Download Excitatory sulphur amino acids evoke a Ca2+

3s
Excitatory
sulphur
amino
acids
evoke
a
Ca2+-independent release of [SHIPASP and [SHIGABA
from primary neuronal cultures by a mechanism which
involves reversal of the high affinity transporters for
L-giu and GABA, respectively.
JOHN DUNLOP', ANGUS GRIEVE', ARNE SCHOUSBOE and ROGER
GRIFFITHS'.
'Department of Biochemistry, University of St. Andrews, St.
Andrews. fife KY16 SAL, U.K.
Royal Danish School of Pharmacy, University of Copenhagen,
Copenhagen, Denmark.
The excitatory sulphur amino acids (SAAs), cysteine sulphinate
(CSA). cysteate (CA), homocysteine sulphinate (HSA),
homocysteate (HCA) and sulpho-cysteine (SC) have been proposed
as possible endogenous ligands acting on excitatory amino acid
( E M ) receptors. This transmitter candidacy has been proposed on
the following grounds. SAAs are, (i) potent neuronal excitants
[I], (ii) endogenous [2,3], (iii) released from brain slices in a
Ca2+-dependent manner in response to high [K+] depolarisation
[3], (iv) substrates for neuronal transport systems [4,5], (v)
mixed agonists at EAA receptor subtypes [6,7], and, (vi)
heterogenously distributed within the CNS [2.3]. We have
recently demonstrated, using primary cultures of neurones as a
model system for transmitter release studies, that SAA occupation
of both NMDA (n-methyl-D-aspartate) and non-NMDA receptors
is coupled to Ca2+-dependent transmitter release 81 Thus,
SAAs-evoke a dosedependent and saturable release of bH]D-ASP
from cerebellar granule cells, and, [3H]GABA from cortical
neurones. In addition to this Ca2+-dependent (neurotransmitter
like) release a significant Ca2+-independent release was
observed.
In this study, the mechanism of the Ca2+-independent component
of release has been examined to reveal an essential requirement
for the activity of the neuronal plasma membrane transporter for
D-ASP in granule cells. and, GABA in cortical neurones.
Primary cultures of neurones [9,10] placed in a superfusion
system [I I ] were employed throughout. Cells were stimulated
using an elevated concentration of SAA (500pM) to exaggerate the
CaP+-independent release. The effect of, (i) transport inhibitors,
(ii) Na+ removal, and, (iii) receptor antagonism, has been
studied in order to evaluate the role of the respective carriers.
Release of [3H]-DASP from cerebellar granule cells evoked by
all 5 SAAs under Ca2+-free conditions was potentiated by
dihydrokainate (2mM) and aspartate-R-hydroxamate (500pM).
Both these compounds are competitive inhibitors of the high
affinity L-glu transporter but are believed to act as substrates for
the carrier 112,131. In addition, evoked release was sensitive to
the removal of Na+ ions (equimolar replacement with choline)
from the superfusion medium (not shown). The extent to which
receptor activation might lead to this component of release was
studied using the EAA receptor antagonist DNQX
(6.7-dinitroquinoxalinedione) at a concentration (200pM)
where both NMDA and non-NMDA responses are antagonised [14].
Co-administration of DNQX with SAA resulted in attenuation of
evoked release suggesting at least an involvement of receptor
activation in the release mechanism. These results are shown in
Fig. 1A with SC shown as a representative SAA agonist.
Release of [3H]GABA from cortical neurones was also sensitive
to the presence of transport inhibitors. SAA-evoked Ca2+independent release of [3H]GABA was potentiated by guvacine
(300pM) and attenuated by SKF-89976A (15pM). both of which
are competitive inhibitors of high affinity GABA uptake but only
the former is a substrate [15]. Sensitivity to removal of Na+ was
also observed in these cells (not shown). Importantly. DNOX
(200pM) was found to completely block this component of release
in cortical neurones suggesting that receptor activation is
essential in this cell type for Ca2+-independent release. These
results are shown in Fig. 1B with CSA as a representative SAA
agonist.
Thus, under conditions where carrier activity is blocked, SAAevoked Ca2+-independent release is attenuated. Conversely, when
carrier activity is stimulated. then a potentiation of this release
component is observed. Studies with the EAA receptor antagonist
DNQX revealed that a primary event of receptor activation was
required in both cell types, although, a differential sensitivity to
receptor antagonism was observed between the two cell types.
-0
15
FRACTION NUMBER
Fig. 1. SAA-ev
cells measured in the presence of 500pM SC (a), or, SC coadministered with 2mM dihydrokainate (b), 500pM aspartate4
-hydroxamate (c) and 200pM DNQX (d). 8. 13H]GABA release
from cortical neurones measured in the presence of 500pM CSA
(a), or, CSA co-administered with 15pM SKF-89976A (b),
300pM guvacine (c) and 200pM DNQX (d). Y-axis is cpm
( X I0 - 3 ) released in individual fractions. Solid line represents
1 min stimulus.
A likely mechanism is that receptor activation and subsequent
depolarisation leads to a sufficient elevation of [Na+]i to drive the
uptake carrier in the reverse direction. Additionally, in granule
cells where some release persists in the presence of DNQX a second
component appears to exist. This may involve a heteroexchange
reaction mediated by direct interaction of SAAs with the high
affinity L-glu carrier. To this end, a heteroexchange of SAAs with
[3H]D-ASP in rat brain synaptosomes has been reported [16,17].
financial support from the Wellcorne Trust and the Mailland
Ramsay Trust (Scholarship to J.D.) is acknowledged.
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