cGMP Flashcards
1
Q
What is cGMP derived from?
A
GTP
2
Q
How are most of cGMP’s effects mediated?
A
Through PKG
3
Q
cAMP can signal via Epacs. Does cGMP have an equivalent?
A
No
4
Q
Which enzyme synthesises cGMP?
A
Guanylyl Cyclase
5
Q
Guanylyl Cyclases are regulated by a diverse range of agonists. Give some examples.
A
- peptide hormones
- bacterial toxins
- free radicals (eg NO)
…also regulated indirectly by intracellular calcium
6
Q
GCs and cGMP-mediated signalling cascades play a central role in the regulation of a diverse range of physiological processes. Give 3 examples.
A
- vascular smooth muscle contractility
- intestinal fluid and electrolyte homeostasis
- phototransduction
7
Q
What are the two main families of Guanylyl Cyclases?
A
- Particulate GCs (membrane bound)
2. Soluble GCs (cytosolic/ NO-sensitive)
8
Q
Both GC families are ubiquitous. What does this mean?
A
They are found in (almost) every cell type
9
Q
Membrane bound (particulate) guanylyl cyclases exist in how many isoforms in mammals?
A
seven
10
Q
How many isoforms of pGC are there? How are they denoted?
A
- GC-A to GC-G
11
Q
What does pCG stand for?
A
Particulate guanylyl cyclase
12
Q
pGCs exhibit a highly conserved domain structure. Give 4 features.
A
- Extracellular binding domain at N-term
- Single TM domain
- Regulatory subunit with significant homology to protein kinases
- C-term catalytic domain
13
Q
Where is the catalytic domain located in pGCs?
A
In the C-teminus
14
Q
How do pGCs become catalytically active?
A
Dimerisation
15
Q
How does the catalytic domain of pGCs compare to that of ACs?
A
Structurally and functionally homologous
16
Q
Based on ligand specificities, pGCs have been classified into three classes. What are these?
A
- Natriuretic peptide receptors
- Intestinal peptide-binding receptors
- Orphan receptors
17
Q
Which class do pGC-A and pGC-B fall into?
A
The class of Natriuretic peptide receptors
18
Q
Which pGCs are in the natriuretic peptide receptor class? What are they activated by?
A
GC-A and GC-B
They are activated by ANP (atrial natriuretic peptide) and BNP (brain natriuretic peptide)
19
Q
Which pGC falls into the Intestinal Peptide-Binding Receptors class? What is it activated by?
A
GC-C
Activated by guanylin, uroguanylin
20
Q
Which class of receptors do the pGC-C, -D, -E, -F, and -G fall into?
A
Orphan receptors
21
Q
What is the structure of soluble GCs? (sGCs)
A
They are heteroDIMERIC proteins consisting of one alpha and one beta subunit
22
Q
How many isoforms of sGC exist?
A
Multiple: 3 alpha subunit isoforms and 3 beta subunit isoforms
23
Q
What do the alpha and beta subunits of GC consist of?
A
N-term regulatory domain (contains heme binding and dimerisation region)
C-term catalytic domain
24
Q
Which domain of sGCs contains the heme binding and dimerisation regions?
A
N-term domain (regulatory)
25
Where are sGCs expressed?
In the cytoplasm of almost all mammalian cells
26
sGCs mediate a wide range of physiological functions. Give 4 examples.
1. inhibition of platelet aggregation
2. relaxation of smooth muscle
3. neuronal signal transduction
4. immunomodulation
27
Which group is required by sGCs in order to be activated by NO?
Heme
28
Where does Heme bind on sGC?
To the regulatory N-terminal domain
29
Which two things are sGCs activated by? Which is more potent.
1. Nitric oxide (NO), a free radical
2. Carbon monoxide (CO)
NO IS FAR MORE POTENT
30
How does NO activate sGCs?
By binding directly to the heme group
31
Give an example of a receptor associated with both PKA and PKG (in different cell types)
IP3
32
Which enzyme mediates the majority of the cellular effects of cGMP?
PKG (serine/threonine kinase)
33
How many classes of PKG exist?
2
34
How do Type I and Type II PKGs differ in expression?
Type I expressed in many cell types
| Type II less common and only expressed in INTESTINE, KIDNEY, BRAIN
35
Type I PKGs are expressed in many cell types. Type II PKGs, however, are only expressed in 3 organs. What are these?
Intestine
Kidney
Brain
36
How many isoforms of Type I PKG exist?
2 (Type I-alpha, and Type I-beta)
37
Type I PKGs exist as homodimers. Which 3 domains does each subunit contain?
1. a dimerisation domain
2. 2 x cGMP-binding domains
3. an autophosphorylation-autoinhibitory domain
38
Where in the cell are Type I PKGs located?
In the soluble fraction
39
Type I PKGs are homodimeric. What are Type II PKGs?
Monomeric kinases
40
What is the structure of Type II PKGs?
1. catalytic domain at C-terminal
2. 2 x cGMP-binding domains at N-terminal
3. generally membrane-bound
41
Where in the cell are Type II PKGs usually found?
Bound to the membrane
42
Give 4 examples of proteins regulated by PKGs
1. IP3 receptor
2. IRAG
3. Calcium-activated maxi K+ channel
4. PP1M (the myosin light chain phosphatase)
43
How does PKG regulate the IP3 receptor?
Causes calcium release from internal stores
44
What is the result of PKG regulation of IRAG?
Inhibits calcium release from internal stores
45
What does IRAG stand for?
IP3-Receptor Associated cGMP kinase substrate
46
PKG has not been found to regulate transcription factors. What is the proposed reason for this?
Too large to enter the nucleus
47
Whilst PKG is too large to enter the nucleus and interact with transcription factors, it is able to affect gene transcription indirectly. How?
By phosphorylating cytoplasmic proteins
48
In addition to PKG, give two other effectors of cGMP
CNG channels
| Phosphodiesterases
49
CNG channels can be activated by different cyclic nucletide to regulate 1) visual transduction and 2) olfaction. Which cyclic nucleotide is each CNG isotope more sensitive to?
1) visual transduction - cGMP
| 2) olfaction - cAMP
50
Which superfamily of ion channels do CNG channels belong to?
Voltage-gated ion channels
51
What are HCN channels?
Hyperpolarisation-activated Cyclic Nucleotide-gated channels
52
Hyperpolarisation-activated Cyclic Nucleotide-gated (HCN) channels are a subfamily of which class of channels?
CNG channels
53
In which two organs are HCN channels expressed?
Brain and Heart
54
How are HCN channels modulated?
1. binding of cyclic nucleotides
| 2. hyperpolarisation
55
Give an example of the function of HCNs.
In pacemaker currents in the sinoatrial (SA) Node
56
How many different families of PDEs exist in mammals?
At least 11
57
How many PDE enzymes exist in humans?
>30
58
Which PDE families hydrolyse:
1. BOTH cGMP and cAMP
2. cAMP only
3. cGMP only
1) 1, 2, 3, 10, 11
2) 4, 7, 8
3) 5, 6, 9
59
Which families of PDEs hydrolyse both cGMP and cAMP?
1, 2, 3, 10, 11
60
Which families of PDEs hydrolyse only cGMP?
5, 6, 9
61
Which families of PDEs hydrolyse only cAMP?
4, 7, 8
62
Give three main factors that can regulate phosphodiesterases
1) substrate availability (e.g. alteration in the rate of hydrolysis of one cyclic nucleotide because of competition by another)
2) intracellular signalling events (e.g. phosphorylation, Ca++)
3) feedback regulation
63
How are PDEs regulated by intracellular signalling events?
1) PDE3 stimulated by insulin
2) PDE6 stimulated by photons through transducin system
3) PDE1 stimulated by Ca++/calmodulin
64
Which PDEs are phosphorylated (and thus regulated) by PKA after cAMP regulation?
PDE1
PDE3
PDE4
65
How does cAMP elevation affect PDEs?
PDE1, -3 and -4 are phosphorylated by PKA after cAMP elevation
66
How does cGMP promote its own breakdown after its elevation?
It binds to allosteric sites on PDE2
67
Which PDE is critical in photo transduction, and which cyclic nucleotide does it act on?
PDE6
| controls cGMP
68
The tone of smooth muscle depends upon a balance between contractile and relaxation mechanisms. Which signalling pathway plays a fundamental role in controlling relaxation?
The NO/cGMP signalling pathway
69
How does the NO/cGMP pathway control relaxation of smooth muscle cells?
By acting at a number of sites concerned with calcium-signalling
70
How do exagenous and endogenous compounds such as nitro-vasodilators, NO and natriuretic peptides produce vasodilation?
Through increases in cGMP
71
What is the effect of cGMP activation of PKG on vascular smooth muscle? How>
It relaxes it by:
1) reducing intracellular calcium and promoting the activity of myosin light chain phosphatase (MLCP) through a direct phosphorylation by PKG, and
2) inhibiting RhoA (which inhibits MLCP through activating ROCK)
3) activates RGS2 which inhibits Galphaq signalling
72
Which RGS protein inhibits G-alpha-q signalling?
RGS2
73
How does activation of PKG by cGMP result in inhibition of G-alpha-q signalling?
It activates RGS2
74
How does inhibition of RhoA help to relax smooth muscle?
Because RhoA inhibits MLCP through the action of the protein ROCK
75
What is the main mechanism through which cGMP reduces Ca++?
It activated PKG, which phosphorylates target proteins
76
cGMP reduces Ca++ principally through the action of PKG phosphorylation of target proteins. What three things does this result in?
1) inhibition of Ca++ influx through L-type calcium channels (by activating BK(Ca) channels)
2) increased Ca++ efflux through activation of Ca2+-pumping and Na+/Ca2+ exchanger
3) decreased Ca2+ mobilisation through inhibition of the IP3 receptor in the sarcoplasmic reticulum by activation of IRAG
77
What does IRAG stand for?
IP3 Receptor-Associated PKG substrate
78
What is the commercial name for sildenafil citrate?
Viagra
79
What is viagra used to treat?
Male erectile dysfuction (impotence) and pulmonary arterial hypertension
80
What does the physiological process of erection involve?
The release of nitric oxide (NO) in the corpus cavernosum of the penis resulting in smooth muscle relaxation and an increased inflow of blood
81
How does nitric oxide cause relaxation of the smooth muscle of the penis? (i.e. result in an erection)
1. NO activates GC
2. GC produces an increase in [cGMP]i
3. cGMP reduces [Ca2+]i (mainly through PKG phosphorylation of target proteins)
4. cGMP desensitises the contractile apparatus to Ca2+
5. blood flows into the penis and it becomes erect
82
How does Sildenafil (VIAGRA) work?
It enhances the level of cGMP
83
How does viagra enhance the level of [cAMP]i?
Inhibiting the cGMP-specific PDE5, which hydrolyses cGMP to 5'GMP
84
Which enzyme does Viagra inhibit to enhance levels of cGMP?
PDE5
85
How does sildenafil (viagra) inhibit PDE5?
It has a structure very similar to cGMP, and acts as a competitive inhibitor
86
What is the structure of PDE5?
It is a homodimer with each subunit containing:
1) a regulatory phosphorylation site (Ser92),
2) two allosteric cGMP binding sites,
3) two Zinc-binding motifs, and
4) a catalytic binding site
87
Which residue is phosphorylated in the regulatory site of PDE5?
Ser92
88
How many allosteric cGMP binding sites are there in PDE5?
2
89
How many zinc-binding motifs are there in PDE5?
2
90
Which cyclic nucleotide does PDE5 act on?
cGMP
91
What is necessary for Ser92 phosphorylation of PDE5 by PKA/PKG?
Occupation of the two allosteric cGMP binding sites
92
Which residue is phosphorylated by PKA/PKG on PDE5?
Ser92
93
How does phosphorylation of Ser92 affect PDE5 activity?
It increases enzyme activity by 50-70%
94
Where is the catalytic domain of PDE5 located?
C-terminus
95
At high doses, viagra can inhibit another enzyme in addition to PDE5. Which is this?
PDE6
96
Which enzyme can be inhibited (in addition to PDE5) by high doses of viagra? Where is this enzyme located?
PDE6. It is found in the retina
97
In addition to male erectile dysfunction (impotence), what has Viagra been approved for the treatment of?
Pulmonary arterial hypertension
98
What does 'Matching' describe?
Normal lung function requires blood to be directed to well ventilated areas of the lungs to ensure optimal gas exchange ('Matching')
99
Normal lung function requires blood to be directed to well ventilated areas of the lungs to ensure optimal gas exchange. What is this called?
Matching
100
What is lung vessel dilation mainly regulated by?
NO/cGMP
101
During disease conditions, malfunction of vasodilation of lung vessels can cause 'Mismatch'. What does this mean?
Blood is directed to poorly ventilated areas
102
Which PDE is highly expressed in lungs?
PDE5
103
What effect can sildenafil have in the lung? Why?
Anti-pulmonary hypertensive effect by preferentially dilating vessels in well oxygenated areas of the lung through targeting PDE5
104
Why is sildenafil sometimes referred to as a 're-matching' drug?
It acts on PDE5 to preferentially dilate vessels in well oxygenated areas of the lung
105
What does cGMP stand for?
cyclic guanosine monophosphate
106
NO has multiple biological effects. List 4.
1. nitrosylation of proteins
2. direct toxic effects
3. induction of protein ADP-ribosylation
4. stimulation of soluble guanylyl cyclase
107
Which type of enzyme does NO act on?
SOLUBLE guanylyl cyclase
108
Which enzyme synthesises NO?
Nitric oxide synthase (NOS)
109
How many types of nitric oxide synthase are there?
3
110
What are the different types of nitric oxide synthase?
iNOS: inducible (toxic)
eNOS: endothelial (signalling)
nNOS: neuronal (signalling)
111
Which NOS produces HIGH NO concentrations that can exhibit direct toxic effects?
iNOS (INDUCIBLE NOS)
112
How does NOS produce NO?
It catalyses oxidation of an amidine nitrogen of L-arginine, generating NO and citrulline
113
NOS produces NO from L-arginine. What else is produced?
Citrulline
114
How does NOS change L-arginine to make NO?
Oxidises an amidine nitrogen
115
How does PKG Type I differ from PKG type II?
1) homodimeric vs monomeric
2) ubiquitous vs intestine/kidney/brain
3) diffuse in soluble part of cell vs membrane-bound
116
Give an example of NO/GC/cGMP/PKG in action
Vasoregulation
117
How does calcium regulate vascular tone?
1) IP3 produced and calcium released from intracellular stores, followed by external calcium entry
2) rise in calcium activates calcium/calmodulin dependent MLCK (phosphorylates MLC resulting in activation of myosin ATPase)
3) fall in calcium inactivates MLCK and causes dephosphorylation of MLC by MLCP (aka PP1M)
118
What is the effect of a rise in calcium on myosin light chain kinase (MLCK)? How is this mediated?
The rise in calcium activates calcium/calmodulin-dependent MLCK
119
What is the effect of Ca/calmodulin-dependent MLCK activation?
Phosphorylation and thus activation of myosin ATPase
120
How does a fall in Ca result in dephosphorylation of MLC (and thus inactivation of myosin ATPase)?
Inactivated MLCK which causes MLCP (PP1M) to dephosphorylate MLC
121
What effect does PKG have on intracellular calcium levels?
Decreases, i.e. through activation of RGS2, which attenuates Galpha(q) signalling
122
Where on PDE5 does viagra bind?
at the CATALYTIC not the allosteric sites (competes with the binding of cGMP)
123
Viagra prompted other companies to produce alternative drugs. Give two examples.
Cialis - long-lasting effect
| Levitra - works in less time than Viagra
124
Viagra has been proven to be effective in treating male impotence and pulmonary arterial hypertension. What else can it be used for?
Altitude sickness
