Mitochondrial sensitivity and altered calcium handling underlie enhanced NMDA-induced apoptosis in YAC128 model of Huntington's disease.

Expansion of a CAG repeat in the Huntington's disease (HD) gene results in progressive neuronal loss, particularly of striatal medium-sized spiny neurons (MSNs). Studies in human HD autopsy brain tissue, as well as cellular and animal models of HD, suggest that increased activity of NMDA-type glutamate receptors and altered mitochondrial function contribute to selective neuronal degeneration. In this regard, the YAC128 mouse model, expressing full-length human huntingtin with 128 glutamine repeats, has been the focus of much interest.

Potentiation of NMDA receptor-mediated excitotoxicity linked with intrinsic apoptotic pathway in YAC transgenic mouse model of Huntington's disease.

Evidence suggests N-methyl-D-aspartate receptor (NMDAR) activation is involved in the degeneration of striatal medium-sized spiny neurons (MSNs) in Huntington's disease (HD). We tested the hypothesis that enhanced NMDAR-mediated excitotoxicity is mediated by the mitochondrial-associated apoptotic pathway in cultured MSNs from YAC transgenic mice expressing full-length huntingtin (htt) with a polyglutamine (polyQ) expansion of 46 or 72 (YAC46 or YAC72). NMDAR-mediated Ca(2+) transients and mitochondrial membrane depolarization were significantly increased in YAC compared to wild-type mice MSNs.

Effects of calcium buffers and calbindin-D28k upon histamine-induced calcium oscillations and calcium waves in HeLa cells.

The effects of the artificial Ca(2+) buffers EGTA and BAPTA upon histamine-induced Ca(2+) oscillations and calcium waves were studied in HeLa cells. These events were also examined in HeLa cell lines transfected with the intracellular calcium-binding protein calbindin-D28k (CaBP; HeLa-CaBP) or the pCINeo vector alone (HeLa-pCINeo). High concentrations of the Ca(2+) indicators fluo-3 and fura-2 significantly influenced the oscillatory pattern of intracellular Ca(2+) in HeLa-pCINeo cells exposed to 1 microM histamine.

Functional NMDA receptor subtype 2B is expressed in astrocytes after ischemia in vivo and anoxia in vitro.

NMDA-type glutamate receptors play a critical role in neuronal synaptogenesis, plasticity, and excitotoxic death. Recent studies indicate that functional NMDA receptors are also expressed in certain glial populations in the normal brain. Using immunohistochemical methods, we detected the presence of the NMDA receptor 2B (NR2B) subunit of the NMDA receptor in neurons but not astrocytes in the CA1 and subicular regions of the rat hippocampus.

Calcium buffering and protection from excitotoxic cell death by exogenous calbindin-D28k in HEK 293 cells.

Calbindin-D28k (CaBP) is a calcium-binding protein found in specific neuronal populations in the mammalian brain that, as a result of its proposed calcium-buffering action, may protect neurons against potentially harmful increases in intracellular calcium. We have stably transfected HEK 293 cells with recombinant human CaBP in order to determine the influence of this protein upon transient increases in intracellular ionic calcium concentration ([Ca(2+)](i)) induced either by transient transfection of the NR1 and NR2A subunits of the N-methyl-D-aspartate (NMDA) receptor and brief exposure to glutamate, photolysis of the caged calcium compound NP-EGTA, or exposure to the Ca(2+)]-ionophore 4-Br-A23187. The presence of CaBP did not significantly reduce the peak [Ca(2+)](i)stimulated by glutamate activation of NMDA receptors but significantly prolonged the recovery to baseline values.

Anoxia-evoked intracellular pH and Ca2+ concentration changes in cultured postnatal rat hippocampal neurons.

The ratiometric indicators 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein and Fura-2 were employed to examine, respectively, intracellular pH (pHi) and calcium ([Ca2+]i) changes evoked by anoxia in cultured postnatal rat hippocampal neurons at 37 degrees C. Under both HCO3-/CO2- and HEPES-buffered conditions, 3-, 5- or 10-min anoxia induced a triphasic change in pHi consisting of an initial fall in pHi, a subsequent rise in pHi in the continued absence of O2 and, finally, a further rise in pHi upon the return to normoxia, which recovered towards preanoxic steady-state pHi values if the duration of the anoxic insult was < or = 5 min. In parallel experiments performed on sister cultures, anoxia of 3, 5 or 10 min duration evoked rises in [Ca2+]i which, in all cases, commenced after the start of the fall in pHi, reached a peak at or just following the return to normoxia and then declined towards preanoxic resting levels.

Inhibition of calcium-dependent NMDA receptor current rundown by calbindin-D28k.

NMDA receptors are regulated by several different calcium-dependent processes. To determine if the presence of the intracellular calcium-binding protein calbindin-D28k can influence the calcium regulation of NMDA receptor activity, human embryonic kidney 293 cells were co-transfected with cDNAs for NMDA receptor subunits and calbindin. Recordings were made using the nystatin perforated patch technique to preserve intracellular contents.

Calcium homeostatic mechanisms operating in cultured postnatal rat hippocampal neurones following flash photolysis of nitrophenyl-EGTA.

1. We examined Ca2+ homeostatic mechanisms in cultured postnatal rat hippocampal neurones by monitoring the recovery of background-subtracted fluo-3 fluorescence levels at 20-22 degrees C immediately following a rapid increase in Ca2+ levels induced by flash photolysis of the caged Ca2+ compound nitrophenyl-EGTA (NP-EGTA). 2.

The effects of artificial calcium buffers on calcium responses and glutamate-mediated excitotoxicity in cultured hippocampal neurons.

After loading cultured rat hippocampal neurons with teh acetoxymethyl ester of the Ca2+ buffer BAPTA, or its dimethyl analogue DMB, the magnitudes of transient (20-25 s) depolarization- or excitatory amino acid-induced Ca2+ responses were reduced, as were the rates of increase and recovery of [Ca2+]i. In contrast, during prolonged (3-30 min) stimulation, the magnitudes of the Ca2+ responses were not reduced in buffer-loaded neurons, even though the rates of increase and recovery were still much slower compared to neurons loaded with the control molecule half-BAPTA-AM. The potential consequences of this action of BAPTA and DMB were then examined in an in vitro model of excitotoxicity in which we found that, in both fetal and postnatal cultures, glutamate-induced excitotoxicity was enhanced, rather than reduced.

Enteropathogenic Escherichia coli markedly decreases the resting membrane potential of Caco-2 and HeLa human epithelial cells.

It is presumed, but not proven, that enteropathogenic Escherichia coli (EPEC) causes secretory diarrhea by altering ion transport in enterocytes. In this study we used the whole-cell, current clamp variant of the patch clamp technique to demonstrate that EPEC infection of HeLa and Caco-2 human epithelial cells reduces cell resting membrane potential. The observed reduction of resting membrane potential in HeLa cells results from EPEC-mediated signal transduction to the host cell but is not dependent upon EPEC-mediated elevation of levels of intracellular free calcium.

Correlation of anoxic neuronal responses and calbindin-D28k localization in stratum pyramidale of rat hippocampus.

Immunohistochemical staining for the calcium-binding protein calbindin-D28k (CaBP) was combined with Lucifer Yellow (LY) identification and intracellular recording of changes in membrane parameters of pyramidal neurons in CA2, CA1, and the subiculum of rat hippocampal slices during brief exposure (4.0 +/- 0.19 min) to N2.

Local circuit neurons immunoreactive for calretinin, calbindin D-28k or parvalbumin in monkey prefrontal cortex: distribution and morphology.

In the cerebral cortex, local circuit neurons provide critical inhibitory control over the activity of pyramidal neurons, the major class of excitatory efferent cortical cells. The calcium-binding proteins, calretinin, calbindin, and parvalbumin, are expressed in a variety of cortical local circuit neurons. However, in the primate prefrontal cortex, relatively little is known, especially with regard to calretinin, about the specific classes or distribution of local circuit neurons that contain these calcium-binding proteins.

Cytosolic free Ca2+ in human syncytiotrophoblast cells increased by gonadotropin-releasing hormone.

Effects of GnRH on free cytosolic Ca2+ concentrations ([Ca2+]i) were examined in individual first trimester human cytotrophoblast and syncytiotrophoblast cells by fura-2 microspectrofluorimetry. GnRH (10(-6) M) did not affect [Ca2+]i in cytotrophoblasts on days 2-9 of culture, with 50 cells tested each day. GnRH (10(-6) M) did not affect [Ca2+]i on days 2-3, but increased [Ca2+]i in 15% of culture-derived syncytiotrophoblasts on day 4 (8 of 52 cells) and in 48% on days 5-9 of culture (158 of 332 cells).

Relative loss of the striatal striosome compartment, defined by calbindin-D28k immunostaining, following developmental hypoxic-ischemic injury.

The striatum is especially vulnerable to hypoxic-ischemic injury, both in adulthood and during development. Striatal injury is likely to play a major role in the chronic abnormalities of motor control which occur as a consequence of developmental hypoxia-ischemia. Previous studies have shown that two striatal neuron phenotypes, cholinergic and NADPH-diaphorase-positive, are resistant to developmental hypoxia-ischemia, but little is otherwise known of patterns of vulnerability among other striatal neurons.

Parvalbumin and calbindin D-28k in the human motor system and in motor neuron disease.

Calbindin D-28k and parvalbumin are neuronal calcium binding proteins of interest in relation to neurodegenerative diseases. Expression of calbindin and parvalbumin may be one of the determinants of selective vulnerability in these disorders. The distribution of these proteins was surveyed in the normal human motor system and in motor neuron disease (MND) using immunocytochemistry in formalin fixed post-mortem tissues.

Changes in parvalbumin-immunoreactive neurons in the rat hippocampus following a kainic acid lesion.

Changes in a sub-population of hippocampal non-pyramidal neurons following a unilateral lesion with kainic acid were examined using an antibody raised against the Ca-binding protein parvalbumin. A loss of 71-97% of the parvalbumin-immunoreactive neurons occurred at the three post-lesion times studied (1, 2 and 4 weeks) in all areas of the ipsilateral hippocampus, but no such loss was observed in the dentate gyrus. Resistant parvalbumin-immunoreactive neurons occurred principally in stratum pyramidale and displayed altered morphology from the normal with swollen dendrites and dendritic varicosities.

Divergent differentiation of rat adrenocortical cells is associated with an interruption of angiotensin II-mediated signal transduction.

The zones of the adrenal cortex contain distinct populations of cells which share a common developmental origin and steroidogenic template. In the rat, zona glomerulosa cells respond to angiotensin II (Ang II) with increased steroidogenesis while zona fasciculata/reticularis cells do not. We have examined Ang II-mediated signal transduction in homogeneous cellular sub-populations derived from either the zona glomerulosa (GLOM) or the zona fasciculata (FASC).

Calbindin-D28K (CaBP28k)-like Immunoreactivity in Ascending Projections.

This study concerns the involvement of calbindin-D28K (CaBP28k)-containing neurons in ascending spinal projections to the brainstem (nucleus of the solitary tract, lateral reticular nucleus area), pontine (parabrachial area) and mesencephalic (periaqueductal grey) structures. All these central structures are important in the processing of visceroception and visceronociception and all are targets for spinal efferents from similar areas. CaBP28k controls the excitability of cells by acting on intrinsic calcium metabolism.

Calbindin-D28K (CaBP28k)-like Immunoreactivity in Ascending Projections.

This study concerns the involvement of calbindin-D28K (CaBP28k)-containing neurons in the efferent projections of both the trigeminal nucleus caudalis and the dorsal vagal complex (nucleus of the solitary tract and area postrema) in rats. Recent evidence has shown that these projections are particularly important for the processing of visceroception and/or nociception at central levels. The trigeminal nucleus caudalis has dense projections to both the nucleus of the solitary tract and the parabrachial area; the dorsal vagal complex is intimately connected to the parabrachial area.

Intracellular calcium and the signaling mechanism of luteinizing hormone-releasing hormone in rat granulosa cells.

Objective: The purpose of these studies was to determine the source(s) of the increase in intracellular free calcium in response to luteinizing hormone-releasing hormone in ovarian granulosa cells.

Study Design: Rat granulosa cells were cultured and loaded with fura-2-acetoxy-methyl ester, a fluorescent calcium indicator dye, and intracellular free calcium measured by microspectrofluorometry. The source of the luteinizing hormone-releasing hormone induced increase in intracellular Ca++ was investigated with various calcium channel blockers (verapamil, diltiazem, and nifedipine), high K+ buffer, and perifusion with media lacking Ca++.

Calcium-binding proteins in the nervous system.

Among the many calcium-binding proteins in the nervous system, parvalbumin, calbindin-D28K and calretinin are particularly striking in their abundance and in the specificity of their distribution. They can be found in different subsets of neurons in many brain regions. Although it is not yet known whether they play a 'triggering' role like calmodulin, or merely act as buffers to modulate cytosolic calcium transients, they are valuable markers of neuronal subpopulations for anatomical and developmental studies.

Glucose- and acetylcholine-induced increase in intracellular free Ca2+ in subpopulations of individual rat pancreatic beta-cells.

The effects of glucose and acetylcholine (ACh) on the intracellular free Ca2+ ion concentration ([Ca2+]i) were measured using fura-2 microspectrofluorimetry in individual rat pancreatic beta-cells prepared by enzymatic digestion and fluorescence-activated cell sorting. The average [Ca2+]i was 139 +/- 2.2 nM (n = 84) in the presence of 4.

Calbindin D-28k and parvalbumin immunohistochemistry in developing rat retina.

The developmental profiles of two calcium-binding proteins, calbindin-D28k (CaBP) and parvalbumin (PV), were investigated immunohistochemically in the developing rat retina. CaBP-immunoreactivity appeared first on embryonic day 17 in the horizontal, amacrine and ganglion cells; on embryonic day 21 in the inner plexiform layer; and on post-natal day 6 in the outer plexiform layer. The reaction intensity had increased to its maximum level by post-natal day 10.

Cytosolic free calcium increased by prostaglandin F2 alpha (PGF2 alpha), gonadotropin-releasing hormone, and angiotensin II in rat granulosa cells and PGF2 alpha in human granulosa cells.

Cytosolic [Ca2+]i was measured using a microspectrofluorimetric technique. Prostaglandin F2 alpha (PGF2 alpha, 10(-6) M) transiently increased the concentration of free cytosolic Ca2+ ([Ca2+]i) in individual rat and human granulosa cells. In a study examining a total of 170 individual rat and human granulosa cells, approximately 100% of rat granulosa cells and 80% of human granulosa cells tested responded to PGF2 alpha (10(-6) M).