Fetal hyperinsulinemia and protein turnover in fetal rat tissues.

The effects of fetal hyperinsulinemia on protein turnover in various tissues of fetal rats were determined after transuteral injection of insulin to rat fetuses at day 19 of gestation. Tissue protein content was measured on the subsequent days of gestation (days 20-22), and protein synthesis was determined at day 20 of gestation in fetal tissues after intravenous injection of [3H]phenylalanine into the maternal circulation, followed by measurements of tissue free and protein-bound phenylalanine specific radioactivity in fetal diaphragm, brain, heart, and liver. Rates of protein degradation in these fetal tissues were calculated by subtracting protein accretion rates from rates of protein synthesis. The injection of insulin to rat fetuses at day 19 of gestation resulted in relative macrosomia versus saline-injected controls from the same litter (body wt at day 20 of gestation, 3.26 +/- 0.15 g for saline-injected fetuses and 3.60 +/- 0.25 g for insulin-injected fetuses, P less than 0.001) and increased protein and RNA content of brain, heart, and liver. Although fractional rates of protein synthesis were not significantly elevated in tissues from the hyperinsulinemic fetuses, absolute rates of protein synthesis were increased in brain, heart, and liver of hyperinsulinemic fetuses. Hyperinsulinemia did not reduce calculated rates of protein breakdown in fetal brain, heart, or liver but did in fetal diaphragm. We conclude that the major effect of fetal hyperinsulinemia on protein turnover in rats is to increase protein synthesis in selected tissues without simultaneously affecting protein breakdown.

Carry-over of sulphadimidine in the faeces and urine of pigs fed medicated feed.

In two experiments sulphadimidine-free pigs were placed in pens previously occupied by pigs fed a diet containing 100 ppm sulphadimidine. Faeces, urine and spilled feed had been removed by scraping the surface of the pens before the new pigs were introduced. The concentration of sulphadimidine in the tissues of the medicated pigs fell below 100 ng/g within 72 hours of withdrawal of the medicated diet and in the fluids the concentration fell below 500 ng/ml within 96 hours. The concentrations in the tissues of the pigs housed in the contaminated pens exceeded 100 ng/g for up to 24 hours but then fell to acceptable concentrations; the concentration of sulphadimidine in body fluids occasionally exceeded 500 ng/ml.

Effects of undernutrition on diaphragm fiber size, SDH activity, and fatigue resistance.

The influence of prolonged nutritional deprivation on the succinate dehydrogenase (SDH) activity and cross-sectional areas of individual fibers in the rat diaphragm and deep portion of the medial gastrocnemius (MGr) muscles was determined. Fatigue resistance of the diaphragm was measured by means of an in vitro nerve-muscle strip preparation. Fiber SDH activity and cross-sectional area were quantified by means of an image processing system. Diaphragm fatigue resistance was significantly improved in the nutritionally deprived (ND) group. In both muscles, nutritional deprivation resulted in a significant decrease in fiber cross-sectional area (both type I and II), type II fibers showing greater atrophy. The SDH activities of type I and II fibers in the diaphragm were not affected by nutritional deprivation. This contrasted with a significant decrease in the SDH activity of both type I and II fibers in the MGr of ND animals. An assessment of the interrelationships between fiber atrophy and fiber SDH activity revealed a greater effect of malnutrition on those diaphragm type II fibers that had the lowest relative SDH activities and the largest cross-sectional areas. By comparison, the effect of malnutrition on type I and II fibers in the MGr was nonselective with regard to fiber SDH activity. We conclude that the enhanced diaphragm fatigue resistance in the ND animals does not result from an increase in the oxidative capacity of muscle fibers and is best explained by the pattern of diaphragm muscle fiber atrophy.

Biochemical characteristics of mammalian diaphragms.

Selected biochemical characteristics of diaphragm muscle were compared among several orders of adult mammals (cattle, swine, rabbit, guinea pig, rat, and mouse) with known differences in resting breathing frequencies (f, range = 15-138). Diaphragms from smaller animals had significantly higher citrate synthase (CS) and phosphofructokinase (PFK) activities and substrate oxidation rates than larger animals. Ranges of activities for CS and PFK were 93-27 and 58-39 mumol.g-1.min-1, respectively; and 34-5 and 19-2 nmol.g-1.min-1 for [U-14C]glucose (GLU) and [1-14C]palmitate (PAL) oxidation, respectively. The percent of native fast myosin (FM) isoforms was significantly different among groups. Mouse diaphragm had the highest % FM (88.6%), whereas the lowest values (7.5%) were observed in cattle diaphragm. Myosin ATPase (M-ATPase, pH 9.8) activity was significantly lower in cattle (0.06 mumol.mg protein-1.min-1) and swine (0.38 mumol.mg protein-1.min-1) diaphragm than in other mammals (range of 1.14-0.67 mumol.mg protein-1.min-1). Correlation coefficients determined among means of measured biochemical parameters and established values of f indicated that CS activity and substrate oxidation rates were significantly correlated with f (r = 0.92, 0.92, 0.86 for CS, GLU, PAL, respectively) and the % FM increased with f. M-ATPase (pH 9.8) was significantly correlated with % FM (r = 0.85), whereas PFK and M-ATPase activities were not closely associated with f. It was concluded that f in mammals is significantly correlated with the biochemical parameters of aerobic capacity and is associated with the percent of FM isoforms in the diaphragm.

Three fast myosin heavy chains in adult rat skeletal muscle.

A new fast myosin heavy chain isoform was electrophoretically detected in adult rat skeletal muscles. It was present at high levels in diaphragm and, therefore, designated as MHCIId. Appreciable amounts of MHCIId were detected in tongue musculature, the extraocular muscles, and in the deep red portions of various fast muscles. Its concentration in fast-twitch muscle was greatly increased by chronic stimulation.

The blood-nerve barrier is rich in glucose transporter.

The glucose transporter of the facilitated diffusion type has been localized in sections of innervated rat diaphragm muscle and sciatic nerve by immunofluorescence, using affinity-purified antibodies against both the entire transporter and the carboxy-terminal peptide. In both tissues the transporter was very abundant in the perineurial sheath of cells surrounding the nerve fibres. The transporter also appeared to be abundant in the endoneurial blood vessels of the sciatic nerve. The identity of the antigen as the glucose transporter was established by extracting sciatic nerve with sodium dodecylsulphate and immunoblotting the extract. A single reactive polypeptide with the expected molecular weight of 55,000 was found. The high concentration of glucose transporter in the cells of the blood-nerve barrier presumably ensures an adequate supply of glucose to the nerve fibres.

Postmortem biochemical indices of antemortem hemorrhagic shock.

The purpose of this study was to determine if the perturbations in two glycolytic metabolites that occur during hemorrhagic shock can be used as discriminatory postmortem indicators of death resulting from severe hemorrhagic shock. Two groups of male albino Sprague-Dawley rats were hemorrhaged by withdrawing either 40% (Group I) or 45% (Group II) of the total blood volume. Glycogen and lactate concentrations were determined at 0 and 48 hr postmortem in the following tissues and organs: diaphragm, heart, liver, kidney cortex, and kidney medulla. The differences in lactate and glycogen in Group I at 0 hr were not significantly different from the nonhemorrhaged controls, with the exception of the lower liver glycogen concentration (58% of control). In Group II glycogen concentration was significantly reduced at 0 hr in the diaphragm (70% of control), liver (37%), and kidney medulla (55%). Lactate concentration was higher in all tissues examined by 270-640%; within 48 hr all tissues for both control and hemorrhaged animals had declined to baseline levels of glycogen concentration, whereas lactate levels had increased as much as 34-fold. There were no highly significant differences in glycogen at 48 hr between the control and hemorrhaged groups. In Group II the lactates were similar for both the control and hemorrhaged animals with the exception of the higher concentrations in the kidney cortex (54%) and medulla (41%). It was concluded from these findings that although significant metabolic perturbations are present at the time of death due to hemorrhage these differences do not persist up to 48 hr postmortem, with the possible exception of the kidney lactate concentrations.

Radioimmunoassay of the anabolic agent zeranol. IV. The determination of zeranol concentrations in the edible tissues of cattle implanted with zeranol (Ralgro).

Rapid solvent extraction combined with a radioimmunoassay using a monoclonal antibody raised against a derivative of zeranol has been used to measure the residues of the anabolic agent zeranol in the edible tissues (muscle, liver, kidney and fat) of cattle treated with Ralgro. Calibration curves, both with and without, tissue extracts exhibit good parallelism. Regression analysis for the extraction of zeranol from tissues dosed with standard amounts of zeranol have correlation coefficients of 0.979, 0.991, 0.986 and 0.985 for muscle, liver, kidney and fat, respectively. The limits of decision defined as the mean value + 3 SD for the concentrations apparently observed (noise) in tissues from animals not treated with Ralgro were 278, 121, 373 and 110 ng/kg for muscle, fat, liver and kidney, respectively. In the tissues of 4 cows implanted with Ralgro (36 mg), and sampled 70 days after implanting, the highest concentration of zeranol in each tissue was 232 ng/kg (muscle), 391 ng/kg (liver), 287 ng/kg (kidney) and 293 ng/kg (fat), and residues were detected in all samples of fat (4), 3 kidney samples and 1 liver sample.

Evaluation of microwave irradiation technique to quantitate acetylcholine in the diaphragm.

This report evaluates rapid enzyme inactivation prior to acetylcholine quantitation in the diaphragm. Methods of sacrifice were decapitation and microwave irradiation. Six-hundred millisecond exposure to microwave irradiation was sufficient to raise diaphragmatic temperature to 91 degrees C and inactivated 90% of the acetylcholinesterase enzyme. The acetylcholine content of the diaphragm was found to be 3.0 nmol/g, independent of the method of sacrifice. The postmortem changes observed in brain acetylcholine content following decapitation did not occur in the diaphragm.

Relationship between the pharmacological effects and the biodisposition of [3H]diisopropylfluorophosphate in mice after inhalation.

The biodisposition of [3H]diisopropylfluorophosphate (DFP) and its metabolites was studied in mice after inhalation administration. In addition, the time course of DFP-induced cholinesterase inhibition in selected tissues, hypothermia, and motor coordination were studied to determine a possible correlation with [3H]DFP, or its metabolites. The time course of tissue concentrations of [3H]DFP showed that [3H]DFP rapidly penetrated all tissues and was quickly hydrolyzed to [3H]diisopropylphosphoric acid (free [3H]DIP) or was covalently bound to tissue (bound [3H]DIP). By 1 hr, the greater portion of the radioactivity was in the form of bound [3H]DIP. Cholinesterase inhibition in brain, lung, diaphragm, and plasma was temporally related to concentrations of bound [3H]DIP between 5 min and 1 day, except at early time points for the lung. Motor incoordination (rotarod test) produced by DFP exposure had a rapid onset, with complete recovery by 10 hr. DFP-induced hypothermia (rectal temperature) had a very similar time-course profile to that of motor incoordination. The time course of hypothermia and motor incoordination was correlated with neither free [3H]DFP nor bound [3H]DIP concentrations in the brain, nor with cholinesterase inhibition in brain. These findings suggest that non-cholinesterase bound [3H]DIP may contribute to the depression of these centrally mediated effects.

Effects of low temperatures on larvae of the genus Trichinella.

We examined the effect of an exposure to -25 degrees C (for 8 days) on the histochemistry and the fine structure of 30-day-old Trichinella larvae from muscle fibres of the diaphragm. The larvae of T. pseudospiralis and T. nelsoni were either destroyed in the muscle fibres, dead, eosinophile, or were not found. The structureless mass of a degenerating, changed sarcoplasm was highly AIP-active, and gave a weak positive reaction for SS-groups of proteins. The wall of the deformed capsule around T. nelsoni, and the cuticle of the larva, stained diffusely; it did not contain AM. In a few muscle fibres exposed to -25 degrees C, histochemical reactions of the capsule surrounding larvae of T. nativa and sometimes of larvae of T. spiralis, and reaction of the changed sarcoplasm, were similar to those of the controls. A few mobile larvae were isolated by digestion only from a diaphragm infected with T. nativa. Deterrent to a prolonged survival of larvae were the formation of ice crystals and a denaturation of proteins by which the sarcoplasm of the infected muscle fibre was changed gradually into both a plasmolytically and karyolytically altered mass. Degenerative changes in the fine structure of infected muscle fibres were demonstrated by the presence of "spheromembranous bodies" in the sarcoplasm resembling myeline formations observed after exposure to poisonous substances, e.g., colchicine.

Heavy metals in fluids and tissues of fetal calves and in young calves of nursing cows exposed or not exposed to anaerobically digested wastewater sludge.

Fetal fluid, diaphragm muscle, heart, kidney, and liver from 14 four- to five-month-old calf fetuses were analyzed for Zn, Cu, Cd, Cr, Ni, Pb, and Fe. The same tissues and bone were examined from postparturient calves 30, 60, and 120 days of age. Weaned calves (8 to 9 months old) were necropsied and diaphragm muscle, heart, kidney, liver, brain, and bone were analyzed. Additional calves from cows exposed or not exposed to anaerobically digested sludge were placed in a feed yard at weaning for a feeding period of 150 days and then were killed and necropsied. Heavy metal content of the aforementioned tissues was compared with the same tissues obtained from peer calves weaned, killed, and necropsied 150 days earlier. Zinc, Cu, and Fe were up to 11 times higher in fetal livers than in control or exposed postparturient calves. These concentrations declined after birth and stabilized at approximately the concentrations observed in adult cattle at about 12 to 14 months of age. Cadmium accumulated to a greater extent in kidneys and livers of exposed calves than in control calves. However, the accumulation did not produce a detectable effect upon the functional or morphologic characteristics of the organs examined. Other metals were present at low levels and were generally not remarkably different in control and exposed animals.

Depletion of total acetylcholine by hemicholinium-3 in isolated rat diaphragm is less in the presence of dexamethasone.

Low concentrations of dexamethasone (Dex) stimulate the initial rate of radioactive choline (Ch) accumulation in the endplate-rich area (EPA) of indirectly stimulated hemidiaphragms, while higher concentrations (greater than 0.6 microM) inhibit. This biphasic concentration-effect curve is found even in the presence of 26 microM hemicholinium-3 (HC-3), an inhibitor of Ch accumulation. In incubations (3 min) where the total hemidiaphragm acetylcholine (ACh) content is not altered by 26 microM HC-3, the inhibition by HC-3 of both the Ch accumulation rate and the incorporation of radioactive Ch into ACh in the EPA of stimulated tissues is less in the presence of 0.2 microM Dex. In 120 min incubations with 15 microM HC-3 and without added Ch, the tissue ACh content is depleted in both stimulated and unstimulated hemidiaphragms. In both cases the depletion of ACh is significantly less in the presence of 0.2 microM Dex. In stimulated tissues a comparable depletion of ACh due to 15 microM HC-3 is also found with 1 and 10 microM Ch added to the medium. It is significantly less when 0.2 microM Dex and 1 microM Ch are added to the medium. In 120 min incubations with stimulated tissue, the amount of "bound' ACh is increased by addition of 30 microM Ch to the medium, decreased in the presence of 0.2 microM Dex, and greatly decreased in the presence of 15 microM HC-3. In the presence of Dex plus HC-3, the decrease in the amount of "bound' ACh due to either Dex or HC-3 alone, is abolished provided that 30 microM Ch is also present.(ABSTRACT TRUNCATED AT 250 WORDS)

Decrease of the spontaneous non-quantal release of acetylcholine from the phrenic nerve in botulinum-poisoned rat diaphragm.

Botulinum type A toxin (BoTx) has been found to diminish by 40% the spontaneous release of acetylcholine (ACh) from normal (acutely denervated) rat diaphragms incubated in the presence of 5 mM K+, while the release of ACh from chronically (4 days) denervated diaphragms was not affected during 2 h incubations. The toxin has been found to rapidly remove (within 10 min) the local depolarization of about 8 mV which developed in the end-plate zones of the diaphragms after the inhibition of cholinesterases; after the administration of BoTx, tubocurarine lost its ability to increase the resting membrane potential (H-response, Katz and Miledi 1977) in the end-plate area of anticholinesterase-treated muscles. It is concluded that BoTx inhibits the non-quantal release of ACh from the motor nerve fibres and that it probably acts directly on the nerve terminal surface membrane (without internalization). The H-response in the rat diaphragm reflects the non-quantal release of ACh from the nerve terminals and not from the muscle fibres.

A comparison of nerve transection and chronic application of beta-bungarotoxin on acetylcholine receptor distribution and other nerve-muscle properties.

beta-Bungarotoxin (beta-BuTX), a snake venom neurotoxin which acts presynaptically to inhibit acetylcholine (ACh) release at the neuromuscular junction, was applied to the rat phrenic nerve-diaphragm muscle preparation to determine its effectiveness to mimic denervation. The distribution of junctional and extrajunctional ACh receptors on the muscle were assayed biochemically by [125I]alpha-bungarotoxin ( [125I]alpha-BuTX) binding and electrophysiologically by iontophoretic application of ACh. Spontaneous transmitter release and muscle membrane potential were measured under conditions of denervation, beta-BuTX treatment, and bee venom phospholipase A2 exposure. Within 7 days after treatment with a single dose (5 micrograms/kg) of enzymatically active beta-BuTX, extrajunctional [125I]alpha-BuTX binding increased fivefold, and there was a decrease in miniature end-plate potential (MEPP) frequency and in resting membrane potential (RMP) to values less than those of control muscles but greater than those of denervated.

The fatty acid composition of the tissues of streptozotocin-diabetic rats.

The authors studied acute changes in the fatty acid composition of the tissues of streptozotocin-diabetic rats. They found that streptozotocin diabetes led to changes in the total lipids fatty acid spectrum in serum and in tissues (liver, adipose tissue, renal cortex diaphragm). After only 7 days' diabetes there was an increase in the percentual proportion of saturated fatty acids and a decrease in the amount of polyene fatty acids in the serum and in all the above tissue of diabetic animals. Palmitic acid (16:0) participated in the increase in the proportion of saturated fatty acids in all the given tissues, while stearic acid (18:0) played a role in the increase in the renal cortex and the serum. Among the monoene acids, there was a drop in the proportion of palmitoleic acid (16:1) in the adipose tissue and serum and in the amount of oleic acid (18:1) in the renal cortex, liver and muscle. Linoleic acid (18:2) played a role in the decrease in the proportion of polyene acids in all the given tissues and the serum, while arachidonic acid (20:4) was involved in the drop in the renal cortex, liver and muscle. The results show that diabetes leads to changes in the fatty acid composition of the renal cortex and muscle, as well as of the liver and adipose tissue. At present it is not yet clear whether there is an absolute decrease in the proportion of essential fatty acids, or whether diabetes is characterized by an increase in the amount of lipids in both serum and tissues.

The effect of glucose loading on steady-state lactate levels and contraction in the isolated rat diaphragm.

Isolated rat diaphragm strips were used to investigate two questions: (a) can the steady-state level of lactate in contracting muscles be increased by glucose loading resulting from the addition of increased external glucose (50 mM) in the presence of insulin (10 mU/mL)? (b) is the isometric tension developed by muscles contracting at different frequencies (0.125 to 1.0 Hz) affected by glucose loading? The results show that lactate levels in contracting muscles are increased by glucose loading over the whole range of contraction frequencies studied. Suppression of isometric contraction increases with contraction frequency but the extent of the suppression is not influenced by glucose loading. Steady-state lactate levels are well correlated with suppression in glucose-loaded muscles (r = 0.89) but not well correlated with suppression in normal muscles (r = 0.46). Isometric tension in the steady-state condition is well correlated with creatine phosphate levels (r = 0.98, 0.92 in glucose-loaded and normal muscles) and reasonably well with ATP (r = 0.88, 0.86, glucose loaded and normal). The increase in resting tension seen during development of steady-state conditions is reduced by glucose loading. It is concluded that several factors may contribute to the suppression of tension in contracting muscle but metabolic product inhibition, at least by products of glycolysis, does not normally play an important part in the isolated rat diaphragm preparation.

Chronic denervation of rat diaphragm: selective maintenance of adult fast myosin heavy chains.

After long-term denervation in mixed rat muscles there is a selective loss of slow myosin. The bidimensional electrophoretic pattern of light chains and the results of preliminary studies on heavy chains have left open the question of whether the nature of the residual fast-like myosin is of the immature or adult type. We have further investigated chronically denervated myosin by (1) electrophoresis in nondissociating conditions; (2) acidic electrophoresis of the heavy chains; and (3) proteolytic mapping of the heavy chains. These techniques clearly distinguish adult myosins from those present in immature muscles. Using these criteria, myosin from the chronically denervated diaphragm is of an adult type, even though the presence of trace amounts of embryonic myosin cannot be excluded. The contractile properties also indicate that chronically denervated hemidiaphragm is more similar to an adult fast muscle than to an immature muscle. The selective maintenance after long-term denervation of fast myosin in adult muscle provides good evidence of the independence of the genetic expression of myosin from a direct neural chemotrophic control.