Agonist-induced down-regulation of type 1 and type 3 inositol 1,4,5-tris-phosphate receptors in A7r5 and DDT 1 MF-2 smooth muscle cells

Abstract

Prolonged stimulation of rat A7r5 aortic smooth muscle cells with 3 μM vasopressin, or of hamster DDT 1 MF-2 smooth muscle cells with 10 μM bradykinin or 100 μM histamine led within 4 h to a 40–50% down-regulation of the type 1 InsP 3 receptor (InsP 3R-1) and of the type 3 InsP 3 receptor (InsP 3R-3). InsP 3R down-regulation was a cell- and agonist-specific process, since several other agonists acting on PLC-coupled receptors did not change the expression level of the InsP 3R isoforms in these cell types and since no agonist-induced down-regulation of InsP 3Rs was observed in HeLa cells. Down-regulation of InsP 3Rs was prevented by an inhibitor of proteasomal protease activity, N-acetyl-Leu-Leu-norleucinal (ALLN). The Ca 2+ channel blocker verapamil (2 μM) also induced InsP 3R-1 down-regulation (43%) in A7r5 cells, which was inhibited by ALLN. In A7r5 cells transiently transfected with a cDNA construct, bearing a luciferase coding sequence under control of the rat InsP3R-1 promoter, reduced luciferase activity could be demonstrated upon stimulation of cells with vasopressin or verapamil. Thus, besides enhanced protein degradation, a reduction of InsP 3R promoter activity might contribute to the down-regulation of InsP 3Rs in A7r5 cells. We next investigated the effect of InsP 3R down-regulation on Ca 2+ responses in A7r5 cells. A rightward shift in the dose-response curve for InsP 3 induced Ca 2 release was observed in permeabilized monolayers of vasopressinpretreated A7r5 cells (EC 50 630 nM and 400 nM for pretreated and non-pretreated cells, respectively). The Ca 2+ responses to threshold doses of vasopressin were markedly reduced in intact vasopressin-pretreated cells. We conclude that prolonged agonist-exposure leads to down-regulation of InsP 3Rs in A7r5 and DDT 1 MF-2 smooth muscle cells. The mechanism of down-regulation likely involves proteasomal degradation and reduction of InsP 3R promoter activity. Moreover, down-regulation of InsP 3Rs resulted in desensitization of Ca 2+ release from InsP 3 sensitive stores.