1. Enzyme linked - plasma membrane surface and cytoplasmic - receptors guanylyl cyclase

3. ANP: Atrial natriuretic peptide

4. cGMP intracellular mediator (1)
1. Plasma membrane receptor with guanylyl cyclase activity
(ANP: atrial natriuetic peptide)
vasodilatation, diuresis, Na uresis,
inhibition of aldosterone secretion
2. Cytosol guanylyl cyclase
(NO)

5. cGMP intracellular mediator (2)
effectors:
Protein kinase G (cGMP dependent protein kinase)
e.g. phosphorylation of IP3 receptor – inhibition,
decrease in Ca2+ concentration in smooth muscle cells
cGMP activated cAMP phosphodiesterase

6. Rhodopsin receptor, a G-protein coupled receptor
similar in structure
differ greatly in amino acid sequences
seven (7) transmembrane alpha helices
alternating cytoplasmic and extracellular loops
N-terminus is extracellular
C-terminus is cytoplasmic
extracellular domain has a unique messenger (ligand) binding site
cytoplasmic domain has a binding site for a specific G-protein
GPCR G protein coupled receptor
X-ray structure of rhodopsin

7. Example of GPCR - rhodopsin
Vertebrates – 2 kinds of photoreceptor cells – rods (function in dim light) and cones (colors recognition).
Rods – receptor is rhodopsin
3 different types of cone cells make slightly different receptors (photopsins) that absorb blue, green or red.
Photoreceptors cells:
photons →conformational change → nerve impulse
rhodopsin 40 kDa transmembrane protein
opsin – protein
11-cis-retinal – prosthetic group (derivate of vitamin A (all trans retinol))
11-cis retinal has broad absorption spectrum with maximum around 500 ns (500 nm = M-1cm-1)
11-cis-retinal

8. Metarhodopsin activates G protein transducin.
Transducin activates enzyme phosphodiesterase (PDE)
phosphodiesterase breaks down cGMP (3', 5' guanosine monophosphate) in cell to 5' GMP
cGMP is ligand for cyclic nucleotide gated Na+ channels in cell membrane, if cGMP present channels open, influx of cations, mainly Na+, but also Ca2+ , when cGMP broken down, closure of Na+ channels reduction in Na+ flow into cell
The rod cell membrane is hyperporized (more negative potential across membrane) and sends a visual signal into a brain

9. Amplification of signal
1 molecule of rhodopsin absorbs 1 photon
1 photoexited rhodopsin activates 500 transducins
500 phosphodiesterase molecules hydrolyses 10 5 cGMP
it prevenst closing of hundred chanels and entering 105 to 107 Na+ /s to the cell
Termination of the light response
GTPase activity of a-GTP transducin converts it to a-GDP transducin
opsin kinase adds ~ 9 phosphate groups to metarhodopsin II
arrestin (a regulatory protein) binds to the phosphorylated metarhodopsin II and prevents transducin from binding

10. Participation of retinal in the visual cycle
all-trans retinal
GDP T T T
Metarhodopsin II
Participation of retinal in the visual cycle
GDP
GTP
GTPase
Transducin
GDP   
GTP 
PDE
PDE
Low Ca via recoverin
stimulates guanylyl cyclase
Low Ca inhibits PDE
Recovery:
Rod cell releases Glu
neurotransmitter in the dark
GMP
(light state)
cGMP
(dark state)
GTP
PDE
PDE
PDE
Lack of cGMP causes decreased intracellular Na+ and Ca2+ -
membrane hyperpolarization lowers release of Glu neurotransmitter to trigger perception of light

11. NO - cGMP
Soluble guanylyl cyclase

12. Hormone Function of NO Intracellular signaling molecule
Regulates blood vessel dilation
Serves as second messenger
Serves as an neurotransmitter
Operates locally, quickly is converted into nitrates or nitrites
Half-life ~ 5 – 10 s
Synthesised from Arg by nitric oxide synthase

13. Nitric oxide is a free radical
It contains an unpaired electron
.N=O
Role in macrophage killing of pathogens
NO also acts as a second messenger that causes relaxation of smooth muscle
It is synthesized as needed, since it cannot be stored in vesicles.

14. Nitric oxide synthase Three isoforms identified in mammals
Neuronal and endothelial NOS - constitutively synthesize basal level of NO
Inducible NOS – is transcriptionaly activated by toxins
All isoforms are homodimer proteins
Each monomer has two domain:
N-terminal domain – catalytic center, contains a heme cofactor, binds oxygen and Arg
C-terminal domain- supplies electron to N-terminal domain, includes FMN and FAD
X-ray structure of N-terminal
domain of NOS
FAD and FMN

15. GTP cGMP +PPi NO NO – receptor - soluble guanylyl cyclase
Binding NO to the heme cofactor in the N-terminal domain
Dimerisation of the C-terminal domain
Activation of C-terminal domain and catalyses of GTP to cGMP conversion
GTP
cGMP – second messenger in the cells
causes smooth muscle relaxation
cGMP +PPi

16. NO – importance in medicine
Angina pectoris: chest pain due to the inadequate flow of the blood
Nitroglycerine: in body is converted to NO
NO relaxes coronary blood vessels and increases blood flow to heart
Ameliorates the symptoms of angina pectoris
Active agent in dynamite
Alfred Nobel suffered from angina pectoris and he wrote
“ It sounds like the irony of fate that I should be ordered by my doctor to take nitroglycerine internally.”

17. How does viagra work
To cause the smooth muscle of the penile blood vessels to relax, the motor nerve first causes the muscle to release a chemical called nitrous oxide;
this chemical in turn activates guanylate cyclase to convert GTP (guanosine triphosphate) to cGMP (cyclic guanosine monophosphate).
Acting as a second messenger the cGMP then acts on the muscle and causes it to relax (consequently allowing blood to enter the penis and erection takes place).
However once cGMP has done its job, it is destroyed by an esterase enzyme (PDE5 (phosphodiesterase 5), and its action upon the muscle is removed.
The action of viagra as a drug is to sustain cGMP by targeting the esterase enzyme; viagra inhibits the esterase enzyme thereby inhibiting the destruction of cGMP (the net effect being to sustain an erection).

18. Viagra - as a PDE inhibitor - is not for patients using nitrates (nitroglycerine)