Solubilization of a mammalian β-adrenergic receptor

Summary Binding sites for iodohydroxybenzylpindolol with characteristics of a ₂-adrenergic receptor have been identified in a crude membrane fraction from guinea-pig lung. The binding sites could be solubilized by treatment of the membrane fraction with digitonin. Upon solubilization receptors retain their ₂-adrenergic type as indicated by the rank order of potencies of agonists in binding-inhibition experiments. The solubilized receptors demonstrate a marked increase in affinity for agonists compared with particulate receptors whereas antagonist affinity remains unchanged. Solubilized receptors are insensitive to divalent cations (Mg²⁺, Mn²⁺, Ca²⁺) which increase the potency of agonists for particulate receptors. The effects of Mg²⁺ can be reversed by Gpp(NH)p in particulate preparations; Gpp(NH)p is ineffective for the solubilized preparation. These experiments establish that -adrenergic receptors can be solubilized even from crude mammalian membrane preparations. They also show that the mammalian -adrenergic receptor in situ is under constraints with respect to agonist affinity and is modulated by divalent cations and guanyl nucleotides in the intact membrane.with technical assistance of L. Braun and C. KonradThis report is part of a dissertation to be presented by J. K. to the Fachbereich Humanmedizin, Justus Liebig-Universität Giessen, in partial fulfillment of the requirements for a Doctor of Medical Science degree     

Combined β-adrenergic and corticosteroid receptor activation regulates AMPA receptor function in hippocampal neurons

Shortly after stress, limbic neurons are exposed to high levels of noradrenaline and corticosterone. These hormones are necessary for optimal behavioural adaptation. Behavioural effects critically depend on noradrenaline acting via β-adrenergic receptors, but these effects are strongly modulated by corticosterone, indicating putative interactions between the two hormones. Since both noradrenaline and corticosterone are known to quickly affect properties of AMPA-type glutamate receptors (AMPAR), we here examined - in hippocampal neurons - three parameters which give insight in the functionality of AMPARs: phosphorylation, surface expression and spontaneous synaptic transmission. In homogenates of adult hippocampal slices, application of corticosterone (30 nM for 15 min) by itself did not affect phosphorylation of the AMPAR GluA1 subunit at S845 or S831. Co-application of the β-adrenergic receptor agonist isoproterenol (10 μM) largely increased S845 (but not S831) phosphorylation. Corticosterone also did not change GluA1 and GluA2 surface expression in hippocampal primary cultures. However, combined administration of corticosterone and 1 μM isoproterenol - which by itself was ineffective - enhanced surface expression. Interestingly, 10 μM isoproterenol alone enhanced GluA1 surface expression, but this was decreased by corticosterone. Finally, in hippocampal primary cultures, the inter-event interval of miniature excitatory postsynaptic currents (mEPSCs) was decreased by the combination of 1 μM isoproterenol and corticosterone (which were ineffective by themselves) while the same combination did not affect the amplitude. We conclude that AMPAR phosphorylation, surface expression and mEPSC inter-event interval respond most strongly to a combination of corticosterone and β-adrenergic receptors. These combined hormonal effects on glutamate transmission might contribute to their memory-enhancing effects.     

Src-kinase-dependent epidermal growth factor receptor transactivation in salivary mucin secretion in response to β-adrenergic G-protein-coupled receptor activation

The principal regulatory factors that control the flow and make-up of salivary secretion are neurotransmitters, released by parasympathetic and sympathetic innervation, that trigger activation of G protein-coupled receptors (GPCRs) on the acinar cells of salivary glands and stimulate the generation of soluble second messengers. In this study, we report that activation of GPCR by -adrenergic agonist leading to stimulation in salivary mucin secretion occurs with the involvement of epidermal growth factor receptor (EGFR). Using [³H]glucosamine-labeled mucous acinar cells of sublingual salivary gland in culture, we show that stimulatory effect of -adrenergic agonist, isoproterenol, on mucin secretion was inhibited in a concentration-dependent manner by EGFR kinase inhibitor, PD153035, as well as wortmannin, an inhibitor of PI3K. Moreover, both inhibitors caused the impedance in the acinar cell mucin secretory responses to -adrenergic agonist-generated second messenger, cAMP, as well as adenylate cyclase activator, forskolin. The acinar cell secretory responses to isoproterenol, furthermore, were blunted in a concentration-dependent fashion by PP2, a selective inhibitor of tyrosine kinase Src responsible for ligand-independent EGFR autophosphorylation. However, no significant alterations in the acinar cell mucin secretory responses to isoproterenol, cAMP or forskolin were attained with an inhibitor of the ERK pathway, PD98059. Our findings underline the role of EGFR as a convergence point in modulation of salivary mucin secretion triggered by -adrenergic agonist GPCR activation and demonstrate the importance of Src kinase in the EGFR transactivation process.     

Regulation of gap-junction protein connexin 43 by β-adrenergic receptor stimulation in rat cardiomyocytes

Aim:

β-adrenergic receptor (β-AR) agonists are among the most potent factors regulating cardiac electrophysiological properties. Connexin 43 (Cx43), the predominant gap-junction protein in the heart, has an indispensable role in modulating cardiac electric activities by affecting gap-junction function. The present study investigates the effects of short-term stimulation of β-AR subtypes on Cx43 expression and gap junction intercellular communication (GJIC) function.

Methods:

The level of Cx43 expression in neonatal rat cardiomyocytes (NRCM) was detected by a Western blotting assay. The GJIC function was evaluated by scrape loading/dye transfer assay.

Results:

Stimulation of β-AR by the agonist isoproterenol for 5 min induces the up-regulation of nonphosphorylated Cx43 protein level, but not total Cx43. Selective β₂-AR inhibitor ICI 118551, but not β₁-AR inhibitor {"type":"entrez-protein","attrs":{"text":"CGP20712","term_id":"874704353","term_text":"CGP20712"}}CGP20712, could fully abolish the effect. Moreover, pretreatment with both protein kinase A inhibitor H89 and G_i protein inhibitor pertussis toxin also inhibited the isoproterenol-induced increase of nonphosphorylated Cx43 expression. Isoproterenol-induced up-regulation of nonphosphorylated Cx43 is accompanied with enhanced GJIC function.

Conclusion:

Taken together, β₂-AR stimulation increases the expression of nonphosphorylated Cx43, thereby enhancing the gating function of gap junctions in cardiac myocytes in both a protein kinase A- and G_i-dependent manner.     

Recent progress in α1-adrenergic receptor research

Alpha1-Adrenergic receptors (AR) play an important role in the regulation of physiological responses mediated by norepinephrine and epinephrine, particularly in the cardiovascular system. The three cloned alpha1-AR subtypes (alpha1A, alpha1B, and alpha1D) are G protein-coupled receptors that signal through the Gq/11 signaling pathway, each showing distinct pharmacological properties and tissue distributions. However, due to the lack of highly subtype-selective drugs, the functional roles of individual subtypes are still not clear. Development of new subtype-specific drugs will greatly facilitate the identification of the functions of each subtype. Conopeptide rho-TIA has been found to be a new alpha1B-AR selective antagonist with different modes of inhibition at alpha1-AR subtypes. In addition, recent studies using genetically engineered mice have shed some light on alpha1-AR functions in vivo, especially in the cardiovascular system and brain. Several proteins have been shown to interact directly with particular alpha1-AR, and may be important in regulating receptor function. Receptor heterodimerization has been shown to be important for cell surface expression, signaling and internalization. These new observations are likely to help elucidate the functional roles of individual alpha1-AR subtypes.     

Regulation of gap-junction protein connexin 43 by β-adrenergic receptor stimulation in rat cardio- myocytes

β-adrenergic receptor （β-AR） agonists are among the most potent factors regulating cardiac electrophysiological properties. Connexin 43 （Cx43）, the predominant gap-junction protein in the heart, has an indispensable role in modulating cardiac electric activities by affecting gap-junction function. The present study investigates the effects of short-term stimulation of β-AR subtypes on Cx43 expression and gap junction intercellular communication （GJIC） function.
Methods： The level of Cx43 expression in neonatal rat cardiomyocytes （NRCM） was detected by a Western blotting assay. The GJIC function was evaluated by scrape loading/dye transfer assay.
Results： Stimulation of β-AR by the agonist isoproterenol for 5 min induces the up-regulation of nonphosphorylated Cx43 protein level, but not total Cx43. Selective β2-AR inhibitor ICI 118551, but not β1-AR inhibitor CGP20712, could fully abolish the effect. Moreover, pretreatment with both protein kinase A inhibitor H89 and Gi protein inhibitor pertussis toxin also inhibited the isoproterenol-induced increase of nonphosphorylated Cx43 expression. Isoproterenol-induced up-regulation of nonphosphorylated Cx43 is accompanied with enhanced GJIC function.
Conclusion： Taken together, β2-AR stimulation increases the expression of nonphosphorylated Cx43, thereby enhancing the gating function of gap junctions in cardiac myocytes in both a protein kinase A-and G1-dependent manner.     

The pharmacogenetics of β-adrenergic receptor antagonists in the treatment of hypertension and heart failure

INTRODUCTION: β-Blockers have an important therapeutic role throughout the cardiovascular continuum. However, there is considerable variation in response to these drugs, which may be related to genetic influences on their pharmacokinetics and pharmacodynamic effects. AREAS COVERED: This review focuses on genetic variations in the drug metabolizing enzymes which influence the pharmacokinetics and potentially the pharmacodynamics of some β-blockers. It also reviews the polymorphisms in the adrenergic receptors (ARs) and their related pathways which are likely to influence the responses to β-blockers. EXPERT OPINION: The CYP2D6 genotypes influence the pharmacokinetics of some β-blockers but the effects on β-blocker responses have been inconsistent and there is currently no general role for CYP2D6 genotyping prior to choosing a particular β-blocker or dose. The common polymorphisms producing changes in the β(1)-ARs, and their signaling pathways, have been associated with clinical outcomes in several studies in hypertension and heart failure. Treatment with β-blockers, especially with higher doses, appears to have greater benefits in patients with the genetic forms of the β(1)-ARs which are more responsive to both agonists and antagonists. However, current data are not sufficiently consistent to support genotyping for these polymorphisms before selecting or initiating β-blocker treatment and further study results are needed to clarify the situation.     

Protective effects of glycyrrhizin against β₂-adrenergic receptor agonist-induced receptor internalization and cell apoptosis

It has been reported that treatment with β? adrenergic receptor (β?AR) agonist bronchodilators may result in airway β?ARs internalization and cardiac muscle cells apoptosis. This could lead to the loss of pharmacological effect of β?AR agonists and increase adverse cardiovascular events in asthma patients receiving β?AR agonist therapy. Glycyrrhizin, the major bioactive component of licorice root extract, has been reported to exhibit protective effect on respiratory system. Here, we investigate the effects of glycyrrhizin against β?AR agonist salbutamol-induced receptor internalization and cell apoptosis. In our study, the live cell confocal imaging and fixed-cell enzyme-linked immunosorbent assay (ELISA) assay revealed that glycyrrhizin significantly inhibited salbutamol-induced surface β?AR internalization. The underlying mechanisms were then identified to be that glycyrrhizin could reduce the association of β?ARs with β-arrestins and clathrin heavy chain as well as the level of G protein-coupled receptor kinase (GRK) mediated phosphorylation of β?ARs. The inhibition of receptor internalization by glycyrrhizin further lead to stabilization of the β?AR mRNA and protein expression, thus amplified the transmembrane signaling via the β?ARs. We also proved that glycyrrhizin could profoundly attenuate salbutamol-induced early cellular apoptosis by regulating the expressions of B-cell lymphoma 2 (Bcl-2) family genes. Taken together, our results suggest that glycyrrhizin exhibits protective effects against β?AR agonist-induced receptor internalization and cell apoptosis. These findings might have practical implications for future strategies of combined application of glycyrrhizin with β?AR receptor agonists to improve the efficacy of bronchodilators in patients with asthma and chronic obstructive pulmonary disease (COPD).     

Does 5-HT restrain panic? A tryptophan depletion study in panic disorder patients recovered on paroxetine

The neurobiological basis of panic disorder has not been clearly established, although a role for serotonin (5-HT) has been postulated. It is clear that drugs which increase 5-HT neurotransmission are effective in treating the condition but how they do so remains a point of debate. The aim of this study was to determine if lowering brain serotonin activity using the technique of tryptophan depletion provoked a short-term relapse of panic symptoms in patients with panic disorder who had responded to drug treatment. Fourteen patients with panic disorder who had responded to treatment with the selective serotonin reuptake inhibitor (SSRI) paroxetine received a tryptophan-free amino acid drink on one occasion and a control drink on the other in a double-blind crossover design. In addition, they received an infusion of flumazenil (used as a pharmacological challenge) and placebo on each day. The tryptophan depleted drink produced an 87% reduction in plasma tryptophan concentration. Flumazenil produced a panic attack (defined by changes in the panic inventory) in seven out of 14 patients when tryptophan depleted and one out of 14 on the control day (p < 0.02). Three patients also experienced temporary depressive symptoms when tryptophan depleted, with no mood changes being seen on the control days. We conclude that rapid lowering of brain serotonin function can allow the precipitation of panic symptoms in response to flumazenil in panic disorder patients who have responded to treatment with an SSRI. This implies that in panic disorder increased 5-HT availability is important in maintaining the response to SSRIs.     

Effects of gender, age and hypertension on β-adrenergic receptor function in rat urinary bladder

β-Adrenoceptors mediate urinary bladder relaxation, and gender, age and hypertension have been linked to bladder dysfunction. Therefore, we have studied whether any of these factors affects the ability of β-adrenoceptor agonists to relax rat bladder detrusor muscle in vitro. For this purpose we have compared male and female Wistar rats, young and old male Wistar rats, and male normotensive and spontaneously hypertensive rats (SHR). Comparisons were done using KCl-precontracted bladder strips (length about 15–20 mm) and the endogenous agonist noradrenaline, the synthetic non-subtype-selective agonist isoprenaline, and the prototypical β₃-adrenoceptor agonists BRL 37,344 and CGP 12,177. While all agonists yielded numerically weaker relaxation in female as compared to male rats (for example for noradrenaline E_max 40±4% vs 53±6% relaxation, pEC₅₀ 5.41±0.13 vs 5.60±0.14), this difference reached statistical significance only for the weak partial agonist CGP 12,177. Responses to all agonists were attenuated in old as compared to young rats, largely due to a reduced maximum effect, although the difference did not reach statistical significance for isoprenaline. The maximum relaxation responses to noradrenaline and isoprenaline were significantly lower in SHR than in normotensive rats, but both strains exhibited similar responses to the partial agonist BRL 37,344. We conclude that factors associated with bladder dysfunction, such as gender, age and hypertension, can be associated with impaired β-adrenoceptor-mediated bladder relaxation. However, these alterations are not always consistent across various agonists, and the extent of the differences can be small. Therefore, we propose that β-adrenoceptor dysfunction may contribute to the pathophysiology of such conditions, but is unlikely to be the only or even the major factor in this regard. We speculate that β-adrenoceptor agonists may be effective in the treatment of bladder dysfunction under all of these conditions.     

Exposure to cigarette smoke downregulates β2-adrenergic receptor expression and upregulates inflammation in alveolar macrophages

Abstract

Cigarette smoke-triggered inflammation is important in the pathophysiology of chronic obstructive pulmonary disease (COPD). β₂-Adrenergic receptor (β₂-AR) is abundantly expressed on inflammatory cells, which is associated with inflammation regulation. To observe alterations in inflammation, pathological changes in lung tissues, and detect changes in β₂-AR expression, rats were exposed for 4 months to cigarette smoke. Pathological changes were observed in lung tissue sections. The levels of inflammatory mediators tumor necrosis factor (TNF)-α, interleukin (IL)-1β in bronchoalveolar lavage fluid (BALF), and lung tissues were measured using enzyme-linked immunosorbent assay (ELISA). Nuclear factor (NF)-κB activity was detected by electrophoretic mobility shift assay (EMSA). Exposure to this regimen of cigarette smoke induced peribronchial and perivascular lymphocytic aggregates and parenchymal accumulation of macrophages in rats. EMSA demonstrated that smoke exposure enhanced NF-κB activation in rats’ alveolar macrophages (AMs). Compared with the control group, smoke exposure induced a notable increase in TNF-α and IL-1β in BALF, lung tissues, and a decrease of β₂-AR expression of AMs. The expression of β₂-AR from AMs was inversely correlated with TNF-α and IL-1β levels of BALF. These data demonstrated that chronic smoke-triggered lung inflammation was accompanied by down-regulation of β₂-AR in rat lungs’ AMs.     

Platelet α2-adrenergic receptor coupling efficiency to Gi protein in subjects with post-traumatic stress disorder and normal controls

Low platelet membrane α₂-adrenergic receptor (α₂AR) density and low basal and forskolin-stimulated cyclic adenosine monophosphate responses, which have been reported in post-traumatic stress disorder (PTSD), suggest either abnormal α₂AR coupling to G_i protein or dysregulation in post-receptor signal transduction mechanisms. α₂AR density in the high- and low-conformational states, agonist affinity in both states and coupling to G_i protein were investigated in 23 drug-free combat PTSD patients and 25 normal controls. α₂AR coupling measures were not different between PTSD patients and controls. Total α₂AR density was higher in PTSD patients than controls, due to a higher density of the receptor in the high-conformational state. There were no differences in agonist affinity to the receptor in either conformational state. Results rule out dysregulation in α₂AR coupling to G_i protein. Studies of post-receptor signal transduction mechanisms are warranted. Received: 8 February 1998/Final version: 15 June 1998     

Comparative pharmacology of human β-adrenergic receptor subtypes—characterization of stably transfected receptors in CHO cells

Although many ₁-receptor antagonists and ₂-receptor agonists have been used in pharmacotherapy for many years their pharmacological properties at all three known subtypes of -adrenergic receptors are not always well characterized. The aim of this study was, therefore, to provide comparative binding characteristics of agonists (epinephrine, norepinephrine, isoproterenol, fenoterol, salbutamol, salmeterol, terbutalin, formoterol, broxaterol) and antagonists (propranolol, alprenolol, atenolol, metoprolol, bisoprolol, carvedilol, pindolol, BRL 37344, CGP 20712, SR 59230A, CGP 12177, ICI 118551) at all three subtypes of human -adrenergic receptors in an identical cellular background. We generated Chinese hamster ovary (CHO) cells stably expressing the three -adrenergic receptor subtypes at comparable levels. We characterized these receptor subtypes and analyzed the affinity of routinely used drugs as well as experimental compounds in competition binding studies, using the non-selective antagonist ¹²⁵I-cyanopindolol as a radioligand. Furthermore, we analyzed the -receptor-mediated adenylyl cyclase activity in isolated membranes from these cell lines. The results from our experiments show that all compounds exhibit distinct patterns of selectivity and activity at the three -receptor subtypes. In particular, a number of ₂- or ₃-receptor agonists that are inverse agonists at the other subtypes were identified. In addition, ₁-receptor antagonists with agonistic activity at ₂- and ₃-receptors were found. These specific mixtures of agonism, antagonism, and inverse agonism at different subtypes may have important implications for the therapeutic use of the respective compounds.     

Metabolism and disposition of a β3-adrenergic receptor agonist LY368842 in male Fisher 344 rats

The metabolism and disposition of LY368842, a β₃-adrenergic receptor agonist, were characterized in F344 rats following oral or intravenous administration of [¹⁴C]LY368842. These studies were conducted as part of the investigation of the mechanism of dark liver pigmentation in LY368842-treated F344 rats. The maximum plasma concentration of LY368842 was reached at 3?h after an oral dose, with an elimination half-life of 4?h. The oral bioavailability of LY368842 was determined as 8%. A tissue distribution study by quantitative whole-body autoradiography indicated high concentrations of radiocarbon in gastrointestinal contents and moderate concentrations in liver. The radiocarbon was rapidly eliminated in rats, with approximately 3% of the dose recovered in urine and 90% in faeces over 168?h. In bile duct-cannulated rats, about 42% of the dose was recovered in bile and 41% remained in the faeces. Metabolites of LY368842 were identified in rat urine, faeces, bile and plasma samples. Oxidative metabolism of LY368842 led to the formation of a hydroxy metabolite, an indole-2,3-dione metabolite and oxidative cleavage products such as amine and diol metabolites. Several glucuronide conjugates were also identified in rat bile. These data suggest that LY368842 is not completely absorbed but is widely distributed, extensively metabolized and rapidly eliminated in rats after oral administration.     

Effects of GABAA receptor ligands on noradrenaline concentration and β-adrenoceptor binding in mouse cerebral cortex

The present experiments investigated changes in -adrenoceptor binding and noradrenaline stores in mouse cerebral cortex after single treatments with drugs which bind to the GABA_A receptor but which attenuate the actions of GABA. Neither the GABA antagonist, securinine, nor the picrotoxin/Cl^– channel ligand, picrotoxin, affected noradrenaline levels or -adrenoceptor binding. However, both the benzodiazepine inverse agonist, DMCM, and pentylenetetrazole increased noradrenaline levels 24 h after injection. Only pentylenetetrazol modified -adrenoceptor binding: there was a significant increase in receptor number 4 days after injection, but a significant decrease after 7 days. The anxiogenic, proconvulsant drug, yohimbine, was without effect. The changes induced by DMCM and pentylenetetrazole do not seem to be related to the behavioural effects of these drugs or to their affinity for binding to benzodiazepine receptors. The possibility that these compounds have actions in addition to those at the GABA_A receptor is discussed.     

Imipramine in panic disorder. 2. Effects on α2-adrenoceptor function

Untreated patients with panic disorder have been shown to have altered α(2)-adrenoceptor sensitivity, as assessed by clonidine challenge testing. We investigated clonidine-induced biochemical, physiological, hor monal and psychological responses in six panic patients, before and after treatment with imipramine. Comparison of the clonidine-induced fall in plasma MHPG pre- and post-treatment showed it to be sig nificantly reduced after imipramine. Significantly smaller clonidine-induced falls in diastolic blood pressure and heart rate occurred after imipramine treatment. However, growth hormone and sedation responses were not altered. These data suggest that reduced sensitivity of some central α(2)-adrenoceptors occurs during treatment of panic disorder with imipramine. However, these changes do not fully explain the therapeutic actions of antidepressants in this condition.     

Functional α1-adrenergic receptor subtypes in human right gastroepiploic artery

AIM: To study the functional alpha1-adrenergic receptor (alpha1-AR) subtypes in human right gastroepiploic artery (RGA). METHODS: The effects of alpha2-AR, alpha1-AR, and alpha1-AR subtype selective antagonists on norepinephrine (NE)-induced vasoconstriction in isolated human RGA were observed by contractile function experiment. RESULTS: Cumulative concentration-response curves for NE were competitively antagonized in RGA by alpha2-AR selective antagonist yohimbine (pA2 6.82+/-0.28, slope 1.12+/-0.40),alpha1-AR selective antagonist prazosin (pA2 9.77+/-0.22, slope 0.90+/-0.22),alpha1A-AR selective antagonists RS17053 (pA2 8.42+/-0.20, slope 0.93+/-0.20) and 5-MU (pA2 8.42+/-0.22, slope 0.88+/-0.18),alpha1D-AR selective antagonist BMY7378 (pA2 6.84+/-0.32, slope 1.05+/-0.17), and alpha1A-,alpha1B-AR selective antagonist WB4101 (pA2 8.88+/-0.20, slope 1.15+/-0.16). The correlation coefficients between these pA2 values of alpha1-AR selective antagonists with pKi values of which obtained from alpha1A-, alpha1B- and alpha1D-AR cloned cells are 0.95, 0.82, and 0.42. After the vessels were pretreated by chlorethylclonidine (CEC), an alpha1B- and alpha1D-AR irreversible alkylating agent, the pD2 values were changed from 5.9+/-0.5 to 5.6+/-0.6 and the maximal contraction was changed from (8.9+/-3.2) g to (8.0+/-3.2) g, respectively. The difference was not significant. CONCLUSION: In human RGA, the contraction response is mainly mediated by alpha1-AR, of which alpha1A-AR plays an important role, whereas alpha1B- and alpha1D-AR are not involved in the contraction response.     

Serotonin function in panic disorder: effect of 1–5-hydroxytryptophan in patients and controls

The responsivity of the serotonergic system to 5-hydroxytryptophan (5-HTP) administration was investigated in seven patients with panic disorder and seven healthy controls. Using cortisol and -endorphin as hormonal probes, no evidence was obtained for a dysfunction of the serotonin system in patients. Behaviorally, controls responded differently from patients, in that controls seemed to be more sensitive to 5-HTP. None of the subjects reported an increase in anxiety.