Regulation of Calcium Concentrations in Synaptosomes: α2‐Adrenoceptors Reduce Free Ca2+by Closure of N‐Type Ca2+Channels

Abstract: The relationship between intrasynaptosomal total (CaT) and free ([Ca2+]i) calcium and 45Ca accumulation was studied under physiological and K+‐depolarised conditions in rat cortical synaptosomes. Under physiological conditions, CaT(10.7 mM) was ∼ 10,000 times higher than [(Ca2+]i(118 nM), showing that there is a large reservoir of sequestered calcium in synaptosomes. 45Ca accumulation was rapid (initial rate, 3.4 nmol/mg protein/min), substantial (7 nmol/mg protein in 2 min), and depolarisation dependent, and reached equilibrium after 5 min. At equilibrium, only 10% of CaTwas freely exchangeable. This pool was much larger than the free Ca2+pool. CaT, [Ca2+]i, and 45Ca accumulations were directly related to the Ca2+concentration in the buffer, suggesting that [Ca2+]iis not highly conserved but is maintained by simple equilibria between the various pools. Clonidine reduced 45Ca accumulation in a time‐ and dose‐dependent manner. Maximum inhibition (40% at 100 μM) occurred at 2 min and the IC50was 80 nMThe reduction caused by clonidine (1 μM) reached equilibrium after 5 min, but this equilibrium value was lower than in controls, suggesting that clonidine changes the exchangeable Ca2+pool size. The effects of clonidine (1 μM) on [Ca2+]i(26% reduction) and on 45Ca accumulation (24% reduction) were most apparent under physiological conditions. However, while it was not dependent on depolarisation, it did not occur in physiological buffer containing low K+concentration (0.1–1 mM). The inhibitory effect of clonidine on 45Ca accumulation is receptor mediated as it was antagonised by idazoxan (1 μM). Under nondepolarised conditions (5 mMK+), ω‐conotoxin fraction GVIA (ω‐CgTx; 0.5 μM) caused a 35% reduction in 45Ca accumulation in a 2‐min incubation, whereas nifedipine (1 μM) had no significant effect. In contrast, both ω‐CgTx and nifedipine effectively blocked 45Ca entry into depolarised synaptosomes. However, the effect of ω‐CgTx was not additive with that of clonidine, whereas that of nifedipine was. Finally, idazoxan (1 μM) did not alter the effect caused by nifedipine. These data show (a) that clonidine decreases 45Ca accumulation by inhibiting N‐ but not L‐type voltage‐sensitive Ca2+channels, (b) that specific Ca2+channels on synaptosomes display a range of voltage sensitivities, and (c) that L‐type Ca2+channels are operational under depolarising conditions.