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PubMed1998

Calbindin D28K and parvalbumin gene expression in rat embryonic ventral forebrain grafts.

The present study characterizes expression of calbindin D28K (CB-D28K) and parvalbumin (PV) in ventral forebrain (VFB) grafts placed in the neocortex of adult rats bearing quisqualic acid lesions to the nucleus basalis magnocellularis. Three to nine months after transplantation surgery, rats were killed for in situ hybridization with probes to CB-D28K or PV and for immunohistochemistry with antibodies to CB-D28K or PV. In addition, an antibody to choline acetyltransferase (ChAT) was used to characterize the cholinergic component in the graft and an antibody to tyrosine hydroxylase (TH) to explore catecholaminergic innervation of the graft. Quantitative analysis of CB-D28K and PV messenger ribonucleic acid (mRNA) was based on counts of silver grains generated by emulsion autoradiography. Cells expressing CB-D28K mRNA were significantly larger than such cells in the adult VFB and the mean number of silver grains per cell was significantly greater than to such cells in the adult VFB. The level of CB-D28K mRNA expression as calculated by ratio of silver grains per unit area was also significantly increased. Quantification of PV mRNA showed no significant differences between the cells in the graft and in the adult VFB. In order to begin to interpret these findings, a comparison was made with such cells in the VFB of developing rats. Brain sections were sampled from embryonic day 17 and postnatal days 1, 5, 12, 19 and adult (6-12 months of age). Cells expressing CB-D28K mRNA were detected in ventral forebrain from postnatal day 5 and cells expressing PV mRNA were detected in ventral forebrain from postnatal day 19. In the course of normal development of the ventral forebrain, no CB-D28K cells were found that were as large or expressed such high levels of CB-D28K mRNA as observed in the grafts. We conclude that changes in grafted cells expressing CB-D28K do not reflect an arrest of developmental processes. TH immunohistochemistry revealed lack of catecholaminergic innervation of the graft, whereas adult mediolateral septal cells that express CB-D28K receive such innervation in addition to other neurotransmitter inputs. Imbalance in neurotransmitter inputs to grafted cells expressing CB-D28K is discussed as a possible factor in their increased size and gene expression.

Animals↗
PubMed2005

Neurogenesis and ontogeny of specific cell phenotypes within the hamster suprachiasmatic nucleus.

The hamster suprachiasmatic nucleus (SCN) is anatomically and functionally heterogeneous. A group of cells in the SCN shell, delineated by vasopressin-ergic neurons, are rhythmic with respect to Period gene expression and electrical activity but do not receive direct retinal input. In contrast, some cells in the SCN core, marked by neurons containing calbindin-D28k, gastrin-releasing peptide (GRP), substance P (SP), and vasoactive intestinal polypeptide (VIP), are not rhythmic with respect to Period gene expression and electrical activity but do receive direct retinal input. Examination of the timing of neurogenesis using bromodeoxyuridine indicates that SCN cells are born between embryonic day 9.5 and 12.5. Calbindin, GRP, substance P, and VIP cells are born only during early SCN neurogenesis, between embryonic days 9.5-11.0. Vasopressin cells are born over the whole period of SCN neurogenesis, appearing as late as embryonic day 12.5. Examination of the ontogeny of peptide expression in these cell types reveals transient expression of calbindin in a cluster of dorsolateral SCN cells on postnatal days 1-2. The adult pattern of calbindin expression is detected in a different ventrolateral cell cluster starting on postnatal day 2. GRP and SP expression appear on postnatal day 8 and 10, respectively, after the retinohypothalamic tract has innervated the SCN. In summary, the present study describes the ontogeny-specific peptidergic phenotypes in the SCN and compares these developmental patterns to previously identified patterns in the appearance of circadian functions. These comparisons suggest the possibility that these coincident appearances may be causally related, with the direction of causation to be determined.

Age Factors↗
PubMedDate not supplied

Calbindin D-28K immunoreactivity of human cone cells varies with retinal position.

Calbindin D-28K is a calcium-binding protein found in the cone but not rod photoreceptor cells in the retinas of a variety of species. Recent studies of the monkey retina indicated that calbindin D-28K may be expressed preferentially in non-foveal regions of the retina. In the current studies of human retinas, immunohistochemical experiments demonstrated that calbindin D-28K is reduced or absent in the fovea and parafovea, but prevalent in the perifovea and periphery. These findings were supported by the quantification of calbindin D-28K in 1-mm trephine punches obtained from different regions of the human retina. The specificity of the anti-calbindin D-28K antibodies used in these studies was confirmed by Western blot analysis using purified calbindin D-28K. The protein was purified from retinal tissue and its identity confirmed by partial amino-acid sequence analysis. The expression of calbindin D-28K did not correlate with the spectral properties of the cones, rather to their position in the retina. The study of spatially expressed genes, like the one encoding calbindin D-28K, may help explain the patterns of retinal degeneration seen in some human cone-rod dystrophies.

Aged↗
PubMed2004

Effects of vitamin D compounds on renal and intestinal Ca2+ transport proteins in 25-hydroxyvitamin D3-1alpha-hydroxylase knockout mice.

BACKGROUND: Vitamin D compounds are used clinically to control secondary hyperparathyroidism (SHPT) due to renal failure. Newer vitamin D compounds retain the suppressive action of 1,25(OH)(2)D(3) on the parathyroid glands and may have less Ca(2+)-mobilizing activity, offering potentially safer therapies. METHODS: This study investigated the effect of a single dose of compound (1,25(OH)(2)D(3), 1,24(OH)(2)D(2), or 1alpha(OH)D(2)) on renal and intestinal Ca(2+) transport proteins, including TRPV5 and TRPV6, and serum Ca(2+), in a novel SHPT model, the 25-OH-D(3)-1alpha-hydroxylase knockout mouse, which lacks endogenous 1,25(OH)(2)D(3) and is severely hypocalcemic. Animals were injected intraperitoneally with compound (100 ng/mouse). RESULTS: Serum levels of 1,25(OH)(2)D(3) and 1,24(OH)(2)D(2) peaked at four hours post-injection (pi), then declined rapidly. 1,25(OH)(2)D(2) generated from 1alpha(OH)D(2) peaked at 12 hours pi and then remained stable. Serum Ca(2+) was increased to near-normal within four hours by 1,25(OH)(2)D(3) and 1,24(OH)(2)D(2), and within 12 hours by 1alpha(OH)D(2). 1,25(OH)(2)D(3) and 1,24(OH)(2)D(2) up-regulated duodenal TRPV5 and TRPV6 mRNA to a similar degree within four hours; mRNA levels decreased by 12 hours after 1,24(OH)(2)D(2) treatment, and by 24 hours after 1,25(OH)(2)D(3) treatment. 1,25(OH)(2)D(3) increased kidney levels of TRPV5, calbindin-D(28K), and calbindin-D(9K) mRNA within four hours; 1,24(OH)(2)D(2) did not change kidney TRPV5 levels and modestly increased calbindin D(9K) by 48 hours. 1alpha(OH)D(2) produced later-onset effects, increasing duodenal TRPV6 and calbindin-D(9K) mRNA levels by 12 hours and TRPV5 by 48 hours. CONCLUSION: In kidney, 1alpha(OH)D(2) increased TRPV5, calbindin-D(28K), and calbindin-D(9K) mRNA levels by 12 hours. This study indicates that Ca(2+) transport proteins, including TRPV5 and TRPV6, are differentially up-regulated by vitamin D compounds.

25-Hydroxyvitamin D3 1-alpha-Hydroxylase↗
PubMed1994

Organization and expression of the mouse spot35/calbindin-D28k gene: identification of the vitamin D-responsive promoter region.

Spot35/calbindin-D28k is transcriptionally induced by 1,25-dihydroxyvitamin D3 in the intestine and kidney but not the brain. The structural organization of the mouse calbindin-D28k gene has now been determined and its transcriptional activation examined. Two direct repeat motifs homologous to other vitamin D-responsive elements were identified at positions -2295 to -2281 and -1160 to -1146. Undirectional deletion analyses of the 5' flanking region indicated that the sequence between -1339 and -1038 conferred vitamin D sensitivity on a reporter construct. Furthermore, the proximal (AGGTGAtgaAAGTCA), but not the distal (AGGTGAacaGGTTCA), direct repeat enhanced 1,25(OH)2D3-stimulated transcription in an orientation-dependent manner when placed upstream of the heterologous SV40 promoter. In conclusion, the proximal direct repeat plays a critical role in mediating the vitamin D-induced expression of calbindin-D28k in the kidney.

Animals↗
PubMed1998

Characterization of neocortical non-pyramidal neurons expressing preprotachykinins A and B: a double immunofluorescence study in the rat.

Neurons expressing preprotachykinin A and preprotachykinin B, which are the precursor prepropeptides of substance P and neurokinin B (neuromedin K), respectively, were characterized immunocytochemically in the rat neocortex. Antibodies raised against C-terminal portions of preprotachykinins were used for labeling cell bodies of preprotachykinin-producing neurons. Neurons immunoreactive for preprotachykinin B were encountered four times more frequently in the neocortex than those immunoreactive for preprotachykinin A. Preprotachykinin A-immunoreactive neurons were scattered more frequently in the deep cortical layers (layers IV-VI) than in the superficial layers (layers I-III), whereas preprotachykinin B-immunoreactive neurons were distributed more frequently in the superficial layers than in the deep layers. Almost all preprotachykinin-expressing neurons were immunoreactive for GABA, suggesting that they were non-pyramidal cells. However, co-expression of the two preprotachykinin immunoreactivities in single neurons was not found. Preprotachykinin-expressing neocortical neurons were further examined with markers for subpopulations of GABAergic cortical neurons. Immunoreactivities for parvalbumin, calbindin and somatostatin were found in 69%, 27% and 11%, respectively, of preprotachykinin A-immunoreactive neurons. Conversely, preprotachykinin A-immunoreactive neurons constituted only 6% of parvalbumin-immunoreactive neurons, 4% of calbindin-immunoreactive neurons and 1% of somatostatin-immunoreactive neurons. Immunoreactivities for calretinin, choline acetyltransferase, vasoactive intestinal polypeptide, corticotropin-releasing factor and cholecystokinin were detected in 13-39% of preprotachykinin B-immunoreactive neurons. Preprotachykinin B immunoreactivity was seen in 33% of calretinin-positive neurons, 45% of cholinergic neurons, 47% of vasoactive intestinal polypeptide-positive neurons, 59% of corticotropin-releasing factor-positive neurons and 83% of cholecystokinin-positive neurons. These results indicate that preprotachykinin A- and preprotachykinin B-expressing neurons constitute separate populations of GABAergic non-pyramidal neurons in the neocortex. Since receptors for substance P and neurokinin B are expressed in GABAergic neurons [Kaneko T. et al. (1994) Neuroscience 60, 199-211] and pyramidal neurons [Ding Y. Q. et al. (1996) J. comp. Neurol. 364, 290-310], respectively, cortical neurons may use two separate lines of tachykinin signals; substance P serves as a signal between GABAergic non-pyramidal neurons, whereas neurokinin B acts as a signal of GABAergic neurons to pyramidal neurons.

Amino Acid Sequence↗
PubMed2006

Calcium-binding proteins in GABAergic calyciform synapses of the reticular nucleus.

Large calyciform synapses in the rat reticular thalamic nucleus are characterized by the presence of gamma-aminobutyric acid. Presynaptic terminals are also loaded with calcium-binding proteins such as parvalbumin, calbindin, calretinin and calcineurin. The number of calyciform terminals containing gamma-aminobutyric acid and parvalbumin is 2005 in young adult rats; calbindin is present in 1,500, calretinin in 850 and calcineurin in 560 calyciform terminals. Developmental studies revealed that gamma-aminobutyric acid and calcium-binding proteins are virtually absent from calyciform terminals at birth but their occurrence increased considerably during postnatal life, suggesting increasing regulation of presynaptic calcium signaling during postnatal life. It is concluded that synaptic activity of large calyciform gamma-aminobutyric acid-containing synapses of the reticular thalamic nucleus is mediated, regulated or accompanied by calcium ions.

Age Factors↗
PubMed1998

Synthesis of calbindin-D28K during mineralization in human bone marrow stromal cells.

1alpha,25-Dihydroxyvitamin D3 [1,25(OH)2D3] is known to modulate Ca2+ metabolism in several cell types. Vitamin-D-dependent calcium binding proteins such as calbindin-D28K (28 kDa calcium binding proteins) have been shown to be regulated by 1,25(OH)2D3 but the mechanisms controlling calbindin synthesis are still poorly understood in human osteoblast cell culture models. The human bone marrow stromal cells (HBMSC) described in this paper developed a calcified matrix, expressed osteocalcin (OC), osteopontin (OP) and responded to 1,25(OH)2D3. The expression of vitamin D receptor mRNA was demonstrated by reverse transcription-PCR. Calbindin-D28K protein was identified only in cells arising from the sixth subculture, which exhibited a calcified matrix and all of the osteoblastic markers, e.g. OC and OP. It was demonstrated by dot-immunodetection using immunological probes, and by in situ hybridization using labelled cDNA probes. Moreover, vitamin D3 enhanced calbindin-D28K synthesis as well as OC synthesis and alkaline phosphatase activity. Uptake of 45Ca induced into the matrix by 1,25(OH)2D3 supports the hypothesis that the calcium-enriched matrix could trap calbindin-D proteins. In conclusion, the studies in vitro described in the present paper indicate, for the first time, a possible role of calbindin-D28K in mineralized matrix formation in HBMSC.

Alkaline Phosphatase↗
PubMed1986

1,25-Dihydroxyvitamin D3-mediated intestinal calcium transport. Biochemical identification of lysosomes containing calcium and calcium-binding protein (calbindin-D28K).

A variety of intestinal cell organelles and proteins have been proposed to mediate 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3)-stimulated calcium absorption. In the present study biochemical analyses were undertaken to determine the subcellular localization of 45Ca after calcium transport in vivo in ligated duodenal loops of vitamin D-deficient chicks injected with 1.3 nmol of 1,25-(OH)2D3 or vehicle 15 h prior to experimentation. Separation of Golgi, mitochondria, basal lateral membrane, and lysosome fractions in the epithelial homogenates was achieved by differential sedimentation followed by centrifugation in Percoll gradients and evaluation of appropriate marker enzyme activities. Both vitamin D-deficient and 1,25-(OH)2D3-treated chicks had the highest levels of 45Ca-specific activity in lysosomal fractions. The lysosomes were also the only organelles to exhibit a 1,25-(OH)2D3-mediated difference in calcium content, increasing to 138% of controls. Lysosomes prepared from 1,25-(OH)2D3-treated chicks also contained the greatest levels of immunoreactive calbindin-D28k (calcium-binding protein). Chloroquine, a drug known to interfere with lysosomal function, was tested and found to inhibit 1,25-(OH)2D3-stimulated intestinal calcium absorption. Neither 1,25-(OH)2D3 nor chloroquine affected [3H]2O transport. In additional experiments, microsomal membranes (105,000 X g pellets) were subjected to gradient centrifugation. The highest levels of 45Ca-specific activity and calcium-binding protein in material from 1,25-(OH)2D3-treated chicks were found in fractions denser than endoplasmic reticulum and may represent endocytic vesicles. In studies on intestinal mucosa of 1,25-(OH)2D3-treated birds fractionated after 30 min of exposure to lumenal Ca2+ or Ca2+ plus chloroquine, 45Ca was found to accumulate in lysosomes and putative endocytic vesicles, relative to controls. A mechanism involving vesicular flow is proposed for 1,25-(OH)2D3-mediated intestinal calcium transport. Endocytic internalization of Ca2+, fusion of the vesicles with lysosomes, and exocytosis at the basal lateral membrane complete the transport process.

Acid Phosphatase↗
PubMed1992

1,25-Dihydroxyvitamin D and not calcium is the major regulator of calbindin-D 9-kDa mRNA levels in vivo.

A possible role of calcium in vivo on intestinal calbindin-D 9-kDa mRNA levels has been studied in rats. In vitamin D-deficient rats, a marked increase in dietary calcium has a small but significant effect on calbindin-D 9-kDa mRNA levels, despite a dramatic increase in serum calcium concentration that clearly resulted from increased intestinal absorption of calcium. On the other hand, vitamin D under all circumstances increased calbindin-D 9-kDa mRNA levels, with the greatest levels found in animals on a low calcium diet where little or no calcium is available for absorption. These results strongly support the idea that 1,25-dihydroxyvitamin D is directly responsible for the induction of calbindin-D 9-kDa.

Animals↗
PubMed2000

Dietary short-chain fructooligosaccharides increase calbindin-D9k levels only in the large intestine in rats independent of dietary calcium deficiency or serum 1,25 dihydroxy vitamin D levels.

Dietary short-chain fructooligosaccharides (Sc-FOS) increase mucosal calbindin-D9k (CaBP) levels in the large intestine whereas levels in the small intestine are decreased in rats. In the present study, we investigated the mechanism by which Sc-FOS induce this increase in CaBP in the large intestine by measuring intestinal CaBP levels in rats fed normal and calcium-deficient diets. Dietary groups included a calcium-containing (0.5%) diet with or without Sc-FOS (100 g/kg diet) and a calcium-deficient (abt. 0.01%) diet with or without Sc-FOS (100 g/kg diet). The rats were fed these diets for 10 days following which they were killed and the intestine removed for collection of the entire mucosa which was divided into four segments, i.e., proximal and distal segments of the small intestine, the cecum and the colorectum. Mucosal CaBP and plasma calcium (Ca), 1,25-dihydroxycholecalciferol (1,25(OH)2D3), 25-hydroxycholecalciferol (25(OH)D3), parathyroid hormone (PTH) and calcitonin levels were measured. Feeding of calcium deficient diet resulted in an increase in CaBP levels in the small intestine, but did not influence levels in the large intestine. Moreover, a significant positive correlation between plasma 1,25(OH)2D3 and CaBP levels in the case of both small intestinal segments (proximal, r = 0.77012, p < 0.00007; distal, r = 0.75056, p < 0.00014) was observed, but not in the case of the large intestinal segments. Sc-FOS increased CaBP levels in the large intestine. These results suggest that the large intestinal CaBP levels do not change in response to dietary calcium conditions and are not regulated by circulating 1,25(OH)2D3 indicating that the effect of Sc-FOS on CaBP levels in the large intestine is independent of the action of 1,25(OH)2D3.

Animals↗
PubMed1989

The presence and estrogen control of immunoreactive calbindin-D9k in the fallopian tube of the rat.

The present study was undertaken to localize and investigate the endocrine control of immunoreactive 9K calbindin-D9k in the fallopian tube (oviduct) of the rat. Rat fallopian tubes were excised with the uterus, immediately fixed by freeze-substitution, and processed for immunoperoxidase staining. Staining employed a rabbit antiserum against purified rat intestinal calbindin-D9k and the streptavidin-biotin technique. Calbindin-D9k immunoreactivity was localized to luminal epithelial cells of the fallopian tube of mature rats, with no staining observed in other tissue layers of the tube. Epithelial cells in both the isthmus and the ampulla were positive for calbindin-D9k. In weanling rats, which have little ovarian function but high levels of 1,25-dihydroxyvitamin D, no immunoreactive calbindin-D9k was observed in any part of the tube. However, after daily injections of estradiol (6 micrograms/day) for 3 days, intense staining was observed in the epithelial cells of the immature rat fallopian tube. Progesterone treatment (1 mg/day for 3 days) of immature rats had no effect on calbindin-D9k in fallopian tube. The lumen of the fallopian tube (oviduct) is the key location for fertilization, a process that requires a narrowly defined concentration of extracellular calcium. By analogy to the intestine, calbindin-D9k may play a role in the transcellular movement of calcium across the fallopian tube epithelium in the fallopian tube lumen.

Animals↗
PubMed1989

Extracellular calcium modulates vitamin D-dependent calbindin-D28K gene expression in chick kidney cells.

The effect of extracellular calcium ion (Ca2+) concentration on 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3)-induction of vitamin D-dependent calcium-binding protein (calbindin-D28K) and its mRNA levels was examined in primary chick kidney cells in vitro. When exposed to normal medium Ca2+ (1.0 mM), 1,25-(OH)2D3 increased calbindin-D28K mRNA, as measured by Northern analysis, by 4-10 fold over basal levels by 12 to 24 h after addition of hormone. In the presence of 0.5 mM Ca2+, 1,25-(OH)2D3 induced calbindin-D28K mRNA by only 2 fold, whereas, when cells were exposed to 2 mM Ca2+, the induction was 10-15 fold. This calcium modulation of 1,25-(OH)2D3 induction was also observed at the level of calbindin-D28K protein concentrations as measured by radioimmunoassay. The alterations in medium Ca2+ were not associated with any change in the rate of total RNA or protein synthesis. These studies suggest that both Ca2+ and 1,25-(OH)2D3 participate in the regulation of calbindin-D28K gene expression in the kidney.

Animals↗
PubMed2002

Activity-dependent expression of calbindin in rabbit floccular Purkinje cells modulated by optokinetic stimulation.

Optokinetic stimulation activates visual climbing fiber pathways that synapse upon contralateral floccular Purkinje cells. Long-term horizontal optokinetic stimulation causes a progressive decrease in gain of the optokinetic reflex and leads to the subsequent genesis of a prolonged negative optokinetic afternystagmus. Since the flocculus is involved in adaptation to optokinetic stimulation, we used the technique of differential display reverse transcription-polymerase chain reaction to explore transcriptional changes in the flocculus evoked by long-term optokinetically evoked climbing fiber discharge. Several differentially transcribed gene products were isolated and sequenced. One of these, calbindin mRNA, was expressed in relatively decreased abundance in the flocculus that received increased climbing fiber input. Decreased transcription of calbindin mRNA was confirmed by northern blots. Hybridization histochemistry was used to localize calbindin mRNA to Purkinje cells and confirmed decreased transcription of calbindin mRNA in Purkinje cells located in folium 1 of the flocculus. Western blots and immunohistochemistry localized the climbing fiber-evoked decreased expression of calbindin to Purkinje cells in folia 1 of the flocculus. The expression of four other calcium-binding proteins in the flocculus was not influenced by optokinetic stimulation. Changes in expression of calbindin could be evoked by decreases in intracellular calcium associated with climbing fiber-evoked decreases in Purkinje cell simple spike activity.The application of differential display reverse transcription-polymerase chain reaction has provided a positive screen for several molecules in addition to calbindin whose expression is affected by naturally evoked activity in a major synaptic pathway to the cerebellum. Further experiments will be required to specify the functional role of each of these molecules.

Animals↗
PubMed1991

Calbindin-D9k. A vitamin-D-dependent, calcium-binding protein in mineralized tissues.

This review summarizes current knowledge on the distribution of the vitamin-D-dependent, calcium-binding protein, calbindin-D9k, an indicator of 1,25(OH)2D3 action, in mineralized tissues, with emphasis on the cellular and subcellular distribution of the protein. Light and electron microscopic immunolocalization studies have shown that calbindin-D9k is present in cartilage and bone. In cartilage, it is restricted to the mature chondrocytes. In bone, it is present in osteoblasts and osteocytes. In both, its synthesis is vitamin D dependent. While calbindin-D9k is a strictly cytosolic protein in soft tissues, it has a specific distribution in mineralized tissues and may even be extracellular. Hence, it is restricted to the lateral edges of the longitudinal septa in the epiphyseal cartilage, the same area in which matrix vesicles are found. Calbindin-D9k lies within the matrix vesicles and is also found in the matrix vesicles near the mineralizing front of trabecular and compact bone. It seems to remain in position over the crystallites formed from the matrix vesicles in fully calcified cartilage and in heavily mineralized bone. The protein is also present in teeth, in the ameloblasts of incisors and molars but not in odontoblasts. The data suggest that calbindin-D9k is required for mineral nucleation in the matrix vesicles of epiphyseal cartilage and bone. The synthesis and distribution of calbindin-D9k in normal and rachitic normal bone and cartilage indicate that vitamin D has a direct action on mineralizing tissues.

Animals↗
PubMed1997

Ca2+ and reactive oxygen species in staurosporine-induced neuronal apoptosis.

Staurosporine (0.03-0.5 microM) induced a dose-dependent, apoptotic degeneration in cultured rat hippocampal neurons that was sensitive to 24-h pretreatments with the protein synthesis inhibitor cycloheximide (1 microM) or the cell cycle inhibitor mimosine (100 microM). To investigate the role of Ca2+ and reactive oxygen species in staurosporine-induced neuronal apoptosis, we overexpressed calbindin D28K, a Ca2+ binding protein, and Cu/ Zn superoxide dismutase, an antioxidative enzyme, in the hippocampal neurons using adenovirus-mediated gene transfer. Infection of the cultures with the recombinant adenoviruses (100 multiplicity of infection) resulted in a stable expression of the respective proteins assessed 48 h later. Overexpression of both calbindin D28K and Cu/Zn superoxide dismutase significantly reduced staurosporine neurotoxicity compared with control cultures infected with a beta-galactosidase overexpressing adenovirus. Staurosporine-induced neuronal apoptosis was also significantly reduced when the culture medium was supplemented with 10 or 30 mM K+, suggesting that Ca2+ influx via voltage-sensitive Ca2+ channels reduces this apoptotic cell death. In contrast, neither the glutamate receptor agonist NMDA (1-10 microM) nor the NMDA receptor antagonist dizocilpine (MK-801; 1 microM) was able to reduce staurosporine neurotoxicity. Cultures treated with the antioxidants U-74500A (1-10 microM) and N-acetylcysteine (100 microM) also demonstrated reduced staurosporine neurotoxicity. These results suggest a fundamental role for both Ca2+ and reactive oxygen species in staurosprine-induced neuronal apoptosis.

Animals↗
PubMed1994

Colocalization of neuropeptides with calbindin D28k and NADPH diaphorase in the enteric nerve plexuses of normal human ileum.

BACKGROUND/AIMS: The chemical coding of enteric neurons differs significantly among species. In the present study, the innervation of normal human ileum was characterized with respect to its chemical coding. METHODS: The submucosa was subdivided into zones 1-3 based on its thickness and distribution of ganglia. The neuropeptides, calbindin D28k, and protein gene product 9.5 were identified by immunocytochemistry. Nitric oxide production was identified by nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase histochemistry. RESULTS: Protein gene product 9.5 staining indicated that cell bodies of the submucosa could be subdivided into zones 1-3. Two major groups of submucosal cell bodies contained either substance P/somatostatin/calcitonin gene-related peptide or vasoactive intestinal peptide/neuropeptide Y/calbindin D28k. Gastrin-releasing peptide-containing cell bodies also colocalized with a subgroup of somatostatin cell bodies. No galanin, met-enkephalin, or NADPH diaphorase-positive cell bodies were present. In the myenteric plexus, the two major groups of cell bodies contained either calbindin or NADPH diaphorase. A proportion of the latter group costained with vasoactive intestinal peptide and met-enkephalin. Cell bodies containing substance P, somatostatin, and calcitonin gene-related peptide were present, forming three different subgroups. CONCLUSIONS: Of the species investigated to date, the chemical coding of human ileal cell bodies most closely resembles that of the rat.

Calbindin 1↗
PubMed2002

Localisation of cannabinoid CB(1) receptor immunoreactivity in the guinea pig and rat myenteric plexus.

Activation of cannabinoid CB(1) receptors inhibits gastrointestinal motility, propulsion, and transit, whereas selective antagonism of these receptors has the opposite effects, suggesting the presence of endocannabinoid tone. Supporting evidence for presynaptic CB(1) receptors on myenteric neurons has been found in vitro. In this study, selective CB(1) receptor antibodies and neuronal markers were used to identify and characterise myenteric neurons expressing cannabinoid receptors. Whole mounts of rat and guinea pig myenteric preparations were dually labelled with antibodies against the CB(1) receptor and choline acetyltransferase, neurofilament proteins, calbindin, calretinin, synapsin I, microtubule-associated protein-2, calcitonin gene-related peptide, or substance P. The pattern of CB(1) receptor labelling and the neurochemical classification of CB(1) receptor-positive cells were markedly influenced by the species and fixation procedure. Virtually all choline acetyltransferase-immunoreactive myenteric neurons expressed CB(1) receptors in ganglia from both species. Subpopulations of neurons identified with calbindin, calretinin, and microtubule-associated protein-2 did not express CB(1) receptors. A few calcitonin gene-related peptide- and substance P-positive somata coexpressed CB(1) receptor immunoreactivity but showed little colocalisation on individual fibres. There was a close association between CB(1) receptor immunoreactivity and fibres labelled for synaptic protein, suggesting a role in the modulation of transmitter release. Functional responses to cannabinoids in the presence of hexamethonium suggest further that CB(1) receptors occur on excitatory motoneurons. In conclusion, CB(1) receptors are expressed on a variety of cholinergic sensory, interneuronal, and motor neurons in myenteric ganglia.

Animals↗