Gen. Pharmac. Vol. 26, No. 3, pp. 589-592, 1995 Copyright !fl 1995ElsevierScienceLtd Printed in Great Britain.All rights reserved 0306-3623/95$9.50+ 0.00
Diazoxide Blocks the Morphine Induced Lengthening of Action Potential Duration on Guinea-pig Papillary Muscle S. A L A R C O N , J. H E R N A N D E Z * a n d M. L. L A O R D E N Department of Physiology and Pharmacology, School of Medicine, Murcia, Spain (Received 19 July 1994)
Almtraet--I. Intracellular microelectrodes were used to evaluate the possible involvement of potassium currents in the action potential prolongation induced by morphine. To this purpose we investigated the electrophysiologicaleffect of morphine on the isolated guinea pig right ventricular papillary muscle in the presence of the potassium channel opener and inhibitor diazoxide and glibenclamide respectively. 2. Diazoxide (1 #M), which is devoid of effect on its own, blocks the lengthening of action potential duration (APD) induced by morphine (5 mM). 3. However, in the presence of glibenclamide (1/IM), morphine (5 mM) prolonged APD in approximately the same proportion as that observed when used alone. 4. These results suggest that diazoxide but not glibenclamide sensitive potassium channels could mediate the APD prolongation induced by morphine. Key WOrds: Electrophysiology, papillary muscle, morphine, diazooxide, glibenclamide
MATERIALS AND METHODS
Previous work (Alarc6n et al., 1992) showed that morphine induces a decrease in the action potential amplitude and a prolongation of the action potential duration. The reduction of the action potential amplitude has been studied elsewhere (Alarc6n et al., 1993). However, the mechanism responsible for the action potential prolongation still remains uncertain. The present study was aimed to evaluate the ionic basis for the action potential prolongation induced by morphine. Since action potential duration is largely dependent on potassium currents (for review see Noble, 1984), we investigated the electrophysiological effect of morphine on the isolated guinea-pig fight ventricular papillary muscle in the presence of the potassium channels opener and inhibitor diazoxide and glibenclamide, respectively (Cook, 1988).
Experiments were performed on guinea-pig fight ventricular papillary muscles. Guinea-pigs, 350-400 g, were stunned by a blow on the head and immediately decapitated. The chest was opened with a midsternal incision and the hearts were rapidly excised. Papillary muscles, 2-3 mm in length and less than 1 mm dia., were isolated from the right ventricle. The muscles were pinned to the silastic base of a recording chamber and superfused continuously at a constant rate of 6 ml min ~with Tyrode solution equilibrated with 95% 02-5% CO2 at a temperature of 37°C (pH 7.4). The composition of the Tyrode solution was (mM): NaCI 136.9, KCI 5.0, MgCl, 1.05, NaH2PO 4 0.4, NaHCO~ 11.9, CaCI 2 1.8 and glucose 5.0. Rectangular pulses, 2 ms in duration and twice threshold voltage, delivered through a bipolar silver electrode connected a multipurpose programmable stimulator (Cibertec CS-20) were used to stimulate the preparation at a rate of I Hz. Transmembrane action potentials were recorded using conventional microelectrode techniques. The variables measured
*To whom all correspondence should be addressed.
S. Alarc6n et al.
-100 mV..J- . . . . .
100 ms i
Control ( ) Morphine 5raM (------)
Control ( ) Morphine 5raM Oiozoxide 1}JM (----) *
Fig. 1. Effects of morphine, in the absence (A) and in the presence (B) of diazoxide (0.1/~M) on the action potential configuration of guinea-pig papillary muscle.
were resting membrane potential (RMP), action potential amplitude (APA), duration at 50, 75 and 90% of repolarization (APD50, APD75 and APD90, respectively). The maximal rate of depolarization (Vmax) of the action potential was obtained by electronic differentiation. The differentiator used had an upper limit of linearity of 1000Vs-I and possesses variable imput filters (I-30kHz). Both action potential and Vm~xwere displayed on a storage oscilloscope (Tektronix 2230) and the oscilloscope traces were recorded on a plotter (Hewlett-Packard 7457 A). In all cases, the preparations were allowed to equilibrate for 1 h before exposure to the drugs. To observe drug effects on preparations, 6-10 action potentials were recorded before and 30rain after addition of mophine (alone or morphine) plus either diazoxide (10 7 M) or glibenclamide (10 -6 M) added to the organ bath 30 min before morphine. The drugs were dissolved in reservoirs of gassed Tyrode to obtain the final bath concentrations. Drugs
The drugs used in this study were: morphine hydrochloride (Alcaliber Laboratories, Spain), Diazoxide (Sigma Chem. Co., Spain) and glibenclamide (Sigma Chem. Co., Spain). Statistics
Data from the electrophysiological studies are expressed as mean values + SEM. The data were analysed by analysis of variance and Student's t-test. Differences of P < 0.05 were considered significant.
RESULTS Figure 1A shows the effect of morphine (5 mM) on the action potential configuration recorded from a papillary muscle of guinea-pig driven at 1 Hz. This concentration was chosen because, as previous work showed its efficacy to increase APD in this preparation (Alarc6n et al., 1992). This effect was reproduced in the present work. Diazoxide was used to determine whether or not this agent modified the effect of morphine. Diazoxide (0.1/~M) was devoid of effect on the action potential recorded from the guinea-pig papillary muscle. However, this concentration of diazoxide blocks the lengthening of
[ ] CONTROL [ ] MORPHINE
[ ] MORPHINE+ DIAZOXlDE
Fig. 2. Effects of morphine alone and in the presence of diazoxide (0.1 #M) on the action potential duration (APD) of guinea-pig ventricular papillary muscle. The increase in APD was measured at 50, 75 and 90% repolarization. *P <0.05 when compared with control or morphine + diazoxide. No statistical difference was obtained between control and morphine + Diazoxide (n = 3-4).
Interaction of morphine and diazoxide on cardiac electrophysiology the action potential duration induced by morphine (Fig. I B). The effect of morphine (5 mM) in the absence and in the presence of diazoxide on the action potential characteristics are summarized in Fig. 2. It is clear that this concentration of morphine increased APD at all percentages of repolarization measured. APD50 by 25.73 + 4.3 msec (P < 0.001), APD75 by 35.21 +3.8 ( P < 0 . 0 0 1 ) and APD90 by 55.45 _+ 5.7 (P < 0.001). Nevertheless, in the presence of diazo×ide (0.1 #M), the morphine induced changes in the APD are devoid of any statistical significance. To ascertain whether or not the electrophysiological effect of morphine were affected by the potassium channel blocker glybenclamide, we tested the effect of morphine (5 mM) in the presence of glybenclamide (1 pM). In these conditions morphine prolonged action potential duration in approximately the same proportion as that observed when used alone. These results suggest that glybenclamide does not potentiate the effect of morphine. DISCUSSION The present results agrees with those previously reported indicating that morphine, at high concentrations (5 mM) prolongs the action potential duration (Alarc6n et al., 1992). This effect is antagonized by the potassium channels opener diazoxide. The prolongation of action potential duration might result from a decreasing of the outward K + currents (Noble, 1984). Previous evidence has shown that opioids could modulate the potassium channels function. In fact, the antinociceptive effect of morphine is antagonized by the potassium channel antagonist glybenclamide (Ocafia et al., 1990). In our results the prolongation of APD induced by morphine (5 mM) is antagonized by the potassium channel opener diazoxide. However it is not affected by the potassium channel blocker glybenclamide. This could be related to different subclasses of ATP sensitive K + channels. In fact, available data for K-ATP channels indicate a diversity of properties in terms of conductance and ATP sensitivity (Ashcroft, 1988). Radio-ligand binding evidence for low affinity sulfonylurea binding sites with different effectiveness K + channel activators and sulfonylureas has been reported (Gopalakrishnan et al., 1991). The morphine induced APD prolongation seems to be mediated by opioid receptors since it is partially blocked by Naloxone (0.1 #M) (Alarc6n et al., 1992). Opioid receptors of the kappa type do not seem to be involved in this effect. Indeed, the selective kappa
agonist U-50,488H shortens, rather than prolongs, the action potential duration, being this effect partially blocked by the kappa antagonist MR-2266 (Alarc6n et al., 1993). On the other hand, only the 6-opioid antagonist 16-methylcyprenorphine, but not the ~ and /~ opioid antagonist, nor-binaltorphimine and naltrexone respectively, shorten the APD (McIntosh et al., 1992). This indicates a possible relation between APD prolongation and 6-opioid receptors. However, the specific type of opioid receptor responsible for this lengthening still remains to be established. Prolongation of APD by interfering with K + channels has some antiarrhythmic potential. In fact, drugs such as Sotalol (Carmeliet, 1985) or Amiodarone (Mason, 1987) are thought to elicit their antiarrhythmic effects by such a mechanism. It has been suggested that conduction inhomogeneities resulting from the opening of ATP-dependent potassium channel probably underlie the occurrence of ventricular fibrillation and subsequent mortality within the first hour after myocardial infarction (Cook, 1988). Antiarrhythmic drugs blocking the Na + channel are generally unable to suppress this arrythmia which is more consistently suppressed by potassium channel blocking drugs (Cook, 1988). Long term studies in patients suffering from ischaemic cardiac disease has also failed to show any reduction of mortality during treatment with class 1 antiarrhythmic agents (CAST, 1989; Mason, 1993). However, this effect have been obtained when these patients were treated with class 3 antiarrhythmic agents such as Amiodarone (Mademanee et al., 1993) or Sotalol (Mason, 1993). It is evident that we, at present, cannot propose morphine for such indication because the APD prolongation is only reached at concentrations far exceeding those considered therapeutic levels. However, developing of new opiates with more potency and selectivity for this effect could have a potential therapeutic role in protecting myocardium of electrical consequences of ischaemia. Acknowledgement--This work was supported by DGYCIT
(PM 91/0149) of Spain.
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