Manuela G. López

Separation and culture of living adrenaline- and noradrenaline-containing cells from bovine adrenal medullae

Separation of viable adrenaline-containing from noradrenaline-containing chromaffin cells in large amounts has been achieved. The procedure involves collagenase digestion of bovine adrenomedullary tissue, isolation of cells through gentle filtration, separation of chromaffin from nonchromaffin cells on discontinuous gradients of the radiopaque contrast Renografin, and separation of adrenaline-enriched from noradrenaline-enriched fractions after centrifugation on self-generated Percoll gradients. Collection of 1-ml Percoll fractions gave two clear-cut catecholamine peaks. The denser peak was enriched in adrenaline and phenylethanolamine-N-methyltransferase (PNMT), suggesting that over 90% of cells were adrenergic. The lighter peak was preferentially enriched in noradrenaline but not in PNMT. With this information, we could collect by gentle aspiration two main fraction layers of larger volumes; one at the bottom of the Percoll gradient, which contained essentially adrenaline-storing cells and the other at the top of the gradient, enriched in noradrenaline cells. Those cells could be maintained viable for at least 1 week in primary monolayer cultures, as shown by neutral red staining and trypan blue exclusion. This method will allow the identification of chemical components, receptors, or ionic channels present in one specific type of cell, to determine their relevance to the regulation of the differential secretion of specific materials present in one but not in the other cell type and to ascertain whether the released materials from one cell type affect the functions of the other.

Novel Tacrine−8-Hydroxyquinoline Hybrids as Multifunctional Agents for the Treatment of Alzheimer’s Disease, with Neuroprotective, Cholinergic, Antioxidant, and Copper-Complexing Properties

Tacrine and PBT2 (an 8-hydroxyquinoline derivative) are well-known drugs that inhibit cholinesterases and decrease beta-amyloid (Abeta) levels by complexation of redox-active metals, respectively. In this work, novel tacrine-8-hydroxyquinoline hybrids have been designed, synthesized, and evaluated as potential multifunctional drugs for the treatment of Alzheimers disease. At nano- and subnanomolar concentrations they inhibit human acetyl- and butyrylcholinesterase (AChE and BuChE), being more potent than tacrine. They also displace propidium iodide from the peripheral anionic site of AChE and thus could be able to inhibit Abeta aggregation promoted by AChE. They show better antioxidant properties than Trolox, the aromatic portion of vitamin E responsible for radical capture, and display neuroprotective properties against mitochondrial free radicals. In addition, they selectively complex Cu(II), show low cell toxicity, and could be able to penetrate the CNS, according to an in vitro blood-brain barrier model.

Galantamine prevents apoptosis induced by β-amyloid and thapsigargin: involvement of nicotinic acetylcholine receptors

Galantamine is currently used to treat Alzheimers disease patients; it behaves as a mild blocker of acetylcholinesterase (AChE) and has an allosteric modulating action on nicotinic acetylcholine receptors (nAChRs). In this study, we observed that galantamine prevented cell death induced by the peptide beta-amyloid(1-40) and thapsigargin in the human neuroblastoma cell line SH-SY5Y, as well as in bovine chromaffin cells. The protective effect of galantamine was concentration-dependent in both cell types; maximum protection was produced at 300 nM. The antiapoptotic effect of galantamine at 300 nM, against beta-amyloid(1-40) or thapsigargin-induced toxicity, was reversed by alpha-bungarotoxin. At neuroprotective concentrations, galantamine caused a mild and sustained elevation of the cytosolic concentration of calcium, [Ca2+]c, measured in single cells loaded with Fura-2. Incubation of the cells for 48 h with 300 nM galantamine doubled the density of alpha7 nicotinic receptors and tripled the expression of the antiapoptotic protein Bcl-2. These results strongly suggest that galantamine can prevent apoptotic cell death by inducing neuroprotection through a mechanism related to that described for nicotine, i.e. activation of nAChRs and upregulation of Bcl-2. These findings might explain the long-term beneficial effects of galantamine in patients suffering of Alzheimers disease.

Q‐ and L‐type Ca2+ channels dominate the control of secretion in bovine chromaffin cells

Potassium‐stimulated catecholamine release from superfused bovine adrenal chromaffin cells (70 mM K+ in the presence of 2 mM Ca2+ for 10 s, applied at 5‐min intervals) was inhibited by the dihydropyridine furnidipine (3 μM) by 50%. ω‐Conotoxin MVIIC (CTx‐MVIIC, 3 μM) also reduced the secretory response by about half. Combined CTx‐MVIIC plus furnidipine blocked 100% catecholamine release. 45Ca2+ uptake and cytosolic Ca2+ concentrations ([Ca2+]i) in K+‐depolarized cells were partially blocked by furnidipine or CTx‐MVIIC, and completely inhibited by both agents. The whole cell current through Ca2+ channels carried by Ba2+ (I Ba) was partially blocked by CTx‐MVIIC. Although ω‐conotoxin GVIA (CTx‐GVIA, 1 μM) and ω‐agatoxin IVA (Aga‐IVA, 0.2 μM) partially inhibited 45Ca2+ entry, I Ba and the increase in [Ca2+]i, the combination of both toxins did not affect the K+‐evoked secretory response. The results are compatible with the presence in bovine chromaffin cells of a Q‐like Ca2+ channel which has a prominent role in controlling exocytosis. They also suggest that Q‐ and L‐type Ca2+ channels, but not N‐ or P‐types are localized near exocytotic active sites in the plasmalemma.

Tacrine–Melatonin Hybrids as Multifunctional Agents for Alzheimer's Disease, with Cholinergic, Antioxidant, and Neuroprotective Properties

Tacrine–melatonin hybrids are potential multifunctional drugs for Alzheimers disease that may simultaneously palliate intellectual deficits and protect the brain against both β‐amyloid peptide and oxidative stress. Molecular modeling studies show that they target both the catalytic active site (CAS) and the peripheral anionic site (PAS) of AChE. They are nontoxic and may be able to penetrate the CNS, according to in vitro PAMPA‐BBB assays.

Antidepressant-like effect of the novel thiadiazolidinone NP031115 in mice.

Glycogen synthase kinase-3beta (GSK-3beta) is an enzyme that phosphorylates glycogen synthase, thereby inhibiting glycogen synthesis. Besides this role, it is now believed that this enzyme plays an important role in the pathophysiology of many brain diseases including depression. Some inhibitors of this enzyme have shown antidepressant effects in animal models. This study investigated the effects of a novel thiadiazolidinone NP031115, a putative GSK-3beta inhibitor, and the well-established GSK-3beta inhibitor AR-A014418 in the mouse forced swimming test (FST), a model widely used to evaluate antidepressant activity. We found that NP031115 had an IC50 of 1.23 and 6.5 microM for GSK-3beta and GSK-3alpha, respectively. NP031115 (0.5 and 5 mg/kg, i.p.), in a way similar to imipramine (15 mg/kg, i.p), fluoxetine (32 mg/kg, i.p), AR-A014418 (9 mg/kg, i.p.), and rosiglitazone (5 microg/site, i.c.v.), significantly reduced immobility time in the FST. NP031115 at the higher dose and AR-A014418 (9 mg/kg, i.p.) reduced locomotion in the open-field test. Rosiglitazone (30 microM), AR-A014418 (1 microM), PG(J2) (10 microM), and NP031115 (1, 10 and 25 microM) activate PPARgamma in CHO transfected cells. GW-9662 (10 microg/site, i.c.v, a PPARgamma antagonist) administered 15 min before NP03115 (5 mg/kg, i.p.) or co-administered with rosiglitazone (5 microg/site, i.c.v.) prevented the antidepressant-like effect of these drugs in the FST. The results of this study show that NP031115 can exhibit an antidepressant effect, likely by inhibiting GSK-3beta and enhancing PPARgamma activity.

Redox Control of Microglial Function: Molecular Mechanisms and Functional Significance

Neurodegenerative diseases are characterized by chronic microglial over-activation and oxidative stress. It is now beginning to be recognized that reactive oxygen species (ROS) produced by either microglia or the surrounding environment not only impact neurons but also modulate microglial activity. In this review, we first analyze the hallmarks of pro-inflammatory and anti-inflammatory phenotypes of microglia and their regulation by ROS. Then, we consider the production of reactive oxygen and nitrogen species by NADPH oxidases and nitric oxide synthases and the new findings that also indicate an essential role of glutathione (γ-glutamyl-l-cysteinylglycine) in redox homeostasis of microglia. The effect of oxidant modification of macromolecules on signaling is analyzed at the level of oxidized lipid by-products and sulfhydryl modification of microglial proteins. Redox signaling has a profound impact on two transcription factors that modulate microglial fate, nuclear factor kappa-light-chain-enhancer of activated B cells, and nuclear factor (erythroid-derived 2)-like 2, master regulators of the pro-inflammatory and antioxidant responses of microglia, respectively. The relevance of these proteins in the modulation of microglial activity and the interplay between them will be evaluated. Finally, the relevance of ROS in altering blood brain barrier permeability is discussed. Recent examples of the importance of these findings in the onset or progression of neurodegenerative diseases are also discussed. This review should provide a profound insight into the role of redox homeostasis in microglial activity and help in the identification of new promising targets to control neuroinflammation through redox control of the brain.

Functional interference between glycogen synthase kinase-3 beta and the transcription factor Nrf2 in protection against kainate-induced hippocampal celldeath

Excitotoxicity mediated by glutamate receptors may underlay the pathology of several neurologic diseases. Considering that oxidative stress is central to excitotoxic damage, in this study we sought to analyze if the transcription factor Nrf2, guardian of redox homeostasis, might be targeted to prevent kainate-induced neuron death. Hippocampal slices from Nrf2 knockout mice exhibited increased oxidative stress and cell death compared to those of control mice in response to kainate, as determined with the redox sensitive probes 2,7-dichlorodihydrofluorescein diacetate (H(2)DCFAC) and propidium iodide and lactate dehydrogenase release, respectively, therefore demonstrating a role of Nrf2 in antioxidant protection against excitotoxicity. In the hippocampus of mice intraperitoneally injected with kainate we observed a rapid activation of Akt, inhibition of GSK-3beta and translocation of Nrf2 to the nucleus, but after 4 h Akt was inactive, GSK-3beta was active and Nrf2 was mostly cytosolic, therefore extending our previous studies which indicate that GSK-3beta excludes Nrf2 from the nucleus. Lithium, a GSK-3beta inhibitor, promoted Nrf2 transcriptional activity towards an Antioxidant-Response-Element (ARE) luciferase reporter and cooperated with sulforaphane (SFN) to induce this reporter and to increase the protein levels of heme oxygenase-1 (HO-1), coded by a representative ARE-containing gene. Conversely, ARE activation by SFN was attenuated by over-expression of active GSK-3beta. Finally, combined treatment with SFN and lithium attenuated oxidative stress and cell death in kainate-treated hippocampal slices of wild type mice but not Nrf2 null littermates. Our findings identify the axis GSK-3beta/Nrf2 as a pharmacological target in prevention of excitotoxic neuronal death.

Tacripyrines, the first tacrine-dihydropyridine hybrids, as multitarget-directed ligands for the treatment of Alzheimer's disease.

Tacripyrines (1-14) have been designed by combining an AChE inhibitor (tacrine) with a calcium antagonist such as nimodipine and are targeted to develop a multitarget therapeutic strategy to confront AD. Tacripyrines are selective and potent AChE inhibitors in the nanomolar range. The mixed type inhibition of hAChE activity of compound 11 (IC(50) 105 +/- 15 nM) is associated to a 30.7 +/- 8.6% inhibition of the proaggregating action of AChE on the Abeta and a moderate inhibition of Abeta self-aggregation (34.9 +/- 5.4%). Molecular modeling indicates that binding of compound 11 to the AChE PAS mainly involves the (R)-11 enantiomer, which also agrees with the noncompetitive inhibition mechanism exhibited by p-methoxytacripyrine 11. Tacripyrines are neuroprotective agents, show moderate Ca(2+) channel blocking effect, and cross the blood-brain barrier, emerging as lead candidates for treating AD.

Combined nimodipine and citicoline reduce infarct size, attenuate apoptosis and increase bcl-2 expression after focal cerebral ischemia

Cerebral ischemia triggers a multitude of pathophysiological and biochemical events that separately affect the evolution of focal ischemia and, therefore, stroke treatment should logically employ all known neuroprotective agents. We hypothesized that a treatment combining nimodipine and citicoline might have a potential neuroprotective effect. To assess this idea, Sprague-Dawley rats underwent transient bilateral common carotid artery ligation with simultaneous middle cerebral artery occlusion for 60 min. Four treatment groups were established. Animals received either: a) saline (control group); b) intracarotid nimodipine infusion during 30 min in the ischemia-reperfusion (nimodipine group); c) i.p. postischemic citicoline injections once daily for 7 days (citicoline group); or d) intracarotid nimodipine bolus during ischemia-reperfusion plus i.p. postichemic citicoline injections (combination group). They were killed after either 7 or 3 days after reperfusion. In the first case, the volume of the infarcted tissue was studied by a stereological procedure and in the second case, in situ end-labeling of nuclear DNA fragmentation (TUNEL) and Bcl-2 expression were employed to determine the level of apoptosis. The infarct volume was significantly reduced in both the nimodipine and the citicoline treatment groups after 7 days of reperfusion; combination of both drugs produced an additive effect. After 3 days of reperfusion, the number of Bcl-2-positive neurons was significantly increased while that of TUNEL-positive cells significantly decreased at the infarct border in the combined-treatment animals. Our findings demonstrate a neuroprotective effect from an acute single dose of nimodipine during ischemia-reperfusion and prolonged post-ischemic treatment with citicoline in a model of focal cerebral ischemia. These results suggest that a possible mechanism of neuroprotective action would be mediated by increased Bcl-2 expression and decreased apoptosis within the boundary zone of the infarct together with neutralization of the ischemia-reperfusion injury.