). After the animals had been sacrificed, hearts and kidneys were immediately removed, carefully cleaned, blotted for 3 min and weighed. Responses to exogenous norepinephrine (0.25, 0.5, 1 and 2/~g/kg) and angiotensin II (10 n g / k g ) were determined at 14 weeks of age in 8 anesthetized animals (sodium pentobarbital, 50 mg/kg, i.p.) randomly selected from each group. Following anesthesia, the trachea was cannulated but the animals breathed spontaneously. Two catheters (PE 10 and PE 50) previously filled with heparinized saline (heparine 50 U / m l ) were introduced in the femoral vein for injections of drugs
and in the carotid artery, respectively. The arterial catheter was connected to a P 50 Statham blood pressure transducer. Blood pressure, recorded on a Gould Brush recorder model 6610-06, was allowed to stabilize for 15 min before starting the experiments and to return to baseline values between the different injections. Body temperature was maintained at 37°C. At the 13th week of age 6 treated and 6 control SHRs randomly selected were housed individually in metabolism cages. Fluid intake and urine volume were measured daily for 5 days and averaged. An aliquot of each urine sample was used for measurement of urinary A D H according to the method of Fressinaud et al. (1974). Immediately after urine collection, two blood samples (1 ml each) were taken from the jugular vein under light ether anesthesia. The first, collected in a tube rinsed with 15% ammonium EDTA solution, pH 6.5 was used to measure plasma renin concentration (PRC) according to the method of Mrnard and Catt (1972). The second, collected in a tube rinsed with heparin was used for determination of plasma Na ÷, K + (flame photometry), urea nitrogen, creatinin and uric acid (Technicon Autoanalyzer). Finally, after discontinuation of treatment (15th week of age), SBP was measured at weekly intervals in the 6 surviving animals of each group up to the age of 25 weeks. All parameters investigated were determined 20 h after the last drug administration.
2.3. Drugs The drugs used were MK 421 (Merck Sharp and Dohme), norepinephrine bitartrate (Levophed R, Winthrop) and angiotensin II (Hypertensin R, Ciba). Doses are expressed in terms of the ester (MK 421), the base (norepinephrine) and the peptide (angiotensin II).
2.4. Statistics Means -+S.E.M. are reported and statistical analysis of the results (treated vs controls) was performed using Student's t-test.
25 2 2C
3. Results
!
3.1. Cardiovascular parameters
2OO
Chronic oral administration of single daily doses of M K 421 at 25 m g / k g to the young SHR markedly inhibited genetic hypertension development (fig. 1). Thus, at the end of the treatment period, SBP measured in the unanesthetized animal was 142.0±2.5 mmHg in the M K 421-treated animals vs. 197.0 ± 3.7 mmHg in the controls. H R was slightly but significantly reduced by M K 421 between the 9th (449.9 ± 8.9 in the treated animals vs. 470.7 ± 10.7 in the controls, P < 0.05) and the 12th week of age (451.4 ± 6.9 in the treated animals vs. 470.3±9.1 in the controls, P < 0 . 0 5 ) but this effect was no longer observed later. In the 14-week old anesthetized SHRs, HR, CI and SI were not treatment-affected (table 1) but SBP was strongly reduced due to the MK 421induced inhibition of PR increase with age. Moreover, RBF was significantly increased in M K 421treated SHRs. Heart weight (HW) was significantly reduced in the treated group at the 14th week of age (table 1). H W / B W ratio also decreased but because of the M K 421-induced lesser age-related BW increase (see below), this decrease did not reach statistical significance. Finally, kidney weight (KW) was not altered by M K 421 (table 1). Ten weeks after discontinuation of treat-
~ 180 a o
q 16o m L)
AGE
(weeks)
Fig. 1. Effect of age on systolic blood pressure of unanesthetized MK 421-treated (25 m g / k g , p.o., daily) ( A ) or control ( 0 ) SHRs. Values depicted are means -+ S.E.M. Values significantly different from respective control values: *** P<0.001.
ment, SBP in the conscious, previously MK 421treated SHRs plateaued at 172.0 ± 2.6 mmHg, a value significantly lower than that observed in controls (216.2 ± 5.5 mmHg, P < 0.001). Fig. 2 depicts the dose-response curves to norepinephrine in both groups. M K 421 produces a shift to the left, significantly so for the two highest doses, indicating an enhanced sensitivity to norepinephrine induced by the chronic converting enzyme blockade. A similar trend, although not
TABL E 1 Effects of MK 421 chronic oral treatment (25 m g / k g per day) on various hemodynamic and biological parameters in the young SHR.
SBPC(mmHg) HR c (beats/mitl) CI c ( m l / m i n per kg) SI~ ( m l / b e a t per kg) P R c ( m m H g / m l per min per kg) RBFC ( m l / m i n per g) H W c (mg) KW ~ (mg) PRC d (rig Ang I / m l per 2 h) ADH d (pg/h)
Control SHRs
MK 421-treated SHRs
165.0 -+ 5.3 428.7 + 9.7 129.1 -+ 11.3 0.30-+ 0.03 1.14-+ 0.11 5.5 + 0.8 1056.4 +26.8 951.2 -+16.4 104.7 ± 9.5 197.9 --+36.3
135.3 --+ 7.0 a 456.4 + 8.8 162.1 -+ 14.5 0.36-+ 0.03 0 . 7 2+- 0.10 a 10.4 -- 1.8 a 937.3 ± 2 5 . 2 b 900.7 -+19.2 220.8 -+32.5 a 282.7 -+40.9
a P < 0 . 0 5 ; b P<0 .0 1 from controls. c Measured in the anesthetized animal at 14 weeks of age (10 weeks of treatment). d Measured on blood or urine samples taken at 13 weeks of age (9 weeks of treatment).
26
302.0 ± 4.0 g (controls), P < 0.05. Furthermore, total fluid intake and urine volume were not drugaffected. Finally, urinary A D H values were not significantly modified (table 1).
A
gE ~4c
4. Discussion UJ < 25
o.'25 . 0.5 NOREPINEPHRINE
~ (~g/kg)
Fig. 2. Mean arterial pressure increases in response to intravenous norepinephrine in anesthetized M K 421-treated (25 m g / k g p.o., daily) ( A ) and control ( 0 ) 14-week old SHRs. Values depicted are m e a n s -4-S.E.M. Values significantly different from respective control values: * P<0.05.
significant, to hyperresponsiveness was also observed with angiotensin II, 10 ng/kg: blood pressure increases were 45.0 - 5.0 in the treated animals and 33.3 ± 5.4 m m H g in the controls, respectively.
3.2. Plasma renin concentration (PRC) and other plasma parameters PRC was significantly augmented in the M K 421-treated SHRs showing a two-fold increase at the 13th week of age (table 1). Plasma Na + (136.6 ± 0.7 in treated vs. 137.4 ± 0.7 mEq/1 in controls), K +, urea nitrogen and creatinine values were not affected by the treatment but plasma uric acid was significantly reduced (39.5 ± 8.8 in treated vs. 73.2 ± 5.7/~mol/1 in controls, P < 0.01).
3.3. Body weight, fluid intake, urinary volume and ADH The age-related increase of the BW was significantly reduced in M K 421-treated SHRs, starting from the 4th week of treatment until its discontinuation. Thus, at 8, 10 and 15 weeks of age (4, 6 and 11 weeks of treatment), mean BW were 157.5 - 2 . 6 g (treated) and 165.7 ± 1.9 (controls), P < 0.05; 212.9 ± 3.1 g (treated) and 226.0 ± 2.4g (controis), P < 0.01 and 269.0-4- 12.0 g (treated) and
M K 421 is a potent and long-lasting oral inhibitor of angiotensin converting enzyme (Patchett et al., 1980; Gross et al., 1981). Its antihypertensive efficacy upon acute administration has been demonstrated in several models of renal hypertension (Sweet et al., 1981), but also in the adult spontaneously hypertensive rat in which a significant decrease of BP has been observed as early as 60 min after a single oral dose of M K 421, with full recovery of initial blood pressure value within 4 h (Sweet et al., 1981). Our experiments demonstrate that M K 421, 25 m g / k g per day chronically administered orally to young SHRs, from their 4th week of age, is also able to prevent genetic hypertension development. Since similar results have already been reported under analogous experimental conditions with a four-fold higher daily oral dose of captopril (Ferrone and Antonaccio, 1979; Giudicelli et al., 1980), our data indicate that M K 421 is at least 4-times more potent on a weight basis than captopril in preventing BP rise in this model. Since the cardiac index is not significantly affected by the drug, it appears that the preventive effect of M K 421 against genetic hypertension development is solely due to the inhibition of the age-related increase in PR which generally develops in SHRs. As previously shown with captopril (Koike et al., 1980; Giudicelli et al., 1981a), early blockade of angiotensin II formation by M K 421 is directly involved and mainly responsible for this inhibition of PR increase, specially in the renal vascular bed which is particularly sensitive to this hormone (Heyndrickx et al., 1976). However, the reduction of norepinephrine release resulting from the blockade of angiotensin II formation (Zimmerman and Gisslen, 1968; Malik and Nasjletti, 1976) may also contribute to the prevention of genetic hypertension development (and perhaps also to the transient bradycardia observed during the first weeks of treatment), specially since sympathetic
27 system hyperactivity plays a dominant role in the first stages of genetic hypertension development (Yamori, 1976; Cutilletta et al., 1977; Richer et al., 1980; Collis et al., 1980). There are contradictory data in the literature regarding the vasopressin secretion in the young SHR as compared to the young Wistar-Kyoto rats. Crofton et al. (1979) have provided evidence for an increased release of vasopressin from the neurohypophysis in the young SHR while Rascher et al. (1981) reported a reduced vasopressin plasma concentration in the young and adolescent stroke-prone SHR. Strain as well as methodology differences may be responsible for this discrepancy but whether vasopressin is involved or not in the early stages of genetic hypertension development in SHRs has not yet been established. However, the fact that in our experiments urinary ADH was not reduced by MK 421 after 9 weeks of treatment, suggests that this mechanism does not participate in the MK 421-induced prevention of genetic hypertension development. Chronic converting enzyme blockade by MK 421 resulted in our experiments in a tendency to an increase in the pressor responses of SHRs to exogenous norepinephrine and angiotensin II. Spertini et al. (1981) also found an enhanced response to angiotensin II but a reduced response to norepinephrine in normotensive rats chronically treated with captopril while Antonaccio and Kerwin (1981) reported a decreased response to both norepinephrine and angiotensin II in SHRs chronically treated with captopril. These data are difficult to reconcile. Increased sensitivity to exogenous angiotensin II, already previously reported with teprotide (Thurston and Laragh, 1975), has been thought to be related to the CEI-induced decrease in circulating angiotensin II which may result in greater availability of vascular receptor sites to exogenous angiotensin II (Thurston and Laragh, 1975; Spertini et al., 1981). Regarding the increased sensitivity to norepinephrine, the same hypothesis could be put forward if one assumes that inhibition of angiotensin II formation leads to a reduction in endogenous norepinephrine release via a presynaptic mechanism (Antonaccio and Kerwin, 1981) and hence possibly to an increased receptor responsiveness to norepinephrine. Fur-
thermore, the fact that the BP was lower in MK 421-treated SHRs than in controls may also have contributed to an enhanced response of the treated rats to the two hormones. However, the data by Antonaccio and Kerwin (1981) do not support this hypothesis and therefore the mechanism of the slight increase in pressor responsiveness to norepinephrine and angiotensin II observed in our experiments remains unclear. Because of the reduction in afterload, the decrease in sympathetic tone and the suppression of the trophic role of angiotensin II, the HW of MK 421-treated SHRs is, as expected, significantly reduced, indicating less myocardial hypertrophy. Thus, like captopril (Giudicelli et al., 1980; Sen et al., 1980; Koike et al., 1980), MK 421 is able to simultaneously prevent the rise in PR and reduce the HW and this parallel effect of the two drugs on the development of functional a n d / o r structural vascular and cardiac abnormalities in the young SHR may account, as previously postulated for captopril (Giudicelli et al., 1981b), for the long-lasting protection which MK-421 and captopril afford against genetic hypertension development, up to 10 weeks or more after discontinuation of treatment. A dissociation between the kinetics of MK 421induced fall in blood pressure and plasma converting enzyme inhibition has been reported by Sweet et al. (1981) in acute experiments performed in the adult SHR. This suggests that the two phenomena might not be related or that the antihypertensive effect could result from blockade of converting enzyme at other sites, e.g. within the vascular wall or the brain. Our experiments, in which MK 421 has been administered chronically to young SHRs only indicate that after 9 weeks of treatment, the drug induces plasma converting enzyme inhibition, as shown by the rise in PRC, and prevention of genetic hypertension development simultaneously. However, one cannot exclude that MK 421 might oppose genetic hypertension development by inhibition of brain converting enzyme, like captopril upon chronic treatment (Mann et al., 1981). The MK 421-induced reduction of BW growth with age and the lack of change in plasma Na + , fluid intake and urine volume after 9weeks of treatment, clearly indicate that MK 421 does not
28
induce sodium and water retention, a property shared by captopril and YS 980, another CEI (Crofton et al., 1979; Unger et al., 1981). In summary, chronic blockade of converting enzyme by MK 421 in the young SHR prevents genetic hypertension development by reducing the age-related increase in PR, vasodilating the renal vascular bed and reducing the cardiac hypertrophy without simultaneously inducing tachycardia and body fluid volume expansion.
Acknowledgements The authors want to express their gratitude to Dr. C. Sweet of Merck Institute for Therapeutic Research, West Point (Pa), U.S.A., for a generous supply of MK 421. They also wish to thank Pr. J. M+nard and Mrs. A. Cottard for ADH measurements.
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verting enzyme inhibitor SQ 14,225 in man, New Engl. J. Med. 298, 991. Giudicelli, J.F., J.L. Freslon, S. Glasson and C. Richer, 1980, Captopril and hypertension development in the SHR. Clin. Exp. Hypertension 2, 1083. Giudicelli, J.F., J.L. Freslon and C. Richer, 1981a, Captopril et prrvention du d~veloppement de l'hypertension grnetique du rat SHR, Nouv. Presse Med. 10, 1547. Giudicelli, J.F., C. Richer and J.L. Freslon, 1981b, Antihypertensive drugs and prevention of genetic hypertension development in SHRs, in: New Trends in Arterial Hypertension, M. Worcel et al., eds., Elsevier, Amsterdam, p. 45. Gross, D.M., C.S. Sweet, E.H. Ulm, E.P. Backlund, A.A. Morris, D. Weitz, D.L. Bohn, H.C. Henger, T.C. Vassil and C.A. Stone, 1981, Effect of N - [ ( S ) - l - c a r b o x y - 3 phenylpropyl]-L-Ala-L-Pro and its ethyl ester (MK 421) on angiotensin converting enzyme in vitro and angiotensin I pressor responses in vivo, J. Pharmacol. Exp. Ther. 216, 552. Heyndrickx, G.R., D.H. Boettcher and S.F. Vatner, 1976, Effects of angiotensin, vasopressin, and methoxamine on cardiac function and blood flow distribution on conscious dogs, Am. J. Physiol. 231, 1579. Koike, H., I. Katsuaki, M. Miyamoto and N. Nishino, 1980, Effects of long-term blockade of angiotensin converting enzyme with captopril (SQ 14,225) on hemodynamics and circulating blood volume in SHR, Hypertension 2, 299. Laffan, R.J., M.E. Goldberg, J.P. High, T.R. Schaeffer, M.H. Waugh and B. Rubin, 1978, Antihypertensive activity in rats of SQ 14,225, an orally active inhibitor of angiotensin I converting enzyme, J. Pharmacol. Exp. Ther. 204, 281. Malik, K.V. and A. Nasjletti, 1976, Facilitation of adrenergic transmission by locally generated angiotensin II in rat mesenteric arteries, Circ. Res. 38, 26. Mann, J.F.E., J. Genest, Th. Unger, B. Scholkens, W. Rascher, A. Schomig and D. Ganten, 1981, A role for the reninangiotensin system in central mechanisms of blood pressure control, in: New Trends in Arterial Hypertension, M. Worcel et al., eds., Elsevier, Amsterdam, p. 101. McDevitt, D.G. and A.S. Nies, 1976, Simultaneous measurement of cardiac output and its distribution with microspheres in the rat, Cardiovasc. Res. 10, 494. M~nard, J. and K.J. Catt, 1972, Measurement of renin activity, concentration and substrate in rat plasma by radioimmunoassay of angiotensin I, Endocrinology 90, 422. Muirhead, E.E., R.L. Prewitt, B. Brooks and W.L. Brosius, 1978, Antihypertensive action of the orally active converting enzyme inhibitor (SQ 14,225) in spontaneously hypertensive rats, Circ. Res. 43 (Suppl. I), 53. Patchett, A., E. Harris, E. Tristram, M. Wyvratt, M. Wu, D. Taub, E. Paterson, T. Ikeler, J. Ten Broeke, L. Payne, T. Ondeyka, E. Thorsett, W. Greenlee, N. Lohr, R. Hoffsommer, H. Joshua, W. Ruyle and C.A. Stone, 1980, A new class of converting enzyme inhibitors, Nature 288, 280. Rascher, W., E. Weidmann and F. Gross, 1981, Vasopressin in the plasma of stroke-prone spontaneously hypertensive rats, Clin. Sci. 61,295.
29 Richer, C., N. Venturini-Souto, J.R. Boissier and J.F. Giudicelli, 1980, fl-adrenoceptor blockade and genetic hypertension development in rats, Clin. Exp. Hypertension 2, 99. Sen, S., R. Tarazi and F.M. Bumpus, 1980, Effect of converting enzyme inhibitor (SQ 14,225) on myocardial hypertrophy in spontaneously hypertensive rats, Hypertension 2, 169. Spertini, F., H.R. Brunner, B. Waeber and H. Gavras, 1981, The opposing effects of chronic angiotensin-converting enzyme blockade by captopril on the responses to exogenous angiotensin II and vasopressin vs norepinephrine in rats, Circ. Res. 48, 612. Sweet, C.S., D.M. Gross, P.T. Arbegast, P.M. Britt, C.T. Ludden, D. Weitz and C.A. Stone, 1981, Antihypertensive activity of N-[(S)-l-(ethoxycarbonyl)-3-phenylpropyl]-LAla-L-Pro (MK 421), an orally active converting enzyme inhibitor, J. Pharmacol. Exp. Ther. 216, 558.
Thurston, H. and J.H. Laragh, 1975, Prior receptor occupancy as a determinant of the pressor activity of infused A II in the rat, Circ. Res. 36, 113. Unger, Th., R.W. Rockhold, G. Brnner, W. Rascher, K. Schaz, G. Speck, A. Schrmig and D. Ganten, 1981, Antihypertensive effects of the novel converting-enzyme inhibitor YS 980 in spontaneously hypertensive rats, Clin. Exp. Hypertension 3, 121. Yamori, Y., 1976, Regulation of blood pressure by the central nervous system, G. Onesti et al. eds., Grunne and Stratton, New York, p. 65. Zimmerman, B.G. and J. Gisslen, 1968, Pattern of renal vasoconstriction and transmitter release during sympathetic stimulation in presence of angiotensin and cocaine, J. Pharmacol. Exp. Ther. 163,320.