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Immunohistochemical cross-reactivity and electrophoretic comigration between calbindin D-27 kDa and visinin.

Calbindin D-27 kDa (previously named vitamin D-CaBP or cholecalcin) and visinin present similitude both for their purification procedure and histochemical localization. We systematically compared by histochemistry calbindin and visinin immunoreactive structures in chick and pigeon retina, in rat cerebellum and kidney and in pigeon cerebellum. The calbindin and visinin immunoreactive structures were identical except in the retina. Preabsorption of anti-visinin with purified chick or rat calbindin suppresses the labelling in every organ studied except in the photoreceptor layer of pigeon and chick retina. Such a persistence of labelling was explained by Western blotting analysis of chick-retina soluble proteins showing a pattern of 7 different proteins recognized by anti-visinin even though only one protein was recognized in rat kidney and cerebellum. Anti-visinin is thus a polyclonal antibody reacting with more than one antigen of the chick retina, one of those antigens being calbindin. Calbindin is the single antigen recognized by anti-visinin in the other tested organs. In conclusion, we present evidence that visinin is a calbindin.

Animals↗

Calcium binding protein in squid brain: biochemical similarity to the 28,000-Mr vitamin D-dependent calcium binding protein (calbindin-D28k).

A calcium binding protein that is biochemically similar to vertebrate 28,000-Mr vitamin D-dependent calcium binding protein (calbindin-D28k) has been purified from squid brain. Squid brain calbindin was found to have an isoelectric point of 5.0, was heat stable up to 60 degrees C, and showed increased electrophoretic mobility in the presence of chelator. Amino acid analysis revealed a high content of glutamic and aspartic acids and a low level of methionine, histidine, and tyrosine, a finding similar but not identical to the composition of vertebrate calbindin-D28k. The molecular weight of the squid protein, determined by Ferguson plot analysis of data obtained from sodium dodecyl sulfate-gel electrophoresis, was calculated to be 25,700, as compared with 27,800 for rat renal calbindin. Immunocytochemical analysis demonstrated immunoreactive protein in a selected population of neurons and fibers in several areas of the molluscan nervous system. This study represents the first purification from an invertebrate of a calcium binding protein that is biochemically similar to vitamin D-dependent calcium binding protein. These results demonstrate that calbindin, although not identical in vertebrates and cephalopods, may be phylogenetically conserved in structure. The restricted distribution of immunoreactive calbindin in both the cephalopod and mammalian brain suggests that the function of neuronal calbindin may also be conserved in evolution.

Amino Acids↗

Neurochemical development of the hippocampal region in the fetal rhesus monkey, III: calbindin-D28K, calretinin and parvalbumin with special mention of cajal-retzius cells and the retrosplenial cortex.

In spite of continuing controversy on the precise function of the calcium-binding proteins expressed in the hippocampal formation, nothing is known about their prenatal development in primates. In this study, calbindin-D28K, calretinin, and parvalbumin were localized in the hippocampal formation of seven rhesus monkey fetuses aged E47 to E90 (term 165 days). All of the three markers were expressed during the first half of gestation in distinct subsets of nonpyramidal neurons: calretinin-containing cells were the most numerous and relatively differentiated contrasting with a more restricted, less mature, parvalbumin-labeled population and a poor calbindin-positive nonpyramidal contingent. The granule cells and pyramidal neurons were calbindin-positive, including the pyramids of CA3 and the subicular complex, in contrast to the situation found in the adult monkey. The presubiculum and retrosplenial cortex, whose merging formed the caudal pole of the hippocampal formation, also expressed precociously the three calcium-binding proteins. A heterogeneous population of Cajal-Retzius-like cells was demonstrated in the marginal zone of the ventral hippocampal formation. The majority co-expressed calbindin-D28K and calretinin and displayed acetylcholinesterase activity but no GABA-like immunoreactivity. Major intrinsic and extrinsic pathways of the hippocampal system (mossy fiber system, alveus, fimbria, angular, and cingular bundles) were immunoreactive for calretinin and/or calbindin. The distinct developmental time course and regional pattern of distribution of calbindin-D28K, calretinin, and parvalbumin in the nonprincipal neurons suggests a precocious but asynchronous prenatal development of different inhibitory circuits in the hippocampal formation of primates. The labeling of several fiber systems in keeping with comparable early events in the entorhinal cortex (Berger et al.: Hippocampus 3:279-305, 1993), suggests the possibility of earlier functional circuits than hitherto inferred from the observations available in rodents, a hypothesis that deserves further investigation.

Animals↗

Localization of parvalbumin, calretinin, and calbindin D-28k in identified extraocular motoneurons and internuclear neurons of the cat.

Calcium-binding proteins have been shown to be excellent markers of specific neuronal populations. We aimed to characterize the expression of calcium-binding proteins in identified populations of the cat extraocular motor nuclei by means of immunohistochemistry against parvalbumin, calretinin, and calbindin D-28k. Abducens, medial rectus, and trochlear motoneurons were retrogradely labeled with horseradish peroxidase from their corresponding muscles. Oculomotor and abducens internuclear neurons were retrogradely labeled after horseradish peroxidase injection into either the abducens or the oculomotor nucleus, respectively. Parvalbumin staining produced the highest density of immunoreactive terminals in all extraocular motor nuclei and was distributed uniformly. Around 15-20% of the motoneurons were moderately stained with antibody against parvalbumin, but their axons were heavily stained, indicating an intracellular segregation of parvalbumin. Colchicine administration increased the number of parvalbumin-immunoreactive motoneurons to approximately 85%. Except for a few calbindin-immunoreactive trochlear motoneurons (1%), parvalbumin was the only marker of extraocular motoneurons. Oculomotor internuclear neurons identified from the abducens nucleus constituted a nonuniform population, because low percentages of the three types of immunostaining were observed, calbindin being the most abundant (28.5%). Other interneurons located within the boundaries of the oculomotor nucleus were mainly calbindin-immunoreactive. The medial longitudinal fascicle contained numerous parvalbumin- and calretinin-immunoreactive but few calbindin-immunoreactive axons. The majority of abducens internuclear neurons projecting to the oculomotor nucleus (80.7%) contained calretinin. Moreover, the distribution of calretinin-immunoreactive terminals in the oculomotor nucleus overlapped that of the medial rectus motoneurons and matched the anterogradely labeled terminal field of the abducens internuclear neurons. Parvalbumin immunostained 42% of the abducens internuclear neurons. Colocalization of parvalbumin and calretinin was demonstrated in adjacent semithin sections, although single-labeled neurons were also observed. Therefore, calretinin is proven to be a good marker of abducens internuclear neurons. From all of these data, it is concluded that parvalbumin, calretinin, and calbindin D-28k selectively delineate certain neuronal populations in the oculomotor system and constitute valuable tools for further analysis of oculomotor function under normal and experimental conditions.

Abducens Nerve↗

Chemically defined neuron groups and their subpopulations in the glomerular layer of the rat main olfactory bulb: III. Structural features of calbindin D28K-immunoreactive neurons.

The present study analyzed three-dimensional structural features and synaptic contacts of morphologically and chemically identified calbindin D28K-immunoreactive neurons in the glomerular layer of the rat main olfactory bulb by means of combined confocal laser scanning light microscopy, high-voltage electron microscopy and electron microscopic serial section/three-dimensional reconstruction. Most of calbindin D28K-immunoreactive neurons were identified as the periglomerular cell type by combined high-voltage electron microscopic and confocal laser scanning light microscopic observations, and the minority were the short-axon cell type and others. The combined confocal laser scanning light microscopic and electron microscopic study revealed that the calbindin D28K-immunoreactive neurons exhibited unique synaptic contact patterns; they received asymmetrical synapses from presumed mitral/tufted dendrites and made conversely symmetrical synapses with them. About 30% of asymmetrical postsynaptic sites and about 40% of symmetrical presynaptic sites formed reciprocal pairs of synapses. Calbindin D28K-immunoreactive dendrites and somata also received synapses from GABA-like-immunoreactive profiles containing numerous pleomorphic, and a few dense-cored, vesicles. On the other hand, surprisingly, calbindin D28K-immunoreactive neurons had almost no synaptic contacts from olfactory nerve terminals. The present study clearly revealed that calbindin D28K-immunoreactive neurons are a type of periglomerular cell involving unique synaptic contacts that have not been reported so far, and thus indicated that so-called periglomerular cells should be heterogeneous in their synaptic connections as well as in their chemical and structural features.

Animals↗

Protein gene product 9.5-like and calbindin-like immunoreactivity in the nasal respiratory mucosa of perinatal humans.

BACKGROUND: Protein gene product 9.5 (PGP) and calbindin-D28k (calbindin) are neuroendocrine markers that have been localized to neuroendocrine cells in the developing tracheobronchial epithelium. Neuroendocrine cells may play some role in the development of the tracheobronchial epithelium. Little is known about the development of the nasal respiratory epithelium (RE). METHODS: Nasal respiratory mucosa from fetal and newborn humans was examined to determine immunoreactivity for PGP and calbindin. RESULTS: At all stages studied, cells of different morphologies displayed PGP-like immunoreactivity (-LI) and calbindin-LI. Columnar immunoreactive cells for both markers predominated, but labeled cells of different shapes were also observed. Most labeled columnar cells were in the RE at its border with olfactory epithelium (OE); a few similarly labeled columnar cells also appeared in this OE. In the lamina propria, PGP-LI was also seen in numerous thin branching fibers. Some of these branches penetrated into the epithelium, where fiber varicosities appeared to contact cells, some of which also exhibited PGP-LI. CONCLUSION: This study demonstrates that during development the human nasal RE contains different cell types, as illustrated by the assortment of epithelial cells displaying PGP-LI and calbindin-LI among unlabeled cells. Because PGP and calbindin immunoreactivities were found within neuroendocrine cells in previous studies, the present results indicate that the developing human nasal RE also may support a number of neuroendocrine cells. Furthermore, at least some of these cells may form synaptic contacts with nerve fibers from outside the epithelium.

Calbindin 1↗

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↗

Calcium-binding protein (28,000 Mr calbindin-D28k) in kidneys of the bullfrog Rana catesbeiana during metamorphosis.

A protein of approximately 28,000 relative molecular mass (Mr) cross-reacting with antiserum against the 28,000-Mr rat renal calcium-binding protein (calbindin-D28k) has been localized in the kidney of a salientian amphibian, Rana catesbeiana. Cells reactive for calbindin-D28k were found in the distal tubule at all stages of metamorphosis by the unlabeled antibody peroxidase-antiperoxidase technique. Adult kidneys appeared to have more calbindin-D28k-positive cells. The renal corpuscle, neck, and proximal tubule were negative. An immunoreactive 28,000-Mr band that comigrated with the band of calbindin-D28k was visualized by the immunoblot technique. The finding of the 28,000-Mr calbindin-D in the anamniotic kidney demonstrates that this calcium-binding protein (CaBP) is phylogenetically older than our previous studies of higher vertebrates had revealed (Rhoten et al., 1985). Although the function of calbindin-D28k in the distal nephron is unknown, this CaBP can now be presumed to have functional significance in the mesonephric as well as the metanephric kidney.

Animals↗

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

In the cerebral cortex, local circuit neurons provide critical inhibitory control over the activity of pyramidal neurons, the major class of excitatory efferent cortical cells. The calcium-binding proteins, calretinin, calbindin, and parvalbumin, are expressed in a variety of cortical local circuit neurons. However, in the primate prefrontal cortex, relatively little is known, especially with regard to calretinin, about the specific classes or distribution of local circuit neurons that contain these calcium-binding proteins. In this study, we used immunohistochemical techniques to characterize and compare the morphological features and distribution in macaque monkey prefrontal cortex of local circuit neurons that contain each of these calcium-binding proteins. On the basis of the axonal features of the labeled neurons, and correlations with previous Golgi studies, calretinin appeared to be present in double-bouquet neurons, calbindin in neurogliaform neurons and Martinotti cells, and parvalbumin in chandelier and wide arbor (basket) neurons. Calretinin was also found in other cell populations, such as a distinctive group of large neurons in the infragranular layers, but it was not possible to assign these neurons to a known cell class. In addition, although the animals studied were adults, immunoreactivity for both calretinin and calbindin was found in Cajal-Retzius neurons of layer I. Dual labeling studies confirmed that with the exception of the Cajal-Retzius neurons, each calcium-binding protein was expressed in separate populations of prefrontal cortical neurons. Comparisons of the laminar distributions of the labeled neurons also indicated that these calcium-binding proteins were segregated into discrete neuronal populations. Calretinin-positive neurons were present in greatest density in deep layer I and layer II, calbindin-immunoreactive cells were most dense in layers II-superficial III, and parvalbumin-containing neurons were present in greatest density in the middle cortical layers. In addition, the relative density of calretinin-labeled neurons was approximately twice that of the calbindin- and parvalbumin-positive neurons. However, within each group of labeled neurons, their laminar distribution and relative density did not differ substantially across regions of the prefrontal cortex. These findings demonstrate that calretinin, calbindin, and parvalbumin are markers of separate populations of local circuit neurons in monkey prefrontal cortex, and that they may be useful tools in unraveling the intrinsic inhibitory circuitry of the primate prefrontal cortex in but normal and disease states.

Animals↗

Effect of parathyroid hormone on renal calbindin-D28k.

The present investigation was conducted to examine the effects of parathyroid hormone (PTH) and parathyroid hormone related peptide (PTHrP) on renal calbindin-D28k in rats. Four groups of studies were performed: (1) parathyroidectomy (PTX) or a sham operation followed by infusion of 1,25-dihydroxyvitamin D (1,25[OH]2D) or vehicle; (2) infusions of PTH(1-34), PTH(1-84), 1,25(OH)2D, or vehicle; (3) infusion of PTHrP(1-34), PTHrP (1-86), PTH(1-34), or vehicle; and (4) injections of calcium or vehicle. PTX reduced renal calbindin-D28k levels even when plasma concentrations of 1,25(OH)2D were kept constant by infusion of 1,25(OH)2D. Infusions of PTH(1-34), PTH(1-84), and 1,25(OH)2D all increased renal calbindin-D28k and plasma calcium, whereas PTHrP(1-34) and PTHrP(1-86) increased renal calbindin-D28k before an increase of plasma calcium took place. Hypercalcemia induced by the injection of calcium did not affect the levels of renal calbindin-D28k. The present data suggest that PTH and PTHrP exert a direct effect on renal calbindin-D28k, which is not mediated by changes of 1,25(OH)2D or calcium.

Animals↗

Calbindin-D28k-containing neurons in the human hypothalamus: relationship to dopaminergic neurons.

Calbindin-D28k is a calcium-binding protein that protects nerve cells from degeneration. It is located in the midbrain dopaminergic neurons that are relatively invulnerable to degeneration in Parkinson's disease. Because the hypothalamic dopaminergic neurons do not degenerate in Parkinson's disease, the present study sought to determine whether these neurons also contain calbindin-D28k. Using immunocytochemical staining with antibodies against calbindin-D28k and tyrosine hydroxylase, and computer imaging techniques, the distributions of calbindin-D28k and tyrosine hydroxylase-containing neurons were mapped. Both neuronal populations were present throughout the rostral-caudal extent of the hypothalamus. However, only in the periventricular region, at the preoptic and anterior hypothalamic levels, was there an overlap in the two cellular distributions. Using the presence of neuromelanin pigment as a marker for dopaminergic neurons, approximately 30% of the dopaminergic neurons contain calbindin-D28k in the periventricular region. These data indicate that a sub-population of hypothalamic dopaminergic neurons contain calbindin-D28k. This finding is discussed in terms of why hypothalamic dopaminergic neurons are resistant to degeneration in Parkinson's disease.

Adult↗

Sexual dimorphism among calbindin-D28K immunoreactive cells in the rat pineal body.

Calbindin antibodies have been used in neuroanatomical studies to give excellent cytoarchitectural staining and visualization of a Golgi-like cellular morphology. Calbindin-D28K immunoreactivity used in rat pineal gland as a marker detected two classes of pineal cells. One class of small cells representing exclusively glial cells was strongly immunoreactive, and presented a large variety of individual shapes. The majority were a pyramidal shape with one or more processes while others displayed a cytoplasmic lipid droplet. Some small cells occurred around pericapillary spaces. The second class of calbindin-D28K positive cells corresponding to type II pinealocytes were characterized by their large size and less intensive labelling. Type II pinealocytes were round or rectangular; the nucleus was infolded and large with a prominent nucleolus. These large cells were preferentially distributed in the vicinity of vessels and assembled in a cluster of more than ten cells. The lack of S-100 and myeloperoxidase immunoreactivities in large calbindin-D28K cells excluded their possible characterization as glial cells and mononuclear phagocytes, while their size (> 15 microns) excluded microglial cells. A sex difference was detected between large calbindin-D28K positive cells. The mean calculated number of large positive cells for males was 6361 +/- 1504 (n = 8) compared to 2162 +/- 1235 (n = 7) for females. No significative difference was detected between males and females for small calbindin-D28K positive cells.

Animals↗

Localization of calbindin-D28k in normal and incised mouse skin: immunohistochemical and immunoblot analysis.

The subcellular localization of the protein calbindin-D28k in normal and incised mouse skin was investigated by immunohistochemical staining and immunoblot analysis. In normal skin, the presence of calbindin-D28k was demonstrated in both the nucleus and the cytoplasm of epidermal keratinocytes. Higher levels of calbindin-D28k were detected in the nucleus than in the cytoplasm. Following incision, the levels of calbindin-D28k were significantly decreased in epidermal keratinocytes, particularly in the nucleus, compared with those in normal skin. The immunohistochemical staining and immunoblot analysis showed nuclear calbindin-D28k to be decreased or absent during a 10-day observation period following skin incision. Based on these findings, it is suggested that calbindin-D28k may be distributed in both the nucleus and cytoplasm of epidermal keratinocytes in mice, and this protein may be involved in active cell proliferation of the epidermis induced by skin incision.

Animals↗

Calbindin D28K as a marker for the degeneration of the striatonigral pathway in Huntington's disease.

An antibody raised against the chick calcium-binding protein calbindin D28K was used in immunohistochemical studies of normal post mortem human brain and the brains of individuals with Huntington's disease. Calbindin D28K immunoreactivity in the caudate nucleus and putamen coincided with the distribution of areas of acetylcholinesterase staining termed the matrix. In the matrix, calbindin D28K immunoreactivity was present in medium-sized neurons, the major neuronal population, however a further minor population of more strongly stained large neurons was detected. In Huntington's disease there was a dramatic loss of the majority of matrix calbindin D28K immunoreactive neurons and a parallel loss of calbindin D28K immunoreactivity from the substantia nigra. In contrast to the medium-sized calbindin D28K immunoreactive neurons which degenerate in Huntington's disease, the larger immunoreactive neurons were relatively preserved.

Acetylcholinesterase↗

Neurofilament M and calbindin D28k are present in mutually exclusive subpopulations of enteric neurons in the rat submucous plexus.

Immunocytochemical methods have been used to examine the localisation of 3 neurofilament proteins and the calcium binding protein, calbindin D28k, in whole mount preparations of the submucous plexus in the Wistar rat. Neurofilament-M (160 kDA protein) was present in 40% of the submucosal neurons, staining fine filaments in the soma and the axonal processes. Calbindin D28k was present in 40% of the submucosal neurons staining both the soma and nerves within the plexus. The neurofilament proteins and calbindin D28k were never observed within the same neurons. Neurofilament-M was co-localised with substance P and calcitonin gene-related peptide but not somatostatin or the other neuropeptides investigated. Calbindin D28k was co-localised with vasoactive intestinal polypeptide and neuropeptide Y. Galanin- and somatostatin-immunoreactive neurons did not contain either the neurofilament proteins or calbindin D28k. The results demonstrate the presence of subsets of submucosal neurons that can be distinguished by the presence of neurofilament-M or calbindin D28k.

Animals↗

Distribution of calbindin-D28K in the brain of the fetal sheep in late gestation.

Immunocytochemical distribution of calbindin-D28K was examined in the brains of fetal sheep at 100-105 and 120-125 days of gestation (dGA) and at term in labor (term approximately 150 dGA) and compared to adult sheep. Brains were perfused, cut frozen and immunostained as freely floating sections with a polyclonal anti-calbindin-D28K antibody. Light microscopy revealed that calbindin-D28K immunogenicity in fetal cerebellum, rhombencephalon, mesencephalon, diencephalon and telencephalon was overall very similar to that seen in the brains of adult sheep. In addition even though fetal sheep peripheral plasma cortisol concentrations increase 10-20 times over the last 3-4 weeks of gestation to peak during labor and delivery, no glucocorticoid dependent increases in calbindin-D28K in the hippocampal formation could be seen in the sheep fetus as have been demonstrated for the adult rat given exogenous glucocorticoids. It is concluded that: (1) the basic pattern of calbindin-D28K distribution seen in adult sheep brains is established by at least 100 dGA in the sheep fetus; and (2) the pattern of calbindin-D28K expression in fetal sheep hippocampal formation is not related to peripheral plasma glucocorticoid concentrations.

Animals↗

Calbindin D28K expression in transfected mouse NIH3T3 cells.

Calbindin D28K (formerly known as vitamin D-dependent calcium binding protein and referred to here as calbindin) is found in a wide variety of tissues, but only in certain cells within those tissues. Apart from its ability to bind calcium, nothing is known about its function in these cells. To investigate its role we have transfected the chick calbindin cDNA into mouse NIH3T3 fibroblasts and established a new cell line where calbindin is permanently expressed. Immunofluorescence studies show that calbindin is distributed throughout the cytoplasm, and treatment of the cells with cycloheximide shows that it has a relatively long half-life within the cell. Measurements of intracellular calcium concentration using Fura-2 suggest that the presence of calbindin within the cells does not affect the increase in intracellular calcium levels which occurs in response to serum stimulation or the rate at which these return to the basal level, but that it may act as a buffer for the entry of extracellular calcium.

3T3 Cells↗

Distribution and co-localization of calbindin D28k with VIP and neuropeptide Y but not somatostatin, galanin and substance P in the enteric nervous system of the rat.

Calbindin D28k, previously demonstrated in the mammalian central nervous system, has been localized to discrete neurons in the enteric nervous system of the rat. Calbindin D28k is present in cell bodies in both the myenteric and submucous plexi and in interganglionic nerve fibers in all regions of the gastrointestinal tract. Immunoreactive nerve fibers were also detected in the mucosal region, although none were observed in the pyloric sphincter, circular or longitudinal muscle layers. The highest concentration of immunoreactivity was present in the submucosal plexus and mucosa of the colon. Western blot analysis of the protein detected by the antiserum confirmed that it comigrated with purified calbindin D28k and the single immunoreactive band seen in extracts from rat brain. The colocalization of calbindin D28k with components of the peptidergic innervation was also investigated. Of the peptides studied the neurons containing both vasoactive intestinal polypeptide and neuropeptide Y in the submucous plexus were seen to exhibit calbindin D28k immunoreactivity. The neurons containing somatostatin, galanin and substance P did not demonstrate co-localization. In the stomach, calbindin D28k was detected within a small number of epithelial cells which were found to correspond to a sub-population of the somatostatin-immunoreactive endocrine cells.

Animals↗