4-acetamido-4--isothiocyanatostilbene-2-2--disulfonic-acid and Acidosis

Acute traumatic or ischemic cerebral lesions are associated with tissue acidosis leading to cytotoxic brain edema, predominantly affecting astrocytes. Glial swelling from acidosis is believed to be the attempt of cells to maintain a physiological intracellular pH (pHi). However, this concept, potentially important for the development of new treatment strategies for cytotoxic brain edema, has not been validated experimentally. In the present study, cell volume and pHi of astrocytes were measured simultaneously in vitro. Exposure of suspended astrocytes to levels of acidosis found in vivo during ischemia and trauma (pH 6.8-6.2) led to a maximal increase in cell volume of 121.2% after 60 min (n = 5, p < 0.05) and to immediate intracellular acidification close to extracellular levels (pH 6.2, n = 5, p < 0.05). Inhibition of membrane transporters responsible for pHi regulation (0.1 mM amiloride for the Na+/H+ antiporter or 1 mM SITS for HCO3- -dependent transporters) inhibited cell swelling from acidosis but did not affect the profound intracellular acidification. In addition, acidosis-induced cell swelling and intracellular acidification were partly prevented by the addition of ZnCl2 (0.1 mM), an inhibitor of selective proton channels not yet described in astrocytes (n = 5, p < 0.05). In conclusion, these data demonstrate that glial swelling from acidosis is not a cellular response to defend the normal pHi, as had been thought. If these results obtained in vitro are transferable to in vivo conditions, the development of blood-brain barrier-permeable agents for the inhibition of acidosis-induced cytotoxic edema might be therapeutically useful, since they do not enhance intracellular acidosis and thus cell damage.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Acidosis; Amiloride; Animals; Animals, Newborn; Astrocytes; Cell Size; Cells, Cultured; Diuretics; Hydrogen-Ion Concentration; Intracellular Fluid; Lactic Acid; Rats; Rats, Sprague-Dawley

The contribution of metabolic bicarbonate to cytosolic pH (pHcyto) regulation was studied on isolated perfused rat liver using phosphorus-31 NMR spectroscopy. Removal of external HCO3- decreased proton efflux from 18.6+/-5.0 to 1.64+/-0.29 micromol/min per g liver wet weight (w.w.) and pHcyto from 7.17+/-0.06 to 6.87+/-0.06. In the nominal absence of bicarbonate, inhibition of carbonic anhydrase by acetazolamide induced a further decrease of proton efflux of 0.69+/-0.26 micromol/min per g liver w.w. reflecting a reduction in metabolic CO2 hydration, and hence a decrease of H+ and HCO3- supplies. Even though 27% of the proton efflux was amiloride-sensitive under bicarbonate-free conditions, amiloride did not change pHcyto, revealing the contribution of additional regulatory processes. Indeed, pH regulation was affected by the combined use of 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) and amiloride since pHcyto decreased by 0.16+/-0.05 and proton efflux by 0.60+/-0.14 micromol/min per g liver w.w. The data suggest that amiloride-sensitive or SITS-sensitive transport activities could achieve, by themselves, pHcyto regulation. The involvement of two mechanisms, most likely Na+/H+ antiport and Na+:HCO3 symport, was confirmed in the whole organ under intracellular and extracellular acidosis. The evidence of Na-dependent transport of HCO3- in the absence of exogenous bicarbonate implies that the amount of metabolic bicarbonate is sufficient to effectively participate to pHcyto regulation.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Acetazolamide; Acidosis; Amiloride; Animals; Bicarbonates; Cytosol; Homeostasis; Hydrogen-Ion Concentration; In Vitro Techniques; Intracellular Fluid; Liver; Magnetic Resonance Spectroscopy; Male; Perfusion; Rats; Rats, Wistar

Exposure of nervous tissue to hypoxia results in interstitial acidification. There is evidence for concomitant decrease in extracellular pH to the increase in tissue lactate. In the present study, we used double-barrelled pH-sensitive microelectrodes to investigate the link between lactate transport and acid-base homeostasis in isolated rat spinal roots. Addition of different organic anions to the bathing solution at constant bath pH caused transient alkaline shifts in extracellular pH; withdrawal of these compounds resulted in transient acid shifts in extracellular pH. With high anion concentrations (30 mM), the largest changes in extracellular pH were observed with propionate > L-lactate approximately pyruvate > 2-hydroxy-2-methylpropionate. Changes in extracellular pH induced by 10 mM L- and D-lactate were of similar size. Lactate transport inhibitors alpha-cyano-4-hydroxycinnamic acid and 4,4'-dibenzamidostilbene-2,2'-disulphonic acid significantly reduced L-lactate-induced extracellular pH shifts without affecting propionate-induced changes in extracellular pH. Hypoxia produced an extracellular acidification that was strongly reduced in the presence of alpha-cyano-4-hydroxycinnamic acid and 4,4'-dibenzamidostilbene-2,2'-disulphonic acid. In contrast, amiloride and 4,4'-di-isothiocyanostilbene-2,2'-disulphonate were without effect on hypoxia-induced acid shifts. The results indicate the presence of a lactate-proton co-transporter in rat peripheral nerves. This transport system and not Na+/H+ or Cl-/HCO3- exchange seems to be the dominant mechanism responsible for interstitial acidification during nerve hypoxia.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Acidosis; Animals; Biological Transport, Active; Carrier Proteins; Coumaric Acids; Extracellular Space; Hydrogen-Ion Concentration; Hypoxia; Lactates; Male; Microelectrodes; Monocarboxylic Acid Transporters; Neural Pathways; Protons; Rats; Rats, Wistar; Spinal Cord

Na(+)-H+ antiport and Na(+)-HCO3- symport are involved in intracellular pH (pHi) homeostasis in cultured hepatocytes. We have studied the occurrence of these transport systems in the intact rat liver by 31P nuclear magnetic resonance. Livers perfused with a Krebs medium (25 mM HCO3-, pH 7.4, 37 degrees C) displayed a cytosolic pH 7.18 +/- 0.05 (n = 32). In response to an acid load (35 mM isobutyric acid), pHi remained constant. The same result was obtained in the presence of 1 mM amiloride (with or without acid load), indicating that the amiloride-sensitive Na(+)-H+ exchanger is inactive at external physiological pH (pHe). Under systemic acidosis (6.5-7.0 pHe range), during the acid load, pHi decreased with increased external proton concentrations and became amiloride sensitive. The pHi set point for the activation of the Na(+)-H+ exchange is 7.0. In the absence of HCO3-, livers showed a constant acidic shift of pHi (0.2 pH unit) in the 6.5-7.5 pHe range. Perfusion with 1 mM stilbene derivative (4-acetamido-4'-isothiocyanostilbene-2-2' disulfonic acid) in the presence of HCO3- and at pHe 7.4 induced a dramatic pHi fall (delta pH 0.15), further accentuated during an acid load (delta pH 0.25). Our results suggest that 1) the symport is always involved in pH homeostasis over a large range of pH variation (6.5-7.5) and 2) the Na(+)-H+ exchanger is activated under systemic acidosis as soon as pHi reaches the set point value.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Acidosis; Amiloride; Animals; Carrier Proteins; Hydrogen-Ion Concentration; Intracellular Membranes; Liver; Magnetic Resonance Spectroscopy; Male; Perfusion; Rats; Rats, Wistar; Sodium-Bicarbonate Symporters; Sodium-Hydrogen Exchangers

To expel the excess protons generated during a cellular acidification and to fully recover basal intracellular pH (pHi), cardiac cells rely on the amiloride-sensitive Na/H antiport. We report that rat single ventricular cardiomyocytes, loaded with the fluorescent pH indicator Snarf-1 and treated with inhibitors of the Na/H antiport, amiloride or its analogues, partially restored their pHi through a bicarbonate-dependent mechanism following an acidosis (imposed by the ammonia-pulse technique). In the presence of ethylisopropylamiloride (10 microM) or amiloride (1 mM) and 25 mM bicarbonate in the extracellular solution, the average time that cells needed to recover half of their pHi, following the removal of 20 mM NH4Cl, was 3.4 min, while the rate of proton efflux was calculated to be 2.0 mM/min. The stilbene derivative, 4-4'-di-isothiocyanostilbene-2,2'-disulphonate (DIDS 200 microM), a known blocker of anion transporters, inhibited this recovery. Both phenylephrine (100 microM, 3 microM propranolol present), an alpha 1-adrenoceptor agonist, and ATP (10 microM), a purinergic agonist, significantly enhanced the rate of proton efflux that was due to this HCO3-dependent alkalinizing mechanism. Phenylephrine and ATP also shortened by three-fold the time that a myocyte needed to recover half of its initial pHi. This bicarbonate-dependent alkalinizing mechanism could provide an additional means by which cardiac cells recover their pHi from acidosis, especially under conditions in which the Na/H antiport is inhibited. Furthermore, catecholamines and ATP, which are released under various pathophysiological conditions often associated with intracellular acidosis, could play an important role in the modulation of pHi under these conditions.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Acidosis; Adenosine Triphosphate; Amiloride; Animals; Bicarbonates; Culture Media; Heart; Hydrogen-Ion Concentration; In Vitro Techniques; Myocardium; Phenylephrine; Rats; Rats, Wistar; Receptors, Adrenergic, alpha

The pHi regulation from intracellular acidosis in the central nervous system appears to be mediated by mechanisms driven by the large inwardly directed Na+ gradient. The involvement of these mechanisms in pHi regulation of neurones and glial cells has been investigated in the leech central nervous system using ion-selective microelectrodes. For recovery from acidification, there appear to be three separate mechanisms: Na+/H+ exchange, Na(+)-dependent Cl-/HCO3- exchange, and Na+-HCO3- cotransport. All three mechanisms have a profound effect on the maintenance of pHi homeostasis in glial cells; whereas in leech neurones, as in other neuronal cells studied previously, the predominant mechanisms are Na+/H+ and Na(+)-dependent Cl-/HCO3- exchange. In addition to acid extrusion mechanisms we also found evidence for Na(+)-independent Cl-/HCO3- exchange. At alkaline pHi this exchanger may mediate some of the pHi recovery from intracellular alkalinization.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Acidosis; Alkalosis; Animals; Bicarbonates; Buffers; Central Nervous System; Electrophysiology; Ganglia; Hydrogen-Ion Concentration; In Vitro Techniques; Ion Exchange; Leeches; Microelectrodes; Neurons; Neurotransmitter Agents; Sodium

We used double-barrelled, neutral carrier, pH-sensitive microelectrodes to study the mechanisms by which the intracellular pH (pHi) is regulated in the connective glial cells of the medicinal leech. In HEPES-buffered, nominally CO2/HCO3(-)-free solutions the recovery of pHi from intracellular acidosis is Na(+)-dependent and reduced by at least half in the presence of amiloride, suggesting the action of Na+:H+ exchange. The rate of pHi recovery by this mechanism can be increased by raising the extracellular buffering power or by increasing extracellular pH. The presence of CO2/HCO3(-)-greatly increases the rate of pHi recovery from intracellular acidosis. This CO2/HCO3(-)-stimulated recovery is also dependent on external Na+, largely Cl(-)-independent, inhibited by DIDS, and accompanied by membrane hyperpolarization. This is consistent with it being mediated by the electrogenic cotransport of Na+ and HCO3- into the cells. A Cl(-)-dependent component to Na(+)- and HCO3(-)-dependent regulation is most easily explained by the added presence of a Na(+)-dependent exchange of HCO3- and Cl-.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Acidosis; Acids; Animals; Bicarbonates; Carbon Dioxide; Chlorides; Connective Tissue; Hydrogen-Ion Concentration; Leeches; Microelectrodes; Neuroglia

The relationships between extracellular pH (pHo), intracellular pH (pHi), and loss of cell viability were evaluated in cultured rat hepatocytes after ATP depletion by metabolic inhibition with KCN and iodoacetate (chemical hypoxia). pHi was measured in single cells by ratio imaging of 2',7'-biscarboxy-ethyl-5,6-carboxyfluorescein (BCECF) fluorescence using multiparameter digitized video microscopy. During chemical hypoxia at pHo of 7.4, pHi decreased from 7.36 to 6.33 within 10 min. pHi remained at 6.1-6.5 for 30-40 min (plateau phase). Thereafter, pHi began to rise and cell death ensued within minutes, as evidenced by nuclear staining with propidium iodide and coincident leakage of BCECF from the cytoplasm. An acidic pHo produced a slightly greater drop in pHi, prolonged the plateau phase of intracellular acidosis, and delayed the onset of cell death. Inhibition of Na+/H+ exchange also prolonged the plateau phase and delayed cell death. In contrast, monensin or substitution of gluconate for Cl- in buffer containing HCO3- abolished the pH gradient across the plasma membrane and shortened cell survival. The results indicate that intracellular acidosis after ATP depletion delays the onset of cell death, whereas reduction of the degree of acidosis accelerates cell killing. We conclude that intracellular acidosis protects against hepatocellular death from ATP depletion, a phenomenon that may represent a protective adaptation against hypoxic and ischemic stress.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Acidosis; Amiloride; Animals; Bicarbonates; Carrier Proteins; Cell Survival; Cells, Cultured; Chlorides; Fluoresceins; Gluconates; Hydrogen-Ion Concentration; Liver; Male; Monensin; Oxygen; Rats; Rats, Inbred Strains; Sodium-Hydrogen Exchangers

Disulfonic stilbenes combine with the carrier protein involved in anion transport and inhibit the exchange of Cl- for HCO3- in a variety of biomembranes. Our aim was to determine whether such a mechanism is operative in the regulation of cerebrospinal fluid (CSF) [HCO3-] in metabolic alkalosis. In anesthetized, curarized, and artificially ventilated dogs either mock CSF (group I, 9 dogs) or mock CSF containing SITS, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (group II, 7 dogs) was periodically injected into both lateral cerebral ventricles. During 6 h of isocapnic metabolic alkalosis, produced by intravenous infusion of Na2CO3 solution, plasma [HCO3-] was increased by approximately 14 meq/l in both groups. In SITS-treated animals the mean cisternal CSF [HCO3-] increased by 7.7 meq/l after 6 h, and this was significantly higher than the respective increment, 3.5 meq/l, noted in the control group. Increments in CSF [HCO3-] in both groups were reciprocated by decrements in CSF [Cl-] with CSF [Na+] remaining unchanged. Cisternal CSF PCO2 and lactate concentrations showed similar increments in both groups. It is hypothesized that in metabolic alkalosis a carrier transports HCO3- out of cerebral fluid in exchange for Cl- and that SITS inhibits this mechanism. The efflux of HCO3- out of CSF in metabolic alkalosis would minimize the rise in CSF [HCO3-] brought about by HCO3-] influx from blood into CSF and therefore contributes to the CSF [H+] homeostasis.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Acid-Base Equilibrium; Acidosis; Animals; Bicarbonates; Blood-Brain Barrier; Chlorides; Dogs; Osmolar Concentration; Phosphates; Sodium; Stilbenes