Age-dependent changes in collagen metabolism and response to hypertonic culture conditions of rat aortic smooth muscle cells and skin fibroblasts

The rates of growth and collagen synthesis of rat aortic smooth muscle cells in culture were decreased with increasing the age of the animals from which the cells were obtained. These rates were further decreased by the presence of a 0.1 M excess concentration of KCl in the culture medium, and the cells obtained from older animals were much more sensitive to the inhibitory effect of the hypertonic culture condition. These tendencies were observed in smooth muscle cells obtained from different phases of in vitro cellular aging and also in skin fibroblasts obtained from animals of different ages. These results indicate that the cells become more sensitive to the environmental changes with increasing age both in vivo and in vitro.     

Phenotypic conversion leads to structural and functional changes of smooth muscle sarcolemma

Continuous changes in the length of smooth muscles require a highly organized sarcolemmal structure. Yet, smooth muscle cells also adapt rapidly to altered environmental cues. Their sarcolemmal plasticity must lead to profound changes which affect transmembrane signal transduction as well as contractility. We have established porcine vascular and human visceral smooth muscle cultures of epithelioid and spindle-shaped morphology and determined their plasma membrane properties. Epithelioid cells from both sources contain a higher ratio of cholesterol to glycerophospholipids, and express a less diverse range of lipid-associated annexins. These findings point to a reduction in efficiency of membrane segregation in epithelioid cells. Moreover, compared to spindle-shaped cells, cholesterol is more readily extracted from epithelioid cells with methyl-beta-cyclodextrin and its synthesis is more susceptible to inhibition with lovastatin. The inability of epithelioid cells to process vasoactive metabolites, such as angiotensin or nucleotides further indicates that contractile properties are impaired. Phenotypic plasticity extends beyond the loss of smooth muscle cell marker genes. The plasma membrane has undergone profound functional changes which are incompatible with cyclic foreshortening, but might be important in the development of vascular disease.     

Effect of estrogen treatment on calcium uptake by the rat uterine smooth muscle

The effect of estrogenization on cellular 45Ca uptake by the isolated uterine strips was studied. Whereas estrogenization 1 h before sacrifice of ovariectomized rats caused no significant change in Ca uptake by the isolated uteri, exposure to estrogen 24 h before sacrifice resulted in a significant increase in uptake. Following continuous exposure for 72 h to estrogen, the uptake of Ca was increasd by more than two-fold. Increased Ca uptake following estrogenization may largely account for the increased contractile responses of the uterine smooth muscle particularly those induced by potassium depolarization.     

Calcium diffusion in uterine smooth muscle sheets

The potassium contracture in the longitudinal muscle of estrogen- treated rat uterus was kinetically investigated. The rates of tension development after Ca addition and relaxation after Ca removal were measured under the high-potassium depolarization. Both rates decreased with an increase in preparation thickness. The relaxation rate had only a slight dependence on temperature. On the contrary, both relaxation and contraction rates in a contraction induced by an electrical stimulation strongly depended on temperature, but not on preparation size. These results suggest that the Ca diffusion process in the extracellular space is the rate-limiting step in relaxation of Ca- dependent contracture under potassium depolarization. The diffusion model, in which the effect of the unstirred layer was considered, could quantitatively explain the experimental results. The apparent diffusion coefficient in the muscle sheet was estimated to be approximately 3 x 10(-7) cm2/s. The difference from that in aqueous solution is discussed.     

Force regulates smooth muscle actin in cardiac fibroblasts

Chronic ventricular pressure overload can regulate expression of alpha-smooth muscle actin (SMA) in cardiac fibroblasts, but it is unclear if force alone or the concomitant activity of angiotensin II is the principal regulatory factor. To test if SMA mRNA and protein in rat cardiac fibroblasts are regulated directly by force, we first induced SMA expression in cultured cells and then applied magnetically generated perpendicular forces through focal adhesions using collagen-coated magnetite beads. Continuous static forces (0.65 pN/micrometer(2)) selectively reduced SMA but not beta-actin mRNA and protein content within 4 h (to 55 +/- 9% of controls); SMA returned to baseline by 8 h. There was no change in SMA content after force application with either plasma or the cellular fibronectin IIIA domain, BSA, or poly-L-lysine beads. The early loss of SMA was apparently due to selective leakage into the cell culture medium. Treatment with angiotensin II (10 nM) abrogated the force-induced reduction of SMA and increased the levels of this protein. The stress kinase p38 was phosphorylated by force, whereas extracellular signal-regulated kinase 1/2 and c-Jun NH(2)-terminal kinase were unaffected. The p38 kinase inhibitor SB-203580 relieved the force-induced SMA reduction. We conclude that force-induced inhibition of SMA is mediated through the p38 kinase pathway, and this pathway antagonizes angiotensin II regulation of SMA.     

Fine structural changes in electrostimulated human skeletal muscle. Evidence for predominant effects on fast muscle fibres

Physical education students were subjected to electrical stimulation of relatively high frequency and current amplitude for 19 days. A quantitative study of several morphological parameters was performed on biopsy samples from gastrocnemius, using stereological methods at both light and electron microscopic levels. The main results were: muscle fibre size was increased; nuclear volume was also increased, suggesting that a proliferation of nuclei had occurred; this was paralleled by an increased content of nuclear DNA. The size of single myonuclei was increased, and their heterochromatin fraction was decreased, these changes being most pronounced in type II fibres. The increase in the mitochondrial fraction was also greatest in type II fibres. It is concluded that this type of electrical stimulation has predominant effects on type II fibres.     

Microinjection of nonmuscle and smooth muscle caldesmon into fibroblasts and muscle cells

Caldesmon is present in a high molecular mass form in smooth muscle and predominantly in a low molecular mass form in nonmuscle cells. Their biochemical properties are very similar. To examine whether these two forms of caldesmon behave differently in cultured cells, we microinjected fluorescently labeled smooth muscle and nonmuscle caldesmons into fibroblasts. Simultaneous injection of both caldesmons into the same cells has revealed that both high and low relative molecular mass caldesmons are quickly (within 10 min) and stably (over 3 d) incorporated into the same structures of microfilaments including stress fibers and membrane ruffles, suggesting that nonmuscle cells do not distinguish nonmuscle caldesmon from smooth muscle caldesmon. The effect of calmodulin on the incorporation of caldesmon has been examined by coinjection of caldesmon with calmodulin. We have found that calmodulin retards the incorporation of caldesmon into stress fibers for a short period (10 min) but not for a longer incubation (30 min). The behavior of caldesmon in developing muscle cells was also examined because we previously observed that caldesmon disappears during myogenesis (Yamashiro, S., R. Ishikawa, and F. Matsumura. 1988. Protoplasma Suppl. 2: 9-21). We have found that, in contrast to its stable incorporation into stress fibers of fibroblasts, caldesmon is unable to be incorporated into thin filament structure (I-band) of differentiated muscle.     

Tensile properties of fibroblasts and vascular smooth muscle cells

Tensile properties of fibroblasts (FBs) and vascular smooth muscle cells (VSMCs) of synthetic and contractile phenotypes were studied using a newly developed micro-tensile tester. FBs were obtained from the rabbit patellar tendon. Synthetic and contractile VSMCs were isolated from the rabbit thoracic aorta with an explant and an enzymatic digestion method, respectively. Each cell was attached to the fine tips of a pair of micropipettes with a cell adhesive and, then, stretched at the speed of 6 microm/sec. Load and length were obtained using a cantilever-type load cell and a VDA, respectively.FBs were broken at the load of 0.9 microN and the elongation to failure of 86 microm, and had the stiffness of 0.02 N/m. VSMCs were not broken even at 2.4 microN. The stiffness of synthetic and contractile VSMCs were 0.09 and 0.17 N/m, respectively. Such large different tensile properties among the three cells are attributable to the differences in components and cytoskeletal structures.     

Emergence of airway smooth muscle functions related to structural malleability

The function of a complex system such as a smooth muscle cell is the result of the active interaction among molecules and molecular aggregates. Emergent macroscopic manifestations of these molecular interactions, such as the length-force relationship and its associated length adaptation, are well documented, but the molecular constituents and organization that give rise to these emergent muscle behaviors remain largely unknown. In this minireview, we describe emergent properties of airway smooth muscle that seem to have originated from inherent fragility of the cellular structures, which has been increasingly recognized as a unique and important smooth muscle attribute. We also describe molecular interactions (based on direct and indirect evidence) that may confer malleability on fragile structural elements that in turn may allow the muscle to adapt to large and frequent changes in cell dimensions. Understanding how smooth muscle works may hinge on how well we can relate molecular events to its emergent macroscopic functions.     

Phosphorylation-dependent structural changes in the regulatory light chain domain of smooth muscle heavy meromyosin.

Smooth muscle heavy meromyosin, a double-headed proteolytic fragment of myosin lacking the COOH-terminal two-thirds of the tail, has been shown previously to be regulated by phosphorylation. To examine phosphorylation-dependent structural changes near the head-tail junction, we prepared five well regulated heavy meromyosins containing single-cysteine mutants of the human smooth muscle regulatory light chain labeled with the photocross-linking reagent, benzophenone-iodoacetamide. For those mutants that generated cross-links, only one type of cross-linked species was observed, a regulatory light chain dimer. Irradiated mutants fell into two classes. First, for Q15C, A23C, and wild type (Cys-108), a regulatory light chain dimer was formed for dephosphorylated but not thiophosphorylated heavy meromyosin. These data provide direct chemical evidence that in the dephosphorylated state, Gln-15, Ala-23, and Cys-108 on one head are positioned near (within 8.9 A) the regulatory light chain of the partner head and that thiophosphorylation abolishes proximity. This behavior was also observed for the Q15C mutant on a truncated heavy meromyosin lacking both catalytic domains. For the actin-heavy meromyosin complex, cross-links were formed in both de- and thiophosphorylated states. S59C and T134C mutants were in a second mutant class, where regulatory light chain dimers were not detected in dephosphorylated or thiophosphorylated heavy meromyosin, suggesting positions outside the region of interaction of the regulatory light chains.     

Platelet factors stimulate fibroblasts and smooth muscle cells quiescent in plasma serum to proliferate

Whole blood serum is widely recognized as essential for the growth of diploid cells in culture. Dermal fibroblasts and arterial smooth muscle cells fail to proliferate in culture in the presence of serum derived from platelet-poor plasma. Platelet-poor plasma serum is capable of maintaining monkey arterial smooth muscle cells quiescent in culture at either low (1.5 x 10(3)) or high (2.0 x 10(4)) population densities. The proportion of cell traversing the cell cycle under these conditions was approximately 3%. Equal numbers of quiescent smooth muscle cells initiated DNA synthesis and cell division when treated with whole blood serum or with an equivalent quantity of platelet-poor plasma serum supplemented with a factor(s) derived from a supernate obtained after exposure of human platelets to purified thrombin in vitro.     

The effect of estrogen and progesterone on structural changes in the uterine glandular epithelium of the ovariectomized sheep.

The development of epithelial cells of the uterine glands of ovariectomized sheep in response to estradiol-17 beta (E) and progesterone (P) was studied using light and electron microscopy. Animals that had been ovariectomized for six weeks were placed in one of three experimental treatment groups. Group I animals (untreated controls) received no steroid treatment. Group II animals (E alone) received one 4-cm E implant (E approximately 5-10 pg/ml) and their uteri were removed after 2, 4, or 6 days. Group III animals (E-primed, P-treated) received an E implant (E approximately 5-10 pg/ml) for 6 days and then were treated with six 13-cm P implants (P approximately 1.5-3 ng/ml), in the continued presence of E, for 2, 4 or 6 days. Six weeks after ovariectomy the epithelial cells of the uterine glands were low cuboidal and morphologically appeared to be synthetically inactive. Following 2 days of E treatment the epithelial cells had significantly increased in cell height, and protein-synthesizing organelles were well developed. Maturation of the secretory apparatus continued throughout E treatment. The Golgi complex and rough endoplasmic reticulum (RER) were abundant. Lysosomal-like granules and granules of varying electron density were present in the cytoplasm. The chronic administration of P to E-primed animals did not result in any further increase in cell height. Elongated mitochondria, a cup-shaped Golgi apparatus, extensive apical microvilli, and irregularly shaped membranous profiles in the supranuclear cytoplasm characterized these uterine epithelial cells. Lysosomal-like granules, small vesicles, and scattered patches of glycogen were seen in the cytoplasm. These data show that the uterine epithelial cells of the ovariectomized sheep undergo morphological alterations in protein-synthesizing organelles and apical specializations that depend on the presence of E and P.     

Contractile responses to angiotensin I and angiotensin II in hamster uterine smooth muscle.

Angiotensin I (A-I) and angiotensin II (A-II) produced dose-dependent increases in isometric tension in isolated strips of uterine smooth muscle prepared from ovariectomized golden hamsters treated with estrogen. Responses to A-II were consistent with receptor--occupancy theory of agonist--receptor interactions. Inhibition of angiotensin-converting enzyme virtually abolished responses to A-I but not those to A-II. Blockade of A-II receptors inhibited responses to both A-I and A-II. Cholinergic or alpha-adrenergic blockade did not alter uterine responses to either A-I or A-II. These findings suggest that contractile responses elicited in the isolated uterus of the hamster are due to its local conversion to A-II and subsequent interactions with specific A-II receptors. Such conversion occurs at least to the extent of 14 to 27 %.     

Diabetes-related changes in cAMP response element-binding protein content enhance smooth muscle cell proliferation and migration

We hypothesized that diabetes and glucose-induced reactive oxygen species lead to depletion of cAMP response element-binding protein (CREB) content in the vasculature. In primary cultures of smooth muscle cells (SMC) high medium glucose decreased CREB function but increased SMC chemokinesis and entry into the cell cycle. These effects were blocked by pretreatment with the antioxidants. High glucose increased intracellular reactive oxygen species detected by CM-H(2)DCFA. SMC exposed to oxidative stress (H(2)O(2)) demonstrated a 3.5-fold increase in chemokinesis (p < 0.05) and accelerated entry into cell cycle, accompanied by a significant decrease in CREB content. Chronic oxidative challenge similar to the microenvironment in diabetes (glucose oxidase treatment) decreases CREB content (40-50%). Adenoviral-mediated expression of constitutively active CREB abolished the increase in chemokinesis and cell cycle progression induced by either high glucose or oxidative stress. Analysis of vessels from insulin resistant or diabetic animals indicates that CREB content is decreased in the vascular stroma. Treatment of insulin-resistant animals with the insulin sensitizer rosiglitazone restores vessel wall CREB content toward that observed in normal animals. In summary, high glucose and oxidative stress decrease SMC CREB content increase chemokinesis and entry into the cell cycle, which is blocked by antioxidants or restoration of CREB content. Thus, decreased vascular CREB content could be one of the molecular mechanisms leading to increased atherosclerosis in diabetes.     

Calcium channel antagonist binding and pharmacology in rat uterine smooth muscle

The calcium channel antagonists altered Ca-dependence of high K+-contractions of the estrogen dominant rat myometrium with the following pA2 values: PN-200-110, 10.63; nitrendipine, 9.56; nifedipine, 9.41; D-600, 9.05; and diltiazem, 7.57. Specific binding of 3H-nitrendipine occurred to the isolated plasma membrane vesicles with Kd of 0.1 to 0.3 nM and was inhibited by PN-200-110, nitrendipine, nifedipine and D-600, and slightly activated by diltiazem. The binding studies and the contractility studies were in excellent agreement for the three dihydropyridines, but not for D-600 and diltiazem.     

Purification and properties of AMP-deaminase from human uterine smooth muscle

AMP-deaminase from human uterine smooth muscle has been isolated, and properties of the enzyme were characterized. At pH 7.0, and in the presence of 100 mM potassium chloride the enzyme manifests a distinctly sigmoidal type of kinetics, with S0.5 parameter value about 12 mM. 1 mM ATP strongly activates the enzyme, and diminishes the value of S0.5 to 1.2 mM. In contrast to that 2.5 mM orthophosphate slightly inhibits the activity of AMP-deaminase studied and increases the S0.5 to about 14 mM. Similarly to ATP, orthophosphate does not influence the maximum velocity of the reaction. Electrophoresis in the presence of sodium dodecyl sulphate revealed that the molecular weight of human smooth muscle AMP-deaminase subunit is close to 37 kDa.