Effects of commonly used protein kinase inhibitors on vascular contraction and L-type Ca2+ current

• Published in: Biochemical pharmacology 2012-10, Vol.84 (8), p.1055-1061
• Main Authors: Saponara, Simona, Fusi, Fabio, Sgaragli, Giampietro, Cavalli, Maurizio, Hopkins, Brian, Bova, Sergio
• Format: Article
• Language: English
• Subjects: Arteries; Calcium channels; Calcium channels (L-type); Kinetics; L-type Ca2+ channel; ML-7; ML-9; Muscle contraction; Myocytes; Myosin light chain kinase; potassium chloride; Protein kinase C; protein kinase inhibitors; Rat tail artery; Rho-associated kinase; Smooth muscle; Tails; Whole-cell patch-clamp; Wortmannin
• ISSN: 0006-2952; 1873-2968
• DOI: 10.1016/j.bcp.2012.07.025

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 [IBa(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 IBa(L) intensity was surprisingly variable. Wortmannin showed negligible effects while HA-1077 and Y-27632 were ineffective. IBa(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 IBa(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 IBa(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.