Blockade by NNC 55-0396, mibefradil, and nickel of calcium and exocytotic signals in chromaffin cells: Implications for the regulation of hypoxia-induced secretion at early life

Adrenal chromaffin cells (CCs) express high voltage activated calcium channels (high-VACCs) of the L, N and PQ subtypes; in addition, T-Lype low-VACCs are also expressed during embryo and neonatal life. Effects of the more frequently used T channel blockers NNC 55-0396 (NNC), mibefradil, and Ni2 + on the whole -cell Ba2 vertical bar current (lBa), the K vertical bar -elicited ICa2 vertical bar]c transients and caLecholamine secretion have been studied in adult bovine CCs (BCCs) and rat embryo CCs (RECCs). NNC, rnibefradil, and Ni2 + blocked BCC lBa with 1050 of 1.8, 4.9 and 70 mu M, while IC50 to block lBa in RECCs were 2.1, 44 and 41 Pronounced blockade of K I -elicited ICa2 I]c transients and secretion was also elicited by the three agents. However, the hypoxia-induced secretion (HIS) of caLecholamine iii RECCs was blocked substantially (75%) with thresholds concentrations of NCC (IC20 to block IBa); this was not the case for mibefradil and Ni2 + that required higher concentrations to block the HIS response. Thus, out of the three compounds, NNC seemed to be an adequate pharmacological tool to discern the contribution of T channels to the HIS response, without a contamination with high-VACC blockade. (C) 2015 Published by Elsevier B.V.Ministerio de Economia y Competitividad, SpainCABICYCUAM/Bioiberica, SpainFundacion Teofilo Hernando, Madrid, SpainUniv Autonoma Madrid, Fac Med, Inst Teofilo Hernando, Madrid 28029, SpainUniv Autonoma Madrid, Fac Med, Dept Farmacol & Terapeut, Madrid 28029, SpainHosp Univ Princesa, Inst Invest Sanitaria, Serv Farmacol Clin, Madrid, SpainTech Univ Dresden, Med Fak Carl Gustav Carus, Dresden, GermanyFed Univ São Paulo UNIFESP, Dept Pharmacol, São Paulo, SP, BrazilFed Univ São Paulo UNIFESP, Dept Pharmacol, São Paulo, SP, BrazilMinisterio de Economia y Competitividad, Spain: SAF 2010-21795Ministerio de Economia y Competitividad, Spain: SAF 2013-44108Web of Scienc

Plasmalemmal sodium-calcium exchanger shapes the calcium and exocytotic signals of chromaffin cells at physiological temperature

The activity of the plasmalemmal Na(+)/Ca(2+) exchanger (NCX) is highly sensitive to temperature. We took advantage of this fact to explore here the effects of the NCX blocker KB-R7943 (KBR) at 22 and 37°C on the kinetics of Ca(2+) currents (ICa), cytosolic Ca(2+) ([Ca(2+)]c) transients, and catecholamine release from bovine chromaffin cells (BCCs) stimulated with high K(+), caffeine, or histamine. At 22°C, the effects of KBR on those parameters were meager or nil. However, at 37°C whereby the NCX is moving Ca(2+) at a rate fivefold higher than at 22°C, various of the effects of KBR were pronounced, namely: 1) no effects on ICa; 2) reduction of the [Ca(2+)]c transient amplitude and slowing down of its rate of clearance; 3) blockade of the K(+)-elicited quantal release of catecholamine; 4) blockade of burst catecholamine release elicited by K(+); 5) no effect on catecholamine release elicited by short K(+) pulses (1-2 s) and blockade of the responses produced by longer K(+) pulses (3-5 s); and 6) potentiation of secretion elicited by histamine or caffeine. Furthermore, the more selective NCX blocker SEA0400 also potentiated the secretory responses to caffeine. The results suggest that at physiological temperature the NCX substantially contributes to shaping the kinetics of [Ca(2+)]c transients and the exocytotic responses elicited by Ca(2+) entry through Ca(2+) channels as well as by Ca(2+) release from the endoplasmic reticulum.We thank the continued support of Fundación Teófilo Hernando, Madrid, Spain. This work was supported by the following grants to to A. G. García: 1) SAF 2010-21795, Ministerio de Economía y Competitividad, Spain; 2) RENEVAS-RETICS-RD06/0026, Instituto de Salud Carlos III, Spain; and 3) CABICYC Bioibérica/UAM. Also by grant from Ministerio de Economía y Competitividad No. 2010-18837 (to L. G. Gandía).Peer reviewe

Antidepressant drugs act by directly binding to TRKB neurotrophin receptors

It is unclear how binding of antidepressant drugs to their targets gives rise to the clinical antidepressant effect. We discovered that the transmembrane domain of tyrosine kinase receptor 2 (TRKB), the brain-derived neurotrophic factor (BDNF) receptor that promotes neuronal plasticity and antidepressant responses, has a cholesterol-sensing function that mediates synaptic effects of cholesterol. We then found that both typical and fast-acting antidepressants directly bind to TRKB, thereby facilitating synaptic localization of TRKB and its activation by BDNF. Extensive computational approaches including atomistic molecular dynamics simulations revealed a binding site at the transmembrane region of TRKB dimers. Mutation of the TRKB antidepressant-binding motif impaired cellular, behavioral, and plasticity-promoting responses to antidepressants in vitro and in vivo. We suggest that binding to TRKB and allosteric facilitation of BDNF signaling is the common mechanism for antidepressant action, which may explain why typical antidepressants act slowly and how molecular effects of antidepressants are translated into clinical mood recovery.Peer reviewe

Antidepressant drugs act by directly binding to TRKB neurotrophin receptors

It is unclear how binding of antidepressant drugs to their targets gives rise to the clinical antidepressant effect. We discovered that the transmembrane domain of tyrosine kinase receptor 2 (TRKB), the brain-derived neurotrophic factor (BDNF) receptor that promotes neuronal plasticity and antidepressant responses, has a cholesterol-sensing function that mediates synaptic effects of cholesterol. We then found that both typical and fast-acting antidepressants directly bind to TRKB, thereby facilitating synaptic localization of TRKB and its activation by BDNF. Extensive computational approaches including atomistic molecular dynamics simulations revealed a binding site at the transmembrane region of TRKB dimers. Mutation of the TRKB antidepressant-binding motif impaired cellular, behavioral, and plasticity-promoting responses to antidepressants in vitro and in vivo. We suggest that binding to TRKB and allosteric facilitation of BDNF signaling is the common mechanism for antidepressant action, which may explain why typical antidepressants act slowly and how molecular effects of antidepressants are translated into clinical mood recovery