Lipoxin A4 activates ALX/FPR2 receptor to regulate conjunctival goblet cell secretion.

Conjunctival goblet cells play a major role in maintaining the mucus layer of the tear film under physiological conditions as well as in inflammatory diseases like dry eye and allergic conjunctivitis. Resolution of inflammation is mediated by proresolution agonists such as lipoxin A4 (LXA4) that can also function under physiological conditions. The purpose of this study was to determine the actions of LXA4 on cultured rat conjunctival goblet cell mucin secretion, intracellular [Ca2+] ([Ca2+]i), and identify signaling pathways activated by LXA4. ALX/FPR2 (formyl peptide receptor2) was localized to goblet cells in rat conjunctiva and in cultured goblet cells. LXA4 significantly increased mucin secretion, [Ca2+]i, and extracellular regulated kinase 1/2 (ERK 1/2) activation. These functions were inhibited by ALX/FPR2 inhibitors. Stable analogs of LXA4 increased [Ca2+]i to the same extent as LXA4. Sequential addition of either LXA4 or resolvin D1 followed by the second compound decreased [Ca2+]i of the second compound compared with its initial response. LXA4 activated phospholipases C, D, and A2 and downstream molecules protein kinase C, ERK 1/2, and Ca2+/calmodulin-dependent kinase to increase mucin secretion and [Ca2+]i. We conclude that conjunctival goblet cells respond to LXA4 to maintain the homeostasis of the ocular surface and could be a novel treatment for dry eye diseases.

Interaction of IFN-γ with cholinergic agonists to modulate rat and human goblet cell function.

Goblet cells populate wet-surfaced mucosa including the conjunctiva of the eye, intestine, and nose, among others. These cells function as part of the innate immune system by secreting high molecular weight mucins that interact with environmental constituents including pathogens, allergens, and particulate pollutants. Herein, we determined whether interferon gamma (IFN-γ), a Th1 cytokine increased in dry eye, alters goblet cell function. Goblet cells from rat and human conjunctiva were cultured. Changes in intracellular [Ca(2+)] ([Ca(2+)](i)), high molecular weight glycoconjugate secretion, and proliferation were measured after stimulation with IFN-γ with or without the cholinergic agonist carbachol. IFN-γ itself increased [Ca(2+)](i) in rat and human goblet cells and prevented the increase in [Ca(2+)](i) caused by carbachol. Carbachol prevented IFN-γ-mediated increase in [Ca(2+)](i). This cross-talk between IFN-γ and muscarinic receptors may be partially due to use of the same Ca(2+)(i) reservoirs, but also from interaction of signaling pathways proximal to the increase in [Ca(2+)](i). IFN-γ blocked carbachol-induced high molecular weight glycoconjugate secretion and reduced goblet cell proliferation. We conclude that increased levels of IFN-γ in dry eye disease could explain the lack of goblet cells and mucin deficiency typically found in this pathology. IFN-γ could also function similarly in respiratory and gastrointestinal tracts.

Resolvin D1 and aspirin-triggered resolvin D1 regulate histamine-stimulated conjunctival goblet cell secretion.

Resolution of inflammation is an active process mediated by pro-resolution lipid mediators. As resolvin (Rv) D1 is produced in the cornea, pro-resolution mediators could be effective in regulating inflammatory responses to histamine in allergic conjunctivitis. Two key mediators of resolution are the D-series resolvins RvD1 or aspirin-triggered RvD1 (AT-RvD1). We used cultured conjunctival goblet cells to determine whether histamine actions can be terminated during allergic responses. We found cross-talk between two types of G protein-coupled receptors (GPRs), as RvD1 interacts with its receptor GPR32 to block histamine-stimulated H1 receptor increases in intracellular [Ca(2+)] ([Ca(2+)]i) preventing H1 receptor-mediated responses. In human and rat conjunctival goblet cells, RvD1 and AT-RvD1 each block histamine-stimulated secretion by preventing its increase in [Ca(2+)]i and activation of extracellular regulated-protein kinase (ERK)1/2. We suggest that D-series resolvins regulate histamine responses in the eye and offer new treatment approaches for allergic conjunctivitis or other histamine-dependent pathologies.

Differential effects of the EGF family of growth factors on protein secretion, MAPK activation, and intracellular calcium concentration in rat lacrimal gland.

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.

Presence of nerves and their receptors in mouse and human conjunctival goblet cells.

PURPOSE: To determine whether neural pathways for controlling goblet cell secretion are present in mouse and human conjunctiva. METHODS: Mouse conjunctiva was homogenized and subjected to electrophoresis and Western blotting to detect PGP 9.5 (indicates nerves), muscarinic receptor subtypes (indicates parasympathetic pathway), and adrenergic receptors (indicates sympathetic pathway). Mouse eyes and human conjunctival tissue were analyzed by immunofluorescence microscopy. Antibodies to vasoactive intestinal peptide (VIP), tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DBH), and muscarinic and alpha(1)- and beta-adrenergic receptor subtypes were used. RESULTS: Western blot demonstrated PGP 9.5, M(1), M(2), and M(3) muscarinic receptors and alpha(1A)-, beta(1)-, beta(2)-, and beta(3)-adrenergic receptors in mouse conjunctiva. Immunoreactivity for VIP, TH, and DBH was found adjacent to mouse and human goblet cells. M(1) and M(2) muscarinic receptors were identified throughout mouse conjunctiva, but M(3) receptor was predominantly on goblet cells. All three muscarinic receptor subtypes were detected on goblet cells in human conjunctiva. alpha(1A)-Adrenergic receptors were found on epithelial cells and on goblet cells in mouse and human conjunctiva. beta(1)- and beta(2)-Adrenergic receptors were found on both epithelial and goblet cells in mouse conjunctiva, but not on human conjunctival cells. beta(3)-Adrenergic receptors were found on both epithelial and goblet cells in human conjunctiva but not on mouse conjunctival cells. CONCLUSIONS: The following conclusions were drawn: parasympathetic nerves and M(1), M(2), and M(3) muscarinic receptors, as well as sympathetic nerves are present on mouse and human goblet cells. The adrenergic receptors beta(1) and beta(2,) but not alpha(1A) and beta(3) are present on mouse conjunctival goblet cells, whereas alpha(1A) and beta(3,) but not beta(1) and beta(2) are present on human conjunctival goblet cells, suggesting that these nerves and receptors could activate goblet cell secretion in mouse and humans.

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.

Cholinergic-induced Ca2+ elevation in rat lacrimal gland acini is negatively modulated by PKCdelta and PKCepsilon.

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.

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.

Lacrimal gland innervation is not altered with the onset and progression of disease in a murine model of Sjögren's syndrome.

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.

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.

Lacrimal gland functions are differentially controlled by protein kinase C isoforms.

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.

Immunolocalization of lacrimal gland PKC isoforms. Effect of phorbol esters and cholinergic agonists on their cellular distribution.

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.

Identification of vasoactive intestinal peptide receptor subtypes in the lacrimal gland and their signal-transducing components.

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.

Lacrimal gland PKC isoforms are differentially involved in agonist-induced protein secretion.

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.

Protein phosphorylation in Golgi, endosomal, and endoplasmic reticulum membrane fractions of lacrimal gland.

Ca2+/calmodulin- and cAMP-dependent protein kinase activities were characterized in two subcellular membrane samples. Membranes from rat lacrimal gland were isolated by differential and density gradient centrifugation into six density windows. The present study focused on membranes from density windows III and V which contain mixtures of apical, Golgi, endosomal, and endoplasmic reticulum membranes in different proportions. Phosphorylation of membrane proteins was measured by incubating the samples in [g-32P]ATP and separating the proteins by discontinuous SDS-PAGE followed by autoradiography. The amount of phosphate incorporated into specific peptide bands was quantified by densitometry. Ca2+/calmodulin-dependent protein kinase phosphorylated a 52,000 MW peptide in membranes from both density windows with a maximal increase from 0.3 to 66 microM free Ca2+. Trifluoperazine and promethazine, two inhibitors of Ca2+/calmodulin-dependent protein kinases, inhibited this phosphorylation. cAMP-dependent protein kinase phosphorylated a 22,000 MW peptide and a 91,000 MW peptide which were present in membranes from density window III only. We conclude that a Ca2+/calmodulin-dependent protein kinase activity is present in membranes from both density window III and V whereas a cAMP-dependent protein kinase activity is present only in membranes from density window III.

Role of protein kinase C in cholinergic stimulation of lacrimal gland protein secretion.

The purpose of this study was to determine the role of protein kinase C (PKC) isozymes in carbachol-induced protein secretion in the lacrimal gland. Three isoforms of PKC are present in rat lacrimal gland; PKC-alpha, -delta and -epsilon. Carbachol translocated PKC-epsilon during 5 s incubation. Pretreatment with PdBu for 0 to 4 h down-regulated PKC-alpha by 31% at 20 min, PKC-epsilon by 36% at 2 h, and PKC-delta by 37% at 4 h. A 2 h phorbol ester treatment inhibited carbachol-induced secretion completely at 1 min and partially at 5, and 20 min, but did not alter the carbachol-induced increase in the intracellular [Ca2+]. We conclude that PKC-alpha and -epsilon, but not PKC-delta, are implicated in cholinergic agonist-induced protein secretion in rat lacrimal gland.

Alpha 1-adrenergic agonist-stimulated protein secretion in rat exorbital lacrimal gland acini.

The alpha-adrenergic stimulation by phenylephrine of lacrimal gland protein secretion was pharmacologically characterized. Acini, prepared from rat exorbital lacrimal glands, were incubated with agonists, antagonists or both for 0-20 min. Peroxidase secretion, an index of protein secretion, was measured spectrophotometrically. Peroxidase secretion was stimulated by the alpha 1-adrenergic agonists clonidine. The non-selective alpha-adrenergic antagonist phentolamine completely inhibited phenylephrine-induced secretion. The selective alpha 1-adrenergic alkylating agent phenoxybenzamine and the selective alpha 1-adrenergic antagonist prazosin partially inhibited phenylephrine-induced secretion. The alpha 2-adrenergic antagonist yohimbine, the beta-adrenergic antagonist timolol, and the dopaminergic antagonist haloperidol also inhibited phenylephrine-induced secretion but were 100-fold less potent than prazosin. It is concluded that phenylephrine activates an alpha 1-adrenergic pathway, but not an alpha 2-adrenergic, beta-adrenergic or dopaminergic pathway, to stimulate lacrimal gland protein secretion from acini.