Influence of PLA2-PG system on purine release and cAMP content in dissociated primary glial cultures from rat striatum.

Purine release and prostaglandin (PG) outflow were simultaneously evaluated from untreated glial primary cultures of rat striatum, at rest and under field electrical stimulation. Purine release was also assayed from sister cultured cells in which a suitable pharmacological treatment with 1 x 10(-6) M dexamethasone or 1 x 10(-4) M indomethacin had produced a complete inhibition of the phospholipase A2-prostaglandin (PLA2-PG) system. Purine release from untreated cells seems to be regulated by specific receptor sites for released adenosine (Ado); A1 receptors exert an inhibitory control on purine release while A2 receptors facilitate it. PG release appears to be related to A1-mediated Ado activity, since culture treatment with 1 x 10(-10) M 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) or 1 x 10(-4) M N-ethylmaleimide (NEM), A1 receptor inhibitory agents able to increase purine release, induced a significant reduction of the evoked PG outflow. Purine amount, released from glial cells with inhibited PLA2-PG system, was remarkably greater than that one assayed from control cultured cells. In so treated cultures, no additive effect, NEM-induced, was detected, while the addition of a mixture of PGs partially reduced the increased purine outflow. An electrically evoked cAMP accumulation, significantly greater than that found in controls, was even detected in cultured cells with inhibited PLA2-PG system. Since 10 micrograms/ml adenosine deaminase (ADA) reduced while DPCPX enhanced the evoked cAMP accumulation, it seems partially due to released Ado and accounts for a prevalent A2-stimulating rather than an A1-inhibitory control on adenylate cyclase activity. Thus, in cultured glial cells, the PLA2-PG system, likely linked to A1 receptor sites, concurs to control purine release and seems to affect less directly cAMP accumulation.

Cultures of glial cells release purines under field electrical stimulation: the possible ionic mechanisms.

Dissociated primary cultures of glial cells released a remarkable amount of purines, at rest and during field electrical stimulation. The HPLC identification of labelled compounds derived from 3H-Adenosine (3H-Ado) (employed to preload the cultures) indicated that nucleotides and nucleosides were represented in the superfusate in equivalent proportions (43.86% and 56.14% respectively). Very much higher amounts of unlabelled purines prevalently constituted by nucleotides compounds (91.10%) were also released and detectable in the superfusate. In all the experimental conditions their evoked release did not result frequency-dependent. Since: a linear increase related to the stimulation frequencies was found for the released labelled compounds; no labelled purines were assayed in 5 x 10-5M Dipyridamole-treated cultures; any significant presence of labelled nucleotides, inosine and hypoxantine was not found in cultures simultaneously treated with 1 x 10-5M 2'-deoxycoformycin and 1 x 10-4M 1-(-5-isoquinolinsulfonyl)-2-methylpiperizine (H7) (3H-Ado amounts resulted more than doubled in these experimental conditions); labelled compounds have been assumed as tracers of a glial purine rate whose release can be connected to electrically-evoked action potentials. Purine outflow from glial cells is not sodium dependent, in fact TTX (5 x 10-7M) did not affect their basal or electrically-evoked release. A remarkable calcium-dependence was also evidentiated by the 1 x 10-4M Verapamil-induced inhibition of basal and evoked release. TEA (1 x 10-2M), a specific inhibitor of potassium efflux throughout calcium-mediated specific channels, strongly reduced the evoked purine outflow and any additive effect of its was not detectable when administered simultaneously to the calcium antagonist. These findings indicate that the frequency-dependent purine release from cultured glial cells is linked to ionic mechanisms, which calcium and potassium are mainly involved in.

Sodium and calcium dependence of purine release from rat cerebral cortical slices.

Electrically evoked purine release from rat cerebral cortical slices was evaluated, using a HPLC analysis combined with radioactivity measurement of the identified fractions. Two different pools of released purines have been identified: one probably related to cell metabolism and the other strictly linked to the nervous transmission. Since a linear increase, due to the stimulation frequencies, was found for the purines released from this second pool, a possible dependence on sodium and calcium transmembrane fluxes was evaluated. Pretreatment of the slices with TTX (5 x 10(-7) M) caused only a partial inhibitory effect on purine release (50%). This effect was probably related to the drug activity on the neuronal component of slices, since TTX induces an almost complete inhibition of purine release from isolated neurons "in cultures" and does not affect it from glial cells. Verapamil (1 x 10(-4) M), a calcium-channel blocker at glial and neuronal level, and TEA (3 x 10(-2) M), a specific inhibitor of calcium-mediated potassium efflux from glial cells, administered to the slices alone or in combination, showed a partial calcium-dependence of purine release. These results suggest a glial role in modulation of electrically-evoked purine release. These cells could exert a "buffering action" that regulates the calcium-mediated potassium availability, by which neuronal activity might be influenced.