Altered Ca2+ handling is observed in different cells in essential hypertension. We investigated the expression of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) and inositol 1,4,5-trisphosphate receptor (IP3R) isoforms in platelets and aortic endothelial cells (EC) isolated from spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) rats by ratio reverse-transcriptase-polymerase chain reaction (RT-PCR) analysis and Western blotting. SERCA2b and SERCA3 were assessed at mRNA (EC and platelets) and at protein level (platelets). IP(3)R1, IP(3)R2, and IP(3)R3 mRNAs were demonstrated in both cell types, but only IP(3)R1 and IP(3)R2 proteins were detected in platelets. Compared with WKY, SHR EC and platelets showed higher SERCA3 and IP(3)R2 expression and lower IP(3)R1 expression. We then investigated the effect of lisinopril (20 mg . kg(-1) . d(-1); 10-week treatment of 4-week-old rats or 2-week treatment of adult rats) and captopril (100 mg . kg(-1) . d(-1); 2-week treatment of adult rats). Consequently, expression patterns of SERCAs and IP(3)Rs were significantly modified. Except for SERCAs mRNA in platelets, all differences between SHR and WKY disappeared. However, SERCA3 remained the predominant isoform. Both EC and platelets demonstrated a high equal expression of IP(3)R2 mRNA. IP(3)R1 was the predominant platelet protein isoform, as it was in untreated WKY. mRNA was also isolated from pancreatic islets of WKY and SHR, but no effect of either rat strain or of lisinopril treatment was observed on the expression of the studied genes. We hypothesize that the identical expression pattern of SERCAs and IP(3)Rs after treatment with ACE inhibitors represents a different nonhypertensive configuration, which, through changes in intracellular Ca2+ handling, improves endothelial and platelet dysfunction in SHR but has no effect in WKY.

The sarco/endoplasmic reticulum Ca2+-transport ATPase (SERCA2) pre-mRNA is alternatively processed in a tissue-specific manner. At its 3′ end, two 5′ splice donor sites compete for the same 3′ acceptor splice site (3′A). While the upstream 5′ donor splice site (5′D1) is used in muscle cells giving rise to the class 1 mRNA, the downstream one (5′D2) is exclusively used in neuronal cells generating the class 4 mRNA. Using a neuroblastoma cell line and a minigene containing the 3′ end of the SERCA2 gene, we have investigated the regulation of the neuronal-type of splicing. We have shown that a strong 3′A is required for splicing because exchanging it for a weaker one abolishes splicing. A second region spanning the entire exon 25 downstream of the 3′A is also necessary for the repression of the muscle-specific splicing in neuronal cells. In addition the tissue-specific (muscle/neuron) selection of the appropriate 5′ donor splice site seems to be determined by at least two distinct but adjacent negative cis-active elements located in the last 237 nt of the optional exon 24. The upstream negative element controls the neuronal splicing while the downstream one represses the muscle-specific splicing in neuronal cells. It is suggested that the cis-active elements in the gene transcript are the target of trans-acting factors that are responsible for the repression of neuronal- or muscle-specific splicing in a tissue-specific manner.

Annals of the New York Academy of SciencesVolume 853, Issue 1 p. 372-375 Regulation of Alternative Splicing of the SERCA2 Pre-mRNA in Muscle FRANK WUYTACK, Corresponding Author FRANK WUYTACK Katholieke Universiteit Leuven, Laboratorium voor Fysiologie, Campus Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium Phone: 32-16-345936; fax: 32-16-345991; e-mail: [email protected]Search for more papers by this authorLUDO VAN DEN BOSCH, LUDO VAN DEN BOSCH Katholieke Universiteit Leuven, Laboratorium voor Fysiologie, Campus Gasthuisberg, Herestraat 49, B-3000 Leuven, BelgiumSearch for more papers by this authorMARK VER HEYEN, MARK VER HEYEN Katholieke Universiteit Leuven, Laboratorium voor Fysiologie, Campus Gasthuisberg, Herestraat 49, B-3000 Leuven, BelgiumSearch for more papers by this authorFAWZIA BABA-AÏSSA, FAWZIA BABA-AÏSSA Katholieke Universiteit Leuven, Laboratorium voor Fysiologie, Campus Gasthuisberg, Herestraat 49, B-3000 Leuven, BelgiumSearch for more papers by this authorLUC RAEYMAEKERS, LUC RAEYMAEKERS Katholieke Universiteit Leuven, Laboratorium voor Fysiologie, Campus Gasthuisberg, Herestraat 49, B-3000 Leuven, BelgiumSearch for more papers by this authorRIK CASTEELS, RIK CASTEELS Katholieke Universiteit Leuven, Laboratorium voor Fysiologie, Campus Gasthuisberg, Herestraat 49, B-3000 Leuven, BelgiumSearch for more papers by this author FRANK WUYTACK, Corresponding Author FRANK WUYTACK Katholieke Universiteit Leuven, Laboratorium voor Fysiologie, Campus Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium Phone: 32-16-345936; fax: 32-16-345991; e-mail: [email protected]Search for more papers by this authorLUDO VAN DEN BOSCH, LUDO VAN DEN BOSCH Katholieke Universiteit Leuven, Laboratorium voor Fysiologie, Campus Gasthuisberg, Herestraat 49, B-3000 Leuven, BelgiumSearch for more papers by this authorMARK VER HEYEN, MARK VER HEYEN Katholieke Universiteit Leuven, Laboratorium voor Fysiologie, Campus Gasthuisberg, Herestraat 49, B-3000 Leuven, BelgiumSearch for more papers by this authorFAWZIA BABA-AÏSSA, FAWZIA BABA-AÏSSA Katholieke Universiteit Leuven, Laboratorium voor Fysiologie, Campus Gasthuisberg, Herestraat 49, B-3000 Leuven, BelgiumSearch for more papers by this authorLUC RAEYMAEKERS, LUC RAEYMAEKERS Katholieke Universiteit Leuven, Laboratorium voor Fysiologie, Campus Gasthuisberg, Herestraat 49, B-3000 Leuven, BelgiumSearch for more papers by this authorRIK CASTEELS, RIK CASTEELS Katholieke Universiteit Leuven, Laboratorium voor Fysiologie, Campus Gasthuisberg, Herestraat 49, B-3000 Leuven, BelgiumSearch for more papers by this author First published: 07 February 2006 https://doi.org/10.1111/j.1749-6632.1998.tb08303.xCitations: 2Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat REFERENCES 1 Van Den Bosch, L., J. Eggermont, H. De Smedt, L. Mertens, F. Wuytack & R. Casteels. 1994. Regulation of splicing is responsible for the expression of the muscle-specific 2a isoform of the sarco/endoplasmic-reticulum Ca2+-ATPase. Biochem. J. 302: 559–566. 2 Mertens, L., L. Van Den Bosch, H. Verboomen, F. Wuytack, H. De Smedt & J. Eggermont. 1995. Sequence and spatial requirements for regulated muscle-specific processing of the sarco/endoplasmic reticulum Ca2+-ATPase 2 gene transcript. J. Biol. Chem. 270: 11004–11011. 3 Van Den Bosch, L., L. Mertens, S. Gijsbers, M. Ver Heyen, F. Wuytack & J. Eggermont. 1997. Sequence elements surrounding the acceptor site suppress alternative splicing of the sarco/endoplamic reticulum Ca2+-ATPase 2 gene transcript. Biochem. J. 322: 885–891. 4 Van Den Bosch, L., L. Mertens, Y. Cavaloc, M. Peterson, F. Wuytack & J. Eggermont. 1996. Alternative processing of the sarco/endoplasmic reticulum Ca2+-ATPase transcripts during muscle differentiation is a specifically regulated process. Biochem. J. 317: 647–651. Citing Literature Volume853, Issue1CARDIAC SARCOPLASMIC RETICULUM FUNCTION AND REGULATION OF CONTRACTILITYSeptember 1998Pages 372-375 ReferencesRelatedInformation

cDNA and genomic clones encoding human sarco/endoplasmic reticulum Ca2+-ATPase 3 (SERCA3) were isolated. The composite nucleotide sequence of the 4.6 kb cDNA, as well as the partial structure of 25 kb of genomic DNA encoding all but the 5´ region of the gene, was determined. The nucleotide sequence coding for the last six amino acids of the pump and the 3´-untranslated region were identified within the sequence of the last exon. Northern blot hybridization analysis using cDNA probes derived from this exon detected a 4.8 kb transcript in several human tissues. Using a cDNA probe derived from the 5´-coding region an unexpected mRNA distribution pattern, consisting of two mRNA species of 4.8 and 4.0 kb, was detected in thyroid gland and bone marrow only. This is the first indication of an alternative splicing mechanism operating on the SERCA3 gene transcript, which most likely generates SERCA3 isoforms with altered C-termini. Human SERCA3 expressed in platelets and in COS cells transfected with the corresponding cDNA was detected with the previously described antibody N89 (directed against the N-terminal region of rat SERCA3) and with a new SERCA3-specific antiserum C91, directed against the extreme C-terminus of the human isoform. A monoclonal antibody PL/IM430, previously assumed to recognize SERCA3 in human platelets, does not react with the 97 kDa human SERCA3 transiently expressed in COS cells. Therefore the 97 kDa isoform detected by PL/IM430 more likely represents a novel SERCA pump, as recently suggested [Kovács, Corvazier, Papp, Magnier, Bredoux, Enyedi, Sarkadi and Enouf (1994) J. Biol. Chem. 269, 6177–6184]. Finally, by fluorescence in situ hybridization and chromosome G-banding analyses, the SERCA3 gene was assigned to human chromosome 17p13.3.

We report the distribution of the sarco(endo)plasmic reticulum Ca2+ ATPase 3 (SERCA3) isoform in the rat brain. Compared to SERCA2 isoform, which is found in all brain regions, SERCA3 is specifically expressed in the Purkinje neurons. This conclusion is based on immunochemical observations using SERCA3- and SERCA2b-specific antibodies, in-situ hybridization using SERCA3-specific oligonucleotide probes and single-cell reverse transcription-polymerase chain reaction (RT-PCR). Immunocytochemistry clearly revealed the expression of SERCA3 in the cell body and in the dentritic processes of the Purkinje neurons. Single-cell ratio RT-PCR showed that Purkinje neurons expressed 3-fold lower levels of SERCA3 mRNA compared to SERCA2 mRNA. SERCA3 expression is very low or absent in the rat cerebrum and brainstem. It is known that the SERCA3 Ca2+ pump has an approximately 5-fold lower affinity for Ca2+ when expressed in COS cells as compared to other SERCA members [15]. If this property is also valid in a neuronal context, the expression of the SERCA3 Ca2+-pump isoform could have important functional implications for the regulation of the cytosolic Ca2+ concentration in Purkinje neurons.

Of the three genes encoding the Ca(2+)transport ATPases of the endoplasmic reticulum, the SERCA2 gene is the major isoform expressed in the mammalian brain. The SERCA2 transcript is alternatively processed generating two protein isoforms: SERCA2a which is expressed in cardiac and slow-skeletal muscle, and SERCA2b, the house-keeping isoform which is ubiquitously expressed. We have studied the expression of SERCA2 in the cat brain, and at a less refined level also in the rat brain, using antibodies specific for either SERCA2a or SERCA2b. The SERCA2a staining was very restricted. The SERCA2a antibody clearly labeled the cell body of the Purkinje neurons and weakly stained the giant cells of the gigantocellular reticular nuclei. In contrast, the SERCA2b isoform was found in most regions of the brain. It appeared to be largely confined to neuronal cells. Neuroglial cells were negative. The antibody stained the cell body. In heavily labeled cells such as the pyramidal cells of the hippocampus and of the cerebral cortex, it also stained the proximal portion of the dendrites. The most intense labeling was observed in the Purkinje neurons, which were stained all over the cell. including the distal ramifications of the dendritic tree. Remarkably the SERCA2b labeling in neuronal cells of the hypothalamic area and the substantia nigra was very weak. The possible physiological significance of these results is discussed.