The requirements for Ca2+, protein phosphorylation and concurrent protein synthesis for zeatin signaling of acidic chitinase transcript accumulation in Cucumis sativus L

We have previously reported that both Ca2+ and staurosporine-sensitive protein kinase(s) are involved in the cytokinin zeatin induction of cucumber chitinase activity and its protein content (Barwe et al. 2001). To further characterize signal transduction events involved in this cytokinin induction of chitinase gene expression, Northern hybridizations of total RNAs prepared from excised, dark-grown cucumber cotyledons treated with cytokinins and/or various agonists and antagonists of signal transduction components, were carried out using a cucumber acidic chitinase (CACHT) cDNA probe (Metraux et al. 1989). CACHT mRNA increased by approximately 5- to 6-fold in response to exogenous zeatin (Z), zeatin riboside (ZR), and benzyladenine (BA) treatment, but failed to accumulate in response to kinetin (K). Among the cytokinins tested, Z was most effective. The Z-induced accumulation of CACHT mRNA was inhibited by a plasma membrane Ca2+ channel blocker verapamil. Treatment of cotyledons with exogenous CaCl2 and calcium ionophore A23187 in the presence and absence of cytokinin enhanced CACHT mRNA accumulation. These two observations suggest the participation of extracellular calcium in signaling Z-induction. Furthermore, the presence of staurosporine (an inhibitor of protein kinase) in Z treatment reduced CACHT mRNA, suggesting the involvement of phosphorylation of one or more cellular proteins. In addition, we provide evidence that the Z-induction of CACHT mRNA is blocked by protein synthesis inhibitor cycloheximide treatment. Taken together, these results suggest that Ca2+ influx from extracellular space, protein phosphorylation, and concurrent protein synthesis events participate in cytokinin signaling during Z-induced CACHT transcript accumulation.

D1/D5 receptor agonists induce a protein synthesis-dependent late potentiation in the CA1 region of the hippocampus.

Agonists of the dopamine D1/D5 receptors that are positively coupled to adenylyl cyclase specifically induce a slowly developing long-lasting potentiation of the field excitatory postsynaptic potential in the CA1 region of the hippocampus that lasts for > 6 hr. This potentiation is blocked by the specific D1/D5 receptor antagonist SCH 23390 and is occluded by the potentiation induced by cAMP agonists. An agonist of the D2 receptor, which is negatively coupled to adenylyl cyclase through G alpha i, did not induce potentiation. Although this slow D1/D5 agonist-induced potentiation is partially independent of N-methyl-D-aspartate receptors, it seems to share some steps with and is occluded by the late phase of long-term potentiation (LTP) produced by three repeated trains of nerve stimuli applied to the Schaffer collateral pathway. Similarly, the D1/D5 antagonist SCH 23390 attenuates the late phase of the LTP induced by repeated trains, and the D1/D5 agonist-induced potentiation is blocked by the protein synthesis inhibitor anisomycin. These results suggest that the D1/D5 receptor may be involved in the late, protein synthesis-dependent component of LTP in the hippocampal CA1 region, either as an ancillary component or as a mediator directly contributing to the late phase.

3-Amino-1,2,4-triazole is an inhibitor of protein synthesis on mitoribosomes in Neurospora crassa

The effects of the herbicide, 3-amino-1,2,4-triazole, an inhibitor of heme synthesis in rat liver, have been examined in the mold Neurospora crassa. The drug is a potent inhibitor of the growth of the mold and produces biochemical changes identical to those produced by chloramphenicol. 3-Amino-1,2,4-triazole, like chloramphenicol, is a direct and specific inhibitor of protein synthesis on mitoribosomes. A decrease in the levels of mitochondrial proteins which are completely or partly made on mitoribosomes and an accumulation in the levels of mitochondrial proteins of cytosolic origin have been observed. Both drugs depress porphyrin and heme levels, but there is actually an elevation in the levels of δ-aminolevulinate dehydratase, the rate-limiting enzyme of the heme-biosynthetic pathway in Neurospora crassa. In liver the enzyme is present in non-limiting amounts and the levels are depressed under conditions of 3-amino-1,2,4-triazole treatment. In Neurospora crassa the ‘derepression’ of δ-aminolevulinate dehydratase under conditions of 3-amino-1,2,4-triazole or chloramphenicol treatment is only partial because the drugs inhibit protein synthesis on mitoribosomes. It is concluded that an optimal rate of protein synthesis on mitoribosomes is necessary to maintain an adequate rate of heme synthesis.

Glucocorticoid receptor and protein/RNA synthesis-dependent mechanisms underlie the control of synaptic plasticity by stress

Learning and memory are exquisitely sensitive to behavioral stress, but the underlying mechanisms are still poorly understood. Because activity-dependent persistent changes in synaptic strength are believed to mediate memory processes in brain areas such as the hippocampus we have examined the means by which stress affects synaptic plasticity in the CA1 region of the hippocampus of anesthetized rats, Inescapable behavioral stress (placement on an elevated platform for 30 min) switched the direction of plasticity, favoring low frequency stimulation-induced decreases in synaptic transmission (long-term depression, LTD), and opposing the induction of long-term potentiation by high frequency stimulation, We have discovered that glucocorticoid receptor activation mediates these effects of stress on LTD and longterm potentiation in a protein synthesis-dependent manner because they were prevented by the glucocorticoid receptor antagonist RU 38486 and the protein synthesis inhibitor emetine. Consistent with this, the ability of exogenously applied corticosterone in non-stressed rats to mimic the effects of stress on synaptic plasticity was also blocked by these agents, The enablement of low frequency stimulation-induced LTD by both stress and exogenous corticosterone was also blocked by the transcription inhibitor actinomycin D, Thus, naturally occurring synaptic plasticity is liable to be reversed in stressful situations via glucocorticoid receptor activation and mechanisms dependent on the synthesis of new protein and RNA, This indicates that the modulation of hippocampus-mediated learning by acute inescapable stress requires glucocorticoid receptor-dependent initiation of transcription and translation.

THE EFFECT OF DIAMPHENETHIDE ON PROTEIN-SYNTHESIS BY THE LIVER FLUKE, FASCIOLA-HEPATICA

The effect of the deacetylated (amine) metabolite of diamphenethide (DAMD, 10 mug ml-1) on the uptake and incorporation by adult Fasciola hepatica of radioactively labelled precursors of DNA, RNA and protein synthesis ([H-3]thymidine, [H-3]uridine and [H-3]leucine, respectively) was measured by liquid scintillation counting. Comparison was made between the effects of DAMD and those of specific inhibitors of DNA, RNA and protein synthesis, namely, 5-fluorouracil, cordycepin and cycloheximide, respectively. DAMD caused a significant decrease in the overall uptake and incorporation of [H-3]uridine by F. hepatica, decreased the incorporation of [H-3]leucine and also caused a significant decrease in the overall protein content of the flukes. The effect of DAMD was similar to that of cycloheximide (I x 10(-3) M), a potent inhibitor of protein synthesis, which also caused a significant decrease in the incorporation of [H-3]leucine by the fluke and a decrease in the overall protein content of the fluke. Cordycepin(100 mug ml-1) caused a significant decrease in the protein content of the fluke, but had no effect on the uptake or incorporation of [H-3]uridine. 5-Fluorouracil (I x 10(-4) m) did not affect the uptake or incorporation of VH]thymidine, nor did it decrease the protein content of the fluke. The results indicate that DAMD inhibits protein synthesis by F. hepatica, possibly by inhibiting RNA synthesis. The results are also consistent with previous morphological investigations involving DAMD.

Signaling of angiotensin II-induced vascular protein synthesis in conduit and resistance arteries in vivo

Affiliation: Faculté de pharmacie, Université de Montréal & Institut de recherches cliniques de Montréal

A channeled tRNA cycle during mammalian protein synthesis.

In earlier studies it was shown that the mammalian translation system is highly organized in vivo and that the intermediates in the process, aminoacyl-tRNAs, are channeled--i.e., they are directly transferred from the aminoacyl-tRNA synthetases to the elongation factor to the ribosomes without dissociating into the cellular fluid. Here, we examine whether spent tRNAs leaving the ribosome enter the fluid phase or are transferred directly to their cognate aminoacyl-tRNA synthetases to complete a channeled tRNA cycle. Using a permeabilized CHO cell system that closely mimics living cells, we find that there is no leakage of endogenous tRNA during many cycles of translation, and protein synthesis remains linear during this period, even though free aminoacyl-tRNA is known to rapidly equilibrate between the inside and outside of these cells. We also find that exogenous tRNA and periodate-oxidized tRNA have no effect on protein synthesis in this system, indicating that they do not enter the translation machinery, despite the fact that exogenous tRNA rapidly distributes throughout the cells. Furthermore, most of the cellular aminoacyl-tRNA synthetases function only with endogenous tRNAs, although a portion can use exogenous tRNA molecules. However, aminoacylation of these exogenous tRNAs is strongly inhibited by oxidized tRNA; this inhibitor has no effect on endogenous aminoacylation. On the basis of these and the earlier observations, we conclude that endogenous tRNA is never free of the protein synthetic machinery at any stage of the translation process and, consequently, that there is a channeled tRNA cycle during protein synthesis in mammalian cells.

Signaling pathways initiated by beta-hydroxy-beta-methylbutyrate to attenuate the depression of protein synthesis in skeletal muscle in response to cachectic stimuli

To investigate the mechanism by which beta-hydroxy-beta-methylbutyrate (HMB) attenuates the depression of protein synthesis in the skeletal muscle of cachectic mice, a study has been carried out in murine myotubes in the presence of proteolysis-inducing factor (PIF). PIF inhibited protein synthesis by 50% within 4 h, and this was effectively attenuated by HMB (25-50 muM). HMB (50 muM) alone stimulated protein synthesis, and this was attenuated by rapamycin (27 nM), an inhibitor of mammalian target of rapamycin (mTOR). Further evidence for an involvement of this pathway was shown by an increased phosphorylation of mTOR, the 70-kDa ribosomal S6 kinase (p70(S6k)), and initiation factor 4E-binding protein (4E-BP1) and an increased association of eukaryotic initiation factor 2 (eIF4E) with eIF4G. PIF alone induced a transient (1-2 h) stimulation of phosphorylation of mTOR and p70(S6k). However, in the presence of HMB, phosphorylation of mTOR, p70(S6k), and 4E-BP1 was increased, and inactive 4E-BP1-eIF4E complex was reduced, whereas the active eIF4G.eIF4E complex was increased, suggesting continual stimulation of protein synthesis. HMB alone reduced phosphorylation of elongation factor 2, but this effect was not seen in the presence of PIF. PIF induced autophosphorylation of the double-strand RNA-dependent protein kinase (PKR), leading to phosphorylation of eIF2 on the alpha-subunit, which would inhibit protein synthesis. However, in the presence of HMB, phosphorylation of PKR and eIF2alpha was attenuated, and this was also observed in skeletal muscle of cachectic mice administered HMB (0.25 g/kg). These results suggest that HMB attenuates the depression of protein synthesis by PIF in myotubes through multiple mechanisms.

Skeletal muscle atrophy, a link between depression of protein synthesis and increase in degradation

Both proteolysis-inducing factor (PIF) and angiotensin II have been shown to produce a depression in protein synthesis in murine myotubes concomitant with an increased phosphorylation of eukaryotic initiation factor 2 (eIF2α). Both PIF and angiotensin II were shown to induce autophosphorylation of the RNA-dependent protein kinase (PKR), and an inhibitor of this enzyme completely attenuated the depression in protein synthesis and prevented the induction of eIF2α phosphorylation. The PKR inhibitor also completely attenuated the increase in protein degradation induced by PIF and angiotensin II and prevented the increase in proteasome expression and activity. To confirm these results myotubes were transfected with plasmids that express either wild-type PKR, or a catalytically inactive PKR variant, PKRΔ6. Myotubes expressing PKRΔ6 showed no increase in eIF2α phosphorylation in response to PIF or angiotensin II, no depression in protein synthesis, and no increase in protein degradation or increase in proteasome expression. Induction of the ubiquitin-proteasome pathway by PIF and angiotensin II has been linked to activation of the transcription factor nuclear factor-κB (NF-κB). Inhibition of PKR prevented nuclear migration of NF-κB in response to both PIF and angiotensin II, by preventing degradation of the inhibitor protein I-κB. Phosphorylation of PKR and eIF2α was also significantly increased in the gastrocnemius muscle of weight losing mice bearing the MAC16 tumor, suggesting that a similar process may be operative in cancer cachexia. These results provide a link between the depression of protein synthesis in skeletal muscle and the increase in protein degradation. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.

Effect of a tumour-produced lipid-mobilizing factor on protein synthesis and degradation

Treatment of murine myoblasts, myotubes and tumour cells with a tumour-produced lipid mobilizing factor (LMF), caused a concentration-dependent stimulation of protein synthesis, within a 24 h period. There was no effect on cell number or [3H] thymidine incorporation, but a similar concentration-dependent stimulation of 2-deoxyglucose uptake. LMF produced an increase in intracellular cyclic AMP levels, which was linearly (r2 = 0.973) related to the increase in protein synthesis. The effect of LMF was attenuated by the adenylate cyclase inhibitor MDL12330A, and was additive with the stimulation produced by forskolin. Both propranolol (10 μM) and the specific β3-adrenergic receptor antagonist SR 59230A (10-5M), significantly reduced the stimulation of protein synthesis induced by LMF. Protein synthesis was also increased by 69% (P = 0.006) in soleus muscles of mice administered LMF, while there was a 26% decrease in protein degradation (P = 0.03). While LMF had no effect on the lysosomal enzymes, cathepsins B and L, there was a decrease in proteasome activity, as determined both by the 'chymotrypsin-like' enzyme activity, as well as expression of proteasome α-type subunits, determined by Western blotting. These results show that in addition to its lipid-mobilizing activity LMF also increases protein accumulation in skeletal muscle both by an increase in protein synthesis and a decrease in protein catabolism. © 2001 Cancer Research Campaign.

Angiotensin II directly inhibits protein synthesis in murine myotubes

Angiotensin II (Ang II) has been implicated in muscle protein loss in cachexia. To determine whether the Ang I/II system directly inhibits protein synthesis in muscle their effect has been monitored in vitro using murine myotubes as a surrogate model system. Ang I inhibited protein synthesis by 40-50% over the concentration range of 0.05-2.5 μM within 30 min of addition, and the inhibition remained relatively constant over 24 h. The effect was attenuated by co-incubation with the angiotensin converting enzyme inhibitor imidaprilat (50 μM) suggesting that inhibition of protein synthesis was due to the formation of Ang II. Ang II also inhibited protein synthesis by 40-50% over the concentration range of 0.1-5 μM, and the inhibition also remained relatively constant between 30 min and 24 h after addition. The effect was attenuated by insulin-like growth factor-1 (IGF-1) (25-100 ng/ml). Thus, Ang I/II have the ability to induce muscle atrophy through inhibition of protein synthesis. © 2005 Elsevier Ireland Ltd. All rights reserved.

Timing and distribution of protein ingestion during prolonged recovery from resistance exercise alters myofibrillar protein synthesis

Quantity and timing of protein ingestion are major factors regulating myofibrillar protein synthesis (MPS). However, the effect of specific ingestion patterns on MPS throughout a 12 h period is unknown. We determined how different distributions of protein feeding during 12 h recovery after resistance exercise affects anabolic responses in skeletal muscle. Twenty-four healthy trained males were assigned to three groups (n = 8/group) and undertook a bout of resistance exercise followed by ingestion of 80 g of whey protein throughout 12 h recovery in one of the following protocols: 8 × 10 g every 1.5 h (PULSE); 4 × 20 g every 3 h (intermediate: INT); or 2 × 40 g every 6 h (BOLUS). Muscle biopsies were obtained at rest and after 1, 4, 6, 7 and 12 h post exercise. Resting and post-exercise MPS (l-[ring-(13)C6] phenylalanine), and muscle mRNA abundance and cell signalling were assessed. All ingestion protocols increased MPS above rest throughout 1-12 h recovery (88-148%, P < 0.02), but INT elicited greater MPS than PULSE and BOLUS (31-48%, P < 0.02). In general signalling showed a BOLUS>INT>PULSE hierarchy in magnitude of phosphorylation. MuRF-1 and SLC38A2 mRNA were differentially expressed with BOLUS. In conclusion, 20 g of whey protein consumed every 3 h was superior to either PULSE or BOLUS feeding patterns for stimulating MPS throughout the day. This study provides novel information on the effect of modulating the distribution of protein intake on anabolic responses in skeletal muscle and has the potential to maximize outcomes of resistance training for attaining peak muscle mass.

Preexercise aminoacidemia and muscle protein synthesis after resistance exercise

PURPOSE We have previously shown that the aminoacidemia caused by the consumption of a rapidly digested protein after resistance exercise enhances muscle protein synthesis (MPS) more than the amino acid (AA) profile associated with a slowly digested protein. Here, we investigated whether differential feeding patterns of a whey protein mixture commencing before exercise affect postexercise intracellular signaling and MPS. METHODS Twelve resistance-trained males performed leg resistance exercise 45 min after commencing each of three volume-matched nutrition protocols: placebo (PLAC, artificially sweetened water), BOLUS (25 g of whey protein + 5 g of leucine dissolved in artificially sweetened water; 1× 500 mL), or PULSE (15× 33-mL aliquots of BOLUS drink every 15 min). RESULTS The preexercise rise in plasma AA concentration with PULSE was attenuated compared with BOLUS (P < 0.05); this effect was reversed after exercise, with two-fold greater leucine concentrations in PULSE compared with BOLUS (P < 0.05). One-hour postexercise, phosphorylation of p70 S6K and rpS6 was increased above baseline with BOLUS and PULSE, but not PLAC (P < 0.05); furthermore, PULSE > BOLUS (P < 0.05). MPS throughout 5 h of recovery was higher with protein ingestion compared with PLAC (0.037 ± 0.007), with no differences between BOLUS or PULSE (0.085 ± 0.013 vs. 0.095 ± 0.010%•h, respectively, P = 0.56). CONCLUSIONS Manipulation of aminoacidemia before resistance exercise via different patterns of intake of protein altered plasma AA profiles and postexercise intracellular signaling. However, there was no difference in the enhancement of the muscle protein synthetic response after exercise. Protein sources producing a slow AA release, when consumed before resistance exercise in sufficient amounts, are as effective as rapidly digested proteins in promoting postexercise MPS.

Sex-based comparisons of myofibrillar protein synthesis after resistance exercise in the fed state

Sex-based comparisons of myofibrillar protein synthesis after resistance exercise in the fed state. J Appl Physiol 112: 1805-1813, 2012. First published March 1, 2012; doi:10.1152/japplphysiol.00170.2012.- We made sex-based comparisons of rates of myofibrillar protein synthesis (MPS) and anabolic signaling after a single bout of high-intensity resistance exercise. Eight men (20 ± 10 yr, BMI = 24.3 ± 2.4) and eight women (22 ± 1.8 yr, BMI = 23.0 ± 1.9) underwent primed constant infusions of L-[ring-13C6]phenylalanine on consecutive days with serial muscle biopsies. Biopsies were taken from the vastus lateralis at rest and 1, 3, 5, 24, 26, and 28 h after exercise. Twenty-five grams of whey protein was ingested immediately and 26 h after exercise. We also measured exercise-induced serum testosterone because it is purported to contribute to increases in myofibrillar protein synthesis (MPS) postexercise and its absence has been hypothesized to attenuate adaptative responses to resistance exercise in women. The exercise-induced area under the testosterone curve was 45-fold greater in men than women in the early (1 h) recovery period following exercise (P < 0.001). MPS was elevated similarly in men and women (2.3- and 2.7-fold, respectively) 1-5 h postexercise and after protein ingestion following 24 h recovery. Phosphorylation of mTORSer2448 was elevated to a greater extent in men than women acutely after exercise (P = 0.003), whereas increased phosphorylation of p70S6K1Thr389 was not different between sexes. Androgen receptor content was greater in men (main effect for sex, P = 0.049). Atrogin-1 mRNA abundance was decreased after 5 h recovery in both men and women (P < 0.001), and MuRF-1 expression was elevated in men after protein ingestion following 24 h recovery (P = 0.003). These results demonstrate minor sex-based differences in signaling responses and no difference in the MPS response to resistance exercise in the fed state. Interestingly, our data demonstrate that exerciseinduced increases in MPS are dissociated from postexercise testosteronemia and that stimulation of MPS occurs effectively with low systemic testosterone concentrations in women.

Rapid aminoacidemia enhances myofibrillar protein synthesis and anabolic intramuscular signaling responses after resistance exercise

Background: Ingestion of whey or casein yields divergent patterns of aminoacidemia that influence whole-body and skeletal muscle myofibrillar protein synthesis (MPS) after exercise. Direct comparisons of the effects of contrasting absorption rates exhibited by these proteins are confounded by their differing amino acid contents. Objective: Our objective was to determine the effect of divergent aminoacidemia by manipulating ingestion patterns of whey protein alone on MPS and anabolic signaling after resistance exercise. Design: In separate trials, 8 healthy men consumed whey protein either as a single bolus (BOLUS; 25-g dose) or as repeated, small, "pulsed" drinks (PULSE; ten 2.5-g drinks every 20 min) to mimic a more slowly digested protein. MPS and phosphorylation of signaling proteins involved in protein synthesis were measured at rest and after resistance exercise. Results: BOLUS increased blood essential amino acid (EAA) concentrations above those of PULSE (162% compared with 53%, P < 0.001) 60 min after exercise, whereas PULSE resulted in a smaller but sustained increase in aminoacidemia that remained elevated above BOLUS amounts later (180-220 min after exercise, P < 0.05). Despite an identical net area under the EAA curve, MPS was elevated to a greater extent after BOLUS than after PULSE early (1-3 h: 95% compared with 42%) and later (3-5 h: 193% compared with 121%) (both P < 0.05). There were greater changes in the phosphorylation of the Akt-mammalian target of rapamycin pathway after BOLUS than after PULSE. Conclusions: Rapid aminoacidemia in the postexercise period enhances MPS and anabolic signaling to a greater extent than an identical amount of protein fed in small pulses that mimic a more slowly digested protein. A pronounced peak aminoacidemia after exercise enhances protein synthesis.