s-3226 and ethylisopropylamiloride

This study evaluated the effects of aldosterone upon Na+/H+ exchange (NHE) activity in immortalized proximal tubular epithelial (PTE) cells from the spontaneously hypertensive rat (SHR) and the normotensive controls (Wistar Kyoto rat; WKY). Increases in NHE activity after exposure to aldosterone occurred in time- and concentration-dependent manner in SHR PTE cells, but not in WKY PTE cells. The aldosterone-induced increases in NHE activity were prevented by spironolactone, but not by the glucocorticoid receptor antagonist Ru 38486. The presence of the mineralocorticoid receptor transcript was confirmed by PCR and NHE1, NHE2, and NHE3 proteins were detected by immunoblot analysis. Cariporide and EIPA, but not S3226, inhibited the aldosterone-induced increase in NHE activity, indicating that NHE1 is the most likely involved NHE isoform. Pretreatment of SHR PTE cells with actinomycin D attenuated the aldosterone-induced increases in NHE activity. The SHR PTE cells had an increased rate of H2O2 production when compared with WKY PTE cells. Treatment of cells with apocynin, a NADPH oxidase inhibitor, markedly reduced the rate of H2O2 production. The aldosterone-induced increase in NHE activity SHR PTE cells was completely prevented by apocynin. In conclusion, the aldosterone-induced stimulation of NHE1 activity is a genomic event unique in SHR PTE cells, which involves the activation of the mineralocorticoid receptor, but ultimately requires the availability of H2O2 in excess.

Topics: Aldosterone; Amiloride; Animals; Cell Line; Cytochalasin B; Dactinomycin; Gene Expression Regulation; Genome; Guanidines; Hydrogen Peroxide; Hydrogen-Ion Concentration; Immunoblotting; Kidney Tubules, Proximal; Methacrylates; Mifepristone; Oxidative Stress; Protein Isoforms; Rats; Rats, Inbred SHR; Receptors, Mineralocorticoid; RNA, Messenger; Sodium-Hydrogen Exchanger 1; Sodium-Hydrogen Exchangers; Spironolactone; Sulfones

Reperfusion after ischemic conditions induces massive endothelial cell (EC) activation, an initial step of reperfusion injury. Reperfusion is characterized by reoxygenation, realkalinization and a localized increase of inflammatory stimuli. In this study, we focused on the influence of extracellular realkalinization on human umbilical vein endothelial cell (HUVEC) activation. We examined intracellular pH (pH(in)) and intracellular free calcium concentration ([Ca(2+)](in)), a second messenger known to mediate von Willebrand factor (VWF) exocytosis in endothelium, upon realkalinization. Furthermore, we measured the agonist-stimulated exocytosis of VWF, Interleukin-8 and soluble P-selectin (sP-Selectin) as markers of EC activation. To verify a morphological correlate of EC activation, we finally observed platelet-endothelial adherence during realkalinization using shear flow. Realkalinization of HUVEC was simulated by switching from bicarbonate buffered Ringer solution of an acidotic pH(ex) of 6.4 to a physiologic pH(ex) of 7.4. Extracellular realkalinization was accompanied by pH(in) recovery from 6.5 to 7.2 within 10 min. Application of cariporide, an inhibitor of the Na(+)/H(+) exchanger subtype 1 (NHE), during extracellular realkalinization significantly delayed the early kinetics of intracellular realkalinization. Histamine stimulated [Ca(2+)](in) was significantly increased upon realkalinization compared to control cells. Also agonist-stimulated release of VWF, Interleukin-8 and sP-Selectin was massively enhanced during pH(in) recovery in comparison to control. Furthermore, we observed an increased platelet binding to endothelium. Interestingly, each of these realkalinization-induced effects were significantly reduced by early application of cariporide. Therefore, delay of acute NHE-dependent pH(in) recovery may represent a promising mechanism for inhibition of EC activation upon reperfusion.

Topics: Acidosis; Amiloride; Calcium; Cell Adhesion; Cells, Cultured; Chloride-Bicarbonate Antiporters; Cyclic AMP; Endothelial Cells; Endothelium, Vascular; Exocytosis; Guanidines; Histamine; Humans; Hydrogen-Ion Concentration; Interleukin-8; Intracellular Fluid; Kinetics; Linear Models; Methacrylates; P-Selectin; Platelet Adhesiveness; Reperfusion Injury; Sodium-Hydrogen Exchangers; Sulfones; Umbilical Veins; von Willebrand Factor

Selective pharmacological Na+/H+ exchange (NHE) inhibitors were used to identify functional NHE isoforms in human small intestinal enterocytes (Caco-2) and to distinguish between direct and indirect effects on transport via the intestinal di/tripeptide transporter hPepT1. The relative potencies of these inhibitors to inhibit 22Na+ influx identifies NHE3 and NHE1 as the apical and basolateral NHE isoforms. The Na+-dependent (NHE3-sensitive) component of apical dipeptide ([14C] Gly-Sar) uptake was inhibited by the selective NHE inhibitors with the same order of potency observed for inhibition of apical 22Na+ uptake. However, 5-(N-ethyl-N-isopropyl) amiloride (EIPA) also reduced [14C]Gly-Sar uptake in the absence of Na+ and this inhibition was concentration and pH (maximal at pH 5.5) dependent. NHE3 inhibition by S1611 and S3226 modulates dipeptide uptake indirectly by reducing the transapical driving force (H+ electrochemical gradient). EIPA (at 100 microM) has similar effects, but at higher concentrations (> 200 microM) also has direct inhibitory effects on hPepT1.

Topics: Amiloride; Analysis of Variance; Biological Transport; Caco-2 Cells; Cation Transport Proteins; Cell Survival; Dipeptides; Dose-Response Relationship, Drug; Epithelial Cells; Guanidines; Humans; Hydrogen-Ion Concentration; Intestine, Small; Membrane Potentials; Membrane Proteins; Methacrylates; Peptide Transporter 1; Protein Isoforms; Protons; Sodium; Sodium-Hydrogen Exchanger 1; Sodium-Hydrogen Exchanger 3; Sodium-Hydrogen Exchangers; Sulfones; Symporters

Previous studies have shown that gastric glands express at least sodium-hydrogen exchanger (NHE) isoforms 1-4. Our aim was to study NHE-3 localization in rat parietal cells and to investigate the functional activity of an apical membrane NHE-3 isoform in parietal cells of rats. Western blot analysis and immunohistochemistry showed expression of NHE-3 in rat stomach colocalizing the protein in parietal cells together with the beta-subunit of the H(+)-K(+)-ATPase. Functional studies in luminally perfused gastric glands demonstrated the presence of an apical NHE isoform sensitive to low concentrations of 5-ethylisopropyl amiloride (EIPA). Intracellular pH measurements in parietal cells conducted in omeprazole-pretreated superfused gastric glands showed an Na+-dependent proton extrusion pathway that was inhibited both by low concentrations of EIPA and by the NHE-3 specific inhibitor S3226. This pathway for proton extrusion had a higher activity in resting glands and was inhibited on stimulation of histamine-induced H(+)-K(+)-ATPase proton extrusion. We conclude that the NHE-3 isoform located on the apical membrane of parietal cells offers an additional pathway for proton secretion under resting conditions. Furthermore, the gastric NHE-3 appears to work under resting conditions and inactivates during periods of H(+)-K(+)-ATPase activity.

Topics: Amiloride; Animals; Blotting, Western; Cell Membrane; Enzyme Inhibitors; Gastric Mucosa; Guanidines; H(+)-K(+)-Exchanging ATPase; Histamine; Hydrogen-Ion Concentration; Immunohistochemistry; In Vitro Techniques; Intracellular Membranes; Isoenzymes; Methacrylates; Omeprazole; Parietal Cells, Gastric; Perfusion; Rats; Rats, Sprague-Dawley; Sodium-Hydrogen Exchanger 3; Sodium-Hydrogen Exchangers

This study assessed the functional role of Na(+)/H(+) exchanger (NHE) isoforms NHE3 and NHE2 in the proximal tubule, loop of Henle, and distal convoluted tubule of the rat kidney by comparing sensitivity of transport to inhibition by Hoe-694 (an agent known to inhibit NHE2 but not NHE3) and S-3226 (an agent with much higher affinity for NHE3 than NHE2). Rates of transport of fluid (J(v)) and HCO(3)(-) (J(HCO3)) were studied by in situ microperfusion. In the proximal tubule, addition of ethylisopropylamiloride or S-3226 significantly reduced J(v) and J(HCO3), but addition of Hoe-694 caused no significant inhibition. In the loop of Henle, J(HCO3) was also inhibited by S-3226 and not by Hoe-694, although much higher concentrations of S-3226 were required than what was necessary to inhibit transport in the proximal tubule. In contrast, in the distal convoluted tubule, J(HCO3) was inhibited by Hoe-694 but not by S-3226. These results are consistent with the conclusion that NHE2 rather than NHE3 is the predominant isoform responsible for apical membrane Na(+)/H(+) exchange in the distal convoluted tubule, whereas NHE3 is the predominant apical isoform in the proximal tubule and possibly also in the loop of Henle.

Topics: Absorption; Amiloride; Animals; Bicarbonates; Biological Transport; Culture Techniques; Guanidines; Kidney Tubules, Distal; Kidney Tubules, Proximal; Loop of Henle; Male; Methacrylates; Rats; Rats, Sprague-Dawley; Sodium-Hydrogen Exchanger 3; Sodium-Hydrogen Exchangers; Sulfones