Abstract

A novel inhibitor of zipper-interacting protein kinase (ZIPK) was used to examine the involvement of ZIPK in the regulation of smooth muscle contraction. Pretreatment of de-endothelialized rat caudal arterial smooth muscle strips with the pyrazolo[3,4-d]pyrimidinone inhibitor 2-((1-(3-chlorophenyl)-4-oxo-4,5-dihydro-1H-pyrazolo [3,4-d]-pyrimidin-6-yl)thio)propanamide (HS38) decreased the velocity of contraction (time to reach half-maximal force) induced by the phosphatase inhibitor calyculin A in the presence of Ca²⁺ without affecting maximal force development. This effect was reversed following washout of HS38 and correlated with a reduction in the rate of phosphorylation of myosin 20-kDa regulatory light chains (LC₂₀) but not of protein kinase C-potentiated inhibitory protein for myosin phosphatase of 17 kDa (CPI-17), prostate apoptosis response-4, or myosin phosphatase-targeting subunit 1 (MYPT1), all of which have been implicated in the regulation of vascular contractility. A structural analog of HS38, with inhibitory activity toward proviral integrations of Moloney (PIM) virus 3 kinase but not ZIPK, had no effect on calyculin A–induced contraction or protein phosphorylations. We conclude that a pool of constitutively active ZIPK is involved in regulation of vascular smooth muscle contraction through direct phosphorylation of LC₂₀ upon inhibition of myosin light chain phosphatase activity. HS38 also significantly attenuated both phasic and tonic contractile responses elicited by phenylephrine, angiotensin II, endothelin-1, U46619, and K⁺-induced membrane depolarization in the presence of Ca²⁺, which correlated with inhibition of phosphorylation of LC₂₀, MYPT1, and CPI-17. These effects of HS38 suggest that ZIPK also lies downstream from G protein–coupled receptors that signal through both Gα_12/13 and Gα_q/11.