Pulmonary Ionocytes Regulate Airway Surface Liquid pH in Primary Human Bronchial Epithelial Cells.

Rationale: Pulmonary ionocytes are a newly discovered airway epithelial cell type proposed to be a major contributor to cystic fibrosis (CF) lung disease based on observations they express the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel at a higher level than any other cell type in the airway epithelia. Moreover, genetically manipulated experimental models that lack ionocytes develop NaCl transport abnormalities and airway surface liquid (ASL) dehydration consistent with CF. However, no direct evidence indicates ionocytes engage in NaCl transport or contribute to ASL formation, questioning the relevance of ionocytes to CF lung disease. Objectives: To determine the ion transport properties of pulmonary ionocytes and club cells in genetically intact healthy and CF airway epithelia. Methods: We measured ion transport at the single-cell level using a self-referencing ion-selective microelectrode technique in primary human bronchial epithelial cell culture. Measurements and Main Results: cAMP-stimulated non-CF ionocytes do not secrete Na+ or Cl- into the ASL, but rather modulate its pH by secreting bicarbonate via CFTR-linked Cl-/bicarbonate exchange. Non-CF club cells secrete Na+ and Cl- to the lumen side after cAMP stimulation. CF ionocytes and club cells do not transport ions in response to cAMP stimulation, but incubation with CFTR modulators elexacaftor/tezacaftor/ivacaftor restores transport properties. Conclusions: We conclude that ionocytes do not contribute to ASL formation but regulate ASL pH. Club cells secrete the bulk of airway fluid. In CF, abnormal ionocyte and club cell function results in acidic and dehydrated ASL, causing reduced antimicrobial properties and mucociliary clearance. This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/).

cAMP triggers Na+ absorption by distal airway surface epithelium in cystic fibrosis swine.

A controversial hypothesis pertaining to cystic fibrosis (CF) lung disease is that the CF transmembrane conductance regulator (CFTR) channel fails to inhibit the epithelial Na+ channel (ENaC), yielding increased Na+ reabsorption and airway dehydration. We use a non-invasive self-referencing Na+-selective microelectrode technique to measure Na+ transport across individual folds of distal airway surface epithelium preparations from CFTR-/- (CF) and wild-type (WT) swine. We show that, under unstimulated control conditions, WT and CF epithelia exhibit similar, low rates of Na+ transport that are unaffected by the ENaC blocker amiloride. However, in the presence of the cyclic AMP (cAMP)-elevating agents forskolin+IBMX (isobutylmethylxanthine), folds of WT tissues secrete large amounts of Na+, while CFTR-/- tissues absorb small, but potentially important, amounts of Na+. In cAMP-stimulated conditions, amiloride inhibits Na+ absorption in CFTR-/- tissues but does not affect secretion in WT tissues. Our results are consistent with the hypothesis that ENaC-mediated Na+ absorption may contribute to dehydration of CF distal airways.

Large pH oscillations promote host defense against human airways infection.

The airway mucosal microenvironment is crucial for host defense against inhaled pathogens but remains poorly understood. We report here that the airway surface normally undergoes surprisingly large excursions in pH during breathing that can reach pH 9.0 during inhalation, making it the most alkaline fluid in the body. Transient alkalinization requires luminal bicarbonate and membrane-bound carbonic anhydrase 12 (CA12) and is antimicrobial. Luminal bicarbonate concentration and CA12 expression are both reduced in cystic fibrosis (CF), and mucus accumulation both buffers the pH and obstructs airflow, further suppressing the oscillations and bacterial-killing efficacy. Defective pH oscillations may compromise airway host defense in other respiratory diseases and explain CF-like airway infections in people with CA12 mutations.

Airway submucosal glands from cystic fibrosis swine suffer from abnormal ion transport across the serous acini, collecting duct, and ciliated duct.

The human airway is protected by an efficient innate defense mechanism that requires healthy secretion of airway surface liquid (ASL) to clear pathogens from the lungs. Most of the ASL in the upper airway is secreted by submucosal glands. In cystic fibrosis (CF), the function of airway submucosal glands is abnormal, and these abnormalities are attributed to anomalies in ion transport across the epithelia lining the different sections of the glands that function coordinately to produce the ASL. However, the ion transport properties of most of the anatomical regions of the gland have never been measured, and there is controversy regarding which segments express CFTR. This makes it difficult to determine the glandular abnormalities that may contribute to CF lung disease. Using a noninvasive, extracellular self-referencing ion-selective electrode technique, we characterized ion transport properties in all four segments of submucosal glands from wild-type and CFTR-/- swine. In wild-type airways, the serous acini, mucus tubules, and collecting ducts secrete Cl- and Na+ into the lumen in response to carbachol and forskolin stimulation. The ciliated duct also transports Cl- and Na+ but in the opposite direction, i.e., reabsorption from the ASL, which may contribute to lowering Na+ and Cl- activities in the secreted fluid. In CFTR-/- airways, the serous acini, collecting ducts, and ciliated ducts fail to transport ions after forskolin stimulation, resulting in the production of smaller volumes of ASL with normal Cl-, Na+, and K+ concentration.

Nebulized hypertonic saline triggers nervous system-mediated active liquid secretion in cystic fibrosis swine trachea.

Inhaled hypertonic saline (HTS) treatment is used to improve lung health in patients with cystic fibrosis (CF). The current consensus is that the treatment generates an osmotic gradient that draws water into the airways and increases airway surface liquid (ASL) volume. However, there is evidence that HTS may also stimulate active secretion of ASL by airway epithelia through the activation of sensory neurons. We tested the contribution of the nervous system and airway epithelia on HTS-stimulated ASL height increase in CF and wild-type swine airway. We used synchrotron-based imaging to investigate whether airway neurons and epithelia are involved in HTS treatment-triggered ASL secretion in CFTR-/- and wild-type swine. We showed that blocking parasympathetic and sensory neurons in airway resulted in ~50% reduction of the effect of HTS treatment on ASL volume in vivo. Incubating tracheal preparations with inhibitors of epithelial ion transport across airway decreased secretory responses to HTS treatment. CFTR-/- swine ex-vivo tracheal preparations showed substantially decreased secretory response to HTS treatment after blockage of neuronal activity. Our results indicated that HTS-triggered ASL secretion is partially mediated by the stimulation of airway neurons and the subsequent activation of active epithelia secretion; osmosis accounts for only ~50% of the effect.

Cystic fibrosis swine fail to secrete airway surface liquid in response to inhalation of pathogens.

Cystic fibrosis is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) channel, which can result in chronic lung disease. The sequence of events leading to lung disease is not fully understood but recent data show that the critical pathogenic event is the loss of the ability to clear bacteria due to abnormal airway surface liquid secretion (ASL). However, whether the inhalation of bacteria triggers ASL secretion and whether this is abnormal in cystic fibrosis has never been tested. Here we show, using a novel synchrotron-based in vivo imaging technique, that wild-type pigs display both a basal and a Toll-like receptor-mediated ASL secretory response to the inhalation of cystic fibrosis relevant bacteria. Both mechanisms fail in CFTR-/- swine, suggesting that cystic fibrosis airways do not respond to inhaled pathogens, thus favoring infection and inflammation that may eventually lead to tissue remodeling and respiratory disease.Cystic fibrosis is caused by mutations in the CFTR chloride channel, leading to reduced airway surface liquid secretion. Here the authors show that exposure to bacteria triggers secretion in wild-type but not in pig models of cystic fibrosis, suggesting an impaired response to pathogens contributes to infection.

Pseudomonas aeruginosa triggers CFTR-mediated airway surface liquid secretion in swine trachea.

Cystic fibrosis (CF) is an autosomal recessive genetic disorder caused by mutations in the gene encoding for the anion channel cystic fibrosis transmembrane conductance regulator (CFTR). Several organs are affected in CF, but most of the morbidity and mortality comes from lung disease. Recent data show that the initial consequence of CFTR mutation is the failure to eradicate bacteria before the development of inflammation and airway remodeling. Bacterial clearance depends on a layer of airway surface liquid (ASL) consisting of both a mucus layer that traps, kills, and inactivates bacteria and a periciliary liquid layer that keeps the mucus at an optimum distance from the underlying epithelia, to maximize ciliary motility and clearance of bacteria. The airways in CF patients and animal models of CF demonstrate abnormal ASL secretion and reduced antimicrobial properties. Thus, it has been proposed that abnormal ASL secretion in response to bacteria may facilitate the development of the infection and inflammation that characterize CF airway disease. Whether the inhalation of bacteria triggers ASL secretion, and the role of CFTR, have never been tested, however. We developed a synchrotron-based imaging technique to visualize the ASL layer and measure the effect of bacteria on ASL secretion. We show that the introduction of Pseudomonas aeruginosa and other bacteria into the lumen of intact isolated swine tracheas triggers CFTR-dependent ASL secretion by the submucosal glands. This response requires expression of the bacterial protein flagellin. In patients with CF, the inhalation of bacteria would fail to trigger ASL secretion, leading to infection and inflammation.

Serotonin triggers cAMP and PKA-mediated intracellular calcium waves in Malpighian tubules of Rhodnius prolixus.

Rhodnius prolixus is a hematophagous insect vector of Chagas disease capable of ingesting up to 10 times its unfed body weight in blood in a single meal. The excess water and ions ingested with the meal are expelled through a rapid postprandial diuresis driven by the Malpighian tubules. Diuresis is triggered by at least two diuretic hormones, a CRF-related peptide and serotonin, which were traditionally believed to trigger cAMP as an intracellular second messenger. Recently, calcium has been suggested to act as a second messenger in serotonin-stimulated Malpighian tubules. Thus, we tested the role of calcium in serotonin-stimulated Malpighian tubules from R. prolixus. Our results show that serotonin triggers cAMP-mediated intracellular Ca(2+) waves that were blocked by incubation in Ca(2+)-free saline containing the cell membrane-permeant Ca(2+) chelator BAPTA-AM, or the PKA blocker H-89. Treatment with 8-Br-cAMP triggered Ca(2+) waves that were blocked by H-89 and BAPTA-AM. Analysis of the secreted fluid in BAPTA-AM-treated tubules showed a 75% reduction in fluid secretion rate with increased K(+) concentration, reduced Na(+) concentration. Taken together, the results indicate that serotonin triggers cAMP and PKA-mediated Ca(2+) waves that are required for maximal ion transport rate.

The cytokines interleukin-1ß and tumor necrosis factor-α stimulate CFTR-mediated fluid secretion by swine airway submucosal glands.

The airway is kept sterile by an efficient innate defense mechanism. The cornerstone of airway defense is mucus containing diverse antimicrobial factors that kill or inactivate pathogens. Most of the mucus in the upper airways is secreted by airway submucosal glands. In patients with cystic fibrosis (CF), airway defense fails and the lungs are colonized by bacteria, usually Pseudomonas aeruginosa. Accumulating evidence suggests that airway submucosal glands contribute to CF pathogenesis by failing to respond appropriately to inhalation of bacteria. However, the regulation of submucosal glands by the innate immune system remains poorly understood. We studied the response of submucosal glands to the proinflammatory cytokines interleukin-1ß and tumor necrosis factor-α. These are released into the airway submucosa in response to infection with the bacterium P. aeruginosa and are elevated in CF airways. Stimulation with IL-1ß and TNF-α increased submucosal gland secretion in a concentration-dependent manner with a maximal secretion rate of 240 ± 20 and 190 ± 40 pl/min, respectively. The half maximal effective concentrations were 11 and 20 ng/ml, respectively. The cytokine effect was dependent on cAMP but was independent of cGMP, nitric oxide, Ca(2+), or p38 MAP kinase. Most importantly, IL-1ß- and TNF-α-stimulated secretion was blocked by the CF transmembrane conductance regulator (CFTR) blocker, CFTRinh172 (100 µmol/l) but was not affected by the Ca(2+)-activated Cl(-) channel blocker, niflumic acid (1 µmol/l). The data suggest, that during bacterial infections and resulting release of proinflammatory cytokines, the glands are stimulated to secrete fluid, and this response is mediated by cAMP-activated CFTR, a process that would fail in patients with CF.

Pseudomonas aeruginosa Homoserine lactone activates store-operated cAMP and cystic fibrosis transmembrane regulator-dependent Cl- secretion by human airway epithelia.

The ubiquitous bacterium Pseudomonas aeruginosa frequently causes hospital-acquired infections. P. aeruginosa also infects the lungs of cystic fibrosis (CF) patients and secretes N-(3-oxo-dodecanoyl)-S-homoserine lactone (3O-C12) to regulate bacterial gene expression critical for P. aeruginosa persistence. In addition to its effects as a quorum-sensing gene regulator in P. aeruginosa, 3O-C12 elicits cross-kingdom effects on host cell signaling leading to both pro- or anti-inflammatory effects. We find that in addition to these slow effects mediated through changes in gene expression, 3O-C12 also rapidly increases Cl(-) and fluid secretion in the cystic fibrosis transmembrane regulator (CFTR)-expressing airway epithelia. 3O-C12 does not stimulate Cl(-) secretion in CF cells, suggesting that lactone activates the CFTR. 3O-C12 also appears to directly activate the inositol trisphosphate receptor and release Ca(2+) from the endoplasmic reticulum (ER), lowering [Ca(2+)] in the ER and thereby activating the Ca(2+)-sensitive ER signaling protein STIM1. 3O-C12 increases cytosolic [Ca(2+)] and, strikingly, also cytosolic [cAMP], the known activator of CFTR. Activation of Cl(-) current by 3O-C12 was inhibited by a cAMP antagonist and increased by a phosphodiesterase inhibitor. Finally, a Ca(2+) buffer that lowers [Ca(2+)] in the ER similar to the effect of 3O-C12 also increased cAMP and I(Cl). The results suggest that 3O-C12 stimulates CFTR-dependent Cl(-) and fluid secretion in airway epithelial cells by activating the inositol trisphosphate receptor, thus lowering [Ca(2+)] in the ER and activating STIM1 and store-operated cAMP production. In CF airways, where CFTR is absent, the adaptive ability to rapidly flush the bacteria away is compromised because the lactone cannot affect Cl(-) and fluid secretion.

The antidiuretic neurohormone RhoprCAPA-2 downregulates fluid transport across the anterior midgut in the blood-feeding insect Rhodnius prolixus.

Osmotic balance in insects is regulated by the excretory system, consisting of Malpighian tubules and the gut under the control of diuretic and antidiuretic factors. Terrestrial insects must conserve water, and antidiuresis is the norm, only interrupted by brief diuretic periods. Surprisingly, little is known about antidiuresis in insects. Two antidiuretic strategies have been described. The first antidiuretic mechanism involves the reabsorption of fluid from the primary urine in the hindgut. More recently, a second antidiuretic strategy was reported, consisting of inhibition of primary urine formation by the Malpighian tubules. Recently, we isolated, characterized, and cloned the gene encoding for the antidiuretic neurohormone (the neuropeptide RhoprCAPA-2) acting on the Malpighian tubules of Rhodnius prolixus. Here we describe a third, novel mechanism central to the antidiuretic strategy of R. prolixus, the inhibition of ion and fluid transport across the anterior midgut by RhoprCAPA-2. Our results show that RhoprCAPA-2 (1 micromol/l) reduces serotonin-stimulated fluid transport from 83 +/- 11 to 12 +/- 12 nl/min and equivalent short-circuit current from 20 +/- 4 to 5 +/- 0.7 microA/cm(2) in diuretic hormone-stimulated anterior midgut. RhoprCAPA-2 appears to function independently of intracellular cGMP or Ca(2+) in the midgut. Thus, the antidiuretic neurohormone RhoprCAPA-2 has multiple target tissues, and we hypothesize that RhoprCAPA-2 functions to coordinate the transport activity of the anterior midgut and Malpighian tubules so that the rate of fluid transport into the haemolymph by the anterior midgut matches the transport rate of Malpighian tubules to maintain the volume and ion composition of haemolymph.

Biological activity of diuretic factors on the anterior midgut of the blood-feeding bug, Rhodnius prolixus.

Probing of a host and ingestion of a blood-meal in a fifth instar Rhodnius prolixus results in a cascade of tightly integrated events, including salivary gland secretion, plasticization of the abdominal cuticle, increased ion and water movement across the anterior midgut (crop) and Malpighian tubules (which rapidly produce urine) and the regular expulsion of urine from the hindgut. In this study we have focussed on the role of the anterior midgut during the rapid postprandial diuresis. The huge blood-meal is pumped into the anterior midgut, during feeding, then modified by diuresis and stored until it is digested. Changes in the anterior midgut activity are rapid. Within minutes of the commencement of feeding there is an increase in the frequency of anterior midgut contractions and diuresis begins with the movement of salt and water across the epithelium of the anterior midgut into the haemolymph. While serotonin, a diuretic hormone in R. prolixus, is known to play a role in the physiological activity of the anterior midgut, we were interested in exploring further the role of serotonin, and other diuretic peptides. We have tested the activity of several peptides, including R. prolixus calcitonin-like diuretic hormone (Rhopr-DH 31), corticotropin-releasing factor (CRF)-like peptide from Zootermopsis nevadensis DH (Zoone-DH) and a kinin from Leucophaea maderae, Leucokinin 1 (LK1). These peptides families are known to be present in the central nervous system of R. prolixus, are putative neurohormones released into the haemolymph after the start of feeding, and have been shown to have activity on a variety of tissues involved in post-feeding diuresis. We show here that both serotonin and Zoone-DH increase the cAMP content of the anterior midgut and that serotonin, Zoone-DH and cAMP analogues increase absorption of water from the anterior midgut, increase the short circuit current and voltage, while decreasing the resistance across the epithelium. While LK1 and Rhopr-DH 31 do not significantly increase absorption, or short circuit current, LK1 does significantly decrease the resistance and transepithelial voltage of the anterior midgut epithelium. All of the factors studied increase the frequency of contractions of the anterior midgut.

Substance P stimulates CFTR-dependent fluid secretion by mouse tracheal submucosal glands.

The mucosa of the proximal airways defends itself and the lower airways from inhaled irritants such as capsaicinoids, allergens, and infections by several mechanisms. Sensory nerves monitor the luminal microenvironment and release the tachykinin substance P (SP) to stimulate mucus secretion. Here, we have studied the role of the cystic fibrosis transmembrane conductance regulator (CFTR) in SP stimulation by comparing mouse airway submucosal gland responses in wild-type (WT) and CFTR-/- mice. Capsaicinoids (chili pepper oil) increased fluid secretion by glands from WT mice five-fold, and this response was abolished by exposing the basolateral aspect of the tracheas to L-732,138 (10 micromol/l), a specific antagonist of the neurokinin-1 receptor. Secretion was also stimulated 25-fold by basolateral application of SP, and this response was strongly inhibited by the CFTR inhibitor CFTR(inh)172. In contrast, submucosal glands from CFTR knockout mice failed to secrete when stimulated by SP (1 micromol/l), although those from wild-type control littermates were responsive. SP stimulation of wild-type glands was also abolished by clotrimazole (25 micromol/l), a blocker of Ca(2+)-activated K(+) channels. These results indicate that SP mediates local responses to capsaicinoids through a mechanism involving coordinated activation of CFTR and K(+) channels. To our knowledge, this is the first study in which CFTR-dependent responses to substance P have been directly demonstrated. Since CFTR regulation is qualitatively similar in human and mouse glands, loss of this local regulation in CF may contribute to reduced innate defenses in CF airways.

Synergistic airway gland mucus secretion in response to vasoactive intestinal peptide and carbachol is lost in cystic fibrosis.

Cystic fibrosis (CF) is caused by dysfunction of the CF transmembrane conductance regulator (CFTR), an anion channel whose dysfunction leads to chronic bacterial and fungal airway infections via a pathophysiological cascade that is incompletely understood. Airway glands, which produce most airway mucus, do so in response to both acetylcholine (ACh) and vasoactive intestinal peptide (VIP). CF glands fail to secrete mucus in response to VIP, but do so in response to ACh. Because vagal cholinergic pathways still elicit strong gland mucus secretion in CF subjects, it is unclear whether VIP-stimulated, CFTR-dependent gland secretion participates in innate defense. It was recently hypothesized that airway intrinsic neurons, which express abundant VIP and ACh, are normally active and stimulate low-level gland mucus secretion that is a component of innate mucosal defenses. Here we show that low levels of VIP and ACh produced significant mucus secretion in human glands via strong synergistic interactions; synergy was lost in glands of CF patients. VIP/ACh synergy also existed in pig glands, where it was CFTR dependent, mediated by both Cl(-) and HCO(3) (-), and clotrimazole sensitive. Loss of "housekeeping" gland mucus secretion in CF, in combination with demonstrated defects in surface epithelia, may play a role in the vulnerability of CF airways to bacterial infections.

Mucus secretion by single tracheal submucosal glands from normal and cystic fibrosis transmembrane conductance regulator knockout mice.

Submucosal glands line the cartilaginous airways and produce most of the antimicrobial mucus that keeps the airways sterile. The glands are defective in cystic fibrosis (CF), but how this impacts airway health remains uncertain. Although most CF mouse strains exhibit mild airway defects, those with the C57Bl/6 genetic background have increased airway pathology and susceptibility to Pseudomonas. Thus, they offer the possibility of studying whether, and if so how, abnormal submucosal gland function contributes to CF airway disease. We used optical methods to study fluid secretion by individual glands in tracheas from normal, wild-type (WT) mice and from cystic fibrosis transmembrane conductance regulator (CFTR) knockout mice (Cftr(m1UNC)/Cftr(m1UNC); CF mice). Glands from WT mice qualitatively resembled those in humans by responding to carbachol and vasoactive intestinal peptide (VIP), although the relative rates of VIP- and forskolin-stimulated secretion were much lower in mice than in large mammals. The pharmacology of mouse gland secretion was also similar to that in humans; adding bumetanide or replacement of HCO(3)(-) by Hepes reduced the carbachol response by approximately 50%, and this inhibition increased to 80% when both manoeuvres were performed simultaneously. It is important to note that glands from CFTR knockout mice responded to carbachol but did not secrete when exposed to VIP or forskolin, as has been shown previously for glands from CF patients. Tracheal glands from WT and CF mice both had robust secretory responses to electrical field stimulation that were blocked by tetrodotoxin. It is interesting that local irritation of the mucosa using chili pepper oil elicited secretion from WT glands but did not stimulate glands from CF mice. These results clarify the mechanisms of murine submucosal gland secretion and reveal a novel defect in local regulation of glands lacking CFTR which may also compromise airway defence in CF patients.

Secretion of water and ions by malpighian tubules of larval mosquitoes: effects of diuretic factors, second messengers, and salinity.

The effects of changes in the salinity of the rearing medium on Malpighian tubule fluid secretion and ion transport were examined in larvae of the freshwater mosquito Aedes aegypti and the saltwater species Ochlerotatus taeniorhynchus. For unstimulated tubules of both species, the K(+) concentration of secreted fluid was significantly lower when larvae were reared in 30% or 100% seawater (O. taeniorhynchus only), relative to tubules from freshwater-reared larvae. The Na(+) concentration of secreted fluid from unstimulated tubules of O. taeniorhynchus reared in 30% or 100% seawater was higher relative to tubules from freshwater-reared larvae. The results suggest that changes in salinity of the larval rearing medium lead to sustained changes in ion transport mechanisms in unstimulated tubules. Furthermore, alterations of K(+) transport may be utilized to either conserve Na(+) under freshwater (Na(+)-deprived) conditions or eliminate more Na(+) in saline (Na(+)-rich) conditions. The secretagogues cyclic AMP [cAMP], cyclic GMP [cGMP], leucokinin-VIII, and thapsigargin stimulated fluid secretion by tubules of both species. Cyclic AMP increased K(+) concentration and decreased Na(+) concentration in the fluid secreted by tubules isolated from O. taeniorhynchus larvae reared in 100% seawater. Interactions between rearing salinity and cGMP actions were similar to those for cAMP. Leucokinin-VIII and thapsigargin had no effect on secreted fluid Na(+) or K(+) concentrations. Results indicate that changes in rearing medium salinity affect the nature and extent of stimulation of fluid and ion secretion by secretagogues.

Effects of cortisol and prolactin on Na+ and Cl- transport in cultured branchial epithelia from FW rainbow trout.

The electrophysiological and ion-transporting properties of cultured gill epithelia from freshwater (FW) rainbow trout were examined in the presence of cortisol and prolactin as media supplements. Epithelia were of the double-seeded insert (DSI) type containing both pavement cells (PVCs) and mitochondria-rich cells (MRCs) and were grown in Leibovitz's L15 media on filters allowing exposure to different apical media conditions. Experiments were carried out in two series after 7-9 days symmetrical (L15 apical-L15 basolateral) culture. In both series, 100% L15 was maintained as the basolateral medium throughout and supplemented with physiologically relevant doses of either prolactin (50 ng/ml), cortisol (500 ng/ml), or cortisol + prolactin (500 + 50 ng/ml, respectively). In series 1, epithelia were exposed to progressively diluted apical media (100, 75, 50, 25, 12.5% L15, and FW) at 24-h intervals. The preparations retained integrity [high transepithelial resistance (TER); low ion efflux rates] during this prolonged dilution protocol. Cortisol, or cortisol + prolactin, resulted in a greater TER and reduced ion efflux rates during dilution, likely an effect on junctional permeability of PVCs, but did not promote active Na+ and Cl- uptake from apical FW. In series 2, epithelia were directly exposed to apical FW and ion fluxes measured over the first 6 h. Under these conditions, cortisol or cortisol + prolactin promoted active uptake of both Na+ and Cl- simultaneously from apical FW, probably attributable to actions on the MRCs. However, Na+-K+-ATPase activities were not significantly altered by any of the treatments in either series. Overall, prolactin alone did not appear to promote FW adaptation but exhibited synergism with cortisol. These results provide further support for the cultured DSI epithelium as an in vitro model for ion transport in FW fish.

Amino acids modulate ion transport and fluid secretion by insect Malpighian tubules.

Insect haemolymph typically contains very high levels of free amino acids. This study shows that amino acids can modulate the secretion of ions and water by isolated Malpighian tubules of Rhodnius prolixus and Drosophila melanogaster. Secretion rates of Rhodnius tubules in amino-acid-free saline increase after addition of serotonin to a peak value, then slowly decline to a plateau. Addition of glutamine, glutamate or aspartate to such tubules increases secretion rates dramatically relative to the controls in amino-acid-free saline, and these increases are sustained for 1-2 h. Seven other amino acids have more modest stimulatory effects, whereas lysine and arginine are inhibitory. Secreted fluid pH and Na(+) concentration increase and K(+) concentration decreases in response to glutamine. Pre-incubation of unstimulated tubules in saline solutions containing amino acids followed by stimulation with serotonin in amino-acid-free saline shows that the effects of amino acids far outlast the duration of exposure to them. Amino acids do not appear to be important as metabolites in Rhodnius tubules, nor do they act to draw significant amounts of water into the lumen by osmosis. Significant stimulation of fluid secretion can be achieved by physiological levels of particular amino acids, whereas those amino acids that inhibit fluid secretion only do so at concentrations much above those at which they occur naturally in the haemolymph. Secretion rates of unstimulated or stimulated Drosophila tubules are increased by pre-incubation in saline solutions containing glutamine or methionine or by continuous exposure to glutamine, methionine or tyrosine. Cysteine dramatically inhibited fluid secretion by Drosophila tubules, but only at concentrations well above the physiological range. We suggest that the amino acids probably function as compatible intracellular osmolytes that are necessary for sustained secretion at high rates by the Malpighian tubules.

Inorganic and organic anion transport by insect renal epithelia.

Insect renal organs typically exhibit high rates of transport of inorganic and organic anions, and therefore provide useful models for the study of epithelial anion transport and its control. Isolated Malpighian tubules of some species secrete a volume of iso-osmotic fluid equal to their own volume in 10-15 s, which means that cellular Cl(-) content is exchanged every 3-5 s. Anion transport can also be achieved against extreme thermodynamic gradients. The concentration of K(+) and Cl(-) in the lumen of the Malpighian tubules of some desert beetles approaches or exceeds saturation. A basolateral Na(+):K(+):2Cl(-) cotransporter plays an important role in vectorial ion transport in Malpighian tubules of many species, but there is also evidence for coupling of Cl(-) transport to the movement of a single cationic species (Na(+) or K(+)). Although an apical vacuolar H(+)-ATPase plays a primary role in energizing transepithelial secretion of chloride via channels or cotransporters in the secretory segment of the Malpighian tubule, several different ATPases have been implicated in reabsorption of Cl(-) by the lower Malpighian tubule or hindgut. Chloride transport is known to be controlled by several neuropeptides, amines and intracellular second messengers. Insect renal epithelia are also important in excretion of potentially toxic organic anions, and the transporters involved may play a role in resistance to insecticides of natural or anthropogenic origin.