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(Hypertension. 1995;26:696-704.)
© 1995 American Heart Association, Inc.

Articles

Regulation of Vascular Smooth Muscle Soluble Guanylate Cyclase Activity, mRNA, and Protein Levels by cAMP-Elevating Agents

Andreas Papapetropoulos; Nandor Marczin; Gloria Mora; Antonio Milici; Ferid Murad; John D. Catravas

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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Abstract Although the biochemical properties of soluble guanylate cyclase (sGC) have been extensively studied, little is known about the regulation of gene expression of sGC subunits by second messengers. cAMP analogues and elevating agents have been previously shown to alter gene expression in vascular cells. The aim of the present study was to investigate the effects of cAMP-elevating agents on sodium nitroprusside–stimulated sGC activity and to correlate activity changes with mRNA and protein levels in cultured rat aortic smooth muscle cells. Pretreatment of cells with 50 to 1000 µmol/L isobutylmethylxanthine or 0.01 to 10 µmol/L forskolin led to a time- and concentration-dependent decrease in sodium nitroprusside–induced cGMP accumulation, first evident after 3 hours of pretreatment with forskolin and 6 hours of pretreatment with isobutylmethylxanthine. Incubation of cells with a protein kinase A–selective inhibitor (H89 or KT 5720) partially or fully prevented the downregulation in sodium nitroprusside–induced cGMP accumulation caused by cAMP-elevating agents. Quantification of reverse transcriptase–polymerase chain reaction products by high-performance liquid chromatography revealed that mRNA for both 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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Cyclic GMP has been recognized as an important intracellular messenger in various tissues.^{1 2 3} In vascular smooth muscle cGMP is formed from GTP by guanylate cyclases that exist in at least two forms: a membrane-bound and a soluble form.³ The membranous form is a single protein that functions both as a receptor for the natriuretic peptide family and as a guanylate cyclase, whereas the soluble form is activated by free radicals such as NO, hydroxyl radicals, and carbon monoxide as well as porphyrins and polyunsaturated fatty acids.^{4 5 6 7 8}

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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Materials
Rats were purchased from Harlan Sprague-Dawley, Inc. Tissue culture plasticware was from Corning Glass Inc; growth medium was from GIBCO Laboratories and fetal calf serum from Hyclone Laboratories Inc. ¹²⁵I was from DuPont-NEN; H89 and KT 5720 were purchased from LC Laboratories, RNAzol from Biotecx Laboratories Inc, the GeneAmp RNA PCR kit from Perkin-Elmer, and the ECL detection system from Amersham International. Protein binding dye, polyvinylidene difluoride membranes, dry milk, Tween 20, and the other immunoblotting reagents were obtained from Bio-Rad; RNAse-free DNAse was purchased from GIBCO BRL. X-ray film was from Eastman Kodak. Carbaprostacyclin was purchased from Cayman Chemical Co. All other chemicals, including penicillin, streptomycin, succinyl tyrosine cGMP methyl ester, IBMX, isoproterenol, forskolin, SNP, bovine serum albumin, NP40, PMSF, aprotinin, EDTA, and others, were from Sigma Chemical Co.

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 (¹²⁵I–succinyl 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 sulfate–7.5% 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 peroxidase–conjugated 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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Intracellular cGMP Accumulation in Cultured Rat Aortic Smooth Muscle Cells
Baseline cGMP values (unstimulated but in the presence of IBMX) of cultured rat aortic smooth muscle cells were expectedly low and ranged from 4 to 12 pmol/mg protein per 15 minutes. In the presence of 0.3 mmol/L IBMX these levels increased 50- to 500-fold on stimulation with 10 µmol/L SNP depending on the passage and confluent state of the cells. Pretreatment of cells with the adenylate cyclase activator forskolin (10 µmol/L) or the nonspecific (both cAMP and cGMP) phosphodiesterase inhibitor IBMX (100 µmol/L) for 24 hours resulted in reduction of the SNP-induced cGMP accumulation in both the absence and presence of phosphodiesterase inhibition (Fig 1). cGMP levels after SNP stimulation in the absence of phosphodiesterase inhibition were reduced from 57.9±5.8 pmol/mg protein per 15 minutes for control to 19.1±1.5 and 22.3±2.8 for forskolin- and IBMX-pretreated cells, respectively; when 0.3 mmol/L IBMX was present during the 15-minute exposure to SNP, cGMP levels were reduced from 799.1±38.2 pmol/mg protein per 15 minutes for control to 211.1±13.1 and 353.3±25.0 for forskolin- and IBMX-pretreated cells, respectively. Similar results were obtained when 1 mmol/L IBMX was used to inhibit cGMP phosphodiesterase activity during the 15-minute SNP exposure. Since SNP-induced cGMP accumulation was reduced in IBMX- and forskolin-pretreated cells to a comparable extent irrespective of the level of phosphodiesterase inhibition, 0.3 mmol/L IBMX was used during the 15-minute SNP stimulation in all subsequent experiments to enhance cGMP levels and improve the sensitivity of the method. Pretreatment of rat aortic smooth muscle cells with 300 µmol/L IBMX (Fig 2A) led to a time-dependent decrease in SNP-induced cGMP accumulation, first evident at 6 hours, that reached a maximum at 24 hours (SNP-stimulated cGMP level for control cells was 549.5±40.1 pmol/mg protein per 15 minutes, decreasing to 75.5±17.5 at 24 hours). Pretreatment of the cells with 50 to 1000 µmol/L IBMX for 12 hours led to a concentration-dependent decrease (Fig 2B) of SNP-stimulated cGMP levels. Similarly, when smooth muscle cells were pretreated with forskolin, SNP-induced cGMP accumulation decreased in a time- and concentration-dependent manner, reaching 26.3±3.3% of control at 24 hours with 10 µmol/L forskolin (Fig 3). In the absence of SNP stimulation, baseline cGMP values were also decreased in IBMX- and forskolin-pretreated cells to 30% to 60% of control. Moreover, pretreatment of cells with 10 µmol/L isoproterenol for 24 hours or 30 µmol/L carbaprostacyclin, a stable prostacyclin analogue, for 12 or 24 hours resulted in decreased SNP-induced cGMP accumulation (Table). Pretreatment of smooth muscle cells with the PKA-selective inhibitors H89 (30 µmol/L) or KT 5720 (5 µmol/L) fully prevented the IBMX-induced and partially prevented the forskolin-induced downregulation in cGMP accumulation after 10 µmol/L SNP (Fig 4).

View larger version (33K): [in this window] [in a new window]   Figure 1. Bar graph shows that downregulation of SNP-induced cGMP accumulation in rat aortic smooth muscle cells pretreated with forskolin or IBMX does not result from alteration in phosphodiesterase activity. Smooth muscle cells were pretreated with 10 µmol/L forskolin or 100 µmol/L IBMX for 12 hours. At the end of pretreatment, cells were washed with Earle's balanced salt solution and incubated for 15 minutes with 10 µmol/L SNP in the absence or presence of IBMX (0.3 mmol/L). Mean±SEM; n=4 wells. *P<.05 from respective vehicle.

View larger version (20K): [in this window] [in a new window]   Figure 2. Bar graphs show time and concentration dependence of IBMX effects on SNP-stimulated sGC activity. Rat aortic smooth muscle cells were pretreated with 300 µmol/L IBMX for the indicated time (A) or 10 to 1000 µmol/L IBMX for 12 hours (B). At the end of the pretreatment period cells were washed twice with Earle's balanced salt solution and incubated for 15 minutes with 10 µmol/L SNP in the presence of 0.3 mmol/L IBMX to prevent cGMP breakdown. cGMP was extracted in HCl and measured by radioimmunoassay. Mean±SEM; n=4 wells. *P<.05 from 0 hour or 0 µmol/L IBMX pretreatment.

View larger version (20K): [in this window] [in a new window]   Figure 3. Bar graphs show time and concentration dependence of the effects of forskolin on SNP-stimulated sGC activity. Rat aortic smooth muscle cells were pretreated with 10 µmol/L forskolin for the indicated time (A) or 0.01 to 10 µmol/L forskolin for 12 hours (B). For details see Fig 2 legend. Mean±SEM; n=4 wells. *P<.05 from 0 hour or 0 µmol/L forskolin pretreatment.

View this table: [in this window] [in a new window]   Table 1. Effects of cAMP-Elevating Agents on Basal or SNP-Stimulated sGC Activity

View larger version (46K): [in this window] [in a new window]   Figure 4. Bar graphs show that selective PKA inhibitors H89 or KT 5720 partially or fully prevent the effects of forskolin and IBMX on SNP-stimulated sGC activity. Smooth muscle cells were pretreated with 10 µmol/L forskolin or 100 µmol/L IBMX for 12 hours. Cells were preincubated with 30 µmol/L H89 (A) or 5 µmol/L KT 5720 (B) for 60 minutes before the addition of forskolin or IBMX. For details see Fig 2 legend. Mean±SEM; n=4 wells. *P<.05 from vehicle; #P<.05 from respective dimethyl sulfoxide (DMSO).

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.

View larger version (20K): [in this window] [in a new window]   Figure 5. Left, Graph shows kinetic analysis of PCR amplification of 1, ß1, and GAPDH sequences. 1 and ß1 (500 ng) or GAPDH (50 ng) were reverse transcribed and amplified for 25 to 31 cycles. PCR products were quantified by HPLC. Right, Graph shows quantitative amplification of sequences for the sGC 1- and ß1-subunits and GAPDH. 1 and ß1 (100 to 500 ng) or GAPDH (25 to 100 ng) total RNA were reverse transcribed and amplified for 28 cycles, and PCR products were quantified by HPLC. A indicates 100 ng 1 and ß1, 25 ng GAPDH; B, 200 ng 1 and ß1, 50 ng GAPDH; C, 300 ng 1 and ß1, 75 ng GAPDH; and D, 500 ng 1 and ß1, 100 ng GAPDH.

View larger version (31K): [in this window] [in a new window]   Figure 6. Gel shows analysis of RT-PCR products for the 1- and ß1-subunits of sGC by agarose gel electrophoresis stained with ethidium bromide. Total RNA (200 ng) was reverse transcribed and amplified for 28 cycles. A marker (M) consisting of DNA fragments in increments of 100 bp (lower band, 100 bp) was used to estimate the size of PCR products. Lanes 1 through 4, 1-subunit; lanes 5 through 8, ß1-subunit. Cells were treated with vehicle (lanes 1 and 5), 10 µmol/L dideoxyforskolin (lanes 2 and 6), 10 µmol/L forskolin (lanes 3 and 7), or 500 µmol/L IBMX (lanes 4 and 8) for 24 hours.

View larger version (12K): [in this window] [in a new window]   Figure 7. Chromatogram of representative peak obtained from HPLC analysis of an RT-PCR sample. Total RNA (200 ng) was reverse transcribed and the ß1-subunit message amplified for 28 cycles; 10 µL of the reaction mixture was injected into the column without any purification. The peak at 2.31 minutes represents the 284-bp PCR product for the sGC ß1-subunit; the peak at 0.63 minute includes unused primers and dNTP.

View larger version (33K): [in this window] [in a new window]   Figure 8. Bar graph shows sGC 1- and ß1-subunit quantification by HPLC after RT-PCR. Cells were pretreated with either vehicle, 10 µmol/L 1,9-dideoxyforskolin, 10 µmol/L forskolin, or 500 µmol/L IBMX for 24 hours. RNA was isolated, and 200 ng for 1 and ß1 or 50 ng for GAPDH were amplified for 28 cycles as described in "Methods." PCR products were quantified by HPLC and calculated as arbitrary units per microliter of RT-PCR. Mean±SEM; n=3 determinations. *P<.05 from respective vehicle.

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.

View larger version (39K): [in this window] [in a new window]   Figure 9. A, Immunoblotting analysis for the 1-subunit of sGC. Cells were pretreated with either vehicle, 10 µmol/L 1,9-dideoxyforskolin, 10 µmol/L forskolin, or 500 µmol/L IBMX for 24 hours. Protein (35 µg) for each group was subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to a membrane. Membranes were incubated with 1:750 dilution of a monoclonal antibody for the 1-subunit (82 kD). Prestained protein standards (Bio-Rad) were used as molecular size markers. B, Membranes were incubated with a monoclonal antibody against tubulin to check for consistency in loading and transfer.

	Discussion

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The major findings of the present study are the following: (1) Pretreatment of rat aortic smooth muscle cells with cAMP-elevating agents reduces basal and SNP-stimulated cGMP accumulation in a time- and dose-dependent manner; (2) reduced cGMP accumulation in these cells results from decreased production of cGMP rather than increased degradation of cGMP, because phosphodiesterase inhibition is unable to restore responsiveness to SNP; (3) PKA mediates the downregulation of SNP-induced cGMP accumulation by cAMP-elevating agents, because the PKA-selective inhibitors partially or fully restored smooth muscle cell responsiveness to SNP; (4) steady-state mRNA levels for both 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

 

HPLC = high-performance liquid chromatography IBMX = isobutylmethylxanthine NO = nitric oxide NOS = nitric oxide synthase PCR = polymerase chain reaction PKA = protein kinase A PMSF = phenylmethylsulfonyl fluoride RT-PCR = reverse transcriptase–polymerase chain reaction sGC = soluble guanylate cyclase SNP = sodium nitroprusside TTBS = Tween 20 in Tris buffered saline

	Acknowledgments

 
This study was supported by National Institutes of Health grant HL-52958. The authors are grateful for the help of Dr Jerry Buccafusco and Jian Wei with the HPLC. We are pleased to acknowledge the expert technical assistance of Connie Snead, Ilona Dienes-Kadi, and Jim Parkerson. We thank Annie Cruz for the preparation of the manuscript.

	Footnotes

 
Reprint requests to Dr John D. Catravas, Vascular Biology Center, Medical College of Georgia, Augusta, GA 30912-2500.

Received March 13, 1995; first decision April 20, 1995; accepted June 14, 1995.

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