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PubMed1996

Slice cultures of the imprinting-relevant forebrain area medio-rostral neostriatum/hyperstriatum ventrale of the domestic chick: immunocytochemical characterization of neurons containing Ca(2+)-binding proteins.

The forebrain area medio-rostral neostriatum/hyperstriatum ventrale, a presumed analogue to the mammalian prefrontal cortex, displays a variety of synaptic changes during auditory filial imprinting. In order to study the underlying basic mechanisms of this synaptic plasticity we developed slice cultures of the medio-rostral neostriatum/hyperstriatum ventrale from newly hatched chicks. As a prerequisite for these investigations and in order to test the suitability of this system for future studies, we performed a thorough characterization of the in vitro tissue, of its cellular components and some of their biochemical features in comparison with in situ tissue. Since in situ the medio-rostral neostriatum/hyperstriatum ventrale has been previously shown to contain three distinct neuron populations characterized by the activity-regulated Ca(2+)-binding proteins parvalbumin, calbindin D28K and calretinin, we used these proteins as neuronal markers to study the survival and preservation of the morphological features of medio-rostral neostriatum/hyperstriatum ventrale neurons in vitro. In agreement with in vivo studies the three Ca(2+)-binding proteins are confined to neuronal cells and they are not colocalized, i.e. they appear to characterize three different neuron populations. The immunoreactive neurons in medio-rostral neostriatum/hyperstriatum ventrale cultures to a certain extent appear to form synaptic contacts with each other, shown by the double immuncytochemical experiments. One difference between cells in vivo and in vitro is their soma size, which is much larger in vitro than in vivo. This and our previous study on neuronal morphology demonstrates that morphologically and biochemically intact neurons can be maintained in medio-rostral neostriatum/hyperstriatum ventrale slice cultures, which may thus provide a suitable in vitro system for further studies of neuronal and synaptic plasticity in vitro.

Animals↗
PubMed2005

The epithelial Ca2+ channel TRPV5 is essential for proper osteoclastic bone resorption.

Bone remodeling involves the interplay of bone resorption and formation and is accurately controlled to maintain bone mass. Both processes require transcellular Ca(2+) transport, but the molecular mechanisms engaged remain largely elusive. The epithelial Ca(2+) channel TRPV5 is one of the most Ca(2+)-selective transient receptor potential (TRP) channels. In this study, the functional role of TRPV5 in bone was investigated. TRPV5 mRNA was expressed in human and murine bone samples and in osteoclasts along with other genes involved in transcellular Ca(2+) transport, including calbindin-D(9K) and calbindin-D(28K), Na(+)/Ca(2+) exchanger 1, and plasma membrane Ca(2+)-ATPase 1b. TRPV5 expression in murine osteoclasts was confirmed by immunostaining and showed predominant localization to the ruffled border membrane. However, TRPV5 was absent in osteoblasts. Analyses of femoral bone sections from TRPV5 knockout (TRPV5(-/-)) mice revealed increased osteoclast numbers and osteoclast area, whereas the urinary bone resorption marker deoxypyridinoline was reduced compared with WT (TRPV5(+/+)) mice. In an in vitro bone marrow culture system, the amount of osteoclasts and number of nuclei per osteoclast were significantly elevated in TRPV5(-/-) compared with TRPV5(+/+) mice. However, using a functional resorption pit assay, we found that bone resorption was nearly absent in osteoclast cultures from TRPV5(-/-) mice, supporting the impaired resorption observed in vivo. In conclusion, TRPV5 deficiency leads to an increase in osteoclast size and number, in which Ca(2+) resorption is nonfunctional. This report identifies TRPV5 as an epithelial Ca(2+) channel that is essential for osteoclastic bone resorption and demonstrates the significance of transcellular Ca(2+) transport in osteoclastic function.

Acid Phosphatase↗
PubMed1995

Regulation of excitatory transmission at hippocampal synapses by calbindin D28k.

Distinct subpopulations of neurons in the brain contain one or more of the Ca(2+)-binding proteins calbindin D28k, calretinin, and parvalbumin. Although it has been shown that these high-affinity Ca(2+)-binding proteins can increase neuronal Ca2+ buffering capacity, it is not clear which aspects of neuronal physiology they normally regulate. To investigate this problem, we used a recently developed method for expressing calbindin D28k in the somatic and synaptic regions of cultured hippocampal pyramidal neurons. Ninety-six hours after infection with a replication-defective adenovirus containing the calbindin D28k gene, essentially all cultured hippocampal pyramidal neurons robustly expressed calbindin D28k. Our results demonstrate that while calbindin D28k does not alter evoked neurotransmitter release at excitatory pyramidal cell synapses, this protein has a profound effect on synaptic plasticity. In particular, we show that calbindin D28k expression suppresses posttetanic potentiation.

Animals↗
PubMed2001

Ionizing radiation-induced, mitochondria-dependent generation of reactive oxygen/nitrogen.

Transient generation of reactive oxygen or nitrogen (ROS/RNS), detected with dihydrodichlorofluoroscein by fluorescence microscopy, occurs within minutes of exposing cells to ionizing radiation. In the 1-10 Gy dose range, the amount of ROS/RNS produced/cell is constant, but the percentage of producing cells increases with dose (20 to 80%). Reversible depolarization of the mitochondrial membrane potential () and decrease in fluorescence of a mitochondria-entrapped dye, calcein, are observed coincidentally. Radiation-induced ROS/RNS, depolarization, and calcein fluorescence decrease are inhibited by the mitochondrial permeability transition inhibitor, cyclosporin A, but not the structural analogue, cyclosporin H. Radiation-stimulated ROS/RNS is also inhibited by overexpressing the Ca(2+)-binding protein, calbindin 28K, or treating cells with an intracellular Ca(2+) chelator. Radiation-induced ROS/RNS is observed in several cell types with the exception of rho(o) cells deficient in mitochondrial electron transport. rho(o) cells show neither radiation-induced ROS/RNS production nor depolarization. We propose that radiation damage in a few mitochondria is transmitted via a reversible, Ca(2+)-dependent mitochondrial permeability transition to adjacent mitochondria with resulting enhanced ROS/RNS generation. Measurements of radiation-induced mitogen-activated protein kinase activity indicate that this sensing/amplification mechanism is necessary for activation of some cytoplasmic signaling pathways by low doses of radiation.

Animals↗
PubMed1994

Parvalbumin, calbindin D-28k, and calretinin immunoreactivity in the ascending auditory pathway of horseshoe bats.

In the subcortical auditory system of Rhinolophus rouxi, antibodies directed against the calcium-binding proteins parvalbumin, calbindin D-28k, and calretinin yield partly overlapping and partly complementary labeling patterns which are described in detail for each nucleus. The most general features of the labeling patterns are that: 1) Parvalbumin is a potent marker for large and heterogeneous populations of cells and puncta (presumed axon terminals) throughout the auditory pathway. 2) Immunostaining with the monoclonal calbindin-antiserum was typically absent or sparse in most auditory brainstem centers, but prominent in auditory nerve fibers and in cells of the medial geniculate body (MGB). 3) Calretinin label is abundant but more restricted to subsets of auditory nuclei or subpopulations of cells than parvalbumin. 4) Calcium-binding proteins are useful markers to define particular subregions or cell types in auditory nuclei: for example, i) different labeling patterns are obtained within the nuclei of the lateral lemniscus and adjacent tegmental zones; ii) in the inferior colliculus both calbindin- and calretinin-antisera yield similar regional specific staining patterns, but label different cell types; iii) subregions of the medial geniculate body have characteristic profiles of calcium-binding proteins; and iv) analyses of different nuclei showed that there is no simple common denominator for cells characterized by the expression of particular calcium-binding proteins, nor does labeling correspond in a straightforward way with specific functional systems. 5) there are profound differences between the calbindin labeling patterns seen in Rhinolophus and those in other mammals.

Animals↗
PubMed1993

A functional and degenerate pair of EF hands contains the very high affinity calcium-binding site of calbindin-D28K.

Calbindin-D28K, a member of the troponin C superfamily of calcium-binding proteins, had six putative EF hand domains containing one very high affinity and two to three lower affinity calcium-binding sites. The location and binding activity of the calcium-binding sites were examined with a recombinant calbindin-D28K protein. This protein (Calb I-II) only contained EF hand domains 1 and 2 of calbindin-D28K. Binding of calcium and calcium analogs, the lanthanides, by the recombinant protein was determined in fluorescence emission experiments. Calb I-II bound 1 mol of terbium/mol of protein. Terbium was displaced from Calb I-II by other lanthanides and calcium. Fluorescence from terbium was not quenched by holmium. These results and Hill plots of calcium binding activity, determined from intrinsic protein fluorescence measurements, indicated the presence of a single high affinity calcium-binding site on Calb I-II. The properties of the binding site indicated that the very high affinity site of calbindin-D28K was located in EF hand domains 1 and 2 of the protein. In addition, these findings indicated the NH2-terminal pair of EF hands in calbindin-D28K did not depend on interactions with other domains in the protein for high affinity calcium binding activity. The results suggested at least one calcium-binding domain of calbindin-D28K can exist as an independent EF hand pair.

Amino Acid Sequence↗
PubMed2005

Calbindin D28k targets myo-inositol monophosphatase in spines and dendrites of cerebellar Purkinje neurons.

The Ca(2+)-binding protein calbindin D28k (CB) is vital for the normal function of the central nervous system but its specific functional role is largely unclear. CB is typically described as a mobile Ca(2+)buffer that shapes the spatiotemporal extent of cellular Ca(2+)signals. Recent biochemical data, however, indicate that CB also has characteristics of a Ca(2+) sensor and activates myo-inositol monophosphatase (IMPase), a key enzyme of the inositol-1,4,5-trisphosphate signaling cascade and an assumed target of mood-stabilizing drugs in the treatment of bipolar disorder. Here, we show that CB interacts with IMPase in cerebellar Purkinje neurons, a cell type well known to rely on inositol-1,4,5-trisphosphate-dependent synaptic integration. Quantification of the mobility of dye-labeled CB with two-photon fluorescence recovery after photobleaching revealed that a substantial fraction of CB is immobilized in spines and dendrites, but not in axons. Immobilization occurs over several seconds, is increased by suprathreshold synaptic activity, and can be relieved by a synthetic peptide that resembles the putative CB-binding site of IMPase, indicating that CB binds to immobilized IMPase. Measurements of the apparent diffusion coefficients of CB imply that CB does not interact with cytosolic IMPase or that the latter is present only in minute amounts in the spiny dendrites of Purkinje neurons. Our results suggest that CB acts as an activity-dependent sensor that targets membrane/cytoskeleton-bound IMPase in central neurons.

Animals↗
PubMed2000

Surviving granule cells of the sclerotic human hippocampus have reduced Ca(2+) influx because of a loss of calbindin-D(28k) in temporal lobe epilepsy.

In mesial temporal lobe epilepsy (mTLE), the predominant form of epilepsy in adults, and in animal models of the disease, there is a conspicuous loss of the intracellular Ca(2+)-binding protein calbindin-D(28k) (CB) from granule cells (GCs) of the dentate gyrus. The role of this protein in nerve cell function is controversial, but here we provide evidence for its role in controlling Ca(2+) influx into human neurons. In patients with Ammon's horn sclerosis (AHS), the loss of CB from GCs markedly increased the Ca(2+)-dependent inactivation of voltage-dependent Ca(2+) currents (I(Ca)), thereby diminishing Ca(2+) influx during repetitive neuronal firing. Introducing purified CB into GCs restored Ca(2+) current inactivation to levels observed in cells with normal CB content harvested from mTLE patients without AHS. Our data are consistent with the possibility of neuroprotection secondary to the CB loss. By limiting Ca(2+) influx through an enhanced Ca(2+)-dependent inactivation of voltage-dependent Ca(2+) channels during prolonged neuronal discharges, the loss of CB may contribute to the resistance of surviving human granule cells in AHS.

Action Potentials↗
PubMed2006

Calbindin-D28K dynamically controls TRPV5-mediated Ca2+ transport.

In Ca(2+)-transporting epithelia, calbindin-D(28K) (CaBP(28K)) facilitates Ca(2+) diffusion from the luminal Ca(2+) entry side of the cell to the basolateral side, where Ca(2+) is extruded into the extracellular compartment. Simultaneously, CaBP(28K) provides protection against toxic high Ca(2+) levels by buffering the cytosolic Ca(2+) concentration ([Ca(2+)](i)) during high Ca(2+) influx. CaBP(28K) consistently colocalizes with the epithelial Ca(2+) channel TRPV5, which constitutes the apical entry step in renal Ca(2+)-transporting epithelial cells. Here, we demonstrate using protein-binding analysis, subcellular fractionation and evanescent-field microscopy that CaBP(28K) translocates towards the plasma membrane and directly associates with TRPV5 at a low [Ca(2+)](i). (45)Ca(2+) uptake measurements, electrophysiological recordings and transcellular Ca(2+) transport assays of lentivirus-infected primary rabbit connecting tubule/distal convolute tubule cells revealed that associated CaBP(28K) tightly buffers the flux of Ca(2+) entering the cell via TRPV5, facilitating high Ca(2+) transport rates by preventing channel inactivation. In summary, CaBP(28K) acts in Ca(2+)-transporting epithelia as a dynamic Ca(2+) buffer, regulating [Ca(2+)] in close vicinity to the TRPV5 pore by direct association with the channel.

Animals↗
PubMed1999

Calbindin and calmodulin localization in the developing vestibular organ of the musk shrew (Suncus murinus).

The distribution of the Ca(2+)-binding proteins, calbindin and calmodulin, in the adult inner ear has been described previously. We undertook immunohistochemical investigation of developmental changes in the distribution of calbindin and calmodulin in the vestibular organ of the musk shrew (Suncus murinus). Expression of calbindin was seen first in the hair cells and the vestibular ganglion on gestational day (GD) 19, in nerve fibres on GD23 and in the otoconia on GD26. On GD19 calmodulin was demonstrable only in the hair cells. On GD26 both Ca(2+)-binding proteins showed a distribution of immunoreactivity in hair cells similar to that seen in adults. The developmental differences in distribution of these binding proteins may suggest different roles in the vestibule. Additionally, Ca(2+)-binding proteins may be a useful index of hair cell maturity.

Animals↗
PubMed2006

Effect of aristolochic acid on intracellular calcium concentration and its links with apoptosis in renal tubular cells.

Aristolochic acid (AA) has been demonstrated to play a causal role in Chinese herbs nephropathy. However, the detailed mechanism for AA to induce apoptosis of renal tubular cells remains obscure. In this study, we show that AA evokes a rapid rise in the intracellular Ca(2+) concentration of renal tubular cells through release of intracellular endoplasmic reticulum Ca(2+) stores and influx of extracellular Ca(2+), which in turn causes endoplasmic reticulum stress and mitochondria stress, resulting in activation of caspases and finally apoptosis. Ca(2+) antagonists, including calbindin-D(28k) (an intracellular Ca(2+) buffering protein) and BAPTA-AM (a cell-permeable Ca(2+) chelator), are capable of ameliorating endoplasmic reticulum stress and mitochondria stress, and thereby enhance the resistance of the cells to AA. Moreover, we show that overexpression of the anti-apoptotic protein Bcl-2 in combination with BAPTA-AM treatment can provide renal tubular cells with almost full protection against AA-induced cytotoxicity. In conclusion, our results demonstrate an impact of AA to intracellular Ca(2+) concentration and its link with AA-induced cytotoxicity.

Animals↗
PubMed2004

Calcium as a mediator of 1,25-dihydroxyvitamin D3-induced apoptosis.

Cellular calcium has been implicated in induction of apoptosis. We have shown that 1,25(OH)(2)D(3)-induced apoptosis is associated with a sustained increase in concentration of intracellular Ca(2+) ([Ca(2+)](i)) resulting from depletion of the endoplasmic reticulum (ER) Ca(2+) stores and activation of the voltage-insensitive Ca(2+) entry pathway [1,25-Dihydroxyvitamin D(3), intracellular Ca(2+) and apoptosis in breast cancer cells, in: A.W. Norman, R. Bouillon, M. Thomasset (Eds.), Vitamin D: Chemistry, Biology and Clinical Applications of the Steroid Hormone, University of California, Riverside, 1997, pp. 473-474; Vitamin D and intracellular calcium, in: P. Quinn, V. Kagan (Eds.), Subcellular Biochemistry: Fat-Soluble Vitamins, Plenum Press, New York, 1998, pp. 271-297; 1,25-Dihydroxyvitamin D(3) and calcium signaling, in: A.W. Norman, R. Bouillon, M. Thomasset (Eds.), Vitamin D Endocrine System: Structural, Biological, Genetic and Clinical Aspects, University of California, Riverside, 2000, pp. 715-718; 1,25-Dihydroxyvitamin D(3) triggers calcium-mediated apoptosis in breast cancer cells, in: A.W. Norman, R. Bouillon, M. Thomasset (Eds.), Vitamin D Endocrine System: Structural, Biological, Genetic and Clinical Aspects, University of California, Riverside, 2000, pp. 399-402; Endocrine 9 (1998) 321]. This study was undertaken to investigate mechanism of 1,25(OH)(2)D(3)-induced apoptosis in breast cancer cells and compare effects of the hormone on Ca(2+) and apoptosis in cancer and normal human mammary epithelial cells. The treatment of MCF-7 breast cancer cells with 1,25(OH)(2)D(3) induced a sustained increase in [Ca(2+)](i) and activated the Ca(2+)-dependent proapoptotic proteases, micro-calpain and caspase-12, as evaluated with antibodies to active (cleaved) forms of the enzymes and the calpain substrate. The selective inhibition of Ca(2+) binding sites of micro-calpain decreased apoptotic indices in the 1,25(OH)(2)D(3)-treated cells. 1,25(OH)(2)D(3) did not induce apoptosis in normal human mammary epithelial cells (HMECs), as evaluated by DNA fragmentation (TUNEL), loss of the plasma membrane asymmetry (Annexin V assay) and morphological criteria. In these cells, 1,25(OH)(2)D(3) triggered a transient Ca(2+) response, which was not accompanied by the calpain and caspase activation. HMEC, but not MCF-7 cells expressed the Ca(2+) binding protein calbindin-D(28k) and buffered Ca(2+) increases induced by a Ca(2+) ionophore ionomycin. In conclusion, we have identified the novel apoptotic pathway in breast carcinoma cells treated with 1,25(OH)(2)D(3): increase in [Ca(2+)](i) -->micro-calpain activation --> caspase-12 activation --> apoptosis. Our findings also imply that differences of Ca(2+) regulatory mechanisms in breast cancer versus normal mammary epithelial cells underlay resistance of normal cells and susceptibility of cancer cells to 1,25(OH)(2)D(3)-induced Ca(2+)-mediated apoptosis.

Apoptosis↗
PubMed2003

Characterization of a murine renal distal convoluted tubule cell line for the study of transcellular calcium transport.

To unravel the molecular regulation of renal transcellular Ca(2+) transport, a murine distal convoluted tubule (mpkDCT) cell line derived from distal convoluted tubules (DCT) microdissected from a SV-PK/Tag transgenic mouse was characterized. This cell line originated from DCT only, as mRNA encoding for the DCT marker thiazide-sensitive Na(+)/Cl(-) cotransporter was expressed, whereas mRNA encoding for the connecting tubule and collecting duct marker aquaporin-2 was not detected, as determined by reverse-transcriptase PCR. mpkDCT cells expressed mRNA encoding the Ca(2+) channels TRPV5 and TRPV6 and other key players necessary for transcellular Ca(2+) transport, i.e., calbindin-D(9k), calbindin-D(28k), plasma membrane Ca(2+)-ATPase isoform 1b, and Na(+)/Ca(2+) exchanger 1. Primary cultures of DCT cells exhibited net transcellular Ca(2+) transport of 0.4 +/- 0.1 nmol.h(-1).cm(-2), whereas net transcellular Ca(2+) transport across mpkDCT cells was significantly higher at 2.4 +/- 0.4 nmol.h(-1).cm(-2). Transcellular Ca(2+) transport across mpkDCT cells was completely inhibited by ruthenium red, an inhibitor of TRPV5 and TRPV6, but not by the voltage-operated Ca(2+) channel inhibitors felodipine and verapamil. With the use of patch-clamp analysis, the IC(50) of ruthenium red on Na(+) currents was between the values measured for TRPV5- and TRPV6-expressing HEK 293 cells, suggesting that TRPV5 and/or TRPV6 is possibly active in mpkDCT cells. Forskolin in combination with IBMX, 1,25-dihydroxyvitamin D(3), and 1-deamino-8-d-arginine vasopressin increased transcellular Ca(2+) transport, whereas PMA and parathyroid hormone had no significant effect. In conclusion, the murine mpkDCT cell line provides a unique cell model in which to study the molecular regulation of transcellular Ca(2+) transport in the kidney in vitro.

Animals↗
PubMed2001

Distribution of transcellular calcium and sodium transport pathways along mouse distal nephron.

The organization of Na(+) and Ca(2+) transport pathways along the mouse distal nephron is incompletely known. We revealed by immunohistochemistry a set of Ca(2+) and Na(+) transport proteins along the mouse distal convolution. The thiazide-sensitive Na(+)-Cl(-) cotransporter (NCC) characterized the distal convoluted tubule (DCT). The amiloride-sensitive epithelial Na(+) channel (ENaC) colocalized with NCC in late DCT (DCT2) and extended to the downstream connecting tubule (CNT) and collecting duct (CD). In early DCT (DCT1), the basolateral Ca(2+)-extruding proteins [Na(+)/Ca(2+) exchanger (NCX), plasma membrane Ca(2+)-ATPase (PCMA)] and the cytoplasmic Ca(2+)-binding protein calbindin D(28K) (CB) were found at very low levels, whereas the cytoplasmic Ca(2+)/Mg(2+)-binding protein parvalbumin was highly abundant. NCX, PMCA, and CB prevailed in DCT2 and CNT, where we located the apical epithelial Ca(2+) channel (ECaC1). Its subcellular localization changed from apical in DCT2 to exclusively cytoplasmic at the end of CNT. NCX and PMCA decreased in parallel with the fading of ECaC1 in the apical membrane. All three of them were undetectable in CD. These findings disclose DCT2 and CNT as major sites for transcellular Ca(2+) transport in the mouse distal nephron. Cellular colocalization of Ca(2+) and Na(+) transport pathways suggests their mutual interactions in transport regulation.

Animals↗
PubMed1994

Transient decrease in calbindin immunoreactivity of the rat fascia dentata granule cells after repeated electroconvulsive shocks.

Changes in hippocampal calbindin immunoreactivity were investigated after repeated electroconvulsive shocks. Adult rats were subjected to 10, 20, or 36 electroconvulsive shocks (50 mA, 0.5 seconds), given as on shock per day, 5 days a week. The rats were sacrificed and processed for calbindin immunohistochemistry 1, 2, and 30 days after the last electroconvulsive shock. In the rats receiving 10 or 20 electroconvulsive shocks, a selective reduction of the calbindin immunoreactivity of the dentate granule cell bodies, dendrites, and mossy fibers was noted 1 and 2 days after the last electroconvulsive shock. After 36 electroconvulsive shocks there was an almost complete loss of calbindin immunoreactivity from the granule cell bodies and dendrites, and the calbindin immunoreactivity of the mossy fibers was markedly reduced. Thirty days after the last of 36 electroconvulsive shocks, the calbindin immunoreactivity was back to normal. Besides demonstrating pronounced changes associated with repeated electroconvulsive shocks, the results confirm the transient nature of these changes.

Animals↗
PubMed2005

Calcium signaling in cancer and vitamin D.

Calcium signals induced by the Ca(2+) regulatory hormone 1,25(OH)(2)D(3) may determine the fate of the cancer cell. We have shown that, in breast cancer cell lines, 1,25(OH)(2)D(3) induces a sustained increase in concentration of intracellular Ca(2+) ([Ca(2+)](i)) by depleting the endoplasmic reticulum (ER) Ca(2+) stores via inositol 1,4,5-trisphosphate receptor/Ca(2+) release channel and activating Ca(2+) entry from the extracellular space via voltage-insensitive Ca(2+) channels. In normal cells, 1,25(OH)(2)D(3) triggered a transient Ca(2+) response via activation of voltage-dependent Ca(2+) channels, which were absent in breast cancer cells. The normal cells, but not breast cancer cells, expressed the Ca(2+) binding/buffering protein calbindin-D(28k) and were capable of buffering [Ca(2+)](i) increases induced by a mobilizer of the ER Ca(2+) stores, thapsigargin, or a Ca(2+) ionophore, ionomycin. The 1,25(OH)(2)D(3)-induced sustained increase in [Ca(2+)](i) in breast cancer cells was associated with induction of apoptotic cell death, whereas the transient [Ca(2+)](i) increase in normal cells was not. The forced expression of calbindin-D(28k) in cytosol or increase in the cytosolic Ca(2+) buffering capacity with the cell-permeant Ca(2+) buffer BAPTA prevented induction of apoptosis with 1,25(OH)(2)D(3) in cancer cells. The sustained increase in [Ca(2+)](i) in breast cancer cells was associated with activation of the Ca(2+)-dependent apoptotic proteases, mu-calpain and caspase-12, as evaluated with antibodies to active (cleaved) forms of the enzymes and the fluorogenic peptide substrates. Selective inhibition of the Ca(2+) binding sites of mu-calpain decreased apoptotic indices in the cancer cells treated with 1,25(OH)(2)D(3), thapsigargin, or ionomycin. The mu-calpain activation preceded expression/activation of caspase-12, and calpain was required for activation/cleavage of caspase-12. Certain non-calcemic vitamin D analogs (e.g., EB 1089) triggered a sustained [Ca(2+)](i) increase, activated Ca(2+)-dependent apoptotic proteases, and induced apoptosis in breast cancer cells in a fashion similar to that of 1,25(OH)(2)D(3). The 1,25(OH)(2)D(3)-induced transient Ca(2+) response in normal mammary epithelial cells was not accompanied by activation of mu-calpain and caspase-12. In conclusion, we have identified the novel apoptotic pathway in breast carcinoma cells treated with 1,25(OH)(2)D(3): increase in [Ca(2+)](i)-->mu-calpain activation-->caspase-12 activation-->apoptosis. Our results support the hypothesis that 1,25(OH)(2)D(3) directly activates this apoptotic pathway by inducing a sustained increase in [Ca(2+)](i). Differences of Ca(2+) regulatory mechanisms in cancer versus normal cells seem to allow 1,25(OH)(2)D(3) and vitamin D analogs to induce Ca(2+)-mediated apoptosis selectively in breast cancer cells. Thus, deltanoids may prove to be useful in the treatment of tumors susceptible to induction of Ca(2+)-mediated apoptosis.

Apoptosis↗
PubMed2003

Complementary distribution of vesicular glutamate transporters 1 and 2 in the nucleus accumbens of rat: Relationship to calretinin-containing extrinsic innervation and calbindin-immunoreactive neurons.

The caudomedial shell of the rat nucleus accumbens exhibits inhomogeneous distribution patterns of the vesicular glutamate transporters 1 (VGLUT1) and 2 (VGLUT2). This paper focuses on the question of whether patterns of VGLUT1 and VGLUT2 correspond to cytoarchitectonically and cytochemically defined subterritories of the caudomedial shell region. VGLUT2 was shown to be coexpressed with calretinin in the dense axonal plexus known to emanate from the paraventricular thalamic nucleus. In regions termed corridors, which are spared by this paraventricular thalamic innervation, axonal terminals were found to be clustered and VGLUT1-immunoreactive. It is assumed that these fibers originate in the prelimbic cortex and/or in the parvicellular basal amygdaloid nucleus known to project to accumbal shell components. Our findings confirm the existence of two well-separated neuronal circuits in the caudomedial shell that are dominated by two different excitatory input systems originating from either thalamic, cortical, or cortex-like amygdaloid sources. The large lateral corridors-which resemble the accumbal core not only in respect to their VGLUT1 immunolabeling but also concerning their content of calbindin-positive cells-may represent a component of the anatomically weakly defined accumbal shore region.

Afferent Pathways↗
PubMed1991

Ca2+ binding in proteins of the calmodulin superfamily: cooperativity, electrostatic contributions and molecular mechanisms.

In a large number of intracellular regulatory proteins of the calmodulin superfamily a pair of closely interacting helix-loop-helix Ca2+ binding sites ('EF hands') constitute the functional unit--an arrangement that enables cooperative binding. We have recently made detailed experimental studies of the binding of Ca2+ ions to calmodulin, its tryptic fragments TR1C and TR2C (which each constitute a globular domain of a pair of EF hands) and calbindin D9k. Macroscopic Ca2+ binding constants have been obtained over a range of ionic strengths (0 to 0.15 M KCl). For calmodulin the measurements indicate that the two separate globular domains TR1C and TR2C retain the Ca2+ binding properties they have in the intact molecule, with positive cooperativity within each domain. The absolute value of the free energy of interaction between the two sites in each domain, a measure of the cooperativity, increases with ionic strength and is greater than or equal to 10 kJ mol-1 at 0.15 M KCl. Two-dimensional 1H NMR studies show that the addition of KCl does not alter the conformation of the protein. In the case of calbindin D9k several categories of mutants have been studied. One group encompasses the effect of protein surface charges 5 to 15 A from the Ca2+ binding sites. Two-dimensional 1H NMR shows that neither the addition of KCl, nor mutations that neutralize the surface charges, change the protein conformation. Although the global structure of calbindin D9k is largely unchanged upon binding of calcium, the structure with only one cation bound is more similar to the (Ca2+)2 form. Interestingly, the dynamical properties of the Ca(2+)-free and the (Ca2+)2-forms of calbindin differ greatly. For example, the rate of NH/ND exchange of the Ca(2+)-free form is on average 200 times faster than that of the (Ca2+)2-form. The results obtained so far point to a non-negligible entropic contribution to the observed cooperativity of Ca2+ binding.

Amides↗