Maintenance and characterization of human myometrial smooth muscle cells in monolayer culture

Human myometrial cells were dispersed from uterine tissue by limited enzymatic digestion of myometrium that was obtained at the time of hysterectomy. The dispersed myometrial cells that are obtained in this manner can be maintained in monolayer culture in the presence of medium that contains fetal bovine serum. In primary culture, as well as after passage, the characteristics of these cells are morphologically and biochemically similar to those of smooth muscle cells and myometrial tissue.     

Effect of progesterone and oestradiol on expression of connexin43 in cultured human myometrium cells

In human myometrium, the formation of gap junctions at various stages of labour and in correlation with the concentration of progesterone and oestradiol in maternal blood was described previously by electron microscopy and laser confocal microscopy of immunohistochemically stained myometrial sections. The present investigation focused on the effect of continuous exposure of isolated myometrial tissue to progesterone and oestradiol on the number of gap junction plaques in human myometrium cells in vitro. The presence of gap junctions was evaluated by immunocytochemistry with antibodies against gap junction protein, connexin43 (Cx43). Human myometrial cells were isolated from biopsies obtained from term pregnant women who had an elective caesarean operation in the 37th or 40th week of pregnancy. The dispersed myometrial cells that were obtained by limited enzymatic digestion of the myometrial samples were maintained in monolayer culture for 1, 3 and 6 days in the presence of medium that contained foetal bovine serum and the steroids at different concentration. In primary culture, as well as after several passages, the characteristics of these cells were morphologically and biochemically similar to those of smooth muscle cells and myometrial tissue. The obtained results showed that the cells in culture responded synchronously to the increased concentrations of oestradiol/progesterone mixtures. The number of gap junctions increased significantly on days 1, 3 and 6 in culture and showed positive correlation (p < 0.05) with the cell number when the concentration of oestradiol was raised to 1 microgram/mL in the progesterone ratio (1.0 microgram/0.5 microgram/mL). No significant correlation, however, in connexin43 gap junction number versus cell number was observed between the six experimental groups treated with progesterone only.     

Biochemical markers of contraction in human myometrial smooth muscle cells in culture

Summary Phosphorylation of a light chain subunit of myosin by Ca²⁺ and calmodulin-dependent myosin light chain kinase is believed to be essential for smooth muscle contraction. The biochemical properties of the myosin phosphorylation system in human myometrial smooth muscle cells in monolayer culture were compared with those of human myometrial tissue and nonmuscle cells in culture. Native myosin was isolated from other cellular proteins of crude homogenates by polyacrylamide gel electrophoresis (in the presence of pyrophosphate) and quantified by densitometry. The myosin content of myometrial smooth muscle cells in culture and that of myometrial tissue were similar and four- to five-fold greater than that of human endometrial stromal cells or skin fibroblasts in culture. The specific activities of myosin light chain kinase in homogenates of myometrial smooth muscle cells that were maintained in culture and in myometrial tissue were similar (2.05±0.18 and 1.60±0.37 nmol phosphate incorporated per min per mg protein, respectively). On the other hand, enzyme activity in skin fibroblasts was only 5% of that in myometrial smooth muscle cells. Myosin light chain kinase activity in myometrial smooth muscle cells was dependent upon Ca²⁺ and was inhibited reversibly by the calmodulin antagonist, calmidazolium. The intracellular Ca²⁺ concentration measured by quin2 fluorescence was 0.12 μM in resting cells and increased in a concentration-dependent manner with KC1 to a maximal value of 0.47 μM. These results indicate that biochemical processes important for smooth muscle contraction are retained in human myometrial smooth muscle cells in culture. This research was supported by grants HL26043, HD11149, and GM07062 from the National Institutes of Health, Bethesda, MD.     

The effect of progesterone on oxytocin-stimulated intracellular mobilization of Ca2+ and prostaglandin E2 and F2alpha secretion from porcine myometrial cells

Our past studies have shown that porcine myometrium produce prostaglandins (PG) during luteolysis and early pregnancy and that oxytocin (OT) and its receptor (OTr) support myometrial secretion of prostaglandins E2 and F2alpha (PGE2 and PGF2alpha) during luteolysis. This study investigates the role of intracellular Ca2+ [Ca2+]i as a mediator of OT effects on PG secretion from isolated myometrial cells in the presence or absence of progesterone (P4). Basal [Ca2+]i was similar in myometrial cells from cyclic and pregnant pigs (days 14-16). OT (10(-7)M) increased [Ca2+]i in myometrial cells of cyclic and pregnant pigs, although this effect was delayed in myometrium from pregnant females. After pre-incubation of the myocytes with P4 (10(-5)M) the influence of OT on [Ca2+]i)was delayed during luteolysis and inhibited during pregnancy. Myometrial cells in culture produce more PGE2 than PGF2alpha regardless of reproductive state of the female. OT (10(-7)M) increased PGE2 secretion after 6 and 12 h incubation for the tissue harvested during luteolysis and after 12 h incubation when myometrium from gravid females was used. In the presence of P4 (10(-5)M), the stimulatory effect of OT on PG secretion was diminished. In conclusion: (1) porcine myometrial cells in culture secrete PG preferentially during early pregnancy and produce more PGE2 than PGF2alpha, (2) OT controls myometrial PGF2alpha secretion during luteolysis, (3) release of [Ca2+]i is associated with the influence of OT on PG secretion, and (4) the effects of OT on PG secretion and Ca2+ accumulation are delayed by P4 during luteolysis and completely inhibited by P4 during pregnancy.     

Effect of a diabetic state on myometrial ultrastructure and isolated uterine contractions in the rat

Alterations in rat myometrial ultrastructure and in vivo uterine contractile responses to oxytocin were examined in estradiol-treated (40 micrograms/kg) euglycemic and streptozotocin-induced (85 mg/kg) diabetic rats. Myometrial morphology was examined 18, 24, and 36 hr after estradiol administration. At the time points examined, nuclei of myometrial cells from euglycemic and diabetic rats were pleomorphic and contained large areas of heterochromatin dispersed throughout the nuclei. Mitochondria were round to oval in shape and contained a dense matrix with cristae that extended across the mitochondria. Myofilaments were found in both euglycemic and diabetic cells but the relative number of myofilaments in diabetic cells appeared to be less than the number found in myometrial cells removed from euglycemic animals. The number of free cytoplasmic ribosomes in diabetic cells also appeared to be less than those found in euglycemic cells. In order to determine if apparent differences in the number of myofilament found in diabetic myometrial cells could be correlated with changes in uterine contractile responses to hormones, oxytocin dose-response curves (10(-8) to 10(-5) M) were examined in isolated uteri removed from saline-injected and estradiol-injected (24-hr pretreatments) euglycemic and diabetic rats. The maximal contractile responses (milligrams tension developed per milligrams tissue) in saline-injected euglycemic and diabetic rats were 49 +/- 5 and 36 +/- 4, respectively, while maximal contractile responses in estradiol-injected euglycemic and diabetic rats were 68 +/- 7 and 45 +/- 5, respectively. Maximal contractile responses induced by oxytocin in estradiol-treated diabetic uteri were significantly smaller than the contractile responses measured in euglycemic estradiol-treated uteri. This study demonstrates that estradiol-induced changes in both myometrial cell morphology and in vitro uterine contractile responses to oxytocin are altered in diabetic rats.     

Evolution of cAMP phosphodiesterase activity in cultured myometrial cells: Effects of steroids and of successive subcultures

Cyclic AMP phosphodiesterase (PDE) activity was characterized in culture of ewe myometrial cells and its sensitivity to steroid hormones was tested. Cultured myometrial cells were maintained from the first to the 20th subculture in the presence of 2% of serum in a medium supplemented with 1 μM of insulin. It was found that myometrial cells possess a PDE activity with atypical kinetics. The nonlinear responses in Lineweaver-Burke plots suggest the presence of high- and low-affinity PDE activities. In cell culture, apparent Km values were similar to those obtained from the original myometrium. Vmax values increased with successive subcultures, revealing an increase in the capacity of the cells to degrade cAMP; in parallel, the growth rate decreased. The PDE specific activity in cultured myometrial cells was inhibited by estra-diol or progesterone. When added together, no synergistic effect was obtained. The rate of inhibition for both steroids was constant during successive passages for both low- and high-affinity conditions. Results obtained in myometrial cell long-term culture were compatible with reports in other species in vivo. Considering the role of cAMP in the regulation of uterine functions, subcultured myometrial cells provided us a useful experimental system with which to study the cAMP metabolism process.     

Selective tonic inhibition of G-6-Pase catalytic subunit, but not G-6-P transporter, gene expression by insulin in vivo

The regulation of glucose-6-phosphatase (G-6-Pase) catalytic subunit and glucose 6-phosphate (G-6-P) transporter gene expression by insulin in conscious dogs in vivo and in tissue culture cells in situ were compared. In pancreatic-clamped, euglycemic conscious dogs, a 5-h period of hypoinsulinemia led to a marked increase in hepatic G-6-Pase catalytic subunit mRNA; however, G-6-P transporter mRNA was unchanged. In contrast, a 5-h period of hyperinsulinemia resulted in a suppression of both G-6-Pase catalytic subunit and G-6-P transporter gene expression. Similarly, insulin suppressed G-6-Pase catalytic subunit and G-6-P transporter gene expression in H4IIE hepatoma cells. However, the magnitude of the insulin effect was much greater on G-6-Pase catalytic subunit gene expression and was manifested more rapidly. Furthermore, cAMP stimulated G-6-Pase catalytic subunit expression in H4IIE cells and in primary hepatocytes but had no effect on G-6-P transporter expression. These results suggest that the relative control strengths of the G-6-Pase catalytic subunit and G-6-P transporter in the G-6-Pase reaction are likely to vary depending on the in vivo environment.     

Tat-mediated protein transduction of human brain pyridoxine-5-P oxidase into PC12 cells

Pyridoxine-5-P oxidase catalyses the terminal step in the biosynthesis of pyridoxal-5-P, the biologically active form of vitamin B6 which acts as an essential cofactor. Here, a human brain pyridoxine-5-P oxidase gene was fused with a gene fragment encoding the HIV-1 Tat protein transduction domain (RKKRRQRRR) in a bacterial expression vector to produce a genetic in-frame Tat-pyridoxine-5-P oxidase fusion protein. Expressed and purified Tat-pyridoxine-5-P oxidase fusion protein transduced efficiently into PC12 cells in a time- and dose-dependent manner when added exogenously to culture media. Once inside the cells, the transduced Tat-pyridoxine-5-P oxidase protein showed catalytic activity and was stable for 48 h. Moreover, the formation of pyridoxal-5-P was increased by adding exogenous Tat-pyridoxine-5-P oxidase to media pre-treated with the vitamin B6 precursor pyridoxine. In addition, the intracellular concentration of pyridoxal-5-P was markedly increased when Tat-pyridoxal kinase was transduced together with Tat-pyridoxine-5-P oxidase into cells.These results suggest that the transduction of Tat-pyridoxine-5-P oxidase fusion protein presents a means of regulating the level of pyridoxal-5-P and of replenishing this enzyme in various neurological disorders related to vitamin B6.     

Introduction and metabolism of pentose and hexose phosphates in permeabilized Morris hepatoma 5123TC cells

Metabolism of arabinose 5-P, ribose 5-P and glucose 6-P in permeabilized and resealed Morris hepatoma 5123TC cells was investigated by measuring the contribution of these compounds to nucleic acid biosynthesis. The level of [14C]-arabinose (non-phosphorylated) incorporation into nucleic acids was slight, presumably due to the low activity of the transport system or the absence or low activity of a specific 'kinase' enzyme. The permeabilizing procedure involved the brief treatment of Morris hepatoma 5123TC cells with lysolecithin and resulted in a cell population which was permeable to charged compounds i.e. sugar phosphates and nucleotides, that otherwise could not cross the plasma membrane. The permeabilized (and resealed cells) retained normal cellular morphology and intactness of specific organelles as judged by the maintenance of functional properties. Following permeabilization, these cells resealed when transferred back to normal growth medium, and continued to divide and increase at the same rates as control non-permeabilized cell cultures. The permeabilized cells incorporated deoxyribonucleotides ([methyl -3H]-TTP) into DNA at a linear rate of 0.047 nmol per 10(7) cells min-1, representing 90-100 per cent of the DNA synthesis rate in vivo. The permeabilization technique, when coupled with procedures to establish cell synchrony, permitted the comparative estimate of the contributions of [14C]-labelled arabinose 5-P, ribose 5-P and glucose 6-P to RNA, DNA, amino acids, CO2, lactate and sugar mono- and bisphosphates. The percentage of [14C]-isotope incorporated into total nucleic acids by these three labelled sugar phosphates were 2.3, 4.9 and 6.3 respectively. Possible reasons for the lower incorporation of 14C from arabinose 5-P are given. The results are consistent with the proposal that arabinose 5-P, an intermediate of the L-type pentose pathway activity of 5123TC cells, was incorporated into nucleic acids by its interconversion with ribulose 5-P and ribose 5-P and thus into PRPP. This study represents the first report of sugar phosphate as opposed to free sugar metabolism by tumour cells in culture.     

Regulation of prostaglandin production by nitric oxide in rat smooth muscle myometrial cells.

Smooth muscle myometrial cells isolated by an enzymatic method from estrogenized rats were used after 7-10 days of culture. They were incubated for 24 h with two distinct competitive nitric oxide (NO) inhibitors: NG-monomethyl-L-arginine (L-NMMA: 300 microM) and L-nitro-arginine methylester (L-NAME: 600 microM, 5 mM and 10 mM). Afterwards, the supernatants were separated in order to measure nitrite production and prostaglandin PGE synthesis. In the present report, we demonstrate that myometrial cells from estrogenized rats are able to produce NO, since all the inhibitors significantly decrease the production of nitrites in the culture media. Furthermore, we report that both inhibitors inhibited PGE synthesis by myometrial cells. We also used a donor of NO in the incubation medium for 24 h, sodium nitroprusside (NP), obtaining an strong (P< 0.001) increase in both nitrite and PGE production. We conclude that myometrial cells can produce NO and that one possible role of the NO synthetized by this cells may be the modulation of PGE production.     

Glucocorticosteroid regulation of prostaglandin biosynthesis in human myometrial smooth muscle cells in monolayer culture

In the present investigation, we evaluated the production of prostaglandins by human myometrial smooth muscle cells maintained in monolayer culture in the absence or presence of glucocorticosteroids. In the presence of cortisol (10(-7) M) or dexamethasone (10(-8) M), the rate of production of prostacyclin (PGI2) by these cells was decreased significantly. The glucocorticosteroid-mediated inhibition of prostaglandin production was attenuated when cortisol-21-mesylate (10(-6) M), a glucocorticosteroid antagonist, was present in the culture medium. The rate of conversion of radiolabeled arachidonic acid to radiolabeled prostaglandins as determined by use of sonicates of myometrial cells and optimal assay conditions, however, was not affected significantly by treatment with cortisol or dexamethasone in concentrations sufficient to inhibit prostaglandin formation by more than 80%. These findings are suggestive that glucocorticosteroids act in human myometrial smooth muscle cells in culture to inhibit prostaglandin formation by way of a receptor-mediated process that does not involve inhibition of enzyme activities that are involved in the biosynthesis of prostaglandins, i.e. the conversion of arachidonic acid to prostaglandin.     

Interaction between nitric oxide and prostaglandin E pathways in rat smooth muscle myometrial cells

Previously, we demonstrated the presence of a nitric oxide (NO) prostaglandin (PG) pathway in myometrial cells obtained from uterine rat tissue. This pathway was modulated by estrogen and one possible function could be to modulate uterine relaxation. In the present study, we investigated the role of progesterone in the regulation of NO synthesis and the uterotonic PGE production by myometrial cells from uterine rat tissue. We worked with two groups of rats: (i) ovariectomizcd (OV) rats, without influence of sex hormones and (ii) OV rats injected with progesterone (4 mg) s.c. Myometrial uterine cells were obtained by a selective enzymatic digestion. In the incubation medium of these cells, nitrite concentration (as a measure of NO production) and PGE production were evaluated. To ensure a specific response, a competitive NOs inhibitor, N(G)-monomethyl-L-arginine; L-NMMA (300 microM) was used. We found that at 48 h of the incubation period, cells obtained from progesterone-primed uterine tissue presented an increase in the nitrite concentration concomitant with a decrease in the PGE production. When L-NMMA was added to the cells, nitrite production and PGE synthesis returned to control values. The fact that this effect had not been observed in the group of cells obtained from OV rats suggests that progesterone was responsible for it. These data provide strong evidence that in spite of the fact that estrogen and progesterone modulate the NO-PG pathway in the uterine rat tissue, the two hormones have opposite effects.     

Structures and metabolism of inositol tetrakisphosphates and inositol pentakisphosphate in bovine adrenal glomerulosa cells

In adrenal glomerulosa cells, angiotensin II stimulates rapid increases in inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) and inositol 1,3,4,5-tetrakisphosphate (Ins-1,3,4,5-P4), followed by slower increases in two additional inositol tetrakisphosphate (InsP4) isomers. One of these InsP4 isomers was previously identified as Ins-1,3,4,6-P4 and shown to be a precursor of inositol pentakisphosphate (InsP5). Analysis of the third InsP4 isomer, purified from cultured bovine adrenal cells labeled with [3H]inositol and stimulated by angiotensin II, revealed that the polyol produced by periodate oxidation, borohydrate reduction, and dephosphorylation was [3H]iditol. This finding is consistent with precursor structures of either Ins-1,4,5,6-P4 or Ins-3,4,5,6-P4 (= L-Ins-1,4,5,6-P4) for the third InsP4 isomer. The [3H]iditol was readily converted to [3H]sorbose by the stereospecific enzyme, L-iditol dehydrogenase, indicating that it originated from Ins-3,4,5,6-P4. Chicken erythrocytes labeled with [3H]inositol also contained high levels of Ins-1,3,4,6-P4 and Ins-3,4,5,6-P4, as well as InsP5, but only small amounts of Ins-1,3,4,5-P4. Both [3H]Ins-1,3,4,6-P4 and [3H]Ins-3,4,5,6-P4, but not [3H]Ins-1,3,4,5-P4, were phosphorylated to form InsP5 in permeabilized bovine glomerulosa cells. In addition, InsP5 itself was slowly dephosphorylated to Ins-1,4,5,6-P4, indicating that its structure is Ins-1,3,4,5,6-P5. These results demonstrate that the higher inositol phosphates are metabolically interrelated and are linked to the receptor-regulated InsP3 response by the conversion of Ins-1,3,4-P3 through Ins-1,3,4,6-P4 to Ins-1,3,4,5,6-P5. The source of Ins-3,4,5,6-P4, the other precursor of InsP5, is not yet known but its elevation in angiotensin II-stimulated glomerulosa cells suggests that its formation is also influenced by agonist-regulated processes.     

Serotonin: an inducer of collagenase in myometrial smooth muscle cells.

Rat myometrial smooth muscle cells in culture actively produce collagenase in medium containing fetal bovine serum, but not in medium containing newborn bovine serum or containing fetal serum adsorbed with dextran-coated charcoal. A dialyzable molecule has been isolated from fetal bovine serum, which restores the ability of the smooth muscle cells to produce collagenase. The molecule has been purified and identified as serotonin (5-hydroxytryptamine). Cells cultured in medium depleted of serotonin for 3 days fail to produce collagenase, as assessed both enzymatically and immunologically. Addition of serotonin promptly restores the ability of the cells to produce the enzyme. The EC50 for serotonin is approximately 2 microM; maximum stimulation of collagenase production is observed at 5 microM. The response is specific for serotonin: a wide variety of compounds tested, either related to serotonin or of potential reproductive significance, were without effect in the induction of collagenase production by the cells. No changes in DNA content, general protein synthesis, or cellular collagen production were observed as a consequence of serotonin depletion or restoration, suggesting a selective effect of the compound on collagenase production. The effect of serotonin was also selective to myometrial smooth muscle cells; collagenase-producing fibroblasts from skin and cervix displayed no serotonin requirement for enzyme production. Studies using specific agonists or antagonists for a variety of serotonin receptor subtypes suggest that the 5-HT-2 receptor mediates the serotonin induction of collagenase in these cells. Preliminary evidence indicates that cultured human myometrial smooth muscle cells are also dependent upon serotonin for collagenase production. The evidence in this study suggests the possibility that serotonin serves as a signal to begin the massive collagen degradation that occurs in the postpartum uterus.     

Induction of platelet-derived growth factor receptor expression in smooth muscle cells and fibroblasts upon tissue culturing

The expression of platelet-derived growth factor (PDGF) receptors in porcine uterus and human skin in situ, was compared with that of cultured primary cells isolated from the same tissues. PDGF receptor expression was examined by monoclonal antibodies specific for the B type PDGF receptor and by RNA/RNA in situ hybridization with a probe constructed from a cDNA clone encoding the B type PDGF receptor. In porcine uterus tissue both mRNA and the protein product for the PDGF receptor were detected in the endometrium; the myometrium, in contrast, contained much lower amounts. Moreover, freshly isolated myometrial cells were devoid of PDGF receptors. However, after 1 d in culture receptors appeared, and after 2 wk of culturing essentially all of the myometrial cells stained positively with the anti-PDGF receptor antibodies and contained PDGF receptor mRNA. Similarly, B type PDGF receptors were not detected in normal human skin, but fibroblast-like cells from explant cultures of human skin possessed PDGF receptors. When determined by immunoblotting, porcine uterus myometrial membranes contained approximately 20% of the PDGF receptor antigen compared with the amount found in endometrial membranes. In addition, PDGF stimulated the phosphorylation of a 175-kD component, most likely representing autophosphorylation of the B type PDGF receptor in endometrial membranes, whereas only a marginal phosphorylation was seen in myometrial membranes. Taken together, these results demonstrate that PDGF receptor expression varies in normal tissues and that fibroblasts and smooth muscle cells do not uniformly express the receptor in situ. Furthermore, fibroblasts and smooth muscle cells that are released from tissues are induced to express PDGF receptors in response to cell culturing. The data suggest that, in addition to the availability of the ligand, PDGF-mediated cell growth in vivo is dependent on factors regulating expression of the receptor.     

A new transient activator of phosphofructokinase during initiation of rapid glycolysis in brain

The tissue contents of previously known allosteric effectors of brain phosphofructokinase (EC 2.7.1.11) (PFK) and the kinetic behavior of isolated PFK were investigated during the initiation of rapid glycolytic flux in freeze-blown rat brain. Comparing 0- with 5-s brains revealed that there was a 4-fold drop in total tissue content of Fru-6-P and a 5.6-fold increase in Fru-1,6-P2 consistent with activation of PFK. Additionally, analysis of brain content showed a 15-fold increase in AMP, a 3-fold decrease in ATP, a 3-fold decrease in Pi, and a 1.6-fold increase in NH4+. There was no change in Fru-2,6-P2, H+, citrate, or Glc-1,6-P2 or the kinetic profiles of isolated PFK for ATP inhibition or Fru-2,6-P2 activation. We concluded that the observed change in PFK activity could be accounted for only partially by changes in the concentrations of adenine nucleotides and other known effectors. High performance liquid chromatography fractions of extracts obtained from 5-s brains showed the activator with a mobility identical to ribose 1,5-P2 and gave 2 nmol/g (wet weight) at 0 s, 10 nmol/g at 5 s, and 2 nmol/g at 20 s. Assay of PFK in the presence of effectors determined to be in tissue at 5 s showed that addition of 10 nmol/ml ribose 1,5-P2 gave a 4-fold activation of PFK. Based on the rapidity of its formation, its potency of activation, and its similarity in chemical properties to authentic ribose 1,5-P2, we conclude that ribose 1,5-P2 served as the initial activator of PFK in brain.     

Transposition of elements of the 412, copia and 297 dispersed repeated gene families in Drosophila.

The stability of elements of three different dispersed repeated gene families in the genome of Drosophila tissue culture cells has been examined. Different amounts of sequences homologous to elements of 412, copia and 297 dispersed repeated gene families are found in the genomes of D. melanogaster embryonic and tissue culture cells. In general the amount of these sequences is increased in the cell lines. The additional sequences homologous to 412, copia and 297 occur as intact elements and are dispersed to new sites in the cell culture genome. It appears that these elements can insert at many alternative sites. We also describe a DNA sequence arrangement found in the D. melanogaster embryo genome which appears to result from a transposition of an element of the copia dispersed repeated gene family into a new chromosomal site. The mechanism of insertion of this copia element is precise to within 90 bp and may involve a region of weak sequence homology between the site of insertion and the direct terminal repeats of the copia element.     

Proliferation and collagen synthesis of human anterior cruciate ligament cells in vitro: effects of ascorbate-2-phosphate, dexamethasone and oxygen tension.

Clinical and experimental studies demonstrate that injured anterior cruciate ligaments (ACL) do not usually heal and that autografts used to repair the ACL rapidly weaken in the early period and take a long time to regain strength. The aim of this study was to develop an in vitro culture system in which environmental and biochemical factors influencing the proliferation and matrix synthesis of cells derived from human anterior cruciate ligaments can be studied. Primary cultures of human ACL cells were obtained by outgrowth from explants of normal ACL obtained at knee replacement for osteoarthritis in Dulbecco's minimum essential medium (DMEM). The effects of the additives 100 microm L-ascorbic acid-2-phosphate (Asc-2-P) and 10 n m dexamethasone (dex) on proliferation and collagen synthesis were assessed after 4, 8 and 12 days in culture. Ligament cells were grown at 0, 5, 10 and 21%p O(2)in the presence of 100 microm asc-2-P and 10 n m dex. DNA content was assessed using the Hoechst dye method and collagen synthesis by the incorporation of 5 mCi/ml [(3)H]proline after 3, 6 and 12 days in culture.At 21%p O(2), the presence of asc-2-P and dex induced significantly greater (P< 0.01, ANOVA) cell proliferation than with either additives alone. Greatest percentage collagen to total protein synthesis was observed when cells were grown in the presence of asc-2-P only. Least proliferation and percentage collagen to total protein synthesis was seen when both additives were omitted. Greatest cell proliferation was seen when cells were grown in 10%p O(2)and 5%p O(2)was associated with increased collagen synthesis.These results suggest that it is possible to study the effects of environmental and biochemical factors on human ACL healing in vitro. Our data suggest oxygen can influence certain biosynthetic activities of ACL cells. Low oxygen tensions lead to an increase in collagen production by ACL cells. However early responses to injury require extensive cell proliferation which may be activated at higher p O(2). Variation of p O(2)in ligaments during healing may therefore be an important modulator of successful repair.     

Rat myometrial smooth muscle cells show high levels of gap junctional communication under a variety of culture conditions

Summary Gap junctional communciation was examined in rat myometrial smooth muscle cells cultured under a variety of conditions. As a functional measure of gap junctional communication, donor cells were microinjected with the fluorescent dye, Lucifer yellow, and the transfer of dye from donor cells to primary neighbor cells was monitored by fluorescence microscopy. In a myometrial smooth muscle cell line established from midgestation (Day 10) rats, high levels of dye transfer, in excess of 90%, were observed in primary cultures and at Passages 1 and 10. A slight decrease in dye transfer to 75% was observed at Passage 5. Similarly, high levels of dye transfer were observed in a smooth muscle cell line established from the myometrium of a late-gestation (Day 19) rat under subconfluent as well as confluent culture conditions. Myometrial smooth muscle cell cultures established from sexually immature 19-day-old rats also exhibited high levels of dye transfer in primary cultures and at Passage 10. Treatment of primary myometrial smooth muscle cell cultures derived from immature 19-day-old rats with 17β-estradiol (50 ng/ml) and 4-pregnen-3,20-dione (150 ng/ml) for 48 h in vitro had no significant effect on the high levels of dye transfer. Thus, extensive dye transfer was observed in the rat myometrial smooth muscle cells under all culture conditions examined, regardless of sexual maturity or gestational stage of the animal, in vitro hormone treatment, or cell density.