Neurochem. Int. Vol.21, No. 2, pp. 293-301, 1992 Printedin Great Britain

0197-0186/92$5.00+0.00 PergamonPress Ltd

POTENTIATION BY Ca 2+ IONOPHORES A N D INHIBITION BY EXTRACELLULAR KC1 OF E N D O T H E L I N - I N D U C E D PHOSPHOINOSITIDE T U R N O V E R IN C6 GLIOMA CELLS WAN-WAN LrN L2 a n d DE-MAW CHUANG1. ~Section on Molecular Neurobiology, BiologicalPsychiatry Branch, National Institute of Mental Health, Bldg I0, Rm 3N 2/2, Bethesda, MD 20892, U.S.A. -*Department of Pharmacology, College of Medicine, National Taiwan University, Taipei, Taiwan, Republic of China (Received 27 September 1991; accepted 22 December 1991)

Al~traet--Interactions between endothelin-I (ET)-induced phosphoinositide (PI) hydrolysis and agents that increase Ca-'+ influx (i.e. A23187 and ionomycin) or induce depolarization (i.e. KCI) were investigated using C6 glioma. A23187 dose-dependently potentiated ET (30 nM)- and ATP (100 #M)-induced [3H]inositol phosphate (IP) accumulation. This potentiation was associated with an increase in the maximal stimulation elicited by both ET and ATP but their ECs0 values were unchanged. This effect of A23187 occurred at concentrations that did not affect basal PI turnover; i.e. 10 nM-3/tM. Ionomycin within the range of 1 nM-I~M also significantlyenhanced ET-induced PI breakdown and this effect was associated with an increase of [Ca2+]~.KC1 in a concentration-dependent manner (14.7-54.7 mM) markedly inhibited PI breakdown elicited by ET and ATP, but had much less inhibition on basal activity and no effect on A23187- and ionomycin-induced responses. In parallel, KC1 added before or after ET, sharply attenuated the increase of ET-induced [Ca2÷], but did not affect basal level or ionomycin-induced [Ca2+]i response. Neither the potentiation by A23 t 87 nor the inhibition by KCI of ET-induced PI turnover was observed in cultured cerebellar astrocytes. Our results suggest that the cell type-specificregulation by Ca2÷ ionophores and KCI on ET-induced PI metabolism is closely related to perturbation of [Ca-'÷]i.

It is well established that stimulation of numerous types of receptors coupled to phospholipase C leads to an increased metabolism of phosphoinositide (PI) and that this membrane signalling pathway plays a fundamental role in a wide spectrum of physiological responses (for reviews, see Berridge, 1987; Chuang, 1989). There is increasing evidence to support a" stimulating effect of calcium on basal PI hydrolysis. Thus, Ca 2÷ ionophore such as A23187 and depolarizing agents such as K + and veratrine have been shown to promote inositol phosphate (IP) formation in brain preparations (Akhtar and Abdel-Latif, 1978 ; Griffin and Hawthorne, 1978; Fisher and Agranoff, 1981 ; Court et aL, 1986; Eva and Costa, 1986; Kendall and Nahorski, 1984; Eberhard and Holz, 1988; Diamant and Atlas, 1989). Emerging evidence also suggests that PI hydrolysis induced by receptor agonists, notably carbachol, can be enhanced by A23187 and high K + (Fisher and Agranoff, 1981 ; Court et al.,

1986; Eva and Costa, 1986; Kendall and Nahorski, 1987 ; Diamant and Atlas, 1989). However, the nature of the enhancement by these agents of receptormediated responses remains largely unknown. Endothelin-I (ET), a potent vasoconstrictor first isolated from endothelial cells, has been shown to be produced in a variety of cell types (for reviews, see Yanagisawa and Masaki, 1989a,b; De Gouville et al., 1989). ET receptors are also widely distributed in many tissues including the CNS and are coupled to PI hydrolysis by phospholipase C as well as influx of Ca 2+. In the established glial cell line C6 glioma and cultured astrocytes, these two ET receptor-mediated events may be involved in increased cell proliferation elicited by ET (Suppattapone et al., 1989; Lin et al., 1990 ; MacCumber et al., 1990 ; Marsault et al., 1990). In this study, we employed C6 glioma and primary cultures of cerebellar astrocytes to investigate effects of two calcium ionophores A23187 and ionomycin and a depolarizing agent KC1 on ET and other agonistinduced PI breakdown. Our results indicate that *Author to whom all correspondence should be addressed. these two groups of agents have opposite effects on 293

294

WAN-WAN LIN and DE-MAw CHUANG

ET-induced PI t u r n o v e r in C6 glioma but have no effect in astrocytes. Moreover, these differential effects appear to result from p e r t u r b a t i o n of intracellular free Ca 2+ level ([Ca2+]i).

EXPERIMENTAL

PROCEDURES

Materials [3H]myo-inositol (16.5 Ci/mmol) was purchased from New England Nuclear (Boston, MA). Dulbecco's Modified Eagle's Medium (DMEM), Basal Eagle's Medium, fetal calf serum were from Gibco (Grand Island, NY). ET was purchased from Peptide Institute Inc. (Osaka, Japan). A23187 and ionomycin were from Calbiochem (San Diego, CA). Fura-2 AM was from Molecular Probes (Eugene, OR). All other chemicals were products of Sigma Chemical Co. (St. Louis, MO). Cell culture Rat C6 glioma cells obtained from American Type Culture Collection (Rockville, MD), with a passage number of 39, were cultured in DMEM as previously described (Lin et aL, 1990). Cells with additional passages of 13 35 were subcultured into 35-mm dishes and assayed for PI turnover upon confluency (4 × 106 cells/dish). Primary cultures of astrocytes were prepared from rat brain cerebella, as previously described (Lin et al., 1990). In brief, cerebella from 8-day-old rats were cleaned of meninges, chopped into small cubes, and digested with trypsin. The dissociated cells were plated at a density of 3 × l06 cells/ 35-mm dish precoated with poly-L-lysine and cultured in Basal Eagle's Medium containing 10% fetal calf serum, 2 mM glutamine and 50 #g/ml gentamicin Culture media were changed twice a week and cells were used for experiments after 10- 12 days in culture. Measurement of phosphoinositide turnover Phosphoinositide hydrolysis was measured as the accumulation of [3H]inositol phosphate (IP) in the presence of LiCI in cells prelabeled overnight with [3H]myo-inositol, as described previously (Linet al., 1990). Stimulants or other testing agents were added to the cells in culture dishes preincubated in physiological saline solution (PSS) (118 mM NaCI, 4.7 mM KCI, 3.0 mM CaCI:, 1.2 mM MgC12, 1.2 mM KH2PO4, 0.5 mM EDTA, 10 mM glucose and 20 mM HEPES, pH 7.4) containing 20 mM LiC1. The reaction was allowed to proceed for 45 min at 37"C and then terminated by addition of ice-cold methanol. The accumulation of [3H]IP was determined by chromatography on an AG1 x 8 column (formate form, 100-200 mesh) eluted with 0.2 N ammonium formate/0.1 N formic acid. lntracellular Ca -'+ measurement Confluent cells grown in cover slips (9 × 35 mm 2) (1 x 106 cells/slip) were loaded with 5 #M Fura-2 and pluronic F-127 (0.25% v/v) in PSS at 37"C for 60 min. Slips were then placed in a continuously stirred thermostatic cuvette at 37°C in 2 ml PSS and fluorescence was monitored on a PTI Delta Scan spectrofluorometer with dual excitation wavelengths of 340 and 380 nm and emission wavelength of 510 nm. [Ca2+]~was calculated using the equation described by Grynkiewiez et

al. (1985). When used, testing agents were added at indicated time to the cuvette in 5 20 F~I. RESULTS

Enhancement of endothelin- I and A TP-induced phosphoinositide turnover by A23187 in C6.qlioma [3H]inositol p h o s p h a t e accumulation in C6 glioma prelabeled with [3H]inositol was increased by the calcium i o n o p h o r e A23187 in a c o n c e n t r a t i o n - d e p e n d e n t manner. The stimulation of IP p r o d u c t i o n induced by 3 a n d 10 # M A23187 was 1 6 8 + 3 8 % ( n = 4 ) and 366 + 6 1 % (n = 13) o f basal activity, respectively. The results in Fig. 1 show t h a t PI t u r n o v e r was markedly stimulated by 30 n M ET a n d 100 p M ATP. Moreover, the presence o f 10 /~M A23187 induced a m a r k e d p o t e n t i a t i o n of Pl responses to ET or ATP. In contrast, a mere additive effect was observed when 1 m M A T P was used in c o m b i n a t i o n with the ionophore. Figure 2(A) d e m o n s t r a t e s a conc e n t r a t i o n - d e p e n d e n t p o t e n t i a t i o n o f ET (30 n M ) induced PI t u r n o v e r by A23187 within the conc e n t r a t i o n of 10 n M - 1 0 #M. The ECs0 value of A23187 was approximately 0.3 ~tM. However, by

T* o E o 4OO0 t_)

D-A23'8' +A23187 (10uM~

f i []

Q.

"6 T

10013

100

--

ET ('30 nM)

ATP (IO0#M)

ATP (lmM)

Fig. 1. Effects of A23187 on basal and agonist-induced [3H]inositol phosphate accumulation in C6 glioma. Cells prelabeled with [3H]myo-inositol were stimulated with 10 #M A23187 in the absence or simultaneous presence of indicated concentrations of agonists for 45 min at 37°C~ Lithiumdependent accumulation of [3H]inositol phosphates was then measured, as described in Experimental Procedures. Data presented are mean+SEM of percentage of basal activity obtained from at least 3 independent triplicate experiments, as shown by the number on the top of each column. *, Synergistic effects observed when agonist was used in combination with A23187.

Ca2+, endothelin and phosphoinositide turnover 3000

295

3OOO

A°

o

+ET ~

"6 2000

2000 ÷A23187 [10~M)

Q.

"6 •~

looo

10130

100

i//

~

-El" ~~1 ~'

I~~IA23187 ,

loo

-~/

8

7

6

5

A23187(-LogM)

4

I

,oo 10 -

co

.4,9

I

I

8 7 ET(-LogM)

Fig. 2. Potentiation of ET-induced PI turnover by A23187 in C 6 glioma. (A) Concentration-dependent potentiation of ET (30 nM) response by A23187; (B) Effect of A23187 on concentration-dependent PI response to ET. Experimental conditions are as described in the Experimental Procedures and legend to Fig. 1. Data shown were mean ___SEM from 3 independent experiments, which were performed in triplicate. --I1--, theoretical additive values of [3H]IP accumulation measured in the co-presence of A23187 and ET.

increasing the concentration of A23187 to 30/~M, no significant potentiation was observed. A23187 did not significantly increase basal PI turnover until its concentration reached 10 #M. In the presence of 10 #M A23187, the potentiation of PI responses to ET was associated with an increase in the maximal extent of stimulation of IP accumulation with no apparent change in the ECso of ET (about 2 nM) [Fig. 2(B)]. The extent of increases was 40-75% above the values of theoretical additivity in the tested concentration range of 0.3-300 nM. Similar to effects on ET-induced responses, A23187 enhanced ATP-induced IP formation in a dose-dependent manner over the range of 10 nM-30 #M [Fig. 3(A)]. At 30 pM, the enhancement was approximately 74% above the theoretical additive value. The potentiation by 10 #M A23187 was observed when ATP-induced PI turnover was measured in the range of 10-300 #M, although no significant enhancement was found with 1 mM ATP [Fig. 3(B)]. The ECso of ATP was approximately 80 #M regardless of the presence or absence of the ionophore.

and 10 #M, the IP accumulation was 106___5% (n = 4) and 860-1-142% (n = 6) of basal level, respectively. As shown in Fig. 4, ET-induced PI response was potentiated by 1 #M ionomycin but was merely additive in the presence of 10 #M of this ionophore. In contrast, ionomycin at either 1 or 10 #M produced only additive effects with ATP (0.1 or 1 mM). Ionomycin, over the concentration range of 1 nM-1 #M, had no effect on basal PI hydrolysis, but dose-dependently potentiated ET-induced response up to 188% of the control (Fig. 5). The EC50 value of ionomycin for enhancing the ET response was about 0.1/aM. In order to relate potentiation of ET response to an increase in intracellular Ca 2÷, [Ca2+]i was measured in C6 glioma cells stimulated with ionomycin. At 0.01, 0.1, 1 and 10 #M of the ionophore, [Ca2+]~ was increased in a dose-dependent manner from 161 + 18 nM up to 1309+ 162 nM [Fig. 6(A) and (B)]. The [Ca2+]~ increase observed with 10 #M was longerlasting than that measured with lower concentrations of this ionophore.

Effects o f ionomycin on agonist-induced phosphoinositide response

Effects o f [K+]o on endothelin-l-induced phosphoinositide turnover and [ Ca2 +] i increase

In C6 glioma, ionomycin produced a greater and steeper stimulation of PI hydrolysis than A23187. At !

Application of external KC1 ranging from 14.7-54.7 mM inhibited ET- and ATP-induced IP formation in

296

WAN-WAN LIN and DE-MAW CHUANG

_A o

+ATP/

c:

o t.J

151)I)

0

0 ! "r

500

100

:a-c/ o o

-,r"~ .a_./~ ~

8 7 6 A23187 (-Log M )

5

~

5

-~23~7

i

J

4 AI"P (-Log M )

3

Fig. 3. Potentiation of ATP-induced P1 turnover by A23187 in C6 glioma. (A) Concentration-dependent potentiation by A23187 of ATP (100 #M) response. (B) Effect of A23187 on concentration-dependent PI response to ATR Experimental conditions are as described in the legend to Fig. 2. Data shown are mean + SEM of a triplicate experiment. Nearly identical results were obtained in another experiment. --I1--, theoretical additive values of [3H]IP accumulation measured in the co-presence of A23187 and ATP shown in (A).

a dose-dependent manner (Table 1). At 54.7 mM, the responses to ET and ATP were inhibited by 54 and 43°/'0, respectively, while ionomycin- and A23187induced PI turnover were unaffected. Basal IP formation was inhibited only by approximately 20% in the range of [K+]o tested. In parallel, [Ca2÷]~ increase elicited by ET was markedly attenuated by high K +, whereas basal [Ca2+]i was not significantly altered (Fig. 7). Application of 10, 30 and 50 mM KCI to cells when ET (10 nM)-induced [Ca2+]~ increase reached its sustained phase, resulted in an instantaneous decline of the Ca 2+ level to 5 6 + 1 % ( n = 4 ) , 30-t-2% ( n = 4 ) , and 12+6% (n = 3) of their control, respectively. Pretreatment of cells with 50 mM KCI (54.7 mM of final [K+]o) also attenuated ET-induced [Ca2+]~ increase from 387+34 nM (n = 18) to 148+23 nM (n = 3). High K ÷ had no apparent effect on the increase of [Ca2÷]i induced by ionomycin. Effects ofA23187 and high K + on endothelin-l-induced phosphoinositide turnover in cerebellar astrocytes When cerebellar astrocytes prepared from 8-day-old rats were exposed to 10 #M A23187, ET- and other

agonist (NE, ATP, angiotensin II, bradykinin and neurotensin)-induced PI hydrolysis were not potentiated (Table 2). In fact, less than additive values were observed in most cases. Moreover, in contrast to inhibition observed in glioma cells, administration of KC1 up to 54.7 mM had no significant effect on ETinduced IP formation (data not shown). DISCUSSION

The present results demonstrate that two calcium ionophores, A23187 and ionomycin, potentiate ETinduced P1 breakdown in C6 glioma cells. Under certain conditions, Ca 2+ ionophore-induced potentiation of PI response to ATP was also observed. Conversely, high potassium attenuated both ET-induced IP formation and increase of free cytosolic Ca 2+. Neither the effect of A23187 nor that of K ÷ on ET-induced response was observed using primary cultures of cerebellar astrocytes. Since ECs0 values of ET and ATP for inducing PI hydrolysis were unchanged by A23187, the potentiation is unlikely due to a modification of the receptors' sensitivity to their agonists. The dose-dependent increase by both ionophores of

Ca2+, endothelin and phosphoinositide turnover [~]--Ioe~mycin

297

2000

J'lTl'l'lo,nomycin IO'6M L~--~-J-lonomycin 10-SM

2000

3 u

1800 o

=

4

1600

+ET

4

u8 •~ 1400

1200

15oo

"6

~1000

4

4 i

soo =*-

800

I

E

100

400

~ I l/

9

~

- E?

X

I

I

I

I

8

7

6

5

Ionomycin {-Log M)

200 100 ET (30nM)

ATP

ATP

( 100 ~M)

(1m M

Fig. 4. Additivity of ionomycin- and agonist-induced PI turnover in C6 glioma. Prelabeled cells were stimulated with ionomycin in the absence, or simultaneous presence, of ET or ATP at indicated concentration for 45 min at 37°C. Data presented are mean __+SEM from at least 3 independent triplicated experiments, as shown by the number on the top of each column. *, Synergistic effect observed when ET was used in combination with 1 #M ionomycin.

ET-induced PI breakdown suggests that the potentiation is directly related to an increase in [Ca2+]i (see below). Accumulating evidence supports the view that basal PI hydrolysis can be enhanced by a rise in [Ca2+]~ in various cell types (Akhtar and Abdel-Latif, 1978 ; Griffin and Hawthorne, 1978; Fisher and Agranoff, 1981; Kendall and Nahorski, 1984; Martin et al., 1986 ; Eberhard and Hoiz, 1988 ; Diamant and Atlas, 1989). Confirming these results, we found that basal [3H]IP accumulation was markedly enhanced by A23187 and ionomycin at approximately 10 #M (Figs 2 and 5). However, it should be stressed that potentiation of ET- and ATP-induced PI turnover by Ca 2+ ionophores is dissociable from their effects on basal PI metabolism. Thus, the synergy of ET-induced PI turnover by either ionophore was observed when their concentrations were in the range of 10 nM to 10 #M with a maximal potentiation found at 1 #M. Conversely, the enhancement of basal PI breakdown occurred when the ionophore concentration was at or above 10 #M. At least in the case of ionomycin, the potentiation of ET response was roughly correlated

Fig. 5. Concentration-dependent potentiation by ionomycin of ET-induced PI turnover in C6 glioma. Experimental conditions are as described in the Experimental Procedures. Data presented are mean ___SEM from 3 independent experiments which were performed in triplicate. Concentrations of ET and ionomycin were as shown in the figure.

with the dose-depenent (10-9-10 -6 M) increase of [Ca2+]~, suggesting that the ionophore's effect is indeed mediated by an influx of extraeellular Ca 2+. The distinct requirement of ionophore concentrations for increasing basal and agonist-induced PI turnover may indicate that basal PI breakdown is less sensitive to a rise in [Ca2+]~. From the experiment of [CaZ+]~ measurement [Fig. 6(A)], it seems that basal phospholipase C activity is stimulated only when its [Ca2+]~ is persistently increased by the ionophore to a level not less than 1 #M. The potentiation by Ca 2+ ionophores on ETinduced PI turnover reported in this study is reminiscent of the effect of A23187 on muscarinic receptormediated PI responses using brain slices and synaptosomes (Fisher and Agranoff, 1981 ; Eva and Costa, 1986). The exact role of Ca 2+ in stimulating these agonist-induced events is unclear. However, one may speculate that Ca 2+ at relatively low concentrations promotes the coupling of receptors to phospholipase C through the purported GTP binding proteins. In this respect, it is noteworthy that a synergy between Ca 2+- and guanine nucleotide-dependent activation of phospholipase C in permeabilized cells also suggests that G-protein is a target of Ca 2+ modulation (Martin et al., 1986 ; Vallar et al., 1987 ; McDonough et al., 1988). Although A23187 and ionomycin both

298

WAN-WAN L]N and DE-MAw CHUANG

A

eoo

A A

rv +

:S600

400

u. el

!

IonomycinIlOnMI =E

iJ~ionomycin (01pM)

loo¢

°cOI

Ionomycin(IOIJM) ,

÷

u. 50o

500

~o

d

'

Iseeonasl

B 1200-

1000, A

t.-om

800, 600.

4m

4OO