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Abstract(s)
The cyclic nucleotides cAMP and cGMP are second messengers whose levels are modulated by
G proteins-coupled receptors, and regulate multiple brain functions, including
neurotransmission, memory and synaptic plasticity. The adenosine A1 receptor is highly
expressed in the hippocampus where it inhibits neurotransmitter release, protects against
excitotoxic insults and regulates synaptic plasticity. Adenosine A1 receptor is coupled to Gi/o
proteins which negatively regulate adenylyl cyclase and thus cAMP formation. Modulation of
cGMP levels by Gi/o proteins-coupled receptors has also been recently reported. In the present
work the ability of adenosine A1 receptors to regulate cyclic nucleotides levels in the
hippocampus was investigated. The role of cyclic nucleotides as mediators of some actions of
adenosine A1 receptor in the hippocampus was also studied.
Activation of adenosine A1 receptor has been shown to decrease cAMP formation in the
cerebral cortex, but its effect on cAMP levels at the hippocampus is not clarified, nor its
interaction with others neuromodulators while regulating cAMP levels. We set forth to
determine the type of interaction found between adenosine A1 and cannabinoids CB1
receptors as negative modulators of cAMP accumulation. Furthermore, we also intend to
explore their combined neuroprotective potential. Quantification of cAMP in hippocampal
slices was performed through an enzymatic immunoassay, while neuroprotection against
NMDA-induced toxicity was assessed by determination of released LDH activity and by
quantification, by fluorescence microscopy, of the uptake of propidium iodide (PI) in cultured
organotypical hippocampal slices. The A1 agonist N6-Cyclopentyladenosine (CPA) decreased
forskolin-stimulated cAMP accumulation in the hippocampal slice with an EC50 of 35 ± 19 nM
and an Emax of 29% ± 5%, whereas for the CB1 agonist, WIN55212-2, an EC50 of 6.6 ± 2.7 μM
and an Emax of 31% ± 2% were obtained. NMDA (50 μM) increased the release of LDH activity by
92% ± 4% (n=4) when compared with control. Application of WIN55212-2 (30 μM) decreased
NMDA-induced LDH activity by 53% ± 11% (n=4), while CPA (100 nM) decreased it by 37% ± 11%
(n=4). The combined inhibitory effect of WIN55212-2 (30 μM) and CPA (100 nM) on cAMP
accumulation (41% ± 6%, n=4) and NMDA-induced LDH release (88% ± 14%, n=4) did not differ
from the sum of the individual inhibitory effects of each agonist (43% ± 8%, n=4, for cAMP
accumulation and 90 % ± 22%, n=4, for LDH release), but was different from the effects of
CPA or WIN55212-2 alone. Similarly, an additive inhibitory effect of co-application of
WIN55212-2 (30μM) and CPA (100nM) on NMDA (50μM)-induced PI uptake was also observed in
CA3 but not in CA1 area of the hippocampal slice. Thus, the combined effect of CB1 and A1
agonists on cAMP accumulation and NMDA-induced neurotoxicity is additive suggesting that
both agonists trigger independent cAMP signalling pathways and produce independent
cumulative neuroprotection against excitotoxic insults in the hippocampus. Previous studies indicate that cGMP produces similar effects to those triggered by adenosine
A1 receptors and, recently, it was reported that cGMP might mediate some actions of
adenosine A1 receptor in the peripheral nervous system. However, the role of cGMP on
adenosine A1 receptor mediated activity at the central nervous system remains obscure. Our
aim was to clarify if cGMP is modulated by A1 receptors at the hippocampus, if this
modulation depends on activation of the soluble form of guanylyl cyclase, and if such
mechanism is identical between male and female rats. Furthermore, the role of cGMP in
mediating the inhibitory effect of adenosine A1 receptor on neurotransmission in the
hippocampus was also investigated. To achieve our objectives, we used two approaches,
enzymatic immunoassays to measure cGMP accumulation and extracellular electrophysiology
to measure synaptic transmission at the rat hippocampal slice. The enzymatic immunoassays
tests reveal that application of CPA increased cGMP accumulation with an EC50 of 4.2 ± 1.4 nM
and an Emax of 17% ± 0.9%. Furthermore, in male rats, the presence of sodium nitroprosside
(SNP, a nitric oxide donor) abolished the effect of CPA on cGMP accumulation. In contrast, in
female rats, SNP failed to modify the increase in cGMP accumulation induced by CPA, but this
increase was reversed by DPCPX, stressing that A1 receptors modulate cGMP accumulation
despite the increase in soluble guanylyl cyclase activity by SNP. Thus, A1 receptors increase
intracellular cGMP levels at the hippocampus, through mechanisms which differ according to
gender. Regarding extracellular electrophysiology studies, we investigated in what extent
blocking the cGMP pathway using nitric oxide synthase (NOS), protein kinase G (PKG) and
soluble guanylyl cyclase (sGC) inhibitors, would interfere with the inhibitory effect of CPA on
synaptic transmission. CPA (15 nM) alone reversibly decreased synaptic transmission by 48% ±
2.1% (n=5) in males and by 54 % ± 5 % in females (n=5). In the presence of the NOS inhibitor LNAME
(300 μM), the sGC antagonist ODQ (10 μM) and the PKG inhibitor KT5823 (1 nM), CPAinduced
inhibition of synaptic transmission was dampened by 57 % ± 9 % (n=5), 23 % ± 7 %
(n=4) and 49 % ± 9 % (n=4), respectively. This attenuation of the effect of CPA was similar in
males and females. These findings suggest that A1 receptor neuromodulatory activity on
synaptic transmission partially depends on the cGMP pathway.
Description
Keywords
Hipocampo - Plasticidade sináptica Transmissão sináptica Neurotransmissão Neuroprotecção Receptor A1 da adenosina