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  • The reduction of the calcium response to AVP could

    2019-11-11

    The reduction of the calcium response to AVP could be also due to a PKA-mediated effect of desensitization of IP3R, as described in rat megakaryocytes [41]. Furthermore, PKA activation inhibits intracellular Ca release in mouse pancreatic acinar cells [42] or in rat cerebellum [43]. The phosphorylation of IP3R by PKA might be involved in these effects. However, there is considerable controversy as it has been described that this phosphorylation could enhance agonist-induced Ca mobilization in various preparations including rat hepatocytes [44] and mouse parotid acinar cells [45]. Phenylephrine contracts vascular smooth by activating α1 adrenoceptors and the subsequent increase of [Ca]c by IP3 mediated release of intracellular Ca and Ca influx through transmembrane channels [46], [47]. We believe that the depletion of intracellular Ca reported here is involved in the reduction of phenylephrine-induced contractions by cAMP-elevating agents in endothelium-denuded Obeticholic Acid in absence of external Ca showed here. The implication of Epac and PKA in such an effect is supported by the ability of their activators to reduce phenylephrine-induced contractions. It is interesting to note that none of the cAMP-elevating agents that increased [Ca]c in RASMC induced significant contractions in isolated endothelium-deprived aorta by themselves, suggesting that Obeticholic Acid a rise in [cAMP]c may deplete Ca from intracellular stores without activating the contractile machinery. It has been reported that thapsigargin-induced [Ca]c increase in RASMC, in absence of extracellular Ca, is not followed by a contraction of rat aorta [10], [11], indicating that the depletion of Ca induced by this drug is not coupled to muscular contraction. Our previous results suggest that the endothelium-independent vasorelaxation induced by nanomolar concentrations of forskolin could be mediated in part by an inhibition of the contraction activated by SOCE [3]. It has been reported that depletion of intracellular Ca stores also activates VOCC by opening of non-selective cation channels and the subsequent depolarization of cell membrane [48]. This activation of L-type VOCC has been also reported in rat aorta [10]. Here, we have probed that cAMP-elevating agents inhibit AVP-induced SOCE, an effect reproduced by separate activation of PKA or Epac that is not inhibited in the presence of the VOCC blocker nifedipine. The implication of this effect on rat aorta contractility is evidenced by the inhibition of the Ca-induced contraction after depletion of internal Ca with phenylephrine by cAMP-elevating agents, an effect that was not reduced by inhibition of L-type VOCC [3], PKA or Epac activation. Although a role of cAMP signalling on the regulation of SOCE has been previously suggested in smooth muscle [49], this is the first work describing an action of Epac on capacitative Ca entry in vascular cells. Moreover, the results described in the literature on the effects of PKA on capacitative Ca entry are very inconsistent depending on the preparation and the agent used for the depletion. Thus, thapsigargin-induced capacitive Ca entry in human lymphocytes can be increased or decreased by different concentrations of H-89, a selective PKA inhibitor [50]. In human pulmonary artery smooth muscle cells, cAMP could reduce capacitative Ca entry independently of PKA activation for short incubation times with forskolin and IBMX and this effect would become PKA-dependent as the incubation time increases [51]. Finally, our experiments shown that depletion of intracellular Ca stores after [cAMP]i elevation is not high and/or fast enough to activate a measurable entry of extracellular Ca through SOCE. In good agreement, Tosun et al. [52] reported that elevated [Ca]c due to store depletion is dissociated from contraction in rat aorta.
    Conclusion Our results suggest that a rise of [cAMP]i increases basal [Ca]c in vascular smooth muscle cells by depletion of intracellular thapsigargin-sensitive Ca stores. This effect reduces the amount of Ca ready to be released from intracellular stores via activation of PKA and Epac. Also, Epac and PKA participate in the inhibition of SOCE by cAMP. Both effects may explain, at least in part, the cAMP endothelium-independent vasorelaxant effects.