Muscle

Recent studies have identified AMP-activated kinase (AMPK) as a target of Ca(2+)/calmodulin-dependent kinase kinase (CaMKKβ) and a negative regulator of myosin light-chain (MLC) kinase (MLCK). The present study examined whether a change in expression or activity of AMPK is responsible for hypercontractility of intestinal longitudinal muscle during inflammation or in response to proinflammatory cytokines. In mouse colonic longitudinal muscle cells, acetylcholine (ACh) stimulated AMPK and MLCK phosphorylation and activity and induced MLC20 phosphorylation and muscle contraction. Blockade of CaMKKβ with STO609 (7-oxo-7H-benzimidazo[2,1-a]benz[de]isoquinoline-3-carboxylic acid acetate) inhibited AMPK and MLCK phosphorylation and augmented MLCK activity, MLC20 phosphorylation, and smooth muscle cell contraction. In muscle cells isolated from the colon of TNBS (2,4,6-trinitrobenzenesulfonic acid)-treated mice or from strips treated with interleukin-1β or tumor necrosis factor-α, nuclear factor κB was activated as indicated by an increase in p65 phosphorylation and IκBα degradation, and AMPK was phosphorylated at a cAMP-dependent protein kinase (PKA)-specific site (Ser(485)) that is distinct from the stimulatory CaMKKβ site (Thr(172)), resulting in attenuation of ACh-stimulated AMPK activity and augmentation of MLCK activity and muscle cell contraction. Inhibition of nuclear factor-κB activity with MG-132 (carbobenzoxy-L-leucyl-L-leucyl-L-leucinal Z-LLL-CHO) or PKA activity with myristoylated PKA inhibitor 14-22 amide blocked phosphorylation of AMPK at Ser(485) and restored MLCK activity and muscle cell contraction to control levels. The results imply that PKA released from IκBα complex phosphorylated AMPK at a PKA-specific site and inhibited its activity, thereby relieving the inhibitory effect of AMPK on MLCK and increasing MLCK activity and muscle cell contraction. We conclude that hypercontractility of intestinal longitudinal muscle induced by inflammation or proinflammatory cytokines is mediated by nuclear factor κB/PKA-dependent inhibition of AMPK and activation of MLCK.

The potential roles of the regulatory proteins actin, tropomyosin (Tm), and caldesmon (CaD), i.e., the components of the thin filament, in smooth muscle have been extensively studied in several types of smooth muscles. However, controversy remains on the putative physiological significance of these proteins. In this study, we intended to determine the functional roles of Tm and CaD in the regulation of smooth muscle contraction by using a reconstitution system of the thin filaments. At appropriate conditions, the thin (actin) filaments within skinned smooth muscle strips of taenia caeci in guinea pigs could be selectively removed by an actin-severing protein, gelsolin, without irreversible damage to the contractile apparatus, and then the thin filaments were reconstituted with purified components of thin filaments, i.e., actin, Tm, and CaD. We found that the structural remodeling of actin filaments or thin filaments was functionally linked to the Ca(2+)-induced force development and reduction in muscle cross-sectional area (CSA). That is, after the reconstitution of the gelsolin-treated skinned smooth muscle strips with pure actin, the Ca(2+)-dependent force development was partially restored, but the Ca(2+)-induced reduction in CSA occurred once. In contrast, the reconstitution with actin, followed by Tm and CaD, restored not only the force generation but also both its Ca(2+) sensitivity and the reversible Ca(2+)-dependent reduction in CSA. We confirmed that both removal of the thin filaments by gelsolin treatment and reconstitution of the actin (thin) filaments with Tm and CaD caused no significant changes in the level of myosin regulatory light chain phosphorylation. We thus conclude that Tm and CaD are necessary for the full regulation of smooth muscle contraction in addition to the other regulatory systems, including the myosin-linked one.

The sarcoplasmic reticulum in situ is an intricate tubular network that surrounds the contractile material in striated muscle cells. Its topographical relationship to other intracellular components, especially the myofibrils, is rather rigidly maintained by a cytoskeleton which enmeshes Z line material and sarcoplasmic reticulum and, ultimately, is anchored at the plasmalemma. As a result, the two main components of the sarcoplasmic reticulum, the junctional SR and the free SR, retain their typical location in the A band region and in the I band region, respectively. The junctional SR, which is thought to be the site for calcium storage and release for contraction, is, thus, always well within one micron of the regulatory proteins associated with the actin filaments. The junctional SR, a synonym for terminal cisterna applying to both skeletal and cardiac muscle, is generally held to be involved in the translation of the action potential into calcium release, mainly because of the close topographic apposition between the junctional SR and the plasmalemma, especially in skeletal muscle. This attractive structure-function correlation is challenged by the observation that in bird cardiac muscle 80% of the junctional SR is spacially far removed from plasmalemma, the site of electrical activity. This anomalous topography is not in conflict with the notion that translation of the action potential into calcium release may be accomplished by a differential transmitter substance, e.g. calcium. Any hypothesis dealing with this problem must account for the anatomy of the bird heart.

Previous studies have shown that high concentrations of ethanol (>/=40%) cause functional damage of the gastrointestinal epithelial barrier by direct cytotoxic effect on the epithelial cells. The effects of lower noncytotoxic doses of ethanol on epithelial barrier function are unknown. A major function of gastrointestinal epithelial cells is to provide a barrier against the hostile substances in the gastrointestinal lumen. The apicolaterally located tight junctions (TJs) form a paracellular seal between the lateral membranes of adjacent cells and act as a paracellular barrier. In this study, we investigated the effects of lower doses of ethanol on intestinal epithelial TJ barrier function using filter-grown Caco-2 intestinal epithelial monolayers. The Caco-2 TJ barrier function was assessed by measuring epithelial resistance or paracellular permeability of the filter-grown monolayers. Ethanol (0, 1, 2.5, 5, 7.5, and 10%) produced a dose-related drop in Caco-2 epithelial resistance and increase in paracellular permeability. Ethanol also produced a progressive disruption of TJ protein (ZO-1) with separation of ZO-1 proteins from the cellular junctions and formation of large gaps between the adjacent cells. Ethanol, at the doses used (</=10%), did not cause cytotoxicity (lactate dehydrogenase release) to the Caco-2 cells. Ethanol produced a disassembly and displacement of perijunctional actin and myosin filaments from the perijunctional areas. On ethanol removal, actin and myosin filaments rapidly reassembled at the cellular borders. Ethanol stimulated the Caco-2 myosin light chain kinase (MLCK) activity but did not affect the MLCK protein levels. Specific MLCK inhibitor ML-7 inhibited both ethanol increases in MLCK activity and TJ permeability without affecting the MLCK protein levels. Consistent with these findings, metabolic inhibitors sodium azide and 2,4-dinitrophenol significantly prevented ethanol-induced increase in Caco-2 TJ permeability, whereas cycloheximide or actinomycin D had no effect. The results of this study indicate that ethanol at low noncytotoxic doses causes a functional and structural opening of the Caco-2 intestinal epithelial TJ barrier by activating MLCK.

It is generally thought that a contraction-relaxation cycle in visceral smooth muscles including vascular smooth muscles is regulated by the cytosolic Ca, i.e. Four Ca ions bind with calmodulin and this complex activates myosin light chain kinase (MLCK), thus causing phosphorylation of 20 kDa protein of myosin light chain (MLC). This phosphorylation of 20 kDa protein triggers actin-myosin cross bridge cycling in the presence of ATP (1–5).

Conventional myosin light chain kinase found in differentiated smooth and non-muscle cells is a dedicated Ca2+/calmodulin-dependent protein kinase which phosphorylates the regulatory light chain of myosin II. This phosphorylation increases the actin-activated myosin ATPase activity and is thought to play major roles in a number of biological processes, including smooth muscle contraction. The catalytic domain contains residues on its surface that bind a regulatory segment resulting in autoinhibition through an intrasteric mechanism. When Ca2+/calmodulin binds, there is a marked displacement of the regulatory segment from the catalytic cleft allowing phosphorylation of myosin regulatory light chain. Kinase activity depends upon Ca2+/calmodulin binding not only to the canonical calmodulin-binding sequence but also to additional interactions between Ca2+/calmodulin and the catalytic core. Previous biochemical evidence shows myosin light chain kinase binds tightly to actomyosin containing filaments. The kinase has low-affinity myosin and actin binding sites in Ig-like motifs at the N- and C-terminus, respectively. Recent results show the N-terminus of myosin light chain kinase is responsible for filament binding in vivo. However, the apparent binding affinity is greater for smooth muscle myofilaments, purified thin filaments, or actin-containing filaments in permeable cells than for purified smooth muscle F-actin or actomyosin filaments from skeletal muscle. These results suggest a protein on actin thin filaments that may facilitate kinase binding. Myosin light chain kinase does not dissociate from filaments in the presence of Ca2+/calmodulin raising the interesting question as to how the kinase phosphorylates myosin in thick filaments if it is bound to actin-containing thin filaments.

The importance of Ca2+calmodulin-dependent myosin light chain kinase (MLCK) for smooth muscle contraction is well documented.1 Until recently, myosin light chain (MLC) phosphatase was thought to be unregulated and constitutively active. However, studies have demonstrated that smooth muscle contraction is dependent on regulation of MLC phosphatase activity.2,3⇓ The inhibition of phosphatase activity increases force at a constant [Ca2+], whereas a stimulation of MLC phosphatase activity reduces force at a constant [Ca2+]. MLC phosphatase isolated from smooth muscle is a holoenzyme consisting of 3 subunits2: a small ≈20-kDa subunit, an ≈38-kDa catalytic subunit, and a myosin-targeting subunit (MYPT) of 110 to 133 kDa. The small subunit has no established function and is not required for either catalytic activity or activation of PP1c.2 The catalytic subunit is a PP1c phosphatase, and the δ isoform is associated with the holoenzyme.4 The large subunit is the MYPT. Isoform diversity of MYPT was first shown in chicken where 2 isoforms (M130 and M133)2 were shown to differ by the presence of a central insert between aa residues 512 to 552.5 Similar isoform diversity has been demonstrated for MYPT isolated from other species.2,6⇓ In addition to the isoform diversity produced by the central insert, alternative splicing of a COOH-terminal exon leads to MYPT isoforms, which differ by the presence or absence of a leucine zipper.7 At any intracellular [Ca+2], force for an agonist stimulated contraction is higher than for depolarization.8,9⇓ This phenomenon has been …

a consistent observation with fatigue in skeletal muscle is a decline in the amplitude of the myoplasmic Ca2+ transient, which is thought to result primarily from a reduced Ca2+ flux through the ryanodine receptor (RyR1) of the sarcoplasmic reticulum (SR) ([Fig. 1][1]). This in turn is thought to

Filamentous myosin is present in both relaxed (myosin light chains unphosphorylated) and contracted (light chains phosphorylated) vascular smooth muscle. The organization of myosin and actin filaments and the insertion of the latter on cytoplasmic and plasma membrane bound dense bodies is consistent with a mini sarcomere-like organization and a sliding filament mechanism of contraction in smooth muscle. Mitochondria are high capacity, low affinity Ca stores in smooth muscle. They do not play a role in the regulation of cytoplasmic Ca2+ at physiological levels. The localization and Ca content of the junctional sarcoplasmatic reticulum (SR) is consistent with this organelle being the major intracellular source of activator Ca released by excitatory transmitters. Repeated contractions in the absence of extracellular Ca2+ (thought to represent recycling of intracellular activator Ca2+) can be demonstrated if the excitatory agent is not allowed to remain in contact with the smooth muscle throughout relaxation.; the demonstration of "recycling" is facilitated if the efflux of cellular Ca2+ is blocked. The rise in total cytoplasmic calcium measured with electron probe analysis during a maintained (30 min) contracture in rabbit portal-anterior mesenteric vein smooth muscle (approximately 0.9 mol/kg dry cytoplasm) is greater than the amount of Ca that could be bound to calmodulin.

Muscle contraction occurs when the thin actin and thick myosin filaments slide past each other. It is generally assumed that this process is driven by cross-bridges which extend from the myosin filaments and cyclically interact with the actin filaments as ATP is hydrolysed. Current biochemical studies suggest that the myosin cross-bridge exists in two main conformations. In one conformation, which occurs in the absence of MgATP, the cross-bridge binds very tightly to actin and detaches very slowly. When all the cross-bridges are bound in this way, the muscle is in rigor and extremely resistant to stretch. The second conformation is induced by the binding of MgATP. In this conformation the cross-bridge binds weakly to actin and attaches and detaches so rapidly that it can slip from actin site to actin site, offering very little resistance to stretch. During ATP hydrolysis by isolated actin and myosin in solution, the cross-bridge cycles back and forth between the weak-binding and strong-binding conformations. Assuming a close correlation between the behaviour of isolated proteins in solution and the cross-bridge action in muscle, Eisenberg and Greene have developed a model for cross-bridge action where, in the fixed filament lattice in muscle, the transition from the weak-binding to the strong-binding conformation causes the elastic cross-bridge to become deformed and exert a positive force, while the transition back to the weak-binding conformation upon binding of MgATP, causes deformation which, during fibre shortening, leads to rapid detachment of the cross-bridge and its re-attachment to a new actin site. From the results of in vitro experiments, it was furthermore suggested that relaxation occurs when the transition from the weak-binding to the strong-binding conformation is blocked. Results of recent mechanical and X-ray diffraction experiments on skinned fibre preparations are consistent with the assumed close correlation between the behaviour of isolated proteins in solution and the behaviour of cross-bridges in muscle. Furthermore, X-ray diffraction experiments allowed to provide experimental evidence for the postulated structural difference between attached weak-binding and attached strong-binding cross-bridges. Finally, recent studies have confirmed the prediction of Eisenberg and Greene that the rate limiting step in vitro determines the rate of force generation in muscle.

SKELETAL MUSCLES adjust their contractile output over an exceptionally wide dynamic range, as needed to produce movements ranging from sitting and standing, to athletics and dance. At the single fiber level, the force generated by skeletal muscles is determined, ultimately, by the formation of cycling cross bridges between actin and myosin. This interaction requires Ca 2 and is governed by the simple relationship: force is proportional to intracellular ionized Ca 2 . Cross-bridge formation is inhibited in resting muscle when Ca 2 is sequestered within the sarcoplasmic reticulum (SR); it is activated when stored Ca 2 is released into the cytosol, allowing actin-myosin interactions to proceed. Contraction stops as Ca 2 is actively resequestered by the Ca 2 -ATPase of the SR (SERCA1). Thus Ca 2 is the central messenger in muscle activation, and the amplitude and time course of the intracellular Ca 2 change is the major determinant of contractile output. The key mechanisms that activate Ca 2 release from the SR are now well defined. This sequence, termed excitation-contraction coupling, begins when an excitatory input from the motor nerve triggers a propagating action potential on the muscle membrane. The action potential rapidly depolarizes the entire sarcolemma, including transverse tubules, which invaginate from the surface membrane and form specialized junctions (triads) with the SR along much of their length. The triad junction serves as a platform for assembling sarcolemmal calcium

The ryanodine receptor type 2 (RyR-2) functions as a Ca2+-induced Ca2+ release (CICR) channel on intracellular Ca2+ stores and is distributed in most excitable cells with the exception of skeletal muscle cells. RyR-2 is abundantly expressed in cardiac muscle cells and is thought to mediate Ca2+ release triggered by Ca2+ influx through the voltage-gated Ca2+ channel to constitute the cardiac type of excitation-contraction (E-C) coupling. Here we report on mutant mice lacking RyR-2. The mutant mice died at approximately embryonic day (E) 10 with morphological abnormalities in the heart tube. Prior to embryonic death, large vacuolate sarcoplasmic reticulum (SR) and structurally abnormal mitochondria began to develop in the mutant cardiac myocytes, and the vacuolate SR appeared to contain high concentrations of Ca2+. Fluorometric Ca2+ measurements showed that a Ca2+ transient evoked by caffeine, an activator of RyRs, was abolished in the mutant cardiac myocytes. However, both mutant and control hearts showed spontaneous rhythmic contractions at E9.5. Moreover, treatment with ryanodine, which locks RyR channels in their open state, did not exert a major effect on spontaneous Ca2+ transients in control cardiac myocytes at E9.5-11.5. These results suggest no essential contribution of the RyR-2 to E-C coupling in cardiac myocytes during early embryonic stages. Our results from the mutant mice indicate that the major role of RyR-2 is not in E-C coupling as the CICR channel in embryonic cardiac myocytes but it is absolutely required for cellular Ca2+ homeostasis most probably as a major Ca2+ leak channel to maintain the developing SR.

To begin the process of forming neural circuits, new neurons first establish their polarity and extend their axon. Axon extension is guided and regulated by highly coordinated cytoskeletal dynamics. Here we demonstrate that in hippocampal neurons, the actin-binding protein caldesmon accumulates in distal axons, and its N-terminal interaction with myosin II enhances axon extension. In cortical neural progenitor cells, caldesmon knockdown suppresses axon extension and neuronal polarity. These results indicate that caldesmon is an important regulator of axon development.

We have used second harmonic generation (SHG) imaging to quantify a strong intrinsic SHG-signal from cellular and subcellular muscle fibre preparations. In isolated single muscle cells,the intrinsic SHG-signal periodically follows the striation pattern and strongly depends on the sarcomere length and the polarization of the illuminating laser beam. At the subcellular level,the SHG signal seems to be located mainly at the overlapping region of the (thin) actin and (thick) myosin filaments. Thus,SHG imaging resolves the arrangement of the contractile structures with high resolution non-invasively and without chromophores. It may also allow to study dynamic molecular interactions of the motor protein myosin with actin filaments during force production and muscle shortening.

Ultrastructural Features of Smooth Muscle and Endothelial Cells of Isolated Isobaric Human Placental and Maternal Arteries