Detection of corticosteroid type I binding sites in heart

Both high affinity (type I) and low affinity (type II) corticosteroid binding sites are detected in cytosolic extracts of atrial and ventricular tissue when [3H]aldosterone is used as the ligand. In the presence of RU-28362, which blocks binding of [3H]hormone to the low affinity type II sites, [3H]aldosterone binds to a single class of high affinity sites. The apparent Kd for binding of the hormone to the type I sites was 1.0 nM in atrial cytosol and 0.75 nM in ventricular cytosol. The concentration of type I sites in atrial (12 fmol/mg protein) and ventricular cytosols (11 fmol/mg protein) is comparable to reported values in renal (17-31 fmol/mg protein) cytosol. Activation of the type I hormone-receptor complexes to the DNA-binding form was examined using chromatography on DEAE-Sephadex anion-exchange resin. The unactivated hormone-receptor complex elutes at a concentration of 400 mM KCl. Following heat treatment (25 degrees C, 30 min) the [3H]aldosterone-receptor complex is transformed to a low salt eluting (200 mM KCl), DNA-binding form. Activation is blocked by inclusion of 10 mM sodium molybdate during heat treatment.     

Involvement of aldosterone and mineralocorticoid receptors in rat mesangial cell proliferation and deformability

We demonstrated recently that chronic administration of aldosterone to rats induces glomerular mesangial injury and activates mitogen-activated protein kinases including extracellular signal-regulated kinases 1/2 (ERK1/2). We also observed that the aldosterone-induced mesangial injury and ERK1/2 activation were prevented by treatment with a selective mineralocorticoid receptor (MR) antagonist, eplerenone, suggesting that the glomerular mesangium is a potential target for injuries induced by aldosterone via activation of MR. In the present study, we investigated whether MR is expressed in cultured rat mesangial cells (RMCs) and involved in aldosterone-induced RMC injury. MR expression and localization were evaluated by Western blotting analysis and fluorolabeling methods. Cell proliferation and micromechanical properties were determined by [3H]-thymidine uptake measurements and a nanoindentation technique using an atomic force microscope cantilever, respectively. ERK1/2 activity was measured by Western blotting analysis with an anti-phospho-ERK1/2 antibody. Protein expression and immunostaining revealed that MR was abundant in the cytoplasm of RMCs. Aldosterone (1 to 100 nmol/L) dose-dependently activated ERK1/2 in RMCs with a peak at 10 minutes. Pretreatment with eplerenone (10 micromol/L) significantly attenuated aldosterone-induced ERK1/2 phosphorylation. Aldosterone (100 nmol/L) treatment for 30 hours increased [3H]-thymidine incorporation and decreased the elastic modulus, indicating cellular proliferative and deforming effects of aldosterone, respectively. These aldosterone-induced changes in cellular characteristics were prevented by pretreatment with eplerenone or an ERK (MEK) inhibitor, PD988059 (100 micromol/L). The results indicate that aldosterone directly induces RMC proliferation and deformability through MR and ERK1/2 activation, which may contribute to the pathogenesis of glomerular mesangial injury.     

Occupancy of aldosterone binding sites in rat kidney cytosol.

The binding sites for aldosterone and a potent aldosterone antagonist (SC-26304) were studied in kidney cytosol from adrenalectomized rats. Preformed cytosol and kidney slices were incubated with 3H-labeled steroids in a wide range of concentrations. The recovery and characteristics of the binding sites were affected by the incubation and homogenization conditions. High-affinity, Type I mineralocorticoid binding was reduced by more than 95% when cytosol was incubated at 25 degrees C in the presence of calcium. Tissue dilution also affected the binding sites. SC-26304 was bound to high- and low-affinity receptors, similar to the binding of aldosterone. The physiologic response to aldosterone could result from binding to either or both sets of sites. Some of the physiologic responses to spirolactones could represent antagonism of the binding of aldosterone to either or both sites. A convenient method is presented for describing the relative occupancy of several different sites by any particular steroid.     

On the role of brain mineralocorticoid (type I) and glucocorticoid (type II) receptors in neuroendocrine regulation

Administrations of the glucocorticoid receptor antagonist (anti-glucocorticoid, RU38486) and the mineralocorticoid antagonist (anti-mineralocorticoid, RU28318) followed by frequent, sequential blood sampling were employed to investigate the possible role the brain mineralocorticoid receptor (MR, type I) and glucocorticoid receptor (GR, type II) have in the regulation of basal and stress-induced adrenocortical secretion in the rat. The anti-mineralocorticoid and anti-glucocorticoid were administered subcutaneously (s.c.) at doses of 2.5 mg and 1.0 mg/100 g body weight, respectively. Both antagonists were also given intracerebroventricularly (i.c.v.) at a dose of 100 ng/rat. Under basal non-stressed conditions (at the diurnal trough in the morning), injections of either saline, anti-glucocorticoid (s.c. or i.c.v.) or anti-mineralocorticoid (s.c.) did not have effect on the plasma corticosterone level. The anti-mineralocorticoid given intracerebroventricularly, however, caused an elevation of plasma corticosterone up to 60 min after the injection. Exposure of the rats to a novel environment resulted in a large increase in the plasma corticosterone level, which was slightly reduced in the rats treated with the anti-glucocorticoid. In vehicle-treated rats, the level returned to basal values at 90 min, while in the anti-glucocorticoid- and anti-mineralocorticoid-treated groups, it remained elevated for prolonged periods. The present study thus shows that (1) the anti-glucocorticoid RU38486 via the brain GR has no effect on the basal plasma corticosterone level in the morning but interferes with a glucocorticoid negative feedback following stress and (2) the anti-mineralocorticoid RU28318 via the brain MR elevates the basal plasma corticosterone level and enhances adrenocortical secretion following stress.(ABSTRACT TRUNCATED AT 250 WORDS)     

Follitropin receptors contain cryptic ligand binding sites

Human choriogonadotropin (hCG) and follitropin (hFSH) have been shown to contact different regions of the extracellular domains of G-protein coupled lutropin (LHR) and follitropin (FSHR) receptors. We report here that hCG and hFSH analogs interact with different regions of an FSHR/LHR chimera having only two unique LHR residues and that binds both hormones with high affinity. hCG and hFSH analogs dock with this receptor chimera in a manner similar to that in which they bind LHR and FSHR, respectively. This shows that although the FSHR does not normally bind hCG, it contains a cryptic lutropin binding site that has the potential to recognize hCG in a manner similar to the LHR. The presence of this cryptic site may explain why equine lutropins bind many mammalian FSHR and why mutations in the transmembrane domain distant from the extracellular domain enable the FSHR to bind hCG. The leucine-rich repeat domain (LRD) of the FSHR also appears to contain a cryptic FSH binding site that is obscured by other parts of the extracellular domain. This will explain why contacts seen in crystals of hFSH complexed with an LRD fragment of the human FSHR are hard to reconcile with the abilities of FSH analogs to interact with membrane G-protein coupled FSHR. We speculate that cryptic lutropin binding sites in the FSHR, which are also likely to be present in thyrotropin receptors (TSHR), permit the physiological regulation of ligand binding specificity. Cryptic FSH binding sites in the LRD may enable alternate spliced forms of the FSHR to interact with FSH.     

Activin-A binds follistatin and type II receptors through overlapping binding sites: generation of mutants with isolated binding activities

Follistatin is a potent extracellular antagonist of members of the TGFbeta superfamily that use activin type II receptors (ActRII/IIB) as part of their signaling complex. A recent crystallographic study indicates that follistatin contacts activin-A residues at both the type I (ALK4) and type II receptor binding interfaces. However, the relative contribution of these two sites on human activin-A to follistatin binding has not been determined. Residues at these sites were mutated to alanine and mutants were screened for their ability to bind follistatin and ActRII and induce FSH secretion from a gonadotrope cell line. Despite extensive mutagenesis across the type I receptor interface, activin-A affinity for follistatin was not significantly diminished. In contrast, mutagenesis of residues at the type II binding interface had pronounced effects on activin's interaction with follistatin. In particular, residues Leu92, Tyr94, Ile100, and Lys102 were critical for high-affinity follistatin binding. Interestingly, mutation of another primary determinant of ActRII/IIB binding, Ser90, did not affect follistatin affinity, suggesting that the interaction surfaces for type II receptors and follistatin were overlapping but not identical. In support, mutation of Asp95, on the opposite edge of the common ActRII/follistatin interface, was disruptive for follistatin binding without affecting ActRII/IIB interactions. Activin-S90A was able to compete with wild-type activin for follistatin binding, whereas activin-D95A, due to its 8-fold lower affinity for follistatin, is a potent activin agonist. These reagents could be used to modulate follistatin antagonism of activin and related ligands in processes such as cancer, wound healing, and reproduction.     

Leptin receptors are down-regulated in uterine implantation sites compared to interimplantation sites

Leptin is a circulating hormone that plays an important role in the regulation of metabolism, obesity, and reproduction. Leptin binds to its receptors on the cell membrane and is involved in the activation of STAT3. Recently, endometrium was suggested to be a novel target for leptin recently. We, therefore, examined the expression of leptin, leptin receptors, and STAT3 in the mouse uterus (implantation and interimplantation sites) to investigate the role of the leptin system during the early implantation period. Leptin mRNA was not detected in mouse uterine tissues or blastocysts, although adipose tissue, the positive control, showed a strong signal. Both of the receptor splice variants were expressed in the uterus and blastocysts, but the mRNA level was much lower in implantation sites compared to interimplantation sites. The mRNA expression of leptin receptors was determined to be higher in stromal cells than in the luminal epithelium using laser capture microdissection (LCM) analysis. Using immunohistochemistry, leptin was detected as a strong signal in the luminal epithelium and embryo, whereas the receptor was detected in subepithelial stromal cells rather than the luminal epithelium. As leptin itself was not detected by RT-PCR, the immunohistologically detected leptin may originate elsewhere, such as in adipose tissue. The differential expression of leptin receptors in implantation sites compared to interimplantation sites suggests that the leptin/leptin receptor system may be a delicate regulator of the implantation process.     

Barbiturate receptor sites are coupled to benzodiazepine receptors.

Barbiturates enhance the binding of [3H]diazepam to benzodiazepine receptor sites in rat brain. This effect occurs at pharmacologically relevant concentrations of barbiturates, and the relative activity of a series of compounds correlates highly with anesthetic activity of the barbiturates and with their ability to enhance postsynaptic inhibitory responses to the neurotransmitter gamma-aminobutyric acid. Barbiturate enhancement of benzodiazepine binding is stereospecific, with the more active anesthetic isomers of N1-methylbarbiturates being also more active than their stereoisomers in enhancing benzodiazepine binding. The active barbiturates produce a reversible enhancement in the affinity of specific benzodiazepine binding with no effect on the number of binding sites. The barbiturate enhancement, but not the baseline benzodiazepine binding, is competitively inhibited by the convulsant picrotoxinin (at 1-10 microM), a drug that has been shown to label barbiturate-sensitive brain membrane sites related to the gamma-aminobutyric acid receptor-ionophore complex. The barbiturate effect is also dependent upon the presence of certain anions, and only those anions, that penetrate the chloride channels regulated by gamma-aminobutyric acid receptors. These results suggest that picrotoxin-sensitive barbiturate binding sites are coupled to benzodiazepine receptors in the gamma-aminobutyric acid receptor-ionophore complex, and that these binding sites have the properties of pharmacologically relevant receptors that mediate at least part of the action of various nervous system depressant and excitatory drugs.     

Distribution of mineralocorticoid receptors in the kidney cells of rats with different levels of aldosterone in their bodies]

A metabolic method was applied to the study of the distribution of mineralocorticoid receptors in the kidney cells of rats at various aldosterone levels in the organism. A great number of binding sites of aldosterone in the cytoplasm and a low aldosterone-H3 binding by the nuclei of kidney cells was revealed in adrenalectomized rats, as well as in the animals kept under conditions of sodium load, when endogenous aldosterone was absent in the organism. A reduction of the number of binding sites in the cytoplasm and an increase of aldosterone-H3 binding in the nuclei was noted in the animals with a high content of the given hormone in the organism (sodium deficiency in food or aldosterone administration to the adrenalectomized animals). The difference in the hormone binding by the nuclei of the kidney cells of rats kept under conditions of sodium loading or deficiency corresponded to an even greater difference in the aldosterone binding by the chromatin proteins of such nuclei. It is supposed that an increased aldosterone concentration in the organism intensified the change from the cytoplasm and the accumulation of specific aldosterone-receptor complexes in the nuclei.     

Glucocorticoid binding to adenohypophysis receptors and its physiological role.

The binding characteristics of glucocorticoids to cytoplasmic and nuclear fractions of the adenohypophysis were determined and an attempt was made to correlate steroid uptake with physiological action. Binding was of high affinity and exhibited a limited number of acceptor sites for dexamethasone (Dx). It was shown that the steroid-receptor complex dissociated rapidly at 37 degree C, but was stable at low temperatures. An inverse relationship was detected between the plasma corticosterone (B) titer and the capacity of receptors to combine 3h-Dx. Shortly after stress or injection of physiological doses of B,50 to 70% of the acceptor sites were saturated. Progressive desaturation of the occupied receptors appeared to depend upon time and the plasma B concentration. Some evidence was also provided supporting a possible correlation between the extent of Dx inhibition of in vitro ACTH secretion and the degree of binding sites' occupancy. These results suggest that glucocorticoid binding to pituitary receptors may be correlated with the physiological action of the steroids on ACTH release.     

Glucocorticoid and mineralocorticoid receptors are involved in the facilitation of anxiety-like response induced by restraint

In previous studies, we have shown that male Wistar rats exposed to a single inescapable stressor session (15 min restraint) exhibited 24 h later an anxiogenic-like behavior in the elevated plus-maze (EPM), which was reversed by inhibition of corticosterone (CS) synthesis with metyrapone (75 mg/kg i.p.) 3 h before stress. Since CS binds to two central corticosteroid receptors, the mineralocorticoid (MR) and the glucocorticoid (GR) receptors, involvement of MR and GR in the modulation of anxiogenic responses was assessed in the EPM. Administration of the GR agonist dexamethasone (Dex, 1.25 microg/kg s.c.) to metyrapone-pretreated rats 1 h before restraint restored the anxiogenic-like response induced by the stressor. Removal of the adrenals also inhibited the anxiogenic-like effect, which was restored by either Dex (1.25 microg/kg s.c.), the MR agonist deoxycorticosterone (0.8 mg/kg s.c.) or CS, the common endogenous agonist of MR and GR (5 mg/kg s.c.) administered 1 h before stress. Intracerebroventricular infusion to intact animals 15 min before restraint of either a selective GR antagonist (A-GR, RU 38486, 100 ng/2 microl), a selective MR antagonist (A-MR, RU 28318, 100 ng/2 microl) or a combination of A-GR and A-MR (100 ng of each one/2 microl), abolished the stress-induced anxiogenic-like effect. The present findings indicate that both MR and GR are involved in the long-term CS modulation of the anxiety response induced by restraint. Both receptors mediate CS effects in an independent manner.     

Functional differences between neurotransmitter binding sites of muscle acetylcholine receptors

A muscle acetylcholine receptor (AChR) has two neurotransmitter binding sites located in the extracellular domain, at αδ and either αε (adult) or αγ (fetal) subunit interfaces. We used single-channel electrophysiology to measure the effects of mutations of five conserved aromatic residues at each site with regard to their contribution to the difference in free energy of agonist binding to active versus resting receptors (ΔG_B1). The two binding sites behave independently in both adult and fetal AChRs. For four different agonists, including ACh and choline, ΔG_B1 is ∼−2 kcal/mol more favorable at αγ compared with at αε and αδ. Only three of the aromatics contribute significantly to ΔG_B1 at the adult sites (αY190, αY198, and αW149), but all five do so at αγ (as well as αY93 and γW55). γW55 makes a particularly large contribution only at αγ that is coupled energetically to those contributions of some of the α-subunit aromatics. The hydroxyl and benzene groups of loop C residues αY190 and αY198 behave similarly with regard to ΔG_B1 at all three kinds of site. ACh binding energies estimated from molecular dynamics simulations are consistent with experimental values from electrophysiology and suggest that the αγ site is more compact, better organized, and less dynamic than αε and αδ. We speculate that the different sensitivities of the fetal αγ site versus the adult αε and αδ sites to choline and ACh are important for the proper maturation and function of the neuromuscular synapse.Receptors at synapses respond to specific chemical signals in the extracellular environment because the active conformation of the protein has a higher affinity for the ligand compared with the resting conformation (1, 2). The active vs. resting difference in binding free energy increases the relative stability of the active state and, hence, the probability of a cellular response. In this report, we describe and distinguish sources of ligand-binding free energy in three kinds of agonist site present in mouse muscle nicotinic acetylcholine receptors (AChRs). Our goal was to use single-channel electrophysiology to assess the relative contribution of significant functional groups to the overall free energy generated by the affinity change at each type of site.At cholinergic synapses, the main chemical signals are ACh released from nerve terminals and choline, which is an ACh precursor, hydrolysis product, and stable component of serum (3). The muscle AChR has central pore surrounded by five subunits of composition α₂βδε in adult-type and α₂βδγ in fetal-type (Fig. 1A) (4). The fetal, γ, subunit is essential for proper synapse maturation, and the adult, ε, subunit is necessary for proper function of mature synapses (5–7). Each AChR pentamer has two agonist binding sites in the extracellular domain, at αδ and either αε (adult) or αγ (fetal) subunit interfaces.Open in a separate windowFig. 1.Ligand binding sites. (A) Side view of a muscle AChR [Torpedo marmorata; PDB ID code 2bg9 (34)] showing an agonist site in the extracellular domain (αW149 and loops A, B, and C are marked). (Inset) Each AChR has two sites (filled circles) at αδ and αε (adult) or αγ (fetal) subunit interfaces. (B) High-resolution view of the ligand binding site of an acetylcholine binding protein occupied by carbamylcholine (CCh) [Lymnaea stagnalis; PDB ID code 1uv6 (11)]. Aromatic residues are labeled using mouse AChR numbering.The change in agonist affinity occurs within the global, resting↔active “gating” conformational change. Structural rearrangements at agonist sites that generate the affinity change are akin to movements of S4 in voltage-gated channels that generate gating currents. Given the central role of receptors at synapses, we thought it important to understand in detail the components of the free energy change that undergird the agonist affinity change. In wild-type AChRs, a large, uphill gating energy without agonists ensures the system will rarely activate constitutively, and a large, downhill free energy generated by affinity increases at the two agonist sites ensures that the protein will be active with a high probability after the release of ACh from the motor nerve terminal (8).We have estimated the free energy contributions of eight functional groups of five conserved residues at three different kinds of muscle AChR agonist site (αδ, αε, and αγ). On the α side of each site, there are four aromatics known to influence agonist affinity: αY190 (in loop C), αY198 (loop C), αY93 (loop A), and αW149 (loop B) (Fig. 1) (9–13). In addition, there is a conserved tryptophan in the nonα subunit, W55 (at position 57 in the δ subunit) (11, 14–16). In fetal AChRs, αW149 and αY198 have been shown to stabilize the quaternary ammonium of the agonist by cation-π forces (10, 13, 17).Previously, estimates of the ACh-binding free energy difference in mouse adult-type receptors after mutations indicated that only three of the mentioned aromatics (αY190, αY198, and αW149) are important (18), and other experiments showed that the free energy difference from both agonist sites combined is greater in fetal vs. adult AChRs (19). Here, we extend and refine these estimates. First, we measured the change in the net binding free energy after a mutation of each aromatic side chain in AChRs having just one functional binding site, so that the αδ, αε, and αγ sites could be probed independently, rather than pairwise. Second, we made some of these measurements using three partial agonists in addition to ACh, including the physiological ligand choline. Third, we estimated the degree of free energy coupling between some of the aromatic side chains at the fetal, αγ, site. Fourth, we used molecular dynamics (MD) simulations to estimate ACh binding energies and suggest structural correlates for differences between the three types of agonist site. We hypothesize that a greater sensitivity of fetal vs. adult AChRs to choline is a reason for the γ→ε subunit swap required for proper maturation of the neuromuscular synapse.