Agonist interactions with chimeric and mutant beta1- and beta3-adrenergic receptors: involvement of the seventh transmembrane region in conferring subtype specificity

beta1- and beta3-adrenergic receptors (AR) are the predominant beta-AR subtypes in adipocytes, and analysis of native and recombinant beta-AR has revealed several pharmacological and biochemical differences between these subtypes. This study used chimeric and mutated rat beta-AR expressed in Chinese hamster ovary cells to examine the basis of certain characteristic differences in the agonist properties of catecholamines and prototypic beta3-AR agonists. The exchange of sequence beyond transmembrane (TM) region 6 between the beta-AR subtypes had dramatic and reciprocal effects on the affinity and efficacy of the prototypic beta3-AR agonists BRL 37,344 and CL 316,243, without affecting the interactions with catecholamines. Mutation of Phe350 and Phe351 in TM7 of the beta1-AR to Ala and Leu found in the beta3-AR was sufficient to allow activation by prototypic beta3-AR agonists. Interestingly, this mutation did not affect catecholamine action and it did not impair the ability of propranolol to block the actions of isoproterenol or the selective beta3-AR agonists. beta1-AR containing beta3-AR sequence from predicted TM5 through TM6 exhibited reduced affinity for catecholamines without altering agonist potency, suggesting enhanced coupling efficiency. Inclusion of the homologous beta1-AR sequence in the beta3-AR, however, did not produce reciprocal effects. These results are the first to define a major determinant of beta3-AR subtype-selective agonism in TM7 and demonstrate that the determinants of selective phenethanolamines, catecholamines, and propranolol action are distinct.     

The contribution of classical (beta1/2-) and atypical beta-adrenoceptors to the stimulation of human white adipocyte lipolysis and right atrial appendage contraction by novel beta3-adrenoceptor agonists of differing selectivities

The role of beta3- and other putative atypical beta-adrenoceptors in human white adipocytes and right atrial appendage has been investigated using CGP 12177 and novel phenylethanolamine and aryloxypropanolamine beta3-adrenoceptor (beta3AR) agonists with varying intrinsic activities and selectivities for human cloned betaAR subtypes. The ability to demonstrate beta1/2AR antagonist-insensitive (beta3 or other atypical betaAR-mediated) responses to CGP 12177 was critically dependent on the albumin batch used to prepare and incubate the adipocytes. Four aryloxypropanolamine selective beta3AR agonists (SB-226552, SB-229432, SB-236923, SB-246982) consistently elicited beta1/2AR antagonist-insensitive lipolysis. However, a phenylethanolamine (SB-220646) that was a selective full beta3AR agonist elicited full lipolytic and inotropic responses that were sensitive to beta1/2AR antagonism, despite it having very low efficacies at cloned beta1- and beta2ARs. A component of the response to another phenylethanolamine selective beta3AR agonist (SB-215691) was insensitive to beta1/2AR antagonism in some experiments. Because no [corrected] novel aryloxypropanolamine had a beta1/2AR antagonist-insensitive inotropic effect, these results establish more firmly that beta3ARs mediate lipolysis in human white adipocytes, and suggest that putative 'beta4ARs' mediate inotropic responses to CGP 12177. The results also illustrate the difficulty of predicting from studies on cloned betaARs which betaARs will mediate responses to agonists in tissues that have a high number of beta1- and beta2ARs or a low number of beta3ARs.     

Examination of ligand-induced conformational changes in the beta2 adrenergic receptor

The environmentally sensitive and cysteine reactive fluorescent probe, IANBD, was used to monitor ligand-induced structural changes in the beta2 adrenergic receptor (beta2AR) by fluorescent spectroscopy. We found that agonists caused a dose-dependent and reversible decrease in fluorescence from the purified IANBD-labeled beta2AR. This suggested that agonists promote a conformational change in the receptor that leads to an increase in the polarity of the environment around one or more IANBD labeled cysteines. The wildtype receptor contains eight free cysteines and mutagenesis and peptide mapping experiments have indicated that several of these sites are accessible for chemical derivatization. Thus, to identify the cysteine(s) involved in the agonist-induced change in fluorescence and thereby map agonist-induced conformational changes in the beta2AR, we generated a series of mutant receptors having limited numbers of cysteines available for fluorescent labeling. Fluorescence spectroscopy analysis of the purified and site-selectively IANBD-labeled mutants showed that IANBD labeled 125Cys and 285Cys are responsible for the observed changes in fluorescence consistent with movements of TM III and VI in response to agonist binding.     

Agonist-induced down-regulation of the beta2-adrenoceptor and its mRNA in human mononuclear leukocytes

Agonist-mediated regulation of beta2-adrenoceptors in mononuclear leukocytes has been examined at the protein but not at the mRNA level. In the present study, incubation of mononuclear leukocytes with the beta-agonist (-)-isoproterenol (10(-6) M) for up to 42 hr led to a maximum decrease in both beta2-adrenoceptor mRNA concentration and total receptor number of ca. 56 and 70%, respectively. The decrease in the mRNA level, however, was slower than for the protein level. After 4 hr of incubation with the beta-agonist, the protein level decreased to a minimum of 65% of the initial amount, while an incubation of 8 hr was necessary to reach a similar decrease in the level of mRNA (69% of the initial level). Measurements of mRNA stability revealed a reduction in the half-life of beta2-adrenoceptor mRNA from 2.7 to 1.1 hr following 4 hr of incubation with (-)-isoproterenol. Our data clearly demonstrate that treatment of human mononuclear leukocytes with (-)-isoproterenol induces a beta2-adrenoceptor down-regulation together with a slower time course of mRNA down-regulation which is partly due to a reduction of mRNA stability.     

Mutation of tyrosine-350 impairs the coupling of the beta 2-adrenergic receptor to the stimulatory guanine nucleotide binding protein without interfering with receptor down-regulation

Long-term stimulation of the beta 2-adrenergic receptor (beta 2AR) leads to an internalization and degradation of the receptor. This down-regulation of the beta 2AR number contributes to the desensitization of the adenylyl cyclase activity induced by chronic exposure to agonists. It was proposed that two tyrosine residues (Tyr-350 and Tyr-354) located in the cytoplasmic tail of the beta 2AR play a crucial role in agonist-induced down-regulation. In addition to perturbation of the down-regulation, the substitution of these tyrosines for alanines also led to a functional uncoupling of the receptor from Gs [Valiquette et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 5089-5093]. To further characterize the relative contribution of Tyr-350 and Tyr-354 to the receptor interaction with Gs and agonist-promoted down-regulation, both tyrosines were individually replaced by alanines and mutant receptors expressed in CHW cells. We show here that mutation of Tyr-350 but not that of Tyr-354 significantly decreased the ability of the beta 2AR to be functionally coupled to Gs and thereby to stimulate the adenylyl cyclase. Moreover, in contrast to the double tyrosine mutation, neither of the single-point mutations affected the agonist-induced down-regulation pattern. These data suggest that the presence of either Tyr-350 or Tyr-354 is sufficient to maintain normal agonist-induced down-regulation whereas the integrity of Tyr-350 is required for an appropriate coupling to Gs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Visualization of agonist-induced sequestration and down-regulation of a green fluorescent protein-tagged beta2-adrenergic receptor

To date, the visualization of beta2-adrenergic receptor (beta2AR) trafficking has been largely limited to immunocytochemical analyses of acute internalization events of epitope-tagged receptors in various transfection systems. The development of a beta2AR conjugated with green fluorescent protein (beta2AR-GFP) provides the opportunity for a more extensive optical analysis of beta2AR sequestration, down-regulation, and recycling in cells. Here we demonstrate that stable expression of beta2AR-GFP in HeLa cells enables a detailed temporal and spatial analysis of these events. Time-dependent colocalization of beta2AR-GFP with rhodamine-labeled transferrin and rhodamine-labeled dextran following agonist exposure demonstrates receptor distribution to early endosomes (sequestration) and lysosomes (down-regulation), respectively. The observed temporal distribution of beta2AR-GFP was consistent with measures of receptor sequestration and down-regulation generated by radioligand-receptor binding assays. Cells stimulated with different beta-agonists revealed time courses of beta2AR-GFP redistribution reflective of the intrinsic activity of each agonist.     

Catecholamines potentiate amyloid beta-peptide neurotoxicity: involvement of oxidative stress, mitochondrial dysfunction, and perturbed calcium homeostasis

Oxidative stress and mitochondrial dysfunction are implicated in the neuronal cell death that occurs in physiological settings and in neurodegenerative disorders. In Alzheimer's disease (AD) degenerating neurons are associated with deposits of amyloid beta-peptide (A beta), and there is evidence for increased membrane lipid peroxidation and protein oxidation in the degenerating neurons. Cell culture studies have shown that A beta can disrupt calcium homeostasis and induce apoptosis in neurons by a mechanism involving oxidative stress. We now report that catecholamines (norepinephrine, epinephrine, and dopamine) increase the vulnerability of cultured hippocampal neurons to A beta toxicity. The catecholamines were effective in potentiating A beta toxicity at concentrations of 10-200 microM, with the higher concentrations (100-200 microM) themselves inducing cell death. Serotonin and acetylcholine were not neurotoxic and did not modify A beta toxicity. Levels of membrane lipid peroxidation, and cytoplasmic and mitochondrial reactive oxygen species, were increased following exposure to neurons to A beta, and catecholamines exacerbated the oxidative stress. Subtoxic concentrations of catecholamines exacerbated decreases in mitochondrial energy charge and transmembrane potential caused by A beta, and higher concentrations of catecholamines alone induced mitochondrial dysfunction. Antioxidants (vitamin E, glutathione, and propyl gallate) protected neurons against the damaging effects of A beta and catecholamines, whereas the beta-adrenergic receptor antagonist propanolol and the dopamine (D1) receptor antagonist SCH23390 were ineffective. Measurements of intracellular free Ca2+ ([Ca2+]i) showed that A beta induced a slow elevation of [Ca2+]i which was greatly enhanced in cultures cotreated with catecholamines. Collectively, these data indicate a role for catecholamines in exacerbating A beta-mediated neuronal degeneration in AD and, when taken together with previous findings, suggest roles for oxidative stress induced by catecholamines in several different neurodegenerative conditions.     

Desensitization of the beta-adrenergic response in human brown adipocytes

Activation of adenylyl cyclase by beta-adrenergic receptors (betaARs) plays a major role in adipose tissue homeostasis. The increase in cAMP promotes lipolysis in white adipose tissue, activates both thermogenesis and lipolysis in brown adipose tissue (BAT), and induces BAT hypertrophy. Previous studies indicated that among the three betaAR subtypes present in adipose tissue, beta3AR could be a potential target for antiobesity treatments in humans. We studied immortalized human brown adipocytes (PAZ6 adipocytes) as a model of beta-adrenergic response in human BAT. PAZ6 adipocytes and freshly isolated mature human brown adipocytes display the same proportions of betaAR subtypes, with beta3AR being the most abundant (approximately 80% of the total). However, beta3AR was poorly coupled to the adenylyl cyclase pathway in PAZ6 cells, contributing to only 10% of the isoproterenol-induced accumulation of cAMP, whereas 20% and 70% of the signal depended on beta1- and beta2-subtypes, respectively. Upon isoproterenol stimulation, beta1- and beta2AR down-regulated with a half-life of about 3 h and the beta3AR with a half-life of 30-40 h. Long term stimulation with both saturating (micromolar) and nonsaturating (nanomolar) concentrations of beta-adrenergic agonists caused a complete desensitization of the beta-adrenergic response at the adenylyl cyclase level and loss of stimulated protein kinase A activity and CREB phosphorylation. These results suggest that cAMP-dependent processes will be desensitized upon permanent treatment with beta3AR agonists. Further studies should establish whether the beta3AR is coupled to other signaling pathways in human brown adipocytes and whether these may contribute to BAT hypertrophy and/or thermogenesis.     

Angiogenesis promoted by vascular endothelial growth factor: regulation through alpha1beta1 and alpha2beta1 integrins

Vascular endothelial growth factor (VEGF), also known as vascular permeability factor, is a cytokine of central importance for the angiogenesis associated with cancers and other pathologies. Because angiogenesis often involves endothelial cell (EC) migration and proliferation within a collagen-rich extracellular matrix, we investigated the possibility that VEGF promotes neovascularization through regulation of collagen receptor expression. VEGF induced a 5- to 7-fold increase in dermal microvascular EC surface protein expression of two collagen receptors-the alpha1beta1 and alpha2beta1 integrins-through induction of mRNAs encoding the alpha1 and alpha2 subunits. In contrast, VEGF did not induce increased expression of the alpha3beta1 integrin, which also has been implicated in collagen binding. Integrin alpha1-blocking and alpha2-blocking antibodies (Ab) each partially inhibited attachment of microvascular EC to collagen I, and alpha1-blocking Ab also inhibited attachment to collagen IV and laminin-1. Induction of alpha1beta1 and alpha2beta1 expression by VEGF promoted cell spreading on collagen I gels which was abolished by a combination of alpha1-blocking and alpha2-blocking Abs. In vivo, a combination of alpha1-blocking and alpha2-blocking Abs markedly inhibited VEGF-driven angiogenesis; average cross-sectional area of individual new blood vessels was reduced 90% and average total new vascular area was reduced 82% without detectable effects on the pre-existing vasculature. These data indicate that induction of alpha1beta1 and alpha2beta1 expression by EC is an important mechanism by which VEGF promotes angiogenesis and that alpha1beta1 and alpha2beta1 antagonists may prove effective in inhibiting VEGF-driven angiogenesis in cancers and other important pathologies.     

Role of clathrin-mediated endocytosis in agonist-induced down-regulation of the beta2-adrenergic receptor

Previous studies have demonstrated that non-visual arrestins function as adaptors in clathrin-mediated endocytosis to promote agonist-induced internalization of the beta2-adrenergic receptor (beta2AR). Here, we characterized the effects of arrestins and other modulators of clathrin-mediated endocytosis on down-regulation of the beta2AR. In COS-1 and HeLa cells, non-visual arrestins promote agonist-induced internalization and down-regulation of the beta2AR, whereas dynamin-K44A, a dominant-negative mutant of dynamin that inhibits clathrin-mediated endocytosis, attenuates beta2AR internalization and down-regulation. In HEK293 cells, dynamin-K44A profoundly inhibits agonist-induced internalization and down-regulation of the beta2AR, suggesting that receptor internalization is critical for down-regulation in these cells. Moreover, a dominant-negative mutant of beta-arrestin, beta-arrestin-(319-418), also inhibits both agonist-induced receptor internalization and down-regulation. Immunofluorescence microscopy analysis reveals that the beta2AR is trafficked to lysosomes in HEK293 cells, where presumably degradation of the receptor occurs. These studies demonstrate that down-regulation of the beta2AR is in part due to trafficking of the beta2AR via the clathrin-coated pit endosomal pathway to lysosomes.     

Metabolic response to various beta-adrenoceptor agonists in beta3-adrenoceptor knockout mice: evidence for a new beta-adrenergic receptor in brown adipose tissue

The beta3-adrenoceptor plays an important role in the adrenergic response of brown and white adipose tissues (BAT and WAT). In this study, in vitro metabolic responses to beta-adrenoceptor stimulation were compared in adipose tissues of beta3-adrenoceptor knockout and wild type mice. The measured parameters were BAT fragment oxygen uptake (MO2) and isolated white adipocyte lipolysis. In BAT of wild type mice (-)-norepinephrine maximally stimulated MO2 4.1+/-0.8 fold. Similar maximal stimulations were obtained with beta1-, beta2- or beta3-adrenoceptor selective agonists (dobutamine 5.1+/-0.3, terbutaline 5.3+/-0.3 and CL 316,243 4.8+/-0.9 fold, respectively); in BAT of beta3-adrenoceptor knockout mice, the beta1- and beta2-responses were fully conserved. In BAT of wild type mice, the beta1/beta2-antagonist and beta3-partial agonist CGP 12177 elicited a maximal MO2 response (4.7+/-0.4 fold). In beta3-adrenoceptor knockout BAT, this response was fully conserved despite an absence of response to CL 316,243. This unexpected result suggests that an atypical beta-adrenoceptor, distinct from the beta1-, beta2- and beta3-subtypes and referred to as a putative beta4-adrenoceptor is present in BAT and that it can mediate in vitro a maximal MO2 stimulation. In isolated white adipocytes of wild type mice, (-)-epinephrine maximally stimulated lipolysis 12.1+/-2.6 fold. Similar maximal stimulations were obtained with beta1-, beta2- or beta3-adrenoceptor selective agonists (TO509 12+/-2, procaterol 11+/-3, CL 316,243 11+/-3 fold, respectively) or with CGP 12177 (7.1+/-1.5 fold). In isolated white adipocytes of beta3-adrenoceptor knockout mice, the lipolytic responses to (-)epinephrine, to the beta1-, beta2-, beta3-adrenoceptor selective agonists and to CGP 12177 were almost or totally depressed, whereas those to ACTH, forskolin and dibutyryl cyclic AMP were conserved.     

An inverted cAMP response element mediates the cAMP induction of the ovine beta 1-adrenergic receptor gene

We identified an inverted, functional cAMP response element (CRE) located at--1599 bp relative to the translation start site within the ovine beta 1-adrenergic receptor (beta 1 AR) gene promoter. In transfection studies with SK-N-MC cells, a 40-bp oligonucleotide containing the potential CRE, beta 1 AR-CRE, conferred a 3- to 4-fold increase in luciferase activity mediated by cAMP. The induction was mimicked by co-transfecting the cells with a vector overexpressing the alpha-catalytic subunit of the cAMP-dependent protein kinase (PKA) without treatment, and was blocked by overexpressing a PKA inhibitor (PKI). In electrophoretic mobility shift assays, a discrete binding pattern was shown in cell nuclear extract probed with the 40 bp beta 1 AR-CRE. The binding was shown to be specific and supershifted by addition of a CRE binding protein (CREB-1) antibody. These data demonstrate that cAMP mediates the induction of beta 1 AR gene expression by interacting with an inverted CRE within the promoter region. This is the first reported functional CRE among all beta 1 AR genes.     

Chromatographic and immunological identification of GnRH (gonadotropin-releasing hormone) variants. Occurrence of mammalian and a salmon-like GnRH in the forebrain of an eutherian mammal: Hydrochaeris hydrochaeris (Mammalia, Rodentia)

Phosphorylation of the beta 2-adrenergic receptor (beta 2AR) is the initial event that underlies rapid agonist-promoted desensitisation. However, the role of phosphorylation in mediating long-term beta 2AR desensitisation is not known. To investigate this possibility, we performed intact cell phosphorylation studies with COS-7 cells transiently expressing an epitope tagged wild-type beta 2AR and found that receptor phosphorylation in cells treated with 1 microM isoproterenol for 24 h was approximately 4-fold over the basal state. This finding suggested that persistent phosphorylation of the receptor might contribute to functional long-term desensitisation which we further explored with mutated beta 2AR lacking the determinants of phosphorylation by the beta AR kinase (beta ARK), PKA or both. In CHW cells expressing the WT beta 2AR, pretreatment with 1 microM isoproterenol for 24 h reduced the isoproterenol-stimulated cAMP response by 82 +/- 5%. Substitution of the PKA sites with alanines had no effect on the extent of desensitisation (77 +/- 6%, P = NS compared to WT). In contrast, desensitisation was only 49 +/- 4% (P < 0.001 compared to WT) when the beta ARK sites were similarly substituted. Removal of both the beta ARK and PKA sites impaired desensitisation to the same extent as the beta ARK mutant. The extent of receptor loss (downregulation) was the same among all of the cell lines used and therefore could not account for the observed differences in desensitisation. Cellular beta ARK activity, assessed by a rhodopsin phosphorylation assay, was equivalent in all cell lines and was unaffected by agonist treatment. PKA activity, however, was dynamically regulated, increasing 4-fold over basal levels after 15 min of isoproterenol and returning to near basal levels after 24 h. The lower level of PKA activity after long-term agonist exposure may therefore have contributed to the apparent lack of effect of removing PKA sites. Nonetheless, long-term desensitisation was clearly attenuated with beta 2AR lacking beta ARK phosphorylation sites. These findings show that in addition to its role in regulating short-term desensitisation, beta ARK-mediated phosphorylation is an important mechanism underlying long-term desensitisation of the beta 2AR as well.     

3-Pyridylethanolamines: potent and selective human beta 3 adrenergic receptor agonists

The 3-pyridylethanolamine L-757,793 is a potent beta 3 AR agonist (EC50 6.3 nM, 70% activation) with 1,300- and 500-fold selectivity over binding to the beta 1 and beta 2 ARs, respectively. L-757,793 stimulated lipolysis in rhesus monkeys (ED50 0.2 mg/kg) with a maximum response equivalent to that elicited by isoproterenol.     

Association of beta-agonists with corresponding beta 2- and beta 1-adrenergic pentapeptide sequences

Synthesized beta 1- and beta 2-pentapeptide sequences corresponding to published adrenoceptor transmembrane activation site subtypes were investigated in vitro for selectivity in association for drug ligands of known selectivity. Both nuclear magnetic resonance spectroscopy and molecular mechanics demonstrated that structural differences among the corresponding pentapeptide activation-site sequences can explain agonist selectivity. Results suggest the agonists bind across the activation site loop on the second transmembrane alpha-helix by dipole/dipole interactions between a ligand and the peptide. Since electrostatic interactions within the membrane may determine the rate of intercellular ion flux, agonist association across the activation site sequence could thereby decrease electrostatic resistance to positive ion flux into the cell. Interactions between the peptides and the ligands may provide insight into the structures and mechanisms involved in association of ligands for the identical sequences on the beta-adrenoreceptors.     

Reconstitution of beta2-adrenoceptor-GTP-binding-protein interaction in Sf9 cells--high coupling efficiency in a beta2-adrenoceptor-G(s alpha) fusion protein

In most studies, coupling of the beta2-adrenoceptor (beta2AR) to the stimulatory, heterotrimeric GTP-binding protein of adenylyl cyclase the (Gs) is studied indirectly by measuring adenylyl cyclase activation. The aim of this study was to establish a model system in which beta2AR-Gs interactions can be studied directly at the level of the G-protein. We expressed the beta2AR alone, in combination with the alpha-subunit of Gs (G(s alpha)), and as fusion protein with G(s alpha) (beta2AR-G(s alpha)) in Sf9 insect cells. The beta2AR expressed alone couples poorly to the endogenous G(s alpha)-like G-protein of Sf9 cells since no high-affinity agonist binding could be detected, and the effects of agonist and inverse agonist on adenylyl cyclase, high-affinity GTPase and guanosine 5'-O-(3-thiotriphosphate) (GTP[S]) binding were small. Beta2AR-G(s alpha) reconstituted high-affinity agonist binding and regulated adenylyl cyclase more effectively than the beta2AR co-expressed with a large excess of G(s alpha). In membranes expressing beta2AR-G(s alpha), highly effective agonist- and inverse agonist regulation of high-affinity GTP hydrolysis and GTP[S] binding was observed. In contrast, agonist and inverse agonist regulation of GTP hydrolysis and GTP[S] binding in membranes expressing beta2AR and G(s alpha) as separate proteins was difficult to detect. Our data show that the beta2AR interacts with G(s alpha) more efficiently when expressed as a fusion protein than when expressed with an excess of non-fused G(s alpha). The beta2AR-G(s alpha) fusion protein provides a very sensitive model system to study the regulation of Gs function by beta2AR agonists and inverse agonists directly at the level of the G-protein.     

Lipolytic action of Buthus occitanus tunetanus venom: involvement of the beta adrenergic pathway

Scorpion venoms contain active neurotoxins known to act selectively at the level of voltage sensitive Na+ and K+ channels on mammal nervous system. In the present report, we show for the first time that the venom of scorpion Buthus occitanus tunetanus (Bot) contains compounds able to activate another cell function in non excitable cells. Addition of this venom to the culture media of 3T3-L1 adipocytes or freshly dissociated rat adipocytes rapidly increases lipolysis as estimated by glycerol release (approximately 3 to 4 fold over basal values) in a dose-dependent manner (EC50 approximately 12 +/- 1.25 micrograms/ml; n = 3). Bot venom effect was lower and not additive to the effect produced by isoproterenol (IPE) (10 microM), a main lipolytic agent, n = 3. In Sephadex G-50 size exclusion chromatography, the lipolytic activity was excluded and not associated to the included neurotoxic fraction. Furthermore, no lipolytic effect could be detected in the Na+ channel specific toxin II purified from Androctonus australis hector (AaHII) or the K+ voltage-dependent channel toxin from Androctonus mauritanicus mauritanicus (KTx). Propranolol (a non selective beta adrenoreceptor (beta AR) antagonist), alprenolol and pindolol (selective beta 1/beta 2 antagonists) totally inhibited in a dose-dependent manner the lipolytic response to Bot venom (IC50 approximately 1 x 10(-7), 7.5 x 10(-8) and 3 x 10(-7)M, respectively), suggesting that venom stimulated lipolysis through the beta AR pathway. The pharmacological profiles of molecules acting more selectively on beta AR subtypes such as CGP 12177 (beta 1/ beta 2 antagonist with beta 3 agonist properties), CGP 20712A (beta 1 antagonist) and ICI 118551 (beta 2 antagonist) strongly suggest that lipolytic action of venom mainly involves the beta 2/beta 1 AR subtypes.     

Effects of beta2 adrenoceptor agonists on T-cell subpopulations

The aim of the present communication is to determine the effects of beta2 adrenoceptor agonists on growth and cytokine secretion using allergen-specific T cells. Four beta2 adrenoceptor agonists were administered at therapeutically relevant doses (salbutamol 1-2 microM; salmeterol 0.03-0.06 microM; terbutaline 0.56-1.12 microM, and fenoterol 0.7-1.4 microM to: a) Cultures of human peripheral mononuclear cells (PBMC) b) Positively selected CD4+ and CD8+ subsets, c) Allergen-specific T-cell lines (TCL). Drug effects on growth kinetics and the secretion of IL-4, IL-5, INF-gamma and IgE following T-cell stimulation were investigated. Comparing the growth inhibitory effect of the 4 beta2 agonists at 2 different concentrations, using 12 PBMC, 10 CD4+ and CD8+ and 10 TCL cultures, the following patterns were observed: PBMC-, CD4+- and CD8+-cultures: salmeterol, followed by salbutamol and fenoterol, was a more potent inhibitor than terbutaline. In long-term TCL-cultures, salmeterol was the most potent drug, followed by fenoterol. No significant differences were observed between salbutamol and terbutaline. TCL secretion of IL-4 and IL-5 (TH2 cytokines) was also significantly inhibited. In one patient, INF-gamma secretion (TH1/THO cytokine) could be enhanced by drug administration. High IgE secretion, from 1% remaining B cells in one of the patients, following PHA+IL-2 stimulation, could be reduced by the drugs. The results showed that the beta2 agonists could influence T-cell growth and function. The changes regarding cell function were individual and related to T-cell phenotypes secreting TH1/THO or TH2 cytokines. These results suggest that administration of beta2 adrenoceptor agonists could be beneficial, not only for bronchodilation, but also for suppressing the underlying inflammatory process dominated by TH2-like cytokine secretion.     

Myocardial signaling defects and impaired cardiac function of a human beta 2-adrenergic receptor polymorphism expressed in transgenic mice

A threonine to isoleucine polymorphism at amino acid 164 in the fourth transmembrane spanning domain of the beta 2-adrenergic receptor (beta 2AR) is known to occur in the human population. The functional consequences of this polymorphism to catecholamine signaling in relevant cells or to end-organ responsiveness, however, are not known. To explore potential differences between the two receptors, site-directed mutagenesis was carried out to mimic the polymorphism. Transgenic FVB/N mice were then created overexpressing wild-type (wt) beta 2AR or the mutant Ile-164 receptor in a targeted manner in the heart using a murine alpha myosin heavy chain promoter. The functional properties of the two receptors were then assessed at the level of in vitro cardiac myocyte signaling and in vivo cardiac responses in intact animals. The expression levels of these receptors in the two lines chosen for study were approximately 1200 fmol/mg protein in cardiac membranes, which represents a approximately 45-fold increase in expression over endogenous beta AR. Myocyte membrane adenylyl cyclase activity in the basal state was significantly lower in the Ile-164 mice (19.5 +/- 2.7 pmol/min/mg) compared with wt beta 2AR mice (35.0 +/- 4.1 pmol/min/mg), as was the maximal isoproterenol-stimulated activity (49.8 +/- 7.8 versus 77.1 +/ 7.3 pmol/min/mg). In intact animals, resting heart rate (441 +/- 21 versus 534 +/- 17 bpm) and dP/dtmax (10,923 +/- 730 versus 15,308 +/- 471 mmHg/sec) were less in the Ile-164 mice as compared with wt beta 2AR mice. Similarly, the physiologic responses to infused isoproterenol were notably less in the mutant expressing mice. Indeed, these values, as well as other contractile parameters, were indistinguishable between Ile-164 mice and nontransgenic littermates. Taken together, these results demonstrate that the Ile-164 polymorphism is substantially dysfunctional in a relevant target tissue, as indicated by depressed receptor coupling to adenylyl cyclase in myocardial membranes and impaired receptor mediated cardiac function in vivo. Under normal homeostatic conditions or in circumstances where sympathetic responses are compromised due to diseased states, such as heart failure, this impairment may have important pathophysiologic consequences.