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Pre-blockade of the sarco-endoplasmic reticulum (ER) calcium ATPase (SERCA) with irreversible thapsigargin depresses exocytosis in adrenal bovine chromaffin cells (BCCs). Distinct expression of voltage-dependent Ca 2+ -channel subtypes and of the Ca 2+ -induced Ca 2+ release (CICR) mechanism in BCCs versus mouse chromaffin cells (MCCs) has been described. We present a parallel study on the effects of the acute SERCA blockade with reversible cyclopizonic acid (CPA), to repeated pulsing with acetylcholine (ACh) at short (15 s) and long intervals (60 s) at 37 °C, allowing the monitoring of the initial size of a ready-release vesicle pool (RRP) and its depletion and recovery in subsequent stimuli. We found (i) strong depression of exocytosis upon ACh pulsing at 15-s intervals and slower depression at 60-s intervals in both cell types; (ii) facilitation of exocytosis upon acute SERCA inhibition, with back to depression upon CPA washout in MCCs; (iii) blockade of exocytosis upon acute SERCA inhibition and pronounced rebound of exocytosis upon CPA washout in BCCs; (iv) basal [Ca 2+ ] c elevation upon stimulation with ACh at short intervals (but not at long intervals) in both cell types; and (v) augmentation of basal [Ca 2+ ] c and inhibition of peak [Ca 2+ ] c amplitude upon CPA treatment in both cell types, with milder effects upon stimulation at 60-s intervals. These results are compatible with the view that while in MCCs the uptake of Ca 2+ via SERCA contributes to the mitigation of physiological ACh triggered secretion, in BCCs the uptake of Ca 2+ into the ER facilitates such responses likely potentiating a Ca 2+ -induced Ca 2+ release mechanism. These drastic differences in the regulation of ACh-triggered secretion at 37 °C may help to understand different patterns of the regulation of exocytosis by the circulation of Ca 2+ at a functional ER Ca 2+ store.

As the peripheral sympathoadrenal axis is tightly controlled by the cortex via hypothalamus and brain stem, the central pathological features of Hunting's disease, (HD) that is, deposition of mutated huntingtin and synaptic dysfunctions, could also be expressed in adrenal chromaffin cells. To test this hypothesis we here present a thorough investigation on the pathological and functional changes undergone by chromaffin cells (CCs) from 2-month (2 m) to 7-month (7 m) aged wild-type (WT) and R6/1 mouse model of Huntington's disease (HD), stimulated with acetylcholine (ACh) or high [K + ] (K + ). In order to do this, we used different techniques such as inmunohistochemistry, patch-clamp, and amperometric recording. With respect to WT cells, some of the changes next summarized were already observed in HD mice at a pre-disease stage (2 m); however, they were more pronounced at 7 m when motor deficits were clearly established, as follows: (i) huntingtin over-expression as nuclear aggregates in CCs; (ii) smaller CC size with decreased dopamine β-hydroxylase expression, indicating lesser number of chromaffin secretory vesicles; (iii) reduced adrenal tissue catecholamine content; (iv) reduced Na + currents with (v) membrane hyperpolarization and reduced ACh-evoked action potentials; (v) reduced [Ca 2+ ] c transients with faster Ca 2+ clearance; (vi) diminished quantal secretion with smaller vesicle quantal size; (vii) faster kinetics of the exocytotic fusion pore, pore expansion, and closure. On the basis of these data, the hypothesis is here raised in the sense that nuclear deposition of mutated huntingtin in adrenal CCs of R6/1 mice could be primarily responsible for poorer Na + channel expression and function, giving rise to profound depression of cell excitability, altered Ca 2+ handling and exocytosis. OPEN PRACTICES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/. Cover Image for this issue: doi: 10.1111/jnc.14201., (© 2018 International Society for Neurochemistry.)

The inhibition of nicotinic acetylcholine receptors (nAChRs) has been proposed as a potential strategy to develop new antidepressant drugs. This is based on the observation that antidepressants that selectively block noradrenaline (NA) or serotonin (5-HT) reuptake also inhibit nAChRs. Dual antidepressants blocking both NA and 5-HT reuptake were proposed to shorten the delay in exerting their clinical effects; whether duloxetine, a prototype of dual antidepressants, also blocks nAChRs is unknown. Here we explored this question in bovine chromaffin cells (BCCs) that express native α 3 , α 5 , and α 7 nAChRs and in cell lines expressing human α 7 , α 3 β 4 , or α 4 β 2 nAChRs. We have found that duloxetine fully blocked the acetylcholine (ACh)-elicited nicotinic currents in BCCs with an IC 50 of 0.86 µ M. Such blockade seemed to be noncompetitive, voltage dependent, and partially use dependent. The ACh-elicited membrane depolarization, the elevation of cytosolic calcium ([Ca 2+ ] c ), and catecholamine release in BCCs were also blocked by duloxetine. This blockade developed slowly, and the recovery of secretion was also slow and gradual. Duloxetine did not affect Na + or Ca 2+ channel currents neither the high-K + -elicited [Ca 2+ ] c transients and secretion. Of interest was that in cell lines expressing human α 7 , α 3 β 4 , and α 4 β 2 nAChRs, duloxetine blocked nicotinic currents with IC 50 values of 0.1, 0.56, and 0.85 µ M, respectively. Thus, in blocking α 7 receptors, which are abundantly expressed in the brain, duloxetine exhibited approximately 10-fold to 100- fold higher potency with respect to reported IC 50 values for various antidepressant drugs. This may contribute to the antidepressant effect of duloxetine., (Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.)

Pre-blockade of the sarco-endoplasmic reticulum (ER) calcium ATPase (SERCA) with irreversible thapsigargin depresses exocytosis in adrenal bovine chromaffin cells (BCCs). Distinct expression of voltage-dependent Ca2+-channel subtypes and of the Ca2+-induced Ca2+ release (CICR) mechanism in BCCs versus mouse chromaffin cells (MCCs) has been described. We present a parallel study on the effects of the acute SERCA blockade with reversible cyclopizonic acid (CPA), to repeated pulsing with acetylcholine (ACh) at short (15 s) and long intervals (60 s) at 37 °C, allowing the monitoring of the initial size of a ready-release vesicle pool (RRP) and its depletion and recovery in subsequent stimuli. We found (i) strong depression of exocytosis upon ACh pulsing at 15-s intervals and slower depression at 60-s intervals in both cell types; (ii) facilitation of exocytosis upon acute SERCA inhibition, with back to depression upon CPA washout in MCCs; (iii) blockade of exocytosis upon acute SERCA inhibition and pronounced rebound of exocytosis upon CPA washout in BCCs; (iv) basal [Ca2+]c elevation upon stimulation with ACh at short intervals (but not at long intervals) in both cell types; and (v) augmentation of basal [Ca2+]c and inhibition of peak [Ca2+]c amplitude upon CPA treatment in both cell types, with milder effects upon stimulation at 60-s intervals. These results are compatible with the view that while in MCCs the uptake of Ca2+ via SERCA contributes to the mitigation of physiological ACh triggered secretion, in BCCs the uptake of Ca2+ into the ER facilitates such responses likely potentiating a Ca2+-induced Ca2+ release mechanism. These drastic differences in the regulation of ACh-triggered secretion at 37 °C may help to understand different patterns of the regulation of exocytosis by the circulation of Ca2+ at a functional ER Ca2+ store. [ABSTRACT FROM AUTHOR]

From a pathogenic perspective, Huntington's disease (HD) is being considered as a synaptopathy. As such, alterations in brain neurotransmitter release occur. As the activity of the sympathoadrenal axis is centrally controlled, deficits in the exocytotic release of catecholamine release may also occur. In fact, in chromaffin cells (CCs) of the adrenal medulla of the R6/1 model of HD, decrease of secretion and altered kinetics of the exocytotic fusion pore have been reported. Those alterations could be linked to mitochondrial deficits occurring in peripheral CCs, similar to those described in brain mitochondria. Here we have inquired about alterations in mitochondrial structure and function and their impact on exocytosis and calcium channel currents (I Ca). We have monitored various parameters linked to those events, in wild type (WT) and the R6/1 mouse model of HD at a pre-disease stage (2 months age, 2 m), and when motor deficits are present (7 months age, 7 m). In isolated CCs from 7 m and in the adrenal medulla of R6/1 mice, we found the following alterations (with respect 7 m WT mice): (i) augmented fragmented mitochondria and oxidative stress with increased oxidized glutathione; (ii) decreased basal and maximal respiration; (iii) diminution of ATP cell levels; (iv) mitochondrial depolarization; (v) drastic decrease of catecholamine release with poorer potentiation by protonophore FCCP; (vi) decreased I Ca inhibition by FCCP; and (vii) lesser potentiation by BayK8644 of I Ca and smaller prolongation of current deactivation. Of note was the fact several of these alterations were already manifested in CCs from 2 m R6/1 mice at pre-disease stages. Based on those results, a plausible hypothesis can be raised in the sense that altered mitochondrial function seems to be an early primary event in HD pathogenesis. This is in line with an increasing number of mitochondrial, metabolic, and inflammatory alterations being recently reported in various HD peripheral tissues. • Here, an investigation is presented on altered mitochondrial function and its impact on calcium and exocytosis in chromaffin cells from R6/1 mice. • Augmented mitochondrial depolarization and oxidative stress, with impaired respiration and ATP synthesis, were found, were found in R6/1 mice. • Drastic diminution of catecholamine release, and poorer potentiation of secretion by FCCP, were observed in R6/1 mice. • Lesser enhancing effects of BayK8644 on I Ca , were noted in R6/1 mice. • Mitochondrial alterations were visible at pre-symptomatic disease stages, suggesting the early initiation of HD pathogenesis. [ABSTRACT FROM AUTHOR]

Adrenal chromaffin cells (CCs) have been used extensively in studies aimed at revealing the intricacies of the Ca2+- dependent early and late steps of regulated exocytosis. They have also served as invaluable models to study the kinetics of single-vesicle exocytotic events to infer the characteristics of opening and closing of the exocytotic fusion pore. We have here tested the hypothesis that stimulation at room temperature of CCs from mice C57BL/6 with physiological acetylcholine (ACh) and with other secretagogues (dimethylphenylpiperazinium, high K+, muscarine, histamine, caffeine), alone or in combination, could trigger amperometric spike events with different kinetics. We found that mean secretory spike events in CCs stimulated with ACh had a fast rise rate of 25 pA/ms and a rapid decay time of 6.2 ms, with a small quantal size (0.31 pC). Surprisingly, these parameters considerably differed from those found in CCs stimulated with all other secretagogues that triggered secretory responses with spike events having smaller rise rates, longer decay times and higher quantal sizes. ACh spikes were unaltered by atropine but mitochondrial protonophore carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone markedly slowed down the rate rise and decay time, and augmented the quantal size of mean secretory events. We conclude that the physiological neurotransmitter ACh triggers a fast and efficient exocytotic response that cannot be mimicked by other secretagogues; such response is regulated by the mitochondrial circulation of calcium ions. [ABSTRACT FROM AUTHOR]