Indacrinone

Abstract: The fluorescence intensity of the dye 1,1′-dipropyloxadicarbocyanine (DiOC3-(5)) has been measured in suspensions of Ehrlich ascites tumor cells in an attempt to monitor their membrane potential (V m ) under different ionic conditions, after treatment with cation ionophores and after hypotonic cell swelling. Calibration is performed with gramicidin in Na+-free K+/choline+ media, i.e., standard medium in which NaCl is replaced by KCl and cholineCl and where the sum of potassium and choline is kept constant at 155mm. Calibration by the valinomycin “null point” procedure described by Lariset al. (Laris, P.C., Pershadsingh, A., Johnstone, R.M., 1976,Biochim. Biophys. Acta 436:475–488) is shown to be valid only in the presence of the Cl−-channel blocker indacrinone (MK196). Distribution of the lipophilic anion SCN− as an indirect estimation of the membrane potential is found not to be applicable for the fast changes inV m reported in this paper. Incubation with DiOC3-(5) for 5 min is demenstrated to reduce the Cl− permeability by 26±5% and the NO 3 − permeability by 15±2%, while no significant effect of the probe could be demonstrated on the K+ permeability. Values forV m , corrected for the inhibitory effect of the dye on the anion conductance, are estimated at −61±1 mV in isotonic standard NaCl medium, −78±3 mV in isotonic Na+-free choline medium and −46±1 mV in isotonic NaNO3 medium. The cell membrane is depolarized by addition of the K+ channel inhibitor quinine and it is hyperpolarized when the cells are suspended in Na+-free choline medium, indicating thatV m is generated partly by potassium and partly by sodium diffusion. Ehrlich cells have previously been shown to be more permeable to nitrate than to chloride. Substituting NO 3 − for all cellular and extracellular Cl− leads to a depolarization of the membrane, demonstrating thatV m is also generated by the anions and that anions are above equilibrium. Taking the previously demonstrated single-file behavior of the K+ channels into consideration, the membrane conductances in Ehrlich cells are estimated at 10.4 μS/cm2 for K+, 3.0 μS/cm2 for Na+, 0.6 μS/cm2 for Cl− and 8.7 μS/cm2 for NO 3 − . Addition of the Ca2+-ionophore A23187 results in net loss of KCl and a hyperpolarization of the membrane, indicating that the K+ permeability exceeds the Cl− permeability also after the addition of A23187. The K+ and Cl− conductances in A23187-treated Ehrlich cells are estimated at 134 and 30 μS/cm2, respectively. The membrane potential is depolarized in hypotonically swollen cells, confirming that the increase in the Cl− permeability following hypotonic exposure exceeds the concommitant increase in the K+ permeability. In control experiments where the membrane potentialV m =E K =E Cl =E Na , it is demonstrated that cell volume changes has no significant effect on the fluorescence signal, apparently because of a large intracellular buffering capacity. The increase in the Cl− conductances is 68-fold when cells are transferred to a medium with half the osmolarity of the standard medium, as estimated from the net Cl− efflux and the change inV m . The concommitant increase in the K+ conductance, as estimated from the net K+ efflux, is only twofold.

Abstract: 3-Amino-5-sulfamoylbenzoic acids and several series of (aryloxy)alkanoic acids were evaluated for their inhibitory effects on two human erythrocyte ion transport systems — the Na+, K+ cotransport system and the DIDS-sensitive anion carrier. Several classic loop diuretics, including the (aryloxy)alkanoic acid-ethacrynic acid and several 3-amino-5-sulfamoylbenzoic acids, like bumetanide and furosemide, displayed relatively strong inhibitory activity versus the cotransport system with relatively weaker action versus the anion carrier. Furthermore, diuretic potency correlated with cotransport inhibitory potency. Another class of (aryloxy)alkanoic acids, namely the [(2,3-dihydro-1 H-inden-5-yl)oxy]acetic acids, such as indacrinone and MK-473, which exhibit less potent loop dacrinone and MK-473, which exhibit less potent loop diuretic activity, were less potent cotransport inhibitors and more effective inhibitors of the anion carrier. Still other (aryloxy)alkanoic acids, with little saliuretic activity, namely a sub-class of [(2,3-dihydro-1 H-inden-5-yl)oxy]alkanoic acids and a series of [(2,3,9,9a-tetrahydro-1 H-fluoren-7-yl)oxy]acetic acids displayed little or no inhibitory action on the cotransport system but enhanced inhibitory action on the anion carrier. Most interestingly, the relative anion carrier inhibitory potency correlated well with the relative inhibitory activity of each compound on bicarbonate-stimulated cell swelling in cat cerebrocortical slices.

Abstract: Purpose The phenoxyacetic acid, ethacrynic acid (ECA), has potential use in glaucoma therapy because it acts to increase aqueous outflow in vivo and in vitro In human trabecular meshwork (HTM) cell culture, ECA acts to change cell shape and attachment, effects that have been correlated with microtubule (MT) alterations and chemical sulfhydryl (SH) reactivity To further explore these actions, we evaluated two non-SH reactive phenoxyacetic acids, indacrinone and ticrynafen, and the MT-disrupting drug vinblastine Methods Excised bovine and porcine eyes were perfused and outflow facility measured Calf pulmonary artery endothelial and HTM cells were grown in culture and cytoskeletal effects evaluated after drug treatment Results Indacrinone, ticrynafen, and vinblastine all caused an increase in outflow facility In contrast with ECA, the outflow effects of indacrinone and ticrynafen were not blocked by excess cysteine Although indacrinone and ticrynafen produced changes in cell shape in vitro, the /3-tubulin staining pattern of treated cells was not altered Vinblastine caused cell shape change and the expected MT disruption Conclusions Phenoxyacetic acids can increase aqueous outflow facility and alter HTM cell shape and attachment in vitro by a non-SH, non-MT mechanism (which is probably shared also by ECA) These findings suggest the possibility of a broader class of glaucoma drugs that may be directed at the HTM An understanding of the cellular target for these drugs has implications both for potential glaucoma therapy and for the cytoskeletal mechanisms involved in normal outflow function Invest Ophthalmol Vis Sci 1997; 38:1526-1534

Abstract: p-Aminohippurate (PAH) and urate are secreted into the proximal tubule lumen across the brush-border membrane. Here we used brush-border membrane vesicles from pig kidney to study PAH and urate transport. Efflux and influx of [3H]PAH were influenced by K+-diffusion potentials indicating electrogenic PAH transport. An outside>inside PAH concentration difference accelerated voltage-sensitive, Na+-coupled d-glucose uptake as efficiently as did an outside>inside Cl– concentration difference, suggesting comparable conductances for PAH and Cl– in brush-border membrane vesicles. Up to 1 mM of the uricosurics indacrinone, tienilic acid, losartan and probenecid, as well as of the stilbenes, DIDS and SITS, and of the loop diuretics furosemide and bumetanide inhibited voltage-driven PAH uptake, but not, or only slightly, voltage-driven Cl– uptake. Voltage-driven [14C]urate uptake, however, was inhibited by 0.1 mM DIDS, 0.2 mM losartan and 0.5 mM probenecid to a similar extent as [3H]PAH uptake. One millimolar pyrazinoic acid, oxonate, xanthine and adenosine inhibited neither [3H]PAH nor [14C]urate uptake. These results suggest that PAH and urate share an anion conductance which is distinct from the Cl– conductance and is probably not the same as a recently identified urate channel (Leal-Pinto E et al. J Biol Chem 272:617–625, 1997).