EFFECTS OF VANADATE ON MGATP STIMULATION OF NA-CA EXCHANGE SUPPORT KINASE-PHOSPHATASE MODULATION IN SQUID AXONS

We have proposed that in squid axons MgATP stimulation of Na-Ca exchan ge involves a phosphorylation-dephosphorylation process catalyzed by a kinase-phosphatase system. In the present work, we used vanadate as a tool to gather further evidence about the mechanism of metabolic cont rol of the Na-Ca exchanger in internally dialyzed and voltage-clamped squid axons. Vanadate, at concentrations up to 100 mu M, stimulated ex tracellular Na (Na-o)-dependent Ca efflux only in the presence of MgAT P but failed to so so when the axons were dialyzed with the nonhydroly zable ATP analogue beta, gamma-methyleneadenosine 5'-triphosphate or w ith CrATP, a MgATP analogue that completely abolishes MgATP stimulatio n of the Na-Ca exchange. In axons fully activated by Mg-adenosine 5'-O -(3-thiotriphosphate), vanadate had no effect on Na-Ca exchange. The d ose-response curve for vanadate stimulation followed Michaelian kineti cs with a K-m of 5.6 +/- 0.4 mu M and a maximum velocity of 216 +/- 10 fmol . cm(-2) . s(-1) (intracellular Ca concentration = 0.8 mu M). Th is coincides with the high affinity of vanadate in inhibiting the in v itro phosphatase activity of an alkaline phosphatase extracted from ra t liver. In addition, vanadate increased fivefold the apparent affinit y for MgATP (K-m from 220 +/- 14 to 40 +/- 4 mu M). Concentrations of vanadate in the millimolar range inhibited the MgATP-stimulated Na-Ca exchange (apparent K-i of 5.7 +/- 0.3 mM) and the in vitro phosphoryla tion by the catalytic subunit of a adenosine 3',5'-cyclic monophosphat e protein kinase (apparent K-i 2.64 +/- 0.04 mM). We conclude that MgA TP stimulation of Na-Ca exchange is proportional to the levels of phos phorylation that result from the balance of the activity of a kinase a nd a phosphatase activity.