Effect of forskolin on endothelin-induced phosphoinositide hydrolysis and calcium mobilization in cultured canine tracheal smooth muscle cells.
1. The effects of increase in intracellular adenosine 3':5'-cyclic monophosphate (cAMP) on endothelin-1 (ET-1)-induced generation of inositol phosphates (IPs) and increase in intracellular Ca2+ ([Ca2+]i) were investigated in canine cultured tracheal smooth muscle cells (TSMCs). 2. Pretreatment of TSMCs with either cholera toxin (CTX; 10 microg ml(-1), 4 h), forskolin (10 microM, 30 min), or dibutyryl cAMP (1 mM, 30 min) inhibited ET-1-stimulated Ca2+ mobilization (by 23 +/- 5%, n = 8) and IPs accumulation (by 32 +/- 6%, n = 4). While after treatment with forskolin for 24 h, the cells retained the ability to respond to ET-1-induced Ca2+ mobilization to the same extent as the control group. 3. Forskolin (1-100 microM) inhibited the ET-1-induced increase in [Ca2+]i, but the lower concentrations had little effect on this response. The inhibitory effects of these agents produced both depression of the maximal response and a shift to the right of the concentration-response curve of ET-1 without changing the -logEC50 values. 4. The water-soluble forskolin analogue L-858051, 7-deacetyl-7beta-(gamma-N-methylpiperazino)-butyryl forskolin, significantly inhibited ET-1-stimulated IPs accumulation. In contrast, the addition of 1,9-dideoxy forskolin, an inactive analogue of forskolin, had little effect on stimulated responses. Moreover, SQ-22536, 9-(tetrahydro-2-furanyl)-9H-purin-6-amine, an inhibitor of adenylate cyclase, and both H-89, N-(2-aminoethyl)-5-isoquinolinesulfonamide, and HA-1004, N-(2-guanidinoethyl)-5-isoquinolinesulfonamide, inhibitors of cAMP-dependent protein kinase (PKA), attenuated the ability of forskolin to inhibit ET-1-induced IPs accumulation. These results suggest that activation of cAMP/PKA was involved in these inhibitory effects of forskolin. 5. The locus of this inhibition of forskolin treatment on AlF4(-)-stimulated IPs accumulation was investigated in canine TSMCs. The AlF4(-)-induced IPs accumulation was inhibited by forskolin, supporting that G protein(s) are directly activated by AlF4- and uncoupled to phospholipase C by forskolin treatment. 6. We conclude that cAMP elevating agents inhibit ET-1-stimulated generation of IPs and Ca2+ mobilization in canine cultured TSMCs. Since generation of IPs and increases in [Ca2+]i are very early events in the activation of ET-1 receptors, attenuation of these events by cAMP elevating agents might well contribute to the inhibitory effect of cAMP on tracheal smooth muscle function.
Yang
C M
CM
Department of Pharmacology, College of Medicine, Chang Gung University, Kwei-San, Tao-Yuan, Taiwan.
Pan
S L
SL
Chiu
C T
CT
Lin
C C
CC
Hsu
Y M
YM
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology*; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004285:Dogs; D019332:Endothelin-1 / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D005260:Female; D019204:GTP-Binding Proteins / Q000187:drug effects / Q000502:physiology; D006868:Hydrolysis; D008297:Male; D009130:Muscle, Smooth / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D010716:Phosphatidylinositols / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D013268:Stimulation, Chemical; D014132:Trachea / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology*
D019332:Endothelin-1; D010716:Phosphatidylinositols; D005576:Colforsin; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D019204:GTP-Binding Proteins; D002118:Calcium