Restini Carolina A, Bendhack Lusiane M
Laboratório de Farmacologia, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, USP, Ribeirão Preto, SP - Brazil.
Eur J Pharmacol. 2006 Dec 28;553(1-3):288-96. doi: 10.1016/j.ejphar.2006.10.024. Epub 2006 Oct 18.
Intracellular pH is a modulator of cellular functions such as smooth muscle contraction. Changes in cytosolic Ca(2+) concentration (Ca(2+)) 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+) 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+) (100%) when compared with the Ca(2+) 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+) 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+) increase due to Ca(2+) influx through non-selective Ca(2+) channels.
细胞内pH是细胞功能（如平滑肌收缩）的调节因子。与收缩相关的胞质Ca(2+)浓度（[Ca(2+)]c）变化是由Ca(2+)内流和肌浆网释放引起的，细胞内pH的改变可影响这两个过程。因此，在本研究中，我们研究了在大鼠肛门尾骨肌平滑肌中，导致碱化剂氯化铵诱导收缩的Ca(2+)内流途径。为此，我们测量了肌肉标本中的等长张力，并通过使用340/380nm比值，对加载有5微摩尔/升FURA2/AM的分离细胞进行[Ca(2+)]c测量。与0.1微摩尔/升去氧肾上腺素诱导的[Ca(2+)]c增加（57.0±12.3%，n = 4）相比，10毫摩尔/升氯化铵诱导的[Ca(2+)]c增加幅度更大（100%）。与在2.5毫摩尔/升Ca(2+)溶液中诱导的收缩（100%）相比，肌肉标本在无Ca(2+)培养基中孵育1分钟后，10毫摩尔/升氯化铵诱导的收缩降低至11.5±5.1%（n = 5）。在无Ca(2+)培养基中孵育3分钟后，氯化铵刺激的收缩几乎完全消失（0.6±0.4%，n = 5）。同样，与10微摩尔/升1-[β-[3[(4-甲氧基苯基)丙氧基]-4-甲氧基苯乙基]-1H-咪唑盐酸盐（SKF96365，一种非选择性Ca(2+)通道阻滞剂）孵育后，氯化铵刺激的收缩降低至47.6±6.7%（n = 7）。另一方面，1微摩尔/升维拉帕米（一种电压门控Ca(2+)通道阻滞剂）和0.05微摩尔/升钙磷蛋白C（一种蛋白激酶C抑制剂）并未改变氯化铵诱导的收缩。在分离细胞上，10微摩尔/升SKF96365可使[Ca(2+)]c降低至72.2±1.7%（n = 4）。综上所述，我们的结果表明，氯化铵可诱导大鼠肛门尾骨肌平滑肌细胞收缩，以及由于Ca(2+)通过非选择性Ca(2+)通道内流导致的[Ca(2+)]c增加。
