Adenosine A₁Receptor-Mediated Activation of Phospholipase C in Cultured Astrocytes Depends on the Level of Receptor Expression

Abstract

Adenosine A 1 receptors induce an inhibition of adenylyl cyclase via G-proteins of the G i/o family. In addition, simultaneous stimulation of A 1 receptors and of receptor-mediated activation of phospholipase C (PLC) results in a synergistic potentiation of PLC activity. Evidence has accumulated that G␤␥ subunits mediate this potentiating effect. However, an A 1 receptormediated increase in extracellular glutamate was suggested to be responsible for the potentiating effect in mouse astrocyte cultures. We have investigated the synergistic activation of PLC by adenosine A 1 and ␣ 1 adrenergic receptors in primary cultures of astrocytes derived from different regions of the newborn rat brain. It is reported here that (1) adenosine A 1 receptor mRNA as well as receptor protein is present in astrocytes from all brain regions, (2) A 1 receptor-mediated inhibition of adenylyl cyclase is of similar extent in all astrocyte cultures, (3) the A 1 receptor-mediated potentiation of PLC activity requires higher concentrations of agonist than adenylyl cyclase inhibition and is dependent on the expression level of A 1 receptor, and (4) the potentiating effect on PLC activity is unrelated to extracellular glutamate.Taken together, our data support the notion that ␤␥ subunits are the relevant signal transducers for A 1 receptor-mediated PLC activation in rat astrocytes. Because of the lower affinity of ␤␥, as compared with ␣ subunits, more ␤␥ subunits are required for PLC activation. Therefore, only in cultures with higher levels of adenosine A 1 receptors is the release of ␤␥ subunits via G i/o activation sufficient to stimulate PLC. It is concluded that variation of the expression level of adenosine A 1 receptors may be an important regulatory mechanism to control PLC activation via this receptor.

Carbamazepine-Induced Upregulation of Adenosine A1-Receptors in Astrocyte Cultures Affects Coupling to the Phosphoinositol Signaling Pathway

“…The results obtained for untreated cultures were similar to those previously obtained by semiquantitative RT-PCR experiments (Biber et al 1997). Signals for adenosine A 1 -receptor mRNA were found in mRNA preparations from astrocytes cultured from all brain regions investigated.…”

“…Adenosine A 1 -receptor mediated PLC activation was only evident in cultures expressing high receptor numbers but was absent in cultures expressing low numbers of adenosine A 1 -receptors (Biber et al 1997). We, therefore, hypothesized that upregulation of A 1 -receptors should alter their coupling to second messenger systems.…”

“…The maximal inhibitory effect was approximately 50% and independent of the brain region from which the astrocytes had been cultured (Biber et al 1997). Ten-day treatment with the A 1 -receptor antagonist CBZ did not alter these inhibitory effects.…”

“…Very often, the coupling of adenosine A 1 -receptors to PLC ␤ is synergistic with the stim- NO . 3 ulation evoked by other receptors, such as ␣ 1 -adrenergic receptors (El-Etr et al 1989;Biber et al 1996Biber et al , 1997, histamine H 1 -receptors (Dickenson and Hill 1994), muscarinic receptors (Biden and Browne 1993), or thyrotropin receptors (Okajima et al 1995). There are, however, also reports suggesting direct activation of PLC by adenosine A 1 -receptors (Freund et al 1994;Murthy and Makhlouf 1995).…”

“…Total RNA was reverse transcribed and PCR experiments were carried out as described previously (Biber et al 1997). Sequences of oligonucleotide primer pairs and PCR conditions: Adenosine A 1 -receptor, # 55 5 ϽϽ -ATT-GCCTTGGTCTCTGTGC and #690 5 ϽϽ -CAGCTCCT-TCCCGTAGTAC, annealing temperature 59 Њ C, 35 cycles.…”