Robin R Hodges

Publications (61) View all

• Article: Signaling pathways used by EGF to stimulate conjunctival goblet cell secretion.

  Robin R Hodges, Jeffrey A Bair, Richard B Carozza, Dayu Li, Marie A Shatos, Darlene A Dartt
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  ABSTRACT: The purpose of this study was to identify the signaling pathways that epidermal growth factor (EGF) uses to stimulate mucin secretion from cultured rat conjunctival goblet cells and to compare the pathways used by EGF with those used by the known secretagogue muscarinic, cholinergic agonists. To this end, goblet cells from rat conjunctiva were grown in culture using RPMI media. For immunofluorescence experiments, antibodies against EGF receptor (EGFR) and ERK 2 as well as muscarinic receptors (M(1)AchR, M(2)AchR, and M(3)AchR) were used, and the cells viewed by fluorescence microscopy. Intracellular [Ca(2+)] ([Ca(2+)](i)) was measured using fura 2/AM. Glycoconjugate secretion was determined after cultured goblet cells were preincubated with inhibitors, and then stimulated with EGF or the cholinergic agonist carbachol (Cch). Goblet cell secretion was measured using an enzyme-linked lectin assay with UEA-I or ELISA for MUC5AC. In cultured goblet cells EGF stimulated an increase in [Ca(2+)](i) in a concentration-dependent manner. EGF-stimulated increase in [Ca(2+)](i) was blocked by inhibitors of the EGF receptor and removal of extracellular Ca(2+). Inhibitors against the EGFR and ERK 1/2 blocked EGF-stimulated mucin secretion. In addition, cultured goblet cells expressed M(1)AchR, M(2)AchR, and M(3)AchRs. Cch-stimulated increase in [Ca(2+)](i) was blocked by inhibitors for the M(1)AchRs, matrix metalloproteinases, and EGF receptors. Inhibitors against the EGF receptor and ERK 1/2 also blocked Cch-stimulated mucin secretion. We conclude that in conjunctival goblet cells, EGF itself increases [Ca(2+)](i) and activates ERK 1/2 to stimulate mucin secretion. EGF-stimulated secretion is dependent on extracellular Ca(2+). This mechanism of action is similar to cholinergic agonists that use muscarinic receptors to transactivate the EGF receptor, increase [Ca(2+)](i), and activate ERK 1/2 leading to an increase in mucin secretion.
  Experimental Eye Research 09/2012; 103:99-113. · 3.26 Impact Factor
• Article: Isolation and characterization of progenitor cells in uninjured, adult rat lacrimal gland.

  Marie A Shatos, Linda Haugaard-Kedstrom, Robin R Hodges, Darlene A Dartt
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  ABSTRACT: The purpose of this study was to investigate the presence of progenitor cells in the uninjured, adult rat lacrimal gland (LG). The presence of progenitor cells was examined in LG sections from male rats using antibodies against selected stem cell markers and α-smooth muscle actin (SMA), which marks myoepithelial cells (MECs), by immunofluorescence microscopy (IF). Small, immature cells were isolated after digestion of LG with collagenase and culture in RPMI 1640 for 2 weeks. Immature cells were examined for expression of stem cell markers by IF. Immature cell were grown in neuronal, epithelial, and myoepithelial cell media, and examined by light morphology and IF using antibodies to markers of different cell lineages. In the intact LGs, MECs expressed the stem cell markers nestin, Musashi 1, ABCG2, Pax6, Chx 10, ΔN p63, and Sox 2. All markers colocalized with SMA. Isolated immature cells contained Ki-67, nestin, Musashi 1, Pax 6, and CHX 10. In neuronal media, immature cells differentiated and assumed a neuronal cell morphology expressing neurofilament 200. In media for human corneal endothelial cells, immature cells differentiated, assumed cobblestone morphology, and labeled with the epithelial marker AE1/AE3. In RPMI media immature cells differentiated into cells with MEC-like morphology, and expressed the MEC markers SMA, α-actinin, adenylate cyclase II, and vimentin. We conclude that uninjured, adult LG contains progenitor cells that may be MECs, which can be isolated and differentiated into multiple lineages.
  Investigative ophthalmology & visual science 03/2012; 53(6):2749-59. · 3.43 Impact Factor
• Article: Increase of intracellular Ca2+ by purinergic receptors in cultured rat lacrimal gland myoepithelial cells.

  Kaori Ohtomo, Marie A Shatos, Joanna Vrouvlianis, Dayu Li, Robin R Hodges, Darlene A Dartt
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  ABSTRACT: To isolate and characterize cultured myoepithelial cells (MECs) from rat lacrimal gland and determine which purinergic receptor subtypes are present and functional in MECs. Rat lacrimal glands were subjected to collagenase digestion, and MECs were grown. RT-PCR was performed for the purinergic receptors P2X(7), P2Y(1), P2Y(11), and P2Y(13) on RNA isolated from the MECs. Immunofluorescence experiments were performed with antibodies against MEC markers and P2X(7), P2Y(1), P2Y(11), and P2Y(13) purinergic receptors. Proteins from MECs were separated using Western blot analysis techniques. In addition, cells were incubated with Fura 2 tetra acetoxymethyl ester, and intracellular [Ca(2+)] ([Ca(2+)](i)) was determined in response to P2 purinergic agonists. MECs expressed the MEC proteins α-smooth muscle actin, vimentin, α-actinin, and adenylyl cyclase II. RT-PCR, Western blot, and immunofluorescence techniques demonstrated the presence of the purinergic receptors P2X(7), P2Y(1), P2Y(11), and P2Y(13). The purinergic agonists ATP, benzoylbenzoyl ATP (BzATP), α,β methylene ATP, UTP, 2-methylthioATP (MeSATP), and ATPγS increased [Ca(2+)](i). As BzATP binds to the P2X(7) receptor, specific characteristics of this receptor were investigated. Neither inhibitors of P2X(7) receptors nor removal of extracellular Mg(2+) or Ca(2+) had an effect on the BzATP-stimulated increase in [Ca(2+)](i). Repeated applications of BzATP desensitized this response. Inhibitors for P2Y(1), P2Y(11), and P2Y(13) each decreased the BzATP-stimulated increase in [Ca(2+)](i) with the P2Y(1) inhibitor most effective. MECs can be isolated from rat lacrimal glands, and they express P2X(7), P2Y(1), P2Y(11), and P2Y(13) purinergic receptors. Surprisingly, BzATP binds the P2Y(1) receptor, which is primarily responsible for the BzATP-stimulated increase in [Ca(2+)](i).
  Investigative ophthalmology & visual science 12/2011; 52(13):9503-15. · 3.43 Impact Factor
• Article: Interaction of alpha1D-adrenergic and P2X(7) receptors in the rat lacrimal gland and the effect on intracellular [Ca2+] and protein secretion.

  Darlene A Dartt, Robin R Hodges
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  ABSTRACT: 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.
  Investigative ophthalmology & visual science 06/2011; 52(8):5720-9. · 3.43 Impact Factor
• Article: Identification of P2X₃ and P2X₇ purinergic receptors activated by ATP in rat lacrimal gland.

  Robin R Hodges, Joanna Vrouvlianis, Rachel Scott, Darlene A Dartt
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  ABSTRACT: PURPOSE. To identify the type of purinergic receptors activated by adenosine triphosphate (ATP) in rat lacrimal gland and to determine their role in protein secretion. METHODS. Purinergic receptors were identified by RT-PCR, Western blot analysis, and immunofluorescence techniques. Acini from rat lacrimal gland were isolated by collagenase digestion. Acini were incubated with the fluorescence indicator fura-2 tetra-acetoxylmethyl ester, and intracellular [Ca(2+)] ([Ca(2+)](i)) was determined. Protein secretion was measured by fluorescence assay. RESULTS. The authors previously showed that P2X(7)receptors were functional in the lacrimal gland. In this study, they show that P2X(1-4) and P2X(6)receptors were identified in the lacrimal gland by RT-PCR, Western blot, and immunofluorescence analyses. P2X(5) receptors were not detected. ATP increased [Ca(2+)](i) and protein secretion in a concentration-dependent manner. Removal of extracellular Ca(2+) significantly reduced the ATP-stimulated increase in [Ca(2+)](i). Repeated applications of ATP caused desensitization of the [Ca(2+)](i) response. Incubation with the P2X(1) receptor inhibitor NF023 did not alter ATP-stimulated [Ca(2+)](i). Incubation with zinc, which potentiates P2X(2) and P2X(4) receptor responses, or lowering the pH to 6.8, which potentiates P2X(2) receptor responses, did not alter the ATP-stimulated [Ca(2+)](i). P2X(3) receptor inhibitors A-317491 and TNP-ATP significantly decreased ATP-stimulated [Ca(2+)](i) and protein secretion, whereas the P2X(3) receptor agonist α,β methylene ATP significantly increased them. The P2X(7) receptor inhibitor A438079 had no effect on ATP-stimulated [Ca(2+)](i) at 10(-6) M but did have an effect at 10(-4) M. CONCLUSIONS. Purinergic receptors P2X(1-4) and P2X(6) are present in the lacrimal gland. ATP uses P2X(3) and P2X(7) receptors to stimulate an increase in [Ca(2+)](i) and protein secretion.
  Investigative ophthalmology & visual science 03/2011; 52(6):3254-63. · 3.43 Impact Factor