Twenty‐Four‐Hour Variation of L‐Arginine/Nitric Oxide/Cyclic Guanosine Monophosphate Pathway Demonstrated by the Mouse Visceral Pain Model

The L‐arginine/nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) pathway is known to be involved in central and peripheral nociceptive processes. This study evaluated the rhythmic pattern of the L‐arginine/NO/cGMP pathway using the mouse visceral pain model. Experiments were performed at six different times (1, 5, 9, 13, 17, and 21 h after light on) per day in male mice synchronized to a 12 h:12 h light‐dark cycle. Animals were injected s.c. with saline, 2 mg/kg L‐arginine (a NO precursor), 75 mg/kg L‐N^G‐nitroarginine methyl ester (L‐NAME, a NOS inhibitor), 40 mg/kg methylene blue (a soluble guanylyl cyclase and/or NOS inhibitor), or 0.1 mg/kg sodium nitroprusside (a nonenzymatic NO donor) 15 min before counting 2.5 mg/kg (i.p.) p‐benzoquinone (PBQ)‐induced abdominal constrictions for 15 min. Blood samples were collected after the test, and the nitrite concentration was determined in serum samples. L‐arginine or L‐NAME caused both antinociception and nociception, depending on the circadian time of their injection. The analgesic effect of methylene blue or sodium nitroprusside exhibited significant biological time‐dependent differences in PBQ‐induced abdominal constrictions. Serum nitrite levels also displayed a significant 24 h variation in mice injected with PBQ, L‐NAME, methylene blue, or sodium nitroprusside, but not saline or L‐arginine. These results suggest that components of L‐arginine/NO/cGMP pathway exhibit biological time‐dependent effects on visceral nociceptive process.     

Participation of the components of L-arginine/nitric oxide/cGMP cascade by chemically-induced abdominal constriction in the mouse

The purpose of this study was to investigate the role of the L-arginine/nitric oxide (NO)/cGMP pathway in p-benzoquinone-induced writhing model in mouse. L-arginine, a NO precursor, displayed antinociceptive effects at the doses of 0.125-1.0 mg/kg. When the doses of L-arginine were increased gradually to 10-100 mg/kg, a dose-dependent triphasic pattern of nociception-antinociception-nociception was obtained. The NO synthase (NOS) inhibitor, NG-nitro-L-arginine methyl ester (L-NAME) (18.7515 mg/kg), possessed antinociceptive activity. Methylene blue (MB), a guanylyl cyclase and/or NOS inhibitor, (5-160 mg/kg) also produced a dose-dependent triphasic response. When L-arginine (50 mg/ kg) was combined with L-NAME (75 mg/kg). L-arginine-induced antinociception did not change significantly. Cotreatment of L-arginine with 5 mg/kg MB significantly decreased MB-induced antinociception and reversed the nociception induced by 40 mg/kg MB to antinociception. It is concluded that the components of L-arginine/nitric oxide/cGMP cascade may participate in nociceptive processes both peripherally and centrally by a direct effect on nociceptors or by the involvement of other related pathways of nociceptive processes induced by NO.     

Cholinergic-NO-cGMP mediation of sildenafil-induced antinociception

Acetylcholine and cholinomimetic agents with predominant muscarinic action are known to increase the concentration of cGMP by activation of nitric oxide signaling pathway in the nociceptive conditions. The present study was aimed to investigate the NO-cGMP-PDE5 pathway in nociceptive conditions in the experimental animals. Nociceptive threshold was assessed by acetic acid-induced writhing assay (chemonociception) or carrageenan-induced hyperalgesia. Sildenafil [1-5 mg/kg, ip, 50-200 microg/paw, intraplantar (ipl)] produced dose dependent antinociception in both the tested models. Coadministration of acetylcholine (50 mcg/paw, ipl) or cholinomimetic agent, neostigmine (0.1 mcg/kg, ip and 25 ng/paw, ipl) augmented the peripheral antinociceptive effect of sildenafil. This effect was sensitive to blockade by L-NAME (20 mg/kg, ip, 100 microg/paw, ipl), a non-selective NOS inhibitor and methylene blue (1 mg/kg, ip), a guanylate cyclase inhibitor, which per se had little or no effect in both the models of nociception. Further, the per se analgesic effect of acetylcholine and neostigmine was blocked by both L-NAME and methylene blue in the models of nociception, suggesting the activation of NO-cGMP pathway. Also, both L-NAME and methylene blue blocked the per se analgesic effect of sildenafil. These results indicate the peripheral accumulation of cGMP may be responsible for antinociceptive effect, and a possible interaction between cholinergic agents and PDE5 system in models of nociception.     

Effect of centrally administered nitric oxide modulators in Brewer's yeast-induced nociception in rats

Possible modulation of Brewer's yeast-induced nociception by centrally (icv) administered nitric oxide (NO) modulators, viz., NO synthase (NOS) inhibitors, NO precursor, donors, scavengers and co-administration of NO donor (SIN-1) with NOS inhibitor (L-NAME) and NO scavenger (Hb) was investigated in rats. Administration of NOS inhibitors and NO scavenger Hb increased the pain threshold capacity significantly, whereas NO donors SIN-1, SNP and NO precursor L-arginine were found to be hyperalgesic. D-arginine, the inactive isomer of L-arginine and methylene blue, inhibitor of soluble guanylate cyclase failed to alter the nociceptive behaviour in rats. Co-administration of SIN-1 with L-NAME and Hb found to increase the nociceptive threshold. The results indicate, that centrally administered NO modulators alter the nociceptive transmission induced by Brewer's yeast in rats.     

Absence of implication of L-arginine/nitric oxide pathway on neuronal cell injury induced by L-glutamate or hypoxia.

L-glutamate, N-methyl-D-aspartate (NMDA), kainate, quisqualate and sodium nitroprusside increased cyclic GMP (cGMP) level on rat whole brain cell culture. The accumulation of cGMP evoked by L-glutamate was inhibited by a NMDA antagonist MK-801, an inhibitor of guanylate cyclase methylene blue and two nitric oxide (NO) synthase inhibitors NG-monomethyl-L-arginine (L-NMMA) and L-NG-nitroarginine (NO2Arg). The inhibition of L-NMMA on cGMP level was reversed partially by addition of L-arginine. Although MK-801 was able to protect cells from neuronal injury induced by L-glutamate or by 5 h hypoxia, L-NMMA and NO2Arg were ineffective. The present study suggests that cGMP elevation mediated by NO following activation by L-glutamate is not involved in neuronal cell injury.     

Nitric oxide and superoxide radical are involved in both initiation and development of adjuvant arthritis in rats

Nitric oxide synthase(NOS) inhibitor, N^ω-nitro-L-arginine methyl ester (L-NAME, 10–300 mg/kg) and L-N^G-monomethyl-arginine (L-NMMA, 30–300 mg/kg) suppressed the swellings of adjuvant-injected paw of rats (25–54%) at day 2 and 8 when dosed intraperitoneally and orally for 4 days from day -1 to day 2 after adjuvant. L-NAME (30–300 mg/kg) also suppressed the edema of the non adjuvant-injected paws (15–42%) at day 28. Local injection of this inhibitor (2 and 10 mg/kg) was without effect. L-arginine (1 g/kg, i.p.), impaired the suppression by L-NAME. Bovine blood Cu, Zn-superoxide dismutase (SOD, 3 mg/kg, i.p.: 28% suppression) and L-NAME (30 mg/kg i.p.: 36% suppression) showed additive effect (52%) in adjuvant-injected paws at day 8 when co-injected. As the effect of 30 mg/kg L-NAME corresponded nearly to that of 10 mg/kg Voltaren^R, this NOS inhibitor would be worth considering as an anti-inflammatory agent. Sodium nitroprusside (NO-donor) and methylene blue (guanylate cyclase inhibitor) had no effect. L-NAME was also suppressive when dosed after adjuvant inoculation and NO is involved in the development and maintenance of swelling.     

Involvement of the nitric oxide-cyclic GMP pathway in the desensitization of bradykinin responses of cultured rat sensory neurons.

Bradykinin (BK) excites a subset of dorsal root ganglion neurons by inducing an inward cation current (IBK) that strongly desensitizes and is accompanied by elevations in cGMP. We have examined the links between cGMP metabolism and IBK. The BK dose dependencies of IBK activation, desensitization, and cGMP production are comparable. Stimulation (with sodium nitroprusside [NP] or 8-bromo-cGMP [8Br-cGMP]) or inhibition (with methylene blue, hemoglobin, and nitric oxide synthase [NOS] inhibitors) of cGMP levels did not mimic or diminish IBK. However, desensitization was affected by the following agents: first, desensitization was enhanced by NP and reduced by NOS inhibitors. Second, the effects of NOS inhibitors could be overcome by 8Br-cGMP or L-arginine. Third, 8Br-cGMP modification of desensitization required receptor occupancy. We conclude that the NO-cGMP pathway affects a component of IBK desensitization at the receptor or G protein level.     

L-arginine protects human heart cells from low-volume anoxia and reoxygenation

Protective effects of L-arginine were evaluated in a human ventricular heart cell model of low-volume anoxia and reoxygenation independent of alternate cell types. Cell cultures were subjected to 90 min of low-volume anoxia and 30 min of reoxygenation. L-Arginine (0-5.0 mM) was administered during the preanoxic period or the reoxygenation phase. Nitric oxide (NO) production, NO synthase (NOS) activity, cGMP levels, and cellular injury were assessed. To evaluate the effects of the L-arginine on cell signaling, the effects of the NOS antagonist N(G)-nitro-L-arginine methyl ester, NO donor S-nitroso-N-acetyl-penicillamine, guanylate cyclase inhibitor methylene blue, cGMP analog 8-bromo-cGMP, and ATP-sensitive K+ channel antagonist glibenclamide were examined. Our data indicate that low-volume anoxia and reoxygenation increased NOS activity and facilitated the conversion of L-arginine to NO, which provided protection against cellular injury in a dose-dependent fashion. In addition, L-arginine cardioprotection was achieved by the activation of guanylate cyclase, leading to increased cGMP levels in human heart cells. This action involves a glibenclamide-sensitive, NO-cGMP-dependent pathway.     

Estrogen ameliorates Nomega-nitro-L-arginine methyl ester-induced blood pressure increment in male spontaneously hypertensive rats: the role of cGMP

Estrogen (17beta-estradiol, or E2) reduces systolic blood pressure (SBP) increment and increases aortic cyclic guanosine monophosphate (cGMP) in male spontaneously hypertensive rats (SHRs). It is unknown, however, whether the E2-enhanced aortic cGMP is essential for the BP-lowering effect or not. Nomega-nitro-L-arginine-methyl ester (L-NAME), an L-arginine analogue and nitric oxide (NO) synthase inhibitor, significantly increases SBP and decreases aortic cGMP in male SHRs. We thus treated male SHRs with vehicle (corn oil) or E2 (s.c, 2 mg/kg/week) with or without L-NAME (20 mg/dl in the drinking water). SBP was measured weekly. Plasma nitrate/nitrite (NOx) concentrations and aortic cGMP levels were all measured at the end of the study. We found that SBP increment was significantly higher in L-NAME group, compared with the controls, and that E2 treatment reduced this L-NAME effect. Plasma 4NOx concentrations were not significantly different among different groups. Basal and acetylcholine-induced aortic cGMP, but not sodium nitroprusside-induced cGMP, were significantly lower in L-NAME group, compared with the controls. E2 co-administration did not modify L-NAME-induced aortic cGMP decrease. These data indicate that E2-induced BP-lowering effect in L-NAME treated male SHRs is not closely associated with the enhancement of vascular cGMP.     

Biological time-dependent difference in effect of peroxynitrite demonstrated by the mouse hot plate pain model

We previously demonstrated the rhythmic pattern of L-arginine/nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) cascade in nociceptive processes. The coupled production of excess NO and superoxide leads to the formation of an unstable intermediate peroxynitrite, which is primarily responsible for NO-mediated toxicity. In the present study, we evaluated the biological time-dependent effects of exogenously administered peroxynitrite on nociceptive processes and peroxynitrite-induced changes in the analgesic effect of morphine using the mouse hot-plate pain model. Experiments were performed at four different times of day (1, 7, 13, and 19 hours after lights on, i.e., HALO) in mice of both sexes synchronized to a 12 h:12 h light-dark cycle. Animals were injected intraperitoneally (i.p.) with saline or 10 mg/kg morphine 30 min before and 0.001 mg/kg peroxynitrite 30 sec before hot-plate testing, respectively. The analgesic effect of morphine exhibited significant biological time-dependent differences in the thermally-induced algesia; whereas, administration of peroxynitrite alone exhibited either significant algesic or analgesic effect, depending on the circadian time of its injection. Concomitant administration of peroxynitrite and morphine reduced morphine-induced analgesia at three of the four different study time points. In conclusion, peroxynitrite displayed nociceptive and antinociceptive when administered alone according to the circadian time of treatment, while it diminished analgesic activity when administered in combination with morphine at certain biological times.     

Role of nitric oxide in lower esophageal sphincter relaxation to swallowing.

Studies were performed in the opossum to define the role of the L-arginine-nitric oxide (NO) pathway in lower esophageal sphincter (LES) relaxation to swallowing and vagal stimulation in viv and intramural nerve stimulation in vitro. In vivo, L-NAME, a water soluble NO synthase (NOS) inhibitor, caused antagonism of LES relaxation due to reflex-induced swallowing. L-NAME (20 mg/kg i.v.) reduced the amplitude of swallow induced relaxation from 88% to 28%. LES relaxation due to electrical stimulation of peripheral end of decentralized vagus nerve was also antagonized. The effects of L-NAME were reversed by L-arginine, but not by D-arginine. L-NAME treatment did not antagonize LES relaxation to intravenous administration of isoproterenol. In vitro, NO and sodium nitroprusside (SNP) caused a decrease in the sphincter tone. The relaxing effect caused by NO and SNP was not antagonized by tetrodotoxin or omega-conotoxin. Inhibitors of NO synthase, L-NMMA and L-NNA, caused slight increase in the spontaneous resting LES tone and concentration-dependent antagonism of electrical field stimulation (EFS) induced LES relaxation. L-NNA (10(-4)M) abolished EFS induced LES relaxation at low frequencies (less than 5 Hz) and antagonized the relaxation to a value 20% of the control at 20 Hz. The antagonistic action of L-NMMA and L-NNA was unaffected by D-arginine but was reversed by L-arginine. The inhibitory effect of NO, SNP, or two other putative inhibitory neurotransmitters (VIP and CGRP) on the LES was not antagonized by L-NNA. These studies show that inhibitors of NO synthase selectively antagonize LES relaxation to all three modes of intramural inhibitory nerve stimulation including physiological swallowing. These studies suggest that the L-arginine-nitric oxide pathway is involved in physiological relaxation of the LES.     

Seasonality in expression and distribution of nitric oxide synthase isoforms in the testis of the catfish, Clarias batrachus: Role of nitric oxide in testosterone production

Nitric oxide (NO) is a well-recognized versatile signaling molecule. It is produced by catalytic action of nitric oxide synthase (NOS) on L-arginine in a variety of animal tissues. Existence of different isoforms of NOS has been shown in mammalian testis, but report on their presence in the testis of ectothermic vertebrates is non-existent. This study demonstrates the differential expressions of two isoforms of nitric oxide synthase (neuronal-nNOS and inducible-iNOS) like molecules in different cell types in the testis of seasonally breeding catfish, Clarias batrachus through immunohistochemistry. Positive immunoprecipitation of nNOS and iNOS like molecules were detected in germ cells as well as interstitial cells only in the recrudescing and fully mature fish. The immunoreactions differed in intensity and varied with changing reproductive status. Treatment of adult male fish with NO donor, sodium nitroprusside, and a NOS inhibitor, N-nitro-L-arginine methyl ester (L-NAME) increased and decreased the total nitrate and nitrite concentration in the testis, respectively. Sodium nitroprusside and L-NAME also induced simultaneous decline and rise in the testicular testosterone level, respectively. These findings, thus, suggest that NOS isoforms are expressed variedly in different cell types in the testis of reproductively active fish. This investigation also suggests that NO inhibits testosterone production in the testis.     

L-NAME causes antinociception by stimulation of the arginine-NO-cGMP pathway

NG-nitro-L-arginine methyl ester (L-NAME) has been used extensively as a paradigmatic inhibitor of NO synthase and has been shown to cause antinociception in several experimental models. We describe here how L-NAME produced a dose-dependent antinociceptive effect when injected intraperitoneally in the mouse after acetic acid induced writhings, or intraplantarly in the rat paw pressure hyperalgesia induced by carrageenin or prostaglandin E2. In contrast another NO synthase inhibitor, NG-monomethyl-L-arginine (L-NMMA), had no significant effect per se but inhibited L-NAME systemic induced antinociception in mice and local induced antinociception in the rat paw hyperalgesia test. D-NAME had no antinociceptive effect upon carrageenin-induced hyperalgesia. Pretreatment of the paws with two inhibitors of guanylate cyclase, methylene blue (MB) and 1H-:[1,2,4]-oxadiazolo-:[4,3-a] quinoxalin-1-one (ODQ) abolished the antinociceptive effect of L-NAME. L-Arginine and the cGMP phosphodiesterase inhibitor, MY 5445 significantly enhanced the L-NAME antinociceptive effect. The central antinociceptive effect of L-NAME was blocked by co-administration of L-NMMA, ODQ and MB. The present series of experiments shows that L-NAME, but not L-NMMA, has an antinociceptive effect. It can be suggested that L-NAME causes the antinociceptive effect by stimulation of the arginine/ NO/ cGMP pathway, since the antinociceptive effect of L-NAME can be antagonized by L-NMMA and abolished by the guanylate cyclase inhibitors (MB and ODQ). In addition, the NO synthase substrate, L-arginine and the cGMP phosphodiesterase inhibitor, MY5445 were seen to potentiate the effects of L-NAME. Thus, L-NAME used alone, has limitations as a specific inhibitor of the arginine-NO-cGMP pathway and may therefore be a poor pharmacological tool for use in characterising participation in pathophysiological processes.     

Does TAME induced contraction involve an endothelium dependent nitric oxide-cyclic GMP mediated pathway?

Two enzyme inhibitors namely L-NAME, a nitric oxide synthase (NOS) inhibitor and methylene blue, a guanylate cyclase inhibitor, were used to elucidate whether N-alpha-tosyl L-arginine methyl ester (TAME)-induced contractions in toad intestinal rings in vitro are mediated through a nitric oxide (NO)- cyclic GMP (c-GMP) pathway. Moreover, a NO precursor, L-arginine was also used to investigate its effect on TAME-induced contractions. Our findings provide evidence that TAME-induced contractions have both an endothelium-dependent and an endothelium-independent component. Based on our findings we now propose that TAME induced contraction involves an endothelium-dependent component mediated through NO and c-GMP.     

The influence of BPC 157 on nitric oxide agonist and antagonist induced lesions in broiler chicks

We describe the effects of nitric oxide (NO) agonists and antagonists and the influence of a novel organoprotective pentadecapeptide BPC 157, on the development of pulmonary hypertension syndrome and tissue lesions in chicks. Acute toxicity, which includes single dose application of saline (1 mL intraperitoneally (ip)), BPC 157 (10 μg/kg bw), L-NAME (NO antagonist, doses 50, 100, 150 mg/kg bw) and L-arginine (NO agonist/100 mg/kg bw with their combination L-NAME + BPC 157; L-NAME + L-arginine) was investigated. In this experiment pathohistological examination of the spleen, heart, liver and lungs and hematological analysis was conducted. In the chronic toxicity experiment, the animals were treated daily for 5 weeks with L-NAME (10 mg/kg bw), L-arginine (100 mg/kg bw), BPC 157 (10 μg/kg bw) and their combinations (L-NAME + BPC 157; L-NAME + L-arginine) ip. Seven animals from each group, including controls (saline 1 mL ip) were killed every week. Application of L-NAME caused pulmonary hypertension syndrome (PHS) in the treated chicks, which was prevented by the simultaneous application of L-arginine and BPC 157. Pathohistological examination of both acute and chronic toxicity revealed that L-NAME caused severe tissue damage (myocardial and hepatic cell necrosis, necrosis of the lymphoid cells in the spleen) while L-arginine provoked predominantly congestion, edema and hemorrhages in all organs. The effect of L-NAME was successfully inhibited by the application of L-arginine and BPC 157 but the latter substance did not cause any tissue or organ damage. Hematological analysis shows significant hemoglobin and leukocyte number decrease in the L-NAME-treated groups of chicks.     

Calcium/calmodulin-dependent kinase II mediates NO-elicited PKG activation to participate in spinal reflex potentiation in anesthetized rats

Calcium/calmodulin protein kinase (CaMK)-dependent nitric oxide (NO) and the downstream intracellular messenger cGMP, which is activated by soluble guanylate cyclase (sGC), are believed to induce long-term changes in efficacy of synapses through the activation of protein kinase G (PKG). The aim of this study was to examine the involvement of the CaMKII-dependent NO/sGC/PKG pathway in a novel form of repetitive stimulation-induced spinal reflex potentiation (SRP). A single-pulse test stimulation (TS; 1/30 Hz) on the afferent nerve evoked a single action potential, while repetitive stimulation (RS; 1 Hz) induced a long-lasting SRP that was abolished by a selective Ca(2+)/CaMKII inhibitor, autocamtide 2-related inhibitory peptide (AIP). Such an inhibitory effect was reversed by a relative excess of nitric oxide synthase (NOS) substrate, L-arginine. In addition, the RS-induced SRP was abolished by pretreatment with the NOS inhibitor, N(G)-nitro-L-arginine-methyl ester (L-NAME). The sGC activator, protoporphyrin IX (PPIX), reversed the blocking effect caused by L-NAME. On the other hand, a sGC blocker, 1H-[1, 2, 4]oxadiazolo[4, 3-alpha]quinoxalin-1-one (ODQ), abolished the RS-induced SRP. Intrathecal applications of the membrane-permeable cGMP analog, 8-bromoguanosine 3',5'-cyclic monophosphate sodium salt monohydrate (8-Br-cGMP), reversed the blocking effect on the RS-induced SRP elicited by the ODQ. Our findings suggest that a CaMKII-dependent NO/sGC/PKG pathway is involved in the RS-induced SRP, which has pathological relevance to hyperalgesia and allodynia.     

Pentadecapeptide BPC 157 Reduces Bleeding and Thrombocytopenia after Amputation in Rats Treated with Heparin,Warfarin, L-NAME and L-Arginine

Background

BPC 157 is a stable gastric pentadecapeptide recently implicated with a role in hemostasis. While NO is largely implicated in hemostatic mechanisms, in tail-amputation-models under heparin- and warfarin-administration, both the NO-synthase (NOS)-blocker, L-NAME (prothrombotic) and the NOS-substrate L-arginine (antithrombotic), were little investigated. Objective. To investigate the effect of L-NAME and L-arginine on hemostatic parameters, and to reveal the effects of BPC 157 on the L-NAME- and L-arginine-induced hemostatic actions under different pathological condition: tail amputation without or with anticoagulants, heparin or warfarin.

Methods

Tail amputation, and/or i.v.-heparin (10 mg/kg), i.g.-warfarin (1.5 mg/kg/day for 3 days) were used in rats. Treatment includes BPC 157, L-NAME, L-arginine, per se and their combination.

Results

After (tail) amputation, with or without i.v.-heparin or i.g.-warfarin, BPC 157 (10 μg/kg, 10 ng/kg, i.p., i.v. (heparin), 10 μg/kg i.g. (warfarin)) always reduced bleeding time and/or haemorrhage and counteracted thrombocytopenia. As for L-NAME and/or L-arginine, we noted: L-arginine (100 mg/kg i.p.)–rats: more bleeding, less/no thrombocytopenia; L-NAME (5 mg/kg i.p.)-rats: less bleeding (amputation only), but present thrombocytopenia; L-NAME+L-arginine-rats also exhibited thrombocytopenia: L-NAME counteracted L-arginine-increased bleeding, L-arginine did not counteract L-NAME-thrombocytopenia. All animals receiving BPC 157 in addition (BPC 157μg+L-NAME; BPC 157μg+L-arginine, BPC 157μg+L-NAME+L-arginine), exhibited decreased haemorrhage and markedly counteracted thrombocytopenia.

Conclusions

L-NAME (thrombocytopenia), L-arginine (increased haemorrhage) counteraction and BPC 157 (decreased haemorrhage, counteracted thrombocytopenia) with rescue against two different anticoagulants, implicate a BPC 157 modulatory and balancing role with rescued NO-hemostatic mechanisms.     

Role of endothelin type B receptor in NO/cGMP signaling pathway in rat median eminence

We studied the effect of endothelins (ETs) on receptor-mediated NO/cGMP signaling in rat arcuate nucleus–median eminence (AN-ME) fragments, an hypothalamic structure known to contain a rich plexus of nitric oxide synthase (NOS)-containing neurons and fibers together with densely arranged ET_B-receptor-like immunoreactive fibers. NOS activity was determined measuring the conversion of [³H] arginine to [³H] citrulline, as an index of NO produced. cGMP production was determined by radio immunoassay. ET-1, ET-3, and the selective ET_B receptor agonist, IRL1620, significantly increased cGMP formation and NOS activity. Preincubation of AN-ME fragment with L-arginine analog, N-nitro-L-arginine (L-NAME), inhibited ET-1 or IRL1620-stimulated cGMP formation. The addition of the selective ET_B receptor antagonist, BQ788, blocked ET-1-, ET-3-, or IRL1620-induced increase in NOS activity and cGMP generation, while BQ123, a selective ET_A receptor antagonist, was ineffective. Our results demonstrate that in whole rat AN-ME fragments, ETs stimulate NO/cGMP signaling pathway through the interaction with the ET_B receptor subtype, supporting the concept that ETs may represent an important regulator of reproductive and neuroendocrine function.     

Effect of nitric oxide on the somatostatinergic system in the rat exocrine pancreas

Nitric oxide (NO) and somatostatin (SS) are two important mediators of the exocrine and endocrine pancreas, exerting opposite effects on this organ. There is strong evidence suggesting an interaction between pancreatic NO and SS. The aim of this study was to determine whether L-arginine (L-Arg), the substrate for NO synthase (NOS), and Nomega-nitro-L-arginine methyl ester (L-NAME), a NOS inhibitor, regulate pancreatic somatostatin-like immunoreactivity (SSLI) content and the SS mechanism of action in pancreatic acinar cell membranes. L-Arg (150 mg/kg, intraperitoneally (i.p.)), L-NAME (50 mg/kg, i.p.) or L-NAME plus L-Arg were injected twice daily at 8 h intervals for 8 days. L-Arg decreased pancreatic SSLI content as well as the number of SS receptors in pancreatic acinar cell membranes whereas L-NAME increased both parameters. The stable SS analogue SMS 201-995 induced a significantly lower inhibition of forskolin-stimulated adenylyl cyclase activity in pancreatic acinar cell membranes from L-Arg-treated rats whereas an increased inhibition was observed in pancreatic acinar membranes from L-NAME-treated rats. These results indicate that the NO system may contribute to the regulation of the pancreatic somatostatinergic system.     

Correlation between nitric oxide and cyclooxygenase-2 pathways in head and neck squamous cell carcinomas

We investigated the interactions between inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) pathways in head and neck squamous cell carcinomas (HNSCCs) and in two carcinoma cell lines. HNSCCs showed an up-regulation of both pathways which were strongly correlated with each other (p=0.02) and with tumor vascularization (p=0.0001 and p=0.008, respectively). In carcinoma cells, Escherichia coli lipopolysaccharide (LPS) and EGF treatment up-regulated both pathways. NOS inhibitor N(G)-monomethyl-L-arginine methyl ester (L-NAME) inhibited this up-regulation. LPS or EGF induced iNOS expression that was not altered by NOS or COX-2 inhibitors. Conversely, LPS or EGF promoted COX-2 expression that was decreased by L-NAME. The NO donor S-nitroso-acetyl-penicillamine (SNAP) up-regulated COX-2 pathway and this effect was reduced by the guanylate cyclase inhibitor methylene blue. Thus, in squamous carcinoma cells, NO increases the activity of COX-2 pathway and this effect is probably mediated by endocellular cGMP level, with potential implications on tumor growth, angiogenesis, and therapy.