Alloxan-induced luminol luminescence as a tool for investigating mechanisms of radical-mediated diabetogenicity.

It is not clear whether the muscle wasting commonly observed in hyperthyroidism is due to alteration in the rate of protein synthesis or degradation. The effect of experimental hyperthyroidism on skeletal-muscle proteolysis in the rat was studied by measuring alanine and tyrosine release from isolated skeletal muscles in vitro and 3-methyl-histidine excretion in vivo. Alanine release from the isolated epitrochlaris-muscle preparation was increased as soon as 24h after a 25 microgram dose of L-tri-iodothyronine in vivo. Conversely, alanine release from muscles of hypothyroid rats was decreased, but restored by L-tri-iodothyronine supplementation before death. Furthermore, 3-methylhistidine excretion was increased in hyperthyroid rats throughout an 18-day treatment period. The increased amino acid release from isolated muscles and the increased 3-methylhistidine excretion in vivo strongly suggests that hyperthyroidism increases skeletal-muscle proteolysis. Furthermore, the thyroid-hormone concentration may be an important factor in regulating muscle proteolysis.     

Time course of the effect of catabolic doses of corticosterone on protein turnover in rat skeletal muscle and liver.

The time course of the response of protein synthesis in muscle and liver to catabolic doses of corticosterone (10 mg/day per 100 g body wt.) was studied in vivo in growing rats over a 12-day period. The rate of protein synthesis in muscle and liver and the rate of actomyosin synthesis in muscle were measured by the phenylalanine-flooding technique, and 3-methylhistidine (N tau-methylhistidine) synthesis was measured by injection of labelled histidine. 3-Methylhistidine concentrations in tissue free pools and urinary excretion were also measured to compare directly with the rate of muscle protein degradation determined as the difference between synthesis and growth each day during the treatment. The overall rate of protein synthesis in muscle fell gradually over the first 4 days, reaching a rate after 5 days that was 36% of the initial rate, and this lower rate was then maintained for the following week. This decrease in the overall rate was accompanied with changes in the relative rate of synthesis in muscle proteins, since during the first 4 days there was a disproportionate decrease in the rate of actomyosin synthesis, and specifically 3-methylhistidine synthesis. In the latter case the synthesis rate was decreased to only 4% of its initial rate after 4 days. These changes in protein synthesis in muscle were accompanied by a transient increase in the rate of protein degradation, which was more than doubled on days 2 and 3 of treatment but which returned to the original rate on day 5, and a similar pattern of response was indicated by urinary 3-methylhistidine excretion, which also exhibited a transient increase. Thus in this case 3-methylhistidine excretion and measured rates of protein degradation in muscle do correlate. The transient effects of the glucocorticoids on degradation compared with the sustained effect on synthesis suggest that these two responses are achieved by different mechanisms. The hepatic size and protein mass were increased by the treatment, and protein synthesis was well maintained until after 12 days, when the rate was suppressed. Although the fractional synthesis rate was transiently increased for 24 h, it is argued that the enlarged liver most likely reflects a decrease in protein degradation resulting from the increased amino acid supply to the liver. This would result from the cessation of muscle growth while dietary supply was maintained.     

Myofibrillar protein degradation in the chicken. 3-Methylhistidine release in vivo and in vitro in normal and genetically muscular-dystrophic chickens

Myofibrillar protein degradation was measured in 4-week-old normal (line 412) and genetically muscular-dystrophic (line 413) New Hampshire chickens by monitoring the rates of 3-methylhistidine excretion in vivo and in vitro. A method of perfusing breast and wing muscles was developed and the rate of 3-methylhistidine release in vitro was measured between 30 and 90min of perfusion. During this perfusion period, 3-methylhistidine release from the muscle preparation was linear, indicating that changes in 3-methylhistidine concentration of the perfusate were the result of myofibrillar protein degradation. Furthermore, the viability of the perfused muscle was maintained during this interval. After 60min of perfusion, ATP, ADP and creatine phosphate concentrations in pectoral muscle were similar to muscle freeze-clamped in vivo. Rates of glucose uptake and lactate production were constant during the perfusion. In dystrophic-muscle preparations, the rate of 3-methylhistidine release in vitro (nmol/h per g of dried muscle) was elevated 2-fold when compared with that in normal muscle. From these data the fractional degradation rates of myofibrillar protein in normal and dystrophic pectoral muscle were calculated to be 12 and 24% respectively. Daily 3-methylhistidine excretion (nmol/day per g body wt.) in vivo was elevated 1.35-fold in dystrophic chickens. Additional studies revealed that the anti-dystrophic drugs diphenylhydantoin and methylsergide, which improve righting ability of dystrophic chickens, did not alter 3-methylhistidine release in vitro. This result implies that changes in myofibrillar protein turnover are not the primary lesion in avian muscular dystrophy. From tissue amino acid analysis, the myofibrillar 3-methylhistidine content per g dry weight of muscle was similar in normal and dystrophic pectoral muscle. More than 96% of the 3-methylhistidine present in pectoral muscle was associated with the myofibrillar fraction. Dystrophic myofibrillar protein contained significantly less 3-methylhistidine (nmol/g of myofibrillar protein) than protein from normal muscle. This observation supports the hypothesis that there may be a block in the biochemical maturation and development of dystrophic muscle after hatching. Free 3-methylhistidine (nmol/g wet wt.) was elevated in dystrophic muscle, whereas blood 3-methylhistidine concentrations were similar in both lines. In summary, the increased myofibrillar protein catabolism demonstrated in dystrophic pectoral muscle correlates with the increased lysosomal cathepsin activity in this tissue as reported by others.     

3-Methylhistidine turnover in the whole body, and the contribution of skeletal muscle and intestine to urinary 3-methylhistidine excretion in the adult rat.

The tissue origin of 3-methylhistidine (N tau-methylhistidine) was investigated in adult female rats. The decay of labelling of urinary 3-methylhistidine was compared with the labelling of protein-bound 3-methylhistidine in skeletal muscle and intestine after the injection of [methyl-14C]methionine. The decay curve for urinary 3-methylhistidine was much steeper than that in muscle or intestine, falling to values lower than those in either tissue after 30 days. The lack of decay of labelling in muscle during the first 30 days is shown to result from the persistence of label in the precursor S-adenosylmethionine. The relative labelling of urinary, skeletal-muscle and intestinal 3-methylhistidine cannot be explained in terms of skeletal muscle accounting for a major proportion of urinary 3-methylhistidine. Measurements were also made of the steady-state synthesis rate of protein-bound 3-methylhistidine in intestinal smooth muscle in vivo in adult female rats. This involved measurement of the overall rate of protein synthesis and measurement of the relative rates of synthesis of 3-methylhistidine and of mixed protein. The synthesis rate of 3-methylhistidine was 29.1%/day, compared with the overall rate of 77.1%/day for mixed, non-mucosal intestinal protein. Measurement of the amount of 3-methylhistidine in skeletal muscle (0.632 +/- 0.024 mumol/g) and in the whole body (0.332 +/- 0.013 mumol/g) indicate that, although the muscle pool is 86% of the total, because of its slow turnover rate of 1.1-1.6%/day, it only accounts for 38-52% of the observed excretion. Measurements of the mass of the intestine (9.95 g/250 g body wt.) and protein-bound 3-methylhistidine content (0.160 mumol/g of tissue) indicate a pool size of 1.59 mumol/250 micrograms rat. Thus 463 nmol of the urinary excretion/day would originate from the intestine, 22% of the total. The tissue source of the remaining urinary excretion is not identified, but other non-muscle sources constituting about 10% of the whole-body pool could account for this with turnover rates of only 6%/day, a much lower value than the turnover rate in the intestine.     

Experimental Methyl Mercury Neurotoxicity: Locus of Mercurial Inhibition of Brain Protein Synthesis In Vivo and In Vitro

Brain cell-free protein synthesis is inhibited by methyl mercury chloride (MeHg) following in vivo or in vitro administration. In this report, we have identified the locus of mercurial inhibition of translation. Intraperitoneal injection of MeHg (40 nmol/g body wt) induced variable inhibition of amino acid incorporation into the post-mitochondrial supernatant (PMS) harvested from the brain of young (10-20-day-old) rats. No mercurial-induced disaggregation of brain polyribosomes nor change in the proportion of 80S monoribosomes was detected on sucrose density gradients. No difference in total RNA was found in the PMS. Initiation complex formation was stimulated by MeHg, as detected by radiolabelled methionine binding to 80S monoribosomes following continuous sucrose density gradient centrifugation. After micrococcal nuclease digestion of endogenous mRNA, both in vivo and in vitro MeHg inhibited polyuridylic acid-directed incorporation of [3H]phenylalanine. However, the in vivo inhibition was no longer observed when [3H]phenylalanyl-tRNAPhe replaced free [3H]phenylalanine in the incorporation assay. The formation of peptidyl[3H]puromycin revealed no difference from controls. There was significant mercurial inhibition of phenylalanyl-tRNA Phe synthetase activity in pH 5 enzyme fractions derived from brain PMS of MeHg-poisoned rats. These experiments revealed that the apparent MeHg inhibition of brain translation in vivo and in vitro is due primarily to perturbation in the aminoacylation of tRNA and is not associated with defective initiation, elongation, or ribosomal function.     

Quantitative aspect of the myofibrillar protein turnover in transient state on dietary protein depletion and repletion revealed by urinary excretion of Nτ-Methylhistidine

The fractional rates of synthesis and breakdown of myosin and actin in skeletal muscle of younn adult male rats were measured during 2 weeks of ad libitum feeding of a protein-free diet, and 8 days of refeeding with an adequate protein diet. Daily urinary excretion of N^τ-Methylhistidine (3-methylhistidine) by the N^τ-methylhistidine pool of the body gave the fractional breakdown rate of the myosin-actin pool. The fractional synthesis rate of the myosin-actin pool was calculated from the fractional breakdown rate and the size of N^τ-methylhistidine pool in the body. The feeding of the protein-free diet resulted in a decreased in body weight and a decrease in daily urinary excretion of N^τ-methylhistidine. Refeeding caused an increase in body weight and a progressive increase in daily urinary excretion of N^τ-methylhistidine. At the start of the experiment, the fractional breakdown rate of the myosin-actin pool was 4% per day and with prolonged protein depletion, the rate decreased to 1.25% per day. The fractional synthesis rate also decreased more rapidly than the breakdown rate. On refeeding for one day with an adequate protein diet, the fractional synthesis rate increased from 0.75 to 5.75% per day. Accumulation of skeletal muscle protein by refeeding was accompanied by a difference between the faster rate of synthesis and slower rate of breakdown even though the fractional breakdown rate increased during the rehabilitation period.     

Effect of biotin on ribonucleic acid synthesis.

A single injection of biotin to biotin-deficient rats produces a two-fold increase in the incorporation, both in vivo and in vitro of precursors into nucleic acids as early as 2 h after the biotin treatment. The specific activity of the precursor pool is not affected by biotin. Analysis of the polysome profile at various times following biotin treatment and a kinetic study of the effect of excess poly(U) on the incorporation of phenylalanine by cell-free amino acid incorporation experiments indicate a marked decrease in messenger-free ribosomes in rat liver after biotin administration.     

Effect of glucocorticoid administration on the rate of muscle protein breakdown in vivo in rats, as measured by urinary excretion of Nτ-methylhistidine

The role of glucocorticoids in regulating the rate of muscle protein breakdown was evaluated by measuring excretion of N(tau)-methylhistidine during administration of various doses of corticosterone to adrenalectomized rats. Groups of rats received daily subcutaneous injections of 0, 0.2, 0.5, 1.0, 5.0 or 10.0mg of corticosterone/day per 100g body wt. for 7 days, followed by 3 days without hormone treatment, after which they were killed. A group with intact adrenal glands served as an additional control. All animals were pair-fed with the untreated adrenalectomized group. No significant differences were noted in growth rate or N(tau)-methylhistidine excretion between the intact or adrenalectomized control groups, or those given 0.2, 0.5 and 1.0mg of corticosterone, whereas growth ceased and N(tau)-methylhistidine excretion rose markedly in the groups receiving 5 and 10mg of corticosterone. After these two high doses of corticosterone, but not after lower doses, there was a loss of weight of the gastrocnemius muscle per 100g of final body wt., but not of the soleus and extensor digitorum longus muscles. The two highest doses of corticosterone also resulted in an increase in liver weight per 100g of final body wt. Lower doses of corticosterone did not cause these changes. Plasma corticosterone concentrations, measured on the final day of injection and again at the time of killing, were decreased to near zero by adrenalectomy and were little raised by doses of 0.2 and 0.5mg daily, but were increased to within the normal range by the 1mg dose. At 5 and 10mg doses, plasma corticosterone concentrations were sustained at 2-3 times those of intact rats, and thus in the range reported for rats exposed to severe stress. Rats given 5 and 10mg doses of corticosterone had glycosuria, and showed considerably elevated concentrations of insulin in the plasma. It is concluded that plasma concentrations of glucocorticoids within the normal range do not regulate the rate of muscle protein breakdown, whereas excessive plasma concentrations of corticosteroids, equivalent to those observed in severe stress, can accelerate muscle protein breakdown.     

An automatic method for determination of urinary and myofibrillar 3-methylhistidine: fractional rate of myofibrillar protein breakdown in rats fed on casein as source of protein

An automatic, more rapid and simplified analytical system for determination of 3-methylhistidine in urine and skeletal muscle is described, which may be applied to more extensive studies of large number of samples within a reasonable period of time and constitutes a powerful tool in understanding the dynamics of protein metabolism in the intact organism. This procedure allows the analysis of 3-Methylhistidine by ion-exchange chromatography in 140 +/- 2.5 min using a single column system. The mean urinary 3-Methylhistidine output of rats weighing about 200 g fed on an adequate diet of casein was 0.84 +/- 0.02 microM/100 g BW, and the mean values for skeletal muscle in these animals were 0.74 +/- 0.03 microM/g tissue. The fractional rate of myofibrillar protein breakdown assessed by the urinary 3-Methylhistidine was 0.028 +/- 0.002%.     

Measurement of concentrations of metabolites in adipose tissue and effects of insulin, alloxan-diabetes and adrenaline

1. Methods are described for the extraction and assay of ATP, ADP, AMP, glucose 6-phosphate, l-glycerol 3-phosphate and citrate in rat epididymal adipose tissue incubated in vitro for 1hr. At this time of incubation rates of glucose uptake and outputs of glycerol, free fatty acids, lactate and pyruvate were shown to be constant. 2. In fat pads incubated in medium containing glucose (3mg./ml.) and albumin (20mg./ml.) the concentrations (in mmumoles/g. wet wt.) were: ATP, 70; ADP, 36; AMP, 9.0; glucose 6-phosphate, 3.0; l-glycerol 3-phosphate, 3.3; citrate, 8.1. 3. The volume of intracellular water calculated from ([(3)H]water space-[(14)C]sorbitol space), ([(14)C]urea space-inulin space) and (weight loss on drying-[(14)C]sorbitol space) was 1.4ml./100g. wet wt. of tissue. The intracellular volume was not changed by insulin, alloxan-diabetes or adrenaline. 4. When compared in terms of mumoles/ml. of intracellular water the concentration of ATP in adipose tissue was less than in heart and diaphragm muscles. The concentrations of ADP and AMP were greater both in absolute terms and relative to ATP. Insulin, alloxan-diabetes and adrenaline had no significant effects on the concentrations of the adenine nucleotides in adipose tissue. 5. The concentration of glucose 6-phosphate was increased by insulin and lowered by alloxan-diabetes and adrenaline. The concentration of l-glycerol 3-phosphate was increased by insulin, unchanged by alloxan-diabetes and lowered by adrenaline. The concentration of citrate was increased by adrenaline and alloxan-diabetes and unchanged by insulin. 6. The effect of glucose concentration in the medium on rates of glucose uptake in adipose tissue from normal rats and alloxan-diabetic rats was investigated. The K(u) of glucose uptake was 29-44mg./100ml. and the V(max.) was 0.77mg./g. wet wt. of tissue/hr. Insulin increased the V(max.) and alloxan-diabetes diminished it, but neither agent significantly altered the K(u). 7. The significance of these results in relation to control of metabolism of adipose tissue is discussed.     

An automatic method for determination of urinary 3-methylhistidine: normal values.

A system for automatic analysis of urinary 3-methylhistidine is described, applying ion-exchange chromatography and using an automatic sample injector, a motoric selector valve, and a diode programmer, which controls the analytical system. The method permits a sampling rate of 22 samples/day. 3-Methylhistidine was completely separated from histidine in 37 min whereas 1-methylhistidine was eluted together with ammonia. The 3-methylhistidine concentration was linear up to 150 nmol/ml and no appreciable sample interaction was found at automatic sequential runs. The error, in a single determination based on duplicate samples, was 4.61% and, in duplicated determinations, 3.26%. The mean urinary 3-methylhistidine output was 299.4 ± 23.8 μmol/day in 12 healthy females and 545.5 ± 35.2 μmol/day in 12 healthy males. The 3-methylhistidine excretion was significantly higher in males than in females, when expressed as the absolute daily output or as the estimated ratio to body weight, body surface area, or creatinine.     

Effect of experimental diabetes on the incorporation of amino acids into protein in rat aorta.

The incorporation of 14C-labelled leucine or phenylalanine into alkali-soluble protein was determined under in vitro conditions in aortic intima-media of normal and streptozotocin-diabetic rats. Two weeks after the induction of diabetes the incorporation of the amino acids into aortic protein was reduced. When determined after diabetes of one week's duration the leucine-14C incorporation was not significantly reduced, while after 5 weeks of diabetes it was severely impaired. After administration of insulin to diabetic rats in vivo for 2 weeks there was no difference in leucine-14C incorporation between normal and diabetic rats. Addition of insulin (0.1 U/ml) in vitro had no effect on the leucine-14C incorporation in either normal or diabetic aorta during incubation times of 3 or 6 h. Elevation of the glucose concentration in vitro from 5.6 to 22.2 mmol/l did not influence the leucine incorporation in diabetic aorta. Both the aortic wet weight and the aortic content of alkali-soluble protein were decreased after 5 weeks of diabetes. The decrease in the protein content of aorta of diabetic animals suggest that the protein synthesis is impaired in vivo.     

Influence of vitamin A status and DDT on vitamin A-dependent protein mannosylation in rat liver

Male Wistar rats of different vitamin A status (total depletion to moderate deficiency) were administered DDT (5 mg/kg/day) or vehicule (corn oil) i.p. daily for 14 days. Vitamin A-dependent protein mannosylation was measured either by in vivo incorporation of [3H]mannose into liver glycoprotein or by in vitro assay of incorporation of [14C]mannose into mannosylretinyl phosphate. Vitamin A deficiency resulted in a significantly impaired in vivo incorporation of mannose in liver glycoprotein but had no effect on the in vitro transport of mannose via retinyl phosphate. Although DDT induced an increase synthesis of liver proteins in smooth endoplasmic reticulum and caused a diminution of the hepatic vitamin A content, it did not affect vitamin A-dependent protein mannosylation.     

Superoxide-forming NADPH oxidase preparation of pig polymorphonuclear leucocyte.

The effect of corticosterone on myofibrillar protein breakdown in diabetic rats was investigated in order to assess the possible counteracting effects of the secondary rise in plasma insulin concentrations which normally accompanies such treatment. N^τ-Methylhistidine excretion, an index of myofibrillar protein breakdown, was compared before and after corticosterone treatment (4.0 mg/100 g body wt. per day) of normal control, adrenalectomized, 10-day-streptozotocin-diabetic and adrenalectomized diabetic rats. Diabetic rats received 1.5 units of insulin/100 g body wt. per day throughout the experiment and showed marked hyperglycaemia and glucosuria during corticosterone treatment, whereas non-diabetic rats had only mild hyperglycaemia but elevated insulin concentrations. Corticosterone treatment increased the average rate of myofibrillar protein breakdown by 68% and 95% respectively in non-diabetic and diabetic rats. Net loss of muscle non-collagen protein for the same 7-day period was greater in diabetic than in non-diabetic animals (4.15 versus 2.84% per day), and the calculated average synthesis rates were lowest in diabetic rats. Adrenalectomy had little effect except to decrease slightly the rate of muscle protein breakdown. These results show that the rise in plasma insulin concentrations that accompanies exogenous corticosterone administration to non-diabetic rats diminishes the catabolic effect of this glucocorticoid on muscle. Insulin appears to antagonize the effects of the glucocorticoid by attenuating the increased rates of myofibrillar protein breakdown and, to a lesser extent, by limiting the decrease in synthesis rates.     

Effect of hyperphenylalaninaemia on lipid synthesis from ketone bodies by rat brain.

The effect of hyperphenylalaninaemia on the metabolism of ketone bodies in vivo and in vitro by developing rat brain was investigated. The incorporation in vivo of [14C]acetoacetate into cerebral lipids was decreased by both chronic (for 3 days) and acute (for 6h) hyperphenylalaninaemia induced by injecting phenylalanine into 1-week-old rats. In studies in vitro it was observed that the incorporation of the radioactivity from [14C]acetoacetate and 3-hydroxy[14C]butyrate into cerebral lipids was inhibited by phenyl-pyruvate, but not by phenylalanine. Phenylpyruvate also inhibited the incorporation of 3H from 3H2O into lipids by brain slices metabolizing either 3-hydroxybutyrate or acetoacetate in the presence of glucose. These findings suggest that the decrease in the incorporation in vivo of [14C]acetoacetate into cerebral lipids in hyperphenylalaninaemic rats is most likely caused by phenylpyruvate and not by phenylalanine. Phenylpyruvate as well as phenylalanine had no inhibitory effects on ketone-body-catabolizing enzymes, namely 3-hydroxybutyrate dehydrogenase, 3-oxo acid CoA-transferase and acetoacetyl-CoA thiolase, in rat brain. Phenylpyruvate but not phenylalanine inhibited the activity of the 2-oxoglutarate dehydrogenase complex from rat and human brain. These findings suggest that the metabolism of ketone bodies is impaired in brains of untreated phenylketonuric patients, and in turn may contribute to the diminution of mental development and function associated with phenylketonuria.     

Effect of corticosterone treatment on muscle protein turnover in adrenalectomized rats and diabetic rats maintained on insulin

The effect of corticosterone on protein turnover in skeletal muscle was investigated in growing rats. Protein synthesis was measured in vivo by the constant infusion of [(14)C]tyrosine. The extent to which any effect of corticosterone is modulated by the hyperinsulinaemia induced by steroid treatment was examined by giving the hormone not only to adrenalectomized rats but also to streptozotocin-induced diabetic rats maintained throughout the treatment period on two dosages of insulin by an implanted osmotic minipump. Approximate rates of protein degradation were also estimated in some cases as the difference between synthesis and net change in muscle protein mass. Measurements were also made of free 3-methylhistidine concentration in muscle and plasma. At 10mg of corticosterone/100g body wt. per day, growth stopped and muscle wasting occurred, whereas at 5 mg of corticosterone/100g body wt. per day no net loss of protein occurred. However, this low dose did induce muscle wasting when insulin concentration was regulated by a dose of 1.2 units/day. Protein synthesis was markedly depressed in all treated groups, the depression in the insulin-maintained rats being marginally more than in the hyperinsulinaemic adrenalectomized rats. The oxidative soleus muscle appeared to be less susceptible to the effect of the corticosterone than was the more glycolytic plantaris or gastrocnemius muscle. Any effect of the corticosterone on protein degradation was much less than its effects on protein synthesis. Where increases in the degradation rates appeared to occur in the rats treated with 10mg of corticosterone/100g body wt. per day, the increases were less than 20%. The free intracellular 3-methylhistidine concentrations were doubled in all groups treated with 5 mg of corticosterone/100g body wt. per day and increased 5-fold in the adrenalectomized rats treated with 10mg of corticosterone/100g body wt. per day, with no change in plasma concentration in any of the groups. It is therefore concluded that: (a) the suppression of protein synthesis is the main effect of glucocorticoids in muscle; (b) marked increases in insulin afford only minor protection against this effect; (c) stimulation of protein degradation may occur, but to a much lesser extent.     

Stimulation of epidermal protein synthesis in vivo by topical triamcinolone acetonide.

The rate of epidermal protein synthesis in vivo was determined in the hairless mouse by a method in which a large dose of [3H]phenylalanine (150 mumol/100 g body wt.) is administered via the tail vein. The epidermal free phenylalanine specific radioactivity rapidly rose to a plateau value which by 10 min approached that of plasma, after which it declined. This dose of phenylalanine did not of itself alter protein synthesis rates, since incorporation of co-injected tracer doses of [3H]lysine and [14C]threonine was unaffected. The fractional rate of protein synthesis obtained for epidermis was 61.6%/day, whereas values for liver and gastrocnemius muscle in the same group of mice were 44%/day and 4.8%/day respectively. When expressed on the basis of RNA content, the value for epidermis (18.6 mg of protein/day per mg of RNA) was approx. 3-fold higher than those for liver and gastrocnemius muscle. Topical administration of 0.1% triamcinolone acetonide increased the epidermal fractional protein synthesis rate by 33% after 1 day and by 69% after 7 days, compared with vehicle-treated controls. These effects were entirely accounted for by the increase in protein synthesis rates per mg of RNA. RNA/protein ratios were unaffected by this treatment.     

INCORPORATION OF PHENYLALANINE, TYROSINE AND TRYPTOPHAN INTO PROTEIN OF HOMOGENATES FROM DEVELOPING RAT BRAIN: KINETICS OF INCORPORATION AND RECIPROCAL INHIBITION

The kinetics of the incorporation into protein of [³H]phenylalanine, [³H]tyrosine and [³H]tryptophan were studied with homogenates prepared from whole brain of 1-, 7-, 21- and 60-day-old rats. The maximal velocities (V_max)of incorporation of phenylalanine and tyrosine decreased and the apparent Michaelis-constants (K_m) for all three amino acids increased with increasing age of the rats. Tyrosine had the smallest and tryptophan the largest K_m values in all age groups. Phenylalanine competitively inhibited the incorporation of tyrosine, but tyrosine inhibited non-competitively the incorporation of phenylalanine. Tryptophan inhibited competitively the incorporation of phenylalanine, but at least partially non-competitively the incorporation of tyrosine. Phenylalanine and tyrosine did not significantly affect the incorporation of tryptophan in homogenates from 60-day-old rats. In 1-day-old rats only a very large excess of phenylalanine or tyrosine inhibited detectably. The K_i for phenylalanine in the incorporation of tyrosine was significantly smaller in 1- than in 60-day-old rats. In every case the inhibition presumably occurred at a single rate-limiting step in the complicated process of incorporation of amino acids into protein.     

Testosterone response of cryptorchid and hypophysectomized rats to human chorionic gonadotrophin (hCG) stimulation

The testosterone responses to a single injection of hCG (100 i.u.) in hypophysectomized (hypox.), cryptorchid or sham-operated rats were followed over a 5-day period. In sham-operated rats, hCG induced a biphasic rise in serum testosterone, peaks being observed at 2 and 72 h. Reduced testis weights, elevated FSH and LH levels and reduced serum testosterone levels were found after 4 weeks of cryptorchidism, but hCG stimulation resulted in a normal 2 h peak in serum testosterone. However, the secondary rise at 72 h in cryptorchid rats was significantly lower than sham-operated rats. Reduced testis weight and undetectable serum FSH and LH levels together with decreased testosterone levels were found 4 weeks after hypophysectomy. Serum testosterone levels rose 2 h after hCG in comparison to hypox. controls but this peak was significantly reduced compared with sham-operated rats. The second rise in serum testosterone began on day 2, peaking on day 4 at levels comparable to that seen in sham-operated rats after hCG. The in vitro basal and hCG stimulated secretion of testosterone by cryptorchid testes was greater than that secreted by normal rat testes (518.0 +/- 45.9 and 3337.6 +/- 304.1 pmol per testis per 4 h compared with 223.6 +/- 24.9 and 1312.9 +/- 141.4 pmol per testis per 4 h for normal rat testes). In cryptorchid animals a single injection of 100 i.u. hCG resulted in a pattern of in vitro refractoriness similar to normal rats, lasting from 12 h to 2 days, during which testosterone secretion was reduced to near basal levels. The in vitro basal and hCG-stimulated secretion of testosterone by hypox. rat testes was severely diminished compared with normal rat testes. The temporal pattern of in vitro secretion of testosterone from hypox. rat testes mimicked the in vivo serum testosterone pattern seen in these animals. This study demonstrates important differences in the in vivo and in vitro testosterone response to hCG after testicular damage.     

Impairment of pancreatic acinar function by reserpine in vivo and in vitro

Summary Chronic reserpine treatment of animals, an experimental model for cystic fibrosis (CF), results in generalized exocrinopathy, impaired pancreatic secretion, and decreased pancreatic content of amylase. The mechanisms of altered acinar function and decreased amylase content in both CF and the reserpine-treated rat are unknown. To examine this alteration, the rate of [³H]phenylalanine (phe) incorporation into cellular protein was determined in pancreatic acinar cells after reserpine treatment of rats in vivo (7 d) and of cells in vitro (1 to 24 h). Acinar cells isolated from control, chronic reserpine-treated, and pair-fed rats were incubated in vitro with 0, 30, 50, or 100 μM reserpine. Reserpine treatment in vitro for 24 h of acinar cells from control rats significantly decreased amylase activity (20 to 70%), an effect similar to that of reserpine treatment in vivo. In vivo, reserpine treatment decreased [³H]phe incorporation (disintegrations per minute per milligram protein) 56% in freshly isolated cells, but did not alter intracellular specific activity (disintegrations per minute per nanomole phe, SA) of [³H]phe. Reserpine treatment (30 and 50 μM) in vitro for 1 h also decreased [³H]phe incorporation by freshly isolated cells from control (53 to 85%) and pair-fed (40 to 68%) rats. Reserpine treatment for 24 h in vitro significantly decreased [³H]phe incorporation by cells from control (82 and 98%), pair-fed (80 and 95%), and chronic reserpine-treated (90 and 97%) rats as compared with cells from respective in vivo treatments cultured with no reserpine. In vitro reserpine treatment also decreased the intracellular SA of [³H]phe in freshly isolated cells from control (14 and 36%) and pair-fed (35 and 39%) rats and in cultured cells from control (11 and 86%), pair-fed (60 and 88%), and chronic reserpine-treated (49 and 76%) rats. However, these alterations of SA by reserpine did not account for the decreased incorporation of [³H]phe into acinar protein, which remained significantly lower (70 to 88%) when expressed as total phe incorporation. These results suggest (a) that reserpine acts directly on acinar cells to alter function and (b) that the ability of the pancreas to synthesize digestive enzymes may be impaired in this model of cystic fibrosis. This study was supported in part by the Cystic Fibrosis Foundation, Bethesda, MD.