Wu C, Bayliss M, Newgreen D, Mundy A R, Fry C H
Institute of Urology & Nephrology, University College London, United Kingdom.
J Urol. 1999 Nov;162(5):1840-7.
The objectives of the study were: i) to examine the ability of carbachol and ATP to raise intracellular [Ca2+] in isolated detrusor myocytes; ii) to determine the origin of the intracellular Ca2+ and iii) to address the question of whether the appearance of purinergic contractions in detrusor from unstable and obstructed human bladders is reflected in the sensitivity of the cell to the two agonists.
Intracellular Ca2+ transients generated by extracellular ATP and carbachol were recorded from isolated human detrusor myocytes. Cells were dissociated by collagenase disruption of the biopsy. Intracellular Ca2+ was measured by epifluorescence microscopy using Fura-2 and electrophysiological recordings were made with patch electrodes.
In cells from stable bladder biopsies the half-maximal concentrations (EC50) for ATP and carbachol to generate Ca2+ transients were 0.10 and 0.25 microM respectively. With cells from unstable bladders the EC50 values for both agonists and the magnitude of the Ca2+ transients were not significantly different from those obtained in cells from normal bladders. The transient in ATP was preceded by a transient depolarisation generated by a large inward current. The carbachol-Ca2+ transient was independent of changes to membrane potential, except in a subset of cells where complex membrane potential changes followed the rise of intracellular Ca2+. The ATP-Ca2+ transient was partially abolished by nicardipine and completely abolished by zero-Ca solutions, the carbachol-Ca2+ transient was unaffected by nicardipine and less completely attenuated by zero-Ca solutions. Prior exposure to caffeine suggested that the carbachol-Ca2+ transient, but not the ATP-Ca2+ transient, originated from intracellular stores.
It is concluded that both agonists are equipotent in increasing intracellular Ca2+, but by different routes. The generation of purinergic contractions in detrusor from unstable bladder is not due to altered sensitivities of the detrusor myocyte to ATP or cholinergic agonists.
本研究的目的是：i）检测卡巴胆碱和ATP升高离体逼尿肌细胞内[Ca2+]的能力；ii）确定细胞内Ca2+的来源；iii）探讨不稳定和梗阻性人类膀胱逼尿肌中嘌呤能收缩的出现是否反映在细胞对这两种激动剂的敏感性上。
从离体人类逼尿肌细胞记录细胞外ATP和卡巴胆碱产生的细胞内Ca2+瞬变。通过胶原酶消化活检组织分离细胞。使用Fura-2通过落射荧光显微镜测量细胞内Ca2+，并用膜片电极进行电生理记录。
在稳定膀胱活检组织的细胞中，ATP和卡巴胆碱产生Ca2+瞬变的半数有效浓度（EC50）分别为0.10和0.25微摩尔。不稳定膀胱细胞中两种激动剂的EC50值以及Ca2+瞬变的幅度与正常膀胱细胞中的值无显著差异。ATP引起的瞬变之前有一个由大内向电流产生的瞬态去极化。卡巴胆碱-Ca2+瞬变与膜电位变化无关，除了一部分细胞，在这些细胞中，复杂的膜电位变化跟随细胞内Ca2+的升高。ATP-Ca2+瞬变部分被尼卡地平消除，完全被无钙溶液消除，卡巴胆碱-Ca2+瞬变不受尼卡地平影响，被无钙溶液减弱的程度较小。预先暴露于咖啡因表明，卡巴胆碱-Ca2+瞬变而非ATP-Ca2+瞬变起源于细胞内储存。
得出结论，两种激动剂在增加细胞内Ca2+方面具有同等效力，但途径不同。不稳定膀胱逼尿肌中嘌呤能收缩的产生并非由于逼尿肌细胞对ATP或胆碱能激动剂的敏感性改变。
