Datensatz

Zusammenfassung

DM-nitrophen (DMN) is a photolabile calcium chelator that has been used extensively to study calcium-triggered exocytosis. Nitrophenyl-EGTA (NPE) is a recently synthesized photolabile calcium chelator that, unlike DMN, selectively binds calcium over magnesium. Here, we compare NPE and DMN for their effectiveness in raising cytosolic calcium ([Ca]i) to trigger exocytosis. The whole cell patch clamp technique was used to monitor membrane capacitance (Cm) and to load both calcium indicator dye and photolabile chelators into rat pituitary melanotrophs prior to flash photolysis. In cells dialysed with DMN, a transient increase in [Ca]; was observed immediately after continuity between the patch pipette and the cell cytosol was achieved. This ‘loading transient’ reflects the release of calcium from DMN during the binding of intracellular magnesium. No such transient was seen with NPE, consistent with the negligible binding of magnesium to this chelator. Following flash photolysis of DMN or NPE, [Ca]i increased, triggering both a rapid exocytic burst and slower sustained phases of exocytosis. When flashes of the same intensity were compared, the photolysis of NPE resulted in smaller increases in [Ca]i and slower exocytic bursts than that of DMN. These findings are in accordance with the properties of the two compounds [Ellis-Davies G.C.R., Kaplan J.H. Nitrophenyl-EGTA, a photolabile chelator that selectively binds Cal+ with high affinity and releases it rapidly upon photolysis. Proc Natl Acad Sci USA 1994; 91: 187–191] and the calcium dependency of the exocytic burst [Thomas P., Wong J.G., Lee A.K., Almers W. A low affinity Ca2+ receptor controls the final steps in peptide secretion from pituitary melanotrophs. Neuron 1993; 11: 93–104]. Although NPE is somewhat less effective than DMN in raising [Ca]i, this chelator promises to be a useful and interesting tool for the time-resolved study of calcium-dependent exocytosis in the presence of physiological concentrations of magnesium