In vitro metabolism of 17beta-estradiol by human liver tissue.

17beta-[6,7- 3H]Estradiol was incubated with adult human liver slices in Krebs-Ringer phosphate buffer containing glucose. Of the identified 3H recovered, 51-76 percent consisted of estrone-3-sulfate (E13S) and 17 beta-estradiol-3-sulfate (E23S). E13S was the main metabolite and was found in both tissue and medium. E23S was present only in the medium. Minor amounts of estrogen glucuronides were formed. When a human liver homogenate was incubated with [3H]E2 in a medium fortified with excess uridine diphosphate glucuronic acid only some 4 percent of conjugation with glucuronic acid was observed. It is suggested that human liver favors sulfurylation as the conjugating mechanism for E2 and E1.     

17-oxidoreduction of 17beta estradiol, estrone and their 3-sulfates by kidney slices from guinea pig and human.

Slices of whole kidney and kidney cortex from the female guinea pig catalyzed a marked reduction of estrone 3-sulfate (E13S) and estrone (E1) to 17beta-estradiol 3-sulfate (E23S) and 17beta-estradiol (E2), respectively, as well as the reverse (dehydrogenation) reactions. Slices of medulla did not appear active in E23S-E13S interconversion but did possess the ability to interconvert E2 and E1, besides possessing considerable sulfatase activity. The use of [3H-55S]E13S and [3H-55S]E23S as substrates, together with a demonstrated lack of estrogen sulfate synthesis by the tissue slices, provided ample evidence that the intact sulfates were involved in direct oxidoreduction. Slices of human kidney cortex catalyzed the reduction of E13S to a very limited extent. Slices of whole kidney and of cortex from guinea pig formed small amounts of estrogen glucuronide(s).     

Metabolism of estrogens and thier sulfates by mouse kidney tissue in vitro.

Whole kidney tissue from mouse was incubated with labelled estrone 3-sulfate (E13S), 17beta-estradiol 3-sulfate (17betaE23S), estrone (E1), and 17beta-estradiol (17betaE2). Considerable reduction of E13S and E1 occurred. E13S gave rise primarily to 17alpha-estradiol 3-sulfate (17 alphaE23S) together with lesser amounts of 17betaE23S. By incubating [3H-35S]E13S with the tissue it was confirmed that formation of the diol sulfates was direct, without accompanying hydrolysis and reconjugation. Conversion of E1 was mainly to 17betaE2 with, on the average, lesser amounts of 17alphaE2. A small degree of direct conversion of 17betaE23S to E13S was found. 17beta-Estradiol was converted to a limited extent to E1 and to much smaller amounts of 17alphaE2.     

The metabolism of estrone and estradiol-17β and their 3-sulfates by female guinea pig liver microsomes

The metabolism, by female guinea pig liver microsomes of estrogen 3-sulfates (estrone-3-sulfate and 17β-estradiol-3-sulfate) was compared to that of the unconjugated estrogens, estrone and estradiol-17β. Metabolites identified indicated that 16β-hydroxylated products (16β hydroxyestrone and 16 epiestriol) arose mainly from the free estrogens while 16α-hydroxy steroid sulfates (16α hydroxyestrone-3-sulfate and estriol-3-sulfate) were predominantly formed from the sulfated estrogens. These results show that the sulfate moiety at position 3 of the steroids directs 16-hydroxylation from the β to the α configuration.     

Effects of Monensin and Colchicine on Myelin Galactolipids

Monensin and colchicine have been used in a variety of systems to disrupt functioning of the Golgi apparatus and transport of Golgi-derived vesicles to the plasma membrane. In this study the effects of monensin and colchicine on the synthesis of cerebroside and sulfatide and their appearance in myelin were examined to determine whether these myelin components are processed through the Golgi apparatus. Brain slices from rats 17 days old were incubated with [3H]galactose and [35S]-sulfate to label cerebroside and sulfatide. Myelin was isolated on sucrose density gradients. Fractions highly enriched in cerebroside and sulfatide were prepared from homogenates and myelin fractions by lipid extraction, alkaline methanolysis, and in some cases TLC. Monensin at 0.1 microM had no significant effect on synthesis of these galactolipids as measured by incorporation of [3H]-galactose into cerebroside or [35S]sulfate into sulfatide in homogenates. However, appearance of [35S]sulfatide in the myelin fraction was reduced to 49% of control, while appearance of [3H]cerebroside was not significantly reduced. Colchicine from 1 mM to 0.1 microM had effects similar to monensin, that is, appearance of [35S]sulfatide in myelin was depressed, but again [3H]cerebroside was not affected. Incorporation of [35S]sulfate into sulfatide in homogenate was 93% of control, while appearance of [35S]sulfatide in the myelin fraction was depressed to 58% of control. The inhibition of appearance of sulfatide in myelin by colchicine and monensin is consistent with the view that sulfation of cerebroside occurs in the Golgi and that sulfatide is transported via Golgi-derived vesicles to the forming myelin membrane.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding of [35S]GTPgammaS stimulated by (+)-pentazocine sigma receptor agonist, is abundant in the guinea pig spleen

Here we measured sigma receptor agonist, [3H](+)-pentazocine binding and (+)-pentazocine-stimulated [35S]GTPgammaS binding throughout brain regions and peripheral organs of mice and guinea pigs to investigate the distribution of G protein-coupled sigma receptors. There was no significant correlationship between both distributions, in which the [3H](+)-pentazocine binding is highest in the liver of each species, while the [35S]GTP-gammaS binding is highest in the guinea pig spleen. The agonist-stimulated [35S]GTPgammaS binding in the spleen was also confirmed by in situ autoradiography using sections. Thus it is suggested that there are at least two subtypes, metabotropic and nonmetabotropic sigma receptors, and the former ones are abundant in the guinea pig spleen.     

Membrane retrieval in the guinea pig neurohypophysis: biochemical characterization of a retrieval structure

[3H]Choline and [35S]methionine injected into the guinea pig hypothalamus in vivo were incorporated into the lipids and proteins, respectively, of secretory vesicles transported to the neural lobe. Prolonged in vivo stimulation of hormone secretion by dehydration decreased the [3H]choline content of secretory vesicles, with a concomitant increase in the [3H]choline content of a membrane fraction isolated on sucrose gradients. After stimulation of neural lobes in vitro in the presence of horseradish peroxidase, this extracellular fluid marker was found in the same membrane fraction. SDS electrophoresis of membrane proteins radiolabelled by [35S]methionine in vivo demonstrated that this fraction contained at least one major protein also present in the secretory vesicle membrane. These results suggest that we have isolated a membrane fraction containing the structure(s) involve in membrane retrieval in the neurohypophysis.     

A source of error in the chromatographic study of 35S-sulfate labeled mucous glycoproteins secreted by the gill epithelium of Mytilus edulis

HPLC combined with [35S]-sulfate/[3H]-glucosamine radiolabeling were employed to study the synthesis and secretion of mucous glycoproteins. The secreted radiolabeled glycoproteins were separated from the medium by precipitation with a mixture of trichloroacetic-phosphotungstic acids (TCA/PTA). The redissolved glycoproteins were chromatographed on an anion exchange protein column at varying pH of the mobile phase and fractions were collected for liquid scintillation counting. Varying the pH of the mobile phase from pH 3 to 7 resulted in a decrease of glycoprotein bound [35S] from 69.5 to 0.5% of the total recovered [35S]-sulfate with the remainder recovered as free [35S]-sulfate. The [3H]-labeled glycoprotein recovered under the uV peaks at this pH range was 99.5%. When high performance size exclusion chromatography was performed the change in mobile phase pH did not affect the 100% recovery of either [35S]-or [3H]-labels under the uV peaks. No free [35S]-sulfate was obtained when [35S]-labeled glycoproteins were separated from the medium using dialysis. These data suggest that the standard method of TCA/PTA precipitation of [35S]-labeled glycoproteins may cleave the [35S]-sulfate ester linkages to the oligosaccharide chains. The [35S]-sulfate may then rebind to the macromolecule by a relatively strong noncovalent bond. This may prove critical in anion exchange protein HPLC studies.     

Is the Adenosine Receptor Modulation of Histamine-Induced Accumulation of Inositol Phosphates in Cerebral Cortical Slices Mediated by Effects on Calcium Ion Fluxes?

In this report, we show that under conditions designed to provide an initially uniform incorporation of [3H]inositol into mouse and guinea pig cerebral cortical slices prior to agonist stimulation, the accumulation of 3H-inositol phosphates (3H-InsPx, x = 1-4) induced by histamine in mouse and guinea pig cerebral cortical slices increased in a quasilinear manner with increasing added calcium. Raising the ambient calcium ion concentration failed to reduce the adenosine receptor-mediated inhibition of the histamine-induced 3H-InsPx response in mouse cerebral cortical slices. Similarly, the potentiation of the histamine response by adenosine receptor activation in guinea pig cerebral cortical slices was unaffected by lowering the added calcium ion concentration. The presence of the calcium ionophore A23187 (33 microM) produced 3H-InsPx responses in both mouse and guinea pig cerebral cortical slices, which were not affected by the presence of the stable adenosine analogue 2-chloroadenosine. A23187 also potentiated the accumulation of 3HInsPx induced by histamine in both species. Both the inhibitory and potentiatory modulations of the histamine response by 2-chloroadenosine in mouse and guinea pig, respectively, were still apparent in the presence of A23187. These results indicate that the histamine-induced 3H-InsPx accumulations in both mouse and guinea pig cerebral cortical slices are sensitive to variations in calcium ion concentrations. However, the adenosine receptor modulations of the histamine responses are relatively insensitive to fluctuations in either extra- or intracellular calcium ion concentrations, and thus cannot be mediated by effects on calcium ion movements.     

Selective depolymerisation of dermatan sulfate: production of radiolabelled substrates for alpha-L-iduronidase, sulfoiduronate sulfatase, and beta-D-glucuronidase

Radiolabelled disaccharide substrates for alpha-L-iduronidase, beta-D-glucuronidase, and sulfoiduronate sulfatase have been prepared from dermatan sulfate by application in sequence of N-deacetylation, deaminative cleavage, and reduction with NaBT4. The yield of disaccharides was approximately 87% of the total oligosaccharide fraction. Five disaccharides were isolated and tentatively identified. The major disaccharide, O-(alpha-L-idopyranosyluronic acid)-(1 leads to 3)-2,5-anhydro-D-[1-3H]talitol 4-sulfate (IdoA-anT4S), represented approximately 75% of the total disaccharide fraction. The other disaccharides were O-(alpha-L-idopyranosyluronic acid 2-sulfate)-(1 leads to 3)-2,5-anhydro-D-[1-3H]talitol 4-sulfate (IdoA2S-anT4S), O-(beta-D-glucopyranosyluronic acid)-(1 leads to 3)-2,5-anhydro-D-[1-3H]talitol 4-sulfate (GlcA-anT4S), O-(beta-D-glucopyranosyluronic acid)-(1 leads to 3)-2,5-anhydro-D-[1-3H]talitol 6-sulfate (GlcA-anT6S), and O-(alpha-L-idopyranosyluronic acid)-(1 leads to 3)-2,5-anhydro-D-[1-3H]talitol (IdoA-anT), which represented approximately 4.5, 11.2, 1.0, and 1.8%, respectively, of the total disaccharide fraction. When incubated with cultured skin-fibroblasts from normal controls, IdoA-anT4S was shown to be a sensitive substrate for alpha-L-iduronidase to produce 2,5-anhydro-D-talitol 4-sulfate (anT4S). Activity toward IdoA-anT4S was not observed with fibroblast homogenates from alpha-L-iduronidase-deficient patients (Mucopolysaccharidosis Type I). Similarly, normal-fibroblast homogenates degraded GlcA-anT6S to anT6S, and GlcA-anT4S to anT4S, at a rate 6 to 8 times greater than found for fibroblasts from beta-D-glucuronidase-deficient patients (Mucopolysaccharidosis Type VII). IdoA-anT4S was hydrolysed by alpha-L-iduronidase at a rate 365 times greater than that for IdoA-anT. Sulfation of the anhydro-D-[1-3H]talitol residues is an important structural determinant in the mechanism of action of alpha-L-iduronidase on disaccharide substrates. IdoA2S-anT4S was degraded to IdoA-anT4S and then to anT4S by normal-fibroblast homogenates, whereas fibroblasts from alpha-L-iduronidase-deficient and sulfoiduronate sulfatase-deficient (Mucopolysaccharidosis Type II) patients produced considerably decreased levels of anT4s and IdoA-anT4S (and anT4S), respectively.     

INTRACELLULAR TRANSPORT OF SECRETORY PROTEINS IN THE PANCREATIC EXOCRINE CELL : I. Role of the Peripheral Elements of the Golgi Complex

It has been established by electron microscopic radioautography of guinea pig pancreatic exocrine cells (Caro and Palade, 1964) that secretory proteins are transported from the elements of the rough-surfaced endoplasmic reticulum (ER) to condensing vacuoles of the Golgi complex possibly via small vesicles located in the periphery of the complex. To define more clearly the role of these vesicles in the intracellular transport of secretory proteins, we have investigated the secretory cycle of the guinea pig pancreas by cell fractionation procedures applied to pancreatic slices incubated in vitro. Such slices remain viable for 3 hr and incur minimal structural damage in this time. Their secretory proteins can be labeled with radioactive amino acids in short, well defined pulses which, followed by cell fractionation, makes possible a kinetic analysis of transport. To determine the kinetics of transport, we pulse-labeled sets of slices for 3 min with leucine-¹⁴C and incubated them for further +7, +17, and +57 min in chase medium. At each time, smooth microsomes ( = peripheral elements of the Golgi complex) and rough microsomes ( = elements of the rough ER) were isolated from the slices by density gradient centrifugation of the total microsomal fraction. Labeled proteins appeared initially (end of pulse) in the rough microsomes and were subsequently transferred during incubation in chase medium to the smooth microsomes, reaching a maximal concentration in this fraction after +7 min chase incubation. Later, labeled proteins left the smooth microsomes to appear in the zymogen granule fraction. These data provide direct evidence that secretory proteins are transported from the cisternae of the rough ER to condensing vacuoles via the small vesicles of the Golgi complex.     

In vitro metabolism of (6,7-H3)estrone and of (6,7-H3) estradiol by the liver in pregnant guinea pigs]

Slices of pregnant guinea pig liver were incubated with (6,7-3H)estrone and with (6,7-3H)estradiol. Free, glucuro- and sulfo-conjugated fractions were isolated by specific extraction and hydrolysis. The radioactivity distribution in these 3 fractions demonstrated a predominance of conjugated compounds (95% of isolated estrogens) with slightly more glucuro-conjugated than sulfo-conjugated compounds. After isolating estrogens by TLC, we were able to determine estrone and estradiol in these 3 fractions from incubations with 3H-estrone or with 3H-estradiol by means of specific activity recrystallisation. Estriol was determined in glucuro-and sulfo-conjugated fractions after incubation with 3H-estrone as well as in sulfo-conjugated fraction after incubation with 3H-estradiol. Glucuro- or sulfo-conjugated estrone was the predominant estrogen after incubation with 3H-estrone just as after incubation with 3H-estradiol. This led us to conclude to an important 17beta-hydroxysteroid-dehydrogenase activity. The 16alpha-hydroxylastic-activity is weaker since estriol represented only 1,43 % of estrogens isolated after incubation with 3H-estrone and 0.82% after incubation with 3H-estradiol.     

ABCG2 transports sulfated conjugates of steroids and xenobiotics

The mechanism for the cellular extrusion of sulfated conjugates is still unknown. In the present study, we investigated whether human wild type ABCG2 transports estrone 3-sulfate (E1S) using membrane vesicles from cDNA-transfected mouse lymphoma cell line (P388 cells). The uptake of [3H]E1S into ABCG2-expressing membrane vesicles was stimulated by ATP, and the Km value for [3H]E1S was determined to be 16.6 microm. The ABCG2-mediated transport of [3H]E1S was potently inhibited by SN-38 and many sulfate conjugates but not by glucuronide and glutathione conjugates or other anionic compounds. Other sulfate conjugates such as [3H]dehydroepiandrosterone sulfate (DHEAS) and [35S]4-methylumbelliferone sulfate (Km = 12.9 microm) and [35S]6-hydroxy-5,7-dimethyl-2-methylamino-4-(3-pyridylmethyl)benzothiazole (E3040) sulfate (Km = 26.9 microm) were also transported by ABCG2. Although [3H]methotrexate, [3H]17beta-estradiol-17beta-D-glucuronide, [3H]2,4-dinitrophenyl-S-glutathione, and [14C]4-methylumbelliferone glucuronide were transported by ABCG2, this took place to a much lesser extent compared with [3H]E1S. It was suggested that ABCG2 preferentially transports sulfate conjugates and that E1S and DHEAS are the potential physiological substrates for this transporter.     

Inhibition of CA V decreases glucose synthesis from pyruvate

The carbonic anhydrase inhibitor acetazolamide reduces citrulline synthesis by intact guinea pig liver mitochondria and also inhibits mitochondrial carbonic anhydrase (CA V) and the more lipophilic carbonic anhydrase inhibitor ethoxzolamide reduces urea synthesis by intact guinea pig hepatocytes in parallel with its inhibition of total hepatocytic carbonic anhydrase activity. Intact hepatocytes from 48-h starved male guinea pig livers were incubated at 37 degrees C in Krebs-Henseleit with 95% O2/5% CO2 at pH 7.1 with 5 mM pyruvate, 5 mM lactate, 3 mM ornithine, 10 mM NH4Cl, 1 mM oleate; with these inclusions both urea and glucose synthesis start with HCO3- -requiring enzymes, carbamyl phosphate synthetase I and pyruvate carboxylase, respectively. Urea and glucose synthesis were inhibited in parallel by increasing concentrations of ethoxzolamide, estimated Ki for each approximately 0.1 mM. In other experiments hepatocytes were incubated at 37 degrees C in Krebs-Henseleit with 95% O2/5% CO2 at pH 7.1 with 10 mM glutamine, 1 mM oleate; with these inclusions glucose synthesis no longer starts with a HCO3- -requiring enzyme. Urea synthesis was inhibited by ethoxzolamide with an estimated Ki of 0.1 mM, but glucose synthesis was unaffected. Intact mitochondria were prepared from 48-h starved male guinea pig livers. Pyruvate carboxylase activity of intact mitochondria was determined in isotonic KCl-Hepes buffer, pH 7.4, 25 degrees C, with 7.5 mM pyruvate, 3 mM ATP, and 10 mM NaHCO3. Inclusion of ethoxzolamide resulted in reduction in the rate of pyruvate carboxylation in intact mitochondria, but not in disrupted mitochondria. It is concluded that carbonic anhydrase is functionally important for gluconeogenesis in the male guinea pig liver when there is a requirement for bicarbonate as substrate.     

Glycosaminoglycan production by bovine aortic endothelial cells cultured in sulfate-depleted medium

Bovine aortic endothelial cells were cultured in medium containing [3H]glucosamine and concentrations of [35S]sulfate ranging from 0.01 to 0.31 mM. While the amount of [3H]hexosamine incorporated into chondroitin sulfate and heparan sulfate was constant, decreasing concentrations of sulfate resulted in lower [35S]sulfate incorporation. Sulfate concentrations greater than 0.11 mM were required for maximal [35S]sulfate incorporation. Chondroitin sulfate was particularly affected so that the sulfate to hexosamine ratio in [3H]chondroitin [35S]sulfate dropped considerably more than the sulfate to hexosamine ratio in [3H] heparan [35S]sulfate. Sulfate concentration had no effect on the ratio of chondroitin 4-sulfate to chondroitin 6-sulfate. The ratios of sulfate to hexosamine in cell-associated glycosaminoglycans were essentially identical with the ratios in media glycosaminoglycans at all sulfate concentrations. DEAE-cellulose chromatography confirmed that sulfation of chondroitin sulfate was particularly sensitive to low sulfate concentrations. While cells incubated in medium containing 0.31 mM sulfate produced chondroitin sulfate which eluted later than heparan sulfate, cells incubated in medium containing less than 0.04 mM sulfate produced chondroitin sulfate which eluted before heparan sulfate and near hyaluronic acid, indicating that many chains were essentially unsulfated. At intermediate concentrations of sulfate, chondroitin sulfate was found in very broad elution patterns suggesting that most did not fit an "all or nothing" mechanism. Heparan sulfate produced at low concentrations of sulfate eluted with narrower elution patterns than chondroitin sulfate, and there was no indication of any "all or nothing" sulfation.     

Extrahepatic synthesis of apolipoprotein E

Apolipoprotein E (apoE) synthesis has been examined in rat and guinea pig tissues using in vitro translation and [35S]methionine labeling of tissue slices. A number of tissues not involved in lipoprotein synthesis synthesize a protein very similar to apoE, including the spleen, adrenal, kidney, testis, ovary, heart, and lung. Although the intestine is involved in lipoprotein synthesis, apoE synthesis could not be detected in intestinal mucosa. The protein synthesized by the extrahepatic tissues was identified as apoE by its electrophoretic mobility, its immunologic reactivity with a monospecific antibody and by limited proteolysis mapping with Staphylococcus aureus V8 protease. ApoE represented between 0.02 and 0.7% of the total protein synthesized in the extrahepatic tissues, indicating that apoE mRNA is a fairly abundant mRNA in these tissues. ApoE mRNA was also detected by hybridization with a rat apoE cDNA clone, which hybridized to a single mRNA 1250 nucleotides in length in rat liver and in extrahepatic tissues. Hybridization of the apoE clone to rat genomic DNA demonstrated that the apoE gene was more heavily methylated in intestinal mucosa, which did not synthesize apoE, than in liver, testis, or kidney. 35S labeling of peritoneal macrophages revealed that both rat and guinea pig macrophages synthesized and secreted apoE in vitro. Rhesus aortic smooth muscle cells also synthesized and secreted apoE. The possible functions of apoE synthesized in the peripheral tissues are considered.     

Enzymic Synthesis of Leukotriene B4 in Guinea Pig Brain

Leukotriene B4 [5(S), 12(R)-dihydroxy-6, 14-cis-8,10-trans-eicosatetraenoic acid] was obtained from endogenous arachidonic acid when slices of the guinea pig brain cortex were incubated with the calcium ionophore A 23187. Enzymes involved in its synthesis, arachidonate 5-lipoxygenase [arachidonic acid to 5(S)-hydroperoxy-6-trans-8,11,14-cis-eicosatetraenoic acid and subsequently to leukotriene A4] and leukotriene A4 hydrolase (leukotriene A4 to B4), were present in the cytosol fraction. Arachidonate 5-lipoxygenase was Ca2+-dependent, and was stimulated by ATP and the microsomal membrane, as was noted for the enzyme from mast cells. The lipid hydroperoxides stimulated 5-lipoxygenase by four- to sixfold. The leukotriene A4 hydrolase activity was rich in brain, and the specific activity (0.4 nmol/min/mg of protein) was much the same as that of guinea pig leukocytes. High activities of these enzymes were detected in the olfactory bulb, pituitary gland, hypothalamus, and cerebral cortex. Since leukotriene B4 is enzymically synthesized in the brain, possible roles related to neuronal functions or dysfunctions deserve to be examined.     

Effect of LH-RH on the release of sulfated lutropin: a potential in vitro bioassay for LH-RH

Sheep pituitary cells prelabelled with radioactive [35S] sulfate (35SO4(2-)) were incubated with different concentrations of LH-RH and the release of LH (lutropin) into the medium was monitored in terms of immunoprecipitable [35S] sulfated LH radioactivity and estimation of LH in the same sample by radioimmunoassay. A dose dependent response was obtained with a maximum of a 16 fold increase in immunoprecipitable 35SO4(2-) -labelled LH radioactivity in the medium which was confirmed by radioimmunoassay. Similar results were also obtained for Buserelin, a well known superactive analogue of LH-RH. However, the half maximal response for Buserelin was obtained at 3-5 nM in comparison to 80.5 nM for LH-RH. After the maximal response to LH-RH as well as Buserelin, a further increase in the concentrations caused a decrease in the release of immunoprecipitable [35S]-sulfate labelled LH into the medium. Differential labelling of stored and newly synthesized LH with radioactive [35S] sulfate and [3H]-labelled leucine revealed that there was a dose dependent increase in the [35S] sulfate labelled LH into the medium whereas the release of [3H]-leucine labelled newly synthesized LH did not show a parallel increase either at different concentrations of LH-RH or at different time intervals. The above observations strongly suggest the possibility of sulfation of LH being the potential signal indicating the storage of LH in sheep pituitary cells. Another important observation in our study was that the dose dependent response of LH-RH in the form of release of [35S]-sulfate labelled LH, which was monitored by immunoprecipitation with specific LH antiserum, can be used in an in vitro bioassay for LH-RH. We believe that a new cheap and sensitive in vitro bioassay could be developed on the basis of this observation.     

Biliary excretion of some anionic derivatives of diethylstilboestrol and phenolphthalein in the guinea pig.

The metabolic fates and modes of excretion of diethylstilboestrol mono[35S]sulphate and diethylstilboestrol di[35S]sulphate were studied in the guinea pig. Comparative studies were also made with [G-3H]diethylstilboestrol and phenolphthalein di[35S]sulphate. Diethylstiboesterol di[35S]sulphate was extensively eliminated in the bile unchanged. After administration of diethylstilboestrol mono[35S]sulphate, extensive biliary elimination of radioactivity was also recorded. Radioactive components were identified as diethylstilboestrol disulphate, diethylstilboestrol monosulphate monoglucuronide and unchanged diethylstilboestrol monosulphate. When [G-3H]diethylstilboestrol was administered, 3H-labelled diethylstilboestrol monoglucuronide, diethylstilboestrol monosulphate monoglucuronide and diethylstilboestrol disulphate appeared in the bile. Phenolphthalein di[35S]sulphate was excreted unchanged in bile. These findings are discussed in relation to studies carried out in the rat [Barford, Olavesen, Curtis & Powell (1977) Biochem. J. 164, 423--430] and species differences are related to differences in enzyme activities in rat and guinea-pig liver.     

Comparison of [3H]Choline and d-[3H]Aspartate Efflux from Guinea Pig and Human Neocortex

The outflow of [3H]choline ([3H]Ch) evoked by electrical field stimulation and the efflux of D-[3H]Asp induced by 35 mM KCl and 1-10 microM ouabain were studied in human and guinea pig cortical slices, kept under identical experimental conditions. [3H]Ch outflow was significantly lower whereas D-[3H]Asp efflux was significantly higher in humans than in guinea pigs. This suggests a different proportion of the two neuronal systems in these two species. Blockade of muscarinic autoreceptors with atropine increased, whereas stimulation of alpha 2 receptors with norepinephrine (NE) reduced, the evoked [3H]Ch outflow to the same extent in human and guinea pig cortical slices. Conversely, NE did not affect ouabain-induced D-[3H]Asp efflux, suggesting that an alpha 2-mediated control is not operative in the glutamatergic cortical structures. Desmethylimipramine, 2-5 microM, was able to increase [3H]Ch outflow through atropine-like mechanisms only in the human. This drug at 20-50 microM inhibited [3H]Ch and D-[3H]Asp efflux in both species, through mechanisms unrelated to its monoamine reuptake blocking properties. Thus, similarities and differences can be detected between humans and guinea pigs with regard to (a) the relative potency of the cholinergic and acidic amino acidergic signals and (b) the modulation of neurotransmitter outflow by drugs acting on auto- and the heteroreceptors.