Caracterización farmacológica y funcional de la subunidad a9a10 del receptor nicotínico de la célula cromafín de la médula de la rata

La reciente identificación de las subunidades nicotínicas a9 y a10 en células sensoriales del aparato auditivo y en neuronas de los ganglios raquídeos, motivaron a investigar la posible expresión de estas subunidades en el parénquima adrenomedular. Mediante la técnica electrofisiológica de “patch-clamp”, se ha medido las corrientes mediadas por los receptores nicotínicos de la acetilcolina en células cromafines de rata (nAChRs). Dos agonistas nicotínicos, la acetilcolina (ACh) y nicotina (Nic) activaron corrientes con una concentración eficaz 50 (CE50) de 63 y 24μM, respectivamente. La colina, agonista selectivo de receptores nicotínicos a7 y a9a10, y la oxotremorina-M, agonista del receptor a9a10, indujeron respuestas con eficacias del 13% y 27%, respectivamente, con respecto a la obtenida con ACh (100μM). La bungarotoxina (BgTx) bloqueó parcial y reversiblemente (CI50 de 3,13nM) las corrientes inducidas por Oxo-M (300μM), sugiriendo la presencia de un nAChR formado por subunidades a9 y a10 en esta preparación. Los efectos bloqueantes de d-tubocurarina, estricnina, atropina y Nic sobre corrientes inducidas por Oxo-M confirmaron esta interpretación. El estudio de la selectividad iónica de los nAChRs a9a10, determinó la siguiente secuencia de permeabilidades: Ca2+ >> Cs+ > Na+. Li+>> Tris+. Los resultados indican que los a9a10 son altamente permeables al Ca2+ y que este catión modula la corriente a través de dicho receptor. Finalmente, cabe señalar que la entrada de Ca2+ a través del receptor 9 10 fue capaz de inducir la respuesta secretora en células cromafines de rata.--- The recent identification of a9 nicotinic acetylcholine receptor (nAChR) subunits and of its interacting partner a10 in sensory cells of the auditory apparatus and dorsal root ganglia neurones, prompted us to investigate whether they could also participate in the efferent synaptic signalling at the adrenal medulla level. We have measured nAChR-mediated currents in rat chromaffin cells by using the whole-cell configuration of the patch-clamp technique. Acetylcholine (ACh) and nicotine (Nic) activated inward currents in chromaffin cells with EC50 of 63 and 24 μM, respectively. Choline, a selective agonist a7 and a9a10 nAChRs, and oxotremorine-M (Oxo-M), a selective agonist of a9a10-containing nAChRs, elicited inward currents that, reached values of 13% and 27%, respectively, of the maximum elicited by ACh (100μM). a-bungarotoxin partially blocked (IC50 of 3,13nM) currents induced by Oxo-M (300 μM) in a reversible manner, suggesting the presence of the a9a10 subtype in chromaffin cells. The inhibitory effects of d-tubocurarine, strychnine, atropine and nicotine on Oxo-M-induced currents confirmed this interpretation. Ionic selectivity of the a9a10 nAChRs study established the following permeability sequence for: Ca2+ >> Cs+ > Na+ Li+>> Tris+. Our results show that the a9a10 receptor is highly permeable to Ca2+ and that is also capable of modulating a9a10-mediated currents. Just, Ca2+ ions entering the cell through a9a10 nAChRs-associated channels were shown to promote exocytosis –as estimated by membrane capacitance monitoring- in chromaffin cells with the membrane potential held at -30mV.Tesi

Farmacoalegría

Sección Deptal. de Historia del Arte III (Contemporáneo)Fac. de Bellas ArtesFALSEsubmitte

The adrenal medulla modulates mechanical allodynia in a rat model of neuropathic pain

We have investigated whether the stress response mediated by the adrenal medulla in rats subjected to chronic constriction injury of the sciatic nerve (CCI) modulates their nocifensive behavior. Treatment with SK29661 (300 mg/kg; intraperitoneal (I.P.)), a selective inhibitor of phenylethanolamine N-methyltransferase (PNMT) that converts noradrenaline (NA) into adrenaline (A), fully reverted mechanical allodynia in the injured hind paw without affecting mechanical sensitivity in the contralateral paw. The effect was fast and reversible and was associated with a decrease in the A to NA ratio (A/NA) in the adrenal gland and circulating blood, an A/NA that was elevated by CCI. 1,2,3,4-tetrahydroisoquinoline-7-sulfonamide (SKF29661) did not a ect exocytosis evoked by Ca2+ entry as well as major ionic conductances (voltage-gated Na+, Ca2+, and K+ channels, nicotinic acetylcholine receptors) involved in stimulus-secretion coupling in chroma n cells, suggesting that it acted by changing the relative content of the two adrenal catecholamines. Denervation of the adrenal medulla by surgical splanchnectomy attenuated mechanical allodynia in neuropathic animals, hence confirming the involvement of the adrenal medulla in the pathophysiology of the CCI model. Inhibition of PNMT appears to be an effective and probably safe way to modulate adrenal medulla activity and, in turn, to alleviate pain secondary to the injury of a peripheral nerveThis research was funded by the SPANISH MINISTER OF SCIENCE AND INNOVATION, grants BFU2011-26253, BFU2015-70067-REDC to A.R.A., and SAF2016-78892 to A.G.G, and by UNIVERSIDAD COMPLUTENSE DE MADRID, grant PR75/18-21593 to A.R.A

The TRPM8 antagonist RGM8-51 displays analgesic activity in different pain models

TRPM8 channels are overexpressed in sensory neurons after nerve injury or inflammation, resulting in enhanced sensitivity (allodynia and hyperalgesia) to physical stimulation, and have been implicated in migraine, but the interest of TRPM8 antagonists is still a matter of controversy (1,2). The aim of our work was to evaluate the analgesic activity of a TRPM8 antagonist, RGM8-51, in different pain models, looking for similarities and differences with other antagonists. To this end, we used the mouse oxaliplatin-induced peripheral neuropathy, the chronic constriction injury of the rat sciatic nerve (CCI) and mouse NTG-induced migraine-like models. Compound RGM8- 51 reduces the cold allodynia induced by oxaliplatin, from 15 to 60 min after administration (0.1-1 μg, i.pl.), decreases the nocifensive responses to cold, heat and mechanical stimuli in the CCI model (10 μg, i.pl., 30 mg/Kg, i.p.), and relief chronic pain associated to migraine in mouse, in a sex-dependent manner (10 or 30 mg/Kg, i.v.). The β–lactam derivative RGM8-51 not only has analgesic activity in all assayed animal models, but also seems to have a different mode of interaction with the TRPM8 channel than other antagonists, as suggested by docking studies

Los adrenoceptores alpha-2 en células cromafines adrenomedulares: papel funcional e implicaciones fisiopatológicas

Chromaffin cells from the adrenal medulla participate in stress responses by releasing catecholamines into the bloodstream. Main control of adrenal catecholamine secretion is exerted both neurally (by the splanchnic nerve fibers) and humorally (by corticosteroids, circulating noradrenaline, etc.). It should be noted, however, that secretory products themselves (catecholamines, ATP, opioids, ascorbic acid, chromogranins) could also influence the secretory response in an autocrine/paracrine manner. This form of control is activity-dependent and can be either inhibitory or excitatory.Among the inhibitory influences, it stands out the one mediated by α2-adrenergic autoreceptors activated by released catecholamines. α2-adrenoceptors areGprotein-coupledreceptors capable to inhibit exocytotic secretion through a direct interaction ofGβγ subunits with voltage-gated Ca2+ channels. Interestingly, upon intense and/or prolonged stimulation, α2-adrenergic receptors become desensitized by the intervention of G protein-coupled receptor kinase 2 (GRK2). In several experimental models of heart failure, there hasbeenreportedthe up-regulationofGRK2 and the loss of functioning of inhibitory α2-adrenoceptors resulting in enhanced release of adrenomedullary catecholamines. Given the importance of circulating catecholamines in the pathophysiology of heart failure, the recovery of α2-adrenergic modulation of the secretory response from chromaffin cells appears as a novel strategy for a better control of the patients with this cardiac disease.Ministerio de Ciencia e Innovación (BFU2011-26253; BFU2015-70067-REDC)Comunidad de Madrid (BRADE-CM S2013/ICE-2958)Sección Deptal. de Farmacología y Toxicología (Veterinaria)Fac. de VeterinariaTRUEpu

Contribution of BK channels to action potential repolarisation at minimal cytosolic Ca2+ concentration in chromaffin cells

BK channels modulate cell firing in excitable cells in a voltage-dependent manner regulated by fluctuations in free cytosolic Ca2+ during action potentials. Indeed, Ca2+-independent BK channel activity has ordinarily been considered not relevant for the physiological behaviour of excitable cells. We employed the patch-clamp technique and selective BK channel blockers to record K+ currents from bovine chromaffin cells at minimal intracellular (about 10 nM) and extracellular (free Ca2+) Ca2+ concentrations. Despite their low open probability under these conditions (V50 of +146.8 mV), BK channels were responsible for more than 25% of the total K+ efflux during the first millisecond of a step depolarisation to +20 mV. Moreover, BK channels activated about 30% faster (τ∈=∈0.55 ms) than the rest of available K+ channels. The other main source of fast voltage-dependent K+ efflux at such a low Ca2+ was a transient K+ (IA-type) current activating with V 50∈=∈-14.2 mV. We also studied the activation of BK currents in response to action potential waveforms and their contribution to shaping action potentials both in the presence and the absence of extracellular Ca2+. Our results show that BK channels activate during action potentials and accelerate cell repolarisation even at minimal Ca2+ concentration, and suggest that they could do so also in the presence of extracellular Ca2+, before Ca2+ entering the cell facilitates their activity. © 2011 Springer-Verlag.This work was supported by grants from the Spanish Ministerio de Ciencia e Innovación (BFU2005-06034; Spanish Ion Channel Initiative (SICI) grant CSD2008-00005) to A.R.A. and (RYC-2009-03979 and SAF2010-20604) to R.S.S.Peer Reviewe