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Effects of maitotoxin on atrial natriuretic factor-mediated accumulation of cyclic GMP in PC12 cells
Life Sciences, Vol. 46, pp. 671-678 Printed in the U.S.A.
Pergamon Press
EFFECTS OF MAITOTOXIN ON ATRIAL NATRIURETIC FACTOR-MEDIATED ACCUMULATION OF CYCLIC GMP IN PC12 CELLS Andrew Schulick,
Fabian Gusovsky, Takeshi Yasumoto* and John W. Daly
Laboratory of Bioorganic Chemistry National Institute of Diabetes, Digestive and Kidney Diseases National Institutes of Health, Bethesda, Maryland, USA and *Faculty of Agriculture Tohoku University, Sendai, Japan (Received in final form January 9, 1990) Summary Maitotoxin (MTX) activates calcium channels and stimulates phosphoinositide breakdown in pheochromocytoma PC12 cells, while having no effect on basal levels of the cyclic nucleotides cAMP and cGMP. Atrial natriuretic factor (ANF) induces a dose-dependent accumulation of cGMP in PC12 cells through the activation of a membrane bound guanylate cyclase. Effects of ANF on cGMP are independent of extracellular concentrations of calcium. Since agents that activate phosphoinositide breakdown can indirectly affect cyclic nucleotide formation, the effects of MTX on ANF-mediated accumulation of cGMP was studied. MTX induces a dose-dependent inhibition of ANFmediated accumulation of cGMP. The inhibition by MTX requires the presence of extracellular calcium, but is unaffected by the calcium channel blocker nifedipine. The inhibitory effect of MTX is not mimicked by the calcium ionophore ionomycin. A phorbol ester, PMA, which stimulates protein kinase C, also inhibits ANF-mediated accumulation of cGMP. Sodium nitroprusside induces large accumulations of cGMP in PC12 cells through the stimulation of a soluble guanylate cyclase. Neither MTX nor PMA inhibit nitroprusside-mediated accumulation of cGMP. The results indicate that in PC12 cells, protein kinase C activation, either directly with PMA, and indirectly with MTX through phosphoinositide breakdown and formation of diacylglycerol, leads to inhibition of ANF-mediated, but not nitroprusside-mediated accumulation of cGMP.
The marine toxin MTX causes an activation of L-type calcium channels that can be blocked by organic calcium channel blockers in pheochromocytoma PC12 cells (1,2). Recently, it was shown that MTX induces phosphoinositide breakdown in PCl2 (3,4) and a wide variety of other cell types (5,6). This action of MTX appears to be 0024-3205/90 $3.00 +.00
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i n d e p e n d e n t of c a l c i u m channel activation, since c a l c i u m channel b l o c k e r s do not inhibit M T X - m e d i a t e d p h o s p h o i n o s i t i d e breakdown
(3). In PC12 cells M T X has no e f f e c t on basal levels of cAMP but, similar to p h o r b o l esters, MTX potentiates forskolin-induced a c c u m u l a t i o n of c A M P (4). The effects of M T X on c A M P a c c u m u l a t i o n a p p e a r to o c c u r t h r o u g h g e n e r a t i o n of d i a c y l g l y c e r o l and s u b s e q u e n t a c t i v a t i o n of p r o t e i n k i n a s e C (4). The e f f e c t s of M T X on r e c e p t o r and n i t r o p r u s s i d e - e l i c i t e d accumulation of c G M P h a v e b e e n now d e t e r m i n e d in PC12 cells. A t r i a l n a t r i u r e t i c factor (ANF) elicits an a c c u m u l a t i o n of cGMP in PC12 cells (7) t h r o u g h i n t e r a c t i o n w i t h a membrane-boundANF r e c e p t o r p r o t e i n that also p o s s e s s e s g u a n y l a t e c y c l a s e a c t i v i t y (8). Nitroprusside stimulates cGMP accumulation t h r o u g h a c t i v a t i o n of a s o l u b l e g u a n y l a t e cyclase. MTX inhibits the r e s p o n s e to ANF, but not the r e s p o n s e to n i t r o p r u s s i d e . Materials
and M e t h o d s
Cell culture: Pheochromocytoma PC12 cells w e r e g r o w n in D u l b e c c o ' s m o d i f i e d Eagle m e d i u m w i t h 6% fetal calf serum 6% horse serum and p e n i c i l l i n (i00 U/ml) and s t r e p t o m y c i n (I00 ~g/ml). Cells w e r e g r o w n at 37°C in an a t m o s p h e r e e n r i c h e d in CO 2. A s s a y of cGMP: Cells w e r e h a r v e s t e d and w a s h e d w i t h 20 ml b u f f e r A (118 m M NaCI, 4.7 ~ M KCl, 3 m M CaCI2, 1.2 m M MgSO4, 1.2 mM KH2PO4, 0.5 m M EDTA, i0 m M g l u c o s e and 20 m M HEPES, pH:7.4). After c e n t r i f u g a t i o n , cells w e r e r e s u s p e n d e d in b u f f e r A to a d e n s i t y of 2-3 m i l l i o n cells/ml. Cells w e r e d i s t r i b u t e d intro p r e w a r m e d (37°C) glass tubes (0.6-0.7 m i l l i o n cells/tube). Cells were preincubated with 3-isobutyl-l-methylxanthine (I00 ~M), a p h o s p h o d i e s t e r a s e inhibitor, for i0 min. Then, agents w e r e added and i n c u b a t i o n s c o n t i n u e d for i0 min at 37°C in a final v o l u m e of 500 ~i. I n c u b a t i o n s w e r e stopped by p l a c i n g tubes in b o i l i n g w a t e r for 4 min. T u b e s w e r e c e n t r i f u g e d and c G M P levels w e r e d e t e r m i n e d in the s u p e r n a t a n t w i t h a commercial r a d i o i m m u n o a s s a y kit (Dupont, Boston, MA). Materials: Cell culture m e d i a and sera w e r e from Gibco (Long Island, NY). M T X was p u r i f i e d from G a m b i e r d i s c u s toxicus as described (9). P h o r b o l - 1 2 - m y r i s t a t e - 1 3 - a c e t a t e (PMA), 4~-phorbol and IBMX w e r e from Sigma (St. Louis, MO). I o n o m y c i n was from C a l b i o c h e m (La Jolla, CA). ANF (rat, t w e n t y eight amino acids) was from P e n i n s u l a (Belmont, CA).
Results A N F induces a d o s e , d e p e n d e n t (ECs0:30 nM) a c c u m u l a t i o n of cGMP in PC12 cells (Fig. i). M T X or h i g h c o n c e n t r a t i o n s of KCI do not affect basal levels of cGMP (Table i). M T X induces a d o s e - d e p e n d e n t i n h i b i t i o n of A N F - m e d i a t e d f o r m a t i o n of cGMP, w i t h an IC50 v a l u e of 1.3 ng/ml (Fig. 2). The M T X - i n d u c e d i n h i b i t i o n was d e p e n d e n t on [Ca2÷]o, r e a c h i n g the m a x i m a l extent of i n h i b i t i o n at 2.5 m M [Ca2÷]0 (Fig. 3). MTX-mediated i n h i b i t i o n of the A N F - r e s p o n s e is not a f f e c t e d by i0 ~M nifedipine, a c a l c i u m channel blocker; and is not mimicked by 1 ~M ionomycin, a calcium ionophore (Table i). I o n o m y c i n at this c o n c e n t r a t i o n induces a rapid and large increase in i n t r a c e l l u l a r c a l c i u m in these cells, as m e a s u r e d w i t h quin-2
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(data not shown). PMA, a p h o r b o l e s t e r a c t i v a t o r of p r o t e i n k i n a s e C (Table I), b u t not an i n a c t i v e 4 ~ - p h o r b o l (data not shown), also inhibits ANF-mediated stimulation of c G M P formation. Sodium nitroprusside induces large a c c u m u l a t i o n s of c G M P t h r o u g h the s t i m u l a t i o n of a s o l u b l e g u a n y l a t e c y c l a s e in m a n y cell types (i0). N e i t h e r M T X nor PMA inhibit n i t r o p r u s s i d e - m e d i a t e d f o r m a t i o n of c G M P in PC12 cells (Table i, Fig. 4).
A W
-m-
4
o u ¢: o
---- 3 E E
o.
2
L u
1
i
i
i
i
i
-9
-8
-7
-6
10g [ANF], M
Figure 1 Dose-dependent s t i m u l a t i o n of a c c u m u l a t i o n of c G M P in PC12 cells. PC12 w e r e incubated w i t h the i n d i c a t e d c o n c e n t r a t i o n s of A N F for i0 min. c G M P was t h e n e x t r a c t e d and a n a l y z e d as d e s c r i b e d in Methods. R e s u l t s are the m e a n s ± SEM of 3 i n d e p e n d e n t experiments.
Discussion M T X causes an a c t i v a t i o n of n i f e d i p i n e - s e n s i t i v e calcium c h a n n e l s in m a n y cell types (1,2, and see Ii). This a c t i o n r e s u l t s in c a l c i u m u p t a k e and h o r m o n e / n e u r o t r a n s m i t t e r r e l e a s e in s e c r e t o r y cells (see Ii). In PC12 cells, M T X induces an i n c r e a s e of [Ca2+]~ as m e a s u r e d w i t h the f l u o r e s c e n t p r o b e quin-2 (4) and w i t h u p t a k e of 45ca2+ (12). s i n c e in n e u r o b l a s t o m a cells, a c t i v a t i o n of v o l t a g e dependent calcium channels through depolarization with high c o n c e n t r a t i o n s of p o t a s s i u m i n c r e a s e s i n t r a c e l l u l a r levels of cGMP, it was of i n t e r e s t to d e t e r m i n e w h e t h e r a c t i v a t i o n of such c a l c i u m channels in PC12 cells by M T X or p o t a s s i u m w o u l d a f f e c t cGMP levels. In PC12 cells, n e i t h e r M T X n o r d e p o l a r i z a t i o n w i t h high p o t a s s i u m c o n c e n t r a t i o n s h a d any effect on basal levels of cGMP (Table i).
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7'
I 6,
!!! !
0
. . . . . . . .
'1
. . . . . . . .
I'0
.TX (rig/m|)
Figure 2 D o s e - d e p e n d e n t i n h i b i t i o n of A N F - s t i m u l a t e d a c c u m u l a t i o n of c G M P by M T X in PC12 cells. PC12 cells w e r e i n c u b a t e d w i t h I00 nM A N F and the i n d i c a t e d c o n c e n t r a t i o n s of M T X for i0 min. c G M P w a s e x t r a c t e d and a n a l y z e d as d e s c r i b e d in Methods. R e s u l t s are the m e a n s ± S E M of 3 i n d e p e n d e n t experiments. 6~
A
_--. 3,
2,
0
~ 0
1
2
3
4
5
[calcium, mM
Figure 3 Calcium-dependent inhibition by M T X of A N F - m e d i a t e d a c c u m u l a t i o n of c G M P in PC12 cells. PC12 c e l l s w e r e i n c u b a t e d for i0 m i n in the p r e s e n c e of b u f f e r (solid t r i a n g l e s ) , i00 n M A N F (open squares), 1 n g / m l M T X (solid squares) or A N F + M T X (open triangles) at t h e i n d i c a t e d concentrations of calcium. Data correspond to a r e p r e s e n t a t i v e e x p e r i m e n t p e r f o r m e d in t r i p l i c a t e , w h i c h w a s r e p e a t e d t h r e e t i m e s w i t h s i m i l a r results.
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Table INHIBITION
1
OF A N F - I N D U C E D c G M P A C C U M U L A T I O N IN PC12 CELLS BY M T X A N D A P H O R B O L E S T E R (PMA). cGMP ( p m o l / m i l l i o n cells ± SEM)
Control
1.98±0.10
MTX
1.70±0.17
(I ng/ml)
ANF (i00 nS)
5.36±0.29
ANF + MTX
2.57±0.41"
A N F + PMA
(i ~M)
ANF + Ionomycin
3.71±0.20" (i ~M)
ANF + MTX + Nifedipine KCI
675
(40 mM)
5.4 +1.2 (i0 ~M)
2.09,
1.60
1.83,
1.84
Nitroprusside
(5 ~M)
5.56,
4.85
Nitroprusside
+ PMA
6.39,
5.15
Nitroprusside
+ MTX
5.34,
5.15
PC12 cells w e r e i n c u b a t e d for i0 m i n at 37°C in the p r e s e n c e of the i n d i c a t e d agents, c G M P w a s e x t r a c t e d and a n a l y z e d as d e s c r i b e d in Methods. PMA, i o n o m y c i n and n i f e d i p i n e alone had no e f f e c t on basal levels of cGMP. V a l u e s c o r r e s p o n d to means ± SEM of at least three i n d e p e n d e n t e x p e r i m e n t s or are from individual experiments each performed in triplicate. *Significant p < 0 . 0 5 v e r s u s A N F alone. The e f f e c t of M T X on s t i m u l a t e d f o r m a t i o n of c G M P in PC12 cells was examined. In PC12 cells A N F e l i c i t s a m a r k e d i n c r e a s e in c G M P levels (7). The A N F r e c e p t o r is a m e m b r a n e p r o t e i n that has g u a n y l a t e c y c l a s e a c t i v i t y (8). U p o n b i n d i n g of ANF, the c y c l a s e a c t i v i t y is s t i m u l a t e d (Fig. i). A N F - m e d i a t e d c G M P f o r m a t i o n is not a f f e c t e d by c h a n g e s in [Ca2÷]o and o c c u r s even in the a b s e n c e of e x t r a c e l l u l a r calcium. M T X induces a d o s e - d e p e n d e n t i n h i b i t i o n of A N F - m e d i a t e d a c c u m u l a t i o n of c G M P (Fig. 2). I n h i b i t i o n by MTX, however, is a b o l i s h e d in the a b s e n c e of e x t r a c e l l u l a r c a l c i u m (Fig. 3). Since the c a l c i u m channel b l o c k e r n i f e d i p i n e does not b l o c k M T X - m e d i a t e d i n h i b i t i o n (Table i), it is u n l i k e l y t h a t a c t i v a t i o n of c a l c i u m c h a n n e l s by M T X m e d i a t e s the e f f e c t s on cGMP. Moreover, the c a l c i u m i o n o p h o r e ionomycin, w h i c h at 1 ~M e l i c i t s a m a r k e d influx of c a l c i u m (data n o t shown), does not inhibit A N F - m e d i a t e d accumulation of c G M P in PC12 cells (Table i). The i n h i b i t o r y e f f e c t of M T X a p p e a r s s p e c i f i c to the A N F - m e d i a t e d response, since nitroprusside-stimulated a c c u m u l a t i o n s of c G M P are u n a f f e c t e d by M T X (Table I).
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120
100
O0 wQ
E
6O
E ~O.
40,
o
20
0
~ 10
100
1000
NPS (JZM)
Figure 4 Lack of i n h i b i t i o n of M T X on s o d i u m n i t r o p r u s s i d e (NPS)m e d i a t e d a c c u m u l a t i o n of c G M P in PCl2 cells. PC12 cells were incubated for i0 min with the indicated c o n c e n t r a t i o n s of NPS in the p r e s e n c e (open squares) or absence (solid triangles) of M T X (i ng/ml). Results c o r r e s p o n d to a r e p r e s e n t a t i v e e x p e r i m e n t p e r f o r m e d in triplicate, w h i c h was r e p e a t e d three times w i t h s i m i l a r results. In a d d i t i o n to c a u s i n g a c t i v a t i o n of c a l c i u m channels, M T X a c t i v a t e s p h o s p h o i n o s i t i d e b r e a k d o w n in every cell t e s t e d thus far (5,6). The e f f e c t of M T X on p h o s p h o i n o s i t i d e b r e a k d o w n a p p e a r s i n d e p e n d e n t of M T X - e l i c i t e d a c t i v a t i o n of c a l c i u m channels, since M T X - e l i c i t e d p h o s p h o i n o s i t i d e b r e a k d o w n in a l m o s t all cases is not a f f e c t e d by c a l c i u m channel b l o c k e r s (3,12), and is a p p a r e n t at lower c o n c e n t r a t i o n s of M T X than those r e q u i r e d for c a l c i u m channel activation (3). However, M T X - m e d i a t e d stimulation of p h o s p h o i n o s i t i d e b r e a k d o w n is d e p e n d e n t on the p r e s e n c e of e x t r a c e l l u l a r c a l c i u m (4). Thus, it remains possible, indeed p e r h a p s likely, that M T X s t i m u l a t e s p h o s p h o i n o s i t i d e b r e a k d o w n t h r o u g h a c t i v a t i o n of a c a l c i u m t r a n s p o r t system r e l a t i v e l y i n s e n s i t i v e to c a l c i u m channel blockers. Recently, MTX-elicited breakdown of p h o s p h o i n o s i t i d e has b e e n shown to result in e i t h e r p o t e n t i a t i o n or inhibition of s t i m u l a t o r y e f f e c t s of o t h e r agents (forskolin, receptor agonists) on c A M P levels (4). In PCI2 cells MTX potentiates forskolin-mediated accumulation of c A M P (4). This p o t e n t i a t i v e r e s p o n s e appears due to M T X - e l i c i t e d s t i m u l a t i o n of p h o s p h o i n o s i t i d e breakdown, r e s u l t i n g in d i a c y l g l y c e r o l f o r m a t i o n and a c t i v a t i o n of p r o t e i n k i n a s e C. Indeed, after M T X t r e a t m e n t a translocation of p r o t e i n k i n a s e C is o b s e r v e d from cytosol to m e m b r a n e s in PC12 cells, an i n d i c a t i o n of the e n z y m e a c t i v a t i o n (13). In order to d e t e r m i n e w h e t h e r i n h i b i t o r y e f f e c t s of MTX on
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c G M P a c c u m u l a t i o n h a v e a s i m i l a r origin, the a c t i o n of an e x o g e n o u s p r o t e i n k i n a s e C a c t i v a t o r was investigated. PMA, a p h o r b o l ester a c t i v a t o r of p r o t e i n k i n a s e C, at 1 ~ M i n h i b i t s A N F - m e d i a t e d cGMP g e n e r a t i o n in PC12 cells (Table i). At this c o n c e n t r a t i o n , PMA induces t r a n s l o c a t i o n of p r o t e i n k i n a s e C in PC12 cells (4), an i n d i c a t i o n of its activation. 4a-Phorbol, w h i c h does not a c t i v a t e p r o t e i n k i n a s e C, does not inhibit the A N F r e s p o n s e (data not shown). T h e r e s u l t s i n d i c a t e that a c t i v a t i o n of p r o t e i n k i n a s e C, e i t h e r i n d i r e c t l y by M T X or d i r e c t l y by an active p h o r b o l ester, r e s u l t s in i n h i b i t i o n of the A N F - s t i m u l a t i o n of g u a n y l a t e cyclase. In o t h e r cell s y s t e m s p h o r b o l esters also inhibit e i t h e r ANF(14,15) or c a r b a m y l c h o l i n e (16) s t i m u l a t e d a c c u m u l a t i o n of cGMP. A l t e r a t i o n s in a c c u m u l a t i o n s of c G M P in cells m a y be e x p l a i n e d through effects on s y n t h e s i s and/or metabolism of the c y c l i c nucleotide. A g e n e r a l effect of M T X on c y c l i c n u c l e o t i d e p h o s p h o d i e s t e r a s e s seems unlikely, since the e f f e c t s of b o t h M T X and a phorbol e s t e r in PC12 cells are p o t e n t i a t i v e w i t h r e s p e c t to accumulations of cAMP (4) and inhibitory with respect to accumulations of c G M P (this study). Furthermore, a selective e f f e c t of p r o t e i n k i n a s e C on a c t i v a t i o n of a c y c l i c G M P - s p e c i f i c phosphodiesterase, seems unlikely, b a s e d on the data for cells treated with nitroprusside. Nitroprusside elicits marked a c c u m u l a t i o n of c G M P in v i r t u a l l y all cells t h r o u g h the a c t i v a t i o n of a c y t o s o l i c g u a n y l a t e c y c l a s e (i0). M T X and a p h o r b o l e s t e r do not i n h i b i t the a c c u m u l a t i o n of c G M P e l i c i t e d by n i t r o p r u s s i d e , i n d i c a t i n g t h a t a g e n e r a l e f f e c t on c G M P m e t a b o l i s m is not i n v o l v e d in the M T X - and p h o r b o l e s t e r - e l i c i t e d i n h i b i t i o n of A N F - m e d i a t e d a c c u m u l a t i o n of cGMP. Thus, it a p p e a r s that a c t i v a t i o n of p r o t e i n kinase C results in a specific inhibition of ANF-mediated s t i m u l a t i o n of c G M P formation. Since the A N F r e c e p t o r itself is a g u a n y l a t e c y c l a s e t h a t is a c t i v a t e d in the p r e s e n c e of ANF, it would appear that phosphorylation of this r e c e p t o r e n z y m e by p r o t e i n k i n a s e C m a y d i m i n i s h its a b i l i t y to be s t i m u l a t e d by ANF. References 1.
2. 3. 4. 5.
6.
7. 8. 9. I0. ii.
M. TAKAHASHI, Y. O H I Z U M I and T. YASUMOTO, J. Biol. Chem. 257 7 2 8 7 - 7 2 8 9 (1982). M. TAKAHASHI, M. TATSUMI, Y. O H I Z U M I and T. YASUMOTO, J. Biol. Chem. 258 1 0 9 4 4 - 1 0 9 4 9 (1983). F. GUSOVSKY, J.W. DALY, T. Y A S U M O T O and E. ROJAS, FEBS Lett. 233 139-142 (1988). F. GUSOVSKY, T. Y A S U M O T O and J.W. DALY, Mol. Pharmacol. 36 4453 (1989). F, SLADECZEK, B.G. SCHMIDT, R. ALONSO, L. VIAN, A. TEP, T. YASUMOTO, R.N. CORY and J. BOCKAERT, Eur. J. Biochem. 174 663670 (1988). F. GUSOVSKY, T. Y A S U M O T O and J.W. DALY, FEBS Lett. 243 307-312 (1989). R.R. FISCUS, B.T. ROBLES, S.A. W A L D M A N and F. MURAD, J. Neurochem. 48 522-528 (1987). S. S C H U L T Z , M. C H I N K E R S a n d D.L. GARBERS, FASEB J. 3 2 0 2 6 - 2 0 3 5 (1989). A. YOKOYAMA, M. MURATA, Y. OSHIMA, T. IWASHITA and T. YASUMOTO, J. Biochem. (Tokyo) 104 184-187 (1988). S.A. W A L D M A N and F. MURAD, Pharmacol. Rev. 39 163-196 (1987). F. G U S O V S K Y and J.W. DALY, Biochem. Pharmacol. (in press).
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12. 13. 14. 15.
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O.H. CHOI, F. GUSOVSKY, T. Y A S U M O T O a n d J.W. DALY, F A S E B J o u r n a l 3 A 1 2 9 8 (1989). T.P. THOMAS, R. GOPALAKRISHNA and W.B. ANDERSON, Meth. Enzymol. 141 3 9 9 - 4 1 1 (1987). P. NAMBI, M. WHITMAN, N. AIYAR, F. S T A S S E N and S.T. CROOKE, Biochem. J. 244 481-484 (1987). R.K. JAISWAL, N. J A I S W A L and R.K. SHARMA, FEBS Lett. 227 47-50
(1988). 16.
W.S. LAI a n d E. 366-373 (1987).
EL-FAKAHANY,
J.
Pharmacol.
Exper.
Ther.
241
Pergamon Press
EFFECTS OF MAITOTOXIN ON ATRIAL NATRIURETIC FACTOR-MEDIATED ACCUMULATION OF CYCLIC GMP IN PC12 CELLS Andrew Schulick,
Fabian Gusovsky, Takeshi Yasumoto* and John W. Daly
Laboratory of Bioorganic Chemistry National Institute of Diabetes, Digestive and Kidney Diseases National Institutes of Health, Bethesda, Maryland, USA and *Faculty of Agriculture Tohoku University, Sendai, Japan (Received in final form January 9, 1990) Summary Maitotoxin (MTX) activates calcium channels and stimulates phosphoinositide breakdown in pheochromocytoma PC12 cells, while having no effect on basal levels of the cyclic nucleotides cAMP and cGMP. Atrial natriuretic factor (ANF) induces a dose-dependent accumulation of cGMP in PC12 cells through the activation of a membrane bound guanylate cyclase. Effects of ANF on cGMP are independent of extracellular concentrations of calcium. Since agents that activate phosphoinositide breakdown can indirectly affect cyclic nucleotide formation, the effects of MTX on ANF-mediated accumulation of cGMP was studied. MTX induces a dose-dependent inhibition of ANFmediated accumulation of cGMP. The inhibition by MTX requires the presence of extracellular calcium, but is unaffected by the calcium channel blocker nifedipine. The inhibitory effect of MTX is not mimicked by the calcium ionophore ionomycin. A phorbol ester, PMA, which stimulates protein kinase C, also inhibits ANF-mediated accumulation of cGMP. Sodium nitroprusside induces large accumulations of cGMP in PC12 cells through the stimulation of a soluble guanylate cyclase. Neither MTX nor PMA inhibit nitroprusside-mediated accumulation of cGMP. The results indicate that in PC12 cells, protein kinase C activation, either directly with PMA, and indirectly with MTX through phosphoinositide breakdown and formation of diacylglycerol, leads to inhibition of ANF-mediated, but not nitroprusside-mediated accumulation of cGMP.
The marine toxin MTX causes an activation of L-type calcium channels that can be blocked by organic calcium channel blockers in pheochromocytoma PC12 cells (1,2). Recently, it was shown that MTX induces phosphoinositide breakdown in PCl2 (3,4) and a wide variety of other cell types (5,6). This action of MTX appears to be 0024-3205/90 $3.00 +.00
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i n d e p e n d e n t of c a l c i u m channel activation, since c a l c i u m channel b l o c k e r s do not inhibit M T X - m e d i a t e d p h o s p h o i n o s i t i d e breakdown
(3). In PC12 cells M T X has no e f f e c t on basal levels of cAMP but, similar to p h o r b o l esters, MTX potentiates forskolin-induced a c c u m u l a t i o n of c A M P (4). The effects of M T X on c A M P a c c u m u l a t i o n a p p e a r to o c c u r t h r o u g h g e n e r a t i o n of d i a c y l g l y c e r o l and s u b s e q u e n t a c t i v a t i o n of p r o t e i n k i n a s e C (4). The e f f e c t s of M T X on r e c e p t o r and n i t r o p r u s s i d e - e l i c i t e d accumulation of c G M P h a v e b e e n now d e t e r m i n e d in PC12 cells. A t r i a l n a t r i u r e t i c factor (ANF) elicits an a c c u m u l a t i o n of cGMP in PC12 cells (7) t h r o u g h i n t e r a c t i o n w i t h a membrane-boundANF r e c e p t o r p r o t e i n that also p o s s e s s e s g u a n y l a t e c y c l a s e a c t i v i t y (8). Nitroprusside stimulates cGMP accumulation t h r o u g h a c t i v a t i o n of a s o l u b l e g u a n y l a t e cyclase. MTX inhibits the r e s p o n s e to ANF, but not the r e s p o n s e to n i t r o p r u s s i d e . Materials
and M e t h o d s
Cell culture: Pheochromocytoma PC12 cells w e r e g r o w n in D u l b e c c o ' s m o d i f i e d Eagle m e d i u m w i t h 6% fetal calf serum 6% horse serum and p e n i c i l l i n (i00 U/ml) and s t r e p t o m y c i n (I00 ~g/ml). Cells w e r e g r o w n at 37°C in an a t m o s p h e r e e n r i c h e d in CO 2. A s s a y of cGMP: Cells w e r e h a r v e s t e d and w a s h e d w i t h 20 ml b u f f e r A (118 m M NaCI, 4.7 ~ M KCl, 3 m M CaCI2, 1.2 m M MgSO4, 1.2 mM KH2PO4, 0.5 m M EDTA, i0 m M g l u c o s e and 20 m M HEPES, pH:7.4). After c e n t r i f u g a t i o n , cells w e r e r e s u s p e n d e d in b u f f e r A to a d e n s i t y of 2-3 m i l l i o n cells/ml. Cells w e r e d i s t r i b u t e d intro p r e w a r m e d (37°C) glass tubes (0.6-0.7 m i l l i o n cells/tube). Cells were preincubated with 3-isobutyl-l-methylxanthine (I00 ~M), a p h o s p h o d i e s t e r a s e inhibitor, for i0 min. Then, agents w e r e added and i n c u b a t i o n s c o n t i n u e d for i0 min at 37°C in a final v o l u m e of 500 ~i. I n c u b a t i o n s w e r e stopped by p l a c i n g tubes in b o i l i n g w a t e r for 4 min. T u b e s w e r e c e n t r i f u g e d and c G M P levels w e r e d e t e r m i n e d in the s u p e r n a t a n t w i t h a commercial r a d i o i m m u n o a s s a y kit (Dupont, Boston, MA). Materials: Cell culture m e d i a and sera w e r e from Gibco (Long Island, NY). M T X was p u r i f i e d from G a m b i e r d i s c u s toxicus as described (9). P h o r b o l - 1 2 - m y r i s t a t e - 1 3 - a c e t a t e (PMA), 4~-phorbol and IBMX w e r e from Sigma (St. Louis, MO). I o n o m y c i n was from C a l b i o c h e m (La Jolla, CA). ANF (rat, t w e n t y eight amino acids) was from P e n i n s u l a (Belmont, CA).
Results A N F induces a d o s e , d e p e n d e n t (ECs0:30 nM) a c c u m u l a t i o n of cGMP in PC12 cells (Fig. i). M T X or h i g h c o n c e n t r a t i o n s of KCI do not affect basal levels of cGMP (Table i). M T X induces a d o s e - d e p e n d e n t i n h i b i t i o n of A N F - m e d i a t e d f o r m a t i o n of cGMP, w i t h an IC50 v a l u e of 1.3 ng/ml (Fig. 2). The M T X - i n d u c e d i n h i b i t i o n was d e p e n d e n t on [Ca2÷]o, r e a c h i n g the m a x i m a l extent of i n h i b i t i o n at 2.5 m M [Ca2÷]0 (Fig. 3). MTX-mediated i n h i b i t i o n of the A N F - r e s p o n s e is not a f f e c t e d by i0 ~M nifedipine, a c a l c i u m channel blocker; and is not mimicked by 1 ~M ionomycin, a calcium ionophore (Table i). I o n o m y c i n at this c o n c e n t r a t i o n induces a rapid and large increase in i n t r a c e l l u l a r c a l c i u m in these cells, as m e a s u r e d w i t h quin-2
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(data not shown). PMA, a p h o r b o l e s t e r a c t i v a t o r of p r o t e i n k i n a s e C (Table I), b u t not an i n a c t i v e 4 ~ - p h o r b o l (data not shown), also inhibits ANF-mediated stimulation of c G M P formation. Sodium nitroprusside induces large a c c u m u l a t i o n s of c G M P t h r o u g h the s t i m u l a t i o n of a s o l u b l e g u a n y l a t e c y c l a s e in m a n y cell types (i0). N e i t h e r M T X nor PMA inhibit n i t r o p r u s s i d e - m e d i a t e d f o r m a t i o n of c G M P in PC12 cells (Table i, Fig. 4).
A W
-m-
4
o u ¢: o
---- 3 E E
o.
2
L u
1
i
i
i
i
i
-9
-8
-7
-6
10g [ANF], M
Figure 1 Dose-dependent s t i m u l a t i o n of a c c u m u l a t i o n of c G M P in PC12 cells. PC12 w e r e incubated w i t h the i n d i c a t e d c o n c e n t r a t i o n s of A N F for i0 min. c G M P was t h e n e x t r a c t e d and a n a l y z e d as d e s c r i b e d in Methods. R e s u l t s are the m e a n s ± SEM of 3 i n d e p e n d e n t experiments.
Discussion M T X causes an a c t i v a t i o n of n i f e d i p i n e - s e n s i t i v e calcium c h a n n e l s in m a n y cell types (1,2, and see Ii). This a c t i o n r e s u l t s in c a l c i u m u p t a k e and h o r m o n e / n e u r o t r a n s m i t t e r r e l e a s e in s e c r e t o r y cells (see Ii). In PC12 cells, M T X induces an i n c r e a s e of [Ca2+]~ as m e a s u r e d w i t h the f l u o r e s c e n t p r o b e quin-2 (4) and w i t h u p t a k e of 45ca2+ (12). s i n c e in n e u r o b l a s t o m a cells, a c t i v a t i o n of v o l t a g e dependent calcium channels through depolarization with high c o n c e n t r a t i o n s of p o t a s s i u m i n c r e a s e s i n t r a c e l l u l a r levels of cGMP, it was of i n t e r e s t to d e t e r m i n e w h e t h e r a c t i v a t i o n of such c a l c i u m channels in PC12 cells by M T X or p o t a s s i u m w o u l d a f f e c t cGMP levels. In PC12 cells, n e i t h e r M T X n o r d e p o l a r i z a t i o n w i t h high p o t a s s i u m c o n c e n t r a t i o n s h a d any effect on basal levels of cGMP (Table i).
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7'
I 6,
!!! !
0
. . . . . . . .
'1
. . . . . . . .
I'0
.TX (rig/m|)
Figure 2 D o s e - d e p e n d e n t i n h i b i t i o n of A N F - s t i m u l a t e d a c c u m u l a t i o n of c G M P by M T X in PC12 cells. PC12 cells w e r e i n c u b a t e d w i t h I00 nM A N F and the i n d i c a t e d c o n c e n t r a t i o n s of M T X for i0 min. c G M P w a s e x t r a c t e d and a n a l y z e d as d e s c r i b e d in Methods. R e s u l t s are the m e a n s ± S E M of 3 i n d e p e n d e n t experiments. 6~
A
_--. 3,
2,
0
~ 0
1
2
3
4
5
[calcium, mM
Figure 3 Calcium-dependent inhibition by M T X of A N F - m e d i a t e d a c c u m u l a t i o n of c G M P in PC12 cells. PC12 c e l l s w e r e i n c u b a t e d for i0 m i n in the p r e s e n c e of b u f f e r (solid t r i a n g l e s ) , i00 n M A N F (open squares), 1 n g / m l M T X (solid squares) or A N F + M T X (open triangles) at t h e i n d i c a t e d concentrations of calcium. Data correspond to a r e p r e s e n t a t i v e e x p e r i m e n t p e r f o r m e d in t r i p l i c a t e , w h i c h w a s r e p e a t e d t h r e e t i m e s w i t h s i m i l a r results.
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Table INHIBITION
1
OF A N F - I N D U C E D c G M P A C C U M U L A T I O N IN PC12 CELLS BY M T X A N D A P H O R B O L E S T E R (PMA). cGMP ( p m o l / m i l l i o n cells ± SEM)
Control
1.98±0.10
MTX
1.70±0.17
(I ng/ml)
ANF (i00 nS)
5.36±0.29
ANF + MTX
2.57±0.41"
A N F + PMA
(i ~M)
ANF + Ionomycin
3.71±0.20" (i ~M)
ANF + MTX + Nifedipine KCI
675
(40 mM)
5.4 +1.2 (i0 ~M)
2.09,
1.60
1.83,
1.84
Nitroprusside
(5 ~M)
5.56,
4.85
Nitroprusside
+ PMA
6.39,
5.15
Nitroprusside
+ MTX
5.34,
5.15
PC12 cells w e r e i n c u b a t e d for i0 m i n at 37°C in the p r e s e n c e of the i n d i c a t e d agents, c G M P w a s e x t r a c t e d and a n a l y z e d as d e s c r i b e d in Methods. PMA, i o n o m y c i n and n i f e d i p i n e alone had no e f f e c t on basal levels of cGMP. V a l u e s c o r r e s p o n d to means ± SEM of at least three i n d e p e n d e n t e x p e r i m e n t s or are from individual experiments each performed in triplicate. *Significant p < 0 . 0 5 v e r s u s A N F alone. The e f f e c t of M T X on s t i m u l a t e d f o r m a t i o n of c G M P in PC12 cells was examined. In PC12 cells A N F e l i c i t s a m a r k e d i n c r e a s e in c G M P levels (7). The A N F r e c e p t o r is a m e m b r a n e p r o t e i n that has g u a n y l a t e c y c l a s e a c t i v i t y (8). U p o n b i n d i n g of ANF, the c y c l a s e a c t i v i t y is s t i m u l a t e d (Fig. i). A N F - m e d i a t e d c G M P f o r m a t i o n is not a f f e c t e d by c h a n g e s in [Ca2÷]o and o c c u r s even in the a b s e n c e of e x t r a c e l l u l a r calcium. M T X induces a d o s e - d e p e n d e n t i n h i b i t i o n of A N F - m e d i a t e d a c c u m u l a t i o n of c G M P (Fig. 2). I n h i b i t i o n by MTX, however, is a b o l i s h e d in the a b s e n c e of e x t r a c e l l u l a r c a l c i u m (Fig. 3). Since the c a l c i u m channel b l o c k e r n i f e d i p i n e does not b l o c k M T X - m e d i a t e d i n h i b i t i o n (Table i), it is u n l i k e l y t h a t a c t i v a t i o n of c a l c i u m c h a n n e l s by M T X m e d i a t e s the e f f e c t s on cGMP. Moreover, the c a l c i u m i o n o p h o r e ionomycin, w h i c h at 1 ~M e l i c i t s a m a r k e d influx of c a l c i u m (data n o t shown), does not inhibit A N F - m e d i a t e d accumulation of c G M P in PC12 cells (Table i). The i n h i b i t o r y e f f e c t of M T X a p p e a r s s p e c i f i c to the A N F - m e d i a t e d response, since nitroprusside-stimulated a c c u m u l a t i o n s of c G M P are u n a f f e c t e d by M T X (Table I).
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120
100
O0 wQ
E
6O
E ~O.
40,
o
20
0
~ 10
100
1000
NPS (JZM)
Figure 4 Lack of i n h i b i t i o n of M T X on s o d i u m n i t r o p r u s s i d e (NPS)m e d i a t e d a c c u m u l a t i o n of c G M P in PCl2 cells. PC12 cells were incubated for i0 min with the indicated c o n c e n t r a t i o n s of NPS in the p r e s e n c e (open squares) or absence (solid triangles) of M T X (i ng/ml). Results c o r r e s p o n d to a r e p r e s e n t a t i v e e x p e r i m e n t p e r f o r m e d in triplicate, w h i c h was r e p e a t e d three times w i t h s i m i l a r results. In a d d i t i o n to c a u s i n g a c t i v a t i o n of c a l c i u m channels, M T X a c t i v a t e s p h o s p h o i n o s i t i d e b r e a k d o w n in every cell t e s t e d thus far (5,6). The e f f e c t of M T X on p h o s p h o i n o s i t i d e b r e a k d o w n a p p e a r s i n d e p e n d e n t of M T X - e l i c i t e d a c t i v a t i o n of c a l c i u m channels, since M T X - e l i c i t e d p h o s p h o i n o s i t i d e b r e a k d o w n in a l m o s t all cases is not a f f e c t e d by c a l c i u m channel b l o c k e r s (3,12), and is a p p a r e n t at lower c o n c e n t r a t i o n s of M T X than those r e q u i r e d for c a l c i u m channel activation (3). However, M T X - m e d i a t e d stimulation of p h o s p h o i n o s i t i d e b r e a k d o w n is d e p e n d e n t on the p r e s e n c e of e x t r a c e l l u l a r c a l c i u m (4). Thus, it remains possible, indeed p e r h a p s likely, that M T X s t i m u l a t e s p h o s p h o i n o s i t i d e b r e a k d o w n t h r o u g h a c t i v a t i o n of a c a l c i u m t r a n s p o r t system r e l a t i v e l y i n s e n s i t i v e to c a l c i u m channel blockers. Recently, MTX-elicited breakdown of p h o s p h o i n o s i t i d e has b e e n shown to result in e i t h e r p o t e n t i a t i o n or inhibition of s t i m u l a t o r y e f f e c t s of o t h e r agents (forskolin, receptor agonists) on c A M P levels (4). In PCI2 cells MTX potentiates forskolin-mediated accumulation of c A M P (4). This p o t e n t i a t i v e r e s p o n s e appears due to M T X - e l i c i t e d s t i m u l a t i o n of p h o s p h o i n o s i t i d e breakdown, r e s u l t i n g in d i a c y l g l y c e r o l f o r m a t i o n and a c t i v a t i o n of p r o t e i n k i n a s e C. Indeed, after M T X t r e a t m e n t a translocation of p r o t e i n k i n a s e C is o b s e r v e d from cytosol to m e m b r a n e s in PC12 cells, an i n d i c a t i o n of the e n z y m e a c t i v a t i o n (13). In order to d e t e r m i n e w h e t h e r i n h i b i t o r y e f f e c t s of MTX on
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c G M P a c c u m u l a t i o n h a v e a s i m i l a r origin, the a c t i o n of an e x o g e n o u s p r o t e i n k i n a s e C a c t i v a t o r was investigated. PMA, a p h o r b o l ester a c t i v a t o r of p r o t e i n k i n a s e C, at 1 ~ M i n h i b i t s A N F - m e d i a t e d cGMP g e n e r a t i o n in PC12 cells (Table i). At this c o n c e n t r a t i o n , PMA induces t r a n s l o c a t i o n of p r o t e i n k i n a s e C in PC12 cells (4), an i n d i c a t i o n of its activation. 4a-Phorbol, w h i c h does not a c t i v a t e p r o t e i n k i n a s e C, does not inhibit the A N F r e s p o n s e (data not shown). T h e r e s u l t s i n d i c a t e that a c t i v a t i o n of p r o t e i n k i n a s e C, e i t h e r i n d i r e c t l y by M T X or d i r e c t l y by an active p h o r b o l ester, r e s u l t s in i n h i b i t i o n of the A N F - s t i m u l a t i o n of g u a n y l a t e cyclase. In o t h e r cell s y s t e m s p h o r b o l esters also inhibit e i t h e r ANF(14,15) or c a r b a m y l c h o l i n e (16) s t i m u l a t e d a c c u m u l a t i o n of cGMP. A l t e r a t i o n s in a c c u m u l a t i o n s of c G M P in cells m a y be e x p l a i n e d through effects on s y n t h e s i s and/or metabolism of the c y c l i c nucleotide. A g e n e r a l effect of M T X on c y c l i c n u c l e o t i d e p h o s p h o d i e s t e r a s e s seems unlikely, since the e f f e c t s of b o t h M T X and a phorbol e s t e r in PC12 cells are p o t e n t i a t i v e w i t h r e s p e c t to accumulations of cAMP (4) and inhibitory with respect to accumulations of c G M P (this study). Furthermore, a selective e f f e c t of p r o t e i n k i n a s e C on a c t i v a t i o n of a c y c l i c G M P - s p e c i f i c phosphodiesterase, seems unlikely, b a s e d on the data for cells treated with nitroprusside. Nitroprusside elicits marked a c c u m u l a t i o n of c G M P in v i r t u a l l y all cells t h r o u g h the a c t i v a t i o n of a c y t o s o l i c g u a n y l a t e c y c l a s e (i0). M T X and a p h o r b o l e s t e r do not i n h i b i t the a c c u m u l a t i o n of c G M P e l i c i t e d by n i t r o p r u s s i d e , i n d i c a t i n g t h a t a g e n e r a l e f f e c t on c G M P m e t a b o l i s m is not i n v o l v e d in the M T X - and p h o r b o l e s t e r - e l i c i t e d i n h i b i t i o n of A N F - m e d i a t e d a c c u m u l a t i o n of cGMP. Thus, it a p p e a r s that a c t i v a t i o n of p r o t e i n kinase C results in a specific inhibition of ANF-mediated s t i m u l a t i o n of c G M P formation. Since the A N F r e c e p t o r itself is a g u a n y l a t e c y c l a s e t h a t is a c t i v a t e d in the p r e s e n c e of ANF, it would appear that phosphorylation of this r e c e p t o r e n z y m e by p r o t e i n k i n a s e C m a y d i m i n i s h its a b i l i t y to be s t i m u l a t e d by ANF. References 1.
2. 3. 4. 5.
6.
7. 8. 9. I0. ii.
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