Hemin Chin

Single Cell Reverse Transcription-Polymerase Chain Reaction Analysis of Rat Supraoptic Magnocellular Neurons: Neuropeptide Phenotypes and High Voltage-Gated Calcium Channel Subtypes

Magnocellular neurosecretory cells (MNCs) in the hypothalamo-neurohypophysial system that express and secrete the nonapeptides oxytocin (OT) and vasopressin (VP) were evaluated for the expression of multiple genes in single magnocellular neurons from the rat supraoptic nucleus using a single cell RT-PCR protocol. We found that all cells representing the two major phenotypes, the OT and VP MNCs, express a small, but significant, amount of the other nonapeptide’s messenger RNA (mRNA). In situ hybridization histochemical analyses confirmed this observation. A third phenotype, containing equivalent amounts of OT and VP mRNA, was detected in about 19% of the MNCs from lactating female supraoptic nuclei. Analyses of these phenotypes for other coexisting peptide mRNAs (e.g. CRH, cholecystokinin, galanin, dynorphin, and the calcium-binding protein, calbindin) generally confirmed expectations from the literature, but revealed cell to cell variation in their coexpression. Our results also show that the high voltage...

Characterization of an L-type calcium channel expressed by human retinal Müller (glial) cells

The traditional notion that glial cells are permeable only to potassium has been revised. For example, glia from various parts of the nervous system have calcium-permeable ion channels. Since characterization of the calcium channels in glia is limited, the purpose of this study was to determine the molecular identity and examine the functional properties of a voltage-gated calcium channel expressed by Müller cells, the predominant glia of the retina. Whole-cell and perforated-patch recordings of human Müller cells in culture revealed a high threshold voltage-activated calcium current that is blocked by dihydropyridines, but not by omega-conotoxin GVIA or omega-conotoxin MVIIC. RT-PCR of cultured human Müller cells using primers specific for the calcium channel subunits demonstrated the expression of an L-type channel composed of the alpha 1D, alpha 2 and beta 3 subunits. The alpha 2 subunit of the Müller cell calcium channel is a splice variant which is distinct from either the skeletal muscle alpha 2s or the brain alpha 2b. Our electrophysiological experiments indicate that the alpha 1D/alpha 2/beta 3 calcium channel is functionally linked with the activation of a potassium channel that may serve as one of the pathways for the redistribution by Müller cells of excess retinal potassium.

Identification of a novel protein containing two C2 domains selectively expressed in the rat brain and kidney

We have isolated and characterized a rat brain cDNA clone which encodes a new protein of 474 amino acids in length which contains two C2 domains structurally homologous to those present in synaptotagmins. The overall amino acid identity in C2 domains between this protein and the synaptotagmins is 36–44%. This protein also contains 3 putative consensus sequences for phosphorylation by cAMP‐dependent protein kinase. RNA blot hybridization revealed a 3.0 kb transcript abundantly expressed only in the rat brain and the kidney. Thus, we called this brain/kidney protein (B/K). In situ hybridization and Northern blot analyses showed that the B/K transcript was found in forebrain including the olfactory bulb, cerebral cortex, hippocampus, and hypothalamus. In the kidney, high levels of B/K transcript were expressed in the papillary region of the inner medulla, the inner stripe of the outer medulla and the cortex. The selective expression in forebrain and kidney suggests that B/K may be involved in similar cAMP‐dependent processes at these very different sites.

A SYSTEMATIC SURVEY OF THE INTERGENIC REGION BETWEEN THE MURINE OXYTOCIN- AND VASOPRESSIN-ENCODING GENES

The genomic region between the oxytocin (OT) and vasopressin (VP) genes in the two strains of mice was independently sequenced by our two groups. In this report, we present our collated sequence data and analyses. The mouse intergenic region (MUIGR) was aligned to that of the rat, which has been reported to contain 6.4-kb long interspersed nuclear element (LINE). The MUIGR sequences in the two mice strains did not contain any LINE sequences. This suggests that the approximately 3.5-kb sequence that is conserved between the rat and mouse intergenic regions is likely to be involved in the regulation of OT and VP expression. We also observed several conserved putative transcription factor recognition sequences. Analysis of the MUIGR revealed the lack of any significant ORFs, but the presence of several repetitive elements.

Agonist regulation of the expression of the delta opioid receptor in NG108-15 cells

Exposure of neuronal cells to the chronic presence of opiates leads to a complex series of biochemical events which reflect the changes that result in tolerance and dependence in animals. To achieve a better understanding of the molecular mechanisms underlying these processes, we have examined the effect of agonist efficacy on the regulation of the δ‐opioid receptor mRNA in NG108‐15 cells. Incubation with various opiates decreased receptor numbers in the order of their efficacy. Northern blot analysis showed that there are 4 size classes of mRNA coding for the δ‐opioid receptor in NG108‐15 cells even though only one known protein species is found. Moreover, the amount of each transcript is coordinately decreased by long‐term etorphine treatment, but not necessarily to the same extent. The etorphine‐induced decrease in receptor mRNA was found to be slow in onset, whereas a much more rapid loss of receptor number was observed. This disparity suggests that the down‐regulation induced by etorphine can occur both at the levels of receptor protein modification and receptor gene expression, and that the mechanisms of the two processes may be different.

Isolation and Characterization of the 5′-Upstream Region of the Human N-type Calcium Channel α1B Subunit Gene CHROMOSOMAL LOCALIZATION AND PROMOTER ANALYSIS

ω-Conotoxin-sensitive N-type Ca2+ channels, unlike dihydropyridine-sensitive L-type channels, are exclusively expressed in nervous tissues. To understand the molecular basis for neuron-specific expression of the N-type channel, we have isolated genomic clones encoding the human α1B subunit gene, localized to the long arm of chromosome 9 (9q34) by fluorescence in situ hybridization, and characterized its 5′-upstream region. The proximal promoter of the α1B subunit gene lacks a typical TATA box, is highly GC-rich, and contains several sequences for transcription factor binding. Primer extension experiments revealed the presence of two transcription start sites. In vitro transfection study of the α1B subunit-luciferase fusion gene showed that the 4.0-kb 5′-flanking region of the α1B gene functions as an efficient promoter in neuronal cells but not in glioma or nonneuronal cells, consistent with the patterns of the endogenous α1B gene expression in these cells. Deletion analysis of α1B subunit-luciferase fusion gene constructs further revealed the presence of several cis-acting regulatory elements, including a potential repressor located in the distal upstream region (−3992 to −1788) that may be important for the neuron-specific expression of the N-type Ca2+ channel α1B subunit gene.

Distribution of B/K protein in rat brain

Abstract. B/K protein is a recently isolated member of the double C2-like-domain protein family, which is highly abundant in rat brain. We generated high-titer rabbit polyclonal antibodies with specificity to the 55-kDa rat B/K protein, and examined the expression pattern of B/K protein in rat brain using an immunohistochemical staining method. Immunoreactivity to B/K protein was widely found in distinct regions of rat brain: strongly in the hypothalamus, most of the circumventricular organs, the locus coeruleus, the A5 neurons of the pons, and the anterior pituitary; moderately in the anterior olfactory nucleus, the raphe nucleus, the subfornical organ, and the median eminence; and faintly in the olfactory bulb, the telencephalon, the substantia nigra pars compacta, and the ventral tegmental area. In contrast, immunoreactivity to B/K protein was not observed in the thalamus, the cerebellum, the posterior pituitary, or the spinal cord. In most of the B/K-expressing neurons, immunoreactivity was expressed mainly in soma but not in nerve fibers. B/K was also expressed in nonneuronal cells such as the tanycytes and the subcommissural organ. In the vasopressin-secreting supraoptic and paraventricular nuclei of the hypothalamus, the site where B/K cDNA was originally isolated from, all of the neurons showing vasopressin immunoreactivity also expressed B/K protein, suggesting an overlap of their expression patterns.

Expression of B/K protein in the hippocampus of kainate-induced rat seizure model.

B/K protein is a newly identified member of double C2-like domain protein family. We examined the expression of B/K protein in the hippocampus of kainate-induced rat seizure model. Intraperitoneal injection of kainate increased the immunoreactivity to B/K protein in the CA1 to CA3 of the hippocampus. B/K protein expression began to increase at 6 h, reached the maximum at 12 h, and then returned nearly to the normal level at 72 h after the injection of kainate (12 mg/kg), and it was also dependent on the dose of kainate between 4 and 16 mg/kg. In electron microscopic and subcellular fractionation studies, B/K protein was localized in the endoplasmic reticulum (ER) of the hippocampus. Kainate also induced the expression of BiP, a typical ER stress marker protein, in the hippocampus and the cortex, and it was coexpressed with B/K protein. Moreover, thapsigargin-induced ER stress caused upregulation of B/K protein expression in PC12 cells. In conclusion, our data showing the induction of both B/K protein expression and ER stress response in the hippocampus of kainate seizure model, and ER-specific expression and ER stress-induced expression of B/K strongly suggest the possible role of B/K protein in epileptogenesis or epilepsy-induced neuronal damage.

Squid Giant Axon Contains Neurofilament Protein mRNA but does not Synthesize Neurofilament Proteins

When isolated squid giant axons are incubated in radioactive amino acids, abundant newly synthesized proteins are found in the axoplasm. These proteins are translated in the adaxonal Schwann cells and subsequently transferred into the giant axon. The question as to whether any de novo protein synthesis occurs in the giant axon itself is difficult to resolve because the small contribution of the proteins possibly synthesized intra-axonally is not easily distinguished from the large amounts of the proteins being supplied from the Schwann cells. In this paper, we reexamine this issue by studying the synthesis of endogenous neurofilament (NF) proteins in the axon. Our laboratory previously showed that NF mRNA and protein are present in the squid giant axon, but not in the surrounding adaxonal glia. Therefore, if the isolated squid axon could be shown to contain newly synthesized NF protein de novo, it could not arise from the adaxonal glia. The results of experiments in this paper show that abundant 3H-labeled NF protein is synthesized in the squid giant fiber lobe containing the giant axon’s neuronal cell bodies, but despite the presence of NF mRNA in the giant axon no labeled NF protein is detected in the giant axon. This lends support to the glia–axon protein transfer hypothesis which posits that the squid giant axon obtains newly synthesized protein by Schwann cell transfer and not through intra-axonal protein synthesis, and further suggests that the NF mRNA in the axon is in a translationally repressed state.

Modulation of the N-type calcium channel gene expression by the α subunit of Go

Abstract Go, a heterotrimeric G-protein, is enriched in brain and neuronal growth cones. Although several reports suggest that Go may be involved in modulation of neuronal differentiation, the precise role of Go is not clear. To investigate the function of Go in neuronal differentiation, we determined the effect of Goα, the α subunit of Go, on the expression of Cav2.2, the pore-forming unit of N-type calcium channels, at the transcription level. Treatment with cyclic AMP (cAMP), which triggers neurite outgrowth in neuroblastoma F11 cells, increased the mRNA level and the promoter activity of the Cav2.2 gene. Overexpression of Goα inhibited neurite extension in F11 cells and simultaneously repressed the stimulatory effect of cAMP on the Cav2.2 gene expression to the basal level. Targeted mutation of the Goα gene also increased the level of Cav2.2 in the brain. These results suggest that Go may regulate neuronal differentiation through modulation of gene expression of target genes such as N-type calcium channels.