Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.24.3 (collagenase)
 18,340 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Release of peroxidase from secretory cells of rat lacrimal gland upon cholinergic stimulation was studied in vitro with single lobules and isolated cells (lacrimocytes). Isolated lobules, kept in Eagle's medium, remain structurally intact and reaction product of peroxidase is confined to cisternae of rough endoplasmic reticulum, elements of the Golgi apparatus, and all secretory granules. Morphologically, exocytosis occurs by membrane fusion and discharge of granule content. The highest rate of peroxidase released from lobules is observed at 10(-4) M carbamylcholine. The specific activity of peroxidase released into the medium is fourfold higher as compared to the lobules. Release of peroxidase is suppressed by atropine when added before or after the addition of carbamylcholine. At 4 degrees C, no peroxidase release occurs upon cholinergic stimulation. The exocytotic release of peroxidase is dependent on energy supply, as indicated by substantial inhibition (at 37 degrees C) under anoxic conditions or in the presence of dinitrophenol, KCN, or carboxyatractyloside. Furthermore, the process is sensitive to colchicine and vinblastine. Isolated lacrimocytes, consiting of 95% secretory acinar cells, are prepared by digestion with collagenase, hyaluronidase, and trypsin. They retain the characteristic polarity of secretory cells in situ, and localization of peroxidase is the same as in lobules. Since isolated lacrimocytes respond to cholinergic stimulation in the same way as lobules, the receptors are not damaged by the isolation procedure and appear to be associated directly with the exocrine cell. Oxygen uptake by isolated lacrimocytes is about 14 nmol O2 X min-1 X 10(-6) cells; it is about doubled by uncoupling with dinitrophenol. Oxygen uptake rises by 20-30% above the resting rate upon cholinergic stimulation. This additional uptake is suppressed by atropine or by added cholinesterase, indicating that continuous receptor occupancy may be required for the energy demand by exocytosis. On the basis of the specific activity of peroxidase in the medium, the energy demand resulting from cholinergic stimulation is estimated to be 0.08 mumol ATP (or energy-rich phosphate bonds) per microgram of protein released from the lacrimocytes.
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PMID:Exocytosis in secretory cells of rat lacrimal gland. Peroxidase release from lobules and isolated cells upon cholinergic stimulation. 95 71

The rabbit has been a useful model for in vivo studies of the pharmacologic control of lacrimal gland fluid secretion. However, by contrast with rodent exorbital lacrimal glands, the rabbit lacrimal gland has not been subjected to detailed cellular, subcellular, or biochemical analyses. Procedures were developed to isolate rabbit lacrimal acini by collagenase digestion and mechanical dispersion. The preparations exhibited good morphology, and trypan blue exclusion rates generally exceeded 90%. The isolated acini responded to carbachol by releasing protein and increasing Na+ unidirectional influx rates. The presence of muscarinic cholinergic and beta-adrenergic receptors was indicated by specific binding of the muscarinic cholinergic antagonist, 3H-N-methylscopolamine (3H-NMS; dissociation constant, Kd, 0.55 nmol/l), and the beta-adrenergic antagonist, 3H-CGP12177 (Kd, 0.34 nmol/l). The maximal binding values measured in crude membrane preparations were 79 fmol/mg for 3H-NMS and 40 fmol/mg for 3H-CGP12177. Subcellular fractionation analyses showed various membrane populations, including a series of Golgi-derived populations admixed with a major endoplasmic reticulum-derived population, a population that may represent the basal-lateral plasma membranes, and a series of populations with characteristics suggesting they are involved in the assembly or recycling of basal-lateral membrane constituents. The authors believe the ability to isolate and analyze acinar preparations from the rabbit lacrimal gland will facilitate various studies of acinar cell biochemistry and physiology that would be impractical with the relatively smaller amounts of material that can be obtained from rat or mouse exorbital lacrimal glands.
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PMID:Isolation and subcellular fractionation analysis of acini from rabbit lacrimal glands. 132 95

Proteolytic enzymes, particularly collagenase, are involved in development of eye cornea chronic ulcers. Analysis of lacrimal fluid obtained from the patients enabled to find not only the collagenase activity but and serine enzymes exhibiting BAEE esterase activity. Plasmin, blood plasma and tissue kallikreins regulated permeability of capillaries in conjunctiva and lacrimal gland as well as they activated latent collagenase. Studies of BAEE esterase activity in lacrimal fluid of the patients are required for prescription of adequate pathogenetic treatment.
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PMID:[Proteolytic enzymes of the lacrimal fluid as pathogenesis factors of chronic corneal ulcer]. 169 16

A recent hypothesis for the cellular mechanism of fluid secretion by lacrimal acini has been based, in part, on the results of subcellular fractionation analyses of lacrimal gland fragments which had been incubated for a brief period in vitro. An important assumption in those studies was that the ion transporters and neurotransmitter receptors measured in isolated subcellular fractions were associated with membranes derived from the acinar cells, since these comprise the bulk of the lacrimal gland mass. This study was undertaken to validate this assumption. Acinar complexes were isolated from rat exorbital lacrimal glands by digestion with collagenase, hyaluronidase, and DNase. Although terminal intralobular duct segments and myoepithelial cells were occasionally noted, the preparations appeared to be free of larger ducts, blood cells, blood vessels, and interstitial cells. Acinar cells were then disrupted, and the homogenates underwent the fractionation procedure used previously for lacrimal gland fragment preparations. This procedure involved a sequence of analyses by differential sedimentation, isopycnic centrifugation on sorbitol gradients, and partitioning in dextran-polyethyleneglycol two-phase systems. Calculated initial specific activities for sodium/potassium adenosinetriphosphatase (Na+/K(+)-ATPase), alkaline phosphatase, acid phosphatase, and succinate dehydrogenase were identical to those obtained from fragment preparations. Major membrane populations resolved by the sequential analyses, including one believed to represent endoplasmic reticulum membranes, two believed to be derived from the acinar cell basal-lateral membrane, and two believed to be derived from the Golgi complex, corresponded closely to populations resolved from lacrimal fragment preparations. In addition to validating the previous use of lacrimal gland fragment preparations in studies of acinar cell function, these results suggest that preparations of isolated lacrimal acini will be useful for future work on neurotransmitter-receptor regulation and basal-lateral plasma membrane dynamics in the lacrimal gland.
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PMID:Analytic subcellular fractionation of acini from rat lacrimal gland. 217 90

Characterization of muscarinic acetylcholine receptors in acinar cells from rat pancreas and lacrimal and parotid glands was achieved by binding of the reversible muscarinic antagonist [3H]quinuclidinyl benzilate (QNB) and the specific alkylating reagent [3H]propylbenzilylcholine mustard (PrBCM) to intact acini or dispersed acinar cells. Binding studies with [3H]QNB showed that acinar cells from pancreas contain 26,400, from parotid 21,400, and from lacrimal gland 25,700 binding sites/cell. To assess molecular size of the receptor in each gland, acini were prepared by digestion with purified collagenase and singly dispersed acinar cells were prepared by a combination of digestion with crude collagenase, hyaluronidase, and alpha-chymotrypsin and divalent cation chelation using EDTA. Muscarinic receptors on acini or dispersed cells were covalently labeled with 5 nM [3H]PrBCM, solubilized directly in hot sodium dodecyl sulfate buffer, and resolved by polyacrylamide gel electrophoresis. When solubilized acini were electrophoresed, a major labeled peak was observed on gels along with a smaller peak of lower apparent molecular weight. For pancreatic acini, the apparent molecular weights of these peaks were 117,600 and 85,700; for parotid acini, 104,800 and 74,500; and for lacrimal acini, 87,200 and 63,100. Addition of muscarinic antagonists to the labeling medium abolished both peaks. When dispersed acinar cells were labeled, the larger peak was eliminated, and all radioactivity was concentrated in a single peak: 87,600 for pancreas, 78,000 for parotid gland, and 62,800 for lacrimal gland. Digestion of prelabeled acini with the mixture of enzymes used to produce dispersed acinar cells similarly shifted all radioactivity into this second peak. Limited digestion of acini or dispersed cells with 1 mg/ml of papain resulted in the disappearance of these higher molecular weight peaks and the appearance of a broad peak at Mr = 40,000. Cells of nonepithelial origin, IM-9 lymphocytes and NG108 neuroblastoma X glioma hybrids, also were labeled with [3H]PrBCM and electrophoresed.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Muscarinic acetylcholine receptor structure in acinar cells of mammalian exocrine glands. 398 Apr 74

To characterize the role of Ca2+ in cholinergic stimulation of lacrimal gland protein secretion, the effects of inhibitors of cellular Ca2+ handling on protein secretion were investigated. Protein secretion was measured from rat exorbital glands using either pieces of gland in perifusion or acini isolated by collagenase digestion. Peroxidase was used as a measure of protein secretion. An inhibitor of Ca2+ influx via voltage sensitive Ca2+ channels (verapamil) at 10(-5) and 5 X 10(-5) M did not alter protein secretion stimulated by the cholinergic agonist carbachol at 10(-5) M. Inhibition of Ca2+ efflux via Na+/Ca2+ exchange by removal of extracellular Na+ or by inhibition of Na+-K+-ATPase activity using ouabain (10(-3) M) or extracellular K+ removal did not stimulate protein secretion. In contrast, inhibition of Ca2+ release from intracellular stores with TMB-8 at 100 micron completely blocked protein secretion stimulated by carbachol at 10(-5) M. Similarly, the Ca2+/calmodulin (CaM) antagonists W-13 and W-12 decreased carbachol-induced protein secretion with potencies similar to those which inhibit Ca2+/CaM dependent processes. We conclude that cholinergic agonists stimulate lacrimal gland protein secretion primarily by mobilizing Ca2+ from intracellular stores and that one mechanism by which this Ca2+ could activate secretion is in conjunction with calmodulin.
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PMID:Role of calcium in cholinergic stimulation of lacrimal gland protein secretion. 401 36

The effect of vasoactive intestinal polypeptide (VIP) on protein secretion from lacrimal gland was investigated by using acini prepared by collagenase digestion of rat exorbital lacrimal glands. Protein secretion was determined by incubating the acini for 0-40 min and analyzing the supernatant for peroxidase, a protein secreted by the rat exorbital lacrimal gland. VIP (10(-10) to 10(-7) M) stimulated secretion in a concentration-dependent manner. A maximum concentration of VIP (10(-8) M) stimulated secretion to the same extent as a maximum concentration of carbachol (10(-5) M). The cholinergic antagonist atropine at a concentration (10(-5) M) that completely abolished carbachol-induced secretion did not alter VIP-stimulated secretion. The secretory effects of maximal concentrations of VIP and carbachol were additive, but decreasing the carbachol concentration potentiated secretion. Unlike carbachol, which had no effect on the acinar cAMP level, VIP increased cAMP content sixfold. Immunohistochemical staining demonstrated VIP-like immunoreactivity in nerve fibers throughout the gland, distributed primarily around acini. We conclude that VIP-like immunoreactive nerves are present in the lacrimal gland and that VIP can stimulate protein secretion but utilizes a pathway separate from, but convergent with, that used by cholinergic agonists.
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PMID:Vasoactive intestinal polypeptide stimulation of protein secretion from rat lacrimal gland acini. 609 81

To study the configuration of myoepithelial cells, we isolated glandular endpieces of various guinea pig glands by collagenase, and visualized the myoepithelial cells by immunohistochemistry for actin, or by Bodipy-phallacidin, under both a light microscope and laser scanning confocal microscopes. In parotid and mandibular glands, the glandular acini were small (about 20-30 microns diameter) and spherical, and each acinus had one or two myoepithelial cells attached that were stellate in shape (central cell body and four to six thin processes). Most of the basal surface of the glandular cells was not covered by myoepithelial cells, and processes often extended to the neighboring acinus. The tubular glandular endpieces of the major sublingual gland, which secretes a mucous substance, were almost fully encircled by band-like myoepithelial cells (about 3-6 microns wide). Although there were many differences between the lacrimal gland and the Harderian gland (e.g., the secretory product of the lacrimal gland was mucous, and glandular lumina were narrow; the Harderian gland secreted lipids and showed wide lumina), the outer contours of both glandular endpieces were the same (about 50-100 microns diameter, ellipsoid or spherical in shape). In both glands, 5-20 stellate myoepithelial cells were attached onto a glandular endpiece, and their arrangement had a lacy appearance. Actin filaments in myoepithelial cells aggregated and formed bundles in the broad processes and cell bodies. The bundles ran across the cell body, but there was no point where the bundles converged. In the arborization, some distal processes reversed their direction. We conclude that the configuration of myoepithelial cells depends on the outer contour of the glandular endpieces rather than on the secretory material or luminal width. The variety of myoepithelial cell configurations in the different exocrine glands we examined suggests that it is quite difficult to assign to myoepithelial cells the general role of expelling secretory products from glandular lumina. These cells seem to maintain the contour of the glandular endpieces, serving as the exoskeleton of the endpieces.
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PMID:Configuration of myoepithelial cells in various exocrine glands of guinea pigs. 804 65

1. We have investigated interactions between intracellular pH (pHi) and the intracellular free calcium concentration ([Ca2+]i) in collagenase-isolated rat lacrimal acinar cells. The fluorescent dyes fura-2 and 2',7'-bis(carboxyethyl)-5-carboxyfluorescein (BCECF) were used to measure [Ca2+]i and pHi, respectively. 2. Application of the weak base NH4Cl alkalinized the cytosol and caused a dose-dependent increase in [Ca2+]i. Trimethylamine (TMA) also alkalinized the cytosol and increased [Ca2+]i. The increase in [Ca2+]i evoked by NH4Cl or TMA was much smaller than that evoked by the secretory agonist acetylcholine (ACh). 3. Application of NH4Cl also increased [Ca2+]i in cells bathed in Ca(2+)-free medium, indicating that NH4Cl released Ca2+ from an intracellular pool. 4. Ammonium chloride had no effect on [Ca2+]i in cells bathed in Ca(2+)-free medium if agonist-sensitive intracellular Ca2+ pools had been depleted with either ACh or the microsomal Ca(2+)-ATPase inhibitor 2,5-di(tert-butyl)hydroquinone. Treatment of cells with NH4Cl in Ca(2+)-free medium reduced the amount of Ca2+ released by ACh. These results suggest that NH4Cl released Ca2+ from the same intracellular pool released by ACh. 5. Calcium release from the agonist-sensitive pool was also triggered when the cytosol was alkalinized by removing the weak acid acetate. 6. Ammonium chloride caused a modest increase in inositol phosphate production, suggesting that NH4Cl may have released stored Ca2+ via an increase in the intracellular inositol 1,4,5-trisphosphate concentration. 7. The increase in [Ca2+]i evoked by NH4Cl was not sustained even in the presence of extracellular Ca2+. In contrast, when a low dose of ACh was used to evoke intracellular Ca2+ release of similar magnitude, sustained Ca2+ entry was observed. 8. Alkalinizing the cytosol appeared to partially inhibit Ca2+ entry triggered by thapsigargin or by ACh. 9. We suggest that alkalinizing the cytoplasm in unstimulated lacrimal acinar cells can release Ca2+ from the intracellular agonist-sensitive Ca2+ pool. However, releasing stored Ca2+ via alkalinization does not appear to trigger significant Ca2+ entry, perhaps because intracellular alkalinization inhibits either the Ca2+ entry pathway or the mechanism which couples the entry pathway to store depletion.
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PMID:Intracellular alkalinization mobilizes calcium from agonist-sensitive pools in rat lacrimal acinar cells. 913 Jan 57

The purpose of this study was to determine the role of p42/p44 mitogen-activated protein kinase (MAPK) in alpha(1)-adrenergically and cholinergically stimulated protein secretion in rat lacrimal gland acinar cells and the pathways used by these agonists to activate MAPK. Acini were isolated by collagenase digestion and incubated with the alpha(1)-adrenergic agonist phenylephrine or the cholinergic agonist carbachol, and activation of MAPK and protein secretion were then measured. Phenylephrine and carbachol activated MAPK in a time- and concentration-dependent manner. Inhibition of MAPK significantly increased phenylephrine- and carbachol-induced protein secretion. Inhibition of EGF receptor (EGFR) with AG1478, an inhibitor of the EGFR tyrosine kinase activity, significantly increased phenylephrine- but not carbachol-induced protein secretion. Whereas phenylephrine-induced activation of MAPK was completely inhibited by AG1478, activation of MAPK by carbachol was not. Phenylephrine stimulated tyrosine phosphorylation of the EGFR, whereas carbachol stimulated p60(Src), and possibly Pyk2, to activate MAPK. We conclude that, in the lacrimal gland, activation of MAPK plays an inhibitory role in alpha(1)-adrenergically and cholinergically stimulated protein secretion and that these agonists use different signaling mechanisms to activate MAPK.
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PMID:Alpha 1-adrenergic and cholinergic agonists activate MAPK by separate mechanisms to inhibit secretion in lacrimal gland. 1238 18


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