Adeno-associated virus vector-mediated interleukin-10 gene transfer inhibits atherosclerosis in apolipoprotein E-deficient mice.

Inflammation is a major contributor to atherosclerosis by its effects on arterial wall biology and lipoprotein metabolism. Interleukin-10 (IL-10) is an anti-inflammatory cytokine that may modulate the atherosclerotic disease process. We investigated the effects of adeno-associated virus (AAV) vector-mediated gene transfer of IL-10 on atherogenesis in apolipoprotein E (ApoE)-deficient mice. A murine myoblast cell line, C2C12, transduced with AAV encoding murine IL-10 (AAV2-mIL10) secreted substantial amounts of IL-10 into conditioned medium. The production of monocyte chemoattractant protein-1 (MCP-1) by the murine macrophage cell line, J774, was significantly inhibited by conditioned medium from AAV2-mIL10-transduced C2C12 cells. ApoE-deficient mice were injected with AAV5-mIL10 into their anterior tibial muscle at 8 weeks of age. The expression of MCP-1 in the vascular wall of the ascending aorta and serum MCP-1 concentration were decreased in AAV5-mIL10-transduced mice compared with AAV5-LacZ-transduced mice. Oil red-O staining of the ascending aorta revealed that IL-10 gene transfer resulted in a 31% reduction in plaque surface area. Serum cholesterol concentrations were also significantly reduced in AAV5-mIL10-transduced mice. To understand the cholesterol-lowering mechanism of IL-10, we measured the cellular cholesterol level in HepG2 cells, resulting in its significant decrease by the addition of IL-10 in a dose-dependent manner. Furthermore, IL-10 suppressed HMG-CoA reductase expression in the HepG2 cells. These observations suggest that intramuscular injection of AAV5-mIL10 into ApoE-deficient mice inhibits atherogenesis through anti-inflammatory and cholesterol-lowering effects.

Macrophage migration inhibitory factor as a redox-sensitive cytokine in cardiac myocytes.

OBJECTIVE: Macrophage migration inhibitory factor (MIF), which plays a pivotal role in the control of inflammatory responses, was first characterized as a T-cell cytokine, but later was also found as a pituitary peptide released in response to infection and stress. However, MIF's role and expression in the myocardium has never been reported. The goal of this study is to examine MIF in the myocardium. METHODS AND RESULTS: MIF protein and mRNA levels were assayed using enzyme-linked immunosorbent assay (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR), respectively. Increased MIF concentrations were detected in the sera of patients with acute myocardial infarction (AMI). In cultured rat cardiac myocytes, significant amounts of MIF were produced in response to hypoxia and hydrogen peroxide (H(2)O(2)), but not to angiotensin II, endothelin-1, interleukin-1beta (IL-1beta) or tumor necrosis factor alpha (TNFalpha). H(2)O(2)-induced MIF production increased in a time- and dose-dependent manner and was completely abolished in the presence of catalase. H(2)O(2) also induced MIF mRNA expression. The H(2)O(2)-induced MIF production was completely inhibited by the protein kinase C (PKC) inhibitor GF109203X, partially inhibited by the tyrosine kinase inhibitor herbimycin A, and uninhibited by calcium chelation or phorbol ester-sensitive PKC down-regulation. This suggests that H(2)O(2)-induced MIF production is mediated by an atypical PKC isoform. DNA microarray analysis revealed that 52 genes were preferentially expressed in response to MIF. Of these, the MIF-induced expression of both glutathione S-transferase (GST) and lipopolysaccharide-induced CXC chemokine (LIX) mRNAs was confirmed using RT-PCR analysis. CONCLUSION: The present results suggest that MIF is expressed by the myocardium in response to redox stress and may play a role in the pathogenesis of myocardial ischemia.

Adeno-associated virus-mediated transfer of endothelial nitric oxide synthase gene inhibits protein synthesis of rat ventricular cardiomyocytes.

We investigated whether nitric oxide (NO) synthase gene transfer could attenuate growth of cultured cardiac myocytes. First, we investigated the effects of exogenous NO and cGMP analog on protein synthesis of cultured neonatal rat cardiac myocytes. The NO donor 3-morpholino-sydnonimine-hydrochloride (SIN-1) and 8-bromo-cGMP caused concentration-dependent decreases in phenylephrine-stimulated incorporation of 3H-leucine into cardiac myocytes. We then transferred endothelial constitutive NO synthase (ecNOS) gene into cultured neonatal rat cardiac myocytes using adeno-associated virus (AAV) vectors. ecNOS gene transfer into cardiac myocytes induced 140 kD ecNOS protein expression and significantly increased cGMP contents of myocytes compared with control cells. ecNOS gene transfer inhibited 3H-leucine incorporation into cardiac myocytes in response to phenylephrine, which was significantly recovered in the presence of the NOS inhibitor N(G)-monomethyl-L-arginine acetate. These results indicate that endogenously generated NO by ecNOS gene transfer using AAV vectors inhibits the alpha-adrenergic agonist-induced cardiac protein synthesis at least partially via cGMP production.

Lipophilic statins augment inducible nitric oxide synthase expression in cytokine-stimulated cardiac myocytes.

Nitric oxide production by inducible nitric oxide synthase (iNOS) may play an important role in the pathogenesis of cardiovascular dysfunction. We investigated the effects of statins on iNOS expression and subsequent nitric oxide synthesis in cardiac myocytes and the mechanism by which statins exert their effects. We measured the production of nitrite, a stable metabolite of nitric oxide, in cultured neonatal rat cardiac myocytes with the Griess reagent. iNOS mRNA and protein expression were assayed by reverse transcription polymerase chain reaction and Western blotting, respectively. The lipophilic statins fluvastatin and lovastatin significantly increased interleukin-1beta-induced nitrite production by cardiac myocytes, whereas hydrophilic pravastatin did not. Increased nitrite production by fluvastatin was accompanied by increased iNOS mRNA and protein accumulation. Exogenous mevalonate, but not squalene, significantly blocked the stimulatory effect of fluvastatin on nitrite production. Cotreatment with geranylgeranyl-pyrophosphate also reversed the effect of fluvastatin. Furthermore, both Rho inhibitor C3 exoenzyme and Rho kinase inhibitor Y-27632 significantly increased interleukin-1beta-induced nitrite accumulation in cardiac myocytes. These results demonstrated that lipophilic statins upregulate iNOS expression and subsequent nitric oxide formation in cardiac myocytes via inhibition of Rho.

Adeno-associated virus-mediated transfer of endothelial nitric oxide synthase gene reduces the vasoconstrictive response.

BACKGROUND: Adeno-associated virus (AAV) has a number of attractive features for gene therapy including the ability to transduce nondividing cells and long term transgene expression. OBJECTIVE: To investigate whether the endothelial constitutive nitric oxide synthase (ecNOS) gene can be efficiently introduced into rat aortic segments using AAV vectors and thereby modulate the vasoconstrictive response. ANIMALS AND METHODS: Excised rat aortas were incubated with medium containing ecNOS-expressing AAV vectors (AAV-ecNOS). Expression of ecNOS in the aortic segments was evaluated by immunohistochemical staining. The isometric tension of the aortic segments transduced with AAV-ecNOS was measured. RESULTS: Adventitial cells in rat aortic segments were efficiently transduced with AAV-ecNOS. The vasoconstrictive response induced by 30 mmol/L K(+) was enhanced in endothelium-denuded aortic segments compared with intact aortic segments. However, in endothelium-denuded aortic segments transduced with AAV-ecNOS, the enhancement of the vasoconstrictive response disappeared. This effect induced by ecNOS gene transfer was abolished in the presence of the nitric oxide synthase inhibitor N(G)-monomethyl-l-arginine acetate. CONCLUSIONS: These results show that ecNOS gene transfer using AAV vectors abolishes the pathological enhancement of the vasoconstrictive response in endothelium-denuded aortic segments.

Serotonin increases interleukin-6 synthesis in human vascular smooth muscle cells.

BACKGROUND: Interleukin-6 (IL-6) is a key molecule in chronic inflammation and has been implicated in the progression of atherosclerosis. Serotonin (5-hydroxytryptamine; 5-HT) causes vascular contraction and proliferation, but its role in atherogenesis has not been clarified. We investigated the effects of 5-HT on IL-6 synthesis in human vascular smooth muscle cells (VSMCs). METHODS AND RESULTS: IL-6 levels in the culture medium of VSMCs were determined by ELISA. IL-6 mRNA accumulation was determined by use of a Quantikine mRNA colorimetric quantification kit. NF-kappaB activation was tested by gel retardation assay. 5-HT induced IL-6 production by VSMCs in a time- and dose-dependent manner, with increased IL-6 mRNA accumulation and nuclear factor-kappaB activation. The effect of 5-HT on IL-6 production was significantly inhibited by the 5-HT(2) receptor antagonist ketanserin and the selective 5-HT(2A) receptor antagonist sarpogrelate. Conversely, the 5-HT(2) receptor agonist alpha-methyl-5-HT increased IL-6 production. The protein kinase C (PKC) inhibitor calphostin C, but not the protein kinase A inhibitor KT5720, suppressed 5-HT-induced IL-6 production. The effect of 5-HT was also abolished in PKC-depleted VSMCs after pretreatment with phorbol 12-myristate 13-acetate for 24 hours. CONCLUSIONS: 5-HT acts on 5-HT(2A) receptors and increases IL-6 synthesis in human VSMCs at least partially through a PKC-dependent pathway. These results suggested that 5-HT may contribute to inflammatory activation of the vessels during atherogenesis.

Amlodipine increases nitric oxide synthesis in cytokine-stimulated cultured vascualar smooth muscle cells.

OBJECTIVE: If calcium channel blockers affect nitric oxide synthesis in the vascular tissue, they could influence disease progression in coronary arteries. We investigated the effects of the calcium channel blocker amlodipine on nitric oxide synthesis by measuring the production of nitrite, a stable metabolite of nitric oxide, in vascular smooth muscle cells. METHODS: We measured the production of nitrate in cultured rat vascular smooth muscle cells with the Griess reagent Inducible nitric oxide synthase protein and mRNA expression were assayed by Western blotting and reverse transcription-polymerase chain reaction, respectively. The levels of NF-kappaB proteins in nuclear extracts were analyzed by gel retardation assay. RESULTS: Incubation of cultures with interleukin-1 , (10 ng/ ml) for 24 h caused a significant increase in nitrite generation. Interleukin-1 l-induced nitrite production by vascular smooth muscle cells was significantly increased by amlodipine in a dose-dependent manner. This augmentative effect of amlodipine was completely abolished in the presence of N(G)-monomethyl-L-arginine or actinomycin D. Amlodipine-induced nitrite production was accompanied by increased inducible nitric oxide synthase mRNA and protein accumulation. Interleukin-1 , induced NF-kappaB activation in vascular smooth muscle cells, and addition of amlodipine further increased this NF-kappaB activation. The effect of amlodipine on nitrite production was maintained in the presence of the calcium channel agonist Bay K 8644. CONCLUSION: Amlodipine enhances nitric oxide synthesis in cytokine-stimulated cultured vascular smooth muscle cells by L-type calcium channel-independent mechanisms.

Adeno-associated virus-mediated vascular endothelial growth factor gene transfer into cardiac myocytes.

Vascular endothelial growth factor (VEGF) is an angiogenic growth factor that stimulates endothelial cell proliferation, increases endothelial permeability, and promotes collateral vessel formation. We transferred human VEGF gene into rat cardiac myocytes using adeno-associated virus (AAV) vectors and investigated whether VEGF secreted from the transduced cardiac myocytes promoted proliferation of endothelial cells. We produced VEGF-expressing AAV vectors (AAV-VEGF) by the adenovirus-free method. Immunoblotting revealed VEGF protein expression in AAV-VEGF-transduced rat cardiac myocytes. More than 60% of cardiac myocytes were stained positively on immunohistochemical staining using anti-VEGF antibody. Concentration of VEGF in the culture medium of AAV-VEGF-transduced myocytes was increased in a vector dose-dependent manner, and VEGF secretion from the transduced myocytes persisted for > or = 14 days. Thymidine incorporation into human vascular endothelial cells was significantly increased by incubation with the conditioned medium from AAV-VEGF-transduced myocytes. This increased thymidine uptake was significantly inhibited by anti-VEGF antibody. We demonstrated here that AAV-mediated VEGF gene transfer into cardiac myocytes induces the secretion of functional VEGF.

Inhibition of vascular endothelial growth factor activity by transfection with the soluble FLT-1 gene.

Vascular endothelial growth factor (VEGF)-induced angiogenesis is involved in the etiology of some cardiovascular diseases. The soluble form of VEGF receptor, FLT-1 (sFLT-1), is a potent antagonist of VEGF. Therefore, we investigated whether transfection with the sFLT-1 gene could inhibit VEGF-induced angiogenesis. Human embryonic kidney (HEK)-293 cells were transfected with plasmids containing VEGF and sFLT-1 (pCMV-VEGF and pCMV-sFLT-1) by the calcium-phosphate co-precipitation method. VEGF- and/or sFLT-1-transfected HEK-293 cells were incubated for 24 h, and then conditioned medium was collected. The effects of conditioned medium on angiogenesis were tested by incorporation of [3H]thymidine into human umbilical vein endothelial cells (HUVECs). Expression of VEGF protein was determined by Western blotting. The conditioned medium from sFLT-1 gene-transfected HEK-293 cells significantly inhibited recombinant VEGF-induced increase in [3H]thymidine incorporation by HUVECs. VEGF gene-transfected HEK-293 cells secreted VEGF protein into conditioned medium. This conditioned medium increased [3H]thymidine incorporation by HUVECs, which was significantly inhibited by co-transfection of sFLT-1 gene with VEGF gene. These observations suggested that sFLT-1 gene transfer could inhibit VEGF-induced DNA synthesis of vascular endothelial cells.

Fluvastatin inhibits matrix metalloproteinase-1 expression in human vascular endothelial cells.

Matrix metalloproteinase-1 (MMP-1), also called interstitial collagenase, may play an important role in the pathogenesis of atherosclerosis and atherosclerotic plaque rupture. We investigated the effects of fluvastatin on MMP-1 expression in human vascular endothelial cells (ECs). The addition of fluvastatin decreased the basal MMP-1 levels in the culture media of ECs in a time-dependent (0 to 48 hours) and dose-dependent (10(-)(8) to 10(-)(5) mol/L) manner. On the other hand, fluvastatin did not affect tissue inhibitor of metalloproteinase-1 levels. Collagenolytic activity in conditioned media of ECs was also dose-dependently reduced by fluvastatin. The effect of fluvastatin on MMP-1 expression was completely reversed in the presence of mevalonate or geranylgeranyl-pyrophosphate, but not in the presence of squalene. Inhibition of Rho by C3 exoenzyme also significantly decreased MMP-1 expression in ECs. Our findings revealed that fluvastatin decreases MMP-1 expression in human vascular ECs through inhibition of Rho.

Gene transfer into rat renal cells using adeno-associated virus vectors.

Adeno-associated virus (AAV) vectors have a number of attractive features, including lack of cytotoxicity, ability to transduce nondividing cells, and long-term transgene expression. We investigated whether rat renal cells could be efficiently transduced with AAV vectors. Rat glomerular mesangial cells were transduced with AAV-lacZ vector containing beta-galactosidase gene in vitro, and the expression of beta-galactosidase was evaluated by X-gal staining and ELISA. For ex vivo experiments, sections of rat kidneys were incubated with AAV-lacZ, and then evaluated by X-gal histochemical staining. The level of beta-galactosidase expression in cultured rat mesangial cells increased in a dose-dependent manner (ranging from 1 x 10(5) to 5 x 10(6) particles/cell). When transduced with 5 x 10(6) vector particles/cell of AAV-lacZ, about 50% of mesangial cells were stained positively with X-gal, and the level of beta-galactosidase expression reached 9.9 +/- 1.5 ng/mg protein. Expression was detectable during the culture period for at least 7 days. X-gal histochemical examination of the ex vivo transduced renal tissue revealed tubular cell and interstitial tissue staining. However, gene transfer was not clearly observed in glomeruli. These findings suggest that AAV vectors have the potential for gene therapy of renal diseases.

Peroxisome proliferator-activated receptor-gamma ligands inhibit nitric oxide synthesis in vascular smooth muscle cells.

Peroxisome proliferator-activated receptor-gamma (PPARgamma) is a key player in glucose metabolism. If PPARgamma ligands modulate nitric oxide (NO) synthesis in the vascular tissue, they may affect the process of plaque formation and postangioplasty restenosis. We investigated the effects of PPARgamma ligands on NO synthesis in vascular smooth muscle cells. Incubation of cultures with interleukin-1beta (10 ng/mL) for 24 hours caused a significant increase in the production of nitrite, a stable metabolite of NO, in cultured rat vascular smooth muscle cells. The PPARgamma agonists troglitazone and 15-deoxy-triangle up(12,14)-prostaglandin J(2) (15d-PG J(2)) dose-dependently inhibited nitrite production by interleukin-1beta-stimulated vascular smooth muscle cells. Decreased interleukin-1beta-induced nitrite production by the PPARgamma agonists was accompanied by decreased inducible NO synthase mRNA and protein accumulation. Interleukin-1beta induced nuclear factor-kappaB activation in vascular smooth muscle cells, and both troglitazone and 15d-PG J(2) markedly suppressed this nuclear factor-kappaB activation. PPARgamma ligands inhibit NO synthesis in cytokine-stimulated vascular smooth muscle cells, suggesting that these agonists may act directly on the vascular smooth muscle and influence the process of atherosclerosis and restenosis.

Effects of aspirin-like drugs on nitric oxide synthesis in rat vascular smooth muscle cells.

The purpose of this study was to investigate the effects of aspirin-like drugs on nitric oxide (NO) synthesis in rat vascular smooth muscle cells (VSMCs). We measured the accumulation of nitrite, a stable oxidation product of NO, and the expression of inducible NO synthase (iNOS) mRNA and protein in rat cultured VSMCs. Sodium salicylate, aspirin, and indomethacin dose-dependently enhanced nitrite production by interleukin (IL)-1beta-stimulated VSMCs at therapeutic plasma concentration ranges. Increased nitrite production by aspirin-like drugs was accompanied by increased iNOS mRNA and protein accumulation in VSMCs. Addition of IL-1beta activated nuclear factor kappaB (NF-kappaB) in VSMCs, but sodium salicylate did not affect IL-1beta-induced NF-kappaB activation. The nonselective lipoxygenase (LO) inhibitor nordihydroguaiaretic acid inhibited sodium salicylate-induced nitrite production, whereas the selective 5-LO inhibitor caffeic acid did not influence production of nitrite. The 12-LO product 12-HETE dose-dependently enhanced nitrite production by IL-1beta-stimulated VSMCs, whereas the 15-LO product 15-HETE did not. Our study demonstrates that aspirin and the aspirin-like drugs, sodium salicylate and indomethacin, increase NO synthesis in IL-1beta-stimulated VSMCs by upregulation of iNOS transcription via a 12-LO pathway. These effects were independent of NF-kappaB activation. In addition to the direct inhibition of platelet function, aspirin-like drugs may contribute to the reduction of atherothrombotic risk in myocardial ischemia via enhancing NO production by VSMCs.

Direct effects of statins on cells primarily involved in atherosclerosis.

Statins are lipid-lowering agents which act by inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase. This enzyme is responsible for the conversion of HMG-CoA to mevalonate. Products of mevalonate metabolism are critical for several cellular processes of eukaryotic cells, and inhibition of the mevalonate pathway by statins has pleiotropic effects. It has been reported that statins inhibit the migration and proliferation of vascular smooth cells (VSMCs) and macrophages, decrease interleukin-6 and inducible nitric oxide synthase expression in VSMCs, improve endothelial function and up-regulate endothelial nitric oxide synthase expression. The above effects of statins are independent of plasma cholesterol levels, and are completely blocked by exogenous mevalonate and some isoprenoids. These findings suggest that, in addition to their effects on plasma lipids, statins exert direct antiatherosclerotic effects on the cells primarily involved in atherosclerosis.

Fluvastatin upregulates inducible nitric oxide synthase expression in cytokine-stimulated vascular smooth muscle cells.

Nitric oxide (NO) production by inducible NO synthase (iNOS) may play an important role in the pathogenesis of atherosclerosis. Although fluvastatin has been shown to reduce progression of atherosclerosis, it is not known whether it regulates iNOS expression. We investigated the effects of fluvastatin on iNOS expression and subsequent NO synthesis in vascular smooth muscle cells (VSMCs) and the mechanism by which fluvastatin exerts its effects. Fluvastatin significantly increased interleukin-1ss (IL-1ss)-induced nitrite production by VSMCs in a time-dependent (0 to 24 hours) and dose-dependent (10(-)(8) to 10(-)(5) mol/L) manner. Increased nitrite production by fluvastatin was accompanied by increased iNOS mRNA and protein accumulation. IL-1ss induced nuclear factor-kappaB activation in VSMCs, which was not affected by fluvastatin. Exogenous mevalonate significantly prevented the stimulatory effect of fluvastatin on nitrite production. Cotreatment with geranylgeranyl-pyrophosphate also reversed the effect of fluvastatin. Furthermore, both Rho inhibitor C3 exoenzyme and Rho kinase inhibitor Y-27632 significantly increased IL-1ss-induced nitrite accumulation in VSMCs. These results demonstrated that fluvastatin upregulates iNOS expression and subsequent NO formation in rat VSMCs through inhibition of Rho.

Endogenously generated nitric oxide by nitric-oxide synthase gene transfer inhibits cellular proliferation.

We investigated whether endothelial nitrite oxide synthase (NOS) gene transfer inhibited cellular proliferation. Endothelial NOS and endothelin type A receptor genes were transferred into 293 cells, a human embryonic kidney cell line, by calcium-phosphate coprecipitation. The cytosolic free Ca(2+) levels ([Ca(2+)](i)) of transfected cells were estimated with fura-2 fluorescence. Thymidine incorporation was increased by endothelin-1 in type A receptor-transfected cells. The endothelial NOS gene transfer did not affect endothelin-1-induced increase in [Ca(2+)](i) of type A receptor-transfected cells, but markedly inhibited mitogen-activated protein kinase and c-fos promoter activities. The endothelial NOS gene transfer also inhibited thymidine incorporation into type A receptor-transfected cells in response to endothelin-1, which was abolished in the presence of the NOS inhibitor N(G)-monomethyl-L-arginine acetate. The endothelin-1-induced increase in cell number was significantly suppressed by endothelial NOS gene transfer as well as by the mitogen-activated protein kinase inhibitor PD98059. These results indicate that endothelial NOS gene transfer inhibits cellular proliferation via inhibition of the mitogen-activated protein kinase cascade.

Efficient gene transfer into cardiac myocytes using adeno-associated virus (AAV) vectors.

Adeno-associated virus (AAV) vectors, derived from a non-pathogenic parvovirus, are considered to be an appropriate gene transfer vehicle for both dividing and non-dividing cells. In the present study, we investigated whether the rat heart could be efficiently transduced with AAV vectors. Rat cardiac myocytes (CM) were infected with AAV-lacZ vector containing beta-galactosidase (beta-gal) gene in vitro, and the expression of beta-gal in CM was evaluated by X-gal staining and beta-gal ELISA. With increasing multiplicities of infection (MOI), more than 60% of CM were stained positively with X-gal, and the beta-gal expression increased to 31.1 +/- 4.6 ng/mg protein in a MOI-dependent manner (MOI: 10(4) to 10(6) particles/cell). The beta-gal expression was also increased in an incubation period-dependent manner (1-24 h). beta-gal expression was maximal at day 3 and then gradually decreased with time. However, beta-gal expression at day 14 was almost the same level as that at day 1 (45.5 +/- 5.9 v 55.2 +/- 6.2 ng/mg protein). Excised rat right ventricular papillary muscles were also infected with AAV-lacZ ex vivo. When the beta-gal expression was evaluated by X-gal staining, more than 80% of CM in the papillary muscles were stained positively, indicating efficient gene transfer into CM using AAV vectors. These findings suggest that AAV vectors are promising for cardiac gene therapy.

Effects of vesnarinone on nitric oxide synthesis in rat cardiac myocytes.

OBJECTIVE: The purpose of this study was to investigate the effects of vesnarinone on nitric oxide (NO) synthesis in cardiac myocytes. METHODS: We measured the accumulation of nitrite, a stable oxidation product of NO synthase (iNOS) protein in cultured neonatal rat cardiac myocytes. RESULTS: Incubation of the cultures with interleukin-1 beta (IL-1 beta; 10 ng/ml) and tumor necrosis factor alpha (TNF alpha; 10 ng/ml) caused a marked increase in nitrite production. Although vesnarinone by itself showed no effect on nitrite accumulation, it enhanced cytokine-induced nitrite production by cardiac myocytes in a dose-dependent manner. The effect of vesnarinone was completely abolished in the presence of NG-monomethyl-L-arginine or actinomycin D. The vesnarinone-induced nitrite production was accompanied by increased iNOS protein expression. In the presence of dibutyryl-cAMP, cytokine-induced nitrite accumulation was further increased, but the stimulatory effect on vesnarinone on nitrite accumulation was diminished. The effect of vesnarinone was also inhibited by Rp-8-Br-cAMPS, a competitive inhibitor of protein kinase A, in a dose-dependent manner. CONCLUSIONS: These findings indicate that vesnarinone increases NO synthesis in cytokine-stimulated cardiac myocytes, at least partially through a cAMP-dependent pathway.

Adenosine stimulates nitric oxide synthesis in rat cardiac myocytes.

We investigated the effects of adenosine on nitric oxide (NO) synthesis by measuring the production of nitrite, a stable metabolite of NO, in cultured neonatal rat cardiac myocytes. Incubation of cultures with interleukin-1 beta (10 ng/ml) for 24 h caused a significant increase in nitrite production. The interleukin-1 beta-induced nitrite production by cardiac myocytes was significantly increased by adenosine or its stable analog 2-chloroadenosine in a dose-dependent manner (10(-7)-10(-4) M). The adenosine A2-receptor antagonist KF-17837 (10(-6) M), but not the A1-receptor antagonist 8-cyclopentyl-1, 3-dipropylxanthine (10(-6) M), significantly inhibited 2-chloroadenosine-mediated nitrite production. The 2-chloroadenosine-induced nitrite production by interleukin-1 beta-stimulated cells was accompanied by inducible NO synthase mRNA and protein accumulation. In the presence of N6, 2'-O-dibutyryladenosine 3',5'-cyclic monophosphate (cAMP) (10(-3) M) or isoproterenol (10(-5) M), interleukin-1 beta-induced nitrite accumulation was further increased, but the effect of 2-chloroadenosine was not additive or synergistic. The protein kinase C inhibitor calphostin C did not inhibit the effect of 2-chloroadenosine. These results indicate that adenosine acts on A2 receptors and augments NO synthesis in interleukin-1 beta-stimulated cardiac myocytes, at least partially through a cAMP-dependent pathway.