Interaction of cyclic GMP and cyclic AMP during neutrophil migration: involvement of phosphodiesterase type III.

In previous experiments, it was shown that migration of electropermeabilized human neutrophils induced by a combination of cGMP and cAMP markedly lower relative to that induced by cGMP or cAMP alone. However, when cGMP was replaced with 8-(para-chlorophenylthio-guanosine-3',5'-cyclic monophosphate (8-pCPT-cGMP), a metabolic stable analogue of cGMP which does not affect the activity of cGMP-regulated phosphodiesterases (PDEs), migration in the presence of cAMP was enhanced in an additive way. To investigate the role of cyclic nucleotide breakdown during neutrophil migration in more detail, specific inhibitors of phosphodiesterase type III (PDE-III) (cGMP-inhibited) were used. Milrinone and cilostamide inhibited migration induced by an optimal concentration of cAMP. This revealed that inhibition of cAMP breakdown, by prolonging the action of an otherwise optimal concentration of cAMP, led to decreased migration, in accordance with the observation that the effect of cAMP on migration of electropermeabilized neutrophils was biphasic. Furthermore, it was found that a combination of 8-pCPT-cGMP and milrinone/cilostamide could substitute for cGMP in both activating cGMP-dependent protein kinase (8-pCPT-cGMP) and inhibiting PDE-III (milrinone/cilostamide). In conclusion, evidence is presented that cGMP and cAMP could interact on the level of PDE-III during neutrophil migration.

Sodium azide enhances neutrophil migration and exocytosis: involvement of nitric oxide, cyclic GMP and calcium.

Azide, in the absence of other stimuli, enhanced neutrophil migration in a chemotactic way. The effect of azide on migration was significant at concentrations > or = 1 microM and maximal at 10 microM azide. Although azide itself could not induce exocytosis, at concentrations > or = 10 microM azide enhanced exocytosis induced by a combination of the chemotactic peptide f-methionyl-leucyl-phenylalanine (fMLP) and cytochalasin B (CB). Azide can be oxidized by catalase and myeloperoxidase in the presence of H2O2, resulting in the generation of nitric oxide (NO). Formation of NO from azide was detected by ESR spectroscopy with carboxy-PTIO as a NO-selective probe, and by measurement of nitrite formation. Azide-induced migration, and the enhancement by azide of fMLP/CB-induced exocytosis, were blocked by pre-incubating cells with aminotriazole, an inhibitor of catalase and myeloperoxidase, suggesting that the effect of azide was mediated by NO. Azide-induced migration, but not the enhancement by azide of fMLP/CB-induced exocytosis, was inhibited to a large extent by inhibitors of soluble guanylate cyclase and by inhibitors of cGMP-dependent protein kinase. These observations suggest that azide-induced migration is mediated via cGMP and cGMP-dependent protein kinase, while the enhancement of fMLP/CB-induced exocytosis is not. Azide caused a sustained elevation of the intracellular Ca2+-concentration of neutrophils stimulated with fMLP/CB, which was not affected by inhibitors of the cGMP-signalling cascade. Since neutrophil exocytosis has been shown to be closely correlated with increases in intracellular Ca2+, a further increase by azide of the intracellular Ca2+-level of cells stimulated with fMLP/CB provides a likely mechanism for the enhancement of fMLP/CB-induced exocytosis by azide.

Intracellular but not extracellular conversion of nitroxyl anion into nitric oxide leads to stimulation of human neutrophil migration.

Considerable controversy exists in the literature with regard to the nature of the agent mediating the biological effects of nitroxyl (NO-) donors. Here it is demonstrated that Angeli's salt (AS), a generator of NO-, enhanced human neutrophil migration. Under aerobic conditions, AS was converted to peroxynitrite to a small extent. However, using methionine, a scavenger of peroxynitrite, it was shown that peroxynitrite was not involved in AS-induced migration. AS equally enhanced human neutrophil migration under aerobic and anaerobic conditions, which strongly suggests that extracellular conversion of NO- to .NO by oxygen was not required. Furthermore, metHb and L-cysteine, which react more readily with NO- than with .NO, inhibited AS-induced migration, whereas the response towards gaseous .NO remained unaffected. AS induced an increase in the intracellular level of cGMP, although the curves for migration and cGMP level appeared to be slightly different in their concentration dependence. An inhibitor of soluble guanylate cyclase and antagonists of cGMP-dependent protein kinase had a more pronounced inhibitory effect on .NO-induced migration than on AS-induced migration. This suggests that the cGMP signalling cascade is partially, but not solely, responsible for AS-induced migration. As it has been demonstrated that soluble guanylate cyclase can only be activated by .NO, and not by NO-, these data indicate that NO- is at least partly converted intracellularly to .NO.

Cyclic nucleotide-induced migration of electropermeabilized neutrophils: the effect of phosphodiesterase-resistant analogues.

OBJECTIVE: To study the effect of cyclic nucleotides and PDE-resistant cyclic nucleotide analogues on neutrophil migration. METHODS: Migration of electropermeabilized neutrophils in Boyden chambers in vitro. RESULTS: Addition of cyclic AMP inhibited migration of electropermeabilized neutrophils in the presence of cGMP, relative to the level of migration in the presence of cGMP alone. However, when cGMP was replaced with 8-pCPT-cGMP or Sp-cGMPS, analogues of cGMP which are not degraded by phosphodiesterases, cAMP synergistically enhanced migration. In contrast, migration in the presence of the phosphodiesterase-resistant cAMP analogue, Sp-cAMPS, was not enhanced by addition of cGMP. CONCLUSIONS: Taking into account reports in the literature which show that cGMP-hydrolysing activity can be enhanced by the catalytic subunit of cAMP-dependent protein kinase, it is hypothesized that breakdown of cGMP in neutrophils may be modulated via cAMP.

Potentiation and inhibition of fMLP-activated exocytosis in neutrophils by exogenous nitric oxide.

Exogenous nitric oxide (NO), not derived from NO-donors, but applied directly, could enhance exocytosis of rabbit peritoneal neutrophils induced by suboptimal concentrations of the chemotactic peptide fMLP. The enhancement was maximal at 30 microM NO. Higher concentrations of NO strongly inhibited fMLP-induced exocytosis. The potentiation of fMLP-induced exocytosis by NO could not be reversed by the inhibitors of guanosine-3',5'-cyclic monophosphate (cGMP) accumulation, LY-83583 and methylene blue, or the antagonists of cGMP-dependent protein kinase, Rp-8-pCPT-cGMPS and Rp-8-Br-cGMPS. The concentration of NO needed to enhance fMLP-induced exocytosis was much higher than the concentration leading to an increase in intracellular cGMP levels. These observations suggest that the enhancement of exocytosis by NO is not likely to be mediated by cGMP. At the concentration which inhibited fMLP-induced exocytosis, NO reduced the intracellular level of glutathione. Since it is known that inactivation of intracellular sulfhydryl groups causes complete inhibition of the exocytotic response, it seems evident that the very strong inhibition of exocytosis by high NO concentrations is due to the reaction of NO with glutathione or with other sulfhydryl group-containing targets.

Carbon monoxide enhances human neutrophil migration in a cyclic GMP-dependent way.

Carbon monoxide (CO) enhanced random migration of human neutrophils. An optimally stimulatory effect was observed with 10 microM CO. CO caused a rapid and transient increase in intracellular level of guanosine-3',5'-cyclic monophosphate (cGMP). The enhancing effect of CO on random migration was reversed to a large extent by inhibitors of cGMP accumulation, and by antagonists of cGMP-dependent protein kinase (G-kinase). These results strongly suggest that the enhancement of random migration by CO is mediated by cGMP and G-kinase. Using hemoglobin, a scavenger of CO, we could show that stimulation of soluble guanylate cyclase over an extended period of time, rather than the observed fast and transient increase in intracellular cGMP levels, is responsible for CO-activated migration. We postulate that CO, like nitric oxide (NO), acts as a biological signal in the immune system.

Modulation of neutrophil migration by exogenous gaseous nitric oxide.

We studied the effect of exogenous nitric oxide (NO) on migration of rabbit peritoneal neutrophils. Exogenous NO enhanced random migration of neutrophils in a concentration-dependent way. An optimally stimulatory effect was observed with 0.5 microM NO, whereas at higher NO concentrations the enhancing effect decreased again. NO caused a rapid and transient increase in intracellular guanosine-3',5'-cyclic monophosphate (cGMP) levels. The enhancing effect of NO on random migration was largely reversed by the inhibitors of cGMP accumulation, LY-83583 and methylene blue, and by the antagonists of cGMP-dependent protein kinase, 8-bromoguanosine-3',5'-cyclic monophosphorothioate, Rp-isomer (Rp-8-Br-cGMPS) and 8-(4-chlorophenylthio)-guanosine-3',5'-cyclic monophosphorothioate (Rp-8-pCPT-cGMPS). These observations strongly suggest that the enhancement of random migration by NO is mediated by cGMP and cGMP-dependent protein kinase. The effect of NO on migration did not occur in the absence of extracellular calcium. Although NO did not induce a measurable elevation of intracellular free calcium, pre-incubation with the intracellular calcium chelator Fura-2/AM abolished the enhancing effect of NO. It appears therefore that a small change in the level of cytoplasmic free calcium does play a role in the enhancement of random migration by NO. High concentrations of NO were found to inhibit chemotaxis induced by an optimal concentration of the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP). This inhibitory effect was also dependent on the presence of extracellular calcium. A role for cGMP in the inhibition of fMLP-induced chemotaxis by NO is not supported by our measurements of intracellular cGMP levels. In contrast to the effects on fMLP, NO did not affect chemotaxis induced by the phorbol ester PMA. In conclusion, we show that NO, not derived from NO donors but applied directly, may stimulate or inhibit neutrophil migration, dependent on the concentration. The enhancing effect of NO on random migration is mediated by cGMP, which emphasizes the importance of this second messenger as a modulator of neutrophil functional.

The role of cyclic nucleotides in neutrophil migration.

1. The literature concerning the effects of cAMP and especially cGMP on neutrophil migration is reviewed. 2. Experiments with agents that enhance cGMP level, and with electroporated neutrophils in which cGMP was introduced, show that the nucleotide has different effects. There is a maximal stimulation at a specific concentration while higher concentrations are less effective or even inhibitory. 3. Some physiologically active peptides such as granulocyte-macrophage colony-stimulating factor (GM-CSF), atrial natriuretic factor, and endothelin appear to modify neutrophil migration via a cGMP-dependent mechanism. 4. Dependent on concentration and conditions (random migration vs. fMLP-activated migration, using nitric oxide (NO), NO donors, and inhibitors of NO synthase), NO has stimulatory or inhibitory effects on neutrophil migration. 5. The differential effects of cGMP and cAMP on neutrophil migration are discussed with regard to intracellular actions, metabolism, interaction with calcium, and relation to structural changes required for cell movement.