M.P. González

NITRIC OXIDE IMPLICATION IN THE CONTROL OF NEUROSECRETION BY CHROMAFFIN CELLS

Abstract: In this work, we have studied the effects of pure nitric oxide (NO) on the regulation of catecholamine (CA) secretion by chromaffin cells, as well as the possible presence of its synthesizing enzyme l‐arginine:NO synthase (NOS) in these cells. Our results show that NO produces a large stimulation of basal CA secretion. This effect was calcium‐ and concentration‐dependent (EC50 = 64 ± 8 µM) and was not due to nonspecific damage of the tissue by NO. NO also modulates the CA secretion evoked by nicotine in a dose‐dependent manner. Although it has a stimulatory effect on the CA secretion evoked by low doses of nicotine (<3 µM; EC50 = 16 ± 3 µM), it produces a dose‐dependent inhibition of the CA secretion induced by high doses of nicotine (≥30 µM; IC50 = 52 ± 6 µM). The mechanism by which NO modulates CA secretion seems to be through the increase in the cyclic GMP levels, because there was a close correlation between the CA secretion and the cyclic GMP levels. The presence of a specific activity of NOS in chromaffin cells has been demonstrated by two independent methods: release of [14C]citruiline from [14C]arginine and formation of an NO‐hemoglobin complex. NOS activity was about 0.5 pmol/min/mg of protein. It was calcium‐ and mainly calmodulin‐dependent and could be specifically blocked by the NOS inhibitor N‐methyl‐l‐arginine. These results suggest that NO could be an important intracellular messenger in the regulation of neurosecretion in chromaffin cells.

Synthesis, biological assessment, and molecular modeling of racemic 7-aryl-9,10,11,12-tetrahydro-7H-benzo[7,8]chromeno[2,3-b]quinolin-8-amines as potential drugs for the treatment of Alzheimer's disease

The synthesis, pharmacological analysis and molecular modeling of the readily available racemic tacrine analogs 21-30, bearing the 7-aryl-9,10,11,12-tetrahydro-7H-benzo[7,8]chromeno[2,3-b]quinolin-8-amine heterocyclic ring system (II), prepared by Friedländer reaction of 2-amino-4-aryl-4H-benzo[h]chromene-3-carbonitriles (11-20) with cyclohexanone, are described in this paper. Molecules 21-30 are potent and selective inhibitors of hAChE, in the low micromolar range, one of the most potent inhibitors, 4-(8-amino-9,10,11,12-tetrahydro-7H-benzo[7,8]chromeno[2,3-b]quinolin-7-yl)-2-methoxyphenol (25), showing a IC(50) (hAChE) = 0.33 ± 0.04 μM. Kinetic studies of compound 25 proved that this compound is a mixed type inhibitor for EeAChE (K(i) = 81 nM). Accordingly, molecular modeling of inhibitor 25 showed that both enantiomers have two major predicted binding modes at the active and at the peripheral anionic sites of AChE. Inhibitor 25 has an excellent antioxidant profile as determined in the ORAC experiment (1.47 ± 0.10 Trolox equiv). Inhibitors 26-28 and 30 are permeable to BBB as determined in the PAMPA assay. Compared to tacrine, selected compounds 26-28 and 30 showed less hepatic toxicity in HepG2 cells. Moreover, cell viability-related studies in cortical neurons in primary cultures show that compounds 26-28 and 30 (0.1-50 μM) have significant neuroprotective effects against mitochondrial chain blockers-induced cell death, and, unlike tacrine, are not neurotoxic at concentrations lower than 50 μM. It is worth highlighting that compound 27 has the best neuroprotective properties out of all assayed compounds and shows no neurotoxicity. To sum up, these tacrine analogs can be considered as attractive multipotent therapeutic molecules on pharmacological receptors playing key roles in the progress of Alzheimers disease.

Segregation of nitric oxide synthase expression and calcium response to nitric oxide in adrenergic and noradrenergic bovine chromaffin cells

Previous work has demonstrated that nitric oxide can be an important intracellular messenger in the regulation of neurosecretion in chromaffin cells. Since standard chromaffin cell cultures are mixed populations of noradrenaline and adrenaline producing cells, it would seem important to understand the functional differences between these individual components. The use of fluorescence imaging techniques for the recording of cytosolic calcium from single chromaffin cells together with the immunoidentification of individual cells with specific antibodies against tyrosine hydroxylase, N-phenyl ethanolamine methyl transferase and nitric oxide synthase, has allowed us to measure single-cell calcium responses in identified adrenergic, noradrenergic and nitrergic chromaffin cells, thus helping us to clarify the differential role of nitric oxide in the function of these chromaffin cell types. 53 +/- 2% of chromaffin cells were able to synthesize nitric oxide (nitric oxidesynthase-positive cells), these cells being mainly noradrenergic (82 +/-2%). Results indicate that nitric oxide donors such as sodium nitroprusside, molsidomine and isosorbide dinitrate evoke [Ca2+]i increases in a 62 +/- 4% of chromaffin cells, the response to nitric oxide donors being between 30 and 50% of that of 20 microM nicotine. Cells responding to nitric oxide donors were mainly adrenergic (68 +/- 5%) although 45 +/- 9% of noradrenergic cells also gave [Ca2+]i increasing responses. The distribution of nitric oxide responding cells between nitric oxide synthase-positive and negative was very similar in the whole population (63 +/- 5 and 60 +/- 7%, respectively), but these differences were more prominent when considering the distribution of nitric oxide response between noradrenergic and adrenergic nitric oxide synthase-positive cells; while 73 6% of adrenergic nitric oxide synthase-positive cells evoke [Ca2+]i increases by nitric oxide stimulation, only 35 +/- 11% of noradrenergic nitric oxide synthase-positive cells respond. Taken together these results seem to indicate that (i) nitric oxide could act within adrenal medulla as both an intracellular and intercellular messenger; and (ii) noradrenergic cells seem to be specialized in nitric oxide synthesis while adrenergic cells with an endocrine function could mainly act as a target of neurosecretory action of this second messenger.

Mechanism through which GABAA receptor modulates catecholamine secretion from bovine chromaffin cells

The actions and mechanism of GABAergic modulation of catecholamine secretion from isolated bovine chromaffin cells were investigated. The GABAA receptor agonist muscimol induced a fast rise in cytosolic [Ca2+]. The mean peak increase was 290 +/- 30 nM over basal levels. The increase in cytosolic [Ca2+] was abolished in the absence of extracellular [Ca2+] and was blocked by the GABAA antagonist bicuculline and the dihydropiridine nifedipine. Muscimol also elicited the release of catecholamines and increased the bisoxonol fluorescence indicating a cell depolarization. The [Ca2+] entry was well correlated with muscimol-evoked catecholamine secretion. When cells were treated with muscimol and a second secretagogue, a biphasic behavior was revealed. Muscimol enhanced the catecholamine release evoked by low concentrations of nicotine or K+, whereas release obtained at high concentrations of nicotine or K+ was actually inhibited. When the muscimol effect on membrane potential was studied in the presence of low K+ or nicotine concentrations, an enhancement of the bisoxonol fluorescence was observed. This effect was reversed at high concentrations of both K+ and nicotine. Measurement of 36Cl- fluxes showed an increase in membrane permeability to Cl- during muscimol stimulation. The influx or efflux in Cl- was dependent on membrane potential. In normal conditions, with a K+ concentration of 5.4 mM, a Cl- efflux was observed by both radiometric techniques, with 36Cl- and by the use of the chloride-sensitive fluorescent probe 6-methoxy-N-(3-sulphopropil)quinolinium, as indicator of intracellular Cl-. At high nicotine (20 mM) or K+ concentrations (105 mM) a Cl- influx was observed using 6-methoxy-N-(3-sulphopropil)quinolinium.(ABSTRACT TRUNCATED AT 250 WORDS)

Transient focal cerebral ischemia significantly alters not only EAATs but also VGLUTs expression in rats: relevance of changes in reactive astroglia.

J. Neurochem. (2010) 113, 1343–1355.

α-Aryl-N-alkyl Nitrones, as Potential Agents for Stroke Treatment: Synthesis, Theoretical Calculations, Antioxidant, Anti-inflammatory, Neuroprotective, and Brain–Blood Barrier Permeability Properties

We report the synthesis, theoretical calculations, the antioxidant, anti-inflammatory, and neuroprotective properties, and the ability to cross the blood-brain barrier (BBB) of (Z)-α-aryl and heteroaryl-N-alkyl nitrones as potential agents for stroke treatment. The majority of nitrones compete with DMSO for hydroxyl radicals, and most of them are potent lipoxygenase inhibitors. Cell viability-related (MTT assay) studies clearly showed that nitrones 1-3 and 10 give rise to significant neuroprotection. When compounds 1-11 were tested for necrotic cell death (LDH release test) nitrones 1-3, 6, 7, and 9 proved to be neuroprotective agents. In vitro evaluation of the BBB penetration of selected nitrones 1, 2, 10, and 11 using the PAMPA-BBB assay showed that all of them cross the BBB. Permeable quinoline nitrones 2 and 3 show potent combined antioxidant and neuroprotective properties and, therefore, can be considered as new lead compounds for further development in specific tests for potential stroke treatment.

Neuronal nitric oxide synthase modulates basal catecholamine secretion in bovine chromaffin cells

The role of endogenously produced nitric oxide (NO) in the regulation of basal catecholamine (CA) secretion was studied in chromaffin cells. Treatment of chromaffin cells with nitric oxide synthase (NOS) inhibitors produced a dose‐dependent increase in basal catecholamine secretion, which paralleled their ability to inhibit NOS activity. This inhibitory profile was similar to that found in neurons, suggesting the constitutive expression of neuronal NOS (nNOS) in these cells, which was confirmed by Western blot analysis. A study of the kinetics and pharmacology of nNOS activity expressed in chromaffin cells in culture indicated that NOS activity is calcium‐dependent, increases with time, and is highly dependent on both intracellular concentrations of L‐arginine (Km ∼ 4 μM, Vmax = 908 ± 60 pmol/hr × 106 cells) and transport of L‐arginine into the cells (exhibiting two affinity constants of k1 = 3.2 ± 0.3 μM and k2 = 126 ± 5.5 μM). The effects of NOS inhibitors on CA secretion were mediated by the L‐arginine‐NO‐cGMP pathway, insofar as exogenous L‐arginine was able to partially block the increase in CA secretion evoked by them, and 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxaline‐1‐one (ODQ), a specific inhibitor of guanylate cyclase, and zaprinast, an inhibitor of the cGMP phosphodiesterase, were able to increase and inhibit, respectively, basal CA secretion in a dose‐dependent manner. These results suggest that chromaffin cells exhibit a tonic production of NO by nNOS that keeps the basal CA secretion at low levels, and this could be necessary for maintaining a normotensive state.

Nitric oxide and peroxynitrite induce cellular death in bovine chromaffin cells: Evidence for a mixed necrotic and apoptotic mechanism with caspases activation

Treatment of chromaffin cells with nitric oxide (NO) donors (SNP and SNAP) and peroxynitrite produces a time‐ and dose‐dependent necrotic and apoptotic cell death. Necrotic cell death was characterized by both an increase in lactate dehydrogenase and ATP release and changes in nuclei and cell morphology (as seen with fluorescence microscopy analysis with propidium iodide and Hoechst 33342). Apoptotic cell death was characterized by nuclear fragmentation and presence of apoptotic cell bodies, by a decrease in DNA content, and by an increase in DNA fragmentation. Treatment of chromaffin cells with lipopolysaccharide (LPS) or cytokines (interferon‐γ, tumor necrosis factor‐α) resulted only in apoptotic cell death. Apoptotic effects of NO‐inducing compounds were specifically reversed, depending on the stimuli, by the NO scavenger carboxy‐PTIO (CPTio) or by the NOS inhibitors L‐NMA and thiocitrulline. NO‐induced apoptotic death in chromaffin cells was concomitant to a cell cycle arrest in G0G1 phase and a decrease in the number of chromaffin cells in the G2M and S phases of cell cycle. All NO‐producing compounds were able to induce activation of caspase 3 and cytochrome c release, and specific inhibitors of caspase 3 and 9, such as Ac‐DEVD‐CHO (CPP32) and Ac‐Z‐LEHD‐FMK, respectively, prevented NO‐induced apoptosis in chromaffin cells. These results suggest that chromaffin cells could be good models for investigating the molecular basis of degeneration in diseases showing death of catecholaminergic neurons, phenomenon in which NO plays an important role.

GABAB receptors modulate catecholamine secretion in chromaffin cells by a mechanism involving cyclic AMP formation

1 The function of γ‐aminobutyric acidB (GABAB) receptors in modulation of catecholamine secretion by chromaffin cells and the possible mechanism involved in this action have been examined. 2 The GABAB agonists (−)‐baclofen and 3‐aminopropylphosphinic acid (3‐APPA) were found to induce a dose‐dependent increase of basal catecholamine secretion. The EC50s were 151 ± 35 μm and 225 ± 58 μm for baclofen and 3‐APPA, respectively. This stimulatory effect was specific since it could be blocked by 0.5 mm of the specific GABAB antagonist CGP‐35348. 3 In contrast, preincubation of chromaffin cells with the GABAB agonists was found to inhibit, in a dose‐dependent manner, the catecholamine secretion evoked by 10 μm nicotine and 200 μm muscimol. 4 The effects of GABAB agonists on both basal and evoked catecholamine secretion were found to be accompanied by parallel changes in intracellular calcium concentration ([Ca2+]i). GABAB agonists produced a dose‐dependent increase in [Ca2+]i which was partially blocked by CGP 35348, but they produced a strong inhibition of the [Ca2+]i increase induced by nicotine and muscimol. 5 The GABAB agonists also produced a dose‐dependent increase in intracellular cyclic AMP levels, there being a direct correlation between both increase in catecholamine secretion and in intracellular cyclic AMP levels. 6 The pretreatment of chromaffin cells with pertussis toxin doubled the catecholamine secretion and increased by four times the intracellular cyclic AMP levels evoked by GABAB agonists. 7 The possible involvement of adenylate cyclase in the mechanism of GABAB receptor modulation of catecholamine secretion is discussed.

Organic silicon protects human neuroblastoma SH-SY5Y cells against hydrogen peroxide effects

BackgroundHydrogen peroxide (H2O2) is a toxic agent that induces oxidative stress and cell death. Silicon (Si) is a biological element involved in limiting aluminium (Al) absorption with possible preventive effects in Alzheimer’s disease. However, Si has not yet been associated with other neuroprotective mechanisms.MethodsThe present experiments evaluated in the SH-SY5Y human neuroblastoma cell line the possible role of different Si G5 (50-1000xa0ng/mL) concentrations in preventing cellular death induced by H2O2 (400xa0μM, 24xa0hours).ResultsOur findings showed that H2O2 promoted cell death in the human SH-SY5Y cell cultures and this could be prevented by Si treatment. The loss in cell viability mediated by H2O2 was due to an apoptotic and necrotic process. Apoptotic death was incurred by regulating caspase-8 activity in the extrinsic pathway. The apoptotic and necrotic cell death induced by H2O2 was almost totally reversed by Si (50-500 ng/mL), indicating that it down-regulates both processes in H2O2 treated cells.ConclusionsAccording to our data, Si is able to increase SH-SY5Y cell survival throughout partially blocking cellular damage related to oxidative stress through a mechanism that would affect H2O2/ROS elimination.