Cigarette smoke inhibition of ion transport in canine tracheal epithelium.

Inhalation of cigarette smoke is known to impair pulmonary mucociliary clearance. Active ion transport by airway epithelium plays an important role in maintaining effective mucociliary clearance by regulating the volume and composition of the airway secretions. To determine the effect of cigarette smoke on airway epithelial ion transport, the electrical properties and transepithelial Na and Cl fluxes were measured in canine tracheal epithelium. In vivo, the inhalation of the smoke from one cigarette acutely and reversibly decreased the electrical potential difference across the tracheal epithelium. In vitro, exposure of the mucosal surface of the epithelium to cigarette smoke decreased the short circuit current and transepithelial resistance. The decrease in short circuit current was due to an inhibition of the rate of Cl secretion with minimal effect on the rate of Na absorption. The effect of cigarette smoke was reversible, was not observed upon exposure of the submucosal surface to smoke, and was most pronounced when secretion was stimulated. The particulate phase of smoke was largely responsible for the inhibitory effect, since filtering the smoke minimized the effect. The effect of cigarette smoke was not prevented by addition of antioxidants to the bathing solutions, suggesting that the inhibition of Cl secretion cannot be entirely attributed to an oxidant mechanism. These results indicate that cigarette smoke acutely inhibits active ion transport by tracheal epithelium, both in vivo and in vitro. This effect may explain, in part, both the abnormal mucociliary clearance and the airway disease observed in cigarette smokers.     

Intracellular chloride activities in canine tracheal epithelium. Direct evidence for sodium-coupled intracellular chloride accumulation in a chloride-secreting epithelium.

Canine tracheal epithelium secretes Cl via an electrogenic transport process that appears to apply to a wide variety of secretory epithelia. To examine the mechanisms involved, intracellular chloride activity, acCl, was measured with Cl-selective intracellular microelectrodes. The results indicate that when the rate of secretion was minimal acCl was 37 mM; with stimulation of secretion the intracellular voltage depolarized, but acCl was not significantly altered, at 39 mM. These findings indicate that: (a) Cl is accumulated across the basolateral membrane under nonsecreting and secreting conditions at an activity 3.8 and 2.4 times, respectively, that predicted for an equilibrium distribution; (b) Cl exit across the apical membrane may be passive with an electrochemical driving force of 22 mV; and (c) stimulation of secretion enhanced the rate of Cl entry across the basolateral membrane, since Cl transport increased without a change in acCl. In the absence of Na in the extracellular fluid, acCl approached the value expected for an equilibrium distribution. This finding suggests that "uphill" entry of Cl into the cell against its electrochemical gradient is dependent upon, and energized by, the entry of Na down its gradient. Submucosal bumetanide, a loop diuretic, also decreased the rate of Cl secretion and decreased acCl, indicating an inhibition of Cl entry. These findings indicate that Cl entry into the cell is directed against its electrochemical gradient and is mediated by a Na-coupled, bumetanide-inhibitable, transport process at the basolateral membrane and that Cl may exit passively down a favorable electrochemical gradient across the apical membrane.     

Energetics of chloride secretion in canine tracheal epithelium. Comparison of the metabolic cost of chloride transport with the metabolic cost of sodium transport

Canine tracheal epithelium secretes Cl and absorbs Na; the energy for both is derived from the activity of the basolateral membrane Na-K-ATPase. These properties allowed us to examine the energetics of Cl secretion by directly comparing the metabolic cost of Cl transport with the metabolic cost of Na transport. We measured the change in short-circuit current and O2 consumption rate that was produced by a variety of maneuvers that alter Na and/or Cl transport rate. The experimental interventions included the use of the secretagogue epinephrine, the Cl transport inhibitor bumetanide, the Na transport inhibitor amiloride, the Na-K-ATPase inhibitor ouabain, and ion substitutions. The O2 consumption rates required for Na and Cl transport were compared in each individual tissue. The results indicate that the oxygen cost of Cl transport is significantly less than the oxygen cost of Na transport: two to four Cl ions are transported for the same metabolic cost that is required to transport one Na ion. These findings suggest that the basolateral membrane Na-dependent Cl entry step couples the entry of more than one Cl ion to each Na ion.     

Proteinases of Pseudomonas aeruginosa evoke mucin release by tracheal epithelium.

We have determined the potential of exoproducts from pathogenic bacteria to stimulate the release of high molecular weight mucins from goblet cells of airway epithelium in a rabbit tracheal explant system. Culture supernatants from proteolytic strains of Pseudomonas aeruginosa and Serratia marcescens, but not supernatants from a number of non-proteolytic strains, released mucins from goblet cells. Highly purified elastase and alkaline proteinase from P. aeruginosa stimulated goblet cell mucin release in a dose-dependent fashion. Lipopolysaccharide, exotoxin A, and alginate of P. aeruginosa did not possess mucin release properties. Proteolytic activity was required for mucin release by P. aeruginosa elastase, but such release in goblet cells was not mediated by cyclic AMP. Morphologic studies suggested rapid release of mucins from goblet cells was response to elastase by a process resembling apocrine secretion. Several nonbacterial proteinases mimicked the effect of Pseudomonas proteases. These studies provide support for the hypothesis that bacterial and other play a role in the pathogenesis of mucus hypersecretion in acute and chronic lung infections.     

Cyclic adenosine monophosphate-dependent kinase in cystic fibrosis tracheal epithelium.

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.     

In vitro alterations of tracheal epithelium of hamsters by carcinogens in organ culture

In vitro organ culture of the hamster's trachea was improved and applied to a carcinogenesis research. The rotary culture enabled explants of tracheal epithelium to survive more than 8 weeks. The study was composed of 2 kinds of culture; untreated and treated with carcinogens. In the untreated culture, Eagle MEM medium had the same culture effect as RPMI 1640 medium. With prolongation of culture time (particularly longer than 5 weeks), irreversible degenerative changes appeared in epithelial cells. Culture for 4 weeks was usually thought to be appropriate for experimental research. In the treated culture, the effect of benzo-(a) pyrene (B(a)P) and B(a)P + cigarette smoking condensate-neutral fraction (CSC-NF) on tracheal epithelium was investigated with light and electron microscopies (TEM and SEM) and autoradiography. Atypical hyperplasia with or without lesions suggesting carcinoma in situ was induced by B(a)P + CSC-NF more evidently and frequently than by B(a)P alone. The present findings corroborated the cocarcinogenetic effect of CSC-NF.     

Adrenoceptor-mediated mechanical responses of canine tracheal smooth muscle

The effect of tone on responses of canine tracheal smooth muscle (TSM) to norepinephrine (NE) was studied to elucidate the role of sympathetic innervation and adrenoceptors in the control of the airways. Electrical field stimulation produced contraction of TSM in vitro which was augmented by eserine, depressed by phentolamine, potentiated by propranolol in the presence of K+ (14 mM) and almost eliminated by tetrodotoxin or atropine. Resting TSM did not contract in response to NE (10(-8) to 10(-4) M) in the presence or absence of propranolol (10(-5)M). The addition of NE (10(-8) to 10(-6) M) at the plateau of contraction produced by K+ (22.8 mM), histamine (10(-6) M) or acetylcholine (5 X 10(-8) M) produced a further phentolamine-sensitive contraction which was potentiated by beta adrenoceptor blockade with propranolol (10(-5) M). The addition of tyramine (10(-5) to 10(-4) M) at the plateau of contraction produced by K+ (22.8 mM) produced a further contraction which was potentiated by propranolol (10(-5) M) and reduced by phentolamine (10(-5) M). Although the response to NE in the presence of elevated tone was contractile at low concentrations of NE (10(-8) to 10(-6) M), a propranolol-sensitive relaxant response was elicited at higher NE concentrations (10(-5) and 10(-4) M). Maximum contractions to NE in the absence or presence of beta-blockade were dependent on the tone of the muscle. These findings suggest a functional adrenergic innervation of canine TSM and the presence of alpha and beta adrenoceptors which mediate contractile and relaxant responses, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relaxant effect of amiloride on canine tracheal smooth muscle

Amiloride, a K+-sparing diuretic, relaxed canine tracheal smooth muscle strips contracted isometrically with high potassium (KCl), carbachol, serotonin and histamine. This indicated that relaxation was not linked to an interaction with an agonist specific receptor. Amiloride-induced relaxation was also not mediated through the production of relaxant prostaglandins, or by the endogenous release of catecholamines. During potassium contractions, amiloride addition produced a slow monophasic, dose-dependent relaxation (IC50 = 12.3 microM). In carbachol contracted strips, 1 and 10 microM amiloride induced a slow monophasic relaxation. With 35 to 250 microM, an initial rapid phase (IC50 = 75.5 microM) was superimposed onto this slow phase (IC50 = 23.5 microM), producing a biphasic relaxation. The rates of relaxation decreased with increased external [Na+] regardless of stimulus, suggesting possible competitive inhibition of a sodium-dependent process. Exposure caused a rapid decline in tension followed by a recovery phase. Tension maintenance during potassium contraction decreased transiently upon the addition of acid to a much lesser extent. Amiloride (100 microM) depressed tension recovery after acid exposure in both cases. Based on the known actions of this drug, inhibition of the Na+-H+ antiporter appears to be consistent with these data. This suggests amiloride may well belong to a new class of smooth muscle relaxants.     

Stimulation of active and passive sodium absorption by sugars in the human jejunum.

The effects of glucose and fructose on water and sodium absorption in the human jejunum were compared to assess the relative contribution of active and passive sugar stimulation of sodium transport. The effect of fructose is assumed to be entirely passive, and the difference between the effects of fructose and glucose is assumed to be a measure of sugar-stimulated, active sodium absorption. Water and sodium movement with mannitol was the base line. Three sets of test solutions with differing sugar concentrations were studied. Fructose stimulated 66-100 per cent as much net sodium and water absorption as glucose. Fructose stimulated potassium absorption, whereas glucose stimulated potassium secretion. Urea absorption was stimulated by both sugars. Glucose and fructose stimulated sodium absorption when chloride was the major anion, but they had relatively little effect on net sodium movement when chloride was replaced by bicarbonate or sulfate. It is concluded that glucose stimulates passive and active sodium transport in the human jejunum. Stimulated active sodium absorption generates an electrical potential across the mucosa that causes sodium (and potassium) secretion and partly or completely nullifies the effect of active sodium transport on net sodium movement. Net sodium absorption sitmulated by glucose is mainly (66-100 per cent) the passive consequence of solvent flow. The accompanying anion determines the degree to which sugars stimulate sodium absorption (C1 greater than SO-4 greater than HCO3). The effects of bicarbonate and sugars on jejunal sodium absorption are not additive.     

Regulation of sodium and water balance by the kidney

Maintenance of consistent internal environment is essential for mammalians to survive in waterless circumstances. Extracellular fluid (ECF) volume is maintained within a narrow range in normal humans, despite day-to-day variations in the dietary intake of salt and water. In general, kidneys are the major organs that play a predominant role in the control of the fluid and electrolyte balance. Recent advances in the molecular biology have provided significant contribution to the identification of kidney specific channels and transporters for sodium and water, leading to better understandings of the regulation of sodium and water balance. Generalized edema formation, a clinical hallmark of ECF volume expansion, represents the accumulation of excessive fluid volumes in the interstitial compartment and is invariably associated with renal sodium retention. In this article, we review the regulation of sodium and water balance in the kidney to understand the renal mechanisms of edema formation.     

Electrogenic sodium absorption in rabbit cecum in vitro.

Regional variations in the ion transport properties of the colon may have significant physiological and pathophysiological implications. However, only limited studies have been performed in cecum, which comprises 50% of the macrosurface of the rabbit colon. In vitro under short-circuit conditions, cecum actively absorbed Na and Cl (JnetNa = 5.6 +/- 0.3, JnetCl = 1.5 +/- 0.3 mu eq.cm-2.h-1) with a short-circuit current (Isc) of 6.29 +/- 0.2 mu eq.cm-2.h-1.Cl substitution with sulfate decreased both JnetNa and Isc by 1.3 mu eq/cm2.h-1.HCO3 removal decreased both JnetNa and Isc 3.3 mu eq.cm-2.h-1. This effect was due primarily to removal of serosal HCO3. There was both a linear correlation between JNanet and Isc (r = 0.845) and a concentration-dependent stimulation of Isc by increasing [Na] in the bathing media. However, 10(-4) M amiloride did not significantly alter either Isc or JnetNa. In contrast, 10(-4) M phenamil, an amiloride analogue highly specific for the Na channel, significantly blocked both Isc and JnetNa. The sulfhydryl reagent PCMBS increased Isc; this response was reversed by phenamil. Electrogenic Cl secretion was stimulated by 1 mM theophylline, 10(-4) M 8BrcAMP and 10(-4) M 8BrcGMP. None of the secretagogues inhibited JnetNa. Epinephrine (5.5 microM) increased JnetNa from 5.9 +/- 1.3 to 7.8 +/- 1.1 (P = 0.02) and JnetCl from 0.1 +/- 1.2 to 2.0 +/- 0.8 (P NS) mu eq.cm-2.h-1. Studies of pH stat demonstrated an epinephrine-stimulated increase in Jm-sHCO3 without a change in Js-mHCO3. Thus, cecum exhibits a distinct type of electrogenic Na electrogenic Na absorption which is partially dependent on the presence of Cl and HCO3, not blocked by amiloride but by phenamil. Because of its large surface area and its novel mechanism of electrogenic Na transport, the cecum exerts an important regulatory role in colonic fluid and electrolyte balance.     

Anti-influenza A virus activity of amantadine hydrochloride and rimantadine hydrochloride in ferret tracheal ciliated epithelium.

The activities and toxicities of amantadine hydrochloride and rimantadine hydrochloride against influenza A/Alaska/6/77 (H3N2) and A/Bangkok/1/79 (H3N2) viruses were compared in organ cultures and ferret tracheal ciliated epithelium. Pretreatment of cultures with concentrations (0.5 and 1 micrograms/ml) comparable to those found in human serum after oral administration of amantadine revealed that rimantadine produced significantly longer protection than amantadine against virus-induced cytopathic effects. Correspondingly, rimantadine produced a comparable protective effect at four- to eight-fold-lower concentrations than amantadine. Both drugs produced increasing and similar effects at higher concentrations, which were comparable to those reported in nasal washings after aerosol administration of amantadine. At the concentrations tested, amantadine was nontoxic. However, at concentrations of 16 and 32 micrograms/ml, rimantadine was toxic to the ciliated epithelium after 10 to 21 days of continuous exposure. When the drugs were added 24 h or more after infection, protection against cytopathic effects decreased markedly. Both drugs moderately suppressed virus production at concentrations of 0.5 to 16 micrograms/ml. However, no dose response or difference between the drugs was observed. Because of comparable antiviral activity at lower concentrations and greater activity at similar concentrations, rimantadine may be more useful than amantadine for oral prophylaxis and therapy of influenza.     

5-Hydroxytryptamine receptor activity of the dopamine receptor agonist fenoldopam in canine tracheal smooth muscle.

Fenoldopam is a new vasodilator undergoing clinical trials for the treatment of hypertensive emergencies. Its pharmacologic effects result from activation of vascular dopamine-1 receptors. In canine tracheal smooth muscle strips, fenoldopam caused a concentration- and calcium-dependent increase in tension, which was not antagonized by atropine, indomethacin or the dopamine-1 receptor antagonist, SCH 23390. The EC50 (1.89 x 10(-6) M) exceeded that of serotonin or acetylcholine (8.38 x 10(-8) and 8.25 x 10(-8) M, respectively). Maximum tension was similar for fenoldopam and serotonin (11.6 +/- 1.5 g, n = 7 and 13.8 +/- 0.8 g, n = 24; P greater than .2) and considerably greater for acetylcholine (20.5 +/- 1.3 g, n = 14; P less than .005). The serotonin antagonists ketanserin and methysergide reversed completely the effect of fenoldopam (5 x 10(-7) M) with IC50 values of 2.5 x 10(-9) and 2.7 x 10(-9) M, respectively. Phentolamine, rauwolscine and chlorpheniramine were also effective, but they were less potent (IC50 values 6.6 x 10(-8), 1.0 x 10(-7) and 1.7 x 10(-7) M, respectively). By contrast, only very high concentrations (IC50, 5.3 x 10(-5) M) of terazosin produced an inhibition of fenoldopam-induced tension increases. The effect of antagonists could be overcome by increasing the fenoldopam concentration. Experiments performed on strips precontracted with serotonin (5 x 10(-8) M) revealed a very similar order of potency for the five antagonists. The addition of serotonin did not increase the tension produced by supramaximal concentrations of fenoldopam (and vice-versa), whereas acetylcholine increased tension further.(ABSTRACT TRUNCATED AT 250 WORDS)     

Augmentation of neutral sodium chloride absorption by increased flow rate in rat ileum in vivo.

Studies in intact animals have shown that intestinal solute absorption is enhanced with increasing flow rates; the mechanism of this phenomenon has not been explored in detail. We used single pass perfusions of rat ileum to study the effect of higher flow rate on electrolyte absorption. Augmenting perfusion rate from 0.5 to 5.0 ml/min resulted in increased rates of sodium (11.0 +/- 0.9 vs. 23.5 +/- 2.7 mueq/min X g) and chloride (12.1 +/- 0.8 vs. 25.0 +/- 2.2 mueq/min X g) absorption, reduction in the estimated unstirred layer thickness (668 +/- 31 vs. 433 +/- 28 micron), minimal changes in intraluminal pressure and transmural potential difference, and a small, though significant, increase in intraluminal volume (19.4 +/- 8.4%). Removal of sodium from the perfusion medium abolished the effect of increased flow rate on chloride absorption as did removal of chloride on sodium absorption; addition of furosemide or acetazolamide to Ringer's solution also inhibited this effect. In separate experiments, stepwise increases in intraluminal volume were induced by elevating the outflow tubing; no effect on electrolyte transport was observed. These studies demonstrate that neutral sodium chloride absorption is enhanced in rat ileum at higher flow rates, perhaps as a result of a decrease in the thickness of unstirred layers.     

EGTA enhancement of adenovirus-mediated gene transfer to mouse tracheal epithelium in vivo

Administration of recombinant adenoviral (AdV) vectors to animals can lead to inflammatory and immune responses. For therapeutic indications in which repeated treatment is necessary, such as cystic fibrosis (CF), these responses can limit the therapeutic usefulness of the vector. In principle, the utility of the vector can be improved by increasing its therapeutic index, that is, by either increasing its efficacy or decreasing its toxicity. A strategy that would enhance the efficacy of an adenoviral approach would allow the use of fewer virus particles to achieve a given level of transgene expression, and thereby also reduce unwanted effects such as immune responses. Following up on our observation that treating polarized normal human bronchial epithelial cells with calcium (Ca(2+))-free medium or the calcium chelator ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) significantly enhanced the subsequent transfection of these cells with cationic lipid:pDNA complexes, we have now asked whether such a treatment protocol might also improve the ability of AdV to infect these cells. Treating polarized airway epithelial cells with EGTA led to a dramatic increase in AdV-mediated transduction, as demonstrated by an approximately 50-fold increase in transgene expression. This strategy was also tested in vivo and resulted in substantial increases (up to 50-fold) in the ability of AdV vectors to infect mouse tracheal epithelium. Transfection of mouse trachea with an AdV aerosol was also significantly increased by pretreatment with EGTA. The enhancing effects of EGTA could not be duplicated with hypo- or hyperosmotic treatments. Light microscopy of mouse trachea that had been EGTA treated and then infected with AdV demonstrated an EGTA-mediated AdV infection of airway epithelial cells. The apparent enhanced potency of AdV for airway cells resulting from this strategy provides a significant increase in the therapeutic index of this gene delivery vector, and may increase the likelihood that it can be used for clinical indications requiring chronic administration of the vector.     

Motility- and blood flow-dependent absorption of amino acids in canine small intestine.

A relationship between L-phenylalanine and L-serine absorption, intestinal motility and blood flow has been studied with a canine in situ isolated jejunal loop in acute experiments and in chronic preparations in conscious dogs. During spontaneous rhythmic contractions, as well as after mechanical and pharmacological stimuli, rhythmic changes in blood flow, related to intestinal contractions, were observed. They had no bearing on mean blood flow or absorption as long as they did not occur together with tonic contractions. The tonic contractions resulted in a prolonged decrease in jejunal blood flow ano diminished amino acid absorption rate. Absorption and motility index, as well as blood flow and motility were negatively correlated. There was a positive rectlinear correlation between intestinal blood flow and absorption, regardless of whether changes in blood flow resulted from the increase in motility or were induced by intestinal artery occlusion. These correlations suggest that changes in amino acid absorption during increased motility depend on changes in blood flow. This relationship may be important in clinical syndromes with hypermotility.     

Effects of sodium bicarbonate in canine hemorrhagic shock

We studied the use of sodium bicarbonate administration in a canine model of hemorrhagic shock to determine its effect on hemodynamics, arterial and venous blood gases, respiratory gases, and blood lactate levels. Thirteen dogs were anesthetized, paralyzed, mechanically ventilated, and hemodynamically monitored. Hypotension was induced and maintained at a mean arterial pressure of 40 to 45 mm Hg using controlled hemorrhage and reinfusion. After 2.5 h of shock, the dogs were randomized into two groups: one group (n = 6) received NaCl infusion; the other (n = 7) received sodium bicarbonate (1 mEq/kg followed by a continuous infusion of 2.5 mEq/kg.h for 2.5 h). CO2 production was increased in the alkali group, but there was no statistically significant difference between groups in any measured hemodynamic, blood gas, or respiratory gas variable. These included heart rate, BP, cardiac output, arterial and venous pH, CO2 production, and bicarbonate levels. Blood lactate levels, however, in the bicarbonate treated animals were significantly (p less than .01) higher than in the group treated with NaCl alone (10.1 +/- 3.2 vs. 5.1 +/- 1.2 mEq/L). These results are similar to the effects of bicarbonate found in other models of lactic acidosis, and suggest that bicarbonate therapy may have limited usefulness in the treatment of lactic acidosis.