Operación del análisis: dos formas de comprender la realidad

f ? i

Inactive Protein Kinase A ■ ■/rif i i

^ u l l I I I - I T . I- i U î k I = . 1, t r ,,7 i i ; . î i ; » r ■ : i ' - ■ - o r r -

Figure 1.1 Simple schematic of the intracellular events following the binding of ligands to a- and P-adrenoreceptors. A b b r e v i a t i o n s : R = R e c e p t o r ( e . g . P- a d r e n e r g i c r e c e p t o r , a - a d r e n e r g i c r e c e p t o r , d o p a m i n e r e c e p t o r o r i n s u l i n r e c e p t o r ) ; L = L i g a n d ( e . g . a d r e n a l i n e o r i n s u l i n ) ; G , = G - s t i m u l a t o r y p r o t e i n ; G j = G - i n h i b i t o r y p r o t e i n ; P D E s = c y c l i c n u c l e o t i d e p h o s p h o d i e s t e r a s e e n z y m e s .

( F i g u r e a d a p t e d f r o m M a t t h e w s a n d V a n H o l d e , 1 9 9 0 )

The enzyme protein kinase was originally discovered by Fischer and Krebs ( 1955) and was thought to be the only molecule known to specifically interact with cAMP. Protein kinase A (PKA) is anchored to different regions of the cytosol by A kinase anchoring proteins (AKAPs) where it is thought to be involved with phosphorylating intracellular proteins to initiate cellularprocesses. Targets for PKA include enzymes as well as transcription factors such as the cAMP response element binding protein (CREB) and cAMP response element

modulator (CREM) (Dwarki et ai, 1990). Dwarki and co-workers (1990) have shown that

the formation of CREB homodimers as well as phosphorylation of CREB by PKA is necessary for the transcriptional activation of a gene. The transcription factor NF-kB is thought to be controlled by cAMP as well as PKA and is involved in the inflammatory response (Carter et ai, 1996; Ollivier et al, 1996). cAMP and PKA are also thought to be

mechanism involved with these nuclear receptors is unclear (Daniel et al, 1998).

1.3 cGMP and guanylyl cyclases

1.3.1 Discovery of cGMP and guanylyl cyclase

The molecule guanosine 3', 5' monophosphate (GMP) was detected in the urine sample of rats in the late 1960s (Hardman et al, 1966 and 1969; Ishikawa, 1969). Hardman and Sutherland (1969) then reported the existence of an enzyme system which catalysed the formation of cGMP from GTP in rat tissues. This enzyme system was distinct from the adenylate cyclase system and was called the guanyl cyclase system. These workers showed that although the guanylyl cyclase was found largely in the soluble component of the cell it was also found in the particulate fraction. It differed in tissue distribution from adenylyl cyclase and it was heat-labile. The membrane form of guanylyl cyclase is a single, transmembrane polypeptide chain. The predicted amino acid sequences of the membrane forms of guanylyl cyclase proteins from sea urchin, rat brain and human kidney revealed that the intracellular (catalytic) domains are highly conserved among these species while the extracellular (ligand binding) domain is highly divergent (Singh et al, 1988; Garbers, 1989; Chinkers et al, 1989). Ligands binding to the extracellular domain of these membrane cyclases activate the cyclase directly to form the second messenger cGMP, a process quite distinct from the activation o f the particulate forms of adenylyl cyclases which are activated indirectly via G-proteins.

1.3.2 Particulate and soluble guanylyl cyclase enzymes

1.3.2.1 Particulate guanylyl cyclase enzymes

The particulate form of guanylyl cyclase belongs to a family of enzymes which comprise o f at least four different monomeric isoenzymes: Type A, B, C and P (Bentley and Beavo, 1992). Type A is activated by atrial natriuretic peptide (ANP) (Winquist et al, 1984; Chinkers et al, 1989,1991) while type B is activated by ANP and brain natriuretic protein (BNP) but at nonphysiologically high concentrations (Schulz et al, 1989). Type C is found almost exclusively in the epithelium o f the gut and is known to be stimulated by the E. coli

enterotoxin (Bentley and Beavo, 1992). Type P is a photoreceptor-specific isoform involved in the visual transduction pathway and is activated by the protein recoverin which is a calcium-sensitive protein (Koch and Stryer, 1988; Koch, 1991; Dizhoor et al, 1991).

1.3.2.2 Soluble guanylyl cyclase enzymes

Soluble guanylyl cyclases are responsible for the endothelium mediated production of cGMP in the vascular smooth muscle. The enzyme itself is composed of two subunits (a and b) with at least one heme group associated with the enzyme (Stone and Marietta, 1995). The carboxy-terminals of the subunits show considerable homology with the known cytoplasmic catalytic domain of the particulate guanylyl cyclases. The main activator of the soluble guanylyl cyclase is nitric oxide (NO) which is released fi'om nerve endings as a neurotransmitter as well as from vascular endothelial cells (Ignarro et al, 1984, 1989; Maggi er a/., 2000).

1.3.3 Signal transduction mechanism involving cGMP

The guanylyl cyclase enzymes are stimulated by a variety of compounds including atrial natriuretic peptides (ANP) and nitrovasodilators such as nitric oxide. The natriuretic peptides bind to the extracellular domain of the membrane forms of monomeric guanylyl cyclases to initiate events leading to natriuresis, diuresis, vasodilation and inhibition of aldosterone secretion (Seidah et al, 1984; Waldman et al, 1984). Nitric oxide released from nerve endings as a neurotransmitter, or vascular endothelial cells, diffuses into smooth muscle cells where it binds to soluble heterodimeric guanylyl cyclases thereby activating these enzymes to ultimately cause muscle relaxation (Figure 1.2). The activated soluble guanylyl cyclases synthesise cGMP from GTP which in turn bind and activate cGMP- dependent protein kinases. These activated kinases then play a key regulatory role leading to muscle relaxation. This action is regulated by the breakdown o f cGMP by cGMP- hydrolysing cyclic nucleotide phosphodiesterases (e.g. PDE5). Inhibition of PDE5, for example, will give rise to a prolongation of the muscle relaxation. The much publicised drug sildenafil is thought to act by this mechanism, inhibiting PDE5 in erectile tissue to give the desired effect (Boolell et a l , 1996). Other targets for the cGMP-dependent protein

inositol triphosphate receptor. e.g. ANP NO Extracellular region Cell membrane pGC Intracellular region GTP sGC PDE1.PDE2.PDE3. PDE5.

PDE6. PDE9. PDE10. PDE11