Michael A. Cousin

Flunarizine inhibits both calcium-dependent and -independent release of glutamate from synaptosomes and cultured neurones.

Flunarizine, an established Ca2+ channel antagonist, blocks both exocytotic glutamate release from mammalian cultured cerebellar granule cells and isolated presynaptic nerve endings (synaptosomes) prepared from two distinct areas of the mammalian brain. This blockade of release displays the same flunarizine concentration dependency in synaptosomes in the presence or absence of Ca2+, with total inhibition at a concentration of 10 microM. In cultured neurones, a selective effect on the L-channel-coupled component of the KCl-evoked rise in intracellular Ca2+, [Ca2+]c, can be demonstrated between flunarizine concentrations of 100 nM and 10 microM, while at concentrations above 10 microM, the remaining residual and transient components are affected. In synaptosomes, flunarizine blocks the KCl-evoked elevation in [Ca2+]c in a concentration-dependent manner. Additionally, 10 microM flunarizine directly antagonises ouabain-induced tetrodotoxin (TTX)-sensitive Na+ influx, glutamate, aspartate and GABA release from synaptosomes, whilst inhibiting veratridine-induced Ca(2+)-independent TTX-sensitive Na+ influx and glutamate release at 15 microM and 10 microM in cells and synaptosomes, respectively. In both cultured neurones and synaptosomes, the ability of flunarizine to block both neurotransmitter and cytoplasmic glutamate release is due to a direct antagonism of both voltage dependent Ca2+ channels and tetrodotoxin-sensitive Na+ channels.

Presynaptic calcium channels and field-evoked transmitter exocytosis from cultured cerebellar granule cells

Regulated exocytosis from cultured rat cerebellar granule cells can be localized by the vesicle specific marker FM2-10 to specific sites, the highest density of which are at visible varicosities coinciding with neurite-neurite contacts. Exocytosis can be evoked by uniform electrical field pulses, which initiate tetrodotoxin-sensitive action potentials, or by elevated KCl. [3H]D-Aspartate is an authentic false transmitter in this preparation, judged by sensitivity of release to bafilomycin A1 and tetanus toxin. The coupling of presynaptic voltage-activated Ca2+ channels to [3H]D-aspartate exocytosis was determined during field stimulation. The peak cytoplasmic free Ca2+ concentration achieved in the varicosities was proportional to Ca2+ entry during a 10 strain of pulses. L-type Ca2+ channels did not contribute to either Ca2+ entry or [3H]D-aspartate exocytosis. The P-type Ca2+ channel antagonist omega-agatoxin-IVA (30 nM) only inhibited at 75% of the varicosities, although a mean 15% inhibition of Ca2+ entry caused a 39% inhibition of exocytosis. In contrast the N-type Ca2+ channel inhibitor omega-conotoxin-GVIA (1 microM), which inhibited at virtually all varicosities, caused mean inhibitions of Ca2+ entry and exocytosis of 26% and 24% respectively. The toxin omega-conotoxin-MVIIC (5 microM), which inhibits N-, P- and Q-type Ca2+ channels, was effective at all varicosities. The Q-type component of Ca2+ entry was calculated to be only 5-10%; however, the additional inhibition of exocytosis was 30%. Thus P-type and particularly Q-type channels appear to be more closely coupled to exocytosis than N-type Ca2+ channels. The residual Ca2+ entry following 5 microM omega-conotoxin-MVIIC is scarcely coupled to release. The omega-agatoxin-IVA and omega-conotoxin-GVIA inhibitions of both Ca2+ entry and exocytosis were additive and varied stochastically between individual varicosities. These results demonstrate that both Q- and P-type Ca2+ channels are highly efficient in their coupling to amino acid exocytosis, with N-type less efficient, and L-type channels not at all. The Ca2+ channel types coupled to exocytosis are also able to support exocytosis when evoked by either brief field-evoked action potentials or prolonged depolarization with KCl, indicating that these presynaptic channels, in contrast to those on the somata of the cells, can respond to widely different patterns of activation.

The calcium channel coupled to the exocytosis of L-glutamate from cerebellar granule cells is inhibited by the spider toxin, Aga-GI.

The increase in cytosolic calcium, [Ca2+]c, evoked with 50 mM KCl in cerebellar granule cells consists of four components; (1) a rapidly inactivating transient or spike; (2) a nifedipine-sensitive non-inactivating plateau; (3) an Aga-GI (spider toxin) sensitive non-inactivating plateau; (4) a residual non-inactivating plateau insensitive to nifedipine and Aga-GI. None of these components is blocked by synthetic arginine polyamine toxin, spermine, (+)-MK-801 hydrogen maleate, D(-)-2-amino-5-phosphonopentanoic acid or omega-conotoxin-GVIA. The proposed P-type channel antagonist, omega-agatoxin-IVA, has a limited but non-significant effect on the elevated plateau [CA2+]c.L-type Ca2+ channels are located primarily on the soma whereas the component of the plateau which is blocked specifically by Aga-GI is localized primarily on the cell neurites. The latter component is coupled to the exocytosis of endogenous glutamate evoked with 50 mM KCl.

Glutamate exocytosis from cerebellar granule cells: The mechanism of a transition to an L-type Ca2+ channel coupling

When cerebellar granule cells in the presence of 1.3 mM calcium chloride (Ca2+) are depolarized by high potassium chloride (KCl), the release of endogenous glutamate is coupled to a high threshold Ca2+ channel blocked by the spider toxin omega Agatoxin-glutamate-release-inhibitor (Aga-GI) and insensitive to the L-type voltage-dependent Ca2+ channel-inhibitor nifedipine. A prolonged KCl depolarization in the absence of Ca2+ followed by addition of 5 mM Ca2+ results in an enhanced nifedipine-sensitive Ca2+ entry; glutamate exocytosis retains sensitivity to tetanus toxin and bafilomycin A1, is now totally inhibited by nifedipine and shows greatly reduced sensitivity to AGA-GI. Single cell Ca2+ imaging indicates that the L-type channel modulating release is preferentially located at somatic regions rather than neurites. A different pattern of vesicle endocytosis monitored with the fluorescent indicator FM1-43 is seen in response to the two depolarization protocols. Furthermore, vesicles loaded during depolarization with high KCl in the presence of 5 mM Ca2+ extensively exocytose dye in a nifedipine-insensitive manner in response to a second similar stimulation but release little dye in response to stimulus with high KCl in the absence of Ca2+ followed by the addition of 5 mM Ca2+. In contrast, vesicles loaded by stimulating with KCl in the absence of Ca2+ followed by the addition of 5 mM Ca2+ can be released by a second similar stimulus and this release is sensitive to nifedipine. Nifedipine sensitivity is not induced in cerebellar synaptosomes subjected to stimulation with high KCl in the absence of Ca2+ followed by the re-addition of 5 mM Ca2+. The results indicate that different populations of channels and vesicles may be functional during two depolarization protocols.

Exocytosis and Selective Neurite Calcium Responses in Rat Cerebellar Granule Cells During Field Stimulation

The free calcium concentration, [Ca2+]c, in fura‐2‐loaded rat cerebellar granule cells was investigated by digital imaging during trains of uniform field stimuli in order to compare the ability of calcium channels in somata and neurites to respond to brief, physiologically relevant depolarizations. Very few somata responded to 20 Hz trains of 1 ms pulses, while virtually all neurites showed an extensive increase which was rapidly reversed when stimulation was terminated. In contrast, both somata and neurites responded when cells were depolarized with 50 mM KCl. The field stimuli evoked a tetrodotoxin‐sensitive increase in Na+ concentration in both somata and neurites. When 4‐aminopyridine, which inhibits delayed K+ currents in these cells, was present during the field stimulus both somata and neurites increased their [Ca2+]c, suggesting that prolongation of the duration of depolarization is required for somatic Ca2+ channel activation. The neurite response did not depend on the orientation of the neurite relative to the applied field. The neurite response was insensitive to nifedipine (1 μM) and ω‐agatoxin‐IVA (30 nM) but was uniformly inhibited by ω‐conotoxin‐GVIA (30% inhibition at 1 μM) and ω‐conotoxin‐MVIIC (44% inhibition at 5 μM). The two inhibitors were not additive. The neurite [Ca2+]c response was insensitive to the combination of ionotropic glutamate receptor antagonists. Field stimulation caused the exocytosis of the fluorescent probe FM1‐43 previously loaded during KCl depolarization, suggesting that presynaptic Ca2+ channels contribute to the field‐evoked neurite response.

Protein kinase C modulates field-evoked transmitter release from cultured rat cerebellar granule cells via a dendrotoxin-sensitive K+ channel

The role of protein kinase C (PKC) in the control of neurotransmitter release from cultured rat cerebellar granule cells was investigated. Release of preloaded [3H]‐d‐aspartate which is incorporated into synaptic vesicles in this preparation was evoked by electrical field stimulation or elevated KCl. PKC activation by phorbol esters resulted in a large facilitation of field‐evoked Ca2+‐dependent [3H]‐d‐aspartate release and a lesser enhancement of KCl‐stimulated release. Inhibition of PKC by Ro 31‐8220 or staurosporine virtually abolished field‐evoked release but had no effect on KCl‐evoked release. Field‐evoked, but not KCl‐evoked, synaptic vesicle exocytosis monitored by the fluorescent vesicle probe FM2‐10 was inhibited by staurosporine. PKC was not directly modulating neurite Ca2+ channels coupled to release, as Ro 31‐8220 did not inhibit these channels. Activation or inhibition of PKC modulated field‐evoked plasma membrane depolarization, but had no effect on KCl‐evoked depolarization, consistent with a regulation of Na+ or K+ channels activated by field stimulation. No modulation of field‐evoked neurite Na+ influx was seen using phorbol esters. Phorbol ester‐induced facilitation of field‐evoked [3H]‐d‐aspartate release and neurite Ca2+ entry was non‐additive with that produced by the specific K+ channel antagonist dendrotoxin‐1, suggesting that PKC modulates transmitter release from field‐stimulated cerebellar granule cells by inhibiting a dendrotoxin‐1‐sensitive K+ channel.