Pregnancy and estradiol modulate myometrial G-protein pathways in the guinea pig.

OBJECTIVE: Coupled to hundreds of receptors, G-proteins modulate signal transduction pathways and are important hormonal targets. The first objective was to determine the effect of pregnancy and estradiol on myometrial guanosine triphosphatase activity. The second objective was to begin dissecting the molecular mechanism(s) underlying alterations in guanosine triphosphatase activity. STUDY DESIGN: Myometrial tissue was obtained from pregnant, nonpregnant, and ovariectomized untreated and estradiol-treated guinea pigs. Myometrial membranes were prepared by homogenization and differential centrifugation. Basal high-affinity specific guanosine triphosphatase activity was quantitated by enzymatic assay and expressed in rhomol 32Pi per milligram protein per minute. Guanosine triphosphatase activity was stimulated using oxytocin, isoproterenol, and prostaglandin F2alpha. Specific G-protein subunits were quantitated using Western blots. G-protein associated gene expression was semiquantitated using HGU133A gene array chips from Affymetrix. RESULTS: Basal myometrial guanosine triphosphatase activity was increased in pregnant compared with nonpregnant animals. Estradiol increased basal myometrial guanosine triphosphatase activity, compared with untreated controls. The effect of estradiol on stimulated activity was agonist dependent. Both Galphas and Galphai isoform 1 protein levels were increased in myometrium from late pregnant compared with nonpregnant animals. By late gestation, the messenger ribonucleic acid levels of those genes were unaltered, compared with the nonpregnant animal. In general, the impact of pregnancy on G-protein family member gene messenger ribonucleic acid expression was modest. Only the small guanosine triphosphatase Rap1b demonstrated altered expression more than 2-fold during either myometrial quiescence (midpregnancy) or activation (term pregnancy) (up 3-fold during quiescence). Genomic network analyses revealed that the expression of another small guanosine triphosphatase, Rab7, was exclusively up-regulated (80%) during quiescence. During late pregnancy, network analysis showed that only G-protein beta was exclusively altered (up-regulated). Estradiol mimicked the pregnancy effect on both transcription and translation of G-protein family members for some but not all potentially relevant genes. CONCLUSION: The increase in functional myometrial guanosine triphosphatase activity during pregnancy may reflect increased synthesis of 1 or more small guanosine triphosphatase.

Duke Scholars

Author Gentzon Hall Medicine, Nephrology