355
Antagonistic Modulatory Roles of Magnesium
and Calcium on Release of
Endothelium-Derived Relaxing Factor and
Smooth Muscle Tone
Michele E. Gold, Georgette M. Buga, Keith S. Wood, Russell E. Byrns,
Gautam Chaudhuri, and Louis J. Ignarro
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
The objective of this study was to elucidate the mechanisms associated with the reciprocal
relation between magnesium and calcium on vascular smooth muscle tone in bovine pulmonary
artery and vein. Rapid removal of magnesium from Krebs-bicarbonate medium used to bathe
isolated rings of precontracted artery or vein caused transient endothelium- and calciumdependent relaxation and cyclic GMP accumulation. Both responses were antagonized by
oxyhemoglobin, methylene blue, or superoxide anion and were enhanced by superoxide
dismutase. The transient relaxation was followed by sustained endothelium-independent
contraction. Endothelium-denuded vascular rings contracted in response to extracellular
magnesium depletion without alteration in cyclic GMP levels. The data suggest that vascular
endothelium-derived nitric oxide is responsible for the calcium-dependent relaxation elicited by
extracellular magnesium depletion. Indeed, in bioassay cascade studies, magnesium removal
from the medium used to perfuse intact artery or vein enhanced the formation and/or release
of an endothelium-derived relaxing factor by calcium-dependent mechanisms. In the absence of
both extracellular magnesium and calcium, calcium readdition caused transient endotheliumdependent relaxation and cyclic GMP accumulation, and both responses were abolished by
oxyhemoglobin or methylene blue. In the presence of magnesium, however, readdition of
calcium to calcium-depleted medium caused only contractile responses. Addition of magnesium
to calcium-containing medium consistently caused endothelium- and cyclic GMP-independent
relaxation that was not altered by oxyhemoglobin or methylene blue. Thus, magnesium and
calcium elicit reciprocal or mutually antagonistic effects at the levels of both endotheliumderived relaxing factor formation and/or release and smooth muscle contraction. This relation
may be of physiological importance, and the possibility that a reduction in circulating
magnesium levels could lead to calcium-mediated vasospasm may be of pathophysiological
concern. (Circulation Research 1990;66:355-366)
Since the initial discovery in 1980 that acetylchocauses endothelium-dependent arterial
k, line
relaxation through the release of an endothelium-derived relaxing factor (EDRF),' much has
been learned about the factors influencing the formation and/or release of EDRF.2-4 Other than prostacyclin, more than one EDRF is likely to exist, but
From the Department of Pharmacology, University of California
Los Angeles, School of Medicine, Los Angeles.
Supported in part by National Institutes of Health grant HL35014 and a grant from the Laubisch Fund for Cardiovascular
Research.
Address for reprints: Dr. Louis J. Ignarro, Department of
Pharmacology, UCLA School of Medicine, Los Angeles, CA
90024.
Received November 14, 1988; accepted August 24, 1989.
only one has been identified. An EDRF released
from perfused cultured aortic endothelial cells5 and
from perfused intact artery and vein6,7 has been
identified as nitric oxide or a closely related labile
nitroso species that spontaneously liberates nitric
oxide on release from endothelial cells. This
endothelium-derived nitric oxide (EDNO) appears
to account for the biological actions of EDRF in
vascular smooth muscle and platelets,5-12 and such
actions are indistinguishable from those first
described for authentic nitric oxide almost a decade
ago.13-16
Extracellular calcium appears to be essential for
endothelium-dependent vascular smooth muscle
relaxation,'7-20 and other studies have shown that
endothelium-dependent relaxation is dependent on
356
Circulation Research Vol 66, No 2, February 1990
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the intracellular concentration of calcium in the endothelial cells.2122 The influx of extracellular calcium
may be one mechanism that couples the interaction of
endothelium-dependent vasodilators at endothelial
cell surface receptors to the synthesis and/or release of
EDRF, although some controversy exists over the
precise mechanisms involved.317-20,22-26 Extracellular
magnesium has been found to inhibit calcium influx at
the vascular smooth muscle membrane27-29 and at the
myocardial sarcolemmal membrane30 and has been
suggested to interfere with calcium release from intracellularly bound sites in vascular smooth muscle.31
Such a reciprocal or antagonistic relation between
magnesium and calcium has been recently described
also with respect to endothelium-dependent vascular
smooth muscle relaxation, in which extracellular magnesium depletion caused endothelium- and calciumdependent arterial relaxation.32 The objective of the
present study was to address the latter observations
and elucidate the mechanism of the apparently mutual
antagonistic relation between extracellular magnesium and calcium on endothelium-dependent relaxation in bovine intrapulmonary artery and vein. To
this end, the influence of extracellular magnesium and
calcium on the formation and/or release of EDRF,
endothelium-dependent and -independent relaxant
responses, and cyclic GMP formation in vascular
smooth muscle was examined.
Materials and Methods
Preparation of Rings of Bovine Intrapulmonary
Artery and Vein
Bovine lungs were obtained from cows 5 years of
age or older and transported to the laboratory in iced
Krebs-bicarbonate solution as described.33 The second
intrapulmonary arterial branch and underlying venous
branch extending into the larger lobe were rapidly
excised, gently cleaned of parenchyma, fat, and adhering connective tissue, and placed in cold preoxygenated Krebs-bicarbonate solution. Segments with outside diameters of 4-6 mm (artery) and 6-8 mm (vein)
were isolated and sliced into rings (4 mm wide) with a
specially designed microtome.34 Rings were prepared
with an intact or functional endothelium as assessed
by 80-100% relaxation to 0.1-1 ,uM acetylcholine in
artery and 10 nM bradykinin in vein. Endothelial cells
were removed from some arterial or venous rings and
are referred to in the text as either endotheliumdenuded or rubbed. Endothelium-denuded arterial or
venous rings were prepared by gently everting the
rings (intimal side out) and rubbing the entire surface
with moistened filter paper for 30 seconds; rings were
then returned to their normal position (intimal side
in). These endothelium-denuded rings contracted in
response to 1 ,uM acetylcholine (artery) or 10 nM
bradykinin (vein).
Mounting Rings and Recording of Muscle Tension
Arterial and venous rings were mounted on
nichrome wires in jacketed, 25-ml capacity, drop-
away bath chambers containing Krebs-bicarbonate
solution (370 C) and gassed with 95%02-5% CO2.34
The upper nichrome wire of each ring was attached
to a force-displacement transducer (model FT03C,
Grass Instrument, Quincy, Massachusetts), and
changes in isometric force were recorded on a Grass
polygraph (model 79D). Arterial and venous rings
were equilibrated for 2 hours at a resting tension of 4
g34 followed by depolarization with 120 mM KCl.
Rings were then washed and allowed to equilibrate
for 45 minutes before initiating any given protocol.
Muscle depolarization stabilizes the subsequent submaximal contraction by phenylephrine or related
contractile agents, presumably by promoting calcium
influx into smooth muscle cells. This procedure has
been used routinely for bovine pulmonary vessels in
this laboratory.33'34 Phenylephrine, a selective aladrenergic receptor agonist was used to elicit precontractile responses in arterial rings, whereas U46619, a
thromboxane A2-mimetic eicosanoid, was used to
precontract venous rings.
Bioassay Cascade Superfusion
A modification35 of the technique developed by
Vane in 1964 was used.36 Briefly, bovine pulmonary
artery or vein with intact endothelium was isolated
and cleaned of adhering connective tissue, and
branches were ligated by means of titanium hemostatic clips (Pilling, Fort Washington, Pennsylvania)
to prevent leakage during vessel perfusion. Polyethylene tubing was fitted at either end of the vessel
segments for perfusion with Krebs-bicarbonate solution at 370 C. The perfusate was allowed to superfuse
three isolated, helically cut, precontracted strips (2.5
cm long, 4 mm wide) of endothelium-denuded artery
or vein arranged in a cascade in which each strip was
separated in flow time by 3 seconds. A second
superfusion line was also positioned over the cascade
(5 ml/min total flow at 370 C). Indomethacin (10 ,uM)
was present in both perfusion and superfusion media
to prevent formation of prostacyclin or other vasodilator prostaglandins. Vessel strips were equilibrated
by superfusion with Krebs-bicarbonate solution for 2
hours before initiating any protocol, at which time
the strips were precontracted with a mixture of
phenylephrine (50 ,uM) and U46619 (50 nM). The
reason for using both phenylephrine and U46619 for
precontraction is that contractile responses were well
maintained and reproducible when the mixture was
used, whereas, phenylephrine alone resulted in a loss
of tone and U46619 alone resulted in marked oscillatory responses. Responses were recorded as
changes in length of the smooth muscle preparations,
measured with auxotonic levers attached to transducers (model 386, Harvard Apparatus, South
Natick, Massachusetts),35 and expressed as centimeters in Figures 5 and 6. Glyceryl trinitrate, a stable
endothelium-independent vasodilator, was superfused over the strips to standardize the preparations
exactly as described previously.35,37 In those experiments in which 1.2 mM magnesium was removed
Gold et al Regulation of EDNO Release by Magnesium and Calcium
ARTERY
VEIN
+Endothel ium
+ Endothelium
,-8 Ach
. M92+
A -M92
BKN
A-Mg92+ +M92+
r- -7
W/E,
5g
10min
5g
t
PE -4
PE -5
-Endothelium
-
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
-5
~
10mm
Endothel ium
W/E
PE
PE-s
357
W/E
t
~
PE-5
PE 4
~~~~~~~~~~~~t
PE -4
FIGURE 1. Effects of extracellular magnesium removal and
readdition on tone and relaxant responses in pulmonary artery.
Unrubbed (+Endothelium) and endothelium-denuded
(-Endothelium) arterial rings were mounted and equilibrated
as described in the text. Contractile responses to phenylephrine
(PE) were 65-75% of maximal. A-Mg2` denotes rapid
replacement of Krebs' bathing medium with Mg2`-free
medium. +Mg`2 denotes readdition of 1.2 mM MgSO4 to the
bathing medium. Acetylcholine (Ach) and glyceryl trinitrate
(GTN) were added at cumulatively increasing concentrations
as indicated. Concentrations are expressed as exponents to the
base power 10 and represent final bath concentrations. W/E
denotes tissue washing followed by 45-minute equilibration.
One tracing representative of sir separate experiments is shown.
FIGURE 2. Effects of extracellular magnesium removal and
readdition on tone and relaxant responses in pulmonary vein.
Unrubbed (+Endothelium) and endothelium-denuded
(-Endothelium) venous rings were mounted and equilibrated
as described in the text. Contractile responses to phenylephrine
(PE) were 65 -75% of maximal. A-Mg2` denotes rapid
replacement of Krebs' bathing medium with Mg2`-free
medium. +Mg2` denotes readdition of 1.2 mM MgSO4 to the
bathing medium. Bradykinin (BKN) and glyceryl trinitrate
(GTN) were added at cumulatively increasing concentrations
as indicated. Concentrations are expressed as exponents to the
base power 10 and represent final bath concentrations. WIE
denotes tissue washing followed by 45-minute equilibration.
One tracing representative offive separate experiments is shown.
from the perfusion medium, the superfusion medium
adjusted to twice the magnesium concentration
to correct for the dilution caused by mixing with the
magnesium-free perfusate. Similar procedures were
followed when the calcium concentration in the
perfusion medium was altered.
interfered directly with antigen-antibody binding in
the radioimmunoassay procedures. Recoveries of
standard amounts of added cyclic nucleotides were
determined periodically, and the values ranged from
92% to 104%. Therefore, no corrections for sample
recoveries were made.
Deternination of Cyclic Nucleotide Levels
Cyclic GMP levels were measured in arterial and
venous rings that had been equilibrated under tension
and depolarized with KCl. Tone was monitored until
the time of freeze-clamping. The rapid drop-away
bath chambers were lowered, and rings were quickly
frozen between brass clamps precooled in liquid nitrogen. Each frozen ring was homogenized in 1 ml of 6%
trichloroacetic acid (ground-glass tissue grinder).
After centrifugation, the supernatant was extracted
with diethyl ether to remove the acid, and aliquots of
the aqueous phase were lyophilized to dryness, reconstituted in buffer, and analyzed by radioimmunoassay.
These procedures were described previously.14'34
None of the test agents added to the bath chambers
Chemicals and Solutions
Acetylcholine chloride, bradykinin triacetate,
A23187, phenylephrine hydrochloride, indomethacin,
hemoglobin, sodium dithionite, methylene blue, pyrogallol, and superoxide dismutase (bovine liver) were
obtained from Sigma Chemical, St. Louis, Missouri.
U46619 ([15S]-hydroxy-lla,9a[epoxymethano]prosta5Z,13E-dienoic acid) was provided by The Upjohn Co,
Kalamazoo, Michigan, and was dissolved in absolute
ethanol at a concentration of 10 mg/ml. Dilutions
were prepared in cold distilled water to a final concentration of 0.1 mM and stored frozen. S-NitrosoN-acetylpenicillamine was synthesized and used as
described previously.38 Glyceryl trinitrate (10% wt/wt
triturated mixture in lactose) was a gift from ICI
was
358
Circulation Research Vol 66, No 2, February 1990
TABLE 1. Influence of Alterations in Magnesium and Calcium Concentrations on Endothelium-Dependent and -Independent Responses
n
Endothelium
Percent relaxation
Percent contraction
Test condition
37+8
+
36
1. A-Mg2+
18+4
30
+
36+6
18
2. 1.2 mM Mg2`
..
38+8
12
..
+
26+3
18
3. A-Mg2+ (vein)
12+2
12
+
31 +4
12
4. 1.2 mM Mg2` (vein)
30+3
12
14+2*
+
18
5. 1 ,uM HbO2, A-Mg2+
12±1*
+
18
6. 10 ,uM MB, I-Mg2+
+
100+0
12
7. 100 units/ml SOD+,-Mg2+
14±2
12
8. 100 units/ml SOD+ A-Mg2+
+
20±3*
12
9. 0.1 mM pyrogallol+A-Mg2+
+
±2*
33±5
18
10. 1 ,uM HbO2, A-Mg2+, 1.2 mM Mg2+
+
30±4
12
11. 10 jsM MB, A-Mg2+, 1.2 mM Mg2+
+
13±2*
24
12. -Ca2+, A_Mg2+
+
55±8
24
13. -Ca2+, A-Mg2+, 1.5 mM Ca'
14. 1 ,uM HbO2, -Ca2+, '-Mg2+, 1.5 mM
+
12
7+1t
Ca2+
15. Acetylcholine
+
11+2
12
0.01 ,uM
44+6
12
0.1 ,uM
89±+11
12
1 ,uM
16. -Ca2+, acetylcholine
+
12
0+0t
0.01 ,uM
+
9± t
12
0.1 ,uM
+
20+2t
12
1 gM
+
24
52+8
17. -Ca2 ,-Mg2+, 1.5 mM Ca2+
18. 1 ,uM HbO2, -Ca2+, -Mg2+, 1.5 mM
+
66±9§
12
Ca2+
19. -Mg2+, acetylcholine
+
12
14±2
0.01 gM
+
43±6
12
0.1 gM
..
..
..
..
..
..
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
1 ,uM
12
Americas, Wilmington, Delaware. Solutions of hygroscopic acetylcholine were prepared in distilled water,
separated into aliquot portions, and stored frozen.
Bradykinin, pyrogallol, A23187, and superoxide dismutase were prepared fresh in distilled water just
before use. Oxyhemoglobin was prepared from hemoglobin by reduction with sodium dithionite in oxygenated Krebs-bicarbonate solution at 40 C, essentially as
described.7 Krebs-bicarbonate solution consisted of
(mM) NaCl 118, KCl 4.7, CaCl2 1.5, NaHCO3 25,
MgSO4 1.2, KH2PO4 1.2, and glucose 11. Depolarizing
KCl solution had a composition similar to Krebsbicarbonate solution except the sodium chloride was
replaced with an equimolar concentration of KCl.
Calculations and Statistical Analysis
Relaxation and contraction were measured as the
decrease and increase, respectively, in tension relative to the tension elicited by precontracting arterial
or venous smooth muscle with phenylephrine or
+
92±12
U46619 as indicated. Values in Figures 7-10 are
expressed as the mean±+-SEM and represent
unpaired data. Comparisons were made by Duncan's
multiple range test39 for comparisons with a common
control (Figures 8-10) or by Student's t test for
unpaired values for all other comparisons where
indicated. The level of statistically significant difference was p<0.05. Figures 1-4 illustrate typical tracings from representative experiments. A summary of
all of the pertinent data related to the experiments
illustrated in Figures 1-4 represented as the
mean±SEM, together with a statistical evaluation of
important differences, is presented in Table 1.
Results
Characteristics of Relaxation ofArtery
and Vein Elicited by Removal and Addition
of Extracellular Magnesium
Rapid removal of magnesium from the extracellular medium produced a rapid and transient
Gold et al Regulation of EDNO Release by Magnesium and Calcium
TABLE 1. Continued
Test condition
20. 1 ,M HbO2, -Mg2+, acetylcholine 1 ,uM
21. -Ca2, -Mg2, 1.5 mM Ca2+
22. SNAP
0.01 ,M
0.1 gM
1 gM
23. -Mg2+, SNAP
0.01 ,M
0.1 ,M
1 gM
24. 1 ,uM HbO2, -Mg2+, SNAP
0.01 ,uM
0.1 ,LM
n
12
24
Endothelium
+
-
Percent relaxation
12
12
12
-
22±3
59±8
99±8
...
12
12
12
-
18±2
54±7
97±6
...
12
12
-
2±1¶
...
14±2T
...
359
Percent contraction
14±111
...
...
82±12
...
...
...
...
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
1 ,uM
12
35±4T
Unrubbed (+) and endothelium-denuded (-) rings of bovine pulmonary artery or vein were mounted and equilibrated as described in
the text. Rings were precontracted (65-75% of maximal) by addition of phenylephrine. Data represent the mean±SEM, and n signifies the
number of separate vascular rings tested. Percent relaxation and contraction, respectively, signify percentage decrease and increase in
phenylephrine-induced tone. Test conditions were as follows: 1, rapid replacement of Krebs' bathing medium with Mg2+-free medium at
time of peak contractile response; 2, added after stabilization of tone in response to -Mg2+; 3, same as 1; 4, same as 2; 5, oxyhemoglobin
(HbO2) added 10 minutes before precontraction, and -Mg2+ added at peak contraction; 6, similar to 5 (MB, methylene blue); 7 and 8,
superoxide dismutase (SOD) included in Mg2+-free medium at -Mg2+; 9, similar to 7; 10, HbO2 added 10 minutes before contraction,
-Mg2+ added at peak contraction, and Mg2+ added after stabilization of tone; 11, similar to 10; 12, replacement of Krebs' bathing medium
with Ca2+-free medium 10 minutes before contraction, and -Mg2+ added at peak contraction; 13, same as 12 except Ca2+ added back after
stabilization of tone; 14, same as 13 except HbO2 added 10 minutes before contraction; 15, cumulative addition of acetylcholine starting at
peak contraction; 16, same as 15 except Krebs' bathing medium replaced with Ca2+-free medium 10 minutes before contraction; 17,
replacement of Krebs' bathing medium with Ca2+- and Mg2+-free medium 10 minutes before contraction and Ca2+ added back at peak
contraction; 18, same as 17 except HbO2 added 10 minutes before contraction; 19, replacement of Krebs' bathing medium with Mg2+-free
medium 10 minutes before contraction and cumulative addition of acetylcholine starting at peak contraction; 20, similar to 19 except HbO2
added 10 minutes before contraction; 21, same as 17; 22, similar to 15 (SNAP, S-nitroso-N-acetylpenicillamine); 23, similar to 19; 24, similar
to 20.
*Significantly different (p<0.05) from values corresponding to test condition 1.
tSignificantly different (p<0.05) from values corresponding to test condition 13.
tSignificantly different (p<0.05) from values corresponding to test condition 15.
§Significantly different (p<0.05) from values corresponding to test condition 17.
IISignificantly different (p<0.05) from values corresponding to test condition 19 (1 ,uM).
TSignificantly different (p<0.05) from values corresponding to test condition 22.
endothelium-dependent relaxation of artery (Figure
1) and vein (Figure 2) followed by return of tone that
was often greater than precontractile tension (Table
1). Endothelium-denuded arterial and venous rings
failed to relax but did contract in response to magnesium removal. Acetylcholine and bradykinin were
used to further assess the endothelium-dependent
relaxation of intrapulmonary artery and vein, respectively, when exposed to magnesium-free medium.
Removal of magnesium from the extracellular
medium had no appreciable inhibitory effect on the
relaxation caused by these endothelium-dependent
relaxants (Table 1). In endothelium-denuded rings
exposed to magnesium-free medium, relaxation
caused by glyceryl trinitrate was intact with maximal
relaxation occurring at 10 nM. Readdition of magnesium to the magnesium-depleted medium resulted in
endothelium-independent relaxant responses. To
further characterize the endothelium-dependent
relaxation produced by magnesium removal and the
endothelium-independent relaxation produced by
magnesium addition, the effects of oxyhemoglobin,
methylene blue, superoxide dismutase, and pyro-
gallol were assessed. Pretreatment of arterial rings
with 1 ,uM oxyhemoglobin or 10 ,uM methylene blue
abolished the endothelium-dependent relaxation
produced by the removal of extracellular magnesium
but had no effect on the relaxation produced by the
readdition of magnesium (Table 1). Superoxide dismutase markedly potentiated, whereas pyrogallol
antagonized, the endothelium-dependent relaxation
produced by removal of extracellular magnesium
(Table 1). The effects of the above test agents on
venous rings were qualitatively similar to those on
arterial rings (data not shown).
Interaction of Magnesium and Calcium in
Endothelium-Dependent Relaxation
The endothelium-dependent relaxation produced
by removal of extracellular magnesium was also
calcium-dependent since relaxation did not occur
when calcium was removed from the bathing medium
(Figure 3). Readdition of calcium resulted in return
of the endothelium-dependent relaxant responses,
and these were inhibited by 1 ,uM oxyhemoglobin
(Figures 3 and 4, Table 1). Figure 4 shows the
360
Circulation Research Vol 66, No 2, February 1990
ARTERY
ARTERY
+ Endothelium
A
Ach
+ Endothelium
+Co2+
Ai
HbO2
-6
-U4-8
5g
10min
- Endothelium
+ Endothelium
-7
W/E
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
-U4-8
U4 -8
AI _-Ca2+
A I _C,02+
FIGURE 3. Effects of extracellular calcium removal and
readdition on relaxant responses to magnesium removal and
acetylcholine (Ach) in pulmonary artery. Unrubbed
(+Endothelium) arterial rings were mounted and equilibrated
as described in the text. A-Mg2` denotes rapid replacement of
Krebs' bathing medium with Mg2+-free medium. A-Ca2'
denotes replacement of Krebs' bathing medium with Ca2+-free
medium 10 minutes before precontraction with U46619 (U4).
Contractile responses to U4 were 65-75% of maximal. +Ca2'
denotes readdition of 1.5 mM CaCl2 to the bathing medium.
Ach was added at cumulatively increasing concentrations as
indicated. Oxyhemoglobin (HbO2) was added after tissue
washing/equilibration at 10 minutes before precontraction with
U4. Concentrations are expressed as exponents to the base
power 10 and representfinal bath concentrations. W/E denotes
tissue washing followed by 45-minute equilibration. One tracing representative offive separate experiments is shown.
reciprocal relation between magnesium and calcium
and the requirement of calcium for endotheliumdependent relaxant responses to extracellular magnesium removal and to acetylcholine. Adding calcium
to arterial rings equilibrated in medium without
magnesium or calcium caused a rapid endotheliumdependent relaxation that did not occur in endothelium-denuded rings. This relaxation was inhibited by 1
,uM oxyhemoglobin. In the presence of oxyhemoglobin,
the subsequent addition of calcium caused a contractile
response. Endothelium-denuded arterial rings contracted in response to calcium, whereas S-nitrosoN-acetylpenicillamine, an unstable nitrosovasodilator
that generates nitric oxide, caused relaxation that was
antagonized by oxyhemoglobin (Figure 4, Table 1).
Influence of Magnesium and Calcium
on
Formation/Release of EDRF
The bioassay cascade technique was used to determine the effects of magnesium (Figure 5) and cal-
iN1
- -U4- 8
M9 2+
AS
1U4 -8
1 _M92+
FIGURE 4. Influence of oxyhemoglobin on relaxant and
contractile responses to calcium and other agents in pulmonary artery. Unrubbed (+Endothelium) and endotheliumdenuded (-Endothelium) arterial rings were mounted and
equilibrated as described in the text. A-Mg2+ and A-Ca2'
denote replacement of Krebs' bathing medium with Mg2+- and
Ca2+-free medium 10 minutes before precontraction with
U46619 (U4). Contractile responses to U4 were 65-75% of
maximal. +Ca2+ denotes cumulative readdition of 1.5 mM
(a), 3 mM (b), and 4.5 mM (c) CaCl2 to the bathing medium.
Acetylcholine (Ach) and S-nitroso-N-acetylpenicillamine
(SNAP) were added at cumulatively increasing concentrations
as indicated. Oxyhemoglobin (HbO2) was added after tissue
washing/equilibration at 10 minutes beforeprecontraction with
U4. Concentrations are expressed as exponents to the base
power 10 and represent final bath concentrations. One tracing
representative of six separate experiments is shown.
cium (Figure 6) on the formation and/or release of
arterial or venous EDRF. EDRF released from
endothelium-intact bovine pulmonary artery that was
perfused with acetylcholine or A23187 was enhanced
when magnesium was removed from the perfusion
medium. Bovine pulmonary vein was perfused with
bradykinin or A23187, and EDRF release was similarly enhanced when magnesium was removed from
the perfusion medium. The magnesium concentration of the superfusion medium was increased when
magnesium-free perfusion medium was used to maintain the fluid bathing the cascade of vascular strips at
a constant magnesium concentration of 1.2 mM.
Glyceryl trinitrate, which was superfused over the
vascular strips, caused relaxation that was not
affected by the removal of magnesium from the
perfusion medium in the presence of superfused
magnesium. The requirement of calcium for EDRF
formation and/or release was further substantiated
by bioassay because removal of calcium from the
Gold et al Regulation of EDNO Release by Magnesium and Calcium
ARTERY
Arterial
Strips
GTN
0.l M
1
ACH
0.3fM
-CO2*
Artery
Perfusion
92
A23
ACH
A23
GTN
Arterial
0.3iMm
0.3j4M
0.3gM
0.1gM
Strips
GTN ACH
IIM
O-lUM
BKN
0.03MM
A23
0.3,MM
ACH
lMm
361
Perfusion
BKN A23 GTN
0.03mM 0.3aM O.luM
1
a/ 1/
2
lSmin
15min
m
1/
7-\/
-
2
i
7
--
VEIN
Venous
Strips
-M
BKN
0.1pM
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
1
A23
BKN
1AM
0.1AM
1
1
/
2+ Porf Fusion|
_| 11RM HbO2
A23
BKN A23
luM
0.1OM
lM
/
/
X
t
-
_
/
\/
FIGURE 5. Influence of extracellular magnesium removal
from perfusion medium on formation/release of endotheliumderived nitric oxide in pulmonary artery and vein. An
endothelium-intact pulmonary artery or vein was perfused.
Endothelium-denuded strips of bovine pulmonary artery
(upper panel) or vein (lower panel) were precontracted with
10 ,MM phenylephrine and 0.01 1iM U46619, respectively,
delivered by superfusion. The numbers 1, 2, and 3 signify the
cascade arrangement of the strips. Responses are monitored as
changes in length (in centimeters). Glyceryl trinitrate (GTN)
was superfused over the strips for 1 minute as indicated.
Breaks in the tracings represent periods of tissue equilibration.
Acetylcholine (ACH), A23187 (A23), and bradykinin (BKN)
were perfused through the artery or vein for 3 minutes as
indicated. -Mg2+ perfusion denotes replacement of Krebs'
perfusion medium with Mg2+-free medium and of Krebs'
superfusion medium with twice (2.4 mM) the normal Mg2`
concentration during the time interval shown. Oxyhemoglobin
(HbO2) was superfused over the venous strips during the time
interval shown. The upper and lowerpanels each represent one
offour separate experiments.
perfusion medium nearly abolished relaxation of
vascular strips of artery (Figure 6) or vein (not
shown) when endothelium-intact vessels were perfused with acetylcholine, bradykinin, or A23187. Calcium (1.5 mM) was always present in the superfusion
fluid bathing the strips. Relaxation of the vascular
strips was restored by readdition of calcium to the
perfusion medium containing bradykinin (Figure 6).
The magnitude of the relaxation was dependent on
the concentration of calcium added. Oxyhemoglobin
abolished the relaxant responses produced by the
+C02+
0.3mM 1.5mM
Arterial
Strips
BKN
0.01MM
1
BKN
0.01UM
BKN
0.01jM
_ +.mM
Co2+
15
+ljiM HbO2
BKN
0.01,M
1
min
2
3
3
-Co02+ _|
Artery
E1
5min
2
........
3
3
--
FIGURE 6. Influence of extracellular calcium removal from
and readdition to perfusion medium on formation/release of
endothelium-derived nitric oxide in pulmonary artery.
Endothelium-denuded strips of bovine pulmonary artery were
precontracted with 10 ,iM phenylephrine and 0.01 gM
U46619 delivered by superfusion. The numbers 1, 2, and 3
signify the cascade arrangement of the strips. Glyceryl trinitrate
(GTN) was superfused over the strips for 1 minute as indicated. Breaks in the tracings represent periods of tissue
equilibration. Acetylcholine (ACH), A23187 (A23), and
bradykinin (BKN) were perfused through the artery or vein for
3 minutes as indicated. - Ca2+ perfusion denotes replacement
of Krebs' perfusion medium with Ca2+-free medium and of
Krebs' superfusion medium containing twice (3.0 mM) the
normal Ca2+ concentration during the time shown. CaCl2 in
0.3 mM and 1.5 mM concentrations was added to Krebs'
perfusion medium during the time interval shown. Oxyhemoglobin (HbO2) was superfused over the arterial strips during
the time interval shown. One tracing is representative offour
separate experiments.
removal of magnesium from (Figure 5) or the addition of calcium to (Figure 6) the perfusion medium.
Mediation by Cyclic GMP of Endothelium-Dependent
Relaxation Elicited by Magnesium Removal and
Calcium Addition
Extracellular magnesium removal produced a rapidly developing, endothelium-dependent fourfold
increase in arterial cyclic GMP levels that was associated with relaxation (Figure 7). Removal of extracellular calcium in the presence of magnesium
caused smooth muscle relaxation, which was greater
in rubbed than intact arterial rings, without appre-
362
Circulation Research Vol 66, No 2, February 1990
100
200
-100
75
150
-75
Artery
+ endothelium
200
150
n
(1<
cl
A'
c
0
100
50
c
25
-50
n
U
50
e.
c
u
c
O
0
c
lt6*6~~*
-25
0
0
0
cs
X
0
200
100
(0
75
150
50
100
0
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
50
25
0
0
C
A-Mg2+
A-
0
R
O
0)
1-
u)
CL
Ca2+
FIGURE 7. Bar graphs showing effects of extracellular magnesium and calcium removal on cyclic GMP levels and tone in
pulmonary artery. Unrubbed (+endothelium) and endothelium-denuded (-endothelium) arterial rings were mounted and
equilibrated as described in the text and precontracted with
phenylephrine to 65-75% of maximal. Control rings (C) were
quick-frozen at the time ofpeak contraction to phenylephrine.
A-Mg2` or A-Ca2' denotes rapid replacement of Krebs' bathing medium at the time ofpeak contraction with Mg2+-free or
Ca2-free medium, respectively; rings were quick-frozen at 2
minutes after medium replacement. Values represent the
mean ±SEM from 16 rings isolated from four animals (four
rings per animal). *Both relaxation and cyclic GMP levels are
significantly different (p<0.01) between +endothelium and
-endothelium. **Only relaxation is significantly different
(p<0.05) between +endothelium and -endothelium.
ciably altering resting cyclic GMP levels. The
endothelium-dependent increase in cyclic GMP levels and relaxation of arterial rings in response to
extracellular magnesium depletion were abolished by
oxyhemoglobin or methylene blue (Figure 8). Both
inhibitors decreased cyclic GMP levels below control
resting levels and reversed relaxation to contractile
responses.
Equilibration of arterial rings in magnesium-free
medium for 15 minutes caused a twofold increase in
cyclic GMP levels, and readdition of magnesium to
the precontracted rings caused cyclic GMPindependent relaxant responses (Figure 9). Neither
oxyhemoglobin nor methylene blue altered such
relaxations although arterial cyclic GMP levels were
lowered significantly. Thus, magnesium-elicited vascular smooth muscle relaxation occurred independent of EDRF (Figures 1 and 2) or cyclic GMP
(Figure 9).
B
50
3
0
E
**
u
x
100
c
+25
+1pM HbO2 +1OM MB
t.
t
A
Mg2+
t
FIGURE 8. Bar graph showing influence of oxyhemoglobin
(HbO2) and methylene blue (MB) on alterations in tone and
cyclic GMP levels elicited by extracellular magnesium removal
in pulmonary artery. Unrubbed (+endothelium) arterial rings
were mounted and equilibrated as described in the text and
precontracted with phenylephrine to 65-75% of maximal. R
denotes resting cyclic GMP levels in arterial rings precontracted in normal Krebs' medium. A-Mg2` denotes rapid
replacement of Krebs' bathing medium at the time of peak
contraction with Mg2`-free medium; arterial rings were quickfrozen at 2 minutes after medium replacement (C). HbO2 or
MB was added 10 minutes before phenylephrine contraction,
and rings were quick-frozen 2 minutes after medium replacement with Mg2+-free medium. Values represent the mean±
SEMfrom 18-24 rings isolated from three to four animals (six
rings per animal). *Significantly different (p<0.01) from
responses designated by R. **Significantly different (p<0.01)
from responses designated by R and C.
In contrast, vascular smooth muscle relaxation
produced by readdition of calcium to magnesiumdepleted extracellular medium was EDRF dependent (Figures 3 and 4) and associated with elevated
cyclic GMP levels (Figure 10). The relaxation and
increase in cyclic GMP levels were abolished by
oxyhemoglobin or methylene blue (Figure 10).
Discussion
One purpose of this study was to develop a better
understanding of the influence of magnesium and
calcium on that component of endotheliumdependent vascular smooth muscle relaxation attributed primarily to EDNO. Nitric oxide or a labile
nitroso compound was identified both chemically and
pharmacologically as one substance that can account
for the biological actions of EDRF released from
intact artery and vein as well as from freshly harvested and cultured aortic endothelial cells.5-7,11,12 In
the present study, acetylcholine and bradykinin were
used as endothelium-dependent relaxants of isolated
precontracted rings of bovine intrapulmonary artery
and vein, respectively, because such effects are known
to be mediated by EDNO and not by cyclooxygenase
Gold et al Regulation of EDNO Release by Magnesium and Calcium
g10010- Artery
§ endothelium
120
.n
363
1200
A
,.C
2
50
05
60 3
25
30
._
~0
C
0
-U
0
c
0
00
+MB
+ HbO2
c
10MM
1MM
,i
3
0
C
to
J.0
~~~tt
+1.2mM Mg2+'
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
FIGURE 9. Bar graph showing influence of oxyhemoglobin
(HbO2) and methylene blue (MB) on alterations in tone and
cyclic GMP levels elicited by magnesium readdition to
magnesium-free extracellular medium. Unrubbed (+endothelium) arterial rings were mounted and equilibrated as
described in the text and precontracted with phenylephrine to
65-75% of maximal. A-Mg2` denotes replacement of Krebs'
bathing medium with Mg2`-free medium 15 minutes before
phenylephrine contraction. Control rings (C) were quickfrozen at the time of peak contraction to phenylephrine. At
peak contraction +1.2 mMMg2+ was added, and arterial rings
were quick-frozen after 5 minutes. HbO2 or MB was added 10
minutes before phenylephrine contraction, and +1.2 mM
Mg2+ was added at peak contraction; rings were quick-frozen
after 5 minutes. Values represent the mean +SEM from 16
rings isolated from four animals (four rings per animal). *Only
relaxation is significantly different (p<0.01) from C. **Cyclic
GMP levels are significantly different (p<0.01) from responses
designated by C and for +Mg2+ control. Relaxant responses
are not significantly different (p>0.05) from +Mg2+ control
responses.
products of arachidonic acid metabolism.40,41 In the
bioassay cascade procedure, acetylcholine or A23187
was used to elicit release of EDRF from perfused
artery and bradykinin, or A23187 was used in perfused vein, but indomethacin was always present in
the perfusion and superfusion media to exclude the
relaxant actions of prostacyclin or other eicosanoids.35 Oxyhemoglobin abolished or nearly abolished
the relaxant effects of EDRF released from both
perfused artery and vein. These observations are
consistent with previous findings that this relaxant
action is attributed to a single EDRF identified
chemically and pharmacologically as nitric oxide or a
chemically related unstable nitroso compound.5'6'11'12
The major findings in this study are that depletion
of extracellular magnesium causes transient endothelium-dependent and calcium-dependent relaxation
not only of artery but also vein, that relaxation is
accompanied by endothelium-dependent cyclic GMP
accumulation, and that such effects are attributed to
the enhanced formation and/or release of an EDRF
FIGURE 10. Bar graph showing influence of oxyhemoglobin
(HbO2) and methylene blue (MB) on alterations in tone and
cyclic GMP levels elicited by calcium readdition to
magnesium- and calcium-free extracellular medium.
Unrubbed (+endothelium) arterial rings were mounted and
equilibrated as described in the text and precontracted with
phenylephrine to 65-75% of maximal. A-Ca2+-Mg2+ denotes
replacement of Krebs' bathing medium with Ca2 - and Mg2+free medium 15 minutes before phenylephrine contraction.
Control rings (C) were quick-frozen at the time of peak
contraction to phenylephrine. At peak phenylephrine contraction +1.5 mM Ca2+ was added, and arterial rings were
quick-frozen after 1 minute. HbO2 or MB was added 10
minutes beforephenylephrine contraction, and +1.5 mM Ca2+
was added at peak contraction; rings were quick-fiozen after 1
minute. Values represent the mean+±SEM from 16 rings
isolated from four animals (four rings per animal).
*Significantly different (p<0.05) from responses designated by
C. **Significantly different (p<0.01) from responses designated by C and for + Ca2+ control.
that is most likely nitric oxide (EDNO). Extracellular
magnesium depletion-elicited relaxation and cyclic
GMP formation are antagonized by oxyhemoglobin,
methylene blue, and superoxide anion generated
from pyrogallol,42 whereas such responses are
enhanced by superoxide dismutase. When the bioassay cascade technique is used, the depletion of
magnesium from the perfusion medium causes a
marked enhancement of formation and/or release of
EDRF. Similar observations were made when simultaneous removal of magnesium from both perfusion
and superfusion media was performed. Thus, magnesium depletion does not appear to alter the responsiveness of smooth muscle to EDRF. The approximate estimated half-life of EDRF is 3-5 seconds,
and relaxant responses in the superfused strips are
antagonized by oxyhemoglobin. Based on earlier
studies from this6,7,11-14 and other5'43-46 laboratories
where similar experimental approaches were taken,
these observations provide evidence that EDNO is
responsible for the vascular actions of extracellular
magnesium depletion.
364
Circulation Research Vol 66, No 2, February 1990
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The above observations on extracellular magnesium depletion in the presence of calcium are mimicked by the addition of calcium to bathing medium
or perfusion medium previously depleted of both
calcium and magnesium. Thus, calcium causes
endothelium-dependent arterial and venous relaxation and cyclic GMP accumulation, and such vascular responses are attributed to EDNO because they
are antagonized by oxyhemoglobin or methylene blue
and because the EDRF from perfused artery and
vein possesses all of the properties of nitric oxide.
These data suggest that extracellular magnesium and
calcium elicit mutually antagonistic or reciprocal
actions at the level of the formation and/or release of
EDNO. Present bioassay procedures as conducted,
however, are incapable of distinguishing between
formation and release of EDNO.
Another clear expression of the opposing biological actions of magnesium and calcium derives from
experiments conducted with endothelium-denuded
rings of artery or vein. Under these conditions extracellular magnesium depletion does not cause relaxation but, rather, causes sustained contractions. Similarly, addition of calcium to media deprived of both
calcium and magnesium causes only contractile
responses. It is notable that even in the presence of a
functional endothelium the characteristic transient
relaxation caused by extracellular magnesium depletion is quickly followed by a sustained contractile
response. The latter contractile response appears to
be attributed to the unopposed smooth muscle
actions of calcium because addition of excess calcium
(greater than 1.5 mM) to endothelium-intact rings in
the presence of extracellular magnesium causes only
contractile responses. Particularly intriguing are the
observations that excess extracellular magnesium
(>1.2 mM) elicits marked endothelium-independent
and cyclic GMP-independent relaxant responses that
are unaltered by oxyhemoglobin, methylene blue, or
indomethacin. One plausible explanation for these
findings is simply that excess magnesium competes
with and overrides the smooth muscle contractile
effects of calcium. The precise mechanism of this
cation interaction or whether it occurs at extracellular surface ion channels or intracellularly is presently
unknown.
Removal of extracellular magnesium caused a
marked endothelium-dependent increase in tissue
cyclic GMP levels within several minutes, which
gradually declined over a 15-minute period but
remained at levels that were about twofold higher
than control basal levels. This was undoubtedly due
to increased EDRF generation or release. Readdition of magnesium did not cause a decrease (within 5
minutes) in cyclic GMP levels to control values, as
one might have expected, due to an antagonistic
action of magnesium on endothelial cell calcium. It
appears that the sustained small elevation in cyclic
GMP levels remaining after magnesium readdition is
due to sustained EDNO action because oxyhemoglobin or methylene blue rapidly lowered cyclic GMP
levels. The reason for this sustained, oxyhemoglobinsensitive, small elevation in cyclic GMP levels is
unknown.
Magnesium and calcium appear to elicit opposing
or antagonistic actions on at least two different levels
of cellular function. One site is at the endothelial cell
on the formation and/or release of EDRF, and the
other site is at the smooth muscle cell on processes
that lead to calcium-mediated contraction. Because
calcium is obligatory for both smooth muscle contraction and EDRF formation and/or release and
because magnesium opposes the actions of calcium at
both sites, vascular smooth muscle responsiveness to
alterations in the extracellular concentration of magnesium and calcium reflects the algebraic sum of the
responses. Thus, the magnitude of the relaxation in
response to sudden extracellular magnesium depletion or addition of excess extracellular magnesium or
removal of extracellular calcium in the presence of
magnesium varies from one experiment to another.
The dual influence of calcium on endothelial and
smooth muscle cells is borne out also by the observations that the removal of extracellular calcium in
the presence of magnesium causes a greater magnitude of relaxation in rubbed than endothelial-intact
arterial rings. That is, in intact rings calcium both
stimulates EDRF formation and/or release and
causes contractions, whereas in rubbed rings calcium
causes only contraction. Thus, the sudden removal of
calcium in the presence of magnesium would be
expected to cause greater relaxation of the rubbed
arterial ring.
In calcium-free extracellular medium acetylcholine
elicited small but significant endothelium-dependent
relaxant responses, whereas magnesium withdrawal
did not. The reason for this difference is unknown
but may be attributed to the release of intracellular
calcium by acetylcholine in the endothelial cells.
There is evidence that endothelium-dependent relaxation elicited by acetylcholine, bradykinin, and other
agonists is dependent also on the presence of both
extracellular and intracellular calcium.3,17,22
Some of the observations made in the present
study are consistent with those reported previously in
which extracellular magnesium depletion caused
endothelium- and calcium-dependent (EDRFmediated) relaxation of isolated canine coronary
artery but caused contraction of endotheliumdenuded artery.32 One difference between the above
study and the present one is that we were unable to
observe contractile effects of magnesium added back
to magnesium-deficient bathing media in which arterial preparations had just relaxed in response to the
removal of magnesium. Instead, we always observed
relaxant responses to added magnesium under any
conditions, and such responses occurred independent of endothelium or the extracellular concentration of magnesium. Furthermore, in our experiments
the relaxations elicited by extracellular magnesium
depletion were transient and were followed by sustained contractions, whereas in the former study32
Gold et al Regulation of EDNO Release by Magnesium and Calcium
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
sustained relaxations were produced. The reasons for
these differences are unknown but may be attributed
to differences in responsiveness of coronary versus
intrapulmonary vessels.
The observations in the present study differ somewhat also from those of another study on coronary
artery47 in that endothelium-dependent relaxation of
bovine intrapulmonary artery and vein was not
dependent on the presence of extracellular magnesium, whereas magnesium appeared to be required
for endothelium-dependent canine coronary arterial
relaxation. Again, the reasons for such differences
are unknown, but perhaps species, blood vessel,
and/or experimental differences account for such
differences in the observations made. It is clear from
the present study that endothelium-dependent relaxation of bovine intrapulmonary artery and vein
occurred unimpaired in the absence of extracellular
magnesium but that extracellular calcium was
required for relaxation. The bioassay cascade technique revealed clearly that endothelium-dependent
vasodilators generate or release EDRF in the
absence of perfused magnesium but not in the
absence of perfused calcium. On the other hand,
endothelium-independent relaxation elicited by glyceryl trinitrate or nitroso compounds occurred independent of extracellular magnesium or calcium.
The present study does not address the cellular site
of interaction of magnesium and calcium. Evidence
exists for the antagonistic actions of magnesium and
calcium at both calcium and magnesium transport
sites associated with the vascular smooth muscle
membrane.48-52 Recently, the existence of calcium
channels in isolated endothelial cells has been
suggested,53 which may become important in
magnesium-calcium interactions.
Magnesium and calcium serve to regulate vascular
smooth muscle tone. Calcium influx into the cytosol
causes smooth muscle contraction, whereas magnesium can control calcium influx into the cell by
competitively interacting at transport sites.49,50 A
decrease in extracellular magnesium concentration is
associated with ischemic heart disease and hypertension,51 whereas infusions of magnesium produce
vasodilation as a result of reduced calcium influx.54
The present study provides evidence that the vascular
endothelium, through the actions of EDNO, is
responsive to changes in the extracellular concentration of magnesium and calcium and that this may
protect against vasoconstriction.
Acknowledgment
The authors wish to thank Diane Rome Peebles
for preparing the illustrations.
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Sjogren A, Edvinsson L: Vasomotor effects of magnesium: A
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KEY WORDS * bovine pulmonary artery * bovine pulmonary vein
* calcium * cyclic GMP * vascular smooth muscle * nitric oxide
* magnesium * endothelium-dependent vasodilation
Antagonistic modulatory roles of magnesium and calcium on release of
endothelium-derived relaxing factor and smooth muscle tone.
M E Gold, G M Buga, K S Wood, R E Byrns, G Chaudhuri and L J Ignarro
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Circ Res. 1990;66:355-366
doi: 10.1161/01.RES.66.2.355
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