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Biomedical subjects

C Peers

Publications and source records attributed to C Peers.

At least 91 records · Page 5Linked to original sources
PubMed1995

Enhancement of Ca2+ channel currents in human neuroblastoma (SH-SY5Y) cells by phorbol esters with and without activation of protein kinase C.

The effects of phorbol esters on Ca2+ channel currents in human neuroblastoma SH-SY5Y cells were studied using whole-cell patch-clamp recordings. Bath application of 12-O-tetradecanoylphorbol-13-acetate (TPA) or phorbol 12,13-dibutyrate (PDBu; 100 nM to 1 microM), known activators of protein kinase C (PKC), enhanced Ca2+ channel currents in a voltage-dependent manner similar to that of Bay K 8644. TPA also enhanced Ca2+ channel currents during cell dialysis with the PKC pseudosubstrate, PKC(19-36), and in cells which had been pre-incubated with 500 nM staurosporine, and which were exposed to staurosporine during recordings. Application of 4 alpha-phorbol-12,13-didecanoate (4 alpha-PDD; 100 nM), which does not activate PKC, caused current enhancement similar to the effects of TPA. However, intracellular dialysis of TPA was without effect on Ca2+ channel currents. Residual Ca2+ channel currents recorded after exposure to 1 microM omega-conotoxin GVIA were still enhanced by TPA, but in the presence of either Bay K 8644 (5 microM) or nifedipine (5 microM), TPA was without effect. When cells were pre-incubated for 10 min at 37 degrees C with 100 nM TPA, currents subsequently recorded in its absence were enhanced as compared to untreated cells; 5 microM nifedipine still inhibited currents to the same degree. This enhancement was not mimicked by 4 alpha-PDD, and was inhibited by staurosporine. Our results indicate that acute applications of phorbol esters (at concentrations commonly used to activate PKC) enhance L-type Ca2+ channel currents in SH-SY5Y cells via a PKC-independent mechanism which appears similar to that induced by Bay K 8644. By contrast, pre-incubation with TPA enhances both L- and N-type currents via activation of PKC.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1995

Inhibition of N-type Ca2+ channel currents in human neuroblastoma (SH-SY5Y) cells by muscarine via stimulation of M3 receptors.

The effects of muscarine on whole-cell Ca2+ channel currents in SH-SY5Y cells were studied using conventional and perforated-patch-clamp techniques, with 10 mM Ba2+ as charge carrier. Muscarine (10-300 microM) caused concentration-dependent inhibitions of Ca2+ channel currents which were only reversible when perforated-patch recordings were used. Inhibition of currents was associated with slowing of activation kinetics in approximately 50% of cells. In the presence of 5 microM nifedipine, muscarine was still able to inhibit currents, but after pre-exposure of cells to 1 microM omega-conotoxin GVIA the inhibitory effects of muscarine were almost completely lost. In the presence of 100 microM muscarine, Bay K 8644 (5 microM) was still able to enhance current amplitudes. Pre-treatment of cells with pertussis toxin (250 ng/ml for 16-24 hr) or inclusion of 1 mM GDP-beta-S in the patch-pipette prevented the inhibitory actions of muscarine. Hexahydrosiladifenidol (0.1-1 microM) antagonized the actions of muscarine (calculated pA2 7.1) but the presence of 10 microM pirenzipine or 0.1 microM methoctramine in the bath solution did not alter the degree of current inhibition caused by 100 microM muscarine. In summary, these results indicate that muscarine in SH-SY5Y cells causes inhibition of N-type Ca2+ channels via a M3 receptor coupled to a pertussis toxin-sensitive G-protein.

Brain Neoplasms↗
PubMed1995

Inhibition of Ca2+ channel currents in human neuroblastoma (SH-SY5Y) cells by neuropeptide Y and a novel cyclic neuropeptide Y analogue.

Whole-cell Ca2+ channel currents were recorded in human neuroblastoma (SH-SY5Y) cells, using conventional and perforated-patch techniques. Neuropeptide Y (NPY, 10-1000 nM) caused concentration-dependent inhibition of Ca2+ channel current amplitudes which was pertussis toxin-sensitive, voltage-dependent and associated with slowing of channel activation kinetics, regardless of which recording configuration was used. Inhibition was observed in all cells tested. Similar current inhibitions were observed with NPY (18-36) and peptide YY, but not with [Leu31, Pro34]NPY, indicating that the effects were mediated by Y2 receptors. Pancreatic polypeptide (100 nM) was without effect on Ca2+ channel currents. The effects of NPY were additive with nifedipine (at a supramaximal concentration of 5 microM), suggesting that NPY predominantly inhibits N-type Ca2+ channels present in these cells, and not L-type. The effects of NPY were mimicked by a novel, cyclic analogue of NPY which is 40-fold more selective for Y2 receptors than other commonly used Y2-selective peptides. The cyclic analogue was also more potent than NPY itself, causing maximal current inhibition at approx 300 nM, whereas the response to NPY was not fully saturated at 1 microM. Our results indicate that SH-SY5Y cells represent an excellent model system for studying the coupling of Y2 receptors to N-type channel inhibition. Furthermore, in the absence of selective antagonists for NPY receptor subtypes, the highly selective Y2 agonist cyclic NPY derivative may prove a useful tool for probing the various roles of Y2 receptors in the nervous system.

Calcium Channels↗
PubMed1995

The use of the human neuroblastoma SH-SY5Y to study the effect of second messengers on noradrenaline release.

1. Recent data suggesting that the human neuroblastoma SH-SY5Y is a suitable cell line in which to study the effect of second messengers on NA release are discussed in the context of current views on exocytosis. 2. Release of NA is evoked by depolarization, as well as activation of muscarinic (M3) and bradykinin (B2) receptors in SH-SY5Y cells which have not been differentiated by the addition of growth factors. 3. Evoked release is enhanced by activation of protein kinase C. 4. Activation of protein kinase C decreases the changes in intracellular calcium evoked by carbachol, bradykinin and 100 mM K+. 5. SH-SY5Y express N-type and L-type voltage sensitive Ca2+ channels. L-Type Ca(2+)-channels are coupled to NA release under conditions of weak depolarization. However with strong depolarization (100 mM K+) both L-type and N-type channels are involved. 6. Muscarinic- and neuropeptide Y receptors are coupled to the inhibition of Ca2+ channel activity.

Cell Line↗
PubMed1994

Bradykinin-evoked release of [3H]noradrenaline from the human neuroblastoma SH-SY5Y.

Bradykinin (BK) evoked [3H]noradrenaline ([3H]NA) release from the human neuroblastoma SH-SY5Y and this was enhanced by pre-treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) for 8 min. This effect of BK was inhibited by 500 microM [D-Phe7]BK and 100 microM [Thi5,8,D-Phe7]BK but not by 500 microM [Des-Arg9,Leu8]BK. The BK (B1)-agonist [Des-Arg9]BK did not evoke [3H]NA release. This suggested that SH-SY5Y expressed BK (B2)-receptors coupled to the release of [3H]NA. BK acting at B2-receptors, also elevated intracellular calcium and depolarized SH-SY5Y cells. Although pre-treatment of SH-SY5Y cells with TPA enhanced BK-evoked [3H]NA release, the elevation of intracellular calcium [Ca2+]; was decreased by about 50%. BK-evoked release of [3H]NA in cells not pre-treated with phorbol ester was only 23% dependent on extracellular calcium. In comparison, following phorbol ester treatment approximately 40% of [3H]NA release was dependent on extracellular calcium. Nifedipine (5 microM), CoCl2 (1 mM) and NiCl2 (1 mM) inhibited NA release in SH-SY5Y cells pre-treated with TPA by 16.0, 47 and 44%, respectively. The results of this study showed that BK, acting at B2-receptors, activated [3H]NA release in SH-SY5Y. Part of this effect appeared to be due to activation of L-type calcium channels but the majority of BK-evoked [3H]NA release in SH-SY5Y cells appeared to depend on [Ca2+]i.

Bradykinin↗
PubMed1994

Calcium channel currents in undifferentiated human neuroblastoma (SH-SY5Y) cells: actions and possible interactions of dihydropyridines and omega-conotoxin.

Ca2+ channel currents were recorded in undifferentiated human neuroblastoma (SH-SY5Y) cells with the whole-cell patch-clamp technique, using 10 mM Ba2+ as charge carrier. Currents were only evoked by depolarizations to -30 mV or more positive (holding potential -80 mV), inactivated partially during 200 ms depolarizing steps, and were abolished by 150 microM Cd2+. Currents could be enhanced by Bay K-8644 and partially inhibited by nifedipine, suggesting that they arose in part due to activation of L-type Ca2+ channels. Currents were also inhibited by the marine snail peptide omega-conotoxin GVIA (omega-CgTx). At a concentration of 10 nM inhibition by omega-CgTx was reversible, but at higher concentrations blockade was always irreversible. Although current inhibition by nifedipine was maximal at 1 microM, supramaximal concentrations reduced the inhibitory actions of omega-CgTx in a concentration-dependent manner. Ca2+ channel currents evoked from a holding potential of -50 mV showed no inactivation during 200 ms depolarizations but declined in amplitude with successive depolarizing steps (0.2 Hz). Current amplitudes could be restored by returning the holding potential to -80 mV. Currents evoked from -50 mV were inhibited by nifedipine and omega-CgTx to a similar degree as those evoked from -80 mV. Our results indicate that undifferentiated SH-SY5Y cells possess L- and N-type Ca2+ channels which can be distinguished pharmacologically but cannot be separated by using depolarized holding potentials. Furthermore, these data suggest that nifedipine has a novel action to inhibit blockade of N-type channels by omega-CgTx.

Brain Neoplasms↗
PubMed1994

Diphenylene iodonium blocks K+ and Ca2+ currents in type I cells isolated from the neonatal rat carotid body.

Diphenylene iodonium (DPI), an inhibitor of NAD(P)H oxidase, blocks hypoxic excitation of the carotid body. We used the whole-cell patch-clamp technique to investigate the actions of DPI on ionic currents in isolated type I carotid body cells. DPI (10 microM) caused reversible blockade of K+ and Ca2+ currents in these cells, indicating that DPI is a non-selective ion channel blocker. Since hypoxic excitation of the carotid body is dependent on Ca2+ influx into type I cells, our observation that DPI blocks Ca2+ currents in type I cells can account for the ability of this compound to inhibit hypoxic excitation of the intact organ.

Animals↗
PubMed1994

Passive transfer of seronegative myasthenia gravis to mice.

Muscle weakness in myasthenia gravis is due to autoantibody-induced loss of functional acetylcholine receptors (AChR). About 15% of myasthenia gravis patients, however, do not have detectable anti-AChR antibodies. To investigate the effect of their plasma immunoglobulins on neuromuscular transmission, mice were injected with plasma (and in some cases purified immunoglobulin G (IgG)) from 7 "seronegative" myasthenia gravis (SMG) patients, and neuromuscular transmission parameters were examined. When injected for 15 days, all patients' plasma caused reductions in miniature endplate potential amplitudes, while endplate potential quantal content was significantly reduced by plasma from 4 of the 7 patients. There were no changes in ACh-induced depolarization or single channel properties, and 125I-alpha-bungarotoxin binding studies showed no effect on AChR number, except in 1 case. Purified IgG injected for 3 days had similar effects to plasma injected for 15 days. Our findings confirm that SMG is autoantibody mediated and that there are pathogenic IgG antibodies. SMG appears to be a heterogeneous disorder and the target(s) for the antibodies may be diverse.

Acetylcholine↗
PubMed1994

Muscarinic (M1) receptor-mediated inhibition of K(+)-evoked [3H]-noradrenaline release from human neuroblastoma (SH-SY5Y) cells via inhibition of L- and N-type Ca2+ channels.

1. Human neuroblastoma (SH-SY5Y) cells were preincubated with [3H]-noradrenaline ([3H]-NA) in the presence of 0.2 mM pargyline to examine the modulation of K(+)-evoked [3H]-NA release by muscarinic agonists. 2. Release of [3H]-NA evoked by 4 min exposure to 100 mM K+ could be partially inhibited by 5 microM nifedipine and partially inhibited by 100 nM omega-conotoxin GVIA (omega-CgTx). When nifedipine and omega-CgTx were added together, evoked release was inhibited by approximately 93%. 3. K(+)-evoked [3H]-NA release was inhibited by > 90% by pretreatment of cells for 2 min with muscarine, carbachol or oxotremorine methiodide (each at 300 microM). For muscarine, inhibition of evoked release was both time- and concentration-dependent and was reversible. Muscarine also inhibited [3H]-NA release evoked by veratridine (28 microM) and replacement of extracellular Ca2+ with Ba2+, but not that evoked by the Ca2+ ionophore, A23187 (19 microM). 4. Residual K(+)-evoked [3H]-NA release measured in the presence of either nifedipine (5 microM) or omega-CgTx (100 nM) was inhibited by muscarine with a similar potency as release evoked in the absence of either Ca2+ channel blocker. Pretreatment of cells for 16-24 h with pertussis toxin (200 ng ml-1) did not affect K(+)-evoked release per se or the ability of muscarine to inhibit such release. 5. Muscarinic inhibition of K(+)-evoked [3H]-NA release was potently antagonized by pirenzepine (pA2 8.14) and by hexahydrosiladiphenidol (pA2 9.03), suggesting the involvement of an M1 receptor. 6. Our results demonstrate that 100 mM K+-evoked release of [3H]-NA from the human neuroblastoma is mediated by activation of both L- and N-type Ca2+ channels. Activation of muscarinic Ml receptors can inhibit release via a pertussis toxin-insensitive mechanism which involves non-selective inhibition of L- and N-type Ca2+ channels.

Calcium Channel Blockers↗
PubMed1994

Diphenyleneiodonium inhibits both potassium and calcium currents in isolated pulmonary artery smooth muscle cells.

Diphenyleneiodonium (DPI) blocks hypoxic vasoconstriction in the pulmonary vasculature. Because one of the actions of DPI is the inhibition of NADPH oxidase, this has led to the suggestion that NADPH oxidase acts as an oxygen tension sensor in pulmonary smooth muscle cells. We investigated the effects of DPI on potassium and calcium currents in freshly isolated pulmonary artery smooth muscle cells by using whole cell patch-clamp recordings, since these ionic currents are known to be involved in hypoxic pulmonary vasoconstriction. DPI (3 and 10 microM) reversibly inhibited potassium currents, and in its presence, residual currents appeared markedly more transient than under control conditions. The actions of DPI could not be reversed by 4.4 mM hydrogen peroxide, the product of NADPH oxidase. Calcium channel currents were also reversibly inhibited by 3 microM DPI. Thus DPI is a nonselective blocker of ionic channels in pulmonary smooth muscle cells, and its mechanism of action does not appear to involve inhibition of hydrogen peroxide formation. The ability of DPI to block calcium currents can explain its inhibition of hypoxic pulmonary vasoconstriction.

Animals↗
PubMed1993

Inhibition of neuronal nicotinic acetylcholine receptors by imipramine and desipramine.

The actions of two structurally related tricyclic antidepressants on neuronal nicotinic acetylcholine receptors were investigated in human neuroblastoma (SY-SY5Y) cells, using whole-cell patch-clamp recordings. Both desipramine and imipramine reversibly inhibited inward currents evoked by application of the nicotinic receptor agonist dimethylphenylpiperazinium iodide (30-300 microM) with IC50 values of 0.17 microM and 1.0 microM respectively (holding potential -70 mV). The degree of current inhibition caused by either tricyclic compound was unaffected by agonist concentration (30-300 microM). The effects of desipramine were voltage-independent over the range -40 mV to -100 mV, and inhibition caused by imipramine only increased very slightly with membrane hyperpolarization over the same range. These results indicate that tricyclic antidepressants can inhibit neuronal nicotinic acetylcholine receptors by mechanisms which are distinct from their actions at non-neuronal nicotinic acetylcholine receptors.

Desipramine↗
PubMed1993

Cross-linking of presynaptic calcium channels: a mechanism of action for Lambert-Eaton myasthenic syndrome antibodies at the mouse neuromuscular junction.

Lambert-Eaton myasthenic syndrome (LEMS) is an autoimmune disorder of neuromuscular transmission. Immunoglobulin G (IgG) autoantibodies act presynaptically to cause functional loss of Ca2+ channels by down-regulation. This in turn causes reduced release of acetylcholine in response to nerve stimulation (i.e. reduced quantal content). We show here that in vitro application of LEMS IgG for 24 h was as effective in causing reduced quantal content in mice as injecting animals with the antibody for 2 days. Furthermore, incubation with divalent (F(ab')2) fragments also significantly reduced quantal content. However, monovalent (Fab) fragments were without significant effect. No antibody or antibody fragment preparation affected spontaneous release. Whole LEMS IgG and F(ab')2 (but not Fab) fragments also inhibited K(+)-evoked 45Ca2+ flux in a small cell carcinoma cell line. These findings present functional evidence that LEMS IgG-induced down-regulation of nerve terminal Ca2+ channels arises due to antibody cross-linking of these channels.

Acetylcholine↗