Messengers, second

The first messengers act from one cell to another, and the second messenger acts within the cell (Rose and Wilkie, 2000). Activation of most membrane-associated receptors/effec-tors generates a diffusable intracellular signal called the second messenger. Major second 

Phosphorylated forms of all the molecules indicated in Table 11.21 may be involved in brain chemistry or nervous activities in some parts of the body. The associated second messengers are phosphorylated. 

It is currently believed that mental conditions such as depression, migraine, manic depression, schizophrenia and so forth are connected in some way with irregularities in the supply or function of neurotransmitters. 

Many hormones, neurotransmitters and so forth act to produce signals at the cell surface which are then transmitted across the membrane to produce intracellular chemical messengers called second messengers . These subsequently trigger various biochemical pathways to produce the cell s eventual response. 

Cyclic AMP. Among the many biochemical reactions of ATP is that of cyclisation to cyclic adenosine monophosphate, c-AMP, which is brought about by the widely distributed enzyme, adenyl cyclase. 

Cyclic AMP was the first second messenger to be discovered [18a]. It acts as a universal messenger in the control and manufacture of enzymes and thus plays a major role as a regulator of many metabolic reactions. The activity of certain protein kinases is stimulated by c-AMP and it is involved in the regulation of glycogen synthesis and breakdown. It is involved in muscle contraction, transcription and the action of many drugs. 

The consequences of transmitter interaction with specific membrane-bound recognition sites have been stressed throughout this chapter. In par- 

A number of criteria must be met to conclude that a cyclic nucleotide is involved in a postsynaptic event. First of all, electrical stimulation of the presynaptic neuron or microiontophoresis of the putative neurotransmitter at the synapse should increase the level of cyclic nucleotide. This increase should be blocked by receptor antagonists or low Ca, which would prevent neurotransmitter release after electrical stimulation. Second, the pres- 

Schematic representation of cAMP synthesis and degradation and effects on protein kinase regulatory and catalytic sid)units. 

8-bromo derivatives of cAMP or cGMP) should mimic the effects of electrical or chemical stimulation. Last, phosphodiesterase inhibitors should potentiate and prolong the effects of the eyclie nucleotides. It is often impossible to measure tissue levels of a cyclic nucleotide in the absence of a phosphodiesterase inhibitor because the turnover of the second messenger is so rapid that the cyclic nucleotide is synthesized and catabolized within seconds. 

Cyclic AMP is catabolized to 5 -adenosine monophosphate by the enzyme cyclic nucleotide phosphodiesterase, which terminates any further cAMP-initiated reactions. This enzyme also requires Mg + for activity. Calcium, again in consort with calmodulin, can stimulate phosphodiesterase activity. Phosphodiesterase appears to exist in multiple forms, each with specificity toward different substrates. Calcium and calmodulin activate only one form of the enzyme. The enzyme is potently inhibited by methyl xanthines, such as caffeine, theophylline, and theobromine. It is believed that at least part of the pharmacological effects of such compounds can be explained through their inhibition of phosphodiesterase and the consequent reduction in the catabolism of cAMP. 

Metabolism. The nucleotide cAMP (adenosine 3, 5 -cyclic monophosphate) is synthesized by membrane-bound adenylate cyclases [1] on the inside of the plasma membrane. The adenylate cyclases are a family of enzymes that cyclize ATP to cAMP by cleaving diphosphate (PPi). The degradation of cAMP to AMP is catalyzed by phosphodiesterases [2], which are inhibited by methylxanthines such as caffeine, for example. By contrast, insulin activates the esterase and thereby reduces the cAMP level (see p. 388). 

Adenylate cyclase activity is regulated by G proteins (Gs and Gi), which in turn are controlled by extracellular signals via 7-helix receptors (see p. 384). Ca -calmodulin (see below) also activates specific adenylate cyclases. 

Action. cAMP is an allosteric effector of protein kinase A (PK-A, [3]). in the inactive state, PK-A is a heterotetramer (C2R2), the catalytic subunits of which (C) are blocked by regulatory units (R autoinhibition). When cAMP binds to the regulatory units, the C units separate from the R units and become enzymatically active. Active PK-A phosphorylates serine and threonine residues of more than 100 different proteins, enzymes, and transcription factors, in addition to cAMP, cCMP also acts as a second messenger, it is involved in sight (see p. 358) and in the signal transduction of NO (see p. 388). 

Type Gq G proteins activate phospholipase C [4]. This enzyme creates two second messengers from the double-phosphorylated membrane phospholipid phosphatidylinositol bisphosphate (PinsP2), i. e., inositol 1,4,5-tris- 

Calcium level. Ca (see p. 342) is a signaling substance. The concentration of Ca ions in the cytoplasm is normally very low (10-100 nM), as it is kept down by ATP-driven Ca pumps and Na /Ca exchangers, in addition, many proteins in the cytoplasm and organelles bind calcium and thus act as Ca buffers. 

In further experiments to determine the rate of PIP2 hydrolysis in these rabbit kidney slices, radiolabel was 

The well-known fact that in irreversibly damaged cells, respiratory control is lost and is accompanied by oxidation of cytochromes a and as, as well as NADH (Taegtmeyer et al., 1985), was originally thoug it to be due to substrate deficiency (Chance and Williams, 1955) but may be due to an enzymatic defect resulting in an inability to metabolize NADH-linked substrates (Pelican etal., 1987). It seems likely therefore that return of function is dependent on preservation of mitochondrial membrane integrity, and the structure and activities of respiratory chain (R.C) complexes I-IV (Chance and Williams, 1955). 

In recent studies on perfused rats hearts (Veitch et al., 1992), it was found that differences in the sensitivity of complexes 1-lV to ischaemic damage were dependent upon the duration of ischaemia and the presence of oxygen. The demonstration that complex 1 is a major defective site dependent upon isolation of mitochondria from homogenates of the tissue by in vitro methods seemed important to us. We therefore decided to attempt to make noninvasive measurements of mitochondrial function soon after reperfusion in transplanted rabbit kidneys by surface fluorescence (for mitochondrial NADH levels) and near infra-red spectroscopy (NIRS) for the redox state of cytaas. 

These are small signaling molecules generated in response to extracellular signals. They amplify and propagate the signal. 

Second messengers relay the primary signal. The distinction between second messengers and normal transducers is that second messengers are small molecules. Extracellular signals of various kinds can activate intracellular pathways that cause an increase in the concentration of a small molecule messenger. All of these pathways involve G-protein coupled receptors. There are two second messengers that you need to know about cyclic nucleotides and calcium. 

Cyclic nucleotides are like regular nucleotides (the things in DNA/RNA) except that the phosphate bridges the 3 and 5 hydroxyl group within 

Cyclic nucleotides are made in response to receptor activation. The receptor activates a G-protein that, in turn, activates adenylyl cyclase to make the cyclic nucleotide. To complete the signaling, the increase in cAMP concentration activates a specific protein kinase (serine/threo-nine), cAMP-dependent protein kinase (A kinase) (Fig. 9-7). To turn off the signaling pathway, the cyclic nucleotides are destroyed by enzymes called phosphodiesterases. These cleave cAMP to AMP. 

Activation of adenylyl cyclase by an activated G-protein coupled receptor results in the synthesis of cAMP. The cAMP activates a downstream kinase, protein kinase A. Phosphodiesterase hydrolyzes and inactivates the cAMP. 

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Earl Sutherland of Vanderbilt University won the 1971 Nobel Prize in physiology or medicine for uncovering the role of cAMP as a second messenger in connection with his studies of the fight or flight hormone epineph rine (Section 27 6)... 

Oxytocin and Vasopressin Receptors. The actions of oxytocin and vasopressin are mediated through their interactions with receptors. Different receptor types as well as different second messenger responses help explain their diverse activities in spite of the hormones stmctural similarities. Thus oxytocin has at least one separate receptor and vasopressin has been shown to have two principal receptor types, and V2. Subclasses of these receptors have been demonstrated, and species differences further compHcate experimental analysis. It is apparent that both oxytocin and receptors function through the GP/1 phosphoHpase C complex (75), while the V2 receptors activate cycHc AMP (76). 

Nitric Oxide. Nitric oxide [10102-43-9] NO, is a ubiquitous intracellular and intercellular messenger serving a variety of functions including vasodilation, cytotoxicity, neurotransmission, and neuromodulation (9). NO is a paramagnetic diatomic molecule that readily diffuses through aqueous and hpid compartments. Its locus of action is dictated by its chemical reactivity and the local environment. NO represents the first identified member of a series of gaseous second messengers that also includes CO. 

Pseudohypoparathyroidism is characterized by end-organ resistance to parathyroid hormone (98,108). This disease takes various forms, including Albright s hereditary osteodystrophy, which has unusual physical features and a generalized resistance to G-protein-linked hormones that function through cAMP as a second messenger. This defect is associated with a deficiency in the levels of the a-subunit of (109). Because this defect may be generalized, such patients also have olfactory dysfunction (110). 

Modulation of second-messenger pathways is also an attractive target upon which to base novel antidepressants. Rolipram [61413-54-5] an antidepressant in the preregistration phase, enhances the effects of noradrenaline though selective inhibition of central phosphodiesterase, an enzyme which degrades cycHc adenosiae monophosphate (cAMP). Modulation of the phosphatidyl iaositol second-messenger system coupled to, for example, 5-HT,, 5-HT,3, or 5-HT2( receptors might also lead to novel antidepressants, as well as to alternatives to lithium for treatment of mania. Novel compounds such as inhibitors of A-adenosyl-methionine or central catechol-0-methyltransferase also warrant attention. 

In addition to the mechanism involving cycHc AMP, nonsugar sweeteners, eg, saccharin and a guanidine-type sweetener, have been found to enhance the production of another second messenger, inositol 1,4,5-trisphosphate (IP3), causing the closure of potassium channels and the release of... 

Excitation of smooth muscle via alpha-1 receptors (eg, in the utems, vascular smooth muscle) is accompanied by an increase in intraceUular-free calcium, possibly by stimulation of phosphoUpase C which accelerates the breakdown of polyphosphoinositides to form the second messengers inositol triphosphate (IP3) and diacylglycerol (DAG). IP3 releases intracellular calcium, and DAG, by activation of protein kinase C, may also contribute to signal transduction. In addition, it is also thought that alpha-1 adrenergic receptors may be coupled to another second messenger, a pertussis toxin-sensitive G-protein that mediates the translocation of extracellular calcium. 

Depletion of ATP in the cells prevents maintenance of the membrane potential, inhibits the functioning of ion pumps, and attenuates cellular signal transduction (e.g., formation of second messengers such as inositol phos phates or cyclic AMP). A marked ATP depletion ultimately impairs the activ-itv of the cell and leads to ceil death. 

Savol,linen, K., Hirvonen, M.-R., and Naarala, J. (1994). Phosphojnositide second messengers m cholinergic e.xcitotoxicity. Neuro toxicology 15(3), 493-502. 

There are numerous second messenger systems such as those utilizing cyclic AMP and cyclic GMP, calcium and calmodulin, phosphoinosiddes, and diacylglerol with accompanying modulatory mechanisms. Each receptor is coupled to these in a variety of ways in different cell types. Therefore, it can be seen that it is impractical to attempt to quantitatively define each stimulus-response mechanism for each receptor system. Fortunately, this is not an... 

FIGURE 2.6 Production of cyclic AMP from ATP by the enzyme adenylate cyclase. Cyclic AMP is a ubiquitous second messenger in cells activating numerous cellular pathways. The adenylate cyclase is activated by the a subunit of Gs-protein and inhibited by the a-subunit of Gj-protein. Cyclic AMP is degraded by phosphodiesterases in the cell. 

FIGURE 2.7 Production of second messengers inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG) through activation of the enzyme phospholipase C. This enzyme is activated by the a- subunit of Gq-protein and also by Py subunits of Gj-protein. IP3 stimulates the release of Ca2+ from intracellular stores while DAG is a potent activator of protein kinase C. 

Some cellular stimulus-response pathways and second messengers are briefly described. The overall efficiency of receptor coupling to these processes is defined as the stimulus-response capability of the cell. 

Group II assays consist of those monitoring cellular second messengers. Thus, activation of receptors to cause Gs-protein activation of adenylate cyclase will lead to elevation of cytosolic or extracellularly secreted cyclic AMP. This second messenger phosphorylates numerous cyclic AMP-dependent protein kinases, which go on to phosphorylate metabolic enzymes and transport and regulatory proteins (see Chapter 2). Cyclic AMP can be detected either radiometrically or with fluorescent probe technology. 

Another major second messenger in cells is calcium ion. Virtually any mammalian cell line can be used to measure transient calcium currents in fluorescence assays when cells are preloaded with an indicator dye that allows monitoring of changes in cytosolic calcium concentration. These responses can be observed in real time, but a characteristic of these responses is that they are transient. This may lead to problems with hemi-equilibria in antagonist studies whereby the maximal responses to agonists may be depressed in the presence of antagonists. These effects are discussed more fully in Chapter 6. 

FIGURE 5.15 Different modes of response measurement, (a) Real time shows the time course of the production of response such as the agonist-stimulated formation of a second messenger in the cytosol, (b) The stop-time mode measures the area under the curve shown in panel A. The reaction is stopped at a designated time (indicated by the dotted lines joining the panels) and the amount of reaction product is measured. It can be seen that in the early stages of the reaction, before a steady state has been attained (i.e., a plateau has not yet been reached in panel A), the area under the curve is curvilinear. Once the rate of product formation has attained a steady state, the stop-time mode takes on a linear character. 

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