Expression of two molecular forms of the complement decay-accelerating factor in the eye and lacrimal gland.

Complement is present in ocular fluids, but the molecular mechanism(s) restricting its activation to exogenous targets and not to autologous ocular cells are currently unknown. To clarify how this control is achieved, monoclonal antibody (mAb)-based techniques were used to examine the eye, the lacrimal gland, and ocular fluids for the decay-accelerating factor (DAF), a membrane regulatory protein which protects blood cells from autologous complement activation on their surfaces. Immunohistochemical staining of tissue sections revealed DAF antigen on corneal and conjunctival epithelia, corneal endothelium, trabecular meshwork, and retina, as well as on lacrimal gland acinar cells and in adjacent lumens. By flow cytometry, cultures of conjunctival epithelium exhibited the highest DAF levels and levels on corneal epithelium greater than corneal endothelium greater than conjunctival fibroblasts. Biosynthetic labeling of corneal endothelium yielded de novo DAF protein with an apparent molecular weight (Mr) of 75 kD, approximating that of blood cell DAF protein, and digestions of conjunctival epithelium with phosphatidylinositol-specific phospholipase C (PI-PLC), an enzyme which cleaves glycoinositolphospholipid membrane anchors, released approximately 70% of the ocular surface DAF protein similar to leukocyte surface DAF protein. Quantitations of DAF by radioimmunometric assay employing mAbs against two DAF epitopes revealed 325 ng/ml (n = 12), 4.8 ng/ml (n = 10), and 22.0 ng/ml (n = 8) of soluble DAF antigen in tears, aqueous humor, and vitreous humor, respectively. Western blot analyses of the tear DAF antigen revealed two DAF forms, one with an apparent Mr of 72 kD resembling membrane DAF forms in other sites, and a second with an apparent Mr of 100 kD, which is previously undescribed. Since DAF activity is essential physiologically in protecting blood cells from autologous complement attack, the identification of DAF on the ocular surface, intraocularly, in the lacrimal gland, and in tears suggests that DAF-mediated control of complement activation is also required in these locations.

Characterization of functional melanotropin receptors in lacrimal glands of the rat.

The specific melanotropin (MSH) binding sites of rat lacrimal glands were characterized with respect to anatomic distribution, peptide specificity and selectivity, and coupling to a biological response. Tissue distribution of MSH binding sites was determined by autoradiography following in situ binding of a radiolabeled, biologically active preparation of a superpotent alpha-MSH analog, [125I]-[Nle4,D-Phe7]-alpha-MSH ([125I]-NDP-MSH). Intense, specific (i.e., alpha-MSH-displaceable) [125I]-NDP-MSH binding was observed throughout lacrimal acinar tissue, but not in ducts or stroma. In freshly isolated lacrimal acinar cells, specific binding of [125I]-NDP-MSH was maximal within 30 min and rapidly reversible, with a dissociation half-time of about 15 min. A number of melanotropins [alpha-MSH, [N,O-diacetyl-Ser1]-alpha-MSH, [des-acetyl-Ser1]-alpha-MSH, beta-MSH, ACTH(1-24) and ACTH(1-39)] were recognized by these binding sites, as assessed by their inhibition of [125I]-NDP-MSH binding; NDP-MSH was the most potent (IC50 = 1.3 x 10(-9) M). In contrast, other peptides, including ACTH(4-10) and the nonmelanotropic peptides VIP, substance P, somatostatin, and ACTH(18-39) (CLIP), had no effects on tracer binding. In isolated lacrimal acinar cells, alpha-MSH and NDP-MSH stimulated intracellular cyclic AMP accumulation. We conclude that lacrimal acinar cells express functional receptors recognizing melanotropins, suggesting that the lacrimal gland may be a target for physiological regulation by endogenous melanotropins.

High-performance liquid chromatographic analyses of porphyrins in hamster Harderian glands.

Porphyrin content and 5-aminolaevulinate synthase activity of the Harderian gland were measured in intact and gonadectomized male and female hamsters; porphyrin profiles were analysed by high-pressure liquid chromatography. The total porphyrin content of the two female groups was similar, but enzyme activity in females ovariectomised for 20 weeks significantly decreased. Intact males have low porphyrin content and enzyme activity, while in castrates (6 weeks) both increased to female levels. Protoporphyrin IX formed 93% of total porphyrins in intact females, compared with 70% of total porphyrins in intact males. The remainder in both sexes was chiefly penta- and hexacarboxylic porphyrins and coproporphyrin and (in females) Harderoporphyrin. Gonadectomy in either sex resulted in protoporphyrin levels intermediate between male and female values.

Localization of atrial natriuretic peptide/cardiodilatin (ANP/CDD)-immunoreactivity in the lacrimal gland of the domestic pig.

The presence of atrial natriuretic peptic/cardiodilatin-immunoreactive material was demonstrated in the lacrimal gland of the domestic pig by high performance liquid chromatography and radioimmunoassay. The immunohistochemical localization revealed a distinct population of cuboid or spindle-shaped ANP/CDD-IR cells in the epithelium of the terminal portion of the secretory tubules. In addition, a moderate number of positive cells was localized intraepithelially in the intralobular ducts as well as the connective tissue between these ducts. Our findings provide a morphological indication that ANP/CDD may play a physiological role in the regulation of sodium transport and secretion in the lacrimal gland.

Composition of long-chain bases in sphingomyelin of the guinea pig Harderian gland.

Sphingomyelin from the guinea pig Harderian gland was isolated and characterized. The purified sphingomyelin gave a broad spot on thin-layer chromatography. The fatty acid composition of the whole sphingomyelin was 71% nonhydroxy acids and 29% 2-hydroxy acids. Methyl-branched fatty acids were only 2% of the total acids. The long-chain bases were composed of straight-chain sphingenines (50%) and sphinganines (6%). Methyl-branched long-chain bases were 44% of the bases. The sphingomyelin was further separated into four fractions (I, II, III, IV) by high-performance liquid chromatography. The ratio of fractions I, II, III, and IV was approximately 2:5:2:1, respectively. The fatty acids of fractions I and II consisted of nonhydroxy acids and those of fractions III and IV were 2-hydroxy acids. The long-chain bases of fractions I and III were sphinganines including 10-, 9-, and 8-methylsphinganines and anteiso-sphinganines. These methyl-branched bases occupied about 70% of the total sphinganines. The long-chain bases of fractions II and IV consisted of sphingenines. The methyl-branched unsaturated bases were only 30% of the total sphingenines, all in the anteiso-form. Thus, the sphingomyelin obtained from guinea pig Harderian gland had complex compositions of fatty acids and long-chain bases, and half the number of long-chain bases had methyl branches. The methyl-branched fatty acids were only a minor component. These characteristics are similar to those of cerebrosides isolated from the same source.

Purification and characterization of a growth factor from guinea pig harderian gland.

A polypeptide growth factor, Harderian gland-derived growth factor (HGDGF), has been purified approximately 43,000-fold from guinea pig Harderian gland by column chromatography on TSK gel DEAE-5PW, blue-Sepharose CL-6B, and Superose 12. The yield was approximately 10%. The Superose 12 fraction was further purified by Aquapore BU-300 reversed-phase chromatography to apparent homogeneity. HGDGF was eluted from TSK gel DEAE-5PW at 0.20-0.35 M NaCl, with a linear gradient of 0.15-0.80 M NaCl and at 2.20 M NaCl from blue-Sepharose CL-6B. The activity of HGDGF toward human embryonic cells (TIG-3) was quantitated, [3H]thymidine incorporation for 48 h being stimulated in a linear and dose-dependent manner. Purified HGDGF has a molecular weight of approximately 13,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and molecular sieve column chromatography. HGDGF is labile to treatment with SH reagents or acetic acid. Both trypsin digestion and boiling decrease the activity of HGDGF. Its pI is 5.1. HGDGF stimulates the multiplication of TIG-3 cells but has no effect on human endothelial cells K2T1 or A2T2 which require fibroblast growth factor for growth. HGDGF appears to differ from other growth factors, suggesting that it is a previously undescribed growth factor.

Electrolyte composition of lacrimal gland fluid and tears of normal and vitamin A-deficient rabbits.

Rabbit tears and lacrimal gland fluid were collected simultaneously during pilocarpine stimulation with the goal of comparing the ionic composition of these fluids at various flow rates. Ions measured were sodium, potassium, calcium, magnesium, zinc, chloride, and bicarbonate. Human tears were also analyzed for purposes of comparison. Generally, tears and lacrimal gland fluid do not differ in ionic composition except for zinc and bicarbonate, which are in higher concentration in tears than in lacrimal gland fluid. The ionic composition of tears and lacrimal gland fluid of vitamin A-deficient rabbits was also analyzed. The maximal flow rate of lacrimal gland fluid was decreased in vitamin A-deficient rabbits as were calcium levels in tears and lacrimal gland fluid, as compared with controls. Concentrations of other ions generally did not differ from normal levels, indicating that vitamin A deficiency has only moderate effects on lacrimal gland function in the rabbit.

Identification of 1-alkyl-2-acyl-3-(2',3'-diacylglycerol)glycerols, a new type of lipid class, in harderian gland tumors of mice.

A new class of alkyl glycerolipids, 1-alkyl-2-acyl-3-(2',3'-diacylglycerol)glycerols, was identified in lipid extracts prepared from harderian gland tumors of mice. After saponification, this lipid class yielded 1-alkyl-3-(1'-glycerol)glycerols. Identification was based on mass spectrometry, proton nuclear magnetic resonance spectroscopy, infrared spectroscopy, and chromatography of various derivatives and appropriate standards that were synthesized. The alkyl moieties of this unique lipid class consisted of saturated aliphatic chains with chain lengths of 14 to 20 carbon atoms. The acyl moieties were mostly saturated and monounsaturated aliphatic chains ranging from 14 to 24 carbon atoms. The alkyl and acyl moieties of 1-alkyl-2-acyl-3-(2',3'-diacylglycerol)glycerols were similar to those of alkyldiacylglycerols present in the same tissue, except for the presence of monounsaturated alkyl moieties in the latter. 1-Alkyl-2-acyl-3-(2', 3'-diacylglycerol)glycerols were only found in trace amounts in the normal harderian glands of mice. The total quantity of the alkyl and acyl moieties with a chain length greater than 20 carbon atoms in the alkyldiacylglycerols from tumors were considerably lower than those found in normal harderian glands of mice. This is the first report of the presence of bisglyceryl ether lipids in mammalian tissue; its unique chemical structure is consistent with the type of ether-linked lipid products that could be synthesized in the reaction catalyzed by alkyldihydroxyacetone-P synthase.

Steroid hormone receptors and the sexual phenotype of the Harderian gland in hamsters.

To investigate the participation of intracellular steroid hormone receptors in the sexual transformation process of the Harderian gland, a series of experiments were undertaken in adult golden hamsters. The invitro labelling of cytosolic steroid-binding sites with appropriate radioligands revealed the presence of androgen, oestrogen and glucocorticoid but not progestin receptors in the glands from animals of both sexes. The androgen receptor of the female gland was further characterized because it was found to be the predominant intracellular steroid receptor. Studies of binding kinetics using [3H]7 alpha,17 alpha-dimethyl-17 beta-hydroxy-4-oestren-3-one (DMNT) as ligand, demonstrated a high affinity androgen-binding site with an apparent dissociation constant (Kd) of 0.7 nmol/l and maximal saturation binding capacity of 84.0 +/- 3.0 (S.D.) fmol/mg protein. Specificity of the androgen receptor was assessed by displacement analysis; DMNT, 5 alpha-dihydrotestosterone, testosterone and 3 alpha-androstanediol were efficient competitors for the androgen-binding site, while oestradiol-17 beta, progesterone and dexamethasone exhibited very little, if any, competitive potency. The sedimentation coefficient of the androgen receptor in sucrose density gradients was 8-9 S. These data indicate that the physicochemical characteristics of the androgen receptor from the female gland are similar to those previously described in the male gland. The striking observation of a complete lack of oestrogen-inducible and oestrogen-insensitive progestin receptors in glands cytosol, even after stimulation with cholera toxin, adds further support to the concept that the androgen receptor is the key molecule mediating the hormone-induced sexual transformation of the Harderian gland in this species.

Nerve fibers showing immunoreactivities for proenkephalin A-derived peptides in the lacrimal glands of the guinea pig.

The extra- and intraorbital lacrimal glands of guinea pigs were studied for the presence and distribution of enkephalin-like immunoreactive nerve fibers. Four specific antisera against the four different enkephalin sequences contained in pre-proenkephalin A (Met-Enk, Met-Enk-Arg-Phe, Met-Enk-Arg-Gly-Leu, and Leu-Enk) were used. All of these immunoreactivities were identically distributed in varicose nerve fibers in both the extra- and intraorbital lacrimal glands. These fibers densely surrounded the glandular acini and also innervated the secretory ducts. Sympathetic denervation had no effect on these nerve fibers. The results suggest that enkephalins derived from proenkephalin A may play a role in the nervous control of the lacrimal secretion.

First demonstration of porphyrins in the lacrimal glands: similarity to the Harderian gland.

The Harderian gland of rodents is the only known tissue with physiological occurrence of high concentrations of porphyrins. In this report we describe the occurrence of considerable concentrations of porphyrins in the extra-orbital and intra-orbital lacrimal glands of the male rat. Similarly as in the Harderian gland, HPLC analysis revealed protoporphyrin as being the prevalent porphyrin homologue in the lacrimal glands. The results may contribute to elucidation of the yet unknown function of the Harderian gland and its porphyrins.

Cellular carbohydrate components in human, rabbit and rat lacrimal gland. Studies using fluorescein and peroxidase labelled lectins.

Orbital lacrimal glands from adult male and female rabbits, rats and humans were examined for the presence of intracellular receptors of four lectins: concanavalin-A agglutinin, lutus tetragonolobus agglutinin, ricinus comunis-60 agglutinin and wheat-germ agglutinin using fluorescein-conjugated lectin and peroxidase labelling methods for fluorescence and electron microscopy, respectively. Lectins were used as specific probes to detect carbohydrate moiety of the lacrimal gland. The pattern of labelling with the lectins suggests that N-acetyl-glucosamine, N-acetyl-D-galactosamine, D-galactose, D-mannose, sialic acid and L-fucose are contained in the lacrimal gland of the three species. The significance of these findings is discussed.

Evidence for an anion exchanger in the mouse lacrimal gland acinar cell membrane.

Anion exchange transport in the mouse lacrimal gland acinar cell membrane was studied by measuring the intracellular H+ (pHi) and Cl- (aCli) activities with double-barreled ion-selective microelectrodes. In a HCO3- -free solution of pH 7.4 (HEPES/Tris buffered), pHi was 7.25 and aCli was 33 mM. By an exposure to a HCO3- (25 mM HCO3-/5% CO2, pH 7.4) solution for 15 min, aCli was decreased to 25 mM, and pHi was transiently decreased to about 7.05 within 1 min, then slowly relaxed to 7.18 in 15 min. Intracellular HCO3- concentration [HCO3-]i, calculated by the Henderson-Hasselbalch's equation, was 11 mM at 1 min after the exposure and then slowly increased to 15 mM. Readmission of the HCO3(-)-free solution reversed the changes in aCli and pHi. The intracellular buffering power was about 40 mM/pH. An addition of DIDS (0.2 mM) significantly inhibited the rates of change in aCli, pHi, and [HCO3-]i caused by admission/withdrawal of the HCO3- solution and decreased the buffer value. Replacement of all Cl- with gluconate in the HCO3- solution increased pHi, and readmission of Cl- decreased pHi. The rates of these changes in pHi were reduced by DIDS by 32-45% but not by amiloride (0.3 mM). In the HCO3- solution, a stimulation of intracellular HCO3- production by exposing the tissue to 25 mM NH4+ increased aCli significantly. While in the HCO3(-)-free solution or in the HCO3- solution containing DIDS, exposure to NH4+ had little effect on aCli.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of very-long-chain fatty acids in rat and mouse harderian gland lipids by capillary gas chromatography-mass spectrometry.

Lipids of Harderian ophthalmic gland were separated by means of thin-layer chromatography with flame ionization detection in an latroscan apparatus. Wax ester and polar lipids (phosphatidylethanolamine and phosphatidylcholine) were detected as the main lipids in rats and glyceryl ether diester and both polar lipids were the main lipids in mice. Fatty acids were determined in individual lipid classes by means of gas chromatography and gas chromatography-mass spectrometry on capillary columns. The content of fatty acids, the positional isomers of monoenoic acids being predominantly C18, C20 and C22, is most interesting. Very-long-chain fatty acids, saturated fatty acids up to C30 and even monoenoic acids up to C28 were detected. Branched-chain fatty acids, predominantly iso and anteiso, are minority components, although their chain length distribution (C15-C27) is broad.

Normal ultrastructure and histochemical characteristics of canine lacrimal glands.

Lacrimal glands of 12 dogs free of ocular disease were examined to determine the normal structure of these glands. The glands consisted of tubuloacinar cells that ultrastructurally and histochemically were of a single type of secretory cell in the tubules and possibly 3 types of secretory cells in the acini. The tubular epithelium contained homogenous electron-dense granules that stained as neutral glycoconjugates (periodic acid-Schiff positive and Alcian blue and high iron diamine negative). The predominant acinar cells contained granules of lesser electron density than those of the tubules, and stained as sialomucin (Alcian blue [pH 2.5] and periodic acid-Schiff-positive, and high iron diamine-negative). A second type of acinar cell was in peripheral lobules that ultrastructurally and histochemically appeared like lipid granules (positive with oil red O and osmium tetroxide). Ultrastructurally, a third type of acinar granule was finely granular, electron-lucent, and frequently coalesced. It was not readily apparent whether the latter was an artifact, a stage in the maturation of the sialomucin granules, or a third type of acinar granule. Individual acinar cells usually had a predominance of 1 granule type, but greater than 1 granule type could be found in some cells. The basal surfaces of the acinar, tubular, and ductal cells were incompletely ensheathed by myoepithelial cells. Plasma cells, lymphocytes, mast cells, endothelial cells, fat cells, and Schwann cells composed the cellular elements of the interstitium. Lymphocytes, mast cells, and nerve endings also were found in the parenchyma.

Melatonin in the lacrimal gland: first demonstration and experimental manipulation.

NAT, HIOMT and melatonin are described in the extra-orbital lacrimal glands. The extra-orbital lacrimal glands of female Syrian hamsters contain higher NAT activity and melatonin levels than those in male glands, while male glands have higher HIOMT activity. Castration did not change melatonin in the lacrimal glands, although NAT and HIOMT activities were altered. The exposure of female hamsters to light in the morning (0600h) was associated with a reduction in both NAT activity and melatonin levels. Porphyrins were not detected in the lacrimal glands of either male or female hamsters.

Identification of 10-methylsphinganine in cerebrosides of the guinea pig harderian gland.

A large amount of branched long chain bases was detected in the cerebrosides of guinea pig Harderian gland. The long chain bases of cerebrosides were analyzed by GLC as trimethylsilyl derivatives. The branched long chain bases were separated into four peaks (I, II, III, IV) according to the number of carbon atoms and the position of branching. In the present work, the structures of long chain bases in the four peaks were analyzed by GLC and GC-MS after conversion of them to aldehydes, alcohols, and fatty acids. Furthermore the main component of long chain bases (Peak II) was isolated by HPLC as N-acetyl derivatives and analyzed by NMR. The structures of branched long chain bases in Peaks I, II, III, and IV are as follows. Branched long chain bases of Peak I are 2-amino-10- (main component), 2-amino-9-, and 2-amino-8-methylhexadecane-1,3-diol. Branched long chain bases of Peak II also consist of a mixture of 2-amino-10-, 2-amino-9-, and 2-amino-8-methyl-heptadecane-1,3-diol. The branched long chain base of Peak III is 2-amino-10-methyl-octadecane-1,3-diol, while that of Peak IV is 2-amino-16-methyloctadecane-1,3-diol. Among these branched long chain bases, 10-methylsphinganines are dominant though the chain lengths are different. These branched long chain bases, in which the substituted positions exist in the middle part of aliphatic chain (10-, 9-, or 8-methylsphinganine) are novel long chain bases in mammals.

The effect of aging on the secretory immune system of the eye.

The objective of the present study was to examine the influence of aging on the ocular secretory immune system of the eye. Levels of IgA and free secretory component (FSC) were measured in lacrimal glands and/or tears of 0.6, 1.3, 3, 8 and 17-month-old male and female rats. In addition, the FSC output of lacrimal tissue cultured in vitro was evaluated. During the period from 0.6 to 1.3 months of age, the content of tear IgA increased nine- and 13-fold in females and males, respectively. This rise was paralleled by changes in the concentration of tear FSC. Prior to the onset of puberty, FSC could be detected in only 7% of tear samples, whereas after pubertal maturation, tear FSC levels had attained adult concentrations. This tear FSC profile was similar to the age-related pattern of FSC output by lacrimal tissue incubated in vitro. Following puberty, tear IgA content continued to increase in both sexes until adulthood (3 months of age) and then plateaued in females from 8 to 17 months of age. In contrast, tear IgA in males appeared to stabilize from 3 to 8 months and then rose significantly to the highest levels at 17 months of age. This increase in males was also reflected in their lacrimal tissue: IgA content underwent a six-fold elevation from 3 to 17 months. Of interest is that the differential kinetics involved in tear IgA and FSC expression resulted in an age-associated decline in the FSC/IgA ratio from post-puberty to senescence. A striking finding in these studies was the persistence of a sexual dimorphism in the secretory immune system of the eye. After pubertal development, IgA and FSC levels were significantly higher in tears of males, compared to those of females, at all ages tested up to 17 months. These gender- and age-related variations in tear IgA and FSC amounts could not be accounted for by changes in either the volume of, or total protein content in, tears.

Distribution of cytoskeletal and contractile proteins in normal and tumour bearing salivary and lacrimal glands.

We have evaluated by means of immunocytochemistry the distribution of various cytoskeletal and contractile proteins (cytokeratins, vimentin, desmin and alpha-smooth muscle actin) in 23 salivary or lacrimal gland primary tumours (15 pleomorphic adenomas and 8 carcinomas in pleomorphic adenoma), one third of which contained areas of normal gland. Normal epithelial luminal cells were stained by cytokeratin antibodies with a general specificity, while myoepithelial cells were selectively stained by a monoclonal antibody (SK2-27) reacting in immunoblots with cytokeratin polypeptides 14, 16 and 17, according to the classification of Moll et al. (1982) and by an antibody directed against alpha-smooth muscle actin (Skalli et al. 1986). In pleomorphic adenomas, both epithelial and myoepithelial cells displayed typical topographic distributions; moreover, myoepithelial cells showed two distinct cytoskeletal phenotypes. These findings could account in part for the heterogeneity of aspects observed in this tumour. In carcinomas, malignant cells were always positive to cytokeratin antibodies with general specificity and myoepithelial cells were absent as judged by anticytokeratin SK2-27 and anti-alpha-smooth muscle actin immunostainings. However, interestingly, there was in all cases a strong positivity for alpha-smooth muscle actin in stromal cells, similarly to what has previously been described for mammary carcinoma (Skalli et al. 1986). Our findings may be useful for the interpretation of the histogenesis of salivary and lacrimal tumour and stromal cells.

Branched long chain bases in cerebrosides of the guinea pig Harderian gland.

Cerebrosides obtained from the guinea pig Harderian gland were analyzed. The purified cerebrosides gave a single spot on thin-layer chromatography, the Rf value being similar to that of phrenosine obtained from whale brain. The cerebrosides consisted of 74.7% of glucosylceramide and 25.3% of galactosylceramide. The fatty acid composition of these cerebrosides was 0.7% of non-hydroxy fatty acids and 99.3% of alpha-hydroxy fatty acids. Among these alpha-hydroxy fatty acids, a small amount of methyl branched acids was detected. The substituted position of methyl branching of alpha-hydroxy fatty acids was the 16th carbon atom from the carboxyl end irrespective of the carbon chain length. The long chain bases were composed of sphinganine (78%) and sphingenine (22%). 4-D-Hydroxysphinganine was not found. The most remarkable feature of the long chain bases of cerebrosides in the Harderian gland was the presence of a large amount of methyl branched sphinganine. The cerebrosides obtained from the cerebrum and cerebellum of the same animal were also analyzed. The sugar, fatty acid, and long chain base compositions of these cerebrosides were similar to those of whale brain cerebrosides. Methyl branched sphinganine was not found in guinea pig brain.