Calcium transport and exchange in mouse 3T3 and SV40-3T3 cells

The kinetics of Ca++ uptake have been evaluated in 3T3 and SV40-3T3 mouse cells. The data reveal at least two exchangeable cellular compartments in the 3T3 and SV40-3T3 cell over a 50-min exposure to 45Ca++. A rapidly exchanging compartment may represent surface-membrane-localized Ca++ whereas a more slowly exchanging compartment is presumably intracellular. The transition of the 3T3 cell from exponential growth (at 3 day's incubation) to quiescence (at 7 days) is characterized by a 7.5-fold increase in the size of the fast component. Quiescence of the 3T3 cell is also characterized by a 3.2-fold increase in the unidirectional Ca++ influx into the slowly exchanging compartment and a 3.6-fold increase in its size. The increase in size of the slow compartment at quiescence may result from a redistribution of intracellular Ca++ to a more readily exchangeable compartment, possibly reflecting a release of previously bound Ca++. In contrast, no significant change in any of these parameters is observed in the proliferatively active SV40-3T3 cells after corresponding period of incubation, even though these cells attained higher growth densities and underwent postconfluence.     

Dependence of intracellular alkali-ion concentrations of 3T3 and SV 40-3T3 cells on growth density.

Intracellular contents of potassium and of sodium are determined for 3T3 and SV 40-3T3 cells in dependence of growth density. In parallel, total cell volume and volume of intracellular water is determined for these cells suspended in physiological buffer. Intracellular potassium concentration thus evaluated for suspended 3T3 cells exhibits a sharp decrease at cellular growth densities which lead to density dependent inhibition of cell proliferation. In the case of SV 40-3T3 cells, this drop of potassium concentration with increasing cellular growth density is not observed, which correlates well with the absence of cell density dependent inhibition of cell growth in the transformed cell line. These results support the notion that processes of stimulation of quiescent 3T3 cells or of cell density dependent inhibition of their proliferation are mediated by processes including changes of potassium transport characteristics leading to increase or decrease respectively of their intracellular potassium concentration. Furthermore, these and other results suggest, that a difference between normal and transformed cells most relevant to their different proliferation behaviour might reside in different transport characteristics for potassium of the plasma membranes of these cells.     

Antioxidants-induced rearrangements of actin cytoskeleton in 3T3 and 3T3-SV40 fibroblasts

Effect of antioxidants on actin cytoskeleton in 3T3 fibroblasts and 3T3 fibroblasts transformed with SV40 virus (3T3-SV40 cells) was studied. Antioxidants used were as follows: N-acetyl-L-cysteine (NAC), (-)-2-oxo-4-thiazolidine-carboxylic acid (OTZ), and glutathione in the reduced form (GSH). Both NAC and OTZ are precursors of GSH in the cell, but, in contrast to NAC, OTZ reduces inside the cell forming L-cysteine. The presence of NAC (5-20 mM) in the culture medium of both cell types resulted in loosening of monolayer, fragmentation of stress fibers, and the appearance of amorphous actin structures. As 3T3-SV40 cells contain less actin stress fibers than 3T3 cells, the NAC-induced rearrangements of actin cytoskeleton were stronger in these cells than in 3T3 cells. In contrast to NAC, OTZ (10-20 mM) did not destroy monolayer and did not induce any visible disappearance of stress fibers either in 3T3 or 3T3-SV40 cells. However, in the presence of OTZ, amorphous actin-containing structures were observed in 3T3-SV40 cells. The effect of glutathione on both cell types was similar to that of NAC. The time required for GSH-induced alterations of actin cytoskeleton (about 5 h) was consistent with the increase in the intracellular level of reactive oxygen species (4 h after addition of GSH to the culture medium). Upon removal of the antioxidants from the medium, actin filament structures were reconstructed. However, within 24 h after withdrawal of NAC or GSH, only a partial reconstruction of stress fibers was observed in 3T3 cells. On the contrary, 3T3-SV40 cells demonstrated formation of well-structured actin fibers similar to normal fibroblasts. These results suggest that GSH can act as a pro-oxidant in the absence of oxidative stress.     

Extension of lifespan of human T lymphocytes by transfection with SV40 large T antigen.

Lymphocytes have a finite and predictable proliferative life span in culture similar to that observed in fibroblasts. In general, the senescence of human fibroblasts is inevitable and irreversible, but their proliferative life span can be extended by certain DNA tumor virus oncogenes, such as the large T antigen of the SV40 virus. Here, we show that human T lymphocytes (HTL) can be stably transfected with SV40 large T and that expression of T antigen extended the life span of T cell cultures. PHA-stimulated HTL were transfected with pSV3neo, an expression vector containing the SV40 early region and the neomycin resistance gene. Transfectants were selected for neomycin (G418) resistance. Control HTL, either mock transfected or transfected with pSV2neo (containing the neomycin resistance gene only), ceased proliferation after about 17 population doublings. In contrast, HTL transfected with pSV3neo underwent more than 170 doublings. pSV3neo-transfected cells expressed SV40 large T RNA, detectable by in situ hybridization, and SV40 T antigen, detectable by immunofluorescence. Greater than 95% of the transfected cells were CD4 positive. These results clearly show that SV40 large T enables HTL to escape senescence. Transfection with SV40 large T may be a valuable method for obtaining long term human T cell lines for studies of both aging and immunology.     

Effects of the tyrosine-kinase inhibitor genistein on DNA synthesis and phospholipid-derived second messenger generation in mouse 10T1/2 fibroblasts and rat liver T51B cells

We have examined the effects of the tyrosine kinase inhibitor genistein on hormone dependent cell proliferation and intracellular signalling in mouse 10T1/2 fibroblasts and rat liver T51B epithelial cells. Genistein inhibits both PDGF and EGF induced mitogenesis with an IC50 of 40 uM and 10 uM respectively. Genistein also inhibits inositol phosphate generation and calcium signalling in response to PDGF, and 1,2-diacylglycerol generation and calpactin II translocation in response to EGF. By contrast genistein does not inhibit inositol phosphate production, Ca2+ signalling or 1,2-diacylglycerol generation in response to ATP or angiotensin II. These data demonstrate that genistein selectively inhibits tyrosine kinase dependent processes without effecting similar responses obtained to hormones which are not dependent upon tyrosine kinase activation.     

An improved method for measuring angiotensin I converting enzyme activity using a highly sensitive angiotensin II radioimmunoassay

A highly sensitive assay for angiotensin I converting enzyme has been developed by using angiotensin I as a substrate. Angiotensin II generated in the reaction mixture was measured by a newly developed specific radioimmunoassay. To protect against angiotensin II destruction, bestatin, an inhibitor of renin, was also used to inhibit plasma renin activity. The reaction was stopped by adding EDTA and MK-521, inhibitors of angiotensin I converting enzyme. The specificity of the antiserum used for the angiotensin II radioimmunoassay was very high. The cross reactivity with angiotensin I was less than 0.5% and none of the proteolytic enzyme inhibitors crossreacted in the assay. The inhibitory effect of pepstatin on plasma renin activity was very high (more than 80%) under the standard assay conditions employed. Serum angiotensinase activity was completely inhibited by the addition of bestatin. An excellent correlation was obtained between this new method and the spectrophotometric method using a synthetic substrate, Hip-His-Leu. The generation of as little as 12 pM of Angiotensin II can be detected. Such low concentration have not been measurable with the usual spectrophotometric method. This new method will facilitate clinical and experimental studies on this unique enzyme, since very low levels of activity can be determined by this highly sensitive radioimmunoassay for angiotensin II.     

A specific effect of external ATP on the permeability of transformed 3T3 cells

Addition of ATP causes a dramatic increase in the rate of p-nitrophenyl phosphate hydrolysis by intact 3T6 and 3T3 cells transformed by polyoma virus and simian virus 40 (SV40). In sharp contrast, untransformed 3T3 cells or secondary mouse embryo fibroblasts, either growing or resting, do not respond to ATP. The activation displays specificity, reversibility and dependence on pH, temperature and ATP concentration. The data suggest that exposure to ATP changes the permeability of transformed cells to p-nitrophenyl phosphate thus revealing an internal, ouabain-insensitive, phosphatase activity.     

Calcium content and distribution as a function of growth and transformation in the mouse 3T3 cell

Total Ca content and that fraction of Ca sensitive to removal by the chelator ethylene glycol-bis(β-aminoethyl ether)N,N,N',N'-tetraacetate (EGTA) have been investigated in the mouse 3T3 cell as a function of growth stage, transformation with SV40 virus, and serum levels of the media. Cells were allowed to grow through several doublings in media containing (45)Ca. The cellular content of (45)Ca was used to access total cell Ca. That fraction of (45)Ca removed by EGTA was presumed to represent primarily surface-localized Ca. The data are expressed on a per cell volume basis to compensate for size differences as a function of growth stage and transformation. During exponential growth phase, the 3T3 cell contains 525pmol Ca/μl cell volume. Of this, approx. 457 pmol/μl is not removable by EGTA and, presumably, is cytoplasmically located. This value is in close agreement with previous studies on the HeLa cell (470 pmol Ca/μl cell water after the removal of the surface Ca). The low level of EGTA- removable Ca present in the 3T3 cell during early exponential growth (68 pmol Ca/μl cell volume) increases progressively with increasing cell density, and upon quiescence it is sevenfold greater. In contrast, SV40- transformed 3T3 cells growing exponentially possess total levels of Ca which are approximately two-thirds the levels of the normal 3T3 cell. However, their EGTA-sensitive Ca is not significantly different from that of exponentially growing, normal 3T3 cells. As the transformed cells continue to grow at high density, their total ca and their sensitivity to EGTA do not change, in contrast to the normal 3T3 cell. Thus, an increase in Ca associated with the cell surface appears to be correlated with growth inhibition. This has been investigated further by regulating growth of the normal and transformed cell with alterations in the serum level of the media. In 4 percent calf serum the normal cell is stopped from continued proliferation. Growth stoppage under these conditions is characterized by a nearly fourfold increase in EGTA-removable Ca, similar to the increase observed upon quiescence in depleted 10 percent serum. Similar treatment of the transformed cell does not reduce its growth rate, nor does it significantly alter Ca distribution. However, at 0.5 percent medium serum levels, the SV40 3T3 growth rate is substantially reduced and, under these conditions, EGTA-removable Ca increases twofold.     

Cardiac fibrogenesis in magnesium deficiency: a role for circulating angiotensin II and aldosterone

Mechanisms underlying cardiac fibrogenesis in magnesium deficiency are unclear. It was reported earlier from this laboratory that serum from magnesium-deficient rats has a more pronounced stimulatory effect on cell proliferation, net collagen production, and superoxide generation in adult rat cardiac fibroblasts than serum from rats on the control diet. The profibrotic serum factors were, however, not identified. This study tested the hypothesis that circulating angiotensin II may modulate cardiac fibroblast activity in hypomagnesemic rats. Male Sprague-Dawley rats were pair-fed a magnesium-deficient (0.0008% Mg) or -sufficient (0.05%) diet for 6 days, and the effects of serum from these rats on [3H]thymidine and [3H]proline incorporation into cardiac fibroblasts from young adult rats were evaluated in the presence of losartan, an angiotensin II type 1 (AT1) receptor antagonist, and spironolactone, an aldosterone antagonist. Losartan and spironolactone markedly attenuated the stimulatory effects in vitro of serum from the magnesium-deficient and control groups, but the inhibitory effects were considerably higher in cells exposed to serum from magnesium-deficient animals. Circulating and cardiac tissue levels of angiotensin II were significantly elevated in magnesium-deficient animals (67.6% and 93.1%, respectively, vs. control). Plasma renin activity was 61.9% higher in magnesium-deficient rats, but serum angiotensin-converting enzyme activity was comparable in the two groups. Furthermore, preliminary experiments in vivo using enalapril supported a role for angiotensin II in magnesium deficiency. There was no significant difference between the groups in serum aldosterone levels. The findings suggest that circulating angiotensin II and aldosterone may stimulate fibroblast activity and contribute to a fibrogenic response in the heart in magnesium deficiency.     

Radioimmunoassay for tubulin: a quantitative comparison of the tubulin content of different established tissue culture cells and tissues

A quantitative estimate of the cellular tubulin concentration can be obtained by the use of a radioimmunoassay based upon the competition between tubulin in cell extracts and a known amount of radioactively labeled homogeneous tubulin during binding to a limited amount of antitubulin antibodies. This assay shows that a variety of widely used tissue culture cells (mouse L cells, mouse 3T3 cells, chick embryo fibroblasts) have a tubulin content which corresponds to approximately 2.5-3.3% of their total protein. Transformation of mouse 3T3 cells by the DNA virus SV40, and of chick embryo cells by the RNA Rous sarcoma virus, does not change the intracellular tubulin concentration. Transformed cells of brain origin, such as some glia tumor cell lines and some neuroblastoma cell lines, have a much lower tubulin content than does normal brain tissue.The intracellular concentration of tubulin in mouse 3T3 cells is discussed in relation to the number of microtubules detected during interphase by immunofluorescence microscopy. These results are also discussed in view of a mechanism of microtubule elongation in vivo driven by self-assembly.     

Analysis of hexose transport in untransformed and sarcoma virus-transformed mouse 3T3 cells by photoaffinity binding of cytochalasin B

The effect of simian virus 40 transformation on the hexose transport system in mouse embryo fibroblast Swiss 3T3 cells was examined. The concentration of hexose transporters was estimated by measuring D-glucose-inhibitable cytochalasin B binding. The binding of cytochalasin B to the plasma membranes of simian virus 40-transformed mouse 3T3 cells (SV3T3 cells) was significantly greater than that of 3T3 cells. On the other hand, cytochalasin B binding to the microsomal membranes of SV3T3 cells was decreased, and the total amount of binding to plasma and microsomal membranes was not significantly changed in both cell lines. The electrophoretic analysis demonstrated that both hexose-transporter components of Mr 46 000 and Mr 58 000 affinity labeled were responsible for an increase in the hexose transport by viral transformation. These results suggested that the higher hexose-transport activity of transformed cells is caused by a redistribution of transporter from intracellular membranes to plasma membranes.     

Hyaluronate degradation in 3T3 and simian virus-transformed 3T3 cells

The cellular control of hyaluronate levels was examined in cultures of simian virus 40-transformed 3T3 (SV3T3) and 3T3 cells which are known to differ in their metabolism of hyaluronate. When [3H]hyaluronate was added to cultures of the two cell lines, four times more ligand was bound per mg of protein by the SV3T3 cells than by the 3T3 cells. Of the bound [3H] hyaluronate, 40% was degraded by the SV3T3 cells to oligosaccharides characteristic of the breakdown of hyaluronate, but only 2% was degraded by 3T3 cells. Hyaluronidase activity was found in the cell layer and medium of the SV3T3 cultures, but was not detectable in 3T3 cells. The SV3T3 enzyme was active only at acidic pH, but at neutral pH the secreted SV3T3 hyaluronidase was thermally more stable then the cell-associated enzyme. In contrast, both cell lines were found to contain similar amounts of beta-glucuronidase and beta-N-acetylglucosaminidase activity. We conclude that the elevated capacity of SV3T3 cells to degrade hyaluronate may be partially responsible for their lack of the hyaluronate-containing pericellular coat which is prominent around 3T3 cells.     

钙调神经磷酸酶在血管紧张素Ⅱ刺激的心脏成纤维细胞增殖中的作用

本研究观察了钙调神经磷酸酶（ＣａＮ）在血管坚张素Ⅱ（ＡｎｇⅡ）刺激的大鼠心脏成纤维细胞增殖中的作用。在培养的大鼠心脏成纤维细胞上,应用双波长荧光 计检测Ｆｕｒａ－２标记的细胞游离Ｃａ＾２＋浓度;应用对硝基苯磷酸（ＰＮＰＰ）作底物测定钙调神经磷酸酶（ＣａＮ）活性;根据＾３Ｈ－胸腺嘧啶掺入法评估ＣａＮ特异性抑制剂环胞素Ａ（ＣｓＡ）对ＡｎｇⅡ刺激的心脏成纤维细胞ＤＮＡ合成的影响。结果表明,ＡｎｇⅡ（１０     

The suppression of cellular proliferation in SV40-transformed 3T3 cells by glucocorticoids

Glucocorticosteroids, when added two hours after cell plating to SV40-transformed, 3T3 mouse fibroblasts in low serum (0.3% v/v), biotin-supplemented medium, suppress cellular proliferation by 24 hours. While some cell death probably occurs, the growth inhibition is not primarily due to cytotoxicity and cytolysis. This conclusion is supported by the following: 1) both dead and viable cell numbers are suppressed, 2) little cell debris is evident in the medium, and 3) very high concentrations of glucocorticoids do not cause an increase in the dead cell count. Furthermore, this growth suppression, which is specific for glucocorticoids since several non-glucocorticoid steroids have no inhibitory effect, is not permanent nor irreversible. Removal of the glucocorticoid and replacement with 10% serum restore rapid proliferation. Although higher concentrations (1% and 10%) of serum afford some protection against glucocorticoid inhibition, this protection is not simply a consequence of faster growth rates. SV3T3 cells can be grown in serum-free medium supplemented with biotin, transferrin, insulin, and epidermal growth factor (EGF). Under these conditions growth rates are comparable to high serum media, yet glucocorticoids are still powerful inhibitors. However, the omission of insulin from serum-free, glucocorticoid cultures does result in observable cell death and lysis. Flow microfluorometry and autoradiographic studies have determined that glucocorticoid-inhibited cells are partially blocked in G1. The proportions of S phase and G2 + M cells are greatly reduced with an accompanying accumulation of G1 cells. These results suggest that glucocorticoids regulate a biochemical step(s) in G1 which is critical for DNA initiation.     

Angiotensin II type 1 receptor expression in two cases of juxtaglomerular cell tumor: correlation to negative feedback of renin secretion by angiotensin II.

The angiotensin II (Ang II) type 1 (AT1) receptor is highly expressed on juxtaglomerular (G) cells and is assumed to be involved in the negative short loop feedback regulation of renin secretion and in the suppression of Ang II-mediated JG cell proliferation and/or growth. However, as JG cell tumor is rare, expression and pathophysiological significance of AT1 receptor expression in JG cell tumor remain unknown. In the present study, we investigated renin responses to various treatments, including the angiotensin converting enzyme inhibitor captopril, and correlated the results with AT1 and Ang II type 2 (AT2) receptor mRNA expression levels in two cases of JG cell tumor. Whereas plasma renin activity (PRA) did not show any significant change in Case 1, it was increased by 72% in Case 2 in response to captopril challenge. In concordance with these results, AT1 receptor mRNA was not detected in tumor tissue of Case 1 but was clearly demonstrated in the tumor of Case 2. AT2 receptor mRNA expression was not detected in either of the cases. In contrast to captopril challenge, PRA was suppressed by 30% in Case 1 and 42% in Case 2 in response to saline infusion, and was increased by 230% in Case 1 and 59% in Case 2 in response to furosemide-upright posture for 2 h. These results suggest that the short loop feedback inhibition of renin secretion by Ang II in JG cell tumor is closely related to AT1 receptor expression levels in the tumor tissue. In addition, the result suggested that despite its autonomy, renin secretion from JG cell tumor is still under physiological regulatory control.     

Intracellular angiotensin II as a regulator of muscle tone in vascular resistance vessels. Pathophysiological implications

The influence of intracellular angiotensin II on the regulation of potassium current and membrane potential of smooth muscle cells of mesenteric arteries and its relevance for the regulation of vascular tone was reviewed. The presence of components of the renin angiotensin system (RAS) in different cells of the cardiovascular system, was discussed including their presence in the nuclei and mitochondria. Emphasis was given to the opposite effects of intracellular and extracellular angiotensin II (Ang II) on the regulation of potassium current, membrane potential and contractility of vascular resistance vessels and its implication to vascular physiology and pathology and the possible role of epigenetic factors on the expression of angiotensin II (Ang II) and renin in vascular resistance vessels as well as its possible pathophysiological role in hypertension and other cardiovascular diseases.     

Cyclic AMP specifically blocks proliferation of rat 3T3 cells transformed by polyomavirus.

Elevated exogenous and intracellular levels of cyclic AMP could totally block proliferation of polyomavirus (PyV) transformants derived from rat 3T3 cells without affecting proliferation of normal cells or simian virus 40 (SV40)-induced transformants. Concanavalin A (ConA) had the opposite effect; it could totally block proliferation of both normal cells and SV40 transformants but reduced proliferation of PyV transformants only twofold. Adenylate cyclase was threefold less active in membranes of PyV transformants, and the number of ConA receptors was similar to that of normal cells. Proliferating PyV transformants contained threefold less cyclic AMP than did proliferating SV40 transformants. The sensitivity to cyclic AMP did not correlate with the degree of transformation: cells transformed by Rous sarcoma virus and tumor cells derived from SV40 transformants were not sensitive to cyclic AMP. The differential effect of cyclic AMP and ConA on proliferation was probably due to the activity of an intact middle t protein. The presence of both large T and small t together with middle t was also required for cyclic AMP sensitivity.     

Cyclic AMP-dependent protein kinases from Balb 3T3 cells and other 3T3 derived lines

Cyclic AMP-dependent protein kinase and 3H-cAMP-binding activities were determined in normal Balb 3T3 cells and compared with the same preparations from SV40, chemical, and spontaneous transformants of 3T3 cells. The cytosolic protein kinase activities and protein kinase activity ratios were similar in all cell lines, although when the normal 3T3 cytosol was prepared by homogenization it contained less 3H-cAMP binding activity than the transformed 3T3 cytosols. The Triton X-100 treated particulate fractions from the normal and transformed 3T3 cells contained similar protein kinase and binding activities. The isozymic profile of cAMP-dependent protein kinases was examined by DEAE-chromatography. The 3T3 cells contained only type II isozyme in either cytosolic or membrane fractions. All transformants of the 3T3 cells contained both type I and type II isozymes. Other cell cultures, including chicken embryo fibroblasts, rat kidney cells, and human or calf endothelial cells contained type I and type II isozymes. Binding of the photoaffinity analogue of cAMP, 8-N3 cAMP, to the regulatory subunits of protein kinases in sonicates obtained from Balb 3T3 and SV 3T3 cells followed by separation on SDS polyacrylamide electrophoresis showed that the amount of RII subunit was approximately equal in the two cell lines. RI in Balb 3T3 cells was detectable but in a much lower quantity than in SV 3T3 cells. The cyclic AMP dependent-protein kinases from Balb 3T3 cells appears to be different from SV 3T3 cells by three criteria: 3H-cAMP binding in homogenates, DEAE chromatographic separation of isozymes, and 8-N3 cAMP binding.     

Characterization of AT2 Receptor Expression in NIH 3T3 Fibroblasts

1. A high expression of angiotensin II receptors and of angiotensin-converting enzyme (ACE) activity was detected in confluent NIH 3T3 fibroblasts.2. Characterization with selective ligands, dithiothreitol, and GTPS, indicated that only the AT₂ subtype was expressed.3. AT₂ receptors and ACE expression were strictly dependent on the cell density and growth phase of the cells, with AT₂ receptors being expressed earlier than ACE. In contrast, high expression of AT₂ receptors irrespective of their growth state was observed in NIH 3T3 cells lacking contact inhibition upon neoplastic transformation with ras.4. Our results imply a possible relation of AT₂ receptors to cell growth and cell–cell contact.