NO Signalling In The CNS

Question 1

What are the different types of nitric oxide synthase (3 genes)

Answer 1

eNOS - endothelial involved in vascular tone
nNOS - within neurons
iNOS - involved in inflammation induced by cytokines

Question 2

Characteristics of nNOS

Answer 2

Associated with NMDAR
needs calcium/ calmodulin to activate
Involved in synaptic plasticity

Question 3

What NOS are regulated by calcium calmodulin

Answer 3

eNOS And nNOS

Question 4

Characteristics of iNOS

Answer 4

Membrane bound
Make up to 1000 items more NO than eNOS
Involved in inflammation

Question 5

What happens to LTP when you KO nNOS

Answer 5

It is attenuated

Question 6

Different domains of nNOS

Answer 6

N terminal to C terminus

Oxygenase domain (contains arg, heme and BH4)
Reductase domain ( contains FAD and NADPH)

May/ may not have PDZ domain

Question 7

What are the splice variants of nNOS and how do they differ?

Answer 7

Alpha, beta, gamma and mew

Have various domains due to varying length
E.g. Only alpha has a PDZ domain (located on N terminus)
Gamma is probably inactive due to partial oxygen domain

Have different subcellular locations as a result alpha at membrane
Beta cytoplasmic

Question 8

Is NOS a monomer, dimer or trimmer

Answer 8

It is a HOMOdimer

Question 9

Why is calcium calmodulin important?

Answer 9

It completes the electron transport chain from the reductase domain to the oxygenase domain also increase the rate of ETC

Question 10

Equation for NO synthesis

Answer 10

Arginine + O2 -> citrulline + NO

Question 11

Functions of NO

Answer 11

Stimulates guanylyl cyclise (soluble)
Nitrosylation of proteins
Free radical formation

Question 12

Soluble guanylyl cyclase contains a heme group…why?

Answer 12

Allows NO to bind to regulatory domain

This causes conformational change which activates the catalytic domain to make cGMP (100-200 fold increase in activity)

Question 13

What can block the function of nNOS?

Answer 13

L-NAME (competitive antagonist of arginine)

Question 14

Name a NO donor which can be used experimentally to mimic the function of NO.

Answer 14

Sodium nitroprusside (SNP)
Broken down in cells to release NO

Question 15

What inhibits sGC?

Answer 15

ODQ

Question 16

What stimulates sGC?

Answer 16

Bay 41-2272

Question 17

Addition of heme experimentally does what to NO?

Answer 17

Decreases it as it will chelate the NO

Question 18

How can we increase the levels of cGMP?

Answer 18

Stimulate production via Bay 41-2272 OR

decrease degradation via inhibition of phosphodiesterase (sildenafil will inhibit PDE5)

Question 19

List the characteristics of a conventional neurotransmitter

Answer 19

Manufactured in cell
Packed as stored in vesicle
Releases of calcium dependant
Acts on receptors on post synaptic memebrane
Exocytosis
Inactivated by reuptake or enzyme activity

Question 20

Why is NO an unconventional NT?

Answer 20

Not packed into vesicles
Cannot be stored
Non released by exocytosis
Although calcium needed for synthesis has nothing to do with release
No specific inactivation system
Very short half life
Receptor is a soluble protein is cytoplasm

Question 21

What does the term ‘volume transmitter’ mean?

Answer 21

Can act as a neurotransmitter in a volume of tissue and not isolated to the post synaptic neuron

Question 22

As a volume transmitter what can NO do?

Answer 22

Retrogradely influence pre synaptic cell (pre synaptic LTP)
Act in neighbouring cells not within its the connectome
Cell trafficking
Gene expression
Nitosylation
Integrate neuronal activity over a limited diffusion space

Question 23

Is nNOS present in the auditory brainstem?

Answer 23

Yes (immunohistochemicak studies say so)

Question 24

What does a synaptic stimulus protocol of the calyx of held do to k+ Kv3 currents on the medial nucleus of the trapezoid body?

Answer 24

It decreases it

As Kv3 is HVA this only occurs at high mV’s

Question 25

What is the synaptic stimulus protocol

Answer 25

60s at 100HZ (50% duty cycle) | Approx 3000 stims per min

Question 26

What happens in an neurone following SSP of a unconnected neurone?

Answer 26

There is a similar decrease in Kv3 activity | Presumably there is a diffusable NT which modulates this unconnected neurone

Question 27

In the connected model of the calyx and MNTB what is the effect of SSP in the presence of 1400w?

Answer 27

No decrease in Kv3 mediated currents as 1400w inhibits nNOS therefore no NO

Question 28

What is the effect of adding DEA to an unconnected neurone?

Answer 28

A similar decrease in kv3 current is seen similar to that of an unconnected neurone following an SSP. Since DEA is a NO donor SSP must have the effect of increasing NO in the unconnected cell

Question 29

Name a Kv3 blocker

Answer 29

Tetraethylammonium (TEA)

Question 30

What happens to Kv3 current on addition of TEA compared to addition of NO donor SNP? Why is this important?

Answer 30

They both show a similar decrease in current. | As TEA at low concentrations is selective for Kv3 it shows that the NO is acting to block Kv3 channels

Question 31

What does 10Hz synaptic stimulation do over 1 hour?

Answer 31

Increases Kv current

Question 32

What happens to Kv current within 20 minutes of NO donor exposure

Answer 32

Kv3 current will decrease

Question 33

What will happen to Kv2 current after NO donor exposure of 1 hour

Answer 33

Kv2 current will increase k+ current to above control (similar pattern to synaptic stimulation for 1 hour)

Question 34

Is TEA is applied at 1 hour following NO donor stimulation what will have to the voltage current relationship

Answer 34

It will stay the same as current mediated by Kv2 which is not blocked by TEA Is inhibited by r-stromatoxin-1 which blocks Kv2 channels

Question 35

Which kinase phosphorylation Kv3 channels

Answer 35

PKC

Question 36

What does Kv3 phosphorylation do to channel conductance?

Answer 36

Decreases conductance | Therefore blocking PKC will increase Kv3 current

Question 37

Over a period of time what happens to Kv currents

Answer 37

Kv3 decreases and Kv2 increases

Question 38

What happens to Kv3 phosphorylation over prolonged period of inactivation?

Answer 38

Becomes more phosphorylated and therefore decrease in activity

Question 39

What happens to phosphorylation of Kv2 over prolonged activation?

Answer 39

It increases This causes and increase in activity Increase in NO over long period = increase in PKG = phosphorylation of Kv2 = increased activity

Question 40

What is the advantage of Kv2 over Kv3?

Answer 40

Kv2 is able to fire at a sustained high frequency for a long period of time, Kv3 is not. Therefore Kv2 takes over for k+ current for prolonged synaptic activity

Question 41

How does the switching of Kv3 to Kv2 related to hearing?

Answer 41

Hypothesis: moderate activity Kv3 = predominant as it is dephosphorylated For prolonged periods of time (high frequency) NO activity is greater than phosphatase activity therefore phosphorylation predominates and Kv3 and Kv2 is phosphorylated which activates Kv2 and inhibits Kv3