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Mechanism of direct cardiostimulating actions of hydralazine
European Journal of Pharmacology, 135 (1987) 137-144
137
Elsevier EJP 00675
Mechanism of direct cardiostimulating actions of hydralazine Junichi Azuma
1,.,
A k i h i k o S a w a m u r a 1, H i s a t o H a r a d a 1, N o b u h i s a A w a t a S u s u m u K i s h i m o t o 1 a n d N i c k Sperelakis 2
1,
1 The Third Department of Internal Medicine, Osaka Unioersity Medical School, Osaka 553, Japan, and 2 Department of Physiology and Biophysics, University of Cincinnati College of Medicine, Cincinnati, OH 45267, U.S.A.
Received 10 November 1986, accepted 16 December 1986
The vasodilator, hydralazine, was reported to also exert a direct positive inotropic effect on the myocardium at high concentrations. In the present study we investigated the mechanism of this positive inotropic action by using the ventricular myocardium of isolated perfused chick hearts. Hydralazine (10-3 M) enhanced contractile force and heart rate, and elevated, the myocardial cyclic AMP level. To study the Ca 2+-dependent slow action potentials, the fast N + channels were voltage-inactivated with elevated K + (25 mM), resulting in a loss of electrical excitability. Hydralazine (10 -4 M) rapidly (< 3 rain) allowed the generation of slow action potentials and accompanying contractions by electrical stimulation. These effects of hydralazine were only partially prevented by propranolol. The results suggest that the increase of myocardial contractility produced by hydralazine is the result, at least in part, of a direct effect on the myocardium to increase Ca 2+ inflow. The increased Ca 2+ influx and inward slow current is due partly to activation of fl-adrenoceptors, with resultant elevation of cyclic AMP, and partly to another mechanism. Inotropic action (positive); Calcium slow channels; Cyclic AMP; Hydralazine; Histamine; Cardiac electrophysiology
1. Introduction Hydralazine has been used as an antihypertensive agent, and recently in the management of congestive heart failure. In addition to reducing arterial pressure by a direct vasodilator effect on the arterioles (.&blad, 1963; Stunkard et al., 1954), hydralazine substantially increases cardiac output and heart rate. These effects have been attributed to reflex activation of the cardiac sympathetic nerves secondary to the decrease in arterial pressure (Brunner et al., 1967). However, some studies in animal models (Brunner et al., 1967; Gershwin and Smith, 1967, Khatri et al., 1977; Songldttinguna and Rand, 1982; Rabinowitz et al., 1986) and in the falling human ventricle (Leier et al., * To whom all correspondence should be addressed: The Third Department of Internal Medicine, Osaka University Medical School, Fukushima 1-1-50, Osaka 553, Japan.
1980) indicate that hydralazine exerts a positive inotropic action directly on the myocardium. The purpose of the present study was to investigate the mechanism of this direct cardiostimulating action of hydralazine. The effect of hydralazine was examined on the contractions, electrophysiological properties and tissue cyclic A M P level of the ventricular myocardium in isolated perfused chick hearts.
2. Materials and methods Hearts were removed from 1 to 5 day old posthatched chicks, and a glass cannula was inserted into the aorta for perfusion of the coronary vessels. The perfusing solutions flowed from two reservoirs (located 60 cm above the heart) that contained either normal Tyrode solution (composition in mM: 137 NaC1, 2.7 KC1, 1.8 CaC12,
0014-2999/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
138
1.0 MgC12, 20 N a H C O 3, 1.0 NaH2PO 4, 5.5 glucose) or 25 mM K ÷ Tyrode solution (isosmolar substitution of K ÷ for Na÷). The solutions were gassed with a mixture of 95% O 2 and 5% CO 2 (pH 7.4), and heated to 37°C. Hydralazine was added in cumulative doses to the perfusing reservoirs to give final concentrations ranging between 10 -5 and 3 × 10 -3 M. A force displacement transducer was used to measure contractions by means of a thread sutured through the apex of the ventricle. The tension developed and its first time derivative ( d T / d t ) were recorded on a penwriter at a slow paper speed. The results with hydralazine were similar whether they were obtained from the tension developed (T) or d T / d t . The data were expressed as a percent change from the control value immediately prior to administration of hydralazine. For electrical stimulation, rectangular current pulses were delivered via platinum plate electrodes. Resting potentials and action potentials were recorded by intracellular micropipettes (filled with 3 M KC1) using the floating microelectrode technique. The maximum rate of rise ( + Vm~x) of the upstroke (phase 0) of the action potential (AP) was determined with a resistance-capacitance differentiator (time constants of 10 -5 and 10 -4 s) calibrated in V / s by means of a ramp generator. + ~rma~ gives a measure of the relative intensity of the inward current. For slow APs, the fast Na + channels were inactivated by partial depolarization produced by perfusion with 25 mM K ÷ Tyrode solution. This rendered the heart unexcitable, and caused mechanical failure despite intense electrical field stimulation. The K+-depolarized hearts were Stimulated at a rate of 50/min. Hydralazine was tested for its ability to induce slow APs and contractions. The stimulator was turned off when required to ascertain whether any slow APs would occur spontaneously in the presence of hydralazine. The d T / d t value for a heart whose excitability had been restored by hydralazine was expressed as a percent of the d T / d t value for the same heart (spontaneously beating) immediately prior to depolarization with 25 mM K ÷. The values for the parameters measured in contraction and electrophysiological experiments were taken after the
hearts achieved a steady state following each concentration of hydralazine (usually 10-15 min). In 3 cases, verapamil (2 × 10 -6 M), a blocker of Ca 2÷ slow channels, was added to test whether it would block the hydralazine-induced slow APs. It was tested in 3 experiments whether hydralazine would potentiate the isoprenaline-induced slow APs and contractions. To test the possibility that histamine release was involved in the positive inotropic action of hydralazine, the hsitamine receptor blocking agents, diphenhydramine (H 1 blocker) (10 -5 M) or cimetidine (H 2 blocker) (5 × 10 -5 M), were added to the perfusing solution. To test the possibility that the hydralazine action was mediated by the fl-adrenoceptors, the antagonist propranolol (10 - 6 M) or pindolol (10 -6 M) was added to the perfusing solution in some experiments. In 3 hearts whose myocardial noradrenaline had been depleted by prior reserpine treatment (1 m g / k g i.p., 24 h prior to the study), it was tested whether hydralazine could still induce slow APs. Noradrenaline levels were measured by gas chromatography-mass spectrometry (Higa et al., 1977): control heart, 0.35 + 0.04 n g / m g tissue ( N = 10); reserpine-treated heart, 0.05 +0.02 n g / m g tissue (N = 10) (P < 0.001). For cyclic AMP analysis, the hearts were perfused with normal Tyrode solution for 10 rain, and hydralazine (3 × 10 -3 M) was then added. After 5 min 20-30 mg of tissue was removed from the apex of the heart, blotted with a filter paper and immediately frozen with a stainless steel clamp precooled in liquid nitrogen. The freeze-clamped tissue was weighed and homogenized for 20 s in 2-3 ml of 6% perchloric acid at 4°C with a Polytron (PT 10-35) homogenizer. The homogenate was centrifuged at 3000 r.p.m, for 10 min at 4°C. The supematant was collected and adjusted to p H 3.0 (with KOH). These samples were stored in a freezer until assayed for cyclic AMP. The cyclic AMP content of the samples was determined in duplicate by radioimmunoassay (Honma et al., 1977) using YAMASA cyclic AMP assay kits (Yamasa Shoyu Co., Ltd.), and expressed as n m o l / m g wet weight of tissue. In some experiments, propranolol (10 -6 M) was added to the perfusing solution to ascertain whether it would
139 affect the cyclic AMP response of the heart to hydralazine or the basal cyclic AMP level (15 rain of exposure). The data were analyzed for statistical significance, depending on the design of the experiments, by: (a) Student's t-test (paired or unpaired), (b) analysis of variance (Scheffe's method was used to compare individual data when a significant F value was shown), or (c) x2-test. Differences were considered significant when the calculated P value was less than 0.05. Values are given as means + S.E.M.
o
o=
+20
O
dT I dt
T |
heart rate
I
2 0
.
.
.
. n=lO mean± S.E.M.
10.5
10.4
10.3
3 x 10.3
Hydralazine ( M )
3. Results 3.1. Contractility and heart rate
The effects of different concentrations of hydralazine on cohtractility (dT/dt) and heart rate associated with the fast action potentials (APs) (hearts perfused with normal Tyrode solution) are summarised graphically in fig. 1. The hearts contracted spontaneously at a rate of approximately 200 beats/min. The heart rate increased significantly with 3 x 10 -3 M hydralazine. Hydralazine (10-3-3 × 10-3 M) significantly increased the contractile force to a peak level of 112 _ 5% (P < 0.05) and 120 + 9% (P < 0.05), respectively, of the control magnitude within 1-2 rain. These positive chronotropic and inotropic actions persisted undiminished until termination of the experiment at 20 min. 3.2. Slow APs and contractions
In hearts in which the fast Na + channels were voltage-inactivated by partial depolarization in elevated K ÷ (25 mM) (fig. 2B), the addition of any agent which increases the density of the slow (Ca2÷-Na ÷) channels available for voltage activation resulted in the generation of slow APs accompanied by contractions. Exposure to hydralazine (10 -3 M) induced a slowly rising overshooting electrical response within 1 min, concomitant with contractions (fig. 2C, C'). The addition of verapamil (2 x 10 -6 M to 3 isolated hearts), a blocker of the transmembrane Ca 2+ slow current,
Fig. i . Graphic summary of the effect of several concentrations of hydralazine on contractility (dT/dt) and heart rate of chick (1-5 day old post-hatched) hearts associated with the fast action potentials (in 2.7 mM [K]0 ). Hydralazine significantly increased the contractile force and heart rate in a dose-dependent fashion. * P < 0.05 vs. control (zero hydralazine); d T / d t = first time derivative of developed tension.
abolished the hydralazine-induced slow APs and contractions (fig. 2D, D'). Hydralazine (10 -3 M) also potentiated the isoprenaline (10 -s M)-induced slow APs and contractions by about 50% (not illustrated). 3.3. Effect of fl-adrenoceptor blockade and histamine receptor blockade on the hydralazine-induced slow responses
Propranolol (10 -6 M) was used to test the possibility that the action of hydralazine involved fl-adrenoceptors. Propranolol did not significantly affect the control contractions (fig. 3). At 10 - 4 M, hydralazine failed to induce contraction (accompanying slow APs) in the presence of propranolol (fig. 3B). However, propranolol did not prevent the induction of contraction by 10- 3 and 3 x 10- 3 M hydralazine but the magnitude of the contractions (accompanying the slow APs) was lower. This suggests that the effect of hydralazine was mediated in part by catecholamine release from the nerve terminals. Concentration-response curves for the magnitude of the contractions (dT/dt) associated with the slow APs induced by hydralazine with and without propranolol (10 -6 M) are given in fig. 4.
140
normal Tyrode
Hydralazine
25 mM K+Tyrode
+
( 10-3M ) 3 rain
Verapamil
( 2xlO-6M )
o-
I,
!
I
10V/s
I
40mV
I
I
I
200 ms A' ~
B'
C'
O'
I1
I-
I
3 rain
Fig. 2. Hydralazine induction of slow action potentials and contractions. (A) Control fast action potential and mechanical recording (A') during perfusion with normal Tyrode solution in a spontaneously beating heart. Upper tracing is the first derivative (+ Vmax) of the action potential (arbitrarily shifted to the right). (B) Elevation of K + to 25 mM decreased the resting potential to about - 4 0 mV and abolished the action potentials and contractions (B') despite intense electrical stimulation. (C) Addition of hydralazine (10 -3 M) produced slowly rising action potentials and contractions (C'). (D) Verapamil (2 x10 -6 M) abolished the action potentials and contractions (D'). T = developed tension.
It c a n b e s e e n t h a t h y d r a l a z i n e p r o d u c e d a g r e a t e r a u g m e n t a t i o n of the c o n t r a c t i l e force i n the a b sence t h a n i n the p r e s e n c e of p r o p r a n o l o l . T h e effect of p r o p r a n o l o l o n the o c c u r r e n c e of the h y d r a l a z i n e - i n d u c e d s p o n t a n e o u s slow res p o n s e s was e x a m i n e d b y t u r n i n g off the electrical
25 mM K+ Tyrode
A
I 10-4M
s t i m u l a t i o n ( t a b l e 1). W i t h o u t p r o p r a n o l o l , 10 - 3 M h y d r a l a z i n e p r o d u c e d s p o n t a n e o u s slow res p o n s e s (25-60 b e a t s . r a i n - 1 ) i n 12 o u t o f 18 hearts, w h e r e a s it d i d n o t p r o d u c e slow r e s p o n s e s i n a n y of the 10 h e a r t s tested w i t h p r o p r a n o l o l (10 - 6 M ) ( P < 0.001).
Hydralazine 10-3M
3 x 10-3M
i
Without Propranolol ,
!
t ~ 5 rain
B
In presense of Propranolol ~ ]0-6M )
!
f
:
o
. . . . . . . . . . . . . . . . . . . .
.
Fig. 3. The effect of propranolol on the contractions (dT/dt = first time derivative of developed tension) accompanying the hydralazine-induced slow action potentials. (A) Without propranolol, hydralazine (10 -4, 10 -3 and 3 x l0 -3 M) induced contractions. (B) In the presence of propranolol (10 -6 M), the magnitude of the contractions (accompanying the slow action potentials) was lower, indicating that the effect of hydralazine was mediated, at least in part, by catecholamine release from the nerve terminals or by direct activation of the ~-adrenoceptors by hydralazine itself.
141 TABLE 1
TABLE 2
Effect of propranolol on the frequency of occurrence of hydralazine-induced spontaneous slow action potentials in 1-5 day old chick hearts. Frequency of occurrence (given in parentheses) was expressed as percent of the number of hearts examined, n.s. = not significant.
Effect of hydralazine on cyclic AMP level of isolated chick hearts perfused with 25 mM K + Tyrode solution with or without propranolol. Data are expressed as means+S.E.M. Number in parentheses represents the number of determinations. Cyclic A M P (nmol •g- 1 wet weight)
Hydralazine concentration (M) 10 - 4
without propranolol With propranolol (10-6M) Statistical significance
10-3
3XlO -3
0/12 (0%) 12/18 (66%) 12/12 (100%) 0/9 (0%) n.s.
0/10 (0%) P < 0.001
3/9
(33%)
Control 0.38 + 0.02 (21) Control with propranolol (10 -6 M) 0.36+0.03 (18) Hydralazine (3 × 10- 3 M) 0.58 ___0.04 (17) a.b Hydralazine with propranolol 0.46 __.0.03 (16) b.¢ Significant difference (P < 0.05) vs. control a, vs. control with propranolol b and vs. hydralazine e.
P < 0.001
It is well k n o w n that p r o p r a n o l o l in high c o n c e n t r a t i o n s has a non-specific local anesthetic action. W e therefore tested the effect o f p i n d o l o l (10 - 6 M), which is a l m o s t d e v o i d of local a n e s t h e t i c activity, in 3 cases. P i n d o l o l d i d n o t p r e v e n t the i n d u c t i o n of c o n t r a c t i o n s b y 10 -3 M h y d r a l a z i n e u n d e r electrical stimulation, b u t the m a g n i t u d e o f the c o n t r a c t i o n s ( a c c o m p a n y i n g the slow A P s ) was p a r t i a l l y d e c r e a s e d b y p i n d o l o l to 65 + 12% of the m a g n i t u d e w i t h o u t pindolol. I n i s o l a t e d h e a r t s f r o m chicks treated with reserpine, h y d r a l a z i n e (3 × 10 -3 M ) still i n d u c e d slow A P s a n d c o n t r a c t i o n s (not illustrated). D i p h e n h y d r a m i n e (10 - s M ; N = 2) a n d c i m e t i d i n e (5 × 10 - s M; N = 7), blockers o f H 1 a n d H 2 receptors, respectively d i d n o t affect the c o n t r a c t i o n s a c c o m p a n y i n g the h y d r a l a z i n e - i n d u c e d slow A P s (not illustrated). This suggests t h a t h i s t a m i n e release is n o t involved in the a c t i o n of h y d r a l a z i n e .
3.~ Cycfic AMP assays A total o f 72 chick h e a r t s were a n a l y z e d for cyclic A M P content. T h e effect of h y d r a l a z i n e (3 × 10 -3 M ) o n the tissue cyclic A M P level in h e a r t s p e r f u s e d either with or w i t h o u t p r o p r a n o l o l 10 - 6 M is s u m m a r i s e d in t a b l e 2. T h e cyclic A M P c o n t e n t in c o n t r o l hearts p e r f u s e d with n o r m a l T y r o d e s o l u t i o n for 15 m i n was 0.38 + 0.02 n m o l . g - 1 wet weight ( N = 21). P r o p r a n o l o l itself d i d
n o t significantly affect the tissue cyclic A M P level (0.36 + 0.03 n m o l . g - Z wet weight, N = 18). This suggests that the hearts were n o t subjected to a fl-adrenergic t o n e d u e to c a t e c h o l a m i n e s released f r o m intrinsic c a r d i a c nerve endings. H y d r a l a z i n e c a u s e d a significant elevation of cyclic A M P to 0.58 ___0.04 n m o l . g - x wet weight ( N = 17, P < 0.05 c o m p a r e d to c o n t r o l a n d p r o p r a n o l o l - t r e a t e d hearts). P r o p r a n o l o l significantly r e d u c e d the lay-
_
100
o
• Hydralazine
T
•
-6
0 Hydralazme + Propranolol ( 10 M )
~ ' ( 12 )
J
E
•-
9)
N
/
~
10-5
10-4
rnean+ S. LM. 10-3
3x10 -3
Hydralazine ( M ) Fig. 4. Graphic summary of the effect of propranolol (10 -6 M)
on the magnitude of contractions associated with the hydralazine-induced slow action potentials (in 25 mM [K]0). Elevation of hydralazine concentration produced an increase in contraction in a concentration-dependent fashion. Propranolol depressed the response to hydralazine. Each number in parentheses gives the number of experiments. * P < 0 . 0 5 v s . + propranolol for 10 -4, 10 -3 and 3×10 -3 M of hydralazine, respectively; dT/dt = first time derivative of developed tension.
142 dralazine-induced elevation of cyclic AMP to 0.46 + 0.03 nmol. g-1 wet weight (N = 16), but this value was still significantly higher than the value for the propranolol-treated hearts. This indicates that the elevation of cyclic AMP by hydralazine was only partially mediated via fl-adrenoceptor activation.
4. Discussion
The present results demonstrate that hydralazine at high concentrations elevates the intracellular cyclic AMP level in the heart. Part of this action is due to activation of the fl-adrenoceptt~rs. The hydralazine induction of Ca 2+-dependent slow action potentials (APs) in myocardial cells (in which elevated K + had abolished excitability), like that by catecholamines, histamine and methylxanthines, probably occurs because of its cyclic AMP-elevating action (Sperelakis and Schneider, 1976; Azuma et al., 1981a). This, in turn, would result in an increase in inward Ca 2+ current via the Ca 2+ slow channels during the cardiac AP. This mechanism could explain the positive inotropic action of hydralazine. Brunner et al. (1967) had proposed that the cardiac stimulation seen after administration of hydralazine to conscious animals might be the result of reflex adjustment to the fall in blood pressure. An increase in sympathetic activity also occurs after hydralazine administration in man (.~blad, 1963). Thus, the cardiac hyperactivity in the whole animal after hydralazine administration might result partly from increased sympathetic stimulation of the heart due to activation of the baroreceptor reflex by the vasodilatation-induced hypotension. In addition, hydralazine was also reported to exert positive inotropic effects directly on the heart itself by releasing noradrenaline from myocardial sympathetic nerve endings or by directly stimulating cardiac fl-adrenoceptors (Gershwin and Smith, 1967; Leier et al., 1980; Rabinowitz et al., 1986). Khatri et al. (1977) demonstrated, by means of direct injection of hydralazine into a coronary artery in dogs, that increased myocardial contractile force was elicited consistently in the area
perfused, and that this local effect of hydralazine was blocked by propranolol. They concluded that the observed increase in myocardial contractility was the result of direct fl-adrenoceptor stimulation of the myocardium. Koch-Weser (1974; 1971) showed that concentrations of hydralazine above 10 -4 M increased the development of tension in kitten ventricular myocardium. Since this positive inotropic effect was blocked by propranolol and was abolished by prior reserpine treatment, it was concluded that the positive inotropic effect was due to the release of noradrenaline from adrenergic nerve endings in the myocardium (Koch-Weser, 1974). Rabinowitz et al. (1986) have recently demonstrated that the positive inotropic action of hydralazine was mediated by the release of catecholamine and resultant activation of the adenylate cyclase system. However, Koch-Weser (1971) suggested that only concentrations of hydralazine far above the therapeutic range produce direct inotropic effects. In the present experiments on chick hearts, release of noradrenaline could not have been the sole mechanism because hydralazine retained some effects in reserpine-pretreated hearts whose noradrenaline content had been depleted. Similar results have been reported for rat atrial preparations (Songldttiguna and Rand, 1982). In addition, the effects of hydralazine, namely, induced slow APs with contractions and elevated cyclic AMP levels, were only partially inhibited by propranolol. Therefore, the increase in myocardial contractility after administration of hydralazine may have been the result of (a) direct fl-adrenoceptor stimulation produced by noradrenaline released from nerve endings, and (b) an increased intracellular cyclic AMP level. The latter could result from phosphodiesterase inhibition as demonstrated for beef heart (Ishii et al., 1979), or from adenylate cyclase stimulation (Rabinowitz, 1986). Gershwin and Smith (1967) observed that hydralazine released histamine from isolated guineapig atria, and that histamine, in turn, released catecholamines. Propranolol blocked the action of hydralazine, as did antihistamines. However, histamine itself is a known positive inotropic agent (Barlet, 1963) which increases cyclic AMP by stimulation of adenylate cyclase (McNeil and
143 M u s c h e k , 1972). T h e positive i n o t r o p i c action of h i s t a m i n e was s h o w n to b e m e d i a t e d b y a c t i v a t i o n of the H 2 receptor, resulting in an increased availa b i l i t y of C a 2+ slow channels ( J o s e p h s o n et al., 1976). However, in the p r e s e n t experiments, h i s t a m i n e - r e c e p t o r b l o c k i n g agents failed to b l o c k the effects o f hydralazine. H e n c e h i s t a m i n e release is p r o b a b l y n o t r e s p o n s i b l e for the positive ino t r o p i c effect of h y d r a l a z i n e . W e showed that h y d r a l a z i n e increased the h e a r t r a t e in hearts p e r f u s e d with n o r m a l T y r o d e solution. Positive c h r o n o t r o p i c agents, such as fla d r e n o c e p t o r agonists, e n h a n c e the rate of pacem a k e r discharge b y s t i m u l a t i n g the p r o d u c t i o n of cyclic A M P b y activating a d e n y l a t e cyclase. A d r e n a l i n e causes a large increase in i n w a r d C a 2+ c u r r e n t ( a n d a small increase in o u t w a r d K + c u r r e n t ) in the S A n o d e (Brown et al., 1979). Since the i n w a r d d e p o l a r i z i n g p a c e m a k e r current in sinus n o d e cells is p a r t l y carried t h r o u g h slow channels ( N o m a a n d Irisawa, 1976), the increased n u m b e r of a c t i v a t e d slow channels in the presence of h y d r a l a z i n e should result in greater a u t o m a t i c i t y . C o n s i s t e n t with this, the s p o n t a n e o u s c o n t r a c t i o n s t h a t a p p e a r e d after h y d r a l a z i n e p e r f u s i o n ( a b o v e 10 - 4 M) were p a r t i a l l y b l o c k e d with p r o p r a n o l o l . Balazs et al. (1981) r e p o r t e d that h y d r a l a z i n e causes m y o c a r d i a l necrosis in rats, which was prev e n t e d b y p r e t r e a t m e n t with p r o p r a n o l o l o r v e r a p a m i l , a n d they suggested that i n c r e a s e d intracellular c a l c i u m p l a y s a role in the p a t h o g e n e s i s o f the necrosis. Thus, o u r p r e s e n t findings that h y d r a l a z i n e increased C a 2 ÷ inflow via slow channels, a n d t h a t this was b l o c k e d b y v e r a p a m i l , m i g h t e x p l a i n the m e c h a n i s m of h y d r a l a z i n e - i n d u c e d m y o c a r d i a l lesions o b s e r v e d b y Balazs et al. Similarly, the e n h a n c e d C a 2÷ inflow c o u l d be a factor in the c a l c i u m o v e r l o a d associated with a d r i a m y c i n - i n d u c e d c a r d i o m y o p a t h y ( A z u m a et al., 1981b). T h e p r e s e n t results suggest that h y d r a l a z i n e has direct c a r d i o s t i m u l a t i n g effects p r o d u c e d b y an i n c r e a s e d C a 2÷ inflow t h r o u g h the C a 2÷ slow channels.
Acknowledgements This work was supported in part by Grant HL-31942 from the National Institutes of Health. We thank Dr. Sadayoshi
Higa for determining noradrenaline, and Miss Y. Ikeda and Y. Ueki for their assistance in preparing the manuscript. We are grateful to Ciba-Geigy (Japan) Limited for a generous supply of hydralazine.
References ,~blad, B., 1963, A study of the mechanism of the hemodynamic effects of hydralazine in man, Acta Pharmacol. Toxicol. 20 (Suppl. 1), 1. Azuma, J., A. Sawamura, H. Harada, T. Tanimoto, T. Ishiyama, Y. Morita, Y. Yamamura and N. Sperelakis, 1981a, Cyclic adenosine monophosphate modulation of contractility via slow Ca 2+ channels in chick heart, J. Mol. Cell. Cardiol. 13, 577. Azuma, J., N. Sperelakis, H. Hasegawa, T. Tanimoto, S. Vogel, K. Ogura, N. Awata, A. Sawamura, H. Harada, T. Ishiyama, Y. Morita and Y. Yamamura, 1981b, Adriamycin cardiotoxicity: Possible pathogenic mechanism, J. Mol. Cell. Cardiol. 13, 381. Balazs, T., V.J. Ferrans, A. El-Hage, S.J. Ehrreich, G.L. Johnson, E.H. Herman, J.C. Atkinson and W.L. West, 1981, Study of the mechanism of hydralazine-induced myocardial necrosis in the rat, Toxicol. Appl. Pharmacol. 59, 524. Barlet, A.L., 1963, The action of histamine on the isolated heart, Br. J. Pharmacol. 21, 450. Brown, H.F., D. DiFrancesco and S.J. Noble, 1979, How does adrenaline accelerate the heart?, Nature 280, 235. Brunner, H., P.R. Hedwall and M. Meier, 1967, Influence of adrenergic betareceptor blockade on the acute cardiovascular effects of hydralazine, Br. J. Pharmac. Chemother. 30, 123. Gershwin, M.E. and N.T. Smith, 1967, Mode of action of hydralazine on guinea pig atria, Arch. Int. Pharmacodyn. Ther. 170, 108. I-Iiga, S., T. Suzuki, A. Hayashi, I. Tsuge and Y. Yamamura, 1977, Isolation of catecholamines in biological fluids by boric acid gel, Anal. Biochem. 77, 622. Honma, M., T. Satoh, J. Yakezawa and M. Ui, 1977, An ultrasensitive method for the simultaneous determination of cyclic AMP and cyclic GMP in small-volume samples from blood and tissue, Biochem. Med. 18, 257. Ishii, A., K. Kubo, T. Deguchi and M. Tanaka, 1979, Ifihibition of cyclic AMP phosphodiesterase activity by ecarazine, hydrochloride, hydralazine and their metabolites, Yakugaku Zasshi 99, 533. Josephson, I., J.F. Renaud, S. Vogel, M. McLean and N. Sperelakis, 1976, Mechanism of the histamine-induced positive inotropic action in cardiac muscle, European J. Pharmacol. 35, 393. Khatri, I., N. Uemura, A. Notagrlacomo and E.D. Freis, 1977, Direct and reflex cardiostimulating effects of hydralazine, Am. J. Cardiol. 40, 38. Koch-Weser, J., 1971, Beta-receptor blockade and myocardial effects of cardiac glycosides, Circ. Res. 28, 109.
144
Koch-Weser, J., 1974, Myocardial inactivity of therapeutic concentrations of hydralazine and diazoxide, Experimentia 30, 170. Leier, C.V., C.E. Desch and R.D. Magorien, D.W. Triffon, D.V. Unverferth, H. Boudoulas and P.P. Lewis, 1980, Positive inotropic effects of hydralazine in human subjects: Comparison with prazosin in the setting of congestive heart failure, Am. J. Cardiol. 46, 1039. McNeil, J.H. and I.D. Muschek, 1972, Histamine effects on cardiac contractility, phosphorylase and adenyl cyclase, J. Mol. Cell. Cardiol. 4, 611. Noma, A. and H. Irisawa, 1976, Membrane currents in the rabbit sinoatrial node cell as studied by the double microelectrode method, Pfliigers Arch. 364, 45.
Rabinowitz, B., W.W. Parmley, Y. Har-zahav, E. Elazar, S. Blumlein, R. Nerinsky and H.N. Neufeld, 1986, Correlation between effects of hydralazine on force and on the adenyl cyclase system of ventricular myocardium in dogs and cats, Cardiovasc. Res. 20, 215. Songldttiguna, P. and M.J. Rand, 1982, The mechanism of the inotropic and chronotropic actions of hydralazine on rat isolated atria, Arch. Int. Pharmacodyn. 255, 269. Sperelakis, N. and LA. Schneider, 1976, A metabolic control mechanism for calcium ion influx that may protect the ventricular myocardial cell, Am. J. Cardiol. 37, 1079. Stunkard, A., L. Wertheimer and W. Redisch, 1954, Studies on hydralazine: evidence for a peripheral site of action, L Clin. Invest. 33, 1047.
137
Elsevier EJP 00675
Mechanism of direct cardiostimulating actions of hydralazine Junichi Azuma
1,.,
A k i h i k o S a w a m u r a 1, H i s a t o H a r a d a 1, N o b u h i s a A w a t a S u s u m u K i s h i m o t o 1 a n d N i c k Sperelakis 2
1,
1 The Third Department of Internal Medicine, Osaka Unioersity Medical School, Osaka 553, Japan, and 2 Department of Physiology and Biophysics, University of Cincinnati College of Medicine, Cincinnati, OH 45267, U.S.A.
Received 10 November 1986, accepted 16 December 1986
The vasodilator, hydralazine, was reported to also exert a direct positive inotropic effect on the myocardium at high concentrations. In the present study we investigated the mechanism of this positive inotropic action by using the ventricular myocardium of isolated perfused chick hearts. Hydralazine (10-3 M) enhanced contractile force and heart rate, and elevated, the myocardial cyclic AMP level. To study the Ca 2+-dependent slow action potentials, the fast N + channels were voltage-inactivated with elevated K + (25 mM), resulting in a loss of electrical excitability. Hydralazine (10 -4 M) rapidly (< 3 rain) allowed the generation of slow action potentials and accompanying contractions by electrical stimulation. These effects of hydralazine were only partially prevented by propranolol. The results suggest that the increase of myocardial contractility produced by hydralazine is the result, at least in part, of a direct effect on the myocardium to increase Ca 2+ inflow. The increased Ca 2+ influx and inward slow current is due partly to activation of fl-adrenoceptors, with resultant elevation of cyclic AMP, and partly to another mechanism. Inotropic action (positive); Calcium slow channels; Cyclic AMP; Hydralazine; Histamine; Cardiac electrophysiology
1. Introduction Hydralazine has been used as an antihypertensive agent, and recently in the management of congestive heart failure. In addition to reducing arterial pressure by a direct vasodilator effect on the arterioles (.&blad, 1963; Stunkard et al., 1954), hydralazine substantially increases cardiac output and heart rate. These effects have been attributed to reflex activation of the cardiac sympathetic nerves secondary to the decrease in arterial pressure (Brunner et al., 1967). However, some studies in animal models (Brunner et al., 1967; Gershwin and Smith, 1967, Khatri et al., 1977; Songldttinguna and Rand, 1982; Rabinowitz et al., 1986) and in the falling human ventricle (Leier et al., * To whom all correspondence should be addressed: The Third Department of Internal Medicine, Osaka University Medical School, Fukushima 1-1-50, Osaka 553, Japan.
1980) indicate that hydralazine exerts a positive inotropic action directly on the myocardium. The purpose of the present study was to investigate the mechanism of this direct cardiostimulating action of hydralazine. The effect of hydralazine was examined on the contractions, electrophysiological properties and tissue cyclic A M P level of the ventricular myocardium in isolated perfused chick hearts.
2. Materials and methods Hearts were removed from 1 to 5 day old posthatched chicks, and a glass cannula was inserted into the aorta for perfusion of the coronary vessels. The perfusing solutions flowed from two reservoirs (located 60 cm above the heart) that contained either normal Tyrode solution (composition in mM: 137 NaC1, 2.7 KC1, 1.8 CaC12,
0014-2999/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
138
1.0 MgC12, 20 N a H C O 3, 1.0 NaH2PO 4, 5.5 glucose) or 25 mM K ÷ Tyrode solution (isosmolar substitution of K ÷ for Na÷). The solutions were gassed with a mixture of 95% O 2 and 5% CO 2 (pH 7.4), and heated to 37°C. Hydralazine was added in cumulative doses to the perfusing reservoirs to give final concentrations ranging between 10 -5 and 3 × 10 -3 M. A force displacement transducer was used to measure contractions by means of a thread sutured through the apex of the ventricle. The tension developed and its first time derivative ( d T / d t ) were recorded on a penwriter at a slow paper speed. The results with hydralazine were similar whether they were obtained from the tension developed (T) or d T / d t . The data were expressed as a percent change from the control value immediately prior to administration of hydralazine. For electrical stimulation, rectangular current pulses were delivered via platinum plate electrodes. Resting potentials and action potentials were recorded by intracellular micropipettes (filled with 3 M KC1) using the floating microelectrode technique. The maximum rate of rise ( + Vm~x) of the upstroke (phase 0) of the action potential (AP) was determined with a resistance-capacitance differentiator (time constants of 10 -5 and 10 -4 s) calibrated in V / s by means of a ramp generator. + ~rma~ gives a measure of the relative intensity of the inward current. For slow APs, the fast Na + channels were inactivated by partial depolarization produced by perfusion with 25 mM K ÷ Tyrode solution. This rendered the heart unexcitable, and caused mechanical failure despite intense electrical field stimulation. The K+-depolarized hearts were Stimulated at a rate of 50/min. Hydralazine was tested for its ability to induce slow APs and contractions. The stimulator was turned off when required to ascertain whether any slow APs would occur spontaneously in the presence of hydralazine. The d T / d t value for a heart whose excitability had been restored by hydralazine was expressed as a percent of the d T / d t value for the same heart (spontaneously beating) immediately prior to depolarization with 25 mM K ÷. The values for the parameters measured in contraction and electrophysiological experiments were taken after the
hearts achieved a steady state following each concentration of hydralazine (usually 10-15 min). In 3 cases, verapamil (2 × 10 -6 M), a blocker of Ca 2÷ slow channels, was added to test whether it would block the hydralazine-induced slow APs. It was tested in 3 experiments whether hydralazine would potentiate the isoprenaline-induced slow APs and contractions. To test the possibility that histamine release was involved in the positive inotropic action of hydralazine, the hsitamine receptor blocking agents, diphenhydramine (H 1 blocker) (10 -5 M) or cimetidine (H 2 blocker) (5 × 10 -5 M), were added to the perfusing solution. To test the possibility that the hydralazine action was mediated by the fl-adrenoceptors, the antagonist propranolol (10 - 6 M) or pindolol (10 -6 M) was added to the perfusing solution in some experiments. In 3 hearts whose myocardial noradrenaline had been depleted by prior reserpine treatment (1 m g / k g i.p., 24 h prior to the study), it was tested whether hydralazine could still induce slow APs. Noradrenaline levels were measured by gas chromatography-mass spectrometry (Higa et al., 1977): control heart, 0.35 + 0.04 n g / m g tissue ( N = 10); reserpine-treated heart, 0.05 +0.02 n g / m g tissue (N = 10) (P < 0.001). For cyclic AMP analysis, the hearts were perfused with normal Tyrode solution for 10 rain, and hydralazine (3 × 10 -3 M) was then added. After 5 min 20-30 mg of tissue was removed from the apex of the heart, blotted with a filter paper and immediately frozen with a stainless steel clamp precooled in liquid nitrogen. The freeze-clamped tissue was weighed and homogenized for 20 s in 2-3 ml of 6% perchloric acid at 4°C with a Polytron (PT 10-35) homogenizer. The homogenate was centrifuged at 3000 r.p.m, for 10 min at 4°C. The supematant was collected and adjusted to p H 3.0 (with KOH). These samples were stored in a freezer until assayed for cyclic AMP. The cyclic AMP content of the samples was determined in duplicate by radioimmunoassay (Honma et al., 1977) using YAMASA cyclic AMP assay kits (Yamasa Shoyu Co., Ltd.), and expressed as n m o l / m g wet weight of tissue. In some experiments, propranolol (10 -6 M) was added to the perfusing solution to ascertain whether it would
139 affect the cyclic AMP response of the heart to hydralazine or the basal cyclic AMP level (15 rain of exposure). The data were analyzed for statistical significance, depending on the design of the experiments, by: (a) Student's t-test (paired or unpaired), (b) analysis of variance (Scheffe's method was used to compare individual data when a significant F value was shown), or (c) x2-test. Differences were considered significant when the calculated P value was less than 0.05. Values are given as means + S.E.M.
o
o=
+20
O
dT I dt
T |
heart rate
I
2 0
.
.
.
. n=lO mean± S.E.M.
10.5
10.4
10.3
3 x 10.3
Hydralazine ( M )
3. Results 3.1. Contractility and heart rate
The effects of different concentrations of hydralazine on cohtractility (dT/dt) and heart rate associated with the fast action potentials (APs) (hearts perfused with normal Tyrode solution) are summarised graphically in fig. 1. The hearts contracted spontaneously at a rate of approximately 200 beats/min. The heart rate increased significantly with 3 x 10 -3 M hydralazine. Hydralazine (10-3-3 × 10-3 M) significantly increased the contractile force to a peak level of 112 _ 5% (P < 0.05) and 120 + 9% (P < 0.05), respectively, of the control magnitude within 1-2 rain. These positive chronotropic and inotropic actions persisted undiminished until termination of the experiment at 20 min. 3.2. Slow APs and contractions
In hearts in which the fast Na + channels were voltage-inactivated by partial depolarization in elevated K ÷ (25 mM) (fig. 2B), the addition of any agent which increases the density of the slow (Ca2÷-Na ÷) channels available for voltage activation resulted in the generation of slow APs accompanied by contractions. Exposure to hydralazine (10 -3 M) induced a slowly rising overshooting electrical response within 1 min, concomitant with contractions (fig. 2C, C'). The addition of verapamil (2 x 10 -6 M to 3 isolated hearts), a blocker of the transmembrane Ca 2+ slow current,
Fig. i . Graphic summary of the effect of several concentrations of hydralazine on contractility (dT/dt) and heart rate of chick (1-5 day old post-hatched) hearts associated with the fast action potentials (in 2.7 mM [K]0 ). Hydralazine significantly increased the contractile force and heart rate in a dose-dependent fashion. * P < 0.05 vs. control (zero hydralazine); d T / d t = first time derivative of developed tension.
abolished the hydralazine-induced slow APs and contractions (fig. 2D, D'). Hydralazine (10 -3 M) also potentiated the isoprenaline (10 -s M)-induced slow APs and contractions by about 50% (not illustrated). 3.3. Effect of fl-adrenoceptor blockade and histamine receptor blockade on the hydralazine-induced slow responses
Propranolol (10 -6 M) was used to test the possibility that the action of hydralazine involved fl-adrenoceptors. Propranolol did not significantly affect the control contractions (fig. 3). At 10 - 4 M, hydralazine failed to induce contraction (accompanying slow APs) in the presence of propranolol (fig. 3B). However, propranolol did not prevent the induction of contraction by 10- 3 and 3 x 10- 3 M hydralazine but the magnitude of the contractions (accompanying the slow APs) was lower. This suggests that the effect of hydralazine was mediated in part by catecholamine release from the nerve terminals. Concentration-response curves for the magnitude of the contractions (dT/dt) associated with the slow APs induced by hydralazine with and without propranolol (10 -6 M) are given in fig. 4.
140
normal Tyrode
Hydralazine
25 mM K+Tyrode
+
( 10-3M ) 3 rain
Verapamil
( 2xlO-6M )
o-
I,
!
I
10V/s
I
40mV
I
I
I
200 ms A' ~
B'
C'
O'
I1
I-
I
3 rain
Fig. 2. Hydralazine induction of slow action potentials and contractions. (A) Control fast action potential and mechanical recording (A') during perfusion with normal Tyrode solution in a spontaneously beating heart. Upper tracing is the first derivative (+ Vmax) of the action potential (arbitrarily shifted to the right). (B) Elevation of K + to 25 mM decreased the resting potential to about - 4 0 mV and abolished the action potentials and contractions (B') despite intense electrical stimulation. (C) Addition of hydralazine (10 -3 M) produced slowly rising action potentials and contractions (C'). (D) Verapamil (2 x10 -6 M) abolished the action potentials and contractions (D'). T = developed tension.
It c a n b e s e e n t h a t h y d r a l a z i n e p r o d u c e d a g r e a t e r a u g m e n t a t i o n of the c o n t r a c t i l e force i n the a b sence t h a n i n the p r e s e n c e of p r o p r a n o l o l . T h e effect of p r o p r a n o l o l o n the o c c u r r e n c e of the h y d r a l a z i n e - i n d u c e d s p o n t a n e o u s slow res p o n s e s was e x a m i n e d b y t u r n i n g off the electrical
25 mM K+ Tyrode
A
I 10-4M
s t i m u l a t i o n ( t a b l e 1). W i t h o u t p r o p r a n o l o l , 10 - 3 M h y d r a l a z i n e p r o d u c e d s p o n t a n e o u s slow res p o n s e s (25-60 b e a t s . r a i n - 1 ) i n 12 o u t o f 18 hearts, w h e r e a s it d i d n o t p r o d u c e slow r e s p o n s e s i n a n y of the 10 h e a r t s tested w i t h p r o p r a n o l o l (10 - 6 M ) ( P < 0.001).
Hydralazine 10-3M
3 x 10-3M
i
Without Propranolol ,
!
t ~ 5 rain
B
In presense of Propranolol ~ ]0-6M )
!
f
:
o
. . . . . . . . . . . . . . . . . . . .
.
Fig. 3. The effect of propranolol on the contractions (dT/dt = first time derivative of developed tension) accompanying the hydralazine-induced slow action potentials. (A) Without propranolol, hydralazine (10 -4, 10 -3 and 3 x l0 -3 M) induced contractions. (B) In the presence of propranolol (10 -6 M), the magnitude of the contractions (accompanying the slow action potentials) was lower, indicating that the effect of hydralazine was mediated, at least in part, by catecholamine release from the nerve terminals or by direct activation of the ~-adrenoceptors by hydralazine itself.
141 TABLE 1
TABLE 2
Effect of propranolol on the frequency of occurrence of hydralazine-induced spontaneous slow action potentials in 1-5 day old chick hearts. Frequency of occurrence (given in parentheses) was expressed as percent of the number of hearts examined, n.s. = not significant.
Effect of hydralazine on cyclic AMP level of isolated chick hearts perfused with 25 mM K + Tyrode solution with or without propranolol. Data are expressed as means+S.E.M. Number in parentheses represents the number of determinations. Cyclic A M P (nmol •g- 1 wet weight)
Hydralazine concentration (M) 10 - 4
without propranolol With propranolol (10-6M) Statistical significance
10-3
3XlO -3
0/12 (0%) 12/18 (66%) 12/12 (100%) 0/9 (0%) n.s.
0/10 (0%) P < 0.001
3/9
(33%)
Control 0.38 + 0.02 (21) Control with propranolol (10 -6 M) 0.36+0.03 (18) Hydralazine (3 × 10- 3 M) 0.58 ___0.04 (17) a.b Hydralazine with propranolol 0.46 __.0.03 (16) b.¢ Significant difference (P < 0.05) vs. control a, vs. control with propranolol b and vs. hydralazine e.
P < 0.001
It is well k n o w n that p r o p r a n o l o l in high c o n c e n t r a t i o n s has a non-specific local anesthetic action. W e therefore tested the effect o f p i n d o l o l (10 - 6 M), which is a l m o s t d e v o i d of local a n e s t h e t i c activity, in 3 cases. P i n d o l o l d i d n o t p r e v e n t the i n d u c t i o n of c o n t r a c t i o n s b y 10 -3 M h y d r a l a z i n e u n d e r electrical stimulation, b u t the m a g n i t u d e o f the c o n t r a c t i o n s ( a c c o m p a n y i n g the slow A P s ) was p a r t i a l l y d e c r e a s e d b y p i n d o l o l to 65 + 12% of the m a g n i t u d e w i t h o u t pindolol. I n i s o l a t e d h e a r t s f r o m chicks treated with reserpine, h y d r a l a z i n e (3 × 10 -3 M ) still i n d u c e d slow A P s a n d c o n t r a c t i o n s (not illustrated). D i p h e n h y d r a m i n e (10 - s M ; N = 2) a n d c i m e t i d i n e (5 × 10 - s M; N = 7), blockers o f H 1 a n d H 2 receptors, respectively d i d n o t affect the c o n t r a c t i o n s a c c o m p a n y i n g the h y d r a l a z i n e - i n d u c e d slow A P s (not illustrated). This suggests t h a t h i s t a m i n e release is n o t involved in the a c t i o n of h y d r a l a z i n e .
3.~ Cycfic AMP assays A total o f 72 chick h e a r t s were a n a l y z e d for cyclic A M P content. T h e effect of h y d r a l a z i n e (3 × 10 -3 M ) o n the tissue cyclic A M P level in h e a r t s p e r f u s e d either with or w i t h o u t p r o p r a n o l o l 10 - 6 M is s u m m a r i s e d in t a b l e 2. T h e cyclic A M P c o n t e n t in c o n t r o l hearts p e r f u s e d with n o r m a l T y r o d e s o l u t i o n for 15 m i n was 0.38 + 0.02 n m o l . g - 1 wet weight ( N = 21). P r o p r a n o l o l itself d i d
n o t significantly affect the tissue cyclic A M P level (0.36 + 0.03 n m o l . g - Z wet weight, N = 18). This suggests that the hearts were n o t subjected to a fl-adrenergic t o n e d u e to c a t e c h o l a m i n e s released f r o m intrinsic c a r d i a c nerve endings. H y d r a l a z i n e c a u s e d a significant elevation of cyclic A M P to 0.58 ___0.04 n m o l . g - x wet weight ( N = 17, P < 0.05 c o m p a r e d to c o n t r o l a n d p r o p r a n o l o l - t r e a t e d hearts). P r o p r a n o l o l significantly r e d u c e d the lay-
_
100
o
• Hydralazine
T
•
-6
0 Hydralazme + Propranolol ( 10 M )
~ ' ( 12 )
J
E
•-
9)
N
/
~
10-5
10-4
rnean+ S. LM. 10-3
3x10 -3
Hydralazine ( M ) Fig. 4. Graphic summary of the effect of propranolol (10 -6 M)
on the magnitude of contractions associated with the hydralazine-induced slow action potentials (in 25 mM [K]0). Elevation of hydralazine concentration produced an increase in contraction in a concentration-dependent fashion. Propranolol depressed the response to hydralazine. Each number in parentheses gives the number of experiments. * P < 0 . 0 5 v s . + propranolol for 10 -4, 10 -3 and 3×10 -3 M of hydralazine, respectively; dT/dt = first time derivative of developed tension.
142 dralazine-induced elevation of cyclic AMP to 0.46 + 0.03 nmol. g-1 wet weight (N = 16), but this value was still significantly higher than the value for the propranolol-treated hearts. This indicates that the elevation of cyclic AMP by hydralazine was only partially mediated via fl-adrenoceptor activation.
4. Discussion
The present results demonstrate that hydralazine at high concentrations elevates the intracellular cyclic AMP level in the heart. Part of this action is due to activation of the fl-adrenoceptt~rs. The hydralazine induction of Ca 2+-dependent slow action potentials (APs) in myocardial cells (in which elevated K + had abolished excitability), like that by catecholamines, histamine and methylxanthines, probably occurs because of its cyclic AMP-elevating action (Sperelakis and Schneider, 1976; Azuma et al., 1981a). This, in turn, would result in an increase in inward Ca 2+ current via the Ca 2+ slow channels during the cardiac AP. This mechanism could explain the positive inotropic action of hydralazine. Brunner et al. (1967) had proposed that the cardiac stimulation seen after administration of hydralazine to conscious animals might be the result of reflex adjustment to the fall in blood pressure. An increase in sympathetic activity also occurs after hydralazine administration in man (.~blad, 1963). Thus, the cardiac hyperactivity in the whole animal after hydralazine administration might result partly from increased sympathetic stimulation of the heart due to activation of the baroreceptor reflex by the vasodilatation-induced hypotension. In addition, hydralazine was also reported to exert positive inotropic effects directly on the heart itself by releasing noradrenaline from myocardial sympathetic nerve endings or by directly stimulating cardiac fl-adrenoceptors (Gershwin and Smith, 1967; Leier et al., 1980; Rabinowitz et al., 1986). Khatri et al. (1977) demonstrated, by means of direct injection of hydralazine into a coronary artery in dogs, that increased myocardial contractile force was elicited consistently in the area
perfused, and that this local effect of hydralazine was blocked by propranolol. They concluded that the observed increase in myocardial contractility was the result of direct fl-adrenoceptor stimulation of the myocardium. Koch-Weser (1974; 1971) showed that concentrations of hydralazine above 10 -4 M increased the development of tension in kitten ventricular myocardium. Since this positive inotropic effect was blocked by propranolol and was abolished by prior reserpine treatment, it was concluded that the positive inotropic effect was due to the release of noradrenaline from adrenergic nerve endings in the myocardium (Koch-Weser, 1974). Rabinowitz et al. (1986) have recently demonstrated that the positive inotropic action of hydralazine was mediated by the release of catecholamine and resultant activation of the adenylate cyclase system. However, Koch-Weser (1971) suggested that only concentrations of hydralazine far above the therapeutic range produce direct inotropic effects. In the present experiments on chick hearts, release of noradrenaline could not have been the sole mechanism because hydralazine retained some effects in reserpine-pretreated hearts whose noradrenaline content had been depleted. Similar results have been reported for rat atrial preparations (Songldttiguna and Rand, 1982). In addition, the effects of hydralazine, namely, induced slow APs with contractions and elevated cyclic AMP levels, were only partially inhibited by propranolol. Therefore, the increase in myocardial contractility after administration of hydralazine may have been the result of (a) direct fl-adrenoceptor stimulation produced by noradrenaline released from nerve endings, and (b) an increased intracellular cyclic AMP level. The latter could result from phosphodiesterase inhibition as demonstrated for beef heart (Ishii et al., 1979), or from adenylate cyclase stimulation (Rabinowitz, 1986). Gershwin and Smith (1967) observed that hydralazine released histamine from isolated guineapig atria, and that histamine, in turn, released catecholamines. Propranolol blocked the action of hydralazine, as did antihistamines. However, histamine itself is a known positive inotropic agent (Barlet, 1963) which increases cyclic AMP by stimulation of adenylate cyclase (McNeil and
143 M u s c h e k , 1972). T h e positive i n o t r o p i c action of h i s t a m i n e was s h o w n to b e m e d i a t e d b y a c t i v a t i o n of the H 2 receptor, resulting in an increased availa b i l i t y of C a 2+ slow channels ( J o s e p h s o n et al., 1976). However, in the p r e s e n t experiments, h i s t a m i n e - r e c e p t o r b l o c k i n g agents failed to b l o c k the effects o f hydralazine. H e n c e h i s t a m i n e release is p r o b a b l y n o t r e s p o n s i b l e for the positive ino t r o p i c effect of h y d r a l a z i n e . W e showed that h y d r a l a z i n e increased the h e a r t r a t e in hearts p e r f u s e d with n o r m a l T y r o d e solution. Positive c h r o n o t r o p i c agents, such as fla d r e n o c e p t o r agonists, e n h a n c e the rate of pacem a k e r discharge b y s t i m u l a t i n g the p r o d u c t i o n of cyclic A M P b y activating a d e n y l a t e cyclase. A d r e n a l i n e causes a large increase in i n w a r d C a 2+ c u r r e n t ( a n d a small increase in o u t w a r d K + c u r r e n t ) in the S A n o d e (Brown et al., 1979). Since the i n w a r d d e p o l a r i z i n g p a c e m a k e r current in sinus n o d e cells is p a r t l y carried t h r o u g h slow channels ( N o m a a n d Irisawa, 1976), the increased n u m b e r of a c t i v a t e d slow channels in the presence of h y d r a l a z i n e should result in greater a u t o m a t i c i t y . C o n s i s t e n t with this, the s p o n t a n e o u s c o n t r a c t i o n s t h a t a p p e a r e d after h y d r a l a z i n e p e r f u s i o n ( a b o v e 10 - 4 M) were p a r t i a l l y b l o c k e d with p r o p r a n o l o l . Balazs et al. (1981) r e p o r t e d that h y d r a l a z i n e causes m y o c a r d i a l necrosis in rats, which was prev e n t e d b y p r e t r e a t m e n t with p r o p r a n o l o l o r v e r a p a m i l , a n d they suggested that i n c r e a s e d intracellular c a l c i u m p l a y s a role in the p a t h o g e n e s i s o f the necrosis. Thus, o u r p r e s e n t findings that h y d r a l a z i n e increased C a 2 ÷ inflow via slow channels, a n d t h a t this was b l o c k e d b y v e r a p a m i l , m i g h t e x p l a i n the m e c h a n i s m of h y d r a l a z i n e - i n d u c e d m y o c a r d i a l lesions o b s e r v e d b y Balazs et al. Similarly, the e n h a n c e d C a 2÷ inflow c o u l d be a factor in the c a l c i u m o v e r l o a d associated with a d r i a m y c i n - i n d u c e d c a r d i o m y o p a t h y ( A z u m a et al., 1981b). T h e p r e s e n t results suggest that h y d r a l a z i n e has direct c a r d i o s t i m u l a t i n g effects p r o d u c e d b y an i n c r e a s e d C a 2÷ inflow t h r o u g h the C a 2÷ slow channels.
Acknowledgements This work was supported in part by Grant HL-31942 from the National Institutes of Health. We thank Dr. Sadayoshi
Higa for determining noradrenaline, and Miss Y. Ikeda and Y. Ueki for their assistance in preparing the manuscript. We are grateful to Ciba-Geigy (Japan) Limited for a generous supply of hydralazine.
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