Characterization of beta-hexosaminidase secretion in rabbit lacrimal gland

The present study was aimed at validating the use of the lysosomal enzyme beta-hexosaminidase as a marker of secretory function in cultured rabbit lacrimal gland acinar cells. The secretory response and morphological characteristics of isolated acinar cells cultured in a serum-free medium supplemented with an extracellular matrix extract were monitored over time as part of optimization of our culturing protocol. Secreted beta-hexosaminidase activity was analyzed and compared with that of another lysosomal enzyme, cathepsin B, as well as protein secreted into the media, w or w/o the presence of secretagogues or protein kinase C activators and inhibitors. Lacrimal gland fluid was obtained from pilocarpine stimulated rabbits, and the activities of beta-hexosaminidase and cathepsin B were measured. A membrane fraction and a soluble fraction were obtained from isolated acinar cells and used for kinetic studies of beta-hexosaminidase in comparison with that released from cultured cells, in the lacrimal gland fluid and in serum. Optimal secretory response was obtained when the cells had been in culture for 2-3 days, coinciding with the formation of acinus-like structures. Stimulation of the cultured cells by carbachol or phorbol esters resulted in a more than 3-fold increase of beta-hexosaminidase release over basal, whereas no effect on cathepsin B release could be detected. Treatment with the protein kinase C inhibitor, chelerythrine chloride, significantly decreased the carbachol and phorbol ester-stimulated secretion. Cathepsin B could not be detected in rabbit lacrimal fluid, but beta-hexosaminidase was easily measured in quantities corresponding to as low as 0.4 microl of tear fluid. Using 4-methylumbelliferyl N-acetyl-beta-D-glucosaminide as a substrate for beta-hexosaminidase, the K(m) in lacrimal gland fluid (1.22+/-0.15 mM) was not significantly different from that of the membrane-associated fraction, the soluble fraction, rabbit serum or activity secreted from cultured cells. Beta-hexosaminidase is secreted by rabbit lacrimal gland, in vivo, and by acinar cells in primary culture, whereas cathepsin B is not secreted under the conditions described. Beta-hexosaminidase therefore provides a versatile marker for secretion in studies of tear production utilizing the rabbit as a model. Our results also indicate that PKC is an important regulator of rabbit lacrimal gland secretion.     

毛果芸香碱促进培养的兔泪腺上皮细胞蛋白合成和分泌

目的研究毛果芸香碱对体外培养兔泪腺上皮细胞蛋白分泌与合成的影响。设计实验研究。研究对象培养的兔泪腺上皮细胞。方法对培养细胞进行广谱细胞角蛋白、波形蛋白免疫组化染色及电镜观察鉴定后,培养的兔泪腺上皮细胞中,分别加入700、70、7、0．7和0．07μmol／L的毛果芸香碱,培养24小时,分别测定培养液和细胞中的β－氨基已糖苷酶活性。在不同浓度毛果芸香碱的培养细胞中加入70μmol／L阿托品,培养24小时,分别测定培养液和细胞中的β－氨基已糖苷酶活性。主要指标β－氨基已糖苷酶活胜（光密度值,OD）。结果毛果芸香碱在700、70、7、0．7和0．07μmol／L浓度时使泪腺上皮细胞培养液β－氨基已糖苷酶活性OD值分别增加17．7％（P＝0．015）、14．7％（P＝0．038）、5．7％（P＝0．399）、413％（P=0．517）和2．0％（P=0．7641,使泪腺上皮细胞冻融液β－氨基已糖苷酶活性OD值分别增加25．0％（P=0.000）、16．8％（P＝0．001）、10．6％（P=0．023）、7．6％（P=0．089）和4．8％（P=0．271）。阿托品抑制毛果芸香碱的作用,使泪腺上皮细胞培养液β-氨基已糖苷酶活性OD值分别减少20．5％（P=0．018）、15．1％（P=0．029）、10．1％（P=0．097）、11．5％（P=0．118）和7．9％（P=0．085）,使泪腺上皮细胞冻融液B一氨基已糖苷酶活性OD值分别减少10．0％（P=0．039）、4．8％（P=0．113）、6．2％（P=0．162）、2．9％（P=0．315）和0％（P=0．300）。结论毛果芸香碱可增加培养的泪腺上皮细胞的蛋白分泌和合成,阿托品可抑制毛果芸香碱的作用。     

Enzymatic studies in lacrimal secretion

The activity of the enzymes: lactate dehydrogenase (LDH), pyruvate kinase (PK), malate dehydrogenase (MDH), amylase (AM) and lysozyme (LZM) was studied during lacrimation in healthy persons. The activity of the enzymes of energy-producing metabolism: LDH, PK and MDH fluctuated in parallel with each other. Fluctuations in AM activity showed no correlation with other enzymes and LZM concentrations remained rather constant. The origin of these enzymes in the process of lacrimal secretion is discussed.     

Surgery of lacrimal secretion

Surgical treatment of lacrimal secretion consists in reducing the product of tears in the lacrimal gland. Denervation of the lacrimal gland was described long ago by Whitwell and remains in current use. Recently, injection of botulinum toxin into the lacrimal gland was successfully carried out. In our department, we have been developing lacrimal duct orifice cauterization for several years. We discuss these three methods and develop the latter in greater detail.     

Retinol secretion by the lacrimal gland

In order to determine the source of the retinol which has been identified in the tear fluid, the lacrimal gland ducts of rabbits and rats were cannulated and the collected lacrimal gland fluid was analyzed by high performance liquid chromatography. Retinol was identified in the lacrimal gland fluid of rabbits and rats, and it is concluded that the lacrimal gland is the source of retinol in the tears. Dose-response studies show that intravenously administered pilocarpine and intra-arterial acetylcholine stimulate secretion of retinol by the lacrimal gland. Intravenous administration of vasoactive intestinal peptide (VIP) also stimulates retinol secretion in a dose-response manner. These observations are similar to the effects of cholinergic drugs and VIP on protein secretion by the lacrimal gland.     

Lacrimal streams: the demonstration of human lacrimal fluid secretion and the lacrimal ductules.

The clinical demonstration of the ductular orifices of the human lacrimal gland is reported. Lacrimal fluid secretion can be shown after instillation of 2% sodium fluorescein. Ductular orifices are visible on biomicroscopy. In keratoconjunctivitis sicca the lacrimal fluid streams appear normal, or to be diminished or absent.     

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.     

Involvement of dopamine D1-like receptors in mediating increases in protein secretion from rabbit lacrimal gland.

To identify and localize the dopamine receptor subtypes in rabbit lacrimal gland which mediate protein secretion, the effects were determined of different dopamine receptor subtype agonists, antagonists, and a beta adrenergic antagonist on this process. Protein secretion into the medium was quantified with the Bradford assay. Dopamine increased protein secretion between 10(-7) and 10(-4)M, and it could be maintained for a subsequent 80 min. The relatively selective D1-like receptor agonist, SKF-38393 (10(-4)M) had a similar effect which was suppressed by the D1-like receptor antagonist, SCH-23390. However, neither the D2-like receptor agonist, quinpirole (10(-4)M), nor the selective D2-like receptor antagonist, sulpiride (10(-4)M) altered either the basal level or the stimulated response to dopamine. The dopamine (10(-4)M)-elicited increases in protein secretion were completely suppressed in the presence of either 10(-4)M propranolol or 10(-4)M bretylium. Protein secretion in rabbit lacrimal gland is mediated by dopaminergic nerves through stimulation of the presynaptic D1-like receptor.     

Adrenocorticotropic hormone stimulation of lacrimal peroxidase secretion

The effect of adrenocorticotropic hormone (ACTH) on secretion of lacrimal gland peroxidase was studied using an in vitro perifusion technique. The peptide stimulated a dose-dependent (1 nM to 100 nM) release of peroxidase, with the maximum level of secretion induced by 20 nM ACTH. Secretion in the presence of submaximal ACTH was potentiated with either 100 microM iso-butylmethylxanthine or 0.3 microM carbachol. In contrast, the combination of ACTH and phenylephrine was additive. Time-dependence studies demonstrated that the stimulation of peroxidase release by ACTH, as with other cyclic adenosine monophosphate mediated secretagogues, showed a latency in reaching the maximum rate which was not evident with either cholinergic or alpha-adrenergic stimulation. Furthermore, where potentiation of the response to ACTH occurred, the time course was distinctly altered from that obtained with either ACTH or the potentiating agonist alone. The data suggest that lacrimal gland function is regulated by a multiple system of neurotransmitters and (or) neuromodulators that involves the activation of peptidergic as well as cholinergic and alpha-adrenergic receptors.     

Comparison of tears and lacrimal gland fluid in the rabbit and guinea pig

The excretory duct of the lacrimal gland of rabbits and guinea pigs was cannulated in situ for collection of pure lacrimal gland fluid, not contaminated by secretions from the Harderian gland or contributions of desquamating cells of the conjunctival and corneal epithelium. Tears as well as lacrimal gland fluid of both species showed a species-specific and molecular weight-dependent pattern after sodium dodecylsulphate-polyacrylamide (SDS-PAA) gradient slab gel electrophoresis. The most striking difference in both species was a protein corresponding to serum albumin present in tears and almost lacking in lacrimal gland fluid. Likewise, a variety of enzymes, total protein and PGE2 were measured in tears and lacrimal gland fluid. For rabbit tears the lacrimal gland is the primary tissue source of lysozyme (LZM), beta-hexosaminidase (beta-hex), angiotensin-converting enzyme (ACE), plasminogen activator (PA) and total protein, while lactate dehydrogenase (LDH) and the greater part of prostaglandin E2 (PGE2) are present in rabbit tears mainly as products from other ocular tissue sources. In guinea pig tears peroxidase (POD), ACE, PA and less PGE2 are exceted by the lacrimal gland, amylase (AMY), LDH and a substantial amount of PGE2 are added to the guinea pig tears by other ocular tissue sources. Beta-hex and total protein are released from the lacrimal gland and from other ocular tissue sources as well.     

Sex hormone regulation of tear lipocalin in the rabbit lacrimal gland

Tear lipocalin (TL) (∼18 kDa), a member of the lipocalin superfamily, has been identified as one of the major proteins present in rabbit lacrimal fluid. The concentration of TL has been found to be decreased in the tears of patients with dry eye disease. Lacrimal gland insufficiency, one of the major causes of dry eye disease, is known to affect mainly postmenopausal women, where there is a significant decrease in the production of androgen and estrogen. These observations suggest that sex hormones might influence dry eye indirectly by regulating the expression of TL. The purpose of this study was to determine: (1) the effect of sexual maturation on the expression of TL; and (2) if the expression of TL is regulated by the estrogen, 17β-estradiol, and/or the androgen, dihydrotestosterone, in sexually mature female rabbits. Lacrimal fluid (LF) and lacrimal gland soluble fraction (Si) was collected from juvenile (2 kg) and sexually mature (4 kg) male and female New Zealand white (NZW) rabbits. In addition, LF and Si were collected from 4 kg rabbits, 7 days after being either sham operated (control), ovariectomized (OVX), ovariectomized treated with estrogen (OVX + E) or ovariectomized treated with dihydrotestosterone (OVX + DHT). Samples were analyzed for protein levels of TL by SDS-PAGE and Western blotting using a polyclonal rat anti-rabbit TL antibody. Densitometry analysis showed that TL protein levels in both LF and Si increased with age in male and female rabbits. In addition, TL protein levels were significantly higher in the sexually mature 4 kg male compared with the 4 kg female, while no significant difference in TL protein levels were seen among the juvenile male and female rabbits. Furthermore, ovariectomy decreased the protein levels of TL in LF and Si fraction by 50% and 20% respectively, compared with control values. Estrogen treatment increased TL protein levels by 30% and 50% in the LF and Si fraction respectively, compared with the sham operated group. DHT treatment also increased TL protein levels by approximately 150% in both LF and Si fraction compared with control values. These results support the hypothesis that sex hormones influence TL protein levels in rabbit lacrimal glands. The possibility of a role of TL in dry eye needs to be further investigated.     

Sympathomimetic protein secretion by young and aged lacrimal gland

The diminished basal tear flow in aged individuals is associated with lymphocytic infiltrations and atrophy of the lacrimal ducts and acini. We have investigated the age-related physiological changes to sympathomimetic stimulation of lacrimal tissue from F344 rats to determine if the responses are uniformly diminished as would be expected by glandular atrophy. The quantitative and temporal pattern of protein and peroxidase secretion by lacrimal gland fragments from young (4 month) and aged (24 month) F344 male rats was examined in a perifusion system. Upon stimulation of tissue from young animals with 0.01 mM phenylephrine for 40 min, secretion above baseline levels of protein was 570.8 micrograms/g tissue and of peroxidase was 45.2 delta A X min-1/g tissue. The response of the aged tissue to phenylephrine was not significantly different from that of the young tissue. beta-adrenergic stimulation by isoproterenol (0.01 mM) evoked only a modest secretion of protein and no consistently measurable peroxidase from young tissue. IBMX alone and in combination with isoproterenol (0.1 mM and 0.01 mM respectively) evoked a large secretion of protein, 1345.7 micrograms/g tissue, and a modest secretion of peroxidase, 9.5 delta A X min-1/g tissue by young tissue. The aged tissue, upon stimulation with the combination of IBMX and isoproterenol, secreted significantly less protein and peroxidase than the young tissue. In separate experiments, the production of cAMP was measured. In young tissue, isoproterenol did not cause a measurable increase of intracellular cAMP. IBMX caused a 2-3 fold increase in cellular cAMP which was not increased further by addition of isoproterenol.(ABSTRACT TRUNCATED AT 250 WORDS)