Corneal permeability in a redesigned corneal holder for the bovine cornea opacity and permeability assay.

The bovine cornea opacity and permeability assay (BCOP) is a proposed alternative to the Draize rabbit test for potential eye irritants. In the standard BCOP, bovine corneas are mounted in a holder on a flat surface between two identical chambers. The flat configuration of the standard holder does not conform to the normal curved shape of the bovine cornea and it comes into direct contact with the cornea tissue. Mounting corneas in this holder causes extensive damage to both epithelial and endothelial corneal cell layers. Our laboratory has designed a new holder that allows the cornea to maintain its natural curvature and does not damage the cornea. Previous tests, using both the new and standard holders, and comparing corneal opacity, hydration and endothelial morphology, have shown that the new holder is a significant improvement over the standard holder. The present study extends the comparisons of the new and standard holders to measurement of corneal fluorescein permeability. The permeability (ng/cm(2)/min) of intact corneas, corneas with no epithelium, and corneas treated with 1% NaOH, isopropanol, acetone, 30% trichloroacetic acid or 30% sodium dodecysulfate for either 1 or 10 min was determined by measuring fluorescence of samples taken from the endothelial chamber after 90 min epithelial exposure to 0.04% sodium fluorescein. In all trials, the redesigned holders yielded not only lower permeability measurements but also decreased measurement variability. The data provide further evidence that the new holder is an improvement over the standard holder and should be incorporated into a new protocol for the BCOP.

A redesigned corneal holder for the bovine cornea opacity and permeability assay that maintains normal corneal morphology.

The bovine cornea opacity and permeability assay (BCOP) has been in use for nearly 10 years but has not been submitted for regulatory approval. In previous reports we have presented corneal hydration and endothelial damage as additional endpoints in this assay and have suggested that the design of the BCOP's corneal holder should be modified. The standard holder used in the BCOP assay induces physical damage to the cornea because it contacts clear cornea causing edge damage to the epithelial, stromal and endothelial layers. Second, by forcing a curved, oval-shaped bovine cornea into a flat, circular opening, corneal wrinkling occurs which can alter the cornea's optical characteristics and, most importantly, induces endothelial damage. We now report on a redesigned BCOP corneal holder that clamps onto the sclera, maintains normal corneal shape and does not cause damage to the endothelium. This ensures that irritancy tests are conducted using healthy, anatomically normal tissue. Tests of this holder using acetone, trichloroacetic acid, isopropanol and benzalkonium chloride show that it is now possible to evaluate effects of chemical substances on the endothelium. The effects of these compounds on corneal opacity and hydration in the new holder are similar to their effects on the cornea in the standard holder.

The eicosanoid, 15-(S)-HETE, stimulates secretion of mucin-like glycoprotein by the corneal epithelium.

PURPOSE: The eicosanoid, 15-(S)-hydroxyeicosa-5Z, 8Z-11Z, 13E-tetraenoic acid (15-(S)-HETE), is known to stimulate production of mucin glycoprotein by airway epithelium. This study investigated the effect of 15-(S)-HETE on the mucin glycoprotein secretion by the corneal epithelium. METHODS: To determine the effect of dose, corneas of anesthetized New Zealand White rabbits were treated with 50, 500, or 5,000 nM 15-(S)-HETE in artificial tears for 120 minutes. To determine the time to onset of the response, corneas were treated with 500 or 1,000 nM 15-(S)-HETE in balanced salt solution for periods ranging from 5 to 120 minutes. Corneas were fixed for electron microscopy in fixative containing 0.5% cetylpyridinium chloride (CPC) to stabilize the layer of mucin-like glycoprotein on the corneal surface. The mucin layer thickness was measured by image analysis of electron micrographs. RESULTS: The layer of CPC-fixed mucin-like glycoprotein on the surface of control corneas was 0.46 +/- 0.04 microm thick. After treatment with 15-(S)-HETE, the thickness of the mucin layer increased to 0.64 +/- 0.1 microm at 50 or 5,000 nM HETE and as much as 1.02 +/- 0.2 microm in response to 500 nM HETE. Mucin thickness reached a statistical maximum of 0.59 +/- 0.1 microm after only 5 minutes of exposure to 500 or 1,000 nM HETE. CONCLUSIONS: Exposure of the cornea to 15-(S)-HETE causes a rapid-onset increase in the thickness of a layer of mucin-like glycoprotein on the surface of the corneal epithelium. This supports previous reports that corneal epithelial cells produce mucin and suggests that treatment with topical 15-(S)-HETE may be effective in treating ocular surface mucin deficiency in dry eye syndrome.

Corneal opacity, hydration and endothelial morphology in the bovine cornea opacity and permeability assay using reduced treatment times.

The purpose of this study was to determine whether the standard bovine cornea opacity and permeability (BCOP) assay exposure time of 10 minutes overestimates the ocular irritancy of chemical substances. Corneas were subjected to BCOP protocol following 30-second and 1-minute exposures to irritants. Corneal opacity and hydration (mg H(2)O/mg cornea) were then measured and compared to data obtained after 10 minute irritant treatments. For most test substances corneal opacity and hydration were lower following reduced exposure times. It is suggested that using shorter exposure times in BCOP protocol may be more predictive of human response to ocular irritants, since irritants are usually in brief contact with the ocular surface during accidental exposure. A second purpose of this study was to examine effects of irritants on the corneal endothelium. Corneas were treated according to BCOP protocol following exposure to irritants for 1 or 10 minutes. The endothelium was stained with Alizarin Red and trypan blue, and examined using light microscopy. Severe irritants, such as NaOH and trichloroacetic acid, cause endothelial cell death. It was also determined that simply mounting the cornea in the BCOP assay holders caused damage to 20% of the endothelial cells. Because the endothelium is essential for normal corneal transparency and hydration, it is suggested that examination of the endothelium be added to the BCOP assay and that optimization of the assay will require modification of the cornea holders.

Intermediate filament, laminin and integrin expression in lacrimal gland acinar cells: comparison of an immortalized cell line to primary cells, and their response to retinoic acid.

PURPOSE: The goal of this study was to characterize intermediate filament, integrin and laminin expression by rabbit lacrimal gland acinar cells in culture, to determine whether retinoic acid (RA) alters expression of these proteins and to compare primary cells to an immortalized rabbit lacrimal gland acinar cell line using flow cytometric analysis. METHODS: Primary cells, maintained in serum free medium, were exposed to 10(-6) M retinoic acid for 24 hours. Immortalized cells were grown in defined medium with Nu-Serum and exposed to retinoic acid. Cells were labeled with monoclonal antibodies to cytokeratins (AE1, AE2, AE3, AE5, CK10/13, CK18), integrins (alpha(3), alpha(6), alpha(V), beta(1), beta(2), beta(3) and beta(4)), laminin, or vimentin and with FITC-conjugated secondary antibodies. Cells were analyzed for antigen expression by flow cytometry and immunocytochemistry. RESULTS: Primary and immortalized cells expressed type I and type II epithelial cytokeratins (AE1 and AE3), cytokeratin 18, and cytokeratin 3 (AE5) Both cell types were negative to AE2 and CK10/13. Primary and immortalized cells expressed vimentin in culture, with immortalized cells expressing this protein at higher levels. Lacrimal acinar cells appear to synthesize laminin which was detected intracellularly in both cells types. Integrins alpha(6) (CD49f) and alpha(V) (CD51) were expressed by primary and immortalized cells. Expression of integrin alpha(6) was 10-fold higher in immortalized cells compared to primary cells. Retinoic acid increased integrin alpha( V) expression by primary and immortalized cells 1.3-fold and 3-fold, respectively, and caused a slight increase in integrin alpha(6) expression by primary cells. Both cell types also expressed integrins beta( 1), beta(2) and beta(3), but beta(4) was detected only in immortalized cells. Lacrimal acinar cells do not express integrin alpha(3). CONCLUSIONS: Expression of cytokeratins, laminin and integrins by primary and immortalized cells was similar, suggesting that the immortalized cell line is a good model for the study of lacrimal structure and function. Since retinoic acid up-regulated only integrin alpha(V), but not cytokeratins, these cells appear to be highly differentiated. Flow cytometry is a useful method for analysis of protein expression by lacrimal acinar cells.

N-linked glycoside and glucuronide conjugates of the retinoid, acitretin, are biologically active in cornea and conjunctiva.

The purpose of this study was to test two water-soluble, synthetic retinoids, glucoseamido acitretin and glucuronamido acitretin, for biological activity in cells of the cornea and conjunctiva. Vitamin A-deficient, xerophthalmic rats were treated topically with these retinoids, and corneas were examined histologically for effects on epithelial keratinization. The effect of these retinoids on the proliferation of rabbit conjunctival fibroblasts in culture was also investigated. Glucoseamido acitretin treatment restored a normal cornea after eight to nine days of treatment, while no improvement was observed in the vehicle-treated corneas. Likewise, glucuronamido acitretin application restored a normal corneal surface and reversed keratinization after eight to ten days of treatment. These retinoids caused no irritation of the eye or ocular adnexa. In culture, exposure of conjunctival fibroblasts to glucoseamide acitretin inhibited cell proliferation. Cultures exposed to glucoseamido acitretin at 10(-8) M or 10(-6) M had cell densities 77.3% and 51.9% of control, respectively, after seven days. Glucuronamido acitretin also inhibited cell proliferation. Cultures exposed to glucuronamido acitretin at 10(-8) M had a cell density of 69.2% of control at day seven, while at 10(-6) M this retinoid completely inhibited cell proliferation. These results show that glucoseamide acitretin and glucuronamido acitretin are biologically active in the cornea and conjunctiva, and may be considered for ophthalmic use in diseases involving abnormalities of ocular surface cell differentiation or hyperproliferation of fibroblasts.

Expression of nuclear retinoic acid receptor and retinoid X receptor mRNA in the cornea and conjunctiva.

PURPOSE: The effects of retinoic acid in cells are mediated by the nuclear retinoic acid receptors (RARs) and retinoid X receptors (RXRs). Although vitamin A is essential for the normal development and maintenance of the ocular surface, the RARs and RXRs have not been studied in cornea and conjunctiva. The purpose of this study was to identify the mRNA for these receptors in corneal and conjunctival cells in culture and to determine whether all-trans retinoic acid is able to induce expression of RAR mRNA. METHODS: Total RNA was extracted from cultured rabbit corneal stroma and conjunctival fibroblasts and rabbit corneal epithelial cells. RNA was analyzed by Northern blotting using the cDNA probes for RAR alpha, RAR beta, RAR gamma, RXR alpha, RXR beta and RXR gamma mRNA. To investigate induction of retinoid receptors, cells were exposed to 10(-6) M all-trans retinoic acid for 2-48 h before preparation of RNA. Effects of retinoic acid on cell proliferation were also investigated. RESULTS: RAR alpha mRNA transcripts (3.7 kb), RAR beta mRNA transcripts (3.3 kb) and RAR gamma mRNA transcripts (3.3 kb) are expressed by all the cell types studied, as are the RXR alpha mRNA transcripts (5.0 kb) and RXR beta mRNA transcripts (3.3 kb). RXR gamma mRNA is not detectable in corneal and conjunctival cells. All-trans retinoic acid induced RAR beta mRNA expression in corneal and conjunctival fibroblasts. Increased mRNA levels were detectable after 4-8 h and peaked by 24 h. RAR beta mRNA was not induced by retinoic acid in corneal epithelial cells. Retinoic acid also inhibited proliferation of conjunctival and corneal fibroblasts but had no effect on growth of corneal epithelial cells. CONCLUSIONS: The expression of RARs and RXRs in the cornea and conjunctiva is similar to that reported in other tissues. The identification of these receptors may lead to a better understanding of gene transcription pathways in the cornea and conjunctiva and of the mechanisms that control keratinization, differentiation and proliferation of the cells of these tissues. The data suggest a relationship between the induction of RAR beta mRNA expression and inhibition of cell proliferation by retinoic acid.

The lacrimal gland expresses nuclear retinoid X receptors.

PURPOSE: The lacrimal gland expresses nuclear retinoic acid receptors. This suggests that retinoids are involved in control of gene expression in the lacrimal gland. Retinoid X receptors (RXRs) form heterodimers with and are required for activation of retinoic acid receptors. The purpose of this study was to identify retinoid X receptors in the lacrimal gland. METHODS: Total RNA was purified from rat, rabbit and human lacrimal glands and from cultured rat lacrimal cells. RNA was analyzed by northern blotting using cDNA probes for RXR alpha, RXR beta and RXR gamma. Nuclear protein extracts from rat and rabbit lacrimal glands were probed for RXR beta by immunoblotting, using a mouse monoclonal antibody. RESULTS: RXR alpha mRNA transcripts (5 kb) and RXR beta mRNA transcripts (3.3 kb) are present in the lacrimal glands of all species studied and in cultured rat lacrimal cells. RXR gamma mRNA (1.9 kb) was detected only in the rabbit lacrimal gland. RXR beta is expressed as a 50 kDa protein in rat and rabbit lacrimal glands. CONCLUSIONS: This study confirms the presence of RXRs in the lacrimal gland, thereby supporting a role for retinoids and their nuclear receptors in the control of gene transcription in the lacrimal gland.

Biological activity of N-(4-hydroxyphenyl) retinamide-O-glucuronide in corneal and conjunctival cells of rabbits and humans.

Previous studies of topical retinoic acid for treatment of ocular surface disease met with limited success due to instability and irritancy of the retinoid and lack of efficacy in keratoconjunctivitis sicca. There has, however, been continued interest in the treatment of mucin deficiency and cicatrizing conjunctival diseases, such as ocular cicatricial pemphigoid (OCP), topically with retinoids. In this study the biological activity of stable, water-soluble, synthetic retinoid, N-(4-hydroxyphenyl) retinamide-O-glucuronide (4-HPROG) was investigated in vivo and in vitro using conjunctival and corneal epithelium and fibroblasts. Vitamin A-deficient rabbits with stage 3-4 corneal xerosis and squamous metaplasia confirmed by conjunctival impression cytology were treated with topical 0.1% 4-HPROG in an artificial tear vehicle for 3 weeks. Impression cytology was repeated at 2 and 3 weeks and at 3 weeks conjunctival biopsies were fixed for histology. Growth curves were generated using conjunctival fibroblasts of rabbits and humans (normals and patients with cicatrizing conjunctival disease including OCP and Stevens-Johnson syndrome) cultured in the 10(-8)-10(-6) M 4-HPROG. In vivo, corneal xerosis cleared in three days. A normal conjunctival epithelium was restored by 2 weeks and goblet cells were present by 3 wk, with no change in vehicle-treated controls. No ocular irritation occurred. In vitro, 10(-6) M 4-HPROG inhibits growth of rabbit conjunctival fibroblasts. The retinoid had no effect on proliferation of conjunctival fibroblasts from normal humans but the doubling time of cells from patients with OCP increased significantly, from 50.9 +/- 10.01 h (control) to 61.5 +/- 8.95 h (retinoid). Proliferation of conjunctival fibroblasts from a patient with Stevens-Johnson syndrome was also inhibited. N-(4-hydroxyphenyl) retinamide-O-glucuronide is biologically active and merits further study to determine its efficacy in controlling conjunctival fibrosis and treating ocular surface squamous metaplasia.

Nuclear retinoic acid receptors in the lacrimal gland.

The lacrimal gland secretes and metabolizes retinoids and responds to retinoic acid in culture. Like other retinoid responsive organs it is expected to express the nuclear retinoid receptors. The goal of this study was to identify the retinoic acid receptors (RAR) in the lacrimal glands of rats, rabbits, and humans. Total RNA was prepared from whole lacrimal glands and rat lacrimal gland acinar cells grown in culture. RNA was subjected to Northern blot analysis and probed for the RAR alpha, RAR beta, and RAR gamma mRNAs. Nuclear extracts of rat and rabbit lacrimal glands were incubated with 3H-all-trans retinoic acid and analyzed by gel filtration chromatography. Western blots of the nuclear extracts were probed using monoclonal antibodies to RAR alpha and RAR beta. Rat lacrimal gland expresses RAR alpha mRNA with two transcripts (3.8 and 3.0 kb), a single RAR beta mRNA transcript (3.3 kb), and a single RAR gamma mRNA transcript (3.3 kb). Cultured rat lacrimal acinar cells also expressed the mRNA for all three RAR subtypes. Rabbit lacrimal glands express mRNAs for RAR alpha (3.7 and 2.9 kb) and RAR beta (3.2 kb) but RAR gamma mRNA is not detectable. Human lacrimal glands also express mRNA for RAR alpha (3.5 and 2.3 kb), RAR beta (3.4 kb) and RAR gamma (3.0 kb). Lacrimal gland nuclear extracts contain proteins in the 50 kDa range that specifically bind retinoic acid with Kd = 1.25 nM in rat lacrimal gland and 0.3 nM in rabbit. The monoclonal antibodies identified RAR alpha and RAR beta in both rat and rabbit lacrimal glands. The results of this study support a role for retinoids in maintaining the structure and function of the lacrimal gland. The presence of RARs suggests potential interactions of these receptors with other members of their superfamily, including androgen and thyroid receptors, which also may be involved in lacrimal function.

Effects of preservative-free artificial tear solutions on corneal epithelial structure and function.

OBJECTIVES: To test the efficacy of a bicarbonate-containing artificial physiologic tear solution (solution PT) in providing an environment in which the damaged corneal epithelium can recover its normal barrier function and to compare this solution with other available artificial tears. Also, to investigate the effects on the corneal mucin layer and epithelial ultrastructure. METHODS: The corneal epithelial permeability of anesthetized rabbits was increased by exposure to 0.1% benzalkonium chloride. The corneas were then exposed to solution PT, with or without bicarbonate, or one of four commercially available artificial tear solutions for 1.5 hours, followed by a 5-minute exposure to 5(6)-carboxyfluorescein. Frozen sections of the corneas were examined by fluorescence microscopy. The fluorescence intensity (FI) of the epithelium was measured by image analysis. Undamaged corneas exposed to tear solutions were examined by transmission electron microscopy after fixation of the mucin layer with cetylpyridinium chloride. RESULTS: The FI of corneas damaged by benzalkonium chloride was increased threefold above those of undamaged controls. Damaged corneas treated with either of two commercial isotonic tear solutions partially recovered their barrier function, but the FI did not reach control levels. Corneas treated with hypotonic solutions containing ethylenediaminetetraacetic acid (EDTA) did not recover. In contrast, the FI of corneas treated with solution PT returned to control levels. This effect was lost in the absence of bicarbonate. Solution PT and the two isotonic solutions maintained normal corneal ultrastructure and mucin layer. Lack of bicarbonate in solution PT resulted in focal damage to superficial epithelial cells, whereas the EDTA-containing solutions destroyed the first two cell layers and reduced the mucin thickness. CONCLUSIONS: Bicarbonate-containing solution PT is superior to the other tear solutions tested in promoting recovery of the damaged corneal epithelial barrier and maintaining normal ultrastructure. The presence of bicarbonate appears to be essential to this process.

The influence of retinoic acid on growth and morphology of rat exorbital lacrimal gland acinar cells in culture.

The lacrimal gland transports and metabolizes retinoids and may require vitamin A for normal function. To study effects of retinoic acid on morphology and growth of the lacrimal gland, rat lacrimal acinar cells were cultured in medium with serum or in serum-free medium in the presence or absence of retinoic acid. In the presence of serum, the acinar cells have a somewhat fibroblastic morphology and form confluent layers. Addition of retinoic acid to these cultures causes formation of tubule-like structures. Retinoic acid inhibits the growth of lacrimal cells in medium with serum and the cells do not reach confluence; however, the labeling of the cells with bromodeoxyuridine is not affected by retinoic acid. In serum-free medium the growth of acinar cells is reduced, but their morphology is epithelial and structures resembling secretory domes are present. Retinoic acid causes a further reduction in growth, domes are absent, and cell spreading and enlargement occurs. The effects of retinoic acid on growth and morphology of lacrimal acinar cells in culture are complex and the relevance of these observations to lacrimal function in vivo is unclear; the study demonstrates, however, that these cells are responsive to retinoic acid.

The rabbit lacrimal gland in vitamin A deficiency.

PURPOSE: To investigate the morphology, ultrastructure, and protein secretion of the vitamin A-deficient rabbit lacrimal gland. METHODS: The lacrimal glands of vitamin A-deficient rabbits and age-matched controls were fixed, processed by standard methods, and examined by light and transmission electron microscopy. Protein secreted by the lacrimal gland was analyzed using gel filtration chromatography and electrophoresis. RESULTS: By light microscopy, the glands of experimental and control rabbits were indistinguishable. Electron microscopy showed little effect of vitamin A deficiency on the lacrimal acini, although occasional pyknotic nuclei were observed. The intralobular ductal epithelium was unaffected. Protein concentration and composition were essentially unchanged in lacrimal gland fluid of vitamin A-deficient rabbits compared to controls. CONCLUSIONS: The rabbit lacrimal gland is minimally affected by vitamin A deficiency, suggesting species differences between rabbits and rats in the vitamin A requirements of the lacrimal gland. Normal lacrimal gland structure and function in vitamin A deficiency allow for the prompt secretion of retinol on the restoration of vitamin A to the diet.

Cellular retinol-binding protein and cellular retinoic acid-binding protein in the lacrimal gland.

Cells that require or metabolize vitamin A contain cellular retinol-binding proteins (CRBP) and cellular retinoic acid-binding proteins (CRABP) which apparently participate in metabolism and transport of retinoids within the cell. Since the lacrimal gland secretes and metabolizes vitamin A, the cellular retinoid-binding proteins and their mRNAs should also be present in this gland. Total RNA from rat and rabbit lacrimal glands was analysed by RNase protection using 32P-labeled antisense cRNA probes. CRBP-I mRNA is present in rat lacrimal gland at 2% of the level in liver but CRBP-I mRNA could not be identified conclusively in rabbit lacrimal gland using the available rat cRNA probe. CRABP-I mRNA is present in rat lacrimal gland at 5% of the level in the 12-day gestation rat fetus, but was not detectable in the rabbit lacrimal gland. Rabbit or rat lacrimal gland cytosol was incubated with [3H]retinol or [3H] retinoic acid followed by ion-exchange chromatography using a Tris buffer and NaCl gradient. CRBP is present in rabbit and rat lacrimal gland at 78.5 +/- 14.5 and 71.1 +/- 11 pmol g-1 protein, respectively. CRABP is present in rat lacrimal gland at 190.6 +/- 13.8 pmol g-1 protein but was not detectable in rabbit lacrimal gland. The presence of CRBP in the lacrimal gland is consistent with its role in secretion of retinol into the lacrimal gland fluid. The apparent lack of CRABP in rabbit lacrimal gland as compared with rat suggests species differences in the role of retinoids in differentiation and maintenance of this organ.

Artificial tear composition and promotion of recovery of the damaged corneal epithelium.

In severe dry eye syndromes the corneal epithelium is compromised with development of punctate erosions and increased permeability. In the present study the ability of artificial tear solutions to promote recovery of the corneal epithelial barrier was determined by measurement of corneal uptake of 5,6 carboxyfluorescein (CF). Corneas of anesthetized rabbits were exposed to 0.01% benzalkonium for 5 min to increase epithelial permeability. The cornea was then exposed to an artificial tear solution for 1.5 h followed by measurement of CF uptake. During exposure to three commercial isotonic, nonpreserved solutions and a solution preserved with polyquaternium-1, CF uptake decreased significantly but did not return to control. No recovery of the epithelial barrier occurred during exposure of corneas to nonpreserved hypotonic solutions. During exposure to an experimental tear solution with an electrolyte composition similar to human tears, buffered with bicarbonate, CF uptake returned to control levels. Bicarbonate is an essential component of this solution because the same formula buffered with borate or without buffer was ineffective in promoting recovery of the damaged corneal epithelium.

Retinyl ester hydrolysis in the rabbit lacrimal gland.

The lacrimal gland stores retinyl esters which are synthesized by the enzyme acyl CoA:retinyl acyl transferase. Retinol is released from retinyl ester reserves by retinyl ester hydrolase (REH). Since the lacrimal gland secretes retinol, this gland should also contain this enzyme. To identify bile salt-dependent REH activity, rabbit lacrimal glands were homogenized in 0.05 M Trismaleate buffer, and enzyme activity was determined in the tissue homogenate, in the membrane fraction and in the cytosolic fraction by measurement of production of retinol from retinyl palmitate (nmol retinol produced/mg protein/h). In the lacrimal gland, production of retinol was optimal in the presence of 200 mM CHAPS at pH 7. The REH activity in the presence of 1000 microM retinyl palmitate was 2.38 +/- 0.18 nmol/mg/h in the homogenate, 1.13 +/- 0.16/nmol/mg/h in membranes and 3.25 +/- 0.26 nmol/mg/h in cytosol. By comparison, REH activity in rabbit liver was 6.58 +/- 0.75 nmol/mg/h. The REH activity in lacrimal gland was not affected by vitamin A deficiency. These data are consistent with the presence of retinyl ester hydrolase activity in the lacrimal gland and provide further evidence that this gland is adapted for metabolism and secretion of retinol.

The lacrimal gland synthesizes retinol-binding protein.

The rabbit lacrimal gland secretes retinol bound to a 20-21 kDa protein. To test the hypothesis that this protein might be retinol-binding protein (RBP) we probed lacrimal gland for RBP mRNA and lacrimal gland fluid for RBP. A rabbit RBP cDNA clone was used to probe rabbit and rat lacrimal gland RNA using RNase protection analysis. The lacrimal gland contains RBP mRNA at a level 0.1 to 0.03% of that observed in the liver. This RBP mRNA was identical to that observed in the liver based on RNase protection analysis, Northern blot analysis and primer extension analysis. The RBP mRNA levels in the lacrimal gland were not altered by the retinol status of the rabbits. We analysed lacrimal gland fluid for RBP by immunoblotting using a monoclonal antibody that recognizes rat, human and rabbit RBP. A single protein band from the rabbit lacrimal fluid bound the antibody, and this protein comigrated with human RBP which also bound the antibody. We conclude that the lacrimal gland contains RBP mRNA and that the lacrimal gland synthesizes and secretes RBP into the lacrimal gland fluid. This is the first demonstration that an extrahepatic tissue containing RBP mRNA synthesizes and secretes the protein in vivo.

Bile salt-independent retinyl ester hydrolase in the rat lacrimal gland.

The lacrimal gland secretes retinol and synthesizes and stores retinyl esters. In this study, presence of retinyl ester hydrolase in rat lacrimal gland was investigated. A bile salt-independent retinyl ester hydrolase was identified in lacrimal gland homogenates and microsomes which were incubated in a tris-maleate buffer with retinyl palmitate as substrate. Retinol produced was measured by HPLC and activity expressed as pmoles retinol produced/mg protein/hr. In homogenates, the enzyme activity had a pH optimum at pH 6 and maximum specific activity of 57.7 pmoles/mg/hr. In microsomes, the activity was optimal at pH 7 and maximum specific activity was 1073 pmoles/mg/hr. In contrast, rat liver retinyl ester hydrolase had a pH optimum at pH 8 and a specific activity of 24.7 pmoles/mg/hr. These observations demonstrate that a complete cycle of retinyl ester metabolism is present in the lacrimal gland.