Vasoconstrictor hormones cause contraction of vascular smooth muscle (VSM) cells by increasing cytosolic free Ca2+ concentration ([Ca2+]i). The best characterized signal transduction pathway leading to elevation of [Ca2+]i involves inositol trisphosphate-mediated release of Ca2+ from the sarcoplasmic reticulum (SR) and Ca2+ entry via non-selective calcium-permeable cation channels. Voltage-sensitive L-type Ca2+ channels are also known to be important in vasoconstrictor action, though the signaling pathways leading to activation of L-type channels are not well characterized. We have previously demonstrated that physiological concentrations of [Arg8]-vasopressin (AVP, 10-100 pM) induce Ca2+ spiking in A7r5 VSM cells by a novel signaling pathway that leads to activation of L-type Ca2+ channels. Membrane depolarization is presumably required for L-type Ca2+ channel activation. We hypothesize, that AVP exerts a depolarizing effect on the smooth muscle cells. Among the most likely candidates to elicit this effect are non-selective cation channels. We have previously provided evidence for two distinct non-L-type divalent cation entry pathways activated by AVP. The molecular natures of the channels that mediate these effects are not known, but members of the transient receptor potential (TRP) family of non-selective cation channels have been implicated. The present proposal seeks to examine the hypothesis that these divalent cation entry pathways play an important role in the Ca2+ spiking response to physiological AVP concentrations.
The specific aims are to: 1) identify which TRP channel homologues are expressed in A7r5 cells and rat mesenteric artery smooth muscle cells (RMASMC) using RT-PCR and Western blotting; 2) characterize the non-selective cation currents activated by 100 pM AVP in A7r5 cells and RMASMC and evaluate their contribution to membrane potential changes using patch clamp techniques; 3) dissociate """"""""capacitative"""""""" store-operated Ca2+ entry from non-capacitative AVP-stimulated Ca2+ entry pathways and determine which is/are important in triggering Ca2+ spiking; 4) knock out or overexpress specific TRP channel homologues to determine their role in AVP-stimulated Ca2+ signaling and their relationship to AVP-stimulated nonselective cation currents and Ca2+ entry pathways.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
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Experimental Cardiovascular Sciences Study Section (ECS)
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Velletri, Paul A
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Loyola University Chicago
Schools of Medicine
United States
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