Adrenoceptor Regulation Research Articles

Introduction: A Short History of Adrenoceptor Research.

This chapter provides a short history of adrenoceptor research starting from the initial discovery of adrenaline. It covers the evolving classification of adrenoceptor subtypes, the cloning of these subtypes from multiple species, and factors such as adrenoceptor regulation, inverse agonism and biased agonism. More details on many of these aspects are provided in other chapters of this volume of Handbook of Experimental Pharmacology.

Adrenoceptor regulation of the mechanistic target of rapamycin in muscle and adipose tissue.

A vital role of adrenoceptors in metabolism and energy balance has been well documented in the heart, skeletal muscle, and adipose tissue. It has been only recently demonstrated, however, that activation of the mechanistic target of rapamycin (mTOR) makes a significant contribution to various metabolic and physiological responses to adrenoceptor agonists. mTOR exists as two distinct complexes named mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) and has been shown to play a critical role in protein synthesis, cell proliferation, hypertrophy, mitochondrial function, and glucose uptake. This review will describe the physiological significance of mTORC1 and 2 as a novel paradigm of adrenoceptor signalling in the heart, skeletal muscle, and adipose tissue. Understanding the detailed signalling cascades of adrenoceptors and how they regulate physiological responses is important for identifying new therapeutic targets and identifying novel therapeutic interventions. LINKED ARTICLES: This article is part of a themed section on Adrenoceptors-New Roles for Old Players. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.14/issuetoc.

Inhibition of prostatic smooth muscle contraction by the inhibitor of G protein-coupled receptor kinase 2/3, CMPD101

Alpha1-adrenoceptors induce prostate smooth muscle contraction, and hold a prominent role for pathophysiology and therapy of lower urinary tract symptoms in benign prostatic hyperplasia. G protein-coupled receptors are regulated by posttranslational regulation, including phosphorylation by G protein-coupled receptor kinases 2 and 3 (GRK2/3). Although posttranslational adrenoceptor regulation has been recently suggested to occur in the prostate, this is still marginally understood. With the newly developed CMPD101, a small molecule inhibitor with assumed specificity for GRK2/3 is now available. Here, we studied effects of CMPD101 on smooth muscle contraction of human prostate tissue. Electric field stimulation caused frequency-dependent contractions, which were inhibited concentration-dependently by CMPD101 (5 µM, 50 µM). 50 µM of CMPD101 did not affect myosin light chain (MCL) phosphorylation or Rho kinase activity, and did not alter contractions induced by highmolar KCl. Noradrenaline, the α1-adrenoceptor agonist phenylephrine, endothelin-1, and the thromboxane A2 analogue U46619 induced concentration-dependent contractions, which were inhibited by CMPD101 (50 µM). CMPD101 (50 µM) did not change phosphorylation of β2-adrenoceptors or β2-adrenergic relaxation of prostate strips. Molecular detection by Western blot and peroxidase staining suggested expression of GRK2 and GRK3 in human prostates. Double labeling in fluorescence staining confirmed that immunoreactivity for GRK2 and GRK3 was located to smooth muscle cells in the prostate stroma. In conclusion, CMPD101 inhibits adrenergic, neurogenic, and non-adrenergic smooth muscle contractions in the human prostate. Underlying mechanisms may be independent from GRK inhibition, and from inhibition of MLC kinase and Rho kinase. This may point to unknown properties of CMPD101.

High Dietary Salt Intake Drives Sympathetically Mediated Alpha‐1 Adrenoceptor Regulation of NCC to Increase Renal Sodium Retention and Blood Pressure via an OXSR1 Dependent Pathway

BackgroundAcute studies have suggested that sympathetic nervous system (SNS) release of norepinephrine (NE) mediates upregulation of the sodium chloride cotransporter (NCC) via synergistic α1 and β adrenergic receptor pathways. Moreover, recent data suggests a key role of oxidative stress responsive 1 (OXSR1), in NCC regulation.HypothesisNorepinephrine (NE) activates an α1 adrenoceptor pathway that is dependent on OXSR1 to mediate increases in NCC activity that drive the development of salt sensitive hypertension (HTN).MethodsMale Sprague‐Dawley (SD) rats receiving a continuous s.c. saline or NE (600ng/min) infusion were fed a 0.6% (NS) or 4% NaCl (HS) diet for 14 days respectively. Separate groups of NE infused SD rats were given terazosin (10mg/kg/day; s.c.) and a NS or HS diet for 14‐days. An OXSR1 targeted lentivirus was injected in the kidneys of NE infused SD rats prior to a 14 day HS diet. On day 14 basal MAP, NCC activity (peak natriuresis to iv hydrochlorothiazide (HCTZ; 2mg/kg) infusion) and peak MAP change in response to ganglionic blockade (iv hexamethonium; 30mg/kg) were assessed. NCC expression, phosphoNCC, and OXSR1 levels were assayed via immunoblotting (N=4/gp).ResultsSD rats remain normotensive when fed a high salt diet and NCC expression and activity is downregulated. NE infusion however, inhibits the NCC downregulation associated with HS intake and evokes salt sensitive HTN. Use of an α1‐adrenoceptor antagonist in NE infused SD rats abolishes salt sensitive HTN and downregulates NCC activity. Significantly, lentiviral knockdown of OXSR1 (confirmed by immunoblot) also eliminates salt sensitive HTN in a similar fashion. Furthermore, ganglionic blockade did not produce any observable differences in NE infused rats on HS or NS that were treated or not treated with the α1‐adrenoceptor antagonist or lentivirus.ConclusionOur data reveals that SNS release of NE can modulate NCC activity via an α1‐adrenoceptor pathway to prevent dietary evoked salt sensitive HTN. Remarkably, both OXSR1 knockdown and α1‐adreoceptor antagonism reverse salt sensitive HTN. As a theoretical model, the SNS release of NE activates a linear α1‐adreoceptor pathway that stimulates OXSR1 mediated regulation of NCC. The PATHWAY‐2 Trail implicates sodium retention as a driver of resistant HTN suggesting that α1‐adreoceptor antagonism and OXSR1 blockade may represent novel therapeutic targets for HTN.Support or Funding InformationThis work was supported by NIH grants R01HL107330 and K02HL112718 and AHA grants 16MM32090001 and AHA17GRNT3367002 to RDW and NIH grant F31DK116501 to AAF.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Antidiuretic and antinatriuretic response to high salt load in normotensive Wistar-Kyoto rats: Role of alpha-1A-adrenoreceptors

Altered renal adrenergic responses have been recognized as pathophysiological responses to high salt intake. This study aims to investigate the influence of 6weeks of high salt diet on α1A -adrenoceptor regulation of renal tubular antinatriuretic and antidiuretic response in normal Wistar Kyoto rats. To achieve the above objective, antinatriuretic and antidiuretic response to phenylephrine was measured in the absence and presence of 5-methylurapidil (5-MeU) using the inulin clearance method. Systemic mean arterial blood pressure and renal haemodynamics were also measured simultaneously. Six weeks of high salt intake in Wistar-Kyoto (WKY) rats did not bring any significant increase in mean arterial blood pressure. WKY rat on high salt diet (WKYHNa) showed an exaggerated increase in absolute and fractional sodium excretion. There was a significant involvement of α1A -adrenoceptor in carrying out renal tubular antinatriuretic and antidiuretic response in Wistar Kyoto rats on normal sodium diet (WKYNNa). However, α1A -adrenoceptor played a minimal role in handling the tubular reabsorptive response in WKY rats on high salt diet.

Abstract P334: Sympathetically Mediated Alpha-1 Adrenoceptor Regulation of the NCC During High Salt Intake: A New Therapeutic Target for Resistant Hypertension

Aim: We hypothesize that excess norepinephrine (NE) modulates NCC activity via an α1 adrenoceptor pathway to drive the development of salt-sensitive hypertension (HTN). Methods: Male Sprague-Dawley (SD) rats receiving a continuous s.c. saline or NE (600ng/min) infusion and naïve Dahl Salt-Sensitive (DSS) rats were fed a 0.6% (NS) or 8% NaCl (HS) diet for 14 or 21 days respectively (N=4/gp). On day 14 (SD) or 21 (DSS) MAP and NCC activity (peak natriuresis to iv hydrochlorothiazide (HCTZ; 2mg/kg) infusion) and expression (via immunoblotting) was assessed. Additional groups of NE infused SD and DSS rats received a propranolol (9.9mg/kg/day; s.c.) or prazosin (2.5mg/kg/day; oral) and a NS or HS diet for 14 or 21 days. Results: SD rats exhibit HS evoked suppression of NCC expression and activity. In contrast, NE infused SD rats and DSS rats exhibit HTN and fail to suppress NCC expression and activity during HS-intake. β-adrenoceptor antagonism (confirmed pharmacologically) reduced MAP in NE infused SD and DSS rats, but failed to decrease NCC activity or expression. In contrast α1-adreoceptor antagonism (confirmed pharmacologically) abolished the salt-sensitive component of HTN and restored dietary sodium evoked suppression of NCC activity and expression in NE infused SD rats and DSS rats. Conclusion: Our data suggests NE activates α, but not β, adrenoceptors to prevent dietary sodium evoked suppression of NCC activity and the development of salt-sensitive hypertension. The PATHWAY-2 Trial reported a primary role of sodium retention in resistant HTN suggesting α1-adreoceptor antagonism represents a new therapeutic approach for resistant and sympathetically mediated HTN.

Rat white adipocytes activate p85/p110 PI3K and induce PM GLUT4 in response to adrenoceptor agonists or aluminum fluoride.

Adipocyte responses to adrenergic and ß-adrenoceptor(-AR) (adrenoceptor) regulation are not sufficiently understood, and information helpful for elucidating the adrenoceptor-responsive machinery is insufficient. Here we show by using immunoprecipitated kinase analysis with a phosphatidylinositol 3-kinase (PI3K) p85 antibody that PI3K activation was induced by treatment with 10 or 100 µM norepinephrine (NE) for 15 min or with 10 mM aluminum fluoride (AF, a guanosine triphosphate (GTP)-binding (G) protein activator) for 20 min in white adipocytes (rat epididymal adipocytes) and that treatment with pertussis toxin (PTX, a G-protein inactivator) inhibited PI3K activation induced by the 20-min treatment with AF in the cells. In addition, western blot analysis revealed that glucose transporter 4 (GLUT4) level in the adipocyte plasma membrane (PM) fraction was increased by treatment with 10 µM NE, 100 µM dobutamine (DOB, a ß1-AR agonist), or 0.1 µM CL316243 (CL, a ß3-AR agonist) for 30 min or with 10 mM AF for 20 min. NE or AF treatment triggered 2-deoxyglucose (2-DG) uptake into adipocytes under the above conditions. Our results advance the understanding of responses to adrenoceptor regulation in white adipocytes and provide possible clues for clarifying the machinery involved in adrenergic and ß-AR responses in the cells.

The sympathetic nervous response in inflammation.

Over the past decades evidence has accumulated clearly demonstrating a pivotal role for the sympathetic nervous system (SNS) and its neurotransmitters in regulating inflammation. The first part of this review provides the reader with an overview showing that the interaction of the SNS with the immune system to control inflammation is strongly context-dependent (for example, depending on the activation state of the immune cell or neuro-transmitter concentration). In the second part we focus on autoimmune arthritis as a well investigated example for sympathetically controlled inflammation to show that the SNS and catecholamines play a differential role depending on the time point of ongoing disease. A model will be developed to explain the proinflammatory effects of the SNS in the early phase and the anti-inflammatory effects of catecholamines in the later phase of autoimmune arthritis. In the final part, a conceptual framework is discussed that shows that a major purpose of increased SNS activity is nourishment of a continuously activated immune system at a systemic level using energy-rich fuels (glucose, amino acids, lipids), while uncoupling from central nervous regulation occurs at sites of inflammation by repulsion of sympathetic fibers and local adrenoceptor regulation. This creates zones of ‘permitted local inflammation’. However, if this ‘inflammatory configuration’ persists and is strong, as in autoimmunity, the effects are detrimental because of the resultant chronic catabolic state, leading to cachexia, high blood pressure, insulin resistance, and increased cardiovascular mortality, and so on. Today, the challenge is to translate this conceptual knowledge into clinical benefit.

Adipose Cells Induce Phospho-Thr-172 AMPK Production by Epinephrine or CL316243 in Mouse 3T3-L1 Adipocytes or MAPK Activation and G Protein-Associated PI3K Responses Induced by CL316243 or Aluminum Fluoride in Rat White Adipocytes

Responses of adipose cells to adrenoceptor regulation, including that of β-adrenoceptor (AR), and the signalling machinery involved in these responses are not sufficiently understood; information that is helpful for elucidating the adrenoceptor (adrenergic and β-AR)-responsive machinery is insufficient. We examined phospho-Thr-172 AMPK production in mouse-derived 3T3-L1 adipocytes treated with epinephrine or CL316243 (a β3-AR agonist) for 15 min. We also examined MAPK activation or G protein-associated PI3K activation or -associated PI3K p85 complex formation in rat epididymal (white) adipocytes treated with CL316243 for 15 min or aluminum fluoride (a G-protein signalling activator) for 20 min. Furthermore, we examined the effect of PTX (a trimeric G-protein inactivator) on p85 complex formation induced by aluminum fluoride treatment. Western blot analysis revealed that epinephrine or CL316243 treatment increased the phospho- Thr-172 AMPK (an active form of AMPK) level in 3T3-L1 adipocytes. Activated kinase analysis with a specific substrate showed that CL316243 or aluminum fluoride treatment activated MAPK in rat adipocytes. Immunoprecipitation experiments with a G-protein β subunit (Gβ) antibody showed that treatment of rat adipocytes with CL316243 activated PI3K and increased the PI3K p85 level in the Gβ antibody immunoprecipitates. Such an increase in the p85 level was similarly elicited by aluminum fluoride treatment in a PTX-sensitive manner. Our results provide possible clues for clarifying the signalling machinery involved in adrenoceptor responses, including those of β3-AR, in mouse-derived adipocytes and rat white adipocytes. Our findings advance the understanding of responses to adrenoceptor regulation in adipose cells and of the cellular signalling machinery present in the cells.

Antinociception occurs with a reversal in α 2-adrenoceptor regulation of TNF production by peripheral monocytes/macrophages from pro- to anti-inflammatory

Tumor necrosis factor-α (TNF) plays a role in neuropathic pain. During neuropathic pain development in the chronic constriction injury model, elevated TNF levels in the brain occur in association with enhanced α 2-adrenoceptor inhibition of norepinephrine release. α 2-Adrenoceptors are also located on peripheral macrophage where they normally function as pro-inflammatory, since they increase the production of the cytokine TNF, a proximal mediator of inflammation. How the central increase in TNF affects peripheral α 2-adrenoceptor function was investigated. Male, Sprague–Dawley rats had four loose ligatures placed around the right sciatic nerve. Thermal hyperalgesia was determined by comparing hind paw withdrawal latencies between chronic constriction injury and sham-operated rats. Chronic constriction injury increased TNF immunoreactivity at the lesion and the hippocampus. Amitriptyline, an antidepressant that is used as an analgesic, was intraperitoneally administered (10 mg/kg) starting simultaneous with ligature placement (day-0) or at days-4 or -6 post-surgery. Amitriptyline treatment initiated at day-0 or day-4 post-ligature placement alleviated hyperalgesia. When initiated at day-0, amitriptyline prevented increased TNF immunoreactivity in the hippocampus and at the lesion. A peripheral inflammatory response, macrophage production of TNF, was also assessed in the current study. Lipopolysaccharide (LPS)-stimulated production of TNF by whole blood cells and peritoneal macrophages was determined following activation of the α 2-adrenoceptor in vitro. α 2-Adrenoceptor regulation of TNF production from peripheral immune-effector cells reversed from potentiation in controls to inhibition in chronic constriction injured rats. This effect is accelerated with amitriptyline treatment initiated at day-0 or day-4 post-ligature placement. Amitriptyline treatment initiated day-6 post-ligature placement did not alleviate hyperalgesia and prevented the switch from potentiation to inhibition in α 2-adrenoceptor regulation of TNF production. Recombinant rat TNF i.c.v. microinfusion reproduces the response of peripheral macrophages from rats with chronic constriction injury. A reversal in peripheral α 2-adrenoceptor regulation of TNF production from pro- to anti-inflammatory is associated with effective alleviation of thermal hyperalgesia. Thus, α 2-adrenoceptor regulation of peripheral TNF production may serve as a potential biomarker to evaluate therapeutic regimens.

Differential regulation on human skin fibroblast by α 1 adrenergic receptor subtypes

Alpha 1 adrenoceptor (α 1-AR) regulation of DNA synthesis was studied in human neonatal foreskin fibroblast. Saturation assay with a specific radioligand for α 1 adrenergic [ 3H]-prazosin revealed two saturated and specific binding sites with high or low affinity. Competitive binding assay with different antagonist subtypes, defined pharmacologically three major types of α 1-AR. The α 1-AR agonists (from 1 × 10 −10 to 1 × 10 −4 M) triggered a biphasic action on DNA synthesis reaching maximal stimulation at 1 × 10 −9 M and maximal inhibition at 1 × 10 −6 M. Prazosin, abolished the stimulatory (pA 2: 9.24) and inhibitory (pA 2: 8.80) actions of α 1-AR agonists. The α 1-AR stimulation resulted in the activation of phosphoinositide turnover (InsP) via phospholipase C (PLC) involving calcium/calmodulin (CaM) and nitric oxide synthase (NOS) that correlates with the DNA synthesis increment; whereas the inhibition resulted in a decrease of cyclic AMP (cAMP) accumulation via adenylate cyclase inhibition. The potency displayed by the specific antagonists tested in binding, DNA synthesis, InsP and NOS at low agonist concentration suggests that they can be elicited by the activation of the same receptor (α 1B-AR subtype); while the decrement in DNA synthesis and cAMP at high concentration account by the activation of α 1D-AR coupled to G i protein. Non-functional α 1A-AR in neonatal human foreskin fibroblast was observed. Results suggest that the expression of α 1-AR subtypes on human skin fibroblast may differentially activate signaling pathways that modulate physiological response of the cells.

Regulation of the HPA axis by alpha 2 adrenoceptors

The hypothalamic-pituitary-adrenal (HPA) axis is regulated by numerous neurotransmitters, including central norepinephrine (NE), which exerts a stimulatory role on the HPA axis. There are numerous adrenoceptors (AR) subtypes involved in the regulation of the effects on NE, including 3 subtypes of alpha (α)2 ARS –α2A, α2B and α2C. While no evidence exists for a specific role of α2B ARs in regulation of central NE release, α2A and α2C ARs exist as presynaptic autoreceptors mediating NE neurotransmission. We investigated the role of the non-selective α2 antagonists RX821002 and idazoxan, a selective α2A antagonist BRL 44408 and a selective α2C antagonist OPC 28326 in blocking increases in rat plasma corticosterone levels produced by the α2 AR agonists clonidine and guanfacine. Corticosterone levels were increased 1 hour after systemic administration of both clonidine and guanfacine, which were dose-dependently blocked by both selective and non-selective α2 AR antagonists. These data suggest that both α2A and α2C ARs regulate the effects of NE on the HPA axis. Further studies on the in vivo effects of α2A and α2C ARs are needed to elucidate the functional activity of α2 ARs.

Noradrenaline overflow in mouse dentate gyrus following locus coeruleus and natural stimulation: real‐time monitoring by in vivo voltammetry

The pattern of catecholaminergic innervation of the dentate gyrus (DG) of the hippocampus, particularly the relatively dense and selective noradrenergic input, creates favourable conditions for real-time monitoring of noradrenaline (NA) release following stimulation of the locus coeruleus (LC) by in vivo voltammetry. Two electrochemically active species with different temporal characteristics were registered in the DG following electrical stimulation of the LC. Several approaches, including testing of anatomical and pharmacological specificity, coating of microelectrodes with Nafion and use of fast cyclic voltammetry, were used to verify the characteristics of electrochemical responses. The first sharp peak that appeared immediately during stimulation was definitely associated with NA overflow. The second late peak was possibly attributable to ascorbic acid. We examined the characteristics of alpha-2 adrenoceptor regulation of NA release in the DG, and showed for the first time that noradrenergic terminals resemble dopaminergic terminals in their mechanisms of increasing the refilling rate of the readily releasable pool following stimulation repeated at short intervals. Amperometric registration of NA in the DG was complicated by interference with electrical activity of hippocampus. This interference could be used, after appropriate filtration, for simultaneous recording from the same microelectrode of NA release and electrical activity of the hippocampus.

Submandibular gland acinar cells express multiple alpha1-adrenoceptor subtypes.

We evaluated an acinar cell line (SMG-C10) cloned from rat submandibular glands as a possible model for alpha(1)-adrenoceptor regulation of submandibular function. alpha(1)-Adrenoceptors are subdivided into three subtypes called alpha(1A), alpha(1B), and alpha(1D), which can be distinguished from one another by their differential affinity values for subtype-selective alpha(1)-adrenoceptor antagonists. Thus, alpha(1)-adrenoceptor subtypes in SMG-C10 cells were characterized with reverse transcription-polymerase chain reaction (RT-PCR) and [(3)H]prazosin binding in side-by-side experiments with native submandibular glands. RT-PCR identified mRNAs for alpha(1A)-, alpha(1B)-, and alpha(1D)-adrenoceptors in SMG-C10 cells and submandibular glands. The inhibition of [(3)H]prazosin binding by 5-methylurapidil (alpha(1A)-selective) was biphasic and fit best to a two-site binding model with 40 +/- 8% high (K(iH))- and 60 +/- 10% low (K(iL))-affinity binding sites in SMG-C10 cells, and 76% high- and 24% low-affinity binding sites in submandibular glands. Respective K(iH) and K(iL) values for 5-methylurapidil were 1.9 +/- 0.4 and 100 +/- 30 nM in SMG-C10 cells and 3.2 +/- 0.8 and 170 +/- 20 nM in submandibular glands. BMY-7378 [8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4.5]decane-7,9-dione dihydrochloride (alpha(1D)-selective)] bound with low affinity in SMG-C10 cells and submandibular glands with K(i) values of 81 +/- 20 and 110 +/- 20 nM, respectively. Chloroethylclondine, an irreversible alkylating agent selective for alpha(1B) adrenoceptors, reduced the density of [(3)H]prazosin binding sites by 42 and 26% in SMG-C10 and submandibular membranes, respectively. Thus, SMG-C10 cells and submandibular glands are similar in expressing receptor protein for alpha(1A)- and alpha(1B)-adrenoceptor subtypes, establishing SMG-C10 cells as a potential model for alpha(1)-adrenoceptor-mediated secretion.

Beta 2-Adrenoceptor regulation of CGRP release from capsaicin-sensitive neurons.

Previous studies have suggested that neurotransmitter substances from the sympatho-adrenomedullary system regulate pulpal blood flow (PBF), in part, by the inhibition of vasoactive neuropeptide release from pulpal sensory neurons. However, no study has evaluated the role of beta-adrenoceptors. We evaluated the hypothesis that activation of beta-adrenoceptors inhibits immunoreactive calcitonin gene-related peptide (iCGRP) release from capsaicin-sensitive nociceptive neurons via in vitro superfusion of bovine dental pulp. Either norepinephrine or epinephrine inhibited capsaicin-evoked iCGRP. The norepinephrine effect was blocked by the selective beta(2)-adrenoceptor antagonist, ICI 118,551, but not by pre-treatment with the selective beta(1)-adrenoceptor antagonist, atenolol. In addition, application of albuterol, a selective beta(2)-adrenoceptor agonist, significantly blocked capsaicin-evoked release of iCGRP. Collectively, these studies demonstrate that activation of beta(2)-adrenoceptors in dental pulp significantly reduces exocytosis of neuropeptides from capsaicin-sensitive nociceptors. This effect may have physiologic significance in regulating PBF. Moreover, since capsaicin selectively activates nociceptors, beta(2)-adrenoceptor agonists may have clinical utility as peripherally acting therapeutics for dental pain and inflammation.

Effect of norepinephrine release on adrenoceptors in severe seizure genetically epilepsy-prone rats

The genetically epilepsy-prone rat (GEPR) seizure model is characterized by extensive abnormalities in brain noradrenergic function. Earlier studies had suggested that GEPRs might not regulate adrenoceptors in a normal fashion. The purpose of the present study was to determine if GEPR-9s are capable of up and down regulation of α 1- and β-adrenoceptors in response to increments or decrements in extracellular norepinephrine. Seizure induction has been shown to increase extracellular norepinephrine. Chronic sound or electroshock-induced seizures caused down regulation of β-adrenoceptors in frontal cortex and in hippocampus from GEPR-9s. Similarly, chronic daily treatment with the norepinephrine reuptake inhibitor desmethylimipramine produced down regulation of β-adrenoceptors in frontal cortex and in hippocampus from GEPR-9s. As is the case in neurologically normal animals, chronic electroshock-induced seizure did not cause down regulation of β-adrenoceptors in 6-hydroxydopamine pretreated GEPR-9s. Chronic electroshock treatment also caused up-regulation of α 1-adrenoceptors in frontal cortex but not in hippocampus. In 6-hydroxydopamine pretreated GEPR-9s, chronic electroshock treatment caused a further up-regulation of α 1-adrenoceptors in frontal cortex but not in hippocampus. Taken together, these results indicate that GEPR-9s are capable of up and down regulation of α 1- and β-adrenoceptors in a manner that is qualitatively similar to the regulation of these receptors in normal animals. Whether the regulation of brain adrenoceptors is quantitatively different in GEPRs from normal animals remains to be established.

Beta adrenoceptor regulation of macrophage arginase activity

Background: Arginase, which metabolizes L-arginine within the urea cycle, is essential for production of polyamines and affects production of nitric oxide by depletion of L -arginine, the common substrate for both arginase and nitric oxide synthase. Having shown that trauma increases splenic macrophage arginase activity, we seek to define the mechanisms for this. RAW macrophage arginase activity and expression are increased by 8-bromo-cAMP in vitro. We hypothesize that since catecholamines increase cAMP, trauma-induced splenic arginase activity may be mediated by post-injury catecholamine release. Methods: RAW 264.7 macrophage arginase activity was measured in vitro in response to 4 catecholamines with or without propranolol or lipopolysaccharide (LPS). C57BL/6 mice underwent laparotomy as a model of moderate trauma after propranolol treatment, with and without intraperitoneal Escherichia coli LPS administration as a simulated pro-inflammatory stimulus. Results: Macrophage arginase activity increased in vitro in response to catecholamines or LPS ( P < .05). Propranolol pretreatment blocked macrophage arginase activity induced by epinephrine (10 μmol/L) in vitro ( P < .05). Trauma or LPS alone increased splenic arginase activity in vivo ( P < .05). Propranolol did not alter LPS-induced splenic arginase activity but did significantly reduce trauma-induced splenic arginase activity ( P < .05). Conclusions: Catecholamines alone increase macrophage arginase activity through β-adrenoceptor activation. Increased splenic arginase activity induced by moderate trauma is decreased by β-adrenoceptor blockade, suggesting that trauma-induced arginase activity is partly mediated by endogenous catecholamines. (Surgery 2000;127:412-8.)

Beta(3)-adrenoceptor regulation and relaxation responses in mouse ileum.

1. This study examines the relationship between beta(3a)- and beta(3b)-adrenoceptor (AR) mRNA levels, beta(3)-AR binding and changes in ileum responses in mice treated with the beta(3)-AR agonist (R, R)-5-[2[[2-(3-chlorophenyl)-2-hydroxyethyl]-amino]-propyl]1, 3-benzodioxole-2,2-dicarboxylate (CL316243), or the beta(3)-AR antagonist 3-(2-ethylphenoxy)-1-[(1S)-1,2,3, 4-tetrahydronapth-1-ylamino]-2S-2-propanol oxalate (SR59230A), or dexamethasone or forskolin. 2. Levels of beta(3a)- and beta(3b)-AR mRNA and the maximum number of binding sites (B(max)) in ileum were unaffected following CL316243 treatment, although responses to CL316243 were reduced by 50% following 4 and 24 h treatment, indicating another desensitization mechanism not involving changes in receptor expression or number. beta(3a)-AR mRNA levels were reduced in both brown (BAT) and white adipose tissue (WAT) but beta(3b)-AR mRNA levels were significantly reduced only in WAT. Levels of beta(3a)- and beta(3b)-mRNA returned towards normal with continued treatment. 3. SR59230A treatment markedly increased beta(3)-AR mRN levels in ileum and BAT but not in WAT. The increase in beta(3)-AR mRNA levels in ileum was associated with increased B(max) levels in binding analysis and increased responses to CL316243, suggesting these as the cause of sensitization. 4. Treatment with forskolin (4 h) or dexamethasone (4 h) significantly reduced beta(3a)-AR mRNA levels in BAT and WAT but did not alter levels in ileum. Responses to CL316243 in ileum were unaffected by either treatment. 5. In summary, the beta(3)-AR is differently regulated in adipose tissue and ileum: Treatment with SR59230A increased beta(3)-AR number, mRNA and responsiveness in ileum, whereas treatment with CL316243 reduced responses without affecting beta(3)-AR number or mRNA levels.

Neutrophil β 2-adrenoceptor function in major depression: G s coupling, effects of imipramine and relationship to treatment outcome

Abnormal β 2-adrenoceptor density and β 2-adrenoceptor-mediated cyclic adenosine monophosphate (cAMP) responses were inconsistently reported in major depressive disorder. Tricyclic antidepressants downregulate β-adrenoceptor density and decrease coupling to G s protein. Abnormal β-adrenoceptor coupling may exist in major depressive disorder and may relate to treatment response. We investigated β 2-adrenoceptor coupling to G s protein in 25 controls, 23 major depressive disorder drug-free patients and 16 major depressive disorder patients after chronic imipramine treatment using agonist displacement experiments. Pretreatment β 2-adrenoceptor coupling and density were normal in patients as a whole. Chronic imipramine induced β 2-adrenoceptor uncoupling. This effect was observed in treatment responders who had increased β 2-adrenoceptor density in the high-conformational state and supercoupling prior to treatment. β 2-adrenoceptor density decreased after imipramine treatment. Treatment non-responders had seemingly normal pretreatment β 2-adrenoceptor function, which was not changed by imipramine. Differences in β 2-adrenoceptor regulation in major depressive disorder may underlie treatment response. The results indirectly implicate abnormal agonist-mediated β 2-adrenoceptor gene expression, protein kinase A, and protein kinase C in major depressive disorder.

Characterisation of the cardiovascular pharmacology of medetomidine in the horse and sheep

Medetomidine was administered to sheep and horses at a dose rate of 5 Pg kg −1 (i.v.). Heart rate and blood pressure were recorded. Medetomidine induced bradycardia and a biphasic blood pressure response consisting of a transient hypertension followed by hypotension. Administration of prazosin (an alpha, adrenoceptor antagonist; 100μg kg 1, i.v.) had no effect on the cardiovascular response to medetomidine (5 Vg kg −1, i.v.), but inhibited the cardiovascular response of methoxamine (an alpha, adrenoceptor agonist; 75 pg kg −1, i.v.). L-659,066 (an alpha2 adrenoceptor antagonist which does not cross the blood brain barrier; 264 μg kg −, i.v.) attenuated the medetomidine induced bradycardia, but had no effect on the cardiovascular response to methoxamine. L-659,066 also reduced the medetomidine induced hypertension in sheep, but had less effect on the horse. It is concluded that both alpha 1 and alpha 2 adrenoceptors are important in the control of cardiovascular function in horses and sheep. Medetomidine appears to act on alpha2 adrenoceptors alone in the sheep. The cardiovascular effects of medetomidine in the horse are complex and may be influenced by central alpha2 adrenoceptor regulation or effects on other receptor subtypes as well as direct stimulation of peripheral alpha 2 adrenoceptors.