Transient cholinergic glycoconjugate secretion from swine tracheal submucosal gland cells TERRY M. DWYER, ATTILA SZEBENI, KATERINA DIVEKI, AND JERRY M. FARLEY Department of Physiology and Biophysics and Department of Pharmacology and Toxicology, University of Mississippi Medical Center, Jackson, Mississippi 39216-4505 Dwyer, Terry, M., Attila Szebeni, Katerina Diveki, and Jerry M. Farley. Transient cholinergic glycoconjugate secretion from swine tracheal submucosal cells. Am J. Physiol. 262 (Lung CeZl. 2MoI. Physiol. 6): L418-L426,1992. By inference, muscarinic stimulation of glycoconjugate release from tracheal submucosal gland cells appears to be a transient, nonequilibrium process (J. M. Farley and T. M. Dwyer, Life Sci. 48: 5967, 1991). To directly characterize the release kinetics of glycoconjugate, we developed an enzyme-linked lectin assay (ELLA) of much improved precision and resolution. To collect secreted products with an improved time resolution, freshly isolated swine tracheal submucosal gland cells were continuously superfused with medium 199 at 37”, buffered with 2v-2hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES) and CO, or bicarbonate; fractions were collected every 15 s to 2 min. A 30-s pulse of 30 nM acetylcholine (ACh) increased the rate of glycoconjugate release by lo- to 25fold for 2-3 min. The peak response averaged 14.2 t 9.0 ng protein ml-l min. 4 million cells-’ or 3.6 t 2.3 fgcell-lo min-’ for 30 nM ACh and 16.2 ~fr 3.0 ng protein ml-‘. min-’ for 100 nM ACh. There was no significant glycoconjugate release following a 30-s pulse of either 10 nM or 1 PM ACh. A second pulse after 7 min had no measurable effect on glycoconjugate release but a full response was obtained after 30 min. A continuous superfusion begun 1 min following the 30-s pulse resulted in a greater release of glycoconjugate than the pulse alone, but the response was not sustained, falling to twice basal levels within 5 min. We conclude that a brief muscarinic stimulation causes a triggered release of mucus glycoprotein followed by a relative refractory period. l

mucus; acetylcholine; say

lectin; trachea;

enzyme-linked

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as-

SECRETED into the airways is composed of a mixture of acidic, highly glycosylated proteins that range from 1.7 to 7 X IO6 Da in mass. On release from the cell into the lumen of the submucosal gland, the glycoconjugate is hydrated by a Donnan equilibrium process (37). The resulting product is ejected from the gland by contraction of the myoepithelial cells lining the mucus gland duct (34). Glycoconjugate secretion has been studied in tissuecultured cells, but no cell lines have been established that respond to muscarinic stimulation (5). Hamster epithelial cells enriched in surface secretory cells exhibit constitutive and triggered glycoconjugate release, but are unresponsive to adrenergic and cholinergic agonists, prostaglandins, leukotrienes, and the Ca2+ ionophore A23187 (16). Type-specific cell lines have been established for bovine serous cells; the secretion product is predominantly proteoglycan and hyaluronic acid, not the mucins typical of mucus cells. Secretion is stimulated by &-adrenergic agonists and prostaglandins in the rank order: prostaglandin E, (PGE1) = PGE, > PGA, > PGD,. Although serous cells can be degranulated by ACh in MUCUS

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situ, cells in culture do not respond (reviewed in 2). Freshly isolated tracheal submucosal gland cells (TSGCs) from weanling swine do respond to muscarinic stimulation (38). Both MI and M3 muscarinic receptor subtypes reside on porcine and human TSGCs (38; 27). A study designed to determine which subtype was associated with mucus glycoconjugate secretion was inconclusive because the pirenzepine block of ACh-induced mucus release yielded results that were inconsistent with equilibrium conditions (12). To better study the nature of this nonequilibrium process, a more specific and sensitive assay for mucus glycoconjugate was developed to enable measurements of secretion from airway submucosal gland cells with greater precision and a better temporal resolution. By this method, tracheal submucosal gland cells transiently secreted glycoconjugate and then became relatively refractory, regardless of whether ACh was continuously present or only briefly applied (9). METHODS Male weanling swine (Yorkshire), 5-15 kg, purchased from local suppliers, were used throughout this study. The pigs were killed by the concussive force of the discharge of a captive bolt pistol directed at the base of the skull and were exsanguinated by cardiac puncture. The trachea was removed immediately and washed three times with N-2-hydroxyethylpiperazine-N’2-ethanesulfonic acid (HEPES) Ringer solution at room temperature. The composition of this solution was (mM): 110 NaCl; 2.0 KCl; 1.0 CaCl,; 2.5 NaHCO,; 0.1 NaH,PO,; 11 glucose; 5 HEPES; penicillin; 100 U/ml; streptomycin, 100 pg/ml. All other steps of the cell isolation and superfusion used medium 199 (Ml99) constituted from Sigma M5017 plus 20 mM HEPES, 26 mM NaHC03 and 5 mM dithiothreitol (DTT), equilibrated with 5% C02-95% air at 37°C. Unless otherwise stated, all chemicals were purchased from Sigma Chemical (St. Louis, MO). Isolation of submucosal gland cells. The gland cells were isolated by an enzymatic method similar to that of Yang et al. (38). The trachea was opened along its anterior aspect, and the surface layer of tissue was dissected free and minced into small pieces. This layer included the surface epithelial cells plus the submucosal gland cells. The pieces were then placed in 20 ml of Ml99 to which was added 1 mg/ml collagenase (Sigma type I), 0.1 mg/ml DNase (Sigma type I), and 0.5 mg/ml bovine serum albumin (BSA, Sigma fraction V). The tissue was placed in a 50-ml centrifuge tube and triturated for 30 min through a lo-ml Pyrex pipette that had previously had its tip sawed off and reformed by fire polishing to an opening of 7.7 mm2. The trituration cycle was 13 s (N. B. Daytner, Stony Brook, NY). The supernatant was discarded and the tissue triturated in 20 ml of fresh enzyme solution for 30 min. The tissue was removed and loose cells were washed free and filtered through 35-~1 nylon mesh with 2 ml Ml99. The wash was combined with supernatant in two l&ml centrifuge tubes and centrifuged for 10 min (setting 5, IEC clinical centrifuge, Needham Heights,

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MA). The pellets were washed with IO ml Ml99 and then resuspended in a total of 6 ml M199. The supernatant was layered onto a preformed discontinuous Percoll density gradient with density layers of 1.033, 1.045, 1.058, and 1.084 g/ml. The gradient was formed in a 50-ml polycarbonate tube and was prewarmed to 37” (29 x 104 mm, IEC 2997). The tube was spun at 500 g for 10 min (1,500 rpm, Mistral 3000E, Curtin Matheson, Houston, TX). To exclude ciliated cells, only cells from the most dense interface (1.058 g/ml) were used (38); these cells were diluted to 8 ml and an aliquot removed for counting. The remainder were immediately diluted to 50 ml and were maintained at 37” until loaded into the superfusion apparatus. Cell viability was determined by trypan blue exclusion. Cell counts were made using a hemacytometer. In these studies, the rate of glycoconjugate secretion from unstimulated tracheal submucosal gland cells depended in part on the conditions of cell isolation. In preliminary experiments, cells were used without a Percoll fractionation step (10); the rate of glycoconjugate release from this population of cells was much greater than for the cells from the 1.058-1.084 g/ml interface. In addition, the rate declined initially, reaching a steady baseline only after 1 h or more. Currently, a visible amount of mucus often accumulates at the 1.033-1.045 g/ml interface during the Percoll fractionation step, indicating that these less-dense cells secrete at a high rate. Superfusion manifold. Four million submucosal gland cells, suspended in -10-20 ml of Ml99/DTT solution, were loaded onto nylon filters housed in Swinney filter holders. The filters were 25 mm in diameter and had 1.2 pm pores (N12SP02500, MSI, Westboro, MA). Both the upper and lower chambers of the Swinney filter holder held 0.9 ml, but only the upper half was filled with solution. The lower half contained a bubble of air, diminishing the time delay between superfusion of the gland cells and collection of the fraction. The filter holders were continuously perfused with test solutions that were maintained at 37” and equilibrated with 5% C02-95% air. The superfusion manifold was optimized for rapid solution changes among up to four solutions, with fractions collected over many time periods (as many as 120 l-min periods); as such, very good resolution was possible for the time course studies. Test solutions were drawn from gassed, water-jacketed reservoirs by a thermostatically heated Sage 4-channel peristaltic pump (375A, Orion Research, Cambridge, MA) to threeway stopcocks attached to a five-way union. The union and filter holder were warmed by a thermostatically controlled air blanket. Thus the solutions were pumped in parallel for the duration of the experiment or for at least 20 min before the solution change. Since three-way stopcocks directed the solution flow, changes could be made with an interruption in flow that lasted no more than 3 s. This arrangement ensured that the different solutions were equilibrated to the same temperature and partial pressure of CO, and that solution changes were presented to the filter holder within 1 s following switching the stopcocks. Effluent from the filter holder was collected by an Eldex fraction collector. Enzyme-linked lectin assay (ELLA). The ELLA required 10 steps. 1) Two hundred microliters of coating buffer plus 50 ~1 of M199, standard or test solution, were added to a well in a microtiter plate and incubated for 18 h at 4’. 2) Each well was rinsed four times with 300 ~1 wash buffer. 3) Two hundred fifty microliters of blocking buffer were added to the wells and incubated for 1 h at 37O. 4) Each well was rinsed four times with 300 ,ul wash buffer. 5) Two-hundred microliters of lectin solution were added to the well and incubated for 1 h at 37”. 6) Each well was rinsed four times with 300 ~1 wash buffer. 7) Two hundred microliters of labeled-avidin were added to the well and incubated for 1 h at 37O. 8) Each well was rinsed four times with 300 ~1 wash buffer. 9) Two hundred microliters of

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substrate solution were added to the well and incubated for 2 h at 37O. 10) The absorbance was read at 405 nm with Thermomax microplate reader (Molecular Devices, Menlo Park, CA). Steps 3, 5, and 7 were performed with an Eppendorf pipette with a Plus-8 casette; steps 2, 4, 6, and 8 were performed with an EL 403 microplate autowasher (Bio-Tek Instruments, Winooski, VT). The solutions were as follows. Coating buffer: 0.1 M sodium bicarbonate, pH 9.2; blocking buffer: 0.1 M phosphate buffer (pH 7.4) containing 0.15 M NaCl, 0.05% Tween-20, and 0.05% gelatin; wash buffer: 0.1 M phosphate buffer pH 7.4 containing 0.05% Tween-20, pH 7.4; lectin solution: 5 mg lectin in IO mM HEPES, 0.15 M NaCl, 0.1 M CaC12, 0.04 % sodium azide (a freshly prepared working solution of 10 rug lectin/ml in wash buffer was used for each binding assay); enzyme labeled-streptavidin solution: 2 mg/ml alkaline phosphatase-labeled streptavidin (Pierce Chemical, Rockford IL; a freshly prepared working solution of 2 pg/ml in wash buffer was used for each binding assay); substrate solution: 2.5 mM (p-nitrophenyl phosphate) (PNPP) in 10 mM diethanolamine buffer (pH 9.5), 0.5 mM MgC12. Easy Wash enzyme-linked immunoabsorbant assay (ELISA) flat bottom plates were used throughout (#25805-96, Corning, Corning, NY). The titer plate was organized by columns of eight wells, giving octuplicate determinations. Column 1 was the blank and contained Ml99; column 2 contained standard mucin and a two-point calibration curve was calculated. Columns 3-12 contained test solutions. The “standard” mucin was obtained by freezing tracheal submucosal gland cells that had been isolated on Percoll, but not used for an experiment. The lysate was centrifuged at 3,000 g for 10 min to remove cellular debris, diluted to -1 pg protein/ ml and stored frozen as aliquots. Sample aliquots were tested for protein by the Coomassie blue assay (4) and for carbohydrate by a micro-PAS assay (80). A lo-rig/ml standard was used because it was on the linear portion of the calibration curve, l-500 ng protein/ml and because the standard allowed for comparisons between preparations. Characterization of the Zectin assay. Calibration curves were first constructed using a commercial preparation of secretory glycoconjugate, mucins from bovine submaxillary gland, (31) (M3895, Sigma, St. Louis, MO). By use of DBA lectin (DoZichos biflorus agglutin, Pierce Chemical, Rockford IL), the assay was linear over the range l-500 ng protein/ml and saturated at -48 pg protein/ml. Two specific substances were tested for their ability to interfere with the ELLA: bovine serum albumin, which is commonly added to media, and chondroitin sulfate, a major secretory product of serous cells (2). Albumin did interfere with the detection of mucus glycoconjugate by lectin, as shown in Fig. 1. In this assay, standard glycoconjugate from submucosal gland cells was assayed in the linear range, 0.2-100 ng protein/ml. Increasing concentrations of bovine serum albumin decreased both the slope and Y-intercept of the linear fit to these data. The Y-intercept declined linearly with albumin concentration; this fall in background signal is consistent with the albumin competing with the blocking buffer for binding sites on the wall of the plastic well, since collagen can bind lectin to a small degree (22). The slope of the calibration line fell sharply at albumin concentrations of 1 and 10 pg/ml, but less so between 10 and 50 pg/ml. By interfering with the slope of the assay, albumin appears to bind at the lectin-recognition sites of the glycoconjugate. Chondroitin sulfate A did not interfere with the lectin assay at concentrations of 0.8-100 pg/ml (Sigma C8529, from bovine trachea). Lectin selectivity was determined by assaying 5-400 ng protein/ml of the standard swine submucosal gland cell glycoconjugate with 0.1-2 pg lectin/well. The slope of the calibration

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as means with standard deviations shown as dotted other values are means t SE. Comparisons were Student’s t test.

1 ug albumin/ml 10 ug albumin/ml 50 ug albumin/ml

lines. All made by

.a RESULTS

The following experiments were designed to investigate the ACh-induced release of glycoconjugate from submucosal gland cells on a time scale of minutes. Individual experiments (MD) are illustrated in Figs. 2-4, whereas averages (*SE) are displayed in Fig. 5. Different portions of the same experiment are illustrated in Figs. 2 and 3. Osmotic shock and glycoconjugate release. Superfusion

.8 n 0 .4

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Fig. 1. Calibration curve: enzyme-linked lectin assay. Solutions of “standard” mucus glycoconjugate in Ml99 were made by serial dilution, from 100 to 0.8 pg/ml. To each dilution was added bovine serum albumin, to a final concentration of 0 (square), 1 (#), 10 (diamond), or 50 (+) lg/ml. Fifty microliters of each solution was added to 200 ~1 of coating buffer, in duplicate, incubated overnight, and assayed by the usual manner. Means t SD are plotted as dotted lines for both 0 and the 50 lg/ml data. Bold solid lines are the linear best fits.

..

. . . . a.

.

-

. .

. . . . . .

. . . . - -.

curve was proportional to the quantity of lectin employed. DBA gave the greatest signal, with the optical densities obtained using BSL (Bandiereae simplicifolia lectin) and SJA (Sophora japonica agglutin) being 0.5 and 0.2 as large as DBA. BPA (Bauhinia purpurea alba), PNA (peanut or Arachis hypogea agglutin), SBA (soybean or Glycine max agglutin), and WGA (wheat germ agglutin) gave little positive signal whatsoever (all biotinylated lectins from Pierce Chemicals, Rockford IL). Experimentalprotocols. In each test, 4 x lo6 cells were loaded onto the filter through a side port in the union above the Swinney filter holder, perfused with Ml99 for 45 min discarding that perfusate, and then fractions of perfusate were collected according to test protocols. Aliqouts of the perfusate were pipetted into 96-well titer plates immediately following the experiment for analysis by ELLA. The performance of the superfusion apparatus was tested by perfusing with distilled water to cause an osmotic shock and nonspecific release of cell contents. Timing of the fractions was adjusted to obtain a maximum number of samples when the glycoconjugate concentration was changing most rapidly: one 60-s sample for baseline, a 30-s sample immediately before the solution change to distilled water, ten 15-s samples beginning at the switch to distilled water and finally two 30-s samples. The fractions were assayed for glycoconjugate (thick solid line). Because the Ml99 included the pH indicator phenol red, the optical density of each fraction was read at 557 nm (Spectronic lOOl+, Milton Roy, Rochester NY) to monitor the time course of the solution change (dotted line). The basic experimental design for applying a test pulse of agonist was to load the cells onto the filter, discard the first 45 min of perfusate, and then to collect six 2-min fractions plus sixteen l-min fractions for a 3O-min control baseline period, perfuse with a test concentration of ACh for 30 s, and collect thirty 1-min samples. Recovery was assayed by presenting a second 30-s superfusion with 30 nM ACh, and the refractory period was tested by a third 30-s superfusion with 30 nM ACh 7 min later. The effect of a continuous exposure to ACh was tested by following the first test pulse of 30 nM ACh with a lomin superfusion with 30 nM ACh. Statistical tests. Data from individual experiments are plotted

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Fig. 2. Osmotic shock and glycoconjugate release. At conclusion of each experiment (data from 19 February 1991 shown), superfusion was switched to distilled water, as marked by thin vertical line. Glycoconjugate was detected by Dolichos biflorus agglutin in assays performed in octuplicate from perfusate collected at 15, 30-, and 60-s intervals with results plotted as a solid line on a greatly expanded horizontal axis. Optical density (OD) of collected fractions was measured at 557 nm, absorption peak of phenol red, pH indicator in Ml99 (dotted line). Based on time course of dilution of phenol red, solution change was rapidly completed. Osmotic shock released a very large quantity of glycoconjugate, estimated at 80% of the total glycoconjugate detected by lectin throughout the 3-h experiment; nevertheless, glycoconjugate was cleared from chamber within 2 min.

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Fig. 3. Burst of glycoconjugate release following a brief ACh pulse. Superfusion of 4 x lo6 tracheal submucosal gland cells was rapidly switched from Ml99 to Ml99 plus 30 nM ACh, as indicated by thin vertical lines. Thin horizontal line indicates mean rate of glycoconjugate secretion for control period (min 46-76) before exposure to ACh; only second half of this period is illustrated in figure. Glycoconjugate was detected by Dolichos biflorus agglutin (DBA) in assays performed in octuplicate from perfusate collected in l-min intervals, with dotted lines giving of: SD. Experiment of 19 February 1991.

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with distilled water. As usual, the glycoconjugate released by osmotic shock was much greater than released by ACh; in this example, 80% of the total DBA specific glycoconjugate detected during the course of the entire experiment was released by the distilled water. Basal release accounted for 12% or a rate of 3.7% h-l. The remaining 8% was released by specific agonists. These calculations assume that a negligible amount of glycoconjugate was synthesized during the 3-h course of the experiment (13). A brief ACh pulse and glycoconjugate secretion. Brief exposures to ACh effectively stimulate the release of glycoconjugate. In the individual experiment illustrated in Fig. 3, the superfusion solution was switched to 30 nM ACh for 30 s beginning at min 76. Glycoconjugate release increased U-fold in the next collection period to a peak of 22.5 t 0.8 ng protein ml-‘. min-‘, but returned to baseline levels of 1.6 t 0.2 ng protein ml-‘. min-’ (n = 16 samples collected for min 60-75) by 4 min. This burst of glycoconjugate was followed by a lo-min period of apparently diminished release, 0.6 t 0.4 ng protein ml-l min-l (n = 10; P > 0.05 vs. basal). The refractory period for glycoconjugate secretion. At very short times after the 30-s superfusion with 30 nM ACh, glycoconjugate secretion can be augmented by additional exposure to ACh. In the individual experiment shown in Fig. 4A, the short pulse of ACh was followed 1 min later by a PO-min continuous superfusion with 30 nM ACh. The initial ACh pulse increased glycoconjugate release N-fold to 11.2 t 1.6 ng proteinml-’ l rein-’ during the following minute. A subsequent continuous superfusion increased the rate still further, to 38-fold greater than basal or 23.4 t 1.7 ng protein ml-‘. min. However, the response was not sustained and fell within 3 or 4 min to a plateau that was 2.2 times baseline, or 1.33 t 0.13 ng protein. ml-‘. min-’ (n = 6; P < 0.01 vs. the basal secretion of 0.64 t 0.19 ng protein/ml with n = 15). Glycoconjugate secretion then fell below the control level following return to superfusion with Ml99 (0.22 t 0.27 ng protein ml-‘. min-‘; n = 15; P < 0.001 vs. basal). Glycoconjugate release becomes refractory to further cholinergic stimulation at intermediate times following a brief exposure to ACh. Figure 4B continues the experiment illustrated in A; at min 105 and 112, 30-s pulses of 30 nM ACh tested the cells’ ability to respond to a cholinergic stimulus. The glycoconjugate release following min 105 was slower to develop than was the case for the initial pulse at min 76, with the rate continuing to increase for 3 min. The 8.4-fold increase to 5.1 t 0.4 ng protein. ml-l min-l was one-half the amplitude of the response following the first 30-s pulse and a one-quarter of the peak amplitude during the continuous superfusion. In sharp contrast, the final pulse at min 112 had no detectable effect on the rate of glycoconjugate release. Concentration dependence of glycoconjugate release. The concentration-response curve for glycoconjugate release rises abruptly when the agonist is applied for 30 s. Figure 5, A-D, plot average responses for triplicate experiments, performed on cells isolated from three animals on separate days, with SEs plotted as dotted lines about the mean. In Fig. 5A, 10 nM ACh perfused for 30 l

l

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Fig. 4. Refractory period. Four million cells from band 3-4 Percoll interface of second collagenase digest were loaded onto nylon filter and perfused with M199. Rates of glycoconjugate secretion observed for min 60 -148 are plotted in A and B. Thin horizontal line gives mean rate of glycoconjugate secretion during initial control period. Dotted lines, t SD. A: superfusion was switched from Ml99 to Ml99 plus 30 nM ACh during periods delimited by thin vertical lines, namely for 30 s at min 76 and for 10 min at min 78-87. B: two 30-s pulses of Ml99 plus 30 nM ACh were perfused during periods delimited by thin vertical lines, at min 105 and 112

with distilled water releases large quantities of DBAspecific glycoconjugate. Thus osmotic shock was used regularly at the conclusion of each experiment to obtain an estim .ate of the freely releasable glycoconju .gate remaining in the gland cells . The effect was also used . to estimate the times required to change solutions and collect the gly coconj ugate that was released by a stimulus In Fig. 2, the switch to distilled water occurred at the vertical thin line. As judged by the optical density at 557 nm, the phenol red was diluted by the distilled water with a time constant of 0.4 min, a rate determined by the volume in the filter holder and the rate of flow. Some mixing occurred in the upper half of the Swinney filter holder, so 90 s were required for a solution change to be 99% complete. The release of glycoconjugate was initiated suddenly when Ml99 concentration had been diluted by 20%. The rate of release increased to a maximum within two 15-s collection periods, continued at this high rate for three 15 s periods and then declined with a time constant of 0.8 min. This treatment took place at the conclusion of an experiment that included three 30-s pulses of 30 nM ACh; 17 min of superfusion with the control Ml99 intervened between the last treatment and the superfusion

l

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Fig. 5. Glycoconjugate release following pulse superfusion of ACh. Experimental conditions were as described in Fig. 4. Thin horizontal line gives mean rate of glycoconjugate secretion during initial control period. ACh was perfused during 30-s periods bounded by thin vertical lines. Dotted lines, *SE; 3 replicates were performed. A: ACh was perfused twice, at 60 and 89 min; 1st was 10 nM and’2nd was 30 nM. B: 3 pulses of 30 nM ACh were given at 76, 106, and 112 min. Basal secretory rate was uniformly low in all 3 experiments. C: same protocol as in C was used, except that 1st pulse was 100 nM ACh D: same protocol as in C was used, except that 1st pulse was 1 PM ACh. Basal secretory rate varied markedly from 1 individual experiment to the next, giving large range of SEs.

s at min 60 had little effect, with the sum of the glycoconjugate secreted during the 3 min following the first pulse being only 1.3 t 0.1 times basal. The standard test pulse of 30 nM ACh for 30 s begun at min 89 had an effect very similar to that illustrated in Fig. 3, with the sum of the glycoconjugate secreted for the 4 min following the pulse being 3.6 t 0.7 times basal or a peak amplitude of 24.6 t 9.4 ng protein ml-‘. min. When 30 nM ACh was perfused for 30 s at both min 78 and 103, glycoconjugate secretion increased to 14.2 t 9.0 ng protein ml-l. min-l (Fig. 5B). The sum of the glycoconjugate secreted during the 4 min following the first pulse was 11.0 t 3.4 times basal. That secreted for the 5 min following the second pulse was 5.6 t 2.2 times basal or 14.1 t 9.5 ng proteinml-’ l rein-1. The large fractional increase in glycoconjugate release is due to the fact that, although the peak amplitude was similar to the other average responses illustrated in Fig. 5, the basal secretion was low in each of the three cell isolates studied in this series. The third pulse of 30 nM ACh for 30 s at min 112 had no effect on the rate of glycoconjugate secretion; as can be seen in Fig. 5, C and D, this lack of response characterized the third pulse for all protocols tested. l

A 30 s superfusion with 100 nM ACh yielded no greater rate of secretion than did 30 nM (13.7 t 2.3 ng protein ml-l . min-l; at the peak), but the release was prolonged over 6 min instead of 3 min (Fig. 5C). The second test pulse of 30 nM ACh at min 106 was smaller than that following 30 nM, yielding a peak release of 10.2 t 3.6 ng protein ml-l. min-l but the release was again prolonged, requiring 5 min to return to basal levels. Following the second and third pulses of ACh, a single large release of glycoconjugate occurred in two of the three cell isolates studied; the first was -20 ng proteinoml-min-l at min 120-121 and the second was 12 ng protein ml-’ min-’ at min 123. These releases were large enough to appear in the mean and SE plotted in Fig 50. The highest concentration of agonist tested, 1 PM ACh, resulted in no additional release of glycoconjugate. This is true for the average shown in Fig. 50 as well as for each of the three individual experiments that comprise Fig. 5. Furthermore, the second and third test pulses of 30 nM at min 106 and 112 yielded no detectable increase in glycoconjugate release. The basal rate of glycoconjugate secretion ranged from 1.5 to 10 ng protein ml-‘. min-‘, as illustrated in Fig. 5, A-D. Within this range, the absolute amount of glycol

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conjugate released by a given dose of ACh was independent of the baseline secretion. However, the relative increase could reach very large numbers when the basal rate was very low; the greatest response in an individual experiment was 25-fold, for the first and second 30-s test pulses of 30 nM ACh, according to the protocol of Fig. 5B. DISCUSSION

Muscarinic stimulation of glycoconjugate release from tracheal submucosal gland cells was quantified by the application of lectin chemistry to the method of enzymelinked immunosorbant assays. Brief, 30-s exposures to ACh acted to increase the rate of glycoconjugate release by lo- to 25fold. The release was transient and was seen only within a narrow concentration range. Detection

of Airway Glycoconjugate by Lectins

Chemistry of mucus. Airway mucus is a complex mixture of glycoconjugates and glycolipids, the precise composition being dependent on the health and autonomic status of the individual (1). Individual classes of the glycoconjugates and glycolipids have characteristic carbohydrate moieties and linkages. For instance, proteoglycans have high molecular masses ( lo6 Da), little branching, and are heavily substituted with carboxy- and sulfate-ester groups, giving the proteins a large anionic net charge. N- and O-linked oligosaccarides are also present. In contrast, glycoconjugates are generally smaller, highly branched, and carry less charge. Glycoconjugates with 0- linkages include the following: mucin, with N-acetyl-D-galactosamine and L-serine or L-threonine linkage; proteoglycan, with a xylose(@l-3)-serine linkage (xyl(@l-3)-ser); and collagen, with a D-galactose5-hydroxy-D-lysine [Gal(pl-5)OH-Lys] (30). Lectins are plant and animal proteins of nonimmune origin that agglutinate cells or precipitate glycoconjugates. The considerable specificity of lectins has been used to define cell types of the airway according to the identity of mucin glycoconjugates in secretory vesicles or attached to cell surfaces (23, 24, 28). Monoclonal antibodies can also characterize cell types according to class of mucin, but in a species-specific manner (23). Histochemical studies of sheep demonstrated that goblet cells and gland cells contained fucose and N-acetyl galactosamine, whereas galactosamine was detected by BSA I only in one type of mucus cell and at basolateral surfaces (23). (See Table 1 for full lectin names and specificities.) In human bronchial mucosa, serous cells were specifically labeled by LCA, PHA, and WGA and mucus cells by HPA and SBA. By this study, the mannose side chain was most specifically characteristic of serous cells and N-acetyl galactosamine of mucus cells (28). Fucose side chains were reported in human, rat, and mouse mucus, but not serous, cells (as measured by UEA I). However, species differences exist, such as the observation that fucose side chains were present in >90% of human samples, but were absent from rat and mouse (as measured by LTA) (35). In contrast, proteoglycans were not detected by lectins, although cartilage proteoglycans could

SECRETION

L423

be made to react with PNA and SBA by treatment with chondroitinase (36). Lectin histochemistry has also demonstrated changes in airway glycoconjugate composition following exposure to environmental irritants and products of inflammation (25). ELLA. As a specific and sensitive probe for glycoconjugates, lectins have the benefit of relative species independence, being chemically defined molecules (22), and being commercially available as conjugates with avidin, streptavidin, or a variety of fluorescent dyes and markers. Airway mucus secretion has been assayed by lectins prior to the development of the ELLA. For instance, doublediffusion plates (Ouchterlony) and hemagglutinin inhibition assays on human sputum and bronchial washings showed the presence of glycoconjugates reactive with UEA I, WGA, SBA, RCA, and PNA, in a patient-specific pattern (21). Two reports of similar assays have been published. In the first (II), lectin was fixed to the walls of a 96-well titer plate; glycoconjugate enzymes were specifically bound by the lectins and enzyme activity assayed. In the second (29), the test protein was immobilized on the wall of a 96-well titer plate and alkaline phosphatase-conjugated lectin was added; the protein was thus indirectly quantified by the enzyme activity of the probe lectin. Glycoconjugate Secretion Airways produce mucus continuously, but the rate of glycoconjugate release can be increased by a variety of stimuli (13). Secretory processes are generally divided into constitutiue (continuous but unregulated) and triggered. Triggered events are typically Ca2+ dependent, caused by a rise in intracellular Ca2+ ( [Ca2+]; ) or an increase in the Ca2+ affinity of the exocytotic event; adenosine 3’,5’-cyclic monophosphate (CAMP) can modulate the Ca2+-induced secretion, and the production of 1,2diacylglycerol increases the Ca2+ sensitivity of the secretory process in many cell types (reviewed in 14 and 18) The rate of unstimulated glycoconjugate secretion from tissue preparations can be modified by altering bath Ca2+. With reduced bath Ca2+, release from canine explants is increased (6), whereas release from feline submucosal glands is decreased (33). Increases in [Ca2+]; and CAMP have been measured in association with airway secretion (19, 20). For the muscarinic stimuli, a significant response was seen at a concentration of methacholine as low as 1 PM (15). This concentration of methacholine is equivalent to -80 nM ACh, given that commercial supplies of the drug are typically racemic and that the + isomer has an affinity constant of 8 X lo6 vs. 5 x lo7 for ACh, (3). Thus the concentrations of ACh that are effective in releasing glycoconjugate in the experiments reported in this paper are at the foot of the concentration-response curve for muscarinic induced increases in feline submucosal gland acinar [Ca”‘];. Muscarinic Release of Glycoconjugate Freshly isolated tracheal submucosal gland cells were chosen to study glycoconjugate secretion because these

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L424 Table

TRANSIENT

SECRETION

1. Lectin specificities Abbreviations

BSA I, BSL BPA DBA HPA LCA LTA PHA PNA RCA SBA SJA UEA I WGA Lectin

CHOLINERGIC

specificities

are from

Source

I

Specificity

of Lectin

(Bandiereae simplicifolia) (Bauhinia purpurea alba) (Horsegram or DoZichos biflorus) (Helix pomatia) (Lens culinaris) (Lotus tetragonolobus) (Phaseolus vulgaris) (Peanut or Arachis hypogea) (Ricinus communis) (Soybean or Glycine max) (Sophora japonica) (Gorse seed or Ulex europaeus) (Wheat germ)

cu-D-gal

gal-/3-1,3-galNac a-D-gal Nat gal Nat a-mannopyranose cu-L-fucose hemagglutinating ,8-D-gal-(1-3)-D-gal P-D-gal, a-D-gal a&D-gal Nat gal Nat and gal a-L-fucose ,&D-&c NAc

Nat

Ref. 22.

cells respond to ACh with increased glycoconjugate secretion and are available in quantity (lo8 cells/animal) (38). The isolated cells were preferred to explants because, once distributed on the nylon filter and continuously perfused, they were free of tissue-derived factors such as inflammatory mediators or neuromediators and were not subject to the myoepithelial contractile elements that are present in the tissue (34). The superfusion apparatus was designed to permit rapid solution changes and collect fractions as often as every 15 s. Control experiments demonstrated that solution changes and glycoconjugate washout occur with a time constant of -1 min, making credible the observation that agonist-induced glycoconjugate release began within 1 min of the 30-s superfusion with ACh and persisted for no more than 3-5 min. However, results shown in Fig. 2 demonstrate the limit to the temporal response of the superfusion apparatus; the time constant of the device was -1 min. Therefore, neither the rising nor the falling phase of the glycoconjugate release was accurately resolved. Concentration dependence. Only a narrow range of ACh concentrations were effective in releasing glycoconjugate; 10 nM gave a barely detectable response and 100 nM gave the maximum burst of secretion. At 1 PM, no response was apparent (Fig. 5). The exact shape of this concentration dependence is likely to depend on two experimental factors. First, the durations and concentrations given are only nominal, since the dead space of the superfusion chamber blunted the concentration profile of the pulse of transmitter. Because of mixing in this antechamber, the gland cells were actually exposed to a delayed and broadened application of agonist. For the 30-s ACh pulse, the peak concentration at the level of the cells was approximately two-thirds of the nominal value. Second, since the ACh pulse gave a different pattern of secretion from the IO-min superfusion, still other stimulus paradigms may have quite different concentration response relationships. Transient release. All statistically significant agonistinduced releases of glycoconjugate were of similar peak amplitudes, regardless of the concentration of ACh during the pulse and regardless of the order the doses were presented. Thus the initial pulses of 30 and 100 nM gave 14.2 t 9.0 and 16.2 t 3.0 ng protein~ml-l~min-l. After the 10,30, and 100 nM ACh pulses, the second test pulse

of 30 nM ACh released 24.6 t 9.4, 14.1 t 9.5, and 12.9 t 3.1 ng protein. ml-‘. min. These three values are not significantly different, although they do trend downward, consistent with development of desensitization. The glycoconjugate release was not sustained, lasting only 2-6 min following a 30-s pulse of ACh. The release was marked by three characteristics. First, the release could be augmented if a second muscarinic stimulus was given very shortly following the initial 30-s ACh pulse (Fig. 4B). Second, the release was not sustained even in the presence of continuous superfusion with ACh, but fell by an order of magnitude (Fig. 4B). Third, there was no measurable effect of a second test pulse of ACh at 7 min following the first conditioning pulse (Fig. 5). Finally, the baseline glycoconjugate release was often visibly depressed following a single test pulse (Figs. 3 and 5A). The cells recovered completely following a recovery period, although recovery tended to be less complete following a conditioning pulse with a high than with a low concentration of ACh. These findings show that TSGCs become relatively refractory to muscarinic stimulation after a conditioning pulse as short as 30 s. This does not represent a depletion of the readily releasable store, since the continuous lomin superfusion gave a peak glycoconjugate release in excess of that seen with a single 30-s pulse. Furthermore, basal and agonist-induced release represented only a small fraction of total glycoconjugate stores, based on the very large quantities of glycoconjugate released by osmotic shock at the conclusion of the experiment. The relative refractory period may be due to desensitization or tachyphylaxis of the muscarinic receptor or a depletion of a second messenger. However, if receptor desensitization or downregulation were the causes of the refractory period, then such a process must be initiated by a brief receptor occupancy, during the 30-s ACh superfusion and then proceed to completion even when the receptors are not occupied by agonist, during the superfusion with Ml99 between test ACh pulses. Alternatively, ACh might release a second secretory product over a slower time scale that interferes with the ELLA. One candidate is chondroitin sulfate from serous gland cells (Z), but in fact chondroitin sulfate does not interfere with the ELLA assay. Other proteins such as bovine serum albumin do interfere, but only at micrograms per milliliter concentrations, and these represent

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TRANSIENT

CHOLINERGIC

quantities of protein that are greater than are present in the 4 X lo6 cells in the superfusion chamber. A final possible explanation is that ACh causes an inhibition as well as a stimulation of glycoconjugate release, with the inhibition being the slower to develop. Such an inhibition would cause the fall in baseline glycoconjugate release as well as the lack of response to superfused ACh at the 7-min posttest pulse and also the apparent lack of a response to the 1 PM ACh superfusion. Residual agonist-induced glycoconjugate release. The rate of glycoconjugate secretion observed during a continuous ACh superfusion was approximately twice basal. This residuum is similar in magnitude to that seen when swine tracheal explants were stimulated with prolonged exposure to ACh (12) and is similar to that observed in other tissues, using the method of radiolabeled secretion product (26). Therefore, the rapid transient made apparent by the increased sensitivity and resolution of the ELLA becomes lost in a collection period that extends for many minutes during which the glycoconjugate is released at a lower rate. Conclusion. ACh induces a transient glycoconjugate secretion, followed by a relative refractory period. When not in the refractory period, the amplitude of the transient is not graded with concentration of agonist; instead, glycoconjugate is released as a burst once a threshold concentration of ACh is reached. As a consequence, vagal reflexes can vigorously generate a quantity of mucus in response to an irritant stimulus. At the same time, prolonged exposure to ACh is inherently less effective, limiting the extent to which the airways are flooded with glycoconjugate in the face of a continued irritation. We are grateful to Angela Ruffin for isolation of the tracheal submucosal gland cells and to Char-Chang Shieh for help in preparing the tissue. This work was supported by grants from the Cystic Fibrosis Foundation (G203), Mississippi Lung Association Henry Harold and Eunice Albriton Memorial Research Grant (to T. M. Dwyer), a Mississippi Lung Association grant (to J. M. Farley), National Heart, Lung, and Blood Institute Grant HL-11678, Biomedical Research Support Grant 2 SO7 RR05386, and National Institute of Drug Abuse Grant DA05094. Address for reprint requests: T. M. Dwyer, Dept. of Physiology and Biophysics, University of Mississippi Medical Center, Jackson, MS 39216-4505. Received

7 June

1991; accepted

in final

form

2 October

1991.

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