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Lithium ions have a potent and selective inhibitory effect on cyclic GMP formation stimulated by neurotensin, angiotensin II and bradykinin
European Journal of Pharmacology, 126 (1986) 111-116
111
Elsevier
L I T H I U M I O N S HAVE A P O T E N T AND SELECTIVE I N H I B I T O R Y EFFECT ON CYCLIC GMP F O R M A T I O N S T I M U L A T E D BY N E U R O T E N S I N , A N G I O T E N S I N II AND BRADYKININ S H I G E N O B U KANBA, M I C H A E L P F E N N I N G , K I Y O K O S. K A N B A and E L L I O T T R I C H E L S O N *
Departments of Psychiatry and Psychology and of Pharmacology, Mayo Clinic and Foundation, Rochester, MN 55905, U.S.A. Received 23 January 1986, revised MS received 11 April 1986, accepted 22 April 1986
S. KANBA, M. PFENNING, K.S. KANBA and E. RICHELSON, Lithium ions have a potent and selective inhibitory effect on cyclic GMP formation stimulated by neurotensin, angiotensin H and bradykinin, European J. Pharmacol. 126 (1986) 111-116. The effect of lithium ion (Li ÷) on receptor-mediated synthesis of cyclic GMP, a putative second messenger, was examined using intact murine neuroblastoma cells (clone N1E-115). Lithium chloride potently inhibited cyclic GMP formation stimulated by the neuropeptides, neurotensin, angiotensin II and bradykinin in an identical concentrationdependent (ICs0s of around 12 mM), saturable and reversible manner. In the presence of veratridine, an alkaloid which by stimulating sodium channels can increase Li + entry into the ceils, Li + inhibited neurotensin-stimulated cyclic GMP formation more potently (ICs0 = 7 mM). No effect of Li ÷ was observed on phosphodiesterase (EC 3.1.4.17) activity. These results suggest that Li + may interfere with the function of these receptors through its inhibitory effect a t a common site in the pathway of receptor-mediated cyclic GMP formation. Lithium
Neurotensin
Angiotensin II
Bradykinin
1. Introduction Effects of lithium ion (Li ÷) on receptor systems which mediate cyclic AMP have been extensively examined (Forn, 1976), while its effects on receptor systems which mediate cyclic G M P as a putative second messenger, the so-called guanylate cyclase system, have been studied minimally to date. Recently, we reported that Li ÷ has a potent inhibitory effect on muscarinic (low-affinity) receptor-mediated synthesis of cyclic G M P by murine neuroblastoma cells (clone N1E-115) (Kanba and Richelson, 1984; Kanba et al., 1985). This inhibition is time- and concentration-dependent and reversible when Li ÷ is removed from the cells (Kanba et al., 1985). However, Li ÷ had no significant effect on muscarinic (high-affinity) receptor-mediated inhibition of the cyclic AMP response to prostaglandin E 1 at corresponding concentrations. These results suggest that Li ÷ has a selective inhibitory effect on the cyclic G M P re* To whom all correspondence should be addressed. 0014-2999/86/$03.50 © 1986 Elsevier Science Publishers B.V.
Cyclic GMP
Phosphodiesterase
sponse mediated by low-affinity muscarinic receptors (Kanba et al., 1985) and have increased interest in the effects of Li ÷ on receptor systems that mediate cyclic GMP. Recently, we showed that the neuropeptides, neurotensin (Gilbert and Richelson, 1984), angiotensin II (Gilbert et al., 1984) and bradykinin (Snider and Richelson, 1984) by activating their respective receptors, stimulate cyclic G M P formation by clone N1E-115. To explore the effect of Li ÷ on the biochemical properties of these receptor systems, we examined its effects on cyclic G M P formation mediated through these neuropeptide receptors. In addition, we examined effects of Li + on phosphodiesterase activity in broken cell preparations.
2. Materials and methods 2.1. Cell culture conditions
Murine neuroblastoma cells (clone N1E-115, subcultures 9-15) were cultured in 20 ml of
112 Dulbecco-Vogt modified Eagle's medium (Grand Island Biological Co., Grand Island, NY) supplemented with 10% (vol/vol) fetal calf serum (Grand Island Biological Co.) (medium I) in 75 cm 2 Corning flasks (Corning Glass Works, Coming, NY). The cells were maintained at 37°C in an atmosphere of 10% CO 2 and 90% humidified air. Subculture was achieved by incubation of cells in a modified Puck D 1 solution and collection of cells by low speed centrifugation (250 × g for 90 s at 4°C). Flasks were inoculated with 1.0-1.5 × 106 cells and beginning on day 5, medium was replenished daily by the addition of 10 ml of fresh medium I and the removal of 10 ml of medium. For the cyclic G M P assays, cells, 10-22 days after subculture, were collected as above for subculturing, washed twice in phosphate-buffered saline (solution I) (in mM: NaC1 110, KC1 5.3, CaC12 1.8, MgC12 1.0, N a 2 H P O 4 2.0, glucose 25, sucrose 70, with adjustments to pH 7.3-7.4 and osmolality 335-340 mOsm) and resuspended in 2.0 ml of solution I.
the assay buffer (40 mM Tris/HC1, pH 8.0) containing 4 mM 2-mercaptoethanol were sonicated for 15 s (setting 3, Ultrasonic Cell Disrupter, Kontes Glass Co., Vineland, N J) and incubated for 5 min at 30°C. The reaction was initiated by the addition of 200/~1 of sonicate to 12 × 75 mm glass tubes containing 100 /~1 cyclic [3H]GMP in the assay buffer containing 20 mM MgC12, 100/~1 cyclic G M P and 100 /~1 of the desired lithium concentration. The incubations were for 15 min at 30°C. The reaction was stopped by placing tubes in a boiling water bath for 45 s and the tubes were cooled in an ice bath. One hundred microliters of 1 m g / m l snake venom solution (Sigma Chemical Co., Cat. No. V0376) was added to each tube which was then incubated for 10 min at 30°C. The reaction was stopped by placing the tubes in an ice bath and adding 1 ml 100% methanol. [3H]Guanosine formed was isolated by chromatography on Dowex l-X8 (200-400 mesh) and quantified by liquid scintillation spectrometry.
2.4. Materials 2.2. Cyclic [3H]GMP assay The details of the assay measuring the relative changes in cyclic G M P using a radioactively labeled precursor and intact cells have been described elsewhere (Richelson et al., 1978). Briefly, cells in solution I were incubated in the presence of 20-30 /~Ci of [3H]guanine for 45 min to radioactively label intracellular stores of GTP, the precursor of cyclic GMP. Pre-labelled cells were further incubated in the presence of lithium chloride or other ions at the indicated concentrations for 30 min. The cells were stimulated by each peptide at 1/~M for 30 s and the reaction was stopped by addition of 50% trichloroacetic acid. The cyclic [3H]GMP formed by the cells was isolated with a cation-exchange resin column and the radioactivity was determined in a Searle Isocap/300 liquid scintillation counter (average efficiency of 40%).
2.3. Phosphodiesterase assay The details of the assay have been described elsewhere (Thompson et al., 1974). Briefly, cells in
Lithium chloride was obtained from Baker (Phillipsburg, N J); sodium chloride, potassium chloride, ammonium chloride, angiotensin II, veratridine and tetrodotoxin from Sigma (St. Louis, MO). [3H]Guanine (7.2 Ci/mmol) and cyclic [3H]GMP (10.6 Ci/mmol) were from Amersham/Searle (Arlington Heights, IL). Bradykinin and neurotensin were from BoehringerMannheim (Indianapolis, IN).
3. Results
When acutely administered, lithium chloride showed a potent inhibitory effect of the cyclic G M P response to the neuropeptides in an identical concentration-dependent manner (fig. 1). The concentration of Li + causing 50% inhibition (IC50) of cyclic G M P formation mediated by neurotensin, angiotensin II and bradykinin were (means + S.E.M.) 14 + 2, 12 + 1 and 12 + 1 raM, respectively. Basal levels of cyclic G M P formation were not significantly affected by Li ÷. These results suggest that Li + acts at a common site in
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-Log[UiCl] (M) Fig. 1. Effects of lithium ions on neuropeptide-stimulated cyclic GMP formation in intact neuroblastoma cells. Clone N1E-115 cells preincubated with [3H]guanine were further incubated in the presence of lithium chloride at the indicated concentrations for 30 min. The cells were stimulated by each peptide at 1 ~M for 30 s. Each point was the mean of duplicate determinations. Data are representative results from one of three independent experiments. In a typical experiment, the basal value was 2.0×104 dpm/106 cells and neurotensin, angiotensin II and bradykinin stimulated cyclic GMP formation more than seven-fold over the basal value. % Neurotensin; ©, angiotensin I|; A, bradykinin.
50 m M Li + for 30 m i n f o l l o w e d b y w a s h i n g in L i + - f r e e m e d i u m r a p i d l y r e c o v e r e d t h e i r a b i l i t y to s y n t h e s i z e cyclic G M P in r e s p o n s e to p e p t i d e agonists. T h i r t y m i n u t e s a f t e r w a s h i n g , the i n h i b i t o r y effect o f Li ÷ was a l m o s t c o m p l e t e l y a b o l i s h e d (data not shown). The alkaloid, veratridine i n c r e a s e d t h e r a t e of t h e i n h i b i t i o n b y Li ÷ o f n e u r o t e n s i n - s t i m u l a t e d cyclic G M P f o r m a t i o n a n d p o t e n t i a t e d this i n h i b i t i o n (fig. 2). T h u s , w h e n v e r a t r i d i n e a c t i v a t e d the s o d i u m c h a n n e l s , t h e IC50 for Li ÷ was 7 + 1 m M (fig. 3). V e r a t r i d i n e ' s effect was c o m p l e t e l y b l o c k e d b y t e t r o d o t o x i n , a s o d i u m c h a n n e l b l o c k e r (fig. 3). V e r a t r i d i n e itself, at 10 /xM for 30 m i n or t e t r o d o t o x i n at 5 × 10 - 7 m g / m l for 30 m i n , h a d n o e f f e c t o n n e u r o t e n s i n - s t i m u l a t e d cyclic G M P f o r m a t i o n . T h e s e results suggest t h a t Li ÷ e n t r y i n t o the cells is n e c e s s a r y to e x e r t t h e i n h i b i t o r y e f f e c t o n the g u a n y l a t e c y c l a s e system. I n a d d i t i o n , t h e i n h i b i t o r y a c t i o n b y Li ÷ of the p e p t i d e - s t i m u l a t e d cyclic G M P f o r m a t i o n was app a r e n t l y n o n - c o m p e t i t i v e . O t h e r m o n o v a l e n t cat i o n s such as Cs ÷, N H ~ - , N a ÷ a n d K ÷ at 20 m M
r e c e p t o r - m e d i a t e d cyclic G M P f o r m a t i o n . T h e i n h i b i t i o n was t i m e - d e p e n d e n t (fig. 2), s a t u r a b l e a n d reversible. Cells p r e i n c u b a t e d w i t h 2.5
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Fig. 2. Time course of inhibition by lithium ions of neurotensin-stimulated cyclic GMP formation in intact neuroblastoma cells. The cells preincubated with [3H]gnanine were further incubated with lithium chloride at 10 mM for the indicated times in the presence or absence of veratridine at 10 /~M. Then, the cells were stimulated by neurotensin at 1 /LM for 30 s. Data are representative results from one of two independent experiments. Each point was the mean of duplicate determinations. O, Control time course; A, time course in the presence of veratridine.
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Fig. 3. Dose-response curves of lithium chloride and inhibition of neurotensin-stimulated cyclic GMP formation. The cells preincubated with [3H]guanine were further incubated with lithium chloride at the indicated concentrations for 30 min in the presence or absence of veratridine at 10 vM and tetrodotoxin at 5×10 -7 mg/ml. Veratridine at 10 /~M or tetrodotoxin at 5 × 10 -7 mg/ml had no significant effect on neurotensin-stimulated cyclic GMP formation, Each point was the mean of triplicate determinations. Data are representative results from one of three independent experiments. Ill, Control; A, veratridine at 10 vM; O, veratridine at 10/xM + tetrodotoxin at 5 × 10 -7 mg/ml.
114 above the concentration in solution I had no significant effect on peptide-stimulated cyclic G M P formation (data not shown). The effect of Li ÷ on phosphodiesterase activity was determined, because the inhibition by Li + of receptor-mediated cyclic G M P formation could be due to stimulation of phosphodiesterase. However, lithium had no significant effect on phosphodiesterase activity even at 20 mM (control cell activity, 4 4 0 0 + 300 dpm p r o d u c t / m i n per 10 6 cells; Li÷-treated cell activity, 4300___ 110 dpm p r o d u c t / m i n per 10 6 cells).
4. Discussion
These results show that Li + inhibited neurotensin, angiotensin II and bradykinin receptormediated cyclic G M P formation in an identical concentration-dependent manner with ICs0s in the range of 12-14 mM (fig. 1). We previously reported that Li ÷ had a similar inhibitory action on carbachol-stimulated cyclic G M P formation with an IC50 of 14 mM (Kanba and Richelson, 1984; Kanba et al., 1985). There was no statistically significant difference among these ICs0s for Li ÷ inhibition of receptor-mediated cyclic G M P formation. The inhibition was time-dependent, saturable and reversible (fig. 2). The time course of the inhibition by Li ÷ of neurotensin-stimulated cyclic G M P formation was similar to that for the entry of Li ÷ into these cells reported by Gorkin and Richelson (1981). For example, the Li + entry was time- and concentration-dependent and its steady state level was achieved after 45 min. Most of intracellular Li ÷ was rapidly lost from the cells by 30 min after the removal of Li ÷. This rapid exit of Li ÷ from cells may explain the rapid recovery of the ability of agonists to stimulate cyclic G M P formation after washing the cells with medium free of Li+. The entry of Li ÷ into these ceils is activated by veratridine, which increases the Li ÷ entry through voltage-dependent sodium channels by activating these channels (Richelson, 1977). Veratridine facilitated the time course (fig. 2) and potentiated the inhibition (fig. 3). The potentiation of Li ÷ inhibition by veratridine was completely abolished
by tetrodotoxin which blocks the activation of sodium channels. These data suggest that Li + may exert inhibition of receptor-mediated synthesis of cyclic GMP, the so-called guanylate cyclase system, by acting somewhere inside the cells. The next question is where and how Li ÷ inhibits the guanylate cyclase system. Although, the mechanism of receptor-mediated cyclic G M P formation remains a matter of hypothesis, several biochemical steps appear to be involved in the guanylate cyclase system (McKinney and Richelson, 1984). One of these steps may involve metabolism of arachidonic acid by action of phospholipase A 2 and a lipoxygenase (Snider et al., 1984). An arachidonic acid metabolite may be responsible for activation of guanylate cyclase. Most receptors which mediate cyclic G M P also stimulate the release of inositol phosphates (Berridge, 1981). Li ÷ has been found to inhibit acutely inositol-l-phosphatase (Hallcher and Sherman, 1980), an enzyme involved in the recycling of inositol into phosphatidylinositol. This inhibition causes the accumulation of more inositol phosphates when receptors are stimulated. However, the chronic administration of Li ÷ depletes inositol necessary to synthesize the polyphosphoinositides (Allison et al., 1976). Depletion of inositol could result in a decline of receptor function (Berridge, 1985). Is this inhibitory effect of Li ÷ on inositol-l-phosphatase responsible for the inhibition by Li ÷ of the guanylate cyclase system? If we assume that the release of inositol phosphates is invovled in the guanylate cyclase system and that the primary site of the Li ÷ action is this enzyme, Li ÷ should acutely augment the cyclic GMP response to receptor stimulation by amplifying agonist-dependent phosphatidylinositol responses and Ca z + mobilization. However, the observation that Li + acutely inhibits the guanylate cyclase system is contradictory to these assumptions. Also, acute onset of Li ÷ inhibition does not appear to be consistent with a depletion of inositol. One may also postulate that the accumulation of inositol phosphates causes the inhibition by Li ÷ of the guanylate cyclase system. We tested this hypothesis in a preliminary way by stimulating the system with sodium nitroprusside which may di-
115 rectly activate g u a n y l a t e cyclase ( K a t s u k i et al., 1977). S o d i u m n i t r o p r u s s i d e d i d not s t i m u l a t e the release of inositol p h o s p h a t e s , b u t s o d i u m nit r o p r o s s i d e - m e d i a t e d cyclic G M P f o r m a t i o n was i n h i b i t e d b y Li ÷ in the identical m a n n e r seen with the agonists. Thus, this p r e l i m i n a r y result does n o t s u p p o r t this hypothesis. Since the i n h i b i t o r y effect of Li ÷ on agonists t i m u l a t e d cyclic G M P f o r m a t i o n could be due to s t i m u l a t i o n of cyclic G M P m e t a b o l i s m , we exa m i n e d Li ÷ effect on p h o s p h o d i e s t e r a s e activity in h o m o g e n a t e s . However, at 20 m M , Li ÷ h a d no significant effect on this enzyme activity. T h e ICs0s of Li ÷ for the g u a n u l a t e cyclase system ( a b o u t 12 m M ) is well b e y o n d the thera p e u t i c b l o o d level (1.2 m M ) for this ion. H o w ever, in t h e presence of veratridine, L i + s IC50 b e c a m e as low as 7 m M , suggesting that in electrically active n e u r o n s Li ÷ could a t t a i n sufficient c o n c e n t r a t i o n s to cause significant i n h i b i t i o n of r e c e p t o r function at clinically relevant c o n c e n t r a tions. A l t h o u g h the function of cyclic G M P as a s e c o n d messenger in n e u r o t r a n s m i s s i o n is less defined as c o m p a r e d with that of cyclic A M P , cyclic G M P could b e involved in changes in the transm e m b r a n e p o t e n t i a l directly or indirectly via cyclic G M P - d e p e n d e n t p r o t e i n kinase activity ( M c K i n ney a n d Richelson, 1984) a n d is k n o w n to be involved in the f u n c t i o n of p h o t o r e c e p t o r s k n o w n as r o d cells. Thus, cyclic G M P is involved in the function of a n u m b e r of different receptors for p u t a t i v e n e u r o t r a n s m i t t e r s a n d inhibition of its synthesis results in i n h i b i t i o n of their function. In a previous p a p e r ( K a n b a et al., 1985), we discussed the possible clinical i m p l i c a t i o n s of the in vitro findings of the i n h i b i t i o n of muscarinic M 1 r e c e p t o r function. Briefly stated, at the m u s c a r i n i c receptor, this i n h i b i t i o n could relate to the thera p e u t i c effects of Li + or to m o r e subtle effects of this ion on m e m o r y a n d cognition. However, it is m o r e likely that i n h i b i t i o n of m u s c a r i n i c M1 rec e p t o r function is responsible, at least in part, for some of the acute toxicity of Li ÷ affecting the nervous system such as b l u r r e d vision, loss of m e m o r y , amnesia, ataxia, confusion a n d delirium. A s the functions o f angiotensin II, b r a d y k i n i n a n d n e u r o t e n s i n receptors in the nervous system be-
c o m e known, we m a y develop insight into the effect of Li + on their function.
Acknowledgements This work was supported by the Mayo Foundation and USPHS Grant MH27692 from N.I.M.H.
References Allison, J.H., M.E. Blisner, W.H. Holland, P.P. Hipps and W.R. Sherman, 1976, Increased brain myo-inositol-l-phosphate in lithium-treated rats, Biochem. Biophys. Res. Commun. 71,664. Berridge, M.J., 1981, Phosphatidylinositol hydrolysis: A multifunctional transducing mechanism, Mol. Cell. Endocrinol. 24, 115. Berridge, M.J., 1985, Calcium-mobilizing receptors: Membrane phosphoinositides and signal transduction, in: Calcium in Biological Systems, eds. R.P. Rubin, G.B. Weiss and J.W. Putney, Jr. (Plenum Press, New York and London) p. 37. Forn, J., 1976,'Lithium and cyclic AMP, in: Lithium Research and Therapy, ed. F.N. Johnson (Academic Press, London, New York and San Francisco) p. 485. Gilbert, J.A., M.A. Pfenning and E. Richelson, 1984, Angiotensin I, II, and III stimulated formation of cyclic GMP in murine neuroblastoma clone N1E-115, Biochem. Pharmacol. 33, 2527. Gilbert, J.A. and E. Richelson, 1984, Neurotensin stimulates formation of cyclic GMP in murine neuroblastoma clone N1E-115, European J. Pharmacol. 99, 245. Gorkin, R.A. and E. Richelson, 1981, Lithium transport by mouse neuroblastoma cells, Neuropharmacology 20, 791. Hallcher, L.M. and W.R. Sherman, 1980, The effects of lithium ion and other agents on the activity of myo-inositol-l-phosphatase from bovine brain, J. Biol. Chem. 255, 10896. Katsuki, S., W.P. Arnold, C.K. Mittal and F. Murad, 1977, Stimulation of guanylate cyclase by sodium nitroprusside, nitroglycerine, and nitric oxide in various tissue preparations and comparison to the effects of sodium azide and hydroxylamine, J. Cycl. Nucl. Res. 3, 1829. Kanba, S., M.A. Pfenning and E. Richelson, 1985, Lithium ions inhibit function of low- but not high-affinity muscarinic receptors of murine neuroblastoma cells (clone N1E-115), Psychopharmacology 86, 413. Kanba, S. and E. Richelson, 1984, Lithium inhibits muscarinic receptor mediated cyclic GMP formation, N. Engl. J. Med. 310, 989. McKinney, M. and E. Richelson, 1984, The coupling of the neuronal muscarinic receptor to responses, Ann. Rev. Pharmacol. Toxicol. 24, 121. Richelson, E., 1977, Lithium ion entry through the sodium channel of cultured mouse neuroblastoma cells: A biochemical study, Science 196, 1001.
116 Richelson, E., F.G. Prendergast and S. Divinetz-Romero, 1978, Muscarinic receptor-mediated cyclic GMP formation by cultured nerve cells: Ionic dependence and effects of local anesthetics, Biochem. Pharmacol. 27, 2039. Snider, R.M., M. McKinney, C. Forray and E. Richelson, 1984, Neurotransmitter receptors mediate cyclic GMP formation by involvement of arachidonic acid and lipoxygenase, Proc. Natl. Acad. Sci. U.S.A. 81, 3905.
Snider, R.M. and E. Richelson, 1984, Bradykinin receptormediated cyclic GMP formation in a nerve cell population (murine neuroblastoma clone N1E-115), J. Neurochem. 43, 1749. Thompson, W.J., G. Brooker and M.M. Appleman, 1974, Assay of cyclic nucleotide phosphodiesterases with radioactive substrates, Meth. Enzymol. 38, 205.
111
Elsevier
L I T H I U M I O N S HAVE A P O T E N T AND SELECTIVE I N H I B I T O R Y EFFECT ON CYCLIC GMP F O R M A T I O N S T I M U L A T E D BY N E U R O T E N S I N , A N G I O T E N S I N II AND BRADYKININ S H I G E N O B U KANBA, M I C H A E L P F E N N I N G , K I Y O K O S. K A N B A and E L L I O T T R I C H E L S O N *
Departments of Psychiatry and Psychology and of Pharmacology, Mayo Clinic and Foundation, Rochester, MN 55905, U.S.A. Received 23 January 1986, revised MS received 11 April 1986, accepted 22 April 1986
S. KANBA, M. PFENNING, K.S. KANBA and E. RICHELSON, Lithium ions have a potent and selective inhibitory effect on cyclic GMP formation stimulated by neurotensin, angiotensin H and bradykinin, European J. Pharmacol. 126 (1986) 111-116. The effect of lithium ion (Li ÷) on receptor-mediated synthesis of cyclic GMP, a putative second messenger, was examined using intact murine neuroblastoma cells (clone N1E-115). Lithium chloride potently inhibited cyclic GMP formation stimulated by the neuropeptides, neurotensin, angiotensin II and bradykinin in an identical concentrationdependent (ICs0s of around 12 mM), saturable and reversible manner. In the presence of veratridine, an alkaloid which by stimulating sodium channels can increase Li + entry into the ceils, Li + inhibited neurotensin-stimulated cyclic GMP formation more potently (ICs0 = 7 mM). No effect of Li ÷ was observed on phosphodiesterase (EC 3.1.4.17) activity. These results suggest that Li + may interfere with the function of these receptors through its inhibitory effect a t a common site in the pathway of receptor-mediated cyclic GMP formation. Lithium
Neurotensin
Angiotensin II
Bradykinin
1. Introduction Effects of lithium ion (Li ÷) on receptor systems which mediate cyclic AMP have been extensively examined (Forn, 1976), while its effects on receptor systems which mediate cyclic G M P as a putative second messenger, the so-called guanylate cyclase system, have been studied minimally to date. Recently, we reported that Li ÷ has a potent inhibitory effect on muscarinic (low-affinity) receptor-mediated synthesis of cyclic G M P by murine neuroblastoma cells (clone N1E-115) (Kanba and Richelson, 1984; Kanba et al., 1985). This inhibition is time- and concentration-dependent and reversible when Li ÷ is removed from the cells (Kanba et al., 1985). However, Li ÷ had no significant effect on muscarinic (high-affinity) receptor-mediated inhibition of the cyclic AMP response to prostaglandin E 1 at corresponding concentrations. These results suggest that Li ÷ has a selective inhibitory effect on the cyclic G M P re* To whom all correspondence should be addressed. 0014-2999/86/$03.50 © 1986 Elsevier Science Publishers B.V.
Cyclic GMP
Phosphodiesterase
sponse mediated by low-affinity muscarinic receptors (Kanba et al., 1985) and have increased interest in the effects of Li ÷ on receptor systems that mediate cyclic GMP. Recently, we showed that the neuropeptides, neurotensin (Gilbert and Richelson, 1984), angiotensin II (Gilbert et al., 1984) and bradykinin (Snider and Richelson, 1984) by activating their respective receptors, stimulate cyclic G M P formation by clone N1E-115. To explore the effect of Li ÷ on the biochemical properties of these receptor systems, we examined its effects on cyclic G M P formation mediated through these neuropeptide receptors. In addition, we examined effects of Li + on phosphodiesterase activity in broken cell preparations.
2. Materials and methods 2.1. Cell culture conditions
Murine neuroblastoma cells (clone N1E-115, subcultures 9-15) were cultured in 20 ml of
112 Dulbecco-Vogt modified Eagle's medium (Grand Island Biological Co., Grand Island, NY) supplemented with 10% (vol/vol) fetal calf serum (Grand Island Biological Co.) (medium I) in 75 cm 2 Corning flasks (Corning Glass Works, Coming, NY). The cells were maintained at 37°C in an atmosphere of 10% CO 2 and 90% humidified air. Subculture was achieved by incubation of cells in a modified Puck D 1 solution and collection of cells by low speed centrifugation (250 × g for 90 s at 4°C). Flasks were inoculated with 1.0-1.5 × 106 cells and beginning on day 5, medium was replenished daily by the addition of 10 ml of fresh medium I and the removal of 10 ml of medium. For the cyclic G M P assays, cells, 10-22 days after subculture, were collected as above for subculturing, washed twice in phosphate-buffered saline (solution I) (in mM: NaC1 110, KC1 5.3, CaC12 1.8, MgC12 1.0, N a 2 H P O 4 2.0, glucose 25, sucrose 70, with adjustments to pH 7.3-7.4 and osmolality 335-340 mOsm) and resuspended in 2.0 ml of solution I.
the assay buffer (40 mM Tris/HC1, pH 8.0) containing 4 mM 2-mercaptoethanol were sonicated for 15 s (setting 3, Ultrasonic Cell Disrupter, Kontes Glass Co., Vineland, N J) and incubated for 5 min at 30°C. The reaction was initiated by the addition of 200/~1 of sonicate to 12 × 75 mm glass tubes containing 100 /~1 cyclic [3H]GMP in the assay buffer containing 20 mM MgC12, 100/~1 cyclic G M P and 100 /~1 of the desired lithium concentration. The incubations were for 15 min at 30°C. The reaction was stopped by placing tubes in a boiling water bath for 45 s and the tubes were cooled in an ice bath. One hundred microliters of 1 m g / m l snake venom solution (Sigma Chemical Co., Cat. No. V0376) was added to each tube which was then incubated for 10 min at 30°C. The reaction was stopped by placing the tubes in an ice bath and adding 1 ml 100% methanol. [3H]Guanosine formed was isolated by chromatography on Dowex l-X8 (200-400 mesh) and quantified by liquid scintillation spectrometry.
2.4. Materials 2.2. Cyclic [3H]GMP assay The details of the assay measuring the relative changes in cyclic G M P using a radioactively labeled precursor and intact cells have been described elsewhere (Richelson et al., 1978). Briefly, cells in solution I were incubated in the presence of 20-30 /~Ci of [3H]guanine for 45 min to radioactively label intracellular stores of GTP, the precursor of cyclic GMP. Pre-labelled cells were further incubated in the presence of lithium chloride or other ions at the indicated concentrations for 30 min. The cells were stimulated by each peptide at 1/~M for 30 s and the reaction was stopped by addition of 50% trichloroacetic acid. The cyclic [3H]GMP formed by the cells was isolated with a cation-exchange resin column and the radioactivity was determined in a Searle Isocap/300 liquid scintillation counter (average efficiency of 40%).
2.3. Phosphodiesterase assay The details of the assay have been described elsewhere (Thompson et al., 1974). Briefly, cells in
Lithium chloride was obtained from Baker (Phillipsburg, N J); sodium chloride, potassium chloride, ammonium chloride, angiotensin II, veratridine and tetrodotoxin from Sigma (St. Louis, MO). [3H]Guanine (7.2 Ci/mmol) and cyclic [3H]GMP (10.6 Ci/mmol) were from Amersham/Searle (Arlington Heights, IL). Bradykinin and neurotensin were from BoehringerMannheim (Indianapolis, IN).
3. Results
When acutely administered, lithium chloride showed a potent inhibitory effect of the cyclic G M P response to the neuropeptides in an identical concentration-dependent manner (fig. 1). The concentration of Li + causing 50% inhibition (IC50) of cyclic G M P formation mediated by neurotensin, angiotensin II and bradykinin were (means + S.E.M.) 14 + 2, 12 + 1 and 12 + 1 raM, respectively. Basal levels of cyclic G M P formation were not significantly affected by Li ÷. These results suggest that Li + acts at a common site in
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Bradyki nin Neurotensin~
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-Log[UiCl] (M) Fig. 1. Effects of lithium ions on neuropeptide-stimulated cyclic GMP formation in intact neuroblastoma cells. Clone N1E-115 cells preincubated with [3H]guanine were further incubated in the presence of lithium chloride at the indicated concentrations for 30 min. The cells were stimulated by each peptide at 1 ~M for 30 s. Each point was the mean of duplicate determinations. Data are representative results from one of three independent experiments. In a typical experiment, the basal value was 2.0×104 dpm/106 cells and neurotensin, angiotensin II and bradykinin stimulated cyclic GMP formation more than seven-fold over the basal value. % Neurotensin; ©, angiotensin I|; A, bradykinin.
50 m M Li + for 30 m i n f o l l o w e d b y w a s h i n g in L i + - f r e e m e d i u m r a p i d l y r e c o v e r e d t h e i r a b i l i t y to s y n t h e s i z e cyclic G M P in r e s p o n s e to p e p t i d e agonists. T h i r t y m i n u t e s a f t e r w a s h i n g , the i n h i b i t o r y effect o f Li ÷ was a l m o s t c o m p l e t e l y a b o l i s h e d (data not shown). The alkaloid, veratridine i n c r e a s e d t h e r a t e of t h e i n h i b i t i o n b y Li ÷ o f n e u r o t e n s i n - s t i m u l a t e d cyclic G M P f o r m a t i o n a n d p o t e n t i a t e d this i n h i b i t i o n (fig. 2). T h u s , w h e n v e r a t r i d i n e a c t i v a t e d the s o d i u m c h a n n e l s , t h e IC50 for Li ÷ was 7 + 1 m M (fig. 3). V e r a t r i d i n e ' s effect was c o m p l e t e l y b l o c k e d b y t e t r o d o t o x i n , a s o d i u m c h a n n e l b l o c k e r (fig. 3). V e r a t r i d i n e itself, at 10 /xM for 30 m i n or t e t r o d o t o x i n at 5 × 10 - 7 m g / m l for 30 m i n , h a d n o e f f e c t o n n e u r o t e n s i n - s t i m u l a t e d cyclic G M P f o r m a t i o n . T h e s e results suggest t h a t Li ÷ e n t r y i n t o the cells is n e c e s s a r y to e x e r t t h e i n h i b i t o r y e f f e c t o n the g u a n y l a t e c y c l a s e system. I n a d d i t i o n , t h e i n h i b i t o r y a c t i o n b y Li ÷ of the p e p t i d e - s t i m u l a t e d cyclic G M P f o r m a t i o n was app a r e n t l y n o n - c o m p e t i t i v e . O t h e r m o n o v a l e n t cat i o n s such as Cs ÷, N H ~ - , N a ÷ a n d K ÷ at 20 m M
r e c e p t o r - m e d i a t e d cyclic G M P f o r m a t i o n . T h e i n h i b i t i o n was t i m e - d e p e n d e n t (fig. 2), s a t u r a b l e a n d reversible. Cells p r e i n c u b a t e d w i t h 2.5
100
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20
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Incubation time, min
Fig. 2. Time course of inhibition by lithium ions of neurotensin-stimulated cyclic GMP formation in intact neuroblastoma cells. The cells preincubated with [3H]gnanine were further incubated with lithium chloride at 10 mM for the indicated times in the presence or absence of veratridine at 10 /~M. Then, the cells were stimulated by neurotensin at 1 /LM for 30 s. Data are representative results from one of two independent experiments. Each point was the mean of duplicate determinations. O, Control time course; A, time course in the presence of veratridine.
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Fig. 3. Dose-response curves of lithium chloride and inhibition of neurotensin-stimulated cyclic GMP formation. The cells preincubated with [3H]guanine were further incubated with lithium chloride at the indicated concentrations for 30 min in the presence or absence of veratridine at 10 vM and tetrodotoxin at 5×10 -7 mg/ml. Veratridine at 10 /~M or tetrodotoxin at 5 × 10 -7 mg/ml had no significant effect on neurotensin-stimulated cyclic GMP formation, Each point was the mean of triplicate determinations. Data are representative results from one of three independent experiments. Ill, Control; A, veratridine at 10 vM; O, veratridine at 10/xM + tetrodotoxin at 5 × 10 -7 mg/ml.
114 above the concentration in solution I had no significant effect on peptide-stimulated cyclic G M P formation (data not shown). The effect of Li ÷ on phosphodiesterase activity was determined, because the inhibition by Li + of receptor-mediated cyclic G M P formation could be due to stimulation of phosphodiesterase. However, lithium had no significant effect on phosphodiesterase activity even at 20 mM (control cell activity, 4 4 0 0 + 300 dpm p r o d u c t / m i n per 10 6 cells; Li÷-treated cell activity, 4300___ 110 dpm p r o d u c t / m i n per 10 6 cells).
4. Discussion
These results show that Li + inhibited neurotensin, angiotensin II and bradykinin receptormediated cyclic G M P formation in an identical concentration-dependent manner with ICs0s in the range of 12-14 mM (fig. 1). We previously reported that Li ÷ had a similar inhibitory action on carbachol-stimulated cyclic G M P formation with an IC50 of 14 mM (Kanba and Richelson, 1984; Kanba et al., 1985). There was no statistically significant difference among these ICs0s for Li ÷ inhibition of receptor-mediated cyclic G M P formation. The inhibition was time-dependent, saturable and reversible (fig. 2). The time course of the inhibition by Li ÷ of neurotensin-stimulated cyclic G M P formation was similar to that for the entry of Li ÷ into these cells reported by Gorkin and Richelson (1981). For example, the Li + entry was time- and concentration-dependent and its steady state level was achieved after 45 min. Most of intracellular Li ÷ was rapidly lost from the cells by 30 min after the removal of Li ÷. This rapid exit of Li ÷ from cells may explain the rapid recovery of the ability of agonists to stimulate cyclic G M P formation after washing the cells with medium free of Li+. The entry of Li ÷ into these ceils is activated by veratridine, which increases the Li ÷ entry through voltage-dependent sodium channels by activating these channels (Richelson, 1977). Veratridine facilitated the time course (fig. 2) and potentiated the inhibition (fig. 3). The potentiation of Li ÷ inhibition by veratridine was completely abolished
by tetrodotoxin which blocks the activation of sodium channels. These data suggest that Li + may exert inhibition of receptor-mediated synthesis of cyclic GMP, the so-called guanylate cyclase system, by acting somewhere inside the cells. The next question is where and how Li ÷ inhibits the guanylate cyclase system. Although, the mechanism of receptor-mediated cyclic G M P formation remains a matter of hypothesis, several biochemical steps appear to be involved in the guanylate cyclase system (McKinney and Richelson, 1984). One of these steps may involve metabolism of arachidonic acid by action of phospholipase A 2 and a lipoxygenase (Snider et al., 1984). An arachidonic acid metabolite may be responsible for activation of guanylate cyclase. Most receptors which mediate cyclic G M P also stimulate the release of inositol phosphates (Berridge, 1981). Li ÷ has been found to inhibit acutely inositol-l-phosphatase (Hallcher and Sherman, 1980), an enzyme involved in the recycling of inositol into phosphatidylinositol. This inhibition causes the accumulation of more inositol phosphates when receptors are stimulated. However, the chronic administration of Li ÷ depletes inositol necessary to synthesize the polyphosphoinositides (Allison et al., 1976). Depletion of inositol could result in a decline of receptor function (Berridge, 1985). Is this inhibitory effect of Li ÷ on inositol-l-phosphatase responsible for the inhibition by Li ÷ of the guanylate cyclase system? If we assume that the release of inositol phosphates is invovled in the guanylate cyclase system and that the primary site of the Li ÷ action is this enzyme, Li ÷ should acutely augment the cyclic GMP response to receptor stimulation by amplifying agonist-dependent phosphatidylinositol responses and Ca z + mobilization. However, the observation that Li + acutely inhibits the guanylate cyclase system is contradictory to these assumptions. Also, acute onset of Li ÷ inhibition does not appear to be consistent with a depletion of inositol. One may also postulate that the accumulation of inositol phosphates causes the inhibition by Li ÷ of the guanylate cyclase system. We tested this hypothesis in a preliminary way by stimulating the system with sodium nitroprusside which may di-
115 rectly activate g u a n y l a t e cyclase ( K a t s u k i et al., 1977). S o d i u m n i t r o p r u s s i d e d i d not s t i m u l a t e the release of inositol p h o s p h a t e s , b u t s o d i u m nit r o p r o s s i d e - m e d i a t e d cyclic G M P f o r m a t i o n was i n h i b i t e d b y Li ÷ in the identical m a n n e r seen with the agonists. Thus, this p r e l i m i n a r y result does n o t s u p p o r t this hypothesis. Since the i n h i b i t o r y effect of Li ÷ on agonists t i m u l a t e d cyclic G M P f o r m a t i o n could be due to s t i m u l a t i o n of cyclic G M P m e t a b o l i s m , we exa m i n e d Li ÷ effect on p h o s p h o d i e s t e r a s e activity in h o m o g e n a t e s . However, at 20 m M , Li ÷ h a d no significant effect on this enzyme activity. T h e ICs0s of Li ÷ for the g u a n u l a t e cyclase system ( a b o u t 12 m M ) is well b e y o n d the thera p e u t i c b l o o d level (1.2 m M ) for this ion. H o w ever, in t h e presence of veratridine, L i + s IC50 b e c a m e as low as 7 m M , suggesting that in electrically active n e u r o n s Li ÷ could a t t a i n sufficient c o n c e n t r a t i o n s to cause significant i n h i b i t i o n of r e c e p t o r function at clinically relevant c o n c e n t r a tions. A l t h o u g h the function of cyclic G M P as a s e c o n d messenger in n e u r o t r a n s m i s s i o n is less defined as c o m p a r e d with that of cyclic A M P , cyclic G M P could b e involved in changes in the transm e m b r a n e p o t e n t i a l directly or indirectly via cyclic G M P - d e p e n d e n t p r o t e i n kinase activity ( M c K i n ney a n d Richelson, 1984) a n d is k n o w n to be involved in the f u n c t i o n of p h o t o r e c e p t o r s k n o w n as r o d cells. Thus, cyclic G M P is involved in the function of a n u m b e r of different receptors for p u t a t i v e n e u r o t r a n s m i t t e r s a n d inhibition of its synthesis results in i n h i b i t i o n of their function. In a previous p a p e r ( K a n b a et al., 1985), we discussed the possible clinical i m p l i c a t i o n s of the in vitro findings of the i n h i b i t i o n of muscarinic M 1 r e c e p t o r function. Briefly stated, at the m u s c a r i n i c receptor, this i n h i b i t i o n could relate to the thera p e u t i c effects of Li + or to m o r e subtle effects of this ion on m e m o r y a n d cognition. However, it is m o r e likely that i n h i b i t i o n of m u s c a r i n i c M1 rec e p t o r function is responsible, at least in part, for some of the acute toxicity of Li ÷ affecting the nervous system such as b l u r r e d vision, loss of m e m o r y , amnesia, ataxia, confusion a n d delirium. A s the functions o f angiotensin II, b r a d y k i n i n a n d n e u r o t e n s i n receptors in the nervous system be-
c o m e known, we m a y develop insight into the effect of Li + on their function.
Acknowledgements This work was supported by the Mayo Foundation and USPHS Grant MH27692 from N.I.M.H.
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116 Richelson, E., F.G. Prendergast and S. Divinetz-Romero, 1978, Muscarinic receptor-mediated cyclic GMP formation by cultured nerve cells: Ionic dependence and effects of local anesthetics, Biochem. Pharmacol. 27, 2039. Snider, R.M., M. McKinney, C. Forray and E. Richelson, 1984, Neurotransmitter receptors mediate cyclic GMP formation by involvement of arachidonic acid and lipoxygenase, Proc. Natl. Acad. Sci. U.S.A. 81, 3905.
Snider, R.M. and E. Richelson, 1984, Bradykinin receptormediated cyclic GMP formation in a nerve cell population (murine neuroblastoma clone N1E-115), J. Neurochem. 43, 1749. Thompson, W.J., G. Brooker and M.M. Appleman, 1974, Assay of cyclic nucleotide phosphodiesterases with radioactive substrates, Meth. Enzymol. 38, 205.