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1.
Exp Eye Res ; 80(3): 379-89, 2005 Mar.
Article in English | MEDLINE | ID: mdl-15721620

ABSTRACT

The purpose of this study was to investigate the expression of the EGF family of growth factors and EGF receptor subtypes (ErbB1-4) present in lacrimal gland and determine the effects of these growth factors on different functions of rat lacrimal gland. RT-PCR was used to detect mRNA expression in the lacrimal gland of selected members of the EGF family of growth factors, namely EGF, transforming growth factor alpha (TGF-alpha), heparin-binding EGF (HB-EGF), and heregulin. The presence of ErbB receptors was investigated by immunofluorescence microscopy and western blot analysis. The effects of EGF, TGF-alpha, HB-EGF, and heregulin on protein secretion from lacrimal gland acini were examined using a fluorescent assay for peroxidase, a marker of protein secretion. Fura-2 tetra-acetoxymethyl ester was used to measure the effects of the growth factors on intracellular [Ca2+] ([Ca2+]i) in acini. MAPK activation in acini by these growth factors was also examined by western blot analysis using antibodies specific to phosphorylated p42/44 MAPK and total p42 MAPK. Rat lacrimal gland expressed EGF, TGF-alpha, HB-EGF, and heregulin mRNA, and all four ErbB receptors were present in the lacrimal gland as detected by western blot analyses. ErbB 1 and ErbB2 were located in basal and lateral membranes of acinar and ductal cells. The location of ErbB3 could not be determined while ErbB4 was found in ductal cells. Heregulin (10(-7) m) significantly increased protein secretion in lacrimal gland acini whereas all growth factors tested significantly increased [Ca2+]i at 10(-7) m. TGF-alpha (10(-9) m), heregulin (10(-7) m), EGF (10(-7) m), and HB-EGF (10(-7) m) significantly increased the amount of phosphorylated MAPK in lacrimal gland acini. We conclude that all members of the EGF family of growth factors studied are synthesised in rat lacrimal gland, could activate all four ErbB receptors that are present in this tissue, and differentially activate lacrimal gland functions.


Subject(s)
Calcium/analysis , Epidermal Growth Factor/analysis , Eye Proteins/metabolism , Lacrimal Apparatus/metabolism , Mitogen-Activated Protein Kinases/metabolism , Animals , ErbB Receptors/analysis , ErbB Receptors/metabolism , Immunohistochemistry/methods , Lacrimal Apparatus/enzymology , Male , Peroxidase/metabolism , RNA, Messenger/analysis , Rats , Rats, Sprague-Dawley , Receptor Protein-Tyrosine Kinases/analysis , Receptor Protein-Tyrosine Kinases/metabolism , Reverse Transcriptase Polymerase Chain Reaction/methods
2.
Auton Autacoid Pharmacol ; 23(5-6): 307-17, 2003.
Article in English | MEDLINE | ID: mdl-15255815

ABSTRACT

1. In the present investigation we examined the regulation of calmodulin (CaM)- and protein kinase C (PKC)-dependent pathways by cytosolic Ca(2+) in the contraction of cat lower oesophageal sphincter (LES). 2. Force developed in response to increasing doses of acetylcholine (ACh) was directly related to the increase of the [Ca(2+)](i) measured by fura-2. Thapsigargin, which depletes Ca(2+) stores, reduced the contraction and the [Ca(2+)](i). In addition, contraction in response to maximal ACh was reduced by the CaM inhibitor CGS9343B but not by the PKC inhibitor chelerythrine. The contraction in response to submaximal ACh was reduced by chelerythrine but not by CGS9343B. 3. In permeabilized cells, the contraction in response to low Ca(2+) (0.54 microm) was also reduced by CGS9343B. 4. The response to high Ca(2+) (1.0 microm) was reduced by CGS9343B. ACh also inhibited PKC activation induced by diacylglycerol, which activation is inhibited by the N-myristoylated peptide inhibitor derived from pseudosubstrate sequences of PKCalphabetagamma (myr-PKC-alphabetagamma), but not of myr-PKC-alpha. 5. These data are consistent with the view that activated CaM-dependent pathways inhibit PKC-dependent pathways, this switch mechanism might be regulated by Ca(2+) in the LES.


Subject(s)
Calcium/physiology , Calmodulin/physiology , Esophagogastric Junction/physiology , Protein Kinase C/physiology , 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine/pharmacology , Acetylcholine/antagonists & inhibitors , Acetylcholine/pharmacology , Alkaloids , Animals , Benzimidazoles/pharmacology , Benzophenanthridines , Calcium/antagonists & inhibitors , Calcium/chemistry , Calmodulin/chemistry , Cats , Cell Membrane Permeability/drug effects , Cell Membrane Permeability/physiology , Diglycerides/pharmacology , Esophagogastric Junction/cytology , Esophagogastric Junction/drug effects , Inositol 1,4,5-Trisphosphate/pharmacology , Male , Muscle Contraction/drug effects , Muscle Contraction/physiology , Muscle Relaxation/drug effects , Muscle Relaxation/physiology , Myocytes, Smooth Muscle/cytology , Myocytes, Smooth Muscle/drug effects , Phenanthridines/pharmacology , Protein Kinase C/antagonists & inhibitors , Protein Kinase C/chemistry , Signal Transduction/physiology , Thapsigargin/antagonists & inhibitors , Thapsigargin/pharmacology
3.
Mol Vis ; 7: 240-6, 2001 Oct 24.
Article in English | MEDLINE | ID: mdl-11702063

ABSTRACT

PURPOSE: To identify the role of PKC-gamma in control of and phosphorylation of connexin 46 (Cx46) in the lens cortex. METHODS: The association between PKC-gamma and Cx46 was determined by co-immunoprecipitation from whole lens. Phosphorylation of Cx46 and activity of PKC-gamma were determined using Western blots, PKC activity assays, and inhibition of PKC activity by addition of isoform-specific PKC pseudosubstrate inhibitors. RESULTS: Co-localization of PKC-gamma and Cx46 was observed in the bow regions and cortical regions of rat lens. PKC-gamma was not observed in the nuclear region and Cx46 was not observed in the epithelial layer. PKC-alpha was not found in lens cortex or nuclear regions. PKC-gamma could be co-immunoprecipitated with Cx46 from lens cortical regions. Cx46 was phosphorylated on both serine and threonine. No tyrosine phosphorylation was observed. The PKC-gamma specific pseudosubstrate inhibitor caused a 73% inhibition of serine phosphorylation on Cx46 at 1 microM, and, 36% inhibition of threonine phosphorylation at the same concentration. Inhibition of phosphorylation of Cx46 with PKC-alpha pseudosubstrate inhibitor was not observed. CONCLUSIONS: PKC-gamma may phosphorylate Cx46, primarily on serine in whole lens. A role for PKC-gamma in control of lens cortical gap junctions is suggested.


Subject(s)
Connexins/metabolism , Isoenzymes/physiology , Lens Cortex, Crystalline/metabolism , Protein Kinase C/physiology , Animals , Blotting, Western , Enzyme Inhibitors/pharmacology , Isoenzymes/antagonists & inhibitors , Lens Cortex, Crystalline/drug effects , Microscopy, Confocal , Peptides/pharmacology , Phosphorylation , Protein Kinase C/antagonists & inhibitors , Rats , Serine/metabolism , Threonine/metabolism
4.
Am J Physiol Cell Physiol ; 281(2): C603-14, 2001 Aug.
Article in English | MEDLINE | ID: mdl-11443060

ABSTRACT

We previously reported an increased secretion of amyloid precursor-like protein 2 (APLP2) in the healing corneal epithelium. The present study sought to investigate signal transduction pathways involved in APLP2 shedding in vitro. APLP2 was constitutively shed and released into culture medium in SV40-immortalized human corneal epithelial cells as assessed by Western blotting, flow cytometry, and indirect immunofluorescence. Activation of protein kinase C (PKC) by phorbol 12-myristate 13-acetate (PMA) caused significant increases in APLP2 shedding. This was inhibited by staurosporine and a PKC-epsilon-specific, N-myristoylated peptide inhibitor. Epidermal growth factor (EGF) also induced APLP2 accumulation in culture medium. Basal APLP2 shedding as well as that induced by PMA and EGF was blocked by a mitogen-activated protein kinase (MAPK) kinase inhibitor, U-0126. Our results suggest that MAPK activity accounts for basal as well as PKC- and EGF-induced APLP2 shedding. In addition, PKC-epsilon may be involved in the induction of APLP2 shedding in corneal epithelial cells.


Subject(s)
Amyloid beta-Protein Precursor/metabolism , Epithelium, Corneal/metabolism , Mitogen-Activated Protein Kinases/physiology , Nerve Tissue Proteins/metabolism , Cell Line, Transformed , Epidermal Growth Factor/pharmacology , Epithelium, Corneal/cytology , Epithelium, Corneal/drug effects , Humans , Mitogen-Activated Protein Kinase Kinases/physiology , Phosphorylation , Protein Kinase C/physiology , Tetradecanoylphorbol Acetate/pharmacology
5.
Invest Ophthalmol Vis Sci ; 42(5): 925-32, 2001 Apr.
Article in English | MEDLINE | ID: mdl-11274068

ABSTRACT

PURPOSE: To determine whether lacrimal and salivary gland nerves of an animal model of Sjögren's syndrome, the MRL/lpr mouse, are able to release acetylcholine. The second purpose was to determine whether activation of the lacrimal gland nerves of the MRL/lpr mouse leads to protein secretion. METHODS: Total saliva was collected for 10 minutes from the oral cavity of male and female MRL/lpr and MRL/+ mice, after intraperitoneal stimulation with pilocarpine and isoproterenol. Lacrimal and salivary gland lobules prepared from 18-week-old MRL/lpr and MRL/+ mice were incubated in the presence of depolarizing KCl (75 mM) solution. Acetylcholine release and peroxidase secretion (a protein secreted by the lacrimal gland) were measured using a spectrofluorometric assay. RESULTS: Female, but not male, MRL/lpr mouse salivary glands were hyper-responsive to in vivo injection of secretagogues. These mice produced significantly higher amounts of saliva than did age-matched MRL/+ mice. Lacrimal and salivary gland nerves from 18-week-old MRL/+ mice released acetylcholine in response to a depolarizing KCl solution. In contrast, nerves in glands from 18-week-old MRL/lpr mice did not increase acetylcholine release in response to the depolarizing solution. Moreover, lacrimal glands from 18-week-old MRL/+ mice were able to secrete peroxidase in response to a depolarizing KCl solution, whereas those from 18-week-old MRL/lpr could not. This was not due to a defect in the secretory process, because addition of an exogenous secretagogue elicited peroxidase secretion from 18-week-old MRL/lpr as well as MRL/+ mice lacrimal glands. CONCLUSIONS: The results show that activation of nerves of lacrimal and salivary glands infiltrated with lymphocytes does not increase the release of neurotransmitters, which results in impaired secretion from these glands.


Subject(s)
Acetylcholine/metabolism , Lacrimal Apparatus/innervation , Parasympathetic Nervous System/metabolism , Salivary Glands/innervation , Sjogren's Syndrome/metabolism , Sympathetic Nervous System/metabolism , Animals , Choline/metabolism , Disease Models, Animal , Female , Injections, Intraperitoneal , Isoproterenol/pharmacology , Lacrimal Apparatus/drug effects , Male , Mice , Mice, Inbred MRL lpr , Peroxidase/metabolism , Pilocarpine/pharmacology , Saliva/metabolism , Salivary Glands/drug effects , Spectrometry, Fluorescence , Tears/metabolism
6.
Exp Eye Res ; 71(6): 619-28, 2000 Dec.
Article in English | MEDLINE | ID: mdl-11095914

ABSTRACT

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.


Subject(s)
Calcium/physiology , Conjunctiva/cytology , Goblet Cells/metabolism , Protein Kinase C/physiology , Animals , Blotting, Western , Chelating Agents , Glycoproteins/metabolism , Ionomycin/pharmacology , Ionophores/pharmacology , Male , Microscopy, Fluorescence , Phorbol 12,13-Dibutyrate/pharmacology , Protein Kinase C/antagonists & inhibitors , Rats , Rats, Sprague-Dawley , Tetradecanoylphorbol Acetate/pharmacology
7.
Invest Ophthalmol Vis Sci ; 41(2): 386-92, 2000 Feb.
Article in English | MEDLINE | ID: mdl-10670466

ABSTRACT

PURPOSE: To investigate the role of protein kinase C (PKC) in cholinergic agonist-induced Ca2+ elevation in lacrimal gland acini. METHODS: Lacrimal gland acini were prepared by collagenase digestion, and changes in intracellular Ca2+ ([Ca2+]i) were measured using fura-2 as a fluorescent probe. RESULTS: Preactivation of PKC by phorbol 12-myristate 13-acetate (PMA), or inhibition of protein phosphatase type 1/2A (PP1/2A) by calyculin A, decreased both the [Ca2+]i transient and the plateau of [Ca2+]i induced by increasing concentrations of carbachol, a cholinergic agonist. Staurosporine, an inhibitor of PKC, completely reversed the effect of PMA. Inhibition of the Ca(2+)-independent PKC isoforms PKCdelta and -epsilon, but not the Ca(2+)-dependent isoform PKCalpha substantially reversed the inhibitory effect of PMA on cholinergic agonist-induced Ca2+ elevation. The inhibitory effect of PMA was obtained only in the presence of extracellular Ca2+, suggesting that PKC inhibits the influx of Ca2+. PMA completely inhibited the cholinergic agonist-induced plateau of [Ca2+]i. PMA and calyculin A decreased both the [Ca2+]i transient and the plateau of [Ca2+]i induced by thapsigargin, further supporting the idea that PKC modulates the entry of Ca2+. CONCLUSIONS: In the lacrimal gland, agonist-induced changes in [Ca2+]i are negatively regulated by PKC-dependent phosphorylation of a target protein(s) that is sensitive to PP1/2A.


Subject(s)
Calcium/metabolism , Carbachol/pharmacology , Cholinergic Agonists/pharmacology , Isoenzymes/metabolism , Lacrimal Apparatus/drug effects , Protein Kinase C/metabolism , Animals , Carbachol/antagonists & inhibitors , Enzyme Activation/drug effects , Enzyme Inhibitors/pharmacology , Fura-2/metabolism , Lacrimal Apparatus/metabolism , Male , Marine Toxins , Oxazoles/pharmacology , Phosphoprotein Phosphatases/antagonists & inhibitors , Phosphorylation , Protein Kinase C-delta , Protein Kinase C-epsilon , Rats , Rats, Wistar , Staurosporine/pharmacology , Tetradecanoylphorbol Acetate/pharmacology , Thapsigargin/pharmacology
8.
FEBS Lett ; 459(3): 285-90, 1999 Oct 15.
Article in English | MEDLINE | ID: mdl-10526151

ABSTRACT

Protein kinases C (PKC) are serine/threonine kinase enzymes involved in the mechanism of cell survival. Their pseudosubstrate sequences are autoinhibitory domains, which maintain the enzyme in an inactive state in the absence of allosteric activators, thus representing an attractive tool for the modulation of different PKC isoforms. Here, we report the use of palmitoylated modified PKC-alpha, -epsilon, and -zeta pseudosubstrate peptides, and determine their intracellular distribution together with their respective PKC isoenzymes. Finally, we propose that the differential distribution of the peptides is correlated with a selective induction of apoptosis and therefore argues for different involvement of PKC isoforms in the anti-apoptotic program.


Subject(s)
Apoptosis , Isoenzymes/metabolism , Protein Kinase C/metabolism , Biological Transport , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , HL-60 Cells , Humans , Isoenzymes/antagonists & inhibitors , Isoenzymes/biosynthesis , Jurkat Cells , Molecular Sequence Data , Palmitates/chemistry , Palmitic Acid/metabolism , Peptides/chemistry , Peptides/pharmacology , Protein Kinase C/antagonists & inhibitors , Protein Kinase C/biosynthesis , Protein Kinase C-alpha , Protein Kinase C-epsilon , Subcellular Fractions/metabolism , Substrate Specificity
9.
Invest Ophthalmol Vis Sci ; 40(6): 1102-11, 1999 May.
Article in English | MEDLINE | ID: mdl-10235543

ABSTRACT

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.


Subject(s)
Conjunctiva/metabolism , Goblet Cells/metabolism , Plant Lectins , Receptors, Muscarinic/physiology , Receptors, Vasoactive Intestinal Peptide/physiology , Animals , Binding Sites/physiology , Cholinergic Agonists/pharmacology , Conjunctiva/cytology , Fluorescent Antibody Technique , Goblet Cells/drug effects , Lectins/metabolism , Male , Rats , Rats, Sprague-Dawley , Receptors, Muscarinic/metabolism , Receptors, Vasoactive Intestinal Peptide/metabolism , Vasoactive Intestinal Peptide/pharmacology
10.
Clin Immunol Immunopathol ; 89(2): 126-33, 1998 Nov.
Article in English | MEDLINE | ID: mdl-9787114

ABSTRACT

The lacrimal glands of patients with Sjögren's syndrome develop extensive lymphocytic infiltration, but also contain a large number of seemingly healthy looking acinar and ductal cells. Despite this, the secretory function of this tissue is impaired, leading to aqueous tear-deficient dry eye. This raises the possibility that there is a defect in the neural innervation of the remaining portion of the lacrimal gland. To test for this possibility, we used antibodies specific to various markers of the parasympathetic, sympathetic, and sensory nerves and performed immunohistochemical analyses of lacrimal glands from a murine model of Sjögren's syndrome, the MRL/Mp-Fas-lpr/lpr (MRL/lpr) and the control mice MRL/Mp-+/+ (MRL/+). Our results show that the MRL/lpr, but not the MRL/+, lacrimal glands become infiltrated with lymphocytes starting at 8 weeks of age which worsens by 12 and 18 weeks. The density and the pattern of parasympathetic, sympathetic, and sensory innervation of the noninflamed acinar tissue of MRL/lpr lacrimal glands, at 4, 8, 12, and 18 weeks, is indistinguishable from that of age-matched control MRL/+ lacrimal glands. We conclude that the loss of the secretory function in Sjögren's syndrome lacrimal glands is not due to a loss or decrease of its innervation.


Subject(s)
Lacrimal Apparatus/innervation , Sjogren's Syndrome/physiopathology , Aging/physiology , Animals , Calcitonin Gene-Related Peptide/physiology , Dacryocystitis/physiopathology , Disease Models, Animal , Lacrimal Apparatus/cytology , Lacrimal Apparatus/metabolism , Lymphocytes/physiology , Mice , Mice, Inbred MRL lpr , Neurons, Afferent/physiology , Neurotransmitter Agents/physiology , Parasympathetic Nervous System/physiopathology , Sjogren's Syndrome/metabolism , Sympathetic Nervous System
11.
Clin Immunol Immunopathol ; 89(2): 134-40, 1998 Nov.
Article in English | MEDLINE | ID: mdl-9787115

ABSTRACT

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.


Subject(s)
Adrenergic alpha-Agonists/pharmacology , Calcium/physiology , Cholinergic Agonists/pharmacology , Lacrimal Apparatus/physiology , Sjogren's Syndrome/physiopathology , Submandibular Gland/physiology , Animals , Dacryocystitis/metabolism , Dacryocystitis/physiopathology , Disease Models, Animal , Lacrimal Apparatus/chemistry , Lymphocytes/pathology , Mice , Mice, Inbred MRL lpr , Signal Transduction , Submandibular Gland/chemistry , Submandibular Gland/cytology , Up-Regulation/drug effects
15.
Ann N Y Acad Sci ; 842: 217-20, 1998 Apr 15.
Article in English | MEDLINE | ID: mdl-9599315

ABSTRACT

Lacrimal gland protein secretion is primarily under the control of cholinergic muscarinic and alpha 1-adrenergic receptors. Cholinergic agonists are coupled to the activation of phospholipase C (PLC), which leads to the production of two second messenger molecules: inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 increases the cytoplasmic concentration of calcium ([Ca2+]i), and DAG activates protein kinase C (PKC), two events that are thought to trigger protein secretion. Lacrimal gland alpha 1-adrenergic receptors are not coupled to the PLC pathway, although their activation leads to a slight increase in [Ca2+]i(3). We have also shown that unlike the cholinergic receptors, alpha 1-adrenergic receptors are not linked to the activation of phospholipase D in lacrimal gland acini. Thus the transduction pathway(s) used by the alpha 1-adrenergic receptors to trigger lacrimal gland protein secretion remains to be identified. PKC was originally described as a Ca2+ and phospholipid-dependent protein kinase activated by DAG produced by the receptor-mediated breakdown of phosphoinositides. Molecular cloning and biochemical techniques have shown that PKC is a family of closely related enzymes consisting of at least eleven different isoforms that has been divided into three categories: (1) conventional PKCs, including PKC alpha, beta I, -beta II and -gamma isoforms have a Ca2+ and DAG-dependent kinase activity; (2) novel PKCs, including PKC epsilon, -delta, -theta, -nu, and -mu isoforms, are Ca(2+)-independent and DAG-stimulated kinases; (3) atypical PKCs, including PKC zeta, and -iota/lambda isoforms, are Ca2+ and DAG-independent kinases. All PKC isoforms, except PKC mu, have a pseudosubstrate sequence in their N-terminal part that is thought to interact with the catalytic domain to keep the enzyme inactive in resting cells. In previous studies, we showed that lacrimal gland acini express three isoforms of PKC: PKC alpha -delta, and -epsilon. In the present study, we report the identification of two other PKC isoforms, namely PKC mu and -iota/lambda. We show that these isoforms are differentially located and that they translocate differentially in response to phorbol esters and cholinergic agonists. We also show that PKC isoforms differentially control lacrimal gland protein secretion and cholinergic-induced Ca2+ elevation. Part of these results has been recently published.


Subject(s)
Isoenzymes/physiology , Lacrimal Apparatus/physiology , Protein Kinase C/physiology , Animals
16.
Kidney Int ; 52(2): 387-92, 1997 Aug.
Article in English | MEDLINE | ID: mdl-9263994

ABSTRACT

To identify kidney biosynthetic abnormalities that may precede the onset of hypertension, we studied the expression of fibronectin (FN) and collagen IV (Coll IV) in young SHR (4 weeks of age) whose systolic blood pressure was normal and similar to that of age-matched control WKY rats. In isolated glomeruli the level of FN protein assessed by immunoblotting tended to be lower in the SHR than in the WKY rats. By Northern analysis the FN/actin mRNA ratio was significantly lower in glomeruli from SHR (0.56 +/- 0.47) than in glomeruli from WKY rats (2.0 +/- 0.8). These abnormalities were maintained in vitro since the expression of FN was significantly lower in SHR than in WKY cultured mesangial cells (FN/actin mRNA ratio = 0.84 +/- 0.46 vs. 1.9 +/- 0.7, P = 0.029). No differences in Coll IV mRNA or protein levels were observed in SHR glomeruli and mesangial cells when compared with WKY rats. The levels of aortic FN and Coll IV mRNAs were not different in SHR and WKY rats. In addition, mesangial cells from SHR showed a significantly higher growth rate than those from WKY. The biosynthetic and proliferative abnormalities observed in the SHR mesangial cells appear to reflect genetic characteristics, and could provide novel insights into cellular mechanisms linking the genetics of hypertension with predisposition to glomerular pathology.


Subject(s)
Glomerular Mesangium/pathology , Hypertension, Renal/pathology , Rats, Inbred SHR/physiology , Actins/genetics , Age Factors , Animals , Aorta/chemistry , Blotting, Western , Cell Division/physiology , Cells, Cultured , Collagen/analysis , Collagen/genetics , Fibronectins/analysis , Fibronectins/genetics , Gene Expression/physiology , Glomerular Mesangium/chemistry , Glomerular Mesangium/cytology , RNA, Messenger/metabolism , Rats , Rats, Inbred WKY
17.
J Membr Biol ; 157(2): 169-75, 1997 May 15.
Article in English | MEDLINE | ID: mdl-9151658

ABSTRACT

In previous studies, we showed that lacrimal gland acini express three isoforms of protein kinase C (PKC): PKCalpha,-delta, and -epsilon. In the present study, we report the identification of two other PKC isoforms, namely PKCmu and -iota/lambda. Using immunofluorescence techniques, we showed that these isoforms are differentially located. PKCalpha and -mu showed the most prominent membrane localization, whereas PKCdelta, -epsilon and -iota/lambda were mainly cytosolic. Using cell fractionation and western blotting techniques, we showed that the phorbol ester, phorbol 12,13-dibutyrate (PdBu, 10(-6) M), translocated all PKC isoforms, except PKCiota/lambda, from the soluble fraction into the particulate fraction. The effect was maximum at 5 min and persisted at 10 min. PKCepsilon was the most responsive to PdBu reaching almost maximal translocation at a PdBu concentration as low as 10(-9) M. The cholinergic agonist, carbachol (10(-5) and 10(-3) M), induced translocation which was transient for PKCdelta, and -mu, but persisted for 10 min for PKCepsilon. Carbachol did not translocate PKCalpha and, like PdBu, did not translocate PKCiota/lambda. We concluded that lacrimal gland PKC isoforms are differentially localized and that they translocate differentially in response to phorbol esters and cholinergic agonists.


Subject(s)
Carbachol/pharmacology , Carcinogens/pharmacology , Isoenzymes/metabolism , Lacrimal Apparatus/enzymology , Muscarinic Agonists/pharmacology , Phorbol 12,13-Dibutyrate/pharmacology , Protein Kinase C/metabolism , Animals , Blotting, Western , Cell Fractionation , Electrophoresis, Polyacrylamide Gel , Fluorescent Antibody Technique, Indirect , Lacrimal Apparatus/drug effects , Male , Rats , Rats, Wistar
18.
Invest Ophthalmol Vis Sci ; 38(3): 610-9, 1997 Mar.
Article in English | MEDLINE | ID: mdl-9071214

ABSTRACT

PURPOSE: To determine the presence of vasoactive intestinal peptide (VIP) receptor (VIPR) subtypes in the lacrimal gland and to determine if the components of the VIP signaling pathway for protein secretion also are present. METHODS: Immunofluorescence studies using conventional fluorescence microscopy or confocal microscopy were performed on fixed sections from rat lacrimal glands using antibodies raised against VIPRs types I and II, and four antibodies against five isoforms of adenylyl cyclase (AC) (II, III, IV, V/VI). Guanine nucleotide binding (G) proteins were detected by Western blotting. Changes in intracellular [Ca2+] ([Ca2+]i) were measured on fura-2-loaded acini in response to VIP. The effect of a myristoylated peptide corresponding to the pseudosubstrate sequence of protein kinase inhibitor (myr-PKI), the endogenous inhibitor of cyclic AMP (cAMP)-dependent protein kinase (PKA), was tested on VIP-stimulated peroxidase secretion. RESULTS: The VIPRs, types I and II, were found on the basolateral membranes of acinar and ductal cells and on myoepithelial cells. Western blotting showed the presence of alpha subunits of Gs, Gi3, G0 and G beta. The AC II was found exclusively on myoepithelial cells; AC IV was located intracellularly in all cells; AC III was found on ducts and possibly nerves; no AC V/VI was detected. The VIP (10(-8) M) caused a small but significant increase in [Ca2+]i of 26 +/- 9 nM. The VIP-stimulated protein secretion was inhibited 71% by myr-PKI. CONCLUSIONS: All components of the VIP signal transduction pathway in the lacrimal gland were present. These findings are consistent with a pathway where VIP released from parasympathetic nerves binds to VIPRs types I and II, activating G proteins, which in turn stimulate AC present on myoepithelial and acinar cells. The AC increases the intracellular cAMP concentration, which activates PKA to stimulate protein secretion. The VIP also stimulated Ca2+ influx, which could play a role in secretion.


Subject(s)
Intracellular Signaling Peptides and Proteins , Lacrimal Apparatus/metabolism , Receptors, Vasoactive Intestinal Peptide/metabolism , Signal Transduction/physiology , Adenylyl Cyclases/metabolism , Animals , Basement Membrane/metabolism , Blotting, Western , Calcium/metabolism , Carrier Proteins/pharmacology , Cyclic AMP-Dependent Protein Kinases/pharmacology , Enzyme Inhibitors/pharmacology , Fluorescent Antibody Technique, Indirect , Fluorescent Dyes/metabolism , Fura-2/metabolism , GTP-Binding Proteins/metabolism , Lacrimal Apparatus/drug effects , Male , Microscopy, Confocal , Peptide Fragments/metabolism , Rats , Rats, Wistar , Receptors, Vasoactive Intestinal Peptide/classification , Vasoactive Intestinal Peptide/pharmacology
19.
Mol Pharmacol ; 51(3): 462-70, 1997 Mar.
Article in English | MEDLINE | ID: mdl-9058601

ABSTRACT

Circular muscle of the esophagus (ESO) is normally relaxed and contracts phasically in response to neural stimuli. In contrast, lower esophageal sphincter (LES) circular muscle maintains spontaneous tone and relaxes in response to neural stimuli. We have previously shown that in vitro, spontaneous LES tone and contraction of ESO in response to acetylcholine (ACh) are antagonized by protein kinase C (PKC) inhibitors, suggesting that PKC activation is responsible for these functions. In the current study, Western blot analysis of LES and ESO revealed PKC-alpha, -betaII, and -gamma isozymes in LES circular muscle, but only PKC-betaII translocated from the cytosolic to the membrane fraction in response to ACh. In contrast, ESO contained PKC-betaII, -gamma, and -epsilon, and only PKC-epsilon translocated to the membrane fraction in response to ACh. In LES single cells isolated by enzymatic digestion and permeabilized by saponin, 1-2-dioctanoylglycerol-mediated contraction was inhibited by preincubation with PKC-betaII antiserum but not by other PKC antisera. In esophageal cells, contraction was inhibited by the PKC-epsilon antiserum but not by antisera against other PKC isozymes. N-Myristoylated peptides derived from the pseudosubstrate sequences of PKC isozymes were used to inhibit saponin, 1-2-dioctanoylglycerol-induced contraction of LES and ESO smooth muscle cells. Contraction of LES cells was reduced by the alpha beta gamma pseudosubstrate but not by the alpha, delta, or epsilon pseudosubstrate. Contraction of ESO cells was reduced by the epsilon pseudosubstrate but not by the alpha, delta, or alpha beta gamma pseudosubstrate. We conclude that different types of contractile activity in the ESO and LES are mediated by different PKC isozymes. LES contraction is mediated by the calcium-dependent PKC-betaII, whereas contraction of ESO is mediated by the calcium-independent PKC-epsilon.


Subject(s)
Esophagogastric Junction/physiology , Esophagus/physiology , Protein Kinase C/physiology , Animals , Cats , Esophagogastric Junction/enzymology , Esophagus/enzymology , Female , Isoenzymes/physiology , Male , Muscle Contraction , Muscle, Smooth/physiology , Protein Kinase C beta , Protein Kinase C-alpha
20.
Am J Physiol ; 272(1 Pt 1): C263-9, 1997 Jan.
Article in English | MEDLINE | ID: mdl-9038832

ABSTRACT

In the present study, we have synthesized and N-myristoylated peptides derived from the pseudosubstrate sequences of protein kinase C (PKC)-alpha, -delta, and -epsilon [Myr-PKC-alpha-(15-28), Myr-PKC-delta-(142-153), and Myr-PKC-epsilon-(149-164)], three isoforms present in rat lacrimal gland, and a peptide derived from the sequence of the endogenous inhibitor of protein kinase A [Myr-PKI-(17-25)]. Lacrimal gland acini were preincubated for 60 min with the myristoylated peptides (10(-10) to 3 x 10(-7) M), then protein secretion was stimulated with a phorbol ester, phorbol 12,13-dibutyrate (10(-6) M); vasoactive intestinal peptide (10(-8) M); a cholinergic agonist, carbachol (10(-5) M); or an alpha 1-adrenergic agonist, phenylephrine (10(-4) M), for 20 min. In intact lacrimal gland acini, Myr-PKC-alpha-(15-28) inhibited phorbol 12,13-dibutyrate-induced protein secretion. This effect was not reproduced by the acetylated peptide or by the myristoylated PKI, which inhibited vasoactive intestinal peptide-induced protein secretion, a response mediated by protein kinase A. Carbachol-induced protein secretion was inhibited by all three peptides. In contrast, phenylephrine-induced protein secretion was inhibited only by Myr-PKC-epsilon-(149-164), whereas Myr-PKC-alpha-(15-28) and Myr-PKC-delta-(142-153) had a stimulatory effect. None of these myristoylated peptides affected the calcium increase evoked by cholinergic or alpha 1-adrenergic agonists. We concluded that phorbol ester- and receptor-induced protein secretion involve different PKC isoforms in lacrimal gland.


Subject(s)
Isoenzymes/physiology , Lacrimal Apparatus/enzymology , Protein Kinase C/physiology , Proteins/metabolism , Adrenergic Agents/pharmacology , Animals , Calcium/metabolism , Cholinergic Agents/pharmacology , Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , Cyclic AMP-Dependent Protein Kinases/metabolism , Enzyme Inhibitors/pharmacology , Isoenzymes/metabolism , Male , Myristates/metabolism , Peptide Fragments/physiology , Phorbol 12,13-Dibutyrate/pharmacology , Phosphorylation , Protein Kinase C/metabolism , Proteins/agonists , Proteins/antagonists & inhibitors , Rats , Rats, Wistar , Vasoactive Intestinal Peptide/pharmacology
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