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Peripheral GABA A Rs: Overview the Lung, Pancreatic Islets, and Lymphocytes

Peripheral GABA A Rs: Overview the Lung, Pancreatic Islets, and Lymphocytes. 王双连 Nov 12, 2012. More Attention Has Been Paid to Peripheral GABA Since 1954. GABAergic Sigals in the CNS. Glutamate. GAD: glutamic acid decarboxylase GABA-T: GABA-transaminase GAT: GABA transporter

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Peripheral GABA A Rs: Overview the Lung, Pancreatic Islets, and Lymphocytes

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  1. Peripheral GABAARs: Overview the Lung, Pancreatic Islets, and Lymphocytes 王双连 Nov 12, 2012

  2. More Attention Has Been Paid to Peripheral GABA Since 1954

  3. GABAergic Sigals in the CNS Glutamate • GAD:glutamicaciddecarboxylase • GABA-T:GABA-transaminase • GAT:GABA transporter • GABAAR is a pentameric chloride channel composed of a combination of various subunits; • (α1-6, β1-3, γ1-3, δ, ε, π, θ, and ρ1-3) • GABABR is a type of G-protein coupled receptors (GPCR) GAD65/67 GABA-T GABA Succinic semialdehyde GAT GABA phasic tonic Cl- Cl- GABAAR GABABR

  4. Characteristics of GABAARs extracellular intracellular Critical Reviews in Biochemistry and Molecular Biology, 42:3–14, 2007

  5. Synaptic and Extrasynaptic GABAAR Currents Glutamate GABA Valerie B. etc. PNAS, 2004 GABA extrasynaptic phasic tonic Cl- Cl- synaptic M. Wallner etc. PNAS, 2003

  6. Modulation of GABAARs • Agonists: GABA and muscimol • Antagonist: bicuculline (competitive inhibition) picrotoxin (non – competitive inhibition) • Benzodiazepines, pentobarbital, and ethanol • Neurosteroids and endocannabinoid 2-arachidonoylglycerol (2-AG) • Ions: Zn2+, Mn2+, Co2+ • Posttranslational modification: protein kinases • Receptor associated proteins:

  7. Overview of the Major GABAAR Subunits • α1: sedative, amnesic and anticonvulsant action of BZ • α 2: Anxiolytic and myorelaxant action of BZ • α 3: anxiolytic and myorelaxant action of BZ • α 4: ethanol sensitivity • α 5: amnesic and myorelaxant action of BZ, memory enhancement • α 6: ethanol sensitivity

  8. Overview of the Major GABAAR Subunits • β1: Salicylidene salicylhydrazide as selective inhibitor • β 2: anesthetic action of etomidate • β 3: anesthetic action of propofol and etomidate • γ1-3: no specific properties • δ: ethanol and neurosteroid sensitivity

  9. Example 1: Benzodiazepines Regulation of GABAARs

  10. Example 2: Ethanol Regulation of GABAARs M. Wallner etc. PNAS, 2003

  11. What Hampers us in Further Understanding GABAAR Functions? • Easy to do: To identify sequence, expression level and localization of indivial subunits in a neuron • Less easier to do: To know subunits collaboration • Hard to do: To identify the subunit arrangement of the pentamer • Experiments have therefore been limited to the role of defined receptor subunit isoforms

  12. GABA and the Lung Story

  13. GABAARs in airway epithelium Expression of GABAARs in airway epithelium Electrophysiological characteristics Physiological and pathophysiological significance

  14. Expression of GAGAARs in lung epithelial cells

  15. Electrophysiological characteristics of GABAARs in lung epithelial cells Perforated Whole- cell Cl- sweep Voltage - clamp recording Current - clamp recording Vholding = -60 mV

  16. Electrophysiological characteristics of GABAARs in lung epithelial cells Voltage - clamp recording Vholding = -60 mV

  17. Electrophysiological characteristics of GABAARs in lung epithelial cells Voltage - clamp recording (Vstep) Vholding = from -60 to 40 mv, △ v = 20 mv, totally 6 sweeps

  18. Electrophysiological Characteristics of GABAARs in Lung Epithelial Cells Perforated Cl- X Voltage - clamp recording Current - clamp recording

  19. Physiological or Pathophysiological Significance of GABAARs in Lung Epithelium

  20. GAD and β2/3 Subunit Upregulation in Asthmatic Lung in Mice and Human Mice Human

  21. IL-13 Mediates the Upregulation of GABA Signals in Asthmatic Mice Lung In vitro β2 subunit GAD65/67

  22. IL-13 Mediates the Up-regulation of GABA Signals in Asthmatic Mice Lung In vivo

  23. Picrotoxin (PTXN) Protects the Lung against OVA-induced Asthma in Mice

  24. Blocking GABAARs does not Affect OVA-induced Inflammation and Hyperreactivity in the Airway

  25. Summary to the GABA-Lung Story Up-regulation of the epithelial GABAergic system occurs downstream of activation of the IL-13 receptor, and that this GABAergic system plays a selective part in goblet cell metaplasia and mucus overproduction.

  26. GABA and the Pancreas Story

  27. Expression of GABAARs in INS-1 cells

  28. GABA-evoked Currents in INS-1 cells Perforated Whole- cell

  29. GABA-evoked Currents in INS-1 cells 0 mmol/l glucose 16.7 mmol/l glucose

  30. Effect of GABAAR activation on the Excitability of INS-1 β cells Reversal potential of IGABA: -43 mV Clinical Pharmacology & Therapeutics, 2008

  31. Effect of GABAAR Activation on the Excitability of INS-1 β cells 0 G: Vm= -50 mV < -43 mV 28 G: Vm= -20 mV > -43 mV outside outside membrane inside inside GABAAR GABAAR Cl- Driving force Driving force Cl-

  32. Effect of GABAAR Activation on the Excitability of INS-1 β cells

  33. Modulation of Ca2+ infux by GABA in INS-1 cells

  34. Regulation of Insulin Secretion by GABA is dependent on Glucose Concentration Lower glucose Higher glucose

  35. Summary to the GABA-INS-1 Story • Activation of GABAARs in beta cells regulates insulin secretion in concert with changes in glucose levels. A mechanism involving Ca2+ movement may underplay. • The GABA system may function as a negative feedback regulating mechanism in the islets.

  36. Insulin release glucose GLUT2 ATP/ADP Ca2+ K+ KATP Ca2+ hypoglycaemia Insulin release hyperglycaemia Insulin release (-) (+) GABA-GABAARs GABA-GABAARs

  37. Insulin Negatively Regulates GABAAR Function and Inhibits GABA- induced beta Cell Secretion

  38. GABA-evoked Currents (IGABA) is Inhibited by Insulin in INS-1 cells

  39. GABA-evoked Currents (IGABA) is Inhibited by Insulin but not Zn2+ in INS-1 cells

  40. Insulin-induced Inhibition on IGABA is PI3-K/Akt Independent Insulin receptor insulin membrane P P IRS active inactive DN: dominant negative PI3-K Wortmannin/ Ly294002 P Akt/PKB P inactive active

  41. Insulin does not Alter the Localization of GABAARs at the INS-1 Plasma Membrane Red: GABAAR beta2/3 Subunit Blue: DIPI

  42. Insulin Sigaling PD98059

  43. Insulin Suppresses IGABA via MEK/ERK Siganling Pathway

  44. Insulin Suppresses GABA-induced Insulin Secretion in INS-1 Cells

  45. Summary GABA - GABAARs (＋) (－) Insulin Insulin release (－) Physiological significance: a feedback mechanism for fine-tuning b-cell secretion Insulin may utilize GABA-GABAARs system to inhibit further release at the peak of the exocytotic event, particularly, at very high local insulin concentration.

  46. The Akt-GABAARs Pathway in Mediating Glucose- Induced Suppression of Glucagon Release in α Cells

  47. GABA Exerts Protective and Regenerative Effects onIslet Beta Cells and Reverses Diabetes

  48. GABAARs in T Lymphocytes

  49. Different Subtypes of GABA-A Receptors Are Expressed in Human, Mouse and Rat T Lymphocytes

  50. Different Subtypes of GABA-A Receptors Are Expressed in Human, Mouse and Rat T Lymphocytes Vh= -80 mV Vh= 60 mV “Tonic” currents

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