Characterization and imaging of A6 epithelial cell clones expressing fluorescently labeled ENaC subunits.

A6 model renal epithelial cells were stably transfected with enhanced green fluorescent protein (EGFP)-tagged alpha- or beta-subunits of the epithelial Na(+) channel (ENaC). Transfected RNA and proteins were both expressed in low abundance, similar to the endogenous levels of ENaC in native cells. In living cells, laser scanning confocal microscopy revealed a predominantly subapical distribution of EGFP-labeled subunits, suggesting a readily accessible pool of subunits available to participate in Na(+) transport. The basal level of Na(+) transport in the clonal lines was enhanced two- to fourfold relative to the parent line. Natriferic responses to insulin or aldosterone were similar in magnitude to the parent line, while forskolin-stimulated Na(+) transport was 64% greater than control in both the alpha- and beta-transfected lines. In response to forskolin, EGFP-labeled channel subunits traffic to the apical membrane. These data suggest that channel regulators, not the channel per se, form the rate-limiting step in response to insulin or aldosterone stimulation, while the number of channel subunits is important for basal as well as cAMP-stimulated Na(+) transport.

Glucocorticoid treatment increases inhibitory m(2) muscarinic receptor expression and function in the airways.

M(2) muscarinic receptors on parasympathetic nerve endings inhibit acetylcholine release in the airways. In this study, the effects of dexamethasone on M(2) receptors in vivo and in primary cultures of airway parasympathetic neurons were tested. Treating guinea pigs with dexamethasone (0.1 mg/kg, daily for 2 d) substantially increased inhibitory M(2) muscarinic receptor function, decreasing airway responsiveness to electrical stimulation of the vagi. At the same time, dexamethasone decreased the response to acetylcholine but not to methacholine, suggesting that cholinesterase activity was increased. When both cholinesterase and M(2) receptors were blocked (using physostigmine and gallamine, respectively) vagally induced bronchoconstriction was increased to control values. In primary cultures of airway parasympathetic neurons, dexamethasone significantly decreased the release of acetylcholine in response to electrical stimulation. Blocking inhibitory M(2) receptors using atropine (10(-5) M) increased acetylcholine release. After the M(2) receptors were blocked there was no difference in acetylcholine release between control and dexamethasone-treated cultures. M(2) receptor gene expression was increased by more than fivefold in dexamethasone-treated cultures. Immunostaining of dexamethasone-treated neurons demonstrated more intense staining. Thus, decreased vagally mediated reflex bronchoconstriction after glucocorticoid treatment may be the result on increased M(2) receptor expression and function as well as increased degradation of acetylcholine by cholinesterase.

LY-294002-inhibitable PI 3-kinase and regulation of baseline rates of Na(+) transport in A6 epithelia.

Blocker-induced noise analysis of epithelial Na(+) channels (ENaCs) was used to investigate how inhibition of an LY-294002-sensitive phosphatidylinositol 3-kinase (PI 3-kinase) alters Na(+) transport in unstimulated and aldosterone-prestimulated A6 epithelia. From baseline Na(+) transport rates (I(Na)) of 4.0 +/- 0.1 (unstimulated) and 9.1 +/- 0.9 microA/cm(2) (aldosterone), 10 microM LY-294002 caused, following a relatively small initial increase of transport, a completely reversible inhibition of transport within 90 min to 33 +/- 6% and 38 +/- 2% of respective baseline values. Initial increases of transport could be attributed to increases of channel open probability (P(o)) within 5 min to 143 +/- 17% (unstimulated) and 142 +/- 10% of control (aldosterone) from baseline P(o) averaging near 0.5. Inhibition of transport was due to much slower decreases of functional channel densities (N(T)) to 28 +/- 4% (unstimulated) and 35 +/- 3% (aldosterone) of control at 90 min. LY-294002 (50 microM) caused larger but completely reversible increases of P(o) (215 +/- 38% of control at 5 min) and more rapid but only slightly larger decreases of N(T). Basolateral exposure to LY-294002 induced no detectable effect on transport, P(o) or N(T). We conclude that an LY-294002-sensitive PI 3-kinase plays an important role in regulation of transport by modulating N(T) and P(o) of ENaCs, but only when presented to apical surfaces of the cells.

Effects of neurokinin receptor antagonists in virus-infected airways.

We investigated the effects of a neurokinin-1 (NK(1)) receptor antagonist (SR-140333) and a NK(2) receptor antagonist (SR-48968) on airway responsiveness and on the function of neuronal M(2) muscarinic receptors, which normally inhibit vagal acetylcholine release, in guinea pigs infected with parainfluenza virus. Antagonists were given 1 h before infection and daily thereafter. Four days later, bronchoconstriction induced by either intravenous histamine (which is partly vagally mediated) or electrical stimulation of the vagus nerves was increased by viral infection compared with control. In addition, the ability of the muscarinic agonist pilocarpine to inhibit vagally induced bronchoconstriction was lost in virus-infected animals, demonstrating loss of neuronal M(2) receptor function. Macrophage influx into the lungs was inhibited by pretreatment with both antagonists. However, only the NK(1) receptor antagonist prevented M(2) receptor dysfunction and inhibited hyperresponsiveness (measured as an increase in either vagally induced or histamine-induced bronchoconstriction). Thus virus-induced M(2) receptor dysfunction and hyperresponsiveness are prevented by a NK(1) receptor antagonist, but not by a NK(2) receptor antagonist, whereas both antagonists had similar anti-inflammatory effects.

Characterization of the ion transport responses to ADH in the MDCK-C7 cell line.

The Madin-Darby canine kidney (MDCK) cell line expresses many characteristics of the renal collecting duct. The MDCK-C7 subclone forms a high-resistance, hormone-responsive model of the principal cells, which are found in distal sections of the renal tubule. The electrophysiological technique of short-circuit current measurement was used to examine the response to antidiuretic hormone (ADH) in the MDCK-C7 clone. Three discrete electrogenic ion transport phenomena can be distinguished temporally and by the use of inhibitors and effectors. Initially the cells exhibit anion secretion through the cystic fibrosis transmembrane conductance regulator (CFTR). The presence of CFTR was confirmed by immunoprecipitation followed by Western blotting. The CFTR-mediated anion secretion is transient and is followed, in time, by a verapamil- and Ba(+)-sensitive anion secretion or cation absorption and, finally, by Na+ reabsorption via epithelial Na+ channels (ENaC). In contrast to other studies of MDCK cells, we see no indication that the presence of CFTR functionally inhibits ENaC. The characterization of the various ion transport phenomena substantiates this cell line as a model renal epithelium that can be used to study the hormonal and metabolic regulation of ion transport.

Ovalbumin sensitization changes the inflammatory response to subsequent parainfluenza infection. Eosinophils mediate airway hyperresponsiveness, m(2) muscarinic receptor dysfunction, and antiviral effects.

Asthma exacerbations, many of which are virus induced, are associated with airway eosinophilia. This may reflect altered inflammatory response to viruses in atopic individuals. Inhibitory M(2) muscarinic receptors (M(2)Rs) on the airway parasympathetic nerves limit acetylcholine release. Both viral infection and inhalational antigen challenge cause M(2)R dysfunction, leading to airway hyperresponsiveness. In antigen-challenged, but not virus-infected guinea pigs, M(2)R dysfunction is due to blockade of the receptors by the endogenous antagonist eosinophil major basic protein (MBP). We hypothesized that sensitization to a nonviral antigen before viral infection alters the inflammatory response to viral infection, so that M(2)R dysfunction and hyperreactivity are eosinophil mediated. Guinea pigs were sensitized to ovalbumin intraperitoneally, and 3 wk later were infected with parainfluenza. In sensitized, but not in nonsensitized animals, virus-induced hyperresponsiveness and M(2)R dysfunction were blocked by depletion of eosinophils with antibody to interleukin (IL)-5 or treatment with antibody to MBP. An additional and unexpected finding was that sensitization to ovalbumin caused a marked (80%) reduction in the viral content of the lungs. This was reversed by the antibody to IL-5, implicating a role for eosinophils in viral immunity.

Ozone-induced hyperresponsiveness and blockade of M2 muscarinic receptors by eosinophil major basic protein.

Control of airway smooth muscle is provided by parasympathetic nerves that release acetylcholine onto M(3) muscarinic receptors. Acetylcholine release is limited by inhibitory M(2) muscarinic receptors. In antigen-challenged guinea pigs, hyperresponsiveness is due to blockade of neuronal M(2) receptors by eosinophil major basic protein (MBP). Because exposure of guinea pigs to ozone also causes M(2) dysfunction and airway hyperresponsiveness, the role of eosinophils in ozone-induced hyperresponsiveness was tested. Animals were exposed to filtered air or to 2 parts/million ozone for 4 h. Twenty-four hours later, the muscarinic agonist pilocarpine no longer inhibited vagally induced bronchoconstriction in ozone-exposed animals, indicating M(2) dysfunction. M(2) receptor function in ozone-exposed animals was protected by depletion of eosinophils with antibody to interleukin-5 and by pretreatment with antibody to guinea pig MBP. M(2) function was acutely restored by removal of MBP with heparin. Ozone-induced hyperreactivity was also prevented by antibody to MBP and was reversed by heparin. These data show that loss of neuronal M(2) receptor function after ozone is due to release of eosinophil MBP.

Phosphoinositide 3-kinase is required for aldosterone-regulated sodium reabsorption.

Aldosterone, a steroid hormone, regulates renal Na+ reabsorption and, therefore, plays an important role in the maintenance of salt and water balance. In a model renal epithelial cell line (A6) we have found that phosphoinositide 3-kinase (PI 3-kinase) activity is required for aldosterone-stimulated Na+ reabsorption. Inhibition of PI 3-kinase by the specific inhibitor LY-294002 markedly reduces both basal and aldosterone-stimulated Na+ transport. Further, one of the products of PI 3-kinase, phosphatidylinositol 3,4,5-trisphosphate, is increased in response to aldosterone in intact A6 monolayers. This increase occurs just before the manifestation of the functional effect of the hormone and is also inhibited by LY-294002. With the use of blocker-induced noise analysis, it has been demonstrated that inhibition of phosphoinositide formation causes an inhibition of Na+ entry in both control and aldosterone-pretreated cultures by reducing the number of open functional epithelial Na+ channels (ENaCs) in the apical membrane of the A6 cells. These novel observations indicate that phosphoinositides are required for ENaC expression and suggest a mechanism for aldosterone regulation of channel function.

Antigen-induced hyperreactivity to histamine: role of the vagus nerves and eosinophils.

M2 muscarinic receptors limit acetylcholine release from the pulmonary parasympathetic nerves. M2 receptors are dysfunctional in antigen-challenged guinea pigs, causing increased vagally mediated bronchoconstriction. Dysfunction of these M2 receptors is due to eosinophil major basic protein, which is an antagonist for M2 receptors. Histamine-induced bronchoconstriction is composed of a vagal reflex in addition to its direct effect on airway smooth muscle. Because hyperreactivity to histamine is seen in antigen-challenged animals, we hypothesized that hyperreactivity to histamine may be due to increased vagally mediated bronchoconstriction caused by dysfunction of M2 receptors. In anesthetized, antigen-challenged guinea pigs, histamine-induced bronchoconstriction was greater than that in control guinea pigs. After vagotomy or atropine treatment, the response to histamine in antigen-challenged animals was the same as that in control animals. In antigen-challenged animals, blockade of eosinophil influx into the airways or neutralization of eosinophil major basic protein prevented the development of hyperreactivity to histamine. Thus hyperreactivity to histamine in antigen-challenged guinea pigs is vagally mediated and dependent on eosinophil major basic protein.

Effects of inflammatory cells on neuronal M2 muscarinic receptor function in the lung.

In the lungs, acetylcholine released from the parasympathetic nerves stimulates M3 muscarinic receptors on airway smooth muscle inducing contraction and bronchoconstriction. The amount of acetylcholine released from these nerves is limited locally by neuronal M2 muscarinic receptors. These neuronal receptors are dysfunctional in asthma and in animal models of asthma. Decreased M2 muscarinic receptor function results in increased release of acetylcholine and in airway hyperreactivity. Inflammation has long been associated with hyperreactivity and the role of inflammatory cells in loss of neuronal M2 receptor function has been examined. There are several different mechanisms for loss of neuronal M2 receptor function. These include blockade by endogenous antagonists such as eosinophil major basic protein, decreased expression of M2 receptors following infection with viruses or exposure to pro inflammatory cytokines such as gamma interferon. Finally, the affinity of acetylcholine for these receptors can be decreased by exposure to neuraminidase.

Hormonal regulation of ENaCs: insulin and aldosterone.

Although a variety of hormones and other agents modulate renal Na+ transport acting by way of the epithelial Na+ channel (ENaC), the mode(s), pathways, and their interrelationships in regulation of the channel remain largely unknown. It is likely that several hormones may be present concurrently in vivo, and it is, therefore, important to understand potential interactions among the various regulatory factors as they interact with the Na+ transport pathway to effect modulation of Na+ reabsorption in distal tubules and other native tissues. This study represents specifically a determination of the interaction between two hormones, namely, aldosterone and insulin, which stimulate Na+ transport by entirely different mechanisms. We have used a noninvasive pulse protocol of blocker-induced noise analysis to determine changes in single-channel current (iNa), channel open probability (Po), and functional channel density (NT) of amiloride-sensitive ENaCs at various time points following treatment with insulin for 3 h of unstimulated control and aldosterone-pretreated A6 epithelia. Independent of threefold differences of baseline values of transport caused by aldosterone, 20 nM insulin increased by threefold and within 10-30 min the density of the pool of apical membrane ENaCs (NT) involved in transport. The very early (10 min) increases of channel density were accompanied by relatively small decreases of iNa (10-20%) and decreases of p.o. (28%) in the aldosterone-pretreated tissues but not the control unstimulated tissues. The early changes of iNa, p.o., and NT were transient, returning very slowly over 3 h toward their respective control values at the time of addition of insulin. We conclude that aldosterone and insulin act independently to stimulate apical Na+ entry into the cells of A6 epithelia by increase of channel density.

Time-dependent stimulation by aldosterone of blocker-sensitive ENaCs in A6 epithelia.

To study and define the early time-dependent response (< or = 6 h) of blocker-sensitive epithelial Na+ channels (ENaCs) to stimulation of Na+ transport by aldosterone, we used a new modified method of blocker-induced noise analysis to determine the changes of single-channel current (iNa) channel open probability (Po), and channel density (NT) under transient conditions of transport as measured by macroscopic short-circuit currents (Isc). In three groups of experiments in which spontaneous baseline rates of transport averaged 1.06, 5.40, and 15.14 microA/cm2, stimulation of transport occurred due to increase of blocker-sensitive channels. NT varied linearly over a 70-fold range of transport (0.5-35 microA/cm2). Relatively small and slow time-dependent but aldosterone-independent decreases of Po occurred during control (10-20% over 2 h) and aldosterone experimental periods (10-30% over 6 h). When the Po of control and aldosterone-treated tissues was examined over the 70-fold extended range of Na+ transport, Po was observed to vary inversely with Isc, falling from approximately 0.5 to approximately 0.15 at the highest rates of Na+ transport or approximately 25% per 3-fold increase of transport. Because decreases of Po from any source cannot explain stimulation of transport by aldosterone, it is concluded that the early time-dependent stimulation of Na+ transport in A6 epithelia is due exclusively to increase of apical membrane NT.

Phosphatidylinositol 3-kinase activation is required for insulin-stimulated sodium transport in A6 cells.

Insulin stimulates amiloride-sensitive sodium transport in models of the distal nephron. Here we demonstrate that, in the A6 cell line, this action is mediated by the insulin receptor tyrosine kinase and that activation of phosphatidylinositol 3-kinase (PI 3-kinase) lies downstream of the receptor tyrosine kinase. Functionally, a specific inhibitor of PI 3-kinase, LY-294002, blocks basal as well as insulin-stimulated sodium transport in a dose-dependent manner (IC50 approximately 6 microM). Biochemically, PI 3-kinase is present in A6 cells and is inhibited both in vivo and in vitro by LY-294002. Furthermore, a subsequent potential downstream signaling element, pp70 S6 kinase, is activated in response to insulin but does not appear to be part of the pathway involved in insulin-stimulated sodium transport. Together with previous reports, these results suggest that insulin may induce the exocytotic insertion of sodium channels into the apical membrane of A6 cells in a PI 3-kinase-mediated manner.

Protein prenylation is required for aldosterone-stimulated Na+ transport.

Aldosterone stimulation of transcellular Na+ flux in polarized epithelial cells is dependent on at least one transmethylation reaction, but the substrate of this signaling step is unknown. Because it is clear that the majority of cellular protein methylation occurs in conjunction with protein prenylation, we examined the importance of prenylation to aldosterone-stimulated Na+ transport in the A6 cell line. Lovastatin, an inhibitor of the first committed step of the mevalonate pathway, inhibits the natriferic effect of aldosterone but does not inhibit insulin-stimulated Na+ flux. The addition of a farnesyl group does not appear to be involved in aldosterone's action. Neither alpha-hydroxyfarne-sylphosphonic acid, an inhibitor of farnesyl:protein transferase, nor N-acetyl-S-farnesyl-L-cysteine, an inhibitor of farnesylated protein methylation, inhibits the hormone-induced increase in Na+ transport. In contrast, N-acetyl-S-geranyl-geranyl-L-cysteine, an inhibitor of geranylgeranyl protein methylation, completely abolishes the aldosterone-induced increase in Na+ flux with no effect on insulin-mediated Na+ transport or cellular protein content. These data indicate that methylation of a geranylgeranylated protein is involved in aldosterone's natriferic action.

Antibody to VLA-4, but not to L-selectin, protects neuronal M2 muscarinic receptors in antigen-challenged guinea pig airways.

Antigen challenge of sensitized guinea pigs decreases the function of inhibitory M2 muscarinic autoreceptors on parasympathetic nerves in the lung, potentiating vagally induced bronchoconstriction. Loss of M2 receptor function is associated with the accumulation of eosinophils around airway nerves. To determine whether recruitment of eosinophils via expression of VLA-4 and L-selectin is critical for loss of M2 receptor function, guinea pigs were pretreated with monoclonal antibodies to VLA-4 (HP1/2) or L-selectin (LAM1-116). Guinea pigs were sensitized and challenged with ovalbumin, and M2 receptor function was tested. In controls, blockade of neuronal M2 muscarinic receptors by gallamine potentiated vagally induced bronchoconstriction, while in challenged animals this effect was markedly reduced, confirming M2 receptor dysfunction. Pretreatment with HP1/2, but not with LAM1-116, protected M2 receptor function in the antigen-challenged animals. HP1/2 also inhibited the development of hyperresponsiveness, and selectively inhibited accumulation of eosinophils in the lungs as measured by lavage and histology. Thus, inhibition of eosinophil influx into the lungs protects the function of M2 muscarinic receptors, and in so doing, prevents hyperresponsiveness in antigen-challenged guinea pigs.

The amiloride-sensitive epithelial Na+ channel: binding sites and channel densities.

The amiloride-sensitive Na+ channel found in many transporting epithelia plays a key role in regulating salt and water homeostasis. Both biochemical and biophysical approaches have been used to identify, characterize, and quantitate this important channel. Among biophysical methods, there is agreement as to the single-channel conductance and gating kinetics of the highly selective Na+ channel found in native epithelia. Amiloride and its analogs inhibit transport through the channel by binding to high-affinity ligand-binding sites. This characteristic of high-affinity binding has been used biochemically to quantitate channel densities and to isolate presumptive channel proteins. Although the goals of biophysical and biochemical experiments are the same in elucidating mechanisms underlying regulation of Na+ transport, our review highlights a major quantitative discrepancy between methods in estimation of channel densities involved in transport. Because the density of binding sites measured biochemically is three to four orders of magnitude in excess of channel densities measured biophysically, it is unlikely that high-affinity ligand binding can be used physiologically to quantitate channel densities and characterize the channel proteins.

Aldosterone-induced proteins in primary cultures of rabbit renal cortical collecting system.

Primary cultures of immunodissected cells from rabbit kidney connecting tubule and cortical collecting duct were used to study aldosterone's action on transcellular Na+ flux. Incubation with 10(-7) M aldosterone stimulated transcellular Na+ transport which was detected as an increase in benzamil-sensitive short-circuit current. The stimulatory response was consistently noted after 2 h of incubation and stabilized after 6 h. 2D-PAGE was used to identify proteins which were induced concurrently with the increase in transcellular Na+ flux after an aldosterone incubation of 15 h. Three aldosterone-induced proteins (AIPs; M(r) = 100, 70-77 and 46-50 kDa) were found in the membrane and microsomal fractions. Two of these appeared to have more than one isoform. A single heterogeneous AIP (M(r) = 77 kDa) was detected in the soluble fraction.

Aldosterone and insulin stimulate amiloride-sensitive sodium transport in A6 cells by additive mechanisms.

The individual effects of aldosterone and insulin on amiloride-sensitive Na+ transport in model renal epithelia have been well characterized. However, in the physiological state, many hormones are present concurrently and their interactions need to be addressed. We have found that, over 5 h, the effects of insulin and aldosterone are additive. This indicates that the biochemical pathways are largely independent. To delineate the signaling pathways, we examined the requirement for tyrosine kinases by using genistein, a tyrosine kinase inhibitor. Genistein blocks basal (constitutive) Na+ transport and inhibits insulin- and aldosterone-stimulated Na+ transport. From these results, we conclude that a tyrosine phosphorylation is an important component of amiloride-sensitive Na+ transport.

Characterization of hormone-stimulated Na+ transport in a high-resistance clone of the MDCK cell line.

The Madin-Darby canine kidney (MDCK) cell line forms an epithelial monolayer which expresses many of the morphological and functional properties of the renal collecting duct. The C7 subclone of the parent line forms an epithelium which expresses many of the characteristics of principal cells. The MDCK-C7 subclone forms a high-resistance epithelium that is capable of vectorial ion transport. We have found that this epithelium responds to aldosterone, antidiuretic hormone (ADH) and insulin like growth factor 1 (IGF1) with increases in amiloride-sensitive Na+ transport. The responses to aldosterone and ADH follow time-courses that are consistent with the action of these hormones in vivo. This is the first demonstration of IGF1-induced Na+ reabsorption in a mammalian model system. Interestingly, a maximal response to any one of these natriferic factors does not inhibit a subsequent response to another hormone. These studies indicate that the C7 subclone retains many of the natriferic responses of the native principal cells and is an ideal model for studying hormonal modulation of Na+ transport.

Differential effects of brefeldin A on hormonally regulated Na+ transport in a model renal epithelial cell line.

Na+ transport in renal epithelia is regulated by a wide variety of endogenous and exogenous cellular factors. Although most natriferic agents have an action on the amiloride-sensitive Na+ channel, the biochemical pathways which precede activation of the channel remain incompletely defined. One approach to dissecting such intricate pathways is to perturb a specific cellular process and determine its importance in the postulated mechanism. The current studies examine the effect of brefeldin A (BFA), an inhibitor of the central vacuolar system, on basal as well as aldosterone-, insulin-, and forskolin-stimulated Na+ transport. In the A6 cell line, BFA had a time-dependent effect on basal transport. Aldosterone-induced Na+ transport was sensitive to BFA while insulin's action was only partially blocked and forskolin-stimulated Na+ transport was relatively resistant to the action of the inhibitor. These studies highlight differences as well as points of convergence in the natriferic pathways.