Paths of ion transport across canine fetal tracheal epithelium

Fluid secretion by the fetal sheep lung is thought to be driven by secretion of Cl- by the pulmonary epithelium. We previously demonstrated Cl- secretion by tracheal epithelium excised from fetal dogs and sheep. In this study we characterized the ion transport pathways across fetal canine tracheal epithelium. The transport of Na+ and Cl- across trachea excised from fetal dogs was evaluated from transepithelial electrical properties and isotope fluxes. Under basal conditions the tissues were characterized by a lumen-negative potential difference (PD) of 11 mV and conductance of 5.2 mS/cm2. The short-circuit current (Isc) was 43 microA/cm2 (1.6 mueq.cm-2.h-1). Basal Na+ flows were symmetrical, but net Na+ absorption (1.1 mueq.cm-2.h-1) could be induced by exposure of the luminal surface to amphotericin B (10(-6) M). Bilateral replacement of Na+ reduced Isc by 85%. Replacement of submucosal Na+ or exposure to submucosal furosemide (10(-4) M) reduced net Cl- secretion by 60-70%. Luminal exposure to indomethacin (10(-6) M) induced a 50% decrease in Isc, whereas isoproterenol (10(-6) M) increased Isc by 120%. The properties of the Cl- secretory pathway across fetal dog trachea are consistent with the model proposed for Cl- secretion across adult dog trachea and other Cl- -secreting tissues (e.g., bullfrog cornea and shark rectal gland). The absence of basal Na+ absorption by fetal dog trachea probably reflects limited apical membrane Na+ permeability.     

Active Na+ uptake in the isolated midgut of larval Sarcophaga bullata

The isolated midgut of larval Sarcophaga bullata actively accumulates Na⁺ from the gut lumen into the haemolymph. The active transport of Na⁺ out of the gut lumen is responsible for the transepithelial potential difference measured across the midgut epithelium, such that the midgut lumen is negative in respect to the haemolymph side. Both the net movement of Na⁺ out of the midgut lumen and the transepithelial potential are inhibited by CN^? and, in addition, the potential in blocked by ouabain.     

Sodium-Sulfate Symport by Aplysia californica Gut

Sulfate transport across plasma membranes has been described in a wide variety of organisms and cell types including gastrointestinal epithelia. Sulfate transport can be coupled to proton, sodium symport or antiport processes involving chloride or bicarbonate. It had previously been observed in Aplysia gut that sulfate was actively absorbed. To understand the mechanism for this transport, short-circuited Aplysia californica gut was used. Bidirectional transepithelial fluxes of both sodium and sulfate were measured to see whether there was interaction between the fluxes. The net mucosal-to-serosal flux of Na(+) was enhanced by the presence of sulfate and it was abolished by the presence of serosal ouabain. Similarly, the net mucosal-to-serosal flux of sulfate was dependent upon the presence of Na(+) and was abolished by the presence of serosal ouabain. Theophylline, DIDS and bumetanide, added to either side, had no effect on transepithelial potential difference or short-circuit current in the Aplysia gut bathed in a Na2SO4 seawater medium. However, mucosal thiosulfate inhibited the net mucosal-to-serosal fluxes of both sulfate and Na(+) and the thiosulfate-sensitive Na(+) flux to that of sulfate was 2:1. These results suggest the presence of a Na-SO4 symporter in the mucosal membrane of the Aplysia californica foregut absorptive cell.     

Anthracene-9-carboxylic acid inhibits an apical membrane,chloride conductance in canine tracheal epithelium

Summary Canine tracheal epithelium secretes Cl from the submucosal to the mucosal surface via an electrogenic transport process that appears to apply to a wide variety of secretory epithelia. Cl exit across the apical membrane is thought to be a passive, electrically conductive process. To examine the cellular mechanism of Cl secretion we studied the effect of anthracene-9-carboxylic acid (9-AC), an agent known to inhibit the Cl conductance of muscle membrane. When added to the mucosal solution, 9-AC rapidly and reversibly decreases short-circuit current and transepithelial conductance, reflecting a reduction in electrogenic Cl secretion. The inhibition is concentration-dependent and 9-AC does not appear to compete with Cl for the transport process. The decrease in current and conductance results from a decrease in the net and both unidirectional transepithelial Cl fluxes without substantial alterations of Na fluxes. Furthermore, 9-AC specifically inhibits a Cl conductance: tissues bathed in Cl-free solutions showed no response to 9-AC. Likewise, when the rate of secretion and Cl conductance were minimized with indomethacin, addition of 9-AC did not alter transepithelial conductance. In contrast, neither removal of Na from the media nor blockade of the apical Na conductance with amiloride prevented a 9-AC-induced decrease in transepithelial conductance. We also found that the effect of 9-AC is independent of transepithelial transport: 9-AC decreases transepithelial conductance despite inhibition of Cl secretion with ouabain or furosemide. Intracellular electrophysiologic techniques were used to localize the effect of 9-AC to a reduction of the electrical conductance of the apical cell membrane: 9-AC hyperpolarizes the electrical potential difference across the apical membrane and decreases its relative conductance. 9-AC also prevents the characteristic changes in the cellular electrical potential profile, transepithelial conductance, and the ratio of membrane conductances produced by a reduction in mucosal bathing solution Cl concentration. These results indicate that 9-AC inhibits Cl secretion in tracheal epithelium by blocking an electrically conductive Cl exit step in the apical cell membrane. Thus, they support a cellular model of Cl secretion in which Cl leaves the cell across a Cl permeable apical membrane driven by its electrochemical gradient.     

Ion transport across the epithelium of the rabbit caecum.

The isolated rabbit caecum was studied in vitro. Under our experimental conditions, the rabbit caecum secreted potassium and chloride and absorbed sodium. To characterize the transport properties of the apical and the basolateral barriers, transepithelial electrical and flux (22Na, 36Cl and 86Rb) measurements and their sensitivity to transport inhibitors (furosemide, DIDS, ouabain and barium) are presented together with intracellular measurements with double-barrelled microelectrodes of intracellular electrical potentials and ionic activities. The fluxes of sodium and chloride were insensitive to DIDS and furosemide. The secretion of potassium and the absorption of sodium were both inhibited by ouabain, indicating that they are coupled through the sodium pump. Ouabain induced a slow fall in the chloride net fluxes, suggesting that these fluxes are also driven by the sodium pump, albeit indirectly. The basolateral to apical fluxes of potassium are insensitive to barium added to the apical side, but are accelerated by the replacement of chloride by gluconate on the apical side, suggesting the presence of a K+/Cl- symport in the apical barrier.     

Sodium-phosphate symport by Aplysia californica gut

Phosphate transport across plasma membranes has been described in a wide variety of organisms and cell types including gastrointestinal epithelia. Phosphate transport across apical membranes of vertebrate gastrointestinal epithelia requires sodium; whereas, its transport across the basolateral membrane requires antiport processes involving primarily chloride or bicarbonate. To decipher the phosphate transport mechanism in the foregut apical membrane of the mollusc, Aplysia californica, in vitro short-circuited Aplysia californica gut was used. Bidirectional transepithelial fluxes of both sodium and phosphate were measured to see whether there was interaction between the fluxes. The net mucosal-to-serosal flux of Na+ was enhanced by the presence of phosphate and it was abolished by the presence of serosal ouabain. Similarly, the net mucosal-to-serosal flux of phosphate was dependent upon the presence of Na+ and was abolished by the presence of serosal ouabain. Theophylline, DIDS and bumetande, added to either side, had no effect on transepithelial difference or short-circuit current in the Aplysia gut bathed in a Na2HPO4 seawater medium. However, mucosal arsenate inhibited the net mucosal-to-serosal fluxes of both phosphate and Na+ and the arsenate-sensitive Na+ flux to that of phosphate was 2:1. These results suggest the presence of a Na-PO4 symporter in the mucosal membrane of the Aplysia californica foregut absorptive cell.     

The Origin of the Short-Circuit Current in the Isolated Skin of the South American Frog Leptodactylus ocellatus

In isolated skins of Leptodactylus ocellatus the short-circuit current is smaller than the sodium net flux and this difference disappears when the skins are bathed in solutions in which the chloride ions have been replaced by sulfate or methylsulfate ions. There is a net movement of chloride ions from outside to inside of the skins in the short-circuit condition with chloride Ringer's solutions bathing the skins. The addition of ouabain to the inside solution markedly reduced not only sodium net flux but also the chloride net influx found. Copper ions added to the outside solutions produced a rise in short-circuit current, as well as the known increase in potential difference. In sodium-free Ringer's (sodium replaced by choline) the orientation of the potential difference across the skins was reversed, the inside being negative instead of positive. The results are interpreted as direct or indirect indications of the presence of a net transfer of chloride ions from outside to inside of these frog skins.     

The cellular mechanism of active chloride secretion in vertebrate epithelia: studies in intestine and trachea

The cellular mechanism of active chloride secretion, as it is manifested in the intestine and trachea, appears to possess the following elements: (1)NaCl cl-transport across the basolateral membrane; (2) Cl- accumulation in the cell above electrochemical equilibrium due to the Na+ gradient; (3) a basolateral Na+-K+ pump that maintains the Na+ gradient; (4) a hormone-regulated Cl- permeability in the apical membrane; (5) passive Na/ secretion through a paracellular route, driven by the transepithelial potential difference; and (6) an increase in basolateral membrane K+ permeability occurring in conjunction with an increase in Na+-K+ pump rate. Electrophysiological studies in canine trachea support this model. Adrenalin, a potent secretory stimulus in that tissue, increases apical membrane conductance through a selective increase in Cl- permeability. Adrenalin also appears to increase basolateral membrane K+ permeability. Whether or not adrenalin also increases paracellular Na+ permeability is unclear. Some of the testable implications of the above secretion model are discussed.     

The phorbol ester, TPA, increases transepithelial epidermal growth factor flux

Exposure of cultured kidney epithelial (LLC-PK1) cell sheets to 10(-7) M TPA, a potent tumor promoter and activator of protein kinase C, initiates within minutes a drop in the transepithelial voltage across these sheets. This fall in potential difference correlates with an over 40-fold increase in the transepithelial flux of 1 mM D-mannitol, suggesting that the intercellular junctions have become leaky. Dual labeling experiments with 1 mM D-[14C]mannitol and 10 nM 125I-EGF show that after promoter treatment, a 7-fold increase in net 125I flux accompanies the increase in mannitol flux. Gel filtration and gel electrophoresis indicate that for control cell sheets only 15% of the transited 125I is actually EGF, whereas with TPA-treated cell sheets, 60% of the 125I which passed across is EGF. These percentages permitted determination of actual EGF flux values, and show that TPA treatment engenders a 35-fold increase in transepithelial EGF flux. Diacylglycerols also increase the junctional permeability of these cells, thereby suggesting the involvement of protein kinase C.     

Calcium secretion in canine tracheal mucosa

Calcium (Ca) affects many cellular functions of the respiratory tract mucosa and might alter the viscoelastic properties of mucus. To evaluate Ca homeostasis in a respiratory epithelium we investigated transport of Ca by the canine tracheal mucosa. Mucosal tissues were mounted in Ussing-type chambers and bathed with Krebs-Henseleit solution at 37 degrees C. Unidirectional fluxes of 45Ca were determined in tissues that were matched by conductance and short-circuit current (SCC). Under short-circuit conditions there was a significant net Ca secretion of 1.82 +/- 0.36 neq . cm-2 . h-1 (mean +/- SE). Under open-circuit conditions, where the spontaneous transepithelial potential difference could attract Ca toward the lumen, net Ca secretion increased significantly to 4.40 +/- 1.14 compared with 1.54 +/- 1.17 neq . cm-2 . h-1 when the preparation was short-circuited. Addition of a metabolic inhibitor, 2,4-dinitrophenol (2 mM in the mucosal bath), decreased tissue conductance and SCC and slightly decreased the unidirectional movement of Ca from submucosa to lumen. Submucosal epinephrine (10 microM) significantly enhanced Ca secretion by 2.0 +/- 0.63 neq . cm-2 . h-1. Submucosal ouabain (0.1 mM) failed to inhibit Ca secretion. The data suggest that canine tracheal mucosa secretes Ca; this secretory process is augmented by epinephrine or by the presence of a transepithelial potential difference as found under in vivo conditions.     

Structural diversity of occluding junctions in the low-resistance chloride-secreting opercular epithelium of seawater-adapted killifish (Fundulus heteroclitus)

The structural features of the chloride-secreting opercular epithelium of seawater-adapted killifish (Fundulus heteroclitus) were examined by thin-section and freeze-fracture electron microscopy, with particular emphasis on the morphological appearance of occluding junctions. This epithelium is a flat sheet consisting predominantly of groups of mitochondriarich chloride cells with their apices associated to form apical crypts. These multicellular groups are interspersed in an otherwise continuous pavement cell epithelial lining. The epithelium may be mounted in Ussing-type chambers, which allow ready access to mucosal and serosal solutions and measurement of electrocal properties. The mean short-circuit current, potential difference (mucosal-side negative), and DC resistance for 19 opercular epithelia were, respectively, 120.0 +/- 18.2 microA/cm2, 12.3 +/- 1.7 mV, and 132.5 +/- 26.4 omega cm2. Short-circuit current, a direct measure of Cl- transport, was inhibited by ouabain (5 micron) when introduced on the serosal side, but not when applied to the mucosal side alone. Autoradiographic analysis of [3H]-ouabain-binding sites demonstrated that Na+,K+-ATPase was localized exclusively to basolateral membranes of chloride cells; pavement cells were unlabeled. Occluding junctions between adjacent chloride cells were remarkably shallow (20-25 nm), consisting of two parallel and juxtaposed junctional strands. Junctional interactions between pavement cells or between pavement cells and chloride cells were considerably more elaborate, extending 0.3-0.5 micron in depth and consisting of five or more interlocking junctional strands. Chloride cells at the lateral margins of crypts make simple junctional contacts with neighboring chloride cells and extensive junctions with contiguous pavement cells. Accordingly, in this heterogeneous epithelium, only junctions between Na+,K+-ATPase- rich chloride cells are shallow. Apical crypts may serve, therefore, as focal areas of high cation conductivity across the junctional route. This view is consistent with the electrical data showing that transmural resistance across the opercular eptihelium is low, and with recent studies demonstrating that transepithelial Na+ fluxes are passive. The simplicity of these junctions parallels that described recently for secretory cells of avian salt gland (Riddle and Ernst, 1979, J. Membr. Biol., 45:21-35) and elasmobranch rectal gland (Ernst et al., 1979, J. Cell Biol., 83:(2, Pt. 2):83 a[Abstr.]) and lends morphological support to the concept that paracellular ion permeation plays a central role in ouabain-sensitive transepithelial NaCl secretion.     

Effects of 0.5 ppm ozone on glycoprotein secretion, ion and water fluxes in sheep trachea

We studied the effects of ozone (O3) exposure on airway mucus secretion. Sheep were exposed in vivo to 0.5 ppm O3, 4 h/day for 2 days (acute, n = 6), 6 wks (chronic, n = 6) or 6 wks + 1 wk recovery (chronic + recovery, n = 6). Secretion of glycoproteins (radiolabeled with 35SO4 and [3H]threonine), and transepithelial fluxes of Cl-, Na+ and water were subsequently measured in tracheal tissues in vitro, and were compared with values from control, unexposed sheep (n = 8). Acute O3 exposure increased basal secretion of sulfated glycoproteins (P less than 0.05), but had no effect on ion fluxes. Chronic exposure reduced basal glycoprotein secretion, but increased net Cl- secretion. Under open-circuit conditions, chronic exposure also induced net water secretion (P less than 0.05). With 7 days recovery, basal glycoprotein secretion (predominantly sulfated) was greatly increased above control, while the increased net secretion of Cl- and of water persisted (P less than 0.05). Histology of the airways indicated that acute exposure induced moderate hypertrophy of submucosal glands in the lower trachea (P less than 0.05), while chronic exposure (with and without recovery) induced a large hypertrophy of submucosal glands in both upper and lower trachea (P less than 0.05). Without recovery, however, the gland cells were devoid of secretory material, whereas with recovery they were full of secretory material. This suggests that the decreased glycoprotein secretion with chronic exposure alone resulted from incomplete replenishment of intracellular stores after 6 wks of stimulation. We conclude that both short- and long-term O3 exposure causes airway-mucus hypersecretion.     

Electrical properties of monolayers cultured from cells of human tracheal mucosa

Dispersed isolated cells were obtained from human tracheal mucosa by digestion with collagenase. Up to 1.5 X 10(8) cells were obtained per trachea and showed up to 95% viability, as judged by trypan blue exclusion. When grown in culture, the cells formed monolayers after approximately 4 days. Electron microscopy of the monolayers revealed a polarized structure. An apical membrane, containing microvilli and a pronounced glycocalyx, was separated from a relatively unspecialized basolateral membrane by typical tight junctions. Monolayers grown on nucleopore filters showed resistances of 44-1,800 omega. cm2 and transepithelial potential differences of 0.1-7.6 mV. Short-circuit current (Isc) was increased by isoproterenol, prostaglandins E2 and F2 alpha, and bradykinin. The loop diuretic, bumetanide, reduced Isc when added to the basolateral (serosal) side but had no effect from the apical (mucosal) side of the monolayers. Furosemide and MK-196 also inhibited Isc. Mucosal amiloride inhibited Isc. Serosal amiloride or mucosal ouabain had no effect on Isc. Serosal ouabain brought Isc to zero after approximately 15 min.     

Intracellular ion concentrations in the frog cornea epithelium during stimulation and inhibition of Cl secretion

Summary The intracellular electrolyte concentrations in the isolated cornea of the American bullfrog were determined in thin freeze-dried cryosections using energy-dispersive X-ray microanalysis. Stimulation of Cl secretion by isoproterenol resulted in a significant increase in the intracellular Na concentration but did not change the intracellular Cl concentration. Similar results were obtained when Cl secretion was stimulated by the Ca ionophore A23187. Inhibition of Cl secretion by ouabain produced a large increase in the intracellular Na concentration and an equivalent fall in the K concentration. Again, no increase or decrease in the intracellular Cl concentration was detectable. Clamping of the transepithelial potential to ±50 mV resulted in parallel changes in the transepithelial current and intracellular Na concentration, but, with the exception of the outermost cell layer, in no changes of the Cl concentration. Only when Cl secretion was inhibited by bumetanide or furosemide, together with a decrease in the Na concentration, was a large fall in the Cl concentration observed. Application of loop diuretics also produced significant increases in the P concentration and dry weight, consistent with some shrinkage of the epithelial cells. The results suggest the existence of a potent regulatory mechanism which maintains a constant intracellular Cl concentration and, thereby, a constant epithelial cell volume. Through the operation of this system any variation in the apical Cl efflux is compensated for by an equal change in the rate of Cl uptake across the basolateral membrane. Cl uptake is sensitive to loop diuretics, directly coupled to an uptake of Na, and dependent on the Na and K concentration gradients across the basolateral membrane. Isoproterenol and A23187 seem to increase the Cl permeability of the apical membrane and thus stimulate Cl efflux. Ouabain inhibits Cl secretion by abolishing the driving Na concentration gradient for Cl uptake across the basolateral membrane.     

A component of fluid absorption linked to passive ion flows in the superficial pars recta

We studied salt and water absorption in isolated rabbit superficial proximal straight tubules perfused and bathed with solutions providing oppositely directed transepithelial anion gradients similar to those which might obtain in vivo. The perfusing solution contained 138.6 mM Cl- 3.8 mM HCO-3 (pH 6.6) while the bathing solution contained 113.6 mM Cl- and 25 mM HCO-3 (pH 7.4); the system was bubbled with 95% O2-5% CO2. At 37 degrees C, net volume absorption (Jv nl min-1 mm-1) was 0.32 +/- 0.03 (SEM); Ve, the transepithelial voltage (millivolts; lumen to bath), was +3.1 +/- 0.2. At 21 degrees C, Ve rose to +3.7 +/- 0.1 and Jv fell to 0.13 +/- 0.01 (significantly different from zero at P less than 0.001); in the presence of 10(-4)M ouabain at 37 degrees C, Ve rose to +3.8 +/- 0.1 and Jv fell to 0.16 +/- 0.01 (P less than 0.001 with respect to zero). In paired experiments, the ouabain- and temperature-insensitive moieties of Jv and Ve became zero when transepithelial anion concentration gradients were abolished. Titrametric determinations net chloride flux at 21 degrees C or at 37 degrees C with 10(-4) M ouabain showed that chloride was the sole anion in an isotonic absorbate. And, combined electrical and tracer flux data indicated that the tubular epithelium was approximately 18 times more permeable to Cl- than to HCO-3. We interpret these results to indicate that, in these tubules, NaCl absorption depends in part on transepithelial anion concentration gradients similar to those generated in vivo and in vitro by active Na+ absorption associated with absorption to anions other than chloride. A quantitative analysis of passive solute and solvent flows in lateral intercellular spaces indicated that fluid absorption occurred across junctional complexes when the osmolality of the lateral intercellular spaces was equal to or slightly less than that of the perfusing and bathing solutions; the driving force for volume flow under these conditions depended on the fact that sigmaHCO3 exceeded sigmaCl.     

The electrical potential difference through the foot epithelium of the snail Achatina achatina, Lameere during mechanical and chemical stimulation

An important electrophysiological variable--the transepithelial potential difference reflects the electrogenic transepithelial ion currents, which are produced and modified by ion transport processes in polarized cells of epithelium. These processes result from coordinated function of transporters in apical and basolateral cell membranes and have been observed in all epithelial tissues studied so far. The experiments were performed on isolated specimens of snail foot. In the experiments, the baseline transepithelial electrical potential difference--PD, changes of transepithelial difference during mechanical stimulation--dPD and the transepithelial resistance were measured with an Ussing apparatus. A total of 60 samples of foot ventral surface of 28 snails were studied. The transepithelial electrical potential difference of isolated foot ranged from -6.0 to 10.0 mV under different experimental conditions. Mechanical stimulation of foot ventral surface caused changes of electrogenic ion transport, observed as transient hyperpolarization (electrical potential difference became more positive). When the transepithelial electrical potential difference decreased during stimulation, the reaction was described as depolarization. When amiloride and bumetanide were added to the stimulating fluid so that the sodium and chloride ion transport pathways were inhibited, prolonged depolarization occurred. Under the influence of different stimuli: mechanical (gentle rinsing), chemical (changes of ion concentrations) and pharmacological (application of ion inhibitors), transient changes of potential difference (dPD) were evoked, ranging from about -0.7 to almost 2.0 mV. Changes in transepithelial potential difference of the pedal surface of the snail's foot related to these physiological stimuli are probably involved in the locomotion of the animal and are under control of the part of the nervous system in which tachykinin related peptides (TRP) act as transmitters.     

Spontaneous reversal of polarity of the voltage across LLC-PK1 renal epithelial cell sheets

While sterilely monitoring transepithelial voltage (potential difference) across LLC-PK cell sheets over a 24-hr period, we noted that the apical-negative, transepithelial voltage, a key property of the LLC-PK1 renal epithelial cell line, reverses polarity to become apical-positive. This spontaneous change of polarity of electrical potential difference (PD) across LLC-PK1 cell sheets cultured on permeable filters was observed to occur approximately 12 hr after refeeding. Unlike the apical (luminal)-negative PD, the apical-positive PD was insensitive to phlorizin and ouabain. Both were insensitive to the diuretics amiloride, furosemide, and 4-acetamido-4-isothiocynato-stilbene-2,2-disulfonic acid (SITS). A pH gradient existed across apical-positive cell sheets (apical medium more acidic by 0.3 units) but an osmotic gradient did not. Unlike the temperature-sensitive apical-negative PD, the apical positive PD was unaffected by brief exposure to 4 degrees C temperature. Junctional disruptive agents such as the tumor promotor, TPA, dissipated both types of PD with similar time courses. The formation of the apical-positive PD correlated in time with apical glucose levels falling below the reported Km of the Na+-sugar contransporter. A high glycolytic rate per se may not be essential for this PD polarity reversal since the reversal could occur in glucose-free medium with a normal time course and magnitude. The lysis with time of floating cells with consequent release of KCl into the apical compartment was also considered as a possible cause of the polarity reversal, but the turnover of even 2 X 10(6) cells in 12 hr was found not to raise apical KCl sufficiently to produce the polarity shift. Although a significant K+ gradient did not exist across cell sheets with apical-positive PD values, a sizable gradient of Cl- did exist, directed apical to basolateral. This gradient, coupled with anion-selective tight junctions, should contribute to the observed apical positive voltage. The voltage polarity shift seen in these cell cultures with time is not unlike the polarity shift occurring in the renal proximal convoluted tubule, with distance from the glomerulus.     

Changes in fluid secretion rate alter net transepithelial transport of MRP2 and P-glycoprotein substrates in Malpighian tubules of Drosophila melanogaster

The effects of stimulants of fluid secretion on net transepithelial transport of the MRP2 substrate Texas Red and the p-glycoprotein substrate daunorubicin were examined in Malpighian tubules of Drosophila melanogaster. Fluid secretion rates were determined using the Ramsay assay and secreted fluid concentrations of Texas Red and daunorubicin were determined using a microfluorometric technique. Nanoliter droplets of secreted fluid were collected in optically flat glass capillaries and dye concentration was determined from fluorescence intensity measured by confocal laser scanning microscopy. Net transepithelial flux of each compound was then calculated as the product of its concentration in the secreted fluid and the fluid secretion rate. Net transepithelial flux of Texas Red increased when fluid secretion was stimulated by tyramine, cyclic AMP or hypoosmotic saline. Net flux decreased when fluid secretion rate of cAMP-stimulated tubules was reduced by elevating saline osmolality with sucrose. Net transepithelial flux of daunorubicin increased when fluid secretion was stimulated by cAMP. Significant increases in dye flux were seen only when the dyes were present at concentrations close to or greater than the concentration required for half maximal transport. Regression analyses showed that 57- 88% of the change in dye flux was attributable to the change in fluid secretion rate when tubules were stimulated with cAMP, cGMP, or tyramine. The results do not suggest that the effects of tyramine and cAMP are mediated through changes in transepithelial potential, nor do they indicate the direct effects of the stimulants on MRP2-like or p-glycoprotein-like transporters (e.g., via protein kinases). Instead, the results suggest that increases in fluid secretion rate minimize diffusive backflux of these dyes and, thus, facilitate higher rates of net transepithelial transport indirectly.     

Electrogenic Na(+)-dependent L-alanine transport in the lizard duodenum. Involvement of systems A and ASC

L-Alanine transport across the isolated duodenal mucosa of the lizard Gallotia galloti has been studied in Ussing chambers under short-circuit conditions. Net L-alanine fluxes, transepithelial potential difference (PD), and short-circuit current (Isc) showed concentration-dependent relationships. Na(+)-dependent L-alanine transport was substantially inhibited by the analog alpha-methyl aminoisobutyric acid (MeAIB). Likewise, MeAIB fluxes were completely inhibited by L-alanine, indicating the presence of system A for neutral amino acid transport. System A transport activity was electrogenic and exhibited hyperbolic relationships for net MeAIB fluxes, PD, and Isc, which displayed similar apparent K(m) values. Na(+)-dependent L-alanine transport, but not MeAIB transport, was partially inhibited by L-serine and L-cysteine, indicating the participation of system ASC. This transport activity represents the major pathway for L-alanine absorption and seemed to operate in an electroneutral mode with a negligible contribution to the L-alanine-induced electrogenicity. It is concluded from the present study that the active Na(+)-dependent L-alanine transport across the isolated duodenal mucosa of Gallotia galloti results from the independent activity of systems A and ASC for neutral amino acid transport.