Biomedical subjects

W Durante

Publications and source records attributed to W Durante.

At least 19 recordsLinked to original sources

PubMed2005

Local administration of carbon monoxide inhibits neointima formation in balloon injured rat carotid arteries.

Recent studies indicate that systemic induction of heme oxygenase-1 (HO-1), which oxidatively degrades heme into iron, biliverdin, and carbon monoxide (CO), or adenoviral-mediated gene transfer of HO-1 inhibits neointima formation after experimental vascular injury. In the present study, we investigated whether the acute, local administration of the HO-1 product, CO, regulates the arterial remodeling response following injury. Immediately after balloon injury of rat carotid arteries, a saturated solution of CO or nitrogen (N2), or phosphate buffered saline (PBS) was incubated luminally within the injured vessels for 30 min. Two weeks after injury, arteries exposed to CO exhibited significantly reduced neointimal area, neointimal area/medial wall area ratio, neointimal thickness, and medial wall area compared to arteries exposed to N2 or PBS. Arteries exposed to CO also demonstrated significantly reduced DNA synthesis in the medial wall two days after injury as suggested by proliferating cell nuclear antigen immunostaining, and this was associated with a decrease in the protein expression of the G1 cyclins, cyclin E and A, and transforming growth factor-beta1. These results indicate that the acute, local delivery of CO blocks the pathophysiological remodeling response to vascular injury, and identifies CO as a potentially important therapeutic agent in the treatment of vasculoproliferative disease.

Animals↗

PubMed2002

Detection of biogenic CO production above vascular cell cultures using a near-room-temperature QC-DFB laser.

We report the first application of pulsed, near-room-temperature quantum cascade laser technology to the continuous detection of biogenic CO production rates above viable cultures of vascular smooth muscle cells. A computer-controlled sequence of measurements over a 9-h period was obtained, resulting in a minimum detectable CO production of 20 ppb in a 1-m optical path above a standard cell-culture flask. Data-processing procedures for real-time monitoring of both biogenic and ambient atmospheric CO concentrations are described.

Air Conditioning↗

PubMed2001

Transforming growth factor-beta 1 stimulates vascular smooth muscle cell L-proline transport by inducing system A amino acid transporter 2 (SAT2) gene expression.

Transforming growth factor-beta1 (TGF-beta 1) is a multifunctional cytokine that contributes to arterial remodelling by stimulating vascular smooth muscle cell (SMC) growth and collagen synthesis at sites of vascular injury. Since l-proline is essential for the synthesis of collagen, we examined whether TGF-beta 1 regulates the transcellular transport of l-proline by vascular SMCs. l-Proline uptake by vascular SMCs was primarily sodium-dependent, pH-sensitive, blocked by neutral amino acids and alpha-(methylamino)isobutyric acid, and exhibited trans-inhibition. Treatment of SMCs with TGF-beta 1 stimulated l-proline transport in a concentration- and time-dependent manner. The TGF-beta 1-mediated l-proline uptake was inhibited by cycloheximide or actinomycin D. Kinetic studies indicated that TGF-beta 1-induced l-proline transport was mediated by an increase in transport capacity independent of any changes in the affinity for l-proline. TGF-beta 1 stimulated the expression of system A amino acid transporter 2 (SAT2) mRNA in a time-dependent fashion that paralleled the increase in l-proline transport. Reverse transcriptase PCR failed to detect the presence of SAT1 or amino acid transporter 3 (ATA3) in either untreated or TGF-beta 1-treated SMCs. These results demonstrate that l-proline transport by vascular SMCs is mediated predominantly by the SAT and that TGF-beta 1 stimulates SMC l-proline uptake by inducing the expression of the SAT2 gene. The ability of TGF-beta 1 to induce SAT2 expression may function to provide SMCs with the necessary levels of l-proline required for collagen synthesis and cell growth.

Amino Acid Transport System A↗

PubMed2001

Adenovirus-mediated heme oxygenase-1 gene delivery inhibits injury-induced vascular neointima formation.

BACKGROUND: Recent studies have demonstrated that systemic pharmacological induction of heme oxygenase-1 (HO-1), the inducible isoform of the initial and rate-limiting enzyme for heme catabolism, attenuates neointima formation after experimental vascular injury. We have now investigated the ability of localized adenovirus-mediated HO-1 (Ad-HO-1) gene delivery to modify arterial remodeling after balloon angioplasty. METHODS AND RESULTS: Two weeks after balloon angioplasty in the rat carotid artery, elevated HO-1 protein was observed in the Ad-HO-1 arteries compared with those exposed to empty adenovirus (Ad-E) or to PBS. The arteries exposed to Ad-HO-1 exhibited significantly reduced neointimal area, medial wall area, neointimal area/medial wall area ratio, and neointimal thickness compared with arteries exposed to Ad-E. The Ad-E vessels showed subtle reductions in each morphometric parameter compared with PBS vessels. In a separate group of animals, concomitant treatment of Ad-HO-1 with the HO-1 inhibitor tin protoporphyrin completely restored each morphometric parameter to control levels. Arteries exposed to Ad-HO-1 demonstrated significantly increased TUNEL labeling of apoptotic nuclei and significantly decreased PCNA labeling of DNA synthesis in the medial wall 48 hours after injury. CONCLUSIONS: These results indicate that HO-1 represents an important in vivo vasoprotective mediator that is capable of attenuating the pathophysiological remodeling response to endovascular injury and suggest that HO-1 may be a novel target for the treatment of vascular disease.

Adenoviridae↗

PubMed2001

Transforming growth factor-beta(1) stimulates L-arginine transport and metabolism in vascular smooth muscle cells: role in polyamine and collagen synthesis.

BACKGROUND: Transforming growth factor-beta(1) (TGF-beta(1)) contributes to arterial remodeling by stimulating vascular smooth muscle cell (VSMC) growth and collagen synthesis at sites of vascular injury. Because L-arginine is metabolized to growth-stimulatory polyamines and to the essential collagen precursor L-proline, we examined whether TGF-beta(1) regulates the transcellular transport and metabolism of L-arginine by VSMCs. METHODS AND RESULTS: TGF-beta(1) increased L-arginine uptake, and this was associated with a selective increase in cationic amino acid transporter-1 (CAT-1) mRNA. In addition, TGF-beta(1) stimulated L-arginine metabolism by inducing arginase I mRNA and arginase activity. TGF-beta(1) also stimulated L-ornithine catabolism by elevating ornithine decarboxylase (ODC) and ornithine aminotransferase (OAT) activity. TGF-beta(1) markedly increased the capacity of VSMCs to generate the polyamine putrescine and L-proline from extracellular L-arginine. The TGF-beta(1)-mediated increase in putrescine and L-proline production was reversed by methyl-L-arginine, a competitive inhibitor of cationic amino acid transport, or by hydroxy-L-arginine, an arginase inhibitor. Furthermore, the formation of putrescine was inhibited by the ODC inhibitor alpha-difluoromethylornithine, and L-proline generation was blocked by the OAT inhibitor L-canaline. L-Canaline also inhibited TGF-beta(1)-stimulated type I collagen synthesis. CONCLUSIONS: These results demonstrate that TGF-beta(1) stimulates polyamine and L-proline synthesis by inducing the genes that regulate the transport and metabolism of L-arginine. In addition, they show that TGF-beta(1)-stimulated collagen production is dependent on L-proline formation. The ability of TGF-beta(1) to upregulate L-arginine transport and direct its metabolism to polyamines and L-proline may contribute to arterial remodeling at sites of vascular damage.

Animals↗

PubMed2001

Heme oxygenase-1 attenuates vascular remodeling following balloon injury in rat carotid arteries.

The heme oxygenase-1 (HO-1) system of heme catabolism has been proposed to exert protective actions upon the cardiovascular system. This investigation examined the influence of HO-1 induction on vascular remodeling following arterial injury. Rats were subjected to left carotid artery (LCA) balloon injury following pre-treatment with either vehicle, the HO-1 inducer hemin (50 mg/kg, SC), or concomitant treatment with hemin and the HO-1 inhibitor tin-protoporphyrin IX (SnPP-IX; 50 micromol/kg, IP). Animals were injected daily for 14 days post-injury, after which animals were sacrificed and tissues obtained. Western blot analyses revealed vascular HO-1 induction after 2 and 16 days of hemin treatment. Positive immunostaining for HO-1 was detected in the endothelial and adventitial layers following 48 h of hemin treatment and positive medial staining for HO-1 after 16 days of hemin treatment. The injured LCA of hemin-treated animals demonstrated significantly attenuated neointimal (NI) area (-57%), NI thickness (-58%), and NI area/medial wall area ratio (-40%) compared to the injured LCA of vehicle controls. The cross-sectional medial wall areas of both LCA and uninjured RCA were also significantly reduced in the hemin-treated animals. SnPP-IX treatment, however, completely restored the NI area, NI thickness, NI area/medial wall area ratio, and partially restored the medial wall area towards control levels. These results directly implicate HO-1 and the products of heme catabolism in attenuating the arterial response to injury and ensuing vascular wall remodeling.

Animals↗

PubMed2001

Real-time measurements of endogenous CO production from vascular cells using an ultrasensitive laser sensor.

Carbon monoxide (CO) has been implicated as a biological messenger molecule analogous to nitric oxide. A compact gas sensor based on a midinfrared laser absorption spectroscopy was developed for direct and real-time measurement of trace levels (in approximate pmol) of CO release by vascular cells. The midinfrared light is generated by difference frequency mixing of two nearinfrared lasers in a nonlinear optical crystal. A strong infrared absorption line of CO (4.61 microm) is chosen for convenient CO detection without interference from other gas species. The generation of CO from cultured vascular smooth muscle cells was detected every 20 s without any chemical modification to the CO. The sensitivity of the sensor reached 6.9 pmol CO. CO synthesis was measured from untreated control cells (0.25 nmol per 10(7) cells/h), sodium nitroprusside-treated cells (0.29 nmol per 10(7) cells/h), and hemin-treated cells (0.49 nmol per 10(7) cells/h). The sensor also detected decreases in CO production after the addition of the heme oxygenase (HO) inhibitor tin protoporphyrin-IX (from 0.49 to 0.02 nmol per 10(7) cells/h) and increases after the administration of the HO substrate hemin (from 0.27 to 0.64 nmol per 10(7) cells/h). These results demonstrate that midinfrared laser absorption spectroscopy is a useful technique for the noninvasive and real-time detection of trace levels of CO from biological tissues.

Animals↗

PubMed2001

Regulation of L-arginine transport and metabolism in vascular smooth muscle cells.

L-Arginine is a semi-essential amino acid that is metabolized to important regulatory molecules. L-Arginine is transported into vascular smooth muscle cells (SMC) by the cationic amino acid transporter (CAT) family of proteins where it is metabolized to nitric oxide (NO), polyamines, or L-proline. Inflammatory mediators, growth factors, and hemodynamic forces stimulate the transport of L-arginine in vascular SMC by inducing CAT gene expression. However, they exert highly specific and divergent regulatory effects on L-arginine metabolism. Inflammatory cytokines induce the expression of inducible NO synthase (iNOS) and direct the metabolism of L-arginine to the antiproliferative gas, NO. In contrast, growth factors stimulate the expression of arginase I and ornithine decarboxylase (ODC) and channel the metabolism of L-arginine to growth stimulatory polyamines. Alternatively, cyclic mechanical strain blocks both iNOS and ODC activity and stimulates arginase I gene expression, directing the metabolism of L-arginine to the formation of L-proline and collagen. Thus, specific biochemical and biophysical stimuli that are found in the circulation regulate the transport and metabolism of L-arginine in vascular SMC. The ability of these physiologically relevant stimuli to upregulate L-arginine transport and generate specific L-arginine metabolites modulates SMC function and may influence the development of vascular disease.

Animals↗

PubMed2000

YC-1, a benzyl indazole derivative, stimulates vascular cGMP and inhibits neointima formation.

The pathobiologic process of arterial stenosis following balloon angioplasty continues to be an enigmatic problem in clinical settings. This research project investigates the ability of YC-1, a benzyl indazole derivative that sensitizes sGC/cGMP, to stimulate endogenous cGMP and attenuate balloon injury-induced neointima (NI) formation in the rat carotid artery. Northern and Western blot analyses revealed enhanced acute expression of iNOS and inducible heme oxygenase (HO-1) mRNA and protein in the injured artery. The contralateral uninjured artery also demonstrated acute HO-1 mRNA and protein induction without detectable iNOS expression. Perivascular application of YC-1 immediately following injury significantly stimulated acute vessel wall cGMP compared to untreated controls. YC-1 treated sections demonstrated significant reduction in NI area (-74%), NI area/medial wall area (-72%), and NI thickness (-76%) 2 weeks post-injury. These results directly implicate YC-1 as a potent new therapeutic agent capable of reducing post-angioplasty stenosis through endogenous CO- and/or NO-mediated, cGMP-dependent processes.

Angioplasty, Balloon↗

PubMed2000

Physiological cyclic stretch directs L-arginine transport and metabolism to collagen synthesis in vascular smooth muscle.

Application of cyclic stretch (10% at 1 hertz) to vascular smooth muscle cells (SMC) increased L-arginine uptake and this was associated with a specific increase in cationic amino acid transporter-2 (CAT-2) mRNA. In addition, cyclic stretch stimulated L-arginine metabolism by inducing arginase I mRNA and arginase activity. In contrast, cyclic stretch inhibited the catabolism of L-arginine to nitric oxide (NO) by blocking inducible NO synthase expression. Exposure of SMC to cyclic stretch markedly increased the capacity of SMC to generate L-proline from L-arginine while inhibiting the formation of polyamines. The stretch-mediated increase in L-proline production was reversed by methyl-L-arginine, a competitive inhibitor of L-arginine transport, by hydroxy-L-arginine, an arginase inhibitor, or by the ornithine aminotransferase inhibitor L-canaline. Finally, cyclic stretch stimulated collagen synthesis and the accumulation of type I collagen, which was inhibited by L-canaline. These results demonstrate that cyclic stretch coordinately stimulates L-proline synthesis by regulating the genes that modulate the transport and metabolism of L-arginine. In addition, they show that stretch-stimulated collagen production is dependent on L-proline formation. The ability of hemodynamic forces to up-regulate L-arginine transport and direct its metabolism to L-proline may play an important role in stabilizing vascular lesions by promoting SMC collagen synthesis.

Amino Acid Transport Systems, Basic↗

PubMed2000

Physiological cyclic stretch causes cell cycle arrest in cultured vascular smooth muscle cells.

Smooth muscle cells (SMC) are the major cellular component of the blood vessel wall and are continuously exposed to cyclic stretch due to pulsatile blood flow. This study examined the effects of a physiologically relevant level of cyclic stretch on rat aortic vascular SMC proliferation. Treatment of static SMC with serum, platelet-derived growth factor, or thrombin stimulated SMC proliferation, whereas exposure of SMC to cyclic stretch blocked the proliferative effect of these growth factors. The stretch-mediated inhibition in SMC growth was not due to cell detachment or increased cell death. Flow cytometry analysis revealed that cyclic stretch increased the fraction of SMC in the G(0)/G(1) phase of the cell cycle. Stretch-inhibited G(1)/S phase transition was associated with a decrease in retinoblastoma protein phosphorylation and with a selective increase in the cyclin-dependent kinase inhibitor p21, but not p27. These results demonstrate that cyclic stretch inhibits SMC growth by blocking cell cycle progression and suggest that physiological levels of cyclic stretch contribute to vascular homeostasis by inhibiting the proliferative pathway of SMC.

Animals↗

PubMed1999

Platelet-derived growth factor stimulates heme oxygenase-1 gene expression and carbon monoxide production in vascular smooth muscle cells.

Recent studies indicate that vascular smooth muscle cells (VSMCs) generate CO from the degradation of heme by the enzyme heme oxygenase-1 (HO-1). Because platelet-derived growth factor (PDGF) modulates various responses of VSMCs, we examined whether this peptide regulates the expression of HO-1 and the production of CO by rat aortic SMCs. Treatment of SMCs with PDGF resulted in a time- and concentration-dependent increase in the levels of HO-1 mRNA and protein. Both actinomycin D and cycloheximide blocked PDGF-stimulated HO-1 mRNA and protein. In addition, PDGF stimulated the production of reactive oxygen species by SMCs. Both the PDGF-mediated generation of reactive oxygen species and the induction of HO-1 protein was inhibited by the antioxidant N-acetyl-L-cysteine. Incubation of platelets with PDGF-treated SMCs resulted in a significant increase in platelet cGMP concentration that was reversed by treatment of SMCs with the HO-1 inhibitor tin protoporphyrin-IX or by addition of the CO scavenger hemoglobin to platelets. In contrast, the nitric oxide inhibitor methyl-L-arginine did not block the stimulatory effect of PDGF-treated SMCs on platelet cGMP. Finally, incubation of SMCs with the releasate from collagen-activated platelets induced HO-1 protein expression that was blocked by a neutralizing antibody to PDGF. These results demonstrate that either administered exogenously or released by platelets, PDGF stimulates HO-1 gene expression and CO synthesis in vascular smooth muscle. The ability of PDGF to induce HO-1-catalyzed CO release by VSMCs may represent a novel mechanism by which this growth factor regulates vascular cell and platelet function.

Animals↗

PubMed1998

Thrombin stimulates vascular smooth muscle cell polyamine synthesis by inducing cationic amino acid transporter and ornithine decarboxylase gene expression.

Thrombin, a serine protease, is a potent mitogen for vascular smooth muscle cells (SMCs), but its mechanism of action is not known. Since L-ornithine is metabolized to growth-stimulatory polyamines, we examined whether thrombin regulates the transcellular transport and metabolism of L-ornithine by vascular SMCs. Treatment of SMCs with thrombin initially (0 to 2 hours) decreased L-ornithine uptake, whereas longer exposures (6 to 24 hours) progressively increased transport. Kinetic studies indicated that thrombin-induced inhibition was associated with a decrease in affinity for L-ornithine, whereas stimulation was mediated by an increase in transport capacity. Thrombin induced the expression of both cationic amino acid transporter (CAT)-1 and CAT-2 mRNA. Furthermore, thrombin stimulated L-ornithine metabolism by inducing ornithine decarboxylase (ODC) mRNA expression and activity. The stimulatory effect of thrombin on both L-ornithine transport and ODC activity was reversed by hirudin, a thrombin inhibitor, and was mimicked by a 14-amino acid thrombin receptor-activating peptide. Thrombin also markedly increased the capacity of SMCs to generate putrescine, a polyamine, from extracellular L-ornithine. The thrombin-mediated increase in putrescine production was reversed by N(G)-methyl-L-arginine, a competitive inhibitor of cationic amino acid transport, or by alpha-difluoromethylornithine (DFMO), an ODC inhibitor. DFMO also inhibited thrombin-induced SMC proliferation. These results demonstrate that thrombin stimulates polyamine synthesis by inducing CAT and ODC gene expression and that thrombin-stimulated SMC proliferation is dependent on polyamine formation. The ability of thrombin to upregulate L-ornithine transport and direct its metabolism to growth-stimulatory polyamines may contribute to postangioplasty restenosis and atherosclerotic lesion formation.

Amino Acid Transport Systems, Basic↗

PubMed1998

Carbon monoxide and vascular cell function (review).

Carbon monoxide (CO) is an endogenously generated gas that may play an important physiological role in the circulation. CO is generated by vascular cells as a byproduct of heme catabolism, in which heme oxygenase (HO) catalyzes the degradation of heme to biliverdin, iron and CO. Two distinct isoforms of HO have been identified in vascular tissue. The HO-2 isoform is constitutively expressed and likely mediates the release of CO under normal physiologic conditions. In contrast, the HO-1 isoform is strongly induced in vascular cells by various stress-associated agents and markedly increases CO synthesis during pathological conditions. The release of CO by vascular cells exerts both paracrine and autocrine effects on vascular smooth muscle cells (SMC) and circulating blood cells. CO regulates blood flow and blood fluidity by inhibiting vasomotor tone, SMC proliferation, and platelet aggregation. These vascular effects of CO are mediated via the activation of soluble guanylate cyclase and the consequent rise in intracellular guanosine 3',5'-cyclic monophosphate levels in target tissues. CO may also play a role in various cardiovascular disorders, including endotoxin shock, ischemia-reperfusion, hypertension, and subarachnoid hemorrhage. This review will focus on the recent progress made in understanding the regulation and function of CO in the vasculature.

Animals↗

PubMed1997

Lysophosphatidylcholine regulates cationic amino acid transport and metabolism in vascular smooth muscle cells. Role in polyamine biosynthesis.

Lysophosphatidylcholine (lyso-PC) is a major component of atherogenic lipids that stimulate vascular smooth muscle cell (SMC) proliferation. Because cationic amino acids are metabolized to growth-stimulatory polyamines, we examined whether lyso-PC regulates the transcellular transport and metabolism of cationic amino acids by vascular SMC. Treatment of SMC with lyso-PC initially (0-2 h) decreased cationic amino acid uptake, whereas longer exposures (6-24 h) progressively increased transport. Kinetic studies indicated that lyso-PC-induced inhibition was associated with a decrease in affinity for cationic amino acids, but the stimulation was mediated by an increase in transport capacity. Lyso-PC strongly induced the expression of cationic amino acid transporter-2 mRNA while modestly elevating the level of cationic amino acid transporter-1 mRNA. In addition, lyso-PC stimulated intracellular cationic amino acid metabolism by inducing ornithine decarboxylase activity and mRNA expression and also by inducing arginase activity in vascular SMC. In contrast, lyso-PC inhibited the catabolism of L-arginine to nitric oxide by blocking inducible nitric oxide synthase expression. Lyso-PC increased markedly the capacity of SMC to generate putrescine, a polyamine, from extracellular L-ornithine and L-arginine. The lyso-PC-mediated increase in the production of putrescine was reversed by NG-methyl-L-arginine, a competitive inhibitor of cationic amino acid transport, or by alpha-difluoromethylornithine, an ornithine decarboxylase inhibitor. The formation of putrescine from L-arginine was also prevented by arginase inhibitor NG-hydroxy-L-arginine. These results demonstrate that lyso-PC stimulates polyamine synthesis in vascular SMC by inducing the expression of the genes that regulate both the transport and metabolism of cationic amino acids. The actions of lyso-PC in stimulating cationic amino acid uptake and directing their metabolism to growth-stimulatory polyamines while simultaneously inhibiting the synthesis of antiproliferative NO, may contribute to lyso-PC-induced SMC proliferation and atherosclerotic lesion formation.

Amino Acid Transport Systems, Basic↗

PubMed1997

Hemodynamic forces induce the expression of heme oxygenase in cultured vascular smooth muscle cells.

Both nitric oxide (NO) and carbon monoxide (CO) are vessel wall-derived messenger molecules that cause platelet inhibition and vasodilation by activating guanylyl cyclase in target cells. Since vascular smooth muscle cells (SMCs) are exposed to shear and tensile stresses, this study examined the effects of these hemodynamic forces on the enzymes that generate NO and CO in SMCs. Monolayers of cultured rat aortic SMCs were subjected to shear stress using a modified cone and plate viscometer, or cyclic elongational stretch using a compliant silastic culture membrane. Shear stress stimulated time-dependent increases in mRNA and protein for inducible heme oxygenase-1 (HO-1), the enzyme which forms CO as a byproduct of heme degradation. The threshold level of shear necessary to induce HO-1 expression was between 5 and 10 dynes/cm2. In contrast, shear stress did not stimulate inducible NO synthase (iNOS) expression. Cyclic stretch also induced the expression of HO-1 but not of iNOS mRNA. Exposure of vascular SMCs to shear stress stimulated the production and release of CO as demonstrated by the CO-dependent increase in intracellular cGMP levels in coincubated platelets. In addition, ADP-stimulated aggregation was inhibited in platelets exposed to sheared SMCs but not in platelets exposed to untreated control SMCs. Treatment of sheared SMCs with the HO-1 inhibitor, tin protoporphyrin-IX, blocked the antiaggregatory effect of the cells, whereas the iNOS inhibitor, methyl--arginine, had no effect. These results indicate that hemodynamic forces induce HO-1 gene expression and CO production in vascular SMCs, and that SMC-derived CO inhibits platelet aggregation. Thus, CO is a novel endogenous vessel wall-derived messenger molecule that may be selectively induced by hemodynamic forces to inhibit platelet reactivity and preserve blood fluidity at sites of vascular injury.

Animals↗

PubMed1997

cAMP induces heme oxygenase-1 gene expression and carbon monoxide production in vascular smooth muscle.

Recent studies indicate that vascular smooth muscle cells generate carbon monoxide (CO) via the action of heme oxygenase (HO). Because adenosine 3',5'-cyclic monophosphate (cAMP) is an important intracellular signaling molecule in the regulation of vascular cell function, we examined whether this second messenger modulates the expression of HO and the production of CO by rat aortic smooth muscle cells. Treatment of smooth muscle cells with the membrane-permeable cAMP derivative dibutyryl cAMP or with compounds that increase intracellular cAMP levels (isoproterenol and forskolin) resulted in a concentration- and time-dependent increase in the levels of HO-1 mRNA and protein, whereas the expression of HO-2 remained unchanged. Both actinomycin D and cycloheximide blocked the basal expression of HO-1 mRNA and protein and prevented the cAMP-mediated induction of HO-1. Incubation of platelets with cAMP-treated smooth muscle cells resulted in a significant increase in platelet cGMP concentration that was partially reversed by treatment of smooth muscle cells with the nitric oxide synthase inhibitor NG-monomethyl-L-arginine or the HO blocker zinc protoporphyrin-IX. However, the combined addition of these two inhibitors to cAMP-treated smooth muscle cells or the addition of the CO and NO scavenger hemoglobin to platelets completely blocked the stimulatory effect on platelet cGMP levels. These results demonstrate that cAMP induces the expression of the HO-1 gene and stimulates the formation of CO and NO in vascular smooth muscle cells. The capacity of cAMP to induce the synthesis of guanylate cyclase-stimulatory CO from smooth muscle cells may represent a novel mechanism by which this nucleotide regulates vascular tone.

Animals↗

PubMed1997

Nitric oxide induces heme oxygenase-1 gene expression and carbon monoxide production in vascular smooth muscle cells.

Since recent studies demonstrate that vascular smooth muscle cells synthesize two distinct guanylate cyclase-stimulatory gases, NO and CO, we examined possible regulatory interactions between these two signaling molecules. Treatment of rat aortic smooth muscle cells with the NO donors, sodium nitroprusside, S-nitroso-N-acetyl-penicillamine, or 3-morpholinosydnonimine, increased heme oxygenase-I (HO-1) mRNA and protein levels in a concentration and time-dependent manner. Both actinomycin D and cycloheximide blocked NO-stimulated HO-1 mRNA and protein expression. Nuclear run-on experiments demonstrated that NO donors increased HO-1 gene transcription between 3- and 6-fold. In contrast, NO donors had no effect on the stability of HO-1 mRNA. Incubation of vascular smooth muscle cells with the membrane-permeable cGMP analogues, dibutyryl cGMP and 8-bromo-cGMP, failed to induce HO-1 gene expression. Treatment of vascular smooth muscle cells with NO donors also stimulated the production and release of CO, as demonstrated by the CO-dependent increase in intracellular cGMP levels in coincubated platelets. Finally, incubating vascular smooth muscle cells with interleukin-1 beta and tumor necrosis factor-alpha induced NO synthesis and also significantly increased the level of HO-1 protein. The cytokine-stimulated production of both NO and HO-1 protein in smooth muscle cells was blocked by the NO synthase inhibitor methyl-L-arginine. These results demonstrate that exogenously administered or endogenously released NO stimulates HO-1 gene expression and CO production in vascular smooth muscle cells. The ability of NO to induce HO-catalyzed CO release from vascular smooth muscle cells provides a novel mechanism by which NO might modulate soluble guanylate cyclase and, thereby, vascular smooth muscle cell and platelet function.

Animals↗