Skeletal Muscle Cytosol Research Articles

Structural Modulation of RyR1 by MgATP and Free Mg2+ in Lipid Membrane using CryoEM

Mg2+ is the most abundant divalent cation in living cells. In the skeletal muscle cytosol, out of the total Mg2+ concentration of 8 mM, around 1 mM is free Mg2+ and around 5 mM exists in the form of MgATP, the biological active form of cellular ATP. Mg2+ is constitutively bound to RyR1 in its free and complexed forms. In fact, preferential binding of Mg2+ to the acidic side chains with similar coordination geometry as Ca2+, allows it to be a potential antagonistic cation of Ca2+, which has much lower cytoplasmic concentrations (<1 μM - 100 μM). Free Mg2+ is thought to inhibit RyRs through its association with Ca2+ binding sites at cytoplasmic or luminal regions, while the effect of MgATP per se is novel in the context of excitation-contraction coupling. We studied the effect of free Mg2+ and MgATP on RyR1 bound to a POPC bilayer enclosed by membrane scaffold protein (nanodisc). CryoEM reconstructions of RyR1 nanodiscs complexed with MgAMPPCP at ∼3.8 Å resolution show that in the absence of any detergents or FKBP, the RyR1 assumes a closed conformation with a pore radius that makes impermeant to Ca2+ ions. Discernible densities for nanodisc and MgATP were observed. Binding of MgATP involves global motion of the central and cytoplasmic domains towards the T-tubule and the fourfold axis with reorganization of the P2 domain. Local motion of the TM helices, U-motif, VSC and EF hands were also observed with concomitant closure of the ATP cavity at the intersubunit interface. With the cryoEM structure of RyR1-MgATP, we seek to explain the mechanistic basis of RyR1 inhibition during the physiological resting state of skeletal muscle. Supported by grants AHA 14GRNT19660003, MDA352845, NIH R01 AR068431, and NIH U24GM116789.

Antidiabetic effects of bitter gourd extracts in insulin-resistant db/db mice

Bitter gourd (BG, Momordica charantia) exerts proven blood glucose- and body weight-lowering effects. To develop an effective and safe application, it is necessary to identify the bioactive compounds and biochemical mechanisms responsible for these effects in type 2 diabetes. A total of forty-five 4-week-old male db/db mice were assigned to five groups of nine each. The mice were given sterile tap water as a control, a whole fruit powder, the lipid fraction, the saponin fraction or the hydrophilic residue of BG at a daily oral dosage of 150 mg/kg body weight for 5 weeks, respectively. Weight gain was significantly decreased in all the BG-treated groups (P ≤ 0.05). Glycated Hb levels were the highest in the control mice compared with all the four BG-treated mice (P = 0.02). The lipid fraction had the strongest effect, and it tended (P = 0.075) to reduce glycated Hb levels from 9.3 % (control mice) to 8.0 % (lipid fraction-treated mice). The lipid and saponin fractions reduced lipid peroxidation of adipose tissue significantly (P ≤ 0.01). Additionally, the saponin fraction and the lipid fraction reduced protein tyrosine phosphatase 1B (PTP 1B) activity in skeletal muscle cytosol by 25 % (P = 0.05) and 23 % (P = 0.07), respectively. PTP 1B is the physiological antagonist of the insulin signalling pathway. Inhibition of PTP 1B increases insulin sensitivity. This is the first study to demonstrate that BG is involved in PTP 1B regulation, and thus explains one possible biochemical mechanism underlying the antidiabetic effects of BG in insulin resistance and type 2 diabetes.

A Novel Approach for Screening the Proteome for Changes in Protein Conformation

Changes in surface hydrophobicity are generally considered as a sensitive indicator for monitoring the structural alterations of proteins that are often associated with changes in function. Currently, no technique has been developed to screen a complex mixture of proteins for changes in the conformation of specific proteins. In this study, we adapted a UV photolabeling approach, using an apolar fluorescent probe, 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (BisANS), to monitor changes in surface hydrophobic domains in either purified rhodanese or skeletal muscle cytosolic proteins by urea-induced unfolding or in response to in vitro metal-catalyzed oxidation. Using two-dimensional polyacrylamide gel electrophoresis (2D PAGE), we identified two specific proteins in skeletal muscle cytosol that exhibited a marked loss of incorporation of BisANS after exposure to in vitro oxidative stress: creatine kinase (CK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). We found that the activities of both enzymes were also reduced significantly in response to oxidative stress. We then determined if this method could detect changes in surface hydrophobicity in specific proteins arising from oxidative stress generated in vivo by muscle denervation. A loss in surface hydrophobic domains in CK and GAPDH was again observed as measured by the BisANS photoincorporation approach. In addition, the CK and GAPDH activity in denervated muscle was markedly reduced. These data demonstrate for the first time that this assay can screen a complex mixture of proteins for alterations in surface hydrophobic domains of individual proteins.

Serotonylation Activates Platelets

Platelets, small anuclear cellular fragments in the circulation, are essential to the clotting process and are implicated in vascular disorders such as thrombosis and atherosclerosis. Platelets adhere to sites of damaged vascular endothelia through a process involving von Willebrand factor (vWF). When stimulated, platelets also release granules that contain various proaggretory factors such as adenosine diphosphate, adenosine triphosphate, serotonin (5-HT), calcium, and multimeric forms of vWF. Platelets also have 5-HT receptors and 5-HT reuptake transporters (SERTs). Using a tryptophan hydroxylase 1 knockout mouse (Tph is the rate-limiting enzyme in 5-HT biosynthesis, and Tph1 is the peripheral isoform), Walther et al . analyzed the role of 5-HT in platelet aggregation and degranulation. Clotting times were prolonged in the Tph1 –/– mice, and the mice were protected from thrombotic vessel occlusion, which suggests a defect in platelet aggregation. Using pharmacological inhibitors of the plasma membrane SERT and the vesicular monoamine transporter, the authors demonstrated that cytosolic 5-HT was essential for effective aggregation of isolated platelets. Platelets treated with radioactive 5-HT showed a pool of unreleasable 5-HT in the aggregated platelets. Secretion of granule contents (vWF) was stimulated in permeabilized platelets from the Tph1 –/– mice by the addition of 5-HT, transglutaminase (TG), and skeletal muscle cytosol. Analysis of the proteins isolated from aggregated platelets showed multiple bands labeled with [ 14 C]5-HT, including the guanosine triphosphatase (GTPase) RhoA. In vitro, Rab4, which controls granule secretion in platelets, was also transamidated by purified liver TGs. Thus, the authors propose that 5-HT stimulates an increase in calcium through its activation of its G q -coupled receptor. The calcium stimulates the activity of TGs, which use 5-HT taken up by the SERT to transamidate GTPases, which renders them constitutively active, promoting changes in platelet shape (RhoA) and degranulation (Rab4). This ultimately produces irreversible platelet aggregation. D. J. Walther, J.-U. Peter, S. Winter, M. Höltje, N. Paulmann, M. Grohmann, J. Vowinckel, V. Alamo-Bethencourt, C. S. Wilhelm, G. Ahnert-Hilger, M. Bader, Serotonylation of small GTPases is a signal transduction pathway that triggers platelet α-granule release. Cell 115 , 851-862 (2003). [Online Journal]

Effect of glutathione on oxymyoglobin oxidation.

The oxidation of oxymyoglobin (OxyMb) to metmyoglobin (MetMb) is responsible for fresh meat discoloration. Glutathione (GSH) is an important tripeptide reductant that can protect lipid and protein from oxidation. The objective of this research was to investigate the effect of GSH on MetMb formation in vitro and in bovine skeletal muscle cytosol. Equine MetMb formation was greater in the presence of GSH than controls at pH 5.6 or 7.2 and 25 or 37 degrees C (p < 0.05); GSH addition to purified bovine OxyMb solution also resulted in more MetMb formation at pH 7.2 and 25 or 37 degrees C (p < 0.05). This effect on MetMb formation was partly or completely inhibited by EDTA or catalase in the GSH-equine OxyMb system (p < 0.05). The addition of GSH to bovine muscle cytosol inhibited MetMb formation at pH 5.6 or 7.2 and 4 or 25 degrees C (p < 0.05); the effect was concentration-dependent. The inhibitory effect was observed in a high molecular weight (HMW) but not low molecular weight fraction of cytosol at pH 7.2 and 25 degrees C (p < 0.05); there was no effect when HMW was heated at 90 degrees C for 15 min. These results suggest the antioxidant effect of GSH on bovine OxyMb is dependent on heat-sensitive HMW cytosolic component(s).

Properties of free and occupied androgen receptor in rat skeletal muscle cytosol: effect of testosterone

Our aim was to investigate the effect of a single testosterone (T) injection on the androgen receptor (AR) in rat skeletal muscle (SM) cytosol. The properties of AR were studied in order to establish the protocol for differential determination of free and hormone-occupied AR in SM cytosols from non-hormone-deficient animals. Using the developed ligand-exchange protocol, we demonstrated that injection of T (1 mg/kg) caused alternating changes of the total AR binding. The binding minimum (23% of the control) was measured 1 h after the injection. It was followed by pronounced and lasting elevation of the AR binding. In the control cytosols, AR complexes constituted ∼25% of the total receptor content. Changes of their relative content immediately after T administration were consistent with rapid nuclear translocation of the AR. Inhibition of protein synthesis by cycloheximide (CHI) injection demonstrated that delayed and lasting increase of the AR binding after T injection partially depended on the stimulated protein synthesis. Altogether, the obtained evidence supports the assumption that the AR mediates elevation of its own gene expression in SM upon administration of T.

Interaction of the Ras-related protein associated with diabetes rad and the putative tumor metastasis suppressor NM23 provides a novel mechanism of GTPase regulation.

Rad is the prototypic member of a new class of Ras-related GTPases. Purification of the GTPase-activating protein (GAP) for Rad revealed nm23, a putative tumor metastasis suppressor and a development gene in Drosophila. Antibodies against nm23 depleted Rad-GAP activity from human skeletal muscle cytosol, and bacterially expressed nm23 reconstituted the activity. The GAP activity of nm23 was specific for Rad, was absent with the S105N putative dominant negative mutant of Rad, and was reduced with mutations of nm23. In the presence of ATP, GDP.Rad was also reconverted to GTP.Rad by the nucleoside diphosphate (NDP) kinase activity of nm23. Simultaneously, Rad regulated nm23 by enhancing its NDP kinase activity and decreasing its autophosphorylation. Melanoma cells transfected with wild-type Rad, but not the S105N-Rad, showed enhanced DNA synthesis in response to serum; this effect was lost with coexpression of nm23. Thus, the interaction of nm23 and Rad provides a potential novel mechanism for bidirectional, bimolecular regulation in which nm23 stimulates both GTP hydrolysis and GTP loading of Rad whereas Rad regulates activity of nm23. This interaction may play important roles in the effects of Rad on glucose metabolism and the effects of nm23 on tumor metastasis and developmental regulation.

Increase of adenylate kinase isozyme 1 protein during neuronal differentiation in mouse embryonal carcinoma P19 cells and in rat brain primary cultured cells.

Adenylate kinase (AK), which catalyzes the equilibrium reaction among AMP, ADP, and ATP, is considered to participate in the homeostasis of energy metabolism in cells. Among three vertebrate isozymes, AK isozyme 1 (AK1) is present prominently in the cytosol of skeletal muscle and brain. When mouse embryonal carcinoma P19 cells were differentiated by retinoic acid into neural cells, the amount of AK1 protein and enzyme activity increased about fivefold concomitantly with neurofilament (NF). Double-immunofluorescence staining showed that both AK1 and NF were located in neuronal processes as well as the perinuclear regions in neuron-like cells, but not in glia-like cells. The amount of brain-type creatine kinase increased only twofold during P19 differentiation. The AK isozyme 2, which was not detected in adult mouse brain, was found in P19 cells and did not increase during the differentiation. Mitochondrial AK isozyme 3, which uses GTP instead of ATP as a phosphate donor, was increased significantly. Immunohistochemical analysis with the primary cultured cells from rat cerebral cortex showed similar cellular localization of AK1 to those observed with differentiated P19 cells. These results suggest an important role of this enzyme in neuronal functions and in neuronal differentiation.

Activated protein kinase C α associates with annexin VI from skeletal muscle

We have previously detected a number of protein kinase C (PKC) alpha-binding proteins in skeletal muscle cytosol by blot overlay assay, and now identify the major, 69 kDa binding protein as annexin VI by immunoblotting and overlay assay of hydroxyapatite chromatography fractions. Annexin VI was also detected in immunoprecipitates of PKC alpha. Annexin VI and PKC alpha are both calcium-dependent phospholipid-binding proteins, and detection of the interaction was dependent on the presence of calcium and phosphatidylserine (PS). The association probably involves specific protein-protein interactions rather than mere bridging by lipid molecules: firstly, detection of PKC alpha-annexin VI complexes by overlay assay was not diminished when PS concentrations were increased over a 10-fold range, while that of other PKC alpha-binding protein complexes was reduced or abolished; secondly, the presence in the overlay assay of a PKC pseudosubstrate peptide, analogous to a PKC sequence previously found to be involved in PKC binding activity, reduced complex formation; thirdly, we were also able to detect annexin VI interaction with PKC beta by overlay of skeletal muscle cytosol, but not with PKC theta, the major novel PKC in this tissue, suggesting sequences specific to calcium-dependent PKC isoenzymes are involved. While other annexin isoforms may be PKC substrates or inhibitors, annexin VI phosphorylation by PKC alpha could not be detected after co-purification, while phosphorylation of subsequently-added histone IIIS was readily observed. Annexin VI is a major skeletal muscle protein and our data are consistent with a role for this isoform in the control of calcium-dependent PKC.

Fatty acyl-CoA-acyl-CoA-binding protein complexes activate the Ca2+ release channel of skeletal muscle sarcoplasmic reticulum.

We previously reported that fatty acyl-CoA esters activate ryanodine receptor/Ca2+ release channels in a terminal cisternae fraction from rabbit skeletal muscle [Fulceri, Nori, Gamberucci, Volpe, Giunti and Benedetti (1994) Cell Calcium 15, 109-116]. Skeletal muscle cytosol contains a high-affinity fatty acyl-CoA-binding protein (ACBP) [Knudsen, Hojrup, Hansen, H.O., Hansen, H.F. and Roepstorff (1989) Biochem. J. 262, 513-519]. We show here that palmitoyl-CoA (PCoA) in a complex with a molar excess of bovine ACBP causes a discrete Ca2+ efflux or allows Ca2+ release from the Ca2+-preloaded terminal cisternae fraction by sub-optimal caffeine concentrations. Both effects were abolished by elevating the free [Mg2+] in the system, which inhibits the Ca2+ release channel activity. Sensitization towards caffeine was a function of both the concentration of the complex and the [PCoA]-to-[ACBP] ratio. In all experimental conditions the calculated free [PCoA] was no more than 50 nM, and such concentrations by themselves were inactive on Ca2+ release channels. The KD for PCoA binding was approx. 2 nM for bovine and yeast ACBP, and slightly higher (8 nM) for rat ACBP. The PCoA-rat ACBP complex behaved in the same manner as the PCoA-bovine ACBP complex, whereas the ester complexed with yeast ACBP was more active in activating/sensitizing Ca2+ efflux. A non-hydrolysable analogue of PCoA bound to (bovine) ACBP also sensitized the Ca2+ release channel towards caffeine. These findings indicate that fatty acyl-CoA-ACBP complexes either interact directly with one or more components in the terminal cisternae membranes or, through interaction with the component(s), donate the fatty acyl-CoA esters to high-affinity binding sites of the membrane, thus affecting (and possibly regulating) Ca2+ release channel activity.

Characterization of two forms of protein kinase C alpha, with different substrate specificities, from skeletal muscle.

We have investigated protein kinase C (PKC) in skeletal muscle cytosol and demonstrated the presence of two major activities. These did not correspond to different PKC isoenzymes but seemed to represent two species of PKC alpha as deduced by: elution during hydroxyapatite chromatography at KH2PO4 concentrations expected of PKC alpha; detection of the two species by three specific but unrelated anti-(PKC alpha) antibodies; immunodepletion of both activities with anti-(PKC alpha) antibody; and demonstration of identical requirements of both Ca2+ ions and lipid for activation. These species, termed PKC alpha 1 and PKC alpha 2, phosphorylated the modified conventional PKC pseudosubstrate peptide (19-31, Ser-25) equally well. Importantly, however, the activities differed in that PKC alpha 1 phosphorylated histone IIIS, and also peptides derived from the EGF receptor and glycogen synthase, to a much greater extent than did PKC alpha 2. Similarly, incubation of crude muscle extracts with either PKC alpha 1 or alpha 2 gave rise to different protein phosphorylation patterns. The involvement of proteolysis, dephosphorylation or oxidative modification in the interconversion of PKC alpha 1 and PKC alpha 2 during preparation was ruled out. Although some PKC-binding proteins were detected in overlay assays, their presence did not explain the anomalous PKC alpha 2 activity. The results suggest that a modification of PKC alpha in situ limits its substrate specificity, and indicate an additional level of control of the kinase that may be a site for modulation of PKC-mediated signal transduction.

Selective binding of FKBP12.6 by the cardiac ryanodine receptor.

The calcium release channels (CRC)/ryanodine receptors of skeletal (Sk) and cardiac (C) muscle sarcoplasmic reticulum (SR) are hetero-oligomeric complexes with the structural formulas (ryanodine recepter (RyR)1 protomer)4(FKBP12)4 and (RyR2 protomer)4(FKBP12.6)4, respectively, where FKBP12 and FKBP12.6 are isoforms of the 12-kDa receptor for the immunosuppressant drug FK506. The sequence similarity between the RyR protomers and FKBP12 isoforms is 63 and 85%, respectively. Using 35S-labeled FKBP12 and 35S-labeled FKBP12.6 as probes to study the interaction with CRC, we find that: 1) analogous to its action in skeletal muscle sarcoplasmic reticulum (SkMSR), FK506 (or analog FK590) dissociates FKBP12.6 from CSR; 2) both FKBP isoforms bind to FKBP-stripped SkMSR and exchange with endogenously bound FKBP12 of SkMSR; and 3) by contrast, only FKBP12. 6 exchanges with endogenously bound FKBP12.6 or rebinds to FKBP-stripped CSR. This selective binding appears to explain why the cardiac CRC is isolated as a complex with FKBP12.6, whereas the skeletal muscle CRC is isolated as a complex with FKBP12, although only FKBP12 is detectable in the myoplasm of both muscle types. Also, in contrast to the activation of the channel by removal of FKBP from skeletal muscle, no activation is detected in CRC activity in FKBP-stripped CSR. This differential action of FKBP may reflect a fundamental difference in the modulation of excitation-contraction coupling in heart versus skeletal muscle.

Structural organisation and lipid composition of the epicuticular accessory layer of infective larvae of Trichinella spiralis

The epicuticle of infective larvae of Trichinella spiralis represents the interface between this intracellular nematode parasite and the cytosol of mammalian skeletal muscle. The macromolecular structures that make up the epicuticle were studied by freeze-fracture electron microscopy and compositional analysis. Three fracture planes were observed: one with a typical plasma membrane-type bilayer organisation which was overlaid by two extended layers of lipid in an inverted cylindrical configuration. This overall structure remained unchanged in response to variations in temperature between 20°C and 45°C. The lipid cylinders were on average 6.8 nm in diameter, with randomly-associated particles that were not dissociated by high-salt treatment, indicative of hydrophobically associated proteins. The majority of the lipids were non-polar, consisting of cholesterol, cholesterol esters, mono- and tri-glycerides, and free fatty acids. Three major classes of phospholipids were identified: phosphatidylethanolamine, phosphatidylglycerol and phosphatidylcholine. Total lipid extracts did not adopt an inverted cylindrical or micellar configuration on isolation, but formed flat sheets of lamellae as did the purified polar and non-polar fractions of the lipids. Isolated lipids did not undergo thermally-induced polymorphism between 20°C and 60°C and there was no pH dependency of the structures adopted. The fatty acid saturation levels of the phospholipids were compatible with the observation that they did not form polymorphic structures on isolation. We suggest that this unusual configuration is probably stabilised by the associated (glyco)proteins and may be required for selective permeation of nutrients from the host cell cytosol and/or for maintaining the high curvature of the parasite within the cell.

Identification and partial characterization of a latent ATP, Mg-dependent protein phosphatase in rabbit skeletal muscle cytosol

Fractionation of rabbit skeletal muscle cytosol on Aminohexyl-Sepharose has resulted in the identification of a latent ATP, Mg-dependent protein phosphatase whose catalytic subunit is in the active conformation, but is inhibited by the presence of more than one modulator unit. The partially purified enzyme is converted to an inactive, kinase FA-dependent form upon incubation at 30 degrees C unless modulator-specific polyclonal antibodies are added to the preparation. The immunoglobulins also relieve the inhibition which is responsible for the low basal phosphatase activity of the enzyme, and they counteract all of the heat-stable inhibitor activity present in the preparation. Addition of free catalytic subunit abolishes the inhibition of the latent enzyme in a dose-dependent way, but cannot prevent the inactivation process. The inactivated phosphatase and the original latent enzyme exhibit the same apparent Mr in sucrose density-gradient centrifugation (70,000) and in gel filtration (110,000).

On the composition of the cytosol of relaxed skeletal muscle of the frog.

This review summarizes a variety of estimates for the concentrations of the principal cytosolic constituents in frog skeletal muscle. From these estimates (listed in the APPENDIX), we chose representative values and used electroneutrality and osmotic considerations to ensure that all major constituents have been considered. Given total cytosolic concentrations of these constituents from the literature, we employed a computer program to calculate the concentrations of all the major ionic species in the cytosol. In relaxed muscle, electroneutrality and osmotic constraints are fulfilled if, in addition to diffusible species, the charge contribution of the myofilaments is considered. Mean buffer power of the diffusible cytosolic species is calculated to be less than one-third of that experimentally determined for whole muscle. Computations indicate that recent estimates of intracellular free magnesium concentration approximately 1 mM are likely to be correct.

Identification of the multifunctional calmodulin-dependent protein kinase in the cytosol, sarcoplasmic reticulum, and sarcolemma of rabbit skeletal muscle

A multifunctional calmodulin-dependent protein kinase (calmodulin kinase) was purified from the cytosol of rabbit skeletal muscle as a subunit of 58 kDa. A 58-kDa protein in sarcoplasmic reticulum (SR) and sarcolemma (SL) of rabbit skeletal muscle was endogenously phosphorylated in a calmodulin-dependent manner. The 58-kDa protein in SR and SL was considered to be identical to the subunit of cytosol calmodulin kinase on the basis of immunoreactivity, calmodulin binding, and autophosphorylation studies and on the patterns of protease-treated phosphopeptides. Calmodulin kinase showed broad substrate specificity and phosphorylated troponins I and T.

Cytosolic glucocorticoid receptors in skeletal muscle of the newborn calf.

A study was conducted to provide information on the characteristics of skeletal muscle glucocorticoid receptors and to determine the response of serum cortisol and thyroxine secretion to colostrum feeding in the neonatal calf. Twenty-four calves (12 males and 12 females) were obtained immediately postpartum and randomly assigned to one of two treatments after being blocked by breed and sex. The treatments consisted of: 1) pooled colostrum and 2) pooled whole milk, with both treatments being force-fed at birth, 12, 24 and 36 h postpartum. Muscle biopsies (20-30 g) were surgically removed from the right semitendinosus at 36 h postpartum from 14 of the 24 calves (7 male and 7 female). There were no glucocorticoid receptor differences in muscle samples from either the colostrum or milk-fed calves. Binding of dexamethasone to skeletal muscle cytosol indicated a moderate to high affinity (Kd = 2.34 X 10(-8)M) and a low capacity (37.61 fmol/mg cytosolic protein) binding receptor. Competition assays indicated that other non-glucocorticoid hormones had relatively high affinity for the glucocorticoid receptor. The average initial cortisol concentration was highest at birth, 127.9 (SE = 25) and 172.4 (SE = 29) ng/ml for colostrum and milk-fed calves, respectively. Following the initial peak at birth, serum cortisol concentrations declined with time (P less than .05) for both treatments. Serum thyroxine concentrations increased (P less than .05) to reach a peak of 23.3 (SE = 3.1) and 21.0 (SE = 2.9) ug/dl for colostrum and milk-fed calves, respectively, then steadily declined.

Androgen receptors in skeletal muscle cytosol from sheep treated with trenbolone acetate.

Female sheep were implanted with the anabolic agent trenbolone acetate and the effects of the treatment on the skeletal muscle androgen receptor studied. In two separate trials the binding capacity/mg skeletal muscle protein was reduced. In trial 1 the animals were implanted (80 mg trenbolone acetate) at the start and after 5 weeks of the trial and samples taken one week later while in the second trial samples were taken 15 days after a single implant (80 mg trenbolone acetate). The ability of a variety of steroids and steroid-like compounds to displace [3H] testosterone and [3H] methyltrienolone was also studied. The androgen receptor was also partially purified using heparin-sepharose chromatography The involvement of the anddrogen receptor in the mode of action of trenbolone is discussed.

Effects of ACTH on the ligand-binding properties of the glucocorticoid receptor exerted not only via elevated levels of corticosteroids

Treatment of mice with ACTH not only increased plasma corticosterone levels, but also reduced the ligand-binding capacity of the glucocorticoid receptor in skeletal muscle cytosol. Non-linear Scatchard plots were obtained for the glucocorticoid receptor following ACTH treatment. This upward concave curvilinearity could not be reproduced by simply adding to the cytosol an amount of corticosterone corresponding to the increase, even though this treatment resulted in reduced binding capacity. The addition of corticosterone resulted in a slower formation of ligand-receptor complexes (lower association rate constant), which offered a partial explanation for the low binding. In addition, ACTH treatment was also found to reduce the binding capacity in adrenalectomized mice that did not respond with corticosterone elevation. However, in this case Scatchard plots were linear.

Binding of glucocorticoid antagonists to androgen and glucocorticoid hormone receptors in rat skeletal muscle

The binding of ten steroids possessing antiglucocorticoid activity has been studied in rat skeletal muscle cytosol. The affinity of these steroids for both the androgen and the glucocorticoid receptors was determined by competition with radioactive R 1881 (methyltrienolone, metribolone) and dexamethasone, respectively. The antiglucocorticoid activity of these compounds was assessed in rat hepatoma (HTC) cells by nuasuring their inhibitory effect on the glucocorticoid-induced tyrosine aminotransferase activity. This led to identification of five novel in vitro glucocorticoid antagonists. All the steroids tested bound to both the glucocorticoid and the androgen receptors in muscle. Four steroids had an affinity for the glucocorticoid receptor higher than for the androgen receptor. The assumption is made that the steroids tested also behave as antagonists when binding to the glucocorticoid receptor in muscle and behave as agonists when binding to the androgen receptor. On this basis, the data allow one to compute a potential anticatabolic (PAG) and a potential anabolic (PAA) index for each compound. These indices might be of predictive value to determine whether these steroids exert their anabolic action in muscle through the glucocorticoid receptor or through the androgen receptor. The data also make it unlikely that satellite cells are a preferential target for anabolic steroids in muscle.