Search

The transport of a hydrolysis-resistant dipeptide, D-phenylalanyl-L-alanine (D-Phe-L-Ala), has been studied by high-performance liquid chromatography in rat lung epithelial cells and apical membrane vesicles. Time-dependent uptake of D-Phe-L-Ala into isolated type II pneumocytes was shown. Uptake was saturable, and Michaelis-Menten kinetics were fitted to the data and gave an apparent Michaelis constant (Km) of 3.4 mM and a maximum velocity (Vmax) of 7.0 nmol.mg protein-1.min-1. However, known peptide transport inhibitors unexpectedly increased intracellular D-Phe-L-Ala concentration when initial rates of peptide uptake were studied. Apical (brush-border) membrane vesicles prepared from rat lung also showed time- and concentration-dependent influx of D-Phe-L-Ala (apparent Km 2.0 mM, Vmax 0.53 nmol.mg protein-1.min-1). Influx of this neutral dipeptide into the vesicles was shown to be both electrogenic and stimulated by an inwardly directed proton gradient. Influx was inhibitable by mercuric chloride and by the amino acid residue modifying compounds N-acetylimidazole and diethylpyrocarbonate. These findings strongly suggest the presence of a proton-coupled peptide transport protein in the apical surface of the type II cell. This transporter may play a role in lung homeostasis.

Active sodium transport contributes to liquid clearance from the alveoli. We hypothesized that the magnitude of active transport of alveolar liquid depends on the extent to which the alveolar epithelium is stretched and, consequently, on the degree of alveolar inflation. In a study on 38 adult rabbits, the left lung was filled in vivo with a solution of glucose (10 mmol/l) made isosmotic with plasma, using sodium chloride, and held at a constant airway pressure of 3, 6, or 9 cmH2O for 6 h. Alveolar liquid clearance was measured directly as a flow into a left main bronchial catheter. Control animals were compared with animals in which active epithelial sodium transport was inhibited by adding amiloride and phloridzin (both 1 mmol/l) to the instillate. At low inflation, active sodium transport reversed a secretion of liquid into the alveoli; at high inflation, active sodium transport made little or no contribution to transepithelial flow. Hydraulic conductance of the left lung was 1.57 microliters.min-1.cmH2O-1.kg body wt-1. The experiments suggest that pulmonary inflation renders active liquid clearance ineffective.

Transport of L-alanyl-D-phenylalanyl-L-alanine was investigated with an in situ vascular perfusion preparation of rat lung and brush border membrane vesicles prepared from type II pneumocytes. In the perfused lung 1 mM tripeptide was transported intact from the alveolar lumen to the vascular perfusate at a mean rate of 25.1 +/- 1.29 (3) nmol/min per g dry weight. D-Phenylalanine also appeared in the vascular perfusate at a rate of 21.9 +/- 1.74 (3) nmol/min per g dry weight indicating that 47% of the absorbed tripeptide was split during passage across the epithelial layer. No dipeptide could be detected in the vascular effluent during perfusions with tripeptide. Rapid L-alanyl-D-phenylalanyl-L-alanine uptake occurred with fresh apical membrane vesicles prepared from type II pneumocytes and this was abolished by treatment with 0.1% triton. The related tripeptide, D-alanyl-L-phenylalanyl-D-alanine, was taken up significantly more slowly by the vesicles. D-phenylalanyl-L-alanine and D-phenylalanyl-D-alanine, were also studied with the vascularly perfused preparation; the mixed dipeptide appeared in the vascular perfusate significantly faster than L-alanyl-D-phenylalanyl-L-alanine whereas D-phenylalanyl-D-alanine appeared more slowly and was not hydrolysed.

We have studied chloride influx and efflux in a highly purified preparation of type II cells freshly isolated from adult guinea-pig lung using 36Cl-. Chloride uptake was time-dependent, saturable (Km < 10 mM) and was inhibited by 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS; Ki approximately 80 microM). In the absence of external chloride (substituted by gluconate), 36Cl- uptake exhibited an overshoot above equilibrium. The rate of 36Cl- entry was strongly inhibited by addition of external nitrate; sulphate was a weaker inhibitor. 36Cl- efflux was stimulated by external bromide > bicarbonate > or = chloride > or = citrate; and was inhibited by propionate > acetate > oxalate. Although the "chloride channel blocker" 4-nitro-2-(3-phenylpropylamino)benzoate (0.14 mM) caused an inhibition, 36Cl- influx did not appear to be electrogenic. These data are compatible with the existence of a substantial electroneutral anion-exchange pathway for chloride transport in freshly isolated adult type II pneumocytes.

Previous in vivo studies of sugar transport across the mature pulmonary epithelium have provided evidence for the existence of a specific phlorizin-inhibitable, sodium-dependent transport process for D-glucose, although no direct evidence for the cellular location of this transport system in fresh cells has been shown to date. With the use of elastase digestion and lectin agglutination, a pure preparation of type II alveolar epithelial cells was isolated from adult guinea pig lung. This preparation always contained >90% type II cells and typically showed approximately 85% cell viability 2-3 h after the isolation procedure had begun. At 37 degrees C, cells showed specific [3H]phlorizin binding that was attenuated by D-glucose and completely abolished by sodium replacement. Substantial accumulation of the hexose [14C]methyl alpha-D-glucopyranoside (14C-labeled AMG), a substrate specific for the sodium-dependent glucose cotransporter was found in the presence of extracellular sodium; this accumulation above equilibrium was abolished on removal of sodium, addition of phlorizin, or in the presence of a saturating concentration (69 mM) of D-glucose. The apparent inhibition constant (Ki) for glucose inhibition of AMG uptake was 0.4 mM and for phlorizin, 0.5 microM. The Hill plot of sodium activation of AMG uptake gave a coefficient of 2.8, suggesting cooperativeness between sodium and AMG transport. 3-O-[14C]methyl-D-glucose (3-OMG) transport was also blocked by phlorizin. Phloretin, in the presence of phlorizin, slowed the initial rate of entry but did not affect the equilibrium that was attained in the presence of phlorizin alone.(ABSTRACT TRUNCATED AT 250 WORDS)

The patch clamp technique was used to record whole cell K+ currents in type II pneumocytes freshly isolated from adult rats. Depolarizing voltage steps evoked outward K+ currents which were distinguished into low and high threshold types, only one type being apparent in any one cell. Low-threshold (LT) currents were activated at test potentials of -40 mV to -20 mV and were reduced in amplitude by 20 mM tetraethylammonium (TEA). High-threshold (HT) currents were activated only at test potentials positive to -20 mV and current noise was always greater than for LT currents. HT currents were also significantly more sensitive than were LT currents to block by TEA. Quinine (1 mM) blocked LT currents reversibly at all activating test potentials. HT currents were also reversibly blocked by 1 mM quinine, but in a voltage-dependent manner, the degree of block increasing with increasing test potential. 4-Aminopyridine (2 mM) further distinguished the two current types: it was virtually without effect on HT currents but caused large reductions in LT current amplitudes, apparently by acting on the open channels underlying this current. These data clearly distinguish type II pneumocytes into two subpopulations and suggest that they may play separate roles in the functioning of the intact alveolar epithelium.

1. In the chronically catheterized sheep fetus between 122 and 143 days gestation the concentration of D-glucose in lung liquid was very low (usually less than 0.01 mM, the lower limit of detection of the analytical method) whereas the mean plasma concentration was 0.19 mM (S.E.M. 0.4, n = 13). 2. When the lung liquid concentration of D-glucose was raised to 1.67-5.00 mM, rapid uptake was observed until the concentration had fallen to its preceding low level. The uptake showed saturation kinetics (Vmax = 2.29-8.78 mumol/min, increasing with gestation; mean Km = 0.14 +/- 0.02 mM, n = 11, no change with gestation). This active uptake of glucose was blocked by phloridzin (10(-4) M). It was associated with a decrease in lung liquid secretion rate from which a change in net sodium flux could be inferred of an order suggesting one-to-one glucose-sodium co-transport. 3. Radiolabelled 3-O-methyl-D-glucose (3-O-meG) - a monosaccharide which is transported but not metabolized - was taken up rapidly from lung liquid and this rapid uptake was inhibited by D-glucose with 50% inhibition at 0.35 mM (+/- 0.08, n = 9). It was also inhibited by phloridzin (10(-4) M). 4. Radiolabelled 2-deoxy-D-glucose - a monosaccharide which is not a substrate for sodium-coupled transport - was taken up only very slowly from lung liquid; the rate of uptake was appropriate for passive diffusional transport and it was unaffected by the addition of D-glucose or phloridzin to lung liquid. 5. Intravenous infusion of D-glucose caused no detectable increase in the concentration of glucose in lung liquid unless phloridzin was added, when a slow increase was observed. 6. In two experiments with active transport blocked by phloridzin in lung liquid (10(-4) M), the rate of entry of labelled 3-O-meG from plasma to lung liquid was measured during intravenous infusion of this tracer for 29 and 23 h. The rates of entry were similar to the rate of efflux of the tracer from lung liquid when uptake was blocked by phloridzin or D-glucose, and similar to the rate expected for a metabolically inert tracer (i.e. it was some two orders of magnitude less than efflux from lung liquid in the absence of an inhibitor).(ABSTRACT TRUNCATED AT 400 WORDS)

Recent studies done with fetal and adult sheep and with monolayers of cultured rat alveolar type II cells suggest that active transport of Na+ across the lung epithelium may contribute to liquid absorption from air spaces, an essential component of the normal switch from placental to pulmonary gas exchange at birth. The goals of this work were 1) to study the ontogeny of cation transport in lung epithelial cells derived from fetal, newborn, and adult rabbits and 2) to determine the influence of premature birth, air breathing, labor, and postnatal lung maturation on K+ uptake in these cells. We harvested granular pneumonocytes by tracheal instillation of proteolytic enzymes followed by centrifugation of the dispersed cells over a discontinuous density gradient of metrizamide. This procedure yielded 65-90% granular pneumonocytes, of which more than 80% excluded vital dye. Using freshly isolated cells, we measured uptake of 86Rb+, which mimics transmembrane movement of K+, in the presence or absence of 10(-4) M ouabain and in the presence or absence of 5 X 10(-4) M furosemide or bumetanide. In adult rabbit studies, 86Rb+ uptake was twice as fast in lung epithelial cells (98 +/- 7 nmol X 10(6) cells-1 X h-1) as it was in alveolar macrophages (51 +/- 6 nmol X 10(6) cells-1 X h-1). Ouabain inhibited 55-60% of the uptake by pneumonocytes, and "loop" diuretics inhibited an additional 15-20%. The rate of 86Rb+ uptake in fetal cells was less than 10% (6 +/- 1 nmol X 10(6) cells-1 X h-1) of the rate in adult cells; ouabain inhibited 80-85% of 86Rb+ uptake in fetal cells.(ABSTRACT TRUNCATED AT 250 WORDS)