Distribution of cholinergic contacts on Renshaw cells in the rat spinal cord: a light microscopic study. (1/134)

1. Cholinergic terminals in the rat spinal cord were revealed by immunohistochemical detection of the vesicular acetycholine transporter (VAChT). In order to determine the relationships of these terminals to Renshaw cells, we used dual immunolabelling with antibodies against gephyrin or calbindin D28k to provide immunohistochemical identification of Renshaw cells in lamina VII of the ventral horn. 2. A total of 50 Renshaw cells were analysed quantitatively using a computer-aided reconstruction system to provide accurate localization of contact sites and determination of somatic and dendritic surface area. Dendrites could be traced for up to 413 microm from the soma in calbindin D28k-identified Renshaw cells and up to 184 microm in gephyrin-identified cells. 3. A total of 3330 cholinergic terminals were observed on 50 Renshaw cells, with a range of 21-138 terminal appositions per cell (mean 66.6 +/- 25.56 contacts per cell). The vast majority (83.5 %) of the terminals were apposed to dendrites rather than the soma. The overall density of cholinergic contacts increased from a little above 1 per 100 microm2 on the soma and initial 25 microm of proximal dendrites to 4-5 per 100 microm2 on the surface of dendritic segments located 50-250 microm from the soma. Single presynaptic fibres frequently formed multiple contacts with the soma and/or dendrites of individual Renshaw cells. 4. VAChT-immunoreactive terminals apposed to Renshaw cells varied in size from 0.6 to 6.9 microm in diameter (mean 2.26 +/- 0.94; n = 986) and were on average smaller than the cholinergic C-terminals apposed to motoneurones, but larger than VAChT-immunoreactive terminals contacting other ventral horn interneurones. 5. The high density and relatively large size of many cholinergic terminals on Renshaw cells presumably correlates with the strong synaptic connection between motoneurones and Renshaw cells. The fact that the majority of contacts are distributed over the dendrites makes the motoneurone axon collateral input susceptible to inhibition by the prominent glycinergic inhibitory synapses located on the soma and proximal dendrites. The relative positions and structural features of the excitatory cholinergic and inhibitory glycinergic synapses may explain why Renshaw cells, although capable of firing at very high frequency following motor axon stimulation, appear to fire at relatively low rates during locomotor activity.  (+info)

Alteration in the expression level of calbindin D28k in the periodontal ligament of the rat molar during experimental tooth movement. (2/134)

The present immunohistochemical study was designed to investigate changes in the distribution and expression level of calbindin D28k in the periodontal ligament during experimental tooth movement in the rat molar to clarify the physiological role of this protein in the ligament. In normal animals, calbindin D28k-like immunoreactivity appeared sparsely in spindle-shaped cells in the alveolar half of the periodontal ligament. Electron microscopic observations showed that these immunoreactive cells were characterized by well-developed rough-surfaced endoplasmic reticulum and phagosomes--which often contained collagen fibers--suggesting that these cells could be categorized as periodontal fibroblasts. Twelve hours following the onset of the experimental tooth movement, cells positive for calbindin D28k increased in number in the periodontal ligament, especially in the alveolar half of the pressured side. Immunoelectron microscopy showed that the calbindin D28k-immunopositive cells had morphological features similar to those of fibroblasts in the normal ligament, and that these cells occasionally made contact with immunonegative macrophage-like cells. Immunopositive cells gradually decreased in number, and the distribution of the cells and intensity of the immunoreactivity returned to normal levels by 14 days following the induction of the experimental tooth movement. The present results suggest that calbindin D28k plays an important role in the homeostasis and cyto-protection of fibroblasts in the periodontal ligament at the initial phase of experimental tooth movement.  (+info)

NMR investigation and secondary structure of domains I and II of rat brain calbindin D28k (1-93). (3/134)

Calbindin D28k, a member of the troponin C superfamily of calcium-binding proteins, contains six putative EF hand domains but binds only four calcium-atoms: one at a binding site of very high affinity and three calcium-atoms at binding sites of lower affinity. The high-affinity site could be located within domain I while domains III, IV, and V bind calcium less tightly. The recombinant protein construct calb I-II (residues 1-93) comprising the first two EF hands affords a unique opportunity to study a pair of EF hands with one site binding calcium tightly and the second site empty. A series of heteronuclear 2D, 3D and 4D high-resolution NMR experiments were applied to calb I-II, and led to the complete assignment of the 1H, 13C and 15N resonances. The secondary structure of the protein was deduced from the size of the 3JHN-Halpha coupling constants, the chemical shift indices of 1Etaalpha, 13Calpha, 13C' and 13Cbeta nuclei and from an analysis of backbone NOEs observed in 3D and 4D NOESY spectra. Four major alpha-helices are identified: Ala13-Phe23, Gly33-Ala50, Leu54-Asp63, Val76-Leu90, while residues Ala2-Leu6 form a fifth, flexible helical segment. Two short beta-strands (Tyr30-Glu32, Lys72-Gly74) are found preceding helices B and D and are arranged in an anti-parallel interaction. Based on these data a structural model of calb I-II was constructed that shows that the construct adopts a tertiary structure related to other well-described calcium-binding proteins of the EF-hand family. Surprisingly, the protein forms a homodimer in solution, as was shown by its NMR characterization, size-exclusion chromatography and analytical ultra-centrifugation studies.  (+info)

Exacerbation of damage and altered NF-kappaB activation in mice lacking tumor necrosis factor receptors after traumatic brain injury. (4/134)

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.  (+info)

Effect of chronic metabolic acidosis on calbindin expression along the rat distal tubule. (5/134)

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.  (+info)

Calbindin D28k-like immunoreactivity during the formation of the enamel-free area in the rat molar teeth. (6/134)

Previous studies have demonstrated the presence of calbindin D28k in the ameloblasts derived from the inner enamel epithelium. The occlusal surfaces of the rodent molars partly lack the enamel covering, which is referred to as enamel-free area (EFA). In the present study, we compared the immunohistochemical localization of calbindin D28k-like immunoreactivity (CB-LI) in the cells at the EFA (EFA cells) and ameloblasts of the rat molar teeth at the light microscopic level. CB-LI was strong in the ameloblasts of the presecretory through the protective stages, while it was faint at the late secretory to transitional stages. However, some mature ameloblasts lacked the immunoreactivity. On the other hand, the majority of EFA cells showed distinct polarization and elongation that were absent in few cells at the early stage of EFA formation. At all stages, the EFA cells adjacent to the ameloblasts showed CB-LI, however, some cells adjacent to the mature ameloblasts lacked the reaction. Intensive CB-LI was demonstrated in EFA cells at the reduced enamel epithelium. These immunohistochemical findings suggest EFA cells have cytochemical properties similar to those of ameloblasts.  (+info)

Calbindin-D28k is expressed in osteoblastic cells and suppresses their apoptosis by inhibiting caspase-3 activity. (7/134)

The rate of osteoblast apoptosis is a critical determinant of the rate of bone formation. Because the calcium-binding protein calbindin-D(28k) has anti-apoptotic properties in neuronal cells and lymphocytes, we searched for the presence of this protein in osteoblastic cells and investigated whether it can modify their response to proapoptotic signals. Calbindin-D(28K) was expressed at low levels in several osteoblastic cell lines and at high levels in primary cultures of murine osteoblastic cells. Transient transfection of rat calbindin-D(28k) cDNA blocked tumor necrosis factor alpha (TNFalpha)-induced apoptosis in osteoblastic MC3T3-E1 cells, as determined by cell viability and nuclear morphology of cells cotransfected with the green fluorescent protein targeted to the nucleus, whereas transfection of the empty vector had no effect. Calbindin-D(28k) levels in several stably transfected MC3T3-E1 lines were directly related to protection from TNFalpha-induced apoptosis. Purified rat calbindin-D(28k) markedly reduced the activity of caspase-3, a critical molecule for the degradation phase of apoptosis, in a cell-free assay. In addition, cell extracts from MC3T3-E1 cells expressing high levels of calbindin-D(28k) decreased caspase-3 activity, compared with extracts from vector-transfected cells. This effect was apparently unrelated to the calcium binding properties of calbindin, as chelation of calcium by EGTA or addition of other calcium-binding proteins such as calbindin-D(9k), S100, calmodulin, and osteocalcin, did not affect caspase-3 activity. Last, calbindin-D(28k) interacts with the active form of caspase-3 as demonstrated by a GST pull-down assay. These results demonstrate that calbindin-D(28k) is a biosynthetic product of osteoblasts with a role in the regulation of apoptosis. They also reveal that the antiapoptotic properties of calbindin-D(28k) may result not only from calcium buffering but also from the ability of the protein to interact with and to inhibit caspase-3 activity, a property that is independent of its calcium binding capability.  (+info)

Calbindin D28k-immunoreactive afferent nerve endings in the laryngeal mucosa. (8/134)

The distribution of the calbindin D28k in the laryngeal sensory structures was studied by immunohistochemistry, immunoelectronmicroscopy, and double immunofluorescence with calretinin-immunoreactivity. Moreover, origin of the nerve endings were observed using retrograde tracer, fast blue. Immunoreactivity for calbindin D28k was found in the various types of nerve endings in the larynx, namely, laminar nerve endings, nerve endings associated with the taste buds, intraepithelial nerve endings, and endocrine cells. The laminar endings with calbindin D28k-immunoreactivity were observed in the subepithelial connective tissue. In some endings, terminals were expanded. The laminar endings were also observed in the perichondrium of the epiglottic cartilage. In the epiglottic and arytenoid epithelia, thick nerve fibers with calbindin D28k-immunoreactivity ascending to taste buds and intragemmal nerve fibers were also observed. Within the epithelial layer, intraepithelial free nerve endings with calbindin D28k-immunoreactivity were observed. Furthermore, diffuse endocrine cells were observed within the laryngeal epithelium. By immunoelectron microscopy, immunoreaction products in the endings mentioned above were localized in the cytoplasm of the axon terminals and nerve fibers which contained with numerous mitochondria. Out of the 100 laminar endings, 18 endings were immunopositive for both calbindin D28k and calretinin, 33 were positive for calbindin D28k but negative for calretinin, and 49 were positive for only calretinin in the double immunofluorescence microscopy. The nerve fibers associated with the taste buds and the free nerve endings, which immunostained for calbindin D28k, were not stained with antibody against calretinin. After injection of the fast blue in the laryngeal mucosa, fast blue-labeled cells were mainly observed in the nodose ganglia. Of the total number of labeled cell in the nodose and dorsal root ganglia at the level C1 to Th2, 65.1% occurred in nodose ganglia (572/879, n = 6). In the nodose ganglia, 79.7% of labeled cells (456/572) were immunoreacted for calbindin D28k. The distribution of calbindin D28k-immunoreactivity may be differnt from that of calretinin. It is suggested that calbindin D28k have regulatory role on intracellular calcium concentration in the laryngeal sensory corpuscles.  (+info)

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