Dynorphin-A(1-17) decreases nitric oxide release and cytotoxicity induced with lipopolysaccharide plus interferon-gamma in murine macrophage cell line J774.

Nitric oxide (NO) is an important mediator of cytotoxicity caused by macrophages or by their resident counterpart in brain-glial cells. Modulation of NO release by both activated macrophages and glial cells has been reported in the presence of endogenous (peptide) and synthetic (non-peptide) agonists with kappa opioid-receptors (KOR) selectivity. The data obtained with macrophages and glial cells are contradictory: enhanced NO release by mouse macrophages was reported in the presence of synthetic agonist of KOR selectivity (Neuropeptides 32 (1998) 287), and decreased NO release by glial cells, in the presence of dynorphin-A((1-8)), endogenous opioid peptide with KOR selectivity (J. Biomed. Sci. 7 (2000) 241). In this study, we used a murine cell line J774 of macrophage origin and examined the effect of dynorphin-A((1-17)), endogenous opioid peptide with selectivity for KOR, on NO release induced with lipopolysaccharide (LPS) plus interferon-gamma (IFN-gamma). Dynorphin-A((1-17)) was chosen since in comparison to dynorphin-A((1-13)), it is more resistant to biodegradation (Peptides 17 (1996) 983), and its effects during prolonged treatment of cells could be more pronounced. The effect of dynorphin-A((1-17)) on NO release was compared to its effect on cytotoxicity, induced with LPS plus IFN-gamma. The data obtained have shown that activation-induced NO release by J774 cells is decreased in the presence of dynorphin-A((1-17)). This was associated with deceased LPS and IFN-gamma-induced cytotoxicity of J774 cells, suggesting their causal relationship. Neither of the observed effects of dynorphin-A((1-17)) could be prevented with the KOR selective antagonist, norbinaltorphimine, suggesting that they are mediated via non-opioid mechanism. By diminishing NO release dynorphin-A((1-17)) may affect cytotoxic ability of macrophages, but may also beneficially influence inflammation-induced damage of local tissue.

Dipeptidyl peptidase IV (DPPIV) enzyme activity on immature T-cell line R1.1 is down-regulated by dynorphin-A(1-17) as a non-substrate inhibitor.

In this study we examined surface expression of CD26 and the corresponding enzyme activity of dipeptidyl peptidase IV (DPPIV) on the cells of immature murine T-cell line, R1.1. The data obtained have shown that R1.1 cells express high density of surface CD26 as compared to normal thymus cells. This was associated with strong enzyme activity, which, based on substrates and inhibitor specificity, corresponded to DPPIV. The DPPIV enzyme activity of R1.1 cells was 10 times stronger than that found on normal murine thymus cells (V(max) = 39 micromol/min/10(6) cells, vs 3.7 micromol/min/10(6) cells, respectively). Upon activation with anti-CD3, up-regulation of both membrane CD26, as well as of DPPIV enzyme activity on R1.1 cells were observed. The finding of strong DPPIV on R1.1 cells makes them suitable model for testing putative substrates/inhibitors of the enzyme in its natural microenvironment. Since in addition to strong DPPIV, R1.1 cells also express kappa opioid receptors (KOR) [European Journal of Pharmacology 227 (1992) 257], we tested the effect of dynorphin-A(1-17), an endogenous opioid peptide with KOR selectivity, on DPPIV of R1.1 cells. Dynorphin-A(1-17) down-regulated DPPIV in a dose-dependent manner, with the potency similar to that of substance P, a known natural DPPIV substrate [Journal of Pharmacology and Experimental Therapeutics 260 (1992) 1257]. DPPIV down-regulation was resistant to bestatin and thiorphan, the inhibitors of two cell surface peptidases (APN and NEP, respectively) with potential of dynorphin-A(1-17) degradation, suggesting that the mechanism underlying the observed effect does not involve degradative products of dynorphin-A(1-17). DPPIV down-regulation was also resistent to KOR antagonist, NBI, suggesting that the mechanism underlying the observed phenomenon involves neither cointernalization of KOR and DPPIV. Collectively, cells of immature T cell line, R1.1 exert strong DPPIV enzyme activity, which could be down-regulated in the presence of dynorphin-A(1-17) by mechanism that presumably includes non-substrate inhibition. By down-regulating DPPIV, dynorphin-A(1-17) may indirectly affect activity and/or specificity of natural substrates of DPPIV, such as substance P, RANTES, and endomorphins.