NF-kappaB is required for TNF-alpha-directed smooth muscle cell migration.

Migration of vascular smooth muscle cells (VSMC) is a crucial event in the formation of vascular stenotic lesions. Tumor necrosis factor-alpha (TNF-alpha) is elaborated by VSMC in atherosclerosis and following angioplasty. We investigated the role of nuclear factor-kappaB (NF-kappaB) in human VSMC migration induced by TNF-alpha. Adenoviral expression of a mutant form of the inhibitor of NF-kappaB, IkappaB-alphaM, suppressed TNF-alpha-triggered degradation of cellular IkappaB-alpha, inhibited activation of NF-kappaB, and attenuated TNF-alpha-induced migration. Further, IkappaB-alphaM suppressed TNF-alpha-stimulated release of interleukin-6 and -8 (IL-6 and IL-8). Neutralization of IL-6 and IL-8 with appropriate antibodies reduced TNF-alpha-induced VSMC migration. Addition of recombinant IL-6 and IL-8 stimulated migration. Collectively, our data provide initial evidence that TNF-alpha-mediated VSMC migration requires NF-kappaB activation and is associated with induction of IL-6 and IL-8 which act in an autocrine manner.

Reactive oxygen and NF-kappaB in VEGF-induced migration of human vascular smooth muscle cells.

Migration and proliferation of vascular smooth muscle cells (VSMC) contribute to angiogenesis and the lesions of atherosclerosis. Since, vascular endothelial growth factor (VEGF) is overexpressed by VSMC in intima of atherosclerotic human coronary arteries, we determined if VEGF could stimulate VSMC migration and the intracellular signals involved. VEGF induced VSMC migration but had no significant activity on proliferation. VEGF increased intracellular reactive oxygen species (ROS), NF-kappaB activation and IL-6 expression. Blockade of the generation of intracellular ROS by antioxidants inhibited VEGF-induced NF-kappaB activation, IL-6 expression, and cell migration indicating that generation of ROS was required for NF-kappaB activation and the chemotactic activity of VEGF. Expression of a mutated, nondegradable form of inhibitor of NF-kappaB (IkappaB-alphaM) suppressed VEGF-triggered activation of NF-kappaB and upregulation of IL-6 as well as VSMC migration. Neutralization of IL-6 by its antibody significantly attenuated the migration stimulated by VEGF. Collectively, our data provide the first evidence that intracellular ROS and NF-kappaB are required for VEGF-mediated smooth muscle cell migration. Further, IL-6 induced by VEGF is involved in the ability of the growth factor to stimulate migration.

Endotoxin stimulated cytokine production in rat vascular smooth muscle cells.

Because inflammatory processes may promote the development of atherosclerosis, we examined the activation of cytokine genes in rat vascular smooth muscle cells in vitro after treatment with bacterial lipopolysaccharide (LPS). Interleukin-1 (IL-1), IL-6 and tumor necrosis factor-alpha (TNF-alpha) mRNA increased in response to LPS. Activation of nuclear factor-kappaB (NF-kappaB) presumably results in NF-kappaB binding to regulatory regions of target genes and activating transcription. We therefore compared the kinetics of NF-kappaB activation, cytokine message production, and TNF-alpha secretion. Maximum active NF-kappaB was found at 30 min after the addition of LPS and decreased thereafter. Increased IL-6 mRNA was detected at 30 min, increased TNF-alpha mRNA at 60 min, and increased IL-1 mRNA at 120 min. Secretion of TNF-alpha was dependent on LPS concentration and was first detected 120 min after LPS addition. Aspirin, which has been shown to inhibit NF-kappaB activation and cytokine secretion in other cell types, did not inhibit NF-kappaB activation or TNF-alpha secretion. However, aspirin reduced the amount of both TNF-alpha and IL-6 mRNA present 30 min after LPS addition by half (P < 0.05).

Stimulation of beta-adrenergic receptors inhibits the release of tumor necrosis factor-alpha from the isolated rat heart.

OBJECTIVES: Beta-adrenergic receptor agonists such as isoproterenol inhibit production of tumor necrosis factor (TNF)-alpha in a number of cell types. Because the heart is a source of TNF-alpha, we hypothesized that isoproterenol would inhibit cardiac production of the cytokine. DESIGN: Analysis of cardiac release of TNF-alpha. SETTING: Medical research laboratory. SUBJECTS: Rats. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: With the approval of the Institutional Animal Care and Use Committee, rats were anesthetized and hearts were removed and perfused. After 30 mins, bacterial lipopolysaccharide (LPS) with or without isoproterenol was infused for 60 mins. At 30, 60, 90, 120, and 150 mins, coronary flow was measured and coronary effluent was analyzed for TNF-alpha. Cardiac production of TNF-alpha was expressed as pg/min. Cyclic adenosine monophosphate (AMP) in the coronary effluent was measured. TNF-alpha messenger RNA was determined in ventricular tissue. After 30 mins, TNF-alpha was undetectable in the coronary effluent However, 60 mins after the initiation of LPS infusion, TNF-alpha release was 875+/-255 pg/min and increased to 2164+/-721 pg/min at 150 mins. Simultaneous infusion of isoproterenol with LPS stimulated cyclic AMP release and inhibited TNF-alpha production. For instance, at 60 and 150 mins, TNF-alpha release was 75+/-38 and 58+/-29 pg/min, respectively (p < .05 vs. LPS alone). Simultaneous infusion of isoproterenol with LPS blocked the induction of TNF-alpha messenger RNA by LPS. Isoproterenol, begun 30 mins after the initiation of LPS infusion, still suppressed LPS-stimulated TNF-alpha release by 95% at 150 mins. Similar results were obtained with norepinephrine. CONCLUSIONS: Activation of beta-adrenergic receptors inhibits cardiac TNF-alpha release. This implies that cytokine production by the heart is inhibited by the sympathetic nervous system. In heart failure, the cardiac response to the sympathetic nervous system is impaired. This impairment may play a role in the high plasma levels of TNF-alpha found in heart failure.

An unusual case of malignant hyperthermia during desflurane anesthesia in an African-American patient.

IMPLICATIONS: Malignant hyperthermia is an uncommon, heritable condition triggered by anesthesia and is followed by an increase in temperature that may be fatal without prompt treatment. It is rare with desflurane and in black individuals of African descent. We present a case of malignant hyperthermia in an African-American patient during desflurane anesthesia.

Isoproterenol inhibits bacterial lipopolysaccharide-stimulated release of tumor necrosis factor-alpha from human heart tissue.

Recent evidence suggests that inflammatory cytokines, particularly tumor necrosis factor alpha (TNF-alpha), may play a role in heart disease. Elevated plasma levels of the cytokine have been reported in congestive heart failure and severe angina and after myocardial infarction. The exact role of TNF-alpha in heart disease and how production is stimulated and regulated in the heart are current areas of investigation. Regarding regulation of production, isoproterenol elevates cyclic AMP and inhibits TNF-alpha release in macrophages. Therefore we hypothesized that stimulation of beta-adrenergic receptors of the sympathetic nervous system would inhibit release of the cytokine from heart tissue. With Institutional Review Board approval and patient consent atrial tissue was obtained during preparation for cardiac bypass. The tissue was divided into segments, placed in culture medium, and incubated for various times in the presence or absence of lipopolysaccharide (LPS) (20 microg/mL) and/or isoproterenol (1 microM). The medium was removed and analyzed for biologically active TNF-alpha by the L929 cell cytotoxicity assay. Tissue samples were weighed and TNF-alpha release was expressed as pg TNF-alpha/mg tissue. Initially, to determine the time course of release, measurements were made at 2, 5, 10, 15, 30, 60, 120, 180, and 360 minutes after the addition of LPS. Elevated TNF-alpha levels in the culture medium were reliably detected at 360 minutes after exposure to LPS. In atrial tissue obtained from seven patients TNF-alpha released into the culture medium at 360 minutes was 6 +/- 3 pg/mg tissue. In the presence of LPS, levels of the cytokine in the culture medium increased to 604 +/- 233 pg/mg tissue (P < 0.05 vs LPS alone). When isoproterenol and LPS were simultaneously added to the culture medium release of TNF-alpha was reduced by 87 per cent to 82 +/- 40 pg/mg tissue (P < 0.05 vs LPS alone). Our results show that activation of the beta-adrenergic receptor inhibits myocardial production of TNF-alpha. This finding suggests that the sympathetic nervous system inhibits production of the cytokine and that impaired sympathetic function in heart failure may play a role in the elevated levels of TNF-alpha.

Thermodiffusion for continuous quantification of hepatic microcirculation--validation and potential in liver transplantation.

Hepatic microcirculation is a main determinant of reperfusion injury and graft quality in liver transplantation. Methods available for the quantification of hepatic microcirculation are indirect, are invasive, or preclude postoperative application. The aim of this study was the validation of thermodiffusion in a new modification allowing long-term use in the clinical setting. In six pigs Doppler flowmeters were positioned around the hepatic artery and portal vein for the measurement of total liver blood flow. Liver perfusion was quantified by thermodiffusion and compared to H(2) clearance as an established technique under baseline conditions, during different degrees of portal venous obstruction and during occlusion of the hepatic artery. Thermodiffusion measurements were recorded for five days postoperatively followed by histological evaluation of the hepatic puncture site. Perfusion data obtained by thermodiffusion were significantly correlated to H(2) clearance (r = 0.94, P < 0. 001) and to liver blood flow (r = 0.9, P < 0.05). The agreement between thermodiffusion and H(2) clearance was excellent (mean difference -2.1 ml/100 g/min; limits of agreement -12.5 and 8.3 ml/100 g/min). Occlusion of the portal vein or hepatic artery was immediately detected by thermodiffusion, indicating a decrease of perfusion by 64 +/- 7% or 27 +/- 5% of baseline, respectively. Perfusion values at baseline and during vascular occlusion were reproducible during the entire observation period. Histological changes of the liver tissue adjacent to the thermodiffusion probes were minute and did not influence long-term measurements. In vivo validation proved that enhanced thermodiffusion is a minimally invasive technique for the continuous, real-time quantification of hepatic microcirculation. Changes in liver perfusion can be safely detected over several days postoperatively. The implication for liver transplantation has led to the clinical application of thermodiffusion.

A reactive oxygen-generating system activates nuclear factor-kappaB and releases tumor necrosis factor-alpha in coronary smooth muscle cells.

BACKGROUND: Recently we reported that bacterial lipopolysaccharide (LPS) stimulates release of tumor necrosis factor alpha (TNF-alpha) from porcine coronary arteries and smooth muscle cells cultured from those vessels. It has also been reported that plasma levels of TNF-alpha are elevated after myocardial infarction. Since it is known that the production of reactive oxygen intermediates (ROI) occurs during ischemia and ROI are suggested activators of the nuclear regulatory factor kappaB (NF-kappaB), we tested the hypothesis that release of TNF-alpha from smooth muscle cells could also be stimulated with a ROI-generating system. MATERIALS AND METHODS: Smooth muscle cells were isolated from porcine coronary arteries. Confluent cells in 48-well culture dishes were treated for 30 min with 0.003 units/ml xanthine oxidase (XO) and 2 mM hypoxanthine (HX) added to the culture medium. The medium was then removed and the cells were washed three times and fresh medium without HX-XO was added. Then, at 1, 3, and 6 h the medium was removed and analyzed for biologically active TNF-alpha. In other experiments, smooth muscle cells were treated with 20 micrograms/ml LPS for 6 h and aliquots of medium analyzed for TNF-alpha. Untreated cells served as controls. Data were analyzed by two-way ANOVA with repeated measures. Extracts of total cell protein were prepared and activation of NF-kappaB was determined by electrophoretic mobility shift assay. RESULTS: Treatment of cells with HX-XO stimulated release of TNF-alpha, which rose to a maximum of 17.5 +/- 1.7 units/mg cell protein at 6 h. This was significantly higher (P < 0. 05) than release stimulated by LPS (10.2 +/- 1.0 units/mg at 6 h) or TNF-alpha detected in the culture medium from untreated control cells (4.2 +/- 0.9 units/mg protein at 6 h). Both HX/XO and LPS activated NF-kappaB. CONCLUSIONS: These results support the conclusion that coronary smooth muscle cells are a potential source of TNF-alpha during events that are associated with formation of ROI such as myocardial ischemia.

Release of tumor necrosis factor-alpha from coronary smooth muscle: activation of NF-kappaB and inhibition by elevated cyclic AMP.

BACKGROUND: Evidence suggests that tumor necrosis factor-alpha (TNF-alpha) is involved in heart diseases such as atherosclerosis. We used porcine coronary arteries and smooth muscle cells cultured from these vessels to study the regulation of production of TNF-alpha. The aims were to determine if bacterial lipopolysaccharide (LPS) could stimulate production; if activation of the nuclear regulatory factor, NF-kappaB, was associated with production; and if intracellular cAMP regulates TNF-alpha in coronary vasculature through a mechanism involving NF-kappaB. MATERIAL AND METHODS: LPS was used to stimulate TNF-alpha production. Forskolin (FSK) and 8-Br-cAMP were added to tissue and cells in order to elevate intracellular cAMP. TNF-alpha release into the bathing medium was measured by the L929 cell cytotoxicity assay. Intracellular cAMP was determined by radioimmunoassay. NF-kappaB activation was determined in whole cell extracts by electrophoretic mobility shift assay. RESULTS: In segments of coronary arteries, LPS stimulated TNF-alpha release which increased with time to a maximum at 6 h (485 +/- 19 units/g tissue) and remained elevated at this level for 24 h. In contrast, the level of TNF-alpha measured at 24 h in medium from coronary tissue not exposed to LPS was 11.1 +/- 4.1 units/g tissue. In the presence of LPS, both FSK and 8-Br-cAMP significantly reduced TNF-alpha release. For instance at 6 h in the presence of LPS and FSK or 8-Br-cAMP, TNF-alpha was 126 +/- 24 and 71.6 +/- 22 units/g tissue, respectively (P < 0.05 vs LPS alone). Tissue levels of cAMP were significantly elevated in the presence of FSK. Similar results were obtained with smooth muscle cells cultured from the coronary arteries; i.e., LPS stimulated TNF-alpha release which was inhibited in a concentration-dependent manner by a rise in intracellular cAMP induced by FSK. In cultured cells release of TNF-alpha stimulated by LPS was associated with activation of NF-kappaB. Neither FSK nor 8-Br cAMP inhibited activation of NF-kappaB by LPS. CONCLUSIONS: Porcine coronary arteries produce TNF-alpha from a smooth muscle cell source. Production stimulated by LPS was inhibited by elevated intracellular cAMP and was associated with activation of NF-kappaB. However, activation of NF-kappaB was not inhibited by elevated cAMP, suggesting that the regulatory action of this cyclic nucleotide could lie downstream from activation of the TNF-alpha gene. These results support the view that coronary vessels can be a source of TNF-alpha possibly involved in heart disease.

Increased intracellular cyclic adenosine 3', 5'-monophosphate inhibits release of tumor necrosis factor-alpha from human vascular tissue and cultured smooth muscle cells.

OBJECTIVES: We recently reported that bacterial lipopolysaccharide stimulates release of tumor necrosis factor (TNF)-alpha from both human vascular tissue and cultured smooth muscle cells. In the current study, we tested the hypothesis that increased intracellular cyclic adenosine 3',5'-monophosphate (cAMP) could inhibit TNF-alpha release. DESIGN: Prospective, repeated-measures analysis. SETTING: Academic research laboratory. SUBJECTS: Segments of internal mammary artery and saphenous vein from patients undergoing coronary artery bypass surgery. MEASUREMENTS AND MAIN RESULTS: Segments of saphenous vein and internal mammary artery and confluent smooth muscle cells cultured from these vessels were incubated in the presence of 20 micrograms/mL bacterial lipopolysaccharide, alone or with the addition of forskolin or 8-Br-cAMP. At 0, 1, 3, 6, 18, and 24 hrs, the incubation medium was removed from vessel segments or cells and was analyzed for biologically active TNF-alpha, using the L929 cell cytotoxicity assay. cAMP was extracted from tissue and cells with 0.1 N HCl and was analyzed by radioimmunoassay. Bacterial lipopolysaccharide stimulated the release of TNF-alpha from internal mammary smooth muscle cells at all time points. For example, at 6 hrs, TNF-alpha concentration in the medium from lipopolysaccharide-stimulated cells was 20 +/- 1.6 U/mg of cell protein, compared with 0.9 +/- 0.5 U/mg of cell protein in control cell medium (p < .05). Forskolin-inhibited bacterial lipopolysaccharide stimulated TNF-alpha release. In the presence of lipopolysaccharide and forskolin, TNF-alpha release at 6 hrs was 8.6 +/- 1.5 U/mg of cell protein (p < .05 vs. in the presence of bacterial lipopolysaccharide alone). Bacterial lipopolysaccharide, alone, had no effect on intracellular cAMP. Forskolin increased intracellular cAMP levels to 74.0 +/- 12 pmol/mg of cell protein at 6 hrs from a control level of 7.7 +/- 0.4 pmol/mg (p < .05). The 8-Br-cAMP, an agent that mimics the action of intracellular cAMP, also inhibited TNF-alpha release stimulated by lipopolysaccharide. Similar inhibition by forskolin and 8-Br-cAMP on TNF-alpha release was obtained with smooth muscle cells from saphenous vein. Finally, in tissue segments from either internal mammary artery or saphenous vein, both forskolin and 8-Br-cAMP inhibited lipopolysaccharide-stimulated TNF-alpha release. CONCLUSIONS: These results are consistent with the conclusion that vascular tissue, particularly the smooth muscle cell, is a source of TNF-alpha. Further, bacterial lipopolysaccharide-stimulated tumor TNF-alpha release can be inhibited by increased intracellular cAMP.

Inhibition of release of tumor necrosis factor-alpha from human vascular tissue and smooth muscle cells by glucocorticoids.

OBJECTIVES: Based on our previous study that bacterial lipopolysaccharide stimulates release of tumor necrosis factor (TNF)-alpha from human vascular tissue and smooth muscle cells, we tested the hypothesis that release of TNF could be inhibited by pretreatment with glucocorticoids. DESIGN: Prospective, repeated-measures analysis of concentration-response relationships. SETTING: Academic anesthesiology research laboratory. SUBJECTS: Segments of internal mammary artery and saphenous vein were obtained during coronary artery bypass surgery. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Confluent human smooth muscle cells, cultured from saphenous vein and internal mammary artery, were exposed to 20 micrograms/mL of bacterial lipopolysaccharide following pretreatment for 18 hrs with either 0.1, 1.0, or 10.0 microM of dexamethasone. At 1, 3, 6, 18, and 24 hrs, the culture medium was removed and analyzed for biologically active TNF-alpha using the L929 cell cytotoxicity assay. Smooth muscle cells exposed to bacterial lipopolysaccharide but not treated with dexamethasone served as controls. In control internal mammary cells, bacterial lipopolysaccharide stimulated TNF-alpha release in a time-dependent manner to a peak of 36 +/- 2.3 U/mg of cell protein at 6 hrs, compared with 0.7 +/- 0.3 U/mg of cell protein in cells not exposed to lipopolysaccharide. Dexamethasone inhibited bacterial lipopolysaccharide-stimulated release at all time points in a concentration-dependent manner. For instance, at 6 hrs, TNF-alpha was 12 +/- 2.2, 6.9 +/- 1.7, and 2.3 +/- 0.9 U/mg of cell protein for cells pretreated with 0.1, 1.0, and 10.0 microM of dexamethasone, respectively (p < .05 vs. control). In separate experiments, segments of internal mammary artery and saphenous vein were obtained from five patients who received 1 g of methylprednisolone intravenously during induction of anesthesia, and from seven patients who did not receive methylprednisolone. Bacterial lipopolysaccharide induced release of TNF-alpha from vascular tissues of untreated patients in a time-dependent manner (e.g., 733 +/- 44 U/g of tissue at 6 hrs in saphenous vein). In contrast, in patients treated with methylprednisolone, bacterial lipopolysaccharide did not stimulate release from vascular tissues incubated for up to 24 hrs. CONCLUSIONS: These results indicate that human vascular tissue, particularly the smooth muscle cell, may be a source of TNF-alpha and that glucocorticoids inhibit release stimulated by bacterial lipopolysaccharide.

Response of cultured cerebral artery smooth muscle cells to the nitric oxide vasodilators, nitroglycerin and sodium nitroprusside.

We characterized the response of soluble guanylyl cyclase in smooth muscle cells cultured from cerebral vessels to the nitric oxide (NO)-producing vasodilators, nitroglycerin (NTG) and sodium nitroprusside (SNP) and determined the ability of these agents to induce tolerance. Smooth muscle cells were isolated from porcine basilar, anterior and middle cerebral, and internal carotid arteries. Following an initial series of experiments using NTG at various concentrations and times of exposure to determine conditions, concentration-response curves of intracellular guanosine 3',5'-cyclic monophosphate (cGMP) to NTG and SNP were determined in cells pretreated for 1 h with 100 mumol NTG to induce tolerance and compared with response curves in control cells. Basal cGMP levels were 2.1 +/- 0.4 pmol/mg cell protein (n = 16). Both NTG and SNP increased cGMP in nontolerant cells, and SNP was more effective. Maximum concentrations of SNP (1 mmol/L) increased cGMP to 163 +/- 5.9 pmol/mg versus 21 +/- 2.4 pmol/mg for 1 mmol/L NTG (p < 0.01). Cells made tolerant to NTG were unresponsive to NTG up to 1 mmol/L but remained responsive to SNP. However, the response curve to SNP was significantly depressed by approximately 25%. Following washout of NTG in tolerant cells, the response of cGMP to SNP returned to control within 12 h, while response to NTG required 36 h. Similar experiments were conducted in cells initially made tolerant to SNP. These results indicate that cerebral artery smooth muscle cells in culture express a functioning soluble guanylyl cyclase and the enzymes that are necessary to metabolize NTG to NO. Prolonged exposure of the cells to NTG induced tolerance as well as cross-tolerance to SNP.

Effect of rat mesenchymal stem cells on development of abdominal adhesions after surgery.

One of the common and most serious side effects of abdominal surgery is the formation of adhesions within the peritoneal cavity during healing. Efforts to prevent adhesion formation have concentrated on inhibiting the inflammatory response, inhibiting the formation or encouraging the lysis of fibrin, and protection of the damaged serosal surface. We are interested in regenerating the serosal surface by providing a source of mesothelial progenitor cells. Rats were divided into groups of 10 each. Abdominal adhesions were created by removing a circle of peritoneum and suturing it back into place. Two weeks later the rats were euthanized and the adhesions scored on a scale of 0-5. A population of mesenchymal stem cells (MSCs) isolated from the skeletal muscle of neonatal rats was tested. The cells were grown in primary culture to expand the population and then trypsinized and frozen at -80 degrees C. They are then thawed and grown in secondary culture before use. The control group were injected with saline i.p. immediately after surgery. The experimental groups received (1) 1.4 X 10(6) MSCs, (2) 5 X 10(6) MSCs, (3) 7.5 X 10(6) dead MSCs, (4) 5 X 10(6) rat smooth muscle cells immediately post-op, and (5) 5 X 10(6) MSCs 4-6 hours after surgery. Only live MSCs given immediately after surgery by i.p. injection significantly decreased the adhesion scores of the rats (mean score of 3.5 vs 0.9). MSCs injected i.p. 4-6 hours after surgery actually increased the adhesion scores (3.5 vs 4.7), and rat smooth muscle cells injected i.p. immediately after surgery had no effect on adhesions. The exact mechanism of action of the MSCs is unknown at this time. However, we postulate that the MSCs have the capacity to differentiate into mesothelial cells capable of repopulating the injured mesothelium.

Human blood vessels release tumor necrosis factor-alpha from a smooth muscle cell source.

OBJECTIVES: In septic shock, the principal source of increased plasma concentrations of tumor necrosis factor alpha (TNF) is considered to be the macrophage. Release from the macrophage is stimulated by bacterial lipopolysaccharide (endotoxin). We tested the hypothesis that vascular tissue also responds to endotoxin by releasing TNF. DESIGN: Prospective repeated measures analysis of timed-release curves. SETTING: Anesthesia research laboratory in an academic medical center. SUBJECTS: With Institutional Review Board approval and patient consent, segments of internal mammary artery and saphenous vein were obtained during coronary artery bypass surgery. INTERVENTIONS: None MEASUREMENTS AND MAIN RESULTS: Segments of saphenous veins were incubated for 24 hrs in the presence or absence of bacterial lipopolysaccharide. At 0.5, 1, 3, 6, and 24 hrs, medium was assayed for TNF. In other experiments, smooth muscle cells were cultured from saphenous veins, incubated with our without bacterial lipopolysaccharide, and a time-course of TNF release determined. Bacterial lipopolysaccharide (20 micrograms/mL) significantly stimulated release of TNF from venous tissue in a time-dependent manner. At 0.5 hrs, TNF was undetectable in untreated tissue and was 48 +/- 8 U/g wet tissue weight in the presence of bacterial lipopolysaccharide. At 3 hrs, TNF was 43 +/- 27 U/g wet tissue weight in untreated and 388 +/- 185 U/g wet tissue weight in treated (p < .01 vs. control) tissue. Segments of internal mammary artery responded in a similar manner. In smooth muscle cells cultured from saphenous vein and internal mammary artery, bacterial lipopolysaccharide triggered the release of TNF. At 3 hrs, the release of TNF in control cells was 0.2 +/- 0.15 U/mg cell protein and 17 +/- 2 U/mg in the presence of 20 micrograms/mL of bacterial lipopolysaccharide (p < .01 vs. control). CONCLUSIONS: Human blood vessels, both artery and vein, produce TNF potentially from a smooth muscle cell source in response to bacterial lipopolysaccharide.

Response of human artery, vein, and cultured smooth muscle cells to atrial and C-type natriuretic peptides.

OBJECTIVES: We determined the response of intracellular cyclic GMP in human arteries and veins and in smooth muscle cells cultured from these vessels to C-type natriuretic peptide in comparison with atrial natriuretic peptide. DESIGN: Repeated-measures analysis of concentration-response curves. SETTING: Anesthesia research laboratory. SUBJECTS: Vascular smooth muscle cells from human blood vessels obtained with Institutional Review Board approval and patient consent. MEASUREMENTS AND MAIN RESULTS: Segments of internal mammary artery and saphenous vein were obtained from patients undergoing coronary artery bypass surgery. Smooth muscle cells were cultured from these vessels. Concentration-response curves of intracellular cyclic GMP were determined and analyzed by two-way analysis of variance with repeated measures. In segments of intact saphenous vein, C-type natriuretic peptide was significantly more effective than atrial natriuretic peptide (16-fold increase in cyclic GMP in response to 1 microM of C-type natriuretic peptide vs. six-fold increase in cyclic GMP in response to 1 microM of atrial natriuretic peptide, p < .05). In rings of intact internal mammary artery, 1 microM of atrial natriuretic peptide (26-fold increase in cyclic GMP over basal value) was more effective than 1 microM of c-type natriuretic peptide (three-fold increase in cyclic GMP over basal value, p < .05). In cultured cells from these vessels, the pattern of response to C-type natriuretic peptide and atrial natriuretic peptide was the same as in the intact vessels. CONCLUSIONS: These results indicated that human smooth muscle cells in arteries and veins express both forms of natriuretic peptide receptors but that atrial natriuretic peptide acts primarily on the artery and C-type natriuretic peptide acts predominantly on the vein. Increased concentrations of C-type natriuretic peptide could contribute to venous pooling in septic shock.

Tolerance to nitroglycerin in vascular smooth muscle cells is not affected by the level of intracellular glutathione or L-cysteine.

A major hypothesis for the mechanism of tolerance to nitroglycerin (NTG) is that continued use causes a decrease in thiol donors within the vascular smooth muscle cell that are essential for the effect of NTG. We tested this idea directly in the target cell. NTG tolerance, measured as reduced formation of intracellular cyclic guanosine monophosphate (cGMP), was induced in pig coronary smooth muscle cells. The consequence of altering intracellular levels of the thiol donors, glutathione (GSH) and L-cysteine (L-cys), was determined. Incubating cells with 100 microM NTG for 1 h caused an 83% reduction in cGMP formation in response to acute readministration of 200 microM NTG for 2 min but was not associated with a reduction in intracellular GSH or L-cys. This result was not altered when intracellular GSH levels were increased three-fold by including 1 mM GSH in the incubation buffer. Also, recovery from tolerance was not affected by supplementation with GSH. Further, the response of cGMP to NTG was not altered by inhibiting the synthesis of GSH and lowering intracellular levels of GSH by 77%. Similar findings were made with supplemental L-cys or N-acetyl-L-cysteine. These results do not support the hypothesis that tolerance to NTG is the result of a reduction of the thiol donors GSH and L-cys within vascular smooth muscle cells.

Protamine does not affect the formation of cGMP or cAMP in pig vascular smooth muscle cells in response to vasodilators.

OBJECTIVES: Protamine has recently been shown to have a direct vasodilator action in isolated vascular tissue. As one possible mechanism for this action, it has been hypothesized that protamine might increase the response of vascular smooth muscle to the endothelium-derived relaxing factor, nitric oxide. In this study, we tested this hypothesis and examined the effect of protamine on other guanosine 3'5'-cyclic monophosphate (cGMP)- and adenosine 3'5'-cyclic monophosphate (cAMP)-dependent processes. DESIGN: Prospective, repeated measures analysis of concentration-response curves. SETTING: Anesthesia research laboratory in an academic medical center. SUBJECTS: Cultured coronary artery smooth muscle cells from pig heart. INTERVENTIONS: Sodium nitroprusside was used to mimic the action of the endothelium-derived relaxing factor by stimulating the soluble guanylyl cyclase and increasing intracellular cGMP. Atrial natriuretic peptide was used to stimulate the particulate guanylyl cyclase. Isoproterenol and forskolin were used to increase intracellular cAMP. The responses to these agents were determined in the presence and absence of protamine. MEASUREMENTS AND MAIN RESULTS: In cultured vascular smooth muscle cells, sodium nitroprusside increased cGMP, the second messenger for endothelium-derived relaxing factor, in a concentration-dependent manner. In cells treated with protamine (32 to 250 micrograms/mL), we could detect no effect of protamine on basal intracellular levels of cGMP until a concentration of 250 micrograms/mL of protamine was used. At this concentration, protamine increased basal cGMP concentrations from 4.2 +/- 0.3 to 9.0 +/- 0.6 pmol/mg protein (p < .001). The response of intracellular cGMP to sodium nitroprusside in cells treated with 250 micrograms/mL or other concentrations of protamine was not different from control. Likewise, we could detect no effect of protamine on intracellular cGMP stimulated with the atrial natriuretic peptide or on cAMP stimulated with the beta-adrenergic receptor agonist, isoproterenol, or with forskolin. CONCLUSIONS: These experiments show that protamine does not alter the responses of the intracellular second messengers, cGMP and cAMP, to the vasodilators sodium nitroprusside, atrial natriuretic peptide, isoproterenol, and forskolin. These results do not support the hypothesis that protamine sensitizes vascular smooth muscle cells to the endothelium-derived relaxing factor, nitric oxide.

C-natriuretic peptide but not atrial natriuretic peptide increases cyclic GMP in cerebral arterial smooth muscle cells.

A-type natriuretic peptide (ANP) is found primarily in the heart and is released into the circulation. C-type (CNP) is found principally in the brain and has also been detected in the systemic circulation. When injected, both peptides produce vasodilatation most likely by elevation of guanosine 3'5'-cyclic monophosphate (cGMP) in smooth muscle cells via two distinct receptors, NPR-A and NPR-B. In this present study, we determined the effects of these two peptides on intracellular cGMP in smooth muscle cells cultured from pig cerebral and peripheral arteries. In smooth muscle cells cultured from the left anterior descending coronary artery, ANP and CNP increased cGMP with equal potency and efficacy (EC50 for ANP and CNP, 3.6 +/- 0.2 x 10(-8) M and 6.7 +/- 0.8 x 10(-8) M, respectively). In contrast, in smooth muscle cells from cerebral arteries, ANP was without effect while CNP increased cGMP in a concentration dependent manner (EC50: 9.6 +/- 1.7 x 10(-8) M). Stimulation of the soluble guanylyl cyclase with either nitroglycerin or nitroprusside was equivalent in the two cell types. The pattern of response of intracellular cGMP to CNP and ANP in isolated intact arteries from brain and heart was similar to that found in the cultured cells. These results suggest that smooth muscle cells in cerebral arteries express only NPR-B while cells from peripheral arteries can express both NPR-A and NPR-B.

Variability of response to atrial natriuretic peptide and sodium nitroprusside in cultured vascular smooth muscle cells from three blood vessels.

OBJECTIVES: Vascular beds vary in their responses to atrial natriuretic peptide and sodium nitroprusside. Both of these agents dilate blood vessels by increasing intracellular guanosine 3',5'-cyclic monophosphate (cyclic-GMP) but activate different enzymes in the vascular smooth muscle cell. We aimed to determine if the response of intracellular cyclic-GMP to atrial natriuretic peptide and sodium nitroprusside varies in smooth muscle cells cultured from different vascular beds. DESIGN: Prospective, repeated measures analysis of concentration-response curves. SETTING: Anesthesia research laboratory of an academic medical center. SUBJECTS: Guinea pigs: Cultured guinea pig smooth muscle cells were obtained from three different blood vessels. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Intracellular cyclic-GMP was measured by radioimmunoassay and concentration-response curves of cyclic-GMP to atrial natriuretic peptide and sodium nitroprusside were determined. Cells from the thoracic aorta were the most responsive to atrial natriuretic peptide. Atrial natriuretic peptide (1 microM) increased cyclic-GMP concentrations to 72 +/- 5 pmol/mg (20-fold increase) with a 50% effective concentration of 3 nM. The concentration-response curve in epicardial coronary smooth muscle cells was to the right with an 50% effective concentration for atrial natriuretic peptide of 20 nM and a maximum response of a ten-fold increase in cyclic-GMP. Coronary resistance vessel cells were unresponsive to atrial natriuretic peptide. In response to sodium nitroprusside, the concentration-response curve in coronary resistance vessel cells was significantly to the left of either thoracic aorta cells or the epicardial coronary cells. The 50% effective concentration for sodium nitroprusside in coronary resistance vessel smooth muscle cells was approximately 1 microM, while in both thoracic aortic cells and epicardial coronary cells, the 50% effective concentration was ten times higher. Coronary resistance cells were the most responsive with a 62-fold increase in cyclic-GMP in response to 1 mM of sodium nitroprusside. The maximum response in thoracic aorta cells and epicardial cells was 20- and 30-fold, respectively. CONCLUSIONS: Smooth muscle cells respond differently to vasodilators. The response of these cultured cells mimics the response of the intact vascular bed from which they were obtained. These findings suggest that phenotypic properties of smooth muscle cells within a blood vessel contribute to the responsiveness of the vessel.

Thermal diffusion probe and instrument system for tissue blood flow measurements: validation in phantoms and in vivo organs.

A minimally invasive probe and instrument system for real-time measurements of temperature, thermal conductivity and tissue blood flow has been designed for research and clinical use. The essence of the probe is a thermistor, located at the tip of catheters or glass and steel needles, and operating in transient self-heated mode at constant temperature increment. Thermal conductivity and tissue blood flow are determined by use of a coupled tissue-probe thermal model. The effects of temporal baseline temperature shifts are minimized by a novel, automatic, analog compensation circuit. Very short heating periods (3 s) and cooling periods (12 s) provided near-continuous measurements (4/min). Calibration experiments performed in media of known thermal conductivity exhibit a linear response with respect to thermal conductivity. In vitro experiments performed in isolated perfused dog liver preparations are presented to evaluate this instrument system. In vivo experiments performed in cat brain, dog liver, and human tumor demonstrate the ability of this instrument system to perform physiologically valid measurements (comparison inter-subjects and intra-subjects). The minimally invasive probes (0.8 mm OD) are capable of long term measurements (several months), with minimal tissue reactions (0.3 mm around the probe).