α2B-ADRENOCEPTORS IN THE REGULATION OF VASCULAR SMOOTH MUSCLE CELL CONTRACTION AND PROLIFERATION

α2-Adrenoceptors (α2-ARs) belong to the large superfamily of G protein-coupled receptors (GPCRs). They mediate important actions of the endogenous catecholamines adrenaline and noradrenaline. All three α2-AR subtypes are involved in the regulation of blood pressure and vascular tone. α2-ARs can regulate both vascular smooth muscle contraction and remodeling of the blood vessel wall, but the intracellular signaling mechanisms involved in these functions have remained largely unknown. The aim of this thesis was to investigate the involvement of the α2B-AR subtype in the regulation of the contraction and proliferation of vascular smooth muscle cells (VSMC), and to clarify the related cellular signaling mechanisms. In order to characterize the effects of α2B-AR activation on VSMC contraction and proliferation and to investigate whether these functions could be altered by drug treatment, a VSMC line stably expressing the human α2B-AR was generated by transfection of rat A7r5 cells. Characterization of the novel A7r5-α2B cell line indicated that the localization and ligand binding properties of the expressed α2B-ARs were in line with earlier studies of α2B-ARs in different host cell environments, and that the receptors had the expected pharmacological characteristics. Therefore, the generated A7r5-α2B cell line was regarded as a useful tool for investigating the functions and regulation of α2B-ARs in VSMCs. α2B-ARs were demonstrated to be capable of mediating VSMC contraction by using a functional assay measuring myosin light chain phosphorylation, which is a biochemical readout of VSMC contraction. The network of signaling pathways involved in α2B-AR-mediated contraction of A7r5 VSMCs appeared to be complex and seemed to involve many mediators, such as Gi proteins, Gβγ subunits, phospholipase C (PLC), protein kinase C (PKC) and L-type Ca2+ channels. Different screening assays, namely DNA microarray, small inhibitor compound library screening and kinase activity profiling, were used to investigate the genetic regulation and intracellular signaling mechanisms involved in α2BAR-evoked proliferation of A7r5 VSMCs. The cellular mechanisms and signal transduction pathways participating in this response appeared to be complex and included redundancy. The employed screening assays and their respective data analysis approaches were found to be useful as tools to map the activation of cellular signaling networks in a situation where the exact mechanisms still remain unknown. These screening tools were considered suitable for hypothesis generation, but additional approaches will be required for further hypothesis testing.α2-Adrenergiset reseptorit (α2-AR) ovat G-proteiinikytkentäisiä reseptoreita (GPCR). Ne aktivoituvat adrenaliinin ja noradrenaliinin vaikutuksesta ja välittävät monia tärkei- tä elimistön säätelytehtäviä. Kaikki kolme α2-AR-alatyyppiä osallistuvat verenpaineen säätelyyn. Ne voivat säädellä sekä verisuonen sileän lihaksen (VSL) supistumista että verisuonen seinämän rakenteessa tapahtuvia muutoksia. Näihin toimintoihin liittyvät solusisäiset viestintämekanismit ovat vielä suurelta osin tuntemattomia. Tämän väi- töstutkimuksen tavoitteena oli selvittää α2B-reseptorialatyypin merkitystä VSL-solujen supistumisen ja proliferaation säätelyssä ja näitä vaikutuksia välittäviä solutason vies- tintämekanismeja. Rotan A7r5-solulinjan VSL-soluihin siirrettiin ihmisen α2B-reseptoria koodittava geeni. Tämä tuotti VSL-solumallin, jonka avulla voitiin tutkia α2B-reseptorien vaikutuksia VSL-so- lujen supistumiseen ja proliferaatioon. Kehitetyn A7r5-α2B -solulinjan reseptorien sijainti ja farmakologiset ominaisuudet olivat odotuksia vastaavat. Tämän validoinnin perusteella A7r5-α2B-solulinjan todettiin soveltuvan α2B-reseptorien toiminnan tutkimiseen VSL-so- luissa. Väitöstutkimuksessa osoitettiin, että α2B-reseptorit välittävät VSL-solujen supistus- vasteita. Tätä tutkittiin mittaamalla myosiinin kevytketjujen fosforylaatiota, sillä myosiinin kevytketjujen fosforylaatio on VSL-solujen supistuksen kannalta keskeinen biokemialli- nen tapahtuma. A7r5-soluissa α2B-välitteiseen supistusvasteeseen liittyvät solunsisäiset viestintämekanismit osoittautuivat monimutkaisiksi ja niihin osallistui monia viestinvälit- täjiä, kuten Gi-proteiinit, Gβγ-alayksiköt, fosfolipaasi C, proteiinikinaasi C ja L-tyypin kal- siumkanavat. DNA-mikrosirumäärityksillä, kinaasi/fosfataasi-inhibiittorikirjaston seulon- nalla ja kinaasiaktivaatiota mittaavilla mikrosirumäärityksillä selvitettiin α2B-välitteiseen VSL-solujen proliferaatiovasteeseen liittyvää geenien ilmentymisen säätelyä ja solunsisäi- siä viestintämekanismeja. VSL-solujen proliferaatiovasteen syntyyn liittyvät mekanismit ja viestintäreitit osoittautuivat monimutkaisiksi ja osittain päällekkäisiksi. Käytetyt seulonta- menetelmät ja niihin sovellettavat analyysit todettiin käytännölliseksi lähestymistavaksi tilanteessa, jossa halutaan kartoittaa ennalta tuntemattomia solunsisäisiä viestintämeka- nismeja. Seulontamenetelmät todettiin hyödyllisiksi uusien havaintojen tuottamisessa, mutta havaintojen tuottamien hypoteesien testaamiseen tarvitaan muita, kohdennettuja menetelmiä

Gene expression profiles and signaling mechanisms in α2B-adrenoceptor-evoked proliferation of vascular smooth muscle cells

Background: alpha(2)-adrenoceptors are important regulators of vascular tone and blood pressure. Regulation of cell proliferation is a less well investigated consequence of alpha(2)-adrenoceptor activation. We have previously shown that alpha B-2-adrenoceptor activation stimulates proliferation of vascular smooth muscle cells (VSMCs). This may be important for blood vessel development and plasticity and for the pathology and therapeutics of cardiovascular disorders. The underlying cellular mechanisms have remained mostly unknown. This study explored pathways of regulation of gene expression and intracellular signaling related to alpha B-2-adrenoceptor-evoked VSMC proliferation.Results: The cellular mechanisms and signaling pathways of alpha B-2-adrenoceptor-evoked proliferation of VSMCs are complex and include redundancy. Functional enrichment analysis and pathway analysis identified differentially expressed genes associated with alpha B-2-adrenoceptor-regulated VSMC proliferation. They included the upregulated genes Egr1, F3, Ptgs2 and Serpine1 and the downregulated genes Cx3cl1, Cav1, Rhoa, Nppb and Prrx1. The most highly upregulated gene, Lypd8, represents a novel finding in the VSMC context. Inhibitor library screening and kinase activity profiling were applied to identify kinases in the involved signaling pathways. Putative upstream kinases identified by two different screens included PKC, Raf-1, Src, the MAP kinases p38 and JNK and the receptor tyrosine kinases EGFR and HGF/HGFR. As a novel finding, the Src family kinase Lyn was also identified as a putative upstream kinase.Conclusions: alpha B-2-adrenoceptors may mediate their pro-proliferative effects in VSMCs by promoting the activity of bFGF and PDGF and the growth factor receptors EGFR, HGFR and VEGFR-1/2. The Src family kinase Lyn was also identified as a putative upstream kinase. Lyn is known to be expressed in VSMCs and has been identified as an important regulator of GPCR trafficking and GPCR effects on cell proliferation. Identified Ser/Thr kinases included several PKC isoforms and the beta-adrenoceptor kinases 1 and 2. Cross-talk between the signaling mechanisms involved in alpha(2) B-adrenoceptor-evoked VSMC proliferation thus appears to involve PKC activation, subsequent changes in gene expression, transactivation of EGFR, and modulation of kinase activities and growth factormediated signaling. While many of the identified individual signals were relatively small in terms of effect size, many of them were validated by combining pathway analysis and our integrated screening approach

Adrenoceptors in GtoPdb v.2023.1

The nomenclature of the Adrenoceptors has been agreed by the NC-IUPHAR Subcommittee on Adrenoceptors [64, 194]. Adrenoceptors, α1 The three α1-adrenoceptor subtypes α1A, α1B and α1D are activated by the endogenous agonists (-)-adrenaline and (-)-noradrenaline. -(-)phenylephrine, methoxamine and cirazoline are agonists and prazosin and doxazosin antagonists considered selective for α1- relative to α2-adrenoceptors. [3H]prazosin and [125I]HEAT (BE2254) are relatively selective radioligands. S(+)-niguldipine also has high affinity for L-type Ca2+ channels. Fluorescent derivatives of prazosin (Bodipy FLprazosin- QAPB) are used to examine cellular localisation of α1-adrenoceptors. α1-Adrenoceptor agonists are used as nasal decongestants; antagonists to treat symptoms of benign prostatic hyperplasia (alfuzosin, doxazosin, terazosin, tamsulosin and silodosin, with the last two compounds being α1A-adrenoceptor selective and claiming to relax bladder neck tone with less hypotension); and to a lesser extent hypertension (doxazosin, terazosin). The α1- and β2-adrenoceptor antagonist carvedilol is used to treat congestive heart failure, although the contribution of α1-adrenoceptor blockade to the therapeutic effect is unclear. Several anti-depressants and anti-psychotic drugs are α1-adrenoceptor antagonists contributing to side effects such as orthostatic hypotension. Adrenoceptors, α2 The three α2-adrenoceptor subtypes α2A, α2B and α2C are activated by (-)-adrenaline and with lower potency by (-)-noradrenaline. brimonidine and talipexole are agonists and rauwolscine and yohimbine antagonists selective for α2- relative to α1-adrenoceptors. [3H]rauwolscine, [3H]brimonidine and [3H]RX821002 are relatively selective radioligands. There are species variations in the pharmacology of the α2A-adrenoceptor. Multiple mutations of α2-adrenoceptors have been described, some associated with alterations in function. Presynaptic α2-adrenoceptors regulate many functions in the nervous system. The α2-adrenoceptor agonists clonidine, guanabenz and brimonidine affect central baroreflex control (hypotension and bradycardia), induce hypnotic effects and analgesia, and modulate seizure activity and platelet aggregation. clonidine is an anti-hypertensive (relatively little used) and counteracts opioid withdrawal. dexmedetomidine (also xylazine) is increasingly used as a sedative and analgesic in human [33] and veterinary medicine and has sympatholytic and anxiolytic properties. The α2-adrenoceptor antagonist mirtazapine is used as an anti-depressant. The α2B subtype appears to be involved in neurotransmission in the spinal cord and α2C in regulating catecholamine release from adrenal chromaffin cells. Although subtype-selective antagonists have been developed, none are used clinically and they remain experimental tools. Adrenoceptors, β The three β-adrenoceptor subtypes β1, β2 and β3 are activated by the endogenous agonists (-)-adrenaline and (-)-noradrenaline. Isoprenaline is selective for β-adrenoceptors relative to α1- and α2-adrenoceptors, while propranolol (pKi 8.2-9.2) and cyanopindolol (pKi 10.0-11.0) are relatively selective antagonists for β1- and β2- relative to β3-adrenoceptors. (-)-noradrenaline, xamoterol and (-)-Ro 363 show selectivity for β1- relative to β2-adrenoceptors. Pharmacological differences exist between human and mouse β3-adrenoceptors, and the 'rodent selective' agonists BRL 37344 and CL316243 have low efficacy at the human β3-adrenoceptor whereas CGP 12177 (low potency) and L 755507 activate human β3-adrenoceptors [88]. β3-Adrenoceptors are resistant to blockade by propranolol, but can be blocked by high concentrations of bupranolol. SR59230A has reasonably high affinity at β3-adrenoceptors, but does not discriminate between the three β- subtypes [332] whereas L-748337 is more selective. [125I]-cyanopindolol, [125I]-hydroxy benzylpindolol and [3H]-alprenolol are high affinity radioligands that label β1- and β2- adrenoceptors and β3-adrenoceptors can be labelled with higher concentrations (nM) of [125I]-cyanopindolol together with β1- and β2-adrenoceptor antagonists. Fluorescent ligands such as BODIPY-TMR-CGP12177 can be used to track β-adrenoceptors at the cellular level [8]. Somewhat selective β1-adrenoceptor agonists (denopamine, dobutamine) are used short term to treat cardiogenic shock but, chronically, reduce survival. β1-Adrenoceptor-preferring antagonists are used to treat cardiac arrhythmias (atenolol, bisoprolol, esmolol) and cardiac failure (metoprolol, nebivolol) but also in combination with other treatments to treat hypertension (atenolol, betaxolol, bisoprolol, metoprolol and nebivolol) [528]. Cardiac failure is also treated with carvedilol that blocks β1- and β2-adrenoceptors, as well as α1-adrenoceptors. Short (salbutamol, terbutaline) and long (formoterol, salmeterol) acting β2-adrenoceptor-selective agonists are powerful bronchodilators used to treat respiratory disorders. Many first generation β-adrenoceptor antagonists (propranolol) block both β1- and β2-adrenoceptors and there are no β2-adrenoceptor-selective antagonists used therapeutically. The β3-adrenoceptor agonist mirabegron is used to control overactive bladder syndrome. There is evidence to suggest that β-adrenoceptor antagonists can reduce metastasis in certain types of cancer [197]

Adrenoceptors in GtoPdb v.2021.3

The nomenclature of the Adrenoceptors has been agreed by the NC-IUPHAR Subcommittee on Adrenoceptors [60, 186]. Adrenoceptors, α1 The three α1-adrenoceptor subtypes α1A, α1B and α1D are activated by the endogenous agonists (-)-adrenaline and (-)-noradrenaline. -(-)phenylephrine, methoxamine and cirazoline are agonists and prazosin and doxazosin antagonists considered selective for α1- relative to α2-adrenoceptors. [3H]prazosin and [125I]HEAT (BE2254) are relatively selective radioligands. S(+)-niguldipine also has high affinity for L-type Ca2+ channels. Fluorescent derivatives of prazosin (Bodipy FLprazosin- QAPB) are used to examine cellular localisation of α1-adrenoceptors. α1-Adrenoceptor agonists are used as nasal decongestants; antagonists to treat symptoms of benign prostatic hyperplasia (alfuzosin, doxazosin, terazosin, tamsulosin and silodosin, with the last two compounds being α1A-adrenoceptor selective and claiming to relax bladder neck tone with less hypotension); and to a lesser extent hypertension (doxazosin, terazosin). The α1- and β2-adrenoceptor antagonist carvedilol is used to treat congestive heart failure, although the contribution of α1-adrenoceptor blockade to the therapeutic effect is unclear. Several anti-depressants and anti-psychotic drugs are α1-adrenoceptor antagonists contributing to side effects such as orthostatic hypotension. Adrenoceptors, α2 The three α2-adrenoceptor subtypes α2A, α2B and α2C are activated by (-)-adrenaline and with lower potency by (-)-noradrenaline. brimonidine and talipexole are agonists and rauwolscine and yohimbine antagonists selective for α2- relative to α1-adrenoceptors. [3H]rauwolscine, [3H]brimonidine and [3H]RX821002 are relatively selective radioligands. There are species variations in the pharmacology of the α2A-adrenoceptor. Multiple mutations of α2-adrenoceptors have been described, some associated with alterations in function. Presynaptic α2-adrenoceptors regulate many functions in the nervous system. The α2-adrenoceptor agonists clonidine, guanabenz and brimonidine affect central baroreflex control (hypotension and bradycardia), induce hypnotic effects and analgesia, and modulate seizure activity and platelet aggregation. clonidine is an anti-hypertensive (relatively little used) and counteracts opioid withdrawal. dexmedetomidine (also xylazine) is increasingly used as a sedative and analgesic in human [31] and veterinary medicine and has sympatholytic and anxiolytic properties. The α2-adrenoceptor antagonist mirtazapine is used as an anti-depressant. The α2B subtype appears to be involved in neurotransmission in the spinal cord and α2C in regulating catecholamine release from adrenal chromaffin cells. Although subtype-selective antagonists have been developed, none are used clinically and they remain experimental tools. Adrenoceptors, β The three β-adrenoceptor subtypes β1, β2 and β3 are activated by the endogenous agonists (-)-adrenaline and (-)-noradrenaline. Isoprenaline is selective for β-adrenoceptors relative to α1- and α2-adrenoceptors, while propranolol (pKi 8.2-9.2) and cyanopindolol (pKi 10.0-11.0) are relatively selective antagonists for β1- and β2- relative to β3-adrenoceptors. (-)-noradrenaline, xamoterol and (-)-Ro 363 show selectivity for β1- relative to β2-adrenoceptors. Pharmacological differences exist between human and mouse β3-adrenoceptors, and the 'rodent selective' agonists BRL 37344 and CL316243 have low efficacy at the human β3-adrenoceptor whereas CGP 12177 (low potency) and L 755507 activate human β3-adrenoceptors [88]. β3-Adrenoceptors are resistant to blockade by propranolol, but can be blocked by high concentrations of bupranolol. SR59230A has reasonably high affinity at β3-adrenoceptors, but does not discriminate between the three β- subtypes [320] whereas L-748337 is more selective. [125I]-cyanopindolol, [125I]-hydroxy benzylpindolol and [3H]-alprenolol are high affinity radioligands that label β1- and β2- adrenoceptors and β3-adrenoceptors can be labelled with higher concentrations (nM) of [125I]-cyanopindolol together with β1- and β2-adrenoceptor antagonists. Fluorescent ligands such as BODIPY-TMR-CGP12177 can be used to track β-adrenoceptors at the cellular level [8]. Somewhat selective β1-adrenoceptor agonists (denopamine, dobutamine) are used short term to treat cardiogenic shock but, chronically, reduce survival. β1-Adrenoceptor-preferring antagonists are used to treat cardiac arrhythmias (atenolol, bisoprolol, esmolol) and cardiac failure (metoprolol, nebivolol) but also in combination with other treatments to treat hypertension (atenolol, betaxolol, bisoprolol, metoprolol and nebivolol) [507]. Cardiac failure is also treated with carvedilol that blocks β1- and β2-adrenoceptors, as well as α1-adrenoceptors. Short (salbutamol, terbutaline) and long (formoterol, salmeterol) acting β2-adrenoceptor-selective agonists are powerful bronchodilators used to treat respiratory disorders. Many first generation β-adrenoceptor antagonists (propranolol) block both β1- and β2-adrenoceptors and there are no β2-adrenoceptor-selective antagonists used therapeutically. The β3-adrenoceptor agonist mirabegron is used to control overactive bladder syndrome. There is evidence to suggest that β-adrenoceptor antagonists can reduce metastasis in certain types of cancer [189]

Adrenoceptors (version 2019.4) in the IUPHAR/BPS Guide to Pharmacology Database

The nomenclature of the Adrenoceptors has been agreed by the NC-IUPHAR Subcommittee on Adrenoceptors [58], see also [180]. Adrenoceptors, α1α1-Adrenoceptors are activated by the endogenous agonists (-)-adrenaline and (-)-noradrenaline. phenylephrine, methoxamine and cirazoline are agonists and prazosin and cirazoline antagonists considered selective for α1- relative to α2-adrenoceptors. [3H]prazosin and [125I]HEAT (BE2254) are relatively selective radioligands. S(+)-niguldipine also has high affinity for L-type Ca2+ channels. Fluorescent derivatives of prazosin (Bodipy PLprazosin- QAPB) are used to examine cellular localisation of α1-adrenoceptors. Selective α1-adrenoceptor agonists are used as nasal decongestants; antagonists to treat hypertension (doxazosin, prazosin) and benign prostatic hyperplasia (alfuzosin, tamsulosin). The α1- and β2-adrenoceptor antagonist carvedilol is used to treat congestive heart failure, although the contribution of α1-adrenoceptor blockade to the therapeutic effect is unclear. Several anti-depressants and anti-psychotic drugs are α1-adrenoceptor antagonists contributing to side effects such as orthostatic hypotension and extrapyramidal effects.Adrenoceptors, α2 α2-Adrenoceptors are activated by (-)-adrenaline and with lower potency by (-)-noradrenaline. brimonidine and talipexole are agonists and rauwolscine and yohimbine antagonists selective for α2- relative to α1-adrenoceptors. [3H]rauwolscine, [3H]brimonidine and [3H]RX821002 are relatively selective radioligands. There is species variation in the pharmacology of the α2A-adrenoceptor. Multiple mutations of α2-adrenoceptors have been described, some associated with alterations in function. Presynaptic α2-adrenoceptors regulate many functions in the nervous system. The α2-adrenoceptor agonists clonidine, guanabenz and brimonidine affect central baroreflex control (hypotension and bradycardia), induce hypnotic effects and analgesia, and modulate seizure activity and platelet aggregation. clonidine is an anti-hypertensive and counteracts opioid withdrawal. dexmedetomidine (also xylazine) is used as a sedative and analgesic in human and veterinary medicine with sympatholytic and anxiolytic properties. The α2-adrenoceptor antagonist yohimbine has been used to treat erectile dysfunction and mirtazapine as an anti-depressant. The α2B subtype appears to be involved in neurotransmission in the spinal cord and α2C in regulating catecholamine release from adrenal chromaffin cells.Adrenoceptors, ββ-Adrenoceptors are activated by the endogenous agonists (-)-adrenaline and (-)-noradrenaline. Isoprenaline is selective for β-adrenoceptors relative to α1- and α2-adrenoceptors, while propranolol (pKi 8.2-9.2) and cyanopindolol (pKi 10.0-11.0) are relatively β1 and β2 adrenoceptor-selective antagonists. (-)-noradrenaline, xamoterol and (-)-Ro 363 show selectivity for β1- relative to β2-adrenoceptors. Pharmacological differences exist between human and mouse β3-adrenoceptors, and the 'rodent selective' agonists BRL 37344 and CL316243 have low efficacy at the human β3-adrenoceptor whereas CGP 12177 and L 755507 activate human β3-adrenoceptors [88]. β3-Adrenoceptors are resistant to blockade by propranolol, but can be blocked by high concentrations of bupranolol. SR59230A has reasonably high affinity at β3-adrenoceptors, but does not discriminate well between the three β- subtypes whereas L 755507 is more selective. [125I]-cyanopindolol, [125I]-hydroxy benzylpindolol and [3H]-alprenolol are high affinity radioligands that label β1- and β2- adrenoceptors and β3-adrenoceptors can be labelled with higher concentrations (nM) of [125I]-cyanopindolol together with β1- and β2-adrenoceptor antagonists. [3H]-L-748337 is a β3-selective radioligand [474]. Fluorescent ligands such as BODIPY-TMR-CGP12177 can be used to track β-adrenoceptors at the cellular level [8]. Somewhat selective β1-adrenoceptor agonists (denopamine, dobutamine) are used short term to treat cardiogenic shock but, chronically, reduce survival. β1-Adrenoceptor-preferring antagonists are used to treat hypertension (atenolol, betaxolol, bisoprolol, metoprolol and nebivolol), cardiac arrhythmias (atenolol, bisoprolol, esmolol) and cardiac failure (metoprolol, nebivolol). Cardiac failure is also treated with carvedilol that blocks β1- and β2-adrenoceptors, as well as α1-adrenoceptors. Short (salbutamol, terbutaline) and long (formoterol, salmeterol) acting β2-adrenoceptor-selective agonists are powerful bronchodilators used to treat respiratory disorders. Many first generation β-adrenoceptor antagonists (propranolol) block both β1- and β2-adrenoceptors and there are no β2-adrenoceptor-selective antagonists used therapeutically. The β3-adrenoceptor agonist mirabegron is used to control overactive bladder syndrome

Expanding our therapeutic options: β-blockers for colon cancer?

Supported by U. Porto/Santander Totta (IJUP) (PP-IJUP2011-320)Colon cancer is the fourth and third most common cancer, respectively in men and women worldwide and its incidence is increasing. Stress response has been associated to the incidence and development of cancer. The catecholamines (CA), adrenaline (AD) and noradrenaline (NA), are crucial mediators of stress response, exerting their effects through interaction with α- and β- adrenergic receptors (AR). Colon cancer cells express β-AR and their activation has been implicated in carcinogenesis and tumor progression. Recently, interest in the efficacy of β-AR blockers as possible additions to cancer treatment paradigms has been gaining strength. The aim of this work was to investigate the effect of several AR agonists and β-blockers, upon cellular proliferation and viability of HT-29 cells, a human colon adenocarcinoma cell line. For this purpose, in the first phase of this work, we determined the EC50 and IC50 values for proliferative and antiproliferative effects, respectively of AR agonists and antagonists. Afterwards, HT-29 cells were incubated in the absence (control) or in the presence of the AR-agonists, AD, NA and isoprenaline (ISO) (0.1-100 μM) for 12 hours or 24 hours. All tested AR agonists revealed proliferative effects upon HT-29 cells. In order to study the effect of several β-blockers upon both proliferation and viability induced by AR activation, cells were treated with propranolol (PRO; 50 μM), carvedilol (CAR; 5μM), atenolol (ATE; 50 μM), or ICI 118,551 (ICI; 5 μM) for 45 minutes prior, and simultaneously, to the incubation with each of the AR agonists, AD and ISO, both at 1 and 10 μM. Our results suggest that adrenergic activation play an important role in colon cancer cells proliferation most probably through β-AR. All the β-blockers under study were able to revert the proliferation induced by AD and ISO, and some of them, per se, significantly decreased the proliferation of HT-29 cells. The elucidation of the intracellular pathways involved in CA-induced proliferation of colon cancer cells, and also in the reversion of this effect by β-blockers, might contribute to reveal promising strategies in cancer treatment.O cancro do cólon é o quarto e terceiro cancro mais comum, respetivamente nos homens e nas mulheres em todo o mundo, e a sua incidência está a aumentar. A resposta ao stresse tem sido associada a um aumento da incidência e desenvolvimento do cancro. As catecolaminas (CA), adrenalina (AD) e noradrenalina (NA) são os principais mediadores da resposta ao stresse, exercendo os seus efeitos através da interação com os recetores adrenérgicos (RA), α e β. As células do cancro do colon expressam predominantemente os RA do tipo β, e a sua ativação está implicada na carcinogénese e na progressão dos tumores. Recentemente, tem sido notório o interesse na eficácia dos RA do tipo β como possíveis adjuvantes para o tratamento do cancro. O objetivo deste trabalho foi investigar o efeito de vários agonistas para os RA, e de β-bloqueadores, na proliferação e viabilidade de uma linha celular de adenocarcinoma de colon humano, as células HT-29. Para este efeito, na primeira fase deste trabalho, determinámos os valores dos EC50 e IC50, respetivamente para o efeito proliferativo e antiproliferativo dos agonistas e antagonistas dos RA em estudo. Posteriormente, as células foram incubadas na ausência (controlo) e na presença dos agonistas, AD, NA e isoprenalina (ISO) (1-100 μM) durante 12 ou 24 horas. Todos os agonistas em estudo aumentaram significativamente a proliferação das células HT-29. Para estudar os efeitos de vários β-bloqueadores na proliferação e na viabilidade induzida pela ativação dos RA, as células foram tratadas com propranolol (50 μM), carvedilol (5 μM), atenolol (50 μM) ou ICI 118,551 (5 μM) 45 minutos antes, e em simultâneo, do tratamento com cada um dos agonistas AD e ISO a 1 e 10 μM. Os nossos resultados sugerem que a ativação adrenérgica desempenha um papel importante na proliferação das células do cancro do cólon, muito provavelmente através dos recetores β. Todos os β-bloqueadores testados foram capazes de reverter a proliferação das células HT-29 induzida pela AD e pela ISO, sendo que alguns deles por si só diminuíram significativamente a proliferação destas células. A elucidação das vias envolvidas na proliferação de células de cancro do cólon induzida pelas CA, e também na reversão deste efeito pelos β-bloqueadores, pode contribuir para revelar estratégias promissoras no tratamento do cancro.U. Porto/Santander Totta (IJUP

CELLULAR TRAFFICKING PROPERTIES AND PHYSIOLOGICAL FUNCTIONS OF THE á1-ADRENOCEPTOR SUBTYPES

The 1-adrenoceptors (1-ARs) serve as an interface between the sympathetic nervous system and the cardiovascular system where they are mediators of systemic arterial blood pressure, initiators of positive inotropy, and regulators of cellular growth responses. There are three subtypes: 1A-, 1B-, and 1D-ARs. This dissertation research investigated the trafficking properties of the 1-ARs at the cellular level as well as physiological relevance of the 1-ARs at the tissue level. In vitro studies using transiently transfected 1-AR/GFP subtypes revealed distinct basal localization patterns and different agonist-mediated activation and desensitization properties. The 1A- and the 1B-AR/GFP subtypes displayed agonist-mediated receptor redistribution, in which rate and degree of redistribution differed. Additionally, redistribution of either of these two receptor subtypes required arrestin-1, a protein often associated with receptor internalization. In contrast, the 1D-AR/GFP did not require arrestin-1 for maintaining the basal receptor orientation pattern. Although these data increase our knowledge of trafficking properties of the 1-AR subtypes, it is of equal importance to determine the role(s) that each subtype contributes to cardiovascular function. The lack of subtype-selective 1-AR pharmacological agents prompted the use of genetically manipulated mouse models with a systemic overexpression of a constitutively active 1B-AR. Echocardiographic analysis of transgenic hearts indicated both an enlarged left ventricular chamber in the absence of hypertrophy and a depressed cardiac function. From isolated transgenic hearts, experimental results suggested a role for the 1B-AR in attenuating the inotropic responses. However, experiments using isolated thoracic aortae from transgenic animals suggested that the 1B-AR does not participate in vascular smooth muscle contractile responses. Additional studies investigated the role of 1D-AR in cardiovascular function by using animals systemically lacking the 1D-AR subtype. Experimental data suggested an 1D-AR participation in vascular smooth muscle function since the deficiency of the 1D-AR subtype affected vasoconstriction in the coronary arteries but not inotropy in the heart. The data presented in this dissertation research suggest subtype specific differences of 1-ARs in cellular localization, signal regulation, and trafficking. Additionally, the data provide an investigation into the physiologic significance of both the 1B- and the 1D-ARs in cardiovascular tissue

Role of pannexins in vasculature

Pannexins are newly discovered proteins that were first discovered by Panchin in 2000. The pannexin family has three isomers, i.e. pannexin-1, pannexin-2, and pannexin-3. In 2011, Billaud et al suggested that pannexin1 channels contribute to the spread of vasoconstriction after activation of α1D-adrenoceptors present on the surface of vascular smooth muscle cell (VSMCs) of thoracodorsal resistance arteries (TDA) isolated from mice. Phenylephrine acting upon the α1D-adrenoceptors activated pannexin1 channels present in the cell to release ATP, which in turn activated P2Y receptors on neighbouring cells to produce a co-ordinated contractile response. This aim of this work was to further investigate the role of pannexin in the regulation of contractile responses in the vasculature. To this end, the present study examined the presence and function of pannexins in the porcine splenic artery (PSA) and the rat aorta (RA), in which α1A- and α1D-adrenoceptors are present respectively. The role of pannexin channels and ATP (via activation of P2 purinoceptors) in the response to exogenous NA-induced contractile responses in the PSA and the RA were investigated, as were responses to sympathetic nerve activation in the PSA. The involvement of pannexin channels was studied using several pannexin inhibitors, i.e. mefloquine (a non-selective pannexin inhibitor), probenecid (a selective pannexin1 inhibitor at low concentrations), carbenoxolone (a selective pannexin1 inhibitor) and Brilliant Blue FCF (a selective pannexin1 inhibitor). Additionally, the involvement of ATP in NA-induced responses was examined using P2 purinoceptor antagonists (PPADS, suramin and NF449). Further experiments examined the role of pannexins in contributing to endothelium-dependent responses in a large vessel i.e. the porcine coronary artery (PCA). The results showed that both pannexin1 and pannexin2 are present in the PSA and the RA. In the PSA, mefloquine and probenecid reduced the responses to both NA-induced contractions and the frequency-dependent response curves generated to sympathetic nerve stimulation, whereas carbenoxolone, suramin and PPADS had no effect on responses to either exogenous NA or those caused by activating the sympathetic nerves. In the RA, mefloquine and probenecid reduced the response to NA-induced contractions, whereas BB-FCF had no effect. Purinoceptor antagonists (suramin, PPADs and NF449) had no effect on responses mediated by either α1A–adrenoceptors in the PSA or α1D–adrenoceptors in the RA, arguing against the role of ATP (via activation of P2 receptors) in mediating NA-induced responses in either the PSA or the RA. Conflicting results were obtained, in some cases, upon the use of three different pannexin inhibitors. The most likely reason for this is that mefloquine demonstrated non-selective inhibitory actions on contractile responses since it was also shown to inhibit responses to KCl, 5-HT, U46619 (the thromboxane mimetic), and responses to re-addition of calcium to depolarised preparations, suggesting that it acts to block L-type Ca2+ currents. Both mefloquine and probenecid demonstrated non-selective inhibitory effects when used at relatively high concentrations. Therefore, mefloquine and probenecid should only be used in low concentrations as pannexin1 inhibitors. It has been suggested that pannexin proteins may be involved in mediating endothelium-derived hyperpolarizing factor (EDHF) responses (Gaynullina, Shestopalov et al 2015). Bradykinin (BK) was shown to induce relaxation in PCA and to a lesser extent in the PSA after inhibition of NO-synthase and cyclooxygenase. The evidence for the involvement of pannexin in mediating an EDHF response was limited in both the PSA and the PCA, since neither carbenoxolone nor probenecid had any effect. While mefloquine reduced EDH-mediated responses to bradykinin in the PCA, the questions about its selectivity make this observation difficult to interpret. The present work therefore provided some evidence for the involvement of pannexin channels in conducting responses to NA-induced α1-adrenoceptor-mediated vasoconstriction in blood vessels in PSA and the RA, although great care must be taken in interpreting this data on the basis of a lack of selectivity of the pharmacological agents currently available as pannexin inhibitors. In addition, there was no evidence that activation of α1-adrenoreceptors causes the release of ATP from inside cells to act as an intercellular messenger, leading to P2 receptor-mediated contractions

Role of pannexins in vasculature

Pannexins are newly discovered proteins that were first discovered by Panchin in 2000. The pannexin family has three isomers, i.e. pannexin-1, pannexin-2, and pannexin-3. In 2011, Billaud et al suggested that pannexin1 channels contribute to the spread of vasoconstriction after activation of α1D-adrenoceptors present on the surface of vascular smooth muscle cell (VSMCs) of thoracodorsal resistance arteries (TDA) isolated from mice. Phenylephrine acting upon the α1D-adrenoceptors activated pannexin1 channels present in the cell to release ATP, which in turn activated P2Y receptors on neighbouring cells to produce a co-ordinated contractile response. This aim of this work was to further investigate the role of pannexin in the regulation of contractile responses in the vasculature. To this end, the present study examined the presence and function of pannexins in the porcine splenic artery (PSA) and the rat aorta (RA), in which α1A- and α1D-adrenoceptors are present respectively. The role of pannexin channels and ATP (via activation of P2 purinoceptors) in the response to exogenous NA-induced contractile responses in the PSA and the RA were investigated, as were responses to sympathetic nerve activation in the PSA. The involvement of pannexin channels was studied using several pannexin inhibitors, i.e. mefloquine (a non-selective pannexin inhibitor), probenecid (a selective pannexin1 inhibitor at low concentrations), carbenoxolone (a selective pannexin1 inhibitor) and Brilliant Blue FCF (a selective pannexin1 inhibitor). Additionally, the involvement of ATP in NA-induced responses was examined using P2 purinoceptor antagonists (PPADS, suramin and NF449). Further experiments examined the role of pannexins in contributing to endothelium-dependent responses in a large vessel i.e. the porcine coronary artery (PCA). The results showed that both pannexin1 and pannexin2 are present in the PSA and the RA. In the PSA, mefloquine and probenecid reduced the responses to both NA-induced contractions and the frequency-dependent response curves generated to sympathetic nerve stimulation, whereas carbenoxolone, suramin and PPADS had no effect on responses to either exogenous NA or those caused by activating the sympathetic nerves. In the RA, mefloquine and probenecid reduced the response to NA-induced contractions, whereas BB-FCF had no effect. Purinoceptor antagonists (suramin, PPADs and NF449) had no effect on responses mediated by either α1A–adrenoceptors in the PSA or α1D–adrenoceptors in the RA, arguing against the role of ATP (via activation of P2 receptors) in mediating NA-induced responses in either the PSA or the RA. Conflicting results were obtained, in some cases, upon the use of three different pannexin inhibitors. The most likely reason for this is that mefloquine demonstrated non-selective inhibitory actions on contractile responses since it was also shown to inhibit responses to KCl, 5-HT, U46619 (the thromboxane mimetic), and responses to re-addition of calcium to depolarised preparations, suggesting that it acts to block L-type Ca2+ currents. Both mefloquine and probenecid demonstrated non-selective inhibitory effects when used at relatively high concentrations. Therefore, mefloquine and probenecid should only be used in low concentrations as pannexin1 inhibitors. It has been suggested that pannexin proteins may be involved in mediating endothelium-derived hyperpolarizing factor (EDHF) responses (Gaynullina, Shestopalov et al 2015). Bradykinin (BK) was shown to induce relaxation in PCA and to a lesser extent in the PSA after inhibition of NO-synthase and cyclooxygenase. The evidence for the involvement of pannexin in mediating an EDHF response was limited in both the PSA and the PCA, since neither carbenoxolone nor probenecid had any effect. While mefloquine reduced EDH-mediated responses to bradykinin in the PCA, the questions about its selectivity make this observation difficult to interpret. The present work therefore provided some evidence for the involvement of pannexin channels in conducting responses to NA-induced α1-adrenoceptor-mediated vasoconstriction in blood vessels in PSA and the RA, although great care must be taken in interpreting this data on the basis of a lack of selectivity of the pharmacological agents currently available as pannexin inhibitors. In addition, there was no evidence that activation of α1-adrenoreceptors causes the release of ATP from inside cells to act as an intercellular messenger, leading to P2 receptor-mediated contractions