Hamster pulmonary endocrine cells with neural cell adhesion molecule (NCAM) immunostaining

Pulmonary endocrine cells of Syrian golden hamster were stained for neural cell adhesion molecule (NCAM) with indirect fluorescent immunostaining and observed with a confocal laser scanning microscope equipped with an argon laser. Sections 100 m thick of hamster lung fixed with phosphate-buffered 4% paraformaldehyde were prepared. The sections were incubated with rat monoclonal antibody against NCAM, followed by fluorescence-labeled antibody against rat immunoglobulin. Some were doubly immunostained for NCAM and one of the following endocrine markers: neuron-specific enolase, calcitonin gene-related peptide and serotonin. Expression of NCAM in the hamster airway epithelium was seen in cell nests resembling neuroepithelial bodies (NEBs). NCAM immunostaining was positive at the lateral cell borders between the cells composing the nest, but negative at the border with the adjacent, presumably non-endocrine cells. Double immunostaining confirmed that the grouped cells with NCAM immunoreactivity were of an endocrine nature, but that single endocrine cells did not show NCAM immunoreactivity. An electron microscopic study with NCAM immunostaining confirmed the light microscopic study. These suggest that NCAM expression could be important for the morphogenesis of NEBs. A confocal laser microscope was used to make theee-dimensional images of NEBs after NCAM immunostaining and the spatial interaction between NEBs and the surrounding microenvironment was studied.     

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

Calbindin antibodies have been used in neuroanatomical studies to give excellent cytoarchitecural 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 m) 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.     

Dual role of calbindin-D28K in vesicular catecholamine release from mouse chromaffin cells

Calbindin-D(28K) is suggested to play a postsynaptic role in neurotransmission and in the regulation of the intracellular Ca(2+) concentration. However, it is still unclear whether calbindin-D(28K) has a role in the regulation of exocytosis, either as Ca(2+) buffer or as Ca(2+) sensor. Amperometric recordings of catecholamine exocytosis from wild-type and calbindin-D(28K) knockout mouse chromaffin cells reveal a strong reduction in the number of released vesicles, as well as in the amount of neurotransmitter released per fusion event in knockout cells. However, Ca(2+) current recordings and Ca(2+) imaging experiments, including video-rate confocal laser scanning microscopy, revealed that the intracellular Ca(2+) dynamics are remarkably similar in wild-type and knockout cells. The combined results demonstrate that calbindin-D(28K) plays an important and dual role in exocytosis, affecting both release frequency and quantal size, apparently without strong effects on intracellular Ca(2+) dynamics. Consequently, the possibility that calbindin-D(28K) functions not only as a Ca(2+) buffer but also as a modulator of vesicular catecholamine release is discussed.     

Effect of calbindin-D28K on cyclosporine toxicity in cultured renal proximal tubular cells

Cyclosporine A (CsA) is known to have direct toxicity to renal tubular cells. Its toxicity may be mediated by intracellular calcium because CsA increases intracellular calcium concentration and enhances the activities of calcium-dependent calpains and caspases. Calbindin-D28k, a cytosolic calcium binding protein, has been used as an intracellular Ca2+ buffer to reduce calcium-mediated cytotoxicity in non-renal cells such as neuronal cells. We investigated the effects of gene transfer of calbindin-D28k cDNA on CsA cytotoxicity and intracellular calcium concentration ([Ca2+]i) in cultured murine proximal tubular (MCT) cells. A plasmid containing calbindin-D28k cDNA under the control of CMV promoter was transfected to MCT cells with liposomes. Cytotoxicity was assessed by LDH release and cell viability assay, and [Ca2+]i was measured ratiometrically with fura-2. Compared with MCT cells, cells transfected with calbindin-D28k cDNA showed a reduction in LDH release by 27, 30, 32, 33, and 19% (all P < 0.05), respectively, after 24 h exposure to 1, 2.5, 5, 10, and 25 microM CsA. Cell viability after CsA treatment was also significantly higher in CB cells. A mock transfection using plasmid without calbindin-D28k cDNA insert did not affect the LDH release or cell viability after CsA treatment. CsA treatment did not affect the protein and mRNA abundance of transfected calbindin-D28k cDNA. The expression of calbindin-D28k did not affect the baseline [Ca2+]i, but significantly suppressed CsA-induced elevation in [Ca2+]i. The expression of calbindin-D28k in renal tubular cells provides cytoprotective effects against CsA toxicity, probably through its buffering effects on [Ca2+]i.     

Retinal innervation of calbindin-D28K cells in the hamster suprachiasmatic nucleus: ultrastructural characterization

The authors have described a subregion of the hamster hypothalamic suprachiasmatic nucleus (SCN) containing cells that are immunopositive for the cytosolic calcium-binding protein, Calbindin-D28K (CaBP). Several lines of evidence indicate that this region may constitute the site of the pacemaker cells that are responsible for the regulation of circadian locomotor rhythms. First, 79% of the CaBP-immunoreactive (ir) neurons express Fos in response to photic stimulation, indicating that they are close to or part of the input pathway to pacemakers. Second, at the light microscopy level, retinal terminals innervate the CaBP subnucleus. Finally, destruction of this subnucleus renders animals arrhythmic in locomotor activity. In this study, the authors examined the ultrastructural relationship between cholera toxin (CTbeta) labeled retinal fibers and the CaBP-ir subregion within the hamster SCN. CTbeta-ir retinal terminals make primarily axo-somatic, symmetric, synaptic contacts with CaBP-ir perikarya. In addition, retinal terminals form synapses with CaBP processes as well as with unidentified profiles. There are also complex interactions between retinal terminals, CaBP perikarya, and unidentified profiles. Given that axo-somatic synaptic input has a more potent influence on a cell's electrical activity than does axo-dendritic synaptic input, cells of the CaBP subregion of the SCN are ideally suited to respond rapidly to photic stimulation to reset circadian pacemakers.     

Ontogeny of calbindin-D28K and calretinin in developing chick kidney

The ontogeny of two calcium-binding proteins (calbindin-D_28k and calretinin) was studied by immunohistochemical techniques in developing chick kidney. This study showed the presence of calbindin on the 5th incubation day and calretinin on the 7th incubation day in mesonephric distal and connecting tubules, and in the medial wall of the Wolffian duct. At later stages, immunostaining for these two proteins, in particular for calretinin, was also demonstrated in some metanephric proximal tubules. Glomeruli and Bowman's capsules were negative both in the mesonephros and metanephros. The presence of calretinin in the developing kidney has thus been demonstrated for the first time. The early expression of calbindin and calretinin in mesonephric distal tubules suggests their role in regulating the final excretion of calcium. The different patterns of immunoreactivity of the walls of the Wolffian duct can be correlated with their different histogenetic and histological features.     

Ontogeny of calbindin-D28K and calbindin-D9K in the mouse kidney, duodenum, cerebellum and placenta.

The appearance of the calcium-binding proteins (CaBP-D28K and CaBP-D9K) in embryonic mice tissues was determined using a sensitive immunohistochemical assay. CaBP-D28K first appears in myenteric nerve plexuses of the duodenum on day E15, in duodenal villus cells on day E16, in Purkinje cells of the cerebellum on day E19, in cells of the mesonephric duct on day E11 and in the metanephric duct on day E12. CaBP-D9K first appears in enterocytes of the duodenum on day E18, in trophoblastic giant cells (TGC) of the placenta on day E10, and in the metanephric duct on day E15. A differential time of appearance and colocalization of the two CaBPs is demonstrated in the embryonic mouse kidney, suggesting either that vitamin D does not control both CaBPs in the foetus or that the vitamin D control is unequal. The early appearance and location of CaBP-D9K in TGCs may suggest that these cells play an important role in transplacental transfer of calcium.     

Immunohistochemical demonstration of calbindin-D 28K (CABP28K) in the spinal cord motoneurons of teleost fish

Summary The distribution and localization of the calciumbinding protein, calbindin-D 28K (CaBP28K), in the spinal cord motoneurons of larvae of the teleost fish, Apteronotus leptorhynchus (Gymnotidae) and Pollimyrus isidori (Mormyridae), and in the adult goldfish, Carassius auratus (Cyprinidae), were determined by means of immunohistochemistry. Sections of whole larvae and goldfish spinal cord were reacted with a polyclonal antibody to rat renal CaBP28K. CaBP28K was located by the PAP technique (Sternberger). It was found in the soma, dendrites, axons and axon terminals of spinal motoneurons but not in those of electromotoneurons of Apteronotus leptorhynchus, whereas it occurred in both motoneurons and electromotoneurons of the larval electric organ of Pollimyrus isidori. In these species CaBP28K was also present in the electromotoneuron axon terminals that make synaptic contacts with the pedicles of the electrocytes. In adult Carassius auratus, CaBP28K was found in the soma, dendrites and axons of certain spinal motoneurons. The results indicate that, in teleosts, the motoneurons containing CaBP28K may represent a well-defined population within the spinal cord; the role of this protein in these cells remains to be determined.     

Acute actions of 1,25-dihydroxyvitamin D3 upon chick pancreatic calbindin-D28K

We have compared the relative responsiveness of pancreatic, intestinal and renal tissue calbindin-D28K protein content to the stimulatory actions of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] in vitamin D-deficient (-D) chicks. Tissue concentrations of calbindin-D28K were undetectable in the -D chick intestine but present, albeit at low concentrations (less than 1 microgram CaBP/mg protein) in the -D kidney and pancreas. Intestinal, pancreatic and renal calbindin-D28K content was stimulated 318, 9.8 and 2.9 fold respectively, 48 hours after -D chicks received a single dose of 1,25(OH)2D3 [6.5 nmol/animal]. The pancreatic calbindin-D28K content could be significantly stimulated as early as 5 hours after 1,25(OH)2D3 administrations in vivo. These findings support the contention that the pancreas is a target for vitamin D, and is consistent with the view that calbindin-D28K plays a role in normal pancreatic functions.     

Structural insights into the calcium-dependent interaction between calbindin-D28K and caspase-3

The regulation of apoptosis involves a complicated cascade requiring numerous protein interactions including the pro-apoptotic executioner protein caspase-3 and the anti-apoptotic calcium-binding protein calbindin-D28K. Using isothermal titration calorimetry, we show that calbindin-D28K binds caspase-3 in a Ca²⁺-dependent fashion. Molecular docking and conformational sampling studies of the Ca²⁺-loaded capase-3/calbindin-D28K interaction were performed in order to isolate potentially crucial intermolecular contacts. Residues in the active site loops of caspase-3 and EF-hands 1 and 2 of calbindin-D28K were shown to be critical to the interaction. Based on these studies, a model is proposed to help understand how calbindin-D28K may deactivate caspase-3 upon binding.

Structured summary of protein interactions

Calbindin-D28K and Caspase-3bind by isothermal titration calorimetry(View interaction)     

Changes in structure and stability of calbindin-D(28K) upon calcium binding

Calbindin-D(28K) is a biologically important protein required for normal neural function and for the transport of calcium in epithelial cells of the intestine and kidney. We have used fluorescence and circular dichroism (CD) spectroscopy to characterize the effects of calcium binding on the structure and stability of calbindin. Ca(2+) titration monitored by fluorescence spectroscopy reveals the presence of two classes of calcium-binding sites with association constants approximately 10(7.5) and approximately 10(8.9)M(-1). CD spectra in the far-UV spectral range show minor changes upon Ca(2+) titration, implying that the secondary structure of calbindin-D(28K) is not greatly affected. On the basis of the CD spectra in the near-UV spectral range, we conclude that the tertiary structure is more sensitive to Ca(2+) addition. The most significant change occurs between pCa 7.0 and pCa 8.0. The variations in the protein thermostability are correlated with those in the near-UV CD spectra. The enthalpy changes upon heat denaturation of calbindin in the apo-state are characteristic of proteins containing several weakly interacting domains with similar thermodynamical properties. Thus, calcium binding by calbindin-D(28K) largely affects the local structure around the aromatic residues and the thermal stability of the protein; the changes in the secondary structure are insignificant.     

Ultrastructural identification of calbindin-D28k-immunoreactive cells in the rat superficial pineal body

The vitamin-D-dependent calcium-binding protein, calbindin-D28k, was detected immunocytochemically in the majority of pericapillary pinealocytes of the rat superficial pineal body. The interstitial cells were calbindin-negative. The significance of these findings is discussed.     

Cloning and analysis of calbindin-D28K cDNA and its expression in the central nervous system

The vitamin D-dependent calcium-binding protein (CaBP), calbindin-D28K (CaBP28K), is present in the central nervous system (CNS), the sensory system, and kidneys of mammals and birds. Recent studies have indicated that several other CaBPs of very similar Mrs are also present in the CNS. This study was carried out to establish the relationship between CaBP28K and other CaBP, particularly spot 35, to provide a basis for further studies on the tissue-specific regulation and distribution of CaBP28K. A cloned pC28 cDNA was isolated from a rat brain expression library using synthetic oligodeoxyribonucleotides (oligos) complementary to rat spot-35 mRNA. This pC28 cDNA had an open reading frame (ORF) of 783 nucleotides (nt) coding for a 261-aa, 30-kDa protein. There was 100% homology between the pC28 sequence and that of the CaBP28K isolated from rat brain cDNA library using a chicken intestinal CaBP28K probe (Hunziker and Schrickel, 1988). Thus the aa and nt sequences of rat CaBP28K and spot 35 are identical. Primer extension studies and Northern analyses show that the major species of CaBP28K mRNA contains a 5'-untranslated region of 132 nt, a coding region of 261 codons and a 3'-untranslated region of 804 nt without the poly(A) tail. The rat CaBP28K probe hybridizes to one major RNA species (1.9 kb) and two minor ones (2.8 and 3.2 kb) in the cerebellum, hippocampus, retina and kidney. This distribution correlates well with the distribution of CaBP28K itself in these organs. Comparison of the genomic organization of the CaBP28K gene with that of other members of the 'EF-hand' CaBP family emphasizes that the CaBP28K gene diverged from the others at the first duplication of the gene encoding one CaBP domain. All the members of the 'EF-hand' gene CaBP family evolved by exon shuffling and specific genomic rearrangements.     

Developmental sex differences in calbindin-D(28K) and calretinin immunoreactivity in the neonatal rat hypothalamus

The proteins calbindin-D(28K) and calretinin buffer intracellular calcium and are speculated to be involved in the integration of neuronal signaling. Using Western blot analysis, we compared the levels of calbindin-D(28K) and calretinin in the developing male and female rat hypothalamus on postnatal days (PN) 0, PN2, PN4, PN6, PN8, and PN10. Analysis of variance (ANOVA) of mean calbindin levels indicated a significant effect of sex (p 相似文献   

Structure, binding interface and hydrophobic transitions of Ca2+-loaded calbindin-D(28K)

Calbindin-D(28K) is a Ca2+-binding protein, performing roles as both a calcium buffer and calcium sensor. The NMR solution structure of Ca2+-loaded calbindin-D(28K) reveals a single, globular fold consisting of six distinct EF-hand subdomains, which coordinate Ca2+ in loops on EF1, EF3, EF4 and EF5. Target peptides from Ran-binding protein M and myo-inositol monophosphatase, along with a new target from procaspase-3, are shown to interact with the protein on a surface comprised of alpha5 (EF3), alpha8 (EF4) and the EF2-EF3 and EF4-EF5 loops. Fluorescence experiments reveal that calbindin-D(28K) adopts discrete hydrophobic states as it binds Ca2+. The structure, binding interface and hydrophobic characteristics of Ca2+-loaded calbindin-D(28K) provide the first detailed insights into how this essential protein may function. This structure is one of the largest high-resolution NMR structures and the largest monomeric EF-hand protein to be solved to date.     

Pulmonary endocrine cells in plexogenic pulmonary arteriopathy

A study of the numbers of pulmonary endocrine cells per cm2 of section of lung obtained at combined heart-lung transplantation in 25 cases of plexogenic pulmonary arteriopathy demonstrated that the peptide which may become unduly prominent in pulmonary arterial disease is bombesin. The type of vascular disease in which bombesin becomes prominent is plexogenic pulmonary arteriopathy, be this primary or secondary to congenital heart disease. The increased prominence of bombesin appears to be related to the stage reached in the arteriopathy. Increased numbers of pulmonary endocrine cells are found in association with classic cellular plexiform lesions with narrow vascular channels. Their numbers are within normal limits when the plexiform lesions are mature with wide vascular channels and narrow intervening septa. The pulmonary endocrine cells are most prominent in the pre-plexiform stage when smooth muscle cells in the inner half of the media of the pulmonary artery show increased electron density, and migrate through gaps in the inner elastic lamina to reach the intima. Here they are transformed into myofibroblasts and proliferate. The migration of muscle cells may be related in some way to long-acting trophic factors released from the pulmonary endocrine cells into the surrounding tissues from which they reach the blood and hence the pulmonary arteries.