Muscarinic signalling affects intracellular calcium concentration during the first cell cycle of sea urchin embryos.

The existence of a response to acetylcholine (ACh) and cholinomimetic drugs in sea urchin eggs and zygotes was investigated in two sea urchin species: Paracentrotus lividus and Lytechinus pictus. The calcium sensitive fluorescent probe, Fura-2 dextran, was employed to investigate the regulation of cytosolic free calcium concentration ([Ca(2+)](i)) by cholinomimetic drugs in unfertilised and fertilised eggs of both the sea urchin species. Exposure to cholinomimetic agonists/antagonists, either extracellularly or intracellularly, had no effect either on resting [Ca(2+)](i) levels in the unfertilised sea urchin egg, or on the transient [Ca(2+)](i) increase at fertilisation. However, following fertilisation, extracellular application of ACh receptors agonists, such as ACh and carbachol, predominantly muscarinic agonist, but not nicotine, induced a significant increase in [Ca(2+)](i), which was partially inhibited by atropine. As a consequence of exposure after fertilisation to the agonists of ACh receptors, chromatin structure was transiently affected. The hypothesis is proposed that muscarinic receptors may be involved in the (presumably Ca(2+)-dependent) modulation of the nuclear status during the first cell cycles.

Chronic ethanol promotes the neuronal differentiation of NG108-15 cells independently of toxin-sensitive G-proteins.

The ability of ethanol to promote neuronal differentiation of NG108-15 mouse neuroblastoma x rat glioma hybrid cells was investigated using morphological, biochemical and electrophysiological techniques. Ethanol concentration-(10-200 mM) and time-(1 h-3 days) dependently reduced cell proliferation, but increased acetylcholinesterase (AChE) activity and cell protein content. Chronic ethanol (200 mM) also time-dependently increased voltage-sensitive Ca(2+) currents in the cells. Similar effects were obtained with chronic treatment of the cells with the standard differentiating agents sodium butyrate or forskolin. Chronic treatment of NG108-15 cells with primary alcohols (0.1-200 mM) of varying chain length all reduced cell proliferation and increased cell protein content and AChE activity with the potency order butanol>propanol>ethanol>methanol. Chronic treatment of NG108-15 cells with cholera toxin (50 ng ml(-1)) or pertussis toxin (50 ng ml(-1)) did not induce differentiation of the cells, nor did it modify the effects of 50 or 200 mM ethanol on cell proliferation, AChE activity or cellular protein content. Chronic cholera toxin did however abolish agonist-stimulated adenylyl cyclase activity in the cells, whereas pertussis toxin abolished receptor-mediated inhibition of adenylyl cyclase activity. Furthermore, inhibitors of protein kinase C (GF 109203X, 5 µM), protein kinase A (H-89, 10 µM) or Ca(2+)/calmodulin-dependent protein kinase II (KN-62, 3 µM) all failed to modify the effects of 200 mM ethanol on cell proliferation, AChE activity and cellular protein content. These experiments indicate that chronic ethanol is able to promote neuronal differentiation of NG108-15 cells independently of toxin-sensitive G-proteins and some protein kinases.

Effects of acute and chronic ethanol on cyclic AMP accumulation in NG108-15 cells: differential dependence of changes on extracellular adenosine.

1. This study investigated the effects of acute and chronic ethanol on basal, agonist- and forskolin-stimulated cyclic AMP formation in NG108-15 mouse neuroblastoma x rat glioma hybrid cells, and examined the role of changes in extracellular adenosine concentrations on the effects observed. 2. NG108-15 cells incubated acutely with ethanol (1-200 mM) displayed concentration-dependent increases in basal and iloprost-stimulated (300 nM; a prostanoid IP receptor agonist) cyclic AMP accumulation but a concentration-dependent decrease in forskolin-stimulated (10 microM) accumulation. 3. Cells treated chronically with ethanol (200 mM) for 48 h displayed increases over control in basal, iloprost- (0.001-10 microM) and forskolin (0.01-100 microM)-stimulated cyclic AMP formation. However, chronic ethanol did not affect [3H]-iloprost binding to cell membranes. 4. Inclusion of adenosine deaminase (ADA; 1 unit ml-1) during the incubation period to measure cyclic AMP accumulation completely abolished the increase in basal accumulation following chronic ethanol, but did not affect the increase in iloprost stimulation. On the other hand ADA partially reversed the increase in forskolin stimulation following chronic ethanol, but even in the presence of high concentrations of ADA (5 units ml-1) the forskolin stimulation remained elevated above control. 5. Cells treated chronically with the adenosine receptor agonist 5'-(N-ethylcarboxamido)-adenosine (NECA; 10 microM for 48 h) displayed a reduction in subsequent NECA- and forskolin-stimulated cyclic AMP accumulation, but iloprost stimulation was not affected. ADA included acutely during the incubation period to measure cyclic AMP accumulation abolished the reduction in forskolin but not NECA stimulation produced by the chronic NECA pretreatment. 6. We have previously noted that ethanol inhibits NG108-15 cell proliferation and alters cell morphology.To mimic this, cells were incubated in the absence of foetal calf serum for 48 h. Following this time, basal, iloprost- and forskolin-stimulated cyclic AMP formation was enhanced over that in cells grown in the presence of serum.7. These results indicate that chronic ethanol enhances cyclic AMP formation in intact NG108-15 cells by more than one mechanism: one involves increased extracellular adenosine concentrations and the other a change in the transduction system beyond the receptor, possibly involving the adenylyl cyclase enzyme. Furthermore the ethanol-induced changes in cyclic AMP accumulation may relate to alterations in NG108-15 cell growth and development.