Characterization of signaling pathways coupled to melatonin receptors in gastrointestinal smooth muscle

Abstract

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. 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. MT1, but not MT2 receptors are expressed in freshly dispersed and cultured gastric smooth muscle cells. Melatonin selectively activated Gq and stimulated phosphoinositide (PI) hydrolysis in freshly dispersed and cultured muscle cells. PI hydrolysis was blocked by the expression of Gq, but not Gi minigene in cultured muscle cells. Melatonin also caused rapid increase in cytosolic Ca(2+) as determined by epifluorescence microscopy in fura-2 loaded single smooth muscle cells, and induced rapid contraction. Melatonin-induced PI hydrolysis and contraction were blocked by a non-selective MT1/MT2 antagonist luzindole (1 μM), but not by a selective MT2 antagonist 4P-PDOT (100 nM), and by the PLC inhibitor U73122. MT2 selective agonist IIK7 (100 nM) had no effect on PI hydrolysis and contraction. We conclude that rabbit gastric smooth muscle cells express melatonin MT1 receptors coupled to Gq. Activation of these receptors causes stimulation of PI hydrolysis and increase in cytosolic Ca(2+), and elicits muscle contraction.

Figure 1. Expression of MT1 receptors and activation of Gα_q by melatonin in gastric smooth muscle cells

(A) RT-PCR. Total RNA isolated from cultured (first passage) rabbit gastric muscle cells and the brain was reverse transcribed, and cDNA was amplified with specific primers for MT₁ or MT₂. Experiments were done in the presence (+ RT) or absence (−RT) of reverse transcriptase (RT). PCR product with predicted size was obtained in the presence of reverse transcriptase with primers for MT₁ (194 bp), but not with primers for MT₂, in smooth muscle cells (SMC), whereas PCR products were obtained with primers for MT₁ (194 bp) and MT₂ (392 bp) in the brain. (B) Western blot. Lysates prepared from dispersed smooth muscle cells (lane 1), cultured gastric smooth muscle cells (lane 2), and the brain (lane 3) of rabbit were run on SDS-PAGE and analyzed by western blot. Proteins were probed with polyclonal antibodies to MT₁ (1:1000) or MT₂ (1:1000). A protein band corresponding to 40 kDa was obtained with only MT₁ antibody in smooth muscle cells, whereas a protein bands corresponding to 40 kDa were obtained with MT₁ and MT₂ antibody in the brain.

Figure 2. Selective activation of G_q proteins by melatonin

Membranes were isolated from dispersed gastric muscle cells and incubated with [³⁵S]GTPγS for 20 min in the presence or absence of melatonin (1 μM). Aliquots were added to wells coated with antibody to Gα_i2, Gα_i3, Gα_s, or Gα_q for 2 h and bound radioactivity from each well was counted by liquid scintillation. The amount of [³⁵S]GTPγS bound to the activated Gα subunit was expressed as counts per minute (cpm) per milligram of protein. Melatonin induced significant increase in the binding of [³⁵S]GTPγS.Gα complexes to wells coated with Gα_q antibody only. Values are mean±SEM of 4 experiments. **p<0.001 significant increase in Gα_q-[³⁵S]GTPγS binding in respone to melatonin.

Figure 3. Stimulation of PLC-β activity and release of Ca²⁺ by melatonin

(A) Phosphoinositide-specific (PI) hydrolysis (PLC-β activity) in response to melatonin was measured in dispersed muscle cells labeled with myo-[³H]inositol. Freshly dispersed muscle cells were treated for 60 s with different concentrations of melatonin and PLC-β activity was measured as increase in water-soluble [³H]inositol formation. The results are expressed as [³H]inositol phosphate formation in counts per minute (cpm) per mg protein above basal levels (basal: 642±99 cpm/mg protein). Values are means±SEM of 4 experiments. (B) Isolated smooth cells were loaded with 5 μM fura-2 and treated with 1 μM melatonin in the absence of extracellular Ca²⁺. The cells were alternately excited at 380 nm and 340 nm. The background and autofluorescence were corrected from images of a cell without the fura 2. Results are expressed as 340/380 ratio and an increase in ratio reflects an increase in cytosolic Ca²⁺. The figure shows results obtained from 38 cells.

Figure 4. Gα_q-dependent activation of PI hydrolysis by melatonin

Cultured gastric muscle cells labeled with myo-[³H]inositol and expressing Gα_q minigene, Gα_i minigene, or control vector were treated with melatonin (1 μM), cholecycstokinin (CCk, 1 nm) or cyclopentyladenosine (CPA, 1 μM) for 60 s. Total [³H]inositol phosphates were separated by ion-exchange chromatography. PI hydrolysis activity stimulated by melatonin or CCK was abolished in cells expressing Gα_q minigene, but was not affected in cells expressing Gα_i minigene. In contrast, PI hydrolysis activity stimulated by CPA was abolished in cells expressing Gα_i minigene, but was not affected in cells expressing Gα_q minigene. Results are expressed as total [³H]inositol phosphate formation in cpm/mg protein. Values are means ± SEM of four experiments. ** Significant inhibition from control response (P<0.01).

Figure 5. Stimulation of muscle contraction by melatonin

Contraction of muscle cells was measured as decrease in basal cell length in response to various concentrations of melatonin. Muscle cells (0.5 ml cell suspension) were treated with melatonin for 30 s and the reaction was terminated with 0.1% acrolein. The mean length of 50 muscle cells was measured by scanning micrometry and was compared with the length of untreated muscle cells (125±4 μm). The contractile response was expressed as the percent decrease in the mean cell length from control cell length. Values are means±SEM of 6 experiments.

Figure 6. Stimulation PI hydrolysis and muscle contraction by melatonin via MT₁ receptors

(A) Phosphoinositide-specific (PI) hydrolysis (PLC-β activity) was measured in dispersed muscle cells labeled with myo-[³H]inositol. Cells were treated for 60 s with melatonin in the presence or absence of a non-selective MT1/MT2 receptor antagonist luzindole (100 nM), a selective MT2 receptor antagonist 4P- PDOT (100 nM), PI hydrolysis inhibitor (10 μM) or MLCK inhibitor ML-9 (1 μM), or with a selective MT2 receptor agonist IIK7 alone (100 nM). PLC-β activity was measured as increase in water-soluble [³H]inositol formation. The results are expressed as [³H]inositol phosphate formation in counts per minute (cpm) per mg protein above basal levels (basal: 562±102 cpm/mg protein). Values are means+SEM of 4 experiments. ** Significant inhibition from control melatonin response (P<0.01). (B) Dispersed muscle cells were treated for 30 s with melatonin in the presence or absence of luzindole (100 nM), 4P-PDOT (100 nM), (10 μM), or MLCK inhibitor ML-9 (1 μM), or with IIK7 alone (100 nM). Contraction of muscle cells was measured as decrease in basal cell length in response to various concentrations of melatonin. Muscle cells (0.5 ml cell suspension) were treated with melatonin (1 μM) in the presence or absence of U73122 (10 μM) or ML-9 (1 μM). The mean length of 50 muscle cells was measured by scanning micrometry and was compared with the length of untreated muscle cells (119±6 μm). The contractile response was expressed as the percent decrease in the mean cell length from control cell length. Values are means±SEM of 6 experiments. ** Significant inhibition from control melatonin response (P<0.01).

Figure 7. Pathway mediating muscle contraction by melatonin

In gastric smooth muscle, melatonin interacts with MT1 receptors, which is coupled to stimulation of phosphoinositide-specific phospholipase C (PLC-β) via Gq. Stimulation of PLC-β activity results in the generation of inositol 1,4,5-trisphosphate (IP₃) and IP₃-dependent Ca²⁺ release leading to muscle contraction.