Role of nitric oxide and carbon monoxide in modulating the ACTH response to immune and nonimmune signals

The role played by nitric oxide (NO) and carbon monoxide (CO) was explored in the adult male rat by determining whether antagonizing the activity of the enzymes responsible for the formation of these gases altered the response of the hypothalamic-pituitary-adrenal (HPA) axis to immune (cytokines) or nonimmune (mild electroshocks) signals. The arginine derivative Nomeganitro-L-arginine-methylester (L-NAME), which inhibits all three NO synthase (NOS) isoforms [inducible (i), endothelial (e) and neuronal (n)] significantly augments the ACTH response to blood-borne cytokines, but decreases it in rats exposed to shocks or other physico-emotional stresses. The effect of L-NAME in both models is mimicked by L-nitroarginine (L-NNA) and L-nitromethylarginine (L-NMMA), which block constitutive (e and n) forms of NOS, but not by aminoguanidine (which blocks iNOS) or 7-nitroindazole (which specifically blocks nNOS). Despite the ability of L-NAME to markedly augment the stimulatory effect of vasopressin on ACTH secretion, removal of this peptide does not interfere with the interaction between L-NAME and systemically administered interleukin-1beta (IL-1beta). In contrast, blockade of prostaglandin formation prevents both the stimulatory effect of IL-1beta on ACTH release, and its potentiation by L-NAME. In contrast to the investigation of the importance of endogenous NO, studies focused on the role of CO remain scarce. Our preliminary results suggest that while blockade of the formation of this gas decreases the ACTH response to various stimuli, it also significantly interferes with the effect of L-NAME in rats systemically administered cytokines, and further decreases the ACTH response to shocks in animals also injected with arginine analogs. These results indicate the possible presence of functional interactions between NO and CO in regulating the activity of the HPA axis. Our present working hypothesis is that in the presence of elevated circulating cytokine levels, endogenous NO acts presynaptically to inhibit the release of ACTH secretagogues from nerve terminals in the infundibulum. As the acute ACTH response to these immune proteins is believed to primarily depend on events taking place within the median eminence, blockade of NO formation results in exaggerated ACTH release. During exposure to shocks and other nonimmune stresses, on the other hand, increased ACTH secretion is primarily due to activation of hypothalamic neurons. In this case, because of the stimulatory influence of endogenous NO on hypothalamic perikarya that manufacture corticotropin-releasing factor (CRF) and/or of the afferents to these neurons, blockade of NOS activity blunts CRF production, and consequently ACTH release. What remains undetermined is the net effect of the opposite influences of NO during long-term exposure to immune or nonimmune stress. Finally, it is possible that the conflicting results reported by investigators who study the role of NO and CO in isolated cell preparations may reflect, at least in part, these opposite effects of NO on different elements of the HPA axis.     

Indomethacin attenuates oxytocin and hypothalamic-pituitary-adrenal axis responses to systemic interleukin-1 beta

Systemic administration of the cytokine IL-1 beta produces a significant release of ACTH into the plasma and activation of hypothalamic oxytocin (OT) and corticotropin releasing factor (CRF) cells. However, the mechanism(s) by which systemic IL-1 beta induces these responses is not clear. In the present study, we have investigated the proposal that catecholamine cells of the ventrolateral medulla (VLM) and nucleus of the solitary tract (NTS) can relay circulating IL-1 signals via a prostaglandin-dependent mechanism to effect the HPA axis responses in the rat. Intra-arterial administration of IL-1 beta (1 pg/kg) to otherwise untreated animals produced a prominent release of ACTH into the plasma, substantial c-fos expression in paraventricular medial parvocellular (mPVN) corticotropin releasing factor (CRF) cells, supraoptic (SON) and paraventricular nucleus (PVN) OT cells, area postrema cells, NTS and VLM catecholamine cells and cells of the central amygdala. Pretreatment with the prostaglandin synthesis inhibitor, indomethacin (10 mg/kg body weight ia) 15 min before IL-1 beta administration (1 pg/kg ia) significantly reduced plasma ACTH release and c-fos expression in PVN and SON OT cells and MPVN CRF cells, in addition, the area postrema, A1 and C1 catecholamine cell groups of the VLM and A2 and C2 catecholamine cell groups of the NTS, all exhibited concomitant reductions in c-fos expression. Conversely indomethacin administration did not alter the IL1 beta-induced expression of c-fos in the central amygdala. These data suggest that central pathways involved in the IL-1 beta-induced activation of the HPA axis and OT cells are, at least in part, dependent upon prostaglandin synthesis. It is proposed that neurons in the area postrema, NTS and VLM might mediate this IL-1 beta-induced activation of hypothalamic CRF and OT cells and release of ACTH into the plasma.     

Altered ACTH and corticosterone responses to interleukin-1 beta in male rats exposed to an alcohol diet: possible role of vasopressin and testosterone

We have previously shown that female rats exposed to an alcohol (ethanol, E) diet exhibited a blunted ACTH response to systemically administered interleukin-1 beta (IL-1 beta). Because of the presence of gender differences in the activity of the hypothalamic-pituitary-adrenal (HPA) axis, and of the possible role played by sex steroids in modulating the inhibitory influence of E in females, we studied the ability of a 10-day E diet to alter ACTH and corticosterone secretion of intact or castrated male rats injected with IL-1 beta or endotoxin, a releaser of endogenous cytokines. Pituitary responsiveness to secretagogues that mediate the endocrine effects of IL-1 beta, namely corticotropin-releasing factor (CRF) and vasopressin (VP), was also investigated. The ACTH responses of animals fed ad libitum (C group) or pair-fed (PF group) to the intravenous administration of IL-1 beta or endotoxin were not statistically different (p > 0.05); therefore, results from these two groups were combined in the initial experiments. Subsequent experiments only used E and C animals. When compared with this latter group, intact E males showed a significant (p < 0.01) decrease in ACTH levels measured 30 and 60 min after the intravenous injection of IL-1 beta or endotoxin. In contrast, E rats released as much corticosterone as C rats in response to IL-1 beta, but significantly (p < 0.05) more following administration of endotoxin (lipopolysaccharide). The stimulatory effect of VP on ACTH release was also measurably blunted by alcohol, whereas that of CRF was not. In none of these experiments were any significant differences observed between C and PF rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Diinosine polyphosphates, a group of dinucleotides with antagonistic effects on diadenosine polyphosphate receptor

Nitric oxide synthase, an enzyme responsible for nitric oxide (NO) formation has been found in the hypothalamic paraventricular nucleus and median eminence, structures closely associated with regulation of the pituitary activity, and the pituitary gland itself. Nitric oxide modulates the stimulated release of CRH from the rat hypothalamus in vitro, which suggests its role in regulating the secretion of ACTH from the pituitary corticotrops and of corticosterone from the adrenal cortex. The purpose of the present study was to elucidate the yet unknown role of endogenous NO in the HPA response to central cholinergic stimulation in conscious rats. Neither L-arginine an NO precursor, nor the NO synthase blockers N omega-nitro-L-arginine methyl ester (L-NAME) and N omega-nitro-L-arginine (L-NNA) caused any consistent changes in the basal serum corticosterone levels. L-arginine, given in higher doses (120-150 mg/kg ip) 15 min prior to icv carbachol (2 micrograms), markedly diminished the carbachol-induced rise in corticosterone secretion. Systemic pretreatment with the nitric oxide synthase inhibitor L-NAME (5 mg/kg) significantly raised the carbachol-elicited corticosterone response, while addition of L-arginine completely blocked the effect of L-NAME. A similar increase in the carbachol-induced corticosterone response was produced by icv pretreatment with L-NAME (2 micrograms), indicating a central site of the NO interaction with cholinergic stimulation of the HPA response. L-NAME is a weak inhibitor of neuronal NOS itself, and must first be de-estrified to N omega-nitro-L-arginine to potently inhibit this enzyme. Systemic (10 mg/kg) and icv (1 microgram) pretreatment with L-NNA enhanced more effectively the carbachol-induced rise in corticosterone secretion than did pretreatment with L-NAME by either route. These results are the first direct evidence that endogenous NO significantly inhibits the HPA response to central cholinergic, muscarinic receptor stimulation under in vivo conditions.     

Role of central interleukin-1 beta in gastrointestinal motor disturbances induced by lipopolysaccharide in sheep

Cytokines are involved in the symptoms of the acute phase response induced by infectious diseases in humans as well as in animals, and interleukin-1 beta (IL-1 beta) has a pivotal role in these changes. The role of central IL-1 beta in the gastrointestinal hypomotility and fever evoked by intravenous administration of lipopolysaccharide (LPS) and the mechanisms involved, were investigated in sheep as an experimental model. LPS (0.1 microgram/kg, intravenously) induced gastrointestinal hypomotility and fever that were significantly reduced by prior intracerebroventricular administration of IL-1 receptor antagonist protein (IL-1ra, 2 micrograms/kg). The effects of LPS were mimicked by intracerebroventricular IL-1 beta (50 ng/kg), whereas IL-1 beta injected intravenously at the same dose only caused a slight and transient fever without modifying the gastrointestinal motility. Prior intracerebroventricular administration of the cyclooxygenase inhibitor indomethacin (100 micrograms/kg) but not the corticotropin-releasing factor (CRF) receptor antagonist alpha-helical CRF9-41 (5 micrograms/kg) blocked all effects by both LPS and IL-1 beta. These results suggest that in sheep, LPS induces digestive motor disturbances through a central release of IL-1 beta and prostaglandins.     

Interleukin 1 beta, but not tumor necrosis factor, enhances neurogenic vasodilatation in the rat skin: involvement of nitric oxide

In phenobarbitone-anesthetized rats the effects of interleukin 1 beta (IL-1 beta) and tumor necrosis factors (TNFs) were examined on the capsaicin-induced increase of plantar cutaneous blood flow in the rat hind paw as measured by laser Doppler flowmetry. IL-1 beta (0.5-500 pg) or TNF alpha or TNF beta (50-500 pg) was injected subcutaneously into the left paws, while the right paws received vehicle (10 microL) only. IL-1 beta was without effect on blood flow by its own but dose dependently enhanced the hyperemia due to capsaicin (0.3 microgram). TNFs failed to enhance the capsaicin-induced vasodilatation although 5000 pg TNF alpha produced a transient increase of local blood flow. Indomethacin (10 mg/kg, i.p.) did not alter the capsaicin-induced vasodilatation but prevented IL-1 beta (50 pg) from augmenting the hyperemic response to capsaicin. Likewise, blockade of nitric oxide formation by NG-nitro-L-arginine methyl ester (L-NAME) failed to affect the capsaicin-evoked vasodilatation but abolished its amplification by IL-1 beta. Systemic pretreatment with a neurotoxic dose of capsaicin reduced the capsaicin-induced hyperemia and prevented the facilitatory effect of IL-1 beta. The hyperemia evoked by intraplantar calcitonin gene related peptide (0.038-3.8 ng) was not altered by IL-1 beta (50 pg). These data indicate that IL-1 beta but not TNF enhances the cutaneous hyperemic response to capsaicin. This proinflammatory action arises from sensitization of afferent nerve endings and depends on nitric oxide and cyclooxygenase products as essential intermediates.     

Modulation of IL-1-induced cartilage injury by NO synthase inhibitors: a comparative study with rat chondrocytes and cartilage entities

Nitric oxide (NO) is produced in diseased joints and may be a key mediator of IL-1 effects on cartilage. Therefore, we compared the potency of new [aminoguanidine (AG), S-methylisothiourea (SMT), S-aminoethylisothiourea (AETU)] and classical [Nomega-monomethyl-L-arginine (L-NMMA), Nomega-nitro-L-arginine methyl ester (L-NAME)] NO synthase (NOS) inhibitors on the inhibitory effect of recombinant human interleukin-1beta (rhIL-1beta) on rat cartilage anabolism. Three different culture systems were used: (1) isolated chondrocytes encapsulated in alginate beads; (2) patellae and (3) femoral head caps. Chondrocyte beads and cartilage entities were incubated in vitro for 48 h in the presence of rhIL-1beta with a daily change of incubation medium to obtain optimal responses on proteoglycan synthesis and NO production. Proteoglycan synthesis was assessed by incorporation of radiolabelled sodium sulphate [Na2(35)SO4] and NO production by cumulated nitrite release during the period of study. Chondrocytes and patellae, as well as femoral head caps, responded concentration-dependently to IL-1beta challenge (0 to 250 U ml(-1) and 0 to 15 U ml(-1) respectively) by a large increase in nitrite level and a marked suppression of proteoglycan synthesis. Above these concentrations of IL-1beta (2500 U ml(-1) and 30 U ml(-1) respectively), proteoglycan synthesis plateaued whereas nitrite release still increased thus suggesting different concentration-response curves. When studying the effect of NOS inhibitors (1 to 1000 microM) on NO production by cartilage cells stimulated with IL-1beta (25 U ml(-1) or 5 U ml(-1)), we observed that: (i) their ability to reduce nitrite level decreased from chondrocytes to cartilage samples, except for L-NMMA and AETU; (ii) they could be roughly classified in the following rank order of potency: AETU > L-NMMA > or = SMT > or = AG > or = L-NAME and (iii) AETU was cytotoxic when used in the millimolar range. When studying the effect of NOS inhibitors on proteoglycan synthesis by cartilage cells treated with IL-1beta, we observed that: (i) they had more marked effects on proteoglycan synthesis in chondrocytes than in cartilage samples; (ii) they could be roughly classified in the following rank order of potency: L-NAME > or = L-NMMA > > AG > SMT > > AETU and (iii) potentiation of the IL-1 effect by AETU was consistent with cytotoxicity in the millimolar range. D-isomers of L-arginine analog inhibitors (1000 microM) were unable to correct nitrite levels or proteoglycan synthesis in IL-1beta treated cells. L-arginine (5000 microM) tended to reverse the correcting effect of L-NMMA (1000 microM) on proteoglycan synthesis, thus suggesting a NO-related chondroprotective effect. However, data with L-NAME and SMT argued against a general inverse relationship between nitrite level and proteoglycan synthesis. Dexamethasone (0.1 to 100 microM) (i) failed to inhibit NO production in femoral head caps and chondrocytes beads whilst reducing it in patellae (50%) and (ii) did not affect or worsened the inhibitory effect of IL-1beta on proteoglycan synthesis. Such results suggested a corticosteroid-resistance of rat chondrocyte iNOS. Data from patellae supported a possible contribution of subchondral bone in NO production. In conclusion, our results suggest that (i) NO may account only partially for the suppressive effects of IL-1beta on proteoglycan synthesis, particularly in cartilage samples; (ii) the chondroprotective potency of NOS inhibitors can not be extrapolated from their effects on NO production by joint-derived cells and (iii) L-arginine analog inhibitors are more promising than S-substituted isothioureas for putative therapeutical uses.     

Distinct pattern of ret oncogene rearrangements in morphological variants of radiation-induced and sporadic thyroid papillary carcinomas in children

We have previously shown that daily injection of alcohol for 3 days induced a significant and long-lasting blunting of the hypothalamic-pituitary-adrenal (HPA) axis response to a subsequent treatment with this drug. The fact that, in contrast, the HPA axis response to footshocks was not altered by prior alcohol administration, suggested the presence of a phenomenon of selective neuroendocrine tolerance. To further test this hypothesis, we determined whether an initial alcohol challenge would alter the ACTH response to immune signals, such as interleukin-1beta (IL-1beta) and/or endotoxin (lipopolysaccharide; LPS). Because of the functional connection between the HPA axis and immune responses, we also determined whether the LPS-induced release of tumor necrosis factor-alpha and interleukin-6, as well as IgG and IgM responses to an antigenic challenge, would be influenced by previous exposure to alcohol. We show here that the intragastric injection of 3 g of alcohol/kg daily for 3 days did not significantly alter the ability of IL-1beta (400 ng/kg) or LPS (1 microg/kg), both injected intravenously 7 days later, to release ACTH. Drug pretreatment did not significantly alter the tumor necrosis factor-alpha response to the low dose of endotoxin used, whereas there was a tendency toward increased circulating interleukin-6 levels in alcohol-pretreated animals. Finally the IgG, but not IgM, response to the antigen phosphocholine-keyhole limpet hemocyanin was significantly (p < 0.05) augmented in rats administered alcohol 7 days before the antigenic challenge. Collectively, these results indicate that an initial exposure to alcohol does not induce long-term changes in the ability of an immune signal (IL-1beta or endotoxin) to activate the HPA axis. In contrast, a small but detectable enhancement of cytokine responses to LPS, and of the IgG response to phosphocholine-keyhole limpet hemocyanin, was observed.     

Cholestatic liver diseases in adults

BACKGROUND: Nitric oxide (NO) blockade by L-nitroarginine methyl ester (L-NAME) inhibits pancreatic secretion in vivo and aggravates caerulein induced pancreatitis. Nitric oxide synthase (NOS) is present in pancreatic islets, endothelium, and nerve fibres. L-NAME blocks all known NOS isoforms. AIM: To investigate the source of NO blocked by L-NAME that inhibits amylase secretion. METHODS: Amylase output was measured in rats in response to caerulein (0.1-50 microg/kg) alone or with indazole. Baseline secretion and the response to supramaximal caerulein were also examined after administration of indazole, L-NAME, haemoglobin, or aminoguanidine under continuous blood pressure measurement. In separate experiments, pancreatic secretion was measured after blockade of afferent nerve fibres by either systemic or local capsaicin. The effect of neural NOS inhibition on caerulein induced pancreatitis was also investigated. RESULTS: L-NAME, haemoglobin, and supramaximal caerulein (10 microg/kg) increased blood pressure, whereas indazole and suboptimal caerulein (0.1 microg/kg) did not. Indazole and capsaicin decreased basal amylase output. L-NAME and haemoglobin reduced basal amylase output to a lesser extent and potentiated the inhibitory response to supramaximal caerulein. In contrast, full neural NOS inhibition by L-NAME partially reversed the expected caerulein induced suppression of amylase output. This effect was reproduced by indazole and capsaicin. Indazole did not alter responses to either optimal (0.25 microg/kg) or suboptimal (0.1 microg/kg) caerulein, nor, in contrast with L-NAME, aggravate the outcome of caerulein induced pancreatitis. CONCLUSIONS: Reduction of circulating NO availability, probably of endothelial origin, is responsible for the decrease in amylase secretion observed in the early response to L-NAME. Nitrergic neurotransmission plays an important role in the control of pancreatic secretion and may induce opposite effects to endothelial NOS activity.     

Nitric oxide synthase inhibitors alter ventilation in isoflurane anesthetized rats

BACKGROUND: Nitric oxide (NO) is present in medullary structures and can modulate respiratory rhythm. The authors determined if spontaneous ventilation at rest and in response to increased carbon dioxide is altered by selective neuronal NO synthase (NOS; 7-nitro-indazole, 7-NI) or nonselective (neuronal plus endothelial) NOS (NG-L-arginine methyl ester [L-NAME] and NG-monomethyl L-arginine [L-NMMA]) inhibitors in rats anesthetized with isoflurane. METHODS: Fifty-four rats received either L-NAME or L-NMMA (1, 10, and 30 mg/kg) or 7-NI (20, 80, and 400 mg/kg) and were compared with time controls (isoflurane = 1.4%), with isoflurane concentrations (1.6%, 1.8%, and 2%) increased consistent with the increased anesthetic depth caused by NOS inhibitors, or with L-arginine (300 mg/kg). Tidal volume (VT), respiratory frequency (f), minute ventilation (VE), and ventilatory responses to increasing carbon dioxide were determined. RESULTS: L-NAME and L-NMMA decreased resting VT and VE, whereas 7-NI had no effect. Increasing concentrations of isoflurane decreased resting f, VT, and VE. L-NAME and L-NMMA decreased VT and VE, whereas 7-NI had no effect at 8%, 9%, and 10% end-tidal carbon dioxide (ETCO2). Increasing concentrations of isoflurane decreased f, VT, and VE at 8%, 9%, and 10% ETCO2. The slope of VE versus ETCO2 was decreased by isoflurane but was unaffected by L-NAME, L-NMMA, or 7-NI. L-arginine alone had no effect on ventilation. CONCLUSIONS: Nonselective NOS inhibitors decreased VT and VE at rest and at increased carbon dioxide levels but did not alter the slope of the carbon dioxide response. Selective neuronal NOS inhibition had no effect, suggesting that endothelial NOS may be the isoform responsible for altering ventilation. Finally, the cause of the decreased ventilation is not a result of the enhanced anesthetic depth caused by NOS inhibitors.     

Regulation of the inducible cyclo-oxygenase pathway in human cultured airway epithelial (A549) cells by nitric oxide

1. In airway epithelium, nitric oxide (NO) is synthesized in the setting of inflammation by inducible nitric oxide synthase (iNOS). Although the role of epithelial derived NO in the regulation of human airways is unknown, prostaglandin E2 (PGE2) is recognised as an important inhibitory mediator in human airways. Cyclo-oxygenase (COX) is the rate limiting enzyme in the production of prostanoids and since inflammatory pathways enhance the expression of an inducible COX (COX-2), both COX-2 and iNOS may be co-expressed in response to an inflammatory stimulus. Although regulation of the COX-2 pathway by NO has been demonstrated in animal models, its potential importance in human airway epithelium has not been investigated. 2. The effect of endogenous and exogenous NO on the COX-2 pathway was investigated in the A549 human airway epithelial cell culture model. Activity of the COX-2 pathway was assessed by PGE2 EIA, and iNOS pathway activity by nitrite assay. A combination cytokine stimulus of interferon gamma (IFNgamma) 100 u ml(-1), interleukin-1beta (IL-1beta) 1 u ml(-1) and lipopolysaccharide (LPS) 10 microg ml(-1) induced nitrite formation which could be inhibited by the competitive NOS inhibitor N(G)-nitro-L-arginine-methyl-ester (L-NAME). IL-1beta alone (1-50 u ml(-1) induced PGE2 formation without significant nitrite formation, a response which was inhibited by the COX-2 specific inhibitor nimesulide. Submaximal stimuli used for further experiments were IFNgamma 100 u ml(-1), IL-1beta 1 u ml(-1) and LPS 10 microg ml(-1) to induce both the iNOS and COX-2 pathways, and IL-1beta 3 u ml(-1) to induce COX-2 without iNOS activity. 3. Cells treated with IFNgamma 100 u ml(-1), IL-1beta I u ml(-1) and LPS 10 microg ml(-1) for 48 h either alone, or with the addition of L-NAME (0 to 10(-2) M), demonstrated inhibition by L-NAME of PGE2 (3.61 +/- 0.55 to 0.51 +/- 0.04 pg/l0(4) cells; P<0.001) and nitrite (34.33 +/- 8.07 to 0 pmol/10(4) cells; P<0.001) production. Restoration of the PGE2 response (0.187 +/- 0.053 to 15.46 +/- 2.59 pg/10(4) cells; P<0.001) was observed after treating cells with the same cytokine stimulus and L-NAME 10(-6) M, but with the addition of the NOS substrate L-arginine (0 to 10(-5) M). 4. Cells incubated with IL-1beta 3 u ml(-1) for 6 h, either alone or with addition of the NO donor S-nitroso-acetyl-penicillamine (SNAP) (0 to 10(-4) M), demonstrated increased PGE2 formation (1.23 +/- 0.03 to 2.92 +/- 0.19 pg/10(4) cells; P< 0.05). No increase in PGE2 formation was seen when the experiment was repeated in the presence of the guanylate cyclase inhibitor methylene blue (50 microM). Cells treated with SNAP alone did not demonstrate an increased PGE2 formation. Cells incubated with IL-1beta 3 u ml(-1) for 6 h in the presence of dibutyryl cyclic guanylate monophosphate (0 to 10(-3) M) also demonstrated an increased PGE2 response (2.56 +/- 0.21 to 4.53 +/- 0.64 pg/10(4) cells; P<0.05). 5. These data demonstrate that in a human airway epithelial cell culture system, both exogenous and endogenous NO increase the activity of the COX-2 pathway in the setting of inflammatory cytokine stimulation, and that this effect is likely to be mediated by guanylate cyclase. This suggests a role for NO in the regulation of human airway inflammation.     

Alteration of the cytokine phenotype in an experimental lung granuloma model by inhibiting nitric oxide

Pulmonary granulomatous inflammation modulated by IFN-gamma and IL-12 is also associated with augmented inducible nitric oxide synthase (NOS II). To address the role of increased nitric oxide synthesis in this model, mice received daily i.p. injections of NG-nitro-L-arginine-methyl ester (L-NAME; 8 mg/kg) during both the 2-wk immunization period with purified protein-derivative (PPD) and the subsequent lung challenge with PPD-coated Sepharose beads. Other groups of animals received saline, L-NAME or NG-nitro-D-arginine-methyl ester (D-NAME; 8 mg/kg) during the pulmonary embolization period and not the PPD sensitization period. On day 4 post-PPD bead challenge, PCR analysis of the whole lung revealed that NOS II expression appeared to be similar in both of the L-NAME treatment protocols. L-NAME-treated mice in both dosing protocols had lung lesions that were significantly larger than granuloma lesions measured in mice that received saline or D-NAME. The enlarged lesions from L-NAME-treated mice contained markedly greater numbers of neutrophils and eosinophils. Equivalent numbers of PPD-activated dispersed cells from whole lungs of L-NAME-treated mice produced significantly higher levels of IL-4 and IL-10 and smaller amounts of IL-12 and IFN-gamma compared with similar lung cultures derived from control or D-NAME-treated mice. Levels of C-C chemokines such as monocyte chemoattractant protein-1 (MCP-1), C10, and macrophage inflammatory protein-1alpha (MIP-1alpha) were also significantly elevated in lung cultures from L-NAME-treated mice compared with controls. Thus, nitric oxide regulates the size and cellular composition of the Th1-type lung granuloma, possibly through its effects on the cytokine and chemokine profile associated with this lesion.     

Early endoscopic realignment as primary therapy for complete posterior urethral disruptions

Circadian changes in the interactions between L-NG-nitroarginine methyl ester (L-NAME), a nitric oxide synthase (NOS) inhibitor, and morphine-induced antinociception were investigated by the mouse hot-plate test. Both the basal pain sensitivity and morphine-induced analgesia undergo significant 24 h variations. L-NAME (40 mg/kg, i.p.) alone did not show any antinociceptive activity, but potentiated morphine-induced analgesia when combined with morphine at all injection times. In terms of percentage absolute potentiation (%AP), L-NAME dramatically augmented the analgesic effect of morphine in the late dark period at 19 hours after lights on (HALO). It is concluded that nitric oxide (NO) is involved in the modulation of the analgesic effect of morphine; thus, the L-NAME and morphine combination might be beneficial in alleviating pain.     

Practical development of re-usable terminologies: GALEN-IN-USE and the GALEN Organisation

Excessive nitric oxide (NO) synthesis, by inducible NO synthase (iNOS), has been implicated in the pathogenesis of inflammatory diseases such as rheumatoid arthritis. We investigated the pathophysiological role of NO using an adjuvant-induced arthritis model. Kinetics of iNOS mRNA expression in paw and spleen showed that it was induced from an early stage of the disease. To further characterize the pathophysiological relevance of iNOS induction in spleen, the mitogenic response of spleen cells was examined. ConA-induced proliferation of spleen cells from arthritic rats was completely suppressed in comparison to normal rats. Elevation of nitrite, which could be converted from NO, was also observed in the culture supernatants. Addition of three NOS inhibitors, S-(2-aminoethyl) isothiouronium bromide (ITU), aminoguanidine (AG) and LNG-nitroarginine methyl ester (L-NAME) all reduced the nitrite level and restored the proliferative response dose-dependently. These NOS inhibitors also showed anti-arthritic effects. Daily subcutaneous administration of either ITU at 50 mg/kg or AG at 200 mg/kg suppressed the paw swelling by 50% in arthritic rats on day 18. Oral administration of L-NAME at 30 mg/kg showed a tendency to suppress the development of arthritis from day 11 to day 15. However, drug-induced hypertension was observed with L-NAME due to poor selectivity for iNOS isozyme. These results suggest that augmented NO synthesis, via iNOS induction, may be partly involved in the pathogenesis of adjuvant-induced arthritis by causing defects in lymphocyte function. Thus, selective inhibition of iNOS might be beneficial for the treatment of immunological abnormalities associated with inflammatory diseases.     

Role of nitric oxide in sepsis-associated pulmonary edema

Transient pulmonary hypertension after inhibition of nitric oxide synthase (NOS) does not alter pulmonary reflection coefficients or lymph flows in endotoxemic sheep. To test the effects of persistent pulmonary hypertension induced by N omega-nitro-L-arginine methylester (L-NAME) and of inhaled NO on pulmonary edema, 18 sheep (three groups) were chronically instrumented with pulmonary artery catheters, femoral arterial fiberoptic thermistor catheters, and tracheostomy. The awake, spontaneously breathing animals received Salmonella typhi endotoxin (lipopolysaccharide; LPS) (10 ng/kg/ min) for 28 h. After 24 h, an airflow of 6 L/min was delivered through the tracheostomy. One group of animals (L-NAME/air) received L-NAME intravenously (25 mg/kg + 5 mg/kg/h) and breathed air. The second group (L-NAME/NO) was given L-NAME and NO (40 ppm) was added to the airflow. The third group was given NaCl 0.9% and breathed air (NaCl/air). Extravascular lung water was measured through the double-indicator dilution technique. Endotoxemia caused pulmonary edema, which was aggravated by L-NAME. Breathing of NO normalized pulmonary artery pressure (Ppa) and ameliorated pulmonary edema. Inhalation of NO may therefore be a therapeutic option for pulmonary edema associated with pulmonary hypertension.     

Mediation by nitric oxide of TRH-, but not metoclopramide-stimulated TSH secretion in humans

In order to establish whether nitric oxide (NO) participates in the regulation of thyroid stimulating hormone (TSH) secretion in humans, seven normal men were treated with a placebo (normal saline) or the NO synthase inhibitor L-NAME, given at doses (40 micrograms kg-1 injected plus 50 micrograms kg-1 infused i.v.) previously found to be unable to change blood pressure. Experiments were carried out either in basal conditions or during stimulation of TSH secretion with an i.v. injection of 200 micrograms thyrotropin releasing hormone (TRH) or 10 mg of the dopaminergic antagonist metoclopramide (MCP). Administration of L-NAME did not change the basal secretion of TSH or the TSH response to MCP, but significantly reduced the TSH increase induced by TRH. These data fail to provide evidence of NO involvement in regulation of basal TSH secretion. NO also appears to be without effects on the dopaminergic control of TSH secretion. In contrast, the inhibitory effect of L-NAME on TRH-induced TSH secretion suggests the mediation by NO of the TSH-releasing action of TRH.     

Effects of nitric oxide synthase inhibition on brain infarction in SOD-1-transgenic mice following transient focal cerebral ischemia

To investigate the role of superoxide in the toxicity of nitric oxide (NO), we examined the effect of nitric oxide synthase (NOS) inhibition on brain infarction in transgenic mice overexpressing CuZn-superoxide dismutase (SOD-1). Male SOD-transgenic mice and non-transgenic littermates (30-35 g) were subjected to 60 min of middle cerebral artery occlusion followed by 24 h of reperfusion. Either NG-nitro-L-arginine methyl ester (L-NAME; 3 mg/kg), a mixed neuronal and endothelial NOS inhibitor, or 7-nitroindazole (7-NI; 25 mg/kg), a selective neuronal NOS inhibitor, was administered intraperitoneally 5 min after the onset of ischemia. At 24 h of reperfusion, the mice were decapitated and the infarct volume was evaluated in each group. In the nontransgenic mice, L-NAME significantly increased the infarct volume as compared with the vehicle, while 7-NI significantly decreased it. In the SOD-transgenic mice, L-NAME-treated animals showed a significantly larger infarct volume than vehicle-treated ones, whereas there were no significant differences between 7-NI- and vehicle-treated mice. Our findings suggest that selective inhibition of neuronal NOS ameliorates ischemic brain injury and that both neuronal and endothelial NOS inhibition may result in the deterioration of ischemic injury due to vasoconstriction of the brain. Since L-NAME increased infarct volume even in SOD-transgenic mice, the protective effect of SOD could result from the vasodilation by increased endothelial NO as well as the reduction of neuronal injury due to less production of peroxynitrite compared to wild-type mice. Moreover, the neurotoxic role of NO might not be dependent on NO itself, but the reaction with superoxide to form peroxynitrite, because of no additive effects of SOD and a neuronal NOS inhibitor.     

The morphogenic/cytotoxic and prostaglandin-stimulating activities of interleukin-1 beta in the rat ovary are nitric oxide independent

Nitric oxide (NO) has been implicated as a mediator of physiologic and pathologic cellular injury. Since the cytokine interleukin-1 beta (IL-1 beta) induces nitric oxide synthase (NOS) activity as well as effects morphogenic/cytotoxic changes and increased prostaglandin (PGE2) levels in cultured whole ovarian dispersates, we set out to determine whether these actions are interrelated. Treatment with IL-1 beta resulted in a marked increase in media nitrite and nitrate accumulation, morphological alterations, and increased release of lactate dehydrogenase (LDH) into media. Addition of IL-1 receptor antagonist (RA) eliminated these IL-1 beta effects. In contrast, specific inhibitors of NOS failed to reverse IL-1 beta-induced morphogenic changes or LDH release in spite of complete reduction of media nitrite to control levels. Similarly, treatment with transforming growth factor beta 1, inhibited IL-1 beta-induced nitrite accumulation, but had no effect on the morphologic or cytotoxic endpoints. Moreover, the addition of sodium nitroprusside, an NO generator, resulted in progressive increments in media nitrite content without a corresponding increase in the IL-1 beta-associated morphogenic changes or media LDH content. Furthermore, IL-1-induced PGE2 accumulation remained unaffected by specific NOS inhibition. These observations support the view that NO does not mediate the morphogenic/cytotoxic or inflammatory-like (e.g., PGE2 inducing) properties of IL-1 beta in cultured whole ovarian dispersates. Although the precise role of NO in ovarian physiology remains unknown, it is possible that NO participates in the periovulatory modulation of ovarian blood flow by virtue of its potent vasodilatory activity.