Effect of ascorbic acid on ACTH-induced cyclic AMP formation and steroidogenesis in isolated adrrenal cells of vitamin E-deficient rats

Isolated adrenal cells from Vitamin E-deficient and control rats were prepared by a trypsin digestion method. Cyclic adenosine 3′,5′-monophosphate (cyclic AMP) formation was studied in response to adrenocorticotropin (ACTH) in the presence and absence of ascorbate by measuring the conversion of prelabeled adenosine 5′-triphosphate [¹⁴C]ATP to cyclic [¹⁴C]AMP. Ascorbate (0.5 mM) inhibited ACTH-induced cyclic [¹⁴C]AMP formation in adrenal cells isolated from Vitamin E-deficient rats but had no effect in the control cells. The inhibitory effect of ascorbate on ACTH-induce cyclic AMP formation in Vitamin E-deficient rats decreased as the concentration of ACTH increased. In Vitamin E-deficient rats ascorbate inhibited ACTH-induced cyclic [¹⁴C]AMP formation after 30 min of incubation. There was no further significant accumulation of cyclic [¹⁴C]AMP at 60 min or 120 min although in the absence of ascorbate cyclic [¹⁴C]AMP continued to be formed. The in vitro addition of α-tocopherol reduced the inhibition of ACTH-induced cyclic [¹⁴C]AMP formation by ascorbate in Vitamin E-deficient rats.These studies suggest that α-tocopherol and ascorbate may affect ACTH-induced cyclic AMP formation through interaction with membrane-bound enzyme adenylate cyclase.     

Comparison of the Ca ++ requirement for the steroidogenic effect of ACTH acid dibutyryl cyclic AMP in rat adrenal cell suspension

Calcium requirement for ACTH and Dibutyryl cyclic AMP (DBCAMP) stimulation of steroidogenesis was compared in rat adrenal cell suspensions. In the absence of added calcium ACTH at low concentrations (< 1 mU/ml) was ineffective; however, the calcium requirement decreased when higher concentrations of ACTH were used. This was not the case with DBCAMP. At all levels of the nucleotide tested, the Ca⁺⁺ requirement was about the same. When the cells were preincubated with EGTA, the Ca⁺⁺ requirement became more pronounced for ACTH than for DBCAMP. The results indicate that the events before the formation of cyclic AMP show a greater dependence on Ca⁺⁺ than the events following its formation.     

Effect of ascorbic acid on ACTH-induced cyclic AMP formation and steroidogenesis in isolated adrenal cells of vitamin E-deficient rats.

Isolated adrenal cells from Vitamin E-deficient and control rats were prepared by a trypsin digestion method. Cyclic adenosine 3',5'-monophosphate (cyclic AMP) formation was studied in response to adrenocorticotropin (ACTH) in the presence and absence of ascorbate by measuring the conversion of prelabeled adenosine 5'-triphosphate [14C]ATP to cyclic [14C]AMP. Ascorbate (0.5 mM) inhibited ACTH-induced cyclic [14C]AMP formation in adrenal cells isolated from Vitamin E-deficient rats but had no effect in the control cells. The inhibitory effect of ascorbate on ACTH-induced cyclic AMP formation in Vitamin E-deficient rats decreased as the concentration of ACTH increased. In Vitamin E-deficient rats ascorbate inhibited ACTH-induced cyclic [14C]AMP formation after 30 min of incubation. There was no further significant accumulation of cyclic [14C]AMP at 60 min or 120 min although in the absence of ascorbate cyclic [14C]AMP continued to be formed. The in vitro addition of alpha-tocopherol reduced the inhibition of ACTH-induced cyclic [14C]AMP formation by ascorbate in Vitamin E-deficient rats. These studies suggest that alpha-tocopherol and ascorbate may affect ACTH-induced cyclic AMP formation through interaction with the membrane-bound enzyme adenylate cyclase.     

Stimulatory effect of gonadotropins on the synthesis of adenosine 3′ :5′-cyclic monophosphate and progesterone by suspensions of rat ovarian interstitial cells

A cell suspension was prepared from immature rat ovaries by treatment with trypsin and collagenase. The isolated cells were capable of converting [8-¹⁴C]adenine to cyclic [¹⁴C]AMP and the rate of this conversion was stimulated in vitro by luteinizing hormone and human chorionic gonadotropine, but not by prolactin, norepinephrine, dopamine or albumin. The accumulation of progesterone was also measured in these cells by radioimmunoassay. In vitro addition of luteinizing hormone stimulated the accumulation of radioimmunoassayable progesterone. The conversion of [8-¹⁴C]adenine to cyclic [¹⁴C]AMP showed a rapid increase during the first 30 min of the incubation period when luteinizing hormone was added to the incubation medium. Progesterone accumulation in response to the same dose of luteinizing hormone showed a lag period for the first 30 min of incubation after which there was an increase up to 2 h. The luteinizing hormone-induced progesterone accumulation was sensitive to puromycin, but there was no effect on the luteinizing hormone-induced increase in cyclic [¹⁴C]AMP formation from [8-¹⁴C]-adenine. Actinomycin D also inhibited the luteinizing hormone-induced progesterone accumulation, with no effect on cyclic AMP formation. The results suggest that the luteinizing hormone-induced progesterone accumulation in rat ovarian interstitial cell suspension is preceded by an increased accumulation of cyclic AMP and that the accumulation of steroid under the influence of luteinizing hormone involve processes sensitive to puromycin and antinomycin D.     

Biogeochemistry of oxalate in the antarctic cryptoendolithic lichen-dominated community

Cryptoendolithic (hidden in rock) lichen-dominated microbial communities from the Ross Desert of Antarctica were shown to produce oxalate (oxalic acid). Oxalate increased mineral dissolution, which provides nutrients, creates characteristic weathering patterns, and may ultimately influence the biological residence time of the community. Oxalate was the only organic acid detectable by HPLC, and its presence was verified by GC/MS. Community photosynthetic metabolism was involved in oxalate production since rates of ¹⁴C-oxalate production from ¹⁴C0₂ were higher in light than in dark incubations. Flaking of the sandstone at the level of the lichen-dominated zone a few millimeters beneath the rock surface can be explained by dissolution of the sandstone cement, which was enhanced by Si, Fe, and Al oxalate complex formation. Added oxalate was observed to increase the solubility of Si, Fe, Al, P, and K. Oxalate's ability to form soluble trivalent metal-oxalate complexes correlated with the observed order of metal oxide depletion from the lichen-dominated zone (Mn > Fe > Al). Thermodynamic calculations predict that Fe oxalate complex formation mobilizes amorphous Fe oxides (ferrihydrite) in the lichen-dominated zone, and where oxalate is depleted, ferrihydrite should precipitate. Hematite, a more crystalline Fe oxide, should remain solid at in situ oxalate concentrations. Oxalate was not a carbon source for the indigenous heterotrophs, but the microbiota were involved in oxalate mineralization to CO₂, since oxalate mineralization was reduced in poisoned incubations. Photooxidation of oxalate to C02 coupled with photoreduction of Fe(Ill) may be responsible for oxalate removal in situ, since rates of ¹⁴C-oxalate mineralization in dark incubations were at least 50% lower than those in the light. Removal of oxalate from Si, Fe, and Al complexes should allow free dissolved Si, Fe, and Al to precipitate as amorphous silicates and metal oxides. This may explain increased siliceous crust (rock varnish or desert varnish) formation near the surface of colonized rocks were light intensity is greatest.Offprint requests to: C.G. Johnston.     

Evidence for 6-keto-PGF1α in adrenal cortex of the rat and effects of 6-keto-PGF1α and PGI2 on adrenal cAMP levels and steroidogenesis

Both intact cortical tissue and isolated cortical cells from the adrenal gland of the rat were analyzed for 6-keto-PGF_1α, the hydrolysis metabolite of PGI₂, using high-performance liquid chromatography and gas chromatography-mass spectrometry. 6-Keto-PGF_1α was present in both incubations of intact tissue and isolated cells of the adrenal cortex, at higher concentrations than either PGF_2α or PGE₂. Thus, the cortex does not depend upon vascular components for the synthesis of the PGI₂ metabolite. Studies $\text{in vitro}$, using isolated cortical cells exposed to 6-keto-PGF_1α (10^?6-10^?4M), show that this PG does not alter cAMP levels or steroidogenesis. Cells exposed to PGI₂ (10^?6-10^?4M), however, show a concentration-dependent increase of up to 4-fold in the levels of cAMP without altering corticosterone production. ACTH (5–200 μU/ml) increased cAMP levels up to 14-fold, and corticosterone levels up to 6-fold, in isolated cells. ACTH plus PGI₂ produced an additive increase in levels of cAMP, however, the steroidogenic response was equal to that elicited by ACTH alone. Adrenal glands of the rat perfused $\text{in situ}$ with PGI₂ showed a small decrease in corticosterone production, whereas ACTH greatly stimulated steroid release. Thus, while 6-keto-PGF_1α is present in the rat adrenal cortex, its precursor, PGI₂, is not a steroidogenic agent in this tissue although it does stimulate the accumulation of cAMP.     

Adrenocorticotropin/α-Melanocyte-Stimulating Hormone (ACTH/MSH)-Like Peptides Modulate Adenylate Cyclase Activity in Rat Brain Slices: Evidence for an ACTH/MSH Receptor-Coupled Mechanism

Abstract: The regulation of adenylate cyclase activity by adrenocorticotropin/α-melanocyte–stimulating hormone (ACTH/MSH)-like peptides was investigated in rat brain slices using a superfusion method. Adenylate cyclase activity was concentration-dependently increased by ACTH-(1–24), α-MSH (EC₅₀ values 16 and 6 nM, respectively), and [Nle⁴,D-Phe⁷]α-MSH (EC₅₀ value 1.6 nM), in the presence of forskolin (1 μM, optimal concentration). 1-9-Dideoxy-forskolin did not augment the response of adenylate cyclase to ACTH-(1–24). Various peptide fragments were tested for their ability to enhance [³H]cyclic AMP production. [Nle⁴,D-Phe⁷]α-MSH increased [³H]cyclic AMP formation with a maximal effect of 30% and was more potent than ACTH-(1–24), ACTH-(1–16)-NH₂, α-MSH, ACTH-(1–13)-NH₂, [MetO⁴]α-MSH, [MetO₂⁴,D-Lys⁸,Phe⁹]ACTH-(4–9), ACTH-(7–16)-NH₂, ACTH-(1–10), and ACTH-(11–24), in order of potency. This structure–activity relationship resembles that found for the previously described peptide-induced display of excessive grooming. ACTH-(1–24) stimulated adenylate cyclase activity in both striatal (maximal effect, ?20%) and septal slices (maximal effect, ?40%), but not in hippocampal or cortical slices. Lesioning of the dopaminergic projections to the striatum did not result in a diminished effect of [Nle⁴,D-Phe⁷]α-MSH on [³H]cyclic AMP accumulation, which indicates that the ACTH/MSH receptor–stimulated adenylate cyclase is not located on striatal dopaminergic terminals. ACTH-(1–24) did not affect the dopamine D₁ or D₂ receptor–mediated modulation of adenylate cyclase activity. Based on the present data, we suggest that the binding of endogenous ACTH or α-MSH to a putative ACTH/MSH receptor in certain brain regions leads to the activation of a signal transduction pathway using cyclic AMP as a second messenger.     

The incorporation of glycerol into mitochondrial lipids during neonatal renal compensatory growth

[¹⁴C]glycerol incorporation into isolated inner and outer mitochondrial membrane is enhanced in the remaining kidney after unilateral nephrectomy. Serum from neonatal rabbits taken 24 hours after unilateral nephrectomy and added to tissue slice incubations appears to stimulate incorporation of [¹⁴C]glycerol into mitochondrial lipids of normal kidney cortex. Post-nephrectomy serum, however, depresses incorporation of [¹⁴C] glycerol and [³H]leucine into mitochondria when added to kidney cortex from animals in which uninephrectomy was performed 24 or 48 hours previously.     

The effect of cyclic adenosine 3′,5′-monophosphate on the uptake of estradiol by the neonatal mouse uterus

Summary Uterine tissue from neonatal mice was incubated in vitro at 0° C for 1 h in a medium containing 1×10^-8 M ³H-estradiol with or without 1×10^-4M dibutyryl cyclic AMP. In some incubations the temperature was raised to 37° C for 15 min after incubation in the cold, in others the temperature was kept at 0° C during this 15 min period. The tissue was frozen in liquid propane cooled in liquid nitrogen, sectioned at 2 or 4 microns, and autoradiograms prepared according to the dry-mount procedure. cAMP increases the cellular uptake of ³H-estradiol in uterine tissue. After rising the temperature to 37° C, grains appeared over the nuclei. cAMP at low temperature increased the cellular uptake of ³H-estradiol, but the grains were not associated with the nuclei. In the autoradiograms the grain number above the epithelium was markedly less than above the stroma.This work has been supported by grants from the Norwegian Research Council for Science and the Humanities, and from the Norwegian Cancer Society (Landsforeningen mot Kreft)     

Prostaglandin synthesis in rat adrenocortical cells.

The biosynthesis of prostaglandins by isolated rat adrenocortical cells has been studied by determinations of products formed during incubations with labeled arachidonic acid and by radioimmunoassays. Analysis by thin-layer chromatographic separation of silicic acid column fractions indicated that PGE2, PGA2, (B2) and PGF2 alpha were the predominant prostaglandins formed by rat adrenocortical cells. Approximately 75% of the incorporated isotope was associated with the prostaglandins of the PGE pathway [PGE2 + PGA2 (B2)]. This was a consistent finding whether cells were incubated directly with arachidonic acid or with cells prelabeled with the substrate prior to study. ACTH did not affect the uptake or oxidation of [1-14C]-arachidonate, but did significantly increase incorporation of labeled substrate into [14C]prostaglandins. Of the ACTH-induced increase, 92% was accounted for by an increase in prostaglandins of the E pathway. Studies with prelabeled cells indicated that 77% of the prostaglandins synthesized in both control and ACTH-stimulated adrenocortical cells was released into the incubation medium during the 2-hr study. These had the same composition [88% PGE2 + PGA2 (B2)] as did the intracellular prostaglandins. Analysis by radioimmunoassays gave comparable data on the distribution of E- and F-type prostaglandins in control cells and cells incubated with ACTH or dibutyryl cyclic AMP. Thus, with these techniques, 88-92% of the increased prostaglandin synthesis due to ACTH or cyclic AMP was produced by the PGE2 rather than the PGF2 alpha pathway.     

A calorimetric method to assess endogenous metabolism and its application to the study of bovine sperm

Calorimetry was used to assess the importance of endogenous metabolism towards total ATP synthesis in bovine sperm in the presence of extracellular glucose. Sperm were incubated in the calorimeter with d-[U-¹⁴C]glucose without or with electron transport inhibitors, rotenone and antimycin A. Steady-state heat production during the incubation was measured for 30 min, the incubations were terminated, and the cell suspensions removed for analysis of radioactive glucose and its metabolic end-products. Heat production (mean±S.E. associated with the metabolism of glucose was calculated, from enthalpies of formation of glucose and its end-product, as ?412±34 mJ/h/10⁸ cells in control incubations and ?263±18 mJ/h/10⁸ cells in incubations with electron transport inhibitors. Measured heat production was ?455±36 and ?263±17 mJ/h/10⁸ cells, respectively. Thus, heat production by endogenous pathways, the difference between measured total heat production and calculated exogenous heat production, was ?43±14 mJ/h/10⁸ cells fro control cells and about ?6 mJ/h/10⁸ cells for inhibited cells. The ration of heat produced per mol of ATP synthesized is similar for all ATP-producing pathways. Therefore, about 10% of total ATP synthesis in control cells and less than 2% in inhibited cells is provided by endogenous pathways when extracellular glucose is present.     

Disposition and metabolism of Ro 24-4736 in the rat

Ro 24-4736, a new platelet activating factor antagonist, is currently under preclinical and clinical development. The tissue distribution of the ¹⁴C-label in male rats following a single intravenous dose of 1.0 mg/kg of ¹⁴C-Ro 24-4736 indicated appreciable uptake by the liver, kidney, heart and gastrointestinal tract. Peak plasma and tissue concentrations were seen at 5 minutes after dosing except for the small intestine (4 hrs) and abdominal fat, stomach and large intestine (4 hrs). Thereafter, the ¹⁴C-label rapidly declined in all tissues. At 48 hours, only 3.5% of the dose was present in the tissues, and 6.1% in the lumen of the gastrointestinal tracts. The excretion of ¹⁴C was essentially completed; 94% of the administered ¹⁴C was excreted in the feces and 4.0% in the urine. Overall recoveries of the administered ¹⁴C label ranged from 96 to 116%. The purified major ¹⁴C-labelled component in the fecal extracts yielded essentially the same NMR spectrum as authentic Ro 24-4736 which accounted for 11% of the dose. In vitro incubations of Ro 24-4736 with rat liver 9S supernatant in an NADPH generating system produced two metabolites. NMR spectra indicated that one metabolite was hydroxylated at carbon-1 while the other one contained a hydroxyl at carbon-10 of the parent molecule. Interestingly, the sites of hydroxylation were at carbons C₁, and C₁₀ bearing the protons guarding the bay area of the phenanthrenoid ring, rather than carbons of the phenyl-methyl-thienotriazolodiazepine moiety.     

UTILIZATION OF ACETATE AND PYRUVATE FOR THE SYNTHESIS OF'TOTAL','BOUND'AND'FREE'ACETYLCHOLINE IN THE ELECTRIC ORGAN OF TORPEDO

—Slices of tissue of the electric organ of Torpedo marmorata were incubated in vitro in a salineurea-sucrose solution containing a labelled precursor of the acetyl moiety of ACh ([1-¹⁴C]glucose, [2-¹⁴C]pyruvate, or [1-¹⁴C]acetate) either alone or in the presence of another unlabelled precursor. The incorporation of ¹⁴C from [1-¹⁴C]acetate into ACh was considerably higher than from the other two substrates. The specific radioactivities (SRA) of the‘total',‘bound’and‘free’ACh were compared in experiments with [2-¹⁴C]pyruvate and [1-¹⁴C]acetate. With both precursors, the SRA of the‘bound’ACh were lower than those of‘total’ACh; consequently, the‘free’ACh pool was more labelled than the‘bound’pool. After short incubations with [2-¹⁴C]pyruvate the SRA of'bound’ACh were closer to the SRA of‘total’ACh than with [1-¹⁴C]acetate. A simple method is described for the labelling of ACh and its separation from other labelled compounds in experiments with the electric organ using [¹⁴C]acetate as the labelled precursor.     

Adrenal cells in tissue culture. The effect of papaverine and amytal on steroidogenesis, respiration and replication

The smooth-muscle relaxant papaverine has been shown to be a potent inhibitor of cyclic AMP phosphodiesterase activity (Kukovetz, W. R., and Poch, G. (1970) Naunyn Schmiedebergs Arch. Pharmakol.267, 189). Because of this, papaverine was tested in monolayer cultures of functional mouse adrenal cortex tumor cells for possible stimulatory effects on Steroidogenesis. At 10^?5m, papaverine was found to inhibit ACTH-stimulated steroidogenesis 50% and at 10^?4m, > 95%. This was associated with a > 10-fold increase in [¹⁴C]lactate production from [¹⁴C]glucose and a 50% reduction in ³²P_i, incorporation into macromolecules. These findings were similar to those observed with the barbiturate amytal, an inhibitor of the mitochondrial electron-transport chain at the level of oxidation of NADH (Site I). Papaverine was 100 times more effective than amytal in inhibiting steroidogenesis and 1000 times more effective in initiating an increase in glycolysis. In intact tumor cells and mitochondria isolated from normal rat adrenals, papaverine (10^?4m) completely inhibits oxygen uptake supported by malate or α-ketoglutarate. Oxygen uptake is restored by the addition of succinate, suggesting that, like amytal, papaverine inhibits respiration at Site I.Papaverine does not inhibit NADPH-supported cholesterol side-chain cleavage in bovine adrenal acetone powders or 11β-hydroxylation in normal rat adrenal cortex mitochondria. By contrast, amytal inhibits both these activities at concentrations comparable to that effective in intact adrenal cells, suggesting a direct interaction of amytal with cytochrome P-450. Both papaverine and amytal inhibit incorporation of thymidine into nuclear DNA to an extent far greater than that observed with either maximally stimulating levels of cyclic AMP or high concentrations of ACTH. Succinate does not reverse the inhibitory effects of either papaverine or amytal on thymidine incorporation into DNA. Papaverine increases intracellular cyclic AMP in both resting and ACTH-treated cells. However, the effects of papaverine on steroidogenesis, glycolysis, ATP-P_i exchange, and DNA synthesis in adrenocortical cells are not directly attributable to this action.     

Diel phytoplankton periodicity in Mikołajskie lake,Poland, as determined by different methods in parallel

Photosynthetic rates as measured by the oxygen light and dark bottle method were highly correlated with estimates using the ¹⁴C technique. The high O₂/¹⁴C ratios found are explained by algal respiration and extracellular release which are included in photosynthetic measurements by the oxygen technique, while the ¹⁴C method yields values close to net photosynthesis. Separation of net- and nannoplankton using a 50 μm plankton net for filtration was not comparable to distinctions made by microscopic examination. Separation of both by filtration caused a significant decrease in the photosynthetic activity of nannoplankton in 24-hour incubations, but had no detectable effect after 4 hours of exposure. “Bottle effects” in 24-hour measurements of photosynthesis were similar using both methods. Asymmetric photosynthetic time-curves as well as vertical phytoplankton migrations were the main reasons for errors in estimates of daily photosynthetic rates from part-day incubations which were extrapolated to the entire day.     

Acetylation and hydroxylation of 5alpha-androstane-3beta,17beta-diol by prostate and epididymis

An unknown radiometabolite, formed in the canine prostate and epididymis after intra-arterial infusion of testosterone-4-¹⁴C in physiologic saline and extraction of the organs with ethyl acetate-acetone, was identified as the 3-monoacetate of 5α-androstane-3β, 17β-diol (3β-diol). Transformation of 3β-diol-¹⁴C to its identified 3-monoacetate derivative could also be demonstrated, if the incubation of the radiosubstrate with minced canine prostate was terminated by ethyl acetate extraction. The formation of polar products in high yield was noted. Whereas minced canine prostate actively converted 5α-androstane-3α,17β-diol-¹⁴C to 17β-hydroxy-5α-androstan-3-one-¹⁴C, the same preparation hydroxylated 3β-diol-¹⁴C predominantly at the 7ξ- and, to a lesser extent, at the 6ξ-positions. Partial identification of the hydroxylated radiometabolites was by crystallization of the CrO₃-oxidation products 5α-androstane-3,6,17-trione-¹⁴C and 5α-androstane-3,7,17-trione-¹⁴C to constant SA and by GLC/MS of the latter derivative. NADPH-supplementation of the preparation enhanced the yield of hydroxylated products derived from 3β-diol-¹⁴C in a 1 hr incubation from 22% to 41%. Analogous supplemented incubations of benign hyperplastic human prostate and canine epididymis produced polar metabolites (in 12.5% and 76% yields, respectively) which gave rise to similar proportions of the same androstanetrione epimers on CrO₃-oxidation.     

Analysis of DNA adducts by accelerator mass spectrometry in human breast tissue after administration of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and benzo[a]pyrene

Epidemiological evidence has suggested an association between meat consumption and the risk of breast cancer. 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a heterocyclic amine found in cooked meat, has been implicated in the aetiology of breast cancer and has been shown to induce tumour formation in rodent mammary glands. In addition, polycyclic aromatic hydrocarbons, such as benzo[a]pyrene (B[a]P) which has also been shown to induce tumour formation at a number of sites in rodents including the breast, are produced during the cooking of meat through the pyrolysis of fats. The aim of this study was to examine the bioavailability of these compounds to human breast tissue and their ability to bind to DNA to form DNA adducts. Patients undergoing breast surgery at York District Hospital were orally administered prior to surgery a capsule containing 20 μg of ¹⁴C PhIP (182 kBq, specific activity 2.05 GBq/mmol) or 5 μg of ¹⁴C B[a]P (36 kBq, specific activity 1.81 GBq/mmol). At surgery, normal and tumour breast tissue was resected and tissue concentrations of carcinogen measured by liquid scintillation counting and DNA adduct levels by accelerator mass spectrometry (AMS) were subsequently determined. It was found that both ¹⁴C PhIP and ¹⁴C B[a]P were able to reach the target organ where they had the ability to form DNA adducts. The level of adducts ranged from 26.22–477.35 and 6.61–208.38 adducts/10¹² nucleotides following administration of ¹⁴C PhIP and ¹⁴C B[a]P, respectively, with no significant difference observed between levels in normal or tumour tissue. In addition, the data obtained in this study were comparable to adduct levels previously found in colon samples following administration of the same compounds to individuals undergoing colorectal surgery. This is the first report that these two carcinogens bind to human breast DNA after administration of a defined low dose.     

Cyclic AMP-induced release of [14C]taurine from pinealocytes.

Pinealocytes were prelabelled with [¹⁴C]taurine. Twenty-four hours later they were treated with derivatives of cyclic AMP. It was found that dibutyryl cyclic AMP and ?-chloro-phenyl-thio cyclic AMP treatment caused a large increase in the release of [¹⁴C]taurine. The effect of dibutryl cyclic AMP on [¹⁴C]taurine release was near maximal fifteen minutes after treatment started. In view of the known stimulatory effects of norepinephrine on pineal cyclic AMP and the recent discovery that norepinephrine causes the release of taurine from pinealocytes, one can conclude that norepinephrine stimulates [¹⁴C]taurine release from pinealocytes by acting through a cyclic AMP mechanism.     

UPTAKE AND RELEASE OF [14C]ADENINE DERIVATIVES AT BEDS OF MAMMALIAN CORTICAL SYNAPTOSOMES IN A SUPERFUSION SYSTEM

(1) Synaptosomal fractions from guinea pig neocortical dispersions prepared in sucrose solutions were deposited from saline media as ‘beds’ on nylon bolting cloth. When incubated with 0.5–10 μm -[¹⁴C]adenine or adenosine in glucose bicarbonate salines, uptake of ¹⁴C from adenosine proceeded at about four times the rate of uptake of [¹⁴C]adenine. This contrasted with the relative uptake of the two compounds to neocortical tissue slices or to beds made from mitochondrial fractions, where uptake was similar with the two precursors. Uptake of both precursors to synaptosome beds was much greater than uptake of inosine. (2) Synaptosome beds, [¹⁴C]adenosine-loaded, contained 88 per cent of the ¹⁴C as 5′-adenine nucleotides, the remainder being present as cyclic AMP, inosine, hypoxanthine and adenosine. When superfused, the ¹⁴C output consisted mainly of adenosine, inosine and hypoxanthine, with some 7 per cent of 5′-nucleotides and 4 per cent of cyclic AMP. (3) Electrical pulses and the addition of 50 mm -KCl each increased the efflux of ¹⁴C from superfused [¹⁴C]adenosine-loaded beds. The superfusates issuing after excitation contained the same ¹⁴C-labelled compounds as issued before, with a small increase in the proportional yield of adenosine. The additional output of ¹⁴C following electrical pulses was diminished by about 50 per cent by 0.5 μm -tetrodotoxin while that following KCl was not affected; it was however prevented when the superfusing fluids were free of Ca²⁺.