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Notes: 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.

Notes: Abstract: Preincubation of D384 cells, derived from the human astrocytoma cell line G-CCM, with dopamine resulted in a time-dependent attenuation of cyclic AMP responsiveness to subsequent dopamine stimulation. This effect was agonist specific because the prostaglandin E1 (PGE,) stimulation of cyclic AMP of similarly treated cells remained unchanged. The attenuation by dopamine was concentration dependent with a maximum observed at 100 μM. A comparison of dopamine concentration-response curves of control and dopamine-preincubated cells revealed no change in the Ka apparent value, but a marked attenuation of the maximal response. Preincubation of cells with dopamine in the presence of D1 but not D2 selective antagonists partially prevented the observed attenuation. Attenuations in dopamine responsiveness were also obtained when D384 cells were preincubated with D1 but not D2 receptor agonists. The level of attenuation attained related to agonist efficiency in stimulating cyclic AMP: SKF38393 〈 3,4-dihydroxynomifensine 〈 fenoldopam 〈 2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene = dopamine. However, increasing the efficiency of 3,4-dihydroxynomifensine stimulation of cyclic AMP, using the synergistic effect of adding a low concentration of forskolin, produced no further change in the attenuation of the subsequent response to dopamine. Thus, the D1 dopamine receptors expressed by D384 cells undergo homologous desensitization. Uncoupling of the D1 dopamine receptor appears to be independent of cyclic AMP formation, analogous to a mechanism proposed for the β-adrenergic receptor.

Notes: 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.

Notes: 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.

Notes: Abstract: Dimethylphenylpiperazinium iodide (a nicotinic agonist) evokes noradrenaline release from human neuroblastoma SH-SY5Y cells that have been pretreated with 12-O-tetradecanoylphorbol 13-acetate for 8 min. This effect of dimethylphenylpiperazinium iodide was inhibited by 1 μM mecamylamine but not by 1 μM atropine, which suggests that SH-SY5Y cells express nicotinic receptors coupled to the release of noradrenaline. Dimethylphenylpiperazinium iodide-evoked release was enhanced by 5 μM Bay K 8644 (an L-type calcium agonist) and inhibited by 1 μM nifedipine. Dimethylphenylpiperazinium iodide depolarised SH-SY5Y cells and enhanced the level of intracellular calcium in cells loaded with fura 2. The effects of dimethylphenylpiperazinium iodide on noradrenaline release, depolarisation, and intracellular calcium levels were all inhibited by 1 μM desmethylimipramine. The results of this study show that nicotinic receptors in SH-SY5Y cells stimulate noradrenaline release by activation of L-type calcium channels.

Notes: 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.

Notes: Abstract: Nordihydroguaiaretic acid (NDGA; a lipoxygenase inhibitor), LY-270766 (an inhibitor of 5-lipoxygenase), and the diacylglycerol lipase inhibitor RG 80267 completely eliminated potassium-evoked release of [3H]noradrenaline ([3H]NA) from the human neuroblastoma clone SH-SY5Y with IC50 values of 10, 15, and 30 μM, respectively. In contrast, these inhibitors only partially inhibited carbachol-evoked release and had little effect on the calcium ionophore A23187-evoked release of NA in this cell line. Arachidonic acid partially inhibited potassium- and A23187-evoked release but did not reverse the inhibition of potassium-evoked release observed in the presence of RG 80267. These studies suggest that arachidonic acid (or its lipoxygenase products) are not important intermediates in the regulation of exocytosis in SH-SY5Y. This conclusion is strengthened by our studies in which SH-SY5Y cells were grown in medium supplemented with bovine serum albumin-linoleic acid (50 μM). Under these conditions there was a selective increase in content of membrane polyunsaturated fatty acids of the ω6 series, including arachidonic acid; however, these changes did not effect potassium-, veratridine-, carbachol-, or calcium ionophoreevoked release of [3H]NA.

Notes: Two cell cultures, NEP2 and NEM2, isolated from human foetal brain have been maintained through several passages and found to express some properties of astrocytes. Both cell cultures contain adenylate cyclase stimulated by catecholamines with a potency order of isoprenaline 〉 adrenaline 〉 salbutamol 〈 noradrenaline, which is consistent with the presence of β2-adrenergic receptors. This study reports that the β2-adrenergic-selective antagonist ICI 118,551 is ˜ 1,000 times more potent at inhibiting isoprenaline stimulation of cyclic AMP (cAMP) formation in both NEP2 and NEM2 than the β1adrenergic-selective antagonist practolol. This observation confirms the presence of β2-adrenergic receptors in these cell cultures. The formation of cAMP in NEP2 is also stimulated by 5′-(N-ethylcarboxamido)adenosine (NECA) more potently than by either adenosine or N6-(L-phenylisopropyl)adenosine (L-PIA), which suggests that this foetal astrocyte expresses adenosine A2 receptors. Further more, L-PIA and NECA inhibit isoprenaline stimulation of cAMP formation, a result suggesting the presence of adenosine A1 receptors on NEP2. The presence of A1 receptors is confirmed by the observation that the A1-selective antagonist 8-cyclopentyl-1,3-dipropylxanthine reverses the inhibition of isoprenaline stimulation of cAMP formation by L-PIA and NECA. Additional evidence that NEP2 expresses adenosine receptors linked to the adenylate cyclase-inhibitory GTP-binding protein is provided by the finding that pretreatment of these cells with pertussis toxin reverses the adenosine inhibition of cAMP formation stimulated by either isoprenaline or forskolin.

Notes: Abstract: 3,4-Dihydroxyphenylethylamine (dopamine) and β-adrenergic receptor agonists and antagonists were assessed for their effects on cyclic AMP accumulation in human astrocytoma derived clone D384 cells. Dopamine, SKP 38393, and 2-amino-6,7-dihydroxy-l,2,3,4-tetrahydronaphthalene increased cyclic AMP content with Ka values of 2.0, 0.2, and 1.6 μM. The D1-selective antagonists SCH 23390 (Ki, 1.2 nM) and SKF 83566 (Ki, 0.8 nM) were over 5,000-fold more potent than the D2-selective antagonist domperidone (Ki, 6.7 μM) at inhibiting dopamine stimulation of cyclic AMP formation. SCH 23388 (Ki, 560 nM; the S-enantiomer of SCH 23390) was 400-fold less potent than SCH 23390. Isopren-aline, adrenaline, salbutamol, and noradrenaline increased cyclic AMP content with Ka values of 0.13, 0.12, 0.22, and 7.60 μM. The β2-selective antagonist ICI 118,551 (Ki, 0.8 nM) was almost 8,000-fold more potent than the β,-selective antagonist practolol (Ki, 5.9 μM) at inhibiting isoprenaline stimulated cyclic AMP accumulation. These results demonstrate that D384 cells express D1-dopamine and β2-adren-ergic receptors linked to adenylate cyclase. Furthermore, the dopamine receptor expressed by D384 cells exhibits a pharmacological profile typical of a mammalian striatal D1-re-ceptor and therefore the use of this clone represents another approach to studying central D1-receptors.

Notes: 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.