Hypotonic swelling stimulates L-type Ca2+ channel activity in vascular smooth muscle cells through PKC

It has been suggested that L-type Ca2+ channels play an important role in cell swelling-induced vasoconstriction. However, there is no direct evidence that Ca2+ channels in vascular smooth muscle are modulated by cell swelling. We tested the hypothesis that L-type Ca2+ channels in rabbit portal vein myocytes are modulated by hypotonic cell swelling via protein kinase activation. Ba2+ currents (I-Ba) through L-type Ca2+ channels were recorded in smooth muscle cells freshly isolated from rabbit portal vein with the conventional whole cell patch-clamp technique. Superfusion of cells with hypotonic solution reversibly enhanced Ca2+ channel activity but did not alter the voltage-dependent characteristics of Ca2+ channels. Bath application of selective inhibitors of protein kinase C (PKC), Ro-31-8425 or Go-6983, prevented I-Ba enhancement by hypotonic swelling, whereas the specific protein kinase A (PKA) inhibitor KT-5720 had no effect. Bath application of phorbol 12,13-dibutyrate (PDBu) significantly increased I-Ba under isotonic conditions and prevented current stimulation by hypotonic swelling. However, PDBu did not have any effect on I-Ba when cells were first exposed to hypotonic solution. Furthermore, downregulation of endogenous PKC by overnight treatment of cells with PDBu prevented current enhancement by hypotonic swelling. These data suggest that hypotonic cell swelling can enhance Ca2+ channel activity in rabbit portal vein smooth muscle cells through activation of PKC.