The cytoskeletal and contractile apparatus of smooth muscle: contraction bands and segmentation of the contractile elements.

Confocal laser scanning microscopy of isolated and antibody-labeled avian gizzard smooth muscle cells has revealed the global organization of the contractile and cytoskeletal elements. The cytoskeleton, marked by antibodies to desmin and filamin is composed of a mainly longitudinal, meandering and branched system of fibrils that contrasts with the plait-like, interdigitating arrangement of linear fibrils of the contractile apparatus, labeled with antibodies to myosin and tropomyosin. Although desmin and filamin were colocalized in the body of the cell, filamin antibodies labeled additionally the vinculin-containing surface plaques. In confocal optical sections the contractile fibrils showed a continuous label for myosin for at least 5 microns along their length: there was no obvious or regular interruption of label as might be expected for registered myosin filaments. The cytoplasmic dense bodies, labeled with antibodies to alpha-actinin exhibited a regular, diagonal arrangement in both extended cells and in cells shortened in solution to one-fifth of their extended length: after the same shortening, the fibrils of the cytoskeleton that showed colocalization with the dense bodies in extended cells became crumpled and disordered. It is concluded that the dense bodies serve as coupling elements between the cytoskeletal and contractile systems. After extraction with Triton X-100, isolated cells bound so firmly to a glass substrate that they were unable to shorten as a whole when exposed to exogenous Mg ATP. Instead, they contracted internally, producing integral of 10 regularly spaced contraction nodes along their length. On the basis of differences of actin distribution two types of nodes could be distinguished: actin-positive nodes, in which actin straddled the node, and actin-negative nodes, characterized by an actin-free center flanked by actin fringes of 4.5 microns minimum length on either side. Myosin was concentrated in the center of the node in both cases. The differences in node morphology could be correlated with different degrees of coupling of the contractile with the cytoskeletal elements, effected by a preparation-dependent variability of proteolysis of the cells. The nodes were shown to be closely related to the supercontracted cell fragments shown in the accompanying paper (Small et al., 1990) and furnished further evidence for long actin filaments in smooth muscle. Further, the segmentation of the contractile elements pointed to a hierarchial organization of the myofilaments governed by as yet undetected elements.

Myosin-I nomenclature.

We suggest that the vertebrate myosin-I field adopt a common nomenclature system based on the names adopted by the Human Genome Organization (HUGO). At present, the myosin-I nomenclature is very confusing; not only are several systems in use, but several different genes have been given the same name. Despite their faults, we believe that the names adopted by the HUGO nomenclature group for genome annotation are the best compromise, and we recommend universal adoption.

Functional analysis of the mutations in the human cardiac beta-myosin that are responsible for familial hypertrophic cardiomyopathy. Implication for the clinical outcome.

More than 30 missense mutations in the beta-cardiac myosin heavy chain gene have been shown to be responsible for familial hypertrophic cardiomyopathy. To clarify the effects of these point mutations on myosin motor function, we expressed wild-type and mutant human beta-cardiac myosin heavy chains in insect cells with human cardiac light chains. The wild-type myosin was well purified with similar enzymatic and motor activities to those of the naturally isolated V3 cardiac myosin. Arg249-->Gln and Arg453-->Cys mutations resulted in decreased actin translocating activity (61 and 23% of the wild-type, respectively) with decreased intrinsic ATPase activity. Arg403-->Gln mutation greatly decreased actin translocating activity (27% of wild type) with a 3.3-fold increased dissociation constant for actin, while intrinsic ATPase activity was unchanged. Val606-->Met mutation only mildly affected the actin translocating activity as well as ATPase activity of myosin. The degree of deterioration by each mutation was closely correlated with the prognosis of the affected kindreds, indicating that myosin dysfunction caused by the point mutations is responsible for the pathogenesis of the disease. Structure/function relationship of myosin is discussed.

Expression of Sialyl-Tn antigens in normal squamous epithelium, dysplasia, and squamous cell carcinoma in the esophagus.

Two monoclonal antibodies, TKH2 and B72.3, directed toward the Sialyl-Tn antigen (SA 2, 6GalNAc alpha-O-Ser/Thr), were examined immunohistochemically to analyze the expression of these antigens in 20 areas of normal squamous epithelium, 12 lesions of dysplasia, and 86 cases of squamous cell carcinoma including 32 with superficial carcinoma in the esophagus. No expression of TKH2 or B72.3 was found in the normal squamous epithelium. Among the 12 lesions of dysplasia only one expressed TKH2. In carcinoma the expression of TKH2 and B72.3 was found in 40 (47%) and 21 (24%) of the 86 carcinomas, respectively; however, the number of positive malignant cells with TKH2 and B72.3 totaled less than half that in the tissue, and no relationship was found between either prognosis or lymph node metastasis and the expression of Sialyl-Tn antigen. These results indicate that Sialyl-Tn antigen appears in the process of malignant transformation or tumor progression in esophageal squamous cell carcinoma; however, the positive expression of Sialyl-Tn antigen was not directly connected to either prognosis or lymph node metastasis.

A novel mechanism for the Ca(2+)-sensitizing effect of protein kinase C on vascular smooth muscle: inhibition of myosin light chain phosphatase.

Mechanisms of Ca2+ sensitization of both myosin light chain (MLC) phosphorylation and force development by protein kinase C (PKC) were studied in permeabilized tonic smooth muscle obtained from the rabbit femoral artery. For comparison, the Ca2+ sensitizing effect of guanosine 5'-O-(gamma-thiotriphosphate) (GTP gamma S) was examined, which had been previously shown to inhibit MLC phosphatase in phasic vascular smooth muscle. We now report that PKC activators (phorbol esters, short chain synthetic diacylglycerols and a diacylglycerol kinase inhibitor) and GTP gamma S significantly increase both MLC phosphorylation and force development at constant [Ca2+]. Major phosphorylation site occurring in the presence of phorbol-12,13-dibutyrate (PDBu) or GTP gamma S at constant [Ca2+] is the same serine residue (Ser-19) as that phosphorylated by MLC kinase in response to increased Ca2+ concentrations. In an ATP- and Ca(2+)-free solution containing 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine (ML-9), to avoid the kinase activity, both PDBu and GTP gamma S significantly decreased the rate of MLC dephosphorylation to half its control value. However, PDBu inhibited the relaxation rate more than did GTP gamma S. In the presence of microcystin-LR to inhibit the phosphatase activity, neither PDBu nor GTP gamma S affected MLC phosphorylation and force development. These results indicate that PKC, like activation of GTP binding protein, increases Ca2+ sensitivity of both MLC phosphorylation and force production through inhibition of MLC phosphatase.

Introduction of high molecular weight (IgG) proteins into receptor coupled, permeabilized smooth muscle.

The permeability to high molecular weight (IgG, 150 kD) proteins of the plasma membrane of receptor-coupled smooth muscles permeabilized with beta-escin was determined using confocal microscopy of immunofluorescent tracers and measurement of lactate dehydrogenase (LDH, 135-140 kD) leakage. Permeabilized strips of rabbit portal vein and guinea pig ileum were incubated in a relaxing solution containing mouse anti-smooth muscle alpha-actin antibody and immunostained with F(ab')2 labeled with tetramethyl rhodamine isothiocyanate. Confocal light microscopy of Triton X-100 and beta-escin permeabilized cells showed homogeneous staining of the cytoplasm, whereas in alpha-toxin treated and intact preparations only damaged cells at the edges of the strips were stained. Both the Ca(2+)-sensitizing effect of phenylephrine, in rabbit portal vein, and Ca2+ release by carbachol in guinea pig ileum, were retained after permeabilization and the treatment with the primary antibody. During the 30 min permeabilization, 38%, and within the next 75 min an additional approximately 30%, of the total LDH leaked out from the beta-escin-treated group, but not from the alpha-toxin-treated group (3.2%). The responsiveness to agonist and maximum contractility was improved if the preparations were incubated during the introduction of proteins at 4 degrees C, rather than 24 degrees C. Ca(2+)-independent myosin light chain kinase (61 kD) contracted the permeabilized portal vein in the absence of free Ca2+ (pCa < 8). In conclusion, permeabilization with beta-escin allows the transmembrane passage of 150 kD proteins under our experimental conditions that also retain receptor-coupled signal transduction.

Inhibition of 20-kDa myosin light chain exchange by monoclonal antibodies against 17-kDa myosin light chain.

Two anti-17,000 Da myosin light chain (LC17) monoclonal antibodies (MM2 and MM10), which increase the actin-activated Mg(2+)-ATPase activity of dephosphorylated smooth muscle myosin, inhibited the exchange of the 20,000 Da regulatory light chain of myosin (LC20). MM2, which shows higher potency of activation of ATPase activity, inhibited the exchange more extensively than MM10, suggesting that there is a correlation between the activation of ATPase activity and the inhibition of the LC20 exchange. The inhibition of the exchange was observed for intact myosin and heavy meromyosin but not subfragment 1, suggesting that the heavy chain at the head-rod junction is involved in the inhibition of LC20 exchange by anti-LC17 antibodies. Alternatively, the interaction between the two heads of the myosin molecule may influence the inhibition of LC20 exchange. These results suggest that LC20 interacts with both LC17 and the heavy chain, and the interaction between LC20 and LC17 is involved in the activation of actin-activated ATPase activity of smooth muscle myosin.

Alteration of the enzymatic properties of smooth muscle myosin by a monoclonal antibody against subfragment 2.

A monoclonal antibody against subfragment 2 (S-2) of smooth muscle myosin, designated MM-9, was generated and characterized. MM-9 potently inhibited subfragment 1 (S-1) release by papain proteolysis of myosin, suggesting that the epitope of MM-9 is at or very close to the S-1/S-2 junction. The depression of Ca2(+)- and Mg2(+)-ATPase activities of myosin at low ionic strength was significantly reduced by MM-9. MM-9 increased the acto dephosphorylated HMM ATPase activity about 3-fold. On the other hand, the antibody had no effect on the KCl-dependence of viscosity of monomeric myosin. These results suggest that the folding of the myosin rod is not the direct determinant of enzymatic activity, and that the subtle conformational change at the S-1/S-2 junction (head-neck region) plays a critical role in determining enzymatic activities.

A novel myosin I from bovine adrenal gland.

A 3.5 kb cDNA clone was isolated from bovine adrenal gland cDNA library. The clone contained a full-length 3.1 kb open reading frame, encoding a novel myosin I. The deduced amino acid sequence was highly homologous to other known myosin Is in the N-terminal 2 kb region which corresponds to the myosin head domain, while no strong homology was detected in the tail region. The head-tail junction contained the Ca(2+)-independent calmodulin binding consensus sequence, suggesting that the novel myosin I binds calmodulin. This was confirmed by calmodulin overlay which showed the binding of 125I-calmodulin to the recombinant myosin I expressed in E. coli. Northern blots with probes from head and tail regions of this myosin I revealed that this novel myosin I is widely distributed among various tissues.

Requirement of the two-headed structure for the phosphorylation dependent regulation of smooth muscle myosin.

It is known for smooth muscle myosin that while acto-HMM ATPase activity is regulated by phosphorylation, acto-S-1 ATPase activity is not regulated. To clarify the heavy chain structure required for the regulation, smooth muscle myosin containing 7 different lengths of the S-2 portion were expressed in Sf9 insect cells using Baculovirus expression system. Myosin containing longer than 991 residues of heavy chain formed a stable two-headed structure while myosin with shorter than 944 residues of heavy chain formed a single-headed structure, indicating that the residues Gln945-Asp991 are critical for the formation of the two-headed structure. The actin activated ATPase activity of myosin mutants having a two-headed structure was activated by phosphorylation while that of myosin mutants that failed to form the two-headed structure was completely independent of phosphorylation. These results suggest that the two-headed structure is critical for the phosphorylation-dependent regulation.

Primary structure required for the inhibition of smooth muscle myosin light chain kinase.

Myosin light chain kinase (MLCK) contains the autoinhibitor sequence right next to the N-terminus side of the calmodulin binding region. In this paper, the structural requirement of the inhibition of MLCK activity was studied using synthetic peptide analogs. Peptides Ala-783-Lys-799 and Ala-783-Arg-798 inhibited calmodulin independent MLCK at the same potency as the peptide Ala-783-Gly-804. Deletion of Arg-797-Lys-799 or substitution of these residues to Ala markedly increased the Ki while the substitution of Lys-792 and Lys-793 to Ala and the deletion of Lys-784-Lys-785 did not affect the inhibitory activity of the peptides. The results suggest that Arg-797-Arg-798 are especially important for the inhibitory activity among other basic residues in the autoinhibitory region.

The inhibitory effects of okadaic acid on platelet function.

Okadaic acid (OA), a potent inhibitor of protein phosphatases type 1 and type 2A, inhibited thrombin-induced platelet aggregation (IC50 = 0.8 microM), [14C]serotonin release and increase in intracellular Ca2+ ([Ca2+]i) in the same dose dependence. In the absence of thrombin OA increased the phosphorylation of 50-kDa protein and 20-kDa myosin light chain (MLC20). The 50-kDa protein phosphorylation was accomplished within a shorter time period and at a lower concentration than was the MLC20. OA decreased the thrombin-induced phosphorylation of 47-kDa protein and MLC20, although phosphorylation of MLC20 reincreased at higher concentrations of OA (5-10 microM). Since type 2A phosphatase is more sensitive to OA than type 1, these results suggest that type 2A phosphatases are involved in the regulation of Ca2+ signaling in thrombin-induced platelet activation.

A second consensus sequence of ATP-requiring proteins resides in the 21-kDa C-terminal segment of myosin subfragment 1.

Previous comparisons of sequence homologies of ATP-requiring enzymes have defined three consensus sequences which appear to be involved in the binding of the nucleotide. One of these was identified in the N-terminal 27-kDa segment of the myosin heavy chain but the other two sequences have not hitherto been located in myosin. The present paper proposes that one of these other two consensus sequences is in the 21-kDa C-terminal portion of S1 and that it may contribute to the ATP binding domain.

Biologically active peptides caged on tyrosine.

We have demonstrated the feasibility of preparing caged peptides by derivatizing a single amino acid side chain in peptides up to 20 amino acids long. Two peptides are illustrated whose activities are reduced by nearly 2 orders of magnitude using this caging approach. The specific strategy described here of derivatizing tyrosine side chains with a charged caging moiety should be generally applicable in the preparation of caged peptides that have a critical tyrosine residue (e.g., LSM1) or that have critical hydrophobic patches (e.g., RS-20). Other amino acid side chains are also accessible via this caging strategy. Derivatives of threonine, serine, lysine, cysteine, glutamate, aspartate, glutamine, and asparagine can be prepared and site specifically inserted into peptides in an analogous manner. The caged peptides synthesized and purified by the methods described here are compatible with biological samples, including living cells, and have been used to demonstrate the central importance of calmodulin, MLCK, and, by inference, myosin II in ameboid locomotion in polarized eosinophil cells. Photoactivation of peptides within cells should provide a wealth of new information in future investigations by allowing specific protein activities to be knocked out in an acute and spatially defined way.

Dynamics of tumor oxygenation, CD31 staining and transforming growth factor-beta levels after treatment with radiation or cyclophosphamide in the rat 13762 mammary carcinoma.

PURPOSE: Tumors are dynamic tissues that undergo marked molecular, biochemical, and physiologic changes in response to cytotoxic anticancer therapies. Understanding the changes in tumor oxygenation and transforming growth factor-beta expression may allow improved treatment regimens to be developed. METHODS AND MATERIALS: The effects of a single dose of radiation therapy (20 Gy) or a single dose of chemotherapy (cyclophosphamide, 250 mg/kg) on several molecular and physiologic parameters of the rat 13762 mammary carcinoma growing subcutaneously in female Fischer 344 rats were explored. RESULTS: Treatment of the tumor-bearing animals with 20 Gy of radiation killed about two logs (99%) of the 13762 tumor cells, and treatment with cyclophosphamide (250 mg/kg) killed about 1.5 logs (95%) of the 13762 tumor cells. Hypoxia, as determined by a pO2 electrode, initially decreased in the tumors of treated animals until 6 h. posttreatment and then increased, so that 24 h. after administration of the radiation therapy or the chemotherapy the number of intratumoral vessels as determined by CD31 staining increased until about 24 h after cytotoxic therapy. Transforming growth factor-beta1, measured by radioimmunoassay, peaked in the serum between 6 h and 18 h and again between 72 h and 96 h after radiation therapy and peaked in the tumor at 24 h and again at 72 h after radiation therapy. The first serum peak after cyclophosphamide was 3 h after drug injection, with second peaks at 36 h and 48 h after drug administration. In the tumor, transforming growth factor-beta1 peaked between 6 h and 8 h after drug administration and again 36 h and 72 h after drug. Apoptosis was maximal 6 h after 20 Gy and 24 h after cyclophosphamide. Vascular endothelial growth factor was also increased in tumors after cytotoxic therapy. CONCLUSIONS: These changes in the tumor physiologic status are sufficient to protect the tumor from a second cytotoxic insult administered days afterwards and to result in a restructuring of the tissue.

Squamous epithelial dysplasia associated with squamous cell carcinoma of the esophagus.

To investigate the relationship between dysplasia and carcinoma of the esophagus, 159 cases of esophageal carcinoma without any preoperative treatment were reviewed retrospectively. There were 75 dysplastic lesions in 32 cases (20.1%). The incidence of co-existence of dysplastic lesions was 0, 58.3, 31.3, 20.8 and 11.4% in intra-epithelial, mucosal and submucosal cancers and those invading the proper muscular layer and adventitia, respectively. Thus, excluding the cases of intra-epithelial carcinoma, the less advanced the lesion, the higher the incidence of dysplasia. Epithelial dysplastic lesions were classified as 12 with mild, 33 with moderate and 30 with severe degrees of dysplasia. Although the continuity of dysplastic lesions to the areas of carcinoma was not so frequent (48.0%), it was more often encountered in severe dysplasia rather than in moderate or mild dysplasia, which suggested some relationship between the severity of dysplasia and carcinoma. In the cases with a dysplastic lesion the multiplicity of squamous cell carcinoma and the intra-epithelial spread of the main lesion were more frequently seen (P < 0.001), suggesting a multicentric occurrence of dysplastic lesions and carcinomas.

Alterations in gene expression between EMT-6 mammary carcinoma monolayers and spheroids identified by differential display.

Differential display was used to define differences in gene expression between murine EMT-6 mammary carcinoma cells grown in monolayer or as spheroids in cell culture. Eighty different combinations of primer sets made of four anchored oligodeo(dT) primers (T(12)MG, T(12)MA, T(12)MT or T(12)MC) and twenty arbitrary ten base primers (AP1-20) were used for RT-PCR of total purified RNA from EMT-6 monolayers or spheroids. After re-screening, fourteen DNA fragments were identified as being selectively expressed in EMT-6 cells grown as spheroids. The fragments were cloned into the pCR II vector. Two of the fourteen cDNA fragments corresponded to mRNA selectively expressed in EMT-6 cells grown as spheroids. These clones were sequenced and found by database searching to correspond to murine heme oxygenase and murine beta(2)-microglobulin. There was a 5.3-fold higher expression of beta(2)-microglobulin and a 5.3-fold greater expression of heme oxygenase in EMT-6 cells grown as spheroids than in the same cells grown as monolayers. These studies reflect the phenotypic flexibility of cells depending upon the growth environment.

Phosphorylation-dependent and ATP-induced changes in structural array in gizzard myosin filament bundles.

We observed the fine structure of myosin filament bundles in 10 mM MgCl2 and studied the effects of light chain phosphorylation on it by electron microscopy. In the filament bundles of dephosphorylated myosin in the presence of ATP, we found a striation pattern of 13.4 nm periodicity running perpendicularly to the long axis of the filaments. However, we did not observe such a pattern in filament bundles of phosphorylated myosin even when exposed to ATP, but many irregularly arranged projections were seen on their surface. The 13.4 nm period of the striation was so close to the dimension of myosin heads that it seems likely that the striation pattern represents the regular array of myosin heads, which is a consequence of conformational changes in dephosphorylated myosin molecules upon reacting with ATP.

Reaction mechanism of Mn2+-ATPase of acto-H-meromyosin in 0.1 M KCl at 5 degrees C: evidence for the Lymn-Taylor mechanism.

The rate constants of a series of elementary steps in the H-meromyosin (HMM) Mn2+-ATPase [EC 3.6.1.3] and acto-HMM Mn2+-ATPase reactions were determined in 0.1 M KCl at 5 degrees C. We found that the rate-limiting step in the HMM Mn2+-ATPase reaction was the liberation of ADP from HMM.ADP, of which the rate constant was estimated to be 0.17 s-1. All the results obtained with the acto-HMM Mn2+-ATPase reaction could be quantitatively explained by a modified Lymn-Taylor mechanism (see Fig. 15). The second-order rate constant for the dissociation of acto-HMM induced by ATP was 3.0 X 10(5) M-1.s-1, and that for the Pi burst in the acto-HMM ATPase reaction was 1.7 X 10(5) M-1.s-1. The second-order rate constant for the binding of HMM.ADP with F-actin was 0.25 s-1.mg-1.ml, and the rate-limiting step in the acto-HMM Mn2+-ATPase reaction was the conversion of HMMPADP into HMM.ADP plus Pi, when the F-actin concentration was high.

Nonlinear dependence of actin-activated Mg2+-ATPase activity on the extent of phosphorylation of gizzard myosin and H-meromyosin.

1. The actin-activated Mg2+-ATPase activity of gizzard HMM increased in proportion to the square of the extent of LC phosphorylation. This result indicates that the LCs of HMM are randomly phosphorylated, and the phosphorylation of both heads of HMM is required for the activation of HMM Mg2+-ATPase by F-actin. 2. In 75 mM KCl, the Mg2+-ATPase activity of gizzard myosin was activated by F-actin only slightly when a half of the total LC was phosphorylated. From 1 to 2 mol LC phosphorylation, the activity was enhanced by F-actin almost linearly. In 30 mM KCl, the activity of acto-gizzard myosin increased sigmoidally with increase in the extent of LC phosphorylation. On electron microscopy, side-by-side aggregates of myosin filaments were observed in 30 mM KCl, but not in 75 mM KCl. It was suggested that the activation of the Mg2+-ATPase activity of acto-gizzard myosin LC phosphorylation is modified by formation of myosin filaments and their aggregates. 3. The relationship between the actin-activated Mg2+-ATPase activity of HMM or myosin and the extent of LC phosphorylation was unaffected by tropomyosin.