Smooth Muscle Cell Expression of Type I Cyclic GMP–dependent Protein Kinase Is Suppressed by Continuous Exposure to Nitrovasodilators, Theophylline, Cyclic GMP, and Cyclic AMP Gerald A. Soff,* Trudy L. Cornwell,‡ Deborah L. Cundiff,* Stephen Gately,* and Thomas M. Lincoln‡ *Department of Medicine, Division of Hematology/Oncology, Northwestern University Medical School, Chicago, Illinois 60611; and ‡Department of Pathology, Division of Molecular and Cellular Pathology, University of Alabama at Birmingham, Birmingham, Alabama 35294 Abstract Introduction A key component of the nitric oxide–cyclic guanosine mono- The nitric oxide (NO)–cGMP1 pathway is a key regulator of phosphate (cGMP) pathway in smooth muscle cells (SMC) vascular tone (1–4). In smooth muscle cells (SMC), NO, also is the type I GMP-dependent protein kinase (PK-G I). Acti- identified as the endothelium-derived relaxing factor, activates vation of PK-G I mediates the reduction of cytoplasmic cal- soluble guanylyl cyclase, leading to an increase in levels of cium concentrations and vasorelaxation. In this manu- cGMP. Atrial natriuretic factor, another mediator of vasodila- script, we demonstrate that continuous exposure of SMC in tion, activates a membrane-bound guanylyl cyclase, also in- culture to the nitrovasodilators S-nitroso-N-acetylpenicil- creasing cGMP levels. cGMP activates a cGMP-dependent lamine (SNAP) or sodium nitroprusside (SNP) results in protein kinase (PK-G), which mediates the vasorelaxant effect z 75% suppression of PK-G I mRNA by 48 h. PK-G I mRNA by phosphorylation of several proteins that regulate intracellu- and protein were also suppressed by continuous exposure to lar Ca21 levels (2, 5–7). As a result, calcium is transported to cGMP analogues 8-bromo- and 8-(4-chlorophenylthio) gua- the endoplasmic and sarcoplasmic reticulum and extruded nosine-3,5-monophosphate or the cAMP analogue dibutyryl from the cell, thus reducing cytoplasmic calcium concentra- cAMP. These results suggest that activation of one or both tions and effecting vasorelaxation. Recent studies indicate that of the cyclic nucleotide–dependent protein kinases mediates PK-G may also catalyze the phosphorylation of the inositol PK-G I mRNA suppression. Using isoform-specific cDNA 1,4,5-triphosphate receptor and, therefore, regulate the mobili- probes, only the PK-G Ia was detected in SMC, either at zation of calcium stores by inositol triphosphate (8, 9). Addi- baseline or after suppression, while PK-G Ib was not de- tional data suggest that NO-induced vasorelaxation may be tected, indicating that isoform switch was not contributing mediated in part by both cGMP-dependent and cGMP-inde- to the gene regulation. Using the transcription inhibitor acti- pendent activation of a K1 channel (10, 11). nomycin D, the PK-G I mRNA half-life in bovine SMC was Two genes encoding mammalian PK-G have been identi- observed to be 5 h. The half-life was not affected by the ad- fied, type I and type II. Type I PK-G exists as two isoforms re- dition of SNAP to actinomycin D, indicating no effect on ferred to as type Ia and type Ib (2, 7, 12, 13). The PK-G Ia and PK-G I mRNA stability. Nuclear runoff studies indicated a Ib isoforms differ only in the initial coding region (89 amino suppression of PK-G I gene transcription by SNAP. PK-G I acids for Ia and 104 for Ib) (12, 13). Although the PK-G I iso- suppression was also observed in vivo in rats given isosor- forms have identical substrate specificity, the Ia isoform re- bide dinitrate in the drinking water, with a dose-dependent quires one-tenth the cGMP concentration for half-maximal ac- suppression of PK-G I protein in the aorta. PK-G I antigen tivity as the Ib isoform (7). In a recent study, the PK-G Ia in whole rat lung extract was also suppressed by adminis- isoform was found in the aorta, heart, kidneys, adrenal glands, tration of isosorbide or theophylline in the drinking water. cerebellum, and lung tissues (14). The PK-G Ib isoform was These data may contribute to our understanding of nitrova- only detected abundantly in the uterus (14). The type II PK-G sodilator resistance, a phenomenon resulting from continu- has been identified in brain and intestinal epithelium (15–17). ous exposure to nitroglycerin or other nitrovasodilators. (J. While much has been learned about the regulation of the Clin. Invest. 1997. 100:2580–2587.) Key words: cyclic GMP– genes for NO synthase (3), little is known about the regulation dependent protein kinases • cyclic GMP • vascular smooth of the gene for PK-G I, despite its critical role for SMC func- muscle • nitroglycerin • drug tolerance tion. Our recent studies have described the growth–state de- pendence of PK-G I expression in SMC in culture (18). We re- ported that PK-G I expression was reduced in subconfluent cultures of low passage aortic SMC and was increased at high Address correspondence to Gerald A. Soff, M.D., Northwestern Uni- density. These results are similar to other findings regarding versity Medical School, 320 East Superior St., Searle 3-565, Chicago, the expression of smooth muscle–specific proteins (19–22). In IL 60611. Phone: 312-503-5132; FAX: 312-503-8104; E-mail: gasoff@ a recent study, PK-G Ia was found to be suppressed by PDGF- merle.acns.nwu.edu Received for publication 30 September 1996 and accepted in re- vised form 20 September 1997. 1. Abbreviations used in this paper: 8-Br-cGMP, 8-bromo-guanosine- 3,5-monophosphate; 8-CPT-cGMP, 8-(4-chlorophenylthio) guano- J. Clin. Invest. sine-3,5-monophosphate; DB-cAMP, dibutyryl cAMP; GAPDH, © The American Society for Clinical Investigation, Inc. glyceraldehyde phosphate dehydrogenase; NO, nitric oxide; PK-A, 0021-9738/97/11/2580/08 $2.00 protein kinase A; PK-G I, type I cGMP–dependent protein kinase; Volume 100, Number 10, November 1997, 2580–2587 SMC, smooth muscle cells; SNAP, S-nitroso-N-acetylpenicillamine; http://www.jci.org SNP, sodium nitroprusside; TBS, Tris-buffered saline. 2580 Soff et al. BB (14). In this study, suppression of PK-G I mRNA and pro- phorimager. Background counts were subtracted from a region of tein has been demonstrated in vascular SMC in vitro and in control DNA (from herring sperm); these counts were equivalent to vivo in response to continuous exposure to nitrovasodilators, counts obtained at areas of the membrane without DNA loading. theophylline, or analogues of cAMP and cGMP. These find- Immunological quantitation of PK-G I from tissue culture. Primary ings suggest that one mechanism that regulates PK-G I gene cultures of rat aortic SMC were plated in 35-mm culture dishes and treated with 8-CPT-cGMP. PK-G I levels were determined from expression is the activation of one or both cyclic nucleotide– crude soluble fractions as reported previously (18). Samples (2 or 5 dependent protein kinases. mg total protein in 0.5 ml) were diluted in Tris-buffered saline (TBS, 20 mM Tris-HCl, pH 7.5, 137 mM NaCl) and applied in triplicate to nitrocellulose membrane in wells of a slot-blot apparatus. Several Methods concentrations of purified bovine lung PK-G Ia in TBS containing 10 mg/ml BSA were also applied to separate wells to construct a stan- Materials. S-nitroso-N-acetylpenicillamine (SNAP) was from Calbio- dard curve of band density (area units) versus nanograms of PK-G I chem Corp. (San Diego, CA). Sodium nitroprusside (SNP), isosor- protein. The primary antibody was affinity-purified rabbit anti–bovine bide dinitrate, 8-bromo-cGMP (8-Br-cGMP), and theophylline were PK-G I (1/100), and the secondary antibody was donkey anti–rabbit obtained from Sigma Chemical Co. (St. Louis, MO). 8-(4-chlorophen- IgG conjugated to horseradish peroxidase (1/10,000). After enhanced ylthio) guanosine-3,5-monophosphate (8-CPT-cGMP) was from Bio- chemiluminescence, data were analyzed by densitometry. Values Log, Inc. (La Jolla, CA). NG-nitro-L-arginine methyl ester, hydro- have been expressed as nanograms of PK-G I per milligram of total chloride (L-NAME) was obtained from Calbiochem Corp. All tissue protein. culture reagents were from Life Technologies, Inc. (Gaithersburg, MD). Animal studies. Male rats of z 400 g, were given water supple- Preparation and culture of SMC. Primary bovine aortic SMC were mented with various doses of isosorbide dinitrate or theophylline for isolated by a modification of the method of Gunther et al. (18, 23, 24). 4 d. The water was changed daily, and the volume of water consumed Rat aortic SMC were prepared from thoracic and abdominal aortae was recorded. The actual dose of drug consumed per animal was cal- of Sprague-Dawley rats by a previously modified protocol of Smith culated based on volume of water consumed, as milligrams of isosor- and Brock (18, 25). Bovine and rat SMC were maintained in a stan- bide dinitrate or theophylline per gram of animal body weight per 24 h. dard humidified 5% CO2 tissue culture incubator in DME and used Control animals were handled in a similar fashion, and were given un- within the first four passages. Bovine SMC were cultured with 20% treated water. Animals were killed on the fourth day by CO2 inhala- calf serum, and rat SMC were cultured with 10% FBS. In all experi- tion. Aortas were excised, then quickly snap-frozen at 2858C. Tissue ments in which the SMC were treated with test compounds (i.e., ni- lysates were prepared by homogenization in 0.5 ml 20 mM sodium trovasodilators, cyclic nucleotides, etc.), the media and test reagents phosphate, pH 6.8, 2 mM EDTA, 0.15 M NaCl, and 0.1 mM PMSF us- were replaced every 24 h with fresh test media or control media for ing an Ultraturax polytron (Tekmar Co., Cincinnati, OH) set at 50%.