Tunicamycin Inhibits Prostaglandin Receptor-Mediated. Phosphoinositide Hydrolysis in Cultured Rat Astrocytes. Jun-ichi Kitanaka, 1 Takako Hamano, Masayuki ...
Neurochemical Research, Vol. 19, No. 12, 1994, pp. 1545-1550
Tunicamycin Inhibits Prostaglandin Receptor-Mediated Phosphoinositide Hydrolysis in Cultured Rat Astrocytes Jun-ichi Kitanaka, 1 Takako Hamano, ~ Masayuki Gotoh, ~ Hitoshi Hashimoto, ~ and Akemichi Baba ~,2
(Accepted July 7, 1994)
Effect of tunicamycin, an inhibitor of N-linked glycosylation, on prostaglandin (PG) F~-stimulated phosphoinositide (PI) hydrolysis was examined in cultured rat astrocytes. Pretreatment of cultured astrocytes with tunicamycin (25-250 ng/ml) inhibited subsequent PGF2, (1 gM)-stimulated PI hydrolysis in concentration- and time-dependent manners. The inhibition completely recovered after removal of tunicamycin and re-incubation for 12 h. Tunicamycin pretreatment (100 ng/ml for 12 h) significantly blocked pS]methionine incorporation into cultured astrocytes, but cell viability was not affected under the condition. Inhibitors of processing of N-linked sugar chains such as bromoconduritol, 1-deoxymannojirimycin, and swainsonine had no effect on PI response to PGF~,. These observations suggest that PGF2~ receptor is N-linked glycosylated. KEY WORDS: Prostaglandin F2~; receptors; tunicamycin; N-Linked glycosylation; phosphoinositide hydrolysis; astrocytes.
INTRODUCTION
sites is essential for the receptor function, no information is available on the contribution of the sugar chains to the PGF2, receptor. In contrast, biochemical studies demonstrated that PGE receptors are N-glycosylated using exoglycosidases (7) and N-linked glycosylation inhibitors (8). In line with these observations, we examined the effect of tunicamycin on PGF2~-stimulated phosphoinositide (PI) hydrolysis in cultured rat astrocytes (9-11). Tunicamycin, a lipophilic analog of uridine diphosphateN-acetylglucosamine (UDP-GlcNAc), blocks the transfer of GlcNAc-I-P from UDP-GlcNAc to dolichyl-P to form dolichyl-PP-GlcNAc (12). As a result, the drug inhibits protein N-glycosylation (13). Thus, it is often used to assess the role of the sugar chains. The results reported here indicate that tunicamycin pretreatment inhibits PGF2,~-stimulated PI hydrolysis and suggest that PGF2~ receptor is N-glycosylated, similar to PGE receptors (7,8).
Prostaglandins (PGs) seem to exert their autocrine and paracrine actions through the specific receptors (1,2). To know the exact signal transduction mechanisms, it is necessary to reveal the structures of the receptors. Structures of PGF2~ receptors from mouse (3), bovine (4), and human (5) have been revealed by molecular cloning. We also cloned cDNA for the rat PGF2~ receptor from cultured astrocytes (6). Based on the results, two potential N-linked glycosylation sites are conserved in the N-termini of PGFz~ receptors among the different species. Although the observation strongly suggests that the addition of sugar chains to the conserved 1 Department of Pharmacology, Faculty of Pharmaceutical Sciences, Osaka University, Suita, Japan. 2 Address reprint requests to: Dr. Akemichi Baba, Department of Pharmacology, Faculty of Pharmaceutical Sciences, Osaka University, 16 Yamadaoka, Suita, Osaka 565, Japan.
1545 0364-3190/94/1200-1545507.00/0 9 1994PlenumPublishingCorporatioa
1546 EXPERIMENTAL
Kitanaka, Hamano, Gotoh, Hashimoto, and Baba PROCEDURE
Materials. myo-[2-3H]Inositol with PT6-271 polymer (707 GBq/ retool) was purchased from Amersham (Tokyo, Japan). L[~sS]Methionine (40.37 TBq/mmol) was from ICN Biomedicals, Inc. (Irvine, CA, U.S.A.). PGF~, was from Funakoshi Pharmaceuticals (Tokyo, Japan). PGF2,, was made up as a stock solution in 100% ethanol and kept in the dark at -200 under a nitrogen atmosphere until use. Final ethanol concentration in the assay medium was less than 0.1% (vol/vol), which did not have any effects on PI hydrolysis. Carbachol, norepinephrine, A23187, cycloheximide, endothelin-1, ATP, bromoconduritol, swainsonine, 1-deoxymannojirimycin, D-glucosamine, and 3-(4,5-dimethylthiazoyl.2-yl)-2,5-diphenyltetrazolium bromide (MTF) were from Sigma Chemical Co. (St. Louis, MO, U.S.A.). All other chemicals used were of the highest purity available. Cell Culture. Astroglial (type-l) (14) cultures were prepared from cerbral cortices of 1-day-old Sprague-Dawley rats as described elsewhere (9,11). Briefly, the enzymatically dissociated brain cells in culture medium [Eagle's minimum essential medium (MEM, Nissui Pharmaceutical Co., Ltd., Tokyo, Japan)] supplemented with 2 mM Lglutamine and 10% fetal calf serum (JRH Bioscienees, Lenexa, KS, U.S.A.) were grown in tissue culture flasks (75 cm2, Coming). After two weeks, oligodendrocytes were removed from the cultures by vigorously shaking the flasks overnight. The cells were trypsinized and grown in Coming 12-wetl plate in 2-3 weeks. The cells were maintained at 37~ in a water-saturated atmosphere of 95% air and 5% CO2, and culture medium was renewed every 3-4 days. At this stage, the morphology was fiat and polygonally shaped. Immunocytochemical staining for glial fibrillary acidic protein demonstrated that these cells were composed of >90% astroeytes (9,11). Phosphoinositide Hydrolysis Assay. Confluent cultures were incubated with myo-[~H]inositol (37 kBq/well) in a culture medium for 24 h, washed three times with lithium-Krebs-Ringer bicarbonate buffer (LiKRB) containing (millimolar) LiC1 10, NaCI 110, KC1 5.5, CaCI2 2.5, MgCI2 1.2, KH2PO4 1.2, NaHC03 20 and D-glucose 11 (pH 7.4), and then incubated with 1 ml of fresh LiKRB at 37~ for 20 rain. For drug pretreatment, drugs were generally added to the culture medium after incubation of the cells with myo-[3H]inositol for 12 h unless otherwise indicated. The control ceils were pretreated with the vehicle in parallel with the dmg-pretreated cells. After 24 h of labeling period, both control and drug-treated cells were rinsed three times with assay buffer. The cells were exposed to agonists in the fresh LiKRB at 37~ for 15 rain. Accumulation of inositol phosphates (IPs) was measured by using Bio-Rad AGI• column chromatography according to the method of Berridge et at. (15) as described previously (11). Inositol phosphate production (% hydrolysis) was calculated as described previously (11) using the following formula: [3H]IPs released (% total amount of [3H] inositol incorporated into the cells) = (dpm in IPs • 100)/(dpm in IPs + dpm in organic fraction), where [3H]IPs is corresponded to [~H]IP1, since the amount of [aH]IP2 plus [3H]IP3 was less than 3% of that of [3H]IP1 (9). [3sS]Methionine Incorporation. Cultured cells were treated with tunicamycin (100 ng/ml) for 8 h, and then coincubated with 3.7 kBq/ ml of [35S]methionine and tunicamycin for 4 h. In control cultures, vehicle was added to the culture medium instead of tunicamycin. The incubation was stopped by removing the assay buffer and immediately washing three times with 4 ml of ice-cold 0.9% NaC1. The cells were extracted for 30 min with 5% trichloroacetic acid (TCA) (wt/vol) at 4~ and washed twice with 95% ethanol (vol/vol). The TCA-insoluble material was extracted with 0.2 N NaOH containing 2% NazCOa for 24 h at 37~ Aliquots of the extracts were counted for radioactivity in scintillation fluid after addition of the same volume of 0,2 N HC1.
The remaining extracts were used for protein content determination by the method of Lowry et al. (16). Lipid Separation. myo-[3H]Inositol-labeled cultures were treated as described for PI hydrolysis assay. Organic phases were pooled and lipids were separated using thin layer chromatography (TLC) plates (silica gel 60, Merck) by the method of Shaikh and Palmer (17) with slight modifications. Briefly, lipid solutions were dried up under nitrogen and dissolved in 25 btl chloroform-methanol-water (20:9:1, by vol). The samples were applied to TLC plates by microsyringe as a small spot and the plates were developed in chloroform-acetone-methanol-acetic acid-water (40:15:13:12:8, by vol). The chromatograms were exposed to iodine vapor and spots corresponded to phosphatidylinositol 4,5-bisphospbate (PIP2) (Rf value of 0.36) were scraped from the plates. The gels were extracted in chloroform-methanol (1:2, vol/vol) and then counted for radioactivity in scintillation fluid. Cell Viability. Cell viability was estimated by MTT assay and lactate dehydrogenase (LDH) release assay. MTT assay was performed according to the method of Mosmann (18). To determine LDH release, the culture medium was removed and analyzed for LDH activity by the method of Wr6blewski and LaDue (19). Total cellular LDH activity (100% release) was determined after addition of 0.1% Triton X100 to the culture medium. Statistical Analysis. Data are presented as the mean -4- SEM of indicated numbers of experiments. Mean values were compared with Student's t test. Differences were considered statistically significant at P
