Serum human chorionic gonadotropin measurement in the diagnosis of ectopic pregnancy when transvaginal sonography is inconclusive

Transition from placental to pulmonary oxygenation at birth depends on a rapid removal of fetal lung fluid from the developing alveoli. Alveolar fluid clearance was examined in ventilated, anesthetized developing guinea pigs of the ages newborn, 2-d-old, 5-d-old, 30-d-old, and 60-d-old (adult). An isosmolar 5% albumin solution was instilled into the lungs of the guinea pigs; the guinea pigs were then studied for 1 h. Alveolar fluid clearance was measured from the increase in alveolar protein concentration as water was reabsorbed. Newborn guinea pigs had a very high alveolar fluid clearance rate that declined rapidly within the first 5 postnatal days towards adult levels. The high alveolar fluid clearance at birth was apparently mediated by the beta-adrenergic system as demonstrated by the elevated plasma epinephrine levels and the increased sensitivity to inhibition by the beta-adrenergic antagonist propranolol immediately after birth. Surprisingly, exogenous addition of epinephrine was not able to stimulate alveolar fluid clearance in the newborn lung, but exogenous epinephrine stimulation increased over time to adult levels. The elevated alveolar fluid clearance at birth was associated with a significantly greater amiloride sensitivity in the newborn guinea pig lung. Northern blot analysis of distal lung tissue as well as isolated alveolar epithelial type II cells showed and confirmed higher levels of the alpha-subunit of the epithelial sodium channel mRNA in the newborn lung that rapidly tapered off toward adult levels. In conclusion, these data demonstrate the importance of the beta-adrenergic system and amiloride-sensitive sodium transporting pathways for clearance of fetal lung fluid at birth.     

Salt and water transport across the alveolar epithelium in the developing lung: correlations between function and recent molecular biology advances (Review)

Significant progress have been made in understanding the mechanisms of alveolar fluid clearance at the time of birth and the transition from placental oxygenation to air breathing. During fetal life, the mammalian lung is a fluid filled secretory organ that fills no respiratory function. Its potential air spaces are filled with fluid that is actively secreted in response to an osmotic force generated by Cl(-)-secretion and the fluid-filled lung is necessary for a proper development of the air-breathing lung. As term approaches, net Cl(-)-secretion decreases, which is accompanied by a decreased secretion rate of the fluid into the air spaces. Concomitantly with the decreased Cl(-)-secretion, the alveolar epithelium begins to absorb Na+ to prepare for fluid absorption and the air breathing life. The causes for the decreased Cl(-)-secretion and the beginning of the Na+ absorption are not clear. Alterations in the hormonal milieu of the lung as well as changes in plasma stress hormone levels have been suggested to play roles. The switch from a placental oxygenation to pulmonary oxygenation requires that the fluid in the air spaces be rapidly removed from the lung lumen. Recent studies have demonstrated that removal of the alveolar fluid at birth is regulated via endogenous plasma epinephrine in the newborn. Molecular, cellular, and whole animal in vivo studies have demonstrated that fluid absorption at birth is related to expression and function of the epithelial sodium channel (ENaC). Several different in vivo and in vitro preparations have been used to investigate the mechanisms of alveolar fluid transport, primarily in adult lungs and have demonstrated that alveolar fluid absorption is driven by active Na+ transport. Both catecholamine-dependent and -independent regulatory mechanisms have been identified, probably acting on ENaC and other apical sodium channels and/or the basolaterally located Na+, K(+)-ATPase. Future studies are needed to integrate new insights to the molecular mechanisms behind fluid clearance with their function in both normal and pathological lungs.     

Epidermal growth factor increases lung liquid clearance in rat lungs

Epidermal growth factor (EGF) has been reported to stimulate the proliferation of epithelial cells and increase Na+ flux and Na+-K+-ATPase function in alveolar epithelial cell monolayers. Increases in Na+-K+-ATPase in alveolar type II cells (AT2) have been associated with increased active Na+ transport and lung edema clearance across the rat alveolar epithelium in a model of proliferative lung injury. Thus we tested whether administration of aerosolized EGF to rat lungs would increase active Na+ transport and lung liquid clearance. Sixteen adult Sprague-Dawley male rats were randomized to three groups. To a group of six rats, an aerosol generated from 20 microgram of EGF in saline was delivered to the lungs, to a second group of five rats only aerosolized saline was delivered, and a third group of five rats without treatment served as the control. Forty-eight hours postaerosolization of rat lungs with EGF there was an approximately 40% increase in active Na+ transport and lung liquid clearance compared with control rats, in the absence of changes in 22Na+, [3H]mannitol, and albumin permeabilities. The Na+-K+-ATPase activity in AT2 cells harvested from these lungs was increased in rats that received aerosolized EGF compared with AT2 cells from both control rats and rats receiving aerosolized saline. These results support the hypothesis that in vivo delivery of EGF aerosols upregulates alveolar epithelial Na+-K+-ATPase and increases lung liquid clearance in rats.     

Dopamine restores lung ability to clear edema in rats exposed to hyperoxia

Exposure to hyperoxia causes lung injury, decreases active sodium transport and lung edema clearance in rats. Dopamine (DA) increases lung edema clearance by stimulating vectorial Na+ flux and Na, K-ATPase function in rat alveolar epithelium. This study was designed to test whether DA (10(-)5 M) would increase lung edema clearance in rats exposed to 100% O2 for 64 h. Active Na+ transport and lung edema clearance decreased by approximately 44% in rats exposed to acute hyperoxia (p < 0.001). DA increased lung edema clearance in room air breathing rats (from 0.50 +/- 0.02 to 0.75 +/- 0.06 ml/h) and in rats exposed to 100% O2 (from 0.28 +/- 0.03 to 0. 67 +/- 0.03 ml/h). Disruption of cell microtubular transport system by colchicine blocked the stimulatory effect of DA on active Na+ transport in control and hyperoxic rats, whereas the isomer beta-lumicolchicine, which does not affect cell microtubular transport, did not inhibit the stimulatory effects of dopamine. The Na,K-ATPase alpha1-subunit protein abundance increased in the basolateral membranes of alveolar type II (ATII) cells incubated with 10(-)5 M DA for 15 min, probably by recruiting Na+ pumps from intracellular pools. Colchicine, but not beta-lumicolchicine, prevented the recruitment of alpha1 subunits to the plasma membrane by DA. Accordingly, DA restored lung ability to clear edema in hyperoxic-injured rat lungs. Conceivably, dopamine induces recruitment of Na+ pumps from intracellular pools to the plasma membrane of alveolar epithelial cells and thus increases lung edema clearance.     

Ventilator-associated lung injury decreases lung ability to clear edema in rats

Ventilator-associated lung injury (VALI) is caused by high tidal volume (VT) excursions producing microvascular leakage and pulmonary edema. However, the effects of VALI on lung edema clearance and alveolar epithelial cells' Na,K-ATPase function have not been elucidated. We studied lung edema clearance in the isolated-perfused rat lung model after ventilation for 25, 40, and 60 min with high VT (peak airway opening pressure [Pao] of approximately 35 cm H2O) and compared them with low VT ventilation (Pao approximately 8 cm H2O), moderate VT ventilation (Pao approximately 20 cm H2O), and nonventilated rats. Lung edema clearance in control rats was 0.50 +/- 0.02 ml/h and decreased after 40 and 60 min of high VT to 0.26 +/- 0.03 and 0.11 +/- 0.08 ml/h, respectively (p < 0.01), but did not change after low VT and moderate VT ventilation at any time point. Lung permeability to small (22Na+, [3H]mannitol) and large solutes (fluorescein isothiocyanate-tagged albumin [FITC-albumin]) increased significantly in rats ventilated for 60 min with high VT, compared with low VT, moderate VT, and control rats (p < 0.01). Paralleling the impairment in lung edema clearance we found a decrease in Na,K-ATPase activity in alveolar type II (ATII) cells isolated from rats ventilated with moderate VT and high VT for 40 min without changes in alpha1 Na,K-ATPase mRNA. We reason that VALI decreases lung ability to clear edema by inhibiting active sodium transport and Na,K-ATPase function in the alveolar epithelium.     

Decreased sodium ion absorption across nasal epithelium of very premature infants with respiratory distress syndrome

OBJECTIVE AND STUDY DESIGN: Successful adaptation to air breathing at birth depends on rapid absorption of fetal lung liquid that is mediated by activation of amiloride-sensitive sodium ion channels. To test the relationship between respiratory epithelial Na+ transport and development of respiratory distress syndrome (RDS), we measured nasal transepithelial potential difference (PD) in 31 very premature (< or = 30 weeks of gestation) newborn infants. Infants were retrospectively assigned to RDS (22 infants) and non-RDS (9 infants) groups on the basis of clinical and chest x-ray criteria. RESULTS: Maximal nasal epithelial PD increased with birth weight (-1.2 mV/100 gm) and was lower in infants with RDS (-16.5 +/- 0.6 mV) than in those without RDS (-22.0 +/- 1.3 mV). Infants without RDS had PD values similar to normal fullterm infants. Amiloride inhibition of PD, an index of Na+ absorption, was significantly lower, within the first 24 hours of life, in infants in whom RDS developed (3.8 +/- 0.2 mV; 29.5% +/- 0.8% inhibition) than in those without RDS (6.1 +/- 0.6 mV; 38.6% +/- 0.5% inhibition). Maximal and amiloride-sensitive PD returned to normal during the recovery phase of RDS. CONCLUSIONS: We conclude that Na+ absorption across nasal epithelium increases with increasing birth weight and that impairment of Na+ absorption across the respiratory epithelia of very premature infants may contribute to the pathogenesis of RDS.     

Calbindin D-28k and monoamine oxidase A immunoreactive neurons in the nucleus basalis of Meynert in senile dementia of the Alzheimer type and Parkinson''s disease

The lungs must be kept "dry" for efficient gas exchange. The mechanisms that contribute to clear alveoli from fetal lung fluid at birth are still present during adult life and allow recovery from alveolar flooding. It has recently been shown with the use of different approaches in vitro, as well as in vivo, that alveolar epithelium performs solute-coupled fluid transport. Fluid absorption from alveoli occurs chiefly as a result of active transepithelial Na+ transport. The mechanisms of Na+ transport have been partly elucidated; Na+ enters alveolar cells through apical Na+ channels and Na(+)-coupled solute transporters and is pumped out at the basolateral membrane by a Na(+)-K(+)-adenosinetriphosphatase (ATPase). Transepithelial Na+ transport and fluid absorption are stimulated by beta-adrenergic agonists, with adenosine 3',5'-cyclic monophosphate being the likely intracellular second messenger. K+ is probably secreted into alveoli because its concentration in the epithelial lining fluid is larger than expected for passive distribution. K+ channels have been described that, in conjunction with Na(+)-K(+)-ATP-ase, might provide pathways for active transport. Active proton secretion or bicarbonate absorption have been reported, which may explain the low pH of the alveolar epithelial lining fluid. It is probable that active solute transports are the main determinants of epithelial lining fluid depth and composition. A challenge for the future is to understand how this homeostasis is achieved.     

Alveolar epithelial fluid transport: basic mechanisms and clinical relevance

New evidence indicates that alveolar fluid clearance is driven by active sodium transport across the alveolar epithelium. Several in vivo as well as some in vitro studies indicate that vectorial sodium transport drives fluid clearance across the alveolar epithelium. This transport process can be upregulated by both catecholamine-dependent and catecholamine-independent mechanisms. Water transport appears to move across the alveolar epithelium primarily via transcellular water channels, recently termed aquaporins. Under some conditions, net alveolar fluid clearance continues even in the presence of acute lung injury. It is now possible to study the rate and mechanisms of alveolar fluid clearance in patients with either hydrostatic or increased permeability pulmonary edema. In addition, it may be possible to increase the rate of alveolar fluid clearance and hence the resolution of pulmonary edema in some patients, using aerosolized beta-adrenergic agonist therapy.     

cAMP-activation of amiloride-sensitive Na+ channels from guinea-pig colon expressed in Xenopus oocytes

Guinea-pig distal colonic mRNA injection into Xenopus laevis oocytes resulted in expression of functional active epithelial Na+ channels in the oocyte plasma membrane. Poly(A)+ RNA was extracted from distal colonic mucosa of animals fed either a high-salt (HS) or a low-salt (LS) diet. The electrophysiological properties of the expressed amiloride-sensitive Na+ conductances were investigated by conventional two-electrode voltage-clamp and patch-clamp measurements. Injection of poly(A)+ RNA from HS-fed animals [from hereon referred to as HS-poly(A)+ RNA] into oocytes induced the expression of amiloride-sensitive Na+ conductances. On the other hand, oocytes injected with poly(A)+ RNA from LS-fed animals [LS-poly(A)+ RNA] expressed a markedly larger amount of amiloride-blockable Na+ conductances. LS-poly(A)+ RNA-induced conductances were completely inhibitable by amiloride with a Ki of 77 nM, and were also blocked by benzamil with a Ki of 1.8 nM. 5-(N-Ethyl-N-isopropyl)-amiloride (EIPA), even in high doses (25 "mu"M), had no detectable effect on the Na+ conductances. Expressed amiloride-sensitive Na+ channels could be further activated by cAMP leading to nearly doubled clamp currents. When Na+ was replaced by K+, amiloride (1 "mu"M) showed no effect on the clamp current. Single-channel analysis revealed slow gating behaviour, open probabilities (Po) between 0.4 and 0.9, and slope conductances of 3. 8 pS for Na+ and 5.6 pS for Li+. The expressed channels showed to be highly selective for Na+ over K+ with a permeability ratio PNa/PK > 20. Amiloride (500 nM) reduced channel Po to values < 0.05. All these features make the guinea-pig distal colon of LS-fed animals an interesting mRNA source for the expression of highly amiloride-sensitive Na+ channels in Xenopus oocytes, which could provide new insights in the regulatory mechanism of these channels.     

Salt restriction induces pseudohypoaldosteronism type 1 in mice expressing low levels of the beta-subunit of the amiloride-sensitive epithelial sodium channel

The amiloride-sensitive epithelial sodium channel (ENaC) is a heteromultimer of three homologous subunits (alpha-, beta-, and gamma-subunits). To study the role of the beta-subunit in vivo, we analyzed mice in which the betaENaC gene locus was disrupted. These mice showed low levels of betaENaC mRNA expression in kidney (approximately 1%), lung (approximately 1%), and colon (approximately 4%). In homozygous mutant betaENaC mice, no betaENaC protein could be detected with immunofluorescent staining. At birth, there was a small delay in lung-liquid clearance that paralleled diminished amiloride-sensitive Na+ absorption in tracheal explants. With normal salt intake, these mice showed a normal growth rate. However, in vivo, adult betaENaC m/m mice exhibited a significantly reduced ENaC activity in colon and elevated plasma aldosterone levels, suggesting hypovolemia and pseudohypoaldosteronism type 1. This phenotype was clinically silent, as betaENaC m/m mice showed no weight loss, normal plasma Na+ and K+ concentrations, normal blood pressure, and a compensated metabolic acidosis. On low-salt diets, betaENaC-mutant mice developed clinical symptoms of an acute pseudohypoaldosteronism type 1 (weight loss, hyperkalemia, and decreased blood pressure), indicating that betaENaC is required for Na+ conservation during salt deprivation.     

Transport characteristics of the colonic epithelium of the Japanese quail (Coturnix coturnix)

The colon of the domestic fowl sustains a reabsorptive Na+ current on both high- and low-sodium diets. However, there is a marked shift in the apical transport step under these two extreme conditions, from amino acid/hexose cotransport on high-salt diets to amiloride-sensitive Na+ channels on low-salt diets. The present experiments were performed to study colonic Na+ transport in another galliform species, the Japanese quail (Coturnix coturnix). Birds were maintained on a commercial game feed containing 0.18% Na+ (78 mumoles/g), an intermediate level of salt intake. Experiments were performed on unstripped colons in standard Ussing chambers with bicarbonate/CO2 buffer solution on both sides. Baseline values (n = 11) for PD (3.13 +/- 0.68 mV) and short circuit current (SCC, 30.87 +/- 7.79 microA/cm2) were lower than those reported for chickens on a similar diet, whereas tissue resistance (76.06 +/- 4.19 omega.cm2) was similar. Addition of amino acids (4 mM leucine + lysine) increased SCC by 10.85 +/- 1.97 microA/cm2. Both phloridzin (1 mM) and amiloride (10(-5) M) decreased SCC, by 7.05 +/- 1.26 and 9.64 +/- 2.68 microA/cm2, respectively. Thus, on this diet the quail colonic epithelium maintains both amino acid/hexose cotransporter activity and amiloride sensitive channel activity. Arginine vasotocin (10(-6) M) caused a small, but consistent decrease in SCC, while acetazolamide increased SCC. Aldosterone (128 micrograms/kg), given 4 hr prior to the experiment (n = 4) significantly reduced the amino acid stimulated SCC. These results confirm, for the Japanese quail, the presence of multiple apical Na+ entry mechanisms in colonic epithelium. Amino acid cotransporter activity, in particular, appears to be highly sensitive to aldosterone suppression.     

Heat shock proteins and arthritis--new readers start here

We have characterised G protein and fatty acid regulation of the Na+ conductance in purified apical membrane vesicles prepared from late gestation fetal guinea-pig lung. Addition of 100 microM GTP gamma S or beta gamma-methylene-GTP, irreversible G protein activators, stimulated conductive 22Na+ uptake (ratio of experimental to control 1.35 +/- 0.02 and 1.34 +/- 0.05, respectively). Conversely, the addition of GDP beta S, an irreversible G protein inhibitor, reduced conductive 22Na+ uptake from 1.00 (control) to 0.79 +/- 0.04. A range of saturated (myristic, palmitic, stearic), monounsaturated (elaidic, oleic) and polyunsaturated (linoleic, arachidonic) fatty acids all stimulated conductive 22Na+ uptake, by between 1.18 +/- 0.05 to 1.56 +/- 0.13 over the control. Both arachidonic acid and GTP gamma S-dependent stimulation were abolished in the presence of 10 microM amiloride. The non-metabolisable analogue of arachidonic acid, eicosa-5,8,11,14-tetraynoic acid also stimulated conductive 22Na+ uptake. Furthermore, addition of indomethacin and nordihydroguairetic acid, inhibitors of cyclooxygenase and lipoxygenase pathways of arachidonate metabolism respectively, did not affect the arachidonic acid stimulation suggesting a direct effect of fatty acid upon the Na+ channel Since mepacrine (50 microM), a phospholipase A2 inhibitor, did not affect the GTP gamma S-stimulated conductive 22Na+ uptake, and inhibition of G protein turnover by GDP beta S did not attenuate the arachidonic acid response we conclude that these two regulatory pathways modulate alveolar Na+ transport directly and independently of each other.     

Measurement of the total concentration of functional Na+, K(+)-pumps in rumen epithelium

Using the technique of vanadate-facilitated [3H]ouabain binding we have developed a simple and reliable assay for measuring the concentration of [3H]ouabain binding sites in small fresh or frozen biopsies of rumen epithelium papillae. In bovine and ovine rumen epithelium obtained from the cranio-ventral rumen sac the concentration of [3H]ouabain binding sites was 1.6-4.9 nmol g dry wt-1 (n = 32) and 3.7-5.2 nmol g dry wt-1 (n = 6), respectively. When incubated in oxygenated Krebs-Ringer bicarbonate buffer fresh biopsies of rumen epithelium maintained a high K+ and low Na+ content for at least 6 h. Na+ loading of the biopsies induced about 20-fold increase of the Na+, K(+)-pump activity based on measurement of ouabain suppressible net [86Rb+] influx. The ouabain suppressible net influx of [86Rb+] measured in Na+ loaded biopsies showed a close correlation to the [3H]ouabain binding capacity (r = 0.80, P < 0.01) and corresponded to 47 +/- 2% (n = 9) of the theoretical maximum flux rate. The ouabain suppressible net influx of K+ and [86Rb+] were linearly related (r = 0.73; P < 0.001). The net Na+ efflux was 1.21 times the net K+ influx. It is concluded that rumen epithelium has a large capacity for active Na+/K+ transport and that there is agreement between the concentration of [3H]ouabain binding sites in the epithelium and the ouabain suppressible rate of net [86Rb+] influx in Na+ loaded biopsies in spite of some uncertainty about the maximum turnover number of the Na+, K(+)-pump in rumen epithelium.     

Dependence of the driving force of the sodium pump on rate of transport

The driving force for active transport of Na+ in the isolated toad bladder, ENa, was measured as the reciprocal slope of the change in conductance with change in short-circuit current after stimulation with antidiuretic hormone. The base-line short-circuit current was altered by change in ambient Na+ concentration or addition of amiloride, maneuvers which alter availability of Na+ at the site of active transport. In the absence of a chemical gradient for Na+ across the bladder, ENa was found to be inversely related to the rate of Na+ transport, a finding incompatible with the simple electrical analogue that has been proposed for the system. The results provide additional support for the view that ENa measured in this way has both energetic and kinetic components.     

Carbachol-induced increase of Na+/H+ antiport and recruitment of Na+,K(+)-ATPase in rabbit lacrimal acini

Parallel arrays of Na+/H+ and Cl-/HCO3- antiporters are believed to catalyze the first step of transepithelial electrolyte secretion in lacrimal glands by coupling Na+ and Cl- influxes across acinar cell basolateral membranes. Tracer uptake methods were used to confirm the presence of Na+/H+ antiport activity in membrane vesicles isolated from rabbit lacrimal gland fragments. Outwardly-directed H+ gradients accelerated 22Na+ uptake, and amiloride inhibited 96% of the H+ gradient-dependent 22Na+ flux. Amiloride-sensitive 22Na+ influx was half-maximal at an extravesicular Na+ concentration of 14 mM. In vitro stimulation of isolated lacrimal acini with 10 microM carbachol for 30 min increased Na+/H+ antiport activity of a subsequently isolated basolateral membrane sample 2.5-fold, but it did not significantly affect Na+/H+ antiport activity measured in intracellular membrane samples. The same treatment increased basolateral membrane Na+,K(+)-ATPase activity 1.4-fold; this increase could be accounted for by decreases in the Na+,K(+)-ATPase activities of intracellular membranes. Thus, it appears that cholinergic stimulation causes recruitment of additional Na+,K(+)-ATPase pump units to the acinar cell basolateral plasma membrane. The mechanistic basis of the increase in basolateral membrane Na+/H+ antiport activity remains unclear.     

Effect of caffeine on K+ efflux in frog skeletal muscle

The exposure of frog skeletal muscle to caffeine (3-4 mM) generates an increase of the K+ (42K+) efflux rate coefficient (kK,o) which exhibits the following characteristics. First it is promoted by the rise in cytosolic Ca2+ ([Ca2+]i), because the effect is mimicked by ionomycin (1.25 microM), a Ca2+ ionophore. Second, the inhibition of caffeine-induced Ca2+ release from the sarcoplasmic reticulum (SR) by 40 microM tetracaine significantly reduced the increase in kK,o (DeltakK,o). Third, charybdotoxin (23 nM), a blocker of the large-conductance Ca2+-dependent K+ channels (BKCa channels) reduced DeltakK,o by 22%. Fourth, apamin (10 nM), a blocker of the small-conductance Ca2+-dependent K+ channels (SKCa channels), did not affect DeltakK,o. Fifth, tolbutamide (800 microM), an inhibitor of KATP channels, reduced DeltakK,o by about 23%. Sixth, Ba2+, a blocker of most K+ channels, did not preclude the caffeine-induced DeltakK,o. Seventh, omitting Na+ from the external medium reduced DeltakK,o by about 40%. Eight, amiloride (5 mM) decreased DeltakK,o by 65%. It is concluded that the caffeine-induced rise of [Ca2+]i increases K+ efflux, through the activation of: (1) two channels (BKCa and KATP) and (2) an external Na+-dependent amiloride-sensitive process.     

Role of gammaENaC subunit in lung liquid clearance and electrolyte balance in newborn mice. Insights into perinatal adaptation and pseudohypoaldosteronism

Genetic evidence supports a critical role for the epithelial sodium channel (ENaC) in both clearance of fetal lung liquid at birth and total body electrolyte homeostasis. Evidence from heterologous expression systems suggests that expression of the alphaENaC subunit is essential for channel function, whereas residual channel function can be measured in the absence of beta or gamma subunits. We generated mice without gammaENaC (gammaENaC -/-) to test the role of this subunit in neonatal lung liquid clearance and total body electrolyte balance. Relative to controls, gammaENaC (-/-) pups showed low urinary [K+] and high urinary [Na+] and died between 24 and 36 h, probably from hyperkalemia (gammaENaC -/- 18.3 mEq/l, control littermates 9.7 mEq/l). Newborn gammaENaC (-/-) mice cleared lung liquid more slowly than control littermates, but lung water at 12 h (wet/dry = 5.5) was nearly normal (wet/dry = 5.3). This study suggests that gammaENaC facilitates neonatal lung liquid clearance and is critical for renal Na+ and K+ transport, and that low level Na+ transport may be sufficient for perinatal lung liquid absorption but insufficient to maintain electrolyte balance by the distal nephron. The gammaENaC (-/-) newborn exhibits a phenotype that resembles the clinical manifestations of human neonatal PHA1.     

Novel benzamil-sensitive sodium-independent choline transport in the cestode Hymenolepis diminuta: evidence for sodium channels uptake at low pH

The uptake of choline by the tegument of Hymenolepis diminuta was investigated. The Q10 at pH 7.0 was 1.7, with an Ea of 90 kJ.mol-1. Choline transport was pH sensitive: At pH 5.0, a Na(+)-independent mechanism predominated, which was inhibited by 100 nM benzamil, 130 mM Na+, and 300 microM verapamil. At pH 7.0, the Na(+)-independent mechanism was inhibited by 130 mM Na+, amiloride, and EIPA with IC50's of 130 microM and 30 microM, respectively, and by benzamil with IC50's of 100 pM (high-potency Benzamil Sensitive Component; HBSC) and 70 microM (low-potency Benzamil Sensitive Component; LBSC). Calcium-free saline enhanced choline uptake non-specifically. Lanthanum3+, Gd3+, gramicidin, nigericin, and high-K+ did not affect choline uptake at pH 5.0 or pH 7.0, and 10 microM verapamil was without effect at pH 5.0, suggesting no significant role for the electrical potential difference across the brush-border membrane, a Na+/H+ antiporter, a Na+/Ca2+ antiporter, or Ca2+ channels in choline uptake. Under physiological conditions, the HBSC accounts for approximately 25% of the total choline taken up at pH 5.0, while the LBSC accounts for approximately 55% of the choline taken up at pH 7.0. The data suggest novel choline transporting mechanisms; an HBSC which displays properties in common with apical Na+ channels, and a unique LBSC of choline transport.     

Pharmacological characterization of stably transfected Na+/H+ antiporter isoforms using amiloride analogs and a new inhibitor exhibiting anti-ischemic properties

A fibroblast mutant cell line devoid of Na+/H+ exchange was used to stably express cDNAs encoding the NHE1, NHE2, and NHE3 Na+/H+ antiporters. Pharmacological studies using amiloride and two of its 5-N-substituted derivatives, 5-N-dimethyl amiloride and 5-N-(methyl-propyl)amiloride (MPA), demonstrate that the NHE1 isoform is the ubiquitously expressed amiloride-sensitive Na+/H+ antiporter (Ki of 0.08 microM for MPA), whereas the NHE2 and NHE3 isoforms exhibit a lower affinity for these inhibitors (Ki of 0.5 microM and 10 microM, respectively, for MPA) and are therefore likely to be members of the epithelial Na+/H+ exchanger's family. In addition, we have used this system to test a new Na+/H+ exchanger inhibitor possessing anti-ischemic properties on myocardial cells [(3-methylsulphonyl-4-piperidinobenzoyl) guanidine methanesulphonate]. This compound inhibits competitively NHE1 (Ki of 0.16 microM) with a much greater affinity than NHE2 and NHE3 (Ki of 5 microM and 650 microM, respectively) and therefore appears to be much more discriminative between these two classes of antiporter isoforms than the amiloride-related molecules. These results suggest an explanation for the observed difference of physiological effects between amiloride and HOE694, and identify this new inhibitor as a useful tool for studies of Na+/H+ exchange.     

Acute bacterial pneumonia in rats increases alveolar epithelial fluid clearance by a tumor necrosis factor-alpha-dependent mechanism

To study the rate and regulation of alveolar fluid clearance in acute pneumonia, we created a model of Pseudomonas aeruginosa pneumonia in rats. To measure alveolar liquid and protein clearance, we instilled into the airspaces a 5% bovine albumin solution with 1.5 microCi of 125I-human albumin, 24 h after intratracheal instillation of bacteria. The concentration of unlabeled and labeled protein in the distal airspaces over 1 h was used as an index of net alveolar fluid clearance. Since there was histologic evidence of alveolar epithelial injury, several methods were used to measure alveolar fluid clearance, including the use of experiments in rats with blood flow and the use of experiments in rats without blood flow, so that movement across the epithelial barrier would be minimized in the latter group. The results with each method were identical. We found that P. aeruginosa pneumonia increased alveolar liquid clearance over 1 h by 48% in studies with blood flow, and by 43% in rats without blood flow, compared with respective controls (P < 0.05). In both studies, this increase was inhibited with amiloride. However, propranolol had no inhibitory effect, thus ruling out a catecholamine-dependent mechanism to explain the increase in alveolar fluid clearance. An antitumor necrosis factor-alpha neutralizing antibody, instilled into the lung 5 min before bacteria, prevented the increase in alveolar liquid clearance in rats with pneumonia (P < 0.05). Also, TNFalpha (5 microg) instilled in normal rats increased alveolar liquid clearance by 43% over 1 h compared with control rats (P < 0.05). In normal rats instilled with TNFalpha, propranolol had no inhibitory effect. In conclusion, gram-negative pneumonia markedly upregulates net alveolar epithelial fluid clearance, in part by a TNFalpha-dependent mechanism. This finding provides a novel mechanism for the upregulation of alveolar epithelial sodium and fluid transport from the distal airspaces of the lung.