NaCl Uptake Research Articles

Na+/H+ and CI-/HCO3-antiporters of bovine pigmented ciliary epithelial cells.

Medical therapy of glaucoma commonly aims at slowing aqueous humor formation by the ocular ciliary epithelial bilayer, but underlying mechanisms are poorly understood. The first step in secretion is NaCl uptake from the stroma into the pigmented ciliary epithelial (PE) cell layer by electroneutral transporters. After crossing gap junctions into the nonpigmented ciliary epithelial (NPE) cell layer, solute is released into the aqueous humor. Published data have indicated that both paired Na+/H+ and Cl-/HCO3- antiporters and the Na+-K+-2Cl- symporter are involved in net uptake. The molecular identities of the paired antiporters have not been elucidated. We have studied continuously cultured bovine PE cells. Acid-activated 22Na+ uptake was inhibited by cariporide, EIPA (ethyl-isopropyl-amiloride) and amiloride, at concentrations characteristic of the NHE-1 isoform. Videomicroscopy of BCECF-loaded PE cells verified the presence of an EIPA-inhibitable Na+/H+ antiporter. Removing external Cl- also triggered an alkalinization, which was Na+-independent and could be inhibited by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS). Application of hypotonicity followed by return to isotonicity triggered a regulatory volume increase, which was pharmacologically similar to the uptake mechanisms described for intact rabbit ciliary epithelium. Reverse transcriptase polymerase chain reaction (RT-PCR) amplification of RNA from the human ciliary body detected expression of the AE2 Cl-/HCO3- exchanger, but not of AE1, cAE3 or bAE3. Immunostaining of bovine PE cells also revealed the presence of AE2 epitope. We conclude that paired NHE-1 Na+/H+ and AE2 Cl-/HCO3- antiporters are important components in the initial step in aqueous humor formation.

Active osmoregulatory ion uptake across the pleopods of the isopod Idotea baltica (Pallas): electrophysiological measurements on isolated split endo- and exopodites mounted in a micro-ussing chamber.

The mechanism of active, osmoregulatory ion uptake was investigated in the pleopods of the marine isopod Idotea baltica (Pallas). Using isolated split half-podites of isopods acclimated to brackish water (20 salinity) mounted in a micro-Ussing chamber and symmetrically superfused with identical haemolymph-like salines, a mean short-circuit current I(sc) of -445 microA cm(-)(2) was measured in endopodites 3-5, corresponding to an inwardly directed transcellular movement of negative charge. Application of ouabain (5 mmol l(-)(1)) to the basolateral superfusate resulted in the almost total abolition of the I(sc) (reduced from -531 to -47 microA cm(-)(2)), suggesting that the Na(+)/K(+)-ATPase is the driving force for active, electrogenic uptake of NaCl. In contrast, mean I(sc) values close to zero were found in preparations of all exopodites and in endopodites 1 and 2. The specific activities of Na(+)/K(+)-ATPase corresponded with these results. Specific activities were highest in posterior endopodites 3-5 and depended on ambient salinity. In all other rami, the activities were much lower and independent of ambient salinity. Activities in posterior endopodites 3-5 were lowest in isopods acclimated to 30 salinity (2-4 micromol P(i )mg(-)(1 )protein h(-)(1)), increased in individuals kept in 20 salinity (8.4 micromol P(i )mg(-)(1 )protein h(-)(1)) and were highest in isopods acclimated to 15 salinity (18.2 micromol P(i )mg(-)(1 )protein h(-)(1)). When specimens were transferred from 30 to 40 salinity, Na(+)/K(+)-ATPase activity increased in the posterior endopodites. The electrophysiological and Na(+)/K(+)-ATPase activity measurements show that active electrogenic ion transport in this species occurs almost exclusively in posterior endopodites 3-5. The endopodite of the fifth pleopod of I. baltica exhibited a microscopic structure remarkably similar to that described for the lamellae of the phyllobranchiae of brachyurans. It is composed of two opposed epithelial monolayers of ionocytes, each covered by cuticle. Bundles of pillar cells are located within the ionocyte layers, which are separated by a fenestrated lamellar septum of connective tissue. The results obtained in this study indicate that endopodites 3-5 play the main role in osmoregulatory ion uptake of the isopod I. baltica. Moreover, the Na(+)/K(+)-ATPase is the only driving force behind active electrogenic ion uptake across the epithelial cells.

Sodium Chloride-Induced Volume Changes of Freshwater Cyanobacterium Synechococcus sp. PCC 7942 Cells Can Be Probed by Chlorophyll a Fluorescence

Freshwater species of the cyanobacterial genus Synechococcus import NaCl passively, and export Na+ actively, by means of primary and secondary extrusion mechanisms. As a result of the ion and water fluxes, cell volumes are enlarged. We show in this paper that the NaCl-induced volume enlargement of Synechococcus sp. PCC 7942 cells is attended by a rapid (k = 0.39 s−1) increase in chlorophyll (Chl) a fluorescence. The cell turgor threshold (measured by osmotic titration of Chl a fluorescence) was lower in the absence of NaCl (0.195 Osm kg−1) than in the presence of 0.4 M NaCl (0.248 Osm kg−1) indicating NaCl uptake by the cells. Turgor thresholds of cells suspended in NaCl-containing medium were enlarged further by protonophoric uncouplers, P-type ATPase inhibitors, and light starvation, conditions that are known to interfere with the active extrusion of Na+ ions. Cell swelling exerts probably a regulation on the distribution of phycobilisome (PBS) excitation between photosystem II (fluorescent Chl a) and photosystem I (nonfluorescent Chl a), since it affects PBS-sensitized Chl a fluorescence, but not directly excited Chl a fluorescence. The dependence of the Chl a fluorescence of cyanobacteria on cell volumes allows probing of bioenergetic phenomena that are related to dynamic osmotic volume changes, transmembrane solute and water fluxes, plasma membrane permeabilities, and internal osmotic conditions of cyanobacterial cells. Thus, cyanobacteria may serve as quite convenient models of aquatic microorganisms in experimental studies directed toward the elucidation of perception mechanisms and defense mechanisms of water and solute stresses.

Energy cost of NaCl transport in isolated gills of cutthroat trout.

Few studies have made direct estimates of the energy required for ion transport in gills of freshwater (FW) and seawater (SW) fish. Oxygen consumption was measured in excised gill tissue of FW-adapted cutthroat trout (Oncorhynchus clarki clarki) to estimate the energy cost of NaCl transport in that osmoregulatory organ. Ouabain (0.5 mM) and bafilomycin A1 (1 microM) were used to inhibit the Na+-K+ and H+ pumps, respectively. Both inhibitors significantly decreased gill tissue oxygen consumption, accounting for 37% of total tissue respiration. On a whole mass basis, the cost of NaCl uptake in the FW trout gill was estimated to be 1.8% of whole animal oxygen uptake. An isolated, saline-perfused gill arch preparation was also used to compare gill energetics in FW- and SW-adapted trout. The oxygen consumption of FW gills was significantly (33%) higher than SW gills. On a whole animal basis, total gill oxygen consumption in FW and SW trout accounted for 3.9 and 2.4% of resting metabolic rate, respectively. The results of both experiments suggest that the energy cost of NaCl transport in FW and SW trout gills represents a relatively small (<4%) portion of the animal's total energy budget.

Coion exclusion properties of polyphosphazene ion-exchange membranes

Equilibrium NaCl uptake studies in crosslinked and non-crosslinked cation-exchange membranes composed of sulfonated poly[bis(3-methylphenoxy)phosphazene] have been performed at 25°C for external salt solutions ranging in concentration from 0.1 to 1.0 M. Membrane swelling, membrane fixed-ion concentration, and membrane coion (anion) concentration were determined for polyphosphazene membranes with ion-exchange capacities of 0.60, 1.22, and 1.40 mmol/g and for a Nafion 117 perfluorosulfonic acid membrane. For the polyphosphazene membranes, coion intrusion decreased with increasing membrane fixed-ion concentration and with polymer crosslinking, but was higher than that with Nafion. Small angle X-ray diffraction was used to model the micro-morphology of the polyphosphazene membranes in terms of a cluster-network structure. Neither the dry polymer ion-exchange capacity nor the wet membrane fixed-ion concentration were accurate measures of the coion exclusion capability of the polyphosphazene membranes, relative to Nafion. It was found, however, that coion exclusion in crosslinked and non-crosslinked polyphosphazene membranes and in Nafion 117 correlated linearly with the fixed-charge/volume ratio of the ionic clusters.

Growth, Water Relations and Accumulation of Organic and Inorganic Solutes in the Halophyte Limonium latifolium cv. Avignon and its Interspecific Hybrid Limonium cas pia x Limonium latifolium cv. Beltlaard During Salt Stress

Summary Plants of two genotypes, Limonium latifolium cv. Avignon and its interspecific hybrid Limonium caspia X Limonium latifolium cv. Beltlaard, grown in a hydroponics culture were submitted to 140 mmol/L NaCl nutrient solution for 20 days (saline treatment). The physiological traits of salt tolerance that differ among these cultivars have been studied. Shoot growth parameters of Beltlaard plants showed that they were affected by salinity, inducing a significant reduction in shoot dry weight and leaf area with respect to the control treatment, while these parameters remained unchanged in Avignon. The behaviour of leaf water relations under saline stress was not different between both genotypes, which indicated that the osmotic effect was similar for both cultivars. At the end of the experiment the cultivars studied were able to achieve the same degree of osmotic adjustment. However, in L. Beltlaard the maintenance of turgor did not permit the maintenance of the growth rate. Leaf growth reductions in response to salinity were associated with higher tissue levels of Na+ and Cl− and changes in the contribution to leaf osmotic potential of K+ and NO3−. The rates of NaCl absorption by roots and the net transfer from roots to shoots was higher in Beltlaard than in Avignon; this fact suggests that the radical exclusion mechanisms are more operative in Avignon. In conclusion, although Avignon showed lower NaCl uptake rates, the growth reduction was more pronounced in Beltlaard because the accumulation of salt in the tissue generated toxicity and nutritional disturbances.

Ionic transport in the fish gill epithelium

The gill of fishes is modified for gas exchange, thereby providing a site for net movement of salts and water down their respective gradients. Specialized cells in the gill epithelium are joined by tight junctions of variable depth and express a variety of transporters and channels. These cells mediate NaCl extrusion in marine fishes and NaCl uptake in freshwater fishes. These transport steps also provide pathways for the extrusion of ammonia and acid vs. base equivalents. J. Exp. Zool. 283:641–652, 1999. © 1999 Wiley-Liss, Inc.

Active excretion of ammonia across the gills of the shore crab Carcinus maenas and its relation to osmoregulatory ion uptake

The mechanism of transbranchial excretion of total ammonia of brackish-water acclimated shore crabs, Carcinus maenas was examined using isolated, perfused gills. Applying physiological gradients of NH4Cl (100–200 μmol · l−1) directed from the haemolymph space to the bath showed that the efflux of total ammonia consisted of two components. The saturable component (excretion of NH4 +) greatly exceeded the linear component (diffusion of NH3). When an outwardly directed gradient (200 μmol · l−1) was applied, total ammonia in the perfusate was reduced by more than 50% during a single passage of saline through the gill. Effluxes of ammonia along the gradient were sensitive to basolateral dinitrophenol, ouabain, and Cs+ and to apical amiloride. Acetazolamide (1 mmol · l−1 basolateral) or Cl−-free conditions had no substantial effects on ammonia flux, which was thus independent of both carbonic anhydrase mediated pH regulation and osmoregulatory NaCl uptake. When an inwardly directed gradient (200 μmol · l−1) was employed, influx rates were about 10-fold smaller and unaffected by basolateral ouabain (5 mmol · l−1) or dinitrophenol (0.5 mmol · l−1). Under symmetrical conditions (100 μmol · l−1 NH4Cl on both sides) ammonia was actively excreted against the gradient of total ammonia, which increased strongly during the experiment and against the gradient of the partial pressure of NH3. The active excretion rate was reduced to 7% of controls by basolateral dinitrophenol (0.5 mmol · l−1), to 44% by basolateral ouabain (5 mmol · l−1), to 46% by Na+-free conditions and to 42% by basolateral Cs+ (10 mmol · l−1), indicating basolateral membrane transport of NH4 + via the Na+/K+-ATPase and K+-channels and a second active, apically located, Na+ independent transport mechanism of NH4 +. Anterior gills, which are less capable of active ion uptake than posterior gills, exhibited even increased rates of active excretion of ammonia. We conclude that, under physiological conditions, branchial excretion of ammonia is a directed process with a high degree of effectiveness. It even allows active extrusion against an inwardly directed gradient, if necessary.

Correlation between transepithelial Na+ transport and transepithelial water movement across isolated frog skin (Rana esculenta).

In the present work the coupling under short-circuited conditions between the net Na(+)-influx across isolated frog skin and the transepithelial transport of water was examined i.e., the short-circuit current (Isc) and the transepithelial water movement (TEWM) were measured simultaneously. It has been shown repeatedly that the Isc across isolated frog skin is equal to the net transepithelial Na+ transport. Furthermore the coupling between transepithelial uptake of NaCl under open-circuit conditions and TEWM was also measured. The addition of antidiuretic hormone (AVT) to skins incubated under short-circuited conditions resulted in an increase in the Isc and TEWM. Under control conditions Isc was 9.14 +/- 2.43 and in the presence of AVT 45.9 +/- 7.3 neq cm-2 min-1 (n = 9) and TEWM changed from 12.45 +/- 4.46 to 132.8 +/- 15.8 nL cm-2 min-1. The addition of the Na+ channel blocking agent amiloride resulted in a reduction both in Isc and TEWM, and a linear correlation between Isc and TEWM was found. The correlation corresponds to that 160 +/- 15 (n = 7) molecules of water follow each Na+ across the skin. In another series of experiments it was found that there was a linear correlation between Isc and the increase in apical osmolarity needed to stop the TEWM. The data presented indicate that the observed coupling between the net transepithelial Na+ transport and TEWM is caused by local osmosis.

Anion binding in bovine corneal stroma

Purpose: The corneal stroma strives to take up water because of the osmotic gradient created by fixed negative charges within the stroma. This fixed negative charge is known to be partly due to glycosaminoglycans and thought to be partly due to bound Cl− ions in the stroma. This study investigates the presence of anion binding within bovine stroma.Methods: Bovine corneas were isolated, de‐epithelialized and de‐endothelialized. The stromal uptake of NaCl, NaAcetate, NaThiocyanate and NaHCO3 solutions at various concentrations was observed using a digital balance. The dry weight of each cornea was noted. Stromal hydrations were calculated and analysed.Results: As the bathing solution concentration increased, for all solutions there was a significant increase in the calculated fixed negative charge values obtained. At physiological concentration of NaCl (150 mM), the fixed negative charge was calculated to be 39.98 ± 1.05 mEq 1−1. For NaAcetate, NaThiocyanate and NaHCO3 at 150 mM the fixed negative charge was calculated to be 38.64 ± 0.51, 43.62 ± 0.51 and 41.98 ± 0.65 mEq 1−1 respectively (n = 10, ± SEM).Conclusions: The degree of anion binding in bovine corneal stroma varies from anion to anion and is dependent on the external bathing solution concentration. At 150 mM, the binding series from this study is NaThiocyanate &gt; NaHCO3 &gt; NaCl &gt; NaAcetate.

CO2 regulation of virulence genes in enteropathogenic Escherichia coli.

Enteropathogenic Escherichia coli (EPEC) are a heterogeneous group of bacteria implicated as a cause of severe persistent enteritis in infants (Robins-Browne 1987). The primary histopathological effect of EPEC infection is the so-called attaching and effacing (AE) lesion. This involves localised destruction of intestinal brush border microvilli and distortion of the apical enterocyte membrane into an actin-rich “pedestal” structure at the point of bacterial contact. Formation of AE lesions is a crucial aspect of the pathogenesis of EPEC. Loss of the absorptive surface of the gut mucosa by effacement of brush border microvilli contributes to disease by reducing NaCl uptake, while enterocyte death as a result of prolonged infection would inflict severe mucosal damage and lead to protracted diarrhoea.

The membrane transporters regulating epithelial NaCl secretion.

Ten years ago, the basic principles operating in one specific, albeit non-mammalian, exocrine gland, the rectal gland of Squalus acanthias, were described in detail. The concept emerging from these studies appeared applicable to almost any other exocrine gland, because it involved membrane transporters which are also present in mammalian epithelial cells. Meanwhile, it has become clear that the mechanisms of NaCl secretion are diverse: the mechanisms of NaCl uptake; the ion channels involved; and also the mechanisms of hormonal control. Nevertheless, several steps in NaCl secretion still appear to be uniform: (1) several signalling pathways converge and act cooperatively, (2) one primary regulatory step is the upregulation of the luminal Cl- conductance, (3) secondarily active NaCl uptake mechanisms are upregulated, (4) increasing evidence links NaCl secretion to membrane trafficking and (5) the entire machinery seems to be primed to secure cellular homeostasis in terms of cytosolic ion concentrations. This brief review summarizes the mechanisms of control of NaCl secretion. The major issues addressed are the NaCl uptake mechanisms, the ion channels involved and the cellular mechanisms coordinating secretion. The major NaCl secreting cells discussed here will be the respiratory epithelial cells, the exocrine cells of pancreatic acini and the cells of colonic crypts.

Active absorption of Na+ and Cl- across the gill epithelium of the shore crab Carcinus maenas: voltage-clamp and ion-flux studies

Mechanisms of active NaCl uptake across the posterior gills of the shore crab Carcinus maenas were examined using radiochemical and electrophysiological techniques. In order to measure short-circuit current (Isc), transepithelial conductance (Gte) and area-related unidirectional fluxes of Na+ and Cl-, single split gill lamellae (epithelium plus cuticle) of hyperregulating shore crabs were mounted in a modified Ussing chamber. The negative short-circuit current measured with haemolymph-like NaCl saline on both sides of the epithelium could be inhibited by application of basolateral ouabain (ouabain inhibitor constant KOua=56&plusmn;10 &micro;mol l-1), 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB; KNPPB=7.5&plusmn;2.5 mmol l-1) or Cs+ (10 mmol l-1). From the apical side, Isc was nearly completely blocked by Cs+ (10 mmol l-1) or Ba2+ (15 &micro;mol l-1), whereas apical addition of furosemide (1 mmol l-1) resulted in only a small current decrease. Cl- influxes were linearly related to negative Isc. The ratio between net influxes of Cl- and Na+ was found to be approximately 2:1. With a single membrane preparation, achieved by permeabilizing the basolateral membrane with amphotericin B, Cl- influxes which were driven by a concentration gradient were shown to depend on the presence of apical Na+ and K+. On the basis of these observations, we propose that active and electrogenic absorption of NaCl across the gill epithelium of hyperregulating shore crabs proceeds as in the thick ascending limb of Henle's loop in the mammalian nephron. Accordingly, branchial NaCl transport is mediated by apical K+ channels in cooperation with apical Na+/K+/2Cl- cotransporters and by the basolateral Na+/K+-ATPase and basolateral Cl- channels.

Role of mitochondria-rich cells in epithelial chloride uptake.

Uptake of NaCl by amphibian tight epithelia, such as skin, urinary bladder and collecting duct, requires considerable thermodynamic work. By calculation it is demonstrated that NaCl absorption from dilute external solutions ([NaCl] approximately 1 mM) demands more energy than can be provided by the Na(+)-K(+)-ATPase alone. Thus, in addition to the Na+ pump, another transport ATPase must be involved. Previously, we have suggested that the other transport ATPase is an apical proton pump in mitochondria-rich (MR) cells. By driving an apical Cl(-)-HCO3- exchange, a rheogenic H+ pump would energize entrance of Cl- across the apical membrane. Since Cl- channels are present in the basolateral membrane, the net result would be a transcellular active rheogenic uptake of Cl-, and secretion of H+ and HCO3-. At higher external concentrations, the Cl- uptake by MR cells is driven by the Na+ pump-generated transepithelial electrical potential difference, which also requires the presence of apical Cl- channels. Electrophysiological methods have been developed by which we have been able to study the polarity of single MR cells and identify apical and basolateral transport systems. We have verified the existence of rheogenic H+ pumps in the apical membrane and Cl- channels in both membranes.

Carbonic anhydrase, a respiratory enzyme in the gills of the shore crabCarcinus maenas

This paper summarizes investigations on the enzyme carbonic anhydrase (CA) in the gills of the osmoregulating shore crabCarcinus maenas. Carbonic anhydrase, an enzyme catalyzing the reversible hydration of CO2 to HCO3 − and H+, is localized with highest activities in the posterior salt-transporting gills of the shore crab- and here CA activity is strongly dependent on salinity. Contrary to the earlier hypothesis established for the blue crabCallinectes sapidus that cytoplasmic branchial CA provides the counter ions HCO3 − and H+ for apical exchange against Na+ and Cl−, the involvement of CA in NaCl uptake mechanisms can be excluded inCarcinus. Differential and density gradient centrifugations indicate that branchial CA is a predominantly membrane-associated protein. Branchial CA was greatly inhibited by the sulfonamide acetazolamide (AZ) Ki=2.4·10−8 mol/l). Using the preparation of the isolated perfused gill, application of 10−4 mol/l AZ resulted in an 80% decrease of CO2/HCO3 − excretion. Thus we conclude that CA is localized in plasma membranes, maintaining the CO2 gradient by accelerating adjustment of the pH-dependent CO2/HCO3 − equilibrium.

How to overcome osmotic stress? Marine crabs conquer freshwater. New insights from modern electrophysiology

In the present article we review our findings on split lamella preparations of crab gills mounted in modified Ussing-chambers with respect to mechanistic and ecophysiological aspects. The leaky gill epithelium of shore crabs adapted to brackish water absorbs Na+ and Cl− in a coupled mode, and shows similarities to other salt-absorbing epithelia exposed to moderately diluted media. The results so far obtained for NaCl uptake across the gills of the shore crab are compatible with a transport model where two cell types operate in parallel, one displaying cotransport-like NaCl absorption, similar to that in the thick ascending limb of Henle's loop of the mammalian mephron, and the other one with characteristics of amiloride-sensitive, channel-mediated Na+ uptake by frog skin. Although there is no clear evidence for the apical mechanisms in this model, it may serve as a good basis for more detailed studies in the future. The moderately tight gill epithelium of freshwater adapted Chinese crabs absorbs Na+ and Cl− independently from each other, and shows similarities to other salt-absorbing epithelia exposed to freshwater. The characteristics of a positive, Na+-dependent short-circuit current with externally Cl−-free saline indicate that active Na+ uptake proceeds in a frog-skin-like mode via apical Na+-channels and the basolateral Na+/K+-pump. The nature of a negative short-circuit current with external Cl−-saline indicates that active and Na+-independent Cl− uptake is driven by an apical V-type H+-pump and proceeds via apical Cl−/ HCO3 −-exchange and basolateral Cl−-channels.

Palmitate transport by rat renal basolateral membrane vesicles in the presence of albumin.

Long chain fatty acids are an important metabolic fuel for renal proximal tubules. Because of their insolubility, they are carried in the blood primarily bound to albumin. Processes involved in the cellular uptake of albumin-bound ligands are incompletely understood. To determine how fatty acids enter renal tubular cells when substrate was presented primarily bound to albumin (molar ratio albumin:palmitate = 1.0), [3H]palmitate uptake by basolateral membrane vesicles (BLMV) isolated from rat renal cortex was measured by a rapid filtration technique. The pattern of uptake was strikingly affected by buffer composition. In low-ionic-strength buffer (300 mM mannitol, 20 mM Tris-N-hydroxethylpiperazine-N'-2-ethanesulfonic acid [HEPES]), [3H]palmitate uptake plateaued in less than 1 min. The calculated volume of distribution of [3H]palmitate indicated extensive binding. In high-ionic-strength buffer with an inwardly directed NaCl, sodium gluconate, or KCl concentration gradient (100 mM) or equal intravesicular and extravesicular NaCl concentration (100 mM), uptake was biphasic with peak values 1.4- to 3-fold higher than plateau values. To test whether uptake could be specifically inhibited, phloretin (200 microM), probenecid (500 microM), or 4,4'-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS; 500 microM) was added to NaCl uptake buffer. These agents were without effect on initial uptake but inhibited the normal biphasic pattern. The biphasic uptake pattern suggested binding followed by translocation to the interior of BLMV with subsequent mediated efflux. Medicated palmitate efflux was demonstrated in BLMV preloaded with [3H]-palmitate in NaCl buffer by: (1) phloretin inhibition of efflux and (2) trans-stimulation of efflux by p-aminohippurate. Results suggest that the translocation of fatty acids derived from albumin:fatty acid complexes occurs only in the presence of high external ion concentration. Subsequent equilibration of fatty acids between albumin, BLMV, and vesicle interior is mediated by the organic anion transport system.

Effects of formate and oxalate on chloride absorption in rat distal tubule.

We previously demonstrated that formate and oxalate stimulate Cl-transport in the rat proximal tubule in accord with a model involving NaCl uptake across the luminal membrane via Cl-/organic anion exchange in parallel with Na+/H+ exchange and organic acid recycling. The purpose of the present study was to test whether similar mechanisms contribute to Cl- transport in the rat distal tubule. In distal tubules microperfused in situ with an isotonic solution, addition of 0.5 mM formate to the luminal perfusate increased Cl- (JCl) and fluid (Jv) absorption by 77 and 93%, respectively. Addition of 5 microM oxalate increased JCl and Jv by 52 and 108%, respectively. In distal tubules perfused with a hypotonic solution, formate stimulated JCl and Jv by 85 and 98%, respectively, and oxalate stimulated JCl and Jv by 80 and 115%, respectively. Addition of 0.5 mM acetate caused no significant change in JCl and Jv. The stimulation of JCl and Jv by formate was largely abolished by addition of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (100 microM) or ethylisopropyl-amiloride (10 microM) to the luminal perfusate. Chlorothiazide (0.5 mM) inhibited baseline JCl and Jv but caused no inhibition of the increments in Jv and JCl induced by formate. Formate stimulation of JCl and Jv was confined to early segments of the distal tubule. We conclude that formate and oxalate markedly enhance JCl and Jv in the early distal tubule by a thiazide-insensitive mechanism involving NaCl entry across the apical membrane by Cl-/organic anion exchange in parallel with Na+/H+ exchange.

Volume-activated Cl(-)-independent and Cl(-)-dependent K+ pathways in trout red blood cells.

1. Swelling of trout erythrocytes can be induced either by addition of catecholamine to the cell suspension, thus promoting NaCl uptake via beta-adrenergic-stimulated Na(+)-H+ exchange (isotonic swelling) or by suspending red blood cells in a hypotonic medium (hypotonic swelling). In both cases cells tend to regulate their volume by losing K+, but the characteristics of the volume-activated K+ pathways are different: after hormonally induced swelling the K+ loss is strictly Cl- dependent; after hypotonic swelling the K+ loss is essentially Cl- independent. 2. In order to determine the nature of these volume regulatory pathways (i.e. whether the net K+ loss was conductive or was by electroneutral K(+)-H+ exchange or KCl co-transport), studies were performed to analyse ion fluxes and associated electrical phenomena. The cell membrane potential and intracellular ionic activities of volume-regulating and volume-static cells were measured by impalement with conventional microelectrodes and double-barrelled ion-sensitive microelectrodes. 3. The information gained from the electrical and ion flux studies leads to the conclusion that both Cl(-)-independent and Cl(-)-dependent K+ loss proceed via electrically silent pathways. 4. Experiments were designed to distinguish between electroneutral K(+)-H+ exchange or KCl co-transport. These were based upon the inhibition of Cl(-)-OH- exchange to evaluate the degree of coupling between K+ and Cl- (KCl stoichiometry, pH change). The experimental observations are consistent with the fact that both Cl(-)-independent and Cl(-)-dependent K+ loss are mediated by coupled K(+)-anion co-transport and not by K(+)-H+ exchange. 5. On the basis of previous data, we suggest that only one type of K(+)-anion co-transport exists in the cell membrane, for which the selectivity for anions varies according to the change in cellular ionic strength induced by swelling.

Alpha 2-adrenergic, but not imidazole, agonists activate NaCl cotransport in rabbit tracheal epithelial cells

The adrenergic agonist clonidine activates NaCl cotransport in rabbit tracheocytes. With the use of the high-affinity analogue p-[125I]iodoclonidine, binding of clonidine to cells was determined to fit a two-site model, with one site of high specificity for alpha 2-adrenergic (alpha 2-AR) and the other with a high affinity for I1-imidazol(in)e (I1) receptors. Total density of binding sites for both receptors was similar at 18 fmol/mg protein. Moxonidine displayed a 166-fold greater specificity for I1 receptors compared with cimetidine. Bumetanide-sensitive Na or Cl transport was stimulated by the alpha 2-AR agonists clonidine or guanabenz but not by the I1 agents cimetidine or moxonidine. I1 agonists-stimulated Na transport was detected only in the presence of bumetanide. Prazosin did not block clonidine-stimulated NaCl uptake or efflux, indicating the presence of an alpha 2A-AR subtype. Addition of clonidine either before or after incubation with l-isoproterenol or forskolin did not attenuate the time- and dose-dependent increase in adenosine 3',5'-cyclic monophosphate (cAMP) levels. Thus clonidine stimulates NaCl cotransport in rabbit tracheocytes through an alpha 2A-AR mechanism that does not require cAMP for signal transduction. In addition, I1-imidazol(in)e receptors stimulate Na transport in rabbit tracheocytes through an unidentified pathway.