Fine tuning by protein kinases of Ca1.2 channel current in rat tail artery myocytes.

Seventeen compounds, rather selective, direct or indirect inhibitors and activators of PKA, PKG, and PKC, were analysed for effects on vascular Ca1.2 channel current (I) by using the patch-clamp technique in single rat tail artery myocytes. The aim was to investigate how PKs regulate I and disclose any unexpected modulation of Ca1.2 channel function by these agents. The cAMP analogues 8-Br-cAMP and 6-Bnz-cAMP partially reduced I in dialysed cells, while weakly increasing it under the perforated configuration. The β-adrenoceptor agonist isoproterenol and the adenylate cyclase activator forskolin concentration-dependently increased I; this effect was reversed by PKA inhibitors H-89 and KT5720, but not by PKI 6-22. The cGMP analogue 8-Br-cGMP, similarly to the NO-donor SNP, moderately reduced I, this effect being reversed to a slight stimulation under the perforated configuration. Among PKG inhibitors, Rp-8-Br-PET-cGMPS decreased current amplitude in a concentration-dependent manner while Rp-8-Br-cGMPS was ineffective. The non-specific phosphodiesterase inhibitor IBMX increased I, while H-89, KT5720, and PKI 6-22 antagonized this effect. The PKC activator PMA, but not the diacylglycerol analogue OAG, stimulated I in a concentration-dependent manner; conversely, the PKCα inhibitor Gö6976 markedly reduced basal I and, similarly to the PKCδ (rottlerin) and PKCε translocation inhibitors antagonised PMA-induced current stimulation. The ensemble of findings indicates that the stimulation of cAMP/PKA, in spite of the paradoxical effect of both 8-Br-cAMP and 6-Bnz-cAMP, or PKC pathways enhanced, while that of cGMP/PKG weakly inhibited I in rat tail artery myocytes. Since Rp-8-Br-PET-cGMPS and Gö6976 appeared to block directly Ca1.2 channel, their docking to the channel protein was investigated. Both compounds appeared to bind the α subunit in a region involved in Ca1.2 channel inactivation, forming an interaction network comparable to that of Ca1.2 channel blockers. Therefore, caution should accompany the use of these agents as pharmacological tools to elucidate the mechanism of action of drugs on vascular preparations.