The influence of polycyclic amines on calcium homeostasis of neuronal cells

Abstract

Ineffective regulation of calcium homeostasis within the central nervous system has been implicated as one of the main factors in the pathology underlying neurodegeneration. Calcium influx is mainly gated through voltage dependent calcium channels (VDCC) as well as N-Methyl-D-Aspartate (NMDA) receptor operated channels. Although the NMDA receptor complex can be modulated by many endogenous compounds, the activation state of both aforementioned channels is regulated through changes in membrane potential. Biological activity attributed to a class of polycyclic amine compounds, the pentacycloundecylamine derivatives, suggest possible intervention in the neurodegenerative cascade through modulation of membrane potential as well as a direct interaction with both voltage dependent and NMDA receptor operated channels. The aim of this study was to synthesise a series of pentacycloundecylamine derivatives and evaluate the effect of these compounds on neuronal membrane potential and calcium homeostasis within the central nervous system. A method was developed to assess the influence of these derivatives on membrane potential of neuroblastoma cells using confocal microscopy. The effects of the test compounds on influx of 45Ca2+ into murine synaptosomes were also evaluated. Compounds were synthesised by reductive amination of the polycyclic "cage", pentacyclo[5.4.0.0 2.6 .O 3.10 .O 5.9]undecane-8,11-dione, and characterised using NMR, MS and IR techniques. A real-time confocal laser microscope (Nikon PCM 2000 Confocal Laser Scanning Microscope [He/Ne laser]) and fluorescent potentiometric indicator, tetramethylrhodarnine methyl ester (TMRM, Molecular Probes, the Netherlands), were used to evaluate the influence of the test compounds on the membrane potential of human SH-SY5Y neuroblastoma cells. Cells were loaded with 500nM TMRM, in HEPES-buffered recording solution and changes in membrane potential, in response to stimulation by high concentration potassium (KCl) solution, were measured in the presence and absence of the pentacycloundecylamine derivatives. Results indicate that 8-benzylamino-8,11-oxapentacyclo[5.4.0. 0 2.6 .O 3.10 .O 5.9 ]undecane (1) and derivatives thereof influence the profile of KCl-induced membrane depolarisation. Test compounds caused an overall reduction in membrane depolarisation with compound 1, compound 5 (8-[(4-aminomethyl)pyridine]-8,ll-oxapentacyclo[5.4.0. 0 2.6 .O 3.10 .O 5.9 ]- undecane) and compound 6 (8-methylamino-8,11-oxapentacyclo[[5.4.0. 0 2.6 .O 3.10 .O 5.9 ]undecane) causing the more prevalent reductions in depolarisation. Compound 3 (8- hepthylamino-8,ll-oxapentacyclo[5.4.0. 0 2.6 .O 3.10 .O 5.9 ]undecane) caused initial stabilisation of membrane potential but failed to provide significant protection over the 870 sec period. Best overall results and significant inhibition of KC1-induced depolarisation were obtained with compound 1. These results indicate that cyclic unsaturated substituents on the pentacycloundecane template are important for the activity of these compounds. Introduction of hydrogen bonding (5) and decreased volume could also be significant. Test compounds were evaluated for antagonism of uptake of 45~a2+i'n murine synaptosomes. Synaptosomes were stimulated with high concentration potassium solution in the presence of 45~a2+T.h e radioactivity of the absorbed 45Ca2+as measured by means of liquid scintillation counting. All the compounds within this series of derivatives significantly antagonise absorption of calcium into neuronal cells. 8-Hepthylamino-8,lloxapentacyclo [5.4.0. 0 2.6 .O 3.10 .O 5.9 ]undecane (3) most extensively antagonised the 45Ca2+ uptake with compound 6 showing poor inhibition. From the results it is suggested that side chain substitution with moieties of larger volume might increase the activity of these derivatives. Hydrogen bonding seems to play a role in activity and compounds with cyclic groups in their side chain also present good activity. Peripheral calcium channel activity described for these derivatives, to a large extent correlate with the central activity indicated in this study. It is suggested that 8-benzylamino-8,ll-oxapentacyclo[5.4.0. 0 2.6 .O 3.10 .O 5.9 ]undecane and derivatives thereof could influence membrane potential of cells within the central nervous system through modulation of voltage activated sodium channels, NMDA receptor operated calcium channels and to some extent VDCC. Screening tests evaluating the antagonism of 45Ca2+ uptake in neuronal cells indicated that all test compounds inhibit influx of calcium into neuronal cells. Taking into consideration the activity indicated for the series of derivatives in this study, it is postulated that these compounds could reduce calcium influx into neuronal cells through modulating membrane depolarisation, and in event of an action potential, inhibiting calcium influx through both VDCC and NMDA calcium channels.