Stimulation Of Protein Secretion Research Articles

A novel chemical chaperone ameliorates osteoblast homeostasis and extracellular matrix in osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is a collagen I-related heritable family of skeletal diseases associated to extreme bone fragility and deformity. Its classical forms are caused by dominant mutations in COL1A1 and COL1A2, which encode for the protein α chains, and are characterized by impairment in collagen I structure, folding, and secretion. Mutant collagen I assembles in an altered extracellular matrix affecting mineralization and bone properties and partially accumulating inside the cells, leading to impaired trafficking and cellular stress. Recently, the chemical chaperone 4-phenylbutyrate (4-PBA) has been proposed as an innovative drug for OI based on its ability to restore intracellular homeostasis, stimulate secretion, and ameliorate collagen-producing cell functions, positively affecting bone properties. However, the limited half-life of the molecule represents a serious hurdle for its use. To efficiently target cellular stress as OI treatment, two new compounds were designed by molecular modelling based on the 4-PBA structure to increase its stability and its ability to implement protein secretion. The short butyryl fatty acid chain of 4-PBA was substituted with a nitro functional group or with a glycine, respectively. The latter, N-benzyl glycine (N-BG), showed the best docking score, less toxicity, and higher stability than 4-PBA. N-BG improved extracellular matrix quality and mineral content together with ameliorating OI cells' homeostasis by increasing ER-associated degradation pathway, reducing apoptosis, and stimulating protein secretion, thus facilitating intracellular clearance from accumulated misfolded proteins. In conclusion, N-BG represents a novel potential available compound to target altered homeostasis in OI with the aim to ameliorate the disease phenotype.

Insights into dynamic evolution of combined scaling-biofouling in reverse osmosis

Although extensive research has focused on individual membrane fouling, there is still a lack of studies regarding combined scaling-biofouling, which is more commonly encountered in practical membrane desalination processes due to the coexistence of inorganic scalants and microorganisms. To our best knowledge, it is the first study that elaborated the dynamic evolution of combined scaling-biofouling in the reverse osmosis (RO) cross-flow filtration process, compared with gypsum scaling and biofouling alone. The results indicated that scaling ions (35 mM CaCl2 + 20 mM Na2SO4) stimulated protein secretion but inhibited that of polysaccharide and total organic carbon (TOC). The gradually formed scaling layer evidently enhanced the hydrophilicity of the fouled membrane, and consequently improved the repulsion of membranes against microbes and the secreted extracellular polymeric substances (EPS), proved by the augmented energy barrier for microbial adhesion through XDLVO interfacial energy analysis, and resulted in the slower flux decline at the later stage of combined scaling-biofouling. In the other perspective, microorganisms and EPS diminished the (020), (021) and (041) facets of gypsum crystals, decreased the crystallinity and size of gypsum crystals, and thus prolonged the formation of inorganic scaling. It was suggested that scaling ions should be effectively controlled through pretreatment as they contributed to more rapid flux decline, as the cleaning efficiency of Ca2+ ions decreased from 84.8 % to 57 % after they combined with EPS, and thus chiefly contributed to reversible membrane fouling resistance. This study fills in the knowledge gap on interactions between gypsum scaling and biofouling during the formation of combined scaling-biofouling in RO filtration, which had significant environmental effects under the strict industrial wastewater discharge standards and zero liquid discharge (ZLD) requirements.

Effects of reflux ratio on the anaerobic sludge and microbial social behaviors in an expanded granular sludge bed reactor: From the perspective of acyl-homoserine lactones-mediated quorum sensing

Performance of anaerobic sludge and microbial social behaviors in an expanded granular sludge bed (EGSB) were evaluated by increasing reflux ratio from 50% to 500% stage by stage, with a constant influent chemical oxygen demand (COD) of 5500 mg/L at hydraulic retention time 12 h. The results indicated that the reflux ratio of 100% − 200% was more favorable for the EGSB with a methane production of 2.4 m3/m3·d. It was found that acyl-homoserine lactones (AHLs)-mediated quorum sensing (QS) could balance various microbial populations in the anaerobic digestion process. C4-HSL and C8-HSL were identified as the specific AHLs in enhancing granulation of anaerobic sludge by stimulating protein secretion into extracellular polymeric substances (EPS). 3-oxo-C6-HSL and 3-oxo-C14-HSL were verified for the enhancement of methanogenesis. The present study showed a novel perspective on the performance of EGSB with reflux ratios based on the AHLs-mediated QS.

Repurposing human kinase inhibitors to create an antibiotic active against drug-resistant Staphylococcus aureus, persisters and biofilms.

New drugs are desperately needed to combat methicillin-resistant Staphylococcus aureus (MRSA) infections. Here we report screening commercial kinase inhibitors for anti-bacterial activity and found the anti-cancer drug sorafenib as major hit effectively killing MRSA strains. Varying the key structural features led to the identification of a potent analogue, PK150, that showed anti-bacterial activity against several pathogenic strains at sub-micromolar concentrations. Furthermore, this antibiotic eliminated challenging persisters as well as established biofilms. PK150 holds promising therapeutic potential as it did not induce in vitro resistance, shows oral bioavailability and in vivo efficacy. Analysis of the mode of action using chemical proteomics revealed several targets, including interference with menaquinone biosynthesis by inhibiting demethylmenaquinone methyltransferase and stimulation of protein secretion by altering the activity of signal peptidase IB. Reduced endogenous menaquinone levels along with enhanced levels of extracellular proteins of PK150-treated bacteria support this target hypothesis. The associated antibiotic effects, especially the lack of resistance development, likely stem from the compound’s polypharmacology.

Physiological cAMP-elevating secretagogues differentially regulate fluid and protein secretions in mouse submandibular and sublingual glands.

The mechanisms underlying the functional differences in sympathetic and parasympathetic regulation of the major salivary glands have received little attention. The acute effects of parasympathetic muscarinic (carbachol)-dependent and combined parasympathetic-dependent plus cAMP-dependent pathways on fluid secretion rates, ion composition, and protein content were assessed using a newly developed ex vivo preparation that allows the simultaneous perfusion of the mouse submandibular (SMGs) and sublingual glands (SLGs). Our results confirm that the muscarinic-dependent pathway accounts for the bulk of salivation in SMGs and SLGs, whereas costimulation with a cAMP-increasing agent (forskolin, isoproterenol, or vasoactive intestinal peptide) did not increase the flow rate. Costimulation with carbachol plus the β-adrenergic agonist isoproterenol decreased the concentration of NaCl and produced a substantial increase in the protein and Ca2+ content of SMG but not SLG saliva, consistent with a sparse sympathetic innervation of the SLGs. On the other hand, forskolin, which bypasses receptors to increase intracellular cAMP by directly activating the enzyme adenylate cyclase, enhanced the secretion of protein and Ca2+ by both the SMGs and SLGs. In contrast, isoproterenol and vasoactive intestinal peptide specifically stimulated protein secretion in SMG and SLG salivas, respectively. In summary, cAMP-dependent signaling does not play a major role in the stimulation of fluid secretion in SMGs and SLGs, whereas each cAMP-increasing agonist behaves differently in a gland-specific manner suggesting differential expression of G protein-coupled receptors in the epithelial cells of SMGs and SLGs.

4-PBA ameliorates cellular homeostasis in fibroblasts from osteogenesis imperfecta patients by enhancing autophagy and stimulating protein secretion

The clinical phenotype in osteogenesis imperfecta (OI) is attributed to the dominant negative function of mutant type I collagen molecules in the extracellular matrix, by altering its structure and function. Intracellular retention of mutant collagen has also been reported, but its effect on cellular homeostasis is less characterized. Using OI patient fibroblasts carrying mutations in the α1(I) and α2(I) chains we demonstrate that retained collagen molecules are responsible for endoplasmic reticulum (ER) enlargement and activation of the unfolded protein response (UPR) mainly through the eukaryotic translation initiation factor 2 alpha kinase 3 (PERK) branch. Cells carrying α1(I) mutations upregulate autophagy, while cells with α2(I) mutations only occasionally activate the autodegradative response. Despite the autophagy activation to face stress conditions, apoptosis occurs in all mutant fibroblasts. To reduce cellular stress, mutant fibroblasts were treated with the FDA-approved chemical chaperone 4-phenylbutyric acid. The drug rescues cell death by modulating UPR activation thanks to both its chaperone and histone deacetylase inhibitor abilities. As chaperone it increases general cellular protein secretion in all patients' cells as well as collagen secretion in cells with the most C-terminal mutation. As histone deacetylase inhibitor it enhances the expression of the autophagic gene Atg5 with a consequent stimulation of autophagy. These results demonstrate that the cellular response to ER stress can be a relevant target to ameliorate OI cell homeostasis.

Luminescence enhancement of the catalytic 19 kDa protein (KAZ) of Oplophorus luciferase by three amino acid substitutions

To characterize the luminescence properties of nanoKAZ, a 16 amino acid substituted mutant of the catalytic 19kDa protein (KAZ) of Oplophorus luciferase, the effects of each mutated amino acid were investigated by site-specific mutagenesis. All 16 single substituted KAZ mutants were expressed in Escherichia coli cells and their secretory expressions in CHO-K1 cells were also examined using the signal peptide sequence of Gaussia luciferase. Luminescence activity of KAZ was significantly enhanced by single amino acid substitutions at V44I, A54I, or Y138I. Further, the triple mutant KAZ-V44I/A54I/Y138I, named eKAZ, was prepared and these substitutions synergistically enhanced luminescence activity, showing 66-fold higher activity than wild-KAZ and also 7-fold higher activity than nanoKAZ using coelenterazine as a substrate. Substrate specificity of eKAZ for C2- and/or C6-modified coelenterazine analogues was different from that of nanoKAZ, indicating that three amino acid substitutions may be responsible for the substrate recognition of coelenterazine to increase luminescence activity. In contrast, these substitutions did not stimulate protein secretion from CHO-K1 cells, suggesting that the folded-protein structure of KAZ might be different from that of nanoKAZ.

Lacritin-Induced Secretion of Tear Proteins From Cultured Monkey Lacrimal Acinar Cells

During inflammation of the ocular surface, increased proinflammatory cytokines depress tear protein secretion, suggesting that a decline in lacrimal cell function contributes to dry eye. Lacritin, a glycoprotein secreted from lacrimal acinar cells, may function as an autocrine factor to stimulate tear protein secretion. The purpose of the present experiment was to investigate lacritin-induced protein secretion in normal and cytokine-pretreated (inflammation model) monkey acinar cells. Acinar cells from monkey lacrimal glands were cultured with or without tumor necrosis factor alpha (TNF-α) plus interferon gamma (IFN-γ). Protein secretion was induced by lacritin or carbachol (Cch, positive control). Proteins were detected and identified by immunoblotting and immunocytochemistry. Intracellular Ca(2+) was measured with the fluorophore Calcium-4, and cell damage was determined by LDH leakage into the culture medium. In cultured monkey acinar cells, lacritin stimulated tear protein secretion in normal cells without elevating intracellular Ca(2+). In contrast, Cch elevated intracellular Ca(2+) and release of tear proteins. This contrast suggested an alternate, calcium-independent mechanism for lacritin-induced protein secretion. TNF-α plus IFN-γ caused LDH leakage from sensitive human corneal epithelial cells, but even higher doses of TNF-α plus IFN-γ did not cause LDH leakage from monkey acinar cells, suggesting a higher tolerance against these cytokines. In cytokine-treated acinar cells, lacritin stimulated protein secretion as much as that in normal cells. In contrast, Cch-induced elevation of Ca(2+) and release of proteins were depressed by cytokines. Lacritin might be a useful biotechnology-based treatment agent against ocular surface diseases where endogenous lacritin is inadequate.

205: Progesterone maintains a balance in collagen metabolism while estrogen drives production in cervical stromal cells

metabolism while estrogen drives production in cervical stromal cells Huiling Ji, Joshua Dahlke, Edward Chien Warren Alpert School of Medicine of Brown University, Obstetrics and Gynecology, Providence, RI OBJECTIVE: Cervical remodeling is associated with extracellular matrix (ECM) remodeling. Collagen is the main ECM component in the cervix contributing to tissue resistance. Collagen remodeling in the cervix during gestation is thought to be hormone regulated and involves both the production of new protein as well as degradation of existing fibers. We have previously shown that estrogen increases collagen secretion. The purpose of this study is to investigate the effects of estradiol and progesterone on both collagen production and degradation in rat cervical stromal cells. STUDY DESIGN: Primary rat cervical stromal cells were cultured using an explant method approved by the IACUC. Passage 2-6 cells were plated on six well plates in serum free media. Cells were treated for 3 days with estradiol or progesterone. Vehicle treated cells were used as controls. Culture medium was collected for soluble collagen assay. The ECM collagen that was adherent to the culture dish was extracted using 0.5M acetic acid. Sircol Red Assay was used to quantify soluble and ECM collagen. Western Blot was performed to evaluate the expression of Type I and III collagen, MMP2, MMP9 and MMP13. Zymography was used to detect MMP2 and MMP9 expression. Data was analyzed using Sigma Stat. RESULTS: Similar to prior studies estradiol increased soluble collagen secretion while no change was observed with progesterone. Estradiol and progesterone had similar effects on ECM collagen. Estradiol stimulated collagen I /III expression and decreased MMP2 expression. Progesterone did not change Type I/III collagen expression but increased MMP13 expression. MMP9 expression did not change in either estradiol or progesterone treated rat cervical stromal cells. CONCLUSION: Estradiol increased overall collagen production by stimulating protein secretion and polymerization while inhibiting the production of collagenases. Collagen content was maintained by progesterone by balancing production and degradation.

Interaction of α1D-Adrenergic and P2X7Receptors in the Rat Lacrimal Gland and the Effect on Intracellular [Ca2+] and Protein Secretion

To determine whether α(1D)-adrenergic receptors (α(1D)-AR) and P2X(7) receptors interact by determining their effect on ATP release, intracellular [Ca(2+)] ([Ca(2+)](i)), and protein secretion in rat lacrimal gland acini. Exorbital lacrimal glands from male Sprague-Dawley rats were divided into pieces or digested with collagenase to form acini. With the use of an imaging system, [Ca(2+)](i) was measured in acini loaded with fura-2. Adenosine triphosphate (ATP) release was determined using the luciferin-luciferase reaction. Peroxidase secretion, our index for protein secretion, was measured spectrophotometrically. Acini were stimulated with the P2X(7) receptor agonist, (benzoylbenzoyl)adenosine 5' triphosphate (BzATP) or the α(1D)-AR agonist phenylephrine with or without antagonist preincubation. Phenylephrine increased ATP release from pieces in a time-dependent manner. The α(1D)-AR antagonist BMY7378 blocked the BzATP-stimulated increase in [Ca(2+)](i) but not in peroxidase secretion. The P2X(7) antagonist A438079 blocked the phenylephrine-stimulated increase in [Ca(2+)](i) but not peroxidase secretion. The increase in [Ca(2+)](i) caused by phenylephrine and BzATP used simultaneously or sequentially was additive, as was the increase in peroxidase secretion. The inhibition of protein kinase C isoforms or calcium calmodulin kinase II did not alter the BzATP-induced increase in [Ca(2+)](i). The authors conclude that activation of α(1D)-AR releases ATP, which induces P2X(7) receptors to increase [Ca(2+)](i) but not to stimulate protein secretion. P2X(7) receptors in turn activate α(1D)-AR to increase [Ca(2+)](i) but not to stimulate protein secretion. Furthermore, α(1D)-AR compared with P2X(7) receptors use different cellular mechanisms to increase [Ca(2+)](i) and cause protein secretion.

Cholinergic Agonists Activate P2X7Receptors to Stimulate Protein Secretion by the Rat Lacrimal Gland

To determine the interaction of M3 muscarinic receptors (M3AChR) and P2X(7) receptors to increase intracellular [Ca2+] ([Ca2+]i) and stimulate protein secretion in rat lacrimal gland cells. Exorbital lacrimal glands from male Sprague-Dawley rats were divided into pieces or digested with collagenase to form acinar clumps. [Ca2+]i was measured using an imaging system in acini incubated with fura-2/AM. Adenosine triphosphate (ATP) release was determined using the luciferin-luciferase reaction. Peroxidase secretion, our index for protein secretion, was measured spectrophotometrically. Acini were stimulated with the P2X7 receptor agonist, (benzoylbenzoyl)adenosine 5' triphosphate (BzATP), cholinergic agonist carbachol, or the activator of conventional and novel PKC isoforms, phorbol 12-myristate 13-acetate (PMA). The increase in [Ca2+]i caused by carbachol and BzATP used simultaneously was less than additive, but the increase in protein secretion was additive. The M3AChR antagonist atropine blocked the BzATP-stimulated increase in [Ca2+]i and peroxidase secretion. The P2X7 antagonist did not alter the carbachol-stimulated increase in [Ca2+]i or peroxidase. PMA- and BzATP-stimulated increases in [Ca2+]i were additive. Neither constitutively active PKCα, dominant-negative PKCα, nor PKCε altered BzATP-stimulated increases in [Ca2+]i. Carbachol increased ATP release from lacrimal gland pieces but not from acini. In lacrimal gland cells, the activation of M3AChRs stimulates P2X7 receptors to increase [Ca2+]i and protein secretion. The underlying mechanisms are unknown but could include the release of ATP or intracellular interactions not mediated by PKC isoforms. In addition, M3AChRs use signaling pathways that overlap with those used by P2X7 receptors to increase [Ca2+]i, but they also use signaling pathways not used by P2X7 receptors to stimulate protein secretion.

Food-Derived Bioactive Peptides Influence Gut Function

Bioactive peptides either present in foods or released from food proteins during digestion have a wide range of physiological effects, including on gut function. Many of the bioactive peptides characterized to date that influence gut motility, secretion, and absorption are opioid agonists or antagonists. The authors review a body of experimental evidence that demonstrates an effect of peptides from food proteins on endogenous (nondietary) protein flow at the terminal ileum of simple-stomached mammals, including adult humans. At least some dietary peptides (1000-5000 Da) significantly enhance the loss of protein from the small intestine, causing an increased amount of protein to enter the colon. Food-derived peptides appear to either stimulate protein secretion into the gut lumen or inhibit amino acid reabsorption or influence both processes simultaneously. The effect of dietary peptides on small-intestine secretory-protein dynamics is discussed in the context of the major components of gut endogenous protein, sloughed cells, enzymatic secretions, mucin, and bacterial protein.

Roles of Protein Kinase C, Ca2+, Pyk2, and c-Src in Agonist Activation of Rat Lacrimal Gland p42/p44 MAPK

Although p42/p44 mitogen-activated protein kinase (MAPK) negatively modulates protein secretion stimulated by cholinergic and alpha(1D)-adrenergic agonists, it does not play a role in epidermal growth factor (EGF)-stimulated protein secretion. Therefore, this study was conducted to determine the roles that protein kinase C (PKC), intracellular Ca(2+) ([Ca(2+)](i)), and nonreceptor tyrosine kinases Pyk2 and Src play in the activation of agonist- and EGF-stimulated MAPK activation. Lacrimal gland acini were isolated by collagenase digestion and incubated with phorbol 12-myristate 13-acetate (PMA) to activate PKC or ionomycin, a Ca(2+) ionophore. Acini were preincubated with the PKC inhibitors calphostin C or Ro-31-8220, EGTA to chelate Ca(2+), or the c-Src inhibitor PP1 before stimulation with the cholinergic agonist carbachol, the alpha(1D)-adrenergic agonist phenylephrine, or EGF. Activated MAPK, Pyk2, and c-Src amounts were measured by Western blot analysis. PMA and ionomycin significantly increased the activation of MAPK in a time- and concentration-dependent manner. Inhibition of PKC partially inhibited carbachol-stimulated MAPK activation while completely inhibiting phenylephrine- and EGF-stimulated MAPK activation. Chelation of Ca(2+) also partially inhibited carbachol-stimulated MAPK with no effect on phenylephrine- and EGF-stimulated MAPK activation. Carbachol increased the phosphorylation of Pyk2 on tyrosine 402 and c-src on tyrosine 416 in a time-dependent manner. The c-src inhibitor PP1 inhibited carbachol-stimulated phosphorylation of Pyk2. It was concluded that cholinergic agonists use Ca(2+) and PKC to phosphorylate Pyk2 and c-Src, which subsequently stimulate MAPK activity. In contrast, alpha(1D)-adrenergic agonists and EGF do not use Pyk2 and Src but do use PKC to activate MAPK.

Effects of α1D-adrenergic receptors on shedding of biologically active EGF in freshly isolated lacrimal gland epithelial cells

Transactivation of EGF receptors by G protein-coupled receptors is a well-known phenomenon. This process involves the ectodomain shedding of growth factors in the EGF family by matrix metalloproteinases. However, many of these studies employ transformed and/or cultured cells that overexpress labeled growth factors. In addition, few studies have shown that EGF itself is the growth factor that is shed and is responsible for transactivation of the EGF receptor. In this study, we show that freshly isolated, nontransformed lacrimal gland acini express two of the three known alpha(1)-adrenergic receptors (ARs), namely, alpha(1B)- and alpha(1D)-ARs. Alpha(1D)-ARs mediate phenylephrine (an alpha(1)-adrenergic agonist)-induced protein secretion and activation of p42/p44 MAPK, because the alpha(1D)-AR inhibitor BMY-7378, but not the alpha(1A)-AR inhibitor 5-methylurapidil, inhibits these processes. Activation of p42/p44 MAPK occurs through transactivation of the EGF receptor, which is inhibited by the matrix metalloproteinase ADAM17 inhibitor TAPI-1. In addition, phenylephrine caused the shedding of EGF from freshly isolated acini into the buffer. Incubation of freshly isolated cells with conditioned buffer from cells treated with phenylephrine resulted in activation of the EGF receptor and p42/p44 MAPK. The EGF receptor inhibitor AG1478 and an EGF-neutralizing antibody blocked this activation of p42/p44 MAPK. We conclude that in freshly isolated lacrimal gland acini, alpha(1)-adrenergic agonists activate the alpha(1D)-AR to stimulate protein secretion and the ectodomain shedding of EGF to transactivate the EGF receptor, potentially via ADAM17, which activates p42/p44 MAPK to negatively modulate protein secretion.

Nitric Oxide and cGMP Mediate α1D-Adrenergic Receptor–Stimulated Protein Secretion and p42/p44 MAPK Activation in Rat Lacrimal Gland

To determine whether alpha(1)-adrenergic receptors use the nitric oxide (NO)/cGMP pathway to stimulate protein secretion by rat lacrimal gland. Identification and cellular location of endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS) were determined by Western blot and immunofluorescence techniques, respectively. Rat lacrimal gland acini were isolated by collagenase digestion, and protein secretion stimulated by phenylephrine, an alpha(1)-adrenergic agonist, was measured with a fluorescence assay system. Acini were preincubated with inhibitors for 20 minutes before addition of phenylephrine (10(-4) M). NO and cGMP were measured in response to phenylephrine stimulation. Activation of p42/p44 MAPK was determined by Western blot analysis with an antibody against phosphorylated (active) p42/p44 MAPK. eNOS and nNOS were both present in lacrimal gland. eNOS appeared to be localized with caveolae, whereas nNOS was present in the nerves surrounding the acini. Inhibition of eNOS with N(G)-nitro-l-arginine methyl ester (l-NAME; 10(-6) M) completely inhibited phenylephrine-stimulated protein secretion, whereas the inactive isomer d-NAME and inhibition of nNOS with S-methyl-l-thiocitrulline did not. Phenylephrine increased NO production in a time- and concentration-dependent manner, but the increase was abolished by the alpha(1D)-adrenergic receptor inhibitor BMY-7378. Inhibition of guanylate cyclase with oxadiazoloquinoxalin (ODQ) also inhibited phenylephrine-induced protein secretion, whereas phenylephrine caused a 2.2-fold increase in cGMP. In addition, preincubation with l-NAME and ODQ inhibited phenylephrine-stimulated p42/p44 MAPK activation. alpha(1D)-Adrenergic agonists stimulate eNOS to produce NO, leading to production of cGMP by guanylate cyclase, to transduce the extracellular signal through the cell and stimulate protein secretion in rat lacrimal gland.

Protein secretion in cockroach salivary glands requires an increase in intracellular cAMP and Ca 2+ concentrations

The salivary glands in the cockroach Periplaneta americana secrete protein-containing saliva when stimulated by serotonin (5-HT) and protein-free saliva upon dopamine stimulation. In order to obtain information concerning the signalling pathways involved in 5-HT-induced protein secretion, we have determined the protein content of saliva secreted after experimental manipulations that potentially elevate intracellular Ca 2+ and cyclic nucleotide concentrations in isolated glands. We have found that 5-HT stimulates the rate of protein secretion in a dose-dependent manner (threshold: 3 × 1 0 - 8 M ; EC 50 1.5 × 1 0 - 6 M ). The maximal rate of 5-HT-induced protein secretion was 2.2 ± 0.2 μg/min. Increasing intracellular Ca 2+ or cAMP by bath application of ionomycin (5 μM), db cAMP (10 mM), forskolin (100 μM) or IBMX (100 μM), respectively, stimulated protein secretion at significantly lower rates, whereas db cGMP (1 mM) did not activate protein secretion. The high rates and the kinetics of 5-HT-induced protein secretion could only be mimicked by either applying forskolin together with IBMX (with or without ionomycin) or by applying IBMX together with ionomycin. Our measurements suggest that 5-HT-induced protein secretion is mediated by an elevation of [cAMP] i and that Ca 2+ may function as a co-agonist and augment the rate of protein secretion.

Amblyomma americanum salivary glands: double-stranded RNA-mediated gene silencing of synaptobrevin homologue and inhibition of PGE 2 stimulated protein secretion

Protein secretion into the saliva from the tick salivary glands is due to exocytosis of vesicular membrane bound granular material regulated by SNARE complex proteins after salivary gland stimulation by PGE 2 [Insect Biochem. Mol. Biol. 32 (2002) 1711]. Proteins associated with vesicles (v-SNAREs) are essential components of the exocytotic process. Synaptobrevin is a key v-SNARE in all secreting cells studied to date. A vesicle-associated synaptobrevin cDNA fragment homologue from the salivary glands of partially fed lone star tick females was cloned and sequenced. Double-stranded (ds) RNA interference (RNAi) is an effective method to silence specific gene expression. The functional role of synaptobrevin in protein secretion in partially fed tick salivary glands was studied with an in vitro RNAi method. Incubation of isolated salivary glands with double-stranded RNA (dsRNA) transcribed from a tick salivary gland synaptobrevin cDNA fragment resulted in decreased expression of the transcript, a reduction in the level of synaptobrevin protein and inhibition of PGE 2 stimulated anticoagulant protein secretion by isolated salivary glands. We demonstrate the applicability of RNAi for studying individual steps in the mechanism of PGE 2 stimulated exocytosis in the salivary glands of ixodid ticks.

Signal transduction pathways used by EGF to stimulate protein secretion in rat lacrimal gland.

To determine the effects of epidermal growth factor (EGF) on lacrimal gland secretion of proteins and characterize its signal-transducing components. Both exorbital lacrimal glands were removed from male Sprague-Dawley rats. Dispersed acini were isolated by collagenase digestion in Krebs-Ringer bicarbonate (KRB) buffer at 37 degrees C. Acini were incubated with EGF (10(-7) M), the cholinergic agonist carbachol (10(-4) M), or the alpha(1)-adrenergic agonist phenylephrine (10(-4) M), and peroxidase secretion was measured by a fluorescence assay. To measure intracellular calcium ([Ca(2+)](i)), acini were incubated in fura-2 tetra-acetoxymethyl ester for 60 minutes at 22 degrees C, and fluorescence was measured at 340 and 380 nm with an emission wavelength of 505 nm. Extracellular Ca(2+) was chelated with KRB-BSA without CaCl(2) and with 2 mM EGTA before measurement of peroxidase secretion. Protein kinase C (PKC) was downregulated by incubating acini overnight, with or without the phorbol ester, phorbol 12-myristate 13-acetate (PMA; 10(-6) M), and peroxidase secretion was measured. EGF-stimulated peroxidase secretion in a concentration-dependent manner with a significant increase at 10(-7) M. EGF-stimulated secretion was inhibited by the EGF receptor (EGFR) inhibitor AG1478, but not by the phosphoinositide-3 kinase inhibitor LY292004 or the mitogen-activated kinase kinase (MEK) inhibitor U0126. EGF increased [Ca(2+)](i), whereas chelation of extracellular Ca(2+) inhibited EGF-induced peroxidase secretion by 90%. Downregulation of PKC also inhibited EGF-stimulated peroxidase secretion. EGF stimulates lacrimal gland secretion of protein by activating the EGFR to increase [Ca(2+)](i) and activate PKC.

Familial combined hyperlipidemia plasma stimulates protein secretion by HepG2 cells

The aim of this study was to evaluate whether soluble factors in plasma of familial combined hyperlipidemia (FCHL) patients affect hepatic protein secretion. Cultured human hepatocytes, i.e., HepG2 cells, were incubated with fasting plasma (20%, v/v, in DMEM) from untreated FCHL patients or normolipidemic controls. Overall protein secretion was 10-15% higher after incubation with FCHL plasma. This was specifically caused by an increase in four secreted proteins, with estimated sizes of 240, 180, 120, and <40 kD (P < 0.001, P < 0.006, P < 0.002, P < 0.02, respectively). The 240 kD protein in the secretion proteome was identified as fibronectin by mass spectrometry. Plasma fibronectin concentrations were elevated in FCHL patients, confirming biological relevance of these data. Overall protein secretion by HepG2 cells correlated with concentrations of triglycerides (r = 0.61, P < 0.001) in the applied plasma samples. VLDL+IDL isolated from FCHL patients, induced a higher protein secretion than lipoproteins isolated from controls (P < 0.001). Remarkably, secretion of apoB, the structural protein of VLDL, was stimulated to a similar extent by FCHL and control plasma. FCHL plasma did not induce excess secretion of apoB by HepG2 cells compared with control plasma. FCHL plasma did stimulate secretion of several distinct hepatic proteins, among which fibronectin was identified.

Nitric oxide and cyclic GMP-mediated protein secretion from cultured lacrimal gland acinar cells.

Nitric oxide (NO) donors and NO synthase (NOS) substrates were tested for their use to stimulate protein secretion from cultured lacrimal gland acinar cells, through activation of guanylate cyclase. Rabbit lacrimal gland epithelial cells (RLG cells) were incubated with NO donors and/or NOS substrates and the protein released into culture medium was determined with bicinchoninic acid assay. Guanylate cyclase activation by NO precursors was determined by measurement of c-GMP produced. Both NO donors and NOS substrates were able to stimulate protein release from RLG cells. Among 6 compounds studied, sodium nitroprusside, isosorbide dinitrate and N(a)-benzoyl L-arginine ethyl ester (BAEE) were most potent to release protein over 100% of the basal release. The guanylate cyclase activity was stimulated by these NO precursors and was inhibited by guanylate cyclase inhibitor, [1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ). NO donors and NOS substrates were able to stimulate protein release from RLG cells via activation of guanylate cyclase and c-GMP release, which was blocked by guanylate cyclase inhibitor, ODQ. It indicates that NO donors and NOS substrates could be used for the treatment of dry eye syndrome if the same holds true in dry eye animal models.