title: Involvement of non-selective Ca2+ channels in the contraction induced by alkalinization of rat anococcygeus muscle cells.
abstract: Intracellular pH is a modulator of cellular functions such as smooth muscle contraction. Changes in cytosolic Ca(2+) concentration ([Ca(2+)](c)) associated with contraction are brought about by Ca(2+) influx and release from the sarcoplasmic reticulum, and alterations in the intracellular pH can affect both processes. In this work, therefore, we have investigated the Ca(2+) influx pathway that contributes to the contraction induced by the alkalinizing agent NH(4)Cl in the rat anococcygeus smooth muscle. For this purpose, we measured the isometric tension in muscle preparations, and [Ca(2+)](c) was measured on isolated cells loaded with 5 micromol/l FURA2/AM by using the ratio 340/380 nm. NH(4)Cl (10 mmol/l) induced a larger increase in [Ca(2+)](c) (100%) when compared with the [Ca(2+)](c) increase induced by 0.1 micromol/l phenylephrine (57.0+/-12.3% n=4). Incubation of the muscle preparations for 1 min in Ca(2+)-free medium reduced the contractions induced by 10 mmol/l NH(4)Cl to 11.5+/-5.1% (n=5), when compared with the contractions induced in 2.5 mmol/l Ca(2+) solution (100%). After 3 min in Ca(2+) free medium, contractions stimulated with NH(4)Cl were almost abolished (0.6+/-0.4%, n=5). In the same way, incubation with 10 micromol/l 1-[beta-[3[(4-methoxyphenyl)propoxyl]-4-methoxy-phenetyl]-1H-imidazole hydrochloride (SKF96365), a non-selective Ca(2+) channels, reduced the contractions stimulated with NH(4)Cl to 47.6+/-6.7% (n=7). On the other hand, 1 micromol/l verapamil, a voltage-operated Ca(2+) channel blocker and 0.05 micromol/l calphostin C, a protein kinase-C inhibitor, did not alter the contractions induced by NH(4)Cl. On isolated cells, [Ca(2+)](c) was reduced to 72.2+/-1.7% (n=4) by 10 micromol/l SKF96365. Taken together, our results suggest that NH(4)Cl induces contraction of rat anococcygeus smooth muscle cells, as well as [Ca(2+)](c) increase due to Ca(2+) influx through non-selective Ca(2+) channels.
authors.affiliation: Laboratório de Farmacologia, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, USP, Ribeirão Preto, SP - Brazil.
authors.forename: Carolina A
authors.initials: CA
authors.lastname: Restini
authors.forename: Lusiane M
authors.initials: LM
authors.lastname: Bendhack
mesh_terms: D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism / Q000502:physiology; D002121:Calcium Channel Blockers / Q000494:pharmacology; D015220:Calcium Channels / Q000187:drug effects / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes; D016257:Fura-2; D007093:Imidazoles / Q000494:pharmacology; D066298:In Vitro Techniques; D008297:Male; D009119:Muscle Contraction / Q000187:drug effects; D009130:Muscle, Smooth / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D009281:Naphthalenes / Q000494:pharmacology; D010656:Phenylephrine / Q000494:pharmacology; D051381:Rats; D014662:Vasoconstrictor Agents / Q000494:pharmacology; D014700:Verapamil / Q000494:pharmacology
chemical_list: D002121:Calcium Channel Blockers; D015220:Calcium Channels; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D007093:Imidazoles; D009281:Naphthalenes; D014662:Vasoconstrictor Agents; D000643:Ammonium Chloride; D010656:Phenylephrine; D014700:Verapamil; C058819:calphostin C; C063159:1-(2-(3-(4-methoxyphenyl)propoxy)-4-methoxyphenylethyl)-1H-imidazole; D002118:Calcium; D016257:Fura-2