Activation of platelet-activating factor (PAF) receptor stimulates nitric oxide (NO) release via protein kinase C-alpha in HEC-1B human endometrial epithelial cell line

Background: 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 vi a activation of isoenzyme-specific protein kinase C (PKC). Materials and methods: Inducible and endothelial NOS was shown by Reverse t ranscription polymerase chain reaction RT-PCR in cDNA synthesized from RNA extract of proliferative and secretory endometrium as well endometrial epit helial 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 determ ined. PAF induced translocation of PKC alpha in HEC-1B cell and its antagon ist effect by Ro 31-8220 was studied by Western immunoblot analysis. PKC is oenzyme regulated by PAF was determined in HEC-1B cell lysate by immunoprec ipitation. Results: PAF-evoked a rapid and concentration-dependent biphasic increase i n total NO in human HEC-1B endometrial epithelial cell line [as measured by a Sievers NOA 280A NO Chemiluminescent Analyser.] This increase in NO rele ase was attenuated by the PAF receptor antagonist, WEB2170. Inhibition of N O synthesis by N-G-monomethyl-L-arginine produced marked dose-dependent att enuation of PAF-mediated NO release, indicating nitric oxide synthase (NOS) activation. PAF-mediated NO release was also inhibited by the PKC inhibito r Ro 31-8220 and by the removal of extracellular calcium, suggesting a depe ndency on PKC and calcium, respectively. RT-PCR analysis showed expression of inducible NOS and endothelial NOS in human endometrium, myometrium and H EC-1B cells. Western immunoblot analysis showed PKC alpha, beta II and iota were the principal isozymes present in the HEC-1B cell line and normal end ometrium, suggesting that both HEC-1B cells and normal endometrium have sim ilar PKC isozymes. PAF induced the translocation of both PKC alpha and PKC iota within the time frame of NO release. The translocation of PKC alpha, b ut not PKC iota, was susceptible to inhibition by Ro 31-8220 that also inhi bited PAF-evoked NO release, suggesting that PKC alpha is the principal iso zyme involved in this process and that eNOS may be a substrate for PKC alph a. Kinase assays performed using immunoprecipitated PKC alpha showed that P AF (1 nM) activated PKC alpha that was inhibited by co-incubation with Ro31 -8220 and Ca2+-free medium. Conclusions: This study demonstrates that PAF-stimulated NO release via PKC alpha in epithelial cells might regulate endometrial functions such as imp lantation and menstruation.