Atrial Natriuretic Factor and Cyclic Guanosine
3',5'-Monophosphate in Vascular Smooth Muscle
MAKITO SATO, KEISHI A B E , KAZUHISA TAKEUCHI, MINORU YASUJIMA, KEN OMATA,
MASAO
HrwATARi,
YUTAKA KASAI, MASAYA TANNO, MASAHIRO KOHZUKI, KEI KUDO,
KAORU YOSHINAGA, AND TADASHI INAGAMI
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SUMMARY To elucidate the molecular mechanism of the vascular action of atrial natriuretic factor
(ANF), we investigated the effects of synthetic ANF and sodium nitroprusside on the levels of intracellular cyclic nucleotides and prostacyclin (measured as its stable metabolite 6-keto-prostaglandin F,a)
in cultured vascular smooth muscle cells from rat mesenteric artery and, in some experiments, from
rat renal artery. Both ANF and sodium nitroprusside increased intracellular cyclic guanosine 3',5'monophosphate (cGMP) levels in a dose-dependent manner but did not affect cyclic adenosine 3',5'monophosphate levels or 6-keto-prostaglandin F,tt synthesis. The stimulatory effects of ANF and
sodium nitroprusside on cGMP levels were additive. Neither the deprivation of extracellular Ca2+ nor
calcium entry blockers affected ANF-stimulated cGMP levels. Preincubation of ANF or sodium
nitroprusside with kallikrein attenuated only the effect of ANF on cGMP levels. The effect of kallikrein was abolished by serine protease inhibitors. In contrast, the oxidant methylene blue inhibited
the effect of sodium nitroprusside on cGMP levels, but not that of ANF. The stimulatory effect of ANF
on cGMP levels was greater in cells from renal artery than in those from mesenteric artery. These
results in cultured vascular smooth muscle cells further support the hypothesis that cGMP mediates
the vasorelaxant action of ANF. (Hypertension 8: 762-771, 1986)
KEY WORDS • atrial natriuretic factor • sodium nitroprusside • cyclic guanosine
3,5-monophosphate • vascular smooth muscle cell • methylene blue • kallikrein
A
PEPTIDE released from atria, generally
termed atrial natriuretic factor (ANF), re. cently has been suggested to play an important role in renal and cardiovascular homeostasis.1"6
This peptide has a potent natriuretic, diuretic, and direct vasorelaxant action, but the molecular mechanisms of its action have not been well elucidated. The
action of ANF seem to be independent of prostaglandins (PGs) 4 ' 5 and Na, + K + -ATPase> 7 Recent studies
have focused on the vascular effects of ANF in terms
of cyclic nucleotides, especially cyclic guanosine
3',5'-monophosphate (cGMP).8"12 Cyclic nucleotides
have been shown to act as mediators for vasorelaxant
actions of various compounds and hormones,13' M and
the vascular action of ANF has been found to be qualitatively similar to that of sodium nitroprusside
(SNP), 15 ' l6 which is thought to relax vascular smooth
muscle through cGMP accumulation.13 '"• "•18 Specific receptors for ANF have been found in vascular tissues19 and cultured vascular smooth muscle cells from
aorta.10 In addition, ANF has been shown to stimulate
guanylate cyclase and to increase cGMP levels in vascular tissues8"12 as well as in cultured aortic smooth
muscle cells.10 However, some controversy exists regarding the vascular action of ANF and its relation to
cGMP. Methylene blue, an oxidant that inhibits SNPinduced arterial guanylate cyclase activation and relaxation,17' 18 has been reported to inhibit12 or not to affect
the vasorelaxant action of ANF. 16
The role of calcium in the vascular action of ANF is
also controversial. Removal of calcium from experimental solutions has been reported to have no effect on
the vasorelaxant action of ANF in rabbit and rat aortic
From the Second Department of Internal Medicine, Tohoku University School of Medicine, Sendai, Miyagi, Japan, and the Department of Biochemistry (T. Inagami), Vanderbilt University School
of Medicine, Nashville, Tennessee.
Supported by grants-in-aid for cardiovascular disease from the
Ministry of Health and Welfare of Japan (58-C-2, 60-C-3) and a
grant-in-aid for scientific research from the Ministry of Education,
Science, and Culture of Japan (59870087).
Portions of this study were presented at the annual meeting of the
American Society of Nephrology, New Orleans, December 1985,
and published in abstract form (Kidney Int 1986;29:258).
Address for reprints: Makito Sato, M.D., Second Department of
Internal Medicine, Tohoku University School of Medicine, Sendai
980, Miyagi, Japan.
Received July 22, 1985; accepted March 4, 1986.
762
ANF AND cGMP IN VASCULAR SMOOTH MUSCLEISato et al.
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strips16 or on the stimulatory effect of ANF on cGMP
levels in rabbit aortic tissues." In the isolated perfused
rat kidney, however, a low calcium solution and the
calcium entry blocker verapamil markedly blunted the
hemodynamic and natriuretic action of ANF. 20 Although these discrepant results may be explained by
the difference in species, experimental conditions,
vascular tissues, sources of ANF (crude or partially
purified atria] extracts or synthetic ANF), or the doses
of ANF, these controversies may weaken the hypothesis that cGMP mediates the vasorelaxant action of
ANF.
It has recently been suggested that serine proteases
(trypsin or kallikrein) modulate the natriuretic and diuretic action of ANF or attenuate the stimulatory effect
of ANF on particulate guanylate cyclase from kidney
or aorta.9'21 However, to our knowledge, the effect of
kallikrein on ANF-stimulated cGMP levels in vascular
smooth muscle cells has not been evaluated. Furthermore, several reports22"23 indicate that renal artery is
more sensitive than other arteries to the vasorelaxant
action of ANF. If cGMP is involved in the vasorelaxant action of ANF, then the cGMP response to ANF
may be greater in cells from renal artery than in those
from other arteries. However, no data are available on
the difference in the cGMP response to ANF among
renal and other arteries.
The purpose of the present experiments was to 1)
confirm existing findings that ANF increases cGMP
levels in vascular tissues, using cultured vascular
smooth muscle cells from mesenteric artery, which has
been known to play an important role in the regulation
of the peripheral vascular resistance;26 2) clarify the
effects of calcium, kallikrein, and methylene blue on
ANF-stimulated intracellular cGMP levels and compare them with those of SNP-stimulated cGMP levels;
and 3) evaluate the cGMP response to ANF in cells
from mesenteric and renal arteries.
Materials and Methods
Materials
Type III collagenase, deoxyribonuclease I, elastase,
soybean trypsin inhibitor, calcium ionophore A23187,
3-isobutyl-l-methylxanthine (IBMX), SNP, and 6keto-PGF la were purchased from Sigma Chemical (St.
Louis, MO, USA). Medium 199, minimal essential
medium (MEM) with or without calcium, antibioticantimycotic, penicillin-streptomycin, L-glutamine, sodium bicarbonate, basal medium Eagle amino acid
solution, and trypsin ethylenediaminetetraacetic acid
(EDTA) were purchased from Gibco Laboratories
(Grand Island, NY, USA), and fetal bovine serum was
obtained from MA Bioproducts (Walkersville, MD,
USA). The ANF was obtained from two sources: a 25
amino acid ANF was synthesized as previously reported,272* and 24 amino acid atriopeptin III29 was purchased from Peninsula Laboratories (Belmont, CA,
USA). Porcine pancreatic kallikrein (130 U/mg protein) and gabexate mesylate, [ethyl-4-(6-guanidinohexanoyloxy)benzoate] methane sulfonate (Foy),30
763
were gifts from Ono Pharmaceutical (Osaka, Japan).
Aprotinin (Trasylol; 6500 KlU/mg) was a gift from
Bayer AG (Leverkusen, West Germany), and angiotensin II (Hypertensin) was obtained from CIBAGeigy (Basel, Switzerland). Methylene blue was purchased from Wako Chemicals (Tokyo, Japan), and
arginine vasopressin was obtained from Peptide Institute (Osaka, Japan). Twelve-well sterile culture dishes
were purchased from Costar (Cambridge, MA, USA),
and six-well dishes were obtained from Nunc (Copenhagen, Denmark). Radioimmunoassay kits for cyclic
adenosine 3',5'-monophosphate (cAMP) and cGMP
were obtained from Yamasa Shoyu (Choshi, Japan).
Antisera for 6-keto-PGF ]a were a gift from Dr.
Michael J. Dunn, Cleveland, OH, USA.
Isolation and Culture of Cells
Vascular smooth muscle cells were isolated from
superior mesenteric arteries and, in some experiments,
from renal arteries of Sprague-Dawley rats (weight,
150-200 g) and were cultured according to the methods of Ives et al.31 and Gunther et al.32 as modified by
Hassid and Williams.33 With the rats under pentobarbital anesthesia, the superior mesenteric or renal arteries
were dissected free of surrounding tissues and placed
in a 35-mm culture dish containing MEM (1.8 mM
Ca 2+ ). The adventitial adipose tissue around the arteries was gently removed with forceps, and the arterial
tissues were incubated for 15 to 20 minutes at 37 °C in a
modified Krebs-Ringer buffer solution of the following composition (in mM): NaCl, 105; KC1, 5;
KH2PO4, 1; N-2-hydroxyethylpiperazine-./V'-2-ethanesulfonic acid, 25; MgSO 4 , 1; glucose, 14; CaCl2, 0.2;
and NaHCO3, 25. The medium was further supplemented with dissociation enzymes (collagenase, 360
U/ml; deoxyribonuclease I, 56 U/ml; elastase, 96
U/ml; soybean trypsin inhibitor, 1 mg/ml). Cells that
were dissociated during the first 15 to 20 minutes were
discarded. The remaining portion was then transferred
to a second aliquot of dissociation medium, and incubation was continued for an additional 30 to 45
minutes. Cells dissociated during the second incubation were collected by centrifugation, passed through a
sterile 150-/iM Nytex gauze filter (Tetko, Elmsford,
NY, USA), and seeded to culture wells in Medium
199. After 2 hours, most cells had attached to the
bottom of wells and were further cultured in Medium
199 containing 10% fetal bovine serum and penicillinstreptomycin. Cells were subcultured by treatment
with 0.08% trypsin EDTA. Morphological examination revealed characteristics of vascular smooth muscle cells with crisscross patterns, hill and valley and
nodular structures in confluent cultures with phasecontrast microscopy and myofilaments and dense bodies with electron microscopy. Cells at passage levels 3
to 9 were used for experiments.
Experiments
All experiments with vascular smooth muscle cells
were conducted at least in triplicate using 12-well Costar culture dishes. Before incubation, cells were rinsed
764
HYPERTENSION
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three times with 0.6 ml of MEM. In most experiments,
cells were incubated with effectors in 0.6 ml MEM
(1.8 mM Ca 2+ ) in the presence or absence of the phosphodiesterase inhibitor IBMX, 10^M, at 37 °C in an
atmosphere of 5% CO 2 , 95% air. A synthetic ANF of
25 amino acids was used only in experiments shown in
Figure 1; atriopeptin III was used in all other experiments. In experiments with different extracellular calcium concentrations, Ca2+-free medium was made
with Ca2+-free MEM fortified with 2 mM ethyleneglycol bis03-aminoethyl ether)-N,A',/V',W,-tetraacetic
acid and the calcium concentration was changed by
adding CaCl2 solution. Cells were preincubated for 30
minutes in media with different extracellular calcium
concentrations or in media containing calcium entry
blockers. In experiments involving kallikrein, ANF or
SNP was preincubated with kallikrein for 1 hour at
37 °C in MEM in the presence or absence of aprotinin
or Foy. In experiments in which interactions of methylene blue with ANF or SNP were evaluated, cells were
preincubated with methylene blue for 15 minutes. To
compare the effect of ANF on cGMP levels in cells
from renal and mesenteric arteries, cells were isolated
from the same rats and cultured in the same dish, and
experiments were performed simultaneously. Experiments were terminated by removal of media, followed
immediately by addition of 0.3 ml of 0.1 N HC1. Cells
were exposed to the HC1 solution for 1 hour, intracellular cyclic nucleotides were extracted into the HC1
fraction, and part of the cellular protein was precipitated and left on the bottom of the culture well.
Assays
Radioimmunoassays of cGMP and cAMP were
done in duplicate after succinylation.34 We found no
cross-reactivity between cGMP or cAMP antisera and
ANF, SNP, IBMX, or cell culture incubation media.
Samples of 0.1 N HC1 solution, diluted 1:5 with 300
mM imidazole buffer, did not interfere with the radioimmunoassay. A 50% displacement of the radioligand was achieved with 30 fmol cGMP or 40 fmol
cAMP. Intra-assay coefficient of variation was 7.8%
for cGMP and 8.1% for cAMP (n = 8). Interassay
coefficient of variation was 11.0% for cGMP (n = 6)
and 12.4% for cAMP (n = 6). The 6-keto-PGF la was
measured by radioimmunoassay of the extracellular
media using antisera as previously described.33 Cellular protein was measured on the precipitate in the
culture well and in the 0.1 N HC1 solution by the
methods of Lowry et al.3* Since precipitated protein
was 56.0 ± 0.3% (n = 25) of total cellular protein and
this percentage was constant in each experiment, we
measured the precipitated protein in the culture well
and corrected the value for total cellular protein by this
percentage in some experiments, essentially as described previously.37
Statistics
The values are expressed as mean ± SE. The statistical significance was evaluated at the 95% confidence
level using Student's t test for unpaired observations or
one-way analysis of variance.
VOL 8, No
200-
c
ffi
'«
o
9,
SEPTEMBER
* P-^0.001
1986
cyclic GMP
150-
Q.
a.
"o
100-
o
3
C
^M
u
50-
cycle AMP
<5
X
/
/
/
/
J
/
•'+, •'+ "'+, '<*?+/+,
ANF, M
FIGURE 1. Effect of atrial natriuretic factor (ANF) on intracellular cyclic nucleotides. Incubations were performed for 10
minutes with ANF (1.1 x I(h9-1.1 X 10-* M) in the presence of
1CH hi 3-isobutyl-l-methylxanthine. ANF increased cGMP levels (p<0.001) but not cAMP levels. Results shown are
means ± SE of eight cultures pooled from two experiments.
Asterisk indicates significant difference compared with basal
values (p<0.001).
Results
A synthetic ANF of 25 amino acids increased intracellular cGMP levels in a dose-dependent manner
(/><0.001) but did not affect cAMP levels (Figure 1).
Threshold stimulation of cGMP levels was observed at
1.1 X 10~9 M, while half-maximal stimulation was
seen around 1.1 x 10~7 M, and maximal stimulation at
1.1 x 10-* to 1.1 x 10-5 M ANF. Basal levels of 6keto-PGFlQ (8.2 ± 0.7 pg//i,g protein/30 min) were not
affected by ANF (1.1 x 10- 9 -l. 1 x 10"3 M). Although
angiotensin II (10^7 M), arginine vasopressin (10~7 M),
and calcium ionophore A23187 (2 x lO^M) stimulated 6-keto-PGF la synthesis about 1.5-fold, 2.0-fold,
and 3.1-fold, respectively, preincubation with ANF,
3.3 x 10"9 to 3.3 x 10-7 M, for 30 minutes did not
affect stimulated 6-keto-PGF la synthesis (data not
shown). As shown in Figure 2, incubation of cells with
3.3 x 10"8 M ANF resulted in a significant increase in
cGMP levels within 1 minute. The response was maximal at 2 minutes and then gradually declined over the
next 30 minutes in the absence of the phosphodiesterase inhibitor IBMX. The presence of 10"4 M IBMX
ANF AND cGMP IN VASCULAR SMOOTH MUSCLE/Sato et al.
765
FIGURE 2. Time course for intracellular cGMP levels in the presence or absence of 3.3 X JCh8 M atrial natriuretic
factor (ANF) or 1(H M 3-isobutyl-lmethylxanthine (IBMX), or both. Results shown are means ± SE of six to
eight cultures pooled from two experiments.
8r
c
£o 6
L_
Q.
"5
Q.
U
-Q
0
1
control
2
D
30
10
Time,
min
potentiated both the peak and the duration of ANFstimulated cGMP levels, while IBMX, 10^or5 X 10"4
M, potentiated basal cGMP levels (from control values
of 0.2 ±0.1 to 0.3 ±0.1 or 0.8 ±0.2 fmol//Lig protein) and ANF (3.3 X 10"* M)-stimulated cGMP levels
(from control values of 1.0 ±0.2 to 12.5 ±3.9 or
70
42.0 ± 6.0 fmoUfjig protein, respectively). The presence of SNP, 10~7 M or above, also increased intracellular cGMP levels (p< 0.001; Figure 3) but did not
affect cAMP levels or 6-keto-PGFla synthesis (data not
shown). Although the amounts of ANF and SNP did
not necessarily achieve maximal stimulation of cGMP
r
ein
60
50
o
Q.
00
40
fmoi/
30
GMP
a.
20
o
cycl
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IBMX alone _
10
basal
10~7M
10" 6 M
10~5M
10" 4 M
10" 3 M
Sodium Nitroprusside
FIGURE 3. Effect of sodium nitroprusside (SNP) on intracellular cGMP levels. Incubations were performed for 5
minutes with SNP (10-7-lCh3 M) in the presence of J(H M 3-isobutyl-l-methylxanthine. At concentrations oflCh7
Morabove, SNP increased cGMP levels (p<0.001). Results shown are means ± SE of eight cultures pooled from
two experiments.
HYPERTENSION
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VOL 8, No
:
640
9,
SEPTEMBER
1986
639±36
620
• | 600
o
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oo 580
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o
E
d
:
62±3
60
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0
ANF
SMP ANF
1X10" !* lxlO"St g ^ p
1
ANF
n
SNP
ANF
3.3X10' M 3 3X10I^H
Expwiment 1 | n - I O )
SMP
Exp» rn^nl 2 (n-«|
ANF
SNP
3.3X10"^ 1X10" M
ANF
SNP
Experment 3 I n - 9 1
FIGURE 4. Additivity ofatrial natriuretic factor (ANF)-stimulated and sodium nitroprusside (SNP)-stimulated
intracellular cGMP levels. Incubations were performed for 5 minutes with ANP, SNP, or both in the presence of
I(H M 3-isobutyl-l-methylxanthine.
levels, the stimulatory effects of ANF, 3.3 X 10"7 to
3.3 x 10-6 M, and SNP, 3.3 x 10-< to 1O~3 M, on
cGMP levels were additive (Figure 4).
To assess the role of extracellular calcium on ANFstimulated cGMP levels, experiments were performed
with Ca2+-free solution or calcium entry blockers. Decreasing extracellular calcium concentration from 1.8
to 0.18 or 0 mM slightly modified the basal cGMP
levels from 0.5 ± 0.1 to 0.5 ± 0.2 or 0.3 ± 0.2 fmol/
fj.g protein (nonsignificant change) and did not affect
ANF (3.3 x lO"7 M)-stimulated cGMP levels (from
13.9 ± 1.6 to 12.1 ±2.0 or 13.5 ±2.1 fmol//xg protein, respectively). Pretreatment of cells with 10~5 M
verapamil or \&* M nifedipine for 30 minutes also
failed to affect ANF-stimulated cGMP levels (data not
shown).
Figure 5 shows the effect of porcine pancreatic kallikrein on ANF-stimulated cGMP levels in the presence or absence of the serine protease inhibitors aprotinin and Foy. Although kallikrein did not change basal
cGMP levels, preincubation of ANF with kallikrein for
1 hour at 37 °C significantly attenuated ANF-stimulated cGMP levels (p<0.0l). The effect of kallikrein
was concentration-dependent and was abolished by
concomitant treatment with aprotinin or Foy in a dosedependent manner. However, kallikrein, in a concentration as high as 10"5 M, did not inhibit SNP-stimulated cGMP levels (basal, 0.9 ± 0 . 1 ; 10"3 M kallikrein,
1.2 ± 0 . 1 ; 3.3 x 10-6 M SNP, 12.4 ±0.5; SNP and
kallikrein, 18.0 ± 1.3 fmol//i.g protein; n — 8). Figure
6 shows the effect of methylene blue on ANF-stimulated or SNP-stimulated cGMP levels. Pretreatment of
cells with methylene blue, 10"7 and 10"6 M, for 15
minutes inhibited the cGMPresponseto SNP but not to
ANF. The inhibitory effect of methylene blue was
concentration-dependent.
The responses of cyclic nucleotides to ANF, SNP,
or isoproterenol were compared in cells from renal and
mesenteric arteries. Cells at passage level 3 were used
for this series of experiments. Basal levels of cGMPor
cAMP were 1.2 ± 0.1 or 21.4 ± 2.1 fmol//xg protein
in cells from mesenteric artery and 1.4 ± 0.1 or
26.9 ± 1.5 fmoiy/u,g protein in cells from renal artery
(no significant difference between the two cell types).
However, cGMP responses to SNP and especially to
ANF were much greater in cells from renal artery than
in those from mesenteric artery (Figure 7), whereas the
cAMP response to isoproterenol did not show any
significant difference between the two cell types
(Figure 8).
Discussion
The present results in cultured vascular smooth muscle cells from rat mesenteric artery are consistent with
previous findings that ANF increases cGMP levels in
vascular tissues.8"12 Although accumulating evidence
indicates that ANF increases cGMP levels in several
tissues, including kidney, liver, and lung, 89 the
mechanism of this increase has not been well elucidated. Harriet et al.8 reported that ANF inhibited cGMP
phosphodiesterase but did not activate soluble or particulate guanylate cyclase in arterial tissues and
cultured aortic smooth muscle cells. In the present
ANF AND cGMP IN VASCULAR SMOOTH MUSCLEJSato et al.
60 ,-
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~. aprotinin 10"'W
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40
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ANF
alone
0.
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+ANF
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0
1
//
1
^ \
1
30
control
300
Kallikrein,
FIGURE 5. Effect of kallikrein on atrial natriuretic factor (ANF)-stimulated intracellular cGMP levels in the
presence or absence of aprotinin or gabexate mesylate (Foy). Incubations were performed for 5 minutes in the
presence ofKh4 M 3-isobutyl-l' -methylxanthine with 3.3 X J0~7MANF that had been preincubated with kallikrein
(3-300 ng/ml)for 1 hour at37°C in the presence ofl(H M aprotinin or Foy (KH-l(h3 M). Kallikrein inhibited the
stimulatory effect of ANF on cGMP levels (p< 0.001), but this effect was blunted by the pretreatment of kallikrein
with aprotinin or Foy. Results shown are means ± SE of eight cultures pooled from two experiments. Single
(p<0.05) and double (p<0.001) asterisks indicate significant difference compared with control values. Dagger
(p<0.05), and section mark(p<0.001) indicate significant difference compared with respective values of ANF
alone.
study, the time course of ANF-stimulated cGMP levels
showed a rapid elevation followed by a gradual decline
after the peak concentration at 2 minutes in the absence
of the phosphodiesterase inhibitor IBMX. In addition,
IBMX markedly potentiated the ANF-stimulated
cGMP levels. These results support the previous findings that ANF-induced intracellular accumulation of
cGMP is due mainly to increased synthesis rather than
inhibition of cGMP degradation by phosphodiesterase. 911
We demonstrated that both ANF and SNP selectively increased intracellular cGMP levels without affecting cAMP levels or 6-keto-PGF la synthesis, suggesting a common molecular mechanism for the vascular
action of ANF and SNP. Although these results provide no direct evidence that cGMP mediates the vasorelaxant action of ANF, it is of note that the threshold
concentration of the 25 amino acid ANF for cGMP
stimulation in the present study was identical to that
shown to relax rabbit aortic strips that were precontracted by norepinephrine.28 Several investigators have
demonstrated that the vasorelaxant action of ANF, like
that of SNP,14 is not dependent on the presence of
vascular endothelial c e l l s , a "• M and the present results
in cultured vascular smooth muscle cells clearly sup-
port these findings. Also, the finding that ANF did not
affect prostacyclin synthesis in vascular smooth muscle cells may be consistent with the finding that PG
synthesis inhibition with indomethacin and aspirin had
no inhibitory effect on renal function and the blood
pressure fall induced by ANF. 5 In addition, it has been
reported that ANF did not change urinary excretion of
PGs in the perfused, hydronephrotic rabbit kidney.4
We also have recently found that chronic infusion of
ANF does not induce any changes in urinary PGE2
excretion in conscious rats.39 Recently, ANF has been
reported to inhibit adenylate cyclase activity in several
tissues, including cultured vascular smooth muscle
cells.40 However, we did not observe any effect of
ANF on intracellular cAMP levels in the present study,
and our experience is consistent with more recent studies in aortic tissues. 9 ' 12 Further studies may be necessary to clarify these discrepancies.
Little is known about the role of calcium in ANFinduced vasorelaxation. It has been reported that the
hemodynamic and natriuretic effects of atrial extracts
were blunted markedly by a low calcium perfusate or
verapamil in the isolated, perfused rat kidney,20 whereas no change was observed in the vasorelaxant action
of ANF on norepinephrine-contracted rabbit or rat aor-
HYPERTENSION
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VOL. 8, No 9, SEPTEMBER
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basal
10"8M
control
10"7M
10~6M
Methylene Blue
FIGURE 6. Effect of methylene blue on atrial natriuretic factor (ANF)-stimulated or sodium nitroprusside
(SNP)-stimulated intracellular cGMP levels. Incubations were performed for 5 minutes in the presence of 10~* M
3-isobutyl-l-methylxanthine with 3.3 X lCh7MANFor3.3 X 10-6 M SNP after a 15-minutepreincubation of cells
in the presence or absence of methylene blue (10-&-I0-6 M). Methylene blue inhibited SNP-stimulated cGMP but
not basal or ANF-stimulated cGMP. Results shown are means ± SE of eight cultures pooledfrom two experiments.
Single (p<0.05) and double (p<0.01) asterisks indicate significant difference compared with control values.
250
r
200
Renal Artery
o
Q.
150
o
100
Q.
Mesenteric Arter
50
o
u
Mesenteric Artery
bisal
3.3x10" 9 M
ANF
3.3x10!M
basal
10"7M
10"6M
SNP
FIGURE 7. The cGMP response to atrial natriuretic factor (ANF) and sodium nitroprusside (SNP) in cultured
vascular smooth muscle cells from renal and mesenteric arteries. Incubations were performed for 5 minutes in the
presence of I(HM 3-isobutyl-l-methylxanthine with ANF (3.3 x 10-*-3.3 x 1(H M) or SNP (10-7-10-* M). Cells
from renal artery showed greater response to both ANF and SNP than did cells from mesenteric artery. Single
(p< 0.05) and double (p< 0.001) asterisks indicate significant differences compared with the respective values of
cells from mesenteric artery. Results shown are means ± SE of eight cultures pooled from two experiments.
1986
ANF AND cGMP IN VASCULAR SMOOTH MVSCLE/Sato et al.
FIGURE 8. The cAMP response to isoproterenol
in cultured vascular smooth muscle cells from
renal and mesenteric arteries. Incubations were
performed for 5 mintues with isoproterenol
(lO-t-lO-5 M) in the presence of 10** M 3-isobutyll-methylxanthine. There was no significant difference between the two cell types. Results shown are
means ± SE of eight cultures pooled from two
experiments.
800
700
§ 600
o
DO
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AMP
400
\1 renal artery
• mesentere artery
300
0
200
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A
100
basal
10"6M
Isoproterenol
tic strips in a calcium-free solution.16 Winquist et al."
found that the ANF-induced increase in cGMP levels
was independent of extracellular calcium in rabbit aortic tissues. We also did not observe any change in
ANF-stimulated cGMP levels in cultured vascular
smooth muscle cells by changing the extracellular calcium concentration or by the addition of the calcium
entry blockers verapamil or nifedipine. These results
suggest that the vasorelaxant action of ANF may be
independent of extracellular calciumlike nitroderivatives.14 However, we cannot rule out the possibility
that the vasorelaxant action of ANF is dependent on
the levels of intracellular calcium, since it has been
recently suggested that cGMP activates the sarcolemmal calcium extrusion adenosine triphosphatase and
relaxes coronary arterial smooth muscle.41
Since it has been suggested that the vasorelaxant
action of ANF is similar to that of SNP, methylene
blue, which inhibits guanylate cyclase activation by
SNP, 17 ' 18 has been used to examine the hypothesis that
the guanylate cyclase-cGMP system is involved in the
vasorelaxant action of ANF. Ohlstein and Berkowitz12
found that methylene blue inhibited the ANF-induced
vascular relaxation and cGMP elevation in rabbit aortic rings, supporting the role of cGMP in the vasorelaxant action of ANF. Conversely, Garcia et al.16 failed to
observe an inhibitory effect of methylene blue on
ANF-induced rabbit mesenteric arterial relaxation, although they speculated that ANF may cause cGMP
accumulation by inhibiting cGMP phosphodiesterase.
In the present study, we found that methylene blue
produced dose-dependent inhibition of SNP-stimulated cGMP but not of ANF-stimulated cGMP. A similar
result recently was described by Winquist et al.42 as an
unpublished observation. These results indicate that
there may be different receptor sites for ANF and SNP,
respectively. This view is furthermore reinforced by
the findings that there was an additivity in ANF-stimulated and SNP-stimulated cGMP levels. The present
findings that methylene blue did not modify ANFstimulated cGMP may be explained by the previous
reports indicating that SNP predominantly stimulates
the soluble form of guanylate cyclase, 1443 which can
be inhibited by methylene blue, 17 ' 18 whereas ANF selectively stimulates the particulate form of guanylate
cyclase,9' "•12 which has not been shown to be inhibited by methylene blue. Thus, there may be a potential
problem in interpreting experiments in which only
methylene blue is employed to evaluate the role of the
guanylate cyclase-cGMP system in the vasorelaxant
action of ANF. Further studies are required to clarify
the effect of methylene blue on particulate guanylate
cyclase.
Renal kallikrein, a serine protease, has been known
to play an important role in the regulation of sodiumwater homeostasis. Recently, Briggs et al.21 reported
that incubation of atrial extracts with pancreatic kallikrein or trypsin, another nonspecific serine protease,
reduced the natriuretic activity of ANF, suggesting a
possible role for kallikrein in peptide inactivation.
Trypsin has also been demonstrated to cleave atrial
extracts of the high molecular weight form to that of
the low molecular weight form, which is biologically
more active,44 and to abolish the ability of ANF to
activate guanylate cyclase from kidney and aorta.9 In
the present study, we also found that incubation of
ANF or SNP with pure porcine pancreatic kallikrein
abolished the ability of ANF, but not of SNP, to increase intracellular cGMP levels in cultured vascular
smooth muscle cells. This effect was concentrationdependent and was inhibited by the serine protease
inhibitors aprotinin or Foy, suggesting that the effect
of kallikrein was due to enzymatic cleavage rather than
to nonspecific effects. Although it is not known wheth-
770
HYPERTENSION
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er kallikrein is physiologically important in the vascular action of ANF, it is of note that the kallikreinlike
enzyme is present in the rat mesenteric artery.45 Inactivation of ANF by vascular kallikreinlike enzyme may
be a local mechanism for in vivo regulation of vasorelaxant activity.
Several reports indicate that renal artery is more
sensitive than other arteries to the vasorelaxant action
of ANF.22"25 In the present study, the cGMP response
to ANF was much greater in cultured cells from renal
artery than in those from mesenteric artery. The difference between two cell types may be explained by the
differences in synthesis, degradation, or cGMP efflux
from the cells; however, this selective cGMP response
to ANF in renal arterial cells should be due mainly to
the increased synthesis rather than to a difference in
phosphodiesterase activity, since the differences in the
cGMP response to ANF between two cell types was
more prominent than that to SNP. Also, the cAMP
response to isoproterenol was similar in two cell types.
Although we did not elucidate the specific mechanism
of this selectivity, the present results may explain the
selective vasodilation of renal artery induced by ANF
if cGMP mediates the vasorelaxant action of ANF.
In conclusion, ANF and SNP share a number of
similarities in their effects on cultured vascular smooth
muscle cells, but their responses to kallikrein and
methylene blue differ. Both ANF and SNP increased
the levels of intracellular cGMP. Kallikrein abolished
the action of ANF but not that of SNP, whereas methylene blue inhibited the action of SNP but not that of
ANF. Our results are consistent with the hypothesis
that cGMP mediates the vasorelaxant action of ANF.
Acknowledgments
We are grateful for the excellent technical assistance of Kaori
Matsuura, Keiko Shiraishi, and Mayumi Nakayama and the secretarial assistance of Junko Okazaki and Emiko Sakai.
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M Sato, K Abe, K Takeuchi, M Yasujima, K Omata, M Hiwatari, Y Kasai, M Tanno, M Kohzuki
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Hypertension. 1986;8:762-771
doi: 10.1161/01.HYP.8.9.762
Hypertension is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 1986 American Heart Association, Inc. All rights reserved.
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