Journal ofNeurochernis1r.v Raven Press, Ltd., New York 0 1990 International Society for Neurochemistry Rapid Communication Evidence for an Astrocyte-Derived Vasorelaxing Factor with Properties Similar to Nitric Oxide Sean Murphy, "Robert L. Minor, Jr., Greg Welk, and "David G. Harrison Departments ojPharmucology and *Internal Medicine, University of Iowa College of Medicine, Iowa City, Iowu, U.S.A. Abstract: To determine whether astrocytes release nonprostanoid vasodilators, cells on microcamer beads were superfused with various agents in the presence of indomethacin, and the effluent was bioas- sayed and also analyzed for nitric oxide by a chemiluminescence technique. Bradykinin and A23187 induced release of a factor that relaxed arterial rings, an effect that was blocked by hemoglobin. The effluent contained either nitric oxide or a related compound that could be reduced to nitric oxide. Production of this factor was com- petitively inhibited by the arginine analogs fl-nitro-L-arginine and p-methyl-L-arginine and could be restored with L-arginine. Quis- qualate and norepinephrine were also effective in causing the release of nitric oxide from astroglial cells. Thus, astrocyte-derivedrelaxing factor has properties similar to those of an endothelium-and neuron- derived relaxing factor. Key Words: Astrocyte-Nitric oxide-Va- sorelaxant-Bradykinin-Arginine-Cerebrum. Murphy S. et al. Evidence for an astrocyte-derived vasorelaxing factor with properties similar to nitric oxide. J. Neurochem. 55, 349-35 1 (1990). Astroglial cells display various surface receptors and per- form important and varied roles in the brain (Murphy and Pearce, 1987). Astrocytes ale intimately associated with neu- rons and, via specialized end-feet, with cerebral blood vessels. Their ability to synthesize and release neuroactive peptides (Shinoda et al., 1989), amino acids (Dutton and Philibert, 1990), and eicosanoids (Murphy et al., 1988) raises the pos- sibility that astrocytes may be involved in bidirectional cell signaling processes in the CNS. Vascular endothelium (Ignarro, 1989) and various other cells, including neurons (Garthwaite et al., 1988, 1989a,b; Bredt and Snyder, 1989), release nonprostanoid, labile va- sodilator factors that have been termed endothelium-derived relaxing factors (EDRFs) and EDRF-related substances. The precise chemical nature of these EDRFs is not certain, but one of them is nitric oxide (NO) (Palmer et al., 1987) or a compound containing an NO moiety (Myers et al., 1989, 1990). Here, we show that astrocytes are a potential source in the CNS of such nonprostanoid factors possessing vaso- dilator activity. MATERIALS AND METHODS Primary cultures of astrocytes from I-day-old rat forebrain were prepared and maintained as previously described (Pearce et al., 1986~). At confluence (1 8 days in vitro; 95% glial fi- brillary acidic protein-positive polygonal astrocytes), cells in TI 50 flasks were resuspended and combined with microcar- rier beads (Cytodex 2; Sigma; - 10' cells/g of beads). After a further 5 days in culture in a spinner flask, the beads were pelleted, washed, resuspended in a small volume of Krebs buffer, and then transferred to the holding chamber for su- perfusion studies (-2 X lo7 cells). Immunocytochemistry of the cells on beads revealed only glial fibrillary acidic protein- positive cells to be present. The bioassay to detect vasorelaxants was essentially that described previously (Myers et al., 1989). Astrocytes on mi- crocamer beads were continuously perfused at 4 ml/min with Krebs buffer aerated with 95% 02/5% C02 and maintained at 37°C. Indomethacin (I pM) was added to the Krebs buffer to block cyclooxygenase activity. Vascular rings were prepared from proximal left circumflex coronary arteries of pigs, de- nuded of endothelium, and mounted on two wire stirrups, one of which was immobile and the other connected to a force transducer (Gould FT03C) for measurement of iso- metric tension development. After the detector ring was stretched to an optimal tension, further preconstricted tension (3.7 * 0.3 g; n = 5) was obtained with prostaglandin FZm (10-7-10-6 M). Denudation was verified by the absence of relaxation to direct application of bradykinin (BK; M), and these vascular rings were insensitive to A23 187. Effluent from the cells initially superfused the detector vessel for re- cording of bioassay responses. At the point of maximal re- laxation, effluent was assayed for nitrogen oxides. Transit times for cell effluent to the bioassay detector ring and the chemiluminescence apparatus were identical (3 s). For the assay of NO and one-electron oxidation products of NO, cell efhent was directed to a collection chamber containing 2% sodium iodide in glacial acetic acid under reflux. NO recov- ered from this reduction preprocessing was then transported in a stream of nitrogen gas under negative pressure into the NO analyzer (model 2108; Dasibi) and exposed to ozone (Myers et al., 1989, 1990). RESULTS AND DISCUSSION BK evoked the release from astroglial cells of a vasorelaxant (Fig. 1 A), which we have termed astrocyte-derived relaxing factor (ADRF). To characterize this factor, a chemilumines- cence assay for NO was used. The assay detects either NO or compounds one oxidation state higher than NO, e.g., nitrite Received April 4, 1990; accepted April 5, 1990. Address correspondenceand reprint requests to Dr. S. Murphy at Department of Pharmacology, BSB, University of Iowa College of Medicine, Iowa City, IA 52242, U.S.A. Abbreviations used: ADRF, astrocyte-derived relaxing factor; BK, bradykinin; EDRF, endothelium-derived relaxing factor; L-NA, p- nitro-i-arginine;NMDA, N-methyl-Baspartate; NMMA, NF-methyl- L-arginine; NO, nitric oxide. 349