A novel choline cotransporter sequestration compartment in cholinergic neurons revealed by selective endosomal ablation.

The sodium-dependent, high affinity choline transporter - choline cotransporter - (ChCoT, aka: cho-1, CHT1, CHT) undergoes constitutive and regulated trafficking between the plasma membrane and cytoplasmic compartments. The pathways and regulatory mechanisms of this trafficking are not well understood. We report herein studies involving selective endosomal ablation to further our understanding of the trafficking of the ChCoT.

Regulation of ANKRD9 expression by lipid metabolic perturbations.

Fatty acid oxidation (FAO) defects cause abnormal lipid accumulation in various tissues, which provides an opportunity to uncover novel genes that are involved in lipid metabolism. During a gene expression study in the riboflavin deficient induced FAO disorder in the chicken, we discovered the dramatic increase in mRNA levels of an uncharacterized gene, ANKRD9. No functions have been ascribed to ANKRD9 and its orthologs, although their sequences are well conserved among vertebrates.

Identification and characterization of syntaxin 1 antisense variants in Limulus polyphemus.

1.A Limulus SMART(TM) cDNA library screening resulted in the cloning of four syntaxin 1 homologs (referred to as Limulus syntaxin [Lim-syn] 1A, 1B, 1C, and 1D) (Wang, Y., Cao, Z.

Cloning and identification of Rab cDNAs from Limulus polyphemus.

Small GTPases of the Rab family are essential for the control of membrane transport between intracellular compartments. Trafficking of the sodium-dependent facilitative insulin responsive glucose transporter (GLUT 4) has been shown to be associated with the intracellular redistribution of Rabs 4, 5 and 11 in adipose and muscle tissues. As a prelude to studies of the endosomal trafficking of the choline cotransporter (ChCoT), we describe herein our initial efforts to identify Rab proteins in Limulus polyphemus central nervous system (CNS) tissue.

The chemorepellent semaphorin is expressed in the horseshoe crab, Limulus polyphemus.

Semaphorins are a family of soluble and membrane-bound proteins that play a critical role in axonal guidance and other processes of neuronal development. Currently, more than twenty semaphorins have been identified, all of which share a conserved 500 amino acid domain near the amino terminus. Semaphorins are divided into eight classes according to species of origin and structural similarities.

Cloning and partial characterization of four plasmalemmal-associated syntaxin isoforms in Limulus.

We describe herein the cloning of a group of syntaxins in Limulus that are associated with the plasma membrane. Initially, multiple degenerate oligonucleotide primers (DOP) and probes were designed from sequences of known plasma membrane associated syntaxins. Combined experiments using reverse transcriptase-polymerase chain reaction (RT-PCR), colony hybridization and reverse dot blot yielded three distinct probes.

Molecular cloning and identification of a putative PKC epsilon cDNA from Limulus polyphemus brain.

The protein kinase C (PKC) family of enzymes is broadly distributed and has been implicated in a diverse array of cellular functions. Recent evidence supporting PKC involvement in the regulation of the Limulus choline cotransporter prompted us to clone PKC from a Limulus central nervous system (CNS) cDNA library. An Aplysia californica calcium independent PKC (Apl II) cDNA probe was used to screen the library and 5' RACE SMART PCR was used to obtain the full-length sequence.

Molecular cloning of a cDNA for a putative choline co-transporter from Limulus CNS.

It is well documented that the sodium dependent, hemicholinium-3 sensitive, high affinity choline co-transporter is rate limiting in the biosynthesis of acetylcholine and is essential to cholinergic transmission. Until recently this transporter had eluded cloning. Okuda et al.

The quantitative distribution of a putative PKC epsilon mRNA in Limulus central nervous system by modified competitive RT-PCR.

Recently, a full length cDNA for the epsilon (epsilon) isoform of protein kinase C (PKC) was cloned and sequenced from a cDNA library for the horseshoe crab, Limulus polyphemus. This multifunctional enzyme has been implicated in the modulation of the choline cotransporter in Limulus and the epsilon isoform has been identified in homogenates from its central nervous system (CNS). RT-PCR has proven to be a very useful method for quantifying even a few molecules of mRNA in tissue samples.

Hemicholinium-3 mustard reveals two populations of cycling choline cotransporters in Limulus.

Cholinergic neurons have both a low-affinity and a high-affinity choline transport process. The high-affinity choline transport is sodium dependent and thus it can be referred to as choline cotransport. Choline cotransport has been shown to be up-regulated by neuronal activity.

The use of competitive PCR mimic to evaluate a Limulus lambda phage genomic DNA library.

1. A lambda phage genomic DNA library for Limulus (L.) polyphemus brain was constructed using the AGEM-12 vector and the host strain KW251.

The involvement of protein kinase C in the regulation of choline cotransport in Limulus.

The involvement of protein kinase C (PKC) in the regulation of [3H]choline cotransport was studied in Limulus brain hemi-slice preparations. The PKC activators, phorbol 12-myristate 13-acetate (PMA) or phorbol 12,13-dibutyrate (PDBu), significantly decreased [3H]choline cotransport. Conversely, the PKC inhibitors, staurosporine (STAURO) and polymyxin B (PMB), each increased [3H]choline cotransport.

Chronic ethanol ingestion produces cholinergic hypofunction in rat brain.

Alterations in cholinergic function due to prolonged ethanol exposure (up to 9 months) were assessed by choline acetyltransferase (ChAT) activity and high-affinity choline uptake (HAChU) in three brain regions of the Long-Evans rat: frontal cortex, parietal cortex, and region of the nucleus basalis of Meynert (NbM). No statistically significant changes were found in ChAT activity in the 3-month group; however, ChAT activity was decreased in both the frontal cortex (-32%) and NbM region (-22%) after 6 months of ethanol exposure. ChAT activity in the parietal cortex was increased 30% after 6 months.

Cloning and sequencing of a cDNA encoding a novel member of the human brain GABA/noradrenaline neurotransmitter transporter family.

Screening of a human hippocampal cDNA library with a rat creatine transporter cDNA-specific probe revealed two types of clones. One identical to the published creatine transporter cDNA sequence (CRT1) and another (CRT2) with four segments of oligodeoxyribonucleotide substitution or addition in different sites of the CRT1 coding sequence. Translation of the CRT2 coding sequence reveals a new protein with regions of perfect homology with the CRT1 amino-acid sequence.

Neurotransmitter and neuropeptide modulation of high affinity choline uptake in Limulus brain.

The role of neurotransmitters in the modulation of the sodium-dependent high affinity choline uptake system (HAChUS) of the horseshoe crab, Limulus polyphemus has been investigated utilizing a tissue slice preparation. Choline uptake was significantly decreased by carbachol but unaffected by atropine and d-tubocurarine. The muscarinic agonist oxotremorine decreased choline uptake by 30.

Differential expression and subcellular localization of protein kinase C isoforms in neuronal and cardiac tissues of Limulus polyphemus.

The presence and subcellular distribution of specific protein kinase C (PKC) isoforms in Limulus neuronal and cardiac tissue extracts were determined using polyclonal antibodies to individual PKC isoforms. Western blot analysis revealed that the brain, abdominal ganglia, cardiac ganglia and heart all contained PKC's alpha, beta, gamma, epsilon and zeta. PKC delta was detected in all neuronal tissues.

An improved invertebrate synaptosomal preparation with cholinergic properties.

Our laboratory has previously characterized a high affinity choline uptake system (HAChUS) in Limulus tissues and synaptosomes. We report here on the characterization of the HAChUS in synaptosomes prepared selectively from central nervous system tissues shown to be enriched for presumed cholinergic functions; namely the protocerebrum, corpora pedunculata and abdominal ganglia. Synaptosomes were prepared from these tissues by means of a modification of the subfractionation procedure developed by Dowdall and Whittaker.

A vinblastine sensitive high affinity choline uptake system.

1. The Limulus cardiac ganglion high affinity choline uptake system (HAChUS) was inhibited 40, 51 and 64% following pre-exposure to 10, 100 and 500 microM vinblastine, respectively. 2.

The status of the study of invertebrate neurons in tissue culture--phylum Arthropoda.

1. Neurobiological studies employing tissue culture are discussed for all invertebrate organisms in phylum Arthropoda and above. Only arthropod species have been investigated in these studies.

A comparative study of high affinity choline uptake and choline utilization in cholinergic and non-cholinergic tissues.

Comparative studies of [3H]choline accumulation were done in the Limulus corpora pedunculata, abdominal ganglia and cardiac ganglion. Dual uptake processes for choline were found in all three tissues. In acute experiments, the corpora pedunculata high affinity choline uptake system showed exclusive sensitivity to ouabain.

Biochemical evidence for cholinergic involvement in the Limulus brain.

The transport of [3H]choline by the corpora pedunculata of the circumoesophageal ring gland (brain) of Limulus polyphemus was studied. Corpora pedunculata slices were incubated individually in Chao's solution containing 0.01 microM [3H]choline at room temperature (25 +/- 2 degrees C) and readily accumulated the radiolabel from the extracellular environment.

On the status of the study of invertebrate neurons in tissue culture--phyla Mollusca and Annelida.

The utilization of tissue culture in neurobiological studies is discussed for all phyla phylogenetically preceding Phylum Arthropoda. Only two phyla, Mollusca and Annelida, are represented in such studies. The members of Phylum Mollusca which have been so investigated are Aplysia, Helisoma and Lymnaea.