Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0022116 (ischemia)
 91,303 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Calcium tolerant pig and rabbit cardiomyocytes were isolated using retrograde aortic perfusion of nominally calcium-free collagenase. Preconditioning protocols used 1 or 3x10-min episodes of ischemic pelleting or pre-incubation with 100 micro M adenosine, followed by a 15-min post-incubation and 180-240-min ischemic pelleting. Control cells were incubated and washed in parallel with the experimental groups. Injury was assessed by determination of cell morphology, trypan blue permeability following osmotic swelling, lactate and HPLC analysis of adenine nucleotides. Preconditioned pig cardiomyocytes had a reduced rate of ischemic contracture, but protection occurred without conservation of ATP. Preconditioned rabbit cardiomyocytes were protected without significant changes in rates of ischemic contracture or ATP depletion. Incubation of ischemic cells with the protein phosphatase inhibitor, fostriecin, at PP2A-selective concentrations (0.1-10 micro M), mimicked preconditioning in both rabbit and pig cardiomyocytes. In rabbits, the KATP channel blocker, 5-hydroxydecanoate (5-HD), did not block preconditioning or fostriecin protection. In the pig, 5-HD blocked both preconditioning and fostriecin protection, with return of the rates of ischemic contracture to control. However, 5-HD was an effective blocker of protection only in early ischemia. Fostriecin mimicked preconditioning in the rabbit and the early responses of the preconditioned pig. Preconditioning appears associated with protein phosphorylation in both the rabbit and the pig, but major pathways leading to protection may differ in the two species.
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PMID:Comparison of in vitro preconditioning responses of isolated pig and rabbit cardiomyocytes: effects of a protein phosphatase inhibitor, fostriecin. 940 76

Calcium-tolerant rabbit cardiomyocytes were isolated using retrograde aortic perfusion with a nominally calcium-free, collagenase buffer. In vitro ischemic preconditioning was induced by a 10-min episode of ischemic pelleting, followed by a 15-min post-incubation and a prolonged period of ischemic pelleting. Injury was assessed by determination of cell contracture and trypan blue permeability following hypotonic swelling and correlated with metabolic assays of lactate and adenine nucleotides. The protein phosphatase PP1/2A inhibitor calyculin A and PP2A-selective fostriecin protected isolated rabbit cardiomyocytes from lethal injury after a 10-min pre-incubation and when added late into ischemic pellets after a delay of 75 min. At the time of late drug addition, cells were severely ATP-depleted and in rigor contracture. Protection with Calyculin A from 1 nM to 1 microM was dose-related. Cells pre-incubated with 10 nM to 10 microM fostriecin 10 min prior to ischemic pelleting were protected with an EC50 approximating 71 nM, implying protection at a PP2A-selective dose. The selective protein kinase C inhibitor, calphostin C, blocked ischemic preconditioning protection but not protection from 1 microM calyculin A. Protection of severely ischemic cardiomyocytes following protein phosphatase inhibition appears not to require PKC activity or ATP conservation. Pre-incubation of cells with calyculin A induced high levels of phosphorylation in p38 mitogen activated protein kinase (MAPK), as compared to the ischemia-induced phosphorylation observed in the untreated group only at 30 min of ischemia, providing evidence of protein phosphatase activity in cardiomyocytes. Pharmacological protection in late ischemia has been demonstrated, but the mechanism of protection is undetermined.
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PMID:Protein phosphatase inhibitors calyculin A and fostriecin protect rabbit cardiomyocytes in late ischemia. 950 Aug 65

Adenosine plays an important role in protection of the heart before, during and after ischemia. Nucleoside transport inhibitors (NTI) increase adenosine concentration without inducing ischemia by preventing its uptake and metabolism in cardiac cells. However, prolonged effects of nucleoside transport inhibitors on adenosine and nucleotide metabolism and its combined effect with nucleotide precursors has not been established in cardiomyocytes. The aim of this study was to investigate the effect of two nucleoside transport inhibitors, dipyridamole (DIPY) and nitrobenzylthioinosine (NBTI) alone or combined with adenine and ribose on adenosine production and ATP content in cardiomyocytes. Rat cardiomyocytes were isolated using collagenase perfusion technique. Isolated cell suspensions were incubated for up to 480 min with different substrates and inhibitors as follows: (1) control; (2) 100 microM adenine and 2.5 mM ribose; (3) 10 microM DIPY; (4) 1 microM NBTI; (5) DIPY, adenine and ribose and (6) NBTI, adenine and ribose. Five microM EHNA (erythro-9(2-hydroxy-3-nonyl)adenine, an inhibitor of adenosine deaminase) was added to all incubations. After incubation, extracts of myocyte suspension were analysed by HPLC for adenine nucleotides and metabolite concentrations. ATP content decreased in cardiomyocytes after 8 h of incubation with DIPY, while no change was observed with NBTI or without inhibitors. Adenosine concentration increased with both DIPY and NBTI. In the presence of adenine and ribose an elevation in ATP concentration was observed, but no significant change in adenosine content. In the presence of DIPY or NBTI together with adenine and ribose, an enhancement in cardiomyocyte ATP concentration was observed together with an increase in adenosine content. This increase in adenosine production was especially prominent with DIPY. In conclusion, dipyridamole causes a decrease in ATP concentration in isolated cardiomyocytes by mechanisms other than nucleoside transport inhibition. Addition of adenine/ribose with dipyridamole prevents the depletion of ATP. Combination of adenine/ribose with nucleoside transport inhibitors may also further enhance adenosine concentration and thus, could be more effective as pharmacological agents for treatment.
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PMID:Effects of nucleoside transport inhibitors and adenine/ribose supply on ATP concentration and adenosine production in cardiac myocytes. 954 46

Previous studies have demonstrated that muscle can take up and express naked DNA or RNA. This study demonstrates that the pDNA can be delivered to and expressed within skeletal muscle when injected rapidly, in a large volume and when all blood vessels leading into and out of the hindlimb are occluded. The additional use of collagenase, papaverine and ischemia raised expression moderately but was not critical. These results demonstrate that a nonviral method can lead to high levels of expression in the muscles of adult animals large than mice.
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PMID:The efficient expression of intravascularly delivered DNA in rat muscle. 957 48

Ischemia/reperfusion events alter the cellular ion homeostasis by intracellular acidosis and a subsequent rise of sodium and calcium concentrations. Since disturbance of intracellular Ca2+ signaling pathways impairs cellular function, we investigated the effect of sodium bicarbonate infusion on hepatocellular Ca2+ dysregulation induced by resuscitation from hemorrhagic shock. Anesthetized Sprague-Dawley rats were bled to a mean arterial blood pressure of 40 mmHg for 60 min. Rats were resuscitated by retransfusion of shed blood (60%) in 20 min and three-fold the bleed out volume as lactated Ringers' during 60 min and received either a bolus infusion of sodium bicarbonate (2 mval/kg body weight) or an equal volume of sodium chloride (0.9%). After hepatocyte isolation by portal collagenase perfusion, the rate of Ca2+ influx (Ca2+in) in the absence and presence of epinephrine (100 nM), cellular Ca2+ uptake (Ca2+up) and membrane Ca2+ flux (Ca2+flux) were determined using 45Ca2+ incubation techniques. Hemorrhage/resuscitation substantially increased hepatocyte Ca2+up (3.44 +/- 0.2 nmol/mg protein) and Ca2+flux (32.8 +/- 5 pmol/mg protein x min) compared to sham-operated controls (2.57 +/- 0.1 and 15.2 +/- 3.5; P < 0.05). Resuscitation with sodium bicarbonate significantly prevented altered hepatocyte Ca2+ regulation (2.31 +/- 0.1 and 14.4 +/- 4.6; P < 0.05). These findings suggested that postischemic hepatocyte Ca2+ overload could partly be due to enhanced membrane Ca2+ movements to correct for altered intracellular pH homeostasis.
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PMID:Effect of sodium bicarbonate infusion on hepatocyte Ca2+ overload during resuscitation from hemorrhagic shock. 966 35

Ultrastructural studies of stunned myocardium have shown disorganization and loss of extracellular collagen and increased collagenase activity early after ischemia and reperfusion. The interplay between matrix metalloproteinase 1 (MMP-1) and tissue inhibitor of metalloproteinase 1 (TIMP-1) regulates the turnover of cardiac extracellular matrix fibrillar collagens. However, the gene expression of MMP-1 and TIMP-1 in stunned myocardium is not known. Here, we determined whether altered expression of MMP-1 and TIMP-1 occurs in globally stunned hearts. An isolated nonworking rabbit heart preparation, perfused with a bovine erythrocyte suspension in modified Krebs solution, was used. Two groups were studied: the stunned group was subjected to 20 min of normothermic global ischemia followed by 120 min of normal reperfusion (n = 8), and the control group underwent 140 min of uninterrupted perfusion (n = 7). The developed pressures at the end of reperfusion for ischemic and control hearts were 67.0 +/- 2.73 and 83.1 +/- 1.52 mm Hg (P < 0. 006) respectively. Ribonuclease protection assays of total left ventricular RNA using riboprobes for MMP-1, TIMP-1, and 18S rRNA were performed. A significant decrease (twofold, P < 0.03) in TIMP-1 gene expression was found in the stunned hearts, while MMP-1 mRNA expression was unchanged. Thus, in early stunning, the decrease in TIMP-1 expression could tip the balance favoring enhanced metalloproteinase activity, promoting collagen turnover, and initiating extracellular matrix remodeling. This may contribute to delayed recovery from myocardial stunning.
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PMID:Decreased expression of tissue inhibitor of metalloproteinase 1 in stunned myocardium. 969 29

We studied the presence of collagen degrading enzymes (matrix metalloproteinases, MMPs) in porcine myocardium following ischemia and late reperfusion. In nine pigs, left anterior descending coronary artery was occluded for 6 h followed by reperfusion for 3 h. Six pigs without coronary occlusion served as controls. After the reperfusion period, transmural biopsies from the anterior (ischemic zone) and posterior wall (non-ischemic myocardium) in the left ventricle were obtained and extracted. Heparin-Sepharose isolated components in extracts were analysed for collagenase (triple-helical collagen degradation) and gelatinase activity (zymography). Immunohistochemistry using anti-human (MMP-1, MMP-2, MMP-9, and fibronectin) antibodies was performed on additional biopsies. Collagenase (MMP-1) and gelatinases (MMP-2, MMP-9) could be demonstrated in the extracts of non-ischemic myocardium from ischemic/reperfused as well as control pigs and MMP-1 and MMP-9 activity was found to be increased in ischemic/reperfused myocardium compared with non-ischemic myocardium. In ischemic/reperfused myocardium from live pigs investigated, myocyte necrosis could be confirmed by fibronectin immunoreaction in myocytes and MMP-1 and MMP-9 immunoreactions were increased. MMP-9 was present in cells likely to be infiltrating leukocytes in a patchy distribution throughout the ischemic myocardium. Quite coincident with MMP-9 positive cells, MMP-1 immunoreaction appeared in necrotic myocytes, in addition to reactions observed in vessel walls, endo- and epicardium, and extracellular matrix in non-ischemic myocardium. Thus, the results showed increased amounts of collagenase (MMP-1) and gelatinase (MMP-9) in ischemic/ reperfused myocardium, indicating the appearance of increased amounts of collagen degrading enzymes very early following ischemia and late reperfusion.
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PMID:Increased amounts of collagenase and gelatinase in porcine myocardium following ischemia and reperfusion. 971 Aug 10

Tumor necrosis factor-alpha (TNF-alpha) is a key mediator of shock-induced cellular and humoral inflammatory cascades. The present study investigated the role of TNF-alpha in oxidative membrane injury and altered hepatocyte Ca2+ regulation, both of which are critical steps in cellular dysfunction during ischemia/reperfusion events. Hemorrhagic shock was induced by bleeding male Sprague-Dawley rats (200-250 g, n=6/group) to a mean arterial blood pressure of 40 mmHg for 60 min. Rats were resuscitated with 60% of shed blood and twice the shed blood volume as Ringers' lactate. At the end of hemorrhage and 60 min after resuscitation, hepatocytes were isolated by liver collagenase perfusion. Hepatocyte Ca2+ uptake (Ca2+up) and Ca2+ membrane flux (Ca2+flux) were determined by 45Ca2+ incubation techniques. Hepatocyte reduced/oxidized glutathione and lipid peroxidation were determined fluorometrically. Both hemorrhage and hemorrhage/resuscitation significantly increased hepatocyte Ca2+up and Ca2+flux. The monoclonal chimeric mouse gamma1 TNF-alpha antibody (TN3gamma1.19.12; 20 mg/kg b.w.) given with resuscitation significantly decreased hepatocyte Ca2+up and Ca2+flux and prevented hepatocyte lipid peroxidation. These findings suggest that oxidative membrane injury could be the result of TNF-alpha modulation of hepatocellular Ca2+ regulation during hemorrhage/resuscitation.
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PMID:Monoclonal antibody to tumor necrosis factor-alpha modulates hepatocellular Ca2+ homeostasis during hemorrhagic shock in the rat. 993 Sep 19

Effective intraductal delivery of the enzyme collagenase into the pancreas is crucial to the subsequent ability to isolate viable islets. Most clinical islet transplant centers load the enzyme into the pancreas by retrograde injection using a syringe following cannulation of the pancreatic duct. An alternative approach is to perfuse the pancreas via the pancreatic duct with collagenase solution using a recirculating perfusion device system. This provides control over perfusion pressures and collagenase temperature. This study reports on our evaluation of the delivery of Liberase-HI into the pancreas of 14 consecutive adult multiorgan cadaveric donors. Alternate glands were procured and processed using an identical protocol with the exception of collagenase delivery. The first group of pancreases was loaded using the perfusion technique where cold (4 degrees C) Liberase-HI was perfused at 80 mmHg for 5 min after which the pressure was increased to 180 mmHg. The collagenase solution was then slowly warmed to 35 degrees C, transferred to the dissociation chamber and mechanically dissociated, and then purified using discontinuous gradients of Ficoll. Pancreases in the second group were loaded with collagenase (28-32 degrees C) using the syringe technique before mechanical dissociation and purification. There were no significant differences in pancreas cold ischemia, donor age, body mass index, maximum blood glucose, or serum amylase of the donors between the two groups. Mean collagenase digestion time in the digestion chamber was not different between the two groups; however, the amount of undigested tissue remaining after dissociation was significantly higher in the syringe-loaded group (15.3 +/- 2.6 g vs. 4.6 +/- 2.1 g, mean +/- SEM, p < 0.05). Postdigestion recovery of islets was 471 +/- 83 x 10(3) IE in the perfusion group compared with 391 +/- 57 x 10(3) IE for the syringe-loaded group. Postpurification recovery was higher in the perfused group (379 +/- 45 vs. 251 +/- 28 x 10(3) IE, p < 0.05, two-tailed paired t-test). No difference in in vitro islet viability was observed between the two groups following glucose perifusion with the calculated stimulation index of 4.6 +/- 0.6 for the perfusion group and 4.2 +/- 0.7 for the syringe-loaded group. Controlled perfusion via the pancreatic duct allows the effective delivery of the enzyme achieving maximal distension to all regions of the pancreas leading to an increased recovery of the islets with no detrimental effect on subsequent in vitro islet function.
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PMID:Intraductal collagenase delivery into the human pancreas using syringe loading or controlled perfusion. 1044 41

We report the average insulin response to acute glucose measured by in vitro perifusion of pancreatic islets isolated from 80 consecutive human organs. Different perifusion parameters were considered [basal release, stimulation index (SI), time to peak, incremental area under the curve delta-AUC alpha)], and the correlation among them was determined. SI positively correlated with delta-AUC alpha (p < 0.001, r = 0.80) while negatively with time to peak (p < 0.05, r = -0.23). We also evaluated several variables of the isolation procedure that might affect responsiveness to glucose by human islets. Sex and age of pancreas donors, cold ischemia time, duration of the digestion, collagenase concentration, and lot characteristics (collagenase, trypsin, clostripain, and proteases activity), and final islet yield were considered. Multivariate regression analysis showed only an independent association between SI and the concentration of collagenase (p = 0.01).
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PMID:Lessons from in vitro perifusion of pancreatic islets isolated from 80 human pancreases. 1070 99


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