Intranasal inoculation with mouse hepatitis virus strain JHM (MHV-JHM) results in acute meningoencephalitis. We found NOS II mRNA expression in brains of acutely infected animals on days 5 through 7 after infection. In situ hybridization and immunohistochemistry demonstrated NOS II message and protein in infiltrating macrophages. Persistent infection with MHV-JHM results in chronic demyelinating encephalomyelitis. NOS II mRNA was detected in persistently infected spinal cords. In situ hybridization and immunohistochemistry showed expression of NOS II in astrocytes in and around demyelinated lesions. These results suggest the role of NO release in acute versus persistent infection with this virus, and its contribution to the resulting pathology, may be different.

Recent studies showed that angiotensin II type 1 receptor blocker (ARB) slows progression of chronic renal disease in patients with type 2 diabetes, regardless of changes in blood pressure. We showed that the imbalance of nitric oxide (NO) and reactive oxygen species (ROS) due to endothelial NO synthase (eNOS) uncoupling contributed to renal dysfunction in the diabetic nephropathy. The aim of this study was to determine the effects of ARB on uncoupled eNOS in rat diabetic nephropathy.

We have previously demonstrated that the neurosteroid dehydroepiandrosterone sulfate (DHEAS) induces functional potentiation of N-methyl-D-aspartate (NMDA) receptors via increases in phosphorylation of NMDA receptor GluN1 subunit (pGluN1). However, the modulatory mechanisms responsible for the expression of the DHEA-synthesizing enzyme, cytochrome P450c17 following peripheral nerve injury have yet to be examined. Here we determined whether oxidative stress induced by the spinal activation of nitric oxide synthase type II (NOS-II) modulates the expression of P450c17 and whether this process contributes to the development of neuropathic pain in rats. Chronic constriction injury (CCI) of the sciatic nerve induced a significant increase in the expression of NOS-II in microglial cells and NO levels in the lumbar spinal cord dorsal horn at postoperative day 5. Intrathecal administration of the NOS-II inhibitor, L-NIL during the induction phase of neuropathic pain (postoperative days 0~5) significantly reduced the CCI-induced development of mechanical allodynia and thermal hyperalgesia. Sciatic nerve injury increased the expression of PKCand PKA-dependent pGluN1 as well as the mRNA and protein levels of P450c17 in the spinal cord at postoperative day 5, and these increases were suppressed by repeated administration of L-NIL. Co-administration of DHEAS together with L-NIL restored the development of neuropathic pain and pGluN1 that were originally inhibited by L-NIL administration alone. Collectively these results provide strong support for the hypothesis that activation of NOS-II increases the mRNA and protein levels of P450c17 in the spinal cord, ultimately leading to the development of central sensitization and neuropathic pain induced by peripheral nerve injury.

Proline-rich tyrosine kinase 2 (PYK2) can be activated by angiotensin II (Ang II) and reactive oxygen species. We report that in endothelial cells, Ang II enhances the tyrosine phosphorylation of endothelial NO synthase (eNOS) in an AT(1)-, H(2)O(2)-, and PYK2-dependent manner. Low concentrations (1-100 micromol/liter) of H(2)O(2) stimulated the phosphorylation of eNOS Tyr657 without affecting that of Ser1177, and attenuated basal and agonist-induced NO production. In isolated mouse aortae, 30 micromol/liter H(2)O(2) induced phosphorylation of eNOS on Tyr657 and impaired acetylcholine-induced relaxation. Endothelial overexpression of a dominant-negative PYK2 mutant protected against H(2)O(2)-induced endothelial dysfunction. Correspondingly, carotid arteries from eNOS(-/-) mice overexpressing the nonphosphorylatable eNOS Y657F mutant were also protected against H(2)O(2). In vivo, 3 wk of treatment with Ang II considerably increased levels of Tyr657-phosphorylated eNOS in the aortae of wild-type but not Nox2(y/-) mice, and this was again associated with a clear impairment in endothelium-dependent vasodilatation in the wild-type but not in the Nox2(y/-) mice. Collectively, endothelial PYK2 activation by Ang II and H(2)O(2) causes the phosphorylation of eNOS on Tyr657, attenuating NO production and endothelium-dependent vasodilatation. This mechanism may contribute to the endothelial dysfunction observed in cardiovascular diseases associated with increased activity of the renin-angiotensin system and elevated redox stress.

Increasing evidences suggest that angiotensin (Ang) II participates in the pathogenesis of endothelial dysfunction (ED) through multiple signaling pathways, including angiotensin type 1 receptor (AT1R) mediated NADPH oxidase (Nox)/reactive oxygen species (ROS) signal transduction. However, the detailed mechanism is not completely understood. In this study, we reported that AngII/AT1R-mediated activated protein phosphatase 2A (PP2A) downregulated endothelial nitric oxide synthase (eNOS) phosphorylation via Nox/ROS pathway. AngII treatment reduced the levels of phosphorylation of eNOS Ser1177 and nitric oxide (NO) content along with phosphorylation of PP2Ac (PP2A catalytic subunit) Tyr307, meanwhile increased the PP2A activity and ROS production in human umbilical vein endothelial cells (HUVECs). These changes could be impeded by AT1R antagonist candesartan (CAN). The pretreatment of 10-8 M PP2A inhibitor okadaic acid (OA) reversed the levels of eNOS Ser1177 and NO content. Similar effects of AngII on PP2A and eNOS were also observed in the mesenteric arteries of Sprague-Dawley rats subjected to AngII infusion via osmotic minipumps for 2 weeks. We found that the PP2A activity was increased, but the levels of PP2Ac Tyr307 and eNOS Ser1177 as well as NO content were decreased in the mesenteric arteries. The pretreatments of antioxidant N-acetylcysteine (NAC) and apocynin (APO) abolished the drop of the levels of PP2Ac Tyr307 and eNOS Ser1177 induced by AngII in HUVECs. The knockdown of p22phox by small interfering RNA (siRNA) gave rise to decrement of ROS production and increment of the levels of PP2Ac Tyr307 and eNOS Ser1177. These results indicated that AngII/AT1R pathway activated PP2A by downregulating its catalytic subunit Tyr307 phosphorylation, which relies on the Nox activation and ROS production. In summary, our findings indicate that AngII downregulates PP2A catalytic subunit Tyr307 phosphorylation to activate PP2A via AT1R-mediated Nox/ROS signaling pathway. The activated PP2A further decreases levels of eNOS Ser1177 phosphorylation and NO content leading to endothelial dysfunction.

1. We have measured extracellular NO/NO(2)(-) concentrations in guinea-pig suprachiasmatic nucleus (SCN) brain slices using fast cyclic voltammetry. A rapid and transient signal equivalent to 2.2+/-0.2 microM NO/NO(2)(-) (mean+/-s.e.mean, n=13) was detected at 1.26 V, the peak oxidation potential for NO, following local electrical stimulation (five pulses of 0.1 ms duration at 100 Hz, delivered every 5 min). 2. The NO/NO(2)(-) signal was inhibited by the non-selective nitric oxide synthase (NOS) inhibitors L-NAME, L-NMMA and the highly selective type II NOS (iNOS) inhibitor 1400 W (Garvey et al., 1997) in a concentration-dependent manner. IC(50) values were 229 microM (65 - 801, n=3, geomean and 95% confidence intervals (C.I.)), 452 nM (88 - 2310, n=5), and 14.2 microM (3.6 - 54.4, n=5), with maximum inhibitions of 82.8+/-6.7, 46.0+/-8.1, and 90.6+/-3.6%, respectively. 3. Exposure of the slices to the protein synthesis inhibitor cyclohexamide or the inhibitor of type II NOS induction dexamethasone immediately following slice cutting, and for a subsequent 4 - 5 h, did not inhibit the NO/NO(2)(-) signal. 4. The evoked NO/NO(2)(-) signal was not reduced following 6 h perfusion in Ca(2+)-free media, consistent with a Ca(2+)-independent type II NOS activity. 5. PCR for type II NOS revealed the presence of this isotype in the SCN, even immediately following removal of the brain. 6. These studies provide the first evidence to suggest a functional, constitutively-active type II NOS within the brain of normal, healthy adult animals, and add type II NOS to the multiple isotypes of NO synthase playing a role within the mammalian SCN.

Arginase II reciprocally regulates endothelial nitric oxide synthase (eNOS) through a p32-dependent Ca2+ control. We investigated the signaling pathway of arginase II-dependent eNOS phosphorylation. Western blot analysis was applied for examining protein activation and [Ca2+]c was analyzed by microscopic and FACS analyses. Nitric oxide (NO) and reactive oxygen species (ROS) productions were measured using specific fluorescent dyes under microscopy. NO signaling pathway was tested by measuring vascular tension. Following arginase II downregulation by chemical inhibition or gene knockout (KO, ArgII-/-), increased eNOS phosphorylation at Ser1177 and decreased phosphorylation at Thr495 was depend on p38 MAPK activation, which induced by CaMKII activation through p32-dependent increase in [Ca2+]c. The protein amount of p32 negatively regulated p38 MAPK activation. p38 MAPK contributed to Akt-induced eNOS phosphorylation at Ser1177 that resulted in accelerated NO production and reduced reactive oxygen species production in aortic endothelia. In vascular tension assay, p38 MAPK inhibitor decreased acetylcholine-induced vasorelaxation responses and increased phenylephrine-dependent vasoconstrictive responses. In ApoE-/- mice fed a high cholesterol diet, arginase II inhibition restored p32/CaMKII/p38 MAPK/Akt/eNOS signaling cascade that was attenuated by p38 MAPK inhibition. Here, we demonstrated a novel signaling pathway contributing to understanding of the relationship between arginase II, endothelial dysfunction, and atherogenesis.

Hypertension is a major cause of heart attack and stroke. Our recent study revealed that gallic acid (GA) exerts protective effects on pressure overload-induced cardiac hypertrophy and dysfunction. However, the role of GA in angiotensin II (Ang II)-induced hypertension and vascular remodeling remains unknown. C57BL/6J mice were subjected to saline and Ang II infusion. Systolic blood pressure was measured using a tail-cuff system. Vascular remodeling and oxidative stress were examined by histopathological staining. Vasodilatory function was evaluated in the aortic ring. Our findings revealed that GA administration significantly ameliorated Ang II-induced hypertension, vascular inflammation, and fibrosis. GA also abolished vascular endothelial dysfunction and oxidative stress in Ang II-infused aortas. Mechanistically, GA treatment attenuated Ang II-induced upregulation of the immunoproteasome catalytic subunits β2i and β5i leading to reduction of the trypsin-like and chymotrypsin-like activity of the proteasome, which suppressed degradation of endothelial nitric oxide synthase (eNOS) and reduction of nitric oxide (NO) levels. Furthermore, blocking eNOS activity by using a specific inhibitor (L-N G-nitroarginine methyl ester) markedly abolished the GA-mediated beneficial effect. This study identifies GA as a novel immunoproteasome inhibitor that may be a potential therapeutic agent for hypertension and vascular dysfunction.

Nitric oxide, produced by the neuronal nitric oxide synthase (nNOS) from L-arginine is an important second messenger molecule in the central nervous system: It influences the synthesis and release of neurotransmitters and plays an important role in long-term potentiation, long-term depression and neuroendocrine secretion. However, under certain pathological conditions such as Alzheimer's or Parkinson's disease, stroke and multiple sclerosis, excessive NO production can lead to tissue damage. It is thus desirable to control NO production in these situations. So far, little is known about the substrate supply to human nNOS as a determinant of its activity. Measuring bioactive NO via cGMP formation in reporter cells, we demonstrate here that nNOS in both, human A673 neuroepithelioma and TGW-nu-I neuroblastoma cells can be fast and efficiently nourished by extracellular arginine that enters the cells via membrane transporters (pool I that is freely exchangeable with the extracellular space). When this pool was depleted, NO synthesis was partially sustained by intracellular arginine sources not freely exchangeable with the extracellular space (pool II). Protein breakdown made up by far the largest part of pool II in both cell types. In contrast, citrulline to arginine conversion maintained NO synthesis only in TGW-nu-I neuroblastoma, but not A673 neuroepithelioma cells. Histidine mimicked the effect of protease inhibitors causing an almost complete nNOS inhibition in cells incubated additionally in lysine that depletes the exchangeable arginine pool. Our results identify new ways to modulate nNOS activity by modifying its substrate supply.

Obesity-associated metabolic alterations are closely linked to low-grade inflammation in peripheral organs, in which macrophages play a central role. Using genetic labeling of myeloid lineage cells, we show that hypothalamic macrophages normally reside in the perivascular area and circumventricular organ median eminence. Chronic consumption of a high-fat diet (HFD) induces expansion of the monocyte-derived macrophage pool in the hypothalamic arcuate nucleus (ARC), which is significantly attributed to enhanced proliferation of macrophages. Notably, inducible nitric oxide synthase (iNOS) is robustly activated in ARC macrophages of HFD-fed obese mice. Hypothalamic macrophage iNOS inhibition completely abrogates macrophage accumulation and activation, proinflammatory cytokine overproduction, reactive astrogliosis, blood-brain-barrier permeability, and lipid accumulation in the ARC of obese mice. Moreover, central iNOS inhibition improves obesity-induced alterations in systemic glucose metabolism without affecting adiposity. Our findings suggest a critical role for hypothalamic macrophage-expressed iNOS in hypothalamic inflammation and abnormal glucose metabolism in cases of overnutrition-induced obesity.

Functionally significant polymorphisms in endothelial nitric oxide synthase (eNOS) and reduced vascular eNOS activity have been associated with increased human diabetic nephropathy (DN), but the pathogenic role of eNOS deficiency in the development of DN has not yet been confirmed. This study characterizes the severity of DN in eNOS(-/-) mice that were backcrossed to C57BLKS/J db/db mice. Although the severity of hyperglycemia was similar to C57BLKS/J db/db mice, by 26 wk, eNOS(-/-) C57BLKS/J db/db mice exhibited dramatic albuminuria, arteriolar hyalinosis, increased glomerular basement membrane thickness, mesangial expansion, mesangiolysis, and focal segmental and early nodular glomerulosclerosis. Even more remarkable, eNOS(-/-) C57BLKS db/db exhibited decreases in GFR to levels <50% of that in eNOS(+/+) C57BLKS db/db, as confirmed by increased serum creatinine. In summary, eNOS(-/-) db/db mice provide the most robust model of type II DN that has been described to date and support a role for deficient eNOS-derived NO production in the pathogenesis of DN.

Here we show that iNOS-deficient mice display enhanced classically activated M1 macrophage polarization without major effects on alternatively activated M2 macrophages. eNOS and nNOS mutant mice show comparable M1 macrophage polarization compared with wild-type control mice. Addition of N6-(1-iminoethyl)-L-lysine dihydrochloride, an iNOS inhibitor, significantly enhances M1 macrophage polarization while S-nitroso-N-acetylpenicillamine, a NO donor, suppresses M1 macrophage polarization. NO derived from iNOS mediates nitration of tyrosine residues in IRF5 protein, leading to the suppression of IRF5-targeted M1 macrophage signature gene activation. Computational analyses corroborate a circuit that fine-tunes the expression of IL-12 by iNOS in macrophages, potentially enabling versatile responses based on changing microenvironments. Finally, studies of an experimental model of endotoxin shock show that iNOS deficiency results in more severe inflammation with an enhanced M1 macrophage activation phenotype. These results suggest that NO derived from iNOS in activated macrophages suppresses M1 macrophage polarization.

We have examined the role of nitric oxide (NO) in a model of functional angiogenesis in which survival of a skin flap depends entirely on angiogenesis to provide an arterial blood supply to maintain tissue viability. The different effects of nitric oxide synthase (NOS) inhibitors on rat skin flap survival appeared to be explained on the basis of their NOS isoform selectivity. Skin flap survival was decreased by iNOS-selective (inducible NOS) inhibitors, S-methyl-isothiourea, aminoguanidine and aminoethylthiorea; unaffected by the non-selective inhibitor nitro-imino-L-ornithine; and enhanced by the cNOS (constitutive NOS, that is endothelial NOS (eNOS) and neuronal NOS (nNOS)) inhibitor, nitro-L-arginine methyl ester. Skin flap survival was reduced in mice with targeted disruption of the iNOS gene (iNOS knockout mice), and the administration of nitro-L-arginine methyl ester significantly increased flap survival in iNOS knockout mice (P<0.05). iNOS immunoreactivity was identified in mast cells in the angiogenic region. Immunoreactive vascular endothelial growth factor (VEGF) and basic fibroblast growth factor were also localized to mast cells. The combination of interferon-gamma and tumour necrosis factor-alpha induced NO production and increased VEGF levels in mast cells cultured from bone marrow of wild-type, but not iNOS KO mice. The increased tissue survival associated with the capacity for iNOS expression may be related to iNOS-dependent enhancement of VEGF levels and an ensuing angiogenic response. Our results provide both pharmacological and genetic evidence that iNOS activity promotes survival of ischaemic tissue.

The angiotensin II type 2 receptor (AT2) is upregulated after tissue damage and mediates protective functions in the renin-angiotensin-aldosterone system (RAAS). One of these is to inhibit inducible nitric oxide synthase (iNOS) in activated macrophages. In the present study, we assessed the effect of AT2 receptor ligands on nitric oxide production in murine macrophages as a potential assay to determine the functional activity of an AT2 receptor ligand. Mouse macrophage J744.2 and RAW264.7 were cultivated in lipopolysaccharide (LPS) to induce M1 differentiation and increase iNOS expression. Using Griess reagent and spectrophotometric analysis, the nitric oxide levels were determined, while employing Western blot and immunocytochemistry to determine basal protein expression. Using the first reported selective non-peptide AT2 receptor agonist, compound C21, we conclude that activation of AT2 receptor reduces nitric oxide production in M1 macrophages. Furthermore, the AT2 receptor selective ligand compound C38, a regioisomer of C21, reported as a selective AT2 receptor antagonist exhibits a similar effect on nitric oxide production. Thus, we propose C38 acts as a partial agonist in the macrophage system. Monitoring nitric oxide attenuation in M1 J744.1 and RAW264.7 macrophages provides a new method for characterizing functional activity of AT2 receptor ligands, foreseen to be valuable in future drug discovery programs.

The inhibitory phosphorylation of endothelial nitric oxide (NO) synthase (eNOS) at Thr497 (eNOSpThr497) by protein kinase C or RhoA-activated kinase is a major regulatory determinant of eNOS activity. The signalling mechanisms involved in the dephosphorylation of eNOSpThr497 have not yet been clarified. This study identifies myosin phosphatase (MP) holoenzyme consisting of protein phosphatase-1 catalytic subunit (PP1c) and MP target subunit-1 (MYPT1) as an eNOSpThr497 phosphatase. In support of this finding are: (i) eNOS and MYPT1 interacts in various endothelial cells (ECs) and in in vitro binding assays (ii) MYPT1 targets and stimulates PP1c toward eNOSpThr497 substrate (iii) phosphorylation of MYPT1 at Thr696 (MYPT1pThr696) controls the activity of MP on eNOSpThr497. Phosphatase inhibition suppresses both NO production and transendothelial resistance (TER) of ECs. In contrast, epigallocatechin-3-gallate (EGCG) signals ECs via the 67 kDa laminin-receptor (67LR) resulting in protein kinase A dependent activation of protein phosphatase-2A (PP2A). PP2A dephosphorylates MYPT1pThr696 and thereby stimulates MP activity inducing dephosphorylation of eNOSpThr497 and the 20 kDa myosin II light chains. Thus an interplay of MP and PP2A is involved in the physiological regulation of EC functions implying that an EGCG dependent activation of these phosphatases leads to enhanced NO production and EC barrier improvement.

Trapezius myalgia is the most common type of chronic neck pain. While physical exercise reduces pain and improves muscle function, the underlying mechanisms remain unclear. Nitric oxide (NO) signaling is important in modulating cellular function, and a dysfunctional neuronal NO synthase (nNOS) may contribute to an ineffective muscle function. This study investigated nNOS expression and localization in chronically painful muscle. Forty-one women clinically diagnosed with trapezius myalgia (MYA) and 18 healthy controls (CON) were included in the case-control study. Subsequently, MYA were randomly assigned to either 10 weeks of specific strength training (SST, n = 18), general fitness training (GFT, n = 15), or health information (REF, n = 8). Distribution of fiber type, cross-sectional area, and sarcolemmal nNOS expression did not differ between MYA and CON. However, MYA showed increased sarcoplasmic nNOS localization (18.8 ± 12 versus 12.8 ± 8%, P = 0.049) compared with CON. SST resulted in a decrease of sarcoplasm-localized nNOS following training (before 18.1 ± 12 versus after 12.0 ± 12%; P = 0,027). We demonstrate that myalgic muscle displays altered nNOS localization and that 10 weeks of strength training normalize these disruptions, which supports previous findings of impaired muscle oxygenation during work tasks and reduced pain following exercise.

CORTICAL GABAERGIC INTERNEURONS IN RODENTS ORIGINATE IN THREE SUBCORTICAL REGIONS: the medial ganglionic eminence (MGE), the lateral/caudal ganglionic eminence (LGE/CGE), and the preoptic area (POA). Each of these neuroepithelial precursor domains contributes different interneuron subtypes to the cortex. Neuronal NOS (nNOS)-expressing neurons represent a heterogenous population of cortical interneurons. We examined the development of these cells in the mouse embryonic cortex and their abundance and distribution in adult animals. Using genetic lineage tracing in transgenic mice we find that nNOS type I cells originate only in the MGE whereas type II cells have a triple origin in the MGE, LGE/CGE, and POA. The two populations are born at different times during development, occupy different layers in the adult cortex and have distinct neurochemical profiles. nNOS neurons are more numerous in the adult cortex than previously reported and constitute a significant proportion of the cortical interneuron population. Our data suggest that the heterogeneity of nNOS neurons in the cortex can be attributed to their multiple embryonic origins which likely impose distinct genetic specification programs.

There is mounting evidence that nitric oxide (NO) may inhibit adrenal steroidogenesis by binding to the heme group of P450 enzymes, particularly the rate-limiting steps cholesterol side-change cleavage P450, aldosterone synthase P450, and 17 alpha-hydroxylase/C(17/20)-lyase P450. Using immunohistochemistry, nitrotyrosine was detectable throughout the ovine adrenal cortex, and endothelial NO synthase (eNOS) was further identified in zona glomerulosa (ZG) and at a higher level throughout the zona fasciculata, increasing toward the medulla. Caveolin-1, 90-kDa heat shock protein, ERK-1/2, and Akt, all known and proposed regulators of eNOS activity, were detected throughout the ovine adrenal cortex. Western immunoblotting confirmed the identity of these proteins as well as the absence of neuronal NOS, inducible NOS, caveolin-2, and caveolin-3. Through dual immunostaining we further identified for the first time a zona intermedia without strong staining for 17 alpha-hydroxylase/C(17/20)-lyase P450 or angiotensin II type 1 receptor, but positive for eNOS. Rhesus adrenals also stained positively for eNOS, but staining was seen only in the ZG and zona reticularis. We conclude that eNOS may play a role in controlling zone-specific aldosterone synthase vs. 11 beta-hydroxylase activities of the single CYP11B gene in sheep. In the rhesus monkey, NO may modulate ZG aldosterone synthase, but it is not needed for control of the distinct 11 beta-hydroxylase in the zona fasciculata. In the zona reticularis, however, eNOS may control C(19) steroid production at the level of 17 alpha-hydroxylase vs. 17,20-lyase activity otherwise unopposed by 3beta-hydroxysteroid dehydrogenase.

1. In the rat, intracerebroventricular (i.c.v.) injection of cadmium, a pollutant with long biological half-life, causes a sustained increase in blood pressure at doses that are ineffective by peripheral route. Since cadmium inhibits calcium-calmodulin constitutive nitric oxide (NO) synthase in cytosolic preparations of rat brain, this mechanism may be responsible for the acute pressor action of this heavy metal. 2. To test this possibility, we evaluated the effect of i.c.v. injection of 88 nmol cadmium in normotensive unanaesthetized Wistar rats, which were i.c.v. pre-treated with: (1) saline (control), (2) L-arginine (L-Arg), to increase the availability of substrate for NO biosynthesis, (3) D-arginine (D-Arg), (4) 3-[4-morpholinyl]-sydnonimine-hydrochloride (SIN-1), an NO donor, or (5) CaCl2, a cofactor of brain calcium-calmodulin-dependent cNOS(I). In additional experiments, the levels of L-citrulline (the stable equimolar product derived from enzymatic cleavage of L-Arg by NO synthase) were determined in the brain of vehicle- or cadmium-treated rats. 3. The pressor response to cadmium reached its nadir at 5 min (43+/-4 mmHg) and lasted over 20 min in controls. L-Citrulline/protein content was reduced from 35 up to 50% in the cerebral cortex, pons, hippocampus, striatus, hypothalamus (P<0.01) of cadmium-treated rats compared with controls. Central injection of N(G) nitro-L-arginine-methylester (L-NAME) also reduced the levels of L-citrulline in the brain. 4. Both the magnitude and duration of the response were attenuated by 1.21 and 2.42 micromol SIN-1 (32+/-3 and 15+/-4 mmHg, P<0.05), or 1 micromol CaCl2 (6+/-4 mmHg, P<0.05). Selectivity of action exerted by SIN-1 was confirmed by the use of another NO donor, S-nitroso-N-acetyl-penicillamine (SNAP). Both L-Arg and D-Arg caused a mild but significant attenuation in the main phase of the pressor response evoked by cadmium. However, only L-Arg reduced the magnitude of the delayed, pressor response. Despite their similarity in ability to attenuate the cadmium-induced pressure effect, L-Arg and its isomer exerted differential biochemical changes in brain L-citrulline, as L-Arg normalized cadmium-induced reduction in L-citrulline levels, whereas i.c.v. D-Arg did not. 5. We conclude that the pressor effect of i.c.v. cadmium is due, at least in part, to reduced NO formation, consequent to inhibition of brain NO synthase. Accumulation of cadmium in the central nervous system could interfere with central mechanisms (including NO synthase) implicated in the regulation of cardiovascular function.

Ketamine is an ionotropic glutamatergic N‑methyl‑D‑aspartate receptor antagonist, which is widely used among recreational drug abusers. Ketamine abusers exhibit substantially reduced bladder capacity, which can lead to urinary frequency. The molecular pathogenesis of ketamine‑induced cystitis has been scarcely reported. Given previous clinical findings, it may be hypothesized that pathological alterations in smooth muscle cells (SMCs) of the urinary bladder serve a crucial role in the mechanism underlying cystitis. In the present study, two lineages of SMCs, one from differentiated foreskin‑derived fibroblast‑like stromal cells and the other from cultured normal aortic SMCs, were used to study ketamine‑induced molecular alterations. Polymerase chain reaction was used to study the effects of ketamine on oxidative stress. The effects of adjuvant chemotherapy with cyclophosphamide (CTX) were also investigated. The results indicated that the expression levels of interleukin‑6 and inducible nitric oxide synthase (iNOS) were decreased, whereas collagen expression and deposition were increased in ketamine‑treated SMCs. Conversely, treatment with CTX restored the expression of iNOS, which may prevent or limit oxidative damage. In conclusion, the present study demonstrated that ketamine may induce several molecular alterations in SMCs and these changes may be associated with the clinical symptoms observed in ketamine abusers. In addition, the specific chemotherapeutic agent CTX may reverse these ketamine‑induced aberrations.