IN-SITU HYBRIDIZATION AND MONOCLONAL-ANTIBODY ANALYSIS OF PLASMA-MEMBRANE CA-PUMP MESSENGER-RNA AND PROTEIN IN SUBMANDIBULAR GLANDS OF RABBIT, RAT AND MAN

The degree of supersaturation of saliva with calcium (Ca) is related t o the mineral phase of enamel in erupted teeth, the incidence of carie s, and the formation of calculus. The mechanisms for regulating saliva ry 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 ant ibody, 5F10. Western blots confirm that the molecular weight of the pr oteins identified by our antibody (135 kDa) is consistent with an appr opriate molecular weight for PMCA antigen (135-150 kDa). Tissue sectio ns were also processed for in situ hybridization to study the distribu tion of the PMCA mRNA isoforms. In mammals, the PMCA1 gene is reported to code for a PMCA protein with a role in maintaining the intracellul ar Ca levels in both epithelial and non-epithelial cells. Other genes including the PMCA2 and PMCA4 genes may code for PMCA proteins specifi c to Ca transporting tissues. Our studies demonstrate cytoplasmic labe ling of PMCA mRNA with hPMCA-1 and hPMCA-4 specific cDNA probes in hum ans, and rPMCA-1 and rPMCA-2 specific oligonucleotide probes in rats. Labeling of PMCA protein and all mRNA isoforms was found in the cytopl asm of the interlobular and intralobular ducts (except for intercalate d ducts). The demonstrated presence of PMCA in SMGs of rabbit, rat, an d man, may suggest a role for PMCA in the regulation of intracellular Ca and in a mechanism for regulating and maintaining the high concentr ation of Ca in saliva.