Immunolocalization of meta-vinculin in human smooth and cardiac muscles

Meta-vinculin, a vinculin-related protein, has been isolated from human uterus smooth muscle. Specific antibodies to meta-vinculin, which distinguish between meta-vinculin and vinculin, were prepared by absorption of anti-meta-vinculin serum on vinculin coupled to nitrocellulose. Meta-vinculin specific antibody demonstrates only smooth and cardiac muscle specificity and is able to cross-react with a small 21-kD fragment of the meta-vinculin polypeptide chain. This antibody does not interact with protease resistant 95-kD core shared by vinculin and meta-vinculin. Meta-vinculin specific antibody was used for the localization of meta-vinculin in smooth and cardiac muscles by the indirect immunofluorescence method. At the light microscopy resolution level it was found that meta-vinculin and vinculin are localized in the same cellular adhesive structures. Meta-vinculin is present in membrane-associated microfilament-bound plaques of smooth muscle, in intercalated discs and costameres of cardiac muscle. In primary culture of smooth muscle cells from human aorta, meta-vinculin and vinculin were found to be present in focal contacts of the cells. During the cultivation of smooth muscle cells, the quantity of meta-vinculin decreased progressively and finally meta-vinculin completely disappeared from the focal contacts. The data show that in smooth and cardiac muscles meta-vinculin could be a structural component of microfilament-membrane attachment sites, defined earlier by the localization of vinculin.     

Properties of smooth muscle vinculin

Vinculin, isolated from turkey gizzard smooth muscle, was purified by chromatography on CM-cellulose after isolation from a DEAE-cellulose column. Two-dimensional gel electrophoretic analysis of crude muscle fractions demonstrated that: 1) much of the approximately 130,000-dalton protein present in smooth muscle did not co-isoelectrically focus with the purified 130,000-dalton vinculin and 2) the purified vinculin consisted of three major, closely spaced isoelectric variants that were present only in small amounts in the original smooth muscle sample. Purified vinculin sedimented as a single peak with a sedimentation coefficient S0 20,w of 5.9. Circular dichroism spectra of purified vinculin indicated a considerable degree of secondary structure, with an alpha-helical content of approximately 50% as measured at 208 nm. The ultraviolet absorption spectrum of vinculin gave a measured E1%(278) of 4.64. Digestion of vinculin, much of which is located at the cytoplasmic surface of the cell membrane, with Ca2+-activated neutral protease purified from skeletal muscle yielded major fragments with molecular weights determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 98,000, 85,000, and 26,000. The factor(s) in DEAE-cellulose-purified vinculin responsible for decreasing the low shear viscosity of actin was removed and found in a crude fraction isolated by CM-cellulose chromatography. The purified vinculin had a small, but positive effect on the MgCl2-induced polymerization of actin as measured by low shear viscometry.     

Expression of meta-vinculin associated with differentiation of chicken embryonal muscle cells

We confirmed the existence of meta-vinculin, which cross-reacts immunologically with vinculin and has a slightly higher molecular weight than the latter. The immunological cross-reactivity between meta-vinculin and vinculin was confirmed with monoclonal antibodies against vinculin. Furthermore, we found that this protein is present only in either smooth or striated muscle, but is absent in non-muscular tissues and that the expression of this protein is associated with a differentiation of muscle cells either in vivo or in vitro. Microinjection experiments of fluorescently labelled vinculin and/or meta-vinculin into the cytoplasm of cultured myotubes suggested that meta-vinculin may be localized at sites similar to vinculin in muscle cells.     

Diversity of vinculin/meta-vinculin in human tissues and cultivated cells. Expression of muscle specific variants of vinculin in human aorta smooth muscle cells

Microheterogeneity of different vinculin and meta-vinculin isoforms in adult human tissues and cultured cells was studied by two-dimensional gel electrophoresis and immunoblotting technique. Four isoforms of vinculin (alpha, alpha', beta, and gamma) and two isoforms of meta-vinculin (alpha and beta) were resolved. alpha-, alpha'-, and beta-isoforms of vinculin were found in all cell types and tissue samples analyzed in the present study. gamma-Isoform of vinculin and both alpha- and beta-isoforms of meta-vinculin were found in smooth (aorta wall and myometrium) and cardiac muscle, rather than in skeletal muscle, liver, foreskin fibroblasts, and macrophages. In the primary culture of human aorta smooth muscle cells, the fractional content of gamma-isoform of vinculin and meta-vinculin was dramatically reduced, and, by the onset of intensive cell division, the proteins could hardly be detected. Subcultured human aorta smooth muscle cells did not contain gamma-vinculin and meta-vinculin. We analyzed the microheterogeneity of vinculin and meta-vinculin in three smooth muscle layers of human aorta wall--media, muscular-elastic (adjacent to media) intima, and subendothelial (juxtaluminal) intima. It was shown that in media the fractional content of gamma-isoform of vinculin was 45% and meta-vinculin, 42%; in muscular-elastic intima the fractional content of gamma-vinculin was 42% and meta-vinculin, 36%. However, in subendothelial intima, the share of these proteins was significantly lower than in adjacent muscular-elastic intima and media. Isoactin pattern that is characteristic of smooth muscle was identical in all aortic layers, thus proving the smooth muscle origin of subendothelial intima cells. These findings demonstrate that human aortic smooth muscle cells in vivo and in vitro undergo coordinated differential expression of smooth muscle specific variants of vinculin, i.e. gamma-vinculin and meta-vinculin.     

Properties of phosphorylated myofibrils from gizzard smooth muscle

Phosphorylation of chicken gizzard myosin light chain in myofibril and its effect on myofibrillar ATPase activity were investigated in the contracted state of myofibrils. When myofibrils were incubated for two hours at 30 degreeds C with ATP, magnesium and calcium, the myosin light chain was phosphorylated by endogenous light-chain kinase. Standing overnight, the phosphorylated light chain was dephosphorylated by endogenous light-chain phosphatase. Control myofibril had much higher ATPase activity than phosphorylated and phosphorylated-dephosphorylated myofibrils. It was very interesting that the phosphorylated and phosphorylated-dephosphorylated myofibrils were quite similar in ATPase activity. However, phosphorylated myofibril differed from phosphorylated-dephosphorylated myofibril in Ca2+ dependency of Mg2+-ATPase activity. The phosphorylated-dephosphorylated myofibril was not affected by the presence or absence of Ca2+. In contrast, phosphorylated myofibril apparently showed a negative Ca2+-sensitivity. On the other hand, the results indicating that the superprecipitation gel formed by phosphorylated-dephosphorylated myosin could not be dissolved in 0.6 M NaCl, suggest that the phosphorylation-dephosphorylation process of the actomyosin system in gizzard myofibril results in stronger actin-myosin interaction.     

Properties of talin from chicken gizzard smooth muscle

This paper describes the structural and biochemical characterization of talin, a protein localized to various cellular sites where bundles of actin filaments attach to the plasma membrane. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the protein has a molecular mass of 225,000 +/- 5,000 daltons. Hydrodynamic measurements at protein concentrations less than 0.72 mg/ml indicate a monomeric protein with a native molecular mass of 213,000 +/- 15,000 daltons. Sedimentation equilibrium experiments indicate self-association at protein concentrations of 0.72 mg/ml and higher. The data suggest that this self-association is a simple monomer:dimer equilibrium over the range of concentrations observed. At low protein concentrations where talin is a monomer, the Stokes radius and sedimentation coefficient vary with ionic strength. Under low ionic strength conditions (5-20 mM NaCl), talin has a Stokes radius of 6.5 nm and a sedimentation value of 9.4, suggesting an asymmetric globular molecule; whereas under high ionic strength conditions (200 mM NaCl), the Stokes radius increases to 7.7 nm and the sedimentation coefficient decreases to 8.8, suggesting a more elongated protein. This conformation change is confirmed by electron microscopy which reveals a more globular protein at low ionic strength which unfolds to become an elongated flexible molecule as the ionic strength is increased to physiological and higher levels. The amino acid composition of talin indicates a low level of aromatic residues, consistent with its relatively low extinction coefficient, talin has an isoelectric point between pH 6.7 and 6.8 based on isoelectric focusing. The detailed purification of talin is described.     

Properties of calcium channels in cardiac muscle and vascular smooth muscle

The voltage-dependent slow channels in the myocardial cell membrane are the major pathway by which Ca²⁺ ions enter the cell during excitation for initiation and regulation of the force of contraction of cardiac muscle. The slow channels have some special properties, including functional dependence on metabolic energy, selective blockade by acidosis, and regulation by the intracellular cyclic nucleotide levels. Because of these special properties of the slow channels, Ca²⁺ influx into the myocardial cell can be controlled by extrinsic factors (such as autonomic nerve stimulation or circulating hormones) and by intrinsic factors (such as cellular pH or ATP level). The slow Ca²⁺ channels of the heart are regulated by cAMP in a stimulatory fashion. Elevation of cAMP produces a very rapid increase in number of slow channels available for voltage activation during excitation. The probability of a slow channel opening and the mean open time of the channel are increased. Therefore, any agent that increases the cAMP level of the myocardial cell will tend to potentiate I_si, Ca²⁺ influx, and contraction. The myocardial slow Ca²⁺ channels are also regulated by cGMP, in a manner that is opposite to that of CAMP. The effect of cGMP is presumably mediated by means of phosphorylation of a protein, as for example, a regulatory protein (inhibitory-type) associated with the slow channel. Preliminary data suggest that calmodulin also may play a role in regulation of the myocardial slow Ca²⁺ channels, possibly mediated by the Ca²⁺-calmodulin-protein kinase and phosphorylation of some regulatory-type of protein. Thus, it appears that the slow Ca²⁺ channel is a complex structure, including perhaps several associated regulatory proteins, which can be regulated by a number of extrinsic and intrinsic factors.VSM cells contain two types of Ca²⁺ channels: slow (L-type) Ca²⁺ channels and fast (T-type) Ca²⁺ channels. Although regulation of voltage-dependent Ca²⁺ slow channels of VSM cells have not been fully clarified yet, we have made some progress towards answering this question. Slow (L-type, high-threshold) Ca²⁺ channels may be modified by phosphorylation of the channel protein or an associated regulatory protein. In contrast to cardiac muscle where cAMP and cGMP have antagonistic effects on Ca²⁺ slow channel activity, in VSM, cAMP and cGMP have similar effects, namely inhibition of the Ca²⁺ slow channels. Thus, any agent that elevates cAMP or cGMP will inhibit Ca²⁺ influx, and thereby act to produce vasodilation. The Ca²⁺ slow channels require ATP for activity, with a K_0.5 of about 0.3 mM. C-kinase may stimulate the Ca²⁺ slow channels by phosphorylation. G-protein may have a direct action on the Ca²⁺ channels, and may mediate the effects of activation of some receptors. These mechanisms of Ca²⁺ channel regulation may be invoked during exposure to agonists or drugs, which change second messenger levels, thereby controlling vascular tone.     

Properties and function of phosphatases from vascular smooth muscle

Myosin light chain phosphatase (MLCP) activity was present in extracts from a wide variety of mammalian tissues. A partially purified preparation of bovine aortic MLCP also showed activity against phosphorylase a and p-nitrophenyl phosphate (PNP). Whether these three activities are ascribable to a single multifunctional phosphatase or to three distinct phosphatases is unknown. The three phosphatase activities coelute during gel filtration both before and after treatment with ethanol showing exclusion volumes corresponding to 240,000 and 35,000 daltons, respectively. This indicates that the enzyme is dissociable into a smaller catalytic subunit. The widespread occurrence of MLCP activity and the close parallel among MLCP, phosphorylase a phosphatase, and PNP phosphatase activities suggest that the enzyme (or enzymes) may participate in physiological processes in addition to dephosphorylation of phosphorylated myosin light chains.     

Properties of the smooth muscle cell endoplasmic reticulum calcium pump

In experiments with 45Ca2+ conducted on digitonin-treated (0.1 mg/ml) myometrium cells suspension, the properties of ruthenium red-insensitive, oxalate- or phosphate-stimulated and thapsigargin- or cyclopiasonic acid-suppressed Mg2+, ATP-dependent calcium pump of myometrium sarcoplasmic reticulum was studied. The Ca2+ accumulation increased linearly in time up to 10 min, the average initial rate was 80-130 pmol Ca2+/10(6) cells per min. In the presence of 10 mM oxalate the values of the activation constant KMg for Mg2+ and K(m) for ATP were 0.6 and 1.0 mM, respectively. The relative efficiency of the different cations in insuring of the ATP-dependent Ca2+ accumulation was Mg2+ > Mn2+ = Co2+ > Ni2+; the Ca2+ accumulation was not observed in the presence of 3 mM Zn2+ or Cu2+. We observed the suppression of calcium pump activity by different inhibitors such as thapsigargin, cyclopiazonic acid, p-chloromercuribenzoic acid, eosin Y ad Na3 VO4: the values of K0.5 were 2.0 nM, 0.3 microM, 0.6 microM, 0.8 microM and 45 microM respectively. The conclusion was made that suspension of myometrial cells treated with digitonin represent a suitable experimental model for studying the properties of myometrium sarcoplasmic reticulum calcium pump.     

Properties of the Ca-pumping ATPase of sarcoplasmic reticulum from vascular smooth muscle

A microsomal fraction enriched in sarcoplasmic reticulum membranes has been isolated from bovine aorta smooth muscle. The properties of the Ca-pumping ATPase were compared to those of the enzymes of skeletal and cardiac sarcoplasmic reticulum. The kinetic (Km and turnover rate) and structural (tryptic digestion pattern) properties of the three ATPases were strikingly similar. The three enzymes, however, displayed (almost) no immunological cross-reactivity. Skeletal muscle sarcoplasmic reticulum and aorta microsomes did not contain phospholamban: their Ca-pumping activity was not regulated by either a cAMP-dependent or a calmodulin-dependent pathway.     

Properties of endothelial cell and smooth muscle cell cultured in ambient pressure

Summary Cultures of umbilical vein endothelial cells and smooth muscle cells were studied in a constant pressure chamber. The following results were obtained: (a) Endothelial cell growth was maximal at 80 mmHg and minimal at 0 mmHg (atmospheric pressure) for the first 2 d of incubation. However, these growth rates were reversed during the following 6 d because of steady increase in growth at 0 mm Hg and a decrease in growth at higher pressures. A degeneration of endothelial cells began at 120 mmHg and marked degeneration was noted at 160 mmHg. Growth of Smooth muscle cells was not influenced by ambient pressure and a steady increase in labeled nuclei continued throughout the period of culture. (b) Elastin, stainable with tannic acid, was noted electronmicroscopically in both endothelial and smooth muscle cells. (c) Production of prostacyclin by endothelial cells was maximal at 0 mmHg and minimal at 80 mmHg, in contrast to the growth pattern of these cells. Production of thromboxane B₂ by endothelial cells and prostacyclin and thromboxane B₂ by smooth muscle cells was very slight and not significantly different. Although it is not known at present what mechanism acts on the vascular cells when cultured in ambient pressure, these results may indicate a new concept of the behavioral relationship between endothelial cell, smooth muscle cell, and blood pressure in vivo.     

Hypertrophic smooth muscle

Summary The ultrastructure of filaments is studied in the hypertrophic musculature of the small intestine of the guinea pig oral to an experimental stenosis. No structural change is observed in the thin and the thick myofilaments. However, there is a remarkable and consistent increase in the number of intermediate (10 nm) filaments; they are the predominant type of filament in many hypertrophic muscle cells. Experiments, in which the force developed in vitro by strips of control and hypertrophic musculature upon stimulation with carbachol, indicate that the force per unit sectional area is slightly less in the hypertrophic muscle than in the control tissue.The author thanks Miss Eva Franke for excellent technical assistance. This work was supported by grants from the Medical Research Council and the Central Research Funds of the University of London     

Hypertrophic smooth muscle

The neurones from the wind-sensitive hairs on the locust head have been filled with cobalt chloride and intensified with silver. All the neurones project through the brain to the suboesophageal ganglion, some continue to the prothoracic ganglion and a few as far as the mesothoracic ganglion. Three different types of projection are described and a regrouping is proposed of Weis-Fogh's five hair fields into three areas. The distribution of the neurones from these areas is described in relation to other structures in the ganglion and is discussed in relation to the function of the hair fields in stability control and grooming.     

Hypertrophic smooth muscle

Summary An extensive hypertrophy of the muscle coat develops in the small intestine of the guinea pig oral to an experimental stenosis. The profiles of smooth muscle cells become larger and irregular in shape. From the analysis of serial sections the arrangement of the muscle cells is less orderly than in control muscles. Many muscle cells are split into two or more branches over part of their length. The average cell volume is 3–4 times that of control muscle cells; the cell surface increases less dramatically and, in spite of the appearance of deep invaginations of the cell membrane, the surface-to-volume ratio falls from 1.4 to 0.8. The average cell length is only slightly increased compared with controls. Smooth muscle cells in mitosis are observed in all the hypertrophic muscles examined, in both muscle layers; in the circular musculature they occur mainly found in the middle part of the layer.The author thanks Miss Eva Franke for excellent technical assistance. This work was supported by grants from the Medical Research Council and the Central Research Funds of the University of London     

Hypertrophic smooth muscle

Summary In adult guinea-pigs, oral to a partial obstruction to the flow of ingesta in the ileum there is a marked increase in the diameter of the intestine and a hypertrophy of the muscle coat. The features of the intramuscular blood vessels and of the extracellular materials were studied by electron microscopy. There is a small increase in the amount of intercellular space measured morphometrically. The basal lamina surrounding the hypertrophic muscle cells is more prominent than in controls. In the intercellular space between muscle cells, in addition to collagen fibrils, there is abundant amorphous material of medium electron density and streak-like, electron-dense material often similar to thickened basal laminae. The total amount of stroma (intercellular materials) present in a unit length of intestine is greatly increased in hypertrophy; a role of the muscle cells in the production of new collagen and other extracellular elements is suggested by the present observations. Many new intramuscular blood vessels (mainly capillaries, some of which are fenestrated) are formed during hypertrophy of the intestinal wall, so that the circular muscle layer remains as well vascularized in the hypertrophic intestine as in the controls. Blood vessels are not formed within the longitudinal muscle layer.     

Hypertrophic smooth muscle

The smooth muscle cells of the circular musculature of the guinea pig ileum are connected by gap junctions (nexuses) which occupy about 0.21% of the cell surface. When the muscle hypertrophies in the portions of the ileum oral to an experimental stenosis, the muscle cells increase in size and number. Gap junctions become markedly larger than in control muscles and occupy 0.49% of the cell surface. While the cells double their surface area, the number of nexuses per unit surface remains unchanged (47--48 per 1000 microns2). The packing density of intramembrane particles (or pits) in the nexuses of hypertrophic muscle cells is 6700 . microns-2, which is slightly less than in control muscle cells (7200 . microns-2). A characteristic grouping of the particles (or the pits) within the nexus is often observed. Nexuses between two processes originating from the same cell are common. Nexuses do not occur in the longitudinal muscle.     

Properties of a collagenase inhibitor partially purified from cultures of smooth muscle cells

1. The metabolism of [14-14C]erucate and [U-14C]palmitate has been investigated in perfused heart from rats fed 0.3% clofibrate for 10 days and from control rats. 2. The total uptake of fatty acids in the heart increased in the clofibrate fed group. Clofibrate increased the oxidation of [14-14C]erucic acid by 100% and the oxidation of [U-14C]palmitic acid by 30% compared to controls. 3. The chain-shortening of erucate to C20:1 and C18:1 fatty acids in the perfused heart was stimulated at least two-fold by clofibrate feeding. 4. The activity of the peroxisomal marker enzyme catalase increased 60%, the activity of cytochrome oxidase increased approx. 16% and the content of total coenzyme A increased 30% in heart homogenates from rats fed clofibrate compared to controls. 5. The isolated mitochondrial fraction from clofibrate fed rats showed an increased capacity for oxidation of palmitoylcarnitine and decanoylcarnitine, while the oxidation of erucoylcarnitine showed little change. 6. It is suggested that clofibrate increases the oxidation of [14-14C]erucic acid in the perfused heart by increasing the capacity for chain-shortening of [14-14C]erucate in the peroxisomal beta-oxidation system.     

Evidence that unphosphorylated smooth muscle myosin supports smooth muscle contraction.

Unphosphorylated smooth muscle myosin filaments do not disassemble in MgATP, provided that the solution is supplemented either by 25% serum albumin or by 6% polyethylene glycol 6000. These filaments are able to support actomyosin retraction but their ATPase activity is not activated by tropomyosin-decorated F-actin.