ryanodine and Neuroblastoma

We synthesized analogs modified in the ribose unit (ribose linked to N1 of adenine) of cyclic ADP-ribose (cADPR), a Ca2+-mobilizing second messenger. The biological activities of these analogs were determined in NG108-15 neuroblastoma x glioma hybrid cells that were pre-loaded with fura-2 acetoxymethylester and subjected to whole-cell patch-clamp. Application of the hydrolysis-resistant cyclic ADP-carbocyclic-ribose (cADPcR) through patch pipettes potentiated elevation of the cytoplasmic free Ca2+ concentration ([Ca2+]i) at the depolarized membrane potential. The increase in [Ca2+]i evoked upon sustained membrane depolarization was significantly larger in cADPcR-infused cells than in non-infused cells and its degree was equivalent to or significantly greater than that induced by cADPR or beta-NAD+. 8-Chloro-cADPcR and two inosine congeners (cyclic IDP-carbocyclic-ribose and 8-bromo-cyclic IDP-carbocyclic-ribose) did not induce effects similar to those of cADPcR or cADPR. Instead, 8-chloro-cADPcR together with cADPR or cADPcR caused inhibition of the depolarization-induced [Ca2+]i increase as compared with either cADPR or cADPcR alone. These results demonstrated that our cADPR analogs have agonistic or antagonistic effects on the depolarization-induced [Ca2+]i increase and suggested the presence of functional reciprocal coupling between ryanodine receptors and voltage-activated Ca2+ channels via cADPR in mammalian neuronal cells.

Topics: Animals; Calcium; Calcium Channel Blockers; Cell Line; Cyclic ADP-Ribose; Cytoplasm; Dose-Response Relationship, Drug; Drug Interactions; Electric Stimulation; Fura-2; Membrane Potentials; Mice; Neural Inhibition; Neuroblastoma; Neurons; Nifedipine; Patch-Clamp Techniques; Ruthenium; Ryanodine

We report that cell survival after neurite transection in a mammalian neuronal model (cultured B104 cells) critically depends on somal [Ca2+]i, a novel result that reconciles separate long-standing observations that somal survival decreases with more-proximal axonal transections and that increased somal Ca2+ is cytotoxic. Using fluorescence microscopy, we demonstrate that extracellular Ca2+ at the site of plasmalemmal transection is necessary to form a plasmalemmal barrier, and that other divalent ions (Ba2+, Mg2+) do not play a major role. We also show that extracellular Ca2+, rather than injury per se, initiates the formation of a plasmalemmal barrier and that a transient increase in somal [Ca2+]i significantly decreases the percentage of cells that survive neurite transection. Furthermore, we show that the increased somal [Ca2+]i and decreased cell survival following proximal transections are not due to less frequent or slower plasmalemmal sealing or Ca2+ entry through plasmalemmal Na+ and Ca2+ channels. Rather, the increased somal [Ca2+]i and lethality of proximal neurite injuries may be due to the decreased path length/increased diameter for Ca2+ entering the transection site to reach the soma. A ryanodine block of Ca2+ release from internal stores before transection has no effect on cell survival; however, a ryanodine- or thapsigargin-induced buildup of somal [Ca2+]i before transection markedly reduces cell survival, suggesting a minor involvement of Ca2+-induced release from internal stores. Finally, we show that cell survival following proximal injuries can be enhanced by increasing intracellular Ca2+ buffering capacity with BAPTA to prevent the increase in somal [Ca2+]i.

Topics: Animals; Cadmium; Calcium; Cell Line, Tumor; Cell Survival; Chelating Agents; Drug Interactions; Egtazic Acid; Enzyme Inhibitors; Fluorescent Dyes; Intracellular Fluid; Neurites; Neuroblastoma; Neurons; Potassium; Rats; Ryanodine; Thapsigargin; Time Factors; Trauma, Nervous System

Neuronal nicotinic acetylcholine receptors (nAChR) can regulate several neuronal processes through Ca2+-dependent mechanisms. The versatility of nAChR-mediated responses presumably reflects the spatial and temporal characteristics of local changes in intracellular Ca2+ arising from a variety of sources. The aim of this study was to analyse the components of nicotine-evoked Ca2+ signals in SH-SY5Y cells, by monitoring fluorescence changes in cells loaded with fluo-3 AM. Nicotine (30 microm) generated a rapid elevation in cytoplasmic Ca2+ that was partially and additively inhibited (40%) by alpha7 and alpha3beta2* nAChR subtype selective antagonists; alpha3beta4* nAChR probably account for the remaining response (60%). A substantial blockade (80%) by CdCl2 (100 microm) indicates that voltage-operated Ca2+ channels (VOCC) mediate most of the nicotine-evoked response, although the alpha7 selective antagonist alpha-bungarotoxin (40 nm) further decreased the CdCl2- resistant component. The elevation of intracellular Ca2+ levels provoked by nicotine was sustained for at least 10 min and required the persistent activation of nAChR throughout the response. Intracellular Ca2+ stores were implicated in both the initial and sustained nicotine-evoked Ca2+ responses, by the blockade observed after ryanodine (30 microm) and the inositoltriphosphate (IP3)-receptor antagonist, xestospongin-c (10 microm). Thus, nAChR subtypes are differentially coupled to specific sources of Ca2+: activation of nAChR induces a sustained elevation of intracellular Ca2+ levels which is highly dependent on the activation of VOCC, and also involves Ca2+ release from ryanodine and IP3-dependent intracellular stores. Moreover, the alpha7, but not alpha3beta2* nAChR, are responsible for a fraction of the VOCC-independent nicotine-evoked Ca2+ increase that appears to be functionally coupled to ryanodine sensitive Ca2+ stores.

Topics: Aniline Compounds; Calcium; Calcium Channels; Calcium Signaling; Enzyme Inhibitors; Fluorescent Dyes; Humans; Intracellular Fluid; Macrocyclic Compounds; Mecamylamine; Neuroblastoma; Neurons; Nicotine; Nicotinic Agonists; Nicotinic Antagonists; Oxazoles; Receptors, Nicotinic; Ryanodine; Tumor Cells, Cultured; Xanthenes

delta-Hexachlorocyclohexane (delta-HCH), a lipophilic neurodepressant agent, has been shown to inhibit neurotransmitter release and stimulate ryanodine-sensitive Ca(2+) channels. However, the effect of delta-HCH on neuronal activity remains unclear, although it may enhance the gamma-aminobutyric acid-induced current. Its effects on ionic currents were investigated in rat pituitary GH(3) cells and human neuroblastoma IMR-32 cells. In GH(3) cells, delta-HCH increased the amplitude of Ca(2+)-activated K(+) current (I(K(Ca))). delta-HCH (100 microM) slightly inhibited the amplitude of voltage-dependent K(+) current. delta-HCH (30 microM) suppressed voltage-dependent L-type Ca(2+) current (I(Ca, L)), whereas gamma-HCH (30 microM) had no effect on I(Ca, L). In the inside-out configuration, delta-HCH applied intracellularly did not change the single channel conductance of large conductance Ca(2+)-activated K(+) (BK(Ca)) channels; however, it did increase the channel activity. The delta-HCH-mediated increase in the channel activity is mainly mediated by its increase in the number of long-lived openings. delta-HCH reversibly increased the activity of BK(Ca) channels in a concentration-dependent manner with an EC(50) value of 20 microM. delta-HCH also caused a left shift in the midpoint for the voltage-dependent opening. In contrast, gamma-HCH (30 microM) suppressed the activity of BK(Ca) channels. Under the current-clamp mode, delta-HCH (30 microM) reduced the firing rate of spontaneous action potentials; however, gamma-HCH (30 microM) increased it. In neuroblastoma IMR-32 cells, delta-HCH also increased the amplitude of I(K(Ca)) and stimulated the activity of intermediate-conductance K(Ca) channels. This study provides evidence that delta-HCH is an opener of K(Ca) channels. The effects of delta-HCH on these channels may partially, if not entirely, be responsible for the underlying cellular mechanisms by which delta-HCH affects neuronal or neuroendocrine function.

Topics: Action Potentials; Animals; Calcium; Central Nervous System Depressants; Dantrolene; Dose-Response Relationship, Drug; Electric Conductivity; Estradiol; Hexachlorocyclohexane; Humans; Indoles; Inositol 1,4,5-Trisphosphate; Kinetics; Large-Conductance Calcium-Activated Potassium Channels; Neuroblastoma; Pituitary Gland; Potassium Channels; Potassium Channels, Calcium-Activated; Rats; Ruthenium; Ryanodine; Tumor Cells, Cultured

Volumes of neuroblastoma x glioma hybrid NG 108-15 cells were electronically measured in order to characterize the mechanisms involved in volume regulation in isosmotic and anisosmotic conditions. The cells behave as perfect osmometers when tonicity was changed at constant chloride concentration by adding sucrose or replacing NaCl with CaCl2 or MgCl2. In contrast, the cell volume was poorly dependent on tonicity when the Cl- concentration was changed by adding NaCl or H2O. Cell shrinkage was induced by cell stirring or after a hypotonicity-induced swelling. These volume decreases were abolished by caffeine but not by ryanodine or EGTA. Shrinkage was also induced by the Ca2+ ionophore ionomycin. The ionomycin-induced volume decrease was abolished by EGTA. Cell swelling induced an outwardly rectifying Cl- current which was blocked by 5-nitro-2-(3-phenylpropylamino)benzoic acid, 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid and dihydroindenyloxy-alkanoic acid. When the tonicity was reduced at constant Cl- concentration by replacing NaCl with CaCl2 or MgCl2, the volume increased and then slowly decreased towards its control value. This regulatory volume decrease was blocked by 5-nitro-2-(3-phenylpropylamino)benzoic acid, 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid and dihydroindenyl-oxy-alkanoic acid. Long-term (hours-days) cell shrinkage was induced by a reduction of the culture medium osmolarity. Long-term cell swelling was induced by an increase of the culture medium osmolarity. These volume changes were abolished by the protein translation inhibitor cycloheximide. The results suggest that NG 108-15 cell volume is regulated by at least four interacting mechanisms controlled, respectively, by intracellular Ca2+, extracellular NaCl, cell volume and intracellular ionic strength. The speculative nature of ionic systems responsible for these volume regulating mechanisms is discussed.

Topics: Animals; Caffeine; Cell Size; Chloride Channels; Culture Media; Egtazic Acid; Glioma; Hybrid Cells; Hypotonic Solutions; Ionomycin; Membrane Potentials; Neuroblastoma; Osmolar Concentration; Ryanodine; Water-Electrolyte Balance

1. The role of cyclic ADP ribose and ryanodine receptors in the inhibition of the M-like current (IK(M,ng)) by acetylcholine was investigated in m1 muscarinic receptor-transformed mouse neuroblastoma-rat glioma hybrid (NG108-15) cells using patch-clamp techniques and calcium microfluorimetry. 2. Acetylcholine (1-100 microM) decreased IK(M,ng) by up to 55 %. Application, via the patch pipette, of the cyclic ADP ribose antagonists 8-amino-cyclic ADP ribose (10-100 microM) and 8-bromo-cyclic ADP ribose (100-1000 microM) reduced this inhibition of IK(M,ng) in a concentration-dependent manner. The half-maximal inhibition concentrations for 8-amino- cyclic ADP ribose and 8-bromo-cyclic ADP ribose were around 40 microM and 1 mM, respectively. 3. Neither of the cyclic ADP ribose antagonists altered the amplitude of IK(M,ng) per se, or the incidence of the concurrent Ca2+-activated K+ current (IIK(Ca)) activation, also mediated by acetylcholine. 4. The ryanodine receptor modulators ryanodine (1-10 microM) and Ruthenium Red (10 microM) did not alter IK(M,ng) amplitude or IK(M,ng) inhibition mediated by acetylcholine. There was a statistically significant increase in the proportion of cells showing outward currents in the presence of Ruthenium Red. 5. Intracellular calcium levels measured with fura-2 microfluorimetry were increased with low concentrations of ryanodine (1 microM), more consistently with caffeine (10 mM), and in almost every case with both bradykinin (300 nM) and acetylcholine (100 microM). Caffeine-, but not bradykinin-evoked responses were abolished by preincubation with ryanodine (10 microM). 6. The fast 'rundown rate' of the M-current recorded in rat superior cervical ganglion cells under whole-cell conditions precluded an investigation of the effects of intracellular dialysis of cyclic ADP ribose. However, when cyclic ADP ribose (5 microM) was applied directly to the cytoplasmic face of inside-out membrane patches excised from rat superior cervical ganglion cells containing M-channels, it had no effect on the main parameters of single channel activity (conductance, mean open time or frequency of opening). 7. These results indicate that cyclic ADP ribose acts on a specific intracellular site to mediate IK(M,ng) inhibition. However, unlike previously established effects of cyclic ADP ribose, the ryanodine receptor is not required, suggesting that another molecular target may be involved. Studies at the single channel level indicate that cyclic ADP

Topics: Acetylcholine; Adenosine Diphosphate Ribose; Animals; Bradykinin; Calcium; Cyclic ADP-Ribose; Glioma; Hybrid Cells; Membrane Potentials; Mice; Neuroblastoma; Neurons; Patch-Clamp Techniques; Rats; Rats, Sprague-Dawley; Receptor, Muscarinic M1; Receptors, Muscarinic; Recombinant Proteins; Ruthenium Red; Ryanodine; Ryanodine Receptor Calcium Release Channel; Superior Cervical Ganglion; Transfection

Recent studies have demonstrated that opioid agonists affect the cytosolic Ca2+ concentration ([Ca2+]i) either by regulating plasma membrane Ca(2+)-channel activity or by mobilizing intracellular Ca2+ stores. The present report documents the [Ca2+]i increase induced by opioid agonists in a human neuroblastoma cell line, SK-N-BE, expressing delta-opioid receptors. In the presence, as well as in the absence, of extracellular Ca2+, opioid agonists enhanced significantly [Ca2+]i, whereas carbachol, known to mobilize specifically inositol 1,4,5-trisphosphate-sensitive intracellular Ca2+ stores, acted only in the presence of extracellular Ca2+. The opioid-induced increase in [Ca2+]i was not affected by treatments modifying the trimeric Gl, Go, and Gs protein transduction mechanisms or the activity of adenylyl cyclase. The Ca(2+)-ATPase pump-inhibiting sesquiterpene lactone, thapsigargin, did not modify the opioid-induced [Ca2+]i response, whereas it abolished the effects of carbachol. The Ryana speciosa alkaloid, ryanodine, at concentrations known to block endoplasmic reticulum ryanodine receptors, decreased significantly the response to opioids without affecting the effects of carbachol. Thus, our results suggest that, in SK-N-BE cells, delta-opioid receptors mobilize Ca2+ from intracellular ryanodine-sensitive stores and the mechanism involved is independent of Gl/Go Gs proteins and protein kinase A activation.

Topics: Calcium; Calcium Channels; Calmodulin-Binding Proteins; Carbachol; Enzyme Inhibitors; GTP-Binding Proteins; Humans; Inositol 1,4,5-Trisphosphate; Muscarinic Agonists; Muscle Proteins; Narcotics; Neuroblastoma; Receptors, Opioid, delta; Ryanodine; Ryanodine Receptor Calcium Release Channel; Thapsigargin; Tumor Cells, Cultured

Intracellular free Ca2+ concentrations ([Ca2+]i) were measured in subclones of NL308 neuroblastoma x fibroblast hybrid cells expressing each of the individual muscarinic acetylcholine receptor (mAChR) subtypes m1, m2, m3 and m4. Application of 100 microM acetylcholine (ACh) increased [Ca2+]i in all four subclones. The increased [Ca2+]i levels were significantly higher in m1- and m3-transformed cells than those in m2- and m4-transformed cells. In more than 95% of m2- and m4-transformed cells, [Ca2+]i showed sinusoidal oscillations. ACh-induced increases in [Ca2+]i were not observed in cells treated with an intracellular Ca2+ chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Removal of extracellular Ca2+ with ethylene-glycol-bis-(beta- aminoethyl)-N,N,N',N'-tetraacetate (EGTA) did not affect the initial [Ca2+]i increases, but reduced the late phases of delta [Ca2+]i in ml- and m3-transformed cells by 20-30%. Oscillations in m2- and m4-transformed cells persisted in EGTA solution (though sometimes slowed in frequency), suggesting that they were of intracellular origin. ACh-induced delta [Ca2+]i and inositol 1,4,5-trisphosphate formation was completely suppressed by pre-treatment with 50-100 ng ml-1 Pertussis toxin (PTX) for 12 h in m2- and m4-transformed cells, but not in m1- and m3-transformed cells. In all cells, extracellular application of caffeine and ryanodine, or intracellular application of cyclic adenosine diphosphate ribose (cAD-PR) produced a rise in [Ca2+]i. ACh-induced [Ca2+]i oscillations were not observed in ryanodine-treated m2-transformed cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Acetylcholine; Animals; Calcium; Cytosol; Egtazic Acid; Fibroblasts; Fluorometry; Fura-2; Hybrid Cells; Inositol 1,4,5-Trisphosphate; Mice; Neuroblastoma; Pertussis Toxin; Radioligand Assay; Receptors, Muscarinic; Ryanodine; Transfection; Type C Phospholipases; Virulence Factors, Bordetella