TP receptor expression in human umbilical vein

ERRASTI A, CESIO C, PELOROSSO F, SOUZA G, DARAY F, ROTHLIN R.

Mar del Plata, Argentina

Congreso; XXXVII Reunion Anual de la Asociacion Argentina de Farmacologia Experimental.; 2005

Asociación Argentina de Farmacología Experimental (SAFE)

TP receptor expression in human umbilical vein (HUV). Errasti, A, Cesio, C, Pelorosso F, Souza, G, Daray, F, Rothlin, R. III Cátedra de Farmacología. Facultad de Medicina. UBA. Paraguay 2155, piso 9, 1121. farmaco3@fmed.uba.ar. Introduction: TXA2 mimetic U-46619 and prostaglandin-like compounds as 8-iso-PGE2 and 8-iso-PGF2a  promote a potent and efficacious constriction of HUV. These effects are blocked by TP receptor antagonists suggesting that TP receptors are involved in this vessel (Daray y col., Br J Pharmacol, 2003, 139; 1409-1416 and Eur. J. Pharmacol., 2004, 19; 499:189-95). Therefore, the aim was to analyze the expression of functionally established TP receptor in HUV by RT-PCR and Western blot. Methods and results: Total RNA (HUV) and proteins (HUV and platelets) were extracted employing Trizol and RIPA lysis buffer, respectively. RNA was quantified at 260/280nm and proteins were measured with Bradford at 595 nm. PCR products were electrophoresed on 2% agarose gels with ethidium bromide and photographed under UV. Endonuclease digestion was used to confirm product identity. Proteins were electrophoresed on 10% SDS-PAGE and electrotransferred onto nitrocellulose membranes which were blocked in TTBS buffer with 5% milk; then incubated overnight with anti human TP rabbit polyclonal antibodies. Membranes were revealed with alkaline phosphatase-conjugated goat anti-rabbit IgG. Immunoreactive bands were detected by chemiluminescence and compared with those obtained in human platelet. Conclusion: The results indicate that whole HUV express aTPa variant at mRNA level and a protein of similar molecular weight that one observed in human platelet, a rich source of TPa receptors.