Estimation of thickness of airway surface liquid in ferret trachea in vitro.

The tracheae of ferrets and rabbits were mounted in vitro in organ baths. While the tracheae were liquid filled, the permeability coefficient ( P) was determined, and then while the tracheae were air filled, the percent clearance for 99mTc-labeled diethylenetriaminepentaacetic acid (DTPA) was determined. The thickness of airway surface liquid (ASL) was estimated by three methods. 1) The initial concentration of 99mTc-DTPA and the total amount of 99mTc-DTPA (the sum of that entering the outside medium, that draining from the trachea, and that washed out at the end of 40 min) gave the initial volume of ASL and thus its thickness. Mean values were 45.7 micron for the ferret and 41.9 micron for the rabbit. 2) Estimates of ASL thickness at the end of the 40-min period, based on the final 99mTc-DTPA concentration and the amount in the washout, were 42.9 micron for ferret and 45.4 micron for rabbit. 3) The ratio of P to percent clearance gave mean ASL thickness values of 49.2 micron for the ferret and 40.3 micron for the rabbit. Thus three separate methods for determining ASL thickness give very similar results, with means in the range 40-49 micron. Administration of methacholine or atropine to ferret tracheae did not significantly change ASL thickness.

H2O2 increases sheep tracheal blood flow, permeability, and vascular response to luminal capsaicin.

Exogenous hydrogen peroxide (H2O2) causes airway epithelial damage in vitro. We have studied the effects of luminal H2O2 in the sheep trachea in vivo on tracheal permeability to low-molecular-weight hydrophilic (technetium-99m-labeled diethylenetriamine pentaacetic acid; 99mTc-DTPA) and lipophilic ([14C]antipyrine; [14C]AP) tracers and on the tracheal vascular response to luminal capsaicin, which stimulates afferent nerve endings. A tracheal artery was perfused, and tracheal venous blood was collected. H2O2 exposure (10 mM) reduced tracheal potential difference (-42.0 +/- 6.4 mV) to zero. It increased arterial and venous flows (56.7 +/- 6.1 and 57.3 +/- 10.0%, respectively; n = 5, P < 0.01, paired t-test) but not tracheal lymph flow (unstimulated flow 5.0 +/- 1.2 microliters.min-1.cm-1, n = 4). During H2O2 exposure, permeability to 99mTc-DTPA increased from -2.6 to -89.7 x 10(-7) cm/s (n = 5, P < 0.05), whereas permeability to [14C]AP (-3,312.6 x 10(-7) cm/s, n = 4) was not altered significantly (-2,565 x 10(-7) cm/s). Luminal capsaicin (10 microM) increased tracheal blood flow (10.1 +/- 4.1%, n = 5) and decreased venous 99mTc-DTPA concentration (-19.7 +/- 4.0, P < 0.01), and these effects were significantly greater after epithelial damage (28.1 +/- 6.0 and -45.7 +/- 4.3%, respectively, P < 0.05, unpaired t-test). Thus H2O2 increases the penetration of a hydrophilic tracer into tracheal blood and lymph but has less effect on a lipophilic tracer. It also enhances the effects of luminal capsaicin on blood flow and tracer uptake.

Sodium metabisulphite causes epithelial damage and increases sheep tracheal blood flow and permeability.

Inhaled sodium metabisulphite (MBS) causes bronchoconstriction, cough and microvascular leakage. We have studied its effects on tracheal blood flow, potential difference (PD) and the permeability from tracheal lumen to venous blood of a low molecular weight hydrophilic tracer, 99mtechnetium-labelled diethylenetriamine penta-acetic acid (99mTc-DTPA) in anaesthetized sheep. Flow was measured in tracheal artery and blood from a cannulated tracheal vein collected for 5 min periods. The tracheal lumen was filled with Krebs-Henseleit solution (KH) containing 99mTc-DTPA for six to eight 15 min periods. During the third or fourth period, MBS (1, 20 or 100 mM) was washed into the tracheal lumen for 15 min. MBS increased tracheal blood flow (venous flow (Q'v), 5-10 min MBS exposure period: 1 mM -9 +/- 18% (n = 3); 20 mM +16 +/- 5% (n = 5; p < 0.05); 100 mM +43 +/- 13% (n = 5; p < 0.05). It decreased PD in a concentration-dependent way. Venous 99mTc-DTPA concentration increased progressively to +266 +/- 176 and +958 +/- 321% 25-30 min after exposure to 20 and 100 mM MBS, respectively (p < 0.05 for both). These effects were not blocked by luminal frusemide (3-7 mM) or flurbiprofen (100-500 microM). Histological sections showed changes to the epithelial cells and large intercellular spaces. Thus, luminal sodium metabisulphite increases tracheal blood flow, reduces transmural potential difference and causes tracheal epithelial damage, leading to an increase in 99mTc-labelled diethylenetriamine penta-acetic acid permeability.

Tracheal epithelial damage alters the effects of luminal histamine on blood flow and tracer flux in anaesthetized sheep.

Epithelial damage greatly increases the flux of a low molecular weight tracer 99mtechnetium-diethylenetriamine penta-acetic acid (99mTc-DTPA) from tracheal lumen to venous-blood in anaesthetized sheep. We have now investigated whether epithelial damage induced by the detergent, Triton X-100, alters the effects of luminal histamine on blood flow and 99mTc-DTPA flux. The cervical trachea was filled with Krebs-Henseleit solution containing 99mTc-DTPA. Tracheal arterial flow was measured and tracheal venous blood collected. The lumen was exposed to 100 microM histamine on two occasions (Hist 1 and Hist 2) for 15 min. In six out of 11 sheep, the lumen was also exposed to 0.2% Triton X-100 between Hist 1 and Hist 2. Triton X-100 increased the baseline 99mTc-DTPA permeability coefficient from -5.3 x 10(-7) to -400 +/- 130 x 10(-7) cm.s-1. After epithelial damage, Hist 2 produced significantly greater changes in arterial and venous flows than Hist 1 (n = 5) (0-5 min: Hist 1 Q1a = +6.4 +/- 0.8%, Q1v = +6.2 +/- 6.2%; Hist 2 Q1a = +36.7 +/- 12.2%, Q1v = +35.4 +/- 8.8%). Similar changes did not occur in the controls. Venous 99mTc-DTPA concentration during Hist 2 after epithelial damage (0-5 min -37.7 +/- 6.9%) was significantly different from Hist 1 (+5.2 +/- 7.0%). Thus, after epithelial damage, luminal histamine produces more rapid and larger changes in blood flow and a greater reduction in venous 99mTc-DTPA concentration.

Tracheal epithelial damage alters tracer fluxes and effects of tracheal osmolaity in sheep in vivo.

Tracheal osmolaity affects blood flow and the flux of a tracer, technetium-99m-labeled diethylenetriamine pentaacetic acid (99mTc-DTPA), from tracheal lumen to venous blood in anesthetized sheep. Hyperosmolar liquids increase blood flow and slightly decrease 99mTc-DTPA flux, whereas hyposmolar liquids have no effect on blood flow and greatly increase 99mTc-DTPA flux. We have now investigated whether epithelial damage induced by exposure of the tracheal lumen to a detergent (0.2% Triton X-100) alters these effects. A tracheal artery was perfused, and tracheal venous blood was collected. The initial tracheal volume was 12.8 +/- 0.7 ml. Triton X-100 greatly increased the permeability coefficient for 99mTc-DTPA from -2.1 x 10(-7) to -240 x 10(-7) cm/s. Hyperosmolar Krebs-Henseleit solution (KH; 739 +/- 6 mosmol/kg) increased arterial (+14.3%) and venous (+21.5%) flows and decreased 99mTc-DTPA output by 51.7%. Water flux into the lumen (+0.3 +/- 0.1 ml) was not significant, and the osmolality decreased by 99 +/- 9 mosmol/kg. Hyposmolar KH (124 +/- 2 mosmol/kg) had no effect on arterial and venous flows (-1.3% for both), and the increase in 99mTc-DTPA output (+8.3%) was small and not significant. The volume decreased by 0.4 +/- 0.1 ml, and the osmolaity increased by 36 +/- 4 mosmol/kg. Thus epithelial damage greatly increases the baseline permeability of the tracheal wall to 99mTc-DTPA. It does not alter the qualitative effects of hypersomolar KH on blood flow and 99mTc-DTPA output but does reduce the effect of hyposmolar KH on 99mTc-DTPA output. The latter effect may be a consequence of the reduced net water movement in response to non-isosmolar solutions after epithelial damage.

Osmolality alters tracheal blood flow and tracer uptake in anesthetized sheep.

Changes in the osmolality of airway surface liquid cause bronchoconstriction, mucus secretion, and ion transport, but little is known about the effects on the permeability of the trachea to drugs applied to the tracheal lumen. Using the anesthetized sheep, we have investigated the effects of hyperosmolar (725 +/- 11 mosmol/kg) and hyposmolar (128 +/- 5 mosmol/kg) Krebs-Henseleit (KH) solution in the tracheal lumen (mean volume 13.6 ml) on the uptake of technetium-99m-labeled diethylenetriamine pentaacetic acid (99mTc-DTPA), a low-molecular-mass hydrophilic tracer that is thought to cross the epithelium via paracellular pathways, and on blood flow. All changes in osmolality were made by altering the NaCl content. We perfused a tracheal artery and collected tracheal venous blood. Hyperosmolar KH increased water movement into the lumen (+2.0 ml) and solute flux out of the lumen. It increased arterial (+24.5%) and venous (+20.6%) flows and decreased 99mTc-DTPA concentration (-26.3%) and output (-12.0%) in venous blood. Hyposmolar KH caused water movement out of the lumen (-0.9 ml) and solute flux into the lumen. It had no effect on arterial (+0.6%) and venous (+5.5%) flows and greatly increased the concentration (+345%) and output (+375%) of 99mTc-DTPA in venous blood. The baseline permeability coefficient for 99mTc-DTPA (-9.1 x 10(-7) cm/s) was not affected by hyperosmolar KH (-8.7 x 10(-7) cm/s) but was increased by hyposmolar KH (-21.4 x 10(-7) cm/s). These results confirm that hyperosmolar liquid in the lumen increases blood flow and indicate that tracer uptake is affected by the bulk flow of water across the airway wall.

The effects of indomethacin and thiorphan on bradykinin-induced albumin output and submucosal gland secretion in the ferret trachea in vivo.

In the ferret liquid-filled trachea in vivo, intraluminal bradykinin (BK, 3-300 microM) produced concentration-dependent increases in the output of lysozyme from submucosal gland serous cells and albumin movement into the lumen. Baseline outputs of albumin and lysozyme were not altered significantly by intraluminal indomethacin (10 microM) or thiorphan (10 microM). However, intraluminal indomethacin completely blocked the BK-induced increase in albumin output. Intraluminal thiorphan (10 microM) did not significantly potentiate BK-induced albumin output, although mean output was higher. Neither indomethacin nor thiorphan significantly altered BK-induced lysozyme output, although mean output was reduced in the presence of indomethacin. Thus BK increases albumin output and may increase lysozyme output via the action of cyclooxygenase products. Inhibition of neutral endopeptidase activity may enhance the action of BK on albumin output.

The effects of intraluminal and extraluminal drug application on secretion and smooth muscle tone in the ferret liquid-filled trachea in vitro.

With the ferret liquid-filled trachea in vitro, intraluminal methacholine (MCh), phenylephrine (PE) and histamine (Hist) increased smooth muscle tone and salbutamol (Salb) decreased tone. Lysozyme output was increased by intraluminal MCh and PE. Albumin transport into the lumen was not altered by intraluminal Hist, Salb or PE. The concentration-response curves for smooth muscle contraction and for lysozyme output to extraluminal MCh lay to the left of those for intraluminal MCh. Indomethacin shifted the smooth-muscle response curves to MCh significantly to the left but did not significantly alter lysozyme output. Extraluminal MCh produced a concentration-dependent increase in albumin output whilst intraluminal MCh did so in one of three studies. Albumin output in response to MCh was not significantly altered by indomethacin. Thus, MCh has a less potent effect on smooth muscle and lysozyme secretion and, to a lesser extent, on epithelial albumin transport when given intraluminally. This may be because the epithelium restricts diffusion of the drug or due to the production of a non-prostanoid factor which inhibits smooth muscle responsiveness. Smooth muscle responsiveness is enhanced by blocking cyclooxygenase activity, suggesting MCh-induced release of a prostanoid with relaxant activity.

An in vivo preparation for measurement of plasma protein and lysozyme output in the ferret tracheal lumen.

An in-vivo ferret tracheal preparation has been developed to study the appearance in the liquid-filled trachea of fluorescein-labelled plasma proteins (FLP) and of lysozyme from submucosal gland serous cells. In order to investigate the influence of nervous activity on the appearance of FLP and lysozyme in the tracheal lumen, the effects of intraluminal bradykinin (an inflammatory mediator and sensory nerve stimulant), intraluminal capsaicin (a stimulant of C-fibres) and electrical stimulation of the cut peripheral end of the right cervical vagus nerve have been measured. Vagal stimulation (10 V, 10 Hz, 1 ms, 90-120 s) increased the secretory rate of lysozyme. It had no effect on FLP rate of output. Intraluminal bradykinin (100 microM) produced a small but significant increase in FLP output but had no effect on lysozyme secretion. Intraluminal capsaicin (33 microM) had no effect on FLP output and had variable effects on lysozyme output. Tracheal pressure was increased by vagal stimulation but was unaffected by bradykinin and capsaicin. Thus, bradykinin increases plasma protein output, probably by an action on the epithelium, whilst vagal stimulation and capsaicin stimulate submucosal glands. This method could be used to determine the factors which alter the rate of movement of plasma proteins into the airway lumen and the secretion of submucosal glands in vivo.

The effect of airflow on mucus secretion into the trachea of the cat.

We have investigated the effects of the passage of air and of instillation of hyperosmolal solutions in a segment of trachea of the pentobarbitone-anaesthetized cat on the release of radiolabelled mucus glycoproteins (mucins) into that segment. Ambient air passed through the segment at 11 min-1 increased the output of both 35S- and 3H-labelled mucins. It also stimulated the output of mucins measured chemically. Passage of ambient air warmed to body temperature caused a similar effect on the output of radiolabelled mucins unless the air had also been saturated with water vapour at that temperature. Instillation of cold Krebs-Henseleit solution into the tracheal segment had no effect on the release of radiolabelled mucins. The action of warmed dry air persisted after extrinsic nerves (sympathetic and parasympathetic) to the trachea had been cut. Hyperosmolal solutions placed in the tracheal segment also increased the output of both 35S- and 3H-labelled mucins. We conclude that passage of ambient air through the trachea of the cat increases mucin output. This is probably by its drying action rather than by the mechanical disturbance from the flow or by cooling. The response might occur during eupnoea but is more likely to be important during hyperpnoea such as occurs during exercise. We discuss the relevance of the response to exercise-induced asthma.

Lateral nasal gland secretion in the anaesthetized dog.

The effects of pharmacological and nervous stimuli on the flow of secretion from the dog lateral nasal gland following catheterization are described. Drugs were injected close-arterially into the arterial supply to the nose, or intravenously. Cholinergic agonists (pilocarpine, methacholine), given intravenously (I.V.) or intra-arterially (I.A.), and stimulation of the vidian nerve produced a copious flow of secretion which was blocked by atropine. The adrenoceptor agonists phenylephrine (alpha) and salbutamol (beta 2), given I.V. or I.A., and stimulation of the vagosympathetic nerve produced a small but consistent flow of secretion. Histamine (50 micrograms), substance P (0.1 micrograms) and prostaglandin E1 (1-5 micrograms), injected I.A., produced small flows of secretion. Bradykinin (25 ng-50 micrograms), 5-hydroxytryptamine (100 ng-50 micrograms) and vasoactive intestinal peptide (VIP) (10 ng-50 micrograms) did not cause secretion. The total protein content, the composition of secretions as revealed by sodium dodecyl sulphate-polyacrylamide agarose gel electrophoresis, and changes in [Na] and [K] in relation to flow of secretion are described. Differences in ion and protein concentrations, and in protein composition, are described for vidian nerve-induced and vagosympathetically induced secretions. Electron microscopy revealed that the gland contains serous cells in the secretory region, and ducts morphologically similar to the intercalated, striated and excretory ducts of salivary glands.

Control of mucoglycoprotein output from the rabbit nose.

A method for the collection of rabbit nasal washings to analyse outputs of mucous glycoproteins is described. The radiolabels sodium [35S]sulphate and [3H]glucose are bound to the glycoproteins. The release of bound 35S and 3H was enhanced by cervical sympathetic nerve stimulation. Adrenoceptor agonists (phenylephrine, dobutamine and salbutamol) given I.V. increased the output of 35S, and the last two drugs increased the output of 3H. The blocking effects of thymoxamine and propranolol on these responses are described. Pilocarpine (given I.V. or intranasally) produced large increases in 35S release; histamine had little effect. Irritants (ammonia and cigarette smoke) and diluted serum or plasma, given intranasally, produced large increases in 3H output, and sometimes enhanced 35S release. Radiolabelled nasal washings fractionated on Sepharose CL-4B gel chromatography columns formed two peaks, with most of the radioactivity in the high molecular weight (mucous glycoprotein) peak.