Bcl-2 prevents apoptotic mitochondrial dysfunction by regulating proton flux

We and others have recently shown that loss of the mitochondrial membrane potential (Deltapsi) precedes apoptosis and chemical-hypoxia-induced necrosis and is prevented by Bcl-2. In this report, we examine the biochemical mechanism used by Bcl-2 to prevent Deltapsi loss, as determined with mitochondria isolated from a cell line overexpressing human Bcl-2 or from livers of Bcl-2 transgenic mice. Although Bcl-2 had no effect on the respiration rate of isolated mitochondria, it prevented both Deltapsi loss and the permeability transition (PT) induced by various reagents, including Ca2+, H2O2, and tert-butyl hydroperoxide. Even under conditions that did not allow PT, Bcl-2 maintained Deltapsi, suggesting that the functional target of Bcl-2 is regulation of Deltapsi but not PT. Bcl-2 also maintained Deltapsi in the presence of the protonophore SF6847, which induces proton influx, suggesting that Bcl-2 regulates ion transport to maintain Deltapsi. Although treatment with SF6847 in the absence of Ca2+ caused massive H+ influx in control mitochondria, the presence of Bcl-2 induced H+ efflux after transient H+ influx. In this case, Bcl-2 did not enhance K+ efflux. Furthermore, Bcl-2 enhanced H+ efflux but not K+ flux after treatment of mitochondria with Ca2+ or tert-butyl hydroperoxide. These results suggest that Bcl-2 maintains Deltapsi by enhancing H+ efflux in the presence of Deltapsi-loss-inducing stimuli.     

Complement inhibition prevents gut ischemia and endothelial cell dysfunction after hemorrhage/resuscitation

BACKGROUND: Complement, a nonspecific immune response, is activated during hemorrhage/resuscitation (HEM/RES) and is involved in cellular damage. We hypothesized that activated complement injures endothelial cells (ETCs) and is responsible for intestinal microvascular hypoperfusion after HEM/RES. METHODS: Four groups of rats were studied by in vivo videomicroscopy of the intestine: SHAM, HEM/RES, HEM/RES + sCR1 (complement inhibitor, 15 mg/kg intravenously given before resuscitation), and SHAM + sCR1. Hemorrhage was to 50% of mean arterial pressure for 60 minutes followed by resuscitation with shed blood plus an equal volume of saline. ETC function was assessed by response to acetylcholine. RESULTS: Resuscitation restored central hemodynamics to baseline after hemorrhage. After resuscitation, inflow A1 and premucosal A3 arterioles progressively constricted (-24% and -29% change from baseline, respectively), mucosal blood flow was reduced, and ETC function was impaired. Complement inhibition prevented postresuscitation vasoconstriction and gut ischemia. This protective effect appeared to involve preservation of ETC function in the A3 vessels (SHAM 76% of maximal dilation, HEM/RES 61%, HEM/RES + sCR1 74%, P < .05). CONCLUSIONS: Complement inhibition preserved ETC function after HEM/RES and maintained gut perfusion. Inhibition of complement activation before resuscitation may be a useful adjunct in patients experiencing major hemorrhage and might prevent the sequelae of gut ischemia.     

Heparin prevents postischemic endothelial cell dysfunction by a mechanism independent of its anticoagulant activity

PURPOSE: Heparin may have protective effects on postischemic vascular endothelial cell function that are distinct from its anticoagulant, antiplatelet, or anticomplement activity. We tested this hypothesis in isolated rat hindlimbs. METHODS: Isolated rat hindlimbs underwent 60 minutes of normothermic ischemia and 10 minutes of reperfusion. Potential heparin interaction with plasma-based proteins or cells was eliminated by perfusion of the hindlimbs with a nonrecirculated albumin-enriched crystalloid buffer. Endothelial function was assessed by measurement of endothelium-derived relaxing factor (EDRF) activity. Limbs perfused at constant pressure were subjected to increasing log dose infusions of acetylcholine and nitroprusside to measure endothelial-dependent (EDRF-mediated) and endothelial-independent vasoreactivity, respectively. Fifty limbs were divided into seven groups: two nonischemic groups (one with heparin) and five ischemia/reperfusion groups treated with increasing doses of heparin (0 to 1.0 U/ml perfusate). RESULTS: The nontreated ischemia/reperfusion group (n = 12) had a 46.2% reduction in endothelial-dependent vasodilation of the rat hindlimb when compared with the nonischemic control (n = 7, p < 0.05). Treatment with heparin 0.5 U/ml (n = 6) nearly abolished this attenuation of endothelial-dependent vasodilation (4.3% reduction, p = not significant vs nonischemic control). The endothelial protective effect of heparin was dose-dependent: groups treated with 0.25 U/ml (n = 6) and 0.1 U/ml heparin (n = 7) showed progressive impairment in postischemic EDRF-mediated vasodilation. Endothelial-independent vasodilation induced by nitroprusside was unchanged by ischemia/reperfusion or heparin treatment, which confirmed that the postischemic damage and its protection by heparin were specific to the endothelium. CONCLUSIONS: Heparin prevented postischemic endothelial cell dysfunction by a mechanism independent of its interactions with plasma-based proteins or cells. This nonanticoagulant protective effect may contribute to the salutary effects of heparinization during acute ischemic events.     

C1-esterase inhibitor prevents early pulmonary dysfunction after lung transplantation in the dog

The success of lung transplantation to a large extent depends on effective protection of the graft from ischemic injury after reperfusion. Although mechanisms have not been clarified, the pathologic findings of ischemic injury after reperfusion are similar to adult respiratory distress syndrome, a condition in which the blood coagulation contact system is activated. This study evaluates the effect of C1-esterase inhibitor (C1-INH), the main inhibitor of the blood coagulation contact system, on short-term lung function in a dog model of orthotopic lung transplantation. Twelve lung transplantations were performed after 24 h of ischemic time. Dogs were randomly assigned to receive either vehicle (Control) or C1-INH. After the lung transplantation in the control group, Pao2 decreased by 84% and both the AaPO2 and the Qs/Qt% increased (340 and 530%, respectively, p < 0.01); these parameters remained unchanged in the C1-INH group. The hypoxemia observed in control animals was associated with decreased blood coagulation contact factors, complement consumption, increased expression of adhesion glycoproteins in leukocytes, and extensive intraalveolar and interstitial neutrophil infiltration. In contrast, C1-INH administration prevented hypoxemia, the decrease in blood coagulation contact factors, the activation of the complement system, the increase in expression of leukocyte adhesion molecules, and inflammatory cell infiltrate. This study has demonstrated that in a dog model of lung transplantation, the administration of C1-INH prevents early pulmonary dysfunction, and it suggests that activation of blood coagulation contact system and complement are important mechanisms causing ischemic injury after reperfusion.     

Nonspecific endothelin-receptor antagonist blunts monocrotaline-induced pulmonary hypertension in rats

Endothelin-1 (ET-1), a potent vasoactive and mitogenic peptide, has been implicated in the pathogenesis of several forms of pulmonary hypertension. We hypothesized that nonspecific blockade of ET receptors would blunt the development of monocrotaline (MCT)-induced pulmonary hypertension in rats. A single dose of the nonspecific ET blocker bosentan (100 mg/kg) given to intact rats by gavage completely blocked the pulmonary vasoconstrictor actions of Big ET-1 and partially blunted hypoxic pulmonary vasoconstriction. After 3 wk, MCT-injected (105 mg/kg sc) rats gavaged once daily with bosentan (200 mg/kg) had lower right ventricular (RV) systolic pressure (RVSP), RV-to-body weight (RV/BW) and RV-to-left ventricular (LV) plus septal (S) weight [RV/(LV+S)] ratios and less percent medial thickness of small pulmonary arteries than control MCT-injected rats. Lower dose bosentan (100 mg/kg) had no effect on these parameters after MCT or saline injection. Bosentan raised plasma ET-1 levels but had no effect on lung ET-1 levels. Bosentan (200 mg/kg) also had no effect on wet-to-dry lung weight ratios 6 days after MCT injection. When given during the last 10 days, but not the first 11 days of a 3-wk period after MCT injection, bosentan reduced RV/(LV+S) compared with MCT-injected controls. We conclude that ET-1 contributes to the pathogenesis of MCT-induced pulmonary hypertension and acts mainly during the later inflammatory rather than the acute injury phase after injection.     

Respiratory muscles during ventilatory support

Knowledge of the fate and behaviour of the respiratory muscles during ventilatory support is important for the guidance of clinical care. Full support facilitates muscle metabolic repletion, but exposes them to the risk of disuse atrophy. The effect of partial support varies according to the selected mode: assisted mechanical ventilation (AMV) and synchronized intermittent mechanical ventilation (SIMV) result in much less respiratory muscle rest than generally anticipated. On the other hand, inspiratory pressure support (IPS) is able to rest the respiratory muscles and to prevent fatiguing contractions. Opposite interventions have been proposed in case of difficult weaning: either to unload the respiratory muscles by using partial support, or to overload them according to a training programme. The optimal strategy is not known and may combine both approaches.     

Long-term treatment in vivo with NOX-101, a scavenger of nitric oxide, prevents diabetes-induced endothelial dysfunction

Substantial evidence exists that diabetes results in impaired endothelial dysfunction suggesting diminished nitric oxide production from diabetic endothelium. It is not known what factors contribute to the development of this defect. In this study, we tested whether chronic treatment in vivo with NOX-101, a water-soluble nitric oxide scavenger, prevents endothelial dysfunction in diabetes. Sprague-Dawley rats were made diabetic by an intravenous injection of streptozotocin. A subgroup of control or diabetic animals received twice daily subcutaneous injections of 80 mg/kg NOX-101 beginning at 48 h after streptozotocin was injected and throughout 8 weeks of diabetes. Body weights and glucose concentrations were monitored weekly. At the end of 8 weeks, blood glucose and glycosylated haemoglobin was raised in diabetic rats but serum insulin concentrations were reduced. Treatment with NOX-101 did not alter glucose or insulin concentrations in control or diabetic rats; however, total glycosylated haemoglobin was partially reduced compared with untreated rats. In a subgroup of 2-week diabetic and age-matched rats fasted for 24 h, NOX-101 abolished total urinary nitrate plus nitrite (an index of nitric oxide production in vivo). In isolated tissue baths, relaxation to the endothelium-dependent vasodilator, acetylcholine, was impaired in diabetic aortic rings and relaxation to nitroglycerin was unaltered. Treatment of control rats with NOX-101 did not alter maximum relaxation to acetylcholine but shifted the response curve slightly to the right. In contrast in diabetic rats, NOX-101 prevented the impairment in endothelium-dependent relaxation but had no effect on relaxation induced by nitroglycerin. These data suggest the possibility that diabetes-induced endothelial dysfunction in diabetes results, in part, from a paradoxical increase in nitric oxide production during the course of the disease. This suggests a novel pathway of vascular complications.     

Options for long-term ventilatory support

Long-term mechanical ventilator support for patients with chronic respiratory failure is becoming more common. This article reviews the common causes for chronic ventilator dependence, and offers an approach to weaning these patients from the ventilator. In addition, the details for preparing these patients for prolonged mechanical ventilation outside of the acute-care hospital setting are discussed. Appropriate education of the patient's caregivers is key to the success of long-term ventilatory support outside of the acute-care hospital.     

EndothelinA receptor blockade improves nitric oxide-mediated vasodilation in monocrotaline-induced pulmonary hypertension

BACKGROUND: Nitric oxide (NO) and endothelin (ET) have been implicated in the pathogenesis of pulmonary hypertension (PH). Chronic ETA antagonist therapy reduces PH in monocrotaline (MCT)-treated rats. Interactions between the L-arginine-NO pathway and the ET system have been described. We therefore studied the effect of long-term treatment with an oral ETA antagonist (LU 135252) on NO-related vasodilation in isolated lungs from control rats and rats with MCT-induced PH. METHODS AND RESULTS: Three weeks after MCT injection, PH was associated with an increase in right ventricular pressure (from 27.4 +/- 0.9 to 66.6 +/- 4.1 mm Hg) and a decrease in endothelium-independent vasodilation in response to sodium nitroprusside (10(-10) to 10(-5) mol/L; delta Emax, from 11.1 +/- 0.9 to 2.7 +/- 0.3 mm Hg). Endothelium-dependent vasodilation in response to acetylcholine (10(-9) to 10(-4) mol/L) and the calcium ionophore A23187 (10(-9) to 10(-7) mol/L) remained unaffected. Treatment with LU 135252 did not significantly affect the endothelium-dependent and -independent vasodilations in control rats. However, in MCT-treated rats, LU 135252 therapy significantly reduced right ventricular pressure (39.7 +/- 2.1 mm Hg), potentiated acetylcholine-induced vasodilatation (delta Emax, from 1.6 +/- 0.2 to 3.7 +/- 0.4 mm Hg), and improved the responses to sodium nitroprusside (delta Emax, from 2.7 +/- 0.3 to 5.6 +/- 0.6 mm Hg). LU 135252 did not significantly alter the non-receptor-mediated endothelium-dependent vasodilation to A23187 or pulmonary constitutive NO synthase activity. CONCLUSIONS: MCT PH is associated with a reduced smooth muscle responsiveness to NO but a maintained endothelium-dependent vasodilatory potency. Long-term ETA antagonist therapy not only restores smooth muscle responsiveness to NO but also increases endothelium-dependent dilation in response to acetylcholine. This mechanism may contribute to the therapeutic benefit of ETA antagonists in PH.     

Regulation of ventilatory muscle blood flow

The ventilatory muscles perform various functions such as ventilation of the lungs, postural stabilization, and expulsive maneuvers (e.g., coughing). They are classified in functional terms as inspiratory muscles, which include the diaphragm, parasternal intercostal, external intercostal, scalene, and sternocleidomastoid muscles; and expiratory muscles, which include the abdominal muscles, internal intercostal, and triangularis sterni. The ventilatory muscles require high-energy phosphate compounds such as ATP to fuel the biochemical and physical processes of contraction and relaxation. Maintaining adequate intracellular concentrations of these compounds depends on adequate intracellular substrate levels and delivery of these substrates by arterial blood flow. In addition to the delivery of substrates, blood flow influences muscle function through the removal of metabolic by-products, which, if accumulated, could exert negative effects on several excitatory and contractile processes. Skeletal muscle substrate utilization is also dependent on the ability to extract substrates from arterial blood, which, in turn, is accomplished by increasing the total number of perfused capillaries. It follows that matching perfusion to metabolic demands is critical for the maintenance of normal muscle contractile function. In this article, I review the factors that influence ventilatory muscle blood flow. Major emphasis is placed on the diaphragm because a large number of published reports deal with diaphragmatic blood flow. The second reason for focusing on the diaphragm is because it is the largest and most important inspiratory muscle.     

The management of neuromuscular ventilatory failure

All patients with respiratory impairment have either primarily ventilatory or primarily oxygenation impairment. Patients with neuromuscular conditions fall into the former category but are all too often managed as though they had the latter with oxygen therapy, bronchodilators, chest physical therapy, intermittent positive pressure breathing, and so on. This approach can only hasten respiratory failure and management by tracheostomy-However, it has been reported that with the use of noninvasive respiratory muscle aids, respiratory morbidity and mortality can be prevented for most patients with neuromuscular disease without resort to tracheostomy or even hospitalization.     

Increasing the colloid osmotic pressure of cardiopulmonary bypass prime and normothermic blood cardioplegia minimizes myocardial oedema and prevents cardiac dysfunction

Various mutations in the fibroblast growth factor receptor 3 (FGFR3) gene have recently been reported in thanatophoric dysplasia (TD). We examined the clinical, radiographic, and histologic findings in 91 cases from the International Skeletal Dysplasia Registry and correlated them with the specific FGFR3 mutation. Every case of TD examined had an identifiable FGFR3 mutation. Radiographically, all of the cases with the Lys650Glu substitution demonstrated straight femora with craniosynostosis, and frequently a cloverleaf skull (CS) was demonstrated. In all other cases, the femora were curved, and CS was infrequently present but was occasionally as severe as TD with the Lys650Glu substitution. Histopathologically, all of the cases shared similar abnormalities, but cases with the Lys650Glu substitution had better preservation of the growth plate. Cases with the Tyr373Cys substitution tended to have more severe radiographic manifestations than the Arg248Cys cases, but there was overlap in the phenotypic spectrum between them. One common classification of TD distinguishes affected infants based on the presence or absence of CS. In contrast, and as originally proposed by Langer et al. [1987: Am J Med Genet 3: 167-179], our data suggest that TD can be divided into at least two groups (TD1 and TD2) based on the presence of straight or curved femora. The variable presence of CS and severity of the radiologic and histologic findings in the other substitutions may be due to other genetic, environmental, or stochastic factors.     

Widespread sites of brain stem ventilatory chemoreceptors

We produced local tissue acidosis in various brain stem regions with 1-nl injections of acetazolamide (AZ) to locate the sites of central chemoreception. To determine whether the local acidosis resulted in a stimulation of breathing, we performed the experiment in chloralose-urethan anesthetized vagotomized carotid-denervated (cats) paralyzed servo-ventilated cats and rats and measured phrenic nerve activity (PNA) as the response index. Measurements of extracellular brain tissue pH by glass microelectrodes showed that AZ injections induced a change in pH at the injection center equivalent to that produced by an increase in end-tidal PCO2 of approximately 36 Torr and that the change in brain pH was limited to a tissue volume with a radius of < 350 microns. We found AZ injections sites that caused a significant increase in PNA to be located 1) within 800 microns of the ventrolateral medullary surface at locations within traditional rostral and caudal chemosensitive areas and the intermediate area, 2) within the vicinity of the nucleus tractus solitarii, and 3) within the vicinity of the locus coeruleus. Single AZ injections produced increases in PNA that were < or = 69% of the maximum value observed with an increase in end-tidal PCO2. We conclude that central chemoreceptors are distributed at many locations within the brain stem, all within 1.5 mm of the surface, and that stimulation of a small fraction of all central chemoreceptors can result in a large ventilatory response.     

Noninvasive management of pediatric neuromuscular ventilatory failure

OBJECTIVE: To evaluate the use of mouth piece/nasal intermittent positive-pressure ventilation (IPPV) as an alternative to intubation or to permit extubation for patients with primarily neuromuscular ventilatory impairment and no ventilator-free breathing ability. DESIGN: A case control study. INTERVENTIONS: Using a protocol in which oxyhemoglobin desaturation was prevented or reversed by the continuous use of noninvasive IPPV and manually and mechanically assisted coughing as needed, patients with neuromuscular ventilatory failure and no ventilator-free breathing ability were managed noninvasively or extubated to continuous use of noninvasive IPPV for ventilatory support on room air. MEASUREMENTS AND MAIN RESULTS: Four of ten patients who presented in acute ventilatory failure were managed without intubation, despite becoming dependent on continuous ventilator use. The six intubated patients were extubated successfully to continuous noninvasive IPPV once normal arterial oxygen saturation levels could be maintained on room air, despite their having no ventilator-free breathing ability. CONCLUSIONS: The use of inspiratory and expiratory aids can decrease the need for intubation for patients with neuromuscular ventilatory failure in the absence of significant lung disease. It can also permit extubation, despite the need for continuous ventilatory support and, thereby, decrease the need to resort to tracheostomy.     

Ibudilast prevents oligodendroglial excitotoxicity

Previously we have demonstrated that cells of oligodendroglial lineage express non-N-methyl-D-aspartate (NMDA) glutamate receptor (GluR) genes and are damaged by kainate induced Ca2+ influx via non NMDA GluR channels of the alpha-amino-3-hydroxy-5-methyl 4 isoxazole propionate (AMPA) type, representing oligodendroglial excitotoxicity. We here present the finding that ibudilast, which is used clinically for treat patients with asthma and cerebrovascular diseases, prevents oligodendroglia excitotoxicity. The oligodendrocyte like cells (OLC), differentiated from the CG-4 cell line established from rat oligodendrocyte-type 2 astrocyte (O-2A) progenitor cells, were exposed to 2 mM kainate for 24 h and cell death was evaluated by measuring activity of lactate dehydrogenase (LDH) released into the culture medium. Kainate induced cell death was prevented by 10 to 100 microM ibudilast, which increased intracellular cAMP. A 45Ca2+ influx study revealed that ibudilast attenuated kainate-induced Ca2+ influx. Inhibition of kainate-induced Ca2+ influx by ibudilast was decreased by H-89, a protein kinase A (PKA) inhibitor, but increased by okadaic acid, an inhibitor of phosphatase 1 and 2A. Therefore, we concluded that ibudilast prevented oligodendroglial excitotoxicity by a PKA-dependent phosphorylation process resulting in decreased kainate-induced Ca2+ influx.