Insulin-like growth factor II increases cytoplasmic free calcium in competent Balb/c 3T3 cells treated with epidermal growth factor

To determine the role of calcium in the action of insulin-like growth factor II (IGF-II), we have examined the effect of multiplication stimulating activity, the rat IGF-II, on cytoplasmic-free calcium concentration, [Ca2+]c, in aequorin-loaded Balb/c 3T3 cells. IGF-II does not cause any change in [Ca2+]c in quiescent cells. By contrast, IGF-II induces changes in [Ca2+]c in platelet-derived growth factor(PDGF) - pretreated competent cells: when competent cells are incubated with epidermal growth factor (EGF) for 10 min, subsequent IGF-II induces an immediate increase in [Ca2+]c. Without EGF treatment, IGF-II does not cause any increase in [Ca2+]c. The priming action of EGF is time dependent, requiring approximately 10 min for the maximum effect. The IGF-II-mediated increase in [Ca2+]c is totally dependent on extracellular calcium and is blocked by lanthanum. When DNA synthesis in PDGF-treated competent cells is assessed by measuring [3H]thymidine incorporation, IGF-II by itself has only a small effect. Likewise, a brief treatment with EGF results in only a small increase in [3H]thymidine incorporation. By contrast, in competent cells briefly treated with EGF, IGF-II causes a marked stimulation of [3H]thymidine incorporation. These results indicate that IGF-II increases [Ca2+]c in competent Balb/c 3T3 cells treated with EGF by stimulating calcium influx and that IGF-II-stimulated calcium influx may be related causally to its action on cell proliferation.     

Evidence that type II insulin-like growth factor receptor is coupled to calcium gating system

In competent Balb/c 3T3 cells primed with epidermal growth factor (primed competent cells), insulin-like growth factor-II (IGF-II) stimulated calcium influx in a concentration dependent manner with the ED50 of 450 pM. When receptor-bound [125I]IGF-II was cross-linked by use of disuccinimidyl suberate, a 240 K-Da protein was radiolabeled. Excess amount of unlabeled IGF-II inhibited the affinity-labeling of the 240 K-Da protein. To further examine whether IGF-II stimulates calcium influx by acting on the type II IGF receptor, we employed polyclonal antibody raised against rat type II IGF receptor, R-II-PABl. This antibody immunoprecipitated the type II IGF receptor and inhibited IGF-II binding in Balb/c 3T3 cell membrane without affecting IGF-I binding. In primed competent cells, R-II-PABl elicited an agonistic action in stimulating [3H]thymidine incorporation. Under the same condition, R-II-PABl elicited a marked stimulation of calcium influx. These results suggest that, in Balb/c 3T3 cells, 1) relatively low concentrations of IGF-II act mainly on the type II IGF receptor; 2) the type II IGF receptor is coupled to a calcium gating system; and 3) binding of a ligand to the type II IGF receptor leads to the stimulation of DNA synthesis.     

Calcium influx: an intracellular message of the mitogenic action of insulin-like growth factor-I

When G0-arrested BALB/c 3T3 cells were treated sequentially with platelet-derived growth factor and epidermal growth factor, cells became responsive to insulin-like growth factor-I (IGF-I). In these primed competent cells, 1 nM IGF-I elicited an approximately 3-fold increase in the calcium influx rate. IGF-I-induced calcium influx was relatively slow in onset and continued for at least 2 h in the presence of IGF-I. When a single Ca2+ channel current was studied by the patch-clamp technique using the cell-attached mode, inward currents with unitary conductance of 19 pS were observed in the presence of 1 nM IGF-I in the patch pipette. IGF-I-sensitive inward current was independent of membrane potential and was activated by a high concentration of insulin. Accordingly, 1 nM IGF-I caused a gradual increase in cytoplasmic free calcium concentration measured by fura2. The action of IGF-I on calcium influx was dependent on extracellular calcium, and IGF-I did not stimulate calcium influx when extracellular calcium concentration was reduced to 10 microM. Both cobalt and tetramethrin blocked the action of IGF-I on calcium influx without affecting the binding of 125I-IGF-I. In primed competent cells, IGF-I-stimulated [3H]thymidine incorporation was dependent on extracellular calcium and was attenuated by cobalt and tetramethrin. When cell-bound 125I-IGF-I was cross-linked by use of disuccinimidyl suberate, a 130-kDa protein was radiolabeled. Affinity labeling of the 130-kDa protein, presumably the alpha-subunit of the IGF-I receptor, was blocked by excess amount of unlabeled IGF-I. These results suggest that relatively low concentrations of IGF-I stimulate calcium influx in primed competent BALB/c 3T3 cells by activating a calcium-permeable cation channel via the IGF-I receptor and that calcium influx may be a critical intracellular message of the progression activity of IGF-I.     

Studies on the cell-cycle dependency of the actions of insulin-like growth factor-I in Balb/c 3T3 cells

The present study was conducted to determine the cell-cycle dependency of various actions of IGF-I in Balb/c 3T3 cells. When autophosphorylation of the IGF-I receptor was determined in [32P]-labelled cells, IGF-I increased radioactivity in a 100 K-Da phosphoprotein, presumably beta-subunit of the IGF-I receptor, both in quiescent and in primed competent cells. Likewise, IGF-I stimulated uptake of [3H]deoxyglucose independent of the cell cycle. In contrast, IGF-I increased calcium entry, radioactivity in [3H]diacylglycerol, and [3H]thymidine incorporation in primed competent cells while these reactions were not induced by IGF-I in quiescent cells. The latter three reactions were attenuated when cells were pretreated with pertussis toxin. These results indicate that some, but not all, of the actions of IGF-I are dependent on the cell cycle in Balb/c 3T3 cells. They also suggest that a pertussis-toxin-sensitive G protein may be involved in the cell-cycle-dependent actions of IGF-I.     

Regulation of transmembrane signal transduction of insulin-like growth factor II by competence type growth factors or viral ras p21

In BALB/c 3T3 cells pretreated with platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) (primed-competent cells), insulin-like growth factors I and II (IGF-I and IGF-II) bind to their own receptors (IGF-IR and IGF-IIR) and stimulate calcium influx and DNA synthesis by a mechanism involving a 40-kDa pertussis toxin substrate. In contrast, these IGFs do not act on unprimed quiescent cells. In this study, the 40-kDa pertussis toxin substrate was identified as Gi-2 alpha using anti-G protein antibodies. We analyzed the quality of signal transduction from IGF-II to Gi-2 alpha. There was no difference in the amount of Gi-2 alpha between quiescent and primed-competent cells, and both of these cells had similar Kd values and numbers of IGF-II-binding sites. Whereas IGF-II did not alter pertussis toxin-catalyzed ADP-ribosylation of Gi-2 alpha in quiescent cells, IGF-II reduced the pertussis toxin substrate activity by 35-50% via the IGF-IIR in primed-competent cells. The action of IGF-II lasted for up to 3 h when IGF-II was present in the medium, and it disappeared when IGF-II was removed. These results suggest that the signaling pathway triggered by IGF-II is uncoupled between the IGF-IIR and Gi-2 alpha in quiescent cells and that PDGF and EGF restore the IGF-IIR-Gi-2 coupling. This study also indicates that low concentrations of IGF-I reduce the pertussis toxin substrate activity of Gi-2 alpha in primed-competent cells in a time course slower than that of IGF-II, but not at all in quiescent cells. However, both of these cells had similar Kd values and numbers of IGF-I binding sites. Therefore, the IGF-I signaling pathway may also be uncoupled between the IGF-IR and Gi-2 alpha in quiescent cells and restored by PDGF and EGF. In BALB/c 3T3 cells transfected with temperature-sensitive Kirsten sarcoma virus bearing the v-Ki-ras gene (ts cells), a 40-kDa pertussis toxin substrate was also identified as Gi-2 alpha. In nonpermissive ts cells, IGF-II was without effect on the pertussis toxin substrate activity of Gi-2 alpha or on calcium influx.(ABSTRACT TRUNCATED AT 400 WORDS)     

Insulin-like growth factor II binding and action in human fetal fibroblasts

To investigate the role of insulin-like growth factor II (IGF-II) in human prenatal growth, IGF-II binding and biological action were studied in four lines of fetal and three lines of postnatal human fibroblasts. Specific binding of IGF-II was similar in both groups: 15.7% and 14.9% for fetal and postnatal fibroblasts, respectively. This was 5-10 times the amount of IGF-I binding found in these cells. IGF-I and IGF-II caused dose-dependent increases in [14C]aminoisobutyric acid (AIB) uptake. IGF-II was sevenfold less potent than IGF-I in stimulating this metabolic response in both fetal and postnatal fibroblasts. The maximal effect of IGF-II in stimulating [14C]AIB uptake approach that of IGF-I. Similar results were obtained when IGF-I and IGF-II stimulation of [3H]thymidine incorporation was compared in fetal and postnatal fibroblasts. Incubation in the presence of alpha IR-3, a monoclonal antibody to the type I IGF receptor, inhibited the ability of both IGF-I and IGF-II to stimulate [14C]AIB uptake and [3H]thymidine incorporation in fetal and postnatal cells. A monoclonal antibody to the insulin receptor did not affect IGF action. These data indicate that IGF-II is a potent metabolic and mitogenic stimulus for human fetal fibroblasts. However, despite the presence of abundant type II IGF receptors on both fetal and postnatal human fibroblasts, IGF-II stimulation of amino acid transport and DNA synthesis appears to be mediated through the type I rather than through its own type II IGF receptor.     

Requirement of phosphatidylinositol 3-kinase activation and calcium influx for leukotriene B4-induced enzyme release

Leukotriene B(4) (LTB(4)) is a potent lipid mediator involved in host defense and inflammatory responses. It causes chemotaxis, generation of reactive oxygen species, and degranulation. However, only little is known of the molecular mechanisms by which LTB(4) induces these biological activities. To analyze the intracellular signaling pathways to mediate lysosomal enzyme release through the cloned LTB(4) receptor (BLT1), we transfected BLT1 to rat basophilic leukemia cells (RBL-2H3). LTB(4) dose-dependently released beta-hexosaminidase, and the release was mostly inhibited when the cells were pretreated with pertussis toxin, indicating that the degranulation is mediated by G(i) proteins. LTB(4) activated phosphatidylinositol 3-kinase (PI3-K) through G(i), and inhibition of PI3-K by wortmannin or LY290042 inhibited degranulation. Granulocytes from PI3-Kgamma-deficient mice showed reduced LTB(4)-induced degranulation, suggesting that this isozyme of PI3-K is involved in the degranulation. LTB(4) also caused calcium release from intracellular stores and calcium influx from the outside milieu through G(i), but only the calcium influx is critical for the lysosomal enzyme release. Calcium influx and PI3-K activation are both downstream events of G(i), since they were inhibited by pertussis toxin. These two events are in essence independent each other, because calcium depletion did not affect PI3-K, and inhibition of PI3-K did not attenuate calcium influx significantly. Thus, our results have clearly shown that LTB(4) binds BLT1 and activates G(i)-like protein, and both PI3-Kgamma activation and a sustained calcium elevation by calcium influx are necessary for enzyme release in these cells.     

Altered expression of inhibitory guanine nucleotide regulatory proteins (Gi-proteins) in experimental hepatocellular carcinoma

Guanine nucleotide regulatory proteins (G-proteins) play an important role in the onset and progression of malignancy. We hypothesized that alterations in inhibitory G-protein (Gi) expression and/or function may contribute to cellular invasion and formation of hepatocellular carcinoma (HCC). H4IIE hepatoma cells were inoculated directly into the liver parenchyma of ACI strain rats, and membranes were prepared from HCC livers and adjacent nonneoplastic livers 12 days following the initial inoculation. Expression of inhibitory Giα proteins was determined by Western blot analysis and changes in the functional activity of these proteins confirmed by pertussis toxin catalyzed ADP ribosylation and adenylyl cyclase activity. Inhibitory Giα1, Giα1/2, and Giα3 protein expression was significantly elevated in HCC when compared to adjacent nonneoplastic liver and sham-operated hepatic tissue. Pertussis toxin catalyzed ADP ribosylation of Giα substrates was significantly enhanced in HCC concomitant with increased basal and stimulated adenylyl cyclase activity following uncoupling of Gi-proteins with manganese ions. The role of Gi-proteins in cellular proliferation was confirmed using cultured H4IIE cells and normal hepatocytes. In quiescent H4IIE cells, mastoparan (Giα activator) increased [³H] thymidine incorporation and cell growth in a dose-dependent manner, whereas both pertussis toxin (a Gi-protein inhibitor) and 8-bromo-cAMP inhibited mitogenesis. In contrast, in isolated cultured hepatocytes, mastoparan inhibited [³H] thymidine incorporation, while pertussis toxin and 8-bromo-cAMP were mitogenic. We conclude that HCC is associated with marked changes in Giα-protein expression in vivo and in vitro, direct activation of which leads to increased mitogenesis in H4IIE cells in vitro. J. Cell. Physiol. 175:295–304, 1998. © 1998 Wiley-Liss, Inc.     

Macrophage-dependent stimulation of T cell-depleted spleen cells by Clostridium difficile toxin A and calcium ionophore

Clostridium difficile toxin A causes severe intestinal inflammation and fluid secretion in rabbit ileum and is chemotactic for neutrophils in vitro. The mechanism of intestinal injury produced by toxin A appears to involve direct epithelial cell damage as well as recruitment of an inflammatory cell response. The current study was undertaken to determine if toxin A can directly stimulate a proliferative response in lymphocytes. Highly purified toxin A, in the presence of the calcium ionophore, ionomycin, stimulated substantial [3H]thymidine incorporation by murine splenic lymphocytes, which was maximal at 10(-9) M toxin A and 800 ng/ml ionomycin. Removal of T cells with anti-Thy-1.2 antibody plus complement had no effect on the proliferative response induced by toxin A. However, [3H]thymidine incorporation in response to toxin A was significantly inhibited (P less than 0.001) by the removal of macrophages from splenocyte suspensions and was restored by the addition of peritoneal macrophages or cell-free supernatant from toxin A-treated macrophage cultures. Analysis of the toxin A-treated macrophage supernatants showed high levels of IL-1, but not IL-2 or IL-4. The combination of recombinant IL-1 plus ionomycin was found to stimulate [3H]thymidine incorporation by T cell-depleted splenic lymphocytes. These results suggest that toxin A stimulates the release of IL-1, and possibly other factors, from macrophages which can costimulate murine B lymphocytes.     

Pertussis toxin does not inhibit muscarinic-receptor-mediated phosphoinositide hydrolysis or calcium mobilization.

Pertussis toxin was used to examine the role of the inhibitory guanine nucleotide regulatory protein, Ni, in muscarinic-receptor-mediated stimulation of phosphoinositide turnover and calcium mobilization. In cultured chick heart cells, pertussis-toxin treatment inhibited muscarinic-receptor-mediated attenuation of isoprenaline-stimulated cyclic AMP accumulation. This finding is consistent with the proposal that pertussis toxin blocks the capacity of Ni to couple muscarinic receptors to adenylate cyclase. In contrast, treatment of chick heart cells or 1321N1 human astrocytoma cells with pertussis toxin did not block muscarinic-receptor-mediated stimulation of phosphoinositide hydrolysis, as measured by [3H]inositol phosphate accumulation in the presence of Li+. Pertussis-toxin treatment also had little effect on basal and muscarinic-receptor-stimulated phosphatidylinositol synthesis, as measured by the incorporation of [3H]inositol into phosphatidylinositol. Activation of muscarinic receptors also enhances the rate of unidirectional 45Ca2+ efflux in 1321N1 cells; this response, like phosphoinositide hydrolysis, was not prevented by pertussis-toxin treatment. Our data suggest that muscarinic receptors are not coupled to phosphoinositide hydrolysis or calcium mobilization through Ni.     

Insulin-like growth factor-I stimulates diacylglycerol production via multiple pathways in Balb/c 3T3 cells

We previously reported that insulin-like growth factor-I (IGF-I) induced sustained calcium cycling across the plasma membrane in primed competent Balb/c 3T3 cells (Kojima, I., Matsunaga, H., Kurokawa, K., Ogata, E., and Nishimoto, I. (1989) J. Biol. Chem. 263, 16561-16567). The present study was conducted to examine whether IGF-I affected cellular metabolism of 1,2-diacylglycerol (1,2-DAG). In primed competent cells prelabeled with [3H]myristate, 1 nM IGF-I caused a 50% increase in [3H]DAG within 10 min. This increase in [3H]DAG was accompanied by 1) a decrease in radioactivity in the glycosylphosphatidylinositol fraction in [3H]glucosamine-labeled cells and a concomitant increase in [3H]inositol-glycan, and 2) a decrease in [3H]phosphatidylcholine and a concomitant elevation of [3H]phosphorylcholine in [3H]choline-labeled cells. When [3H]choline-labeled cells were treated with 10 nM 12-O-tetradecanoylphorbol-4-acetate (TPA), [3H]phosphatidylcholine was reduced by 50%. The TPA-induced reduction of [3H]phosphatidylcholine was completely blocked by 50 microM sphingosine and 50 microM H-7, inhibitors of protein kinase C. Both sphingosine and H-7 attenuated IGF-I-mediated reduction of [3H]phosphatidylcholine. In addition, treatment with IGF-I for 3 h or more resulted in sustained increase in 1,2-DAG mass, which was attenuated by cycloheximide. The increase in DAG mass was accompanied by enhanced incorporation of [14C]glucose into 1,2-DAG. These results indicate that, in primed competent Balb/c 3T3 cells, IGF-I stimulates 1,2-DAG production via multiple pathways and that IGF-I may induce breakdown of phosphatidylcholine by a mechanism involving protein kinase C.     

Muscarinic stimulation of calcium influx and norepinephrine release in PC12 cells

Muscarinic cholinergic receptor stimulation evokes catecholamine secretion from some cell types, but the mechanism has not been well characterized. Using pheochromocytoma (PC12) cells, we show that the muscarinic agonist methacholine stimulates 45Ca2+ influx and [3H]norepinephrine release in a dose-dependent manner. Experiments performed in Na+-free medium or with inhibitors of voltage-dependent Ca2+ channels suggest the involvement of a receptor-activated Ca2+ channel which differs significantly from the voltage-dependent Ca2+ channel involved in nicotinic receptor-stimulated release. Furthermore, both influx and release were inhibited by pertussis toxin (0.5-2.0 ng/ml, 21 h) with a dose dependency which paralleled the dose dependency of pertussis toxin-dependent in vivo ADP-ribosylation of a 41-kDa protein. These experiments provide the first evidence that muscarinic stimulation evokes neurotransmitter secretion by opening a receptor-activated Ca2+ channel which is controlled by a pertussis toxin-sensitive protein.     

Steroidal and growth factor regulation of [3H]thymidine incorporation by cultured endosalpingeal cells of the bovine oviduct

Summary Cultured cells from the bovine endosalpinx were used to evaluate effects of estradiol-17β, progesterone, epidermal growth factor, and insulinlike growth factors I and II on [³H]thymidine incorporation. Cells were treated with hormones and growth factors when approximately 50% confluent. After 24 h, DNA synthesis was quantified by pulsing cells with [³H]thymidine for 12 h and determining uptake into DNA. Cells prepared by mechanical dispersal incorporated more [³H]thymidine than cells dispersed with collagenase. However, hormonal responses were the same for both types of cells. As compared to plastic, cells on a Matrigel substratum exhibited lower incorporation of [³H]thymidine and were unresponsive to hormones. Estradiol-17β increased [³H]thymidine incorporation slightly at 10⁻¹⁰ mol/liter and higher. Epidermal growth factor, insulinlike growth factor-I, and insulinlike growth factor-II also stimulated [³H]thymidine incorporation. Effects of insulinlike growth factor-I were greater for cells treated with estradiol-17β. In the absence of estradiol, progesterone inhibited [³H]thymidine incorporation at 1, 10, and 100 ng/ml. When estradiol-17β was present, progesterone stimulated [³H]thymidine incorporation at 1 ng/ml and reduced incorporation at 100 ng/ml. In conclusion, [³H]thymidine incorporation by cultured oviductal endosalpingeal cells can be regulated by ovarian steroids and growth factors. These molecules may represent signals through which the ovary, embryo, and oviduct regulate oviductal growth. Work conducted while on a sabbatical leave supported by the Deutsche Forschungsgemeinschaft.     

Evidence for the Implication of Phosphoinositol Signal Transduction in μ-Opioid Inhibition of DNA Synthesis

An opioid receptor agonist, [D-Ala2,Me-Phe4,Glyol5]enkephalin (DAMGE), decreased [3H]thymidine incorporation into DNA of fetal rat brain cell aggregates. This action proved to depend on the dose of this enkephalin analog and the interval the aggregates were maintained in culture. The opioid antagonist naltrexone and the mu-specific antagonist cyclic D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr amide (CTOP) reversed the DAMGE effect, arguing for a receptor-mediated mechanism. The mu-opioid nature of this receptor was further established by inhibiting DNA synthesis with the highly mu-selective agonist morphiceptin and blocking its action with CTOP. Several other opioids, pertussis toxin, and LiCl also diminished DNA synthesis, whereas cholera toxin elicited a modest increase. Naltrexone completely reversed the inhibition elicited by the combination of DAMGE and low doses of LiCl but not by that of high levels of LiCl alone. The enkephalin analog also reduced basal [3H]inositol trisphosphate and glutamate-stimulated [3H]inositol monophosphate and [3H]inositol bisphosphate accumulation in the aggregates. These DAMGE effects were reversed by naltrexone and were temporally correlated with the inhibition of DNA synthesis. A selective protein kinase C inhibitor, chelerythrine, also inhibited thymidine incorporation dose-dependently. The effect of DAMGE was not additive in the presence of chelerythrine but appeared to be consistent with their actions being mediated via a common signaling pathway. These results suggest the involvement of the phosphoinositol signal transduction system in the modulation of thymidine incorporation into DNA by DAMGE.     

Lysophosphatidic acid is a mediator of Trp-Lys-Tyr-Met-Val-d-Met-induced calcium influx

Intracellular calcium (Ca(2+)) homeostasis is very strictly regulated, and the activation of G-protein-coupled receptor (GPCR) can cause two different calcium changes, intracellular calcium release, and calcium influx. In this study, we investigated the possible role of lysophosphatidic acid (LPA) on GPCR-induced Ca(2+) signaling. The addition of exogenous LPA induced dramatic Ca(2+) influx but not intracellular Ca(2+) release in U937 cells. LPA-induced Ca(2+) influx was not affected by pertussis toxin and phospholipase C inhibitor (U73122), ruling out the involvement of pertussis toxin-sensitive G-proteins, and phospholipase C. Stimulation of U937 cells with Trp-Lys-Tyr-Met-Val-D-Met (WKYMVm), which binds to formyl peptide receptor like 1, enhanced phospholipase A(2) and phospholipase D activation, indicating LPA formation. The inhibition of LPA synthesis by phospholipase A(2)-specific inhibitor (MAFP) or n-butanol significantly inhibited WKYMVm-induced Ca(2+) influx, suggesting a crucial role for LPA in the process. Taken together, we suggest that LPA mediates WKYMVm-induced Ca(2+) influx.     

Insulin-like growth factor I (IGF-I) production and the presence of IGF-I receptors in rat medullary thyroid carcinoma cell line 6-23 (clone 6)

To clarify whether insulin-like growth factor I (IGF-I) is an autocrine growth factor of rat medullary thyroid carcinoma (MTC) cell line, 6-23 (clone 6), IGF-I binding to MTC cell membranes, IGF-I levels in the conditioned culture medium of MTC cells and the effects of IGF-I on methyl-[3H]thymidine incorporation to MTC cells were examined. Scatchard analysis of saturation binding studies revealed the association constant and the maximal binding capacity were 1.0 x 10(9) M-1 and 199 fmol/mg of membrane protein, respectively. The binding of [125I]IGF-I to MTC cell membranes was inhibited by unlabeled IGF-I, IGF-II and insulin; the relative potencies were IGF-I greater than IGF-II much greater than insulin, suggesting the presence of type I IGF receptors in MTC cells. IGF-I levels in the conditioned culture medium of MTC cells were 120 +/- 3 pM (mean + SE). IGF-I (10(-10) to 10(-8) M) dose-dependently stimulated methyl-[3H]thymidine incorporation to MTC cells. These findings suggest a possible role of IGF-I as an autocrine growth factor for MTC cells.     

A monoclonal antibody which inhibits epidermal growth factor binding has opposite effects on the biological action of epidermal growth factor in different cells

An epidermal growth factor (EGF) receptor-interactive monoclonal antibody (151-IgG) that inhibits EGF binding to PC12 rat pheochromocytoma cells and to various other cell types has been produced. The hybridoma clone was obtained by fusing Sp2/O-Ag14 myeloma cells with splenocytes from Balb/C mice which had been immunized with n-octyl glucoside-solubilized protein from isolated PC12 cell plasma membranes. The antibody is an IgG which binds to protein A. 151-IgG did not bind EGF. At 0.5 degrees C 151-IgG was directly competitive for EGF binding to PC12 cells. It also inhibited EGF binding to bovine corneal endothelial cells, rabbit corneal fibroblasts, human foreskin fibroblasts, and normal rat kidney cells, and it slightly enchanced EGF binding to SW 3T3 cells. PC12 cells have the same number of binding sites for 151-IgG as for EGF (approximately 27,000 sites/cell). 151-IgG inhibited the photoactivatable cross-linking of EGF to a protein of Mr 170,000 in PC12 cells. 151-IgG inhibited the EGF-stimulated incorporation of [3H]thymidine into quiescent bovine corneal endothelial cells, rabbit corneal endothelial cells, epithelial normal rat kidney cells, and SW 3T3 cells while it enhanced the EGF-stimulated [3H]thymidine incorporation into quiescent human foreskin fibroblasts. 151-IgG by itself possessed intrinsic EGF-like activity for human fibroblasts but not for the other cells tested. This suggests that there is a difference in EGF receptors and/or processing in these normal cell types.     

Nongenomic mechanism of glucocorticoid inhibition of bradykinin-induced calcium influx in PC12 cells: possible involvement of protein kinase C

Many stimulants, including bradykinin (BK), can induce increase in [Ca(2+)](i) in PC12 cells. Bradykinin induces an increase in [Ca(2+)](i) via intracellular Ca(2+) release and extracellular Ca(2+) influx through the transduction of G protein, but not through voltage-sensitive calcium channels. In this experiment, We analyzed how corticosterone (Cort) influences BK-induced intracellular Ca(2+) release and extracellular Ca(2+) influx, and further studied the mechanism of glucocorticoid's action. To dissociate the intracellular Ca(2+) release and extracellular Ca(2+) influx induced by BK, the Ca(2+)-free/Ca(2+)- reintroduction protocol was used. The results were as follows: (1) The Ca(2+) influx induced by BK could be rapidly inhibited by Cort, but intracellular Ca(2+) release could not be affected significantly. (2) The inhibitory effect of Cort-BSA (BSA -conjugated Cort) on Ca(2+) influx induced by BK was the same as the effect of free Cort. (3) Protein kinase C (PKC) activator (phorbol 12-myristate 13-acetate) could mimic and PKC inhibitor G?6976 could reverse the inhibitory effect of Cort. (4) There was no inhibitory effect of Cort on Ca(2+) influx induced by BK when pretreated with pertussis toxin. The results suggested, for the first time, that Cort might act via a putative membrane receptor and inhibit the Ca(2+) influx induced by BK through the pertussis toxin -sensitive G protein-PKC pathway.     

Soluble insulin-like growth factor II/mannose 6-phosphate receptor inhibits DNA synthesis in insulin-like growth factor II sensitive cells

The soluble form of the insulin-like growth factor II (IGF-II)/mannose 6-P (IGF-II/M6P) receptor is released by cells in culture and circulates in the serum. It retains its ability to bind IGF-II and blocks IGF-II-stimulated DNA synthesis in isolated rat hepatocytes. Because these cells are not normally stimulated to divide by IGF-II in vivo, the effect of soluble IGF-II/M6P receptor on DNA synthesis has been further investigated in two cell lines sensitive to IGF-II; mouse 3T3(A31) fibroblasts, stimulated by low levels of IGF-II following priming by epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) and Buffalo rat liver (BRL) cells, which secrete IGF-II and proliferate in the absence of exogenous growth factors. Soluble IGF-II/M6P receptor (0.2-2.0 microgram/ml) purified from a rat hepatoma cell line inhibited DNA synthesis (determined by dThd incorporation) in both cell lines. Basal DNA synthesis was very low in serum-free 3T3 cells, but high in serum-free BRL cells, possibly as a result of autocrine IGF-II production. The inhibitory effect was reversible in cells preincubated with soluble receptor prior to incubation with growth factors and could also be overcome by excess IGF-II. Soluble receptor was more potent in IGF-II-stimulated 3T3 cells and serum-free BRL cells than in BRL cells incubated with serum. Mean inhibition by four preparations of soluble receptor (1 microgram/ml) was 34.7% +/- 4.4% in BRL cells stimulated with fetal calf serum (FCS) (5%) compared to 54.8% +/- 4.2% in serum-free BRL cells (P = 0.05) and 60.6% +/- 6.5% (P = 0.02) in 3T3 cells stimulated by PDGF, EGF, and IGF-II. Soluble receptor had no effect on DNA synthesis in 3T3 cells stimulated with IGF-I. These results demonstrate that soluble receptor, at physiological concentrations, can block proliferation of cells by IGF-II and could therefore play a role in blocking tumor growth mediated by IGF-II.     

Possible involvement of a GTP-binding protein in a late event during endogenous ganglioside-modulated cellular proliferation

The B subunit of cholera toxin, a protein which binds specifically to ganglioside GM1 on the cell surface, stimulates DNA synthesis in quiescent Swiss 3T3 fibroblasts as measured by an increase in [3H]thymidine incorporation. Pertussis toxin pretreatment markedly inhibits B subunit-induced DNA synthesis. The inhibitory effects of pertussis toxin were observed even in the presence of insulin which greatly potentiates the mitogenic response to the B subunit. Treatment with either pertussis toxin or insulin did not alter the binding of the B subunit to the cells. The dose-response for pertussis toxin-induced inhibition of DNA synthesis correlated closely with the dose-response for ADP-ribosylation of a 41-kDa membrane protein, suggesting the involvement of a GTP-binding protein that is a substrate for pertussis toxin (Gi) in mitogenesis induced via cross-linking of endogenous gangliosides. Pertussis toxin, in a similar concentration-dependent manner, also inhibited the mitogenic response to unfractionated fetal calf serum and to bombesin in the absence or presence of insulin. The inhibitory effect of pertussis toxin was clearly unrelated to any effects on known G proteins coupled to adenylate cyclase or phospholipase C. In addition, pertussis toxin did not impair the early increase in cytosolic free Ca2+ induced by the B subunit or bombesin. Pertussis toxin-induced inhibition of DNA synthesis could still be observed even when the toxin was added as late as 6 h after addition of the growth-promoting agents. This suggests the involvement of a GTP-binding protein in a late step of the B subunit- and bombesin-mediated pathways of mitogenesis. The possibility that other growth factors bypass this pathway is shown by their lack of sensitivity to pertussis toxin.