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(Hypertension. 1996;27:49-55.)
© 1996 American Heart Association, Inc.

Articles

Vascular Structure and Expression of Endothelin-1 Gene in L-NAME–Treated Spontaneously Hypertensive Rats

Pavol Sventek; Jin-S. Li; Kevin Grove; Christian F. Deschepper; Ernesto L. Schiffrin

From the MRC Multidisciplinary Research Group on Hypertension, Clinical Research Institute of Montréal, University of Montréal, Québec, Canada.

	Abstract

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Abstract Inhibition of nitric oxide synthase by L-arginine analogues is associated with elevation of blood pressure in rats. Deoxycorticosterone acetate (DOCA)-salt hypertensive rats and DOCA-salt–treated spontaneously hypertensive rats (SHR) overexpress the endothelin-1 gene in blood vessels, and this is associated with severe vascular hypertrophy, whereas SHR do not overexpress endothelin-1 and exhibit limited vascular hypertrophy. In this study malignant hypertension was induced in SHR by chronic administration of the L-arginine analogue N^G-nitro-L-arginine methyl ester (L-NAME), a potent inhibitor of nitric oxide synthase, to determine whether malignant hypertension would result in endothelin-1 gene overexpression in blood vessels and in greater severity of vascular hypertrophy, as found in malignant DOCA-salt–treated SHR. L-NAME treatment induced malignant hypertension in SHR, with a systolic blood pressure of 246±2 mm Hg, compared with 211±2 mm Hg (P<.01) in untreated SHR. Plasma renin activity was very high in L-NAME–treated SHR, and their plasma immunoreactive endothelin concentration was slightly but significantly elevated (P<.01). After 3 weeks of treatment, aortic and to a lesser degree mesenteric artery weights were significantly increased in L-NAME–treated SHR compared with untreated SHR. However, cardiac weight and the media cross-sectional area or media width–to–lumen diameter ratio of small arteries from the coronary, renal, mesenteric, or femoral vasculature were not increased in L-NAME–treated SHR in comparison with untreated SHR. The abundance of endothelin-1 mRNA measured by Northern blot analysis was significantly increased in L-NAME–treated SHR in aorta and with less magnitude in the mesenteric arterial tree. The absence of accentuation of cardiac and small artery hypertrophy in malignant hypertension in L-NAME–treated SHR, despite enhanced expression of the endothelin-1 gene in blood vessels, may suggest a direct or indirect inhibitory effect of L-NAME on cardiovascular growth, probably independent of its effects on nitric oxide synthase, counterbalanced in aorta and large mesenteric arteries by the hypertrophic effect of enhanced vascular endothelin-1 gene expression. These results also suggest a role for blood pressure and potentially for nitric oxide in the regulation of endothelin-1 gene expression in blood vessels.

Key Words: aorta • resistance arteries • nitric oxide • endothelium-derived relaxing factor

	Introduction

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The role of endothelins in hypertension is not well understood.^{1 2 3} Recent studies have shown that the endothelin-1 gene is overexpressed in blood vessels from different vascular beds of DOCA-salt hypertensive rats,^{4 5 6 7} as well as malignant DOCA-salt–treated SHR.⁸ Both these models respond with slight but significant blunting in the severity of elevated blood pressure when treated chronically with endothelin receptor antagonists,^{9 10} suggesting that endothelin-1 gene overexpression may play a role in blood pressure elevation under these particular conditions. We recently demonstrated that DOCA administration is not mandatory to induce enhanced vascular expression of endothelin-1. In 1K1C Goldblatt hypertensive rats, 1K1C similarly elevated levels of endothelin-1 mRNA could be detected, whereas they were not elevated in the early, renin-dependent phase of 2K1C Goldblatt hypertension (authors' unpublished observations, 1995). Thus, vascular expression of endothelin-1 appears to be increased in DOCA-salt hypertensive rats,^{4 5} DOCA-salt SHR,⁸ and 1K1C hypertensive rats (authors' unpublished observations), all of which exhibit severe vascular hypertrophy,^{8 11 12} but not in 2K1C hypertensive rats (authors' unpublished observations) or in SHR,^{4 13} which have less severe vascular hypertrophy.^{11 14} In the models that overexpress endothelin-1 in the endothelium^{6 7} of blood vessels, chronic endothelin receptor antagonist treatment resulted in less severe vascular hypertrophy in small resistance-sized arteries (Reference 9 and authors' unpublished observations, 1995), whereas in SHR, which do not overexpress endothelin-1, there was no effect on vascular hypertrophy.^{15 16} On this basis, we proposed that enhanced endothelin-1 gene expression in blood vessels may accentuate vascular hypertrophy.³ However, whether severely elevated blood pressure could be sufficient by itself to increase endothelin-1 gene expression in blood vessels was not resolved.

We therefore decided to examine vascular structure and vascular endothelin-1 gene expression in a model of malignant hypertension not receiving DOCA. To induce malignant hypertension, a rat model was chosen in which vascular hypertrophy could be predicted not to be severe. Administration of the nitric oxide synthase inhibitor L-NAME is known to result in blood pressure elevation unaccompanied by severe vascular changes.^{17 18 19 20} It was speculated that chronic treatment of SHR with L-NAME would result in malignant hypertension. In this study, therefore, the relationship between vascular hypertrophy and expression of the endothelin-1 gene in the vasculature was investigated in SHR treated chronically with L-NAME.

	Methods

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Animal Experiments
The protocol was approved by the Animal Care Committee of the Clinical Research Institute of Montreal and followed the recommendations of the Canadian Council for Animal Care. SHR were bought from Taconic Farms (Germantown, NY) and were received at 12 weeks of age. Rats were housed under conditions of constant temperature (22°C) and humidity (60%) and exposed to a 12-hour dark-light cycle. At age 13 weeks they were offered either tap water or water containing L-NAME at a concentration adjusted daily such that rats received 30 mg·kg^-1·d^-1 (this dose was shown to be relatively well tolerated by SHR in preliminary experiments, whereas higher doses, as we used in Sprague-Dawley rats,^{17 18} were associated with high mortality of treated SHR within a few days). Systolic blood pressure was measured indirectly in prewarmed restrained rats by the tail-cuff method and recorded on a Grass model 7 polygraph (Grass Medical Instruments) fitted with a 7P8 preamplifier and a model PCPB photoelectric pulse sensor before treatment was started and at 8 and 16 days of treatment. The average of three pressure readings was recorded. Rats were killed by decapitation after 16 days of treatment. Of 9 SHR treated with L-NAME, 7 survived up to day 16 of the experiment, but all exhibited reduced growth, and during the last week, 5 of these experienced significant weight loss and apathy, ate much less, and had a hunched appearance typical of that of rats with malignant hypertension. Because blood pressure measurements by the tail-cuff method were thought not to be as reliable as usual on day 16 of treatment with L-NAME as a consequence of a reduction in pulse pressure that rendered the detection of the level of systolic blood pressure less precise, an additional small group of SHR of the same age (13 weeks) was implanted with telemetric transmitters (TA11PA-C40), and a catheter was placed into the distal portion of the descending aorta under anesthesia with methohexital sodium (Brietal Sodium, Lilly) 50 mg/kg IP for telemetric recording of arterial pressure (Data Sciences International).²¹ Hourly averages of 10-second samples obtained every 5 minutes were used to obtain blood pressures of untreated and L-NAME–treated rats.

Preparation of Small Arteries for Morphometry
The technique for dissection of small vessels from the heart, the kidney cortex, and the femoral region of the rat has been described.^{15 16} The heart and the kidneys were removed and placed in ice-cold Krebs solution. The rat was then placed in the supine position, and the skin of the right hind leg was incised. An artery in the popliteal region approximately 2 mm long was dissected. To dissect coronary vessels, the right ventricle was opened to expose coronary arteries on the interventricular septum. The interventricular artery was followed to the cardiac apex, and then the chordae tendineae and the myocardium were separated and a vessel 2 mm long was isolated. For the isolation of renal cortical arteries, the renal capsule was first removed. The kidney was sectioned, and renal arteries were dissected close to the renal cortex and then followed distally. A renal arcuate artery approximately 2 mm long was isolated. Mesenteric small arteries were obtained as previously described.^{11 12 14} Superior mesenteric arteries were taken from the part of the mesenteric vascular bed that feeds the jejunum 8 to 10 cm distal to the pylorus. A third-order branch 1 mm distant from the intestine and approximately 2 mm long was isolated. The vessels were mounted as ring preparations on an isometric myograph (Living Systems Instrumentation). The dissection and mounting were performed in PSS at room temperature. PSS had the following composition (in mmol/L): NaCl 120, NaHCO₃ 25, KCl 4.7, KH₂PO₄ 1.18, MgSO₄ 1.17, CaCl₂ 2.5, EDTA 0.026, and glucose 5.5. All solutions were bubbled with 95% O₂/5% CO₂ to give a pH of 7.40 to 7.45 and maintained at 37°C.

Protocol for Study of Small Arteries
After mounting, the vessels were warmed to 37°C and allowed to equilibrate in PSS for approximately 30 minutes with the vessel internal circumference set to give a wall tension of 0.2 mN/mm. Then media width was measured with a Leitz-Diavert inverted light microscope (Wild Leitz) at x320 magnification at 12 different sites along the wall, which were then averaged. The vessels were then set to L₀, where L₀=0.9 L₁₀₀ and L₁₀₀ is the internal circumference (calculated from the distance between the wires) that the vessels would have had in vivo when relaxed and under a transmural pressure of 100 mm Hg. After this, the vessels were maintained in PSS at 37°C for a further 90 minutes. After the rest period, the vessels were stimulated with 10 µmol/L methoxamine or 10 nmol/L arginine ([Arg⁸])-vasopressin (in the case of coronary small arteries) to ensure that the vessels isolated were arteries and that they developed a tension of more than 2 mN/mm.

Northern Blot Analysis
A 1.5-cm-long segment of thoracic aorta and the complete mesenteric arterial tree were removed and dissected free of fat. Tissues were then snap-frozen in liquid nitrogen and stored at -70°C until extraction of total RNA was performed. Total RNA was extracted from frozen tissues by a guanidine isothiocyanate–phenol-chloroform method.²² Total RNA samples (20 µg) were denatured in 1x running buffer (20 mmol/L MOPS, pH 7.0, 6 mmol/L sodium acetate, 1 mmol/L EDTA), 6% formaldehyde, and 50% formamide for 15 minutes at 65°C. RNA samples were run on a 1.0% agarose gel containing 1x running buffer for 4 to 5 hours. The samples were transferred from the gel to a nylon membrane, Hybond-N (Amersham), by capillary action with 3 mol/L NaCl/0.3 mol/L sodium citrate (20x SSC). After blotting, the membranes were dried by baking at 80°C for 2 hours. The locations of the 18S and 28S rRNA species were revealed by staining with 0.02% methylene blue in 0.3 mol/L sodium acetate (pH 5.5). Membranes were prehybridized at 60°C for 2 hours (42°C for the ³²P-labeled oligonucleotide probe for the 18S rRNA) in 400 mmol/L sodium phosphate buffer (pH 7.2) containing 5% SDS, 1 mmol/L EDTA, 0.1% bovine serum albumin, and 50% formamide. Hybridization with the ³²P-labeled probe was carried out for 18 to 20 hours at 60°C. The membranes were washed in 12.5 mmol/L NaCl/0.1% SDS three times at 72°C for 20 minutes. The membranes were exposed to Reflection films (Dupont) with intensifying screens at -70°C for 6 days (2 to 4 hours for the 18S rRNA). The autoradiograms were analyzed with a Bio-Rad imaging densitometer and MOLECULAR ANALYST software version 1.1 (Bio-Rad Laboratories).

The rat endothelin-1 probe was prepared from rat lung RNA by reverse transcriptase–PCR.⁵ A 319-bp rat preproendothelin-1 PCR product was obtained by use of a 5' forward primer: 5'-CTAGGTCTAAGCGATCCTTG-3' and a 3' reverse primer: 5'-TTCTGGTCTCTGTAGAGTTC-3' located at nucleotides 266 to 285 and 565 to 584, respectively, of the coding sequence of the rat endothelin-1 cDNA.²³ This PCR product was then cloned into pGEM-7zf(+) plasmid (Promega). The radiolabeled antisense riboprobe was prepared as previously described⁵ with [-³²P]UTP (800 Ci/mmol; Dupont). 18S rRNA was analyzed by use of a specific oligonucleotide probe (5'-CTTCCTCTAGATAGTCAAGTTCGACCGTCT-3)²⁴ labeled with T4 polynucleotide kinase (Pharmacia) and [-³²P]ATP (3000 Ci/mmol, Dupont). The ³²P-labeled probes were purified by chromatography using a Sephadex G-50 column (Pharmacia) or NACS cartridges (Gibco-BRL) for the riboprobe and the oligonucleotide probe, respectively.

Measurement of Plasma Endothelin and Plasma Renin Activity
Blood was obtained from the neck during the first few seconds after decapitation in tubes containing potassium EDTA for measurement of plasma endothelin-1 and plasma renin activity. Immunoreactive endothelin-1 was extracted from plasma by passage through C18 Sep-Pak cartridges (Waters Associates) and measured by radioimmunoassay as previously described.²⁵ The antibody against endothelin-1 was from Peninsula. The minimum detectable concentration of endothelin was 0.4 pmol/L, and recovery of 5 pmol/L of endothelin-1 added to plasma was 75%. The cross-reactivity of the antibody was 10% with big endothelin and 7% with endothelin-3. Plasma renin activity was measured by radioimmunoassay of angiotensin I generated during a 2-hour incubation in the presence of 8-hydroxyquinoline and sodium edetate as angiotensinase inhibitors at pH 6.5 and at 37°C as previously described.⁴

Analysis of Data
The media cross-sectional area of small arteries (A) was obtained from the media thickness (m) and the circumference of vessels (L), all measured with the vessel relaxed and under no passive stretch (wall tension of 0.2 mN/mm), and was calculated as A=Lm+m². Using L₀ and the calculated media cross-sectional area and assuming a constant media volume, the standardized media thickness of blood vessels (at L₀) was then calculated. The lumen diameter was obtained as L₀/. Since the weight of the rats was very different at the end of the experiment as a result of weight loss in the last few days, the cross-sectional area of the media of small arteries of smaller rats is also presented after correction by dividing by the square root of the ratio between the body weight of the lighter rats before rats lost weight and the mean weight of untreated rats (square root of body weight at 8 days divided by 349=0.9128). We previously showed that this correction in normally growing rats most closely approximates the media cross-sectional area of small mesenteric arteries of rats of very different body weights.¹⁵

Values are given as mean±SEM. Statistical differences were evaluated by Student's t test. Results were considered significantly different when P<.05.

	Results

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L-NAME treatment induced a significantly faster rise in blood pressure and a reduced increase in body weight in SHR that was already measurable after 8 days, whereas untreated SHR exhibited their usual increase in blood pressure and body weight (Table 1). At 16 days of treatment, 7 of 9 L-NAME–treated SHR survived, and of these 7, 5 had lost weight significantly, demonstrated apathy, and had a hunched appearance as found in malignant hypertension. Whereas blood pressure continued to rise slowly in untreated SHR, systolic blood pressure rose steeply in L-NAME–treated rats, and because pulse pressure was reduced, it could not be measured as reliably as is usual with the tail-cuff method in L-NAME–treated SHR. Accordingly, a small additional group of SHR were implanted with intravascular catheters and treated (n=3) or not treated (n=2) with L-NAME in a similar way. Results of their intravascular blood pressures monitored continuously by telemetry are also shown in Table 1. Systolic blood pressure thus rose to approximately 200 mm Hg in SHR and to 240 mm Hg in L-NAME–treated SHR. Tracings of the mean arterial pressures of an SHR and an L-NAME–treated SHR are shown in Fig 1, demonstrating that mean arterial pressure remained stable within the experimental period, varying between 145 and 160 mm Hg in the SHR, with a systolic blood pressure of approximately 190 to 200 mm Hg and diastolic of 125 to 140 mm Hg. In the SHR that received L-NAME, depicted in Fig 1, mean blood pressure rose immediately as of day 2, when L-NAME was started, to 175 to 200 mm Hg for the first 8 days, rising after that progressively to a mean arterial pressure of 210 to 230 mm Hg, at which time rats started losing weight. Mean arterial pressure presented important oscillations, however, particularly on day 15 to 16 of experimentation, which were also found in untreated SHR. Toward the end of the experimental period, the pulse pressure of L-NAME–treated rats was reduced, with a systolic blood pressure of 240 to 250 mm Hg (which was in agreement with the measurements by the tail-cuff method, Table 1) and diastolic blood pressure of 190 to 210 mm Hg.

View this table: [in this window] [in a new window]   Table 1. Body Weight, Blood Pressure, Heart and Vascular Weights, Plasma Renin Activity, and Plasma Endothelin Concentration

View larger version (26K): [in this window] [in a new window]   Figure 1. Tracings of continuous mean arterial pressure (MAP) obtained telemetrically in an untreated SHR and in an L-NAME–treated SHR. Samples were obtained for 10 seconds every 5 minutes for 16 days.

Although heart weight was not increased in SHR receiving L-NAME, heart weight–to–body weight ratio was (Table 1). However, rats not only gained less weight up to day 8 of the experiment but actually lost weight from day 8 to day 16. For this reason, the heart weight–to–body weight ratio was also calculated from the body weight of rats before they started losing weight, and this ratio, which probably is more representative, was similar in SHR and L-NAME–treated SHR, suggesting that cardiac hypertrophy was not more severe in L-NAME–treated SHR despite the malignant levels of blood pressure elevation attained.

The weights of 1.5-cm-long segments of thoracic aorta and of the complete mesenteric vascular tree dissected free of fat were greater in L-NAME–treated SHR than in untreated SHR (Table 1) despite the lower body weight of the former (without any correction for body weight). This suggested that hypertrophy of conduit arteries and large arteries was more severe in L-NAME–treated SHR than in untreated SHR. Since SHR treated with L-NAME grow less and then lose weight after day 8, it may be more appropriate to compare weights of vessels after correction for these events. If the weight of vessels of L-NAME–treated SHR was corrected as described in "Analysis of Data" by their body weight before they started losing weight, at day 8, then the increase in the severity of hypertrophy of aorta and mesenteric arteries of L-NAME SHR was shown to be quite important (Table 1).

Small arteries from the coronary, renal, mesenteric, and femoral circulations with a lumen diameter of approximately 200 µm were investigated on a wire myograph under standardized conditions. There were no differences in lumen diameter, media width, media cross-sectional area, or ratio of media width to lumen diameter in any of the vascular beds between SHR and L-NAME–treated SHR (Table 2). Since differences of 100 g in body weight of rats may significantly influence cross-sectional area of the media of small vessels but not other parameters,¹⁶ the media cross section was corrected by dividing this parameter by the square root of the ratio between the body weight at 8 days of rats treated with L-NAME before rats lost weight and the mean weight of untreated rats (square root of body weight at 8 days divided by 349=0.9128). We previously showed that this correction in normally growing rats most closely approximates the media cross-sectional area of small mesenteric arteries of rats of very different body weights. Even after this correction, shown in Table 2 in parentheses, the media cross-sectional area of L-NAME–treated SHR was not significantly increased in any vascular bed except in the mesenteric circulation, in which it was slightly but significantly larger (P<.05).

View this table: [in this window] [in a new window]   Table 2. Morphometric Parameters of Small Arteries

Northern blot analysis was performed on RNA extracted from aorta and mesenteric arteries. The abundance of endothelin-1 mRNA was greater in vessels of L-NAME–treated SHR than in SHR, particularly in aorta, in which it was increased eightfold, and to a lesser degree in the mesenteric arterial tree, in which it was increased twofold (Figs 2 and 3).

View larger version (45K): [in this window] [in a new window]   Figure 2. Representative Northern blot of total RNA (20 µg per lane) extracted from the aorta of SHR and SHR treated with L-NAME (SHR+L-NAME). Top, Single band of 2.3 kb corresponding to the rat endothelin-1 mRNA transcript. The analysis was done with a specific 32P-labeled complementary RNA probe for rat preproendothelin-1. Bottom, Band of the 18S ribosomal RNA on the same blots, obtained by hybridizing with a specific 32P-labeled oligonucleotide probe and used to normalize the abundance of endothelin-1 mRNA (see Fig 3). Similar results were obtained in at least two different membranes with material from different rats.

View larger version (16K): [in this window] [in a new window]   Figure 3. Bar graphs show the endothelin-1 (ET-1) mRNA content expressed as the ratio of the optical density of the endothelin-1 mRNA to the 18S rRNA bands (in arbitrary units, mean±SEM) from samples of RNA extracted from the aorta and the mesenteric arterial bed (Mesenteric arteries) of 6 SHR and 7 L-NAME–treated SHR examined by Northern blot analysis.

	Discussion

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Previous studies from our laboratory have shown that the vascular contents of endothelin-1 mRNA transcripts and immunoreactive endothelin-1 are elevated in some models of experimental hypertension, particularly in DOCA-salt hypertensive rats,^{4 5 6} DOCA-salt SHR,⁸ and more recently in 1K1C Goldblatt hypertensive rats, but not in 2K1C Goldblatt hypertensive rats (authors' unpublished observations, 1995) or in untreated SHR.^{4 13} The increase in expression of endothelin-1 in blood vessels appears to be localized to the endothelium.^{6 7} In the hypertensive rat models that overexpress endothelin-1, there is concomitant severe vascular hypertrophy. This is apparent in DOCA-salt hypertensive rats,¹² DOCA-salt SHR,⁸ and 1K1C Goldblatt hypertensive rats.¹¹ In contrast, the experimental hypertensive models that do not overexpress endothelin-1 in blood vessels exhibit less severe vascular hypertrophy, as in the case of SHR¹⁴ and 2K1C Goldblatt hypertensive rats.¹¹ Interestingly, chronic endothelin antagonism results in slight but significant lowering of blood pressure and at the same time in a very important attenuation, beyond what would be expected of the small fall in blood pressure, of the development of hypertrophic remodeling of vessels, particularly of small arteries, in DOCA-salt hypertensive rats⁹ and in DOCA-salt SHR (authors' unpublished observations, 1995), both of which exhibit vascular overproduction of endothelin-1. No effect of chronic endothelin antagonist administration was found in SHR,^{15 16} which do not produce excess endothelin-1 in the vasculature. Also of interest is the fact that the attenuation of vascular hypertrophy seen after chronic treatment with an endothelin antagonist is greater in small mesenteric arteries⁹ than in conduit arteries such as aorta (which is only mildly affected) or large mesenteric arteries (which do not appear to respond to endothelin antagonism²⁶ ). The proposal that overexpression of endothelin-1 in blood vessels could play a role in vascular hypertrophy is supported by the demonstration that endothelin-1 has hypertrophic properties in vitro.^{27 28 29} In the present study, we show that vascular endothelin-1 expression is significantly enhanced in L-NAME–induced malignant hypertension in SHR. However, previous reports have demonstrated that in L-NAME–induced hypertension, there is little vascular hypertrophy.^{17 18 19 20} The present study shows that the severity of vascular hypertrophy is increased in aorta and to a lesser extent in the mesenteric arterial tree but that there is no enhancement of vascular hypertrophy in small coronary, renal, femoral, and mesenteric arteries (except perhaps in the latter, when the cross section of the media is evaluated after correction for body weight differences) in L-NAME–treated SHR compared with untreated SHR. Thus, this study provides findings that could be deemed somewhat surprising: accentuation of vascular hypertrophy in conduit and large arteries in L-NAME–treated SHR but little or no accentuation of vascular hypertrophy in small resistance-sized arteries in four vascular beds, together with absence of increased severity of cardiac hypertrophy, at the same time that endothelin-1 expression in blood vessels, which should have hypertrophic effects, is enhanced in aorta and to a lesser degree in mesenteric arteries. These apparently contradictory findings occur in the presence of a systolic blood pressure that is elevated to malignant levels in L-NAME–treated SHR, 246±2 mm Hg, versus 211±2 mm Hg in untreated SHR, and that would therefore be expected to be associated with severe blood pressure–dependent cardiovascular hypertrophy.

Recent work has emphasized the surprising finding that L-NAME–induced hypertension is not accompanied by left ventricular hypertrophy³⁰ and is associated with little^{17 18 20} or no vascular hypertrophy of resistance-sized arteries,¹⁹ this despite the fact that the renin-angiotensin system may be activated in this model,³¹ which should contribute to hypertrophy. We now additionally find in L-NAME–treated SHR that not only is the renin-angiotensin system activated (plasma renin activity is extremely elevated) but also endothelin-1 expression in blood vessels and plasma endothelin immunoreactivity are increased, which should also contribute to hypertrophy.^{27 28 29} Furthermore, since nitric oxide has been shown to inhibit growth of smooth muscle cells in vitro,³² it would be expected that under L-NAME, which inhibits nitric oxide synthase, hypertrophy would be enhanced, since the potential tonic inhibitory effect of nitric oxide on smooth muscle cell growth is no longer present. It could thus be expected that in L-NAME–treated SHR, vascular hypertrophy would be exaggerated, particularly considering that another potentially hypertrophic system, the renin-angiotensin system, is also activated in this model. Since hypertrophy of blood vessels and the heart does not occur to the degree expected, this suggests (as proposed previously²⁰ ) that L-NAME could have a direct or indirect inhibitory effect on cell growth completely independent of its inhibitory effect on nitric oxide synthase, which in fact would be expected to potentiate growth.³² This potential direct or indirect inhibitory effect of L-NAME on cardiovascular growth remains to be demonstrated, but this possibility should be considered in the light of studies that have shown that L-NAME has other actions independent of its ability to inhibit nitric oxide synthesis.³³ The reported antimuscarinic action of L-NAME could be one underlying mechanism for a direct inhibitory effect of L-NAME on cell growth, since muscarinic receptor–mediated stimulation of DNA synthesis and growth factor–like activity has been described in brain-derived cells and other cells.^{34 35} Alternatively, since muscarinic receptors play a crucial role in the hypothalamus, stimulating growth hormone release,³⁶ the antimuscarinic effect of L-NAME could exert itself indirectly by interfering with growth hormone secretion or through other mechanisms that remain to be established. The putative direct or indirect inhibitory effect of L-NAME on growth could be exerted in the heart and in small arteries and to a lesser degree in large arteries, perhaps because of a countervailing hypertrophic influence²⁹ of the enhanced expression of endothelin-1, predominantly in the latter. It appears from the present results that the enhanced expression of endothelin-1 in aorta and large mesenteric arteries is still able to accentuate aortic and mesenteric artery hypertrophy in this model. In part, the persistence of the hypertrophy of large arteries potentially induced by endothelin-1 may be related to our previous observation that the attenuation of vascular hypertrophy seen after chronic treatment of DOCA-salt hypertensive rats with an endothelin antagonist is greatest in small mesenteric arteries⁹ and less in conduit arteries such as aorta, whereas there is no attenuation of the hypertrophy of large mesenteric arteries.²⁶ This could suggest that endothelin-1–dependent vascular hypertrophy may be antagonized more easily in small arteries, perhaps by the potential direct inhibitory effect of L-NAME. The effects of endothelin could be more difficult to block in large arteries either pharmacologically (with an endothelin antagonist²⁶ ) or physiopathologically (in L-NAME–induced hypertension, present study). Alternatively, there may be a decreasing severity of enhancement of endothelin-1 gene expression distally from the aorta, which appears to be the case, since mesenteric artery endothelin-1 mRNA expression is only doubled, in contrast to the eightfold increase in aorta. Preliminary experiments using in situ hybridization histochemistry have suggested that the abundance of mRNA transcripts may be lower in small arteries of L-NAME–treated SHR than in untreated SHR, which may indicate that the results of this study could also be interpreted to be a consequence of absence of upregulation of endothelin-1 synthesis in small resistance-sized mesenteric arteries, in agreement with the apparent decreasing gradient of overexpression of endothelin-1 from the aorta distally to mesenteric arteries. Since enhanced expression of endothelin-1 in the heart occurs in the endothelium of coronary arteries and not in the myocardium in hypertensive models overexpressing endothelin-1⁷ and since long-term endothelin receptor antagonist treatment (which attenuates vascular hypertrophy in DOCA-salt hypertensive rats) does not affect left ventricular hypertrophy, a role of endothelin-1 in cardiac hypertrophy in L-NAME–treated SHR would not be expected. Thus, the absence of accentuation of cardiac hypertrophy in L-NAME–treated SHR in the present study despite activation of the vascular expression of the endothelin-1 gene and malignant levels of blood pressure is probably unrelated to changes in endothelin-1 expression.

The mechanisms involved in the enhancement of expression of the endothelin-1 gene in L-NAME–treated SHR were not investigated in this study. Vasopressin, angiotensin II, thrombin, epinephrine, and transforming growth factor-ß^{37 38 39 40} and pressure^{41 42} are some of the factors that could play a role in enhanced endothelin-1 production and secretion. The renin-angiotensin system is activated in L-NAME–treated rats.³¹ In 2K1C Goldblatt hypertensive rats, in which the renin-angiotensin system is activated, there is no enhancement of expression of endothelin-1 in blood vessels (authors' unpublished observations, 1995), suggesting that angiotensin II, which may stimulate endothelin-1 expression in endothelial cells,^{37 39} is not involved. Mineralocorticoids, which could be stimulated by the increased circulating levels of angiotensin II, are also probably not involved, since endothelin-1 vascular overexpression also occurs in 1K1C Goldblatt hypertensive rats, in which mineralocorticoids do not play a role (authors' unpublished observations, 1995). Since blood pressure rises to malignant levels in L-NAME–treated SHR (as well as in DOCA-salt SHR, which also overexpress endothelin-1 in blood vessels⁸ ), very high blood pressure could participate directly or indirectly in the mechanisms underlying the elevated expression of endothelin-1 in the vasculature.^{41 42} However, blood pressure may not be the only factor, since SHR^{4 13} and 2K1C Goldblatt hypertensive rats (unpublished observations, 1995) have elevated blood pressure but no enhancement of vascular expression of the endothelin-1 gene. Finally, since under L-NAME treatment nitric oxide synthesis is abrogated, a potential role of the latter in inhibiting endothelin-1 synthesis could result in enhanced expression of endothelin-1 when nitric oxide synthase is inhibited by L-NAME. The possibility of a regulatory role of nitric oxide in endothelin production has already been proposed on the basis of in vitro experiments.^{43 44} Thus, nitric oxide, which is already known to be a vasorelaxant and inhibitor of smooth muscle cell growth,³² could also exert antihypertensive effects through inhibition of endothelin-1 production.

In conclusion, this study shows that in the malignant form of hypertension induced by L-NAME administration to SHR, vascular expression of endothelin-1 mRNA transcripts is elevated in aorta and to a lesser extent in the mesenteric arterial tree. Associated with this is an accentuation of hypertrophy of aorta and of the mesenteric arterial bed, in agreement with endothelin-1 overexpression at these levels of the vasculature, but there is no increase in the severity of hypertrophy of the heart or of small resistance-sized arteries of the coronary, renal, femoral, and mesenteric circulations, despite the presence of malignant levels of elevated blood pressure. These data are compatible with a vascular hypertrophic effect of endothelin-1 gene overexpression in blood vessels, but they also suggest that there may be a direct or indirect inhibitory action of L-NAME on cardiovascular growth, independent of its inhibitory effects on nitric oxide synthase, which, on the contrary, would in fact be expected to favor growth of vascular smooth muscle cells. This potential inhibitory effect of L-NAME on cardiovascular growth may be counterbalanced by endothelin-1 overexpression in vessels such as the aorta and large mesenteric arteries. Finally, the results of this study may suggest a role for nitric oxide in the regulation of endothelin-1 production by the endothelium of blood vessels.

	Selected Abbreviations and Acronyms

 

1K1C = one-kidney, one clip 2K1C = two-kidney, one clip DOCA = deoxycorticosterone acetate L-NAME = NG-nitro-L-arginine methyl ester PCR = polymerase chain reaction PSS = physiological salt solution SHR = spontaneously hypertensive rats

	Acknowledgments

 
This work was supported by a group grant from the Medical Research Council of Canada to the Multidisciplinary Research Group on Hypertension and by grants from the Fondation des maladies du cur du Québec.

	Footnotes

 
Reprint requests to Ernesto L. Schiffrin, MD, PhD, Clinical Research Institute of Montreal, 110 Pine Ave W, Montreal, Quebec, Canada H2W 1R7.

Received June 19, 1995; first decision July 28, 1995; accepted September 18, 1995.

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