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 μM, n = 6, respectively. Stimulation of ASMCs by 5-HT produced an initial transient peak followed by a sustained, concentration-dependent elevation in [Ca²⁺](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-HT(2A) 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 metoclopramide (5-HT(1A) and 5-HT₃ receptor antagonists, respectively) were increased to as high as 1 μM 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-HT(2A) 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. Copyright (C) 1999 Elsevier Science Inc.