Regulation of conjunctival goblet cell secretion by Ca(2+)and protein kinase C.

Conjunctival goblet cells secrete mucus in response to cholinergic (muscarinic) agonists, but the underlying signaling pathways activated in this tissue are not well understood. Cholinergic agonists usually activate phospholipase C to produce inositol 1,4,5 trisphosphate and diacylglycerol. Inositol 1,4,5 trisphosphate increases the intracellular Ca(2+)concentration ([Ca2(+)](i)) while diacylglycerol activates protein kinase C (PKC). PKC and Ca(2+), either by itself or with calmodulin, activate cellular functions. Goblet cell glycoprotein secretion, our index of mucin secretion, was measured from pieces of rat conjunctiva with an enzyme-linked lectin assay using the lectin Ulex europaeus agglutinin I (UEA-I). UEA-I selectively recognizes high molecular weight glycoproteins secreted by the goblet cells. Increasing the [Ca(+)](i)with the Ca(2+)ionophore ionomycin stimulated glycoprotein secretion from conjunctival goblet cells. Cholinergic agonist-induced secretion was completely blocked by chelation of extracellular Ca(2+)and by the Ca(2+)/calmodulin-dependent protein kinase inhibitors KN93 and W7 as well as their inactive analogs KN92 and W5. Activation of classical and novel PKC isozymes by phorbol 12-myristate 13-acetate and phorbol 12,13-dibutyrate stimulated goblet cell glycoprotein secretion. When ionomycin and PMA were added simultaneously, secretion was additive. PKC isozymes were identified by Western blotting analyses with antibodies specific to nine of the 11 PKC isozymes (PKCgamma and zeta were not tested). All nine PKC isozymes were identified in the conjunctival epithelium. The cellular location of the PKC isozymes was determined by immunofluorescence microscopy. Goblet cells contained the classical PKC isozymes PKCalpha, -betaI and -betaII, the novel PKC isozymes PKCepsilon, -theta;, and - mu, and the atypical PKC isozyme PKCzeta. We were unable to determine if PKC activation is required for cholinergic-agonist induced secretion because the PKC inhibitors chelerythrine and staurosporine alone greatly increased secretion. We conclude that Ca(2+)plays a major role in cholinergic agonist-induced conjunctival goblet cell secretion, but this agonist appears not to use Ca(2+)/calmodulin-dependent protein kinases. We also conclude that activated PKC can stimulate goblet cell secretion and that seven different PKC isoforms are present in the goblet cells.

Immunolocalization of muscarinic and VIP receptor subtypes and their role in stimulating goblet cell secretion.

PURPOSE: To determine the subtypes of cholinergic muscarinic receptors and receptors for vasoactive intestinal peptide (VIP) present in rat conjunctival goblet cells and whether cholinergic agonists and VIP stimulate goblet cell secretion. METHODS: Immunofluorescence studies were performed using antibodies against the m1, m2, and m3 muscarinic receptor subtypes and VIP receptors 1 and 2 (VIPR1 and VIPR2). The lectin Ulex europeus agglutinin I was used to measure glycoconjugate secretion, the index of secretion, from goblet cells in an enzyme-linked lectin assay. In this assay, pieces of conjunctiva were placed on filter paper and incubated for 15 to 120 minutes, with or without increasing concentrations of the cholinergic agonist carbachol or VIP. The muscarinic antagonist atropine and the muscarinic receptor-subtype-selective antagonists pirenzepine (M1), gallamine (M2), and 4-4-diphenylacetoxy-N-(2-chloroethyl)-piperidine hydrochloride (4-DAMP mustard; M3) were incubated with carbachol to determine specificity of receptor activation. RESULTS: Immunoreactivity to M2 and M3 receptors was found on goblet cell membranes subjacent to the secretory granules. Immunoreactivity to M1 receptor was not on goblet cells but was on the stratitfied squamous cells. Immunoreactivity to VIPR2 was found on goblet cells with a localization similar to that of the M2 and M3 receptors. VIPR1 was not found on goblet cells or on the stratified squamous cells. Carbachol and VIP induced a time- and concentration-dependent stimulation of glycoconjugate secretion. Carbachol, at 10(-4) M, induced a threefold increase in glycoconjugate secretion, which was completely inhibited by atropine (10(-5) M). Carbachol-induced secretion was inhibited 54% +/- 8% by pirenzepine (10(-5) M), 69% +/- 14% by gallamine (10(-5) M), and 72% +/- 11% by 4-DAMP mustard (10(-5) M). A twofold increase in glycoconjugate secretion was obtained with VIP at 10(-8) M. CONCLUSIONS: Cholinergic agonists, through M2 and/or M3 muscarinic receptors, and VIP, through VIPR2, regulate conjunctival goblet cell secretion, suggesting that goblet cell secretion in vivo is under the control of parasympathetic nerves.

Ca2+ signaling by cholinergic and alpha1-adrenergic agonists is up-regulated in lacrimal and submandibular glands in a murine model of Sjögren's syndrome.

Innervation of the lacrimal gland of MRL/Mp-Fas-lpr/lpr (MRL/lpr), a murine model for Sjögren's syndrome, is unaltered with the onset or progression of the lymphocytic infiltration. To determine whether lacrimal and submandibular gland cells are able to respond to external stimuli, acini were prepared from MRL/lpr (diseased) and MRL/Mp-+/+ (MRL/+, control) mice at 4, 8, and 12 weeks of age and loaded with the fluorescent dye fura-2 to monitor changes in the intracellular Ca2+ concentration ([Ca2+]i) in response to cholinergic and alpha1-adrenergic stimulation, two major stimuli of lacrimal gland protein secretion. Cholinergic-induced [Ca2+]i increase was up-regulated 3- and 4-fold in lacrimal gland acini isolated from 8- and 12-week-old MRL/lpr mice, respectively, compared to 4-week-old animals, but was not up-regulated in age-matched MRL/+ control mice. Similarly, alpha1-adrenergic-induced [Ca2+]i increase was up-regulated 7- and 12-fold in acini isolated from 8- and 12-week-old MRL/lpr mice, respectively, compared to 4-week-old animals, but was not up-regulated in MRL/+ mice. Cholinergic-induced [Ca2+]i increase in submandibular gland acini of MRL/lpr and MRL/+ mice was the same at all ages. In contrast, alpha1-adrenergic-induced [Ca2+]i increase was up-regulated 3-fold in acini from 12-week-old MRL/lpr mice, compared to 4-week-old mice, but was not up-regulated in age-matched MRL/+ mice. We conclude that the Ca2+ signaling portion of cholinergic and alpha1-adrenergic pathway in the lacrimal gland and the Ca2+ signaling portion of alpha1-adrenergic pathway in the submandibular gland is up-regulated with the onset and progression of the lymphocytic infiltration in the MRL/lpr murine model of Sjögren's syndrome.

X-ray diffraction and transmission electron microscopy of Morquio syndrome type A cornea: a structural analysis.

PURPOSE: This case report describes the structural characterization of the corneal stroma from a patient with Morquio syndrome type A. METHODS: A left penetrating keratoplasty was performed, and the cornea was examined using transmission electron microscopy and synchrotron x-ray diffraction. The interfibrillar proteoglycans were visualized in the electron microscope by using cuprolinic blue. RESULTS: Stromal collagen fibrils showed a bimodal distribution of diameters: 70% had a distribution comparable to that in normal tissue (20-30 nm) and 30% contained larger fibrils (30-42 nm) as seen by electron microscopy. Both electron microscopy and x-ray diffraction showed that the bulk numeric density of fibrils per unit area in cross-section (number density) was higher than normal in the Morquio syndrome cornea. The arrangement of proteoglycans throughout most of the Morquio syndrome cornea appeared normal, but many of the filaments were twice their normal length. In the anterior stroma, very large proteoglycan filaments (< or = 400 nm long) were found. Other ultrastructural differences also were noted, including abnormal keratocytes and long spacing collagen. CONCLUSION: The variation in fibril diameter and number density were modeled to account for only a 5% decrease in light scattering compared with the normal cornea. The extensive corneal clouding seen in the Morquio syndrome cornea cannot therefore be attributed to the variation in fibril diameters; collagen-free areas and expanded cells seem to be the most likely cause.

Beta-ig. Molecular cloning and in situ hybridization in corneal tissues.

PURPOSE: To identify a protein that copurifies with type VI collagen from rabbit cornea and to determine its cell source in rabbit corneal tissues by in situ hybridization. METHODS: Type VI collagen was extracted from cornea with urea and purified by ammonium sulfate precipitation and gel chromatography. The purity of the collagen was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). On reduction with mercaptoethanol or dithiothreitol, the alpha chains of type VI collagen ran into the gel. In addition to the type VI collagen polypeptides, an extra 68-kDa protein band appeared, suggesting that this protein is present as a large molecular weight component before reduction. Amino acid sequencing indicated that protein was related to beta ig-h3 from humans. Western blot analysis was used to determine immunologic similarity to this human protein. A rabbit stromal cell cDNA library was screened with human beta ig-h3 cDNA probe. Positive clones were sequenced and analyzed for sequence homology. Oligonucleotide probes prepared from rabbit cDNA sequences were used for Northern blot analysis and in situ hybridization of corneal tissues. RESULTS: Electroblotting of the SDS-PAGE and amino acid sequence analysis of the first 10 N-terminal amino acids of the 68-kDa band gave 100% homology with a known protein produced by human adenocarcinoma cells, beta ig-h3. This 68-kDa protein was identical immunologically to beta ig-h3 by Western blot analysis. Sequence analysis of a rabbit cDNA clone contained the whole coding region and had high identity with both human beta ig-h3 and mouse beta ig-m3. The deduced amino acid sequence had 92% identity with these species. An oligonucleotide probe from the rabbit cDNA sequence detected a single band of mRNA from cultures of stromal cells consistent in size with human beta ig-h3 mRNA. The authors refer to the rabbit form of beta ig-h3 as beta ig because the protein was obtained from normal rabbit cornea and the mRNA comes from primary cultures of rabbit stromal cells and not from a cloned cell line. In situ hybridization of rabbit corneal tissue indicated that the beta ig mRNA is located primarily in the epithelium of normal adult cornea, in fetal stromal cells, and both endothelium- and stroma-derived cells in healing corneal wounds. Normal adult endothelium and stroma did not show beta ig mRNA label. CONCLUSIONS: The highly conserved amino acid sequence homology between the human, mouse, and rabbit proteins and the temporal expression of beta ig message during corneal healing and development suggest this protein plays an important role in the morphogenesis of corneal tissues.

Structure of corneal scar tissue: an X-ray diffraction study.

Full-thickness corneal wounds (2 mm diameter) were produced in rabbits at the Schepens Eye Research Institute, Boston. These wounds were allowed to heal for periods ranging from 3 weeks to 21 months. The scar tissue was examined using low- and wide-angle x-ray diffraction from which average values were calculated for 1) the center-to-center collagen fibril spacing, 2) the fibril diameter, 3) the collagen axial periodicity D, and 4) the intermolecular spacing within the collagen fibrils. Selected samples were processed for transmission electron microscopy. The results showed that the average spacing between collagen fibrils within the healing tissue remained slightly elevated after 21 months and there was a small increase in the fibril diameter. The collagen D-periodicity was unchanged. There was a significant drop in the intermolecular spacing in the scar tissues up to 6 weeks, but thereafter the spacing returned to normal. The first-order equatorial reflection in the low-angle pattern was visible after 3 weeks and became sharper and more intense with time, suggesting that, as healing progressed, the number of nearest neighbor fibrils increased and the distribution of nearest neighbor spacings reduced. This corresponded to the fibrils becoming more ordered although, even after 21 months, normal packing was not achieved. Ultrastructural changes in collagen fibril density measured from electron micrographs were consistent with the increased order of fibril packing measured by x-ray diffraction. The results suggest that collagen molecules have a normal axial and lateral arrangement within the fibrils of scar tissue. The gradual reduction in the spread of interfibrillar spacings may be related to the progressive decrease in the light scattered from the tissue as the wound heals.

Proteoglycan and collagen morphology in superficially scarred rabbit cornea.

We have examined the changes in collagen and proteoglycan morphology in superficial lamellar keratectomy wounds produced in rabbit corneas. The ultrastructural location within the tissue of keratan sulphate and chondroitin sulphate proteoglycans was demonstrated using the cationic dye Cuprolinic Blue under critical electrolyte conditions. Large proteoglycan filaments (up to 500 nm long) appeared in the early stages of wound healing; these were most common after two weeks' wound healing, after which they decreased both in number and size. At these early stages of scar formation, spaces containing proteoglycans were present amongst bundles of collagen fibrils. As proteoglycans play an important role in controlling corneal hydration, the presence of the large proteoglycan-filled spaces would result in an abnormally high water content which is found in early scar tissue.

A morphological study of rabbit corneas after laser keratectomy.

We have examined the morphology of the collagen and proteoglycans in rabbit corneas that have undergone excimer laser photorefractive keratectomy using a clinical, 193 nm excimer laser. The photoablation was carried out to a stromal depth of 100 microns and a diameter of 6 mm. All ablated corneas developed a haze that was most intense between week 4 and week 8 and which showed no improvement after week 16. The corneas were stained with the cationic dye cuprolinic blue to visualise proteoglycans and were then processed for transmission electron microscopy. The ultrastructural location of proteoglycans (keratan sulphate and dermatan sulphate) was observed in the corneal wounds at different time intervals. Corneas that had undergone steroid treatment post-operatively were also examined. In the healing tissue proteoglycan filaments of abnormal size were observed, which became most prominent after 2 weeks. As healing progressed these abnormal filaments decreased but after 45 weeks some were still present, indicating that the proteoglycan content had not returned to normal.