Cardiac microvascular endothelial cells express a functional Ca 2+-sensing receptor

The mechanism whereby extracellular Ca2+ 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 Ca2+-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 Ca2+-sensitive fluorochrome, Fura-2/AM. A number of CaSR agonists, such as spermine, Gd 3+, La3+ and neomycin, elicited a heterogeneous intracellular Ca2+ signal, which was abolished by disruption of inositol 1,4,5-trisphosphate (InsP3) signaling and by depletion of intracellular stores with cyclopiazonic acid. The inhibition of the Na +/Ca2+ exchanger upon substitution of extracellular Na+ unmasked the Ca2+ signal triggered by an increase in extracellular Ca2+ levels. Finally, aromatic amino acids, which function as allosteric activators of CaSR, potentiated the Ca2+ response to the CaSR agonist La3+. These data provide evidence that CMEC express CaSR, which is able to respond to physiological agonists by mobilizing Ca2+ from intracellular InsP3-sensitive stores.Facultad de Ciencias Exacta

Cardiac microvascular endothelial cells express a functional Ca 2+-sensing receptor

The mechanism whereby extracellular Ca2+ 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 Ca2+-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 Ca2+-sensitive fluorochrome, Fura-2/AM. A number of CaSR agonists, such as spermine, Gd 3+, La3+ and neomycin, elicited a heterogeneous intracellular Ca2+ signal, which was abolished by disruption of inositol 1,4,5-trisphosphate (InsP3) signaling and by depletion of intracellular stores with cyclopiazonic acid. The inhibition of the Na +/Ca2+ exchanger upon substitution of extracellular Na+ unmasked the Ca2+ signal triggered by an increase in extracellular Ca2+ levels. Finally, aromatic amino acids, which function as allosteric activators of CaSR, potentiated the Ca2+ response to the CaSR agonist La3+. These data provide evidence that CMEC express CaSR, which is able to respond to physiological agonists by mobilizing Ca2+ from intracellular InsP3-sensitive stores.Facultad de Ciencias Exacta

Cardiac microvascular endothelial cells express a functional Ca 2+-sensing receptor

The mechanism whereby extracellular Ca2+ 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 Ca2+-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 Ca2+-sensitive fluorochrome, Fura-2/AM. A number of CaSR agonists, such as spermine, Gd 3+, La3+ and neomycin, elicited a heterogeneous intracellular Ca2+ signal, which was abolished by disruption of inositol 1,4,5-trisphosphate (InsP3) signaling and by depletion of intracellular stores with cyclopiazonic acid. The inhibition of the Na +/Ca2+ exchanger upon substitution of extracellular Na+ unmasked the Ca2+ signal triggered by an increase in extracellular Ca2+ levels. Finally, aromatic amino acids, which function as allosteric activators of CaSR, potentiated the Ca2+ response to the CaSR agonist La3+. These data provide evidence that CMEC express CaSR, which is able to respond to physiological agonists by mobilizing Ca2+ from intracellular InsP3-sensitive stores.Facultad de Ciencias Exacta

Ca2+ signalling in damaged endothelium: do connexin hemichannels aid in filling the gap?

A monolayer of endothelial cells (ECs) lines the lumen of blood vessels and forms a multifunctional transducing organ that regulates cardiovascular homeostasis. Disruption of endothelial integrity, as a result of either angioplasty or stent deployment, may produce a late in-stent restenosis and limit the beneficial outcome of reconstructive vascular surgery. Restoration of endothelial lining requires spreading, migration and proliferation of ECs nearby the lesion site. Intracellular Ca2+ signalling plays a major regulatory role in stimulating wound healing, however, the mechanism whereby injury increases Ca2+ levels at the wound edge is still unclear. The analysis of Ca2+ signals elicited by scraping an endothelial monolayer in vitro suggested the involvement of intracellular Ca2+ release from InsP3-sensitive stores and Ca2+ entry through unknown ion channels in the plasma membrane. Recent studies carried out by our group on excised rat aorta highlighted a novel role for connexin hemichannels (ChHcs) in mediating Ca2+ entry in injured endothelium. This observation sheds new light on the notion that the rate of wound repair is reduced in ECs transfected with dominant negative connexin inhibitors. Understanding the signal transduction pathway leading to EC activation is likely to provide novel targets to design therapeutic applications aiming at restoring endothelial integrity and treating cardiovascular diseases

Ca2+ signaling in injured in situ endothelium of rat aorta

The inner wall of excised rat aorta was scraped by a microelectrode and Ca2+ signals were investigated by fluorescence microscopy in endothelial cells (ECs) directly coupled with injured cells. The injury caused an immediate increase in the intracellular Ca2+ concentration ([Ca2+]i), followed by a long-lasting decay phase due to Ca2+ influx from extracellular space. The immediate response was mainly due to activation of purinergic receptors, as shown by the effect of P2X and P2Y receptors agonists and antagonists, such as suramin, alpha,beta-MeATP, MRS-2179 and 2-MeSAMP. Inhibition of store-operated Ca2+ influx did not affect either the peak response or the decay phase. Furthermore, the latter was: (i) insensitive to phospholipase C inhibition, (ii) sensitive to the gap junction blockers, palmitoleic acid, heptanol, octanol and oleamide, and (iii) sensitive to La3+ and Ni2+, but not to Gd3+. Finally, ethidium bromide or Lucifer Yellow did not enter ECs facing the scraped area. These results suggest that endothelium scraping: (i) causes a short-lasting stimulation of healthy ECs by extracellular nucleotides released from damaged cells and (ii) uncouples the hemichannels of the ECs facing the injury site; these hemichannels do not fully close and allow a long-lasting Ca2+

Hydrogen sulfide promotes calcium signals and migration in tumor-derived endothelial cells

Hydrogen sulfide (H(2)S) is a gasotransmitter that plays several roles in various tissues, including the cardiovascular system. Because it has been recently proposed to act as a mediator of angiogenesis progression, here we investigate the effects of H(2)S in a well-established model of tumor angiogenesis: endothelial cells obtained from human breast carcinoma (B-TECs). Ca(2+) imaging and patch-clamp experiments reveal that acute perfusion with NaHS, a widely employed H(2)S donor, activates cytosolic calcium (Ca(c)) increase, as well as potassium and nonselective cationic currents, in B-TECs. Stimulation with NaHS in the same concentration range (1 nM-200 μM) evoked Ca(c) signals also in "normal" human microvascular endothelial cells (HMVECs), but the amplitude was significantly lower. Moreover, although NaHS failed to promote either migration or proliferation on HMVECs, B-TEC migration was enhanced at low-micromolar NaHS concentrations (1-10 μM). Remarkably H(2)S mediates tumor proangiogenic signaling triggered by vascular endothelial growth factor (VEGF). B-TECs pretreated with dl-propargylglycine (5mM, 30 min), an inhibitor of the H(2)S-producing enzyme cystathionine γ-lyase, showed drastically reduced migration and Ca(c) signals induced by VEGF (20 ng/ml). We conclude that H(2)S plays a role in proangiogenic signaling of tumor-derived but not normal human ECs. Furthermore the ability of this gasotransmitter to interfere with B-TEC responsiveness to VEGF suggests that it could be an interesting target for antiangiogenic strategies in tumor treatment

Cardiac microvascular endothelial cells express a functional Ca2+-sensing receptor

The mechanism whereby extracellular Ca(2+) 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(2+)-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(2+)-sensitive fluorochrome, Fura-2/AM. A number of CaSR agonists, such as spermine, Gd(3+), La(3+) and neomycin, elicited a heterogeneous intracellular Ca(2+) signal, which was abolished by disruption of inositol 1,4,5-trisphosphate (InsP(3)) signaling and by depletion of intracellular stores with cyclopiazonic acid. The inhibition of the Na(+)/Ca(2+) exchanger upon substitution of extracellular Na(+) unmasked the Ca(2+) signal triggered by an increase in extracellular Ca(2+) levels. Finally, aromatic amino acids, which function as allosteric activators of CaSR, potentiated the Ca(2+) response to the CaSR agonist La(3+). These data provide evidence that CMEC express CaSR, which is able to respond to physiological agonists by mobilizing Ca(2+) from intracellular InsP(3)-sensitive stores