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1.
Transplant Proc ; 44(8): 2300-3, 2012 Oct.
Article in English | MEDLINE | ID: mdl-23026579

ABSTRACT

To study whether treatment with heparin (HEP) attenuates intestinal dysfunction caused by ischemia (I) and reperfusion (R), rats were treated with HEP (100 U/kg intravenously) or saline solution (SS) before I (60 min), which was produced by occlusion of the superior mesenteric artery, and R (120 min). After I or I/R, we mounted 2-cm jejunal segment in an organ bath to study neurogenic contractions stimulated by electrical pulses or KCl, using a digital recording system. Thin jejunal slices were stained with hematoxylin and eosin for optical microscopy. Compared with the sham group, jejunal contractions were similar in the I + HEP and the I/R + HEP groups, but reduced in the I + SS and the I/R + SS groups. The jejunal enteric nerves were damaged in the I + SS and the I/R + SS, but not in the I + HEP and the I/R + HEP cohorts. These results suggested that HEP attenuated intestinal dysfunction caused by I and I/R.


Subject(s)
Gastrointestinal Agents/pharmacology , Heparin/pharmacology , Jejunum/blood supply , Jejunum/drug effects , Reperfusion Injury/prevention & control , Animals , Cytoprotection , Disease Models, Animal , Electric Stimulation , Enteric Nervous System/drug effects , Enteric Nervous System/physiopathology , Gastrointestinal Motility/drug effects , Jejunum/innervation , Jejunum/pathology , Jejunum/physiopathology , Male , Potassium Chloride/pharmacology , Rats , Rats, Wistar , Reperfusion Injury/pathology , Reperfusion Injury/physiopathology
2.
Transplant Proc ; 44(8): 2304-8, 2012 Oct.
Article in English | MEDLINE | ID: mdl-23026580

ABSTRACT

To study whether ischemic preconditioning (IPC) attenuated intestinal dysfunction caused by ischemia (I) and reperfusion (R), rats were underwent 60 minutes of I which was produced by occlusion of the superior mesenteric artery, and/or 120 minutes R. The IPC group had the I procedure previously stimulated for 5 minutes and the R for 10 minutes. IPC and sham groups were injected with saline solution (SS) via the femoral vein 5 minutes before the I and R, and for R. After I or I/R, 2-cm jejunal segments were mounted in an organ bath to study neurogenic contractions stimulated by electrical pulses or KCl using a digital recording system. Thin jejunal slices were stained with hematoxylin and eosin for optical microscopy. Compared with the sham group, jejunal contractions were similar in the IPC + I and the IPC + I/R groups, but reduced in the I + SS and the I/R + SS groups. The jejunal enteric nerves were damaged in the I + SS and the I/R + SS groups, but not in the IPC groups. These results suggested that ischemic preconditioning attenuated intestinal dysfunction caused by I and I/R.


Subject(s)
Ischemic Preconditioning , Jejunum/blood supply , Reperfusion Injury/prevention & control , Animals , Disease Models, Animal , Electric Stimulation , Enteric Nervous System/physiopathology , Gastrointestinal Motility , Jejunum/drug effects , Jejunum/innervation , Jejunum/pathology , Jejunum/physiopathology , Male , Potassium Chloride/pharmacology , Rats , Rats, Wistar , Reperfusion Injury/pathology , Reperfusion Injury/physiopathology
3.
Transplant Proc ; 44(8): 2309-12, 2012 Oct.
Article in English | MEDLINE | ID: mdl-23026581

ABSTRACT

To examine whether treatment with L-arginine (ARG), a substrate of nitric oxide biosynthesis, attenuated intestinal dysfunction caused by ischemia (I) and reperfusion (R), we treated rats with ARG (100 mg/kg intravenously) or saline solution (SS) before 60 minutes of I produced by occlusion of the superior mesenteric artery and/or during 120 minutes of R. After I or I/R, we isolated 2-cm jejunal segments for mounting in an organ bath to study neurogenic contractions stimulated by electrical pulses or KCl with the use of a digital recording system. Thin jejunal slices were stained with hematoxylin and eosin for optical microscopy. Jejunal contractions were similar in the sham and I+ARG, but reduced in I+SS, I/R+SS, and I/R+ARG groups. Jejunal enteric nerves were damaged in I+SS, IR+SS, and IR+ARG, but not in the I+ARG group, suggesting that ARG attenuate intestinal dysfunctions due to I but not to R.


Subject(s)
Arginine/pharmacology , Gastrointestinal Agents/pharmacology , Jejunum/blood supply , Jejunum/drug effects , Reperfusion Injury/drug therapy , Animals , Cytoprotection , Disease Models, Animal , Electric Stimulation , Enteric Nervous System/drug effects , Enteric Nervous System/physiopathology , Gastrointestinal Motility/drug effects , Jejunum/innervation , Jejunum/pathology , Jejunum/physiopathology , Male , Potassium Chloride/pharmacology , Rats , Rats, Wistar , Reperfusion Injury/pathology , Reperfusion Injury/physiopathology
4.
Transplant Proc ; 44(8): 2317-20, 2012 Oct.
Article in English | MEDLINE | ID: mdl-23026583

ABSTRACT

To study whether treatment with adenosine (ADO), an agonist of adenosine receptors, attenuates intestinal dysfunction caused by ischemia (I) and reperfusion (R), we treated rats with ADO (15 mg/kg or saline solution (SS) intravenously before 60 minutes occlusion of the superior mesenteric artery (I) and/or 120 minutes after its release (R). After I or I/R, isolated jejunal segments (2 cm) were mounted in an organ bath to study nerve-mediated contractions stimulated by electrical pulses or KCI with the use of a digital recording system. Thin jejunal slices were stained with hematoxylin and eosin for optical microscopy. Compared with the sham group, jejunal contractions were reduced in I+SS and IR+SS but similar after treatment with ADO (I+ADO and IR+ADO groups). We concluded that rat jejunal enteric nerves were damaged in I+SS and IR+SS but not in the I+ADO and IR+ADO groups. These results suggested that ADO attenuated intestinal dysfunction due to I and R.


Subject(s)
Adenosine/pharmacology , Gastrointestinal Agents/pharmacology , Jejunum/blood supply , Jejunum/drug effects , Reperfusion Injury/prevention & control , Animals , Cytoprotection , Disease Models, Animal , Electric Stimulation , Enteric Nervous System/drug effects , Enteric Nervous System/physiopathology , Gastrointestinal Motility/drug effects , Jejunum/innervation , Jejunum/pathology , Jejunum/physiopathology , Potassium Chloride/pharmacology , Rats , Rats, Wistar , Reperfusion Injury/pathology , Reperfusion Injury/physiopathology
5.
Transplant Proc ; 44(8): 2313-6, 2012 Oct.
Article in English | MEDLINE | ID: mdl-23026582

ABSTRACT

To study whether treatment with the beta-blocker atenolol (AT) attenuates intestinal dysfunction caused by ischemia (I) and reperfusion (R), rats were treated with AT (1.5 mg · kg(-1), intravenously) or saline solution (SS) prior to I (60 minutes), which was produced by occlusion of the superior mesenteric artery, and/or R (120 minutes). After I or I/R, 2-cm jejunal segments were mounted in an organ bath to study neurogenic contractions stimulated by electrical pulses or KCl using a digital recording system. Thin jejunal slices were stained with hematoxylin and eosin for optical microscopy analysis. Compared to the sham group, jejunal contractions were similar in the I + AT and the I/R + AT groups, but reduced in the I + SS and the I/R + SS groups. The jejunal enteric nerves were damaged in the I + SS and the I/R + SS groups, but not in the I + AT and the I/R + AT. These results suggest that AT may attenuate intestinal dysfunction caused by I and I/R.


Subject(s)
Adrenergic beta-1 Receptor Antagonists/pharmacology , Atenolol/pharmacology , Gastrointestinal Agents/pharmacology , Jejunum/blood supply , Jejunum/drug effects , Reperfusion Injury/prevention & control , Animals , Cytoprotection , Disease Models, Animal , Electric Stimulation , Enteric Nervous System/drug effects , Enteric Nervous System/physiopathology , Gastrointestinal Motility/drug effects , Jejunum/innervation , Jejunum/pathology , Jejunum/physiopathology , Male , Potassium Chloride/pharmacology , Rats , Rats, Wistar , Reperfusion Injury/pathology , Reperfusion Injury/physiopathology
6.
Transplant Proc ; 42(2): 448-50, 2010 Mar.
Article in English | MEDLINE | ID: mdl-20304161

ABSTRACT

To study whether treatment with L-arginine (ARG), a substrate of nitric oxide biosynthesis, attenuates intestinal dysfunction caused by ischemia (I) and reperfusion (R), rabbits treated with ARG (100 mgxkg(-1), intravenously) or saline solution (SS) prior to I (60 minutes) by occlusion of superior mesenteric artery and/or during R (120 minutes). After I or I/R, 2-cm jejunal segments were isolated and mounted in an organ bath to study of neurogenic contractions stimulated by electrical pulses or KCl using a digital recording system. Thin jejunal slices were stained (hematoxylin and eosin) for analysis by optical microscopy. Compared to the sham group, jejunal contractions were similar in I + ARG, but reduced in I + SS, I/R + SS, and I/R + ARG groups. The jejunal enteric nerves were damaged in I + SS, I/R + SS, and I/R + ARG, but not in I + ARG group, suggesting that ARG can attenuate intestinal dysfunctions due to I, but not to R.


Subject(s)
Arginine/pharmacology , Intestines/blood supply , Nitric Oxide/biosynthesis , Reperfusion Injury/prevention & control , Animals , Arginine/therapeutic use , Blood Circulation , Femoral Vein/physiology , Gastrointestinal Motility/drug effects , Gastrointestinal Motility/physiology , Ischemia/physiopathology , Jejunum/blood supply , Jejunum/drug effects , Jejunum/pathology , Mesenteric Artery, Superior/physiology , Rabbits , Reperfusion/adverse effects , Reperfusion Injury/physiopathology
7.
Transplant Proc ; 42(2): 454-6, 2010 Mar.
Article in English | MEDLINE | ID: mdl-20304163

ABSTRACT

To study if the treatment with adenosine (ADO), an agonist of adenosine receptors, attenuates intestinal dysfunction caused by ischemia (I) and reperfusion (R), we treated rabbits with ADO (15 mg x kg(-1), intravenously) or saline solution (SS) to I (60 minutes) before occlusion of superior mesenteric artery and/or R (120 min). After I or I/R, isolated jejunal segments (2 cm) were mounted in an organ bath to study nerve-mediated contractions stimulated by electrical pulses or KCl using a digital recording system. Thin jejunal slices were stained (hematoxylin and eosin) for analysis by optical microscopy. Compared to the sham group, the jejunal contractions were similar in I + ADO, but reduced in I + SS, I/R + SS, and I/R + ADO groups. We concluded that the jejunal enteric nerves were damaged in I + SS, I/R + SS, and I/R + ADO, but not in I + ADO group. These results suggested that ADO attenuated intestinal dysfunction due to I, but not to R.


Subject(s)
Adenosine/pharmacology , Intestines/blood supply , Reperfusion Injury/drug therapy , Animals , Blood Circulation , Electric Stimulation , Femoral Vein/drug effects , Femoral Vein/physiology , Jejunum/blood supply , Jejunum/drug effects , Jejunum/physiology , Male , Mesenteric Artery, Superior/physiology , Muscle Contraction/drug effects , Muscle Contraction/physiology , Potassium Chloride/pharmacology , Purinergic P1 Receptor Agonists , Rabbits , Sodium Chloride/pharmacology
8.
Transplant Proc ; 42(2): 457-60, 2010 Mar.
Article in English | MEDLINE | ID: mdl-20304164

ABSTRACT

To study whether treatment with L-nitro-arginine methyl ester (L-NAME), an inhibitor of nitric oxide biosynthesis, attenuates intestinal dysfunction caused by ischemia (I) and/or reperfusion (R), rabbits were treated with L-NAME (15 mgxkg(-1), intervenously) or saline olution (SS) prior to I (60 minutes) induced by occlusion of superior mesenteric artery and/or R (120 minutes). After I or I/R, isolated jejunal segments (2 cm) were mounted in an organ bath to study nerve-mediated contractions stimulated by electrical pulses or KCI using a digital recording system. Thin jejunal slices were stained (hematoxylin and eosin) for analysis by optical microscopy. Compared with a sham group, the jejunal contractions were similar in the I/R + L-NAME, but reduced in I + SS, I/R + SS, and I + L-NAME groups. The jejunal enteric nerves were damaged in the I + SS, I/R + SS, and I + L-NAME cohorts, but not among the I/R + L-NAME cohort. These results suggested that L-NAME attenuated intestinal dysfunction caused by R but not by I.


Subject(s)
Gastrointestinal Motility/drug effects , Intestinal Diseases/prevention & control , NG-Nitroarginine Methyl Ester/pharmacology , Reperfusion Injury/prevention & control , Reperfusion Injury/physiopathology , Animals , Electric Stimulation , Ischemia/physiopathology , Jejunum/drug effects , Jejunum/innervation , Jejunum/physiology , Male , Muscle Contraction/drug effects , Muscle Contraction/physiology , Rabbits , Sodium Chloride/pharmacology
9.
Transplant Proc ; 42(2): 461-4, 2010 Mar.
Article in English | MEDLINE | ID: mdl-20304165

ABSTRACT

To study whether treatment with 5'-adenosine triphosphate (ATP), an agonist of P2 purine receptors, attenuated intestinal dysfunction caused by ischemia (I) and/or reperfusion (R), rabbits were treated with ATP (15 mgxkg(-1), intravenously) or saline solution (SS) 60 minutes before I by occlusion of the superior mesenteric artery and/or R (120 minutes). After I or I/R isolated 2-cm jejunal segments were mounted in an organ bath to study nerve-mediated contractions stimulated by electrical pulses or KCl using a digital recording system. Thin jejunal slices were stained (hematoxylin and eosin) for optical microscopy. Compared to a sham group, the jejunal contractions were similar to sham hosts among I + ATP, but reduced in I + SS, I/R + SS, and I/R + ATP groups. The jejunal-enteric nerves were damaged in I + SS, I/R + SS, and I/R + ATP, but not the I + ATP group. These results suggested that ATP attenuated intestinal dysfunction produced by I, but not that caused by R.


Subject(s)
Adenosine Triphosphate/pharmacology , Intestines/blood supply , Ischemia/physiopathology , Jejunum/blood supply , Reperfusion Injury/physiopathology , Animals , Blood Circulation/drug effects , Blood Circulation/physiology , Ischemia/drug therapy , Jejunum/drug effects , Jejunum/innervation , Male , Mesenteric Artery, Superior/drug effects , Mesenteric Artery, Superior/physiopathology , Rabbits , Sodium Chloride/pharmacology
10.
Transplant Proc ; 41(3): 807-11, 2009 Apr.
Article in English | MEDLINE | ID: mdl-19376358

ABSTRACT

In this work, we evaluate the effects of adenosine 5' triphosphate (ATP) on hepatic lesions caused by ischemia/reperfusion (I/R) in liver rabbit. Rabbits were pretreated with ATP (15 mg/kg IV) or saline solution 0.9% (SS), before the hepatic I/R procedure. We evaluated the effects of ATP on hepatic injury before and after I/R. The warm hepatic I/R procedure caused profound acute liver injury, as indicated by elevated serum aspartate aminotransferase, alanine aminotransferase, and lactic dehydrogenase levels, as well as a high apoptotic cell count. All these changes were attenuate by ATP treatment before the hepatic I/R procedure. These results suggested that ATP exerted protective effects on hepatic I/R lesions in the rabbit. This ATP effect may be related to improved energy metabolism during reperfusion in ischemic livers protecting against functional damage of cellular and subcellular membranes during lipid peroxidation.


Subject(s)
Liver Diseases/physiopathology , Purines/metabolism , Reperfusion Injury/physiopathology , Adenosine Triphosphate/therapeutic use , Alanine Transaminase/drug effects , Alanine Transaminase/metabolism , Animals , Aspartate Aminotransferases/drug effects , Aspartate Aminotransferases/metabolism , Ischemia/physiopathology , L-Lactate Dehydrogenase/drug effects , L-Lactate Dehydrogenase/metabolism , Liver/drug effects , Liver/physiopathology , Liver Diseases/prevention & control , Male , Mitochondria, Liver/drug effects , Mitochondria, Liver/metabolism , Rabbits , Reperfusion Injury/prevention & control
11.
Transplant Proc ; 41(3): 816-9, 2009 Apr.
Article in English | MEDLINE | ID: mdl-19376360

ABSTRACT

We evaluated the effects of a substrate in the biosynthesis of nitric oxide (NO)-l-arginine (LARG)-on hepatic lesions caused by ischemia/reperfusion (I/R) injury in rabbit livers. Rabbits were pretreated with LARG (150 mg/kg IV) or saline solution 0.9% (SS) before the hepatic I/R procedure. The effects of LARG on hepatic injury were evaluated before and after I/R. The warm hepatic I/R procedure produced profound acute liver injury, as indicated by elevated values of serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactic dehydrogenase (LDH), as well as a high apoptotic cell count. All changes were attenuated by treatment with LARG before the hepatic I/R procedure. These results suggested that LARG produced protective effects on hepatic I/R lesions. This protective effect of LARG was probably associated with blocking generation of superoxide anions during the hepatic I/R procedure.


Subject(s)
Arginine/therapeutic use , Liver Diseases/prevention & control , Reperfusion Injury/prevention & control , Alanine Transaminase/blood , Alanine Transaminase/drug effects , Animals , Aspartate Aminotransferases/blood , Aspartate Aminotransferases/drug effects , L-Lactate Dehydrogenase/blood , L-Lactate Dehydrogenase/drug effects , Liver Circulation/drug effects , Male , Nitric Oxide/metabolism , Rabbits , Reperfusion Injury/enzymology , Reperfusion Injury/pathology , Vasoconstriction/drug effects
12.
Transplant Proc ; 41(3): 812-5, 2009 Apr.
Article in English | MEDLINE | ID: mdl-19376359

ABSTRACT

Because the role of heparin (HEP) in hepatic ischemia/reperfusion (I/R) injury is still not fully understood, we investigated the effects of treatment with HEP on hepatic I/R injury in rabbits. For I/R procedures, the portal vein and hepatic artery were occluded by a metallic clamp to promote ischemia. The clamp was removed after 30 minutes to allow reperfusion. Rabbits undergoing the I/R procedure were treated with HEP (100 U/kg) or saline solution 0.9% (SS). When compared with levels before I/R, the serum aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase, levels were increased by the hepatic I/R procedure, among rabbits treated with SS or HEP. However, the increase in these enzymes was lower among rabbits treated with HEP. Histologic analysis of hepatic tissue of rabbits undergoing I/R and treated with SS showed marked lesions in the central lobule with significant inflammatory infiltration. In contrast, a significant reduction in lesions caused by I/R was observed in the livers of rabbits treated with HEP. After starting reperfusion, we visualized apoptotic cells with nuclear staining among rabbits submitted to I/R and treated with SS, but not those treated with HEP. These results suggested that HEP was able to attenuate hepatic lesions caused by I/R in the livers of rabbits.


Subject(s)
Heparin/therapeutic use , Ischemia/drug therapy , Liver Diseases/drug therapy , Reperfusion Injury/prevention & control , Alanine Transaminase/drug effects , Alanine Transaminase/metabolism , Animals , Aspartate Aminotransferases/drug effects , Aspartate Aminotransferases/metabolism , Disease Models, Animal , Fibrinolytic Agents/therapeutic use , Ischemia/enzymology , L-Lactate Dehydrogenase/drug effects , L-Lactate Dehydrogenase/metabolism , Liver/enzymology , Liver Diseases/enzymology , Male , Rabbits , Reperfusion Injury/enzymology
13.
Transplant Proc ; 41(3): 820-3, 2009 Apr.
Article in English | MEDLINE | ID: mdl-19376361

ABSTRACT

In this work, we evaluated the effects of allopurinol (ALO), an inhibitor of xanthine oxidase (XO), on hepatic lesions caused by ischemia/reperfusion (I/R) in the rabbit liver. Rabbits were pretreated with ALO (10 mg/kg IV) or saline solution 0.9% before the hepatic I/R procedure. The effects of ALO on hepatic injury were evaluated before and after I/R. A standard, warm hepatic I/R procedure caused profound acute liver injury, as indicated by elevated serum aspartate aminotransferase, alanine aminotransferase, and lactic dehydrogenase levels, as well as a high apoptotic cell count. All of these changes were reversed by the administration of ALO before the hepatic I/R procedure. In conclusion, ALO exerted protective effects on hepatic I/R lesions. This protective effect of ALO was probably associated with blocking the generation of superoxide anions during the hepatic I/R procedure by inhibiting XO activity.


Subject(s)
Allopurinol/therapeutic use , Liver Diseases/prevention & control , Reperfusion Injury/prevention & control , Alanine Transaminase/blood , Alanine Transaminase/drug effects , Animals , Aspartate Aminotransferases/blood , Aspartate Aminotransferases/drug effects , Enzyme Inhibitors/therapeutic use , L-Lactate Dehydrogenase/blood , L-Lactate Dehydrogenase/drug effects , Male , Rabbits , Xanthine Oxidase/antagonists & inhibitors
14.
Nitric Oxide ; 10(1): 1-10, 2004 Feb.
Article in English | MEDLINE | ID: mdl-15050529

ABSTRACT

Among the many initiating events that lead to apoptosis or programmed cell death, loss of contact between the cell and the extracellular matrix has been extensively studied. Adhesion-related apoptosis referred to as anoikis is initiated by the action of anti-adhesive substances. Nitric oxide is one of these anti-adhesive substances that have the capacity to signal and trigger pro-apoptotic events in a variety of cell types. Nitric oxide can inhibit cell adhesion, interfere with the assembly of focal adhesion complexes, and disrupt the cell-extracellular matrix interactions. These actions occur in cell that exhibit a dissociation of growth factor signals from alterations in the cytoskeleton, ultimately leading to apoptosis. Since this involves anti-adhesive events, nitric oxide can be considered as causing anoikis. This review article summarizes the available evidence of how nitric oxide participates in apoptosis induced by loss of anchorage (anoikis).


Subject(s)
Anoikis/drug effects , Nitric Oxide/pharmacology , Animals , Cell Adhesion/drug effects , Humans , Mitochondria/chemistry , Mitochondria/metabolism , Mitochondria/physiology , Signal Transduction
15.
Braz J Med Biol Res ; 35(2): 181-90, 2002 Feb.
Article in English | MEDLINE | ID: mdl-11847521

ABSTRACT

Previous studies have shown that exogenously generated nitric oxide (NO) inhibits smooth muscle cell proliferation. In the present study, we stimulated rabbit vascular smooth muscle cells (RVSMC) with E. coli lipopolysaccharide (LPS), a known inducer of NO synthase transcription, and established a connection between endogenous NO, phosphorylation/dephosphorylation-mediated signaling pathways, and DNA synthesis. Non-confluent RVSMC were cultured with 0, 5, 10, or 100 ng/ml of the endotoxin. NO release was increased by 86.6% (maximum effect) in low-density cell cultures stimulated with 10 ng/ml LPS as compared to non-stimulated controls. Conversely, LPS (5 to 100 ng/ml) did not lead to enhanced NO production in multilayered (high density) RVSMC. DNA synthesis measured by thymidine incorporation showed that LPS was mitogenic only to non-confluent RVSMC; furthermore, the effect was prevented statistically by aminoguanidine (AG), a potent inhibitor of the inducible NO synthase, and oxyhemoglobin, an NO scavenger. Finally, there was a cell density-dependent LPS effect on protein tyrosine phosphatase (PTP) and ERK1/ERK2 mitogen-activated protein (MAP) kinase activities. Short-term transient stimulation of ERK1/ERK2 MAP kinases was maximal at 12 min in non-confluent RVSMC and was prevented by preincubation with AG, whereas PTP activities were inhibited in these cells after 24-h LPS stimulation. Conversely, no significant LPS-mediated changes in kinase or phosphatase activities were observed in high-density cells. LPS-induced NO generation by RVSMC may switch on a cell density-dependent proliferative signaling cascade, which involves the participation of PTP and the ERK1/ERK2 MAP kinases.


Subject(s)
DNA/biosynthesis , Escherichia coli , Lipopolysaccharides/pharmacology , Mitogen-Activated Protein Kinases/metabolism , Muscle, Smooth, Vascular/drug effects , Nitric Oxide/metabolism , Protein Tyrosine Phosphatases/metabolism , Animals , Blotting, Western , Cell Count , ErbB Receptors , Muscle, Smooth, Vascular/cytology , Phosphorylation , Precipitin Tests , Rabbits
16.
Braz. j. med. biol. res ; 35(2): 181-190, Feb. 2002. ilus, graf
Article in English | LILACS | ID: lil-303545

ABSTRACT

Previous studies have shown that exogenously generated nitric oxide (NO) inhibits smooth muscle cell proliferation. In the present study, we stimulated rabbit vascular smooth muscle cells (RVSMC) with E. coli lipopolysaccharide (LPS), a known inducer of NO synthase transcription, and established a connection between endogenous NO, phosphorylation/dephosphorylation-mediated signaling pathways, and DNA synthesis. Non-confluent RVSMC were cultured with 0, 5, 10, or 100 ng/ml of the endotoxin. NO release was increased by 86.6 percent (maximum effect) in low-density cell cultures stimulated with 10 ng/ml LPS as compared to non-stimulated controls. Conversely, LPS (5 to 100 ng/ml) did not lead to enhanced NO production in multilayered (high density) RVSMC. DNA synthesis measured by thymidine incorporation showed that LPS was mitogenic only to non-confluent RVSMC; furthermore, the effect was prevented statistically by aminoguanidine (AG), a potent inhibitor of the inducible NO synthase, and oxyhemoglobin, an NO scavenger. Finally, there was a cell density-dependent LPS effect on protein tyrosine phosphatase (PTP) and ERK1/ERK2 mitogen-activated protein (MAP) kinase activities. Short-term transient stimulation of ERK1/ERK2 MAP kinases was maximal at 12 min in non-confluent RVSMC and was prevented by preincubation with AG, whereas PTP activities were inhibited in these cells after 24-h LPS stimulation. Conversely, no significant LPS-mediated changes in kinase or phosphatase activities were observed in high-density cells. LPS-induced NO generation by RVSMC may switch on a cell density-dependent proliferative signaling cascade, which involves the participation of PTP and the ERK1/ERK2 MAP kinases


Subject(s)
Animals , Rabbits , DNA , Lipopolysaccharides , Mitogen-Activated Protein Kinases , Muscle, Smooth, Vascular , Nitric Oxide , Protein Tyrosine Phosphatases , Blotting, Western , Cell Count , Escherichia coli , Phosphorylation , Precipitin Tests , ErbB Receptors
17.
Atherosclerosis ; 157(2): 505-12, 2001 Aug.
Article in English | MEDLINE | ID: mdl-11472753

ABSTRACT

Increased monocyte adherence to the vessel wall is one of the earliest events in atherosclerosis. The mechanism by which hypercholesterolemia causes alterations in endothelial adhesiveness for monocytes is unclear. This study sought to determine if monocyte adhesion molecule expression is affected by low-density lipoprotein (LDL)-cholesterol levels. Patients with hypercholesterolemia and stable coronary artery disease were compared with those without major cardiovascular risk (control). Patients with hypercholesterolemia were treated with simvastatin 20--40 mg/day for 8--10 weeks. Blood samples were examined with flow cytometry assays at baseline and after cholesterol-lowering therapy. Monocyte CD11b and CD14 adhesion molecule expression, measured as fluorescence intensity, were significantly (P<0.0001) higher in hypercholesterolemic patients before the study (176.9+/-9.8 and 138.0+/-4.8, respectively) when compared with that in control subjects (97.2+/-8.1 and 84.0+/-6.4, respectively). Both decreased markedly with treatment: to 118.8+/-6.9 and 103.1+/-3.9, respectively. Monocyte L-selectin expression was significantly lower in patients with hypercholesterolemia before treatment (43.0+/-3.0) when compared with control subjects (79.9+/-2.7), and it increased markedly with treatment (54.2+/-2.5). LDL levels correlated directly with both CD11b and CD14 expression and correlated inversely with L-selectin expression. These data show that hypercholesterolemia affects monocyte adhesion molecule expression which, in turn, decreases with statin-induced plasmatic cholesterol reduction. Such perturbations in monocyte function likely represent a proinflammatory response to hypercholesterolemia and may have a role in the early progression of atherogenesis.


Subject(s)
Anticholesteremic Agents/therapeutic use , Cell Adhesion Molecules/blood , Hypercholesterolemia/blood , Hypercholesterolemia/drug therapy , Monocytes/metabolism , Simvastatin/therapeutic use , Adult , Coronary Disease/complications , Female , Humans , Hypercholesterolemia/complications , L-Selectin/blood , Lipopolysaccharide Receptors/blood , Macrophage-1 Antigen/blood , Male , Middle Aged , Reference Values
18.
Exp Parasitol ; 99(4): 190-7, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11888245

ABSTRACT

Insulin-like growth factor (IGF)-I constitutively present in the skin is one of the first growth factors that Leishmania parasites encounter after transmission to the vertebrate host. We have previously shown that IGF-I is a potent growth-promoting factor for Leishmania parasites. IGF-I binds specifically to a single-site putative receptor at the parasite membrane, triggering a cascade of phosphorylation reactions. In the present article we characterize the receptor for IGF-I on Leishmania (Leishmania) mexicana promastigotes. The receptor is a monomeric glycoprotein with a molecular mass of 65 kDa and is antigenically related to the alpha chain of human type 1 IGF-I receptor. Upon IGF-I stimulation the receptor undergoes autophosphorylation on tyrosine residues with activation of its signaling pathway. Activation of the IGF-I receptor also leads to phosphorylation of an 185-kDa molecule that is homologous to the substrate of the insulin receptor present in human cells, the insulin receptor substrate 1 (IRS-1).


Subject(s)
Leishmania mexicana/metabolism , Receptors, Somatomedin/chemistry , Animals , Chromatography, Affinity , Chromatography, Gel , Molecular Weight , Phosphorylation , Precipitin Tests , Receptors, Somatomedin/immunology , Receptors, Somatomedin/metabolism
19.
Free Radic Res ; 35(6): 843-56, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11811535

ABSTRACT

Alpha-tocopherol augmentation in human neutrophils was investigated for effects on neutrophil activation and tyrosine phosphorylation of proteins, through its modulation of protein kinase C (PKC) and tyrosine phosphatase activities. Incubation of neutrophils with alpha-tocopherol succinate (TS) resulted in a dose-dependent incorporation into cell membranes, up to 2.5 nmol/2x10(6) cells. A saturating dose of TS (40 micromol/l) inhibited oxidant production by neutrophils stimulated with phorbol myristate acetate (PMA) or opsonized zymosan (OZ) by 86 and 57%, as measured by luminol-amplified chemiluminescence (CL). With PMA, TS inhibited CL generation to a similar extent to staurosporine (10 nmol/l) or genistein (100 micromol/l), and much more than Trolox (40 micromol/l). With OZ, TS inhibited CL to a similar extent to Trolox. Neutrophil PKC activity was inhibited 50% or more by TS or staurosporine. The enzyme activity was unaffected by genistein or Trolox, indicating a specific interaction of alpha-tocopherol. TS or Trolox increased protein tyrosine phosphorylation in resting neutrophils, and as with staurosporine further increased tyrosine phosphorylation in PMA-stimulated neutrophils, while the tyrosine kinase (TK) inhibitor genistein diminished phosphorylation. These effects in resting or PMA-stimulated neutrophils were unrelated to protein tyrosine phosphatase (PTP) activities, which were maintained or increased by TS or Trolox. In OZ-stimulated neutrophils, on the other hand, all four compounds inhibited the increase in tyrosine-phosphorylated proteins. In this case, the effects of pre-incubation with TS or Trolox corresponded with partial inhibition of the marked (85%) decrease in PTP activity induced by OZ. These results indicate that alpha-tocopherol inhibits PMA-activation of human neutrophils by inhibition of PKC activity, and inhibits tyrosine phosphorylation and activation of OZ-stimulated neutrophils also through inhibition of phosphatase inactivation.


Subject(s)
Neutrophils/drug effects , Neutrophils/metabolism , Phosphotyrosine/metabolism , Protein Kinase C/antagonists & inhibitors , Protein Tyrosine Phosphatases/metabolism , alpha-Tocopherol/pharmacology , Chromans/pharmacology , Enzyme Activation/drug effects , Humans , Immunoblotting , Neutrophils/enzymology , Oxidants/metabolism , Ozone/pharmacology , Phosphorylation/drug effects , Tetradecanoylphorbol Acetate/pharmacology
20.
Free Radic Biol Med ; 28(2): 174-82, 2000 Jan 15.
Article in English | MEDLINE | ID: mdl-11281284

ABSTRACT

Nitric oxide (NO) can participate in cellular signaling. In this study, monoclonal antibodies against proteins from the growth factor-mediated signalling pathway were used to identify a set of 126-, 56-, 43-, and 40-kDa proteins phosphorylated on tyrosine at NO stimulation of murine fibroblasts overexpressing the human epidermal growth factor receptor. The band corresponding to the 126-kDa protein was FAK. The 56-kDa protein was Src kinase, and the doublet 43- and 40-kDa protein corresponded to the extracellular-regulated MAP kinases (ERK1/ERK2). The effects of NO on focal adhesion complexes were also investigated. FAK was constitutively associated with the adapter protein Grb2 in HER14 cells. Treatment of the cells with the NO donor, sodium nitroprusside, or with EGF did not change this association. We also detected a basal constitutive association of Src kinase with FAK in HER14 cells. In NO-treated cells, this association was stimulated. The doublet 43/40-kDa protein was identical to the ERK1/ERK2 MAP kinases. NO stimulated an increase in ERK1/ERK2 phosphorylation as assessed by a shift in its eletrophoretic mobility and by increased phosphotyrosine immunoreactivity. Furthermore, NO-dependent activation of ERK1/ERK2 depended on the intracellular redox status. Inhibition of glutathione synthesis was necessary to promote activation of the kinases.


Subject(s)
Adaptor Proteins, Signal Transducing , Mitogen-Activated Protein Kinases/metabolism , Nitric Oxide Donors/pharmacology , Nitric Oxide/physiology , Nitroprusside/pharmacology , Protein-Tyrosine Kinases/metabolism , src-Family Kinases/metabolism , Animals , Buthionine Sulfoximine/pharmacology , Cell Line , Epidermal Growth Factor/pharmacology , Fibroblasts/enzymology , Focal Adhesion Kinase 1 , Focal Adhesion Protein-Tyrosine Kinases , GRB2 Adaptor Protein , Mice , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3 , Molecular Weight , Penicillamine/analogs & derivatives , Penicillamine/pharmacology , Phosphoproteins/isolation & purification , Phosphorylation , Phosphotyrosine/metabolism , Proteins/metabolism , Signal Transduction , Triazenes/pharmacology
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