Receptor-mediated modulation of avian caecal muscle contraction by melatonin: Role of tyrosine protein kinase

Abstract

Melatonin receptors in the quail caecum were studied by 2[125I]iodomelatonin binding assay and the involvement of tyrosine protein kinase in the melatonin-induced contraction was explored. The binding of 2[125I]iodomelatonin in the quail caecum membrane preparations was saturable, reversible and of high affinity with an equilibrium dissociation constant (Kd) of 24.6 ± 1.1 pm (n = 7) and a maximum number of binding sites (Bmax) of 1.95 ± 0.09 fmol (mg/protein) (n = 7). The relative order of potency of indoles in competing for 2[125I]iodomelatonin binding was: 2-iodomelatonin > melatonin > 2-phenylmelatonin > 6-chloromelatonin > 6-hydroxymelatonin > N-acetylserotonin, indicating that ML1 receptors are involved. The binding was inhibited by Mel1b melatonin receptor antagonists, luzindole and 4-phenyl-2-propionamidotetralin (4-P-PDOT) as well as by non-hydrolyzable analogs of GTP like GTPγS and Gpp(NH)p but hot by adenosine nucleotides. The latter suggests that the action ofmelatonin on the caecum is G-protein linked. Cumulative addition of melatonin (1-300 nM) potentiated both the amplitude and frequency of spontaneous contractions in the quail caecum. The potentiation of rhythmic contractions was blocked by both luzindole and 4-P-PDOT. Antagonists of tyrosine kinase, genistein(2 μM) and erbstatin(4 μM) suppressed the modulation of spontaneous contractions by melatonin, but hot inhibitors of protein kinase C (PKC) or protein kinase A (PKA). Melatonin-induced increment in spontaneous contraction was blocked by nifedipine (0.4 nM). Thus, we suggest that melatonin potentiates spontaneous contraction in the quail caecum via interacting with G-protein-coupled Mel1b receptor which may activate L-type Ca2+ channels by mobilizing tyrosine kinases.