The EIGER detector for low-energy electron microscopy and photoemission electron microscopy.

EIGER is a single-photon-counting hybrid pixel detector developed at the Paul Scherrer Institut, Switzerland. It is designed for applications at synchrotron light sources with photon energies above 5 keV. Features of EIGER include a small pixel size (75 µm × 75 µm), a high frame rate (up to 23 kHz), a small dead-time between frames (down to 3 µs) and a dynamic range up to 32-bit. In this article, the use of EIGER as a detector for electrons in low-energy electron microscopy (LEEM) and photoemission electron microscopy (PEEM) is reported. It is demonstrated that, with only a minimal modification to the sensitive part of the detector, EIGER is able to detect electrons emitted or reflected by the sample and accelerated to 8-20 keV. The imaging capabilities are shown to be superior to the standard microchannel plate detector for these types of applications. This is due to the much higher signal-to-noise ratio, better homogeneity and improved dynamic range. In addition, the operation of the EIGER detector is not affected by radiation damage from electrons in the present energy range and guarantees more stable performance over time. To benchmark the detector capabilities, LEEM experiments are performed on selected surfaces and the magnetic and electronic properties of individual iron nanoparticles with sizes ranging from 8 to 22 nm are detected using the PEEM endstation at the Surface/Interface Microscopy (SIM) beamline of the Swiss Light Source.

The expression and function of Ca(2+)-sensing receptors in rat mesenteric artery; comparative studies using a model of type II diabetes.

BACKGROUND AND PURPOSE: The extracellular calcium-sensing receptor (CaR) in vascular endothelial cells activates endothelial intermediate-conductance, calcium-sensitive K(+) channels (IK(Ca)) indirectly leading to myocyte hyperpolarization. We determined whether CaR expression and function was modified in a rat model of type II diabetes. EXPERIMENTAL APPROACH: Pressure myography, western blotting, sharp microelectrode and K(+)-selective electrode recordings were used to investigate the functional expression of the CaR and IK(Ca) in rat mesenteric arteries. KEY RESULTS: Myocyte hyperpolarization to the CaR activator calindol was inhibited by Calhex 231. U46619-induced vessel contraction elevated the extracellular [K(+)] around the myocytes, and inhibition of this 'K(+) cloud' by iberiotoxin was needed to reveal calindol-induced vasodilatations. These were antagonized by Calhex 231 and significantly smaller in Zucker diabetic fatty rat (ZDF) vessels than in Zucker lean (ZL) controls. Myocyte hyperpolarizations to calindol were also smaller in ZDF than in ZL arteries. In ZDF vessels, endothelial cell CaR protein expression was reduced; IK(Ca) expression was also diminished, but IK(Ca)-generated hyperpolarizations mediated by 1-EBIO were unaffected. CONCLUSIONS AND IMPLICATIONS: The reduced CaR-mediated hyperpolarizing and vasodilator responses in ZDF arteries result from a decrease in CaR expression, rather than from a modification of IK(Ca) channels. Detection of CaR-mediated vasodilatation required the presence of iberiotoxin, suggesting a CaR contribution to vascular diameter, that is, inversely related to the degree of vasoconstriction. Compromise of the CaR pathway would favour the long-term development of a higher basal vascular tone and could contribute to the vascular complications associated with type II diabetes.

Stimulation of cell proliferation by calcium and a calcimimetic compound.

Some mesenchymal cells respond to stimulation by specific cations with increased cell proliferation. In the present study we have investigated whether the parathyroid/kidney/brain calcium-sensing receptor (PCaR) can mediate such mitogenic responses. We have expressed the recombinant rat PCaR in CCL39 hamster fibroblasts, which do not express a detectable endogenous cation sensor. The transfected cells responded to increased extracellular calcium concentrations ([Ca2+]e) with strong inositol phosphate (IP) formation, which was insensitive to pertussis toxin treatment of cells. We could not detect negative coupling of the receptor to adenylyl cyclase. The calcimimetic NPS R-568 left-shifted the concentration-response curve for [Ca2+]e-induced IP formation and increased the maximal response. In [3H]thymidine incorporation experiments, increasing [Ca2+]e from 1 to 4 mM was found to stimulate DNA synthesis weakly, but significantly. A strong potentiation of this response was observed in the presence of NPS R-568. [Ca2+]e and NPS R-568 also synergized to increase cell numbers in cultures maintained in defined medium. In contrast to our expectations, no significant stimulation of IP formation or cell proliferation could be observed after stimulation of cells with the reported PCaR agonist gadolinium (Gd3+) or with aluminum (Al3+), which stimulates osteoblast proliferation. Gd3+ actually inhibited IP formation stimulated by increased [Ca2+]e as well as by thrombin and AlF4-, indicating toxicity. However, submaximal receptor stimulation by Gd3+ was evident when intracellular calcium transients were measured in fluo-3-loaded cells. Our data show that PCaR can stimulate cell proliferation when expressed in an appropriate cellular context. However, it is unlikely that PCaR mediates the strong mitogenic effects elicited by the cations Gd3+ and Al3+ observed in osteoblasts.

Iodoaminopotentidine and related compounds: a new class of ligands with high affinity and selectivity for the histamine H2 receptor.

The synthesis and biological evaluation of a new class of histamine H2 antagonists with N-cyano-N'-[omega-[3-(1-piperidinylmethyl)phenoxy] alkyl]guanidine partial structure are described as part of an extensive research program to find model compounds for the development of new radioligands with high H2 affinity and specific activity. High receptor affinity is achieved by an additional (substituted) aromatic ring, which is connected with the third guanidine N by a carbon chain spacer and an amine, carboxamide, ester, or sulfonamide link ("polar group"). In functional studies for H2 antagonistic activity and other pharmacological actions [e.g. H1 antihistaminic, antimuscarinic, antiadrenergic (alpha 1, beta 1), 5-HT2 blocking activity] in the isolated guinea pig atrium and ileum and rat aorta and tail artery, the compounds proved to be highly potent and selective histamine H2 receptor antagonists. The H2 antagonistic activity is mainly depending on the length of both the N'-alkyl chain (chain A) and the N"-spacer (chain B). Compounds with a C3 chain A and a C2 chain B are most potent in the preferred group of substances, i.e., the carboxamide series. A wide variety of substituents at the aromatic ring is tolerated, among them iodine, amino, and azido groups. These compounds are up to 32 times more potent than cimetidine in the isolated guinea pig right atrium. The replacement of the carboxamide by an ester group (44c) is well tolerated, while replacement of the cyanoguanidine by an urea group results in nearly 100-fold decrease in activity (46c,e). The iodinated benzamides are among the most potent H2 antagonists known so far. The [125I]-labeled form of 31f ([125I]iodoaminopotentidine, [125I]-N-[2-(4-amino-3-iodobenzamido) ethyl]-N'-cyano-N"-[3-[3-(1-piperidinylmethyl) phenoxy]propyl]guanidine) and its photolabile analogue 31h ([125I]iodoazidopotentidine, [125I]-N-[2-(4-azido-3- iodobenzamido)ethyl]-N'-cyano-N"-[3-[3-(1-piperidinyl-methyl)pheno xy] propyl]guanidine) proved to be useful probes for reversible and irreversible labeling of the histamine H2 receptor. Radioligand binding studies in guinea pig cerebral membranes revealed considerably higher H2 receptor affinity for 31f (pKi = 9.15), 31h (pKi = 8.58), and some analogues than functional experiments (guinea pig atrium), presumably reflecting an easier access to the H2 receptors in membranes.

Distribution of bone morphogenetic protein and bone morphogenetic protein receptor transcripts in the rodent nervous system and up-regulation of bone morphogenetic protein receptor type II in hippocampal dentate gyrus in a rat model of global cerebral ischemia.

Bone morphogenetic proteins belong to the transforming growth factor-beta superfamily and act through serine/threonine kinase type I and type II receptors such as bone morphogenetic protein receptor type I and type II. In order to further understand the roles that these factors exert in the nervous system, we have examined the expression pattern of seven bone morphogenetic proteins and bone morphogenetic protein receptor type I and II transcripts in the brain and spinal cord of rodent. Whereas bone morphogenetic protein receptor type I expression was low in rat brain, in situ hybridization studies performed with specific digoxigenin-labelled riboprobes revealed the presence of bone morphogenetic protein receptor type II-positive cells throughout the brain, with a notable localization in dopaminergic cells of the substantia nigra. Bone morphogenetic protein receptor type II transcripts were also expressed by large motoneuron-like cells located in the ventral horn of the spinal cord and by sensory neurons of dorsal root ganglia. In addition, we observed a significant up-regulation of bone morphogenetic protein receptor type II in the granule cells of the dentate gyrus 48 h after transient global cerebral ischemia in rat suggesting that modulation of this receptor intervenes during neuronal plasticity or repair that occur upon brain injury. Among the potential ligands for this receptor, bone morphogenetic protein-6 and bone morphogenetic protein-7 were expressed in meninges and the choroid plexus, while bone morphogenetic protein-4-expressing cells were spatially and temporally regulated in myelinated structures during development and in the adult suggesting its expression in oligodendrocytes. These data clearly indicate that besides their roles in bone and embryonic tissues, bone morphogenetic proteins and their receptors may have also important functions in adult neural tissues.

A detailed mapping of histamine H1-receptors in guinea-pig central nervous system established by autoradiography with [125I]iodobolpyramine.

[125I]Iodobolpyramine, a potent and selective histamine H1-receptor antagonist derived from mepyramine, was used to generate light microscopic autoradiograms on sections of guinea-pig brain and spinal cord. Histamine H1-receptors were labelled with high sensitivity over a low background as determined using mianserin or other H1-receptor antagonists as competing agents. An atlas of H1-receptors was established using five sagittal sections and 39 frontal sections, the latter serially prepared at 50 micron intervals. Labelled areas were identified by comparison with corresponding, classically stained sections and their density was rated according to an arbitrary scale. Autoradiographic grains were detected in a large variety of gray matter areas whereas they were generally absent from white matter areas. In the cerebral cortex, H1-receptors are present in all areas and layers with a higher density in lamina IV. In the hippocampal formation, H1-receptors display a laminated pattern of distribution and are the most abundant in the dentate gyrus (hilus and molecular layer) and in several areas of the subiculum and commissural complex. In the amygdaloid complex, the highest densities are found in the medial group of nuclei. In the basal forebrain, the striatum is moderately labelled whereas the nucleus accumbens, islands of Calleja and most septal nuclei are highly labelled. In the thalamus, H1-receptors are present in high density, particularly in the anterior, median and lateral groups of nuclei. In the hypothalamus the labelling is highly heterogeneous with high densities in, for example, medial preoptic area, dorsomedial, ventromedial and most posterior nuclei, including the tuberomammillary complex in which histaminergic perikarya and short axons are present.(ABSTRACT TRUNCATED AT 250 WORDS)

Detailed mapping of the histamine H2 receptor and its gene transcripts in guinea-pig brain.

Autoradiographic studies of the distribution of the histamine H2 receptor and its messenger RNAs were performed on serial frontal and a few sagittal sections of guinea-pig brain using [(125)I]iodoaminopotentidine for radioligand binding and a 33P-labelled complementary RNA probe for in situ hybridization, respectively. Both probes were validated by assessing non-specific labelling using non-radioactive competing H2 receptor ligands and a sense probe for binding sites and gene transcripts, respectively. In some areas, e.g., cerebral cortex, hippocampal complex or cerebellum, such studies were completed by identification of neurons expressing the H2 receptor messenger RNAs on emulsion-dipped sections. Nissl-stained sections from comparable levels were used to localize brain structures. In many brain areas, the distribution of the H2 receptor and its messenger RNAs appeared to parallel that known for histaminergic axons. For instance. high levels of both H2 receptor markers were detected in striatal and limbic areas known to receive abundant histaminergic projections. In contrast, in septum, hypothalamic, pontine and several thalamic nuclei, a comparatively low density of both H2 receptor markers was detected, suggesting that histamine actions in these areas are mediated by H1 and/or H3 receptors. Generally, the distribution of H2 receptor messenger RNA correlates well with that of [(125)I]iodoaminopotentidine binding sites, although some differences were observed. In a few regions (e.g., substantia nigra, locus coeruleus) high or moderate densities of binding sites were accompanied by a much more restricted expression of H2 receptor transcripts. Conversely, the mammillary region and the pontine nucleus exhibited higher levels of hybridization than of binding sites. In hippocampus, cerebral and cerebellar cortex there was a selective localization of the H2 receptor messenger RNA in the granule cells of dentate gyrus, pyramidal cells of the Ammon's horn and cerebral cortex, and Purkinje cells of cerebellum, whereas [(125)I]iodoaminopotentidine binding sites were located in layers where the dendritic trees of these messenger RNA-expressing neurons extend. The same discrepancy between messenger RNAs and binding sites suggests that striatonigral endings are endowed with the H2 receptor. The histamine H1 and H2 receptors both appear to be present in several brain areas, in some cases in a way suggesting their potential co-expression by the same neuronal populations, e.g., in granule and pyramidal cells in the hippocampal formation. This co-expression accounts for synergic responses, e.g., on cAMP generation, previously observed upon co-stimulation of both receptor subtypes. The widespread distribution of the H2 receptor, namely in thalamic nuclei or in telencephalic areas such as most layers of the cerebral cortex, together with its excitatory role previously established in electrophysiological studies, support its alleged function in mediating the histamine-driven control of arousal mechanisms. In addition, the detection of H2 receptor expression in brainstem areas from which other monoaminergic pathways involved in the control of states of sleep and wakefulness emanate, e.g., several raphe nuclei, locus coeruleus or substantia innominata, suggests possible interrelationships between all of these systems with highly divergent projections to the thalamus and telencephalon. The present mapping of the H2 receptor and its gene transcripts should facilitate neurochemical, neurophysiological and behavioural studies aimed at clarifying the role of histaminergic systems in brain.

Three histamine receptors (H1, H2 and H3) visualized in the brain of human and non-human primates.

The distribution of histamine H1, H2 and H3 receptors in postmortem human and rhesus monkey brain was examined using receptor autoradiography. [125I]Iodobolpyramine, [125I]iodoaminopotentine and [3H](R) alpha-methylhistamine were used as ligands to label H1, H2 and H3 receptors respectively. The 3 receptor subtypes were identified in the human and monkey brains. Each receptor presented comparable distribution in the two primate brains. H1 and H2 receptors were particularly enriched in the caudate and putamen and observed in other brain areas such as the neocortex and hippocampus. H3-receptors were found to predominate in the basal ganglia where the highest densities were localized in the two segments of the globus pallidus. They were also observed in the hippocampus and cortical areas. The distribution of these 3 histamine receptors in the primate brain suggests the involvement of histaminergic mechanism in the functions of many brain areas. In particular, H2 and H3 receptors could play a role in the regulation of the basal ganglia functions in primates.

Interaction of mianserin, amitriptyline and haloperidol with guinea pig cerebral histamine H2 receptors studied with [125I]iodoaminopotentidine.

Tricyclic antidepressants were previously shown to potently inhibit the histamine-induced activation of H2 receptors linked to adenylate cyclase in cerebral membranes, and this effect was postulated to represent the mechanism of their therapeutic activity. However, these drugs were found to be much less potent (up to more than hundred-fold) at H2 receptors mediating cyclic AMP responses in intact cells. We have presently assessed whether this large difference in potency of antidepressants, also observed with haloperidol, results from a modified pharmacological specificity of the H2 receptor according to the medium composition. We have studied the binding of [125I]iodoaminopotentidine (125I-APT) to striatal or hippocampal membranes under various experimental conditions. At equilibrium the Kd of 125I-APT, a highly selective ligand for H2 receptors, was six times higher in a supplemented Tris buffer used for adenylate cyclase assays in cell-free systems than in a Krebs-Ringer medium used in studies with intact cells. The medium composition also variously affected the Ki values of the four compounds studied amitriptyline, mianserin, haloperidol and tiotidine. Whereas the Ki value of amitriptyline was little affected, that of the other compounds was four to five times lower in the supplemented Tris buffer than in the Krebs-Ringer medium. With the exception of tiotidine, the Ki values of other compounds in the binding test performed in this medium, were intermediate between those derived from the antagonism of histamine-induced cyclic AMP responses in membranes and intact cells. These data indicate that the difference between the two test responses is diversely attributable to several factors according to the compounds, the main ones being medium composition and possibly cell disruption.

Characterization of 5-ht6 receptor and expression of 5-ht6 mRNA in the rat brain during ontogenetic development.

We have determined the pharmacological characteristics of the rat 5-ht6 receptor stably expressed in CHO cells. Moreover, using RT-PCR experiments the in vivo expression of the gene encoding this receptor was studied in rat at various embryonic days (ED) starting from ED10 to birth (PN0) and at post-natal days (PN) up to PN36. The pharmacological analysis of the [3H]5-HT binding in stably transfected CHO cells expressing rat 5-ht6 receptors revealed the presence of a single class of high affinity saturable binding sites for 5-HT corresponding to an affinity constant: Kd=27.2+/-3.4 nM. This receptor also exhibited a high affinity for a number of typical and atypical antipsychotics, tricyclic antidepressant drugs and ergot alkaloïds. In stably transfected CHO cells, serotonin elicited a potent stimulation of adenylyl cyclase activity which was blocked by antipsychotic and antidepressant drugs. These results confirm the hypothesis that 5-ht6 receptors may correspond to an important target for atypical antipsychotics and reveal an original pharmacological profile for this receptor. The study of the ontogeny of the 5-ht6 mRNA in rat developing brain showed that 5-ht6 mRNA were first detectable with a high level on ED12, slighly decreased up to ED17 and then remained stable at high level until the adult age. The ontogenetic pattern of 5-ht6 mRNA expression appeared to correlate with the occurence of the first cell bodies of serotonergic neurons; the early expression of 5-ht6 mRNA and the fact that this receptor is positively coupled to the production of cAMP may suggest a role for 5-ht6 receptor in the early growth process involving the serotonergic system.

Molecular characterization of the family of choline transporter-like proteins and their splice variants.

We show here that the choline transporter-like (CTL) family is more extensive than initially described with five genes in humans and complex alternative splicing. In adult rat tissues, CTL2-4 mRNAs are mainly detected in peripheral tissues, while CTL1 is widely expressed throughout the nervous system. During rat post-natal development, CTL1 is expressed in several subpopulations of neurones and in the white matter, where its spatio-temporal distribution profile recalls that of myelin basic protein, an oligodendrocyte marker. We identified two major rat splice variants of CTL1 (CTL1a and CTL1b) differing in their carboxy-terminal tails with both able to increase choline transport after transfection in neuroblastoma cells. In the developing brain, CTL1a is expressed in both neurones and oligodendroglial cells, whereas CTL1b is restricted to oligodendroglial cells. These findings suggest specific roles for CTL1 splice variants in both neuronal and oligodendrocyte physiology.

Photoaffinity labeling and electrophoretic identification of the H1-receptor: comparison of several brain regions and animal species.

The photoaffinity probe [125I]iodoazidophenpyramine was used to label irreversibly the H1-receptor in membranes of several guinea pig brain regions and of the cerebral cortex of the rat, mouse, and pig. Following sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, two main bands were specifically labeled in all tissues: a 56-kilodalton (kDa) peptide and a 41-47-kDa peptide whose relative importance diminished in the presence of protease inhibitors. This indicates that, in all tissues examined, in spite of evidence for pharmacological heterogeneity, the ligand recognition domain of the H1-receptor resides in a 56-kDa peptide.

Developmental and adult expression of rat calcium-sensing receptor transcripts in neurons and oligodendrocytes.

The calcium-sensing receptor (CaSR) is a member of a growing family of heptahelical receptors with an unusually large extracellular domain. To further delineate its functions in neurons and glia, we have investigated the expression pattern of CaSR transcripts in the postnatal and adult rat brain, spinal cord and dorsal root ganglia by in situ hybridization. CaSR-expressing cells were spatially and temporally regulated in myelinated structures with a caudo-rostral pattern that paralleled that of myelin basic protein, a marker of myelination, with a downregulation observed in the adult. Double-labelling studies demonstrated that CaSR mRNA colocalizes with myelin basic protein-expressing cells within fibre tracts, suggesting that CaSR is expressed by mature oligodendrocytes. In cultured rat oligodendrocytes, Ca2+ induced stimulation of phosphatidylinositol hydrolysis with an EC50 of 1.4 mM and increased intracellular calcium. NPS R-568 (1 microM), a calcimimetic, significantly stimulates the inositol phosphate response, whereas a less potent stereoisomer, NPS S-568 (1 microM), was without effect. These data suggest that a functional CaSR is expressed in mature oligodendrocytes with a potential role in myelination. CaSR expression was also developmentally regulated in neurons of the orbital cortex and in the CA2 region of the hippocampus, and present in olfactory nuclei, hypothalamic areas and in the area postrema through postnatal days to adulthood. This expression is consistent with a role of CaSR in olfactory or gustatory signal integration, and with the regulation of fluid and mineral homeostasis. CaSR expression in a subpopulation of small cells in dorsal root ganglia suggests additional roles for extracellular Ca2+ in sensory nerves.

Regional distribution of Sonic Hedgehog, patched, and smoothened mRNA in the adult rat brain.

In vertebrates, Sonic Hedgehog (Shh), Desert Hedgehog (Dhh), and Indian Hedgehog (Ihh) genes encode a family of morphogen proteins that are implicated in a wide range of signaling activities, particularly during embryonic development. These secreted proteins are proposed to mediate their effects on target cells by interacting with their putative receptor, Patched (Ptc), and with a seven-pass transmembrane protein, Smoothened (Smo). However, the roles that these signaling molecules may play in adult tissues, particularly in brain, are not yet clearly defined. Therefore, we investigated the expression of these genes in adult rat tissues. Northern blot analysis revealed expression of Shh, Dhh, and Ihh genes in peripheral tissues, whereas Shh transcript was also identified in brain. It is interesting that northern blot analysis with probes derived from the mouse Ptc and Smo genes revealed the expression of a 7.9-kb and a 3.7-kb transcript, respectively, in all brain tissues examined. In situ hybridization experiments using specific digoxigenin-labeled riboprobes showed expression of Ptc and Smo transcripts in discrete brain areas. Shh-positive cells were observed in restricted regions of the brain. Within the cerebellum, Shh, Ptc, and Smo transcripts were colocalized in the Purkinje cell layer. These data suggest that, besides its roles in determining cell fate and patterning during embryogenesis, the hedgehog signaling pathway may have also important roles in the adult brain.

Molecular cloning of rat mast cell protease 1 and development of specific probes for its gene transcript.

Rat mast cell protease of type 1 (RMCP1) is a specific marker of connective tissue mast cells selectively occurring in some tissues, e.g., the tongue. Its amino acid sequence is known (Le Trong et al., Biochem. 1987, 26, 6988-6994) but not the corresponding nucleotide sequence. Amplification of mRNAs from rat tongue was performed by reverse transcriptase-polymerase chain reaction (RT-PCR) using oligonucleotide primers corresponding to the translated region of rat mast cell protease 2 (RMCP2) gene. The cDNA obtained was subcloned and sequenced, leading to an amino acid sequence which matched the known 227 amino acid sequence. In addition there was, however, two sequences of 11 amino acids at the N-terminus and 13 amino acids at the C-terminus. The amino acid identity was of 74% with RMCP2, and of 76%, 65% and 90% with the mouse proteases MMCP1, MMCP2 and MMCP4, respectively. Based on the sequence of RMCP1 or RMCP2 cDNAs, selective oligoprobes were designed and their specificity established by Northern blot analysis of mRNAs purified from tongue and jejunum, two tissues containing selectively type 1 and 2 protease, respectively. Single 1.2 and 1.0 kb transcripts were evidenced in tongue and jejunum, respectively. In addition, a RT-PCR method was developed to amplify selectively each transcript which may serve as reliable markers in the analysis of mast cell heterogeneity, differentiation and function.

N1-Arylsulfonyl-N2-(1-aryl)ethyl-3-phenylpropane-1,2-diamines as novel calcimimetics acting on the calcium sensing receptor.

The synthesis and calcimimetic properties of N1-arylsulfonyl-N2-(1-aryl)ethyl-3-phenylpropane-1,2-diamines are described. The most active compound of the series (3n, used at 10 microM) produced 97+/-11% of the maximal stimulation of [3H]IP production obtained by 10 mM Ca2+ in CHO cells expressing the calcium sensing receptor (CaSR). This calcimimetic activity was due to a specific interaction of this compound with the CaSR.

Calcium sensing receptor: molecular cloning in rat and localization to nerve terminals.

We have molecularly cloned a calcium sensing receptor (CaSR) from a rat striatal cDNA library. Rat CaSR displays 92% overall homology to its bovine counterpart with seven putative transmembrane domains characteristic of the superfamily of guanine nucleotide-binding proteins and significant homology with the metabotropic glutamate receptors. Northern blot analysis reveals two transcripts in thyroid, kidney, lung, ileum, and pituitary. In brain highest regional expression of the RNA occurs in the hypothalamus and the corpus striatum. Immunohistochemistry reveals discrete punctate localizations throughout the brain that appear to be associated with nerve terminals. No staining is evident in cell bodies of neurons or glia. Cerebral arteries display an intense network of CaSR immunoreactive fibers associated with vessel innervation. CaSR on nerve terminal membranes may regulate neurotransmitter disposition in response to Ca2+ levels in the synaptic space.

Cloned and expressed rat Ca2+-sensing receptor.

We have stably expressed cDNA for the rat brain Ca2+ sensing receptor in Chinese hamster ovary cells. Stimulation of phosphatidylinositol hydrolysis and arachidonic acid (AA) release displayed markedly cooperative responses to Ca2+ with Hill coefficients of 4-5. Both phosphatidylinositol and AA responses were not detected below a threshold of 1.5 mM Ca2+. Mg2+ behaved as a partial agonist with only half the maximal inositol phosphate and AA responses displayed by Ca2+ and with a more shallow concentration-response slope. The potency of Mg2+ in augmenting inositol phosphate and AA responses, in the presence of 1.5 mM Ca2+, implies that serum Mg2+ concentrations attained in clinical conditions will influence the Ca2+-sensing receptor.