Salient effects of L-carnitine on adenine-nucleotide loss and coenzyme A acylation in the diabetic heart perfused with excess palmitic acid. A phosphorus-31 NMR and chemical extract study.

The beneficial effects of L-carnitine perfusion on energy metabolism and coenzyme A acylation were studied in isolated hearts from control and diabetic rats. All hearts were perfused at a constant flow rate with a glucose/albumin buffer which contained 2.0 mM palmitate. 31P-NMR was utilized to assess sequential phosphocreatine and ATP metabolism during 1 h of recirculation perfusion. L-Carnitine (5.0 mM final concentration) was added after 12 min of baseline recirculation perfusion. Frozen samples were taken after 1 h of recirculation perfusion for spectrophotometric analysis of high-energy phosphates and the free and acylated fractions of coenzyme A. L-Carnitine perfusion of diabetic hearts attenuated or prevented the reduction of ATP observed in untreated diabetic hearts. It also attenuated the accumulation of long-chain fatty-acyl coenzyme A. Although L-carnitine improved myocardial function in diabetic hearts, this was independent of any direct effect on physiological indices. Thus, the salutory effect of acute perfusion with L-carnitine on energy metabolism in the isolated perfused diabetic rat heart appears to be a direct effect on lipid metabolism.

Lipid-mediated impairment of normal energy metabolism in the isolated perfused diabetic rat heart studied by phosphorus-31 NMR and chemical extraction.

The relationship between extracellular palmitate and the accumulation of long-chain fatty-acyl coenzyme A with that of high-energy phosphate metabolism was investigated in the isolated perfused diabetic rat heart. Hearts were perfused with a glucose/albumin buffer supplemented with 0, 0.5, 1.2 or 2.0 mM palmitate. 31P-NMR was used to analyze phosphocreatine and ATP metabolism during 1 h of constant-flow recirculation perfusion. At the end of perfusion, frozen samples were taken for chemical analysis of high-energy phosphates and the free and acylated fractions of coenzyme A and carnitine. Perfusion of diabetic hearts with palmitate, unlike control hearts, caused a time-dependent and concentration-dependent reduction in ATP, despite normal and constant phosphocreatine. Concentrations of acid-soluble coenzyme A, long-chain-acyl coenzyme A and total tissue coenzyme A were elevated in palmitate-perfused diabetic hearts, while the total tissue carnitine pool was decreased. Increases in long-chain-acyl coenzyme A correlated with the reduction in myocardial ATP. This reduction in ATP could not be adequately explained by alterations in heart rate, perfusion pressure or vascular resistance.

Syngeneic pancreatic islet transplantation reverses endothelial dysfunction in experimental diabetes.

Diabetes is known to cause impaired endothelium-dependent relaxation of blood vessels. The purpose of this study was to determine whether this endothelial dysfunction is a permanent defect or is reversible after acute arginine supplementation in vitro or by surgical intervention in vivo using syngeneic pancreatic islet transplantation. Lewis rats were injected with streptozotocin to induce diabetes and were studied either 8 or 12 weeks later. Another group received syngeneic islets via intraportal injection at 8 weeks of diabetes and were allowed to become euglycemic for 4 weeks before study. Thoracic aortic rings were tethered in isolated muscle baths, contracted with a submaximal concentration of norepinephrine, and challenged with either the endothelium-dependent vasodilator acetylcholine or the endothelium-independent vasodilator nitroglycerin. Relaxation to acetylcholine (but not nitroglycerin) was reduced in both 8- and 12-week diabetic rings compared with age-matched control rings. Preincubation of diabetic rings in vitro with L-arginine (but not D-arginine) restored relaxation to acetylcholine to normal to rings from 8-week but not 12-week diabetic animals. Plasma basic amino acids (arginine, lysine, and histidine) were reduced by diabetes, whereas other neutral or acidic amino acids were unchanged (phenylalanine, proline, and glutamate), reduced (serine, cysteine, threonine, tyrosine, tryptophan, and aspartate), or elevated (isoleucine, leucine, and valine). Islet transplantation restored to normal the changes in plasma amino acids. Elevation in blood glucose and total glycosylated hemoglobin in diabetic animals was normalized after islet transplantation. Furthermore, islet transplantation completely restored the defective endothelium-dependent relaxation to acetylcholine in diabetic rings.(ABSTRACT TRUNCATED AT 250 WORDS)

Peroxidative stress in diabetic blood vessels. Reversal by pancreatic islet transplantation.

Diabetic complications are believed to arise, in part, through an increase in oxidative stress. We characterized antioxidant status in vascular tissue in untreated diabetic rats and in diabetic rats rendered euglycemic by pancreatic islet transplantation. Three key endogenous antioxidant enzymes (e.g., superoxide dismutase, catalase, and glutathione peroxidase) were measured. Sprague-Dawley rats with streptozotocin-induced diabetes were killed after 8 weeks of untreated hyperglycemia and compared with age-matched controls. Eight weeks of untreated diabetes resulted in a significant increase of tissue catalase in aorta, iliac artery, and femoral artery as compared with controls. No significant changes in either superoxide dismutase or glutathione peroxidase were observed in aorta, iliac artery, or femoral artery of diabetic animals. This increase in catalase in diabetic vascular tissue suggests increased oxidative stress due to chronic exposure to H2O2 in vivo. To assess the impact of islet transplantation on oxidative stress in vascular tissue, inbred Lewis strain rats were rendered diabetic with streptozotocin. After 8 weeks of untreated diabetes, rats received an intraportal islet isograft and were monitored for 4 subsequent weeks of euglycemia. Islet transplantation improved weight gain and normalized blood glucose and total glycosylated hemoglobin. While catalase was significantly increased in aorta and iliac artery at 8 and 12 weeks of diabetes, vascular catalase was restored to normal by islet transplantation. These data suggest that islet transplantation is an effective treatment strategy to minimize increased oxidative stress in diabetic vasculature.

Superoxide dismutase plus catalase improves post-ischaemic recovery in the diabetic heart.

Streptozotocin induced diabetic rat hearts were perfused under constant flow conditions with or without 1 x 10(5) U.litre-1 each of superoxide dismutase and catalase (SOD + CAT). Total global ischaemia was produced for 20 min followed by 30 min of reperfusion at pre-ischaemic flow rates. After 5 min of reperfusion, isovolumic LV developed pressure was reduced in diabetic hearts, at 22 (SEM 11)% of baseline v 67(12)% in controls, with increased frequency of ventricular fibrillation (VF) (3/10 v 10/11 hearts). SOD + CAT improved isovolumic LV developed pressure to 67(8)% of baseline during early reperfusion of diabetic hearts but did not affect non-diabetic hearts. SOD + CAT also increased the adenylate energy charge potential in post-ischaemic diabetic hearts to 0.826(0.011) v 0.781(0.012) in diabetic controls, and reduced the incidence and duration of reperfusion induced VF in diabetic hearts. SOD + CAT augmented the production of prostacyclin in coronary effluents during early reperfusion of diabetic hearts, from (baseline) 11.5(1.7) to 18.1(3.0) ng.min-1.g-1 at 2 min, compared with 11.1(1.6) to 12.5(1.9) ng.min-1.g-1 at same interval in diabetic controls. Indomethacin prevented the protective effect of the free radical scavengers on function and VF. In contrast, perfusion with the prostacyclin analogue, iloprost (3 x 10(-8) M), alone completely prevented early post-ischaemic dysfunction and reduced VF from 559(172) to 16(8) s. Oxygen derived free radicals may mediate reperfusion induced contractile dysfunction and VF in acutely diabetic hearts following brief episodes of myocardial ischaemia. The beneficial effects of SOD + CAT appear to be mediated mainly by an increase in prostacyclin production during early reperfusion.

Glucose elevations alter bradykinin-stimulated intracellular calcium accumulation in cultured endothelial cells.

OBJECTIVE: Diabetes selectively injures receptor-mediated endothelium-dependent relaxation. In this study, we investigated the effect of elevated glucose concentrations on intracellular calcium (Ca2+i) signal transduction in response to stimulants of EDRF/nitric oxide release in cultured bovine aortic endothelial cells. METHODS: [Ca2+i] was measured in cell suspensions using Fura-2 and fluorescence spectroscopy while nitric oxide production was evaluated using radioimmunoassay of cGMP production. RESULTS: After 24 h exposure to 25 mM glucose in Ham's F-12 media, the increase in endothelial cell [Ca2+i] in response to 100 nM bradykinin was attenuated by 40% while the response to ionomycin was unaltered. When RMPI medium was used, no reduction in response to bradykinin was observed at 25 mM glucose, but a significant reduction in [Ca2+i] signal was observed after exposure to 35 mM glucose for a similar time period. Defective [Ca2+i] signaling was also seen in cells using MEM medium. [Ca2+i] signal responses to ionomycin and NaF, a G-protein activator of extracellular calcium entry via calcium channels, were unaltered by elevated glucose exposure. The defect in [Ca2+i] signal was not mimicked by either mannose or sucrose, but was prevented by co-incubation with cytochalasin B to inhibit glucose uptake. Neither superoxide dismutase nor catalase nor the extracellular hydroxyl radical scavenger, mannitol, blocked the reduction in the bradykinin-induced increase of [Ca2+i] in elevated glucose-exposed cells; however, the reduction was completely blocked by the cell-permeable hydroxyl radical scavenger, dimethylthiourea. Bradykinin-stimulated (but not ionomycin-stimulated) cGMP production within endothelial cells or in RFL-6 detector cells was attenuated by elevated glucose exposure. CONCLUSIONS: Hyperglycemia may contribute to defective endothelium-dependent relaxation in diabetes via an attenuated increase in Ca2+i signal transduction for the release of nitric oxide by endothelial cells. This defect possibly arises as a consequence of hydroxyl radicals formed intracellularly.

Diabetic-induced endothelial dysfunction in rat aorta: role of hydroxyl radicals.

OBJECTIVE: Previous studies suggest a role of superoxide anion radicals (.O2-) in impaired endothelium-dependent relaxation of diabetic blood vessels; however, the role of secondary reactive oxygen species remains unclear. In the present study, we investigated a role of various potential reactive oxygen species in diabetic endothelial dysfunction. METHODS: Thoracic aortic rings from 8-week streptozotocin-induced diabetic and age-matched control rats were mounted in isolated tissue baths. Endothelium-dependent relaxation to acetylcholine (ACH) and endothelium-independent relaxation to nitroglycerin (NTG) were assessed in precontracted rings. RESULTS: ACH-induced relaxation was impaired in diabetic compared to control rings and was not improved with either indomethacin or daltroban. ACH-induced relaxation in both control and diabetic rings was completely blocked with the nitric oxide synthase inhibitors, L-nitroarginine methyl ester or L-nitroarginine (L-NA). NTG-induced relaxation was insensitive to L-NA and was unaltered by diabetes. Pretreatment with superoxide dismutase (SOD) at activities which did not alter contractile tone failed to alter response to ACH in diabetic rings. Similar results were obtained using either catalase or mannitol. In contrast, the combination of SOD plus catalase or DETAPAC, an inhibitor of metal-facilitated hydroxyl radical (.OH) formation, markedly enhanced relaxation to ACH in diabetic but not in control rings. Neither the combination of SOD plus catalase nor DETAPAC altered the sensitivity or relaxation to NTG in control rings with or without endothelium. In diabetic rings with endothelium, both DETAPAC or SOD plus catalase increased sensitivity but not maximum relaxation to NTG. In diabetic rings without endothelium, relaxation and sensitivity to NTG were unaltered by either treatment. In L-NA-treated diabetic rings with endothelium, sensitivity and relaxation to NTG was unaltered by either DETAPAC or SOD plus catalase. CONCLUSION: Diabetic endothelium produces increases in both .O2- and H2O2 leading to enhanced intracellular production of .OH. Thus, .OH are implicated in diabetes-induced endothelial dysfunction.

Cardioprotective effects of monophosphoryl lipid A, a novel endotoxin analogue, in the dog.

OBJECTIVE: The major objective of the present study was to determine the effects of a new endotoxin analogue, monophosphoryl lipid A (MLA), on myocardial infarct size in dogs. A second aim was to determine if potential cardioprotective effects of MLA might be mediated via an enhancement of antioxidant defence mechanisms. METHODS: Barbiturate anaesthetised dogs were subjected to 60 min left circumflex coronary artery occlusion followed by 5 h reperfusion. Either of two different doses of MLA (30 and 100 micrograms.kg-1) or an equivalent volume of vehicle were given intravenously 24 h prior to the infarct experiments. Transmural myocardial blood flow was measured at 30 min of occlusion by the radioactive microsphere technique and infarct size was determined at the end of 5 h of reperfusion by triphenyltetrazolium staining. Tissue catalase and myeloperoxidase activities were measured at 5 h of reperfusion as indices of antioxidant activity and neutrophil infiltration, respectively. RESULTS: There were no significant differences between groups in systemic haemodynamic variables, myocardial oxygen demand, ischaemic bed size, or coronary and collateral blood flow to the ischaemic region. However, administration of MLA produced a marked dose dependent reduction in myocardial infarct size: 19.8(SEM 3.7)% and 14.1(2.5)%, respectively, v 32.7(2.9)% in the vehicle control group, p < 0.05. Pretreatment with either 30 or 100 micrograms.kg-1 of MLA resulted in small increases in tissue catalase activity in the non-ischaemic region of the heart: 0.169(0.033) and 0.197(0.013) K.g-1, respectively, v 0.136(0.013) K.g-1 tissue in the control; however, the increases were not statistically significant by ANOVA. Myeloperoxidase activity in the border zone immediately adjacent to the infarct was markedly decreased in both MLA treated groups: MLA 30 micrograms.kg-1, 2.69(0.82); MLA 100 micrograms.kg-1, 2.49(0.47), v control group, 5.81(1.20) units.g-1 tissue; p < 0.05. CONCLUSIONS: These data are the first to show a marked cardioprotective effect of a lipid A derivative of endotoxin in an in vivo model of myocardial infarction. Although the mechanism responsible for the reduction in infarct size by MLA is unknown, a reduction in neutrophil migration at the site of ongoing tissue injury, the border zone, may be partially responsible.

Temporal changes in endocardial energy metabolism following propranolol and the metabolic basis for protection against isoprenaline cardiotoxicity.

Propranolol infusions in open-chested, anesthetised dogs increased endocardial levels of ATP and phosphocreatine. It reduced ADP, AMP, glucose-6-phosphate and lactate in this region. Endocardial glycogen was transiently increased. Metabolic perturbations in the endocardium produced by cardiotoxic doses of isoprenaline were prevented or reduced by propranolol pretreatment. Thus, propranolol appears to have a more selective action on endocardial versus midmyocardial or epicardial metabolism.

Attenuation of myocardial acidosis by propranolol during ischaemic arrest and reperfusion: evidence with 31P nuclear magnetic resonance.

31P nuclear magnetic resonance (NMR) spectroscopy was used to ascertain whether propranolol could reduce the development of myocardial acidosis during periods of ischaemic arrest and were studied. Cardiac pH progressively declined during ischaemia from a normal 6.97 +/- 0.02 (n = 23) to 6.09 +/- 0.04 or 5.96 +/- 0.04, respectively. Normalisation of pH following reperfusion occurred only in the 35 min ischaemic hearts. Propranolol (1 mg. litre-1) given prior to arrest significantly reduced the magnitude of developing acidosis regardless of the length of ischaemia. Furthermore, it aided in the normalisation of intramyocardial pH upon reperfusion in both groups. Propranolol significantly reduced the magnitude of phosphocreatine (PCr loss normally seen during ischaemic arrest alone, but it did not protect against depletion of ATP. Restoration of PCr reperfusion was virtually complete in all cases, while transient increases in ATP were seen only in those hearts protected by propranolol. In summary, this NMR study demonstrated the first direct evidence that a significant component of the myocardial acidosis caused by global ischaemia and arrest can be blocked by propranolol.

An ESR study of the nitroxide radical of pentastarch-conjugated deferoxamine.

At higher concentrations, deferoxamine (DFO) reacts with hydroxyl radicals to produce a stable nitroxide free radical. Formation and decay of this nitroxide radical was investigated and compared with a novel modified pentastarch conjugate of DFO (MPS-DFO). Photolytic generation of hydroxyl radicals from H2O2 in the presence of free DFO produced a nitroxide radical with coupling constants of aN = 8.0 G and aH = 6.5 G. Under the same experimental conditions, equimolar concentrations of MPS-DFO produced an ESR signal of reduced intensity while iron-saturated MPS-DFO produced no signal. Incubation of free DFO with pentastarch (i.e., without conjugation) greatly decreased the intensity of the nitroxide radical signal. Using a spin-trapping technique with 5,5-dimethyl-1-pyrroline N-oxide (DMPO), the pentastarch vehicle was shown to inhibit the DMPO-OH adduct formation. The decay of the DFO nitroxide radical decayed with a second-order rate constant while that of MPS-DFO decayed with a first-order rate constant. Thus, a novel derivative of DFO may provide some additional benefit in limiting DFO nitroxide radical formation and might explain the reported reduced in vivo toxicity of MPS-DFO relative to free DFO.

Detection by ESR of DMPO hydroxyl adduct formation from islets of Langerhans.

Electron spin resonance (ESR) spectroscopy together with spin trapping techniques and the application of state-of-the-art loop gap resonators was used to provide a direct measure of spontaneous oxygen radical production by homogenates of freshly isolated and cultured rat pancreatic islets. Using the spin trap agent, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), we were able to detect production by islets of an ESR-sensitive radical signal consisting of a quartet with intensity ratio of 1:2:2:1 and hyperfine splitting of aN = aH = 14.9 Gauss, which is consistent with the DMPO-OH adduct. The amplitude of the signal was decreased by decreasing amount of islets and not detected in the absence of islets. Formation of the DMPO-OH adduct was diminished by the hydroxyl radical scavengers (e.g., ethanol, dimethylsulfoxide, and dimethylthiourea). Only partial attenuation of signal was produced by incubation with an iron chelator or using chelex-treated buffers. The ESR signal was insensitive to the xanthine oxidase inhibitor, oxypurinol, or to superoxide dismutase, but was eliminated in a concentration-dependent manner by either potassium cyanide or catalase (but not heat-inactivated catalase). These observations suggest that the origin of the DMPO-OH arose not from free hydroxyl radicals but primarily from endogenous hydrogen peroxide production perhaps of mitochondrial origin. The development of this technology has implications for the potential measure of oxygen radical production in islet homogenates under pathologic conditions as well as to the application of other cell culture systems.

Electron spin resonance analysis of heme-nitrosyl and reduced iron-sulfur centered complexes in allogeneic, heterotopic cardiac transplants: effects of treatment with pyrrolidine dithiocarbamate.

Inhibition of inducible nitric oxide synthase (iNOS) prolongs allograft survival suggesting a role for nitric oxide (.NO) in allograft rejection. Induction of iNOS is regulated by the oxidant-sensitive, nuclear factor kappa B (NF-kappaB) in many cell types. In the present study using electron spin resonance (ESR) spectroscopy, we evaluated whether pyrrolidine dithiocarbamate (PDTC), a metal chelator and antioxidant, might limit .NO production during the development of rejection in cardiac allografts. We performed either isogeneic (Lewis to Lewis) or allogeneic (Wistar-Furth to Lewis) heterotopic abdominal cardiac transplantation. Allograft recipients received daily injections of PDTC or aminoguanidine (a known inhibitor of iNOS). At postoperative days 4 or 6, grafted and native hearts of transplant recipients were flushed with cardioplegic solution to remove blood contamination. ESR data of allografts revealed a triplet nitrogen signal (aN=17.5 G) and centered at g=2.012 and an additional broad signal at g=2.08. This signal was not seen in either isografts or native hearts of either isograft or allograft recipients. Based upon these parameters, these signals are attributed to nitrosomyoglobin. This signal was inhibited by treatment with aminoguanidine or PDTC. Under these conditions, PDTC also prolonged graft survival from 6.6+/-0.2 to 11.7+/-0.3 days. Thus, it is conceivable that nitrosylmyoglobin formation precedes rejection in cardiac allografts and inhibition of nitrosomyoglobin with agents such as PDTC contribute to improved graft survival.

Antioxidant treatment inhibits activation of myocardial nuclear factor kappa B and inhibits nitrosylation of myocardial heme protein in cardiac transplant rejection.

Nitric oxide production via inducible nitric oxide synthase (iNOS) is believed to play a role in cardiac allograft rejection. Previously, we showed that antioxidants can significantly prolong cardiac graft survival, but the nature of this protection is unknown. In the present study, we examined the protective effect of another antioxidant, dimethylthiourea (DMTU), in a model of cardiac allograft rejection. Specifically, we hypothesized that DMTU would prolong graft survival and decrease activation of nuclear factor-kappa B (NF-kappa B), an important redox-sensitive transcription factor necessary for iNOS gene expression. NF-kappa B was activated by twofold as early as postoperative day 2 in allografts. NF-kappa B activation in allografts progressed to a peak of ninefold by postoperative day and remained increased until postoperative day 6. No activation of NF-kappa B was observed in isografts for comparable time periods. Treatment with DMTU resulted in a significant prolongation of graft survival. This beneficial effect was associated with diminished activation of myocardial NF-kappa B. Treatment with DMTU also resulted in decreased formation of iron-nitrosylprotein complexes as evidenced by electron paramagnetic resonance spectroscopy. These studies provide evidence that reactive oxygen plays a significant role in signal transduction for activation via the transcription factor, NF-kappa B, thereby modulating distal actions and consequences of iNOS-derived nitric oxide.

Delayed cardiac allograft rejection due to combined cyclosporine and antioxidant therapy.

The effectors of cell death in allograft rejection are poorly understood. Oxygen derived free radicals (ODFR) may participate in graft destruction. We examined the impact of the antioxidants ascorbic acid (AA) and alpha-tocopherol (AT) with low dose CsA on rat cardiac allograft survival. Lewis rats that had undergone heterotopic abdominal cardiac transplantation with Wistar-Furth allografts (day 0) were divided into 6 groups. Group 1 was the control group; groups 2 and 3 received AA (1200 mg/kg), and groups 4 and 5 received AT (800 IU/kg) by gavage daily until rejection. Groups 3, 5, and 6 were given CsA (2.5 mg/kg i.m.) days 1-15. Allograft rejection times (in days) were 7.7 +/- 1, 10.3 +/- 1.5 (P < 0.01 vs. group 1), 37.1 +/- 6.4 (P < 0.01 vs. group 1, P = 0.0004 vs. group 6), 9.0 +/- 1.4, 26.5 +/- 3.6 (P < 0.01 vs. group 1, P < 0.03 vs. group 6), and 20 +/- 4.9 (P < 0.01 vs. group 1) for groups 1, 2, 3, 4, 5, and 6. To assess the impact of AA on ODFR production, chemiluminescence was performed on zymosan-activated Lewis whole blood from control rats and rats administered AA. AA significantly decreased peak chemiluminescence (P < 0.05) as compared with nontreated rats indicating effective ODFR scavenging. To determine whether AA and AT inhibit lymphocyte stimulation, mixed lymphocyte response testing was performed with irradiated Wistar-Furth lymphocytes as stimulator cells for Lew responder cells from rats treated as groups 3, 5, and 6. CsA significantly suppressed (P < .05) proliferation as compared with untreated controls. Neither AA nor AT enhance CsA's immunosuppressive effect by mixed lymphocyte response testing. In summary, prolongation of allograft survival with antioxidants AA and AT does not result from abrogation of lymphocyte responsiveness or alteration in CsA bioavailability. Rather, these data suggest that ODFR are involved in allograft destruction and support a role for effective antioxidant therapy in the treatment of allograft rejection.

NOX 100, a nitric oxide scavenger, enhances cardiac allograft survival and promotes long-term graft acceptance.

BACKGROUND: We examined the role of nitrosative stress in allograft destruction. METHODS: Rats undergoing cardiac transplants received NOX-100, a water-soluble nitric oxide (NO) scavenger with antioxidant properties, with or without low-dose cyclosporine (CsA). Graft survival, NO production, and nuclear factor kappa B (NF-kappaB) activity were studied. RESULT: Using NOX-100 daily until rejection prolonged graft survival (11.6+/-0.6 vs. 7.4+/-0.2 days; P<0.05). Daily low-dose CsA (2.5 mg/kg im) for 7 days or until rejection also prolonged survival (12.6+/-0.5 and 21.6+/-1.6 days, respectively; P<0.01 vs. Controls). Low-dose CsA for 7 days and NOX-100 for 30 days prolonged graft survival (45.0+/-4.7 days; P<0.01 vs. all groups.). NOX-100 had no effect on whole blood CsA levels. Combination therapy until Day 100 resulted in 1 graft loss at Day 116 and indefinite survival in 3 animals (>300 days), which accepted a second WF strain heart without further immunosuppressive therapy but promptly rejected a third party (ACI) cardiac allograft. NOX-100 and CsA reduced nitrate and nitrite, and combination therapy completely normalized NO through to Day 30. Electron paramagnetic resonance spectroscopic analysis demonstrated reduction of signals for nitrosylmyoglobin and nitrosyl-heme with NOX-100 and elimination of signals with CsA alone or combination therapy. Activity of myocardial NF-kappaB decreased with monotherapy vs. untreated allografts. Combination therapy resulted in further inhibition of NF-kappaB up to Day 30. The extent of graft survival correlated with the extent of NO scavenging and NF-kappaB inhibition. Short-term combination therapy had no effect on graft lymphocytic infiltrate on Days 15, 20, and 30. CONCLUSION: These data support a role for both oxidative and nitrosative stress in rejection and the immunoregulatory potential of antioxidant therapy after transplantation.

Myocardial nuclear factor-kappaB activity and nitric oxide production in rejecting cardiac allografts.

BACKGROUND: Nuclear factor-kappaB (NF-kappaB) is a rapid response transcription factor for genes whose products are critical for inflammation and immunity. In a rat model of heterotopic cardiac transplantation, we studied NF-kappaB DNA binding activity and nitric oxide (.NO) production in untreated allografts and whether inhibition of NF-kappaB suppresses .NO production and prolongs graft survival. METHODS: In allograft recipients and isograft controls, NF-kappaB was assayed by electrophoretic mobility shift assay, daily from transplant until rejection. Myocardial .NO was directly detected in explanted allografts by electron spin resonance spectroscopy on day 6 after transplant. The potent inhibitor of NF-kappaB, pyrrolidine dithiocarbamate (PDTC; 250 mg/kg s.c.) was administered daily from transplant until day of rejection. The extent of graft lymphocytic infiltrate was assessed by routine hematoxylin and eosin staining. Immunohistochemical staining of NF-kappaB was per formed to identify the cell type responsible for NF-kappaB activity. RESULTS: A time-dependent increase in myocardial NF-kappaB activity was seen in untreated allografts as compared with isografts as determined by PhosphorImage analysis. Peak NF-kappaB activity occurred in allografts on day 4 with a ninefold increase as compared with isografts (24.0+/-3.7% vs. 2.7+/-0.5; P<0.05). On posttransplant day 6, electron spin resonance spectroscopy analysis of allografts demonstrated .NO identified by a triplet nitrogen signal centered at g=2.012 with hyperfine splitting of 17.5 Gauss, which is consistent with nitrosoheme formation and low-field signals at g=2.08 and g=2.03 consistent with nitrosomyoglobin. These signals were not seen in native hearts of allograft recipients. With PDTC administration, a threefold decrease in NF-kappaB activity within the transplanted heart was observed on posttransplant day 5 as compared with untreated allografts (9.7+/-1.6% vs. 23.5+/-2.5%; P<0.01). PDTC prolonged graft survival as compared with untreated allografts (11.7+/-0.3 vs. 6.6+/-0.2 days; P<0.05) and reduced the intensity of the nitrosoheme and nitrosomyoglobin signals. Allograft mononuclear cell infiltrate correlated with peak NF-kappaB activity with peak infiltrate on posttransplant day 4. PDTC treatment had no effect on the extent of infiltrate. Immunohistochemical staining localized NF-kappaB to the infiltrating mononuclear cells on posttransplant day 5. CONCLUSION: These data support a role for NF-kappaB in allograft rejection.

Effects of nicorandil on coronary circulation and myocardial ischemia.

The effect of the new antianginal drug, nicorandil, was studied in several models of myocardial ischemia in anesthetized dogs. In animals subjected to an acute or chronic coronary artery occlusion, nicorandil produced increases in collateral perfusion when changes in aortic pressure were minimized. In a model of irreversible ischemia, nicorandil produced a marked (50%) decrease in myocardial infarct size. In several models of reversible ischemia-reperfusion injury, the "stunned myocardium," nicorandil was shown to enhance the recovery of systolic segment shortening after a brief period (15 to 30 minutes) of coronary occlusion. Other vasodilators such as nitroglycerin or nifedipine were not as efficacious as nicorandil. In a model of multiple (n = 3) coronary occlusion (5 minutes) with intermittent (30 minutes) reperfusion, nicorandil improved the recovery of systolic segment shortening during reperfusion and prevented the loss of adenosine triphosphate and tissue edema that occurred in untreated hearts. The beneficial effects of nicorandil on functional and metabolic recovery after recurrent ischemia was shown to be partially the result of an energy-sparing effect of nicorandil to reduce free fatty acid use during the ischemic period. Cyclooxygenase blockade with indomethacin did not block the beneficial effects of nicorandil in the stunned myocardium. These results suggest that nicorandil does not promote an increase of prostacyclin. Finally, nicorandil was found to inhibit superoxide anion production by human neutrophils stimulated by formyl-methionyl-leucyl-phenylalanine plus cytochalasin B. These results suggest that part of the beneficial actions of nicorandil may occur during reperfusion and may be the result of a reduction in oxygen free radical production.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of amlodipine on myocardial ischemia-reperfusion injury in dogs.

The effects of the dihydropyridine calcium channel blocker amlodipine on subendocardial segment shortening (%SS), regional myocardial blood flow, myocardial high-energy phosphate levels and tissue water content were compared with those of a saline-treated group of barbital-anesthetized dogs subjected to a 45-minute coronary artery occlusion followed by 60 minutes of reperfusion. Saline or amlodipine (200 micrograms/kg administered intravenously) was given 15 minutes before coronary occlusion. There were no significant differences between groups in ischemic bed size or hemodynamics although dP/dt was higher after amlodipine administration. Subepicardial collateral blood flow was higher in the amlodipine group during coronary occlusion. After occlusion, %SS in the ischemic region was markedly decreased in both series and passive systolic lengthening resulted. Despite similar decreases in %SS during occlusion, the amlodipine-treated dogs showed a marked improvement in myocardial segment function of the ischemic reperfused region throughout 60 minutes of reperfusion compared with saline-treated dogs. In addition, amlodipine prevented the rebound increase in phosphocreatine and attenuated the loss of adenine nucleotides and increase in tissue water in the ischemic reperfused area at 60 minutes of reperfusion. These results suggest that amlodipine has a favorable effect on the functional and metabolic recovery of the ischemic reperfused myocardium and may have potential as a therapeutic agent for the treatment of coronary artery disease. The mechanism of action of amlodipine in this model is unknown but may be partially related to a drug-induced increase in coronary collateral blood flow or a decrease in afterload.

Pancreatic transplantation reverses endothelial dysfunction in experimental diabetes mellitus.

BACKGROUND: There is insufficient evidence whether transplantation of the whole pancreas can reverse vascular complications associated with diabetes. In this study we investigated whether pancreatic transplantation in experimental diabetes reverses established defects in endothelium-dependent relaxation. METHODS: Streptozotocin-induced diabetic rats underwent whole-pancreas transplantation after 12 weeks of disease. Endothelial function was evaluated 4 weeks after transplantation and compared with that of control- and age-matched diabetic animals. Blood was taken for analysis of glucose, insulin, total glycosylated hemoglobin, and plasma amino acid levels. Descending thoracic aortas were isolated, sectioned into rings, and mounted in isolated tissue baths. In precontracted rings, endothelium-dependent relaxation to acetylcholine was performed and compared with endothelium-independent relaxation to nitroglycerin as a control. RESULTS: Pancreatic transplantation normalized the increases in glucose and total glycosylated hemoglobin levels and the decrease in serum insulin levels. Diabetes resulted in impaired relaxation to acetylcholine without altering relaxation to nitroglycerin. Pancreatic transplantation completely restored the defective relaxation to acetylcholine without altering the relaxation to nitroglycerin. CONCLUSIONS: These results suggest that pancreatic transplantation selectively improved endothelium-dependent relaxation as opposed to a generalized improvement in vascular smooth muscle reactivity. Furthermore, these studies suggest for the first time that one aspect of vascular complications (i.e., endothelial dysfunction) is amenable to this surgical intervention.