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PubMed1991

Active Ca2+ transport in primary cultures of rabbit kidney CCD: stimulation by 1,25-dihydroxyvitamin D3 and PTH.

Rabbit connecting tubules and cortical collecting ducts, which represent 79 +/- 5% of the calbindin-D28k-containing kidney cells, were isolated by immunodissection from the rabbit kidney superficial cortex and seeded on permeable filters. After 6 days in culture the monolayers developed a potential difference (PD) of -24 +/- 3 mV (lumen negative) and a transepithelial resistance (R) of 284 +/- 19 omega.cm2. Addition of 10(-6) M amiloride to or removal of Na+ from the mucosal side reversed the PD to +6 +/- 4 mV and concomitantly increased R to 660 +/- 122 omega.cm2. The cells developed functional parathyroid hormone (PTH) and arginine vasopressin receptors, but calcitonin receptors were absent. The monolayer actively absorbed Ca2+ against an electrochemical gradient with a rate of 121 +/- 13 nmol.h-1.cm-2. Removal of serosal Na+ inhibited Ca2+ absorption by 63 +/- 8%. Exposure to 10(-7) M 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] for 48 h or 10(-7) M bovine PTH (bPTH)-(1-34) for 1 h, increased transcellular Ca2+ absorption by 53 +/- 13% or 24 +/- 8%, respectively. The effects of 1,25(OH)2D3 and PTH in combination were neither additive nor potentiating. In addition, the cultured cells expressed calbindin-D28k, and, after exposure to 10(-7) M 1,25(OH)2D3 for 48 h, the calbindin-D28k content increased fourfold.

Adrenocorticotropic Hormone↗
PubMed1995

1 alpha, 25-dihydroxyvitamin D3 receptor ontogenesis in fetal renal development.

We used immunohistochemical techniques to examine the distribution of 1 alpha, 25-dihydroxyvitamin D3 receptors (VDR) in developing rat and mouse kidneys and murine metanephric organ culture. In vivo, the patterns of expression in the two species were similar despite the slight difference in gestational periods (rat, 22 days; mouse, 19 days). Starting at gestational day 15, epitopes for VDR were found in cells of branching ureteral buds and in surrounding mesenchyme and at later developmental stages in glomerular visceral and parietal epithelial cells and proximal and distal tubules (DT). Epitopes for the 28-kDa calcium-binding protein (calbindin D28k) were found exclusively in DTs starting at gestational day 19. The pattern of VDR expression during in vitro nephrogenesis in serum-free murine metanephric organ culture paralleled that seen in vivo. At the time of explantation into organ culture (gestational day 13), VDR epitopes were not detected. By 3 days of in vitro development, VDR expression was identical to that found in gestational day 15 metanephroi in vivo. VDR expression after 5 days of in vitro development mirrored the pattern of gestational day 17 metanephroi in vivo. No calbindin D28k epitopes were seen at any in vitro developmental stage studied. We demonstrate for the first time that VDR are present in specific areas of the developing rat and mouse kidney early in gestation. Calbindin D28k appears later in developing rat and mouse kidney and is distributed differently than the VDR. Metanephric organ culture may be a useful model for studying the regulation and function of VDR during early renal development.

Animals↗
PubMed2004

Inactivation of calcium-binding protein genes induces 160 Hz oscillations in the cerebellar cortex of alert mice.

Oscillations in neuronal populations may either be imposed by intrinsically oscillating pacemakers neurons or emerge from specific attributes of a distributed network of connected neurons. Calretinin and calbindin are two calcium-binding proteins involved in the shaping of intraneuronal Ca2+ fluxes. However, although their physiological function has been studied extensively at the level of a single neuron, little is known about their role at the network level. Here we found that null mutations of genes encoding calretinin or calbindin induce 160 Hz local field potential oscillations in the cerebellar cortex of alert mice. These oscillations reached maximum amplitude just beneath the Purkinje cell bodies and are reinforced in the cerebellum of mice deficient in both calretinin and calbindin. Purkinje cells fired simple spikes phase locked to the oscillations and synchronized along the parallel fiber axis. The oscillations reversibly disappeared when gap junctions or either GABA(A) or NMDA receptors were blocked. Cutaneous stimulation of the whisker region transiently suppressed the oscillations. However, the intrinsic somatic excitability of Purkinje cells recorded in slice preparation was not significantly altered in mutant mice. Functionally, these results suggest that 160 Hz oscillation emerges from a network mechanism combining synchronization of Purkinje cell assemblies through parallel fiber excitation and the network of coupled interneurons of the molecular layer. These findings demonstrate that subtle genetically induced modifications of Ca2+ homeostasis in specific neuron types can alter the observed dynamics of the global network.

Action Potentials↗
PubMed2004

Analysis of calcium binding protein immunoreactivity in the claustrum and the endopiriform nucleus of the rabbit.

The present paper describes parvalbumin, calbindin-D28k and calretinin immunoreactivity in the claustrum and endopiriform nucleus of adult rabbits. Studied neuronal populations are characterized by morphological heterogeneity. Four types were identified in each subpopulation of cells containing calcium binding proteins on the basis of the number of processes and their branching pattern. There were no spatial differences in the distribution of cells containing either parvalbumin or calbindin-D28k in the claustrum and endopiriform nucleus. Well documented presence of the various projective zones in the rabbit claustrum did not reflect the specific distribution of neurons containing calcium binding proteins, except those containing calretinin. Their localization may correspond with the limbic zone. We have found that the rabbit claustrum and endopiriform nucleus have different pattern of parvalbumin and calretinin immunoreactivity. The former was more intense in the claustrum and the distribution of cell types was significantly different from that in the endopiriform nucleus. Calretinin-positive cells were observed in the claustrum, while in the endopiriform nucleus they were scarce. The distribution of neither calbindin-D28k-ir neurons nor fibers allowed differentiation of claustrum and endopiriform nucleus. Significant differences between the claustrum and endopiriform nucleus observed in the rabbit might be related with ontogenetic as well as other (functional) factors.

Animals↗
PubMed1999

Early development and composition of the human primordial plexiform layer: An immunohistochemical study.

The early expression of reelin, calcium-binding proteins (calretinin, calbindin, and parvalbumin), and neurofilament proteins have been explored in the developing central nervous system of human embryos and fetuses during the first trimester of gestation. Our objective has been to determine further the nature, developmental roles, and contributions of the early neurons and fibers of the original subpial neuropil, i.e., the primordial plexiform layer (PPL). In young embryos (4-5 weeks old), neurofilament protein-labeled fibers run through the subpial neuropil of the caudal portion of the neural tube, reaching the mesencephalon rostrally. At this age, calretinin-immunoreactive and calbindin-immunoreactive neurons are also found among cells already detached from the ventricular zone. The expression of neurofilament protein, calretinin, and calbindin follows an ascending caudorostral gradient, reaching the cerebral vesicles by the 6th-7th week of gestation. In the cerebral cortex, this timing coincides with the initial expression of reelin in the PPL. The reelin immunoreactivity throughout the most superficial cellular population of the cortical PPL supports the early genesis of Cajal-Retzius cells, around the 6th week of gestation. After the splitting of the PPL by the formation of the cortical plate (7-8 weeks of gestation), reelin-immunoreactive cells remain only in the newly established layer I. This study proposes that an initial PPL may be a universal feature of the developing central nervous system.

Calbindin 2↗
PubMed2002

I(h) channels contribute to the different functional properties of identified dopaminergic subpopulations in the midbrain.

Dopaminergic (DA) midbrain neurons in the substantia nigra (SN) and ventral tegmental area (VTA) are involved in various brain functions such as voluntary movement and reward and are targets in disorders such as Parkinson's disease and schizophrenia. To study the functional properties of identified DA neurons in mouse midbrain slices, we combined patch-clamp recordings with either neurobiotin cell-filling and triple labeling confocal immunohistochemistry, or single-cell RT-PCR. We discriminated four DA subpopulations based on anatomical and neurochemical differences: two calbindin D28-k (CB)-expressing DA populations in the substantia nigra (SN/CB+) or ventral tegmental area (VTA/CB+), and respectively, two calbindin D28-k negative DA populations (SN/CB-, VTA/CB-). VTA/CB+ DA neurons displayed significantly faster pacemaker frequencies with smaller afterhyperpolarizations compared with other DA neurons. In contrast, all four DA populations possessed significant differences in I(h) channel densities and I(h) channel-mediated functional properties like sag amplitudes and rebound delays in the following order: SN/CB- --> VTA/CB- --> SN/CB+ --> VTA/CB+. Single-cell RT-multiplex PCR experiments demonstrated that differential calbindin but not calretinin expression is associated with differential I(h) channel densities. Only in SN/CB- DA neurons, however, I(h) channels were actively involved in pacemaker frequency control. In conclusion, diversity within the DA system is not restricted to distinct axonal projections and differences in synaptic connectivity, but also involves differences in postsynaptic conductances between neurochemically and topographically distinct DA neurons.

Action Potentials↗
PubMed2003

Immunocytochemical localization of neurons containing the AMPA GluR2/3 subunit in the hamster visual cortex.

AMPA glutamate receptors play a crucial role in brain functions such as synaptic plasticity and development. We have studied the chemo-architecture of the AMPA glutamate receptor subtype GluR2/3 in the hamster visual cortex by immunocytochemistry and compared it with the distribution of the calcium-binding proteins, calbindin D28K and calretinin. Anti-GluR2/3-immunoreactive (IR) neurons were predominantly located in layers II/III, V, and VI, and the majority of the labeled neurons were round or oval. However, many pyramidal cells in layer V were also labeled. Two-color immunofluorescence revealed that none of the GluR2/3-IR neurons contained calbindin D28 K or calretinin. Thus specific layers of neurons express the GluR2/3 subunit and these do not correlate with expression of calbindin D28K and calretinin.

Animals↗
PubMed2006

Immunocytochemical evidence that rod-connected horizontal cell axon terminals remodel in response to experimental retinal detachment in the cat.

PURPOSE: Cats have two types of horizontal cell (HC); one is axon-bearing (B-type), the other is axonless (A-type). We have previously described neurite sprouting from HCs in response to experimental retinal detachment. Here we sought to determine whether one or both types elaborate these outgrowths. METHODS: Sections as well as wholemounts of retinas detached for 3, 7 and 28 days together with control retinas were double or triple labeled with antibodies to the calcium binding proteins calretinin and calbindin, to the synaptic vesicle-associated membrane protein 2 (VAMP2), and to the 70 and 200 kDa subunits of the neurofilament protein. Digital immunofluorescence images were collected by both confocal and two-photon microscopy. RESULTS: In control retina, both HC types label with antibodies to calretinin and calbindin D, but only the A-type also intensely labels with the neurofilament protein antibody. After 3, 7 and 28 days of detachment, these staining patterns persist, but there is a moderate upregulation of neurofilament protein in the B-type cell. In the detached retina, HC processes sprout neurites that appear most commonly as a loose array of fine beaded processes rising from the outer plexiform layer (OPL) into the outer nuclear layer (ONL), or, especially at 28 days, as stout unbranching processes that often cross the ONL en route to the subretinal space where some expand and arborize. Both types are strongly calretinin-positive while being somewhat less positive for antibodies to calbindin D and neurofilament protein. Moreover, they all arise from similarly labeled processes in the distal-most domain of the OPL where the narrowly stratified field of axon terminal boutons of the B-type HC normally innervates rod spherules, two to three thousand per cell. Our data indicate that the HC sprouts apparently arise specifically from the axon terminal of the B-type cell since outgrowths were never seen arising from either type of HC perikaryon or from processes identifiable as A-type dendrites. CONCLUSIONS: The data described here point to the specific remodeling of the rod-connected axon terminals of the B-type cell through neurite outgrowth. Rods respond to detachment by withdrawing synaptic terminals from the OPL while cones do not. Those HC outgrowths that terminate within the ONL appear to retain their connection with the retracted terminals. Others apparently have lost their presynaptic targets and cross the ONL in association with hypertrophied Müller cell processes.

Animals↗
PubMed1995

On the structure of the pretectal nuclei of the rat: an immunocytochemical and tracer study.

We have studied the distribution of the calcium-binding proteins parvalbumin, calbindin and calretinin, the NADPH diaphorase activity and the morphology of the commissural neurons, revealed by the stereotactic applications of fluorogold in the pretectal complex of the rat. The histochemical differentiation of the pretectal complex shows a complementary pattern of parvalbumin and calbindin containing cells. Only a few of the neurons in the pretectal complex contain calbindin. Calretinin immunoreactivity is scant and diffuse. The NADPH-diaphorase activity is restricted to neurons and terminals in the nucleus of the optic tract and the dorsal terminal nucleus. Due to numerous active fibers which traverse these nuclei they display a reticular appearance. Commissural neurons constitute 20% of the cell number of the pretectal complex and are restricted to the dorsal and lateral terminal nuclei.

Animals↗
PubMed1998

Hippocampal interneurons expressing glutamic acid decarboxylase and calcium-binding proteins decrease with aging in Fischer 344 rats.

Aging leads to alterations in the function and plasticity of hippocampal circuitry in addition to behavioral changes. To identify critical alterations in the substrate for inhibitory circuitry as a function of aging, we evaluated the numbers of hippocampal interneurons that were positive for glutamic acid decarboxylase and those that expressed calcium-binding proteins (parvalbumin, calbindin, and calretinin) in young adult (4-5 months old) and aged (23-25 months old) male Fischer 344 rats. Both the overall interneuron population and specific subpopulations of interneurons demonstrated a commensurate decline in numbers throughout the hippocampus with aging. Interneurons positive for glutamic acid decarboxylase were significantly depleted in the stratum radiatum of CA1, the strata oriens, radiatum and pyramidale of CA3, the dentate molecular layer, and the dentate hilus. Parvalbumin interneurons showed significant reductions in the strata oriens and pyramidale of CA1, the stratum pyramidale of CA3, and the dentate hilus. The reductions in calbindin interneurons were more pronounced than other calcium-binding protein-positive interneurons and were highly significant in the strata oriens and radiatum of both CA1 and CA3 subfields and in the dentate hilus. Calretinin interneurons were decreased significantly in the strata oriens and radiatum of CA3, in the dentate granule cell and molecular layers, and in the dentate hilus. However, the relative ratio of parvalbumin-, calbindin-, and calretinin-positive interneurons compared with glutamic acid decarboxylase-positive interneurons remained constant with aging, suggesting actual loss of interneurons expressing calcium-binding proteins with age. This loss contrasts with the reported preservation of pyramidal neurons with aging in the hippocampus. Functional decreases in inhibitory drive throughout the hippocampus may occur due to this loss, particularly alterations in the processing of feed-forward information through the hippocampus. In addition, such a profound alteration in interneuron number will likely alter inhibitory control of excitability and neuronal synchrony with behavioral states.

Aging↗
PubMed1994

Identification and characterization of striatal cell subtypes using in vivo intracellular recording and dye-labeling in rats: III. Morphological correlates and compartmental localization.

In the first two reports of this series, in vivo intracellular recording techniques were used to characterize the electrophysiological properties of two types of striatal neurons that had been identified by their distinct response patterns to stimulation of corticostriatal afferents. In this paper, we examined whether cells showing Type I or Type II response patterns also differed with respect to their morphology or compartmental localization by combining intracellular recording and Lucifer yellow staining with immunocytochemical localization of calbindin 28 kd immunoreactivity. In the majority of cases, both Type I and Type II neurons exhibited similar morphological characteristics, with 80% of the Type I cells (13/16) and all of the Type II cells (n = 40) being small or medium spiny neurons. In each case where the morphological class of the cell was different than the spiny cell class, the cell exhibited a Type I response pattern. These spiny neurons had somata that averaged 17.1 +/- 1.3 microns in diameter and gave rise to between four and eight primary dendrites. The axons typically arose from cell bodies (7/13 for Type I and 25/40 for Type II cells) and emitted extensive local axonal collaterals. However, the axons of Type I cells more frequently originated from the dorsal surface of the somata (9/13; 69%), whereas Type II axons more frequently arose from the ventral surface of the somata (25/35; 71%), which may account for their different extracellular waveforms. In coronally sectioned tissue (n = 18), the axons always projected laterally when the somata were located in the medial striatum and projected medially when the somata were in the lateral striatal region. In a subset of experiments (N = 22), Lucifer yellow-stained neurons were localized with respect to their position within the patch and matrix compartments of the striatum using subsequent staining for calbindin 28 kd immunoreactivity. Of the 20 labeled medium spiny neurons examined (Type II: N = 13; Type I: N = 7), 19 were located in the calbindin-positive matrix compartment. The only neuron localized to the patch compartment was a medium spiny cell that exhibited a Type II paired impulse response pattern. In addition, of the two aspiny neurons from this group with beaded dendrites, one was localized to the border between adjacent patch and matrix compartments, whereas the other was located completely within the matrix compartment. Therefore, despite their distinct paired impulse response patterns, the majority of both Type I and Type II neurons were medium spiny cells located in the matrix compartment of the striatum.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1995

Sources of inputs to longitudinal muscle motor neurons and ascending interneurons in the guinea-pig small intestine.

Light- and electron-microscopic studies were used to investigate connections between specific subgroups of neurons in the myenteric plexus of the guinea-pig small intestine. Inputs to two classes of calretinin-immunoreactive (IR) nerve cells, longitudinal muscle motor neurons and ascending interneurons, were examined. Inputs from calbindin-IR primary sensory neurons and from three classes of descending interneurons were studied. Electron-microscopic analysis showed that calbindin-IR axons formed two types of inputs, synapses and close contacts, on calretinin-IR neurons. About 40% of inputs to the longitudinal muscle motor neurons and 70% to ascending interneurons were calbindin-IR. Approximately 50% of longitudinal muscle motor neurons were surrounded by bombesin-IR dense pericellular baskets and 40% by closely apposed varicosities. At the electron-microscope level, the bombesin-IR varicosities were found to form synapses and close contacts with the motor neurons. Dense pericellular baskets with bombesin-IR surrounded 36% of all ascending interneurons, and a further 17% had closely apposed varicosities. Somatostatin- and 5-HT-IR descending interneurons provided no dense pericellular baskets to calretinin-IR nerve cells. Thus, calretinin-IR, longitudinal muscle motor neurons and ascending interneurons receive direct synaptic inputs from intrinsic primary sensory neurons and from non-cholinergic, bombesin-IR, descending interneurons.

Animals↗
PubMed1993

Total numbers of neurons in myenteric ganglia of the guinea-pig small intestine.

Two techniques that are thought to stain all of the neurons in the myenteric ganglia of the intestine are NADH diaphorase histochemistry and immunohistochemistry using a "nerve cell body" antiserum. However, this assumption has never been directly verified. In the present study myenteric ganglia of the guinea-pig ileum were prepared as whole-mounts and stained with either of these techniques. All nerve cells that could be identified in the whole-mounts were counted. The whole-mounts were then embedded flat in resin and serially sectioned at 1 micron. Nerve cells were identified and counted from the serial sections, and the data compared to those obtained from the whole-mounts. NADH diaphorase histochemistry did not reveal all the neurons at incubation times that gave selective staining. In contrast, "nerve cell body" antiserum stained the entire neuronal population. To determine the total number of nerve cell bodies/ganglion and the proportion of nerve cell bodies with calbindin immunoreactivity, whole-mounts that had been processed for calbindin immunohistochemistry were serially sectioned and reconstructed. The total number of neurons per myenteric ganglion was 105 +/- 10 (SE). Calbindin-immunoreactive neurons comprised about 20% of the myenteric neurons, which is considerably less than previous estimates, because previously the total population has been underestimated. The spatial density of myenteric neurons in the undistended ileum of the guinea-pig is 17,300 nerve cells/cm2.

Animals↗
PubMed1992

GABAergic neuronal populations in monkey primary auditory cortex defined by co-localized calcium binding proteins and surface antigens.

The primary auditory cortex (A1) of monkeys was investigated by immunohistochemistry, using antibodies to gamma-aminobutyric acid (GABA), to the calcium binding proteins parvalbumin and calbindin, and to certain proteoglycan epitopes. The two calcium binding proteins were found to be localized in subpopulations of GABAergic neurons. Parvalbumin immunoreactive cells were mostly found in the middle layers of the cortex. Parvalbumin immunoreactivity was found in fibres in the white matter underlying A1 and a particularly dense concentration of parvalbumin immunoreactive fibers and terminals occurred in layer IV suggesting that a significant population of geniculocortical fibers is also parvalbumin positive. Calbindin positive cells were mostly located in superficial layers and in these layers the neuropil staining was also dense. Two monoclonal antibodies (MAbs) raised against monkey brain tissue and which had previously been shown to recognize neuronal surface antigens stained overlapping subpopulations of GABAergic cells. Occasional pyramidal cells were also immunoreactive. Most of the MAb positive cells were found in the middle layers and all were parvalbumin but not calbindin immunoreactive. Although the physiological roles in the brain for calcium binding proteins and the relevant cell surface markers have not yet been clarified, the presence of these markers in selected subpopulations of cells suggests the existence of functionally distinct circuits in AI cortex.

Animals↗
PubMed2005

Calcium transport in human salivary glands: a proposed model of calcium secretion into saliva.

Salivary calcium plays a vital role in bio-mineralization of dental enamel and exposed dentin. In order to elucidate the yet unknown cellular and molecular mechanisms of calcium secretion in human salivary glands the presence of various relevant plasma membrane transport systems for calcium were investigated. Using an RT-PCR approach, expression of the epithelial calcium channel (CaT-Like), the calcium binding protein (calbindin-2), the endoplasmic reticulum pumps (SERCA-2 and -3), and the plasma membrane calcium ATPases (PMCA-1, -2, and -4), were found in parotid and submandibular glands. Immunohistochemistry revealed that CaT-Like is located in the basolateral plasma membrane of acinar cells; while calbindin-2, SERCA-2 and SERCA-3 were found inside the acinar cells; and PMCA-2 was found in the apical membrane and in the secretory canaliculi between the cells. Based on these findings, we propose the following model of calcium secretion in human salivary glands: (1) calcium enters the acinar cell at the basolateral side via calcium channel CaT-Like (calcium influx); (2) intracellular calcium is taken up into the endoplasmic reticulum by SERCA-2 and possibly SERCA3 or bound to calbindin-2 (intracellular calcium pool); and (3) calcium is secreted by PMCAs at the apical plasma membrane (calcium efflux).

Adult↗
PubMed2003

Immunohistochemical analysis of intracardiac ganglia of the rat heart.

The neurochemistry of intracardiac neurons in whole-mount preparations of the intrinsic ganglia was investigated. This technique allowed the study of the morphology of the ganglionated nerve plexus found within the atria as well as of individual neurons. Intracardiac ganglia formed a ring-like plexus around the entry of the pulmonary veins and were interconnected by a series of fine nerve fibres. All intracardiac neurons contained immunoreactivity to PGP-9.5, choline acetyl transferase (ChAT) and neuropeptide Y (NPY). Two smaller subpopulations were immunoreactive to calbindin or nitric oxide synthase. Furthermore, a subpopulation (approximately 6%) of PGP-9.5/ChAT/NPY-immunoreactive cells lacking both calbindin and nitric oxide synthase (NOS) was surrounded by pericellular baskets immunoreactive to ChAT and calbindin. Vasoactive intestinal peptide (VIP), calcitonin gene-related peptide (CGRP), pituitary adenylate cyclase-activated peptide (PACAP), substance P and tyrosine hydroxylase (TH) immunoreactivity was observed in nerve fibres within the ganglion, but never in neuronal somata. Furthermore, immunoreactivity for NPY was not observed in pericellular baskets surrounding intracardiac neurons, despite being present in all intrinsic neuronal cell bodies. Taken together, the results of this study indicate a moderate level of chemical diversity within the intracardiac neurons of the rat. Such chemical diversity may reflect functional specialisation of neurons in the intracardiac ganglia.

Animals↗
PubMed1989

Calmodulin binding to the intestinal brush-border membrane: comparison to other calcium-binding proteins.

The intestinal brush-border membrane contains a high concentration of calmodulin bound to a 105,000 dalton (105 kDa) protein. Binding of radioiodinated calmodulin to this protein does not require calcium but is inhibited by trifluoperazine and excess unlabelled calmodulin. Recent evidence suggests that the 105 kDa protein in conjunction with calmodulin may be involved in the regulation of calcium transport across the brush-border membrane. In this report, we evaluated the binding of the 105 kDa protein to other radioiodinated calcium-binding proteins including the vitamin D-dependent intestinal calcium-binding protein. We observed that troponin C and S100 beta protein both bound strongly to the 105 kDa protein. The binding of S100 beta was inhibited by EGTA, but was little affected by trifluoperazine and excess unlabelled S100 beta, whereas that of troponin C was inhibited by trifluoperazine and excess unlabelled troponin C, but was little affected by EGTA. Both troponin C and S100 beta bound to a large number of proteins to which calmodulin did not bind. The vitamin D-dependent calcium-binding protein (calbindin) from chick intestine and rat kidney also bound to the 105 kDa protein, albeit more weakly than troponin C, S100 beta and calmodulin. The binding of the calbindins was increased by EGTA and was little affected by trifluoperazine and excess unlabelled calbindin. Parvalbumin, rat osteocalcin, and alpha-lactalbumin showed little binding to any brush-border membrane protein. Our results indicate that the 105 kDa calmodulin-binding protein of the intestinal brush border can bind to a variety of calcium-binding proteins all of which contain homologous regions thought to be the calcium-binding sites. Only the binding of troponin C resembles the binding of calmodulin, however, in being inhibited by trifluoperazine and excess unlabelled ligand. The functional significance of these observations in terms of regulating calcium transport across the brush-border membrane remains to be established.

Animals↗
PubMed1994

Cortical neurons containing calretinin are selectively resistant to calcium overload and excitotoxicity in vitro.

Calbindin and the more recently identified protein calretinin are structurally related calcium-binding proteins having a broad distribution in the brain. Recent evidence supports a neuroprotective role for calbindin in regulating calcium homeostasis during periods of heightened Ca2+ influx. It is not known if calretinin might have a similar function. We investigated if calretinin-containing neurons have a survival advantage in rat neocortical cultures treated with a calcium ionophore or excitatory amino acids. Neuronal cultures were challenged with the calcium ionophore A23187 at different concentrations to produce a broad range of cell death. Cell loss was quantified for both the calretinin immunopositive and the calretinin immunonegative populations of neurons. We found that 3 h after exposure to 2 microM A23187 there was a 48% loss of the calretinin immunonegative population of neurons whereas the calretinin immunopositive set of neurons was reduced by only 18%. Calretinin positive neurons were still relatively spared after treatment with 3 microM A23187. The ionophore had no cytotoxic effect when calcium ions were removed from the extracellular medium. We also studied glutamate excitotoxicity by treating the neuronal cultures with the excitatory amino acids glutamate, N-methyl-D-aspartate or kainate for 5 min and examining survival three hours later. We found again that calretinin-containing neurons were relatively spared after exposure to the excitatory amino acids; at doses of N-methyl-D-aspartate and kainate that produced a 32-40% loss of calretinin immunonegative neurons, only 2-10% of calretinin immunopositive neurons died. Similar results were obtained for glutamate. These results demonstrate that neurons containing calretinin are better able to survive disturbances in calcium homeostasis than cells not containing this calcium-binding protein. The fact that this effect was observed with ionophore treatment, as well as excitatory amino acids, suggests that neither the density nor distribution of glutamate receptors on the different cell types was a factor in determining selective vulnerability. We hypothesize that the neuroprotective effect of calretinin is due to the buffering capacities of the protein in a manner analogous to that suggested for calbindin.

Animals↗