Adrenoceptor Desensitization Research Articles

Adrenoceptor Desensitization: Current Understanding of Mechanisms.

G-protein coupled receptors (GPCRs) transduce a wide range of extracellular signals. They are key players in the majority of biologic functions including vision, olfaction, chemotaxis, and immunity. However, as essential as most of them are to body function and homeostasis, overactivation of GPCRs has been implicated in many pathologic diseases such as cancer, asthma, and heart failure (HF). Therefore, an important feature of G protein signaling systems is the ability to control GPCR responsiveness, and one key process to control overstimulation involves initiating receptor desensitization. A number of steps are appreciated in the desensitization process, including cell surface receptor phosphorylation, internalization, and downregulation. Rapid or short-term desensitization occurs within minutes and involves receptor phosphorylation via the action of intracellular protein kinases, the binding of β-arrestins, and the consequent uncoupling of GPCRs from their cognate heterotrimeric G proteins. On the other hand, long-term desensitization occurs over hours to days and involves receptor downregulation or a decrease in cell surface receptor protein level. Of the proteins involved in this biologic phenomenon, β-arrestins play a particularly significant role in both short- and long-term desensitization mechanisms. In addition, β-arrestins are involved in the phenomenon of biased agonism, where the biased ligand preferentially activates one of several downstream signaling pathways, leading to altered cellular responses. In this context, this review discusses the different patterns of desensitization of the α 1-, α 2- and the β adrenoceptors and highlights the role of β-arrestins in regulating physiologic responsiveness through desensitization and biased agonism. SIGNIFICANCE STATEMENT: A sophisticated network of proteins orchestrates the molecular regulation of GPCR activity. Adrenoceptors are GPCRs that play vast roles in many physiological processes. Without tightly controlled desensitization of these receptors, homeostatic imbalance may ensue, thus precipitating various diseases. Here, we critically appraise the mechanisms implicated in adrenoceptor desensitization. A better understanding of these mechanisms helps identify new druggable targets within the GPCR desensitization machinery and opens exciting therapeutic fronts in the treatment of several pathologies.

C-Reactive Protein Causes Blood Pressure Drop in Rabbits and Induces Intracellular Calcium Signaling.

Systemic diseases characterized by elevated levels of C-reactive protein (CRP), such as sepsis or systemic inflammatory response syndrome, are usually associated with hardly controllable haemodynamic instability. We therefore investigated whether CRP itself influences blood pressure and heart rate. Immediately after intravenous injection of purified human CRP (3.5 mg CRP/kg body weight) into anesthetized rabbits, blood pressure dropped critically in all animals, while control animals injected with bovine serum albumin showed no response. Heart rate did not change in either group. Approaching this impact on a cellular level, we investigated the effect of CRP in cell lines expressing adrenoceptors (CHO-α1A and DU-145). CRP caused a Ca2+ signaling being dependent on the CRP dose. After complete activation of the adrenoceptors by agonists, CRP caused additional intracellular Ca2+ mobilization. We assume that CRP interacts with hitherto unknown structures on the surface of vital cells and thus interferes with the desensitization of adrenoceptors.

Identification of proteasome subunit beta type 3 involved in the potential mechanism of corticosteroid protective effectiveness on beta-2 adrenoceptor desensitization by a proteomics approach.

Asthma is a chronic inflammatory disease characterized by airway inflammation with mucus hypersecretion and hyperresponsiveness to various nonspecific stimuli. Corticosteroids are usually used to prevent β2 adrenoceptor (β2AR) desensitization in clinical and experimental practice. But the exact mechanism of corticosteroid effectiveness on β2AR desensitization is unclear. To find the potential mechanisms related to the protective effects of corticosteroid on salbutamol induced β2AR desensitization by a proteomics approach. Thirty-two BALB/c (6-8 weeks old) mice were divided into four groups: group A, control group, phosphate buffered saline (PBS)-treated group; group B, asthmatic group, treated by ovalbumin (OVA); group C, β2AR desensitized asthmatic group, treated by OVA and salbutamol (SBT) and group D, corticosteroid-treated β2AR desensitized asthmatic group, treated with OVA, SBT and Dexamethasone (DEX). After administrated with those drugs, their serum total IgE, bronchoalveolar lavage fluid (BALF) cytokine concentration, airway resistance and membrane receptor number of β2AR were evaluated. After then, the mice of group C and D were sacrificed, their protein from lung tissue were extracted and then seperated by two-dimensional gel electrophoresis (2DE). Then, the isolated protein spots were analyzed by ImageMaster software and mass spectrometry. Bioinformatic tools were used to search these protein spots and find interesting protein spots associated with corticosteroid protective effect on β2AR desensitization. Finally, these protein spots were confirmed by Western blotting. With inflammatory cell count, cytokine concentration of BALF, pathological sections, total serum IgE, airway resistance, membrane receptor number and β2AR total amount changes, asthmatic mouse model and β2AR desensitization asthmatic mouse model were successfully established. Seventeen protein spots were found different expression between group C and group D, 4 protein spots were down-regulated and 13 protein spots were up-regulated compared to group C. Proteasome subunit beta type 3 was down-regulated. Increased proteasome subunit beta type 3 expression may be responsible for salbutamol-induced β2AR desensitization in asthmatic disease, and DEX possibly render the β2AR resensitization partially by decreasing the content of proteasome.

Characterising the Mechanism of Airway Smooth Muscle β2 Adrenoceptor Desensitization by Rhinovirus Infected Bronchial Epithelial Cells

Rhinovirus (RV) infections account for approximately two thirds of all virus-induced asthma exacerbations and often result in an impaired response to β2 agonist therapy. Using an in vitro model of RV infection, we investigated the mechanisms underlying RV-induced β2 adrenoceptor desensitization in primary human airway smooth muscle cells (ASMC). RV infection of primary human bronchial epithelial cells (HBEC) for 24 hours produced conditioned medium that caused β2 adrenoceptor desensitization on ASMCs without an effect on ASMCs viability. Less than 3 kDa size fractionation together with trypsin digestion of RV-induced conditioned medium did not prevent β2 adrenoceptor desensitization, suggesting it could potentially be mediated by a small peptide or lipid. RV infection of BECs, ASMCs and fibroblasts produced prostaglandins, of which PGE2, PGF2α and PGI2 had the ability to cause β2 adrenoceptor desensitization on ASMCs. RV-induced conditioned medium from HBECs depleted of PGE2 did not prevent ASMC β2 adrenoceptor desensitization; however this medium induced PGE2 from ASMCs, suggesting that autocrine prostaglandin production may be responsible. Using inhibitors of cyclooxygenase and prostaglandin receptor antagonists, we found that β2 adrenoceptor desensitization was mediated through ASMC derived COX-2 induced prostaglandins. Since ASMC prostaglandin production is unlikely to be caused by RV-induced epithelial derived proteins or lipids we next investigated activation of toll-like receptors (TLR) by viral RNA. The combination of TLR agonists poly I:C and imiquimod induced PGE2 and β2 adrenoceptor desensitization on ASMC as did the RNA extracted from RV-induced conditioned medium. Viral RNA but not epithelial RNA caused β2 adrenoceptor desensitization confirming that viral RNA and not endogenous human RNA was responsible. It was deduced that the mechanism by which β2 adrenoceptor desensitization occurs was by pattern recognition receptor activation of COX-2 induced prostaglandins.

Enhancement of μ-Opioid Receptor Desensitization by Nitric Oxide in Rat Locus Coeruleus Neurons: Involvement of Reactive Oxygen Species

It has previously been shown that nitric oxide (NO) synthase is involved in the development of opioid tolerance. The aim of the present work was to study the effect of NO on μ-opioid receptor (MOR) desensitization. Furthermore, we explored the possible role of reactive oxygen species (ROS) in this effect. Single-unit extracellular and whole-cell patch-clamp recordings were performed on locus coeruleus (LC) neurons from rat brain slices. Perfusion with high concentrations of Met(5)-enkephalin (ME) caused a concentration-related reduction of opioid effect, reflecting the induction of homologous MOR desensitization. The NO donors sodium nitroprusside and diethylamine NONOate markedly enhanced the ME-induced MOR desensitization, although the acute effect of ME on K(+) conductance was not affected by sodium nitroprusside. Continuous perfusion with the antioxidants melatonin, trolox, 21-[4-(2,6-di-1-pyrrolidinyl-4-pyrrimidinyl)-1-piperazinyl]-pregna-1,4,9(11)-triene-3,20-dione(Z)-2-butenedioate (U74389G), and diethyldithiocarbamate prevented the effect of sodium nitroprusside on MOR desensitization, but they did not themselves alter the desensitization. Like sodium nitroprusside, the ROS-generating molecule H(2)O(2) enhanced MOR desensitization induced by ME. However, α(2)-adrenoceptor desensitization induced by noradrenaline was not modified by H(2)O(2), suggesting a selective action of ROS on MOR. Our results suggest that elevated levels of NO, which may be reached in pathological processes, enhance homologous desensitization of MOR in the LC, probably through a mechanism involving ROS generation.

Chronic treatment in vivo with β‐adrenoceptor agonists induces dysfunction of airway β2‐adrenoceptors and exacerbates lung inflammation in mice

Inhalation of a β-adrenoceptor agonist (β-agonist) is first-line asthma therapy, used for both prophylaxis against, and acute relief of, bronchoconstriction. However, repeated clinical use of β-agonists leads to impaired bronchoprotection and, in some cases, adverse patient outcomes. Mechanisms underlying this β(2) -adrenoceptor dysfunction are not well understood, due largely to the lack of a comprehensive animal model and the uncertainty as to whether or not bronchorelaxation in mice is mediated by β(2) -adrenoceptors. Thus, we aimed to develop a mouse model that demonstrated functional β-agonist-induced β(2) -adrenoceptor desensitization in the context of allergic inflammatory airway disease. We combined chronic allergen exposure with repeated β-agonist inhalation in allergen-treated BALB/C mice and examined the contribution of β(2) -adrenoceptors to albuterol-induced bronchoprotection using FVB/NJ mice with genetic deletion of β(2) -adrenoceptors (KO). Associated inflammatory changes - cytokines (ELISA), cells in bronchoalevolar lavage and airway remodelling (histology) and β(2) -adrenoceptor density (radioligand binding) - were also measured. KEY RESULTS β(2) -Adrenoceptors mediated albuterol-induced bronchoprotection in mice. Chronic treatment with albuterol induced loss of bronchoprotection, associated with exacerbation of the inflammatory components of the asthma phenotype. This animal model reproduced salient features of human asthma and linked loss of bronchoprotection with airway pathobiology. Accordingly, the model offers an advanced tool for understanding the mechanisms of the effects of chronic β- agonist treatment on β-adrenoceptor function in asthma. Such information may guide the clinical use of β-agonists and provide insight into development of novel β-adrenoceptor ligands for the treatment of asthma.

Sphingosine 1-phosphate-mediated α1B-adrenoceptor desensitization and phosphorylation. Direct and paracrine/autocrine actions

Sphingosine-1-phosphate-induced α1B-adrenergic receptor desensitization and phosphorylation were studied in rat-1 fibroblasts stably expressing enhanced green fluorescent protein-tagged adrenoceptors. Sphingosine-1-phosphate induced adrenoceptor desensitization and phosphorylation through a signaling cascade that involved phosphoinositide 3-kinase and protein kinase C activities. The autocrine/paracrine role of sphingosine-1-phosphate was also studied. It was observed that activation of receptor tyrosine kinases, such as insulin growth factor-1 (IGF-I) and epidermal growth factor (EGF) receptors increased sphingosine kinase activity. Such activation and consequent production of sphingosine-1-phosphate appear to be functionally relevant in IGF-I- and EGF-induced α1B-adrenoceptor phosphorylation and desensitization as evidenced by the following facts: a) expression of a catalytically inactive (dominant-negative) mutant of sphingosine kinase 1 or b) S1P1 receptor knockdown markedly reduced this growth factor action. This action of sphingosine-1-phosphate involves EGF receptor transactivation. In addition, taking advantage of the presence of the eGFP tag in the receptor construction, we showed that S1P was capable of inducing α1B-adrenergic receptor internalization and that its autocrine/paracrine generation was relevant for internalization induced by IGF-I. Four distinct hormone receptors and two autocrine/paracrine mediators participate in IGF-I receptor–α1B-adrenergic receptor crosstalk.

Mechanisms involved in α1B‐adrenoceptor desensitization

α(1B)-Adrenergic receptors mediate many of the actions of the natural catecholamines, adrenaline and noradrenaline. They belong to the seven transmembrane domains G protein-coupled receptor superfamily and exert their actions mainly through activation of Gq proteins and phosphoinositide turnover/calcium signaling. Many hormones and neurotransmitters are capable of inducing α(1B)-adrenergic receptor phosphorylation and desensitization; among them: adrenaline and noradrenaline, phorbol esters, endothelin-I, bradykinin, lysophosphatidic acid, insulin, EGF, PDGF, IGF-I, TGF-β, and estrogens. Key protein kinases for these effects are G protein coupled receptor kinases and protein kinase C. The lipid/protein kinase, phosphoinositide-3 kinase also appears to play a key role, acting upstream of protein kinase C. In addition to the agents employed for cells stimulation, we observed that paracrine/autocrine mediators also participate; these processes include EGF transactivation and sphingosine-1-phosphate production and action. The complex regulation of these receptors unlocks opportunities for therapeutic intervention.

Sex Hormones Modulate Salbutamol-Elicited Long-Term Relaxation in Isolated Bovine Tracheal Strips

Sex hormones are of interest regarding gender differences in the clinical manifestations of airway diseases. No conclusive data are available on the sex hormone modulation of β-adrenoceptor-mediated responses on airways. To this aim, isolated preparations of bovine trachea were used to establish the sex hormone influence on salbutamol-elicited relaxation. This had 2 components, a full acute relaxation followed by a loss of efficacy, close to half of the effect. The remaining half was reverted by the β-blocker, propranolol. The loss of salbutamol-elicited relaxation might reflect the receptor desensitization, as shown by the lack of effect by subsequent administration of salbutamol, and the decrease in the immunostaining of β₂-adrenoceptors. Sex hormones differently modified the salbutamol-elicited response. Testosterone, but not other androgens or estradiol, had a synergic effect, facilitating the acute relaxation and decreasing the loss of spasmolytic effect, associated with an increase in the latency of desensitization and a decrease in the time taken to reach long-term steady-state tone. These effects, not modified by the antiandrogen flutamide or epithelium removal, seem to be independent of a modulation of β₂-adrenoceptor desensitization. Testosterone also relaxed preparations with desensitized β-adrenoceptor. Therefore, testosterone modulates tracheal smooth muscle tone, facilitating bronchodilation caused by β₂-adrenoceptor agonists which might be of pharmacological interest.

Relationships of Adrenoceptor Polymorphisms with Obesity

Obesity, hypertension, and type 2 diabetes are rapidly growing public health problems. Heightened sympathetic nerve activity is a well-established observation in obesity, hypertension, and type 2 diabetes. Human obesity, hypertension, and diabetes have strong genetic as well as environmental determinants. Reduced energy expenditure and resting metabolic rate are predictive of weight gain, and the sympathetic nervous system participates in regulating energy balance through thermogenesis. The thermogenic effects of catecholamines in obesity are mainly mediated via the β2, and β3-adrenergic receptors in humans. Further, β2-adrenoceptors importantly influence vascular reactivity and may regulate blood pressure. β-adrenoceptor polymorphisms have also been associated with adrenoceptor desensitization, increased adiposity, insulin resistance, and enhanced sympathetic nervous activity. Many epidemiological studies have shown strong relationships between adrenoceptor polymorphisms and obesity, but the observations have been discordant. This paper will discuss the current topics involving the influence of the sympathetic nervous system and β2- and β3-adrenoceptor polymorphisms in obesity.

Mediators of sympathetic activation in metabolic syndrome obesity

The metabolic syndrome represents a major public health burden because of its high prevalence in the general population and its association with cardiovascular disease and type 2 diabetes. Accumulated evidence based on biochemical, neurophysiologic, and indirect measurements of autonomic activity indicate that visceral obesity and the metabolic syndrome are associated with enhanced sympathetic neural drive and vagal impairment. The mechanisms linking metabolic syndrome with sympathetic activation are complex and not completely understood, and cause-effect relationships need further clarification from prospective trials. Components of the metabolic syndrome that may directly or indirectly enhance sympathetic drive include hyperinsulinemia, leptin, nonesterified fatty acids, proinflammatory cytokines, angiotensinogen, baroreflex impairment, and obstructive sleep apnea. beta-Adrenoceptor polymorphisms have also been associated with adrenoceptor desensitization, increased adiposity, insulin resistance, and enhanced sympathetic activity. Because chronic sympathetic activation contributes to hypertension and its target-organ damage, sympathoinhibition remains an important goal in the therapeutic management of the metabolic syndrome.

Ageing and vascular adrenoceptor desensitization: too little, too late?

Ageing and vascular adrenoceptor desensitization: too little, too late?

Simultaneous alpha2B- and beta2-adrenoceptor activation sensitizes the alpha2B-adrenoceptor for agonist-induced down-regulation.

We recently reported that alpha(2A)-adrenoceptor (AR) desensitization and down-regulation occurs after 24-h treatment with epinephrine (EPI) (0.3 microM) in BE(2)-C cells that express both alpha(2)- and beta(2)-ARs. The same concentration of norepinephrine (NE) has no effect. The effect of EPI is prevented by beta(2)-AR blockade and is associated with an increase in G protein-coupled receptor kinase 3 (GRK3) expression. Because differences in agonist-induced down-regulation of the alpha(2A)-versus alpha(2B)-ARs have been reported, the present study examines the effects of simultaneous activation of alpha(2B)- and beta(2)-ARs on alpha(2B)-AR number and signaling. We studied NG108 cells that naturally express alpha(2B)-ARs, and BN17 cells, NG108 cells transfected to express the human beta(2)-AR. In NG108 cells, alpha(2B)-AR desensitization and down-regulation require treatment with 20 microM EPI or NE; GRK expression was not changed. In BN17 cells expressing beta(2)-ARs, the threshold EPI concentration for alpha(2B)-AR desensitization and down-regulation was reduced to 0.3 microM; 10 microM NE was required for the same effect. Furthermore, 24-h EPI or NE treatments that produced desensitization also resulted in a selective 2-fold up-regulation of GRK3; GRK2 was unchanged. The beta-AR antagonist alprenolol (1 microM) and GRK3 antisense (but not sense) DNA blocked 0.3 microM EPI- and 10 microM NE-induced desensitization and down-regulation of the alpha(2B)-AR as well as GRK3 up-regulation. In conclusion, simultaneous activation of alpha(2B)- and beta(2)-ARs results in a 67-fold decrease in the threshold concentration of EPI required for alpha(2B)-AR down-regulation. This lower threshold for down-regulation is associated with alpha(2B)- and beta(2)-AR dependent up-regulation of GRK3 expression.

Glucocorticoids reverse IL‐1β‐induced impairment of β‐adrenoceptor‐mediated relaxation and up‐regulation of G‐protein‐coupled receptor kinases

1. The aim of the present study was to examine the effects of glucocorticoid dexamethasone on airway responsiveness to albuterol after intratracheal instillation of saline or IL-1beta in Brown-Norway rats in vivo and to elucidate the molecular mechanism of this effect. 2. IL-1beta caused a significant reduction in albuterol-mediated relaxation to protect against MCh-induced bronchoconstriction. Dexamethasone attenuated the IL-1beta-induced impaired relaxation while alone had no effect when compared to rats treated identically with saline. 3. The density of beta(2)-adrenoceptors was significantly reduced in lung membranes harvested from IL-1beta-treated rats, which was associated with impaired isoproterenol- and forskolin-stimulated cyclic AMP accumulation and adenylyl cyclase (AC) activity ex vivo. Dexamethasone did not prevent IL-1beta-induced down-regulation of beta(2)-adrenoceptors but completely blocked IL-1beta-induced impairment of cyclic AMP accumulation and AC activity stimulated by isoproterenol and forskolin. 4. The inhibitory G-protein subtypes, G(ialpha1), G(ialpha2) and G(ialpha3), were detected in lung membranes prepared from all groups of rats but the intensity of G(ialpha1) and G(ialpha2) was markedly increased in IL-1beta-treated rats, which were not prevented by dexamethasone. 5. The activity of cytosolic GRK and the expression of GRK2 and GRK5 were elevated in the lung of IL-1beta-treated rats, which were completely abolished by dexamethasone. 6. These results indicate that treatment of rats with IL-1beta results in desensitization of pulmonary beta(2)-adrenoceptors. In light of data obtained in this study, we propose that both the decrease in AC activity and the increase in GRK activity, which are reversed by dexamethasone, may underlie beta(2)-adrenoceptor desensitization.

Chronic systemic administration of salmeterol to rats promotes pulmonary beta(2)-adrenoceptor desensitization and down-regulation of G(s alpha).

1. The aim of the present study was to examine the effects of chronic infusion of the long-acting agonist salmeterol on pulmonary beta(2)-adrenoceptor function in Sprague-Dawley rats in vivo and to elucidate the molecular basis of any altered state. 2. Systemic administration of rats with salmeterol for 7 days compromised the ability of salmeterol and prostaglandin E(2) (PGE(2)), given acutely by the intravenous route, to protect against ACh-induced bronchoconstriction when compared to rats treated identically with vehicle. 3. beta(1)- and beta(2)-adrenoceptor density was significantly reduced in lung membranes harvested from salmeterol-treated animals, which was associated with impaired salmeterol- and PGE(2)-induced cyclic AMP accumulation ex vivo. 4. Three variants of G(s alpha) that migrated as 42, 44 and 52 kDa peptides on SDS polyacrylamide gels were detected in lung membranes prepared from both groups of rats but the intensity of each isoform was markedly reduced in rats that received salmeterol. 5. The activity of cytosolic, but not membrane-associated, G-protein receptor-coupled kinase was elevated in the lung of salmeterol-treated rats when compared to vehicle-treated animals. 6. The ability of salmeterol, administered systemically, to protect the airways of untreated rats against ACh-induced bronchoconstriction was short-acting (t(off) approximately 45 min), which contrasts with its long-acting nature when given to asthmatic subjects by inhalation. 7. These results indicate that chronic treatment of rats with salmeterol results in heterologous desensitization of pulmonary G(s)-coupled receptors. In light of previous data obtained in rats treated chronically with salbutamol, we propose that a primary mechanism responsible for this effect is a reduction in membrane-associated G(s alpha). The short-acting nature of salmeterol, when administered systemically, and the reduction in beta-adrenoceptor number may be due to metabolism to a biologically-active, short-acting and non-selective beta-adrenoceptor agonist.

Cross-talk between receptors with intrinsic tyrosine kinase activity and α1b-adrenoceptors

The effect of epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) on the phosphorylation and function of α1b-adrenoceptors transfected into Rat-1 fibroblasts was studied. EGF and PDGF increased the phosphorylation of these adrenoceptors. The effect of EGF was blocked by tyrphostin AG1478 and that of PDGF was blocked by tyrphostin AG1296, inhibitors of the intrinsic tyrosine kinase activities of the receptors for these growth factors. Wortmannin, an inhibitor of phosphoinositide 3-kinase, blocked the α1b-adrenoceptor phosphorylation induced by EGF but not that induced by PDGF. Inhibition of protein kinase C blocked the adrenoceptor phosphorylation induced by EGF and PDGF. The ability of noradrenaline to increase [35S]guanosine 5´-[γ-thio]triphosphate ([35S]GTP[S]) binding in membrane preparations was used as an index of the functional coupling of the α1b-adrenoceptors and G-proteins. Noradrenaline-stimulated [35S]GTP[S] binding was markedly decreased in membranes from cells pretreated with EGF or PDGF. Our data indicate that: (i) activation of EGF and PDGF receptors induces phosphorylation of α1b-adrenoceptors, (ii) phosphatidylinositol 3-kinase is involved in the EGF response, but does not seem to play a major role in the action of PDGF, (iii) protein kinase C mediates this action of both growth factors and (iv) the phosphorylation of α1b-adrenoceptors induced by EGF and PDGF is associated with adrenoceptor desensitization.

Cross-talk between receptors with intrinsic tyrosine kinase activity and alpha1b-adrenoceptors.

The effect of epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) on the phosphorylation and function of alpha(1b)-adrenoceptors transfected into Rat-1 fibroblasts was studied. EGF and PDGF increased the phosphorylation of these adrenoceptors. The effect of EGF was blocked by tyrphostin AG1478 and that of PDGF was blocked by tyrphostin AG1296, inhibitors of the intrinsic tyrosine kinase activities of the receptors for these growth factors. Wortmannin, an inhibitor of phosphoinositide 3-kinase, blocked the alpha(1b)-adrenoceptor phosphorylation induced by EGF but not that induced by PDGF. Inhibition of protein kinase C blocked the adrenoceptor phosphorylation induced by EGF and PDGF. The ability of noradrenaline to increase [(35)S]guanosine 5'-[gamma-thio]triphosphate ([(35)S]GTP[S]) binding in membrane preparations was used as an index of the functional coupling of the alpha(1b)-adrenoceptors and G-proteins. Noradrenaline-stimulated [(35)S]GTP[S] binding was markedly decreased in membranes from cells pretreated with EGF or PDGF. Our data indicate that: (i) activation of EGF and PDGF receptors induces phosphorylation of alpha(1b)-adrenoceptors, (ii) phosphatidylinositol 3-kinase is involved in the EGF response, but does not seem to play a major role in the action of PDGF, (iii) protein kinase C mediates this action of both growth factors and (iv) the phosphorylation of alpha(1b)-adrenoceptors induced by EGF and PDGF is associated with adrenoceptor desensitization.

Albuterol-induced downregulation of Gsalpha accounts for pulmonary beta(2)-adrenoceptor desensitization in vivo.

The aim of the present study was to develop a chronic in vivo model of pulmonary beta(2)-adrenoceptor desensitization and to elucidate the nature and molecular basis of this state. Subcutaneous infusion of rats with albuterol for 7 days compromised the ability of albuterol, given acutely, to protect against acetylcholine-induced bronchoconstriction. The bronchoprotective effect of prostaglandin E(2), but not forskolin, was also impaired, indicating that the desensitization was heterologous and that the primary defect in signaling was upstream of adenylyl cyclase. beta(2)-Adrenoceptor density was reduced in lung membranes harvested from albuterol-treated animals, and this was associated with impaired albuterol-induced cyclic adenosine monophosphate (cAMP) accumulation and activation of cAMP-dependent protein kinase ex vivo. Gsalpha expression was reduced in the lung and tracheae of albuterol-treated rats, and cholera toxin-induced cAMP accumulation was blunted. Chronic treatment of rats with albuterol also increased cAMP phosphodiesterase activity and G protein-coupled receptor kinase-2, but the extent to which these events contributed to beta(2)-adrenoceptor desensitization was unclear given that forskolin was active in both groups of animals and that desensitization was heterologous. Collectively, these results indicate that albuterol effects heterologous desensitization of pulmonary Gs-coupled receptors in this model, with downregulation of Gsalpha representing a primary molecular etiology.

Lack of β adrenoceptor desensitization in brain following the dual noradrenaline and serotonin reuptake inhibitor venlafaxine

Venlafaxine, a dual amine reuptake inhibitor, was utilized to delineate the role of the individual aminergic components of the `serotonin/noradrenaline link' in modifying receptor-linked second messenger cascades. Venlafaxine (20 mg/kg i.p. bid for 10 days) failed to alter in normal animals either the density of β adrenoceptors or the response of the β adrenoceptor-coupled adenylate cyclase system to noradrenaline but significantly decreased the cyclic AMP response to noradrenaline in the brain of rats with selective depletion of brain serotonin by p-chlorophenylalanine. The studies provide evidence for a cross-talk between noradrenergic and serotonergic receptor cascades at the level of mechanisms involved in the desensitization of the β adrenoceptor-coupled adenylate cyclase system.

Response of large and small vessels to alpha and beta adrenoceptor stimulation in heart failure: effect of angiotensin converting enzyme inhibition.

The increased sympathetic drive in chronic heart failure (CHF) might provoke vascular adrenoceptor desensitization, which, together with endothelial dysfunction, could contribute to the altered vasomotor tone seen in CHF. We investigated 1) whether CHF alters the responses mediated by alpha and beta adrenoceptors in small and large peripheral arteries, and 2) the effect of angiotensin-converting enzyme (ACE) inhibition. Rats with CHF (coronary artery ligation) were treated with placebo or the ACE inhibitor lisinopril (10 mg/kg/d) starting 7 days after ligation. Responses to phenylephrine (alpha 1 agonist), salbutamol (beta 2 agonist) as well as acetylcholine (endothelium-dependent), were assessed after 3 months in isolated and pressurized segments of the abdominal aorta, the femoral and the mesenteric arteries. In animals with hemodynamic signs of CHF, neither the vasoconstrictor responses to phenylephrine nor the vasodilator response to salbutamol were affected. In contrast, the dilator response to acetylcholine of both small arteries, but not that of the aorta, was impaired. Furthermore, CHF did not modify vessel structure. While lisinopril did not modify the responses to adrenergic agonists, it normalized the response to acetylcholine. Furthermore, ACE inhibition reduced vascular media cross sectional area and collagen density. Thus, the unchanged arterial responsiveness to adrenoceptor agonists does not indicate any vascular adrenoceptor desensitization, while endothelial dependent vasodilation of small arteries is impaired in CHF. ACE inhibition does not modify the response to adrenergic stimuli, prevents endothelial dysfunction and induces both cardiac and vascular remodeling, which probably contribute to the effect ACE inhibitors have on exercise tolerance and survival.