Role of Phosphatidylinositol 3-Kinase (PI3K), Mitogen-Activated Protein Kinase (MAPK), and Protein Kinase C (PKC) in Calcium Signaling Pathways Linked to the α ₁-Adrenoceptor in Resistance Arteries

Insulin resistance plays a key role in the pathogenesis of type 2 diabetes and is also related to other health problems like obesity, hypertension, and metabolic syndrome. Imbalance between insulin vascular actions via the phosphatidylinositol 3-Kinase (PI3K) and the mitogen activated protein kinase (MAPK) signaling pathways during insulin resistant states results in impaired endothelial PI3K/eNOS- and augmented MAPK/endothelin 1 pathways leading to endothelial dysfunction and abnormal vasoconstriction. The role of PI3K, MAPK, and protein kinase C (PKC) in Ca ²⁺ handling of resistance arteries involved in blood pressure regulation is poorly understood. Therefore, we assessed here whether PI3K, MAPK, and PKC play a role in the Ca ²⁺ signaling pathways linked to adrenergic vasoconstriction in resistance arteries. Simultaneous measurements of intracellular calcium concentration ([Ca ²⁺] _i) in vascular smooth muscle (VSM) and tension were performed in endothelium-denuded branches of mesenteric arteries from Wistar rats mounted in a microvascular myographs. Responses to CaCl ₂ were assessed in arteries activated with phenylephrine (PE) and kept in Ca ²⁺-free solution, in the absence and presence of the selective antagonist of L-type Ca ²⁺ channels nifedipine, cyclopiazonic acid (CPA) to block sarcoplasmic reticulum (SR) intracellular Ca ²⁺ release or specific inhibitors of PI3K, ERK-MAPK, or PKC. Activation of α ₁-adrenoceptors with PE stimulated both intracellular Ca ²⁺ mobilization and Ca ²⁺ entry along with contraction in resistance arteries. Both [Ca ²⁺] _i and contractile responses were inhibited by nifedipine while CPA abolished intracellular Ca ²⁺ mobilization and modestly reduced Ca ²⁺ entry suggesting that α ₁-adrenergic vasoconstriction is largely dependent Ca ²⁺ influx through L-type Ca ²⁺ channel and to a lesser extent through store-operated Ca ²⁺ channels. Inhibition of ERK-MAPK did not alter intracellular Ca ²⁺ mobilization but largely reduced L-type Ca ²⁺ entry elicited by PE without altering vasoconstriction. The PI3K blocker LY-294002 moderately reduced intracellular Ca ²⁺ release, Ca ²⁺ entry and contraction induced by the α ₁-adrenoceptor agonist, while PKC inhibition decreased PE-elicited Ca ²⁺ entry and to a lesser extent contraction without affecting intracellular Ca ²⁺ mobilization. Under conditions of ryanodine receptor (RyR) blockade to inhibit Ca ²⁺-induced Ca ²⁺-release (CICR), inhibitors of PI3K, ERK-MAPK, or PKC significantly reduced [Ca ²⁺] _i increases but not contraction elicited by high K ⁺ depolarization suggesting an activation of L-type Ca ²⁺ entry in VSM independent of RyR. In summary, our results demonstrate that PI3K, ERK-MAPK, and PKC regulate Ca ²⁺ handling coupled to the α ₁-adrenoceptor in VSM of resistance arteries and related to both contractile and non-contractile functions. These kinases represent potential pharmacological targets in pathologies associated to vascular dysfunction and abnormal Ca ²⁺ handling such as obesity, hypertension and diabetes mellitus, in which these signaling pathways are profoundly impaired.