Effects of [Na+] and [Ca2+] on the responses to milrinone in rat cardiac preparations

European Journal of Pharmacology, 120 (1986) 267-274 Elsevier

EFFECTS OF [Na +1 A N D PREPARATIONS i

ICa2+l ON

267

T H E R E S P O N S E S T O M 1 L R I N O N E IN RAT CARDIAC

RAMESH K. GOYAL 2 and JOHN H. MCNE1LL 3 Division of Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, The University of British Columbia, 2146 East Mall, Vancouver, B.C., Canada V6T IW5 Received 15 October 1985, accepted 29 October 1985

R.K. GOYAL and J.H. MCNEILL, Effects of [Na +1 and [Ca 2+] on the responses of rnilrinone in rat cardiac preparations, European J. Pharmacol. 120 (1986) 267-274. In the present investigation we have studied the influence of changing the [Ca 2÷] and [Na ÷] on the cardiac responses to milrinone in various preparations of rat heart. Milrinone (5 x 10 5 to 8 x 10 -4 M) produced a dose-dependent positive chronotropic effect on right atrium and a positive inotropic effect on left atrium and papillary muscle of the rat. A decrease in [Ca2+] (from 2.2 to 1.1 mM) or an increase in [Na +] (from 120 to 60 mM) increased the milrinone-induced inotropic effect in left atrium and papillary muscle. However, in right atrium the chronotropic effect of milrinone was significantly decreased under these conditions. Opposite changes to milrinone-induced responses were observed when [Ca 2÷] was increased (to 3.3 mM) or when the [Na +] was decreased to 60 mM. Nifedipine (3 x 10 3 M), a selective Ca 2÷ channel blocker, significantly inhibited the chronotropic response to milrinone in right atrium. However, the inotropic response to milrinone was found to be significantly greater in the presence of nifedipine. A veratridine-induced positive inotropic effect in the left atrium was also significantly increased in the presence of nifedipine. Tetrodotoxin (TTX, 1 x 10 -6 M), a fast sodium channel blocker, significantly reduced the inotropic response to milrinone in left atrium and papillary muscle. A milrinone-induced dose-dependent increase in the baseline tension was observed in the right atrium which was abolished in low [Ca 2+ ] and significantly increased in high [Ca2+]. Our data suggest the possibility that milrinone increases Ca 2÷ influx in the right atrium to cause the chronotropic effect. Milrinone also may possess an action like veratridine, involving an increased influx of Na ÷ through fast Na ÷ channels in left atrium and papillary muscle, and this action is possibly involved in the positive inotropic effect. Rat atria and papillary muscle Na +-Ca2+ exchange

Milrinone

Fast Na + channel

I. Introduction Milrinone, [1,6,dihydro-2-methyhl-6 oxo(3,4' bip y r i d i n e ) 5-carbonitrile], is a b i p y r i d i n e c o m p o u n d which possesses significant positive i n o t r o p i c and c o r o n a r y v a s o d i l a t o r p r o p e r t i e s (Alousi et al., 1983a). The sustained h a e m o d y n a m i c and clinical effects, as studied b y Bairn et al. (1983) and M a s k i n et al. (1983), reveal that the drug is well tolerated 1 Supported by a grant from the B.C. Heart Foundation. 2 Post-doctoral Fellow of the B.C. Heart Foundation. 3 To whom all correspondence should be addressed. 0014-2999/86/$03.50 © 1986 Elsevier Science Publishers B.V.

Nifedipine

Tetrodotoxin

Calcium

a n d is devoid of certain side effects seen with a m r i n o n e , such as t h r o m b o c y t o p e n i a , gastro-intestinal d i s t u r b a n c e s and aggravation of ventricular ectopy. Studies on the m e c h a n i s m of the c a r d i o t o n i c activity of milrinone on e x p e r i m e n t a l a n i m a l s reveal that it does not involve an action on a u t o n o m i c receptors, release of e n d o g e n o u s catecholamines, h i s t a m i n e or p r o s t a g l a n d i n s a n d does not affect N a + K + A T P a s e (Alousi et al., 1983a). Milrinone, like a m r i n o n e , inhibits c a r d i a c a d e n o s i n e 3 ' , 5 ' - m o n o p h o s p h a t e ( c A M P ) phosphodiesterase, with a resultant increase in c a r d i a c c A M P levels (Alousi et al., 1983a). However, the

26~

time course for this increase does not seem to correspond to the increase in muscle developed tension and therefore it is unlikely to be responsible for the initiation of the inotropic response (Alousi et al., 1983b). Thus the mechanism of action of milrinone is still not clear. It is generally accepted that the ionic environment of the cell profoundly affects the cellular responses of the tissue. For example, the presence of Na + in the extracellular medium is necessary for the maintenance of normal function in a variety of excitable tissues, including heart (Fozzard, 1977). Existence of the Na*-Ca e~ exchange system, and the competitive interactions of sodium and calcium in cardiac membranes have also been documented (Miller and Moisescu, 1976; Benninger et al., 1976; Reeves and Sutko, 1983). The present investigation was undertaken to study the influence of various ions on milrinone-induced cardiac effects on various preparations of rat heart so as to elucidate its mechanism of action. 2. Materials and methods

Young albino female rats of Wistar strain, weighing 165-185 g were killed by a sharp blow to the head and decapitation. Hearts were quickly excised and right atria, left atria and left papillary muscles were dissected out. They were mounted in 15 ml organ baths containing Chenoweth-Koelle (CK) buffer (pH 7.4), which was maintained at 37°C and constantly bubbled with carbogen (95% O~ + 5% C02). The composition of CK was (in raM): NaC1 120, KCI 5.6, CaCI, 2.2, MgC12 2.1, glucose 10.0, NaHCO~ 19.2, EDTA 0.03. Right atria were allowed to beat spontaneously whereas left atria and papillary muscles were stimulated with 5 ms square-wave impulses at 2 x threshold voltage, at 1 Hz using a Grass stimulator, model 5D-9. The responses to various drugs were recorded using a Grass force displacement transducer (model FT.03) which was coupled to a Grass 79D polygraph. All the preparations were stabilized for 1 h in normal CK with a resting tension of 1.0 g. In the first series of experiments graded doses of milrinone ( 5 × 1 0 5 to 8 × 1 0 4 M) were added in a cumulative manner with a time interval of

5 rain between two successive doses. After taking a control dose-response curve of milrinone, preparations were kept in the CK containing low ('a :" (1.1 mM CaC12) or high Ca 2 t (3.3 mM CaC12) for 15 rain and the responses to milrinone were recorded again. The experiments were also done using low Na t (60 mM NaC1) or high Na ~ (240 mM NaCI). Isosmolarity in the low Na ~ solution was maintained using sucrose (79.0 raM), while the high Na + solution was hypertonic and for which control experiments were done using a hypertonic solution which contained an equimolar concentration (156 raM) of sucrose. Solutions VII and VIII contained different [Ca :* ] as well as [Nat]. In the second series of experiments, after taking the control dose-dependent responses to milrinone ( 5 × 1 0 5 to 8 × 1 0 4 M), tetrodotoxin (TTX, 10 ~' M) or nifedipine (3 × 10 s M) was added to the bath and after 20 rain the responses to milrinone were again recorded. In yet another series of experiments the responses to veratridine (5 x 10 7 to 5 × 10 ~' M) were measured in the presence and absence of nifedipine (3 × 10 ~ M). The data have been analysed to assess statistical significance using one-way analysis of variance followed by the Neuman-Keul's test. In cases where only two groups were compared, the paired Student's t-test has been used. The level of significance was set at P < 0.05. Milrinone was generously provided by SterlingWinthrop Research Institute (U.S.A.). It was dissolved in 0.1 N HC1 and the pH was adjusted to 4.5 using 0.1 N NaOH. The working solution was diluted in CK buffer. Nifedipine was generously provided by Miles Laboratories (Canada) and was dissolved in dimethylsulfoxide. Tetrodotoxin was dissolved in citrate buffer (pH 4.8) and veratridine was dissolved in ether.

3. Results

3.1. Right atrium Milrinone produced a dose-dependent increase in the rate of spontaneously beating right atrium (fig. la). Decreasing the [Ca 2+] in the bathing fluid

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significantly increased the basal-rate (table 1), however the responses to milrinone were decreased significantly. Increasing the [Ca 2+] did not significantly alter the basal-rate (table 1), but the

milrinone-induced positive chronotropic effect was increased. This increase was significantly different from the responses obtained in the low [Ca -~+] buffer (fig. la).

TABLE 1 Basal-tension and rate in different solutions. Solution

Right atrium rate (beats/min)

Left atrium tension (g)

Left papillary muscle tension (g)

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270

3.2. Le[t atrium

Decreasing the [Na ~] in the buffer significantly reduced tile basal-rate (table 1 ). Milrinone-induced responses were found to be significantly greater in low [Na ~ ] as compared to control and high [Na ~] buffer (fig. lb). The responses to milrinone were also significantly decreased in the hypertonic solution. The basal-rate was not decreased (table 1) when [ C a : ' ] was increased in the buffer with low Na ~. The responses to milrinone were still found to be significantly greater in the preparations bathed in this medium. The responses to milrinone were significantly decreased in solutions with low [Ca -~' ] and high [Na~]. N i f e d i p i n e ( 3 × 1 0 ~ M) significantly decreased the milrinone-induced positive chronotropic response in right atrium (fig. la). Milrinone also produced a dose-dependent increase in baseline tension in right atrium (fig. 2). This was enhanced when the preparation was bathed in a high [Ca =~ ] solution, whereas it was completely abolished in a low [Ca :~ ] solution (fig. 2a). A change in the [Na ~ ] did not significantly alter the milrinone-induced change in baseline tension (fig. 2b).

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Fig. 2. The effect of milrinone on the baseline tension of milrinone in the right atrium of rat in CK buffer with different [Na* ] and [Ca2+ I. Each point represents the mean and the bar indicates S.E.M, of 5-12 experiments, * Denotes significantly different from control; § depicts significantly different from the other group (P < 0.05).

271

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the [Ca 2+] was increased. Tetrodotoxin (10 6 M), a fast N a + channel blocker antagonized the response to milrinone in the left atrium (fig. 4b). Nifedipine (3 × 10 s M) significantly decreased

the basal-tension of left atrium (table 1). However, the responses to milrinone were significantly potentiated in the presence of nifedipine (fig. 4b). Veratridine (fig. 4a) also produced a dose-depen-

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272 dent inotropic effect on left atrium and, like that of milrinone, this effect was significantly potentiated in the presence of nifedipine.

3.3. Left papillary muscle As in the left atrium, milrinone produced a dose-dependent inotropic response in the left papillary muscle. The left papillary muscle was found to be more sensitive to alterations in ionic concentrations. The basal-tension in left papillary muscle was drastically reduced when [Ca -'+ ] was lowered or [Na 4 ] was increased (table 1). The milrinone-induced inotropic effect was significantly increased in low [Ca 2+] or in the presence of nifedipine. An increase in [Ca `,+ ] slightly decreased the responses to milrinone. A decrease in [Na ~] completely abolished the positive inotropic response to milrinone in left papillary muscle. In solution VIII, in which [Na +] was increased and [Ca 2 +] was decreased, the responses were found to be significantly increased. Tetrodotoxin (10 ~' M) also inhibited the responses to milrinone in this preparations.

4. Discussion The data from the present investigation provide evidence for the involvement of [Ca -'+ ] and [Na + ] in the chronotropic and intropic effects of milrinone. The chronotropic response to milrinone in right atrium was increased when [Ca -'+ ] was increased and the decrease in [Ca 2+ ] decreased this effect (fig. la). These results indicate that the mechanism of chronotropic action of milrinone is due to an increased availability of Ca 2~ from extracellular sites. Further, nifedipine, a selective Ca 2+ channel blocker significantly inhibited the chronotropic response to milrinone in right atrium (fig. la). A decrease in [Na +] increased the response to milrinone in right atrium while an increase in [Na ~] inhibited the chronotropic effect in the same preparation (fig. lb). The decrease in the milrinone-induced chronotropic effect seen in low [Na~] does not seem to be due to a change in osmolarity since there was an equimolar replace-

me,at of Na ' with sucrose. The decreased milrinone chronotropic response in high [ N a ' ] may be attributed to the increase in osmolarity of the solution since in hypertonic solution the chronotropic effect of milrinone was decreased to a similar extent. The change in basal-rate may have an influence on the effect of various drugs (Simpson and McNeill, 1980). Since the basal-rate of right atrium was significantly decreased in low [Na +] (table 1), it might be expected that the increase in the milrinone-induced response in low [Na +] could be at least partly due to the lowering of the basal-rate. To rule out this possibility [Ca 2 ~ ] was increased in a solution containing low [Na- ]. While the basal-rate was not altered significantly in this medium (table 1), the milrinone-induced chronotropic effect was still enhanced. In a relatively similar situation Simpson et al. (1981) reported that hypothyroidism alters the cardiac responsiveness to adrenergic amines and these effects did not seem to be entirely related to the reduced basalrate. Thus the alteration to the milrinone-induced chronotropic effect seen in low [Na +] appears to be due to the effect of Na* ion. The results of the above studies on the milrinone-induced chronotropic effect in right atrium also indicate the existence of the Na ~-Ca-~ exchange system in this preparation. The existence of a transport system that mediates the movement of both Na+ and Ca 2 + across the cell membrane was first postulated in an effort to explain the antagonistic actions of extracellular Na ~ and Ca 2 on the force generated by the contraction of cardiac muscles (Liattgau and Niedergerke, 1958). Similar results were observed by other workers (Wilbrandt and Koller, 1948; Chapman and Tunstall, 1971: Miller and Moisescu, 1976). Evidence was later presented which demonstrated that the presence of Na t inhibited 45Ca uptake by cardiac tissues (Langer, 1964). Studies on the Na + dependence of 45Ca fluxes in guinea-pig atria (Jundt et al., 1975) and squid giant axons (Baker et al., 1969; Blaustein and Russell, 1975) provided evidence for the existence of a Na-Ca exchange system and the competitive nature of Na ~ and Ca 2 + for this transport system. The opposite effects of [Na +] and [Ca 2~ ] on the milrinone-induced chronotropic effects could be explained on the basis of a competi-

273 rive interaction between Na + and Ca 2+ in the Na-Ca exchange system. It is possible that milrinone increases intracellular [Ca 2+] by activating the Na-Ca exchange system. Low [Na +] or high [Ca R+] facilitates the effect while high [Na +] or low [Ca 2+] has the opposite effect. Milrinone was also found to increase the end-diastolic tension in right atrium. This was completely abolished in low [Ca R+ ] and enhanced in high [Ca 2+ ] (fig. 2). These results further support the hypothesis that the milrinone-induced increase in the chronotropic effect could be due to its ability to increase movement of extracellular Ca 2+ ions and the activation of Na-Ca exchange. The positive inotropic effect of milrinone in left atrium and papillary muscle appears to be mediated through a mechanism different from that of right atrium. The end-diastolic tension of left atrium and papillary muscle was not altered by milrinone. The inotropic response to milrinone was increased when [Ca 2+ ] was decreased, whereas a decrease in the response was observed when [Ca 2+] was increased (fig. 3a). These results indicate that milrinone does not increase the influx of extracellular [Ca 2+] in these preparations and that Ca 2+, in fact, opposes the milrinone-induced inotropic effect in these preparations. The inotropic effect of milrinone was significantly potentiated when [Na +] was increased, whereas it was abolished when [Na +] was decreased to 50% in both left atrium and papillary muscle. The increase in the inotropic response in high [Na +] could not be due to a change in osmolarity or basal-tension because in the identical situation, when the osmolarity was increased by sucrose, the basal-tension (table 1) as well as the milrinone-induced responses (fig. 3b) were decreased significantly. Thus it appears that milrinone needs Na + for the inotropic action in these preparations. The inotropic responses to milrinone were still increased in high [Na +] when [Ca 2+] was lowered and were abolished in low [Na +] even when [Ca 2+] was increased. Further, the observation that tetrodotoxin, a fast Na + channel blocker (Narahashi, 1974) antagonized the milrinone-induced positive inotropic effect in left atrium (fig. 4b) and papillary muscle suggests the possibility of an action of milrinone similar to that

of veratridine. The veratrum alkaloids are reported to exert a positive inotropic action and produce arrhythmias (Krayer et al., 1953; Benforado, 1957). Horackova and Vassort (1974) found that veratrine failed to produce an inotropic response in frog hearts perfused with Na-free Ringer solution. They also found that the positive inotropic action to veratrine was suppressed by tetrodotoxin. These workers and others suggested that veratrine possibly acts by increasing Na + influx (Horackova and Vassort, 1973). It has been reported that the positive inotropic action of veratrine is dependent on an intracellular store of Ca 2+ (Horackova and Vassort, 1973). Increased intracellular [Na + ] causes an increase in the intracellular Ca R+, either by releasing Ca 2+ from the intracellular sites or via the Na-Ca exchange system at the surface membrane similar to that observed with squid axon membrane (Baker et al., 1969). It also appears that there is competition between Na + and Ca 2+. Such an interaction between Na + and Ca 2+ has also been reported by various other workers (Blaustein, 1977; Philipson and Nishimoto, 1980; Reeves and Sutko, 1979; 1980; 1983). This competitive interaction can also explain the decrease in response to high doses of milrinone seen at higher calcium levels in left atrium (fig. 3). Competition between Na + and Ca ~-+ for entry into the cell could result in a reduced inotropic effect. The inotropic response to milrinone and veratridine was significantly increased in the presence of nifedipine (fig. 4). This effect could partly be attributed to a significant decrease in basal tension seen in the presence of nifedipine (table 1). However, it is also possible that in the presence of nifedipine, the influx of extracellular Ca 2+ is reduced and there is a concomitent increase in intracellular Na +. This intracellular Na + causes an increased release of Ca 2+ from the intracellular sites as described before for veratridine and may be responsible for increased inotropic effect. In conclusion, the mechanism of positive inotropic action of milrinone appears to be similar to that of veratridine in that both these drugs cause an increased influx of Na +, possibly through fast N a + channels, resulting in an increased intracellular [Na + ]. The increased intracellular [Na + ] produces a positive inotropic action probably

274

through increasing intracellular Ca-"+. The mechanism of the chronotropic action of milrinone in right atrium appears to be mediated through an increased influx of extracellular Ca 2 ' through the voltage-sensitive Ca channels.

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