Related Experiment Videos
A3 adenosine receptors in human astrocytoma cells: agonist-mediated desensitization, internalization, and
M L Trincavelli1, D Tuscano, M Marroni
1Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, Universitá Pisa, Pisa, Italy.
Abstract:
A(3) adenosine receptor activation has been previously demonstrated to result in both neuroprotective and neurodegenerative effects, depending upon specific pathophysiological conditions. This dual effect may depend on receptor regulation mechanisms that are able to change receptor availability and/or function. In the present study, we investigated desensitization, internalization, and down-regulation of native A(3) adenosine receptors in human astrocytoma cells after exposure to the agonist 2-chloro-N6-(3-iodobenzyl)-N-methyl-5'-carbamoyladenosine (Cl-IBMECA). Cl-IBMECA induced a concentration-dependent inhibition of adenylyl cyclase activity with an EC(50) value of 2.9 +/- 0.1 nM. The effect was suggested to be mediated by A(3) adenosine receptor subtype by the use of selective adenosine receptor antagonists. Cell treatment with pertussis toxin abolished Cl-IBMECA-mediated inhibition of adenylyl cyclase activity, evidencing an A(3) receptor coupling to inhibitory G protein. Short-term exposure to the agonist Cl-IBMECA (100 nM) caused rapid receptor desensitization, within 15 min. Agonist-induced desensitization was accompanied by receptor internalization: A(3) adenosine receptor internalized with rapid kinetics, within 30 min, after cell exposure to 100 nM Cl-IBMECA. The localization of A(3) adenosine receptors on the plasma membrane and in intracellular compartments was directly revealed by immunogold electron microscopy. After desensitization, the removal of agonist led to the restoration of A(3) adenosine receptor functioning through receptor recycling to the cell surface within 120 min. Prolonged agonist exposure (1-24 h) resulted in a marked down-regulation of A(3) adenosine receptors that reached 21.9 +/- 2.88% of control value after 24 h. After down-regulation, the recovery of receptor functioning was slow (24 h) and associated with the restoration of receptor levels close to control values. In conclusion, our results demonstrated that A(3) receptors, in astrocytoma cells, are regulated after short- and long-term agonist exposure.
Insights
Activation of A(3) adenosine receptors in astrocytoma cells leads to desensitization and internalization. Receptor function is restored upon agonist removal, but prolonged exposure causes down-regulation and slow recovery.
Area of Science:
- Neuroscience
- Cell Biology
- Pharmacology
Background:
- A(3) adenosine receptors exhibit dual neuroprotective and neurodegenerative effects.
- Receptor regulation mechanisms influence A(3) adenosine receptor availability and function.
- Understanding these mechanisms is crucial for targeted therapeutic interventions.
Purpose of the Study:
- To investigate the regulation of native A(3) adenosine receptors in human astrocytoma cells.
- To examine receptor desensitization, internalization, and down-regulation following agonist exposure.
- To elucidate the kinetics and recovery of A(3) adenosine receptor function.
Main Methods:
- Exposure of human astrocytoma cells to the A(3) adenosine receptor agonist Cl-IBMECA.
- Measurement of adenylyl cyclase activity and G protein coupling.
- Utilizing immunogold electron microscopy to track receptor localization.
- Assessing receptor desensitization, internalization, down-regulation, and recovery kinetics.
Main Results:
- Cl-IBMECA inhibited adenylyl cyclase activity, confirming A(3) receptor-mediated effects coupled to inhibitory G proteins.
- Short-term agonist exposure induced rapid receptor desensitization and internalization.
- Prolonged agonist exposure led to significant receptor down-regulation.
- Receptor function was restored via recycling after desensitization, but recovery was slow after down-regulation.
Conclusions:
- A(3) adenosine receptors in astrocytoma cells are dynamically regulated by agonist exposure.
- Both short-term and long-term agonist treatments trigger distinct regulatory responses.
- These findings provide insights into the complex modulation of A(3) adenosine receptor signaling in cellular contexts.