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Contraction of smooth muscle involves myosin light chain (MLC) kinase catalyzed phosphorylation of the regulatory MLC, activation of myosin, and the development of force. However, this cannot account for all aspects of a smooth muscle contraction, suggesting that other regulatory mechanisms exist. One potentially important technique to study alternative sites of contractile regulation is the use of small interfering RNA (siRNA). The goal of this study was to determine whether siRNA technology can decrease the levels of a specific protein and allow for the determination of how that protein affects contractile regulation. To achieve this goal, we tested the hypothesis that casein kinase 2 (CK2) is part of the complex regulatory scheme present in vascular smooth muscle. Using intact strips of swine carotid artery, we determined that siRNA against CK2 produced a tissue that resulted in a ~60% knockdown after 4 days in organ culture. Intact strips of vascular tissue depleted of CK2 produced greater levels of force and exhibited an increased sensitivity to all stimuli tested. This was accompanied by an increase in cross-bridge cycling rates but not by a change in MLC phosphorylation levels. α-Toxin-permeabilized vascular tissue depleted of CK2 also showed an increased sensitivity to calcium compared with control tissues. Our results demonstrate that siRNA is a viable technique with which to study regulatory pathways in intact smooth muscle tissue. Our results also demonstrate that CK2 plays an important role in the mechanism(s) responsible for the development of force and cross-bridge cycling by a MLC phosphorylation-independent pathway. [ABSTRACT FROM AUTHOR]

Abstract: Actin polymerization and depolymerization plays a central role in controlling a wide spectrum of cellular processes. There are many actin-binding proteins in eukaryotic cells. Their roles in the remodeling of the actin architecture and whether they work cooperatively await further study. Caldesmon (CaD) is an actin-binding protein present in nearly all mammalian cells. Cortactin is another actin-binding protein found mainly in the cell cortex. There have been no reports suggesting that CaD and cortactin interact with each other or work as partners. Here, we present evidence that CaD binds cortactin directly by overlay, pull-down assays, ELISA, and by column chromatography. The interaction involves the N-terminal region of cortactin and the C-terminal region of CaD, and appears to be enhanced by divalent metal ions. Cortactin competes with both full-length CaD and its C-terminal fragment for actin binding. Binding of cortactin partially alleviates the inhibitory effect of CaD on the actomyosin ATPase activity. Not only can binding be demonstrated in vitro, the two proteins also co-localize in activated cells at the cortex. Whether such interactions bear any functional significance awaits further investigation. [Copyright &y& Elsevier]

We have reported that 15-hydroxyeicosatetraenoic acid (15-HETE) induces pulmonary artery (PA) contraction in rats exposed to hypoxia by activating extracellular signal-regulated kinase 1/2 (ERK1/2). In this study, we investigated the characteristics of 15-HETE mediating phosphorylation of ERK1/2 and caldesmon in rat pulmonary arterial smooth muscle cells (PASMCs). Our data showed that 15-HETE upregulated ERK1/2 phosphorylation in a dose-dependent manner, which could be blocked by ERK pathway inhibitors U0126 and PD98059. ERK1/2 phosphorylation was attenuated by inhibiting endogenous 15-HETE formation with lipoxygenase inhibitor, cinnamyl 3,4-dihydroxy-[alpha]-cyanocinnamate (CDC), in both normoxic and hypoxic PASMCs. ERK1/2 phosphorylation in response to 15-HETE was detected in cytosol as well as in nucleus and phosphorylatd ERK1/2 partly translocated into nucleus, which could be blocked by PD98059. In addition, caldesmon was phosphorylated in 15-HETE-stimulated cells; this could be inhibited by PD98059. These data demonstrated that 15-HETE is associated with ERK1/2 activation and caldesmon phosphorylation in PASMCs and that 15-HETE is at least partly involved in mediating activation of hypoxia-initiated ERK pathway, possibly leading to hypoxic pulmonary vasoconstriction. [ABSTRACT FROM AUTHOR]

Abstract: New data on the movements of tropomyosin singly labeled at α- or β-chain during the ATP hydrolysis cycle in reconstituted ghost fibers have been obtained by using the polarized fluorescence technique which allowed us following the azimuthal movements of tropomyosin on actin filaments. Pronounced structural changes in tropomyosin evoked by myosin heads suggested the “rolling” of the tropomyosin molecule on F-actin surface during the ATP hydrolysis cycle. The movements of actin-bound tropomyosin correlated to the strength of S1 to actin binding. Weak binding of myosin to actin led to an increase in the affinity of the tropomyosin N-terminus to actin with simultaneous decrease in the affinity of the C-terminus. On the contrary, strong binding of myosin to actin resulted in the opposite changes of the affinity to actin of both ends of the tropomyosin molecule. Caldesmon inhibited the “rolling” of tropomyosin on the surface of the thin filament during the ATP hydrolysis cycle, drastically decreased the affinity of the whole tropomyosin molecule to actin, and “freezed” tropomyosin in the position characteristic of the weak binding of myosin to actin. [Copyright &y& Elsevier]

Abstract: Hybrid contractile apparatus was reconstituted in skeletal muscle ghost fibers by incorporation of skeletal muscle myosin subfragment 1 (S1), smooth muscle tropomyosin and caldesmon. The spatial orientation of FITC-phalloidin-labeled actin and IAEDANS-labeled S1 during sequential steps of the acto-S1 ATPase cycle was studied by measurement of polarized fluorescence in the absence or presence of nucleotides conditioning the binding affinity of both proteins. In the fibers devoid of caldesmon addition of nucleotides evoked unidirectional synchronous changes in the orientation of the fluorescent probes attached to F-actin or S1. The results support the suggestion on the multistep rotation of the cross-bridge (myosin head and actin monomers) during the ATPase cycle. The maximal cross-bridge rotation by 7° relative to the fiber axis and the increase in its rigidity by 30% were observed at transition between A**·M**·ADP·Pi (weak binding) and A∧·M∧·ADP (strong binding) states. When caldesmon was present in the fibers (OFF-state of the thin filament) the unidirectional changes in the orientation of actin monomers and S1 were uncoupled. The tilting of the myosin head and of the actin monomer decreased by 29% and 90%, respectively. It is suggested that in the “closed” position caldesmon “freezes” the actin filament structure and induces the transition of the intermediate state of actomyosin towards the weak-binding states, thereby inhibiting the ATPase activity of the actomyosin. [Copyright &y& Elsevier]

Abstract: Cytoskeleton modulating compounds have been shown to lower intraocular pressure (IOP) and increase outflow facility. Caldesmon is one protein that is involved in the regulation of actin stress fiber formation. The effects of rat non-muscle caldesmon (Cald) gene over-expression on focal adhesions in human trabecular meshwork (HTM) cells and on outflow facility in organ-cultured human and monkey anterior segments were determined. Treatment of HTM cells with adenovirus-delivered caldesmon (AdCaldGFP) resulted in characteristic changes in the actin cytoskeleton and matrix adhesions within 24–48hr post-transduction. Stress fibers gradually disappeared and novel actin structures were formed (see manuscript by Grosheva et al., this issue). In cells with disrupted stress fibers, vinculin-containing focal adhesions were also disrupted. In organ-cultured anterior segments, baseline outflow facility (μlmin−1 mmHg−1) for all anterior segments averaged (mean±sem): human, 0.19±0.03 (n=12); monkey, 0.36±0.02 (n=19). In human anterior segments, transduction with 107 plaque forming units of AdGFPCald increased outflow facility by 43±21% (p≤0.11, n=6) at 66hr compared to baseline and corrected for the changes in outflow facility of the contralateral vehicle treated segment. Using the same time point, i.e. 2–3 days after injection, outflow facility in monkey anterior segments, transduced with 1.5×107 plaque forming units of AdGFPCald was increased by 35±18%, p<0.2, n=10 compared to baseline and corrected for the change in outflow facility in the contralateral AdGFP treated segment. Combining human (66hr) and monkey (2–3 days) data, outflow facility was increased by 38±13%, p<0.02, n=16. Additional analysis of maximum responses in monkey anterior segments from 1 to 6 days after transduction showed outflow facility was increased by 66±18%, p<0.01, n=10. Caldesmon over-expression, which relaxes cultured HTM cells and disrupts their actin cytoskeleton and cell–matrix adhesions, also appears to increase outflow facility in organ-cultured human and monkey anterior segments. This suggests that over-expression of the caldesmon gene in the TM may be an effective approach for the gene therapy of glaucoma. [Copyright &y& Elsevier]

Abstract: Caldesmon is an actin-binding protein that is capable of stabilizing actin filaments against actin-severing proteins, inhibiting actomyosin ATPase activity, and inhibiting Arp2/3-mediated actin polymerization in vitro. Caldesmon is a substrate of cdc2 kinase and Erk1/2 MAPK, and phosphorylation by either of these kinases reverses the inhibitory effects of caldesmon. Cdc2-mediated caldesmon phosphorylation and the resulting dissociation of caldesmon from actin filaments are essential for M-phase progression during mitosis. Cells overexpressing the actin-binding carboxyterminal fragment of caldesmon fail to release the fragment completely from actin filaments during mitosis, resulting in a higher frequency of multinucleated cells. PKC-mediated MEK/Erk/caldesmon phosphorylation is an important signaling cascade in the regulation of smooth muscle contraction. Furthermore, PKC activation has been shown to remodel actin stress fibers into F-actin-enriched podosome columns in cultured vascular smooth muscle cells. Podosomes are cytoskeletal adhesion structures associated with the release of metalloproteases and degradation of extracellular matrix during cell invasion. Interestingly, caldesmon is one of the few actin-binding proteins that is associated with podosomes but excluded from focal adhesions. Caldesmon also inhibits the function of gelsolin and Arp2/3 complex that are essential for the formation of podosomes. Thus, caldesmon appears to be well positioned for playing a modulatory role in the formation of podosomes. Defining the roles of actin filament-stabilizing proteins such as caldesmon and tropomyosin in the formation of podosomes should provide a more complete understanding of molecular systems that regulate the remodeling of the actin cytoskeleton in cell transformation and invasion. [Copyright &y& Elsevier]

The actin-binding protein caldesmon (CaD) exists both in smooth muscle (the heavy isoform, h-CaD) and non-muscle cells (the light isoform, l-CaD). In smooth muscles h-CaD binds to myosin and actin simultaneously and modulates the actomyosin interaction. In non-muscle cells l-CaD binds to actin and stabilizes␣the actin stress fibers; it may also mediate the interaction between actin and non-muscle myosins. Both h- and l-CaD are phosphorylated in vivo upon stimulation. The major phosphorylation sites of h-CaD when activated by phorbol ester are the Erk-specific sites, modification of which is attenuated by the MEK inhibitor PD98059. The same sites in l-CaD are also phosphorylated when cells are stimulated to migrate, whereas in dividing cells l-CaD is phosphorylated more extensively, presumably by cdc2 kinase. Both Erk and cdc2 are members of the MAPK family. Thus it appears that CaD is a downstream effector of the Ras signaling pathways. Significantly, the phosphorylatable serine residues shared by both CaD isoforms are in the C-terminal region that also contains the actin-binding sites. Biochemical and structural studies indicated that phosphorylation of CaD at the Erk sites is accompanied by a conformational change that partially dissociates CaD from actin. Such a structural change in h-CaD exposes the myosin-binding sites on the actin surface and allows actomyosin interactions in smooth muscles. In the case of non-muscle cells, the change in l-CaD weakens the stability of the actin filament and facilitates its disassembly. Indeed, the level of l-CaD modification correlates very well in a reciprocal manner with the level of actin stress fibers. Since both cell migration and cell division require dynamic remodeling of actin cytoskeleton that leads to cell shape changes, phosphorylation of CaD may therefore serve as a plausible means to regulate these processes. Thus CaD not only links the smooth muscle contractility and non-muscle motility, but also provides a common mechanism for the regulation of cell migration and cell proliferation. [ABSTRACT FROM AUTHOR]

Abstract: The function of the ubiquitous actin-binding protein, caldesmon (l-CaD) in mammalian non-muscle cells remains elusive. During mitosis, l-CaD becomes markedly phosphorylated at Ser497 and Ser527 (in the rat sequence), therefore, it has been suggested that l-CaD is involved in cytokinesis by inhibiting the actomyosin interaction until it is phosphorylated, although direct in vivo evidence is still missing. In the present study, we used F-actin staining and specific antibodies against these two phosphorylation sites of l-CaD to simultaneously monitor actin assembly and l-CaD phosphorylation. Our observations demonstrated that the level of l-CaD phosphorylation undergoes dynamic changes during the cell cycle. The spatial and temporal distributions of phospho-CaD do not correlate with cytokinesis per se, but rather, with the level of actin bundles in a reciprocal manner. The highest l-CaD phosphorylation level coincides with the disassembly of actin cytoskeleton during mitotic cell rounding. Ser-to-Ala mutations at these two positions prevent stress fibers from disassembly upon migratory stimulation. In addition, phospho-CaD appears to colocalize with nascent focal adhesion complexes during postmitotic spreading. These findings suggest that l-CaD phosphorylation plays an important role not only in cytoskeleton remodeling during cell shape changes, but also in cell spreading and migration. [Copyright &y& Elsevier]

Abstract: Caldesmon (CaD) is thought to regulate smooth muscle contraction, because it binds actin and inhibits actomyosin interactions. A synthetic actin-binding peptide (GS17C) corresponding to Gly666–Ser682 of chicken gizzard CaD has been shown to induce force development in permeabilized smooth muscle cells. The mechanism of GS17C’s action remains unclear, although a structural effect was postulated. By photo-crosslinking and fluorescence quenching experiments with a gizzard CaD fragment (H32K; Met563-Pro771) and its mutants, we showed that GS17C indeed dissociated the C-terminal region of H32K from actin, in a manner similar to extracellular signal-regulated kinase-mediated phosphorylation, thereby reversing the CaD-imposed inhibition and enabling the actomyosin interaction. [Copyright &y& Elsevier]

We previously reported that caldesmon (CaD), together with tropomyosin (TM), effectively protects actin filaments from gelsolin, an actin-severing protein. To elucidate the structure/function relationship of CaD, we dissected the functional domain of CaD required for the protection. The basic C-terminal half of rat nonmuscle CaD (D3) inhibits gelsolin activity to the same degree as intact CaD, although a smaller C-terminal region of D3 does not. This smaller C-terminal region contains the minimum regulatory domain responsible for the inhibition of actomyosin ATPase, and for the binding to actin, calmodulin and TM. These results suggest that the domain responsible for the inhibition of gelsolin activity lies outside the minimum regulatory domain, and that the positive charge possessed by the C-terminal half of CaD is important for its interaction with actin. Moreover, while the D3 fragment promotes the aggregation of F-actin into bundles as reported previously, this bundle formation is inhibited by the acidic N-terminal half of CaD, as well as by poly-l-glutamate. It seems likely that the acidic N-terminal half of CaD neutralizes the superfluous basic feature of the C-terminal half. A comparison between D3 and calponin, another actin-binding protein that is also basic and has similar actin-regulatory activities, is also discussed. [ABSTRACT FROM PUBLISHER]

Both protein kinase C (PKC) and extracellular signal-regulated kinases (ERK1/2) are involved in mediating vascular smooth muscle contraction. We tested the hypotheses that in addition to PKC activation of ERK1/2, by negative feedback ERKs modulate PKC-induced contraction, and that their interactions modulate both thick and thin myofilament pathways. In ovine middle cerebral arteries (MCA), we measured isometric tension and intracellular free calcium concentration ([Ca2+]i) responses to PKC stimulation [phorbol 12,13-dibutyrate (PDBu), 3 ⊗ 10-6 M] in the absence or presence of ERK1/2 inhibition (U-0126, 10-5 M). After PDBu z ERK1/2 inhibition, we also examined by Western immunoblot the levels of total and phosphorylated ERK1/2, caldesmonSer789, myosin light chain20 (MLC20), and CPI-17. PDBu induced significant increase in tension in the absence of increased [Ca2+]i. PDBu also increased phosphorylated ERK1/2 levels, a response blocked by U-0126. In turn, U-0126 augmented PDBu-induced contractions. PDBu also was associated with significant increases in phosphorylated caldesmonSer789 and MLC20 levels, each of which peaked at 5 to 10 min. PDBu also increased phosphorylated CPI-17 levels, which peaked at 2 to 3 min. Rho kinase inhibition (Y-27632, 3 ⊗ 10-7 M) did not alter PDBu-induced contraction. These results support the idea that PKC activation can increase CPI-17 phosphorylation to decrease myosin light chain phosphatase activity. In turn, this increases MLC20 phosphorylation in the thick filament pathway and increases Ca2+ sensitivity. In addition, ERK1/2-dependent phosphorylation of caldesmonSer789 was not necessary for PDBu-induced contraction and appears not to be involved in the reversal of caldesmon's inhibitory effect on actin-myosin ATPase. [ABSTRACT FROM AUTHOR]

Abstract: Caldesmon (CaD) is an actin-binding protein that is capable of inhibiting the actomyosin ATPase activity in vitro. CaD has a single gene that is alternatively spliced to generate the smooth muscle-specific form, h-CaD, and a shorter isoform, l-CaD, that is present only in non-muscle cells. The difference between h- and l-CaD is a highly charged repeating sequence, corresponding to a 35nm-long single helical region that separates the N-terminal domain from the C-terminal domain of h-CaD. To test whether such an elongated h-CaD is essential for smooth muscles to function properly, we have specifically abrogated its expression in the mouse by targeting h-CaD without affecting the expression of l-CaD. After genotyping, we have obtained homozygous knockout mice that indeed lack h-CaD, but nevertheless express varying amounts of l-CaD in a tissue-dependent fashion. The contractility of smooth muscles isolated from the knockout animals is currently under investigation. [Copyright &y& Elsevier]

Summary: Stromal tumours of the endometrium can be difficult to distinguish from smooth muscle tumours of the uterus when evaluating conventional morphology alone. This review focuses on the value of immunohistochemical markers for diagnosing endometrial stromal tumours with emphasis on the recent literature. Low-grade endometrial stromal sarcomas show consistent expression of CD10 but the specificity of staining is low, since approximately 50% of highly cellular leiomyomas and almost all leiomyosarcomas are also positive for CD10. Therefore, a panel of markers must be used for distinguishing an endometrial stromal tumour from a uterine smooth muscle tumour, including antibodies against CD10, desmin, h-caldesmon and oxytocin receptors. On rare occasions, additional markers such as inhibin, CD117 and HMB-45 may be of help in the differential diagnosis. [Copyright &y& Elsevier]

A thin-filament-regulated latch-bridge model of smooth muscle contraction is proposed to integrate thin-filament-based inhibition of actomyosin ATPase activity with myosin phosphorylation in the regulation of smooth muscle mechanics. The model included two latch-bridge cycles, one of which was identical to the four-state model as proposed by Hal and Murphy (Am J Physiol Cell Physiol 255: C86-C94, 1988), whereas the ultraslow cross-bridge cycle has lower cross-bridge cycling rates. The model-fitted phorbol ester induced slow contractions at constant myosin phosphorylation and predicted steeper dependence of force on myosin phosphorylation in phorbol ester-stimulated smooth muscle. By shifting cross bridges between the two latch-bridge cycles, the model predicts that a smooth muscle cell can either maintain force at extremely low-energy cost or change its contractile state rapidly, if necessary. Depending on the fraction of cross bridges engaged in the ultraslow latch-bridge cycle, the model predicted biphasic kinetics of smooth muscle mechanics and variable steady-state dependencies of force and shortening velocity on myosin phosphorylation. These results suggest that thin-filament-based regulatory proteins may function as tuners of actomyosin ATPase activity, thus allowing a smooth muscle cell to have two discrete cross-bridge cycles with different cross-bridge cycling rates. [ABSTRACT FROM AUTHOR]

This paper reviews the contractility and the expression of contractile and regulatory proteins in the detrusor smooth muscle (DSM) following partial bladder outlet obstruction (PBOO) in rabbits. PBOO was surgically induced by partial ligation of the urethra in adult male New Zealand White rabbits. The force generated by DSM strips from normal and obstructed bladders which showed bladder dysfunction, despite detrusor hypertrophy (decompensated bladder, DB) was measured. The expression of contractile and regulatory proteins was analyzed by reverse transcriptase-polymerase chain reaction and Western blotting. The DSM from obstructed DB revealed an overexpression of SM-A myosin heavy chain isoform (associated with decreased maximum velocity of shortening). DSM from sham-operated rabbits showed phasic contractions, whereas the detrusor from DB was tonic, exhibiting slow development of force, a longer duration of force maintenance, and slow relaxation. Rho-kinase inhibitor Y-27632 enhanced the relaxation of precontracted (with 125 mM KCl) DSM strips from DB. The enhancement of relaxation of DB by Y-27632 was associated with dephosphorylation of myosin light chain. The detrusor from normal bladders expresses predominantly the smooth muscle caldesmon (h-CaD), a thin filament-associated protein. However, the DSM from DB shows an overexpression of l-CaD, the non-muscle isoform of CaD. The l-CaD colocalizes with myosin in the cytoplasmic filaments in myocytes. These results show that the alteration of contractility of the detrusor following PBOO is associated with changes in the expression of proteins that form the contractile apparatus and regulate the actomyosin ATPase activity and contraction. [ABSTRACT FROM AUTHOR]

We have previously shown that thrombin induces endothelial cell barrier dysfunction via cytoskeleton activation and contraction and have determined the important role of endothelial cell myosin light chain kinase (MLCK) in this process. In the present study we explored p38 MAP kinase as a potentially important enzyme in thrombin-mediated endothelial cell contractile response and permeability. Thrombin induces significant p38 MAP kinase activation in a time-dependent manner with maximal effect at 30 min, which correlates with increased phosphorylation of actin- and myosin-binding protein, caldesmon. Both SB-203580 and dominant negative p38 adenoviral vector significantly attenuated thrombin-induced declines in transendothelial electrical resistance. Consistent with these data SB-203580 decreased actin stress fiber formation produced by thrombin in endothelium. In addition, dominant negative p38 had no effect on thrombin-induced myosin light chain diphosphorylation. Thrombin-induced total and site-specific caldesmon phosphorylation (Ser789) as well as dissociation of caldesmon-myosin complex were attenuated by SB-203580 pretreatment. These results suggest the involvement of p38 MAP kinase activities and caldesmon phosphorylation in the MLCK-independent regulation of thrombin-induced endothelial cell permeability. [ABSTRACT FROM AUTHOR]

We previously generated an isoformspecific gene knockout mouse in which SM-B myosin is permanently replaced by SM-A myosin. In this study, we examined the effects of SM-B myosin loss on the contractile properties of vascular smooth muscle, specifically peripheral mesenteric vessels and aorta. The absence of SM-B myosin leads to decreased velocity of shortening and increased isometric force generation in mesenteric vessels. Surprisingly, the same changes occur in aorta, which contains little or no SM-B myosin in wild-type animals. Calponin and activated mitogenactivated protein kinase expression is increased and caldesmon expression is decreased in aorta, as well as in bladder. Light chain-17b isoform (LC17b) expression is increased in aorta. These results suggest that the presence or absence of SM-B myosin is a critical determinant of smooth muscle contraction and that its loss leads to additional changes in thin filament regulatory proteins. [ABSTRACT FROM AUTHOR]

The present study tested the hypothesis that ERK activation is an essential step in the onset of labor in a rat model of preterm labor. The administration of RU-486, an antiprogesterone agent, to rats induced preterm delivery 22.2 ± 0.24 h after treatment. Changes in basal signaling events were studied in myometrial tissue from CO2-euthanized rats. Rats treated with RU-486 displayed a dramatically increased in vitro uterine contractility compared with gestational stagematched, sham-treated rats. In vitro contractility was not significantly different from that during spontaneous labor. During RU-486-induced preterm labor, as previously described for spontaneous labor, ERK phosphorylation levels increased, as did phosphorylation of caldesmon at Ser789, an ERK phosphorylation site. Also, a small but significant increase in 20-kDa myosin light chain phosphorylation was seen at a constant intracellular pCa of 7. When rats were chronically treated with an agent that prevents ERK activation, U-0126, the onset of RU-486-induced preterm labor was delayed in a statistically significant manner. Chronic in vivo treatment with U-0126 also significantly inhibited the RU-486-induced increase in in vitro contractility and ERK and caldesmon phosphorylation but did not alter the RU486-induced increase in 20-kDa myosin light chain phosphorylation. These data indicate that ERK activation is a component of the multiple events leading to the development of labor in this rat model. We suggest that the ERK pathway could possibly be used to identify targets for the development of a novel class of tocolytic agents. [ABSTRACT FROM AUTHOR]

Despite intensive in vitro studies, little is known about the regulation of caldesmon (CAD) by Ca2+-calmodulin (Ca2+-CaM) in vivo. To investigate this regulation, a mutant was generated of the C-terminal fragment of human fibroblast CaD, termed CaD39-AB, in which two crucial tryptophan residues involved in Ca2+-CaM binding were each replaced with alanine. The mutation abolished most CaD39-AB binding to Ca2+-CaM in vitro but had little effect on in vitro binding to actin filaments and the ability to inhibit actin/tropomyosin-activated heavy meromyosin ATPase. To study the functional consequences of these mutations in vivo, we transfected an expression plasmid carrying CaD39-AB cDNA into Chinese hamster ovary (CHO) cells and isolated several clones expressing various amounts of CaD39-AB. Immunofluorescence microscopy revealed that mutant CaD39-AB was distributed diffusely throughout the cytoplasm but also concentrated at membrane ruffle regions. Stable expression of CaD39-AB in CHO cells disrupted assembly of stress fibers and focal adhesions, altered cell morphology, and slowed cell cycle progression. Moreover, CaD39-AB-expressing cells exhibited motility defects in a wound-healing assay, in both velocity and the persistence of translocation, suggesting a role for CaD regulation by Ca2+-CaM in cell migration. Together, these results demonstrate that CaD plays a crucial role in mediating the effects of Ca2+-CaM on the dynamics of the actin cytoskeleton during cell migration. [ABSTRACT FROM AUTHOR]

Caldesmon is a component of smooth muscle thin filaments which inhibits their interaction with myosin. We have used polarized fluorescence technique to study the behavior of caldesmon during the interaction of myosin subfragment 1 (S1) with thin filaments reconstituted in rabbit skeletal muscle ghost fibers by incorporation of smooth muscle tropomyosin and caldesmon labeled with acrylodan at cysteine residue located in the C-terminal region. Significant changes in acrylodan fluorescence intensity upon addition of skeletal muscle S1 reflected substantial displacement of caldesmon from thin filaments, while alterations in the calculated fluorescence parameters indicated the simultaneous rearrangement of the remaining caldesmon fraction. The orientation of caldesmon in the S1-thin filament complex relative to the fiber axis changes by approximately 7° and the mobility of the fluorescent probe by about 9%. The alterations in caldesmon orientation were proportional to the strength of S1 binding and diminished respectively upon addition of ADP and ADP-Vi. The changes in orientation of acrylodan-caldesmon evoked by the interaction of S1 with thin filaments were more pronounced than that in AEDANS-F-actin which suggests that the spatial arrangement of caldesmon in the complex is governed not only by F-actin but also by S1. The results may indicate that the changes in spatial arrangement of caldesmon are adjusted to the conformation of F-actin and S1 characteristic for particular steps of the ATP hydrolysis cycle. [Copyright &y& Elsevier]

Abstract Background: The purpose of the present study was to investigate morphological changes in bladder smooth muscle of rats with partial outlet obstruction. We investigated smooth muscle cell phenotypic changes and implication of synthetic phenotype in contractility decrease and bladder compliance after bladder outlet obstruction. Methods: Partial bladder outlet obstruction was introduced in female rats. Bladder were removed at 1, 3, 6, 10 and 20 weeks after the obstruction. Temporal pattern of changes in bladder mass, light microscopic pathogenesis and phenotypic expression of the bladder smooth muscle cells in the electron micrographs were investigated. Expression of contractile protein was also investigated by the immunoblotting method. Results: Marked increase in bladder mass with marked thickening of smooth muscle layer was observed at 1 week after obstruction. The ratio of myocytes exhibiting contractile and synthetic phenotypes was almost constant until 6 weeks after the obstruction, but thereafter, synthetic phenotypes gradually increased and the ratio (synthetic/contractile phenotype) was 1.5-fold at 20 weeks after the obstruction. Caldesmon was most markedly expressed after the obstruction among contractile proteins examined by the immunoblotting method. Conclusion: Phenotypic changes were confirmed in bladder smooth muscle, and the decrease of the ratio of contractile phenotype was observed after long-term obstruction of the bladder outlet. Among the contractile proteins in the bladder smooth muscle cell, caldesmon was considered a reliable marker for predicting the pathogenetic conditions of the bladder. [ABSTRACT FROM AUTHOR]

Aims: To assess the frequency of expression and potential diagnostic utility of calponin and caldesmon in synovial sarcomas. Methods and results: Immunohistochemistry with antibodies to calponin and h-caldesmon was undertaken in paraffin sections from 50 synovial sarcomas (19 biphasic and 31 monophasic, of which one biphasic and eight monophasic tumours had poorly differentiated areas), 10 each of malignant peripheral nerve sheath tumour (MPNST), solitary fibrous tumour (SFT), dermatofibrosarcoma (DFSP), Ewing's sarcoma (ES/PNET), and neuroblastoma, eight alveolar rhabdomyosarcomas, five adult fibrosarcomas and five carcinosarcomas. Nine of 19 biphasic synovial sarcomas were positive for calponin in spindle and glandular areas, nine in spindle cells only, and one in glands only. All monophasic synovial sarcomas and poorly differentiated areas expressed calponin; in monophasic tumours this was focally (29% of cases), moderately (39%), or diffusely (32%) positive and the poorly differentiated areas were usually moderately or diffusely positive. Four synovial sarcomas showed focal reactivity for h-caldesmon. Calponin was found in 4/10 MPNST, 7/10 SFT, 4/10 DFSP, 3/5 fibrosarcomas and the spindle component of the carcinosarcomas. H-caldesmon was weakly positive in 1/10 MPNST, 4/10 SFT, 0/10 DFSP, 0/5 fibrosarcomas and 1/5 carcinosarcomas (glands only). Both markers were negative in the other small round cell tumours. Conclusions: Calponin can be used as an additional marker for synovial sarcoma. Its absence argues against the diagnosis. The presence of calponin might be useful in distinguishing poorly differentiated synovial sarcoma from other small round cell tumours. H-caldesmon is not helpful in diagnosis of synovial sarcoma. [ABSTRACT FROM AUTHOR]

Summary Background The structural integrity of the blood vessels such as small arteries and veins is studied less frequently in malignant tumours than is angiogenesis. Objectives To clarify the characteristics of small arteries and small veins of melanoma lesions. Methods We immunohistochemically investigated various types of melanocytic tumours using antibodies specific for endothelial and vascular smooth muscle cells, and analysed the relationship between the expression of these molecules in the blood vessels and the biological characteristics of the tumours. Formalin-fixed, paraffin-embedded sections of 15 cases of benign melanocytic tumours and 64 cases of malignant melanomas were investigated. Results Significant suppression of expression of h-caldesmon (h-CD) and calponin h1 (CNh1) was observed in the blood vessels of malignant melanomas compared with both benign melanocytic tumours and normal tissues. In particular, the level of h-CD expression was inversely correlated with the frequency of metastasis and positively correlated with the survival rate in patients with malignant melanoma. Conclusions These findings suggest that alterations of the tumour vessels are an important factor for the prognosis of malignant melanoma, and that suppression of h-CD and CNh1 in the blood vessels in malignant melanoma reflects a structural fragility of the vessels, leading to their easy penetration by tumour cells. Defective expression of these molecules is likely to be an important marker for metastatic potential and for poor prognosis of melanoma. [ABSTRACT FROM AUTHOR]

Relay of information from the extracellular environment into the cell often results from a peptide growth factor binding to its cognate cell surface receptor; this event is an integral mechanism by which many cellular functions occur, including cell growth, motility, and survival. In recent years, however, this requirement for ligand binding has been shown to be surpassed by several distinct mechanisms, including cell surface receptor cross-talk (e.g., between epidermal growth factor receptor [EGFR] and G-coupled receptors), receptor-extracellular matrix interactions (e.g., EGFR: integrin complexes), and finally by structural mutations within the receptor itself. While all of these pathways result in so-called ligand-independent signaling by the EGF receptor, to date, only structural mutations in the receptor have been shown to result in qualitative changes in downstream targets of the receptor, which specifically result in oncogenic signaling, transformation, and tumorigenicity. In this review, we describe aspects of the known signaling properties of the retroviral oncogene v-ErbB as a model of ligand-independent oncogenic signaling, and compare these properties to results emerging from ongoing studies on structurally related EGF receptor mutants originally identified in human tumors. A better understanding of the signaling pathways used by these uniquely oncogenic receptor tyrosine kinase mutants may ultimately reveal new targets for the development of novel therapeutics selective for the inhibition of tumor cell growth. [Copyright &y& Elsevier]

The calcineurin-mediated pathway is involved in skeletal and cardiac hypertrophy and vascular development in vivo, but the relationship between this pathway and the phenotype of smooth muscle cells (SMCs) remains unknown. Using visceral SMCs in culture as a model system of differentiated SMCs, we investigated the role of the calcineurin-mediated pathway in maintaining the differentiated phenotype of SMCs, which depends on the insulin-like growth factor (IGF-I)-triggered activation of the phosphatidylinositol 3-kinase (PI3-K)/protein kinase B (PKB(Akt)) pathway. Treatment with calcineurin inhibitors, cyclosporin A or FK506, or the forced expression of the natural calcineurin inhibitor, CAIN, induced SMC dedifferentiation. Notably, suppression of the promoter activities of the SMC molecular markers caldesmon and α1 integrin by blocking the PI3-K/PKB(Akt) pathway was rescued by the forced expression of constitutively active calcineurin Aα, suggesting that the calcineurin-mediated pathway is critical for maintaining the differentiated phenotype of SMCs and works downstream of the PI3-K/PKB(Akt) pathway. [Copyright &y& Elsevier]

Smooth muscles exist in the wall of hollow organs in our body and are responsible for controlling the flow of vital fluids that are essential for the normal function of the cardiovascular, respiratory, digestive, and reproductive systems. Many diseases, such as hypertension, asthma, indigestion, and premature birth, may attribute to malfunction of smooth-muscle contraction. It is therefore important to decipher how smooth-muscle contraction is regulated. This review attempts to give a brief overview of current understanding about the molecular mechanisms of smooth-muscle regulation and, in particular, to discuss possible roles of caldesmon in this regulatory process. [ABSTRACT FROM AUTHOR]

The interaction of caldesmon with different Ca2+-binding proteins has been analyzed, and it is supposed that one of the conformers of calmodulin might be an endogenous regulator of caldesmon. The arrangement of caldesmon and Ca2+-binding proteins within their complexes has been analyzed by different methods. The central helix of calmodulin is supposed to be located near the single Cys residue in the C-terminal domain of caldesmon. The N-terminal globular domain of calmodulin interacts with sites A and B" of caldesmon, whereas the C-terminal globular domain of calmodulin binds to site B of caldesmon. The complex of calmodulin and caldesmon is very flexible; therefore, both parallel and antiparallel orientation of polypeptide chains of the two proteins is possible in experiments with short fragments of caldesmon and calmodulin. The length, flexibility, and charge of the central helix of calmodulin play an important role in its interaction with caldesmon. Phosphorylation of caldesmon by different protein kinases in vitro has been analyzed. It was shown that phosphorylation catalyzed by casein kinase II of sites located in the N-terminal domain decreases the interaction of caldesmon with myosin and tropomyosin. Caldesmon and calponin may interact with phospholipids. The sites involved in the interaction of these actinbinding proteins with phospholipids have been mapped. It is supposed that the interaction of calponin and caldesmon with phospholipids may play a role in the formation of cytoskeleton. Calponin interacts with 90-kD heat shock protein (hsp90) that may be involved in transportation of calponin and its proper interaction with different elements of cytoskeleton. Calponin, filamin, and a-actinin can simultaneously interact with actin filaments. Simultaneous binding of two actin-binding proteins affects the structure of actin bundles and their mechanical properties and may be of great importance in formation of different elements of cytoskeleton. [ABSTRACT FROM AUTHOR]

Our recent study of developing myoepithelial cells (MECs) in rat salivary glands demonstrated that developing MECs begin to express α-smooth muscle actin (αSMA) first and, thereafter, keratin 14. Therefore, it is unlikely that duct basal cells expressing keratin 14 alone are immature or undifferentiated MECs. In this study we carried out immunohistochemistry of pleomorphic adenomas and adenoid cystic carcinomas including normal salivary glands using monoclonal antibodies to keratin 14, smooth muscle proteins and keratin 19. The smooth muscle proteins examined included αSMA, h-caldesmon and h1-calponin; h1-calponin was observed in keratinocytes and nerve fibers, indicating that the protein is not specific to smooth muscle, whereas αSMA and h-caldesmon turned out to be highly specific markers for smooth muscle cells in normal tissues. In normal glands, MECs were positive for both keratin 14 and smooth muscle proteins (αSMA and h-caldesmon). Non-MEC cells were essentially devoid of smooth muscle proteins. Non-MEC duct basal cells expressed keratin 14 with or without keratin 19, and luminal cells keratin 19 with or without keratin 14. This suggests that the keratin 14-positive, smooth muscle proteins-negative duct basal cells are luminal cell progenitors. Luminal cells in tubular structures of both tumors were positive for keratin 19 with or without keratin 14. Nonluminal peripheral cells of pleomorphic adenomas were mostly positive for keratin 14, and a small fraction of them expressed smooth muscle proteins. Conversely, peripheral cells of adenoid cystic carcinomas were mostly positive for smooth muscle proteins, and some of them expressed keratin 14. These results strongly suggest (1) that the luminal cell progenitors transform into major constituents of pleomorphic adenoma cells with keratin 14 but not smooth muscle proteins, and (2) that the peripheral cells of adenoid cystic carcinoma are derived from undifferentiated MECs. Solid structures of pleomorphic adenomas were formed by proliferation of the peripheral cells. MECs were observed only occasionally in the periphery. Solid and cribriform structures of adenoid cystic carcinomas were formed by proliferation of the luminal cells. MECs were observed in the periphery and around the pseudocyst. [ABSTRACT FROM AUTHOR]

An immunohistochemical study of developing glomeruli in human fetal kidneys. Background. In the glomerulonephritis, mesenchymal cells frequently repeat the expression of fetal immunohistochemical phenotypes. However, in human glomerulogenesis the phenotypic alteration of mesangial and other types of glomerular cells has not been clearly defined. Our aim was to clarify the characteristics of fetal mesangial cells and glomerular capillary endothelial cells, as well as their changes during glomerulogenesis using immunohistochemistry. Methods. We examined the renal tissues of 34 autopsied fetuses and neonates, 5 children, and 5 adults using immunohistochemistry and immunoelectron microscopy, using antibodies for cytoskeletons, contraction-associated proteins, and endothelial cell markers. Results. In the V and S stages, there were no cells showing mesangial and endothelial features within the vesicles and the S-shaped bodies. In the S stage, small blood vessels, consisting of endothelial cells (CD31+ , CD34+ ) and primitive perivascular mesenchymal cells (α-smooth muscle actin+ , low molecular caldesmon+ , vimentin+ ), were branched from developing interlobular arteries and appeared to extend to the lower clefts of the S-shaped bodies. In the C stage, the perivascular mesenchymal cells aggregated at the root of the immature glomeruli. In the M stage, they migrated toward the periphery of immature glomeruli and gradually lost their fetal immunohistochemical features. Similarly, with further maturation, the fetal glomerular capillary endothelial cells gradually lost the immunostaining for CD34, while the strong staining intensity of CD31 remained unchanged, just as that in the adult glomerular capillary endothelial cells. Conclusions. In human glomerulogenesis, we demonstrate that fetal mesangial and capillary endothelial cells change their immunohistochemical phenotypes with maturation. They gradually lose... [ABSTRACT FROM AUTHOR]

Studies normal salivary, mammary, and sweat glands with markers of smooth muscle differentiation. Markers useful for identifying myoepithelial cells; Role of the differential expression of smooth muscle contractile proteins in reflecting different functions of myoepithelial cells in the various sites.

Immunohistochemical and clinicopathological features of 58 gastrointestinal stromal tumors (GIST) were studied. One occurred in the esophagus, 41 in the stomach, nine in the small intestine, and seven in the large intestine. By using indirect immunoperoxidase staining for Cajal cell markers (c-kit protein and CD34), smooth muscle markers (α-smooth muscle actin, desmin, heavy caldesmon and calponin) and Schwann cell markers (S-100 protein and Leu 7), GIST were classified into five groups, such as Cajal cell type (n = 9), myogenic type (n = 5), Schwann cell type (n = 2), mixed cell type (n = 40) and undifferentiated type (n = 2). c-kit Protein (42/58; 72%) and CD34 (45/58; 78%) were commonly and diffusely expressed in GIST. Novel smooth muscle markers, caldesmon (29/58; 50%) and calponin (18/58; 31%), were useful in detecting myogenic characters of GIST. S-100 protein was expressed in 16 (28%) tumors, two of which were also reactive with Leu 7 (CD57). Three small bowel tumors with skeinoid fibers expressed the Cajal cell markers, and were categorizable in GIST. Clinicopathological analyses using aggressive (n = 21) and non-aggressive (n = 21) GIST indicated that the malignant potential was correlated with the intestinal location, large tumor size, high cellularity, necrosis, solid (non-interlacing bundled) pattern of growth, negativity of c-kit protein and/or CD34, high mitotic count, and high MIB-1 labeling. [ABSTRACT FROM AUTHOR]

Background. Phenotypic changes in glomerular mesangial cells and interstitial cells are considered to be closely associated with the progression of glomerulosclerosis and renal fibrosis in IgA nephropathy. To further evaluate the relevance of phenotypic changes, we examined the expression of four different contractile proteins as markers of the phenotypic changes. Methods. The expression of nonmuscle myosin heavy chain isoform (SMemb), α-smooth muscle actin (α-SMA), caldesmon (CaD), and tropomyosin (TM) was evaluated by indirect immunofluorescent studies in renal biopsy specimens from 21 patients with IgA nephropathy. The relationships with the histological parameters and clinical parameters were analyzed. Results. Pronounced expression (score of 2 or more) of contractile proteins was observed in the majority of the patients (57%–90%) compared with controls. There was no significant relationship between the glomerular expression score of any contractile protein and the proliferation index or sclerotic index. Daily urinary protein excretion in the group expressing low levels of caldesmon in the glomerulus was significantly lower than that in the group expressing high levels ( P < 0.05). There were significant correlations among the glomerular expressions of these contractile proteins, except for TM ( P < 0.05). All contractile proteins were expressed in tubulointerstitial lesions, and only SMemb expression was detected in tubular epithelial cells. The interstitial scores of all contractile proteins increased in parallel with the degree of tubulointerstitial lesion ( P < 0.05). Conclusions. Contractile proteins appeared to be coordinately expressed in the glomerulus and the interstitium in IgA nephropathy, which may reflect the mechanical stress involved with glomerulonephritis. [ABSTRACT FROM AUTHOR]

The basic structure and functional properties of smooth muscle thin filaments were established about 10 years ago. Since then we and others have been working on the details of how tropomyosin, caldesmon and the Ca2+-binding protein regulate actin interaction with myosin. Our work has tended to emphasize the similarities between caldesmon and troponin function whilst others have been more concerned with the differences. The need to resolve the resulting differences has stimulated us to find new and more direct ways of investigating the mechanism of thin filament regulation. In recent years an apparent divergence has opened up between functional measurements, which indicate an allosteric-cooperative regulatory mechanism in which caldesmon and Ca2+-binding protein control actin-tropomyosin state in the same way as troponin, and structural measurements which show thin filament structures unlike striated muscle thin filaments. The challenge is to interpret function in terms of structure. We have combined functional studies with expression and mutagenesis of caldesmon and with structural methods including X-ray crystalography of tropomyosin- caldesmon crystals, electron microscopy and helical reconstruction of actin-tropomyosin-caldesmon complexes and high resolution nuclear magnetic resonance spectroscopy of the C-terminus of caldesmon in interaction with actin and calmodulin. We have used this information to propose a structural mechanism for caldesmon regulation of the smooth muscle thin filament. [ABSTRACT FROM AUTHOR]

This review is focused on the cross-bridge interaction of the organized contractile system of smooth muscle fibres. By using chemically skinned preparations the different enzymatic reactions of actinmyosin interaction have been associated with mechanical events. A rigor state has been identified in smooth muscle and the binding of ATP causes dissociation of rigor cross-bridges at rates slightly slower than those in skeletal muscle, but fast enough not to be rate-limiting for cross-bridge turn over in the muscle fibre. The release of inorganic phosphate (Pi) is associated with force generation, and this process is not rate-limiting for maximal shortening velocity (Vmax) in the fully activated muscle. The binding of ADP to myosin is strong in the smooth muscle contractile system, a property that might be associated with the generally slow cross-bridge turn over. Both force and Vmax are modulated by the extent of myosin light chain phosphorylation. Low levels of activation are considered to be associated with the recruitment of slowly cycling dephosphorylated cross-bridges which reduces shortening velocity. The attachment of these cross-bridge states in skinned smooth muscle can be regulated by cooperative mechanisms and thin filament associated systems. Smooth muscles exhibit a large diversity in their Vmax and the individual smooth muscle tissue can alter its Vmax under physiological conditions. The diversity and the long-term modulation of phenotype are associated with changes in myosin heavy and light chain isoform expression. [ABSTRACT FROM AUTHOR]

Protein kinase C (PKC) is now known to play an important physiological role in essentially all cell types. This review will focus on what is known about the kinase in contractile differentiated smooth muscle. Current knowledge on the molecular structure of PKC isoforms will be discussed as they relate to mechanisms of translocation and targeting of the kinase within smooth muscle cells. Studies performed on PKC-dependent signalling pathways in differentiated smooth muscle cells will be discussed with emphasis on studies from our laboratory, especially discussing thin filament linked pathways. Thick filament linked PKC-dependent pathways will be described in more detail elsewhere in this monograph. [ABSTRACT FROM AUTHOR]

The actin binding protein caldesmon inhibits the actin-activation of myosin ATPase activity. The steps in the cycle of ATP hydrolysis that caldesmon could inhibit include: (1) the binding of myosin to actin, (2) the transition between any two actin–myosin states and (3) the distribution between inactive and active states of actin. The analysis of these possibilities is complicated because caldesmon binds to both myosin and actin and because each caldesmon molecule binds to several actin monomers. This paper reviews procedures for analysing these interactions and summarizes current information on the stability and dynamics of the interaction of caldesmon with actin and myosin. Possible effects of caldesmon on transitions within the ATPase cycle of actomyosin are also discussed. [ABSTRACT FROM AUTHOR]

Phenotypic modulation of smooth muscle cells is closely associated with vasculogenesis, enterogenesis and some diseases such as atherosclerosis, hypertension and leiomyogenic tumorigenicity. During phenotypic modulation, smooth muscle cells change their morphology, cell function and biochemical characteristics. Recent studies have focused on the regulation mechanism of smooth muscle cell-specific genes at the levels of transcription and/or alternative splicing in a phenotype-dependent manner. Typical examples of such genes include caldesmon, α-tropomyosin, myosin heavy chain, SM22, calponin and α1 integrin. Cell adhesion molecules and growth factors/cytokines also play a critical role for controlling phenotype of smooth muscle cells via signal transduction pathways such as phosphoinositide 3-kinase and mitogen-activated protein kinases. [ABSTRACT FROM AUTHOR]

Calmodulin, the ubiquitous and multifunctional Ca-binding protein, mediates many of the regulatory effects of Ca, including the contractile state of smooth muscle. The principal function of calmodulin in smooth muscle is to activate crossbridge cycling and the development of force in response to a [Ca] transient via the activation of myosin light-chain kinase and phosphorylation of myosin. A distinct calmodulin-dependent kinase, Ca/calmodulin-dependent protein kinase II, has been implicated in modulation of smooth-muscle contraction. This kinase phosphorylates myosin light-chain kinase, resulting in an increase in the calmodulin concentration required for half-maximal activation of myosin light-chain kinase, and may account for desensitization of the contractile response to Ca. In addition, the thin filament-associated proteins, caldesmon and calponin, which inhibit the actin-activated MgATPase activity of smooth-muscle myosin (the cross-bridge cycling rate), appear to be regulated by calmodulin, either by the direct binding of Ca/calmodulin or indirectly by phosphorylation catalysed by Ca/calmodulin-dependent protein kinase II. Another level at which calmodulin can regulate smooth-muscle contraction involves proteins which control the movement of Ca across the sarcolemmal and sarcoplasmic reticulum membranes and which are regulated by Ca/calmodulin, e.g. the sarcolemmal Ca pump and the ryanodine receptor/Ca release channel, and other proteins which indirectly regulate [Ca] via cyclic nucleotide synthesis and breakdown, e.g. NO synthase and cyclic nucleotide phosphodiesterase. The interplay of such regulatory mechanisms provides the flexibility and adaptability required for the normal functioning of smooth-muscle tissues. [ABSTRACT FROM AUTHOR]

The patterns of expression of the smooth muscle regulatory proteins caldesmon and myosin light chain kinase were investigated in the developing chicken gizzard. Immunofluorescent studies revealed that both proteins were expressed as early as E5 throughout the mesodermal gizzard anlage, together with actin, α-actinin and a small amount of nonmuscle myosin. These proteins appear to form the scaffold for smooth muscle development, defined by the onset of smooth muscle myosin expression. During E6, a period of extensive cell division, smooth muscle myosin begins to appear in the musculi laterales close to the serosal border and, later, also in the musculi intermedii. Until about E10, myosin reactivity expands into the pre-existing thin filament scaffold. Later in development, the contractile and regulatory proteins co-localize and show a regular uniform staining pattern comparable to that seen in adult tissue. By using immunoblotting techniques, the low-molecular mass form of caldesmon and myosin light chain kinase were detected as early as E5. During further development, the expression of caldesmon switched from the low-molecular mass to the high-molecular mass form; in neonatal and adult tissue, high-molecular mass caldesmon was the only isoform expressed. The level of expression of myosin light chain kinase increased continously during embryonic development, but no embryospecific isoform with a different molecular mass was detected. [ABSTRACT FROM AUTHOR]

The exertion of periodic dynamic strain on the arterial wall is hypothesized to be relevant to smooth muscle cell morphology and function. This study has investigated the effect of cyclic mechanical stretching on rabbit aortic smooth muscle cell proliferation and expression of contractile phenotype protein markers. Cells were cultured on flexible-bottomed dishes and cyclic stretch was applied (frequency 30 cycles/min, 15% elongation) using a Flexercell Strain unit. Cyclic stretch potentiated smooth muscle cell proliferation in serum-activated cultures but not in cultures maintained in 0.5% fetal calf serum. Stretching induced a serum-independent increase of h-caldesmon expression and this effect was reversible following termination of mechanical stimulation. Strain was without effect on smooth muscle myosin or calponin expression. In cells grown on laminin stretch-induced h-caldesmon expression was more prominent than in cells cultured on collagen types I and IV, poly-L-lysine and gelatin. These data suggest that cyclic mechanical stimulation possesses dual effect on vascular smooth muscle cell phenotype characteristics since it: 1) potentiates proliferation, an attribute of a dedifferentiated phenotype; and 2) increases expression of h-caldesmon considered a marker of a differentiated smooth muscle cell state. [ABSTRACT FROM AUTHOR]

We studied the effect of caldesmon peptides on the regulation of shortening of single guinea-pig taenia coli cells permeabilised with saponin. When contraction was initiated by Ca2+ and MgATP shortening rate at pCa 4.5 was 0.17±0.04 cell lengths s–1 and half-maximal rate was at pCa 5.6. Following thiophosphorylation with 1 mM adenosine 5′- O-(3-thiotriphosphate) (ATP[γ- S]) at pCa 4.5 for 10 min, on addition of ATP these cells contracted at of 0.25±0.04 cell lengths s–1 independently of pCa. If thiophosphorylated cells were preincubated with H1 (domains 3 and 4 of caldesmon), shortening speed was reduced (ID50=2 µM). Shortening speed was also reduced by 658C (domain 4b) at higher concentrations (ID50=400 µM). H13 (domain 4a), which does not block weak binding but inhibits actin-tropomyosin, inhibited cell shortening (ID50=6 µM). H2, which blocks weak binding but does not inhibit actin-tropomyosin, did not inhibit shortening. Western blots of the cells showed that the peptides were tightly bound within the cell but the native caldesmon was not displaced. These results indicate that exogenous caldesmon peptides added to smooth muscle cells may be incorporated into the thin filaments and produce effects on shortening, as expected if it were involved in tropomyosin-dependent inhibition of the actin filament in the cell. [ABSTRACT FROM AUTHOR]

In smooth muscle, the state of prolonged contraction (latch state) is associated with very slow energy turnover and cycling of crossbridges that are dephosphorylated. A similar state may be reproduced in skinned fibres when the calcium-induced contraction is terminated by calcium removal with ethylenebis(oxonitrilo)tetraacetate (EGTA) and, during the slow relaxation that follows, force is maintained by dephosphorylated crossbridges that cycle slowly or not at all and may cooperatively reattach after detachment (Khromov et al. 1995, Biophys J 69:2611–2622). In guinea-pig skinned taenia coli that has been pretreated by prolonged incubation with caldesmon (5 μM), the rate of relaxation is approximately 1.6 times greater than in untreated controls, with half-times of relaxation being 1.3 and 2.1 min, respectively. In contrast, preloading the fibres with calponin does not accelerate relaxation. Preloading the fibres with caldesmon also accelerates the relaxation of skinned fibres from the state of rigor contraction when the latter is terminated by immersion into an ATP-containing relaxing solution or, in the presence of inorganic phosphate (Pi), also by flash-photolytic release of ATP from caged-ATP. Even in the latter case, relaxation is comparatively slow, possibly because of cooperative reattachment of dephosphorylated crossbridges which delays net crossbridge detachment and hence relaxation. We propose that by inhibition of cooperative reattachment caldesmon accelerates relaxation, even in the presence of Pi, and that the latch-like state of skinned fibres is supported by dephosphorylated cooperatively attaching crossbridges and may be regulated by the activity of caldesmon in the smooth muscle cell. [ABSTRACT FROM AUTHOR]

The role of caldesmon in the regulation of smooth muscle contraction was investigated in chemically skinned smooth muscle fibres from the guineapig taenia coli. A 19-kDa C-terminal fragment of caldesmon gave a minor (<5%) reduction of force in fully thiophosphorylated fibres, but reduced force by about 50% at intermediate activation levels without affecting the level of light chain phosphorylation. An extraction procedure was developed using incubation in solutions containing high Mg concentrations. Protein analysis revealed a selective decrease in the amount of caldesmon in the fibres. Maximal active force per cross-sectional area was unaffected. The Ca dependence of active force was shifted towards lower Ca concentrations and became less steep. The effects of extraction of caldesmon could in part be reversed by incubation in a solution containing purified caldesmon. The results are consistent with the hypothesis that caldesmon in smooth muscle thin filaments inhibits force generation and plays a role in regulating cooperative attachment of cross-bridges at sub-maximal levels of activation in smooth muscle. [ABSTRACT FROM AUTHOR]