Biomedical subjects

M Kanzaki

Publications and source records attributed to M Kanzaki.

At least 73 records · Page 4Linked to original sources

PubMed1997

Activation of a calcium-permeable cation channel CD20 expressed in Balb/c 3T3 cells by insulin-like growth factor-I.

CD20 functions as a calcium-permeable cation channel. When expressed in Balb/c 3T3 cells, CD20 accelerates the G1 progression induced by insulin-like growth factor-I (IGF-I). To further characterize how CD20 modulates the action of IGF-I, we investigated whether the activity of CD20 channel was affected by IGF-I. In quiescent cells expressing CD20, IGF-I increased cytoplasmic free calcium concentration, [Ca2+]c, which was reversed by the removal of extracellular calcium. In contrast, IGF-I did not increase [Ca2+]c in cells that did not express CD20. In perforated patch clamp recordings, addition of IGF-I to the bath solution augmented the Ca2+ permeability, which was reversed by anti-CD20 antibody. In cell-attached patch, calcium-permeable channel activity with unitary conductance of 7 picosiemens was detected, which was abolished by anti-CD20 antibody. The single channel activities were markedly enhanced when IGF-I was included in the pipette solution, whereas IGF-I added to the bath solution was ineffective. When cells were first exposed to pertussis toxin, activation of the channel by IGF-I was blocked. Transfection of cDNA for Gip2, a constitutive active form of alphai2, activated the CD20 channel. These results indicate that the CD20 channel is regulated by the IGF-I receptor by a mechanism involving pertussis toxin-sensitive G protein.

3T3 Cells↗

PubMed1997

Assessment of the role of activin A and transforming growth factor beta in the regulation of AML12 cell growth.

The present study was conducted to determine the role of two autocrine factors, activin A and transforming growth factor beta (TGF-beta), in the growth regulation of AML12 hepatocytes. We overexpressed truncated type II activin and/or TGF-beta receptors in AML12 cells. In AML12 cells overexpressing truncated type II activin receptors (AML-tAR cells), the inhibitory effect of activin A on DNA synthesis was completely blocked. AML-tAR cells proliferated faster than parental cells, both in the presence and absence of epidermal growth factor (EGF). However, AML-tAR cells could not grow in soft agar. Follistatin augmented EGF-induced DNA synthesis in AML12 cells, whereas it was ineffective in AML-tAR cells. In AML12 cells overexpressing truncated type II TGF-beta receptor (AML-tTR cells), the inhibitory effect of TGF-beta on DNA synthesis was blocked. AML-tTR cells proliferated faster than parental cells, both in the presence and absence of EGF, but at a slower rate than that of AML-tAR cells. AML-tTR cells did not grow in soft agar. The growth rate of cells overexpressing both types of truncated receptors was identical to that of AML-tAR cells, and these cells did not grow in soft agar. These results indicate that both activin A and TGF-beta act as autocrine inhibitors of DNA synthesis in AML12 cells, and that the blocking of the actions of two factors does not lead to transformation. Activin A is a predominant autocrine factor in these cells.

Activin Receptors↗

PubMed1997

Y chromosome (Yq11) microdeletions in idiopathic azoospermia.

BACKGROUND: Cytogenetic anomalies and molecular deletions of the Y chromosome in idiopathically sterile men suggest that genetic factor(s) controlling spermatogenesis are located in the distal portion of Yq11. We studied Y chromosome microdeletions in the Yq11.23 region in idiopathic azoospermia. METHODS: We studied 25 azoospermic male patients with a cytogenetically normal 46XY karyotype; 13 exhibited Sertoli-cell-only syndrome and 12 exhibited maturation arrest. Microdeletions in the Yq11 region were examined using the PCR technique with 4 pairs of primers from DNA loci in Yq11.23. RESULTS: Microdeletions in Yq11.23 were detected in 4 of the 25 azoospermic men. The most common deletion was of the Y6HP52pr sequence, which was detected in 3 of 13 men with Sertoli-cell-only syndrome but in only 1 of 12 with maturation arrest. CONCLUSION: Detection of microdeletions within the Yq11 sequence is an important clue to the genetic factor(s) underlying azoospermia.

Adult↗

PubMed1997

Nitric oxide production by cultured rat Leydig cells.

Nitric oxide (NO) has emerged as an intracellular and intercellular messenger in a number of biological systems. In the present study, we demonstrated that NO was produced by cultured rat Leydig cells, and that inducible nitric oxide synthase (iNOS) messenger RNA (mRNA) was expressed in Leydig cells. NO was measured as nitrite with the method of Griess. Although unstimulated Leydig cells produce little NO, interleukin-1 beta (IL-1 beta) markedly increased NO production. NO production was inhibited by the NOS inhibitor, NG-monomethyl-L-arginine. Northern blot analysis showed that iNOS mRNA was little expressed in freshly isolated immature Leydig cells, but that iNOS mRNA levels were increased by the addition of IL-1 beta in a dose-dependent manner at the concentration up to 10 ng/ml. The levels of iNOS mRNA were increased as early as 3 h after the addition of IL-1 beta and persisted for up to 24 h. In adult Leydig cells, IL-1 beta stimulated iNOS mRNA expression. Immunocytochemical analysis demonstrated iNOS-like immunoreactivity in the cytoplasm of Leydig cells. These results indicate that NO is produced in Leydig cells and suggest that NO might be involved in the physiological function of Leydig cells.

Animals↗

PubMed1997

Formation of reactive oxygen species by spermatozoa from asthenospermic patients: response to treatment with pentoxifylline.

PURPOSE: We determined the incidence of reactive oxygen species formation by spermatozoa from asthenospermic patients, and the relationship between reactive oxygen species formation and sperm motion parameters. We also assessed the efficacy of in vitro and in vivo pentoxifylline treatment of asthenospermic patients whose spermatozoa generated high reactive oxygen species levels. MATERIALS AND METHODS: Reactive oxygen species formation by spermatozoa from asthenospermic patients and fertile volunteers was measured by chemoluminescence. Reactive oxygen species formation by the sperm preparations was investigated without stimulation (steady state), or after stimulation with N-formyl-methionyl-leucyl-phenylalanine (f-MLP) or phorbol-12-myristate-13-acetate. Spermatozoa from 15 asthenospermic patients whose spermatozoa produced high levels of reactive oxygen species at steady state were treated in vitro with pentoxifylline to determine its effect on reactive oxygen species generation and sperm motion parameters. These same 15 patients and 18 with asthenospermia whose spermatozoa did not produce reactive oxygen species at steady state were treated with pentoxifylline at 2 different dosages (300 and 1,200 mg. daily) to determine its effect on reactive oxygen species generation, sperm motion parameters and sperm fertilizing ability in vivo. RESULTS: When reactive oxygen species formation was detected in the steady state that was not stimulated by f-MLP, the source of reactive oxygen species could be attributed to the spermatozoa themselves. Spermatozoa from 15 of 71 asthenospermic patients generated reactive oxygen species at steady state. Pentoxifylline decreased reactive oxygen species generation by spermatozoa in these patients, and preserved the decrease of curvilinear velocity and beat cross frequency for 6 hours in vitro. For these patients orally administered pentoxifylline failed to decrease reactive oxygen species generation by spermatozoa, and had no effect on sperm motility, sperm motion parameters and sperm fertilizing ability at low dosage (300 mg. daily). However, it increased motility and beat cross frequency at high dosage (1,200 mg. daily) but it had no effect on sperm fertilizing ability. CONCLUSIONS: Stimulation of sperm preparations with f-MLP can identify the source of reactive oxygen species generated at steady state. Among asthenospermic patients there were some whose spermatozoa produced detectable steady state levels of reactive oxygen species. In this group pentoxifylline appeared to be effective for decreasing reactive oxygen species formation and preserving sperm motion parameters in vitro. Orally administered pentoxifylline had no effect at low dosage but it increased sperm motility and some sperm motion parameters without altering sperm fertilizing ability at high dosage.

Fertilization↗

PubMed1997

[A case report of left postero-lateral thoracotomy for simultaneous CABG and left lower lobectomy].

Surgical management of patients with concomitant resectable lung lesions and critical cardiac disease is controversial. We report a case of concomitant pulmonary and cardiac surgery via a left thoracotomy. A 67-year-old male was admitted to our hospital complaining of recurrent bloody sputum and an abnormal shadow on chest X-ray. Chest CT and MRI showed a tumor in the left lower lobe (S10), with invasion of the diaphragm. A diagnosis of squamous cell carcinoma was obtained by transbronchial lung biopsy. The patient had a history of angina pectoris, and stress testing was positive. Coronary angiography showed 90% stenosis at segment 5, suggesting a risk of perioperative or postoperative myocardial infarction. This necessitated simultaneous surgical treatment for lung cancer and ischemic heart disease. A lobectomy of the left lower lung was performed, followed by coronary artery bypass grafting (CABG), using the great saphenous vein. The postoperative course was uneventful except for the occurrence of cholecystitis. Lung cancer and ischemic heart disease can be safely treated simultaneously via a single incision, with and benefit for selected patients.

Aged↗

PubMed1996

Calcium as a second messenger of the action of transforming growth factor-beta on insulin secretion.

In MIN6 insulinoma cells, transforming growth factor-beta (TGF-beta) induced the oscillatory elevation of the cytoplasmic free calcium concentration, [Ca2+]c, in the presence of 5.5 mM glucose. The increase in [Ca2+]c induced by TGF-beta was totally dependent on calcium entry and attenuated by nifedipine or nickel chloride. In contrast, carbachol elevated [Ca2+]c in the presence of nickel chloride. When the plasma membrane was hyperpolarized by diazoxide, TGF-beta did not raise [Ca2+]c, whereas both carbachol and depolarizing concentration of potassium elevated [Ca2+]c under the same conditions. TGF-beta did not affect either the cellular cyclic AMP or inositol trisphosphate levels. In the presence of 5.5 mM glucose, TGF-beta induced a 3-fold increase in insulin secretion and the effect of TGF-beta was blocked by either nifedipine or nickel chloride. TGF-beta did not stimulate insulin secretion in the presence of 100 microM diazoxide, whereas both carbachol and 40 mM potassium chloride significantly increased insulin secretion. These results suggest that TGF-beta induces the oscillatory elevation of [Ca2+]c in MIN6 cells by stimulating calcium entry via voltage-dependent calcium channels. Calcium is an intracellular messenger of the action of TGF-beta on insulin secretion.

Calcium↗

PubMed1996

Norepinephrine reverses the effects of activin A on DNA synthesis and apoptosis in cultured rat hepatocytes.

Activin A, an autocrine factor produced by hepatocytes, inhibits mitogen-stimulated DNA synthesis and induces apoptotic death of cultured rat hepatocytes. Several lines of evidence indicate that norepinephrine (NE), as a comitogenic growth factor, alters the balance between growth stimulation and inhibition and acts as a trigger for the initiation of hepatocyte proliferation. In the present study, we examined whether NE modulated the effects of activin A on rat hepatocytes in primary culture. Activin A, at a concentration of 10(-9) mol/L, blocked the effect of epidermal growth factor (EGF) on DNA synthesis, that was assessed by measuring [3H] thymidine incorporation and nuclear labeling, almost completely, and NE reversed the inhibitory effect of activin A on DNA synthesis. This effect of NE was dose-dependent, being significant at concentrations of 10(-6) mol/L and above, but was overcome by higher concentrations of activin A, and was attenuated by prazosin, but not by yohimbine or propranolol. NE exerted its effect during the first 24 hours of culture, but was ineffective when added after 24 hours. EGF augmented the release of follistatin, an activin-binding protein known to block the action of activin A, by hepatocytes and NE did not affect the amount of follistatin they released. In addition to inhibiting DNA synthesis by hepatocytes cultured with EGF, activin A induced death of hepatocytes cultured in the absence of EGF. The nuclear morphology of cells cultured with activin A alone was strikingly changed compared with untreated control cells and marked identation of the nuclear membranes and moderate chromatin condensation were observed. Fragmentation of DNA was also observed, suggesting that activin A induced apoptosis, and activin-mediated cell death was prevented significantly by NE. These results indicate that NE, acting on alpha 1-adrenergic receptors, attenuates the effects of activin A on DNA synthesis by and apoptosis of cultured rat hepatocytes.

Activins↗

PubMed1996

Intravenous administration of follistatin: delivery to the liver and effect on liver regeneration after partial hepatectomy.

When 1 microgram 125I-follistatin was administered into a rat intravenously, radioactivity levels in serum decreased rapidly. Analysis with a biexponential equation showed that the initial half-life and the terminal half-life were 4.0 and 130.8 minutes, respectively. After 2 hours of infusion, approximately 9% of the follistatin infused remained in the liver, which was much more than that in kidney, spleen, pancreas, intestine, or lung. Autoradiography of the liver obtained at 24 hours of infusion revealed that numerous grains were located in parenchymal cells. Radioactivity of 125I-follistatin in the liver remained elevated until 72 hours and declined markedly thereafter. When a booster shot of 125I-follistatin was administered at 72 hours, radioactivity in the liver at 120 hours was markedly increased compared with that in rats that received a single shot of 125I-follistatin. We then examined the effect of intravenous infusion of follistatin on liver regeneration after hepatectomy of 70%. Immediately after the hepatectomy, either 1 microgram follistatin or saline was infused intravenously. In some rats, a booster shot was infused at 72 hours. After 120 hours of hepatectomy of 70%, remnant liver weight, liver regeneration rate, and DNA content were significantly (P < .05) higher in rats that received a booster shot of follistatin at 72 hours than those in control rats. These results indicate that follistatin administered intravenously accumulates in the liver and promotes liver regeneration after partial hepatectomy.

Animals↗

PubMed1996

Characterization of the activin receptor in cultured rat hepatocytes.

Activin A is an autocrine inhibitor of initiation of DNA synthesis in rat hepatocytes. The present study was conducted to characterize the cell-surface receptors for activin A in cultured rat hepatocytes by measuring 125I-activin A binding. Scatchard analysis of 125I-activin A binding indicated the existence of two classes of binding sites with apparent Kd values of 3 x 10(-10) mol/L and 3.5 x 10(-9) mol/L. Pretreatment of the cells with heparitinase reduced the number of low-affinity binding sites, whereas pretreatment with excess exogenous follistatin increased the number of low-affinity binding sites. Affinity cross-linking of 125I-activin A to hepatocytes revealed distinct protein complexes with molecular weights of approximately 48, 65, and 85 kd, which may represent cross-linked cell-bound follistatin, type I and type II activin receptors, respectively. Another band with a molecular weight of 180 kd was also found, which may represent the type III activin receptor. When hepatocytes were cultured with epidermal growth factor (EGF), both high- and low-affinity binding sites increased at 12 hours without altering their affinities. At 60 hours of the incubation with EGF, the high-affinity binding sites decreased while the number of low-affinity binding sites increased slightly. These results indicate that two classes of 125I-activin A binding sites exist in cultured hepatocytes: the high-affinity binding site may represent oligomeric complex of the type I and type II receptors, and at least part of the low-affinity binding site may represent cell-bound follistatin. The number of activin receptors in hepatocytes is increased after the stimulation with EGF.

Activin Receptors↗

PubMed1996

Metenkephalin in seminal plasma of infertile men.

BACKGROUND: Enkephalin is one of the opioids, which is expressed widely in reproductive organs. However, the function of enkephalin in male reproduction is not completely understood. The effect of metenkephalin on sperm motility remains especially controversial. In this study we examined the level of metenkephalin in seminal plasma from men with normal sperm production and patients with asthenospermia, oligospermia, and azoospermia to investigate the role of metenkephalin in seminal plasma on sperm function. We also investigated the effect of metenkephalin on sperm motility in vitro. METHODS: Sixty nine infertile patients (31 oligospermic, 21 asthenospermic, and 17 azoospermic) were included in this study. The level of metenkephalin in seminal plasma of these men was measured and the effect of the peptide on the motility of human sperm was examined in vitro. Seventeen men with normal seminograms were a control group. RESULTS: The level of metenkephalin in the seminal plasma of semen from asthenospermic men was significantly lower than that from the controls (P < 0.05). No significant correlations between the level of metenkephalin and the mean pathing or progressive velocity of sperm, or serum hormone levels were observed. In the in vitro study, which used semen from the controls, treatment of sperm with metenkephalin (50-200 pg/mL) maintained sperm motility for 4 hours. On the other hand, motility of sperm incubated without metenkephalin began to decrease at 3 hours. Metenkephalin levels of 50 pg/ mL in seminal plasma is considered to be necessary for maintaining sperm motility. CONCLUSION: These results suggest that metenkephalin in seminal plasma is an important clue in the investigation of decreased sperm motility.

Enkephalin, Methionine↗

PubMed1996

Expression and regulation of neuropeptide Y messenger ribonucleic acid in cultured immature rat Leydig and Sertoli cells.

Neuropeptide Y (NPY) potentiates the release of gonadotropins from the pituitary in response to GnRH in the hypothalamus and modulates reproductive function. In the present study, we showed that 1) specific organs in the male rat reproductive tract express NPY messenger RNA (mRNA); 2) the multifactorial regulation of NPY mRNA in rat Leydig and Sertoli cells is temporally and hormonally regulated in vitro; 3) both Sertoli cell factor(s) and germ cell factor(s) potentiated to stimulate NPY gene levels in Leydig cells; and 4) intense NPY immunoreactivity was detected in cultured Leydig cells. Using the RT-PCR method, we found that Leydig cells, Sertoli cells, epididymis, and vas deferens expressed NPY mRNA, whereas germ cells, seminal vesicle, and prostate did not. Northern blot analyses showed that NPY mRNA was not expressed in freshly isolated immature Leydig cells, but that NPY mRNA levels were increased by the addition of LH, cytokines such as interleukin-1 alpha and -1 beta, forskolin, or phorbol 13-myristate 12-acetate. Npy mRNA levels in immature Sertoli cells were also increased by FSH. In addition, a germ cell factor(s) secreted from pachytene spermatocytes or round spermatids purified by centrifugal elutriation as well as a Sertoli cell factor(s) stimulated by FSH increased NPY gene levels in Leydig cells. Immunocytochemical analyses showed that the immunostaining was more marked in Leydig cells than in Sertoli cells in vitro. These findings indicate that testicular NPY gene expression is induced in Leydig cells or Sertoli cells by gonadotropins or cytokines within the testes, and that factors secreted from Sertoli cells or germ cells affect NPY gene expression in Leydig cells in vitro. Our findings suggest that NPY expressed in the reproductive system may modulate reproductive function as well as that in the nervous system.

Animals↗

PubMed1996

Growth of differentiation: determination by FSH of the action of insulin-like growth factor-I in cultured rat granulosa cells.

Insulin-like growth factor-I (IGF-I) is a potent mitogen in many cell systems. In cultured rat granulosa cells, however, IGF-I is known to be an inducer of differentiation. The present study was conducted to identify the factor which determines the direction of IGF-I action: either DNA synthesis or LH receptor expression. When granulosa cells were incubated with IGF-I in the presence of various concentrations of follicle-stimulating hormone (FSH), DNA synthesis as assessed by [3H]thymidine incorporation was increased only in the presence of low doses of FSH. The stimulatory effect of FSH on DNA synthesis was observed in a very narrow range of FSH concentration between 2 and 10 ng/ml. At higher concentrations, FSH had little effect on DNA synthesis but instead induced expression of receptors for luteinizing hormone (LH), a marker of granulosa cell differentiation. At 5 ng/ml, FSH elicited maximal stimulation of DNA synthesis and simultaneously induced LH receptor expression to some extent. In these cells, DNA synthesis peaked at 36 h but expression of LH receptor occurred later than 36 h, peaking at 60 h. The ability of IGF-I to stimulate DNA synthesis was enhanced by the long term pretreatment with FSH: when FSH was added from the beginning and IGF-I was added after 36 h or later, IGF-I-mediated DNA synthesis was approximately twice as great, and was accompanied by a two-fold increase in the number of bromodeoxyuridine-labeled nuclei. Under these conditions, LH receptor expression was reduced to approximately 50%. Finally when cells were incubated for 12 h with or without FSH, washed extensively with the medium and then IGF-I was added, DNA synthesis was augmented only in FSH-primed cells. Forskolin, an activator of adenylate cyclase, reproduced the effect of FSH. These results indicate that, in the presence of FSH, IGF-I has the ability to induce both DNA synthesis and differentiation and that FSH determines the action of IGF-I on promotion of either growth or differentiation. Furthermore, priming with FSH renders granulosa cells responsive to IGF-I in terms of DNA synthesis.

Animals↗

PubMed1996

Two distinct signaling pathways activated by activin A in glucose-responsive pancreatic beta-cell lines.

Activin A stimulates insulin secretion in pancreatic beta-cells by a calcium-dependent mechanism. The present study was conducted to further characterize the effects of activin A in two glucose-responsive insulinoma cell lines, MIN6 and HIT-T15 cells. In HIT-T15 cells, activin A evoked an increase in cytoplasmic free calcium concentration, stimulated insulin secretion, maintained glucose responsiveness of the cells and inhibited DNA synthesis. However, activin A did not have any effect in MIN6 cells. Measurement of 125I-labeled activin A binding in MIN6 cells revealed that the number of binding sites was markedly reduced, suggesting that the refractoriness was due, at least partly, to the reduced numbers of the activin receptor. Stable transfectants of MIN6 cells that overexpressed the type II activin receptor were then developed. The transfected cells (MIN6-ActR cells) expressed ten times more 125I-labeled activin A-binding sites than parental cells and the apparent Kd was 1.15 nM, which was nearly identical to that in parental cells. Affinity cross-linking in MIN6-ActR cells showed that a 90 kDa type II receptor as well as a 52 kDa protein, presumably follistatin, was markedly labeled with 125I-labeled activin A. Although MIN6-ActR cells expressed significant numbers of activin receptors, activin A did not induce immediate calcium-dependent responses in these cells. In contrast, activin A was capable of inducing long-term effects in MIN6-ActR cells; thus, reduction of the glucose concentration in culture medium from 25 to 5.5 mM for 4 days resulted in a remarkable loss of insulin response to glucose stimulation but this decline in response to glucose was prevented by the addition of activin A during culture. In addition, activin A inhibited DNA synthesis in MIN6-ActR cells. Hence, although activin A did not induce calcium-dependent responses, it evoked some calcium-independent effects in MIN6-ActR cells. Taken together, activin A elicits various effects in beta-cells by both calcium-dependent and -independent mechanisms.

Activin Receptors↗

PubMed1996

Blockade of DNA synthesis induced by platelet-derived growth factor by tranilast, an inhibitor of calcium entry, in vascular smooth muscle cells.

The present study was conducted to establish a pharmacological method of controlling growth of vascular smooth muscle cells (VSMC) by blocking calcium entry. In cultured rat VSMC, 1 nM platelet-derived growth factor (PDGF) induced a biphasic elevation of cytoplasmic free calcium concentration, ([Ca2+]c). The second sustained phase of [Ca2+]c was dependent on extracellular calcium. At lower concentrations, PDGF induced oscillatory changes in [Ca2+]c, and reduction of extracellular calcium attenuated the oscillation. An antiallergic compound, tranilast, abolished the sustained phase of [Ca2+]c induced by 1 nM PDGF. Tranilast also inhibited the oscillatory changes in [Ca2+]c induced by 200 pM PDGF. In addition, PDGF-induced calcium influx in the late G1 phase, as assessed by measuring the initial uptake of 45Ca, was inhibited by tranilast in a concentration-dependent manner. Tranilast also inhibited PDGF-augmented DNA synthesis; the ID50 for the inhibition of DNA synthesis was nearly identical to that for calcium influx. Although tranilast blocked PDGF-induced calcium entry, it did not affect PDGF-mediated autophosphorylation of the PDGF receptor, activation of phosphatidylinositol 3-kinase, activation of Ras or mitogen-activated protein kinase. Similarly, PDGF-induced elevation of diacylglycerol was not affected by tranilast. These results suggest that the antiallergic drug tranilast inhibits PDGF-induced DNA synthesis by blocking PDGF-mediated calcium entry. Tranilast may be of use in controlling PDGF-induced DNA synthesis in VSMC.

Animals↗

PubMed1995

Expression of calcium-permeable cation channel CD20 accelerates progression through the G1 phase in Balb/c 3T3 cells.

CD20 is a transmembrane protein that functions as a Ca(2+)-permeable cation channel (Bubien, J. K., Zhou, L. J., Bell, P. D., Frizzel, R. A., and Tedder, T. F. (1993) J. Cell Biol. 121, 1121-1132) and is involved in growth regulation of B lymphocytes. In order to further investigate the role of calcium entry in cell cycle progression, we introduced the cDNA encoding a Ca(2+)-permeable cation channel, CD20, into Balb/c 3T3 cells. Balb/c 3T3 cells transfected with a vector containing cDNA encoding CD20 expressed the CD20 protein, which was detected by assaying the binding of a monoclonal antibody against CD20. Calcium-permeable cation channel activity was detected in CD20-expressing cells by whole cell patch clamp recording and microfluorometric determination of the cytoplasmic Ca2+ concentration using fura-2. The expression of CD20 induced significant alterations in the responses of the cells to insulin-like growth factor-I (IGF-I). IGF-I induced DNA synthesis by control cells only when they had been pretreated with both platelet-derived growth factor (PDGF) and epidermal growth factor (EGF). In contrast, DNA synthesis by 30% of the quiescent CD20-expressing cells was initiated in response to IGF-I in the absence of priming with PDGF and EGF. When control quiescent cells were primed with PDGF and EGF, the addition of IGF-I led to the initiation of DNA synthesis after 14 h or more, whereas it induced DNA synthesis by CD20-expressing cells primed with PDGF and EGF 4 h earlier. The IGF-induced DNA synthesis was dependent on extracellular Ca2+, and expression of CD20 reduced the concentration of extracellular Ca2+ required for it. Furthermore, DNA synthesis by approximately 25% of the CD20-expressing cells was initiated after priming with PDGF and EGF, even in the absence of the progression factor IGF-I. These results indicate that CD20 expressed in Balb/c 3T3 cells functions as a constitutively active Ca(2+)-permeable cation channel and that expression of CD20 accelerates G1 progression in a Ca(2+)-dependent manner.

3T3 Cells↗

PubMed1995

Production of activin A and follistatin in cultured rat vascular smooth muscle cells.

Activin A, a member of the transforming growth factor beta supergene family, modulates DNA synthesis in cultured rat vascular smooth muscle cells (VSMC) (Kopma et al. (1993) Exp. Cell. Res. 206, 152-156). In the present study, we studied the production of activin A and follistatin in VSMC. When VSMCs cultured in a 24-well plate were cultured with 10% fetal calf serum (FCS) for 24 h, 0.94 +/- 0.20 pmol/well (mean +/- SE, n = 6) of bioactive activin was released into the culture media. Reverse-transcription polymerase chain-reaction revealed the expression of mRNA for the beta A subunit of inhibin but not for either the beta B or alpha subunit. Bioactivity of activin was increased in quiescent cells treated with FCS or platelet-derived growth factor (PDGF) but not with angiotensin II (Ang II) or insulin-like growth factor-I (IGF-I). Ang II or IGF-I did not stimulate DNA synthesis by itself but, when these two agents were combined, they increased nuclear labeling by 16.4% and release of bioactive activin by 170% of basal. The dose-response relationship and time course study indicated that PDGF-mediated release of activin correlated with initiation of DNA synthesis. Steady state expression of mRNA for the beta A subunit was markedly elevated 12 h after the addition of PDGF and was reduced thereafter. To assess the significance of autocrine activin, the effect of PDGF was determined in the presence and absence of excess of exogenous follistatin. The PDGF-mediated DNA synthesis was enhanced by the addition of excess follistatin.(ABSTRACT TRUNCATED AT 250 WORDS)

Activins↗

PubMed1995

Production of activin A in human intestinal epithelial cell line.

Production of activin was studied in four cell lines of epithelial cells: FRTL-5, JCT-12, GH4C1, and FHs74Int cells. Bioactivity of activin was detected in conditioned media of FRTL-5, JCT-12, and FHs74Int cells. Among these three cell lines, FHs74Int cells, which were derived from human embryonic intestine, released a relatively f1p4e amount of bioactive activin. In these cells, serum and epidermal growth factor (EGF), which were capable of stimulating DNA synthesis, augmented release of bioactive activin in middle to late G1 phase. In addition, basic FGF (bFGF), which had no effect on DNA synthesis in these cells, also increased release of activin. In bFGF-treated FHs74Int cells, bioactive activin was released within 4 hr of the addition of bFGF. The reverse-transcription polymerase chain reaction reveals that mRNA for only the beta A subunit of activin is expressed in these cells. Immunoblotting of lysate from serum-treated cells using anti-human activin A antibody indicated the existence of a 12.5-kDa protein under a reducing condition. FHs74Int cells did not express binding site for [125I]activin A and exogenous activin A did not affect DNA synthesis in these cells. These results indicate that FHs74Int cells derived from human embryonic intestine synthesize and release activin A. Activin A released from intestinal epithelial cells might be a modulatory factor in cells in intestinal mucosa.

Activins↗