Phosphorylation of the translational repressor PHAS-I by the mammalian target of rapamycin.

The immunosuppressant rapamycin interferes with G1-phase progression in lymphoid and other cell types by inhibiting the function of the mammalian target of rapamycin (mTOR). mTOR was determined to be a terminal kinase in a signaling pathway that couples mitogenic stimulation to the phosphorylation of the eukaryotic initiation factor (eIF)-4E-binding protein, PHAS-I. The rapamycin-sensitive protein kinase activity of mTOR was required for phosphorylation of PHAS-I in insulin-stimulated human embryonic kidney cells. mTOR phosphorylated PHAS-I on serine and threonine residues in vitro, and these modifications inhibited the binding of PHAS-I to eIF-4E. These studies define a role for mTOR in translational control and offer further insights into the mechanism whereby rapamycin inhibits G1-phase progression in mammalian cells.

Mutational analysis of sites in the translational regulator, PHAS-I, that are selectively phosphorylated by mTOR.

Results obtained with PHAS-I proteins having Ser to Ala mutations in the five known phosphorylation sites indicate that mTOR preferentially phosphorylates Thr36 and Thr45. The effects of phosphorylating these sites on eIF4E binding were assessed in a far-Western analysis with a labeled eIF4E probe. Phosphorylation of Thr36 only slightly attenuated binding of PHAS-I to eIF4E, while phosphorylation of Thr45 markedly inhibited binding. Phosphorylation of neither site affected the electrophoretic mobility of the protein, indicating that results of studies that rely solely on a gel-shift assay to assess changes in PHAS-I phosphorylation must be interpreted with caution.

Mucosa-dependent muscarinic liberation of prostaglandins from rat isolated trachea.

1. The present study examined whether cholinoceptor stimulation modulates the release of arachidonic acid-derived mediators from rat isolate tracheae. 2. Tracheae were preincubated with [3H]-arachidonic acid and the outflow of 3H-compounds was determined. Acetylcholine and the muscarinic agonist, carbachol but not nicotine, increased the rate of tritium outflow maximally by about 30%. The M3 receptor-preferring antagonist rho-fluoro-hexahydrosiladiphenidol was more effective than pirenzepine and methoctramine in antagonizing the effect of acetylcholine. 3. High performance liquid chromatography analysis (methanol gradient) of the released 3H-compounds showed that one peak, co-eluting with [14C]-prostaglandin E2([14C]-PGE2) and [3H]-PGD2, was enhanced almost 10 fold following muscarinic receptor activation, whereas the outflow of [3H]-arachidonic acid remained unaffected. 4. Using an acetonitril gradient separation it was shown that [3H]-PGE2, [3H]-PGD2 and [3H]-PGF2alpha are released spontaneously, but [3H]-PGE2 represented the major fraction of 3H-prostaglandins. Acetylcholine enhanced the release of all three 3H-prostaglandins, but the effect on PGE2 was most pronounced and most consistent. 5. After removal of the mucosa the muscarinic effect of acetylcholine on total tritium and on that of the 3H-prostaglandins ([3H]-PGE2/PGD2 peak) was abolished. 6. Acetylcholine also enhanced the outflow of radioimmunologically determined PGE2 in a mucosa-dependent manner. 7. After inhibition of cyclo-oxygenase by 3 microM indomethacin, the outflow of 3H-prostaglandins was reduced to almost undetectable levels and acetylcholine evoked a marked release [3H]-arachidonic acid. The phospholipase A2 inhibitor, quinacrine (up to 100 microM) also blocked the effect of acetylcholine on the outflow of 3H-prostaglandins, but this was not followed by a compensatory increase in the outflow of [3H]-arachidonic acid. 8. In conclusion, activation of muscarinic receptors which have characteristics of the M3 subtype can evoke release of prostaglandins from the airway mucosa.

Characterization of endogenous noradrenaline release from intact and epithelium-denuded rat isolated trachea.

1. Overflow of endogenous noradrenaline (NA) from the in vitro incubated rat trachea evoked by two periods of electrical field stimulation (S1, S2 at 3 or 15 Hz) or by high potassium (60 mM) was determined by high performance liquid chromatography (h.p.l.c.) with electrochemical detection. 2. In the presence of the neuronal uptake inhibitor desipramine, the alpha 2-adrenoceptor antagonist, yohimbine, enhanced the overflow of NA evoked by stimulation at 3 Hz by about 100% suggesting the presence of presynaptic inhibitory autoreceptors on the sympathetic nerves innervating the trachea. 3. When desipramine and yohimbine were present throughout the experiments, the overflow of NA evoked by the second period of electrical stimulation (S2) was significantly smaller than that evoked by the first (S1). This decline of overflow was prevented when the NA precursor, tyrosine, was additionally present throughout the experiments. 4. After removal of the epithelium, the tissue content of NA was reduced by about 30%, suggesting that part of the NA may be present and released within the epithelium. However, the overflow of NA evoked by stimulation at 3 Hz or 15 Hz was reduced by 70-80%, indicating that the epithelium may additionally exert a permissive role on the release of NA within the airways, possibly by suppressing inhibitory factors. 5. Stimulation by high potassium (60 mM for 10 min) caused a large overflow of NA (about 45% of the tissue NA), both from epithelium-free and epithelium-denuded tracheae. Thus the 'endogenous inhibition' of NA release after removal of the epithelium is surmountable when a high potassium stimulus is applied.

Beta-adrenoceptors mediate inhibition of [3H]-acetylcholine release from the isolated rat and guinea-pig trachea: role of the airway mucosa and prostaglandins.

1. Rat or guinea pig isolated tracheae were labelled with [3H]-choline to measure evoked tritium outflow, which reflects neuronal release of [3H]-acetylcholine. Tritium outflow was evoked either by electrical stimulation of the extrinsic vagal nerve (rat tracheae) or by 27 mM potassium (guinea pig tracheae). 2. In rat tracheae isoprenaline (0.01, 0.1 microM) inhibited evoked [3H]-acetylcholine release, whereas beta 2-adrenoceptor-selective agonists (fenoterol, formoterol, salbutamol) were ineffective. 3. The inhibitory effect of isoprenaline was abolished under the following conditions: (i) presence of propranolol (1 microM) or of the beta 1-selective antagonist CGP 20712 A (0.1 microM); (ii) removal of the mucosa at the start of the experiments; (iii) blockade of cyclooxygenase activity by 3 microM indomethacin. 4. In rat isolated tracheae prelabelled with [3H]-arachidonic acid, isoprenaline (0.1 microM) but not formoterol (0.01 microM) enhanced the outflow of [3H]-prostaglandins (PGD2, PGE2). This effect was blocked by 0.1 microM CGP 20712 A. 5. In guinea pig tracheae electrical stimulation of the extrinsic vagal nerve did not cause a constant release of [3H]-acetylcholine, but 27 mM potassium elicited a reproducible release of [3H]-acetylcholine. In this species both isoprenaline (0.1 microM) and formoterol (0.01 microM) inhibited evoked [3H]-acetylcholine release. Inhibition was abolished under the following conditions: (i) presence of propranolol (1 microM) or of the beta 2-selective antagonist ICI 118551 (0.3 microM); (ii) removal of the mucosa at the start of the experiments; (iii) blockade of cyclooxygenase activity by 3 microM indomethacin. 6. In conclusion, the present experiments have demonstrated that activation of beta-adrenoceptors localized in the mucosa mediates inhibition of [3H]-acetylcholine release from the neuroeffector junctions of the pulmonary, parasympathetic nerves most probably by the liberation of inhibitory prostaglandins from the airway mucosa. The adrenoceptor subtype involved differs in rat (beta 1 subtype) and guinea pig (beta 2 subtype) airways.

Cellular pharmacology of D-3-azido-3-deoxy-myo-inositol, an inhibitor of phosphatidylinositol signaling having antiproliferative activity.

D-3-Azido-3-deoxy-myo-inositol (3AMI) is an inhibitor of the growth of v-sis-transformed NIH 3T3 cells but not of wild-type NIH 3T3 cells, whose effects may be mediated through the phosphatidylinositol-3'-kinase pathway. We studied some properties of the cellular pharmacology of 3AMI using high-specific-activity [3H]-3AMI. The uptake of [3H]-3AMI by wild-type NIH 3T3 and v-sis NIH 3T3 cells was similar. [3H]-3AMI was a substrate for phosphatidylinositol synthetase, with the maximal velocity (Vmax) being 1.0 nmol min-1 mg-1 and the Michaelis constant (Km) being 23 mM. Corresponding values obtained for [3H]-myo-inositol as a substrate were 5.5 nmol min-1 mg-1 and 3.2 mM. [3H]-3AMI was incorporated into the cellular inositol lipids of v-sis NIH 3T3 cells to a similar extent as that observed for [3H]-myo-inositol but was not incorporated into the inositol lipids of wild-type NIH 3T3 cells. The [3H]-3AMI incorporated by the v-sis NIH 3T3 cells was present in the phosphatidylinositol and phosphatidylinositol phosphate fractions but not in bisphosphorylated phosphatidylinositol. myo-Inositol antagonized the growth-inhibitory effects of 3AMI. The v-sis NIH 3T3 cells were found to be more sensitive than the wild-type NIH 3T3 cells to growth inhibition (without 3AMI) caused by the removal of myo-inositol from the medium. The results of the study suggest that 3AMI is an antimetabolite of myo-inositol. The relative sensitivity of v-sis NIH 3T3 and some other cells to 3AMI may be a reflection of increased myo-inositol requirements for the growth of these cells as compared with wild-type NIH 3T3 cells.

Endogenous nitric oxide inhibits leukotriene B4 release from rat alveolar macrophages.

Effects of endogenous nitric oxide (NO) on the release of mediators of the lipoxygenase and cyclo-oxygenase pathway from rat alveolar macrophages were studied. Alveolar macrophages, freshly isolated or after 18-h culture, were incubated in (amino acid-free) Krebs medium and labelled with [3H]arachidonic acid. The release of [3H]leukotriene B4 and [3H]prostanoids (separated by high performance liquid chromatography) was determined. A 23187 was used as stimulus, as rising intracellular Ca2+ activates directly the phospholipase A2 and lipoxygenase pathway. A 23187 (10 microM) enhanced [3H]leukotriene B4 release from freshly prepared alveolar macrophages about 65-fold, but only 5- to 6-fold from cultured alveolar macrophages. Evoked [3H]leukotriene B4 release and spontaneous [3H]prostanoid release were inhibited when L-arginine (300 microM) was added to the Krebs incubation medium of alveolar macrophages, in which marked NO synthase had been induced by culture with lipopolysaccharides (10 microg/ml). Inhibitory effects of L-arginine were prevented by N(G)-monomethyl-L-arginine (L-NMMA, 100 microM). Inhibition of NO synthase during the culture period by L-NMMA (culture medium, in contrast to Krebs medium, already contains the substrate of NO synthase, L-arginine), resulted in attenuation of the 'culture-dependent' decline of the evoked release of [3H]leukotriene B4 and allowed lipopolysaccharides to cause an increase in spontaneous [3H]prostanoid release (i.e., to induce cyclo-oxygenase activity). In conclusion, in rat alveolar macrophages, endogenous NO appears to inhibit the release of mediators of the cyclo-oxygenase and lipoxygenase pathway through multiple sites of action.

Effects of indomethacin on muscarinic inhibition of endogenous noradrenaline release from rat isolated trachea.

The release of endogenous noradrenaline from rat isolated tracheae was evoked by electrical field stimulation (3 Hz, 540 pulses) in the presence of yohimbine, desipramine and tyrosine. The muscarine receptor agonist oxotremorine concentration-dependently inhibited the evoked release of noradrenaline by 95% at 1 mumol/l, EC50 values in two series of experiments 41 and 57 nmol/l, respectively. The effect of oxotremorine was antagonized by the non-selective muscarine receptor antagonist scopolamine (10-1000 nmol/l) in a manner suggesting a simple competitive interaction (slope of Schild plot -0.94; pA2 value 8.88). However, the M2 selective muscarine receptor antagonist methoctramine (0.1-10 mumol/l) affected the action of oxotremorine in a manner suggesting a complex interaction (slope of Schild plot -0.47). Addition of indomethacin (3 mumol/l) caused an increase of the evoked release of noradrenaline by 45% and low concentrations of oxotremorine (0.01 and 0.1 mumol/l, but not 1 mumol/l) became less effective resulting in a slight shift to the right of the concentration response curve (EC50 169 nmol/l). Moreover, in the presence of indomethacin methoctramine (0.1-10 mumol/l) antagonized the effects of oxotremorine in a manner suggesting a simple competitive interaction (slope of Schild plot -0.93, pA2 value 7.61). In the presence of indomethacin, the concentration response curve of oxotremorine was only slightly shifted to the right in the presence of the M1 receptor selective antagonist pirenzepine (1 mumol/l, -log KB 6.1) and not significantly affected by the M3 receptor selective antagonist p-fluoro-hexahydrosiladifenidol (1 mumol/l). In conclusion, the release of noradrenaline in the rat trachea is inhibited via presynaptic muscarine heteroreceptors of the M2 subtype.(ABSTRACT TRUNCATED AT 250 WORDS)

Beta-adrenoceptor-mediated facilitation of endogenous noradrenaline release from rat isolated trachea.

Overflow of endogenous noradrenaline from rat isolated trachea was evoked by electrical field stimulation (3 Hz, 540 pulses) in the presence of yohimbine, desipramine and tyrosine. Isoprenaline 100 nmol/l increased the evoked overflow of noradrenaline by about 65%. This effect was antagonized by propranolol (100 nmol/l) and the beta 2-selective adrenoceptor antagonist ICI 118,551 (100 nmol/l), but not by the beta 1-selective adrenoceptor antagonist CGP 20712 A (100 nmol/l). The beta 2-selective adrenoceptor agonist formoterol (1-100 nmol/l) also facilitated the evoked overflow of noradrenaline, but maximally by only about 25% at 10 nmol/l, i.e. formoterol behaved as a partial agonist at these facilitatory beta-adrenoceptor. This assumption is also supported by the observation that formoterol (10 nmol/l) acted as antagonist against isoprenaline (100 nmol/l). Mechanical removal of the mucosa resulted in a 30% decrease in tissue noradrenaline and a 55% reduction of the evoked overflow of noradrenaline. In mucosa-denuded preparations isoprenaline failed to facilitate noradrenaline overflow. In the presence of indomethacin (3 mumol/l) the evoked overflow of noradrenaline from mucosa containing preparations was increased by about 50%, but isoprenaline still further facilitated the evoked noradrenaline overflow by about 40%. In conclusion, the overflow of noradrenaline in the rat trachea is facilitated via beta 2-adrenoceptors, an effect which requires an intact airway mucosa.

Pretreatment with drug-specific antibody reduces desipramine cardiotoxicity in rats.

The effect of a drug-specific antibody on desipramine (DMI) cardiotoxicity was studied in rats. Animals were pretreated i.v. with 4.2 g/kg of a monoclonal antibody (anti-TCA) followed by DMI HCl 30 mg/kg i.p. (molar ratio of anti-TCA binding sites to DMI = 0.56). Peak QRS complex prolongation was substantially lower after pretreatment with anti-TCA than after control antibody (70 +/- 14 v. 21 +/- 4%, p less than 0.001). Time to peak toxicity was the same in both groups. Binding of DMI by anti-TCA was demonstrated by a higher serum total DMI concentration and increased DMI binding in serum after anti-TCA compared to controls. The DMI concentration in anti-TCA treated animals was lower in some organs (brain, lung, liver, spleen), but not in others (heart, muscle, kidney, fat). The calculated fraction of the DMI dose bound by anti-TCA was 19.9%. The steepness of the DMI dose-response curve was examined by administering DMI alone (without antibody) at various doses to rats. Compared to 30 mg/kg DMI, a dose reduction of 30-50% was needed to reduce QRS duration to the same extent as anti-TCA pretreatment. We conclude that DMI cardiotoxicity was markedly reduced by the binding of a relative small fraction of the DMI body burden to anti-TCA. This disproportionate effect of DMI binding was not due to the steepness of the DMI dose-response curve, nor to slowing of the rate of DMI distribution to tissues.

First-in-human study demonstrating pharmacological activation of heme oxygenase-1 in humans.

Heme oxygenase (HO)-1 degrades heme and protects against oxidative stress, but it has not been pharmacologically induced in humans. In this randomized study of 10 healthy volunteers, hemin (3 mg/kg intravenously in 25% albumin) was shown to increase plasma HO-1 protein concentration four- to fivefold and HO-1 activity ~15-fold relative to baseline at 24 and 48 h (placebo -56.41 +/- 6.31 (baseline), 69.79 +/- 13.00 (24 h), 77.44 +/- 10.62 (48 h) vs. hemin -71.70 +/- 9.20 (baseline), 1,126.20 +/- 293.30 (24 h), 1,192.20 +/- 333.30 (48 h)) in four of five subjects as compared with albumin alone (P

Analysis of the genomic organization of the human cationic amino acid transporters CAT-1, CAT-2 and CAT-4.

By screening nucleotide databases, sequences containing the complete genes of the human cationic amino acid transporters (hCATs) 1, 2 and 4 were identified. Analysis of the genomic organization revealed that hCAT-2 consists of 12 translated exons and most likely of 2 untranslated exons. The splice variants hCAT-2A and hCAT-2B use exon 7 and 6, respectively. The hCAT-2 gene structure is closely related to the structure of hCAT-1, suggesting that they belong to a common gene family. hCAT-4 consists of only 4 translated exons and 3 short introns. Exons of identical size and highly homologous to exon 3 of hCAT-4 are present in hCAT-1 and hCAT-2.

Rapamycin inhibition of interleukin-2-dependent p33cdk2 and p34cdc2 kinase activation in T lymphocytes.

The immunosuppressant rapamycin (RAP) is a potent inhibitor of the entry of interleukin (IL)-2-stimulated T cells into S-phase. Earlier results indicated that RAP treatment arrested the growth of the murine IL-2-dependent T cell line CTLL-2 in late G1-phase. To explore further the interactions of RAP with the cell cycle control machinery in T cells, we examined the effects of RAP treatment on the activation of the cyclin-dependent kinases p34cdc2 and p33cdk2 in G1-phase CTLL-2 cells. Stimulation of factor-deprived cells with IL-2 led to the assembly of high molecular weight complexes containing active p34cdc2 and p33cdk2. The appearance of these complexes was explained, at least in part, by the association of both cyclin-dependent kinases with IL-2-induced cyclin A. RAP treatment profoundly inhibited both cyclin A expression and the appearance of active cyclin A-cyclin-dependent kinase complexes in IL-2-stimulated, late G1-phase CTLL-2 cells. Although p34cdc2 activation was largely dependent on association with cyclin A, a significant proportion of the active p33cdk2 pool was complexed with cyclin E. In contrast to cyclin A, the IL-2-induced accumulation of cyclin E in G1-phase cells was only partially suppressed by RAP, and cyclin E-p33cdk2 complexes were readily detected in drug-treated cells. These cyclin E-cyclin-dependent kinase complexes were nonetheless devoid of histone H1 kinase activity. The inhibitory effects of RAP on the activation of cyclin E- and cyclin A-associated cyclin-dependent kinases suggest that one or both events participate in the regulation of T cell entry into S-phase.

Reversal of desipramine toxicity in rats using drug-specific antibody Fab' fragment: effects on hypotension and interaction with sodium bicarbonate.

The effect of drug-specific antibody Fab' fragment on desipramine (DMI) toxicity was studied in anesthetized rats to determine 1) whether DMI-induced hypotension can be reversed, and 2) whether the effect of this Fab' fragment can be enhanced by the concurrent administration of hypertonic NaHCO3. DMI (60 mg/kg) was administered i.p. to produce marked hypotension. Antitricyclic antidepressant (TCA) Fab' (molar Fab'/DMI ratio = 0.11) or control Fab' was administered 15 min later as a 10 min i.v. infusion. The mean arterial pressure was higher at the end of anti-TCA Fab' infusion than after control Fab' (58 +/- 8 vs. 17 +/- 7 mm Hg, P less than .001). In a second protocol, DMI (30 mg/kg) was administered to prolong QRS duration. Anti-TCA Fab' alone (molar Fab'/DMI ratio = 0.09) and NaHCO3 alone both reduced QRS prolongation compared to control treatment, and combined therapy was more effective than either one alone. In both protocols, anti-TCA Fab' markedly increased the total DMI concentration and the bound fraction of DMI in serum, but did not alter the unbound DMI concentration. In the low DMI dose protocol, anti-TCA Fab' also reduced the cardiac DMI concentration. Concurrent treatment with anti-TCA Fab' and NaHCO3 substantially increased urinary DMI and anti-TCA Fab' excretion compared to treatment with anti-TCA Fab' alone.(ABSTRACT TRUNCATED AT 250 WORDS)

The mammalian target of rapamycin phosphorylates sites having a (Ser/Thr)-Pro motif and is activated by antibodies to a region near its COOH terminus.

The eukaryotic initiation factor 4E (eIF4E)-binding protein, PHAS-I, was phosphorylated rapidly and stoichiometrically when incubated with [gamma-32P]ATP and the mammalian target of rapamycin (mTOR) that had been immunoprecipitated with an antibody, mTAb1, directed against a region near the COOH terminus of mTOR. PHAS-I was phosphorylated more slowly by mTOR obtained either by immunoprecipitation with other antibodies or by affinity purification using a rapamycin/FKBP12 resin. Adding mTAb1 to either of these preparations of mTOR increased PHAS-I phosphorylation severalfold, indicating that mTAb1 activates the mTOR protein kinase. mTAb1-activated mTOR phosphorylated Thr36, Thr45, Ser64, Thr69, and Ser82 in PHAS-I. All five of these sites fit a (Ser/Thr)-Pro motif and are dephosphorylated in response to rapamycin in rat adipocytes. Thus, our findings indicate that Pro is a determinant of the mTOR protein kinase specificity and that mTOR contributes to the phosphorylation of PHAS-I in cells.

Protein-tyrosine kinase-dependent activation of STAT transcription factors in interleukin-2- or interleukin-4-stimulated T lymphocytes.

The proliferation of activated T lymphocytes is critically dependent on the binding of the T-cell growth factors, interleukin (IL)-2 and IL-4, to distinct but evolutionarily related cell surface receptors. Previous results suggest that the IL-2 receptor (IL-2R) and IL-4R are coupled to both overlapping and distinct intracellular signaling pathways in T lymphocytes. In this study, we demonstrate that activation of Janus tyrosine kinases (JAKs) and STAT transcription factors is rapidly induced by exposure of factor-dependent murine T-cell lines to IL-2 or IL-4. Both IL-2 and IL-4 stimulated the rapid activation of JAK1 and JAK3, whereas JAK2 activity was unaffected by either cytokine. These responses were accompanied by the appearance in cell nuclei of 3 DNA binding activities that recognized a high-affinity binding site for STAT factors. In transient transfection assays, this STAT factor target sequence conferred IL-2 and IL-4 inducibility on a synthetic luciferase reporter gene. Antibody supershifting experiments indicated that IL-2 induces the formation of STAT dimers containing STAT3 and STAT1 alpha. Although IL-4 also activated STAT1 alpha, the major IL4-induced STAT factor is not STAT3 and remains undefined. Pretreatment of the T-cells with the protein-tyrosine kinase inhibitor herbimycin A blocked both the nuclear translocation of STAT factors and STAT-dependent reporter gene transcription. Immunoblot analyses confirmed that cytoplasmic STAT3 was heavily phosphorylated on tyrosine in IL-2-stimulated cells, and that phosphorylated STAT3 appeared in the nuclei of these cells. These results indicate that identical JAKs and partially overlapping sets of STATs are activated by IL-2 and IL-4 in T lymphocytes.

Rapamycin-induced inhibition of p34cdc2 kinase activation is associated with G1/S-phase growth arrest in T lymphocytes.

The macrolide rapamycin (RAP) is a potent inhibitor of interleukin-2 (IL-2)-induced T-cell proliferation. Current models suggest that RAP, when complexed to its intracellular receptor, FK506-binding protein, interferes with an IL-2 receptor-coupled signaling pathway required for cell-cycle progression from G1- to S-phase. Here we show that RAP treatment inhibits the growth of an IL-2-dependent cytotoxic T-cell line, CTLL-2, in late G1-phase, just prior to entry of the cells into S-phase. In contrast, RAP-treated CTLL-2 cells retained the ability to respond to IL-2 with enhanced cytolytic activity, indicating that RAP was not a general suppressant of cellular responsiveness to IL-2. Subsequent studies revealed that IL-2 stimulation triggered a delayed activation of the p34cdc2 kinase, the timing of which correlated with the G1- to S-phase transition. The IL-2-dependent increase in p34cdc2 kinase activity was blocked by RAP. The RAP sensitivity of the p34cdc2 activation mechanism implicates this signaling pathway in the control of S-phase commitment in IL-2-stimulated T-cells.