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

The mechanism(s) of electrolyte transport across the human colonic contraluminal domain is not well understood. Current studies were undertaken to develop a technique for the isolation and purification of the human colonic basolateral membrane vesicles (BLMV) and to examine the presence of a Na+-H+ exchange process in these membranes. BLMV were purified from mucosal scrapings of organ donor proximal colons utilizing a Percoll density gradient centrifugation technique, and Na+ transport was examined utilizing a rapid filtration, technique. Our data demonstrate that purified basolateral membranes were enriched 10- to 11-fold in Na+, K+-ATPase activity compared to crude homogenate. Results consistent with the Na+-H+ exchange in BLMV are as follows: (1) an outwardly directed H+ gradient stimulated 22Na uptake; (2) 22Na uptake was markedly inhibited by EIPA and amiloride; (3) H+-gradient-stimulated 22Na uptake was not inhibited by bumetanide, SITS, DIDS, acetazolamide, phenamil and benzamil; (4) 22Na uptake was voltage insensitive; (5) 22Na uptake demonstrated saturation kinetics; (6) 22 Na uptake was markedly inhibited by Na+ and Li+ but was unaffected by N-methyl glucamine+, choline+, and NH4+. Immunoblotting studies demonstrated this Na+-H+ exchanger isoform to be represented by NHE1. In conclusion, a technique has been established for the purification of functional human proximal colonic BLMV, and an electroneutral Na+-H+ exchange process has been demonstrated in these membranes.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Acetazolamide; Amiloride; Biological Transport; Bumetanide; Carbonic Anhydrase Inhibitors; Cell Membrane; Colon; Humans; Hydrogen; In Vitro Techniques; Ion Exchange; Sodium; Sodium-Potassium-Exchanging ATPase; Tissue Donors

The trophectoderm of the mouse blastocyst is a fluid transporting epithelium that is responsible for generating a fluid-filled cavity called the blastocoel. Vectorial transport of ions from the medium into the blastocoel generates an osmotic gradient that drives fluid across this epithelium. We report here that substitution of Na+ or Cl-, but not K+, in the medium halves the rate of blastocoel expansion in the mouse blastocyst. Entrance of Na+ into the trophectoderm may involve several routes, since both blastocoel expansion and 22Na+ uptake are decreased in the presence of the highly specific Na+/H+ exchanger inhibitor, 5-(N-ethyl-N-isopropyl)amiloride, and to a lesser extent with the amiloride-sensitive Na+-channel blocker, benzamil. Uptake of 22Na+ manifests saturation kinetics as a function of extracellular Na+ concentration, whereas uptake of 36Cl- is linear. Furthermore, neither 4,4-diisothiocyanostilbene-2,2-disulfonic acid, which is an inhibitor of the Cl-/HCO3- exchanger, nor 2-(3,4-dichlorobenzyl)-5-nitrobenzoic acid, which is a Cl- -channel blocker, affect either blastocoel expansion or 36Cl- uptake. These results suggest that Na+ entry into the mouse blastocyst is carrier-mediated and probably involves several routes that include the Na+/H+ exchanger and possibly the Na+-channel. Chloride entry, however, may not be carrier-mediated and may occur through a paracellular route, i.e., between the trophectodermal cells.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Adenosine Triphosphate; Amiloride; Animals; Biological Transport; Blastocyst; Carrier Proteins; Chloride Channels; Chloride-Bicarbonate Antiporters; Chlorides; Cytochalasin D; Cytochalasins; Ectoderm; Epithelium; Kinetics; Membrane Proteins; Mice; Nitrobenzoates; Potassium; Sodium; Sodium Channels; Sodium-Hydrogen Exchangers