981 resultados para rat sciatic nerved K-pNPPase


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We previously showed that 16-day-old rats exposed to a relatively high dose of ethanol at 10-15 postnatal days of age have fewer neurons in the hilus region of the hippocampus compared with controls. Dentate gyrus granule cell numbers, however, showed no statistically significant changes attributable to the ethanol treatment. It is possible that some of the changes in brain morphology, brought about as a result of the exposure to ethanol during early life, may not be manifested until later in life. This question has been further addressed in an extension to our previous study. Wistar rats were exposed to a relatively high daily dose of ethanol on postnatal days 10-15 by placement in a chamber containing ethanol vapour, for 3 h/day. The blood ethanol concentration was found to be similar to430 mg/dl at the end of the period of exposure. Groups of ethanol-treated (ET), separation control (SC), and mother-reared control (MRC) rats were anaesthetised and killed either at 16 or 30 days of age by perfusion with phosphate-buffered 2.5% glutaraldehyde. The Cavalieri principle and the physical disector methods were used to estimate, respectively, the regional volumes and neuron cell numerical densities in the hilus and granule cell regions of the dentate gyrus. The total numbers of neurons in the hilus region and granule cell layer were computed from these estimates. It was found that 16-day-old animals had 398,000-441,000 granule cells, irrespective of group. The numbers of granule cells increased such that by 30 days of age, rats had 487,000-525,500 granule cells. However, there were no significant differences between ethanol-treated rats and their age-matched controls in granule cell numbers. In contrast, ethanol-treated rats had slightly but significantly fewer neurons in the hilus region than did control animals at 16 days of age, but not at 30 days of age. Therefore, it appears that a short period of ethanol exposure during early life can have effects on neuron numbers of some hippocampal neurons, but not others. The effects on hilar neuron numbers, observed as a result of such short periods of ethanol treatment, appeared to be transitory. (C) 2003 Wiley-Liss, Inc.

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Percutaneous transluminal coronary angioplasty is a frequently used interventional technique to reopen arteries that have narrowed because of atherosclerosis. Restenosis, or renarrowing of the artery shortly after angioplasty, is a major limitation to the success of the procedure and is due mainly to smooth muscle cell accumulation in the artery wall at the site of balloon injury. In the present study, we demonstrate that the antiangiogenic sulfated oligosaccharide, PI-88, inhibits primary vascular smooth muscle cell proliferation and reduces intimal thickening 14 days after balloon angioplasty of rat and rabbit arteries. PI-88 reduced heparan sulfate content in the injured artery wall and prevented change in smooth muscle phenotype. However, the mechanism of PI-88 inhibition was not merely confined to the antiheparanase activity of this compound. PI-88 blocked extracellular signal-regulated kinase-1/2 (ERK1/2) activity within minutes of smooth muscle cell injury. It facilitated FGF-2 release from uninjured smooth muscle cells in vitro, and super-released FGF-2 after injury while inhibiting ERK1/2 activation. PI-88 inhibited the decrease in levels of FGF-2 protein in the rat artery wall within 8 minutes of injury. PI-88 also blocked injury-inducible ERK phosphorylation, without altering the clotting time in these animals. Optical biosensor studies revealed that PI-88 potently inhibited (K-i 10.3 nmol/L) the interaction of FGF-2 with heparan sulfate. These findings show for the first time the capacity of this sulfated oligosaccharide to directly bind FGF-2, block cellular signaling and proliferation in vitro, and inhibit injury-induced smooth muscle cell hyperplasia in two animal models. As such, this study demonstrates a new role for PI-88 as an inhibitor of intimal thickening after balloon angioplasty. The full text of this article is available online at http://www.circresaha.org.

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Real-time Taqman(TM) RT-PCR was used to make quantitative comparisons of the levels of PrRP mRNA expression in micropunch brain samples from rats at different stages of the oestrous cycle and in lactation. The nucleus of the solitary tract and ventrolateral reticular nuclei of the medulla oblongata contained significantly (P < 0.05) greater levels of PrRP mRNA than any hypothalamic region. Within the hypothalamus, the highest level of PrRP expression was localised to the dorsomedial aspect of the ventromedial hypothalamus. All other hypothalamic regions exhibited significantly (P < 0.05) lower levels of expression, including the rostral and caudal dorsomedial hypothalamus. Very low levels of PrRP expression were observed in the arcuate nucleus, paraventricular nucleus, medial preoptic nucleus and ventrolateral aspect of the ventromedial hypothalamus. No significant changes in PrRP expression were noted in any sampled region between proestrus, oestrus or dioestrus. Similarly, PrRP expression in hypothalamic regions did not differ between lactating and non-lactating (dioestrous) animals. During validation of RT-PCR techniques we cloned and sequenced a novel splice variant of PrRP from the hypothalamus. This variant arises from alternative splicing of the donor site within exon 2, resulting in an insert of 64 base pairs and shift in the-codon:reading frame with the introduction of an early stop codon. In the hypothalamus and brainstem, mRNA expression of the variant was restricted to regions that expressed PrRP. These results suggest that PrRP expression in the hypothalamus may be more Widespread than previously reported. However, the relatively low level of PrRP in the hypothalamus and the lack of significant changes in expression during the oestrous cycle and lactation provides further evidence that PrRP is unlikely to be involved in the regulation of prolactin, secretion. (C) 2003 Elsevier Science B.V. All rights reserved.

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We are interested in determining whether low maternal vitamin D-3 affects brain development in utero. Whilst the vitamin D receptor (VDR) has been identified in embryonic rat brains, the timing and magnitude of its expression across the brain remains unclear. In this study we have quantitated VDR expression during development as well correlated the timing of its appearance with two vital developmental events, apoptosis and mitosis. Brains from embryonic rats (embryonic days 15-23) were examined. We show that the well-described increase in apoptotic cells and decrease in mitotic cells during development correlates with the appearance of the VDR in brain tissue. Given that vitamin D-3 regulates mitosis and apoptosis in non-neuronal tissue we speculate that the timing of VDR expression in embryonic brain may directly or indirectly mediate features of neuronal apoptosis and mitosis.

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We have previously shown that exposing rats to a relatively high dose of ethanol during early postnatal life can result in an alteration in spatial learning ability. The hippocampal formation is known to be involved in the control of this ability. The purpose of the present study was to determine whether exposure of rats to ethanol during early postnatal life had either immediate or delayed effects on the numbers of pyramidal cells in the CA1-CA3 subregion of the hippocampus. Wistar rats were exposed to a relatively high daily dose of ethanol at postnatal day 10-15 by placing them for 3 h/day in a chamber containing ethanol vapor. Groups of ethanol-treated (ET), separation control (SC), and mother-reared control (MRC) rats were anesthetized and killed at 16 and 30 days of age by perfusion with phosphate-buffered 2.5% glutaraldehyde. The Cavalieri principle was used to determine the volumes of the CA1 and CA2+CA3 regions. The physical disector method was used to estimate the numerical density of neurons in each of the subdivisions. The total number of pyramidal cells was calculated by multiplying the appropriate estimates of the numerical density by the volume. There were significant age-related reductions in the total numbers of pyramidal cells at 16-30 days of age irrespective of the groups examined. Ethanol treated rats were found to have slightly but significantly fewer pyramidal cell neurons than either the MRC or SC groups. These observations indicate that pyramidal cells in the hippocampus may be vulnerable to a relatively high dose of ethanol exposure during this short period of early postnatal life. (C) 2003 Wiley-Liss, Inc.

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A method was developed that allows conversion of changes in maximum Ca2+-dependent fluorescence of a fixed amount of fluo-3 into volume changes of the fluo-3-containing solution. This method was then applied to investigate by confocal microscopy the osmotic properties of the sealed tubular (t-) system of toad and rat mechanically skinned fibers in which a certain amount Of fluo-3 was trapped. When the osmolality of the myoplasmic environment was altered by simple dilution or addition of sucrose within the range 190-638 mosmol kg(-1), the sealed t-system of toad fibers behaved almost like an ideal osmometer, changing its volume inverse proportionally to osmolality However, increasing the osmolality above 638 to 2,550 mosmol kg(-1) caused hardly any change in t-system volume. In myoplasmic solutions made hypotonic to 128 mosmol kg(-1), a loss of Ca2+ from the sealed t-system of toad fibers Occurred, presumably through either stretch-activated cationic channels or store-operated Ca2+ channels. In contrast to the behavior of the t-system in toad fibers, the volume of the sealed t-system of rat fibers changed little (by

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The 19-amino acid conopeptide (rho-TIA) was shown previously to antagonize noncompetitively alpha(1B)-adrenergic receptors (ARs). Because this is the first peptide ligand for these receptors, we compared its interactions with the three recombinant human alpha(1)-AR subtypes (alpha(1A), alpha(1B), and alpha(1D)). Radioligand binding assays showed that rho-TIA was 10-fold selective for human alpha(1B)- over alpha(1A)- and alpha(1D)-ARs. As observed with hamster alpha(1B)-ARs, rho-TIA decreased the number of binding sites (B-max) for human alpha(1B)-ARs without changing affinity (K-D), and this inhibition was unaffected by the length of incubation but was reversed by washing. However, rho-TIA had opposite effects at human alpha(1A)-ARs and alpha(1D)-ARs, decreasing KD without changing Bmax, suggesting it acts competitively at these subtypes. rho-TIA reduced maximal NE-stimulated [H-3] inositol phosphate formation in HEK293 cells expressing human alpha(1B)-ARs but competitively inhibited responses in cells expressing alpha(1A)- or alpha(1D)-ARs. Truncation mutants showed that the amino-terminal domains of alpha(1B)- or alpha(1D)-ARs are not involved in interaction with rho-TIA. Alanine-scanning mutagenesis of rho-TIA showed F18A had an increased selectivity for alpha(1B)-ARs, and F18N also increased subtype selectivity. I8A had a slightly reduced potency at alpha(1B)-ARs and was found to be a competitive, rather than noncompetitive, inhibitor in both radioligand and functional assays. Thus rho-TIA noncompetitively inhibits alpha(1B)-ARs but competitively inhibits the other two subtypes, and this selectivity can be increased by mutation. These differential interactions do not involve the receptor amino termini and are not because of the charged nature of the peptide, and isoleucine 8 is critical for its noncompetitive inhibition at alpha(1B)-ARs.

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Diadromous freshwater shrimps are exposed to brackish water both as an obligatory part of their larval life cycle and during adult reproductive migration; their well-developed osmoregulatory ability is crucial to survival in such habitats. This study examines gill microsomal Na,K-ATPase (K-phosphatase activity) kinetics and protein profiles in the freshwater shrimp Macrobrachium amazonicum when in fresh water and after 10-days of acclimation to brackish water (21 parts per thousand salinity), as well as potential routes of Na(+) uptake across the gill epithelium in fresh water. On acclimation, K-phosphatase activity decreases 2.5-fold, Na,K-ATPase alpha-subunit expression declines, total protein expression pattern is markedly altered, and enzyme activity becomes redistributed into different density membrane fractions, possibly reflecting altered vesicle trafficking between the plasma membrane and intracellular compartments. Ultrastructural analysis reveals an intimately coupled pillar cell-septal cell architecture and shows that the cell membrane interfaces between the external medium and the hemolymph are greatly augmented by apical pillar cell evaginations and septal cell inviginations, respectively. These findings ire discussed regarding the putative movement of Na(+) across the pillar cell interfaces and into the hemolymph via the septal cells, powered by the Na,K-ATPase located in their invaginations. (C) 2008 Elsevier Inc. All rights reserved.

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Statement of the study: Based on data from ecological and analytic epidemiological studies, we have proposed that low prenatal vitamin D is a candidate risk-modifying factor for schizophrenia. Previously, we demonstrated that low prenatal vitamin D adversely affected brain development in neonatal rats (Eyles et al, 2003). Here we examine the impact of both prenatal and early life hypovitaminosis D on various outcomes in the adult rat brain. Methods: Female Sprague-Dawley rats were made vitamin D deficient via the use of a special diet (Dyets CA) and lighting conditions that excluded UVB radiation. Animals were kept under these conditions for 6 weeks then mated with males kept under normal conditions. Vitamin deplete dams were kept under these conditions during pregnancy. Offspring from two test groups were examined. Offspring were either reared with dams repleted with vitamin D at birth or remained under deplete conditions till weaning. Both test groups were weaned under normal vitamin D conditions and remained so till testing at adulthood. We compared the brains of adult offspring kept under both test conditions with animals from control environments. Summary of results: We found a significant persistent dose-related increase in lateral ventricle volume and alterations in anterior cingulate and prefrontal cortical cell densities (consistent with the known prodifferentiation properties of this steroid). In both test groups we observed a reduced expression of NGF as well as a down-regulation of transcripts coding for GABAA alpha 4 receptor and two neuronal structural elements; MAP2 and Neurofilament L. Conclusion: These findings provide further evidence that vitamin D is involved in brain development. An increase in prefrontal cortical cell density, a reduction neuronal structural elements and persistent ventriculomegaly are all common anatomical findings in the brains of patients with schizophrenia. The specific reduction in transcripts for neuronal structural proteins but not GFAP is also in accordance with the proposal that frontal cortical architecture in schizophrenia reflects a reduction in connectivity rather than a reduction in glial processes(Goldman-Rakic and Selemon, 1997). These findings confirm the biological plausibility of early life hypovitaminosis D as a risk factor for schizophrenia.

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The blue crab, Callinectes danae, tolerates exposure to a wide salinity range employing mechanisms of compensatory ion uptake when in dilute media. Although the gill (Na(+), K(+))-ATPase is vital to hyperosmoregulatory ability, the interactions occurring at the sites of ATP binding on the molecule itself are unknown. Here, we investigate the modulation by Na(+) and K(+) of homotropic interactions between the ATP-binding sites, and of phosphoenzyme formation of the (Na(+),K(+))-ATPase from the posterior gills of this euryhaline crab. The contribution of the high- and low-affinity ATP-binding sites to maximum velocity was similar for both Na(+) and K(+). However, in contrast to Na(+), a threshold K(+) concentration triggers the appearance of the high-affinity binding sites, displacing the saturation curve to lower ATP concentrations. Further, a low-affinity site for phosphorylation is present on the enzyme. These findings reveal notable differences in the catalytic mechanism of the crustacean (Na(+),K(+))-ATPase compared to the vertebrate enzyme. (C) 2008 Elsevier Inc. All rights reserved.

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Different stoichiometries are observed between alpha and beta subunits of Na,K-ATPase that depend on the method employed to solubilize and purify the enzyme. It is not known whether this variability is due to loss of protein-protein association, or is a result of the replacement of essential phospholipids by detergent molecules. With the aim of understanding the effect of enzyme/surfactant ratio on both the catalytic activity and the enzyme structure, we have investigated the bulk and surface properties of the enzyme. The circular dichroism (CD) spectra, surface tension and dilatational surface elasticity results were compared with the residual ATPase activity of the Na,K-ATPase in different surfactant and protein concentrations. Na,K-ATPase in the (alpha beta)(2) form dissociated to the alpha beta form on dilution, and associated to the (alpha beta)(4) form when concentrated. These different stoichiometries have similar ATPase activities and are in equilibrium at C(12)E(8) concentrations below the CIVIC (0.053 mg mL(-1)). At detergent concentrations above the CIVIC the ATPase activity of all forms was abolished, which is concomitant with the dissociation of the a and subunits. (C) 2008 Elsevier Inc. All rights reserved.

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We have characterized the kinetic properties of ectonucleoside triphosphate diphosphohydrolase 1 (E-NTPDase1) from rat osseous plate membranes. A novel finding of the present study is that the solubilized enzyme shows high- and low-affinity sites for the substrate in contrast with a single substrate site for the membrane-bound enzyme. In addition, contrary to the Michaelian chraracteristics of the membrane-bound enzyme, the site-site interactions after solubilization with 0.5% digitonin plus 0.1% lysolecithin resulted in a less active ectonucleoside triphosphate diphosphohydrolase, showing activity of about 398.3 nmol Pi min(-1) mg(-1). The solubilized enzyme has M(r) of 66-72 kDa, and its catalytic efficiency was significantly increased by magnesium and calcium ions; but the ATP/ADP activity ratio was always < 2.0. Partial purification and kinetic characterization of the rat osseous plate E-NTPDase1 in a solubilized form may lead to a better understanding of a possible function of the enzyme as a modulator of nucleotidase activity or purinergic signaling in matrix vesicle membranes. The simple procedure to obtain the enzyme in a solubilized form may also be attractive for comparative studies of particular features of the active sites from this and other ATPases.

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A glycosylphosphatidylinositol (GPI)-anchored enzyme (rat osseous plate alkaline phosphatase-OAP) was studied as monolayer (pure and mixed with lipids) at the air-water interface. Surface pressure and surface potential-area isotherms showed that the enzyme forms a stable monolayer and exhibits a liquid-expanded state even at surface pressure as high as 30 mN m(-1). Isotherms for mixed dimyristoylphosphatidic acid (DMPA)-OAP monolayer showed the absence of a liquid-expanded/liquid-condensed phase transition as observed for pure DMPA monolayer. In both cases, pure or mixed monolayer, the enzyme preserves its native conformation under compression at the air-water interface as observed from in situ p-polarized light Fourier transform-infrared reflection-absorption spectroscopic (FT-IRRAS) measurements. Changes in orientation and conformation of the enzyme due to the presence or absence of DMPA, as well as due to the surface compression, are discussed. (C) 2008 Published by Elsevier Inc.