Environmental salinity regulates the in vitro production of [3H]-1,25-dihydroxyvitamin D3 and [3H]-24,25 dihydroxyvitamin D3 in rainbow trout (Oncorhynchus mykiss)

Previous studies have shown that specific binding of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) to enterocyte basolateral membranes (BLM), as well as circulating concentrations, is affected in response to changes in environmental salinity. It is not known if the production of 1,25(OH)2D3 and 24,25(OH)2D3 is affected by environmental salinity. The aim of the present study was to measure the in vitro production of [3H]-1,25(OH)2D3 and [3H]-24,25(OH)2D3 in fresh water (FW) and after 1, 2, 3, and 7 days after transfer to seawater (SW). Pooled sub-cellular fractions (mitochondria and microsomes) from liver or kidney was incubated with [3H]-25(OH)D3 and the produced metabolites were separated using HPLC. Hepatic production of [3H]-1,25(OH)2D3 was decreased after 24h in SW. This was followed by an up-regulation after 48h and a second, slower decrease in production rate which leveled out after 7 days in SW. The production rate in SW was lower than the original rate in FW-adapted fish. For hepatic [3H]-24,25(OH)2D3 production the pattern was reversed. Renal production of [3H]-24,25(OH)2D3 increased significantly during the period of SW acclimation. These results suggest that environmental salinity regulates the production rate of the two antagonizing calcium regulatory hormones; 1,25(OH)2D3 and 24,25(OH)2D3. This gives further evidence to the hypothesis that there is a physiological regulation and a differentiated importance of 1,25(OH)2D3 and 24,25(OH)2D3 in relation to environmental calcium concentrations.     

Comparative studies of the intestinal absorption of [3H]pteroylmonoglutamate and [3H]pteroylheptaglutamate in man.

Comparative efficiencies of absorption of crystalline folic acid polyglutamate and monoglutamyl folic acid were determined in 11 normal subjects by measurement of the excretion of radioisotope in the urine after oral administration of [3H]pteroylheptaglutamic acid ([3H]PteGlu7) synthesized in our laboratory and of [3H]pteroylglutamic acid ([3H]PGA). Following ingestion of 0.6 mumole of [3H]PteGlu7, urinary excretion of radioactivity over 48 hr averaged 56.1 +/- 11.2% of the total dose. By comparison the ingestion of 0.6 mumole of [3H]PGA resulted in an average urinary excretion of 70.8 +/- 13.0% for the same time period. Approximately 90% of the urinary radioactivity was excreted during the initial 24-hr collection period. The mean recovery of radioactivity in urine and stool was 94% and recovery exceeded 84% in all subjects. The principal radioactive compound in the urine chromatographed with standard pteroylmonoglutamates. By chromatography, urinary folates were monoglutamates whether [3H]PGA or [3H]PteGlu7 was administered. The time course of folate absorption for the study compounds was compared by measuring the rise in serum radioactivity after the oral folate dose. Peak values in serum folate radioactivity following [3H]PGA occurred at 1 hr, whereas the peak values after [3H]PteGlu7 more often occurred at 2 hr. Only monoglutamyl folate was detected in the serum. These studies demonstrate that in normal subjects physiological doses of crystalline monoglutamyl and crystalline heptaglutamyl folates are both absorbed with high degrees of efficiency.     

An alternative method for the synthesis of 11-[3H]ketotestosterone and 11β-[3H]hydroxytestosterone from [3H]cortisol

Experimental conditions for the synthesis of radioactive 11-ketotestosterone and 11β-hydroxytestosterone from cortisol of high specific activity are described. For the synthesis of 11-ketotestosterone, cortisol is first oxidized to cortisone with 0.1% aqueous chromic acid. The use of hydroxysteroid dehydrogenases for reduction of intermediates in the reaction sequence from C₂₁ to C₁₉ steroid provides consistently high yields of product.     

Adenosine receptors in brain membranes: binding of N6-cyclohexyl[3H]adenosine and 1,3-diethyl-8-[3H]phenylxanthine.

N6-Cyclohexyl[3H]adenosine ([3H]CHA) and 1,3-diethyl-8-[3H]phenylxanthine ([3H]DPX) to bind to adenosine receptors in brain membranes. The agonist [3H]CHA has high affinity in both bovine and guinea pig brain (Kd, 0.7 nM and 6 nM, respectively). [3H]CHA binding kinetics are slow (dissociation t1/2;60 min); binding is much higher at 25 degrees C than at 0 degrees C and is inhibited by guanine nucleotides. Potencies of nucleosides and xanthines in competing for [3H]CHA sites indicate that specific binding is entirely to A1 adenosine receptors. In bovine brain, the antagonist [3H]DPX exhibits high-affinity binding (Kd, 5 nM) to the same A1 receptors that bind [3H]CHA. Binding kinetics are rapid (dissociation t1/2, 1 min), and binding is moderately higher at 0 degrees C than at 25 degrees C. In guinea pig brain, [3H]DPX binding has only moderate affintiy (Kd 50 nM), and about 60% of specific binding is to sites that resemble A2 adenosine receptors.     

Glucose uptake and pulsatile insulin infusion: euglycaemic clamp and [3-3H]glucose studies in healthy subjects

To test the hypothesis that insulin has a greater effect on glucose metabolism when given as pulsatile than as continuous infusion, a 354-min euglycaemic clamp study was carried out in 8 healthy subjects. At random order soluble insulin was given intravenously either at a constant rate of 0.45 mU/kg X min or in identical amounts in pulses of 1 1/2 to 2 1/4 min followed by intervals of 10 1/2 to 9 3/4 min. Average serum insulin levels were similar during the two infusion protocols, but pulsatile administration induced oscillations ranging between 15 and 62 microU/ml. Glucose uptake expressed as metabolic clearance rate (MCR) for glucose was significantly increased during pulsatile insulin delivery as compared with continuous administration (270-294 min: 8.7 +/- 0.7 vs 6.8 +/- 0.9 ml/kg X min, P less than 0.01, and 330-354 min: 8.9 +/- 0.5 vs 7.4 +/- 0.9 ml/kg X min, P less than 0.05). The superior efficacy of pulsatile insulin delivery on glucose uptake was not consistently found until after 210 min of insulin administration. In both infusion protocols, endogenous glucose production as estimated by the [3-3H]glucose infusion technique was suppressed to insignificant values. Finally, the effect of insulin on endogenous insulin secretion and lipolysis as assessed by changes in serum C-peptide and serum FFA was uninfluenced by the infusion mode. In conclusion, insulin infusion resulting in physiological serum insulin levels enhances glucose uptake in peripheral tissues in healthy subjects to a higher degree when given in a pulsed pattern mimicking that of the normal endocrine pancreas than when given as a continuous infusion.     

Catechol estrogen formation by brain tissue: a comparison of the release of tritium from [2-3H]estradiol with [6,7-3H]2-hydroxyestradiol formation from [6,7-3H]estradiol by rabbit hypothalami in vitro

In the course of establishing an assay for estrogen-2-hydroxylase activity, a detailed comparison was made between the formation of tritiated water (3H2O) and [6,7-3H]2-hydroxyestradiol (2-OHE2) by rabbit hypothalami in vitro from 2-3H- and 6,7-3H-labeled estradiol, respectively. The amounts of both 3H2O and [6,7-3H]2-OHE2 formed were stimulated several-fold by the nonionic detergent Tween-80. Maximum activity for both functions was associated with the soluble fractions (S2, 17,500 X g supernatant, for tritium release; S3, 100,000 X g supernatant, for 2-OHE2 formation). In contrast, maximal 3H2O formation by rat liver was associated with the microsomal (P3, 100,000 X g pellet) fraction and was virtually abolished by Tween-80. The amount of 3H2O formed exceeded, up to severalfold, the amount of 2-OHE2 produced under all conditions examined and in all subcellular fractions. The bulk of the excess 3H2O formation, unrelated to the production of 2-OHE2, could be eliminated by adding ascorbic acid (10 mM) to the incubation medium. However, a second, smaller component of spurious 3H2O release could not be suppressed. This component was responsible for a persistent lack of stoichiometry between the formation of 3H2O and 2-OHE2, with the former exceeding the latter by up to 2-fold. This discrepancy was unaffected by ascorbic acid (up to 20 mM), unlabeled 2-OHE2 (up to 10 microM), and reducing the temperature of incubation from 37 to 30 C, measures that prolonged the t1/2 of 2-OHE2 during incubation with hypothalamic tissue from under 3 min to over 100 min. These findings 1) raise doubts about the validity of using 3H2O formation from [2-3H]estradiol as a quantitative index of estrogen-2-hydroxylase activity, and 2) establish conditions under which further metabolism of 2-OHE2 is inhibited, thereby making it practical to quantify enzyme activity on the basis of the amount of catechol estrogen formed. Evidence is also presented that the release of 3H2O from [2-3H]estradiol by hypothalamic tissue, unrelated to 2-OHE2 formation, may be enzymatically mediated.     

Neuroanatomical localization of sex steroid-concentrating cells in the Japanese quail (Coturnix japonica): autoradiography with [3H]-testosterone, [3H]-estradiol, and [3H]-dihydrotestosterone

Steroid autoradiography was undertaken to determine the neuroanatomical loci which might be involved in the activation of steroid-sensitive behaviors in the Japanese quail (Coturnix japonica). Male and female quail were either surgically gonadectomized or photically regressed and implanted with androgen or estrogen to restore normal sexual and courtship behavior. After gonadectomy or implant removal, each quail was injected with 250 microCi of [3H]-testosterone (3H-T), [3H]-estradiol (3H-E2), or [3H]-dihydrotestosterone (3H-DHT), sacrificed, processed for autoradiography, and the telencephalon, diencephalon, mesencephalon, and rhombencephalon were examined for labelled cells. Following 3H-T or 3H-E2 injection and autoradiography, labelled cells were found in nucleus septalis lateralis (SL), nucleus preopticus medialis (POM), nucleus paraventricularis (PVN), regio lateralis hypothalami (LHy), nucleus inferior hypothalami (IH), nucleus infundibuli (IN), nucleus intercollicularis (ICo), substantia grisea centralis (GCt), nucleus taeniae (Tn), and in the reticular formation near nucleus motorius nervi trigemini (MV). In addition, following 3H-E2 autoradiography, labelled cells were found around nucleus accumbens (Ac). Following 3H-DHT autoradiography, labelled cells were found only in SL, PVN, Tn, LHy, ICo, and CGt. No labelled cells were found in Ac, POM, IH, IN, or MV even after long exposure times. These results suggest that the nuclei labelled following 3H-E2 but not 3H-DHT administration bind exclusively the aromatized metabolites of T. Since quail show a sex difference in male-typical copulatory behavior in response to E2, labelled cells were counted in POM, LHy, IH, and Tn of male and female quail following 3H-E2 injection and autoradiography. No sex differences in the number of labelled cells were found in POM, LHy, or IH. Males were found to have more labelled cells than females in Tn. These results show that sex differences in male-typical copulatory behavior are not due to sex differences in the number of cells binding estrogens in POM. The results reported here constitute the most neuroanatomically extensive report of steroid binding cells to date for a galliform brain, the first comparison in a galliform bird of the distributions of cells labelled following injection of 3H-T, 3H-E2, and 3H-DHT and the first analysis of sex differences in numbers of estrogen-binding cells in four nuclei in the avian brain.     

Remethylation and transsulfuration of methionine in cirrhosis: studies with L-[H3-methyl-1-C]methionine

Disturbances of the methionine cycle may result in liver injury. Patients with alcohol-induced liver disease often exhibit hypermethioninemia and a delayed clearance (CL) of methionine, but the extent to which transsulfuration and remethylation pathways of the cyclic methionine metabolism are affected is unknown. Methionine turnover was determined in 7 healthy volunteers and 6 patients with alcohol-induced cirrhosis after oral administration of 2 mg/kg [(2)H(3)-methyl-1-(13)C]methionine, which permitted us to follow transsulfuration by its decarboxylation to (13)CO(2) and remethylation by replacement of the labeled methyl group by an unlabeled one. Basal plasma concentrations of endogenous methionine (50 +/- 5 vs. 25 +/- 2 micromol/L, mean +/- SEM, P <.001) were significantly higher in patients with cirrhosis and its CL was significantly decreased (774 +/- 103 vs. 2,050 +/- 141 mL/min, P <.001). Methionine turnover amounted to 42 +/- 4 vs. 27 +/- 3 micromol/kg/h (P <.05) in controls and patients with cirrhosis, respectively. The fraction of administered methionine undergoing remethylation was lower in patients with cirrhosis (7.6 +/- 1.5 vs. 14.1 +/- 1.1%, P <.005). However, because of the larger pool of circulating methionine, the total flux of methionine through the remethylation pathway was similar in both groups. A significantly lower fraction of the administered dose appeared in the form of (13)CO(2) in breath in patients with cirrhosis (2.2 +/- 0.4 vs. 11.0 +/- 0.8%, P <.001). In conclusion, the data indicate that the liver with cirrhosis compensates for a decreased activity of remethylating enzymes by operating at higher concentrations of methionine. In contrast, transsulfuration is impaired in patients with alcohol-induced cirrhosis such that an assessment of transsulfuration by a simple breath test may provide a clinically useful estimate of hepatic function.     

Sigma opiates and certain antipsychotic drugs mutually inhibit (+)-[3H] SKF 10,047 and [3H]haloperidol binding in guinea pig brain membranes.

The relationship between binding of antipsychotic drugs and sigma psychotomimetic opiates to binding sites for the sigma agonist (+)-[3H]SKF 10,047 (N-allylnormetazocine) and to dopamine D2 sites was investigated. In guinea pig brain membranes, (+)-[3H]SKF 10,047 bound to a single class of sites with a Kd of 4 X 10(-8) M and a Bmax of 333 fmol/mg of protein. This binding was different from mu, kappa, or delta opiate receptor binding. It was inhibited by opiates that produce psychotomimetic activities but not by opiates that lack such activities. Some antipsychotic drugs inhibited (+)-[3H]SKF 10,047 binding with high to moderate affinities in the following order of potency: haloperidol greater than perphenazine greater than fluphenazine greater than acetophenazine greater than trifluoperazine greater than molindone greater than or equal to pimozide greater than or equal to thioridazine greater than or equal to chlorpromazine greater than or equal to triflupromazine. However, there were other antipsychotic drugs such as spiperone and clozapine that showed low affinity for the (+)-[3H]SKF 10,047 binding sites. Affinities of antipsychotic drugs for (+)-[3H]SKF 10,047 binding sites did not correlate with those for [3H]spiperone (dopamine D2) sites. [3H]-Haloperidol binding in whole brain membranes was also inhibited by the sigma opiates pentazocine, cyclazocine, and (+)-SKF 10,047. In the striatum, about half of the saturable [3H]haloperidol binding was to [3H]spiperone (D2) sites and the other half was to sites similar to (+)-[3H]SKF 10,047 binding sites.     

Autoradiographic binding studies with [3H]oestradiol and [3H]dihydrotestosterone in the autonomic genital ganglion (plexus of Frankenh?user) of the mouse

Male, female and Tfm mice (testicular feminization) were injected with [3H]oestradiol or [3H]dihydrotestosterone, and autoradiograms prepared of male accessory sex organs and of the cervico-vaginal portion of the female reproductive tract. After injection of [3H]oestradiol in male, female and Tfm animals a nuclear concentration of radioactivity was found in a subpopulation--about 20-30%--of the neurons of the genital ganglion. No such concentration was seen after [3H]dihydrotestosterone. The results suggest a direct genomic effect of oestradiol on certain neurons of the autonomic genital ganglion in both sexes.     

Renal Aldosterone Receptors: Studies with [3H]Aldosterone and the Anti-Mineralocorticoid [3H]Spirolactone (SC-26304)

In vivo, a spirolactone (SC-26304) inhibited the effects of aldosterone on urinary K(+):Na(+) ratios and the binding of [(3)H]aldosterone to renal cytoplasmic and nuclear receptors. Cytoplasmic binding of [(3)H]aldosterone and [(3)H]spirolactone (SC-26304) was similar in magnitude and involved the same set of sites. Under three sets of conditions-(i) in the intact rat, (ii) in kidney slices, and (iii) in reconstitution studies (mixing prelabeled cytoplasm with either purified renal nuclei or chromatin), [(3)H]spirolactone (SC-26304) did not yield specific nuclear complexes in contrast to the reproducible generation of these complexes with [(3)H]aldosterone. In glycerol density gradients, cytoplasmic [(3)H]aldosterone receptor complexes sedimented at 8.5 S and 4 S in low concentrations of salt and at 4.5 S in high concentrations of salt. Cytoplasmic [(3)H]spirolactone (SC-26304) receptor complexes sedimented at 3 S in low concentrations of salt and 4 S in high concentrations of salt. These results are discussed in terms of an allosteric model of the receptor system.     

Differential diagnosis of hydroxydicarboxylic aciduria based on release of3H2O from [9,10-3H]myristic and [9,10-3H]palmitic acids by intact cultured fibroblasts

Summary Intact cultured fibroblasts from patients with deficiency of long-chain 3-hydroxyacyl-CoA dehydrogenase release³H₂O from [9,10-³H]myristic acid and [9,10-³H]palmitic acid more slowly than normal. The ratio of activity (palmitate/myristate) is also low and the expression (rate with palmitate)²/(rate with myristate) gives good differentiation between affected and unaffected cells. In some patients who have shown hydroxydicarboxylic aciduria when unwell there is reduced³H₂O production from [9,10-³H]myristic and [9,10-³H]palmitic acids by intact cultured fibroblasts but normal 3-hydroxyacyl-CoA dehydrogenase activities in disrupted cells. The palmitate/myristate ratio is higher than in long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency. The basic defect in these patients is still unknown but it is suggested that caution be used over the administration of medium-chain triglyceride.