IJUP08 - Universidade do Porto

Calcium uptake by rat hippocampal synaptosomes: regulation by
endogenous adenosine and different depolarizing agents
A. Rocha, M.G.B. Lobo, and P. Correia-de-Sá
Laboratório de Farmacologia e Neurobiologia, UMIB, Instituto de Ciências Biomédicas Abel
Salazar, Universidade do Porto, Portugal.
Adenosine is an endogenous modulator of the CNS refraining neuronal excitability (e.g. [1]).
Because endogenous levels of adenosine rise markedly during seizure activity, this nucleoside has
been proposed as an “endogenous anticonvulsant”. The anticonvulsant effect of adenosine may be
due to A receptors activation [2] leading to suppression of voltage-dependent Ca
1 2+
entry [3].
Animal models and human tissue (from epileptics submitted to neurosurgery) exhibit similar
mechanisms underlying epileptic disorders [4]. Synaptosomes – isolated nerve endings that
maintain physiological properties such as the ability to release neurotransmitters in a Ca 2+
-
dependent manner - are throughly used to study pathogenesis of epilepsy. Veratridine (VT, a
selective Na +
opener), 4-aminopyridine (4-AP, a nonselective K +
channel blocker) and high K +
concentrations are the most common strategies to depolarise synaptosomal plasma membrane as
they allow to differentiate between presynaptic Na +
channel-mediated responses; these compounds
increase intracellular Ca 2+
accumulation in the rat hippocampus as well as in the human brain.
Nevertheless, we recently showed that the choice of depolarising agent (VT vs high [K +
]) may be
crucial for evidencing paradoxical effects of drugs regulating Ca 2+
influx into hippocampal
synaptosomes, such as nifedipine [5]. Therefore, we investigated whether the same occurred using
4-AP as compared with VT. We also tested the modulatory role of endogenous adenosine on
neuronal Ca 2+
uptake by incubating hippocampal synaptosomes with adenosine deaminase (ADA),
which inactivates adenosine into inosine.
Synaptosomes were prepared from rat hippocampal homogenates [5]. Samples (50 μl) of
synaptosomal suspension were pre-incubated with test drugs for 10 min, at 37 ºC. Synaptosomal
loading was performed during 3 min with 45
CaCl (0.32 μCi/μmol) in the presence of the
2
depolarizing agent. Adding ice-cold Tris-EGTA solution followed by filtration under negative
pressure through GF/C glass fibre filters, terminated 45
Ca uptake. The radioactivity associated with
the synaptosomes was determined by liquid scintillation spectrometry.
Inactivation of endogenous adenosine with ADA (0.5 UmL -1
) caused a global increase on 45
Ca
uptake into the rat hippocampal synaptosomes, indicating that there is a significant adenosine
inhibitory tonus regulating depolarisation-induced Ca 2+
influx into nerve terminals. VT (5 μM)
increased 45
Ca uptake into hippocampal synaptosomes (67±9 %, n=4). 4-AP (10 mM) increased
45
Ca uptake to a lesser extent (23±4%, n=4). Co-application of VT (5 μM) together with 4-AP (10
mM) increased 45
Ca uptake (138±15 % (n=4) above the control level. This higher-than-additive
effect, contrasts with the additive result obtained upon applying VT (1-10 μM) together with high
[K +
] (15-30 mM) [5], indicating the existence of synergism between VT and 4-AP.
[1] Cunha, R. Neurochem. Int. 38, 107-125, 2001
[2] Zhang, G., Franklin, P.H. & Murray, Eur. J. Pharmacol.,255, 239-243,1994
[3] Wu, L.G. & Sagau, P, Neuron, 12, 1139-1148, 1997
[4] Avoli,M., Louvel,J., Pumain,R. & Kohling,R., Prog. Neurobiol., 22, 166-200, 2005
[5] Costa, J. Lobo, M.G.B. & Correia-de-Sá., Eur. J. Pharmacol., 544, 39-48, 2006
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