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`Proc. Nati. Acad. Sci. USA
`Vol. 86, pp. 3375-3378, May 1989
`Medical Sciences
`
`Role of endothelium-derived nitric oxide in the regulation of
`blood pressure
`(L-arginine/hypertension/vascular endothelium/endothelium-derived relaxing factor)
`
`D. D. REES, R. M. J. PALMER, AND S. MONCADA
`Wellcome Research Laboratories, Langley Court, Beckenham, Kent BR3 3BS, United Kingdom
`
`Communicated by George H. Hitchings, January 27, 1989
`
`The role of endothelium-derived nitric oxide
`ABSTRACT
`in the regulation of blood pressure in the anesthetized rabbit
`was studied with Na-monomethyl-L-arginine (L-NMMA), a
`specific inhibitor of its formation from L-arginine. L-NMMA
`(3-100 mg-kg-'), but not its D-enantiomer, induced a dose-
`dependent long-lasting (15-90 min) increase in mean systemic
`arterial blood pressure. L-NMMA (100 mg kg-') also inhibited
`significantly the hypotensive action of acetylcholine, without
`affecting that of glyceryl trinitrate. Both these actions of
`L-NMMA were reversed by L-arginine (300 mg-kg-'), but not
`by D-arginine (300 mg-kg-'), indomethacin (1 mg-kg-'), pra-
`zosin (0.3 mg-kg-'), or by vagotomy. The effects of L-NMMA
`in vivo were associated with a significant inhibition of the
`release of nitric oxide from perfused aortic segments ex vivo.
`This inhibition was reversed by infusing L-arginine through the
`aortic segments. These results indicate that nitric oxide for-
`mation from L-arginine by the vascular endothelium plays a
`role in the regulation of blood pressure and in the hypotensive
`actions of acetylcholine.
`
`Although endothelium-dependent vascular relaxation and the
`release of endothelium-derived relaxing factor in vitro has
`been clearly established (1-4), there is only circumstantial
`evidence to indicate their occurrence in vivo. Alterations in
`vessel diameter that follow changes in blood flow are endo-
`thelium dependent (5). Furthermore, damage to the endothe-
`lium (6, 7) or treatment with methylene blue (8) or gossypol
`(9), two nonspecific inhibitors of endothelium-dependent
`relaxation, all abolish the response to endothelium-depend-
`ent vasodilators in vivo without affecting the response to the
`endothelium-independent vasodilators, sodium nitroprusside
`or glyceryl trinitrate (n3Gro).
`Nitric oxide (NO) accounts for the biological actions of
`endothelium-derived relaxing factor (10-15) and is formed by
`vascular endothelial cells from the terminal guanido nitrogen
`atom(s) of the amino acid L-arginine (16, 17). This biosyn-
`thetic process, the endothelium-dependent relaxation of
`vascular rings, and the vasodilatation induced by acetylcho-
`line (ACh) in the coronary circulation of the rabbit heart are
`inhibited by the L-arginine analogue, Na-monomethyl-
`L-arginine (L-NMMA; refs. 4 and 18-20). These results
`indicate that L-arginine is the physiological precursor for NO
`synthesis by the vascular endothelium.
`We have now used L-NMMA to investigate the role of NO
`in the regulation of blood pressure in the anesthetized rabbit.
`
`MATERIALS AND METHODS
`Methods. Male New Zealand White rabbits (2.0-2.2 kg)
`were anesthetized with sodium pentobarbitone (40-50
`mg-kg-1). Anesthesia was then maintained by a continuous
`
`infusion of sodium pentobarbitone (15 mg kg-' hr-1) via the
`left marginal ear vein and the rabbits were ventilated with
`room air via a tracheotomy tube.
`Mean arterial blood pressure was monitored with a pres-
`sure transducer (Bell & Howell, Ashford, U.K.) connected,
`via a cannula containing heparinized (10 units-ml-1) saline, to
`the right carotid artery. Phenylephrine was administered as a
`continuous infusion into the right marginal ear vein. All other
`drugs were administered as 15-sec infusions via a cannula in
`the right femoral vein. Blood pressure and heart rate were
`monitored continuously on a four-channel polygraph (Grass).
`For ex vivo experiments, L-NMMA (100 mg kg-1; i.v.) was
`administered and the animals were sacrificed 10 min later.
`The thoracic aorta was rapidly removed and perfused intralu-
`minally with Krebs' buffer within 30 min of treatment. The
`release of NO induced by ACh was determined by cascade
`bioassay and chemiluminescence (4).
`Materials. NO (>99.98% pure, British Oxygen, Guildford,
`U.K.) solutions were prepared as described (11).
`n3Gro (Wellcome), phenylephrine, ACh, atropine sulfate,
`indomethacin, L- and D-arginine (Sigma), U46619 (9,11-
`dideoxy-9a,lla-methanoepoxy prostaglandin F2,) (Cayman
`Chemicals, Ann Arbor, MI), sodium pentobarbitone (May &
`Baker, Dagenham, U.K.), and prazosin (Pfizer Diagnostics)
`were obtained as indicated. L-NMMA and Nw-monomethyl-
`D-arginine (D-NMMA) were synthesized as described (21).
`All drugs used were administered in saline.
`Statistical Evaluation. Results, expressed as means ± SEM
`of n experiments, were analyzed statistically by Student's t
`test for paired data. A value of P < 0.05 was considered
`statistically significant.
`
`RESULTS
`L-NMMA (3-100 mg-kg-'), but not D-NMMA (100 mg-kg-'),
`caused a dose-dependent increase in mean arterial blood
`pressure (Fig. 1). The hypertension induced by L-NMMA
`was rapid in onset, reaching a plateau within 5 min (Fig. 2A).
`Its duration was also dose dependent, so that the hyperten-
`sion induced by 3 mg-kg-' and 100 mg-kg-' of L-NMMA
`lasted 10-15 and 60-90 min, respectively (n = 5). The
`increase in blood pressure induced by L-NMMA (100
`mg-kg-1) was always accompanied by a slight bradycardia
`(Fig. 2; n = 5). Administration of L-arginine (300 mg-kg-'),
`which had no direct effect on either mean arterial blood
`pressure or on heart rate, reversed the hypertension and the
`decrease in heart rate induced by L-NMMA within 10 min
`(Fig. 2B).
`ACh (0.1-3.0 ,g-kg-1) and n3Gro (1-30 ,uggkg-') caused a
`dose-dependent fall in mean arterial blood pressure (ED50
`0.20 ± 0.04 and 2.4 ± 0.3 ,ugkg-', respectively; n = 3)
`without affecting the heart rate significantly. Phenylephrine
`
`The publication costs of this article were defrayed in part by page charge
`payment. This article must therefore be hereby marked "advertisement"
`in accordance with 18 U.S.C. §1734 solely to indicate this fact.
`
`Abbreviations: L-NMMA, N'-monomethyl-L-arginine; D-NMMA,
`N'O-monomethyl-D-arginine; n3Gro, glyceryl trinitrate; ACh, acetyl-
`choline.
`
`3375
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`3376
`
`Medical Sciences: Rees et al.
`
`Proc. Natl. Acad. Sci. USA 86 (1989)
`
`Effect of L-NMMA on the hypotensive responses
`Table 1.
`induced by ACh and n3Gro
`
`Fall in blood pressure, mmHg
`(mean ± SEM)
`Phenylephrine
`L-NMMA
`
`13.3 ± 2.8
`23.3 ± 2.7
`33.1 ± 2.6
`37.4 ± 3.0
`
`2.6 ± 1.7*
`10.1 ± 2.0*
`22.6 ± 1.9*
`28.5 ± 1.1*
`
`Vasodilator
`ACh, &gkg-1
`0.1
`0.3
`1
`3
`n3Gro, .ggkg-'
`6.8 ± 1.9
`6.1 ± 1.5
`1
`16.4 ± 3.1
`16.7 ± 4.7
`3
`30.8 ± 3.0
`30.0 ± 3.9
`10
`37.3 ± 5.0
`37.0 ± 5.2
`30
`The vasodilator effects of ACh and n3Gro were compared in
`animals treated with L-NMMA and in animals whose blood pressure
`had been elevated to a comparable level with phenylephrine. The
`blood pressure in the L-NMMA (100 mg kg-')- and phenylephrine
`(300 ig-kg4-hr-1)-treated animals was 84.6 ± 3.2 and 88.6 ± 7
`mmHg, respectively. L-NMMA inhibited the vasodilator effects of
`ACh but not those of n3Gro.
`*P < 0.05.
`
`abolished within 15 min this inhibition by L-NMMA (100
`mgkg-; n = 3).
`When the blood pressure was raised by phenylephrine, the
`hypotensive actions of similarly effective doses of ACh (0.3
`,ugkg-1) and n3Gro (3 zg-kg-') were accompanied by an
`increase in the heart rate of 11 ± 2 and 10 + 2 beats per min,
`respectively (n = 3). These doses of ACh and n3Gro caused
`a similar increase in heart rate when the blood pressure was
`raised by L-NMMA (9 ± 1 and 12 ± 2 beats per min,
`respectively; n = 3).
`The blood pressure was not affected by indomethacin (1
`mg-kg-') but was slightly reduced by prazosin (0.3 mg-kg-').
`Bilateral vagotomy caused a small increase in mean arterial
`blood pressure. None of these interventions affected the
`actions of L-NMMA (n = 3 for each).
`Infusion ofACh (0.1-3.0 ,uM) for 1 min through the excised
`aortae from untreated animals induced a concentration-
`dependent release of NO detected by bioassay (Fig. 3A) or by
`chemiluminescence (Fig. 4A). In contrast, the release of NO
`induced by ACh (0.1-3.0 ,uM) from aortae obtained from
`animals treated with L-NMMA (100 mg-kg-') was inhibited
`significantly when measured by bioassay (Fig. 3B) or
`chemiluminescence (Fig. 4B). Infusion of L-arginine (100
`,uM) through the aortae from control animals did not affect
`significantly the release of NO induced by ACh but restored
`fully the release observed in aortae from L-NMMA-treated
`animals (n = 3).
`
`DISCUSSION
`A physiological role for endothelium-derived NO in the
`control of vascular tone in vivo has not been clearly demon-
`strated. This has mainly been due to the difficulties associ-
`ated with removal of the endothelium in vivo, the extremely
`labile nature of NO, and the absence of a specific inhibitor of
`its synthesis. There is, however, some evidence to support
`the proposal that the endothelium-dependent vasodilator
`responses observed in conduit arteries in vitro also occur in
`vivo (5-9).
`The formation of NO in the coronary circulation in vitro
`accounts for the vasodilatation induced by ACh in the rabbit
`heart (22) and by bradykinin in the guinea pig heart (23),
`indicating that NO also plays a role in regulating the tone of
`resistance arteries. The demonstration that L-arginine is the
`physiological precursor for NO synthesis by the vascular
`
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`
`Effect of L-NMMA (3-100 mg-kg-; i.v.) on the mean
`FIG. 1.
`arterial blood pressure (B.P.) of the rabbit. o, Increase in blood
`pressure in each animal; *, mean. Measurements were made 5 min
`after administration of L-NMMA. Resting jlood pressure was 68 +
`3 mmHg (n = 5).
`(300 ,ug kg-1 hr-') raised the blood pressure to a level similar
`to that induced by L-NMMA (100 mg-kg-') and significantly
`enhanced the hypotensive actions of ACh and n3Gro. When
`the enhanced responses to ACh and n3Gro in phenylephrine-
`treated animals were taken as controls, then L-NMMA (100
`mg-kg-'), but not D-NMMA (100 mg-kg-'), caused a signif-
`icant inhibition of the ACh-induced hypotension, without
`affecting that induced by n3Gro (Table 1). Administration of
`L-arginine (300 mg-kg-'), but not D-arginine (300 mg-kg-'),
`
`A
`
`30 min.
`30 min.
`120_///
`
`B.P.
`(mm Hg) 60
`
`90
`
`30-
`28Q
`
`;
`
`200
`
`H .R
`(bpm)
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`B
`
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`(mm Hg) 60
`
`30_
`
`280r
`
`24
`
`200L
`
`H.R.
`
`(bpm)
`
`L-NMMA
`100mg kg1
`
`1min
`
`__
`
`1min
`
`VII
`
`L-NMMA
`100mg kg91
`
`L-Arginine
`300mg kg-1
`
`(A) Effect of L-NMMA (100 mg-kg-; i.v.) on blood
`FIG. 2.
`pressure (B.P.) and heart rate (H.R.). Trace is representative ofthree
`experiments, in which the duration of these effects of L-NMMA was
`between 60 and 90 min. (B) Reversal of the effect of L-NMMA (100
`mgkg-'; i.v.) on blood pressure and heart rate by L-arginine (300
`mg-kg-'; i.v.). Trace is representative of three experiments.
`
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`Medical Sciences: Rees et aL
`
`Proc. Natl. Acad. Sci. USA 86 (1989)
`
`3377
`
`0.1
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`ACh MM
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`C.
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`t
`5OnM
`N3Gro O.T.
`
`0.1 0.3
`
`1
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`3
`0.1 0.3
`ACh tiM T.D.
`
`1
`
`3
`
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`N3Gro O.T.
`
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`B.
`
`Aorta
`4 sec
`
`i
`
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`
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`L-ARGININE lOOjM T.D.
`
`10min
`7 2cm
`
`t
`
`5OnM
`N3Gro O.T.
`
`t
`t
`0-1 0.3
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`1
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`0.1 0.3
`3
`ACh UM T.D.
`
`1
`
`3
`
`5OnM
`N3Gro O.T
`
`Effect of L-NMMA on the ex vivo release of NO induced
`FIG. 3.
`by ACh. A segment (6 cm) of the thoracic (donor) aorta was removed
`10 min after treatment with L-NMMA (100 mg-kg-'; i.v.), placed in
`a perspex chamber, and perfused at 5 ml-min1 with Krebs' buffer.
`The effluent was used to superfuse in a cascade two spiral strips of
`rabbit aorta denuded of endothelium (RbA) and submaximally
`contracted with U46619 (30 nM). Atropine (200 nM) was infused over
`the tissues (O.T.) to inhibit the direct effects of ACh administered as
`1-min infusions. (A) Control, untreated rabbit. ACh (0.1-3.0 AM)
`infused through the donor aorta (T.D.) induced the release of NO as
`shown by the relaxations of the bioassay tissues. This release was not
`significantly affected by a continuous infusion of L-arginine (100 AuM)
`through the donor aorta. (B) Rabbit treated with L-NMMA (100
`mgkg-'; i.v.). The release of NO by ACh (0.1-3.0 AM) infused
`through the donor aorta was greatly reduced and was restored by a
`continuous infusion of L-argilnine (100 AM) through the donor aorta.
`Trace is representative of three experiments.
`endothelium and the identification of L-NMMA as a specific
`inhibitor of this pathway (4, 18, 19) has allowed the investi-
`gation of the role of NO in the regulation of blood pressure.
`Intravenous administration of L-NMMA induced a dose-
`dependent enantiomer-specific hypertension and partially
`inhibited the hypotensive action of ACh, but not that of
`n3Gro. Whether L-NMMA affects the uptake or the utiliza-
`tion of arginine by cells is not yet known; however, the
`present data are quantitatively and qualitatively similar to
`those obtained in vitro. Indeed, in vascular strips (4, 18, 19),
`and in the isolated perfused heart ofthe rabbit (20), L-NMMA
`induces an increase in vascular tone and an inhibition of
`endothelium-dependent responses. These actions are due to
`the inhibition of the release of NO, suggesting that the results
`obtained in vivo are also attributable to the same mechanism.
`
`Release of NO from the perfused rabbit aorta detected by
`FIG. 4.
`chemiluminescence. The effluent of the donor aorta was infused
`continuously into a reaction vessel containing 75 ml of 1% sodium
`iodide in glacial acetic acid under reflex. NO was removed under
`reduced pressure in a stream of N2 and mixed with ozone; the
`chemiluminescent product was quantified by reference to NO stan-
`dards. (A) Control, untreated rabbit. Release of NO by ACh (0.1-3.0
`,LM; 1-min infusions) in the absence (u) and presence (X) of a
`continuous infusion of L-arginine (100 ILM). (B) Rabbit treated with
`L-NMMA (100 mg-kg-'; i.v.) 10 min prior to removal of the aorta.
`Release of NO by ACh (0.1-3.0 tiM) in the absence (r) and presence
`(n) ofa continuous infusion of L-arginine (100 tiM). Each value is the
`mean ± SEM of three separate experiments. *, P < 0.05.
`
`The effect of L-NMMA in vivo was slow to disappear
`unless accelerated by a 3-fold molar excess of exogenous
`L-arginine, reinforcing our suggestion (4, 18) that L-NMMA
`is a competitive inhibitor of the NO-forming enzyme(s).
`Furthermore, this effect could also be demonstrated ex vivo
`where the inhibition of the formation of NO observed after
`treatment with L-NMMA was reversed by an infusion of
`L-arginine. These findings suggest that there is a continuous
`utilization of L-arginine for the enzymic formation of NO by
`resistance arteries and provide the first evidence that NO
`formation contributes to the regulation of blood pressure.
`The failure of L-arginine to affect blood pressure directly is
`also consistent with observations in conduit and in resistance
`vessels in vitro (4, 20, 24) and endothelial cells in culture (16),
`which suggest that under normal conditions there is sufficient
`endogenous L-arginine to saturate the NO-forming enzyme.
`More L-NMMA was necessary to inhibit the hypotension
`induced by ACh than to increase the blood pressure, sug-
`gesting that during stimulation with ACh there is an increased
`mobilization of L-arginine, the antagonism of which requires
`more L-NMMA. These results are consistent with those
`obtained in vascular rings in vitro, where more L-NMMA
`was required to inhibit ACh-induced relaxation than to
`increase basal tone (4).
`
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`3378
`
`Medical Sciences: Rees et al.
`
`Proc. Natl. Acad. Sci. USA 86 (1989)
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`26.
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`
`29.
`
`The hypertension induced by L-NMMA was accompanied
`by bradycardia and the hypotension induced by ACh and
`n3Gro, in the presence of L-NMMA, was accompanied by
`tachycardia. These effects were small and were also observed
`when the blood pressure was raised by phenylephrine and
`were not affected by the a,-antagonist prazosin or by vagot-
`omy, suggesting that they are reflex in nature rather than a
`direct effect of L-NMMA on heart rate.
`Our results indicate that the vascular endothelium, by
`synthesizing NO from L-arginine, plays a significant role in
`the control of blood pressure. The precise regional hemody-
`namic changes that occur as a result of preventing the
`synthesis of NO by the vascular endothelium require eluci-
`dation.
`A decrease in endothelium-dependent relaxation has been
`observed in vessels from hypertensive animals (25-29),
`although the precise mechanism has not been established. In
`view of our data, it is tempting to speculate that changes in
`the synthesis or the actions of NO in the vasculature may
`either be involved in some forms of hypertension or be one
`of the mechanisms involved in the genesis of hypertension in
`general.
`Whether administration of arginine would be beneficial for
`the treatment of hypertension and whether populations
`consuming a low arginine diet exhibit an increased incidence
`of hypertension should also be investigated. Further work is
`also needed to determine the long-term pathophysiological
`consequences of this way of changing vascular reactivity and
`inducing experimental hypertension.
`
`We are indebted to Dr. Harold Hodson and Richard Beams for the
`synthesis of L- and D-NMMA.
`
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`
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