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Biomedical subjects

D G Lambert

Publications and source records attributed to D G Lambert.

At least 127 records · Page 7Linked to original sources
PubMed1994

Studies on the mechanism of [3H]-noradrenaline release from SH-SY5Y cells: the role of Ca2+ and cyclic AMP.

1. The roles of both Ca2+ and adenosine 3':5'-cyclic monophosphate (cyclic AMP) in carbachol and K(+)-stimulated [3H]-noradrenaline release from SH-SY5Y human neuroblastoma cells were examined. 2. Both carbachol and K+ caused a time- and dose-related stimulation of [3H]-noradrenaline release. The release event in perfused cells was monophasic. Half-maximum stimulation measured in statically incubated (3 min) cells was 38 +/- 4 microM and 63 +/- 4 mM respectively. K+ (100 mM, added)-evoked release was greater than that produced by carbachol (1 mM). 3. Both carbachol and K+ caused a time- and dose (measured at 3 min)-related stimulation of cyclic AMP formation with half-maximum stimulation occurring at 5 +/- 1 microM and 49 +/- 2 mM respectively. In contrast to its effects on release, carbachol produced a greater stimulation of cyclic AMP formation than K+. 4. K(+)-stimulated [3H]-noradrenaline release was entirely dependent on Ca2+ entry as 2.5 mM Ni2+ abolished release. However, carbachol-evoked (1 mM) release appeared to be unaffected by Ni2+ pretreatment. 5. These data suggest that in SH-SY5Y cells, elevated cyclic AMP levels are not directly involved in [3H]-noradrenaline release. In addition, carbachol-stimulated release is largely independent of extracellular Ca2+ possibly implying a role for intracellular stored Ca2+ in the release process.

Calcium↗
PubMed1993

Effects of morphine and its metabolites on opiate receptor binding, cAMP formation and [3H]noradrenaline release from SH-SY5Y cells.

Opiate receptor occupation leads to a variety of intracellular events including inhibition of adenylyl cyclase and cAMP formation. We have examined the opiate binding characteristics, effects on cAMP formation and [3H]noradrenaline release of morphine, morphine-6 (M6G) and -3 (M3G)-glucuronides, and fentanyl in SH-SY5Y human neuroblastoma cells. M6G and M3G are the major metabolites of morphine formed in vivo whose cellular action remains to be fully elucidated. In binding experiments morphine (affinity, K50 = 96 nM) and fentanyl (K50 = 99 nM) were more potent than M6G (K50 = 393 nM), while M3G was inactive. However, for cAMP inhibition morphine (half maximum inhibition, IC50 = 193 nM) and M6G (IC50 = 113 nM) were roughly equipotent, with fentanyl (IC50 = 27 nM) being more potent and producing a greater maximum inhibition (56%). M3G was inactive. These in vitro data are in general agreement with the in vivo effects of these glucuronides. Moreover, all of the opiates tested failed to inhibit K(+)-evoked release of [3H]noradrenaline. Whilst these data do not support a role for cAMP in neurotransmitter release, alterations in cAMP formation may still have a role to play in the mechanism of analgesia.

Cell Line↗
PubMed1993

Fentanyl inhibits the uptake of [3H]noradrenaline in cultured neuronal cells.

We have examined how fentanyl modulates [3H]noradrenaline uptake in two cultured neuronal cell preparations, the human neuroblastoma SH-SY5Y and the rat phaeochromocytoma PC12. Fentanyl produced a significant, dose-dependent inhibition of [3H]noradrenaline uptake at concentrations in excess of 0.1 mumol litre-1 (P < 0.05) and 0.3 mumol litre-1 (P < 0.05) for PC12 and SH-SY5Y cells, respectively. However, these values exceed the serum concentration of fentanyl required to produce analgesia. At the maximum concentration examined (100 mumol litre-1), fentanyl produced 85-95% inhibition of uptake. This effect was not antagonized by naloxone, implying a nonopioid mechanism of action. Imipramine 1 mumol litre-1 reduced [3H]noradrenaline uptake by 65-70% but morphine, in contrast to fentanyl, had no effect (P > 0.1).

Adrenal Gland Neoplasms↗
PubMed1992

Muscarinic receptors, phosphoinositide metabolism and intracellular calcium in neuronal cells.

1. We have utilised SH-SY5Y human neuroblastoma cells and primary cultures of rat neonatal cerebellar granule cells, both expressing M3 muscarinic receptors, to examine agonist driven polyphosphoinositide hydrolysis and alterations in intracellular calcium. 2. Stimulation of SH-SY5Y cells leads to a biphasic increase in intracellular calcium, the initial peak being due to the release of calcium from an intracellular store and the second maintained phase being due to calcium entry across the plasma membrane. The channel involved does not appear to be voltage sensitive, to involve a pertussis toxin sensitive G protein, or be opened by inositol polyphosphates. 3. Muscarinic receptor stimulation also leads to increased inositol polyphosphate formation in SH-SY5Y cells. Ins(1,4,5)P3 mass formation was biphasic in profile whereas Ins(1,3,4,5)P4 mass formation was slower and monophasic in profile. These data are consistent with substantial activity of 5-phosphatase (dephosphorylating Ins(1,4,5)P3 to Ins(1,4)P2) and 3-kinase (phosphorylating Ins(1,4,5)P3 to Ins(1,3,4,5)P4) in SH-SY5Y cells. 4. In order to better understand the role of Ins(1,4,5)P3 and its metabolites in calcium homeostasis we have examined the ability of a variety of natural and synthetic analogues to release intracellular sequestered calcium. The Ins(1,4,5)P3 calcium mobilizing receptor displays a remarkable degree of stereo- and positional selectivity with the most potent agonist to date being Ins(1,4,5)P3 (EC50 = 0.09 microM). 5. As an alternative to the continuous SH-SY5Y neuroblastoma (tumour derived) cell line we have used the primary cultured cerebellar granule cell. These cells also display a biphasic increase in Ins(1,4,5)P3 mass and a subsequent release of intracellular stored calcium. In our hands carbachol appears to increase calcium influx, a response which is only visible in the absence of magnesium.

Animals↗
PubMed1992

Carbachol-stimulated calcium entry in SH-SY5Y human neuroblastoma cells: which route?

M3 muscarinic receptors expressed on SH-SY5Y human neuroblastoma cells are linked to phosphoinositide turnover and rises in [Ca2+]i. The rise in [Ca2+]i is biphasic with the peak phase being due to release from an intracellular Ins(1,4,5)P3-sensitive site and the plateau phase being due to Ca2+ entry across the plasma membrane. Ca2+ entry does not appear to involve voltage sensitive Ca2+ channels, a pertussis toxin sensitive G-protein-operated Ca2+ channel or Ins(1,4,5)P3/Ins(1,3,4,5)P4-operated Ca2+ channel. We suggest that carbachol-stimulated Ca2+ entry in SH-SY5Y human neuroblastoma cells occurs via receptor operated Ca2+ channels and through capacitive refilling.

Calcium↗
PubMed1992

Elevation of cytosolic calcium by cholinoceptor agonists in SH-SY5Y human neuroblastoma cells: estimation of the contribution of voltage-dependent currents.

1. Muscarinic but not nicotinic receptor stimulation in SH-SY5Y human neuroblastoma cells induces a concentration-dependent increase in [3H]-inositol phosphate formation and a biphasic increase in [Ca2+]i. The latter involves release from both an intracellular store and Ca2+ entry across the plasma membrane. Here we examine the possibility that this agonist-stimulated Ca2+ entry occurs indirectly, as a consequence of depolarization. 2. Electrophysiological characterization, by whole cell patch-clamp techniques revealed that SH-SY5Y cells possess a tetrodotoxin-sensitive inward sodium current, a dihydropyridine-insensitive calcium current and an outward potassium current which was blocked by tetraethylammonium, 4-aminopyridine and intracellular caesium ions. The outward potassium current showed voltage-dependent activation and inactivation, similar to that seen for A-currents. 3. Application of nicotinic agonists evoked an inward current in cells voltage-clamped at negative holding potentials, but this current rectified, resulting in little or no outward current flow at positive potentials. The mean amplitude at a holding potential of -60 mV was -1.14 nA. Extrapolation of the current-voltage relation gave a reversal potential of +8 mV, indicative of a non-specific cationic permeability. 4. Application of muscarinic agonists had no detectable effect in most of the cells tested. However, in one third of cells studied, a small slowly activating inward current was observed. The mean amplitude of this current at a holding potential of -60 mV was -8.3 pA.5. This study confirms that SH-SY5Y cells possess voltage-dependent sodium, potassium and calcium currents. In addition, these cells are strongly depolarized by nicotinic agonists, which produce little change in [Ca2t]1. On the other hand, muscarinic agonists produce profound changes in [Ca2+1J with only a small inward current (depolarization). The contrasting effects of these two cholinoceptor agonists strongly implies that the Ca2+ entry after muscarinic receptor activation is not primarily due to activation of voltage-dependent calcium channels.

Calcium↗
PubMed1992

Rapid desensitization of muscarinic m3 receptor-stimulated polyphosphoinositide responses.

Chinese hamster ovary (CHO) cells transfected with human m3 muscarinic receptor cDNA (Bmax, 1343 +/- 46.8 fmol/mg of protein) were used to investigate agonist-mediated muscarinic receptor desensitization. Stimulation of CHO-m3 cells with a maximal dose of carbachol resulted in a biphasic production of mass inositol-1,4,5-trisphosphate [Ins(1,4,5)P3], measured by radioreceptor binding assay. The first phase comprises a rapid 8-10-fold increase in Ins(1,4,5)P3 that peaks after 10 sec and falls to levels 3-4-fold over basal within 1 min Ins(1,4,5)P3 rises again over the next 20 min to approximately 8-10-fold above basal, where levels are sustained for at least 2 hr. This later phase is, therefore, considered to be a desensitization-resistant component of m3 receptor activation. A 5-min pre-exposure of CHO-m3 cells to carbachol resulted in attenuation of the initial peak Ins(1,4,5)P3 response to a subsequent application of agonist. The attenuation of the Ins(1,4,5)P3 response was reversible with a t1/2 of approximately 7.5 min. Desensitization and recovery of the peak Ins(1,4,5)P3 response correlated with a decrease and subsequent recovery of m3 receptor-mediated mobilization of intracellular calcium stores, suggesting that the consequence of peak Ins(1,4,5)P3 desensitization is a reduced calcium mobilization response. N-[3H]Methylscopolamine binding to intact cells revealed that there was no change in cell surface m3 receptors during the 5-min pre-exposure to agonist, indicating that the mechanism of muscarinic receptor desensitization described here is not sequestration or internalization of receptors.

Animals↗
PubMed1991

Accumulation and metabolism of Ins(1,4,5)P3 and Ins(1,3,4,5)P4 in muscarinic-receptor-stimulated SH-SY5Y neuroblastoma cells.

Stimulation of M3 muscarinic receptors expressed by SH-SY5Y cells induced a dose- and time-related increase in the mass of Ins(1,4,5)P3 (basal 38.3 +/- 5.8 pmol/mg of protein) and Ins(1,3,4,5)P4 (basal 6.1 +/- 1.2 pmol/mg of protein). Comparison of radioreceptor mass assays with [3H]inositol labelling showed higher-fold stimulations with the former protocol. The later accumulation of Ins(1,4,5)P3 and Ins(1,3,4,5)P4 mass was dependent upon extracellular Ca2+.

Animals↗