Expression and functional characterization of the mt1 melatonin receptor from rat brain in Xenopus oocytes: evidence for coupling to the phosphoinositol pathway.
Melatonin-sensitive receptors were expressed in Xenopus laevis oocytes following an injection of mRNA from rat brain. The administration of 0.1-100 micromol/L melatonin to voltage-clamped oocytes activates calcium-dependent chloride currents via a pertussis toxin-sensitive G protein and the phosphoinositol pathway. To determine which melatonin receptor type (mt1, MT2, MT3) is functionally expressed in the Xenopus oocytes, we used (i) agonists and antagonists of different receptor types to characterize the pharmacological profile of the expressed receptors and (ii) a strategy of inhibiting melatonin receptor function by antisense oligonucleotides. During pharmacological screening administration of the agonists 2-iodomelatonin and 2-iodo-N-butanoyl-5-methoxytryptamine (IbMT) to the oocytes resulted in oscillatory membrane currents, whereas the administration of the MT3 agonist 5-methoxycarbonylamino-N-acetyltryptamine (GR135,531) exerted no detectable membrane currents. The melatonin response was abolished by a preceding administration of the antagonists 2-phenylmelatonin and luzindole but was unaffected by the MT3 antagonist prazosin and the MT2 antagonist 4-phenyl-2-propionamidotetralin (4-P-PDOT). In the antisense experiments, in the control group the melatonin response occurred in 45 of 54 mRNA-injected oocytes (83%). Co-injection of the antisense oligonucleotide, corresponding to the mt1 receptor mRNA, caused a marked and significant reduction in the expression level (13%; P < 0.001). In conclusion, the results demonstrate that injection of mRNA from rat brain in Xenopus oocytes induced the expression of the mt1 receptor which is coupled to the phosphoinositol pathway.
Blumenau
C
C
Institute of Physiology, Münster, Germany.
Berger
E
E
Fauteck
J D
JD
Madeja
M
M
Wittkowski
W
W
Speckmann
E J
EJ
Musshoff
U
U
D000818:Animals; D001921:Brain / Q000378:metabolism*; D017931:DNA Primers / Q000737:chemistry; D004594:Electrophysiology; D015870:Gene Expression / Q000502:physiology; D008550:Melatonin / Q000031:analogs & derivatives / Q000494:pharmacology; D008564:Membrane Potentials / Q000187:drug effects; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D009865:Oocytes / Q000187:drug effects / Q000502:physiology*; D018408:Patch-Clamp Techniques; D010716:Phosphatidylinositols / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D011956:Receptors, Cell Surface / Q000819:agonists / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D018160:Receptors, Cytoplasmic and Nuclear / Q000819:agonists / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D044094:Receptors, Melatonin; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D014982:Xenopus laevis / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; D016376:Oligonucleotides, Antisense; D010716:Phosphatidylinositols; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; D018160:Receptors, Cytoplasmic and Nuclear; D044094:Receptors, Melatonin; D008550:Melatonin