Biomedical subjects

S Christakos

Publications and source records attributed to S Christakos.

At least 55 records · Page 3Linked to original sources

PubMed1994

Two vitamin D3-dependent calcium binding proteins increase calcium reabsorption by different mechanisms. II. Effect of CaBP 9K.

The distal tubule is a major site of hormone regulated Ca2+ reabsorption. We have previously shown that in the rabbit, vitamin D depletion decreases both the Ca2+ uptake by the luminal membrane of nephron distal segments and the ATP-dependent Ca2+ transport by the basolateral membrane of these same segments. We also reported that in in vitro experiments, the vitamin D-dependent calcium binding protein (CaBP) 28K enhances Ca2+ uptake by the high-affinity Ca2+ transport system of the distal luminal membranes. The purpose of the present study was to investigate the role of CaBP 9K in Ca2+ transport by the two polar membranes of the proximal and distal nephron segments. Preloading the luminal membranes of the two series of tubules with 10 microM CaBP 9K did not modify 0.5 mM Ca2+ uptake by these membranes. In contrast, preincubation of distal tubule basolateral membranes with 10 microM CaBP 9K strongly enhanced ATP-dependent Ca2+ transport by these membranes. A similar although more modest effect was observed in proximal basolateral membranes. This effect was present only in calmodulin depleted membranes. Calmodulin also increased the ATP-dependent Ca2+ uptake by the depleted membranes. The two actions were not additive. A dose-response curve showed that the maximal CaBP 9K effect was obtained at 10(-5) M. CaBP 9K increased the Vmax of ATP-dependent uptake from 0.43 +/- 0.03 to 0.79 +/- 0.06 nmol/mg/10 sec (P < 0.005), leaving the Km unchanged. It is concluded that CaBP 9K activates the Ca2+ pump in the basolateral membrane of both the proximal and distal tubule.(ABSTRACT TRUNCATED AT 250 WORDS)

Absorption↗

PubMed1994

1,25-Dihydroxyvitamin D3 and pancreatic beta-cell function: vitamin D receptors, gene expression, and insulin secretion.

Previous studies have indicated that the pancreas has receptors specific for 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] and that 1,25-(OH)2D3 increases insulin secretion in vitamin D-deficient rats. In this study we report that in vitamin D-replete, but calcium-deficient, rats in which 1,25-(OH)2D3 levels are elevated, insulin secretion is not altered. In addition, in in vitro studies 1,25-(OH)2D3 at concentrations of 10(-10)-10(-7) M was consistently found to inhibit insulin secretion from islets of vitamin D-replete rats or from the rat insulinoma beta-cell line RIN 1046-38. The RIN cell line was found to contain both vitamin D receptors and calbindin-D28k (CaBP-D28k) protein and mRNA. In RIN cells, treatment with sodium butyrate (2 mM for 3 days) induces a more islet phenotype, as indicated by increased insulin content and secretion and increased insulin gene expression. 1,25-(OH)2D3 treatment (50-100 nM for 48 or 72 h) had no effect on the enhanced levels of insulin secreted in the presence of butyrate. However, 2 mM sodium butyrate induced CaBP-D28k protein (4-fold; control, 0.8 +/- 0.2; sodium butyrate, 3.5 +/- 0.1 microgram/mg protein) and mRNA (3-fold) in the RIN cell line, in accord with the induction by butyrate of insulin content and secretion and beta-cell differentiation, suggesting a possible role for CaBP-D28k in these processes. Although 1,25-(OH)2D3, unlike butyrate, did not enhance insulin secretion, both 1,25-(OH)2D3 (100 nM) and butyrate (2 mM) inhibited RIN cell growth (to 69% and 28% of the control, respectively), and butyrate and 1,25-(OH)2D3 in combination led to a further inhibition of cell growth (to 13% of the control). In response to 1,25-(OH)2D3 (10 nM for 72 h), vitamin D receptors were up-regulated 313% in RIN cells [control, 37 +/- 2; 1,25-(OH)2D3 treated, 115 +/- 5 fmol/mg protein]. In conclusion, 1) contrary to previous studies in the vitamin D-deficient rat, our findings indicate that 1,25-(OH)2D3 action does not necessarily result in enhanced insulin secretion; 2) inhibition of cell growth and up-regulation of vitamin D receptors by 1,25-(OH)2D3 suggest that parameters in addition to insulin secretion can be affected by 1,25-(OH)2D3 in the beta-cell; 3) the RIN beta-cell line provides a novel in vitro system for studying the effect of the vitamin D endocrine system on pancreatic islet physiology.

Aging↗

PubMed1993

Identification of sequence elements in mouse calbindin-D28k gene that confer 1,25-dihydroxyvitamin D3- and butyrate-inducible responses.

We have examined the 5' flanking region of the mouse calbindin-D28k gene and identified a 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]-responsive element by deletion mutant analysis of the native promoter as well as by studies with a heterologous thymidine kinase (TK) promoter. The segment between residues -200 and -169 was found to confer a dose-dependent 1,25-(OH)2D3 responsiveness through the TK promoter in Ros 17/2.8 cells as well as in CV-1 cells cotransfected with pAV-hVDR (human vitamin D receptor expression vector). This region contains sequences homologous to the rat osteocalcin vitamin D response element (VDRE). Incubation of this element with nuclear extracts from 1,25-(OH)2D3-treated Ros 17/2.8 cells or from 1,25-(OH)2D3-treated COS cells that had been transfected with pAV-hVDR resulted in a specific protein-DNA interaction. In addition to 1,25-(OH)2D3, sodium butyrate, a differentiating agent, has also been found to modulate expression of calbindin-D28k. Deletion analysis of the mouse calbindin-D28k promoter as well as studies with a heterologous TK promoter resulted in identification of a butyrate-responsive element between -180 and -150 that was found to bind specifically to nuclear factors from butyrate-treated Ros 17/2.8 cells. This butyrate-responsive element may represent a genetic element acted upon by enhancer binding proteins. In summary, the 5' flanking region of the mouse calbindin-D28k gene contains responsive elements that interact with nuclear factors and may mediate, at least in part, the enhanced expression of this gene by 1,25-(OH)2D3 and butyrate.

Animals↗

PubMed1993

Effect of 1,25,28-trihydroxyvitamin D2 and 1,24,25-trihydroxyvitamin D3 on intestinal calbindin-D9K mRNA and protein: is there a correlation with intestinal calcium transport?

Although analogs and metabolites of vitamin D have been tested for their calciotropic activity, very little information has been available concerning the effects of these compounds on gene expression. In this study one analog of vitamin D, 1,25,28-trihydroxyvitamin D2 [1,25,28-(OH)3D2], and one metabolite, 1,24,25-trihydroxyvitamin D3 [1,24,25-(OH)3D3], were tested for their effect on intestinal calbindin-D9K mRNA and protein as well as for their effect on intestinal calcium absorption and bone calcium mobilization. These compounds were also evaluated for their ability to compete for rat intestinal 1,25-(OH)2D3 receptor sites and to induce differentiation of human leukemia (HL-60) cells as indicated by reduction of nitro blue tetrazolium. In vivo studies involved intrajugular injection of 12.5 ng 1,25-(OH)2D3 or test compound to vitamin D-deficient rats and sacrifice after 18 h. 1,25,28-Trihydroxyvitamin D2 had no effect on intestinal calcium absorption, bone calcium mobilization, or intestinal calbindin-D9K protein and mRNA. Competitive binding to 1,25-(OH)2D3 receptors was 0.8% of that observed using 1,25-(OH)2D3. However, 20- and 40-fold higher doses of 1,25,28-(OH)3D2 (250 and 500 ng) resulted in significant inductions in calbindin-D9K protein and mRNA (3.5 to 7.4-fold), although doses as high as 800 ng were found to have no effect on intestinal calcium absorption or bone calcium mobilization.(ABSTRACT TRUNCATED AT 250 WORDS)

25-Hydroxyvitamin D 2↗

PubMed1993

Apoptosis and signal transduction: clues to a molecular mechanism.

Apoptosis, or programmed cell death, plays an essential role in specific cell deletion during normal embryonic and adult development in vertebrate and invertebrate species. Recent evidence suggests that signal transduction pathways governing cellular proliferation and cell cycle progression also mediate the physiological response to changes in the extracellular environment that trigger the anti-proliferative state characteristic of apoptosis.

Animals↗

PubMed1993

Calbindin-D28k gene expression in the developing mouse kidney.

Calbindin-D28k appears in the metanephric kidney during embryogenesis. We studied the temporal appearance and spatial distribution of calbindin-D28k mRNA in the developing kidneys of 12-day fetal through 21-day postnatal mice by in situ hybridization. 35S-UTP-labeled antisense (cRNA) probe to calbindin-D28k mRNA hybridized to the ureteric buds of 12-day embryos, whereas adjacent metanephrogenic tissue was unlabeled. By embryonic day 13, Y-shaped bodies of "advancing" ureteric buds were labeled intensely. In 16-day embryos, ampullae of ureteric buds were located immediately beneath the renal capsule and labeled strongly, in contrast to metanephric tubules and S-shaped bodies. The former were unlabeled and the latter were labeled only at points of contact with the ampullae. Subsequently, the ampullae of the metanephric ureteric buds hybridized with the cRNA probe, and from the 18th embryonic to the 21st postnatal day, this labeling was intense. The cRNA probe did not hybridize with the renal vesicles, proximal tubules, or tubular segments of Henle's loop derived from nephrogenic blastema, but it did label distal nephron segments. By the 21st postnatal day, collecting ducts and ureter no longer were labeled. In conclusion, calbindin-D28k mRNA is present in the developing mouse kidney, and its distribution during nephrogenesis is identical to that of calbindin-D28k per se. Collectively, these findings show that the calbindin-D28k gene is transcribed and its message is translated by the cells of the ureteric bud during the initial stage of renal morphogenesis.

Animals↗

PubMed1993

Differential localization of class III, beta-tubulin isotype and calbindin-D28k defines distinct neuronal types in the developing human cerebellar cortex.

This immunohistochemical study compares the localization of the neuronal class III beta-tubulin isotype (beta III) to that of calbindin-D28k in 40 human fetal and postnatal cerebella ranging from 12 weeks gestation to adulthood. In the external granule layer of the developing cerebellar cortex, beta III staining was present in the premigratory (postmitotic) zone of horizontal neurons but was absent in "epithelioid" cells of the subpial proliferative mitotic zone. In the molecular layer, intense beta III staining was associated with parallel fibers, stellate/basket neurons and migrating fusiform granule neurons. beta III staining was also present in internal granule neurons. In contrast, beta III was not detectable in fetal and neonatal Purkinje neurons and Golgi II neurons, but was evident in these neurons from juvenile and adult cerebella. Calbindin-D28k staining was present in Purkinje neurons also delineating their somatic spines ("pseudopodia"), lateralizing and apical dendrites (including dendritic spines), subpopulations of small to intermediate-sized Golgi II neurons in the internal granule layer ("synarmotic cells" of Landau), large to medium-sized subcortical Golgi II neurons and neurons of cerebellar roof nuclei, at various gestational stages and postnatally. It was absent in the external granule layer, parallel fibers, stellate/basket and internal granule neurons. Variable degrees of beta III and calbindin-D28k staining were detected in subpopulations of immature neuroepithelial cells of the ventricular matrix at the roof of the fourth ventricle. Glial (including Bergmann glia) and mesenchymal cells were not stained for either antigenic determinants. The differential expression of calbindin-D28k and beta III defines distinct populations of neurons in the developing human cerebellar cortex and supports the ontogenetic concept of Ramon y Cajal.

Adolescent↗

PubMed1993

Calcium buffering properties of calbindin D28k and parvalbumin in rat sensory neurones.

1. We have examined the ability of the Ca(2+)-binding proteins (CABP) calbindin D28k and paravalbumin to modulate increases in the intracellular free Ca2+ concentration ([Ca2+]i), produced by brief depolarizations, in rat dorsal root ganglion (DRG) neurones. 2. In order to obtain good voltage control, we replated DRG neurones prior to performing these experiments. Immunocytochemical staining of these cells revealed that approximately 10% stained for CABPs. 3. Using fluorescently labelled parvalbumin, we demonstrated that in the whole-cell voltage clamp mode the protein freely entered the cell soma with a mean half-life t0.5 of 6 min 22 s +/- 54 s. 4. Analysis of the effects of calbindin D28k (370 microM) and parvalbumin (1 mM) on Ca2+ currents in the whole-cell voltage clamp mode, revealed that neither protein changed the rate of inactivation of the Ca2+ current or its rate of run-down. 5. Introducing either calbindin D28k (370 microM) or parvalbumin (1 mM) into the cell soma did not significantly alter the basal [Ca2+]i when compared to control cells. 6. Compared to control cells, both CABPs significantly reduced the peak [Ca2+]i obtained for a Ca2+ influx of an equivalent charge density, whereas lysozyme (1 mM), a protein with low affinity for Ca2+, failed to do so. 7. Calbindin D28k caused an 8-fold decrease in the rate of rise in [Ca2+]i and altered the kinetics of decay of [Ca2+]i to a single slow component. Parvalbumin also slowed the rate of rise in [Ca2+]i. Parvalbumin selectively increased a fast component in the decay of the Ca2+ signal. 8. These data demonstrate that both calbindin D28k and paravalbumin effectively buffer Ca2+ in a cellular environment and may therefore regulate Ca(2+)-dependent aspects of neuronal function.

Animals↗

PubMed1992

Nerve growth factor increases calcium binding protein (calbindin-D28K) in rat olfactory bulb.

Calbindin-D28K (CaBP28K) is a soluble intracellular protein capable of sequestering micromolar concentrations of calcium. The in vivo regulation of CaBP28K by recombinant human nerve growth factor (rhNGF) was studied in adult, male rats. Via Alzet 2002 pumps, each rat received, for 14 days, a lateral ventricle infusion (i.c.v.; n = 5-6/group) of 12 microliters PBS/day containing 1.0 microgram cytochrome C (control) or an equal amount of rhNGF. Six other animals received a vehicle or rhNGF infusion into the central neostriatum. CaBP28K was elevated by 75% (P less than 0.01) in the olfactory bulb following i.c.v. rhNGF in each of two experiments and was not altered in the temporal cortex, hippocampus, olfactory tubercle, cerebellum, or neostriatum. Direct striatal injections of rhNGF did not alter CaBP28K in the neostriatum or other regions (including the olfactory bulb). The increases in olfactory bulb CaBP28K protein levels were verified via Western blot analysis. CaBP28K immunocytochemistry revealed that 33% of olfactory bulb neurons are immunoreactive for CaBP28K and that the number or proportion of immunoreactive neurons did not change with i.c.v. infusions of rhNGF, suggesting that exogenously delivered rhNGF augments the content of CaBP28K in olfactory bulb neurons that normally express the protein. Endogenous NGF may function as a neuroprotective factor by enhancing the ability of these cells to sequester cytoplasmic calcium and retard calcium-mediated neurodegeneration.

Animals↗

PubMed1992

Specific 1,25-dihydroxyvitamin D3 binding sites in choroid plexus.

Quantitative autoradiographic analysis of [3H] 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) binding in vitamin D deficient mice provided evidence for high levels of specific binding in choroid plexus and, to a lesser extent, ventral hippocampus. Sucrose gradient analysis yielded a 3-4S peak of specific [3H]1,25(OH)2D3 binding in bovine choroid plexus, but not amygdala or hippocampus. Scatchard analysis of [3H]1,25(OH)2D3 binding in bovine choroid plexus yielded KD = 0.23 +/- 0.06 nM and Nmax = 43.5 +/- 0 fmol/g tissue (n = 5). This result indicates the presence of significant receptor-like [3H]1,25(OH)2D3 binding sites in the choroid plexus and, thus, suggests roles for this hormone in regulating the entry of calcium into the brain and/or in the central regulation of calcium homeostasis.

Animals↗

PubMed1992

In vitro enzyme activation with calbindin-D28k, the vitamin D-dependent 28 kDa calcium binding protein.

Purified porcine erythrocyte membrane Ca(2+)-ATPase and 3':5'-cyclic nucleotide phosphodiesterase were stimulated in a dose-dependent, saturable manner with the vitamin D-dependent calcium binding protein from rat kidney, calbindin-D28k (CaBP-D28k). The concentration of CaBP-D28k required for half-maximal activation (K0.5 act.) of the Ca(2+)-ATPase was 28 nM compared to 2.2 nM for calmodulin (CaM), with maximal activation equivalent upon addition of either excess CaM or CaBP-D28k. 3':5'-Cyclic nucleotide phosphodiesterase (PDE) also showed equivalent maximum saturable activation by calbindin (K0.5 act. = 90 nM) or calmodulin (K0.5 act. = 1.2 nM). CaBP-D28k was shown to effectively compete with CaM-Sepharose for PDE binding. Immunoprecipitation with CaBP-D28k antiserum completely inhibited calbindin-mediated activation of PDE but had no effect on calmodulin's ability to activate PDE. While the physiological significance of these results remains to be established, they do suggest that CaBP-D28k can activate enzymes and may be a regulator of yet to be identified target enzymes in certain tissues.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1992

Calbindin-D28K-containing neurons in animal models of neurodegeneration: possible protection from excitotoxicity.

Brain levels of the calcium binding protein Calbindin-D28K (CaBP28K) and CaBP28K mRNA were measured for various animal models of neurodegenerative diseases (MPTP-treated C57BL/6J mice and Sprague-Dawley rats receiving striatal/intraperitoneal kainic acid or quinolinic acid into the nucleus basalis magnocellularis). Brain areas were tested (radioimmunoassay, Western blot, slot blot, and Northern blot) for levels of CaBP28K and CaBP28K mRNA. The various models did not exhibit any changes in protein or mRNA levels from the controls, suggesting that CaBP28K-containing neurons were not lost after exposure to these neurotoxins. Immunocytochemical characterization of the substantia nigra of the MPTP-treated mice revealed that there was significant dopaminergic cell loss in this brain area after MPTP treatment. The majority of dopaminergic neurons that degenerated did not contain CaBP28K. The small percentage of surviving neurons were CaBP28K-positive. These results suggest that the presence of CaBP28K may protect neurons from calcium-mediated neurotoxicity.

Animals↗

PubMed1992

Dietary calcium modifies concentrations of lead and other metals and renal calbindin in rats.

We studied the effects of dietary calcium on kidney, femur, testis, liver, heart and brain concentrations of lead, magnesium, iron, copper, calcium and zinc in rats exposed to lead for 1 y. Renal levels of the 28,000 Da, vitamin D-dependent, calcium-binding protein calbindin-D28K were also measured. Seventy-two weanling male Sprague-Dawley rats were randomly assigned to one of nine treatment groups. Rats were fed diets containing 0.1, 0.5 or 2.5% Ca for 52 wk and were simultaneously given either 0, 50 or 100 mg lead/L in their drinking water. Rats fed the 0.1% Ca diet had organ lead concentrations that were two- to 20-fold greater than the corresponding animals fed 0.5% Ca. Rats fed diets containing 2.5% Ca had the lowest organ lead concentrations. Despite substantial effects of diet Ca on organ lead concentrations, Ca did not significantly influence concentrations of most other divalent metals studied with the exception of kidney calcium and magnesium, testis iron, plasma ionic calcium and magnesium, and several femur metals. Kidney calcium concentrations were lower in rats fed 2.5% Ca diets than in those fed 0.1 or 0.5% Ca diets. For rats not given lead, renal calbindin concentrations were highest in rats fed 0.1% Ca, and lowest in rats fed 2.5% Ca. Lead inhibited an increase in renal calbindin in the rats fed 0.1% Ca, but paradoxically increased renal calbindin levels in animals fed 2.5% Ca.

Administration, Oral↗

PubMed1992

Molecular aspects of the calbindins.

Studies from our laboratory concerning regulation of calbindin include regulation by 1,25-dihydroxycholecalciferol [1,25(OH)2D3], receptor regulation as a possible mechanism for modulating calbindin's response to hormone, tissue specific regulation and regulation by factors other than 1,25(OH)2D3. With regard to receptor regulation, we found that the induction of calbindin mRNA in intestine and kidney by 1,25(OH)2D3 is not accompanied by a corresponding alteration in vitamin D receptor (VDR) mRNA in the vitamin D-deficient, low calcium rat. However, in the vitamin D-replete rat, administration of 1,25(OH)2D3 results in an induction of both calbindin and VDR mRNA in these tissues. These results suggest the presence of an inhibitor of 1,25(OH)2D3-mediated receptor up-regulation in the vitamin D-deficient, low calcium animal. Glucocorticoids can also regulate calbindin gene expression. Dexamethasone treatment (50 micrograms.100 g body weight-1.d-1 for 4 d) results in a 75% decrease in rat intestinal calbindin-D9k mRNA. This decrease may be related to the inhibition of intestinal calcium absorption previously observed after glucocorticoid administration. Kidney calbindin-D28k mRNA is unaffected by glucocorticoid treatment, indicating tissue specificity of the glucocorticoid response. To evaluate more precisely the means whereby 1,25(OH)2D3 and other modulators can influence calbindin gene expression, we isolated the chromosomal gene for calbindin-D28k by screening a mouse genomic library in cosmid. Ros 17/2.8 cells were transfected with recombinant plasmids in which the mouse calbindin promoter is fused to the reporter gene encoding chloramphenicol acetyltransferase. Deletion studies have enabled us to identify sequence elements in the mouse calbindin-D28k gene that confer basal activation and a hormone inducible response.

Animals↗

PubMed1992

Age-related alterations in calbindin-D28K induction by 1,25-dihydroxyvitamin D3 in primary cultures of rat renal tubule cells.

In vivo studies have indicated that renal calbindin-D28K protein and mRNA levels decrease in adult and old rats, and this decrease parallels the age-associated decline in serum 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] levels. However, diminished renal responsiveness to 1,25-(OH)2D3 with advancing age could also contribute to decreased calbindin-D28K expression. To study renal responsiveness with age, primary cell cultures were established from the kidney cortices of young (1 month old), adult (10-12 months old), and old (20-24 months old) rats. Cells were incubated in medium K-1 containing 2% fetal calf serum. Calbindin-D28K protein levels were determined by Western blot and enzyme-linked immunosorbent assay. In young animals, the levels of calbindin-D28K declined from 12.1 +/- 1.3 micrograms/mg protein in the intact kidney to 1.6 +/- 0.07 micrograms/mg protein in cells that had been cultured for 3 days in the absence of 1,25-(OH)2D3. This sharp decline in calbindin-D28K protein concentration moderated by days 6-8. The continuous presence of 10(-7) M 1,25-(OH)2D3 in the medium did not abolish the decline. The low levels of calbindin-D28K in the cells cultured in the absence of 1,25-(OH)2D3 provided an excellent experimental system in which to compare the response of the cells to 1,25-(OH)2D3 between age groups. In cultured cells treated with 1,25-(OH)2D3 for 72 h, calbindin-D28K induction was greater in cells from adult and old animals compared to cells from young animals. The ratios of calbindin-D28K content (with vitamin D/without vitamin D) were 2.2 +/- 0.2, 4.7 +/- 0.5, and 7.1 +/- 1.5 for young, adult, and old cells, respectively. These studies suggested that the observed in vivo decrease in renal calbindin-D28K with age is primarily due to the lowered circulating 1,25-(OH)2D3.

Aging↗

PubMed1991

Cerebellar Purkinje cell markers are expressed in retinal bipolar neurons.

Previous studies have been directed at the elucidation of neuron-specific gene expression in the mammalian central nervous system. In particular, we have identified a series of marker molecules that are expressed in cerebellar Purkinje cells with varying degrees of specificity. Here, we show by light microscopic immunocytochemistry and Northern transfer and hybridization that two of these markers, namely, L7 and PEP19, are expressed in the retina of mouse and rabbit, while a third marker, cerebellin, is absent. Light and electron microscopic immunocytochemistry proves that L7-like immunoreactivity is restricted to rod bipolar cells, while PEP 19-like immunoreactivity is distributed in both rod and cone bipolars. PEP19 is also expressed by subsets of amacrine and ganglion cells. The density of PEP19-positive bipolar cells is greater than that of L7-positive bipolar cells, although the density of each is approximately equal in central and peripheral portions of the retina. An antiserum to a fourth Purkinje cell marker, vitamin D-dependent calcium-binding protein-28 kD (CaBP), reveals primarily axonless horizontal cells, but also subsets of rod bipolar, amacrine, and, in the mouse but not in the rabbit, ganglion cells. The processes of immunoreactive cell bodies form discrete bands in the internal plexiform layer, and mixtures of the antisera help distinguish their identity. Thus, these Purkinje cell markers can be used at the electron microscopic level to unravel the extremely complex neuropil of this retinal layer. Furthermore, knowledge of the retinal distribution of this panel of molecules is of general value for future studies of retinal neuronal typology and can serve to map the densities of subsets of bipolar cells throughout the retina. The expression of L7 and PEP19 in bipolar cells and in Purkinje cells suggests a biochemical relationship between these two spatially distant neuronal populations.

Animals↗

PubMed1991

Brain calbindin-D28k and an Mr 29,000 calcium binding protein in cerebellum are different but related proteins: evidence obtained from sequence analysis by tandem mass spectrometry.

A calcium binding protein of Mr 29,000 which cross-reacts with antibodies raised against chick calbindin-D28k was previously reported to be present in rat cerebellum [Pochet, R., Parmentier, M., Lawson, D. E. M., & Pasteels, J. L. (1985) Brain Res. 345, 251-254]. It was suggested that the Mr 29,000 protein represents another form of calbindin-D28k. In our laboratory we were able to identify Mr 28,000 and 29,000 proteins in rat, human, and chick cerebellum by their ability to bind 45Ca in a 45Ca blot assay. Two calcium binding proteins of Mr 27,680 and 29,450 were isolated from rat cerebelli by the use of gel permeation chromatography and preparative gel electrophoresis. After reverse-phase high-performance liquid chromatography (HPLC) the proteins were sequenced. Sequence analysis by tandem mass spectrometry indicated only 52% identity between the rat cerebellar Mr 28,000 and 29,000 proteins. Thus they are not different forms of the same protein, as previously suggested. Eighty-nine percent identity was observed between the rate cerebellar Mr 29,000 protein and chick calretinin [Rogers, J. H. (1987) J. Cell Biol. 105, 1343-1353]. The difference in identity between the rat cerebellar Mr 29,000 protein and chick calretinin may be due to species differences, and thus this protein is most likely rat calretinin. However, barely detectable levels of mRNA and weak immunoreactivity have been reported for calretinin in cerebellum. Since we found the Mr 29,000 calcium binding protein to be an abundant protein in rat cerebellum, these results suggest either posttranscriptional regulation of calretinin in cerebellum or species differences.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1991

Immunoreactive calcium-binding protein (calbindin-D28k) in interneurons and trigeminothalamic neurons of the rat nucleus caudalis localized with peroxidase and immunogold methods.

Calbindin-D28k is a highly abundant protein found in neurons in selected brain regions, including cells in sensory systems of the brainstem. Because of its capacity to bind cytosolic Ca++, calbindin-D28k is thought to contribute to the regulation of compartmental Ca++ concentrations in neurons. In this study of the rat spinal trigeminal nucleus, calbindin-D28k was localized with immunoperoxidase and immunogold methods. Results showed that immunoreactive calbindin-D28k neurons were widely distributed to all regions of the nucleus, but were particularly numerous in the substantia gelatinosa. Some trigemino-thalamic neurons that were identified by retrograde labeling of a conjugated wheat-germ agglutinin with horseradish peroxidase also contained calbindin-D28k immunoreactivity. Most of the calbindin-D28k labeling was found in cell bodies and dendrites. Axon terminals were rarely stained. More discrete labeling with a gold-conjugated second antibody showed that the predominant site of calbindin-D28k was the matrix of the cytoplasm. Gold label was also heavily associated with euchromatin within nuclei. These findings show that immunoreactive calbindin-D28k is localized to both interneurons and projecting neurons of the spinal trigeminal nucleus. Many of these cells are likely to receive glutamatergic afferent inputs, which may act in part by increasing Ca++ flux into the neurons. Calbindin-D28k has a high capacity for buffering Ca++ and under some conditions may protect neurons against glutamate-induced excitotoxicity. We speculate that calbindin-D28k may function to regulate calcium concentrations in spinal trigeminal neurons.

Animals↗