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

ANP: Atrial natriuretic peptide

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)

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

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

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 = 40 000 M-1cm-1) 11-cis-retinal

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

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

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

NO - cGMP Soluble guanylyl cyclase

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

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.

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

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

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.”

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).

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