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 via activation of isoenzyme-specific protein kinase C (PKC).


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. 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.


RESULTS
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.


CONCLUSIONS
This study demonstrates that PAF-stimulated NO release via PKCalpha in epithelial cells might regulate endometrial functions such as implantation and menstruation.
Dearn
S
S
Department of Reproductive and Vascular Biology, Birmingham Women's Hospital, University of Birmingham, Edgbaston, UK.
Rahman
M
M
Lewis
A
A
Ahmed
Z
Z
Eggo
M C
MC
Ahmed
A
A
D001381:Azepines / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D002460:Cell Line; D004717:Endometrium / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*; D004789:Enzyme Activation; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism; D005260:Female; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D007247:Infertility, Female; D007527:Isoenzymes / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; D010972:Platelet Activating Factor / Q000378:metabolism*; D010980:Platelet Membrane Glycoproteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D015398:Signal Transduction; D014230:Triazoles / Q000494:pharmacology; D019323:omega-N-Methylarginine / Q000494:pharmacology
D001381:Azepines; D007211:Indoles; D007527:Isoenzymes; D010972:Platelet Activating Factor; D010980:Platelet Membrane Glycoproteins; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D014230:Triazoles; C039351:platelet activating factor receptor; D019323:omega-N-Methylarginine; D009569:Nitric Oxide; C063964:bepafant; C496319:NOS2 protein, human; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; C496628:PRKCA protein, human; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D002118:Calcium; C064758:Ro 31-8220