Transcript Peripheral GABAARs: View from the Liver, Lung, Pancreatic

Peripheral GABAARs: 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
GAD: glutamic acid decarboxylase
Glutamate
GAD65/67
GABA-T: GABA-transaminase
GABA-T
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)
GABA
Succinic
semialdehyde
GAT
GABA
GABABR is a type of G-protein coupled
receptors (GPCR)
phasic
tonic
ClGABAAR
ClGABABR
Characteristics of GABAARs
extracellular
intracellular
Critical Reviews in Biochemistry and
Molecular Biology, 42:3–14, 2007
Synaptic and Extrasynaptic GABAAR Currents
Glutamate
GABA
GABA
Valerie B. etc. PNAS, 2004
extrasynaptic
tonic
Cl-
synaptic
M. Wallner etc. PNAS, 2003
Cl-
phasic
Modulation of GABAARs
• Agonists: GABA and muscimol
• Antagonist: bicuculline (competitive inhibition)
picrotoxin (non – competitive inhibition)
• Benzodiazepines, pentobarbital, and ethanol
• Neurosteroids and endocannabinoid 2arachidonoylglycerol (2-AG)
• Ions: Zn2+, Mn2+, Co2+
• Posttranslational modification: protein kinases
• Receptor associated proteins:
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
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
Example 1: Benzodiazepines Regulation of GABAARs
Example 2: Ethanol Regulation of GABAARs
M. Wallner etc. PNAS, 2003
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
GABA and the Lung Story
GABAARs in airway epithelium
Expression of GABAARs in airway epithelium
Electrophysiological characteristics
Physiological and pathophysiological significance
Expression of GAGAARs in lung epithelial cells
Electrophysiological characteristics of
GABAARs in lung epithelial cells
Perforated
Whole- cell
Cl-
sweep
Voltage - clamp recording Current - clamp recording
Vholding = -60 mV
Electrophysiological characteristics of GABAARs
in lung epithelial cells
Voltage - clamp recording
Vholding = -60 mV
Electrophysiological characteristics of
GABAARs in lung epithelial cells
Voltage - clamp recording (Vstep)
Vholding = from -60 to 40 mv, △ v = 20 mv, totally 6 sweeps
Electrophysiological Characteristics of
GABAARs in Lung Epithelial Cells
Perforated
Cl-
X
Voltage - clamp recording
Current - clamp recording
Physiological or Pathophysiological
Significance of GABAARs in Lung Epithelium
Human
Mice
GAD and β2/3 Subunit Upregulation in Asthmatic
Lung in Mice and Human
IL-13 Mediates the Upregulation of GABA
Signals in Asthmatic Mice Lung
In vitro
GAD65/67
β2 subunit
In vivo
IL-13 Mediates the Up-regulation of GABA
Signals in Asthmatic Mice Lung
Picrotoxin (PTXN) Protects the Lung against
OVA-induced Asthma in Mice
Blocking GABAARs does not Affect OVA-induced
Inflammation and Hyperreactivity in the Airway
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.
GABA and the Pancreas Story
Expression of GABAARs in INS-1 cells
GABA-evoked Currents in INS-1 cells
Perforated
Whole- cell
GABA-evoked Currents in INS-1 cells
0 mmol/l glucose
16.7 mmol/l glucose
Effect of GABAAR activation on the
Excitability of INS-1 β cells
Reversal potential of IGABA: -43 mV
Clinical Pharmacology &
Therapeutics, 2008
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
GABAAR
Driving force
GABAAR
Cl-
Driving force
inside
Cl-
Effect of GABAAR Activation on the
Excitability of INS-1 β cells
Modulation of Ca2+ infux by GABA in INS-1 cells
Regulation of Insulin Secretion by GABA is dependent
on Glucose Concentration
Lower glucose
Higher glucose
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.
Insulin release
glucose
GLUT2
ATP/ADP
K+
KATP
hyperglycaemia
Insulin release
(-)
GABA-GABAARs
Ca2+
Ca2+
hypoglycaemia
Insulin release
(+)
GABA-GABAARs
Insulin Negatively Regulates GABAAR Function and
Inhibits GABA- induced beta Cell Secretion
GABA-evoked Currents (IGABA) is Inhibited by
Insulin in INS-1 cells
GABA-evoked Currents (IGABA) is
Inhibited by Insulin but not Zn2+ in INS-1
cells
Insulin-induced Inhibition on IGABA is
PI3-K/Akt Independent
Insulin receptor
insulin
membrane
P
P
IRS
DN: dominant negative
inactive active
PI3-K
Wortmannin/
Ly294002
P
Akt/PKB
inactive
P
active
Insulin does not Alter the Localization of GABAARs at
the INS-1 Plasma Membrane
Red: GABAAR beta2/3 Subunit
Blue: DIPI
Insulin Sigaling
PD98059
Insulin Suppresses IGABA via MEK/ERK Siganling
Pathway
Insulin Suppresses GABA-induced Insulin
Secretion in INS-1 Cells
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.
The Akt-GABAARs Pathway in Mediating GlucoseInduced Suppression of Glucagon Release in α Cells
GABA Exerts Protective and Regenerative Effects on
Islet Beta Cells and Reverses Diabetes
GABAARs in T Lymphocytes
Different Subtypes of GABA-A Receptors Are Expressed in
Human, Mouse and Rat T Lymphocytes
Different Subtypes of GABA-A Receptors Are Expressed in
Human, Mouse and Rat T Lymphocytes
Vh= -80 mV
Vh= 60 mV
“Tonic” currents
An Intrinsic GABAergic System in Human Lymphocytes
(10 mM)
(10 mM)
Whole cell currents,
Vh = -50 mV
GABA Channels are Activated with
1M GABA+ 100 nM THDOC on CD8+ DR+/+ T Cells
Single-channel currents
GABA Channels are Activated with
1M GABA/100 nM THDOC on CD4+/CD8+ DR+/+ T Cells
GABA
GABA +THDOC
GABA
Single-channel currents
CD8+ DRlyp/lyp T cells but not CD4+ DRlyp/lyp T cells
Express Higher Level of GABAA Channel Subunits
DRlyp/lyp: diabetic prone rats
DR+/+: diabets resistant rats
CD8+ DRlyp/lyp T Cells Express More GABAAR Subunits
as Compared with CD4+ DRlyp/lyp T cells
DRlyp/lyp: diabetic prone rats
DR+/+: diabets resistant rats
CD4+ and CD8+ T cell Proliferation is
Decreased by Sub-M GABA Concentration
CD8+ DRlyp/lyp T cells
?
Conclusions
• Animals that do develop type-1 diabetes do express higher levels of GABAA
channel subunits in CD8+ T cells than their wild-type counterparts.
• GABA in the islets of Langerhans may diminish inflammation by inhibition of
activated T lymphocytes
• Our results raise the question of whether an enhanced GABAA channel
activity might delay or possibly prevent the development of type-1 diabetes.