Neurochcm. 1#ll. Vol. 21, No. 4, pp. 573 579. 1992

Printed in Great Britain

0197+0186/9255.00+0.00 Pergamon Press Lid

R E G U L A T I O N OF B R A D Y K I N I N - I N D U C E D P H O S P H O I N O S I T I D E T U R N O V E R IN C U L T U R E D C E R E B E L L A R A S T R O C Y T E S : POSSIBLE ROLE OF PROTEIN KINASE C WAN-WAN LIN 1"2 a n d D E - M A W CHUANG I* t Section on Molecular Neurobiology, Biological Psychiatry Branch, NIMH, Bldg. 10, Rm 3N212, Bethesda, MD 20892, U.S.A. -~Department of Pharmacology, College of Medicine, National Taiwan University, Taipei, Taiwan ( Receiz'ed 16 Janua O' 1992 ; accepted 20 March 1992)

Abstract Phosphoinositide hydrolysis was studied in primary cultures of rat cerebellar astrocytes prelabeled with [3H]myo-inositol. Among the agonists examined, the rank order of efficacies in causing phosphoinositide hydrolysis was bradykinin > endothelin-I > ATP > norepinephrine. The bradykinin response was robust (24-1bid increase) with EC~,~ value of 30 nM and saturating concentration of 1 I~M. Preincubation of cells with pertussis toxin did not affect the activation of phosphoinositide turnover by bradykinin. Although short-term (within 90 min) treatment of cells with phorbol dibutyrate attenuated bradykinin-induced phosphoinositide breakdown, the inhibitory effect was lost after 3 6 h of phorbol dibutyrate treatment. Extended (24 h) preincubation resulted in a potentiation of bradykinin response. Homologous desensitization of bradykinin response was observed in cells prestimulated with bradykinin for up to 6 h. However, similar to the effect of phorbol dibutyrate, 24-h pretreatment with bradykinin selectively sensitized the response to bradykinin. Up-regulation of the bradykinin response was also observed in cells prestimulated with endothelin-1 or norepinephrine for 24 h, although these treatments resulted in only homologous desensitization to their own response. Our results suggest that cultured cerebellar astrocytes express bradykinin receptors coupled to phospholipase C and in these cells protein kinase C plays a more prominent role in the negative-feedback regulation of bradykinin-evoked phosphoinositide response.

Increasing evidence indicates that glial cells in the CNS are not merely a passive supporting substance. Instead, they interact with neurons extensively to modulate the neural circuits. Astrocytes in the brain have been found to possess a large variety of receptors for classical ncurotransmitters and neuropeptides (for reviews, see Murphy and Pearce, 1987; Pearce and Murphy, 1988; Hansson, 1988). Phosphoinositidc (PI) hydrolysis by phospholipase C in astrocytes is stimulated by many receptor agonists including norepinephrine (NE) (Pearce et al., 1986a), carbachol (Pearcc et al., 1986a), glutamate (Pearce et al., 1986b ; Nicoletti et al., 1990), A T P (Pearce et al., 1989), bradykinin (BK) (Cholewinski et al., 1988), platelet activating factor (Murphy and Welk, 1989), arachi-

donic acid (Murphy and Welk, 1989), angiotensin II (Lin ct al., 1990), substance P (Torrens et al., 1986; Cholewinski et al., 1988) and endothelin-I (ET-1) (Lin et al,, 1990; M a c C u m b e r et al., 1990). Thus, these phospholipase C-coupled receptors may be linked to some of the neurophysiological functions endowed to astrocytes. For example, ET-1 appears to stimulate mitogenesis in astrocytes and C~, glioma by a mechanism dependent on diacylglycerol formation ( M a c C u m b e r et al., 1990; Zhang et al., 1991). Most studies on receptor-mediated Pi turnover in astrocytes were conducted using preparations derived from a cerebral cortex. However, it should be noted that numerous reports have indicated regional variations in biochemical profiles of astroglia (Cholewinski et al., 1988; Cholewinski and Wilkin, 1988; El-Etr et al., 1989; Hansson, 1988; Wilkin et al., * Author to whom all correspondence should be addressed. 1990). In this context, Nicoletti et al. (1986) have Ahbre~'iations: BK, bradykinin; ET-I, endothelin-l; IP, reported that glutamate evokes a weak (10 20%) inositol phosphates ; NE, norepinephrine ; PDBu, phorincrease ofinositol phosphate (IP) production in cercbol 12,13-dibutyrate ; PI, phosphoinositide ; PKC, protein kinase C. bellar astrocyte cultures, whereas Pearce et al., 573

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1986b) showed t h a t g l u t a m a t e is a m u c h m o r e robust stimulus (3-fold) in cerebral astrocytes. F u r t h e r investigation o f this receptor-mediated activity is therefore w a r r a n t e d . In preliminary studies we f o u n d t h a t primary cultures o f cerebellar astrocytes express muliple types o f receptors coupled to p h o s p h o l i p a s e C, such as receptors for ET-1, angiotensin II, NE, ATP, neurotensin, c a r b a c h o l a n d g l u t a m a t e (Lin et al., 1990). In addition, dual time effects o f p h o r b o l esters o n these agonist responses were observed. S h o r t - t e r m (10 min) t r e a t m e n t with a p h o r b o l ester significantly reduced the responses to all agonists tested. However, long-term (24 h) p h o r b o l ester p r e t r e a t m e n t potentiated PI responses to ET-I a n d A T P , but this sensitization p h e n o m e n o n did n o t occur for angiotensin II, NE, n o r n e u r o t e n s i n - i n d u c e d effects. Here, we report for the first time t h a t bradykinin, the naturally occurring n o n a p e p t i d e , at physiological c o n c e n t r a t i o n s robustly stimulates PI t u r n o v e r in cultured cerebellar astrocytes. Moreover, this BKinduced effect is particularly susceptible to sensitizalion by persistent p r e t r e a t m e n t with a p h o r b o l ester a n d receptor agonists including BK, ET-1 a n d NE.

EXPERIMENTAL PROCEDURES

Materials ET-[ was purchased from Peptide Institute Inc. (Osaka, Japan). The following were purchased from Sigma Chemical Co. (St. Louis, MO): NE, BK, ATP, phorbol 12,13-dibutyrate (PDBu), and pertussis toxin. [3H]myo-inositol (16.5 Ci/mole) was a product of New England Nuclear (Boston, MA). Primary cultures of cerebellar astrocytes Glial cell cultures were prepared from the cerebellum of 8-day-old Sprague-Dawley rats as previously described (Lin et al., 1990). Briefly, cerebella were dissected and dissociated by mechanical chopping and trypsinization to obtain a cell suspension. Cells were plated at a density of 3 × 106 cells per 35 mm dish precoated with poly-L-lysine. Cultures were maintained in Basal Eagle's Medium plus 10% fetal calf serum, 2 mM glutamine and 50 ltg/ml gentamicin, which was changed twice weekly. The cells were grown in a humidified 5% CO2 atmosphere at 37°C, and after 10-12 days in vitro were used to study PI hydrolysis. Cultures prepared in this manner contained a confluent monolayer of flat polygonal glial cells of which more than 90% of the cell population stained positive for glial fibrillary acidic protein. Measurement o f P1 hydrolysis P1 hydrolysis was assessed by measuring accumulation of [3H]IP in cells prelabeled for 24 h with [3H]myo-inositol (2.5 #Ci/dish), as described in our previous publications (Chuang and Dillon-Carter, 1988; Lin et al., 1990). At the end of incubation, culture dishes were washed three times with 1 ml physiological salt solution containing 20 mM LiC1 and

incubated at 37°C for 45 min in the same buffer. The reaction was initiated by addition of indicated agonists and allowed to proceed for another 45 min at 37°C; ice-cold methanol was then added to terminate the reaction. The accumulation of [3H]IP was determined by an anion-exchange chromatography on an AG 1 x 8 column (Berridge et al., 1982). Basal values of [3H]IP were in the range of 1750 2400 DPM/dish. Drug pretreatment of cerebellar astrocytes PDBu (500 nM) or indicated concentration of a receptor agonist (BK, ET- I or NE) was added to the growth medium for variable periods of time during [3H]myo-inositol labeling of cells. Drugs were added sequentially to culture dishes so that all cells were harvested at 24 h after [3H]inositol addition. Cells in dishes were washed three times with 1 ml of physiological salt solution (Chuang and Dillon-Carter, 1988) and then stimulated with indicated agonists in the presence of lithium to induce the accumulation of [3H]IP. Data analysis Student's t-test was used for statistical evaluation. P < 0.05 was considered significant. The n value represents the number of triplicate experiments. RESULTS Agonist-induced P I turnover in cerebellar astrocytes Exposure o f p r i m a r y cultures of cerebellar astrocytes to BK, ET-1, A T P a n d N E resulted in a dosed e p e n d e n t increase in [3H]IP f o r m a t i o n with various efficacies a n d potencies (Fig. I), The ECs0 values for BK, E T - I , A T P a n d N E - i n d u c e d increase in IP form a t i o n were a p p r o x i m a t e l y 30 n M , 1.7 n M , 0.2 m M a n d 2/~M, respectively. The r a n k order o f efficacy in stimulating PI hydrolysis was B K > ET-I > A T P > NE. A t a s a t u r a t i o n c o n c e n t r a t i o n (1 #M), BKinduced IP a c c u m u l a t i o n was 2 3 7 7 _ 191% (n = 15) o f basal activity.

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Fig. 1. Concentration dependence of agonist-stimulated PI turnover in primary cultures of cerebellar astrocytes. Experimental conditions are as described in the methods. Each value represents the mean + SEM from at least 3 independent experiments; each was performed in triplicate. 0 - - 0 , BK (n = 5); O - - O , ET-1 (n = 4); A - - A , NE (n = 3); ~ - - A , ATP (n = 5). The value of basal (100%) [3H]IP was in the range of 1750 to 2150 DPM/dish.

Bradykinin-induced phosphoinositide turnover

Effects of PDBu and pertussis toxin on BK-induced PI response As shown in Fig. 2, the time course for modulation of PI response to 1 /~M B K by P D B u (500 nM) was clearly biphasic. Short-term (15 and 90 min) pretreatment with P D B u inhibited the PI response to BK by 43 + 7% (n = 3) and 18___2% (n = 3), respectively. At 3 and 6 h after pretreatment, the inhibitory effect of P D B u was lost and the BK response returned to its control value. However, with an extended P D B u preincubation time of 24 h, a marked potentiation (204+ 35%, n = 6) of the PI response occurred. This 24 h pretreatment with P D B u resulted in a concentration-dependent potentiation of the response to BK ( l / t M ) with a saturation concentration and ECs0 value of 30 and 1.4 nM, respectively (Fig. 3). P D B u pretreatment only weakly attenuated basal PI activity to 94_+6% (n = 3) and 8 2 + 4 % (n = 6) of the control after 15 min and 24 h, respectively, indicating that the potentiation observed at 24 h is not due to a decrease in basal activity. In another experiment, pretreatment of cells with pertussis toxin (500 ng/ml) for 24 h was found not to alter PI hydrolysis induced by BK (117_+6% of the control, n = 4, data not shown).

Desensitization and sensitization As shown in Fig. 4, BK-induced PI turnover was attenuated by prestimulation with BK (1 #M) for a time period ranging from 30 min to 6 h, whereas it was increased with extended prestimulation of 24 h. BK prestimulation for 30 min markedly increased basal activity, presumably due to residual [3H]IP gen-

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Fig. 3. Dose-dependent effects of PDBu on BK-induced [3H]IP formation. Cells were preincubated with indicated concentration of PDBu or vehicle (ethanol,