Physiological characterization of the Na +/Ca 2+ exchanger (NCX) in hepatopancreatic and antennal gland basolateral membrane vesicles isolated from the freshwater crayfish Procambarus clarkii

Abstract

The purpose of this study was to physiologically characterize the basolateral Na +/Ca 2+ exchanger (NCX) in basolateral membrane vesicles (BLMVs) of hepatopancreas and antennal gland of intermolt crayfish. Conditions were optimized to measure Na +-dependent Ca 2+ uptake and retention in the BLMV including use of intravesicular (IV) oxalate and measuring initial uptake rates at 20 s. Na +-dependent Ca 2+ uptake rate into BLMV was temperature insensitive. Na +-dependent Ca 2+ uptake rate was dependent upon free Ca 2+ with saturable Michaelis–Menten kinetics determined as follows: hepatopancreas, maximal uptake rate ( J max)=2.45 nmol/mg per min, concentration at which carrier operates at half-maximal uptake rate ( K m)=0.69 μM Ca 2+; antennal gland, J max=13.2 nmol/mg per min, K m=0.59 μM Ca 2+. The two vesicle populations exhibited different sensitivity to putative NCX inhibitors. Benzamil had no effect on Na +-dependent Ca 2+ uptake rate in hepatopancreas; in antennal gland it was inhibitory at concentrations up to 30 μM and was stimulatory at higher concentrations. Conversely the inhibitor quinacrine was inhibitory at 10 μM in hepatopancreas and was stimulatory at 1000 μM; meanwhile it was ineffective in antennal gland BLMV. Short circuiting the BLMV had no effect on Na +-dependent Ca 2+ uptake rate suggesting that the process may be electroneutral. Compared with another prominent basolateral transporter in hepatopancreas the plasma membrane Ca 2+ ATPase (PMCA), the NCX has 70-fold greater J max (at comparable temperature) and a lower affinity. In antennal gland the NCX has 40-fold greater J max and a lower affinity. In hepatopancreas and antennal gland BLMV NCX appears to determine the rate of basolateral Ca 2+ efflux in intermolt.