The effects of ACTH on adrenal steroidogenesis and blood corticosteroid levels in the echidna (Tachyglossus aculeatus).

1. The effects of short-term (S.T., 30 min) and long-term (L.T., 4 days) administration of ACTH on peripheral blood corticosteroid levels and on in vitro steroidogenesis were investigated. 2. Control levels of cortisol, corticosterone and aldosterone were 58 +/- 12, 130 +/- 26 and 10 +/- 6 (SEM) ng/100 ml respectively. 3. Corticosterone was 70% higher after S.T. and 150% higher after L.T., when cortisol was 800% higher. 4. Adrenal homogenates from control echidnas converted [14C]progesterone predominantly to 11-deoxycorticosterone (45%) and 11-deoxycortisol (12%). 5. After L.T. the principal product was corticosterone (25%), but S.T. had no effect. 6. In control echidnas the Km and V for 11 beta-hydroxylation of 11-deoxycorticosterone were 20 microM and 2.8 rho mol/min/mg respectively. After L.T. V increased to 10 rho mol/min/mg.     

Changes in the activity of the adrenal cortex in fasted echidnas (Tachyglossus aculeatus) exposed to low ambient temperatures

• 1.1. The adrenal cortex is necessary for survival of echidnas at low ambient temperatures. In this study, adrenal gland activity was investigated in echidnas exposed to 2 days of cold (14°C) and fasting, alternating with 2 days at room temperature and feeding ad lib.
• 2.2. In the cold. 2.75 ± 0.29% (SD) of the initial body weight was lost daily. Plasma amino acid concentration did not change while glucose concentration decreased from 2.6 ± 0.3 to 1.5 ± 0.3 mmol/l with consecutive sessions of cold.
• 3.Plasma concentrations of corticosterone (7.2 ± 1.4 nmol/l) and cortisol (4.4 ± 1.9 nmol/l) were unchanged by repeated cold exposure. However, the adrenal response to ACTH stimulation decreased and the clearance of corticosteroids increased after cold exposure.
• 4.It was concluded that exposure to cold increases the utilization of glucocorticoids and decreases the capacity for their biosynthesis.

     

Localization of aldosterone and corticosterone in the central nervous system,assessed by quantitative autoradiography

Nuclear localization of tritiated aldosterone in the CNS was studied in rats by numerical evaluation of silver grains, deposited over neuronal cell nuclei in thawmounted autoradiograms, and compared with the localization obtained after prior administration of a 100-fold excess of radioinert aldosterone, corticosterone or 18-hydroxy-11-deoxycorticosterone (18-OH-DOC). Corticosterone and 18-OH_DOC completely prevented nuclear localization in most regions examined. However, in contrast to pretreatment with aldosterone, pretreatment with corticosterone and 18-OH-DOC did not completely prevent the concentration of radio-activity in the cell nuclei of the indusium griseum. Traces of radioactivity were, furthermore, retained in areas CA₁ and CA₂ and the dentate gyrus in rats exposed to corticosterone, but not to 18-OH-DOC, prior to [³H]aldosterone. A similar profile of silver grain distribution to that noted with aldosterone was found for corticosterone except that with tritiated corticosterone the most intense concentration of radioactivity occurred in hippocampal areas CA₁ and CA₂ and not in the indusium griseum. Prior administration of excess deoxycorticosterone acetate abolished nuclear accumulation of tritiated corticosterone. Dihydrotestosterone, on the other hand, failed to compete with tritiated corticosterone at a dose 200-fold in excess of the tritiated steroid.We conclude that (1) a receptor readily shared by aldosterone, corticosterone, 18-OH-DOC and DOC, but not by dihydrotestosterone, is widely distributed throughout the CNS, (2) a receptor shared by aldosterone and 18-OH-DOC, but not by corticosterone may be present in hippocampal areas CA₁ and CA₂, (3) that both these as well as the receptor accepting dihydrotestosterone can be located within the same cell.Dedicated to K. A. C. Elliott on his 80th birthday.     

An adrenocortical tumor secreting weak mineralocorticoids

An adrenocortical carcinoma (15.5 g) secreting excessive amounts of steroids with weak mineralocorticoid activity in a 25-year-old woman was studied with particular reference to its in vivo and in vitro secretions of steroids. Severe hypertension, occasional low serum potassium and suppressed PRA were the major clinical findings, and were improved with removal of the tumor. In the preoperative stage, plasma levels of 11-deoxycorticosterone, 18-hydroxy-11-deoxycorticosterone, corticosterone and 18-hydroxycorticosterone were all increased. However, the plasma level of aldosterone was repeatedly normal. Although plasma levels of pregnenolone, 17-hydroxypregnenolone, progesterone and 17-hydroxyprogesterone were very high, those of other late step steroids, i.e. 11-deoxycortisol, cortisol, dehydroepiandrosterone, androstenedione and testosterone were almost normal. From these findings, a major etiological role of weak mineralocorticoids such as 11-deoxycorticosterone, 18-hydroxycorticosterone and corticosterone in her hypertension was suggested. Pregnenolone and 17-hydroxypregnenolone in tumor tissue were increased, but 11-deoxycorticosterone, corticosterone, aldosterone, cortisol and adrenal androgens such as dehydroepiandrosterone, androstenedione and testosterone were below normal or low normal. In vitro production of 11-deoxycorticosterone, aldosterone or cortisol by the tumor tissue slices was very low and scarcely responded to synthetic ACTH.     

Melatonin down-regulates steroidal hormones,thymocyte apoptosis and inflammatory cytokines in middle-aged T. cruzi infected rats

Chagas disease, triggered by the flagellate protozoan Trypanosoma cruzi (T. cruzi) plays a potentially threat to historically non-endemic areas. Considerable evidence established that the immuno-endocrine balance could deeply influence the experimental T. cruzi progression inside the host's body. A high-resolution multiple reaction monitoring approach (MRM^HR) was used to study the influence of melatonin on adrenal and plasma steroidal hormones profile of T. cruzi infected Wistar rats. Young (5 weeks) and middle-aged (18 months) male Wistar rats received melatonin (5 mg/Kg, orally) during the acute Chagas disease. Corticosterone, 11-dehydrocorticosterone (11-DHC), cortisol, cortisone, aldosterone, progesterone and melatonin concentration were evaluated. Interleukin-1 alpha and β (IL-1α and β), IL-6 and transforming growth factor beta (TGF-β) were also analyzed. Our results revealed an increased production of corticosterone, cortisone, cortisol and aldosterone in middle-aged control animals, thus confirming the aging effects on the steroidal hormone profile. Serum melatonin levels were reduced with age and predominantly higher in young and middle-aged infected rats. Melatonin treatment reduced the corticosterone, 11-DHC, cortisol, cortisone, aldosterone and progesterone in response to T. cruzi infection. Decreased IL-1 α and β concentrations were also found in melatonin treated middle-aged infected animals. Melatonin treated middle-aged control rats displayed reduced concentrations of TGF-β. Melatonin levels were significantly higher in all middle-aged rats treated animals. Reduced percentages of early and late thymocyte apoptosis was found for young and middle-aged melatonin supplemented rats. Finally, our results show a link between the therapeutic and biological effects of melatonin controlling steroidal hormones pathways as well as inflammatory mediators.     

Metoclopramide,a dopamine antagonist,stimulates aldosterone secretion in rhesus monkeys but not in dogs or rabbits

This study was designed to investigate the role of dopamine in the control of aldosterone secretion in three frequently used laboratory animals. Five New Zealand rabbits, five mongrel dogs and five rhesus monkeys received metoclopramide (MCP) (200 μg/kg iv) and blood samples were collected at 0,5,15,30 and 45 minutes after drug administration. MCP had no effect on plasma aldosterone concentrations at any sampling time in the rabbits or dogs. However, MCP produced a rapid and marked increase in plasma aldosterone from 6.5±0.6 ng/dl to 18.1±2.8 ng/dl at 5 min. and a maximum level of 40.5±4.4 ng/dl at 10 min. after drug administration in the monkeys. MCP had no significant effect on plasma cortisol or plasma renin activity levels in the three species. Prolactin rose in the monkeys from 8.6±1.2 ng/ml to a maximum of 123.5±8.5 ng/ml at 15 min. after MCP. Administration of MCP resulted in a rise in plasma 18-hydroxycorticosterone in the monkeys from 12.5±1.4 ng/dl to a maximum concentration of 50.0±5.1 ng/dl 15 min. after drug administration. Plasma corticosterone, 11-deoxycorticosterone, and 18-hydroxydeoxycorticosterone were not altered by MCP. Although unlikely, it is possible that ketamine may have accounted for some of the changes in plasma aldosterone and 18-hydroxycorticosterone observed after metoclopramide in the monkeys. The findings suggest that dopamine modulates aldosterone biosynthesis in the monkey probably by regulating glomerulosa 18-hydroxylase activity.     

Acyl CoA:Cholesterol acyl transferase activity in human placental microsomes: Inhibition by progesterone

The characteristics of acyl CoA:cholesterol acyltransferase (ACAT; EC 2.3.1.26) in microsomes prepared from human term placenta were studied and the rate of incorporation of [1-¹⁴C] oleoyl CoA into cholesteryl esters was measured. The apparent K_m of the enzyme for [1-¹⁴C] oleoyl CoA was 38 ± 9 μm and the V for the reaction was 15 ± 6 pmol × mg^? protein × min^?1. The Hill coefficient for the reaction was 1.2, indicative of some degree of positive cooperativity. Cholesterol, added to the incubation mixture, did not influence ACAT activity, indicating that endogenous microsomal cholesterol served as an effective substrate for the placental ACAT enzyme. However, [1,2-³H]cholesterol in the presence of oleoyl CoA was incorporated into cholesteryl esters by placental microsomes. When progesterone was present in the incubation mixture at a concentration of 20 μm, ACAT activity was inhibited 50%. Pregnenolone, 5α-dihydroprogesterone, 17α-hydroxyprogesterone, deoxycorticosterone, dehydroisoandrosterone, androstenedione, testosterone, and estradiol-17β also inhibited ACAT activity, whereas corticosterone, cortisol, and estriol had little effect. These results are supportive of the view that ACAT activity in human placenta may be regulated by endogenously synthesized steroid hormones.     

Evaluation of adrenal function in rats by the measurement of urinary free corticosterone, free aldosterone and free 11-deoxycorticosterone.

Methods für the determination of urinary free corticosterone, free aldosterone and free 11-deoxycorticosterone (DOC) in rats are described. The free corticosteroids were measured in urine samples of 0.1–0.5 (2.0) ml by radioimmunoassay after purification by column chromatography. The validity of the methods is demonstrated by the data of the free urinary corticoids under basal conditions and after adrenal suppression and various forms of adrenal stimulation. The basal excretion of free corticosterone, free aldosterone and free DOC was 123.71 ± 15.31 (x? ± SD), 3.87 ± 1.29 and 10.61 ± 2.24 ng/day, respectively, exhibiting a decrease to 26.20 ± 5.21, 1.05 ± 0.47 and 1.35 ± 1.20 ng/day after adrenal suppression by dexamethasone. Irrespective of the mode of adrenal stimulation i.e., synthetic ACTH and systemic (cold, hunger) or neurotrophic (ether, reserpine) stress stimuli free corticosterone increased to about 450 ng/day, while free aldosterone excretion decreased during hunger and cold and was strongly enhanced after the application of reserpine. Furthermore, determination of urinary free DOC, which increased by a factor of 4, may be applied in the metyrapone test. There was a good correlation between the excretion of free corticosterone and that of free aldosterone and free DOC under basal conditions and after ACTH application, demonstrating that ACTH is responsible for the secretion of all the 3 corticoids measured. It is concluded, that the measurement of the urinary excretion of corticosterone, aldosterone and DOC is a valuable parameter of adrenal function in rats. Furthermore, in small laboratory animals like rats steroid measurements in urine are often more advantageous than Measurements in plasma.     

Temperature and pressure dependent equilibrium studies of 1, 4, 8, 11-tetramethyl-1, 4, 8, 11-tetraazacyclotetradecane nickel(ii) in coordinating solvents

In coordinating solvents, the complex 1, 4, 8, 11- tetramethyl-1, 4, 8, 11-tetraazacyclotetradecane nickel(II) bisperchlorate exists as an equilibrium mixture involving four coordinate R,S,R,S-[Ni(tmc)]²⁺ and five coordinate R,S,R,S-[Ni(tmc)(solvent)]²⁺ species. Spectrophotometric measurements of this equilibrium in a number of solvents have been conducted over a range of temperatures and pressures. The stability order for the five coordinate complex in the solvents investigated is CH₃CN>DMF>DMSO>C₆H₅CN> H₂O>ClCH₂CN at 25 °C. Differences in stability are considered in terms of the measured thermodynamic parameters ΔH° and ΔS°. Both steric and electronic factors were found to influence solvent coordination with the macrocyclic complex.For the equilibrium in CH₃CN, C₆H₅CN, DMF and H₂O, reaction volumes, ΔV°, of −3.2±0.5, −4.2±0.5, −0.2±0.5 and −0.5±0.5 cm³ mol⁻¹ respectively have been determined. Each is significantly smaller than the corresponding solvent molar volume. The ΔV° for the equilibrium in CH₃CN is comparable with the previously determined activation volume for exchange of this solvent on R, S, R, S- [Ni(tmc)(CH₃CN)]²⁺. The equilibrium and measured volume parameters are discussed in relation to the mechanism for solvent exchange.     

Conversion of 11-deoxycorticosterone and corticosterone to aldosterone by cytochrome P-450 11 beta-/18-hydroxylase from porcine adrenal

Highly purified cytochrome P-450 11 beta-/18-hydroxylase and the electron carriers adrenodoxin and adrenodoxin reductase were prepared from porcine adrenal. When the enzyme was incubated with the electron carriers, 11-deoxycorticosterone (DOC) and NADPH, the following products were isolated and measured by HPLC: corticosterone, 18-hydroxy-11-deoxycorticosterone (18-hydroxyDOC), 18-hydroxycorticosterone and aldosterone. All of the DOC consumed by the enzyme can be accounted for by the formation of these four steroids. Aldosterone was identified by mass spectroscopy and by preparing [3H]aldosterone from [3H]corticosterone followed by recrystallization at constant specific activity after addition of authentic aldosterone. Corticosterone and 18-hydroxycorticosterone were also converted to aldosterone. Conversion of corticosterone and 18-hydroxycorticosterone to aldosterone required P-450, both electron carriers, NADPH and substrate. The reaction is inhibited by CO and metyrapone. Moreover, all three activities of the purified enzyme decline at the same rate when the enzyme is kept at room temperature for various periods of time and when the enzyme is treated with increasing concentrations of anti-11 beta-hydroxylase (IgG) before assay. It is concluded that cytochrome P-450 11 beta-/18-hydroxylase can convert DOC to aldosterone via corticosterone and 18-hydroxycorticosterone. The stoichiometry of this conversion was found to be 3 moles of NADPH, 3 moles of H+ and 3 moles of oxygen per mole of aldosterone produced.     

Facilitated diffusion and active transport systems for glucose in metacercariae of Clinostomum marginatum (Digenea)

Metacercariae of Clinostomum marginatum excysted from yellow perch, Perca flavescens, appear to have two systems for transporting glucose across the tegument, facilitated diffusion and active transport. These systems were distinguished by their differential sensitivities to Na⁺, phlorizin and phloretin. In Ringer's saline for cold-blooded vertebrates, 0.1 mm phlorizin and phloretin were incomplete, but similarly effective inhibitors of glucose uptake in 3 min incubations; worms accumulated in 1 h nonmetabolized 3-O-methylglucose against an apparent concentration difference demonstrating the active transport component. In Na⁺-free saline, phlorizin sensitivity and active transport capacity disappeared, but a phloretin sensitive, Na⁺-independent component remained. The V_max and K₁ of the Na⁺-independent system were 3.0 ± 0.54 μmol/g ethanol-extracted dry wt/h, and 0.8 ± 0.36 mm, respectively. V_max and K₁ of the Na⁺-dependent system, estimated by subtracting the Na⁺-independent values from those obtained in Ringer's saline, were 1.3 ± 0.27 μ mol/g ethanol-extracted dry wt/h, and0.7 ± 0.36mm, respectively.     

Functional expression of the cDNAs encoding rat 11 beta-hydroxylase [cytochrome P450(11 beta)] and aldosterone synthase [cytochrome P450(11 beta, aldo)]

Expression plasmids containing two cDNAs of a rat cytochrome P450(11 beta) family, pcP450(11 beta)-62 [Nonaka, Y., Matsukawa, N., Morohashi, K., Omura, T., Ogihara, T., Teraoka, H. & Okamoto, M. (1989) FEBS Lett. 255, 21-26] and pcP450(11 beta, aldo)-46 [Matsukawa, N., Nonaka, Y., Ying, Z., Higaki, J., Ogihara, T. & Okamoto, M. (1990) Biochem. Biophys. Res. Commun. 169, 245-252], were constructed and introduced into COS-7 cells by electroporation. Enzymatic activities of the expressed cytochromes P450(11 beta) and P450(11 beta, aldo) were determined by using 11-deoxycorticosterone, corticosterone, 18-hydroxy-11-deoxycorticosterone, 18-hydroxycorticosterone, or 19-hydroxy-11-deoxycorticosterone as a substrate. Cytochrome P450(11 beta) catalyzed 11 beta-, 18- and 19-hydroxylations of 11-deoxycorticosterone and 19-oxidation or 19-hydroxy-11-deoxycorticosterone at substantial rates, 18-hydroxylation of corticosterone at a very low rate, but no aldosterone production. Cytochrome P450(11 beta, aldo) catalyzed 11 beta- and 18-hydroxylations of 11-deoxycorticosterone, 18-hydroxylation of corticosterone and aldosterone production from 11-deoxycorticosterone or corticosterone. But neither 19-hydroxylation of 11-deoxycorticosterone nor 19-oxidation of 19-hydroxy-11-deoxycorticosterone was catalyzed by cytochrome P450(11 beta, aldo).     

Glucose transport and metabolism in the perfused hindquarters of lean and obese-hyperglecemic (db/db) mice effects of insulin and electrical stimulation

Changes in glucose transport and metabolism in skeletal muscles of the obese-diabetic mice (db/db) was characterized using the perfused mouse hindquarter preparation. Metabolism of [5-³H]glucose, uptake of 3-O-[methyl-³H]glucose (methylglucose) and [2-¹⁴C]deoxyglucose (deoxyglucose) was studied under resting, electrically stimulated contracting, and insulin-stimulated conditions. Basal rate of methylglucose uptake was 255 ± 18 and 180 ± 9 μl/15 min per ml intracellular fluid space for lean and db/db mice, respectively. The V_? of methylglucose transport was decreased with no change in K_m in the db/db mice. Both electrical stimulation and insulin (1/mU/ml) increased methylglucose uptake rate 2-fold in both lean and obese mice. We observed no significant change in insulin sensitivity in the db/db mice in stimulating methylglucose uptake which was subnormal under all conditions. Similar results were obtained using deoxyglucose. Likewise, uptake of glucose and ³H₂O production from [5-³H]glucose were significantly reduced, both at rest and during electrically stimulated contraction in the db/db mouse. However, lactate production in the electrically stimulated db/db mouse preparations was not significantly different from that in the lean mice. These data suggest a major contribution from an impaired glucose transport activity to the reduction in glucose metabolism in the db/db mouse skeletal muscle.     

Purification and characterization of aminoglycoside phosphotransferase APH(6)-Id,a streptomycin-inactivating enzyme

As part of an overall project to characterize the streptomycin phosphotransferase enzyme APH(6)-Id, which confers bacterial resistance to streptomycin, we cloned, expressed, purified, and characterized the enzyme. When expressed in Escherichia coli, the recombinant enzyme increased by up to 70-fold the minimum inhibitory concentration needed to inhibit cell growth. Size-exclusion chromatography gave a molecular mass of 31.4 ± 1.3 kDa for the enzyme, showing that it functions as a monomer. Activity was assayed using three methods: (1) an HPLC-based method that measures the consumption of streptomycin over time; (2) a spectrophotometric method that utilizes a coupled assay; and (3) a radioenzymatic method that detects production of ³²P-labeled streptomycin phosphate. Altogether, the three methods demonstrated that streptomycin was consumed in the APH(6)-Id-catalyzed reaction, ATP was hydrolyzed, and streptomycin phosphate was produced in a substrate-dependent manner, demonstrating that APH(6)-Id is a streptomycin phosphotransferase. Steady-state kinetic analysis gave the following results: K _m(streptomycin) of 0.38 ± 0.13 mM, K _m(ATP) of 1.03 ± 0.1 mM, V _max of 3.2 ± 1.1 μmol/min/mg, and k _cat of 1.7 ± 0.6 s^?1. Our study demonstrates that APH(6)-Id is a bona fide streptomycin phosphotransferase, functions as a monomer, and confers resistance to streptomycin.     

Effect of alpha-MSH on the corticosteroid production of isolated zona glomerulosa and zona fasciculata cells

α-MSH (10^?9 ? 6×10^?7M) potentiates the effect of ACTH (10^?11 ? 5×10^?9M) on adrenocortical steroidogenesis decreassng ED₅₀ of ACTH from 220 to 183 pg/ml on zona fasciculata corticosterone-, and from 739 to 437 pg/ml on zona glomerulosa aldosterone production. α-MSH alone increases aldosterone production of zona glomerulosa cells in doses (10^?9 ? 6×10^?7M) that do not stimulate zona fasciculata corticosterone production. The response of zona glomerulosa aldosterone production to α-MSH can be characterized by a bi-phase dose-response curve.     

Kinetics of imidazole addition to the axial site of the iron(II) complex of 2, 3, 9,10-tetraphenyl-1,4, 8,11-tetraaza-1,3, 8,10-cyclotetradecatetraene in dimethyl sulfoxide. Evidence for a dissociative-interchange mechanism

The axial adduct formation of the iron(II) complex of 2,3,9,10-tetraphenyl-l,4,8,11-tetraaza-1,3,8,10-cyclotetradecatetraene (L) with imidazole in dimethyl sulfoxide has been investigated spectrophotometrically at various temperatures and pressures. In the presence of a large excess of imidazole the reaction with the two phases has been observed. The first faster reaction is the formation of the monoimidazole complex of FeL²⁺, and the second slower reaction corresponds to the formation of the bisimidazole complex. Activation parameters are as follows: for the first step with k₁ (25.0°C) = (6.8 ±0.2)×10⁵ mol⁻¹ kg s⁻¹, ΔH³₁ = 47.5 ± 4.9 kJ mol⁻¹, ΔS³₁ = 26±16 J K⁻¹ mol⁻¹, and ΔV³₁ (30.0°C) = 27.2±1.5 cm³ mol⁻¹; for the second step with k₂ (25.0°C) = 26.8±0.8 mol⁻¹ kg s⁻¹, ΔH³₂ = 91.6± 0.8 kJ mol⁻¹, ΔS³₂ = 90±3 J K⁻¹ mol⁻¹, and ΔV³₂ (35.0°C) = 21.8±0.9 cm³ mol⁻¹. The large positive activation volumes strongly indicate a dissociative character of the activation process.     

THE UPTAKE OF BIOGENIC MONOAMINES BY THE ISOLATED AURICLE OF THE SNAIL (HELIX POMATIA)

—The uptake of [³H]5HT, [³H]dopamine, [³H]noradrenaline and [³H]octopamine into the auricle of Helix pomatia was studied. When tissues were incubated at 25°C in media containing radioactive amines, tissue:medium ratios of about 49:1, 14:1 and 5:1 for 5-HT, dopamine, noradrenaline, and octopamine respectively were obtained after a 20–30 min incubation time. Tissues incubated at 25°C in media containing radioactive amines for 20–30 mins showed that almost all (96%) the radioactivity was present as unchanged [³H]5-HT, [³H]dopamine, [³H]octopamine or [³H]noradrenaline. The high tissue:medium ratios for 5-HT and dopamine, but not for noradrenaline and octopamine, showed saturation kinetics which were dependent upon temperature and sodium ions. From the Lineweaver–Burk plots, two uptake mechanisms for 5-HT at 25°C were resolved; the high affinity uptake process having a K_m1 value of 6.0 ± 10^?8m and a V_m1 value of 0.115 nmol/g/min while the lower affinity process had a K_m2 value of 1.04 ± 10^?6m and a V_m2 value of 0.66nmol/g/min. At 0°C a single uptake mechanism for 5-HT occurred which gave a K_m value of 5.02 ± 10^?8m and a V_m value of 0.0165 nmol/g/min. In the case of dopamine, the Lineweaver–Burk plot at 25°C showed a single uptake process with values for K_m and V_m of 1.55 ± 10^?7m and 0.086 nmol/g/min respectively. This process did not function at 0°C. The effect of various agents and ions upon the accumulation processes for all amines was also studied, and the data indicate that the same neurons probably accumulate more than one amine type. It is concluded that 5-HT and dopamine uptake in the auricle is a mechanism for inactivating these substances at 25°C and that an uptake mechanism for 5-HT also functions at 0°C. The results are discussed from the point of view of 5-HT's being the cardioexcitatory substance in the snail heart.     

Partial purification and some properties of rat liver sulfotransferase I,a glucocorticoid sulfotransferase usually restricted to female rats

Three rat liver enzymes, sulfotransferases I, II, and III, sulfate glucocorticoids. This report describes the purification of sulfotransferase I, the glucocorticoid sulfotransferase usually restricted to females, 227 ± 63-fold from cytosol. As shown, this represents a probable 1000- to 1250-fold enrichment compared to liver homogenates. Highly purified sulfotransferase I exhibits a molecular weight of approximately 156,000, determined by sedimentation velocity experiments. Its pH optimum is pH 6.0 ± 0.1. However, its properties are routinely measured at pH 6.8 for reasons enumerated in the text. At this pH it exhibits a sequential mechanism for cortisol sulfation. Its K_m values for cortisol and its coenzyme, 3′-phosphoadenosine-5′-phosphosulfate, are 7.09 ± 0.65 and 10.6 ±1.1 μm, respectively. Sulfotransferase I also appears to contain essential sulfhydryl groups, shown by p-hydroxymercuribenzoate inhibition studies. Furthermore, it is activated by divalent Co, Cr, Mg, or Mn and inhibited by 100 μm Cd²⁺ or 50 μm Zn²⁺. Comparison of the ability of purified sulfotransferase I to sulfate 40 μm cortisol, dehydroepiandrosterone, deoxycorticosterone, estradiol-17β, and testosterone showed that sulfotransferase I is not as specific a glucocorticoid sulfotransferase as is sulfotransferase III (described earlier). Sulfotransferase I was inhibited strongly by a variety of steroids and diethylstilbestrol at 1.0 μm concentrations. Extensive comparisons of the properties of sulfotransferases I and III and their potential significance are made throughout the text.     

Trypanosoma lewisi: alterations in membrane function in the rat.

DEAE-cellulose-purified Trypanosoma lewisi from 4-day (dividing trypanosomes) and 7-day (non-dividing trypanosomes) infections in rats were compared for initial uptake of glucose, leucine, and potassium. Glucose entered the parasitic cells by mediated (saturable) processes, whereas leucine and K⁺ entered by mediated processes and diffusion. Glucose entry was significantly elevated in 4-day cells (V_max 4.00 ± 1.02 nmoles/ 1 × 10⁸ cells/min) with respect to 7-day cells (V_max 1.83 ± 0.62 nmoles 1 × 10⁸ cells/min). Likewise, the affinity of the glucose carrier was significantly greater in 4-day cells (K_m = 0.30 ± 0.02 mM) than in 7-day cells (K_m = 0.59 ± 0.11 mM). When leucine and K⁺ transport were compared in 4- and 7-day populations, significant elevations in the rate of entry (V_max) of both substrates were observed for 4-day cells; K_m values for leucine and K⁺ were not altered by the stage of infection. For leucine, the V_max and K_m for 4-day cells were 2.40 ± 0.50 nmoles/1 × 10⁸ cells/30 sec and 78 ± 7 μM, respectively; corresponding values in 7-day cells were 1.06 ± 0.02 nmoles/1 × 10⁸ cells/30 sec and 66 ± 11 μM. For K⁺, the V_max and K_m for 4-day cells were 15.97 ± 0.38 nmoles/1 × 10⁸ cells/min and 1.2 mM, respectively; corresponding values in 7-day cells were 4.76 ± 1.82 nmoles/1 × 10⁸ cells/min and 1.05 mM. The observed increase in the rate of K⁺ entry into 4-day cells was attributable to enhanced influx; no significant difference in the rate of K⁺ efflux was noted when 4- and 7-day cells were compared ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of K⁺ leak for 4- and 7-day cells were 68.1 ± 9.3 and 67.9 ± 15.2 min, respectively). Potassium influx was ouabain insensitive. Membrane function in 7-day cells was not uniformly inhibited. No significant difference in the activity of the membrane-bound enzyme, 5′-nucleotidase, was observed when 4- and 7-day cells were compared.