Who Wins the Competition: TRPV5 or Calbindin-D28K?
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Effect of chronic metabolic acidosis on calbindin expression along the rat distal tubule.
Calbindin D28k has been reported to be involved in the transcellular calcium transport along the rat distal tubule. It has also been shown that chronic metabolic acidosis (CMA) induces significant hypercalciuria. The present study investigated whether CMA affects the mRNA and the protein expression of calbindin D28k along isolated distal tubule (DT) of rats. The animals were made acidotic by adding 0.28 mol/L NH4Cl to the drinking water for 7 d. This maneuver was associated with an increase in plasma ionized calcium. Inulin clearance experiments demonstrated that metabolic acidosis did not affect GFR, but it significantly increased both total and fractional urinary calcium excretion. To define the role of calbindin D28k, total RNA was extracted from DT, identified, and microdissected from collagenase-treated kidneys. cDNA was synthesized from RNA using reverse transcriptase and oligo(dT)(12-18) primers. Calbindin D28k mRNA abundance was semiquantified by a competitive reverse transcription-PCR, using an internal standard of cDNA that differed from the wild-type calbindin D28k by a deletion of 86 bp. The reverse transcription-PCR was performed starting from the same amount of total RNA. For each set of experiments, control and acidotic rats were studied in parallel. The identity of the DT was further verified by the presence of the thiazide-sensitive NaCl cotransporter (rTSC1) mRNA. Calbindin D28k mRNA abundance was 0.89 +/- 0.21 amol/ng total RNA in DT of CMA rats (n = 5) compared with 0.30 +/- 0.12 amol/ng total RNA of control rats (n = 5) (P < 0.05). Using specific rabbit polyclonal anti-calbindin D28k antibody, Western blotting was performed starting from thin slices of outer cortex. Densitometric analysis revealed that in acidotic rats (n = 7) there was a 17 +/- 5% (P < 0.05) increase in calbindin D28k protein abundance compared with controls (n = 7). These results indicate that in the rat, ammonium chloride loading induces an increase in filtered ionized calcium load that is associated with a significant upregulation of calbindin D28k both at the mRNA and protein level. These last effects will help to reduce the concomitant hypercalciuria, thus mitigating the consequence of CMA on calcium metabolism.
Acidosis↗
Human cortical distal nephron: distribution of electrolyte and water transport pathways.
The exact distributions of the different salt transport systems along the human cortical distal nephron are unknown. Immunohistochemistry was performed on serial cryostat sections of healthy parts of tumor nephrectomized human kidneys to study the distributions in the distal convolution of the thiazide-sensitive Na-Cl cotransporter (NCC), the beta subunit of the amiloride-sensitive epithelial Na channel (ENaC), the vasopressin-sensitive water channel aquaporin 2 (AQP2), and aquaporin 3 (AQP3), the H(+) ATPase, the Na-Ca exchanger (NCX), plasma membrane calcium-ATPase, and calbindin-D28k (CaBP). The entire human distal convolution and the cortical collecting duct (CCD) display calbindin-D28k, although in variable amounts. Approximately 30% of the distal convolution profiles reveal NCC, characterizing the distal convoluted tubule. NCC overlaps with ENaC in a short portion at the end of the distal convoluted tubule. ENaC is displayed all along the connecting tubule (70% of the distal convolution) and the CCD. The major part of the connecting tubule and the CCD coexpress aquaporin 2 with ENaC. Intercalated cells, undetected in the first 20% of the distal convolution, were interspersed among the segment-specific cells of the remainder of the distal convolution, and of the CCD. The basolateral calcium extruding proteins, Na-Ca exchanger (NCX), and the plasma membrane Ca(2+)-ATPase were found all along the distal convolution, and, in contrast to other species, along the CCD, although in varying amounts. The knowledge regarding the precise distribution patterns of transport proteins in the human distal nephron and the knowledge regarding the differences from that in laboratory animals may be helpful for diagnostic purposes and may also help refine the therapeutic management of electrolyte disorders.
Aged↗
Calbindin-D9k and parvalbumin are exclusively located along basolateral membranes in rat distal nephron.
There is strong evidence that vitamin D-dependent Ca(2+)-binding proteins, i.e., calbindin-D9k and calbindin-D28k, facilitate diffusion of Ca2+ through the cytosolic compartment of renal and intestinal cells, which transport Ca2+ transcellularly. In the study presented here, parvalbumin, calbindin-D9k, and calbindin-D28k were localized precisely by immunocytochemistry in rat kidney. Antisera recognizing specifically the thick ascending loop of Henle, the connecting tubules and collecting ducts, and the intercalated cells of the collecting ducts were used to identify different cell types. In rat kidney cortex, parvalbumin and calbindin-D9k colocalized in the thick ascending loop of Henle, the distal convoluted tubule, the connecting tubule, and the intercalated cells of the collecting duct. Strikingly, in all responsive cells, parvalbumin and calbindin-D9k were exclusively present in a thin layer along the basolateral membrane. In contrast, calbindin-D28k was only present in the distal convoluted and connecting tubule, where it was evenly distributed through the cytosol. In conclusion, the exclusive localization of parvalbumin and calbindin-D9k at the basolateral membrane of immunopositive renal cells implies their involvement in the regulation of transport processes located in these membranes rather than a role as intracellular Ca2+ buffer and Ca2+ shuttle between the two opposing membranes.
Animals↗
Renal and intestinal calcium-binding proteins in normal and uraemic rats.
The effect of chronic uraemia on the concentrations of the 28 kDa renal and 9 kDa intestinal calcium-binding proteins (calbindin-D28K and calbindin-D9K) was investigated in rats. Calbindin-D9K was measured by a competitive enzyme-linked immunoadsorbent assay and calbindin-D28K by rocket immunoelectrophoresis. Chronic uraemia was induced by 5/6 nephrectomy and the results were compared to sham-operated animals. Rats were fed on a diet containing 0.9% calcium and 1.2% phosphorous. Plasma creatinine and plasma urea were elevated in the nephrectomized rats (p less than 0.001), while plasma-1,25-dihydroxycalciferol vitamin D and fractional calcium absorption were unchanged. Plasma parathyroid hormone was significantly elevated in the uraemic rats. The concentration of calbindin-D28K in renal tissue was increased (p less than 0.001) in rats with chronic uraemia and a direct correlation was found between renal calbindin-D28K and plasma urea (p less than 0.05). Intestinal calbindin-D9K correlated inversely with plasma creatinine (p less than 0.05), but the mean level of calbindin-D9K was unchanged in this model of moderate chronic uraemia. Thus, different regulatory mechanisms control levels of calbindin-D9K and calbindin-D28K.
Animals↗
Immuno-localization of the calcitriol receptor, calbindin-D28k and the plasma membrane calcium pump in the human eye.
Using immunohistochemical methods, we detected epitopes of the calcitriol receptor, the ATP-dependent plasma membrane calcium pump and the 28kD vitamin D-dependent calcium-binding protein in sections of the human eye. In retinal photoreceptors, vitamin D receptor, plasma membrane calcium pump and calcium-binding protein epitopes were detected in the outer nuclear layer. Epitopes for the vitamin D receptor and the calcium-binding protein were present in the inner and outer segments of the photoreceptors, where visual transduction occurs. All three proteins were detected in some cells of the ganglion cell layer, the inner nuclear layer, and the retinal pigment epithelium. Epitopes for these proteins also were noted in the ciliary body epithelium. VDR epitopes were seen in lens epithelium. Some immunostaining for VDR, PMCA and calbindin-D28k also was present in the endothelium and in the basal epithelium of the cornea. The presence of these proteins in several tissues of the human eye suggests that the proteins may play a role in the cellular physiology of the eye. Their exact functions in the eye remain undetermined.
Antibodies, Monoclonal↗
In situ hybridization of calbindin-D 28 k transcripts in undecalcified sections of the rat continuously erupting incisor.
Calbindin-D9k and calbindin-D-28k genes are useful systems to investigate the tissue- and stage-specificity as well as the hormonal control of gene expression. Since they regulate cellular calcium mobilization, their study may be of interest in mineralized tissues. However, thus far, immunocytochemical labelling has been mainly realized in these systems. In order to set up methods for mRNA investigation, in situ hybridization of calbindin-D28k mRNAs was performed in the continuously erupting incisor of Sprague-Dawley rats (15-, 30-, and 56-day-old). 35S UTP labelled antisense and sense riboprobes specific for brain calbindin-D 28k were used for in situ hybridization. Specific and non-specific signals could not be discerned when studying decalcified samples. In contrast, on sections not pretreated with EDTA, calbindin-D 28k transcripts (in tooth and kidney) appeared strongly labelled with antisense probes, while sense probes provided a negligible background. In ameloblasts, the signal (i.e., calbindin-D 28k mRNA levels) increased during the presecretory stage. Different mRNA gradients and subcellular distribution patterns characterized the secretory and maturation stages. A nuclear labelling was observed, associated with the highest levels of transcripts. These data suggest a developmental control of calbindin-D28k mRNA transcription. Calbindin-D28k gene expression appears to be up-regulated during the initiation of both secretory and maturation stages of enamel mineralization.
Ameloblasts↗
A new histochemical double-stain method using three-dimensional analysis with confocal laser scanning microscopy.
We describe a new technique for immunohistochemical and enzyme-histochemical double staining using confocal laser scanning microscopy in the reflection mode. As an example, we investigated the immunoreactivity for Spot 35-calbindin-D28K, a vitamin D-dependent calcium binding protein, and the enzyme activity for Ca(2+)-ATPase in the rat kidney. The lead precipitation method for Ca(2+)-ATPase was initially used to process kidney slices. Each specimen was then dehydrated and embedded in a water soluble resin. Thin sections were cut from the resin block, and an indirect immunocolloidal gold method with silver enhancement for Spot 35-calbindin-D28K antigen was carried out on the glass slides. Results were then observed by confocal laser scanning microscopy in the reflection mode. The three-dimensional distribution of the reaction products was detected by serial optic slice images. Lead phosphate particles, which represented the location of Ca(2+)-ATPase, were distributed deep in the section. The most intense signals from the silver particles were detected from the surface slice of the section. A stereoscopic image generated from the serial optic slices clearly showed the differences in their distribution.
Animals↗
Comparative immunolocalization of the plasma membrane calcium pump and calbindin D28K in chicken retina during embryonic development.
The immunolocalization of the plasma membrane calcium pump (PMCA) was studied in 4-week-old chick retina in comparison with calbindin D28K (CaBP) immunostaining. We have demonstrated that the monoclonal anti-PMCA antibody SF10 from human erythrocyte plasma membrane cross-reacts with a Ca2+ pump epitope of the cells from the neural retina. The immunolocalization of both proteins was also studied during the embryonic development of the chicken retina. At age 4.5 days, the cells of the retina were faintly immunoreactive to PMCA and CaBP antibodies, but the lack of cellular aggregation and differentiation did not allow discrimination between the two proteins. A clear difference in the localization was seen from the tenth day of development through post-hatching with slight variation. PMCA localized mainly in the outer and inner plexiform layers, in some cells in the ganglion layer, in the nerve fiber layer and slightly in the photoreceptor cells. CaBP was intensely stained in cones, cone pedicles and some amacrine cells. The number of CaBP positive amacrine cells declined after hatching. A few ganglion cells and several nerve fibers were CaBP immunoreactive. The role of these proteins in the early stages of retinal development is unknown, but the results suggest that Ca2+ homeostasis in the retina is well regulated, probably to avoid excessive accumulation of Ca2+, which often leads to neurodegeneration.
Animals↗
Adult-generated neurons in the dentate gyrus send axonal projections to field CA3 and are surrounded by synaptic vesicles.
The subgranule zone of the dentate gyrus in rats has been shown to be proliferative into adulthood and senescence. However, the connectivity of newly generated, identified neurons in the adult has not been definitively described. In the present study, 9 weeks after a series of intraperitoneal injections of 5-bromo-2'-deoxyuridine (BrdU), animals received stereotaxic iontophoretic injections of Fluoro-Gold (FG) into field CA3. Three weeks after FG injections, sections were analyzed for BrdU immunoreactivity (proliferative label), FG retrograde label, and either calbindin-D28k or synaptophysin immunohistochemistry. A large proportion (up to 44%) of BrdU-labeled cells in the dentate gyrus within regions of FG retrograde label incorporated FG. All of the doubly labeled (BrdU-FG) neurons also immunolabeled with the antibody to calbindin-D28k. Many doubly labeled (BrdU-FG) cells were also surrounded in three planes by synaptophysin immunoreactivity. We conclude that newly generated neurons in the dentate gyrus have the correct immunohistochemical profile, send appropriate axonal projections to field CA3, and are surrounded by profiles containing synaptic vesicle proteins.
Animals↗
Calcium binding proteins immunohistochemistry and identification of neurons in the mammalian pineal gland of the African giant rat: Cricetomys gambianus.
The presence of true neurons in the rodent pineal gland is still a matter of controversy. In this work, by using immunohistochemistry with five antibodies against calcium-binding proteins (calbindin-D28k, calretinin, calmodulin, neurocalcin and S-100 beta) and Cricetomys gambianus, a rodent belonging to Muridae family living in Africa, we were able to illustrate the presence of neurons in the pineal gland. Anti-calbindin-D28k and anti-calretinin labelled neurons belonging to two neural ganglia. One ganglion was localized in the anterior part of the gland near the pineal stalk and the other one in the posterior portion of the organ. Immunoreactive neurons are medium in size (15-20 microns) and have long thick processes running towards the stalk. Calretinin and calbindin-D28k positive neurons stained with different intensities. Thin processes were detected by anti-calretinin whereas thick processes were preferentially calbindin-D28k positive. Neurocalcin labelled a few smaller neurons and many thin processes within the ganglion. Calmodulin could not be detected immunochemically. Within the ganglia many astrocytic processes were S-100 beta positive. The afferent and the efferent pathways of the pineal ganglia remain to be elucidated.
Animals↗
Exacerbation of damage and altered NF-kappaB activation in mice lacking tumor necrosis factor receptors after traumatic brain injury.
Tumor necrosis factor alpha (TNFalpha) is widely expressed in both neurons and glia and has been shown to be upregulated after traumatic brain injury (TBI). TNFalpha receptor activation results in activation of the transcription factor nuclear factor kappaB (NF-kappaB), which may serve an antiapoptotic role via the induction of target genes manganese superoxide dismutase (MnSOD) and/or calbindin. In the present study, we used a controlled cortical impact model of TBI with pertinent lines of transgenic mice to combine both morphological characterization and molecular analysis to elucidate the role of TNFalpha after TBI. Measurements of both the lesion volume and the blood-brain barrier breach indicated exacerbations in mice rendered genetically deficient in both the p55 and p75 TNFalpha receptors (TNFR-KO) compared with wild-type animals. Additionally, animals genetically altered to overexpress MnSOD showed a significant decrease in lesion volume compared with that of control littermates, whereas no alterations were observed in mice lacking the calcium-binding protein calbindin D28k. Analysis of NF-kappaB activation and relative levels of MnSOD revealed delayed responses in the injured cortex of TNFR-KO animals compared with wild-type animals, implying that endogenous TNFalpha may be neuroprotective after TBI.
Animals↗
Tissue transglutaminase is increased in Huntington's disease brain.
The polyglutamine-expanded N-terminal region of mutant huntingtin causes neurodegeneration in Huntington's disease (HD). Neuronal intranuclear and cytosolic inclusions composed of mutant huntingtin are found in brains of HD patients. Because tissue transglutaminase cross-links proteins into filamentous aggregates and polypeptide-bound glutamines are primary determining factors for tissue transglutaminase-catalyzed reactions, it has been hypothesized that tissue transglutaminase may contribute to the formation of these aggregates. In this report immunohistochemical and biochemical methods were used to demonstrate that tissue transglutaminase expression and transglutaminase activity are elevated in HD brains in a grade-dependent manner. In the striatum, tissue transglutaminase activity was significantly increased in the grade 3 HD cases compared with controls. When normalized to the neuronal marker calbindin D28k, immunoblot analysis revealed that in the striatum the levels of tissue transglutaminase were significantly increased in all HD cases compared with controls. Immunohistochemical staining of the HD striatum revealed that tissue transglutaminase immunoreactivity was markedly increased in all grades as compared with controls. In the superior frontal cortex, tissue transglutaminase activity was significantly higher in all HD cases as compared with controls. Quantitative analysis of immunoblots demonstrated that tissue transglutaminase levels were elevated in HD grades 2 and 3 cases. Tissue transglutaminase immunoreactivity within the superior frontal neocortex was also greater in all the HD cases compared with controls. These data clearly indicate that tissue transglutaminase is elevated in HD brain and may play a role in the disease process.
Aged↗
Calbindin D28k and calretinin in oxytocin and vasopressin neurons of the rat supraoptic nucleus. A triple-labeling immunofluorescence study.
The aim of the present study was to examine quantitatively whether two calcium-binding proteins, calbindin D28k and calretinin, are localized in oxytocin and vasopressin neurons of the supraoptic nucleus of the male rat. We used a triple-labeling immunofluorescence method with a confocal laser scanning microscope. Of the oxytocin-labeled cells, 70% were stained for both calbindin D28k and calretinin, 15% were stained for only calbindin D28k, 13% were stained for only calretinin, and 2% were stained for neither protein. Of the vasopressin-labeled cells, 73% were stained for neither calbindin D28k nor calretinin, 21% were stained for only calbindin D28k, 4% were stained for only calretinin, and 2% were stained for both proteins. Calbindin D28k and calretinin have been shown previously to contribute to calcium homeostasis by buffering [Ca2+]i. Therefore, these findings suggest that most of the oxytocin neurons may have a higher Ca(2+)-buffering capacity than most of the vasopressin neurons.
Animals↗
Neurones in the adult rat anterior medullary velum.
The presence of neurones in the rat anterior medullary velum (AMV) has been investigated by using antibodies to the calcium-binding proteins, parvalbumin (PV), calretinin (CR), and calbindin-D28k (CB). Disparate populations of mainly GABAergic neurones were located in the rostral and caudal regions of the AMV. The rostral region of the AMV was characterised by GABAergic CR-labelled or PV-labelled neurones. CR-labelled neurones were bipolar or multipolar with round to ovoid somata (diameters between 8 and 12 microm), and rostrocaudally running dendrites forming a network. PV-labelled neurones had round somata (diameters between 6 and 10 microm) and were bi-tufted, with beaded dendrites. Both CR-labelled and PV-labelled dendrites formed punctate pericellular associations with unlabelled somatic profiles. In the caudal region of the AMV, PV-labelled neurones were GABAergic, multipolar cells, having round somata (diameters between 9 and 12 microm), with either beaded or nonbeaded dendrites forming a network of interconnecting dendrites. PV-labelled pericellular associations were made around both PV-labelled and unlabelled somatic profiles. CR labelled unipolar brush cells (UBCs) were not GABAergic. UBCs were characterised by a round to oval somata (10-15 microm in diameter) from which a single primary dendrite emerged to form a distal expansion having small terminal dendrites. From the distal expansion, there also appeared to be CR-labelled processes emanating and extending for up to 250 microm. CB occasionally labelled "Purkinje-like cells" (PLCs). The rat AMV is a more complex structure than first envisaged with the presence of predominantly inhibitory neurones expressing different calcium-binding proteins. Functional and anatomic aspects of this circuitry are further discussed.
Animals↗
Cortical localization of dopamine D4 receptors in the rat brain--immunocytochemical study.
Using polyclonal antibody against dopamine D4 receptor we investigated cortical distribution of D4 receptors, with the special emphasis on regions of the prefrontal cortex. Prefrontal cortex is regarded as a target for neuroleptic drugs, and engaged in the regulation of the psychotic effects of various substances used in the experimental modeling of schizophrenia. Western blot analysis performed on samples from the rat cingulate, parietal, piriform cortices and also striatum revealed that antibody recognized one main band of approximately 40 kD, which corresponds to the predicted molecular weight of D4 receptor protein. In immunocytochemical studies we found D4 receptor-positive neurons in all regions of prefrontal cortex (cingulate, agranular/insular and orbital cortices) and all cortical regions adjacent to prefrontal cortex, such as frontal, parietal and piriform cortex. Substantial number of D4 receptor-positive neurons has also been observed within the striatum and nucleus accumbens. In general, a clear stratification of the D4 receptor-positive neurons was observed in the cortex with the highest density seen in layers II/III and V/VI. D4 immunopositive material was also found in the dendritic processes, particularly clearly visible in the layer II/III. At the cellular level D4 receptor immunoreactivity was seen predominantly on the periphery of the cell body, but a certain population of neurons with clear cytoplasmatic localization was also identified. In addition to cortical distribution of D4 receptor-positive neurons we tried also to define types of neurons expressing D4 receptor protein. In double-labeling experiments, D4 receptor protein was found in nonphosphorylated neurofilament H-positive, calbindin-D28k-positive, as well as parvalbumin-positive cells. Since, used proteins are markers of certain populations of pyramidal neurons and GABA-ergic interneurons, respectively, our data indicate that D4 receptors are located on cortical pyramidal output neurons and their dendritic processes as well as on interneurons. Above localization indicates that D4 receptors are not only directly influencing excitability of cortical inter- and output neurons but also might be engaged in dendritic spatial and temporal integration, required for the generation of axonal messages. Additionally, our data show that D4 receptors are widely distributed throughout the cortex of rat brain, and that their cortical localization exceeds the localization of dopaminergic terminals.
Animals↗
Cholinergic endings on various neurones containing calcium binding proteins and glutamic acid decarboxylase in the hippocampus of the rat.
Immunohistochemical study of the cholinergic innervation of the hippocampal cells containing glutamic acid decarboxylase (GAD) and calcium binding proteins: parvalbumin (PV), calbindin D28k (CB) and calretinin (CR) was conducted on 5 adult rat brains. Analysis of sections with double immunostaining for vesicular acetylcholine transporter (VAChT; the marker of cholinergic cells, fibres and terminals) and respectively either GAD or PV, CB, CR, using confocal laser-scanning microscope shows that the intensive cholinergic innervations receive GAD, PV and CB-positive hippocampal cells. Cholinergic afferentiations of the CR-positive neurones are considerably fewer.
Acetylcholine↗
Differential calbindin-immunoreactivity in dopamine neurons projecting to the rat striatal complex.
The calcium-binding protein calbindin-D28K is an anatomical marker that has been associated with resistance to neurodegeneration and with the electrophysiological characteristics of neurons. In this study, we compared the presence of calbindin in dopamine neurons projecting to three distinct functional regions of the striatal complex: the striatum, and the core and the shell of the nucleus accumbens. After iontophoretic injections of Fluoro-Gold in the dopaminergic terminal fields, the presence of tyrosine hydroxylase and calbindin were immunohistochemically assessed in the mesencephalon. It was found that the proportion of cells expressing calbindin was highest in the dopamine cells projecting to the core (72%), intermediate in the cells projecting to the shell (51%) and lowest in the cells projecting to the dorsolateral striatum (2.6%). These results do not support the idea that calbindin is a sufficient condition to confer resistance to neurodegeneration because shell-projecting neurons seem the most resistant to it. The present data also raise the question of the role of calbindin in the differences in firing characteristics among dopamine neurons projecting to the striatal complex.
Animals↗