A Small Molecule Pyrazolo[3,4-d]pyrimidinone Inhibitor of Zipper-Interacting Protein Kinase Suppresses Calcium Sensitization of Vascular Smooth Muscle.

摘要

A novel inhibitor of zipper-interacting protein kinase (ZIPK) was utilized to examine the involvement of ZIPK in the regulation of smooth muscle contraction. Pre-treatment of de-endothelialized rat caudal arterial smooth muscle strips with the pyrazolo[3,4-d]pyrimidinone inhibitor 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 LC20 (myosin 20-kDa regulatory light chains) but not of CPI-17 (protein kinase C-potentiated inhibitory protein for myosin phosphatase of 17 kDa), Par-4 (prostate apoptosis response-4) or MYPT1 (myosin phosphatase targeting subunit 1), all of which have been implicated in the regulation of vascular contractility. A structural analog of HS38, with inhibitory activity towards PIM3 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 of G protein-coupled receptors that signal through both Gα12/13 and Gαq/11.