High glucose attenuates peptide agonist-evoked increases in cytosolic free [Ca²⁺ ] in rat aortic smooth muscle cells

Incubation of cultured rat aortic smooth muscle cells (ASMCs) in a medium containing high glucose concentrations (25 mM) did not affect the basal cytosolic free calcium ([Ca²⁺]_i) but led to significant reductions in peak [Ca²⁺]_i response evoked by arginine vasopressin, angiotensin II, and endothelin-1 (ET-1). This was observed in both the presence and absence of extracellular Ca²⁺. Maintenance of rat ASMCs in a medium containing mannose (an osmotic control for high glucose) did not affect either the basal or peptide agonist-evoked increase in [Ca²⁺]_i. However, pretreatment with either the nonselective protein kinase C (PKC) inhibitor staurosporine or the selective PKC inhibitor 2,6-diamino-N-([1-(1-oxotridecyl)-2 piperidinyl] methyl) hexanamide reversed the attenuating effect of high glucose on peak [Ca²⁺]_i response evoked by ET-1. Also, short-term incubation of ASMCs with the active phorbol ester, phorbol 12-myristate 13-acetate, led to a reduction in peak [Ca²⁺]_i response to all three agonists, whereas the inactive phorbol ester, 4α-phorbol 12,13-didecanoate, which does not activate PKC, had no such effect. Although high-glucose treatment of rat ASMCs led to significant reductions in the maximal number of binding sites to the extent of 39% of [¹²⁵I]ET-1 specific binding, no significant differences in the affinity (K_d ∼110 pM) characteristics were evident between control and high-glucose treatment groups. It is proposed that incubation of rat ASMCs with high glucose enhances the de novo synthesis of diacylglycerol and activates membrane-bound PKC and that this, in turn, impairs agonist-mediated intracellular Ca²⁺ mobilization.