Mercedes Villarroya

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.

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.

Neuroprotective and Cholinergic Properties of Multifunctional Glutamic Acid Derivatives for the Treatment of Alzheimer’s Disease

Novel multifunctional compounds have been designed, synthesized, and evaluated as potential drugs for the treatment of Alzheimers disease (AD). With an L-glutamic moiety as a suitable biocompatible linker, three pharmacophoric groups were joined: (1) an N-benzylpiperidine fragment selected to inhibit acetylcholinesterase by interacting with the catalytic active site (CAS), (2) an N-protecting group of the amino acid, capable of interacting with the acetylcholinesterase (AChE)-peripheral anionic site (PAS) and protecting neurons against oxidative stress, and (3) a lipophilic alkyl ester that would facilitate penetration into the central nervous system by crossing the blood-brain barrier. At submicromolar concentration, they inhibit AChE and butyrylcholinesterase (BuChE) of human origin, displace the binding of propidium iodide from the PAS of AChE, and could thus inhibit Abeta aggregation promoted by AChE. They also display neuroprotective properties against mitochondrial free radicals, show low toxicity, and could be able to penetrate into the CNS.

The nicotinic acetylcholine receptor of the bovine chromaffin cell, a new target for dihydropyridines.

The effects of 1,4-dihydropyridine derivatives on divalent cation transients and catecholamine release stimulated by either high K+ or the nicotinic receptor agonist dimethyl-phenyl-piperazinium (DMPP) have been compared in bovine adrenal chromaffin cells. The activation of Ca2+ entry pathways was followed by measuring 45Ca2+ or Mn2+ uptake, or by the changes of [Ca2+]i in fura-2-loaded chromaffin cells. Various dihydropyridine Ca2+ channel blockers (nimodipine, PCA50938, nifedipine, nitrendipine, furnidipine) abolished the DMPP-mediated effects, but prevented only partially the activation by high [K+]0 of 45Ca2+ uptake. The IC50 for DMPP-induced activation was around 1 microM. The L-type Ca2+ channel activator Bay K 8644 potentiated the uptake of 45Ca2+ induced by K+ depolarization at concentrations between 10 nM and 1 microM, but completely inhibited the uptake of 45Ca2+ by DMPP (IC50, 0.9 microM). Both high [K+]0 and DMPP produced membrane depolarization as measured using bis-oxonol. The DMPP-evoked, but not the K(+)-evoked membrane depolarization was prevented by Na+ removal, suggesting that the depolarization was due to Na+ entry through the acetylcholine receptor ionophore. Nimodipine at 10 microM abolished the depolarization induced by DMPP, leaving the K(+)-evoked depolarization unaffected. Tetrodotoxin (2 microM) did not affect the DMPP- or high K(+)-mediated cell depolarization. Whole-cell inward current evoked by 100 microM DMPP (IDMPP) was measured in cells voltage-clamped at -80 mV. Nimodipine (10 microM) reduced IDMPP by 36%; Bay K 8644 (10 microM) inhibited IDMPP by 67%. DMPP-evoked catecholamine release from superfused chromaffin cells was reduced by over 90% with 10 microM nimodipine; in contrast, K(+)-evoked release was decreased by 20%.(ABSTRACT TRUNCATED AT 250 WORDS)

Chondroitin Sulfate Protects SH-SY5Y Cells from Oxidative Stress by Inducing Heme Oxygenase-1 via Phosphatidylinositol 3-Kinase/Akt

We investigated the mechanism of the neuroprotective properties of chondroitin sulfate (CS), an endogenous perineuronal net glycosaminoglycan, in human neuroblastoma SH-SY5Y cells subjected to oxidative stress. Preincubation with CS for 24 h afforded concentration-dependent protection against H2O2-induced toxicity (50 μM for 24 h) measured as lactic dehydrogenase released to the incubation media; cell death was prevented at the concentrations of 600 and 1000 μM. Cell death caused by a combination of 10 μM rotenone plus 1 μM oligomycin-A (Rot/oligo) was also reduced by CS at concentrations ranging from 0.3 to 100 μM; in this toxicity model, maximum protection was achieved at 3 μM (48%). No significant protection was observed in a cell death model of Ca2+ overload (70 mM K+, for 24 h). H2O2 and Rot/oligo generated reactive oxygen species (ROS) measured as an increase in the fluorescence of dichlorofluorescein diacetate-loaded cells. CS drastically reduced ROS generation induced by both H2O2 (extracellular ROS) and Rot/oligo (intracellular ROS). CS also increased the expression of phosphorylated Akt and heme oxygenase-1 by 2-fold. The protective effects of CS were prevented by chelerythrine, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), cycloheximide, and Sn(IV)-protoporphyrin IX. Taken together, these results show that CS can protect SH-SY5Y cells under oxidative stress conditions by activating protein kinase C, which phosphorylates Akt that, via the phosphatidylinositol 3-kinase/Akt pathway, induces the synthesis of the antioxidant protein heme oxygenase-1.

Endothelium-independent relaxation by 17-α-estradiol of pig coronary arteries

We have studied the effects of 17-α-estradiol, a non-estrogenic steroid, on pig coronary arteries contracted by K+, Ca2+ or serotonin. Experiments were performed on helicoidal strips and rings of left circumflex coronary arteries from freshly slaughtered white pigs and on helicoidal strips of rat thoracic aorta. The strips and rings were suspended inside a water-jacketed muscle chamber in an oxygenated Krebs solution at 37°C. From the initial K+-evoked contraction, 17-α-estradiol caused a relaxation with an IC50 (15 μM) which was in the range of the IC50s obtained for nitroglycerin (1.3 μM) and nicorandil (50 μM). Contractions evoked by Ca2+ were inhibited by 17-α-estradiol, but full blockade could not be achieved. Serotonin-evoked contractions were also blocked by 17-α-estradiol with an IC50 of 2.1 μM; 17-β-estradiol also inhibited the serotonin-evoked contractions. In the presence of 100 μM of the nitric oxide synthase inhibitor N-nitro-L-arginine methyl ester, the relaxing properties of 17-α-estradiol in pig coronary arteries and rat thoracic aorta were unaffected, suggesting that endothelial NO release was unrelated to these effects. 17-α-Estradiol also relaxed denuded pig coronary artery strips, suggesting that other endothelial-derived relaxing factors were not involved in its vascular effects. The results are compatible with the idea that 17-α-estradiol causes relaxation of coronary vessels by acting directly on the cell membrane of smooth muscle cells; these effects seem to be unrelated to the genomic physiological effects of estrogens. These acute vasorelaxant effects can best be explained by blockade of voltage-dependent Ca2+ channels and the ensuing restriction of extracellular Ca2+ availability to the contractile machinery. This is in line with the recent hypothesis that estrogens behave as ‘endogenous Ca2+ channel antagonists’.

Voltage inactivation of Ca2+ entry and secretion associated with N‐ and P/Q‐type but not L‐type Ca2+ channels of bovine chromaffin cells

1 In this study we pose the question of why the bovine adrenal medullary chromaffin cell needs various subtypes (L, N, P, Q) of the neuronal high‐voltage activated Ca2+ channels to control a given physiological function, i.e. the exocytotic release of catecholamines. One plausible hypothesis is that Ca2+ channel subtypes undergo different patterns of inactivation during cell depolarization. 2 The net Ca2+ uptake (measured using 45Ca2+) into hyperpolarized cells (bathed in a nominally Ca2+‐free solution containing 1·2 mM K+) after application of a Ca2+ pulse (5 s exposure to 100 mM K+ and 2 mM Ca2+), amounted to 0·65 ± 0·02 fmol cell−1; in depolarized cells (bathed in nominally Ca2+‐free solution containing 100 mM K+) the net Ca2+ uptake was 0·16 ± 0·01 fmol cell−1. 3 This was paralleled by a dramatic reduction of the increase in the cytosolic Ca2+ concentration, [Ca2+]i, caused by Ca2+ pulses applied to fura‐2‐loaded single cells, from 1181 ± 104 nM in hyperpolarized cells to 115 ± 9 nM in depolarized cells. 4 A similar decrease was observed when studying catecholamine release. Secretion was decreased when K+ concentration was increased from 1·2 to 100 mM; the Ca2+ pulse caused, when comparing the extreme conditions, the secretion of 807 ± 35 nA of catecholamines in hyperpolarized cells and 220 ± 19 nA in depolarized cells. 5 The inactivation by depolarization of Ca2+ entry and secretion occluded the blocking effects of combined ω‐conotoxin GVIA (1 μM) and ω‐agatoxin IVA (2 μM), thus suggesting that depolarization caused a selective inactivation of the N‐ and P/Q‐type Ca2+ channels. 6 This was strengthened by two additional findings: (i) nifedipine (3 μM), an L‐type Ca2+ channel blocker, suppressed the fraction of Ca2+ entry (24 %) and secretion (27 %) left unblocked by depolarization; (ii) FPL64176 (3 μM), an L‐type Ca2+ channel ‘activator’, dramatically enhanced the entry of Ca2+ and the secretory response in depolarized cells. 7 In voltage‐clamped cells, switching the holding potential from ‐80 to ‐40 mV promoted the loss of 80 % of the whole‐cell inward Ca2+ channel current carried by 10 mM Ba2+ (IBa). The residual current was blocked by 80 % upon addition of 3 μM nifedipine and dramatically enhanced by 3 μM FPL64176. 8 Thus, it seems that the N‐ and P/Q‐subtypes of calcium channels are more prone to inactivation at depolarizing voltages than the L‐subtype. We propose that this different inactivation might occur physiologically during different patterns of action potential firing, triggered by endogenously released acetylcholine under various stressful conditions.