Intracellular Buffering Capacity in Molluscan Muscle: Superfused Muscle versus Homogenates

Abstract

In situ estimates of intracellular pH (pHi) in isolated bands of radula protractor muscles from the whelk Busycon canaliculatum were made using phosphorus nuclear magnetic resonance spectroscopy (31P-NMR). The 31P-NMR spectra contained distinct resonances for arginine phosphate and the α, β, and γ phosphates of ATP. In addition, initial spectra displayed a small inorganic phosphate (Pi) peak, which disappeared upon continued superfusion under aerobic conditions. Addition of 2 mM 2-deoxyglucose to the medium resulted in the generation of an additional resonance corresponding to 2-deoxyglucose-6-phosphate (2DGP). Using the chemical shift differences of both the Pi and 2DGP peaks relative to the internal reference arginine phosphate, the resting aerobic pH, was found to be 7.29 ± 0.07 SD (n = 3) and 7.32 ± 0.04 SD (n = 5), respectively. Intracellular buffering capacity (β) was estimated in situ using NH₄Cl prepulse and weak acid loading techniques. The β values were 30 ± 3.9 SD (n = 7) and 31 ± 3.8 SD (n = 7) μ mol·pH⁻¹·g intracellular water⁻¹, respectively. A third method, by titration of tissue homogenates, yielded a β value of 85.8 ± 17.5 SD (n = 8) μ mol·pH⁻¹·g intracellular water⁻¹. This large discrepancy can be partially explained by the rapid hydrolysis of arginine phosphate immediately after homogenization, generating free inorganic phosphate, an excellent buffer in the physiological pH range. The chemical nature of intracellular buffers in B. canaliculatum radula protractor muscle was examined through crude acid fractionation and subsequent amino acid analysis. The total histidine content in this muscle accounted for 72% of the in situ buffering capacity. Unlike some vertebrate systems where large amounts of histidine-containing dipeptides function as buffers, most of the histidine is associated with the protein fraction of the tissue.