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1.
J Nutr ; 131(10): 2628-35, 2001 Oct.
Article in English | MEDLINE | ID: mdl-11584082

ABSTRACT

Vitamin A and its active metabolite retinoic acid (RA) modulate host-pathogen interactions by interfering with the host immune and inflammatory response including prostaglandin (PG) biosynthesis. The effects of RA on phospholipase A(2) (PLA(2)) and cyclooxygenase (COX) isoforms in vitro are controversial, and few in vivo studies exist. We investigated the in vivo effects of RA on PG biosynthesis in the presence or absence of lipopolysaccharide (LPS) in rats. RA alone [10 mg/(kg. d) for 5 d] increased plasma and liver PG concentrations by increasing COX-1 protein expression (twofold that of control rats). RA acted synergistically with LPS to increase plasma (400-fold) and liver (15-fold) concentrations of prostaglandin E(2) (PGE(2)) and significantly, but to a lesser extent, other PG compared with RA rats, in the absence of major differences in PLA(2) expression or activity or COX-1 and COX-2 mRNA or protein expression. The RA + LPS-mediated increase in PGE(2) was significantly attenuated (97%) by aminoguanidine (AG), a relatively specific inhibitor of the inducible nitric oxide synthase (NOS2), consistent with the previously reported synergistic effect of RA and LPS on NOS2 expression and activity. In addition, RA and LPS induced the expression of the microsomal isoform of PGE synthase (mPGES). In conclusion, in vivo, RA and LPS increased PG and especially PGE(2) concentrations. The PGE(2) increase was associated with NOS2-mediated activation of COX and induction of mPGES. These results contribute to the characterization of the effects of vitamin A on the host inflammatory response.


Subject(s)
Isoenzymes/metabolism , Keratolytic Agents/pharmacology , Lipopolysaccharides , Prostaglandin-Endoperoxide Synthases/metabolism , Prostaglandins/biosynthesis , Tretinoin/pharmacology , Animals , Cyclooxygenase 2 , Drug Synergism , Isoenzymes/genetics , Liver/drug effects , Liver/enzymology , Male , Prostaglandin-Endoperoxide Synthases/genetics , Prostaglandins/blood , Rats , Rats, Inbred WKY
2.
Circulation ; 104(12 Suppl 1): I197-201, 2001 Sep 18.
Article in English | MEDLINE | ID: mdl-11568055

ABSTRACT

BACKGROUND: Cardiac dysfunction after brain death has been documented, but its mechanisms remain unclear. Myocardial ischemia has been suggested as a possible cause. The aim of the present study was to investigate the existence of an imbalance between myocardial oxygen delivery and demand as a possible cause of myocardial dysfunction in brain-dead pigs. METHODS AND RESULTS: Interstitial myocardial lactate and adenosine concentrations were assessed with cardiac microdialysis in 2 groups of animals: brain-dead pigs (n=7) and brain-dead pigs treated with labetalol (10+/-3 mg/kg) (n=7). Heart rate (HR), left ventricular (LV) dP/dt(max), rate-pressure product (RPP), cardiac output (CO), and left anterior descending coronary artery blood flow (QLAD) were continuously monitored. Brain-dead pigs exhibited a transient significant increase in HR, LV dP/dt(max), RPP, and CO and a limited increase in QLAD. This resulted in functional myocardial ischemia attested to by the significantly increased adenosine and lactate microdialysate concentrations. In brain-dead pigs treated with labetalol, there was a moderate increase in HR, QLAD, and adenosine microdialysate concentrations; LV dP/dt(max), RPP, CO, and myocardial lactate concentrations remained stable, confirming the preservation of aerobic metabolism. CONCLUSIONS: Brain death was associated with an increase in myocardial interstitial adenosine and lactate concentrations, as well as with myocardial dysfunction; all were attenuated by labetalol, suggesting an imbalance between oxygen consumption and oxygen delivery as a possible cause of myocardial dysfunction after brain death.


Subject(s)
Brain Death/physiopathology , Cardiomyopathies/physiopathology , Myocardial Ischemia/diagnosis , Myocardial Ischemia/physiopathology , Adenosine/metabolism , Animals , Blood Flow Velocity/drug effects , Blood Gas Analysis , Blood Pressure , Cardiac Output/drug effects , Cardiomyopathies/complications , Coronary Circulation/drug effects , Heart Rate/drug effects , Labetalol/pharmacology , Lactic Acid/metabolism , Microdialysis , Myocardial Ischemia/complications , Myocardium/metabolism , Oxygen/metabolism , Oxygen Consumption , Swine , Sympatholytics/pharmacology , Ventricular Function, Left/drug effects
3.
J Mol Cell Cardiol ; 33(5): 933-45, 2001 May.
Article in English | MEDLINE | ID: mdl-11343416

ABSTRACT

S. Grosjean, Y. Devaux, C. Seguin, C. Meistelman, F. Zannad, P.-M. Mertes, R. A. Kelly and D. Ungureanu-Longrois. Retinoic Acid Attenuates Inducible Nitric Oxide Synthase (NOS2) Activation in Cultured Rat Cardiac Myocytes and Microvascular Endothelial Cells. Journal of Molecular and Cellular Cardiology (2001) 33, 933-945. The inducible NO synthase (NOS2) in cardiac tissue contributes to myocardial and coronary inflammation and dysfunction. Several natural (endogenous) hormones such as retinoic acid, the active metabolite of vitamin A, have the ability to attenuate NOS2 activation in inflammatory cells. The aim of this study was to investigate the effect of RA on NOS2 activation in cultured cardiac microvascular endothelial cells (CMEC) and adult rat ventricular myocytes (ARVM). CMEC were stimulated either with a combination of 10 microg/ml lipopolysaccharide (LPS) and 50 IU/ml interferon- gamma (IFN- gamma) or with a combination of 1 ng/ml interleukin-1 beta (IL-1 beta)+IFN- gamma whereas ARVM were stimulated with 1 ng/ml IL-1 beta and 50 IU/ml IFN- gamma in the absence or presence of all-trans retinoic acid (atRA). Activation of the NOS2 pathway was estimated by measurement of mRNA (Northern blot) and protein (Western blot) expression, enzyme activity by conversion of [(3)H]L -arginine to [(3)H]L -citrulline, and nitrite accumulation. NOS2 mRNA half-life was studied in CMEC and ARVM in the presence of actinomycin D. In CMEC and ARVM stimulated with a combination of LPS and/or cytokines, atRA (10(-6), 10(-5)M) significantly (P<0.05) attenuated NOS2 mRNA and protein expression, enzymatic activity and reduced supernatant nitrite concentration. Upon stimulation with LPS/IFN- gamma, atRA significantly decreased NOS2 mRNA half-life. This was not seen after stimulation with IL-1 beta/IFN- gamma. These results document for the first time an effect of RA on NOS2 activation in cardiac cells. They may contribute to the characterization of the immunomodulatory effects of retinoids in myocardial and coronary inflammatory disorders.


Subject(s)
Endothelium, Vascular/metabolism , Microcirculation/metabolism , Nitric Oxide Synthase/metabolism , Tretinoin/pharmacology , Animals , Arginine/chemistry , Blotting, Northern , Blotting, Western , Cells, Cultured , Citrulline/chemistry , Dactinomycin/pharmacology , Enzyme Activation , Interferon-gamma/pharmacology , Interleukin-1/pharmacology , Lipopolysaccharides/pharmacology , Macrophages, Alveolar/metabolism , Male , Nitric Oxide Synthase Type II , Nitrites/metabolism , Protein Synthesis Inhibitors/pharmacology , RNA, Messenger/metabolism , Rats , Rats, Wistar , Time Factors , Tretinoin/metabolism
4.
J Chromatogr B Biomed Sci Appl ; 745(2): 279-86, 2000 Aug 18.
Article in English | MEDLINE | ID: mdl-11043747

ABSTRACT

Complex interactions between the L-arginine/nitric oxide synthase (NOS) pathway and the sympathetic nervous system have been reported. Methods capable of measuring L-arginine and norepinephrine (NE) have mainly been reported for plasma. We report the use of the microdialysis technique combined with high-performance liquid chromatography (HPLC) for measurement of both L-arginine and NE within the same tissue microdialysis sample. The microdialysis probe consisted of linear flexible probes (membrane length: 10 mm, outside diameter: 290 microm, molecular weight cut-off 50 kDa). The method used for L-arginine measurement was HPLC with fluorescence detection, giving a within-run and a between-day coefficient of variation of 2.9 and 12.8%, respectively. The detection limit was 0.5 pM/20 microl injected for L-/D-arginine. The method used for NE measurement was HPLC with electrochemical detection. The coefficients of variation were 4% for within-assay precision and 7.5% for between-assay precision. The detection limit for NE was 1 fmol/20 microl injected. The microdialysis technique coupled with HPLC system was validated in vivo to measure muscular interstitial concentrations of both arginine and NE under baseline conditions and after intravenous infusion of 500 mg/kg of L-arginine or D-arginine. In conclusion, the microdialysis technique coupled to HPLC allows the simultaneous measurements of both L-arginine and NE within the same tissue microenvironment and will enable the study of the complex interactions between the L-arginine/NO pathway and sympathetic nervous system within the interstitial space of different organs.


Subject(s)
Arginine/analysis , Chromatography, High Pressure Liquid/methods , Muscles/chemistry , Norepinephrine/chemistry , Animals , Electrochemistry , Hemodynamics , Male , Microdialysis , Rats , Rats, Wistar , Reproducibility of Results
5.
Am J Physiol Endocrinol Metab ; 279(5): E1045-53, 2000 Nov.
Article in English | MEDLINE | ID: mdl-11052959

ABSTRACT

Vitamin A and its metabolite retinoic acid modulate the host response to pathogens through poorly characterized mechanisms. In vitro studies have suggested that retinoic acid decreases inducible NO synthase (NOS2, or iNOS) expression, a component of innate immunity, in several cell types stimulated with lipopolysaccharide (LPS) or cytokines. This study investigated the effect of retinoic acid on LPS-stimulated NOS2 expression in vivo. Wistar-Kyoto rats received all-trans retinoic acid (RA, 10 mg/kg) or vehicle intraperitoneally daily for 5 days followed by LPS (4 mg/kg) or saline intraperitoneally and were killed 6 h later. NOS2 activation was estimated by mRNA (RT-PCR) and protein (Western-blot) expression and plasma nitrate/nitrite accumulation. In sharp contrast to previous in vitro study reports, RA significantly enhanced NOS2 mRNA, protein expression, and plasma nitrate/nitrite concentration in LPS-injected rats but not in saline-injected rats. This was associated with increased expression of interleukin-2, interferon (IFN)-gamma and IFN regulatory factor-1 mRNAs in several organs and increased IFN-gamma plasma concentration. RA significantly increased mortality in LPS-injected rats. The NOS inhibitor aminoguanidine (50 mg/kg before LPS injection) significantly attenuated the RA-mediated increase in mortality. These results demonstrate for the first time that RA supplementation in vivo enhances activation of the LPS-triggered NOS2 pathway.


Subject(s)
Lipopolysaccharides/pharmacology , Nitric Oxide Synthase/metabolism , Tretinoin/pharmacology , Animals , Blotting, Western , DNA-Binding Proteins/genetics , Gene Expression , Interferon Regulatory Factor-1 , Interferon-gamma/genetics , Interleukin-1/genetics , Interleukin-2/genetics , Male , Nitrates/blood , Nitric Oxide Synthase/analysis , Nitric Oxide Synthase/genetics , Nitric Oxide Synthase Type II , Nitrites/blood , Phosphoproteins/genetics , RNA, Messenger/analysis , Rats , Rats, Inbred WKY , Reverse Transcriptase Polymerase Chain Reaction , Salmonella typhimurium , Tumor Necrosis Factor-alpha/genetics
6.
J Mol Cell Cardiol ; 32(3): 493-504, 2000 Mar.
Article in English | MEDLINE | ID: mdl-10731447

ABSTRACT

Adenosine is a potent vasodilator whose concentration has been shown to increase in cardiac tissue in response to hypoxia. However, the time-dependent relationship between the levels of myocardial interstitial adenosine and tissue oxygenation has not yet been completely established. Therefore, the purpose of this study was to investigate the complex relationship between tissue myocardial oxygen tension (PtiO(2)) and interstitial myocardial adenosine and lactate concentrations by developing a new technique which combines a cardiac microdialysis probe and a Clark-type P O(2)electrode. The combined and the single microdialysis probes were implanted in the left ventricular myocardium of anesthetized pigs. The consequences of the combined use of microdialysis and P O(2)probes on myocardial PtiO(2)and microdialysis performances against glucose were evaluated. A moderate but significant reduction in the relative recovery against glucose of the combined probe was observed when compared to that of the single microdialysis probe (42+/-2 v 32+/-1%, mean+/-S.E. M.n=5 P<0.05), at 2microl/min microdialysis probe perfusion flow. Similarly, myocardial oxygen enrichment, measured by the P O(2)electrode, was negligible when microdialysis probe perfusion flow was 2microl/min. Systemic hypoxia (FiO(2)=0.08) resulted in a significant decrease in PtiO(2)from 30+/-4 to 11+/-2 mmHg, limited increase in coronary blood flow (CBF), and a significant increase in myocardial adenosine and lactate concentrations from 0.34+/-0.05 to 0.98+/-0.06micromol/l and from 0.45+/-0.05 to 0.97+/-0.06 mmol/l respectively (P<0.05). Increasing the FiO(2)to 0.3 restored the PtiO(2)and hemodynamic parameters to baseline values with no changes in interstitial adenosine and lactate concentrations. Nevertheless, myocardial interstitial adenosine remained significantly higher than baseline values. In conclusion, this study demonstrates the ability of a combined probe to measure simultaneously regional myocardial PtiO(2)and metabolite concentration during hypoxia. The hypoxia-induced increase in myocardial adenosine persists after correction of hypoxia. The physiological significance of this observation requires further studies.


Subject(s)
Adenosine/metabolism , Lactates/metabolism , Myocardium/metabolism , Oxygen/metabolism , Animals , Blood Gas Analysis , Electrodes , Glucose/metabolism , Hemodynamics , Microdialysis , Swine
8.
Ann Transplant ; 5(4): 54-60, 2000.
Article in English | MEDLINE | ID: mdl-11499363

ABSTRACT

OBJECTIVES: Cardiac dysfunction following brain death is associated with highly increased myocardial norepinephrine, lactate and adenosine concentrations. Administration of labetalol, a mixed alpha-, beta-adrenergic receptor antagonist, attenuates metabolic disturbances and improves myocardial function. The purpose of this study was to investigate beta-adrenergic receptor (beta AR) density and affinity in the presence or absence of labetalol administration, as a possible mechanism of the protective effects of this drug. METHODS: Experimental animals were divided into three groups: sham-operated, brain-dead pigs, and brain-dead pigs treated with labetalol (10 +/- 3 mg/kg). The maximum number of binding sites (Bmax) and the dissociation constant (Kd) of beta AR were determined with (-)-[125I]cyanopindolol on myocardial samples harvested 3 hours after brain death. RESULTS: Left ventricular beta AR density and affinity were identical in brain-dead and sham-operated animals. Labetalol-treated pigs exhibited a significant decrease of Bmax and an increase of Kd as compared with brain-dead pigs. Bmax decrease was due to the persistence of labetalol in the membrane preparations. Increased Kd was too low to be biologically significant. Therefore, beta AR number and affinity can be considered as unchanged after adrenergic blockade with labetalol. CONCLUSIONS: The protective mechanism of labetalol on brain death-induced myocardial dysfunction cannot be explained by changes in beta AR density and affinity but is probably related to a preservation of the oxygen consumption/oxygen delivery balance during the autonomic storm.


Subject(s)
Brain Death/metabolism , Labetalol/administration & dosage , Myocardium/metabolism , Pindolol/analogs & derivatives , Receptors, Adrenergic, beta/drug effects , Adrenergic alpha-Antagonists/pharmacology , Adrenergic beta-Antagonists/pharmacology , Animals , Brain Death/physiopathology , Heart/drug effects , Hemodynamics/drug effects , In Vitro Techniques , Kinetics , Pindolol/metabolism , Swine
9.
Med Biol Eng Comput ; 37(2): 196-201, 1999 Mar.
Article in English | MEDLINE | ID: mdl-10396823

ABSTRACT

Microdialysis is an established technique for measuring the kinetics of various neurotransmitters within the extracellular space in the field of neurochemistry. Recently, its use has been extended to sampling in other tissues, including liver, kidney and the heart. A persistent problem in cardiac microdialysis concerns two parameters related to myocardial function: pressure and frequency (heart rate). The aim of the study is to evaluate the consequences of pressure and frequency on transmembrane exchanges. Linear flexible microdialysis probes (membrane length: 12 mm, outside diameter: 390 microns, MWCO 50,000 Daltons) were designed in our laboratory. The probes, perfused at 2 microL/min with sterile water, were placed in a system filled with a glucose solution (2 g/L) and able to generate either static: 0 to 400 mmHg (0 to 53.31 kPa) or pulsatile pressure: 0-100; 0-200; 0-300 mmHg (0-13.32; 0-26.65; 0-39.98 kPa) at different frequencies: 1, 2 and 3 Hz. At 2 mu litre min-1 perfusion rate, the pressure inside the probe is estimated to be 80 mmHg (10.66 kPa). Under static pressure conditions, the glucose recovery rate can be expressed as an exponential function, and the outflow rate can be expressed as a linear function of the external pressure level. Under dynamic conditions, the external mean pressure must be accounted for. When external mean pressure exceeds 80 mmHg (10.66 kPa) (pressure generated by the flow rate of perfusion inside the probe), the recovery rate increases with frequency. Conversely, if the outer mean pressure is lower than 80 mmHg (10.66 kPa), the recovery rate decreases with frequency. Theoretical and experimental modelling results in a nomogram that can be used to estimate in vivo recovery. In conclusion, mass transfer across a microdialysis membrane is dependent on the direction of the transmembrane pressure gradient and increases with heart rate. These findings must be taken into account when in vivo recovery rates during cardiac microdialysis are determined.


Subject(s)
Heart/physiology , Microdialysis , Models, Cardiovascular , Calibration , Heart Rate , Humans , Microdialysis/instrumentation , Ventricular Pressure
10.
Transplantation ; 66(10): 1278-84, 1998 Nov 27.
Article in English | MEDLINE | ID: mdl-9846509

ABSTRACT

BACKGROUND: Brain death-related cardiovascular dysfunction has been documented; however, its mechanisms remain poorly understood. We investigated changes in myocardial function and metabolism in brain-dead and control pigs. METHODS: Heart rate, systolic (SAP) and mean (MAP) arterial pressure, left ventricular (LV) dP/dtmax, rate-pressure product, cardiac output (CO), left anterior descending coronary artery blood flow, lactate metabolism, and interstitial myocardial purine metabolite concentrations, monitored by cardiac microdialysis, were studied. A volume expansion protocol was performed at the end of the study. RESULTS: After brain death, a transient increase in heart rate (from 90 [67-120] to 158 [120-200] beats/min) (median, with range in brackets), MAP (82 [74-103] to 117 [85-142] mmHg), LV dP/dtmax (1750 [1100-2100] to 5150 [4000-62,000] mmHg x sec(-1), rate-pressure product (9100 [7700-9700] beats mmHg/min to 22,750 [20,000-26,000] beats mmHg/min), CO (2.2 [2.0-4.0] to 3.3 [3.0-6.0] L/min), and a limited increase in left anterior descending coronary artery blood flow (40 [30-60] to 72 [50-85] ml/min) were observed. Net myocardial lactate production occurred (27 [4-40] to -22 [-28, -11] mg/L, P<0.05) and persisted for 2 hr. A 6-7-fold increase in adenosine dialysate concentration was observed after brain death induction (2.9 [1.0-5.8] to 15.8 [7.0-50.7] micromol/L), followed by a slow decline. Volume expansion significantly increased MAP, CO, and LV dP/dtmax in control animals, but decreased LV dP/dtmax and slightly increased CO in brain-dead animals. A significant increase in adenosine concentration was observed in both groups, with higher levels (P<0.05) in brain-dead animals. CONCLUSIONS: Brain death increased oxygen demand in the presence of a limited increase in coronary blood flow, resulting in net myocardial lactate production and increased interstitial adenosine concentration consistent with an imbalance between myocardial oxygen demand and supply. This may have contributed to the early impairment of cardiac function in brain-dead animals revealed by rapid volume infusion.


Subject(s)
Adenosine/metabolism , Brain Death/metabolism , Lactic Acid/metabolism , Myocardium/metabolism , Animals , Blood Gas Analysis , Brain Death/physiopathology , Hemodynamics , Lactic Acid/blood , Microdialysis , Swine , Time Factors
13.
Article in English | MEDLINE | ID: mdl-11253781

ABSTRACT

Contractile dysfunction of the respiratory muscles plays an important role in the genesis of respiratory failure during sepsis. Nitric oxide (NO), a free radical that is cytotoxic and negatively inotropic in the heart and skeletal muscle, is produced in large amounts during sepsis by a NO synthase inducible (iNOS) by LPS and/or cytokines. The aim of this study was to investigate whether iNOS was induced in the diaphragm of Escherichia coli endotoxemic rats and whether inhibition of iNOS induction or of NOS synthesis attenuated diaphragmatic contractile dysfunction. Rats were inoculated intravenously (IV) with 10 mg/kg of E. coli endotoxin (LPS animals) or saline (C animals). Six hours after LPS inoculation animals showed a significant increase in diaphragmatic NOS activity (L-citrulline production, P < 0.005). Inducible NOS protein was detected by Western-Blot in the diaphragms of LPS animals, while it was absent in C animals. LPS animals had a significant decrease in diaphragmatic force (P < 0.0001) measured in vitro. In LPS animals, inhibition of iNOS induction with dexamethasone (4 mg/kg IV 45 min before LPS) or inhibition of NOS activity with N(G)-methyl-L-arginine (8 mg/kg IV 90 min after LPS) prevented LPS-induced diaphragmatic contractile dysfunction. We conclude that increased NOS activity due to iNOS was involved in the genesis of diaphragmatic dysfunction observed in E. coli endotoxemic rats.


Subject(s)
Diaphragm/physiopathology , Endotoxemia/physiopathology , Escherichia coli Infections/physiopathology , Muscle Contraction/physiology , Nitric Oxide/physiology , omega-N-Methylarginine/pharmacology , Animals , Dexamethasone/pharmacology , Diaphragm/drug effects , Endotoxins/pharmacology , Enzyme Inhibitors/pharmacology , Glucocorticoids/pharmacology , Male , Nitric Oxide Synthase/antagonists & inhibitors , Rats , Rats, Sprague-Dawley
14.
J Mol Cell Cardiol ; 29(2): 677-87, 1997 Feb.
Article in English | MEDLINE | ID: mdl-9140825

ABSTRACT

Nitric oxide (NO) has been shown to be a ubiquitous intercellular autacoid in the heart and, in cultured rat ventricular myocytes, to decrease the contractile responsiveness to isoproterenol (ISO). The aim of the present study was to investigate whether exogenous (sodium nitroprusside, SNP) or endogenous nitric oxide generated from L-arginine modulated the response to ISO in cultured chick embryo ventricular myocytes. SNP 1 microM or L-arginine 1 mM had no effect on baseline contractile function. Superfusion with ISO 100 nM significantly increased myocyte amplitude of shortening to 1.31 +/- 0.06 (ratio to baseline amplitude). Initial superfusion with SNP 1 microM or L-arginine 1 mM attenuated the response to ISO to 0.89 +/- 0.05 and 1.09 +/- 0.07 respectively (P < 0.05). Potassium ferrocyanide which is not a NO donor and D-arginine the inactive substrate of NO synthase did not attenuate the response to ISO. Myocyte cGMP content was significantly increased by incubation with SNP 1 microM (31.65 +/- 3 fmol/well) but not by L-arginine 1 mM (11.1 +/- 0.3 fmol/well) as compared to myocytes incubated in control medium (11 +/- 0.9 fmol/well). Preincubation with SNP 1 microM or L-arginine 1 mM significantly attenuated the ISO mediated-increase in cAMP content from 4.33 +/- 0.2 pmol/well (ISO 100 nM alone) to 1.48 +/- 0.36 fmol/well and 1.72 +/- 0.21 pmol/well respectively. Potassium ferrocyanide and D-arginine had no effect on myocyte cGMP or cAMP content. Chick embryo myocytes have measurable and LNMMA-inhibited NO synthase activity as measured by the conversion of [3H] L-arginine to [3H] L-citrulline. In conclusion, these results demonstrate that in cultured chick embryo ventricular myocytes both exogenous and endogenous NO elevate cGMP. This may account for the inhibition of beta-adrenergic agonist-stimulated increases in cAMP and amplitude of shortening via an unidentified intracellular negative coupling.


Subject(s)
Heart Ventricles/cytology , Myocardial Contraction/drug effects , Nitric Oxide/metabolism , Nitric Oxide/pharmacology , Adrenergic beta-Agonists/pharmacology , Animals , Arginine/metabolism , Arginine/pharmacology , Calcium/metabolism , Calcium/pharmacology , Chick Embryo , Cyclic AMP/chemistry , Cyclic AMP/metabolism , Ferrocyanides/pharmacology , Heart Ventricles/embryology , Isoproterenol/pharmacology , Nitric Oxide Synthase/metabolism , Nitroprusside/pharmacology , Nucleotides, Cyclic/chemistry , Nucleotides, Cyclic/metabolism , Substrate Specificity , Vasodilator Agents/pharmacology
15.
Am J Physiol ; 272(2 Pt 2): H958-68, 1997 Feb.
Article in English | MEDLINE | ID: mdl-9124460

ABSTRACT

Basic fibroblast growth factor (bFGF; fibroblast growth factor-2) and angiotensin II (ANG II), among other peptide signaling autacoids (cytokines), are known to regulate the phenotypic adaptation of cardiac muscle to physiological stress. The cell type(s) in cardiac muscle responsible for ANG II synthesis and secretion and the role of endogenous cytokines in the regulation of bFGF induction remain unclear. With the use of confluent, serum-starved, low-passage cultures of cardiac microvascular endothelial cells (CMEC), ANG II could be detected in cellular lysates and in medium conditioned by these cells with the use of high-performance liquid chromatography followed by radioimmunoassay. The secretion of angiotensins by individual CMEC could be detected with a cell-blot assay technique. ANG II secretion was decreased by brefeldin A, an agent that interrupts constitutive and regulated secretory pathways for peptide autacoid/ hormone synthesis, suggesting de novo synthesis, activation, and secretion of angiotensins by CMEC. In primary isolates of adult rat ventricular myocytes (ARVM) and CMEC, ANG II, acting at ANG II type 1 receptors in both cell types, was found to increase bFGF mRNA levels measured by ribonuclease protection assay. Endothelin-1 (ET-1), which is known to be synthesized by CMEC, and bFGF itself, which has been detected in both ARVM and CMEC, increased bFGF transcript levels in both cell types. Interleukin-1beta (IL-1beta), which like ANG II and ET-1 is known to activate mitogen-activated protein kinases in both ARVM and CMEC, increased bFGF mRNA levels only in cardiac myocytes. Thus cytokines such as ANG II, ET-1, bFGF, and IL-1beta locally generated by cellular constituents of cardiac muscle, including CMEC, regulate bFGF mRNA levels in a cell type-specific manner.


Subject(s)
Angiotensin II/metabolism , Coronary Circulation , Endothelium, Vascular/metabolism , Fibroblast Growth Factor 2/metabolism , Myocardium/metabolism , Angiotensin II/pharmacology , Animals , Cells, Cultured , Endothelin-1/pharmacology , Endothelium, Vascular/cytology , Fibroblast Growth Factor 2/genetics , Fibroblast Growth Factor 2/pharmacology , Heart Ventricles , Microcirculation , Myocardium/cytology , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley
16.
J Clin Invest ; 98(7): 1550-9, 1996 Oct 01.
Article in English | MEDLINE | ID: mdl-8833903

ABSTRACT

Nitric oxide (NO), a free radical that is negatively inotropic in the heart and skeletal muscle, is produced in large amounts during sepsis by an NO synthase inducible (iNOS) by LPS and/or cytokines. The aim of this study was to examine iNOS induction in the rat diaphragm after Escherichia Coli LPS inoculation (1.6 mg/kg i.p.), and its involvement in diaphragmatic contractile dysfunction. Inducible NOS protein and activity could be detected in the diaphragm as early as 6 h after LPS inoculation. 6 and 12 h after LPS, iNOS was expressed in inflammatory cells infiltrating the perivascular spaces of the diaphragm, whereas 12 and 24 h after LPS it was expressed in skeletal muscle fibers. Inducible NOS was also expressed in the left ventricular myocardium, whereas no expression was observed in the abdominal, intercostal, and peripheral skeletal muscles. Diaphragmatic force was significantly decreased 12 and 24 h after LPS. This decrease was prevented by inhibition of iNOS induction by dexamethasone or by inhibition of iNOS activity by N(G)-methyl-L-arginine. We conclude that iNOS was induced in the diaphragm after E. Coli LPS inoculation in rats, being involved in the decreased muscular force.


Subject(s)
Diaphragm/drug effects , Endotoxins/pharmacology , Muscle Contraction/drug effects , Nitric Oxide Synthase/biosynthesis , Abdominal Muscles/enzymology , Animals , Cyclic GMP/analysis , Dexamethasone/pharmacology , Diaphragm/enzymology , Diaphragm/physiopathology , Enzyme Induction , Heart Ventricles/enzymology , Immunohistochemistry , Intercostal Muscles/enzymology , Male , Muscle, Skeletal/enzymology , Myocardium/enzymology , Rats , Rats, Sprague-Dawley , Stereoisomerism , Tissue Distribution , omega-N-Methylarginine/pharmacology
17.
J Biol Chem ; 271(39): 23928-37, 1996 Sep 27.
Article in English | MEDLINE | ID: mdl-8798625

ABSTRACT

The cytokine-inducible isoform of nitric oxide synthase (iNOS or NOS2) plays an important role in the immune response to some pathogens. Within the heart, increased activity of NOS2 in cardiac microvascular endothelial cells (CMEC) also can diminish the contractile function of adjacent cardiac myocytes. Glucocorticoids, which are known to suppress cytokine induction of NOS2 in many cell types, caused only a moderate (approximately 20%) decline in NOS2 protein content and maximal activity measured in homogenates of cytokine-treated CMEC, but almost completely inhibited synthesis of nitrogen oxides (NOx) by intact cells. To determine whether glucocorticoids were inhibiting cellular NOx production by limiting the availability of NOS co-factors or substrate, the effect of dexamethasone on tetrahydrobiopterin (BH4) and L-arginine availability in cytokine-treated CMEC was examined. Dexamethasone prevented the coordinate induction of GTP cyclohydrolase I with NOS2 after exposure to interleukin-1beta and interferon-gamma and also the increase in intracellular BH4 content in cytokine-treated CMEC. Addition of BH4 overcame dexamethasone-mediated suppression of nitrite production. Dexamethasone also prevented a cytokine-mediated increase in L-arginine uptake into CMEC by suppressing the induction of the high affinity cationic amino acid transporters CAT-1 and CAT-2B and the low affinity CAT-2A transporter. In addition, dexamethasone also inhibited cytokine induction in CMEC of argininosuccinate synthase, the rate-limiting enzyme for the de novo synthesis of arginine from citrulline. Thus, glucocorticoids regulate NOx production following cytokine exposure in cardiac microvascular endothelial cells primarily by limiting BH4 and L-arginine availability.


Subject(s)
Arginine/metabolism , Biopterins/analogs & derivatives , Dexamethasone/pharmacology , Glucocorticoids/pharmacology , Nitric Oxide Synthase/biosynthesis , Nitric Oxide/metabolism , Animals , Argininosuccinate Lyase/metabolism , Argininosuccinate Synthase/metabolism , Biological Transport/drug effects , Biopterins/biosynthesis , Biopterins/metabolism , Cells, Cultured , Cytokines/pharmacology , GTP Cyclohydrolase/genetics , Gene Expression Regulation, Enzymologic/drug effects , Interferon-gamma/pharmacology , Interleukin-1/pharmacology , Male , Nitrites/metabolism , Rats , Rats, Sprague-Dawley , Recombinant Proteins
18.
Circ Res ; 77(3): 486-93, 1995 Sep.
Article in English | MEDLINE | ID: mdl-7543825

ABSTRACT

Unlike large-vessel endothelial cells in cell culture, cardiac microvascular endothelial cells (CMEC) isolated from adult rat ventricular muscle exhibit little detectable constitutive nitric oxide (NO) synthase activity after isolation in vitro but respond to specific combinations of inflammatory mediators with an increase in inducible NO synthase (iNOS; type 2 NO synthase) activity. CMEC iNOS is induced by soluble inflammatory mediators in lipopolysaccharide-activated rat alveolar macrophage-conditioned medium at 24 hours, and this induction can be partially prevented by either interleukin-1 (IL-1) receptor antagonist or a polyclonal anti-rat tumor necrosis factor-alpha (TNF-alpha) antiserum. Interferon-gamma (IFN-gamma), which by itself does not induce iNOS in CMEC, potentiates and accelerates iNOS induction by IL-1 beta. Transforming growth factor-beta (TGF-beta) decreases iNOS activity, protein content, and mRNA abundance in IL-1 beta- and IFN-gamma-pretreated CMEC. To determine whether NO released by CMEC would affect myocyte contractile function in vitro, freshly isolated ARVM were allowed to settle onto confluent, serum-starved CMEC that had been pretreated for 24 hours with IL-1 beta, a cytokine that alone does not affect myocyte contractile function in vitro. Baseline contractile amplitude, at 2 Hz and 37 degrees C, of myocytes in heterotypic culture with IL-1 beta-pretreated CMEC was not different from that of myocytes in control, homotypic myocyte cultures. However, cocultured myocytes exhibited decreased contractile responsiveness to 2 nmol/L isoproterenol compared with control cells, and this could be reversed by the addition of 1 mmol/L NG-monomethyl-L-arginine, an inhibitor of NOS.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Amino Acid Oxidoreductases/biosynthesis , Endothelium, Vascular/enzymology , Isoproterenol/pharmacology , Myocardial Contraction/drug effects , Animals , Cells, Cultured , Heart Ventricles , Interleukin-1/pharmacology , Lipopolysaccharides/pharmacology , Male , Nitric Oxide/physiology , Nitric Oxide Synthase , Rats , Rats, Sprague-Dawley , Transforming Growth Factor beta/pharmacology
19.
Circ Res ; 77(3): 494-502, 1995 Sep.
Article in English | MEDLINE | ID: mdl-7543826

ABSTRACT

Recent evidence has documented that increased activity of an inducible nitric oxide synthase (iNOS; type 2 NO synthase) in primary isolates of adult rat ventricular myocytes after exposure to soluble mediators in medium conditioned by lipopolysaccharide-activated macrophages is associated with a decrease in their contractile responsiveness to beta-adrenergic agonists. It remained unclear which specific inflammatory cytokines in this medium contribute to the induction of iNOS activity in myocytes and whether induction of iNOS would result in an obligatory decline in contractile function. Interleukin (IL)-1 beta and tumor necrosis factor-alpha (TNF-alpha) were both present in the lipopolysaccharide-activated macrophage-conditioned medium. However, only IL-1 receptor antagonist and not an anti-rat TNF-alpha antiserum diminished the extent of iNOS induction in myocytes exposed to this medium and prevented a decline in contractile responsiveness to isoproterenol. When recombinant cytokines were used, IL-1 beta, TNF-alpha, and IFN-gamma each induced iNOS activity in cardiac myocytes at 24 hours. However, only the combination of IL-1 beta and IFN-gamma reproducibly caused contractile dysfunction in cardiac myocytes. Among the constituents of the defined medium routinely used for maintenance of adult rat ventricular myocytes in primary culture, it was noted that insulin (10(-7) mol/L) was required for NO production, as detected by nitrite release in cytokine-pretreated myocytes, although insulin had no effect on the extent of induction of iNOS mRNA or maximal enzyme activity in myocyte cell lysates.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Adrenergic beta-Agonists/pharmacology , Amino Acid Oxidoreductases/biosynthesis , Cytokines/pharmacology , Myocardial Contraction/drug effects , Myocardium/enzymology , Animals , Insulin/pharmacology , Interferon-gamma/pharmacology , Interleukin 1 Receptor Antagonist Protein , Lipopolysaccharides/pharmacology , Macrophages/physiology , Male , Nitric Oxide Synthase , Rats , Rats, Sprague-Dawley , Sialoglycoproteins/pharmacology , Tumor Necrosis Factor-alpha/pharmacology
20.
Am J Physiol ; 268(3 Pt 2): H1293-303, 1995 Mar.
Article in English | MEDLINE | ID: mdl-7535006

ABSTRACT

There are important phenotypic differences between endothelial cells of large vessels and the microvasculature and among microvascular endothelial cells isolated from different tissues and organs. In contrast to most macrovascular endothelial cells, we demonstrate that cultured cardiac microvascular endothelial cells (CMEC) have no detectable constitutive NO synthase (NOS) activity but have a robust increase in NOS activity in response to specific inflammatory cytokines. To determine the identity of the inducible NOS (iNOS) isoform(s) induced by cytokines, we used reverse-transcription polymerase chain reaction techniques to clone and sequence a 217-bp cDNA fragment from CMEC cultures pretreated with interleukin-1 beta (IL-1 beta) and interferon-gamma (IFN-gamma) that was identical to the corresponding portion of the murine macrophage iNOS cDNA. By use of this CMEC iNOS cDNA as a probe in Northern analyses, IL-1 beta, but not IFN-gamma, increased iNOS mRNA content in CMEC, although IFN-gamma markedly potentiated iNOS induction in these cells. In IL-1 beta- and IFN-gamma-pretreated CMEC, dexamethasone only minimally suppressed the rise in iNOS mRNA, protein abundance, or maximal iNOS enzyme activity in whole cell lysates but suppressed nitrite production by 60% in intact CMEC. Dual labeling of cytokine-pretreated CMEC in primary culture with an anti-iNOS antiserum and a fluorescein-labeled lectin specific for the microvascular endothelium of rat heart (GS-1) confirmed the presence of iNOS expression in these cells. iNOS was also detected in microvascular endothelium in situ in ventricular muscle from lipopolysaccharide-, but not sham-injected, rat hearts.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Amino Acid Oxidoreductases/biosynthesis , Interferon-gamma/pharmacology , Interleukin-1/pharmacology , Myocardium/enzymology , Amino Acid Oxidoreductases/genetics , Animals , Base Sequence , Cells, Cultured , Cloning, Molecular , DNA, Complementary/genetics , Endothelium, Vascular/drug effects , Endothelium, Vascular/enzymology , Enzyme Induction/drug effects , Immunohistochemistry , Mice , Microcirculation/drug effects , Microcirculation/enzymology , Molecular Sequence Data , Nitric Oxide Synthase , Polymerase Chain Reaction , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats
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