Angiotensin II type 2 receptor-coupled nitric oxide production modulates free radical availability and voltage-gated Ca2+ currents in NTS neurons

The medial region of the nucleus tractus solitarius (mNTS) is a key brain stem site controlling cardiovascular function, wherein ANG II modulates neuronal L-type Ca(2+) currents via activation of ANG II type 1 receptors (AT(1)R) and production of reactive oxygen species (ROS). ANG II type 2 receptors (AT(2)R) induce production of nitric oxide (NO), which may interact with ROS and modulate AT(1)R signaling. We sought to determine whether AT(2)R-mediated NO production occurs in mNTS neurons and, if so, to elucidate the NO source and the functional interaction with AT(1)R-induced ROS or Ca(2+) influx. Electron microscopic (EM) immunolabeling showed that AT(2)R and neuronal NO synthase (nNOS) are coexpressed in neuronal somata and dendrites receiving synapses in the mNTS. In the presence of the AT(1)R antagonist losartan, ANG II increased NO production in isolated mNTS neurons, an effect blocked by the AT(2)R antagonist PD123319, but not the angiotensin (1-7) antagonist D-Ala. Studies in mNTS neurons of nNOS-null or endothelial NOS (eNOS)-null mice established nNOS as the source of NO. ANG II-induced ROS production was enhanced by PD123319, the NOS inhibitor N(G)-nitro-l-arginine (LNNA), or in nNOS-null mice. Moreover, in the presence of losartan, ANG II reduced voltage-gated L-type Ca(2+) current, an effect blocked by PD123319 or LNNA. We conclude that AT(2)R are closely associated and functionally coupled with nNOS in mNTS neurons. The resulting NO production antagonizes AT(1)R-mediated ROS and dampens L-type Ca(2+) currents. The ensuing signaling changes in the NTS may counteract the deleterious effects of AT(1)R on cardiovascular function.     

Amyloid-beta (25-35) increases activity of neuronal NO-synthase in rat brain

Nitric oxide (NO) is a free radical with multiple functions in the nervous system. NO plays an important role in the mechanisms of neurodegenerative diseases including Alzheimer's disease. The main source of NO in the brain is an enzymatic activity of nitric oxide synthase (NOS). The aim of the present study was to analyze the expression and activity of both neuronal (nNOS) and inducible (iNOS) isoenzymes in the cerebral cortex and hippocampus of rats after intracerebroventricular administration of amyloid-beta (A beta) peptide fragment A beta(25-35). NADPHd histochemistry as well as immunohistochemistry were also used to investigate nNOS and iNOS expression in rat brain. The data presented here show that A beta(25-35) did not influence levels of nNOS or iNOS mRNA or protein expression in both structures studied. A beta(25-35) activated nNOS in the cerebral cortex and hippocampus without effect on iNOS activity. A beta(25-35) decreased the number of NADPHd-expressing neurons in the neocortex, but it did not significantly influence the number NADPHd-positive cells in the hippocampus. The peptide had no effect on the number of nNOS containing cells. We hypothesize that increased synthesis of NO induced by A beta(25-35) is related to qualitative alterations of nNOS molecule, but not to changes in NOS protein expression.     

Pulmonary NO synthase expression is attenuated in a fetal baboon model of chronic lung disease

Nitric oxide (NO), produced by NO synthase (NOS), serves multiple functions in the perinatal lung. In fetal baboons, neuronal (nNOS), endothelial (eNOS), and inducible NOS (iNOS) are all primarily expressed in proximal respiratory epithelium. In the present study, NOS expression and activity in proximal lung and minute ventilation of NO standard temperature and pressure (VeNO(STP)) were evaluated in a model of chronic lung disease (CLD) in baboons delivered at 125 days (d) of gestation (term = 185 d) and ventilated for 14 d, obtaining control lung samples from fetuses at 125 or 140 d of gestation. In contrast to the normal 73% increase in total NOS activity from 125 to 140 d of gestation, there was an 83% decline with CLD. This was related to marked diminutions in both nNOS and eNOS expression and enzymatic activity. nNOS accounted for the vast majority of enzymatic activity in all groups. The normal 3.3-fold maturational rise in iNOS protein expression was blunted in CLD, yet iNOS activity was elevated in CLD compared with at birth. The contribution of iNOS to total NOS activity was minimal in all groups. VeNO(STP) remained stable in the range of 0.5-1.0 nl x kg(-1) x min(-1) from birth to day 7 of life, and it then rose by 2.5-fold. Thus the baboon model of CLD is characterized by deficiency of the principal pulmonary isoforms, nNOS and eNOS, and enhanced iNOS activity over the first 2 wk of postnatal life. It is postulated that these alterations in NOS expression and activity may contribute to the pathogenesis of CLD.     

eNOS involved in colitis-induced mucosal blood flow increase

The role of NO in inflammatory bowel disease is controversial. Studies indicate that endothelial nitric oxide synthase (eNOS) might be involved in protecting the mucosa against colonic inflammation. The aim of this study was to investigate the involvement of nitric oxide (NO) in regulating colonic mucosal blood flow in two different colitis models in rats. In anesthetized control and colitic rats, the distal colon was exteriorized and the mucosa visualized. Blood flow (laser-Doppler flowmetry) and arterial blood pressure were continuously monitored throughout the experiments, and vascular resistance was calculated. Trinitrobenzene sulfonic acid (TNBS) or dextran sulfate sodium (DSS) was used to induce colitis. All groups were given the NOS inhibitor N(omega)-nitro-l-arginine (l-NNA) or the inducible NOS (iNOS) inhibitor l-N(6)-(1-iminoethyl)-lysine (l-NIL). iNOS, eNOS, and neuronal NOS (nNOS) mRNA in colonic samples were investigated with real-time RT-PCR. Before NOS inhibition, colonic mucosal blood flow, expressed as perfusion units, was higher in both colitis models compared with the controls. The blood flow was reduced in the TNBS- and DSS-treated rats during l-NNA administration but was not altered in the control group. Vascular resistance increased more in the TNBS- and DSS-treated rats than in the control rats, indicating a higher level of vasodilating NO in the colitis models. l-NIL did not alter blood pressure or blood flow in any of the groups. iNOS and eNOS mRNA increased in both colitis models, whereas nNOS remained at the control level. TNBS- and DSS-induced colitis results in increased colonic mucosal blood flow, most probably due to increased eNOS activity.     

Discoordinate regulation of renal nitric oxide synthase isoforms in ovariectomized mRen2. Lewis rats

Estrogen depletion markedly exacerbates hypertension in female congenic mRen2. Lewis rats, a model of tissue renin overexpression. Because estrogen influences nitric oxide synthase (NOS) and NO may exert differential effects on blood pressure, the present study investigated the functional expression of NOS isoforms in the kidney of ovariectomized (OVX) mRen2. Lewis rats. OVX-mRen2. Lewis exhibited an increase in systolic blood pressure (SBP) of 171 +/- 5 vs. 141 +/- 7 mmHg (P < 0.01) for intact littermates. Renal cortical mRNA and protein levels for endothelial NOS (eNOS) were reduced 50-60% (P < 0.05) and negatively correlated with blood pressure. In contrast, cortical neuronal NOS (nNOS) mRNA and protein levels increased 100 to 300% (P < 0.05). In the OVX kidney, nNOS immunostaining was more evident in the macula densa, cortical tubules, and the medullary collecting ducts compared with the intact group. To determine whether the increase in renal nNOS expression constitutes a compensatory response to the reduction in renal eNOS, we treated both intact and OVX mRen2. Lewis rats with the selective nNOS inhibitor L-VNIO from 11 to 15 wk of age. The nNOS inhibitor reduced blood pressure in the OVX group (185 +/- 3 vs. 151 +/- 8 mmHg, P < 0.05), but pressure was not altered in the intact group (146 +/- 4 vs. 151 +/- 4 mmHg). In summary, exacerbation of blood pressure in the OVX mRen2. Lewis rats was associated with the discoordinate regulation of renal NOS isoforms. Estrogen sensitivity in this congenic strain may involve the influence of NO through the regulation of both eNOS and nNOS.     

Renal cortical and medullary blood flow responses to L-NAME and ANG II in wild-type, nNOS null mutant, and eNOS null mutant mice

Experiments in wild-type (WT; C57BL/6J) mice, endothelial nitric oxide synthase null mutant [eNOS(-/-)] mice, and neuronal NOS null mutant [nNOS(-/-)] mice were performed to determine which NOS isoform regulates renal cortical and medullary blood flow under basal conditions and during the infusion of ANG II. Inhibition of NOS with N(omega)-nitro-l-arginine methyl ester (l-NAME; 50 mg/kg iv) in Inactin-anesthetized WT and nNOS(-/-) mice increased arterial blood pressure by 28-31 mmHg and significantly decreased blood flow in the renal cortex (18-24%) and the renal medulla (13-18%). In contrast, blood pressure and renal cortical and medullary blood flow were unaltered after l-NAME administration to eNOS(-/-) mice, indicating that NO derived from eNOS regulates baseline vascular resistance in mice. In subsequent experiments, intravenous ANG II (20 ng x kg(-1) x min(-1)) significantly decreased renal cortical blood flow (by 15-25%) in WT, eNOS(-/-), nNOS(-/-), and WT mice treated with l-NAME. The infusion of ANG II, however, led to a significant increase in medullary blood flow (12-15%) in WT and eNOS(-/-) mice. The increase in medullary blood flow following ANG II infusion was not observed in nNOS(-/-) mice, in WT or eNOS(-/-) mice pretreated with l-NAME, or in WT mice administered the nNOS inhibitor 5-(1-imino-3-butenyl)-l-ornithine (1 mg x kg(-1) x h(-1)). These data demonstrate that NO from eNOS regulates baseline blood flow in the mouse renal cortex and medulla, while NO produced by nNOS mediates an increase in medullary blood flow in response to ANG II.     

Distribution of NADPH Diaphorase-Exhibiting Primary Afferent Neurons in the Trigeminal Ganglion and Mesencephalic Trigeminal Nucleus of the Rabbit

1. Nitric oxide (NO) is highly reactive gaseous molecule to which many physiological and pathological functions have been attributed in the central (CNS) and peripheral (PNS) nervous system. The present investigation was undertaken to map the distribution pattern of the enzyme responsible for the synthesis of NO, nitric oxide synthase (NOS), and especially its neuronal isoform (nNOS) in the population of primary afferent neurons of the trigeminal ganglion (TG) and mesencephalic trigeminal nucleus (MTN) of the rabbit.2. In order to identify neuronal structures expressing nNOS we applied histochemistry to its specific histochemical marker nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd).3. We found noticeable amount of NADPHd-exhibiting primary afferent neurons in TG of the rabbit under physiological conditions. The intensity of the histochemical reaction was highly variable reaching the maximum in the subpopulation of small-to-medium-sized neurons. The large-sized neurons were only weakly stained or actually did not posses any NADPHd-activity. In addition, NADPHd-positive nerve fibers were detected between clusters of the ganglionic cells and in the peripheral branches of the trigeminal nerve (TN). NADPHd-exhibiting MTN neurons were noticed in the whole rostrocaudal extent of the nucleus even though some differences were found concerning the ratio of NADPHd-positive versus NADPHd-negative cell bodies. Similarly, we observed striking diversity in the intensity of NADPHd histochemical reaction in the subpopulations of small-, medium-, and large-sized MTN neurons.4. The predominant localization of NADPHd in the subpopulation of small-to-medium-sized TG neurons which are generally considered to be nociceptive suggests that NO probably takes part in the modulation of nociceptive inputs from the head and face. Furthermore, we tentatively assume that NADPHd-exhibiting MTN neurons probably participate in transmission and modulation of the proprioceptive impulses from muscle spindles of the masticatory muscles and mechanoreceptors of the periodontal ligaments and thus provide sensory feedback of the masticatory reflex arc.     

Spatiotemporal Alterations of the NO/NOS Neuronal Pools Following Transient Abdominal Aorta Occlusion: Morphological and Biochemical Studies in the Rabbit

1. Brief interruption of spinal cord blood flow resulting from transient abdominal aortic occlusion may lead to degeneration of specific spinal cord neurons and to irreversible loss of neurological function. The alteration of nitric oxide/nitric oxide synthase (NO/NOS) pool occurring after ischemic insult may play a protective or destructive role in neuronal survival of affected spinal cord segments.2. In the present study, the spatiotemporal changes of NOS following transient ischemia were evaluated by investigating neuronal NOS immunoreactivity (nNOS-IR), reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) histochemistry, and calcium-dependent NOS (cNOS) conversion of [³H] l-arginine to [³H] l-citrulline.3. The greatest levels of these enzymes and activities were detected in the dorsal horn, which appeared to be most resistant to ischemia. In that area, the first significant increase in NADPHd staining and cNOS catalytic activity was found immediately after a 15-min ischemic insult.4. Increases in the ventral horn were observed later (i.e., after a 24-h reperfusion period). While the most intense increase in nNOS-IR was detected in surviving motoneurons of animals with a shorter ischemic insult (13 min), the greatest increase of cNOS catalytic activity and NADPHd staining of the endothelial cells was found after stronger insult (15 min).5. Given that the highest levels of nNOS, NADPHd, and cNOS were found in the ischemia-resistant dorsal horn, and nNOS-IR in surviving motoneurons, it is possible that NO production may play a neuroprotective role in ischemic/reperfusion injury.     

Nitric oxide produced via neuronal NOS may impair vasodilatation in septic rat skeletal muscle

Impaired vascular responsiveness in sepsis may lead to maldistribution of blood flow in organs. We hypothesized that increased production of nitric oxide (NO) via inducible nitric oxide synthase (iNOS) mediates the impaired dilation to ACh in sepsis. Using a 24-h cecal ligation and perforation (CLP) model of sepsis, we measured changes in arteriolar diameter and in red blood cell velocity (V(RBC)) in a capillary fed by the arteriole, following application of ACh to terminal arterioles of rat hindlimb muscle. Sepsis attenuated both ACh-stimulated dilation and V(RBC) increase. In control rats, arteriolar pretreatment with the NO donors S-nitroso-N-acetylpenicillamine or sodium nitroprusside reduced diameter and V(RBC) responses to a level that mimicked sepsis. In septic rats, arteriolar pretreatment with the "selective" iNOS blockers aminoguanidine (AG) or S-methylisothiourea sulfate (SMT) restored the responses to the control level. The putative neuronal NOS (nNOS) inhibitor 7-nitroindazole also restored the response toward control. At 24-h post-CLP, muscles showed no reduction of endothelial NOS (eNOS), elevation of nNOS, and, surprisingly, no induction of iNOS protein; calcium-dependent constitutive NOS (eNOS+nNOS) enzyme activity was increased whereas calcium-independent iNOS activity was negligible. We conclude that 1) AG and SMT inhibit nNOS activity in septic skeletal muscle, 2) NO could impair vasodilative responses in control and septic rats, and 3) the source of increased endogenous NO in septic muscle is likely upregulated nNOS rather than iNOS. Thus agents released from the blood vessel milieu (e.g., NO produced by skeletal muscle nNOS) could affect vascular responsiveness.     

Neurochemical plasticity of nitric oxide synthase isoforms in neurogenic detrusor overactivity after spinal cord injury

Nitric oxide (NO) participates in the neural pathways controlling the lower urinary tract (LUT). Expression of NO synthase (NOS) can be upregulated after spinal cord injury (SCI), and altered NOS activity may participate in resulting LUT dysfunction. To investigate distribution of NOS-immunoreactivity (NOS-IR) in neurons of rats following SCI and the possible effects of NOS inhibitors. Expression of neuronal and inducible NOS-IR in lumbosacral spinal cord was assessed in rats. Cystometry was performed to examine effects of intrathecal injection of NOS inhibitor. There was increased expression of neuronal NOS-IR after trauma. Maximum bladder capacity was increased by neuronal NOS (nNOS) inhibitors. Upregulation of nNOS may facilitate emergence of the spinal micturition reflex following SCI; nNOS inhibitor suppressed SCI-induced urinary incontinence by increasing bladder capacity. Our results indicate manipulation of NO production could help treat LUT dysfunction after SCI.     

Increased nitric oxide concentrations in posterior hypothalamus and central sympathetic function on nitrate tolerance following subcutaneous nitroglycerin.

The present study was to examine the distributions of nitric oxide (NO) in the brain regions and peripheral vessels following subcutaneously administered nitroglycerin (NTG) and determine the noradrenergic activity and the role of central sympathetic function in acute nitrate tolerance. Tolerance to NTG was produced by subcutaneous (sc) administration of 4.0 mg NTG as four separate hourly pulse injections of 1.0 mg each in male (5-8 months) Sprague-Dawley rats. Rats in sham-treated group received sc injections of saline. Rats were killed by sodium pentobarbital (150 mg/kg, ip) at 10 min after last sc injection. The brain, gracilis muscle, aorta, superior mesenteric artery, coronary artery, and pulmonary vessels were quickly removed. Concentrations of nitrite (NO2-), nitrate (NO3-), and total NO2- plus NO3- (NOx-) were quantified in the micropunches of the anterior hypothalamus, the posterior hypothalamus (PH), the nucleus tractus solitarius, the lateral reticular nucleus, and the vessels in a blinded fashion. The central actions of acute tolerance to NTG were also determined using blockades of sympathetic functions in conscious rats. Four separate hourly pulse sc injections of 1.0 mg NTG produced a marked shift of the dose-response curve for arterial pressure depression induced by intravenous injection of the challenge doses of NTG. The same doses of sc NTG caused increases in NOx- [92+/-16% (mean +/- SE)] and NO3- productions (77+/-15%) in the PH, but did not significantly change in other brain regions (n = 6). NOx- and NO3- productions were significantly enhanced in the superior mesenteric artery, aorta, coronary artery, and pulmonary vessels following sc NTG, but were not altered in gracilis muscle by the treatment. The tolerance responses to arterial pressure depression were attenuated by intravenous administration of either prazosin (300 microg/kg), an alpha1-adrenoceptor antagonist, or chlorisondamine (10 mg/kg), a sympathetic ganglion blockading agent (n = 5-6). The results suggest that acute NTG tolerance predominately increases NO production in the PH. NO production was also markedly enhanced in the large and middle vessels but not in small vessels during acute NTG tolerance. The arterial pressure tolerance to NTG was reversed by blockade of central sympathetic function. We conclude that NO formation is increased in the PH following systemically administered NTG and NO in the PH may facilitate central sympathetic functions which contribute to nitrate tolerance.     

Contributions of nitric oxide synthase isozymes to exhaled nitric oxide and hypoxic pulmonary vasoconstriction in rabbit lungs

We investigated the source(s) for exhaled nitric oxide (NO) in isolated, perfused rabbits lungs by using isozyme-specific nitric oxide synthase (NOS) inhibitors and antibodies. Each inhibitor was studied under normoxia and hypoxia. Only nitro-L-arginine methyl ester (L-NAME, a nonselective NOS inhibitor) reduced exhaled NO and increased hypoxic pulmonary vasoconstriction (HPV), in contrast to 1400W, an inhibitor of inducible NOS (iNOS), and 7-nitroindazole, an inhibitor of neuronal NOS (nNOS). Acetylcholine-mediated stimulation of vascular endothelial NOS (eNOS) increased exhaled NO and could only be inhibited by L-NAME. Selective inhibition of airway and alveolar epithelial NO production by nebulized L-NAME decreased exhaled NO and increased hypoxic pulmonary artery pressure. Immunohistochemistry demonstrated extensive staining for eNOS in the epithelia, vasculature, and lymphatic tissue. There was no staining for iNOS but moderate staining for nNOS in the ciliated cells of the epithelia, lymphoid tissue, and cartilage cells. Our findings show virtually all exhaled NO in the rabbit lung is produced by eNOS, which is present throughout the airways, alveoli, and vessels. Both vascular and epithelial-derived NO modulate HPV.     

Neuronal and Endothelial Nitric Oxide Synthases in the Paraventricular Nucleus Modulate Sympathetic Overdrive in Insulin-Resistant Rats

A central mechanism participates in sympathetic overdrive during insulin resistance (IR). Nitric oxide synthase (NOS) and nitric oxide (NO) modulate sympathetic nerve activity (SNA) in the paraventricular nucleus (PVN), which influences the autonomic regulation of cardiovascular responses. The aim of this study was to explore whether the NO system in the PVN is involved in the modulation of SNA in fructose-induced IR rats. Control rats received ordinary drinking water, whereas IR rats received 12.5% fructose-containing drinking water for 12 wks to induce IR. Basal SNA was assessed based on the changes in renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) in response to chemicals administered to the PVN. We found an increased plasma norepinephrine level but significantly reduced NO content and neuronal NOS (nNOS) and endothelial NOS (eNOS) protein expression levels in the PVN of IR rats compared to Control rats. No difference in inducible NOS (iNOS) protein expression was observed between the two groups. In anesthetized rats, the microinjection of sodium nitroprusside (SNP), an NO donor, or Nω-nitro-L-arginine methyl ester (L-NAME), a non-selective inhibitor of NOS, into the PVN significantly decreased and increased basal SNA, respectively, in both normal and IR rats, but these responses to SNP and L-NAME in IR rats were smaller than those in normal rats. The administration of selective inhibitors of nNOS or eNOS, but not iNOS, to the PVN significantly increased basal SNA in both groups, but these responses were also smaller in IR rats. Moreover, IR rats exhibited reduced nNOS and eNOS activity in the PVN. In conclusion, these data indicate that the decreased protein expression and activity levels of nNOS and eNOS in the PVN lead to a reduction in the NO content in the PVN, thereby contributing to a subsequent enhancement in sympathoexcitation during IR.     

Interaction between endogenous nitric oxide and carbon monoxide in the pathogenesis of recurrent febrile seizures

The aim of the study was to investigate the interaction between nitric oxygenase (NOS)/nitric oxide (NO) and heme oxygenase (HO)/carbon monoxide (CO) system in the pathogenesis of recurrent febrile seizures (FS). On a rat model of recurrent FS, the ultrastructure of hippocampal neurons was observed under electron microscopy, and expression of neuronal NOS (nNOS) in hippocampus and NO formation in plasma were examined after treatment with ZnPP-IX, an HO-1 inhibitor. In the ultrastructure of hippocampal neurons, the expression of HO-1 in hippocampus and CO formation in plasma were examined after treatment with L-NAME, a NOS inhibitor. We found that hippocampal neurons were injured after recurrent FS. The gene and protein expression of nNOS and HO-1 increased markedly in hippocampus in FS rats, while CO formation in plasma increased markedly and the concentration of NO in plasma increased slightly. ZnPP-IX could worsen the neuronal damage of recurrent FS rats. However, it further increased the expression of nNOS and endogenous production of NO obviously. L-NAME alleviated the neuronal damage of recurrent FS rats, but decreased the expression of HO-1 and CO formation. The results of this study suggested that endogenous NOS/NO and HO/CO systems might interact with each other and therefore play an important regulating role in recurrent FS brain damage.     

Influence of estradiol on NADPH diaphorase/neuronal nitric oxide synthase activity and colocalization with progesterone or type II glucocorticoid receptors in ovine hypothalamus

Nitric oxide (NO) has been shown to play an important role in both the neuroendocrine reproductive and stress axes, which are closely linked. Because progesterone (P4) receptors (PRs) and glucocorticoid receptors (GRs) are not found in GnRH neurons and the NOergic system has been implicated in the control of GnRH secretion, this study aimed to ascertain whether steroids altered the NOergic system. Our first objective was to map the distribution of NO synthase (NOS) cells in the ovine preoptic area (POA) and hypothalamus and to determine whether NOS activity is enhanced by estradiol (E2) treatment. Using NADPH diaphorase (NADPHd) histochemistry, we found that NADPHd-positive neurons were spread throughout the ovine POA and hypothalamus, and that all NADPHd cells were immunoreactive for NOS. In response to estradiol, a significant increase in the number of NADPHd cells was noted only in the ventrolateral region of the ventromedial nucleus (VMNvl), with no significant difference in the POA or arcuate nucleus. Progesterone and glucocorticoid receptors were colocalized with NADPHd reactive neurons in the POA, arcuate nucleus, and VMNvl of ewes in both treatment groups. In ewes receiving estradiol, the number of NADPHd-positive cells containing steroid receptors in the POA (PR, 81%; GR, 79%) and arcuate nucleus (PR, 89%; GR, 84%) was similar, but in the VMNvl, fewer NADPHd-positive cells contained GR (PR, 88%, GR, 31%). These data show that estradiol up-regulates NOS activity in a site-specific manner and that the influence and possible interaction of progesterone and corticosteroids on NO producing cells may differ according to the neural location.     

Acute depletion of heme by succinylacetone alters vascular responses but does not induce hypertension

Heme plays a critical role in blood pressure regulation because it is required by a number of enzymes that synthesize vascular modulators, including nitric oxide (NO), carbon monoxide (CO), guanosine 3',5'-cyclic monophosphate (cGMP), endothelium-derived hyperpolarizing factor (EDHF), and prostacyclin. The goal of this study was to examine the vascular effects of a short-term depletion of heme achieved through administration of the heme-synthesis inhibitor succinylacetone (SA), an irreversible inhibitor of aminolevulinic acid dehydratase (ALAD). Rats were depleted of heme by using a 4-day treatment with SA. This treatment impacted hemoenzyme function, decreasing renal nitric oxide synthase (NOS) activity (as indicated by decreased in vitro NOS activity), and increasing kidney microsomal heme oxygenase (HO) activity by 27%. SA treatment also resulted in enhanced reduction in blood pressure after infusions of exogenous NO donor MAHMA NONOate (at high dose) and acetylcholine (at low doses). Nevertheless, this SA treatment was insufficient to produce an overt elevation of basal arterial pressure. This latter lack of effect may be the result of multiple compensatory mechanisms for the regulation of blood pressure.     

Progressive dysfunction of nitric oxide synthase in a lamb model of chronically increased pulmonary blood flow: a role for oxidative stress

Cardiac defects associated with increased pulmonary blood flow result in pulmonary vascular dysfunction that may relate to a decrease in bioavailable nitric oxide (NO). An 8-mm graft (shunt) was placed between the aorta and pulmonary artery in 30 late gestation fetal lambs; 27 fetal lambs underwent a sham procedure. Hemodynamic responses to ACh (1 microg/kg) and inhaled NO (40 ppm) were assessed at 2, 4, and 8 wk of age. Lung tissue nitric oxide synthase (NOS) activity, endothelial NOS (eNOS), neuronal NOS (nNOS), inducible NOS (iNOS), and heat shock protein 90 (HSP90), lung tissue and plasma nitrate and nitrite (NO(x)), and lung tissue superoxide anion and nitrated eNOS levels were determined. In shunted lambs, ACh decreased pulmonary artery pressure at 2 wk (P < 0.05) but not at 4 and 8 wk. Inhaled NO decreased pulmonary artery pressure at each age (P < 0.05). In control lambs, ACh and inhaled NO decreased pulmonary artery pressure at each age (P < 0.05). Total NOS activity did not change from 2 to 8 wk in control lambs but increased in shunted lambs (ANOVA, P < 0.05). Conversely, NO(x) levels relative to NOS activity were lower in shunted lambs than controls at 4 and 8 wk (P < 0.05). eNOS protein levels were greater in shunted lambs than controls at 4 wk of age (P < 0.05). Superoxide levels increased from 2 to 8 wk in control and shunted lambs (ANOVA, P < 0.05) and were greater in shunted lambs than controls at all ages (P < 0.05). Nitrated eNOS levels were greater in shunted lambs than controls at each age (P < 0.05). We conclude that increased pulmonary blood flow results in progressive impairment of basal and agonist-induced NOS function, in part secondary to oxidative stress that decreases bioavailable NO.     

Neuronal NO modulates spontaneous and ANG II-stimulated fetal swallowing behavior in the near-term ovine fetus

Spontaneous fetal swallowing occurs at a markedly higher rate compared with spontaneous adult drinking activity. This high rate of fetal swallowing is critical for amniotic fluid volume regulation. Central NO is critical for maintaining the normal rate of fetal swallowing, as nonselective inhibition of NO (with central N(G)-nitro-L-arginine methyl ester) suppresses spontaneous and angiotensin II (ANG II)-stimulated swallowing. We sought to differentiate the contributions of central endothelial vs. neuronal NO in the regulation of spontaneous and stimulated fetal swallowing, using a selective neuronal NO synthase (nNOS) inhibitor. Six time-dated pregnant ewes and fetuses were chronically prepared with fetal vascular and intracerebroventricular (i.c.v.) catheters and electrocorticogram (ECoG) and esophageal electromyogram electrodes and studied at 130 +/- 1 days of gestation. After an initial 2-h baseline period (0-2 h), the selective nNOS inhibitor N-propyl-L-arginine (NPLA) was injected i.c.v. (2-4 h). At 4 h, the dose of NPLA was repeated, together with ANG II, and fetal swallowing was monitored for a final 2 h. Four fetuses also received an identical control study (on an alternate day) in which NPLA was replaced with artificial cerebrospinal fluid (aCSF). Suppression of nNOS by i.c.v. NPLA significantly reduced mean (+/- SE) spontaneous fetal swallowing (1.35 +/- 0.12 to 0.50 +/- 0.07 swallows/min; P < 0.001). Injection of ANG II in the presence of NPLA had no dipsogenic effect on fetal swallowing (0.68 +/- 0.09 swallows/min). In the aCSF study, i.c.v. aCSF did not change fetal swallowing (0.93 +/- 0.10 vs. 0.95 +/- 0.09 swallows/min), whereas i.c.v. ANG II resulted in a significant increase in the rate of fetal swallowing (2.0 +/- 0.04 swallows/min; P = 0.001). We speculate that the suppressive dipsogenic effects of central NPLA indicate that spontaneous and ANG II- stimulated fetal swallowing is dependent on central nNOS activity.     

Specific inhibition of nitric oxide synthases at different time points in a murine model of pulmonary sepsis

Excessive production of nitric oxide (NO) by NO synthase (NOS) and a subsequent oxidative stress reaction are thought to be critically involved in the pathophysiology of sepsis. Previous studies suggested that NO production by neuronal NOS (nNOS) and inducible NOS (iNOS) is implemented in the disease process at different time points after the injury. Here we tested the roles of selective pharmacological inhibition of nNOS and iNOS at different time points in a murine model of pulmonary sepsis. The injury was induced by intranasal administration of live Pseudomonas aeruginosa (3.2 × 10⁷ colony-forming units) in C57BL/6 wild-type mice. The animals received no treatment (control) or treatment with a specific nNOS inhibitor (4 or 8 h), iNOS inhibitor (4 or 8 h), or non-specific NOS inhibitor (4 or 8 h). In controls, the injury was associated with excessive releases of pro-inflammatory cytokines in the plasma, enhanced tissue lipid peroxidation, and decreased survival. Non-specific NOS inhibition at either time point did not influence survival and was not further investigated. While nNOS inhibition at 4 h was associated with a trend toward improved survival and significantly reduced contents of lung nitrite/nitrate (NO_x) and liver malondialdehyde, the blockade of nNOS at 8 h had no effect on these parameters. In contrast, early iNOS inhibition was associated with a trend toward decreased survival and no effects on lung NO_x and liver malondialdehyde contents, whereas later iNOS blockade was associated with decreased malondialdehyde content in liver homogenates. In conclusion, pulmonary sepsis in mice may be beneficially influenced by specific pharmacological nNOS inhibition at an earlier time point and iNOS inhibition at a later time points post-injury. Future investigations should identify the time changes of the expression and activation of NOS isoforms.     

Localization of nitric oxide synthase-like immunoreactivity in the developing nervous system of the snail Ilyanassa obsoleta

Production of nitric oxide (NO), an evolutionarily conserved, intercellular signaling molecule, appears to be required for the maintenance of the larval state in the gastropod mollusc Ilyanassa obsoleta. Pharmacological inactivation of endogenous nitric oxide synthase (NOS), the enzyme that generates NO, can trigger metamorphosis in physiologically competent larvae of this species. Neuropils in the brains of these competent larvae display histochemical reactivity for NADPH diaphorase (NADPHd), an indication of neuronal NOS activity. The intensity of NADPHd staining is greatest in the neuropil of the apical ganglion (AG), a region of the brain that contains the apical sensory organ and that innervates the bilobed ciliated velum, the larval swimming and feeding organ. Once metamorphosis is initiated, the intensity of NADPHd staining in the AG and presumably, concomitant NO production, decline. The AG is finally lost by the end of larval metamorphosis, some 4 days after induction. To determine if the neurons of the AG are a source of larval NO, we conducted immunocytochemical studies on larval Ilyanassa with commercially available antibodies to mammalian neuronal NOS. We localized NOS-like immunoreactivity (NOS-IR) to 3 populations of cells in competent larvae: somata of the AG and putative sensory neurons in the edge of the mantle and foot. Immunocytochemistry on pre-competent larvae demonstrated that numbers of NOS-IR cells in the AG increase throughout the planktonic larval stage.