ATP-dependent control of steady-state cytosolic calcium in cultured gastric smooth muscle.

The regulation of intracellular calcium uptake and release in cultured gastric smooth muscle cells was studied in saponin-permeabilized cells derived from the rabbit antrum. Cells were studied in an ATP-regenerating medium in which the value of the ATP-to-ADP ratio was fixed by variation of the relative concentrations of creatine and creatine phosphate in the presence of a constant concentration of adenine nucleotides and creatine kinase. Free calcium in the medium was measured through the use of the fluorescent probe fura-2. As the ratio of ATP/ADP was increased (8.5, 55.0, and 155.0), the rate of calcium sequestration was increased, resulting in a decrease of steady-state free calcium (275.2, 178.4, and 98.1 nM, respectively). The addition of glucose (5 mM) and hexokinase (15 U/ml), which results in an increase of ADP due to the phosphorylation of glucose in the medium, caused an increase of free calcium concentration to a new set point of approximately 400 nM. Mitochondrial blockade with antimycin A before permeabilization had no effect on calcium sequestration or the resultant free calcium concentration, indicating that under physiological conditions calcium is sequestered predominantly into nonmitochondrial storage sites. Specific variation of ATP/ADP had no effect on the concentration dependence of inositol trisphosphate-induced calcium efflux, suggesting the functional independence of intracellular calcium influx and efflux pathways. These results indicate a significant role for cytoplasmic ATP/ADP in the control of intracellular calcium sequestration and the regulation of steady-state calcium concentration in cultured gastrointestinal smooth muscle cells.