| The Melatonin, a close derivative of serotonin, is involved in physiological regulation of circadian rhythms. In the gastrointestinal (GI) system, melatonin exhibits endocrine, paracrine and autocrine actions and is implicated in the regulation of GI motility. Generally, melatonin actions oppose the stimulatory actions of serotonin on motility. However, it is not known whether melatonin can also act directly on GI smooth muscle cells. The aim of the present study was to determine the expression of melatonin receptors in smooth muscle and identify their signaling pathways. Muscle cells were isolated from rabbit distal stomach by enzymatic digestion, filtration and centrifugation and cultured in DMEM-10. Expression of melatonin receptors, MT1 and MT2, was determined by RT-PCR and Western blot. G protein activity was measured by melatonin-induced increase in Galpha binding to [35S]GTPgammaS. Phosphoinositide (PI)-specific phospholipase C (PLC-beta) activity was measured by ion-exchange chromatography. Cytosolic Ca2+ was measured in fura-2 loaded cells and muscle contraction was measured by scanning micrometry. In cultured gastric smooth muscle cells MT1 was detected by RT-PCR and western blot. Melatonin activated Galphaq, but not Galphas, Galpha i1, Galphai2, or Galphai3. Consistent with activation of Galphaq, melatonin stimulated PLC-beta activity (PI hydrolysis), increased cytosolic Ca2+, and elicited muscle contraction. Stimulation of PLC-beta activity was blocked by the expression of Galphaq minigene and contraction was blocked by the PLC-beta inhibitor, U73122. We conclude that gastric smooth muscle cells express receptors for melatonin (MT1) coupled to Gq. The receptors mediate stimulation of PLC-beta activity and increase in cytosolic Ca2+, and elicit muscle contraction. |
