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Hierarchical Arrangement of Contractile Mechanisms in Resistance Arteries
Author(s) -
Haghbin Nadia,
Welsh Donald
Publication year - 2021
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2021.35.s1.02524
Subject(s) - calphostin c , myograph , g protein coupled receptor , calphostin , protein kinase c , biophysics , chemistry , microbiology and biotechnology , phenylephrine , mesenteric arteries , biology , endocrinology , medicine , signal transduction , contraction (grammar) , artery , blood pressure
Contraction/relaxation of vascular smooth muscle cells (VSMCs) in resistance arteries tunes tissue blood flow delivery. G‐protein coupled receptors (GPCRs) are integral to set this dynamic balance and they work by controlling two general signaling pathways. The first centers on coupling between V M and cytosolic [Ca 2+ ] via L‐type Ca 2+ channels, whereas the second operates independently of voltage through Ca 2+ sensitizing mechanisms. While untested, it is presumed that agonists activate both signaling pathways with their relative contributions remaining static across a full concentration range. In contrast, we propose a hierarchical arrangement with voltage‐dependent mechanisms preceding voltage‐independent mechanisms. To define the order and relative importance of voltage‐dependent and independent signaling to GPCR‐induced constrictions, we employed a combination of functional, pharmacological, and molecular techniques. Mouse mesenteric arteries were isolated and mounted in a pressure myograph and GPCR agonists (Thromboxane A2 mimetic and Phenylephrine) were applied globally or discretely. Arterial tone increased in a concentration dependent manner, with the subsequent application of nifedipine (L‐type Ca 2+ channel blocker) attenuating these responses, particularly at the lower concentration range. The remaining voltage‐independent constriction was blocked by calphostin C, a Protein Kinase C (PKC) inhibitor. Voltage‐independent constriction was also observed when agonists were discretely applied to a small portion of the vessel such that there was not enough charge to change membrane potential. This local change was also sensitive to calphostin C pre‐treatment. Ongoing work is focused on isolating the downstream PKC targets involved in voltage‐independent constriction. Using the highly sensitive three‐step western blot approach to probe key PKC phosphorylation sites (MYPT1‐T855, ‐T697, and CPI‐17), we have found the CPI‐17 phosphorylation site to be particularly promising in enabling agonist‐induced responses. In summary, our findings indicate a hierarchical arrangement with a shift from voltage‐dependent to voltage‐independent signaling as GPCR receptors are increasingly activated. These findings have translational application as to pathobiology of cerebral vasospasm and to its effective amelioration.