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      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

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          Abstract

          The primary structure of frog neurotensin ( fNT) has recently been determined and it has been shown that fNT is a potent stimulator of α-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<sup>–7</sup>  M) provoked an increase in the action current discharge followed by an arrest of spike firing. In the gramicidin-perforated patch configuration, fNT (10<sup>–7</sup>  M) induced a depolarization accompanied by an increase in action potential frequency and a decrease in membrane resistance. Administration of graded concentrations (10<sup>–10</sup> to 10<sup>–6</sup>  M) of fNT or the C-terminal hexapeptide NT(8–13) caused a dose-dependent increase in the frequency of action potentials with EC<sub>50</sub> of 2 × 10<sup>–8</sup> and 5 × 10<sup>–9</sup>  M, respectively. The stimulatory effect of fNT was mimicked by various pseudopeptide analogs, with the following order of potency: Boc-[Trp<sup>11</sup>]NT(8–13) > Boc-[ D-Trp<sup>11</sup>]NT(8–13) > Boc-[Lys<sup>8,9</sup>, Nal<sup>11</sup>]NT(8–13) > Boc-[Ψ11,12]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<sup>–5</sup>  M) stimulated the spike discharge but did not block the response to fNT. In contrast, SR 142948A (10<sup>–5</sup>  M), another NTS1 receptor antagonist and nts2 receptor agonist, inhibited the excitatory effect of fNT. The specific nts2 receptor ligand levocabastine (10<sup>–6</sup>  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<sup>–4</sup>  M), fNT caused an irreversible stimulation of the action potential discharge. Conversely, dialysis of melanotropes with guanosine-5′- O-(2-thiodiphosphate) (10<sup>–4</sup>  M) completely blocked the effect of fNT. Pretreatment of cells with cholera toxin (1 µg/ml) or pertussis toxin (0.2 µg/ml) did not affect the electrical response to fNT. Intracellular application of the G<sub>o/i/s</sub> protein antagonist GPAnt-1 (3 × 10<sup>–5</sup>  M) had no effect on the fNT-evoked stimulation. In contrast, dialysis of melanotropes with the G<sub>q/11</sub> protein antagonist GPAnt-2A (3 × 10<sup>–5</sup>  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<sub>q/11</sub>-protein-coupled receptor subtype whose pharmacological profile shares similarities with those of mammalian NTS1 and nts2 receptors.

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          Most cited references 4

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          Cloning and expression of a complementary DNA encoding a high affinity human neurotensin receptor.

           D Gully,  D Caput,  X Dumont (1993)
          A human neurotensin receptor (hNTR) cDNA was cloned from the colonic adenocarcinoma cell line HT29. The cloned cDNA encodes a putative peptide of 418 amino acids with 7 transmembrane domains. The amino acid sequence of the hNTR is 84% identical to the rat NTR [Neuron, 4 (1990) 847-854]. Transfection of this cDNA into COS cells results in the expression of receptors with pharmacological properties similar to those found with HT29 cells. Northern blot analysis using the hNTR cDNA probe indicated a single transcript of 4 kb in the brain, the small intestine and blood mononuclear cells.
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            Neurotensin-like immunoreactivity and neurotensin receptors in the rat hypothalamus and in the neurointermediate lobe of the pituitary gland

            In the rat hypothalamus, cell bodies containing neurotensin-like immunoreactivity were mainly found in the medial preoptic area, the periventricular nucleus, the paraventricular nucleus, the supraoptic nucleus and the arcuate nucleus. [3H]neurotensin binding sites were observed throughout the hypothalamus with a dense accumulation of silver grains over the paraventricular nucleus, the arcuate nucleus and the median eminence region. By radioimmunoassay neurotensin-like immunoreactivity was also found in the neurointermediate lobe of the pituitary gland of various mammalian species and in human postmortem posterior pituitary glands. In the rat studies involving pituitary stalk transections and the neurotoxin monosodium glutamate indicated the presence of a neurotensinergic pathway from the arcuate nucleus to the neurointermediate lobe of the pituitary gland. [3H]neurotensin binding sites were found to be concentrated over the intermediate lobe of the pituitary gland and their presence was not affected by pituitary stalk transection, indicating their localization on endocrine cells of the intermediate lobe of the pituitary gland.
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              Effect of acetylcholine on the electrical and secretory activities of frog pituitary melanotrophs.

              The activity of melanotroph cells of the amphibian pars intermedia is regulated by multiple factors including classical neurotransmitters and neuropeptides. In this study, we have examined the possible involvement of acetylcholine (ACh) in the regulation of electrical and secretory activities of frog pituitary melanotrophs. Electrophysiological recordings were conducted on cultured cells by using the patch-clamp technique in the whole-cell configuration. In parallel, alpha-MSH release from acutely dispersed pars intermedia cells was studied by means of the perifusion technique. In all cells tested in the current-clamp mode, superfusion with ACh (10(-6) M) gave rise to a depolarization associated with an enhanced frequency of action potentials. Administration of ACh (10(-6) M) to perifused cells also induced stimulation of alpha-MSH release. These results indicate that the neurotransmitter ACh exerts a direct stimulatory effect on pituitary melanotrophs. The action of ACh on electrical and secretory activities was mimicked by muscarine (10(-5) M), while ACh-induced alpha-MSH secretion was completely abolished by the muscarinic antagonist atropine (10(-6) M). The depolarizing effect of muscarine was suppressed by the specific M1 muscarinic antagonist pirenzepine (10(-5) M), indicating the existence of a M1 subtype muscarinic receptor in frog pars intermedia cells. In addition, using a monoclonal antibody against calf muscarinic receptors, we have visualized, by the immunofluorescence technique, the presence of muscarinic receptor-like immunoreactivity in cultured intermediate lobe cells. Electrophysiological recordings showed that nicotine (10(-5) M) induces membrane depolarization associated with an increase of the frequency of action potentials.(ABSTRACT TRUNCATED AT 250 WORDS)
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                Author and article information

                Journal
                NEN
                Neuroendocrinology
                10.1159/issn.0028-3835
                Neuroendocrinology
                S. Karger AG
                0028-3835
                1423-0194
                2000
                December 2000
                22 December 2000
                : 72
                : 6
                : 379-391
                Affiliations
                aEuropean Institute for Peptide Research (IFRMP 23), Laboratory of Cellular and Molecular Neuroendocrinology, Institut National de la Santé et de la Recherche Médicale U-413, Centre National de la Recherche Scientifique, University of Rouen, Mont-Saint-Aignan, and bLaboratory of Amino Acids, Peptides and Proteins, Centre National de la Recherche Scientifique UMR 5810, University of Montpellier 2, Montpellier, France
                Article
                54607 Neuroendocrinology 2000;72:379–391
                10.1159/000054607
                11146421
                © 2000 S. Karger AG, Basel

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                Page count
                Figures: 8, References: 49, Pages: 13
                Categories
                Pituitary Cell Biology

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