Melittin stimulates Na entry,Na-K pump activity and DNA synthesis in quiescent cultures of mouse cells

Melittin, an amphipatic polypeptide, increases several fold the activity of Na-K pump in quiescent Swiss 3T3 cells. As with other growth factors, melittin increases the activity of the pump by increasing Na entry into the cell. In contrast, other early responses are not elicited by the toxin. At concentrations that promote ion fluxes, melittin stimulates DNA synthesis in quiescent mouse cells acting synergistically with insulin, epidermal growth factor and with the growth factor released by SV40 BHK cells. In contrast, melittin does not interact synergistically with either phorbol esters or vasopressin. The cellular effects of melittin are consistent with the proposal that ion fluxes signal the initiation of mitogenesis in quiescent cells.     

Parathyroid hormone induction of creatine kinase activity and DNA synthesis is mimicked by phospholipase C, diacylglycerol and phorbol ester

Parathyroid hormone (PTH), which increases cAMP levels, also induces an increase in the activity of the brain isozyme of creatine kinase and in DNA synthesis in osteoblast-enriched bone cell cultures by a cAMP-independent mechanism. The following results lead us to the conclusion that PTH induction of brain isozyme of creatine kinase activity and DNA synthesis occurs by activation of membranal phospholipid metabolism leading to increased protein kinase C activity and Ca2+ mobilization, a mechanism demonstrated for several growth factors and other hormones. (1) Binding of membranal phospholipids by agents such as gentamycin or antiphospholipid antibodies abolishes the stimulation by PTH of creatine kinase activity and DNA synthesis but not of cAMP production. (2) Treatment of cell cultures with exogenous phospholipase C increases brain isozyme of creatine kinase activity and DNA synthesis, but not cAMP production; these stimulations are also blocked by serum containing anti-phospholipid antibodies. PTH has no additional effect on stimulation of creatine kinase activity by phospholipase C (and only a slight effect on DNA synthesis). (3) A synthetic diacylglycerol (1-oleyl-2-acetyl glycerol) or phorbol ester (phorbol 12-myristate 13-acetate) or Ca2+ ionophore, A23187 induces creatine kinase activity and DNA synthesis in the cultures. However, this effect is not blocked by antiphospholipid sera and PTH has no additional effect. (4) Inhibition of protein kinase C activity by drugs reported to inhibit the enzyme (retinoic acid, quercetin) abolishes the stimulation of brain isozyme of creatine kinase activity and of DNA synthesis by PTH.     

Synergistic stimulation of DNA synthesis by cyclic AMP derivatives and growth factors in mouse 3T3 cells

Addition of the cAMP derivatives butcAMP or 8BrcAMP to quiescent cultures of Swiss 3T3 causes synergistic stimulation of DNAk synthesis with insulin, phorbol esters, vasopressin, epidermal growth factor, or fetal bovine serum (2-5%). In the presence of insulin, 8BrcAMP, and butcAMP stimulate [3H]-thymidine incorporation into acid-precipitable material in a dose-dependent manner. The effect of these agents is specific since 8Br5'AMP, 5'AMP, butyrate, or 8BrcGMP fail to stimulate DNA synthesis under identical experimental conditions. Furthermore, the mitogenic effects of the cAMP derivatives were markedly potentiated by 1-methyl-3-isobutyl xanthine and 4-(3-butoxy-4-methoxy benzyl)-2-imidazolidine, both of which are potent inhibitors of cyclic nucleotide phosphodiesterase activity. The growth-promoting effects of the cAMP derivatives were demonstrated by [3H]-thymidine incorporation (either by scintillation counting or by autoradiography), by flow cytofluorometric analysis, and by increase in cell number. When quiescent Swiss 3T3 cells were exposed to butcAMP and insulin, DNA synthesis began after a lag of 17h. The result of sequential additions of cAMP derivatives and insulin to quiescent 3T3 cells suggest that these agents must act simultaneously in G0/G1 to stimulate entry into DNA synthesis in these cells. The findings support the proposition that an increase in cellular levels of cAMP (but not cGMP) act sas a mitogenic stimulus for confluent and quiescent Swiss 3T3 cells.     

Synergistic stimulation of S6 ribosomal protein phosphorylation and DNA synthesis by epidermal growth factor and insulin in quiescent 3T3 cells

Using an improved method to quantify the level of phosphorylation of the S6 ribosomal protein, we have analyzed the effect of growth stimuli on S6 phosphorylation in quiescent murine Swiss/3T3 cells to see if it can be dissociated from the later increase in DNA synthesis. Saturating concentrations of epidermal growth factor (EGF), insulin and serum each stimulate phosphorylation of the S6 ribosomal protein to the same maximal level; this is not so for DNA synthesis. Subsaturating concentrations of EGF and insulin act synergistically to stimulate both S6 phosphorylation and DNA synthesis, but qualitatively the two synergistic interactions are expressed differently. Insulin increases the maximal response of DNA synthesis to EGF, whereas it decreases the concentration of EGF required for half-maximal stimulation of S6 phosphorylation. We conclude that S6 phosphorylation is not a principal regulator of DNA synthesis, and that insulin and EGF regulate both S6 phosphorylation and DNA synthesis through different, but interacting, pathways of action.     

Vasopressin rapidly stimulates NA entry and NA-K pump activity in quiescent cultures of mouse 3T3 cells

Addition of (Arg) vasopressin to quiescent cultures of Swiss 3T3 cells rapidly stimulates an ouabain-sensitive ⁸⁶Rb uptake. In contrast the hormone has no significant effect on the rate of efflux of this cation from preloaded cells. The stimulation of ⁸⁶Rb uptake is cycloheximide-insensitive, occurs within minutes of hormone addition and results from an increase in the Vmax of the uptake system. Vasopressin stimulates ion uptake in a concentration-dependent fashion (1-100 ng/ml); oxytocin also stimulated the Na-K pump but at significantly higher concentrations. The stimulation of the Na-K pump by vasopressin is apparently mediated by an increase in Na entry into the cells, since the hormone (1) strikingly shifts the concentration dependence on Na⁺ of the Na-K pump, (2) increases ²²Na uptake, and (3) increases intracellular Na contents when the efflux of this ion is blocked by ouabain. Since vasopressin is a potent mitogen for Swiss 3T3 cells, the results provide further evidence in support of a possible role of monovalent ion fluxes in signalling the initiation of growth stimulation.     

Stimulation of phosphatidylcholine breakdown and diacylglycerol production by growth factors in Swiss-3T3 cells.

The effect of a number of growth factors on phosphatidylcholine (PtdCho) turnover in Swiss-3T3 cells was studied. Phorbol 12-myristate 13-acetate (PMA), bombesin, platelet-derived growth factor (PDGF) and vasopressin rapidly stimulated PtdCho hydrolysis, diacylglycerol (DAG) production, and PtdCho synthesis. Insulin and prostaglandin F2 alpha (PGF2 alpha) stimulated PtdCho synthesis, but not its breakdown, whereas epidermal growth factor (EGF) and bradykinin were without effect. Stimulation of PtdCho hydrolysis by the above ligands resulted in increased production of phosphocholine and DAG (due to phospholipase C activity) and significant amounts of choline, suggesting activation of a phospholipase D as well. CDP-choline and glycerophosphocholine levels were unchanged. Down-regulation of protein kinase C with PMA (400 nM, 40 h) abolished the stimulation of PtdCho hydrolysis and PtdCho synthesis by PMA, bombesin, PDGF and vasopressin, but not the stimulation of PtdCho synthesis by insulin and PGF2 alpha. PtdCho hydrolysis therefore occurs predominantly by activation of protein kinase C (either by PMA or PtdIns hydrolysis) leading to elevation of DAG levels derived from non-PtdIns(4,5)P2 sources. PtdCho synthesis occurs by both a protein kinase C-dependent pathway (stimulated by PMA, PDGF, bombesin and vasopressin) and a protein kinase C-independent pathway (stimulated by insulin and PGF2 alpha). DAG production from PtdCho hydrolysis is not the primary signal to activate protein kinase C, but may contribute to long-term activation of this kinase.     

Insulin induces epidermal growth factor (EGF) receptor clustering and potentiates EGF-stimulated DNA synthesis in swiss 3T3 cells: a mechanism for costimulation in mitogenic synergy

In many cellular systems, activation with more than one ligand can produce a cellular response that is greater than the sum of the individual responses to the ligands. This synergy is sometimes referred to as coactivation. In Swiss 3T3 fibroblasts, activation of the epidermal growth factor (EGF) receptor produces a weak induction of DNA synthesis. Insulin has no stimulatory effect on this response. However, in combination, EGF and insulin synergize to cause a large induction of S phase. The underlying cellular biochemistry of this effect has been examined. The data indicate that phospholipase C activation is a major component of agonist-induced DNA synthesis. In contrast, activation of p70 S6 kinase by single agonists was inversely related to their ability to stimulate DNA synthesis. Therefore, it was examined whether stimulation of Swiss 3T3 cells with insulin causes changes in the subcellular distribution of EGF receptors and phospholipase Cgamma1 that could potentially explain the observed synergy or costimulation. It was found that insulin effectively induced the accumulation of EGF receptors on the actin arc of cells without activation of the EGF receptor. In contrast, EGF, when added for several hours, did not cause accumulation of the EGF receptor at this site. However, both EGF and insulin stimulated the accumulation of phospholipase Cgamma1 at the actin arc, which was coincident with the EGF receptor in the case of insulin- stimulated cells. Therefore, it is suggested that the insulin-induced coclustering of the EGF receptor with phospholipase Cgamma1 at the actin arc may allow for greater efficiency of signal transduction, resulting in the synergy observed for these two hormones in stimulation of DNA synthesis.     

On the mechanisms of the growth-promoting effect of prostaglandins in hepatocytes: The relationship between stimulation of DNA synthesis and signaling mediated by adenylyl cyclase and phosphoinositide-specific phospholipase C

While many observations indicate that prostaglandins may act as positive regulators of hepatocyte proliferation, the underlying mechanisms are not known. We have examined some of the signal pathways in the growth response induced by prostaglandins in hepatocytes, with particular focus on adenylyl cyclase and phosphoinositide-specific phospholipase C. Adult rat hepatocytes were cultured as primary monolayers in serum-free medium in the presence of EGF and insulin. PGE₂ or PGF_2α (added 0-3 h after plating) enhanced the incorporation of [³H]-thymidine into DNA (measured at 50 h); at 100 γM the stimulation was about threefold. PGI₂ and PGD₂ also showed significant but smaller stimulatory effects. No significant increase in the level of cyclic AMP (cAMP) was detected in response to any of the prostaglandins. Low concentrations of glucagon (0.1-10 nM), a potent activator of hepatic adenylyl cyclase, or 8-bromo-cAMP (0.1-10 γM) enhanced the DNA synthesis. When 8-bromo-cAMP was used in maximally effective concentrations, no further stimulation was obtained by combining it with glucagon, whereas the effects of PGE₂ and 8-bromo-cAMP were completely additive. All the prostaglandins also showed additivity with the effect of glucagon on the DNA synthesis. PGE₂, PGF_2α, PGI₂, and PGD₂ increased intracellular inositol-1,4,5-trisphosphate (InsP₃), with a relative order of efficacy roughly corresponding to their activity as stimulators of DNA synthesis. Increases in cytosolic free Ca²⁺, as measured in single cells, were elicited in a majority of the hepatocytes by all these prostaglandins at 1 γM. Supramaximal concentrations of vasopressin, a strong activator of phospholipase C in hepatocytes, acted additively with PGE₂ on the DNA synthesis. Pretreatment of the hepatocytes with a concentration of pertussis toxin that prevented the inhibitory effect of PGE₂ on glucagon-induced cAMP accumulation did not abolish the ability of PGE₂ to stimulate the DNA synthesis. The results do not support a role for adenylyl cyclase activation in the stimulatory effect of prostaglandins on hepatocyte growth. While the data are compatible with an involvement of phosphoinositide-specific phospholipase C in the growth-promoting effect of prostaglandins in cultured rat hepatocytes, they suggest this may not be the sole mechanism. © 1995 Wiley-Liss, Inc.     

Ionic responses rapidly elicited by porcine platelet-derived growth factor in Swiss 3T3 cells.

Addition of porcine platelet-derived growth factor (PDGF) to quiescent cultures of Swiss 3T3 cells caused a marked, dose-dependent stimulation of Na+ influx and Na-K pump-mediated 86Rb+ uptake. Porcine PDGF (a single component in SDS polyacrylamide gels) stimulated ion fluxes to the same maximal extent as partially purified preparations, and exhibited half-maximal effect at 6 ng/ml (2 X 10(-10) M). Maximal effect was achieved at 30 ng/ml (10(-9) M). In the presence of insulin, PDGF elicited mitogenesis at comparable concentrations. PDGF stimulated ion uptake in a time-dependent fashion; maximal effect was obtained after 5 min of exposure to the growth factor. PDGF stimulates Na+ influx via an amiloride-sensitive pathway, suggesting that PDGF enhances the activity of a Na+/H+ antiport system. In accordance with this possibility, the mitogen caused an increase of intracellular pH by 0.15 pH units, as judged by the steady-state distribution of labelled 5,5-dimethyloxazolidine-2,4-dione (DMO). Porcine PDGF stimulated E-type prostaglandin synthesis and cAMP accumulation but these events could be dissociated from the stimulation of the ionic fluxes, which was detected within minutes and was not blocked by indomethacin. It is suggested that PDGF elicits multiple signals to stimulate cell proliferation in 3T3 cells.     

Stimulation of hepatocyte DNA synthesis by neurotensin

Epidermal growth factor and transforming growth factor alpha stimulated DNA synthesis in primary cultures of adult rat hepatocytes. Neurotensin amplified epidermal growth factor-stimulated or transforming growth factor alpha-stimulated DNA synthesis by three- to eightfold. Neurotensin by itself did not stimulate DNA synthesis. Amplification of DNA synthesis by neurotensin was observed as low as 10^?10 M, and it was increased in a dose-dependent manner with maximal effects at 10^–8 M. These results were obtained when hepatocytes were cultured in Williams' medium E, but not in Leibovitz L-15 medium, suggesting that a minor component(s) in the medium is required for hepatocytes to fully respond to neurotensin. Neurotensin effect on DNA synthesis was observed not only in normal rat hepatocytes but also in partially hepatectomized rat hepatocytes, although its effect was stronger in normal hepatocytes. Amplified DNA synthesis was inhibited by transforming growth factor β. Secondary mitogens (co-mitogens) such as insulin, vasopressin, or angiotensin II interacted additively with low concentrations of epidermal growth factor as well as with neurotensin. Neurotensin-related peptides such as kinetensin or neuromedin-N, which was released from blood plasma by pepsin digestion, did not have this amplifying effect on DNA synthesis at any concentrations tested. Neurotensin mRNA was found in several organs including brain and intestine, but not liver. These results suggest that neurotensin can be regarded as a new secondary mitogen and that it may be involved in cell proliferation, including regenerating liver as a gastrointestinal hormone and/or a neurotransmitter. © 1994 Wiley-Liss, Inc.     

On the source of the vasopressin-induced increases in diacylglycerol in hepatocytes

In hepatocytes pre-labelled with [3H]glycerol, vasopressin increased by 20% the amount of radioactivity present in diacylglycerols. The effect of vasopressin was partially dependent on Ca2+. The magnitude of the increase in [3H]diacylglycerol was 5-times the sum of the radioactivity present in phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate. No stimulation by vasopressin of the initial rate of incorporation of radioactivity into diacylglycerols was observed in cells incubated in the presence of 10 mM [3H]glycerol. Treatment of hepatocytes labelled with either [3H]ethanolamine or [3H]choline with vasopressin, ionophore A23187 or phospholipase C increased the amount of radioactivity present in trichloroacetic acid extracts of the cells. The effect of vasopressin was dependent on extracellular Ca2+. It is concluded that in hepatocytes vasopressin increases diacylglycerols by a process which does not principally involve the conversion of phosphoinositides to diacylglycerol or the de novo synthesis of diacylglycerol from glycerol 3-phosphate, but does involve the Ca2+-dependent conversion of phosphatidylethanolamine and phosphatidylcholine to diacylglycerol.     

Growth-promoting effects of Ca2+-mobilizing agents in hepatocytes: Lack of correlation between the acute activation of phosphoinositide-specific phospholipase C and the stimulation of DNA synthesis by angiotensin II,vasopressin, norepinephrine,and prostaglandin F2α

Although several hormones that promote hepatocyte proliferation also activate phosphoinositide-specific phospholipase C (PI-PLC) and mobilize Ca²⁺, the role of PI-PLC in the growth-stimulating effect of these agents is not clear. We have investigated this issue further, by exposing freshly isolated adult rat hepatocytes to vasopressin, angiotensin II, norepinephrine (in the presence of the β-adrenoceptor blocker timolol) or PGF_2α, and examined both acute responses and the subsequent DNA synthesis when the cells were grown in monolayer culture. All the agonists elevated the level of inositol 1,4,5-trisphosphate (InsP₃) and enhanced the DNA synthesis, amplifying the response to epidermal growth factor (EGF), and this comitogenic effect could be exerted by a single exposure of the cells 24 h prior to the addition of EGF. The acute activation of PI-PLC, measured as the early rise (peak 15–60 s) in InsP₃, was 8–10-fold with vasopressin or angiotensin II, 3–4-fold with norepinephrine, and ∼︁2-fold with PGF_2α. For all the agonists, a rise in cytosolic free Ca²⁺ in 100% of the cells and a maximal increase in glycogen phosphorylase activity were evoked at concentrations that approximately doubled the level of InsP₃. However, the growth-stimulatory effects of these agonists showed a different order of efficacy as compared to the activation of PI-PLC; in terms of the maximal stimulation of DNA synthesis, the effects were: norepinephrine ≈︂ PGF_2α > angiotensin II > vasopressin. Also, norepinephrine, PGF_2α, and angiotensin II, but not vasopressin, further enhanced the DNA synthesis when their concentrations were increased above those yielding maximal elevation of InsP₃. In experiments where vasopressin and angiotensin II were combined, their effects on the DNA synthesis were additive while the InsP₃ responses were not. The results show that the extent of the initial activation of PI-PLC is not the determinant for the magnitude of the growth effects of Ca²⁺-mobilizing hormones in hepatocytes. This suggests either (a) that the proliferative response to these agents is determined by the activity of PI-PLC at a later time, or its integral over an extended part of the prereplicative period, rather than by the acute activation, or (b) that additional, PI-PLC-independent, mechanisms are required. © 1996 Wiley-Liss, Inc.     

Synergistic stimulation of DNA synthesis by bradykinin and vasopressin in swiss 3T3 cells

Vasopressin and bradykinin bind to receptors coupled to GTP-binding proteins and rapidly induce polyphosphoinositide breakdown leading to Ca²⁺ mobilization and activation of protein kinase C. Both peptides are known to induce mitogenesis in the presence of growth factors that act through receptors with intrinsic tyrosine kinase activity. Surprisingly, addition of a combination of vaso-pressin and bradykinin to Swiss 3T3 cells synergistically stimulates DNA synthesis in the absence of any other growth factors. This effect is induced at nanomolar concentrations of the peptides and could be inhibited by addition of specific receptor antagonists or broad spectrum neuropeptide antagonists. Bradykinin, which stimulates transient activation of protein kinase C, induces DNA synthesis in synergy with substances that cause long-term activation of protein kinase C, like vasopression or phorbol 12, 13-dibutyrate. Down-regulation of protein kinase C inhibited the induction of mitogenesis by the combination of vasopressin and bradykinin, thus demonstrating the importance of long-term activation of this enzyme for DNA synthesis. Analysis of tyrosine phosphorylated proteins of M_r = 110,000–130,000 and M_r = 70,000–80,000 revealed a biphasic response after stimulation with bradykinin, whereas the response induced by vasopressin declined after the initial maximum. The combination of bradykinin with vasopressin caused an enhanced and prolonged increase in tyrosine phosphorylation of these proteins as compared with the individual peptides. Inhibition of tyrosine phosphorylation by tyrphostin was paralleled by inhibition of DNA synthesis. Together, these results demonstrate synergistic stimulation of DNA synthesis by bradykinin and vasopressin via prolonged stimulation of multiple signaling pathways and imply that the interactive effects of Ca²⁺ -mobilizing peptides on mitogenesis may be more general than previously thought. © 1994 Wiley-Liss, Inc.     

Thrombin induces proliferation of osteoblast-like cells through phosphatidylcholine hydrolysis

We examined the effect of thrombin on phosphatidylcholine-hydrolyzing phospholipase D activity in osteoblast-like MC3T3-E1 cells. Thrombin stimulated the formation of choline dose dependently in the range between 0.01 and 1 U/ml, but not the phosphocholine formation. Diisopropylfluorophosphate (DFP)-inactivated thrombin had little effect on the choline formation. The combined effects of thrombin and 12-O-tetradecanoylphorbol-13-acetate, a protein kinase C-activating phorbol ester, on the choline formation were additive. Staurosporine, an inhibitor of protein kinases, had little effect on the thrombin-induced formation of choline. Combined addition of thrombin and NaF, an activator of heterotrimeric GTP-binding protein, did not stimulate the formation of choline further. Pertussis toxin had little effect on the thrombin-induced formation of choline. Thrombin stimulated Ca²⁺ influx from extracellular space time and dose dependently. The depletion of extracellular Ca²⁺ by EGTA exclusively reduced the thrombin-induced choline formation. Thrombin had only a slight effect on phosphoinositide-hydrolyzing phospholipase C activity. Thrombin induced diacylglycerol formation and DNA synthesis, and increased the number of MC3T3-E1 cells, but DFP-inactivated thrombin did not. Thrombin suppressed both basal and fetal calf serum-induced alkaline phosphatase activity in these cells. Propranolol, an inhibitor of phosphatidic acid phosphohydrolase, inhibited both the thrombin-induced diacylglycerol formation and DNA synthesis. These results suggest that thrombin stimulates phosphatidylcholine-hydrolyzing phospholipase D due to self-induced Ca²⁺ influx independently of protein kinase C activation in osteoblast-like cells and that its proliferative effect depends on phospholipase D activation. © 1996 Wiley-Liss, Inc.     

Insulin and prolactin synergistically stimulate beta-casein messenger ribonucleic acid translation by cytoplasmic polyadenylation

Previous studies have shown that the synthesis and stability of milk protein mRNAs are regulated by lactogenic hormones. We demonstrate here in cultured mouse mammary epithelial cells (CID 9) that insulin plus prolactin also synergistically increases the rate of milk protein mRNA translation. Insulin alone stimulates synthesis of both milk and nonmilk proteins, whereas prolactin alone has no effect, but insulin plus prolactin selectively stimulate synthesis of milk proteins more than insulin alone. The increase in beta-casein mRNA translation is also reflected in a shift to larger polysomes, indicating an effect on translational initiation. Inhibitors of the phosphatidylinositol 3-kinase, mammalian target of rapamycin, and MAPK pathways block insulin-stimulated total protein and beta-casein synthesis but not the synergistic stimulation. Conversely, cordycepin abolishes synergistic stimulation of protein synthesis without affecting insulin-stimulated translation. The poly(A) tract of beta-casein mRNA progressively increases from approximately 20 to about 200 A residues over 30 min of treatment with insulin plus prolactin. The 3'-untranslated region of beta-casein mRNA containing an unaltered cytoplasmic polyadenylation element is sufficient for the translational enhancement and mRNA-specific polyadenylation, based on transient transfection of cells with a reporter construct. Insulin and prolactin stimulate cytoplasmic polyadenylation element binding protein phosphorylation with no increase of cytoplasmic poly(A) polymerase activity.     

A partially purified serum fraction synergistically enhances the mitogenic activity of epidermal growth factor and insulin in quiescent cultures of 3T3 cells.

Epidermal growth factor and insulin need a low concentration of serum to effectively stimulate quiescent 3T3 cells into DNA synthesis. We partially purify a polypeptide component of serum which has no activity by itself but which acts synergistically with epidermal growth factor and insulin to stimulate cultures of 3T3 cells into DNA synthesis as effectively as whole serum. The active fraction is separated from serum by gel chromatography on Sephadex G-100, under acid dissociating conditions, and chromatographs with a molecular weight of 18,000 daltons.We suggest as a general technique, the use of pure growth factors to assay for growth promoting fractions from serum or other sources. Fractions which are not mitogenic by themselves can be detected when assayed together with their complementary pure factors.     

Insulin-stimulated phosphoinositide metabolism in isolated fat cells

Treatment of isolated fat cells with insulin produced increases of up to 4.8-fold in the incorporation of [3H]inositol into phosphatidylinositol. This effect of insulin was both time- and dose-dependent with half-maximal stimulation at 30 microunits/ml of insulin. Insulin increased the labeling of phosphatidylinositol and phosphatidylinositol 4,5-bisphosphate but not phosphatidylinositol 4-monophosphate in cells which had been preincubated with [3H]inositol for 90 min. Incubation of the cells in a Ca2+-free buffer increased the basal level of phosphatidylinositol labeling and enhanced the effect of insulin. Glucagon and isoprenaline, both of which stimulate lipolysis, had no effect on phosphatidylinositol labeling but did potentiate insulin-stimulated incorporation of [3H]inositol into phosphatidylinositol. Phosphoinositide breakdown was measured by the accumulation of inositol phosphates. Insulin did not increase the level of the inositol phosphates at all concentrations of the hormone tested. By comparison, phenylephrine and vasopressin were able to stimulate phosphoinositide breakdown. Pretreatment of the cells with insulin enhanced the effect of phenylephrine on inositol phosphates' accumulation, suggesting that insulin may potentiate phenylephrine-mediated phosphoinositide turnover. From these data we conclude that insulin stimulates the de novo synthesis of phosphatidylinositol and phosphatidylinositol 4,5-biphosphate, but has no effect on phosphoinositide breakdown.     

Phorbol esters inhibit alpha1 adrenergic stimulation of glycogenolysis in isolated rat hepatocytes

Tumor promoting phorbol esters can stimulate Ca⁺⁺-phospholipid-dependent protein kinase. It has been suggested that this enzyme may mediate the effects of calcium-dependent hormones. In this paper the effects of phorbol 12-myristate 13-acetate (TPA) on isolated rat hepatocyte metabolism were studied. Phorbol esters completely blocked alpha₁-adrenergic stimulation of glycogenolysis. This effect is quite specific for alpha₁-adrenergic actions, as the stimulations of glycogenolysis by vasopressin, angiotensin II, ionophore A-23187 and glucagon were unaffected by TPA. The potencies of the different phorbol esters used in this study suggests that the inhibitory effects of these agents may be due to activation of protein kinase C. The effect of phorbol esters on alpha₁-adrenergic actions seems to occur at an early step of the alpha₁-adrenergic action. TPA (10^?11–10^?6M) was unable to stimulate glycogenolysis. Urea synthesis, which is stimulated by vasopressin and alpha₁-adrenergic agents, was not stimulated by phorbol ester, neither alone nor in combination with the Ca⁺⁺ ionophore A-23187.     

Inorganic phosphate is necessary for the stimulation of DNA synthesis in swiss 3T3 cells by pure mitogenic agents

We compared the ability of dialysed fetal bovine serum and of combinations of purified growth-promoting factors such as insulin, epidermal growth factor (EGF), vasopressin, fibroblast-derived growth factor and antitubulin agents to stimulate DNA synthesis in 3T3 cells maintained in the absence or presence of inorganic phosphate (P_i). When DNA synthesis was stimulated by serum in the absence of P_i the level induced was 70% of that observed in P_i-containing medium. In contrast, combinations of growth-promoting factors in the absence of P_i stimulated less than 8% of the DNA synthesis which they induced in complete medium. Addition of as little as 50 μM P_i fully restored the ability of the factors to stimulate DNA synthesis. Cells stimulated by purified mitogens in the absence of P_i became blocked in early G1, and for up to 48 h the block was reversible by readdition of p_i. The effectiveness of dialysed serum to stimulate DNA in the absence of P_i suggest that dialysed serum might contain a component capable of supplying P_i to support DNA synthesis. Indeed, delipidization of serum by solvent extraction resulted in loss of ability to stimulate DNA synthesis in the absence of p_i, but delipidized serum stimulated DNA synthesis virtually, as well as dialysed serum in the presence of P_i. Previous conclusions suggesting that P_i is not essential for DNA synthesis appear to require re-evaluation.     

Inhibition of mechanical strain-induced fetal rat lung cell proliferation by gadolinium,a stretch-activated channel blocker

Normal growth of the fetal lung is dependent upon fetal breathing movements. We have previously demonsrated that mechanical strain, simulating fetal breathing movements, stimulated DNA synthesis and cell division by reaggregated alveolar-like structures of fetal rat lung cells. Herein, we report that both intracellular and extracellular calcium modulate strain-induced proliferative activity. Strain-induced cell proliferation was inhibited by BAPTA/AM, an intracellular calcium chelator. The intracellular calcium modulators, cyclopiazonic acid and 2,5-di-(tert-butyl)-1, 4-benzohydroquinone, increased DNA synthesis of unstrained cultures and partially reduced strain-induced cell growth activity. A similar effect was noted with the calcium ionophore A23187. Extracellular Ca²⁺ increased DNA synthesis in unstrained cultures in a concentration-dependent fashion. The stimulatory effect of strain on DNA synthesis was also dependent on the calcium concentration in the medium. Furthermore, strain-enhanced DNA synthesis was inhibited by the presence of a divalent ion chelator, EGTA, in the medium. Mechanical strain increased ⁴⁵Ca²⁺ influx within 1 min after the onset strain. This rapid entry of calcium was not affected by calcium channel blockers, such as verapamil or Ni²⁺. Calcium channel blockers verapamil, nifedipine, Ni²⁺, Co²⁺, or La³⁺ also did not inhibit strain-induced cell growth activity. In contrast, gadolinium, a stretch-activated channel blocker, inhibited strain-induced ⁴⁵Ca²⁺ influx and suppressed strain-enhanced DNA synthesis. We conclude that the entry of calcium into cells through stretch-activated ion channels plays a critical role in strain-induced fetal lung cell proliferation. © 1994 Wiley-Liss, Inc.