Oxygen sensor of the heart.

How oxygen is sensed by the heart and what mechanisms mediate its sensing remain poorly understood. As recent reports show that low PO2 levels are detected by the cardiomyocytes in a few seconds, the rapid and short applications of low levels of oxygen (acute hypoxia), which avoid multiple effects of chronic hypoxia, may be used to probe the oxygen-sensing pathway of the heart. Here, we explored the oxygen-sensing pathway, focusing primarily on cellular surface membrane proteins that were first exposed to low PO2. Such studies suggest that acute hypoxia primarily targets the cardiac calcium channels, where either the channel itself or moieties closely associated with it, for instance heme-oxygenase-2 (HO-2) interacting through kinase phosphorylation, signal the α-subunit of the channel to the altered levels of PO2. Amino acids 1572-1651, the CaMKII phosphorylation sites (S1487 and S1545), CaM-binding sites (I1624 and Q1625), and Ser1928 of the carboxyl tail of the α-subunit appear to be critical residues that sense oxygen. Future studies on HO-2 knockout mice or CRISPR/Cas9 gene-edited human-induced pluripotent stem-cell-derived cardiomyocytes that reduce CaM-binding affinity are likely to provide deeper insights into the O2-sensing mechanisms.

Mutation in RyR2-FKBP Binding site alters Ca2+ signaling modestly but increases "arrhythmogenesis" in human stem cells derived cardiomyocytes.

AIMS: To gain insights into FKBP regulation of cardiac ryanodine receptor (RyR2) and Ca2+ signaling, we introduced the point mutation (N771D-RyR2) corresponding to skeletal muscle mutation (N760D-RyR1) associated with central core disease (CCD) via CRISPR/Cas9 gene-editing in the RyR2 FKBP binding site expressed in human induced pluripotent stem cell-derived cardiomyocytes (hiPSCCMs). Patients inflicted with CCD and other hereditary skeletal muscle diseases often show higher incidence of atrial or ventricular arrhythmias. METHODS AND RESULTS: Ca2+ imaging of voltage-clamped N771D-RyR2 mutant compared to WT hiPSCCMs showed: (1) ∼30% suppressed ICa with no significant changes in the gating kinetics of ICa; (2) 29% lower SR Ca2+ content and 33% lower RyR2 Ca2+ leak; (3) higher CICR gain and 30-35% increased efficiency of ICa-triggered Ca2±release; (4) higher incidence of aberrant SR Ca2+ releases, DADs, and Ca2+ sparks; (5) no change in fractional Ca2+-release, action potential morphology, sensitivity to isoproterenol, and sarcomeric FKBP-binding pattern. CONCLUSIONS: The more frequent spontaneous Ca2+ releases and longer Ca2+ sparks underlie the increased incidence of DADs and cellular arrhythmogenesis of N771D-RyR2 mutant. The smaller RyR2 Ca2±leak and SR content result from suppressed ICathat is compensated by higher CICR gain.

Regulation of Ca2+ signaling by acute hypoxia and acidosis in cardiomyocytes derived from human induced pluripotent stem cells.

AIMS: The effects of acute (100 s) hypoxia and/or acidosis on Ca2+ signaling parameters of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) are explored here for the first time. METHODS AND RESULTS: 1) hiPSC-CMs express two cell populations: rapidly-inactivating ICa myocytes (τi<40 ms, in 4-5 day cultures) and slowly-inactivating ICa (τi ≥ 40 ms, in 6-8 day cultures). 2) Hypoxia suppressed ICa by 10-20% in rapidly- and 40-55% in slowly-inactivating ICa cells. 3) Isoproterenol enhanced ICa in hiPSC-CMs, but either enhanced or did not alter the hypoxic suppression. 4) Hypoxia had no differential suppressive effects in the two cell-types when Ba2+ was the charge carrier through the calcium channels, implicating Ca2+-dependent inactivation in O2 sensing. 5) Acidosis suppressed ICa by ∼35% and ∼25% in rapidly and slowly inactivating ICa cells, respectively. 6) Hypoxia and acidosis suppressive effects on Ca-transients depended on whether global or RyR2-microdomain were measured: with acidosis suppression was ∼25% in global and ∼37% in RyR2 Ca2+-microdomains in either cell type, whereas with hypoxia suppression was ∼20% and ∼25% respectively in global and RyR2-microdomaine in rapidly and ∼35% and ∼45% respectively in global and RyR2-microdomaine in slowly-inactivating cells. CONCLUSIONS: Variability in ICa inactivation kinetics rather than cellular ancestry seems to underlie the action potential morphology differences generally attributed to mixed atrial and ventricular cell populations in hiPSC-CMs cultures. The differential hypoxic regulation of Ca2+-signaling in the two-cell types arises from differential Ca2+-dependent inactivation of the Ca2+-channel caused by proximity of Ca2+-release stores to the Ca2+ channels.

Regulation of Ca2+ signaling by acute hypoxia and acidosis in rat neonatal cardiomyocytes.

Ischemic heart disease is an arrhythmogenic condition, accompanied by hypoxia, acidosis, and impaired Ca2+ signaling. Here we report on effects of acute hypoxia and acidification in rat neonatal cardiomyocytes cultures. RESULTS: Two populations of neonatal cardiomyocyte were identified based on inactivation kinetics of L-type ICa: rapidly-inactivating ICa (τ~20ms) myocytes (prevalent in 3-4-day cultures), and slow-inactivating ICa (τ≥40ms) myocytes (dominant in 7-day cultures). Acute hypoxia (pO2<5mmHg for 50-100s) suppressed ICa reversibly in both cell-types to different extent and with different kinetics. This disparity disappeared when Ba2+ was the channel charge carrier, or when the intracellular Ca2+ buffering capacity was increased by dialysis of high concentrations of EGTA and BAPTA, suggesting critical role for calcium-dependent inactivation. Suppressive effect of acute acidosis on ICa (~40%, pH6.7), on the other hand, was not cell-type dependent. Isoproterenol enhanced ICa in both cell-types, but protected only against suppressive effects of acidosis and not hypoxia. Hypoxia and acidosis suppressed global Ca2+ transients by ~20%, but suppression was larger, ~35%, at the RyR2 microdomains, using GCaMP6-FKBP targeted probe. Hypoxia and acidosis also suppressed mitochondrial Ca2+ uptake by 40% and 10%, respectively, using mitochondrial targeted Ca2+ biosensor (mito-GCaMP6). CONCLUSION: Our studies suggest that acute hypoxia suppresses ICa in rapidly inactivating cell population by a mechanism involving Ca2+-dependent inactivation, while compromised mitochondrial Ca2+ uptake seems also to contribute to ICa suppression in slowly inactivating cell population. Proximity of cellular Ca2+ pools to sarcolemmal Ca2+ channels may contribute to the variability of inactivation kinetics of ICa in the two cell populations, while acidosis suppression of ICa appears mediated by proton-induced block of the calcium channel.

Electrophysiological properties and augmented catecholamine release from chromaffin cells of WKY and SHR rats contributing to the hypertension development elicited by chronic EtOH consumption.

It is known that chronic ethanol (EtOH) consumption leads to hypertension development and has been associated with deleterious effects on the cardiovascular system. Whether this condition alters calcium (Ca2+) signaling and exocytosis in adrenal chromaffin cells (CCs) as the case is for genetic hypertension, is unknown. We explored this question in four randomized experimental groups, male Wistar Kyoto (WKY/EtOH) and Spontaneously Hypertensive (SHR/EtOH) rats were subjected to the intake of increasing EtOH concentrations (5-20%, for 30 days) and their respective controls (WKY/Control and SHR/Control) received water. WKY/EtOH developed hypertension and cardiac hypertrophy; blood aldehyde dehydrogenase (ALDH) and H2O2 were also augmented. In comparison with WKY/Control, CCs from WKY/EtOH had the following features: (i) depolarization and higher frequency of spontaneous action potentials; (ii) decreased Ca2+ currents with slower inactivation; (iii) decreased K+ currents; (iv) augmented K+-elicited cytosolic Ca2+ transients ([Ca2+]c); (v) enhanced K+-elicited catecholamine release. These cardiovascular, blood and CCs changes were qualitatively similar to those undergone by SHR/Control and SHR/EtOH. The results suggest that the hypertension elicited by chronic EtOH has pathogenic features common to genetic hypertension namely, augmented [Ca2+]c transients and catecholamine release from their CCs.

Calcium Channel Subtypes and Exocytosis in Chromaffin Cells at Early Life.

Here we review the contribution of the various subtypes of voltage-activated calcium channels (VACCs) to the regulation of catecholamine release from chromaffin cells (CCs) at early life. Patch-clamp recording of inward currents through VACCs has revealed the expression of high-threshold VACCs (high-VACCs) of the L, N, and PQ subtypes in rat embryo CCs and ovine embryo CCs. Low-threshold VACC (low-VACC) currents (T-type) have also been recorded in rat embryo CCs and rat neonatal slices of adrenal medullae. Near full blockade by nifedipine and nimodipine of the K(+)-elicited secretion as well as the hypoxia induced secretion (HIS) supports the dominant role of L-VACC subtypes to the regulation of exocytosis at early life. Partial blockade by ω-conotoxin GVIA and ω-agatoxin IVA suggests a transient participation of N and PQ high-VACCs to the regulation of the HIS response at early stages of CC exposure to hypoxia. T-type low-VACC current did not elicit exocytosis triggered by electrical depolarising pulses applied to rat embryo CCs in one study, but largely contributed to the HIS response in neonatal rat adrenal slices in another. In spite of scarce available data, the sequence of events driving the HIS response in CCs at early life could be established as follows: (i) hypoxia blocks one or more K(+) channels; (ii) as a consequence, mild membrane depolarisation occurs; (iii) T-type low-VACCs open at membrane potentials more hyperpolarised than those required to recruit the high-VACCs; (iv) firing of action potentials then occurs; (v) fast-inactivating N and PQ high-VACCs transiently open and low-inactivating L high-VACCs remain open along the hypoxia stimulus; (vi) increase of cytosolic Ca(2+) takes place; and (vii) the exocytotic release of catecholamine occurs in two phases, an explosive initial phase, driven by Ca(2+) entry through L, N and PQ channels, followed by a more sustained catecholamine release at a slower rate driven by L-type channels.

Positive allosteric modulation of alpha-7 nicotinic receptors promotes cell death by inducing Ca(2+) release from the endoplasmic reticulum.

Positive allosteric modulation of α7 isoform of nicotinic acetylcholine receptors (α7-nAChRs) is emerging as a promising therapeutic approach for central nervous system disorders such as schizophrenia or Alzheimer's disease. However, its effect on Ca(2+) signaling and cell viability remains controversial. This study focuses on how the type II positive allosteric modulator (PAM II) PNU120596 affects intracellular Ca(2+) signaling and cell viability. We used human SH-SY5Y neuroblastoma cells overexpressing α7-nAChRs (α7-SH) and their control (C-SH). We monitored cytoplasmic and endoplasmic reticulum (ER) Ca(2+) with Fura-2 and the genetically encoded cameleon targeting the ER, respectively. Nicotinic inward currents were measured using patch-clamp techniques. Viability was assessed using methylthiazolyl blue tetrazolium bromide or propidium iodide staining. We observed that in the presence of a nicotinic agonist, PNU120596 (i) reduced viability of α7-SH but not of C-SH cells; (ii) significantly increased inward nicotinic currents and cytosolic Ca(2+) concentration; (iii) released Ca(2+) from the ER by a Ca(2+) -induced Ca(2+) release mechanism only in α7-SH cells; (iv) was cytotoxic in rat organotypic hippocampal slice cultures; and, lastly, all these effects were prevented by selective blockade of α7-nAChRs, ryanodine receptors, or IP3 receptors. In conclusion, positive allosteric modulation of α7-nAChRs with the PAM II PNU120596 can lead to dysregulation of ER Ca(2+) , overloading of intracellular Ca(2+) , and neuronal cell death. This study focuses on how the type II positive allosteric modulator PNU120596 (PAM II PNU12) affects intracellular Ca(2+) signaling and cell viability. Using SH-SY5Y neuroblastoma cells overexpressing α7-nAChRs (α7-SH) and their control (C-SH), we find that PAM of α7-nAChRs with PNU120596: (i) increases inward calcium current (ICa ) and cytosolic Ca(2+) concentration ([Ca(2+) ]cyt ); (ii) releases Ca(2+) from the ER ([Ca(2+) ]ER ) by a Ca(2+) -induced Ca(2+) release mechanism; and (iv) reduces cell viability. These findings were corroborated in rat hippocampal organotypic cultures. [Ca(2+) ]cyt , cytosolic Ca(2+) concentration; [Ca(2+) ]ER , endoplasmic reticulum Ca(2+) concentration; α7 nAChR, α7 isoform of nicotinic acetylcholine receptors; α7-SH, SH-SY5Y stably overexpressing α7 nAChRs cells; C-SH, control SH-SY5Y cells; Nic, nicotine; PNU12, PNU120596.

Depressed excitability and ion currents linked to slow exocytotic fusion pore in chromaffin cells of the SOD1(G93A) mouse model of amyotrophic lateral sclerosis.

Altered synaptic transmission with excess glutamate release has been implicated in the loss of motoneurons occurring in amyotrophic lateral sclerosis (ALS). Hyperexcitability or hypoexcitability of motoneurons from mice carrying the ALS mutation SOD1(G93A) (mSOD1) has also been reported. Here we have investigated the excitability, the ion currents, and the kinetics of the exocytotic fusion pore in chromaffin cells from postnatal day 90 to postnatal day 130 mSOD1 mice, when motor deficits are already established. With respect to wild-type (WT), mSOD1 chromaffin cells had a decrease in the following parameters: 95% in spontaneous action potentials, 70% in nicotinic current for acetylcholine (ACh), 35% in Na(+) current, 40% in Ca(2+)-dependent K(+) current, and 53% in voltage-dependent K(+) current. Ca(2+) current was increased by 37%, but the ACh-evoked elevation of cytosolic Ca(2+) was unchanged. Single exocytotic spike events triggered by ACh had the following differences (mSOD1 vs. WT): 36% lower rise rate, 60% higher decay time, 51% higher half-width, 13% lower amplitude, and 61% higher quantal size. The expression of the α3-subtype of nicotinic receptors and proteins of the exocytotic machinery was unchanged in the brain and adrenal medulla of mSOD1, with respect to WT mice. A slower fusion pore opening, expansion, and closure are likely linked to the pronounced reduction in cell excitability and in the ion currents driving action potentials in mSOD1, compared with WT chromaffin cells.

Hypoxia-elicited catecholamine release is controlled by L-type as well as N/PQ types of calcium channels in rat embryo chromaffin cells.

At early life, the adrenal chromaffin cells respond with a catecholamine surge under hypoxic conditions. This response depends on Ca(2+) entry through voltage-activated calcium channels (VACCs). We have investigated here three unresolved questions that concern this response in rat embryo chromaffin cells (ECCs): 1) the relative contribution of L (α1D, Cav1.3), N (α1B, Cav2.2), and PQ (α1A, Cav2.1) to the whole cell Ca(2+) current (ICa); 2) the relative contribution of L and N/PQ channels to the cytosolic Ca(2+) elevations triggered by hypoxia (Δ[Ca(2+)]c); and 3) the role of L and non-L high-VACCs in the regulation of the catecholamine surge occurring during prolonged (1 min) hypoxia exposure of ECCs. Nimodipine halved peak ICa and blocked 60% the total Ca(2+) entry during a 50-ms depolarizing pulse to 0 mV (QCa). Combined ω-agatoxin IVA plus ω-conotoxin GVIA (Aga/GVIA) blocked 30% of both ICa peak and QCa. This relative proportion of L- and non-L VACCs was corroborated by Western blot that indicated 55, 23, and 25% relative expression of L, N, and PQ VACCs. Exposure of ECCs to hypoxia elicited a mild but sustained Δ[Ca(2+)]c; the area of Δ[Ca(2+)]c was blocked 50% by nifedipine and 10% by Aga/GVIA. Exposure of ECCs to 1-min hypoxia elicited an initial transient burst of amperometric secretory spikes followed by scattered spikes along the time of cell exposure to hypoxia. This bulk response was blocked 85% by nimodipine and 35% by Aga/GVIA. Histograms on secretory spike frequency vs. time indicated a faster initial inactivation when Ca(2+) entry took place through N/PQ channels; more sustained secretion but at a lower rate was associated to Ca(2+) entry through L channels. The results suggest that the HIS response may initially be controlled by L and P/Q channels, but later on, N/PQ channels inactivate and the delayed HIS response is maintained at lower rate by slow-inactivating L channels.

Murine muscle engineered from dermal precursors: an in vitro model for skeletal muscle generation, degeneration, and fatty infiltration.

Skeletal muscle can be engineered by converting dermal precursors into muscle progenitors and differentiated myocytes. However, the efficiency of muscle development remains relatively low and it is currently unclear if this is due to poor characterization of the myogenic precursors, the protocols used for cell differentiation, or a combination of both. In this study, we characterized myogenic precursors present in murine dermospheres, and evaluated mature myotubes grown in a novel three-dimensional culture system. After 5-7 days of differentiation, we observed isolated, twitching myotubes followed by spontaneous contractions of the entire tissue-engineered muscle construct on an extracellular matrix (ECM). In vitro engineered myofibers expressed canonical muscle markers and exhibited a skeletal (not cardiac) muscle ultrastructure, with numerous striations and the presence of aligned, enlarged mitochondria, intertwined with sarcoplasmic reticula (SR). Engineered myofibers exhibited Na(+)- and Ca(2+)-dependent inward currents upon acetylcholine (ACh) stimulation and tetrodotoxin-sensitive spontaneous action potentials. Moreover, ACh, nicotine, and caffeine elicited cytosolic Ca(2+) transients; fiber contractions coupled to these Ca(2+) transients suggest that Ca(2+) entry is activating calcium-induced calcium release from the SR. Blockade by d-tubocurarine of ACh-elicited inward currents and Ca(2+) transients suggests nicotinic receptor involvement. Interestingly, after 1 month, engineered muscle constructs showed progressive degradation of the myofibers concomitant with fatty infiltration, paralleling the natural course of muscular degeneration. We conclude that mature myofibers may be differentiated on the ECM from myogenic precursor cells present in murine dermospheres, in an in vitro system that mimics some characteristics found in aging and muscular degeneration.

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.

Chondroitin sulfate, a major component of the perineuronal net, elicits inward currents, cell depolarization, and calcium transients by acting on AMPA and kainate receptors of hippocampal neurons.

Chondroitin sulfate (CS) proteoglycans (CSPGs) are the most abundant PGs of the brain extracellular matrix (ECM). Free CS could be released during ECM degradation and exert physiological functions; thus, we aimed to investigate the effects of CS on voltage- and current-clamped rat embryo hippocampal neurons in primary cultures. We found that CS elicited a whole-cell Na(+)-dependent inward current (ICS) that produced drastic cell depolarization, and a cytosolic calcium transient ([Ca(2+)]c). Those effects were similar to those elicited by α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) and kainate, were completely blocked by NBQX and CNQX, were partially blocked by GYKI, and were unaffected by MK801 and D-APV. Furthermore, ICS and AMPA currents were similarly potentiated by cyclothiazide, a positive allosteric modulator of AMPA receptors. Because CSPGs have been attributed Ca(2) (+) -dependent roles, such as neural network development, axon pathfinding, plasticity and regeneration after CNS injury, CS action after ECM degradation could be contributing to the mediation of these effects through its interaction with AMPA and kainate receptors.

Novel features on the regulation by mitochondria of calcium and secretion transients in chromaffin cells challenged with acetylcholine at 37°C.

From experiments performed at room temperature, we know that the buffering of Ca(2+) by mitochondria contributes to the shaping of the bulk cytosolic calcium transient ([Ca(2+)]c) and secretion transients of chromaffin cells stimulated with depolarizing pulses. We also know that the mitochondrial Ca(2+) transporters and the release of catecholamine are faster at 37°C with respect to room temperature. Therefore, we planned this investigation to gain further insight into the contribution of mitochondrial Ca(2+) buffering to the shaping of [Ca(2+)]c and catecholamine release transients, using some novel experimental conditions that have not been yet explored namely: (1) perifusion of bovine chromaffin cells (BCCs) with saline at 37°C and their repeated challenging with the physiological neurotransmitter acetylcholine (ACh); (2) separate blockade of mitochondrial Ca(2+) uniporter (mCUP) with Ru360 or the mitochondrial Na(+)/Ca(2+) exchanger (mNCX) with CGP37157; (3) full blockade of the mitochondrial Ca(2+) cycling (mCC) by the simultaneous inhibition of the mCUP and the mNCX. Ru360 caused a pronounced delay of [Ca(2+)]c clearance and augmented secretion. In contrast, CGP37157 only caused a tiny delay of [Ca(2+)]c clearance and a mild decrease in secretion. The mCC resulting in continued Ca(2+) uptake and its release back into the cytosol was interrupted by combined Ru360 + CGP37157 (Ru/CGP), the protonophore carbonyl cyanide-p-trifluoromethoxyphenylhydrazone, or combined oligomycin + rotenone (O/R); these three treatments caused a mild but sustained elevation of basal [Ca(2+)]c that, however, was not accompanied by a parallel increase in basal secretion. Nevertheless, all treatments caused a pronounced augmentation of ACh-induced secretion, with minor changes of the ACh-induced [Ca(2+)]c transients. Combined Ru/CGP did not alter the resting membrane potential in current-clamped cells. Additionally, Ru/CGP did not increase basal [Ca(2+)]c near subplasmalemmal sites and caused a mild decrease in the size of the readily releasable vesicle pool. Our results provide new functional features in support of the view that in BCCs there are two subpopulations of mitochondria, M1 underneath the plasmalemma nearby exocytotic sites and M2 at the core cell nearby vesicle transport sites. While M1 serves to shape the ACh-elicited exocytotic response through its efficient Ca(2+) removal by the mCUP, M2 shapes the lower [Ca(2+)]c elevations required for new vesicle supply to the exocytotic machinery, from the large reserve vesicle pool at the cell core. The mCUP of the M1 pool seems to play a more prominent role in controlling the ACh responses, in comparison with the mNCX.

Stabilizers of neuronal and mitochondrial calcium cycling as a strategy for developing a medicine for Alzheimer's disease.

For the last two decades, most efforts on new drug development to treat Alzheimer's disease have been focused to inhibit the synthesis of amyloid beta (Aß), to prevent Aß deposition, or to clear up Aß plaques from the brain of Alzheimer's disease (AD) patients. Other pathogenic mechanisms such as the hyperphosphorylation of the microtubular tau protein (that forms neurofibrillary tangles) have also been addressed as, for instance, with inhibitors of the enzyme glycogen synthase-3 kinase beta (GSK3ß). However, in spite of their proven efficacy in animal models of AD, all these compounds have so far failed in clinical trials done in AD patients. It seems therefore desirable to explore new concepts and strategies in the field of drug development for AD. We analyze here our hypothesis that a trifunctional chemical entity acting on the L subtype of voltage-dependent Ca(2+) channels (VDCCs) and on the mitochondrial Na(+)/Ca(2+) exchanger (MNCX), and having additional antioxidant properties, may efficiently delay or stop the death of vulnerable neurons in the brain of AD patients. In recent years, evidence has accumulated indicating that enhanced neuronal Ca(2+) cycling (NCC) and futile mitochondrial Ca(2+) cycling (MCC) are central stage in activating calpain and calcineurin, as well as the intrinsic mitochondrial pathway for apoptosis, leading to death of vulnerable neurons. An additional contributing factor to neuronal death is the excess free radical production linked to distortion of Ca(2+) homeostasis. We propose that an hybrid compound containing a dihydropyridine moiety (to block L channels and mitigate Ca(2+) entry) and a benzothiazepine moiety (to block the MNCX and slow down the rate of Ca(2+) efflux from the mitochondrial matrix into the cytosol), as well as a polyphenol moiety (to sequester excess free radicals) could break down the pathological enhanced NCC and MCC, thus delaying the initiation of apoptosis and the death of vulnerable neurons. In so doing, such a trifunctional compound could eventually become a neuroprotective medicine capable of delaying disease progression in AD patients.

Resveratrol augments nitric oxide generation and causes store calcium release in chromaffin cells.

The cardiovascular protecting effect of the grape fruit trans-resveratrol has been explained among other factors, through augmentation of nitric oxide (NO) production in cardiovascular tissues. Another effect of low resveratrol concentration is the inhibition of single-vesicle quantal release of catecholamine from bovine adrenal chromaffin cells, that was recently suggested to be an additional factor contributing to its beneficial cardiovascular effects. We have investigated here the effects of a low concentration of trans-resveratrol (1 µM) on Ca(2+) and NO signaling pathways in bovine chromaffin cells, in an attempt to understand the mechanism underlying its previously reported inhibitory effects on quantal secretion. In cells loaded with fura-2 acetoxymethyl ester (fura-2), we have found that 1 µM resveratrol produces a transient elevation of the cytosolic Ca(2+) concentration ([Ca(2+)](c)). This Ca(2+) transient was drastically reduced when the Ca(2+) store was depleted by ryanodine and dantrolene; it was also inhibited by N(ω)-nitro-l-arginine methyl ester hydrochloride (L-NAME) and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). Furthermore, the Ca(2+) transient was mimicked by NO donor S-nitroso-N-acetyl-penicillamine (SNAP). Resveratrol also enhanced the production of nitrites and NO, and L-NAME blocked both responses; in contrast, augmentation by SNAP of nitrites and NO was unaffected by ODQ and was only partially inhibited by L-NAME. On the basis of these results, we are proposing that resveratrol is mitigating the catecholamine surge occurring during stress, through its ability to elicit mild local [Ca(2+)](c) transients and enhanced NO production, that blocks the last steps of exocytosis.

Blockade by nanomolar resveratrol of quantal catecholamine release in chromaffin cells.

The cardiovascular protecting effects of resveratrol, an antioxidant polyphenol present in grapes and wine, have been attributed to its vasorelaxing effects and to its anti-inflammatory, antioxidant, and antiplatelet actions. Inhibition of adrenal catecholamine release has also been recently implicated in its cardioprotecting effects. Here, we have studied the effects of nanomolar concentrations of resveratrol on quantal single-vesicle catecholamine release in isolated bovine adrenal chromaffin cells. We have found that 30 to 300 nM concentrations of resveratrol blocked the acetylcholine (ACh) and high K(+)-evoked quantal catecholamine release, amperometrically measured with a carbon fiber microelectrode. At these concentrations, resveratrol did not affect the whole-cell inward currents through nicotinic receptors or voltage-dependent sodium and calcium channels, neither the ACh- or K(+)-elicited transients of cytosolic Ca(2+). Blockade by nanomolar resveratrol of secretion in ionomycin- or digitonin-treated cells suggests an intracellular site of action beyond Ca(2+)-dependent exocytotic steps. The fact that nanomolar resveratrol augmented cGMP is consistent with the view that resveratrol could be blocking the quantal secretion of catecholamine through a nitric oxide-linked mechanism. Because this effect occurs at nanomolar concentrations, our data are relevant in the context of the low circulating levels of resveratrol found in moderate consumers of red wines, which could afford cardioprotection by mitigating the catecholamine surge occurring during stress.