The binding of the Ca'+-channel blocker d-cis-[3H]diltiazem to guinea pig skeletal muscle microsomes is temperature-dependent. At 2°C the KD is 39 nM and B max is 11 pmol/mg protein. The binding is fully reversible (K-r = 0.02 min-'). The binding sites discriminate between the diastereoisomers l-and d-cis-diltiazem, reconize verapamil, gallopamil and tiapamil, and are sensitive to La'+-inhibition. At 30°C the KD is 37 nM and the Bmpx is 2.9pmol/mg protein. D-cis-diltiazem-labelling is regulated by the 1 +dihydropyridine Ca'+-channel blockers and a novel Ca'+-channel activator in a temperature-dependent manner. At 30°C an enhancement of d-cis-diltiazem binding by the channel blockers is observed. This is attributed to a B, increase. ECse-values for enhancement and the maximal enhancement differ for the individual 1 ,Cdihydropyridines. At 2°C 1 ,Cdihydropyridines inhibit d-cis-['H]diltiazem binding. This is attributed to a B,, decrease. We have directly labelled one of the drug receptor sites within the Ca2+-channel which can allosterically interact with the 1 ,4-dihydropyridine binding sites. Skeletal muscle

A 1,4‐dihydroypyridine arylazide photoaffinity ligand, [3H]azidopine (50.6 Ci/mmol), has been synthesized. [3H]Azidopine binds reversibly with a K d of 350 pM to guinea‐pig skeletal muscle membranes in the absence of ultraviolet light. The reversible [3H]azidopine binding is inhibited stereoselectively by 1,4‐dihydropyridines, phenylalkylamine Ca2+ channel blockers and La3+. Covalent incorporation into membrane proteins after photolysis was investigated by sodium dodecyl sulfate polyacrylamide slab gel electrophoresis. [3H]Azidopine is photoincorporated specifically into a protein of M r∼145 000. The covalent labelling of the M r ∼145 ooo band is inhibited stereoselectively by drugs and cations which block the reversible [3H]azidopine binding. It is suggested that [3H]azidopine is photoincorporated into a subunit of the putative Ca2+ channel.

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The functional role of alpha-adrenoceptors was investigated in different parts of the rabbit heart. Phenylephrine (PE) caused a marked increase in force of contraction (Fc) and a prolongation of the action potential (AP) in preparations from the left atrium and the right ventricle. The response was less pronounced in the right atrium and in the left ventricle, whereas APs of spontaneously beating sinoatrial preparations remained completely unchanged. Phentolamine as well as the diesters phorbol 12,13 dibutyrate (PDBu) or 12-O-tetradecanoyl-phorbol-13-acetate (TPA) eliminated the effects of PE. The contribution of alpha-adrenoceptors to the effects of adrenaline (Adr) and noradrenaline (NA) on Fc was determined in preparations from the right ventricle. Phentolamine and the phorbol diesters reduced the effects of Adr and NA by about 30 to 60%; the remaining response was abolished by propranolol. It can be derived from our experiments that, in some parts of the rabbit heart, a considerable amount of the effects of Adr and NA is due to the stimulation of alpha-adrenoceptors. The present findings therefore support the view that, in the rabbit heart, the maximally effective drive of the heart requires the stimulation of both alpha- and beta-adrenoceptors. The inhibitory effects of phorbol diesters on the alpha-adrenoceptor-mediated response indicate that the activation of protein kinase C (PKC) specifically uncouples alpha-adrenoceptors from the effector system, whereas the response to beta-adrenoceptor stimulation remains unchanged.