Phosphorus nuclear magnetic resonance spectroscopy was used to evaluate the impact of experimental reductions of intracellular pH on in vitro preparations of the radula protractor muscle of the marine gastropod, Busycon canaliculatum. The intracellular pH of radula refractor muscle bundles superfused with buffered artificial sea water (pH = 7.8) was 7.29. It was possible to clamp muscle intracellular pH at various acidotic states by changing the superfusate to 5, 10, and 15 mmol.l-1 5,5-dimethyl-oxazolidine-2,4-dione in buffered artificial sea water (pH = 6.5). Consistent and temporally stable reductions of intracellular pH were achieved (intracellular pH = 6.98, 6.79, and 6.62, respectively). During the acidotic transitions,
arginine phosphate concentrations decreased and
inorganic phosphate concentrations increased in a reciprocal manner and remained essentially constant after the intracellular pH stabilized. The extent of changes in
arginine phosphate and
inorganic phosphate was directly proportional to the magnitude of the imposed
acidosis. Total
adenosine triphosphate concentrations remained unchanged in all treatments. However, the
magnesium adenosine triphosphate to total
adenosine triphosphate ratio declined in direct relation to the extent of the
acidosis. Intracellular free Mg2+ fell incrementally with reduced intracellular pH. All of the above effects were rapidly reversed when the 5,5-dimethyl-oxazolidine-2,4-dione was washed out by changing the superfusate to buffered artificial sea water (pH = 7.8). Mg-
adenosine diphosphate concentrations were calculated in all treatments using equilibrium constants for the
arginine kinase reaction corrected for pH and intracellular free [Mg2+]. The metabolite, intracellular pH, and [Mg2+] data were used to estimate the effective free energy of hydrolysis of
adenosine triphosphate (dG/d xi
ATP) under most experimental conditions. Experimental
acidosis resulted in dramatic reductions in dG/d xi
ATP which were fully reversible upon wash-out of 5,5-dimethyl-dioxazolidine-2,4-dione and recovery to normal intracellular pH conditions.
Acidosis resulted in net hydrolysis of
arginine phosphate, likely via a complex mechanism involving enhancement of rate of
adenosine triphosphate hydrolysis and/or inhibition of
adenosine triphosphate synthesis.