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Circulation. 1995;91:1229-1235

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(Circulation. 1995;91:1229-1235.)
© 1995 American Heart Association, Inc.

Articles

Direct Measurements of Endothelium-Derived Nitric Oxide Release by Stimulation of Endothelin Receptors in Rat Kidney and Its Alteration in Salt-Induced Hypertension

Yasunobu Hirata, MD; Hiroshi Hayakawa, MD; Etsu Suzuki, MD; Kenjiro Kimura, MD; Kazuya Kikuchi, PhD; Tetsuo Nagano, PhD; Masaaki Hirobe, PhD; Masao Omata, MD

From the Second Department of Internal Medicine (Y.H., H.H., E.S., K. Kimura, M.O.) and Faculty of Pharmaceutical Sciences (K. Kikuchi, T.N., M.H.), University of Tokyo, Japan.

Correspondence to Yasunobu Hirata, MD, the Second Department of Internal Medicine, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan.


*    Abstract
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 *Abstract
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Background Stimulation of endothelin subtype B (ETB) receptors has been proposed to induce release of endothelium-derived nitric oxide (EDNO).

Methods and Results To obtain direct evidence of its release and its alteration in deoxycorticosterone acetate (DOCA)-salt hypertension, EDNO released from renal vessels by ET stimulation was assayed by a highly sensitive chemiluminescence method. Kidneys were isolated from DOCA-salt and control rats, and renal perfusion pressure (RPP) and EDNO (by hydrogen peroxide–luminol chemiluminescence) in the perfusate were monitored simultaneously during perfusion of ET-1, ET-3, an ETA receptor antagonist (BQ-123), and an ETB receptor agonist (BQ-3020). In control rats, ET-1 and ET-3 dose-dependently increased both RPP and NO release. Although the vasoconstricting effects of ET-1 were greater, their NO-releasing effects were comparable. The increase in NO release by ETs was inhibited by NG-monomethyl-L-arginine. After 10-6 mol/L BQ-123 treatment, ET-1 decreased RPP and increased NO release in control kidneys. DOCA-salt rats responded to these agents with much less NO release. BQ-3020 at up to 10-10 mol/L caused vasodilation (RPP, 10-11 mol/L, -5.4±1.7%, P<.01) associated with increased NO release in control kidneys (+9.0±2.7 fmol · min-1 · g-1 kidney wt, P<.01). However, in DOCA-salt kidneys, BQ-3020 caused renal vasoconstriction (RPP, +5.4±2.4%, P<.01 versus control) and a much smaller NO release (+1.1±0.4 fmol · min-1 · g-1 kidney wt, P<.01 versus control). Northern blot analysis revealed that renal ETB mRNA was significantly decreased in DOCA-salt rat kidneys compared with controls (0.36±0.13 versus 1.00±0.23, P<.05).

Conclusions These results suggest that ET-1 and ET-3 release EDNO via ETB receptors in renal vessels. ETB-mediated NO release was reduced in DOCA-salt rats, which may modulate renal function and thus blood pressure regulation in DOCA-salt hypertensive rats.


Key Words: endothelium-derived factors • endothelin • hypertension • kidney


*    Introduction
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 up arrowAbstract
 *Introduction
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The vascular endothelium plays a critical role in regulating the vascular tone by secreting endothelium-derived constricting and relaxing factors.1 Among these factors, endothelin (ET) and endothelium-derived nitric oxide (EDNO) have opposing effects on vascular tone and a close relation to each other. ET-1 is one of the most potent known vasoconstrictors in vivo and has been implicated in the pathogenesis of diseases with elevated vascular tone,2 including hypertension.3 However, bolus intravenous administration of the peptide causes a transient but marked reduction followed by a long-lasting elevation of blood pressure.4 An isopeptide, ET-3, shows more hypotensive effects,5 possibly due to ET-induced release of EDNO.6

The molecular cloning of ET receptors has strongly suggested that the biphasic effect of ET can be attributed to a combination of vasoconstriction mediated by ETA receptors on vascular smooth muscle cells7 and vasodilation mediated by ETB receptors on vascular endothelial cells.8 ET-1 binds to both ETA and ETB receptors, whereas ET-3 binds mostly to ETB receptors.7 8 The ETB receptor is an isopeptide nonselective receptor that is thought to cause release of NO when bound.8 9 10 The effect of ETB binding on NO release has been assayed by measurements of NO metabolites, NOx,9 10 and of the second messenger of NO, cGMP, mostly in cultured cells.9 10 11 12 This hypothesis is also supported by data that the vasodilatory effects of ET-1 can be suppressed by NO synthase inhibitors, such as NG-monomethyl-L-arginine (L-NMMA), and by a soluble guanylate cyclase inhibitor, methylene blue, in various vasculature model systems.13 14 15 However, since these experiments did not measure NO directly, simultaneous changes in vascular tone and NO release caused by ETs have not been demonstrated. For example, it is not known whether the transient vasodilation induced by ET is due to a transient release of NO. Furthermore, the involvement of EDNO in ET-induced vasodilation remains controversial. Baydoun et al16 showed that ET-1–induced coronary vasodilation in isolated rat heart was independent of prostaglandin (PG)I2 or EDNO, because neither cyclooxygenase inhibitors nor EDNO inhibitors suppressed ET-induced vasodilation. Ohlstein et al17 observed similar phenomena in a rat hindquarter model. Moreover, Okamura et al18 showed that ET-3–induced vasodilation in dog coronary artery was inhibited by PGI2 synthesis inhibitors but not by EDNO inhibitors.

Endothelium-dependent vasodilation is generally impaired in hypertensive vessels, suggesting the existence of endothelial damage. We have reported that acetylcholine-evoked NO release was markedly reduced in DOCA-salt–induced hypertension.19 20 Various mechanisms may underlie this reduction in DOCA-salt hypertension, including a reduction in NO synthesis from the NO synthase substrate, L-arginine, and an alteration in endothelial receptors. We noted that L-arginine supplementation partially reversed the reduction in NO release and in endothelium-dependent vasodilation by acetylcholine in DOCA-salt hypertensive rats.21 On the other hand, receptor-mediated NO release may also be impaired. Although the response to exogenously administered acetylcholine was reduced in DOCA-salt hypertension, it is unlikely that cholinergic stimulation actually plays a role in endothelium-dependent vasodilation in the kidney in vivo. In contrast, endogenous ET is likely to participate in the pathophysiological regulation of EDNO release via autocrine or paracrine mechanisms.

We recently developed a highly sensitive assay system for EDNO using a chemiluminescent reaction and applied it to the isolated perfused rat kidney to monitor the NO concentration of the perfusate simultaneously with renal perfusion pressure.20 22 In the present study, we used this assay to examine the effects of ET receptor stimulation on renal vascular resistance and NO release in normal and DOCA-salt hypertensive rat kidney.


*    Methods
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 *Methods
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Preparation of DOCA-Salt and Control Rats
DOCA-salt rats were prepared as previously described.23 Wistar rats at the age of 6 weeks were unilaterally nephrectomized, and silicone rubber containing 200 mg DOCA/kg body wt was implanted subcutaneously under light ether anesthesia. Rats were given 0.9% saline to drink for 8 weeks. Control rats were also unilaterally nephrectomized but were not given DOCA or saline. Blood pressure in all rats was measured weekly by a tail-cuff method.

Isolated Perfused Kidney
On the day of the experiment, rats were anesthetized with 30 mg/kg pentobarbital IP, and the right kidney was isolated and perfused as previously described.24 In brief, after an abdominal incision, we punctured the mesenteric artery with an 18-gauge double-lumen needle and positioned the tip in the right renal artery. Perfusion was then started at 5 mL/min with a Krebs-Henseleit buffer, and the kidney was isolated without ischemia. Buffer was saturated with 95% O2/5% CO2 at 37°C and contained 10-6 mol/L phenylephrine to maintain perfusion pressure at about 100 mm Hg. The renal vein was also cannulated to drain the perfusate into the NO assay system.

Measurement of NO Release
NO concentration in the perfusate was measured with a chemiluminescence assay.20 22 The venous effluent was introduced at 2 mL/min by a double-head plunger pump into a rotary mixer for thorough mixing with a chemiluminescence probe of 10 mmol/L hydrogen peroxide, 18 µmol/L recrystallized luminol (Sigma Chemical Co), 2 mmol/L potassium carbonate, and 150 mmol/L desferrioxamine to chelate iron ions derived from hemoglobin. The probe was pumped at 0.5 mL/min by another plunger pump. The mixture of the perfusate and probe then entered a chemiluminescence detector. The chemiluminescent signal was measured continuously and recorded on a standard pen recorder. Renal perfusion pressure (RPP) was monitored at the renal artery through the double-lumen needle connected to a pressure transducer. The dead volume of the system between the renal vein and the detector was 0.5 mL, introducing a 15-second lag time between perfusion pressure changes and chemiluminescent signals. The NO signal was calibrated with an NO solution or horseradish peroxidase, and the zero point was adjusted by use of the perfusate.20 22

Study Protocol
After a 60-minute period of perfusion for equilibration, we assayed the effects of vehicle, ET-1 (Peptide Institute), and L-NMMA (Sigma) on RPP and NO chemiluminescence. The agents were administered by infusion pumps at 0.25 mL/min in this order: vehicle; 10-12 mol/L, 10-11 mol/L, 10-10 mol/L, and 10-9 mol/L ET-1; and 10-4 mol/L L-NMMA at 10-minute intervals. The effects of ET-3 (Peptide Institute) were measured in the same way. To study the role of ETA in the effects of ET-1, we assayed the effects of an ETA antagonist, BQ-123 (Banyu Tsukuba Research Institute),25 on ET-1–induced changes in RPP and NO chemiluminescence. In a preliminary study, BQ-123 dose-dependently inhibited 10-10 mol/L ET-1–induced vasoconstriction with an IC50 value of 10-8 mol/L, a value similar to the 2.2x10-8 mol/L in the binding assay of the original report.25 Infusion of 10-6 mol/L BQ-123 began 10 minutes before the graded doses of ET-1 were infused. Finally, to compare the responses to pure ETB stimulation, we studied the effects of an ETB agonist, BQ-3020 (Banyu).26 These effects of ET-1, ET-3, L-NMMA, BQ-123, and BQ-3020 on RPP and NO release were compared for control and DOCA-salt–treated rats.

ET Levels in the Plasma and Perfusate
Blood was collected from the abdominal aorta of similarly treated DOCA-salt rats and control rats (n=5 each) under pentobarbital anesthesia (30 mg/kg IP). Blood was immediately centrifuged at 4°C in a tube containing disodium EDTA and aprotinin. The release rate of ET into the perfusate from the isolated kidney was determined. The kidneys from 8 DOCA-salt rats and 8 control rats were isolated and perfused with the Krebs-Henseleit buffer at 5 mL/min as described above. After a 60-minute equilibration period, the venous effluent was collected for 60 minutes. Collecting tubes were changed every 15 minutes. Plasma and perfusate concentrations of ET were measured by radioimmunoassay after extraction with a Sep-Pak C18 column as previously reported.27

Northern Blot Analysis of ETA and ETB Receptor mRNA
RNA was extracted from the kidneys of DOCA-salt rats (blood pressure, 204±8 mm Hg, mean±SEM) and control rats (blood pressure, 132±4 mm Hg; n=4 each) by the acid guanidinium thiocyanate–phenol–chloroform method,28 as reported previously.29 30 Poly(A+) RNA was obtained by use of oligo dT latex (Oligotex-dT30, Takara Shuzo Co). Two micrograms per lane of Poly(A+) RNA was subjected to electrophoresis on a 1.5% agarose gel and transferred to a nylon membrane. After ultraviolet cross-linking, the membrane was prehybridized at 37°C, followed by hybridization overnight at 37°C with 32P-labeled rat ETA and ETB cDNAs. cDNAs were prepared by the reverse transcription–polymerase chain reaction (RT-PCR) and subcloned by the method of Eguchi et al.31 The washed membrane was exposed to x-ray film at -80°C for 24 to 72 hours. ß-Actin mRNA was used as a loading control with a commercially available cDNA probe (Takara Shuzo Co). ETA, ETB, and ß-actin mRNA signals were quantified on a laser densitometer, and the ratios of ETA and ETB to ß-actin signals were calculated.

Statistical Analysis
Values are expressed as mean±SEM. The effects of the agents tested were assessed by ANOVA for repeated measures followed by a modified t test (Dunnett's test). Comparisons between DOCA-salt and control rats were assessed by an unpaired Student's t test. P<.05 was considered to be statistically significant.


*    Results
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*Results
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The TableDown lists baseline data for DOCA-salt and control rats in each study. The DOCA-salt rat group showed much higher blood pressures and greater heart and kidney weights. However, baseline RPP did not differ between the two groups.


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  		Table 1. Baseline Data of DOCA-Salt Hypertensive and Control Rats Used in Each Experiment

Fig 1Down demonstrates the representative tracings of RPP and NO signals during ET-1, ET-3, and acetylcholine infusion. ET-1 dose-dependently increased RPP. NO signals rose almost in parallel with RPP in control rats, whereas DOCA-salt–treated rats showed a low baseline and little response in NO chemiluminescence to ET-1. As shown in Fig 1CDown, although the rise in RPP caused by ET-3 was smaller than that by ET-1, ET-3 caused similar increases in NO signals. Both ET-1 and ET-3 caused rapid increases in NO chemiluminescence but only gradual elevations of RPP. Particularly when ET-1 was given at concentrations less than 10-10 mol/L, the RPP increase to reach the plateau levels lagged behind the NO release. Addition of L-NMMA further increased RPP and diminished NO chemiluminescence. The NO-releasing activity of ET-1 and ET-3 was smaller than that of acetylcholine (Fig 1DDown). As summarized in Fig 2Down, ET-1 caused a dose-dependent elevation of RPP and NO signals in the controls. However, baseline NO release was much higher in the control rats than in DOCA-salt rats (26.9±12.3 versus 3.5±1.8 fmol · min-1 · g-1 kidney wt, P<.01), and the response of NO chemiluminescence to ET-1 treatment was negligible in DOCA-salt rats. On the other hand, ET-3 had different effects on RPP than ET-1 (Fig 3Down). In kidneys from control rats, lower concentrations of ET-3 tended to decrease RPP, and doses higher than 10-9 mol/L were required to increase RPP. Despite the bimodal response of RPP, NO chemiluminescence increased in an ET-3 dose-dependent fashion. The NO-releasing activity of ET-3 in control rats was comparable to that of ET-1. In DOCA-salt–treated rats, ET-3–induced increases in NO chemiluminescence were minimal, and doses as low as 10-11 mol/L increased RPP, resulting in significant differences in pressor responses to 10-11 and 10-10 mol/L ET-3 from those in control rats.



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  		Figure 1. Representative tracings of nitric oxide chemiluminescence in the renal perfusate and of renal perfusion pressure (RPP) during the administration of endothelin-1 (ET-1) and NG-monomethyl-L-arginine (L-NMMA) in control (A) and deoxycorticosterone acetate (DOCA)-salt (B) rat kidneys. The effects of ET-3 (C) and acetylcholine (D) in control kidneys are also shown. KW indicates kidney weight.



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  		Figure 2. Bar graphs showing effects of endothelin-1 (ET-1) on renal perfusion pressure (RPP) and release rates of nitric oxide into the perfusate of isolated control and deoxycorticosterone acetate (DOCA)-salt kidneys. *P<.05, {dagger}P<.01, §P<.001 vs baseline. KW indicates kidney weight.



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  		Figure 3. Bar graphs showing effects of endothelin-3 (ET-3) on renal perfusion pressure (RPP) and release rates of nitric oxide into the perfusate of isolated control and deoxycorticosterone acetate (DOCA)-salt kidneys. *P<.05, {dagger}P<.01, §P<.001 vs baseline. KW indicates kidney weight.

Fig 4Down compares the effects of ET-1 in the presence of the ETA antagonist BQ-123. Although BQ-123 alone influenced neither RPP nor NO chemiluminescence, pretreatment with 10-6 mol/L BQ-123 abolished ET-1–induced increases in RPP at concentrations of ET-1 lower than 10-10 mol/L. BQ-123 significantly increased NO release at concentrations of ET-1 higher than 10-10 mol/L, compared with NO release in its absence (P<.05). BQ-123 also reduced the effects of ET-1 on RPP in DOCA-salt rats. However, the increase in NO release in DOCA-salt rats was minimal. Fig 5Down compares the effects of an ETB agonist, BQ-3020, on DOCA-salt and control rat kidneys. In the controls, BQ-3020 reduced RPP at concentrations lower than 10-11 mol/L. At 10-9 mol/L, BQ-3020 increased RPP. BQ-3020 also caused a dose-dependent increase in NO release despite its biphasic effects on RPP. DOCA-salt rat kidneys responded differently to BQ-3020 than did controls. In DOCA-salt rat kidneys, there was no depressor response to any dose of BQ-3020, and doses higher than 10-11 mol/L increased RPP significantly. Furthermore, the increase in NO chemiluminescence caused by BQ-3020 was much smaller in DOCA-salt rats.



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  		Figure 4. Bar graphs showing effects of pretreatment with an endothelin (ET)A antagonist, BQ-123 (10-6 mol/L), on ET-1–induced vasoconstriction and nitric oxide release in deoxycorticosterone acetate (DOCA)-salt and control kidneys. *P<.05, {dagger}P<.01, §P<.001 vs baseline. RPP indicates renal perfusion pressure; KW, kidney weight.



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  		Figure 5. Bar graphs showing effects of an endothelin (ET)B agonist, BQ-3020, on renal perfusion pressure (RPP) and nitric oxide release in the perfusate of deoxycorticosterone acetate (DOCA)-salt and control kidneys. *P<.05, {dagger}P<.01 vs baseline. KW indicates kidney weight.

Plasma immunoreactive ET concentration did not differ between the two groups of rats (DOCA-salt, 3.6±0.9 pg/mL versus control, 2.7±0.5 pg/mL, P=NS). The release rate of ET into the perfusate was stable during the 60-minute perfusion period. The average rate in DOCA-salt rats was 4.3±0.8 pg/min, similar to that in controls (4.3±1.0 pg/min).

Northern blot analysis detected ETB mRNA from rat kidneys at about 4.8 kb, consistent with a previous report.31 As shown in Fig 6Down, the ratio of renal ETB mRNA to ß-actin mRNA was significantly lower in DOCA-salt rats than in control rats. ETA mRNA was detected at about 5.2 kb, and the ratio of ETA to ß-actin mRNA was comparable in the two groups of kidneys (Fig 7Down).



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  		Figure 6. Northern blot analysis of renal endothelin (ET)B mRNA and ß-actin mRNA and the ratio of ETB mRNA to ß-actin mRNA in deoxycorticosterone acetate (DOCA)-salt and control rat kidneys.



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  		Figure 7. Northern blot analysis of renal endothelin (ET)A mRNA and ß-actin mRNA and the ratio of ETA mRNA to ß-actin mRNA in deoxycorticosterone acetate (DOCA)-salt and control rat kidneys.


*    Discussion
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 up arrowIntroduction
 up arrowMethods
up arrowResults
*Discussion
down arrowReferences
 
In the present study, simultaneous real-time measurements of perfusion pressure and NO release in the isolated rat kidney were used to explore the relation between the changes in vascular tone and NO release induced by ETs. This sensitive assay system demonstrated that both ET-1 and ET-3 quickly increased NO release from the isolated kidney and that this increase was sustained throughout the ET infusion. This increase in NO release was diminished by the addition of L-NMMA. ET-1–induced vasoconstriction, particularly at concentrations less than 10-10 mol/L, was evoked via ETA receptors, since a specific antagonist for ETA, BQ-123, substantially decreased it. BQ-123 also augmented the ET-1–evoked increase of NO release. Although the mechanism is unclear, this may have caused a further reduction in the vasoconstrictive activity of ET-1. Our data added direct evidence that ET-1 and ET-3 stimulate EDNO release via ETB receptors equipotently. The increase in NO release induced by ETs was about one third to one fifth of that caused by acetylcholine.20 21 22 These findings may help in understanding the overall effects of ET-1 and ET-3 on vascular tone.

Possible pathogenetic roles of ET in hypertension have frequently been discussed because of its potent vasoconstrictive activity.3 However, whether the vascular reactivity to ET is increased in DOCA-salt–treated animals remains controversial. Nguyen et al32 showed that ET-1–induced mesenteric vasoconstriction was reduced in DOCA-salt hypertension. Deng and Schiffrin33 reported further that this reduced response was selective for ET-1. On the other hand, other studies have reported increased vasoconstrictive responses to ET in DOCA-salt rats.34 35 A report from the former group also demonstrated decreases in the intracellular Ca2+ concentration responses, inositol triphosphate turnover, and ET binding to the vasculature in DOCA-salt rats,36 suggesting the downregulation of ET receptors. Despite the controversy, it has remained unclear which receptor subtype was altered in these rats. Our data showed that ET-3–induced renal vasoconstriction was significantly increased in the DOCA-salt group, associated with reduced NO release by ET-3. Furthermore, the response to a specific ETB agonist, BQ-3020, was similar to that to ET-3 in both rat groups. We therefore conclude that ETB-mediated effects are impaired in DOCA-salt rats. It is unclear whether this alteration was due to downregulation of ETB receptors. However, we have already reported that DOCA-salt rats were markedly hyporesponsive to acetylcholine in both renal vasodilation and NO release and showed endothelial damage by histology.20 21 22 DOCA-salt rats were also hyporesponsive to bradykinin, histamine, and ADP (unpublished observations). Furthermore, plasma and perfusate ET levels in DOCA-salt rats were comparable to those of controls in the present study. These findings suggest that intact ETB receptors on the endothelium may be reduced by mechanical or metabolic insults rather than by functional receptor downregulation in DOCA-salt rats due to elevated ET concentrations. This is compatible with the results of the Northern blot analysis of ETB mRNA, although we did not determine the cells of origin of these mRNAs within the kidney.

The response of RPP to ET-3 and BQ-3020 was quite distinct in the two groups of kidneys. An ETB agonist at low concentrations decreased RPP in control kidneys, whereas it increased RPP in DOCA-salt kidneys. This may be due to the difference in the NO release induced by ETs in these groups. In DOCA-salt hypertension, plasma ET-like immunoreactivity has consistently been reported as normal,32 34 37 whereas the arterial ET-1 contents and expression of its mRNA were increased.38 39 Moreover, although the origin of circulating ET-3 remains undetermined, the plasma concentration of ET-3 has been reported to be normal in DOCA-salt rats.37 Therefore, even though ET levels are comparable between DOCA-salt rats and controls, a decrease in ETB receptors in the endothelial cells of DOCA-salt rats may contribute to renal vasoconstriction.

As noted above, decreases in the endothelial ETB receptors may lead to the potentiation of ET-1–induced vasoconstriction. Furthermore, it has been reported that ET induces EDNO release, while EDNO seems to suppress ET release. It has been shown that ET secretion from porcine aorta and from cultured endothelial cells was increased by L-NMMA40 and by oxyhemoglobin,41 respectively. Therefore, the decreased release of EDNO by DOCA-salt rat kidney may potentiate the vasoconstrictive action of ET-1. Nevertheless, the pressor response of DOCA-salt rat kidney vasculature to ET-1 was comparable to that of controls. Recent studies suggest that ETB receptors expressed in the vascular smooth muscle cells are involved in its contraction.42 43 In the present study, a high concentration of BQ-3020 caused a substantial increase in RPP even in the control rat kidneys. This effect seems to be due to the stimulation of not only ETA but also ETB receptors in the smooth muscle cells. Therefore, the lack of changes in ETA mRNA and a decrease in ETB mRNA in the smooth muscle cells may explain the lack of differences in the pressor response to ET-1 between DOCA-salt rats and controls.

We observed generalized alteration of endothelial function, as reflected by NO release, in DOCA-salt hypertension. Recent studies suggest that EDNO not only regulates vascular tone but also facilitates sodium excretion through direct effects on glomerular hemodynamics and tubular reabsorption.44 45 46 47 De Nicola et al46 showed that L-NMMA decreased proximal reabsorption. Stoos et al47 also reported that NO decreased solute transport and increased cGMP concentrations in the cortical collecting ducts. These data imply that a decrease in EDNO may increase sodium retention and contribute to maintenance of hypertension, particularly salt-sensitive hypertension.

In conclusion, both ET-1 and ET-3 stimulate EDNO release via ETB receptors. ETB-mediated NO release and renal vasodilation are altered in DOCA-salt–treated rats, suggesting that impaired agonist-induced NO release may modulate the renal function and thus blood pressure regulation in salt-dependent hypertension.


*    Acknowledgments
 
This study was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas: "Vascular Endothelium–Smooth Muscle Coupling" and by Grant-in-Aid 05670948 from the Japanese Ministry of Education, Culture, and Science. The authors wish to thank Drs M. Ihara and M. Yano (Banyu Tsukuba Research Institute) for supplying BQ-123 and BQ-3020 and Dr T. Imai (Tokyo Medical and Dental University) for providing rat ETA and ETB cDNAs.

Received June 9, 1994; revision received September 6, 1994; accepted September 23, 1994.


*    References
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 up arrowAbstract
 up arrowIntroduction
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up arrowDiscussion
*References
 

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