Omega-conotoxin- and nifedipine-insensitive voltage-operated calcium channels mediate K(+)-induced release of pro-thyrotropin-releasing hormone-connecting peptides Ps4 and Ps5 from perifused rat hypothalamic slices.
The rat thyrotropin-releasing hormone (TRH) precursor (prepro-TRH) contains five copies of the TRH progenitor sequence linked together by intervening sequences. Recently, we have shown that the connecting peptides prepro-TRH-(160-169) (Ps4) and prepro-TRH-(178-199) (Ps5) are released from rat hypothalamic neurones in response to elevated potassium concentrations, in a calcium-dependent manner. In the present study, the role of voltage-operated calcium channels in potassium-induced release of Ps4 and Ps5 was investigated, using a perifusion system for rat hypothalamic slices. The release of Ps4 and Ps5 stimulated by potassium (70 mM) was blocked by the inorganic ions Co2+ (2.6 mM) and Ni2+ (5 mM). In contrast, the stimulatory effect of KCl was insensitive to Cd2+ (100 microM). The dihydropyridine antagonist nifedipine (10 microM) had no effect on K(+)-evoked release of Ps4 and Ps5. Furthermore, the response to KCl was not affected by nifedipine (10 microM) in combination with diltiazem (1 microM), a benzothiazepine which increases the affinity of dihydropyridine antagonists for their receptor. The dihydropyridine agonist BAY K 8644, at concentrations as high as 1 mM, did not stimulate the basal secretion of Ps4 and Ps5. In addition, BAY K 8644 had no potentiating effect on K(+)-induced release of Ps4 and Ps5. The marine cone snail toxin omega-conotoxin, a blocker of both L- and N-type calcium channels had no effect on the release of Ps4 and Ps5 stimulated by potassium. Similarly, the omega-conopeptide SNX-111, a selective blocker of N-type calcium channels, did not inhibit the stimulatory effect of potassium. The release of Ps4 and Ps5 evoked by high K+ was insensitive to the non-selective calcium channel blocker verapamil (20 microM). Amiloride (1 microM), a putative blocker of T-type calcium channels, did not affect KCl-induced secretion of the two connecting peptides. Taken together, these results indicate that two connecting peptides derived from the pro-TRH, Ps4 and Ps5, are released by K(+)-induced depolarization through activation of voltage-sensitive calcium channels. The calcium channels appear to have a pharmacological profile different from that of L- and N-type channels. Although, their insensitivity to low Cd2+ concentrations and sensitivity to Ni2+ ions would support the involvement of T-type calcium channels, the lack of effect of amiloride suggests that they belong to a yet undefined class of calcium channels.
Valentijn
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K
European Institute for Peptide Research, CNRS URA 650, UA INSERM University of Rouen, Mont-Saint-Aignan, France.
Tranchand Bunel
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D
Vaudry
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H
D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D002104:Cadmium / Q000494:pharmacology; D002121:Calcium Channel Blockers / Q000494:pharmacology*; D015220:Calcium Channels / Q000187:drug effects / Q000502:physiology*; D003035:Cobalt / Q000494:pharmacology; D007031:Hypothalamus / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D066298:In Vitro Techniques; D007700:Kinetics; D008297:Male; D009532:Nickel / Q000494:pharmacology; D009543:Nifedipine / Q000494:pharmacology*; D010446:Peptide Fragments / Q000378:metabolism*; D010456:Peptides, Cyclic / Q000494:pharmacology*; D011188:Potassium / Q000494:pharmacology*; D011498:Protein Precursors / Q000378:metabolism*; D011863:Radioimmunoassay; D051381:Rats; D017208:Rats, Wistar; D013973:Thyrotropin-Releasing Hormone / Q000378:metabolism*; D014700:Verapamil / Q000494:pharmacology; D020960:omega-Conotoxins
D002121:Calcium Channel Blockers; D015220:Calcium Channels; D010446:Peptide Fragments; D010456:Peptides, Cyclic; D011498:Protein Precursors; D020960:omega-Conotoxins; D002104:Cadmium; C053423:Conus magus toxin; C057639:prepro-thyrotropin releasing hormone (160-169); C057640:prepro-thyrotropin-releasing hormone (178-199); D003035:Cobalt; D013973:Thyrotropin-Releasing Hormone; D000584:Amiloride; D009532:Nickel; D014700:Verapamil; D009543:Nifedipine; D011188:Potassium