Protease-activated receptor isoform expression in pregnant and nonpregnant rat myometrial tissue.

OBJECTIVE: Three protease-activated receptor (PAR1, 3, and 4) isoforms have been shown to be responsible for the cellular effects of thrombin; another PAR isoform (PAR2) is responsible for the cellular effects of trypsin. The present studies sought to test the hypothesis that one (or more) of these PAR isoforms is expressed in myometrial tissue, thereby accounting for the uterotonic effects of these novel agonists. METHODS: The rat PAR3 and 4 isoforms were cloned from a rat spleen cDNA library. PAR isoform mRNA expression was determined by using reverse-transcriptase polymerase chain reactions (PCR) in Sprague-Dawley rats. Confirmation of the identity of the amplified mRNA was done by sequence analysis. Relative quantification of the PAR1 and PAR2 isoforms was performed using a real-time quantitative reverse transcriptase PCR (RT-PCR) technique. PAR protein expression was confirmed by Western blots using polyclonal antibodies. RESULTS: The rat PAR3 and 4 homologues showed significant sequence homology to the mouse and human amino acid and nucleotide sequences. The RT-PCR studies confirmed PAR1-4 expression in myometrium from rats in estrus. PAR3 was expressed in uterus, spleen, kidney, liver, lung, brain, and heart. PAR4 was expressed in uterus, spleen, and lung. Messenger RNA for the PAR1 and 2 isoforms was expressed during the second half of gestation in myometrium from timed-pregnant rats. In contrast, mRNA for the PAR3 and 4 isoforms was not detected in gestational myometrium. PAR protein expression appeared to match tissue mRNA expression patterns. CONCLUSION: These RT-PCR studies confirmed ubiquitous expression of the PAR1 and PAR2 isoforms in myometrium and other rat tissues; in contrast, the PAR3 and PAR4 isoforms are expressed in a tissue-specific and gestationally related pattern.

Intrauterine expression of prothrombin in the sprague-dawley rat.

Thrombin appears to underlie myometrial contractions in response to intrauterine bleeding. In a similar fashion, thrombin generated within the uterus in the absence of active bleeding could also produce contractions. These studies sought to determine whether functionally active prothrombin is expressed in the pregnant and nonpregnant rat uterus. Uteri were obtained from proestrus/estrus and timed-pregnant Sprague-Dawley rats. Western blots were performed using antithrombin antibodies. Immunohistochemical studies were performed using the same antibodies along with the Vector Elite ABC kit. Qualitative reverse transcriptase-polymerase chain reaction studies were performed using rat prothrombin-specific oligonucleotide primers. In vitro uterine contraction studies were performed using Taipan snake venom (an exogenous prothrombinase) and components of the plasma prothrombinase complex (Factors Xa and V) with and without pretreatment with thrombin inhibitors (heparin or hirudin). The Western blots demonstrated prothrombin peptides in myometrial tissue from estrus and pregnant rats. The immunohistochemical studies confirmed prothrombin peptides in both the circular and longitudinal myometrium, along with the endometrium. The reverse transcriptase-polymerase chain reaction studies demonstrated prothrombin mRNA in the endometrium and placenta, but not in the myometrial smooth muscle. The Taipan snake venom stimulated a significant increase in contractions, which were suppressed by pretreatment with heparin and hirudin. The Factor Xa and V complex also significantly stimulated uterine contractions, which were likewise inhibited by hirudin. These studies provide evidence supporting the expression of functionally active prothrombin in the pregnant and nonpregnant rat uterus. Based on the presence of its mRNA, prothrombin appears to be synthesized in the endometrium and placenta; in contrast, the myometrial smooth-muscle cells appear to sequester preformed prothrombin. These results support the hypothesis that intrauterine thrombin could play an autocrine/paracrine role in the regulation of contractile activity.