Diurnal variations in tear glycoproteins: evidence for an epithelial origin for the major non-reducible > or = 450 kDa sialoglycoprotein(s).

PURPOSE: To characterize the nature and origin of changes in tear glycoproteins accompanying eye closure. METHODS: Reflex (R) and overnight closed (C) eye tears collected by capillary tubes were centrifuged with the resulting R pellets (primarily desquamated epithelial cells) and C pellets (primarily PMN and some epithelial cells) extracted in acidic PBS. Extracts and supernatants were separated by size-exclusion HPLC and/or SDS-PAGE. Gels were stained or blotted and immune- or lectin-probed. An HPLC glycoprotein fraction of > or = 450 kDa isolated from all four sources was characterized before and after partial deglycosylation, using antibodies specific to known mucin and carbohydrate epitopes. Immunofluorescence microscopy was carried out on human conjunctiva, using as probe a MAb to salivary mucin specific for a sialyl Lea epitope, which was found to cross-react specifically with the major non-reducible high molecular weight sialoglycoproteins (SGs) in tears. These SGs were immunoprecipitated and blot-probed along with tissue extracts. RESULTS: R fluid contained minor amounts of numerous glycoproteins, including probably several of inducible lacrimal secretory origin. Results confirmed sIgA as the principal source of the intense reducible glycoprotein bands common to C fluid. Smaller amounts of free secretory component and serum glycoproteins were also visualized. The HPLC fraction (> or = 450 kDa) consisted of four major non-reducible glycoproteins. In R fluid, this fraction (< 1% total protein) consisted primarily of two entities: a 450-500 kDa SG and a larger asialoglycoprotein. The SG accounts for as much as 85% of the total protein in the R pellet extract. C fluid was associated with a selective increase in SGs and a shift in distribution to two SGs > 500 kDa. All SGs exhibited a common antigenicity reacting specifically with the MAb for the sialyl Lea epitope. SGs indistinguishable in size and antigenicity were recovered in epithelial extracts. Immunofluorescence microscopy revealed that reactivity was localized to the epithelial plasma membrane, increasing in intensity from basal to apical cells. Although these SGs exhibited some properties in common with MUC1, immunological and other data suggest a unique SG. CONCLUSIONS: Tear glycoproteins are derived from four principal sources. In R fluid, an inducible lacrimal secretion predominates. In C fluid, a constitutive sIgA secretion predominates, augmented by a serum exudate and SGs derived at least in part from the epithelium. In R fluid and pellet extracts, the SGs consist primarily of a 450-500 kDa species that is most probably derived from the plasma membrane. Larger antigenically related SGs are prevalent in C fluid.

High-performance liquid chromatographic fractionation and partial characterization of cystic fibrosis serum ultrafiltrates.

Analytical separation of serum ultrafiltrates by high-performance liquid chromatography produces a distinctive peak with a retention time of 18.5-21 min (subfraction 18.5) from cystic fibrosis serum ultrafiltrates and obligate heterozygote serum ultrafiltrates, but not in significant concentrations from control or asthmatic serum ultrafiltrates. Semipreparative separation of control serum ultrafiltrates produced a small peak with similar retention time that was approximately 1% of the arbitrary absorbance units found in this cystic fibrosis subfraction. Subfraction 18.5 had biological activity only when separated from cystic fibrosis serum ultrafiltrate, but did not contain measurable amounts of C3a des-arginine and C4a des-arginine. Subfraction 18.5 is a low-molecular-weight material (1000-1400 daltons) that contains 14.9 micrograms orcinol positive material per 50 micrograms protein. The spectrum of subfraction 18.5 indicates that it has to be purified to homogeneity.

Biological activities of cystic fibrosis serum. IV. Stimulation of the calcium mediated K+ efflux from rat submandibular gland fragments.

Cystic fibrosis (CF) and heterozygote sera stimulate a significant K+ efflux from rat submandibular gland fragments in the presence of 1 mM ouabain. This sensitive parameter can be maximally stimulated by as little as 0.5% CF serum and is inhibited by the calcium channel blocker D600 and EGTA. Specific receptor blockers propranolol, phenoxybenzamine or atropine do not inhibit the CF serum-stimulated K+ efflux and agonists do not supramaximally stimulate K+ efflux when added with serum. CF serum-induced K+ efflux did not result in the leakage of lactic dehydrogenase into the bathing media nor did it mimic the action of the calcium ionophore A23187 when added in the presence of D600. In addition, ultrafiltrates of CF serum (less than 10,000 daltons) also stimulated K+ efflux from rat submandibular gland tissue fragments.

Agonist-induced secretions and potassium release from rat submandibular gland slices.

Secretory activity induced by stimulation of alpha-adrenergic, beta-adrenergic, and muscarinic cholingeric receptors and by dibutyryl cAMP and 8-bromo cGMP has been investigated in rat submandibular tissue slices. Isoproterenol produced a sialic acid secretion from the acinar cells that was inhibited by propranolol, but not by phenoxybenzamine or atropine. Dibutyryl cAMP produced a sialic acid secretion from the acinar cells that was not inhibited by propranolol, phenoxybenzamine, or atropine. Both norepinephrine and acetylcholine produced significant secretion of sialic acid but at a reduced efficacy. Norepinephrine stimulated both peptide hydrolase secretion from the granular duct cells and a release of K+ from the acinar cells. Both actions were inhibited by phenoxybenzamine, but not by propranolol or atropine. Acetylcholine stimulated a minimal secretion of peptide hydrolase from the granular duct cells and a significant release of K+ from the acinar cells. The norepinephrine- and acetylcholine-stimulated release of K+ was increased after the addition of 1 mM ouabain. High concentrations of 8-bromo cGMP induced a K+ efflux that was not inhibited by phenoxybenzamine or atropine. Vacuolation of the acinar cells was correlated with K+ release.

The biologic activities of cystic fibrosis serum. II. Ultrastructural aspects of the effect of cystic fibrosis sera and calcium ionophore A23187 on rabbit tracheal explants.

Ultrastructural and cytochemical observations indicate that both cystic fibrosis (CF) sera and calcium ionophore A23187 induce a swelling or an increase in the size and possibly the number of secondary lysosomes and an increase in mucus secretion in epithelium of the rabbit tracheal bioassay system. Extended incubation of the rabbit tracheal explants with either CF or control sera produces a cytotoxic effect on the tracheal epithelium, but only after the termination of the normal bioassay time period. Comparative ultrastructural study of the effect of both CF sera and calcium ionophore A23187 on the rabbit tracheal bioassay system indicates that increased membrane permeability to calcium may be important in the production of the ciliary dyskinesia response by CF serum factor(s) in the rabbit tracheal bioassay system.

Electron microscopic autoradiographic analysis of the uptake and intracellular transport of H3-leucine by the rat submandibular gland acinar cells in tissue slices.

Uptake of H3-leucine into secretory product and its subsequent intracellular transport was analyzed by electron microscopic autoradiographic techniques in the rat submandibular gland acinar cells in vitro. The route and kinetic timetable of intracellular transport was established for the acinar cell secretory product by calculating the present of silver grains and relative grain density associated with the various organelles on a time sequence basis. Radioactivity was first associated with the rough endoplasmic reticulum; then the convex surface of the Golgi apparatus; the concave surface of the Golgi apparatus; and finetics of intracellular transport in the rat submandibular gland acinar cell with other established systems revealed only a difference in the exit of radioactivity from the concave surface of the Golgi apparatus.

The biologic activities of cystic fibrosis serum. I. The effects of cystic fibrosis sera and calcium ionophore A 23187 on rabbit tracheal explants.

An ionophore A23187-induced increase in membrane permeability to calcium ions in culture medium produced a rabbit tracheal mucociliary response indistinguishable from that caused by cystic fibrosis (CF) sera on three different occasions. Specific chelation of calcium ions with EGTA in the basal medium Eagle (BME) media with no additive or in native CF sera abolished the mucociliary disturbances in all cases. Increased membrane permeability to calcium may be important in the production of the mucociliary response by CF serum factor(s) in the tracheal assay system.

Peroxisomes in inner adrenocortical cells of fetal and adult guinea pigs.

Abundant membrane-bounded granules, 0.1-0.45 microm in diameter, occur among the elements of the smooth-surfaced endoplasmic reticulum in zona fasciculata and zona reticularis adrenocortical cells of guinea pigs. Acid phosphatase cannot be cytochemically demonstrated in them, and they are therefore distinct from lysosomes. Incubation in medium containing 3,3'-diaminobenzidine results in dense staining of the granules, identifying them as peroxisomes. These small peroxisomes increase in number as fetal adrenocortical cells differentiate, and they appear to arise from dilated regions of endoplasmic reticulum. They maintain interconnections with the smooth endoplasmic reticulum and with one another.