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Increases in Ca2+ concentration in the nucleus of neurones modulate gene transcription and may be involved in activity-dependent long-term plasticity, apoptosis, and neurotoxicity. Little is currently known about the regulation of Ca 2+ in the nuclei of neurones. Investigation of neuronal nuclei is hampered by the cellular heterogeneity of the brain where neurones comprise no more than 10% of the cells. The situation is further complicated by large differences in properties of different neurones. Here we report a method for isolating nuclei from identified central neurones. We employed this technique to study nuclei from rat cerebellar Purkinje and granule neurones. Patch-clamp recording from the nuclear membrane of Purkinje neurones revealed numerous large-conductance channels selective for monovalent cations. The nuclear membrane of Purkinje neurones also contained multiple InsP 3 -activated ion channels localized exclusively in the inner nuclear membrane with their receptor loci facing the nucleoplasm. In contrast, the nuclear membrane of granule neurones contained only a small number of mainly anion channels. Nuclear InsP 3 receptors (InsP 3 Rs) were activated by InsP 3 with EC 50 = 0.67 µM and a Hill coefficient of 2.5. Ca 2+ exhibited a biphasic effect on the receptors elevating its activity at low concentrations and inhibiting it at micromolar concentrations. InsP 3 in saturating concentrations did not prevent the inhibitory effect of Ca 2+ , but strongly increased InsP 3 R activity at resting Ca 2+concentrations. These data are the first evidence for the presence of intranuclear sources of Ca 2+in neurones. Ca 2+ release from the nuclear envelope may amplify Ca 2+ transients penetrating the nucleus from the cytoplasm or generate Ca 2+ transients in the nucleus independently of the cytoplasm.

1. Single K+ channel currents and membrane potential were recorded in the endothelium of excised intact rat aorta.2. Two types of K+ channel were found in excised patches, KCh and KAP. With Na+ and K+ as the main external and internal cations, outward conductances were 6-7 pS (KCh) and 2-8 pS (KAP) In symmetric 150 mM K+, the inward conductances were 18 and 9-1 pS. 4. Apamin at concentrations > 1 nm inhibited KAP channels. Block was complete at 10 nM.KAP channels were insensitive to charybdotoxin. KCh channels were inhibited by charybdotoxin at concentrations > 50 nm, but were insensitive to apamin. 5. d-Tubocurarine (dTC) evoked flickering activity of KAP channels at concentrations > 5/SM and complete block at 100 /M. At these doses, dTC did not affect KCh channels, but at concentrations > 1 mM it decreased the single channel amplitude. 6. Hyperpolarization evoked by acetylcholine was unaffected by apamin or dTC at low concentrations (< 100 /M), but inhibited by high concentrations of charybdotoxin (>50 nM) or dTC (> 1 mM).7. These data suggest that KCh channels are novel Ca2+-activated K+ channels responsible for the ACh-evoked hyperpolarization in the endothelium of rat aorta.

SUMMARY1. The passive electrical properties and the effects of acetylcholine on the membrane potential of the endothelium of intact rat aorta were investigated using the whole cell mode of the patch clamp technique.2. Unstimulated endothelium had a membrane potential of -58 + 8 mV (S.E.M., n = 193; range -47 to -76 mV). The input resistance was 43 + 13 MQ (S.E.M., n = 8; range 26-64 MQ). KCl and BaCl2, but not tetraethylammonium (2 mM), 4-aminopyridine (5 mM) or 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS; 100 /M) depolarized the endothelium.3. Acetylcholine (0-2A4 gM) evoked in most preparations a biphasic response with a transient hyperpolarization to a value close to the K+ reversal potential, followed by depolarization beyond the resting potential. In 46 % of recordings, the depolarization was followed by oscillations in membrane potential. The duration of the hyperpolarization and magnitude of the depolarization was similar in all recordings from a given aorta, but varied greatly between different preparations.4. Hyperpolarization of the endothelium below the K+ reversal potential reversed the direction of the first phase of the acetylcholine-evoked response, which was unaffected by tetraethylammonium, 4-aminopyridine, or DIDS.5. The removal of extracellular Ca2+ evoked a depolarization of the endothelium from -61 + 3 to -34 + 3 mV (S.E.M., n = 9) over 2-15 min. Restoration of external Ca2+ evoked a transient hyperpolarization.6. ACh applied in nominally Ca2+-free medium shortly after Ca2+ removal evoked only a transient hyperpolarization. After the establishment of a stable membrane potential in Ca2+-free medium, acetylcholine was without effect. 7. NiCl2 (2 mM) evoked a small depolarization of the endothelium (6 + 2 mV; S.E.M., n = 7). The subsequent removal of Ni2+ evoked a transient hyperpolarization.8. In the presence of Ni2+, acetylcholine evoked a short-lived hyperpolarization. Both the application of Ni2+ and the removal of extracellular Ca2+ immediately blocked oscillations in membrane potential evoked by acetylcholine. S. M. MARCHENKO AND S. 0. SAGE membrane potential, depolarization of the endothelium alone, by current injection or application of KCl, did not evoke oscillations.11. The activator of protein kinase C, phorbol 12, 13-dibutyrate (200 nM) depolarized and greatly increased the input resistance of the endothelium, presumably due to an effect on gap junctions. The blockers of protein kinase C, staurosporine (200 nM) and H-7 (200 nM), were without effect on acetylcholineevoked responses.12. These results suggest that acetylcholine evokes complex changes in endothelial membrane potential following changes in cytosolic calcium concentration. The initial agonist-evoked hyperpolarization appeared dependent on the release of Ca2+ from intracellular stores, whilst the prolonged phase of the hyperpolarization, depolarization and oscillations in membrane potential are dependent upon the entry of Ca2+ through Ni2+-sensitive channels. Protein kinase C does not appear to be involved in...

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