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  • The interferences between thapsigargin and


    The interferences between thapsigargin- and forskolin-induced Ca release indicate that these drugs deplete the same intracellular stores in RASMC. In fact, after partial depletion of thapsigargin-sensitive stores, the forskolin-induced increases in [Ca]c were significantly reduced. Similarly, a previous application of cAMP-elevating agents reduced the increase in [Ca]c by a subsequent application of thapsigargin. We hypothesize that cAMP-induced depletion of Ca may reduce the amount of Ca available to be released by a subsequent stimulation with an agonist. In our experiments, cAMP-elevating agents reduced the AVP-induced increase of [Ca]c in absence of external Ca. It has been reported that forskolin or isoprenaline transiently increase basal [Ca]c levels in RASMC, and reduce the subsequent increase of [Ca]c induced by AVP. However, contrary to our observations, this effect was completely dependent on the presence of extracellular Ca [15]. In addition, a previous depletion of calcium reservoirs with AVP significantly reduced the amount of intracellular Ca released by forskolin or db-cAMP. This fact reinforces the idea that cAMP depletes Ca stores that participate in the agonist-induced contractions mediated in part by a release of IP3-sensitive calcium stores. From our experiments we cannot suggest a mechanism for the cAMP-mediated depletion of Ca stores, but it could be related to the phosphorylation of phospholamban by PKA, which leads to an increased Ca leak from the sarcoplasmic reticulum in cardiomyocytes [21]. cAMP may also reduce phosphoinositide hydrolysis or phosphorylate IP3 receptors (IP3R) via PKA activation [22] although the consequences of these effects are unclear and controversial results have been published [23]. Moreover, Epac facilitates internal Ca mobilization by sensitizing IP3R in mice neurons [24] and Ca-mobilizing actions of 8-pCPT-2′-O-Me-cAMP exist in mouse pancreatic beta 873 [25], [26] and rat inner medullary collecting duct cells [27], [28]. In view of these reports, we intended to study the implication of PKA and Epac on the cAMP-induced depletion of internal Ca. PKA expression in vascular myocytes, including RASMC, is widely recognized [29] and the presence of Epac has been reported in rat aortic homogenates [1], [30] and RASMC [31], [32], [33]. Our results shown that forskolin-induced Ca mobilization is diminished by Rp-cAMPs, a specific competitive inhibitor of PKA [34], [35] and by 873 ESI-09, at concentrations that specifically inhibits Epac (although there is considerable controversy regarding its selectivity, see below), and reproduced by the joint activation of PKA (with 6-Bnz-cAMP) and Epac (with 8-pCPT-2′-O-Me-cAMP). However, the separate activation of these proteins did not significantly modify basal [Ca]c. 8-pCPT-2′-O-Me-cAMP has been widely accepted as a selective activator of Epac at the concentrations used in this study [4], [36], [37] and higher concentrations of this compound (300μM) have been reported to selectively activate smooth muscle Epac [5]. Also, this compound does not phosphorylate vasodilator-stimulated phosphoprotein (VASP), a common substrate of PKA [7]. Concerning 6-Bnz-cAMP, we have demonstrated its ability to phosphorylate VASP in rat aortic smooth muscle [1], according to previous reports suggesting that this compound selectively activates PKA (see, i.e., Zieba et al. [7]). On the other hand, the results obtained with ESI-09 should be taken with caution, since we have found only one study evaluating the effects of this compound in a biological system [38], and it has been suggested that this compound may not act selectively on Epac [39]. An interpretation of these results is that activation of both PKA and Epac could be necessary to deplete internal Ca stores. In fact, both proteins operate synergistically to negatively regulate cell proliferation via a Rap1-independent mechanism [32] or to inhibit platelet aggregation (unpublished observations). However, we believe that PKA and Epac may induce separately an increment of basal [Ca]c not detected with our methodology. In this connection, an augmentation of IP3-induced Ca release from the endoplasmic reticulum is mediated by PKA, but not by Epac, in blowfly salivary glands [40]. Furthermore, Purves et al. [30] reported that 8-pCPT-2′-O-Me-cAMP induces a transient increase of basal [Ca]c in RASMC in the absence of extracellular Ca, supporting a role for Epac alone in the release of internal Ca in these cells. Also, preincubation of RAMSC with PKA or Epac activators inhibited AVP- or thapsigargin-induced increase of [Ca]c in absence of external Ca, thus reproducing the results obtained with cAMP-elevating agents. This suggests that both proteins participate in the depletion of Ca reservoirs by cAMP, and that a simultaneous activation of both proteins is not necessary for this action. In this sense, both PKA and Epac inhibitors separately reduce the effects of forskolin on AVP-induced contractions.