No abstract

Neuronal nicotinic acetylcholine receptors are members of the ligand-gated ion channel receptor superfamily and may play important roles in modulating neurotransmission, cognition, sensory gating, and anxiety. Because of its distribution and abundance in the CNS, the alpha 7 nicotinic receptor is a strong candidate to be involved in some of these functions. In this paper we describe the synthesis and in vitro profile of AR-R17779, (-)-spiro[1-azabicyclo[2.2. 2]octane-3,5'-oxazolidin-2'-one] (4a), a potent full agonist at the rat alpha 7 nicotinic receptor, which is highly selective for the rat alpha 7 nicotinic receptor over the alpha 4 beta 2 subtype. Preliminary SAR of AR-R17779 presented here indicate that there is little scope for modification of this rigid molecule as even minor changes result in significant loss of the alpha 7 nicotinic receptor affinity.

Several lines of evidence indicate that a rapid loss of protein kinase C (PKC) activity may be important in the delayed death of neurons following cerebral ischemia. However, in primary neuronal cultures, cytotoxic levels of glutamate have been reported not to cause a loss in PKC as measured by immunoblot and conventional activity methods. This apparent contradiction has not been adequately addressed. In this study, the effects of cytotoxic levels of glutamate, NMDA, and α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid (AMPA) on membrane PKC activity was determined in cortical neurons using an assay that measures only PKC that is active in isolated membranes, which can be used to differentiate active enzyme from that associated with membranes in an inactive state. A 15‐min exposure of day 14–18 cortical neurons to 100 µM glutamate, AMPA, or NMDA caused a rapid and persistent loss in membrane PKC activity, which by 4 h fell to 30–50% of that in control cultures. However, the amount of enzyme present in these membranes remained unchanged during this period despite the loss in enzyme activity. The inactivation of PKC activity was confirmed by the fact that phosphorylation of the MARCKS protein, a PKC‐selective substrate, was reduced in intact neurons following transient glutamate treatment. By contrast, activation of metabotropic glutamate receptors by trans‐(1S,3R)‐1‐amino‐1,3‐cyclopentanedicarboxylic acid was not neurotoxic and induced a robust and prolonged activation of PKC activity in neurons. PKC inactivation by NMDA and AMPA was dependent on extracellular Ca2+, but less so on Na+, although cell death induced by these agents was dependent on both ions. The loss of PKC activity was likely effected by Ca2+ entry through specific routes because the bulk increase in intracellular free [Ca2+] effected by the Ca2+ ionophore ionomycin did not cause the inactivation of PKC. The results indicate that the pattern of PKC activity in neurons killed by glutamate, NMDA, and AMPA in vitro is consistent with that observed in neurons injured by cerebral ischemia in vivo.

The level of cytoplasmic calcium has been proposed to act as a regulator of acetylcholine receptor synthesis (Betz, H., and J. P. Changeaux (1979) Nature 278: 749-751). However, there is little known about the effect of altered calcium levels on the metabolism of the acetylcholine receptor. We have investigated the effect of decreased extracellular calcium on the metabolism of acetylcholine receptors in cultured rat myotubes. Our results show that the acetylcholine receptor levels on the surface of myotubes were decreased 25 to 30% following overnight incubation in calcium-deficient medium. In contrast, creatine phosphokinase activity levels and total protein synthesis were unaffected. Calcium depletion did not change the rate of receptor degradation significantly (0.037 hr-1, compared to 0.033 hr-1 for control cells) but dramatically decreased the rate of incorporation of new acetylcholine receptors into the plasma membrane. The time course for incorporation of new acetylcholine receptors into the plasma membrane of calcium-depleted cells was similar to control cells treated with cycloheximide, suggesting that de novo receptor synthesis was inhibited. These results indicate that intracellular calcium levels and acetylcholine receptor synthesis are not related in a simple reciprocal fashion and suggest that the regulation of acetylcholine receptor levels involves more than one intracellular compartment of calcium.