Nitric Oxide: The Coming of the Second Messenger
The greatest benefit of second messengers is speed and amplification. There are certain key parts inside the second messenger systems. The first is a G-Protein. Kimball J. "Second messengers". Retrieved February 10, Animation: Second Messenger: cAMP. There are hundreds of different GPCR proteins, and nearly a third of all drugs target this A diverse set of ligands bind to this type of receptor, including peptide hormones, Different G-alpha proteins activate different second messenger pathways This reduces signaling by preventing the association with the G-protein.
We also knew that certain hormones were capable of elevating cGMP levels in intact tissues such as heart and blood vessels but no such an effect was seen in cell-free systems. To fully understand hormone—cyclase coupling, it was imperative to have a cell-free system.
With such a system one could add individual components and see the effect on the system.G Protein Coupled Receptors - Nervous system physiology - NCLEX-RN - Khan Academy
Having experience with working on cAMP as a student, I decided to add azide to different tissues and found that the effect was tissue-specific and that it only activated the soluble enzyme and not the particulate. In addition, this reaction required oxygen, and there was also a time-lag of several minutes before the rate of the reaction became maximal.
We were able to enhance the effect with thiols such as cystine. All this suggested that the effects of azide, hydroxylamine, and of nitrite were indirect.
Second Messenger Systems
They were not proximal activators of guanylyl cyclase but were probably being converted to something else or influencing some other molecule.
We also noticed that when azide was added to cell cultures or liver and brain slices, the levels of cGMP would rise. That enabled us to use azides instead of hormones to achieve a similar effect. We took two crude supernatant fractions: We also had a non-responsive heart fraction. When we mixed liver and heart supernatants, the azide effect disappeared. We assumed that the reason was that the heart tissue possessed an inhibitor.
That was a simple but very important experiment. We later went on to purify the inhibitor which turned out to be the proteins hemoglobin and myoglobin. When we mixed liver with cerebral cortex, the azide effect was potentiated. We therefore assumed the existence of an activator in the liver tissue that is absent in the cerebral cortex.
- Second messenger system
- G-protein Coupled Receptors
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We discovered that the activating factor t was a catalase. The inhibitors, hemoglobin and myoglobin and the activator, catalase are all heme proteins. The fact that NO was both inhibited and activated by heme proteins was intriguing, but at the time we did not know the significance of this discovery. We found that azide elevated cGMP levels in smooth muscle and caused muscle relaxation.
Dose—response curves, time-courses, and other tests convinced us that one of the effects of cGMP was smooth muscle relaxation. We later found that cyclic GMP induced relaxation in vascular smooth muscle as well. Using my medical background, I contemplated whether other smooth muscle relaxants such as nitroglycerine and nitroprusside had similar effects on cGMP levels and indeed they did. We ended up with a long category of compounds we called nitro-vasodilators, although not all were nitro compounds.
The list included azide, hydroxylamine, nitrite, phenylhydrazine, nitroprusside, nitrosamines, and others. We assumed that all those compounds were converted to an intermediate. We postulated that the activator must be nitric oxide because we knew that hemoglobin and myoglobin blocked the effects of all these activators, and we knew from the literature back in the s that nitric oxide had high affinity for the heme prosthetic group in hemoglobin and myoglobin.
We synthesized nitric oxide in the fume hood, combined sodium nitrite, sulphuric acid, and ferrous sulphate with a stir bar and vented the gas into our cyclase incubations. The nitric oxide activated every preparation we tested.
Nitric oxide was indeed the intermediate that we were looking for. The fact that we had an activator that is also a free radical was a radical idea. This idea led to a thought that other enzyme systems might be regulated by free radicals as well. This is what a nitro-vasodilator does in smooth muscle. It quickly elevates cGMP, causing relaxation, whereby the messenger goes away, but relaxation persists.
The reason for this is that cGMP has a short half-life; it is degraded and released from the cell. The physiologic effect downstream persists because the cyclic GMP-activated protein kinases and the phosphorylated proteins have a longer half-life and a slower turn-over.
Nitroglycerine had been used for a hundred years, and no one understood how it worked. The levels of PIs in cells are dynamically regulated by extracellular signals. In response to some signals e. As we discuss later in this chapter, PIs act as membrane docking sites for signaling molecules and also, in some cases, stimulate catalytic activity. Proteins bind to PIs through a PH domain.
Different PH domains show different phosphoinositide binding specificities. Figure Several second messengers are derived from phosphatidylinositol PI. Each membrane-bound PI kinase places a phosphate on a specific hydroxyl group on the inositol ring, producing more Phosphoinositides can be cleaved by the membraneassociated enzyme phospholipase C PLC to generate yet other second messengers.
These cleavage reactions produce 1,2- diacylglycerol DAGa lipophilic molecule that remains linked to the membraneand free phosphorylated inositols, which can diffuse into the cytosol. Signaling pathways involving any of these second messengers sometimes are referred to as inositol- lipid pathways.
Since it is water soluble, IP3 can diffuse within the cytosol carrying a hormone signal from the cell surface to the ER surface. Binding of a hormone to its receptor leads to activation of the G protein Gqwhich in turn more This simple experiment demonstrates the specificity of the IP3 effect. In addition, not all cells respond identically to IP3. Because of this variability, different types of cells may exhibit very different responses to the same extracellular signal. In skeletal muscle cells, these receptors are located in the membrane of the sarcoplasmic reticulum SR and associate with the cytoplasmic domain of the dihydropyridine receptora voltage-sensing protein in the plasma membrane.
Voltagesensing dihydropyridine receptors in the plasma membrane contact ryanodine receptors located in the membrane of the sarcoplasmic reticulum. In response to a change more The principal function of DAG is to activate a family of plasma-membrane protein kinases collectively termed protein kinase C.
In the absence of hormone stimulation, protein kinase C is present as a soluble cytosolic protein that is catalytically inactive. The activation of protein kinase C in different cells results in a varied array of cellular responses, indicating that it plays a key role in many aspects of cellular growth and metabolism. In liver cells, for instance, protein kinase C helps regulate glycogen metabolism by phosphorylating glycogen synthase, yielding the inactive form of this enzyme.
Protein kinase C also phosphorylates various transcription factors; depending on the cell type, these induce or repress synthesis of certain mRNAs. Synthesis of cGMP is catalyzed by two types of guanylate cyclase: Binding of ligand to the extracellular domain of these receptors promotes the activity of the intracellular guanylate cyclase catalytic domain, leading to formation of cGMP see Figure dleft.
Soluble guanylate cyclases are activated by a gas, nitric oxide NO. These enzymes are heterodimers and contain a bound heme molecule that interacts with both subunits Figure a. Binding of nitric oxide to the heme leads to a conformational change in the enzyme and stimulates its catalytic activity. Figure cGMP mediates local signaling by nitric oxide. NO synthase, acting constitutively or in response to specific signals, catalyzes the formation of nitric oxide from arginine and O2.
Once formed, nitric oxide diffuses only locally through tissues and is highly labile with a half-life of from 2 to 30 seconds. It plays an important role in mediating many local cellular interactions, as exemplified by the local control of arterial smooth muscle contractility Figure b.
The nitric oxide that is formed diffuses from the endothelial cell and into neighboring smooth muscle cells where it binds to and activates soluble guanylate cyclase.
The subsequent increase in cGMP then leads to muscle relaxation and dilation of the vessel. Nitric oxide also helps control communication at certain synapses in the central nervous system Section