In situ hybridization and monoclonal antibody analysis of plasma membrane Ca-pump mRNA and protein in submandibular glands of rabbit, rat and man

The degree of supersaturation of saliva with calcium (Ca) is related to the mineral phase of enamel in erupted teeth, the incidence of caries, and the formation of calculus. The mechanisms for regulating salivary Ca concentration are therefore of relevance to dentistry. Sections of rabbit, rat and human submandibular gland (SMG) were processed for immuno- histochemistry with a specific anti-plasma membrane Ca-pump antibody, 5F10. Western blots confirm that the molecular weight of the proteins identified by our antibody (135 kDa) is consistent with an appropriate molecular weight for PMCA antigen (135-150 kDa). Tissue sections were also processed for in situ hybridization to study the distribution of the PMCA mRNA isoforms. In mammals, the PMCA1 gene is reported to code for a PMCA protein with a role in maintaining the intracellular Ca levels in both epithelial and non-epithelial cells. Other genes including the PMCA2 and PMCA4 genes may code for PMCA proteins specific to Ca transporting tissues. Our studies demonstrate cytoplasmic labeling of PMCA mRNA with hPMCA-1 and hPMCA-4 specific cDNA probes in humans, and rPMCA-1 and rPMCA-2 specific oligonucleotide probes in rats. Labeling of PMCA protein and all mRNA isoforms was found in the cytoplasm of the interlobular and intralobular ducts (except for intercalated ducts). The demonstrated presence of PMCA in SMGs of rabbit, rat, and man, may suggest a role for PMCA in the regulation of intracellular Ca and in a mechanism for regulating and maintaining the high concentration of Ca in saliva.