Acquired transient stuttering during a migraine attack.

Stuttering is an abnormality in the fluency of speech, which is characterized by interruption of the normal rhythm due to involuntary repetition and prolongation, or arrest, of uttered letters or syllables. The aphasic syndrome and dysarthria can be associated with classic migraine, but, to our knowledge, no study has so far described stuttering as the only neurological symptom accompanying an attack.

Expression of the ryanodine receptor type 3 calcium release channel during development and differentiation of mammalian skeletal muscle cells.

In vertebrate skeletal muscles, the type 1 isoform of ryanodine receptor (RyR1) is essential in triggering contraction by releasing Ca2+ from the sarcoplasmic reticulum in response to plasma membrane depolarisation. Recently, the presence of another RyR isoform, RyR3, has been detected in mammalian skeletal muscle cells, raising the question of the eventual relevance of RyR3 for muscle cell physiology. The expression of RyR3 was investigated during differentiation of skeletal muscle cells. Using antibodies able to distinguish the different RyR isoforms and Western blot analysis, the RyR3 protein was detected in the microsomal fractions of differentiated skeletal muscle cells but not of undifferentiated cells. Accordingly, blocking muscle differentiation by the addition of either transforming growth factor-beta or basic fibroblast growth factor prevented the expression of the RyR3 protein. In differentiated skeletal muscle cells, RyR3 was expressed independent of cell fusion and myotube formation. The expression of RyR3 was also investigated during development of the diaphragm muscle. The RyR3 content in the diaphragm muscle increased between the late stage of fetal development and the first postnatal days. However, at variance with RyR1, which reached maximum levels of expression 2-3 weeks after birth, the expression of RyR3 was found to be higher in the neonatal phase of the diaphragm muscle development (2-15 days after birth) than in the same muscle from adult mice. The differential content of RyR3 in adult skeletal muscles was found not to be mediated by neurotrophic factors or electrical activity. These findings indicate that RyR3 is preferentially expressed in differentiated skeletal muscle cells. In addition, during skeletal muscle development, its expression is regulated differently from that of RyR1.

Activation of delta-opioid receptors inhibits neuronal-like calcium channels and distal steps of Ca(2+)-dependent secretion in human small-cell lung carcinoma cells.

Human small-cell lung carcinoma (SCLC) cells express neuronal-like voltage-operated calcium channels (VOCCs) and release mitogenic hormones such as serotonin (5-HT). Opioid peptides, on the other hand, have been shown to reduce SCLC cell proliferation by an effective autocrine pathway. Here we show that in GLC8 SCLC cells, only delta-opioid receptor subtype mRNA is expressed. Consistently, the selective delta-opioid agonist [D-Pen2-Pen5]-enkephalin (DPDPE), but not mu and kappa agonists, potently and dose-dependently inhibits high-threshold (HVA) VOCCs in these cells. As in peripheral neurons, this modulation is largely voltage-dependent, mediated by pertussis toxin (PTX)-sensitive G-proteins, cAMP-independent, and mainly affecting N-type VOCCs. With the same potency and selectivity, DPDPE also antagonizes the Ca(2+)-dependent release of [3H]serotonin ([3H]5-HT) from GLC8 cells. However, DPDPE inhibits not only the depolarization-induced release, but also the Ca(2+)-dependent secretion induced by thapsigargin or ionomycin. This suggests that besides inhibiting HVA VOCCs, opioids also exert a direct depressive action on the secretory apparatus in GLC8 cells. This latter effect also is mediated by a PTX-sensitive G-protein but, contrary to VOCC inhibition, it can be reversed by elevations of cAMP levels. These results show for the first time that opioids effectively depress both Ca2+ influx and Ca(2+)-dependent hormone release in SCLC cells by using multiple modulatory pathways. It can be speculated that the two mechanisms may contribute to the opioid antimitogenic action on lung neuroendocrine carcinoma cells.

Calcium pools in Ehrlich carcinoma cells. A major, high affinity Ca2+ pool is sensitive to both inositol 1,4,5-trisphosphate and thapsigargin.

To investigate the presence and the size of different non-mitochondrial Ca2+ pools of Ehrlich ascites tumor cells (EATCs), digitonin-permeabilized cells were allowed to accumulate Ca2+ in the presence of mitochondrial inhibitors and treated with the reticular Ca(2+)-ATPase inhibitor thapsigargin, IP3 and the Ca2+ ionophore A23187. Emptying of thapsigargin-sensitive Ca2+ stores prevented any Ca2+ release by IP3, and, after IP3 addition, little or no Ca2+ was released by thapsigargin. In both instances, a further Ca2+ release was accomplished by A23187. The IP3-thapsigargin-sensitive pool and the residual A23187-sensitive one corresponded to approximately 60 and 37% of non-mitochondrial stored Ca2+, respectively. In intact EATCs, IP3-dependent agonists and thapsigargin discharged Ca2+ pools almost completely overlapping, and A32187 released a minor residual Ca2+ pool. The IP3-insensitive pool appeared to have a relatively low affinity for Ca2+ (below 600 nM). The high affinity, IP3-sensitive Ca2+ pool was discharged in a 'quantal' manner following step additions of sub maximal [IP3], and the IP3-induced fractional Ca2+ release was more marked at higher concentrations of stored (luminal) Ca2+, The IP3-sensitive Ca2+ pool appeared to be devoid of the Ca(2+)-activated Ca2+ release channel since caffeine did not released any Ca2+ in intact and permeabilized EATCs, and Western blot analyses of EATC microsomal membranes failed to detect any known ryanodine receptor isoform.

Anti-beta 2 subunit antisense oligonucleotides modulate the surface expression of the alpha 1 subunit of N-type omega-CTX sensitive Ca2+ channels in IMR 32 human neuroblastoma cells.

High voltage activated Ca2+ channels are heteropolymeric complexes in which the alpha 1 subunit forms the channel, while the alpha 2-delta and beta subunits are important for the assembly and regulation of the biophysical properties of the channel. We have tested the role of the beta 2 subunit on the expression and electrophysiological properties of the omega-conotoxin GVIA-sensitive Ca2+ channel expressed in the IMR 32 human neuroblastoma cell line. Anti-beta 2 subunit antisense oligonucleotides supplied to the cells in culture induced a time-dependent increase in the number of [125I]-omega-conotoxin binding sites on the cell surface, which was not paralleled by an increase in current amplitude. We suggest that a reduction in the expression of beta 2 stimulates the transport to the plasma membrane of non-functioning Ca2+ channels and, in particular, of the alpha 1 omega-conotoxin binding subunit.

Serotonin release and cell proliferation are under the control of alpha-bungarotoxin-sensitive nicotinic receptors in small-cell lung carcinoma cell lines.

Neuronal type nicotinic acetylcholine receptors (nAchRs) have recently been identified in small-cell lung carcinoma. We here show that both nicotine and cytisine stimulate [3H]serotonin release in a dose-dependent manner; this effect is antagonized by alpha-bungarotoxin (alpha Bgtx) and alpha-conotoxin MI (alpha Ctx). Nicotine and cytisine stimulate in vitro SCLC proliferation and this effect is completely antagonized by both alpha Bgtx and alpha Ctx. By PCR analysis, we demonstrate the presence in SCLC of both the alpha 7 and the beta 2 nAchR subunits mRNA. These data show that nAchRs play an important role in the biology of SCLC, and that alpha Bgtx-sensitive receptors of the alpha 7 subtype are crucially involved in both the secretagogue and mitogenic effects of nicotinic agonists.

Expression of three alternative acetylcholinesterase messenger RNAs in human tumor cell lines of different tissue origins.

To study the molecular mechanisms underlying the intensive expression of acetylcholinesterase (AChE) in different tumor types, we characterized levels and composition of its messenger RNA (mRNA) sequences in heterologous tumor cell lines, primary tumor biopsies, and normal fetal and adult tissues and determined their exon-intron origin within the corresponding ACHE gene. Reverse transcription followed by polymerase chain reaction (RT-PCR) revealed three alternatively spliced ACHE mRNAs in NT2/D1 teratocarcinoma, NCI-N-592 small cell lung carcinoma, TE671 medulloblastoma, K-562 erythroleukemia, and 293 transformed embryonal kidney cells. The three ACHE mRNAs include the principal species expressed in brain and muscle and two additional transcripts containing insertions of 751 or 829 residues downstream from the exon 4 domain. The inserted region, which represents an intron in brain and muscle, is expressed in the tumor cell lines either as a "readthrough" form or with 78 residues deleted from its 5' end. A major band of 2.5 kb was labeled with ACHE cDNA in poly(A)+ RNA blots from medulloblastoma cells or brain tissue, whereas a PCR-amplified probe from the inserted domain labeled a 3.4-kb band but not the 2.5-kb band in poly(A)+ RNA from small cell lung carcinoma. The ACHE mRNAs including the alternative insertions were found only in cell lines with levels of the principal ACHE mRNA species equal to or higher than those in brain (1-10 molecules/cell), determined by following the kinetics of mRNA PCR amplification. Genomic DNA sequencing revealed that the inserted domains in the ACHE mRNAs expressed in the tumor cell lines encode C-terminal peptides of 40 and 14 residues. These include a free cysteine, terminate with the consensus HG element, and continue by a 29-residue-long C-terminal hydrophobic cleavable peptide, properties characteristic of precursors to phosphoinositide (PI)-linked proteins. In extension of the reported expression of PI-linked AChE in hemopoietic cells including K-562, our findings demonstrate the existence of ACHE mRNAs with the potential to encode one hydrophilic and two PI-linked forms of AChE in tumor cells from both hemopoietic and nonhemopoietic origins.

Calcium channel subtypes controlling serotonin release from human small cell lung carcinoma cell lines.

Small cell lung carcinoma is an aggressive neuroendocrine tumor that secretes several hormones, some of which act as autocrine growth factors. In order to obtain more information on the process of hormone secretion from this tumor, we have studied the role of intracellular free Ca2+ concentrations and voltage-operated calcium channels in the control of [3H]serotonin release from in vitro growing cell lines. We found that the Ca2+ ionophore ionomycin and the Ca(2+)-ATPase antagonist thapsigargin induced a dose-dependent increase of intracellular Ca2+ and a parallel enhancement of [3H]serotonin release. KCl-induced depolarization also stimulated a dose- and Ca(2+)-dependent [3H]serotonin release that in the GLC8 cell line was effectively inhibited by Ca2+ channel antagonists (Cd2+, nitrendipine, verapamil, omega-conotoxin GVIA, and omega-agatoxin IVA) and potentiated by the Ca2+ channel agonist BayK8644. Autoantibodies against Ca2+ channels present in the sera of Lambert-Eaton myasthenic patients antagonized KCl- but not ionomycin-induced [3H]serotonin release. Polymerase chain reaction analysis indicated that GLC8 cells express L-, N-, and P-type neuronal Ca2+ channel alpha 1 subunits, together with two types of Ca2+ channel beta subunits. The presence of three functionally distinct high threshold Ca2+ channels was also revealed by patch clamp experiments; high threshold Ca2+ channels were identified as dihydropyridine-sensitive (L-type), omega-conotoxin GVIA-sensitive (N-type), and omega-agatoxin IVA-sensitive (P-type). Our data demonstrate that [3H]serotonin is released by small cell lung carcinoma cells in a Ca(2+)-dependent manner and that depolarization-induced [3H]serotonin release is mediated by Ca2+ influx through distinct, neuron-like, Ca2+ channel subtypes.

Splicing of an anti-sense Alu sequence generates a coding sequence variant for the alpha-3 subunit of a neuronal acetylcholine receptor.

In this report we demonstrate that an alpha-3 acetylcholine receptor subunit transcriptional variant originates through alternative splicing of a complementary sequence of the right arm of an Alu element. This element is located within the 5.1 Kb intron found between exons 5 and 6 of the alpha-3 acetylcholine receptor subunit gene. The transcriptional variant originates from the normal splicing process and carries an in-frame stop codon. If translated, it should encode for a peptide lacking the 4th transmembrane domain of the normal subunit.

Tumor necrosis factor alters synaptic transmission in rat hippocampal slices.

The effects of human recombinant tumor necrosis factor (TNF-alpha) on the synaptic transmission were studied in rat hippocampal slices by using extracellular field potential recordings. Population spikes and/or excitatory postsynaptic potentials were extracellularly recorded in hippocampus CA1 region from stratum pyramidale and stratum radiatum, respectively, and synaptic transmission was examined in the Schaffer collateral/commissural-CA1 pathway. Basal neurotransmission slightly and promptly increased in slices acutely exposed to TNF-alpha (1-100 nM). Examination of the long-term potentiation (LTP) revealed that a brief treatment with the cytokine did not influence LTP, while a long-lasting application of TNF-alpha (50 min or more) inhibited LTP in a dose-dependent way in the range of 1-100 nM. A role for TNF-alpha as a peptide of immunological significance belonging to the family of brain neuromodulators is discussed.

Neuronal-type nicotinic receptors in human neuroblastoma and small-cell lung carcinoma cell lines.

A beta subunit of the neuronal nicotinic receptor, sharing 88% homology with the rat beta 4 subunit, has been cloned from a human neuroblastoma cell line. The gene encoding the human beta 4 subunit is expressed in association with the alpha 3 gene in neuroblastoma and small-cell lung carcinoma cell lines. Patch-clamp experiments and radioligand binding assays confirm that these neuroendocrine tumor cell lines express functional neuronal nicotinic receptors. We suggest that these receptors might play a crucial role in the control of neurotransmitter and hormone secretion from neurosecretory human tumors.

Isoform-selective deficit of glycine receptors in the mouse mutant spastic.

The mutant mouse spastic (spa) develops a characteristic motor disorder about 2 weeks after birth, with symptoms resembling sublethal poisoning by the glycinergic antagonist strychnine. Correspondingly, adult homozygotic mutants (spa/spa) exhibit a severe reduction of inhibitory glycine receptors in spinal cord and brain. Here we show that the spastic mutation selectively interferes with the postnatal accumulation of the adult isoform of the glycine receptor protein, whereas perinatal expression of the neonatal receptor isoform is not detectably affected. Heterologous expression in X. laevis oocytes of poly(A)+ RNA and Northern blot analysis indicate normal levels of glycine receptor alpha 1 subunit transcripts in spinal cord of adult spastic mutants. Thus, the age-dependent manifestation of spastic symptoms after birth reflects a selective effect of the mutation on the developmental expression of the adult glycine receptor isoform.

Molecular cloning of human neuronal nicotinic receptor alpha 3-subunit.

Neuronal nicotinic receptors (nAchRs) have been isolated or cloned in insect, bird and mammalian neurons, but no information exists on the primary structure of human neuronal nAchRs. By screening a cDNA library from the human neuroblastoma cell line IMR 32 with a cDNA probe corresponding to the full length of rat alpha 3-nicotinic subunit, we have identified an open reading frame encoding a protein of 502 amino acids. This protein shows all the features of members of the ligand-gated receptor superfamily and has two cysteine residues at positions 192, 193 which are typical of the nicotinic alpha-subunits. Because of its high homology to rat alpha 3 (93% amino acid identity), we conclude that we have cloned the human alpha 3-nicotinic subunit.