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Defining the roles of Ca2+ entry in endothelin-1 and thromboxane A2 receptor mediated vascular contractile responses

Y.Y. Chan, N. Scrimgeour, G.Y. Rychkov and D.P. Wilson, Discipline of Physiology, School of Medical Sciences, University of Adelaide, SA 5000, Australia.

Introduction: Ca2+ is an important mediator of vascular contractility, which can enter the cytosol through voltage-gated L- and T-channels and intracellular SR Ca2+ release. Upon depletion of the SR Ca2+ store, Ca2+-release activated Ca2+ (CRAC) channels, which are composed of plasma membrane bound Orai1 and SR-bound STIM1, form functional channels, allowing store refilling. Although endothelin-1 (ET-1) and thromboxane A2 are both potent vasoconstrictors implicated in various vascular disease states, they mediate Ca2+ entry and vasoconstriction through uniquely different mechanisms.

Aim and Method: To identify the role of IP3 receptors, CRAC-, L- and T-channels in ET-1 and thromboxane A2-mediated vasoconstriction. Using an in vitro rat artery model, functional vascular myography coupled with patch clamp analysis were used to identify the activation and inhibition of Ca2+ entry pathways mediated by agonists and pharmacological inhibitors of ion channels, respectively.

Results: Brief sequestration of extracellular Ca2+ using EGTA (5mM) revealed that approximately 20% ET-1-mediated vasoconstriction involved IP3-mediated SR Ca2+ (p<0.05; n=4). Following SR Ca2+ depletion using cyclopiazonic acid (10mM) (a SERCA pump inhibitor) and 2-aminoethyl diphenyl borate (100mM) (which is known to block IP3 receptors, CRAC channels and potentially non-selective cation channels), vascular contractility was abrogated (P<0.05; n=4), indicating a role for both IP3 receptors and CRAC channels. Blocking extracellular Ca2+ entry using combined L-/T-channel blockers, mibefradil (1mM) (p<0.05; n=7) and efonidipine (0.021mM) (p<0.05; n=13) attenuated approximately 65% ET-1-mediated vasoconstriction in the microvasculature (Ball et al,, 2009). Patch clamp analysis of ICRAC has revealed that in addition to blocking L- and T-channels, both mibefradil and efonidipine also inhibited CRAC channels. In contrast, thromboxane A2-mediated vasoconstriction only involved Ca2+ entry through L-channels and RhoA-Rho kinase Ca2+-independent sensitization and does not involve IP3 receptors, T or CRAC channels.

Conclusion: In the microvasculature, ET-1 mediates Ca2+ entry via L, T, IP3 receptors and CRAC channels. In contrast to the traditional L-type Ca2+ channel blockers, the more recently developed combined L-/T-channel blockers may provide additional benefit through blockade of CRAC channels, which may effectively enable clinical modulation of SR Ca2+ release.

Ball CJ, Wilson DP, Turner SP, Saint DA, Beltrame JF. (2009) Heterogeneity of L- and T-channels in the vasculature: Rationale for the efficacy of combined L- and T-blockade. Hypertension 53: 654-660.