Am. J. Physiol. Regulatory Integrative Comp. Physiol.
278: R628–R639, 2000.
Mg2⫹-induced endothelium-dependent relaxation of blood
vessels and blood pressure lowering: role of NO
ZHI-WEI YANG,1 ASEFA GEBREWOLD,1 MAJA NOWAKOWSKI,2
BELLA T. ALTURA,1,3 AND BURTON M. ALTURA1,3,4
Departments of 1Physiology, 2Pathology, and 4Medicine and 3Center
For Cardiovascular and Muscle Research, State University of New York,
Health Science Center at Brooklyn, Brooklyn, New York 11203
rat aorta; microvessels; calcium; endothelium-derived relaxing factor; vasodilatation; guanosine 38,58-cyclic monophosphate
The costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
R628
FOR MORE THAN 100 YEARS, it has been known that
systemic administration of magnesium ions (Mg2⫹) to
most vertebrates and all mammals produces concentration-dependent falls in arterial blood pressure (for
review see Ref. 31). Ever since the classic studies of
Hazard and Wurmser in 1932 (21), it has been known
that Mg2⫹ is a potent vasodilator agent and relaxant of
smooth muscle. Although it often has been assumed
that these important hemodynamic effects are brought
about by some direct actions on vascular smooth muscle
cells (31), a number of recent studies on isolated blood
vessels and single vascular smooth muscle cells have
indicated that Mg2⫹ can exert multiple actions on
vascular muscle (for recent review see Ref. 6). Over the
last two decades, it has been shown that Mg2⫹ can
modulate agonist actions and hormone-receptor binding on smooth muscle cells (for review see Ref. 6) and
may be a requirement for the action of various relaxant
vasodilator substances via as yet unknown effects on
endothelial cells (11, 25, 41).
The endothelium has been shown to play a functional
role in the regulation of vasomotor tone by generating
and releasing some endothelial factors (19, 23). It is
generally accepted that the relaxant response of arterial vessels to diverse vasodilators (9, 15, 19) is mediated by a so-called endothelium-derived relaxing factor
(EDRF) (18), and several reports proposed that the
EDRF is nitric oxide (NO) (18, 30). Basal and stimulated release of NO and an associated activation of
guanylate cyclase via an increased cellular content of
guanosine 38,58-cyclic monophosphate (cGMP) are now
established endogenous regulatory mechanisms of cardiovascular homeostasis (30).
A little over 10 years ago, it was demonstrated that
acetylcholine (ACh)-induced relaxation of vascular
smooth muscle depends on the presence of extracellular
Mg2⫹ concentration ([Mg2⫹]0) (11). Elevation in [Mg2⫹]0
above physiological levels inhibits spontaneous mechanical activity and lowers baseline tension of several
types of blood vessels, whereas lowering [Mg2⫹]0 (below
0.6 mM) exerts opposite effects, i.e., produces an increase in tension and contractility (2, 5–6, 8, 11). In
addition, a variety of agonists that induce contraction
in vascular smooth muscle exhibit depressed or attenuated contractile activity as [Mg2⫹]0 is elevated (2, 5, 6,
33). These effects of Mg2⫹ are related to modulation of
Ca2⫹ permeability, binding, and translocation in vascu-
0363-6119/00 $5.00 Copyright r 2000 the American Physiological Society
http://www.ajpregu.org
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Yang, Zhi-Wei, Asefa Gebrewold, Maja Nowakowski,
Bella T. Altura, and Burton M. Altura. Mg2⫹-induced
endothelium-dependent relaxation of blood vessels and blood
pressure lowering: role of NO. Am. J. Physiol. Regulatory
Integrative Comp. Physiol. 278: R628–R639, 2000.—In vitro
extracellular Mg2⫹ concentration ([Mg2⫹]0) produces endothelium-dependent and endothelium-independent relaxations in
rat aorta in a concentration-dependent manner. These relaxant effects of Mg2⫹ on intact rat aortic rings, but not denuded
rat aortic rings, were suppressed by either NG-monomethyl-Larginine (L-NMMA), N␻-nitro-L-arginine methyl ester (L-NAME),
or methylene blue. The inhibitory effects of L-NMMA and
L-NAME could be reversed partly by L-arginine. [Mg2⫹]0induced dilatation in vivo in rat mesenteric arterioles and
venules was almost completely inhibited by NG-nitro-Larginine and L-NMMA. Removal of extracellular Ca2⫹ concentration ([Ca2⫹]0) or buffering intracellular Ca2⫹ concentration
in endothelial cells, with 10 µM 1,2-bis(2-aminophenoxy)ethane-N,N,N8,N8-tetraacetic acid-AM, markedly attenuated
the relaxant effects of Mg2⫹. Mg2⫹ produced nitric oxide (NO)
release from the intact aortic rings in a concentrationdependent manner. Removal of [Ca2⫹]0 diminished the increased NO release induced by elevated levels of [Mg2⫹]0. In
vivo infusion of increasing doses (1–30 µM/min) of MgSO4,
directly into the femoral veins of anesthetized rats, elicited
significant concentration-dependent sustained increases in
serum total Mg and concomitant decreases in arterial blood
pressure. Before and after employment of various doses of
MgSO4, intravenous administration of either L-NMMA (10
mg/kg) or L-NAME (10 mg/kg) increased (i.e., reversed) the
MgSO4-lowered blood pressure markedly, and intravenous
injection of L-arginine restored partially the increased blood
pressure effects of both L-NMMA and L-NAME. Our results
suggest that 1) small blood vessels are very dependent on NO
release for Mg2⫹ dilatations and 2) the endothelium-dependent relaxation induced by extracellular Mg2⫹ is mediated by
release of endothelium-derived relaxing factor-NO from the
endothelium, and requires Ca2⫹ and formation of guanosine
38,58-cyclic monophosphate.
MG2⫹-INDUCED RELAXATION AND BLOOD PRESSURE LOWERING
lar smooth muscle (7). However, the vasodilatative
effects of extracellular Mg2⫹ may be complex and are
less well understood, and the relationships among
extracellular Mg2⫹, vascular endothelium, and smooth
muscle have not been elucidated. The present study
was designed to determine whether Mg2⫹-induced endothelium-dependent vasorelaxation and its blood pressure lowering actions are mediated by NO release from
endothelium and to gain insight into the relationship
between Mg2⫹, EDRF, Ca2⫹, and relaxation of vascular
smooth muscle.
MATERIAL AND METHODS
precontracted by 0.2 µM PE fail, however, to relax in the
presence of 10⫺8 –10⫺6 M ACh.
For the extracellular Ca2⫹-free experiments, aortic rings
were equilibrated in Ca2⫹-free Krebs-Ringer bicarbonate
solution containing 0.2 mM ethylene glycol-bis(␤-aminoethyl
ether)-N,N,N8,N8-tetraacetic acid (EGTA) for at least 90 min.
Pretreatment of the aortic vessels with extracellular Ca2⫹free medium decreased about 70% of the aortic maximal
contractions induced by 80 mM KCl or 0.2 µM PE and
suppressed significantly the relaxations of precontracted
aortic rings in the presence of 10⫺8-10⫺6 M ACh. To obtain
similar degrees of tone, the concentrations of PE required to
induce precontractions for untreated vessels were lowered.
Determination of nitrite release induced by Mg2⫹. Nitrite
was measured by using a modification of the Griess reaction
as described previously (20, 24). Rat thoracic aortic rings
were cut into rings (about 2.0 cm), and four rings were placed
in a test tube containing 3 ml NKR solution (1.2 mM Mg2⫹)
gassed with 95% O2 and 5% CO2 at 37°C. Samples for basal
accumulation of nitrite formed from released NO were taken
first for control levels. The buffer was then replaced and the
rings were stimulated with Mg2⫹ at concentrations of 2.4, 4.8,
7.2, and 9.6 mM. Each concentration of Mg2⫹ was administered every 30 min. NO synthase (NOS) antagonists (L-NMMA
and L-NAME) were employed 20 min before addition of Mg2⫹.
The incubation solutions were assayed for the stable end
products of NO, nitrite. Briefly, 50-µl samples of incubation
solutions (in quadruplicate) were mixed in the wells of a
96-well microtiter plate with 100 µl of the Griess reagent,
containing a 1:1 (vol/vol) mixture of 1% (wt/vol) sulfanilamide
in 30% acetic acid and 0.5% (wt/vol) of N-(l-naphthyl)ethylenediamine dihydrochloride in 60% acetic acid. The chromophore
generated by the reaction with nitrite was detected spectrophotometrically (550 nm) using a microtiter plate reader
(BioTek, Winooski, VT). The concentration of nitrite was
calculated by using calibration with known concentrations of
NaNO2.
Measurement of femoral artery blood pressure in anesthetized rats and blood Mg levels. Experiments were performed
in accordance with the Guiding Principles in the Care and
Use of Animals approved by the Council of the American
Physiological Society (1980). Male rats were sedated and
anesthetized with 50 mg/kg pentobarbital sodium (im). A
polyethylene catheter was inserted into the femoral artery
and connected to a fluid-filled pressure transducer (Gould,
Statham) to measure femoral arterial blood pressure (34) and
serum Mg levels by atomic absorption spectrophotometry (8).
Another polyethylene catheter was inserted into a femoral
vein to allow systemic infusion of drugs (34). Systemic
vascular responses to Mg2⫹, L-NMMA, L-NAME, or L-arginine
were elicited in two different protocols: in the first set of
experiments, increasing doses (1, 10, 20, and 30 µM/min, rate
0.04 ml/min) of MgSO4 were infused into the femoral vein;
after stable femoral arterial blood pressure was obtained for
10 min, 10 mg/kg L-NMMA or L-NAME were infused into the
femoral vein. Simultaneously, the systolic and diastolic arterial blood pressures in the femoral artery were measured
continuously. In the second set of experiments, 10 mg/kg
L-NMMA or L-NAME were infused into a femoral vein; after
stable femoral arterial blood pressure was recorded for 10
min increasing doses (1, 10, 20, and 30 µM/min, rate 0.04
ml/min) of MgSO4 were then infused into the femoral vein.
Consequently, 15 mg/kg L-arginine was infused into the
femoral vein, and simultaneously the systolic and diastolic
arterial blood pressure were measured continuously.
Measurement of responses of in vivo mesenteric arteriolar
and venular lumen sizes to exogenously applied Mg2⫹. In vivo
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General procedures. Male, adult Wistar rats (350–450 g)
were killed by stunning and subsequent decapitation. The
thoracic aortas were removed carefully, immediately placed
in normal Krebs-Ringer bicarbonate (NKR) solution at pH 7.4
containing (in mM) 118 NaCl, 4.7 KCl, 1.2 KH2PO4, 1.2
MgSO4, 2.5 CaCl2, 10 dextrose, and 25 NaHCO3 (4, 41). These
segments were mounted on stainless steel pins under 2 g
resting tension in organ baths, attached to force transducers
(Grass model FT 03), and connected to Grass model 7
polygraphs. The organ bath containing normal Krebs-Ringer
bicarbonate solution was gassed continuously with 95% O2-5%
CO2 and warmed to 37°C (pH 7.4). Incubation media were
routinely changed every 15 min as a precaution against
interfering metabolites (3). Stimulation of rings with 80 mM
KCl was repeated every 35–40 min, two to three times, until
contractile responses were stable. The successful removal of
endothelium was assessed by showing that ACh (10⫺8 –10⫺6
M) failed to relax the ring segments precontracted by 0.2 µM
phenylephrine (PE), while 10⫺8 –10⫺6 M ACh did relax the
intact endothelium segments (42).
Inasmuch as NG-monomethyl-L-arginine (L-NMMA), N␻nitro-L-arginine methyl ester (L-NAME), and methylene blue
potentiate vessel tone by blocking the synthesis and activation of basal nitric oxide, respectively (27), the ring segments
treated with L-NMMA, L-NAME, and methylene blue were
precontracted initially with lower concentrations of PE.
The level of ionization of magnesium in Mg2⫹-modified
Krebs-Ringer bicarbonate solution was monitored by NOVA
Biomedical (Waltham, MA) ion-selective electrodes (12). After
testing with KCl and incubation in normal Krebs-Ringer
bicarbonate solution for 45 min, the rings were exposed to
Mg2⫹-modified Krebs-Ringer bicarbonate solutions and then
the data were obtained.
Intracellular Ca2⫹ buffering and extracellular Ca2⫹ removal. For intracellular Ca2⫹-buffered experiments, arterial
segments were first precontracted by PE. When stable contraction of the arterial rings was obtained, 10 µM acetyl methyl
ester of 1,2-bis(2-aminophenoxy)ethane-N,N,N8,N8-tetraacetic acid (BAPTA-AM, a membrane permeable Ca2⫹ chelator)
were added to the bath medium. Previous studies have
indicated that 5–20 µM BAPTA-AM can completely buffer
intracellular Ca2⫹ concentration ([Ca2⫹]i) in vascular endothelial cells (1, 13), but more than 50 µM BAPTA-AM is needed to
buffer [Ca2⫹]i in smooth muscle cells (39). According to these
studies, we investigated the effects of pretreatment of the
ring segments with 10 µM of BAPTA-AM on KCl- or PEinduced contractions and on the ACh-induced endotheliumdependent relaxation. After incubation of the arterial segments with 10 µM of BAPTA-AM for 15 min, 80 mM KCl or
0.2 µM PE retains ability to contract the intact vascular
tissues, but the maximal responses of the vessels are suppressed by about 25%; 10 µM BAPTA-AM pretreated rings
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MG2⫹-INDUCED RELAXATION AND BLOOD PRESSURE LOWERING
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Fig. 1. Effects of extracellular Mg2⫹ concentration ([Mg2⫹]0) on phenylephrine (PE)-precontracted isolated rat aortic
rings with (⫹E) or without (⫺E) endothelium. In endothelium-denuded rings, 50 nM PE was employed to induce
similar degree of tone to that of endothelium-containing rings induced by 0.2 µM PE (A and B). A: tracings of typical
bioassay curves for extracellular Mg2⫹. B: each point represents means ⫾ SE expressed as percentage of PE-induced
contraction. *P ⬍ 0.01 compared with control, #P ⬍ 0.05 and ##P ⬍ 0.01 compared with endothelium-denuded rings; n ⫽
6 each. For control X-axis data is time (min) only. Concentrations of [Mg2⫹]0 indicate final bath concentration.
studies were carried out using direct in vivo microscopic
observation of arterioles and venules in mesenteries of pentobarbital sodium (Nembutal, 40 mg/kg im)-anesthetized male
rats. In selected studies, ketamine HCl (60 mg/kg im) was
also used as an anesthetic to be certain that the effects of
Mg2⫹, NG-nitro-L-arginine (L-NNA), and L-NMMA were not a
reflection of barbiturate anesthesia. After induction of anesthesia, tracheostomies were performed and polyethylene
MG2⫹-INDUCED RELAXATION AND BLOOD PRESSURE LOWERING
Calculations and statistical analyses. The percent relaxation was expressed as means ⫾ SE. Statistical evaluation of
the results was carried out by analysis of the Newman-Keuls
test, which took into account that some vessels were from the
same animal. Mg salt-induced changes from control or initial
arteriolar and venular lumen size (before Mg salt application)
were assessed for statistical significance by paired t-test
and/or ANOVA with Scheffé’s contrast test. The results were
considered significant at P ⬍ 0.05.
RESULTS
[Mg2⫹]0 produces concentration-dependent relaxation
of isolated rat aorta; intact endothelium vs. endotheliumdenuded responses. After precontraction with PE,
[Mg2⫹]0 induces concentration-dependent relaxations
of isolated rat aortic rings at concentrations of 2.4–9.6
mM (Fig. 1, A and B), compared with the untreated,
paired, and timed controls [the rings incubated with
NKR solution containing physiological concentration
(1.2 mM) of Mg2⫹]. Removal of endothelium results in
about a 25–35% attenuation of the Mg2⫹-induced relaxations (Fig. 1B). The effective concentration producing
approximately 50% of the maximal relaxation responses (EC50 value) for Mg2⫹ is about 2.46 ⫾ 0.06 mM
in endothelium-containing rat aorta rings; removal of
the endothelium increases significantly the EC50 value
for Mg2⫹ to about 3.89 ⫾ 0.08 mM (P ⬍ 0.05).
Mg2⫹-induced relaxation and EDRF (NO). In the
presence of 150 µM L-NMMA or 50 µM L-NAME,
Mg2⫹-induced relaxation in intact ring segments is
suppressed significantly; the EC50 value increases to a
value of approximately 4.23 ⫾ 0.09 mM, and the
relaxant response of the intact rings to [Mg2⫹]0 is
restored partly by addition of 50 µM L-arginine to the
bathing medium (Fig. 2). The presence of 150 µM
L-NMMA or 50 µM L-NAME did not affect the concentration-response curves of endothelium-denuded rat aorta
rings to [Mg2⫹]0 (not shown).
Involvement of Ca2⫹ in Mg2⫹-induced vasorelaxation.
A clear requirement of calcium for the endothelium-
Fig. 2. Effects of NG-monomethyl-L-arginine (LNMMA, 150 µM), N␻-nitro-L-arginine methyl ester (L-NAME, 50 µM), and L-arginine (L-Arg, 50
µM) on [Mg2⫹]0-induced relaxation of endothelium-intact rat aortic segments. To achieve similar levels of PE-induced contractions, concentrations of PE were lowered from 0.2 µM to 40 or 30
nM in intact rings and from 50 to 12 or 10 nM in
denuded rings, respectively, for rings treated
with L-NMMA or L-NAME. Each point represents
means ⫾ SE expressed as tension (g). * P ⬍ 0.01
compared with control; n ⫽ 6 each.
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catheters were placed in a left carotid artery (PE-90), a left
external jugular vein (PE-20), and a branch of the ileocolic
artery (PE-10) for direct arterial pressure monitoring and
drug administration.
The rat mesenteries were prepared and kept under physiological conditions according to procedures described previously (8, 33, 34). The mesenteric tissues were superfused with
a Ringer-gelatin bicarbonate solution (pH 7.4), maintained at
37–37.5°C (8, 33, 34). The composition of the Ringer-gelatin
bicarbonate has been reported (8).
In vivo microscope observations for discrete, quantitative
changes in microvascular lumen sizes of arterioles and venules were made at magnifications up to ⫻3,000 using the
image-splitting television microscope recording system (8, 33,
34). During topical (perivascular) applications of magnesium
salt solutions, the superfusion of the Ringer-gelatin bicarbonate solution was temporarily interrupted and the change in
arteriolar (15–25 µm) and/or venular (18–40 µm) lumen sizes
were recorded for 3 min after topical application (0.1 ml
volumes). Systemic (intravenous and intra-ileocolic arterial)
administration (1–40 µmol/min) was made using an infusion
pump (Harvard Apparatus, model 600–910; 0.04 ml/min).
Ringer-gelatin bicarbonate solution, by itself (placebo control), caused no significant changes in baseline microvascular
lumen sizes, microvascular flow patterns, or reactivity (8,
33, 34).
Drugs. The following pharmacological agents were purchased from Sigma Chemical (St. Louis, MO): L-NNA, LNAME, L-arginine, ACh, EGTA, methylene blue, sulfanilamide, N-(l-naphthyl)ethylenediamine dihydrochloride, and
propranolol HCl. L-NMMA was purchased from Calbiochem
(La Jolla, CA). PE HCl and atropine sulfate were bought from
Mann Research Laboratories (New York, NY). BAPTA-AM
was purchased from Molecular Probes (Eugene, OR). Cimetidine HCl and diphenhydramine HCl were received from
Smith Kline and French Laboratories (Welwyn Garden City,
Herts, UK). Indomethacin was received from Merck (Rahway,
NJ). Methysergide maleate was purchased from Sandoz
Pharmaceuticals (Hanover, NJ). Naloxone HCl was purchased from Dupont (Wilmington, DE). Pentobarbital sodium
injection was purchased from Abbott Laboratories (North
Chicago, IL). All other organic and inorganic chemicals were
obtained from Fisher Scientific (Fair Lawn, NJ) and were of
the highest purity.
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MG2⫹-INDUCED RELAXATION AND BLOOD PRESSURE LOWERING
dependent relaxation of rat aorta produced by Mg2⫹
was found in this study. Removal of extracellular Ca2⫹
had an appreciable, significant (P ⬍ 0.01) inhibitory
effect on the relaxation caused by all concentrations of
Mg2⫹ tested (Fig. 3, A and B). The relaxant effects of
Mg2⫹ on intact, aortic rings were inhibited, almost
completely, when the intracellular Ca2⫹ in endothelial
cells was buffered with 10 µM membrane-permeable
BAPTA-AM (Fig. 3, A and B).
NO release induced by Mg2⫹ and its inhibition with
NOS antagonists as well as removal of extracellular
Ca2⫹ concentration. Figure 4 shows the peak concentrations of NO release from the intact rat aortic rings
evoked by administration of Mg2⫹ at concentrations of
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Fig. 3. Concentration effects of [Mg2⫹]0 obtained in absence and presence of either extracellular Ca2⫹ or in presence
of 10 µM 1,2-bis(2-aminophenoxy)ethane-N,N,N8,N8-tetraacetic acid (BAPTA-AM) in PE-precontracted intact rat
aortic segments. A: tracings of typical bioassay curves for effects of [Mg2⫹]0 in presence (a) and absence of [Ca2⫹] (b)
or in presence of 10 µM BAPTA-AM (c). B: each point represents means ⫾ SE expressed as tension (g). * P ⬍ 0.01
compared with control; n ⫽ 10.
MG2⫹-INDUCED RELAXATION AND BLOOD PRESSURE LOWERING
R633
2.4, 4.8, 7.2, and 9.6 mM. The NO concentration rose
markedly with increasing concentration of Mg2⫹ and
reached a maximum (14.5 ⫾ 1.35 µM) at a [Mg2⫹]0
concentration of 9.6 mM. Preincubation of the intact
rings with 150 µM L-NMMA or 50 µM L-NAME significantly reduced (P ⬍ 0.001) the 9.6 mM Mg2⫹-induced
NO release from 14.5 ⫾ 1.35 µM to 3.8 ⫾ 0.3 µM and
3.45 ⫾ 0.3 µM, respectively. Using denuded rat aortic
rings, we could not detect any measurable NO products
under our experimental conditions (data not shown,
n ⫽ 6). Interestingly, removal of extracellular Ca2⫹
concentration ([Ca2⫹]0) resulted in a complete inhibition of NO release in response to 4.8 and 7.2 mM
[Mg2⫹]0 (Fig. 4).
Effects of diverse pharmacological agents on Mg2⫹
vascular relaxation. As shown in Fig. 5, the extracellular Mg2⫹-induced relaxations of intact rat aortic rings
were antagonized significantly by pretreatment of the
vessels with 5.0 µM methylene blue, an antagonist of
soluble guanylate cyclase activation (22). Pretreatment
of the denuded vessels with 5.0 µM methylene blue did
not, however, attenuate the relaxant responses of the
vessels to extracellular Mg2⫹ (data not shown, n ⫽ 6
each). A muscarinic ACh receptor antagonist (atropine,
0.5 µM), an antagonist of prostaglandin formation
(indomethacin, 5.0 µM), an antagonist of histamine H1
receptors (diphenhydramine, 5.0 µM), an antagonist of
histamine H2 receptors (cimetidine, 5.0 µM), a ␤-adrenoceptor antagonist (propranolol, 5.0 µM), an opiate
receptor antagonist (naloxone, 5.0 µM), and an antagonist of serotonin receptors, methysergide (5.0 µM), did
not modify the response of intact rat aorta rings to
extracellular Mg2⫹ (data not shown, n ⫽ 6 each).
Effects of intravenous Mg2⫹ on arterial blood pressure
and serum Mg level: modulation by L-NMMA, L-NAME,
and L-arginine. Figure 6, A and B, demonstrates that
Fig. 5. Concentration effects of extracellular
Mg2⫹ on endothelium-intact rat aortic rings
in absence and presence of methylene blue
(MB, 5.0 µM). To obtain similar levels of
active, developed forces in experiments, concentration of PE was lowered from 0.2 µM to
40 nM in intact rings and from 50 to 12 nM in
denuded rings, respectively, for rings treated
with MB. Each point represents means ⫾ SE
expressed as tension (g). * P ⬍ 0.01 compared
with control; n ⫽ 6 each.
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Fig. 4. Action of [Mg2⫹]0 on release of
nitric oxide (NO; nitrite, µM) from isolated, intact rat aorta and effects of LNMMA, L-NAME, and removal of extracellular Ca2⫹ on Mg2⫹-induced NO release. A,
control (1.2 mM [Mg2⫹]0); B, 2.4 mM [Mg2⫹]0;
C, 4.8 mM [Mg2⫹]0; D, 7.2 mM [Mg2⫹]0; E,
9.6 mM [Mg2⫹]0; F, 9.6 mM [Mg2⫹]0 plus
150 µM L-NMMA, G, 9.6 mM [Mg2⫹]0 plus
50 µM L-NAME; H, 7.2 mM [Mg2⫹]0 in
absence of extracellular Ca2⫹; I, 4.8 mM
[Mg2⫹]0 in absence of extracellular Ca2⫹.
Each point represents means ⫾ SE expressed as NO production (µM). # P ⬍ 0.05
and * P ⬍ 0.01 compared with control; n ⫽
8 each.
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MG2⫹-INDUCED RELAXATION AND BLOOD PRESSURE LOWERING
intravenous infusion of increasing doses of MgSO4 (1,
10, 20, and 30 µM/min, rate 0.04 ml/min) directly into
the rat femoral vein produced sustained decreases in
the femoral arterial blood pressure. Mean measured
serum concentrations of Mg in rat blood, for infusion
doses of 1, 10, 20, and 30 µM/min, were 1.33 ⫾ 0.03,
2.45 ⫾ 0.05, 3.70 ⫾ 0.06, and 4.95 ⫾ 0.09 mM,
respectively. The femoral systolic blood pressure was
reduced decrementally from 148 ⫾ 0.86 to 96 ⫾ 0.69
mmHg (Fig. 6A), and the femoral diastolic blood pressure was decreased decrementally from 98 ⫾ 0.75 to
56 ⫾ 0.62 mmHg (Fig. 6B) after infusion of 1–30
µM/min of MgSO4. Intravenous administration of 10
mg/kg L-NMMA or L-NAME after infusion of 30
µM/min magnesium sulfate resulted in a significant
reversal of the femoral arterial blood pressure decrements; the MgSO4-decreased femoral systolic blood
pressure was increased from 96 ⫾ 0.69 to 135.8 ⫾
0.78 mmHg or 138.5 ⫾ 0.72 (Fig. 6A) for the respective
NOS antagonists. The MgSO4-decreased femoral diastolic blood pressure was raised from 56 ⫾ 0.62 to 85 ⫾
0.82 mmHg or 88.6 ⫾ 0.92 (Fig. 6B). The inhibitory
effect of L-NMMA on the Mg2⫹ infusion-induced decrement of blood pressure was antagonized partially
by infusion of 15 mg/kg L-arginine. As indicated in Fig.
7, A and B, preinjection of 10 mg/kg L-NMMA or
L-NAME into the rat femoral vein significantly suppressed the dose-dependent relaxant action of Mg2⫹ on
systemic arterial blood pressure in vivo. The decrements of rat femoral arterial blood pressure, observed
in vivo to increased doses of Mg2⫹, were restored partly
by direct injection of 15 mg/kg L-arginine into the rat
femoral vein in the L-NMMA- and L-NAME-treated
groups.
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Fig. 6. Dose-response relations of intravenous
infusion of magnesium sulfate (MgSO4, µM/min)
on femoral arterial systolic (A) and diastolic (B)
blood pressure in anesthetized rats, and effects of
infusion of L-NAME (10 mg/kg), L-NMMA (10
mg/kg), and L-Arg (15 mg/kg) on MgSO4-induced
decrements of systolic (A) and diastolic (B) arterial blood pressure. Each bar represents means ⫾
SE expressed as mmHg. * P ⬍ 0.01 and # P ⬍ 0.05
compared with 0-MgSO4 employment. For LNAME, L-NMMA, and L-Arg test experiments, 30
µM/min MgSO4 was utilized; n ⫽ 10 each.
MG2⫹-INDUCED RELAXATION AND BLOOD PRESSURE LOWERING
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Relationships between NO production and relaxation
of intact rat aortic rings and between [Mg2⫹] in serum
and reduction of arterial blood pressure. Figure 8A
indicates that the NO production is intimately associated with the relaxation of intact rat aortic rings
(induced by elevation of [Mg2⫹]0). A proportional relationship clearly exists between the NO release and the
percentage of relaxation of intact rat aortic rings (r ⫽
0.973, P ⬍ 0.001). With respect to arterial blood pressure, it is clearly and directly related to the [Mg] level
in the serum (r ⫽ 0.952, P ⬍ 0.001); the higher the [Mg]
in the serum, the lower the arterial blood pressure
(Fig. 8B).
[Mg2⫹]0-induced vasodilatation of intact mesenteric
arterioles and venules is inhibited by L-NNA and LNMMA. Using intact anesthetized rats and in situ
qualitative video microscopy, we found that perivascularly MgCl2-induced vasodilatation of intact mesenteric
arterioles could be almost completely inhibited by prior
administration of either L-NNA or L-NMMA (10 mg/kg,
Table 1). Although not shown, similar results could be
obtained using MgSO4 or Mg. Aspartate, HCl (n ⫽ 5–6
each, data not shown); intact mesenteric venules responded very similarly (n ⫽ 12 each, data not shown).
MgCl2- or MgSO4-induced mesenteric arteriolar and
venular vasodilatation produced by close intra-arterial
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Fig. 7. Effects of preinjection of L-NAME (10
mg/kg) or L-NMMA (10 mg/kg) and postinjection of L-Arg (15 mg/kg) on decreased rat
femoral arterial systolic (A) and diastolic (B)
blood pressure caused by continuous infusion
of magnesium sulfate (MgSO4). Each point
represents means ⫾ SE expressed as mmHg.
* P ⬍ 0.01 compared with control; n ⫽ 10 each.
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MG2⫹-INDUCED RELAXATION AND BLOOD PRESSURE LOWERING
(via ileocolic artery) or intravenous administration of
these Mg salts could also be inhibited almost completely by prior administration of L-NNA or L-NMMA
(data not shown, n ⫽ 12). Intravenous infusion of 20
mg/kg L-arginine was found to restore 70–90% of the
responses to MgCl2 (or MgSO4) that were inhibited by
L-NNA and L-NMMA (n ⫽ 4 each, data not shown).
DISCUSSION
Up to the present time it has been thought that
extracellular Mg2⫹ induces vasodilatation by a direct
action on vascular smooth muscle (31). The results of
the present study demonstrate that the relaxant responses to extracellular Mg2⫹ were attenuated in arterial rings after removal of endothelium, suggesting that
such aortic relaxant responses induced by Mg2⫹ are in
part (about 40%) endothelium dependent and that
generation and release of some mediator, such as the
cGMP-associated EDRF, may be involved in [Mg2⫹]0induced relaxation of blood vessels.
Previous studies indicate that magnesium deficiency,
rather than excess Mg2⫹, impairs vasorelaxation (5, 6,
10). In isolated canine coronary arteries, Altura and
Altura (11) demonstrated that endothelium-dependent
cGMP-mediated vasodilatation (response to ACh) required the presence of extracellular Mg2⫹. Ku and Ann
(25), also using isolated canine coronary arteries, demonstrated that magnesium deficiency produced a dysfunctional response to several endothelium-dependent
vasodilators, including ACh and thrombin. Under our
experimental conditions it was found that preincubation of endothelium-intact rat aortic rings with either
L-NAME or L-NMMA (both being antagonists of NOS)
almost abolished the endothelium-dependent component of extracellular Mg2⫹-induced relaxation at every
concentration tested. The effects of NOS antagonists
were specific because their actions could be reversed
largely by the presence of high concentrations of Larginine, a physiological substrate for NOS (35). The
inhibitory effects of NOS antagonists on [Mg2⫹]0-
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Fig. 8. Linear regression analyses of NO production
and relaxation of intact rat aortic rings (A) (both induced by elevation of [Mg2⫹]0), serum Mg2⫹ concentration and reduction of rat arterial blood pressure (B).
Each point for aortic relaxation to [Mg2⫹]0 and for
percentage reduction in blood pressure represents
means ⫾ SE; n ⫽ 6 each.
MG2⫹-INDUCED RELAXATION AND BLOOD PRESSURE LOWERING
Table 1. Effects of perivascularly applied MgCl2 in the
absence and presence of NOS antagonists on
arteriolar lumen sizes in rat mesentery
Lumen Size, µm
Groups Dose, µmol
After Mg2⫹
%Increase
in Lumen
Size
10
7
8
18.5 ⫾ 0.6
17.9 ⫾ 1.0
18.8 ⫾ 0.8
20.3 ⫾ 0.4*
22.4 ⫾ 0.9*
25.0 ⫾ 1.1†
9.7*
25.1*
32.9†
8
6
8
18.6 ⫾ 0.5
19.2 ⫾ 0.8
17.8 ⫾ 0.6
18.8 ⫾ 0.6
20.4 ⫾ 0.8
19.8 ⫾ 0.8
1.0‡
6.3‡
11.2‡
6
8
6
18.4 ⫾ 0.8
18.0 ⫾ 0.8
17.6 ⫾ 0.8
18.8 ⫾ 0.8
19.2 ⫾ 0.8
19.8 ⫾ 0.6
2.1‡
6.7‡
12.5‡
Values are means ⫾ SE; n, no. of rats. NOS, nitric oxide synthase;
N G-nitro-L-arginine; L-NMMA, N G-monomethyl-L-arginine.
Significantly different from controls (before MgCl2 ; paired t-test,
* P ⬍ 0.05 and † P ⬍ 0.01). Significantly different from MgCl2 before
L-NNA and L-NMMA (t-test, ‡ P ⬍ 0.01).
L-NNA,
induced relaxation in smaller, intact resistance vessels
were much stronger because L-NNA and L-NMMA
(antagonists of NOS) almost totally suppressed [Mg2⫹]0induced dilatation in rat mesenteric arterioles and
venules. These in vivo microcirculatory data become
more compelling when viewed in light of our findings
showing that administration of L-arginine almost restores the Mg2⫹-induced arteriolar and venular dilatations despite the presence of the NOS antagonists.
Some previous studies of others could be considered
pertinent to our present findings: 1) Mg2⫹-induced
relaxation of aortas from DOCA-salt hypertensive rats
is much less in vessels denuded of endothelium, suggesting a mediation by EDRF-NO (26) and 2) high concentrations of [Mg2⫹]0 added to physiological salt solutions
improve coronary arterial endothelium-dependent relaxation in perfused rat hearts (14).
The relaxations herein induced by extracellular Mg2⫹
were not modified by a variety of antagonists of endogenous neuro-humoral vasodilators but were blocked by
methylene blue, a selective antagonist of soluble guanylate cyclase (22), activation of which is responsible for
the vasorelaxant action of NO (40). The present experimental data thus point to the probable involvement of
[Mg2⫹]0 stimulation of guanylate cyclase within the
vessel wall. These results strongly suggest that the
endothelium-dependent component of relaxant action
of extracellular Mg2⫹ is mediated through endogenous
NO. This conclusion is definitively supported by our
new, quantitative NO chemical data. The chemical
measurement of NO release of isolated, intact rat aortic
rings made in the present study is, as far as we are
aware, the first to be made in a study of Mg2⫹-induced
vasorelaxation. The data clearly indicate that [Mg2⫹]0
produces a concentration-dependent NO release from
the intact rat aortic rings. The release of NO that
accumulated during exposure of the intact rat aortic
rings to each elevated concentration of Mg2⫹ employed
closely correlated with the Mg2⫹-induced relaxation of
the intact rings (Figs. 1, 4, and 8A). Because NO
products of denuded rat aortic rings could not be
detected in the present study and NOS antagonists
(L-NMMA and L-NAME) suppressed markedly 9.6 mM
Mg2⫹-induced NO release of intact rat aortic rings (Fig.
4), the NO products stimulated by Mg2⫹ clearly derive
from the endothelium of the vessels. These new data
provide us with direct, quantitative evidence that it is
the NO released from the endothelium that mediates
the Mg2⫹-produced endothelium-dependent relaxation.
The probability that extracellular Mg2⫹-induced relaxation in the endothelium-containing blood vessels is
mediated by endogenous NO is supported also by the in
vivo experimental data in the present study. Our in vivo
results demonstrate that infusion of increasing doses
(1–30 µM/min) of MgSO4 directly into femoral veins of
anesthetized rats produced sustained, concentrationdependent decreases in femoral arterial systolic and
diastolic blood pressures; systemic administration of
L-NAME or L-NMMA before and after infusion of
MgSO4 could, however, restore the decreased blood
pressure. Infusion of L-arginine partially reversed the
increased blood pressure caused by either L-NAME or
L-NMMA. The results clearly were more impressive
with the in vivo findings, indicating that L-NNA and
L-NMMA (antagonists of NOS) almost suppressed completely Mg2⫹-induced dilatation in rat mesenteric arterioles and venules. The microcirculatory and blood
pressure findings suggest that the smaller the blood
vessel, the more dependent the Mg2⫹-induced vasodilatation on endothelial cell release of NO. In this context,
the present findings suggest that the level of [Mg] in rat
serum is intimately associated with the magnitude of
the reduction of rat arterial blood pressure (Fig. 8B).
Our in vivo experimental results are also consistent
with some findings presented previously on the intact
microcirculation by others, which indicate that various
magnesium salts, including MgSO4, infused intravenously in increasing doses increase peripheral and
cerebral flows and decrease cerebral and systemic
vascular resistances (32, 37). The present in vivo
experiments may imply physiologically that Mg2⫹ is a
mediator of endothelial function and pharmacologically
that higher concentrations of Mg2⫹ stimulate endothelial function. Thus decreased blood pressure responses
to exogenous Mg2⫹ infusion appear to be a combination
of physiological and pharmacological effects of Mg2⫹.
Our present findings suggest that Mg2⫹-mediated
endothelium-dependent relaxation and NO release in
rat aorta appear to be Ca2⫹ dependent, because either
removal of extracellular Ca2⫹ or complete buffering of
intracellular free Ca2⫹ in endothelial cells almost completely inhibited the relaxant response of intact rat
aorta to all concentrations of [Mg2⫹]0 tested. Furthermore, the absence of extracellular Ca2⫹ completely
suppressed both NO release and production of NO in
intact rat aortas by 4.8 and 7.2 mM [Mg2⫹]0. Previous
investigations indicated that 1) Ca2⫹-activated K⫹ channels are responsible for the ACh-evoked hyperpolarization in endothelium of rat aorta (28) and cell hyperpolar-
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MgCl2 alone
1.0
10.0
40.0
MgCl2 ⫹ L-NNA
1.0
10.0
40.0
MgCl2 ⫹ L-NMMA
1.0
10.0
40.0
n
Before Mg2⫹
(Control)
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MG2⫹-INDUCED RELAXATION AND BLOOD PRESSURE LOWERING
Perspectives
Our present study suggests that in vitro [Mg2⫹]0 can
induce endothelium-dependent and endothelium-independent relaxations in rat aortas in a concentrationdependent manner; in vivo infusion of Mg2⫹ produces
concentration-dependent decreases in arterial blood
pressure concomitant with dilatation of intact microvessels. Administration of NOS antagonists reversed
[Mg2⫹]0-induced, endothelium-dependent relaxations
in rat aortas and [Mg2⫹]0-induced dilatation in rat
mesenteric arterioles and venules as well as Mg2⫹lowered blood pressure. These inhibitory effects of NOS
antagonists could be reversed partially by L-arginine.
Therefore, we tentatively propose the following new
mechanisms for Mg2⫹-induced endothelium-dependent
relaxation and blood pressure lowering: 1) activation of
Ca2⫹-activated K⫹ channels and elevation of [Ca2⫹]i as
well as release of EDRF (NO) appear to account for the
larger part of the peripheral vasodilatation evoked by
elevated [Mg2⫹]0 and 2) large conduit vessels, to fully
relax to [Mg2⫹]0, recruit endothelial-derived relaxant
factors (NO) for approximately 50% of the response, the
remainder being via direct actions on vascular smooth
muscle cells.
Some of this work was supported by the National Institute on
Alcohol Abuse and Alcoholism Research Grant AA-08674 to B. M.
Altura.
Address for reprint requests and other correspondence: B. M.
Altura, Box 31, SUNY Health Science Center at Brooklyn, 450
Clarkson Ave., Brooklyn, New York 11203.
Received 18 February 1999; accepted in final form 29 September
1999.
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