Adenosine A1 receptors and mitochondria: targets of remote ischemic preconditioning.

Adenosine is involved in classic preconditioning in most species and acts especially through adenosine A1 and A3 receptors. The aim of the present study was to evaluate whether remote ischemic preconditioning (rIPC) activates adenosine A1 receptors and improves mitochondrial function, thereby reducing myocardial infarct size. Isolated rat hearts were subjected to 30 min of global ischemia and 60 min of reperfusion [ischemia-reperfusion (I/R)]. In a second group, before isolation of the heart, a rIPC protocol (3 cycles of hindlimb I/R) was performed. Infarct size was measured with tetrazolium staining, and Akt/endothelial nitric oxide (NO) synthase (eNOS) expression/phosphorylation and mitochondrial function were evaluated after ischemia at 10 and 60 min of reperfusion. As expected, rIPC significantly decreased infarct size. This beneficial effect was abolished only when 8-cyclopentyl-1,3-dipropylxanthine (adenosine A1 receptor blocker) and NG-nitro-l-arginine methyl ester (NO synthesis inhibitor) were administered during the reperfusion phase. At the early reperfusion phase, rIPC induced significant Akt and eNOS phosphorylation, which was abolished by the perfusion with an adenosine A1 receptor blocker. I/R led to impaired mitochondrial function, which was attenuated by rIPC and mediated by adenosine A1 receptors. In conclusion, we demonstrated that rIPC limits myocardial infarct by activation of adenosine A1 receptors at early reperfusion in the isolated rat heart. Interestingly, rIPC appears to reduce myocardial infarct size by the Akt/eNOS pathway and improves mitochondrial function during myocardial reperfusion. NEW & NOTEWORTHY Adenosine is involved in classic preconditioning and acts especially through adenosine A1 and A3 receptors. However, its role in the mechanism of remote ischemic preconditioning is controversial. In this study, we demonstrated that remote ischemic preconditioning activates adenosine A1 receptors during early reperfusion, inducing Akt/endothelial nitric oxide synthase phosphorylation and improving mitochondrial function, thereby reducing myocardial infarct size.

Effect of Sodium Nitrite and L-NNA on the Outcome of Experimental Ischemic Stroke.

We studied the effect of sodium nitrite in doses of 5 and 50 mg/kg and NO synthase inhibitor L-NNA in a dose of 20 mg/kg on the course of experimental ischemic stroke caused by occlusion of both carotid arteries. Sodium nitrite and NO synthase inhibitor were administered 1 h prior to occlusion of еру carotid arteries and 5 sec after brain ischemia. Sodium nitrite in a dose of 5 mg/kg had a protective effect on the time course of neurological disorders and reduced animal mortality. NO synthase inhibitor L-NNA aggravated the neurological symptoms.

Role of nitric oxide in the pathogenesis of alloxan diabetes.

We studied the effects of N(w)-nitro-L-arginine (L-NNA), a nonselective inhibitor of NO synthases, on the severity of type 1 diabetes mellitus induced by subcutaneous injection of 130 mg/kg alloxan in August rats with high activity of NO system and in Wistar rats. Five days after alloxan injection, hyperglycemia levels after overnight fasting in August and Wistar rats were 27.1±3.7 and 22.0±1.1 mmol/liter, respectively (p<0.03). The mortality over 15 days after alloxan injection in August rats was higher than in Wistar rats (36 and 26%, respectively). L-NNA normalized glucose levels in diabetics of both groups. It completely prevented mortality in August and reduced it to 13% in Wistar rats. Body weight loss and polydipsia after L-NNA injection were also less pronounced in August rats. Plasma nitrite/nitrate concentrations in August rats were 32% higher than in Wistar rats, both in intact and diabetic rats. These data attest to an important role of NO in the pathogenesis of alloxan diabetes.

17ß-estradiol-linked nitro-L-arginine as simultaneous inducer of apoptosis in melanoma and tumor-angiogenic vascular endothelial cells.

Aggressive melanoma is commonly associated with rapid angiogenic growth in tumor mass, tumor cells acquiring apoptosis resistance, inhibition of cellular differentiation etc. Designing a single anticancer molecule which will target all these factors simultaneously is challenging. In the pretext of inciting anticancer effect through inhibiting nitric oxide synthase (NOS) via estrogen receptors (ER) in ER-expressing skin cancer cells, we developed an estrogen-linked L-nitro-arginine molecule (ESAr) for inciting anticancer effect in melanoma cells. ESAr showed specific anticancer effect through diminishing aggressiveness and metastatic behavior in melanoma cells and tumor. In comparison, ESAr showed significantly higher antiproliferative effect than parent molecule L-nitroarginine methyl ester (L-NAME, a NOS inhibitor) through induction of prominent apoptosis in melanoma cells. ESAr-pretreated aggressive melanoma cells could not form tumor possibly because of transformation/differentiation into epithelial-type cells. Furthermore, its antiangiogenic effect was demonstrated through ESAr-induced antiproliferation in HUVEC cells and apoptosis-induction in tumor-associated vascular endothelial cells, thereby significantly restricting severe growth in melanoma tumor. The targeting moiety, estrogen, at the therapeutic concentration of ESAr has apparently no effect in tumor-growth reduction. Albeit, no specific NOS-inhibition was observed, but ESAr could simultaneously induce these three cancer-specific antiaggressiveness factors, which the parent molecule could not induce. Our data rationalize and establish a new use of estrogen as a ligand for potentially targeting multiple cellular factors for treating aggressive cancers.

[Effects of a nitric oxide donor, the substratum of NO-synthase, and an inhibitor of NO-synthase on the ROS-generation activity of phagocytes in the course of ascites tumor growth].

Experiments in vitro were performed to investigate the effects of the nitric oxide donor (SNP), the substratum of NO-synthase (L-arginine), and the inhibitor of NO-synthase (nitroarginine) on the ROS-generating activity of blood plasma polymorphonuclear leucocytes and ascitic fluid macrophages isolated at different times of tumor (Zaidel hepatoma) growth in animal organism. It was found that in the initial period of tumor growth the nitric oxide donor at a concentration of 8 x 10(-5) M reduced the potential ROS-generating activity of macrophages by 38.5 +/- 9.0% and that of polymorphic-nuclear leucocytes of plasma by 27.6 +/- 7.0 %. However, the dynamics of this process during tumor growth was conservative: variations in the production of ROS by phagocytes were 10 +/- 3.0%. L-arginine induced a decrease in the ROS-generating activity of granulocytes and mononucleares by 25-30%. This fact points to an inducible inhibiting effect of NO-synthase on the ROS-generating activity of NADPH-oxidase in the course of tumor growth. The inhibitor of NO-synthase, nitroarginine, produced a monotonous increase in the ROS-generating activity of phagocytes isolated from the tumor at different periods of its growth. The use NO-synthase inhibitors for increasing ROS levels in the region of tumor growth may favor the suppression of tumor cell growth in vivo.

Bradykinin receptors antagonists and nitric oxide synthase inhibitors in vincristine and streptozotocin induced hyperalgesia in chemotherapy and diabetic neuropathy rat model.

PURPOSE: The influence of an irreversible inhibitor of constitutive NO synthase (L-NOArg; 1.0 mg/kg ip), a relatively selective inhibitor of inducible NO synthase (L-NIL; 1.0 mg/kg ip) and a relatively specific inhibitor of neuronal NO synthase (7-NI; 0.1 mg/kg ip), on antihyperalgesic action of selective antagonists of B2 and B1 receptors: D-Arg-[Hyp3,Thi5,D-Tic7,Oic8] bradykinin (HOE 140; 70 nmol/kg ip) or des Arg10 HOE 140 (70 nmol/kg ip) respectively, in model of diabetic (streptozotocin-induced) and toxic (vincristine-induced) neuropathy was investigated. METHODS: The changes in pain thresholds were determined using mechanical stimuli--the modification of the classic paw withdrawal test described by Randall-Selitto. RESULTS: The results of this paper confirm that inhibition of bradykinin receptors and inducible NO synthase but not neuronal NO synthase activity reduces diabetic hyperalgesia. Pretreatment with L-NOArg and L-NIL but not 7-NI, significantly increases antihyperalgesic activity both HOE 140 and des Arg10 HOE 140. It was also shown that both products of inducible NO synthase and neuronal NO synthase activation as well as bradykinin are involved in hyperalgesia produced by vincristine. Moreover, L-NOArg and 7-NI but not L-NIL intensify antihyperalgesic activity of HOE 140 or des-Arg10HOE 140 in toxic neuropathy. CONCLUSIONS: Results of these studies suggest that B1 and B2 receptors are engaged in transmission of nociceptive stimuli in both diabetic and toxic neuropathy. In streptozotocin-induced hyperalgesia, inducible NO synthase participates in pronociceptive activity of bradykinin, whereas in vincristine-induced hyperalgesia bradykinin seemed to activate neuronal NO synthase pathway. Therefore, concomitant administration of small doses of bradykinin receptor antagonists and NO synthase inhibitors can be effective in alleviation of neuropathic pain, even in hospital care.

[Using NO-synthase inhibitors derived from L-arginine for preventing acute experimental lung edema development in mice].

The preventive and curative action of NO-synthase inhibitors derived from L-arginine was investigated on the model of toxic lung edema induced by phosgene (LCt50 - 84) in mice. The most pronounced decrease in the phosgene-induced lung edema was observed for aminoguanidine, NG-nitro-L-arginine (L-NNA), and L-nitroarginine methyl ester (L-NAME). Aminoguanidine was effective in cases of both preventive and curative administration. L-NNA, an inhibitor of the constitutive isoform of NO-synthase, was effective only after preventive injection, while L-NAME, an inhibitor of both inducible and constitutive isoforms of NO-synthase, was effective only after curative use. Therefore, the NO-synthase inhibitors are a promising group of pharmacological agents for the treatment of toxic lung edema induced by phosgene.

Detrimental role of endogenous nitric oxide in host defence against Sporothrix schenckii.

We earlier demonstrated that nitric oxide (NO) is a fungicidal molecule against Sporothrix schenckii in vitro. In the present study we used mice deficient in inducible nitric oxide synthase (iNOS-/-) and C57BL/6 wild-type (WT) mice treated with Nomega-nitro-arginine (Nitro-Arg-treated mice), an NOS inhibitor, both defective in the production of reactive nitrogen intermediates, to investigate the role of endogenous NO during systemic sporotrichosis. When inoculated with yeast cells of S. schenckii, WT mice presented T-cell suppression and high tissue fungal dissemination, succumbing to infection. Furthermore, susceptibility of mice seems to be related to apoptosis and high interleukin-10 and tumour necrosis factor-alpha production by spleen cells. In addition, fungicidal activity and NO production by interferon-gamma (IFN-gamma) and lipopolysaccharide-activated macrophages from WT mice were abolished after fungal infection. Strikingly, iNOS-/- and Nitro-Arg-treated mice presented fungal resistance, controlling fungal load in tissues and restoring T-cell activity, as well as producing high amounts of IFN-gamma Interestingly, macrophages from these groups of mice presented fungicidal activity after in vitro stimulation with higher doses of IFN-gamma. Herein, these results suggest that although NO was an essential mediator to the in vitro killing of S. schenckii by macrophages, the activation of NO system in vivo contributes to the immunosuppression and cytokine balance during early phases of infection with S. schenckii.

Roles of nitric oxide synthase inhibition and vascular endothelial growth factor receptor-2 inhibition on vascular morphology and function in an in vivo model of pancreatic cancer.

PURPOSE: Both nitric oxide (NO) and vascular endothelial growth factor (VEGF) mediate tumor vascular function. Because these molecules regulate one another's expression, we hypothesized that NO synthase (NOS) inhibition produces effects comparable to those of anti-VEGF therapy on human pancreatic cancer xenografts. EXPERIMENTAL DESIGN: L3.6pl human pancreatic cancer cells were s.c. implanted in nude mice. On day 6, mice were randomized to receive (a) PBS (control), (b) DC101 [VEGF receptor 2 (VEGFR-2) antibody] by i.p. injection, (c) N-nitro-l-arginine (NNLA; NOS inhibitor) in the drinking water, or (d) both DC101 and NNLA. Mice were killed on day 20. RESULTS: DC101 and NNLA as single agents inhibited tumor growth by approximately 50% to 60% (P < 0.008 for both). Furthermore, combined therapy inhibited mean tumor growth by 89% (P < 0.008). Combined inhibition of VEGFR-2 and NOS also decreased mean vessel counts by 65% (P < 0.03) and vessel area by 80% versus controls (P < 0.001). In contrast to DC101 where vessel diameter was similar to control, NNLA decreased mean vessel diameter by 42% (P < 0.001). NNLA also led to a 54% (P < 0.03) decrease in tumor uptake of the perfusion marker Hoechst 33342 versus controls whereas DC101 decreased Hoechst 33342 staining by 43% (P < 0.03). The combination of inhibitors decreased perfusion by 73% (P < 0.03). CONCLUSIONS: Although VEGFR-2 can mediate NOS activity, the combination of VEGFR-2 and NOS inhibition significantly increased the antivascular effect over single agent therapy. The addition of NOS inhibition led to an even further alteration of tumor vessel morphology and vascular perfusion compared with VEGFR-2 blockade, suggesting that NO and VEGFR-2 have distinct but complementary effects on the tumor vasculature.

The effects of tempol, 3-aminobenzamide and nitric oxide synthase inhibitors on acoustic injury of the mouse cochlea.

Oxygen free radicals have been implicated in the pathogenesis of acoustic injury of the cochlea. The purpose of this study was to evaluate the effects of tempol (a superoxide anion scavenger), 3-aminobenzamide (a poly (ADP-ribose) synthetase (PARS) inhibitor), N-nitro-l-arginine (a non-selective nitric oxide synthase (NOS) inhibitor), 7-nitroindazole (a selective neuronal NOS inhibitor) and aminoguanidine (a selective inducible NOS inhibitor) on acoustic injury. Mice were exposed to a 4 kHz pure tone of 110-128 dB SPL for 4h. Tempol, 3-aminobenzamide or N-nitro-l-arginine was intraperitoneally administered immediately before the onset of acoustic overexposure, while 7-nitroindazole or aminoguanidine was intraperitoneally administered every 12h starting immediately before the onset of acoustic overexposure. The threshold shift of the auditory brainstem response (ABR) and hair cell loss were then evaluated one and two weeks after acoustic overexposure. Tempol and 3-aminobenzamide significantly protected the cochlea against acoustic injury, whereas the NOS inhibitors did not exert any protective effect. These findings suggest that reactive oxygen species and PARS are involved in acoustic injury of the cochlea. However, further study is necessary to elucidate the roles of nitric oxide and nitric oxide synthase in acoustic injury.

NOS isoenzyme content in brain nuclei as related to food intake in experimental cancer cachexia.

Evidence implies that nitric oxide (NO) in the central nervous systems mediates anorexia in tumor-bearing hosts. We have therefore evaluated, by immunohistochemical image analyses, net alterations of nitric oxide synthases (nNOS, eNOS, iNOS) in brain nuclei [paraventricular hypothalamic nucleus (PVN), medial habenular nucleus (MHB), lateral habenular nucleus (LHB), paraventricular thalamic nucleus (PV), lateral hypothalamic area (LHA), ventromedial hypothalamic nucleus (VMH), nucleus of the solitary tract (NTS)] of tumor-bearing mice (TB) with prostanoid-related anorexia. Pair-fed (PF) and freely fed (FF) non-tumor-bearing mice were used as controls. c-fos was analyzed as indicator of neuronal activation. nNOS was significantly increased in VMH and PVN from TB mice, while eNOS was significantly increased in LHB and LHA. iNOS was significantly increased in LHA and PVN nuclei, but decreased in MHB, LHB and VMH from tumor-bearers. However, several of these alterations were similarly observed in brain nuclei from pair-fed controls. Provision of unspecific NOS-antagonists to TB mice increased nNOS, eNOS and iNOS in several brain nuclei (PVN, LHA, VMH), but left tumor-induced anorexia unchanged. c-fos was significantly increased in all brain nuclei in PF mice except for NTS, LHA and PVN compared to controls, while tumor-bearing mice had increased c-fos in LHA and PVN only compared to controls. Our results demonstrate a complex picture of NOS expression in brain areas of relevance for appetite in tumor-bearing hosts, where most changes seemed to be secondary to stress during negative energy balance. By contrast, NOS content in PVN and LHA nuclei remains candidate behind anorexia in tumor disease. However, nitric oxide does not seem to be a primary mediator behind tumor-induced anorexia. NO may rather secondarily support energy intake in conditions with negative energy balance.

Protein and DNA oxidation in spinal injury: neurofilaments--an oxidation target.

This study measured the time courses of protein and DNA oxidation following spinal cord injury (SCI) in rats and characterized oxidative degradation of proteins. Protein carbonyl content-a marker of protein oxidation-significantly increased at 3-9 h postinjury and the ratio 8-hydroxy-2-deoxyguanosine/deoxyguanosine-an indicator of DNA oxidation-was significantly higher at 3-6 h postinjury in the injured cords than in the sham controls. This suggests that oxidative modification of proteins and DNA contributes to secondary damage in SCI. Densities of selected bands on coomassie-stained gels indicated that most proteins were degraded. Neurofilament protein (NFP) was particularly evaluated immunohistochemically; its light chain (NFP-68) was gradually degraded in nerve fibers, neuron bodies, and large dendrites following SCI. A mixture of Mn (III) tetrakis (4-benzoic acid) porphyrin (10 mg/kg)-a novel SOD mimetic-and nitro-L-arginine (1 mg/kg)-an inhibitor of nitric oxide synthase-injected intraperitoneally, increased NFP-68 immunoreactivity and the numbers of NFP-positive nerve fibers post-SCI, correlating NFP degradation in SCI to free radical-triggered oxidative damage for the first time. Therefore, blockage of protein and DNA oxidation in the secondary injury stage may improve long-term recovery-important information for development of the SCI therapies.

Killing of Leishmania donovani amastigotes by poly ICLC in hamsters.

In vitro as well as in vivo studies suggest that cytokine-induced synthesis of nitric oxide (NO) from L-arginine is a major effector mechanism against intracellular pathogens. In this study, we demonstrate that golden hamsters infected with Leishmania donovani amastigotes upon treatment with polyinosinic-polycytidylic acid stabilized with polylysine and carboxymethylcellulose (poly ICLC), a potent interferon inducer and immune enhancer, in combination with L-arginine, develop the capacity to eliminate intracellular pathogens. This antileishmanial activity of poly ICLC was suppressed by N w nitro-L-arginine (N w NLA), an inhibitor of inducible NO synthase. Furthermore, prolonged treatment of infected animals with L-arginine alone for 5 days more after 5 day treatment with poly ICLC plus L-arginine increased the antileishmanial activity compared with 5 day treatment with poly ICLC plus L-arginine, suggesting that inducible NO synthase, once activated, produces NO for 5 days more. Our results suggest that an L-arginine-dependent, NO-mediated mechanism is probably responsible for the antileishmanial action of poly ICLC.

Serotonergic mediation of the antidepressant-like effects of nitric oxide synthase inhibitors.

Recent studies indicate that nitric oxide (NO) synthase inhibitors have antidepressant-like potential in various animal models. In the present study the behavioural activity of the NO synthase inhibitors, N(G)-nitro-L-arginine (L-NA) and 7-nitroindazole (7-NI), were assessed in a modified rat forced swimming test (FST). Both L-NA and 7-NI, dose dependently reduced immobility and increased swimming behaviour in the rat FST. This behavioural profile parallels the one previously shown with selective serotonin re-uptake inhibitors and serotonergic agonists. Thus, we examined the role of serotonin mediating the behavioural effects of L-NA and 7-NI in the rat FST. Depletion of endogenous serotonin using para-chlorophenylalanine (pCPA; 3 x 150 mg/kg, i.p.) completely blocked L-NA (20 mg/kg, i.p.) and 7-NI (20 mg/kg, i.p.)-induced reductions in immobility and increases in swimming behaviour during the FST. In conclusion these observations suggest that NO synthase inhibitors elicit their antidepressant-like activity in the modified swimming test through a serotonin dependent mechanism.

Antidystonic efficacy of nitric oxide synthase inhibitors in a rodent model of primary paroxysmal dystonia.

In a hamster model (genetic symbol dt(sz)) of primary paroxysmal non-kinesiogenic dystonic choreoathetosis, recent studies have shown beneficial effects of glutamate and dopamine receptor antagonists. Nitric oxide (NO), synthesized from L-arginine by NO synthase in response to glutamate receptor activation, elicits cyclic GMP and modulates glutamate-mediated processes and striatal dopamine release. Therefore, the effects of NO synthase inhibitors and of L-arginine on severity of dystonia were investigated in dt(sz) hamsters in which dystonic attacks, characterized by twisting movements and postures, can be induced by stress. The NO synthase inhibitors N(G)-nitro-L-arginine (L-NNA), N(G)-nitro-L-arginine methyl ester (L-NAME) and 7-nitroindazole significantly reduced the severity of dystonia. At antidystonic effective doses neither L-NNA nor L-NAME caused observable side effects, whereas 7-nitroindazole exerted moderate reduction of locomotor activity. The antidystonic effect of L-NAME was reversed by co-administration of the NO precursor L-arginine. However, L-arginine administered alone did not exert any effect on severity of dystonia. Cerebellar cyclic GMP levels in brains of mutant hamsters in comparison to non-dystonic control hamsters did not significantly differ, but the cerebellar cyclic GMP levels tended to be increased in dt(sz) hamsters during a dystonic attack. L-NAME significantly decreased the cerebellar cyclic GMP levels in both dt(sz) and control hamsters. Although an overproduction of NO is probably not critically involved in the pathogenesis of paroxysmal dystonia, it may contribute to the manifestation of dystonic attacks, as indicated by the antidystonic effects of NO synthase inhibitors. Peripheral side effects may limit the clinical use of NO synthase inhibitors, but more selective inhibitors of the neuronal NO synthase should be considered as interesting candidates for the treatment of paroxysmal dystonia.

Prevention of the hemorrhagic hypotension-induced hepatic arterial vasoconstriction by L-arginine and naloxone.

The possible involvement of the L-arginine-nitric oxide pathway and endogenous opioid mechanisms in the hemorrhagic hypotension- (HH) induced changes of hepatic arterial blood flow and vascular resistance was studied in cats. During HH hepatic arterial blood flow was significantly higher both in L-arginine- and naloxone-treated animals than in controls. Furthermore, HH induced a significant increase of the hepatic vascular resistance in the control group, which was prevented by L-arginine or naloxone treatment. Because inhibition of the nitric oxide synthesis by N(G)-nitro-L-arginine in normotensive cats induced a similar increase of the hepatic vascular resistance to that observed during HH in the control group, our results indicate that impairment of the endothelial function may be responsible for the hemorrhage-induced L-arginine- and naloxone-reversible hepatic arterial vasoconstriction. This hypothesis is consistent with our previous observations demonstrating the development of endothelial dysfunction in the feline hepatic artery during HH.

Modulation of extracellular glutamate concentration by nitric oxide synthase inhibitor in rat transient forebrain ischemia.

The purpose of the present study was to clarify the effect of topical administration of a nitric oxide synthase inhibitor on extracellular glutamate concentration in transient forebrain ischemia. Two microdialysis probes were inserted into the bilateral striata of Wistar rats. NG-Nitro-L-arginine (L-NNA) with or without L-arginine was topically administered into the unilateral striatum through one of the microdialysis probes, while Ringer's solution was perfused into the contralateral striatum as the control, and 14 minutes of forebrain ischemia was applied. The extracellular glutamate concentration during ischemia and subsequent reperfusion was statistically significantly higher on the 100 microM L-NNA-perfused side than on the control side, but 1 mM L-NNA was ineffective. When 100 microM L-NNA was perfused together with 500 microM L-arginine, the glutamate concentration did not differ from that on the control side. Moreover, administration of 500 microM L-arginine significantly suppressed the glutamate elevation after reperfusion. The fact that the lower dose of L-NNA increased the accumulation of glutamate during ischemia and reperfusion without altering blood flow may indicate that nitric oxide affords protection against ischemia neuronal damage. However, since the higher dose of L-NNA did not affect the glutamate concentration, it appears that the effect of nitric oxide on extracellular glutamate concentration in forebrain ischemia differs, depending on the degree of the inhibition of NOS activity.

Nitric oxide synthase inhibitor reduces delayed neuronal death in gerbil hippocampal CA1 neurons after transient global ischemia without reduction of brain temperature or extracellular glutamate concentration.

We planned a study to determine whether or not the mechanism of nitric oxide (NO) neurotoxicity involves the elevation of extracellular glutamate or changes of brain temperature in the pathogenesis of delayed neuronal death of gerbil hippocampal CA1 neurons following 5-min transient forebrain ischemia. Intraventricular injection of 5 microliters of 5.0 mg/ml N omega-nitro-L-arginine (LNNA) significantly preserved neuronal density in the central part of the CA1 region examined 7 days after 5-min ischemia [188.5 +/- 8.5/mm: 90.0% of the 209.5 +/- 11.1/mm density in the sham-operated controls vs. 16.7 +/- 6.4/mm in those injected with artificial cerebrospinal fluid (CSF) only]. There was no difference between these two groups in hippocampal temperature before, during or after 5-min ischemia. The glutamate concentration ([Glu]) during 5-min ischemia measured by a microdialysis technique was similar in the two groups (peak [Glu.] = 2.76 +/- 0.62 pmol/microliters dialysate in the artificial CSF group and = 2.93 +/- 0.64 pmol/microliters dialysate in the LNNA group). It was found that the neuronal toxicity of NO does not involve hyperthermia or the increase of extracellular glutamate concentration in the hippocampal CA1 region during 5-min ischemia.

Effects of aminoguanidine on systemic inflammatory response syndrome induced by platelet activating factor and by lipopolysaccharide in rats.

We investigated the effects of aminoguanidine, a relatively selective inhibitor of inducible nitric oxide (NO) synthase, on the systemic inflammatory response syndrome induced by platelet activating factor (PAF) and by lipopolysaccharide in rats, with emphasis on NO production in vivo. Aminoguanidine treatment improved survival rates after lipopolysaccharide challenge, whereas it aggravated the lethality caused by PAF. Lipopolysaccharide induced a marked increase in the concentrations of nitrate and nitrite in plasma compared with vehicle administration, and the increase was prevented by aminoguanidine. In contrast, PAF challenge with or without aminoguanidine did not affect the concentrations of nitrate and nitrite in plasma compared with vehicle administration. These results suggest that NO derived from inducible NO synthase is not a major participant in the systemic inflammatory response syndrome induced by PAF. Aminoguanidine is not likely to provide beneficial effects in conditions where PAF is produced and the concentrations of nitrate and nitrite in plasma are not significantly increased.

Potentiation of photodynamic therapy antitumor activity in mice by nitric oxide synthase inhibition is fluence rate dependent.

The effects of systemic administration of the nitric oxide synthase (NOS) inhibitor NG-nitro-L-arginine (L-NNA) in combination with photodynamic therapy (PDT) on tumor response, tumor oxygenation and tumor and normal skin perfusion were studied in C3H mice bearing subcutaneous radiation-induced fibrosarcoma tumors. Photodynamic therapy was carried out using the photosensitizer Photofrin (5 mg/kg) in conjunction with a low fluence rate (30 mW/cm2) and a high fluence rate (150 mW/cm2) protocol at a total fluence of 100 J/cm2. Low fluence rate PDT produced approximately 15% tumor cures, a response not significantly altered by administration of 20 mg/kg L-NNA either 5 min before or after PDT. In contrast, high fluence rate PDT produced no tumor cures by itself, but addition of L-NNA either pre- or post-PDT resulted in approximately 30% and approximately 10% tumor cures, respectively. The L-NNA by itself tended to decrease tumor pO2 levels and perfusion, but statistically significant differences were reached only at one time point (1 h) with one of the oxygenation parameters measured (% values < 2 mm Hg). Photodynamic therapy by itself decreased tumor oxygenation and perfusion more significantly. Addition of L-NNA before PDT further potentiated this effect. The L-NNA exerted its most striking effects on the PDT response of the normal skin microvasculature. Low fluence rate PDT caused severe and lasting shut-down of skin microvascular perfusion. With high fluence rate PDT, skin perfusion was initially decreased but recovered to persistent normal levels within 1 h of treatment. Administration of L-NNA reversed this response, converting it to complete and lasting vascular shut-down identical to that achieved with low fluence rate PDT. This effect was somewhat L-NNA dose dependent but was still marked at a dose of 1 mg/kg. It occurred whether L-NNA was given before or after PDT. The L-NNA did not alter the long-term vascular response of skin to low fluence rate PDT. The ability of L-NNA to correspondingly improve tumor response and severely limit skin vascular perfusion following high fluence rate PDT, while providing no benefit for the low fluence rate protocol, suggests that vascular changes in the tumor surrounding normal tissue contribute to the enhanced tumor curability with adjuvant L-NNA treatment.