4-acetamido-4--isothiocyanatostilbene-2-2--disulfonic-acid and Disease-Models--Animal

T-wave alternans, characterized by a beat-to-beat change in T-wave morphology, amplitude, and/or polarity on the ECG, often heralds the development of lethal ventricular arrhythmias in patients with left ventricular hypertrophy (LVH). The aim of our study was to examine the ionic basis for a beat-to-beat change in ventricular repolarization in the setting of LVH. Transmembrane action potentials (APs) from epicardium and endocardium were recorded simultaneously, together with transmural ECG and contraction force, in arterially perfused rabbit left ventricular wedge preparation. APs and Ca(2+)-activated chloride current (I(Cl,Ca)) were recorded from left ventricular myocytes isolated from normal rabbits and those with renovascular LVH using the standard microelectrode and whole cell patch-clamping techniques, respectively. In the LVH rabbits, a significant beat-to-beat change in endocardial AP duration (APD) created beat-to-beat alteration in transmural voltage gradient that manifested as T-wave alternans on the ECG. Interestingly, contraction force alternated in an opposite phase ("out of phase") with APD. In the single myocytes of LVH rabbits, a significant beat-to-beat change in APD was also observed in both left ventricular endocardial and epicardial myocytes at various pacing rates. APD alternans was suppressed by adding 1 microM ryanodine, 100 microM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), and 100 microM 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS). The density of the Ca(2+)-activated chloride currents (I(Cl,Ca)) in left ventricular myocytes was significantly greater in the LVH rabbits than in the normal group. Our data indicate that abnormal intracellular Ca(2+) fluctuation may exert a strong feedback on the membrane I(Cl,Ca), leading to a beat-to-beat change in the net repolarizing current that manifests as T-wave alternans on the ECG.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Action Potentials; Animals; Arrhythmias, Cardiac; Calcium Signaling; Cardiac Pacing, Artificial; Chloride Channels; Disease Models, Animal; Electrocardiography; Endocardium; Hypertrophy, Left Ventricular; Myocardial Contraction; Myocytes, Cardiac; Patch-Clamp Techniques; Pericardium; Rabbits; Ryanodine; Time Factors

Gallstone disease remains a leading cause of morbidity and mortality in humans. Despite extensive research into the physiology of the gallbladder, little is known about mucosal events that precede and contribute to stone formation. Here, we describe and partially characterize a cultured epithelial model of human gallbladder mucosa.. Cells originally obtained from a well-differentiated gallbladder mucosal carcinoma were cultured in modified Eagle's minimum media (supplemented with fetal calf serum and antibiotics) on polycarbonate supporting matrices.. Cell cultures were observed to come to confluence with 6 to 9 days. Light and transmission electron microscopy demonstrated the resultant epithelia to be predominantly one cell thick, to be polar in orientation, and to have apical villi. Epithelia exhibited cytokeratin markers consistent with their epithelia origin, functionally acidified the mucosal bathing solutions, and secreted mucin. Further experiments demonstrated transepithelial potential differences, mucosal-to-serosal transfer of sodium which could be inhibited with amiloride and 4-acetamido-4'-isothiocyanatostilbene-2-2'-disulfonic acid, and paracellular movement of neutral molecular probes inversely related to size.. This culture model of human gallbladder mucosal carcinoma cells exhibits parameters consistent with native gallbladder and may offer a convenient new research tool for the study of the pathophysiology of gallstone formation.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Amiloride; Biological Transport; Disease Models, Animal; Epithelium; Gallbladder; Gallbladder Neoplasms; Humans; Keratins; Methods; Microscopy, Electron; Mucins; Mucous Membrane; Sodium; Tumor Cells, Cultured

We have developed a model of reperfusion injury in Krebs buffer-perfused rabbit lungs, characterized by pulmonary vasoconstriction, microvascular injury, and marked lung edema formation. During reperfusion there was a threefold increase in lung superoxide anion (O2-) production, as measured by in vivo reduction of nitroblue tetrazolium, and a twofold increase in the release of O2- into lung perfusate, as measured by reduction of succinylated ferricytochrome c. Injury could be prevented by the xanthine oxidase inhibitor allopurinol, the O2- scavenger SOD, the hydrogen peroxide scavenger catalase, the iron chelator deferoxamine, or the thiols dimethylthiourea or N-acetylcysteine. The protective effect of SOD could be abolished by the anion channel blocker 4,4'-diisothiocyano-2,2'-stilbene disulfonic acid, indicating that SOD consumes O2- in the extracellular medium, thereby creating a concentration gradient favorable for rapid diffusion of O2- out of cells. Our results extend information about the mechanisms of reperfusion lung injury that have been assembled by studies in other organs, and offer potential strategies for improved organ preservation, for treatment of reperfusion injury after pulmonary thromboembolectomy, and for explanation and therapy of many complications of pulmonary embolism.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Antioxidants; Cytochrome c Group; Disease Models, Animal; Free Radicals; Ion Channels; Lung Diseases; Male; Nitroblue Tetrazolium; Rabbits; Reperfusion Injury; Superoxide Dismutase; Superoxides; Xanthine Oxidase

Swelling of brain slices is shown to occur in response to elevated potassium levels or glutamate, which is accompanied by astrocytic swelling. Cl-/HCO3- anion exchange inhibitors, such as SITS (4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid) or furosemide, but not the specific cotransport inhibitor bumetanide, inhibit swelling or increased ion uptake in rat brain slices caused by elevated potassium although there were marked species differences in sensitivity. A novel anion exchange inhibitor, L-644,711, inhibits swelling and increased ion uptake caused by glutamate in rat and cat brain slices, as well as inhibiting [3H]glutamate uptake in primary rat astrocyte cultures. Possible mechanisms of action of the inhibitors are discussed. L-644,711 was also found to be effective in promoting recovery from a trauma plus hypoxia head injury model in cats. Marked perivascular astrocytic swelling is associated with this head injury model, and L-644,711 also inhibited such astroglial swelling as determined ultrastructurally. The significance of these findings in relation to possible connections between astrocytic swelling and brain pathology is discussed.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Animals; Astrocytes; Biological Transport; Brain; Brain Injuries; Cats; Cells, Cultured; Chlorides; Disease Models, Animal; Fluorenes; Glutamates; Glutamic Acid; Hypoxia, Brain; Ouabain; Potassium; Rats

Pancreatic acini of control and reserpine-treated rats were incubated with the isotopic tracer 36Cl to compare Cl accumulation in the absence and presence of secretagogues and transport inhibitors. Two phases of Cl accumulation were ascertained in resting control cells: an initial rate (0-5 min) and a steady state level (10-30 min) of accumulation. Both phases were enhanced by acetylcholine (1 microM) and caerulein (10 nM), but not by 10 nM vasointestinal peptide or 10 microM forskolin. Exposure to 1 mM DIDS (4,4'-diisothiocyano-2,2'-stilbene disulfonic acid) inhibited both phases of Cl accumulation, whereas exposure to 1 mM amiloride had a delayed effect on the initial rate and reduced the steady state phase in both resting (unstimulated) or acetylcholine-stimulated cells. Furosemide (1 mM) had no effect on Cl accumulation when added to the cells just before tracer, but reduced it when added 10 min before. Neither the initial phase nor the steady state level of Cl accumulation were enhanced by acetylcholine in acini of reserpine-treated rats and the effect of DIDS on the initial phase was smaller than in control cells. Continued exposure to this inhibitor resulted, furthermore, in a significantly larger steady state Cl content. The inhibitory effects of amiloride and of a 10-min preincubation with furosemide were similar to those observed in control cells. These results suggest that Cl accumulates in rat pancreatic acini by way of DIDS-sensitive mechanisms that are activated by Ca2+-mediated, but not by cAMP-mediated, secretagogues. These mechanisms are altered in acini of reserpine-treated rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Acetylcholine; Amiloride; Animals; Biological Transport; Ceruletide; Chlorides; Colforsin; Cystic Fibrosis; Disease Models, Animal; Furosemide; Kinetics; Male; Pancreas; Rats; Rats, Inbred Strains; Reserpine; Vasoactive Intestinal Peptide