Mammalian beta-adrenergic receptors. Structural differences in beta 1 and beta 2 subtypes revealed by peptide maps

Photoaffinity labeling techniques using p-azido-m-[125I]iodobenzylcarazolol have recently demonstrated that both the beta 1- and beta 2-adrenergic receptor-binding subunits from mammalian tissues including heart, lung, and erythrocytes reside on peptides of Mr approximately equal to 62,000-64,000. In this study, a two-dimensional gel electrophoresis method for peptide mapping was used to investigate and compare the structure of beta 1 - and beta 2-adrenergic receptor subtypes. When the photoaffinity labeled Mr approximately equal to 62,000 peptides from the beta 2-adrenergic receptors of rat lung and erythrocyte are subjected to simultaneous proteolysis using Staphylococcus aureus V8 proteinase or papain, exactly the same peptide fragments are generated from each subunit. In contrast, when the Mr approximately equal to 62,000 peptide containing the beta 1-adrenergic receptor-binding subunit derived from the rat heart is proteolyzed simultaneously with the Mr approximately equal to 62,000 peptide containing the beta 2-adrenergic receptors from either lung or erythrocyte, the peptide fragments generated are distinctly different. Peptide maps of beta 1-adrenergic receptors from the myocardial tissue of different species (pig versus rat) yield slightly different maps while the maps derived from the beta 2-adrenergic receptors of hamster lung and rat lung or erythrocytes reveal no interspecies differences. These data suggest: 1) alterations in the primary structure of the beta-adrenergic receptor may be responsible for the pharmacological specificities characteristic of beta 1- and beta 2-adrenergic receptor subtypes; and 2) alterations in the primary structure of similar beta-adrenergic receptor subtypes across different species may relate to the magnitude of their phylogenetic differences.     

The fat cell beta-adrenergic receptor. Purification and characterization of a mammalian beta 1-adrenergic receptor

The beta 1-adrenergic receptor of rat fat cells was effectively solubilized with digitonin and purified by affinity chromatography and steric exclusion high pressure liquid chromatography (HPLC). The purification strategy described permits an approximately 24,000-fold purification of the beta 1-adrenergic receptor of fat cells with an overall recovery of approximately 70%. Purified receptor preparations demonstrate a specific activity for (-) [3H]dihydroalprenolol binding of 12 nmol/mg of protein. The purified receptor was shown to migrate in steric exclusion HPLC as a Mr = 67,000 protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of radioiodinated purified receptor revealed a single, major peptide of Mr = 67,000. The binding of (-) [3H]dihydroalprenolol to purified receptor preparations displayed stereoselectivity and affinities for antagonists similar in nature to the membrane-bound and digitonin-solubilized beta 1-adrenergic receptor. In addition to the Mr = 67,000 component, a Mr = 140,000 form of the receptor was identified in HPLC runs of freshly prepared, affinity chromatographed receptor preparations that had not been frozen. This larger form of the receptor yielded binding activity of Mr = 67,000 on sequential HPLC runs and was shown to contain the Mr = 67,000 peptide. The beta 1-receptor from this mammalian source, composed of a single Mr = 67,000 peptide, is clearly quite distinct from the purified avian beta 1-, amphibian beta 2-, and mammalian beta 2-adrenergic receptors described by others.     

Site-directed anti-peptide antibodies define the topography of the beta-adrenergic receptor

Molecular cloning has revealed the primary structure of a number of G-protein-linked receptors. The organization and topography of these proteins predicted to have seven hydrophobic membrane-spanning domains, in contrast, have not been established. Antibodies were prepared against 11 peptides corresponding to each of the hydrophilic sequences of the hamster beta 2-adrenergic receptor. Each of the anti-peptide antibodies displayed immunoreactivity for its synthetic peptide antigen and beta 2-adrenergic receptor (Mr 65,000) on blots of cell membranes and of purified receptor. All but three anti-peptide antisera also displayed immunoreactivity toward human placental and rat fat cell beta 1-adrenergic receptors, reflecting the level of sequence identity that exists between the two subtypes, Chinese hamster ovary cells stably transfected with an expression vector harboring the cDNA encoding the hamster beta 2-adrenergic receptor provided a cell type with 2 million receptors/cell, suitable for in situ localization of the sequences used as antigens. Indirect immunofluorescence of intact and permeabilized cells performed with these site-directed anti-peptide antibodies permitted the assignment of the general topography of each of the hydrophilic sequences of this G-protein-linked receptor. The results support the predictive value of hydropathy analysis for one class of membrane proteins with multiple transmembrane-spanning domains.     

Identification of a novel Mr = 76-kDa form of beta-adrenergic receptors

The structure of the human beta-adrenergic receptor in purified basal membranes of human placental syncytiotrophoblast was probed using photoaffinity labeling. Basal membranes display a high specific activity of receptors (4-5 pmol/mg protein) and possess both beta 1- and beta 2-adrenergic receptors subtypes. Autoradiography of membranes that were incubated with the beta-adrenergic antagonist [125I]iodoazidobenzylpindolol, photolyzed and then subjected to sodium dodecylsulfate-polyacrylamide gel electrophoresis, identified four radiolabeled peptides, Mr = 65-kDa, 54-kDa, 43-kDa and a novel higher molecular weight 76-kDa form of the receptor. Photoaffinity labeling of each of these four peptides displayed the pharmacological properties expected for true beta-adrenergic receptors. The 76-kDa photoaffinity labeled receptor peptide observed in human placenta basal membranes has not been reported elsewhere. Competition studies with the beta1-selective ligand CGP-20712A demonstrate that the photoaffinity labeled receptor peptides are composed of both beta 1- and beta 2-adrenergic receptor subtypes.     

The mammalian beta 2-adrenergic receptor: purification and characterization

The beta 2-adrenergic receptors from hamster, guinea pig, and rat lungs have been solubilized with digitonin and purified by sequential Sepharose-alprenolol affinity and high-performance steric-exclusion liquid chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography of iodinated purified receptor preparations reveal a peptide with an apparent Mr of 64 000 in all three systems that coincides with the peptide labeled by the specific beta-adrenergic photoaffinity probe (p-azido-m-[125I]iodobenzyl)carazolol. A single polypeptide was observed in all three systems, suggesting that lower molecular weight peptides identified previously by affinity labeling or purification in mammalian systems may represent proteolyzed forms of the receptor. Purification of the beta-adrenergic receptor has also been assessed by silver staining, iodinated lectin binding, and measurement of the specific activity (approximately 15 000 pmol of [3H]dihydroalprenolol bound/mg of protein). Overall yields approximate 10% of the initial crude particulate binding, with 1-3 pmol of purified receptor obtained/g of tissue. The purified receptor preparations bind agonist and antagonist ligands with the expected beta 2-adrenergic specificity and stereoselectivity. Peptide mapping and lectin binding studies of the hamster, guinea pig, and rat lung beta 2-adrenergic receptors reveal significant similarities suggestive of evolutionary homology.     

The cardiac beta-adrenergic receptor. Structural similarities of beta 1 and beta 2 receptor subtypes demonstrated by photoaffinity labeling

The beta-adrenergic receptor photoaffinity ligand p-azido-m-[125I]iodobenzylcarazolol has been used to covalently label the beta 1 and beta 2 adrenergic receptor binding subunits present in left ventricular myocardial membranes derived from mammalian (including human) and nonmammalian species. Covalent incorporation of the photoaffinity ligand into membrane proteins was followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the case of the human, canine, porcine, rabbit, and rat left ventricle, all of which contain predominantly or exclusively beta 1-adrenergic receptors, two peptides of Mr approximately equal to 62,000 (major component) and Mr approximately equal to 55,000 (minor component) were specifically labeled and visualized by autoradiography. Photoincorporation into these two bands could be blocked with the appropriate drugs to display a beta 1-adrenergic receptor pharmacological specificity. Simultaneous sodium dodecyl sulfate-polyacrylamide gel electrophoresis of samples from each species revealed that all of the Mr = 62,000 peptides co-migrated suggesting similarity in the beta 1-adrenergic receptor binding subunit peptides in all of these species. The minor component Mr approximately equal to 55,000 appears to be a proteolytic degradation product of the Mr = to 62,000 peptide. Its formation could be decreased by proteinase inhibitors. This suggests that the heterogeneity of the labeling pattern observed in mammalian tissues in this and previous studies may be the result of proteolytic degradation of the receptor subunit which occurs during membrane preparation. Photoaffinity labeling of frog ventricular membranes which contain predominantly beta 2-adrenergic receptors also revealed two peptides of Mr approximately equal to 62,000 (major component) and 55,000 (minor component) with the pharmacological selectivity of a beta 2-adrenergic receptor. These data suggest marked similarities in the beta 1- and beta 2-adrenergic receptor binding subunits of different species and suggest that the pharmacological subtype might be determined by the detailed structure, i.e. amino acid sequence, at the ligand binding sites of the receptor peptide.     

Antibodies to peptide determinants in transforming growth factor beta and their applications

Polyclonal antibodies have been raised to a series of synthetic peptides which correspond to essentially all regions of the transforming growth factor beta 1 (TGF-beta 1) molecule. All antisera were evaluated for their abilities to react with TGF-beta 1 and TGF-beta 2 in either the native or reduced form in enzyme-linked immunosorbent assays, Western blots, and immunoprecipitation assays. While all antisera demonstrated some ability to recognize TGF-beta 1 in these systems, there was limited cross-reactivity with TGF-beta 2, suggesting that substantial sequence or conformational differences exist between the two growth factors. On Western blots 5-10 ng of purified human platelet TGF-beta 1 could be detected when probed with affinity-purified peptide antisera generated against peptides corresponding to residues 48-77, 50-75, and 78-109 of the 112 amino acid TGF-beta 1 monomer. Antisera raised against peptides 50-75 and 78-109 were most effective in immunoprecipitating reduced and native 125I-TGF-beta 1, respectively. The antisera also were tested for their effectiveness in blocking the binding of 125I-TGF-beta 1 to its receptor. Anti-peptide 78-109 and anti-peptide 50-75 blocked 80% and 40% of the binding, respectively, while antibodies against amino-terminal peptides were without effect. These data suggest that the carboxyl-terminal region of TGF-beta 1 may play a significant role in the binding of the native ligand to its receptor.     

Deglycosylated mammalian beta 2-adrenergic receptors: effect on radioligand binding and peptide mapping

Mammalian beta-adrenergic receptors are glycoproteins containing both high-mannose and complex-type carbohydrate chains [G. L. Stiles, J. L. Benovic, M. G. Caron, and R. J. Lefkowitz (1984) J. Biol. Chem. 259, 8655-8663]. Endoglycosidase F treatment of beta 2-adrenergic receptors results in the removal of at least two N-linked oligosaccharides, resulting in an increased mobility of the receptor peptide on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Mr 62,000 to 49,000). In the present study the properties of the deglycosylated beta 2-adrenergic receptor were assessed. Following deglycosylation, the beta 2-adrenergic receptor recognized both agonists and antagonists with the same potency order and affinities as the glycosylated form. In addition, two-dimensional gel electrophoresis peptide mapping techniques applied to control and deglycosylated beta 2-adrenergic receptors (both within and between species) demonstrated that there was a marked homology of the beta 2-adrenergic receptor between species which are closely related phylogenetically. In addition, the glycan component of the receptor did not appear to interfere with the ability of proteinases to generate accurate peptide maps.     

Age-related changes in the control of hepatic cyclic AMP levels by alpha 1- and beta 2-adrenergic receptors in male rats

Hepatocytes from juvenile male rats (80-110 g) showed a 12-fold elevation of cAMP in response to epinephrine, which was mediated by beta 2-adrenergic receptors. In these cells, either alpha 1- or beta 2-adrenergic stimulation alone activated phosphorylase and glucose release although the alpha 1-phosphorylase response was 10-fold more sensitive to epinephrine and resulted in more rapid (by 10-20 s) activation of the enzyme. This suggests that the beta 2-adrenergic response is functionally unimportant for glycogenolysis, even in juvenile rats. beta 2-Adrenergic stimulation did, however, produce an increase in the rate of gluconeogenesis from [U-14C] lactate in these cells. Aging in the male rat was associated with attenuation of the beta 2-adrenergic cAMP response coupled with the emergence of an alpha 1-receptor-mediated accumulation of cAMP. The order of potency displayed by the alpha 1-adrenergic/cAMP system to adrenergic agonists and antagonists was identical with that of the alpha 1-adrenergic/Ca2+ system. These data suggest that, in maturity, hepatic alpha 1-receptors become linked to 2 separate transduction mechanisms, namely Ca2+ mobilization and cAMP generation. Calcium depletion of hepatocytes from adult, but not juvenile, male rats increased the alpha 1-component of the cAMP response to epinephrine, but under these conditions, alpha 1-activation of phosphorylase occurred more slowly than in calcium-replete cells. Blockade of alpha 2-adrenergic receptors did not significantly modify catecholamine effects on hepatocyte cAMP or phosphorylase a levels in male rats at any age studied, suggesting a lack of functional significance for these receptors in the regulation of glycogenolysis.     

Novel mutants of the human beta1-adrenergic receptor reveal amino acids relevant for receptor activation

Activation of G protein-coupled receptors like the beta(1)-adrenergic receptor results in conformational changes that ultimately lead to signal propagation through a G protein to an effector like adenylyl cyclase. In this study we identified amino acids that seem to be critical for activation of the human beta(1)-adrenergic receptor. Activation patterns of mutant receptors were analyzed using two structurally different ligands for beta-adrenergic receptors that both are mixed agonist/antagonists. Broxaterol and terbutaline are agonists at beta(2)- and beta(3)-receptors; however, they act as antagonists at the beta(1)-subtype. We reasoned that this functional selectivity may be reflected by a corresponding sequence pattern in the receptor subtypes. Therefore, we exchanged single amino acids of the beta(1)-adrenergic receptor for residues that were identical in the beta(2)- and beta(3)-subtypes but different in the beta(1)-receptor. Pharmacological characterization of such receptor mutants revealed that binding of a panel of agonists and antagonists including broxaterol and terbutaline was unaltered. However, two of the mutants (I185V and D212N) were activated by broxaterol and terbutaline, which acted as antagonists at the wild-type receptor. Two additional mutants (V120L and K253R) could be activated by terbutaline alone, which is structurally more closely related to endogenous catecholamines like epinephrine than to broxaterol. A model of the human beta(1)-adrenergic receptor showed that the four gain-of-function mutations are outside of the putative ligand-binding domain substantiating the lack of an effect of the mutations on binding characteristics. These results support the notion that Val-120, Ile-185, Asp-212, and Lys-253 are critically involved in conformational changes occurring during receptor activation.     

Human fat cell beta-adrenergic receptors: beta-agonist-dependent lipolytic responses and characterization of beta-adrenergic binding sites on human fat cell membranes with highly selective beta 1-antagonists

Beta-adrenergic receptors were characterized in human fat cell membranes using 125I-labeled cyanopindolol (125I-labeled CYP) and highly selective beta 1-antagonists. The iodinated radioligand bound saturably and specifically to a single class of high affinity binding sites. The number of binding sites determined with 125I-labeled CYP closely agreed with that determined with two other tritiated radioligands: [3H]dihydroalprenolol and [3H]CGP-12,177. Since 125I-labeled CYP does not discriminate between beta 1- and beta 2-adrenoceptors, the densities of the two receptor subtypes were determined from the competition curves of 125I-labeled CYP by highly selective beta 1-antagonists (bisoprolol, ICI-89,406, CGP-20,712A, and LK-204,545). Moreover, in order to enable correlation with binding data, the regulation of adenylate cyclase activity and of lipolysis was tested with various beta-agonist and antagonist compounds. The results obtained on fat cell membranes from abdominal subcutaneous adipose tissue demonstrated the following. 1) 125I-labeled CYP represents a valuable tool for the quantification and the delineation of beta-receptor subtypes. 2) The presence of sodium ions in binding buffers causes a modification of the affinity of beta-sites for some beta-antagonists. 3) The human fat cell beta adrenergic receptor population defined by nonselective radioligands is composed of two subtypes that can be interpreted in terms of classic beta 1- and beta 2-adrenergic receptor subtypes as assessed by competition studies with highly selective antagonists; beta 2-sites are predominant (60-70% of 125I-labeled CYP sites) in the adipocytes of slightly overweight women. 4) Results support the idea that beta 1- as well as beta 2-adrenergic receptors are coupled with adenylate cyclase and involved in the induction of lipolysis. 5) The results focus on the interest in some beta 2-agonist drugs (zinterol, clenbuterol) as partial inductors of lipolysis, with the lipolytic efficacies of these compounds being well correlated with their efficacies at 125I-labeled CYP sites.     

Identification of the Subunit Structure of Rat Pineal Adrenergic Receptors by Photoaffinity Labeling

The adrenergic receptors of rat pineal gland were investigated using radiolabeled ligand binding and photoaffinity labeling techniques. 125I-2-[beta-(4-hydroxyphenyl)ethylaminomethyl]tetralone (125I-HEAT) and 125I-cyanopindolol (125I-CYP) labeled specific sites on rat pineal gland membranes with equilibrium dissociation constants (KD) of 48 (+/- 5) pM and 30 (+/- 5) pM, respectively. Binding site maxima were 481 (+/- 63) and 1,020 (+/- 85) fmol/mg protein. The sites labeled by 125I-HEAT had the pharmacological characteristics of alpha 1-adrenergic receptors. 125I-CYP-labeled beta-adrenergic receptors were characterized as a homogeneous population of beta 1-adrenergic receptors. The alpha 1- and beta 1-adrenergic receptors were covalently labeled with the specific photoaffinity probes 4-amino-6,7-dimethoxy-2-(4-[5-(4-azido-3-[125I]iodophenyl) pentanoyl]-1-piperazinyl) quinazoline (125I-APDQ) and 125I-p-azidobenzylcarazolol (125I-pABC). 125I-APDQ labeled an alpha 1-adrenergic receptor peptide of Mr = 74,000 (+/- 4,000), which was similar to peptides labeled in rat cerebral cortex, liver, and spleen. 125I-pABC labeled a single beta 1-adrenergic receptor peptide with a Mr = 42,000 (+/- 1,500), which differed from the 60-65,000 peptide commonly seen in mammalian tissues. Possible reasons for these differences are discussed.     

Regulation of diamine oxidase expression by beta 2-adrenoceptors in normal and hypertrophic rat kidney

The administration of preferential adrenergic receptor antagonists to uninephrectomized rats revealed the beta 2-adrenergic mediation in diamine oxidase activity increase that occurs in the remaining kidney undergoing compensatory hypertrophy. In fact, beta 1, beta 2- or beta 2, but not alpha 1-, alpha 2-, or beta 1-receptor-blocking agents prevented this enzyme enhancement. Further studies with adrenoceptor agonists, such as epinephrine (alpha 1, alpha 2, beta 1, beta 2), isoproterenol (beta 1, beta 2) or terbutaline (beta 2) showed that also in normal rat kidney diamine oxidase activity is under the control of catecholamine-beta 2-receptors through a mechanism that involves new synthesis of mRNA and protein. Theophylline, an inhibitor of phosphodiesterase, or forskolin, an activator of adenyl cyclase, increased diamine oxidase activity as does epinephrine or nephrectomy. Thus, catecholamine-triggered beta 2-receptors coupled to adenyl cyclase are involved in the regulation of diamine oxidase activity in normal and hypertrophic rat kidney.     

Purification and immunochemical interrelationship of insulin receptors from rat brain and liver.

Insulin receptors from rat brain and liver were purified. Brain purified receptor exhibited protein bands of apparent Mr = 135,000 and 95,000 molecular weight corresponding to alpha- and beta-subunits, retained a tyrosine specific protein kinase activity and demonstrated phosphorylation that is hormonally sensitive. Antisera were raised against both insulin receptor preparations and enzyme-linked immunosorbent assay was developed. The comparison of two insulin receptors was based on a displacement enzyme-linked immunosorbent assay where antisera were interchanged on predetermined optimal dilutions. This indicated that both insulin receptors possess some unique antigenic determinants thereby implying a structural difference.     

Subtype-Selective Immunoprecipitation of the β2-Adrenergic Receptor

Most antibodies known to interact with beta-adrenergic receptors do not exhibit subtype selectivity, nor do they provide quantitative immunoprecipitation. A monoclonal antibody, G27.1 raised against a synthetic peptide corresponding to the C-terminus of the beta 2-adrenergic receptor of hamster, is selective for the beta 2 subtype. G27.1 provides nearly quantitative immunoprecipitation of the beta 2-adrenergic receptor from hamster lung that has been photoaffinity-labeled and solubilized with sodium dodecyl sulfate. Immunoprecipitation is completely blocked by nanomolar concentrations of the immunizing peptide. This antibody interacts with beta 2-adrenergic receptors from three rodent species, but not with those from humans. When C6 glioma cells, which contain both beta 1- and beta 2-adrenergic receptors, are photoaffinity-labeled in the absence or presence of subtype-selective antagonists, subtype-selective photoaffinity-labeling results. G27.1 can immunoprecipitate beta 2-, but not beta 1-, adrenergic receptors from these cells. Similar results were obtained following subtype-selective photoaffinity-labeling of membranes from rat cerebellum and cerebral cortex. The beta-adrenergic receptors from C6 glioma cells and rat cerebral cortex exist as a mixture of two molecular weight species. These species differ in glycosylation, as shown by endoglycosidase F digestion of crude and immunoprecipitated receptors.     

Quantitative assessment of beta 1- and beta 2-adrenergic receptor homo- and heterodimerization by bioluminescence resonance energy transfer

Quantitative bioluminescence resonance energy transfer (BRET) analysis was applied to the study of beta(1)- and beta(2)-adrenergic receptor homo- and heterodimerization. To assess the relative affinity between each of the protomers, BRET saturation experiments were carried out in HEK-293T cells. beta(1)- and beta(2)-adrenergic receptors were found to have similar propensity to engage in homo- and heterotropic interactions suggesting that, at equivalent expression levels of the two receptor subtypes, an equal proportion of homo- and heterodimers would form. Analysis of the data also revealed that, at equimolar expression levels of energy donor and acceptor, more than 80% of the receptor molecules exist as dimers and that this high incidence of receptor dimerization is insensitive to receptor density for expression levels varying between 1.4 and 26.9 pmol of receptor/mg of membrane protein. Taken together, these results indicate that most of the receptors expressed in cells exist as constitutive dimers and that, at least in undifferentiated fibroblasts, the proportion of homo- and heterodimers between the closely related beta(1)- and beta(2)-adrenergic receptors is determined by their relative levels of expression.     

Mediation of isoproterenol-induced thirst in rats by beta2-adrenergic receptors.

Isoproterenol-induced thirst in rats has been attributed to the activation of beta-adrenergic receptors. Since these receptors can be further differentiated pharmacologically into beta1 and beta2 types, experiments were performed using several beta-adrenergic agonists and antagonists to determine the receptor type initiating the isoproterenol-induced thirst. The beta1- and beta2-adrenergic antagonist, d,l-propranolol (1 mg/kg, ip), blocked the increase in water intake usually accompanying acute subcutaneous administration of isoproterenol (25 microgram/kg) to female rats. Since l-propranolol is known to stabilize membranes and to possess anesthetic-like properties, d-propranolol was also used. This isomer has little beta-adrenergic-blocking activity but possesses anesthetic-like activity. Administration of d-propranolol (1 mg/kg, ip) failed to affect the drinking response to acute administration of isoproterenol (25 microgram/kg). Practolol (125 mg/kg), a beta1-adrenergic antagonist with little anesthetic properties, also had no effect on water intake of isoproterenol-treated rats. Butoxamine, a selective beta2-adrenergic antagonist, attenuated the drinking response to isoproterenol. Salbutamol (150 microgram/kg), a beta2-adrenergic agonist, mimicked the effect of isoproterenol on water intake. These results are consistent with the suggestion that beta2-adrenergic receptors mediate the isoproterenol-induced thirst in rats.     

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.     

Noradrenergic stimulation of BDNF synthesis in astrocytes: mediation via alpha1- and beta1/beta2-adrenergic receptors

Brain-derived neurotrophic factor (BDNF) synthesis in astrocytes induced by noradrenaline (NA) is a receptor-mediated process utilizing two parallel adrenergic pathways: beta1/beta2-adrenergic/cAMP and the novel alpha1-adrenergic/PKC pathway. BDNF is produced by astrocytes, in addition to neurons, and the noradrenergic system plays a role in controlling BDNF synthesis. Since astrocytes express various subtypes of alpha- and beta-adrenergic receptors that have the potential to be activated by synaptically released NA, we focused our present study on the mediatory role of adrenergic receptors in the noradrenergic up-regulation of BDNF synthesis in cultured neonatal rat cortical astrocytes. NA (1 microM) elevates BDNF levels by four-fold after 6 h of incubation. Its stimulation was partly inhibited by either the beta1-adrenergic antagonist atenolol, the beta2-adrenergic antagonist ICI 118,551, or by the alpha1-adrenergic antagonist prazosin, while the alpha2-adrenergic antagonist yohimbine showed no effect. BDNF levels in astrocytes were increased by the specific beta1-adrenergic agonist dobutamine and the beta2-adrenergic agonist salbutamol, as well as by adenylate cyclase activation (by forskolin) and PKA activation (by dBcAMP). However, none of the tested agonists or mediators of the intracellular beta-adrenergic pathways were able to reach the level of NA's stimulatory effect. BDNF cellular levels were also elevated by the alpha1-adrenergic agonist methoxamine, but not by the alpha2-adrenergic agonist clonidine. The increase in intracellular Ca2+ by ionophore A23187 showed no effect, whereas PKC activation by phorbol 12-myristate 13-acetate (TPA) potently stimulated BDNF levels in the cells. The methoxamine-stimulated BDNF synthesis was inhibited by desensitizing pretreatment with TPA, indicating that the alpha1-stimulation was mediated via PKC activation. In conclusion, the synthesis of astrocytic BDNF stimulated by noradrenergic neuronal activity is an adaptable process using multiple types (alpha1 and beta1/beta2) of adrenergic receptor activation.