PhD Scientific Days 2024

Poster Session B - Pharmaceutical Sciences and Health Technologies 1.

Optimization of a synaptosome model to study the effects of inhibition of angiotensin receptor 1 and  2 on synaptic glutamate release

Text of the abstract

Introduction:
Central nervous system effects of angiotensin receptor blockers (ARBs) gained a massive interest recently. Previous findings from our research group have demonstrated the anti-allodynic effect of ARBs in neuropathic animal model. Furthermore, telmisartan was found capable of enhancing the antinociceptive effect of morphine and reducing opioid tolerance.

Aims:
In this study, we intended to examine the impact of ARBs on synaptic glutamate transmission using a synaptosome model. Our aim was to transfer the previously applied synaptosome model into a suspension format and its optimization for a more sensitive and robust assessment of neurotransmitter release. We aimed at investigating the effect of ARBs on synaptic glutamate release using the optimized method.

Methods:
Freshly prepared synaptosomes and cryopreserved ones were examined. Their viability was assessed using resazurin reduction and LDH release assays. The amount of released glutamate was quantified using a glutamate oxidase enzyme-coupled reaction with fluorescence detection method.

Results:
Our results indicated that synaptosomes maintained their viability following resuspendation. Compared to the previously applied method significantly lower amount of synaptosome can be used, as approximately 3 µM of glutamate was released from 2 mg of synaptosomes. Suspension method is characterized by a more uniform distribution of synaptosomes resulting more reproducible and robust assay. Based on the results of storage stability experiments of freshly prepared suspension, viability decline can be avoided using snap freezing in HEPES-sucrose buffer containing 10% FBS without addition of DMSO.
Using the optimized method, we examined the effect of ARBs on glutamate release, showing a concentration-dependent reduction in 4-aminopyridine-induced glutamate release.
Interestingly angiotensin II itself could not increase the glutamate release suggesting the involvement of multiple receptors in its effect.

Conclusions:
The optimized model provides a more physiological method for studying the effects of various compounds on neurotransmitter release. This approach significantly reduces the required tissue quantity, thereby minimizing the number of animals to be sacrificed. Reduced glutamate release in response to ARBs may contribute to their previously observed antinociceptive effect.