Potassium depolarisation markedly enhances muscarinic receptor stimulated inositol tetrakisphosphate accumulation in rat cerebral cortical slices

Vol. 141, No, 3, 1986

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1130-1137

December 30, 1986

POTASSIUM DEPOLARISATION MARKEDLY ENHANCESMUSCARINIC RECEPTORSTIMULATED INOSlTOL TETRAKISPHOSPHATEACCUMULATION IN RAT CEREBRAL CORTICAL SLICES John G BAIRD and Stefan R NAHORSKI Department of Pharmacology and Therapeutics, Medical Sciences Building, University of Leicester, Leicester, LEI 7RH, United Kingdom Received November 5, 1986

SUMMARY: Rat cerebral cortical slices labelled with [3H]-inositol were incubated with the musc~rinic agonist carbachol in media containing normal 5.9 mM or elevate~ 24 mM K- ions. Over the f i r s t few minutes both carbachol and elevated K stimulated the production of [3H]-inositol phosphates. The very rapid formation of [3H]-inositol t e t r a k i s , t r i s and bisphosphate was+followed by accumulation of [3H]-inositol monophosphate. However, elevated K resulted in a r e l a t i v e l y larger stimulation of [3H]-inositol bisphosphate than muscarinic receptor stimul~tion. When carbachol effects were examined in media containing elevated K , production of [3H]-inositol trisphosphate was apparently additive whereas the mono and bisphosphate displayed somewhat synergistic responses a f t e r 1-2 minutes. In contrast, [3H]-inositol tetrakisphosphate K+ production was greatly enhanced and marked synergy was observed between the and carbachol responses. The production of the tetrakisphosphate under these conditions was dependent on e x t r a c e l l u l a r Ca2+ and a stimulatory effect of this divalent ion on the 3-kinase is discussed. ® 1986AcademicPress, Inc. I t is now generally accepted that various hormones and neurotransmitters enhance the metabolism of phosphoinositides resulting in the production of the potential second messengers Ins 1,4,5 P3 and diacylglycerol

(1,2).

Although

the details of these systems in the central nervous system are only beginning to be elucidated, much work has been performed on receptor-mediated inositol phosphate accumulation using lithium to block the degradation of Ins PI (see 3,4 for reviews).

Depolarising s t i m u l i , such as elevated e x t r a c e l l u l a r K+ or

opening Na2+ channels with the alkaloid veratrine, also stimulates [3H] Ins P1 under these conditions and evidence from this laboratory suggest that this may be secondary to the entry of Ca2+ through dihydropyridine and voltage-sensitive gates (5,6). Abbreviations: Iris PI : inosqtol monophosphate, Ins P2 : inositol bisphosphate, Ins P3 : inositol trisphosphate, Ins 1,4,5 P3 : inositol 1,4,5 trisphosphate, Ins 1,3,4 P3 : inositol 1,3,4 trisphosphate, Ins 1,3,4, 5 ~ / I n s ~ : inositol 1,3,4,5 tetrakisphosphate, KRB : Krebs Ringer Bicarbonate, PCA : Percholoric acid, EGTA : ethyleneglycolbis-(amino ethylether) t e t r a - a c e t i c acid. 0006-291 X/86 $1.50 Copyright © 1986 by Academic' Press, Inc'. All rights of reproduction in any .form reserved.

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Recent studies i n i t i a l l y

from t h i s laboratory (7) and subsequently con-

firmed elsewhere (8-10), have revealed that receptor stimulation in several tissues also leads to the production of the novel polyphosphate, Ins P4 and current evidence favours the view that this compound is produced from Ins 1,4,5 P3 by a specific kinase (11,12).

The discovery of t h i s phosphate and kinase

pathway has added a new dimension to our understanding of phosphoinositide metabolism and cell signalling and a potential second messenger role for Ins 1,3,4,5 P4 is being intensively examined. In the present experiments, we examine the effects of the muscarinic agonist and elevated K+ on the production of [3H]-inositol cerebral cortex slices.

phosphates in rat

The remarkable synergy between these two stimuli may

provide clues to the control and function of Ins P4 formation.

MATERIALS AND METHODS Cerebral cortical slices (350 ~m x 350 ~m) were prepared from male Sprague-Dawley rats and pre-incubated for 60 minutes in Krebs-Henseleit buffer as previously described (13). Aliquots (50 ~I) of packed slices (1-2 m9 of protein) were then incubated at 37 ° in flat-bottomed vials with myo [2-JH] inositol (NEN), 2-5 uCi/vial for 60 minutes. After t h i s period, the agonist was added giving a total final volume of 300 ~I. Incubations were terminated with 300 ~I of 7.0% cold PCA. After 20 minutes and thorough mixing, the tissue was sedimented by centrifugation. The inositol phosphates were extracted by the method of Downes et al (14). B r i e f l y , after centrifugation the supernatant was removed and EDTA (I0 mM) was added (125 ~I of EDTA/500 ul of supernatant). An equal volume of the supernatant of I : I Tri-n-octylamine/Freon ( l , l , 2 - t r i c h l o r o t r i f l u r o e t h a n e ) was added and mixed thoroughly. After c e n t r i fugation the upper phase was removed. Following neutralisation with sodium bicarbonate (5 mM) the inositol phosphates were separated on columns of Dowex l-anion exchange resin (mesh size 200-400, formate form) (7). Ins PI, Ins P2 and Ins P3 were eluted sequentially with a total volume of 16 ml of 0.25 M ammonium formate, 0.5 M ammonium formate/O.l M formic acid and 0.8 M ammonium formate/O.l M formic acid respectively. Ins P4 was eluted with 2.0 ml of 2.0 M ammonium formate/O.l M formic acid. For Ins PI, Ins ~ and Ins P3 the f i r s t I0 ml eluting off was kept and the r a d i o a c t i v i t y in a 2 ml aliquot determined. The extra 6 mls, which constituted the t a i l end of the peak was washed through with the appropriate solution. These elution patterns have been c a r e f u l l y evaluated with authentic standards and each fraction f a i t h f u l l y elutes following re-application to the columns (see 7).

RESULTS Figure 1 shows data cumulated from a number of separate experiments showing the production of Ins PI' Ins P2' Ins P3 and Ins P4 in response to the addition of either carbachol K+ (24 mM) or K+ and carbachol together, during 5 1131

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

InsP 1

2800 260C 2400,

2400-

2200,

2200-

2000

T

2000-

1800-

1800-

= ~ 1600-

1600-

~

1400-

1400-

1200-

1200-

1000-

1000-

800-

800-

600-

600-

400-

400-

200-

200-

O-

O-

o

TIME (MINS)

TIMES (MINS)

< InsP4

2800. 26002400220020001800bsP 3 1600-

1600-

1400-

1400-

1200-

1200-

1000-

1000-

.~

800-

800-

o

600-

600-

400 -

400-

200-

200O-

G

4

~

~

;

k

TIME (MINS)

TIME (MINS)

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Vol, 141, No. 3, 1986 minutes incubation.

BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS Both K+ and carbachol were each able to increase the

production of i n o s i t o l

phosphates with the K+ stimulated production of Ins P2

being greater than that e l i c i t e d by carbachol.

A s i m i l a r pattern was obtained

with Ins P3 (a mixture of the two known isomers, shown previously (7) to be approximately 70% Ins (1,4,5) P3), although the d i f f e r e n c e between K+ and carbachol stimulation was not so marked.

As reported elsewhere (7,9) Ins P4'

P3 and P2 are rapidly elevated whereas Ins PI only accumulates a f t e r a delay of a minute or so.

Incubation of K+ and carbachol together caused an

approximately additive response f o r Ins P3 with a synergistic response apparent in the other i n o s i t o l

phosphates.

This was especially notable f o r Ins P4 where

synergy was apparent at 15 seconds and t h i s increase becoming maximal a f t e r 3 to 5 minutes.

The increase in Ins P4 represented an approximate 60-fold

increase over basal values a f t e r 3 minutes and as such is the largest increase obtained for any of the i n o s i t o l initially

phosphates.

The other i n o s i t o l

phosphates

produced an additive response to K+ and carbachol with a s y n e r g i s t i c

response occurring in Ins PI and Ins P2 only a f t e r 2 minutes and 1 minute respectively and then to a much lesser extent than that observed f o r Ins P4" The calcium dependency of these e f f e c t s was investigated (Figures 2 and 3) by incubating the slices in the absence of added calcium and with increasing concentrations of the chelator EGTA. the a b i l i t y

In the absence of added Ca2+ (Figure 2)

of K+ to stimulate Ins P2 and Ins P4 production was reduced (by

approximately 45% and 60% r e s p e c t i v e l y ) , whereas IP 3 production was unaffected. In contrast the a b i l i t y of carbachol to stimulate Ins P2 and Ins P4 in the presence or absence of elevated K+ was not s i g n i f i c a n t l y these experiments, e x t r a c e l l u l a r Ca2+ is s t i l l

altered.

Since in

present at s i g n i f i c a n t

levels

(I0 ~M) (5), f u r t h e r experiments with increasing concentrations of the chelator EGTA were performed (Figure 3). pressed [ 3 H ] - i n o s i t o l

Increasing concentrations of t h i s agent sup-

phosphates stimulated by carbachol in the presence of 24

Figure I. Time course showing the effects of addition of K+ (total concen~ 4 mM) ( A ) , carbachol (1 mM) ( • ) and K + carbachol (24 and 1 mM respectively) (Q) on production of Ins PI, Ins P2, Ins P3 and Ins P4 in cerebral cortical slices. Data are expressed as mean ± SEM of at least 4 separate experiments. 1133

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS InsP4

InsP2

1800 1600

800

1400

700

1200

600

~1000

500 c

"N

400

800

InsP 3

400i

600

300

300

40O

E ._= 200

2OO

0 100

200

0

Control

IO0

K+ Carbachol K++ 24raM (lmM) Carbachol (lmM)

Control

K÷ Carbachol

24ram (lmM)

K++

Control

(1raM)

K++

K+ Carbachol

24raM (1raM)

Carbachol

Carbachol

(1raM)

Comparison of incubating cerebraI cortical slices is KRB containing roximately 1.3 mM) or no added Ca2" ('calcium f r e e ' ) shown as hatched markings, for one minute in the conditions shown. Data for Ins P. is not shown as r e l a t i v e l y l i t t l e change was noted at this time. Data is mean±± SEM from at least 4 separate determinations.

mM K+.

However, stimulated Ins P4 appeared to be most s e n s i t i v e to EGTA with

the K+ and carbachol stimulated production of Ins P4 being v i r t u a l l y abolished at 50 ~M EGTA. 100[ ] InsP3 ' ~

~

• InsP4

§ so

6

1'0

2'0

3'0

CONCENTRATION

4'0

gO I10~

OF EGTA(IJM)

Figure 3. The effects of increasing EGTA concentration on Ins P3 and ~ns P4 production after a 5 minute incubation stimulated in the presence of K- and carbacho] (24 mM and 1 mM respectively) using cerebral cortical slices in 'calcium free' KRB. Data are expressed as a per cent of the control value (ie no EGTA) and are the mean ± SEM of at least 3 separate determinations.

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Vol. 141, No. 3, 1986

BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS DISCUSSION

Recent experiments have established that activation of cell surface receptors can not only stimulate the production of Ins 1,4,5 P3 but also Ins 1,3,4,5 P4 and Ins 1,3,4 P3 (7-10).

I t now seems probable that these new

polyphosphates arise from the action of a specific 3-kinase on Ins 1,4,5 P3 to form Ins 1,3,4,5 P4 (11,12) which can then be metabolised to Ins 1,3,4 P3 by a 5-phosphatase (7).

Following our i n i t i a l

identification

of Ins 1,3,4,5 P4 in

carbachol-stimulated cerebral cortex slices (7), the present experiments demonstrate that generation of this polyphosphate is dramatically enhanced when muscarinic receptors are stimulated under conditions of K+-induced depolarisationo

I t is significant that the synergistic enhancement between these two

stimuli is observed at early times and increases markedly over the f i r s t minutes.

5

In contrast, no synergistic response was observed for Ins P3 (a

probable mixture over the f i r s t

few minutes of 70% Ins 1,4,5 and 30% Ins 1,3,4

P3) (7) and although there was a small degree of synergy between K+ and carbachol in terms of Ins P2 and ins P1 production, t h i s was only observed at somewhat l a t e r times after stimulation.

Therefore, Ins P4 production is

increased the most in terms of rate and extent in the presence of both carbachol and K+.

At least two possible mechanisms should be considered.

F i r s t l y , K+ could increase the apparent efficiency of coupling between the receptor, G-protein and phospholipase C°

This has been suggested by Eva and

Costa (15) to explain a K+ dependent potentiation of carbachol induced Ins P1 accumulation in rat hippocampus.

Unfortunately, these investigators did not

assay the other polyphosphates and only examined very prolonged incubations. The present observations indicate that no enhancement of Ins P3 could be detected at early time points and, therefore, t h i s argues strongly against this interpretation,

i f i t is assumed that the primary substrate of receptor

mediated phosphoinositide metabolism is phosphatidylinositol (1).

4,5 bisphosphate

However, separation of the Ins P3 isomers under these conditions is

crucial to support this argument and t h i s is under investigation. 1135

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

A second p o s s i b i l i t y should include effects of K+ on calcium entry through voltage sensitive channels.

Work from our laboratory (6) and recently con-

firmed elsewhere (16) indicates that the inositol phosphate response to K÷ is secondary to the entry of calcium through dihydropyridine sensitive gates.

The

present results support, at least in part, a direct effect of calcium on phosphoinositide metabolism in brain since K+ leads to a different pattern of accumulated inositol phosphates to that seen with the receptor agonist carbachol.

Ins P2 accumulates predominantly after elevated K+ and t h i s

response is very sensitive to a lowering of e x t r a c e l l u l a r calcium. be the result of calcium-activated phosphatidylinositol

This could

4-phosphate hydrolysis

as suggested by Kolesnick and Gershengorn (17) to explain the action of the calcium ionophore A23187 in GH3 c e l l s ,

Furthermore, studies using permea-

bilised cells have shown that Ins P3 formation can be triggered by calcium entry even though receptor agonists may not require elevated i n t r a c e l l u l a r concentrations of this ion (18,19). On the basis of this interpretation one could argue that elevated i n t r a c e l l u l a r calcium entering through voltage sensitive channels may divert the metabolism of carbachol stimulated Ins 1,4,5 P3 to Ins 1,3,4,5, P4 perhaps by a c t i v i t y of the 3-kinase.

A recent study (20) reports stimulation of 3-kinase

a c t i v i t y by t h i s bivalent ion in RINm5F cells and also interconversion of Ins 1,4,5 P3 to Ins 1,3,4,5 P4 has been shown to be calcium and ATP-dependent in permeabilised adrenal glomerulosa cells (21).

In the present studies, the

enhanced Ins P4 response in the presence of carbachol and elevated K+ was p a r t i c u l a r l y sensitive to chelation of e x t r a c e l l u l a r calcium by EGTA and strongly suggests that the synergism between K+ and carbachol depends, at least in part, on entry of t h i s bivalent ion from the e x t r a c e l l u l a r medium. This amplified production of Ins P4 may be of particular significance in excitable tissues such as neurones which contain a high density of voltage sensitive calcium channels.

Whether this represents an e f f i c i e n t mechanism to

remove Ins 1,4,5 P3 or whether newly formed Ins 1,3,4,5 P4 has another functional role in cells remains to be established. 1136

Vol. 141, No. 3, 1 9 8 6

BIOCHEMICAL AND BIOPHYSICALRESEARCHCOMMUNICATIONS ACKNOWLEDGEMENTS

We thank Caroline Kirkpatrick for manuscript preparation and SERC for financial support. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. I0. II. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.

Berridge, M.J. and Irvine, R.F. (1984) Nature, 302, 315-321 Nishizuka, Y. (1984) Nature, 308, 693-698 Downes, C.P. (1986) Neurochem I n t , (in press) Nahorski, S.Ro, Kendall, D.A. and Batty, I (1986) Biochem Pharmacol, 35, 2447-2453 Kendall, D.A. and Nahorski, S.R. (1984) J Neurochem, 42, 1388-1394 Kendall, D.A. and Nahorski, S.R. (1986) Eur J Pharmacol, 115, 31-37 Batty, I . , Nahorski, S.R. and Irvine, R.F. (1985) Biochem J, 232, 211-215 Heslop, J.P., Irvine R.Fo, Tashjian, A. and Berridge, M.J. (1985) J Exp Biol, 119, 395-401 Hawkins, P.T., Stephens, L. and Downes, C.P. (1986) Biochem J, 238, 507-516 Turk, J., Wolf, B.A. and McDaniel, M.L. (1986) Biochem J, 237, 259-263 Irvine, R.F., Letcher, J., Heslop, J.P. and Berridge, M.J. (1986) Nature, 320, 631-634 Hansen, C.A., Mah, S. and Williamson, J.R. (1986) J Biol Chem, 261, 8100-8103 Brown, E., Kendall, D.A. and Nahorski, S.R. (1984) J Neurochem, 42, 1379-1387 Downes, C.P., Hawkins, P.T. and Irvine, R.Fo (1986) Biochem J, 238, 501-506 Eva, C. and Costa, E. (1986) J Neurochem, 46, 1429-1435 Zergnig, G, Moshammer, T. and Glossman, H. (1986) Eur J Pharmacol, 128, 221-229 Kolesnick, R.N. and Gershengorn, M.C. (1984) J Biol Chem, 259, 9514-9403 Best, L. (1986) Biochem J, 238, 773-779 Taylor, C.W., Merritt, J.E., Putney, J.W. and Rubin, R.Po (1986) Biochem J, 238, 765-772 Biden, T. and Wollheim, C.B. (1986) J Biol Chem, (in press) Rossier, M.F., Dentand, I . A . , Lew, P.D., Capponi, A.M. and Vallotton, M.B. (1986) Biochem Biophys Res Comm, 139, 259-265

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