5‐Hydroxytryptamine receptor‐mediated phosphoinositide hydrolysis in canine cultured tracheal smooth muscle cells

Chuen Mao Yang*, Ying‐Ling ‐L Yo, Jen‐Tsung ‐T Hsieh, Richard Ong

*Corresponding author for this work

Research output: Contribution to journalJournal Article peer-review

30 Scopus citations

Abstract

5‐Hydroxytryptamine (5‐HT) has been shown to induce contraction of tracheal smooth muscle. However, the mechanisms of action of 5‐HT are not known. We therefore investigated the effects of 5‐HT on phospholipase C (PLC)‐mediated phosphoinositide (PI) hydrolysis and its regulation in canine cultured tracheal smooth muscle cells (TSMCs) labelled with [3H]‐inositol. 5‐HT‐induced inositol phosphates (IPs) accumulation was time‐ and dose‐dependent with a half‐maximal response (EC50) and a maximal response at 0.38 ± 0.05 and 10 μm, respectively. Ketanserin and mianserin (10 and 100 nm), 5‐HT2 receptor antagonists, were equipotent in blocking the 5‐HT‐induced IPs accumulation with pKB values of 8.46 and 8.21, respectively. In contrast, the dose‐response curves of 5‐HT‐induced IPs accumulation were not shifted until the concentrations of NAN‐190 and metoclopramide (5‐HT1A and 5‐HT3 receptor antagonists, respectively) were increased up to 10 μm. Pretreatment of TSMCs with pertussis toxin or cholera toxin did not inhibit the 5‐HT‐induced IPs accumulation, but partially inhibited the A1F4‐induced IPs response. Stimulation of IPs accumulation by 5‐HT required the presence of external Ca2+ and was blocked by EGTA. The addition of Ca2+ (3–620 nm) to digitonin‐permeabilized TSMCs directly stimulated IPs accumulation. A further Ca2+‐dependent increase in IPs accumulation was obtained by inclusion of either guanosine 5′‐O(3‐thiotriphoshate) (GTPγS) or 5‐HT. The combination of GTPγS and 5‐HT elicited an additive effect on IPs accumulation. Treatment with phorbol 12‐myristate 13‐acetate (PMA, 1 μm, 30 min) abolished the 5‐HT‐induced IPs accumulation. The concentrations of PMA that gave a half‐maximal and maximal inhibition of 5‐HT‐induced IPs accumulation were 2.2 ± 0.4 nm and 1 μm, n = 3, respectively. The protein kinase C (PKC) activator, 4α‐phorbol 12,13‐didecanoate, at 1 μm, did not influence this response. The inhibitory effect of PMA was reversed by staurosporine, a PKC inhibitor, suggesting that the inhibitory effect of PMA is mediated through the activation of PKC. The site of this inhibition was further investigated by examining the effect of PMA on AlF4‐induced IPs accumulation in canine TSMCs. AlF4‐stimulated IPs accumulation was inhibited by PMA treatment, suggesting that the effect of PMA is distal to the 5‐HT receptor. Acetylcholine‐induced IPs accumulation was completely inhibited by atropine, but not affected by ketanserin or mianserin, suggesting that 5‐HT‐induced IPs accumulation is not due to release of acetylcholine. These results demonstrate that 5‐HT directly stimulates PLC‐mediated PI hydrolysis via a pertussis toxin‐ and cholera toxin‐insensitive GTP binding protein in canine TSMCs and that this coupling process is negatively regulated by PKC. 5‐HT2 receptors may be predominantly mediating IPs accumulation and presumably IP‐induced Ca2+ release may function as the transducing mechanism for 5‐HT‐stimulated contraction of tracheal smooth muscle. 1994 British Pharmacological Society

Original languageEnglish
Pages (from-to)777-786
Number of pages10
JournalBritish Journal of Pharmacology
Volume111
Issue number3
DOIs
StatePublished - 03 1994
Externally publishedYes

Keywords

  • 5‐Hydroxytryptamine
  • G protein
  • inositol phosphates
  • phorbol ester
  • protein kinase C

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