Cytoplasmic calcium in individual proximal tubular cells in culture

The development of high quantal yield Ca2+-sensitive fluorescent indicators has made microspectrofluorometric monitoring of cytoplasmic calcium feasible. The detailed technique to monitor cytoplasmic calcium concentration in individual proximal tubular cells grown on glass cover slips is described. Manipulations of cytoplasmic calcium concentration by means of a Ca2+ ionophore or Ca2+-free medium resulted in corresponding changes of fura-2 fluorescence. Parathyroid hormone elicited a fivefold increase in cytoplasmic calcium concentration, with the subsequent complete recovery of this parameter in 3 min. This effect of parathyroid hormone was abolished by perfusion of the cells with Ca2+-free medium. Repeated pulses of parathyroid hormone spaced at an interval of 20 min, caused refractoriness of adenosine 3',5'-cyclic monophosphate response, whereas cytoplasmic calcium transients remained unaltered. When the frequency of the sequential pulses with parathyroid hormone was increased (5 min intervals), the amplitude of calcium transients was diminished, and the recovery of basal level of cytoplasmic calcium was incomplete and protracted. These observations may have application to disordered renal cell calcium metabolism in hyperparathyroid states.