Prolonged in vitro exposure of rat brain slices to adenosine analogues: Selective desensitization of adenosine A1 but not A2 receptors

doi:10.1016/0922-4106(92)90010-S

European Journal of Pharmacology: Molecular Pharmacology

Volume 227, Issue 3, 2 November 1992, Pages 317-324

https://doi.org/10.1016/0922-4106(92)90010-S Get rights and content

Abstract

Agonist-induced desensitization of adenosine A₁ and A₂ receptors was studied in rat striatum slices maintained in carbo-oxygenated Krebs buffer. Slices were exposed to adenosine analogues (either cyclo-penlyl-adenosine or N-ethyl-carboxamido-adenosine) for selected time periods (15–60 min) and repeatedly washed at the end of agonist exposure. Agonist-induced changes of adenosine receptors were then evaluated in P2 fractions prepared from slices by measuring A₁ and A₂ rcccptor-regulated adenylatc cyclase. A₁ receptors were rapidly desensitized by agonist exposure, as shown by a gradual loss of A₁ receptor-mediated inhibition of basal cyclase activity and cAMP formation, which was evident within 15–30 min after addition of the adenosine analogue. Agonist-induced desensitization of A₁ receptors was dose- and time-dependent, and seemed quicker in onset with cyclo-pentyl-adenosine, according to the higher A₁ selectivity of this receptor agonist, with respect to N-ethyl-carboxamido-adenosine. Binding of the A₁-sclective agonist [³H]cyclo-hexyl-adenosine was unaffected by the desensitization procedure at any of the exposure periods utilized, suggesting that an uncoupling of A₁ receptors from their transduction system is indeed responsible for the loss of functional activity. Loss of A₁ receptor function was accompanied by a time-dependent amplification of A₂ receptor-mediated stimulation of adenylete cyciase activity, likely due to an ‘unmasking’ of A₂ stimulatory receptor function as a consequence of the desensitizalion of A₁ inhibitory receptors. All these effects could be completely counteracted by the concomitant exposure to an adenosine receptor antagonist, and specifically involved the coupling mechanisms of adenosine receptors with their effector system. It is therefore concluded that the prolonged exposure of brain slices to adenosine analogues rapidly and selectively desensitizes A₁₄ receptors in the absence of any loss of A2 receptor function. These receptor adaptive changes might be relevant to brain pathological conditions characterized by over-exposure to endogenous adenosine.

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A number of studies have examined the effect of chronic adenosine receptor stimulation on adenosine receptor-linked signal transduction pathways (Abbracchio et al., 1992; Von Lubitz et al., 1994; Hettinger-Smith et al., 1996; Ciruela et al., 1997; Ruiz et al., 1996, 2005). The in vitro studies show that chronic exposure of neuronal or smooth muscle cell cultures and brain slice cultures to adenosine A1 receptor agonists result in a reduced density of A1 receptors (Abbracchio et al., 1992; Hettinger-Smith et al., 1996; Ciruela et al., 1997). Other reports show unchanged or reduced A1 receptor binding values after chronic in vivo treatment with A1 receptor agonists in whole brain preparations (Von Lubitz et al., 1994; Ruiz et al., 1996, 2005).
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In fact, the use of selective adenosine receptor antagonists showed that the blockade during the hypoxic period of A1Rs (with 8PT), but not of A2ARs (with SCH 58261), prevented the hypoxia-induced desensitization of A1Rs. This is in agreement with previous studies showing the existence of a homologous desensitization of A1Rs in brain tissue caused by a prolonged administration of A1R agonists both in vitro (e.g. Abbracchio et al., 1992; Hettinger et al., 1998; Vendite et al., 1998) and in vivo (e.g. Fernandez et al., 1996; Ruiz et al., 1996). The present study, however, focused on the ability of the endogenous agonist, released on hypoxia, to induce A1R desensitization rather than on the ability of exogenously added A1R agonists to desensitize A1Rs.
Activation of A₁ adenosine receptors is important for both the neuromodulatory and neuroprotective effects of adenosine. However, short periods of global ischemia decrease A₁ adenosine receptor density in the brain and it is not known if a parallel loss of functional efficiency of A₁ adenosine receptors occurs. We now tested if hypoxia leads to changes in the density and efficiency of A₁ adenosine receptors to inhibit excitatory synaptic transmission in rat hippocampal slices. In control conditions, the adenosine analog 2-chloroadenosine, inhibited field excitatory post-synaptic potentials with an EC₅₀ of 0.23μM. After hypoxia (95% N₂ and 5% CO₂, for 60 min) and reoxygenation (30 min), the EC₅₀ increased to 0.73μM. This EC₅₀ shift was prevented by the presence of the A₁ adenosine receptor antagonist 8-phenyltheophyline, but not by the A_2AR antagonist 7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4-triazolo[1,5-c] pyrimidine, during the hypoxic period. This decreased efficiency of A₁ adenosine receptors was not paralleled by a global change of A₁ adenosine receptor density or affinity (as evaluated by the binding parameters obtained in nerve terminal membranes). However, the density of biotinylated A₁ adenosine receptors at the plasma membrane of nerve terminals was reduced by 30% upon hypoxia/reoxygenation, in a manner prevented by the A₁ adenosine receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine and mimicked by prolonged (60 min) supra-maximal activation of A₁ adenosine receptors with 2-chloroadenosine (10μM). These results indicate that hypoxia leads to a rapid (<90 min) homologous desensitization of A₁ adenosine receptor-mediated inhibition of synaptic transmission that is likely due to an internalization of A₁ adenosine receptors in nerve terminals.

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Prolonged in vitro exposure of rat brain slices to adenosine analogues: Selective desensitization of adenosine A1 but not A2 receptors

Abstract

Eur. J. Pharmacol.

Pharmacol. Res. Commun.

Neuroscience

Brain Res.

Anal. Biothem.

Trends Pharmacol. Sci.

Neurosci. Lett.

J. Biol. Chem.

J. Biol. Chem.

Brain Res.

Anal. Biochem.

J. Biol. Chem.

Exp. Neurol.

Anal. Biochem.

Anal. Biochem.

Eur. J. Pharmacol.

Neurosci. Lett.

Adenosine receptors in rat basophilic leukaemia cells: transductional mechanisms and effects on 5-hydroxytryptamine release

Br. J. Pharmacol.

Prolonged in vitro exposure of rat brain slices to adenosine analogues: Selective desensitization of adenosine A₁ but not A₂ receptors