Effect of subchronic lithium treatment on citalopram-induced increases in extracellular concentrations of serotonin in the medial prefrontal cortex

We investigated the effect of citalopram [a selective serotonin (5-HT) reuptake inhibitor; SSRI] and MKC-242 (a selective 5-HT1A agonist), following treatment with subchronic lithium (p.o., 1 week) on extracellular 5-HT concentrations in the medial prefrontal cortex (mPFC). Acute treatment with citalopram (3 and 30 mg/kg) led to significant increases in extracellular 5-HT concentrations. The subchronic lithium group showed significantly higher basal levels of extracellular 5-HT than normal diet controls. Acute citalopram (3 and 30 mg/kg) treatment together with subchronic lithium treatment showed significant increases in the extracellular 5-HT concentrations, compared with citalopram treatment alone. Acute MKC-242 (1 mg/kg) treatment showed significant decreases in extracellular 5-HT concentrations, in both the normal diet and lithium diet groups to the same extent. The addition of lithium did not change the effect of the 5-HT1A agonist on extracellular 5-HT concentrations. This study suggests that lithium augmentation of the antidepressant effect of SSRI is mediated by the additional increases in extracellular 5-HT concentrations following the co-administrations of lithium and SSRI.     

Effects of Modafinil on Clonic Seizure Threshold Induced by Pentylenetetrazole in Mice: Involvement of Glutamate,Nitric oxide,GABA, and Serotonin Pathways

Epilepsy is the third most common chronic brain disorder. Modafinil is an awakening agent approved for narcolepsy. In addition to its clinical uses some reports revealed that modafinil was associated with some alterations in seizure threshold. The purpose of this study was to clarify the effect of acute administration of modafinil in clonic seizure threshold (CST) induced by pentylenetetrazole in mice and the involvement of glutamate, nitric oxide, gamma amino butyric acid (GABA), and serotonin systems in this feature. Modafinil at 80 and 150 mg/kg showed anti- and pro-convulsant effects respectively and expressed maximum anti- and pro-convulsant activities at 30 min after injection. Both modulatory effects were blunted by pretreatment of l-NAME [nonspecific nitric oxide synthase (NOS) inhibitor; 10 mg/kg, i.p.], 7-nitroindazole (a neuronal NOS inhibitor; 40 mg/kg, i.p.), and aminoguanidine (an inducible NOS inhibitor; 50 mg/kg, i.p.). Injection of the NOS precursor l-arginine (60 mg/kg, i.p.) before modafinil did not change the anti-convulsant effect, while thoroughly reversed the pro-convulsant one. Our experiments displayed that administration of diazepam (a GABA_A receptor agonist; 0.02 mg/kg, i.p.) and MK-801 (a NMDA receptor antagonist; 0.05 mg/kg, i.p.) before different doses of modafinil significantly increased CST. Finally, pretreatment of citalopram (a selective serotonin reuptake inhibitor; 0.1 mg/kg, i.p.) did not modify the convulsant activities of modafinil. Therefore, nitric oxide system may mediate anti-convulsant activity, while glutamate, nitric oxide, and GABA pathways may involve in pro-convulsant property. Serotonin receptors have no role on convulsant effects of modafinil.     

Chronic inhibition of nitric oxide synthase activity by N(G)-nitro-L-arginine induces nitric oxide synthase expression in the developing rat cerebellum

Studies on chronic inhibition of nitric oxide synthase (NOS) in the CNS suggest a plastic change in nitric oxide (NO) synthesis in areas related to motor control, which might protect the animal from the functional and behavioral consequences of NO deficiency. In the present study, the acute and chronic effect of the substrate analogue inhibitor N(G)-nitro-l-arginine (l-NNA) was examined on NO production, NO-sensitive cyclic guanosine monophosphate (cGMP) levels and the expression of NOS isoforms in the developing rat cerebellum. Acute intraperitoneal administration of the inhibitor (5-200mg/kg) to 21-day-old rats reduced NOS activity and NO concentration dose dependently by 70-90% and the tissue cGMP level by 60-80%. By contrast, chronic application of l-NNA between postnatal days 4-21 diminished the total NOS activity and NO concentration only by 30%, and the tissue cGMP level by 10-50%. Chronic treatment of 10mg/kg l-NNA induced neuronal (n)NOS expression in granule cells, as revealed by in situ hybridization, NADPH-diaphorase histochemistry and Western-blot, but it had no significant influence on tissue cGMP level or on layer formation of the cerebellum. However, a higher concentration (50mg/kg) of l-NNA decreased the intensity of the NADPH-diaphorase reaction in granule cells, significantly reduced cGMP production, and retarded layer formation and induced inducible (i)NOS expression & activity in glial cells. Treatments did not affect endothelial (e)NOS expression. The administration of the biologically inactive isomer D-NNA (50mg/kg) or saline was ineffective. The present findings suggest the existence of a concentration-dependent compensatory mechanism against experimentally-induced cronich inhibition of NOS, including nNOS or iNOS up-regulation, which might maintain a steady-state NO level in the developing cerebellum.     

Acute effects of combining citalopram and pindolol on regional brain serotonin synthesis in sham operated and olfactory bulbectomized rats

The olfactory bulbectomized (OBX) rat is considered to be a good model of the pathology of human depression and also of the functional actions of antidepressant drug therapy. It has been proposed that antidepressant effects of selective serotonin reuptake inhibitors (SSRIs) can be accelerated by blocking 5-HT_1A/B autoreceptors with pindolol. The underlying mechanism is thought to involve acute unrestricting of 5-HT release and, consequently, relatively enhanced 5-HT turnover throughout the forebrain serotonergic networks. The effect of this combination on 5-HT turnover in sham operated or OBX rats can be assessed at the level of 5-HT synthesis, a very important presynaptic step in serotonergic neurotransmission, using the α-[¹⁴C]methyl-l-tryptophan autoradiography method. In sham rats, acute citalopram (20 mg/kg) treatment increased synthesis at almost all serotonergic terminal regions but slightly decreased synthesis at serotonergic cell body regions (i.e. dorsal and median (not significant) raphe; ～16%). Combining pindolol (10 mg/kg) with citalopram further increased synthesis at many regions in sham rats (relative to treatment with only citalopram). In OBX rats, citalopram decreased synthesis at a few terminal regions and greatly decreased synthesis at the dorsal and median raphe (～45%; relative to OBX rats treated with saline). Combining pindolol with citalopram greatly increased synthesis at almost all regions in OBX rats (relative to treatment with only citalopram). These results suggest that acute citalopram effects result in elevated terminal 5-HT synthesis, but these effects are restrained by 5-HT_1A/B autoreceptor feedback to different degrees in sham and OBX rats. Moreover, 5-HT_1A/B autoreceptor feedback is stronger in OBX rats and may underlie the delay of SSRI effects in OBX rats and, correspondingly, in human depression. Pindolol acceleration and augmentation of SSRI antidepressant therapy for human depression may be mediated by attenuation of 5-HT_1A/B autoreceptor feedback, permitting unhindered SSRI effects on serotonergic terminals.     

Role of nitric oxide in the nicotine-induced pituitary-adrenocortical response.

Nitric oxide (NO) is a major signaling molecule and biological mediator of the hypothalamic-pituitary-adrenal (HPA) axis. We investigated the role of NO formed by endothelial (e), neuronal (n) and inducible (i) nitric oxide synthase (NOS) in the stimulatory effect of nicotine on the HPA axis in rats under basal conditions. Also possible interaction of NOS systems with endogenous prostaglandins (PG) in that stimulation was assessed. NOS and cyclooxygenase inhibitors were administered i.p. 15 min prior to nicotine (2, 5 mg/kg i.p.). Plasma ACTH and serum corticosterone levels were measured 1 h after nicotine injection. NOS blockers given alone did not markedly affect the resting ACTH and corticosterone levels. L-NAME (2-10 mg/kg), a broad spectrum NOS inhibitor considerably and dose dependently enhanced the nicotine-induced ACTH and corticosterone secretion. L-NNA (2 mg/kg) and 7-nitroindazole (7-NI 20 mg/kg), neuronal NOS inhibitors in vivo also significantly augmented the nicotine-induced ACTH and corticosterone levels. L-arginine greatly impaired the nicotine-induced hormone responses and reversed the L-NNA elicited enhancement of the nicotine-evoked ACTH and corticosterone response. In contrast to the constitutive eNOS and nNOS antagonists, an inducible NOS antagonist guanethidine (50-100 mg/kg i.p.) did not substantially affect the nicotine-elicited pituitary-adrenocortical responses. Indomethacin (2 mg/kg i.p.), a non-selective cyclooxygenase blocker abolished the L-NAME and L-NNA-induced enhancement of the nicotine-evoked ACTH and corticosterone response. These results indicate that NO is an inhibitory mediator in the HPA axis activity. Inhibition of its generation by eNOS and nNOS significantly enhances the nicotine-induced HPA response. Under basal conditions iNOS is not involved in the nicotine-induced ACTH and corticosterone secretion. Prostaglandins play an obligatory role in the response of HPA axis to systemic nicotine administration.     

Baclofen and NOS inhibitors interact to evoke synergistic hypothermia in rats

Our laboratory recently demonstrated that a drug combination of baclofen and L-NAME, a nonspecific nitric oxide synthase (NOS) inhibitor, evokes synergistic hypothermia in rats. These data are the first demonstration of synergy between a GABA agonist and NOS inhibitor. While the hypothermic synergy suggests a role for NOS in baclofen pharmacology, it is unclear whether the super-additive hypothermia is specific for baclofen and L-NAME or extends to drug combinations of baclofen and other NOS inhibitors. The site of action (central or peripheral) and isoforms of NOS that mediate the synergy are also unknown. Here, we confirm the hypothermic synergy with additional data and discuss potential mechanisms of the drug interaction. Baclofen (2.5, 3.5, 5 and 7.5 mg/kg, i.p.) was administered to rats by itself or with 7-nitroindazole (7-NI), a neuronal NOS inhibitor. 7-NI (10 mg/kg, i.p.) did not affect body temperature. For combined administration, 7-NI (10 mg/kg, i.p.) increased the relative potency of baclofen (F=18.9, P<0.05). The present data validate the hypothermic synergy caused by the drug combination of baclofen and L-NAME and implicate nNOS in the synergy. In a context broader than thermoregulation, NO production and transmission may play an important role in baclofen pharmacology.     

Effects of Milnacipran in Animal Models of Anxiety and Memory

Serotonin (5-HT) and noradrenaline (NA) are involved in both pathogenesis and recovery from depression and anxiety. We examined the effects of acute and chronic treatment with milnacipran, a serotonin/noradrenaline reuptake inhibitors (SNRIs) antidepressant, on anxiety and memory retention in rats. Male Wistar rats received acute or chronic administration of milnacipran (12.5, 25 or 50 mg/kg) or saline (control group). The animals were separately submitted to elevated plus-maze, inhibitory avoidance and open-field tasks 1 h after injection, in the acute group, or 23 h after last injection, in the chronic group. Our results showed an anxiolytic-like effect after chronic administration of milnacipran at doses of 25 and 50 mg/kg. The treatment does not interfere in memory retention and habituation to a novel environment at any doses studied. These findings support that milnacipran, an established SNRIs antidepressant, can also be useful in the treatment of anxiety disorders.     

Effect of combination of nitric oxide synthase and cyclooxygenase inhibitors on carrageenan-induced pleurisy in rats

In the present study, we examined the effects of L-nitroarginine methylester (L-NAME), a non-selective nitric oxide synthase (NOS) inhibitor, indomethacin (IND), a non-selective COX inhibitor and a combination of these agents (L-NAME+IND) on carrageenan-induced pleurisy in rats. Exudate volume, albumin leakage, leukocyte influx, exudate and plasma nitrite/nitrate (NO(x)) levels and exudate PGE(2) levels increased markedly 6 h after an intrapleural injection of 2% carrageenan. First, the effects of L-NAME and IND alone were investigated. L-NAME non-significantly reduced exudate volume by 26% at 10 mg/kg (i.p.), and significantly by 45% at 30 mg/kg. IND dose-dependently decreased the exudate volume at 0.3-10 mg/kg (p.o.) and the effect reached the maximal level at 1 mg/kg (33%). Second, the effects of L-NAME (10 mg/kg, i.p.), IND (1 mg/kg, p.o.) and L-NAME+IND were examined. L-NAME and IND alone at the dose employed significantly reduced the exudate volume and albumin levels by 21-26%. L-NAME but not IND tended to reduce the increased exudate and plasma NO(x) by 18% and 19%, respectively. IND but not L-NAME significantly reduced leukocyte numbers and PGE(2) levels in the exudates by 25% and 77%, respectively. L-NAME+IND significantly reduced exudate volume, albumin leakage, leukocyte number, PGE(2) and NO(x) by 43%, 41%, 31%, 80% and 37%, respectively. The inhibitory effects of L-NAME+IND on exudate volume, albumin leakage and NO(x) levels were greater than those of L-NAME and IND alone. In conclusion, a non-selective NOS inhibitor and COX inhibitor showed anti-inflammatory effects at the early phase of carrageenan-induced pleurisy, and a combination of both inhibitors had a greater effect than each alone probably via the potentiation of NOS inhibition. The simultaneous inhibition of NOS and COX could be a useful approach in therapy for acute inflammation.     

Nitric oxide synthase-independent generation of nitric oxide in rat skeletal muscle ischemia-reperfusion injury.

We have used electron paramagnetic resonance to investigate the time course of nitric oxide (NO) generation and its susceptibility to inhibitors of nitric oxide synthase (NOS) in ischemia-reperfusion (IR) injury to rat skeletal muscle in vivo. Significant levels of muscle nitroso-heme complexes were detected 24 h postreperfusion, but not after at 0.05, 3, and 8 h of reperfusion. The levels of muscle nitroso-heme complexes were not decreased by the NOS inhibitor N-nitro-L-arginine methyl ester as a single dose (30 mg/kg) prior to reperfusion or as multiple doses continued throughout the reperfusion (total administered, 120 mg/kg) or by the potent NOS inhibitor S-methylisothiourea (3 mg/kg). In contrast, nitroso-heme levels were reduced by the glucocorticoid dexamethasone (2.5 mg/kg). Muscle necrosis in vitro did not result in the formation of nitroso-heme complexes. The finding that reperfusion after ischemia is necessary for NO formation suggests that an inflammatory pathway is responsible for NOS-independent NO formation in IR injury to skeletal muscle.     

Chronic treatment with zinc and antidepressants induces enhancement of presynaptic/extracellular zinc concentration in the rat prefrontal cortex

Zinc exhibits antidepressant-like activity in preclinical tests/models. Moreover, zinc homeostasis is implicated in the pathophysiology of affective disorders. The aim of the present study was to examine the effect of chronic zinc, citalopram and imipramine intraperitoneal administration on the presynaptic and extracellular zinc concentration in the rat prefrontal cortex and hippocampus. We used two methods: zinc–selenium histochemistry (which images the pool of presynaptic-vesicle zinc) and anodic stripping voltammetry (ASV) for zinc determination in microdialysate (which assays the extracellular zinc concentration). We report that chronic (14×) zinc (hydroaspartate, 10 and 65 mg/kg) and citalopram (20 mg/kg) administration increased the pool of presynaptic zinc (by 34, 50 and 37%, respectively) in the rat prefrontal cortex. The 21% increase induced by imipramine (20 mg/kg) was marginally significant. Likewise, zinc (hydroaspartate, 65 mg/kg), citalopram and imipramine increased the extracellular zinc (although with a different pattern: time point, area under the curve and/or basal level) in this brain region. Furthermore, zinc induced an increase in presynaptic (by 65%) and extracellular zinc (by 90%) in the hippocampus, while both citalopram and imipramine did not. These results indicate that all of the treatments increase presynaptic/extracellular zinc concentrations in the rat prefrontal cortex, which may then contribute to their antidepressant mechanisms. Alterations induced by zinc (but not antidepressants) administration in the hippocampus may be related to specific zinc mechanisms. All the data (previous and present) on the effect of antidepressant treatments on the presynaptic/extracellular zinc concentrations suggest the involvement of this biometal presynaptic/synaptic homeostasis in the antidepressant mechanism(s).     

Role of prostanoids and nitric oxide inhibition in rats with experimental hepatic fibrosis

Nitric oxide (NO) and prostaglandins have been proposed as vasodilator substances involved in peripheral vasodilatation characteristic of the liver cirrhosis. A link between NO and prostanoids has been suggested. The present study investigated the effect of simultaneous blockade of both, NO synthase (NOS) and cyclooxigenase (COX) in sham-operated (SO), or rats with bile-duct ligation (BDL) in the development of liver fibrosis. Animals were distributed in two groups SO (n=15) or BDL (n=15). Treatments (5 days) started three weeks after surgical procedure. Both, SO and BDL animals were treated with indomethacin (INDO) (5 mg/kg/day) alone, with NG-nitro-L-arginine-methyl-ester (NAME) (4 mg/kg/day) alone or with INDO and NAME combination at the same doses. At the end of follow-up body weight, packed cell volume, mean arterial blood pressure (MAP) and heart rate were measured. Liver tissue was processed for histological studies. In this study, BDL animals showed a decreased MAP. Treatment with L-NAME in BDL rats increased MAP. The chronic COX inhibition alone did not play an important role in the haemodynamic changes. The BDL produced a loss of hepatic structure, with ductular metaplasia that occupied the greater part of the hepatic parenchyma. Also, an important degree of fibrosis was observed. Both NO and PG synthesis inhibitors, alone or in combination, induced enhancing collagen fiber deposition in the hepatic parenchyma. These findings support the notion that the interaction between the NOS and COX pathways should be relevant in hepatic cirrhosis in which both NOS and COX are induced.     

The effect of alpha-melanocyte stimulating hormone on colonic inflammation in the rat

The effect of alpha-melanocyte stimulating hormone (alpha-MSH) on colonic inflammation in the rat. In this study, we investigated the effects of alpha-MSH administration on trinitrobenzene sulfonic acid-induced colitis and the role of nitric oxide and prostaglandins in this response. alpha-MSH treatment (25 microg/rat, intraperitoneally; twice daily for 3 days) reduced the colonic macroscopic lesions compared to untreated ones in both acute and chronic colitis groups. This effect was reversed by pretreatment with the nitric oxide donor, sodium NP (4 mg/kg, intravenously) or cyclooxygenase-1 selective antagonist indomethacin (5 mg/kg, subcutaneously) in the acute group and with the cyclooxygenase-2 selective antagonist nimesulide (3 mg/kg, subcutaneously) in the chronic group. alpha-MSH had no effect on colonic wet weight and myeloperoxidase activity compared to the untreated colitis group. However, protein oxidation was markedly elevated in the alpha-MSH-treated group compared to untreated ones. Nitroprusside and indomethacin reversed the effect of alpha-MSH on macroscopic lesions in the acute groups, whereas nimesulide showed a similar effect in the chronic group. In conclusion, the results of our study show a protective role of alpha-MSH on colonic lesions which partially involves nitric oxide and prostaglandins.     

Chronic antidepressants and GABA "B" receptors: a GABA hypothesis of antidepressant drug action

Amitryptyline (10 mg/kg), desipramine (5 mg/kg), citalopram (10 mg/kg) and viloxazine (10 mg/kg) were administered to rats either acutely (decapitation 1 hr after i.p. injection) or subacutely (by subcutaneous minipump implantation for 18 days followed by decapitation 24 hr after removal). After acute administration there was not any consistent alteration in GABA levels, GAD activity, 3H GABA "A" or 3H-GABA "B" receptor binding or 3H-nipecotic acid binding to the recognition site for GABA uptake in the frontal cortex or hippocampus. Upon subacute antidepressant drug infusion, GABA levels, GAD activity and 3H-GABA-"A" binding showed only scattered differences in drug treated animals as compared to saline treated rats. However, 3H-GABA "B" binding in the frontal cortex was consistently elevated after all drug treatments (in % of control: amitryptyline = 155%; desipramine = 151%; citalopram = 173%; viloxazine = 189%). Scatchard analysis showed that this was due to a Bmax increase without an effect in Kd. These findings were reproduced by subacute administration of pargyline, a MAO inhibitor. These data suggest that GABA "B" receptors may be involved in the mechanism of action of antidepressant drugs and provide a link between GABAergic and monoaminergic hypotheses of depression.     

Effects of nitric oxide synthase inhibitors on the febrile response to lipopolysaccharide and muramyl dipeptide in guinea pigs

We investigated the effect of N-nitro-L-arginine methyl ester (L-NAME), an unspecific nitric oxide synthase (NOS) inhibitor, and aminoguanidine, a relatively selective inhibitor of the inducible NOS enzyme, on both gram-negative lipopolysaccharide (LPS) and gram-positive muramyl dipeptide (MDP) fever in guinea pigs. Intraperitoneal injection of either 10 mg/kg L-NAME or 25 mg/kg aminoguanidine inhibited the febrile response to an intramuscular injection of 50 microg/kg MDP. However, LPS fever (20 microg/kg) was inhibited only by L-NAME. The development of LPS fever may therefore occur independently of the synthesis of nitric oxide by the inducible NOS enzyme, while MDP fever may involve synthesis of nitric oxide by both the inducible and the constitutively expressed NOS enzymes.     

Supplemental L-arginine HCI augments bacterial phagocytosis in human polymorphonuclear leukocytes

That L-arginine (L-Arg) augments the host response to acute bacterial sepsis suggests that this amino acid intervenes early in the immune response, perhaps via the nitric oxide synthetase (NOS) pathway. The effect of L-Arg supplementation on in vitro phagocytosis of fluorescein-labeled, heat-killed Staphylococcus aureus by peripheral blood neutrophils (PMNs) from 12 normal human volunteers was studied. Separated PMNs were incubated for 2 h with labeled bacteria, with and without supplemental L-Arg, D-arginine, glycine, and/or the NOS inhibitors L-canavanine, aminoguanidine, or L-N^G-nitroarginine methyl ester. PMNs were fixed and extracellular fluorescence quenched with crystal violet. By flow cytometry and confocal microscopy, L-Arg supplementation was shown to result in a highly significant increase in PMN bacterial phagocytosis, the maximal effect being seen with L-Arg 380 μM and falling off with higher concentrations. This augmentation was completely abrogated by NOS inhibitors in molar excess, but inhibitors alone did not suppress phagocytosis below that of unsupplemented controls. Neither D-arginine nor glycine affected phagocytosis; the L-Arg effect was stereospecific and not related to utilization of L-Arg as an energy source. L-Arg supplementation significantly enhances bacterial phagocytosis in human neutrophils, perhaps by effects on cytoskeletal phenomena, and this appears to be mediated through NOS activity. Phagocytosis by nonspecific immune cells which intervene early in the response to sepsis is critically important, and beneficial effects of L-Arg on the clinical course of sepsis may be due at least in part to augmentation of phagocyte function. © 1996 Wiley-Liss, Inc.     

Effect of chronic lithium administration on endothelium-dependent relaxation of rat mesenteric bed: role of nitric oxide

The mechanism of action of lithium, an effective treatment for bipolar disease, is still unknown. In this study, the mesenteric vascular beds of control rats and rats that were chronically treated with lithium were prepared by the McGregor method, and the mesenteric vascular bed vasorelaxation responses were examined. NADPH-diaphorase histochemistry was used to determine the activity of NOS (nitric oxide synthase) in mesenteric vascular beds. We demonstrated that ACh-induced vasorelaxation increased in the mesenteric vascular bed of rats treated with lithium. Acute No-nitro-L-arginine methyl ester (L-NAME) administration in the medium blocked ACh-induced vasorelaxation in the control group more effectively than in lithium-treated rats, while the vasorelaxant response to sodium nitroprusside, a NO donor, was not different between lithium-treated and control groups. Acute aminoguanidine administration blocked ACh-induced vasorelaxation of lithium-treated rats, but had no effect in the control rats. Furthermore, NOS activity, determined by NADPH-diaphorase staining, was significantly greater in the mesenteric vascular beds from chronic lithium-treated rats than in those from control rats. These data suggest that the enhanced ACh-induced endothelium-derived vasorelaxation in rat mesenteric bed from chronic lithium-treated rats might be associated with increased NOS activity, likely via iNOS. Simultaneous acute L-NAME and indomethacin administration suggests the possible upregulation of EDHF (endothelium-derived hyperpolarizing factor) in lithium-treated rats.     

PI3K/Akt-Independent NOS/HO Activation Accounts for the Facilitatory Effect of Nicotine on Acetylcholine Renal Vasodilations: Modulation by Ovarian Hormones

We investigated the effect of chronic nicotine on cholinergically-mediated renal vasodilations in female rats and its modulation by the nitric oxide synthase (NOS)/heme oxygenase (HO) pathways. Dose-vasodilatory response curves of acetylcholine (0.01–2.43 nmol) were established in isolated phenylephrine-preconstricted perfused kidneys obtained from rats treated with or without nicotine (0.5–4.0 mg/kg/day, 2 weeks). Acetylcholine vasodilations were potentiated by low nicotine doses (0.5 and 1 mg/kg/day) in contrast to no effect for higher doses (2 and 4 mg/kg/day). The facilitatory effect of nicotine was acetylcholine specific because it was not observed with other vasodilators such as 5′-N-ethylcarboxamidoadenosine (NECA, adenosine receptor agonist) or papaverine. Increases in NOS and HO-1 activities appear to mediate the nicotine-evoked enhancement of acetylcholine vasodilation because the latter was compromised after pharmacologic inhibition of NOS (L-NAME) or HO-1 (zinc protoporphyrin, ZnPP). The renal protein expression of phosphorylated Akt was not affected by nicotine. We also show that the presence of the two ovarian hormones is necessary for the nicotine augmentation of acetylcholine vasodilations to manifest because nicotine facilitation was lost in kidneys of ovariectomized (OVX) and restored after combined, but not individual, supplementation with medroxyprogesterone acetate (MPA) and estrogen (E2). Together, the data suggests that chronic nicotine potentiates acetylcholine renal vasodilation in female rats via, at least partly, Akt-independent HO-1 upregulation. The facilitatory effect of nicotine is dose dependent and requires the presence of the two ovarian hormones.     

Neuronal nitric oxide synthase is resistant to ethanol.

To test for a possible role of nitric oxide (NO) in the neurotoxicity of ethanol, we studied the effects of ethanol on the neuronal NO synthase (nNOS) both in vitro and in vivo. Ethanol, up to 200 mM, did not change the NOS activity in the cerebellar homogenate or the production of NO by the cultured cerebellar granule cells. The number of NADPH diaphorase-positive cells in the culture did not change after the exposure to 200 mM ethanol in vitro. The NOS activity in the various brain regions of mice remained similar to the controls after the acute (3 g/kg) and the chronic (33 g/kg/day, 3.5 days) administration of ethanol. N(omega)-nitro-L-arginine, a NOS inhibitor, did not affect the ethanol-withdrawal behavior. These results indicate that nNOS is resistant to ethanol at clinically relevant concentrations and that ethanol affects the NO-operated system in the brain through a pathway other than that of nNOS.     

Acute and conditioned hypoxic tolerance augmented by endothelial nitric oxide synthase inhibition in mice.

To identify a possible role for nitric oxide (NO) in acute hypoxic tolerance (HT) we measured hypoxic survival time (HST), effect of hypoxic conditioning (HC), and survival following hypoxic conditioning while blocking or mimicking the action of nitric oxide synthase (NOS). To inhibit NOS, CD-1 mice were given supplemental endogenous NOS inhibitor asymmetrical dimethylarginine (ADMA) or a synthetic NOS inhibitor N(omega)-nitro-L-arginine (L-NNA), both of which nonselectively inhibit three of the isoforms of NOS [inducible (iNOS), neuronal (nNOS), and endothelial NOS (eNOS)]. ADMA (10 mg/kg i.p.) or saline vehicle was given 5 min before HST testing. L-NNA was given orally at 1 g/l in drinking water with tap water as the control for 48 h before testing. Both ADMA and L-NNA significantly increased HST and augmented the HC effect on HST. Neither the nNOS selective inhibitor 7-nitroindazole (7-NI) nor the iNOS selective inhibitor N-{[3-(aminomethyl)phenyl]methyl}-enthanimidamide (1400W) had a statistically significant effect on HST or HT. The NO donor, 3-morpholinosydnoeimine, when given alone did not significantly decrease HT, but it did mitigate the increased HT effect of L-NNA. These data confirm that acute hypoxic conditioning increases HT and that NOS inhibition by endogenous (ADMA) and a synthetic NOS inhibitor (L-NNA) further increases HT, whereas iNOS and nNOS inhibition does not, suggesting that it is the inhibition of eNOS that mediates enhancement of HT.