Abstract

The effects of insulin (10 nM) and sodium orthovanadate (SOV, 25 microM) pretreatment for 24 h on endothelin-1 (ET-1) and IRL-1620 (ETB agonist)-evoked increases in intra-cellular free calcium levels [Ca2+]i were determined by fura-2 fluorescence methodology using cultured rat aortic smooth-muscle cells (ASMCs). Whereas insulin preincubation led to an increase (p < 0.05) in [Ca2+]i responses to ET-1, SOV enhanced [Ca2+]i responses to both ET-1 (p < 0.05) and IRL-1620 (p < 0.01). Saturation binding studies revealed that insulin pretreatment led to a 2.2-fold (p < 0.01) increase in [125I]ET-1 binding sites, and SOV pretreatment led to a 2.4-fold (p < 0.01) and 6.6-fold (p < 0.01) increase in the maximal number of binding sites labeled by [125I]ET-1 and [125I]IRL-1620, respectively. Northern blot analysis revealed that insulin upregulated only ETA mRNA levels, and SOV enhanced both ETA and ETB mRNA levels. Pretreatment with the tyrosine kinase inhibitor genistein abolished these effects. These data suggest that insulin pretreatment of ASMCs selectively upregulates ETA receptor expression and ETA-mediated [Ca2+]i, signaling, and SOV upregulates both ETA and ETB receptor expression and exaggerates [Ca2+]i responses mediated by both ETA and ETB in rat ASMCs.

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