(1/179) Fluid secretion by the malpighian tubules of the tsetse fly Glossina morsitans: the effects of ouabain, ethacrynic acid and amiloride.
The effects of three inhibitors of sodium transport on the secretion of fluid by the Malpighian tubules of Glossina morsitans have been observed. The cardiac glycoside, ouabain, affects neither the rate of secretion nor the sodium concentration of the fluid secreted when isolated tubules are bathed by solutions containing a range of sodium and potassium concentrations. Secretion is inhibited, however, by ethacrynic acid and amiloride. The results confirm that fluid secretion by the Malpighian tubules of this insect is dependent on the active transport of sodium ions and show that Na+/k+ exchange pumps are not involved in this process. (+info)
(2/179) Enthacrynic and acid effects on inner wall pores in living monkeys.
PURPOSE: The influence of the inner wall of Schlemm's canal on aqueous outflow facility remains poorly understood. We examined the relationship between inner wall pore characteristics and outflow facility in living primate eyes in which facility had been pharmacologically increased by ethacrynic acid (ECA) infusion and in contralateral control eyes. METHODS: Outflow facility (two-level constant pressure perfusion) was measured in eight pairs of living monkey eyes before and after administration of a bolus dose of either 0.125 mM ECA or vehicle. After exsanguination, eyes were fixed in situ under constant-pressure conditions (mean fixation pressure approximately 19 mm Hg). The density and diameter of inner wall pores and the number and area of platelet aggregates on the inner wall of Schlemm's canal were measured by scanning electron microscopy. RESULTS: In ECA-treated eyes, outflow facility increased 63% (P < 0.0001), intracellular pore density decreased 46% (P = 0.0094), intracellular pore size increased 27% (P = 0.049), platelet aggregate density increased 158% (P < 0.0001), and area covered by platelets increased 210% (P = 0.012) relative to contralateral controls. Although the average density and size of intercellular pores were essentially unaffected by ECA, an increased density of large (> or = 1.90 microm) intercellular pores was seen in ECA-treated eyes. The density of intracellular pores increased with the duration of fixative perfusion. Other than a weak negative correlation between outflow facility and intracellular pore density in ECA-treated eyes (P = 0.052), facility was not correlated with inner wall pore features. CONCLUSIONS: Our data are most consistent with a scenario in which ECA promotes formation of large intercellular pores in the inner wall of Schlemm's canal, which are then masked by platelet aggregates. Masking of intercellular pores, combined with fixation-induced alteration of inner wall pore density, greatly complicates attempts to relate facility to inner wall structure and suggests that in vivo pore density is smaller than in fixed tissue. Additionally, facility-influencing effects of ECA on the juxtacanalicular tissue cannot be excluded. (+info)
(3/179) Effects of ethacrynic acid on Schlemm's canal inner wall and outflow facility in human eyes.
PURPOSE: The role of the inner wall of Schlemm's canal in determining aqueous outflow facility is poorly understood. To quantify the relationship between inner wall pore characteristics and aqueous outflow facility in human eyes, both control eyes and eyes in which facility had been pharmacologically increased by ethacrynic acid (ECA) infusion were studied. METHODS: Outflow facility was measured in enucleated human eyes before and after delivery of 0.25 mM ECA (one eye of each of 6 pairs) or 2.5 mM ECA (one eye of each of 13 pairs). ECA, and vehicle in contralateral eyes, was delivered into Schlemm's canal by retroperfusion, thereby largely avoiding drug exposure to the trabecular meshwork. After facility measurement, eyes were fixed under conditions of either constant pressure (physiological intraocular pressure, 13 pairs) or "equal flow" (6 pairs) and were microdissected to expose the inner wall of Schlemm's canal. The density and diameter of intercellular and intracellular inner wall pores were measured using scanning electron microscopy. RESULTS: Retroperfusion with 2.5 mM ECA increased facility by 73% (P < 0.001), whereas 0.25 mM ECA increased facility by 19% (not statistically significant). The density of intercellular pores in the inner wall of Schlemm's canal was increased by 520% in 2.5 mM ECA-retroperfused eyes (P < 0.00004), whereas intracellular pore density remained approximately constant. Large pores (size > or = 1.1 microm) were particularly enhanced in ECA retroperfused eyes. The net change in facility due to ECA was not correlated with changes in pore density or other inner wall pore statistics. CONCLUSIONS: Our data are most consistent with a model in which pores in the inner wall of Schlemm's canal indirectly influence facility. However, measured changes in facility due to changes in inner wall properties did not agree with quantitative predictions of the pore funneling theory, suggesting that changes in facility may instead be due to gel leakage from the extracellular spaces of the juxtacanalicular tissue. More definitive experiments are required to confirm this hypothesis. (+info)
(4/179) Bimolecular glutathione conjugation kinetics of ethacrynic acid in rat liver: in vitro and perfusion studies.
The conjugation kinetics of glutathione (GSH) and ethacrynic acid (EA) were studied in rat liver perfusion studies, where efficient removal occurred (steady-state extraction ratio E(ss), approximately 0.8-0.4 at concentrations ranging from 10-200 microM) despite the appreciable plasma protein binding. The declining E(ss) paralleled the saturation in GSH conjugate (EA-SG) formation; EA-SG primarily appeared in bile as the unchanged glutathionyl adduct (90%) and minimally as cleavage products. The GSH conjugation of EA in perfused liver was described by the constants K(m)(overall) of 67 microM and V(max)(overall) of 0.23 micromol/min/g liver. These differed from those observed for the bimolecular nonenzymatic (constant of 126 microM(-1) min(-1)) and enzymatic (K(m) for GSH and EA were 1.2 mM and 94 microM, respectively; V(max) of 533 nmol/min/mg liver cytosolic protein or 32 micromol/min/g liver) GSH conjugation of EA in vitro. But they were similar to those estimated for EA uptake in isolated rat hepatocytes by saturable (K(m)(uptake) = 57 microM, and V(max)(uptake) = 0.55 micromol/min/g liver) and nonsaturable (0.015 ml/min/mg) processes. At increasing EA concentrations (>25 microM), time-dependent changes were observed for E(ss) and EA-SG formation, which rapidly decreased with time after the attainment of steady state due to the rapid loss of cellular GSH. The composite data were described adequately by a physiological model that accounted for transport and the GSH-dependent conjugation of EA. The results suggest that the rate-limiting process for hepatic EA GSH conjugation is cellular uptake, but cosubstrate availability controls the rate of metabolism when GSH becomes depleted. (+info)
(5/179) The role of tyrosine-9 and the C-terminal helix in the catalytic mechanism of Alpha-class glutathione S-transferases.
Glutathione S-transferases (GSTs) play a key role in the metabolism of drugs and xenobiotics. To investigate the catalytic mechanism, substrate binding and catalysis by the wild-type and two mutants of GST A1-1 have been studied. Substitution of the 'essential' Tyr(9) by phenylalanine leads to a marked decrease in the k(cat) for 1-chloro-2,4-dinitrobenzene (CDNB), but has no affect on k(cat) for ethacrynic acid. Similarly, removal of the C-terminal helix by truncation of the enzyme at residue 209 leads to a decrease in k(cat) for CDNB, but an increase in k(cat) for ethacrynic acid. The binding of a GSH analogue increases the affinity of the wild-type enzyme for CDNB, and increases the rate of the enzyme-catalysed conjugation of this substrate with the small thiols 2-mercaptoethanol and dithiothreitol. This suggests that GSH binding produces a conformational change which is transmitted to the binding site for the hydrophobic substrate, where it alters both the affinity for the substrate and the catalytic-centre activity ('turnover number') for conjugation, perhaps by increasing the proportion of the substrate bound productively. Neither of these two effects of GSH analogues are seen in the C-terminally truncated enzyme, indicating a role for the C-terminal helix in the GSH-induced conformational change. (+info)
(6/179) Uptake and glutathione conjugation of ethacrynic acid and efflux of the glutathione adduct by periportal and perivenous rat hepatocytes.
We assessed the impact of zonal factors on the hepatic reduced glutathione (GSH) conjugation of ethacrynic acid (EA). Uptake of EA by enriched periportal (PP) and perivenous (PV) rat hepatocytes was characterized by both saturable (V(max)(uptake) = 3.4 +/- 1.7 and 3. 2 +/- 0.8 nmol/min/mg protein and K(m)(uptake) = 51 +/- 13 and 44 +/- 15 microM) and nonsaturable (12 +/- 5 and 12 +/- 3 microl/min/mg protein) components. Values for the overall GSH conjugation rates of EA (200 microM) were similar among the zonal hepatocytes and resembled those for the influx transport rates. In the absence of the hepatocyte membrane, GSH conjugation in PV and PP hepatocyte cytosol was similar, but a higher perivenous GSH conjugation activity toward EA (PV/PP of 2.4) that mirrored the higher PV/PP ratios of immunodetectable GSTs Ya (1.7) and Yb2 (2.5) was found in cell lysates obtained by the dual-digitonin-pulse perfusion technique. The GSH conjugation rates in the subcellular fragments were, however, much greater than those observed for intact hepatocytes. Efflux rates of the glutathione conjugate EA-SG from zonal hepatocytes were similar, as were levels of the immunodetectable multidrug-resistance protein 2/canalicular multispecific organic anion transporter (Mrp2/cMoat) in the 100,000g pellets. The composite results suggest that the GSTs responsible for EA metabolism are more abundant in the PV region, albeit that the gradient of enzymatic activities is shallow. Despite the existence of zonal metabolic activity, the overall GSH conjugation rate of EA is homogeneous among cells because the reaction is rate limited by uptake, which occurs evenly. Results on EA-SG efflux suggest the acinar homogeneity in Mrp2/cMoat function for canalicular transport. (+info)
(7/179) Dehydroascorbic acid uptake in a human keratinocyte cell line (HaCaT) is glutathione-independent.
Vitamin C plays an important role in neutralizing toxic free radicals formed during oxidative metabolism or UV exposure of human skin. This study was performed to investigate the mechanisms that regulate the homoeostasis of vitamin C in HaCaT cells by identifying the events involved in the transport and in the reduction of dehydroascorbic acid. Dehydroascorbic acid accumulated to a greater extent and faster compared with ascorbic acid; its transport appeared to be mediated by hexose transporters and was entirely distinct from ascorbic acid transport. Dehydroascorbate reductase activity was unaffected by glutathione depletion, although it was sensitive to thiol protein reagents. These observations, as well as the subcellular distribution of this enzymic activity and the cofactor specificity, indicate that thioredoxin reductase and lipoamide dehydrogenase play an important role in this reduction process. HaCaT cells were able to enhance their dehydroascorbic acid reductase activity in response to oxidative stress. (+info)
(8/179) Effect of organic mercurials and sulfhydryl compounds on the urease activity of Proteus: inhibition by urine and ascorbic acid.
Meralluride, mercaptomerin, ethacrynic acid, and penicillamine inhibited urease activity of Proteus mirabilis. The activity of the organic mercurials and ethacrynic acid was markedly inhibited by human and dog urine. Antiurease activity could not be detected in the urine of a human and a dog given meralluride by injection. Urine from patients receiving penicillamine also failed to inhibit urease activity. Ascorbic acid inhibited, whereas dehydroascorbic acid enhanced, the activity of the mercurials, but neither agent altered the inhibitory effect of urine. The lethal effect of meralluride against Proteus occurred at the same concentration at which urease activity was inhibited, but penicillamine inhibited the enzymatic activity without affecting viability of the organism. The data suggest that these sulfhydryl-reactive compounds will not be useful against Proteus infections of the urinary tract. (+info)