Adenosine‐dependent regulation of cyclic AMP accumulation in primary cultures of rat astrocytes and neurons

Abstract

The regulation of intracellular cyclic AMP (cAMP) formation by adenosine (Ado) and its analogues has been examined in primary cultures of rat-brain astrocytes and neurons. In the presence of the phosphodiesterase inhibitor, Ro 20-1724, basal levels of cAMP ranged from 40-120 pmol/mg protein in both cell types. Levels were not altered by treating the cells with Ado deaminase, which suggested that they did not produce appreciable amounts of endogenous Ado under standard culture conditions. In the astrocytes, microM quantities of agonists increased cAMP up to 30-fold higher than basal values; the relative potencies were typical of an A2 Ado receptor (NECA greater than Ado greater than R-PIA). Neuron-enriched cultures exhibited a maximum fourfold increase in cAMP in response to NECA; this was decreased a further eightfold when the cultures had prolonged exposure to the antimitotic agent, c-Ara, to eliminate greater than 98% of the nonneuronal cells. Low (nM) amounts of the Ado agonists inhibited cAMP formation in both cell types. In the astrocytes, the order of potency of inhibition of isoproterenol-stimulated cAMP formation was typical of an A1 receptor (R-PIA greater than Ado greater than NECA); maximum inhibition was 55-65%. Isoproterenol did not increase cAMP in the neuronal cultures. However, forskolin-stimulated formation was effectively (approximately 50%) inhibited by A1 Ado agonists; inhibition was not affected by prolonged treatment with c-Ara. From this study we tentatively concluded that rat astrocytes and neurons both contain inhibitory A1 Ado receptors, but that the stimulatory "A2" subtype is localized mainly on astrocytes.

Adenosine A2B‐receptor‐mediated cyclic AMP accumulation in primary rat astrocytes

“…Under the same conditions, however, the A2A-selective compound, CGS 21680 Jarvis et al, 1989) caused no significant accumulation at concentrations up to 10 lAM (n = 3) ( Figure 3, Table 1). The ECm values calculated for NECA and adenosine were in close agreement with the values previously reported for stimulation of cyclic AMP accumulation in rat astrocytes (Murphy et al, 1991) PD 115,199 (Bruns et al, 1987b) and xanthine amine congener ( Figure 6). The KD values determined from individual inhibition curves for these antagonists were similar to those previously reported at the A2B-receptor site ( and A2B (Gurden et al, 1993).…”

“…The KD values determined from individual inhibition curves for these antagonists were similar to those previously reported at the A2B-receptor site ( and A2B (Gurden et al, 1993). The presence of Al-and A2-receptors in cultures of human foetal (Woods et al, 1989), murine (Van Calker et al, 1979;Ebersolt et al, 1983) and rat astrocytes (Murphy et al, 1991) has previously been suggested on the basis of pharmacological studies using, primarily adenosine receptor agonists and antagonists with little or no subtype selectivity. In the present study we have used (aLosinski et al, 1993;bAlexander et al, 1989b;dAlexander et al, 1989a), antagonism of A2B-receptor-mediated relaxation of guinea-pig aorta (cLosinksi et al, 1993) or antagonism of adenylyl cyclase stimulation in rat cerebral cortex (eBazil & Minneman, 1986).…”

“…A number of groups have reported that adenosine and derivatives such as NECA, 2-chloroadenosine and phenylisopropyladenosine can stimulate cyclic AMP accumulation in rat, mouse and human glial preparations (Van Calker et al, 1979;Ebersolt et al, 1983;Elfman et al, 1984;Woods et al, 1989;Murphy et at., 1991 …”

Early growth response gene 1 (Egr-1) is required for new and reactivated fear memories in the lateral amygdala

“…Little is known about the mechanisms by which EGR-1 contributes to long-term synaptic plasticity, memory consolidation, and reconsolidation. In cultured neurons, both glutamatergic signaling (Condorelli et al 1994) and L-type voltage-gated calcium channels (Murphy et al 1991) have been shown to regulate EGR-1 expression. Further, EGR-1, along with the nuclear transcription factors Elk-1 and CREB, have been shown to be regulated by ERK/MAPK signaling during long-term potentiation (LTP) in the dentate gyrus (Davis et al 2000), suggesting that one or both of these transcription factors plays a critical role in ERK-driven EGR-1 transcription.…”

Modulation of adenosine A1 and A2A receptors in C6 glioma cells during hypoxia: involvement of endogenous adenosine

“…The protocol described by Murphy et al (1991) was followed with modifications. Briefly, cells were pre-incubated in serum-free DMEM buffered with 20 mM HEPES pH 7.4 supplemented with 0.1 mM Ro 20-1724 and 2 U/mL ADA, for 10 min at 37°C, after which different drugs were added to the wells and incubated for 15 min at 37°C in a final volume of 250 lL.…”