5-Hydroxytryptamine-induced phosphoinositide hydrolysis and Ca²⁺ mobilization in canine cultured aorta smooth muscle cells

The effect of 5-HT on phospholipase C (PLC)-mediated phosphoinositide (PI) hydrolysis and intracellular Ca²⁺ ([Ca²⁺]<inf>i</inf>) 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<inf>50</inf>) and a maximal response at 6.4 and 10 μM, n=9, respectively. Stimulation of ASMCs by 5-HT produced an initial transient peak followed by a sustained, concentration-dependent elevation in [Ca²⁺]<inf>i</inf>. The half-maximal response (pEC<inf>50</inf>) of 5-HT for the peak and sustained plateau were 6.8 and 6.4, respectively. Ketanserin and mianserin, 5-HT<inf>2A</inf> antagonists, were equipotent and had high affinity in antagonizing the 5-HT-induced IP accumulation and [Ca²⁺]<inf>i</inf> change with pK<inf>B</inf> values of 8.9 and 8.8, respectively. In contrast, the concentration-effect curves of 5-HT-induced IP and [Ca²⁺]<inf>i</inf> responses were not shifted until the concentrations of NAN-190 and metoclopramide (5-HT<inf>1A</inf> and 5-HT<inf>3</inf> receptor antagonists, respectively) were increased up to 10 μM with pK<inf>B</inf> values of 5.8 and 5.7, indicating the 5-HT, receptor-mediated responses had low affinity for these antagonists. Pretreatment of ASMCs with pertussis toxin caused a significant inhibition of 5-HT-induced IP accumulation and [Ca²⁺]<inf>i</inf> change in ASMCs. Incubation of ASMCs in the absence of external Ca²⁺ led to a significant attenuation of IP accumulation and [Ca²⁺]<inf>i</inf> change induced by 5-HT. Influx of external Ca²⁺ was required for the 5-HT-indcued responses, since Ca²⁺-channel blockers, verapamil, nifedipine, and Ni²⁺, partially inhibited the 5-HT-induced IP accumulation and Ca²⁺ mobilization. The sustained elevation of [Ca²⁺]<inf>i</inf> 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⁺]<inf>i</inf> to lower than the resting level. The sustained elevation of [Ca²⁺]<inf>i</inf> 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-mcdiated PI hydrolysis and Ca²⁺ mobilization via a pertussis toxin-sensitive G protein in canine ASMCs. 5-HT<inf>2A</inf> receptors may be predominantly mediating IP accumulation and subsequently IP-induced Ca²⁺ mobilization may function as the transducing mechanism for 5-HT-stimulated contraction of aorta smooth muscle.