s-3226 and cariporide

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

The mouse blastocyst consists of the trophectoderm, the inner cell mass, and a fluid-filled cavity, the blastocoel. Formation and subsequent expansion of this cavity is important for further differentiation of the inner cell mass and successful implantation. Previous work provided evidence that vectorial transport of Na+ and CL- ions through the trophectoderm into the blastocoel generates an osmotic gradient that drives fluid across this epithelium. As the activity of the Na+ / H+ exchanger (NHE) has been implicated as the exchanger responsible for facilitating the transtrophectodermal Na+ flux, the functional role of NHE in mouse blastocoel development was determined. Embryos were cultured in the presence of subtype-specific NHE inhibitors to examine the role of NHEs in blastocoel development. When 2-cell stage embryos were treated continuously with a specific inhibitor of NHE-1, cariporide, the embryos passed beyond the 8-cell stage and became blastocysts. However, in the presence of a specific inhibitor of NHE-3, S3226, the 2-cell stage embryos developed to the morula stage but formation of the blastocyst were inhibited in a dose-dependent manner. Cariporide did not inhibit the formation of the blastocoel cavity from the morula stage whereas S3226 did inhibit that process. S3226 also reduced the rate of re-expansion of blastocysts collapsed by cytochalasin D upon transfer to the control medium. An immunofluorescence study showed that NHE-3 was detected in the vicinity of the cell membrane of the trophectoderm, especially in the apical cell margins of the trophectoderm. These results suggest that NHE-3 is likely involved in blastocyst formation.

Topics: Analysis of Variance; Animals; Blastocyst; Dose-Response Relationship, Drug; Fluorescent Antibody Technique; Guanidines; Immunohistochemistry; Methacrylates; Mice; Microscopy, Confocal; Sodium-Hydrogen Exchangers; Sulfones

It was recently demonstrated that ammonia inhibits sodium absorption in the proximal colon of rats. In order to investigate the effect of luminal ammonia in the distal colon, sodium and chloride transport were measured in Ussing chambers. Under short-circuit conditions, distal colon absorbed sodium and chloride. When luminal ammonia (30 mmol l(-1)) was present, sodium and chloride absorption was diminished. Inhibition of the two Na(+)-H(+) exchanger isoforms NHE2 and NHE3, which are known to be located in the apical membrane of the distal colon epithelium, failed to influence the effect of ammonia on transepithelial sodium and chloride fluxes. The inhibitory effect of ammonia was eliminated under the following conditions: after block of carbonic anhydrases with acetazolamide, in the presence of an unspecific blocker of Na(+)-H(+) exchangers, and under chloride-free conditions. Ammonia did not alter electrogenic sodium absorption. These results demonstrate that luminal ammonia inhibits sodium and chloride absorption in rat distal colon. We suggest that ammonia inhibits NaCl absorption by interfering with a Na(+)-H(+) exchanger that is not NHE2 or NHE3

Topics: Ammonia; Animals; Buffers; Chlorides; Colon; Electrophysiology; Guanidines; In Vitro Techniques; Intestinal Absorption; Male; Methacrylates; Patch-Clamp Techniques; Rats; Rats, Sprague-Dawley; Rats, Wistar; Sodium; Sodium-Hydrogen Exchangers; Solutions; Species Specificity; Sulfones

The influence of ammonia on sodium and chloride fluxes in rat proximal colon was studied in Ussing chamber experiments. Under short-circuit conditions, the proximal colon absorbed sodium and secreted chloride. The presence of ammonia (30 mmol 1(-1) mucosal) diminished Na+ absorption, but had hardly any influence on Cl- fluxes. Blocking the apical Na+/H+ exchanger isoform NHE2 by amiloride or HOE642 diminished absorptive Na+ and Cl- fluxes. In contrast, the NHE3-specific antagonist S3226 was ineffective. Amiloride and HOE642 also inhibited the effect of ammonia on net sodium absorption. In bicarbonate-free buffer solution, ammonia failed to alter the absorptive fluxes of sodium and chloride, but increased the secretory fluxes of Na+ and Cl-. The latter effect was blocked by HOE642. These results suggest that basal NaCl absorption in rat proximal colon depends to a large extent on NHE2. Mucosal ammonium decreases Na+ absorption and this effect can be antagonized by blocking NHE2. This observation suggests that ammonium interacts with the apical Na+/H+ exchanger, thereby diminishing sodium absorption.

Topics: Amiloride; Ammonia; Animals; Chlorides; Colon; Guanidines; Intestinal Absorption; Male; Methacrylates; Rats; Rats, Sprague-Dawley; Sodium; Sodium-Hydrogen Exchangers; Sulfones

Inhibition of Na+/H+ exchange (NHE) subtypes has been investigated in a study of the mouse fibroblast L cell line (LAP1) transfected with human (h) NHE1, rabbit (rb) NHE2, rat (rt) or human (h) NHE3 as well as an opossum kidney cell line (OK) and porcine renal brush-border membrane vesicles (BBMV). S3226 ¿3-[2-(3-guanidino-2-methyl-3-oxo-propenyl)-5-methyl-phenyl]-N-isopro pylidene-2-methyl-acrylamide dihydro-chloride¿ was the most potent and specific NHE3 inhibitor with an IC50 value of 0.02 micromol/l for the human isoform, whereas its IC50 value for hNHE1 and rbNHE2 was 3.6 and approximately = 80 micromol/l, respectively. In contrast, amiloride is a weak NHE3 inhibitor (IC50>100 micromol/l) with a higher affinity to hNHE1 and rbNHE2. Cariporide (4-isopropyl-3-methylsulphonyl-benzoyl-guanidine methane-sulphonate), which has an IC50 for NHE3 of approximately 1 mmol/l, is a highly selective NHE1 inhibitor (0.08 micromol/l). Therefore, S3226 is a novel tool with which to investigate the physiological and pathophysiological roles of NHE3 in animal models.

Topics: Amiloride; Animals; Cell Line; Dose-Response Relationship, Drug; Guanidines; Humans; Ion Channels; Methacrylates; Mice; Microvilli; Opossums; Rabbits; Rats; Sodium-Hydrogen Exchanger 3; Sodium-Hydrogen Exchangers; Stereoisomerism; Sulfones; Swine