Selective protection of RBCs against photodynamic damage by the band 3 ligand dipyridamole.

BACKGROUND: All studied photosensitizers for virus inactivation impair RBCs. To reduce damage to the RBCs without affecting virucidal activity, selective protection of the RBCs is necessary. The ability of the band 3 ligand, dipyridamole, to react with singlet oxygen and to increase the selectivity of photosterilization was investigated. STUDY DESIGN AND METHODS: Solutions of dipyridamole were illuminated in the presence of tetrasulfonated aluminum phthalocyanine (AlPcS(4)) and dimethylmethylene blue (DMMB). Solutions of amino acids, RBCs, and vesicular stomatitis virus (VSV) in RBC suspensions were photodynamically treated in the presence or absence of dipyridamole. RESULTS: Illumination of a solution of dipyridamole in the presence of AlPcS(4) or DMMB resulted in changes in the optical spectrum of dipyridamole. The photooxidation of dipyridamole was inhibited by azide and augmented by D(2)O, which suggests the involvement of singlet oxygen. Photooxidation of amino acids and photodamage to RBCs was strongly reduced in the presence of dipyridamole. In contrast, photoinactivation of VSV in RBC suspensions was only slightly affected by dipyridamole. CONCLUSION: Dipyridamole can improve the specificity of photodynamic sterilization of RBC concentrates, thereby increasing the practical applicability of this photodecontamination method.

Erythrocytes reduce extracellular ascorbate free radicals using intracellular ascorbate as an electron donor.

Ascorbate is readily oxidized in aqueous solution by ascorbate oxidase. Ascorbate radicals are formed, which disproportionate to ascorbate and dehydroascorbic acid. Addition of erythrocytes with increasing intracellular ascorbate concentrations decreased the oxidation of ascorbate in a concentration-dependent manner. Concurrently, it was found, utilizing electron spin resonance spectroscopy, that extracellular ascorbate radical levels were decreased. Control experiments showed that these results could not be explained by leakage of ascorbate from the cells, inactivation of ascorbate oxidase, or oxygen depletion. Thus, this means that intracellular ascorbate is directly responsible for the decreased oxidation of extracellular ascorbate. Exposure of ascorbate-loaded erythrocytes to higher levels of extracellular ascorbate radicals resulted in the detection of intracellular ascorbate radicals. Moreover, efflux of dehydroascorbic acid was observed under these conditions. These data confirm the view that intracellular ascorbate donates electrons to extracellular ascorbate free radical via a plasma membrane redox system. Such a redox system enables the cells to effectively counteract oxidative processes and thereby prevent depletion of extracellular ascorbate.

Plasma membrane properties involved in the photodynamic efficacy of merocyanine 540 and tetrasulfonated aluminum phthalocyanine.

Merocyanine 540 (MC540)-mediated photodynamic damage to erythrocytes was strongly reduced when illumination was performed at pH 8.5 as compared to pH 7.4. This could be explained by high pH-mediated hyperpolarization of the erythrocyte membrane, resulting in decreased MC540 binding at pH 8.5. In accordance, the MC540-mediated photooxidation of open ghosts was not inhibited at pH 8.5. Photoinactivation of vesicular stomatitis virus (VSV) was not inhibited at pH 8.5. This suggests that illumination at increased pH could be an approach to protect red blood cells selectively against MC540-mediated virucidal phototreatment. With tetrasulfonated aluminum phthalocyanine (AIPcS4) as photosensitizer, damage to erythrocytes, open ghosts and VSV was decreased when illuminated at pH 8.5. A decreased singlet oxygen yield at high pH could be excluded. The AIPcS4-mediated photooxidation of fixed erythrocytes was strongly dependent on the cation concentration in the buffer, indicating that the surface potential may affect the efficacy of this photosensitizer. This study showed that altering the environment of the target could increase both the efficacy and the specificity of a photodynamic treatment.

Photodynamic sterilization of red cells and its effect on contaminating white cells: viability and mechanism of cell death.

BACKGROUND: Phthalocyanines are useful sensitizers for photodynamic sterilization of red cell concentrates. Various lipid-enveloped viruses can be inactivated with only limited red cell damage. Because white cells are involved in the immunomodulatory effects of blood transfusions, the study of the effect of photodynamic treatment on these cells is imperative. STUDY DESIGN AND METHODS: White cell-enriched red cell suspensions were photodynamically treated with either the hydrophobic Pc4 (HOSiPcOSi-(CH3)2(CH2)3N(CH3)2) or water-soluble aluminum phthalocyanine tetrasulfonate (AIPCS4) under virucidal conditions. Viability of white cell subpopulations on Days 0, 1, and 4 after treatment was determined by fluorescence-activated cell sorting by flow cytometric analysis of propidium iodide uptake. Apoptosis induction was studied by DNA ladder formation and staining for an early marker of apoptosis (annexin V). RESULTS: Treatment with Pc4 causes a significant decrease in cell viability of all white cells, as shown by prodidium iodide uptake. Monocytes and granulocytes are the most sensitive, and lymphocytes are relatively more resistant. Some of the cells die by apoptosis, which is induced within 30 minutes after treatment. Treatment with AIPCS4 damages only monocytes; other cell populations are not affected. CONCLUSIONS: Physicochemical properties of the photosensitizers partly determine their effect on white cells. Differences in intracellular localization are likely to be responsible for the effects observed.

Inhibition of various steps in the replication cycle of vesicular stomatitis virus contributes to its photoinactivation by AlPcS4 or Pc4 and red light.

Vesicular stomatitis virus (VSV) was used as a model virus to study the processes involved in photoinactivation by aluminum phthalocyanine tetrasulfonate (AlPcS4) or silicon phthalocyanine HOSiPcOSi(CH3)2(CH2)3N(CH3)2 (Pc4) and red light. Previously a very rapid decrease in the intracellular viral RNA synthesis after photodynamic treatment was observed. This decrease was correlated to different steps in the replication cycle. Binding of VSV to host cells and internalization were only slightly impaired and could be visualized by electron microscopy. The capability of the virus to fuse with membranes in an acidic endosomal environment was studied using both pyrene-labeled liposomes and a hemolysis assay as a model. These tests indicate a rapid decrease of fusion capacity after AlPcS4 treatment, which correlated with the decrease in RNA synthesis. For Pc4 treatment no such correlation was found. The fusion process is the first step in the replication cycle, affected by AlPcS4 treatment, but also in vitro RNA polymerase activity was previously shown to be inhibited. Inactivation of VSV by Pc4 treatment is apparently caused by damage to a variety of viral components. Photodynamic treatment of virus suspensions with both sensitizers causes formation of 8-oxo-7,8-dihydroguanosine in viral RNA as measured by HPLC with electrochemical detection. This damage might be partly responsible for inhibition of the in vitro viral RNA polymerase activity by photodynamic treatment.

Transfusion-transmitted diseases: risks, prevention and perspectives.

During the past decades major improvements in blood safety have been achieved, both in developed and developing countries. The introduction of donor counseling and screening for different pathogens has made blood a very safe product, especially in developed countries. However, even in these countries, there is still a residual risk for the transmission of several pathogens. For viruses such as the human immunodeficiency virus (HIV), and the hepatitis viruses B and C, this is due mainly to window-period donations. Furthermore, the threat of newly emerging pathogens which can affect blood safety is always present. For example, the implications of the agent causing new variant Creutzfeld-Jakob disease for transfusion practice are not yet clear. Finally, there are several pathogens, e.g. CMV and parvo B19, which are common in the general donor population, and might pose a serious threat in selected groups of immunosuppressed patients. In the future, further improvements in blood safety are expected from the introduction of polymerase chain reaction for testing and from the implementation of photochemical decontamination for cellular blood products. The situation in transfusion medicine in the developing world is much less favorable, due mainly to a higher incidence and prevalence of infectious diseases.

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.

Effect of membrane potential on the binding of merocyanine 540 to human erythrocytes.

Illumination of erythrocytes in the presence of merocyanine 540 (MC540) resulted in changed binding characteristics of MC540, i.e. a red shift in the emission maximum of bound dye with an increase in the relative fluorescence quantum yield. Aluminum phthalocyanine tetrasulfonate-mediated photodynamic treatment, before addition of MC540, resulted in a comparable change in the MC540-binding characteristics with, in addition, an increase in the concentration of MC540 in the membrane. Both photodynamic treatments induce depolarization of the red cell membrane, with a dose dependency comparable to that of changed MC540 binding. Also depolarization, induced by incubation of the cells with A23187 in the presence of Ca2+ in high [K+] buffer, resulted in similar changes in the MC540 binding characteristics. These results indicate a relation between photodynamically induced membrane depolarization and changed MC540-binding characteristics. Hyperpolarization induced by incubation with A23187 in low [K+] buffer resulted in decreased binding of MC540. In accordance, the MC540-mediated photodamage to red cells decreased upon hyperpolarization of the cells. The results indicate that the binding of MC540 to erythrocytes is strongly dependent on the membrane potential and that hyperpolarization of the membrane could be a possible protection mechanism for erythrocytes against MC540-mediated photodynamic damage.

Ascorbate stimulates ferricyanide reduction in HL-60 cells through a mechanism distinct from the NADH-dependent plasma membrane reductase.

The impermeable oxidant ferricyanide is reduced by the plasma membrane redox system of HL-60 cells. The rate of reduction is strongly enhanced by ascorbate or dehydroascorbate. The aim of this study was to determine the mechanism by which ascorbate and dehydroascorbate accelerate ferricyanide reduction in HL-60 cells. Addition of ascorbate or dehydroascorbate to cells in the presence of ferricyanide led to the intracellular accumulation of ascorbate. Control experiments showed that extracellular ascorbate was rapidly converted to dehydroascorbate in the presence of ferricyanide. These data suggest that intracellular ascorbate originates from extracellular dehydroascorbate. Accumulation of ascorbate was prevented by inhibitors of dehydroascorbate transport into the cell. These compounds also strongly inhibited ascorbate-stimulated ferricyanide reduction in HL-60 cells. Thus, it is concluded that the stimulation of ferricyanide reduction is dependent on intracellular accumulation of ascorbate. Changing the alpha-tocopherol content of the cells had no effect on the ascorbate-stimulated ferricyanide reduction, showing that a nonenzymatic redox system utilizing alpha-tocopherol was not involved. p-Chloromercuribenzenesulfonic acid strongly affected ferricyanide reduction in the absence of ascorbate, whereas the stimulated reaction was much less responsive to this compound. Thus, it appears that at least two different membrane redox systems are operative in HL-60 cells, both capable of reducing ferricyanide, but through different mechanisms. The first system is the ferricyanide reductase, which uses NADH as its source for electrons, whereas the novel system proposed in this paper relies on ascorbate.

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.

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.

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.

The role of protein kinase C activity in the killing of Chinese hamster ovary cells by ionizing radiation and photodynamic treatment.

In several recent studies it has been shown that protein kinase C (PKC) activity may either potentiate or antagonize cell killing by different cytotoxic agents. These apparently conflicting observations suggest that the effects of PKC activity on cell survival may depend on the different properties of different cell types but do not exclude the possibility that the effects may also depend on the nature of the cytotoxic agent. In this context the effects of PKC activation and PKC inhibition or down-regulation on Chinese hamster ovary (CHO) cell survival after photodynamic treatment and ionizing radiation were studied. It appeared that PKC activation by short-term incubation with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) protected CHO cells against ionizing radiation but, in contrast, sensitized the cells to photodynamic treatment. Conversely, inhibition of PKC by H7 and down-regulation of PKC activity by prolonged incubation with TPA sensitized CHO cells to ionizing radiation but protected the cells against photodynamic treatment. These results demonstrate that in one particular cell type PKC activity may have opposite effects on cell survival following cellular damage, depending on the nature of the cytotoxic agent.

Shelf-life of photodynamically sterilized red cell concentrates with various numbers of white cells.

BACKGROUND: Phthalocyanines are useful sensitizers for the photodynamic sterilization of red cell concentrates. The use of the phthalocyanine Pc4 (HOSiPcOSi(CH3)2(CH2)3N(CH3)2) and red light is very efficient in killing various viruses. The addition of scavengers of Type I photodynamic reactions and the use of cremophor to deliver Pc4 give protection to the red cells. STUDY DESIGN AND METHODS: Various red cell components, either white cell-enriched, buffy coat-removed, or white cell-reduced, have been used to study the effect of photodynamic treatment with Pc4 on hemoglobin and potassium leakage and on ATP and glucose levels after prolonged storage. RESULTS: After treatment, storage interval-dependent damage to the red cells could be observed. In components with 26 x 10(9) white cells per L, virus inactivation was less efficient than that in components with no or 2 x 10(9) white cells per L. Similarly, red cells were less affected by the treatment in components with a large number of white cells. Pretreatment storage and use within 1 week after photodynamic treatment induce less damage to the red cells at the moment of transfusion. CONCLUSION: Various improvements in the treatment protocol may ultimately lead to the implementation of photodynamic treatment in transfusion practice. In this respect, the white cell content of the red cell concentrates should be taken into account.

Effect of hydrogen peroxide on the binding of Merocyanine 540 to human erythrocytes.

The fluorescent dye Merocyanine 540 (MC540) is often used as a probe to monitor the molecular packing of phospholipids in the outer leaflet of biomembranes. In a previous study we showed that the increased staining of erythrocytes with a perturbed membrane structure was mainly due to an increase in the fluorescence yield of cell-bound MC540, rather than to an increase of the number of bound molecules. Erythrocytes and ghosts exposed to continuous fluxes of H2O2 exhibited pronounced lipid peroxidation. Further, red blood cells subjected to this form of oxidative stress also showed increased staining with MC540. It appeared that this was caused by a strong increase in binding of MC540, together with a slight red shift of the fluorescence emission maximum and a small increase in the fluorescence yield of bound MC540. The changed MC540 binding characteristics were not observed when lipid peroxidation was suppressed by the presence of the antioxidant BHT in the incubation medium. However, open ghosts exposed to H2O2 showed no increase of MC540 binding, excluding a direct involvement of lipid peroxidation. Measurement of fluorescence emission spectra and gel filtration studies showed that MC540 can bind to H2O2-exposed hemoglobin. Experiments with erythrocytes lysed in hypotonic medium after exposure to H2O2 revealed that peroxidation of lipids with H2O2 induced a non-specific permeabilization of the plasma membrane to MC540, thereby allowing MC540 to bind to the oxidatively denatured, more hydrophobic hemoglobin. These results indicate that conclusions about packing of phospholipids in the outer leaflet of the membrane based on increased MC540-staining should be drawn with care.

Primary targets for photoinactivation of vesicular stomatitis virus by AIPcS4 or Pc4 and red light.

Phthalocyanines are useful sensitizers for the photodynamic sterilization of red blood cell concentrates. The mechanism of photoinactivation of lipid-enveloped viruses is not completely understood. Vesicular stomatitis virus (VSV) was used as a model virus to study the primary targets of photoinactivation by aluminum phthalocyanine tetrasulfonate (AIPcS4) or silicon phthalocyanine HOSiPcOSi(CH3)2(CH2)3N(CH3)2 (Pc4) and red light. Inactivation conditions for VSV in buffer were determined using an end point dilution assay, and viral RNA synthesis in host cells was measured to determine the loss of infectivity in a direct way. The very rapid decrease in the viral RNA synthesis after photodynamic treatment was correlated with respect to different potential primary targets that are involved in different steps of the viral replication cycle. Damage to the viral proteins, induced by treatment with AIPcS4 or Pc4 and analyzed by gel electrophoresis, could not account for the observed loss of infectivity. Binding of VSV to host cells was only slightly impaired after photodynamic treatment with both sensitizers and could therefore not be responsible for the rapid decrease in viral RNA synthesis in cells. A very strong inhibition of viral RNA polymerase activity after treatment with AIPcS4 and red light was detectable using an in vitro assay. This decrease correlated well with the loss of infectivity, indicating that either the RNA or the viral RNA polymerase is the primary target for photoinactivation of VSV with AIPcS4. Treatment with Pc4 did not cause inhibition of viral RNA polymerase activity to an extent that could account for the observed very rapid loss of infectivity. It was therefore concluded that neither the viral proteins nor the binding to the host cells nor the RNA or RNA polymerase are the primary targets for photoinactivation of VSV by Pc4.

In vitro fluence rate effects in photodynamic reactions with AIPcS4 as sensitizer.

It has been shown previously that the efficiency of photodynamic therapy (PDT) both in vivo and in vitro is dependent on fluence rate. In this study, different in vitro experiments showed that tetrasulfonated aluminum phthalocyanine (AIPcS4) is more efficient in photosensitization if the light is delivered at low fluence rate. Erythrocyte damage, virus inactivation and photooxidation of reduced glutathione (GSH) and histidine were all enhanced if light was delivered at 100 W/m2 as compared to 500 W/m2. Bleaching did not occur under these conditions. Oxygen depletion, shown to be important in fluence rate effects observed in vivo, does not seem to be involved. On theoretical grounds saturation of the triplet state is not likely under these conditions. A possible explantation for the observed fluence rate effects might be found in different reaction pathways, that are favored under high or low fluence rate illuminations. These reactions might involve uni- or bimolecular reactions of intermediate products, resulting in less efficiency at higher fluence rate. It proves to be important, under all circumstances, to monitor fluence rate, because a change in fluence rate, even with similar total fluences, might influence photobiological results in an unexpected way.

Synergistic interaction of photodynamic treatment with the sensitizer aluminum phthalocyanine and hyperthermia on loss of clonogenicity of CHO cells.

When CHO cells were exposed to hyperthermia and subsequently to photodynamic treatment, the combined effects were additive but in the reverse sequence the interaction was synergistic. The synergistic interaction comprised two quite different components: (1) photodynamically induced sensitization of cellular proteins and/or supramolecular structures for thermal inactivation and (2) a photodynamically induced inhibition of the cellular repair system for sublethal thermal damage. The first component of the synergistic interaction was reflected by a change of the Arrhenius parameters of thermal cell killing. A lowering of the activation energy of this process was responsible for the synergistic interactions, whereas a concomitant decrease of the frequency factor, opposing this effect, actually caused a much lower degree of synergism at higher temperatures. This component of the synergistic interaction did not respond to the insertion of an intermediate incubation period between the two treatments. The second component of the synergistic interaction, viz the interference with the ability of cells to survive sublethal thermal damage, was reversible, as an intermediate incubation between photodynamic treatment and hyperthermia resulted in its repair. The photodynamically induced inhibition of the ability of cells to survive sublethal thermal damage was not related to ATP or glutathione depletion, inhibition of de novo protein synthesis or impairment of degradation of damaged protein molecules. Restoration of the repair system for sublethal damage depended on a metabolic process and required free intracellular Ca2+, suggesting that a cell signaling pathway may be involved. Thus, in a practical sense the magnitude of the synergistic interaction between photodynamic treatment and hyperthermia depends on the length of the interval between the two treatments and on the temperature and duration of the subsequent thermal treatment. This may have significant consequences for the development of clinical protocols for the combined application of photodynamic therapy and hyperthermia in the treatment of tumors.

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.