Enhanced epithelial sodium channel activity in neonatal Scnn1b mouse lung attenuates high oxygen-induced lung injury.

Prolonged oxygen therapy leads to oxidative stress, epithelial dysfunction, and acute lung injury in preterm infants and adults. Heterozygous Scnn1b mice, which overexpress lung epithelial sodium channels (ENaC), and their wild-type (WT) C57Bl6 littermates were utilized to study the pathogenesis of high fraction inspired oxygen ([Formula: see text])-induced lung injury. Exposure to high [Formula: see text] from birth to postnatal (PN) day 11 was used to model oxidative stress. Chronic exposure of newborn pups to 85% O2 increased glutathione disulfide (GSSG) and elevated the GSH/GSSG redox potential (Eh) of bronchoalveolar lavage fluid (BALF). Longitudinal X-ray imaging and Evans blue-labeled-albumin assays showed that chronic 85% O2 and acute GSSG (400 µM) exposures decreased alveolar fluid clearance (AFC) in the WT lung. Morphometric analysis of WT pups insufflated with GSSG (400 µM) or amiloride (1 µM) showed a reduction in alveologenesis and increased lung injury compared with age-matched control pups. The Scnn1b mouse lung phenotype was not further aggravated by chronic 85% O2 exposure. These outcomes support the hypothesis that exposure to hyperoxia increases GSSG, resulting in reduced lung fluid reabsorption due to inhibition of amiloride-sensitive ENaC. Flavin adenine dinucleotide (FADH2; 10 µM) was effective in recycling GSSG in vivo and promoted alveologenesis, but did not impact AFC nor attenuate fibrosis following high [Formula: see text] exposure. In conclusion, the data indicate that FADH2 may be pivotal for normal lung development, and show that ENaC is a key factor in promoting alveologenesis, sustaining AFC, and attenuating fibrotic lung injury caused by prolonged oxygen therapy in WT mice.

Study of the photolytic and photocatalytic transformation of amiloride in water.

The diffusion of drug residues in wastewaters and surface waters as rivers and streams may constitute a problem for the environment, with consequences on the ecosystem and also on the human health. This paper deals with the study of the photo-induced transformation of amiloride, an orally administered diuretic agent, under simulated solar light. Direct photolysis and photocatalyzed degradation processes, using titanium dioxide as a photocatalyst, were investigated. The study involved the monitoring of the drug decomposition, the identification of intermediate compounds of the decomposition, the assessment of mineralization, as well as the evaluation of the toxicity associated to the degradation products. Amiloride underwent complete degradation within 30min of irradiation (heterogeneous photocatalysis) or 4h (homogeneous photolysis). HPLC coupled to HRMS, via ESI interface, demonstrated to be a powerful tool to identify and measure degradation products of the studied drug. By considering the photocatalytic process, the identified intermediates are formed through: (1) dechlorination and hydroxylation of the heteroaromatic ring; (2) the detachment of the guanidinic moiety; (3) cleavage of the heteroaromatic ring. The drug photomineralization was a rather slow process and after 4h of irradiation 25% of the total organic carbon (TOC) was still present. Chlorine was stoichiometrically released as chloride ions within the considered irradiation times (4h), while nitrogen was only partially converted into ammonium ions. This was due to the formation of guanidine, known to be hardly mineralized photocatalytically, and some other small molecules still containing the nitrogen. Acute toxicity, measured with a Vibrio fischery assay, showed that amiloride transformation proceeded through the formation of toxic compounds.

Effects of Na+/H+ exchanger inhibitors on subcellular localisation of endocytic organelles and intracellular dynamics of protein transduction domains HIV-TAT peptide and octaarginine.

Protein transduction domains such as those derived from the HIV protein TAT have great potential as vectors for delivery of therapeutic entities such as genes and proteins into cells. Extensive studies have shown that a major fraction of the most studied variants enters cells via an endocytic mechanism. However, controversy surrounds the exact uptake mechanism and whether a specific pathway is utilised. Studies showing inhibition of uptake of protein transduction domains in the presence of ion-transport inhibitors such as amiloride and its more potent analogue 5-(N-ethyl-N-isopropyl) amiloride (EIPA) suggest a link between peptide internalisation and macropinocytosis. In this study, using immunolabelling of early and late components of the endocytic pathway, we show that treatment of cells with EIPA and to a lesser extent amiloride affects the morphology and subcellular location of early, late endosomes and lysosomes. Enlarged early and late endocytic structures were observed in EIPA-treated cells, and these organelles accumulated in a perinuclear region. Results from experiments investigating the effects of EIPA on distribution of fluorescent octaarginine were in agreement with the immunolocalisation studies. Treatment of the CD34(+) leukaemia cell line KG1a with EIPA in the presence of fluorescent conjugates of HIV-TAT peptide and octaarginine showed distinct vesicular staining in agreement with untreated cells but EIPA-treated cells were additionally characterized by increased localization of the peptides in the cytosol. At levels previously shown to inhibit uptake of HIV-TAT peptide and octaarginine in other cell lines, EIPA was without major effect on uptake of both peptides in KG1a cells.

Disrupting the establishment of polarizing activity by teratogen exposure.

Between days 9.5 and 10, the forelimb buds of developing murine embryos progress from stage 1 which are just beginning to express shh and whose posterior mesoderm has only weak polarizing activity to stage 2 limbs with a distinguishable shh expression domain and full polarizing activity. We find that exposure on day 9.5 to teratogens that induce the loss of posterior skeletal elements disrupts the polarizing activity of the stage 2 postaxial mesoderm and polarizing activity is not subsequently restored. The ontogeny of expression of the mesodermal markers shh, ptc, bmp2, and hoxd-12 and 13, as well as the ectodermal markers wnt7a, fgf4, fgf8, cx43, and p21 occurred normally in day 9.5 teratogen-exposed limb buds. At stage 3, the treated limb apical ectodermal ridge usually possessed no detectable abnormalities, but with continued outgrowth postaxial deficiencies became evident. Recombining control, stage matched limb bud ectoderm with treated mesoderm prior to ZPA grafting restored the duplicating activity of treated ZPA tissue. We conclude that in addition to shh an early ectoderm-dependent signal is required for the establishment of the mouse ZPA and that this factor is dependent on the posterior ectoderm.

Intracellular Ca2+ mediates the cytotoxicity induced by bepridil and benzamil in human brain tumor cells.

The effects of bepridil and benzamil, known Na(+)-Ca2+ exchange blockers, on the growth of human brain tumor cells were evaluated using SK-N-MC human neuroblastoma and U-373 MG human astrocytoma cells as model cellular systems. These drugs induced cytotoxicity in both cells in a dose-dependent manner. Agonist (2% fetal bovine serum) alone induced a rapid increased intracellular Ca2+ concentration and then it returned to the basal level. However, the pretreatments of these drugs resulted in a more sustained high intracellular Ca2+ concentration mobilized by an agonist. Moreover, BAPTA/AM, an intracellular Ca2+ chelator, significantly blocked the cytotoxicity induced by these drugs. These results suggest that bepridil and benzamil act as effective inhibitors of in vitro growth of human brain tumor cells and that intracellular Ca2+ may be involved in the mechanism of actions of these agents.

Intracellular pH and heat sensitivity in two human cancer cell lines.

BACKGROUND AND PURPOSE: The majority of studies which have demonstrated a linear correlation between intracellular pH (pHi) and thermosensitivity have used rodent cell lines. In order to understand the therapeutic potential of this strategy, it is necessary to determine whether similar observations can be obtained with human cancer cells. MATERIALS AND METHODS: Human breast cancer MCF-7, and nasopharyngeal carcinoma CNE-1 cell lines were heated at 43 degrees C under extracellular pH (pHe) conditions of 7.2 or 6.8, +/- NaHCO3. We studied the function of the Na+/H+ antiport, one of the primary membrane regulators of pHi, pHi level, and clonogenic survival after these treatments. RESULTS: After 2-h exposure to 43 degrees C at pHe 7.2, antiport activity in MCF-7 cells was > 50% relative to that of unheated cells, in contrast to < 20% relative activity for CNE-1 cells. Respective survival levels under these conditions were 0.25 and 0.04. The addition of 5-(N-ethyl-N-isoproply) amiloride (EIPA), a potent inhibitor of Na+/H+ antiport, had no effect on MCF-7 cells, but enhanced cytotoxicity for CNE-1 cells, when heated at pHe 6.8 without NaHCO3. Analysis of the correlation between log surviving fraction and pHi demonstrated that this relationship was much steeper for CNE-1 compared to MCF-7 cells. CONCLUSION: The relationship between pHi levels and thermosensitivity observed in rodent cells can also apply to two human cancer cell lines: MCF-7 and CNE-1, with the latter cells being apparently more amenable to manipulations of pHi regulation compared to the former.

Experimental models of distal renal tubular acidosis.

A number of potential defects may impair acidification either directly or indirectly in the CCT, the OMCT, the IMCD, or in all segments. These defects are summarized in Table 1. Findings from studies in animal models of DRTA have enhanced out understanding of the pathophysiological basis of these disorders. Nevertheless, considerable effort needs to be directed in the future toward defining the cellular basis of these defects, especially in the inherited forms of classical hypokalemic DRTA, for which an adequate experimental model does not yet exist.

The binding site on cochlear stereocilia for antisera raised against renal Na+ channels is blocked by amiloride and dihydrostreptomycin.

The mechanoelectrical transduction channels on hair cells have been suggested to be operated by tip links that are stretched when the hair bundle is deflected in the direction of the tallest row of stereocilia. Localising these channels is therefore an important test of this hypothesis. The transduction channels are known to be amiloride-sensitive and immunogold labelling with antibodies raised against the amiloride-sensitive epithelial Na+ channel from kidney (alpha NaCh), has suggested that sites with similar characteristics are located in the region where the tips of the shorter stereocilia appear to come into contact with the sides of the adjacent taller stereocilia rather than being associated directly with the tip links. Now, further immunocytochemical experiments have been performed to determine if amiloride and dihydrostreptomycin, both of which can block transduction, can affect this labelling. Immunofluorescent labelling of the stereocilia is obtained when surface preparations of the organ of Corti are fixed and incubated with alpha NaCh followed by an appropriate secondary antibody. This labelling is abolished by trypsinization prior to fixation but retained if the tissue is pretreated with amiloride and then trypsinized in its presence. Because amiloride is known to protect amiloride-binding sites from degradation by trypsin, these results suggest that alpha NaCh is revealing amiloride-binding sites on the stereocilia. Similarly, immunofluorescent labelling of the stereocilia is abolished if cochlear tissue is pretreated with dihydrostreptomycin (DHS) and fixed in its presence prior to incubation with alpha NaCh. Quantitative analysis of colloidal gold labelling using transmission electron microscopy shows that DHS treatment produces a significant reduction in the number of gold particles on stereocilia, especially in the region of contact between them. These results suggest that anti-Na+ recognises a site with characteristics similar to the mechanoelectrical transduction channels.

Exacerbation of acetazolamide teratogenesis by amiloride and its analogs active against Na+/H+ exchangers and Na+ channels.

Postaxial forelimb ectrodactyly induced by acetazolamide given on Day 9.5 of murine gestation is thought to be mediated by reduced intracellular pH (pHi) within the limb bud. Coadministration of amiloride increases the incidence and severity of acetazolamide-induced forelimb malformations and further reduces limb bud pHi. These findings were hypothesized to be attributable to the action of amiloride as an inhibitor of Na+/H+ exchangers (NHEs), plasma membrane-localized proteins involved in the maintenance of cellular pH homeostasis. Here, we explored this hypothesis further by coadministering with acetazolamide, amiloride, or analogs known to preferentially inhibit NHEs 5-(N-methyl-N-isobutyl)-amiloride, 5-(N, N-hexamethylene)-amiloride, 5-(N, N-dimethyl)-amiloride, and 5-(N-ethyl-N-isopropyl)-amiloride or amiloride-sensitive Na+ channels (benzamil). The coadministration of either amiloride, benzamil, 5-(N, N-dimethyl)-amiloride, 5-(N-ethyl-N-isopropyl)-amiloride, or 5-(N-methyl-N-isobutyl)-amiloride all dose responsively increased the frequency and severity of forelimb malformations compared to acetazolamide alone. None of the analogs given alone induced forelimb ectrodactyly. The data are consistent with the original hypothesis that the exacerbation of acetazolamide teratogenesis is due to NHE inhibition. Surprisingly, benzamil was the most potent potentiator of acetazolamide teratogenesis. This result strongly suggests that amiloride-sensitive Na+ channels are also present within the murine embryo and are likely to play a role in pHi homeostasis.

Effects of chronic and in utero treatment with diuretics on mouse exocrine cells studied by X-ray microanalysis.

In an attempt to develop an animal model for the disease cystic fibrosis, mice were chronically treated with diuretics. In addition, pregnant mice were treated with diuretics and the effect of this treatment in utero on the newborn mice was studied. Pancreas and submandibular gland acinar cells were investigated by X-ray microanalysis and transmission electron microscopy. Long-term treatment with furosemide (up to 13 months) caused transient changes in the elemental content of the pancreatic acinar cells: a decrease in chloride and sulfur, and an increase in phosphorus, potassium and magnesium. All changes normalized with prolonged treatment. Some morphological changes were found in the zymogen granules. Treatment with amiloride or furosemide in utero caused a decrease in cellular sodium and chloride levels, indicative of inhibition of transepithelial ion and fluid transport. Also treatment of adult animals for two months with amiloride caused lower intracellular sodium and chloride levels. In adult animals only minor effects of diuretic treatment on submandibular gland acinar cells were noted. In utero treatment with amiloride caused an increase in sodium and chloride content indicative of cell damage.

A cell-permeant amiloride derivative induces caspase-independent, AIF-mediated programmed necrotic death of breast cancer cells.

Amiloride is a potassium-sparing diuretic that has been used as an anti-kaliuretic for the chronic management of hypertension and heart failure. Several studies have identified a potential anti-cancer role for amiloride, however the mechanisms underlying its anti-tumor effects remain to be fully delineated. Our group previously demonstrated that amiloride triggers caspase-independent cytotoxic cell death in human glioblastoma cell lines but not in primary astrocytes. To delineate the cellular mechanisms underlying amiloride's anti-cancer cytotoxicity, cell permeant and cell impermeant derivatives of amiloride were synthesized that exhibit markedly different potencies in cancer cell death assays. Here we compare the cytotoxicities of 5-benzylglycinyl amiloride (UCD38B) and its free acid 5-glycinyl amiloride (UCD74A) toward human breast cancer cells. UCD74A exhibits poor cell permeability and has very little cytotoxic activity, while UCD38B is cell permeant and induces the caspase-independent death of proliferating and non-proliferating breast cancer cells. UCD38B treatment of human breast cancer cells promotes autophagy reflected in LC3 conversion, and induces the dramatic swelling of the endoplasmic reticulum, however these events do not appear to be the cause of cell death. Surprisingly, UCD38B but not UCD74A induces efficient AIF translocation from the mitochondria to the nucleus, and AIF function is necessary for the efficient induction of cancer cell death. Our observations indicate that UCD38B induces programmed necrosis through AIF translocation, and suggest that its cytosolic accessibility may facilitate drug action.

[Hyponatremia--should it be ignored or diagnosed?--Case 8/2011]. / Hyponatriämie--ignorieren oder diagnostizieren?--Fall 8/2011.

UNLABELLED: HISTORY, CLINICAL FINDINGS: A 72-year-old dehydrated female was admitted to our emergency department. She presented with a decreased level of consciousness and had experienced a fall. Her medication included hydrochlorothiazide and amiloride. DIAGNOSTIC: Laboratory findings showed a severe hyponatremia with a serum sodium concentration of 107 mmol/l and a reduced serum osmolality. Urine sodium and potassium excretion were > 30 mmol/l. A CT scan of the head did not show any signs of trauma. DIAGNOSIS, THERAPY AND CLINICAL COURSE: Using a diagnostic algorithm, the diagnosis of a hypotonic hypovolemic hyponatremia due to the intake of diuretics was confirmed. By intravenous infusion of physiological sodium chloride solution and cessation of diuretics, serum sodium concentration was raised gradually. Hereby, the patient`s state of consciousness completely normalized. CONCLUSIONS: Hyponatremia represents the most frequent electrolyte disturbance of hospitalized patients. It correlates with neurological deficits, proneness to falling and intrahospital mortality. Due to diagnostic insecurity of many physicians, the finding of a hyponatremia is often ignored or misclassified. Standardized approaches using diagnostic algorithms improve diagnostic accuracy. The here presented algorithm is based on only few parameters: serum and urine osmolality, urine sodium and potassium. Besides gradual raise of serum sodium, therapy of the underlying cause is essential, for example cessation of diuretics. For patients with syndrome of inadequate secretion of antidiuretic hormone (SIADH; hypotonic isovolemic hyponatremia), selective arginin-vasopressin-receptor 2-antagonists (vaptans) are a new therapeutic option. However, due to high costs, we only see an indication for patients with SIADH who are not able to consequently comply with fluid restriction.

Amiloride derivatives induce apoptosis by depleting ER Ca(2+) stores in vascular endothelial cells.

BACKGROUND AND PURPOSE: Amiloride derivatives are blockers of the Na(+)/H(+) exchanger (NHE) and at micromolar concentrations have protective effects on cardiac and brain ischaemia/reperfusion injury but at higher concentrations also induce apoptosis. Here, we aimed to elucidate the mechanism related to this cytotoxic action. EXPERIMENTAL APPROACH: We quantified the expression of genes associated with endoplasmic reticulum (ER) stress and measured changes in luminal ER Ca(2+) concentration ([Ca(2+)](ER)) with a 'cameleon' indicator, D1ER. KEY RESULTS: Amiloride derivatives induced apoptosis in vascular endothelial cells, an effect that increased at alkaline extracellular pH. The potency order for cytotoxicity was 5-(N,N-hexamethylene)-amiloride (HMA) > 5-(N-methyl-N-isobutyl) amiloride > 5-(N-ethyl-N-isopropyl) amiloride (EIPA) >> amiloride. HMA dose-dependently increased the transcription of the ER stress genes GADD153 and GADD34 and rapidly depleted [Ca(2+)](ER), mimicking the effects of the sarco/endoplasmic reticulum ATPase (SERCA) inhibitor thapsigargin. The NHE1-specific inhibitor HOE 694 inhibited NHE activity by 87% but did not alter [Ca(2+)](ER). The decrease in [Ca(2+)](ER) evoked by amiloride derivatives was also observed in HeLa cells and was mirrored by an increase in cytosolic Ca(2+) concentration. CONCLUSIONS AND IMPLICATIONS: Amiloride derivatives disrupt ER and cytosolic Ca(2+) homeostasis by a mechanism unrelated to NHE inhibition, most likely by interfering with the activity of SERCA. We propose that ER Ca(2+) depletion and subsequent ER stress provide a rationale framework for the apoptotic effects of amiloride derivatives.

Amiloride uptake and toxicity in fission yeast are caused by the pyridoxine transporter encoded by bsu1+ (car1+).

Amiloride, a diuretic drug that acts by inhibition of various sodium transporters, is toxic to the fission yeast Schizosaccharomyces pombe. Previous work has established that amiloride sensitivity is caused by expression of car1+, which encodes a protein with similarity to plasma membrane drug/proton antiporters from the multidrug resistance family. Here we isolated car1+ by complementation of Saccharomyces cerevisiae mutants that are deficient in pyridoxine biosynthesis and uptake. Our data show that Car1p represents a new high-affinity, plasma membrane-localized import carrier for pyridoxine, pyridoxal, and pyridoxamine. We therefore propose the gene name bsu1+ (for vitamin B6 uptake) to replace car1+. Bsu1p displays an acidic pH optimum and is inhibited by various protonophores, demonstrating that the protein works as a proton symporter. The expression of bsu1+ is associated with amiloride sensitivity and pyridoxine uptake in both S. cerevisiae and S. pombe cells. Moreover, amiloride acts as a competitor of pyridoxine uptake, demonstrating that both compounds are substrates of Bsu1p. Taken together, our data show that S. pombe and S. cerevisiae possess unrelated plasma membrane pyridoxine transporters. The S. pombe protein may be structurally related to the unknown human pyridoxine transporter, which is also inhibited by amiloride.

Influence of dietary amiloride supplement on the zinc status of growing rats with marginal zinc supply.

We divided 36 male pathogen-free Sprague-Dawley rats with an average live mass (LM) of 51 g into four treatment groups of nine animals each. They received for a period of 28 trial days a semisynthetic purified diet based on casein for ad libitum consumption, supplemented with 5 ppm zinc (groups 1-3) or 57 ppm zinc (group 4) as zinc sulfate. In addition to the diet, groups 2 and 3 were given a diuretic supplement of amiloride at the therapeutic dose rate (0.4 mg amiloride/kg LM0.75 per day) or in a dosage corresponding to the chronic toxicity level (maximum tolerated dose; 0.8 mg amiloride/kg LM0.75 per day). The supplementation with amiloride, acting as a potential Zn-binding ligand at the selected dosage levels, had no influence on the animals' live weight during the 28-day trial period; weight gain was determined solely by the dietary Zn concentration. Amiloride administered at the therapeutic or the maximum tolerated dose produced no evidence of a diminished Zn status in terms of the alkaline phosphatase activity in the serum or the Zn concentration in the serum, femur and testes. Medication with amiloride at the maximum tolerated dose even exerted a positive effect on the zinc supply status as demonstrated by the raised Zn concentration in the serum. This suggests that zinc supplementation may not be required during medication with amiloride in human medicine.

Effect of chronic treatment with diuretics on mouse liver: a morphological and microanalytical investigation.

In an attempt to produce an animal model for the disease cystic fibrosis (CF), mice were treated chronically with the diuretics amiloride and furosemide, in order to cause chronic inhibition of transepithelial ion transport. Experiments were carried out on adult mice (2 months treatment); in addition, pregnant mice were treated with diuretics, and tissue from offspring 2 and 7 days post partum was investigated. Since biliary cirrhosis is a common occurrence in CF, hepatocytes in the treated mice were investigated by X-ray microanalysis and by light and electron microscopy. Treatment with amiloride caused a significant decrease in cellular Na concentration in adult animals and in in utero treated mice 2 days after birth. The decrease in Na was paralleled by a decrease in Cl, but K levels were not affected. Furosemide caused a slight increase of cellular Na concentrations, especially in animals aged 7 days. In the adult animals, both amiloride and furosemide caused a significant decrease of the cellular Na and Cl levels. No signs of cirrhosis could be observed. Inconsistent changes in the accumulation of lipid droplets in hepatocytes of adult animals treated with amiloride were observed by electron microscopy. It can be concluded that chronic treatment with diuretics, even though it causes some, possibly pathological, changes of the liver, is only of very limited value for generating an animal model to study liver disease in CF.

Abnormalities in ureter and kidney development in mice given acetazolamide-amiloride or dimethadione (DMO) during embryogenesis.

These experiments more accurately define the effects of the combination acetazolamide-amiloride or a single dose of dimethadione (DMO), the active metabolite of trimethadione, on the development of the ureter. When acetazolamide-amiloride was administered in C57BL/6NCrlBR mice on day 9, 9.5, or 10 of gestation (plug = day 0) a second ureter was formed, anterior to the original ureter, inducing a second kidney. The second ureter then fails to make a connection with the developing bladder and remains attached to the mesonephric duct. The mesonephric duct becomes the vas deferens in the male and deteriorates completely in the female leading to either a restricted ureter or a blocked ureter depending on the sex of the fetus. Administration of a single dose of DMO between gestational day 9 and 10.3 produced both renal agenesis and ureters of varying lengths. Some ureters were of normal length with a tuft of one or two nephrons at their tip, while others were one half or one quarter of their normal length. In some instances the ureter was completely absent. The reason for this strong effect on the ureter is unknown.

The effect of amiloride and sodium chloride on rat renal and hepatic 11 beta-hydroxysteroid dehydrogenase activities.

The ability of glucocorticoid hormones to interact with glucocorticoid or mineralocorticoid receptors is modulated by 11 beta-hydroxysteroid dehydrogenases, interconverting active 11 beta-hydroxyglucocorticoids to inactive 11-ketones. This is, amongst others, important in maintaining a normal salt-water homeostasis. In this study, we determined the effect of treating rats for 4 days with the potassium sparing diuretic amiloride (5 mg/kg subcutaneously) or with 3% NaCl in drinking water on renal and hepatic microsomal oxidative and reductive 11 beta-hydroxysteroid dehydrogenase activities and immunoreactive 11 beta-hydroxysteroid dehydrogenase 1 protein. Treatment with amiloride resulted in a 1.5-fold rise of microsomal corticosterone 11 beta-oxidation rates in kidney (using NAD and NADP as cofactors) and in liver (for NADP only), but had no effect on microsomal 11-dehydrocorticosterone reduction. Renal 11 beta-hydroxysteroid dehydrogenase 1 immunoreactive protein was increased 1.6-fold by amiloride. NaCl treatment appeared to have no effect.

Estimating intracellular pH in developing rodent embryos using a computer imaging technique: changes in embryonic pH and proliferation rates following maternal treatment with acetazolamide.

Using the transplacental distribution of the weak acid 5,5-dimethyloxazolidine-2,4-dione (DMO), a computer assisted imaging technique has been developed to permit the estimation of intracellular pH (pHi) in very specific areas of the developing rodent embryo. The study reported here demonstrates the heterogeneity of radiolabeled DMO distribution in the developing mouse forelimb. The pattern of pHi distribution shifts from one of high pHi values in the proximal core of the mesoderm on day 10 of gestation to one of higher pHi values in the mesoderm just underlying the ectoderm on day 11. Studies [Scott et al. (1990) Toxicol. Appl. Pharmacol. 103:238-254] in which DMO concentration was monitored following treatment with acetazolamide or acetazolamide plus amiloride were done in whole embryo homogenates or pooled limb samples which allow for the calculation of an average pHi but may not reflect the pHi in very specific locations of the limb. Two hours after acetazolamide administration, the pHi pattern was not significantly changed from control. Intracellular pH was raised above control levels but was not significant statistically except in the peripheral mesoderm in the ventral third of the forelimb. Fifteen hours after acetazolamide treatment, there was a significant decrease in pHi values with no change in pattern. However, treatment with acetazolamide plus amiloride for 15 hr produced a marked reduction of pHi values throughout the forelimb bud. Changes in bromodeoxyuridine labeling index (an indication of proliferative activity) following treatment with acetazolamide or acetazolamide plus amiloride are reported. The combination treatment reduced the labeling index by approximately 15% below that of control embryos in the limb region where absence of digit(s) will occur. However, we found no overall correlation of proliferative rate and pHi of limb bud mesoderm in treated embryos. Consequently, we were unable to causally associate reduced pHi with decreased proliferative rate.

Acute cardiovascular and pulmonary effects of intravenous and aerosolized amiloride in the dog.

Active Na+ absorption by the epithelia that line the airways can drive volume from the airway surface. Exposure of the lumen-facing surface of airway epithelia to amiloride inhibits Na+ transport. Consequently, aerosolized amiloride may help hydrate the desiccated surface liquid that characterizes lung diseases such as cystic fibrosis. We studied the acute cardiovascular and pulmonary effects of amiloride in anesthetized dogs. Intravenous amiloride reduced blood pressure and increased ventilation frequency and airways resistance of the dog. The effects were dose-related (ED50 = 1.3 X 10(-6) mol/kg), resembled those of injected histamine, and were antagonized by methapyrilene. These results were compatible with the release of endogenous histamine by amiloride, but the graded dose-effect relationship for amiloride differed from the quantal relationship induced by the prototypical releaser, 48/80. Aerosolization of a Ringer's solution that was nearly saturated with amiloride deposited, in upper airway surface liquid, drug concentrations which in previous studies, were sufficient to inhibit Na+ transport by canine airways (ED50 approximately 10(-6) M). Cardiovascular function, airways resistance, and indices of pulmonary tissue water, gas exchange, and perfusion were not affected. Since circulating amiloride after aerosolization was estimated to fall below the concentration that induced vasodepression and correct circled bronchoconstriction after intravenous injection, we conclude that the potency of aerosolized amiloride to induce acute effects in tissues other than airway epithelia is no greater than that of intravenous drug.