monensin and Hypoxia

Sickle cell disease (SCD) is characterized by sickle hemoglobin (HbS) which polymerizes under deoxygenated conditions to form a stiff, sickled erythrocyte. The dehydration of sickle erythrocytes increases intracellular HbS concentration and the propensity of erythrocyte sickling. Prevention of this mechanism may provide a target for potential SCD therapy investigation. Ionophores such as monensin can increase erythrocyte sodium permeability by facilitating its transmembrane transport, leading to osmotic swelling of the erythrocyte and decreased hemoglobin concentration. In this study, we treated 13 blood samples from patients with SCD with 10 nM of monensin ex vivo. We measured changes in cell volume and hemoglobin concentration in response to monensin treatment, and we perfused treated blood samples through a microfluidic device that permits quantification of blood flow under controlled hypoxia. Monensin treatment led to increases in cell volume and reductions in hemoglobin concentration in most blood samples, though the degree of response varied across samples. Monensin-treated samples also demonstrated reduced blood flow impairment under hypoxic conditions relative to untreated controls. Moreover, there was a significant correlation between the improvement in blood flow and the decrease in hemoglobin concentration. Thus, our results demonstrate that a reduction in intracellular HbS concentration by osmotic swelling improves blood flow under hypoxic conditions. Although the toxicity of monensin will likely prevent it from being a viable clinical treatment, these results suggest that osmotic swelling should be investigated further as a potential mechanism for SCD therapy.

Topics: Anemia, Sickle Cell; Erythrocytes; Hemoglobin, Sickle; Humans; Hypoxia; Ionophores; Monensin

The purpose of the present study was to characterize the role of Na+, pH and cellular swelling in the pathogenesis of hypoxic injury to rat livers.. When livers were perfused with hypoxic Krebs-Henseleit bicarbonate buffer (KHB) containing 143 mM Na+, release of LDH began after 30 min and was maximal after 60 min. In livers perfused with choline-substituted low-Na+ KHB (25 mM Na+), LDH release began after 60 min and peaked after 120 min or longer. Supplementation of KHB with mannitol, a permeant sugar with antioxidant properties, suppressed LDH release, whereas sucrose, an impermeant disaccharide, did not afford protection. At the end of hypoxic perfusions with KHB and low-Na+ KHB, liver weight was not different, whereas mannitol but not sucrose increased liver weight after hypoxia. At pH 7.4, monensin, a Na+-H+ ionophore, reversed protection against hypoxia by low-Na+ KHB (10 mM Na+) but had no effect at pH 6.8. As measured directly by confocal microscopy of biscarboxyethylcarboxyfluorescein fluorescence, pH was lower during perfusion with low-Na+ KHB than KHB.. Cytoprotection by low Na+ was not mediated by prevention of Na+-dependent tissue swelling. Rather, promotion of intracellular acidification likely mediates cytoprotection in low-Na+ buffer.

Topics: Animals; Cell Survival; Diuretics, Osmotic; Edema; Glucose; Hepatocytes; Hydrogen-Ion Concentration; Hypoxia; In Vitro Techniques; Ionophores; Liver; Male; Mannitol; Monensin; Organ Preservation; Organ Preservation Solutions; Organ Size; Perfusion; Rats; Rats, Sprague-Dawley; Rats, Wistar; Sodium; Sucrose; Tromethamine

Maintenance of low coronary flow (1 ml/min) during 40 or 70 min of anoxia maintained function and prevented Ca2+ overload during reoxygenation in isolated rat hearts. In comparison, recovery from 40 min of global ischemia resulted in only 20% of preischemic function and an increase in end-diastolic pressure (LVEDP) to 39 mmHg. Reperfusion Ca2+ uptake rose from 0.6 to 10.2 mumol/g dry tissue. Intracellular Na+ (Nai+) increased from 13 to 61 mumol/g dry tissue after 40 min of global ischemia, but was unchanged in hearts with low flow anoxia. When glucose and pyruvate were omitted from buffer used for anoxic perfusion, recovery was only 15% of preanoxic values, LVEDP rose to 32 mmHg, and reperfusion Ca2+ uptake was 7.2 mumol/g dry. In addition, Nai+ increased (47.4 mumol/g dry tissue) and ATP was depleted (1.0 mumol/g dry tissue) in the absence of substrate. In anoxic hearts supplied substrate, Nai+ stayed low (12 mumol/g dry tissue) and ATP was preserved (11.6 mumol/g dry tissue). Addition of ouabain (100 or 200 microM) and provision of zero-K+ buffer increased Nai+ and resulted in impaired functional recovery, increased LVEDP, and greater reperfusion Ca2+ uptake. These interventions also decreased energy availability in anoxic hearts. To distinguish between effects of Na+ accumulation and ATP depletion, monensin, a Na+ ionophore, was added during low flow anoxia. Monensin increased Nai+, decreased functional recovery and increased reperfusion Ca2+ uptake in a dose-dependent manner (1-10 microM) without changing ATP content. These results suggested that reduction of Nai+ accumulation by maintenance of Na+, K+ pump activity was the major mechanism of the beneficial effects of low coronary flow on reperfusion injury.

Topics: Adenosine Triphosphate; Animals; Biological Transport, Active; Calcium; Coronary Circulation; Coronary Disease; Energy Metabolism; Hypoxia; In Vitro Techniques; Male; Monensin; Myocardial Reperfusion Injury; Myocardium; Perfusion; Rats; Rats, Inbred Strains; Sodium; Sodium-Potassium-Exchanging ATPase

The effects of the Na+ ionophore monensin on contractile responses were investigated in guinea-pig aorta in normal and high K+ solutions. In normal K+ (5.4 mM) solution, monensin (2 X 10(-5) M) produced a rapid increase in tension followed by slow relaxation. This contraction was markedly inhibited by phentolamine (10(-5) M) or prazosin (10(-6) M) and was accompanied by an increase in tritium efflux from tissue preloaded with [3H]norepinephrine. In the presence of phentolamine, monensin (1-2 X 10(-5) M) or ouabain (1-2 X 10(-5) M) caused only a small and slowly developing contraction. Simultaneous application of these agents caused a more rapid and greater contraction. Either monensin or ouabain gradually increased cellular Na+ and decreased cellular K+ content. When monensin was applied simultaneously with ouabain, there was a rapid increase in cellular Na+ and loss of cellular K+. In high K+ (65.4 mM) solution, monensin (10(-6) M) slightly reduced the increased tension level but when external glucose was omitted monensin markedly inhibited the contraction. A significant decrease in tissue ATP content was observed only when monensin was applied in glucose-free solution. Similarly, hypoxia (N2 bubbling) markedly inhibited the high K+ contraction and decreased the tissue ATP content only in the absence of glucose. These results suggest that monensin produces a neurogenic contraction due to the release of endogenous catecholamines and also produces a myogenic contraction by a decrease in transmembrane Na+ and K+ gradients when the Na+-K+ pump is inhibited by ouabain, and that monensin inhibits aerobic energy metabolism of vascular smooth muscle.

Topics: Adenosine Triphosphate; Animals; Aorta, Thoracic; Furans; Glucose; Guinea Pigs; Hypoxia; In Vitro Techniques; Male; Monensin; Muscle Contraction; Muscle, Smooth, Vascular; Norepinephrine; Ouabain; Phentolamine; Potassium; Prazosin; Sodium; Verapamil