Juliana Redondo

Role of lipid peroxidation and the glutathione‐dependent antioxidant system in the impairment of endothelium‐dependent relaxations with age

Age‐related changes in the blood prooxidant‐antioxidant state, as well as its influence on the relaxant responses to acetylcholine (ACh) were studied in the tail artery from 6‐, 24‐ and 30‐month‐old Sprague‐Dawley (SD) rats. Malondialdehyde (MDA) plasma levels increased 2 and 3 times in 24‐ and 30‐month‐old rats, respectively, when compared with 6‐month‐old rats (0.43±0.09 μM). This increase was accompanied by an induction of 6‐phosphogluconate dehydrogenase (6PG‐DH) and glutathione reductase (GR) activities in red blood cells from 24‐month‐old rats. In 30‐month‐old rats, a further induction of these enzymatic activities, as well as glucose‐6‐phosphate dehydrogenase (G6P‐DH) and glutathione peroxidase (GPx) activities was observed. No differences with age were found in the concentration‐response curves to ACh in isolated tail artery segments from 6‐ and 24‐month‐old rats precontracted with 0.3 μM noradrenaline (NA). However, a decrease in sensitivity to ACh‐induced relaxation was observed in 30‐month‐old rats; EC30 values were 3.5 (1.3–8.0)×10−7 M and 18.1 (8.9–30.1)×10−7 M for 6‐ and 30‐month‐old rats, respectively. Moreover, a decrease in maximum ACh relaxation (10 μM) was found in 30‐month‐old rats in comparison with that obtained in 6‐month‐old rats (58.5±3.9% and 42.5±3.4% of previous NA contraction, respectively). Incubation of tail artery segments with MDA (0.5, 1 or 10 μM) caused a reduction of ACh‐induced relaxations that was different in the three ages. Thus, the reduction of ACh‐induced relaxations became significant with 0.5 μM MDA in 6‐, with 1 μM MDA in 24‐, and with 10 μM MDA in 30‐month‐old rats. In addition, MDA did not cause a shift in the concentration‐response curve to ACh, but a decrease in the maximum response. Superoxide dismutase (SOD; 150 u ml−1, a superoxide anion scavenger) reversed the inhibitory effect of MDA on ACh‐induced relaxations at all ages studied. We conclude that: (1) ageing produces an increase in lipid peroxidation process, as indicated by the increase in MDA plasma levels, that is accompanied by an induction of lipid peroxide detoxification enzymes; (2) the changes in prooxidant‐antioxidant equilibrium with age contribute, at least partially, to the impairment of the relaxant responses evoked by ACh; and (3) the effect of MDA appears to be mediated by superoxide anion at all ages studied.

Vascular sodium pump: endothelial modulation and alterations in some pathological processes and aging

The vascular Na+ pump maintains intracellular ionic concentration and controls membrane potential. Its inhibition by cardiac glycosides enhances the intracellular Na+ concentration. This in turn activates the Na+-Ca2+ exchange mechanism, which induces intracellular Ca2+ increase, membrane depolarization, and noradrenaline release from perivascular adrenergic nerve endings; mechanisms that promote vasoconstriction. This article reviews the relevance of the Na+ pump in vascular tone regulation and the modulation of its activity by the endothelium. The endothelium negatively modulates the vasoconstriction elicited by Na+ pump inhibition by the release of nitric oxide, according to some authors, or an unknown factor, as suggested by others. The possible existence of endogenous digitalis-like factors is also reviewed, as is the involvement of the vascular Na+ pump in some cardiovascular disorders and aging.

Purkinje Cell Pathology and Loss in Multiple Sclerosis Cerebellum.

Cerebellar ataxia commonly occurs in multiple sclerosis, particularly in chronic progressive disease. Previous reports have highlighted both white matter and grey matter pathological changes within the cerebellum; and demyelination and inflammatory cell infiltrates appear commonly. As Purkinje cell axons are the sole output of the cerebellar cortex, understanding pathologic processes within these cells is crucial to develop strategies to prevent their loss and thus reduce ataxia. We studied pathologic changes occurring within Purkinje cells of the cerebellum. Using immunohistochemic techniques, we found changes in neurofilament phosphorylation states within Purkinje cells, including loss of dephosphorylated neurofilament and increased phosphorylated and hyperphosphorylated neurofilament. We also found Purkinje axonal spheroids and Purkinje cell loss, both of which occurred predominantly within areas of leucocortical demyelination within the cerebellar cortex. These changes have important implications for the study of cerebellar involvement in multiple sclerosis and may help design therapies to reduce the burden of ataxia in the condition.

Comparison of the vasoconstrictor responses induced by endothelin and phorbol 12,13-dibutyrate in bovine cerebral arteries.

The vascular effects of endothelin-1 (ET-1) were compared with those elicited by phorbol 12,13-dibutyrate (PDB), an activator of the protein kinase C (PKC), to analyze the involvement of this enzyme on ET-1 responses. PDB and ET-1 caused slow-developing contractions (sustained and transient, respectively), which were reduced by the PKC inhibitor, staurosporine (1 and 10 nM). Only the contractile effects evoked by ET-1 were reduced in Ca-free medium and by the Ca channel antagonist, nifedipine (1 microM), and increased by the Ca channel agonist, BAY K 8644 (10 nM). PDB (10 and 30 nM) preincubation reduced the vasoconstriction elicited by 5-hydroxytryptamine (5-HT; 0.01, 0.1 and 1 microM) in a way dependent on phorbol concentration and preincubation time, whereas ET-1 (1 nM) increased the contractile response to 5-HT (0.1 microM). Furthermore, PDB (0.1 microM) also reduced the responses elicited by ET-1 (30 microM) and vice versa. ET-1 (0.1 microM) induced transient translocation of PKC activity from the cytosol to the membrane, which was less than that produced by PDB (0.1 microM). Electrical stimulation induced [3H]noradrenaline (NA) release, which was increased by PDB (10 and 100 nM) and not affected by ET-1 (10 nM). These results indicate: (1) the responses induced by PDB and ET-1 were independent and dependent on extracellular Ca, respectively; (2) PKC is involved in NA release and 5-HT responses, but mainly in desensitization of these responses, and (3) PKC is activated by ET-1 and is implicated in vascular actions of ET-1, but other mechanisms, such as the activation of ET-1 receptors and opening of dihydropyridine-sensitive Ca channels also appear to be involved.

Endothelial Stimulation of Sodium Pump in Cultured Vascular Smooth Muscle

We studied vascular sodium pump activity and its regulation by vasoactive agents and endothelium in cultured aortic vascular smooth muscle cells from normotensive Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). Baseline sodium pump activity (ouabain-inhibitable 86Rb+ uptake) was similar in cells from both rat strains. Angiotensin II and endothelin-1 increased ouabain-inhibitable 86Rb+ uptake more in SHR than WKY cells, whereas no effects were obtained with sodium nitroprusside, 8-bromo-cGMP, or iloprost. We examined the influence of endothelium on vascular sodium pump activity either by coculturing smooth muscle and endothelial cells or by using conditioned medium. Both coculture for 24 hours with endothelial cells and treatment with conditioned medium increased smooth muscle cell sodium pump activity, this effect being higher in SHR cells. These results suggest that the endothelium may modulate sodium pump activity in the underlying smooth muscle by releasing a diffusible compound, which is more active on SHR smooth muscle. The conditioned medium obtained in the presence of inhibitors of angiotensin-converting enzyme, endothelin-1-converting enzyme, cyclooxygenase, lipoxygenase, and nitric oxide synthase had no effect on the ability of conditioned medium to increase sodium pump activity, suggesting that angiotensin II, endothelin-1, eicosanoids, and nitric oxide are not involved in this stimulatory effect. The nature of the possible endothelial factor involved is still unknown, but it possesses a molecular weight between 25 and 50 kD, is heat stable, and is sensitive to trypsin treatment. We propose it could be a growth factor.

Nitric oxide synthase induction by ouabain in vascular smooth muscle cells from normotensive and hypertensive rats.

Objective To investigate the effect of ouabain on inducible nitric oxide synthase (iNOS) activity and expression in cytokine-stimulated vascular smooth muscle cells (VSMC) from normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). Methods VSMC were treated for 24 h and afterwards, nitric oxide (NO) release was determined by the production of nitrite, a stable metabolite of NO. Activity of iNOS was measured by the conversion of [3H]-L-arginine to [3H]-L-citrulline and iNOS protein expression by Western blotting. Results Ouabain (0.01–1 mmol/l) further enhanced interleukin-1β(II-1β)-induced nitrite production by WKY and SHR VSMC, although a more pronounced effect was observed in SHR cells (maximum response 52.1 ± 5.2 and 71.2 ± 6.4% of II-1β effect in WKY and SHR cells, respectively). Such response on NO release was mimicked by the calcium ionophore A 23187 (0.01–1 μmol/l) and abolished by the voltage-operated calcium channels (VOCC) nifedipine (0.1 μmol/l). Expression of iNOS showed that ouabain increased the synthesis of the enzyme in WKY and SHR VSMC stimulated with II-1β, and this effect was higher in SHR cells. The increased iNOS expression was significantly reduced by nifedipine. Conclusions Ouabain stimulation of iNOS expression and activity in Il-1β-stimulated VSMCs from WKY rats and SHR seems to be related to increased intracellular calcium influx through VOCC. The more pronounced effect observed in SHR VSMC could be explained by an altered calcium entry in the hypertensive strain.

Purkinje cell injury, structural plasticity and fusion in patients with Friedreich's ataxia

Purkinje cell pathology is a common finding in a range of inherited and acquired cerebellar disorders, with the degree of Purkinje cell injury dependent on the underlying aetiology. Purkinje cells have an unparalleled resistance to insult and display unique regenerative capabilities within the central nervous system. Their response to cell injury is not typical of most neurons and likely represents both degenerative, compensatory and regenerative mechanisms. Here we present a pathological study showing novel and fundamental insights into Purkinje cell injury, remodelling and repair in Friedreich’s ataxia; the most common inherited ataxia. Analysing post-mortem cerebellum tissue from patients who had Friedreichs ataxia, we provide evidence of significant injury to the Purkinje cell axonal compartment with relative preservation of both the perikaryon and its extensive dendritic arborisation. Axonal remodelling of Purkinje cells was clearly elevated in the disease. For the first time in a genetic condition, we have also shown a disease-related increase in the frequency of Purkinje cell fusion and heterokaryon formation in Friedreichs ataxia cases; with evidence that underlying levels of cerebellar inflammation influence heterokaryon formation. Our results together further demonstrate the Purkinje cell’s unique plasticity and regenerative potential. Elucidating the biological mechanisms behind these phenomena could have significant clinical implications for manipulating neuronal repair in response to neurological injury.

Reductions in neuronal peroxisomes in multiple sclerosis grey matter

Background: Peroxisomes are organelles in eukaryotic cells with multiple functions including the detoxification of reactive oxygen species, plasmalogen synthesis and β-oxidation of fatty acids. Recent evidence has implicated peroxisomal dysfunction in models of multiple sclerosis (MS) disease progression. Objectives: Our aims were to determine whether there are changes in peroxisomes in MS grey matter (GM) compared to control GM. Methods: We analysed cases of MS and control GM immunocytochemically to assess peroxisomal membrane protein (PMP70) and neuronal proteins. We examined the expression of ABCD3 (the gene that encodes PMP70) in MS and control GM. Analyses of very long chain fatty acid (VLCFA) levels in GM were performed. Results: PMP70 immunolabelling of neuronal somata was significantly lower in MS GM compared to control. Calibration of ABCD3 gene expression with reference to glyceraldehyde 3-phsophate dehydrogenase (GAPDH) revealed overall decreases in expression in MS compared to controls. Mean PMP70 counts in involved MS GM negatively correlated to disease duration. Elevations in C26:0 (hexacosanoic acid) were found in MS GM. Conclusions: Collectively, these observations provide evidence that there is an overall reduction in peroxisomal gene expression and peroxisomal proteins in GM neurons in MS. Changes in peroxisomal function may contribute to neuronal dysfunction and degeneration in MS.

Axonal motor protein KIF5A and associated cargo deficits in multiple sclerosis lesional and normal-appearing white matter

Understanding the causes of axonal pathology remains a key goal in the pursuit of new therapies to target disease progression in multiple sclerosis (MS). Anterograde axonal transport of many proteins vital for axonal viability is mediated by the motor protein KIF5A, which has been linked to several neurological diseases. This study aimed to investigate the expression of KIF5A protein and its associated cargoes: amyloid precursor protein (APP) and neurofilament (NF) in post mortem MS and control white matter (WM) and to determine if KIF5A expression is influenced by the presence of MS risk single nucleotide polymorphisms (SNPs) identified in the region of the KIF5A gene.

Reductions in kinesin expression are associated with nitric oxide-induced axonal damage

Axonal injury is often characterized by axonal transport defects and abnormal accumulation of intra‐axonal components. Nitric oxide (NO) has a key role in mediating inflammatory axonopathy in many neurodegenerative diseases, but little is known about how nitrosative/oxidative stress affects axonal transport or whether reductions in kinesin superfamily protein (KIF) expression correlate with axon pathology. KIFs are molecular motors that have a key role in axonal and dendritic transport, and impairment of these mechanisms has been associated with a number of neurological disorders. This study shows that rat cortical neurons exposed to NO display both a time‐dependent decrease in KIF gene/protein expression and neurofilament phosphorylation in addition to a reduction in axonal length and neuronal survival. Because mesenchymal stem cells (MSCs) represent a promising therapeutic candidate for neuronal/axonal repair, this study analyzes the capacity of MSCs to protect neurons and axonal transport mechanisms from NO damage. Results show that coculture of MSCs with NO‐exposed neurons results in the preservation of KIF expression, axonal length, and neuronal survival. Altogether, these results suggest a potential mechanism involved in the disruption of axonal transport and abnormal accumulation of proteins in axons during nitrosative insult. We hypothesize that impaired axonal transport contributes, per se, to progression of injury and provide further evidence of the therapeutic potential of MSCs for neurodegenerative disorders.