Growth factor-stimulated phosphorylation of Akt and p70(S6K) is differentially inhibited by LY294002 and Wortmannin.

The phosphoinositide 3-kinase (PI3K) inhibitors, LY294002 (LY) and wortmannin (WM), are widely used to examine the role of PI3K in growth factor signaling. These compounds inhibit the kinase action of PI3K, thus preventing the accumulation of PI(3,4,5)P3 and PI(3,4)P2 (PIs) and subsequent phosphorylation and activation of the downstream effectors of PI3K, Akt and p70(S6K). The efficacy of these inhibitors has been demonstrated by measuring cellular levels of PIs or the kinase activity of immunoprecipitated PI3K. However, their effects on activation of Akt and p70(S6K), more widely used markers of PI3K activation, has not been formally tested. We have examined the effects of LY and WM on phosphorylation of Akt and p70(S6K) by insulin-like growth factor-I, insulin, and platelet-derived growth factor in skeletal muscle cells. LY is much less effective in blocking the phosphorylation of Akt than p70(S6K); at concentrations which completely inhibit phosphorylation of p70(S6K), phosphorylation of Akt is only partially inhibited by LY. WM also inhibits IGF-I-stimulated phosphorylation of Akt and p70(S6K) with unequal potency but is equally effective in blocking insulin-stimulated phosphorylation of these peptides. Our data demonstrate that inhibiting PI3K signaling through one of its downstream mediators (p70(S6K)) may not indicate complete blockage of the PI3K pathway which may be signaling through an alternate downstream branch (Akt). These findings indicate that the efficacy of LY and WM in blocking PI3K-activation of Akt and p70(S6K) must be tested within the context of every experiment, and that the results obtained with the use of these inhibitors must be interpreted according to their specific effects on the PI3K/Akt and PI3K/p70(S6K) signaling pathways.

Functional inactivation of the IGF-I receptor delays differentiation of skeletal muscle cells.

Skeletal myoblasts are inherently programmed to leave the cell cycle and begin the differentiation process following removal of exogenous growth factors. Serum withdrawal results in a marked induction of IGF production which is essential for skeletal muscle differentiation in vitro. However, the potential role of the tyrosine kinase IGF-I receptor (thought to be the principal mediator of both IGF-I and II signaling in skeletal muscle) in the decision of myoblasts to begin differentiation following serum withdrawal is unknown. To explore the role of the IGF-I receptor in this decision by skeletal myoblasts, we functionally inactivated endogenous IGF-I receptors in mouse C2C12 cells using a dominant negative, kinase-inactive IGF-I receptor in which the ATP-binding site lysine (K) at residue 1003 has been mutated to alanine (A). Cell lines with the greatest degree of mutant IGF-I receptor expression (A/K cells) demonstrated functional inactivation of endogenous IGF-I receptors as determined by their impaired ability to phosphorylate the principal substrate of the IGF-I receptor, IRS-1, in response to treatment with IGF-I. In addition, the proliferative response of myoblasts to IGF-I was completely abolished in A/K cells. Following withdrawal of exogenous growth factors, A/K cells demonstrated a marked delay in the induction of the gene expression of myogenin, a skeletal muscle-specific transcription factor essential for differentiation, and a subsequent delay in the induction of muscle creatine kinase activity. Delayed differentiation in A/K cells was associated with prolonged phosphorylation of the cell cycle regulatory retinoblastoma (Rb) protein; it is the un- (or hypo-) phosphorylated form of Rb which is known to promote differentiation in skeletal myoblasts. Thus, the IGF-I receptor regulates the timing of myoblast differentiation induced by serum withdrawal. The delayed differentiation of skeletal myoblasts with functionally inactive IGF-I receptors may result, at least in part, from delayed induction of myogenin gene expression and prolonged phosphorylation of the Rb protein.

Opposing early inhibitory and late stimulatory effects of insulin-like growth factor-I on myogenin gene transcription.

Insulinlike growth factors (IGFs) stimulate skeletal muscle cell differentiation in association with an increase in the mRNA of myogenin, a member of the MyoD family of skeletal muscle-specific transcription factors that plays an essential role in the differentiation process. However, this is a relatively late effect, requiring treatment periods of >24 h. In contrast, IGFs initially inhibit skeletal muscle cell differentiation, associated with a marked reduction in myogenin mRNA. The mechanisms by which IGF-I initially inhibits and subsequently stimulates myogenin expression are unknown. In the first 24 h, we find that IGF-I inhibits myogenin gene transcription by >80% but has no effect on myogenin mRNA stability. Similarly, in the first 24 h, IGF-I markedly inhibits myogenin promoter activity; the sequence -145 to -9 of the myogenin gene is sufficient to confer this inhibitory effect of IGF-I. In contrast, 48 h of treatment with IGF-I results in an increase in myogenin promoter activity that parallels the increase in myogenin steady-state mRNA. This increase in promoter activity is completely prevented in constructs lacking the sequence -1,565 to -375 of the myogenin gene. These data indicate that the early inhibitory and late stimulatory effects of IGF-I on myogenin expression are mediated at the level of transcription, and that these time-dependent, opposing effects of IGF-I on myogenin transcription are mediated by distinct regions of the myogenin gene. To our knowledge, this is the first demonstration of a gene whose promoter activity is initially inhibited and subsequently stimulated by IGF-I.

Effects of d-amphetamine on the frequency of the spontaneous contraction of the portal vein in rat.

1. Effects of d-amphetamine on the frequency of spontaneous contraction of the longitudinal muscle of the portal vein were studied in Wistar rats. Its effects on the circular muscles of the pulmonary artery and stomach also were tested. 2. d-Amphetamine increased the frequency of the spontaneous contraction of the portal vein. The ratio of the frequency of the spontaneous contraction of the portal vein before and after d-amphetamine treatment also was increased. The effect was not affected in the presence of prazocin, (+)-lysergic acid diethylamide, atropine and haloperidol. These results that the d-amphetamine-elicited response was not due mainly to the activation of adrenergic, serotoninergic (5-HT), cholinergic or dopaminergic receptors. 3. Increasing extracellular calcium or sodium ion concentrations decreased the frequency of the spontaneous contraction of the portal vein. However, the ratios of the frequency of the spontaneous contraction of the rat portal vein before and after d-amphetamine treatment in media containing different extracellular calcium or sodium concentrations were not significantly altered. Tetrodotoxin did not alter the effect of d-amphetamine on the frequency of spontaneous contractions. It appeared that calcium and sodium ions may not take part in the effects of d-amphetamine on the frequency of the portal vein. 4. An increase in extracellular potassium ion concentrations increased the frequency of the spontaneous contractions of the portal vein. In addition, the ratios of the frequency of the spontaneous contractions of the rat portal vein before and after d-amphetamine treatment in media containing different extracellular potassium ion concentrations were significantly altered. Tetraethylammonium chloride (TEA) and 4-aminopyridine (4-AP) increased the spontaneous contractions of the portal vein. However, TEA and 4-AP did not increase the d-amphetamine-elicited increasing effect on the frequency of spontaneous muscle contractions. 5. Levochromakalim, a potassium channel opener, decreased the frequency of the spontaneous contractions of the portal vein. Levochromakalim also decreased the effect of d-amphetamine on the frequency of spontaneous contractions of the muscle. It appeared that potassium ion may be associated with the effects of d-amphetamine on the activity of the portal vein. 6. d-Amphetamine potentiated, whereas prazosin decreased, the noradrenaline-elicited contracture of the rat pulmonary artery in a dose-dependent manner. 7. d-Amphetamine elicited contracture of the circular muscle of rat stomach, whereas it did not alter the frequency of the spontaneous contraction of the muscle. 8. Both 5-HT and d-amphetamine elicited the contracture of the circular muscle of rat stomach. Ketanserin decreased the 5-HT-elicited response, whereas it did not alter the d-amphetamine-elicited response in the muscle. d-Amphetamine did not alter the frequency of the spontaneous contraction of the stomach. 9. It is concluded that d-amphetamine has different effects on the frequency of spontaneous smooth muscle contractions. It increased the frequency in the portal vein, but it did not alter the frequency in stomach circular muscle.

Regulation of pathways of extramitochondrial fatty acid oxidation and liver fatty acid-binding protein by long-chain monocarboxylic fatty acids in hepatocytes. Effect of inhibition of carnitine palmitoyltransferase I.

The regulation of the extramitochondrial fatty acid oxidation pathways located in the peroxisomes and the endoplasmic reticulum is not fully understood. Although both long-chain dicarboxylic fatty acids, which are poorly metabolized in hepatocytes, and non-beta-oxidizable fatty acid analogs induce peroxisomal beta-oxidation and liver fatty acid-binding protein (L-FABP) by a pretranslational mechanism, monocarboxylic long-chain fatty acids, which are rapidly esterified and oxidized, do not. To establish whether impaired utilization and, hence, sustained intracellular levels of monocarboxylic long-chain fatty acids increase their efficacy as inducers, the effect of oleic acid on cytochrome P-450 4A1, peroxisomal beta-oxidation, and L-FABP during inhibition of mitochondrial beta-oxidation was determined. In primary hepatocyte cultures, oleic acid had no inducing effect, but in the presence of 2-tetradecylglycidic acid (TDGA), an inhibitor of carnitine palmitoyltransferase I, it induced P-450 4A1, peroxisomal beta-oxidation, and L-FABP pretranslationally. An increase in peroxisomal beta-oxidation was also noted in the presence of etomoxir, another inhibitor of carnitine palmitoyltransferase I. Exposure of hepatocytes to TDGA for 1 h led to an expected decrease in incorporation of radiolabel from [1-14C]oleate into CO2 and water-soluble products. In contrast, long-term exposure to TDGA increased incorporation of [1-14C]oleate into oxidation products, most likely due to an adaptive induction of peroxisomal beta-oxidation. Both acute and long-term exposure of hepatocytes to TDGA decreased incorporation of oleic acid into triglycerides, an effect that may have contributed to the intracellular accumulation of fatty acids. These results provide support for a mechanism by which long-chain fatty acids or specific metabolites, including long-chain acyl-CoA esters and long-chain dicarboxylic acids, act as signals in the induction of P-450 4A1, peroxisomal beta-oxidation, and L-FABP under conditions in which long-chain fatty acids accumulate due to impaired entry into the mitochondrial beta-oxidation pathway.

[The stress distribution of prefabricated screw posts. Part III: Techniques].

Some commonly used endodontic screw posts were analysed for various techniques of post insertion by photoelastic stress distribution. These endodontic dowels were installed in assumed clinical conditions. These conditions included: (1) no backing off during post installation, (2) the channelling of the post without cleaning, (3) a smaller drill was used prepare the post channel, and (4) an axis deviated contrangle was used to prepare the post channel. According to this photoelastic analysis, the techniques of post insertion may affect the stress distribution. Suggestions for post insertion are as follows: 1. The stress can be reduced by counterrotating the dowel one-fourth turn after resistance to installation has been detected. 2. The channel should be cleaned thoroughly before post insertion. 3. Because accurate post fit is important to maximize both retention and support, the screw post should be used with the matched post/reamer system; and accurate contrangle drilling should be maintained.

[Stress distribution of prefabricated screw posts. Part I: Post size].

A commonly used prefabricated screw post was analysed for its various lengths and diameters. When endodontic dowels were installed in standardized models, direct comparisons of stress distributing properties were analysed through photoelastic stress analysis. According to the analysis, installation of the post produced severe lateral stress. At shorter lengths, these stress concentrations were much more severe. However, increasing the diameter of the dowel may reduce the stress slightly. Under load conditions, the stress-producing characteristics of the dowel increased with shorter lengths and smaller diameters. It seems that increasing the surface area of insertion with the dentin improves the distribution of the load caused by the insertion of the post.

Phase transition in a lipid bilayer. II. Influence of adamantane derivatives.

The influence of thirty-four adamantane, protoadamantane, and homoadamantane derivatives on the phase transition characteristics of the bilayer in dipalmitoyl lecithin liposomes has been determined by differential scanning calorimetry. Each of these compounds induces a broadening of the phase transition profile of the lipid bilayer that is dependent upon the concentration of the solute and its molecular structure. The concentration--response curves obtained for these solutes suggest that the cage compound derivatives modify the phase properties and under some conditions may induce a phase separation in the doped bilayer. The relative activity sequences obtained for the compounds examined cannot be accounted for by simple considerations of lipid/water partition coefficients, substitution constants based on free energy relationships, or the relative polarities or sizes of substituent groups. The observations are consistent with the hypothesis that the position and orientation of a solute within the bilayer are critical factors in determining its relative potency. The position of a solute within the bilayer is significantly controlled by the presence of polar substituents and by the relative geometric relationships of these groups. For a given substituent group, the shape and size of the hydrocarbon cage becomes increasingly important. It is apparent that seemingly minor modifications in the structure of a solute can significantly alter its influence on the phase transition behavior of a bilayer.