Guanosine, a guanine-based purine nucleoside, has been described as a neuromodulator that exerts neuroprotective effects in animal and cellular ischemia models. However, guanosine's exact mechanism of action and molecular targets have not yet been identified. Here, we aimed to elucidate a role of adenosine receptors (ARs) in mediating guanosine effects. We investigated the neuroprotective effects of guanosine in hippocampal slices from A 2A R-deficient mice (A 2A R −/− ) subjected to oxygen/glucose deprivation (OGD). Next, we assessed guanosine binding at ARs taking advantage of a fluorescent-selective A 2A R antagonist (MRS7396) which could engage in a bioluminescence resonance energy transfer (BRET) process with NanoLuc-tagged A 2A R. Next, we evaluated functional AR activation by determining cAMP and calcium accumulation. Finally, we assessed the impact of A 1 R and A 2A R co-expression in guanosine-mediated impedance responses in living cells. Guanosine prevented the reduction of cellular viability and increased reactive oxygen species generation induced by OGD in hippocampal slices from wild-type, but not from A 2A R −/− mice. Notably, while guanosine was not able to modify MRS7396 binding to A 2A R-expressing cells, a partial blockade was observed in cells co-expressing A 1 R and A 2A R. The relevance of the A 1 R and A 2A R interaction in guanosine effects was further substantiated by means of functional assays (i.e., cAMP and calcium determinations), since guanosine only blocked A 2A R agonist-mediated effects in doubly expressing A 1 R and A 2A R cells. Interestingly, while guanosine did not affect A 1 R/A 2A R heteromer formation, it reduced A 2A R agonist-mediated cell impedance responses. Our results indicate that guanosine-induced effects may require both A 1 R and A 2A R co-expression, thus identifying a molecular substrate that may allow fine tuning of guanosine-mediated responses.
Fluorescence-detected linear dichroism microscopy allows observing various molecular processes in living cells, as well as obtaining quantitative information on orientation of fluorescent molecules associated with cellular features. Such information can provide insights into protein structure, aid in development of genetically encoded probes, and allow determinations of lipid membrane properties. However, quantitating and interpreting linear dichroism in biological systems has been laborious and unreliable. Here we present a set of open source ImageJ-based software tools that allow fast and easy linear dichroism visualization and quantitation, as well as extraction of quantitative information on molecular orientations, even in living systems. The tools were tested on model synthetic lipid vesicles and applied to a variety of biological systems, including observations of conformational changes during G-protein signaling in living cells, using fluorescent proteins. Our results show that our tools and model systems are applicable to a wide range of molecules and polarization-resolved microscopy techniques, and represent a significant step towards making polarization microscopy a mainstream tool of biological imaging.
The interactions between TRPV1 and µ-opioid receptors (MOR) have recently attracted much attention because these two receptors play important roles in pain pathways and can apparently modulate each other’s functioning. However, the knowledge about signaling interactions and crosstalk between these two receptors is still limited. In this study, we investigated the mutual interactions between MOR and TRPV1 shortly after their activation in HEK293 cells expressing these two receptors. After activation of one receptor we observed significant changes in the other receptor’s lateral mobility and vice versa. However, the changes in receptor movement within the plasma membrane were not connected with activation of the other receptor. We also observed that plasma membrane β-arrestin 2 levels were altered after treatment with agonists of both these receptors. Knockdown of β-arrestin 2 blocked all changes in the lateral mobility of both receptors. Furthermore, we found that β-arrestin 2 can play an important role in modulating the effectiveness of ERK1/2 phosphorylation after activation of MOR in the presence of TRPV1. These data suggest that β-arrestin 2 and ERK1/2 are important mediators between these two receptors and their signaling pathways. Collectively, MOR and TRPV1 can mutually affect each other’s behavior and β-arrestin 2 apparently plays a key role in the bidirectional crosstalk between these two receptors in the plasma membrane.
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