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PubMed1997

Modulation and phosphorylation of calbindin-D28K correlates with protein kinase C activation1.

Calbindin-D28K is a vitamin D3 dependent calcium-binding protein expressed in renal distal tubules. We previously reported that 24 h treatment with 1 alpha, 25-dihydroxyvitamin D3 (1,25-D3), the active form of vitamin D3, induces calbindin-D28K and activates protein kinase C (PKC) in MDBK (Madin-Darby bovine kidney) cells. In contrast, 24 h treatment with the phorbol ester 12-O-tetradecanoylphorbol-3-acetate (TPA) downregulates calbindin-D28K and PKC activity. In the present studies, we demonstrate that TPA rapidly enhances calbindin-D28K expression in MDBK cells in the absence of 1,25-D3. The enhancement of calbindin-D28K expression is preceded by activation and translocation of PKC alpha. Further, we show that PKC directly phosphorylates calbindin-D28K in a calcium- and phospholipid-dependent manner in vitro. In MDBK cells, the calbindin-D28K antibody immunoprecipitates a 28 kDa protein for which phosphorylation is enhanced after treatment for 1 h with TPA or 24 h with 1,25-D3. Consistent with amino acid sequence analysis of calbindin-D28K indicating two threonine residues that fit the consensus for PKC phosphorylation, TPA-treated MDBK cells exhibit enhanced expression of a phosphothreonine-containing protein that co-migrates with calbindin-D28K. These studies offer the first report that calbindin-D28K is a phosphoprotein and implicate the PKC signal transduction pathway in its regulation.

Animals↗
PubMedDate not supplied

Ultrastructural morphometric analysis of lipofuscin in pyramidal cells of the human Ammon's horn.

Lipofuscin is a waste product of autolysosomal metabolism. The amount of lipofuscin in the cytoplasm depends on cell type, cell function and age. In most studies, either the fluorescent or the stained component of lipofuscin was investigated. Quantitative morphological investigations of the lipofuscin composition separated into the vacuolar and granular component were missing. For the hippocampal pyramidal cells we have determined the lipofuscin quantity and, separately, the vacuolar and granular component at the ultrastructural level. The hippocampal subfields CA 1, CA 2, CA 3 and CA 4 were observed at the ages 20, 40, 60 and 80 years (+/-3 years). Quantitative determinations of the vacuolar and granular component of neuronal lipofuscin in pyramidal cells were performed with a semi-automatic image analysis system. In CA 1 pyramidal cells the lipofuscin content was significantly lower than in the other sectors, which did not differ significantly in their lipofuscin content. The amount of the granular component in relation to the vacuolar component in CA 1 was larger than in the other sectors. With advancing age the lipofuscin content per cell increased. The vacuolar component of all hippocampal subfields experienced a larger increase than the granular component. Consequently the relation of the vacuolar and granular component changed; the relative amount of the vacuolar component increased, while that of the granular component decreased with age. The differences between sector CA 1 and the other hippocampal subfields were discussed with reference to differences of metabolic and functional activity of the neurons. Cytoprotective factors like Calbindin D28k were discussed for CA 1.

Journal Article↗
PubMed1997

Estrogen responsiveness of renal calbindin-D28k gene expression in rat kidney.

In women, calcium excretion in the urine rises after menopause and falls with estrogen replacement therapy. The amount of calcium lost in the urine following estrogen therapy is less than should occur based on changes in serum calcium and the amount of calcium filtered by the kidney. This suggests there may be a direct effect of estrogen therapy to increase renal calcium reabsorption. Calbindin D28k is a putative calcium ferry protein located in the distal renal tubules which has been shown to increase transcellular calcium transport. We proposed that estrogen loss after menopause may diminish gene expression of renal calbindin D28k and subsequently diminish renal calcium reabsorption. We used the ovariectomized rat model of estrogen deficiency to investigate changes at the messenger RNA level of calbindin D28k in ovariectomized rats (OVX), sham ovariectomized rats (S-OVX), and estrogen treated ovariectomized rats (E-OVX). We have demonstrated that ovariectomy in rats diminishes the gene expression of renal calbindin D28k. The mRNA levels were approximately three times lower in OVX rats than S-OVX rats. Administration of 17 beta estradiol to OVX rats produced a significant increase in mRNA level to greater than the S-OVX rats by 4 h. Measurement of serum 1,25 dihydroxyvitamin D3 showed lower levels in OVX rats than S-OVX rats but no significant change in E-OVX animals. In conclusion, our results indicate that estrogen increases renal calbindin D28k mRNA levels, by a mechanism independent of changes in 1,25 dihydroxyvitamin D3. This may result in increased expression of calbindin D28k protein which may have a role in reducing renal calcium excretion.

Animals↗
PubMed1996

Effects of recombinant human growth hormone and insulin-like growth factor-I, with or without 17 beta-estradiol, on bone and mineral homeostasis of aged ovariectomized rats.

This study aimed to evaluate whether recombinant human growth hormone (rhGH) or insulin-like growth factor-I (rhIGF-I) can reverse or prevent further bone loss in aged osteopenic ovariectomized (OVX) rats and to compare their effects with those of 17 beta-estradiol (E2). Twelve-month-old rats were OVX, remained untreated for 8 weeks, and subsequently received daily subcutaneous (SC) injections of rhGH (75 micrograms/day), rhIGF-I (250 micrograms/day), E2 (1.5 micrograms/day), and their respective combinations during 8 weeks, and were then compared with sham-operated, pretreatment OVX, and saline-treated OVX rats. A single sc injection of rhGH resulted in peak hGH concentrations after 90 minutes, with a half-life of 124 minutes; the highest plasma IGF-I concentrations were reached 45 minutes after rhIGF-I injection (+57% vs. baseline) with a gradual decline thereafter. Measurements included: biochemical parameters of bone remodeling (plasma osteocalcin and urinary pyridinolines); histomorphometry of proximal tibial metaphysis; DXA of femur; biomechanical analysis of femur and fifth lumbar vertebra (L5); plasma 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), and calbindin-D9K in duodenal mucosa. Whereas all E2-treated OVX rats had much suppressed bone remodeling, rhGH or rhIGF-I had no effect on any biochemical or histomorphometrical parameter of remodeling. The bone mineral density (BMD) at the distal femoral metaphysis as well as parameters of strength at L5 were maintained at pretreatment values in OVX rats treated with E2, GH, or IGF-I, but not in saline-treated OVX rats; their effects were not additive, however. Trabecular bone volume in the tibial metaphysis was also higher in rats treated with these agents than in saline-treated rats, but this was more apparent at the primary than at the secondary spongiosa, suggesting that their mechanism of action is on primary spongiosa formation or breakdown. E2 alone was ineffective to augment the BMD at the femoral diaphysis; however, the diaphyseal BMD was 12-14% higher (p < 0.01) after 8 weeks of GH treatment than in pretreatment or saline-treated OVX rats and sham-operated rats, while IGF-I was less effective than GH, GH or IGF-I treatment had no effect on plasma 1,25(OH)2D3 or duodenal calbindin-D9K concentrations, but the combination of GH or IGF-I with E2 potentiated the effect of E2 to stimulate calbindin-D9K concentrations and urinary calcium excretion, indicating "hyperabsorption hypercalciuria." In conclusion, the administration of rhGH and rhIGF-I, like that of E2, into aged OVX rats prevents further loss of bone mass and strength at sites containing trabecular bone. In addition, rhGH increases cortical bone mass above pretreatment values.

Absorptiometry, Photon↗
PubMed1992

Evaluation of a putative vitamin D response element in the avian calcium binding protein gene.

A primary response of the avian intestine to 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] is increased synthesis of a 28-kD calcium-binding protein, calbindin-D28k (CaBP). This study examined whether 1,25-(OH)2D3 regulates CaBP gene transcription by an interaction of the vitamin D receptor (VDR) with a vitamin D-responsive element (VDRE) in the CaBP promoter. A genomic clone of CaBP containing about 1 kb of 5'-flanking DNA and 13 kb of the structural gene was isolated. 5'-Flanking DNA from -320 to -306 had considerable sequence similarity to VDREs identified in other genes. Indeed, a portion of the CaBP gene containing this region (-743 to +47) linked to a growth hormone reporter construct elicited a 1,25-(OH)2D3-dependent, VDR-dependent increase in reporter expression in transiently transfected chicken embryo fibroblasts. However, deletion analysis demonstrated that the sequences responsible for this induction reside 3' to -133 and the putative VDRE at -320 to -306 was not involved in the response. Furthermore, transfection of heterologous promoter constructs consisting of a Ban I fragment (-354 to -252) linked to the Herpes simplex thymidine kinase promoter revealed no effect of this region on reporter expression. Gel mobility shift analysis confirmed that this putative VDRE in the CaBP promoter was not a high-affinity binding site for VDR. Consequently, functional significance with respect to the primary induction of CaBP by 1,25-(OH)2D3 cannot be ascribed to this region of the CaBP promoter.

Animals↗
PubMed1988

Analysis of rat vitamin D-dependent calbindin-D28k gene expression.

We report the use of a cloned cDNA for mammalian calbindin-D28k (28-kDa vitamin D-dependent calcium-binding protein) to study the expression of the rat calbindin gene. Tissue distribution studies, using Northern analysis, indicated that calbindin-D28k-mRNA is detected in rat kidney and brain but is not detected in rat intestine, testes, bone, pancreas, liver, lung, or skeletal muscle. Both rat kidney and brain contain three RNA species (1.9, 2.8, and 3.2 kilobase pairs). The regulation of the gene was characterized by both Northern and slot blot analysis. Hormonal regulation, developmental expression of calbindin-D28k-mRNA, and the effect of dietary alteration were examined. In the kidney all three species of mRNA were dependent on the presence of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) for their induction. The time course of induction of renal calbindin-D28k-mRNA indicated that a significant increase in calbindin-D-mRNA was detectable as early as 2 h following a single injection of 1,25-(OH)2D3 (200 ng/100 g of body weight), reaching a maximum at 12 h. Unlike the kidney high levels of calbindin-D28k-mRNA were observed in the brain of vitamin D-deficient rats. The concentration of calbindin-D28k-mRNA in brain was unchanged after 1,25-(OH)2D3 administration. Developmental studies indicated that calbindin-D-mRNA in rat kidney and brain is present prior to birth but is developmentally regulated in a tissue-specific manner. The most pronounced changes in the abundance of renal calbindin-D28k-mRNA occur between birth and 1 week of age. Unlike the kidney a large increase in brain calbindin-D28k-mRNA occurs at a later time, between 1 and 2 weeks of age (the period of major synapse formation). In dietary alteration studies results of Northern blot analysis indicate that low dietary phosphorus results in increased calbindin-D-mRNA in kidney but not in brain. These studies represent the first analysis of the rat calbindin-D28k gene and its regulation in vivo. Our findings suggest that in rat kidney and brain there are significant differences both in the expression of the gene for calbindin-D28k and its regulation by 1,25-(OH)2D3.

Aging↗
PubMed2005

Protein biomarkers of nephrotoxicity; a review and findings with cyclosporin A, a signal transduction kinase inhibitor and N-phenylanthranilic acid.

Biomarkers of nephrotoxicity range from plasma and urine biochemistry, enzymic assays for brush border and lysosomal markers plus new protein markers by immunoassay. Because of the complexity of the nephron and regional sensitivity to xenobiotics, it is important to co-localise sites of marker release with pathological lesions. Han Wistar rats were treated p.o.for up to 14 days with compounds causing selective nephrotoxicity. Compounds used were cyclosporin A ,a signal transduction inhibitor and N-phenylanthranylic acid (NPAA). Plasma and urine was collected for biochemistry and urinalysis (including proteomics and metabonomics) and at termination kidneys were fixed for standard H&E pathology and immunohistochemistry examinations for D28 k calbindin, calmodulin, phospho-erk, Cox 1, Cox 2 and other markers. Cyclosporin A treatment caused injury to the thick ascending limb (TAL) of the nephron and was associated with a down-regulation of calbindin protein expression in cortical distal tubules (mean score 75% reduction) and TALs (21% reduction). Inhibition of signal transduction used p-erk as a downstream marker of activity. P-erk was highly expressed in the collecting ducts and inhibition of signalling caused a 39% reduction in IHC score. There was no evidence of direct renal injury by there was a hypercalcaemia (9% increase) and hyperphosphataemia (24% increase) at 24 hrs post-dose and metastatic calcification by 7 days. NPAA treatment caused renal papillary necrosis in some treated rats (sometimes unilateral) with some secondary dilation of distal tubules. Unlike NSAID treatment, there was no evidence of Cox 1 or 2 dysregulation on IHC and the Cox1 positive interstitial cells did not loose integrity before the onset of necrosis. There were a number of urinary proteomic and metabonomic alterations which are being characterised. The 3 model nephrotoxicants studied demonstrated the linkage of protein expression on IHC to nephron segment-specific sites as important for urinary biomarker validation and linkage to mechanisms.

Animals↗
PubMed1988

Relationship between the levels of calbindin synthesis and calbindin mRNA in chick intestine. Quantitation of calbindin mRNA.

An RNA-excess filter hybridization assay was established to measure the absolute amount of calbindin mRNA in chick tissues. The tissue with the highest level of mRNA is intestine, followed by kidney and cerebellum; the mRNA was not detected in liver and skin. Calbindin mRNA in intestine and kidney is vitamin D-dependent. The maximum concentration of calbindin and its mRNA found after dosing vitamin D-deficient chicks with dihydroxyvitamin D3 (1,25-(OH)2D3) is less than 5% of that found with vitamin D dosing. Secondary 1,25-(OH)2D3 stimulation produced greatly increased amounts of both calbindin mRNA and the protein, at least reaching levels similar to those found after vitamin D dosing. In this last case, each mucosal cell contains about 2000 calbindin mRNA molecules which are translated at a rate sufficient to account for the levels of calbindin found. Calbindin mRNA is translated most rapidly in the very short time periods after its release into the cytoplasm. 1,25-(OH)2D3 has two effects on calbindin mRNA formation: first, to permit the expression of the calbindin gene and a second effect, of slower onset but more persistent, which increases either the rate of calbindin gene transcription or the stability of calbindin mRNA.

Animals↗
PubMed2004

Chronic cortisol exposure promotes the development of a GABAergic phenotype in the primate hippocampus.

Glucocorticoids regulate plasticity and survival of hippocampal neurons. Aberrant exposure to this steroid hormone can result in neurodegeneration, perhaps secondary to disruption of calcium homeostasis. Calbindin, a calcium-binding protein that buffers excess calcium, may protect against neurodegeneration resulting from overabundance of intracellular calcium. In this study, we examined whether chronic treatment (1 year) with cortisol enhances hippocampal calbindin expression in primates. Calbindin is a marker for inhibitory neurons and the dentate gyrus is known to adopt an inhibitory phenotype in response to extreme conditions such as seizures. Thus, we hypothesized that chronic cortisol exposure may also promote a GABAergic phenotype. Therefore, we examined the expression of the GABA-synthesizing enzyme glutamic acid decarboxylase. The expression of brain-derived neurotrophic factor, which is responsive to glucocorticoids, was also examined. Our results demonstrate significant increases in calbindin, glutamic acid decarboxylase and brain-derived neurotrophic factor in several regions of the primate hippocampus, including the dentate gyrus and CA3, in response to chronic cortisol exposure. These results suggest that chronic cortisol exposure may shift the balance towards a GABAergic phenotype, perhaps as part of a compensatory feedback mechanism to dampen the initial excitatory effects of glucocorticoids in the hippocampus.

Animals↗
PubMed2002

Chemical coding and electrophysiology of enteric neurons expressing neurofilament 145 in guinea pig gastrointestinal tract.

Electrophysiologic recording and indirect immunofluorescence were combined to study localization of the medium-sized neurofilament 145 (NF145) component of the cytoskeleton in morphologically identified neurons in the myenteric and submucosal plexuses of the guinea pig enteric nervous system. Neuronal localization of chemical markers, including calbindin DK28, calretinin, nitric oxide synthase, choline-acetyltransferase, neuropeptide Y, serotonin, neurokinin 1 receptor protein, and somatostatin, was integrated with electrophysiologic and morphologic results for a more complete assessment. NF145 immunoreactivity (-IR) was present in ganglion cells with Dogiel type I morphology in the myenteric plexus of the stomach and small and large intestine. NF145-IR was not found in myenteric ganglion cells with Dogiel type II morphology. NF145-IR was not present in any of the ganglion cells in the submucosal plexus. NF145 was expressed in nerve fibers in both myenteric and submucosal plexuses. The majority of these fibers were identified as sympathetic postganglionic axons based on their disappearance in organotypic culture and on their expression of tyrosine hydroxylase. The myenteric ganglion cells with NF145-IR had electrophysiologic properties of S-type enteric neurons. NF145-IR was found in neurons with vasoactive intestinal peptide, serotonin, nitric oxide synthase, somatostatin, and neurokinin 1 receptor but not with neuropeptide Y or calbindin. The results in general suggest that NF145 is localized to distinct subsets of myenteric motor neurons and interneurons. Absence of NF145 from ganglion cells in the submucosal plexus is an example of differences between myenteric and submucosal components of the enteric nervous system.

Action Potentials↗
PubMed1992

Local chromatin changes accompany the expression of the calbindin-D28K gene: tissue specificity and effect of vitamin D activation.

The high affinity calcium-binding protein calbindin-D28K is one of the known proteins transcriptionally up-regulated by the hormonally active form of vitamin D3, 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. This regulation is tissue specific, since in the absence of 1,25-(OH)2D3, the expression of calbindin-D28K is virtually abolished in intestine, whereas it is decreased, but clearly detectable, in kidney, and it remains present at its highest level in cerebellum. Several studies have shown that there is a strong correlation between an increase in the sensitivity to nuclease digestion of a given gene locus and its potential for transcription. Furthermore, hypersensitive sites have often been mapped to regions of DNA including or surrounding sequences known to be important for the regulation of gene transcription. In this study we have scanned the 5'-end and flanking DNA of the calbindin-D28K gene for the presence of DNase-I-hypersensitive (DH) sites in order to localize possible regulatory regions involved in the tissue-specific and hormone-dependent regulation of this gene. We have found that in tissues where calbindin is not expressed, such as liver, no DH sites could be detected. In cerebellum, the same set of DH sites was observed in the presence or absence of 1,25-(OH)2D3 treatment, reflecting the vitamin D-independent expression of the calbindin gene in this tissue. A more complex pattern of DH sites was found in intestine, independently of the vitamin D status of the animal.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Regulation of calbindin-D28K gene expression in response to acute and chronic morphine administration.

The effect of acute and chronic morphine administration on calbindin-D28K (calbindin) gene expression has been studied. One group of adult male rats received a single injection of morphine (10 mg/kg, s.c.) or saline and were sacrificed 1 or 4 h later. Another group was injected with escalating doses of morphine sulfate twice daily for 15 days to induce tolerance and physical dependence. Rats were sacrificed 1 h after the last injection. In a third group, the effect of naloxone-precipitated withdrawal on gene expression in morphine-addicted rats was also analyzed 1 h after naloxone (1 mg/kg, i.p.). The cerebellum and remaining brain (minus the cerebellum) were removed, and total RNA was extracted and used for analysis. Calbindin mRNA levels in cerebellum were decreased to 30%-40% control at 1 and 4 h after a single morphine injection. Co-administration of the opiate antagonist, naloxone, reversed the effect of morphine. Tolerance developed to the acute effects in that levels were not altered significantly 1 h after morphine injection in chronically-treated rats. Unlike the cerebellum, calbindin mRNA in the remainder of the brain (minus the cerebellum) was unchanged 1 and 4 h following morphine administration to drug-naive rats, but was increased more than 2-fold compared to controls 1 h after morphine injection in chronically treated animals. Naloxone-precipitated withdrawal caused a small (20%) but significant decrease in calbindin mRNA in the cerebellum, with no change in the brain (minus the cerebellum).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2004

Identification of estrogen-responsive genes in the GH3 cell line by cDNA microarray analysis.

To identify estrogen-responsive genes in somatolactotrophic cells of the pituitary gland, a rat pituitary cell line GH3 was subjected to cDNA microarray analysis. GH3 cells respond to estrogen by growth as well as prolactin synthesis. RNAs extracted from GH3 cells treated with 17beta-estradiol (E2) at 10(-9) M for 24 h were compared with the control samples. The effect of an antiestrogen ICI182780 was also examined. The array analysis indicated 26 genes to be up-regulated and only seven genes down-regulated by E2. Fourteen genes were further examined by real-time RT-PCR quantification and 10 were confirmed to be regulated by the hormone in a dose-dependent manner. Expression and regulation of these genes were then examined in the anterior pituitary glands of female F344 rats ovariectomized and/or treated with E2 and 8 out of 10 were again found to be up-regulated. Interestingly, two of the most estrogen-responsive genes in GH3 cells were strongly dependent on E2 in vivo. #1 was identified as calbindin-D9k mRNA, with 80- and 118-fold induction over the ovariectomized controls at 3 and 24 h, respectively, after E2 administration. #2 was found to be parvalbumin mRNA, with 30-fold increase at 24 h. Third was c-myc mRNA, with 4.5 times induction at 24 h. The levels were maintained after one month of chronic E2 treatment. Identification of these estrogen-responsive genes should contribute to understating of estrogen actions in the pituitary gland.

Animals↗
PubMed2006

Sequence of information processing for emotions based on the anatomic dialogue between prefrontal cortex and amygdala.

The prefrontal cortex and the amygdala have synergistic roles in regulating purposive behavior, effected through bidirectional pathways. Here we investigated the largely unknown extent and laminar relationship of prefrontal input-output zones linked with the amygdala using neural tracers injected in the amygdala in rhesus monkeys. Prefrontal areas varied vastly in their connections with the amygdala, with the densest connections found in posterior orbitofrontal and posterior medial cortices, and the sparsest in anterior lateral prefrontal areas, especially area 10. Prefrontal projection neurons directed to the amygdala originated in layer 5, but significant numbers were also found in layers 2 and 3 in posterior medial and orbitofrontal cortices. Amygdalar axonal terminations in prefrontal cortex were most frequently distributed in bilaminar bands in the superficial and deep layers, by columns spanning the entire cortical depth, and less frequently as small patches centered in the superficial or deep layers. Heavy terminations in layers 1-2 overlapped with calbindin-positive inhibitory neurons. A comparison of the relationship of input to output projections revealed that among the most heavily connected cortices, cingulate areas 25 and 24 issued comparatively more projections to the amygdala than they received, whereas caudal orbitofrontal areas were more receivers than senders. Further, there was a significant relationship between the proportion of 'feedforward' cortical projections from layers 2-3 to 'feedback' terminations innervating the superficial layers of prefrontal cortices. These findings indicate that the connections between prefrontal cortices and the amygdala follow similar patterns as corticocortical connections, and by analogy suggest pathways underlying the sequence of information processing for emotions.

Amygdala↗
PubMed1994

Development of specific populations of interneurons in the ventral horn of the embryonic chick lumbosacral spinal cord.

The development, morphological and neurochemical properties of specific populations of interneurons were investigated in the ventral horn of the embryonic and mature chick lumbosacral spinal cord by using pre- and post-embedding immunocytochemical as well as anterograde axonal tracing techniques. We have identified and traced the morphological maturation of the following cell groups: (1) Neurons immunoreactive for calbindin-D 28k (CaB), a calcium-binding protein that has been reported to be a marker of certain subsets of excitatory spinal neurons. We have distinguished and traced the maturation of three CaB-immunoreactive cell groups in the ventral horn; (2) Neurons immunoreactive for GABA and glycine, the two putative inhibitory amino acid neurotransmitters in the spinal cord; (3) Neurons within the nucleus marginalis, a cell group located in the ventrolateral aspect of the white matter in close proximity to the lateral motor column. The characteristic features of the development of these neurons are discussed and correlated with previous neuroanatomical and physiological studies concerning motor functions in the developing chick spinal cord.

Animals↗
PubMed1991

Distribution of the calcium-binding proteins parvalbumin and calbindin-D28k in the sensorimotor cortex of the rat.

This study examined and compared the immunocytochemical distribution of the two calcium-binding proteins parvalbumin and calbindin-D28k in the primary motor and somatosensory areas of the rat neocortex. Parvalbumin-immunoreactive cells were found in all layers of the cortex except layer 1 and reached their peak density in the middle layers. The two cortical areas differed markedly in the number, cell size and morphology of immunoreactive cells. Parvalbumin-positive cells were more than twice as numerous in the somatosensory cortex compared to the motor cortex. In addition, the average size of their cell bodies was 25-30% larger in the somatosensory area. Parvalbumin cells in the motor area represented several classes of nonpyramidal cells, while the somatosensory cortex contained in addition many large cells with thick vertically oriented primary dendrites. Some of these cells resembled regular or inverted pyramidal neurons. Punctate neuropil labeling was much heavier in the upper layers of the somatosensory than in the motor cortex and was especially heavy in layer 4. Dense parvalbumin-positive perisomatic puncta surrounded large, unstained pyramidal cells in layer 5B of the motor cortex. Calbindin-D28k neuronal staining in both areas was confined to two populations. The most prominent was darkly labeled, small nonpyramidal cells confined to two bands in layers 2/3 and 5/6. There was also a lighter stained population composed of many pyramidal cells distributed throughout layers 2 and 3. In addition, the motor area contained a band of lightly stained, large pyramidal cells in layer 5B. Calbindin-D28k neuropil labeling was heaviest in layers 1 to 3. In contrast to parvalbumin, we found only minor differences in distribution, size and morphology of calbindin-D28k cell body or neuropil staining in the two cortical areas. Double-labeling immunocytochemistry showed that the large majority of immunoreactive cells contained only calbindin-D28k or parvalbumin, but a distinct population of multipolar cells in the upper layers of the somatosensory cortex contained both. The clear parcellation of parvalbumin immunoreactivity in the rat neocortex suggests that parvalbumin is preferentially associated with specific neuronal populations and terminals in the somatosensory cortex. The more general and homogeneous labeling of the upper layers of the cortex indicates that calbindin-D28k could be related to the relatively high density of calcium channels or N-methyl-D-aspartate receptors in the superficial layers of the rat cortex.

Animals↗
PubMed1991

Sensitive enzyme immunoassay for human 28 kDa calbindin-D.

A sandwich-type enzyme immunoassay for human 28 kDa vitamin D-dependent calcium binding protein (calbindin-D) was established with a sensitivity of 1 pg/tube. Antisera were generated in rabbits injected with highly purified human kidney calbindin-D, and specific antibodies to calbindin-D were purified by the use of a column of calbindin-D-coupled Sepharose. The purified antibodies showed a single band at the position corresponding to calbindin-D on an immunoblotting test with a crude extract of human kidney. The assay system consisted of polystyrene balls with immobilized F(ab')2 antibodies and the same antibodies labeled with beta-D-galactosidase from Escherichia coli. The assay was specific to 28 kDa calbindin-D, showing no cross-reactivity with other calcium binding proteins such as S-100a0 (alpha alpha), S-100b (beta beta), parvalbumin and calmodulin. The assay was also reproducible (coefficients of variation between assays were less than 10%). With the present method, immunoreactive calbindin-D could be detected in various human tissues, with major concentrations in kidney and brain. The values for immunoreactive calbindin-D in various body fluids of healthy subjects varied from undetectable in serum and semen to 3.8 +/- 2.0 (SD) micrograms/g creatinine in urine and 2.9 +/- 0.8 (SD) micrograms/l in cerebrospinal fluid. Immunohistochemically, the calbindin-D in human kidney was localized in epithelial cells of distal tubules.

Adult↗
PubMedDate not supplied

Age and gender effects on 1,25-dihydroxyvitamin D3-regulated gene expression.

Several factors involved in regulation of bone mineral metabolism were compared in male and female Fischer 344 rats of different ages (1, 2.5, 6, and 18 months). Plasma 1,25-(OH)2D3 concentrations decreased with age in rats of both genders. Abundance of calbindin-D28K and its mRNA in kidney and calbindin-D9K and its mRNA in duodenum also decreased with age in both male and female rats. Renal 24-hydroxylase activity and 24-hydroxylase mRNA content were elevated significantly in 18-month-old males and females, compared with younger ages. These data suggest that increased renal catabolism of 1,25-(OH)2D3 may be responsible for low plasma 1,25-(OH)2D3 concentrations observed in older animals. Plasma PTH and 1,25-(OH)2D3 concentrations, renal 24-hydroxylase enzyme activity and 24-hydroxylase mRNA content, duodenal 24-hydroxylase mRNA abundance, and duodenal calbindin-D9K and calbindin-D9K mRNA content were greater in males than in females at 2.5 months of age. Lower plasma 1,25-(OH)2D3 concentrations in females seem to explain observed gender differences in expression of 1,25-(OH)2D3-stimulated genes. The combined effects of these gender differences at ages when peak bone density is being developed may contribute to the greater incidence of osteoporosis in females than in males.

Aging↗