Amiloride Hydrochloride Research Articles

Spectrophotometric determination of rhenium as perrhenate by extraction with amiloride hydrochloride

Rhenium (0–20 μg) may be determined as perrhenate spectrophotometrically at 362 nm after its extraction at pH 4 with amiloride hydrochloride into 4-methyl-2-pentanone. The relative standard deviation for 7 determinations of 10 μg of perrhenate is 1.5%. The effects of pH and diverse ions are reported. Only perchlorate, periodate and iodide interfere seriously. The system has been applied to the determination of rhenium on alumina and on carbon catalysts.

Suppression by amiloride of bombesin‐enhanced peritoneal metastasis of intestinal adenocarcinomas induced by azoxymethane

The effects of concomitant administration of bombesin and of the diuretic drug amiloride on the development of large and small intestinal tumors induced by azoxymethane (AOM), the incidence of their metastasis to the peritoneum and the labeling index of intestinal adenocarcinomas were investigated in inbred Wistar rats. From the start of the experiment, rats were given weekly s.c. injections of AOM for 10 weeks and s.c. injections of bombesin and/or a higher or lower dose of amiloride hydrochloride (amiloride) every other day until the end of the experiment in week 45. Administration of bombesin significantly increased the incidence of intestinal tumors and cancer metastasis to the peritoneum in week 45. It also significantly increased the labeling index of intestinal adenocarcinomas. Although administration of both doses of amiloride with bombesin had little or no influence on the enhancement of intestinal tumorigenesis by bombesin, the location, histological type, depth of involvement or labeling index of intestinal adenocarcinomas, a higher dose of amiloride significantly reduced the incidence of cancer metastasis to the peritoneum. Our findings indicate that amiloride possesses an anti-metastatic activity.

Modulators of growth in primary culture of rat proximal tubular cells II

Summary: The present studies assessed the effects of manipulating extracellular sodium (Na) concentration and Na transport on cellular hypertrophy and hyperplasia in primary culture of rat proximal tubular cells. A concentration‐dependent effect on thymidine incorporation and protein content was observed with cell culture media Na concentration of 130, 140 and 150 mmol/L. This effect was independent of osmolality (matched with mannitol) and no stimulatory effect occurred if choline was substituted for Na. Cells derived from sham‐operated (Sx) animals exposed to a higher media concentration of Na (150 vs 140 mmol/L) had both stimulated thymidine incorporation to 186.8 ± 35.41% (P<0.05) and enhanced cell protein content to 134.7 ± 135% (P<0.05). This effect was more pronounced in cell cultures derived from unilaterally nephrectomized (Nx) animals, being 212.8 ± 31.5% (P<0.01) for thymidine incorporation (P<0.05 vs cells from sham‐operated animals grown in high Na media) and 114.4 ± 3.2% (P<0.001) for protein content (P=0.11 vs sham‐operated cells grown in similar conditions). the addition of 10−4 mmol/L ethylisopropyl amiloride hydrochloride (EIPA) to Nx cells in a normal or high Na concentration media resulted in a decrease in cellular protein content to 82.6 ± 6.8% (P<0.05) and 85.5 ± 0.2% (P<0.0001) compared to respective controls. 10−4 mol/L EIPA in media supplemented with insulin‐like growth factor (IGF‐1) blocked the proliferative response normally seen in response to this growth factor from 156.6 ± 13.7 to 27.5 ± 3.1% (P<0.0001) compared to control. However, the presence of EIPA did not abrogate the hypertrophic response elicited by IGF‐1 (cell protein content 128.1 ± 13.1% of control with IGF‐1 vs 124.9 ± 12.5 with IGF‐1 and EIPA; P= n.s.). Addition of 10−4 mol/L EIPA to 10% serum derived from either Sx or Nx animals blocked the growth response to the sera, limiting the cellular protein content to 76.6 ± 5.5% (P<0.0001) and 89.7 ± 4.4% (P<0.0001) and thymidine incorporation to quiescent levels of 0.2 ± 0.1% (P<0.0001) and 0.4 ± 0.1% (P<0.0001) compared to respective controls. In summary, rat renal proximal tubular cell growth is influenced by Na concentrations in the cell culture environment and inhibited in the presence of EIPA. This supports a role for altered epithelial transport in the cellular growth response to a number of stimuli.

Sodium intake linked to amiloride-sensitive gustatory transduction in C57BL/6J and 129/7 mice

Mice of 129/7 and C57BL/6J inbred strains received two-bottle, 48-h preference tests of NaCl solutions vs. distilled water. 129/J mice exhibited a greater preference for 0.08 M NaCl than did C57BL/ 6J mice. To determine if this strain difference was mediated by taste, the integrated neural responses of the chorda tympani nerve to a concentration range of NaCl and KCI were examined. Gustatory neural responses to NaCl were similar for 129/J and C57BL/ 6J mice. However, lingual application of 0.5 mM amiloride hydrochloride significantly suppressed chorda tympani responses to a range of NaCl concentrations in C57BL/6J mice but did not do so consistently in 129/J mice. Amiloride failed to significantly suppress responses to a range of KC1 concentrations in both mouse strains. The results suggest that for 129/J mice, sodium reception and transduction are primarily amiloride insensitive, whereas for C57BL/6J mice, both amiloride-sensitive and amiloride-insensitive components are present. The strain difference in NaCl intake may be mediated, in part, through gustatory mechanisms, with reduced preference for NaCl influenced by amiloride-sensitive sodium transduction mechanisms.

Increased ion transport in cultured nasal polyp epithelial cells.

To measure transepithelial bioelectric properties of cultured human nasal polyp and turbinate epithelial cells to test the hypothesis that nasal polyps have increased rates of ion transport. Cohort study. Private referral center. Individuals undergoing surgery for symptomatic nasal obstruction due to polyps caused by cystic fibrosis, nonatopic rhinosinusitis, or allergic rhinosinusitis. Epithelial cells were removed from separated polyp and turbinate samples by protease disaggregation and cultured on permeable collagen matrix supports. Transepithelial potential difference and resistance were measured daily. At the time of maximal transepithelial potential difference, the cultures were mounted in modified Ussing chambers and exposed to a sodium-positive channel blocker (amiloride hydrochloride) and to selected chloride-negative channel agonists (isoproterenol bitartrate, adenosine triphosphate). Maximal transepithelial potential difference, resistance, and equivalent short-circuit current. Bioelectric responses to amiloride, isoproterenol, and adenosine triphosphate. Polyp cultures had higher transepithelial potential difference and equivalent short-circuit current than turbinate cultures. The mediator responses were greater for polyp than for turbinate cultures. Polyp epithelia have increased Na+ absorption and Cl- permeability relative to turbinate epithelia. These results are consistent with the hypothesis that increased epithelial fluid absorption contributes to the development of nasal polyps.

Colorimetric Determination of Guanethidine Sulfate, Guanoxan Sulfate, and Amiloride Hydrochloride in Tablets

Simple colorimetric methods for the determination of guanethidine sulfate (I), guanoxan sulfate (II), and amiloride hydrochloride (III) are described. These methods are based on the reaction of each drug with 9,10-phenanthraquinone in the presence of either 1-naphthol, resorcinol, or 3,5-dihydroxybenzoic acid with 3 M potassium hydroxide aqueous solution. Green to blue colors were obtained with maximum absorptions at 648, 590, and 615 nm, respectively. Conditions for maximum color development with respect to reagent concentrations, reaction times, color stabilities, and the suitable concentration of potassium hydroxide solution were studied. Concentration ranges for I are 12-22, 4-24, and 6-14 μg/ml; for II they are 4-14, 4-16, and 4-16 μg/ml; and for III they are 10-24 and 14-30 μg/ml. With an application of this proposed method, the above three drugs in tablets were estimated within a relative standard deviation of less than 2%.

Suppression of neointimal thickening and smooth muscle cell proliferation after arterial injury in the rat by inhibitors of Na(+)-H+ exchange.

Replication of vascular smooth muscle cells is a key event in the pathogenesis of restenosis following angioplasty. Little is known about early biochemical events involved in the proliferation of smooth muscle cells following arterial injury. In the present study, the effect of Na(+)-H+ exchange inhibitors on neointima formation after balloon injury of the rat carotid artery was investigated. Neointima formation was quantified 14 days after injury by morphometric measurement of cross-sectional neointimal area and by fluorometric determination of DNA content. The specific Na(+)-H+ exchange inhibitor 3-methylsulfonyl-4-piperidino-benzoyl guanidine mesylate (Hoe 694) dose-dependently reduced neointimal area and DNA content, the latter finding indicating a true antiproliferative effect. The structurally different Na(+)-H+ exchange blocker 5-(N-ethyl-N-isopropyl)amiloride hydrochloride had comparable inhibitory effects on neointimal area and DNA content, whereas 5-methylsulfonyl-2-piperidino-benzoyl guanidine hydrochloride, a position isomer of Hoe 694 lacking Na(+)-H+ exchange blocking properties, did not suppress neointima formation. The effect of Na(+)-H+ exchange blockers on neointima formation depended on the duration of drug application. Maximal suppression was achieved only when Hoe 694 was applied throughout the entire experiment for 14 days. This inhibitory effect of Na(+)-H+ exchange blocker application for the first 2 weeks following injury lasted for 2 months. In conclusion, the results of the present study reveal a potential role of Na(+)-H+ exchange for smooth muscle cell proliferation in vascular disease.

Spectrofluorimetric determination of guanethidine sulphate, guanoxan sulphate and amiloride hydrochloride in tablets and in biological fluids using 9,10-phenanthraquinone

A highly sensitive spectrofluorimetric method for the determination of some drugs of the monosubstituted guanidine derivatives in laboratory made tablets, in spiked human serum and in urine samples is presented. The method is based on the reaction of guanethidine sulphate (I), guanoxan sulphate (II) and amiloride hydrochloride (III) with 9,10-phenanthraquinone (IV) to give highly fluorescent derivatives. The linearity ranges were found to be 0.06–0.96 μg/ml for (I) and (II) and 0.04–0.28 μg/ml for (III), with relative standard deviation less than 2%. Mean percentage recoveries for tablets were found to be 99.9 ± 1.3, 100.5 ± 1.1 and 100.0 ± 1.6 for I, II and III, respectively. For I and III the results are highly correlated with the B.P. methods. Using the synchronous fluorimetry, differentiation between I and II was possible. Chloroform, dichloromethane and ethyl acetate have been used to extract I, II and III, respectively from serum and urine at basic pH, followed by applying the proposed fluorimetric method. Percentage recoveries were found to be 95.7–102.2%. The limit of detection is 0.04 μg/ml for I and II and 0.02 μg/ml for III.

Suppression of human alveolar macrophage-derived cytokines by amiloride.

Various human alveolar macrophage (AM)-derived cytokines in the lungs have been shown to be present under conditions of normal homeostasis as well as during the pathogenesis of inflammation. Although extensive investigation has demonstrated the induction of cytokines from AM, relatively little is known regarding endogenous and exogenous regulation of their production. Several pharmacologic agents, including corticosteroids, cyclooxygenase inhibitors, prostaglandins, and methyl-xanthines have been examined for their role in the modulation of mononuclear phagocyte-derived cytokines. In this study, we examine the role of amiloride for the regulation of AM-derived interleukin (IL)-8, tumor necrosis factor (TNF), IL-6, and IL-1 beta. Amiloride in concentrations of 10(-4) to 10(-6) M, concentrations capable of being achieved in the distal airways via nebulization, were shown to inhibit lipopolysaccharide-stimulated, AM-derived IL-8 and TNF in both a time- and dose-dependent fashion. In addition, 5-(N,N-hexamethylene) amiloride hydrochloride, an amiloride analogue with specific sodium channel antiport inhibition, resulted in a similar dose-dependent suppression of lipopolysaccharide-stimulated, AM-derived IL-8 production. Furthermore, the suppressive effect of amiloride appeared to be at the level of mRNA for IL-8, TNF, IL-1 beta, and IL-6, whereas steady-state levels of beta-actin mRNA remained unaltered. These findings would suggest that amiloride has a potentially important modulating influence for the regulation of AM-derived cytokines.

Amiloride sensitivity of the chorda tympani response to sodium chloride in sodium-depleted Wistar rats.

Peripheral gustatory mechanisms that may contribute to the expression of sodium (Na) appetite have been a focus of interest for many years. Because amiloride-sensitive Na transport is involved in the generation of neural signals in response to NaCl stimulation, the present study assessed whether changes in amiloride sensitivity of the neural response to NaCl accompany the induction of a Na appetite in the rat. Na deprivation was achieved by acute depletion with the diuretic furosemide. The magnitude of the whole-nerve chorda tympani response to 0.5 M NaCl was reduced in Na-depleted, compared with Na-replete, rats, which provides qualified support for previous reports that the induction of a Na appetite is associated with reduced neural responses to NaCl. However, changes in sensitivity to the specific Na channel blocker amiloride hydrochloride as a result of Na depletion were not evident. These findings suggest that the behavioral and neural changes that occur after Na depletion are not based on changes in amiloride sensitivity in the taste bud.

Felodipine: A New Dihydropyridine Calcium-Channel Antagonist

Felodipine, a dihydropyridine calcium-channel antagonist, significantly reduces systolic and diastolic blood pressure (BP) in patients with hypertension and has been associated with beneficial hemodynamic effects in patients with chronic stable angina pectoris or congestive heart failure (CHF). In hypertensive patients, felodipine does not appear to significantly affect glomerular filtration rate, creatinine clearance, glucose tolerance, or plasma lipoprotein concentrations. Studies comparing felodipine with other agents as monotherapy in mild to moderate hypertension have demonstrated felodipine to be at least as efficacious as hydrochlorothiazide (HCTZ) and HCTZ plus amiloride hydrochloride in combination. Comparisons of felodipine with other agents as adjuncts to beta-blocker or diuretic therapy have shown felodipine to be at least as effective as HCTZ, propranolol hydrochloride, prazosin hydrochloride, and nifedipine. Evaluations of patients with chronic stable angina are limited, and additional studies are needed before felodipine can be recommended for the routine management of angina pectoris. Similarly, additional studies are essential to delineate the role of felodipine, if any, in the management of CHF. In the management of hypertension, felodipine 5-40 mg/d significantly reduces systolic and diastolic BP. Although some patients may be controlled throughout the entire dosing interval when felodipine is administered bid, many patients will require more frequent dosing to obtain adequate BP control. Adverse effects associated with felodipine are similar to those of other dihydropyridine calcium-channel antagonists and include peripheral edema, headache, dizziness, flushing, and fatigue. A potentially clinically important drug interaction was observed when felodipine was administered concomitantly with theophylline aminopropanol; significant decreases in theophylline concentrations were noted. In summary, felodipine appears to be safe and effective for the management of hypertension when used alone or in combination with other antihypertensive agents. The efficacy of felodipine in the management of chronic stable angina pectoris and CHF requires further investigation.

Amiloride sensitivity of chorda tympani response to NaCl in Fischer 344 and Wistar rats

Fischer 344 (F-344) rats fail to prefer NaCl solutions to water at any concentration and avoid NaCl solutions preferred by other strains, including Wistar rats. Behavioral and electrophysiological responses of the mammalian gustatory system to NaCl have been shown to depend on a sodium transport system that is specifically blocked by lingual application of the sodium-transport blocker amiloride. The present study examined whether strain differences exist between F-344 and Wistar rats in the amiloride sensitivity of the chorda tympani (CT) electrophysiological response to NaCl. Whole nerve CT recordings were obtained from adult F-344 and Wistar rats during chemical stimulation of the anterior tongue. Responses to NaCl solutions ranging from 0.01 to 1.0 M were examined both before and after pretreatment with amiloride hydrochloride. Integrated whole nerve responses to NaCl solutions were expressed relative to the response to 0.5 M NH4Cl. Strain differences in the response to NaCl solutions emerged, with F-344 animals showing a significantly larger amplitude of the tonic response to NaCl, relative to NH4Cl, than Wistars. F-344 rats were also more sensitive to the sodium-channel blocker amiloride. These results suggest that strain differences in amiloride sensitive signals mediated by the CT nerve may contribute to the NaCl aversion displayed by F-344 rats.

Hypertension in the elderly: a study of a combination of atenolol, hydrochlorothiazide and amiloride hydrochloride

SummaryThe anti‐hypertensive effects of atenolol (Tenormin) 50 mg, a potassium‐sparing diuretic (half‐strength Moduretic) comprising hydrochlorothiazide 25 mg plus 2.5 mg amiloride hydrochloride, and the ‘free’ combination of atenolol and diuretic were compared in elderly hypertensive patients aged 60‐79 years.After a four‐week run‐in period on placebo, patients were randomly assigned, in a double‐blind manner, to atenolol or diuretic treatment, each for four weeks. Thereafter patients were given the ‘free’ combination for a further four weeks and this treatment was continued for six months.Blood pressure and heart rate were measured after the patient had rested for five minutes supine and after two minutes standing. These blood pressure measurements were made at least 24 hours after the preceding dose using a Random Zero sphygmomanometer. Results from 26 of the 27 patients entered into the study showed an advantage for combination therapy combined with either atenolol or diuretic treatment alone.No significant difference was found between treatments in the frequency of supraventricular and ventricular ectopic beats occurring in six patients who underwent 24‐hour ambulatory ECG monitoring. However, ectopic activity was reduced in some patients during beta‐blocker treatment.Few adverse effects occurred with any treatment. Three patients withdrew during the placebo period and three withdrew while taking active treatment.This study has shown that the combination of atenolol, hydrochlorothiazide and amiloride hydrochloride is an effective, safe, well‐tolerated antihypertensive drug regimen when used once daily in elderly hypertensive patients.

Analysis of Planar Chromatograms by Fast Atom Bombardment and Laser Desorption Mass Spectrometry

Abstract The applications of fast atom bombardment mass spectrometry coupled with thin-layer chromatography in a study of phospholipids, alkaloids derived from bloodroot rhizomes, and diuretics are summarized. Use of the mass spectrometric data to trace compounds via characteristic fragment ions increases the effective resolution of the chromatographic resolution. Quantitative values derived in TLC/FAB experiments for the diuretic amiloride hydrochloride is linear over two orders of magnitude encompassing the clinically useful range. The use of a laser desorption ionization method and Fourier transform mass analysis for TLC plate detection is also described; matrix and concentration effects are significant in this case, but several successful applications are described. An interface device for the linkage

Inhibitory Effects of Amilorides on Pinealocyte Adenosine 3′,5′-Monophosphate and Guanosine 3′,5′- Monophosphate Accumulation: Possible Involvement of Postreceptor Mechanisms*

In rat pinealocytes, alpha 1-adrenergic receptor activation increases intracellular pH (pHi) through Ca2+/protein kinase-C-dependent activation of the Na+/H+ antiporter. Using a series of amiloride analogs, norepinephrine stimulation of cGMP accumulation is also found to be pHi dependent. In this study, we examined the postreceptor mechanisms involved in the amiloride effects on cyclic nucleotide accumulation using agents that simulate alpha 1-adrenoceptor activation. Four amiloride analogs, with a 500-fold difference in their inhibitory potency of the Na+/H+ antiporter, were used. 5-(N,N-Hexamethylene)amiloride (HA), the most active inhibitor of the Na+/H+ antiporter, had a stimulatory effect on isoproterenol (ISO)-stimulated cAMP, while its effect on cGMP was inhibitory. The other three amiloride derivatives had no effect on the ISO-stimulated cAMP or cGMP responses. All four amilorides (at 10 microM) had no effect on the phenylephrine potentiation of cAMP responses in beta-adrenergically stimulated cells, while they inhibited the potentiation of cGMP accumulation according to their inhibitory potency on the Na+/H+ antiporter. Using depolarizing concentrations of K+, it was found that HA was additive to the submaximal potentiation by K+ on ISO-stimulated cAMP, while its effect on cGMP was inhibitory. Amiloride hydrochloride dihydrate, the amiloride that is least potent in its inhibitory action on the Na+/H+ antiporter, had no effect on the K+ potentiation of either cAMP or cGMP. Using 4 beta-phorbol 12-myristate 13-acetate in cells treated with 10 mM K+ and ISO, it was found that HA was additive to phorbol 12-myristate 13-acetate and K+ potentiation of the cAMP response, while its effect on the cGMP response was inhibitory. Amiloride hydrochloride had no effect on either the cAMP or cGMP response. It can be concluded from these studies that 1) HA has a stimulatory effect on the beta-adrenoceptor-Gs-adenylate cyclase pathway that is independent of inhibition of the Na+/H+ antiporter; 2) postreceptor mechanisms are involved in HA's effects on cAMP and cGMP accumulation; and 3) the action of HA on cGMP is likely to be related to its effect on the Na+/H+ antiporter.

Peripheral taste responses in genetically hypertensive rats

In two-bottle preference-aversion tests, the spontaneously hypertensive rat (SHR) tolerates higher concentrations of NaCl than the normotensive Wistar-Kyoto (WKY). In contrast, the inbred Dahl salt-sensitive (S/JR) and inbred Dahl salt-resistant (R/JR) rat show similar preferences for NaCl. In order to determine if taste receptor function was also altered between the hypertensive rat and its normotensive control, we recorded electrophysiological taste responses from the chorda tympani (CT) nerve in SHR, WKY, S/JR and R/JR rats. Responses to a concentration series (0.05 M to 0.5 M) of NaCl, NaAcetate, KCl, NH 4Cl and CaCl 2 were recorded before and after lingual application of amiloride hydrochloride, an epithelial sodium transport blocker. When expressed relative to the 0.5 M NH 4Cl response, responses to the majority of stimuli were equivalent between the SHR and WKY. By comparison, relative responses to NaCl were greater in the R/JR than S/JR; however, the magnitude of amiloride suppression was equivalent between these two strains. Relative responses to the majority of the remaining salts did not differ between the S/JR and R/JR. These results suggest that taste receptor function may be equivalent between the hypertensive rat and its normotensive control.

Perceptual characteristics of the amiloride-suppressed sodium chloride taste response in the rat.

The contribution of amiloride-sensitive membrane components to the perception of NaCl taste was assessed by using a conditioned taste aversion procedure. Eight independent groups of adult rats were conditioned to avoid either 0.1M NaCl, 0.5M NaCl; 0.1M NH4Cl, or 1.0M sucrose while their tongues were exposed either to water or to the sodium transport blocker amiloride hydrochloride. In contrast to rats exposed to water during conditioning, rats exposed to amiloride were unable to acquire a conditioned taste aversion to 0.1M NaCl. Differences in the acquisition of taste aversions between the amiloride- and nonamiloride-treated groups were not apparent when the conditioned stimulus (CS) was 0.5M NaCl, 0.1M NH4Cl, or 1.0M sucrose. Although the magnitude of the 0.5M NaCl aversion was similar between amiloride- and non-amiloride-treated rats, the perceptual characteristics of the CS differed between groups. Analyses of stimulus generalization gradients revealed that amiloride-treated rats generally avoided all monochloride salts after conditioning to 0.5M NaCl but not nonsodium salts or nonsalt stimuli. In contrast, rats not treated with amiloride only generalized the 0.5M NaCl aversion to sodium salts. No differences in generalization gradients occurred between groups when the CS was 0.1M NH4Cl or 1.0M sucrose. These findings suggest that the "salty" taste of NaCl is primarily related to the amiloride-sensitive portion of the functional taste response in rats. Conversely, the portion of the NaCl response insensitive to amiloride appears to have "sour-salty" perceptual characteristics and does not appear to be perceived as being salty.

Application of p- N, N-dimethylphenylenediamine dihydrochloride for the determination of some diuretics

A spectrophotometric method is described for the determination of acetazolamide, frusemide, polythiazide, benzthiazide, hydrochlorothiazide, hydroflumethiazide, trichlormethiazide and amiloride hydrochloride (AML) either in pure form or in pharmaceutical formulations. The method is based on the reaction with p- N, N-dimethylphenylenediamine dihydrochloride and chloramine-T, to give a coloured product having maximum absorbance at 660 nm (for AML) or at 540 nm for the others, and reproducible within ± 1.0%.

Hydrochlorothiazide With or Without Amiloride for Hypertension in the Elderly

To the Editor. —In a recent article by Myers1published in the June 1987 issue of theArchives, the following points need further clarification. 1. One of the study's objectives was to monitor the effects of increasing doses of hydrochlorothiazide, yet it is unclear from the study design whether both the dose of hydrochlorothiazide and amiloride, or just the hydrochlorothiazide alone, was increased. The author states that patients were randomly allocated to 12 weeks of increasing doses of hydrochlorothiazide either alone or in combination with amiloride. Tablets contained either hydrochlorothiazide, 25 mg, or hydrochlorothiazide, 25 mg, plus amiloride hydrochloride, 2.5 mg, with the tablets being identical to each other and placebo in both appearance and taste.... If the goal blood pressure (supine diastolic pressure,

Amiloride and amiloride analogs inhibit Na +/K +-transporting ATPase and Na +-coupled alanine transport in rat hepatocytes

Amiloride, a commonly used inhibitor of Na +-H + exchange, has been shown to exhibit a variety of nonspecific effects. Recently, the more potent amiloride analogs, 5-(N,N- dimethyl)amiloride hydrochloride (DMA) and 5-(N- ethyl-N- isopropyl)amiloride (EIA), have been used to control for the nonspecific effects of the parent compound. In the present study, we have explored the effects of these analogs on Na +/K +-transporting ATPase (Na +/K +-ATPase) and Na +-coupled alanine transport in primary rat hepatocyte cultures and rat liver plasma membranes, and we have compared the effects of these analogs with the effects of amiloride and ouabain. Amiloride, DMA, and EIA increased staedy-state Na + content and inhibited ouabain-sensitive 86Rb + uptake in a reversible, concentration-dependent, ouabain-like manner, with estimated 50% inhibitory concentrations (IC 50) of 3.0 · 10 −3 M, 5.2 · 10 −4 M, and 1.2 · 10 −4 M, respectively. Amiloride, DMA and EIA also inhibited ouabain-sensitive ATP hydrolysis in rat liver plasma membranes with similar potency (IC 50 values of 2.2 · 10 −3 M, 2.2 · 10 −3 M, and 1.7 · 10 −4 M, respectively). In separate experiments, amiloride (5 · 10 −3 M), DMA (10 −3 M), and EIA (2.5 · 10 −4 M) decreased the uptake into hepatocytes of alanine by 20%, 61%, and 59%, respectively, and further studies with DMA (10 −3 M) demonstrated that this inhibition was largely due to a decrease in the Na +-dependent fraction of alanine uptake. These findings indicate that amiloride, DMA, and EIA inhibit hepatic Na +/K +-ATPase directly, reversibly, and with a relative rank order potency of EIA > DMA > amiloride . All three compounds also inhibit the hepatic uptake of alanine, and presumably could indirectly inhibit other Na +-coupled transport processes as well.