Cyclic GMP activation

MLCK itself is phosphorylated by cyclic-AMP activated protein kinase, (protein kinase A) and cyclic-GMP activated protein kinase, (protein kinase G). Protein kinase A will phosphorylate MLCK at two sites and protein kinase G at one in some cases and two in others. These differences seem to be important in how the individual smooth muscle cells are regulated. 

A partially purified cyclic GMP-activated protein kinase from lobster muscle was later found to have a for cyclic GMP of about 0.08 /x.M and for cyclic AMP of about 4 /aM. A cyclic AMP-activated protein kinase isolated from the same tissue had an apparent Ka for cyclic AMP of about 0.02 /J.M and for cyclic GMP of about 1.2 ptM [76]. Cyclic GMP-dependent phosphorylation of endogenous protein has been demonstrated in membranes of mammalian smooth muscle [78]. In the presence of 10 mM Mn, a half-maximal increase in the phosphorylation of these proteins occurred with 20-30 nM cyclic GMP, but ten-fold higher concentrations of cyclic AMP were required to produce the same increase in phosphorylation. 

A protein inhibitor of cyclic AMP-activated protein kinase has been isolated from mammalian tissue and this promotes a five-fold increase in the binding constant of cyclic AMP to protein kinase [79]. The inhibitor is assumed to interact with the catalytic subunit at the regulatory subunit binding site. Thus, inhibitor protein and regulatory subunit modulate the catalytic subunit activity by an identical mechanism. However, it may be more complex, as a protein kinase modulator protein has been isolated which alters the substrate specificity both of cyclic AMP and of cyclic GMP-activated protein kinases, increasing the phosphorylation of some protein substrates and decreasing that of others [80]. 

For cyclic GMP, a cyclic GMP-activated protein kinase from lobster muscle is utilised. The cyclic GMP assay is somewhat less sensitive than the radioimmunoassay but has the advantage of the short time needed for the preparation of the binding protein 0.5 to 1 pmol of cyclic GMP can be estimated [155]. 

The method is based upon the ability of low concentrations of cyclic nucleotides to activate protein kinases which catalyse the phosphorylation of protein substrates, such as histone, by ATP [156,157]. The extent of phosphorylation is proportional to the amount of the cyclic nucleotides. The limits of sensitivity of the method are about 0.3 pmol for cyclic AMP and 0.5 pmol for cyclic GMP. Purification on a Dowex 50 column separates the two cyclic nucleotides from each other and removes any substances, such as ATP, which might interfere with the assay. Cyclic GMP is further purified by column chromatography on aluminum oxide and Dowex 1. Cyclic AMP-activated protein kinase is prepared from bovine heart and cyclic GMP-activated protein kinase from lobster tail. 

The conditions for the cyclic GMP assay are the same, except that cyclic GMP samples or standards up to 20 pmol are used with the cyclic GMP-activated protein kinase preparation. 

In most cases, cyclic AMP functions by activating the isoforms of cyclic AMP-dependent protein kinase (PKA), and cyclic GMP activates a PKG. Recently, a number of additional actions of cyclic nucleotides have been described, all with pharmacological relevance. 

We evaluated whether the Mn(ii)-based SOD mimics could potentiate the levels of nitric oxide, a potent vasorelaxant. Superoxide reacts with nitric oxide in a diffusion-controlled manner to produce peroxynitrite. By catalyzing the dismu-tation of superoxide, SOD mimics would be expected to increase nitric oxide levels. The SOD mimic SC-52608 enhanced nitric oxide levels (as assessed by cyclic GMP activity) in rat lung fibroblasts in a dose-dependent manner. SC-52608 induced the relaxation of preconstricted rat aortic rings. The aortic ring relaxation was endothelium-dependent and inhibitable by a nitric oxide synthase inhibitor. Intravenous administration of SC-52608 into conscious rats resulted in a transient, dose-dependent decrease in blood pressure. The results are consistent with the SOD mimic SC-52608 potentiating levels of nitric oxide, which causes the observed relaxation of the aortic rings and the decrease in blood pressure. 

CREB stands for cyclic-AMP response element (CRE) binding protein and is a transcription factor. When phosphorylated by cyclic AMP- and cyclic GMP-dependent Protein Kinases or other protein kinases it binds to gene promoters that contain a specific binding site. After binding, the respective transcription activity is modulated. 

Synthesized by soluble guanylyl cyclase and particulate guanylyl cyclase from guanosine triphosphate (GTP). Nitric oxide activates soluble guanylyl cyclase to enhance cyclic GMP production that contributes to various NO actions. Cyclic GMP is hydrolyzed by phosphodiesterases. Cyclic GMP binds to and activates cGMP-dependent protein kinase, phosphodiesterases, and Cyclic Nucleotide-regulated Cation Channels. 

Guanylyl cyclases (GC) are a family of enzymes (EC 4.6.1.2) that catalyse the formation of the second messenger cyclic GMP (cGMP) from guanosine triphosphate (GTP). GCs are subdivided in soluble GCs and GCs that are membrane-bound and linked to a receptor. Activation occurs by nitric oxide (NO) and pqrtide hormones, respectively [1,2]. 

Cyclic GMP is made from GTP by the enzyme gua-nylyl cyclase, which exists in soluble and membrane-bound forms. Each of these isozymes has unique physiologic properties. The atriopeptins, a family of peptides produced in cardiac atrial tissues, cause natriuresis, diuresis, vasodilation, and inhibition of aldosterone secretion. These peptides (eg, atrial natriuretic factor) bind to and activate the membrane-bound form of guanylyl cyclase. This results in an increase of cGMP by as much as 50-fold in some cases, and this is thought to mediate the effects mentioned above. Other evidence links cGMP to vasodilation. A series of compounds, including nitroprusside, nitroglycerin, nitric oxide, sodium nitrite, and sodium azide, all cause smooth muscle re-... 

SCHROEDER, H., LEITMAN, D. C., Hayward, L. D., Bennett, B. M., Murphy, F., Cultured rat lung fibroblasts as a model for organic nitrate-induced cyclic GMP accumulation and activation of guanylate cyclase. J. Appl. Cardiol. 2 (1987), p. 301-311... 

Changes in the concentration of arginine, via arginase activity, can play a role in regulating the rate of nitric oxide synthesis. Nitric oxide is a messenger molecule that has several roles. One is to increase the activity of the enzyme guanyl cyclase, which increases the concentration of cyclic GMP. The latter causes vasolidation in peripheral... 

A further characteristic of this principle is that, if the activity of phosphodiesterase is decreased, the concentration of cyclic GMP will increase to an extent dependent upon the extent of the decrease in activity. This characteristic has been made use of by the pharmaceutical industry. Cyclic GMP has a vasodilatory effect and this is the case for the arterioles that supply blood to the corpus cavemosum in the penis, which controls the erection of the penis. Drugs were developed (e.g. sildenafil) that inhibits cyclic GMP phosphodiesterase and hence increases the cyclic GMP level which resnlts in vasodilation of the arterioles and an increase in the snpply of blood to the spongy tissue of the corpus cavemosum, which expands resulting in erection. This dmg has been found to be effective in some patients snffering from erectile dysfunction. This can be a particular problem in diabetic patients and more elderly men (Chapter 19). 

An increase in the activity of cyclic GMP phosphodiesterase produces a decrease in the concentration of cyclic GMP only if the activity of the enzyme guanyl cyclase remains constant. The principle underlying this requirement is discussed in Chapter 12,... 

Relaxation of smooth mnscles is controlled by the concentration of cyclic GMP in the muscle. This is regulated by the activities of the enzyme that forms cyclic GMP (i.e. gnanyl cyclase) and the enzyme that degrades cyclic GMP, that is, cyclic GMP phosphodiesterase (see Box 12.2). This is analogons to the enzyme system that regulates the concentration of cyclic AMP, by the activities of adenyl cyclase and phosphodiesterase ... 

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