Integrative aspects of renal epithelial calcium transport in crayfish: temporal and spatial regulation of PMCA

The molting cycle of the freshwater (FW) crayfish, Procambarus clarkii, has been used as a model to study the cellular physiology and molecular biology of proteins (channels, exchangers and pumps) that effect epithelial Ca transport (ECT). Specifically, periods of net Ca flux (typically postmolt influx or premolt efflux) have been compared with periods of net Ca balance (intermolt). In the present study, we further explore the spatial and temporal regulation of plasma membrane Ca ATPase (PMCA) in the antennal gland (kidney). Crayfish are uniquely adapted to produce dilute urine through reabsorption of filtered Ca at the nephridial canal; reabsorption increases in pre/postmolt to compensate for the freshwater absorbed at ecdysis to elicit shedding. Prior work has suggested that PMCA mRNA and protein increase in pre- and postmolt stages compared with intermolt at the antennal gland. In the present paper, we used state-of-the-art techniques to increase the spatial and temporal resolution of this observation. Real-time PCR indicated that the PMCA mRNA expression increased 10-fold in premolt with a further doubling in postmolt. In situ hybridization confirmed that the PMCA mRNA was expressed primarily within the nephridial canal and labyrinth of the antennal gland, and that labelling increased in pre- and postmolt. Immunolocalization with confocal visualization confirmed that the PMCA protein is primarily membrane associated in the nephridial canal cells (basolateral but also apical).