Release of phosphatidylinositol phosphodiesterase by electrical stimulation of the rat phrenic nerve

after thawing they were first diluted and washed by centrifugation in the cold with buffered 5 mM-EDTA (pH 7.4), to remove all Mg’+, followed by two washes with 5 mM-Hepes (adjusted to pH 7.4 with NaOH). After resuspension, the membranes were divided into several aliquots, with or without MgClz or other additions. The progress of the ATP[ I HIADP exchange and [y”P]ATPase reactions was then measured in the same samples with and without EGTA, either with ‘initial’ and ‘final’ tubes in triplicate or as timecourses. This procedure makes it possible to allow for the effect of changing nucleotide concentrations, due to ATP hydrolysis, in the calculation of the ATP-ADP exchange rate (Cavieres. 1983). The separation of the nucleotides and P, in the boiled samples was performed by thin-layer chromatography on polyethyleneimine-cellulose sheets. A time-course experiment conducted with and without I .5 mM-MgClz, with and without 500 PM-EGTA and with and without 200pM-LaC1,, showed a Ca’+-dependent ATP ADP exchange of (mmolih per litre of original cells) 0.426 f 0.027 (mean f s.E.. eight time points) with Mg‘+ and 0.017 f 0.006 without. The Ca’+-dependent ATPase activities were 0.655 f 0.013 and -0.017 f 0.016, respectively. La‘+ inhibited the Ca’+ -dependent ATP-ADP exchange and ATPase activities by 28% and 75%. respectively, in the presence of Mg” , but was without effect on the small exchange activity remaining without Mg’+. The ME’+ and La’+ effects on the Ca’+ -dependent ATPase activity are similar to those reported by others (see Schatzmann. 1982). The stimulation of the enzyme by CaM did not change the Mg2+ requirement for ATP-ADP exchange. At 16pg/ml, CaM stimulated the small Ca’+ -dependent ATP-ADP exchange in the absence of Mg” by a factor of 3, but 1.5mM-MgC1, did in turn multiply this by a factor larger than 30. I t was possible that the Mg’+ effect was an artefact arising from the binding of some EDTA to the membranes, after their initial wash. Thus, only those aliquots receiving Mg2+ during the 37°C incubation would have had enough Caz+ available by displacement from the contaminating EDTA. This possibility was tested by including two washes with I mM-CaCl’ after the EDTA wash and before the Hepes washes, a procedure which should remove all contaminant EDTA. The resulting Ca’+-dependent ATP-ADP exchanges (same units) were 0.807 f 0.029 with 1.5mM-MgClz and 0.045 f 0.01 1 without (triplicate initials and finals). Thus, the Mg’+ stimulation of the exchange seems to be a genuine effect. One possible interpretation for the Mg’+ requirement of ATP-ADP exchange is that, at 37”C, ADP is released mainly from E,P. In other words, that a reversible conformational transition in the phosphoenzyme occurs with mainly the ADP-bound form, i.e. E,P(ADP) g E2P(ADP). The small but consistent ATP-ADP exchange found in the absence of Mg2+ would then be due to ADP release from E, P and this may presumably be the only path available for reversal at 0°C. This scheme would also explain the partial inhibition observed with La7+ ions. It is then possible that ADP and P, release, as well as the unloading of Ca’+ at the external aspect of the membrane, occur all in relation to the same enzyme form of the Ca pump.