Alpha1-adrenoceptors in the rat cerebral cortex: new insights into the characterization of alpha1L-and alpha1D-adrenoceptors

36 p., figuras y tablas, bibliografíaAmong the three alpha1-adrenoceptor subtypes (alpha1A, alpha1B and alpha1D) a peculiar intracellular localization and poor coupling to membrane signals of cloned alpha1D-adrenoceptor has been reported. In addition, the alpha1L-adrenoceptor (low affinity for prazosin), a functional phenotype of alpha1A, has been described. The purpose of this work was to analyze the expression, cellular localization and coupling to membrane signalling (inositol phosphate accumulation) of alpha1-adrenoceptor subtypes in a native tissue, the rat cerebral cortex. mRNA for the three subtypes was quantified by real-time RT-PCR (alpha1D>alpha1B>>alpha1A). Alpha1-adrenoceptors were also detected by immunoblotting, revealing alpha1A- and alpha1B-adrenoceptors to be predominantly expressed in the membrane fraction and the alpha1D-adrenoceptor to be localized in the cytosolic fraction. Competitive radioligand binding studies revealed the presence of alpha1D-adrenoceptor in tissue homogenates, whereas only alpha1A- and alpha1B-subtypes were detected in membranes. The proportion of alpha1A-adrenoceptor increased after treatment with noradrenaline, suggesting differences in agonist-mediated trafficking. Saturation experiments detected high- and low (alpha1A/L)-prazosin binding sites, the latter of which disappeared on incubation with GppNHp. The alpha1A/L-adrenoceptor was heavily implicated in the inositol phosphate response, while the alpha1D-subtype did not play a relevant role. These results suggest that the predominant cytosolic localization of alpha1D-adrenoceptor lies behind its poor coupling to membrane signalling such as inositol phosphate pathway. The fact that the alpha1Ladrenoceptor detected in radioligand binding studies disappeared in the presence of GppNHp implies that it represents a conformational state of the alpha1A-adrenoceptor coupled to G protein.This study was supported by the Spanish Dirección General de Programas y Transferencia de Conocimiento del Ministerio de Ciencia e Innovación (Grant SAF2004-01541 and SAF2007-62120); and Instituto de Salud Carlos III, Fondo de Investigaciones Sanitarias (Grant FIS PI070509). Vanessa Segura, Nicla Flacco and Eduardo Oliver are financed by the Spanish Ministry of Education and Science (Fellowships BES-2005-108383, AP-2005-5076 and AP-2004-3536).Peer reviewe

Correlation between mRNA levels and functional role of 1-adrenoceptor subtypes in arteries: evidence of 1L as a functional isoform of the 1A-adrenoceptor

10 pages, 3 figures, 10 tables.-- PMID: 15951348 [PubMed]The mRNA levels for the three alpha1-adrenoceptor subtypes, alpha1A, alpha1B, and alpha1D, were quantified by real-time RT-PCR in arteries from Wistar rats. The alpha1D-adrenoceptor was prominent in both aorta (79.0%) and mesenteric artery (68.7%), alpha1A predominated in tail (61.7%) and small mesenteric artery (73.3%), and both alpha1A- and alpha1D-subtypes were expressed at similar levels in iliac artery. The mRNA levels of the alpha1B-subtype were a minority in all vessels (1.7-11.1%). Concentration-response curves of contraction in response to phenylephrine or relaxation in response to alpha1-adrenoceptor antagonists on maximal sustained contraction induced by phenylephrine were constructed from control vessels and vessels pretreated with 100 micromol/l chloroethylclonidine (CEC) for 30 min. The significant decrease in the phenylephrine potency observed after CEC treatment together with the inhibitory potency displayed by 8-{2-[4-(2-methoxyphenyl)-1-piperazinyl]-8-azaspiro (4,5) decane-7-dionedihydrochloride} (BMY-7378, an alpha1D-adrenoceptor antagonist) confirm the relevant role of alpha1D-adrenoceptors in aorta and iliac and proximal mesenteric arteries. The potency of 5-methylurapidil (an alpha1A-adrenoceptor antagonist) and the changes in the potency of both BMY-7378 and 5-methylurapidil after CEC treatment provided evidence of a mixed population of alpha1A- and alpha1D-adrenoceptors in iliac and distal mesenteric arteries. The low potency of prazosin (pIC50 < 9) as well as the high 5-methylurapidil potency in tail and small mesenteric arteries suggest the main role of alpha1A/alpha1L-adrenoceptors with minor participation of the alpha1D-subtype. The mRNA levels and CEC treatment corroborated this pattern and confirmed that the alpha1L-adrenoceptor could be a functional isoform of the alpha1A-subtype.This work was supported by a research grant from the Spanish Comisión Interministerial de Ciencia y Tecnología (SAF2001-2656). Daniel Martí Canet received a fellowship from Universidad Cardenal Herrera-CEU.Peer reviewe

The impact of alpha1-adrenoceptors up-regulation accompanied by the impairment of beta-adrenergic vasodilatation in hypertension

9 pages, 7 figures, 3 tables.-- PMID: 19060223 [PubMed]In human and animal hypertension models, increased activity of G-protein-coupled receptor kinase (GRK) 2 determines a generalized decrease of beta-adrenergic vasodilatation. We analyzed the possibility of differential changes in the expression and functionality of alpha(1A), alpha(1B), alpha(1D), beta(1), beta(2), and beta(3)-ARs also being involved in the process. We combined the quantification of mRNA levels with immunoblotting and functional studies in aortas of young and adult spontaneously hypertensive rats (SHRs) and their controls (Wistar Kyoto). We found the expression and function of beta(1)-adrenoceptors in young prehypertensive SHRs to be higher, whereas a generalized increase in the expression of the six adrenoceptors and GRK2 was observed in aortas of adult hypertensive SHRs. alpha(1D)- and beta(3)-adrenoceptors, the subtypes that are more resistant to GRK2-mediated internalization and mostly expressed in rat aorta, exhibited an increased functional role in hypertensive animals, showing two hemodynamic consequences: 1) an increased sensitivity to the vasoconstrictor stimulus accompanied by a decreased sensitivity to the vasodilator stimulus (alpha(1D)-ARs are the most sensitive to agonists, and beta(3)-ARs are the least sensitive to agonists); and 2) a slower recovery of the basal tone after adrenergic stimulus removal because of the kinetic characteristic of the alpha(1D) subtype. These functional changes might be involved in the greater sympathetic vasoconstrictor tone observed in hypertension.This work was supported by the Spanish Comisión Interministerial de Ciencia y Tecnología [Grant SAF2004-01541]; Generalitat Valenciana [Grants GV2004-B-085, GRUPOS05-038]; and Instituto de Salud Carlos III, Fondo de Investigaciones Sanitarias [Grant FIS PI070509]; and by the Spanish Ministry of Education and Science [Fellowships AP-2004-3536 and AP-2005-5076].Peer reviewe

Discovery Patterns of Drugs Approved for Treating Bipolar Disorder by Applying Operational Criteria of Serendipity: A Historical Analysis

While the scientific community has long acknowledged the significant role of serendipity in shaping the field of contemporary psychopharmacology, its impact has not undergone a comprehensive analysis using operational methodologies. Serendipity, originally defined as discoveries stemming from a blend of luck and astuteness by Walpole, has been operationalized by our research group. Our definition builds upon Walpole’s notion of fortuitous accidents and keen insight leading to the unexpected revelation of something unintended. We have discerned four unique serendipitous attributability patterns. In this paper, we delve into the historical aspect of how serendipity has played a role in the discovery and advancement of drugs approved for addressing different stages of bipolar disorder. The revelation of valproate’s antimanic properties aligns with the pure serendipitous pattern (type I). Conversely, the discovery of lithium’s antimanic effects corresponds to the mixed type II pattern, where the initial serendipitous observation of sedation in research animals, prompted by adding lithium salts to enhance uric acid solubility, led to the planned discovery of its calming impact on manic patients. On the contrary, the recognition of the mood-stabilizing attributes of lamotrigine aligns with the type III archetype. This particular pattern is defined by serendipity emerging as a consequence of a discovery that was initially nonserendipitous, as mood-stabilizing efficacy was incidentally observed during the treatment of epileptic patients for whom the drug was originally developed. Finally, carbamazepine and, notably, atypical antipsychotics fall within the type IV pattern. In this category, serendipity is entirely absent, as the antimanic use of carbamazepine was a straightforward extension of the use of other antiepileptic drugs. Atypical antipsychotics were introduced into bipolar disorder therapy through a deliberate and targeted design process, seeking drugs capable of acting on specific biological targets.Sección Deptal. de Farmacología y Toxicología (Veterinaria)Depto. de Farmacología y ToxicologíaFac. de VeterinariaFac. de FarmaciaTRUEpu

Uric acid treatment after stroke modulates the Krüppel-like factor 2-VEGF-A axis to protect brain endothelial cell functions: Impact of hypertension

Uric acid (UA) is a promising protective treatment in ischaemic stroke, but the precise molecular targets underlying its in vivo beneficial actions remain unclear. High concentrations of UA inhibit angiogenesis of cultured endothelial cells via Krüppel-like factor 2 (KLF)-induced downregulation of vascular endothelial growth factor (VEGF), a pro-angiogenic mediator that is able to increase blood–brain barrier (BBB) permeability in acute stroke. Here, we investigated whether UA treatment after ischaemic stroke protects brain endothelial cell functions and modulates the KLF2-VEGF-A axis. Transient intraluminal middle cerebral artery (MCA) occlusion/reperfusion was induced in adult male spontaneously hypertensive (SHR) rats and corresponding normotensive Wistar-Kyoto (WKY) rats. Animals received UA (16 mg/kg) or vehicle (Locke's buffer) i.v. at reperfusion. BBB permeability was evaluated by Evans blue extravasation to the brain and in human cerebral endothelial hCMEC/D3 cells under oxygen-glucose deprivation/re-oxygenation. Circulating VEGF-A levels were measured in rats and acute ischaemic stroke patients from the URICO‐ICTUS trial. Angiogenesis progression was assessed in Matrigel-cultured MCA. Worse post-stroke brain damage in SHR than WKY rats was associated with higher hyperaemia at reperfusion, increased Evans blue extravasation, exacerbated MCA angiogenic sprouting, and higher VEGF-A levels. UA treatment reduced infarct volume and Evans blue leakage in both rat strains, improved endothelial cell barrier integrity and KLF2 expression, and lowered VEGF-A levels in SHR rats. Hypertensive stroke patients treated with UA showed lower levels of VEGF-A than patients receiving vehicle. Consistently, UA prevented the enhanced MCA angiogenesis in SHR rats by a mechanism involving KLF2 activation. We conclude that UA treatment after ischaemic stroke upregulates KLF2, reduces VEGF-A signalling, and attenuates brain endothelial cell dysfunctions leading to neuroprotection.This work was supported by Ministerio de Ciencia e Innovación [SAF2014-56111-R]; Generalitat de Catalunya [2017-SGR-645]; Instituto Carlos III [FIS PI13/0091, RIC RD12/0042/0006]; and Instituto de Salud Carlos III (Spain) co-funded by EU FEDER funds Redes Temáticas de Investigación Cooperativa Sanitaria RETICS-INVICTUS-RD16/019