Comparison of Effect of Endothelin Antagonism and Angiotensin-Converting Enzyme Inhibition on Blood Pressure and Vascular Structure in Spontaneously Hypertensive Rats Treated With Nω-Nitro-l-Arginine Methyl Ester
Correlation With Topography of Vascular Endothelin-1 Gene Expression
Inhibition of nitric oxide synthase by l-arginine analogues such as Nω-nitro-l-arginine methyl ester (L-NAME) in spontaneously hypertensive rats (SHR) is associated with malignant hypertension and enhanced expression of the endothelin-1 gene in some blood vessels. In this study, SHR treated chronically with L-NAME (SHR–L-NAME) were given the angiotensin I–converting enzyme inhibitor cilazapril or the endothelin-A/endothelin-B receptor antagonist bosentan for 3 weeks. Systolic pressure was lowered slightly by cilazapril (213±2 versus 229±2 mm Hg in untreated SHR–L-NAME, P<.01) but was not significantly lowered by bosentan (223±2 mm Hg). Hypertrophy of aorta and small arteries (coronary, renal, mesenteric, and femoral) was decreased by cilazapril treatment and unaffected by bosentan. Expression of the endothelin-1 gene was evaluated in SHR–L-NAME by in situ hybridization histochemistry, which showed that endothelin-1 expression was enhanced in the endothelium of aorta but not in small mesenteric arteries in these rats. The absence of enhancement of endothelin-1 gene expression in small arteries may account for the absence of increased severity of hypertrophy of small vessels in SHR–L-NAME and may be a mechanism whereby L-NAME inhibits cardiovascular growth. These results suggest that in the absence of enhanced small-artery endothelin-1 expression, endothelin antagonism does not lower blood pressure. The blood pressure–lowering effect of angiotensin I–converting enzyme inhibition suggests a role for the renin-angiotensin system in the malignant form of hypertension that develops in SHR treated with L-NAME.
Endothelins may participate in mechanisms leading to hypertension via vasoconstrictor or vascular hypertrophic effects.1 Enhanced ET-1 gene expression has been demonstrated in arteries from different vascular beds of DOCA-salt hypertensive rats,2 3 4 5 malignant DOCA-salt–treated SHR,6 and recently in one-kidney, one clip (1K1C) Goldblatt hypertensive rats7 as well as in the severe hypertension induced by treatment with the nitric oxide synthase inhibitor L-NAME in SHR.8 DOCA-salt hypertensive rats9 and DOCA-salt SHR10 exhibit a slight but significant reduction in the severity of elevated BP when treated chronically with endothelin receptor antagonists; this has also been shown in acute experiments with intravenous administration of selective ETA receptor antagonists.11 12 This suggests that enhancement of vascular ET-1 gene expression may play a role in some forms of experimental hypertension, such as DOCA-salt hypertensive rats, DOCA-salt SHR,6 and 1K1C hypertensive rats,7 in which severe vascular hypertrophy is present,6 13 14 but not in 2K1C hypertensive rats7 or in SHR,4 15 in which small-artery hypertrophy is less severe.14 16 In addition, endothelin antagonism did not affect BP acutely in SHR,11 nor did long-term treatment prevent the development of hypertension or affect the maintenance of elevated BP,17 18 in agreement with the lack of excess vascular expression of ET-1.
We recently demonstrated that when SHR are treated with L-NAME, they develop malignant hypertension associated with some enhancement of the aortic hypertrophy already present in SHR but show little increase in the severity of small-artery hypertrophy, although in larger blood vessels, increased ET-1 gene expression is detectable.8 To determine whether the model of malignant hypertension induced by L-NAME in SHR exhibited an endothelin-dependent component, we examined the response of BP and vascular structure to endothelin antagonism with the ETA/ETB receptor antagonist bosentan19 in this hypertensive model. L-NAME induces hypertension with few vascular changes,20 21 22 23 even though endothelin gene expression is enhanced. Thus, the response to bosentan might demonstrate not only the presence of endothelin dependency but also the potential role on BP elevation of vascular structure altered by endothelin overexpression. The effect of bosentan on BP and vascular structure was compared with that of the ACE inhibitor cilazapril because L-NAME treatment is known to stimulate the renin-angiotensin system,24 another potential cause of vascular hypertrophy, and ACE inhibition has been shown to affect favorably vascular structure in hypertension.25 Hence, this model should be more susceptible to the BP-lowering action of these drugs. In addition, we carried out in situ hybridization histochemistry to further clarify the potential relationship of results with topographical localization of sites of ET-1 gene overexpression in the vasculature and to establish whether small arteries participated in the overexpression of ET-1 detected previously by Northern blot analysis in larger vessels.8
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 13 weeks of age, they received tap water or water containing L-NAME at a concentration adjusted daily such that rats received 30 mg/kg per day. This dose is relatively well tolerated by SHR, whereas higher doses, used in Sprague-Dawley rats,20 21 have been associated with a high mortality in treated SHR within a few days. Since the day L-NAME administration was begun, half of the rats also received 100 mg/kg per day bosentan mixed with powdered chow. It has been shown that this oral dose blocks the pressor action of intravenously injected ET-1 for more than 24 hours.17 18 Bosentan was a kind gift of Dr Martine Clozel, Pharma Research, F Hoffmann–La Roche Ltd, Basel, Switzerland. Systolic BP was measured indirectly in prewarmed, restrained rats by the tail-cuff method and recorded on a polygraph (model 7, Grass Medical Instruments) fitted with a 7P8 preamplifier and a model PCPB photoelectric pulse sensor before treatment was started and at 7 and 14 days of treatment. The average of three pressure readings was recorded. Rats were killed by decapitation after the last BP measurement. Because BP measurements by the tail-cuff method may not be precise when systolic BP is above 220 mm Hg, particularly in malignant hypertensive rats,8 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 with rats under methohexital sodium anesthesia (50 mg/kg IP, Eli Lilly) for telemetric recording of arterial pressure (Data Sciences International).26 These SHR were then offered L-NAME and some of them received bosentan, as had the previous groups of SHR. Hourly averages of 10-second samples obtained every 5 minutes were used to obtain BPs of these SHR–L-NAME and bosentan-treated SHR–L-NAME. L-NAME treatment is associated with renin stimulation, and SHR are sensitive to treatment with ACE inhibitors; therefore, for comparison with the bosentan-treated SHR–L-NAME, a group of rats similar to those subjected to bosentan treatment was treated with the ACE inhibitor cilazapril (kindly provided by Dr J.-P. Clozel, F Hoffmann–La Roche Ltd) given at a daily dose of 10 mg/kg body wt in their drinking water.
Preparation of Small Arteries for Morphometry
The technique for dissection of small vessels from the heart, kidney cortex, and femoral region of the rat has been previously described.17 18 The heart and 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 of about 2 mm in length was dissected. For dissection of coronary vessels, the right ventricle was opened to expose coronary arteries on the interventricular septum. The interventricular artery was followed to the cardiac apex; the chordae tendinae and myocardium were separated, and a 2-mm-long vessel was isolated. For 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 followed distally. A renal arcuate artery of about 2 mm in length was isolated. Mesenteric small arteries were obtained as previously described.13 14 16 17 18 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 from the intestine and about 2 mm in length was isolated. The vessels were mounted as ring preparations on an isometric myograph (Living Systems Instrumentation). The dissection and mounting were performed in room-temperature physiological salt solution (PSS) of the following composition (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 were 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 about 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 ×320 magnification at 12 different sites along the wall, which were averaged. The vessels were set to L0, where L0=0.9·L100 and L100 is the internal circumference that the vessels would have had in vivo when relaxed and under a transmural pressure of 100 mm Hg. Then 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 to ensure that the vessels isolated were arteries and developed a tension of greater than 2 mN/mm.
In Situ Hybridization Histochemistry
Mesenteric vessels and thoracic aortas were immediately removed from rats, frozen in isopentane cooled to −35°C, and stored at −80°C until processed for in situ hybridization histochemistry. Frozen sections of arteries (10 μm thick) were obtained on a Bright-Hacker cryostat, thaw-mounted on polylysine-coated glass slides, and stored at −80°C until processed for in situ hybridization as described previously.4 5 Tissue sections from both SHR–L-NAME and SHR were placed together on the same glass slide, thus permitting a direct comparison of the labeled sections and eliminating interslide variability. Frozen sections were fixed in 0.1 mol/L phosphate-buffered 4% formaldehyde solution, pH 7.2, for 60 minutes and then washed three times in 0.05 mol/L phosphate-buffered saline, pH 7.4, for 10 minutes each. Deproteination was carried out with proteinase K (0.1 μg/mL) for 5 minutes at 37°C. Slides were transferred to 0.1 mol/L triethanolamine, pH 8.0, and incubated in the same solution containing acetic anhydride (0.25% vol/vol) for 10 minutes at room temperature. Sections were then rinsed in 2× SSC and dehydrated in ethanol (50% to 100%). Sense and antisense strand radioactive ET-1 RNA probes were diluted in hybridization buffer (75% formamide, 10% dextran sulfate, 3× SSC, 50 mmol/L NaPO4 [pH 7.4], 1× Denhardt's solution [0.02% each of Ficoll 400, polyvinylpyrrolidone, and bovine serum albumin], and 0.1 mg/mL yeast RNA) to a final concentration of 40×103 disintegrations per minute per microliter. Dithiothreitol was added to a final concentration of 10 mmol/L. Hybridization mix (25 μL per slide) was applied, and sections were coverslipped. The tissues were incubated in a hybridization oven at 55°C for 16 hours. The next day, coverslips were removed in 2× SSC. Sections were treated with RNase A (40 μg/mL) at 37°C for 45 minutes to remove single-stranded RNA molecules. Successive washes at room temperature followed in 2×, 1×, and 0.5× SSC for 10 minutes each and in 0.1× SSC at 60°C for 1 hour. The tissue was then dehydrated, and sections were dipped in Kodak NTB2 nuclear emulsion (diluted 1:1 with water) and stored at 4°C. After 6 weeks of exposure, autoradiograms were developed in Kodak D19 at full strength for 2 minutes and fixed in 30% sodium thiosulfate for 4 minutes. For identification of cellular structures, the sections were counterstained with cresyl violet. Specificity of labeling was established by incubation of adjacent sections with the sense strand ET-1 RNA probe of the same size and specific activity as the antisense strand probe. Tissues from eight rats were sectioned (four SHR–L-NAME and four SHR), and multiple sections for each group were examined (minimum of 12 tissue sections).
Preparation of Rat ET-1 Probe
The rat ET-1 sense and antisense RNA probes were prepared by RNA transcription reaction with T7 or SP6 RNA polymerase, respectively, and a cDNA plasmid construction previously described.4 5 Briefly, a polymerase chain reaction product, obtained by amplification of cDNA from rat lung with specific oligonucleotide primers3 derived from the cloned rat prepro-ET-1 sequence,27 was subcloned into pGEM7zf(+). From this construction, single strand sense or antisense ET-1 RNA probes of 319 bp were generated. Radiolabeled riboprobes were prepared with 35S-UTP and 35S-CTP (1250 Ci/mmol, Amersham) in the same labeling reaction. Transcription reaction mixtures contained 250 μCi (200 pmol) each of 35S-UTP and 35S-CTP, 150 μmol/L each of ATP and GTP, 12.5 mmol/L dithiothreitol, 20 U RNAse inhibitor, the appropriate linearized plasmid preparation in 1 μL (1 μg/μL concentration), and 6 U of SP6 or T7 RNA polymerase in a total volume of 10 μL. The reactions were carried out for 60 to 90 minutes at 37°C. The specific activity of each of these probes is estimated to be approximately 2×105 Ci/mmol.
Observation and Photography
Labeled tissue sections were observed with a Zeiss Axiophot microscope equipped with a Darklite Illuminator (Micro Video Instruments, Inc). All photographs were taken with double exposure settings. The autoradiographic grains were first exposed to Kodak Tungsten 64 ASA slide film under dark light illumination, thus resulting in the white grain appearance. The second exposure was taken under brightfield illumination with the lamp temperature set at 3200°K and the use of two neutral gray filters. This second exposure shows the underlying tissue structure counterstained with cresyl violet. The camera was set on automatic exposure at 800 ASA for the darkfield exposure and 200 ASA for the brightfield exposure. Use of a double exposure also permits the proper focusing adjustment on the autoradiographic grains of the underlying tissue structure. Color prints were produced with the color slides obtained as negatives.
Measurement of Plasma Endothelin and Plasma Renin Activity
During the first few seconds after rats were decapitated, blood was collected from the neck in tubes containing potassium EDTA for measurement of plasma ET-1 and plasma renin activity. Immunoreactive ET-1 was extracted from plasma by passage through C18 Sep-Pak cartridges (Waters Associates) and measured by radioimmunoassay as previously described.2 7 The antibody against ET-1 was from Peninsula Laboratories. The minimum detectable endothelin concentration was 0.4 pmol/L, and recovery of 5 pmol/L of ET-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 37°C as previously described.2
The media cross-sectional area of small arteries (A) was obtained from the media thickness (m) and 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+πm2. Using L0 and the calculated media cross-sectional area and assuming a constant media volume, we then calculated the standardized media thickness of blood vessels (at L0). The lumen diameter was obtained by L0/π.
Values are given as mean±SE. Statistical differences were evaluated by one-way or repeated measures ANOVA as appropriate, followed by Newman-Keuls post hoc test. Results were considered significantly different at a value of P<.05.
BP, Body Weight, Plasma Renin Activity, and Plasma Immunoreactive Endothelin
Compared with SHR not receiving L-NAME, SHR–L-NAME showed a significantly faster rise in BP and reduced increase in body weight, particularly during the second week of treatment with L-NAME, regardless of whether they received the endothelin antagonist bosentan or ACE inhibitor cilazapril (Fig 1⇓, Table 1⇓). However, SHR–L-NAME that received cilazapril achieved a slightly but significantly lower (P<.01) systolic BP than SHR–L-NAME not receiving antihypertensive treatment or those treated with bosentan; they also had slightly higher body weight. To confirm that bosentan-treated SHR–L-NAME did not exhibit diastolic or mean BP differences compared with untreated rats and that tail-cuff systolic BP results were reliable, we implanted 10 SHR with intravascular catheters and treated them with L-NAME (n=4) and bosentan (n=4) or did not treat them (n=2), similar to the previous group. Systolic intravascular BP at the end of the experiment is shown in Table 1⇓ for comparison with tail-cuff BP values. Twenty-four-hour systolic and diastolic BPs on selected days under treatment are also shown in Fig 2⇓. Systolic and diastolic BPs were already significantly higher than baseline (P<.001) on the day after L-NAME administration (significance not indicated on figure for clarity). No differences were present between the means of 24-hour systolic and diastolic intravascular BPs of the SHR–L-NAME and bosentan-treated SHR–L-NAME throughout the treatment period.
Plasma renin activity was very high in SHR–L-NAME (Table 1⇑), as found in previous studies.8 Cilazapril-treated rats also exhibited very high renin levels, as expected under ACE inhibitor treatment. Plasma renin activity was not measured in rats treated with bosentan because plasma volume from rats of this group was insufficient. Endothelin plasma levels were significantly lower in cilazapril-treated SHR–L-NAME than in other groups of SHR–L-NAME.
Heart and Conduit Artery Weights
Heart weight was not increased in SHR–L-NAME, nor was it affected by antihypertensive treatment (Table 1⇑). The weight per unit length of segments of thoracic aorta was greater in SHR–L-NAME than in untreated SHR despite the lower body weight of the former, suggesting that hypertrophy of conduit arteries was more severe in SHR–L-NAME (Table 1⇑). Cilazapril treatment resulted in complete correction of aortic hypertrophy, whereas bosentan did not affect it.
Investigation on a wire myograph of small arteries from the coronary, renal, mesenteric, and femoral circulations (lumen diameter of approximately 200 μm) showed that lumen diameter, media width, media cross-sectional area, and media width–to–lumen diameter ratio did not differ in any of the vascular beds between SHR and SHR–L-NAME treated without or with bosentan (Table 2⇓). However, SHR–L-NAME treated with cilazapril exhibited significant reductions in media width, cross-sectional area of the media, and media-to-lumen ratio of small arteries of the four vascular beds.
In Situ Hybridization Histochemical Demonstration of Topographical Variations in Vascular ET-1 Gene Expression
In a previous study,8 SHR–L-NAME exhibited enhanced vascular expression of the ET-1 gene compared with untreated SHR, to a greater degree in aorta than mesenteric arteries. In that study and the current one, SHR–L-NAME had accentuated aortic hypertrophy but no excess mesenteric small-artery hypertrophy. To determine whether this and the absence of BP lowering by bosentan were the result of differences in the extent of vascular ET-1 gene overexpression in large and small arteries, we performed in situ hybridization histochemistry. In agreement with Northern blot analysis data of the previous study,8 Table 3⇓, Fig 3⇓, and Fig 4⇓ show that the abundance of ET-1 mRNA transcripts demonstrated by in situ hybridization with the ET-1 antisense probe was increased in the endothelium of the aorta of SHR–L-NAME (Figs 3B and 4B⇓⇓) compared with that of SHR (Figs 3A and 4A⇓⇓). Little reaction occurred with the sense probe, shown only for SHR–L-NAME, in which grains had no specific anatomic localization (Fig 3C⇓). However, the density of grains corresponding to hybridization of the ET-1 antisense probe with ET-1 mRNA was lower in the endothelium of small mesenteric arteries of SHR–L-NAME (Fig 3F⇓, and at greater magnification, Fig 4D⇓) than of untreated SHR (Fig 3D⇓, and at greater magnification, Fig 4C⇓). Fig 3E⇓ shows results with the sense probe control of Fig 3D⇓. Table 3⇓ confirms these data, showing the results of grain counting in slides from different rats for both aorta and the smaller branches of mesenteric arteries.
The present study demonstrates that BP elevation in SHR–L-NAME is endothelin independent based on the absence of response to chronic endothelin receptor antagonism. On the other hand, there is a renin-dependent component of the hypertension, as demonstrated by BP lowering by cilazapril, an ACE inhibitor. The absence of endothelin dependency is surprising because it was previously shown that in SHR–L-NAME ET-1 expression was enhanced in large arteries.8 Studies from our laboratory have shown that when the vascular contents of ET-1 mRNA transcripts and immunoreactive ET-1 are elevated in DOCA-salt hypertensive rats4 5 or in DOCA-salt SHR,10 long-term treatment (3 weeks) with the endothelin antagonist bosentan resulted in some lowering of BP (20 mm Hg).9 10 Together with this BP lowering, there was a regression of vascular hypertrophy.9 28 On the basis of these data, we proposed that ET-1, which exhibits mitogenic and hypertrophic properties on smooth muscle cells,29 30 could play a direct role in the development of vascular hypertrophy in some models of experimental hypertension.1 9 In contrast to those data,9 10 28 the present study shows that chronic endothelin receptor antagonism in SHR–L-NAME neither lowered BP nor induced regression of vascular hypertrophy. This resembles results in SHR, which do not overexpress ET-1 in blood vessels2 6 and do not respond to endothelin antagonism with lowering of BP or regression of vascular hypertrophy.11 17 18
Hypertensive models that overexpress ET-1 have severe vascular hypertrophy (eg, DOCA-salt hypertensive rats,13 DOCA-salt SHR,6 and 1K1C Goldblatt hypertensive rats14 ). In contrast, experimental hypertensive models that do not overexpress ET-1 in blood vessels exhibit less-severe vascular hypertrophy, as in the case of SHR16 and 2K1C Goldblatt hypertensive rats.14 In L-NAME–induced hypertension, there is little vascular hypertrophy.20 21 22 23 In SHR–L-NAME, the severity of hypertrophy of aorta and to a lesser extent of the mesenteric arterial tree was greater than in untreated SHR, but vascular hypertrophy in small coronary, renal, femoral, and mesenteric arteries was not enhanced.8 Thus, if ET-1 expression in blood vessels correlated with vascular hypertrophy in SHR–L-NAME, vascular expression of ET-1 could present regional variations. The present study shows with in situ hybridization histochemistry that enhanced ET-1 expression is limited to large blood vessels, which exhibit a greater degree of hypertrophy in SHR–L-NAME than in untreated SHR. However, overexpression of ET-1 was not found in small vessels of SHR–L-NAME, which do not present enhanced hypertrophy. Overexpression of ET-1 limited to large arteries in SHR–L-NAME may explain the absence of enhancement of small-artery hypertrophy in these rats compared with untreated SHR and may suggest that the lack of a BP-lowering effect of bosentan is causally related to the absence of endothelin-dependent hypertrophy of small arteries in SHR–L-NAME. This may also indicate that endothelin-dependent small-artery hypertrophy plays a role in an endothelin-dependent component of BP elevation.
L-NAME–induced hypertension not only presents little vascular hypertrophy of resistance-sized arteries20 21 22 23 but is also paradoxically associated with the absence of significant left ventricular hypertrophy.31 Whereas the absence of ET-1 gene overexpression in small arteries may be part of the mechanism whereby L-NAME treatment abrogates the development of vascular hypertrophy in hypertensive models, ET-1 overexpression in the heart, when it occurs in hypertension, is localized in the endothelium of large and small coronary arteries and appears not to occur to a significant degree in the myocardium.5 Long-term treatment of DOCA-salt hypertensive rats9 or DOCA-salt SHR28 with the endothelin receptor antagonist bosentan did not blunt the progression of cardiac hypertrophy, although it prevented the progression of small-artery hypertrophy. This suggests that if L-NAME has an inhibitory effect on growth in the heart, as we have previously suggested,8 23 it is not related to a direct or indirect inhibition of ET-1 production, whereas in small arteries it may be partly related to the absence of enhanced endothelin expression.
In SHR–L-NAME, plasma renin activity is elevated (present results and Reference 8), and previous reports have shown that L-NAME–induced hypertension is partially renin dependent.24 It is therefore not surprising that cilazapril treatment lowered the BP of SHR–L-NAME. Despite the fact that the BP lowering by cilazapril was not very dramatic, regression of vascular hypertrophy (of aorta and small arteries in the four circulations examined) was marked. This may indicate that regression was a direct consequence of the pharmacological effect of cilazapril, whether via inhibition of angiotensin II generation, increased accumulation of kinins, or other effects of the ACE inhibitor but independent of the lowering of BP. Plasma renin activity was not measured in bosentan-treated rats because plasma volume was insufficient for measurement of endothelin immunoreactivity and renin and determination of the former appeared more critical in this study. Although bosentan treatment could further enhance the already high renin production of SHR–L-NAME, which could have antagonized the lowering of BP by long-term endothelin antagonism in these rats, recent studies suggest that endothelins do not affect renin secretion in either normal or 2K1C rats32 or in 1K1C (normal renin) or 2K1C (high renin) hypertensive rats.33 It is therefore unlikely in the present study that plasma renin activity was altered in SHR–L-NAME treated with bosentan compared with untreated SHR–L-NAME, and accordingly, the missing data are less relevant.
Plasma immunoreactive endothelin of SHR–L-NAME was normalized by cilazapril treatment. Since the model examined has a severe or malignant form of hypertension, it is possible that endothelial damage together with increased endothelin production in some vessels is associated with enhanced spillover, leading to elevated circulating levels of endothelin. Cilazapril, by lowering BP and perhaps through a direct beneficial effect on endothelial function,34 may reduce this endothelin spillover into the bloodstream. It is also possible that ACE inhibitors reduce endothelin gene expression, but this remains to be proved. Finally, there is evidence of pituitary contribution to circulating endothelin immunoreactivity,35 which may increase under some pathological conditions. Since plasma vasopressin may be increased in malignant hypertension36 and plasma endothelin and vasopressin may rise and fall in parallel,35 it is also possible that lowering of plasma endothelin immunoreactivity in SHR–L-NAME treated with cilazapril is secondary to effects on the pituitary. This requires further investigation.
Since endothelial ETB receptors may antagonize the vasopressor effects of endothelins by stimulating nitric oxide and prostacyclin release,37 blockade of endothelial ETB receptors by bosentan, a combined ETA/ETB receptor antagonist,19 could reduce the BP-lowering effect of endothelin receptor antagonism. However, L-NAME inhibits nitric oxide generation, leaving only blockade of the potential ETB receptor–mediated stimulation of prostacyclin production as a factor. Moreover, bosentan administered chronically lowered BP in DOCA-salt hypertensive rats9 and DOCA-salt SHR,10 which is also observed with ETA receptor antagonists given intravenously,11 12 and failed to lower BP in SHR,17 18 similar to findings with an intravenous ETA receptor antagonist.11 The potential pathophysiological participation of endothelial ETB receptors is still uncertain. Furthermore, bosentan, although a combined ETA/ETB receptor antagonist, may act predominantly as an ETA antagonist when given chronically, since its affinity for ETB receptors is in the high nanomolar range, whereas that for ETA receptors is in the low nanomolar range.19 Thus, it is unlikely that bosentan failed to lower BP because of the blockade of endothelial ETB receptors. However, this remains to be further explored through long-term administration of orally active endothelin receptor antagonists that are more selective for the ETA receptor.
In conclusion, this study shows with in situ hybridization histochemistry that in the malignant form of hypertension induced by L-NAME administration in SHR, vascular expression of ET-1 mRNA transcripts is elevated in the aorta but not small arteries of the mesenteric circulation. This may explain the absence of enhanced vascular hypertrophy of small arteries, the absence of a BP-lowering effect of the endothelin antagonist bosentan despite increased conduit artery ET-1 expression, and the lack of effect of bosentan on vascular structure in SHR–L-NAME. In contrast, ACE inhibition slightly lowered BP and caused significant regression of large- and small-artery hypertrophy in SHR–L-NAME, demonstrating that the renin-angiotensin system but not endothelin plays a significant role in this form of experimental malignant hypertension. Finally, the elevation of BP in SHR to malignant levels after receiving L-NAME without accentuation of hypertrophy of small arteries in several vascular beds (Reference 8 and this study) may cast doubts on the necessary participation of hypertrophy of small, resistance-sized arteries in BP elevation in some forms of hypertension.
Selected Abbreviations and Acronyms
|ACE||=||angiotensin I–converting enzyme|
|ETA, ETB||=||endothelin A, endothelin B|
|L-NAME||=||Nω-nitro-l-arginine methyl ester|
|SHR||=||spontaneously hypertensive rat(s)|
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 coeur du Québec. The authors thank André Turgeon and Micheline Vachon for expert technical help.
Reprint requests to Ernesto L. Schiffrin, MD, PhD, Clinical Research Institute of Montreal, 110, Pine Ave W, Montreal, Quebec, Canada H2W 1R7.
- Received November 20, 1995.
- Revision received January 9, 1996.
- Accepted April 4, 1996.
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