H-2 gene complex and corticosteroid responsiveness: evidence that the corticosteroid hormone signal transduction pathway in the adult mouse lung is not associated with haplotype-specific responses to corticosteroids.

Differential responsiveness to corticosteroids (CORT) has been shown to be related to HLA haplotype. A strong association between the mouse homolog to the human HLA complex, the H-2 complex, and intrauterine responses to CORT have also been demonstrated; haplotype differences alter CORT-induced susceptibility to cleft palate and temporal differences in lung maturation. Since variation in the glucocorticoid receptor (GR) is associated with tissue specific responses to CORT, we hypothesize that haplotype-specific CORT responsiveness may be regulated by H-2 associated modification of GR expression and/or function. Given that H-2 congenic mice are genetically identical except at the H-2 complex on mouse chromosome 17 and the GR structural gene is encoded on chromosome 18, the GR gene is identical in these mice. However, any step in the GR signal transduction pathway may be regulated by gene(s) at or near the H-2 complex and result in haplotype-specific differences in CORT responsiveness. We have investigated differences in qualitative and quantitative characteristics of the adult B10 (H-2b) and B10.A (H-2a) pulmonary GR by Scatchard analysis, immunochemical and biochemical assays. No differences in the GR binding parameters (BMAX and Kd), receptor form and level, or ligand-GR complex binding to glucocorticoid response element (GR-GRE) were detected, leading us to conclude that H-2 associated factors do not regulate the relative intrauterine responses to CORT by modulating the adult GR.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of mineralocorticoid type I and glucocorticoid type II receptors in astrocyte glia as a function of time in culture.

In the present study we have examined the expression of mineralocorticoid Type I and glucocorticoid Type II receptors in astrocyte glia maintained in culture for different periods of time. Cytosolic mineralocorticoid Type I receptors were labeled with [3H]aldosterone (ALDO) in the presence of a 500-fold molar excess of the potent Type II receptor ligand RU 28362. [3H]Dexamethasone (DEX) was used to label cytosolic Type II receptors. Both Type I and Type II receptor binding was saturable in astrocyte glia that had been maintained in culture for 20 and 30 days following final plating (i.e. 20- and 30-day-old cultures). Scatchard analysis of [3H]ALDO binding revealed a single class of Type I receptors, with dissociation constants (Kd) of 0.45 +/- 0.13 nM and 0.53 +/- 0.07 nM, respectively, in 20- and 30-day-old cultures. The number of Type I receptors in 30-day-old cultures was nearly half that found in 20-day-old cultures (22.06 vs 42.64 fmol/mg protein). Linear Scatchard plots were also obtained for [3H]DEX binding to cytosol prepared from 20- and 30-day-old cultures. There were no significant differences in the Kd or Bmax values for [3H]DEX binding in 20- or 30-day-old cultures, i.e. 2.06 +/- 0.15 nM and 247.36 +/- 18.16 fmol/mg protein for 20-day-old cells and 2.3 +/- 0.74 nM and 261.02 +/- 3.08 fmol/mg protein for 30-day-old cells. These Bmax values are more than double the Bmax value for [3H]DEX binding observed in our previous studies in 10-day-old astrocyte glial cultures. Switching cultured astrocyte glial from serum-supplemented to serum-free medium had no significant effects on the Kd values of Type I or Type II receptors in all the cultures tested. However, treatment with serum-free medium increased the number of Type I receptors in 30-day-old cultures to a level similar to that found in 20-day-old cultures. Taken together, these binding data suggest that Type I and Type II receptors are expressed differently in astrocyte glia as a function of time in culture.

Characterization of Glucocorticoid Type II Receptors in Neuronal and Glial Cultures from Rat Brain.

Abstract The purpose of this study was to characterize and compare the properties of glucocorticoid Type II receptors in neuronal and astrocyte glial cultures prepared from rat brain. Type II receptors in cytosol prepared from cultured cells were labeled with [(3) H]dexamethasone (DEX) at 0 degrees C. The binding was saturable and specific, with a complete displacement by unlabeled DEX or RU 28362 (a pure glucocorticoid). Scatchard analysis of [(3) H]DEX binding suggested a single class of receptors with a slightly lower dissociation constant (K(d)) in neuronal (1.13 nM) versus astrocyte glial (1.64 nM) cytosol. The number of binding sites (B(max)) in astrocyte glial cultures was four times that in neuronal cultures on a per milligram protein basis (120.3 versus 29.3 fmol/mg protein). The presence of Type II receptors in cultured neurons and astrocyte glia was further confirmed by immunofluorescent staining with a monoclonal antibody against this receptor (BuGR-2). The steroid specificity of Type II receptors was studied by examining the displacement of [(3) H]DEX binding to cytosol with unlabeled steroids. For both types of cultures, the potency series for competition was RU 28362> DEX> corticosterone> > aldosterone. Switching cultured cells from serum-supplemented to serum-free medium reduced [(3) H]DEX binding at low concentrations (0.5 to 5 nM) of the ligand in both types of culture, thus resulting in a decrease in the apparent affinity. This treatment did not, however, have any significant effect on the total number of binding sites. In summary, these results demonstrate that both neuronal and astrocyte glial cells in culture contain specific glucocorticoid Type II receptors, which resemble those seen in the brain and peripheral tissues.

Neither chronic exposure to ethanol nor aging affects type I or type II corticosteroid receptors in rat hippocampus.

Both chronic exposure to ethanol and aging are reported to result in a loss of hippocampal pyramidal neurons and an elevation in plasma corticosteroid concentration. Aging has also been reported to result in a reduction in corticosterone receptors and corticosterone-concentrating cells in the hippocampus. Since these aging-associated effects have been hypothesized to be due to the cumulative exposure to corticosteroids over the life span, in the present studies, we investigated the hypothesis that corticosteroids play a similar role in the loss of hippocampal neurons during chronic ethanol ingestion. In contrast to our expectations, when a liquid diet containing either ethanol or sucrose was administered to male Long-Evans rats for 20-24 weeks, a period of treatment found previously to result in hippocampal neuronal loss, there were no ethanol-associated effects on the specific binding of [3H]-aldosterone to Type I or of [3H]dexamethasone to Type II receptors in cytosol derived from either whole hippocampus or dorsal versus ventral hippocampus. Allowing the rats to withdraw from the chronic ethanol exposure for 12 weeks also failed to reveal any ethanol-associated effects on corticosteroid receptor concentrations in the hippocampus. The basal morning concentrations of corticosterone in blood plasma were not affected by any of these treatments. In view of these null findings we next investigated the effects of aging on hippocampal corticosteroid receptors in male Long-Evans and Fischer 344 rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Presence of type I and type II/IB receptors for adrenocorticosteroid hormones in the inner ear.

Aldosterone-Type I and dexamethasone-Type II/IB receptor complexes were identified in cytosol prepared from both cochlear and vestibular tissue samples. The specific binding capacity of Type I receptors in the cochlear tissues was approximately equal to that in the vestibular tissues. Likewise, the binding capacity of Type II/IB receptors in the cochlea was approximately equal to that in the vestibular endorgans. Based on the total specific binding measured with dexamethasone, the Type II/IB receptors appeared to outnumber the Type I receptors in cochlear and vestibular tissues by a factor of approximately 2.6; however, when adjustments were made for the probable cross-binding of dexamethasone to Type I receptors, these ratios were decreased to approximately 1.6. The existence of protein receptors for adrenocorticosteroid hormones demonstrated in the present study clearly suggests a mechanism whereby such hormones may directly regulate fluid and ionic gradients in the inner ear.

High affinity binding and regulatory actions of dexamethasone-type I receptor complexes in mouse brain.

It is often implied that the various molecular, physiological, and behavioral responses to the glucocorticoid dexamethasone (DEX) are mediated in brain exclusively via the interactions of this synthetic steroid with the classical glucocorticoid (type II) receptor. The results reported in this study, however, suggest this generalization may, at least for the female mouse, be too restrictive. In the first experiment we compared the efficacy of the mineralocorticoid aldosterone (ALDO) with that of DEX to measure the classical mineralocorticoid (type I) receptor in brain cytosol. Since both of these steroids also bind to type II receptors, our assays included the type II receptor-selective ligand, RU26988. Whereas the specific binding of ALDO to type I receptors was largely unaffected by a 10-fold increase in the concentration of RU26988 (50- vs. 500-fold excess), there was a dramatic reduction in the specific binding of DEX. In a follow-up experiment, Scatchard analyses were used to confirm the differential affinity of RU26988 for DEX- vs. ALDO-type I receptor-binding sites and to reveal that the affinity of type I receptors for DEX (Kd approximately 0.83 nM) was nearly as high as it was for ALDO (Kd approximately 0.46 nM). A series of competition studies indicated that the competitive affinity (Kdc) of DEX for the ALDO-binding site was equivalent to the Kd computed in the saturation analyses, thus suggesting that the high affinity binding sites for DEX and ALDO on type I receptors may be equivalent or at least overlapping. The binding of DEX to these high affinity sites may prove to be important, since the systemic administration of this steroid was found to down-regulate both type I and type II receptors in a number of brain regions. Because coadministration of the type I receptor antagonist RU26752 was shown to block these actions on type I, but not type II receptors, the formation of the DEX-type I receptor complex appears to be required for DEX-induced type I receptor down-regulation. An analysis of the in vitro efficacy of ALDO- vs. DEX-type I receptor transformation suggests that whereas there is a significant increase in the binding of both complexes to DNA-cellulose after treatment with thiocyanate, there is also a dramatic decrease in the stability of DEX- but not ALDO-type I receptor binding. We contend that it is this decrease in binding stability that mediates the DEX-induced down-regulation of type I receptors.(ABSTRACT TRUNCATED AT 400 WORDS)

Aldosterone-stimulated down-regulation of both type I and type II adrenocorticosteroid receptors in mouse brain is mediated via type I receptors.

The concentrations of type I and type II adrenocorticosteroid receptors in brain cytosol obtained from adrenalectomized-ovariectomized female mice were measured with five different assay conditions. Among the five brain regions studied, hippocampus had the highest concentration of type I receptors, whereas cerebral cortex had the highest concentration of type II receptors. The value of properly correcting for dexamethasone cross-binding to type I receptors when type II receptors are being assayed was demonstrated using the type II receptor-selective ligand RU28362. A time-course study revealed a transient up-regulation of both receptor classes in most brain regions after adrenalectomy-ovariectomy, with maximal values achieved 3-5 days postsurgery and a reduction to near-intact levels by 16 days postsurgery. A single sc injection of aldosterone given to adrenalectomized-ovariectomized mice produced a profound down-regulation of type I receptors in hippocampal, cerebral cortex, hypothalamic, brain stem, and cerebellar samples, whereas it down-regulated type II receptors only in hippocampal and cerebral cortical samples. A similar injection of RU28362 failed to down-regulate type I receptors in any brain region, but it did reduce the concentration of type II receptors in all brain regions except cerebellum. The actions of aldosterone appear to be mediated solely through type I receptors, since injections of the type I receptor antagonist RU26752 prevented aldosterone-induced down-regulation of both type I and type II receptors, whereas RU26752 had no effect on the down-regulatory actions of RU28362. The ability of aldosterone to down-regulate type I, but not type II, receptors in hypothalamic, brain stem, and cerebellar samples suggests that type I and type II receptors are concentrated in separate populations of cells in these brain regions, whereas in hippocampus and cerebral cortex there is a sufficient degree of colocalization to permit type II receptor down-regulation via the action of aldosterone-type I receptor complexes. We speculate that this action is mediated at least in part at the genomic level by the suppression of type I and type II receptor mRNA synthesis brought about by the interactions of transformed aldosterone-type I receptor complexes with the DNA regulatory elements upstream from the genes for these receptors.

Treatment of mouse brain cytosol with dextran-coated charcoal and high salt does not reveal a new glucocorticoid binder.

Incubation of steroid-free whole mouse brain cytosol from adrenalectomized-ovariectomized mice with saturating concentrations of tritiated dexamethasone was found to label all Type I as well as all Type II adrenocorticosteroid receptors. The quantitative and brain regional distribution of residual dexamethasone binding in cytosols pre-treated with dextran-coated charcoal (DCC) and 300 mM KCl was indistinguishable from that for tritiated aldosterone-Type I receptor complexes under the same conditions. We therefore conclude that the dexamethasone binding sites remaining after DCC and KCl treatment of steroid-free brain cytosol are due to the presence of Type I receptors. The differential sensitivity of Type I and Type II receptors to the DCC/KCl treatment paradigm may be useful in the purification of Type I receptors.

Initial characterizations of nontransformed and transformed [3H ]aldosterone-type I receptor complexes in brain cytosol.

Incubation of [3H]aldosterone-type I receptor complexes in mouse brain cytosol with the chaotropic anion thiocyanate increased the fraction of receptors retained by DNA-cellulose from less than 10% to over 40%, whereas it decreased the fraction retained by protamine sulfate from more than 90% to less than 10%. Thiocyanate-induced transformation to the DNA-binding species was also accompanied by a 2.1-fold decrease in the rate of [3H]aldosterone dissociation from type I receptors as well as by an increase in the apparent positive charge and hydrophobicity of the surface of these receptors, as revealed by DEAE Bio-Gel ion exchange and pentyl agarose hydrophobic interaction chromatography. Sucrose density gradient sedimentation and Sephacryl S-300 gel exclusion chromatography revealed a reduction in the sedimentation coefficient and Stokes radius of the steroid-receptor complex from 9.6S and 8.0 nm before to 4.7S and 6.1 nm after transformation, respectively. These changes in hydrodynamic parameters were found to correspond to a 2.8-fold reduction in the apparent molecular mass from 331,000 before to 120,000 after transformation. In view of these various findings as well as the known differential affinity of protamine sulfate for the 90K heat shock protein, we suggest that thiocyanate-induced transformation is initiated by the dissociation of two molecules of heat shock protein from each steroid/DNA-binding type I receptor subunit.

Differential up- and down-regulation of type I and type II receptors for adrenocorticosteroid hormones in mouse brain.

Adult female mice were adrenalectomized and ovariectomized and the concentration of Type I and Type II receptors in whole brain, kidney, and liver cytosol determined at various time thereafter by incubation with [3H]aldosterone (+ RU 26988 to prevent binding to Type II receptors) or [3H]dexamethasone, respectively. Type I receptor binding in brain was found to undergo a dramatic biphasic up-regulation, with levels six times that of intact levels by 24 h post-surgery and a doubling again by 4-8 days post-surgery. By 16 days, however, Type I specific binding had returned to intact levels. Similar, but less dramatic fluctuations were seen in kidney and liver, whereas much smaller fluctuations were seen for Type II receptors in all three tissues. In a follow-up study with Scatchard analyses we observed a similar transient up- and down-regulation in maximal binding for Type I, and to a lesser extent Type II receptors in all three tissues. As expected, the apparent binding affinity for both receptors increased after surgical removal of competing endogenous steroids. Radioimmunoassays revealed that plasma concentrations of corticosterone were reduced to near undetectable levels by 24 h post-surgery. A direct comparison of male and female mice revealed no sex-related differences in Type I receptor binding capacity fluctuations in brain cytosol after adrenalectomy-gonadectomy. Lastly, treatment with exogenous aldosterone or corticosterone was found to prevent adrenalectomy-gonadectomy-induced up-regulation of Type I and, to a lesser extent, Type II receptors in brain. Somewhat surprisingly, the potency of these two adrenocorticosteroids appeared to be very similar for both receptor types.

A novel effect of molybdate on the binding of [3H]aldosterone to gel-filtered type I receptors in brain cytosol.

Recently we reported that adding molybdate to crude steroid-free cytosol at 0 degree C results in a dose-dependent reduction in the binding of [3H]aldosterone ([3H]ALDO) to Type I adrenocorticosteroid receptors. In the experiments outlined here, we found that addition of molybdate to steroid-free brain cytosol produces a 30-50% increase in the subsequently measured maximal specific binding capacity (BMAX) of [3H]ALDO-Type I receptors if the cytosol is subjected to Sephadex G-25 gel filtration prior to steroid addition. These manipulations were found to have no effect on the equilibrium dissociation constant (Kd) of the receptors. In contrast, when gel filtration of steroid-free cytosol was performed in the absence of molybdate, there was a 2-fold increase in the Kd and over a 50% reduction in the subsequently measured BMAX of [3H]ALDO-Type I receptors. When molybdate was added to this steroid-free cytosol immediately following gel filtration, there was no reduction (or increase) in Type I receptor [3H]ALDO binding capacity compared with non-gel-filtered controls. The addition of as little as 2 mM molybdate to crude steroid-free cytosol was found to stabilize the binding capacity of Type I receptors during exposure to 22 degrees C incubations; however, when gel-filtered steroid-free cytosol was exposed to these conditions at least 10 mM molybdate was required to stabilize Type I receptor binding capacity. Adding the sulfhydryl reducing reagent, dithiothreitol, to the various steroid-free cytosols had little effect on [3H]ALDO-Type I receptor binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Further chemical differentiation of type I and type II adrenocorticosteroid receptors in mouse brain cytosol: evidence for a new class of glucocorticoid receptors.

There are at least two classes of intracellular receptors for adrenocorticosteroid hormones in brain. Type I receptors have a high affinity for the naturally occurring gluco- and mineralocorticoids, corticosterone (CORT) and aldosterone (ALDO), respectively, and a very low affinity for synthetic glucocorticoids such as dexamethasone (DEX). type II receptors have a high affinity for the synthetic glucocorticoids, a lower affinity for CORT and a very low affinity for ALDO. In recent studies with mouse brain cytosol we have found a number of other biochemical differences between these two receptor types. In the present study, brain cytosol from adrenalectomized mice was prepared in HEPES buffer and subjected to various potentially inactivating treatments prior to assessment of Type I and Type II receptor specific binding capacity by incubation for 24 h at 0 degrees C with [3H]ALDO +/- [1H]RU 26988 (to prevent or permit the cross-binding of [3H]ALDO to Type II receptors) or [3H]DEX +/- [1H]Prorenone (to prevent or permit the cross-binding of [3H]DEX to Type I receptors), respectively. These studies revealed that 10-20% of the high-affinity (Kd = 3 nM) [3H]DEX specific binding capacity remained even after extensive, high concentration and repeated pretreatments with dextran-coated charcoal (DDC. to remove endogenous sulfhydryl-reducing reagents and other biochemicals). These procedures had little effect on Type I receptors. Further analyses revealed that DCC-resistant [3H]DEX binders were not Type I receptors since they were not saturated by [1H]Prorenone. These binders were also not inactivated by aging steroid-free cytosol at 0 degree C or by treating it with buffers containing 0.3 M KCl. Since these

Effects of polyhydric and monohydric compounds on the stability of type I receptors for adrenal steroids in brain cytosol.

We have shown previously that unoccupied type I receptors for adrenal steroids in brain cytosol lose their capacity to bind [3H]aldosterone ([3H]ALDO) in a time- and temperature-dependent manner. Based on reports that sugars and polyvalent alcohols are capable of stabilizing a variety of globular proteins, we attempted in the present study to stabilize type I receptors by including polyhydric compounds in our brain cytosol preparations. However, contrary to expectations, adjusting cytosol to a 10% (g/dl) concentration of ethylene glycol, glycerol, erythritol, xylitol, ribitol, or sorbitol failed to stabilize these receptors at 0 degree C and in fact produced a slight reduction in [3H]ALDO binding capacity. The magnitude of this reduction was greater when cytosol was incubated for 2 h at 22 degrees C prior to incubation with [3H]ALDO. In contrast to these results, when brain cytosol was adjusted to a 10% (g/dl) concentration of the monohydric compound, ethanol, a significant increase in [3H]ALDO binding to type I receptors was found. Under identical conditions, methanol and propanol failed to have a significant effect on the binding capacity of these receptors. When cytosol was aged for 2 h at 22 degrees C, all three of these monohydric compounds produced a marked loss in the [3H]ALDO binding capacity of type I receptors. An investigation of various doses of ethanol at 0 degree C on the subsequent binding of [3H]ALDO yielded an inverse U-shaped curve with 10% ethanol producing the highest level of specific binding, as reflected by an increase in maximal binding in Scatchard plots, and 40% ethanol producing a complete loss in type I receptor binding capacity.(ABSTRACT TRUNCATED AT 250 WORDS)

Activation of glucocorticoid-type II receptor complexes in brain cytosol leads to an increase in surface hydrophobicity as determined by hydrophobic interaction chromatography.

Hydrophobic interaction chromatography has been used to demonstrate an increase in the surface hydrophobicity of [3H]triamcinolone acetonide ([3H]TA)-labeled type II receptors in mouse brain cytosol following transformation of these receptor complexes to the activated DNA-binding form. After removing unbound [3H]TA and molybdate (which prevents activation) by gel filtration, [3H]TA-type II receptors were activated by incubation at 22 degrees C for 20 min. Gel filtration was then used to remove newly dissociated steroid and to readjust the molybdate and/or KCl concentration. Unactivated and activated receptors were then added to propyl, butyl, pentyl, hexyl, octyl, decyl, and dodecyl alkyl agarose, phenyl agarose, or unmodified agarose columns equilibrated and eluted with buffers of various molybdate and KCl concentrations and/or other additions, including glycerol, ethylene glycol, and urea. Under high-salt conditions, activated receptors were retained longer than unactivated receptors run on butyl, pentyl, hexyl, and phenyl agaroses. With the longer alkyl chain columns, essentially none of the [3H]TA was eluted in association with receptor macromolecules. Removal of the remaining steroid required receptor denaturation with urea. Under low-salt conditions, both receptor forms were retained more avidly on all alkyl agarose columns; however, on phenyl agarose only activated receptors displayed this increased retention. Further studies revealed that optimal separation and subsequent recovery of unactivated and activated [3H]TA-type II receptor complexes were achieved on pentyl agarose columns equilibrated and eluted with buffers containing 50 mM molybdate and 600-1,200 mM KCl.(ABSTRACT TRUNCATED AT 250 WORDS)

Activated type II receptors in brain cannot rebind glucocorticoids: relationship to progesterone's antiglucocorticoid actions.

Exchange assays have often been used to quantitate steroid receptors when endogenous ligands are present; however, there are no reports of their successful application to activated glucocorticoid-Type II receptor complexes. In addition to investigating the reasons for this failure, the present study also examined the effects of progesterone on glucocorticoid dissociation from, and reassociation with unactivated and activated Type II receptors. Molybdate-stabilized brain cytosol from adrenal-ovariectomized mice was incubated with [3H]dexamethasone ( +/- [1H]DEX) for 40 h at 0 degree C. Afterwards free steroid was removed on Sephadex G-25 columns in the presence (unactivated receptors) or absence (activated receptors) of molybdate. Activation, as measured by DNA-cellulose binding, was achieved by incubating molybdate-free cytosol at 22 degrees C for 20 min followed by G-25 filtration in the presence of molybdate. The rates of dissociation and reassociation were then measured by incubating cytosol with [1H]triamcinolone acetonide (TA) or [3H]TA ( +/- [1H]TA) at 12 degrees C. An exchange assay was also employed in which cytosol was incubated first with [1H]DEX for 40 h at 0 degree C followed by bound-free steroid separations and 12 degrees C incubations with [3H]TA ( +/- [1H]TA). Both approaches revealed that even though activation reduced the rate of DEX dissociation from Type II receptors by 40%, it eliminated the ability of the newly unoccupied receptors to rebind glucocorticoid. Adding [1H]progesterone to occupied receptor preparations increased dissociation rate constants by nearly 3-fold, for both unactivated and activated Type II receptors. Since [1H]TA failed to prevent this effect, progesterone appears to act at an allosteric site(s) which cannot be occupied by glucocorticoids. Exchange assays revealed that progesterone-facilitated dissociation increased the rate of glucocorticoid rebinding to unactivated, but not activated Type II receptors. These results suggest that spontaneous and progesterone-facilitated termination of glucocorticoid genomic actions could be mediated by steroid dissociation since unoccupied activated Type II receptors do not rebind agonist steroid.

Linearization of two ligand-one binding site scatchard plot and the "IC50" competitive inhibition plot: application to the simplified graphical determination of equilibrium constants.

A simple procedure for linearizing the curved, two ligand-one binding site Scatchard plot resulting from the presence of a constant concentration of competitive inhibitor is proposed; the same procedure may also be applied to the analysis of data derived from the "IC50" competitive inhibition experimental design. Furthermore, a useful generalization of the Cheng-Prusoff correction is presented.

Transformation of the [3H]aldosterone-type I receptor complex to a DNA-binding species.

For most steroid receptor complexes, the transformation to a DNA-binding species can be achieved readily in vitro by incubation at elevated temperatures and/or salt concentrations. Although the aldosterone-Type I receptor complex forms a clear exception to this generalization, a marked increase in its transformation can be achieved by incubation with the chaotropic anion, thiocyanate. Time and concentration-response analyses with brain cytosol revealed that over 40% of the complexes were retained in DNA-cellulose assays after a 15 min pre-incubation at 0 degree C with 100 mM thiocyanate. As expected, molybdate prevented this transformation; however, in contrast to results with heat- and/or salt-induced transformation of other steroid receptors, the molybdate effect was only partially removed by gel filtering the cytosol prior to thiocyanate addition. Thiocyanate-induced transformation should prove useful in the biochemical characterization and purification of non-transformed and transformed aldosterone-Type I receptor complexes.

Chemical differentiation of type I and type II receptors for adrenal steroids in brain cytosol.

Studies outlined here compare the properties of mineralocorticoid (Type I) and glucocorticoid (Type II) receptors in cytosol from adrenalectomized mouse brain. Pretreating cytosol with dextran-coated charcoal (DCC) produced a 4.7-fold increase in the subsequent macromolecular binding of the mineralocorticoid, [3H]aldosterone (20 nM ALDO, in the presence of a 50-fold molar excess of the highly specific synthetic glucocorticoid, RU 26988), whereas it produced a 55% decrease in the binding of the glucocorticoid, [3H]triamcinolone acetonide (20 nM TA). Scatchard analyses revealed that DCC pretreatment had no effect on the affinity or maximal binding of Type I receptors for [3H]ALDO (in the presence of a 0-, 50- or 500-fold excess of RU 26988), whereas it produced a 3- to 6-fold increase in the Kd, and an 8-43% decrease in the maximal binding, of Type II receptors for [3H]TA and [3H]dexamethasone. Optimal stability of unoccupied Type I receptors at 0 degree C was found to be achieved in buffers containing glycerol, but lacking molybdate. Although the addition of molybdate was found to reduce the loss in Type I receptor binding observed after incubating unlabelled cytosol at 12 or 22 degrees C, this stabilization was accompanied by a concentration-dependent reduction in the binding of [3H]ALDO at 0 degree C. Scatchard analyses showed that this reduction was due to a shift in the maximal binding, and not the affinity, of the Type I receptors for [3H]ALDO. The presence or absence of dithiothreitol in cytosol appeared to have little effect on the stability of Type I receptors. In contrast to our finding for Type I receptors, it was possible to stabilize the binding capacity of unoccupied Type II receptors, even after 2-4 h at 12 or 22 degrees C, if the glycerol containing buffers were supplemented with both molybdate and dithiothreitol. In summary, these results indicate distinct chemical differences between Type I and Type II receptors for adrenal steroids.

Effect of chronic administration of deoxycorticosterone acetate on salt appetite of captopril-treated rats.

Chronic dietary administration of the angiotensin I-converting enzyme inhibitor, captopril (1.0 g/kg food), induced an appetite for 0.15 M NaCl solution relative to distilled water in a two-bottle ad libitum access paradigm. Graded doses of deoxycorticosterone acetate (DOCA) were administered to the captopril-treated rats via Silastic tubes implanted sc. A U-shaped dose-response relationship was observed between dose of DOCA and intake of 0.15 M NaCl solution. The lowest intake of NaCl occurred with a dose of 102.1 micrograms DOCA/day (333 micrograms/kg X day). Water and food intakes were not affected significantly. At the end of the first week of treatment with DOCA, pilocarpine (3.0 mg/kg, ip.)-stimulated salivary chloride concentration was measured. The relationship between salivary chloride concentration and mean intake of 0.15 M NaCl solution was also U shaped, with the minimal NaCl intake associated with a salivary chloride concentration of 54 meq/liter. During the second week of treatment with DOCA, all rats were offered a choice between 0.25 M NaCl solution and distilled water. Rats receiving DOCA at doses of 102.1 micrograms/day or greater had a significantly lower intake of NaCl solution than those receiving either captopril alone or 66.5 micrograms DOCA/day in combination with captopril. These results indicate that the appetite for NaCl solution induced by captopril can be inhibited by concurrent administration of DOCA. They also suggest that changes in the concentration of electrolytes in saliva may be associated with changes in the appetite for NaCl solution.

ATP-dependent stabilization against microsomal factor-induced loss of unoccupied glucocorticoid receptors.

ATP stabilizes the unoccupied glucocorticoid receptor from brain at 12 degrees C, but only in the presence of a destabilizing microsomal factor. This stabilization is optimal at an ATP concentration of about 1 mM, higher concentrations resulting in an increase in the rate of heat inactivation. Other nucleotides, including CTP, GTP, UTP, ADP, cAMP and cGMP were ineffective in stabilizing receptors, although additions of some of these nucleotides actually resulted in further destabilization of the unoccupied glucocorticoid receptor.