The effects of l-thyroxine and dexamethasone on steroid dynamics in male cynomologous monkeys

Male cynomologous monkeys (M. fascicularis) were infused with [3H]androgens, [14C]estrogens and [3H]cortisol before and after the administration of l-thyroxine, (l-T4) 150 micrograms/day for 6 wk, dexamethasone 8 mg every 8 h for 3 doses and dexamethasone 1.0 mg/day for 8 days. Blood samples were obtained before each of the infusions and analyzed for endogenous T, A, E1, E2 and F concentrations, % free T and % free E2, sex hormone-binding globulin (SHBG) and cortisol binding globulin (CBG) capacity. When l-T4 was being administered, T4 and triiodothyronine (T3) concentrations were also measured. Blood samples were obtained during the infusions and analyzed for radioactivity as testosterone (T), androstenedione (A), dihydrotestosterone (DHT), estradiol (E1), estrone (E2), and cortisol (F). All urine was collected for 96 h and an aliquot of the pooled urine was analyzed for radioactivity as estrone and estradiol glucuronide. The administration of l-T4 for 6 wk to 3 monkeys resulted in a marked rise in T4 and T3 levels, from 4.8 +/- 0.4 micrograms/dl and from 136 +/- 6 to 515 +/- 71 ng/dl, respectively. MCRT, MCRE2 and MCRE1 did not change, but MCRA values increased slightly and MCRF increased 2-3 fold. [rho]T.E2 did not change but [rho]A.E1BM showed a slight but significant increase. The inter-conversions between the androgens and between the estrogens were not altered. There was a 2-3-fold increase in SHBG and a decrease in %FT but no change in %FE2 or CBG. The concentrations of T, A and DHT rose but there was no trend in the levels of the estrogens. The administration of dexamethasone 8 mg every 8 h for 3 doses or 1 mg/day for 8 days caused no changes in the MCRs for T, A, E1 and E2 but did cause a significant decrease in MCRF. Measurement of splanchnic and peripheral tissue extractions before and after acute dexamethasone administration in 1 monkey showed that the decrease in MCRF was the result of a marked decrease, 11-2%, in splanchnic extraction of F. The extractions of T and E2 were relatively unaffected. The concentrations of T and F fell but E2 remained the same. % FT and % FE2 rose slightly and the concentrations of SHBG and CBG were unchanged. The androgen interconversions and estrogen interconversions were not affected but [rho]T,E2BM and [rho]A,E1BM showed slight decreases.(ABSTRACT TRUNCATED AT 400 WORDS)     

Evaluation of radiation dose resulting from the ingestion of [3H]- and [14C]thymidine in the rat

Average doses to rat tissues from the ingestion of 2-[14C]thymidine were compared with those from methyl-[3H]thymidine or 6-[3H]thymidine. Among the three precursors, [14C]thymidine gave the highest dose to spleen and small intestine. The doses to other tissues from [14C]thymidine were almost the same or lower as compared with those from [3H]thymidine, irrespective of the 9 times higher beta-ray energy of 14C than that of 3H. In the case of [14C]thymidine, most of the dose was given by radioactivity incorporated into the organic tissue constituents (non-volatile radioactivity). In the case of [3H]thymidine, however, the dose contributions by non-volatile radioactivity were very small and the major contributions were rather from volatile radioactivity (3HHO), formed by degradation of [3H]thymidine. No significant difference in their total doses was found between the two [3H]precursors, but the dose from non-volatile radioactivity alone was 2-3 times higher with methyl-[3H]thymidine than with 6-[3H]thymidine. Estimates of the dose to cell nuclei in various tissues after the ingestion of [3H]thymidine were also made in order to predict more precisely possible radiation hazards.     

Effects of pleural pressure and abdominal pressure on diaphragmatic blood flow

Alveolar transfer of prostaglandin E2 (PGE2) was characterized in isolated perfused guinea pig lungs (n = 19) by measuring radioactivity appearing in the venous effluent during 30 min after intratracheal instillation of [3H]PGE2, [14C]-mannitol, and [125I]iodoantipyrine. Recovery of lipid-soluble [125I]iodoantipyrine [91 +/- 3% (SE)] after 30 min was used to estimate total 3H and 14C delivered to the exchanging region of lung at time 0. In seven control lungs, 58 +/- 4% of [14C]mannitol and 16 +/- 4% of [3H]PGE2 was retained 10 min after instillation. Neither perfusion with diphloretin phosphate (10 micrograms/ml; n = 4) nor hypothermia (5 degrees C; n = 5) significantly affected the amount of [14C]mannitol retained; however, [3H]PGE2 remaining in these lungs increased significantly to 36 +/- 4 and 53 +/- 2%, respectively. Addition of unlabeled PGE2 (200 micrograms) to the instilled solution (n = 3) increased retention of both [14C]mannitol (80 +/- 3%) and [3H]PGE2 (65 +/- 4%). Alveolar transfer of [3H]PGE2 was calculated as the difference in percent retention of [14C]mannitol and [3H]PGE2 and normalized to that of [14C]mannitol. After 10 min, alveolar transfer of [3H]PGE2 was 71 +/- 8% in control lungs but was decreased to 26 +/- 7, 10 +/- 5, and 19 +/- 6% by diphloretin phosphate, hypothermia, or unlabeled PGE2, respectively. These data suggest that alveolar clearance of PGE2 involves a saturable drug- and temperature-sensitive process.     

Characterization of the transport properties of cloned rat multidrug resistance-associated protein 3 (MRP3).

We have previously cloned rat MRP3 as an inducible transporter in the liver (Hirohashi, T., Suzuki, H., Ito, K., Ogawa, K., Kume, K., Shimizu, T., and Sugiyama, Y. (1998) Mol. Pharmacol. 53, 1068-1075). In the present study, the function of rat MRP3 was investigated using membrane vesicles isolated from LLC-PK1 and HeLa cell population transfected with corresponding cDNA. The ATP-dependent uptake of both 17beta estradiol 17-beta-D-glucuronide ([3H]E217betaG) and glucuronide of [14C] 6-hydroxy-5, 7-dimethyl-2-methylamino-4-(3-pyridylmethyl) benzothiazole (E3040), but not that of [3H]leukotriene C4 and [3H]2, 4-dinitrophenyl-S-glutathione, was markedly stimulated by MRP3 transfection in both cell lines. The Km and Vmax values for the uptake of [3H]E217betaG were 67 +/- 14 microM and 415 +/- 73 pmol/min/mg of protein, respectively, for MRP3-expressing membrane vesicles and 3.0 +/- 0.7 microM and 3.4 +/- 0.4 pmol/min/mg of protein, respectively, for the endogenous transporter expressed on HeLa cells. [3H]E217betaG had also a similar Km value for MRP3 when LLC-PK1 cells were used as the host. All glucuronide conjugates examined (E3040 glucuronide, 4-methylumbelliferone glucuronide, and naphthyl glucuronide) and methotrexate inhibited MRP3-mediated [3H]E217betaG transport in LLC-PK1 cells. Moreover, [3H]methotrexate was transported via MRP3. The inhibitory effect of estrone sulfate, [3H]2,4-dinitrophenyl-S-glutathione, and [3H]leukotriene C4 was moderate or minimal, whereas N-acetyl-2,4-dinitrophenylcysteine had no effect on the uptake of [3H]E217betaG. The uptake of [3H]E217betaG was enhanced by E3040 sulfate and 4-methylumbelliferone sulfate. Thus we were able to demonstrate that several kinds of organic anions are transported via MRP3, although the substrate specificity of MRP3 differs from that of MRP1 and cMOAT/MRP2 in that glutathione conjugates are poor substrates for MRP3.     

Metabolism and excretion of peptide leukotrienes in the anesthetized rat

The metabolism and excretion of the peptide leukotrienes C4, D4, E4 and N-acetylleukotriene E4 have been studied in the anesthetized rat. The intravenous administration of [3H]leukotriene C4 (2.6 X 10(-11) mol/kg) showed a rapid clearance of radioactivity from the blood and a time-related biliary excretion, recovering 69 +/- 1.6% (n = 6) over 60 min. Less than 1% of total radioactivity was recovered in the urine over the same time period. Similarly, the intravenous administration of [3H]leukotriene D4 (2.5 X 10(-11) mol/kg), [3H]leukotriene E4 (2.5 X 10(-11) mol/kg) and N-acetyl[3H]leukotriene E4 (2.1 X 10(-11) mol/kg) showed a 62 +/- 7.5% (n = 4), 52 +/- 1.5% (n = 4) and 37 +/- 4.6% (n = 5) biliary recovery of radioactivity, respectively, after 60 min. Examination of bile identified leukotriene D4 and N-acetylleukotriene E4 as the main products, although substantial radioactivity, which probably represents unidentified polar products, was present at the solvent fronts of the reverse-phase HPLC. Time course studies indicated a relatively rapid conversion of leukotriene C4 to leukotriene D4, while leukotriene D4 metabolism appeared to be much slower. Leukotriene E4 was a minor product, suggesting that the N-acetylation process is rapid. Incubation of [3H]leukotriene C4 in rat plasma and whole blood in vitro resulted in a slow conversion of leukotriene C4 to leukotriene D4 and leukotriene E4 only. These data suggest that the majority of the leukotriene metabolism and excretion in vivo in the anesthetized rat occurs predominantly in the hepatic system. We conclude that this model is suitable for the measurement of in vivo production of peptide leukotrienes.     

Androgen and estrogen dynamics in the female baboon (Papio anubis)

Androgen and estrogen dynamics were studied in 5 female baboons (Papio anubis) using constant infusions of [3H]androstenedione/[14C]estrone and [3H]testosterone/[14C]estradiol. Blood samples were obtained prior to the infusions and both blood and plasma was used for measurements of androstenedione (A), testosterone (T), dihydrotestosterone (DHT), estrone (E1), estradiol (E2). Plasma was used for measurements of sex-hormone binding globulin (SHBG), and the percents of T and E2 free, bound to SHBG, and to albumin. Blood samples obtained during the infusions were analyzed for radioactivity as purified androgens and estrogens. Metabolic clearance rates (MCR), and transfer factors ([rho]BB; fraction of steroid infused which is converted to and measured in blood as product) and blood production rates were calculated from whole blood data. All urine was collected for 96 h and an aliquot analyzed for radioactivity as the glucuronides of estrone and estradiol and the % peripheral aromatization calculated. The MCR's, calculated in whole blood, of A, E1, E2 and T were 53 +/- 6 1/day/kg, 39.3 +/- 3 1/day/kg, 29.9 +/- 5.2 1/day/kg and 10.1 +/- 2.3 1/day/kg, respectively. Each MCR was different (P less than 0.05) from the others. The PB of E1 was 15 +/- 2 micrograms/day and was not different from that of E2 (12 +/- 3 micrograms/day). The PB of A, 231 +/- 55 micrograms/day, was greater than that of T, 13 +/- 5 micrograms/day. The interconversions of both the androgens (18.9 +/- 3.4% vs 3.9 +/- 1.0%) and the estrogens (48.8 +/- 10.7% vs 4.0 +/- 0.8%) favored the oxidative pathway, i.e. conversion of 17-OH to 17-oxo steroids. The conversion ratio of A to DHT was greater than that of T to DHT (16.4 +/- 2.1% vs 5.3 +/- 0.7%), and A is a more important source of DHT than is T. The percent of T bound to SHBG (80.7 +/- 0.9%) was greater than percent of E2 (36.9 +/- 9.8%) and inversely the percents of T bound to albumin and free (17.5 +/- 0.8% and 1.65 +/- 0.16%) were less than the respective percents for estradiol (60.5 +/- 9.5% and 2.40 +/- 0.27%). The mean SHBG concentration was 54 +/- 6 nM. The peripheral aromatization of androstenedione, 1.36 +/- 0.05%, was greater than of testosterone, 0.18 +/- 0.02%. This difference is, in part, due to the lack of SHBG-binding of androstenedione. The general pattern of androgen and estrogen dynamics is similar to that in women. This similarity is due, in part, to the presence of SHBG in both baboons and women.     

Evidence for coupling of bradykinin receptors to a guanine-nucleotide binding protein to stimulate arachidonate liberation in the osteoblast-like cell line, MC3T3-E1.

The effect of bradykinin on the activation production of inositol 1,4,5-trisphosphate and prostaglandin E2 (PGE2) was examined in the murine osteoblastic cell line, MC3T3-E1. Bradykinin, at concentrations ranging from 1 to 1000 nM, stimulated the production of inositol 1,4,5-trisphosphate 2.5- to 3-fold within 10 s, and elevated cytosolic-free Ca2+, even in the absence of external Ca2+. This process is mediated through the activation of phospholipase C. Bradykinin at the same concentration also stimulated the production of PGE2 and caused a release of 3H radioactivity from the cells prelabeled with [3H]arachidonic acid, probably via the activation of phospholipase A2. Pretreatment of the cells with pertussis toxin inhibited the stimulation of PGE2 production and 3H radioactivity release, while the elevation in cytosolic Ca2+ and the production of inositol 1,4,5-trisphosphate were not altered by toxin-pretreatment. The addition of an unhydrolyzable analog of GTP, guanosine 5'-[gamma-thio]triphosphate (GTP gamma S) to the beta-escin-permeabilized cells prelabeled with [3H]arachidonic acid enhanced the release of 3H radioactivity. The simultaneous presence of bradykinin with GTP gamma S further activated the 3H radioactivity release in the beta-escin-permeabilized cells. These results provide evidence that receptors for bradykinin in the MC3T3-E1 couple stimulating arachidonate release, probably via the activation of phospholipase A2, through a guanine nucleotide binding protein sensitive to pertussis toxin.     

Characterization of Met-139 as the photolabeled amino acid residue in the steroid binding site of sex hormone binding globulin using delta 6 derivatives of either testosterone or estradiol as unsubstituted photoaffinity labeling reagents.

Immunopurified human sex hormone binding globulin (SHBG) was photoinactivated and photolabeled by radioinert and radioactive photoaffinity labeling steroids delta 6-testosterone (delta 6-T) and delta 6-estradiol (delta 6-E2). The maximal levels of specific incorporation of these two reagents were 0.50 and 0.33 mol of label/mol of SHBG, respectively. Covalently labeled SHBG fractions were citraconylated, reduced, carboxymethylated, and cleaved by trypsin. Separation of tryptic digests by reverse-phase liquid chromatography gave single radioactive peaks at the same retention times with both steroid reagents. However, the two labeled peptidic fractions could be distinguished by capillary electrophoresis and immunodetection with anti-steroid antibodies, whereas the covalent attachment of radioactivity was confirmed by thin-layer chromatography on silica gel. Edman degradation of the two labeled peptides showed a single sequence His-Pro-Ile-([3H]X)-Arg corresponding to the pentapeptide His-Pro-Ile-Met-Arg 136-140 of SHBG sequence. The coincidence, in both cases, of the absence of an identifiable amino acid residue and of the elution of the most intense peak of radioactivity at the fourth cycle of Edman degradation suggests that the same Met-139 residue was labeled by delta 6-[1,2-3H2]T or by delta 6-[17 alpha-3H]E2. Liquid secondary ion mass spectrometry of the two peptides showed [M+H]+ ions at m/z 939.8 or 923.8, corresponding respectively to the addition of delta 6-T or delta 6-E2 to the pentapeptide. The presence of the steroid molecule in the delta 6-[3H]T-pentapeptide conjugate was confirmed by the difference of 2 mass units with the [M+H]+ peak of the delta 6-[4-14C]T-pentapeptide conjugate.     

Utilization of circulating androstenedione and testosterone for estradiol production during gestation in the rat

The placenta provides androgen precursors for ovarian estradiol (E2) production during the second half of gestation in the rat. However, no studies have measured E2 synthesis in vivo from circulating testosterone (T) or androstenedione (A) before or after Day 12 of gestation. In addition, it is not known whether the placenta near term continues to serve as the major source of androgens. Therefore, we measured the ovarian conversion of circulating T and A to E2 in vivo on Days 11, 16, and 21 of gestation (term = Day 23). Rats (N = 6-8/group) were anesthetized with pentobarbital and a constant infusion of [3H]T or [3H]A initiated via a jugular vein. After isotopic equilibrium was achieved at 60 min, blood samples were obtained from the contralateral jugular (J) vein and a uterine-ovarian (UO) vein, and the ovaries were removed. In a second group of rats on Day 16 of gestation, either the gravid uterus or both ovaries were removed after initiation of isotope infusion, and blood samples obtained 60 min later. Radiolabeled T, A, and E2 were isolated and purified by sequential paper chromatography. The concentration of [3H]E2 following infusion of either androgen was greater in the UO vein than in the J vein on Days 16 and 21 (p less than 0.02), but not on Day 11, of gestation. In animals infused with [3H]T, [3H]E2 (cpm/ml) in UO vein increased (p less than 0.001) from 84 +/- 33 (mean +/- SE) on Day 11 to 357 +/- 30 and 312 +/- 46 on Days 16 and 21, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of hexose transport in respiration deficient hamster lung fibroblasts

The transport of [3H]2-deoxy-D-glucose (2DG) and [3H]3-O-methyl-D-glucose (3-OMG) was elevated in a respiration deficient (NADH coenzyme Q [Co Q] reductase deficient) Chinese hamster lung fibroblast cell line (G14). This sugar transport increase was related to an increased Vmax for 2DG transport, 26.9 +/- 4.2 nmoles 2DG/mg protein/30 sec in the G14 cell line vs 9.5 +/- 0.6 nmoles 2DG/mg protein/30 sec in the parental V79 cell line. No differences were observed in their respective Km values for 2DG transport (3.9 +/- .6 vs. 3.0 +/- .13 mM). Factors which increase sugar transport (e.g., glucose deprivation, serum or insulin exposure) or decrease sugar transport (e.g., serum deprivation) in the parental V79 cell line had little effect on sugar transport in the G14 respiration deficient cell lines. Amino acid transport, specific 125I-insulin binding to cells, and insulin-stimulated DNA synthesis, however, were similar in both cell lines. Exposure of both cell lines to varying concentrations of cycloheximide (0.1-50 micrograms/ml) for 4 h resulted in differential effects on 2DG transport. In the parental cell line (V79) low cycloheximide concentrations resulted in decreased 2DG transport, while higher concentrations (greater than or equal to 1 microgram/ml) resulted in elevated 2DG transport. In the G14 cell line, 2DG transport decreased at all concentrations of cycloheximide (up to 50 micrograms/ml). The data indicate that the G14 mutant has been significantly and specifically affected in the expression of sugar transport activity and in the regulatory controls affecting sugar transport activity.     

Selective permeability of the blood-uterine lumen barrier in rats: importance of molecular size

To investigate the influence of molecular size on the abilities of polar, nonelectrolytic substances to diffuse passively across the blood-uterine lumen barrier, the abilities of [3H] mannitol, [3H] sucrose and [3H] inulin (mol. wt. 182, 342 and 5200, respectively) to enter uterine luminal fluid from blood were compared in immature, ovariectomized rats implanted for 3 days with Silastic capsules containing estradiol. Relatively constant serum radioactivity concentrations were achieved for the period of 1-4 h after intravenous injection of the test substances by tightly ligating the renal pedicles of all animals prior to injection. Although uterine fluid radioactivity concentrations for [3H] inulin increased significantly between 1 and 4 h after injection, those for [3H] sucrose and [3H] mannitol did not change significantly with time, thus preventing calculation of conventional permeability indices. Therefore, the ratios of uterine fluid to serum radioactivity concentrations 2 h after intravenous injection of the test substances into animals with ligated renal pedicles were determined; the ratios (means +/- SEM) for [3H] mannitol, [3H] sucrose and [3H]-inulin were 0.11 +/- 0.015, 0.038 +/- 0.011 and 0.037 +/- 0.012, respectively. As indicated by these ratios, the rate of transfer into the uterine lumen of [3H] mannitol relative to that of [3H] sucrose was markedly greater than predicted by the ratio of their respective aqueous diffusion coefficients at body temperature. This disproportionality suggested that diffusion across the blood-uterine lumen barrier by some substances is governed by molecular sieving (sterically restricted diffusion) and therefore that this barrier is selectively permeable to these substances on the basis of molecular size.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of deoxyribonuclease I and micrococcal nuclease on the release of dexamethasone-receptor complex from nuclei of sensitive and insensitive hepatoma cell lines

(1) Fu5 cells were sensitive to the glucocorticoid inhibition of cell growth and the hormonal induction of tyrosine aminotransferase (but not fructose-1,6-bisphosphatase and glycogen synthase). AH-130 and AH-7974 cells were insensitive to both effects. (2) The release of [3H]dexamethasone radioactivity from the nuclei of Fu5 and AH-130 cells preincubated with [3H]dexamethasone increased as the KCl concentration increased from 0 to 0.4 M, with no significant difference between the two cell lines. (3) The radioactivity was more sensitively released in Fu5 nuclei than in AH-130 nuclei upon treatment with DNAase I. The release of radioactivity was always larger than the release of DNA in both cell nuclei. In contrast to DNAase I, micrococcal nuclease treatment did not show any difference between the two cell lines in the release of radioactivity from nuclei, always showing a release of radioactivity equal to that of DNA.     

Nuclear interactions of zearalanol-oestrogen receptor complexes in rat liver: a comparison with oestradiol-17 beta

Nuclear interactions of alpha-zearalanol (P-1496) and oestradiol-17 beta (E2) were compared following binding of these compounds to cytosolic oestrogen receptor. A single dose of P-1496 (400 micrograms) or E2 (25 micrograms) given subcutaneously to ovariectomized female rats resulted in two peaks of nuclear oestrogen-receptor concentrations at approx. 0.5 and 4.5 h and showed no qualitative differences between the two compounds. Under in vitro cell-free conditions, [3H]P-1496 was also able to cause oestrogen receptor retention by liver nuclei. Moreover, analysis of salt-extracted nuclear-bound receptor on sucrose gradients gave similar results to those obtained using [3H]E2 with a main peak of radioactivity sedimenting at 5S. Using [3H]P-1496, the time-course of nuclear retention was examined in both males and females. These studies showed no sex difference with nuclear-bound radioactivity reaching a plateau between 20-30 min. The ability of oestrogen-receptor complexes to bind to DNA was examined by DNA-cellulose chromatography. Using either [3H]E2 or [3H]P-1496 as the ligand, qualitative sex differences were shown in the number of peaks present. A comparison of chromatographic patterns obtained with the two ligands suggested close similarities in non-covalent DNA binding between the two compounds, in both males and females. These studies indicate that P-1496 is capable of causing retention of oestrogen receptor by liver nuclei and it binds to DNA in a manner similar to E2. Hence, our data do not explain the marked difference in oestrogenic activity observed in vivo between E2 and P-1496.     

Stimulation of choline release from NG108-15 cells by 12-O-tetradecanoylphorbol 13-acetate.

The effects of the potent tumour-promoting phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) on phosphatidylcholine (PtdCho) metabolism were investigated in the neuroblastoma X glioma hybrid cell line NG108-15. TPA (100 nM) stimulated by 150-200% the release into the medium of 3H radioactivity from cells that had been pre-labelled with [3H]choline. H.p.l.c. analysis of the medium revealed that TPA stimulated the release of only free [3H]choline (212 +/- 11% of control), without affecting such other labelled metabolites as [3H]phosphocholine and [3H]glycerophosphocholine. This effect was concentration-dependent, with a half-maximal effect obtained at 27.5 +/- 6.8 nM, and was observable as early as 5-10 min after exposure to TPA. The TPA-induced release of [3H]choline into the medium was accompanied by a small and variable decrease in cellular [3H]PtdCho (to 93 +/- 4% of control). However, the radioactivity associated with water-soluble cellular choline metabolites (mainly [3H]phosphocholine and [3H]glycerophosphocholine) remained unchanged. TPA also stimulated the release of [3H]choline derived from [3H]PtdCho that had been produced via the methylation pathway from [3H]methionine. These data suggest that phosphatidylcholine may serve as the source of free choline released from the cells in response to TPA. The possible enzymic mechanisms underlying this response are discussed.     

Asymmetric distribution of exogenous thymidine among pyrimidine isostichs suggests compartmentalization of replicative DNA synthesis during regeneration in rat liver

The distribution of radioactivity among pyrimidine isostichs (or isoplyths) of DNA from 24-h regenerating rat liver was studied with [3H]Thd, [14C]orotate or with inorganic 32Pi. Expression of incorporated radioactivity as log10% of total radioactivity recovered for each of the 11 pyrimidine isostichs detected showed that radioactivity from [3H]Thd was asymmetrically distributed among the isostichs, i.e., 3H radioactivity failed to access regions of DNA yielding lower molecular weight pyrimidine isostichs as efficiently as it accessed regions yielding higher molecular weight pyrimidine isostichs. The thymine (T) content of isostichs exceeded that of cytosine (C), i.e., T/C ratios for the first 10 isostichs averaged 1.43 +/- 0.08 and 1.28 +/- 0.05, depending on the method of analysis; furthermore, the T/C ratio for isostich 1 was significantly higher than ratios for isostichs 2 through 10. Asymmetric distributions of [3H]Thd radioactivity also were seen at 18 or 30 h post-partial hepatectomy. Thus, radioactivity from [3H]Thd, a DNA precursor from the salvage pathway, failed to efficiently access lower molecular weight isostichs despite thymine enrichment, suggesting that thymine moieties were supplied from additional sources. Radioactivity from [14C]orotate accessed lower molecular weight pyrimidine tracts more efficiently than [3H]Thd, but less efficiently than it accessed higher molecular weight isostichs, resulting in an asymmetric distribution of 14C radioactivity. This result suggested that appreciable quantities of thymine and cytosine moieties utilized for DNA synthesis were supplied de novo, but other sources also were utilized. Radioactivity from 32Pi, a de novo precursor, was distributed symmetrically, i.e., the slope among lower molecular weight isostichs increased enough that it was indistinguishable from slopes for intermediate and higher molecular weight isostichs. Since 32P radioactivity among lower molecular weight isostichs reflects appreciable contributions of de novo phosphate moieties from both pyrimidine- and purine-containing deoxynucleoside triphosphates, opportunities for observing contributions of 32P radioactivity from pathways other than the de novo pathways appeared to lie beyond limits of detectability. The distribution of radioactivity from labeled DNA precursors among lower molecular weight pyrimidine tracts (a) indicate that thymine moieties are contributed by both salvage and de novo pathways; (b) support the possibility that cytosine moieties also are contributed by both pathways; and (c) support the 'replitase' concept for channeling dNTPs to replicating forks.     

The effect of ascorbic acid on collagen polypeptide synthesis and proline hydroxylation during the growth of cultured fibroblasts

The rate of collagen synthesis relative to the rate of synthesis of noncollagen protein was determined in several lines of cultured fibroblasts using an assay which measures [¹⁴C]proline incorporation into the polypeptide chains of collagen. In this assay procedure, collagen is degraded by protease-free collagenase regardless of whether proline and lysine residues are hydroxylated, thus separating the process of polypeptide synthesis from hydroxylation. It was found that the relative rate of collagen synthesis in L-929 cells was approximately 0.8–1% at all stages of growth. There was no significant increase in the relative rate of collagen synthesis in stationary phase compared to log phase cells in the lines Balb 3T3, 3T6, 3T12, and Swiss mouse 3T6. In all cases, the absolute incorporation of [¹⁴C]proline into both collagen and noncollagen proteins expressed as radioactivity incorporated per milligram of cellular protein, was 2–10 times higher in log phase cells, depending on the line examined.     

Estrogen metabolism as measured in blood and urine in female rhesus monkeys

In order to measure the interconversions of estrone (E1) and estradiol (E2) and their conversion to the 16 alpha-hydroxylated estrogens, 16 alpha-hydroxy estrone (16 alpha-OHE1) and estriol (E3), we infused 11 female rhesus monkeys with [3H]E2 and [14C]E1 and measured radioactivity in the blood as E1, E2 and 16 alpha-OHE1 (n = 9) and in the urine as the glucuronides of E1, E2, 16 alpha-OHE1, and E3 (n = 11). The mean conversion of E1 to E2 as measured in blood (percent of infused E1 measured in blood as E2, [rho]1.2BB) was 29.2 +/- 1.6% and as measured in the urine of the same animals, [rho]1.2BM, was 77.4 +/- 5.9%. The mean conversion of E2 to E1, [rho]2.1BB was 21.5 +/- 1.0% and as measured in urine, [rho]2.1BM was 67.7 +/- 4.6%. Thus for both estrone and estradiol only 30-35% of the interconversions occurred in pools which were in equilibrium with the blood pool of these estrogens. The remaining 65-70% occurred in a pool, probably liver, in which glucuronidation occurred immediately after conversion. The conversion ratios (the ratio of the concentration in the blood of radioactivity as 16 alpha-OHE1 to its precursor, CRPrec,16 alpha-OHE1) was 0.036 +/- 0.008 for CRE1,16 alpha-OHE1 and 0.0039 +/- 0.0010 for CRE2,16 alpha-OHE1. The percentages of administered E1 excreted in the urine as the glucuronides of E1, E2, 16 alpha-OHE1 and E3 were 20.1 +/- 1.5, 1.6 +/- 0.2, 0.96 +/- 0.20 and 0.76 +/- 0.07 respectively. The percentages of administered E2 excreted in the urine as E1, E2, 16 alpha-OHE1 and E3 were 14.4 +/- 1.0, 2.2 +/- 0.3, 0.57 +/- 0.05 and 0.68 +/- 0.11 respectively. Thus there are minor differences in the patterns of excreted metabolites of E1 and E2. Furthermore, 16 alpha-OHE1 and E3 are not major metabolites of E1 or E2 in the female rhesus monkey.     

Metabolism of arachidonic acid in vitro by ovine conceptuses recovered during early pregnancy

Metabolism of [3H] arachidonic acid ([3H] AA) and synthesis of prostaglandins were examined with ovine conceptuses and endometrial slices collected on various days after mating. Tissues were incubated for 24 hr with or without 5 microCi of [3H] AA and with 200 micrograms radioinert AA. In experiment 1, results of chromatography indicated that conceptuses collected on days 14 and 16 after mating metabolized [3H] AA to PGE2, PGF2 alpha, PGFM, 6-keto-PGF1 alpha, and to unidentified compounds in three chromatographic regions. One of these regions (region I) contained triglycerides. Endometrial slices metabolized only small amounts of the [3H] AA to prostaglandins. In experiment 2, results of radioimmunoassays indicated that day 14 conceptuses released somewhat similar amounts (ng/mg tissue) of PGF2 alpha (32.1 +/- 17.9), PGFM (8.4 +/- 6.2), PGE2 (12.3 +/- 7.5) and 6-keto-PGF1 alpha (41.4 +/- 4.8), whereas day 16 conceptuses released more (P less than .05) PGF2 alpha (9.0 +/- 4.1) and 6-keto-PGF1 alpha (15.9 +/- 2.7) than PGE2 (0.9 +/- 0.2) or PGFM (0.5 +/- 0.08). Day 14 and 16 endometrial slices released (ng/mg tissue) more (P less than .05) PGFM (3.0 +/- 0.2) and 6-keto-PGF1 alpha (4.0 +/- 0.4) than PGF2 alpha (0.5 +/- 0.08) or PGE2 (0.05 +/- 0.02). In experiment 3, conceptuses were recovered on days 16, 20 and 24 of pregnancy and incubated with [3H] AA to determine the effects of indomethacin on [3H] AA metabolism. In general, indomethacin (Id; 4 X 10(-4) M) reduced (P less than .05) the percentage of total dpm recovered as prostaglandins, but Id increased the release of chromatographic region I. Experiment 4 was conducted with day 16, 20 and 24 conceptuses to evaluate the time course of metabolism of [3H] AA, and the appearance of region I and of prostaglandins. In general, the percentage of total dpm in region I increased as the percentage of dpm as [3H] AA decreased. The percentage of dpm as prostaglandins increased as the percentage of dpm in region I decreased. Prostaglandins, probably essential for embryonal survival and development, were synthesized in vitro by ovine conceptuses.     

Generation of Arachidonic Acid and Diacylglycerol Second Messengers from Polyphosphoinositides in Ischemic Fetal Brain

Intracerebral administration of [3H]arachidonic acid ([3H]ArA) into 19-20-day-old rat embryos, resulted in a rapid incorporation of label into brain lipids. One hour after injection, 55.6 +/- 8.2, 18.0 +/- 3.4, and 13.7 +/- 1.3% of the total radioactivity was associated with phosphatidylcholine, phosphatidylinositol, and phosphatidylethanolamine, respectively. Approximately 10% of radioactivity was found acylated in neutral lipids of which free ArA comprised only 1.5 +/- 0.2% of the total radioactivity. Complete restriction of the maternal-fetal circulation for < or = 40 min did not affect the rate of [3H]ArA incorporation (t1/2 = 2 min) into fetal brain lipids, suggesting an effective acylation mechanism that proceeds irrespective of the impaired blood flow. After a short restriction period (5 min), the radioactivity in diacylglycerol was elevated by 50%. After a longer restriction period (20 min), the radioactivity in the free fatty acid and diacylglycerol fractions increased to values of 130 and 87%, respectively. Polyphosphoinositides prelabeled with either [3H]ArA or 32P were rapidly degraded after 5 min of ischemia. After 20 min, the decrease in phosphatidylinositol-4-phosphate and phosphatidylinositol-4,5-bisphosphate radioactivity was 47 and 70%, respectively. Double labeling of phospholipids with [14C]palmitic acid and [3H]ArA indicated a preferential loss of [3H]ArA within the polyphosphoinositide species after 20 min, but not after 5 min of ischemia. The specific activity of [14C]palmitate remained unchanged. The current data suggest phospholipase C-mediated diacylglycerol formation at the beginning of the insult followed by a phospholipase A2-mediated ArA liberation at a later time, both enzymes presumably acting preferentially on polyphosphoinositide species.     

Cellular distribution and clearance of aerosolized dipalmitoyl lecithin.

Wistar-Lewis rats were anesthetized anc connected to a 3-MHz nebulizer which aerosolized 250 muCi l-alpha-1-palmitoyl-2palmitoyl-[9-10-3H]phosphatidylcholine ([3H]DPL) for 3 min. Appleton frozen-section autoradiographs showed greater than 4 times background radioactivity in approximately 30% of alveoli at 1 min and 2 h after aerosol. As tritium content in the lung decreased, it increased in liver, spleen, kidney, blood, and urine. Percentage of radioactivity from [3H]phosphatidylcholine in the liver declined with time, while [3H]phosphatidylethanolamine doubled between 2 and 12 h. One minute postaerosol 2,500 +/- 500 (SE) type I cells/mm3 lung and 2,500 +/- 750 type II cells/mm3 lung had greater than 20 times background radioactivity; 2 h later only 950 +/- 250 type I cells/-m3 lung still had levels of radioactivity greater than 20 times background while 3,150 +/- 600 type II cells/mm3 lumg now had this level of 3HIDPL. Corresponding numbers of alveolar macrophages were 450 +/- 250 1 min postaerosol and 1,100 +/- 200 after 2 h. Aerosolized DPL as a synthetic surfactant is hampered by significantly faster clearance from the alveolar surface as compared with normal in vivo DPL.