Investigating the Role of PKC‐delta in Voltage‐Independent Contractile Pathways in Mouse Resistance Arteries

Abstract

Vascular smooth muscle cells (VSMCs) tune blood flow delivery through G protein‐coupled receptor (GPCR) associated signaling pathways and two general contractile mechanisms. The first, electromechanical coupling ties membrane potential (VM) to cytosolic [Ca2+] via voltage‐sensitive L‐type Ca2+ channels. The second—independent of voltage—centers on mechanisms of Ca2+ sensitization via Rho kinase and Protein Kinase C (PKC). While untested, agonists presumably activate both, with their relative contribution remaining static across concentration range. We hypothesize that voltage‐dependent mechanisms proceed voltage‐independent, and this order is contingent on the mode of agonist application.Mouse mesenteric/cerebral arteries mounted in a pressure myograph (60 mmHg) were globally or focally exposed to GPCR agonists (U46619 and Phenylephrine) and vasomotor responses, intercellular [Ca2+] levels, and VM monitored. Global application of either agonists increased arterial tone in a concentration dependent manner. Subsequent Nifedipine (L‐type Ca2+ channel blocker) application attenuated constrictive responses, particularly at the lower agonist concentrations. The remaining voltage‐independent constriction was blocked by Calphostin C (PKC inhibitor). While these findings highlight a hierarchical arrangement (voltage‐dependent preceding voltage‐independent), this order is not static. Stimulating a reduced number of VSMCs through focal agonist application, induces localized vasomotor responses that do not conduct across the arteriole or alter intercellular [Ca2+] or VM. Such focal responses could bypass electromechanical coupling owing to insufficient charge to change VM. Thus, this response is heavily dependent on Ca2+ sensitization mechanisms. Western blot analysis indicates that PKCα and PKCδ are likely the key regulatory isoforms in agonist‐induced constriction. Ongoing work is focused on translocation of PKCα and PKCδ and phosphorylation state of their key downstream targets including MYPT1, CPI‐17, Caldesmon, and HSP27.In summary, our findings reveal a hierarchical but malleable contractile arrangement in VSMCs whereby voltage‐dependent signaling precedes voltage‐independent, as long as sufficient smooth muscle cells are activated. These findings are of particular translational value to the pathobiology/management of cerebral vasospasm.