THE RAT PLATELET 97-KDA CA2-3 PROTEIN(ATPASE ISOFORM IS THE SARCOENDOPLASMIC RETICULUM CA2+ATPASE)

We recently showed that human and rat platelets express two types of S ERCAs (Sarco Endoplasmic Reticulum Ca2+ ATPases): a 100-kDa SERCA2b is oform and a 97-kDa SERCA isoform. Here, we explored the possibility th at the rat 97-kDa isoform is identical to the SERCA3 protein. For this purpose, we first attempted to detect SERCA3 mRNA in rat platelet tot al RNA by reverse transcription-polymerase chain reaction using SERCA3 -specific primers, and demonstrated the presence of this mRNA species by sequencing the amplification product. We then searched for a relati onship between the expression of the SERCA3 mRNA and of the 97-kDa pro tein using either rat aortic smooth muscle cells, previously found not to express the 97-kDa SERCA isoform (negative model), or platelets of spontaneously hypertensive rats (SHR), which overexpress this isoform (overexpression model) but express the 100-kDa SERCA2b isoform normal ly. No expression of SERCA3 mRNA was detectable by analysis of smooth muscle cell RNA, but comparison by reverse transcription-polymerase ch ain reaction of the SERCA2b and SERCA3 mRNAs from the platelets of nor motensive (Wistar-Kyoto, WKY) rats and SHR clearly demonstrated a 238 +/- 43% increase in the expression of the SERCA3 mRNA in SHR platelets only. Last, by comparative Western blotting of WKY rat and SHR platel et membranes using a recently developed polyclonal anti-SERCA3 antibod y, we established that the 97-kDa SERCA and the SERCA3 protein are ide ntical, as immunostaining of the 97-kDa protein revealed a 230 +/- 25% increase in the expression of this protein in SHR versus WKY rat plat elets. It is concluded that the 97-kDa platelet SERCA isoform, which i s up-regulated in SHR, is the SERCA3 protein. As far as we know, this constitutes the first demonstration of the actual presence of this Ca2 + ATPase isoform in normal cells, in addition to the artificial transf ection systems.