5-Hydroxytryptamine-induced phosphoinositide hydrolysis and Ca2+ mobilisation in canine cultured aorta smooth muscle cells.
The effect of 5-hydroxytryptamine (5-HT) on phospholipase C (PLC)-mediated phosphoinositide (PI) hydrolysis and intracellular Ca2+ ([Ca2+]i) changes was investigated in canine cultured aorta smooth muscle cells (ASMCs). 5-HT-stimulated inositol phosphate (IP) accumulation was time and concentration dependent with a half-maximal response (pEC50) and a maximal response at 6.4 and 10 microM, n = 6, respectively. Stimulation of ASMCs by 5-HT produced an initial transient peak followed by a sustained, concentration-dependent elevation in [Ca+]i. The half-maximal response (pEC50) values of 5-HT for the peak and sustained plateau were 7.1 and 6.9, respectively. Ketanserin and mianserin (1 and 3 nM), 5-HT2A antagonists, were equipotent and had high affinity in antagonising the 5-HT-induced IP accumulation and [Ca2+]i change with pK(B) values of 8.6-9.1 and 8.6-9.4, respectively. In contrast, the concentration-effect curves of 5-HT-induced IP and [Ca2+]i responses were not shifted until the concentrations of NAN-190 and metoctopramide (5-HT1A and 5-HT3 receptor antagonists, respectively) were increased to as high as 1 microM with pK(B) values of 5.7-6.3 and 6.1-6.6, respectively, indicating that the 5-HT receptor-mediated responses had low affinity for these antagonists. Pre-treatment of ASMCs with pertussis toxin (100 ng/mL, 24 h) caused a significant inhibition of 5-HT-induced IP accumulation and [Ca2+]i change in ASMCs. Depletion of external Ca2+ or removal of Ca2+ by addition of EGTA led to a significant attenuation of IP accumulation and [Ca2+]i change induced by 5-HT. Influx of external Ca2+ was required for the 5-HT-induced responses, because Ca2+-channel blockers--verapamil, nifedipine and Ni2+--partly inhibited the 5-HT-induced IP accumulation and Ca2+ mobilisation. The sustained elevation of [Ca2+]i response to 5-HT was dependent on the presence of external Ca2+. Removal of external Ca2+ by addition of 5 mM EGTA during the sustained phase caused a rapid decline in [Ca2+]i to lower than the resting level. The sustained elevation of [Ca2+]i could then be evoked by addition of 1.8 mM Ca2+ in the continued presence of 5-HT. These results demonstrate that 5-HT directly stimulates PLC-mediated PI hydrolysis and Ca2+ mobilisation, at least in part, through a pertussis toxin-sensitive G protein in canine ASMCs. 5-HT2A receptors may be predominantly mediating IP accumulation, and subsequently IP-induced Ca2+ mobilisation may function as the transducing mechanism for 5-HT-stimulated contraction of aorta smooth muscle.
Chiu
C T
CT
Department of Pharmacology, College of Medicine, Chang Gung University, Tao-Yuan, Taiwan.
Tsao
H L
HL
Fan
L W
LW
Wang
C C
CC
Chien
C S
CS
Yang
C M
CM
D000818:Animals; D001011:Aorta / Q000166:cytology; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D004285:Dogs; D005260:Female; D006868:Hydrolysis; D007295:Inositol Phosphates / Q000378:metabolism; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism*; D037342:Pertussis Toxin; D010716:Phosphatidylinositols / Q000378:metabolism*; D012701:Serotonin / Q000378:metabolism* / Q000494:pharmacology; D012702:Serotonin Antagonists / Q000494:pharmacology; D017366:Serotonin Receptor Agonists / Q000494:pharmacology; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D007295:Inositol Phosphates; D010716:Phosphatidylinositols; D012702:Serotonin Antagonists; D017366:Serotonin Receptor Agonists; D010566:Virulence Factors, Bordetella; D012701:Serotonin; D037342:Pertussis Toxin; D002118:Calcium