Bibliothek

Notizen: The regulation of intracellular calcium by cholinergic agonists was investigated in the human neuroblastoma SH-SY5Y, loaded with fura-2. The resting free Ca2+ concentration in this cell line was 199 ± 14 nM (mean ± SEM, n = 19). At 1 mM extracellular Ca2+, high concentrations of carbachol and acetylcholine evoked a biphasic change in intracellular Ca2+ concentration, consisting of a transient initial peak followed by a decline to a plateau that was significantly higher than the basal level. Carbachol (0.5 mM) and acetylcholine (10 μM) caused a maximal increase in the intracellular Ca2+ concentration, reaching a peak of 465 ± 52 (mean ± SEM, n = 12) and 422 ± 48 nM (mean ± SEM, n = 7), respectively, in 〈4 s. This initial calcium transient declined to a plateau of 268 ± 36 and 240 ± 27 nM for carbachol and acetylcholine, respectively, in ∼40 s. The plateau persisted until the agonist was displaced by the addition of antagonist. Atropine, hexahydrosiladifenidol (HHSD), pirenzepine, and methoctramine inhibited the carbachol-evoked initial calcium transient with Ki values of 0.85 ± 0.05, 8.3 ± 1.6, 411 ± 36, and 240 ± 46 nM (mean ± SEM, n = 3), respectively, and the acetylcholine-induced initial calcium transient with Ki values of 0.48 ± 0.18, 13.5 ± 8.5, 192 ± 32, and 414 ± 25 nM (mean ± SEM of two experiments), respectively, results suggesting that an M3 muscarinic receptor was predominantly mediating these effects. Furthermore, atropine, HHSD, and pirenzepine inhibited the plateau phase of the carbachol- evoked change in intracellular Ca2+ concentration with Ki values of 0.25, 1, and 861 nM, respectively, which are consistent with an M3 receptor being coupled to this effect. The plateau was abolished in the presence of EGTA, thereby converting the biphasic signal into a monophasic response. Under these conditions, the initial calcium transient or its pharmacology was not altered. However, if the cells were washed in calcium-free buffer for 3 min, the initial peak was decreased by∼30%. The subsequent readdition of calcium caused a further increase in fura-2 fluorescence. The influx of calcium and hence the plateau were also blocked by nickel but were insensitive to verapamil. We may conclude, therefore, on pharmacological grounds, that an M3 muscarinic receptor is coupled both to the peak response, presumably due to inositol trisphosphate mobilization of intracellular calcium, and to the plateau due to an influx of extracellular calcium.

Notizen: Abstract: The human neuroblastoma clone SH-SY5Y expresses potassium-, carbachol-, and calcium ionophore A23187-evoked, calcium-dependent release of [3H]noradrenaline. Release in response to carbachol and potassium was greater than additive. Atropine (Ki= 0.33 nM), hexahydrosiladifenidol (Ki= 18 nM), and pirenzepine (Ki= 1,183 nM) completely inhibited the carbachol-evoked noradrenaline release, an order of potency suggesting that an M3 receptor was linked to release. In contrast, noradrenaline release was only partially inhibited by the M2-selective antagonists meth-octramine (10-4M) and AFDX-116 (10-4M), by ∼14 and 46%, respectively. The nicotinic antagonist d-tubocurarine (10-4M) resulted in a partial inhibition of release, a finding suggesting that a nicotinic receptor may also be involved. SH-SY5Y provides a suitable cell line in which to study the biochemical mechanisms underlying the cholinergic receptor regulation of noradrenaline release.

Notizen: Abstract: Clones have been isolated from the human astrocytoma cell line G-CCM. Homogenates of clone D384 contain an adenylate cyclase that is stimulated by 3,4-dihydroxyphenylethylamine (dopamine), noradrenaline, and isoprenaline with Ka apparent values of 4, 56, and 2.7 μM, respectively. The Ka apparent value for dopamine was increased by the D-l antagonist cis-flupenthixol, 25 and 100 nM, to 23 and 190 μM, respectively, but was unaffected by propranolol (1 μf. Noradrenaline stimulation of adenylate cyclase was only partially inhibited by either propranolol (10 μM) or cis-flupenthixol (1 μM). Propranolol (10 μM), but not cis-flupenthixol (1 μM), prevented stimulation by isoprenaline. The stimulation of adenylate cyclase by dopamine and noradrenaline remained unchanged in the presence of phentolamine (1 μM) and sulpiride (1 μM). These results suggest that clone D384 contains both D-l dopaminergic and β-adrenergic receptors coupled to adenylate cyclase. Dopamine stimulates D384 adenylate cyclase through D-1 receptors, isoprenaline via β-receptors, and noradrenaline through both receptors.

Notizen: Abstract: The effect of hypoglycaemic, hypoxic, and ischaemic conditions on high-affinity neurotransmitter transport was studied in the human astrocytoma clone D384 and the human neuroblastoma clone SH-SY5Y. Both cell lines expressed a sodium-dependent glutamate/aspartate transporter. Km values for d-[3H]aspartate uptake were 6.1 ± 0.9 µM for D384 cells and 5.3 ± 0.3 µM for SH-SY5Y cells (mean ± SEM of three experiments). In addition, SH-SY5Y, but not D384, expressed a sodium-dependent noradrenaline transporter with Km = 0.6 ± 0.1 µM (mean ± SEM of three experiments). Up to 3 h of hypoglycaemic conditions had no effect on neurotransmitter uptake or on ATP levels of each cell line. In sharp contrast, during hypoxic conditions, the uptake of d-[3H]aspartate and [3H]noradrenaline declined by 43–56% within 5 min. These reduced rates of neurotransmitter uptake were maintained over 30 min of hypoxic conditions. Five minutes of ischaemic conditions caused similar reductions in neurotransmitter uptake rates. A correlation between reductions in rates of neurotransmitter uptake and in ATP levels was observed for each cell line. Results are discussed in relation to other brain preparations, which are used as models of the nervous system to study the effects of ischaemic conditions on neurotransmitter and energy metabolism.

Notizen: Abstract: Noradrenaline (NA) and the α2-adrenergic agonists clonidine, BHT-920, and UK 14304-18 inhibit potassiumevoked release of [3H]NA from rat occipital cortex tissue chops with similar potencies. NA (10−5M) was most effective as up to 85% inhibition could be observed compared with 75%, 55%, and 35% for UK 14304-18, clonidine, and BHT-920, respectively, all at 10−5M. Potassium-evoked release was enhanced by both forskolin (10−5M) and 1 mM dibutyryl cyclic AMP. Pretreatment of tissue chops with 1 mM dibutyryl cyclic AMP in the presence of 3-isobutyl-1-methylxanthine partially reversed the α2-adrenergic agonist inhibition of NA release. No reversal of inhibition was observed following pretreatment with 10−5M forskolin. The effects of clonidine, BHT-920, UK-14308–18, and NA on cyclic AMP formation stimulated by (a) forskolin, (b) isoprenaline, (c) adenosine, (d) potassium, and (e) NA were examined. Only cAMP formation stimulated by NA was inhibited by these α2-adrenergic agonists. These results suggest that only a small fraction of adenylate cyclase in rat occipital cortex is coupled to α2-adrenergic receptors. These results are discussed in relation to recent findings that several α2-adrenergic receptor subtypes occur, not all of which are coupled to the inhibition of adenylate cyclase, and that α2-adrenergic receptors inhibit NA release in rat occipital cortex by a mechanism that does not involve decreasing cyclic AMP levels.

Notizen: Abstract: The effects of steroid hormones on the cyclic AMP responses to stimulation of human astrocytoma cells (D384) by dopamine, prostaglandin E1 (PGE1), and isoprenaline were investigated. Incubation of D384 cells with dexamethasone resulted in a potentiation of the PGE1 and isoprenaline responses and a marked attenuation of the dopamine response. The time courses of the effects of dexamethasone on dopamine and PGE1 responses were similar, requiring long-term (at least 18 h) incubation of cells with the steroid. Concentration-response curves of dexamethasone effects on dopamine and PGE1 responses yielded similar Ka apparent values, suggesting a common mechanism. Cycloheximide, a protein synthesis inhibitor, prevented the effects of dexamethasone. Only steroids with glucocorticoid activity reproduced the dexamethasone effects. Direct stimulation of Gs with 5-guanylylimidodiphosphate and adenylate cyclase with forskolin revealed no significant differences in their activities in dexamethasone-treated and untreated cells. Furthermore, a comparison of the dopamine and PGE1 concentration-response curves obtained from dexamethasone-treated and untreated cells suggested that the affinity of the receptors for their agonists remained unchanged. These results suggest that glucocorticoids may alter protein synthesis and thereby the number of receptors expressed by D384 cells.

Notizen: © 2000 American Institute of Physics.

Notizen: We show that changes in the relative mole fractions of Li2O and Na2O in alkali metaphosphate glasses lead to "anomalies" in the specific heat and structural relaxations. The heat capacity change between the liquid and glassy states, Δcp(Tg), at the calorimetric glass transition temperature, Tg, exhibits a minimum when the mole fractions of Li2O and Na2O are comparable. Moreover, systematic changes in the temperature dependence of the viscosity, η, i.e., changes in the "fragility" of the system, accompany these changes in mole fraction. This observed dependence of the "fragility" on the mixed alkali ion composition occurs in the absence of apparent changes in the covalent network connectivity which normally accounts for this behavior in glasses. © 1998 American Institute of Physics.

Notizen: The high resolution spectrum of the 000 vibronic band of the C˜ 2A1–X˜ 2A1 transition of CaNH2 was recorded with a laser ablation/supersonic molecular beam spectrometer. Approximately 140 lines of the Ka′=0←Ka″=0 and the Ka′=1←Ka″=1 sub–bands were measured and combined with the previous A˜ 2B2–X˜ 2A1 and B˜ 2B1–X˜ 2A1 results. A global fit of the data was carried out and the effective spectroscopic constants for the X˜, A˜, B˜, C˜ states are reported. A complete set of spin–rotation constants (εαα's) are now available for the A˜ 2B2, B˜ 2B1 and C˜ 2A1 states. The unpaired electron in these three excited states can be considered to be located in three p–orbitals (px,py,pz) centered on the metal atom. The simple pure precession model provides estimates for the 9 spin–rotation parameters in the A˜, B˜, and C˜ states. © 1997 American Institute of Physics.

Notizen: Abstract: The effect of inhibition and down-regulation of protein kinase C (PKC) subtypes α, ε, and ζ on noradrenaline (NA) secretion from human SH-SY5Y neuroblastoma cells was investigated. The PKC inhibitor Ro 31-7549 inhibited carbachol-evoked NA release (IC50 0.6 µM) but not 100 mM K+-evoked release. In addition, Ro 31-7549 inhibited the enhancement of carbachol- and K+-evoked release after pretreatment with 12-O-tetradecanoylphorbol 13-acetate (TPA; 100 nM) for 8 min, with IC50 values of 0.7 and 2.4 µM, respectively. Immunoblotting studies showed that prolonged exposure (48 h) of SH-SY5Y cells to phorbol 12,13-dibutyrate (PDBu) or bryostatin-1 caused down-regulation of PKC-α and PKC-ε but not PKC-ζ. Under these conditions, the acute TPA enhancement of NA release was inhibited. Moreover, the inhibition of TPA-enhanced secretion was also apparent after only 2-h exposure to either PDBu or bryostatin-1, conditions that caused down-regulation of PKC-α, but not PKC-ε or ζ. The PKC inhibitor Gö-6976 (2 µM), which has been shown to inhibit selectively PKC-α and β in vitro, also inhibited the TPA enhancement of carbachol- and K+-evoked NA release by 〉50%. These data suggest that in SH-SY5Y cells, the ability of TPA to enhance carbachol- and K+-evoked NA secretion is due to activation of PKC-\ga.