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Stroke. 1995;26:1079-1085

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(Stroke. 1995;26:1079-1085.)
© 1995 American Heart Association, Inc.

Articles

Cerebral Blood Flow During Inhibition of Brain Nitric Oxide Synthase Activity in Normal, Hypertensive, and Stroke-Prone Rats

Makoto Izuta, MD; Nathalie Clavier, MD; Jeffrey R. Kirsch, MD Richard J. Traystman, PhD

From the Department of Anesthesiology and Critical Care Medicine, The Johns Hopkins Medical Institutions, Baltimore, Md.


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Background and Purpose Because tonic production of nitric oxide (NO) is important in regulating cerebrovascular tone and NO may be important in the mechanism of brain injury from focal ischemia, we speculated that stroke predisposition in spontaneously hypertensive stroke-prone rats (SHR-SP) may be related to impaired tonic production of NO. This study was designed to test the hypothesis that the cerebral blood flow (CBF) response to inhibition of NO synthase in SHR-SP would be different than that observed in normal Wistar-Kyoto (WKY) rats and non–stroke-prone spontaneously hypertensive rats (SHR).

Methods Pentobarbital-anesthetized, mechanically ventilated rats were tested for CBF response to saline, 5 or 20 mg/kg IV of NG-monomethyl-L-arginine (L-NMMA), or 20 mg/kg IV of N{omega}-nitro-L-arginine (L-NA). In addition, specificity for an NO-dependent mechanism was assessed by determining the ability to reverse any alteration in CBF with L-arginine. Hemorrhage was used to minimize any increase in mean arterial blood pressure (MABP) from NO synthase inhibition. In a separate cohort of rats, differential sensitivity of NO synthase for inhibition by nitro-arginine analogues was determined.

Results Baseline MABP was greater in SHR-SP (185±3, n=38) and SHR (169±3, n=38) compared with WKY rats (101±2 mm Hg, n=38, P<.05). Baseline CBF was similar between strains; however, cerebrovascular resistance was higher in SHR-SP (2.16±0.09, n=27) and SHR (1.94±0.07, n=27) compared with WKY rats (1.23±0.06 mm Hg/mL per minute per 100 g, n=27, P<.05). CBF was unchanged with 5 mg/kg L-NMMA or with L-arginine in the absence of L-NMMA in each strain. CBF decreased similarly in SHR and SHR-SP (n=9 each) in response to 20 mg/kg L-NMMA (SHR, 85±6 to 67±6; SHR-SP, 87±7 to 69±5 mL/min per 100 g) and was completely reversed by L-arginine. CBF did not decrease with 20 mg/kg L-NMMA in WKY rats. Administration of L-NA (n=5 each) produced similar reduction of CBF (WKY rats, 67±6%; SHR, 49±9%; SHR-SP, 61±6% of baseline) and inhibition of NO synthase in each strain ({approx}80% inhibition).

Conclusions There was no difference in the cerebrovascular response to NO synthase inhibition in SHR-SP and non–stroke-prone SHR. Therefore, it is unlikely that an altered sensitivity of NO synthase to inhibition can explain predisposition to stroke in SHR-SP.


Key Words: cerebral blood flow • cerebral ischemia • nitric oxide • rats


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
There are several different scenarios by which an alteration in a nitric oxide (NO)–mediated cerebral vasodilation can be implicated in the mechanism of cerebral injury in spontaneously hypertensive stroke-prone rats (SHR-SP). One possible scenario involves insufficient tonic production of NO, which could result in increased basal cerebral vascular tone and focal ischemia. This possibility is supported by the finding that NO appears to contribute to vasodilation under both basal and pathological conditions.1 2 3 In addition, NO inhibits platelet adhesion and aggregation and platelet and monocyte activation. Therefore, decreased tonic NO production may cause platelet adhesion and aggregation, which may be a cause of stroke in SHR-SP.4 Evidence supporting decreased NO-mediated mechanisms in SHR-SP includes the findings that in SHR-SP, acetylcholine-induced production of cGMP is reduced in isolated cerebral blood vessels5 and bradykinin-induced NO production is reduced from cultured endothelium.6 Accumulation of monocytes and platelets in cerebral perforating arteries and morphological alterations of cerebral vessels, including endothelium, in SHR-SP have also been reported.4 7 8 Decreased NO availability may be due to decreased production by NO synthase or increased consumption.9 We have recently found that whole-brain NO synthase activity, measured under optimal conditions, is not impaired in SHR-SP compared with control Wistar-Kyoto (WKY) or non–stroke-prone spontaneously hypertensive rats (SHR).10 If decreased NO production or increased NO consumption was the cause of increased incidence of stroke in SHR-SP, we would expect less reduction in cerebral blood flow (CBF) after administration of the pharmacological NO synthase inhibitors NG-monomethyl-L-arginine (L-NMMA) or N{omega}-nitro-L-arginine (L-NA).

Another possible scenario for the involvement of NO in the predisposition to brain injury in SHR-SP involves increased production of NO and production of other toxic metabolites in the setting of decreased cerebral perfusion. Evidence for increased production of NO by SHR-SP compared with WKY rats has recently been demonstrated in vascular smooth muscle that has been exposed to ultraviolet light.11 Consistent with a role for NO in the mechanism of brain injury from ischemia, several studies have demonstrated that inhibiting production of NO may improve outcome from either permanent12 or transient13 focal ischemia in cats. The exact mechanisms involved in NO-mediated brain injury have not been defined, but it has been hypothesized that increased production of excitatory amino acids during ischemia results in increased intracellular calcium and stimulation of NO synthase to produce excessive amounts of NO.14 Once formed, NO is believed to react with other metabolites (eg, superoxide anion) to produce toxic byproducts.9 If this mechanism is important in the production of brain injury in SHR-SP, we would expect a greater reduction in CBF during NO synthase inhibition with L-NA or L-NMMA.

The present study was designed to test whether differences in cerebral responses to NO synthase inhibition with either L-NMMA or L-NA correlate with the differences in predisposition to stroke in three related strains of rat: control (WKY), SHR, and SHR-SP. Sensitivity of brain NO synthase to inhibition was determined by measuring the effect of intravenous L-NMMA and L-NA on CBF and maximal NO synthase activity measured in brain biopsy samples under optimal conditions in vitro.


*    Materials and Methods
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 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
This study was conducted in accordance with National Institutes of Health guidelines for the use of experimental animals and the protocols approved by the Institutional Animal Care and Use Committee.

A colony of SHR-SP was bred in our institution from three pairs of male and female rats obtained from the National Institutes of Health (Laboratory Sciences Section, Veterinary Resources Program, National Center for Research Resources, Bethesda, Md). For each SHR-SP, age-matched SHR and WKY rats were obtained (Charles River, Kingston, NY) at the time of weaning (3 to 4 weeks of age). All rats were kept in the same virus-free environment. All but one group of rats was maintained on a regular diet (NIH 31, Zeigler Bros, Inc) with water ad libitum. One group was fed a Japanese-style diet (Zeigler Bros, Inc) with saline for drinking from the time of weaning until the time of measurement of brain NO synthase activity. Each group of rats was 16 to 20 weeks old at the time of the CBF and brain NO synthase study.

Genetic integrity in the three strains was previously assessed.10 In this previous study we only evaluated basal and calcium-stimulated NO synthase activity in the three strains of rats.10 In the present study two cohorts of animals were studied. In the first protocol we evaluated the effect of systemically administered L-NMMA or L-NA on brain NO synthase activity measured in brain biopsy samples in vitro under optimal conditions. In the second protocol we evaluated the effect of systemically administered L-NMMA or L-NA on regional CBF.

In both protocols, rats were anesthetized with sodium pentobarbital (65 mg/kg IP) and their lungs ventilated by a tracheostomy with oxygen-enriched air (FIO2, {approx}0.3). Polyethylene catheters (PE-50) were placed into the tail artery for measurement of mean arterial blood pressure (MABP) and into the abdominal aorta via the femoral artery for controlled hemorrhage (blood pressure control) during administration of L-NA or L-NMMA and to obtain the microsphere reference sample (CBF measurement protocol). A catheter (PE-50) placed into the femoral vein was used for administration of fluid, blood, and drugs as needed throughout each protocol. In the CBF protocol, radiolabeled microspheres were injected by means of a catheter (PE-10) placed in the left ventricle via the right subclavian artery.

Thermistors were placed into the rectum as an estimate of core temperature and into the temporalis muscle as an estimate of brain temperature. Both temperatures were maintained at 37°C to 38°C with the use of a heating pad and heat lamp. After completion of surgery, pancuronium bromide (0.1 mg/kg IV) was administered.

Arterial blood pressure was continuously monitored. Arterial pH, PaCO2, and PaO2 were measured with a self-calibrating Radiometer electrode system (ABL 3). Hemoglobin and arterial oxygen content were measured with a hemoximeter (model OSM3, Radiometer). Blood glucose was measured with a glucose analyzer (model 2300A, Yellow Springs Instruments).

In the CBF measurement protocol, regional CBF was measured with radiolabeled microspheres (16±0.5 µm diameter; Du Pont–NEN Products) by means of the reference withdrawal method.15 16 Three radioactive isotopes (153Gd, 113Sn, and 46Sc) were injected in random sequence in each animal. Approximately 0.2 million microspheres in 0.3 mL saline were injected into the left ventricle during a 20-second period. The microsphere reference sample was withdrawn from the abdominal aorta catheter at a rate of 0.68 mL/min starting 0.5 minutes before the microsphere injection and ending 1.5 minutes after the injection was completed. Heparinized blood from a donor of the same strain was transfused into the femoral vein at 0.68 mL/min simultaneously with the arterial reference withdrawal.

In the brain NO synthase activity protocol, NO synthase activity was determined with modification of the techniques described by Dwyer et al17 and Bredt and Snyder18 before and after the administration of 20 mg/kg of either L-NMMA or L-NA. Brain biopsy samples ({approx}50 mg each) were obtained from parietal cortex through a previously constructed craniectomy site. In all experiments and for each tissue studied, samples from age-matched rats of each strain were always processed in parallel. Samples were assayed in vitro for NO synthase activity with the use of modifications of previously described techniques18 by determining the conversion of [14C]L-arginine to [14C]L-citrulline, the formation of which is stoichiometric with NO synthesis.

Briefly, biopsy samples were quickly rinsed in ice-cold buffer (50 mmol/L Tris-HCl [pH 7.4] and 2 mmol/L EDTA) and placed on ice for processing. Samples were sonicated in 20 vol (wt/vol) of buffer and centrifuged at 10 000g for 15 minutes at 4°C, and the supernatant was assayed in duplicate. The reaction, conducted at 20°C, was initiated by adding 25 µL of brain supernatant to 100 µL of reaction mixture containing 1 µmol/L [14C]L-arginine, 1 mmol/L NADPH, and 1 mmol/L CaCl2. The reaction was terminated after 30 minutes by adding 2 mL of stop buffer (30 mmol/L HEPES [pH 5.2] and 3 mmol/L EDTA). [14C]L-Citrulline was eluted on chromatography columns (resin AG-50WX8, Na+ form, pH 7.0) and quantified by liquid scintillation spectroscopy (Beckman LS 1800). Total citrulline recovered was calculated from specific activity of the [14C]L-arginine, correcting for counting efficiency, and was expressed as picomoles per minute per milligram protein. To assess that citrulline production was due to NO synthase activity, parallel samples were processed in the presence of 100 µmol/L N{omega}-nitro-L-arginine methyl ester (L-NAME), demonstrating complete (>99%) inhibition of L-arginine conversion. Protein concentration was measured by the method of Bradford.19 Values are expressed in picomoles per milligram protein per minute.

In the brain biopsy protocol, we evaluated the effect of L-NMMA (20 mg/kg IV in 1 mL saline) in WKY rats, SHR, SHR-SP, and SHR-SP fed a Japanese-style diet for 12 weeks and L-NA (20 mg/kg IV in 1 mL saline) in WKY rats, SHR, and SHR-SP. In each of these groups physiological variables were recorded and a baseline biopsy sample obtained 30 minutes after surgery. Immediately after hemostasis was obtained at the biopsy site, each animal received a 1-mL intravenous bolus of either saline, L-NMMA, or L-NA. MABP was maintained at baseline values by controlled hemorrhage after drug administration. Physiological variables were recorded and a second biopsy sample obtained from the parietal cortex contralateral to the first biopsy 20 minutes after administration of each drug.

In the CBF measurement protocol, we evaluated the effect of saline, L-NMMA (5 or 20 mg/kg IV in 1 mL saline), or L-NA (20 mg/kg IV in 1 mL saline) and reversal with L-arginine (200 mg/kg IV in 1 mL saline) in WKY rats, SHR, and SHR-SP. Baseline physiological variables were recorded and CBF measurements obtained in each species 30 minutes after the completion of surgery. Baseline measurements were followed by a 1-mL intravenous injection that contained either saline, L-NMMA, or L-NA. After drug administration, MABP was maintained at baseline values by controlled hemorrhage into heparinized syringes. CBF and all physiological variables were measured 20 minutes later. In each group, we then administered L-arginine and after 20 minutes repeated measurements of physiological variables and CBF.

At the end of each protocol, the rat was killed with intravenous potassium chloride. The brain was removed and sectioned to determine blood flow to whole brain and to cerebrum. After the tissue was sectioned, it was weighed and placed in 15-mL poly-Q vials for analysis in an autogamma scintillation spectrometer (Minaxi Auto-Gamma 5000 series, Packard Instruments). The energy windows were set (in kiloelectron volts) at 68 to 170 for 153Gd, 360 to 440 for 113Sn, and 830 to 1200 for 46Sc. The overlap of activity from high-energy isotopes into low-energy windows was corrected by differential spectroscopy. Blood flows are expressed in milliliters per minute per 100 g tissue by correcting for tissue weight. Cerebrovascular resistance (CVR; MABP/blood flow to cerebrum) was calculated at the time of each CBF measurement.

Each variable is expressed as the mean±SE. Within each protocol, the effect of species and drug treatment for each variable was determined by two-way ANOVA. If a significant drug effect was demonstrated, subsequent one-way ANOVA (repeated measures) was performed to determine the effect of L-NMMA or L-NA and L-arginine on the individual variable. If a significant group effect or a groupxdrug interaction was demonstrated, one-way ANOVA was performed for the individual treatment between strains. Post hoc comparisons were made with the Newman-Keuls test. Statistical significance was declared at P<=.05.


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 
Body weight was similar for WKY rats (442±5 g, n=38) and SHR (413±5 g, n=38), and both were greater than the body weight for SHR-SP (353±3 g, n=38). Each of these groups weighed more than SHR-SP fed a Japanese-style diet (248±14 g, n=6). The TableDown demonstrates MABP in each protocol for each strain. For each protocol, MABP was similar in SHR and SHR-SP, which were both greater than in WKY rats. In each strain MABP was well controlled at values similar to baseline throughout the protocol. Throughout the protocols arterial hemoglobin ranged between 13 and 15 g/dL, pH between 7.3 and 7.5, PaCO2 between 31 and 39 mm Hg, PaO2 between 160 and 215 mm Hg, and glucose between 66 and 110 mg/dL. There were no physiologically significant differences for any of these variables between strains or as a result of drug treatment in any of the protocols.


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  		Table 1. Mean Arterial Blood Pressure in Each of the Strains During Treatment With Saline or Nitric Oxide Synthase Inhibitor Followed by L-Arginine

Fig 1Down demonstrates the effect of 20 mg/kg IV L-NMMA or 20 mg/kg L-NA on brain NO synthase activity. Baseline NO synthase activity was similar between groups (WKY rats, 3.27±0.46 [n=11]; SHR, 3.43±0.49 [n=11]; SHR-SP, 3.34±0.51 [n=11]; SHR-SP on a Japanese-style diet, 3.65±0.76 pmol/mg protein per minute [n=6]). Intravenous treatment with 20 mg/kg L-NMMA reduced NO synthase activity in any strain. In contrast, intravenous treatment with 20 mg/kg L-NA produced a profound reduction in NO synthase activity that was similar between groups.



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  		Figure 1. Bar graphs show nitric oxide synthase (NOS) activity presented as a percentage of baseline values in rats treated with 20 mg/kg IV of NG-monomethyl-L-arginine (L-NMMA) (top) and rats treated with 20 mg/kg IV of N{omega}-nitro-L-arginine (L-NA). Groups tested included Wistar-Kyoto (WKY) rats, spontaneously hypertensive rats (SHR), spontaneously hypertensive stroke-prone rats (SHR-SP), and SHR-SP fed a Japanese-style diet and salt loaded (SHR-SP DIET). Values are mean±SE. *P<=.05 for basal NOS activity vs post–L-NMMA or post–L-NA values.

Baseline blood flow was similar between groups (eg, forebrain: WKY rats, 84±4; SHR, 87±4; SHR-SP, 89±4 mL/min per 100 g [n=27 each]). However, baseline CVR was highest in SHR-SP (2.16±0.09), next highest in SHR (1.94±0.07), and least in WKY rats (1.23±0.06 mm Hg/mL per minute per 100 g [n=27 each]). Fig 2Down demonstrates CBF and CVR response to NO synthase inhibition. Treatment with saline or 5 mg/kg L-NMMA did not result in a change in CBF or CVR in any strain. Administration of L-arginine produced no change in CBF or CVR compared with values obtained after saline administration. Treatment with 20 mg/kg L-NMMA did not alter CBF in WKY rats but produced a similar small reduction of CBF in SHR and SHR-SP. CVR increased in response to 20 mg/kg L-NMMA to a similar extent in all strains. In each strain administration of L-arginine resulted in return of CBF and CVR to values that were not different from baseline. Administration of 20 mg/kg L-NA produced a similar reduction in CBF and an increase in CVR in all strains. For both CBF and CVR the changes were more pronounced after 20 mg/kg L-NA than with 20 mg/kg L-NMMA, which correlated with the degree of NO synthase inhibition produced by each of these drugs. In each strain administration of L-arginine increased CBF and decreased CVR compared with the post–L-NA values. In WKY rats both CBF and CVR returned to values similar to baseline after treatment with L-arginine. In SHR, after treatment with L-arginine CBF remained lower than baseline (72±10% of baseline), but CVR was not different from baseline (147±22% of baseline). In SHR-SP CBF remained lower than (76±5% of baseline) and CVR remained higher than (130±10% of baseline) baseline values after treatment with L-arginine. Between-group analysis did not reveal any difference between strains in degree of vasoconstriction produced by L-NA or subsequent reduction in vascular tone after L-arginine.



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  		Figure 2. Bar graphs show cerebral blood flow (CBF; top) and cerebral vascular resistance (CVR; bottom) expressed as a percentage of baseline value. Each rat was treated with either saline (black bars), 5 (narrowly striped bars) or 20 (widely striped bars) mg/kg of NG-monomethyl-L-arginine (L-NMMA), or 20 mg/kg IV of N{omega}-nitro-L-arginine (L-NA) (white bars). Groups tested included Wistar-Kyoto (WKY) rats, spontaneously hypertensive rats (SHR), and spontaneously hypertensive stroke-prone rats (SHR-SP). Values are mean±SE. *P<=.05 for basal value vs post–drug treatment value.


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
In this study we confirmed that baseline NO synthase activity is similar among WKY rats, SHR, and SHR-SP10 and added the finding that, although salt loading and feeding SHR-SP a Japanese-style diet predispose these animals to stroke,20 cortical NO synthase activity is not changed. There was no difference between strains in sensitivity of NO synthase for inhibition by L-NMMA or L-NA. In all strains L-NA was a much more potent inhibitor of in vitro NO synthase activity than L-NMMA, which was correlated with a greater effect of L-NA on basal cerebral vascular tone.

The mechanisms involved in predisposition to stroke in SHR-SP have not been clearly defined. Some investigators have suggested that stroke predisposition in spontaneous hypertension may be related to impaired endothelium-dependent vasodilation, which appears to be linked to activation of the prostaglandin H2–thromboxane A2 receptor.21 22 In these same studies pial arteriolar dilation to nitroglycerin was normal.21 22 However, other investigators have found attenuated vasodilation and cGMP production with local application of acetylcholine in an isolated carotid artery strip preparation in SHR-SP compared with WKY rats.5 Although provocative, these studies did not address the possibility that predisposition to stroke in SHR-SP may be related to differences between groups in the amount of basal NO-mediated cerebral vascular tone. Our finding of similar NO synthase inhibition and similar alteration of CBF and CVR between strains with L-NMMA and L-NA suggests that it is unlikely that an alteration in sensitivity of whole-brain NO synthase to pharmacological inhibition accounts for predisposition to stroke in SHR-SP. However, because our technique for measuring NO synthase does not allow us to separate the contribution of various brain cell types, it is possible that either neuronal or endothelial NO synthase23 may be altered without an effect on overall whole-brain sample NO synthase activity. For example, NO synthase is regionally heterogeneous in brain.23 24 25 In addition, although NO synthase is consistently present in endothelium, endothelium represents a relatively small component of overall brain volume.26

Our study suggests that the degree of alteration of cerebral vascular tone is correlated with the degree of inhibition of NO synthase. This is consistent with the dose-response relation we found using L-NMMA alone in piglets.2 In this previous study we did not directly measure NO synthase activity but observed CBF changes at a dose of L-NMMA similar to that observed in the present study. The exact mechanism for the increase in cerebral vascular tone produced by NO synthase inhibition is unclear. The decrease in CBF produced by NO synthase inhibition under normocapnic conditions does not appear to be metabolically mediated.2 27 28 29 It is possible that L-NMMA and L-NA may affect NO synthase present in endothelium, astrocytes, perivascular neurons, or parenchymal neurons.23 30 No study has yet determined the effect of more specific inhibitors of neuronal NO synthase (eg, 7-nitro-indazole and related substituted indazoles).31 However, L-NA causes constriction of isolated cerebral penetrating arterioles in vitro,32 suggesting that astrocytes and parenchymal neurons are not necessary for the cerebral vascular constriction that is observed after systemic administration of NO synthase inhibitors. In addition, Toda et al33 have presented data that suggest that cerebral vascular constriction after NO synthase inhibition is due to suppression of NO synthesis in nitroxidergic nerves innervating the cerebral arterial wall rather than an elimination of basal release of NO from the endothelium. Although the exact site of NO production that affects cerebral vascular tone and the segment of the cerebral vasculature involved were not addressed in our study, specificity for an NO-mediated mechanism was confirmed by the observation that cerebral vascular tone returns toward baseline in L-NMMA– and L-NA–treated rats after administration of L-arginine.

In the present study we evaluated two different inhibitors of NO synthase to avoid the possibility that a difference in response between strains after administration of one of these agents would be due to one of the known or unknown side effects rather than to its effect on NO synthase. For example, L-NMMA has inhibitory effects on cytochrome C reduction by ferrous iron.34 The degree of alteration of cerebral vascular tone with L-NMMA was small, as could be predicted from other studies in rats with the use of this same NO synthase inhibitor.35 Compared with L-NMMA, administration of L-NA produced a much greater alteration in cerebral vascular tone (20 mg/kg L-NMMA versus 20 mg/kg L-NA). Therefore, we believe that investigators must be cautious not to extrapolate the dose of one inhibitor of NO synthase from that of another inhibitor of NO synthase.

We found that at baseline, blood flow to the cerebral hemispheres was not decreased in SHR-SP or SHR compared with WKY rats, although CVR was similarly increased in SHR-SP and SHR. These data are similar to those presented by others who have evaluated CBF in SHR and WKY rats.36 37 However, Yamori and Horie38 found that cortical blood flow was not altered in SHR-SP until after 5 months of age, when MABP was consistently greater than 200 mm Hg. It is possible that the different finding in the present study is due to a difference in age, in the degree of hypertension, in experimental conditions (our rats were anesthetized), or in the techniques used for CBF measurement (H2 clearance versus radiolabeled microspheres). Our rats were 5 months old at the time of study, and MABP was less than 200 mm Hg under pentobarbital anesthesia. We have not measured MABP in any of these strains of rats in the unanesthetized state. Therefore, we cannot exclude the possibility that MABP may be higher in our rats in the unanesthetized state. In this context it may be that the effects of NO synthase inhibition would be different if we had waited longer after development of hypertension or after some duration of feeding the rats a Japanese-style diet. We believe that this is unlikely because basal NO synthase activity and response to NO synthase inhibition were similar in rats treated with a high-salt diet. We did not measure CBF in the rats fed a Japanese-style diet with salt loading because their small size prevented us from being able to insert the microsphere injection catheter.

Although baseline NO synthase activity was similar between groups, it was approximately one third of values reported in cortex from our previous study.10 We believe that this is accounted for by differences in experimental protocol. For example, in the present study all animals were anesthetized with pentobarbital before a biopsy sample was obtained. On the contrary, in the previous study brain biopsy samples were obtained after decapitation of rats without use of anesthetic agents.10 This is an important difference in experimental technique. Pentobarbital does not decrease NO synthase activity in vitro when experiments are performed at 22°C with optimal substrate conditions.39 However, it may have inhibitory effects on NO synthase activity and on the NO-mediated signal transduction pathway in vivo.40 Differences in response to pentobarbital in in vivo and in vitro systems may be related to differences in experimental temperature (22°C in vitro, 37°C to 38°C in vivo) or availability of substrates and cofactors (optimal in vitro, not controlled in vivo). Similarly, we cannot exclude the possibility that the effect of NO synthase inhibition may be different among the three strains of rats in the unanesthetized state.

We did not assess the effect of L-arginine on in vitro NO synthase activity. However, we did assess the effects of L-arginine on CBF. In these rats we measured CBF under baseline conditions, 20 minutes after saline administration, and then 20 minutes after L-arginine administration (200 mg/kg) in all three strains. In WKY rats L-arginine caused an increase in CBF compared with baseline conditions (101±5 compared with 88±4 mL/min per 100 g) but not compared with postsaline conditions (96±5 mL/min per 100 g). Despite the CBF difference between baseline and post–L-arginine conditions, there was no difference in CVR. In addition, L-arginine did not change CBF or CVR in SHR or SHR-SP. Others have demonstrated that 300 mg/kg L-arginine increases cortical blood flow (laser-Doppler flowmetry) to 120% of baseline in Sprague-Dawley rats.41 The lack of cerebral hyperemia produced by L-arginine in the present study may be related to the lower dose of L-arginine used, the fact that our blood flow measurement included both cortical and subcortical structures, or the possibility that there are important differences between species in the cerebral vascular effects of L-arginine.

The smaller effect of L-NMMA compared with L-NA on in vitro NO synthase activity may be due to a more reversible effect of L-NMMA, compared with L-NA, on enzyme inhibition. The assay is performed with an excess of L-arginine, as in the normal intracellular environment. Therefore, if L-NMMA results in a reversible inhibition of NO synthase, then the addition of excess of "fresh" L-arginine in the reaction media may reverse this inhibition, despite an in vivo NO synthase inhibition. In contrast, L-NA may exert a partially irreversible inhibition of NO synthase, which would not be reversed by an excess of L-arginine in the reaction media.

In conclusion, there was no difference among strains in basal NO synthase activity or in sensitivity of NO synthase for inhibition by L-NMMA or L-NA. In all strains L-NA was a much more potent inhibitor of NO synthase than L-NMMA, which was correlated with a greater effect of L-NA on basal cerebral vascular tone. Our data do not support the hypothesis that increased predisposition to stroke in SHR-SP is due to an alteration in sensitivity of NO synthase to pharmacological inhibition.


*    Acknowledgments
 
This study was supported by grant NS-20020 from the US Public Health Service, National Institutes of Health, and by a Lavoisier scholarship from the French government. The authors would like to thank the technicians of the Department of Anesthesiology and Critical Care Medicine Research Laboratories for their excellent technical assistance and Meagan Williams for her expertise in performing the NO synthase assay.


*    Footnotes
 
Reprint requests to Jeffrey R. Kirsch, MD, Department of Anesthesiology and Critical Care Medicine, The Johns Hopkins Hospital, 600 N Wolfe St, Blalock 1410, Baltimore, MD 21287-4963.

Received August 8, 1994; revision received February 1, 1995; accepted February 27, 1995.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 up arrowDiscussion
 *References
 

  1. Clavier N, Kirsch JR, Hurn PD, Traystman RJ. Cerebral blood flow is reduced by Nw-nitro-L-arginine methyl ester during delayed hypoperfusion in cats. Am J Physiol. 1994;267:H174-H181. [Abstract/Free Full Text]
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