Dr Anja Teschemacher Flashcards

Question

Angiotensin II

Answer 1

Chromaffin cell AngII receptors couple to multiple G-protein pathways Results show that activation of multiple types of G-proteins + their pathways by a single modulator acting through a single receptor (ATR I) can produce concentration-dependent, bi-directional regulation of exocytosis! Low concentration = AngI receptor, Gi/o, inhibit VGCC current = inhibit exocytosis High concentration = AngI receptor, Gq, increase IC [Ca], increase capacitance = promote exocytosis -requires Ca release from IC stores

Answer 2

BIS/CalC = PKC blocker (catalytic/regulatory domain) PMA phorbol esters = PKC activator (DAG analogue) ``` CPA = blocks Ca release from IC stores Caffeine = high concentrations can release Ca from IC stores ```

Answer 3

Chromaffin cell Gq pathway which engages munc13 to increase stimulus-coupled secretion by recruiting vesicles to the RRP GPCR is therefore able to regulate exocytosis at the level of the SNARE complex to produce rapid STP of the secretory output ``` H1 receptor = Gq Potentiates exocytosis (increase capacitance), but decreases Ca ``` Activation of Gq, but DAG does not activate PKC - it activates Munc13 - Activation by PMA phorbol esters/DAG - CalC blocks (regulatory domain: PKC + Munc13) - BIS does not block (catalytic domain of PKC) - Therefore not PKC dependent Activation of Munc13 = primes vesicles by regulating Munc18 binding to syntaxin's closed conformation Histamine - increase the size of the RRP of vesicles!

Answer 4

Angiotensin II - ATR I Low = Gi/o - decrease exocytosis High = Gq - increase exocytosis Activation of multiple types of G-proteins/pathways by a single modulator acting at 1 receptor can produce concentration-dependent bi-directional regulation of exocytosis Histamine - HI R - Gq Activation increases the activity of Munc13 (via DAG; PKC-independent) - GPCR is therefore able to regulate exocytosis at the level of the SNARE complex to produce rapid STP of the secretory output Both act to increase the size of the RRP by shifting vesicles from the reserve pool --> RRP (primed)

Answer 5

Electrochemical detection of catecholamine release in real-time Dopamine, adrenaline, noradrenaline - oxidised at positive potentials Carbon fibre electrode charged to positive potential - close to release sites (~5um) - detection of catecholamine release in 'diffusion spikes'

Answer 6

~200-500nm = larger pores, therefore pore diameter can be regulated Capable of kiss + run fusion Amperometry - see 'foot signals' = a fraction of the vesicle content is released through the unstable fusion pore before pore closure or collapse Dynamin regulates fusion events! Dynamin - normally phosphorylated High stimulation - calcineurin is activated = deP dynamin, reveals binding sites for accessory proteins Recruits myosin II = dilates the fusion pore to facilitate peptide release LINK SCB: Dynamin = involved in CME (GTPase; PICK1 binding stimulates polymerisation + pinching off the newly forming vesicle) Myosin = MyoVb involved in post-synaptic exocytosis of AMPARs; Ca binds (sedimentation assay: folded-to-extended), exposes binding domains for RabII = recruit recycling endosomal into the dendritic spine

Answer 7

SCV - mainly full fusion LDCV - both! Kiss + run fusion = increases with age Age - the kinetics of full fusion and kiss + fun fusion differ with age - alter differentially - implies that there are different regulatory mechanisms

Answer 8

~50nm = very small therefore always expand to full fusion | Small pore - due to the physics of membrane curvature, most are full fusion

Answer 9

Treatment = L-DOPA - enhanced DAergic neurotransmission via increased pool of vesicular dopamine (RRP) = increased release events seen on amperometry VMAT2 = drug target for enhanced DAergic transmission - either increasing VMAT2 or enhancing current protein VMAT2 dysfunction has been identified in PD brains! Reserpine PD-model = bind to VMAT2 and reversible inhibit its' function

Answer 10

Detection of single secretory vesicles in individual cells Positively charged carbon fibre except 5um tip! Close proximity required to pick up diffusion of transmitters when it is released via exocytosis Tracer of release events = higher spike, larger area under diffusion curves, more catecholamine released

Answer 11

RVLM = rostral ventrolateral medulla - A2 + C1 region Electron microscopy = larger size of vesicles in C1 Microamperometry = larger quanta of catecholamines released per exocytic event in C1 neurones Electrophysiology = low AngII (Gi/o; decrease IC [Ca]) - less inhibition of Ca influx therefore less inhibition of exocytosis Leads to increased sympathetic activity = increase blood pressure in SHR!

Answer 12

NAergic neurones in A1, A2 + OSC Noradrenaline in peripheral noradrenergic nerves is released exclusively from LDCVs by an exocytotic mechanism TTX (VGSC blocker) eliminated majority of exocytic events - mainly, but not entirely, AP driven SCV, DCV, LDCV Main population = SCV + DCV

Answer 13

High neuronal activity = increased K+ uptake (depolarise) + increased glutamate (EAAT1/2) Trigger glyogenolysis (glycogen --> glucose) Glucose --> pyruvate --> lactate Lactate released via MCT1 (astrocyte) Lactate taken up via MCT2 (neurones) Oxidised to pyruvate = enter Krebs cycle for ATP! EC [lactate] increases with stimulation ***MCT used H+ as a co-substrate!

Answer 14

Hypothalamus Adrenaline binds to alpha-1 receptors on astrocytes - release ATP - bind to P2X7 receptors on glutamatergic cells, cause an increase in AMPAR insertion Gliotransmitter contributes directly to the regulation of postsynaptic efficacy at glutamatergic synapses in the CNS

Answer 15

Glutamate Exo = BoTx/tetanus application decreased Ca-dependent glutamate release or Activation of P2X receptor, increase [Ca], activate VRAC receptor, release glutamate ATP Hemichannel blocker inhibits ATP release in astrocytic hippocampal slice TIRF = observation of exocytosis of secretory lysosomes following activation

Answer 16

SNAP-25 (neurone) + SNAP-23 (astrocyte) Synaptotagmin-1 (neurone) + synaptotagmin 4, 7 or 11 (astrocyte) Synaptobrevin-2 (neurone) + synaptobrevin-3 (astrocyte) Slower kinetics in astrocytes - involves GPCR signalling + IP3-mediated Ca release from IC stores!!! (Neuronal = VGCCs)

Answer 17

Synaptotagmin in astrocytes Synaptotagmin 4 K/O = reduced SNARE-mediated exocytosis of glutamate BUT mRNA expression study (qRT-PCR) = astrocytes express little synaptotagmin 4 and lots of synaptotagmin 11 BUT - mRNA expression does not equal protein expression Maybe look at Western blots?

Answer 18

Astrocyte connected cell network via gap junctions - each astrocyte contributes a hemichannel (6x connexins; gap junction = 2 hemichannels) IC signalling network - propagate Ca waves throughout the network Paracrine via ATP release (exocytic??) - acts via gap junctions Highly controversial field

Answer 19

Astrocytes in the cortex can modulate the inhibitory activity of neurones in the neocortex Astrocytes release ATP, bind to P2X receptors, cause a decrease in IPSP in pyramidal neurones Down-regulation of inhibitory synaptic currents (IPSPs) in the neocortex due to astrocytic ATP release binding to P2XRs

Answer 20

Lactate released via MCT1 from astrocytes - neurones take it up via MCT2 - enter Krebs cycle for ATP In vivo: Disrupt MCT1 = causes amnesia; rescued by EC lactate Disrupt MCT2 = causes amnesia; cannot be rescued by EC lactate Lactate shuffle = important for L+M - long-term memory formation and maintenance of LTP (disrupt = amnesia) Disrupting the expression of astrocytic MCT1 = causes amnesia + LTP impairment

Answer 21

Age-related disease in astrocytic Ca signalling - decrease in exocytosis of gliotransmitters, particularly ATP Cortical astrocytes - decrease of astrocytic modulation of synaptic transmission (ie. modulating fast synaptic transmission) could contribute to age-related impairment synaptic plasticity and cognitive impairment

Answer 22

Ventral medulla surface (VMS) in brainstem Astrocytes = chemosensitive; blood acidosis = activate paracrine ATP signalling in astrocytes (ATP binds P2X/YRs, IC [Ca], release ATP) ATP activates phrenic nerve/central inspiratory drive = activate diaphragm muscles (major respiratory muscle) = increase breathing! Experimentally: In vivo artificially anaesthetised + hyperventilated rat Optogenetically stimulate astrocytes - activates phrenic nerve activity = increase breathing

Answer 23

Astrocytes release lactate (MCT1) Lactate shuffle not involved! Bind to Gs-coupled receptors on NAergic neurones in the locus coeruleus - release noradrenaline = NAergic input into the cortex - arousal, alertness, motivation, sleep/wake cycle Micro-injections of lactate into LC = arousal in anaesthetised rats - Increased arterial blood pressure - Power changes in the cortex (sleeping state --> awake state) Lactate = acting as a gliotransmitter to control arousal!

Answer 24

Brainstem hypoxia contributes to the development of hypertension in SHR Hypoxia = lower O2 in brainstem Astrocytes = activated (low pH) - increased paracrine ATP signalling (Ca waves) Increased release of lactate Act as a gliotransmitter to increase central inspiratory drive (via increasing phrenic nerve activity) to increase breathing Increased sympathetic drive Increased exocytosis in RVLM: -Larger LDCV (EM) + catecholamine release (microamperometry) = larger NA release = increased BP!!!

Answer 25

SNAREs do not form a complex/zipper before the Ca signal arrives Ca signal triggers cross-linking of the vesicle + PM = close contact! Close proximity allows for rapid binding of vSNAREs + tSNAREs Nucleation triggered = SNAREs zipper + fusion/exocytosis occurs Entire control of fusion is up-stream of SNARE nucleation - no pausing of the highly exergonic process (the mechanism of complexin arresting SNARE zippering in the middle is difficult to understand because SNARE assembly precedes along a steep downhill energy gradient; strongly exergonic reaction) SUPPORT: Simple model - therefore highly conserved throughout evolution Mutated SNAREs affect zippering due to increases in 'misfiring' - assembly without fusion Explains why sucrose triggers Ca-independent exocytosis of RRP - Sucrose causes water efflux = negative pressure in the cell - Negative pressure draws docked vesicles closer to the plasma membrane = triggers SNARE firing

Answer 26

Ca binds, causes an "electrostatic switch" - shield negatively charged residues on the phospholipid binding site, therefore facilitating binding to negatively charged phospholipids