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Abstract: Regulation of smooth muscle contraction is driven by a number of protein kinases: the evidence for this often originates from studies that investigate the effects of extracellularly added specific protein kinase inhibitors. Six compounds, thought to be selective inhibitors of various kinases, were analysed for their effects on vascular L-type Ca2+ channels because this potential subsidiary activity could strongly influence our understanding of the pathways involved in smooth muscle contraction. Whole-cell L-type Ba2+ currents [I Ba(L)] were recorded in single myocytes, and contractile responses were measured from endothelium-denuded rings taken from the rat tail artery. Although ML-7, ML-9, and wortmannin (MLCK inhibitors), HA-1077 and Y-27632 (Rho-associated kinase inhibitors), and GF-109203X (PKC inhibitor) relaxed rings pre-contracted with high KCl in a concentration-dependent manner, their effect on I Ba(L) intensity was surprisingly variable. Wortmannin showed negligible effects while HA-1077 and Y-27632 were ineffective. I Ba(L) was partly inhibited by GF-109203X and blocked by ML-7 and ML-9 in a concentration-dependent manner, with the blockade by ML-7 being voltage-dependent. Whilst ML-7, ML-9, and GF-109203X sped up the inactivation kinetics of I Ba(L), GF-109203X did not modify ML-7- or ML-9-induced effects, with both intensity and kinetics of the current remaining unchanged. In contrast, application of Bay K 8644 on myocytes pre-treated with ML-7 or ML-9 raised I Ba(L) beyond control values. In conclusion, ML-7 and ML-9 inhibit L-type Ca2+ channels via a mechanism independent of MLCK, PKC or Rho kinase activities, and as such caution should be used in employing these agents to elucidate the role of kinases in smooth muscle contraction. [Copyright &y& Elsevier]

Abstract: In the present study, the effects of 3,5-di-t-butylcatechol (DTCAT) on ryanodine receptor Ca2+ channel (RyRC) of skeletal muscle sarcoplasmic reticulum (SR) vesicles were investigated, both by monitoring extravesicular Ca2+ concentration directly with the Ca2+ indicator dye arsenazo III and by studying the high-affinity [3H]ryanodine binding. DTCAT stimulated Ca2+ release from junctional (terminal cisternae) vesicles in a concentration-dependent manner, with a threshold activating concentration of 30μM and a pEC50 value of 3.43±0.03M. The release of Ca2+ induced by DTCAT was antagonized in a concentration-dependent manner by ruthenium red, thus indicating that RyRC is involved in the mechanism of stimulation. A structure–activity relationship analysis carried out on a limited number of compounds suggested that both hydroxy and t-butyl groups in DTCAT were important for the activation of RyRC. DTCAT inhibited [3H]ryanodine binding to SR vesicles with a K i of 232.5μM, thus indicating that it acted directly at the skeletal muscle ryanodine receptor binding site to stimulate Ca2+ release. In conclusion, the ability of DTCAT to release Ca2+ from TC vesicles of skeletal muscle is noteworthy in view of its possible use as an alternative compound to either caffeine or halothane for performing the “In vitro contracture test” to diagnose the susceptibility of some patients to develop malignant hyperthermia under particular pharmacological treatments. [Copyright &y& Elsevier]

Ca V 1.2 channels play a fundamental role in the regulation of vascular smooth muscle tone. The aim of the present study was to synthesize morin derivatives bearing the nitrophenyl moiety of dihydropyridine Ca 2+ antagonists to increase the flavonoid vasorelaxant activity. The effects of morin and its derivatives were assessed on Ca V 1.2 and K Ca 1.1 channels, both in vitro and in silico, as well as on the contractile responses of rat aorta rings. All compounds were effective Ca V 1.2 channel blockers, positioning in the α 1C subunit region where standard blockers bind. Among the four newly synthesized morin derivatives, the penta-acetylated morin-1 was the most efficacious Ca 2+ antagonist, presenting a vasorelaxant profile superior to that of the parent compound and, contrary to morin, antagonized also the release of Ca 2+ from the sarcoplasmic reticulum; surprisingly, it also stimulated K Ca 1.1 channel current. Computational analysis demonstrated that morin-1 bound close to the K Ca 1.1 channel S6 segment. In conclusion, these findings open a new avenue for the synthesis of valuable multi-functional, vasorelaxant morin derivatives capable to target several pathways underpinning the pathogenesis of hypertension., (Copyright © 2021 Elsevier Inc. All rights reserved.)