Neu differentiation factor stimulates phosphorylation and activation of the Sp1 transcription factor. (1/4103)

Neu differentiation factors (NDFs), or neuregulins, are epidermal growth factor-like growth factors which bind to two tyrosine kinase receptors, ErbB-3 and ErbB-4. The transcription of several genes is regulated by neuregulins, including genes encoding specific subunits of the acetylcholine receptor at the neuromuscular junction. Here, we have examined the promoter of the acetylcholine receptor epsilon subunit and delineated a minimal CA-rich sequence which mediates transcriptional activation by NDF (NDF-response element [NRE]). Using gel mobility shift analysis with an NRE oligonucleotide, we detected two complexes that are induced by treatment with neuregulin and other growth factors and identified Sp1, a constitutively expressed zinc finger phosphoprotein, as a component of one of these complexes. Phosphatase treatment, two-dimensional gel electrophoresis, and an in-gel kinase assay indicated that Sp1 is phosphorylated by a 60-kDa kinase in response to NDF-induced signals. Moreover, Sp1 seems to act downstream of all members of the ErbB family and thus may funnel the signaling of the ErbB network into the nucleus.  (+info)

(S)-(-)-Cotinine, the major brain metabolite of nicotine, stimulates nicotinic receptors to evoke [3H]dopamine release from rat striatal slices in a calcium-dependent manner. (2/4103)

Cotinine, a major peripheral metabolite of nicotine, has recently been shown to be the most abundant metabolite in rat brain after peripheral nicotine administration. However, little attention has been focused on the contribution of cotinine to the pharmacological effects of nicotine exposure in either animals or humans. The present study determined the concentration-response relationship for (S)-(-)-cotinine-evoked 3H overflow from superfused rat striatal slices preloaded with [3H]dopamine ([3H]DA) and whether this response was mediated by nicotinic receptor stimulation. (S)-(-)-Cotinine (1 microM to 3 mM) evoked 3H overflow from [3H]DA-preloaded rat striatal slices in a concentration-dependent manner with an EC50 value of 30 microM, indicating a lower potency than either (S)-(-)-nicotine or the active nicotine metabolite, (S)-(-)-nornicotine. As reported for (S)-(-)-nicotine and (S)-(-)-nornicotine, desensitization to the effect of (S)-(-)-cotinine was observed. The classic nicotinic receptor antagonists mecamylamine and dihydro-beta-erythroidine inhibited the response to (S)-(-)-cotinine (1-100 microM). Additionally, 3H overflow evoked by (S)-(-)-cotinine (10-1000 microM) was inhibited by superfusion with a low calcium buffer. Interestingly, over the same concentration range, (S)-(-)-cotinine did not inhibit [3H]DA uptake into striatal synaptosomes. These results demonstrate that (S)-(-)-cotinine, a constituent of tobacco products and the major metabolite of nicotine, stimulates nicotinic receptors to evoke the release of DA in a calcium-dependent manner from superfused rat striatal slices. Thus, (S)-(-)-cotinine likely contributes to the neuropharmacological effects of nicotine and tobacco use.  (+info)

Retrotransposons transcribed preferentially in proximal tubules of salt-hypertensive rats. (3/4103)

BACKGROUND: The kidney is considered to play an important etiologic role in salt-sensitive hypertension. The aim of the present study was to isolate genes whose expression differs between the kidneys of salt-hypertensive and control rats using an mRNA differential display method. METHODS: Dahl salt-sensitive (DS) and control salt-resistant rats (DR) were fed a 0.3% or 8% NaCl diet. Renal RNA was amplified by RNA arbitrarily primed polymerase chain reaction (RAP-PCR) and compared among DR 0.3%, DR 8%, DS 0.3%, and DS 8%. Gene expression and localization were examined by Northern blotting, RNase protection assay, and in situ hybridization. Full-length nucleotide sequence was determined by screening a DS rat kidney cDNA library. RESULTS: We identified one differentially displayed clone, and its expression was greater in DS than DR, which was not affected by salt loading. The sequence was 90% homologous to the 3'-noncoding region of the nicotinic acetylcholine receptor alpha7 subunit gene. Its expression was kidney-specific, and was localized in the proximal tubules. The transcript level was markedly increased precedent to the development of hypertension. Its expression was also high in other salt-sensitive rats, and low in normotensive Sprague-Dawley and Wistar rats. The full-length cDNA contained elements homologous to the retroviral pol gene, a primer binding site sequence for reverse transcriptase, and long-terminal repeats. CONCLUSION: These results demonstrated that the newly identified transcripts (REPT1) belong to a novel retrotransposon family, which showed unique strain-, age-, tissue-, and cell type-specific expression pattern.  (+info)

N-type voltage-dependent calcium channels mediate the nicotinic enhancement of GABA release in chick brain. (4/4103)

The role of voltage-dependent calcium channels (VDCCs) in the nicotinic acetylcholine receptor (nAChR)-mediated enhancement of spontaneous GABAergic inhibitory postsynaptic currents (IPSCs) was investigated in chick brain slices. Whole cell recordings of neurons in the lateral spiriform (SpL) and ventral lateral geniculate (LGNv) nuclei showed that cadmium chloride (CdCl2) blocked the carbachol-induced increase of spontaneous GABAergic IPSCs, indicating that VDCCs might be involved. To conclusively show a role for VDCCs, the presynaptic effect of carbachol on SpL and LGNv neurons was examined in the presence of selective blockers of VDCC subtypes. omega-Conotoxin GVIA, a selective antagonist of N-type channels, significantly reduced the nAChR-mediated enhancement of gamma-aminobutyric acid (GABA) release in the SpL by 78% compared with control responses. Nifedipine, an L-type channel blocker, and omega-Agatoxin-TK, a P/Q-type channel blocker, did not inhibit the enhancement of GABAergic IPSCs. In the LGNv, omega-Conotoxin GVIA also significantly reduced the nAChR-mediated enhancement of GABA release by 71% from control values. Although omega-Agatoxin-TK did not block the nicotinic enhancement, L-type channel blockers showed complex effects on the nAChR-mediated enhancement. These results indicate that the nAChR-mediated enhancement of spontaneous GABAergic IPSCs requires activation of N-type channels in both the SpL and LGNv.  (+info)

Light-induced calcium influx into retinal axons is regulated by presynaptic nicotinic acetylcholine receptor activity in vivo. (5/4103)

Visual activity is thought to be a critical factor in controlling the development of central retinal projections. Neuronal activity increases cytosolic calcium, which was hypothesized to regulate process outgrowth in neurons. We performed an in vivo imaging study in the retinotectal system of albino Xenopus laevis tadpoles with the fluorescent calcium indicator calcium green 1 dextran (CaGD) to test the role of calcium in regulating axon arbor development. We find that visual stimulus to the retina increased CaGD fluorescence intensity in retinal ganglion cell (RGC) axon arbors within the optic tectum and that branch additions to retinotectal axon arbors correlated with a local rise in calcium in the parent branch. We find three types of responses to visual stimulus, which roughly correlate with the ON, OFF, and SUSTAINED response types of RGC reported by physiological criteria. Imaging in bandscan mode indicated that patterns of calcium transients were nonuniform throughout the axons. We tested whether the increase in calcium in the retinotectal axons required synaptic activity in the retina; intraocular application of tetrodotoxin (10 microM) or nifedipine (1 and 10 microM) blocked the stimulus-induced increase in RGC axonal fluorescence. A second series of pharmacological investigations was designed to determine the mechanism of the calcium elevation in the axon terminals within the optic tectum. Injection of bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid-AM (BAPTA-AM) (20 mM) into the tectal ventricle reduced axonal calcium levels, supporting the idea that visual stimulation increases axonal calcium. Injection of BAPTA (20 mM) into the tectal ventricle to chelate extracellular calcium also attenuated the calcium response to visual stimulation, indicating that calcium enters the axon from the extracellular medium. Caffeine (10 mM) caused a large increase in axonal calcium, indicating that intracellular stores contribute to the calcium signal. Presynaptic nicotinic acetylcholine receptors (nAChRs) may play a role in axon arbor development and the formation of the topographic retinotectal projection. Injection of nicotine (10 microM) into the tectal ventricle significantly elevated RGC axonal calcium levels, whereas application of the nAChR antagonist alphaBTX (100 nM) reduced the stimulus-evoked rise in RGC calcium fluorescence. These data suggest that light stimulus to the retina increases calcium in the axon terminal arbors through a mechanism that includes influx through nAChRs and amplification by calcium-induced calcium release from intracellular calcium stores. Such a mechanism may contribute to developmental plasticity of the retinotectal system by influencing both axon arbor elaboration and the strength of synaptic transmission.  (+info)

NMR spatial structure of alpha-conotoxin ImI reveals a common scaffold in snail and snake toxins recognizing neuronal nicotinic acetylcholine receptors. (6/4103)

A 600 MHz NMR study of alpha-conotoxin ImI from Conus imperialis, targeting the alpha7 neuronal nicotinic acetylcholine receptor (nAChR), is presented. ImI backbone spatial structure is well defined basing on the NOEs, spin-spin coupling constants, and amide protons hydrogen-deuterium exchange data: rmsd of the backbone atom coordinates at the 2-12 region is 0.28 A in the 20 best structures. The structure is described as a type I beta-turn (positions 2-5) followed by a distorted helix (positions 5-11). Similar structural patterns can be found in all neuronal-specific alpha-conotoxins. Highly mobile side chains of the Asp-5, Arg-7 and Trp-10 residues form a single site for ImI binding to the alpha7 receptor. When depicted with opposite directions of the polypeptide chains, the ImI helix and the tip of the central loop of long chain snake neurotoxins demonstrate a common scaffold and similar positioning of the functional side chains, both of these structural elements appearing essential for binding to the neuronal nAChRs.  (+info)

Selective effects of a 4-oxystilbene derivative on wild and mutant neuronal chick alpha7 nicotinic receptor. (7/4103)

1. We assessed the pharmacological activity of triethyl-(beta-4-stilbenoxy-ethyl) ammonium (MG624), a drug that is active on neuronal nicotinic receptors (nicotinic AChR). Experiments on the major nicotinic AChR subtypes present in chick brain, showed that it inhibits the binding of [125I]-alphaBungarotoxin (alphaBgtx) to the alpha7 subtype, and that of [3H]-epibatidine (Epi) to the alpha4beta2 subtype, with Ki values of respectively 106 nM and 84 microM. 2. MG624 also inhibited ACh elicited currents (I(ACh)) in the oocyte-expressed alpha7 and alpha4beta2 chick subtypes with half-inhibitory concentrations (IC50) of respectively 109 nM and 3.2 microM. 3. When tested on muscle-type AChR, it inhibited [125I]-alphaBgtx binding with a Ki of 32 microM and ACh elicited currents (I(ACh)) in the oocyte-expressed alpha1beta1gammadelta chick subtype with an IC50 of 2.9 microM. 4. The interaction of MG624 with the alpha7 subtype was investigated using an alpha7 homomeric mutant receptor with a threonine-for-leucine 247 substitution (L247T alpha7). MG624 did not induce any current in oocytes expressing the wild type alpha7 receptor, but did induce large currents in the oocyte-expressed L247T alpha7 receptor. The MG624 elicited current (I(MG62)) has an EC50 of 0.2 nM and a Hill coefficient nH of 1.9, and is blocked by the nicotinic receptor antagonist methyllycaconitine (MLA). 5. These binding and electrophysiological studies show that MG624 is a potent antagonist of neuronal chick alpha7 nicotinic AChR, and becomes a competitive agonist following the mutation of the highly conserved leucine residue 247 located in the M2 channel domain.  (+info)

Regulation of alpha4beta2 nicotinic receptor desensitization by calcium and protein kinase C. (8/4103)

Neuronal nicotinic acetylcholine receptor (nAChR) desensitization is hypothesized to be a trigger for long-term changes in receptor number and function observed after chronic administration of nicotine at levels similar to those found in persons who use tobacco. Factors that regulate desensitization could potentially influence the outcome of long-lasting exposure to nicotine. The roles of Ca2+ and protein kinase C (PKC) on desensitization of alpha4beta2 nAChRs expressed in Xenopus laevis oocytes were investigated. Nicotine-induced (300 nM; 30 min) desensitization of alpha4beta2 receptors in the presence of Ca2+ developed in a biphasic manner with fast and slow exponential time constants of tauf = 1.4 min (65% relative amplitude) and taus = 17 min, respectively. Recovery from desensitization was reasonably well described by a single exponential with taurec = 43 min. Recovery was largely eliminated after replacement of external Ca2+ with Ba2+ and slowed by calphostin C (taurec = 48 min), an inhibitor of PKC. Conversely, the rate of recovery was enhanced by phorbol-12-myristate-13-acetate (taurec = 14 min), a PKC activator, or by cyclosporin A (with taurec = 8 min), a phosphatase inhibitor. alpha4beta2 receptors containing a mutant alpha4 subunit that lacks a consensus PKC phosphorylation site exhibited little recovery from desensitization. Based on a two-desensitized-state cyclical model, it is proposed that after prolonged nicotine treatment, alpha4beta2 nAChRs accumulate in a "deep" desensitized state, from which recovery is very slow. We suggest that PKC-dependent phosphorylation of alpha4 subunits changes the rates governing the transitions from "deep" to "shallow" desensitized conformations and effectively increases the overall rate of recovery from desensitization. Long-lasting dephosphorylation may underlie the "permanent" inactivation of alpha4beta2 receptors observed after chronic nicotine treatment.  (+info)