Spatiotemporal dynamics and roles of K+, Cl- and H+ fluxes in regulatory volume decrease in nasopharyngeal carcinoma cells

Linjie Yang1, Shuang Peng2, Shuanfeng Teng1, Zhenfeng Liu2, Bingxue Li1, Linyan Zhu1, Liwei Wang2, Lixin Chen1. 1Department of Pharmacology, Medical College, Jinan University, Guangzhou, China, 2Department of Physiology, Medical College, Jinan University, Guangzhou, China.

Cell volume regulation is fundamental for various cell functions including cell proliferation, apoptosis and migration among others (Lang F et al. 1998). The ion channels that are involved in the processes of volume regulation have been studied extensively by using the invasive patch clamp technique (Chen et al. 2002). However, the dynamic activities of the ion channels involved have not been clarified in intact cells. In this study, the spatiotemporal dynamics of transmembrane K+, Cl- and H+ transport in the process of regulatory volume decrease (RVD) induced by hypotonic stimulation were investigated using the ion-selective non-invasive micro-test technique (Smith PJ et al. 2001) in human nasopharyngeal carcinoma CNE-2Z cells. Measurements with this technique revealed the separated K+ and Cl- effluxes accompanied by a steady H+ efflux and extracellular pH decrease during the RVD process. The activation of K+ efflux induced by 47% hypotonic stimulation was about 5 min ahead of Cl- efflux. The K+ efflux declined gradually within a few minutes, but the Cl- efflux was maintained in a relatively high level when cells remained exposed to the hypotonic solutions. The K+ efflux was inhibited by the potassium channel blocker Clotrimazole (100 μM). The chloride channel blocker Tamoxifen (10 μM) suppressed the Cl- efflux. Whole-cell patch-clamp recordings demonstrated that the volume-activated K+ or Cl- currents had similar Clotrimazole and Tamoxifen inhibition with the ion effluxes recorded by non-invasive micro-test technique. These results indicate that the K+ and Cl- effluxes are mainly carried by the potassium channel and the chloride channel respectively. The activation of H+ efflux induced by hypotonic stimulation was similar to that of Cl- efflux, with a latency of 5-8 min, and did not decrease with time. Both the H+ efflux and extracellular pH decrease could be partly inhibited by the proton inhibiter Omeprazole. The intracellular pH was also significantly decreased from 7.2 ± 0.05 to 6.6 ± 0.06 (Mean ± SEM, n = 5, P < 0.01) after hypotonic stimulation, and followed by a gradual recovery to about 7.0 in 30 min. Decreases of extracellular pH from 7.4 to 6.5, 6.0 or 5.0 inhibited K+ currents (by 62.1±10.5% at pH 6.5, n = 5, P<0.05) and efflux (by 70.2±13.9% at pH 6.5, n = 5, P<0.05), while moderate decrease of extracellular pH activated Cl- currents (by 11.7±1.3% at pH 6.5, n = 5, P<0.05). Inhibition of H+ efflux by Omeprazole promoted K+ efflux. In conclusion, the RVD process was inhibited by extracellular acidification, and enhanced by Omeprazole. The data in this study suggest that the decrease of K+ efflux, when exposed to hypotonic solution, may be associated with the H+ efflux. Differential sensitivity of K+ and Cl- channels to extracellular pH may accounts for the dynamic difference of K+, Cl- in the process of RVD induced by hypotonic stimulation. Changes in extracellular pH may modulate cell volume recovery through regulation of pH sensitive K+ and Cl- channels, which are crucial in cell function.

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Chen L et al. (2002) Am. J. Physiol. Cell Physiol., 283, C1313-1323.

Smith PJ et al. (2001) Am. J. Physiol. Cell Physiol., 280, C1-11.