Antagonistic effect of NG-monomethyl-l-arginine (l-NMMA), low Ca2+ and Ro31-8220 on PAF mediated NO release by HEC-1B cell due to activation of NOS and PKC. (A) Effect of inhibition of NO synthesis by l-NMMA on PAF-mediated NO release. HEC-1B cells were stimulated for 30 min with 1 nM platelet-activating factor in the presence of increasing concentrations of NG-monomethyl-l-arginine. The media was removed and NO levels determined using a Sievers NOA 270B chemiluminescent analyser. The results are expressed as means ± S.E.M. of a typical experiment (triplicate determinations per experiment; 3 similar experiments). Basal NO release was 2150 pmol/well and PAF-mediated NO release in the absence of l-NMMA was 2995 pmol/well. These values have not subtracted from the data. (B) Effect of inhibition of protein kinase C and low external Ca2+ and on PAF-mediated NO release from HEC-1B cells. Following serum starvation, confluent monolayers were washed with sterile PBS and pretreated at 37°C with low Ca2+ buffer (150 nM external Ca2+) or with Ro 31-8220 at 1 µM 30 min. Cells were then stimulated with 1 nM PAF in standard media, in the presence of low Ca2+, in the presence of 1 µM Ro 31-8220 for 30 min. The results are expressed as means ± SEM of a representative experiment (n = 9; 2 similar experiments).