Platelet Ca2+ ATPases - A plural, species-specific, and multiple hypertension-regulated expression system

Gaining insight into nonmuscle Ca2+ signaling requires basic knowledge of t he major structures involved, We investigated the expression of platelet Ca (2+)ATPases in normal and hypertension-associated abnormal Ca2+ signaling. First, overall identification of normotensive Wistar-Kyoto rat Ca(2+)ATPase s was attempted by looking for newly described human platelet 3'-end altern atively spliced sarco/endoplasmic reticulum Ca(2+)ATPases (SERCA) 3b mRNA a nd plasma membrane Ca(2+)ATPase (PMCA) 1b and 4b proteins, in addition to S ERCA2b and SERCA3a isoforms, For SERCAs, comparative analyses of human and Wistar-Kyoto rat SERCA3 platelet mRNA by reverse transcription-polymerase c hain reaction (RT-PCR) followed by sequencing established that human platel ets coexpressed SERCA3b and a third SERCA3c, while rat cells were devoid of them but expressed a still unknown splice variant that we termed rSERCA3b/ 3c, Its identification using 3'-end SERCA3 gene and rapid amplification of cDNA ends (RACE)-PCR studies showed that it results from an additional SERC A3 alternative splicing process, which uses a second alternative polyadenyl ation site located in the last intron. For PMCAs, with the use of gene-spec ific RT-PCR followed by sequencing and Western blotting using 5F10 monoclon al antibody, expression of human and rat platelet PMCA1b and PMCA4b was sim ilar. Second, comparative analysis of these newly identified Ca(2+)ATPases and SERCA3a in age-matched spontaneously hypertensive rat platelets demonst rated (1) a marked downregulation of rSERCA3b/3c, which became null, and a 1.71-fold increase in SERCA3a and (2) an opposite regulation of the 2 PMCAs , namely, a 3.3-fold decrease in PMCA1b mRNA and a 3.7-fold increase in PMC A4b mRNA, Hence, platelets coexpress multiple, diverse, and species-specifi c Ca(2+)ATPases, including a novel fourth SERCA3. Moreover, expression of P MCA (1b and 4b), SERCA3a, and rSERCA3b/3c was modulated in rat hypertension . Hence, Ca(2+)ATPases should be regarded as constituting a new rational ba sis for the understanding of nonmuscle cell Ca2+ signaling.