Effect of chronic excess of tumour necrosis factor-alpha on contractile proteins in rat skeletal muscle.

The effect of chronic tumour necrosis factor-alpha (TNF-alpha) treatment on the synthesis of specific myofibrillar proteins such as heavy chain myosin, light chain myosin and G-actin in rat diaphragm were evaluated. Muscles (diaphragm) from control and experimental groups (TNF-alpha i.v. at 50 microg/kg body wt for 5 days) were incubated in the presence of 35S-methionine for 2 h. Myofibrillar protein extracts were prepared and protein was electrophoresed on sodium dodecyl sulphate-polyacrylamide gels. Heavy chain myosin, light chain myosin and G-actin were identified by Western blot analysis using specific monoclonal antibodies. Polyacrylamide gel electrophoresis (PAGE) followed by Western blot analysis revealed two types of heavy chain myosin (206 and 212 kD), all four types of light chain myosin (15, 16.5, 18 and 20 kD) and a single type of G-actin (42 kD). Chronic TNF-alpha treatment produced a significant decline in the synthesis of all types of myofibrillar proteins, namely heavy chain myosin, light chain myosin and G-actin. TNF-alpha impaired peptide-chain initiation in diaphragm muscle which was reversed by the branched-chain amino acids (BCAA) therapy of TNF-alpha treated rats. These findings indicate a significant role for TNF-alpha in the translational regulation of protein synthesis in skeletal muscle.

Effect of tumour necrosis factor-alpha on total myofibrillar and sarcoplasmic protein synthesis and polysomal aggregation in rat skeletal muscles.

The total sarcoplasmic and myofibrillar protein synthesis was reduced in incubated fast-twitch extensor digitorum longus (EDL) and slow-twitch soleus of rat after in vivo tumour necrosis factor-alpha treatment at 50 micrograms/kg/day for 5 days. The rate of protein synthesis in the myofibrillar fraction was inhibited more severely (41% in EDL and 34% in soleus) than that in the sarcoplasmic fraction (23% in EDL and 14% in soleus). Sucrose density gradient centrifugation analysis indicated that TNF-alpha treatment impaired polysomal aggregation in rat diaphragm muscle. Compared with the control muscles, the ratio of 40S and 60S subunits to polysomes was higher in TNF-alpha treated muscles. These findings suggest a role for TNF-alpha in the translational regulation of protein synthesis in rat skeletal muscle.

The effect of histone-mRNA interaction in protein synthesis.

The effect of lysine-rich and arginine-rich histones on selective inhibition of translation of mRNAs of tobacco mosaic virus (TMV), brome mosaic virus (BMV) and luciferase, in rabbit reticulocyte (RRL) was evaluated. Nuclease-treated RRL were supplemented with an appropriate mRNA and incubated with lysine-rich or arginine-rich histones at 5 and 2.5 micrograms/100 microliters lysate at 30 degrees C. Protein synthesis was measured as incorporation of 3H-leucine into protein for 24 min. The lysine-rich histone was more inhibitory than arginine-rich histone. At 5 and 2.5 micrograms/100 microliters lysate concentration, the lysine-rich histone inhibited translation of TMV mRNA by 57% and 35%, respectively, after 24 min. The corresponding values for arginine-rich histone were 31% and 13%. Both histones had a more inhibitory effect on the translation of viral mRNA than luciferase mRNA. Arginine-rich histone at 2.5 micrograms/100 microliters lysate did not cause any inhibition of luciferase mRNA. The spectrophotometric analysis of histone-mRNA mixtures at 280 nm indicated that, compared with arginine-rich histone, lysine-rich histone had a stronger affinity for mRNA.

Effect of glucocorticoids on tumor necrosis factor induced suppression of protein synthesis in rat aortic smooth muscle: comparison with extensor digitorum longus.

Protein synthesis was measured in incubated aortic smooth muscle and fast-twitch extensor digitorum longus (EDL) in both sham operated and adrenalectomized (ADX) rats. The studies were performed on four groups: a) untreated controls; b) rats treated with tumor necrosis factor (TNF) @ 50 micrograms/kg. b.w.; c) rats treated with cortisone @ 100 mg/kg/day for 5 days; or d) rats treated with cortisone plus TNF. Both TNF and cortisone suppressed protein synthesis in the aortic smooth muscle and EDL in intact animals. TNF given together with cortisone, induced a significant additional decrease in protein synthesis in both muscle types as compared to cortisone-treated rats. The rate of protein synthesis in aortic smooth muscle from sham operated rats was control > TNF > cortisone > Cortisone+TNF; in the case of EDL, rate was control > cortisone > TNF > Cortisone+TNF. In ADX animals, TNF alone did not affect protein synthesis in both aortic smooth muscle and EDL. Though cortisone alone produced a significant inhibition of protein synthesis, there was no significant further decline in protein synthesis when cortisone was given together with TNF. These findings suggest that the inhibitory effect of TNF on muscle protein synthesis is mediated through glucocorticoids.

Effect of fasting, branched-chain amino acids, and glucocorticoids on histone H1 extractability from rat skeletal muscle.

The effect of 72 h fasting, nutritional therapy of fasted rats, and acute and chronic glucocorticoid treatment on the yield of histone H1 from rat hind limb muscles was determined. Fasting significantly enhanced the extractability of muscle H1. The effect of treating starved rats with glucose alone, or with glucose supplemented with branched-chain amino acids (BCAA), or with two commercial preparations of mixtures of essential and non-essential amino acids was evaluated. Treatment of starved rats with glucose alone significantly decreased H1 extractability from muscles, but isocaloric treatment with glucose supplemented with BCAA or two commercial preparations of amino acid mixtures was more effective. Glucocorticoid treatment for 5 days enhanced the yield of H1 from muscles less than starvation. The enhanced H1 extractability from muscles noted in starved rats is similar to that reported in rats with insulinopenic diabetes and may reflect changes in nuclear fragility.

Effect of parvalbumin and S-100 protein on protein synthesis in rabbit reticulocyte lysate.

The effect of two high affinity Ca+ binding acidic proteins, parvalbumin and S-100 protein on protein synthesis in rabbit reticulocyte lysates (RRL), was investigated. Nuclease-treated RRL, supplemented with yeast mRNA, and 3H-leucine were incubated at 37 degrees C, and incorporation of 3H-leucine into protein was determined for 24 min. At 20 micrograms/100 microliters lysate concentration, both parvalbumin and S-100 protein caused a marked inhibition of protein synthesis compared with the control lysate. At a lower concentration parvalbumin was less inhibitory than histone H1; the effect of S-100 protein was not significant. The combined inhibitory effect of parvalbumin and H1 was not additive probably due to strong interaction between them as was evidenced by the enhanced absorbance of parvalbumin-H1 mixture. Spectrophotometric profiles of parvalbumin-tRNA mixture indicated that, unlike H1, parvalbumin did not inhibit protein synthesis by binding with nucleic acids. These results suggest an important role for parvalbumin in translational regulation.

A translational inhibitor from muscles of diabetic rats: identification as histone H1.

A heat- and acid-stable protein fraction that inhibited peptide chain initiation in rabbit reticulocyte lysates was extracted from frozen, powdered rat skeletal muscles by stepwise trichloroacetic acid precipitation. Streptozotocin-induced diabetes increased the inhibitory activity; this was prevented by insulin therapy. Size-exclusion high-performance liquid chromatography resolved four inhibitory fractions; only one was consistently increased (approximately 2-fold) in muscle extracts from diabetic rats. Polysome profiles of lysates incubated with this fraction indicated peptide chain initiation inhibition. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis the purified inhibitory fraction migrated with apparent Mr 30 and 32 kDa, which on Western blot immunostained with antisera against histone H1/H1(0). Perchloric acid extraction of muscle homogenates yielded approximately twofold more H1 from diabetic than from control rats; yield from diabetics decreased to control values 5 h after subcutaneous insulin injection. Inclusion of detergent during homogenization increased H1 yield more from muscles of control than from diabetic rats and abolished the difference between them. Because H1 affects several biochemical reactions, its facilitated extraction from insulin-deprived tissues can bias interpretation of studies of insulin action.

Histone-H1 inhibits translation by reticulocyte lysates with relative mRNA selectivity.

Histone-H1 purified from rat skeletal muscle is a relatively potent inhibitor of peptide chain initiation in a cell free system, the rabbit reticulocyte lysate (50% inhibition at approximately 0.4 microM). H1 does not inhibit formation of the ternary complex nor its attachment to 40S ribosomes; the data are compatible with H1 binding to mRNA. The inhibition shows mRNA selectivity: translation of beta-globin mRNA is more affected than that of alpha-globin mRNA and hepatic albumin mRNA more than total hepatic mRNA. Whether or not histone-H1 plays a role in translational regulation in intact cells is conjectural, it may serve as a useful model for protein-mRNA interactions.

Evaluation of isomers of octopamine for in vitro alpha-adrenergic stimulation of the aortic smooth muscle from spontaneously hypertensive rats.

Isomers of octopamine were tested for in vitro alpha-adrenergic stimulation of aortic smooth muscle of spontaneously hypertensive rats (SHR). In order to test the response of alpha 1-adrenoceptors to meta-, para-, and ortho-octopamine, alpha 2-adrenoceptors were blocked with 10(-7) M yohimbine, and to measure the response of alpha 2-adrenoceptors the alpha 1-adrenoceptors were blocked with 10(-7) M prazosin. The contractile response of aortic smooth muscle of SHR to stimulation by phenylephrine, m-, p-, and o-isomers of octopamine in the presence of yohimbine was not appreciably altered. However, administration of prazosin severely attenuated the response of muscles of these compounds indicating that like phenylephrine, the isomers of octopamine stimulate mainly alpha 1-adrenoceptors. The attenuation of contractile response to isomers of octopamine in the presence of prazosin was not as pronounced as in the case of phenylephrine. The comparative potencies of phenylephrine, m-, p-, and o-octopamine in the presence of 10(-7) M prazosin were 1:1.2:2.5:0.75, respectively. Thus, it appears that the isomers of octopamine, especially para- and meta-octopamine, play a much more important role in the physiology of vascular smooth muscle than has been thus far perceived.

The effect of hyperthyroidism and hypothyroidism on alpha 1- and alpha 2-adrenergic responsiveness in rat aortic smooth muscle.

We report the role of thyroid hormones on in vitro responsiveness of rat aortic smooth muscle to alpha-adrenergic stimulation. Four groups of rats: hypothyroid, hyperthyroid, thyroxine (0.1 mg/kg) treated hypothyroid and controls were employed. Response of alpha 1- and alpha 2-adrenoceptors was evoked with 6 incremental doses (10(-9) to 10(-4) M) of preferential alpha 1-agonist, phenylephrine and alpha 2-agonist, clonidine respectively. alpha 1-Adrenoceptors were also evoked by phenylephrine after blockade of alpha 2-adrenoceptors with 10(-7) M yohimbine. Similarly, alpha 2-adrenoceptors were stimulated with clonidine after blocking alpha 1-adrenoceptors with selective antagonists prazosin (10(-7) M). Aortic responsiveness to alpha-agonist norepinephrine was compared between the aortae of hypothyroid and euthyroid rats after blockade of alpha 2-adrenoceptors with 10(-4) M corynanthine. We report that in hypothyroid aortae, alpha 1-adrenergic response was significantly decreased, the dose response curve shifted to the right and the maximal response was 30% less than the normal; alpha 2-adrenergic response was completely inhibited in hypothyroid state; also, IP injections of 0.1 mg/kg thyroxine twice in 48 h to thyroidectomized rats reversed the effects of hypothyroidism on both alpha 1- and alpha 2-adrenergic response. Hyperthyroidism did not alter alpha 1- and alpha 2-adrenergic response. These results signify the role of thyroid hormones in the regulation of alpha-adrenergic response in rat aortae.

Regulatory effects of S-100 protein and parvalbumin on protein kinases and phosphoprotein phosphatases from brain and skeletal muscle.

In the eluted fractions of histone-treated crude extracts separated by Sephadex G-200 filtration, multiple protein kinase (PK) activities, including three from brain and two from skeletal muscle, were augmented by both S-100 protein and parvalbumin on the phosphorylation of endogenous substrates. One additional PK activity suppressed by both S-100 and parvalbumin was also found in muscle. In comparison, phosphoprotein phosphatases (PPase), which were also prepared by the same procedure of initial step of histone-treatment followed by the steps of Bio-Gel P-6DG for brain and DNA-cellulose for muscle, were all activated by S-100 while inhibited by parvalbumin and phosphatidylserine.

Muscle protein synthesis: regulation of a translational inhibitor.

Insulin and branched-chain amino acids are known to stimulate protein synthesis in skeletal muscle. Extracts prepared from rat diaphragms after incubation in balanced salt solution and glucose alone yielded heat- and acid-stable, TCA-precipitable, nondialyzable factor(s) that inhibit protein synthesis when added to rabbit reticulocyte lysates. Polyribosomal profiles of inhibited lysates were consistent with a defect in peptide-chain initiation. Addition of insulin and amino acids to the diaphragm incubation media partially removed the inhibition seen with the muscle extract and was accompanied by an increase in polysomes and decreased subunits. Similarly, extracts prepared from rat hindlimb muscle 48 h after induction of diabetes were much more inhibitory in rabbit reticulocyte lysates than extracts from control rats. Polyribosomal profiles were consistent with defective peptide-chain initiation. Trypsin treatment before assay abolished the inhibitory activity of muscle extracts from diabetic rats. Because translation-inhibiting peptide(s) appear to be under metabolic and/or hormonal control, their possible role in muscle protein homeostasis warrants further study.