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
Authors: Belmeguenai Amor , Vaudry Hubert , Leprince Jérôme , Vivet Bertrand , Cavelier Florine , Martinez Jean , Louiset Estelle 
Published: April 2003

The primary structure of frog neurotensin (<i>f</i>NT) has recently been determined and it has been shown that <i>f</i>NT is a potent stimulator of α-MSH secretion by frog pituitary melanotropes. In the present study, we have investigated the effects of <i>f</i>NT 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 <i>f</i>NT. In the cell-attached configuration, <i>f</i>NT (10<sup>–7</sup> <i>M</i>) provoked an increase in the action current discharge followed by an arrest of spike firing. In the gramicidin-perforated patch configuration, <i>f</i>NT (10<sup>–7</sup> <i>M</i>) 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> <i>M</i>) of <i>f</i>NT 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> <i>M</i>, respectively. The stimulatory effect of <i>f</i>NT was mimicked by various pseudopeptide analogs, with the following order of potency: Boc-[Trp<sup>11</sup>]NT(8–13) > Boc-[<i>D</i>-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-<i>D</i>-Trp-Ile-Leu and Lys-Lys-Pro-<i>D</i>-Trp-Glu-Leu-OH, did not affect the electrical activity. The NTS1 receptor antagonist and nts2 receptor agonist SR 48692 (10<sup>–5</sup> <i>M</i>) stimulated the spike discharge but did not block the response to <i>f</i>NT. In contrast, SR 142948A (10<sup>–5</sup> <i>M</i>), another NTS1 receptor antagonist and nts2 receptor agonist, inhibited the excitatory effect of <i>f</i>NT. The specific nts2 receptor ligand levocabastine (10<sup>–6</sup> <i>M</i>) had no effect on the basal electrical activity and the response of melanotropes to <i>f</i>NT. In cells which were dialyzed with guanosine-5′-<i>O</i>-(3-thiotriphosphate) (10<sup>–4</sup> <i>M</i>), <i>f</i>NT caused an irreversible stimulation of the action potential discharge. Conversely, dialysis of melanotropes with guanosine-5′-<i>O</i>-(2-thiodiphosphate) (10<sup>–4</sup> <i>M</i>) completely blocked the effect of <i>f</i>NT. Pretreatment of cells with cholera toxin (1 µg/ml) or pertussis toxin (0.2 µg/ml) did not affect the electrical response to <i>f</i>NT. Intracellular application of the G<sub>o/i/s</sub> protein antagonist GPAnt-1 (3 × 10<sup>–5</sup> <i>M</i>) had no effect on the <i>f</i>NT-evoked stimulation. In contrast, dialysis of melanotropes with the G<sub>q/11</sub> protein antagonist GPAnt-2A (3 × 10<sup>–5</sup> <i>M</i>) abrogated the response to <i>f</i>NT. The present data demonstrate that <i>f</i>NT is a potent stimulator of the electrical activity of frog pituitary melanotropes. These results also reveal that the electrophysiological response evoked by <i>f</i>NT 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.