Prostanoid formation in primary astroglial cell cultures: Ca(2+)-dependency and stimulation by A 23187, melittin and phospholipases A(2) and C.
Prostaglandin (PG) and thromboxane B(2) (TXB(2)) biosynthesis was studied in cultured astrocytes from neonatal rat brain hemispheres. After two weeks of cultivation, prostanoids were formed with the spectrum: PGD(2) > TXB(2) > PGF(2?) > PGE(2), as measured by specific radioimmunoassays. Under basal conditions PGD(2) biosynthesis (9.55 ng/mg protein/15 min) was in the same order of magnitude as the sum of the other prostanoids. The formation of prostanoids was stimulated in a concentration dependent manner (up to 6-10 fold) by the calcium ionophore A 23187 (0.01-10 ?M) as well as by melittin (0.01-5 ?g/ml), phospholipase A(2) (10-40 U/ml) and phospholipase C (0.01-1 U/ml). Basal and evoked PG and TXB(2) biosynthesis depended on the availability of Ca(2+), as demonstrated in Ca(2+) free incubation medium containing Na(2)EDTA (1 ?M), or with verapamil (100 ?M) and 3,4,5-trimethoxybenzoic acid-8-(diethylamino)-octylester-HCl (TMB-8, 1-100 ?M). Indomethacin (10 ?M), mepacrine (100 ?M) and p-bromophenacylbromide (50 ? M) inhibited basal and evoked PG formation. Thin-layer chromatography (TLC) detection after incubation of the cells with [(3)H]arachidonic acid (1 ?Ci/ml, for 60 min) confirmed the results obtained by radioimmunoassay. Incubation of [(3)H]arachidonic acid labelled cells with inonophore or phospholipases, followed by lipid extraction and TLC, showed that A 23187 liberated [(3)H]arachidonic acid predominantly from phosphatidylethanolamine, whereas phospholipase A(2) and C reduced mainly the labelling of the phosphatidyl-inositol/-choline fraction. Potassium depolarization of the cells did not enhance prostanoid formation. Similarly, drugs with affinity to ?- or ?-adrenoceptors, or to dopamine-, 5-hydroxytryptamine-, muscarine-, histamine-, glutamate-, aspartate-, GABA, adenosine- and opioid-receptors failed to stimulate prostanoid biosynthesis. Also compounds like angiotensin, bradykinin and thrombin were ineffective in this respect. In conclusion, our results confirm that cultured astrocytes possess the complete pattern of enzymes necessary for prostanoid formation and hence might play a crucial role in brain prostanoid biosynthesis. Stimulation of prostanoid biosynthesis involves Ca(2+)-dependent activation of phospholipase A(2), cyclooxygenase reaction and further PG metabolism. However, the endogenous stimulus for enhanced prostanoid synthesis in the brain still has to be established.
Keller
M
M
Department of Pharmacology, University of Freiburg, Hermann-Herder-Str. 5, D-7800 Freiburg, F.R.G.
Seregi
A
A
Hertting
G
G
Jackisch
R
R