(Hypertension. 1995;26:696-704.)
© 1995 American Heart Association, Inc.
Articles |
From the Vascular Biology Center (A.P., J.D.C.), Department of Pharmacology and Toxicology (A.P., N.M., A.M., J.D.C.), and Department of Physiology and Endocrinology (G.M.), Medical College of Georgia, Augusta, and Molecular Geriatrics Corp, Lake Bluff, Ill (F.M.).
| Abstract |
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1- and
ß1-subunits of sGC were decreased in cells pretreated
with isobutylmethylxanthine and forskolin but not with dideoxyforskolin
(inactive analogue). Moreover, protein levels for the sGC
1-subunit of cells pretreated with
isobutylmethylxanthine and forskolin but not with dideoxyforskolin were
decreased as indicated by Western blot analysis. These data
indicate that cAMP-elevating agents decrease sGC activity, possibly by
decreasing mRNA or protein levels or both.
Key Words: guanylate cyclase cyclic AMP cyclic GMP nitroprusside
| Introduction |
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sGC is a heterodimer composed of a large (
) and small (ß) subunit
containing 1 mole heme bound per mole of holoenzyme.9
Whereas basal sGC activity is heme independent, NO stimulation of sGC
activity requires the presence of heme for the formation of a
nitrosyl-heme complex that is the active paramagnetic species
responsible for the enzyme activation.5 10 11 Three cDNA
sequences for the
-subunit (
1 through
3) and ß-subunit (ß1 through
ß3) have already been cloned and
sequenced.12 13 14 15 16 However, it is still unclear which
subunits represent real isoforms of the sGC protein and which
are just species variants. The rat and bovine lung enzymes are composed
of one
1- and one ß1-subunit;
ß2 is preferentially expressed in the rat kidney; the
human brain enzyme is an
3ß3 dimer; and a
different
-subunit (
2) was cloned from fetal
human brain. Recent cloning and expression experiments have revealed
that although the
- and ß-subunits each appear to possess a
catalytic domain, expression of enzymatic activity requires the
presence of both subunits.17 Substitution of
1 but not ß1 with
2 in the
1ß1 complex yields a functional
2ß1 enzyme.15
At least two important biological roles for cGMP in mammalian vascular cells have been elucidated. cGMP is the intracellular messenger for NO-mediated smooth muscle relaxation18 and inhibition of platelet aggregation.19 Endothelium-derived NO is probably the most important endogenous activator of sGC. Under physiological conditions it is synthesized by the endothelium from L-arginine in a reaction catalyzed by the endothelial NOS.20 Another form of NOS, namely, inducible or type II NOS, is found in the smooth muscle as well as other cell types; this form of NOS is not constitutively expressed but is synthesized on stimulation of the smooth muscle with cytokines.21 22 23 24 Recent evidence suggests that basally released endothelium-derived NO contributes to vascular tone regulation because the L-arginine analogues, which serve as competitive inhibitors of endothelium-derived NO synthesis, increase blood pressure.25
In addition, exogenous nitrovasodilators that release NO (ie, SNP and glycerin trinitrate) produce smooth muscle relaxation by activating sGC and increasing cGMP formation.26 27 Although the mechanism of action of cGMP with respect to vasodilation has not been fully elucidated, it is known that cGMP lowers intracellular Ca2+ levels and alters the phosphorylation pattern of cellular proteins involved in contraction.1 28 Although some of the actions of cGMP are mediated by activation of cGMP-dependent protein kinase,29 cGMP may also exert its effects through activation of class II and/or inhibition of class III cAMP phosphodiesterases.3 The aim of the present study was to investigate the effects of cAMP-elevating agents on sGC activity, mRNA, and protein levels in cultured rat vascular smooth muscle cells.
| Methods |
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Cell Culture
Rat aortic smooth muscle cells were isolated from 325- to 350-g
Wistar rats, four to five rats per isolation, with the use of
previously published procedures.30 Animal handling and
euthanasia were in accordance with guidelines from the Institutional
Committee on Animal Use for Research and Education. Cells were
positively identified as smooth muscle by indirect
immunofluorescent staining for
-actin with the use
of a mouse anti
-actin antibody and anti-mouse IgG
fluoroisothiocyanate conjugate. Smooth muscle cells were grown in 50%
F12 and 50% Dulbecco's modified Eagle's medium supplemented with
10% fetal bovine serum, 0.2 g/L L-glutamine, 100 U/mL
penicillin, and 0.1 mg/mL streptomycin. For the present study cells
between passages 1 and 5 were used.
Radioimmunoassay for cGMP
The radioligand (125Isuccinyl
cGMP tyrosine methyl ester) was prepared in our laboratory. Stock
solutions of the succinyl tyrosine methyl ester of cGMP were made up in
50 mmol/L sodium acetate buffer, pH 4.75, and iodinated
with carrier-free 125I.31 The iodination
reaction products were separated by reversed-phase
HPLC.32 With the use of a monoclonal antibody for cGMP,
radioimmunoassay was performed in the Gammaflo automated
radioimmunoassay system.33 Standard stock solutions of
cGMP (20 µmol/L) were prepared in 0.1N HCl, and the absorbance of the
solution was routinely monitored spectrophotometrically (UV 160U,
Shimadzu). Standard dilutions (0.63 to 80 nmol/L) were made fresh from
the stock solution. The HCl extract containing cGMP was used for
radioimmunoassay directly.
Determination of Intracellular cGMP in Cultured Rat Aortic Smooth
Muscle Cells
Cells were treated with IBMX (10 to 1000 µmol/L) or forskolin
(0.01 to 10 µmol/L) for 3 to 24 hours. At the end of the incubation
time cells were washed with Earle's balanced salt solution and then
incubated with this solution containing 10 µmol/L SNP for 15 minutes
in the presence of IBMX (0.3 mmol/L) to prevent cGMP breakdown unless
otherwise noted. After the 15-minute incubation with SNP, medium was
rapidly aspirated, and 500 µL of 0.1N HCl was added to each well to
stop enzymatic reactions and extract cGMP. Thirty minutes later the HCl
extract was collected, and cell remnants were removed from the wells by
adding hot 1.0N NaOH and scraping the well with a rubber policeman. The
HCl extract was analyzed for cGMP by radioimmunoassay, and
NaOH-solubilized samples were used for protein determination. In a
separate series of experiments designed to evaluate the involvement of
phosphodiesterase in the downregulation of cGMP accumulation by
cAMP-elevating agents after pretreatment with IBMX or forskolin,
cells were stimulated with 10 µmol/L SNP for 15 minutes in the
absence or presence of IBMX (0.3 and 1 mmol/L). When H89 or KT 5720 was
used to block cAMP-dependent protein kinase,34 35 cells
were pretreated for 1 hour with 30 µmol/L H89 or 5 µmol/L KT 5720
before the 12-hour incubation with IBMX or forskolin. cGMP accumulation
in response to 10 µmol/L SNP was then determined in the presence of
IBMX as described above.
Protein Determination
Protein content of the supernatant of the centrifuged
(2000 rpm for 5 minutes at room temperature) NaOH-solubilized samples
was measured by the Bradford method.36 Sample aliquots
were combined with the protein binding dye, and optical density was
determined at 630 nm with a multiwell plate reader (Dynetech
Laboratories Inc). Bovine albumin, fraction V, was used as the
standard.
Isolation of Total RNA and RT-PCR
Rat aortic smooth muscle cells were cultured in 100-mm dishes
and incubated with either vehicle, 10 µmol/L 1,9-dideoxyforskolin
(inactive analogue of forskolin), 10 µmol/L forskolin, or 500
µmol/L IBMX for 24 hours. Total RNA was isolated with a commercially
available kit (RNAzol), quantified by absorbance at 260 nm, and stored
at -70°C in a mercaptoethanol/ethanol/ammonium acetate
solution. With the use of published sequences,37 38 39
primers were synthesized for the sGC
1-subunit (forward,
base position 1071 5'-GAAATCTTCAAGGGTTATG-3' and reverse, base
position 1896 5'-CACAAAGCCAGGACAGTC-3'), ß1-subunit
(forward, base position 1450 5'-GGTTTGCCAGAACCTTGTATCCACC and reverse,
base position 1733 5'-GAGTTTTCTGGGGACATGAGACACC-3'), and GAPDH
(forward, base position 35 5'-TGAAGGTCGGTGTCAACGGATTTGGC-3' and
reverse, base position 1017 5'-CATGTAGGCCATGAGGTCCACCAC-3') in an
automated DNA synthesizer with phosphoramidite chemistry. RNA was
reverse transcribed and amplified with a commercially available kit
(GeneAmp RNA PCR kit) in a DNA Thermal Cycler 480 (Perkin-Elmer). RNA
samples were precipitated and resuspended in 10 mmol/L Tris, 10 mmol/L
NaCl, and 10 mmol/L EDTA (pH 8.0) and were incubated with 3 U
RNAse-free DNAse per 35 µg total RNA for 30 minutes at 37°C to
digest traces of genomic DNA. Total RNA (50 to 500 ng) was combined
with 50 U Moloney murine leukemia virus reverse transcriptase in 5
mmol/L MgCl2, 50 mmol/L KCl, 10 mmol/L Tris-HCl, 1
mmol/L deoxyribonucleoside triphosphates, 10 U RNAse
inhibitor, and 2.5 µmol/L random hexamers to prime the
cDNA formation in a reaction volume of 20 µL for 15 minutes at
42°C. Samples were then heated for 5 minutes at 99°C to destroy
reverse transcriptase activity before the PCR reaction. cDNA was
amplified (25 through 31 cycles) at 92°C for 1 minute, 58°C for 1.5
minutes, and 72°C for 3 minutes (melting, annealing, and extension
temperatures, respectively). For the PCR reaction, primers were used at
1 µmol/L for the
1- and ß1-subunits and
0.2 µmol/L for GAPDH. MgCl2 concentration was 2 mmol/L,
and 2.5 U polymerase was used per reaction. After the amplification 10
µL of the PCR reaction mixture was electrophoresed on 0.9% agarose
gels, stained with ethidium bromide, visualized on a UV
transilluminator, and photographed. A molecular weight standard
consisting of 100-bp increments between 100 and 2600 bp was used to
confirm the predicted PCR product size.
HPLC Quantification of RT-PCR Products
Five to 25 µL PCR reaction mixture was used for HPLC
analysis40 in a Bio-Rad model 1350 HPLC system
with an on-line 1706 UV-visible photometer, driven by a Bio-Rad
Series 800 and HRLC software package to control flow rate and gradient
production and to calculate the peak area based on the UV
absorbance (260 nm). The mobile phase consisted of buffer A (1 mol/L
NaCl in 25 mmol/L Tris-HCl, pH 9.0) and buffer B (25 mmol/L Tris-HCl,
pH 9.0). Flow through the Perkin-Elmer TSK DEAE-NPR column was set at 1
mL/min. The gradient used was 35% A in B for 2 minutes, 35% to 54% A
for 0.1 minute, 54% to 60% A for 2.9 minutes, 60% to 75% A for 1
minute, 75% to 100% A for 2 minutes, and 100% to 35% A for 0.1
minute. The relative amounts of PCR products were calculated as the
area under the curve in arbitrary units divided by the volume of the
RT-PCR reaction injected into the column and presented as
arbitrary units per microliter.
Immunoblotting
Rat aortic smooth muscle cells were cultured in 60-mm dishes and
incubated with vehicle, 10 µmol/L 1,9-dideoxyforskolin, 10
µmol/L forskolin, or 500 µmol/L IBMX for 24 hours. After the
24-hour period cells were lysed in lysis buffer (1% NP40, 150 mmol/L
NaCl, 20 mmol/L HEPES [pH 7.0], 1 mmol/L EDTA, 1% aprotinin, and 1
mmol/L PMSF). Cell lysates were centrifuged at 20 000 rpm, the
supernatant fraction was collected, and protein concentration was
measured by the Bradford method.36 Thirty-five
micrograms per lane was electrophoresed in sodium dodecyl
sulfate7.5% polyacrylamide gels and transferred to a
polyvinylidene difluoride membrane at 60 V for 1.5 hours at
4°C in a buffer containing 25 mmol/L Tris and 700 mmol/L glycine.
Membranes were incubated overnight at 4°C with 5% dry milk in buffer
containing 0.1% (vol/vol) TTBS to block nonspecific binding. The
following day membranes were incubated with 1:750 of a monoclonal
antibody (H6) against the
1-subunit of
sGC41 in 5% milk in TTBS with 1 mol/L glucose and 10%
glycerol for 1 hour at room temperature, washed three times with TTBS
for 20 minutes each time, blocked for an additional hour with 5% milk
in TTBS, and finally incubated for 1 hour with a horseradish
peroxidaseconjugated anti-mouse IgG (1:10 000 dilution,
Amersham). Immunoreactive protein bands were visualized with the use of
the ECL system after 15 minutes of exposure to x-ray film. To check
for equality in loading and transfer, membranes were subsequently
incubated with a monoclonal antibody against tubulin, and
immunoreactive bands were visualized after exposure to x-ray film
for 30 seconds.
Data Analysis
Data are presented as mean±SEM of the indicated number
of individual observations. cGMP values are expressed either as
picomoles per milligram protein per 15 minutes or as a percentage of
the control value. Statistical comparisons between groups were
performed with one-way ANOVA or Student's t test, as
appropriate. Differences among means were considered significant at a
value of P<.05.
| Results |
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Quantification of mRNA for sGC by RT-PCR Followed by
HPLC
To investigate the effects of cAMP-elevating agents on sGC subunit
mRNA, we isolated total RNA from cells treated with vehicle,
dideoxyforskolin, forskolin, and IBMX and then reverse transcribed,
amplified, and quantified the total RNA by HPLC. To ensure that the PCR
reaction had not reached a plateau phase under the conditions used to
amplify and quantify sGC subunit mRNA, we performed kinetic
analysis for the
1-, ß1-, and
GAPDH-amplified sequences (Fig 5A) with
500 ng per reaction total RNA as the template for the
1-
and ß1-subunits and 50 ng per reaction for GAPDH. Smaller
amounts of total RNA were used as the template for GAPDH amplification
because the use of 500 ng total RNA for GAPDH amplification led to a
plateau at a lower cycle number compared with
1 and
ß1. The RT-PCR product peak area (in arbitrary units)
as determined by absorbance at 260 nm by HPLC increased in a
logarithmic fashion between 25 and 29 cycles (r=.99, .97,
and .99 for
1, ß1, and
GAPDH, respectively) and started reaching a plateau at 31 cycles. The
slopes of the linear regression analysis for
1
and ß1 were similar (0.178 and 0.173, respectively),
revealing similar amplification efficiencies. Thus, for mRNA
quantification, 28 cycles were routinely used (Fig 5B).
Under these conditions RNA was linearly amplified in the range of 100
to 500 ng for
1 and ß1 and 25 to 100 ng
for GAPDH, with respective r values from linear regression
analyses of .99, .98, and .99. To be able to detect both
increases and decreases in mRNA levels for the sGC subunits, we used
200 ng total RNA from control and pretreated cells per reaction for 28
cycles. Fifty nanograms per reaction of GAPDH was amplified in a
separate tube as an internal standard for reverse transcription and PCR
amplification. Fig 6 shows
representative gel electrophoresis of PCR products
for the sGC
1-subunit (826 bp) and
ß1-subunit (284 bp); mRNA for both sGC subunits was not
altered by pretreatment with the inactive forskolin analogue, whereas
cells pretreated with either forskolin or IBMX showed a decrease in
mRNA for both subunits. Fig 7 is a
representative chromatogram of an HPLC
separation of an sGC ß1-subunit PCR product. Unused
primers, dNTPs, and enzymes were eluted in the 0.63-minute peak. HPLC
quantification of the PCR products (Fig 8) confirmed the observations made with
gel electrophoresis. The
1-subunit mRNA, while remaining
unaltered on pretreatment with dideoxyforskolin, was undetectable in
forskolin- and IBMX-pretreated cells. The ß1 mRNA of
IBMX- and forskolin-pretreated cells was affected to a smaller but
significant extent. GAPDH levels were not significantly changed, with
the exception of the IBMX group, in which they were slightly
increased.
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Immunoblotting
Immunoblotting of cell lysates from cultured rat aortic smooth
muscle cells with a monoclonal antibody against the
1-subunit of sGC revealed a single 82-kD band (Fig 9). Although pretreatment with
dideoxyforskolin had no effect on
1 protein levels,
pretreatment with forskolin or IBMX for 24 hours decreased the
1-subunit to undetectable levels.
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| Discussion |
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1- and
ß1-subunits of sGC are reduced in smooth muscle cells
pretreated with IBMX and forskolin; and (5) the amount of protein for
the
1-subunit of sGC is reduced in IBMX- and
forskolin-pretreated smooth muscle cells. In a recent report42 ß1-subunit sGC mRNA levels were decreased on exposure to cAMP-elevating agents in RFL-6 rat fetal lung fibroblasts. Interestingly, forskolin, unlike dibutyryl cAMP and IBMX, produced a transient decrease in ß1 mRNA RFL-6 cells, and cGMP accumulation following SNP stimulation was found to be unaltered after a single dose of 10-6 mol/L forskolin for 1 to 24 hours. However, after exposure to 10-6 mol/L forskolin for 1 or 2 days, with forskolin replenished every 12 hours, SNP-induced cGMP accumulation was decreased by 39% to 63% compared with control. In the present study pretreatment of rat aortic smooth muscle cells with the nonspecific phosphodiesterase inhibitor IBMX produced a significant decrease in the ability of the cells to accumulate cGMP in response to SNP that was first evident at 6 hours, whereas single exposure to the adenylate cyclase activator forskolin (10 µmol/L) elicited a significant decrease as early as 3 hours. The apparent discrepancies between the results presented here and those previously published42 may be attributed to the different cell types used or differences in cell culture conditions. In any event, cAMP-elevating agents were found in both studies to inhibit sGC responses. It should be noted that a seemingly related but opposite phenomenon has already been described. In a study by Hu et al43 prolonged exposure of vessels to catecholamines, which are known to reduce cAMP levels, leading to vasoconstriction, resulted in the development of enhanced sensitivity to SNP. To investigate whether the reduced cGMP levels in response to SNP stimulation in cells pretreated with cAMP-elevating agents is due to decreased synthesis or increased degradation of cGMP, we determined cGMP accumulation in both the presence and absence of phosphodiesterase inhibition. Since cGMP levels in response to SNP were reduced irrespective of phosphodiesterase inhibition, we concluded that decreased cGMP levels result from attenuated synthesis of cGMP.
Induction of type II NOS (inducible NOS) activity in vascular smooth muscle cells has been shown to lead to a decrease in relaxation of preconstricted bovine mesenteric rings and cGMP accumulation in response to SNP.44 Similar results were obtained in cultured rat aortic smooth muscle cells, in which nitrovasodilator-induced cGMP accumulation was found to be attenuated in cells pretreated with endotoxin or interleukin-1ß compared with control.45 In addition, elevation of intracellular cAMP levels has been shown to lead to induction of type II NOS or to potentiate the induction by interleukin-1ß in several cell types.46 47 48 49 In rat vascular smooth muscle cells it is still controversial whether increased cAMP is a sufficient stimulus for induction of NO production or whether it simply potentiates the responses of other inducers, such as endotoxin and interleukin-1ß. In our experiments there was no evidence that NOS activity was induced in cells pretreated with cAMP-elevating agents. However, simultaneous exposure of cells to IBMX or isoproterenol and endotoxin leads to potentiation of the endotoxin response by twofold to threefold (unpublished data, 1994). The inability of cAMP-elevating agents to induce NO production by themselves rules out the possibility that the observed downregulation in SNP-induced cGMP accumulation produced by IBMX and forskolin is mediated through the induction of NOS. To study the role of PKA in the downregulation of cGMP accumulation in response to SNP in IBMX- and forskolin-pretreated cells, we exposed rat aortic smooth muscle cells to PKA-selective inhibitors before IBMX and forskolin pretreatment. Exposure to H89 or KT 5720 partially or fully restored the ability of cells to synthesize cGMP on stimulation with SNP, suggesting that PKA activation plays a critical role in decreasing sGC activity in IBMX- and forskolin-pretreated cells.
To further investigate the mechanism of reduction of sGC activity by
cAMP-elevating agents, we determined alterations in steady-state
mRNA levels of forskolin- and IBMX-pretreated cells. To detect changes
in the amounts of mRNA for the sGC subunits, we developed an
RT-PCR/HPLC method. Since the yield of PCR products reflects the
initial template levels only under conditions of exponential
amplification, we performed a kinetic analysis to determine the
number of cycles and the amount of starting template to be used for all
target sequences (
1, ß1, and
GAPDH). After 28 cycles of amplification steady-state mRNA levels
for both the
1- and ß1-subunits of sGC
were decreased in cells pretreated with IBMX (500 µmol/L) and
forskolin (10 µmol/L) but not dideoxyforskolin (10 µmol/L) for 24
hours. RT-PCR data for the sGC ß1-subunit were confirmed
by the more conventional Northern blot analysis. The
coordinated regulation of expression of the
- and ß-subunits
comes as no surprise, as the human sGC
3- and
ß3-subunits have been shown to colocalize around region
4q32 on chromosome 4.50 Although no data are available for
the promoter regions of the sGC subunit genes, it is possible that the
genes coding for the
1- and ß1-subunits
share a common promoter, as is the case for the mouse surf-1
and surf-2 genes.51 To correlate changes in sGC
mRNA and activity with changes at the protein level, we performed
Western blot analysis. The
1 protein levels were
decreased in cells pretreated for 24 hours with 500 µmol/L IBMX or 10
µmol/L forskolin but not in those pretreated with 10 µmol/L
dideoxyforskolin. Since the presence of both
- and ß-subunits
is required for the expression of sGC activity and since
1 protein levels were found to be decreased in cells
pretreated with cAMP-elevating agents, reduced cGMP accumulation
in response to SNP in IBMX- and forskolin-pretreated cells
should result from decreased amounts of sGC rather than reduced
affinity of the enzyme for its substrate. The mechanism by which
elevation in intracellular cAMP levels leads to decreased sGC activity
needs to be further investigated. Decreased steady-state mRNA and
protein levels may result either from inhibition of transcription and
protein synthesis or from decreased half-life of the mRNA and
protein, respectively. Observations by Shimouchi et al42
suggest that sGC subunits have a prolonged half-life in the cell.
In agreement with this, SNP-induced cGMP accumulation does not decrease
in cells exposed to cycloheximide or actinomycin D for 12 to 36 hours
compared with control cells (unpublished data, 1994). It is therefore
reasonable to speculate that since sGC activity is reduced as early as
3 to 6 hours after exposure to cAMP-elevating agents, given the much
longer half-life of sGC, reduced activity, at least at the early
phase, cannot result from inhibition of synthesis and must therefore be
caused by increased protein degradation.
In conclusion, increases in intracellular cAMP lead to decreased sGC activity through reduction of sGC subunit mRNA and protein levels. Since sGC is the intracellular "receptor" for NO in vascular smooth muscle cells, reduction of sGC gene expression in vivo could lead to decreased biological effectiveness of NO under conditions of prolonged elevation of cAMP, which may serve as a homeostatic mechanism counteracting the vasorelaxant actions of cAMP-elevating agents.
| Selected Abbreviations and Acronyms |
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| Acknowledgments |
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| Footnotes |
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Received March 13, 1995; first decision April 20, 1995; accepted June 14, 1995.
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