The rat platelet 97-kDa Ca2+ATPase isoform is the sarcoendoplasmic reticulum Ca2+ATPase 3 protein.
We recently showed that human and rat platelets express two types of SERCAs (Sarco Endoplasmic Reticulum Ca2+ATPases): a 100-kDa SERCA2b isoform and a 97-kDa SERCA isoform. Here, we explored the possibility that the rat 97-kDa isoform is identical to the SERCA3 protein. For this purpose, we first attempted to detect SERCA3 mRNA in rat platelet total 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 relationship between the expression of the SERCA3 mRNA and of the 97-kDa protein 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 normally. No expression of SERCA3 mRNA was detectable by analysis of smooth muscle cell RNA, but comparison by reverse transcription-polymerase chain reaction of the SERCA2b and SERCA3 mRNAs from the platelets of normotensive (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 platelet membranes using a recently developed polyclonal anti-SERCA3 antibody, we established that the 97-kDa SERCA and the SERCA3 protein are identical, as immunostaining of the 97-kDa protein revealed a 230 +/- 25% increase in the expression of this protein in SHR versus WKY rat platelets. It is concluded that the 97-kDa platelet SERCA isoform, which is 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 transfection systems.
Bobe
R
R
U348 Institut National de la Santé et de la Recherche Médicale, Hôpital Lariboisière, Paris, France.
Bredoux
R
R
Wuytack
F
F
Quarck
R
R
Kovàcs
T
T
Papp
B
B
Corvazier
E
E
Magnier
C
C
Enouf
J
J
D000818:Animals; D001792:Blood Platelets / Q000201:enzymology*; D015153:Blotting, Western; D000252:Calcium-Transporting ATPases / Q000235:genetics / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D015870:Gene Expression; D006973:Hypertension / Q000201:enzymology; D008970:Molecular Weight; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D011921:Rats, Inbred WKY; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*
D012333:RNA, Messenger; D000252:Calcium-Transporting ATPases