Endomorphin 1 activates nitric oxide synthase 2 activity and downregulates nitric oxide synthase 2 mRNA expression

Endomorphins 1 and 2 are newly discovered opioid tetrapeptides whose structure is more resistant to enzymatic degradation than that of other opioid peptides. Endomorphins 1 and 2 are considered as endogenous ligands with a high affinity for mu receptors. A number of studies have shown that opioid peptides per se can induce release of nitric oxide from rodent and human immune cells. Endomorphins seemed to be involved in the process of vasodilatation by stimulating release of nitric oxide. In our study we stimulated in vitro J774 macrophages with different concentrations of endomorphin 1 or 2 for measuring nitric oxide release and nitric oxide synthase 2 (NOS 2) mRNA expression. Results showed that 48 h incubation did not enhance nitric oxide release when measured with the Griess method. On the other hand, using real-time amperometric detection of nitric oxide release shortly after challenge with endomorphins, we showed that only 10(-6) M endomorphin 1 was able to stimulate nitric oxide release from a J774 macrophage cell line by activation of NOS 2 isoenzyme. The peak release was 1000-1500 s after stimulation and was in the range of nitric oxide release stimulated with 10 microg/ml lipopolysaccharide. In contrast to this, endomorphin 2 failed to induce nitric oxide release in all tested concentrations. Using a specific inhibitor of nitric oxide synthase 2 (N-(3-[aminomethyl]benzyl)acetamidine, 1400W) we eliminated the stimulatory effect of endomorphin 1 on nitric oxide release. The expression of mRNA for NOS 2 in J774 macrophages, after 30 min incubation with either lipopolysaccharide or 10(-6) M endomorphin 1 was not upregulated. As expected, lipopolysaccharide induced de novo NOS 2 transcription within 4 h. At the same time, in contrast to lipopolysaccharide, mRNA expression of cells treated with endomorphin 1 was downregulated. Since a mu-opioid receptor specific antagonist beta-funaltrexamine hydrochloride inhibited nitric oxide release from endomorphin 1-treated cells, the effect seemed to be mu-opioid receptor mediated.