5-Hydroxytryptamine-Induced Phosphoinositide Hydrolysis and Ca 2+ Mobilisation in Canine Cultured Aorta Smooth Muscle Cells

Abstract

The effect of 5-hydroxytryptamine (5-HT) on phospholipase C (PLC)-mediated phosphoinositide (PI) hydrolysis and intracellular Ca 2+ ([Ca 2+] 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 (pEC 50) and a maximal response at 6.4 and 10 μM, n = 6, respectively. Stimulation of ASMCs by 5-HT produced an initial transient peak followed by a sustained, concentration-dependent elevation in [Ca 2+] i. The half-maximal response (pEC 50) 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-HT 2A antagonists, were equipotent and had high affinity in antagonising the 5-HT-induced IP accumulation and [Ca 2+] i change with p K 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 [Ca 2+] i responses were not shifted until the concentrations of NAN-190 and metoclopramide (5-HT 1A and 5-HT 3 receptor antagonists, respectively) were increased to as high as 1 μM with p K 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 [Ca 2+] i change in ASMCs. Depletion of external Ca 2+ or removal of Ca 2+ by addition of EGTA led to a significant attenuation of IP accumulation and [Ca 2+] i change induced by 5-HT. Influx of external Ca 2+ was required for the 5-HT-induced responses, because Ca 2+-channel blockers—verapamil, nifedipine and Ni 2+—partly inhibited the 5-HT-induced IP accumulation and Ca 2+ mobilisation. The sustained elevation of [Ca 2+] i response to 5-HT was dependent on the presence of external Ca 2+. Removal of external Ca 2+ by addition of 5 mM EGTA during the sustained phase caused a rapid decline in [Ca 2+] i to lower than the resting level. The sustained elevation of [Ca 2+] i could then be evoked by addition of 1.8 mM Ca 2+ in the continued presence of 5-HT. These results demonstrate that 5-HT directly stimulates PLC-mediated PI hydrolysis and Ca 2+ mobilisation, at least in part, through a pertussis toxin-sensitive G protein in canine ASMCs. 5-HT 2A receptors may be predominantly mediating IP accumulation, and subsequently IP-induced Ca 2+ mobilisation may function as the transducing mechanism for 5-HT-stimulated contraction of aorta smooth muscle.