Side specific effect of 5-hydroxytryptamine on NaCl transport in the apical and basolateral membrane of rat tracheal epithelia

5-Hydroxytryptamine (5-HT) can be released from mast cells and platelets through an IgE-dependent mechanism and may play a role in the pathogenesis of allergic bronchoconstriction. However, the effect of 5-HT on ion transport by the airway epithelium is still controversial. The objective of this study was to determine whether 5-hydroxytryptamine (5-HT) regulates NaCl transport by different mechanisms in the apical and basolateral membrane of tracheal epithelia. We studied the rat tracheal epithelium under short-circuit conditions in vitro. Short-circuit current (I(sc)) was measured in rat tracheal epithelial monolayers cultured on porous filters. 5-HT inhibited Na(+) absorption [measured via Na(+) short-circuit current (I(Na)(sc))] in the apical membrane and stimulated Cl(-) secretion [measured via Cl(-) short-circuit current (I(Cl)(sc))] in the basolateral membrane. Functional localization using selective 5-HT agonists and antagonists suggest that I(Cl)(sc)is stimulated by the basolateral membrane-resident 5-HT receptors, whereas I(Na)(sc) is inhibited by the apical membrane-resident 5-HT2 receptors. The basolateral addition of 5-HT increases intracellular cAMP content, but its apical addition does not. The addition of BAPTA/AM blocked the decrease of I(Na)(sc)which was induced by the apical addition of 5-HT, and 5-HT increased intracellular Ca concentrations. These results indicate that 5-HT differentially affects I(Na)(sc)and I(Cl)(sc)across rat tracheal monolayers through interactions with distinct receptors in the apical and the basolateral membrane. These effects may result in an increase of water movement towards the airway lumen.     

Interaction between the basolateral K+ and apical Na+ conductances in Necturus urinary bladder

Experimental modulation of the apical membrane Na+ conductance or basolateral membrane Na+-K+ pump activity has been shown to result in parallel changes in the basolateral K+ conductance in a number of epithelia. To determine whether modulation of the basolateral K+ conductance would result in parallel changes in apical Na+ conductance and basolateral pump activity, Necturus urinary bladders stripped of serosal muscle and connective tissue were impaled through their basolateral membranes with microelectrodes in experiments that allowed rapid serosal solution changes. Exposure of the basolateral membrane to the K+ channel blockers Ba2+ (0.5 mM/liter), Cs+ (10 mM/liter), or Rb+ (10 mM/liter) increased the basolateral resistance (Rb) by greater than 75% in each case. The increases in Rb were accompanied simultaneously by significant increases in apical resistance (Ra) of greater than 20% and decreases in transepithelial Na+ transport. The increases in Ra, measured as slope resistances, cannot be attributed to nonlinearity of the I-V relationship of the apical membrane, since the measured cell membrane potentials with the K+ channel blockers present were not significantly different from those resulting from increasing serosal K+, a maneuver that did not affect Ra. Thus, blocking the K+ conductance causes a reduction in net Na+ transport by reducing K+ exit from the cell and simultaneously reducing Na+ entry into the cell. Close correlations between the calculated short-circuit current and the apical and basolateral conductances were preserved after the basolateral K+ conductance pathways had been blocked. Thus, the interaction between the basolateral and apical conductances revealed by blocking the basolateral K+ channels is part of a network of feedback relationships that normally serves to maintain cellular homeostasis during changes in the rate of transepithelial Na+ transport.     

Transport-related modulation of the membrane properties of toad urinary bladder epithelium.

Impedance analysis and transepithelial electrical measurements were used to assess the effects of the apical membrane Na+ channel blocker amiloride and anion replacement on the apical and basolateral membrane conductances and areas of the toad urinary bladder (Bufo marinus). Mucosal amiloride addition decreased both apical and basolateral membrane conductances (Ga and Gbl, respectively) with no change in membrane capacitances (Ca and Cbl). Consequently, the specific conductances of these membranes decreased without significant changes in membrane area. Following amiloride removal, an increase was obtained in the steady-state rate of sodium transport compared to values before amiloride addition. This increase was independent of the initial transport rate, suggesting activation of a quiescent pool of apical sodium channels. Chloride replacement by acetate or gluconate had no significant effects on apical or basolateral membrane capacitances. The effects of these replacements on membrane conductances depended on the anion species. Gluconate (which induces cell shrinkage) decreased both membrane conductances. In contrast, acetate (which induces cell swelling) increased Ga and had no effect on Gbl. The increase in the apical membrane conductance was due to an increase in the amiloride-sensitive Na+ conductance of this membrane. In summary, mucosal amiloride addition or chloride replacements led to changes in membrane conductances without significant effects on net membrane areas.     

Domain-specific purinergic signaling in polarized rat cholangiocytes

In cholangiocytes, adenine nucleotides function as autocrine/paracrine signals that modulate ductular ion transport by activation of purinergic receptors. The purpose of these studies was to identify cellular signals that modulate ATP release and nucleotide processing in polarized normal rat cholangiocytes. In Ussing chamber studies, selective exposure of the apical and basolateral membranes to ATP or adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS) stimulated increases in short-circuit current. Apical purinergic receptor agonist preference was consistent with the P2Y(2) subtype. In contrast, basolateral ADP was more potent in stimulating transepithelial currents, consistent with the expression of different basolateral P2 receptor(s). Luminometric analysis revealed that both membranes exhibited constitutive ATP efflux. Hypotonic exposure enhanced ATP release in both compartments, whereas decreases in ATP efflux during hypertonicity were more prominent at the apical membrane. Increases in intracellular cAMP, cGMP, and Ca(2+) also increased ATP permeability, but selective effects on apical and basolateral ATP release differed. Finally, the kinetics of ATP degradation in apical and basolateral compartments were distinct. These findings suggest that there are domain-specific signaling pathways that contribute to purinergic responses in polarized cholangiocytes.     

Prolactin, an activator of epithelial Na+ channel, inhibits basolateral K+ channels in adult tree frog skin

Adult amphibian skin actively transports Na+ from its apical to basolateral side while in turn, K+ is recycled through Na+, K+-ATPase and K+ channels located in the basolateral membrane. We previously found that PRL stimulates Na+ transport in the skin of the adult tree frog (Hyla arborea japonica) via an increase in the open-channel density of the epithelial Na+ channel (ENaC). If PRL also activates basolateral K+ channels, this activation would help to stimulate Na+ transport, too. Whether PRL does indeed stimulate basolateral K+ channels in the adult tree frog was examined by measuring the short-circuit current across nystatin-treated skin. Both tolbutamide, a K(ATP) channel blocker, and tetrapentylammonium (TPA), a KCa channel blocker, blocked the current, the effect of TPA being more powerful than that of tolbutamide. Contrary to expectation, PRL inhibited the basolateral K+ channels in this skin. In the presence of basolateral amiloride, PRL still inhibited the basolateral K+ current, suggesting that the (Na+)-H+ exchanger located in the basolateral membrane does not mediate the inhibitory effect of PRL on the basolateral K+ channels in Hyla.     

Modulation of basal and peptide hormone-stimulated Na transport by membrane cholesterol content in the A6 epithelial cell line.

These studies examined the effect of altering plasma membrane cholesterol on basal Na+ flux as well as on the natriferic responses to the peptide hormones, insulin and anti-diuretic hormone (ADH) in the A6 model renal cell line. Membrane cholesterol concentrations were depleted or enriched using methyl-beta-cyclodextrin (MbetaCD) or a MbetaCD/cholesterol inclusion complex respectively. Effects of changes in the apical and basolateral plasma membranes were examined independently. Apical membrane cholesterol removal or supplementation had no effect on the basal Na+ transport rate. Short-term apical membrane cholesterol supplementation also had no effect on insulin-stimulated Na+ transport or on the initial phase of the ADH response. Interestingly, the additional apical membrane cholesterol had an inhibitory effect on the ADH response after 30 minutes. Apical membrane cholesterol depletion partially inhibited the responses to both insulin and ADH. Conversely, supplementation of basolateral cholesterol caused a significant increase in basal Na+ flux. Removal of cholesterol from the basolateral plasma membrane caused a decrease in basal Na+ flux with a time course analogous to channel turnover and completely inhibited peptide hormone responses. None of the changes in membrane cholesterol content decreased transcellular resistance. These results indicate an important role for membrane cholesterol content in the regulation of ENaC-mediated Na+ uptake.     

Electrophysiological effects of basolateral [Na+] in Necturus gallbladder epithelium

In Necturus gallbladder epithelium, lowering serosal [Na+] ([Na+]s) reversibly hyperpolarized the basolateral cell membrane voltage (Vcs) and reduced the fractional resistance of the apical membrane (fRa). Previous results have suggested that there is no sizable basolateral Na+ conductance and that there are apical Ca(2+)-activated K+ channels. Here, we studied the mechanisms of the electrophysiological effects of lowering [Na+]s, in particular the possibility that an elevation in intracellular free [Ca2+] hyperpolarizes Vcs by increasing gK+. When [Na+]s was reduced from 100.5 to 10.5 mM (tetramethylammonium substitution), Vcs hyperpolarized from -68 +/- 2 to a peak value of -82 +/- 2 mV (P less than 0.001), and fRa decreased from 0.84 +/- 0.02 to 0.62 +/- 0.02 (P less than 0.001). Addition of 5 mM tetraethylammonium (TEA+) to the mucosal solution reduced both the hyperpolarization of Vcs and the change in fRa, whereas serosal addition of TEA+ had no effect. Ouabain (10(-4) M, serosal side) produced a small depolarization of Vcs and reduced the hyperpolarization upon lowering [Na+]s, without affecting the decrease in fRa. The effects of mucosal TEA+ and serosal ouabain were additive. Neither amiloride (10(-5) or 10(-3) M) nor tetrodotoxin (10(-6) M) had any effects on Vcs or fRa or on their responses to lowering [Na+]s, suggesting that basolateral Na+ channels do not contribute to the control membrane voltage or to the hyperpolarization upon lowering [Na+]s. The basolateral membrane depolarization upon elevating [K+]s was increased transiently during the hyperpolarization of Vcs upon lowering [Na+]s. Since cable analysis experiments show that basolateral membrane resistance increased, a decrease in basolateral Cl- conductance (gCl-) is the main cause of the increased K+ selectivity. Lowering [Na+]s increases intracellular free [Ca2+], which may be responsible for the increase in the apical membrane TEA(+)-sensitive gK+. We conclude that the decrease in fRa by lowering [Na+]s is mainly caused by an increase in intracellular free [Ca2+], which activates TEA(+)-sensitive maxi K+ channels at the apical membrane and decreases apical membrane resistance. The hyperpolarization of Vcs is due to increase in: (a) apical membrane gK+, (b) the contribution of the Na+ pump to Vcs, (c) basolateral membrane K+ selectivity (decreased gCl-), and (d) intraepithelial current flow brought about by a paracellular diffusion potential.     

Cl Secretagogues Reduce Basolateral K Permeability in the Rabbit Corneal Epithelium

The stromal-to-tear transport of Cl by the rabbit corneal epithelium is increased by pharmacological effectors (secretagogues) that raise cAMP. It is well established that such secretagogues increase the apical membrane permeability to Cl and thus facilitate the efflux of the anion. However, we and others have found that cAMP-elevating agents frequently decrease the transepithelial potential difference across the rabbit cornea. The mechanism underlying this latter phenomenon had not been characterized. In this report, transepithelial and microelectrode studies were combined with measurements of unidirectional fluxes of 36Cl, 22Na and 86Rb to show that secretagogues known to act via cAMP also decrease the K permeability of the basolateral membrane, which by cellular depolarization would decrease apical Cl secretion. This effect was increasingly pronounced as a function of concentration when agents (e.g., epinephrine, isoproterenol) were applied to the apical side of the preparations. The addition of these agonists to the basolateral bathing solution, or of forskolin to the apical side, solely elicited inhibitions of basolateral K permeability. It seems that apical Cl and basolateral K conductances are independently and inversely regulated by cAMP. The opposite effects that cAMP could have on fluid secretion and epithelial thickness, by increasing apical Cl permeability but decreasing basolateral K permeability, may serve as a mechanism to maintain epithelial thickness within a narrow range.     

Tetracyclines reduce Na+/K+ pump capacity in Calu-3 human airway cells.

We studied the effect of tetracyclines on the Na+/K+ pump activity in Calu-3, a human airway cell line. To estimate Na+/K+ pump capacity on the basolateral membrane, an ouabain-sensitive component of the short-circuit current (Isc) was measured in the presence of nystatin, an ionophore of Na+. The application of ouabain (1 mM) to the basolateral solution completely inhibited the Isc generated by adding nystatin (50 microM) to the apical solution. Tetracycline (TC), minocycline (MC), or demethylchlortetracycline (DC) at 0.5 mM applied to the apical but not to the basolateral solution also decreased the nystatin-induced Isc. Neither phlorizin- nor diphenylamine-2-carboxylic acid-sensitive Isc was affected by TC, MC, or DC. These results indicate that tetracyclines may permeate only through the apical membrane with the result that the Na+/K+ pump's capacity for Na+ extrusion should be suppressed without a decrease in Cl- transport.     

Effects of basolateral ouabain, amphotericin B, cyanide and potassium on amiloride noise during voltage clamp of Rana pipiens skin support sodium-amiloride competition

In a previous study, the amiloride-induced corner frequency (fc) was found to decrease as apical sodium was increased. This effect was small or absent when the basolateral surface was exposed to high potassium. It has been suggested that the apical sodium effect may be indirect, due either to increased intracellular [Na+] which repelled amiloride or to an increased potential at the apical surface which reduced amiloride affinity. High basolateral K+ might then suppress the sodium effect either by preventing intracellular [Na+] from increasing or by allowing a better clamp of the apical membrane potential by reducing basolateral membrane resistance and potential. We checked the effects of basolateral [K+], of cyanide and of ouabain at concentrations known to increase intracellular [Na+]. We found only negligible effects on fc. In addition, amphotericin B added to the basolateral bathing solution either in 115 mM Na+ or in 120 mM K+ had no significant effect on fc. We found that relatively wide variation in clamp potential under all conditions, even with active transport severely inhibited, left fc virtually constant. Since the amiloride kinetics were independent of clamp potential, we were able to measure paracellular and transcellular conductances separately by examining the voltage dependence of clamp current (linear) and amiloride noise power (quadratic). This made possible estimation of channel density and single-channel current.     

Na+-Ca2+ exchange and calcium permeability in canine basolateral membrane vesicles: the effects of dibutyryl cAMP and specific inhibitors

The role of dibutyryl 3',5'-cyclic adenosine monophosphate (dibutyryl cAMP) as putative second messenger for parathyroid hormone (PTH) in regulating canine proximal tubular basolateral membrane Na+-Ca2+ exchange and passive calcium permeability was assessed, as was the nature of this passive calcium permeability. Dibutyryl cAMP (50 mg) infused in vivo over 30 min increased fractional phosphate excretion from 4.9 +/- 1.8% to 20.5 +/- 4.6%, P less than 0.05, n = 6, but had no effect on either passive Ca2+ efflux or sodium-stimulated Ca2+ efflux from Ca2+-preloaded basolateral membrane vesicles (BLMV). Both of these mechanisms have been previously shown to be stimulated by PTH. Further studies were performed to investigate the mechanism of the passive calcium flux. Calcium uptake by BLMV was blocked by lanthanum (La3+) but not by the calcium-channel blocker verapamil. La3+ blocked efflux of Ca2+ from preloaded vesicles when it was placed in the external solution. This La3+-blockable efflux was larger in potassium equivalent BLMV prepared from normal dogs than in BLMV prepared from thyroparathyroidectomized dogs. Benzamil produced 50% inhibition of sodium-stimulated Ca2+ uptake at 250 microM whereas neither amiloride nor diltiazem achieved 50% inhibition at the maximal doses studied. Benzamil, 1 mM, had no effect on passive calcium efflux and neither did the substitution of sucrose for potassium, which has been shown to affect Ca2+-Ca2+ exchange by the Na+-Ca2+ exchanger. This suggests that the calcium flux under potassium equivalent conditions was not mediated by Ca2+-Ca2+ exchange by the Na+-Ca2+ exchanger. These results demonstrate that the basolateral membrane of proximal tubular cells possesses both a Na+-Ca2+ exchanger inhibitable by benzamil and a passive calcium permeability not inhibited by benzamil nor by verapamil but by La3+. Neither of these two mechanisms of calcium flux was affected by dibutyryl cAMP whereas both have been shown to be stimulated by PTH.     

Effects of intracellular signals on Na+/K(+)-ATPase pump activity in the frog skin epithelium.

The effects of intracellular signals (pHi, Na+i, Ca2+i, and the electrical membrane potential), on Na+ transport mediated by the Na+/K+ pump were investigated in the isolated Rana esculenta frog skin. In particular we focussed on pHi sensitivity since protons act as an intrinsic regulator of transepithelial Na+ transport (JNa) by a simultaneous control of the apical membrane Na+ conductance (gNa) and the basolateral membrane K+ conductance (gK). pHi changes which modify JNa, gNa and gK, do not affect the Na+ transport mediated by the pump as shown by kinetic and electrophysiological studies. In addition, no changes were observed in the number of 3H-ouabain binding sites in acid-loaded epithelia. Our attempts to modify cellular Ca2+ (by using Ca(2+)-free/EGTA Ringer solution or A23187 addition) also failed to produce any significant effects in the Na+ pump turnover rate or the number of 3H-ouabain binding sites. The Na+ pump current was found to be sensitive to the basolateral membrane potential, saturating for very positive (cell) potentials and a reversal potential of -160 mV was calculated from I-V relationships of the pump. Changes in Na+i considerably affected the Na+ pump rate. A saturating relationship was found between pump rate and Nai+ with maximal activation at Nai+ greater than 40 mmol/l; a high dependence of the pump rate and of the number of 3H-ouabain binding sites was observed in the physiological range of Nai+. We conclude that protons (in the physiological pH range) which act directly and simultaneously on the passive transport pathways (gNa and gK), have no direct effect on the Na+/K+ pump rate. After an acid load, the inhibition of JNa is primarily due to the reduction of gNa. This results in a reduction of Nai and the pump turnover rate then becomes dependent on other pathways of Na+ entry such as the basolateral membrane Na+/H+ exchanger.     

Coupling of volume and Na+ transport in frog skin epithelium

Whole skins and isolated epithelia were bathed with isotonic media (congruent to 244 mOsm) containing sucrose or glucose. The serosal osmolality was intermittently reduced (congruent to 137 mOsm) by removing the nonelectrolyte. Transepithelial and intracellular electrophysiological parameters were monitored while serosal osmolality was changed. Serosal hypotonicity increased the short-circuit current (ISC) and the basolateral conductance, hyperpolarized the apical membrane (psi mc), and increased the intracellular Na+ concentration. The increases in apical conductance and apical Na+ permeability (measured from Goldman fits of the relationship between amiloride-sensitive current and psi mc) were not statistically significant. To verify that the osmotically induced changes in ISC were mediated primarily at the basolateral membrane, the basolateral membrane potential of the experimental area was clamped close to 0 mV by replacing the serosal Na+ with K+ in Cl--free media. The adjoining control area was exposed to serosal Na+. Serosal hypotonicity produced a sustained stimulation of ISC across the control, but not across the adjoining depolarized tissue area. The current results support the concept that hypotonic cell swelling increases Na+ transport across frog skin epithelium by increasing the basolateral K+ permeability, hyperpolarizing the apical membrane, and increasing the electrical driving force for apical Na+ entry.     

Cyclic AMP inhibits Na+/H+ exchange at the apical membrane of Necturus gallbladder epithelium

The effects of elevating intracellular cAMP levels on Na+ transport across the apical membrane of Necturus gallbladder epithelium were studied by intracellular and extracellular microelectrode techniques. Intracellular cAMP was raised by serosal addition of the phosphodiesterase inhibitor theophylline (3 mM) or mucosal addition of either 8-Br-cAMP (1 mM) or the adenylate cyclase activator forskolin (10 microM). During elevation of intracellular cAMP, intracellular Na+ activity (alpha Nai) and intracellular pH (pHi) decreased significantly. In addition, acidification of the mucosal solution, which contained either 100 or 10 mM Na+, was inhibited by approximately 50%. The inhibition was independent of the presence of Cl- in the bathing media. The rates of change of alpha Nai upon rapid alterations of mucosal [Na+] from 100 to 10 mM and from 10 to 100 mM were both decreased, and the rate of pHi recovery upon acid loading was also reduced by elevated cAMP levels. Inhibition was approximately 50% for all of these processes. These results indicate that cAMP inhibits apical membrane Na+/H+ exchange. The results of measurements of pHi recovery at 10 and 100 mM mucosal [Na+] and a kinetic analysis of recovery as a function of pHi suggest that the main or sole mechanism of the inhibitory effect of cAMP is a reduction in the maximal rate of acid extrusion. In conjunction with the increase in apical membrane electrodiffusional Cl- permeability, produced by cAMP, which causes a decrease in net Cl- entry (Petersen, K.-U., and L. Reuss, 1983, J. Gen. Physiol., 81:705), inhibition of Na+/H+ exchange contributes to the reduction of fluid absorption elicited by this agent. Similar mechanisms may account for the effects of cAMP in other epithelia with similar transport properties. It is also possible that inhibition of Na+/H+ exchange by cAMP plays a role in the regulation of pHi in other cell types.     

Apical adenosine regulates basolateral Ca2+-activated potassium channels in human airway Calu-3 epithelial cells

In airway epithelial cells, apical adenosine regulates transepithelial anion secretion by activation of apical cystic fibrosis transmembrane conductance regulator (CFTR) via adenosine receptors and cAMP/PKA signaling. However, the potent stimulation of anion secretion by adenosine is not correlated with its modest intracellular cAMP elevation, and these uncorrelated efficacies have led to the speculation that additional signaling pathways may be involved. Here, we showed that mucosal adenosine-induced anion secretion, measured by short-circuit current (Isc), was inhibited by the PLC-specific inhibitor U-73122 in the human airway submucosal cell line Calu-3. In addition, the Isc was suppressed by BAPTA-AM (a Ca2+ chelator) and 2-aminoethoxydiphenyl borate (2-APB; an inositol 1,4,5-trisphosphate receptor blocker), but not by PKC inhibitors, suggesting the involvement of PKC-independent PLC/Ca2+ signaling. Ussing chamber and patch-clamp studies indicated that the adenosine-induced PLC/Ca2+ signaling stimulated basolateral Ca2+-activated potassium (KCa) channels predominantly via A2B adenosine receptors and contributed substantially to the anion secretion. Thus, our data suggest that apical adenosine activates contralateral K+ channels via PLC/Ca2+ and thereby increases the driving force for transepithelial anion secretion, synergizing with its modulation of ipsilateral CFTR via cAMP/PKA. Furthermore, the dual activation of CFTR and KCa channels by apical adenosine resulted in a mixed secretion of chloride and bicarbonate, which may alter the anion composition in the secretion induced by secretagogues that elicit extracellular ATP/adenosine release. Our findings provide novel mechanistic insights into the regulation of anion section by adenosine, a key player in the airway surface liquid homeostasis and mucociliary clearance.     

Adenosine stimulates anion secretion across cultured and native adult human vas deferens epithelia

Experiments were conducted to determine the responsiveness of human vas deferens epithelial cell monolayers to adenosine and related agonists. Human abdominal vas deferens epithelial cells have been isolated from adult tissues and grown to confluence on permeable supports. All cells exhibit intense ZO-1 and cytokeratin immunoreactivity. Cultured cell monolayers exhibit high electrical resistance with a lumen-negative potential difference and short circuit current (I(sc)) indicative of anion secretion and/or cation absorption. A portion of the basal I(sc) is inhibited by amiloride. Amiloride-sensitive I(sc) is enhanced by exposure to glucocorticoids and is Na(+) dependent, indicating the presence of epithelial sodium channel-mediated Na(+) absorption. Epithelial anion secretion and intracellular generation of cAMP are acutely stimulated by adenosine and the adenosine receptor agonist 5'-(N-ethylcarboxamido)adenosine (NECA), with these effects being fully blocked by 8-phenyltheophylline. Adenosine receptors are localized to the apical membrane of the epithelial cells, as basolateral adenosine is without effect. Freshly excised human vas deferens recapitulate observations made on cultured epithelia when evaluated with the self-referencing vibrating probe: amiloride inhibition of basal ion transport, stimulation by adenosine, and inhibition by 8-phenyltheophyline. These results demonstrate that adult human vas deferens epithelium actively transports ions to generate the luminal environment of the deferent duct. Thus, vas deferens epithelium likely plays an active role in male fertility, and interventions that modulate epithelial function might be exploited to treat male-factor infertility or in contraception.     

Cyclic AMP modulation of ion transport across frog retinal pigment epithelium. Measurements in the short-circuit state

In the frog retinal pigment epithelium (RPE), the cellular levels of cyclic AMP (cAMP) were measured in control conditions and after treatment with substances that are known to inhibit phosphodiesterase (PDE) activity (isobutyl-1-methylxanthine, SQ65442) or stimulate adenylate cyclase activity (forskolin). The cAMP levels were elevated by a factor of 5-7 compared with the controls in PDE-treated tissues and by a factor of 18 in forskolin-treated tissues. The exogenous application of cAMP (1 mM), PDE inhibitors (0.5 mM), or forskolin (0.1 mM) all produced similar changes in epithelial electrical parameters, such as transepithelial potential (TEP) and resistance (Rt), as well as changes in active ion transport. Adding 1 mM cAMP to the solution bathing the apical membrane transiently increased the short-circuit current (SCC) and the TEP (apical side positive) and decreased Rt. Microelectrode experiments showed that the elevation in TEP is due mainly to a depolarization of the basal membrane followed by, and perhaps also accompanied by, a smaller hyperpolarization of the apical membrane. The ratio of the apical to the basolateral membrane resistance increased in the presence of cAMP, and this increase, coupled with the decrease in Rt and the basolateral membrane depolarization, is consistent with a conductance increase at the basolateral membrane. Radioactive tracer experiments showed that cAMP increased the active secretion of Na (choroid to retina) and the active absorption of K (retina to choroid). Cyclic AMP also abolished the active absorption of Cl across the RPE. In sum, elevated cellular levels of cAMP affect active and passive transport mechanisms at the apical and basolateral membranes of the bullfrog RPE.     

An analytical model of ionic movements in airway epithelial cells.

A new mathematical model of ion movements in airway epithelia is presented, which allows predictions of ion fluxes, membrane potentials and ion concentrations. The model includes sodium and chloride channels in the apical membrane, a Na/K pump and a cotransport system for Cl- with stoichiometry Na+:K+:2Cl- in the basolateral membrane. Potassium channels in the basolateral membrane are used to regulate cell volume. Membrane potentials, ion fluxes and intracellular ion concentration are calculated as functions of apical ion permeabilities, the maximum pump current and the cotransport parameters. The major predictions of the model are: (1) Cl- concentration in the cell is determined entirely by the intracellular concentration of negatively charged impermeable ions and the osmotic conditions; (2) changes in intracellular Na+ and K+ concentrations are inversely related; (3) cotransport provides the major driving force for Cl- flux, increases intracellular Na+ concentration, decreases intracellular K+ concentration and hyperpolarizes the cell interior; (4) the maximum rate of the Na/K pump, by contrast, has little effect on Na+ or Cl- transepithelial fluxes and a much less pronounced effect on cell membrane polarization; (5) an increase in apical Na+ permeability causes an increase in intracellular Na+ concentration and a significant increase in Na+ flux; (6) an increase in apical Cl- permeability decreases intracellular Na+ concentration and Na+ flux; (7) assuming Na+ and Cl- permeabilities equal to those measured in human nasal epithelia, the model predicts that under short circuit conditions, Na+ absorption is much higher than Cl- secretion, in agreement with experimental measurements.     

Agonist-induced polarized trafficking and surface expression of the adenosine 2b receptor in intestinal epithelial cells: role of SNARE proteins

Adenosine, acting through the A2b receptor, induces vectorial chloride and IL-6 secretion in intestinal epithelia and may play an important role in intestinal inflammation. We have previously shown that apical or basolateral adenosine receptor stimulation results in the recruitment of the A2b receptor to the plasma membrane. In this study, we examined domain specificity of recruitment and the role of soluble N-ethylmaleimide (NEM) attachment receptor (SNARE) proteins in the agonist-mediated recruitment of the A2b receptor to the membrane. The colonic epithelial cell line T84 was used because it only expresses the A2b-subtype adenosine receptor. Cell fractionation, biotinylation, and electron microscopic studies showed that the A2b receptor is intracellular at rest and that apical or basolateral adenosine stimulation resulted in the recruitment of the receptor to the apical membrane. Upon agonist stimulation, the A2b receptor is enriched in the vesicle fraction containing vesicle-associated membrane protein (VAMP)-2. Furthermore, in cells stimulated with apical or basolateral adenosine, we demonstrate a complex consisting of VAMP-2, soluble NEM-sensitive factor attachment protein (SNAP)-23, and A2b receptor that is coimmunoprecipitated in cells stimulated with adenosine within 5 min and is no longer detected within 15 min. Inhibition of trafficking with NEM or nocodazole inhibits cAMP synthesis induced by apical or basolateral adenosine by 98 and 90%, respectively. cAMP synthesis induced by foskolin was not affected, suggesting that generalized signaling is not affected under these conditions. Collectively, our data suggest that 1) the A2b receptor is intracellular at rest; 2) apical or basolateral agonist stimulation induces recruitment of the A2b receptor to the apical membrane; 3) the SNARE proteins, VAMP-2 and SNAP-23, participate in the recruitment of the A2b receptor; and 4) the SNARE-mediated recruitment of the A2b receptor may be required for its signaling.     

Effect of a NaCl gradient on the transport of para-aminohippuric acid into the vesicles of the basolateral membrane of the kidney cortex

Basolateral membrane vesicles were isolated from the rat kidney cortex by a modified method of cation precipitation. Different steps of preparation were analysed using the marker enzymes: Na+,K+-ATPase (for basolateral membrane), alkaline phosphatase (for apical membrane), glucose-6-phosphatase (for membranes of endoplasmic reticulum) and succinate dehydrogenase (for mitochondria). The basolateral membrane was purified by a 8-9-fold treatment with Na+,K+-ATPase, while other membrane contaminations were as low as 2% (as compared to homogenate). The transport of 3H-p-aminohippurate (3H-PAH) by basolateral membrane vesicles was measured under different experimental conditions. The 3H-PAH uptake was found to be Na-gradient dependent. The initial rate of 3H-PAH uptake in the presence of NaCl gradient (500 pM/mg X min) was higher than without the gradient (88 pM/mg X min). It is concluded that the PAH transfer across the basolateral membrane may be energized by the Na+ chemical gradient.