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During fluid absorption induced by amphotericin B, the lateral intercellular spaces (LIS) of the dog tracheal epithelium were widely dilated as compared to untreated time controls. When fluid absorption was inhibited by ouabain, or by replacement of luminal Na by choline, amphotericin B failed to cause dilation of the LIS. These data suggest that, as in other epithelia, a significant amount of transepithelial fluid flow passes down the LIS, and that these spaces may provide the local osmotic compartment which is responsible for linking transepithelial fluid movement to active ion transport.

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.

Under short-circuit conditions, vasoactive intestinal peptide (VIP) did not alter net Na+ movement but selectively stimulated net Cl- secretion across dog tracheal epithelium with a high affinity (Km congruent to 10(-8) M). The increase in Cl- secretion was not different from the rise in short-circuit current (Isc). However, stimulation of Cl- secretion was not maximal, because the addition of isoproterenol (10(-6) M) to VIP-treated tissues further increased the Isc by 54%. The effect of exogenous VIP was not blocked by a combination of atropine, phentolamine, propranolol (10(-5) or 10(-6) M), or tetrodotoxin (10(-6) M). Under open-circuit conditions, VIP caused an increase in the net secretion of Cl- and Na+, but the changes did not reach statistical significance. We conclude that VIP acts directly on receptors on the surface of epithelial cells to stimulate active Cl- secretion. The abundance of VIP nerves in the submucosa suggests that VIP may be important in regulation of fluid movement across the epithelium.

Airway epithelia possess transepithelial ion transport processes which may help to regulate the fluid content of airway secretions. Chloride secretion promotes fluid movement from blood to airway lumen. Active absorption of Na favours fluid movement in the opposite direction. The balance between these two processes can be altered by a number of agents which stimulate Cl secretion. The importance of ion transport for normal mucociliary clearance is suggested by the finding that airway epithelia in patients with cystic fibrosis are unable to secrete chloride. This defect may cause the characteristically sticky and tenacious mucous secretions which are the major cause of death in this disease.

The sections in this article are: 1 Airway Secretions 1.1 Surface Mucus 1.2 Submucosal Glands 1.3 Effects of Drugs and Mediators 1.4 Hypertrophy of Mucus-Secreting Cells 2 Ion Transport and Water Movement 2.1 Introduction 2.2 Mechanisms of Ion Transport 2.3 Regulation of Ion Transport 2.4 Relationship Between Ion Transport and Water Movement

1. Tissues with raised intracellular Na levels, produced by incubation in K-free media, were used throughout. The uptake of 42K by these Na-loaded tissues was followed for 10 min in the presence and absence of 1-37 X 10(-4) M ouabain, this being sufficient to inhibit Na pumping maximally. Subtraction of the uptake seen in the presence from that seen in the absence of ouabain gave estimates of the pumped ouabain-sensitive K uptake. 2. In Na-free (MgCl2) medium this depended on the [K]0 in a sigmoidal fashion with a half maximal [K]0 for activation of some 4mM. The maximal uptake of K was 3 m-mole/kg.min corresponding to a transmembrane flux of some 12-5 p-mole. cm-2.sec-1. 3. In the presence of Na the K activation curve became more obviously sigmoid and higher concentrations of K were needed to achieve a given active K influx. The results were well fitted by assuming that Na and K competed for two identical, non-interacting sites on the external pump face. 4. Addition of K during the efflux of 24Na into a Na-free (MgCl2) medium led to an increased rate of tracer loss. The magnitude of this increase depended on the [K] used in a hyperbolic fashion and it was abolished by addition of ouabain. The [K] causing half-maximal activation of ouabain-sensitive Na efflux was in the order of 1-2 mM. 5. When the [K] in the uptake media was 1-5 mM; Na, Li, Rb and Cs all inhibited ouabain-sensitive K uptake, the order of effectiveness being Rb greater than Cs greater than Na greater than Li. With a E1TKA10 OF 0-15 MM low concentrations of Cs and Rb were shown to stimulate K uptake. Such an effect is predicted by assuming two ion binding sites on the pump's outer face, and that the pump can translocate mixtures of K and either Rb or Cs...

Uptake of tritiated ouabain by cells isolated from dog tracheal epithelium showed two components: a saturable component with a Km of 5.1 X 10(-8) M and a maximal uptake of 8.3 X 10(5) molecules/cell and a nonsaturating component of uptake that was linear with concentration. Several criteria indicated that the saturable uptake component represented binding to the Na+-K+-ATPase. To estimate the average surface area per cell, a known number of cells were pelleted and weighed, and the average surface area was calculated, assuming the cells to be perfectly spherical. The validity of this assumption was confirmed by comparing the calculated surface areas of cells in isotonic and hypotonic media. From the values for maximal saturable uptake and average surface area, a pump density of approximately 2,400 sites/micron2 was calculated. Given that the apical membrane lacks Na pumps and accounts for only approximately 5% of the total surface area, this value corresponds to the pump density of the basolateral cell membrane. The pump densities of ciliated, goblet, and basal cells were compared by autoradiography. The three cell types had approximately the same density of pump sites.

We studied the effects of neurokinin A (NKA) and neurokinin B (NKB), the mammalian-derived tachykinins, on the electrical and ion transport properties of canine tracheal epithelium. Both tachykinins dose-dependently increased short-circuit current (Isc) when added to the mucosal (NKA: delta Isc(max) = 24.2 +/- 2.4 microA/cm2, KD = 9 nM; NKB: delta Isc(max) = 1.42 +/- 2.2 microA/cm2, KD = 32 nM) or submucosal (NKA: delta Isc(max) = 10.5 +/- 1.2 microA/cm2, KD = 45 nM; NKB: delta Isc(max) = 2.2 +/- 1.4 microA/cm2, KD = 80 nM) bath. Isc responses to mucosal addition of tachykinins consisted of transient and subsequent steady-state components, whereas submucosal addition elicited only steady-state responses. Inhibition of Cl transport with bumetanide or substitution of Cl reduced the maximal changes in Isc. In paired tissues, NKA increased net 36Cl flux toward the mucosa from 1.83 +/- 0.49 to 2.71 +/- 0.46 mu eq.cm-2.h-1 (p less than 0.05), without affecting net 22Na flux toward the submucosa. The increases in Isc induced by tachykinins were not modified by prior tissue incubation with phentolamine, propranolol, atropine, tetrodotoxin, or indomethacin, but were effectively inhibited by (D-Pro2, D-Trp7,9)substance P. The cyclic AMP (cAMP) levels in the surface epithelium were increased by the addition of NKA and NKB. These findings suggest that NKA and NKB selectively stimulate the secretion of Cl across canine tracheal epithelium, probably by acting directly on the tachykinin receptors, and that these effects are associated with the increased production of intracellular cAMP.

The tracheal mucosa from a 12-year-old girl was digested with collagenase 4 hr after her death from cystic fibrosis. Forty million viable cells were obtained. The cells, plated at 10(6) per cm2 onto four Nuclepore filters coated with human placental collagen, formed confluent monolayers after 1 day. Their ultrastructure was similar to that of normal human cells. They were studied in conventional Ussing chambers or with intracellular microelectrodes on days 5-7 after plating. The monolayers displayed resistance of 380 +/- 50 omega X cm2 and short-circuit current (Isc) of 1.8 +/- 0.4 microA X cm-2. This resistance is similar to that obtained for dog or normal human monolayers. The Isc is less than normal human (approximately 3 microA X cm-2) or dog (approximately 10 microA X cm-2) cells. The cystic fibrosis cells resembled normal monolayers in that serosal ouabain and mucosal amiloride inhibited Isc, while mucosal ouabain or serosal amiloride had no effect. They differed from normal human or dog cells in that Isc was not inhibited by bumetanide and the stimulation of Isc by isoproterenol or prostaglandin E2 was greatly reduced or abolished. Addition of isoproterenol depolarized apical membrane potential (psi a) and decreased fractional resistance (fR) in normal human and dog but had no effect on psi a or fR in cystic fibrosis cells. Reduction of mucosal chloride from 120 to 5 mM by replacement with gluconate increased fR of dog and normal human monolayers and depolarized psi a by 22 (dog) or 30 (human) mV. In cystic fibrosis monolayers, chloride replacement hyperpolarized psi a by 2 mV and had little effect on fR. These results suggest that the primary defect in cystic fibrosis is reduced apical membrane chloride conductance.

The uptake of α-aminoisobutyric acid (AIB) by guinea pig taenia coli was linear with time over a 5-h period. There was a saturable component with Km of 0.7 mM and a maximal value of 4.75 mmol/kg tissue per hour. A second nonsaturable component of uptake became important at high concentrations of AIB (> 5 mM). Preincubation in K+-free or Na+-free (high-K+) media greatly reduced uptake. The dependence of AIB uptake on outside [Na+] ([Na+]0) was approximately linear over the range 0–140 mM. With [Na+]0 equal to 25 mM the uptakes with various substitutes were in the order sucrose > Mg2+ > choline+ > Li+ > Cs+ > Rb+ = K+. Addition of ouabain or removal of K+ during efflux of AIB led to marked increases in the efflux rate, ouabain being the more effective treatment. Changes in tissue Na+ and K+ levels were found to be slightly greater with ouabain than with K+-free medium. Lanthanum (5 mM) prevented the uptake of Na+ seen in K+-free medium and also abolished the increase in the rate of loss of AIB. It also reduced the rise in tissue Na+ produced by ouabain; there was a corresponding reduction in the ouabain-induced increase in the rate of loss of AIB. It is concluded that both the influx and efflux of AIB in this tissue are Na+ dependent, and that the accumulation of AIB relies on the transmembrane Na+ gradient.

Epithelial cells from dog trachea, when grown in tissue culture, formed confluent monolayers 5-6 days after plating. After 8-10 days, domes [mean diam 356 +/- (SE) 53 micron] appeared in monolayers grown in collagen-coated flasks. When grown on polycarbonate filters coated with collagen, a detectable resistance (greater than 5 omega X cm2) and transepithelial potential difference (PD) (greater than 0.1 mV) developed 6 days after plating and increased to approximately 15 omega X cm2 and 15 mV at 10 days. Serosal ouabain (10(-4) M) abolished PD and short-circuit current (Isc). Luminal ouabain had no effect. Luminal amiloride (10(-4) M) and serosal bumetanide (10(-4) M) each decreased PD and Isc. However, a combination of both of these drugs did not abolish Isc. Isoproterenol (10(-5) M), dibutyryl adenosine 3',5'-cyclic monophosphate (10(-3) M), vasoactive intestinal peptide (10(-7) M), prostaglandin (PG) E2 (10(-5) M), PGF2 alpha (10(-5) M), and bradykinin (10(-5) M) each increased PD and Isc. Thus these monolayer cultures maintain electrical properties resembling those of the original tissue. This preparation may prove useful for the study of water and ion transport by airway epithelia.

Cells from the dog's tracheal mucosa formed confluent epithelial sheets in culture. Typical tight junctions separated the apical from the basolateral portion of the cell membrane. The apical portion of the cell contained numerous short microvilli and a pronounced glycocalyx. The basolateral portion of the plasma membrane was unspecialized except for extensive gap junctions between cells. Freeze-fracture showed that the cultured cells lacked the basolateral square (orthogonal) arrays of the original tissue, particles previously implicated in ion transport. Formation of domes indicated the presence of active fluid absorption. Domes appeared between days 4 and 8 of culture and persisted for about 1 week. Cell sheets showed a transepithelial resistance of approximately equal to 400 omega X cm2 and a short-circuit current (Isc) of approximately 5 microA X cm-2. The effects of transport inhibitors indicated that both active Na absorption and active Cl secretion were present. Isc was increased by isoproterenol, prostaglandins E2 and F2 alpha, vasoactive intestinal peptide, dibutyryl cyclic AMP, leukotrienes C4 and D4, and bradykinin. These changes were probably due to stimulation of active Cl secretion.

We have measured the fluxes of 82Br or 131I across short-circuited dog tracheal epithelium following complete replacement of chloride in the bathing medium by either bromide or iodide. In addition, we have partly replaced the bathing chloride with bromide, iodide, or thiocyanate and studied the effects of these anions on 36Cl fluxes. Our results show that the affinity sequence for the chloride transport process is I > Br congruent to Cl, that Br is transported, but that I is not. Thiocyanate is a potent inhibitor of chloride transport, and its effects are greater than those predicted by a simple competitive interaction with chloride for transport sites.

Eosinophil major basic protein (MBP) is a granule-associated cytotoxic protein found in sputum and deposited on airway tissues of patients with acute asthma. We therefore studied the effect of human MBP on ion transport in dog tracheal epithelium. We mounted the posterior tracheal membranes of mongrel dogs in Ussing chambers and measured potential differences across the membranes and short-circuit current. Using 22Na+ and 36Cl- as tracers, we determined net ion movements. The addition of MBP (5 X 10(-6) M) to the mucosal, but not to the serosal, side of the membranes produced an increase in short-circuit current from 2.25 +/- 0.19 (mean +/- SE) to 2.78 +/- 0.23 muEq.cm-2h-1 (p less than 0.0001) and in net chloride secretion from 1.57 +/- 0.22 to 2.31 +/- 0.24 muEq.cm-2h-1 (p less than 0.01). There was no change in net sodium movement. Pretreatment with indomethacin (10(-5) M) attenuated, but did not abolish, the increase in short-circuit current. After exposure to MBP, prostaglandin E2 release into the serosal bathing solution increased from 10.0 +/- 4.2 to 17.0 +/- 6.9 ng.cm-2h-1 (p less than 0.05). The results of this study indicate that MBP simulates prostaglandin E2 production and chloride secretion by dog tracheal epithelium. Thus, eosinophils in the airways, through release of MBP, may affect mucociliary clearance by changing the volume and composition of respiratory tract fluid.

Fluid and electrolyte transport by epithelial cells in vitro can be recognized by the ability of cultured cells to form domes and by the electrical properties of monolayer cultures. Pulmonary alveolar epithelial cells are thought to be partially responsible for fluid movement in the fetal lung, but their role in electrolyte transport in the adult lung is not known. We isolated alveolar type II cells from adult rat lung and maintained them on plastic culture dishes alone, on plastic culture dishes coated with an extracellular matrix, and on collagen-coated Millipore filters. Numerous large domes were formed on culture dishes coated with the extracellular matrix; smaller domes were formed on uncoated plastic culture dishes. Sodium butyrate (3 mM) stimulated dome formation. Transmission electron microscopy showed that the epithelial cells had flattened but still retained lamellar inclusions and that the cells were polarized with microvilli on the apical surface facing the culture medium. The electrical properties of the monolayers maintained on collagen-coated Millipore filters were tested in two laboratories. The transepithelial potential differences were 0.7 +/- 0.1 mV (24 filters, seven experiments) and 1.3 +/- 0.1 mV (13 filters, two experiments) apical side negative, and the corresponding resistances were 217 +/- 11 ohm X cm2 and 233 +/- 12 ohm X cm2. Terbutaline (10 microM) produced a biphasic response with a transient decrease and then a sustained increase in potential difference. Amiloride (0.1 mM) completely abolished the potential difference when it was added to the apical side but not when it was added to the basal side, whereas 1 mM ouabain inhibited the potential difference more effectively from the basal side. Thus, type II cells form a polarized epithelium in culture, and these cells actively transport electrolytes in vitro.

Binding of [3H]ouabain by the dog's tracheal epithelium shows a nonspecific component depending linearly on ouabain concentration, and a specific saturable component with a Km of 10(-7) M. Control experiments showed that the tracer taken up was not trapped within the extracellular space nor bound to tissue collagen. Inhibition of the saturable uptake by high K, metabolic inhibition, low Na, and low temperature indicated that binding was to Na/K ATPase. One-sided exposures of tissue sheets to tracer showed that the submucosal side took up 10 X as much tracer as the luminal. Autoradiography localized tracer uptake under all conditions to the cells' basolateral membranes.

In culture, cells from the dog tracheal epithelium form confluent cell sheets that have ion transport processes similar to those of the original tissue. Cell sheets grown from "normal" human tracheal mucosa resemble those of the dog in the presence of active chloride secretion, which can be stimulated by endogenous mediators. Tracheal cells cultured from patients with cystic fibrosis lack chloride secretion because their apical membranes are impermeable to this anion.

The smooth muscle of the monkey oviduct is similar to that described for other species and organs. Cell-to-cell contacts, in the form of simple appositions, were numerous. The adrenergic nerve supply was sparse, and close contacts between nerve and muscle were extremely rare. There was no difference in the density of innervation between the isthmus and ampulla. The rabbit appeared to have a greater denisty of innervation than the monkey.

Cl transport in apical membrane vesicles derived from bovine tracheal epithelial cells was studied using the Cl-sensitive fluorescent indicator 6-methoxy-N-(3-sulfopropyl) quinolinium. With an inwardly directed 50 mM Cl gradient at 23°C, the initial rate of Cl entry (J Cl) was increased significantly from 0.32±0.12 nmol · sec−1 · mg protein−1 (mean±sem) to 0.50±0.07 nmol · sec−1 · mg protein−1 when membrane potential was changed from 0 to +60 mV with K/valinomycin. At 37°C, with membrane potential clamped at 0 mV, there was a 34±7% (n=5) decrease inJ Cl from a control value of 0.37±0.03 nmol · sec−1 · mg protein−1 upon addition of 0.2mm diphenylamine-2-carboxylate. The following did not alterJ Cl significantly (J Cl values gives as percent change from control): 50mm cis Na (−1±5%), 0.1mm furosemide (−3±4%), 0.1mm furosemide in the presence of 50mm cis Na (−5±2%), 0.1mm H2DIDS (−18±9%), a 1.5 pH unit inwardly directed H gradient (−7±7%), and 0.1mm H2DIDS in the presence of a 1.5 unit pH gradient (4±18%). With inward 50mm anion gradients, the initial rates of Br and I entry (J Br andJ 1, respectively) were not significantly different fromJ Cl.J Cl was a saturable function of Cl concentration with apparentK d of 24mm and apparentV max of 0.54 nmol · sec−1 · mg protein−1. Measurement of the temperature dependence ofJ Cl yielded an activation energy of 5.0 kcal/mol (16–37°C). These results demonstrate that Cl transport in tracheal apical membrane vesicles is voltage-dependent and inhibited by diphenylamine-2-carboxylate. There is no significant contribution from the Na/K/2Cl, Na/Cl, or Cl/OH(H) transporters. The conductive pathway does not discriminate between Cl, Br, and I and is saturable. The low activation energy supports a pore-type mechanism for the conductance.

Confluent cell sheets were cultured from the tracheal epithelium of normal humans or from tracheal and nasal epithelia of patients with cystic fibrosis (CF). Changes in short-circuit current (Isc) or cyclic AMP (cAMP) levels in response to 10(-5) M isoproterenol were measured. In CF tracheal cells the response to isoproterenol was transient, and the maximal increase in Isc was one-tenth normal. In CF nasal cells, isoproterenol or epinephrine caused only small transient increases in Isc. However, in both CF nasal and tracheal cells, the Ca ionophore, A23187, caused relatively large increases in Isc that were inhibited by the Cl transport blocker, bumetanide, suggesting that Cl secretion can be induced by raising intracellular levels of Ca. In normal tracheal cell sheets, cAMP levels increased within 15 s of isoproterenol addition and continued to increase for up to 20 min. Resting levels of cAMP in CF tracheal cells were not statistically different from those of normal cells and showed linear increases for up to 4 min after addition of isoproterenol. Changes in cAMP in CF nasal cells were similar to the changes in CF tracheal cells. After 2 min, all three cell types showed cAMP levels elevated approximately equal to 10-fold. These results suggest that receptor-activated stimulation of adenylate cyclase is normal in CF. However, though raised cAMP levels stimulate Cl secretion in normal, they are unable to do so in CF airway epithelial cells.

Smooth muscles actively accumulate Cl. Abolition of the transmembrane Na gradient has been shown to lead to a passive distribution of Cl. This suggested to us that Na and Cl may move together across the smooth muscle cell membrane, Cl accumulation resulting due to [Na]i being less than [Na]o. In favour of this view are the findings that Cl uptake is reduced in Na-free medium, Na uptake is reduced in Cl-free medium, reducing [Cl]i reduces Na efflux, and increasing [Na]i increases Cl efflux.

Ion fluxes or fluid flow were measured across sheets of dog tracheal epithelium mounted in Ussing chambers or a special apparatus, respectively. Under short-circuit conditions, luminal amphotericin B (3 X 10(-5) M) caused an inhibition of net Cl secretion and an increase in net Na absorption across paired tissues. In paired tissues under resting open-circuit conditions, there was no significant net transepithelial flux of either Cl or Na. Amphotericin B induced significant net fluxes of both Cl and Na toward the serosal side. In separate tissues from the same animals, there was no significant transepithelial fluid movement under resting conditions. Amphotericin B caused a net absorption of fluid. The absorption of salt and fluid in amphotericin B-treated tissues was abolished by ouabain. We conclude that stimulation of active Na transport by amphotericin B leads to fluid absorption. In vivo, the movement of fluid across the dog tracheal epithelium may be dependent on a balance between active Cl secretion and active Na absorption.

1. Binding of tritiated ouabain to the smooth muscle of the guinea-pig taenia coli showed two components, one saturable at lower glycoside concentrations and the other linear with increasing concentrations.2. The saturable component alone was affected by extracellular potassium concentrations. This component seems to be bound to sodium pumping sites, and when completely saturated binds 1.1 x 10(11) molecules per mg fresh wt. of tissue, or 250-300 molecules per square micron of membrane, assuming a volume:surface area ratio of 1.5 mum.3. Only a fraction of (42)K uptake by the cells can be blocked by ouabain at maximal concentrations. In normal Krebs solution two thirds can be blocked. The remaining one third is presumably passive uptake. The fraction blocked is reduced as the extracellular potassium concentration, and thus passive uptake, is increased.4. The amount of potassium pumped into the cells at various concentrations of extracellular potassium has been calculated. In normal Krebs solution the amount pumped in 45 min was 20.0 m-mole/kg fresh wt., and this was increased at higher potassium concentrations.5. On the assumption of a stoichiometry of 3Na: 2K, the pump sites in normal Krebs solution have a turnover rate of 1320 min(-1).6. Indirect calculations of sodium movements suggest that the sodium permeability may be about 0.9 x 10(-8) cm sec(-1) and the pump may generate a current of 0.9 x 10(-7) A cm(-2). This crossing an Ohmic membrane resistance of 30-60 kOmega cm(2) would be equivalent to a potential difference of 3-5 mV.

Cl-impermeability in cystic fibrosis (CF) tracheal epithelium derives from a deficiency in the beta-adrenergic regulation of apical membrane Cl- channels. To test the possibility that cAMP-dependent kinase is the cause of this deficiency, we assayed this kinase in soluble fractions from cultured airway epithelial cells, including CF human tracheal epithelial cells. Varying levels of cAMP were used in these assays to derive both a Vmax and apparent dissociation constant (Kd) for the enzymes in soluble extracts. The cAMP-dependent protein kinase from CF human tracheal epithelial cells has essentially the same Vmax and apparent Kd as non-CF human, bovine, and dog tracheal epithelial cells. Thus, the total activity of the cAMP-dependent kinases and their overall responsiveness to cAMP are unchanged in CF.

The pathways of ion movement across canine tracheal epithelium, a Cl-secreting tissue, were examined by three techniques. First, the measurement of simultaneous, unidirectional fluxes of Na or Cl and mannitol, a large hydrophilic molecule that serves as a marker of the paracellular pathway, indicated that a significant fraction of both the Na flux from submucosa to mucosa (J Na sm) and the flux of Cl from mucosa to submucosa (J Cl ms) traverse the cellular pathway. The ratio of the Na-to-Cl diffusion coefficients through the paracellular pathway was 0.23, in contrast to the free solution ratio of 0.63. Second, in voltage-clamp experiments we examined the effect of transepithelial voltage differences on the unidirectional fluxes of Na and Cl. The results agree with the previous findings, suggesting that there are voltage-independent, or transcellular, backfluxes of Na and Cl, and that the relative permeability of Na to Cl through the voltage-dependent (presumably paracellular) pathway was 0.28. Third, measurement of transepithelial diffusion potentials gave a Na-to-Cl permeability ratio of 0.31 +/- 0.02 (mean +/- SE). These results suggest that there are significant transcellular backfluxes of Na and Cl and that the paracellular pathway in the canine trachea is anion selective. An anion-selective pathway would tend to shunt the secreted Cl back through the paracellular pathway, thus minimizing the net ion and fluid movement across the tissue in the open-circuit condition.

The "loop" diuretics MK-196, bumetanide, piretanide, and furosemide are all potent inhibitors of Cl transport by the dog's tracheal epithelium. In short-circuited tissues, the drugs caused significant decreases in both unidirectional Cl fluxes and in the net flux of Cl toward the lumen; the change in net Cl flux was not significantly different from the change in short-circuit current. The drugs had no effect on active Na absorption. All drugs caused a significant fall in tissue conductance. All drugs, except MK-196, were more potent from the serosal bath; MK-196 was equipotent from either side of the tissue. In experiments with isolated cells, the diuretics caused no significant changes in intracellular Na and K concentrations, a fall in intracellular Cl concentration, and approximately equal falls in Na and Cl influxes. These results suggest that the site of action of these drugs is on a basolateral linked Na-Cl entry process. Additional evidence for such a linked entry process was provided by experiments in which removal of Cl reduced Na influx and removal of Na reduced Cl influx.

We measured the rate of fluid movement across the tracheal epithelium of the dog by an electrical method, modified from that originally described by Wiedner (Rev. Sci. Instrum. 47: 775-6, 1976). The base-line volume flow (Jv) from the submucosa to the mucosa was 44 +/- 23 nl.min-1.cm-2 (mean +/- SE; n = 6). In only three of the tissues was the resting Jv significantly greater than zero. Stimulation with 2 X 10(-3) M aminophylline (a drug that we have shown stimulates Cl secretion under short-circuit conditions) increased Jv in all tissues to a mean of 137 +/- 29 nl.min-1.cm-2 (P < 0.001). We also measured the unidirectional fluxes of Cl with the tissue at its spontaneous electrical potential. During the base-line period, the net flux of Cl was not significantly different from zero. During stimulation with aminophylline, Cl secretion increased to a mean of 1.44 +/- 0.50 microneq.cm-2.h-1. Comparison of the stimulated values of Jv and net Cl secretion suggests that fluid secretion by the trachea is isotonic during stimulation with aminophylline.

Sheets of trachea from ferret and cat were mounted in Ussing chambers and continuously short circuited. Under resting conditions, in both the cat and ferret there was little or no Cl secretion, and Na absorption accounted for most of the short-circuit current (Isc). Ouabain (10(-4) M, serosal bath) reduced Isc to zero in 30–60 min. This decline was matched by a decrease in net Na absorption. Amiloride (10(-4) M, luminal bath) caused a significant decrease in Isc and conductance (G) in both species. Bumetanide (10(-4) M, serosal bath) had negligible effects on Isc and G. In both species, isoproterenol increased Isc by stimulating Cl secretion. Methacholine induced equal amounts of Na and Cl secretion, with little change in Isc. In the cat, prostaglandins E2 and F2 alpha and bradykinin increased Isc, responses which were abolished in Cl-free medium. In open-circuited cat tissues, Na flux from the serosal to mucosal side was measured simultaneously with the secretion of nondialyzable 35S. Prostaglandins E1, E2, and F2 alpha, histamine, bradykinin, methacholine and isoproterenol all increased both Na and 35S-mucin secretion.

We have developed a preparation of isolated cells from dog tracheal mucosa. The three major cell types of the intact epithelium (ciliated, secretory, and basal) are present in approximately the same proportions as in the intact tissue. These cells show high viability as judged by exclusion of vital dyes, high O2 consumption, and incorporation of amino acids into protein. The intracellular ion contents (in mmol/l cell H2O) are [K]i, 150; [Na]i, 20; and [Cl]i, 50. Ouabain (10(-4) M) causes a rise in [Na]i and a reciprocal loss of intracellular K.

The idea that the fluid component of cat tracheal submucosal gland secretions is produced by Na-linked secretion of Cl was tested. Gland secretion was stimulated with phenylephrine; gland fluid flow, net salt movement, and output of 35SO4-labeled macromolecules were measured. With CI, I, NO3, or Br as the major anion, phenylephrine caused equal increases in gland flow and output of 35S-labeled macromolecules while increasing net transepithelial 22Na movement from 0 to about 10 mu eq X cm-2 X h-1. With the impermeant gluconate as a major anion, phenylephrine caused the same increase in output of 35S label, a smaller increase in gland flow, and had no effect on net 22Na movement. Short circuiting in the presence of Cl, or high concentrations of the loop diuretics, furosemide or bumetanide, did not alter the actions of phenylephrine. Ouabain or replacement of Na by choline or Li abolished all secretory effects of phenylephrine. We conclude that active Cl secretion is not responsible for the transepithelial flows of salt and water induced by phenylephrine. Instead, these flows may be secondary to the release of osmotically active components of the secretory granules.

1. Tissues were allowed to equilibrate in a Tris-buffered Krebs solution and were then exposed to similar solutions containing up to 5 mM-La. La caused shrinkage and significant losses of tissue K, Na, Mg and Ca. The shrinkage was exactly accountable for by a reduction in the extracellular space (e.c.s.) as measured by [14C]sucrose. No significant change was seen in tissue Cl content. Exposure for 1 hr to 5 mM-La led to a total cation loss of 24-3 +/- 1-6 m-equiv/kg or, correcting for the small change in Cl content, a loss of positive charge of 23-8 +/- 2-2 m-equiv/kg fresh wt. 2. Using the radioisotope 140La it was shown that this loss of cation was balanced by an uptake of La3+. 3. Subtraction of the ions in the measured [14C]sucrose space from the total tissue ion contents led to estimates of the "cellular" ion contents. The effects of 1 hr exposure to 5 mM-La on these were a loss of 12-9 +/- 2-4 m-equiv/kg of cation and a gain of 10-0 +/- 2-6 m-equiv/kg of Cl. 4. Similar changes in ion content were produced by La on "Na-loaded" and "K-loaded" tissues, these being tissues which by exposure to K-free or Na-free (high K) solutions had replaced all their K with Na or vice versa. 5. The uptakes of 24Na and 36Cl by Na-loaded tissues were both describable as the sum of two exponentail processes: a fast component (t 1/2 congruent to 1/2 min), which was presumed to be extracellular and a slower, presumed transmembrane, component. La reduced the rapid component of uptake of 24Na by an amount greater than that predicted by the reduction in the e.c.s., the extra amount lost being some 10--15 m-equiv/kg. La also reduced the amount of rapidly exchanging 36Cl, bwt this reduction was entirely accounted for by the change in the e.c.s. La reduced the rate constant of the slow component of 24Na uptake. 6. La reduced the rapidly exchanging component of 42K uptake by normal tissues by an amount equivalent to about 0-5 m-mole/kg fresh wt. of K in excess of the change in the extracellular space. 7. La had little effect on the effluxes of 36Cl and 42K from normal tissues. However, it reduced the size of the fastest component of exchange of 42K efflux from K-loaded tissues by an amount equal to some 10-15 m-equiv/kg in excess of the reduction in the e.c.s. A similar reduction in the rapidly exchanging component of 24Na efflux from normal tissues was also seen. La slowed the efflux of 24Na from Na-loaded tissues at times when the tracer lost could safely be regarded as intracellular. 8. The taenia coli when exposed to K-free solutions gains Na and loses K. In the presence of La the gain in Na was completely blocked. K was still lost, however, being accompanied by Cl and increased shrinkage. La also prevented the uptake of Na from high Na media by ion-depleted tissues (produced by exposure to sucrose media), while having little effect on the uptake of K from high K media by such tissues. 9 Tedia by such tissues. 9. The cation displaced by La in excess of that lost due to the reduction of the e.c.s...

The effects of stimulating protein kinase C on Cl- secretion across dog tracheal epithelium were investigated. The phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), and the synthetic diacylglycerol, 1-oleolyl-2-acetylglycerol (OAG), which stimulate protein kinase C (PKC), both stimulated short-circuit current (Isc) with Kd of 10 nM and 1 microM, respectively. In Cl(-)-free solution, the increases in Isc were virtually abolished, suggesting that these compounds stimulate Cl- secretion, a hypothesis confirmed for TPA by measurement of 36Cl- fluxes. The stimulations of Cl- secretion were not sensitive to indomethacin, nor were cAMP levels elevated during stimulation. In addition to its transient stimulatory effect, TPA at high doses caused the eventual lowering of the base-line Isc and a block of subsequent stimulation by cAMP-mediated agonists. This was probably not the result of toxicity or an effect on adenylate cyclase or on cAMP-dependent protein kinase. Cell extracts from both cultured and native dog tracheal epithelial cells showed strong PKC activities. These results suggest that PKC may play a role in regulating Cl- secretion across dog tracheal epithelium.

Confluent monolayers of polygonal epithelioid cells were obtained from enzymatically and mechanically dispersed bovine anterior pituitaries (AP) and pars tuberali (PT). The ultrastructure of the cells composing the monolayer was consistent with the follicular or folliculostellate cells (FC) of the pituitary, i.e., lack of secretory granules; formation of follicles in culture; interdigitations with neighboring cells with numerous tight junctions; presence of extensive microfilaments; and sparse rough endoplasmic reticulum and Golgi apparatus. Culture media from monolayers of first passage cultures contained little if any of the AP hormones' luteinizing hormone, prolactin, and ACTH. Shortly after reaching confluency, regions of the monolayer bulge away from the surface of the culture dish to form domes. Dome formation has been described only with cultures of cells that function as transport epithelia in vivo. FC cultured on polycarbonate filters were placed in Ussing chambers. A transepithelial potential difference of approximately 1.1 mV and a resistance greater than 300 omega cm2 were detectable 4–5 days after plating. The short-circuit current (Isc) was decreased 70% by amiloride applied to the mucosal surface and further decreased by the addition of ouabain at the serosal surface. The beta-adrenergic agonist isoproterenol increased the Isc and this action was prevented by a beta-antagonist. These observations indicate that pituitary FC in culture behave as a transport epithelium. Considering the organization of FC in the AP and PT, they suggest a regulatory role for FC in the maintenance of the ionic composition of the interstitial fluid of the pituitary gland.

Release of cyclooxygenase products from primary cultures of dog or human tracheal epithelium was measured by radioimmunoassay. In both species, bradykinin, platelet-activating factor (PAF), and A23187 (a calcium ionophore) caused increases in the rate of release of prostaglandin (PG) E2 and smaller increases in PGF2 alpha, 6-keto-PGF1 alpha, and thromboxane B2 output. Isoproterenol, vasoactive intestinal peptide (VIP), methacholine, and leukotrienes C4 and D4 had no effect on release of these cyclooxygenase products. Gas chromatography-mass spectrometry showed that the ratio of PGE2 to PGD2 released from dog cells by A23187 was 30:1. Short-circuit current across dog cells was stimulated by bradykinin, A23187, PAF, VIP, methacholine, and isoproterenol. Only the responses to bradykinin and A23187 were reduced by pretreatment with indomethacin.

A quantitative fluorescence assay has been developed to measure Cl flux across liposomal membranes for use in chloride transporter reconstitution studies. A Cl-sensitive fluorophore [6-methoxy-N-(3-sulfopropyl)quinolinium; SPQ] was entrapped into phospholipid/cholesterol liposomes formed by bath sonication, high-pressure extrusion, and detergent dialysis. Liposomes containing entrapped SPQ were separated from external SPQ by passage down a Sephadex G25 column. There was less than 10% leakage of SPQ from liposomes in 8 h at 4 degrees C and in 2 h at 23 degrees C. Cl influx (JCl in millimolar per second or nanomoles per second per centimeter squared) was determined from the time course of SPQ fluorescence, measured by cuvette or stopped-flow fluorometry, in response to inward Cl gradients. In 90% phosphatidylcholine (10% cholesterol liposomes at 23 degrees C, JCl in response to a 50 mM inward Cl gradient was 0.06 +/- 0.01 mM.s-1 (SD, n = 3) in the absence and 0.27 +/- 0.02 mM.s-1 in the presence of a K/valinomycin voltage clamp (0 mV), showing that the basal Cl "leak" is conductive; JCl increased (1.7 +/- 0.1)-fold in the presence of a 60-mV inside-positive diffusion potential. Accuracy of chloride influx rates determined by the SPQ method was confirmed by measurement of 36Cl uptake. In liposomes voltage-clamped to 0 mV, JCl was linear with external [Cl] (0-100 mM), independent of pH gradients, and strongly dependent on temperature (activation energy 18 +/- 1 kcal/mol, 12-42 degrees C) as predicted for channel-independent Cl diffusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Muscarinic receptor distribution in smooth muscle, exocrine glands, and epithelium of the ferret trachea was determined using [3H]propylbenzilylcholine mustard ([3H]PrBCM) binding and autoradiography. Specific, atropine-sensitive [3H]PrBCM binding was quantified autoradiographically in the trachealis muscle (approximately 21 binding sites/microns2), surface epithelium (approximately 6 binding sites/microns2), and submucosal glands (approximately 5 binding sites/microns2). Serous and mucous cells in the glands did not differ in receptor density. Binding sites on gland and epithelial cells were associated with basolateral membranes. In the trachealis muscle, a gradient in receptor density was observed, with outer layers of muscle containing 3 to 10 times more receptors per unit area than inner layers. Receptor distribution in both glands and muscle paralleled the distribution of cholinergic axons. However, at the light microscope level, there was no evidence for the presence of receptor “hot spots” related to the position of individual axons. The parallelism in the distribution of axons and receptors suggests the possibility of neural control of the genesis and/or maintenance of receptor distribution in these tissues.

Bradykinin increased mean short-circuit current (Isc) when added either to the mucosal (KD = 1.1 nM; delta Imaxsc = 29.8 +/- 4.4 microA/cm2) or submucosal bath (KD = 108 nM; delta Imaxsc = 27.1 +/- 4.9 microA/cm2). Bumetanide or replacement of Cl reduced the maximal change in Isc. In paired tissues, the increase in net 36Cl flux toward the mucosa equaled the change in Isc. Net 22Na flux toward the submucosa was unchanged. Involvement of intramural nerves was ruled out because bradykinin-induced increases in Isc were not inhibited by phentolamine, propranolol, atropine, or tetrodotoxin. A direct action of bradykinin on the epithelium was also made probable by the autoradiographic demonstration of specific bradykinin binding sites. The antagonists of arachidonic acid metabolism, indomethacin and eicosa-5,8,11,14-tetraynoic acid, inhibited the increase in Isc induced by bradykinin. Finally, prostaglandin E2 release was significantly increased by submucosal addition of bradykinin, and this effect was abolished by pretreatment with indomethacin. We conclude that bradykinin stimulates Cl secretion in canine tracheal epithelium by increasing prostaglandin release.

We examined whether the leukotrienes (LT) B4, C4, D4, and E4, and 5-, 12-, and 15-hydroxyeicosatetraenoic (HETE) acids alter the electrical properties of canine tracheal epithelium. Short-circuit current (ISC) increased by 20.3 +/- 4.3 and 25.9 +/- 6.5 microA/cm2 after LTC4 and LTD4 addition, was unchanged following LTB4, LTE4, 5-, or 15-HETE addition, and was decreased by 10 +/- 3% after 12-HETE addition. LTC4- and LTD4-induced increases in ISC were dependent on the presence of Cl and were equal to the increases in net 36Cl flux toward the mucosa. The cyclooxygenase inhibitor indomethacin (10(-5) M) or the phospholipase inhibitor mepacrine (10(-5) M) diminished increases in ISC. The release rate of prostaglandins E2 and F2 alpha and thromboxane B2 rose from 8.2 +/- 2.3, 2.3 +/- 0.5, and 1.1 +/- 0.5 to 15.5 +/- 3.3, 6.0 +/- 1.4, and 4.0 +/- 0.8 ng . cm-2 . h-1, respectively, after LTC4 addition, and from 6.9 +/- 1.4, 2.5 +/- 1.1, and 1.7 +/- 1.3 to 20.2 +/- 3.1, 10.5 +/- 3.1, and 5.2 +/- 2.4 ng . cm-2 . h-1, respectively, after LTD4 addition. The release rates of 6-keto-prostaglandin F1 alpha were unchanged. A thromboxane A2 mimetic, U46619, failed to increase ISC and thromboxane synthetase inhibitors OKY-046 and dazmegrel failed to inhibit the induced increases in ISC, suggesting that thromboxane has little effect on ISC. Thus lipoxygenase products of arachidonic acid can modulate Cl secretion by the canine tracheal epithelium and do so through the release of cyclooxygenase products, including prostaglandins E2 and F2 alpha.