Expression and tissue distribution of the mRNAs encoding the human thromboxane A2 receptor (TP) alpha and beta isoforms

of the in of TP TP b and approximately of TP a TP b confirmed in the spontaneously transformed derived cell line. Expression of TP mRNAs in the various correlated with protein expression, as by radioligand binding using the selective TP antagonist H] expression of the a or b isoforms. Similarly, at the protein level, TP selective radioligands currently available, such as [ 3 H]SQ29,549 (30,37) or [ 3 H]S-145 (31) do not discriminate between either receptor isoform and sufficiently sensitive receptor specific antibodies that will allow for detection of low amounts of TP expressed on cell membranes are not currently available. As a necessary prelude to studies which may elucidate the possible differential physiologic roles of the TPs, it is necessary to establish the expression and tissue distribution of the individual TP isoforms. Hence, in this study, a semi quantitative reverse-transcriptase PCR (RT-PCR) approach has been developed that allows for the identification of and the assessment of relative expression and abundance of the mRNAs for the TP a and TP b isoforms from different tissue and cell types. Where possible, TP receptor expression was validated by radioligand binding studies using the highly selective TP antagonist SQ29,548. This study will provide a starting foundation for elucidating the roles of TP isoforms in physiologic and pathophysiologic states. carried reactions reactions Following amplifications, products of the PCR reactions (7 m l) were analysed by agarose gel electrophoresis (39); the gels were then subjected to densitometry by scanning on a UVP GDS800 gel documentation system. The levels of TP a and TP b mRNA expression were determined by measurement of PCR product band intensities on the densitometric scans; in each case, expression levels, in arbitrary units, are represented as a ratio relative to GA3PDH expression (i.e TP a /GA3PDH – standard error of the mean (S.E.M.); TP b /GA3PDH – S.E. M. , in arbitrary units) and the relative levels of TP a to TP b are represented as a ratio of these values in each case, i.e TP a /TP b – S.E. M. , arbitrary units. For each cell or tissue type screened, at least three independent experiments were carried out. Results are expressed as mean – S.E. M. where n ‡ 3. For each independent cell / tissue type screened, the ratio of TP a / GA3PDH and TP b / GA3PDH were analysed using the unpaired Student t-test. P values of less than or equal to ( £ ) 0.05 were considered to indicate a statistically significant difference and, where values were found to be significant, are indicated in the Figures. g P] Carrier primers 3M absolute -20 then 20 dH used as probe for hybridization of SDS and 100 m g/ml denatured, sonicated salmon sperm DNA overnight at 44 C. Membranes washed as previously Phosphor GS 250 Molecular examine the relative expression of the individual TP a and TP b isoforms. In all, 17 different human cell / tissue types were examined, including 6 whole tissue samples and 5 primary cell lines. In the absence of available tissue or primary cells, a number of representative immortalised cell lines were also examined including platelet-like megakaryocytes, adult and foetal aortic and uterine SMC’s, human umbilical vein endothelial cells and hepatoblastoma cell lines which may usefully reflect the relative expression of the TP isoforms in the progenitor tissues / cells. TP b encoding mRNA’s. We found that both TP isoforms are expressed at the mRNA level in thymic tissue, with 3-fold greater expression of TP a relative to TP b . The relative role of each TP isoform in DNA fragmentation and apoptosis remains to be determined.