26th Oct Flashcards
What is the function of guanylyl cyclases?
Convert GTP to cGMP
What are the three main effectors of cGMP?
Cyclic nucleotide gated channels
PKG
PDE
What are the two main families of guanylyl cyclases?
Particulate GC
Soluble GC
How many mammalian isoforms are there of particulate GC?
7
Outline the domain structure of particulate guanylyl cyclase
Extracellular binding domain Single TM domain Regulatory domain with homology to protein kinases C-terminal catalytic domain Dimerisation domain
Are particulate GCs homodimers?
Yes
What are the three groups of particulate GCs?
Natriuretic Peptide Receptors
Intestinal peptide binding receptors
Orphan receptors
What ligands activate Natriuretic peptide receptors (group of particulare GCs)?
Atrial naturetic peptide
Brain naturetic peptide
What are the effects of activation of natriuretic peptide receptors?
Increased natriuresis (increased sodium excretion) Decreased systemic vascular resistance
What are the effects of activation of intestinal peptide binding receptors (group of GCs)?
Regulate electrolyte and water transport in intestinal and renal epithelium
Are soluble GCs homodimers?
No they are heterodimers
What are the subunits of soluble GCs?
Alpha and beta
Outline the basic domain structure of each subunit of soluble GC
N-terminal regulatory domain - with heme and dimerisation domain
C-terminal catalytic domain
What are the ligands that activate soluble GCs?
NO and CO
Who identified NO as an active substance in 1977?
Murad
When did Furchgott and Zawadzki find that the endothelial derived relaxing factor (EDRF) causes smooth muscles to relax?
1980
Who found that Endothelial derived relaxing factor (EDRF) is NO?
Moncada and Igarro
Who won the Nobel Prize in 1998 for their work on NO?
Furchgott, Ignaro and Murad
Why must NO be made as and when needed, rather than being stored in vesicles?
It is a gaseous compound therefore it only remains stable for a few seconds
What are the three major physiological functions of NO?
Smooth muscle relaxation
Platelet aggregation
Neurotransmission
What are the molecular functions of NO?
Promote soluble guanylyl cyclase
Nitrosylate proteins such as L type calcium channels
Promote ADP-ribosylation
Be used as a non-specific immune response in high concentrations
Which enzyme synthesises NO?
Nitric oxide synthase
What are the three types of nitric oxide synthase?
inducible NOS
endothelial NOS
neuronal NOS
Outline the reaction that nitric oxide synthase catalyses
L-arginine –> Citrulline and NO
What ion is NO activity dependent upon?
Calcium
What residues does PKG phosphorylate?
Serine/threonine
What are the two maintypes of PKG?
Type 1
Type 2
Describe Type 1 PKG
Soluble homodimers present in many cell types 2 isoforms (alpha and beta) Functions = smooth muscle relaxation, vasorelaxation and platelet aggregation
What are the domain sof each subunit in type I PKG?
Dimerisation domain
2cGMP binding domains
Autophosphorylation and autoinhibitory domain
Describe Type II PKG
Present in intestine, kidney and brain
Generally membrane bound
Functions = Intestinal secretion, bone growht, renin secretion and circadian rhythm
Describe the conformation of inactive PKG
It is folded over so that the pseudosubstrate domain is blocking the kinase domain, preventing ligand from binding.
Outline the pathway from L-arginine to PKG
L-arginine –> Citrulline +NO –> sGC –> cGMP –> PKG
Outline the main physiological functions of PKG
Cardiac Protection Smooth muscle relaxation Neuronal plasticity Endothelial permeability Gene transcription Urinary tract function
Outline the process of regulating vascular tone
Gq activated –> iP3 release –> Calcium release from ER –> Further calcium release from L-type calcium channels –> Calmodulin activation –> MLCK activation –> MLC phosphorylation –> contraction
Simultaneously Galpha 12/13 activation –> RhoA –> ROCK –| MLCP
Outline how NO causes vasorelaxation
ENDOTHELIAL CELL (Gq receptor –> Increase in calcium –> eNOS activation –> Citrulline and NO production), NO diffuses into SMOOTH MUSCLE CELL (Stimulates sGC –> cGMP –> PKG activation –> Potassium channel opening therefore reduction in calcium entry, RGS2 activation therefore inhibition of Galphaq, IRAG activation therfore inhibition of iP3 receptors on sarcoplasmic reticulum, inhibition of RhoA therefore stopping ROCK from inhibiting MLCP, activation of MLCP) –> MLC dephosphorylation –> Vasorelaxation
Outline some important physiological functions of cyclic nucleotide gated channels
Vision, olfaction, cardiac pacemaker cells, renal collecting duct cells
Outline the reactions catalysed by PDE
cAMP –> 5’AMP
cGMP –> 5’GMP
How many different PDE families are there in mammals
11
What are the three main types of PDEs?
Those that hydrolyse both cGMP and cAMP
Those that hydrolyse cGMP
Those that hydrolyse cAMP
How can cGMP regulate PDEs?
They can alter the rate of hydrolysis by competition at the catalytic active site in PDE 1, 2 and 3
They can regulate enzymatic activity through direct binding to allosteric sites
What is the chemical name of viagra?
Sidenafil citrate
How does viagra work (not the molecular pathway)?
It causes relaxation of the smooth muscle in the penis and thus a rush of blood to the corpa cavernosum, causing it’s cavernous sinuses to fill with blood causing an erection.
Outline the pathway for an erection
Psychogenic/reflexogenic stimulus –> neural depolarisation –> increae in intracellular calcium –> Calmodulin activated neuronal NOS –> NO causes vasorelaxation and shear stress –> activates PI3K –> Akt –> endothelial NOS –> NO –> more relaxation
How does viagra cause an erection?
It blocks PDE5, thus preventing cGMP being converted to 5’GMP, leading to increased stimulation of PKG –> decrease in calcium –> corpus cavernosal smooth muscle relaxation –> blood inflow
How does PKG cause PDE activation?
PKG will phosphorylate PDE5 at ser92 if GTP is bound to PDE5’s allosteric binding sites, leading to further activation
What is another clinical use for viagra?
Treats pulmonary hypertension
How does PKG1 stimulate apoptosis in response to NO?
PKG1 activation in colon cells results in phosphorylation and activation of MEKK1 leading to activation of SGK1 and the JNK pathway –> apoptosis
