Attenuation of MPTP-induced dopaminergic neurotoxicity by TV3326, a cholinesterase-monoamine oxidase inhibitor

(R)-[(N-propargyl-(3R) aminoindan-5-yl) ethyl methyl carbamate] (TV3326) is a novel cholinesterase and brain-selective monoamine oxidase (MAO)-A/-B inhibitor. It was developed for the treatment of dementia co-morbid with extra pyramidal disorders (parkinsonism), and depression. On chronic treatment in mice it attenuated striatal dopamine depletion induced by MPTP and prevented the reduction in striatal tyrosine hydroxylase activity, like selective B and non-selective MAO inhibitors. TV3326 preferentially inhibits MAO-B in the striatum and hippocampus, and the degree of MAO-B inhibition correlates with the prevention of MPTP-induced dopamine depletion. Complete inhibition of MAO-B is not necessary for full protection from MPTP neurotoxicity. Unlike that seen after treatment with other MAO-A and -B inhibitors, recovery of striatal and hippocampal MAO-A and -B activities from inhibition by TV3326 did not show first-order kinetics. This has been attributed to the generation of a number of metabolites by TV3326 that cause differential inhibition of these enzymes. Inhibition of brain MAO-A and -B by TV3326 resulted in significant elevations of dopamine, noradrenaline and serotonin in the striatum and hippocampus. This may explain its antidepressant-like activity, resembling that of moclobemide in the forced-swim test in rats.     

Mitochondrial permeability transition mediates apoptosis induced by N-methyl(R)salsolinol,an endogenous neurotoxin,and is inhibited by Bcl-2 and rasagiline,N-propargyl-1(R)-aminoindan

The role of mitochondrial permeability transition (PT) in apoptosis induced by an endogenous neurotoxin, N-methyl(R)salsolinol [NM(R)Sal], was studied by use of dopaminergic neuroblastoma SH-SY5Y cells. NM(R)Sal reduced mitochondrial membrane potential, DeltaPsim, in the early phase of apoptosis, which was not suppressed by a pan-caspase inhibitor, but was antagonized by Bcl-2 and cyclosporin A, suggesting the involvement of the PT in NM(R)Sal-induced loss of DeltaPsim. NM(R)Sal-induced apoptosis was completely inhibited not only by Bcl-2 and a pan-caspase inhibitor, but also by cyclosporin A, suggesting the essential role of the PT in NM(R)Sal-induced apoptosis. In mitochondria isolated from rat liver, NM(R)Sal induced swelling and reduced DeltaPsim, which was inhibited by cyclosporin A and Bcl-2 overexpression. These results indicate that NM(R)Sal induced the PT by direct action on the mitochondria. Rasagiline, N-propargyl-1(R)-aminoindan, which is a now under a clinical trial for Parkinson's disease, suppressed the DeltaPsim reduction, release of cytochrome c, and apoptosis induced by NM(R)Sal in SH-SY5Y cells. Rasagiline also inhibited the NM(R)Sal-induced loss of DeltaPsim and swelling in the isolated mitochondria, proving that rasagiline directly targets the mitochondria also. Altogether, mitochondrial PT plays a key role both in NM(R)Sal-induced cell death and the neuroprotective effect of rasagiline.     

Efficacité et innocuité du prélèvement percutané de sperme testiculaire dans la prise en charge de l’infertilité masculine

Objective

To assess the efficacy and safety of percutaneous testicular biopsy to provide sperm cells for ICSI in male patients with azoospermia not amenable to surgical treatment.

Materials and methods

From October 1995 to December 2001, 175 biopsies were performed in men with azoospermia to provide material for intracytoplasmic sperm injection. Azoospermia was obstructive (OA) in 41 cases and non-obstructive (NOA) in 134 cases. Open biopsy was performed in the first 15 patients in the series and percutaneous biopsy was performed on an outpatient basis, under local anesthesia, with a Biopty Gun® (14G needle), in the subsequent patients as the first step in management. Open surgical biopsies were performed in another 15 patients following a sperm cell-negative percutaneous biopsy.

Results

All biopsies performed for OA were positive, but only 51/134 biopsies (38%) were positive in the NOA group. The material provided by percutaneous biopsy, when positive for sperm cells, was always sufficient to perform ICSI. When percutaneous biopsy was negative, open surgical biopsy failed to give better results. Five men developed minor complications (acute hematocele) following percutaneous biopsies requiring reoperation for hemostasis (3.12%). No major complications were observed. Results were comparable in terms of fertilization and pregnancy rates whether fresh or frozen-thawed sperm was used.

Conclusion

Percutaneous testicular sperm extraction is a safe, well-tolerated and cost-effective procedure in the management of male-factor infertility related to azoospermia.     

A pleckstrin homology domain specific for phosphatidylinositol 4, 5-bisphosphate (PtdIns-4,5-P2) and fused to green fluorescent protein identifies plasma membrane PtdIns-4,5-P2 as being important in exocytosis

Kinetically distinct steps can be distinguished in the secretory response from neuroendocrine cells with slow ATP-dependent priming steps preceding the triggering of exocytosis by Ca(2+). One of these priming steps involves the maintenance of phosphatidylinositol 4, 5-bisphosphate (PtdIns-4,5-P(2)) through lipid kinases and is responsible for at least 70% of the ATP-dependent secretion observed in digitonin-permeabilized chromaffin cells. PtdIns-4,5-P(2) is usually thought to reside on the plasma membrane. However, because phosphatidylinositol 4-kinase is an integral chromaffin granule membrane protein, PtdIns-4,5-P(2) important in exocytosis may reside on the chromaffin granule membrane. In the present study we have investigated the localization of PtdIns-4,5-P(2) that is involved in exocytosis by transiently expressing in chromaffin cells a pleckstrin homology (PH) domain that specifically binds PtdIns-4, 5-P(2) and is fused to green fluorescent protein (GFP). The PH-GFP protein predominantly associated with the plasma membrane in chromaffin cells without any detectable association with chromaffin granules. Rhodamine-neomycin, which also binds to PtdIns-4,5-P(2), showed a similar subcellular localization. The transiently expressed PH-GFP inhibited exocytosis as measured by both biochemical and electrophysiological techniques. The results indicate that the inhibition was at a step after Ca(2+) entry and suggest that plasma membrane PtdIns-4,5-P(2) is important for exocytosis. Expression of PH-GFP also reduced calcium currents, raising the possibility that PtdIns-4,5-P(2) in some manner alters calcium channel function in chromaffin cells.     

Intracellular pH regulation by Na(+)/H(+) exchange requires phosphatidylinositol 4,5-bisphosphate

The carrier-mediated, electroneutral exchange of Na(+) for H(+) across the plasma membrane does not directly consume metabolic energy. Nevertheless, acute depletion of cellular ATP markedly decreases transport. We analyzed the possible involvement of polyphosphoinositides in the metabolic regulation of NHE1, the ubiquitous isoform of the Na(+)/H(+) exchanger. Depletion of ATP was accompanied by a marked reduction of plasmalemmal phosphatidylinositol 4,5-bisphosphate (PIP(2)) content. Moreover, sequestration or hydrolysis of plasmalemmal PIP(2), in the absence of ATP depletion, was associated with profound inhibition of NHE1 activity. Examination of the primary structure of the COOH-terminal domain of NHE1 revealed two potential PIP(2)-binding motifs. Fusion proteins encoding these motifs bound PIP(2) in vitro. When transfected into antiport-deficient cells, mutant forms of NHE1 lacking the putative PIP(2)-binding domains had greatly reduced transport capability, implying that association with PIP(2) is required for optimal activity. These findings suggest that NHE1 activity is modulated by phosphoinositides and that the inhibitory effect of ATP depletion may be attributable, at least in part, to the accompanying net dephosphorylation of PIP(2).     

Visualization of Phosphoinositides That Bind Pleckstrin Homology Domains: Calcium- and Agonist-induced Dynamic Changes and Relationship to Myo-[3H]inositol-labeled Phosphoinositide Pools

Phosphatidylinositol 4,5-bisphosphate (PtdIns[4,5]P₂) pools that bind pleckstrin homology (PH) domains were visualized by cellular expression of a phospholipase C (PLC)δ PH domain–green fluorescent protein fusion construct and analysis of confocal images in living cells. Plasma membrane localization of the fluorescent probe required the presence of three basic residues within the PLCδ PH domain known to form critical contacts with PtdIns(4,5)P₂. Activation of endogenous PLCs by ionophores or by receptor stimulation produced rapid redistribution of the fluorescent signal from the membrane to cytosol, which was reversed after Ca²⁺ chelation. In both ionomycin- and agonist-stimulated cells, fluorescent probe distribution closely correlated with changes in absolute mass of PtdIns(4,5)P₂. Inhibition of PtdIns(4,5)P₂ synthesis by quercetin or phenylarsine oxide prevented the relocalization of the fluorescent probe to the membranes after Ca²⁺ chelation in ionomycin-treated cells or during agonist stimulation. In contrast, the synthesis of the PtdIns(4,5)P₂ imaged by the PH domain was not sensitive to concentrations of wortmannin that had been found inhibitory of the synthesis of myo-[³H]inositol– labeled PtdIns(4,5)P₂. Identification and dynamic imaging of phosphoinositides that interact with PH domains will further our understanding of the regulation of such proteins by inositol phospholipids.     

Supression of hemin-mediated oxidation of low-density lipoprotein and subsequent endothelial reactions by hydrogen sulfide (H2S)

Heme-mediated oxidative modification of low-density lipoprotein (LDL) plays a crucial role in early atherogenesis. It has been shown that hydrogen sulfide (H₂S) produced by vascular smooth muscle cells is present in plasma at a concentration of about 50 µmol/L. H₂S is a strong reductant which can react with reactive oxygen species like superoxide anion and hydrogen peroxide. The current study investigated the effect of H₂S on hemin-mediated oxidation of LDL and oxidized LDL (oxLDL)-induced endothelial reactions. H₂S dose dependently delayed the accumulation of lipid peroxidation products—conjugated dienes, lipid hydroperoxides (LOOH), and thiobarbituric acid reactive substances—during hemin-mediated oxidation. Moreover, H₂S decreased the LOOH content of both oxidized LDL and lipid extracts derived from soft atherosclerotic plaque, which was accompanied by reduced cytotoxicity. OxLDL-mediated induction of the oxidative stress responsive gene, heme oxygenase-1, was also abolished by H₂S. Finally we have shown that H₂S can directly protect endothelium against hydrogen peroxide and oxLDL-mediated endothelial cytotoxicity. These results demonstrate novel functions of H₂S in preventing hemin-mediated oxidative modification of LDL, and consequent deleterious effects, suggesting a possible antiatherogenic action of H₂S.     

New Insights into the Methodology of L-Arginine-Induced Acute Pancreatitis

Animal models are ideal to study the pathomechanism and therapy of acute pancreatitis (AP). The use of L-arginine-induced AP model is nowadays becoming increasingly popular in mice. However, carefully looking through the literature, marked differences in disease severity could be observed. In fact, while setting up the L-arginine (2×4 g/kg i.p.)-induced AP model in BALB/c mice, we found a relatively low rate (around 15%) of pancreatic necrosis, whereas others have detected much higher rates (up to 55%). We suspected that this may be due to differences between mouse strains. We administered various concentrations (5–30%, pH = 7.4) and doses (2×4, 3×3, or 4×2.5 g/kg) of L-arginine-HCl in BALB/c, FVB/n and C57BL/6 mice. The potential gender-specific effect of L-arginine was investigated in C57BL/6 mice. The fate of mice in response to the i.p. injections of L arginine followed one of three courses. Some mice (1) developed severe AP or (2) remained AP-free by 72 h, whereas others (3) had to be euthanized (to avoid their death, which was caused by the high dose of L-arginine and not AP) within 12 h., In FVB/n and C57BL/6 mice, the pancreatic necrosis rate (about 50%) was significantly higher than that observed in BALB/c mice using 2×4 g/kg 10% L–arginine, but euthanasia was necessary in a large proportion of animals, The i.p. injection of lower L-arginine concentrations (e.g. 5–8%) in case of the 2×4 g/kg dose, or other L-arginine doses (3×3 or 4×2.5 g/kg, 10%) were better for inducing AP. We could not detect any significant differences between the AP severity of male and female mice. Taken together, when setting up the L-arginine-induced AP model, there are several important factors that are worth consideration such as the dose and concentration of the administered L arginine-HCl solution and also the strain of mice.     

Measurement of Inositol 1,4,5-Trisphosphate in Living Cells Using an Improved Set of Resonance Energy Transfer-Based Biosensors

Improved versions of inositol-1,4,5-trisphosphate (InsP₃) sensors were created to follow intracellular InsP₃ changes in single living cells and in cell populations. Similar to previous InsP₃ sensors the new sensors are based on the ligand binding domain of the human type-I InsP₃ receptor (InsP₃R-LBD), but contain a mutation of either R265K or R269K to lower their InsP₃ binding affinity. Tagging the InsP₃R-LBD with N-terminal Cerulean and C-terminal Venus allowed measurement of InsP₃ in single-cell FRET experiments. Replacing Cerulean with a Luciferase enzyme allowed experiments in multi-cell format by measuring the change in the BRET signal upon stimulation. These sensors faithfully followed the agonist-induced increase in InsP₃ concentration in HEK 293T cells expressing the Gq-coupled AT1 angiotensin receptor detecting a response to agonist concentration as low as 10 pmol/L. Compared to the wild type InsP₃ sensor, the mutant sensors showed an improved off-rate, enabling a more rapid and complete return of the signal to the resting value of InsP₃ after termination of M3 muscarinic receptor stimulation by atropine. For parallel measurements of intracellular InsP₃ and Ca²⁺ levels in BRET experiments, the Cameleon D3 Ca²⁺ sensor was modified by replacing its CFP with luciferase. In these experiments depletion of plasma membrane PtdIns(4,5)P₂ resulted in the fall of InsP₃ level, followed by the decrease of the Ca²⁺-signal evoked by the stimulation of the AT1 receptor. In contrast, when type-III PI 4-kinases were inhibited with a high concentration of wortmannin or a more specific inhibitor, A1, the decrease of the Ca²⁺-signal preceded the fall of InsP₃ level indicating an InsP₃-, independent, direct regulation of capacitative Ca²⁺ influx by plasma membrane inositol lipids. Taken together, our results indicate that the improved InsP₃ sensor can be used to monitor both the increase and decrease of InsP₃ levels in live cells suitable for high-throughput BRET applications.