Intercellular calcium waves in primary cultured rat mesenteric smooth muscle cells are mediated by connexin43.

Intercellular Ca(2+) wave propagation between vascular smooth muscle cells (SMCs) is associated with the propagation of contraction along the vessel. Here, we characterize the involvement of gap junctions (GJs) in Ca(2+) wave propagation between SMCs at the cellular level. Gap junctional communication was assessed by the propagation of intercellular Ca(2+) waves and the transfer of Lucifer Yellow in A7r5 cells, primary rat mesenteric SMCs (pSMCs), and 6B5N cells, a clone of A7r5 cells expressing higher connexin43 (Cx43) to Cx40 ratio. Mechanical stimulation induced an intracellular Ca(2+) wave in pSMC and 6B5N cells that propagated to neighboring cells, whereas Ca(2+) waves in A7r5 cells failed to progress to neighboring cells. We demonstrate that Cx43 forms the functional GJs that are involved in mediating intercellular Ca(2+) waves and that co-expression of Cx40 with Cx43, depending on their expression ratio, may interfere with Cx43 GJ formation, thus altering junctional communication.

Innervation of putative rapidly adapting mechanoreceptors by calbindin- and calretinin-immunoreactive primary sensory neurons in the rat.

Calbindin and calretinin are two homologous calcium-binding proteins that are expressed by subpopulations of primary sensory neurons. In the present work, we have studied the distribution of the neurons expressing calbindin and calretinin in dorsal root ganglia of the rat and their peripheral projections. Calbindin and calretinin immunoreactivities were expressed by subpopulations of large- and small-sized primary sensory neurons and colocalized in a majority of large-sized ones. The axons emerging from calbindin- or calretinin-immunoreactive neurons innervated muscle spindles, Pacini corpuscles and subepidermal lamellar corpuscles in the glabrous skin, formed palisades of lanceolate endings around hairs and vibrissae, and gave rise to intraepidermal nerve endings in the digital skin. Since most of these afferents are considered as rapidly adapting mechanoreceptors, it is concluded that calbindin- or calretinin-expressing neurons innervate particular mechanoreceptors that display physiological characteristics of rapid adaptation to stimuli.

Glutamine synthesis rate in the hyperammonaemic rat brain using simultaneous localized in vivo H-1 and N-15 MRS

Objectives: Glutamine synthetase is a critical step in the glutamate-glutamine cycle, the major mechanism of glutamate neurotransmission and is implicated in the mechanism of ammonia toxicity. 15N MRS is an alternative approach to 13C MRS in studying glutamate- glutamine metabolism. 15N MRS studies allow to measure an apparent glutamine synthesis rate (Vsyn) which reflects a combination of the glutamate- glutamine cycle activity (Vnt) and net glutamine accumulation. The net glutamine synthesis (Vsyn-Vnt) can be directly measured from 1H NMR. Therefore, the aim of this study was to perform in vivo localized 1H MRS interleaved with 15N MRS to directly measure the net glutamine synthesis rate and the apparent glutamine synthesis rate under 15N labeled ammonia infusion in the rat brain, respectively. Methods: 1H and 15N MRS data were acquired interleaved on a 9.4T system (Varian/Magnex Scientific) using 5 rats. 15NH4Cl solution was infused continuously into the femoral vein for up to 10 h (4.5 mmol/h/kg).1 The plasma ammonia concentration was increased to 0.95±0.08 mmol/L (Analox GM7 analyzer). 1H spectra were acquired and quantified as described previously.2 15N unlocalized and localized spectra were acquired using the sequence;3 and quantified using AMARES and an external reference method.4 The metabolic model used to analyze the total Gln and 5-15N labeled Gln time courses is shown on Figure 1A. Results: Glutamine concentration increased from 2.5±0.3 to 15±3.3 mmol/kg whereas the total glutamate concentrations remained unchanged (Figure 1B). The linear fit of the time-evolution of the total Gln from the 1H spectra gave the net synthesis flux (Vsyn-Vnt), which was 0.021± 0.006 mmol/min per g (Figure 1D). The 5-15N Gln peak (_271 ppm) was visible in the first and all subsequent scans, whereas the 2-15N Gln/Glu peak (_342 ppm) appeared after B1.5 h (Figure 1C). From the in vivo 5-15N Gln time course, Vsyn = 0.29±0.1 mmol/min per g and a plasma NH3 fractional enrichment of 71%±6% were calculated. Vnt was 0.26±0.1 mmol/min/g, obtained assuming a negligible Gln efflux.5 Vsyn and Vnt were within the range of 13C NMR measurements.6 Conclusion: The combination of 1H and 15N NMR allowed for the first time a direct and localized measurement of Vnt and apparent glutamine synthesis rate. Vnt is approximately one order of magnitude faster than the net glutamine accumulation.

Gene expression profiles in rat mesenteric lymph nodes upon supplementation with Conjugated Linoleic Acid during gestation and suckling

Background Diet plays a role on the development of the immune system, and polyunsaturated fatty acids can modulate the expression of a variety of genes. Human milk contains conjugated linoleic acid (CLA), a fatty acid that seems to contribute to immune development. Indeed, recent studies carried out in our group in suckling animals have shown that the immune function is enhanced after feeding them with an 80:20 isomer mix composed of c9,t11 and t10,c12 CLA. However, little work has been done on the effects of CLA on gene expression, and even less regarding immune system development in early life. Results The expression profile of mesenteric lymph nodes from animals supplemented with CLA during gestation and suckling through dam's milk (Group A) or by oral gavage (Group B), supplemented just during suckling (Group C) and control animals (Group D) was determined with the aid of the specific GeneChip® Rat Genome 230 2.0 (Affymettrix). Bioinformatics analyses were performed using the GeneSpring GX software package v10.0.2 and lead to the identification of 89 genes differentially expressed in all three dietary approaches. Generation of a biological association network evidenced several genes, such as connective tissue growth factor (Ctgf), tissue inhibitor of metalloproteinase 1 (Timp1), galanin (Gal), synaptotagmin 1 (Syt1), growth factor receptor bound protein 2 (Grb2), actin gamma 2 (Actg2) and smooth muscle alpha actin (Acta2), as highly interconnected nodes of the resulting network. Gene underexpression was confirmed by Real-Time RT-PCR. Conclusions Ctgf, Timp1, Gal and Syt1, among others, are genes modulated by CLA supplementation that may have a role on mucosal immune responses in early life.

Lack of hypotriglyceridemic effect of gemfibrozil as a consequence of age-related changes in rat liver PPARalpha.

We have investigated if changes in hepatic lipid metabolism produced by old age are related to changes in liver peroxisome proliferator-activated receptor alpha (PPARalpha). Our results indicate that 18-month-old rats showed a marked decrease in the expression and activity of liver PPARalpha, as shown by significant reductions in PPARalpha mRNA, protein and binding activity, resulting in a reduction in the relative mRNA levels of PPARalpha target genes, such as liver-carnitine-palmitoyl transferase-I (CPT-I) and mitochondrial medium-chain acyl-CoA dehydrogenase (MCAD). Further, in accordance with a liver PPARalpha deficiency in old rats, treatment of old animals with a therapeutic dose of gemfibrozil (GFB) (3mg/kg per day, 21 days) was ineffective in reducing plasma triglyceride concentrations (TG), despite attaining a 50% reduction in TG when GFB was administered to young animals at the same dose and length of treatment. We hypothesize that the decrease in hepatic PPARalpha can be related to a state of leptin resistance present in old animals.

The auditory pathway in cat corpus callosum.

The cortical auditory fields of the two hemispheres are interconnected via the corpus callosum. We have investigated the topographical arrangement of auditory callosal axons in the cat. Following circumscribed biocytin injections in the primary (AI), secondary (AII), anterior (AAF) and posterior (PAF) auditory fields, labelled axons have been found in the posterior two-thirds of the corpus callosum. Callosal axons labelled by small individual cortical injections did not form a tight bundle at the callosal midsagittal plane but spread over as much as one-third of the corpus callosum. Axons originating from different auditory fields were roughly topographically ordered, reflecting to some extent the rostro-caudal position of the field of origin. Axons from AAF crossed on average more rostrally than axons from AI; the latter crossed more rostrally than axons from PAF and AII. Callosal axons originating in a discrete part of the cortex travelled first in a relatively tight bundle to the telo-diencephalic junction and then dispersed progressively. In conclusion, the cat corpus callosum does not contain a sector reserved for auditory axons, nor a strictly topographically ordered auditory pathway. This observation is of relevance to neuropsychological and neuropathological observations in man.

Comparative genomics of the vertebrate insulin/TOR signal transduction pathway: A network-level analysis of selective pressures

Complexity of biological function relies on large networks of interacting molecules. However, the evolutionary properties of these networks are not fully understood. It has been shown that selective pressures depend on the position of genes in the network. We have previously shown that in the Drosophila insulin/target of rapamycin (TOR) signal transduction pathway there is a correlation between the pathway position and the strength of purifying selection, with the downstream genes being most constrained. In this study, we investigated the evolutionary dynamics of this well-characterized pathway in vertebrates. More specifically, we determined the impact of natural selection on the evolution of 72 genes of this pathway. We found that in vertebrates there is a similar gradient of selective constraint in the insulin/TOR pathway to that found in Drosophila. This feature is neither the result of a polarity in the impact of positive selection nor of a series of factors affecting selective constraint levels (gene expression level and breadth, codon bias, protein length, and connectivity). We also found that pathway genes encoding physically interacting proteins tend to evolve under similar selective constraints. The results indicate that the architecture of the vertebrate insulin/TOR pathway constrains the molecular evolution of its components. Therefore, the polarity detected in Drosophila is neither specific nor incidental of this genus. Hence, although the underlying biological mechanisms remain unclear, these may be similar in both vertebrates and Drosophila.

Role of salt-inducible kinase 1 in the activation of MEF2-dependent transcription by BDNF.

Substantial evidence supports a role for myocyte enhancer factor 2 (MEF2)-mediated transcription in neuronal survival, differentiation and synaptic function. In developing neurons, it has been shown that MEF2-dependent transcription is regulated by neurotrophins. Despite these observations, little is known about the cellular mechanisms by which neurotrophins activate MEF2 transcriptional activity. In this study, we examined the role of salt-inducible kinase 1 (SIK1), a member of the AMP-activated protein kinase (AMPK) family, in the regulation of MEF2-mediated transcription by the neurotrophin brain-derived neurotrophic factor (BDNF). We show that BDNF increases the expression of SIK1 in primary cultures of rat cortical neurons through the extracellular signal-regulated kinase 1/2 (ERK1/2)-signaling pathway. In addition to inducing SIK1 expression, BDNF triggers the phosphorylation of SIK1 at Thr182 and its translocation from the cytoplasm to the nucleus of cortical neurons. The effects of BDNF on the expression, phosphorylation and, translocation of SIK1 are followed by the phosphorylation and nuclear export of histone deacetylase 5 (HDAC5). Blockade of SIK activity with a low concentration of staurosporine abolished BDNF-induced phosphorylation and nuclear export of HDAC5 in cortical neurons. Importantly, stimulation of HDAC5 phosphorylation and nuclear export by BDNF is accompanied by the activation of MEF2-mediated transcription, an effect that is suppressed by staurosporine. Consistent with these data, BDNF induces the expression of the MEF2 target genes Arc and Nur77, in a staurosporine-sensitive manner. In further support of the role of SIK1 in the regulation of MEF2-dependent transcription by BDNF, we found that expression of wild-type SIK1 or S577A SIK1, a mutated form of SIK1 which is retained in the nucleus of transfected cells, is sufficient to enhance MEF2 transcriptional activity in cortical neurons. Together, these data identify a previously unrecognized mechanism by which SIK1 mediates the activation of MEF2-dependent transcription by BDNF.

Circadian clock-coordinated 12 Hr period rhythmic activation of the IRE1alpha pathway controls lipid metabolism in mouse liver.

The mammalian circadian clock plays a fundamental role in the liver by regulating fatty acid, glucose, and xenobiotic metabolism. Impairment of this rhythm has been shown to lead to diverse pathologies, including metabolic syndrome. Currently, it is supposed that the circadian clock regulates metabolism mostly by regulating expression of liver enzymes at the transcriptional level. Here, we show that the circadian clock also controls hepatic metabolism by synchronizing a secondary 12 hr period rhythm characterized by rhythmic activation of the IRE1alpha pathway in the endoplasmic reticulum. The absence of circadian clock perturbs this secondary clock and provokes deregulation of endoplasmic reticulum-localized enzymes. This leads to impaired lipid metabolism, resulting in aberrant activation of the sterol-regulated SREBP transcription factors. The resulting aberrant circadian lipid metabolism in mice devoid of the circadian clock could be involved in the appearance of the associated metabolic syndrome.

Defining the level of evidence for technology adoption in the localized prostate cancer pathway.

New technologies in prostate cancer are attempting to change the current prostate cancer pathway by aiming to reduce harms while maintaining the benefits associated with screening, diagnosis, and treatment. In this article, we discuss the optimal evaluation that new technologies should undergo to provide level 1 evidence typically required to change the practice. With this in mind, we focus on feasible and pragmatic trials that could be delivered in a timely fashion by many centers while retaining primary outcomes that focus on clinically meaningful outcomes.

Myelination in rat brain aggregating cell cultures.

Aggregates of fetal rat brain were maintained in rotating culture for 30-40 days and were analyzed morphologically and biochemically. At 4 days in culture all cells were undifferentiated. At 26 days in vitro over 90% of all cells within the aggregates could be identified as neurons, astrocytes or oligodendrocytes. Myelinated axons and morphologically mature synapses were present at 26 days. Myelination started between 18 and 19 days in culture as determined biochemically. Myelin basic protein sulphatide synthesis and 2′,3′-cyclic nucleotide 3′-phosphohydrolase activity increased with in vitro age. The amount of myelin observed within the aggregates was much lower than observed at the corresponding age in vivo. Neurons and neuronal processes were undergoing severe degeneration in the 40-day aggregates and synaptic contacts were not maintained. There were no normal myelinated axons at 40 days although multilammellar membranes were found intra- and extracellularly. The ganglioside pattern of the aggregates were qualitatively similar to rat whole brain. Quantitatively the GM3ganglioside was elevated in comparison to whole rat brain. Our results indicate that aggregating rat brain cultures provide a useful in vitro system for the biochemical and morphological analysis of myelin formation.

Effect of manganese chloride on the neurochemical profile of the rat hypothalamus.

Manganese (Mn(2+))-enhanced magnetic resonance imaging studies of the neuronal pathways of the hypothalamus showed that information about the regulation of food intake and energy balance circulate through specific hypothalamic nuclei. The dehydration-induced anorexia (DIA) model demonstrated to be appropriate for studying the hypothalamus with Mn(2+)-enhanced magnetic resonance imaging. Manganese is involved in the normal functioning of a variety of physiological processes and is associated with enzymes contributing to neurotransmitter synthesis and metabolism. It also induces psychiatric and motor disturbances. The molecular mechanisms by which Mn(2+) produces alterations of the hypothalamic physiological processes are not well understood. (1)H-magnetic resonance spectroscopy measurements of the rodent hypothalamus are challenging due to the distant location of the hypothalamus resulting in limited measurement sensitivity. The present study proposed to investigate the effects of Mn(2+) on the neurochemical profile of the hypothalamus in normal, DIA, and overnight fasted female rats at 14.1 T. Results provide evidence that γ-aminobutyric acid has an essential role in the maintenance of energy homeostasis in the hypothalamus but is not condition specific. On the contrary, glutamine, glutamate, and taurine appear to respond more accurately to Mn(2+) exposure. An increase in glutamine levels could also be a characteristic response of the hypothalamus to DIA.

Effect of chronic ethanol feeding on rat hepatocytic glutathione. Compartmentation, efflux, and response to incubation with ethanol.

Hepatocytes from rats that were fed ethanol chronically for 6-8 wk were found to have a modest decrease in cytosolic GSH (24%) and a marked decrease in mitochondrial GSH (65%) as compared with pair-fed controls. Incubation of hepatocytes from ethanol-fed rats for 4 h in modified Fisher's medium revealed a greater absolute and fractional GSH efflux rate than controls with maintenance of constant cellular GSH, indicating increased net GSH synthesis. Inhibition of gamma-glutamyltransferase had no effect on these results, which indicates that no degradation of GSH had occurred during these studies. Enhanced fractional efflux was also noted in the perfused livers from ethanol-fed rats. Incubation of hepatocytes in medium containing up to 50 mM ethanol had no effect on cellular GSH, accumulation of GSH in the medium, or cell viability. Thus, chronic ethanol feeding causes a modest fall in cytosolic and a marked fall in mitochondrial GSH. Fractional GSH efflux and therefore synthesis are increased under basal conditions by chronic ethanol feeding, whereas the cellular concentration of GSH drops to a lower steady state level. Incubation of hepatocytes with ethanol indicates that it has no direct, acute effect on hepatic GSH homeostasis.