Pluricellular organisms are characterized by the existence of tissues, i.e. sets of cells which have acquired specialized functions. This specialization is advantageous in the sense that it permits a distribution of work but it necessitates means of controlling and integrating the activities of various tissues. Hormones constitute precisely a means of communication between territories distant from one another.
It was suggested that hormones act on the activity of enzyme (as activators or inhibitors) or on the repression and induction of the synthesis of enzymes. In fact, although the physiological effects of hormones are generally well known, their primary site of action and their mechanism of action — in molecular terms — have not yet been clearly explained.
Furthermore, the control of the synthesis of hormones, and of their secretion by endocrine glands, in response to a change of the concentration of a compound (food or waste) in the extra-cellular fluids, represents a domain which is not yet understood, though it is known that secretion can be stimulated either by a nervous impulse originating from the brain, or by a hormone secreted by another gland (e.g., an antepituitary stimuline).
1. Control of the Metabolism of Glycogen:
Glycogen is the principal source of energy for muscle contraction. Its degradation and synthesis are subjected to a control which modifies the degree of phosphorylation of glycogen phosphorylase and glycogen synthetase respectively, by the action of numerous proteins, especially 5 different protein kinases, 4 protein phosphatases and various regulatory proteins like calmoduline, troponine C, etc.
Thanks to these proteins, the metabolism of glycogen can be controlled by Ca2+ ions and by cyclic AMP (cAMP), denoted “second messengers” because they relay the action of the “first messengers” which are respectively electric stimulation and β-adrenergic stimulation (by adrenaline).
The hormonal control of glycogen metabolism involves adrenaline (in the muscle and liver) and glucagon (in the liver).
Glucagon is a peptide hormone (29 amino acids) secreted by the α cells of Langerhans islets of the pancreas (the β cells secrete insulin). Adrenaline has a simpler structure and derives from tyrosine. The physiological effect of these two hormones can be easily shown: they cause an increase of the blood glucose and of the glucose which enters the oxidation processes.
This increase of available glucose takes place at the expenses of glycogen but, as we will see in the following, the primary site of action of these two hormones is a reaction rather distant from the degradation reaction of glycogen.
In the muscle, the adrenaline — called first messenger by some authors — brought by the circulating blood, binds to the membrane of the target-cell and activates adenyl-cyclase, an enzyme catalyzing the scission of ATP into PP + cyclic AMP (or adenosine 3′, 5′ cyclic monophosphate or cAMP).
The latter formed on the internal surface of the cell membrane, will diffuse into the cell; it is the second messenger which has taken the relay from the first. It will act on a protein kinase which needs cyclic AMP for its activity. This protein kinase transforms an inactive non-phosphorylated phosphorylase b kinase into active phosphorylated phosphorylase b kinase.
Phosphorylase b kinase can then catalyze the phosphorylation of phosphorylase b (which contains 2 subunits) into phosphorylase a (which contains 4 subunits); this phosphorylation modifies 4 serine residues (one per polypeptide chain) which are transformed into phosphoserine by the action of phosphorylase b kinase.
Phosphorylase a thus formed constitutes the active form of glycogen phosphorylase which permits, as mentioned earlier, the glycogenolysis, i.e. thephospliorolysis of glycogen. All these reactions are schematically represented in figure 8-17. In the liver — under the influence of adrenaline or glucagon — there is a set of similar reactions, although there are some differences, especially at the level of the phosphorylases.
In addition to this activating action on glycogenolysis, cyclic AMP also has an inhibiting action on the synthesis of glycogen. As shown by figure 8-17, the protein kinase activated by cyclic AMP also has an action on glycogen synthetase and catalyzes — with other kinases — the transformation of active non-phosphorylated glycogen synthetase (form I) into inactive phosphorylated glycogen synthetase (form D).
Conversely, when cyclic AMP concentration decreases, there is dephosphorylation of phosphorylase a into phosphorylase b, and of glycogen synthetase D into glycogen synthetase I, which brings about an arrest of the degradation of glycogen and a resumption of its synthesis.
It must be remembered that other factors also influence the synthesis and degradation of glycogen, especially oxygen content: thus, a decrease of oxygen content results in a decrease of ATP concentration with an increase of ADP (or AMP) and Pi concentrations, which is favourable to the phosphorolysis of glycogen and unfavourable to its synthesis.
On the other hand, when the muscle is stimulated electrically, the Ca2+ ions liberated from the sarcoplasmic reticulum not only trigger muscle contraction, but also activate phosphorylase kinase and consequently glycogen phosphorylase, which accelerates glycogenolysis, providing the ATP required for muscle contraction.
Phosphorylase kinase is therefore not only under the dependence of the cAMP-dependent protein kinase but also under that of Ca2+ions. It has an oligomeric structure (αβγδ)4: the sub-units α and β are phosphorylated by the protein kinase-cyclic AMP complex, sub-unit γ is responsible for the catalytic activity, and sub-unit δ is the one which binds the Ca2+ions.
This δ sub-unit is identical to calmoduline, a protein involved in the regulation of numerous Ca2+-dependent enzymes. Phosphorylase kinase also seems to be activated by troponine C (the protein of muscle fibers which confers calcium sensitivity to the contractile system), which resembles calmoduline by its structure and property to bind Ca2+ ions.
Contrary to glycogen phosphorylase, which has only one site (a serine) phosphorylated during its activation by phosphorylase kinase, most enzymes controlled by reversible phosphorylation undergo phosphorylations on several sites.
Thus phosphorylase kinase, under the action of adrenaline, has two serine residues phosphorylated by the cAMP-dependent protein kinase, one on the a sub-unit, the other on the β subunit, and glycogen synthetase has seven serine residues phosphorylated (on the C-terminal side of the protein) under the action of 5 protein kinases (the cAMP-dependent protein kinase, phosphorylase kinase and the glycogen synthetase kinases 3, 4 and 5).
Severalprotein-phosphatases (1, 2A, 2B, 2C), capable of dephosphorylating the enzymes of the metabolism of glycogen, were characterized, but it is interesting to note that protein-phosphatase 1, dephosphorylates the β sub-unit of phosphorylase kinase, glycogen phosphorylase and glycogen synthetase, and therefore only one enzyme catalyzes the dephosphorylation reactions leading to the inhibition of glycogenolysis and activation of glycogenogenesis.
Insulin controls the metabolism of glycogen but apparently without calling upon cAMP or CA2+ ions. By interacting with its receptor in the plasmic membrane, this hormone triggers modifications (increase or decrease) of the degree of phosphorylation of diverse intracellular proteins (perhaps through a second messenger). It thus controls the metabolism of glycogen by stimulating the activity of glycogen synthetase.
2. Control of Other Cellular Processes:
It soon became obvious that the cAMP-dependent protein kinase must have activities other than the one first shown (activation of the phosphorylase kinase) to explain the various effects of cAMP.
According to a hypothesis that was advanced, the specificity of a hormone is determined by the presence of the corresponding receptor in the membrane of the target-cell and by the nature of the substrate (or substrates) of the cAMP-dependent protein kinase present in this cell.
When these substrates are enzymes, the effect of phosphorylation is often to modify the constants of affinity for a substrate, an enhancer or an inhibitor (this is particularly the case with the enzymes of the metabolism of glycogen).
Numerous enzymes are controlled by the cAMP-dependent protein kinase, which explains the great diversity of action of hormones in various cells. Thus, triglyceride lipase is activated and glycerol-phosphate acyl transferase is inactivated, which permits the respective control of the catabolism and synthesis of triglycerides in the adipose tissues under the action of adrenaline in the same way as activation of phosphorylase kinase and inactivation of glycogen sythetase permit the control respectively of the catabolism and synthesis of glycogen under the action of adrenaline (in the muscle) or glucagon (in the liver).
The activation of cholesterol esterase increases the pool of cholesterol available for the steroidogenesis in the adrenal cortex under the action of ACTH. The phosphorylation of acetyl-coenzyme A carboxylase decreases its activity, which explains the inhibition of the synthesis of fatty acids in the adipose tissues under the action of adrenaline and in the liver under the action of glucagon.
The inactivation of pyruvate kinase and the activation of fructose 1, 6 bisphosphatase play an important role in the stimulation of gluconeogenesis by glucagon in the liver. But the hormonal control of many other processes seems to involve the phosphorylation of proteins, especially during growth, cellular differentiation, muscle contraction, transmembrane transports, etc., although the corresponding molecular mechanisms have not yet been explained.
It must be noted that the cAMP-dependent protein kinase is a very specific enzyme, phosphorylating at a significant rate only few proteins, and only on one or two sites among the large number of residues of serine and threonine. It appears that its physiological substrates always comprise two adjacent basic amino acids on the N-terminal side of the phosphorylated residue.
Numerous biological effects of Ca2+ ions involve a protein, calmoduline, which, after binding Ca2+ ions, undergoes conformational changes and thus acquires new possibilities of interaction. About twelve enzymes activated by calmoduline have already been identified, especially proteins kinases (particularly phosphorylase kinase) and proteins phosphatases which affect the activity of key-enzymes in various tissues.
For example, in the brain a cairn oduline-dependent protein kinase phosphorylates tryptophan hydroxylase and tyrosine hydroxylase, thus controlling the synthesis of serotonine and dopamine (two neuromediators).
A number of analogies are observed between the activation of proteins (by phosphorylation) under the influence of cAMP on the one hand, and calmoduline on the other. The same process can be activated by either of these control systems, depending on the tissue considered.
Besides, the cAMP-dependent protein kinase and the calmoduline-Ca2+-dependent proteins kinases often control the phosphorylation of the same proteins. We have seen that the cAMP-dependent protein kinase can phosphorylate the phosphorylase kinase, which is also a calm oduline-dependent Ca2+ enzyme.
The fact that the concentration of cAMP-dependent protein kinase is high in tissues where glycogen metabolism is not important was one of the first observations made in favour of the role of this enzyme in metabolisms other than that of glycogen. The same observation was made for various protein phosphatases (1,2A, 2B, 2C), suggesting that they also play a role in the control of processes other than the metabolism of glycogen.
These phosphatases act particularly in the liver on pyruvate kinase, acetyl coenzyme A carboxylase, hydroxymethylglutaryl coenzyme A reductase, and therefore seem to be involved in the control of neoglucogenesis, synthesis of fatty acids and synthesis of cholesterol.
These phosphatases also act on the light chains of myosin, which suggests that they are involved in the control of muscle contraction. Protein phosphatases 1 and 2A act on the initiation factor eIF-2 and can therefore be considered as stimulating protein synthesis.
Processes of phosphorylation and dephosphorylation of proteins occupy an important place in numerous control mechanisms of cellular activity. In addition to those already mentioned, one may cite the phosphorylation of nuclear proteins (especially the histones) or that of ribosomal proteins which probably act in the control of the organization and expression of genes, but whose mechanisms and role are not yet known exactly.
We have mentioned that cyclic AMP plays a role in the control mechanisms in microorganisms, particularly in the phenomenon of catabolic repression. In higher organisms, it constitutes, as just mentioned, the relay of various hormones and therefore acts in the hormonal control of numerous cellular processes.