Simultaneous fluorescence measurement of calcium and membrane potential responses to endothelin

Vasopressin stimulates calcium signaling and chloride-dependent depolarization in glomerular mesangial cells. We describe a technique whereby both calcium and membrane potential changes can be simultaneously monitored with fluorescent probes. This technique was validated by comparison with single parameter measurements in basal and vasopressin-stimulated mesangial cells. It was shown that the calibration for calcium is unaffected by that for membrane potential, whereas the calibration for membrane potential is affected by prior calcium calibration. Accordingly, it was necessary to calibrate for the former first. The technique was then applied to investigate the effects of endothelin, which was found to elicit a concentration-dependent calcium release response and a chloride-dependent depolarization of mesangial cells. The interaction between the calcium signaling response to vasopressin and endothelin was also investigated. When vasopressin stimulation occurred subsequent to endothelin stimulation, and vice versa, a calcium response was still evident. However, these agonists displayed partial heterologous desensitization in that prior stimulation with vasopressin attenuated the subsequent response to endothelin, and vice versa. This suggests the presence of functionally distinct hormone-responsive calcium pools. The technique of double-parameter fluorescent measurement outlined could potentially be applied to other cellular signaling parameters by the use of the appropriate probes.