Inhibition of neuronal nitric oxide synthase attenuates the development of morphine tolerance in rats

Our previous results have shown the involvement of nitric oxide in acute opioid desensitization of mu-opioid receptors in vitro. In the present study, we investigated the effect of repeated administration of 7-nitroindazole (7-NI; 30 mg/kg/12 h, i.p., 3 days), an inhibitor of neuronal nitric oxide synthase in vivo, on mu-opioid receptor tolerance induced by subchronic treatment with morphine in rats. The inhibitory effect of the opioid agonist Met5-enkephalin (ME) on the cell firing rate was evaluated by single-unit extracellular recordings of noradrenergic neurons in the locus coeruleus from brain slices, and the antinociceptive effect of morphine was measured by tail-flick techniques. In morphine-treated animals, concentration-effect curves for ME in the locus coeruleus were shifted by 5-fold to the right as compared to those in sham-treated animals, which confirmed the induction of mu-opioid receptor tolerance. However, tolerance to ME in morphine-treated rats was fully prevented by co-administration of 7-NI when compared to the vehicle-morphine group. Likewise, the antinociceptive effect of morphine was reduced in morphine-treated animals as compared to the sham group, whereas the antinociceptive tolerance was partially prevented by co-administration of 7-NI in morphine-treated rats (when compared to the vehicle-morphine group). Finally, 7-NI administration in sham-treated rats failed to change the effect induced by ME on the locus coeruleus or by morphine in the tail-flick test as compared to vehicle groups. These results demonstrate that subchronic administration of a neuronal inhibitor of nitric oxide synthase attenuates the development of morphine tolerance to the cellular and analgesic effects of mu-opioid receptor agonists.     

Role of nitric oxide in rat locus coeruleus in hypoxia-induced hyperventilation and hypothermia

The locus coeruleus modulates the ventilatory and thermoregulatory response to hypoxia and contains nitric oxide synthase. Therefore, we examined the effects of L-NAME unilaterally microinjected into the locus coeruleus on hypoxic hyperventilation and hypothermia. Ventilation and body temperature were measured before and after microinjection of L-NAME (100 nmol/0.5 microl) into the locus coeruleus, followed by hypoxia. Control rats received microinjection of D-NAME (an inactive enantiomer of L-NAME). Under normoxia, L-NAME treatment did not affect ventilation or body temperature. D-NAME did not affect hypoxia-induced hyperventilation and hypothermia. L-NAME treatment reduced the ventilatory response to hypoxia but did not affect hypoxia-induced hypothermia. These data suggest that nitric oxide in the locus coeruleus is involved in the ventilatory response to hypoxia, exercising an inhibitory modulation on the locus coeruleus neurons, but plays no role in hypoxia-induced hypothermia.     

Enhancement of μ-opioid receptor desensitization by orexin-A in rat locus coeruleus neurons

Opioids have always been used in clinical practice for pain management. However, development of tolerance to their effects following long term administration, seriously restricts further clinical use of these drugs. In this regard, μ-opioid receptor (MOR) desensitization, as an initial step in development of opioid tolerance, is of particular significance. Previous studies support the involvement of orexinergic system in development of opioid tolerance. Locus coeruleus (LC) nucleus has been shown to modulate pain and development of tolerance. Opioid receptors (particularly μ) are densely expressed within the LC. Moreover, it receives widespread orexinergic inputs and orexin type 1 receptors (OX1Rs) are also highly expressed in this brain region. In the present study, the effect of orexin-A (OXA) on met-enkephalin (ME)-induced MOR desensitization was investigated in locus coeruleus neurons of male Wistar rats (2–3 weeks of age). ME (30 μM), as a potent MOR agonist, was applied for 10 min and the outward K⁺ current was recorded using whole cell patch clamp recording. The percentage of decrease in ME-induced K⁺ current was considered as the degree of MOR desensitization. Results indicated that OXA (100 nM) enhances ME-induced MOR desensitization via affecting OX1Rs in rat locus coeruleus neurons and this effect is mediated by a protein kinase C dependent mechanism within the LC. The activity of orexinergic system might potentiate the signaling pathways underlying opioid-induced receptor desensitization.     

Transient homologous mu-opioid receptor desensitization in rat locus coeruleus neurons.

Opioid agonists hyperpolarize neurons of the locus coeruleus (LC) in the slice preparation. When opioids were applied at concentrations that caused a maximum hyperpolarization, the membrane potential hyperpolarized to a peak (about 30 mV) in the first minute and then declined over a period of 5 min. In addition, following the washout, the amplitude of the hyperpolarization induced by a lower concentration of opioid was significantly reduced as compared to control. The original response to both the low and the high concentrations of opioid recovered after removal of opioids for about 20 min. The decline in response, termed "acute desensitization," was observed only with concentrations of opioids that caused a maximum hyperpolarization and was dependent on the concentration of opioid applied (EC50 for [Met5]-enkephalin (ME), between 3 and 5 microM). The response to ME (300 nM) was reduced to 6% of control following washout of a 5-min application of ME (30 microM), whereas the response to noradrenaline (300 nM) was reduced to 75% of control. The acute desensitization therefore was selective for the opioid receptor with marginal cross-desensitization to the alpha 2-adrenoceptor-mediated hyperpolarization. The desensitization still occurred following treatment with beta-chlornaltrexamine (beta-CNA), to decrease receptor reserve, as well as in cells taken from animals treated chronically with morphine. The mechanism for the acute desensitization was investigated using agents thought to alter kinase activity. This acute desensitization may represent an initial stage in the development of tolerance produced by chronic administration of opioids.     

Bio-imaging of nitric oxide-producing neurones in slices of rat brain using 4,5-diaminofluorescein

4,5-Diaminofluorescein (DAF-2) was used to identify individual nitric oxide (NO)-producing neurones in brain slices in vitro. Coronal slices of midbrain or hippocampus, 300 microm thick from young adult rats, were incubated for 30 min in 1 microM DAF-2 diacetate (DAF-2 DA) and maintained in ACSF at 33 degrees C. Illumination at 450-490 nm revealed punctate fluorescence in neurones in the lateral tegmental nucleus, dorsal raphe nucleus, dorsolateral periaqueductal grey matter, deep collicular layers and cortical areas. Neurones in the hippocampal pyramidal cell layer, molecular layer of the dentate gyrus and the hilus fluoresced also. The fluorescence was abolished by pre-incubation of slices with L-NAME (100 microM-1 mM), the inhibitor of constitutive nitric oxide synthase (NOS), but not by D-NAME (100 microM) or L-NIL (5-50 microM), an inhibitor of inducible NOS. In some superficially located arterioles, there were small regions of bright fluorescence close to the outer smooth muscle wall and diffuse fluorescence within the adjacent smooth muscle cells. A diffuse fluorescence was also seen in some superficially located capillaries. Basal production of NO was not seen within deeper blood vessels. DAF-2 DA offers a sensitive indicator for visualising basal production of NO with high spatial resolution and could provide a means of identifying NOS-containing neurones in brain slices in vitro prior to neurophysiological study.     

Ng-nitro-L-arginine,an NOS inhibitor,reduces tolerance to morphine in the rat locus coeruleus

Ng-nitro-L-arginine (L-NArg), a potent nitric oxide synthase inhibitor, has been implicated as a potential mechanism for attenuating the development of tolerance to opioid drugs and for suppressing opioid withdrawal. Neurons in the locus coeruleus (LC) express opioid receptors and these neurons exhibit both tolerance to chronic administration of opioids and antagonist-precipitated withdrawal hyperactivity. This study tested the hypothesis that L-NArg would attenuate the development of opioid tolerance in LC neurons. Challenge doses of morphine were administered while recording single-cell extracellular activity in brain slices from rats who had been concurrently treated for 5 days with morphine (75 mg morphine sulfate base pellets) and L-NArg (10 mg/kg, ip, bid). The average ED₅₀ for morphine of cells from rats who received L-NArg injections and morphine pellets was similar to that in cells from rats who had been implanted with sham pellets (14.5–18 nM). In contrast, the average ED₅₀ of cells from morphine pelleted animals who received saline injections was substantially higher (34.5 nM). These results demonstrate that L-NArg attenuates the development of tolerance to morphine in LC neurons. Synapse 29:233–239, 1998. © 1998 Wiley-Liss, Inc.     

Nitric oxide regulates swimming in the jellyfish Aglantha digitale

The cnidarian nervous system is considered by many to represent neuronal organization in its earliest and simplest form. Here we demonstrate, for the first time in the Cnidaria, the neuronal localization of nitric oxide synthase (NOS) in the hydromedusa Aglantha digitale (Trachylina). Expression of specific, fixative-resistant NADPH-diaphorase (NADPH-d) activity, characteristic of NOS, was observed in neurites running in the outer nerve ring at the base of the animal and in putative sensory cells in the ectoderm covering its tentacles. At both sites, diphenyleneiodonium (10(-4) M) abolished staining. Capillary electrophoresis confirmed that the NO breakdown products NO2- and NO3- were present at high levels in the tentacles, but were not detectable in NADPH-d-negative areas. The NADPH-d-reactive neurons in the tentacles send processes to regions adjacent to the inner nerve ring where swimming pacemaker cells are located. Free-moving animals and semi-intact preparations were used to test whether NO is involved in regulating the swimming program. NO (30-50 nM) and its precursor L-arginine (1 mM) stimulated swimming, and the effect was mimicked by 8-Br-cGMP (50-100 microM). The NO scavenger PTIO (10-100 microM) and a competitive inhibitor of NOS, L-nitroarginine methyl ester (L-NAME, 200 microM), significantly decreased the swimming frequency in free-moving animals, while its less-active stereoisomer D-nitroarginine methyl ester (D-NAME, 200 microM) had no such effect. 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ, 5-20 microM), a selective inhibitor of soluble guanylyl cyclase, suppressed spontaneous swimming and prevented NO-induced activation of the swimming program. We suggest that an NO/cGMP signaling pathway modulates the rhythmic swimming associated with feeding in Aglantha, possibly by means of putative nitrergic sensory neurons in its tentacles.     

The NO‐cGMP pathway in the rat locus coeruleus: electrophysiological, immunohistochemical and in situ hybridization studies

The effect of two nitric oxide (NO) donors, SIN-1 and DEA/NO, as well as of the inactive SIN-1 derivative molsidomin, was studied on locus coeruleus (LC) neurons in a slice preparation using intracellular recordings. In addition, the effect of the guanylate cyclase inhibitor ODQ was analysed. Furthermore, the effect of NO donors on cyclic guanosine monophosphate (GMP) levels in the LC was studied using the indirect immunofluorescence technique, and the expression of soluble guanylyl cyclase with in situ hybridization. In 36 of 66 LC neurons extracellular application of SIN-1 and DEA/NO caused a hyperpolarization and a decrease in apparent input resistance. In almost 20% of neurons SIN-1 increased the firing rate. No effect could be recorded with the brain-inactive SIN-1 derivative molsidomin. The membrane permeable cGMP analogue 8-bromo-cGMP imitated the action of SIN-1. The hyperpolarizing effect of SIN-1 and DEA/NO was attenuated by preincubation with the guanylyl cyclase inhibitor ODQ. The immunohistochemical analysis revealed lack of cGMP immunostaining in non-stimulated slices, whereas SIN-1 dramatically increased this staining in about 40% of the LC neurons, and these neurons were all tyrosine hydroxylase positive, that is noradrenergic. A large proportion of the LC neurons expressed soluble guanylyl cyclase mRNA. The present and previous results suggest that NO, released from a small number of non-noradrenergic neurons in the LC, mainly has an inhibitory influence on many noradrenergic neurons, by upregulating cGMP levels via stimulation of soluble guanylyl cyclase. As nitric oxide synthase is present only in a small number of non-noradrenergic neurons ( Xu et al. 1994 ), a few neurons may influence a large population of noradrenergic LC neurons, which in turn may control activity in many regions of the central nervous system.     

Hypocretin/orexin depolarizes and decreases potassium conductance in locus coeruleus neurons

Recent studies demonstrated that noradrenergic locus coeruleus (LC) neurons are a particularly strong target of the novel neuropeptide, hypocretin (orexin). The present study sought to elucidate the action of hypocretin-B (HCRT) on LC neurons recorded intracellularly in rat brain slices. Bath (1.0 microM) or local puff application (50-100 microM in pipette) of HCRT depolarized LC neurons in rat brain slices and increased their spontaneous discharge rate. Depolarization evoked by HCRT was persistent in the presence of tetrodotoxin (TTX, 1 microM) and Co2+ (1 mM), indicating that HCRT directly activated LC neurons, and that its effect on the postsynaptic cell was not due to activation of TTX-sensitive sodium channels or Co2+-sensitive calcium channels. The apparent input resistance was significantly increased in the majority of LC neurons during the HCRT-evoked depolarization. Moreover, the HCRT-evoked depolarization was decreased in amplitude with hyperpolarization of membrane. The present results indicate that decreased potassium conductance is involved in the effect of HCRT on LC neurons.     

Nitric oxide modulates striatal neuronal activity via soluble guanylyl cyclase: an in vivo microiontophoretic study in rats

It is now well established that nitric oxide (NO) acts as a neuromodulator in the central nervous system. To assess the role of NO in modulating striatal activity, single-unit recording was combined with iontophoresis to study presumed spiny projection neurons in urethane-anesthetized male rats. Striatal neurons recorded were essentially quiescent and were therefore activated to fire by the iontophoretic administration of glutamate, pulsed in cycles of 30 sec on and 40 sec off. In this study, iontophoresis of 3-morpholinosydnonimine hydrochloride (SIN 1), a nitric oxide donor, produced reproducible, current-dependent inhibition of glutamate-induced excitation in 12 of 15 striatal neurons, reaching its maximal inhibitory effect (76.2 +/- 5.6% below baseline) during the application of a 100 nA current. Conversely, microiontophoretic application of N-omega-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase, produced clear and reproducible excitation of glutamate evoked firing in 7 of 10 cells (51.4 +/- 2.3%, at 100 nA). To evaluate the involvement of cyclic guanosine monophosphate (cGMP) in the electrophysiological effects produced by the NO donor, the effects of methylene blue, an inhibitor of guanylyl cyclase, on the responses of nine neurons to SIN 1 were tested. In six of nine neurons the effect of SIN 1 was significantly reduced during continuous iontophoretic administration (50 nA) of methylene blue. Taken together, these data show that NO modulates the striatal network and that inhibitory control of the output neurons is involved in this effect. These results also suggest that the effects of nitric oxide on striatal neurons are partially mediated via cGMP.     

Nitric oxide modulates low-Mg2+-induced epileptiform activity in rat hippocampal-entorhinal cortex slices

The production of nitric oxide (NO) during low-Mg2+-induced epileptiform activity in rat hippocampal-entorhinal cortex slices was investigated by real-time monitoring using 1,2-diaminoanthraquinone (DAQ). NO reacts with the aromatic amino groups of DAQ at neutral pH and in the presence of oxygen to form the fluorescence product 1H-anthra-[1,2d]-[1,2,3]triazole-6,11-dione (ATD). The DAQ-induced formation of ATD required NO and was insensitive to radical oxygen species. Removal of Mg2+ ions from the artificial cerebrospinal fluid (ACSF) induced a significant elevation in the ATD fluorescence signal. The application of L-arginine (2 mM), a substrate of nitric oxide synthase (NOS), caused a comparable increase in the ATD fluorescence signal. Furthermore, ATD signal increase induced either by low-Mg2+ ACSF or by L-arginine was sensitive to N-nitro-L-arginine methyl ester (L-NAME), a NOS inhibitor. The application of L-NAME (200 microM) caused a complete blockade of low-Mg2+-induced epileptiform activity. Under this condition, increasing NO concentration by addition of the NO donor S-nitroso-N-acetylpenicillamine (200 microM) reinduced the epileptiform activity. It has been concluded that onset and maintenance of low-Mg2+-induced spontaneous epileptiform activity are modulated by NO concentration. Further NO imaging studies may help to elucidate the role of NO in detail and may bring to light new means for epilepsy therapy.     

Involvement of the nitric oxide pathway in the anticonvulsant effect of tramadol on pentylenetetrazole-induced seizures in mice

In the present study, the effects of tramadol on pentylenetetrazole (PTZ)-induced seizures and involvement of nitric oxide (NO) were assessed in mice. To determine the threshold for clonic seizures, PTZ was administered intravenously. Tramadol was administered intraperitoneally (0.5-50mg/kg) 30 minutes prior to induction of seizures. The effects of the nitric oxide synthase (NOS) inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME; 0.5, 1, 5, and 10mg/kg), the nitric oxide precursor L-arginine (10, 30, and 60 mg/kg), and the nonspecific opioid receptor antagonist naloxone (0.1, 0.5, 1, and 5mg/kg) on the anticonvulsant effect of tramadol were investigated. Administration of tramadol (1mg/kg) increased the threshold for seizures induced with PTZ in a monophasic, dose-independent, and time-dependent manner. Acute administration of L-NAME (5 and 10mg/kg) inhibited the anticonvulsant effect of tramadol (1mg/kg), whereas L-arginine, in the noneffective dose range (30 and 60 mg/kg), potentiated the seizure threshold when co-administered with a subeffective dose of tramadol (0.5mg/kg). Naloxone partially and dose-independently antagonized the anticonvulsant effect of tramadol (1mg/kg). These results indicate that the anticonvulsant effect of tramadol is mediated by the nitric oxide pathway and also by classic opioid receptors.     

Adenosine inhibits locus coeruleus neurons: An intracellular study in a rat brain slice preparation

Intracellular recording was used to study the effect of adenosine (3-100 microM) on rat locus coeruleus (LC) neurons in a brain slice preparation. Bath application of adenosine (100 microM) reduced the rate of spontaneous firing in 88% of LC neurons. In some LC neurons adenosine also caused membrane hyperpolarization (2-10 mV) and reductions in input resistance of 9-24%. Adenosine effects were dose-dependent and antagonized by the adenosine receptor antagonist theophylline.     

Role of nitric oxide in modulating neurotransmitter release from rat striatum

The effect of the nitric oxide synthase inhibitor N-nitro- -arginine methyl ester (L-NAME) on the basal and stimulation-evoked release of dopamine (DA) and acetylcholine (ACh) was investigated in rat striatum. The experiments were carried out in isolated superfused striatal slices, loaded with either [³H]-dopamine or [³H]-choline.We have found that L-NAME reduced the elecrical field stimulation-evoked release of DA, while its enantiomer N-nitro-D-arginine methyl ester (D-NAME) was ineffective. In the presence of the nitric oxide (NO) precursor -arginine L-NAME failed to influence DA release. Furthermore, treatment with the N-methyl- -aspartate (NMDA) receptor antagonist MK-801 completely reversed the effect of L-NAME on striatal DA release. In contrast, L-NAME had no effect on either the basal or the stimulation-evoked ACh release in any experimental conditions studied.Our data indicate that endogenously produced NO is involved in the modulation of striatal DA, but not in ACh release. Furthermore, it seems likely that the modulatory effect of NO is linked to activation of presynaptic NMDA receptors located on the striatal dopaminergic nerve terminals.     

Excitatory action of N-methyl- -aspartate on the rat locus coeruleus is mediated by nitric oxide: an in vivo voltammetric study

Biochemical, electrophysiological and behavioural studies have provided evidence that activation of N-methyl- -aspartate (NMDA) receptors contributes to the hyperactivity of noradrenergic neurons of the locus coeruleus (LC) in precipitated opioid withdrawal. Recently, it was demonstrated that central administration of nitric oxide (NO) synthase inhibitors suppresses this hyperactivity suggesting that NO mediates the NMDA receptor activation of LC in opioid withdrawal. Using a combination of microdialysis and in vivo voltammetry, this study examined whether local application of NMDA to the LC in opioid naive animals mimics the NO-dependent LC response seen in opioid withdrawal. In the urethane anaesthetized rat, perfusion of the LC (2 μl min⁻¹) with a solution of NMDA (5 mmol) via a microdialysis probe for 9 min resulted in a rapid and robust increase (290.1±32.2% above baseline) in the catechol oxidation current (CA·OC) recorded from the LC using differential normal pulse voltammetry (DNPV). The NMDA microdialysis also produced a large increase in the blood pressure (150.4±6.9% above baseline). An injection of the non-competitive NMDA receptor antagonist (+)MK-801 (0.5 mg kg⁻¹ i.v.), given 45 min after the start of NMDA application, rapidly returned both the CA·OC signal and the blood pressure response to baseline levels. Pretreatment of animals with intraventricular nitric oxide synthase (NOS) inhibitor, N^ω-nitro- -arginine methyl ester ( -NAME) (100 μg) significantly inhibited NOS activity in the LC, PAG-PVG and cerebellum. This dose of -NAME, administered prior to application of NMDA by microdialysis abolished the NMDA-induced rise in the CAOC recorded in the LC and the increase in systolic blood pressure. The results show that in voltammetry experiments, NMDA produces hyperactivity of LC and hypertension, responses that are dependent upon the synthesis of NO. Thus, in opioid naive rats, regional NMDA application via microdialysis mimics characteristics of the LC response that occur during the antagonist-precipitated opioid withdrawal.     

Long-term glutamate desensitization in locus coeruleus neurons and its role in opiate withdrawal

During opiate withdrawal, there is an elevated and prolonged efflux of glutamate and aspartate in the locus coeruleus (LC). The enhanced excitatory amino acid (EAA) release is thought to contribute to the withdrawal-induced activation of LC neurons and to the expression of the physical withdrawal syndrome. In this study, prolonged bath applications of glutamate to LC neurons in brain slices resulted in a slowly developing long-term glutamate desensitization (LTGD). LTGD was observed during extracellular recordings or in neurons voltage-clamped to −60 mV, in both cases reaching a maximum of about a 50% reduction in the glutamate response. Responses in the desensitized cells gradually recovered within 3 h. Cyclothiazide, an inhibitor of rapid glutamate receptor desensitization did not prevent LTGD. LTGD could not be induced by prolonged applications of EAA agonists other than glutamate, either alone or in various combinations. However, after induction by glutamate, there was cross-desensitization to quisqualate but not to AMPA or NMDA. LTGD was blocked by either lowering extracellular Ca²⁺ concentrations or by treatment with the protein kinase C inhibitor chelerythrine but not by inhibitors of calcium/calmodulin-dependent kinase or nitric oxide synthase. Applications of the protein kinase C activator phorbol diacetate did not cause a decrease in glutamate responses indicating that an activation of protein kinase C may not be sufficient for desensitization to occur. A decrement of the glutamate response resembling LTGD occurred after treatment by the protein phosphatase inhibitors okadaic acid or calyculin A. LC neurons in brain slices prepared from opiate-withdrawn rats exhibited glutamate responses that were initially desensitized and recovered within 3 h after withdrawal. These results suggest that LTGD in LC neurons may occur during opiate withdrawal and could contribute to the time course of LC hyperactivity and the associated behavioral withdrawal syndrome.     

A role for nitric oxide in the median eminence and arcuate nucleus response to capsaicin treatment in rats.

This study examined a possible functional involvement of nitric oxide (NO) in the median eminence (ME) and arcuate nucleus (ARC) after capsaicin treatment in rats. Subcutaneous injection of capsaicin increased nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) activity in the ARC-ME compared with vehicle treatment. Fos expression was increased in the ARC after capsaicin injection compared with vehicle-treated rats. Pretreatment with the NO synthase (NOS) inhibitor, N(omega)-nitro-L-arginine methyl ester (L-NAME) attenuated the effect of capsaicin on Fos expression and NADPH-d reactivity in the ARC-ME in comparison with rats injected with D-NAME, the inactive stereoisomer of L-NAME. These observations suggest that NO makes a major contribution to the response of the ARC-ME to a stressor such as capsaicin.     

Noradrenergic regulation in mouse supraoptic nucleus involves a nitric oxide pathway only to regulate arginine-vasopressin expression and not oxytocin expression

Noradrenalin (NA) regulates the expression of arginine-vasopressin (AVP) and oxytocin (OT) by magnocellular neurons in the supraoptic nucleus (SON) of the hypothamalus. Nitric oxide (NO) may be one of the factors involved in the NA signaling pathway regulating AVP and OT expression. To test this possibility, we used an ex vivo experimental model of mouse hypothalamus slices. Increases in AVP and OT levels in the SON were detected by immunohistochemistry and immunoenzyme assays after 1 hr and 4 hr incubations with NA (10(-4) M). There was also an increase in the expression and activity of neuronal NOS and inducible NOS in the SON as assessed by immunohistochemical and histoenzymological analysis of NADPH-diaphorase, whereas endothelial NOS was undetectable. To specify the role of NO, the slices were treated with NA and L-arginine methyl ester (L-NAME, an NOS inhibitor; 3 microM). This treatment for 1 hr abolished the NA-induced increase in AVP. Treatment with sodium nitroprusside (SNP, an NO donor; 0.1 mM) increased AVP levels, confirming that NO regulates AVP expression. Addition of 1 mM EGTA during the incubation with NA reduced the AVP increase by half, indicating that both nNOS and iNOS activities are involved in the regulation. A 1-hr treatment with L-NAME did not prevent the increase in OT induced by NA; similarly, treatment with SNP had no effect. These findings show that NO is involved in the regulation of AVP expression by NA and that NA control of OT expression is independent of NO.     

Distribution of c-fos mRNA in the brain following intracerebroventricular injection of nitric oxide (NO)-releasing compounds: possible role of NO in central cardiovascular regulation

We injected nitric oxide (NO)-releasing compounds and NO synthase (NOS) inhibitors into the brains of conscious, freely moving rats and measured the effects on mean arterial blood pressure (MAP) and heart rate, as well as on the expression of c-fos mRNA, neuronal NOS (nNOS) mRNA and NADPH-diaphorase, an indicator of NOS activity. When administered i.c.v., the NO donor, NOC-18, caused a significant fall in MAP and heart rate, whereas the NOS inhibitor, NG-nitro-L-arginine methyl ester (L-NAME), induced a significant rise in MAP. The same dose of NOC-18 or L-NAME when administered i.v. did not affect MAP and heart rate. Centrally administered NOC-18 induced c-fos mRNA expression in several regions of the brain involved in the baroreceptor response, including the nucleus of the solitary tract, the area postrema and the rostral ventrolateral medulla, as well as areas involved in the integration of autonomic, neuroendocrine and behavioural responses, including the medial preoptic area, the organum vasculosum lamina terminalis, the bed nucleus of stria terminalis, the paraventricular nucleus (PVN), the supraoptic nucleus (SON), the central nucleus of amygdala (CeA) and the locus coeruleus. Most of the areas that expressed c-fos also contained nNOS mRNA and/or NADPH-d-positive neurones and fibres. i.c.v. injection of L-NAME induced c-fos mRNA expression in PVN, SON, locus coeruleus and NTS, suggesting a tonic inhibition of neuronal activity by NO or stimulation of neuronal activity by endogenous NO. i.v. injection of NOC-18 or L-NAME did not induce any significant c-fos mRNA expression in rat brain. These results demonstrate that NO acts directly in the brain to reduce the systemic blood pressure, and that the endogenous NO pathway may play a role in cardiovascular and autonomic regulation by modulating neuronal activities in discrete regions of the brain.     

Cerebral nitric oxide concentration and microcirculation during hypercapnia, hypoxia, and high intracranial pressure in pigs

Intracerebral nitric oxide (NO) concentration was measured to establish the technique and to investigate the response of the NO concentration to CO(2)variations, hypoxia, and reduced cerebral perfusion pressure. An intracerebral nitric oxide sensor was used in 10 pigs. Cerebral microcirculation was measured by laser Doppler flowmetry. Five pigs received 40 mg/kg nitro-1-arginine methyl ester (L-NAME). Baseline NO concentration was 246 +/- 42 nM. Hypercapnia increased cerebral microcirculation (P< 0.05) and NO concentration (P< 0.05). Hypoxia decreased NO concentration (P< 0.05). During high intracranial pressure, cerebral microcirculation decreased (P< 0.05) before the NO concentration decreased (P< 0.05), and after normalisation of the intracranial pressure the NO concentration increased, but more slowly than the cerebral microcirculation. L-NAME caused a decrease in cerebral microcirculation (P< 0.05) and NO concentration (P< 0.05) to a new steady state, and L-NAME attenuated the changes in NO concentration after hypoxia (P< 0.05) and high intracranial pressure (P< 0.05). In conclusion, the electrochemical sensor appears to reliably detect changes in localised intracerebral NO concentration and seems to be a promising tool for direct measurement of this chemically unstable substance.