UTP activates multiple second messenger systems in cultured rat astrocytes

Neuroscience Letters, 162 (1993) 105 108 © 1993 Elsevier Scientific Publishers lreland Ltd. All rights reserved 0304-3940/93/$ 06.00

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UTP activates multiple second messenger systems in cultured rat astrocytes G r e t c h e n Bruner, Sean M u r p h y * Department of Pharmacology, College of Medicine, UniversiO, {~flowa, Iowa City, IA 52242, USA (Received 29 May 1993; Revised version received 3 August 1993; Accepted 3 August 1993)

Kuy words: Astrocyte; ATP: UTP: Purinergic; Eicosanoid; Inositol phosphate In astrocytes, a number of second messenger systems are activated upon stimulation with ATE Recently, UTP has been demonstrated to have effects similar to ATP in some cell types. To determine if this was also true in astrocytes, cultured cells were stimulated with UTP which was lbund to evoke thromboxane release, stimulate inositol phospholipid turnover and increase intracellular free calcium concentration. A 53 kDa protein was identified on astrocyte membranes by immunoblotting with an antibody raised against a putative rat fibroblast P2~ receptor. These data indicate that astrocytes possess a UTP-sensitive receptor which may be distinct from the Pzv-purinergic receptor.

Astrocytes maintain close proximity to many other cell types in the central nervous system. Release of active compounds by surrounding cells can affect astrocyte cell function, and astrocytes in culture express receptors for a number of neurotransmitters and neuromodulators which are functionally coupled to a variety of second messenger systems. Among these active agents are the purines ATP and ADP. The idea that ATP can act as an intercellular messenger has become widely accepted, and purinergic receptors have been described in a variety of tissues [3]. Four categories of P2-purinergic receptors have been proposed based on the relative ability of ATP analogs to elicit the response of interest [2]. However, it is becoming clear that these categories are somewhat limiting and that in some tissues, responses to ATP analogs do not fit into the established receptor subtypes [17]. ATP stimulates inositol phospholipid turnover, evokes eicosanoid release and increases free intracellular calcium ([Ca2+]i) in cultured cortical rat astrocytes [1, 5, 8, 18]. Based on the current classification system, these responses are thought to be via activation of a P2v-purinergic receptor. However, we have demonstrated that phospholipase A 2 and phospholipase C are independently coupled to the purinergic receptor [1], and a possible explanation for this is that two distinct receptors are i,lvolved. Recently, UTP has been demonstrated to have receptor-mediated effects which are similar to those seen *Corresponding author. Fax: (I) (319) 335-7958.

with ATP [11, 13, 16]. To determine if UTP alters astrocyte function we investigated its effects on eicosanoid production, inositol phosphate accumulation and changes in [Ca2+]i. Primary astrocyte cultures were derived from the cerebral cortex of neonatal rats as previously described [14]. The cells were grown in large T-flasks in Eagle's minimal essential medium supplemented with 22 mM glucose, 2 mM glutamine, 50/lg/ml gentamicin, and 10% (v/v) fetal bovine serum (EMEM) with medium changes every 3-4 days until confluent (14 DIV). The cells were then subcultured by mild trypsinization into multiwell plates and used 5 to 7 days later. These cultures contained > 95% glial fibrillary acidic protein-positive cells. Cultures were switched to serum-free EMEM 18 24 h prior to experiments, and, in indicated studies, 100 ng/ml pertussis toxin (PTx) was added at this time. Eicosanoid production was assessed by determination of thromboxane (TX) release. Confluent cultures in 12well plates were washed and incubated in 0.5 ml Earle's balanced salt solution supplemented with 20 mM glucose and then stimulated in fresh medium with agonists or vehicle for 30 min at 37°C. The medium was removed and assayed for TX by radioimmune assay as previously described [15]. UTP stimulated TX production in cultured astrocytes in a concentration-dependent manner with an ECs0 of approximately 50 stM (Fig. 1A). This is similar to the response seen with ATP [18]. UTP-stimulated TX production could be completely inhibited by pre-treatment

106 o f the cells with 100 ng/tnl PTx (111 + 11% inhibition, n = 4 at 500 a M U T P ) , a c o n c e n t r a t i o n o f P T x which also c o m p l e t e l y inhibits A T P - e v o k e d T X release [1]. C o n c u r r e n t s t i m u l a t i o n with m a x i m a l c o n c e n t r a t i o n s o f A T P a n d U T P (500 a M each) h a d n o a d d i t i v e effect on T X p r o d u c t i o n ( d a t a n o t shown). I n o s i t o l p h o s p h o l i p i d t u r n o v e r was d e t e r m i n e d as described p r e v i o u s l y [18]. Briefly, cultures in 6-well plates

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Fig. 2. Additive effects of ATP and UTP on InsP accumulation. Astrocytes labeled with [3H]inositol were stimulated with ATP 500/IM. UTP 500/tM or a combination of both for 30 min in the presence of 5 mM LiCI. After removal of the medium, total lnsPs were determined as described in the text. Results are expressed as a percentage of basal InsP values and are the average + S.E,M. of 4 observations. Basal lnsP accumulation was 181 + 25 cpm Aq/10,000 cpm lipid, n ,-- 4. **Significantly different from ATP or UTP alone based on analysis of ~.~triancefollowed by Tukey test, P < 0.05.

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Fig. 1. Concentration dependent effects of UTP. A: astrocyte cultures were stimulated with UTP for 30 min and the medium removed and assayed for TX by RIA. Results are expressed as percent basal release and are the average _+S.E.M. of 4 observations. Basal TX release was 64 + 3 pg/well, n = 4. B: astrocytes labeled with [3H]inositolwere stimulated with UTP for 30 min in the presence of 5 mM LiCI. After removal of the medium, total InsPs were determined as described in text. Results are expressed as a percentage of basal InsP values and are the average + S.E.M. of 4 observations. Basal InsP accumulation was 176 + 13 cpm Aq/10,000 cpm lipid, n = 4.

were l a b e l e d for 18-24 h in serum-free E M E M c o n t a i n ing 1 a C i / m l myo-[2-3H]inositol (20 C i / m m o l , A m e r sham, A r l i n g t o n Heights, IL), w a s h e d with p h y s i o l o g i c a l saline s o l u t i o n a n d p r e i n c u b a t e d for 15 min in fresh solution c o n t a i n i n g 5 m M LiCI a n d then s t i m u l a t e d with agonists o r vehicle for 30 min. I n c u b a t i o n s were then termin a t e d with the r e m o v a l o f the m e d i u m a n d a d d i t i o n o f 0.7 ml ice-cold m e t h a n o l . T h e cells were harvested a n d the [3H]inositol p h o s p h a t e s (InsP) were e x t r a c t e d a n d s e p a r a t e d b y ion exchange c h r o m a t o g r a p h y . T h e 3H rec o v e r e d in I n s P ( a q u e o u s phase) was s t a n d a r d i z e d to the [3H]inositol i n c o r p o r a t i o n into cell lipids. PTx p r e - t r e a t m e n t d i d n o t affect lipid i n c o r p o r a t i o n o f [3H]inosito]. U T P h a d little if a n y effect on I n s P a c c u m u l a t i o n except at very high c o n c e n t r a t i o n s (Fig. 1B): This is in contrast to A T P w h e r e the ECs0 is a p p r o x i m a t e l y 100 a M [18]. P T x p r e t r e a t m e n t (100 ng/ml) did n o t significantly a t t e n u a t e U T P s t i m u l a t i o n (500 a M ) o f I n s P f o r m a t i o n unlike t h a t seen with A T P s t i m u l a t i o n [1]. A d d i t i o n a l l y , A T P a n d U T P at m a x i m a l c o n c e n t r a t i o n s were additive in their effects o n I n s P p r o d u c t i o n (Fig. 2). To d e t e r m i n e U T P - e v o k e d changes in [Ca2+]i, astrocytes g r o w n o n glass coverslips were l o a d e d w i t h 2 a M F u r a - 2 - a c e t o x y m e t h y l ester in serum,free E M E M cont a i n i n g 0.1% b o v i n e s e r u m a l b u m i n for 60 min at 370C a n d a l l o w e d to m e t a b o l i z e the ester in m e d i u m alone for 45 m i n at 37°C. T h e cells were then w a s h e d with H a n k ' s b a l a n c e d salt s o l u t i o n ( p H 7.2) a n d m a i n t a i n e d at 25°C

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Fig. 3. Change in [Ca~+]~ in response to UTP. Astrocyte cultures grown on glass coverslips and loaded with Fura-2 were stimulated with 100 HM U T P and the change in [Ca2+]1 was determined as described in text. U T P was added at arrow. Tracing is representative of the response seen with three separate coverslips. Average increase in [Ca-~+]~ was 260_+ 81 nM, n = 3.

until use. Changes in the ratio of 505 nm emission at 340 and 380 nm excitation of the cells were measured in a SPEX ARCU/COM-2-A. Values for [Ca2+]~ were determined using the method of Grynkiewicz et al. [7] based on saturating and zero calcium conditions. U T P stimulated an increase in [CaZ+]i (Fig. 3) in a concentrationdependent manner which was not inhibited by PTx pretreatment (data not shown), results which parallel those seen with ATP stimulation [1]. To provide additional evidence for a U T P receptor in astrocytes, immunoblotting of astrocyte membranes was performed using antiserum to a 53 kDa protein described to be a P_~t, receptor in 3T6 fibroblasts [4]. Astrocytederived membrane proteins were prepared from cells grown in a T- 150 culture flask. Cells were lysed with 1 ml of 10 mM Tris buffer pH 8.0 containing 0.5 mM phenyl methyl sultbnyl fluoride. The lysed cells were collected and homogenized with 5 strokes of a glass-teflon homogenizer. After centrifugation at 25,000 x g for 20 min, the supernatant was removed and the pellet was resuspended in 200/,tl of lysis buffer, and 200/,tl of sample buffer containing SDS and bromophenol blue was added and the sample was boiled for 10 min. The final concentration of protein was approximately 2.5 /lg//A. Approximately 75/lg of protein per lane was then loaded onto a 7.5% polyacrylamide gel and separated according to the method of Laemmli [10] and then transferred to nitrocellulose for immunoblotting analysis. After blocking with 0.5% milk, alternating lanes were incubated with rabbit preimmune serum or antiserum to the putative P2u receptor. The antibody is non-inhibitory and identifies the

glyco-portion of the antigen [4]. A secondary goat antirabbit antibody conjugated to alkaline phosphatase was used to determine proteins cross-reacting to the primary antibody. A band at approximately 59 kDa was seen in both the lane incubated with preimmune serum and with the P2t antibody (Fig. 4). In addition, two bands were seen in the lane probed with the antibody which were not visible in the preimmune lane. One band corresponded to a protein at approximately 53 kDa. The other appeared at approximately 110 kDa. The larger molecular weight band is not detected by the antibody in 3T6 cells, but it has been seen in some other cell lines and is not merely a dimeric form of the 53 kDa protein (Dr. Laurie Erb, personal communication). The antibody, therefore, may be crossreacting with two different nucleotide receptors in astrocytes, but whether the receptor(s) is/are coupled A

B -

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21.5 14.3 Fig. 4. I m m u n o b l o t of a putative P2~, receptor in astrocyte membranes. Astrocyte m e m b r a n e proteins (75 ,ug per lane) were separated onto a 7.5% polyacrylamide gel and transferred to nitrocellulose where they were probed with either an antibody to a 53 kDa purinergic receptor (A), or with preimmune serum (B). Molecular weights in kDa arc indicated at the right. Arrowheads indicate l ltl kDa and 59 kDa proteins. Arrow indicates 53 kDa putative P~t, receptor protein. Blot is representative of results seen in three separate membrane preparations.

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to eicosanoid release or inositol phospholipid turnover (or both) is unknown. Evidence for ATP as an extracellular signaling molecule is growing. However, determination of the particular receptor subclass at which ATP is acting has proved problematic. Although purinergic analogs are helpful in determining potential receptor classes, discrepancies still remain in the classification system [17]. Without selective antagonists to specific receptor subclasses, positive identification remains equivocal. To further complicate the classification system, UTP has now been described to have extracellular signaling effects [ 11, 13, 16] which may or may not be due to activation of the same receptors at which ATP acts. The study of UTP as an extracellular agent is just now beginning. Hence, its physiological significance as a cell signaling molecule is not well defined. High concentrations of uridine nucleotides are found in platelet storage granules [6] and in brain and kidney [9], but the conditions under which UTP might be released into the extracellular space is still unknown. Clearly, as more cells are demonstrated to respond to extracellular UTP, these conditions will need to be determined. In astrocytes, ATP stimulates a number of second messenger systems, but it has not been determined whether these effects are due to one type of receptor coupled to multiple second messenger pathways, or separate receptors each with its own effector system. The studies with UTP suggest that activation of PLA2 and PLC may indeed be via two distinct receptor subtypes. First, the concentration dependencies of UTP on TX and InsP accumulation are different. Secondly, UTP and ATP are additive with respect to InsP accumulation whereas they are not in regard to TX production. This suggests that a nucleotide receptor in addition to a PzY receptor may indeed be expressed in cultured astrocytes. The recent cloning of the P2u [12] and P2Y [19] receptors will aid in the investigation of such a possibility. We wish to thank Dr. Gary A. Weisman and Dr. Laurie Erb (University of Missouri) for the gift of the P2u antibody and Nori Slonneger and Hsin Lee Lin for technical assistance. This work is supported in part by NIH Grant NS 24621. 1 Bruner, G. and Murphy, S., Purinergic P2v purinergic receptors on astrocytes are directly coupled to Phospholipase A~, Gila, 7 (1993) 219--224. 2 Burnstock, G., Purinergic mechanisms, Ann. NY Acad. Sci. 603 (1990) 1-8.

3 EI-Moatassim, C., Dornand, J. and Mani, .I.-~ . [:xtracellular A l P and cell signalling, Biochim. Biophys. Acta, 1t 34 il992) 31 4 5 4 Erb, L., Hoover, R.K. and Weisman, G.A. Identification of cell surface, nucleotide-binding proteins in mouse fibroblasts using BzATP and BzUTP: correlation with P~ receptor activity, hat. 3. Purine Pyrimidine Res., 3 (1992) 74. 5 Gebicke-Haerter, P. J., Wurster, S., Schobert, A. and Hertting, G,, Pz-purinoceptor induced prostaglandin synthesis in primary rat astrocyte cultures, Arch. Pharmacol., 338 (1988) 704 707. 6 Goetz, U., Da Prada, M. and Pletscher, A. Adenine-. guanine-, and uridine-5'-phosphonucleotides in blood platelets and storage organelles of various species, J. Pharmacol. Exp. Ther., 178 (1971) 210 215. 7 Grynkiewicz, G., Poenie, M. and Tsien, R.Y.. A new generation of Ca -'+ indicators with greatly improved fluorescence properties, J. Biol. Chem., 260 (1985) 3440 3450. 8 Kastritsis, CH.C., Salm, A.K. and McCarthy, K., Stimulation of the P2v purinergic receptor on type 1 astroglia results in inositol phosphate formation and calcium mobilization, J. Neurochem., 58 (1992) 1277 1284. 9 Keppler, D., Rudigier, J. and Decker, K. Enzymatic determination of uracil nucleotides in tissues, Anal. Biochem., 38 (1970) 105-114. 10 Laemmli, U.K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature, 227 (1970)680-685. I 1 Lustig, K.D., Sportiello, M.G., Erb, L. and Weisman, G.A., A nucleotide receptor in vascular endothelial cells is specifically activated by the fully ionized forms of ATP and UTP, Biochem. J , 284 (1992) 733-739. 12 Lustig, K.D., Shiau, A.K., Brake, A.J. and Julius, D. Expression cloning of an ATP receptor from mouse neuroblastoma cells, Proc. Natl. Acad. Sci. USA, 90 (1993) 5113-5117. 13 Motte, S., Pirotton, S. and Boeynaems, J.M., Heterogeneity of ATP receptors in aortic endothelial cells: Involvement of P2Y and P2~ receptors in inositol phosphate response, Circ. Res., 72 (1993) 504~ 510. 14 Murphy, S., Generation of astrocyte cultures from normal and neoplastic central nervous system. In RM. Conn (Ed.), Methods in Neurosciences, Vol. 2, Academic Press, Orlando, 1990, pp. 33:47. 15 Murphy, S., Welk, G. and Thwin, S.S., Stimulation of thromboxane release from primary cell cultures derived from human astrocytic glioma biopsies, Glia, 3 (19901)205--211. 16 Needham, L., Cusack, N.J., Pearson, J.D. and Gordon, J.L., Characteristics of the P2 purinoceptor that mediates prostacyclin production by pig aortic endothelial cells, Eur. J. Pharrnacol., 134 (1987) I99 209. t 7 O'Connor, S.E., Dainty, I.A. and Left P., Further subclassification of ATP receptors based on agonist studies, Trends Pharmacol. Sci., 12 (1991) t37 141. 18 Pearce, B., Murphy, S., Jeremy, J., Morrow, C. and Dandona, E, ATP-evoked Ca 2+ mobilization and prostanoid release from astrocytes: Pz-purinergic receptors linked to phosph0inositide hydrolysis, J. Neurochem., 52 (1989) 971 977. 19 Webb, T.A., Simon, J., Krishek, B.J., Bateson, A.N, Smart, T:G., King, B.F., Burnstock, G. and Barnard, E.A., Cloning and functional expression of a brain G-protein-coupled ATP receptor, FEBS Lett., 324 (1993) 219 225.