A 3a-hydroxy A-ring-reduced metabolite of progesterone, 3a-hydroxy-5a-pregnan-20-one (allopregnanolone), and one of deoxycorticosterone (DOC), 3a,21-dihydroxy-5a-pregnan-20-one (ailotetrahydroDOC), are among the most potent known ligands of y-aminobutyric acid (GABA) receptors designated GABAA in the central nervous system. With specific radioimmunoassays, rapid (<5 min) and robust (4-to 20-fold) increases of allopregnanolone and allotetrahydroDOC were detected in the brain (cerebral cortex and hypothalamus) and in plasma of rats after exposure to ambient temperature swim stress. Neither steroid was detectable in the plasma of adrenalectomized rats either before or after swim stress. However, allopregnanolone, but not allotetrahydro-DOC, was still present in the cerebral cortex (>3 ng/g) after adrenalectomy. These data demonstrate the presence of allopregnanolone and allotetrahydroDOC in brain and show that acute stress results in a rapid increase of these neuroactive steroids to levels known to modulate GABAA receptor function.It has been almost 50 years since Selye (1) reported the anesthetic and sedative properties of several 3a-hydroxy A-ring-reduced pregnane steroids, including the major metabolites of progesterone, 3a-hydroxy-5a-pregnan-20-one (allopregnanolone) and 3a-hydroxy-5p-pregnan-20-one (pregnanolone), and the major metabolite of deoxycorticosterone (DOC), 3a,21-dihydroxy-5a-pregnan-20-one (allotetrahydroDOC). In addition to their sedative-hypnotic and anesthetic effects, these steroids have subsequently been shown to produce a number of other behavioral effects when administered to laboratory animals, including anticonflict (2), anticonvulsant (3), and analgesic actions (4). The behavioral effects of these 3a-hydroxysteroids do not appear to involve genomic events that are mediated by steroid receptors since they occur quite rapidly (seconds to minutes) after parenteral administration, and since neither steroid binds with appreciable affinity to progesterone receptors (5). Recently, we and others (6-13) have reported that allopregnanolone and allotetrahydroDOC bind with high affinity to recognition sites associated with the y-aminobutyric acid (GABA) receptor complex. The GABAA receptor is one of two known receptors for GABA, the principal inhibitory neurotransmitter in brain, and has been shown to mediate the anxiolytic, sedative, and hypnotic actions of many drugs, including benzodiazepines and anesthetic barbiturates (14, 15). Radioligand binding and Cl-flux studies (6-12) as well as electrophysiologic experiments in cultured embryonic neurons (16) have provided evidence for the presence of unique and perhaps multiple steroid recognition sites (13) associated with the GABAA receptor macromolecular complex. In experiments using recombinantly expressed GABAA receptors, both allopregnanolone and allotetrahydroDOC were effective in potentiating GABA-activated Cl-currents at low nanomolar concentrations, comparable to the most potent benzodiazepines (17).The relatively high affinity of th...
Nondenaturing gradient gel electrophoresis continues to be used widely for resolution and characterization of lipoprotein subclasses. Methods for making such gels in the laboratory have been published, but occasionally samples do not display uniform mobilities for all lanes in a laboratory-made gel.To help overcome this limitation, we recommend a modification-addition of a sucrose gradient-that significantly improves within-gel variation in protein mobility. Extensive literature supports the hypothesis that knowledge of lipoprotein size distributions provides a more detailed indication of lipoprotein metabolism and a more accurate prediction of cardiovascular disease risk (1). Lipoprotein size distributions have been measured by a number of physicochemical methods, perhaps most commonly by gradient gel electrophoresis. Previously, we have described a highly reproducible method designed to make nondenaturing acrylamide gradient gels for the electrophoretic separation of plasma HDLs (2) and LDLs (3). More recently, we described a composite gradient gel that enabled the simultaneous analysis of both LDLs and HDLs (4). Estimates of peak diameter and fractional absorbance made from the composite gel were strongly correlated with those made using the respective dedicated gel formats (4). Recently, however, we began to notice that the gels have run unevenly on occasion; that is, samples appeared to run different distances depending on location in the gel (generally, samples in the outside lanes tended to run faster than those in the interior lanes, leading to a "frowning" appearance). The problem was observed in the LDL portion of the gradient and did not seem to affect mobilities of HDLs in the areas of higher acrylamide concentrations.In a previous study, Rhigetti et al. (5) reported that convective flows are imprinted in the final gel structure, but this effect can be minimized by use of a sucrose gradient during the gelling process. Accordingly, we modified our published protocol and tested whether the addition of a sucrose gradient might solve the problem of differential mobilities for different lanes in a gel.Composite acrylamide gradient gels were made exactly as described previously (4) except that, to some high limit solutions, we added sucrose (10 g/l). The two types of gels will be referred to as Ϫ sucrose and ϩ sucrose. Briefly, the gradient was made with two solutions: a low limit solution (3% acrylamide with 4% cross-linker) and a high limit solution (31% acrylamide with 5.5% cross-linker and Ϯ 10% sucrose). Gels were cast in groups of 16 and allowed to polymerize for 3 h prior to storage at 4 Њ C for no more than 1 month before use. Serum and plasma samples from baboons and humans, respectively, were subjected to electrophoresis at 3,000 v · h (using electrophoresis chambers supplied by CBS Scientific) and staining with Sudan black B. These samples were run as part of ongoing studies in this laboratory. Following restoration of the original gel shape and size in the electrophoresis buffer, we su...
The levels of 3 alpha-hydroxy-5 alpha-pregnan-20-one (allopregnanolone) and the epimeric 3 alpha-hydroxy-5 beta-pregnan-20-one (pregnanolone) were studied in women with prospectively confirmed premenstrual syndrome (n = 15) and in a group of asymptomatic control women (n = 12) during the luteal phase of the menstrual cycle. Single late luteal phase plasma samples were selected to make comparisons of plasma hormone levels between patients and controls in the following measures: allopregnanolone, pregnanolone, the ratio of allopregnanolone to pregnanolone, the ratio for each of these anxiolytic steroids to the parent compound progesterone, and the ratio of the sum of allopregnanolone and pregnanolone to progesterone. Differences in these measures were compared by analysis of variance. Additionally, correlations were performed among the various hormone measures and between the hormone measures and the symptom self-ratings. Analysis of variance showed no significant between group differences in the plasma levels of allopregnanolone, pregnanolone, and progesterone. Plasma levels of both allopregnanolone and pregnanolone were correlated with plasma progesterone levels. However, there were no significant correlations between the severity of mood and behavioral symptoms and plasma levels of progesterone, allopregnanolone, and pregnanolone. These data suggest that symptoms of premenstrual syndrome are not associated with a simple deficiency state of either progesterone or its anxiolytic steroid metabolites.
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