title: Neurotensin modulates the electrical activity of frog pituitary melanotropes via activation of a G-protein-coupled receptor pharmacologically related to both the NTS1 and nts2 receptors of mammals.
abstract: The primary structure of frog neurotensin (fNT) has recently been determined and it has been shown that fNT is a potent stimulator of alpha-MSH secretion by frog pituitary melanotropes. In the present study, we have investigated the effects of fNT on the electrical activity of cultured frog melanotropes by using the patch-clamp technique and we have determined the pharmacological profile of the receptors mediating the effect of fNT. In the cell-attached configuration, fNT (10(-7) M) provoked an increase in the action current discharge followed by an arrest of spike firing. In the gramicidin-perforated patch configuration, fNT (10(-7) M) induced a depolarization accompanied by an increase in action potential frequency and a decrease in membrane resistance. Administration of graded concentrations (10(-10) to 10(-6) M) of fNT or the C-terminal hexapeptide NT(8-13) caused a dose-dependent increase in the frequency of action potentials with EC(50) of 2 x 10(-8) and 5 x 10(-9) M, respectively. The stimulatory effect of fNT was mimicked by various pseudopeptide analogs, with the following order of potency: Boc-[Trp(11)]NT(8-13) > Boc-[D-Trp(11)]NT(8-13) > Boc-[Lys(8,9), Nal(11)]NT(8-13) > Boc-[Psi11,12]NT(8-13). In contrast, the cyclic pseudopeptide analogs of NT(8-13), Lys-Lys-Pro-D-Trp-Ile-Leu and Lys-Lys-Pro-D-Trp-Glu-Leu-OH, did not affect the electrical activity. The NTS1 receptor antagonist and nts2 receptor agonist SR 48692 (10(-5) M) stimulated the spike discharge but did not block the response to fNT. In contrast, SR 142948A (10(-5) M), another NTS1 receptor antagonist and nts2 receptor agonist, inhibited the excitatory effect of fNT. The specific nts2 receptor ligand levocabastine (10(-6) M) had no effect on the basal electrical activity and the response of melanotropes to fNT. In cells which were dialyzed with guanosine-5'-O-(3-thiotriphosphate) (10(-4) M), fNT caused an irreversible stimulation of the action potential discharge. Conversely, dialysis of melanotropes with guanosine-5'-O-(2-thiodiphosphate) (10(-4) M) completely blocked the effect of fNT. Pretreatment of cells with cholera toxin (1 microg/ml) or pertussis toxin (0.2 microg/ml) did not affect the electrical response to fNT. Intracellular application of the G(o/i/s) protein antagonist GPAnt-1 (3 x 10(-5) M) had no effect on the fNT-evoked stimulation. In contrast, dialysis of melanotropes with the G(q/11) protein antagonist GPAnt-2A (3 x 10(-5) M) abrogated the response to fNT. The present data demonstrate that fNT is a potent stimulator of the electrical activity of frog pituitary melanotropes. These results also reveal that the electrophysiological response evoked by fNT can be accounted for by activation of a G(q/11)-protein-coupled receptor subtype whose pharmacological profile shares similarities with those of mammalian NTS1 and nts2 receptors.
authors.affiliation: European Institute for Peptide Research (IFRMP 23), Laboratory of Cellular and Molecular Neuroendocrinology, INSERM U-413, Centre National de la Recherche Scientifique, University of Rouen, Mont-Saint-Aignan, France.
authors.forename: A
authors.initials: A
authors.lastname: Belmeguenai
authors.forename: H
authors.initials: H
authors.lastname: Vaudry
authors.forename: J
authors.initials: J
authors.lastname: Leprince
authors.forename: B
authors.initials: B
authors.lastname: Vivet
authors.forename: F
authors.initials: F
authors.lastname: Cavelier
authors.forename: J
authors.initials: J
authors.lastname: Martinez
authors.forename: E
authors.initials: E
authors.lastname: Louiset
mesh_terms: D000218:Adamantane / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D002478:Cells, Cultured; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D020962:Heterotrimeric GTP-Binding Proteins / Q000378:metabolism*; D007093:Imidazoles / Q000494:pharmacology; D008024:Ligands; D008297:Male; D008322:Mammals; D008544:Melanocytes / Q000187:drug effects / Q000378:metabolism*; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D009496:Neurotensin / Q000378:metabolism / Q000494:pharmacology*; D018408:Patch-Clamp Techniques; D010446:Peptide Fragments / Q000378:metabolism / Q000494:pharmacology*; D010902:Pituitary Gland / Q000166:cytology*; D011720:Pyrazoles / Q000494:pharmacology; D011804:Quinolines / Q000494:pharmacology; D011895:Rana ridibunda; D018028:Receptors, Neurotensin / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D013873:Thionucleotides / Q000494:pharmacology
chemical_list: D007093:Imidazoles; D008024:Ligands; D010446:Peptide Fragments; D011720:Pyrazoles; D011804:Quinolines; D018028:Receptors, Neurotensin; C104418:SR 142948A; D013873:Thionucleotides; C114036:neurotensin type 1 receptor; D006153:Guanosine Diphosphate; C079087:SR 48692; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D009496:Neurotensin; C054381:neurotensin (8-13); C023427:guanosine 5'-O-(2-thiodiphosphate); D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020962:Heterotrimeric GTP-Binding Proteins; D000218:Adamantane