Loop diuretic derivative L-644, 711 inhibits K+-stimulated cellular injury in neonatal guinea pig cortical astrocytes

Abstract

An early pathological rise in extracellular K+ following acute hypoxia results in Cl- uptake into astrocytes through the Cl/HCO3- exchanger with an osmotic equivalent of water. This study addressed effects of the anion transport inhibitor, L-644,711, (5,6,-dichloro-2,3, 9,9a-tetrahydro-3-oxo-9a-propyl-1H-fluroen-7-yl)oxyacetic acid. Confluent primary cultures from neonatal guinea pigs, characterized as > 95% astrocytes with antiserum to glial fibrillary acidic protein, were manipulated by incubation in either basal buffer (BB) with the ionic composition of Dulbecco's minimum essential media (DMEM) or one with high extracellular K+ (HiK). Incubation in 27 or 60 mM Hik significantly reduced cell viability and precipitated a time-dose dependent increase in lactate dehydrogenase (LDH) efflux (30 min to 4 h). L-644,711 was not cytotoxic, and significantly inhibited HiK-stimulated LDH efflux. The optimal effective dose of L-644,711 for preventing injury in guinea pig astrocytes was 10(-11)M when administered simultaneously with the HiK paradigm or in reversing injury when administered 30 min after exposing cells to HiK. These findings indicate the potential usefulness of agents which modify ion transport processes in hypoxic-ischemic cerebral injury.

DPOFA, a Cl-/HCO3 -exchanger antagonist, stimulates fluid absorption across basolateral surface of the retinal pigment epithelium

“…Human, cat, guinea pig, and rabbit constitute responsive species with similar high levels of DPOFA inhibitory activities [18,19,26], while rodents (rat and mouse) represent non-responsive species [20,27]. Before conducting experiments on fluid absorption in bovine RPE-choroid system we wanted to confirm that isoform specificity of Cl - /HCO 3 - transporter in bovine RPE is similar to that of the human RPE cells, to assure that data obtained in bovine RPE system would be predictive of outcomes in the human retina.…”

“…Basolateral Cl - /HCO 3 - exchanger in the retinal pigment epithelium is a reasonable drug target for up-regulation of fluid reabsorption as it recycles Cl - back to the RPE thus reducing the rate of fluid absorption from subretinal space. DPOFA (shown in Figure 1), also known in the literature as B-3(+) [20] and L-644,711 [18], is a non-diuretic small molecule Cl - /HCO3 - exchanger antagonist that has been systemically administered to humans in clinical trials for trauma-induced brain damage [15-17]. It has been shown that DPOFA exhibits remarkable species specificity in regard of inhibiting its main target, Cl - /HCO 3 - exchanger.…”

“…While the IC 50 of 2 × 10 -11 M in the K + -induced swelling assay is reported in one of the responsive species, cats, the IC 50 in a similar rat assay is only 2 × 10 -7 M [16]. It has been shown that human, cat, guinea pig, and rabbit constitute responsive species with similar levels of DPOFA inhibitory activities [18,19,26], while rodents (rat and mouse) represent non-responsive species [20,27]. Bovine RPE-choroid system is a widely used experimental tool for assessing the effect of drug treatment on reabsorption of fluid across the RPE [31-33].…”

“…DPOFA, (R)-(+)-(5,6-dichloro 2,3,9,9a-tetrahydro 3-oxo-9a-propyl-1H-fluoren-7-yl)oxy]acetic acid, is an abandoned fluorenone drug that has been systemically administered to humans in clinical trials for trauma-induced brain damage [15-17]. While the primary molecular target for DPOFA is thought to be a Cl - /HCO 3 - exchanger [18-20], a Cl - channel blocker activity has also been suggested for this drug [21]. In the present study we conducted preliminary analysis of the effect of DPOFA on fluid transport using the bovine choroid-RPE ex vivo system.…”