Neutral metalloendopeptidase associated with the smooth muscle cells of pregnant rat uterus

The pregnant rat uterus contains a membrane-bound metalloendopeptidase that is biochemically and immunologically similar to kidney enkephalinase (E.C.3.4.24.11). The uterus enzyme readily cleaved specific neutral endopeptidase substrates and oxytocin as well as the synthetic elastase substrate, Suc(Ala)3-pNA, yet did not digest native elastin. Using specific inhibitors, the uterus endopeptidase was identified as a metallopeptidase and not a serine protease, having an absolute requirement for zinc and perhaps calcium for maximal activity. The uterus endopeptidase cross-reacted with polyclonal antiserum to kidney microvillar endopeptidase and a monoclonal antibody to common acute lymphocytic leukemia antigen. Immunohistochemical localization of the enzyme in a 17 day pregnant uterus indicated that the enzyme was localized on the smooth muscle bundles of the myometrium and the endometrial epithelium. Total enzyme activity was 25 times higher in the late-term pregnant uterus (17th day of pregnancy) than in the nonpregnant uterus. Enzyme levels dropped rapidly prior to parturition and within 4 days after delivery the enzyme activity had returned to control levels. Inhibition of NEP in uterine strips with phosphoramidon resulted in a marked potentiation of oxytocin-induced contractions. Our results suggest that the uterine endopeptidase may have an important role in regulating uterine smooth muscle cell contraction during the later stages of pregnancy through its action on oxytocin and perhaps other biologically active peptides.     

Type V collagen regulates the assembly of collagen fibrils in cultures of bovine vascular smooth muscle cells

Vascular smooth muscle cells (SMCs), the major cellular constituent of the medial layer of an artery, synthesize the majority of connective tissue proteins, including fibrillar collagen types I, III, and V/XI. Proper collagen synthesis and deposition, which are important for the integrity of the arterial wall, require the antioxidant vitamin C. Vitamin C serves as cofactor for the enzymes prolyl and lysyl hydroxylase, which are responsible for the proper hydroxylation of collagen. Here, the role of type V collagen in the assembly of collagen fibrils in the extracellular matrix (ECM) of cultured vascular SMCs was investigated. Treatment of SMCs with vitamin C resulted in a dramatic induction in the levels of the cell-layer associated pepsin-resistant type V collagen, whereas only a minor induction in the levels of types I and III collagen was detected. Of note, the deposition of type V collagen was accompanied by the formation of striated collagen fibrils in the ECM. Immunohistochemistry demonstrated that type V collagen, but not type I collagen, became masked as collagen fibrils matured. Furthermore, the relative ratio of type V to type I collagen decreased as the ECM matured as a function of days in culture, and this decrease was accompanied by an increase in the diameter of collagen fibrils. Together these results suggest that the masking of type V collagen is caused by its internalization on continuous deposition of type I collagen on the exterior of the fibril. Furthermore, they suggest that type V collagen acts as framework for the initial assembly of collagen molecules into heterotypic fibrils, regulating the diameter and architecture of these fibrils.     

Location of enzymes of acetylcholine hydrolysis in smooth muscle cells of the breast and uterus

The data on distribution of cholinesterase activity in smooth myocytes of the mammary ducts and in the tissue of normal and pathologically altered myometrium are presented. In the myometrium tumorous tissue, as demonstrate hystochemical investigation results, terminals of the nervous fibers are practically absent, while in the norm they are present in the intermuscular plexuses and accompany blood vessels. At the myomic alteration of the uterine smooth musculature, in myocytes a high enzymatic activity of the acetylcholine hydrolysis enzymes is revealed. An essentially activity of cholinesterases is found in the smooth myocytes of the mammary ducts. Participation of the acetylcholine hydrolysis and synthesis mechanisms in organization of rhythmic contractile activity is discussed.     

Involvement of phospholipase D in store-operated calcium influx in vascular smooth muscle cells

In non-excitable cells, sustained intracellular Ca2+ increase critically depends on influx of extracellular Ca2+. Such Ca2+ influx is thought to occur by a 'store-operated' mechanism, i.e. the signal for Ca2+ entry is believed to result from the initial release of Ca2+ from inositol 1,4,5-trisphosphate-sensitive intracellular stores. Here we show that the depletion of cellular Ca2+ stores by thapsigargin or bradykinin is functionally linked to a phosphoinositide-specific phospholipase D (PLD) activity in cultured vascular smooth muscle cells (VSMC), and that phosphatidic acid formed via PLD enhances sustained calcium entry in this cell type. These results suggest a regulatory role for PLD in store-operated Ca2+ entry in VSMC.     

Phenotypic heterogeneity influences the behavior of rat aortic smooth muscle cells in collagen lattice

Phenotypic modulation of vascular smooth muscle cells (SMCs) in atherosclerosis and restenosis involves responses to the surrounding microenvironment. SMCs obtained by enzymatic digestion from tunica media of newborn, young adult (YA) and old rats and from the thickened intima (TI) and underlying media of young adult rat aortas 15 days after ballooning were entrapped in floating populated collagen lattice (PCL). TI-SMCs elongated but were poor at PCL contraction and remodeling and expressed less alpha2 integrin compared to other SMCs that appeared more dendritic. During early phases of PCL contraction, SMCs showed a marked decrease in the expression of alpha-smooth muscle actin and myosin. SMCs other than TI-SMCs required 7 days to re-express alpha-smooth muscle actin and myosin. Only TI-SMCs in PCL were able to divide in 48 h, with a greater proportion in S and G2-M cell cycle phases compared to other SMCs. Anti-alpha2 integrin antibody markedly inhibited contraction but not proliferation in YA-SMC-PLCs; anti-alpha1 and anti-alpha2 integrin antibodies induced a similar slight inhibition in TI-SMC-PCLs. Finally, TI-SMCs rapidly migrated from PCL on plastic reacquiring their epithelioid phenotype. Heterogeneity in proliferation and cytoskeleton as well the capacity to remodel the extracellular matrix are maintained, when SMCs are suspended in PCLs.     

The degradation of endogenous and exogenous proteins in cultured smooth muscle cells

The pathways of degradation followed by endogenous proteins in cultured smooth muscle cells were compared with the well-characterized lysosomal pathway involved in the degradation of apolipoprotein B of endocytosed LDL. Under conditions in which lysosomal activity towards ¹²⁵I-labeled LDL was almost completely inhibited by chloroquine and/or ammonium chloride, the degradation of short-lived and abnormal proteins, assessed by the release of [³H]phenylalanine, was reduced by only 10–17%. The basal rate of degradation of long-lived proteins was reduced by about 30% by the same inhibitors while the accelerated proteolysis found under nutrient-poor conditions could be completely accounted for by the lysosomal system as defined by these lysosomotrophic agents. Temperature studies indicated differences between the mechanisms involved in the degradation of long-lived proteins (E_a = 18 kcal/mol) and short-lived proteins (E_a = 10 kcal/mol). Arrhenius plots for the degradation of endogenous proteins showed no transitions between 15 and 37°C in contrast to the breakdown of LDL which ceased below 20°C. The results indicate that the degradation of rapid-turnover proteins is largely extralysosomal and that a significant breakdown of long-lived proteins occurs also outside lysosomes.     

Complex regulation of collagen gene expression in cultured bovine aortic smooth muscle cells

Cultured bovine aortic smooth muscle cells display an increase in production of type I and type III collagen as a function of the number of days in second passage (Beldekas, J. C., Gerstenfeld, L. C., Sonenshein, G. E., and Franzblau, C. (1982) J. Biol. Chem. 257, 12552-12556). In this study, we report that the regulation of these events is highly complex and relates to the growth state of the cells. Cultures, seeded at 1.5 X 10(6) cells/75-cm2 flask, produced very little collagenous protein early when the cells were proliferating rapidly. As they approached confluence at day 6, collagen synthesis began to increase. Maximal collagen synthesis was observed at day 14. In contrast, the levels of the mRNAs for type I and type III collagen increased only up to the 10th day and thereafter decreased. Cell-free translation analyses indicated that the translational activity of the collagen mRNAs was increasing over the time course. These results suggest that both translational and pretranslational sites are involved in the control of collagen production by aortic smooth muscle cells, and that collagen synthesis is inversely related to the proliferative state of the cells in culture.     

Involvement of calcium in calcium-current inactivation in smooth muscle cells from rat vas deferens

Summary Ca-channel currents were recorded in Cs-loaded single smooth muscle cells from rat vas deferens to define the dependence of the inactivation time course on Ca concentration. The decay of Ca-channel current obtained in a Ba²⁺- or Sr²⁺-containing external solution during long voltage-clamp pulses was much slower than that in a Ca-containing solution. The difference was not due to a change in the surface potential of the membrane as judged from the steady-state activation and inactivation curves. When Ca was the charge carrier, increasing external Ca concentration slightly accelerated the rate of inactivation. In addition, the rate of inactivation of Ca-channel current in 10.8mm Ba was also accelerated by adding Ca to the external solution in a concentration-dependent manner. The time course of Ca-current inactivation was slowed when the cells were dialyzed with a high concentration of citrate, a Ca-chelating agent. From these results, we concluded that a mechanism regulated by intracellular Ca activity plays a role in the inactivation of Ca channels in smooth muscle. The Ca-dependent process may protect against Ca overload by regulating Ca entry in smooth muscle cells.     

Involvement of phospholipase D in oxidative stress-induced necrosis of vascular smooth muscle cells.

Phospholipase D (PLD) has been associated with necrosis. However, it is not clear whether PLD plays a causative role in this cellular process. We investigated the role of PLD in oxidative stress-induced necrosis of vascular smooth muscle cells (VSMCs). Pervanadate (hydrogen peroxide plus orthovanadate) but not hydrogen peroxide alone activated PLD in a dose- and time-dependent manner. Exposure of VSMCs to pervanadate resulted in necrosis. Pretreatment with butan-1-ol, a PLD inhibitor, attenuated both pervanadate-induced necrosis and increase of intracellular Ca(2+). Removal of extracellular Ca(2+) inhibited pervanadate-induced necrosis by 50%. These results suggest that PLD activation mediates pervanadate-induced necrosis of VSMCs, which is at least partly due to Ca(2+) toxicity.     

Synthesis of collagen by smooth muscle in the hyertrophic intestine

During hypertrophy of the small intestine of the guinea-pig at the oral side of an experimental stenosis, the individual intestinal segments do not increase in length but undergo a remarkable increase in weight (up to 7 times that of control segments). Concomitantly an increase in collagen content is found; this is usually larger than the increase in weight, and the concentration of collagen is therefore also increased. This effect is more noticeable in the muscle coat than in the wall as a whole. It is suggested, on the basis of histological observations, that some of this collagen is synthesized by smooth muscle cells.     

Heparin regulates the collagen phenotype of vascular smooth muscle cells: induced synthesis of an Mr 60,000 collagen

The effect of heparin on the biosynthetic phenotype of rat vascular smooth muscle cells (SMC) was investigated in vitro. Addition of heparin to the culture medium of early passage rat SMC resulted in a marked (3-15-fold) increase of a cell layer-associated Mr 60,000 protein that was sensitive to digestion by purified bacterial collagenase and contained significant amounts of hydroxyproline. Pulse-chase analysis of heparin-treated SMC revealed that the Mr 60,000 collagen was a primary and abundant product of these cells and was not processed extracellularly to a smaller form. The inductive effect of heparin could be mimicked by iota carrageenan or dextran sulfates but not by hyaluronic acid, dermatan sulfate, or chondroitin sulfates. The induction was concentration dependent with a maximal effect observed at a heparin concentration of 10 micrograms/ml. Synthesis of the Mr 60,000 collagen increased 18-24 h after addition of heparin to the cultures. Following induction and subsequent removal of heparin, synthesis of the protein remained maximal for at least 12 h and required 72 h to return to a basal level. These data demonstrate that the biosynthetic phenotype of vascular SMC in vitro can be controlled, at least in part, by heparin and related polyanions and suggest a role for similar molecules endogenous to the vessel wall in the regulation of SMC function.     

Glucocorticoids stimulate collagen and noncollagen protein synthesis in cultured vascular smooth muscle cells

The effect of glucocorticoids on collagen synthesis was examined in cultured bovine aortic smooth muscle (BASM) cells. BASM cells treated with 0.1 microM dexamethasone during their proliferative phase (11 d) were labeled with [3H]proline for 24 h, and the acid-precipitable material was incubated with bacterial collagenase. Dexamethasone produced an approximate twofold increase in the incorporation of proline into collagenase-digestible protein (CDP) and noncollagen protein (NCP) in the cell layer and medium. The stimulation was present in both primary mass cultures and cloned BASM. An increase in CDP and NCP was detected at 0.1 nM, while maximal stimulation occurred at 0.1 microM. Only cells exposed to dexamethasone during their log phase of growth (1-6 d after plating) showed the increase in CDP and NCP when labeled 11 d after plating. The stimulatory effect was observed in BASM cells treated with the natural bovine glucocorticoid, cortisol, dexamethasone, and testosterone, but was absent in cells treated with aldosterone, corticosterone, cholesterol, 17 beta-estradiol, and progesterone. The increase in CDP and NCP was absent in cells treated with the inactive glucocorticoid, epicortisol, and totally abolished by the antagonist, 17 alpha-hydroxyprogesterone, suggesting that the response was mediated by specific cytoplasmic glucocorticoid receptors. Dexamethasone-treated BASM cells showed a 4.5-fold increase in the specific activity of intracellular proline, which was the result of a twofold increase in the uptake of proline and depletion of the total proline pool. After normalizing for specific activity, dexamethasone produced a 2.4- and 2.8-fold increase in the rate of collagen and NCP synthesis, respectively. Cells treated with dexamethasone secreted 1.7- fold more collagen protein in 24 h compared to control cultures. The BASM cells secreted 70% Type I and 30% Type III collagen into the media as assessed by two-dimensional gel electrophoresis. The ratio of these two types was not altered by dexamethasone. The results of the present study demonstrate that glucocorticoids can act directly on vascular smooth muscle cells to increase the synthesis and secretion of collagen and NCP.     

Aortic smooth muscle cells in collagen lattice culture: effects on ultrastructure, proliferation and collagen synthesis

Adult pig smooth muscle cells (SMC) were isolated from the aortic media by collagenase digestion, subcultured as monolayer, and then re-integrated into a three-dimensional network of type I collagen. The contractile state characteristic for resident arterial wall SMC changed to the synthetic, fibroblast-like state. The cells reorganized the randomly orientated collagen fibrils causing the lattice to shrink. The influence of the extracellular matrix on the ultrastructure, the proliferation, and the collagen synthesis of these SMC embedded in the collagen lattice was investigated and compared to cells cultured in monolayer. The amount of total protein and collagens synthesized by SMC embedded in lattices was lowered as compared to monolayer cultures. Whereas total protein synthesis decreased continuously during the culture period, the proportion of collagen synthesis remained at a constant level. Although cells proliferated in lattices, proliferation was clearly slowed down as compared to monolayer cultures. The ultrastructure of entrapped synthetic state SMC was comparable to that of monolayer-cultured cells. Their cytoplasm was largely filled by elements of the endoplasmic reticulum, Golgi complexes and abundant mitochondria. With prolonged culture time, electron-dense granules as well as bodies containing whorled membranes could be found in the cytoplasm. These results indicate that synthetic state SMC can exhibit differential biosynthetic activity dependent on the actual matrix environment; cells seem to be able to sense the macromolecular composition of the extracellular matrix and to modify their production of matrix components accordingly.     

Phagocytosis of cytoplasmic blebs derived from smooth muscle cells by fibroblast-like cells and macrophages in the post-partum rat uterus

Numerous blebs were observed in contact with smooth muscle cells (SMC) by light microscopy in the myometrium of the rat uterus after parturition. Electron-microscopically the cell surface of SMC showed bulbous protrusions, which often lacked a basement membrane and were less electron-dense than the surrounding cytoplasm or sometimes nearly electron-lucent. Many bulbous protrusions were separated from SMC and became the isolated structures which we called cytoplasmic blebs. These bulbous protrusions and cytoplasmic blebs were often found to be phagocytosed by fibroblast-like cells and macrophages. A series of these tissue changes in the uterine myometrium after delivery, possibly due to hypoxic conditions, contribute to a rapid involution of SMC which have enlarged during pregnancy.