Structure and Function of the Rat Basilar Artery During Chronic Nitric Oxide Synthase Inhibition
Background and Purpose Nitric oxide is an important regulator of vascular tone and may also be implicated in the modulation of vascular growth and structure. This study presents the effects of chronic nitric oxide inhibition, with or without antihypertensive treatment, on the structure and function of the basilar artery in rats.
Methods Rats were treated for 6 weeks with Nω-nitro-l-arginine methyl ester (50 mg/kg per day) alone or in combination with verapamil (100 mg/kg per day) or with trandolapril (1 mg/kg per day). Untreated rats served as controls. The structure and reactivity of perfused and pressurized basilar arteries were analyzed in vitro using a video dimension analyzer.
Results Systolic arterial pressure increased only in the nitro-arginine–treated group, as did the media-to-lumen ratio of the basilar artery. This structural alteration, which was prevented by verapamil and trandolapril, was mainly due to remodeling and not to growth. Chronic inhibition of the l-arginine pathway increased the response of the basilar artery to serotonin, while the opposite was found for endothelin. Verapamil and trandolapril prevented these functional alterations that seemed related to the changes in the vascular structure.
Conclusions The remodeling and functional alterations of the basilar artery seem to depend mainly on the elevation of arterial pressure with little contribution of the l-arginine pathway. Furthermore, nitric oxide does not seem to be implicated in the modulation of normal cerebral vascular growth in vivo. However, hypertension-induced changes in vascular reactivity and structure could alter cerebral blood flow and eventually contribute to the development of stroke in this model of hypertension.
The basal production of NO by the endothelium is an important vasodilator mechanism that counteracts tonic vascular constriction of neuronal, endocrine, or local origin.1 Indeed, chronic inhibition of NO synthesis by the administration of analogues of l-arginine, the natural precursor of NO, leads to hypertension in normotensive rats2 3 and in dogs.4 Growing evidence, mostly produced in vitro, suggests that endothelium-derived NO inhibits growth.5 6 7 However, at least in mesenteric resistance arteries, chronic inhibition of the l-arginine/NO pathway produces only remodeling and not growth.8 Moreover, this treatment does not produce hypertrophy of the heart, unless the activity of the renin-angiotensin system is also increased.9 A detailed microscopic examination of the central nervous system of rats chronically treated with an inhibitor of NO synthesis shows, besides vascular damage and multiple infarcts, thickening of the media of cerebral arteries.10 However, it was not determined if this was due to remodeling or to actual growth of the vessel wall. Therefore, the structural changes of cerebral vessels during prolonged inhibition of NO synthesis have yet to be determined.
Functional and structural changes in large and small cerebral arteries have been suggested to contribute to ischemic or hemorrhagic cerebrovascular events, a most important complication of hypertension.11 12 In the vessel wall, endothelium-derived NO acts as a vasodilator, inhibitor of platelet function, and possible antiproliferative mediator and therefore is likely to exert a protective effect.1 Indeed, chronic (up to 11 weeks) inhibition of the l-arginine pathway produces spinal cord and, with a lower incidence, brain infarcts.10 The rapid onset and the different distribution of these infarcts compared with other hypertension models suggest that endothelium-derived NO, and not just the increase in arterial pressure, plays a role in the infarction process.10 We were therefore interested to evaluate structural and functional alterations of the basilar artery in rats chronically treated with l-name, an analogue of l-arginine that inhibits NO synthesis. However, since the arterial pressure rise associated with such a treatment may also produce structural and functional modifications on its own, we evaluated these parameters in the presence or the absence of hypertension. We therefore also studied subgroups of L-NAME–treated rats that received concomitant antihypertensive therapy with either the calcium antagonist verapamil or the angiotensin-converting enzyme inhibitor trandolapril.
Materials and Methods
Animal Preparation and Monitoring
Six-week-old male Wistar Kyoto rats (IFFA Credo) were divided into four different treatment groups, each composed of seven rats: one group served as control (and received normal rat chow with free access to drinking water), one group was given L-NAME in its drinking water, a third group was treated with L-NAME and trandolapril (both in the drinking water), and the last group received L-NAME in its drinking water and verapamil in its otherwise normal rat chow. The actual dose given for each of these agents was calculated according to the water or food intake, which was measured three times weekly. Accordingly, the respective groups received an average of 58.0±1.1 mg/kg per day of L-NAME, 1.2±0.1 mg/kg per day of trandolapril, and 107.6±5.5 mg/kg per day of verapamil. Before the beginning of treatments and every week thereafter, the rats were weighed, and their systolic arterial pressure and heart rate were measured by a tail-cuff method with a pulse transducer (Model LE 5000, Letica). The average of three measurements was taken at each occasion. The treatments were carried out for 6 weeks, at which time they were discontinued for a period of 2 to 3 days before the experiments in an attempt to avoid short-term effects of the antihypertensive drugs during the in vitro experiments. All these procedures were approved by the Commission for Animal Research of the canton of Bern.
Vessel Preparation and Measurements
To allow for the dissection of the basilar arteries, the rats were anesthetized (thiopental, 50 mg/kg IP) and decapitated. The skull was then opened, the brain was gently removed, and the brain stem was put into ice-cold Krebs solution of the following composition (mmol/L; control solution): NaCl 118.6, KCl 4.7, CaCl2 2.5, KH2PO4 1.2, MgSO4 1.2, NaHCO3 25.1, edetate calcium disodium 0.026, and glucose 10.1. The basilar artery (internal diameter of approximately 200 μm) was excised under a dissection microscope from its proximal end to approximately half of its total length. The artery was then pulled and sutured onto a small glass cannula (afferent cannula) positioned in a vessel chamber (Living Systems Instrumentation) and superfused with control solution maintained at 37°C and oxygenated (95% O2, 5% CO2). The other end of the vessel was inserted in an efferent cannula as previously described,13 and the vessel was perfused intraluminally by gravity with control solution containing 1% BSA at a pressure of 35 mm Hg. This pressure was found to be optimal for serotonin contractions in rat basilar arteries. Under these conditions, intraluminal flow was approximately 0.2 mL/min.
The vessel perfusion chamber was positioned on the stage of an inverted microscope (Nikon, TSM-F), and the amplified image was transmitted by a video camera to a monitor and a video dimension analyzer (V91, Living Systems Instrumentation), allowing for continuous on-line measurements of changes of the vessel lumen diameter and media thickness. The signal of the internal diameter was then documented with a pen recorder (Gould). Before the experiments, the vessels were allowed to equilibrate for 45 minutes. At the end of the equilibration period, the resting lumen diameter and media thickness of each vessel were measured in the absence of any pharmacological agents.
In a pilot study, the structure of the basilar artery was determined in the absence and presence of calcium in five control and five L-NAME–treated rats. After 45 minutes of incubation in a calcium-free control solution, the dimensions were measured and the vessels were equilibrated for an additional 45 minutes in a control solution containing calcium (see above). A small contraction of 5% to 10% occurred in the presence of calcium in the basilar arteries of both control and L-NAME–treated animals. The difference in the wall-to-lumen ratio between the two groups was therefore maintained (data not shown). In fact, the linear regression between the media-to-lumen ratio in calcium-free and normal calcium conditions had a slope of 0.87 (r=.88, P<.001). Therefore, the structural determination was made only in control solution in the present study.
Vascular Reactivity Experiments
To assess vascular reactivity in basilar arteries obtained from each group of rats, the following protocol was performed. All drugs were applied extraluminally, and each part of the protocol was preceded by a washout period with control solution of 45 minutes. First, a single dose of Ang II (10−7 mol/L) was administered. Then, a concentration-response curve to serotonin (10−9 to 3×10−6 mol/L) was constructed. Third, a concentration-response curve to acetylcholine (10−9 to 10−5 mol/L) was obtained after a 30% precontraction of the vessel with serotonin. Similar conditions were then used to obtain a concentration-response curve to sodium nitroprusside (10−10 to 10−6 mol/L). Finally, a concentration-response curve was recorded with ET-1 (10−11 to 10−8 mol/L). Relaxations to acetylcholine or sodium nitroprusside were not obtained from the group treated with L-NAME and trandolapril, since the arteries could not be contracted with serotonin, norepinephrine, or prostaglandin F2α. In four additional control and L-NAME–treated rats, the relaxation to acetylcholine was studied in the presence of a 30-minute preincubation with l-arginine (10−4 mol/L). The structure-related parameters were measured in these arteries as well as in those from two additional control and L-NAME–treated rats in which no functional analysis was performed.
Histology of the Brain
After removal of the brain stem, the remainder of the cerebral tissue was fixed in 4% buffered formaldehyde. Coronal sections of the whole brain, cut at 1.5-mm intervals, were then embedded on their frontal face in paraffin and cut with a nominal microtome setting of 4 μm. Van Gieson’s elastic, periodic acid–Schiff’s reagent, and hemalum-eosin stains were used to evaluate cerebral parenchymal lesions, arteries, and arterioles. The pericallosal artery, present on an average of four coronal sections per rat, was chosen for enumeration of arterial cells of the medial and adventitial layers. The number of cells was averaged for each animal per cross section. A morphometric analysis of vessel wall components was not considered useful because the brains were not fixed by controlled pressure perfusion. Indeed, the use of such a procedure was prevented by the functional study of the basilar artery. Evaluation of the lesions was performed on coded slides by two independent observers, and differences in assessment were resolved by a review session that also served to enumerate the cells. Brain tissue appropriate for morphological evaluation was obtained from five to seven animals per group.
Verapamil and trandolapril were kindly provided by Knoll Pharmaceuticals; L-NAME was purchased at Calbiochem-Novabiochem. All the following drugs were obtained from Sigma Chemical Co: Ang II, serotonin, acetylcholine chloride, sodium nitroprusside, and l-arginine. All the above-mentioned drugs were dissolved in distilled water and diluted in control solution. ET-1 was obtained from Calbiochem-Novabiochem, dissolved in water containing 0.1% BSA, and diluted in control solution containing 0.05% BSA.
Data Expression and Statistical Analysis
The growth index was calculated from the cross-sectional area (CSA) with the equation (CSAL−CSAC)/CSAC∗100, where the index L stands for L-NAME and C for control. The theoretical background for the calculation of the remodeling index is described in detail elsewhere.14 The equation is (LDC−LDremodel)/(LDC−LDL)∗100, where LD is the lumen diameter and LDremodel is calculated from the external diameter of the media (ED) of the L-NAME group as Sqrt ([EDL]2−4∗CSAC/π). Contractions are expressed as the percentage of decrease in lumen diameter from the baseline diameter. Relaxations are expressed as the percentage of increase in lumen diameter from the extent of precontraction. For each individual concentration-response curve, the maximum and the half maximum effective concentration (EC50) were calculated by nonlinear regression. Values are expressed as mean±SEM, except for correlation analysis, which shows the actual data. The concentration-response curves of the different groups and the data shown in Fig 1⇓ were compared by ANOVA for repeated measurements with Bonferroni’s correction for multiple comparisons.15 Morphological measurements, maximum responses, and EC50 values were compared with one-way ANOVA with the same correction. Cell numbers were compared using Wilcoxon’s test for two samples. Pearson correlation coefficients were calculated by linear regression. P<.05 was considered significant.
Weight, Arterial Pressure, and Heart Rate
The weight, systolic arterial pressure, and heart rate changes during the 6-week treatment period are depicted in Fig 1⇑. The weight gain was similar in control and L-NAME–treated animals but significantly less in rats receiving the antihypertensive treatments. The sustained hypertension produced by the L-NAME treatment was completely prevented by verapamil and trandolapril. The chronic administration of L-NAME induced a discrete bradycardia, possibly due to a baroreflex-mediated decrease of the sympathetic drive, which was further enhanced by verapamil and trandolapril (Fig 1⇑).
Basilar Artery Structure
The basilar artery of L-NAME–treated rats had a significantly smaller internal diameter and an increased thickness of the media compared with that of control rats, resulting in an increased media-to-lumen ratio (Table 1⇓). The calculation of the remodeling index gave a value of 76%, suggesting that three quarters of the reduction of the internal diameter could be accounted for by remodeling. On the other hand, the growth index, calculated with the cross-sectional area, was not increased significantly (Table 1⇓).
The concomitant administration of L-NAME and either verapamil or trandolapril prevented the occurrence of these structural changes (Table 1⇑), suggesting that elevated arterial pressure per se, and not NO synthase inhibition, contributed mostly to remodeling. Accordingly, we found a significant relationship between systolic arterial pressure and media-to-lumen ratio (Fig 2⇓). In addition to its preventive effect, trandolapril significantly decreased the media thickness of the basilar artery without modifying the lumen diameter, thus producing a relative atrophy of the vessel (reduction of the cross-sectional area, Table 1⇑).
Basilar Artery Reactivity
The maximal effect and the half maximal effective concentration of several vasoactive substances are given in Table 2⇓.
The maximal response and the sensitivity of the basilar artery to serotonin were significantly enhanced in rats treated with L-NAME but were normalized by the administration of verapamil (NS versus control rats, Table 2⇑ and Fig 3A⇓). Trandolapril had further effects and inhibited nearly completely the vasoconstriction induced by serotonin.
The half maximal effective concentration of ET-1 (EC50) did not differ significantly between the groups, although there was a tendency for greater sensitivity with antihypertensive treatments (Table 2⇑ and Fig 3B⇑). In contrast to what was observed with serotonin, the maximal contraction to ET-1 tended to be smaller in L-NAME–treated rats compared with control animals. Verapamil normalized the response, whereas trandolapril significantly increased the vasoconstriction induced by ET-1 (Table 2⇑).
The response to a single maximal concentration of Ang II (10−7 mol/L) tended to be increased by chronic L-NAME treatment (NS, Table 2⇑). Verapamil, in contrast to its effect with other vasoconstrictors, could not prevent this alteration. The vasoconstriction induced by Ang II was even further enhanced in the rats chronically treated with L-NAME and trandolapril.
Relationship Between Structure and Function
In an attempt to link the functional alterations to structural changes of the basilar artery, the relationship between maximal vasoconstriction and media thickness was analyzed in vessels obtained from all groups of rats.
For serotonin, a positive correlation was observed (Fig 4A⇓), suggesting that contraction to this vasoactive monoamine increases with medial thickness. In contrast, a negative correlation was found between maximal vasoconstriction and media thickness for ET-1 (Fig 4B⇓). There was no relationship between the contractile effect of Ang II and the media thickness of the basilar artery (r=−.22, P=.28, data not shown).
Endothelium-dependent relaxation induced by acetylcholine (10−9 to 10−5 mol/L) was reduced by 50% in rats receiving chronic L-NAME therapy (Table 2⇑). In contrast, the endothelium-independent relaxation to sodium nitroprusside was not significantly altered by L-NAME. When arteries obtained from the L-NAME group were preincubated with l-arginine (10−4 mol/L), the maximal vasodilatation produced by acetylcholine was normalized (relaxation of 32±9%, n=4), whereas it was not changed in vessels obtained from the control group treated in a similar fashion (relaxation of 27±8%, n=4, data not shown)
In rats treated with L-NAME and verapamil, the endothelium-dependent relaxation induced by acetylcholine was similar to that obtained in rats treated with L-NAME alone, suggesting that verapamil did not improve the endothelial function in the basilar artery. Data on endothelium-dependent relaxation could not be obtained in rats treated with trandolapril, since no relevant level of precontraction could be obtained in these vessels (see above).
The histological examination of the brain slices revealed parenchymal lesions only in L-NAME–treated animals (4 of 5 rats). They consisted of small infarcts with mononuclear infiltrate (2 of 5), focal glial proliferation (3 of 5), and fresh hemorrhage (1 of 5). Arterial or arteriolar lesions were not discernible beyond doubt in any animal. The medial cell count of the pericallosal artery was highest for the control group (29±4 per cross section) and slightly lower (NS) for the L-NAME and L-NAME plus antihypertensive treatments (L-NAME, 24±1; L-NAME+verapamil, 26±4; L-NAME+trandolapril, 21±2). The adventitial cell number was equal in the control and L-NAME groups (19±2 and 16±1, respectively), slightly lower in the L-NAME+verapamil group (13±1, NS versus control), and significantly lower in the L-NAME+trandolapril group (9±1, P<.01 versus control and L-NAME alone).
The present results demonstrate that chronic inhibition of NO synthesis in rats leads to changes in the structure and reactivity of the basilar artery. However, because these modifications are prevented by concomitant antihypertensive therapy with either verapamil or trandolapril, the contribution of the elevation of arterial pressure seems to be of greater importance than the inhibition of NO synthesis.
The structural changes of the basilar artery induced by L-NAME treatment involves primarily remodeling with minimal growth. The calculated remodeling index (76%) is similar to what is described for the pial arteries of stroke-prone spontaneously hypertensive rats16 and for other vascular beds of several models of experimental hypertension, such as genetic,17 aortic coarctation,18 one-kidney, one clip,19 two-kidney, one clip,20 and deoxycorticosterone acetate (DOCA) salt,21 as well as in essential hypertension.22 23 A reduced distensibility of the vessel wall by L-NAME treatment could account for the altered vascular structure that was determined at a single low perfusion pressure. Although it may be different in the basilar artery, pressure-diameter curves done in mesenteric arteries of similar diameter do not show any difference in distensibility between control and L-NAME–treated rats (H.T., P.M., T.F.L., unpublished data, 1995). Tonic vascular contraction by L-NAME could also be responsible for an increased media-to-lumen ratio. However, as described in “Materials and Methods,” similar observations were obtained in vessels perfused with a calcium-free solution.
The nonsignificant growth index of 7% that we found in L-NAME–treated rats is lower than those reported for other models of experimental hypertension, which are usually above 20%.17 18 19 20 21 However, it is closer to the values found in essential hypertension.22 23 Quantitative assessment of the cellularity of the pericallosal artery, the only large extraparenchymal vessel studied by histology, corroborates the absence of any proliferative response in the media of L-NAME–treated rats. Such a weak effect on growth by the chronic inhibition of NO synthesis, which is also reported for the mesenteric resistance arteries8 and for the heart9 in this model of acquired hypertension, suggests that NO may not be involved in the basal regulation of growth. Indeed, in this model, cardiac hypertrophy can only be observed when the renin-angiotensin system is activated,9 24 probably in response to renal damage produced by prolonged hypertension.25 The discrepancy concerning the inhibitory effect of NO on growth between these in vivo results and those obtained in vitro5 6 7 could be due to the phenotypic alteration of smooth muscle cells in culture, acquiring the ability to reduce their proliferation with an increased intracellular cyclic GMP level.7 It may also be possible that in vivo the l-arginine pathway influences growth both positively and negatively, thus preventing its chronic blockade to show any net effect.26 This aspect of endothelium-mediated growth modulation deserves further investigation.
Both antihypertensive treatments prevent totally the rise in arterial pressure induced by the chronic L-NAME treatment. In previous studies using either angiotensin-converting enzyme inhibitors24 27 or angiotensin receptor antagonists,3 27 28 arterial pressure was also reduced or normalized, suggesting that the renin-angiotensin system is importantly involved in this model of hypertension. However, in this study, the calcium antagonist verapamil was equally effective in reducing blood pressure. Verapamil and trandolapril, apart from normalizing blood pressure, also prevent the structural alterations produced by chronic L-NAME administration. This observation, together with the significant positive relationship between systolic arterial pressure and the media-to-lumen ratio, suggests that vascular remodeling is primarily dependent on the arterial pressure level with little contribution of the l-arginine pathway. Indeed, the formation of NO is also inhibited in the L-NAME+verapamil–treated group, as shown by the blunted endothelium-dependent relaxation to acetylcholine, despite a normal vascular structure. Since intra-arterial pressure was not measured, it is not possible to determine which component of arterial pressure is better correlated with the changes in vascular structure. In stroke-prone spontaneously hypertensive rats,29 pulse pressure is more closely related to the alteration of cerebral arteriolar structure than are mean or systolic blood pressures.
Trandolapril has complex effects on the vascular structure, producing atrophy of the vessels that, according to the histological examination, seems to be due to a reduced cellularity of the adventitia and to a lesser extent of the media. This cannot be explained by the lower body weight of the animals, since verapamil treatment did not have such an effect on structure despite a similar body weight. Therefore, trandolapril seems to inhibit normal vascular growth in these young rats. Although this may be due to the inhibition of Ang II formation, trandolaprilat has been shown to decrease the proliferation of cultured vascular smooth muscle cells by delaying the initiation of the mitotic process.30
In addition to producing structural alterations, the chronic inhibition of NO formation also induces heterogeneous changes in the reactivity of the basilar artery to vasoconstrictor agents. Indeed, contractions to serotonin and Ang II (NS) are enhanced in L-NAME–treated rats, whereas ET-1 contractions tend to be weaker (NS). The enhanced responsiveness and sensitivity to serotonin may result from the amplifying effect of vascular remodeling.31 In the case of ET-1, such a decreased contraction of resistance vessels has also been reported in spontaneous hypertension of the rat,32 in DOCA-salt hypertensive rats,21 and in patients with mild essential hypertension.33 It may therefore represent a secondary mechanism to a sustained increase in blood pressure and to the structural alterations induced by the hypertensive state (see below).
Verapamil treatment also prevents the functional alterations induced by L-NAME, except for Ang II–induced responses, which remain elevated. Trandolapril has additional effects, as it blunts nearly completely the contraction to serotonin and enhances the responses to ET-1. These additional effects are surprising but may be explained, at least in part, by the structural alterations that trandolapril produces. Indeed, there is a linear relationship between the media thickness, which is reduced by trandolapril, and the efficacy of vasoconstrictors. In that respect, serotonin and ET-1 seem to be affected significantly but differently by a change in wall thickness, suggesting that the vascular structure has some influence on vascular reactivity. This phenomenon may also explain that contractions to prostaglandin F2α and norepinephrine could not be elicited in arteries from this treatment group. Receptor downregulation or upregulation could also be involved in the alterations of vasoconstriction efficacy during such chronic treatments, and this may be particularly important to explain the enhanced contraction to Ang II in rats treated with L-NAME and trandolapril.
Rats treated with L-NAME alone show a 50% decrease in endothelium-dependent relaxation of the basilar artery, as assessed by acetylcholine concentration-response curves. This blunted response is restored by acute incubation of the vessels with l-arginine, demonstrating that it is due to a decreased production of NO. However, verapamil cannot restore the dilatation, suggesting that the endothelium-dependent relaxation is still impaired despite the normalization of blood pressure. Verapamil may therefore lower blood pressure through a nonspecific mechanism. On the other hand, other vascular beds, also contributing to peripheral vascular resistance, may show an improved responsiveness to acetylcholine under verapamil treatment. Indeed, the endothelium-dependent relaxation of the mesenteric artery obtained from L-NAME–treated rats can be normalized by both verapamil and trandolapril (H.T., P.M., T.F.L., unpublished data, 1995).
Infarction or ischemic lesions found in L-NAME–treated rats confirm the results of another histological evaluation of cerebrovascular lesions in rats treated for up to 11 weeks with L-NAME.10 In that study, infarcts were observed in the brain but most predominantly in the spinal cord. The authors also reported vascular lesions that were not discernible in our study, a difference that can be related to the shorter (6 weeks) treatment period. Hence, it is most likely that structural and functional alterations of the basilar artery precede vascular damage in the brain. Because no lesions are found in brains from rats treated with L-NAME and antihypertensive treatments, these agents therefore exert a beneficial effect on ischemic events, probably through their hypotensive effect, which in turn normalizes the vascular structure and reactivity of cerebral arteries. Further studies will determine whether stroke-related mortality in L-NAME–treated rats is reduced by antihypertensive therapy.
The present results demonstrate that prolonged inhibition of NO synthesis in rats leads to an increased media-to-lumen ratio of the basilar artery by remodeling of a similar amount of tissue. This remodeling may be responsible, at least in part, for the changes in the reactivity of the basilar artery to vasoconstrictor agents. However, since these modifications are prevented by concomitant antihypertensive therapy with either verapamil or trandolapril, the contribution of the elevation of arterial pressure seems to be of greater importance than the inhibition of NO synthesis. Furthermore, the lack of increase in cross-sectional area of the basilar artery suggests that NO is not involved in the normal regulation of vascular growth in this vessel. The reduction of blood pressure by the antihypertensive agents may therefore reduce the incidence of stroke by preventing vascular functional and structural alterations that seem to depend on the level of blood pressure.
Selected Abbreviations and Acronyms
|Ang II||=||angiotensin II|
|BSA||=||bovine serum albumin|
|L-NAME||=||Nω-nitro-l-arginine methyl ester|
This work was supported by a grant from the Swiss National Research Foundation (grant 32-32541.91) and from Knoll Pharmaceuticals, Ludwigshafen, Germany, and by a fellowship from the Medical Research Council of Canada (Dr Moreau) and a stipend from the Senglet Foundation, Basel/Switzerland (Dr Küng). Dr J. Gries and Dr M. Kirchengast from Knoll, Germany, are acknowledged for the supply of verapamil and trandolapril, together with the appropriate chronic dosing regimen.
- Received March 13, 1995.
- Revision received June 26, 1995.
- Accepted June 30, 1995.
- Copyright © 1995 by American Heart Association
Ribeiro MO, Antunes E, de Nucci G, Lovisolo SM, Zatz R. Chronic inhibition of nitric oxide synthesis: a new model of arterial hypertension. Hypertension. 1992;20:298-303.
Manning RD Jr, Hu L, Mizelle HL, Montani JP, Norton MW. Cardiovascular responses to long-term blockade of nitric oxide synthesis. Hypertension. 1993;22:40-48.
Garg UC, Hassid A. Nitric oxide-generating vasodilators and 8-bromo-cyclic guanosine monophosphate inhibit mitogenesis and proliferation of cultured rat vascular smooth muscle cells. J Clin Invest. 1989;83:1774-1777.
Assender JW, Southgate KM, Newby AC. Does nitric oxide inhibit smooth muscle proliferation? J Cardiovasc Pharmacol. 1991;17(suppl 3):S104-S107.
Deng LY, Thibault G, Schiffrin EL. Effect of hypertension induced by nitric oxide synthase inhibition on structure and function of resistance arteries in the rat. Clin Exp Hypertens. 1993;15:527-537.
Arnal JF, el Amrani AI, Chatellier G, Menard J, Michel JB. Cardiac weight in hypertension induced by nitric oxide synthase blockade. Hypertension. 1993;22:380-387.
Blot S, Arnal J-F, Xu Y, Gray F, Michel J-B. Spinal cord infarcts during long-term inhibition of nitric oxide synthesis in rats. Stroke. 1994;25:1666-1673.
Whisnant JP. Extracranial and intracranial arterial disease. Hypertens Res. 1994;17(suppl 1):S43-S46.
Hennerici MG, Schwartz A. Hypertension related cerebral lesions: are patterns of cerebral infarction topography of diagnostic relevance? Hypertens Res. 1994;17(suppl 1):S33-S36.
Dohi Y, Thiel MT, Bühler FR, Lüscher TF. Activation of endothelial l-arginine pathway in resistance arteries: effect of age and hypertension. Hypertension. 1990;15:170-179.
Heagerty AM, Aalkjaer C, Bund SJ, Korsgaard N, Mulvany MJ. Small artery structure in hypertension: dual process of remodeling and growth. Hypertension. 1993;21:391-397.
Wallenstein S, Zucker CL, Fleiss JL. Some statistical methods useful in circulation research. Circ Res. 1980;47:1-9.
Baumbach GL, Heistad DD. Remodeling of cerebral arterioles in chronic hypertension. Hypertension. 1989;13:968-972.
Mulvany MJ, Baandrup U, Gundersen HJG. Evidence for hyperplasia in mesenteric resistance vessels of spontaneously hypertensive rats using a 3-dimensional dissector. Circ Res. 1985;57:794-800.
Korsgaard N, Mulvany MJ. Cellular hypertrophy in mesenteric resistance vessels from renal hypertensive rats. Hypertension. 1988;12:162-167.
Deng LY, Schiffrin EL. Morphological and functional alterations of mesenteric small resistance arteries in early renal hypertension in rats. Am J Physiol. 1991;261:H1171-H1177.
Deng LY, Schiffrin EL. Effects of endothelin on resistance arteries of DOCA-salt hypertensive rats. Am J Physiol. 1992;262:H1782-H1787.
Izzard AS, Cragoe EJ, Heagerty AM. Intracellular pH in human resistance arteries in essential hypertension. Hypertension. 1991;17:780-786.
Falloon BJ, Heagerty AM. In vitro perfusion studies of human resistance artery function in essential hypertension. Hypertension. 1994;24:16-23.
Baylis C, Mitruka B, Deng A. Chronic blockade of nitric oxide synthesis in the rat produces systemic hypertension and glomerular damage. J Clin Invest. 1992;90:278-281.
O’Connor KJ, Knowles RG, Patel KD. Nitrovasodilators have proliferative as well as antiproliferative effects. J Cardiovasc Pharmacol. 1991;17(suppl 3):S100-S103.
Pollock DM, Polakowski JS, Divish BJ, Opgenorth TJ. Angiotensin blockade reverses hypertension during long-term nitric oxide synthase inhibition. Hypertension. 1993;21:660-666.
Jover B, Herizi A, Ventre F, Dupont M, Mimran A. Sodium and angiotensin in hypertension induced by long-term nitric oxide blockade. Hypertension. 1993;21:944-948.
Baumbach GL, Siems JE, Heistad DD. Effects of local reduction in pressure on distensibility and composition of cerebral arterioles. Circ Res. 1991;68:338-351.
Folkow B. Physiological aspects of primary hypertension. Physiol Rev. 1982;62:347-503.
Dohi Y, Lüscher TF. Endothelin in hypertensive resistance arteries: intraluminal and extraluminal dysfunction. Hypertension. 1991;18:543-549.