Roberto Berra Romani a , Abdul Raqeeb b, f , Umberto Laforenza c , Manuela Federica Scaffino c , Francesco Moccia d , Josè Everardo Avelino-Cruz b , Amanda Oldani b , Daniela Coltrini e , Veronica Milesi f , Vanni Taglietti b , Franco Tanzi b
24 June 2008
The mechanism whereby extracellular Ca<sup>2+</sup> exerts the endothelium-dependent control of vascular tone is still unclear. In this study, we assessed whether cardiac microvascular endothelial cells (CMEC) express a functional extracellular Ca<sup>2+</sup>-sensing receptor (CaSR) using a variety of techniques. CaSR mRNA was detected using RT-PCR, and CaSR protein was identified by immunocytochemical analysis. In order to assess the functionality of the receptor, CMEC were loaded with the Ca<sup>2+</sup>-sensitive fluorochrome, Fura-2/AM. A number of CaSR agonists, such as spermine, Gd<sup>3+</sup>, La<sup>3+</sup> and neomycin, elicited a heterogeneous intracellular Ca<sup>2+</sup> signal, which was abolished by disruption of inositol 1,4,5-trisphosphate (InsP<sub>3</sub>) signaling and by depletion of intracellular stores with cyclopiazonic acid. The inhibition of the Na<sup>+</sup>/Ca<sup>2+</sup> exchanger upon substitution of extracellular Na<sup>+</sup> unmasked the Ca<sup>2+</sup> signal triggered by an increase in extracellular Ca<sup>2+</sup> levels. Finally, aromatic amino acids, which function as allosteric activators of CaSR, potentiated the Ca<sup>2+</sup> response to the CaSR agonist La<sup>3+</sup>. These data provide evidence that CMEC express CaSR, which is able to respond to physiological agonists by mobilizing Ca<sup>2+</sup> from intracellular InsP<sub>3</sub>-sensitive stores.