Active Ion Transport Processes Research Articles

Basolateral Cl−uptake mechanisms in Xenopus laevis lung epithelium

A thin liquid layer covers the lungs of air-breathing vertebrates. Active ion transport processes via the pulmonary epithelial cells regulate the maintenance of this layer. This study focuses on basolateral Cl(-) uptake mechanisms in native lungs of Xenopus laevis and the involvement of the Na(+)/K(+)/2 Cl(-) cotransporter (NKCC) and HCO(3)(-)/Cl(-) anion exchanger (AE), in particular. Western blot analysis and immunofluorescence staining revealed the expression of the NKCC protein in the Xenopus lung. Ussing chamber experiments demonstrated that the NKCC inhibitors (bumetanide and furosemide) were ineffective at blocking the cotransporter under basal conditions, as well as under pharmacologically stimulated Cl(-)-secreting conditions (forskolin and chlorzoxazone application). However, functional evidence for the NKCC was detected by generating a transepithelial Cl(-) gradient. Further, we were interested in the involvement of the HCO(3)(-)/Cl(-) anion exchanger to transepithelial ion transport processes. Basolateral application of DIDS, an inhibitor of the AE, resulted in a significantly decreased the short-circuit current (I(SC)). The effect of DIDS was diminished by acetazolamide and reduced by increased external HCO(3)(-) concentrations. Cl(-) secretion induced by forskolin was decreased by DIDS, but this effect was abolished in the presence of HCO(3)(-). These experiments indicate that the AE at least partially contributes to Cl(-) secretion. Taken together, our data show that in Xenopus lung epithelia, the AE, rather than the NKCC, is involved in basolateral Cl(-) uptake, which contrasts with the common model for Cl(-) secretion in pulmonary epithelia.

Acid–base regulatory ability of the cephalopod (Sepia officinalis) in response to environmental hypercapnia

Acidification of ocean surface waters by anthropogenic carbon dioxide (CO(2)) emissions is a currently developing scenario that warrants a broadening of research foci in the study of acid-base physiology. Recent studies working with environmentally relevant CO(2) levels, indicate that some echinoderms and molluscs reduce metabolic rates, soft tissue growth and calcification during hypercapnic exposure. In contrast to all prior invertebrate species studied so far, growth trials with the cuttlefish Sepia officinalis found no indication of reduced growth or calcification performance during long-term exposure to 0.6 kPa CO(2). It is hypothesized that the differing sensitivities to elevated seawater pCO(2) could be explained by taxa specific differences in acid-base regulatory capacity. In this study, we examined the acid-base regulatory ability of S. officinalis in vivo, using a specially modified cannulation technique as well as (31)P NMR spectroscopy. During acute exposure to 0.6 kPa CO(2), S. officinalis rapidly increased its blood [HCO(3)(-)] to 10.4 mM through active ion-transport processes, and partially compensated the hypercapnia induced respiratory acidosis. A minor decrease in intracellular pH (pH(i)) and stable intracellular phosphagen levels indicated efficient pH(i) regulation. We conclude that S. officinalis is not only an efficient acid-base regulator, but is also able to do so without disturbing metabolic equilibria in characteristic tissues or compromising aerobic capacities. The cuttlefish did not exhibit acute intolerance to hypercapnia that has been hypothesized for more active cephalopod species (squid). Even though blood pH (pHe) remained 0.18 pH units below control values, arterial O(2) saturation was not compromised in S. officinalis because of the comparatively lower pH sensitivity of oxygen binding to its blood pigment. This raises questions concerning the potentially broad range of sensitivity to changes in acid-base status amongst invertebrates, as well as to the underlying mechanistic origins. Further studies are needed to better characterize the connection between acid-base status and animal fitness in various marine species.

Characterization of active ion transport across primary rabbit corneal epithelial cell layers (RCrECL) cultured at an air-interface

Previously, we reported the development of a primary culture model of tight rabbit corneal epithelial cell layers (RCrECL) characterizing bioelectric parameters, morphology, cytokeratin, and passive permeability. In the present study, we specifically evaluated the active ion transport processes of RCrECL cultured from either pigmented or albino rabbits. Primary cultured RCrECL were grown at an air-interface on Clear–Snapwells™ precoated with collagen/fibronectin/laminin and mounted in a modified Ussing-type chamber for the evaluation of their active ion transport processes under short-circuited conditions. Contribution of active Na + and Cl − transport to overall short-circuit current ( I sc) was evaluated by removing Na + and Cl −, respectively, from bathing fluids of RCrECL and measurements of net fluxes of Na + and Cl − using 22Na and 36Cl, respectively. Amiloride and benzamil were used to determine the role of apical Na +-channel activities to net Na + fluxes. N-phenylanthranilic acid (NPAA), ouabain, BaCl 2 and bumetanide were used to determine the role of basolateral Na,K-ATPase, apical Cl −-channel, and basolateral K +-channel and Na +(K +)2Cl −-cotransporter activities, respectively, in active ion transport across RCrECL. I sc of RCrECL derived from pigmented rabbits was comprised of 64±2% and 44±5% for active Na + and Cl − transport, respectively, consistent with net Na + absorption and Cl − secretion of 0.062±0.006 and 0.046±0.008 μEq/cm 2/hr estimated from radionuclide fluxes. Apical amiloride and benzamil inhibited I sc by up to ∼50% with an IC 50 of 1 and 0.1 μ m, respectively, consistent with participation of apical epithelial Na +-channels to net Na + absorption across RCrECL cultured from pigmented rabbits. Addition of ouabain to the basolateral, NPAA to the apical, BaCl 2 to the basolateral and bumetanide to basolateral fluid decreased I sc by 86±1.5%, 53±3%, 18±1.8% and 13±1.9% in RCrECL cultured from pigmented rabbits, while 85±0.7%, 36±1.6%, 38±1.8% and 15±3.5% decrea ses are observed for RCrECL from albino rabbits, respectively. Air-interface cultured RCrECL from either pigmented or albino rabbits exhibited active ion transport properties similar to those present in excised tissues. This primary culture system may be a reliable in-vitro model for mechanistic characterization of corneal epithelial function and regulation of transport properties.

Anion secretion by the inner medullary collecting duct. Evidence for involvement of the cystic fibrosis transmembrane conductance regulator.

It is well established that the terminal renal collecting duct is capable of electrogenic Na+ absorption. The present experiments examined other active ion transport processes in primary cultures of the rat inner medullary collecting duct. When the amiloride analogue benzamil inhibited electrogenic Na+ absorption, cAMP agonists stimulated a transmonolayer short circuit current that was not dependent on the presence of Na+ in the apical solution, but was dependent on the presence of Cl- and HCO3-. This current was not inhibited by the loop diuretic bumetanide, but was inhibited by ouabain, an inhibitor of the Na+/K+ pump. The current was reduced by anion transport inhibitors, with a profile similar to that seen for inhibitors of the cystic fibrosis transmembrane conductance regulator (CFATR) Cl- channel. Using several PCR strategies, we demonstrated fragments of the predicted lengths and sequence identity with the rat CFTR. Using whole-cell patch-clamp analysis, we demonstrated a cAMP-stimulated Cl- current with characteristics of the CFTR. We conclude that the rat inner medullary collecting duct has the capacity to secrete anions. It is highly likely that the CFTR Cl- channel is involved in this process.

Air-interface cultures of guinea pig airway epithelial cells: effects of active sodium and chloride transport inhibitors on bioelectric properties.

To systematically study airway epithelial barrier properties and active ion transport processes during challenge to environmental toxicants, in vitro models that closely resemble airway epithelium in vivo are required. Guinea pig tracheobronchial epithelial (GPTE) cells cultured in an air interface form a tight, confluent monolayer, which may be a more suitable model for the studies of the airway epithelial barrier in vivo. In the present study, bioelectric properties of such GPTE cell monolayers were characterized with the use of pharmacological agents. Treatment of the basolateral side with ouabain completely abolished the short-circuit current (SCC), while apical addition had little effect. Apical addition of amiloride abolished 75% of the SCC, while basal addition had little effect. Treatment of the basolateral side with furosemide or bumetanide reduced the SCC by 20%, while apical addition had no effect. Apical or basolateral terbutaline increased SCC threefold with identical time courses; results with furosemide suggest a preferential stimulation of Cl- secretion. In contrast to GPTE monolayers, canine tracheal epithelial monolayers, under short-circuit conditions, primarily secrete Cl-, possibly due to apparent species differences. The predominance of Na+ reabsorption suggests that GPTE monolayers cultured in an air interface may share similar transport properties to human airway epithelium.

Ion transport processes and middle ear physiopathology. An experimental approach using cell culture.

The role of the middle ear (ME) epithelium is primarily to keep the cavities air-filled and fluid-free. This condition requires elimination of mucus and cellular debris from the tympanic cavity by the ciliary activity. This clearance activity may be related to active ion transport processes in the epithelium. This paper presents a review of the experimental data suggesting the existence of ion transports in ME, and demonstrating the contribution of the culture of ME epithelial cells to the investigation of ion transport properties. Primary cultures of ME epithelial cells from Mongolian gerbils (Meriones unguiculatus) were undertaken. Polarized monolayers exhibited domes on non-porous supports. On porous supports, the absorptive function was shown to be related to a Na+ absorption from apical to basal sides of the epithelium, by means of apical amiloride-sensitive Na+ channels and basolateral Na+, K(+)-ATPase. In turn, this sodium absorption would generate an osmotic gradient and drive a water flow. An amiloride-induced chloride transport is also suggested. A hormonal modulation of these ion transport properties might play a role in the development of the mucociliary impairment that initiates chronic otitis media. The establishment of a middle ea cell line using wild-type SV 40 infection is in progress. This might provide further information on the physiology of the ME.

NO2Decreases Paracellular Resistance to Ion and Solute Flow in Alveolar Epithelial Monolayers

Primary cultured monolayers of rat alveolar epithelial cells grown on tissue culture-treated Nuclepore filters were exposed to 2.5 ppm nitrogen dioxide (NO2) for 2-20 min. Changes in monolayer bioelectric properties and solute permeabilities were subsequently measured. Exposure to NO2 produced a dose-dependent decrease in monolayer transepithelial electrical resistance (Rt), whereas monolayer short-circuit current was unaffected. Post-exposure monolayer permeability to 14C-sucrose (which primarily crosses alveolar epithelium via the paracellular pathway) increased markedly. That for 3H-glycerol (which permeates through both paracellular and transcellular pathways) increased to a lesser extent. Partial recovery of Rt and solute permeabilities was noted by 48-h post-exposure. The time courses of the decrease in Rt and increase in solute permeabilities were similar. These results suggest that NO2 primarily impairs passive alveolar epithelial barrier functions in vitro, probably by altering intercellular junctions, and does not appear to directly affect cell membrane active ion transport processes. When correlated with results obtained from experimental approaches, studies of in vitro alveolar epithelial monolayers may facilitate investigations of dosimetry, sites, and mechanisms of oxidant injury in the lung.

Impaired intestinal sodium and chloride transport in the blind loop syndrome of the rat

Self-filling blind loops of rat jejunum were used as a model for the blind loop syndrome in humans. Electrical resistance, short circuit current, and unidirectional sodium and chloride fluxes were measured using the Ussing technique. Whereas net fluxes for sodium and chloride did not differ significantly from zero in the blind loop or in the control, unidirectional fluxes of either direction were decreased and electrical resistance was increased, indicating an increase in the tightness of the intestinal wall. Measurements of alternating current impedance and micropuncture experiments revealed that this was due to an increase in epithelial resistance from 9 ± 1 Ω · cm2 (n = 15, results of both methods) to 27 ± 4 Ω · cm2 (n = 15) and in subepithelial resistance from 40 ± 2 Ω · cm2 (n = 15) to 76 ± 7 Ω · cm2 (n = 15). As the ratio of epithelial to subepithelial resistance was similar in the blind loop and in the control, lower transport rates in the blind loop are indicative of impaired epithelial transport function. Subsequently, two different transport systems were characterized. First, the 3-omethyl-glucose-induced, phlorizin-reversible increase in short circuit current, representing glucosecoupled sodium absorption, showed a 77% decrease in maximum velocity in the blind loop and no change in Km. Second, the chloride-induced, bumetanide-reversible increase in short circuit current in tissues stimulated simultaneously by prostaglandin E1 and theophylline, representing rheogenic chloride secretion, also showed a decrease in maximum velocity (of 83%) and no change in Km. A morphometric analysis revealed that the crypt surface area increased by 100% in the blind loop, whereas the villous surface area was not significantly different between blind loops and controls. We conclude that the jejunal self-filling blind loop is characterized by impaired active ion transport processes and an increase in epithelial and subepithelial resistance.

The effects of independent sodium and chloride depletion on ion balance in freshwater mussels

Ligumia subrostrata maintained in choline chloride or Na2SO4 exhibited a loss of either sodium or chloride from the blood at rates similar to those observed in mussels kept in deionized water. When the ion-depleted mussels were returned to a medium containing the depleted ion, the influx of the depleted ion was increased while the efflux was depressed compared with control mussels. Blood sodium concentrations, in mussels undergoing sodium depletion, decreased linearly to approximately 50% of the normal condition, while calcium levels increased. Blood chloride concentrations decreased exponentially during depletion, with the mussel capable of withstanding an 80% reduction. Blood bicarbonate levels increased in mussels undergoing chloride depletion. The elevation of the blood bicarbonate in chloride-depleted and calcium in the sodium-depleted mussels resulted in partial compensation for the sodium chloride lost from the blood, by helping maintain the total solute and the balance of ionic charge, and may act to "prime" the active ion transport processes. When returned to media containing the depleted ion, the mussels reaccumulated sodium and chloride within 2 to 4 days. These data provide evidence for the presence of independent regulatory mechanisms for sodium and chloride.

Mechanisms of action of cholera and Escherichia coli enterotoxins

Current information is reviewed on the mechanism of secretion in small intestine, including how it is altered by cyclic 3′, 5′-adenosine monophosphate and on the structures and properties of cholera and both heat-labile and heat-stable Escherichia coli enterotoxins. Two separate active ion transport processes are altered by cyclic 3′,5′-adenosine monophosphate: 1) coupled absorption of NaCl is inhibited in villus cells and 2) active anion secretion is stimulated, probably in crypt cells. Cholera and heat-labile E. coli toxins exert their secretory effect by stimulating intestinal mucosal adenylate cyclase. This stimulation results from the A1 subunitcatalyzed transfer of adenosine diphosphate ribose from NAD to a membrane-bound guanosine triphosphatase, thereby inhibiting this enzyme, which normally represses adenylate cyclase. Heatstable E. coli enterotoxin stimulates intestinal mucosal guanylate cyclase, which appears to be the basis for its enterotoxicity.

ATPase activities in retinal pigment epithelium and choroid

The Mg 2−-, the Na +-K +- and the HCO 3 −-dependent ATPase activities of retinal pigment epithelium-choroid tissue from grassfrog, bullfrog and rabbit have been measured. In the frogs, Mg 2+ ATPase activity was between 1 and 2 μmols P i liberated hr −1 mg −1 protein, while Na +-K + ATPase and HCO 3 − ATPase were about 20 and 25% of this activity, respectively. Corresponding values for the rabbit were 8·5 μmol P i hr −1 mg −1 protein, 8% and 9%. Na +-K + ATPase activity in each species was completely inhibited by 2 m m-calcium, while HCO 3 − ATPase was unaffected by calcium or by ouabain. The pigment epithelium-choroid preparations of bullfrog and rabbit were scraped to yield suspensions of isolated epithelial cells, relatively free of other tissue components, and a residual fraction which contained some epithelial cells, choroidal elements and red blood cells. Na −-K + ATPase activity was present in the epithelial cells of both species and absent from the choroid of the rabbit. HCO 3 − ATPase and Mg 2+ ATPase activities were roughly equally distributed in the bullfrog fractions, but in the rabbit were concentrated in the choroid. The results are discussed in relation to the postulated active ion transport processes of the pigment epithelium.

The influence of ouabain on the elimination of injected and orally applied dyes in Drosophila hydei

The sour dyes azocarmine and indigocarmine are excreted through the Malpighian tubules and the midgut after injection into the body cavity of third instar Drosophila hydei larvae. After injection, the other organs are free of dyes. The epithelium of the midgut does not allow orally applied dyes to pass into the haemolymph. Ouabain diminishes significantly the content of dyes in the cavity of the Malpighian tubules and of the midgut. The maximal concentration of azocarmine decreases in the Malpighian tubules to about 65 per cent and in the midgut to about 70 per cent. Indigocarmine decreases in the Malpighian tubules to about 55 per cent. The content of indigocarmine of the midgut does not change significantly after ouabain injections. As ouabain inhibits active ion transport, the decrease of the concentration of dyes is seen as proof of the coupling of active ion transport processes and of excretion of the dyes. Moreover, this decrease points to an ouabain-sensitive transport mechanism, which is localized in the epithelia of the Malpighian tubules and midgut.