Metabolic regulation by alpha 1- and alpha 2-adrenoceptors.

The role of alpha-adrenoceptors in the mediation of autonomic function, particularly in the control of the cardiovascular system, is widely known. However, alpha-adrenoceptors are also important in the regulation of a variety of metabolic processes that occur in the body either through direct action or by stimulation of the release of other mediators that control metabolic function. Thus, alpha 2-adrenoceptor activation by circulating or neuronally released catecholamines inhibits the release of insulin from pancreatic islet beta-cells and, by inhibiting this response, alpha 2-adrenoceptor antagonists have been shown to have an antihyperglycemic effect. The alpha-adrenoceptor-mediated regulation of the release of pituitary hormones is indirect, with alpha-adrenoceptors being located on peptidergic neurons in the hypothalamus that secrete releasing hormones into the hypophysial portal system to regulate the secretion of hormones from the anterior pituitary gland. Thus, the increase in cortisol secretion from the adrenal glands following a meal is produced, at least in part, by an alpha 1-adrenoceptor-mediated increase in vasopressin and CRF-41 secretion from neurons on the hypothalamus that stimulate the release of adrenocorticotrophic hormone secretion from the pituitary gland, which subsequently stimulates the synthesis and release of cortisol from the adrenal medulla. In addition to metabolic regulation by alpha 1- and alpha 2-adrenoceptors within the endocrine system, alpha-adrenoceptors are also a component of the system that regulates certain aspects of metabolism within autonomic effector cells, such as the control of smooth muscle cell division and growth during periods of continued alpha-adrenoceptor activation as a result of activation of second messenger systems.     

Inhibition of cell surface receptor-bound plasmin by alpha 2-antiplasmin and alpha 2-macroglobulin.

The purpose of this investigation was to characterize the reaction of alpha 2-antiplasmin (alpha 2AP) and alpha 2-macroglobulin (alpha 2M) with human plasmin bound to rat C6 glioma cells and human umbilical vein endothelial cells (HUVECs). Binding of plasmin (0.1 microM) to C6 cells at 4 degrees C did not cause cell detachment, decrease viability or change cell morphology. The KD and Bmax for the binding of diisopropyl phosphoryl plasmin (DIP-plasmin) to C6 cells were 0.9 microM and 2.6 x 10(6) sites/cell. The dissociation rate constants (koff) for 125I-plasmin were 9.7 x 10(-4) and 4.0 x 10(-4) s-1 at 4 degrees C in the presence and absence of 0.3 microM DIP-plasmin, respectively. Similar constants were determined for 125I-plasminogen and 125I-DIP-plasmin. Neither alpha 2AP nor alpha 2M affected the dissociation of DIP-plasmin. C6 cell-associated 125I-plasmin reacted slowly with alpha 2AP; however, the inhibition rate constants exceeded the koff. alpha 2AP-plasmin complex formed after the plasmin dissociated into solution (reaction pathway 1) and by direct reaction of alpha 2AP with cell-associated enzyme (reaction pathway 2). High concentrations of alpha 2AP favored pathway 2. C6 cell-associated plasmin was also protected from inhibition by alpha 2M. While the same pathways were probably involved in this reaction, alpha 2M was less effective than alpha 2AP as an inhibitor of nondissociated plasmin (pathway 2). When C6 cell-bound plasmin reacted with alpha 2AP, alpha 2AP-plasmin complex was recovered primarily in the medium, suggesting dissociation of complexes formed on the cell surface. Plasmin-receptor dissociation and inhibition experiments were performed at 22 degrees and 37 degrees C, confirming the conclusions of the 4 degrees C studies. Comparable results were also obtained using HUVEC cultures. These studies demonstrate that cell-associated plasmin is protected from inhibition by alpha 2M as well as alpha 2AP. At least two reaction pathways may be demonstrated for the inhibition of plasmin that is initially receptor-bound; however, neither pathway is highly effective, accounting for the "plasmin-protective" activity of the cell surface.     

Gene transfer mediated by alpha2-macroglobulin.

alpha2-Macroglobulin covalently linked to poly(L)-lysine can be used as a vehicle for receptor-mediated gene transfer. This modified alpha2-macroglobulin maintains its ability to bind to the alpha2-macroglobulin receptor, and was shown to introduce a luciferase reporter gene plasmid into HepG2 human hepatoma cells in vitro. The alpha2-macroglobulin receptor is a very large and multifunctional cell surface receptor, whose rapid and efficient internalization rate makes it attractive for gene therapy, e.g. for hepatic gene targeting via injection into the portal vein.     

Inhibition of aspartic proteinases by alpha 2-macroglobulin.

The effect of alpha 2-macroglobulin, one of the major antiproteinases in the plasma of vertebrates, on the action of the aspartic proteinases chymosin, cathepsin D and cathepsin E towards peptide and protein substrates at pH 6.2 was examined. Activities towards protein substrates were blocked, thus demonstrating that alpha 2-macroglobulin can inhibit aspartic proteinases, in addition to serine proteinases, cysteine proteinases and metalloproteinases.     

Prothymosin alpha is phosphorylated by casein kinase-2.

Prothymosin alpha (ProT alpha) is a 12.5 kDa acidic polypeptide that is considered to have a nuclear function related to cell proliferation. Inspection of its amino acid sequence revealed the presence of sequences that may serve as targets for phosphorylation by casein kinase-2 (CK-2). ProT alpha isolated from calf thymocytes was phosphorylated in vitro by CK-2. The phosphorylation sites are Ser and Thr residues located among the first 14 amino acid residues in the ProT alpha sequence. Another site that is theoretically suitable for phosphorylation by CK-2, at the C-terminus of the polypeptide, is not, in fact, phosphorylated. Thymosin alpha 1 (T alpha 1), a peptide whose sequence corresponds to the first 28 amino acids of ProT alpha, is also phosphorylated by CK-2 at the same phosphorylation sites as ProT alpha. In cultured splenic lymphocytes ProT alpha was phosphorylated at Thr residues located at positions 7, 12 and/or 13. Based on these observations we conclude that CK-2, or another cellular kinase with similar sequence specificity, is responsible for phosphorylation of ProT alpha in vivo.     

Reversal of mu-opioid-mediated respiratory depression by alpha 2-adrenoceptor antagonism.

The present study was performed in order to evaluate the effects of the selective alpha 2-adrenoceptor antagonist 6-chloro-2,3,4,5-tetrahydro-3-methyl-1H-3-benzazepine (SK&F 86466) on dermorphin-induced analgesia, respiratory depression and inhibition of locomotor activity in the conscious rat. Intracerebroventricular (icv) administration of dermorphin (3 nmol/rat) decreased respiration rate and relative ventilatory minute volume maximally by 38% and 50% of baseline respectively. SK&F 86466 dose-dependently reversed the dermorphin-induced depression of ventilatory parameters, while SK&F 86466 exerted no effect on dermorphin-induced analgesia or depression of locomotor activity due to catalepsia. It appears, therefore, that alpha 2-adrenoceptors selectively interact with mu 2-opioid-receptor mediated effects, such as respiratory depression, but are not involved in the modulation of mu 1-opioid-related effects, such as supraspinal analgesia and depression of locomotor activity.     

Differential scanning calorimetry of alpha 2-macroglobulin and alpha 2-macroglobulin-proteinase complexes.

Differential scanning calorimetry is shown to detect substantial structural alterations occurring on the association of proteinases with the serum glycoprotein alpha 2-macroglobulin. At pH 7.5, the thermally induced unfolding of the macroglobulin occurs at approx. 60 degrees C with a transition enthalpy of 17 J/g. Association of active thermolysin, trypsin and papain shifts the transition temperature to 77 degrees C (transition enthalpy 5 J/g), indicating that a substantial conformational change accompanies the binding event. The stoicheiometry of the thermolysin--alpha 2-macroglobulin association producing this change appears to be unity, implying the presence of co-operative subunit interactions in the mechanism of association. The calorimetric method provides a novel approach for the evaluation of conformational variants induced on protein-protein association or pre-existing in the purified macroglobulin.     

Platelet alpha2-macroglobulin and alpha1-antitrypsin.

Subcellular membrane and granule fractions derived from human platelets contain immunologically identifiable alpha2-macroglobulin and alpha1-antitrypsin. These platelet-derived inhibitors show a reaction of immunologic identity when compared to alpha2-macroglobulin and alpha1-antitrypsin purified from human plasma. Further, the platelet protease inhibitors possessed a similar subunit polypeptide chain structure to their plasma counterparts as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis. Studies of the binding of radiolabeled trypsin to the various solubilized platelet subcellular fractions suggest that the granule-associated alpha2-macroglobulin and alpha1-antitrypsin, as well as membrane-associated alpha2-macroglobulin were functionally active. Quantitatively, circulating platelets contain relatively small concentrations of these inhibitors as compared to platelet-associated fibrinogen and factor VIIIAGN. Platelet protease inhibitors may modulate the protease-mediated events involved in the formation of hemostatic plugs and thrombi.     

Immunoaffinity purification of GABAA receptor alpha-subunit iso-oligomers. Demonstration of receptor populations containing alpha 1 alpha 2, alpha 1 alpha 3, and alpha 2 alpha 3 subunit pairs.

Novel methods for the isolation of gamma-aminobutyric acidA (GABAA) receptor alpha subunit iso-oligomers have been developed. Thus, populations of GABAA receptors containing the GABAA receptor alpha 1 subunit, the alpha 2 subunit, and the alpha 3 subunit have been purified from sodium deoxycholate extracts of bovine cerebral cortex with the retention of specific [3H]flunitrazepam-binding activity by anti-alpha 1 324-341, anti-Cys alpha 2 414-424, or anti-Cys alpha 3 454-467 antibody affinity chromatography, respectively. The relative abundance of the different specificity alpha subunits in these preparations was compared with benzodiazepine affinity chromatography-purified GABAA receptors by immunoblotting. In each case, it was found that although the immunoreactivity with the specific alpha subunit antibody that was used for purification was enriched in immunoaffinity-purified receptors, reactivity with the other alpha subunit specificity antibodies, together with anti-gamma 2 1-14 Cys immunoreactivity was found. Immunoprecipitation of GABAA receptors purified by anti-alpha 1 324-341 antibody affinity chromatography by all three anti-alpha subunit antibodies employed, together with the use of anti-alpha 1 324-341 and anti-Cys alpha 2 414-424 antibody affinity columns in series, further substantiated the partial co-purification of the different polypeptides. These results demonstrate the copurification of the gamma 2 subunit with each population of alpha 1, alpha 2, alpha 3 subunit-enriched GABAA receptors. They also show the existence of minor populations of GABAA receptors that contain alpha 1 alpha 2, alpha 1 alpha 3, and alpha 2 alpha 3 subunit pairs within single oligomers.     

Disappearance of prostaglandins F2alpha and 15-methyl F2alpha from amniotic fluid as measured by radiommunoassay.

A sensitive and relatively specific radioimmunoassay for 15 (S) 15 methyl prostaglandin F2alpha was used to determine the levels of the drug in amniotic fluid after it had been injected intra-amniotically for termination of second trimester pregnancy. The disappearance of the free acid (tham salt) and methyl ester of the prostaglandin analogue were similar. The results of this preliminary study suggest that the drug rapidly equilibrates in the fluid and this followed by a slow removal from the amniotic sac. A comparison with a similar study with PGF2Alpha revealed that the analogue had a longer half-life in the amniotic fluid.     

Inactivation of alpha- and beta-thrombin by antithrombin-III, alpha 2-macroglobulin and alpha 1-proteinase inhibitor.

Inactivation of alpha- and beta-thrombin by alpha 2-macroglobulin, by alpha 1-proteinase inhibitor and by antithrombin-III and heparin was studied. The amount of alpha- and beta-thrombin inactivated by antithrombin-III was proportional to the concentration of the inhibitor, but the inactivation rates of the two forms of thrombin were different. Heparin facilitated complex-formation between alpha-thrombin and antithrombin-III, whereas inactivation of beta-thrombin by antithrombin was only slightly influenced, even at a heparin concentration two orders of magnitude higher. alpha 2-Macroglobulin inhibited both alpha- and beta-thrombin activity similarly, i.e. the amount of alpha- and beta-thrombin inactivated as well as the rates of their inhibition were the same. alpha 1-Proteinase inhibitor also formed a complex with alpha- and beta-thrombin, similarly to antithrombin-III, although the inactivation of the enzyme needed high inhibitor concentration and long incubation time. These results suggest that the inactivation of beta-thrombin, if it occurs in the plasma, is also controlled by plasma inhibitors.     

Identification of alpha 2-macroglobulin conformational intermediates by electron microscopy and image analysis. Comparison of alpha 2-macroglobulin-thrombin and alpha 2-macroglobulin reacted with cis-dichlorodiammineplatinum(II) and trypsin.

Human alpha 2-macroglobulin (alpha 2M) exists in two well defined, highly distinct conformations and in less well described intermediate conformations. In this study, previously characterized reactions were used to partially or completely transform the conformation of alpha 2M. Electron micrographs of each preparation were subjected to image analysis. Ternary alpha 2M-trypsin (2 mol of trypsin/mol of alpha 2M) was analyzed as a control for the fully transformed state. Correspondence analysis (CORAN) and hierarchical ascendant classification (HAC) generated five image clusters from 330 aligned alpha 2M-trypsin complexes. Average images of each cluster resembled the letter "H" with four nearly equivalent lateral arms. Abnormally shaped lateral arms were not demonstrated by HAC, using a variety of factor sets. In a native polyacrylamide gel electrophoresis system, alpha 2M-thrombin migrated in a diffuse band partially behind alpha 2M-trypsin, suggesting conformational heterogeneity. CORAN and HAC of 733 alpha 2M-thrombin complexes identified two neighboring clusters, the average images of which showed an H-like structure in which one arm was replaced by a globular stain-excluding body. The two alpha 2M-thrombin clusters included 125 images (17.1% of image population). The complete absence of atypical lateral arm structure in the alpha 2M-trypsin clusters suggests that this variation is not the result of orientation or staining artifact. Native alpha 2M was reacted with cis-dichlorodiammineplatinum(II) and then with trypsin to form alpha 2M-Pt-trypsin, a preparation that includes partially transformed alpha 2M structures. CORAN and HAC of 580 alpha 2M-Pt-trypsin complexes generated five clusters, the average images of which showed atypical lateral arm structure equivalent to that demonstrated with alpha 2M-thrombin. The five alpha 2M-Pt-trypsin clusters accounted for 15.2% of the image population. These studies suggest that alpha 2M conformational change intermediates demonstrate common structural characteristics, permitting an elucidation of the steps involved in this complex transformation.     

Mechanism of hypochlorite-mediated inactivation of proteinase inhibition by alpha 2-macroglobulin.

The proteinase-proteinase inhibitor balance plays an important role in mediating inflammation-associated tissue destruction. alpha 2-Macroglobulin (alpha 2M) is a high-affinity, broad-spectrum proteinase inhibitor found abundantly in plasma and interstitial fluids. Increased levels of alpha 2M and proteinase-alpha 2M complexes can be demonstrated in patients with sepsis, emphysema, peridontitis, rheumatoid arthritis, and other inflammatory diseases. Despite these increased levels, proteolysis remains a significant problem. We hypothesized that a mechanism for inactivating alpha 2M-mediated proteinase inhibition must exist and recently demonstrated that alpha 2M isolated from human rheumatoid arthritis synovial fluid is oxidized and has decreased functional activity. The oxidant responsible for alpha 2M inactivation and the mechanism of such destruction were not studied. We now report that while hypochlorite and hydroxyl radical both modify amino acid residues on alpha 2M, only hypochlorite can abolish the ability of alpha 2M to inhibit proteinases. Hydrogen peroxide, on the other hand, has no effect on alpha 2M structure or function. Protein unfolding with increased susceptibility to proteolytic cleavage appears to be involved in alpha 2M inactivation by oxidation. The in vivo relevance of this mechanism is supported by the presence of multiple cleavage fragments of alpha 2M in synovial fluid from patients with rheumatoid arthritis, where significant tissue destruction occurs, but not in patients with osteoarthritis. These results provide strong evidence that hypochlorite oxidation contributes to enhanced tissue destruction during inflammation by inactivating alpha 2M.     

Metabolism of prostaglandins E2 and F2 alpha by human fetal membranes.

Prostaglandin E2 (PGE2) is important in the early stages of human labour, leading particularly to cervical ripening and dilatation. The source of PGE2 is thought to be either the amnion or the decidua, but the chorion interposes between the amnion and the target tissues, namely the myometrium and cervix. In order to investigate the role of the chorion in modulating prostanoid production, [3H]PGE2 was added to the amnion side of fetal membranes, and the production of metabolites on both sides of the fetal membrane followed by HPLC. The major metabolite was 13,14-dihydro-15-oxo-PGE2 with smaller amounts of 13,14-dihydro-15-oxo-PGA2 and PGB2. The production of all metabolites of PGE2 was time dependent. [3H]PGF2 alpha, which is normally produced by the decidua, was also added to fetal membranes and found to be metabolised to 13,14-dihydro-15-oxo-PGF2 alpha and PGE2. These results suggest that the metabolic enzymes in the chorion may determine intra-uterine levels of prostaglandins, and may also determine the identity of the eicosanoids released by intact fetal membranes.     

Subtypes of alpha 1- and alpha 2-adrenergic receptors.

The adrenergic receptors are members of the superfamily of G protein-coupled receptors. There are three major types of adrenergic receptors: alpha 1, alpha 2, and beta. Each of these three major types can be divided into three subtypes. Within the alpha 1-adrenergic receptors, alpha 1A and alpha 1B subtypes have been defined pharmacologically on the basis of reversible antagonists, such as WB4101 and phentolamine, and the irreversible antagonist chloroethylclonidine. In at least some tissues the mechanism of action of the alpha 1A subtype is related to activation of a calcium channel, whereas the alpha 1B receptor exerts its effect through the second messenger inositol trisphosphate. Both of these receptor subtypes as well as a third, the alpha 1C, have been identified by molecular cloning. Three pharmacological subtypes of the alpha 2-adrenergic receptor have also been identified. Prototypic tissues and cell lines in continuous culture have been developed for each of these subtypes, which facilitated their study. The definition of the alpha 2 subtypes has been based on radioligand binding data and more limited functional data. All three subtypes have been shown to inhibit the activation of adenylate cyclase and thus reduce the levels of cAMP. Three alpha 2-adrenergic receptor subtypes have been identified by molecular cloning in both the human and rat species. There is reasonable agreement between the pharmacological identified subtypes and those identified by molecular cloning.     

Subtype-selective desensitization of alpha 2-adrenergic receptors. Different mechanisms control short and long term agonist-promoted desensitization of alpha 2C10, alpha 2C4, and alpha 2C2.

We have recently shown that the alpha 2C10 adrenergic receptor (AR) undergoes short term agonist-promoted desensitization, mediated by phosphorylation of sites in the third intracellular loop. There is significant divergence in the third loop amino acid sequences between alpha 2C10 and the other subtypes, alpha 2C4 and alpha 2C2. We therefore explored the mechanisms of alpha 2AR subtype desensitization by expressing each human subtype in Chinese hamster ovary cells and subjecting them to short and long term epinephrine exposures. After 30 min of agonist exposure, alpha 2C10 and alpha 2C2 displayed desensitization characterized by rightward shifts in the curves for epinephrine-mediated inhibition of adenylyl cyclase (EC50 = alpha 2C10, 0.24 +/- 0.02 microM increasing to 1.1 +/- 0.1 microM; alpha 2C2, 1.3 +/- 0.3 increasing to 2.6 +/- 0.3 microM). Coincident with alpha 2C10 and alpha 2C2 desensitizations were decreases in agonist high affinity binding. In contrast, alpha 2C4 underwent no functional desensitization after short term agonist exposure, nor were there any changes in agonist high affinity binding. Agonist-promoted receptor sequestration was clearly greater with alpha 2C10 (approximately 26%) and alpha 2C2 (approximately 35%) as compared to alpha 2C4 (approximately 12%), but such sequestration did not play a significant role in short term desensitization, as blockade with concanavalin A had no effect on desensitization patterns. In contrast to these findings, all alpha 2AR subtypes underwent desensitization after prolonged (24 h) agonist exposure. However, alpha 2C10 and alpha 2C2 displayed substantially more desensitization (approximately 20-fold increase in EC50) as compared to alpha 2C4 (approximately 5-fold increase). The primary mechanism of desensitization during long term agonist exposure was found to be a decrease in the amount of cellular Gi, which was equivalent in magnitude in cells expressing all three subtypes. However, in addition to a decrease in Gi, alpha 2C10 and alpha 2C2 underwent down-regulation of receptor levels during long term agonist exposure, while alpha 2C4 did not. Given that all three subtypes bind endogenous catecholamines with high affinity and inhibit adenylyl cyclase efficiently, the significance of multiple subtypes has heretofore been obscure. Our results show that alpha 2AR undergo subtype-selective desensitization to agonists and suggest that alpha 2AR subtypes may have evolved to meet differing needs for adaptive regulation.     

Expression of mutant eukaryotic initiation factor 2 alpha subunit (eIF-2 alpha) reduces inhibition of guanine nucleotide exchange activity of eIF-2B mediated by eIF-2 alpha phosphorylation.

The inhibition of protein synthesis that occurs upon phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF-2 alpha) at serine 51 correlates with reduced guanine nucleotide exchange activity of eIF-2B in vivo and inhibition of eIF-2B activity in vitro, although it is not known if phosphorylation is the cause of the reduced eIF-2B activity in vivo. To characterize the importance of eIF-2 alpha phosphorylation in the regulation of eIF-2B activity, we studied the overexpression of mutant eIF-2 alpha subunits in which serine 48 or 51 was replaced by an alanine (48A or 51A mutant). Previous studies demonstrated that the 51A mutant was resistant to phosphorylation, whereas the 48A mutant was a substrate for phosphorylation. Additionally, expression of either mutant partially protected Chinese hamster ovary (CHO) cells from the inhibition of protein synthesis in response to heat shock treatment (P. Murtha-Riel, M. V. Davies, J. B. Scherer, S. Y. Choi, J. W. B. Hershey, and R. J. Kaufman, J. Biol. Chem. 268:12946-12951, 1993). In this study, we show that eIF-2B activity was inhibited in parental CHO cell extracts upon addition of purified reticulocyte heme-regulated inhibitor (HRI), an eIF-2 alpha kinase that phosphorylates Ser-51. Preincubation with purified HRI also reduced the eIF-2B activity in extracts from cells overexpressing wild-type eIF-2 alpha. In contrast, the eIF-2B activity was not readily inhibited in extracts from cells overexpressing either the eIF-2 alpha 48A or 51A mutant. In addition, eIF-2B activity was decreased in extracts prepared from heat-shocked cells overexpressing wild-type eIF-2 alpha, whereas the decrease in eIF-2B activity was less in heat-shocked cells overexpressing either mutant 48A or mutant 51A. While the phosphorylation at serine 51 in eIF-2 alpha impairs the eIF-2B activity, we propose that serine 48 acts to maintain a high affinity between phosphorylated eIF-2 alpha and eIF-2B, thereby inactivating eIF-2B activity. These findings support the hypothesis that phosphorylation of eIF-2 alpha inhibits protein synthesis directly through reducing eIF-2B activity and emphasize the importance of both serine 48 and serine 51 in the interaction with eIF-2B and regulation of eIF-2B activity.     

Genetic polymorphism of alpha 2HS-glycoprotein.

A genetic polymorphism of the human serum glycoprotein, alpha 2HS-glycoprotein, can be recognized using isoelectric focusing in polyacrylamide, followed by silver-stain immunofixation. In a North American Caucasian population, two common alleles and one rare allele have been recognized, with frequencies as follows: AHSG*1: .6419, AHSG*2: .3535, and AHSG*3: .0046; polymorphism information content (PIC): .36. A black population from various islands of the Caribbean has the two most common alleles, plus a variant (B) not found in the white population. Allele frequencies in the blacks were: AHSG*1: .6901, AHSG*2: .2606, AHSG*B: .0493; PIC: .396. Family studies confirmed the allele designations. Alleles in both populations were in Hardy-Weinberg equilibrium. This polymorphism will be useful as a marker on chromosome 3q and for forensic studies. The serum concentration associated with AHSG*1 may be somewhat greater than that associated with AHSG*2. Differences between the allele products remained after removal of sialic acid from the glycoprotein with neuraminidase. The silver-stain immunofixation technique used for this polymorphism has wide application for the study of polymorphisms where the protein is present in low concentration or where only low titer antiserum is available.