BACKGROUND AND PURPOSEPrevious studies have pointed to the plant flavonoids myricetin and quercetin as two structurally related stimulators of vascular Cav1.2 channel current (ICa1.2). Here we have tested the proposition that the flavonoid structure confers the ability to modulate Cav1.2 channels. EXPERIMENTAL APPROACHTwenty-four flavonoids were analysed for their effects on ICa1.2 in rat tail artery myocytes, using the whole-cell patch-clamp method. KEY RESULTSMost of the flavonoids stimulated or inhibited ICa1.2 in a concentration-and voltage-dependent manner with EC50 values ranging between 4.4 mM (kaempferol) and 16.0 mM (myricetin) for the stimulators and IC50 values between 13.4 mM (galangin) and 100 mM [(Ϯ)-naringenin] for the inhibitors. Key structural requirements for ICa1.2 stimulatory activity were the double bond between C2 and C3 and the hydroxylation pattern on the flavonoid scaffold, the latter also determining the molecular charge, as shown by molecular modelling techniques. Absence of OH groups in the B ring was key in ICa1.2 inhibition. The functional interaction between quercetin and either the stimulator myricetin or the antagonists resokaempferol, crysin, genistein, and 5,7,2′-trihydroxyflavone revealed that quercetin expressed the highest apparent affinity, in the low mM range, for Cav1.2 channels. Neither protein tyrosine kinase nor protein kinase Ca were involved in quercetin-induced stimulation of ICa1.2. CONCLUSIONS AND IMPLICATIONSQuercetin-like plant flavonoids were active on vascular Cav1.2 channels. Thus, the flavonoid scaffold may be a template for the design of novel modulators of vascular smooth muscle Cav1.2 channels, valuable for the treatment of hypertension and stroke. AbbreviationsBay K 8644, (S)-(-)-methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)pyridine-5-carboxylate; ICa1.2, Cav1.2 channel current; Vh, holding potential

Findings show that smooth muscle contractile activity and the biophysical and pharmacological features of L-type Ca(2+) channels are similar in VSMC24h and VSMC0h. The fact that reproducible results were obtained in vascular myocytes up to 24 h after dissociation may facilitate vascular smooth muscle cell investigation by increasing throughput and reducing the number of animals required.

BACKGROUND AND PURPOSEThe carbazole alkaloid murrayafoline A (MuA) enhances contractility and the Ca 2+ currents carried by the Ca v 1.2 channels [I Ca1.2 ] of rat cardiomyocytes. As only few drugs stimulate I Ca1.2 , this study was designed to analyse the effects of MuA on vascular Ca v 1.2 channels. EXPERIMENTAL APPROACHVascular activity was assessed on rat aorta rings mounted in organ baths. Ca v 1.2 Ba 2+ current [I Ba1.2 ] was recorded in single rat aorta and tail artery myocytes by the patch-clamp technique. Docking at a 3D model of the rat, α 1c central pore subunit of the Ca v 1.2 channel was simulated in silico. KEY RESULTSIn rat aorta rings MuA, at concentrations ≤14.2 μM, increased 30 mM K + -induced tone and shifted the concentration-response curve to K + to the left. Conversely, at concentrations >14.2 μM, it relaxed high K + depolarized rings and antagonized Bay K 8644-induced contraction. In single myocytes, MuA stimulated I Ba1.2 in a concentration-dependent, bell-shaped manner; stimulation was stable, incompletely reversible upon drug washout and accompanied by a leftward shift of the voltage-dependent activation curve. MuA docked at the α 1C subunit central pore differently from nifedipine and Bay K 8644, although apparently interacting with the same amino acids of the pocket. Neither Bay K 8644-induced stimulation nor nifedipine-induced block of I Ba1.2 was modified by MuA. CONCLUSIONS AND IMPLICATIONSMurrayafoline A is a naturally occurring vasoactive agent able to modulate Ca v 1.2 channels and dock at the α 1C subunit central pore in a manner that differed from that of dihydropyridines.

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