Adrenoceptor Desensitization Research Articles

Catecholamine Receptors and Signal Transduction

Recent research in the area of catecholamine receptors has focused on structure, classification, and tissue localization of adrenoceptor subtypes, intracellular mechanisms influenced by catecholamine receptor occupation, including negative feedback loops responsible for receptor desensitization, pharmacology of neurological, psychiatric, and cardiovascular drugs acting at catecholamine receptors, roles of catecholamine receptor subtypes in behavioral and physiological processes, and catecholamine receptors in pathophysiological states. Distinctions based on genetic sequences for identifying types and subtypes of catecholamine receptors have outpaced those based on pharmacological techniques such as ligand binding. As a result, the functional importance of many of these distinctions remains unclear. This chapter includes summaries of classification schemas for these subtypes, lists of the associated G-proteins and intracellular messengers that couple catecholamine receptor occupation with cellular functional changes, and notes on some of the specific agonists and antagonists. In response to pharmacological doses of agonists, many studies have focused on refining understanding of mechanisms of adrenoceptor desensitization. Adrenoceptor occupation presumably results in conformational changes that initiate the signaling process. The α 1A subtype predominates in human heart, liver, brain, and prostate. Transcriptional regulation appears to underlie tissue-specific expression of this subtype. In humans, α 1 -adrenoceptors play relatively minor roles in mediating catecholamine effects in the heart, and kidneys play an important role in regulation of vascular tone.

Phosphodiesterase 4 and tolerance to β2-adrenoceptor agonists in asthma

β 2 -Adrenoceptor agonists provide a mainstay in the treatment of asthma worldwide. However, despite their ability to provide symptomatic relief, chronic or repeated exposure to β 2 -adrenoceptor agonists does not resolve asthmatic inflammation, because of the rapid development of tolerance by pro-inflammatory and immune cells of the lung. The prevailing belief is that tolerance to the so-called non-bronchodilator actions of β 2 -adrenoceptor agonists is largely attributable to direct receptor desensitization mediated by G protein receptor-coupled kinases and/or cAMP-dependent protein kinase. Here, Mark Giembycz suggests another, largely ignored, explanation for β 2 -adrenoceptor desensitization that is based on the accelerated degradation of cAMP by Phosphodiesterase.

Mechanisms of beta adrenoceptor desensitisation in the failing human heart.

Journal Article Mechanisms of β adrenoceptor desensitisation in the failing human heart Get access Sian E Harding, Sian E Harding Department of Cardiac Medicine, National Heart and Lung Institute, Dovehouse Street, London SW3 6LY, United Kingdom: S E Harding, L A Brown, D G Wynne, C H Davies, P A Poole-Wilson. Correspondence to Dr Harding. Search for other works by this author on: Oxford Academic PubMed Google Scholar Lesley A Brown, Lesley A Brown Department of Cardiac Medicine, National Heart and Lung Institute, Dovehouse Street, London SW3 6LY, United Kingdom: S E Harding, L A Brown, D G Wynne, C H Davies, P A Poole-Wilson. Correspondence to Dr Harding. Search for other works by this author on: Oxford Academic PubMed Google Scholar Dylan G Wynne, Dylan G Wynne Department of Cardiac Medicine, National Heart and Lung Institute, Dovehouse Street, London SW3 6LY, United Kingdom: S E Harding, L A Brown, D G Wynne, C H Davies, P A Poole-Wilson. Correspondence to Dr Harding. Search for other works by this author on: Oxford Academic PubMed Google Scholar Crispin H Davies, Crispin H Davies Department of Cardiac Medicine, National Heart and Lung Institute, Dovehouse Street, London SW3 6LY, United Kingdom: S E Harding, L A Brown, D G Wynne, C H Davies, P A Poole-Wilson. Correspondence to Dr Harding. Search for other works by this author on: Oxford Academic PubMed Google Scholar Philip A Poole-Wilson Philip A Poole-Wilson Department of Cardiac Medicine, National Heart and Lung Institute, Dovehouse Street, London SW3 6LY, United Kingdom: S E Harding, L A Brown, D G Wynne, C H Davies, P A Poole-Wilson. Correspondence to Dr Harding. Search for other works by this author on: Oxford Academic PubMed Google Scholar Cardiovascular Research, Volume 28, Issue 10, October 1994, Pages 1451–1460, https://doi.org/10.1093/cvr/28.10.1451 Published: 01 October 1994 Article history Received: 03 February 1994 Accepted: 21 April 1994 Published: 01 October 1994

S-4-5 - Pheochromocytoma (PHEO)-harboring New England Deaconess Hospital (NEDH) rats: A model for hypertension and in vivo adrenoceptor desensitization.

S-4-5 - Pheochromocytoma (PHEO)-harboring New England Deaconess Hospital (NEDH) rats: A model for hypertension and in vivo adrenoceptor desensitization.

Beta Adrenergic receptor desensitisation may serve a cardioprotective role.

The aim was to test the hypothesis that beta adrenoceptor desensitisation is a form of cardioprotection whereby the myocardium is protected from the injurious effects of excessive adrenergic stimulation by isoprenaline. A new sensitive and specific method of identifying cardiac myocyte necrosis with monoclonal antimyosin was employed. Antibody labelled necrotic cardiomyocytes from male Sprague-Dawley rats (190-300 g) were identified by immunofluorescence. Homologous beta adrenoceptor desensitisation was induced by 9 d pretreatment with isoprenaline infusion (20 mg.kg-1.d-1), and heterologous desensitisation by treatment with 0.15% propylthiouracil in the diet for six weeks. The effects of isoprenaline induced cardiomyocyte injury in these animals were compared with those in control rats. In euthyroid control rats, treatment with the beta adrenergic agonist, isoprenaline, produced prolonged tachycardia and hypotension, and a significant amount of cardiac myocyte necrosis subendocardially. When the same isoprenaline challenge was given to rats pretreated for 9 d with an isoprenaline infusion, the haemodynamic response was markedly attenuated and very little myocyte necrosis was noted. In the model of heterologous desensitisation occurring in hypothyroidism, isoprenaline challenge produced markedly diminished haemodynamic response and little cardiac myocyte necrosis. In both homologous and heterologous models of beta adrenoceptor desensitisation, the susceptibility of the myocardium to isoprenaline induced cytotoxicity is markedly reduced.

Determination of the role of noradrenergic and 5-hydroxytryptaminergic neurones in postsynaptic alpha 2-adrenoceptor desensitization by desipramine and ECS.

1. Experiments were conducted to determine the respective roles which noradrenergic and 5-hydroxytryptaminergic neurones play in the down-regulation of postsynaptic alpha 2-adrenoceptors by desipramine and electroconvulsive shock (ECS). The functional status of these receptors was monitored by use of clonidine-induced mydriasis in conscious mice. 2. Mydriasis to clonidine (0.1 mg kg-1, i.p.) was markedly attenuated by administration of either desipramine (10 mg kg-1, i.p.) for 14 days or ECS (200 V, 2s) given five times over ten days confirming our previous observations. 3. The neurotoxin, DSP-4 (100 mg kg-1, i.p. X 2), reduced brain noradrenaline levels by 64% and abolished the mydriasis induced by the noradrenaline releasing agent and reuptake inhibitor, methamphetamine, without significantly altering the response to clonidine, confirming our earlier results. This lesion prevented the attenuation of clonidine mydriasis by repeated administration of desipramine, but not ECS. 4. Lesioning of central 5-hydroxytryptaminergic neurones with 5,7-dihydroxytryptamine (75 micrograms, i.c.v.) had no influence on the reduction in clonidine mydriasis produced by repeated administration of either desipramine or ECS. 5. Since noradrenergic neurones are essential for the desensitization of postsynaptic alpha 2-adrenoceptors by desipramine, it indicates that this effect is probably the result of increased synaptic noradrenaline levels. This mechanism is not responsible for the change induced by ECS because this adaptation is independent of an intact noradrenergic input. 5-HT-containing neurones do not play a permissive role in the down-regulation of postsynaptic alpha 2-adrenoceptors by either antidepressant treatment.

Adriamycin cardiomyopathy in the rabbit: alterations in contractile proteins and myocyte function

The aim was to determine the mechanism of the cardiotoxic effect of adriamycin, particularly at the level of the function of the cardiac myocyte. After chronic exposure to adriamycin, the contractile responses of single isolated cardiac myocytes to increasing calcium and isoprenaline were measured, as well as oxygen consumption of myocyte suspensions. Creatine kinase and myosin isoforms were investigated in whole ventricle. The degree of fibrosis of the ventricle was quantified using histological methods. 24 white male New Zealand rabbits were treated with adriamycin (1 mg.kg-1) twice a week for eight weeks, and allowed to recover for two weeks. There were 29 untreated controls. Six further rabbits were implanted with mini osmotic pumps delivering a constant infusion of isoprenaline for one week; five controls had pumps containing saline. Cardiac myocytes were enzymatically isolated, and their contraction amplitude and velocity monitored. Cells isolated from adriamycin treated rabbits had a lower contraction amplitude than those from controls when maximally activated with calcium, at 11.1(0.9)% shortening (n = 11) v 13.6(0.5)% (n = 14), p less than 0.02; or with isoprenaline, at 11.6(0.6)% shortening v 13.1(0.4)%, p less than 0.05. Contraction, but not relaxation, velocity in maximum calcium or isoprenaline was also significantly lower in cells from adriamycin treated animals. Oxygen consumption per 10(6) cells was lower in preparations from treated animals (p less than 0.05), but the relative effects of glucose, acetate, 2,4-DNP, and cyanide were unaffected. There was no significant change in creatine kinase or myosin isozyme composition or in amounts of fibrosis following adriamycin treatment. However, the quantity of myosin per g wet weight of tissue was significantly reduced from 8.04(0.45) mg.g-1 wet tissue, n = 4, in controls to 5.76(1.55) mg.g-1, n = 6, in adriamycin treated animals (p less than 0.001). The EC50 for isoprenaline was unchanged in cells from treated animals. Together with the unaltered maximum isoprenaline/calcium ratio, this implies that there is no change in beta adrenoceptor sensitivity following adriamycin treatment. To confirm that it was possible to desensitise rabbit cardiac beta adrenoceptors, and to detect changes in sensitivity on single cells, rabbits were treated with isoprenaline for one week. Such treatment decreased the maximum isoprenaline/calcium contraction amplitude ratio from 0.97(0.15), n = 5, to 0.47(0.12), n = 6 (p less than 0.05), and increased the EC50 from 7.9 to 224 nM (p less than 0.05). Single cardiac myocytes isolated from the hearts of adriamycin treated rabbits show a decrease in contraction amplitude, velocity, and oxygen consumption compared to controls. The decreased contractility of individual myocytes may relate to their low myosin content, and could contribute to the reduced cardiac output produced by adriamycin treatment. Heart failure induced by adriamycin in the rabbit is not accompanied by beta adrenoceptor desensitisation.

Protein kinase A and/or C inhibitors potentiate isoproterenol-induced cyclic AMP accumulation in intact human lymphocytes

The objective of the present study was to investigate the roles of protein kinase A and/or C in agonist-induced beta adrenoceptor activation in intact human lymphocytes. Lymphocytes from healthy subjects were incubated with isoproterenol and phophodiesterase inhibitor (IBMX, 1.0 mM) after 20 minutes of preincubation with (or without) various compounds possessing protein kinase A and/or C inhibitory activities. These compounds included the relatively selective protein kinase C (PK-C) inhibitors (W-7, calmidazolium, polymyxin B, neomycin, tamoxifen and clomiphene), purified protein inhibitors of protein kinase A (PK-A) (obtained synthetically, or purified from bovine hearts and porcine hearts) and the two compounds (H-7, H-9), which have been found to inhibit both PK-A and PK-C. The results showed that all PK-C inhibitors alone decreased cellular basal cAMP levels while inhibitors of PK-A as well as both H-7 and H-9 increased basal cAMP levels in a dose dependent manner at certain concentrations. All inhibitors studied potentiated isoproterenol-induced cAMP accumulation. The protein kinase A and C inhibitor, H-7, also potentiated PGE 1 (but not forskolin)-induced cAMP accumulation. In contrast, the protein kinase C activator, PMA, inhibited isoproterenol- and PGE 1- (but not forskolin) induced cAMP accumulation. These data suggest that the potentiating effects of PK-A and/or C inhibitors may be related to the inhibition of PK-A and/or PK-C, both of which have been shown to be involved in beta 2 adrenoceptor desensitization and phosphorylation.

Time Course of α<sub>2</sub>-Adrenoceptor Desensitization Induced by Adrenaline Infusion in Man

After a rapid increase in infusion rate of adrenaline to elevate the initial heart rate by more than 15%, a continuous infusion was sustained in order to maintain elevated adrenaline plasma levels. At the end of the first phase of the experiment, the thrombocyte alpha 2-adrenoceptor number (determined by radioligand binding) was significantly lowered. After the second part of the experiment, a further significant decrease in the density of binding sites was observed, while elevated adrenaline plasma levels were kept constant. The decrease in the number of binding sites was accompanied by a slight increase in affinity of the radioligand. Serum potassium concentration significantly decreased during the experiment, while other serum electrolytes showed no significant alterations.

Active sensitisation modifies β-adrenoceptor reactivity in guinea-pig trachea

The occurrence of beta-adrenoceptor desensitisation in clinical experience, after long term therapy with specific beta 2-agonists, is still an open question, even if this phenomenon is easily observed in different experimental models. Since the majority of these experiments have been performed in normal animals, we investigated the possible occurrence of beta-adrenoceptor desensitisation in the ovalbumin actively sensitised guinea-pig model of experimental asthma. The isoproterenol concentration-response curves performed in pilocarpine contracted guinea-pig trachea in vitro were shifted to the right by the beta-adrenoceptor desensitisation procedure, which was achieved by the in vitro administration of isoproterenol (10(-5) M x 2 times x 20 min each) both in normal and ovalbumin sensitised tissues. The same desensitisation procedure markedly affected epinephrine-relaxing capacity in both normal and ovalbumin actively sensitised guinea-pig tracheae. However, the ovalbumin sensitised tissue seemed to be more sensitive than normal to the specific beta 2-agonist procaterol; in parallel the beta 2-mediated relaxation was more impaired by isoproterenol-induced beta-adrenoceptor down regulation in ovalbumin sensitised trachea when compared to normal. Similar results have been obtained using salbutamol as the beta 2-adrenoceptor desensitising agent. The changes in beta-adrenoceptor reactivity between normal and ovalbumin sensitised guinea-pig tracheae seemed to depend on the active sensitisation process. No difference in the degree of beta-adrenoceptor down regulation was observed in passively ovalbumin sensitised guinea-pig trachea as compared to normal. These data suggest that, in this model of experimental asthma, beta-adrenoceptor reactivity is in some way modified and that this phenomenon might contribute to the genesis of asthma.

Ketotifen increases cyclic adenosine 3′,5′-monophosphate in intact human lymphocyte and potentiates other adenylate cyclase activating agents

We have investigated the effects of ketotifen on the cyclic adnosine 3′, 5′-monophosphate (cyclic AMP) response of intact human lymphocyte and its interaction with adenylate cyclase activating agents. In the presence of cyclic AMP phosphodiesterase inhibitor (3-isobutyl-1-methyl -xanthine), ketotifen (10 −8-10 −4 M) caused an 80% increase in cyclic AMP content of human lymphocyte, a magnitude similar to that observed with hydrocortisone. The cyclic AMP level peaked at about 15 minutes and remained elevated for at least 45 minutes. In addition, ketotifen (10 −6 -10 −4 M) markedly potentiated the effect of several adenylate cyclase stimulating agents, including L-isoproterenol, prostaglandin E 1 and forskolin. The biochemical mechanisms underlying these effects are unknown. It may be at least partly related to the ability of ketotifen to reverse and prevent beta 2 adrenoceptor desensitization and to promote the formation of hormone - nucleotide - high affinity receptor complex. These effects may contribute to its prophylactic effect in the treatment of bronchial asthma.

Mechanisms and time course of beta 1 adrenoceptor desensitisation in mammalian cardiac myocytes.

To study the mechanisms and time course of beta1 adrenoceptor desensitisation in mammalian heart tissue neonatal rat cardiac myocytes (greater than 90% pure) were cultured in serum free medium. Cells were exposed to 1 mumol X litre-1 (-)-isoprenaline for 30 min, 4 h, and 16 h. In myocyte membranes mean(SEM)) 125I-iodocyanopindolol binding was 167(46) pmol X litre-1 (n = 5) and did not differ at 30 min, 4 h, or 16 h in control compared with (-)-isoprenaline treated cells. The maximum number of binding sites was 84(32) fmol X mg protein-1 and was unchanged at 30 min, but (-)-isoprenaline stimulated adenylate cyclase activity significantly decreased from 221(62) to 103(37) pmol X mg protein-1 30 min-1. (-)-Isoprenaline competition curves at 30 min showed a significant increase in the proportion of low affinity binding sites from 46% to 62% (n = 5). By 4 h the maximum number of binding sites was significantly decreased by 54%, adenylate cyclase activity remained depressed, and agonist affinity decreased threefold in the (-)-isoprenaline treated cells. At 16 h (-)-isoprenaline treated cells showed alterations similar to the 4 h values in the maximum number of binding sites, adenylate cyclase activity, and affinity for (-)-isoprenaline. (-)-Isoprenaline stimulated adenylate cyclase activity took 72 h to recover after desensitisation. Overnight ultracentrifugation of the cytosol showed a significant 40% increase in beta adrenoceptor density in cells exposed to (-)-isoprenaline for 4 h (n = 5), suggesting receptor internalisation.(ABSTRACT TRUNCATED AT 250 WORDS)

Autoinhibition of noradrenaline release from the rat heart as a function of the biophase concentration. Effects of exogenous alpha-adrenoceptor agonists, cocaine, and perfusion rate.

Rat isolated perfused hearts with the right sympathetic nerves intact were loaded with 3H-(-)-noradrenaline. The nerves were stimulated with trains of 180 pulses at 3 Hz and at 10 min intervals. The overflow of 3H-noradrenaline and 3H-metabolites was determined by liquid scintillation spectrometry. Clonidine (IC50 17 nM), oxymetazoline (IC50 63 nM), and alpha-methylnoradrenaline (apparent IC50 35 nM, determined in the presence of cocaine and propranolol) decreased the stimulation-evoked overflow of 3H-noradrenaline by 26, 49, and 78%, respectively, but not methoxamine up to 100 microM (propranolol present). Oxymetazoline and alpha-methyl-noradrenaline did not cause desensitization of the presynaptic adrenoceptors when present at their IC80 for 33 min. At a perfusion rate of 7 ml/min, yohimbine 1 microM enhanced the stimulation-evoked 3H-noradrenaline overflow by 26% in the absence, and by 58% in the presence of cocaine. Phentolamine 1 microM increased it by 69% when the neuronal reuptake was blocked. The increase by the antagonists faded with successive period of nerve stimulations, and was positively correlated with the biophase concentration of noradrenaline as reflected by the amount of 3H-noradrenaline released into the perfusate per nerve stimulation. At a perfusion rate of 1.8 ml/min (neuronal reuptake blocked), yohimbine 1 microM increased the overflow by 127%. The results indicate that the alpha 2-adrenoceptor-mediated autoinhibition in the rat perfused heart depends on the clearance of transmitter from the biophase via neuronal reuptake and diffusion into the vascular space. Reduction of either elimination pathway enhances the biophase concentration of noradrenaline, thus increasing the autoinhibition of release.

Modulation of noradrenergic transmission in the guinea-pig mesenteric artery: an electrophysiological study.

We carried out electrophysiological experiments on guinea-pig mesenteric arteries in an attempt to clarify the modification of noradrenaline (NA) release from noradrenergic nerve terminals by the action of prejunctional adrenoceptors. NA (10(-7)-10(-5) M) suppressed the amplitude of the first excitatory junction potential (e.j.p.(f], the facilitation process, and the e.j.p. after facilitation was completed (e.j.p.(s]with no change in the post-junctional membrane properties of smooth muscles. These actions of NA on e.j.p.s were antagonized by high concentrations of extracellular Ca, [Ca]o, but not in a simple competitive manner. NA (10(-7) M) suppressed the appearance but not the amplitude of the miniature e.j.p.s. These effects of NA on transmission indicate that NA acts on prejunctional nerve terminals and suppresses the release of NA from nerve terminals rather than producing a desensitization of post-junctional adrenoceptors. Prazosin and phentolamine (10(-6) M) did suppress the NA-induced contraction (greater than 10(-6) M) but did not suppress the contraction evoked by perivascular nerve stimulation, below a frequency of 1.0 Hz. At a dose of 10(-7) M, yohimbine, clonidine, prazosin and phentolamine had no effect on the muscle membrane potential and resistance. Yet on the e.j.p.(f), yohimbine and clonidine caused suppression, phentolamine enhancement and prazosin had no effect. On the e.j.p.(s), yohimbine and phentolamine caused enhancement, clonidine suppression and prazosin had no effect. These results indicate that at least three different adrenoceptors are distributed on the neuromuscular junction in this tissue, i.e. alpha 1-extrajunctional, alpha 2-prejunctional, and an unknown subtype of intrajunctional adrenoceptors. Furthermore, the feedback mechanism on NA release is mediated by suppression of the influx of Ca. Nonselective and non-specific actions of alpha-adrenoceptor agonists and antagonists were also elucidated.

Adrenoceptor desensitization after immobilization stress or repeated injection of isoproterenol.

Immobilization stress (2.5 h daily) or repeated injection of isoproterenol (1 mg/kg, 3 times daily) for 1 wk caused a subsensitivity to the chronotropic and pressor effect of epinephrine in pithed rats. Propranolol (1 mg/kg) inhibited, to a greater extent in control than in immobilized rats, the chronotropic effect of epinephrine. The residual heart rates did not differ significantly among control, immobilized, and isoproterenol-treated rats. Practolol (1 mg/kg) but not butoxamine (5 mg/kg) mimicked the effect of propranolol. The subsensitivity in the blood pressure responses was not abolished by injection of either of the beta-adrenoceptor blockers. Butoxamine or propranolol shifted the epinephrine dose-blood pressure response curves to the left. The degree of shift was similar in control and immobilized or isoproterenol-treated rats. Daily injection of quinacrine (16 mg/kg), an antimalarial agent that inhibits phospholipase A2, blocked the subsensitivity to the chronotropic effect of epinephrine, but not that to the pressor effect of epinephrine. These observations suggest that immobilization stress causes desensitization of alpha- and beta 1-receptors but not beta 2-receptors. The mechanism of desensitization of beta 1-receptors by immobilization appears to be similar to that after isoproterenol treatment, possibly due to increased turnover of phospholipids.