Abstract
ABSTRACTObjective:To characterize the calcium influx pathways implicated in the sustained elevation of endothelial intracellular calcium concentration, required for the synthesis and release of relaxing factors.Methods:We evaluated the effect of the newly synthesized pyrazole derivatives, described as selective inhibitors for ORAI (BTP2/Pyr2 and Pyr6) and TRPC3 (Pyr3 and Pyr10) channels, upon endothelium- and extracellular calcium-dependent relaxations stimulated by acetylcholine and thapsigargin, in pre-constricted rat thoracic aortic rings.Results:Acetylcholine and thapsigargin responses were completely reverted by Pyr2 and Pyr6 (1 to 3μM). Pyr3 (0.3 to 3μM) caused a rapid reversal of acetylcholine (6.2±0.08mg.s−1) and thapsigargin (3.9±0.25mg.s−1) relaxations, whereas the more selective TRPC3 blocker Pyr10 (1 to 3μM) had no effect. The recently described TRPC4/5 selective blocker, ML204 (1 to 3μM), reverted completely acetylcholine relaxations, but minimally thapsigargin induced ones. Noteworthy, relaxations elicited by GSK1016790A (TRPV4 agonist) were unaffected by pyrazole compounds or ML204. After Pyr2 and Pyr6 pre-incubation, acetylcholine and thapsigargin evoked transient relaxations similar in magnitude and kinetics to those observed in the absence of extracellular calcium. Sodium nitroprusside relaxations as well as phenylephrine-induced contractions (denuded aorta) were not affected by any of pyrazole compounds (1 to 3μM).Conclusion:These observations revealed a previously unrecognized complexity in rat aorta endothelial calcium influx pathways, which result in production and release of nitric oxide. Pharmacologically distinguishable pathways mediate acetylcholine (ORAI/TRPC other than TRPC3/TRPC4 calcium-permeable channels) and thapsigargin (TRPC4 not required) induced calcium influx.
Highlights
Accumulated evidence indicates that extracellular calcium is required for the continuous production of nitric oxide (NO) implying that calcium influx is involved.[1,2,3,4,5] For instance, many endogenous and synthetic substances that cause endothelium- and NO-dependent relaxations in isolated arteries, or stimulate NO production in cultured endothelial cells (EC), induce a rapid rise in intracellular calcium concentration ([Ca2+]i) that is dependent on external Ca2+ concentration
The three agents caused sustained relaxations, which were strictly endothelium-dependent and abolished by L-NNA (Figures 1A to 1E); the onset of these responses was rapid for ACh/Thap (2 minutes) and they reached a steady state in 4-6 minutes, which persisted for at least 10 minutes
The endothelium-dependent relaxations elicited by ACh and Thap were transformed into transient relaxations while those elicited by GSK101 were abolished
Summary
Accumulated evidence indicates that extracellular calcium is required for the continuous production of nitric oxide (NO) implying that calcium influx is involved.[1,2,3,4,5] For instance, many endogenous and synthetic substances that cause endothelium- and NO-dependent relaxations in isolated arteries, or stimulate NO production in cultured endothelial cells (EC), induce a rapid rise in intracellular calcium concentration ([Ca2+]i) that is dependent on external Ca2+ concentration. A correlation between changes in endothelial [Ca2+]i and NO production has been shown.[4,5] Despite these data indicating that calcium influx in EC is necessary for the synthesis and release of relaxing factors, the identity of the channels involved in this influx, and whether distinct extracellular Ca2+-dependent stimuli of EC involve the same or a different set of channels remain unknown. We took advantage of the recent availability of pyrazole derivatives, which cause selective blockade of ORAI (pyr; pyr6) and TRPC3 (pyr; pyr10) channels, as well as of the TRPC4 blocker ML204,(8-11) and investigated their effects upon acetylcholine and thapsigargin induced endothelium dependent relaxations Of special interest are the recently discovered ORAI proteins, as well as some members of non-selective cation permeable TRP channel family, which can mediate either storeoperated calcium entry (SOCE) (capacitative) or noncapacitative Ca2+ influx in a variety of cells.[6,7] Since ORAI and TRP channels are found in EC, these molecules have emerged as putative mediators of Ca2+ influx required for production of NO by these cells.
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