Effects of Chronic Nitric Oxide Synthase Inhibition on Cerebral Arterioles in Rats
Abstract We examined the effects of nitric oxide (NO) synthase inhibition on the structure and mechanics of cerebral arterioles. We measured pressure, diameter, and cross-sectional area of the vessel wall (histologically) in maximally dilated cerebral arterioles in Sprague-Dawley rats that were untreated or treated for 3 months with the NO synthase inhibitor NG-nitro-l-arginine methyl ester (L-NAME; 10 mg/kg per day). Treatment with L-NAME increased cerebral arteriolar mean (87±6 versus 42±2 mm Hg, P<.05) and pulse (25±2 versus 13±2 mm Hg, P<.05) pressures, as well as cross-sectional area of the vessel wall (1839±70 versus 1019±58 μm2, P<.05) and external diameter (101±4 versus 87±2 μm, P<.05). These findings suggest that hypertension induced by NO synthase inhibition is accompanied by hypertrophy of the vessel wall and enlargement of cerebral arterioles in rats. To determine the role of cerebral arteriolar pulse pressure in hypertrophy of cerebral arterioles during inhibition of NO synthase, we measured the cross-sectional area of the vessel wall in rats treated with L-NAME that underwent unilateral carotid clipping. Unilateral carotid clipping failed to prevent increases in cross-sectional area of the vessel wall (1507±173 and 1613±148 μm2 in the clip and sham sides, respectively) in rats treated with L-NAME, even though increases in pulse pressure were prevented (16±1 and 27±1 mm Hg in the clip and sham sides, respectively, P<.05). These findings suggest that inhibition of NO synthase may promote hypertrophy of cerebral arterioles independently of increases in arteriolar pulse pressure.
Cerebral arterioles undergo structural alterations in several models of chronic hypertension. In SHR and SHRSP, arterioles undergo hypertrophy of the vessel wall accompanied by a paradoxical increase in distensibility and, at the same time, remodeling with a reduction in external diameter.1 2 Cerebral arterioles in one-kidney, one clip renal hypertensive rats also undergo hypertrophy accompanied by an increase in distensibility, but they do not undergo remodeling.2 Thus, structural alterations of cerebral arterioles may vary depending on the model of chronic hypertension.
Several determinants appear to contribute to alterations in vascular structure during chronic hypertension. Determinants implicated in hypertrophy include increases in arterial pressure,3 in particular pulse pressure,4 5 6 sympathetic nerves,7 8 the renin-angiotensin system,9 10 and the endothelium-derived factor endothelin.11 12 Determinants that may contribute to remodeling include genetic factors2 and the renin-angiotensin system.4 In contrast to their apparent contributions to hypertrophy, increases in pulse pressure,5 sympathetic nerves,7 and endothelin11 apparently do not play a role in remodeling. Thus, determinants of hypertrophy and remodeling apparently differ.
Another determinant that may contribute to alterations in vascular structure during chronic hypertension is the endothelium-derived relaxing factor NO. This suggestion is based on the following observations. First, inhibitors of NO synthase induce hypertension in rats.13 14 15 In addition, topical application of NO synthase inhibitors in vivo produces constriction of cerebral blood vessels in Sprague-Dawley rats.16 17 These findings indicate that production and release of NO by vascular endothelium may contribute to the regulation of resting vascular tone both systemically and in the brain. Second, blood pressure in Dahl salt-sensitive rats on a high salt diet is normalized by treatment with l-arginine, the substrate for NO production.18 Thus, in at least one model of chronic hypertension, reduced availability or release of NO may contribute to the maintenance of chronically elevated arterial pressure. Finally, it has been observed that vasodilator drugs that generate NO inhibit mitogenesis and proliferation of vascular smooth muscle in culture.19 This observation suggests that in addition to regulation of resting vascular resistance, NO also may contribute to regulation of vascular structure.
The first goal of this study was to examine the effects of chronic treatment with the NO synthase inhibitor L-NAME on wall mass of cerebral arterioles in rats. Because treatment with L-NAME causes hypertension, we placed a clip on one carotid artery in some rats to normalize cerebral arteriolar pulse pressure and thus separate the effects of increased pressure per se from nonpressor effects of L-NAME. Our hypothesis was that chronic hypertension induced by inhibition of NO production may produce cerebral vascular hypertrophy independently of increases in cerebral arteriolar pressure. The second goal was to determine whether treatment with L-NAME results in increases in cerebral arteriolar distensibility. In previous studies, we found that hypertrophy is associated with increases in distensibility of cerebral arterioles in SHR and renal hypertensive rats.2 Thus, it seemed likely that L-NAME–induced hypertension is accompanied by increases in arteriolar distensibility. The third goal was to determine whether L-NAME results in remodeling of cerebral arterioles. Based on previous findings that cerebral arterioles undergo remodeling in SHR but not in Sprague-Dawley rats with renal hypertension, we suggested that genetic factors may play a role in remodeling.2 It seemed plausible, therefore, that treatment with L-NAME would not result in remodeling of cerebral arterioles in Sprague-Dawley rats.
Experiments were conducted on male Sprague-Dawley rats. Animals were allowed free access to food and tap water, and they were housed at 25°C and exposed to 12 hours of light each day. Procedures followed in this study were in accordance with institutional guidelines for the care and use of experimental animals at the University of Iowa.
To examine the effects of inhibition of NO synthase on cerebral arterioles, a group of rats was treated with L-NAME (100 mg/L) in the drinking water beginning at 4 weeks of age. Rats that drank tap water served as controls. Water intake in the L-NAME–treated groups decreased from ≈200 mL/kg per day at the beginning of treatment to ≈100 mL/kg per day at the end. Thus, intake of L-NAME during the treatment period decreased from ≈20 mg/kg per day to ≈10 mg/kg per day.
To determine whether the effects of L-NAME on cerebral arterioles resulted from increases in arterial pressure, a second group of rats underwent unilateral carotid clipping before treatment with L-NAME was begun. Rats were anesthetized with sodium pentobarbital (2.5 mg/100 g body wt, IP), and a clip was placed on the left common carotid artery. Clips with a gap size of 0.30 mm were made from 2-mm strips of silver sheet. The right carotid artery was exposed but not clipped. Because we could not exclude damage to sympathetic nerves caused by the carotid clip, the superior cervical ganglia were removed on both sides. Rats that underwent exposure of both carotid arteries and removal of both superior cervical ganglia and drank tap water served as controls for L-NAME–clipped rats.
After about 3 months of treatment, we examined the mechanics of cerebral arterioles. Animals were weighed and anesthetized with sodium pentobarbital (5 mg/100 g body wt, IP), intubated, and mechanically ventilated with room air and supplemental oxygen. Paralysis of skeletal muscle was obtained with gallamine triethiodide (20 mg/kg IV). Because the animals were paralyzed, we evaluated them frequently for adequacy of anesthesia. Additional anesthesia (1.7 mg/100 g body wt, IV) was administered when pressure to a paw evoked a change in blood pressure or heart rate.
A catheter was inserted into a femoral vein for injection of drugs and fluids. A catheter was inserted into a femoral artery to record systemic arterial pressure and obtain blood samples for measurement of arterial blood gases, and a catheter was inserted into the other femoral artery to withdraw blood to produce hypotension.
Measurement of Cerebral Arteriolar Pressure and Diameter
We measured pressure and diameter in first-order arterioles on the cerebrum20 through an open skull preparation.21 The head was placed in an adjustable head holder, and a 1-cm incision was made in the skin to expose the skull. The skin edges were retracted with sutures, and ports were placed for inflow and outflow of artificial CSF. A craniotomy was made over the left parietal cortex, and the dura was incised to expose cerebral vessels. In rats with unilateral carotid clipping, a dam of acrylic was constructed along the exposed portion of the superior sagittal suture, and craniotomies were made over the parietal cortex of the left and right cerebral hemispheres.5 The exposed brain was continuously suffused with artificial CSF, warmed to 37°C to 38°C, and equilibrated with a gas mixture of 5% CO2/95% N2. The composition of the CSF was (mmol/L): KCl 3.0, MgCl2 0.6, CaCl2 1.5, NaCl 131.9, NaHCO3 24.6, urea 6.7, dextrose 3.7.21
Systolic, diastolic, mean, and pulse pressures were measured continuously in cerebral arterioles with a micropipette connected to a servo-null pressure-measuring device (model 5, Instrumentation for Physiology and Medicine, Inc). Pipettes were sharpened to a beveled tip of 3 to 5 μm in diameter, filled with 1.5 mol/L sodium chloride, and inserted into the lumen of a cerebral arteriole with a micromanipulator. The presence of the pipette tip in the vessel had no discernible effect on the diameter of cerebral arterioles.
Arterioles were monitored through a microscope connected to a closed-circuit video system with a final magnification of ×356. Images of arterioles were digitized using a video frame grabber installed in a Macintosh computer (Quadra 900, Apple Computer). Arteriolar diameter was measured from the digitized images using image-analysis software (NIH Image, National Institutes of Health, Research Services Branch, NIMH). The precision of this system is 0.4 to 0.6 μm.
Approximately 20 to 30 minutes after surgery, measurements of cerebral arterioles were obtained under baseline conditions. Vascular smooth muscle was then deactivated by suffusion of cerebral vessels with artificial CSF containing EDTA (67 mmol/L), which produces complete deactivation of smooth muscle in cerebral arterioles.21 Pressure-diameter relationships were obtained in deactivated cerebral arterioles between cerebral arteriolar pressures of 50 and 10 mm Hg in the hypertensive rats and 40 and 10 mm Hg in the normotensive rats. Hemorrhage was used to reduce cerebral arteriolar pressure in decrements of 10 mm Hg at pressures down to 20 mm Hg of cerebral arteriolar pressure and decrements of 5 mm Hg at pressures between 20 and 10 mm Hg. After each pressure step, arteriolar diameter achieved a steady state within 15 seconds. Inner diameter was measured ≈30 seconds later. Maximally dilated arterioles were fixed at physiological pressure in vivo by suffusion of vessels with glutaraldehyde fixative (0.0225 glutaraldehyde in 0.10 mol/L cacodylate buffer) while cerebral arteriolar pressure were maintained at baseline levels. Arterioles were considered to be adequately fixed when blood flow through the arteriole ceased. After the animal was killed by an injection of potassium chloride, the arteriolar segment used for pressure-diameter measurements was removed, processed for electron microscopy, and embedded in Spurr’s low-viscosity resin while cross-sectional orientation was maintained.
Cross-sectional area of the arteriolar wall was determined histologically from 1-μm sections using a light microscope interfaced with the video image analyzing system described above. Luminal and total (lumen plus vessel wall) cross-sectional areas of the arteriole were measured by tracing the inner and outer edges of the vessel wall. Cross-sectional area of the vessel wall was calculated by subtracting luminal cross-sectional area from total cross-sectional area.
Calculation of Mechanical Characteristics
Circumferential stress (ς) was calculated from cerebral arteriolar pressure (P), inner diameter of cerebral arterioles (Di), and wall thickness (WT): ς=(P×Di)/(2WT). Cerebral arteriolar pressure was converted from millimeters of mercury to newtons per square meter (1 mm Hg=1.334×102 N/m2). Wall thickness was calculated from the cross-sectional area of the vessel wall (CSA) and inner cerebral arteriolar diameter: WT=[(4CSA/π+Di2)1/2–Di]/2. External diameter of cerebral arterioles (De) was calculated as De=Di+2WT. Histological determinations of cross-sectional area were used in all calculations of wall thickness and circumferential stress. Circumferential strain (ε) was calculated as ε=(Di–Do)/Do, where Do is original diameter. We defined original diameter as the diameter at 10 mm Hg pressure.
To obtain tangential elastic modulus, the stress-strain data from each animal were fitted to an exponential curve (y=aebx) using least-squares analysis: ς=ςoeβε, where ςo is stress at original diameter and β is a constant that is related to the rate of increase of the stress-strain curve. Tangential elastic modulus (ET) was calculated at several different values of stress from the derivative of the exponential curve: ET=dς/dε=βςoeβε.
Determination of Wall Composition
Volume density of smooth muscle, elastin, collagen, basement membrane associated with endothelium and smooth muscle, and endothelium was quantitated from electron micrographs of the vessel wall using a method we have described previously.22 Ultrathin sections of the arteriolar wall were cut on an ultramicrotome and stained with phosphotungstic acid (0.0025). Electron micrographs were taken at a standard magnification of ×9000 and enlarged photographically by a factor of 3 for a final magnification of ×27 000. To ensure uniform sampling, the vessel wall was divided into four quadrants of equal size. At least two electron micrographs were taken randomly in each quadrant for a total of 10 electron micrographs per vessel.
A standard point-counting grid (double square lattice test system D16)23 was used to count the number of points contained within profiles of smooth muscle, elastin, collagen, basement membrane, and endothelium. Volume density (VV) of each component was calculated from the number of points in each component (Pa) and the total number of points contained within the vessel wall (PT): VV=Pa/PT. Cross-sectional area of individual wall components (CSAC) was calculated from VV of each component and total cross-sectional area (CSAT) of the wall measured histologically: CSAC=CSAT×VV.
Assay of NO Synthase Activity by l-[14C]Citrulline Production
Portions of cerebellum from untreated and L-NAME–treated Sprague-Dawley rats were frozen under liquid nitrogen and stored at −70°C. Activity of NO synthase was determined as the rate of conversion of l-[14C]arginine (DuPont NEN) to l-[14C]citrulline following a method previously described.24 Brain tissue samples were homogenized in 20 vol (wt/vol) of ice-cold buffer [50 mmol/L tris(hydroxymethyl)aminomethane, 2 mmol/L EDTA, pH 7.4] and centrifuged at 10 000g for 15 minutes at 4°C. Enzyme activity was assayed at 20°C in duplicate using the supernatant. The reaction was initiated by adding 25 μL of brain supernatant to 100 μL of a mixture containing 3 μmol/L l-[14C]arginine, 1 mmol/L NADPH, and 1 mmol/L CaCl2. The reaction was terminated after 15 minutes by adding 1.8 mL of stop buffer [30 mmol/L N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid and 3 mmol/L EDTA, pH 5.5].
Samples were applied to a 0.5-mL Dowex resin column (Sigma No. 50X-400, Na+ form) to remove l-[14C]arginine. The columns were then washed with 1 mL of water, and l-[14C]citrulline was quantified in the flow-through fraction using a scintillation counter. Enzyme activity is expressed as picomoles per milligram protein per minute. The concentration of l-[14C]citrulline was calculated after subtracting the blank value, which represents nonspecific radioactivity in the absence of enzyme activity. To determine whether the measured citrulline production was due to activity of constitutive NO synthase (calcium-dependent NO synthase), parallel samples were processed in the absence of calcium and in the presence of a potent inhibitor of activity of NO synthase (L-NAME, 1 mmol/L). Protein concentrations of the samples were measured using the Bradford assay.
ANOVA was used to compare systemic mean pressure, arteriolar pressures, diameters, cross-sectional area of the vessel wall, cross-sectional area and volume density of individual components, ratios of nondistensible to distensible components, and slope of tangential elastic modulus versus stress. Probability values were calculated using Student’s t test. Statistics were determined using JMP statistics software (SAS Institute Inc) on a Macintosh computer.
No deaths were observed in L-NAME–treated rats until the final month of treatment, during which time 37% of the rats died. None of the untreated rats died during the 3 months of observation. Treatment with L-NAME had no significant effect on body weight in rats (430±6 g in control rats versus 417±13 g in L-NAME–treated rats).
Systemic mean arterial pressure and cerebral arteriolar systolic, diastolic, mean, and pulse pressures were significantly greater in L-NAME–treated rats than in untreated rats (Table 1⇓). Diameter before deactivation with EDTA was not significantly different in cerebral arterioles of rats treated with L-NAME than in untreated rats (Table 1⇓). During deactivation with EDTA, internal and external diameters and cross-sectional area of the vessel wall were greater in cerebral arterioles in rats treated with L-NAME than in cerebral arterioles in untreated rats (Table 1⇓). Thus, during L-NAME–induced hypertension in rats, cerebral arterioles underwent hypertrophy of the vessel wall.
Although carotid clipping in L-NAME–treated rats significantly reduced both mean and pulse pressure in cerebral arterioles, it was more effective in reducing pulse pressure than mean pressure (Table 2⇓). Pulse pressure was similar in clipped cerebral arterioles in L-NAME–treated rats and cerebral arterioles in untreated rats. In contrast, mean pressure was significantly higher in clipped arterioles in L-NAME–treated rats than in arterioles in untreated rats (Table 2⇓). Active and passive diameters and cross-sectional area of the vessel wall were not significantly different in sham and clipped cerebral arterioles of L-NAME–treated rats (Table 2⇓). Thus, carotid clipping in L-NAME–treated rats did not prevent hypertrophy of cerebral arterioles, even though it normalized arteriolar pulse pressure.
Internal diameter in cerebral arterioles during maximal dilatation was larger in L-NAME–treated rats than in untreated rats at all levels of arteriolar pressure between 10 and 40 mm Hg (Fig 1⇓, left panel). The stress-strain curve in cerebral arterioles in L-NAME–treated rats was similar to the curve in cerebral arterioles in control rats (Fig 1⇓, right panel). In addition, the slope of tangential elastic modulus versus stress was not significantly different in L-NAME–treated rats and untreated rats (5.4±0.4 versus 5.1±0.5). These findings suggest that L-NAME–induced hypertension was not accompanied by alterations in passive distensibility of cerebral arterioles, despite hypertrophy of the vessel wall.
Unilateral carotid clipping had no effect on the relationship between pressure and internal diameter (Fig 2⇓, left panel) or on the stress-strain curve (Fig 2⇓, right panel) and the slope of tangential elastic modulus versus stress (4.7±0.3 versus 5.1±0.4 for the sham arterioles) in cerebral arterioles of L-NAME–treated rats. These findings suggest that increases in cerebral arteriolar diameter in L-NAME–treated rats occurred independently of increases in cerebral arteriolar pulse pressure.
Cross-sectional areas of smooth muscle, elastin, and basement membrane associated with smooth muscle and endothelium were significantly greater in rats treated with L-NAME than in untreated rats (Table 3⇓). Cross-sectional area of collagen was not significantly increased, whereas cross-sectional area of endothelium was similar in rats treated with L-NAME compared with untreated rats (Table 3⇓).
To relate composition of the vessel wall to passive distensibility, we calculated the ratio of nondistensible to distensible components in the arteriolar wall, as discussed previously.7 22 When basement membrane (BaseM) was combined with collagen (C), and when smooth muscle (SM) and endothelium (Endo) were combined with elastin (E), the ratio of nondistensible to distensible components ([C+BaseM]/[E+SM+Endo]) was similar in untreated and treated rats (Table 3⇑). Thus, when all of the major components of the arteriolar wall were taken into account, hypertrophy of cerebral arterioles in rats with L-NAME–induced hypertension was not accompanied by a modification in the proportion of the components of the arteriolar wall.
NO Synthase Activity
Treatment with L-NAME for 3 months decreased NO synthase activity in cerebellum of Sprague-Dawley rats, as expressed by the rate of conversion of l-[14C]arginine to l-[14C]citrulline (9.28±0.72 versus 45.10±3.95 pmol/mg per minute, P<.05). This finding indicates that chronic treatment with L-NAME was sufficient to inhibit NO synthase activity in the brain, and thus suggests that availability of NO was diminished in cerebral blood vessels of L-NAME–treated rats.
There were three major new findings in this study. First, chronic hypertension induced by treatment with L-NAME resulted in hypertrophy of cerebral arterioles of Sprague-Dawley rats. Carotid clipping during treatment with L-NAME in Sprague-Dawley rats did not prevent hypertrophy in cerebral arterioles, and it normalized arteriolar pulse pressure but not mean or systolic pressure. These findings suggest that treatment with L-NAME promotes hypertrophy of cerebral arterioles even in the absence of increases in arteriolar pulse pressure. To our knowledge, this study is the first to demonstrate that inhibition of NO synthase may result in vascular hypertrophy in cerebral arterioles independently of increases in arterial pulse pressure. Second, whereas distensibility of cerebral arterioles is increased in SHRSP, SHR, and renal hypertensive rats,2 21 distensibility of cerebral arterioles in rats with L-NAME–induced hypertension is unchanged. Third, in contrast to remodeling with a reduction of external diameter in cerebral arterioles of SHRSP and SHR,1 2 treatment with L-NAME did not result in remodeling of cerebral arterioles in Sprague-Dawley rats. This finding provides additional support for the concept that genetic factors not present in Sprague-Dawley rats may contribute to remodeling of cerebral arterioles during chronic hypertension.2
Cerebral arterioles undergo hypertrophy in several models of experimental hypertension including SHRSP, SHR, and renal hypertension.2 21 Determinants that may contribute to cerebral vascular hypertrophy during chronic hypertension include increases in pressure,3 25 neurohumoral factors,7 8 9 10 genetic factors,26 27 and endothelial factors, such as endothelin.11 12
Another endothelium-derived product that may contribute to regulation of vessel growth is NO. Consideration for this possibility first emerged with the finding that NO suppresses mitogenesis and proliferation of vascular smooth muscle cells in tissue culture.19 On the other hand, an important role for NO in vascular growth has not been supported by more recent studies in which NO synthase inhibitors were used to suppress production of NO in living animals. Dunn and Gardiner28 found that treatment with L-NAME produced no change in cross-sectional area of the vessel wall in mesenteric resistance arteries in Brattleboro rats. Although Schiffrin and colleagues29 30 found an increase in cross-sectional area of the vessel wall in mesenteric resistance arteries in L-NAME–treated rats, the increase was smaller in magnitude (ΔCSA of ≈7% to 10%) than often found by other investigators in other models of hypertension that have levels of arterial pressure similar to those in rats treated with L-NAME, such as SHR31 (ΔCSA of ≈70%) and renal hypertensive rats (ΔCSA of ≈40%).32
We found in this study that treatment of rats with L-NAME not only resulted in an increase in cross-sectional area of the vessel wall in cerebral arterioles, but the magnitude was greater (ΔCSA of ≈50% to 80%) than in small mesenteric arteries28 29 30 and was similar to, or greater than, increases found in cerebral arterioles in other models of hypertension that have similar increases in arterial pressure, including SHR2 (ΔCSA of ≈58%), SHRSP21 (ΔCSA of ≈30%), and renal hypertensive rats2 (ΔCSA of ≈43%). Thus, in contrast to previous studies,28 29 30 the findings in this study suggest that chronic hypertension induced by inhibition of NO synthase may produce substantial hypertrophy in the cerebral circulation. Furthermore, the findings also support the concept that NO may play an important role in the regulation of vascular growth.
We have considered three possibilities to account for the discrepancy between the magnitude of L-NAME–induced hypertrophy found in this study and previous studies.28 29 30 First, L-NAME may inhibit growth of vascular smooth muscle directly when given in higher doses,33 which in turn would tend to negate any vascular growth that might otherwise occur as a consequence of reduced availability of NO and its antiproliferative effects. The dose of L-NAME used to induce hypertension was substantially higher in previous studies28 29 30 (50 to 100 mg/kg per day) than in this study (10 mg/kg per day). It is possible, therefore, that if a higher dose of L-NAME had been used in this study, less hypertrophy might have resulted in cerebral arterioles.
Another possibility is that the effects of L-NAME–induced hypertension on vascular growth are time-dependent. In this study, rats were treated with L-NAME for 12 weeks as opposed to 2 to 4 weeks in previous studies.28 29 30 If the degree of vascular hypertrophy increases as a function of exposure to L-NAME and hypertension, it would not be surprising to find a greater magnitude of vascular hypertrophy in this study than in studies in which duration of treatment was shorter.
A third possibility is that the effects of L-NAME–induced hypertension on vascular growth may depend on vessel size and region. We examined cerebral arterioles with an average inner diameter of ≈90 μm (maximally dilated), whereas investigators in previous studies examined mesenteric resistance arteries with an average inner diameter of ≈220 μm.28 29 30
An important consideration in this study is whether hypertrophy of cerebral arterioles in L-NAME–treated rats resulted directly from increases in arteriolar pressure per se rather than a nonpressor effect of L-NAME. To test this possibility, we examined the effects of unilateral carotid clipping, which we previously found to normalize pulse pressure and prevent hypertrophy in cerebral arterioles of SHRSP.5 In this study, carotid clipping did not prevent hypertrophy of cerebral arterioles in L-NAME–treated rats, even though increases in arteriolar pulse pressure were prevented. On the other hand, carotid clipping failed to normalize systolic and mean pressures in cerebral arterioles. Thus, we cannot rule out the possibility that hypertrophy in cerebral arterioles of L-NAME–treated rats was the result of increases in systolic or mean pressure. There are two reasons to consider this possibility less likely, however. First, carotid clipping prevents cerebral arteriolar hypertrophy in SHRSP, even though clipping only partially attenuates increases in arteriolar systolic and mean pressures.5 Second, arteriovenous fistulae in rats increases pulse pressure, but not mean pressure, in cerebral arterioles and results in hypertrophy of the arteriolar wall.34
Another factor that may have contributed to hypertrophy of cerebral arterioles in rats treated with L-NAME is an interaction with the renin-angiotensin system. Angiotensin-converting enzyme inhibitors or angiotensin II receptor blockers prevent or attenuate increases in blood pressure in L-NAME–treated rats.35 36 37 At the same time, however, treatment with L-NAME apparently does not increase plasma angiotensin II levels35 36 37 and either has no effect on plasma renin activity35 36 or results in increases in plasma renin only after 4 to 6 weeks of treatment.37 These findings suggest an interaction between angiotensin II AT1 receptors and NO synthase inhibition. In this study, therefore, it is possible that treatment of rats with L-NAME may have resulted in activation of AT1 receptors, which in turn may have stimulated hypertrophy of cerebral arterioles.
Finally, it has been suggested that NO may play a role in the regulation of endothelin-1 gene expression in blood vessels.38 This suggestion is based on the finding that treatment with L-NAME results in increases in severity of hypertrophy in aorta and large mesenteric arteries in SHR together with increases in endothelin-1 mRNA in the vessel wall.38 In contrast, neither severity of hypertrophy nor endothelin-1 mRNA is increased in small mesenteric resistance arteries of SHR during treatment with L-NAME.39 These findings suggest that L-NAME stimulates production of endothelin-1 in large, but not small, arteries and thus results in additional hypertrophy only in large arteries as a consequence of the trophic effects of endothelin-1.40 41 On the basis of this assumption, one might conclude that in this study, endothelin-1 did not contribute to hypertrophy of cerebral arterioles in L-NAME–treated rats. On the other hand, we are unaware of any evidence to indicate that L-NAME does not increase endothelin-1 mRNA in cerebral arterioles. Thus, one cannot rule out absolutely a role for endothelin-1 in the development of cerebral arteriolar hypertrophy during treatment with L-NAME.
Mechanics and Composition
Delacrétaz and colleagues42 observed that hypertension caused by chronic inhibition of NO synthesis in Wistar-Kyoto rats was not associated with altered distensibility in carotid artery, in contrast to SHR in which distensibility of carotid artery was increased. We found in this study that L-NAME–induced hypertension did not alter passive distensibility of cerebral arterioles in rats. These findings suggest that L-NAME–induced hypertension does not alter passive distensibility in large or small cerebral arteries.
We have proposed previously that increases in distensibility that accompany hypertrophy of cerebral arterioles in SHRSP, SHR, and rats with one-kidney, one clip renal hypertension may be due to a reduction in the proportion of stiff (collagen and basement membrane) to compliant (smooth muscle, elastin, and endothelium) components in cerebral arterioles.2 22 If proportional composition influences vascular distensibility, one might anticipate that an absence of altered vascular distensibility during hypertension would be accompanied by no change in proportional composition of the vessel wall. The findings in this study appear to support this concept. The ratio of nondistensible to distensible components, as well as distensibility, was not altered in cerebral arterioles of L-NAME–treated rats.
Cerebral arterioles in SHRSP1 and SHR,2 but not in renal hypertensive rats,2 undergo remodeling of the vessel wall with a reduction in external diameter. Based on these findings, we suggested that genetic factors may play a role in the remodeling of cerebral arterioles with a reduction in external diameter during chronic hypertension.2 The findings of the present study provide additional support for the possibility that genetic factors, present in SHRSP and SHR but not in Sprague-Dawley rats, contribute to the remodeling of cerebral arterioles during chronic hypertension. In this study, we found that treatment of Sprague-Dawley rats with L-NAME does not result in a reduction of external diameter in cerebral arterioles. Instead, we found that L-NAME–induced hypertension was accompanied by an increase in external diameter. Increases in external diameter have been described previously in atherosclerotic arteries in monkeys43 and humans.44 The factors responsible for increases in external diameter of large arteries in atherosclerosis and cerebral arterioles in L-NAME–induced hypertension are not yet well defined. Nonetheless, it is tempting to speculate that reductions in availability of NO may play a role in both cases. In atherosclerosis, for example, availability of NO in large arteries may be reduced by increased destruction of NO, perhaps by release of oxygen radicals from leukocytes in the arterial wall.45
Selected Abbreviations and Acronyms
|L-NAME||=||NG-nitro-l-arginine methyl ester|
|SHR||=||spontaneously hypertensive rats|
|SHRSP||=||stroke-prone spontaneously hypertensive rats|
This work was supported by National Institutes of Health grants HL-22149 and NS-24621 and funds from the Iowa Affiliate of the American Heart Association. J.M.C. is the recipient of a Fellowship Award from the Iowa Affiliate of the American Heart Association. We thank Dr Frank Faraci for critical review of this manuscript.
- Received October 16, 1996.
- Revision received December 10, 1996.
- Accepted April 23, 1997.
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