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

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

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

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

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

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

Opioid-receptor-mediated inhibition of [3H]dopamine but not [3H]noradrenaline release from rat mediobasal hypothalamus slices.

The modulation of the electrically evoked release of [3H]dopamine (DA) and [3H]noradrenaline (NA) by opioid receptor activation was examined in superfused slices of rat mediobasal hypothalamus (MBH). [3H]DA release was inhibited (maximally by 30-35%) by both the selective kappa-agonist U 50,488 (1 nM to 1 microM) and the selective mu-agonist DAGO (0.01-1 microM) but not by the delta-selective agonist DPDPE (1 microM). Naloxone partly antagonized the inhibitory effect of U 50,488 and completely that of DAGO, whereas the selective kappa-antagonist norbinaltorphimine (nor-BNI) only antagonized the inhibition caused by U 50,488. The dopamine D2 receptor agonist quinpirole as well as the alpha 2-adrenoceptor agonist oxymetazoline both decreased (by 25-30%) the evoked overflow of [3H]DA. The evoked release of [3H]NA was not modulated by any of the opioid agonists nor by quinpirole. However, the alpha 2-adrenoceptor agonist oxymetazoline inhibited the release of [3H]NA by 30-40%. Activation of alpha 2-adrenoceptors by oxymetazoline prevented the inhibitory effect of U 50,488, but not DAGO, on evoked [3H]DA release, whereas the selective kappa-antagonist nor-BNI antagonized the inhibition by oxymetazoline of [3H]DA, but not [3H]NA, release. In conclusion, activation of both kappa- and mu-opioid receptors results in an inhibition of evoked DA release from MBH slices but does not modulate NA release. Therefore, several of the reported effects of opioids on hormone secretion may be an (indirect) consequence of a reduction of DA release.(ABSTRACT TRUNCATED AT 250 WORDS)     

Increases in [3H]FK-506 and [3H]L-NG-Nitro-Arginine Binding in the Rat Brain After Nigrostriatal Dopaminergic Denervation

Receptor autoradiographic technique was studied to investigate sequential changes in FK-506 binding proteins, nitric oxide synthase and dopamine uptake sites in the brain 1 week to 8 weeks after unilateral 6-hydroxydopamine injection of the medial forebrain bundle in rats. [³H]FK-506, [³H]L-N^G-nitro-arginine and [³H]mazindol were used to label FK-506 binding proteins (immunophilin), nitric oxide synthase and dopamine uptake sites, respectively. [³H]FK-506 binding showed about 13-25% increase in the ipsilateral striatum from 2 to 8 weeks after degeneration of nigrostriatal pathway. However, no significant change in [³H]FK-506 binding was observed in the ipsilateral substantia nigra during the postlesion periods. In the contralateral side, [³H]FK-506 binding also showed about 13-25% increase in the striatum from 2 to 8 weeks postlesion. The substantia nigra showed a 21% increase in [³H]FK-506 binding only 2 weeks after the lesioning. On the other hand, [³H]L-N^G-nitro-arginine binding showed about 21-31% increase in the parietal cortex and striatum 1 week or 2 weeks postlesion. In the contralateral side, a 21% increase in [³H]L-N^G-nitro-arginine binding was found in the dorsolateral striatum only 1 week postlesion. In contrast, degeneration of nigrostriatal pathway caused a conspicuous loss of [³H]mazindol binding in the ipsilateral striatum (87-96%), substantia nigra (36-73%) and ventral tegmental area (91-100%) during the postlesion periods. In the contralateral side, no significant changes in [³H]mazindol binding were observed in these areas upto 8 weeks after the postlesion. The present study demonstrates that unilateral injection of 6-hydroxydopamine into the medial forebrain bundle of rats can cause a significant increase in [3H]FK-506 and [³H]L-N^G-nitro-arginine bindings in the brains. In contrast, a marked reduction in [³H]mazindol binding is observed in the brains after the lesioning, indicating severe damage to nigrostriatal dopaminergic pathway. These results suggest that immunophilin and nitric oxide synthase may play some role in the pathogenesis of neurodegenerative disorders such as Parkinson's disease.     

Accumulation of [3H]Sialyl-conjugates in sialidosis (sialidase-deficient) fibroblasts cultured in the presence of [3H]-N-acetylmannosamine

Skin fibroblasts from normal individuals and a patient with the infantile form of sialidosis were cultured for up to 72h in medium containing [³H]-N-acetylmannosamine. The sialidosis fibroblasts consistently accumulated more labeled compound(s) than the control cells, i.e. 37–88% more cpm per mg protein. Precipitation of sonicates of these cells with 10% trichloracetic acid, TCA, demonstrated that the excess radioactivity in the sialidosis fibroblasts was in one or more TCA soluble compounds. There was no detectable difference in the amount of label in the TCA insoluble material.The TCA soluble, labeled, material from the sialidosis and the control fibroblasts was separated, isolated and purified on AG1-X8, QAE Sephadex A-25 and Bio-Gel P-4 chromatography columns. Analysis of the isolated material showed the excess radioactivity in the sialidosis fibroblasts to be due to increased levels of [³H]sialic acid covalently bound to a variety of anionic sialyl conjugates. These compounds have been separated and partially purified.Finally, acid hydrolysis and chromatographic analysis of the TCA insoluble fractions showed that greater than 80% of the label in this material was also due to [³H]sialic acid. There was no detectable difference between the control and the sialidosis patient in the amount of label in this fraction.     

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

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

Metabolism of [3H]equilin-[35S]sulfate and [3H]equilin sulfate after oral and intravenous administration in normal postmenopausal women and men

The absorption of equilin sulfate and equilin from the gastrointestinal tract was determined in normal men after the ingestion of [3H]equilin-[35S]sulfate or a mixture of [3H]equilin and equilin-[35S]sulfate, while the metabolism of equilin sulfate was investigated after iv administration of [3H]equilin sulfate to postmenopausal women. After the oral administration of [3H]equilin-[35S]sulfate, equilin sulfate containing both 3H and 35S was isolated from plasma; however, only in the first sample taken at 10 min was the 3H/35S ratio the same as that of the [3H]equilin-[35S]sulfate ingested. The 3H/35S ratio then increased, and by 12 h only traces of equilin-[35S]sulfate were detectable. Similarly, after the ingestion of [3H]equilin and equilin-[35S]sulfate, [3H]equilin-[35S]sulfate was isolated from plasma. The 3H/35S ratio was at all time points greater than the 3H/35S ratio of the ingested mixture. Analysis of urine indicated that over 98% of 35S was not associated with any steroid and was most likely inorganic sulfate. After iv administration of [3H] equilin sulfate to postmenopausal women, equilin, equilenin, 17 beta-dihydroequilin, and 17 beta-dihydroequilenin were isolated from the urine. These results indicate that 1) some of the orally administered equilin sulfate was absorbed from the gut without prior hydrolysis, 2) some equilin sulfate was hydrolyzed in the gut before absorption; 3) equilin was absorbed more efficiently than equilin sulfate; 4) equilin absorbed was readily sulfated and circulated in this form; and 5) equilin sulfate was extensively metabolized, and the metabolites were excreted in the urine mainly conjugated with glucuronic acid.     

Distribution of [3H]-d-penicillamine in mouse kidney

Summary ³H-labeled d-penicillamine has been injected in mice in order to study by autoradiography its distribution in the kidney. The results show heavy accumulation of the drug in the epithelial cells of the proximal tubules of the kidney, whereas glomeruli and distal tubules do not show any significant accumulation. This peculiar tropism of penicillamine to proximal tubules is entirely consistent with the Heymann's nephritis model suggested by Bacon as a pathogenetic model for penicillamine-induced nephropathy.     

Unreliability of tritiated cholesterol: studies with [1,2-3H]-cholesterol and [24,25-3H]cholesterol in humans.

Over the past 18 months different lots of [1,2-3H]cholesterol and [24,25-3H]cholesterol were found to be radiochemically acceptable by conventional chemical and other in vitro tests, yet, when co-administered with [4-14C]cholesterol to human subjects, an abrupt fall in the 3H/14C specific activity ratio in plasma cholesterol was discovered in every case. We have concluded that all batches of [3H]cholesterol should be regarded as radiochemically unreliable unless they are shown to behave identically in all respects to [4-14C]cholesterol in an appropriate in vivo assay system.     

Extensive in situ activation of nuclear estrogen receptors after exposure of murine uteri to [3H]estradiol or [3H]4-hydroxytamoxifen

Estrogen receptor activation has been examined in murine uteri by characterizing binding to ATP-Sepharose. Determinations were performed under conditions in which specific binding to estrogen receptors was demonstrated by both agonist and antagonist without participation by nonreceptor antiestrogen-binding components. Cell-free activation of estrogen receptors in cytosol was more effectively promoted by [3H]estradiol estradiol than by [3H]4-hydroxytamoxifen or [3H]tamoxifen aziridine. However, when in situ activation was examined after intact uteri were exposed to [3H]estradiol or [3H]4-hydroxytamoxifen, virtually all extracted nuclear receptors demonstrated activated binding to ATP-Sepharose within 20 min of hormone exposure. Profiles of nuclear receptor activation were remarkably similar after exposure to either agonist or antagonist. Estrogen receptors in cytosol prepared after exposing intact uteri to 3H-labeled ligands were characterized by much less ability to bind to ATP-Sepharose than nuclear receptors. After uteri were exposed to [3H]estradiol, the activated receptor fraction in the cytosol progressively increased in contrast to preparations obtained after uteri were exposed to [3H]4-hydroxytamoxifen, which demonstrated a constant level of activation. Thus, even when activation has occurred within the intact uterus, agonists and antagonists may be characterized by different apparent levels of receptor activation in the cytosol fraction. These differences in the cytosol, however, are considerably overshadowed by the extensive activation occurring within the nuclear fraction, which we have observed to be similar with agonist and antagonist. Since estrogen receptors appear to act within chromatin, and cytosol receptors may be produced by receptor redistribution during preparation, we interpret these observations to indicate that in situ receptor activation is very similar after exposure to either agonist or antagonist. Consequently, antagonism does not appear to be associated with antiestrogens that impede receptor activation within intact murine uteri.     

Influence of steroid hormone treatments or pregnancy on [3H]prazosin and [3H]rauwolscine binding to myometrial alpha-adrenoceptors in the ewe.

The adrenergic antagonists [3H]prazosin and [3H] rauwolscine were used to identify alpha 1- and alpha 2-adrenoceptors respectively in the ovine myometrium. Ewes were allocated to four groups according to steroid hormone treatments or physiological status, namely ovariectomized ewes either as untreated controls, treated with oestradiol-17 beta or progestagen plus oestradiol-17 beta, and pregnant ewes at mid-gestation. Binding of both [3H]prazosin and [3H]rauwolscine to membrane preparations from the ovine myometrium was saturable, of high affinity and rapidly reversed by phentolamine (10 mumol/l). Based on the relative order of potency of selected adrenergic agonists and antagonists, the myometrial binding sites labelled by [3H]prazosin and [3H]rauwolscine were characterized as alpha 1- and alpha 2-adrenoceptors respectively. Saturation binding studies with [3H]prazosin showed that the number of alpha 1-adrenoceptors was low (maximal binding capacity, Bmax, between 19 and 24 fmol/mg protein) and there were no noticeable differences between the animal groups. Moreover, the equilibrium dissociation constant (Kd) did not vary significantly between groups (Kd between 0.10 and 0.17 nmol/l). In contrast, saturation binding studies with [3H]rauwolscine revealed the presence of a high number of alpha 2-adrenoceptors. Values of Bmax were far higher in the pregnant ewes (1096 +/- 241 fmol/mg protein; means +/- S.D.) than in any of the non-pregnant ovariectomized ewes. For these latter groups, the highest Bmax values were found in the group treated with both progestagen and oestrogen (382 +/- 77 fmol/mg protein) compared with treatment with oestrogen alone (101 +/- 8 fmol/mg protein) or with controls (82 +/- 12 fmol/mg protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

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

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

Multiple forms of nuclear estrogen receptor in the immature rat uterus after in vitro exchange with [3H]estradiol or [3H] antiestrogens

We have compared the properties of salt-extracted uterine nuclear estrogen receptors (ER) labeled with [³H]estradiol (E₂) or [³H]antiestrogens after in vivo exposure of immature rats to these compounds or after in vitro exchange. As expected, nuclear ER labeled during a l h in vivo treatment with [³H]E₂ migrated predominantly at 5S, although this peak was skewed towards the lighter side. [³H]4-Hydroxytamoxifen ([³H]OH tamoxifen, the active metabolite of tamoxifen)-nuclear ER complexes showed comparable sedimentation rates (4.6 ± 0.05S) after injection of the [³H]antiestrogen. On the other hand, when nuclear extracts from rats treated with unlabeled E₂ in vivo were subjected to exchange with [³H]E₂ or [³H]antiestrogens for 16 h at 23°C, a marked difference. in sedimentation profiles was observed. While the [³H]E₂ -nuclear ER complex sedimented primarily as a 3S species with variable amounts of a heavier (4.0–4.5S) shoulder, complexes between nuclear ER and [³H]tamoxifen, [³H]OH tamoxifen or [³H]CI628M formed sharp, symmetrical peaks at 4S. Both the 3S and 4S components represented forms of ER as they were eliminated in the presence of excess unlabeled DES, and they were displaced to 7–8S after reaction with specific anti-ER antibodies. The 3S peak was also abolished by addition of excess nonradioactive nafoxidine or OH tamoxifen during exchange with [³H]E₂. These results suggest that more rapidly sedimenting forms of uterine nuclear ER (4–5S) may be converted to a 3S species by the action of endogenous proteases and that association of these large forms with antiestrogens may stabilize them in a conformation less or differentially susceptible to cleavage.     

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

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

The nuclear accumulation of [3H]testosterone and [3H]estradiol in the brain of the female primate: evidence for the aromatization hypothesis

To identify the metabolites of estradiol (E2) and testosterone (T) in nuclei obtained from the female primate brain and, hence, to investigate the mechanism of their actions on behavior, 9 ovariectomized adult rhesus monkeys were studied. Two of these females were injected with 5.5 mCi [3H]T, and 30 min later, samples of 14 brain areas, pituitary gland, and peripheral tissues were removed and homogenized. Purified cell nuclei and a crude cytosol fraction were prepared, extracted with ether, and fractionated by HPLC to identify steroid metabolites. In nuclei from the hypothalamus, preoptic area, and amygdala, [3H]E2 formed locally was the major form of radioactivity. In nuclei from the clitoris, [3H]dihydrotestosterone was the major form of radioactivity, and in nuclei in all other brain samples and in the pituitary gland and uterus, [3H]T predominated. Two females (controls) were pretreated for 5 days with oil sc, injected with 1 mCi [3H]E2, and killed 60 min later. In these females, elevated nuclear concentrations of [3H]E2 were found in the hypothalamus, preoptic area, amygdala, pituitary gland, and uterus. Similar results were obtained in 2 females that were pretreated for 5 days with 2 mg/day dihydrotestosterone propionate, sc, and then injected with 1 mCi [3H]E2. In 3 females that were pretreated for 5 days with 2 mg/day T propionate, sc, and then injected with 1 mCi [3H]E2, levels of [3H]E2 were reduced by 100% (P less than 0.01) in nuclei from preoptic area and amygdala compared with control values and by 78% (P less than 0.05) in nuclei from the hypothalamus. There were no comparable reductions in steroid levels in cerebral cortex, pituitary gland, or uterus. This is the first direct evidence in the brain of a female primate that the actions of T and E2 involve the same receptor systems.     

[3H]Bradykinin receptor binding in mammalian tissue membranes.

[3H]Bradykinin binds to membranes from a variety of mammalian tissues in a saturable fashion with a dissociation constant of about 5 nM. Highest levels of binding are detected in guinea pig ileum, colon, and duodenum and in the oestrus rat uterus. The relative potencies of bradykinin derivatives in competing for these binding sites in guinea pig ileum membranes correlate with their contractile potencies in the ileum better than with contractile effects in the uterus. Thus, receptor recognition sites may differ in ileum and uterus. Monovalent and divalent cations at physiological concentrations reduce [3H]bradykinin binding. Of the monovalent cations, medium lowers binding 50% at about 80 mM. Among the divalent cations, calcium lowers binding about 50% at 5 mM, suggesting a link to the calcium conductance channel.     

Binding of [3H]forskolin to rat brain membranes.

[12-3H]Forskolin (27 Ci/mmol) has been used to study binding sites in rat brain tissue by using both centrifugation and filtration assays. The binding isotherm measured in the presence of 5 mM MgCl2 by using the centrifugation assay is described best by a two-site model: Kd1 = 15 nM, Bmax1 (maximal binding) = 270 fmol/mg of protein; Kd2 = 1.1 microM; Bmax2 = 4.2 pmol/mg of protein. Only the high-affinity binding sites are detected when the binding is determined by using a filtration assay; Kd = 26 nM, Bmax = 400 fmol/mg of protein. Analogs of forskolin that do not activate adenylate cyclase (EC 4.6.1.1) do not compete effectively for [3H]forskolin binding sites. Analogs of forskolin that are less potent than forskolin in activating adenylate cyclase are also less potent in competing for forskolin binding sites. The presence of 5 mM MgCl2 or MnCl2 was found to enhance binding. In the presence of 1 mM EDTA the amount of high-affinity binding is reduced to 110 fmol/mg of protein with no change in Kd. There is no effect of CaCl2 (20 mM) or NaCl (100 mM) on the binding. No high-affinity binding can be detected in membranes from ram sperm, which contains an adenylate cyclase that is not activated by forskolin. It is proposed that the high-affinity binding sites for forskolin are associated with the activated complex of catalytic subunit and stimulatory guanine nucleotide binding protein.     

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

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