Norbert Weiss

Overexpression of cellular glutathione peroxidase rescues homocyst(e)ine-induced endothelial dysfunction

Homocyst(e)ine (Hcy) inhibits the expression of the antioxidant enzyme cellular glutathione peroxidase (GPx-1) in vitro and in vivo, which can lead to an increase in reactive oxygen species that inactivate NO and promote endothelial dysfunction. In this study, we tested the hypothesis that overexpression of GPx-1 can restore the normal endothelial phenotype in hyperhomocyst(e)inemic states. Heterozygous cystathionine β-synthase-deficient (CBS(−/+)) mice and their wild-type littermates (CBS(+/+)) were crossbred with mice that overexpress GPx-1 [GPx-1(tg+) mice]. GPx-1 activity was 28% lower in CBS(−/+)/GPx-1(tg−) compared with CBS(+/+)/GPx-1(tg−) mice (P < 0.05), and CBS(−/+) and CBS(+/+) mice overexpressing GPx-1 had 1.5-fold higher GPx-1 activity compared with GPx-1 nontransgenic mice (P < 0.05). Mesenteric arterioles of CBS(−/+)/GPx-1(tg−) mice showed vasoconstriction to superfusion with β-methacholine and bradykinin (P < 0.001 vs. all other groups), whereas nonhyperhomocyst(e)inemic mice [CBS(+/+)/GPx-1(tg−) and CBS(+/+)/GPx-1(tg+) mice] demonstrated dose-dependent vasodilation in response to both agonists. Overexpression of GPx-1 in hyperhomocyst(e)inemic mice restored the normal endothelium-dependent vasodilator response. Bovine aortic endothelial cells (BAEC) were transiently transfected with GPx-1 and incubated with dl-homocysteine (HcyH) or l-cysteine. HcyH incubation decreased GPx-1 activity in sham-transfected BAEC (P < 0.005) but not in GPx-1-transfected cells. Nitric oxide release from BAEC was significantly decreased by HcyH but not cysteine, and GPx-1 overexpression attenuated this decrease. These findings demonstrate that overexpression of GPx-1 can compensate for the adverse effects of Hcy on endothelial function and suggest that the adverse vascular effects of Hcy are at least partly mediated by oxidative inactivation of NO.

Endothelial dysfunction and atherothrombosis in mild hyperhomocysteinemia

Mildly elevated plasma homocysteine levels are an independent risk factor for atherothrombotic vascular disease in the coronary, cerebrovascular, and peripheral arterial circulation. Endothelial dysfunction as manifested by impaired endothelium-dependent regulation of vascular tone and blood flow, by increased recruitment and adhesion of circulating inflammatory cells to the endothelium, and by a loss of endothelial cell antithrombotic function contributes to the vascular disorders linked to hyperhomocysteinemia. Increased vascular oxidant stress through imbalanced thiol redox status and inhibition of important antioxidant enzymes by homocysteine results in decreased bioavailability of the endothelium-derived signaling molecule nitric oxide via oxidative inactivation. This plays a central role in the molecular mechanisms underlying the effects of homocysteine on endothelial function. Supplementation of folic acid and vitamin B12 has been demonstrated to be efficient in lowering mildly elevated plasma homocysteine levels and in reversing homocysteine-induced impairment of endothelium-dependent vasoreactivity. Results from ongoing intervention trials will determine whether homocysteine-lowering therapies contribute to the prevention and reduction of atherothrombotic vascular disease and may thereby provide support for the causal relationship between hyperhomocysteinemia and atherothrombosis.

Influence of Hyperhomocysteinemia on the Cellular Redox State - Impact on Homocysteine-Induced Endothelial Dysfunction

Abstract Hyperhomocysteinemia is an independent risk factor for the development of atherosclerosis. An increasing body of evidence has implicated oxidative stress as being contributory to homocysteines deleterious effects on the vasculature. Elevated levels of homocysteine may lead to increased generation of superoxide by a biochemical mechanism involving nitric oxide synthase, and, to a lesser extent, by an increase in the chemical oxidation of homocysteine and other aminothiols in the circulation. The resultant increase in superoxide levels is further amplified by homocysteinedependent alterations in the function of cellular antioxidant enzymes such as cellular glutathione peroxidase or extracellular superoxide dismutase. One direct clinical consequence of elevated vascular superoxide levels is the inactivation of the vasorelaxant messenger nitric oxide, leading to endothelial dysfunction. Scavenging of superoxide anion by either superoxide dismutase or 4,5-dihydroxybenzene 1,3-disulfonate (Tiron) reverses endothelial dysfunction in hyperhomocysteinemic animal models and in isolated aortic rings incubated with homocysteine. Similarly, homocysteine-induced endothelial dysfunction is also reversed by increasing the concentration of the endogenous antioxidant glutathione or overexpressing cellular glutathione peroxidase in animal models of mild hyperhomocysteinemia. Taken together, these findings strongly suggest that the adverse vascular effects of homocysteine are at least partly mediated by oxidative inactivation of nitric oxide.

Transient Receptor Potential Canonical Type 1 (TRPC1) Operates as a Sarcoplasmic Reticulum Calcium Leak Channel in Skeletal Muscle

Extensive studies performed in nonexcitable cells and expression systems have shown that type 1 transient receptor potential canonical (TRPC1) channels operate mainly in plasma membranes and open through phospholipase C-dependent processes, membrane stretch, or depletion of Ca2+ stores. In skeletal muscle, it is proposed that TRPC1 channels are involved in plasmalemmal Ca2+ influx and stimulated by store depletion or membrane stretch, but direct evidence for TRPC1 sarcolemmal channel activity is not available. We investigated here the functional role of TRPC1 using an overexpressing strategy in adult mouse muscle fibers. Immunostaining for endogenous TRPC1 revealed a striated expression pattern that matched sarcoplasmic reticulum (SR) Ca2+ pump immunolabeling. In cells expressing TRPC1-yellow fluorescent protein (YFP), the same pattern of expression was observed, compatible with a longitudinal SR localization. Resting electric properties, action potentials, and resting divalent cation influx were not altered in TRPC1-YFP-positive cells. Poisoning with the SR Ca2+ pump blocker cyclopiazonic acid elicited a contracture of the fiber at the level of the overexpression site in presence and absence of external Ca2+ which was not observed in control cells. Ca2+ measurements indicated that resting Ca2+ and the rate of Ca2+ increase induced by cyclopiazonic acid were higher in the TRPC1-YFP-positive zone than in the TRPC1-YFP-negative zone and control cells. Ca2+ transients evoked by 200-ms voltage clamp pulses decayed slower in TRPC1-YFP-positive cells. In contrast to previous hypotheses, these data demonstrate that TRPC1 operates as a SR Ca2+ leak channel in skeletal muscle.

Design of a disulfide-less, pharmacologically inert, and chemically competent analog of maurocalcine for the efficient transport of impermeant compounds into cells

Maurocalcine is a 33-mer peptide initially isolated from the venom of a Tunisian scorpion. It has proved itself valuable as a pharmacological activator of the ryanodine receptor and has helped the understanding of the molecular basis underlying excitation-contraction coupling in skeletal muscles. Because of its positively charged nature, it is also an innovative vector for the cell penetration of various compounds. We report a novel maurocalcine analog with improved properties: (i) the complete loss of pharmacological activity, (ii) preservation of the potent ability to carry cargo molecules into cells, and (iii) coupling chemistries not affected by the presence of internal cysteine residues of maurocalcine. We did this by replacing the six internal cysteine residues of maurocalcine by isosteric 2-aminobutyric acid residues and by adding an additional N-terminal biotinylated lysine (for a proof of concept analog) or an N-terminal cysteine residue (for a chemically competent coupling analogue). Additional replacement of a glutamate residue by alanyl at position 12 further improves the potency of these analogues. Coupling to several cargo molecules or nanoparticles are presented to illustrate the cell penetration potency and usefulness of these pharmacologically inactive analogs.

Determination of Total Homocysteine in Human Plasma by Isocratic High-Performance Liquid Chromatography

A simple, sensitive and precise isocratic HPLC method for the determination of total homocysteine in human plasma is described. The thiol compounds were liberated from plasma proteins by reduction with tri-n-butylphosphine and derivatized with a thiol-specific fluorogenic marker, 7-fluoro-benzo-2-oxa-1,3-diazole-4-sulphonate. The derivatives were separated isocratically within 7 min by reversed-phase HPLC using a Superspher 100 RP-18 column as stationary phase. By using this approach more than 200 samples a day can be assayed for total homocysteine. The method was linear up to 100 mumol/l and proved to be sensitive with a detection limit of 0.1 mumol/l and the lowest limit of reliable quantification of 0.5 mumol/l for homocysteine in buffer. Intra- and inter-assay coefficients of variation were both < 4% at a concentration of 10 mumol/l homocysteine. Similar results were obtained for homocysteine concentrations between 0.5 and 100 mumol/l. The analytical recovery for these concentrations ranged from 94.9 to 117.0%. As compared to other protocols published so far, this modified method is less complicated but equally sensitive and reproducible and allows a rapid determination of total homocysteine and cysteine in human plasma under routine conditions.

[Voltage-dependent calcium channels at the heart of pain perception]

Voltage-dependent calcium channels represent a major pathway of calcium entry into neurons, where they participate actively to cell excitability and to the molecular processes of synaptic transmission. For that reason, they have been the direct or indirect pharmacological targets of analgesics and this long before their implication in the physiology of nociception had been demonstrated. These last years, the still more refined molecular characterization of these channels and their associated regulatory subunits and the demonstration of their implication in nociceptive processes indicates that these structures are prime pharmacological targets for the management of pain. Herein, we detail the recent breakthroughs on calcium channel structure, function and pharmacology, review the implication of calcium channels in the transmission of nociception, and evaluate their importance as targets for the treatment of pain perception. The search for specific inhibitors of voltage-dependent calcium channels appears as a prelude to the development of new promising analgesic molecules.

Reply to Tajeddine et al.: TRPC1: Subcellular Localization?

This is a response to a letter by Tajeddine et al. (1). Tajeddine et al. (1) dispute the localization of TRPC1 in skeletal muscle sarcoplasmic reticulum (SR) by evoking non-specificity of the anti-TRPC1 Chemicon antibody we used in our study to localize TRPC1 (2). They show that immunolabeling of muscle fibers with this antibody gave the same striated intracellular pattern of expression in wild-type and TRPC1-deficient mice. It would have been more convincing if the authors were able to show a total extinction of TRPC1 labeling in TRPC1−/− mouse fibers using the antibodies they utilized in their previous studies (3, 4). Yet, we obtained a similar striated pattern of expression using a TRPC1 antibody purchased from Sigma in wild-type muscle (see Fig. 1). Our main concern is that the consequence of the knock-out strategy on TRPC1 protein translation is not ascertained by Dietrich et al. (5) who published the knock-out. Four ATG codons are present downstream from the stop codon in position 2 of exon 9, generated by targeted disruption of TRPC1 mRNA, which can all represent putative novel start codons. The sequence downstream of these four start codons is in frame with the encoding region of TRPC1. Therefore, in the absence of any biochemical characterization in TRPC1−/− muscle (5, 6), it cannot be excluded that truncated TRPC1 proteins translated from one of these initiation codons accumulate. Since the Chemicon antibody epitope (QLYDKGYTSKEQKDC) sits downstream from the four starting methionines, the production of these truncated proteins could explain the positive signal observed by Tajeddine et al. (1) in TRPC1−/− mouse muscle. Without further characterization of TRPC1−/− mouse muscles, data obtained by Tajeddine et al. (1) do not convincingly challenge localization of TRPC1 in the longitudinal SR, which is moreover confirmed by the localization and the functional activity of the transfected TRPC1-YFP (2). Fig. 1.