Pei J M, Yu X C, Bian J S, Wong T M
Department of Physiology, and Institute of Cardiovascular Sciences and Medicine, Faculty of Medicine, The University of Hong Kong, Hong Kong, China.
Am J Physiol. 1999 Sep;277(3):C492-500. doi: 10.1152/ajpcell.1999.277.3.C492.
To study the effects of kappa-opioid receptor stimulation on intracellular Ca2+ concentration ([Ca2+]i) homeostasis during extracellular acidosis, we determined the effects of kappa-opioid receptor stimulation on [Ca2+]i responses during extracellular acidosis in isolated single rat ventricular myocytes, by a spectrofluorometric method. U-50488H (10-30 microM), a selective kappa-opioid receptor agonist, dose dependently decreased the electrically induced [Ca2+]i transient, which results from the influx of Ca2+ and the subsequent mobilization of Ca2+ from the sarcoplasmic reticulum (SR). U-50488H (30 microM) also increased the resting [Ca2+]i and inhibited the [Ca2+]i transient induced by caffeine, which mobilizes Ca2+ from the SR, indicating that the effects of the kappa-opioid receptor agonist involved mobilization of Ca2+ from its intracellular pool into the cytoplasm. The Ca2+ responses to 30 microM U-50488H were abolished by 5 microM nor-binaltorphimine, a selective kappa-opioid receptor antagonist, indicating that the event was mediated by the kappa-opioid receptor. The effects of the agonist on [Ca2+]i and the electrically induced [Ca2+]i transient were significantly attenuated when the extracellular pH (pHe) was lowered to 6.8, which itself reduced intracellular pH (pHi) and increased [Ca2+]i. The inhibitory effects of U-50488H were restored during extracellular acidosis in the presence of 10 microM ethylisopropyl amiloride, a potent Na+/H+ exchange blocker, or 0.2 mM Ni2+, a putative Na+/Ca2+ exchange blocker. The observations indicate that acidosis may antagonize the effects of kappa-opioid receptor stimulation via Na+/H+ and Na+/Ca2+ exchanges. When glucose at 50 mM, known to activate the Na+/H+ exchange, was added, both the resting [Ca2+]i and pHi increased. Interestingly, the effects of U-50488H on [Ca2+]i and the electrically induced [Ca2+]i transient during superfusion with glucose were significantly attenuated; this mimicked the responses during extracellular acidosis. When a high-Ca2+ (3 mM) solution was superfused, the resting [Ca2+]i increased; the increase was abolished by 0.2 mM Ni2+, but the pHi remained unchanged. Like the responses to superfusion with high-concentration glucose and extracellular acidosis, the responses of the [Ca2+]i and electrically induced [Ca2+]i transients to 30 microM U-50488H were also significantly attenuated. Results from the present study demonstrated for the first time that extracellular acidosis antagonizes the effects of kappa-opioid receptor stimulation on the mobilization of Ca2+ from SR. Activation of both Na+/H+ and Na+/Ca2+ exchanges, leading to an elevation of [Ca2+]i, may be responsible for the antagonistic action of extracellular acidosis against kappa-opioid receptor stimulation.
为研究κ-阿片受体激动在细胞外酸中毒期间对细胞内钙离子浓度（[Ca2+]i）稳态的影响，我们采用荧光分光光度法，测定了κ-阿片受体激动对分离的单个大鼠心室肌细胞在细胞外酸中毒期间[Ca2+]i反应的影响。选择性κ-阿片受体激动剂U-50488H（10 - 30μM）剂量依赖性地降低电诱导的[Ca2+]i瞬变，该瞬变由Ca2+内流以及随后肌浆网（SR）中Ca2+的释放引起。U-50488H（30μM）还增加静息[Ca2+]i，并抑制咖啡因诱导的[Ca2+]i瞬变，咖啡因可从SR释放Ca2+，这表明κ-阿片受体激动剂的作用涉及Ca2+从其细胞内储存库释放到细胞质中。5μM的选择性κ-阿片受体拮抗剂nor-binaltorphimine消除了对30μM U-50488H的Ca2+反应，表明该事件由κ-阿片受体介导。当细胞外pH（pHe）降至6.8时，激动剂对[Ca2+]i和电诱导的[Ca2+]i瞬变的作用显著减弱，而细胞外pH降低本身会降低细胞内pH（pHi）并增加[Ca2+]i。在存在10μM乙基异丙基氨氯吡咪（一种有效的Na+/H+交换阻滞剂）或0.2 mM Ni2+（一种假定的Na+/Ca2+交换阻滞剂）的情况下，细胞外酸中毒期间U-50488H的抑制作用得以恢复。这些观察结果表明，酸中毒可能通过Na+/H+和Na+/Ca2+交换拮抗κ-阿片受体激动的作用。当加入已知可激活Na+/H+交换的50 mM葡萄糖时，静息[Ca2+]i和pHi均升高。有趣的是，葡萄糖灌注期间U-50488H对[Ca2+]i和电诱导的[Ca2+]i瞬变的作用显著减弱；这模拟了细胞外酸中毒期间的反应。当用高钙（3 mM）溶液灌注时，静息[Ca2+]i升高；0.2 mM Ni2+可消除该升高，但pHi保持不变。与高浓度葡萄糖灌注和细胞外酸中毒的反应一样，[Ca2+]i和电诱导的[Ca2+]i瞬变对30μM U-50488H的反应也显著减弱。本研究结果首次证明，细胞外酸中毒拮抗κ-阿片受体激动对SR中Ca2+释放的作用。Na+/H+和Na+/Ca2+交换的激活导致[Ca2+]i升高，可能是细胞外酸中毒对κ-阿片受体激动拮抗作用的原因。
