The ability of divalent cations to enhance ethanol-induced sleeping time

41cohol. Vol. 3, pp. 6%72, 1986. ©Ankho InternationalInc. Printed in the U.S.A.

0741-8329/86 $3.00 + .00

The Ability of Divalent Cations to Enhance Ethanol-Induced Sleeping Time DEN'ETSU

SUTOO,* KAYO AKIYAMA* AND KIMIKO IIMURA¢

*Division of Biochemistry, Institute of Medical Science, University of Tsukuba and tDivision of Pathology, Tsukuba University Hospital, Sakura-Mura, lbaraki-Ken 305, Japan R e c e i v e d 11 F e b r u a r y 1985 SUTOO, D., K. AKIYAMA AND K. IIMURA. The ability of divalent cations to enhance ethanol-induced sleeping time. ALCOHOL 3(1) 69--72, 1986.--This investigation was carried out to determine if prolongation of ethanol-induced sleep by divalent cations is mediated by calmodulin (CAM) and biogenic amine. The effects of CaM antagonist, W-7:[N-(6Aminohexyl)-5-chloro-l-naphthalenesulfonamide],serotonin (5-HT) synthesizing enzyme inhibitor, p-chlorophenylalanine (PCPA), and catecholamine synthesizing enzyme inhibitor, c~-methyltyrosine (aMPT) on ethanol-induced sleeping time enhanced by divalent cations were studied in ddY male mice. The ethanol-induced sleeping time was increased by 70, 200, 180, 70, and 45% by intraventricular (IVT) injection of CaCI2 (10/~mol/kg), MnCI~ (15/zmol/kg), ZnCI2 (2.5 p.mol/kg), CdCI~ (1 p.mol/kg), and HgCI._,(1 ~mol/kg), respectively, compared to the saline group. On the other hand, when mice were treated IVT with W-7 and their divalent cation, the sleeping time induced by ethanol was decreased compared to that of the cation without W-7 treated mice. Also, when mice were injected simultaneously with either PCPA or aMPT and CaCI2, ZnCI2, CdCI~, or HgCIz, the ethanol-induced sleeping time was less compared to those given saline together with their cation, respectively. These results would suggest a probable mechanism in which Ca ++, Zn +÷, Cd ++, and Hg ++ prolong ethanolinduced sleeping time by activating biogenic amine synthesizing enzymes through cerebral CaM and CaM-dependent protein kinase. Sleeping time Ethanol Divalent cation Calmodulin W-7: [N-(6-Aminohexyl)-5-chloro-l-naphthalenesulfonamide] p-Chlorophenylalanine

THE interrelationship between ethanol-induced sleep and the levels of biogenic amines in the brain has been extensively investigated. These studies have shown that the administration of monoamine oxidase inhibitors will increase the levels of serotonin (5-HT), dopamine (DA), and norepinephrine (NE) and will also prolong ethanol-induced sleep [19]. Also, it has been reported that the administration of 5-HT, DA, and NE to mice will increase ethanol-induced sleep [10, 20, 21]. Moreover, the administration of L3,4-dihydroxyphenylalanine to mice has been said to markedly increase ethanol-induced sleep and to raise the DA level in the brain [3]. In addition to these findings, workers have observed that the duration of ethanol-induced sleep in mice and rats was prolonged by the administration of divalent cations such as Ca ÷+, Mn +÷, Cd ÷+, and Zn +÷ [11, 12, 14]. This ability of divalent cations to prolong ethanol-induced sleeping time was markedly increased by ionophores. However, this effect was reduced to the original duration by the intraventricular (IVT) injection of a chelating agent [14,30]. Earlier, we reported that the prolongation of ethanolinduced sleep by calcium ions was antagonized by the administration of an inhibitor of the 5-HT synthesizing enzyme (p-chlorophenylalanine (PCPA)), and catecholamine synthesizing enzyme inhibitors (a-methyltyrosine (aMPT) and diethyldithiocarbamate) [24]. Also, a similar modification to the action of ethanol and calcium ions was found with pretreatment with calmodulin (CAM) antagonists, W-7:[N-(6Aminohexyl)-5-chioro-l-naphthalenesulfonamide] and tri-

~-Methyityrosine

fluoperazine [26]. Furthermore, DA level in the cerebrum was increased by 25% by administration of CaC12 [24]. Therefore, we suggest the following mechanism: Ca ++ ions activate 5-HT, DA, and NE synthesizing enzyme via cerebral CaM and CaM-dependent protein kinase, thus raising the levels of 5-HT, DA, and NE and thereby enhancing ethanol-induced sleep. Furthermore, it is surmised that 5-HT, DA, and NE synthesizing enzyme activity is regulated via CaM by not only Ca ++ but also other divalent cations. Because it has been reported that Mg ÷+, Mn ++ [29], Zn ++ [2], and Cd ++ [18] also bound to CaM, and ethanol-induced sleeping time was prolonged by Mn ++, Zn ÷+, and Cd ++. Also, this is supported by the in vitro test [8] that CaMdependent phosphodiesterase was activated by various metal cations. The present study was carried out in order to clarify how divalent cations are able to prolong ethanol-induced sleeping time. For this purpose PCPA, t~MPT, and W-7 were administered to mice in order to inhibit the 5-HT, DA, and N E synthesizing enzyme activities and CaM in the brain; the sleeping time was then measured following the administration of ethanol, both with and without CaCI2, MgCI2, MnCI2, ZnCI.,, CdC12, and HgCI2. METHOD Male mice of the ddY strain (Doken, Ibaraki, Japan) were housed at room temperature (22-2°C) and were exposed to a 12 hr light/dark schedule (6:00 a.m.-6:00 p.m.) for more than

69

7O

SUTOO, AKIYAMA AND IIMURA TABLE 1

TABLE 2

THE EFFECTS OF VARIOUS CATIONS AND CALMODULIN ANTAGONIST, W-7, ON ETHANOL-INDUCED SLEEPING TIME IN MICE

THE EFFECTS OF PCPA AND ~MPT ON ETHANOL-INDUCED SLEEPING TIME ENHANCED BY DIVALENT CATIONS

Treatment* Saline Saline + W-7 CaCle CaCI,_, + W-7 MgCI~ MgCIe MnCI=, MnCI._, + W-7 ZnCle ZnCI., + W-7 CdC1,_, CdCI2 + W-7 HgCI., HgCle + W-7

Dose of cation (p.mol/kg, IVT)

N

l0 10 20 40 15 15 2.5 2.5 I 1 1 1

11 9 9 10 10 10 9 8 8 8 9 9 11 11

Sleeping time (min _+ S.E.) 86.8 88.4 148.6 97.7 90.3 92.2 258.8 162.6 241.1 166.3 149.2 99.6 126.5 98.2

+ 7.0 ± 7.3 ± 8.7+ _+ 7.0§ _~ 7.0 ± 9.1 ± 13.7+ ± 9.3¢§ ± 18.4t ± 16.5+~ ± 8.7~" ± 5,9§ ± 4,6¢ -~: 8,2:~

*Cations and W-7 (4.2 ~g/mouse) were injected IVT 30 min before the injection of 30% ethanol in saline (4.5 g/kg IP). All IVT injection volumes were 5 tzl, tp<0.01 (compared to saline by the Dunnett's t-test). Sp<0.01; §p<0.001 (compared to cation itself respectively by the Student's t-test).

o n e w e e k b e f o r e use in the e x p e r i m e n t s . T h e mice weighed 20-25 g ( a p p r o x i m a t e l y 35 d a y s old) at the start of the experi m e n t , a n d all a n i m a l s were used only o n c e . P C P A m e t h y l ester and aMPT methyl ester (Nakarai Chemicals Ltd., K y o t o , J a p a n ) were d i s s o l v e d in water. W-7 (Seikagaku K o g y o Co. L t d . , T o k y o , J a p a n ) , CaCI2.2H~O, MgCI2"6H20, MnCI~.4H.,O, ZnCI2, CdC12.2.5H20, a n d HgCI2 ( N a k a r a i C h e m i c a l s L t d . ) w e r e d i s s o l v e d in physiological saline. E t h a n o l was diluted to 30% (v/v) in saline f r o m 95% e t h a n o l . T o d e t e r m i n e e t h a n o l - i n d u c e d sleeping time, mice w e r e i n j e c t e d with P C P A , 300 mg/kg, i n t r a p e r i t o n e a l l y (IP), a M P T (100 mg/kg, IP), W-7 (4.2 izg/mouse, IVT), or c a t i o n s (dosages are s h o w n in T a b l e 1, I V T ) at 48 hr, 24 hr, 30 rain, or 30 min, r e s p e c t i v e l y , b e f o r e t h e i n j e c t i o n o f e t h a n o l (4.5 g/kg, IP). T h e i r i n h i b i t o r a n d c a t i o n d o s a g e s w e r e b a s e d o n studies b y J o u v e t [16], S m i t h et al. [23], King et al. [17] a n d E r i c k s o n et al. [11]. D o s e of W-7 w a s c h o s e n b e c a u s e t h e c o n c e n t r a t i o n o f W-7 r e q u i r e d to d i s p l a c e 50% o f the labelled W-7 f r o m C a M or to p r o d u c e 50% i n h i b i t i o n o f p h o s p h o d i e s t e r a s e a c t i v a t i o n has b e e n s h o w n to b e 31 ~ M a n d 28 p.M r e s p e c t i v e l y for W-7 [15]. T h e b r a i n v o l u m e o f mice in this s t u d y was m e a s u r e d to b e a b o u t 0.37 ml. T h e c o n c e n t r a t i o n o f W-7 a t t a i n e d w a s a p p r o x i m a t e l y 30 p,M w h e n diffused u n i f o r m l y t h r o u g h o u t t h e brain. I n t r a v e n t r i c u l a r injections were p e r f o r m e d in c o n s c i o u s mice as d e s c r i b e d by Clark et al. [9] using a n i n j e c t i o n v o l u m e of 5 p,1/mouse. S l e e p i n g time d u r a t i o n was defined as t h e t i m e f r o m t h e loss o f the righting reflex to t h e time w h e n the righting reflex o c c u r r e d twice within 30 s e c - - r o o m t e m p e r a t u r e , e x t r a n e o u s s o u n d s , food intake, and ethanol concentration were controlled. T h e liver a l c o h o l d e h y d r o g e n a s e ( L A D H ) activity was m e a s u r e d a c c o r d i n g to t h e m e t h o d o f B o n n i c h s e n a n d B r i n k

Treatment* (Dose) IP (mg/kg) IVT (tzmol/kg)

N

Sleeping time (min + S.E.)

Saline, Saline. Saline, Saline, Saline, Saline,

Saline CaCI2(10) MnCI~ (151 ZnCI., (2.5) CdCI.2 (I) HgCL(I)

12 14 10 7 12 9

84.2 161.2 234.2 267.0 148.6 122.6

-* 3.8 ~ 3.8 ± 21.2 ±: 15.2 ± 8.8 ~_ 4.4

PCPA (300), PCPA(300), PCPA (300), PCPA (300), PCPA(300), PCPA(300),

Saline CaCI~(10) MnCle (15) ZnCle (2.5) CdCI:~(I) HgCI~(I)

tl I1 11 II 13 15

92.tl 121.2 223.2 185.6 115.6 91.4

+ ~ + ± :~ t

(~MPT (100), aMPT(100). ~MPT(100), ~MPT (100), c~MPT (100), ~MPT (100),

Saline CaCI~ (10) MnCI~(15) ZnCI:~ (2.5) CdCI=, (I) HgCI~ (1)

12 12 II 10 10 ~3

97.6 124.4 241.4 198.0 102.2 98.4

-_: 10.0 ± 10.2; :: 19.8 :~- 22.2 + + 13.05 ~ 5.0~

7.2 12.45 22.8 23.6 + 5.6+ 7.2~:

*PCPA, aMPT or each cations was injected 48 hr, 24 hr or 30 rain, respectively, before the injection of ethanol (4.5 g/kg, IP). -p<0.05:Sp<0.01 (compared to cation alone respectively by the Dunnett's t-test).

TABLE 3 THE EFFECTS OF W-7, PCPA, aMPT, AND VARIOUS CATIONS ON THE MICE LIVER ALCOHOL DEHYDROGENASE ACTIVITY

Drugs Saline W-7 PCPA ceMPT CaCI._, MgCI~ MnCI~ ZnCI=, CdCI~ HgCI~

Dose

4.2 txg/mouse 300 mg/kg 100 mg/kg 10/xmol/kg 40/zmol/kg 15 txmol/kg 2.5 gmol/kg 1 ,umol/kg 1 p.mol/kg

N 10 9 10 10 10 11 I1 10 8 7

LADH Activity* (mean ± S.E.) 34.1 33.1 34.9 34.8 34.7 31.7 26.9 32.5 31.6 31.5

± + + ± ± ~ ± ± ± ~

1.5 t.7 2.6 1.1 2.0 1.4 3.3 ~, 1.5 1.4 1.4

*Expressed as nmoles NADH formed/mg protein/min. +Significant difference from saline [/)<0.01 by Dunnett's t-test). [6]. Mice w e r e injected with W-7, P C P A , a M P T , a n d e a c h c a t i o n ( d o s a g e s are s h o w n in T a b l e 3) at 1 hr, 48 hr. 24 hr, a n d 1 hr, r e s p e c t i v e l y , b e f o r e sacrifice by d e c a p i t a t i o n . T h e liver (1 g) was h o m o g e n i z e d with 4 ml of 0.1 M glycines o d i u m h y d r o x i d e b u f f e r (pH 9.6) at 4°C a n d c e n t r i f u g e d for 15 min at 10,000xg (4°C). The s u p e r n a t a n t fluid was used for the L A D H activity assay. D a t a were a n a l y z e d with S t u d e n t ' s t-test. An a n a l y s i s of

D I V A L E N T CATIONS A N D E T H A N O L - I N D U C E D S L E E P variance and the Dunnett's t-test were used to compare several experimental groups with the same control. A probability of 0.05 or less was considered significant. RESULTS

As shown in Table 1, ethanol-induced sleep was increased by approximately 70, 200, 180, 70, and 45% by IVT injection of 10/~mol/kg CaCI~, 15/zmol/kg MnC12, 2.5 ~mol/kg ZnCI~, 1 /xmol/kg CdCI._,, and 1 t~mol/kg HgCI2, respectively, compared to the control mice group. This result agrees with the previous reports [12,30]. On the other hand, when mice were treated IVT with W-7 and CaCI._,, MnCI._,, ZnCI2, CdCI2, and HgCI2, the sleeping time induced by ethanol was decreased by approximately 35, 40, 30, 35 and 20%, respectively, compared to that of the cation without W-7 treated mice. However, pretreatment of mice with MgCI=, (40/~mol/kg) did not increase the sleeping time. The effects of PCPA and a M P T on the sleeping time after ethanol both with and without various cations are shown in Table 2. The ethanol-induced sleeping time was not significantly changed by PCPA or c~MPT which is in agreement with previous results [10,19]. When mice were injected simultaneously with PCPA and CaCI2, ZnCI2, CdCI2, or HgCI._,, the ethanol-induced sleeping time was decreased by approximately 25, 30, 20, and 25% of those given saline together with their cation, respectively. Also, the ethanol-induced sleeping time was approximately 25, 20, 30, and 20% less when pretreated with c~MPT and CaCI2, ZnCI2, CdCI2, or HgCI2, respectively, compared to that of the saline with cation treated mice. However, the ethanol-induced sleeping time was not changed when pretreated simultaneously with PCPA and MnCI2 or c~MPT and MnCI.,. As shown in Table 3, the L A D H activity was decreased by approximately 21Y'A~by the administration of MnCI., as compared with the saline group. W-7, PCPA, aMPT, and other cations did not affect the L A D H activity markedly in this concentration. DISCUSSION

It has been shown that IVT, IP, and intravenous administration of Ca +~, Mn ~+, Zn ~ , and Cd ~÷ prolongs ethanolinduced sleeping time in mice and rats [11, 12, 14, 24]. We have been investigating the mechanism by which divalent cations enhance ethanol-induced sleep in relationship to the biogenic amine level in the brain. As the first step, the mechanism of Ca "~ in enhancing ethanol's effects was suggested in a previous study [24,26J--namely, Ca ++ facilitates the synthesis of 5-HT, DA, and NE through cerebral CaM and CaM-dependent protein kinase, together with which increases their biogenic amine release, thus ethanol-induced sleep is consequently enhanced. The present study investigated the mechanism of prolongation of the ethanol-induced sleeping time by other various divalent cations. In this present study, ethanol-induced sleep was increased by IVT administration of CaCI2, MnCI.,, ZnCI~, CdCI=, and HgCI2; however, when mice were given IVT W-7, and CaCI2, CdCI2 or HgC12, there was no enhancement. Also, when mice were treated IVT with W-7 and MnCI2 or ZnC12, the sleeping time was decreased compared to that of the cation without W-7 treated mice. This result suggests that Ca ++, Mn ++, Zn ++, Cd ++, and Hg++-sensitive ethanol-induced sleep is being blocked by CaM antagonist. We have also observed [26] that when mice were treated IVT with W-7 together with 5-HT (15 nmol/mouse, IVT), DA (30 nmol/mouse, IVT), and NE

71 (30 nmol/mouse, IVT), the sleeping time induced by ethanol and calcium was enhanced when compared to those treated with only W-7. This finding suggests that the prolongation of ethanol-induced sleeping time by Ca ++ is at first blocked by W-7 and is subsequently increased by 5-HT, DA, and NE. Namely, W-7 selectively inhibits the level of 5-HT, DA, and NE, but does not affect other neuronal functions. Pretreatment of mice with MgCI2 (40 tzmoYkg) did not increase the sleeping time. This finding is in agreement with the report of 'H NMR spectroscopy that the binding patterns of Ca ++ and Mg ÷~ to CaM are different [22]. In addition to these findings, • we have observed that in the aromatic and aliphatic regions of 400 MHz 'H NMR spectrum of bovine brain CaM with one molar ratio of cation per CaM, Zn +÷, Cd ÷÷, Hg ÷+, and Mn +÷ induced spectral changes which were very similar to those seen for Ca++; however, Mg÷+-induced change was different [25,27]. Therefore, it may be suggested that Zn ~~, Cd +÷, Hg ÷', and Mn 4. bind with cerebral CaM like Ca ~÷, hence raising a large conformational change. Incidentally, ethanol-induced sleeping time of mice treated with W-7 plus CaC12, CdC1..,, or HgCI.., was not significantly different from the saline mice group; however, the ethanol-induced sleeping time of mice treated with MnCI., or ZnCI., plus W-7 was enhanced compared to the saline mice group. These results suggest that not only CaM but also some other mechanism may be found that prolongs ethanolinduced sleeping time by Mn + ~ or Zn ÷ ~. One possible mechanism may be related to the decrease in the enzymatic activity of L A D H by M n " . On the other hand, the ability of Ca ~+, Zn ~ ', Cd ~ ÷, and Hg ~ to prolong the ethanol-induced sleeping time was antagonized by PCPA and c~MPT. This result indicates that the increase in ethanol-induced sleeping time due to CaCI2, ZnCI2, CdCI2, and HgCI2 results from the increase of 5-HT, DA, and NE in the brain. This result is supported by brain slice tests that show tyrosine hydroxylase [7] and tryptophan hydroxylase [4, 5, 13] activities may be regulated by a CaMdependent protein kinase through calcium ions. Also, we have reported that IV injection of CaCI., (20 p,mol/kg) raised the cerebral level of DA by 25% [24]. Based on these findings, we have suggested that the prolongation of ethanolinduced sleeping time induced by divalent cations results from an increase in tyrosine hydroxylase or tryptophan hydroxylase activities in the brain through the action of a divalent cation-CaM-dependent protein kinase. However, the prolongation of ethanol-induced sleeping time by Mn ~ was not changed by PCPA or aMPT. In future investigations, manganese should be reexamined by other techniques. W-7 has been used in pharmacological studies Which show it has a very high affinity for CaM and small toxicity to other cellular constituents [1]. However, the calmodulin antagonists are not specific in their pharmacologic actions [1,28]. Also, Cd ÷÷ and Hg ++ are documented nonspecific in pharmacologic activity, thus a possible complication in interpreting the data. Therefore, our conclusions on the mechanism of divalent cations in enhancing ethanol's effects must be confirmed by other techniques. Finally, W-7, PCPA, and a M P T did not induce a marked change in the L A D H activity in this concentration. Therefore, it may be said that the modifications of the ethanol-induced sleeping time by these agents observed in the present study are not related to a change in the enzymatic activity of LADH. The results and the above discussion of the present study would suggest a probable mechanism in which Ca *÷, Zn ÷+, Cd "~, and Hg +" prolong ethanol-induced sleeping time by

72

SUTOO, AKIYAMA AND IIMURA

activating tyrosine hydroxylase and tryptophan hydroxylase t h r o u g h i n t r a c e r e b r a l C a M a n d C a M - d e p e n d e n t protein

k i n a s e w h i c h s u b s e q u e n t l y raise the levels of 5-HT, DA, a n d NE.

ACKNOWLEDGEMENTS We wish to express our thanks to Dr. Rona N. Fulton, Youngstown Hospital Association, OH, for reading the manuscript; to Dr. Hitoshi Takita, professor of University of Tsukuba, to Mr. Yosoji Kobayashi, the president of the Yomiuri Shimbun (Japanese newspaper), and to Dr. Yoshio Matsumoto, professor of Kitasato University, who kindly gave us encouragement throughout our study.

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