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Ca²⁺ signaling in human fetoplacental vasculature: effect of CGRP on umbilical vein smooth muscle cytosolic Ca²⁺ concentration

Department of Obstetrics and Gynecology, University of Texas Medical Branch, Galveston, Texas

Submitted 21 January 2005 ; accepted in final form 23 March 2005

CGRP is a potent vasodilator with increased levels in fetoplacental circulation during late pregnancy. We have recently demonstrated that acute CGRP exposure to fetoplacental vessels in vitro induced vascular relaxation, but the signaling pathway of CGRP in fetoplacental vasculature remains unclear. We hypothesized that CGRP relaxes fetoplacental vasculature via regulating smooth muscle cytosolic Ca²⁺ concentrations. In the present study, by using human umbilical vein smooth muscle (HUVS) cells (HUVS-112D), we examined CGRP receptors, cAMP generation, and changes in cellular Ca²⁺ concentrations on CGRP treatment. These cells express mRNA for CGRP receptor components, calcitonin receptor-like receptor, and receptor activity-modifying protein-1. Direct saturation binding for ¹²⁵I-labeled CGRP to HUVS cells and Scatchard analysis indicate specificity of the receptors for CGRP [dissociation constant (K_D) = 67 nM, maximum binding capcity (B_max) = 2.7 pmol/million cells]. Exposure of HUVS cells to CGRP leads to a dose-dependent increase in intracellular cAMP accumulation, and this increase is prevented by CGRP antagonist CGRP_8–37. Using fura-2-loaded HUVS cells, we monitored the effects of CGRP on intracellular Ca²⁺ concentration ([Ca²⁺]_i). In the presence of extracellular Ca²⁺, bradykinin (10^–6 M), a fetoplacental vasoconstrictor, increases HUVS cells [Ca²⁺]_i concentration. CGRP (10^–8 M) abolishes bradykinin-induced [Ca²⁺]_i elevation. When the cells were pretreated with glibenclamide, an ATP-sensitive potassium channel blocker, the CGRP actions on bradykinin-induced Ca²⁺ influx were profoundly inhibited. In the absence of extracellular Ca²⁺, CGRP (10^–8 M) attenuated the increase of [Ca²⁺]_i induced by a sarcoplasmic reticulum Ca²⁺ pump ATPase inhibitor thapsigargin (10^–5 M). Furthermore, Rp-cAMPS, a cAMP-dependent protein kinase A inhibitor, blocks CGRP actions on thapsigargin-induced Ca²⁺ release from sarcoplasmic reticulum. Our results suggested that CGRP relaxes human fetoplacental vessels by not only inhibiting the influx of extracellular Ca²⁺ but also attenuating the release of intracellular Ca²⁺ from the sarcoplasmic reticulum, and these actions might be attributed to CGRP-induced intracellular cAMP accumulation.

vascular smooth muscle cells; fetoplacental circulation; calcitonin gene-related peptide; cytosolic calcium mobilization