KCC3 and KCC4 expression in rat adult forebrain.

Potassium chloride ion cotransporters (KCCs) are part of a family of transporters classically described as being involved in cell volume regulation. Recently, KCC2 has been shown to have a role in the development of the inhibitory actions of amine transmitters, whereas KCC3 also plays a fundamental role in the development and function of the central and peripheral nervous system. We have re-assessed the expression of each of the known KCCs in the rat forebrain using RT-PCR and in situ hybridisation histochemistry. As well as confirming the widespread expression of KCC1 and KCC2 throughout the brain, we now show a more restricted expression of KCC3a in the hippocampus, choroid plexus and piriform cortex, as well as KCC4 in the choroid plexus and the suprachiasmatic nucleus of the hypothalamus. The expression of KCC4 in the latter and KCC2 in the lateral hypothalamic and ventromedial hypothalamic nuclei suggests that these cotransporters may have selective roles in neuroendocrine or homeostatic functions. Finally, we demonstrate the existence of a truncated splice variation of KCC3a in the rat that appears to be expressed exclusively in neurons (as is KCC2), whereas the native form of KCC3a and KCC4 appears to be expressed in glial cells.

Neuromedin U neurones in the rat nucleus of the tractus solitarius are catecholaminergic and respond to peripheral cholecystokinin.

Centrally administered neuromedin U (NMU) has profound effects on food intake and energy expenditure. In the rat, central expression of NMU mRNA is confined to the brainstem and the hypothalamus/pituitary, while mRNA for the receptor NMU2R is expressed in the hypothalamus and hippocampus, as well as in the lining of the ventricular system, but not in the brainstem. We demonstrate that a subpopulation of catecholaminergic neurones in the brainstem nucleus of the tractus solitarius contain NMU and are activated by the gut-derived peptide, cholecystokinin. This is consistent with NMU neurones having an anorectic action, probably via their interaction with other neurones in the paraventricular hypothalamus.

Imidazoline receptor antisera-selected (IRAS) cDNA: cloning and characterization.

The imidazoline-1 receptor (IR1) is considered a novel target for drug discovery. Toward cloning an IR1, a truncated cDNA clone was isolated from a human hippocampal lambda gt11 cDNA expression library by relying on the selectivity of two antisera directed against candidate IR proteins. Amplification reactions were performed to extend the 5' and 3' ends of this cDNA, followed by end-to-end PCR and conventional cloning. The resultant 5131-basepair molecule, designated imidazoline receptor-antisera-selected (IRAS) cDNA, was shown to encode a 1504-amino acid protein (IRAS-1). No relation exists between the amino acid sequence of IRAS-1 and proteins known to bind imidazolines (e.g., it is not an alpha2-adrenoceptor or monoamine oxidase subtype). However, certain sequences within IRAS-1 are consistent with signaling motifs found in cytokine receptors, as previously suggested for an IR1. An acidic region in IRAS-1 having an amino acid sequence nearly identical to that of ryanodine receptors led to the demonstration that ruthenium red, a dye that binds the acidic region in ryanodine receptors, also stained IRAS-1 as a 167-kD band on SDS gels and inhibited radioligand binding of native I1 sites in untransfected PC-12 cells (a source of authentic I1 binding sites). Two epitope-selective antisera were also generated against IRAS-1, and both reacted with the same 167-kD band on Western blots. In a host-cell-specific manner, transfection of IRAS cDNA into Chinese hamster ovary cells led to high-affinity I1 binding sites by criteria of nanomolar affinity for moxonidine and rilmenidine. Thus, IRAS-1 is the first protein discovered with characteristics of an IR1.

Characterization of a partial cDNA clone detected by imidazoline receptor-selective antisera.

A cDNA clone has been isolated from a human hippocampal cDNA expression library by relying on the selectivity of two antisera that are specific for imidazoline binding proteins. A 1789 bp cDNA clone was sequenced and shown to contain a single open-reading frame that predicts a 66 kDa polypeptide, but it is truncated based on its lack of a stop codon and poly-A+ tail. Two regions of homology exist for the predicted amino acid sequence in common with chromogranin-A and B proteins, a zinc finger protein, and the ryanodine receptor. Northern blot analyses of poly-A+ mRNA from 36 human tissues indicated two differentially expressed transcripts of 6.0 and 9.5 kb. The 6.0 kb mRNA form was enriched in brain and endocrine tissues as compared to other tissues, but not in strict concordance with I1-imidazoline binding sites. The highest overall amounts of the combined transcripts were found in pituitary. In situ hybridization histochemistry revealed an enrichment of the message in neuronal cell bodies of the rat hippocampus and cerebellar cortex. This clone has some of the properties expected of an imidazoline receptor.

Imidazoline receptor proteins are regulated in platelet-precursor MEG-01 cells by agonists and antagonists.

The I1-imidazoline receptor is a novel brainstem modulator of sympathetic outflow that is elevated on platelets and in brains of depressed patients. A positive correlation has been reported (accompanying manuscript) between plasma norepinephrine (NE) concentrations and the densities (Bmax) of platelet I1 binding sites (I1 sites). I1-candidate proteins of 33 kDa and 85 kDa are now identified on Western blots probed with anti-imidazoline receptor antiserum (IRBP antiserum), that correlate with Bmax values for I1 sites. Furthermore, a human megakaryoblastoma cell line (MEG-01) has been used to study the regulation of these proteins on megakaryocytic cells, while bovine adrenal chromaffin cells provide a standard I1 cell type for comparison. Both the 33 kDa and 85 kDa IRBP-immunoreactive bands were enriched in plasma membrane fractions. IRBP antiserum did not cross-react with I2 imidazoline binding sites located on platelet mitochondrial membranes. The 85 kDa band was enhanced under conditions lacking fetal bovine serum (FBS) from the culture medium 6 h prior to harvesting. Conversely, 33 kDa protein was enhanced on MEG-01 cells grown in the presence of 10% FBS; suggesting that a precursor (85 kDa) and product (33 kDa) relationship might be induced by serum. The 85 kDa band was robustly up-regulated in response to imidazoline receptor-sensitive ligands; moxonidine, idazoxan and agmatine (10 microM each for 6 h). NE also up-regulated the 85 kDa IRBP-immunoreactive protein on MEG-01 membranes, but to a lesser extent. Idazoxan, an imidazoline alpha 2-antagonist, off-set its induction of 85 kDa protein by reducing the 33 kDa band. Yohimbine, a non-imidazoline alpha 2-antagonist, was ineffective alone, or in combination with moxonidine (up to 40 microM), but yohimbine blocked NE's induction of the 85 kDa band. Therefore, a rise in either plasma NE and/or endogenous I-site ligands (i.e. agmatine) could explain an elevation of imidazoline receptors observed in depression.

Co-detection by two imidazoline receptor protein antisera of a novel 85 kilodalton protein.

Imidazoline receptors (I-receptors) are considered as potential therapeutic targets for a spectrum of stress-induced illnesses. Yet, I-receptors remain poorly defined at the molecular level. In this study, candidate imidazoline receptor proteins were compared using two imidazoline receptor-selective antisera of diverse origins. One antiserum was derived from affinity-purified imidazoline-binding protein. The second antiserum was produced as an anti-idiotypic antiserum, from purified IgG selective for imidazolines. Despite such diverse origins, both antisera co-identified an 85 kDa band on western blots from a variety of tissues. The integrity of the 85 kDa band was dependent on protection by eight different protease inhibitors. Other proteolytic breakdown products (obtained after homogenization with only one protease inhibitor) were comparable in size to previously reported smaller immunoreactive bands. The full-size 85 kDa band was also enriched in plasma membrane fractions and abundant in rat PC12 cells and brain regions known to be abundant in I1 binding sites. Furthermore, the immunodensity of the 85 kDa band, against anti-idiotypic antiserum, was linearly correlated with reported I1 site radioligand Bmax values (r2 = 0.8736, P = 0.0002) across nine rat tissues. Therefore, a possible candidate for the full-length imidazoline receptor(s) appears to be an 85 kDa protein.

Characterization of DNase I hypersensitive sites in the mouse 68-kDa neurofilament gene.

Four brain-specific DNase I hypersensitive sites (HSS) have previously been identified flanking the mouse 68-kDa neurofilament gene within a 1.7 kb upstream sequence which confers neuronal specificity of expression of this gene in transgenic mice. Previously several DNA-binding factors were detected at the HSS closest to the transcription start site (HSS1). However, no major brain-specific factors were identified, suggesting a possible role for the three remaining HSS in conferring tissue-specificity to the NF-L gene. Sequence analysis of the NF-L promoter region demonstrated the presence of an extensive CT repeat and several potential binding sites which are also found in other neurofilament promoters. Gel mobility shift assays revealed a similar but not identical banding pattern with brain and liver nuclear extracts at HSS2, and HSS3, however the banding pattern for HSS4 was predominantly brain-specific. DNase I footprinting revealed several factors binding to the upstream HSS regions in brain and liver nuclear extracts. These include a CCAAT box at HSS2, a novel brain-specific footprint near an adenovirus promoter element E2aE-C beta and a single liver-specific footprint associated with an POU/octamer binding site at HSS4. The presence of brain-specific gel shift bands and tissue-specific footprints associated with HSS4, suggest that this region may play an important role in the regulation of the NF-L gene.

Interaction of multiple nuclear proteins with the promoter region of the mouse 68-kDa neurofilament gene.

Four brain-specific, DNase I hypersensitive sites (HSS) have been mapped to the 5' flanking region of the mouse 68-kDa neurofilament gene. These sites are contained within a 1.7-kb sequence that confers neuronal specificity of expression of this gene in transgenic mice. To identify DNA sequences that might be involved in gene regulation, the HSS situated near the promoter region has been analyzed by gel mobility shift assays and DNase I footprinting to investigate protein binding sequences. Of particular interest are two footprints localized to a 9-nucleotide sequence that flanks both the light and medium neurofilament gene in mouse and to a sequence that demonstrates partial homology to several promoter regions, including element-1, a motif required for neuron specificity in Drosophila. A prominent footprint was also detected at a sequence that contains a near-perfect palindrome centered at a PstI restriction site.

Genes expressed in cortical neurons--chromatin conformation and DNase I hypersensitive sites.

DNase I sensitivity experiments were performed utilizing DNA probes to genes which are either transcribed in rat cortical neurons (the 68 kDa neurofilament gene and the neuron-specific enolase gene) or are transcriptionally silent (albumin). Results suggest that unlike liver, in which a hierarchy in chromatin conformation exists between transcribed and nontranscribed genes, the majority of protein coding sequences in cortical neurons may be relatively sensitive to nuclease digestion. This supports our previous observation of an increased DNase I sensitivity of total chromatin in cortical neurons. Nuclease sensitivity experiments also revealed the presence of brain-specific DNase I hypersensitive sites associated with the two neuron-specific genes.

Developmental changes in the synthesis of nonhistone nuclear proteins relative to the appearance of a short nucleosomal DNA repeat length in cerebral hemisphere neurons.

Fluctuations in the pattern of synthesis of nonhistone nuclear proteins were noted in cerebral hemisphere neurons during early postnatal development of the rat. Noteworthy changes included the synthesis of an acidic nuclear protein of relative molecular weight 41,000 (41K), two chromatin-associated basic proteins (37K and 38K) and several high molecular weight chromatin acidic proteins. These changes in the synthesis of nonhistone nuclear proteins occur at a developmental stage when a short nucleosomal DNA repeat length has appeared in cerebral hemisphere neurons.