Abstract
Impairment of the fertility in the platelet-activating factor (PAF) receptor transgenic female mice suggests changes in PAF functions can influence uterine receptivity. We hypothesized that vasodilatory actions of PAF in the uterus was exerted by PAF-mediated nitric oxide (NO) release via activation of isoenzyme-specific protein kinase C (PKC). Inducible and endothelial NOS was shown by Reverse transcription polymerase chain reaction RT-PCR in cDNA synthesized from RNA extract of proliferative and secretory endometrium as well endometrial epithelial cell lines HEC-1B. The effect of WEB2170, N(G)-monomethyl-L-arginine (L-NMMA) and Ro31-8220 on PAF mediated NO release by HEC-1B cell was determined. PAF induced translocation of PKCalpha in HEC-1B cell and its antagonist effect by Ro 31-8220 was studied by Western immunoblot analysis. PKC isoenzyme regulated by PAF was determined in HEC-1B cell lysate by immunoprecipitation. PAF-evoked a rapid and concentration-dependent biphasic increase in total NO in human HEC-1B endometrial epithelial cell line [as measured by a Sievers NOA 280A NO Chemiluminescent Analyser.] This increase in NO release was attenuated by the PAF receptor antagonist, WEB2170. Inhibition of NO synthesis by N(G)-monomethyl-L-arginine produced marked dose-dependent attenuation of PAF-mediated NO release, indicating nitric oxide synthase (NOS) activation. PAF-mediated NO release was also inhibited by the PKC inhibitor Ro 31-8220 and by the removal of extracellular calcium, suggesting a dependency on PKC and calcium, respectively. RT-PCR analysis showed expression of inducible NOS and endothelial NOS in human endometrium, myometrium and HEC-1B cells. Western immunoblot analysis showed PKCalpha, betaII and iota were the principal isozymes present in the HEC-1B cell line and normal endometrium, suggesting that both HEC-1B cells and normal endometrium have similar PKC isozymes. PAF induced the translocation of both PKCalpha and PKCiota within the time frame of NO release. The translocation of PKCalpha, but not PKCiota, was susceptible to inhibition by Ro 31-8220 that also inhibited PAF-evoked NO release, suggesting that PKCalpha is the principal isozyme involved in this process and that eNOS may be a substrate for PKCalpha. Kinase assays performed using immunoprecipitated PKCalpha showed that PAF (1 nM) activated PKCalpha that was inhibited by co-incubation with Ro31-8220 and Ca(2+)-free medium. This study demonstrates that PAF-stimulated NO release via PKCalpha in epithelial cells might regulate endometrial functions such as implantation and menstruation.
Highlights
Platelet-activating factor (PAF) is a potent, inflammatory lipid mediator that increases vascular permeability and vasodilatation processes that accompany human implantation and men-Molecular Medicine, Volume 6, Number 1, January 2000 struation [1,2]
Addition of platelet-activating factor (PAF) receptor antagonist WEB2170 after 15 min of PAF stimulation inhibited PAF-evoked nitric oxide (NO) release in a time-dependent manner (Fig. 2) indicating that PAF-mediated NO release appears to be sustained over the time period studied
PAF did not activate protein kinase C (PKC)␣ when cells were cultured in medium containing 1m M EGTA. In both Ca2ϩ-free salt solution and in EGTA-supplemented medium, PKC␣ kinase activity was higher in the control incubations than the PAF-stimulated samples. This is the first study to conclusively demonstrate that activation of PAF receptor stimulates the release of NO from a human endometrial epithelial cell line and that this response is dependent on PKC␣ activation
Summary
Platelet-activating factor (PAF) is a potent, inflammatory lipid mediator that increases vascular permeability and vasodilatation processes that accompany human implantation and men-Molecular Medicine, Volume 6, Number 1, January 2000 struation [1,2]. Materials and methods: Inducible and endothelial NOS was shown by Reverse transcription polymerase chain reaction RT-PCR in cDNA synthesized from RNA extract of proliferative and secretory endometrium as well endometrial epithelial cell lines HEC-1B. PAF induced translocation of PKC␣ in HEC-1B cell and its antagonist effect by Ro 31-8220 was studied by Western immunoblot analysis. Results: PAF-evoked a rapid and concentrationdependent biphasic increase in total NO in human HEC-1B endometrial epithelial cell line [as measured by a Sievers NOA 280A NO Chemiluminescent Analyser.] This increase in NO release was attenuated by the PAF receptor antagonist, WEB2170. Inhibition of NO synthesis by NG–monomethyl– L–arginine produced marked dose-dependent attenuation of PAF-mediated NO release, indicating nitric oxide synthase (NOS) activation. RT-PCR analysis showed expression of inducible NOS and endothelial NOS in human endometrium, myometrium and HEC-1B cells. Conclusions: This study demonstrates that PAFstimulated NO release via PKC␣ in epithelial cells might regulate endometrial functions such as implantation and menstruation
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
