Cytoplasmic pH in the action of insulin-like growth factor-I in cultured porcine thyroid cells

The effects of insulin-like growth factor-I (IGF-I) on cytoplasmic pH (pHi) and [3H]thymidine incorporation were studied in primary cultures of porcine thyroid cells. IGF-I alkalinized thyroid cells and stimulated thymidine incorporation in a dose-dependent manner; the effects of IGF-I on alkalinization (the maximal rates of change of cytoplasmic pHi/min ((dpHi/dt)max)) and thymidine incorporation were observed at 2 ng/ml and were maximal at 100 ng/ml, with half-maximal stimulation at approximately 10 ng/ml. The results indicate that Na+/H+ exchange or cell alkalinization may function as a transmembrane signal transducer in the action of IGF-I on thyroid cell proliferation. Several mitogens and comitogens which activate sodium hydrogen exchange, including epidermal growth factor, platelet-derived growth factor and nerve growth factor, have been listed. Activation with IGF-I has not, however, been presented before. Thus the present study constitutes the first demonstration of IGF-I-stimulated activation of Na+/H+ exchange or cell alkalinization.     

Presence of epidermal growth factor, platelet-derived growth factor, and their receptors in human myometrial tissue and smooth muscle cells: their action in smooth muscle cells in vitro.

Immunohistochemical observations indicate that human myometrial smooth muscle cells express epidermal growth factor (EGF) and platelet-derived growth factor (PDGF)-AB and contain EGF and PDGF-beta receptors with no variation in intensity with phases of the menstrual cycle. Furthermore, immunofluorescent microscopic studies revealed that primary myometrial smooth muscle cell cultures also express EGF, PDGF-AB, and contain EGF and PDGF-beta, but not alpha-receptor. Incubation of subconfluent smooth muscle cells in serum-free medium leads to quiescence within 48 h as demonstrated by 3H-thymidine incorporation and labeling index. Exposure of quiescent cells to 10% fetal bovine serum stimulates resumption of DNA synthesis and proliferation in a time-dependent manner with a doubling time of 41.6 h. EGF (1.5-50 ng/ml) and PDGF-AB (1-10 ng/ml) in a dose- and time-dependent manner significantly stimulated 3H-thymidine incorporation by quiescent myometrial smooth muscle cells (P less than 0.05). Combinations of EGF (15 ng/ml) and PDGF-AB (10 ng/ml) significantly increased 3H-thymidine incorporation induced by either growth factor alone (P less than 0.05). PDGF-BB at 10 ng/ml also stimulated 3H-thymidine incorporation and its effect was similar to that induced by PDGF-AB at the same concentration. 17 beta-Estradiol (E2) at 1 microM inhibited 3H-thymidine incorporation by the smooth muscle cells (P less than 0.05). E2 also reduced the stimulatory effect of EGF (15 ng/ml) and PDGF (3 ng/ml). Progesterone at 1 microM either alone or in combination with E2 did not have any effect on 3H-thymidine incorporation or alter the mitogenic action of EGF and PDGF. The effect of EGF and PDGF on cell growth and 3H-thymidine incorporation by myometrial smooth muscle cells was independent of phases of the menstrual cycle. In summary, the results of present studies indicate that human myometrial tissue and myometrial smooth muscle cells in primary culture locally produce EGF and PDGF-AB and contain EGF and PDGF-beta, but not alpha-receptors. Moreover, the myometrial smooth muscle cells in culture respond to the mitogenic action of EGF and PDGF.     

Endothelin is a potent mitogen for rat vascular smooth muscle cells

The effect of endothelin (ET), a novel endothelium-derived vasoconstrictive peptide, on DNA synthesis was studied in cultured rat vascular smooth muscle cells (VSMC). ET stimulated incorporation of [3H]thymidine into DNA of the quiescent VSMC in a dose-dependent manner; the approximate half-maximal and maximal stimulation for DNA synthesis was induced with 2 x 10(-10) M and 10(-9) M, respectively. The stimulatory effect by ET on DNA synthesis was completely inhibited by the calcium channel blocker nifedipine. ET combined with epidermal growth factor and transforming growth factor-alpha, but not with platelet-derived growth factor, had synergistic effects. These data indicate that ET is a potent mitogen as well as a constrictor for VSMC, suggesting its potential role in the development of vascular disease.     

Direct inhibition of rat granulosa cell inhibin production by epidermal growth factor in vitro

The effect of epidermal growth factor (EGF) on inhibin production by rat granulosa cells has been investigated using a recently developed inhibin radioimmunoassay (RIA). Granulosa cells from intact immature diethylstilbestrol (DES)-treated rats were exposed to EGF (1-100 ng/ml) in the presence or absence of FSH for varying periods in vitro. An inhibitory effect of EGF on basal inhibin secretion was evident at day 2 of culture and was sustained over the subsequent 2 days. This action on basal inhibin secretion was dose-dependent, and maximal inhibition to 50% of control was observed at a dose of 100 ng EGF/ml at day 4. EGF also inhibited basal progesterone secretion in a similar manner. EGF caused a dose-dependent inhibition of FSH-stimulated inhibin secretion, with an ID50 (0.5 ng/ml, 0.08 nM) about one-eighth that in the absence of FSH. In addition, EGF also inhibited the stimulation of inhibin production by 8-Br-cAMP and prostaglandin E2. To exclude the possibility that EGF was toxic to the granulosa cells, several biochemical parameters related to cell growth were measured. EGF treatment did not alter cell number but slightly increased [3H]thymidine incorporation into cellular DNA. The effect of EGF on [35S]methionine incorporation into cellular protein was biphasic, being stimulatory at doses less than 10 ng/ml but inhibitory at 100 ng/ml. The present data have demonstrated a direct inhibitory effect of EGF on basal and FSH-stimulated inhibin production by granulosa cells suggesting an important regulatory role of this growth factor in the differentiation of ovarian function.     

Hormonal stimulation of DNA synthesis in primary cultures of adult rat hepatocytes.

Adult rat hepatocytes have been previously isolated and maintained in monolayer culture, but attempts to stimulate DNA synthesis have been unsuccessful. Hormonal conditions are now described which induce DNA synthesis in cultured hepatocytes from partially hepatectomized rats. DNA synthesis was determined autoradiographically by the incorporation of [3H]thymidine into nuclei of morphologically distinct hepatocytes. Insulin (4-4000 nM) or epidermal growth factor (10 ng/ml) alone caused significant increases in the labeling index. The two hormones together acted synergistically to produce labeling indices of 35-50% on the third day of culture, compared with 2-7% in control cultures. The addition of glucagon (400 nM) further increased the labeling indes. Dexamethasone (80 ng/ml) inhibited DNA synthesis but, under certain conditions, enhanced cell attachment. Growth hormone and triiodothyronine had no significant effect on DNA synthesis. The mixture of epidermal growth factor, insulin, and glucagon also stimulated incorporation of [3H]thymidine into phenol-extracted DNA. Although DNA synthesis was stimulated, cell division occurred infrequently. These data suggest a prominent role for epidermal growth factor in promoting hepatic DNA synthesis by acting in concert with insulin and glucagon.     

Stimulation of aortic smooth muscle cell mitogenesis by serotonin.

Bovine aortic smooth muscle cells in vitro responded to 1 nM to 10 microM serotonin with increased incorporation of [3H]thymidine into DNA. The mitogenic effect of serotonin was half-maximal at 80 nM and maximal above 1 microM. At a concentration of 1 microM, serotonin stimulated smooth muscle cell mitogenesis to the same extent as human platelet-derived growth factor (PDGF) at 12 ng/ml. Tryptamine was approximately 1/10th as potent as serotonin as a mitogen for smooth muscle cells. Other indoles that are structurally related to serotonin (D- and L-tryptophan, 5-hydroxy-L-tryptophan, N-acetyl-5-hydroxytryptamine, melatonin, 5-hydroxyindoleacetic acid, and 5-hydroxytryptophol) and quipazine were inactive. The stimulatory effect of serotonin on smooth muscle cell DNA synthesis required prolonged (20-24 hr) exposure to the agonist and was attenuated in the presence of serotonin D receptor antagonists. When smooth muscle cells were incubated with submaximal concentrations of serotonin and PDGF, synergistic rather than additive mitogenic responses were observed. These data indicate that serotonin has a significant mitogenic effect on smooth muscle cells in vitro, which appears to be mediated by specific plasma membrane receptors.     

Stimulation of insulin-like growth factor I production in primary cultures of chicken hepatocytes by chicken growth hormone

Extracts of conditioned media from primary cultures of chicken hepatocytes stimulated [3H]thymidine incorporation into chick embryo fibroblasts demonstrating that the cells released mitogen(s) into the medium. There was also a simultaneous release of insulin-like growth factor I (IGF-I) immunoreactivity into the medium. Acid chromatography of freeze-dried extracts of conditioned media by high performance gel permeation chromatography demonstrated that IGF-I immunoreactivity eluted in a major peak with a molecular weight of 7500 Da and a minor peak with a molecular weight of 55,000 Da. The release of IGF-I immunoreactivity was increased by pituitary-derived chicken growth hormone (cGH) in a dose-dependent manner with half-maximum stimulation occurring at a cGH concentration of 40 ng/ml and maximum stimulation at cGH concentrations greater than 800 ng/ml. These results demonstrate that cultured chicken hepatocytes produce IGF-I and that this can be stimulated by cGH in vitro.     

Platelet-derived growth factor suppresses and fibroblast growth factor enhances cytokine-induced production of nitric oxide by cultured smooth muscle cells. Effects on cell proliferation.

Stimulation of thymidine incorporation by basic fibroblast growth factor or epidermal growth factor treatment of cultured quiescent smooth muscle cells (rat and human) was attenuated by the concomitant treatment with interleukin-1 beta in the presence of indomethacin. Platelet-derived growth factor-AB and -BB-induced thymidine incorporation was not inhibited by the presence of the cytokine under similar experimental conditions. Elevation of nitrite levels in the conditioned medium of cultures exposed to interleukin-1 beta correlated with the inhibition of thymidine incorporation. Platelet-derived growth factor-AB and -BB inhibited the production of nitric oxide (measured as nitrite levels in conditioned medium) by cells treated simultaneously with interleukin-1 beta and growth factor. However, platelet-derived growth factor-AA neither affected nitrite production nor thymidine incorporation by smooth muscle cells. Levels of cytokine-stimulated nitrite production by smooth muscle cells were increased synergistically by the presence of fibroblast growth factors or epidermal growth factor. The inhibition of thymidine incorporation and concomitant elevation of nitrite production was abolished in the presence of nitro-L-arginine. Cultures maintained in the presence of low levels of the cytokine for 9 days were growth-inhibited, and this was reversed when culture medium was supplemented with nitro-L-arginine. The treatment of smooth muscle cells, which were grown in coculture inserts with the cytokine to induce nitric oxide production, before their combination with other quiescent layers of cells resulted in the inhibition of thymidine incorporation by this second layer of cells regardless of the growth factor used for stimulation. Nitric oxide may act as an endogenous inhibitor of smooth muscle cell proliferation in the vessel wall, and impairment of its production may be one action of potent vascular mitogens such as platelet-derived growth factor.     

Effects of transforming growth factor-beta on the production of immunoreactive insulin-like growth factor I and progesterone and on [3H]thymidine incorporation in porcine granulosa cell cultures

Transforming growth factor-beta (TGF beta) has been reported to enhance many FSH-stimulated functions in rat granulosa cell cultures. We therefore, investigated the actions of TGF beta on cultured porcine granulosa cells. We evaluated the production of immunoreactive insulin-like growth factor I (iIGF-I) and progesterone in short term (3-day) and in longer term (7-day) cultures using porcine TGF beta 1 (pTGF beta 1). TGF beta had a biphasic effect on epidermal growth factor (EGF)-stimulated iIGF-I production in short term cultures. A modest stimulatory effect was apparent at 10 pg/ml; however, this end point was completely inhibited by 1-10 ng/ml. TGF beta also had a slight stimulatory effect on basal iIGF-I production at 1 pg/ml, but not at higher levels. In longer term cultures TGF beta did not have a significant effect on either basal or FSH-stimulated iIGF-I production. In both short and longer term cultures TGF beta markedly inhibited basal and FSH-stimulated progesterone production. We also evaluated the effects of TGF beta on the incorporation of [3H]thymidine into DNA and found that basal and growth factor-stimulated [3H]thymidine incorporation were inhibited. No stimulatory effects of TGF beta on progesterone production or [3H]thymidine incorporation could be detected over the dose range tested (1 pg/ml to 10 ng/ml). The effects of human TGF beta 1 and pTGF beta 2 were compared with those of pTGF beta 1 on basal and EGF-stimulated [3H]thymidine incorporation. Effects of the peptides were qualitatively similar, but pTGF beta 2 was somewhat less inhibitory to EGF-stimulated [3H]thymidine incorporation than pTGF beta 1. The present studies show that in contrast to the well documented stimulatory actions of TGF beta in cultured rat granulosa cells, this growth factor is a predominantly negative regulator of porcine granulosa cells. With the exception of a modest stimulation of iIGF-I production at very low doses, the effects of TGF beta were to potently inhibit both growth and differentiated function. The inhibitory nature of TGF beta should not be overlooked when considering the possible role of this peptide in ovarian development and differentiation.     

Mitogenic action of interleukin-1 alpha on vascular smooth muscle cells mediated by PDGF

We have investigated the effect of interleukin-1 (IL-1) on the growth of vascular smooth muscle cells (VSMC) isolated from rat aortae. Murine recombinant IL-1 alpha increased tritiated leucine incorporation into VSMC. IL-1 also stimulated tritiated thymidine uptake by VSMC in a dose-dependent manner. On the other hand, Ca2(+)-channel blocker, verapamil, inhibited the IL-1-induced thymidine uptake by VSMC with an IC50 of 10(-8) M. Antibody specific for platelet-derived growth factor (PDGF) also totally inhibited the IL-1-induced thymidine uptake. IL-1 showed no effects on the intracellular Ca2+ level in VSMC. Above results support the premise that IL-1 promotes the growth of VSMC via induction of endogenous PDGF production and might thus participate in the abnormal proliferation of VSMC that occurs early in atherogenesis.     

Tumor necrosis factor-alpha inhibits growth factor-mediated cell proliferation through SHP-1 activation in endothelial cells

Src homology 2-containing protein-tyrosine phosphatase 1 (SHP-1) is known to regulate signal transduction through the dephosphorylation of tyrosine kinases. In this study, we addressed the role of SHP-1 under tumor necrosis factor-alpha (TNF-alpha) stimulation in endothelial cells. The addition of recombinant vascular endothelial growth factor (50 ng/mL) or epidermal growth factor (50 ng/mL) significantly increased thymidine incorporation and c-fos promoter activity, whereas TNF-alpha (5 ng/mL) attenuated these effects in human or bovine aortic endothelial cells. In bovine aortic endothelial cells, we confirmed endogenous SHP-1 expression and that TNF-alpha activated SHP-1. Importantly, overexpression of dominant-negative SHP-1 attenuated the effect of TNF-alpha on thymidine incorporation and c-fos promoter activity. In addition, TNF-alpha attenuated vascular endothelial growth factor- and epidermal growth factor-induced extracellular signal-regulated kinase phosphorylation, whereas overexpression of dominant-negative SHP-1 prevented this inhibitory effect of TNF-alpha. Taken together, our results suggested that TNF-alpha inhibited growth factor-mediated cell proliferation through SHP-1 activation.     

Epidermal growth factor stimulates cell proliferation and inhibits functional differentiation of mouse mammary epithelial cells in culture

Mouse mammary epithelial cells cultured on collagen gels multiplied and produced casein and alpha-lactalbumin in response to insulin, cortisol, and PRL. The addition of epidermal growth factor (EGF) at 50 ng/ml increased the total number of epithelial cells by 30-40% and thymidine incorporation into DNA 4.7-fold after 5 days of culture. In contrast, EGF inhibited hormonal induction of the synthesis of casein and alpha-lactalbumin in those cells by about 45% and 55%, respectively, without inhibiting total protein synthesis. Furthermore, EGF decreased casein mRNA activity by 55% and increased total mRNA activity by 66% in cells cultured with the three hormones. These effects of EGF were apparent at 0.1 ng/ml and were maximal at 50-100 ng/ml and could be reversed by its removal from the medium, followed by the addition of anti-EGF antibody. The inhibition of casein synthesis by EGF was unaffected by the concentrations of insulin, cortisol, and PRL. Other growth factors, such as fibroblast growth factor, multiplication-stimulating activity, nerve growth factor, and platelet-derived growth factor, did not simulate the effects of EGF. Cytarabine (1 microgram/ml), which inhibited thymidine incorporation into DNA by 94%, did not block the inhibitory action of EGF on casein synthesis. These results suggest that EGF serves as a regulator of hormone-dependent growth and differentiation of mammary epithelial cells.     

Adipocyte-derived plasma protein adiponectin acts as a platelet-derived growth factor-BB-binding protein and regulates growth factor-induced common postreceptor signal in vascular smooth muscle cell

BACKGROUND: Vascular smooth muscle cell proliferation plays an important role in the development of atherosclerosis. We previously reported that adiponectin, an adipocyte-specific plasma protein, accumulated in the human injured artery and suppressed endothelial inflammatory response as well as macrophage-to-foam cell transformation. The present study investigated the effects of adiponectin on proliferation and migration of human aortic smooth muscle cells (HASMCs). Methods and Results- HASMC proliferation was estimated by [(3)H] thymidine uptake and cell number. Cell migration assay was performed using a Boyden chamber. Physiological concentrations of adiponectin significantly suppressed both proliferation and migration of HASMCs stimulated with platelet-derived growth factor (PDGF)-BB. Adiponectin specifically bound to (125)I-PDGF-BB and significantly inhibited the association of (125)I-PDGF-BB with HASMCs, but no effects were observed on the binding of (125)I-PDGF-AA or (125)I-heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) to HASMCs. Adiponectin strongly and dose-dependently suppressed PDGF-BB-induced p42/44 extracellular signal-related kinase (ERK) phosphorylation and PDGF beta-receptor autophosphorylation analyzed by immunoblot. Adiponectin also reduced PDGF-AA-stimulated or HB-EGF-stimulated ERK phosphorylation in a dose-dependent manner without affecting autophosphorylation of PDGF alpha-receptor or EGF receptor. CONCLUSIONS: The adipocyte-derived plasma protein adiponectin strongly suppressed HASMC proliferation and migration through direct binding with PDGF-BB and generally inhibited growth factor-stimulated ERK signal in HASMCs, suggesting that adiponectin acts as a modulator for vascular remodeling.     

Proliferation and Na+/H+ exchange activation by endothelin in vascular smooth muscle cells.

Initiation and development of proliferative responses to growth factors are often associated to an activation of the Na+/H+ exchange. The present work examined the effect of endothelin (ET-1) on cell proliferation and Na+/H+ exchange in cultured vascular smooth muscle cells. In rat aortic vascular smooth muscle, ET-1 (0.1 to 10 nmol/L) increased the [3H] thymidine uptake in a dose-dependent manner. This effect was enhanced in presence of insulin (0.1 micrograms/mL to 10 micrograms/mL) as a function of concentration. The Na+/H+ exchange, which is a necessary response for mitogenesis, was dose-dependently stimulated by increasing concentrations of ET-1 (1 to 1000 nmol/L) and presented a biphasic response: a transient acidification followed by a sustained alkalinization. Alkalinization induced by ET-1 was similar to that obtained by the phorbol 12-myristate 13-acetate (PMA). An inhibitor of protein kinase C, H7, or a long-term pretreatment of cells with PMA for 24 h inhibited the effect of ET-1 and PMA on Na+/H+ exchange. These results confirm that ET-1 could act as a growth factor for vascular smooth muscle cells and suggest that its mode of action depends for a large part to protein kinase C activation.     

Interaction of receptors for insulin-like growth factor I, platelet-derived growth factor, and fibroblast growth factor in rat aortic cells

Serum contains various growth factors which regulate the proliferation of cells. We investigated the growth of cultured arterial smooth muscle cells under the influence of insulin-like growth factor I (IGF I), fibroblast growth factor (FGF), and platelet-derived growth factor (PDGF), and examined the effect of these growth factors on the binding of [125I] IGF I and on the binding of [125I]PDGF to these cells. IGF I, FGF, and PDGF stimulated [6-3H]thymidine incorporation into DNA of confluent cultures of cells which were incubated in modified Dulbecco's modified Eagle medium. However, the effect of these growth factors on DNA synthesis was much more potent in Dulbecco's modified Eagle medium with 1% fetal calf serum. FGF and PDGF potentiated the growth-promoting effect of IGF I. The binding of [125I]IGF I to the cells was increased after a preincubation with FGF and PDGF. The binding was potently increased by FGF (100 ng/ml) after a preincubation time of 30 min. There was an increase in binding during the first 3 h of preincubation followed by a decrease after 4-5 h. PDGF (10-1000 ng/ml) stimulated [125I]IGF I binding only after 2 h of preincubation. The stimulation was dose dependent. Maximal stimulation of the binding was observed after 3 h of preincubation followed by a decrease after 4-5 h of preincubation. Specific binding sites for PDGF on smooth muscle cells could be demonstrated too. A preincubation of confluent cells with IGF I caused a dose-dependent increase in [125I]PDGF binding. These results support the hypothesis that the regulation of the binding of a specific growth factor by a second growth factor is important for the control of cell growth.     

Effects of insulin-like growth factor I, platelet-derived growth factor, fibroblast growth factor, and transforming growth factor-beta on thymidine incorporation into fetal liver cells

We have studied the effect of different growth factors on thymidine incorporation in cell cultures of erythroid cells from fetal calf and rat livers, a system which has been used in the past as a bioassay for the purification of erythropoietin and erythropoietin-like factors. Insulin-like growth factor I significantly stimulated thymidine incorporation in calf and rat liver cells. Its action in both cell types was practically identical to the effect of bovine serum erythrotropin, a peptide structurally similar to insulin-like growth factor II. The synergistic effect between insulin-like growth factor I and erythropoietin could be observed with partially purified sheep plasma erythropoietin but not with recombinant human erythropoietin. The highest thymidine incorporation was observed when both erythropoietin and insulin-like growth factor I were added simultaneously. Platelet-derived growth factor had a lower thymidine incorporation-stimulating activity than insulin-like growth factor and did not have any synergistic effect with erythropoietin in rat liver cells. Fibroblast growth factor (0.4-6.0 ng/ml) was completely inactive in the thymidine incorporation assay of calf liver cells. Transforming growth factor-beta alone at a concentration of 1 ng/ml significantly inhibited thymidine incorporation into rat liver cells. It seems that from all growth factors tested so far, those belonging to the insulin family of peptides are the most likely to be detected in the thymidine incorporation assays using calf or rat liver cells.     

Factor XII-induced mitogenesis is mediated via a distinct signal transduction pathway that activates a mitogen-activated protein kinase.

Clotting factor XII (Hageman factor) contains epidermal growth factor (EGF)-homologous domains and is reported to be a potent mitogen for human hepatoma (HepG2) cells. In this study, we tested whether factor XII exhibits growth factor activity on several other EGF-sensitive target cells, including fetal hepatocytes, endothelial cells, alveolar type II cells, and aortic smooth muscle cells. We found that factor XII significantly enhanced [3H]thymidine incorporation in aortic smooth muscle cells (SMCs) and all other cells tested. Tyrphostin, a growth factor receptor/tyrosine kinase antagonist, inhibited both EGF- and factor XII-induced responses. However, differences in the levels of magnitude of DNA synthesis, the observed synergism between EGF and factor XII, and the differential sensitivity to tyrphostin suggest that the EGF receptor and the factor XII receptor may be nonidentical. The factor XII-induced mitogenic response was achieved at concentrations that were 1/10th the physiologic range for the circulating factor and was reduced by popcorn inhibitor, a specific factor XII protease inhibitor. Treatment of aortic SMCs with factor XII, as well as activated factor XII, resulted in a rapid and transient activation of a mitogen-activated/extracellular signal-regulated protein kinase with peak activity/tyrosine phosphorylation observed at 5 to 10 min of exposure. Taken together, these data (i) confirm that clotting factor XII functions as a mitogenic growth factor and (ii) demonstrate that factor XII activates a signal transduction pathway, which includes a mitogen-activated protein kinase.     

Cyclic AMP: a mitogenic signal for Swiss 3T3 cells.

Addition of cholera toxin (100 ng/ml) to quiescent cultures of Swiss 3T3 cells acts synergistically with serum (2-4%), insulin, phorbol esters, epidermal growth factor, and fibroblast-derived growth factor to stimulate DNA synthesis. In the presence of insulin, cholera toxin caused a dose-dependent increase in cumulative [3H]thymidine incorporation into acid-insoluble material and in the intracellular cyclic AMP (cAMP) level. The dose--response curves for the two processes were similar. Furthermore, addition of 1-methyl-3-isobutylxanthine (15--500 microM) or of 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (5--100 microM), both of which are potent inhibitors of cyclic nucleotide phosphodiesterase which are potent inhibitors of cyclic nucleotide phosphodiesterase activity, stimulated DNA synthesis and increased cAMP levels in Swiss 3T3 cells. These compounds strikingly potentiated the effect of cholera toxin on DNA synthesis and on cAMP levels. When quiescent Swiss 3T3 cells were exposed to cholera toxin (100 ng/ml) and insulin at 10 micrograms/ml (4- to 7-fold increase in cAMP level) or to these agents and 1-methyl-3-isobutyl xanthine at 50 microM (35-fold increase in cAMP level), DNA synthesis began after a lag of 16 hr. These results indicate that cAMP acts as a mitogenic signal for Swiss 3T3 cells and differ from the widely held view that cyclic AMP inhibits the proliferation of fibroblast cells.     

血小板衍生生长因子—BB对血管内皮细胞、平滑肌细胞及成纤维细胞增殖的影响

目的 观察血小板衍生生长因子—BB对培养的人血管内皮细胞、兔平滑肌细胞和人成纤维细胞增殖的影响。方法 采用培养的人脐静脉血管内皮细胞、兔动脉血管平滑肌细胞和人血管成纤维细胞，应用^3H—TdR掺入方法，观察血小板衍生生长因子—BB对三种细胞DNA合成的影响。结果 血小板衍生生长因子—BB可促进处于静止状态的三种细胞DNA的合成，并呈现出明显的浓度依赖关系，在30ng／ml的浓度时成纤维细胞DNA的合成达到高峰，在40ng／ml的浓度时内皮细胞、平滑肌细胞DNA的合成达到高峰。成纤维细胞、平滑肌细胞分别在PDCF-BB作用24h和36h年到DNA合成的高峰，内皮细胞在48h时DNA合成量最高。结论 血小板衍生生长因子—BB可明显促进培养的人脐静脉血管内皮细胞、兔动脉血管平滑肌细胞和人血管成纤维细胞的增殖。     

Increased thymidine incorporation into fetal rat cartilage in vitro in the presence of human somatomedin, epidermal growth factor and other growth factors

The incorporation of [3H]thymidine by rat costal cartilage in vitro was studied at different fetal and postnatal ages and the effect of partially purified human somatomedin, mouse epidermal growth factor, platelet secretion products, insulin and growth hormone on thymidine uptake by fetal cartilage was examined. Thymidine uptake in plasma-free medium was many times greater in late fetal life than after birth. The incorporation of [3H]thymidine into costal cartilage from 21-day fetuses was significantly (P less than 0.05) increased above control values in the presence of 10 micrograms somatomedin/1, and when cartilage was incubated in medium containing somatomedin and diluted human plasma there was a synergistic action. Epidermal growth factor at a concentration of 1 ng/l was a potent stimulator of thymidine uptake. Secretion products from human platelets after their aggregation by thrombin stimulated [3H]thymidine uptake at a concentration of 2% (v/v), but were inhibitory at high concentrations. High concentrations of platelet secretion products stimulated the incorporation of [35S]sulphate by cartilage. A pharmacological concentration of 10 mu. insulin/ml stimulated [3H]thymidine uptake, but not concentrations of 1 or 100 mu./ml. Growth hormone had no effect. The results showed that fetal cartilage had a greater endogenous mitogenic activity than postnatal cartilage. While somatomedins may be important in the regulation of fetal body growth, other protein growth factors also stimulate fetal skeletal tissues.