1 Beating of aggregates of embryonic chick myocytes, in primary culture, was quantified by use of a motion-detector and video-recorder technique. Interactions of palmitoyl carnitine, a putative endogenous ligand at Ca2 + channels, with calcium antagonists were investigated. 2 Bay K 8644 (1-100nM) and palmitoyl carnitine (0.2-30 M) increased edge movement of the aggregates; beats fused so that there was an increase in baseline 'tone'. The concentrations required to produce a 50% increase in edge movement were 2.5 nm for Bay K 8644 and 2Mm for palmitoyl carnitine. Higher concentrations (20-30pM) of palmitoyl carnitine caused tachycardia of abrupt onset but resulted in cessation of beating. The effects of palmitoyl carnitine were not stereo-selective in that the (+)-and (-)-isomers were equieffective. Lysophosphatidyl choline (LPC) had no effect in concentrations up to 1OMm but higher concentrations caused tachycardia followed by cessation of beating. High concentrations of both palmitoyl carnitine and LPC (100pM) caused break-up of the aggregates, presumably as a result of detergent effects.3 Palmitoyl carnitine (1-100pM) reversed the inhibitory effects of nisoldipine (0.3/jiM), diltiazem (10Mm) and verapamil (1 gM). Ouabain was ineffective in reversing the effects of nisoldipine, differentiating the effects of palmitoyl carnitine from those of Na+/K+ATPase inhibition. In contrast, palmitoyl carnitine did not reverse the inhibitory effects of pimozide (2 Mm) or lidoflazine (7jiM); palmitoyl carnitine showed a similar profile to Bay K 8644 in this respect. 4 These findings indicate that the effects of palmitoyl carnitine closely resemble those of Bay K 8644 and can be differentiated from those of lysophospholipids. As palmitoyl carnitine accumulates in the sarcolemma during myocardial ischaemia, the mode of action in the Ca2 + channel may have clinical relevance for the use of calcium antagonists in ischaemia.

1 Primary aggregate cultures of embryonic chick heart have been used to investigate the effects of calcium channel antagonists and facilitators on myocardial contractility. 2 The number of aggregates showing movement was inhibited in a concentration-dependent manner by calcium antagonists from different subgroups with negative log concentrations inhibiting movement in 50% of aggregates as follows: Class 1-nisoldipine (7.20); Class 2-verapamil (6.36), diltiazem (5.83); Class 3-lidoflazine (5.68), pimozide (6.25). 3 The effects of the dihydropyridine facilitators Bay K 8644 and CGP 28392 on aggregate beating were investigated by evaluating the interaction between calcium channel facilitators and antagonists from the three subgroups of calcium antagonists. Concentrations of antagonists that inhibited beating in 85% of aggregates were used. Both Bay K 8644 and CGP 28392 reversed nisoldipine-, diltiazem-or verapamil-induced inhibition of beating. 4 Bay K 8644 was approximately 10 times more potent than CGP 28392 in reversing nisoldipine-, diltiazem-or verapamil-induced inhibition of beating. 5 For each facilitator the concentrations causing 50% reversal of inhibition of aggregate beating against the three antagonists were similar. There was little evidence for differential modulation by verapamil or diltiazem of the action of the dihydropyridine facilitators. 6 Bay K 8644 did not reverse lidoflazine-or pimozide-induced inhibition of beating, indicating that these drugs may act at a site distinct from the dihydropyridine site on the calcium channel.

1 The protective effects of some calcium antagonists against different forms of calcium overload contracture were investigated in embryonic chick cardiac myocytes. 2 Tetrodotoxin-sensitive sodium currents were recorded from the myocytes by the whole-cell voltage-clamp technique. Although the peak current was attenuated by veratrine, the inactivation process was markedly inhibited, resulting in a large increase in the total inward current. Action potentials were prolonged by veratrine, automaticity was inhibited and the membrane potential depolarized from -79 to around -45 mV. 3 Measurements of contraction were made from aggregates of myocytes using a video edge detection technique which quantified edge movement. Veratrine caused an initial positive inotropism then inhibited automaticity of aggregates with subsequent development of a tonic contracture to around 300% of the twitch contraction. 4 Veratrine-induced contractures were not significantly affected by 10Mm diltiazem or verapamil.Nifedipine (5 yM), nimodipine (5 uM) and ryanodine (5 yM) also had little effect whilst nicardipine and flunarizine caused a concentration-dependent inhibition of veratrine-induced contractures with IC5 s of 3 pM and 2 yM respectively. 5 Veratrine-induced contractures were found to be very sensitive to extracellular calcium concentration with an EC50 of 32 Mm. Edge movement associated with beating of the myocytes was much less sensitive to calcium (EC5o = 1 mM). Submaximal veratrine contractures in 20-50Mm extracellular calcium were not potentiated by 1 yM Bay K 8644. 6 Tetrodotoxin also inhibited veratrine-induced contractures but did not affect contractions induced by ouabain in the presence of IO yM diltiazem. 7 Ouabain-induced contractures were also inhibited by nicardipine and flunarizine indicating that these drugs can protect against calcium overload in embryonic chick heart by a mechanism independent of the normal form of voltage-sensitive sodium or calcium channels.

1 A series of dihydropyridine derivatives has been evaluated for calcium channel agonist activity using reversal of nisoldipine-induced inhibition of beating of aggregates of embryonic chick myocytes. This test appears to be specific for calcium channel agonists since isoprenaline and cardiac glycosides are inactive. 2 RS 30026 was the most potent of the series, was significantly more potent than CGP 28392 and of similar potency to Bay K 8644 (pEC50 = 7.45, 6.16 and 7.20, respectively 6 RS 30026 appears to be the most potent and effective calcium channel agonist described to date.

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