Specific Pregnancy-Induced Angiotensin II Type-1 Receptor Expression in Ovine Uterine Artery Does Not Involve Formation of Alternate Splice Variants or Alternate Promoter Usage

Recently we reported that pregnancy is associated with a dramatic increase in angiotensin II type-1 receptor (AT1-R; both protein and mRNA) in ovine uterine artery endothelial cells (UAEC), which far exceeds that seen in omental (systemic) arteries. Recent reports also suggest that alternate splicing of AT1-R mRNA may play a role in regulation of AT1-R expression in humans. Herein, we have investigated the possibility of alternate transcript splicing/promoter usage in UAEC from pregnant ewes by 5ʹ-RACE (rapid amplification of cDNA 5ʹ-ends). To provide our control "reference" sequences, we first performed 5ʹ-RACE analysis of AT1-R mRNA transcripts in liver, kidney, and adrenal cortex. Analysis of 17 resultant clones showed exceptional homology, indicating that a single identically spliced mRNA product is observed in all three ovine tissues. Homology of the 5ʹ-untranslated region to that of the human was low (34.2%), but four in-context start/stop codons and the beginning of human exons 1 and 5 were highly conserved. Subsequently we isolated 30 individual clones using UAEC RNA from three pregnant ewes and found no evidence of any sequence formed through unique splicing or promoter usage. We conclude that the pregnancy-induced increase in AT1-R expression unique to UAEC during pregnancy is not mediated by splicing of a unique transcript or unique promoter usage.