Contraction phosphorylation

In the presence of calcium, the primary contractile protein, myosin, is phosphorylated by the myosin light-chain kinase initiating the subsequent actin-activation of the myosin adenosine triphosphate activity and resulting in muscle contraction. Removal of calcium inactivates the kinase and allows the myosin light chain to dephosphorylate myosin which results in muscle relaxation. Therefore the general biochemical mechanism for the muscle contractile process is dependent on the avaUabUity of a sufficient intraceUular calcium concentration. 

Phosphodiesterase Inhibitors. Because of the complexity of the biochemical processes involved in cardiac muscle contraction, investigators have looked at these pathways for other means of dmg intervention for CHF. One of the areas of investigation involves increased cycHc adenosine monophosphate [60-92-4] (cAMP) through inhibition of phosphodiesterase [9025-82-5] (PDE). This class of compounds includes amrinone, considered beneficial for CHF because of positive inotropic and vasodilator activity. The mechanism of inotropic action involves the inhibition of PDE, which in turn inhibits the intracellular hydrolysis of cAMP (130). In cascade fashion, cAMP-catalyzed phosphorylation of sarcolemmal calcium-channels follows, activating the calcium pump (131). A series of synthetic moieties including the bipyridines, amrinone and milrinone, piroximone and enoximone, [77671-31-9], C22H22N2O2S, all of which have been shown to improve cardiac contractiUty in short-term studies, were developed (132,133). These dmgs... 

The procedure described is essentially that of Shioiri and Yamada. Diphenyl phosphorazidate is a useful and versatile reagent in organic synthesis. It has been used for racemlzatlon-free peptide syntheses, thiol ester synthesis, a modified Curtius reaction, an esterification of a-substituted carboxylic acld, formation of diketoplperazines, alkyl azide synthesis, phosphorylation of alcohols and amines,and polymerization of amino acids and peptides. - Furthermore, diphenyl phosphorazidate acts as a nitrene source and as a 1,3-dipole.An example in the ring contraction of cyclic ketones to form cycloalkanecarboxylic acids is presented in the next procedure, this volume. 

Smooth muscle contractions are subject to the actions of hormones and related agents. As shown in Figure 17.32, binding of the hormone epinephrine to smooth muscle receptors activates an intracellular adenylyl cyclase reaction that produces cyclic AMP (cAMP). The cAMP serves to activate a protein kinase that phosphorylates the myosin light chain kinase. The phosphorylated MLCK has a lower affinity for the Ca -calmodulin complex and thus is physiologically inactive. Reversal of this inactivation occurs via myosin light chain kinase phosphatase. 

Smooth muscle myosin contains two myosin light chains. Phosphorylation of the regulatory light chain by myosin light chain kinase is a mandatory step to induce contraction. 

Obara, K. de Lanerolle, P. (1989). Isoproterenol attentuates myosin phosphorylation and contraction of tracheal muscle. J. Appl. Physiol. 66,2017-2022. 

Although the fundamental chemomechanical transduction processes seem to be the same in all types of vertebrate muscle, contraction in smooth muscle is characterized by much greater involvement of enzymatically catalyzed control reactions. In smooth muscle the control reactions themselves involve the use of phosphorylation-dephosphorylation cycles. Moreover, they are futile in the sense they cause the expenditure of bond energy without a tangible work resultant, i.e., compounds synthesized or external work done. 

Although in in vivo circumstances an intracellular free calcium increase apparently acts as the primary modulator of contraction, it can be bypassed in highly permeabilized smooth muscle preparations where the active subunit of MLCK can be introduced to phosphorylate myosin and induce contraction. The MLCK catalyzed phosphorylation of serine-19 is seen as the necessary event in the activation of smooth muscle myosin to form crossbridges. Thus, the rising phase of force during an isometric smooth muscle contraction follows an increase in the degree of phosphorylation of myosin, and that in turn follows the transient rise of (a) cytosolic free Ca, (b) Ca-calmodulin complexes, and (c) the active form of MLCK. The regulation of the intracellular calcium is discussed below. The dynam-... 

If MLCK activates contraction by increasing myosin phosphorylation, then an increase in the activity of myosin light chain phosphatase, MLCP, by decreasing the fraction of myosin which is phosphorylated, should lead to relaxation from the active (contractile) state. Cyclic adenosine monophosphate (AMP) is a strong inhibitor of smooth muscle contraction and it has been suggested that activation of MLCP could result from its phosphorylation via cAMP activated protein kinase (see Figure 5). 

One should note overall, that while some of these suggested mechanisms may in the future prove to have a role in the control of smooth muscle contraction, in chemically skinned preparations maximum force development follows activation by the MLCK active subunit in extremely low Ca " ion concentrations. The conclusion can hardly be avoided that phosphorylation alone is sufficient for activation, and if another mechanism is involved, it is not necessary for the initial genesis of force. If such mechanisms are operative, then they might be expected to run in parallel or consequent to myosin phosphorylation. A possible example of this category of effect is that a GTP-dependent process (G-protein) shifts the force vs. Ca ion concentration relationship to lower Ca ion concentrations. This kind of mechanism calls attention to the divergence of signals along the intracellular control pathways. 

Figure 12-14. The creatine phosphate shuttle of heart and skeletal muscle. The shuttle allows rapid transport of high-energy phosphate from the mitochondrial matrix into the cytosol. CKg, creatine kinase concerned with large requirements for ATP, eg, muscular contraction CIC, creatine kinase for maintaining equilibrium between creatine and creatine phosphate and ATP/ADP CKg, creatine kinase coupling glycolysis to creatine phosphate synthesis CK, , mitochondrial creatine kinase mediating creatine phosphate production from ATP formed in oxidative phosphorylation P, pore protein in outer mitochondrial membrane.

The general picture of muscle contraction in the heart resembles that of skeletal muscle. Cardiac muscle, like skeletal muscle, is striated and uses the actin-myosin-tropomyosin-troponin system described above. Unlike skeletal muscle, cardiac muscle exhibits intrinsic rhyth-micity, and individual myocytes communicate with each other because of its syncytial nature. The T tubular system is more developed in cardiac muscle, whereas the sarcoplasmic reticulum is less extensive and consequently the intracellular supply of Ca for contraction is less. Cardiac muscle thus relies on extracellular Ca for contraction if isolated cardiac muscle is deprived of Ca, it ceases to beat within approximately 1 minute, whereas skeletal muscle can continue to contract without an extraceUular source of Ca +. Cyclic AMP plays a more prominent role in cardiac than in skeletal muscle. It modulates intracellular levels of Ca through the activation of protein kinases these enzymes phosphorylate various transport proteins in the sarcolemma and sarcoplasmic reticulum and also in the troponin-tropomyosin regulatory complex, affecting intracellular levels of Ca or responses to it. There is a rough correlation between the phosphorylation of Tpl and the increased contraction of cardiac muscle induced by catecholamines. This may account for the inotropic effects (increased contractility) of P-adrenergic compounds on the heart. Some differences among skeletal, cardiac, and smooth muscle are summarized in... 

Phosphorylation of Myosin Light Chains Initiates Contraction of Smooth Muscle... 

When smooth muscle myosin is bound to F-actin in the absence of other muscle proteins such as tropomyosin, there is no detectable ATPase activity. This absence of activity is quite unlike the situation described for striated muscle myosin and F-actin, which has abundant ATPase activity. Smooth muscle myosin contains fight chains that prevent the binding of the myosin head to F-actin they must be phosphorylated before they allow F-actin to activate myosin ATPase. The ATPase activity then attained hydrolyzes ATP about tenfold more slowly than the corresponding activity in skeletal muscle. The phosphate on the myosin fight chains may form a chelate with the Ca bound to the tropomyosin-TpC-actin complex, leading to an increased rate of formation of cross-bridges between the myosin heads and actin. The phosphorylation of fight chains initiates the attachment-detachment contraction cycle of smooth muscle. 

Figure 49-14. Regulation of smooth muscle contraction by Ca. pL-myosin is the phosphorylated light chain of myosin L-myosin is the dephosphorylated light chain. (Adapted from Adelstein RS, Eisenberg R Regulation and kinetics of actin-myosin ATP interaction. Annu Rev Biochem 1980 49 921.)...

The calmodulin-4Ca +-activated light chain kinase phosphorylates the hght chains, which then ceases to inhibit the myosin-F-actin intetaction. The contraction cycle then begins. 

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