Follicular fluid lipoproteins in the mare: Evaluation of HDL transfer from plasma to follicular fluid

Using a density gradient ultracentrifugal procedure, we have separated equine plasma and follicular fluid high-density lipoproteins (HDL). The density distribution of the follicular fluid HDL was clearly displaced towards the highest densities in comparison with that of plasma HDL. Similarly, an analysis of size distributions showed a decrease in follicular fluid HDL diameters (4.2 to 9.2 nm) compared to plasma HDL (5.5 to 9.5 nm). HDL were isolated into three subfractions on the basis of the disposition of the Sudan Black stained bands in the centrifuge tubes. Concentrations of each subfraction were clearly lower in the follicular fluid, and the relative percentages with regard to the plasma equivalents were inversely proportional to the molecular weights (23.8% for HDL-1, 49.9% for HDL-2 and 63.7% for HDL-3). The cholesterol/phospholipid molar ratio and the esterified/free cholesterol molar ratio were clearly increased in the follicular HDL-2 and HDL-3 subfractions. The apolipoprotein distribution in follicular fluid HDL was very close to that in plasma HDL. LCAT activity measured in human as well as equine samples was weaker in follicular fluid compared to plasma in both species (4.0 nmol of free cholesterol esterified per h per ml vs. 24 nmol per h per ml). Theoretical concentrations of follicular fluid HDL were calculated assuming that the HDL particles would be merely a filtration product undergoing no detectable metabolic modifications. Biochemical measurements showed that the lightest particules (HDL-1) were less numerous than suggested by the theoretical calculation. Thus, although follicular fluid HDL appear to be a filtration product of plasma HDL, they undergo metabolic transformations that we suggest may be linked to hormonal synthesis and reverse cholesterol transport.     

Formation of Polyamines in the Rumen of Goats During Growth

Ornithine decarboxylase (E.G. 4.1.1.17) and S-adenosylmethionine decarboxylase (E.G. 4.1.1.50) and their products putrescine, spermidine and spermine were estimated in the rumen liquid from 3 groups of growing kids and 23 adult goats. Polyamines were also estimated in the feedstuff used. Marked differences in polyamine synthesis in rumen liquid were observed between the different groups of kids. Two groups of kids growing up together with adult goats had at an age of 2–4 months a peak of a few days duration in enzyme activity as well as in polyamine concentration. In these groups ornithine decarboxylase activity reached maximal values of 158±79 s (n = 4) and 100 (66–117) (n = 3) nmol[14CO2]/ml rumen liquid/h at an age of 120 and 77 days, respectively. The corresponding activity in rumen liquid from kids who were isolated from other animals was only about 1/10 of this value. By comparison ornithine decarboxylase activity in adult goats was 30.7±20 (n = 43) nmol[14CO]/ml/h. In rumen liquid from kids grown up together with adults, concentrations of the polyamines reached maximum at about the same time as ornithine decarboxylase activity. The mean maximal concentration of putrescine in the 2 groups was about 350 and 500 nmol/ml, while the corresponding value for spermidine was about 200 nmol/ml in both groups. Relatively constant and high concentration of polyamines were present in the feedstuff used. However, in growing kids the ruminai putrescine and spermidine concentration at times far exceeded those that could be accounted for by the estimated intake of polyamines by the food. The results therefore strongly indicate that polyamines are formed in considerable amounts in rumen content of kids during the phase of rapid growth. Results from a few experiments with calves also indicate that this may be true for cattle. polyamines; putrescine; spermidine; spermine; ornithine-decarboxylase; rumen liquid.     

Tissue Choline Studied Using a Simple Chemical Assay

Abstract: An enzymatic-radioisotopic assay was used to measure free choline in unextracted tissue. The lowest concentration of free choline in any tissue studied was present in human cerebrospinal fluid (mean, 5.7 μM; range, 1.8–31.2 μM). A postmortem increase in concentration of free choline occurred in blood (O.2 nmol/min ml), kidney (13 nmol/min·g), and liver (22 nmol/min·g) of mice. The concentration of free choline in these tissues was estimated by extrapolation to be 5, 77, and 29 nmol/g (or ml), respectively. Several treatments were found to increase the concentration of free choline. For example, intraperitoneal administration of choline or 2-amino-2-methyl-propanol (a choline oxidase inhibitor) induced an increase in the level of choline in blood, kidneys, liver, and brain of mice, and administration of 2-dimethylaminoethanol (deanol) caused an increase in kidney and liver choline. The level of choline in blood was increased when rats were treated orally with either antibiotics or esters of choline such as phosphorylcholine, glycerylphos-phorylcholine, laroylcholine, or propionylcholine. The results show that the concentration of free choline may be regulated by intestinal metabolism, availability of esterified precursors, and activity of enzymes that metabolize choline.     

Simultaneous determination of nefiracetam and its metabolites by high-performance liquid chromatography

A reversed-phase high-performance liquid chromatographic assay was developed to simultaneously quantitate nefiracetam (NEF), a novel nootropic agent, and its three known oxidized metabolites (N-[(2,6-dimethylphenylcarbamoyl)methyl]succinamic acid (5-COOH-NEF), 4-hydroxy-NEF and 5-hydroxy-NEF) in human serum and urine. The quantitative procedure was based on solid-phase extraction with Sep-Pak C₁₈ and ultraviolet detection at 210 nm. The calibration curves of NEF and the metabolites were linear over a wide range of concentrations (0.5–21.5 nmol/ml for NEF and 0.4–9.5 nmol/ml for metabolites in serum and 4–86 nmol/ml for NEF and 8–190 nmol/ml for metabolites in urine). Intra- and inter-day assay coefficients of variation for the compounds were less than 10%. The limit of detection was 0.1 nmol/ml for NEF, 5-COOH-NEF and 4-hydroxy-NEF, and 0.2 nmol/ml for 5-hydroxy-NEF in both serum and urine. This method is applicable for the determination of NEF and its metabolites in human serum and urine with satisfactory accuracy and precision.     

Effects of intracerebroventricular injection of delta-opioid receptor agonist TAN-67 or antagonist naltrindole on acute cerebral ischemia in rat

This work was performed to determine the role of delta-opioid receptor (DOR) in protection against acute ischemia/reperfusion injury. Transient (1 h) focal cerebral ischemia was induced by middle cerebral artery occlusion (MCAO). DOR agonist TAN-67 (30 nmol, 60 nmol, 200 nmol), DOR antagonist naltrindole (20 nmol, 50 nmol, 100 nmol) or artificial cerebral spinal fluid (aCSF) was injected respectively into the lateral cerebroventricle of the rat 30 min before the induction of brain ischemia. Neurological deficits were assessed by the five-grade system (Longa's methods). The brain infarct was measured by cresyl violet (CV) staining and infarct volume was analyzed by an image processing and analysis system. The expression of DOR was detected by Western blot. The results showed that 60 nmol TAN-67 significantly reduced the infarct volume (P<0.05), attenuated neurological deficits (P<0.05) and tended to increase the expression of about 60 kDa DOR protein (P>0.05), while 100 nmol naltrindole aggravated ischemic damage and decreased about 60 kDa DOR protein expression (P<0.05). These results suggest that DOR activation protects the brain against acute ischemia/reperfusion injury in rat.     

Some biochemical characteristics and preservation of epididymal camel spermatozoa (Camelus dromedarius)

Testicles were isolated from thirty five apparently healthy dromedary camels (Camelus dromedarius), aged between 5 to 18 years, in a local slaughterhouse during the rutting season. Epididymal fluid was collected from one epididymis for determination of twelve biochemical and antioxidant parameters using ELISA commercial kits. Spermatozoa were harvested from each region of the other epididymis (head, body and tail) and stored in SHOTOR^®, Green buffer^® + 20% egg yolk and INRA-96^® extenders at 5 and 30 °C. Results revealed that, in the epididymal fluid, concentrations of testosterone, glucose, albumin, total protein, cholesterol, fatty acids, iron, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were 5.19 ± 1.69 ng/mL, 3.10 ± 0.41 mmol/L, 6.26 ± 1.26 g/dL, 0.50 ± 0.07 mg/dL, 1.74 ± 0.09 mmol/L, 6.62 ± 0.81 nmol/ul, 926.20 ± 100.18 ug/dL, 51.17 ± 7.74 mIU/ml, and 143.16 ± 18.67 mIU/ml, respectively. The antioxidants activities of catalase, glutathione peroxidase (GPx) and superoxide dismutase (SOD) in the epididymal fluid were 121.55 ± 6.57 nmol/min/ml, 59.35 ± 10.98 nmol/min/ml and 0.18 ± 0.03 U/ml, respectively. Epididymal sperm motility and concentration were higher (P < 0.05) in the body and tail than the head. The viability indices of total and forward sperm motility, at 5 and 30 °C, obtained from the tail region were superior (P < 0.05) in both SHOTOR^® and INRA-96^® extenders than Green buffer extender. It may be concluded that INRA-96^® extender is the best for storing dromedary epididymal spermatozoa at 5 and 30 °C.     

Changes in the levels of growth hormones, insulin, cortisol, thyroxine and somatomedin-C/IGF-1, with increasing gestational age in the fetal pig, and the effect of thyroidectomy in utero

1. Blood samples were taken from 30 chronically catheterized pig fetuses in utero. Levels of growth hormone, insulin, cortisol, thyroxine and somatomedin-C/IGF-1 were measured in the plasma of intact fetuses and the plasma of thyroidectomized fetuses at various gestational ages during the latter part of pregnancy. 2. Growth hormone levels were high (mean +/- SEM: 83 +/- 9 ng/ml and remained constant throughout this period. 3. Insulin levels were also constant and ranged between 4 and 14 mU/l. 4. Cortisol levels showed a general increase from 400 nmol/l at 97 days to 1200 nmol/l at term and this increase was not affected by thyroidectomy. 5. IGF-1 levels were lower than in the sows (48.0 +/- 3.0 ng/ml) and did not change throughout this period. 6. Thyroxine levels were also unchanged at about 92 +/- 4 nmol/l. 7. Thyroidectomy resulted in lower (P less than 0.001) thyroxine levels (28 +/- 3 nmol/l) but had no effect on the levels of any other hormone.     

Determination of serum and plasma uridine levels in mice,rats, and humans by high-pressure liquid chromatography

Serum and plasma uridine levels in mice, rats, and humans were conveniently measured by reverse-phase high-pressure liquid chromatography. Human serum levels were in the range of 1.9 to 8.4 nmol/ml, rat serum levels ranged from 3.7 to 9.4 nmol/ml, and mouse serum levels measured 8.0 to 11.8 nmol/ml. Levels of uridine were the same in human plasma and serum; however, plasma from mice and rats was somewhat lower than serum in uridine content ranging from 1.7 to 4.1 nmol/ml in rats and 1.5 to 4.7 nmol/ml in mice. There was some variation in the individual human serum and rat plasma uridine levels throughout the day, but the values were within the normal range, and the variations had no set pattern. Withholding food for 16–24 h had no observable effect on serum uridine levels in mice and humans or on rat plasma levels. These results suggest that uridine levels are regulated and are not a direct reflection of dietary intake of uridine.     

Occurrence of sulfatide as a major glycosphingolipid in WHHL rabbit serum lipoproteins

Glycosphingolipids in serum and lipoproteins from Watanabe hereditable hyperlipidemic rabbit (WHHL rabbit), which is an animal model for human familial hypercholesterolemia (FH), were analyzed for the first time in this study. Chylomicrons and very low density, low density, and high density lipoproteins contained sulfatide as a major glycosphingolipid (12 nmol/mumol total phospholipids (PL) in chylomicrons, 19 nmol/mumol PL in VLDL, 18 nmol/mumol PL in LDL, and 14 nmol/mumol PL in HDL) with other minor glycosphingolipids such as glucosylceramide, galactosylceramide, GM3 ganglioside, lactosylceramide, and globotriaosylceramide. The concentration of sulfatide as a major glycosphingolipid in WHHL rabbit serum (121 nmol/ml) was much higher than that in normal rabbit serum (3 nmol/ml). Fatty acids of the sulfatides comprised mainly nonhydroxy fatty acids (C22, 23, and 24) and significant amounts of hydroxy fatty acids (about 10%) whereas long chain bases of the sulfatides comprised mostly (4E)-sphingenine with a significant amount of 4D-hydroxysphinganine (about 10%). Furthermore, sulfatides in the liver and small intestine from normal and WHHL rabbits (where serum lipoproteins are produced) were determined to amount to 260 nmol/g liver in WHHL rabbit, 104 nmol/g liver in control rabbit, 99.6 nmol/g small intestine in WHHL rabbit, and 31.2 nmol/g small intestine in control rabbit. Ceramide portions of the sulfatides in the liver were mainly composed of (4E)-sphingenine and nonhydroxy fatty acids, while those in the small intestine were mainly composed of 4D-hydroxysphinganine and hydroxy fatty acids. These results indicated that the sulfatides of serum lipoproteins were mostly derived from the liver (90% of the total), and that the remaining sulfatides (10% of the total) might be derived from the small intestine. These two sulfatides, which have different ceramide portions, could be useful markers for metabolic and biosynthetic studies of various lipoproteins in WHHL rabbit, and thus would be helpful to further elucidate the relationship between hypercholesterolemia and atherosclerosis in the rabbit.     

Enrichment and isolation of a ruminal bacterium with a very high specific activity of ammonia production

When mixed ruminal bacteria were inoculated into semicontinuous cultures (25% transfer every other day) containing lactate, dulcitol, pectin, or xylose and Trypticase (1 g/liter) as the sole nitrogen source, the specific activity of ammonia production increased. The greatest enrichment was observed with lactate and xylose, and in these cases the specific rate of ammonia production was eightfold higher than that of the ruminal fluid control (approximately 35 nmol of ammonia per mg of protein per min). Isolates with different morphologies were obtained from each of the enrichments, but in no case did the specific activity of any isolate exceed that of the mixed ruminal bacteria. If Trypticase (15 g/liter) was used as the only energy and nitrogen source, there was an even greater increase in ammonia production, and two monensin-sensitive bacteria, a Peptostreptococcus species and a Clostridium species, were obtained. The Peptostreptococcus species was unable to grow on any of 25 carbohydrate or carbohydrate derivatives tested; but the Clostridium species was able to use glucose, maltose, fructose, cellobiose, trehalose, sorbitol, and salicin as energy sources. Neither organism was able to grow in the absence of an amino acid source, but growth rates on Trypticase were greater than 0.35/h. The specific activities of ammonia production were 346 and 427 nmol/mg of protein per min for strains of Peptostreptococcus and Clostridium, respectively. Megasphaera elsdenii and Bacteroides ruminicola, previously isolated ruminal ammonia producers, had specific activities of only 11 and 19 nmol of ammonia per mg of protein per min, respectively. The most probable number of Clostridium species in ruminal fluid was less than 10(3)/ml, but the Peptostreptococcus species was present at 10(8)/ml.(ABSTRACT TRUNCATED AT 250 WORDS)     

Determination of l-carnitine,acetyl-l-carnitine and propionyl-l-carnitine in human plasma by high-performance liquid chromatography after pre-column derivatization with 1-aminoanthracene

A new sensitive high-performance liquid chromatographic procedure for the determination of l-carnitine (LC), acetyl-l-carnitine (ALC) and propionyl-l-carnitine (PLC) in human plasma has been developed. Precolumn derivatization with 1-aminoanthracene (1AA), performed in phosphate buffer in the presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) as catalyst, is involved. The fluorescent derivatives were isocratically separated on a reversed-phase column (C₁₈). The eluate was monitored with a fluorimetric detector set at 248 nm (excitation wavelength) and 418 nm (emission wavelength). Because of the presence of endogenous carnitines, the validation was performed using dialyzed plasma. The identity of the derivatized compounds was assessed by mass spectrometry and the purity of the chromatographic peaks was confirmed by HPLC-tandem mass spectrometry. The limits of quantitation were 5 nmol/ml for LC, 1 nmol/ml for ALC and 0.25 nmol/ml for PLC. The recovery of the extraction procedure was in the range 82.6%–95.4% for all 3 compounds. Good linearity (R≈0.99) was observed within the calibration ranges studied: 5–160 nmol/ml for LC, 1–32 nmol/ml for ALC and 0.25–8 nmol/ml for PLC. Precision was in the range 0.3–16.8% and accuracy was always lower than 10.6%.     

Enrichment and isolation of a ruminal bacterium with a very high specific activity of ammonia production.

When mixed ruminal bacteria were inoculated into semicontinuous cultures (25% transfer every other day) containing lactate, dulcitol, pectin, or xylose and Trypticase (1 g/liter) as the sole nitrogen source, the specific activity of ammonia production increased. The greatest enrichment was observed with lactate and xylose, and in these cases the specific rate of ammonia production was eightfold higher than that of the ruminal fluid control (approximately 35 nmol of ammonia per mg of protein per min). Isolates with different morphologies were obtained from each of the enrichments, but in no case did the specific activity of any isolate exceed that of the mixed ruminal bacteria. If Trypticase (15 g/liter) was used as the only energy and nitrogen source, there was an even greater increase in ammonia production, and two monensin-sensitive bacteria, a Peptostreptococcus species and a Clostridium species, were obtained. The Peptostreptococcus species was unable to grow on any of 25 carbohydrate or carbohydrate derivatives tested; but the Clostridium species was able to use glucose, maltose, fructose, cellobiose, trehalose, sorbitol, and salicin as energy sources. Neither organism was able to grow in the absence of an amino acid source, but growth rates on Trypticase were greater than 0.35/h. The specific activities of ammonia production were 346 and 427 nmol/mg of protein per min for strains of Peptostreptococcus and Clostridium, respectively. Megasphaera elsdenii and Bacteroides ruminicola, previously isolated ruminal ammonia producers, had specific activities of only 11 and 19 nmol of ammonia per mg of protein per min, respectively. The most probable number of Clostridium species in ruminal fluid was less than 10(3)/ml, but the Peptostreptococcus species was present at 10(8)/ml.(ABSTRACT TRUNCATED AT 250 WORDS)     

Determination of l-carnitine, acetyl-l-carnitine and propionyl-l-carnitine in human plasma by high-performance liquid chromatography after pre-column derivatization with 1-aminoanthracene

A new sensitive high-performance liquid chromatographic procedure for the determination of l-carnitine (LC), acetyl-l-carnitine (ALC) and propionyl-l-carnitine (PLC) in human plasma has been developed. Precolumn derivatization with 1-aminoanthracene (1AA), performed in phosphate buffer in the presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) as catalyst, is involved. The fluorescent derivatives were isocratically separated on a reversed-phase column (C₁₈). The eluate was monitored with a fluorimetric detector set at 248 nm (excitation wavelength) and 418 nm (emission wavelength). Because of the presence of endogenous carnitines, the validation was performed using dialyzed plasma. The identity of the derivatized compounds was assessed by mass spectrometry and the purity of the chromatographic peaks was confirmed by HPLC-tandem mass spectrometry. The limits of quantitation were 5 nmol/ml for LC, 1 nmol/ml for ALC and 0.25 nmol/ml for PLC. The recovery of the extraction procedure was in the range 82.6%–95.4% for all 3 compounds. Good linearity (R≈0.99) was observed within the calibration ranges studied: 5–160 nmol/ml for LC, 1–32 nmol/ml for ALC and 0.25–8 nmol/ml for PLC. Precision was in the range 0.3–16.8% and accuracy was always lower than 10.6%.     

Determination of ofloxacin and moxifloxacin and their penetration in human aqueous and vitreous humor by using high-performance liquid chromatography fluorescence detection

Information on comparing the penetration of ofloxacin and moxifloxacin in the human eye is unavailable, although these two antibiotics are commonly used in ophthalmic surgery. There is a need for a rapid, reliable, and sensitive methodology for their determination in ocular fluids. We developed a robust HPLC procedure with fluorescence detection for simultaneous analysis of ofloxacin and moxifloxacin in human and rabbit aqueous and vitreous samples. The linearity of the method ranged from 10 ng/ml to 100 microg/ml with r(2) > 0.996. Most inter- and intrabatch imprecision was about 5% (range 1.6-7.6%), recoveries between 95 and 104%, and accuracies between 93 and 104% at 0.1 and 1 microg/ml. The detection limits of both compounds were 10 ng/ml (0.028 nmol/ml for ofloxacin and 0.023 nmol/ml for moxifloxacin). No sample treatment was necessary for aqueous humor and only acetonitrile precipitation was required for vitreous humor. The chromatographic time was short, 22 min. We applied this method to study penetrations of ofloxacin and moxifloxacin in aqueous and vitreous humors of human and rabbits. There was no significant difference of penetration between the two antibiotics into aqueous and vitreous but ofloxacin was found at significantly higher concentrations in aqueous than in vitreous. We also detected contralateral transfer of the antibiotics in rabbit eyes.     

Species characterization of plasma nitrite/nitrate (NOx) concentration

Experiments were designed to detect and determine differences between nitrite/nitrate concentration ([NOx]) in plasma across 15 species selected from seven classes of vertebrates. Blood collected in syringes was placed immediately into ethylenediaminetetraacetic acid (EDTA)-containing tubes and was centrifuged. Plasma [NOx] was determined by measurement of chemiluminescence. Across classes of vertebrates, baseline plasma [NOx] ranged from 0.6 to 171.3 nmol/ml. Mean +/- SD plasma [NOx] was highest in a fresh-water, jawless fish (lamprey, 95.5 +/- 9.1 nmol/ml) and lowest in a saltwater cartilaginous fish (skates, 1.1 +/- 0.4 nmol/ml). Both amphibians tested had a wide range in plasma [NOx], which was explained partly by temporal changes during the year. Within the mammalian class, plasma [NOx] ranged from 3.8 to 43.2 nmol/ml. Results of this study indicate that NO is detectable in plasma of all classes of vertebrates and that baseline concentration varies among species.     

Glucuronidation of benzo[a]pyrene in hamster embryo cells

The formation of water-soluble metabolites of tritium-labeled benzo[a]pyrene (BP) by cultured hamster embryo cells was studied. The ratio of the radioactivity in the aqueous phase to that in the organic phase increased with the incubation period. After incubation for 48 h with 3.75 nmol/ml of [3H] BP in the medium more than 90% of the 3H-radioactivity was found in the aqueous phase, whereas with 10-fold more BP about half the radioactivity remained in the organic phase. The main metabolites extracted from the medium at 37.5 nmol/ml BP with ethyl acetate by high pressure liquid chromatography (HPLC) were 9,10-diol and 7,8-diol; but after treatment of the medium with beta-glucuronidase the main oxygenated metabolites were phenols, the amount of 9-OH BP being more than that of 3-OH BP. beta-Glucuronidase also released 9,10-diol and 7,8-diol, but most of these diols were in the free form in the medium. The medium from cells treated with 3.75 nmol/ml BP has a quantitatively different profile, and most of the radioactivity obtained by extraction with organic solvent and digestion with beta-glucuronidase was eluted in the regions of phenols. These results show that in hamster embryo cells BP is mainly metabolised to conjugates of phenols with glucuronic acid.     

Quantitative analysis of sulpiride in body fluids by high-performance liquid chromatography with fluorescence detection

A high-performance liquid chromatographic method for the analysis of sulpiride, N-ethyl-2-(2-methoxy-5-sulphonamido-benzamido-methyl)-pyrrolidine, in body fluids is described. A structurally related compound, N-ethyl-2-(2,4-dimethoxy-benzamido-methyl)-pyrrolidine, was used as internal standard.A fluorescence detector with excitation maximum at 299 nm and emission maximum at 342 nm was used for the quantitation. The detection limit was about 10 ng/ml in serum and cerebrospinal fluid and about 200 ng/ml in urine. The experimental error was 5–10% in the concentration range 25–100 ng/ml. Some preliminary data from a pharmacokinetic study in healthy volunteers are presented. The half-life for sulpiride in serum was about 8 h. Sulpiride was also measured in cerebrospinal fluid from five drug-treated psychotic patients.     

Difference in the inhibitory effect of linoleic acid hydroperoxide on prostacyclin biosynthesis between cultured endothelial cells from human umbilical cord vein and cultured smooth muscle cells from rabbit aorta

The biosynthesis of prostacyclin in cultured endothelial cells from human umbilical cord vein was inhibited by linoleic acid hydroperoxide at a concentration of 0.1 nmol/ml (in terms of malondialdehyde), while that in cultured smooth muscle cells from rabbit aorta was inhibited only at a higher concentration of the hydroperoxide (5.0 nmol/ml).     

Characteristics of the response of dispersed guinea-pig adrenal cells to low doses (1-50 pg/ml) of alpha 1-24 adrenocorticotrophin

Isolated adrenal cells prepared by tryptic digestion of the guinea-pig adrenal gland are sensitive to low concentrations (less than 25 pg/ml) of adrenocorticotrophin (ACTH). Cell which have been pre-incubated for 2 h. centrifuged and resuspended in fresh culture medium prior to the introduction of 10 pg/ml ACTH for 60 min show a marked increase (328 +/- 109 nmol/l; mean +/- SD) in cortisol secretion over the control compared to freshly dispersed cells (75 +/- 45 nmol/l). Further potentiation of the ACTH effect was seen with the pre-incubated cells by suplementing the medium with calcium (8 mM) and ascorbate (2 mM) but not with theophylline (1 mM). Basal cortisol secretion was not affected by any of the additives. In the presence of 8 mM calcium and after 60 min incubation 10 pg/ml ACTH stimulated cortisol secretion from 328 nmol/l over the control to 839 +/- 382 nmol/l. The effect of ascorbate (2 mM) was to further increase the effect of ACTH at all dose levels tested (1-25 pg/ml). The concentration of ACTH required to provoke half maximal cortisol secretion decreased from 95 pg/ml with normal medium to 12 pg/ml with calcium -ascorbate supplemented medium. Using this supplemented medium the cells were sensitive to 1 pg/ml and cortisol secretion was stimulated 10-fold over the control with 50 pg/ml, a dose which saturated the system.     

In vitro calcium deposition by rachitic rat matrix vesicles: nucleoside triphosphate supported calcium deposition.

The present study was designed to test whether ATP at serum levels can support matrix vesicle-mediated Ca deposition while the final Ca x P ion product is maintained at or below serum or cartilage fluid levels. Rachitic rat epiphyseal cartilage matrix vesicles (40 micrograms protein/ml) in a simple calcifying solution (without exogenously added Pi) containing 50 mM Tris, pH 7.6 at 37 degrees C, 0.1 M NaCl, 1.35 mM CaCl2, 1 mM ATP, deposited about 500 nmol Ca/mg protein after 5 h. The amount of Ca deposited increased with increases in incubation time, concentrations of ATP, Ca2+, hydroxide, and matrix vesicle protein. UTP, GTP, and CTP were equally effective in supporting Ca deposition by matrix vesicles. ATP-alpha,beta-methylene and ATP-beta,gamma-methylene were inhibitory for ATP-dependent Ca deposition. Experiments with limiting amounts of ATP and Ca2+ available in the calcifying solution indicated that ATP concentration at serum levels, in the presence of Ca x P ion products at serum or cartilage fluid levels, can support matrix vesicle-mediated Ca deposition.