An allosteric modulator binds to a conformational hub in the ß2 adrenergic receptor.

Most drugs acting on G-protein-coupled receptors target the orthosteric binding pocket where the native hormone or neurotransmitter binds. There is much interest in finding allosteric ligands for these targets because they modulate physiologic signaling and promise to be more selective than orthosteric ligands. Here we describe a newly developed allosteric modulator of the ß2-adrenergic receptor (ß2AR), AS408, that binds to the membrane-facing surface of transmembrane segments 3 and 5, as revealed by X-ray crystallography. AS408 disrupts a water-mediated polar network involving E1223.41 and the backbone carbonyls of V2065.45 and S2075.46. The AS408 binding site is adjacent to a previously identified molecular switch for ß2AR activation formed by I3.40, P5.50 and F6.44. The structure reveals how AS408 stabilizes the inactive conformation of this switch, thereby acting as a negative allosteric modulator for agonists and positive allosteric modulator for inverse agonists.

Probing the Existence of a Metastable Binding Site at the ß2-Adrenergic Receptor with Homobivalent Bitopic Ligands.

Herein, we report the development of bitopic ligands aimed at targeting the orthosteric binding site (OBS) and a metastable binding site (MBS) within the same receptor unit. Previous molecular dynamics studies on ligand binding to the ß2-adrenergic receptor (ß2AR) suggested that ligands pause at transient, less-conserved MBSs. We envisioned that MBSs can be regarded as allosteric binding sites and targeted by homobivalent bitopic ligands linking two identical pharmacophores. Such ligands were designed based on docking of the antagonist (S)-alprenolol into the OBS and an MBS and synthesized. Pharmacological characterization revealed ligands with similar potency and affinity, slightly increased ß2/ß1AR-selectivity, and/or substantially slower ß2AR off-rates compared to (S)-alprenolol. Truncated bitopic ligands suggested the major contribution of the metastable pharmacophore to be a hydrophobic interaction with the ß2AR, while the linkers alone decreased the potency of the orthosteric fragment. Altogether, the study underlines the potential of targeting MBSs for improving the pharmacological profiles of ligands.

Carvedilol inhibits EGF-mediated JB6 P+ colony formation through a mechanism independent of adrenoceptors.

Carvedilol is reported to prevent cancers in humans and animal models. However, a molecular mechanism has yet to be established, and the extent to which other ß-blockers are chemopreventive remains relatively unknown. A comparative pharmacological approach was utilized with the expectation that a mechanism of action could be devised. JB6 Cl 41-5a (JB6 P+) murine epidermal cells were used to elucidate the chemopreventative properties of ß-blockers, as JB6 P+ cells recapitulate in vivo tumor promotion and chemoprevention. The initial hypothesis was that ß-blockers that are GRK/ß-arrestin biased agonists, like carvedilol, are chemopreventive. Sixteen ß-blockers of different classes, isoproterenol, and HEAT HCl were individually co-administered with epidermal growth factor (EGF) to JB6 P+ cells to examine the chemopreventative properties of each ligand. Cytotoxicity was examined to ensure that the anti-transformation effects of each ligand were not due to cellular growth inhibition. Many of the examined ß-blockers suppressed EGF-induced JB6 P+ cell transformation in a non-cytotoxic and concentration-dependent manner. However, the IC50 values are high for the most potent inhibitors (243, 326, and 431 nM for carvedilol, labetalol, and alprenolol, respectively) and there is no correlation between pharmacological properties and inhibition of transformation. Therefore, the role of α1- and ß2-adrenergic receptors (AR) was examined by standard competition assays and shRNA targeting ß2-ARs, the only ß-AR expressed in JB6 P+ cells. The results reveal that pharmacological inhibition of α1- and ß2-ARs and genetic knockdown of ß2-ARs did not abrogate carvedilol-mediated inhibition of EGF-induced JB6 P+ cell transformation. Furthermore, topical administration of carvedilol protected mice from UV-induced skin damage, while genetic ablation of ß2-ARs increased carvedilol-mediated effects. Therefore, the prevailing hypothesis that the chemopreventive property of carvedilol is mediated through ß-ARs is not supported by this data.

Identification of Alprenolol Hydrochloride as an Anti-prion Compound Using Surface Plasmon Resonance Imaging.

Prion diseases are transmissible neurodegenerative disorders of humans and animals, which are characterized by the aggregation of abnormal prion protein (PrPSc) in the central nervous system. Although several small compounds that bind to normal PrP (PrPC) have been shown to inhibit structural conversion of the protein, an effective therapy for human prion disease remains to be established. In this study, we screened 1200 existing drugs approved by the US Food and Drug Administration (FDA) for anti-prion activity using surface plasmon resonance imaging (SPRi). Of these drugs, 31 showed strong binding activity to recombinant human PrP, and three of these reduced the accumulation of PrPSc in prion-infected cells. One of the active compounds, alprenolol hydrochloride, which is used clinically as a ß-adrenergic blocker for hypertension, also reduced the accumulation of PrPSc in the brains of prion-infected mice at the middle stage of the disease when the drug was administered orally with their daily water from the day after infection. Docking simulation analysis suggested that alprenolol hydrochloride fitted into the hotspot within mouse PrPC, which is known as the most fragile structure within the protein. These findings provide evidence that SPRi is useful in identifying effective drug candidates for neurodegenerative diseases caused by abnormal protein aggregation, such as prion diseases.

Novel Paradigms Governing ß1-Adrenergic Receptor Trafficking in Primary Adult Rat Cardiac Myocytes.

The ß1-adrenergic receptor (ß1-AR) is a major cardiac G protein-coupled receptor, which mediates cardiac actions of catecholamines and is involved in genesis and treatment of numerous cardiovascular disorders. In mammalian cells, catecholamines induce the internalization of the ß1-AR into endosomes and their removal promotes the recycling of the endosomal ß1-AR back to the plasma membrane; however, whether these redistributive processes occur in terminally differentiated cells is unknown. Compartmentalization of the ß1-AR in response to ß-agonists and antagonists was determined by confocal microscopy in primary adult rat ventricular myocytes (ARVMs), which are terminally differentiated myocytes with unique structures such as transverse tubules (T-tubules) and contractile sarcomeres. In unstimulated ARVMs, the fluorescently labeled ß1-AR was expressed on the external membrane (the sarcolemma) of cardiomyocytes. Exposing ARVMs to isoproterenol redistributed surface ß1-ARs into small (∼225-250 nm) regularly spaced internal punctate structures that overlapped with puncta stained by Di-8 ANEPPS, a membrane-impermeant T-tubule-specific dye. Replacing the ß-agonist with the ß-blocker alprenolol, induced the translocation of the wild-type ß1-AR from these punctate structures back to the plasma membrane. This step was dependent on two barcodes, namely, the type-1 PDZ binding motif and serine at position 312 of the ß1-AR, which is phosphorylated by a pool of cAMP-dependent protein kinases anchored at the type-1 PDZ of the ß1-AR. These data show that redistribution of the ß1-AR in ARVMs from internal structures back to the plasma membrane was mediated by a novel sorting mechanism, which might explain unique aspects of cardiac ß1-AR signaling under normal or pathologic conditions.

Antilipolytic effects of 1,8-naphthyridine derivatives ß-adrenoceptor antagonists in rat white adipocytes.

The rat fat cell ß-adrenoceptors were investigated by studying the effects of new 1,8-naphthyridine derivatives synthesized starting from 7-amino-2-chloro-3-phenyl-1,8-naphthyridine on lipolysis induced by isoprenaline, and alprenolol. Lipolysis induced by isoprenaline agonist was competitively antagonized by the 1,8-naphthyridine analogue with a 7-hydroxy-2-(4'-methoxybenzylamine)-6-nitro-3-phenyl substituent designated as 3. In contrast, 10, 50, and 100 µm of 7-methoxy and 7-ethoxy derivatives did not modify the concentration-response curve of isoprenaline. A rightward shift of the curve was, however, observed with 50 µm of a 7-methoxy-2-(4'-methoxybenzylamine)-6-amino-3-phenyl substituent designated as 10. The selective ß1 -AR antagonist, 7-hydroxy-4-morpholinomethyl-2-piperazino-1,8-naphthyridine slightly reduced isoprenaline-induced lipolysis only at high doses. Alprenolol-mediated lipolytic effect was antagonized by derivative 3, 10 and the selective ß3 -AR antagonist SR 59,230A, but resistant to the selective ß1 -AR antagonist 7-hydroxy-4-morpholinomethyl-2-piperazino-1,8-naphthyridine. The results provide preliminary pharmacological evidence for the antilipolytic effect of the newly synthesized 1,8-naphthyridine derivatives on rat fat cells. The analogues designated as 3 and 10 were the most potent antagonists of this series.

Flow Cytometric Quantification of Peripheral Blood Cell ß-Adrenergic Receptor Density and Urinary Endothelial Cell-Derived Microparticles in Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is a heterogeneous disease characterized by severe angiogenic remodeling of the pulmonary artery wall and right ventricular hypertrophy. Thus, there is an increasing need for novel biomarkers to dissect disease heterogeneity, and predict treatment response. Although ß-adrenergic receptor (ßAR) dysfunction is well documented in left heart disease while endothelial cell-derived microparticles (Ec-MPs) are established biomarkers of angiogenic remodeling, methods for easy large clinical cohort analysis of these biomarkers are currently absent. Here we describe flow cytometric methods for quantification of ßAR density on circulating white blood cells (WBC) and Ec-MPs in urine samples that can be used as potential biomarkers of right heart failure in PAH. Biotinylated ß-blocker alprenolol was synthesized and validated as a ßAR specific probe that was combined with immunophenotyping to quantify ßAR density in circulating WBC subsets. Ec-MPs obtained from urine samples were stained for annexin-V and CD144, and analyzed by a micro flow cytometer. Flow cytometric detection of alprenolol showed that ßAR density was decreased in most WBC subsets in PAH samples compared to healthy controls. Ec-MPs in urine was increased in PAH compared to controls. Furthermore, there was a direct correlation between Ec-MPs and Tricuspid annular plane systolic excursion (TAPSE) in PAH patients. Therefore, flow cytometric quantification of peripheral blood cell ßAR density and urinary Ec-MPs may be useful as potential biomarkers of right ventricular function in PAH.

ß-Blockers have differential effects on the murine asthma phenotype.

BACKGROUND AND PURPOSE: Our previous studies have shown the ß2 -adrenoceptor and its endogenous ligand, adrenaline, are required for development of the asthma phenotype in murine asthma models. Chronic administration of some, but not other, ß-blockers attenuated the asthma phenotype and led us to hypothesize that biased signalling was the basis of their differential effects, experimentally and clinically. EXPERIMENTAL APPROACH: We used mice with no detectable systemic adrenaline (PNMT(-/-) ) and wild-type (WT) mice to study the effects of four ß-blockers, alprenolol, carvedilol, propranolol and nadolol, in an ovalbumin sensitization and challenge (Ova S/C) murine model of asthma. The parameters measured were inflammatory cell infiltration, mucous metaplasia and airway hyperresponsiveness. To interpret the pharmacological action of these ligands quantitatively, we conducted computer simulations of three-state models of receptor activation. KEY RESULTS: Ova S/C PNMT(-/-) mice do not develop an asthma phenotype. Here, we showed that administration of alprenolol, carvedilol or propranolol in the absence of interference from adrenaline using Ova S/C PNMT(-/-) mice resulted in the development of an asthma phenotype, whereas nadolol had no effect. Ova S/C WT mice did develop an asthma phenotype, and administration of alprenolol, propranolol and carvedilol had no effect on the asthma phenotype. However, nadolol prevented development of the asthma phenotype in Ova S/C WT mice. Computer simulations of these four ligands were consistent with the isolated three-state receptor model. CONCLUSION AND IMPLICATIONS: ß-Blockers have different effects on the murine asthma phenotype that correlate with reported differences in activation or inhibition of downstream ß2 -adrenoceptor signalling pathways.

Label-free functional selectivity assays.

G protein-coupled receptors (GPCRs) represent the largest class of drug targets. Ligand-directed functional selectivity or biased agonism opens new possibility for discovering GPCR drugs with better efficacy and safety profiles. However, quantification of ligand bias is challenging. Herein, we present five different label-free dynamic mass redistribution (DMR) approaches to assess ligand bias acting at the ß2-adrenergic receptor (ß2AR). Multiparametric analysis of the DMR agonist profiles reveals divergent pharmacology of a panel of ß2AR agonists. DMR profiling using catechol as a conformational probe detects the presence of multiple conformations of the ß2AR. DMR assays under microfluidics, together with chemical biology tools, discover ligand-directed desensitization of the receptor. DMR antagonist reverse assays manifest biased antagonism. DMR profiling using distinct probe-modulated cells detects the biased agonism in the context of self-referenced pharmacological activity map.

Modulation of ß-adrenergic receptor in rat lung after gamma irradiation.

OBJECTIVES: To study the effect of γ irradiation on ß-adrenergic receptors of the lung. MATERIALS AND METHODS: Healthy Sprague-Dawley rats were used as an animal model. Cell membrane proteins of lung tissue were harvested after the whole lung received 20 Gy of 60Co γ irradiation. 125I-labeled iodopindolol (125I-IPIN) was used as a ligand of ß-adrenergic receptors. The numbers of the ß-adrenergic receptors were determined by radioligand-receptor binding assay (RBA). Data were compared with irreversible blockage using antagonist bromoacetylalprenololmenthan (BAAM). RESULTS: The post-radiation RBA assay showed that the number of ß-adrenergic receptors in lung tissue decreased at a steady rate. It decreased to 48% of the normal level at the 15th day after irradiation. At 40 days after radiation the level of ß-adrenergic receptors started to increase at a steady rate and reached to the normal level around 70 days after radiation. There were significant differences in receptor synthesis, degradation and regeneration rates between irradiation group and BAMM group. CONCLUSIONS: The whole lung irradiation could severely affect the levels of ß-adrenergic receptors. The potential clinical implications of radiation-induced changes of ß-adrenergic receptors warrant further investigation.

Noradrenergic but not cholinergic modulation of olfactory bulb during processing of near threshold concentration stimuli.

Neuromodulatory systems such as noradrenaline (NE), acetylcholine (ACh), and serotonin (5HT) serve important functions in sensory perception. We use the olfactory bulb (OB) as a model system to study the roles of individual neuromodulators in sensory perception. Using a spontaneous, nonreward motivated detection task, as well as a reward-motivated task, we show that rats can easily respond to odorants at very low concentrations when motivated to do so in a food-rewarded task, despite not showing spontaneous responses to these low concentration odorants. Using the same tasks paired with local bulbar infusions of noradrenergic and cholinergic drugs, we then show that rats engage their noradrenergic, but not their cholinergic system, to better respond to near threshold odorants. These results suggest that while cholinergic modulation of OB function is mostly important for odor decorrelation and discrimination, noradrenergic modulation is important for signal-to-noise modulation.

Synthesis and characterization of high-affinity 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene-labeled fluorescent ligands for human ß-adrenoceptors.

The growing practice of exploiting noninvasive fluorescence-based techniques to study G protein-coupled receptor pharmacology at the single cell and single molecule level demands the availability of high-quality fluorescent ligands. To this end, this study evaluated a new series of red-emitting ligands for the human ß-adrenoceptor family. Upon the basis of the orthosteric ligands propranolol, alprenolol, and pindolol, the synthesized linker-modified congeners were coupled to the commercially available fluorophore BODIPY 630/650-X. This yielded high-affinity ß-adrenoceptor fluorescent ligands for both the propranolol and alprenolol derivatives; however, the pindolol-based products displayed lower affinity. A fluorescent diethylene glycol linked propranolol derivative (18a) had the highest affinity (log K(D) of -9.53 and -8.46 as an antagonist of functional ß2- and ß1-mediated responses, respectively). Imaging studies with this compound further confirmed that it can be employed to selectively label the human ß2-adrenoceptor in single living cells, with receptor-associated binding prevented by preincubation with the nonfluorescent ß2-selective antagonist 3-(isopropylamino)-1-[(7-methyl-4-indanyl)oxy]butan-2-ol (ICI 118551) ( J. Cardiovasc. Pharmacol.1983, 5, 430-437. ).

Involvement of ß-adrenergic receptor in synaptic vesicle swelling and implication in neurotransmitter release.

Secretory vesicle swelling is required for vesicular discharge during cell secretion. The G(αo) -mediated water channel aquaporin-6 (AQP-6) involvement in synaptic vesicle (SV) swelling in neurons has previously been reported. Studies demonstrate that in the presence of guanosine triphosphate (GTP), mastoparan, an amphiphilic tetradecapeptide from wasp venom, activates G(o) protein GTPase, and stimulates SV swelling. Stimulation of G proteins is believed to occur via insertion of mastoparan into the phospholipid membrane to form a highly structured α-helix that resembles the intracellular loops of G protein-coupled adrenergic receptors. Consequently, the presence of adrenoceptors and the presence of an endogenous ß-adrenergic agonist at the SV membrane is suggested. Immunoblot analysis of SV using ß-adrenergic receptor antibody, and vesicle swelling experiments using ß-adrenergic agonists and antagonists, demonstrate the presence of functional ß-adrenergic receptors at the SV membrane. Since a recent study shows vH(+) -ATPase to be upstream of AQP-6 in the pathway leading from G(αo) -mediated swelling of SV, participation of an endogenous ß-adrenergic agonist, in the binding and stimulation of its receptor to initiate the swelling cascade is demonstrated.

Noradrenergic modulation of behavioral odor detection and discrimination thresholds in the olfactory bulb.

The mammalian main olfactory bulb (MOB) receives a significant noradrenergic input from the locus coeruleus. Norepinephrine (NE) is involved in the acquisition of conditioned odor preferences in neonatal animals and in some species-specific odor-dependent behaviors. Thus far, the role of NE in odor processing in adult rats remains less studied. We investigated the role of noradrenergic modulation in the MOB on odor detection and discrimination thresholds using behavioral and computational modeling approaches. Adult rats received bilateral MOB injections of vehicle, NE (0.1-1000 microM), noradrenergic receptor antagonists and NE + receptor antagonists combined. NE infusion improved odor detection and discrimination as a function of NE and odor concentration. The effect of NE on detection and discrimination magnitude at any given odor concentration varied in a non-linear function with respect to NE concentration. Receptor antagonist infusion demonstrated that alpha1 receptor activation is necessary for the modulatory effect of NE. Computational modeling showed that increases in the strength of alpha1 receptor activation leads to improved odor signal-to-noise ratio and spike synchronization in mitral cells that may underlie the behaviorally observed decrease of detection and discrimination thresholds. Our results are the first to show that direct infusion of NE or noradrenergic receptor antagonists into a primary sensory network modulates sensory detection and discrimination thresholds at very low stimulus concentrations.

The interactivities with lipid membranes differentially characterize selective and nonselective beta1-blockers.

BACKGROUND AND OBJECTIVE: beta-Adrenoceptor-blocking agents have been used for perioperative management during anaesthesia, in which selective beta1-blockers are advantageous over nonselective beta-blockers. Apart from the different affinity for beta-adrenoceptors, beta1-blockers were differentially characterized in light of their different interaction with lipid membranes. METHODS: Selective (atenolol, metoprolol and esmolol) and nonselective (alprenolol, oxprenolol and propranolol) beta1-blockers were reacted at 0.2-1 mmol l with 1,2-dipalmitoylphosphatidylcholine liposomes and biomimetic membranes consisting of phospholipids, sphingolipid and cholesterol. Their membrane interactivities were comparatively determined using the potency to modify membrane fluidity by measuring fluorescence polarization. Their relative hydrophobicities were evaluated by reversed-phase liquid chromatography. RESULTS: The chromatographic evaluation divided the tested drugs into more hydrophobic ones containing nonselective beta-blockers and less hydrophobic ones containing selective beta1-blockers. Nonselective beta-blockers, but not selective beta1-blockers, fluidized liposomal membranes, with the potency being oxprenolol < alprenolol < propranolol. Membrane-active alprenolol preferentially acted on the hydrophobic deeper regions of phospholipid bilayers. The potency of nonselective beta-blockers to fluidize biomimetic membranes was greatest in propranolol, followed by alprenolol and oxprenolol, whereas all selective beta1-blockers were inactive. CONCLUSION: The membrane-fluidizing effects of beta-blockers are correlated with their relative hydrophobicities and their respective conformations to perturb the alignment of phospholipid acyl chains. The membrane-interacting characteristics differentiate beta-blockers as nonselective propranolol, alprenolol and oxprenolol vs. beta1-selective atenolol, metoprolol and esmolol. Such differentiation reflects not only the structural difference but also the beta-adrenoceptor-blocking difference. The membrane fluidization may be partly responsible for the nonselective blockade of beta-adrenoceptors.

The importance of valine 114 in ligand binding in beta(2)-adrenergic receptor.

G-protein coupled receptors (GPCRs) are transmembrane signaling molecules, with a majority of them performing important physiological roles. beta(2)-Adrenergic receptor (beta(2)-AR) is a well-studied GPCRs that mediates natural responses to the hormones adrenaline and noradrenaline. Analysis of the ligand-binding region of beta(2)-AR using the recently solved high-resolution crystal structures revealed a number of highly conserved amino acids that might be involved in ligand binding. However, detailed structure-function studies on some of these residues have not been performed, and their role in ligand binding remains to be elucidated. In this study, we have investigated the structural and functional role of a highly conserved residue valine 114, in hamster beta(2)-AR by site-directed mutagenesis. We replaced V114 in hamster beta(2)-AR with a number of amino acid residues carrying different functional groups. In addition to the complementary substitutions V114I and V114L, the V114C and V114E mutants also showed significant ligand binding and agonist dependent G-protein activation. However, the V114G, V114T, V114S, and V114W mutants failed to bind ligand in a specific manner. Molecular modeling studies were conducted to interpret these results in structural terms. We propose that the replacement of V114 influences not only the interaction of the ethanolamine side-chains but also the aryl-ring of the ligands tested. Results from this study show that the size and orientation of the hydrophobic residue at position V114 in beta(2)-AR affect binding of both agonists and antagonists, but it does not influence the receptor expression or folding.

Beta2-adrenoceptor signaling is required for the development of an asthma phenotype in a murine model.

Chronic regular use of beta(2)-adrenoceptor (beta(2)-AR) agonists in asthma is associated with a loss of disease control and increased risk of death. Conversely, we have found that administration of beta(2)-AR inverse agonists results in attenuation of the asthma phenotype in an allergen-driven murine model. Besides antagonizing agonist-induced signaling and reducing signaling by empty receptors, beta-AR inverse agonists can also activate signaling by novel pathways. To determine the mechanism of the beta-AR inverse agonists, we compared the asthma phenotype in beta(2)-AR-null and wild-type mice. Antigen challenge of beta(2)-AR-null mice produced results similar to what was observed with chronic beta(2)-AR inverse agonist treatment, namely, reductions in mucous metaplasia, airway hyperresponsiveness (AHR), and inflammatory cells in the lungs. These results indicate that the effects of beta(2)-AR inverse agonists are caused by inhibition of beta(2)-AR signaling rather than by the induction of novel signaling pathways. Chronic administration of alprenolol, a beta-blocker without inverse agonist properties, did not attenuate the asthma phenotype, suggesting that it is signaling by empty receptors, rather than agonist-induced beta(2)-AR signaling, that supports the asthma phenotype. In conclusion, our results demonstrate that, in a murine model of asthma, beta(2)-AR signaling is required for the full development of three cardinal features of asthma: mucous metaplasia, AHR, and the presence of inflammatory cells in the lungs.

Beta-blockers alprenolol and carvedilol stimulate beta-arrestin-mediated EGFR transactivation.

Recent evidence suggests that binding of agonist to its cognate receptor initiates not only classical G protein-mediated signaling, but also beta-arrestin-dependent signaling. One such beta-arrestin-mediated pathway uses the beta(1)-adrenergic receptor (beta(1)AR) to transactivate the EGFR. To determine whether beta-adrenergic ligands that do not activate G protein signaling (i.e., beta-blockers) can stabilize the beta(1)AR in a signaling conformation, we screened 20 beta-blockers for their ability to stimulate beta-arrestin-mediated EGFR transactivation. Here we show that only alprenolol (Alp) and carvedilol (Car) induce beta(1)AR-mediated transactivation of the EGFR and downstream ERK activation. By using mutants of the beta(1)AR lacking G protein-coupled receptor kinase phosphorylation sites and siRNA directed against beta-arrestin, we show that Alp- and Car-stimulated EGFR transactivation requires beta(1)AR phosphorylation at consensus G protein-coupled receptor kinase sites and beta-arrestin recruitment to the ligand-occupied receptor. Moreover, pharmacological inhibition of Src and EGFR blocked Alp- and Car-stimulated EGFR transactivation. Our findings demonstrate that Alp and Car are ligands that not only act as classical receptor antagonists, but can also stimulate signaling pathways in a G protein-independent, beta-arrestin-dependent fashion.

Neuropeptide Y and glucocorticoid secretion from guinea pig adrenal gland: an in vivo and in vitro study.

The effects of neuropeptide Y (NPY) on adrenal glucocorticoid secretion are controversial, and we have investigated this issue in guinea pigs, where, like in humans and cows, the main glucocorticoid hormone is cortisol. In vivo experiments showed that prolonged NPY administration markedly lowered cortisol plasma concentration not only in normal guinea pigs, but also in animals whose hypothalamic-pituitary-adrenal axis and renin-angiotensin system had been pharmacologically interrupted by the simultaneous administration of dexamethasone and captopril. In vitro experiments ruled out the possibility that in vivo glucocorticoid anti-secretagogue action of NPY can ensue from a direct effect on the adrenal gland. In fact, NPY did not affect cortisol secretion from dispersed guinea pig inner adrenocortical cells. In contrast, NPY raised cortisol production from adrenal slices containing medullary tissue, and this effect was blocked by the beta-adrenoceptor antagonist l-alprenolol. This finding, coupled with the demonstration that NPY enhanced catecholamine release from guinea pigadrenomedullary tissue, strongly suggests that NPY may stimulate glucocorticoid secretion in this species through an indirect mechanism involving catecholamines, that in a paracrine manner promote the secretion of inner adrenocortical cells. In light of these observations, the conclusion is drawn that the in vivo effects of NPY are mediated by mechanism(s) independent of either the suppression of the main adrenal agonists ACTH and angiotensin-II or the direct inhibition of adrenal secretion. The possibility merits an investigation into whether NPY enhances the production of peptides, which, like leptin, inhibit adrenal glucocorticoid secretion acting as circulating hormones.

The role of Gi proteins in reduced vasorelaxation response to beta-adrenoceptor agonists in rat aorta during maturation.

Beta-adrenoceptor mediated vasorelaxation and cAMP production decline during maturation and aging in rat aorta. beta-adrenoceptor-stimulated vasorelaxation is mainly triggered by Gsalpha-mediated activation of adenylyl cyclase. beta(2)-adrenoceptors can also activate Gi protein which inhibits adenylyl cyclase activity. In this study, we examined the role of Gi proteins in the decreased beta-adrenoceptor mediated responses during maturation. Pertussis toxin treatment of aortic rings to inhibit Gialpha activation completely restored age related decline in isoproterenol-stimulated maximal vasorelaxation in 3-month old rats. This treatment increased the potency, but not the maximal response of isoproteronol to produce vasorelaxation in 6 month old rats. The maximal isoproteronol stimulated cAMP responses were also partially restored in pertussis toxin-treated rings from 3 or 6-month old rats. We also examined beta-adrenoceptor stimulated binding of (35)[S]GTPgammaS to Gsalpha and Gialpha1/2 in aortic membranes from 1, 3 and 6-month old rats. In 1-month old rats, isoproterenol-stimulated (35)[S]GTPgammaS binding to Gsalpha was significantly higher than that of 3 or 6-month old rats. Isoproterenol-stimulated (35)[S]GTPgammaS binding to Gialpha1/2 was found to be significantly increased in 3 or 6-month old rats compared to 1-month old rats. The results of this study showed that beta-adrenoceptor-mediated activation of Gs and Gi proteins was declined and increased, respectively, and inhibition of the Gi mediated activity by pertussis toxin treatment partially restored impaired vasorelaxation and cAMP response to beta-adrenoceptor stimulation during maturation in rat aorta. The decrease in beta-adrenoceptor mediated activation of Gs gradually increased during maturation. All together these results indicated that beta-adrenoceptor mainly activates Gs protein in aorta from 1-month old rats, while it activates Gi and with a certain degree of decline it also activates Gs in aorta from 3 and 6-months old rats and not only the increase in beta-adrenoceptor coupling to Gi but also the decrease in its coupling to Gs play a role in the impaired beta-adrenoceptor responses in rat aorta during maturation.