Bagnasco S M, Montrose M H, Handler J S
Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Am J Physiol. 1993 May;264(5 Pt 1):C1165-70. doi: 10.1152/ajpcell.1993.264.5.C1165.
Madin-Darby canine kidney (MDCK) cells accumulate the nonperturbing osmolytes myo-inositol and betaine when grown in hypertonic medium. When returned to isotonic conditions, there is a transient basolateral efflux of these osmolytes, contributing to regulatory volume decrease. Using fura-2 fluorescence, we estimated intracellular calcium concentrations after switching MDCK cells from 500 to 300 mosM medium. Cell calcium increased 565 +/- 93 nM within 5 min. Lowering extracellular calcium inhibited the increase in cell calcium and osmolyte efflux when cells were shifted from 500 to 300 mosM medium. The calcium channel blockers lanthanum and nifedipine also inhibited osmolyte efflux after the shift from 500 to 300 mosM. In the absence of change in medium tonicity, increasing cell calcium by exposure to 1 microM ionomycin did not alter osmolyte efflux. As in PAP-HT25 cells, the cytochrome P-450 inhibitors ketoconazole and SKF-525A inhibited the efflux of both osmolytes caused by a reduction in osmolarity. Thus an early rise in cell calcium that is dependent on extra-cellular calcium and a pathway blocked by inhibitors of cytochrome P-450 oxidase are critical in regulation of osmolyte efflux when MDCK cells are shifted from hypertonic to isotonic medium.
当在高渗培养基中生长时，犬肾传代细胞（MDCK）会积累非干扰性渗透溶质肌醇和甜菜碱。当恢复到等渗条件时，这些渗透溶质会出现短暂的基底外侧外流，导致调节性容积减小。我们使用fura-2荧光法，在将MDCK细胞从500 mosM培养基转换为300 mosM培养基后估计细胞内钙浓度。细胞内钙在5分钟内增加了565±93 nM。当细胞从500 mosM培养基转换为300 mosM培养基时，降低细胞外钙可抑制细胞内钙的增加和渗透溶质的外流。钙通道阻滞剂镧和硝苯地平在从500 mosM转换为耐300 mosM后也抑制了渗透溶质的外流。在培养基张力无变化的情况下，通过暴露于1 microM离子霉素增加细胞内钙并不会改变渗透溶质的外流。与PAP-HT25细胞一样，细胞色素P-450抑制剂酮康唑和SKF-525A抑制了由渗透压降低引起的两种渗透溶质的外流。因此，当MDCK细胞从高渗培养基转换为等渗培养基时，依赖于细胞外钙的细胞内钙早期升高以及被细胞色素P-450氧化酶抑制剂阻断的途径在渗透溶质外流的调节中至关重要。
