Inhibition of the effects of alfathesin and other steroid anesthetics by catatoxic steroids in rats.

In rats, the sleeping time induced by overdosage with eight steroid anesthetics--alfathesin, 3-(3-oxo-17beta-hydroxy-19-nor-4-androsten-17alpha-yl)-propionic acid-lactone (SC-8109), 21-hydroxy=5alpha-pregnane-3,20-dione (P-234), 4-pregnene-3,11,20-trione (Bio.66), 17-hydroxy-3-oxo-4-androstene-17alpha-propionic acid-gamma-lactone(SC-5233),3alpha-hydroxy-5beta-pregnane-11,20-dione, 5beta-pregnane-3,11,20-trione (U-1373), and hydroxydione--was abolished or considerably reduced by a variety of catatoxic compounds, particularly 3beta-hydroxy-20-oxo-5-pregnene-16alpha-carbonitrile (PCN), 9alpha-fluoro-11beta,17-dihydroxy-3-oxo-4-androstene-17alpha-propionic acid potassium salt (CS-1), prednisolone, ethylestrenol and spironolactone. Phenobarbital and diphenylhydantoin, two non-steroidal stimulators of hepatic microsomal drug metabolism, were also highly effective. In contrast, triamcinolone, estradiol,progesterone, desoxycorticosterone and hydroxydione, which exert little or no catatoxic activity, failed to significantly diminish anesthesia or sedation.     

Multiple structural features of steroids mediate subtype-selective effects on human alpha4beta3delta GABAA receptors

Neurosteroids have been shown to mediate some of their physiological effects via a modulatory site on type A inhibitory gamma-aminobutyric acid (GABAA) receptors. In particular, recent evidence has implicated selective potentiation of the delta subunit of GABAA receptors as an important mediator of in vitro and in vivo neurosteroid activity. However, this has been demonstrated for only a very small number of steroids, so both the generality of this finding, and the structural features of steroids which mediate functional delta-selectivity, are unclear. We have used a potentiometric assay based on fluorescence resonance energy transfer to measure GABA-activated responses in L(tk-) cells stably transfected with human GABAA receptor alpha4beta3delta and alpha4beta3gamma2 receptor subtypes. A set of 28 steroids were evaluated on these subtypes to characterise their functional potency and efficacy in modulating GABA responses. For most compounds there was a clear separation of their efficacy profiles between the receptor subtypes, with a substantially larger maximal response at the alpha4beta3delta receptor. 5beta-Pregnan-3beta-ol-20-one, 5beta-pregnane-3alpha,20beta-diol and 5beta-pregnane-3alpha,17alpha-diol-11,20-dione showed particularly high efficacy for alpha4beta3delta. No compounds were identified that simply inhibited responses at delta-containing receptors. However, 5beta-pregnane-3alpha,17alpha,20beta-triol, prednisolone 21-acetate, 4-pregnene-17alpha,20alpha-diol-3-one-20-acetate, 4-pregnen-20alpha-ol-3-one, and 5beta-pregnane-3alpha,17alpha,21-triol-20-one inhibited, though did not abolish, GABA responses at the alpha4beta3gamma2 subtype, while evoking modest-amplitude potentiation of alpha4beta3delta responses. Molecular modelling on this compound series using principal components analysis indicates that several structural features of steroids underlie their relative functional selectivity for potentiation of delta-containing GABAA receptors.     

Delta 5-3beta-hydroxysteroid dehydrogenase (3 beta HSD) from Digitalis lanata. Heterologous expression and characterisation of the recombinant enzyme

During the biosynthesis of cardiac glycosides, Delta (5)-3beta-hydroxysteroid dehydrogenase (3 beta HSD, EC 1.1.1.51) converts pregnenolone (5-pregnen-3beta-ol-20-one) to isoprogesterone (5-pregnene-3,20-dione). A 3 beta HSD gene was isolated from leaves of Digitalis lanata. It consisted of 870 nucleotides containing a 90 nucleotide long intron. A full-length cDNA clone that encodes 3 beta HSD was isolated by RT-PCR from the same source. A SPH I /KPN I 3 beta HSD cDNA was cloned into the pQE30 vector and then transferred into E. COLI strain M15[pREP4]. 3 beta HSD cDNA was functionally expressed as a His-tagged fusion protein (pQ3 beta HSD) composed of 273 amino acids (calculated molecular mass 28,561 Da). pQ3 beta HSD was purified by metal chelate affinity chromatography on Ni-NTA. Pregnenolone and other 3beta-hydroxypregnanes but not cholesterol were 3beta-oxidised by pQ3 beta HSD when NAD was used as the co-substrate. Testosterone (4-androsten-17beta-ol-3-one) was converted to 4-androstene-3,17-dione indicating that the pQ3 beta HSD has also 17beta-dehydrogenase activity. pQ3 beta HSD was able to reduce 3-keto steroids to their corresponding 3beta-hydroxy derivatives when NADH was used as the co-substrate. For comparison, 3 beta HSD genes were isolated and sequenced from another 6 species of the genus DIGITALIS. Gene structure and the deduced 3 beta HSD proteins share a high degree of similarity.     

Synthesis and evaluation of potential radioligands for the progesterone receptor

Several steroidal analogues were synthesized as potential gamma-emitting radioligands for the progesterone receptor. Each of these compounds was tested as an inhibitor of the specific binding of [3H]-17 alpha,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione (R5020) to the progesterone receptor in rabbit uterine cytosol. R5020 is a well-known progestin with high affinity for the receptor. Of the compounds synthesized, aromatic N-substituted C-17 steroidal carboxamides inhibited the binding only poorly. Three compounds, 16 alpha-iodo-4-estren-17 beta-ol-3-one, 17 alpha-[2(E)-iodovinyl]-4-estren-17 beta-ol-3-one, and 17 alpha-[2(Z)-iodovinyl]-4-estren-17 beta-ol-3-one were excellent competitors, each having a Ki less than or equal to that of the natural progestin, progesterone. Since similar iodinated analogues of estrogens have been shown to be extremely stable both in vivo and in vitro, these compounds are potentially useful ligands for the progesterone receptor.     

Effects of digitoxigenin, digoxigenin, and various cardiac glycosides on cardenolide accumulation in shoot cultures of Digitalis lanata

Various cardenolide genins and cardenolide glycosides were administered to light-grown and dark-grown Digitalis lanata shoot cultures to investigate conversion reactions related to the formation and rearrangement of the sugar side chain of Digitalis glycosides. Digitoxigenin was converted to digitoxigen-3-one, 3-epidigitoxigenin, and digoxigenin. In addition, various cardiac glycosides were formed, including mono-glycosides with glucose, glucomethylose, fucose, and digitalose, as well as the corresponding diglycosides, all containing a terminal glucose. Digitoxosylated cardenolides were not formed, although the light-grown shoot cultures were capable of producing these compounds. Exogenous cardenolide fucosides were not converted into cardenolide digitoxosides. Administration of evatromonoside (digitoxigenin monodigitoxoside) did not force the formation of cardenolide di- or tridigitoxosides. Our results support the hypothesis that cardenolide fucosides and digitoxosides are formed via different biosynthetic routes and that cardenolide genins can be fucosylated but not digitoxosylated, indicating that digitoxosylation may only occur at an earlier stage in the cardenolide pathway.     

5α-△9(11)-16β-甲基-3β,17α,21-三羟基-孕甾烯-3β,21-双醋酸酯-20酮和5α,17α-甲基-17β-羟基-雄甾-3酮的微生物转化

用诺卡氏菌与节杆菌混合菌种转化从蕃麻皂素制得的中间体5α-△⁹⁽¹¹⁾-16-16β-甲基-3β,17α,21三羟基孕甾烯-3β,21-双醋酸酯-20酮(Ⅰ)得50%的16β-甲基-△^1,4,9(11)-孕甾三烯-20酮(Ⅱ)和少量的16β-甲基-9,11α环氧-△¹,4孕甾二烯-20酮(Ⅲ)。另外,又用同样的混合菌种转化从剑麻皂素制得的中间体5α,17α甲基-17β羟基-雄甾-3酮(Ⅳ)得50%17α甲基-17β羟基-△^1,4-雄甾二烯-3酮(Ⅴ)。如改变培养基则得3,17β-羟基-17α-甲基-9酮基-9,10开环-1,3,5(10)雄甾三烯化合物。     

Differential modulation of the gamma-aminobutyric acid type C receptor by neuroactive steroids.

Gamma-aminobutyric acid type C receptor channels (GABA(C)Rs) composed of rho subunits are pharmacologically distinct from GABA(A) receptor channels (GABA(A)Rs). This difference is illustrated by the insensitivity of homo-oligomeric rho(1) receptor channels to many known modulators of GABA(A)Rs, such as barbiturates and benzodiazepines. A number of endogenous metabolites of corticosterone and progesterone, known as neuroactive steroids, compose yet another class of compounds that can modulate GABA(A)Rs. Here, several neuroactive steroids are shown to also modulate the rho(1) receptor channel. 5alpha-Pregnane-3alpha,21-diol-20-one (allotetrahydrodeoxycorticosterone), 5alpha-pregnane-3alpha-ol-11, 20-dione (alphaxalone), and 5alpha-pregnane-3alpha-ol-20-one (allopregnanolone) potentiated the GABA-evoked currents from rho(1) receptor channels and concomitantly altered the deactivation kinetics by prolonging the decay time. In contrast, 5beta-pregnane-3alpha-ol-20-one (pregnanolone), 5beta-pregnane-3, 20-dione (5beta-dihydroprogesterone), and 5beta-pregnane-3alpha, 21-diol-20-one (tetrahydrodeoxycorticosterone), all potentiators of GABA(A)Rs, inhibited the GABA-elicited currents of the rho(1) receptor channel. In comparison to GABA(A)Rs, the modulation of rho(1) receptor channels by these neuroactive compounds occurred with relatively high concentrations of the neuroactive steroids and was more prominent in the presence of low concentrations of GABA, equivalent to fractions of the EC(50) value of the rho(1) receptor channel. Structural comparison of these six neuroactive steroids reveals that the key parameter in determining the mode of modulation for the rho(1) receptor channel is the position of the hydrogen atom bound to the fifth carbon, imposing a trans- or cis-configuration in the backbone structure. This is the first demonstration of isomeric compounds that can differentially modulate the activity of the rho(1) receptor channel.     

5α-△9(11)-16α-甲基-3β,17α,21-三羟基-孕甾烯-3β,21-双醋酸酯-20酮的微生物转化

简单节杆菌A₁及耻垢杆菌MS₁两种菌混合培养转化地塞米松中间体5α-△⁹⁽¹¹⁾-16α-甲基-3μ,17μ,21-三羟基-孕甾烯-3μ,21-双醋酸酯-20酮(Ⅲ)可得到16α-甲基-△^1.4.0(11)-孕甾烯-17α,21-双羟基-3,20双酮(Ⅳ)(65%收率)及少量的16α-甲基-△^1.4孕甾烯-9α,11α-环氧-17α,21双羟基-3,20双酮(Ⅴ)。     

Increased behavioral neurosteroid sensitivity in a rat line selectively bred for high alcohol sensitivity.

Acute administration of a neurosteroid 5beta-pregnan-3alpha-ol-20-one induced a greater impairment in motor performance of the selectively bred alcohol-sensitive (ANT) than alcohol-insensitive (AT) rats. This difference was not associated with the sensitivity of gamma-aminobutyrate type A (GABA(A)) receptors, as 5alpha-pregnan-3alpha-ol-20-one (allopregnanolone) decreased the autoradiographic signals of t-butylbicyclophosphoro[35S]thionate binding to GABA(A) receptor-associated ionophores more in the brain sections of AT than ANT rats. Nor was the difference associated with baseline levels of neuroactive progesterone metabolites, as 5alpha-pregnan-3,20-dione (5alpha-DHP) and 5alpha-pregnan-3alpha-ol-20-one were lower in the ANT rats. After ethanol (2 g/kg, i.p.) administration and the subsequent motor performance test, the increased brain concentrations of these metabolites were still lower in the ANT than AT rats, although especially in the cerebellum the relative increases were greater in the ANT than AT rats. The present data suggest that the mechanisms mediating neurosteroid-induced motor impairment are susceptible to genetic variation in rat lines selected for differences in ethanol intoxication.     

Synthesis of 2-Oxacortexone and its Effect on Ca2+ Uptake in Bovine Spermatozoa

Low temperature base catalyzed autoxidation (BCA) of the A-ring of 21-acetoxypregn-5-ene-3,20-dione 20-ethylene ketal ( 7 ) resulted in the saponification of the ester with the concomitant formation of 2,21-dihydroxypregna-1,4-diene-3,20-dione 20-ethylene ketal ( 8 ) Continued BCA at ambient temperature, converts the latter to 1,21-dihydroxy-2-oxaprogesterone 20-ethylene ketal ( 9 ), which is reduced by NaBH₄ to the 2-oxasteroid, 21-hydroxy-2-oxaprogesterone 20-ethylene ketal ( 10 ). Treatment of enol 8 , lactol 9 , 9 , and lactone 10 with aqueous acid generates the corresponding deprotected analogs 2,21-dihydroxypregna-1,4-diene-3,20-dione (enol 11 ), 1,21-dihydroxy-2-oxaprogesterone (lactol 12 ), and 2-oxacortexone (2-oxadesoxycorticosterone, 21-hydroxy-2-oxaprogesterone, lactone 13 ). In bovine spermatozoa, neither 2-oxasteroid ketal 10 nor its deprotected analog 13 stimulated Ca²⁺ uptake. In high concentration (0.5 mM), the inhibition of Ca²⁺ uptake is only 37% for 13 , as compared to 83% found with the parent steroid, cortexone (desoxycorticosterone, 21-hydroxyprogesterone, 5 ). The difference in molecular structure between 13 and 5 indicates the importance of the oxygen atom in ring A in achieving the protective effect of the steroid. Ketalization of the C-20 carbonyl is not important for protection. Thus it seems that by replacing C-2 by an oxygen atom we can reduce the biological damage caused by relatively high concentrations of steroid treatment. These results are highly significant when treatment of patients with high doses of steroids is considered.     

Enhancement of [3H]muscimol binding to brain synaptic membranes by progesterone and related pregnanes

Progesterone (a delta 4-3 keto-pregnane) as well as a series of pregnanes enhanced [3H]muscimol binding to rat cerebral cortex membranes. This effect was increased by preincubation in the presence of the drugs, progesterone being effective only after a 30 min preincubation. The effect of progesterone was dose-dependent from 1 nM to 100 microM reaching a maximum of 140% over control. Its 20 alpha and 20 beta reduced metabolites (20 alpha- and 20 beta-OH-4-pregnen-3-one) had no effect. Ring A reduction in either the 5 alpha or 5 beta position resulted in steroids (5 alpha- and 5 beta-pregnane-3,20-di-one) producing moderate but consistent facilitation of binding (30-40% increase at 10 microM). The presence of a hydroxyl group in C3 had variable results in the potency of pregnanes to facilitate muscimol binding. Pregnanes with a 3 beta-OH group showed weaker activity than those having a 3 alpha-OH group, 5 alpha-pregnan-3 alpha-ol-20-one being the most potent (100% stimulation). Pregnenolone, a delta 5-3 beta-OH-pregnane, was only effective at low concentrations (10 nM to 10 microM). Scatchard analysis of [3H]muscimol binding in the presence of progesterone and 5 alpha-pregnan-3 alpha-ol-20-one revealed that the observed effect was due to an increase in binding sites rather than to a change in affinity.     

Modulation of the GABAA receptor by depressant barbiturates and pregnane steroids.

1. The modulation of the gamma-aminobutyric acidA (GABAA) receptor by reduced metabolites of progesterone and deoxycorticosterone has been compared with that produced by depressant barbiturates in: (a) voltage-clamp recordings from bovine enzymatically isolated chromaffin cells in cell culture, and (b) an assay of the specific binding of [3H]-muscimol to a preparation of porcine brain membranes. 2. The progesterone metabolites 5 alpha- and 5 beta-pregnan-3 alpha-ol-20-one (greater than or equal to 30 nM) reversibly and dose-dependently enhanced the amplitude of membrane currents elicited by locally applied GABA (100 microM), and over the concentration range 30 nM-100 microM stimulated the binding of [3H]-muscimol. In contrast, 5 alpha- and 5 beta-pregnan-3 beta-ol-20-one (30 nM-100 microM) had little effect in either assay, indicating a marked stereoselectivity of steroid action. 3. Scatchard analysis of the ligand binding data suggested an apparent increase in the number, rather than the affinity, of detectable [3H]-muscimol binding sites as the principle action of the active steroid isomers. 4. GABA-evoked currents were also potentiated by androsterone (1 microM) and the deoxycorticosterone metabolite 5 alpha-pregnane-3 alpha,21-diol-20-one (100 nM). 5. Secobarbitone (10-100 microM), pentobarbitone (10-300 microM) and phenobarbitone (100-500 microM) reversibly and dose-dependently potentiated the amplitude of GABA-evoked currents in the absence of any change in their reversal potential. 6. At relatively high concentrations (greater than or equal to 30 microM) secobarbitone and pentobarbitone directly elicited a membrane current. It is concluded that such currents result from GABAA receptor-channel activation since they share a common reversal potential with GABA-evoked responses (approximately 0 mV), are reversibly antagonized by bicuculline (3 microM), and potentiated by either diazepam (1 microM) or 5 beta-pregnan-3 alpha-ol-20-one (500 nM). 7. Secobarbitone (1 microM-1 mM) dose-dependently enhanced the binding of [3H]-muscimol. In common with the active steroids, an increase in the apparent number of binding sites was responsible for this effect. 8. A saturating concentration (1 mM) of secobarbitone in the ligand binding assay did not suppress the degree of enhancement of control binding produced by 5 beta-pregnan-3 alpha-ol-20-one (30 nM-100 microM). Similarly the steroid, at a concentration of 100 microM, did not influence the enhancement of [3H]-muscimol binding by secobarbitone (1 microM-1 mM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of serine proteases by steroids

Proteolysis of ¹⁴C-labeled globin, as well as the hydrolysis of the specific substrate benzoyl tyrosine ethyl ester, by purified bovine chymotrypsin was found to be inhibited by several steroid hormones. The inhibition of chymotrypsin by the steroids was of a competitive nature, with K_i values of 9.9 × 10^?5 M for triamcinolone (9-fluoro-11β, 16α,17,21-tetrahydroxy-1,4-pregnadiene-3,20-dione), 1.6 × 10^?4 M for cortisol (11β,17α,21-trihydroxypregn-4-ene-3,20-dione), 3.7 × 10^?4 M for testosterone (17β-hydroxy-4-androsten-3-one), 5.0 × 10^?4 M for dexamethasone (9-fluoro-11β,17,21-trihydroxy-16α-methyl-1,4-pregnadiene-3,20-dione), and 1.0 × 10^?4 M for epicortisol (11α,17,21-trihydroxy-4-pregnene-3,20-dione). The activity of purified bovine trypsin on its specific substrate, TAME (tosyl arginine methyl ester), also showed a similar pattern of inhibition by steroids. Both chymotrypsin and trypsin were found to bind ³H-labeled dexamethasone and cortisol. This binding was markedly inhibited by the general protease inhibitor, PMSF (phenylmethanesulfonyl fluoride), whereas the chymotrypsin-specific inhibitor, TPCK (l-[1-tosyl-amido-2-phenyl]ethylchloromethyl ketone), inhibited only the steroid binding to chymotrypsin but not to trypsin. These observations indicate that serine proteases recognize steroid hormones in a fashion similar to the recognition of their specific substrates and that the steroids inhibit activity of these enzymes at their binding sites.     

Mometasone furoate degradation and metabolism in human biological fluids and tissues

The in vitro metabolic and non-metabolic degradation kinetics of mometasone furoate (MF) was investigated in selected human biological fluids and subcellular fractions of tissues. Qualitative and quantitative differences in transformation profiles of MF were observed among human biological media. Degradation was the major event in plasma and urine with four new degradation products identified; A: 21-chloro-17alpha-hydroxy-16alpha-methyl-9beta,11beta-oxidopregna-1,4-diene-3,20-dione 17-(2-furoate), B: 9alpha,21beta-dichloro-11beta,21alpha-dihydroxy-16alpha-methylpregna-1,4,17,20-tetraen-3-one 21-(2-furoate), C: 21beta-chloro-21alpha-hydroxy-16alpha-methyl-9beta,11beta-oxidopregna-1,4,17,20-tetraen-3-one 21-(2-furoate), and D: 21-chloro-17alpha-hydroxy-16alpha-methyl-9beta,11beta-oxidopregna-1,4-diene-3,20-dione. A, B and C were predominant and D was minor in plasma while A and C were predominant in urine. Hydrolysis of the 17-ester bond of MF was not a major event in plasma. The turnover of MF in plasma was faster than that in phosphate buffers of pH 7.4.Metabolism of MF occurred primarily and rapidly in liver, appreciably in intestine, but negligibly in in vitro lung tissue. While 6beta-hydroxylation was a major metabolic pathway for MF in microsomes of both human liver and intestine, other parallel and subsequent metabolism pathways could also be involved. If these degradation and metabolic products are also formed and active in humans in vivo, both MF and its 'active' products need to be taken into account when determining the systemic bioavailability of MF and in establishing concentration-effect relationships with this drug.     

Effect of neurosteroids on glutamate binding sites and glutamate uptake in rat hippocampus

Effects of some neurosteroids on the binding of [3H]-glutamate, [3H]-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and [3H]-MK-801, as well as on the [3H]-glutamate uptake were examined in rat hippocampus. The following compounds were evaluated: (a) positive modulators of the GABA(A) receptor: 5alpha-pregnan-3alpha-ol-20-one (allopregnanolone), 5alpha-pregnane-3alpha,21-diol-20-one (allotetrahydrodeoxycorticosterone), 5alpha-pregnan-3alpha-ol-11,20-dione (alphaxalone) and 5alpha-androstan-3alpha-ol-17-one (androsterone); (b) compounds showing GABA(A)-antagonistic and/or N-methyl-D-aspartic acid (NMDA)-agonistic properties: dehydroepiandrosterone sulfate and pregnenolone sulfate; (c) a substance which, apart from its GABA(A)-agonistic potency, has a NMDA-antagonistic action: 5beta-pregnan-3alpha-ol-20-one. None of those neurosteroids tested at concentrations of 0.001-100 microM affected the binding of [3H]-glutamate, [3H]-AMPA and [3H]-MK-801 or the glutamate uptake. The present study suggests that the previously reported inhibitory effects of neurosteroids on excitatory amino acid-induced seizures and neurotoxicity can be linked neither to the direct interaction of these compounds with the above binding sites on glutamate receptor complexes, nor to the glutamate uptake mechanism.     

Effect of epipregnanolone and pregnenolone sulfate on chronic tolerance to ethanol

The aim of the present study was to investigate the influence of neurosteroids on the development of tolerance to ethanol. Male Swiss mice were injected daily with the positive allosteric modulator of the gamma amino butyric acid-A (GABA(A)) receptor epipregnanolone (5beta-pregnan-3beta-ol-20-one; 0.15 mg/kg i.p.) or pregnenolone sulfate (5-pregnen-3beta-ol-20-one sulfate sodium; 0.08 mg/kg i.p.) - considered a negative allosteric modulator of this receptor and/or positive allosteric modulator of the N-methyl-D-aspartate (NMDA) receptor - 30 min before ethanol (2.5 g/kg i.p.). They were tested on the rota-rod apparatus, under continuous acceleration (1rpm/s), at 30, 60 and 90 min after ethanol injections for 5 days. The results showed that tolerance to the motor incoordinating effect of ethanol occurred on the fifth day of treatment when this effect was blocked by pretreatment with epipregnanolone. On the other hand, ethanol tolerance was enhanced by pretreatment with pregnenolone sulfate from the second to the fifth days of treatment. Taken together, our results suggest that neurosteroids can either stimulate or block the development of chronic tolerance to ethanol. Moreover, since neurosteroids can interact with GABA(A) or NMDA receptor systems, our results suggest the involvement of these systems in the actions of neurosteroids upon ethanol tolerance.     

Modulation of the GABAA receptor by barbiturates and pregnane steroids: differential effects of the influence of assay temperature

The effect of temperature on the modulation of the GABAA receptor by barbiturates and steroids has been investigated in-vitro using a radioreceptor binding assay. Displaceable [3H]muscimol binding to a crude membrane preparation from rat cerebral cortex was enhanced by the endogenous steroid metabolite, 5 beta-pregnan-3 alpha-ol-20-one, by the synthetic steroid, alphaxalone, and by pentobarbitone in a dose-dependent manner. Hydrocortisone and corticosterone had no significant effect on [3H]muscimol binding. Analysis of binding data using a curve-fitting program ('Ligand') showed that both pentobarbitone (1 mM) and 5 beta-pregnan-3 alpha-ol-20-one (10 microM) increased the apparent number of high affinity binding sites in the membrane but had no effect on the affinity of [3H]muscimol binding (Kd approx. 11 nM). Increasing the assay temperature from 0 degrees C to 35 degrees C decreased [3H]muscimol binding and decreased the enhancement of binding by pentobarbitone but had no effect on 5 beta-pregnan-3 alpha-ol-20-one enhancement of binding. 5 alpha-Pregnan-3 alpha-ol-20-one increased the apparent rate of association of [3H]muscimol binding to its receptor whereas pentobarbitone had no effect. These different effects on the apparent association rate and the different responses to temperature, suggest that the barbiturate and steroid may interact with the GABAA receptor through different binding sites.     

Investigation into the rapid effects of 17 beta-estradiol and neuroactive steroids upon beta-amyloid(25-35)-induced activation of phosphoinositide-specific phospholipase C in human platelets

In the present study, the rapid effects of five steroids (17 beta-estradiol, progesterone, allopregnanolone, 3 alpha-hydroxy-5 alpha-pregnan-20-one and 3 alpha-hydroxy-5 beta-pregnan-20-one) and the plant steroid trans-resveratrol upon the calcium response to beta-amyloid(25-35) peptide (A beta(25-35)) in human platelets was measured. A beta(25-35) produced a robust increase in intracellular calcium due to a direct activation of phosphoinositide-specific phospholipase C. None of the steroids significantly affected the response to A beta(25-35). In contrast, trans-resveratrol appeared to increase the response to A beta(25-35) at a concentration that decreased the response to thrombin, although the possibility that these changes are artifactual could not be ruled out. It is concluded that although steroids affect human platelet Ca2+ homeostasis, this is not a rapid event, unless very high concentrations are used.     

Cardenolide analogues. 4. (20R)- and (20S)-Cardanolides: on the roles of the 20(22)-ene and 14beta-hydroxyl in genin activity.

(20R)-20,22-Dihydrodigitoxigenin (3a) and (20S)-20,22-dihydrodigitoxigenin (3b) were isolated from (20R,S)-20,22-dihydrodigitoxigenin (3) by three fractional crystallizations each from ethyl acetate. The two diastereomers have distinct NMR spectra and similar (Na+,K+)ATPase inhibitory activities (I50 = 1.1-1.4 X 10(-5) M)--about 1/100 as active as digitoxigenin (1). Their activity compared with other cardenolide analogues suggests a passive geometric role for the 20(22) double bond in eliciting (Na+,K+)ATPase inhibition, keeping the lactone carbonyl in the proper orientation. (20S)-3 beta,14 beta-Dihydroxy-22-methylene-5 beta,14 beta-cardanolide (7a) was then synthesized from 3a, and (20R)-3 beta,14 beta-dihydroxy-22-methylene-5 beta,14 beta-cardanolide (7b) from 3b. They were found to be equivalently active in inhibiting (Na+,K+)ATPase, with I50 values of 7.0 x 10(-5) M. Although it has been usually believed that the 14 beta-hydroxyl of cardenolides increases binding to the receptor, 2b (the 14-ene derivative of 7b) was more than twice as active (I50 = 3.0 X 10(-5)) than either 7a or 7b.     

Terrestrinins A and B, two new steroid saponins from Tribulus terrestris

Two new steroid saponins, named terrestrinins A (1) and B (2), along with six known compounds were isolated from the Chinese medicine herb Tribulus terrestris, and their chemical structures were elucidated as 26-O-beta-D-glucopyranosyl-(25S)-furostan-4(5),20(22)-diene-3,12-dione (1) and 26-O-beta-D-glucopyranosyl-(25S)-5alpha-furostane-3beta,22alpha,26-triol-3-O-beta-D-xylopyranosyl(1 --> 3)-[(beta-D-xylopyranosyl(1 --> 2)]-beta-D-glucopyranosyl(1 --> 4)-[alpha-L-rhamnopyranosyl(1 --> 2)]-beta-D-galactopyranoside (2) on the basis of spectroscopic techniques.