Brain Nitric Oxide Synthase Research Articles

Expression of nitric oxide synthase isoforms in human pregnant myometrium at term

To determine whether nitric oxide synthase isoforms are expressed in human myometrium, to define their localization, and to examine the possibility that one or more may play a role in initiating labor. Human myometrial biopsies were collected from term laboring and nonlaboring patients. The mRNA expression was assessed using in situ hybridization. The relative expression levels were also assessed by Affymetrix GeneChip hybridization. The protein expression was determined using Western blot analysis, and localization was shown using immunostaining. Both inducible nitric oxide synthase and brain nitric oxide synthase were expressed in myometrial cells and blood vessel endothelial cells. Endothelial nitric oxide synthase was expressed exclusively in blood vessel endothelial cells. There were no differences in distribution or quantity of nitric oxide synthase expression between term labor and nonlabor myometrium. All three isotypes of nitric oxide synthases were expressed in human pregnant term uterus. Their expression did not change in the myometrium of the laboring uterus.

Use of an electrochemical nitric oxide sensor to detect neuronal nitric oxide production in conscious, unrestrained rats

Introduction: Amperometric sensors that directly measure nitric oxide (NO) are readily employed in pharmacologic research. While several of these sensors have been developed, none has been investigated for use in conscious, freely moving animals. An approach was developed and validated for real-time quantitation of neuronal NO production in rats without restricting locomotor activity or other potentially useful behavioral endpoints. Methods: Male Sprague–Dawley rats were equipped with a femoral vein or intraperitoneal cannula. A guide cannula and an amperometric NO sensor were placed in the left and right hippocampus, respectively. Following recovery, rats received a 6-h intravenous infusion of saline, l-arginine (an NO precursor; 250 or 500 mg/kg/h), or incremental intraperitoneal 7-nitroindazole (an NO synthase inhibitor; 200-mg/kg loading dose and 100 mg/kg every 2 h). The sensor recorded NO production continuously and microdialysis samples were collected incrementally throughout the experiment. Griess assay analysis of microdialysate samples was compared to sensor readings in vivo. In vitro degradation of an NO donor also was used to validate sensor performance. Results: Exogenous administration of l-arginine resulted in incremental increases in the neuronal NO signal. A reduction in NO production was observed during administration of 7-nitroindazole, a selective neuronal NO synthase inhibitor. A significant correlation was observed in vitro between the Griess assay analysis, an indirect analytical approach, and the NO sensor readings. The lack of a strong correlation between these measures in vivo is consistent with the indirect nature of the Griess assay. Discussion: The current approach allows real-time determination of neuronal NO production in unrestrained rats. This model will be invaluable in evaluating pharmacologic issues regarding brain tissue NO synthesis, assessing brain NO synthase as a molecular target, and establishing the effects of pharmacologic agents on neuronal NO production.

Decreased Expression of Brain Nitric Oxide Synthase in Macula Densa Cells and Glomerular Epithelial Cells of Rats with Mercury Chloride-Induced Acute Renal Failure

To elucidate the mechanisms responsible for the development of HgCl 2-induced acute renal failure (ARF), we examined the expression of brain type (b) nitric oxide synthase (NOS), which is involved in the generation of the vasodilator nitric oxide (NO), in the renal cortex of rats at 20 h after exposure to 7.5 mg/kg HgCl 2. Both blood urea nitrogen and serum creatinine were significantly increased in rats exposed to HgCl 2 relative to control rats, indicating the induction of ARF resulting from HgCl 2 exposure. Histopathological analysis demonstrated that, in addition to necrosis of proximal tubule epithelial cells, necrosis of macula densa cells and swelling of glomerular epithelial cells were observed in the renal cortex of rats with HgCl 2-induced ARF. Consequently, the number of pars maculata segments was decreased by 42% in rats with HgCl 2-induced ARF compared to control rats. The primary sites of bNOS mRNA and protein expression were macula densa cells and glomerular epithelial cells in the renal cortex of control rats and rats with HgCl 2-induced ARF. The abundance of the bNOS mRNA and protein was significantly decreased in rats with HgCl 2-induced ARF relative to control rats. These observations suggest that the production of the vasodilator NO derived from bNOS is decreased at the glomerulus level in the HgCl 2-induced ARF setting. Thus, the reduction in bNOS expression may in part contribute to the progression of HgCl 2-induced ARF through the deterioration of glomerular hemodynamics. In addition, the decrease in bNOS expression may be primarily the result of cell injury caused by the cytotoxic effect of HgCl 2.

Physiologic pH changes modulate calcium ion dependence of brain nitric oxide synthase in Carassius auratus

Species of the fish genus Carassius survive prolonged anoxia. Nitric oxide (NO) regulates cerebral blood flow in these fish during normoxic conditions whereas adenosine is the main vasoregulating molecule during anoxia. We investigated the calcium ion dependence of Carassius auratus brain NO synthase (NOS) as a function of pH. The physiological pH decrease from 7.2 to 6.8, which takes place during anoxia, greatly decreases NOS activity. This strong pH dependence is mainly due to variation of the calcium sensitivity of the enzyme. The EC 50 is 0.15 μM at pH 7.2 and 2.1 μM at pH 6.8 for the soluble enzyme. The particulate enzyme is also dependent on pH variations. The reduced sensitivity to calcium ions at acidic pH decreases both NO and H 2O 2 production, saving the cells by suppression of the formation of potentially toxic nitrogen and oxygen species. Modulation of NOS activity by variation of its calcium affinity within the range of physiological pH constitutes an important and rapid mechanism to control the formation of NO and H 2O 2 during normoxia–anoxia and anoxia–normoxia transitions.

Bcl-2-linked apoptosis due to increase in NO synthase in brain of SAMP10

We examined the linkage of nitric oxide (NO)-induced apoptosis to acceleration of brain aging of senescence-accelerated mouse prone 10 (SAMP10). The expression of neuronal nitric oxide synthase (nNOS) increased in the cerebral cortex of the brain of SAMP10 in an age-dependent manner and significantly higher levels of neuronal nitric oxide synthase (nNOS) were observed in both young and old SAMP10 as compared to age-matched controls. Moreover, a lower level of anti-apoptotic protein Bcl-2 and a higher level of pro-apoptotic protein cytochrome c in cytosol were observed in SAMP10 compared to the control. However, there was no significant difference in the expression of pro-apoptotic protein p53 between SAMP10 and the control. Furthermore, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL)-positive apoptotic cells were more abundant in the cerebral cortex of aged SAMP10 than in the control. The present results suggest that an age-dependent increase of NO by up-regulation of nNOS promotes the Bcl-2-linked apoptosis in the cerebral cortex of SAMP10 and this may cause the acceleration of brain aging of SAMP10.

Effect of Intrauterine Ischemia-Hypoxia on Endothelial Nitric Oxide Synthase in Fetal Brain in Rats

Nitric oxide (NO) plays important roles in the regulation of cerebral blood flow (CBF) in the perinatal period. The present study was undertaken to investigate the influence of intrauterine ischemia-hypoxia (IH) on the expression of endothelial NO synthase (eNOS) in fetal brains in rats. To induce intrauterine IH insult, bilateral uterine arteries were ligated on day 17 of pregnancy. Activities and mRNA levels in the brain of the fetuses were examined on days 17–21 of pregnancy. The IH insult caused the increase in both activities and mRNA levels of eNOS on day 21 of pregnancy, whereas there were no significant changes in neuronal NOS mRNA levels. Endothelial NOS expression in the fetal brains was increased by intrauterine IH insult, suggesting that eNOS may contribute to the maintenance of CBF against ischemia or hypoxia conditions in the fetal rats.

Is dopamine the answer? Other systems in the neurochemical effects of psychostimulants

For over 20 years, the focus of studies examining the neurochemical and behavioral effects of cocaine and other psychostimulants has been on dopamine. Many behavioral studies have shown that dopamine plays an important role in the reinforcing and behavioral effects of cocaine. Cocaine binds to the dopamine transporter and inhibits dopamine uptake. While there are some effects of cocaine on dopamine receptors, dopamine levels, and the dopamine transporter, these neurochemical studies have not been able to account fully for the altered behavioral effects of cocaine following chronic cocaine administration. Recent studies by Kantak et al. have shown that the reinforcing effects of psychostimulants depend upon activation of brain nitric oxide synthase. In addition, Rocha et al. have reported that cocaine is self‐administered in animals lack dopamine transporters. This finding suggests that other neurochemical components are necessary for the reinforcing effects (and hence the abuse) of cocaine. Since cocaine binds to dopamine, norepinephrine and serotonin transporters, it is likely that a combination of effects on these systems may be responsible for the behavioral effects of cocaine. Mu‐ and kappa‐opioids regulate dopamine and serotonin and this regulation plays a role in the effects of cocaine (Izenwasser et al.). Unterwald and colleagues have shown that there are large effects of cocaine on opioid receptors and second messenger regulation. These studies show that there are interactions between multiple systems and that these interactions are important factors in the effects of abused drugs, perhaps more important than activation of dopaminergic systems alone. These findings will be discussed in terms of the implications for the development of treatments for cocaine abuse.

Presence and activity of nitric oxide synthase isoforms in ischemia-reperfusion-injured flaps.

Nitric oxide is produced from the amino acid L-arginine by nitric oxide synthase, which has three known isoforms: (1) endothelial nitric oxide synthase and (2) brain nitric oxide synthase, both of which are constitutive nitric oxide synthase; and (3) inducible nitric oxide synthase. The authors' hypothesis is that after reperfusion injury, endothelial cell dysfunction leads to disruption of nitric oxide synthase-mediated nitric oxide production and that this may in part explain the deleterious effects of ischemia-reperfusion injury on tissue survival and blood reflow in flaps. An experiment was designed to study the effects of ischemia-reperfusion injury on the bioactivity of all three isoforms of nitric oxide synthase. Buttock skin flaps and latissimus dorsi myocutaneous flaps were elevated in eight pigs. Flaps on one side of the animal were randomized to receive 6 hours of arterial ischemia, whereas flaps on the other side served as controls. At 6 hours of ischemia and at 1, 4, and 18 hours after reflow, tissue biopsy specimens were obtained and were processed for both constitutive nitric oxide synthase and inducible nitric oxide synthase enzyme activity on the basis of the L-citrulline assay. In addition, specimens were processed for Western blot analysis of the three isoforms. The authors' results revealed three key findings: first, there was a statistically significant (p < 0.001) decrease in constitutive nitric oxide synthase activity of ischemia-reperfusion-injured flaps as compared with controls in both skin and muscle for all time intervals measured. Second, Western blot analyses of endothelial nitric oxide synthase and brain nitric oxide synthase showed a significant decrease in the signal intensity in ischemic and reperfused tissue as compared with controls. Third, the inducible nitric oxide synthase isoform's activity and protein remained undetectable in both tissue types for all time points measured. The authors' data demonstrated that following ischemia-reperfusion injury in the pig flap model there was a disruption of constitutive nitric oxide synthase expression and activity, which may lead to decreased nitric oxide production. The significant decrease in nitric oxide synthase activity found in the current study may partly explain the mechanism of tissue damage in flaps subjected to ischemia-reperfusion injury. Knowledge of the kinetics of nitric oxide synthase activity under conditions of ischemia-reperfusion injury has important implications for the choice and timing of delivery of therapeutic agents whose goal is to increase the bioavailability of nitric oxide in reperfused tissue.

The complex role of nitric oxide in the regulation of glomerular ultrafiltration

The complex role of nitric oxide in the regulation of glomerular ultrafiltration. Nitric oxide is an important neurohumoral modulator of glomerular ultrafiltration and renal hemodynamics. Multiple nitric oxide synthase (NOS) isoforms are present within the kidney. However, it is difficult to discern which NOS is most active from prior studies using non-selective NOS blockers. It is recently apparent that NOS activity is important to the activity of tubuloglomerular feedback (TGF) systems, systems that relate tubular reabsorption to the regulation of glomerular ultrafiltration. Neuronal (nNOS) or brain NOS (bNOS; NOS I) is present within the macula densa, the sensing element of TGF systems. Inhibition of NOS activity and specifically bNOS enhances TGF activity. The TGF system also adapts temporally and these events appear to be dependent upon up-regulation of activity of bNOS within the kidney. Temporal adaptation occurs within one to three hours whereby activation of TGF is followed by a gradual return of the glomerular filtration rate (GFR) and renal blood flow toward normal levels. After 24 hours of benzolamide treatment and withdrawal of this agent, glomerular filtration actually increases to supranormal levels and this is prevented by inhibition of bNOS activity. Factors regulating bNOS activity have not been fully clarified. We have recently observed N-methyl-d-aspartate (NMDA) receptors within the kidney that, when inhibited, result in major reductions in renal blood flow and GFR, suggesting an important role for the NMDA receptor in regulation of renal hemodynamics. Future studies will determine whether NMDA receptor is also an important regulator of bNOS activity and the TGF system.

Developmental regulation of brain nitric oxide synthase expression in the ferret thalamic reticular nucleus

We have found that cells in the ferret thalamic reticular nucleus (TRN) express brain nitric oxide synthase (bNOS) in a transient pattern during early postnatal development. Similar to our previous findings in the lateral geniculate nucleus (LGN), bNOS expression in the TRN is first observed at postnatal day 7 (P7) and continues to P35. Quantitative measures show a significant change in the relative numbers of bNOS+ cells from P7–P35, and suggest there is a transition in morphology from a bipolar shape with two primary dendrites, to a more complex, multipolar arrangement. During TRN development, the pattern of bNOS expression shifts from the somatodendritic localization seen during the first postnatal month to expression within axon fibers in the adult. Expression of bNOS within TRN cells demonstrates an additional source of nitric oxide in the developing visual thalamus, perhaps indicating a common function for thalamic nitergic neurons as cellular mediators in the establishment of central topography both in the LGN and the TRN.

Inhibition of fibrin-induced neurogenic pulmonary edema by previous unilateral left-vagotomy correlates with increased levels of brain nitric oxide synthase in the nucleus tractus solitarii of rats

In the course of investigations on mechanisms underlying development of neurogenic pulmonary edema (NPE), we have evaluated effects of nitric oxide (NO) in the central nervous system on incidence and severity in the fibrin-induced pulmonary edema model. Rats were left-unilaterally vagotomized 1, 2 and 4 weeks before injections of fibrinogen and thrombin into the cisterna magna, after cutting the right vagus nerve, grouped as LV1W, LV2W or LV4W, respectively. The brain NO synthase (NOS) mRNA level in the left medulla oblongata was elevated in the LV2W group, compared to the control, but decreased in the LV4W rats. Incidences of pulmonary edema were 100% in the control group, decreasing to 78% in LV1W group, 17% in LV2W group, and back to 72% in LV4W group. The lung water ratio, a parameter of severity, demonstrated a similar pattern of change as the incidence. The lowered incidence and severity obtained in the LV2W group were reversed by intracisternal injection of N ω-nitro- l-arginine methyl ester ( l-NAME). From these results, we propose that an increase in nitric oxide, possibly in the nucleus tractus solitarius 2 weeks after left vagotomy, may have an inhibitory action on the development of neurogenic pulmonary edema in rats.

A brain nitric oxide synthase study in the rat: production of a nitroso-compound NA and absence of nitric oxide synthesis

The products of brain NO-synthase (NOS) were studied by different analytical techniques with the same incubation conditions. Voltammetric techniques used a micro cell containing NOS and its substrate (10 mM arginine). Using porphyrin microelectrodes with differential pulse amperometry nitric oxide (NO) was not detected when nafion membrane was present (less than 0.3 μM). Nitrite was detected with the same microelectrode without membrane (0.42 mM). Differential pulse voltammetry (DPV) with micro carbon electrode detected a nitroso-compound (NA) in reduction (1 mM) and not NO. In oxidation the observed DPV peak was due to nitrite (0.43 mM). Citrulline was detected by high performance liquid chromatography (0.51 mM). Using Diels Alder reaction in NOS preparation a NA-cycloadduct was observed by capillary electrophoresis (0.2 mM) and mass spectrometry (0.22 mM). Diels Alder reaction is the reaction of the identification of the nitroso group. NA-cycloadduct degradation by retro Diels Alder reaction gave equimolar concentrations of citrulline and nitrite without NO production. These observations lead us to affirm that NOS synthesizes NA.

Brain nitric oxides synthase in major pelvic ganglia of aged (LETO) and diabetic (OLETF) rats.

This study was conducted to evaluate the effects of aging and diabetes mellitus (DM) on brain nitric oxide synthase (bNOS) expression in major pelvic ganglia (MPG) of rats. Otsuka Long Evans Tokushima Fatty rats (12, 30, and 70 weeks old), which are genetic models with non-insulin-dependent DM (NIDDM), and age-matched nondiabetic Long Evans Tokushima Otsuka controls were used. The MPG of all rats in this study were subjected to cryo-sectioning and staining with bNOS polyclonal AB and rhodamine-conjugated rabbit IgG. Fluorescence intensities of the stained neurons were assessed in randomly selected fields per each specimen. Animals of both groups revealed significant decline in the staining intensity of their neurons with aging and the progress of DM, but diabetic rats showed more decline than controls. In conclusion, both aging and NIDDM could decrease bNOS expression in rat MPG. However, NIDDM has a more evident effect than aging on that expression. The decrease in bNOS may cause a disturbance in functions of the target pelvic structures of these ganglia under both conditions.

What is brain nitric oxide synthase doing in the kidney?

Brain, or type I, nitric oxide synthase is expressed strongly in the macula densa of the kidney. Functional studies show that it blunts the tubuloglomerular feedback response that causes vasoconstriction of the renal afferent arteriole in response to sodium chloride reabsorption at this site, and regulates renin release from the juxtaglomerular apparatus. Studies published this year have made great progress in defining the mechanism of action of type I nitric oxide synthase on tubuloglomerular feedback. Macula densa-derived nitric oxide can act via both the autocrine and probably the paracrine routes. Nitric oxide bioactivity in the juxtaglomerular apparatus is strongly curtailed by oxidative stress in hypertensive models. The signalling pathways within macula densa cells have been examined in detail. Nitric oxide synthase type I in the macula densa probably adapts renal hemodynamics and possibly renin secretion to changes in blood pressure and salt intake.

CONTRIBUTION OF CEREBRAL NITRIC OXIDE TO BLADDER OVERACTIVITY AFTER CEREBRAL INFARCTION IN RATS

We investigated the contribution of cerebral nitric oxide to neurogenic voiding dysfunction after cerebral infarction. The left mid cerebral artery in female Sprague-Dawley rats was occluded with 4-zero monofilament nylon thread. Bladder activity was monitored during infusion cystometrography. Time or dose dependent effects of intracerebral ventricular administration of the nonselective nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME), were investigated in conscious, sham operated and cerebral infarcted rats. The selective neuronal nitric oxide synthase inhibitor 1-(2-trifluoromethylphenyl) imidazole was also administered to determine the participation of nitric oxide synthase subtypes. Cross-sectional infarct area was measured and infarct volume was calculated 12 hours after mid cerebral artery occlusion. Bladder capacity was reduced by 54% 30 minutes after mid cerebral artery occlusion. L-NAME significantly increased bladder capacity in a dose and time dependent manner in cerebral infarcted rats but had no effect on sham operated rats. L-NAME (50 microg./kg.) administered 3 or 5 hours after occlusion significantly increased bladder capacity. This effect of L-NAME was reversed by injecting 250 microg. L-arginine per rat, which alone did not produce any significant change in bladder capacity in cerebral infarcted rats. Administration of 1-(2-trifluoromethylphenyl) imidazole also significantly increased bladder capacity in these rats. On the other hand, 5 microg. of the nitric oxide donor FK-409 per rat reduced bladder capacity for 10 to 15 minutes. None of the drugs affected infarct volume. These results indicate that supraspinal nitric oxide has an important role in bladder overactivity after cerebral infarction but it does not affect normal micturition in rats. This finding suggests a central mechanism sensitive to nitric oxide for bladder overactivity after cerebral infarction.

Basic classical, peptidergic and nitrergic innervation pattern of human nasal glands--a histochemical and immunohistochemical study

Seromucous glands are important components of the human nasal mucosa. The innervation patterns are relevant for understanding the control of the different physiological and pathophysiological glandular functions. Beside classic neurotransmitters some neuropeptides seem to influence the glandular secretion. Tissue samples of 35 human inferior turbinates were taken during nasal surgery and preserved. Serial cryosections or paraffin sections were cut and incubated with antibodies either to Tyrosinhydroxilase or to Vasoactive Intestinal Peptide (VIP), Calcitonin Gene-Related Peptide (CGRP) and endothelial or brain Nitric Oxide Synthase (eNOS or bNOS). AChE- and NADPH-diaphorase-histochemistry were performed. Immunoreactive nerve fibers were found in the periglandular tissue around the acini, ducts and in the periglandular connective tissue. The density of positive immunoreactive structures depended on the different antibodies. VIP was found in contact to acinus cells, CGRP in the connective tissue around glandular cells. Particular immunoreactions to VIP and CGRP-antibodies could be detected near the glandular duct system. The eNOS-reactions were found in small capillaries near the acinus cells. Immunohistochemical and histochemical methods allow a detailed marking of nerval structures in nasal mucosa. The localization of neurons with different neurotransmitters and neuropeptides in the periglandular tissue confirms the direct nerval control of the diverse glandular functions. The detection of bNOS- and NADPH-d-positive structures around glandular cells and eNOS in the endothelium of periglandular capillaries suggests that NO takes an additional part in the regulation of nasal glands.

Structural requirements of benzodiazepines for the inhibition of pig brain nitric oxide synthase

Nitric oxide synthases (NOS) are heme-containing enzymes which catalyse the oxidation of l-arginine to nitric oxide and l-citrulline. Some nitrogenated compounds have been reported to coordinate with the iron atom from the heme group, thus inhibiting NOS. 1,4-Benzodiazepines are nitrogenated compounds which have many physiological effects such as antianxiety, antiepileptic, hypnotic, and muscle relaxation properties. The aim of this paper was to measure the effect of different benzodiazepines on NOS activity in pig brain extracts. Medazepam, pinazepam, diazepam, oxazepam and alprazolam competitively inhibited NOS with IC 50 in the micromolar range. Other benzodiazepines showed no effect at concentrations as high as 200 μM. Due to the structural similarity of the benzodiazepine ring nucleus with l-arginine, we propose a benzodiazepine–enzyme interaction to explain the competitive inhibitions. By comparing benzodiazepine effects and their structures, the inhibitory effect of benzodiazepines on NOS is related to the absence of substituents on N4 and to the absence of a halogen substituent on C5 phenyl group. Although benzodiazepine’s inhibitions observed in this study are not in the physiological range in normal cases, these inhibitions could be significant in drug abuse situations and should be taken into account for the rational design of drugs which specifically inhibit NOS.

Repeated, short-term ischemia augments bradykinin-mediated opening of the blood–tumor barrier in rats with RG2 glioma

The objective of this study was to investigate the effects of repeated, short-term ischemia on bradykininmediated permeability of the blood-brain barrier (BBB) and the blood-tumor barrier (BTB). The mechanism by which bradykinin transiently opens the BTB, involves B2 receptors, Ca2+ flux, nitric oxide (NO) and cyclic GMP (cGMP). Since global and focal cerebral ischemia are known to increase levels of brain nitric oxide synthase (bNOS) and endothelial nitric oxide synthase (eNOS) we tested the hypothesis that bradykinin may increase the BTB permeability to a greater extent under ischemic rather than nonischemic conditions. The vertebral arteries in female Wistar rats were coagulated immediately after intracerebral implantation of RG2 glioma. Short-term ischemia was produced in some rats by a modification of the fourvessel occlusion procedure for incomplete forebrain ischemia, in which the common carotid arteries were clamped daily for 15 min on days 7, 8 and 9 after tumor implantation, after which reperfusion was allowed. On day 10 after tumor implantation, bradykinin (10 µg kg-1 min-1) or phosphate-buffered saline (PBS) was infused for 15 min into the right carotid artery of anesthetized, sham-operated (nonischemic controls) and ischemic rats, followed by an intravenous bolus (100 µCi kg-1) each of [14C]-iodo-antipyrine (IAP), [14C]dextran or [14C]-aminoisobutyric acid (AIB) to measure regional cerebral blood flow (rCBF), blood volume, or unidirectional transfer constant Ki, respectively, by quantitative autoradiography. A single 15-min ischemic episode significantly decreased rCBF in the tumor center (158.9 ± 17.33 in control vs. 58.78 ± 24.45 ml 100 g-1 min-1 in ischemic group; p < 0.01) and in the tumor periphery (106.82 ± 7.34 in control vs. 70.55 ± 26.66 ml 100 g-1 min-1 in ischemic group; p < 0.05). Respective mean blood volume in tumors (11.7 ± 13.3, 12.7 ± 14.0, and 13.3 ± 14.5 µlg-1) from ischemic-PBS, nonischemic-bradykinin, and ischemic-bradykinin groups, respectively, was not significantly different; mean blood volume in normal brain (3.7, 3.1 and 3.8 µlg-1) was not significantly different among these groups either. Intracarotid infusion of bradykinin following repeated ischemia significantly increased mean Ki, as compared to bradykinin infusion in nonischemic controls, in both the tumor center (36.60 ± 8.4 vs. 22.90 ± 4.61 µlg-1 min-1, p < 0.05) and in tumor periphery (17.70 ± 5.93 vs. 8.50 ± 4.42 µlg-1 min-1, p < 0.05). Mean Ki values for tumor center and tumor periphery of ischemic rats receiving intracarotid bradykinin were 3-fold greater than those of nonischemic rats infused with PBS. Immunohistochemical and Western blot analyses showed that repeated, short-term ischemia significantly increased the levels of bNOS in tumor cells and eNOS in tumor capillaries, but neither induced iNOS nor affected B2 receptor levels in tumor cells in vivo, as compared with nonischemic controls. Taken together, these results demonstrate for the first time that repeated, short-term ischemia augments bradykinin-mediated opening of the BTB. We conclude that the elevated intratumoral levels of bNOS and eNOS may 'prime' the NO generating capacity of tumor cells. Consequently, increased de novo synthesis and a correspondingly elevated concentration of NO within the tumor, therefore, may be one mechanistic explanation for the significantly increased, bradykinin-mediated BTB opening under ischemic conditions, reported here. [Neurol Res 2001; 23: 631-640]

Effects of chronic L-NAME treatment on rat focal cerebral ischemia and cerebral vasoreactivity

Nitric oxide has been shown to be involved in the regulation of cerebral blood flow and the consequences of cerebral ischemia. Short-term inhibition of its synthesis induces hypertension and increases the cortical infarct volume in focal ischemia. Our purpose was to investigate the influence of the long-term inhibition of nitric oxide synthase on infarct volume due to middle cerebral artery (MCA) occlusion and on the reactivity of cerebral arteries. Sprague Dawley rats were given N ω-nitro-L-arginine methyl ester (L-NAME) for 2 or 6 weeks and compared to untreated normotensive rats and untreated spontaneously hypertensive rats (SHRs). Brain nitric oxide synthase activity was measured by the 14C-L-arginine assay. Arterial blood pressure was measured in each group. Independently, the reactivity of MCA trees was studied in vitro by a perfusion technique. Cortical infarct volume was not significantly modified by either 2-week or 6-week L-NAME treatment, despite induced hypertension, whereas it was significantly higher in SHRs than in normotensive rats. The reactivity of the MCA tree was significantly affected by the treatment with a clearcut time-dependency. Compared to normotensive controls, contractility to noradrenaline and serotonin was reduced, more severely at 6 weeks, and while dilatation to acetylcholine and nitroprusside was moderately reduced at 6 weeks, dilatation to papaverine was then increased. A major difference of treated animals compared to SHRs was the decreased response to 5-hydroxytryptamine. We conclude that infarct expansion may be limited in treated animals by a progressive reduction in cerebral artery response to vasoconstrictory neurotransmitters, concomitant with augmented non-guanylate cyclase dilator responses (cf. papaverine) and some recovery of dilatation to acetylcholine.

Prolonged suppression of brain nitric oxide synthase activity by 7-nitroindazole protects against cerebral hypoxic–ischemic injury in neonatal rat

Nitric oxide mediates glutamate-induced excitotoxicity associated with cerebral hypoxia–ischemia through production in the brain by several isoforms of nitric oxide synthase (NOS). We examined the influence of the selective neuronal NOS inhibitor, 7-nitroindazole (7-NI), on brain NOS activity and its neuroprotective effects against cerebral hypoxic–ischemic injury in the postnatal day (PND) 7 rat. In the first set of experiments, 7-NI (50 mg/kg) administered intraperitoneally (i.p.) transiently inhibited NOS activity to 40% below the vehicle control level at 1 h after injection ( P<0.001, analysis of variance (ANOVA)). In contrast, 7-NI (100 mg/kg, i.p.) inhibited NOS activity to 56% below the control level at 1 h with prolonged suppression of NOS activity at 3, 6, 9 and 12 h after injection. Two-factor ANOVA revealed an overall effect on NOS activity of 7-NI treatment ( P<0.001) and time after injection ( P<0.001). In the second set of experiments, 7-NI (50, 100 mg/kg) or an equal volume of vehicle was administered after unilateral carotid artery ligation, but 30 min before hypoxia in PND 7 rats. 7-NI (100 mg/kg) significantly protected against cerebral hypoxic–ischemic injury (100 mg/kg of 7-NI, 1.7±1.0% damage; control, 8.7±1.6%, P<0.05). 7-NI administered 15 min after cerebral hypoxia–ischemia was not neuroprotective. The data suggest that the protective effect of 7-NI is dose dependent, and is related to the duration of suppressed NOS activity.