Cytotoxic activity of tumor necrosis factor is inhibited by amiloride derivatives without involvement of the Na+/H+ antiporter

AbstractCytotoxicity of tumor necrosis factor (TNF) on L929s cells was efficiently blocked by several amiloride analogs but not by amiloride itself. This protection did not require RNA or protein synthesis. Na+/H+ antiporter-negative L-M(TK−) cells (LAP) could be killed by TNF, showing that the Na+/H+ exchanger is not required for TNF-cytotoxicity. Similar protection against TNF-mediated cell lysis by amiloride derivatives was found for LAP and L929s cells, excluding a blockade of the Na+/H+ antiporter as the cause of the protection against TNF by these agents

The cellular pool of Na+ channels in the amphibian cell line A6 is not altered by mineralocorticoids. Analysis using a new photoactive amiloride analog in combination with anti-amiloride antibodies.

An amiloride-sensitive Na+ channel is found in the apical plasma membrane of high resistance, Na+ transporting epithelia. We have developed a method for the identification of this channel based on the use of a new high affinity photoreactive amiloride analog, 2'-methoxy-5'-nitrobenzamil (NMBA), and anti-amiloride antibodies to identify photolabeled polypeptides. NMBA specifically labels the putative Na+ channel in bovine kidney microsomes. A 130-kDa polypeptide is detected on immunoblots with anti-amiloride antibodies. NMBA is a potent inhibitor of Na+ transport in the established amphibian kidney epithelial cell line A6, and specifically labels a 130-kDa polypeptide. We utilized both NMBA photolabeling and [3H]benzamil binding in order to examine the cellular pool of putative channels following hormonal regulation of Na+ transport. This pool is not significantly altered by the mineralocorticoid agonist aldosterone or antagonist spironolactone, despite a 3.8-fold difference in transepithelial Na+ transport

Amiloride analogues induce responses in isolated rat cardiovascular tissues by inhibition of Na+/Ca2+ exchange

The role of inhibition of Na+/Ca2+ exchange in the positive inotropic, negative chronotropic and vasorelaxant responses to amiloride and some of its analogues was investigated in isolated cardiovascular tissues from female Wistar rats. The compounds tested were amiloride, 5-(N-ethyl-N-isopropyl)amiloride (EIPA, a potent inhibitor of Na+/H+ exchange), phenamil and 2′,4′-dimethylbenzamil (DMB), both potent Na+ channel inhibitors with activity against Na+/Ca2+ exchange, and 5-(N-4-chlorobenzyl)-2′,4′-dimethylbenzamil (CBDMB), a potent inhibitor of Na+/Ca2+ exchange with reduced activity against Na+ channels compared with its parent compound DMB. Phenamil, DMB and CBDMB increased the force of contraction of right ventricular papillary muscles with similar potencies (-log EC50 values: 4.77 ± 0.06, 5.09 ± 0.09, 4.97 ± 0.17 respectively), while amiloride and EIPA gave small negative inotropic responses. All compounds gave negative chronotropic responses at similar concentrations to those which exerted inotropic effects. Inhibition of KCl contraction of endothelium-free aortic rings was observed with all compounds tested. Phenamil, DMB and CBDMB but not amiloride or EIPA showed a shift to the left of the concentration-response curves in the presence of intact endothelium. These results provide further evidence for positive inotropic and endothelium-dependent vasorelaxant effects of amiloride analogues mediated by inhibition of Na+/Ca2+ exchange

Control of ion transport in the thyroid: prostaglandin E2 activates cation transport on the basal membrane of cultured porcine thyroid cell monolayers

Confluent monolayers of cultured porcine thyroid cells transport fluid from the apical to the basal surface, forming circumscribed zones of detachment (domes) from the culture dish substrate. Stimulation of fluid transport by prostaglandin E (PGE: 1 μmol/l) was associated with an increase in transepithelial potential (TEP). Intracellular potentials (equal to the potential difference across the apical membrane of the cell, E(apical)) and the TEP were measured in individual domes so that the potential difference across the basal membrane of the cell (E(basal)) could be calculated from the relationship TEP = E(apical) - E(basal). The PGE-induced increase in TEP was associated with hyperpolarization of the basal membrane, accompanied by a slight depolarization of the apical membrane. Lines of best fit by least-squares regression showed E(apical) = -20.3 mV + 0.219 TEP (correlation coefficient r = 0.627, P < 0.001) and E(basal) = -20.3 mV - 0.781 TEP (r = 0.944; P < 0.001). Phenamil (1 μmol/l), a Na channel selective amiloride analogue, reduced the TEP from 13.25 ± 0.58 (S.E.M.; n = 56) to 2.39 ± 0.16 mV (n = 51; P < 0.001) and hyperpolarized the apical membrane potential from -20.7 ± 0.68 (n = 60) to -32.2 ± 0.83 mV (n = 105; P < 0.001). The response of the TEP to phenamil was immediate, and was promptly reversed on washing; in contrast, addition of 5-(N-ethyl-N-isopropyl)amiloride (20 μmol/l; selective for Na/H antiporters) resulted in a slow depolarization over 30 min with a slow recovery after washout. Exposure of the cultures to media of pH 7.04 (compared with the normal pH of 7.34) resulted in a reduced response to PGE, and a reduction in magnitude of E(basal). It was concluded that stimulation of ion transport by PGE in thyroid monolayers involves activation of cation transport across the basal membrane

Bidirectional ion transport in thyroid: Secretion of anions by monolayer cultures that absorb sodium

Cultured porcine thyroid cell monolayers transport Na in an apical-to-basal direction, resulting in the development of a basal-positive transepithelial potential difference (TEP) and the formation of domes (fluid-filled elevations of the cell layer above the culture dish substrate). Stimulation by prostaglandin E (PGE) increases the magnitude of the TEP, the short-circuit current (I(sc)) measured in Transwell Ussing chambers, and the height of domes in cultures grown on impermeable substrates. A phenamil-resistant, PGE-stimulated component of the I(sc) in Transwells and of the TEP in monolayers in conventional culture dishes was inhibitable by bumetanide, a diuretic drug that blocks NaKCl symporters, mediating active transport of Cl. The rate of decrease in height of domes in cultures after addition of phenamil, presumably indicative of transport of fluid in a basal-to-apical direction, was also reduced by bumetanide. Studies with Transwells in Cl-free, HCO-free or Cl- and HCO-free media indicated that thyroid cells transported HCO as well as Cl in a basal-to-apical direction. It was concluded that the thyroid epithelium is both sodium absorbing and anion secreting

Inhibitory effects of amiloride and its analogues on prostaglandin E2-stimulated fluid transport by cultured porcine thyroid cells: Evidence for apical membrane Na+ channels

Confluent monolayers of cultured porcine thyroid cells transport fluid from the apical to the basal surface, forming circumscribed zones of detachment (domes) from the culture dish substrate. Fluid transport, as measured by increase in dome height, was stimulated by prostaglandin E (PGE; 1μmol/l) and inhibited by amiloride (0.1-100 μmol/l). Values of the inhibition constant [K(i)] with 95% confidence limits for each of a series of amiloride analogues were: 3',4'-dichlorobenzamil (DCB), 0.090(0.045-0.18) μmol/l; 2',4'-dimethylbenzamil (DMB), 0.14 (0.074-0.27) μmol/l; amiloride, 0.72 (0.33-1.8) μmol/l; 5-(N,N-hexamethylene)amiloride (HMA), 71 (5.9-43) μmol/l; 5-(N-ethyl-N-isopropyl)amiloride (EIPA), 33 (15-17) μmol/l; and 2-guanidinobenzimidazole, 243 (110-570) μmol/l. Triaminopyrimidine was ineffective at concentrations up to 1 mmol/l. Since DCB and DMB are known to have a higher affinity for Na channels, while HMA and EIPA show higher affinity for Na/H antiports, it was concluded that PGE-stimulated fluid transport involved an apical membrane Na channel

Regulation of cytoplasmic pH of cultured bovine corneal endothelial cells in the absence and presence of bicarbonate

Intracellular pH (pHi) in confluent monolayers of cultured bovine corneal endothelial cells was determined using the pH-dependent absorbance of intracellularly trapped 5(and 6)carboxy-4',5'-dimethylfluorescein. Steady-state pH was 7.05 +/- 0.1 in the nominal absence of bicarbonate, and 7.15 +/- 0.1 in the presence of 28 mM HCO3-/5% CO2. Following an acid load imposed by a NH4Cl prepulse, pHi was regulated in the absence of HCO3- by a Na+-dependent process inhibitable to a large extent by 1 mM amiloride and 0.1 mM dimethylamiloride. In the presence of 28 mM HCO3-/5% CO2, this regulation was still dependent on Na+, but the inhibitory potency of amiloride was less. DIDS (1 mM) partially inhibited this regulation in the presence, but not in the absence of bicarbonate. With cells pretreated with DIDS, amiloride was as effective in inhibiting recovery from acid load as in the absence of HCO3-. The presence of intracellular Cl- did not appreciably affect this recovery, which was still sensitive to DIDS in the absence of Cl-. Removal of extracellular Na+ led to a fall of pHi, which was greatly attenuated in the absence of HCO3-. This acidification was largely reduced by 1 mM DIDS, but not by amiloride. Cl removal led to an intracellular alkalinization in the presence of HCO3-. The presence of a Cl-/HCO3- exchanger was supported by demonstrating DIDS-sensitive 36Cl- uptake into confluent cell monolayers. Thus, bovine corneal endothelial cells express three processes involved in intracellular pH regulation: an amiloride-sensitive Na+/H+ antiport, a Na+-HCO3- symport and a Cl-/HCO3- exchange, the latter two being DIDS sensitive