Agonist-induced down-regulation of type 1 and type 3 inositol 1,4,5-trisphosphate receptors in A7r5 and DDT1 MF-2 smooth muscle cells.
Prolonged stimulation of rat A7r5 aortic smooth muscle cells with 3 microM vasopressin, or of hamster DDT1 MF-2 smooth muscle cells with 10 microM bradykinin or 100 microM histamine led within 4 h to a 40-50% down-regulation of the type 1 InsP3 receptor (InsP3R-1) and of the type 3 InsP3 receptor (InsP3R-3). InsP3R 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 InsP3R isoforms in these cell types and since no agonist-induced down-regulation of InsP3Rs was observed in HeLa cells. Down-regulation of InsP3Rs was prevented by an inhibitor of proteasomal protease activity, N-acetyl-Leu-Leu-norleucinal (ALLN). The Ca2+ channel blocker verapamil (2 microM) also induced InsP3R-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 InsP3R promoter activity might contribute to the down-regulation of InsP3Rs in A7r5 cells. We next investigated the effect of InsP3R down-regulation on Ca2+ responses in A7r5 cells. A rightward shift in the dose-response curve for InsP3-induced Ca2+ release was observed in permeabilized monolayers of vasopressin-pretreated A7r5 cells (EC50 630 nM and 400 nM for pretreated and non-pretreated cells, respectively). The Ca2+ 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 InsP3Rs in A7r5 and DDT, MF-2 smooth muscle cells. The mechanism of down-regulation likely involves proteasomal degradation and reduction of InsP3R promoter activity. Moreover, down-regulation of InsP3Rs resulted in desensitization of Ca2+ release from InsP3 sensitive stores.
Sipma
H
H
Laboratorium voor Fysiologie, Campus Gasthuisberg O/N, KU Leuven, Belgium.
Deelman
L
L
Smedt
H D
HD
Missiaen
L
L
Parys
J B
JB
Vanlingen
S
S
Henning
R H
RH
Casteels
R
R
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001011:Aorta / Q000166:cytology; D001920:Bradykinin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D015220:Calcium Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002217:Carbachol / Q000494:pharmacology; D006224:Cricetinae; D015536:Down-Regulation / Q000187:drug effects*; D015870:Gene Expression / Q000187:drug effects; D017930:Genes, Reporter; D006367:HeLa Cells; D006632:Histamine / Q000494:pharmacology; D006801:Humans; D053496:Inositol 1,4,5-Trisphosphate Receptors; D009131:Muscle, Smooth, Vascular / Q000737:chemistry* / Q000166:cytology / Q000187:drug effects; D010277:Parasympathomimetics / Q000494:pharmacology; D011401:Promoter Regions, Genetic / Q000502:physiology; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014667:Vasopressins / Q000494:pharmacology; D014700:Verapamil / Q000494:pharmacology
D002121:Calcium Channel Blockers; D015220:Calcium Channels; C506926:ITPR1 protein, human; D053496:Inositol 1,4,5-Trisphosphate Receptors; D010277:Parasympathomimetics; D018160:Receptors, Cytoplasmic and Nuclear; D014667:Vasopressins; D006632:Histamine; D000255:Adenosine Triphosphate; D002217:Carbachol; D014700:Verapamil; D001920:Bradykinin; D002118:Calcium