lourriulof ,Vrurorhrm,rr?. 1976 Val 27. pp 635-636 Pergamon Prcss. Printed m Great Britain
NOILVJINnMIN03 LXOHS
SHORT COMMUNICATION
Effects of guanidino compounds on rabbit brain microsomal Na’-K’ ATPase activity (Received 31 December 1975. Accepted 16 March 1976)
SOMCG U A N I U I N O compounds induced convulsions in rabbits when administered intracisternally; among them are taurocyamine. glycocyamine. N-acetylarginine. y-guanidinobutyric acid. guanidine and methylguanidine (JINNAI C’I d . 1966. 1969; MATSLMOTO t’f ul., 1976; Mizcho ei u l , 1975). The mechanism by which these compounds induce conv~lsioiihas not yet been clarified. It may he possible that guanidino compounds cause convulsion in the same manner as ouabain. a specific inhibitor of Na’-K’ activated ATPase activity. causing convulsion in rats through the disruption of the membrane potential (BIGNAMI & PALLANDI. 1966; CORNOGet ul.. 1967). It was the purpose of this study to examine whether convulsions induced by guanidino compounds are associated with an inhibition of rabbit brain microsomal ATPase activity. An adult white rabbit (body weight 2.0 kg) was killed with a strong blow to the head without use of anesthesia and the whole hrain except for the cerebellum taken out. Thc brain tissues were homogenized with 9 vol. of 0.25 M-sucrose. I mM-EDTA (pH 7.0) using a Teflon-glass homogenizer. The whole procedure was carried out at 0 -4‘C. The homogenate was centrifuged at 1O.oOOy for 70 min to precipitate unbroken cells, nuclei and mitochondrial fractions. The supernatant was centrifuged at 105.000y for 60 min to obtain a microsomal fraction. The precipitate was washed twice with 0.2.5 M-sucrose-l mhi-
TAFIL~ I EFFKTS
EDTA and repeating the centrifugation at 105.000g for 60 min. The final sediment was suspended in the same sucrose-EDTA medium to give a protein concentration of 4 5 rng/ml and kept at -2O’C until further. Protein concentration was assayed by the method of LOWRY et ol. (1951). ATPase assay was carried out as follows: the complete reaction mixture contained 50 mM-imidazole buffer (pH 7.0). 5 mM-MgCl,, 100 mM-NaCI. 20 mM-KCI and 5&100 pg enzyme protein in a total volume of 1.0 ml. Ouabain concentration was, where used 0.2 mM. The mixture was preincubated at 37’C for 5 min. The reaction was started with the addition of 0.1 ml of a 40mM-ATP solution. After 10 min at 37°C the reaction was terminated by the addition of 1.0 ml of cold 16% perchloric acid. The precipitated protein was removed by centrifugation. The liberated inorganic phosphate content was determined by the method of MARTIN& D ~ T (1949). Y Enzyme activity was expressed as pmol P, released in I h per mg protein. All assays were carried out in triplicate. The average specific activity of total ATPase (14 experiments) in the preparation was found to be 47 5pmol/mg protein/h and 17 3 for Mg2+ ATPase. 30 7 for Na’-K+ ATPase respectively. ERects of guanidino compounds o n ATPase were examined by adding each compound to the reaction mixture
+
OF GUANIDINO COMPOLINDSou RABBIT BRAIN MICROSOMAL
5.0 S'O SO S'O
S'O
s
S'O
5'0
i
05 5'0 s '0 5'0 S'O
E
E P El 8
* Slgnificantly differenc from control ( P < 0 I ” J 635
+
ATPASEA C T I ~ I T I
616
Short communication
to produce a final concentration of 0.5 mM. The examined gusnidino compounds %ere taurocyamine. glycocyamine. guanidinosuccinic acid. ,V-acetylarginine. ,6-guanidinopropionic acid, ;-guanidino-B-hydroxybutyric acid, 7-guanidinobutyric acid. arginine. homoarginine. guanidine and methylguan idine. Out of the eleven guanidino compounds. only methylguanidine reduced Na+-K' ATPase activity (20 2 7 pmol, mg protein:h) to 67"" of control values ( P < 0.1"" for the difference between the control and the methylguanidrne added group). None of the other ten guanidino compounds, including taurocyamine. glycocyamine, N-acetylarginine, 7-guanidinobutyric acid and guanidine, which have convulsive effects in rabbits, inhibited Na+-K+ ATPase activity. None of the eleven compounds tested showed any effect on Mg" ATPase activity (Table I ) . Methylguanidine behaved as a noncompetitive inhibitor. Its inhibitory activity was much lower than that of ouabain Our results suggest that the basic mechanism involved in convulsive eRects of guanidino compounds is not connected with brain microsomal Na'-K + ATPase activity.
1,lirirurr /or
Nuiirohiolog?. Okuyuim L:iiirrrsily A f r4icul School. Sltikoro-clro 2-5-1. Okajarriu. Jupurr
BIGNAMI
A. &
REFERENCES G . (1966)
PALLAND1
M MATS~'MUTO A Mo~i
A"1ttri-t..
Lnrid. 209.
4 13-414.
COKNOCJ. L.. JR.. CONATAS N. K. & Fi IrKMAl J. R . (19671 . 4 1 i i w . J . Parhol. 51. 573-590. JlNNAl D . SAWAl A. & MORIA. (1966) ~Vofiirc.. Loiitl. 212. 61 7 JINNAI D.. MOW A,. MCKAWA3 . OHKLISL H.. HOSOTASI M.. MIZCWO A. & TYEL. C. (1969) Jap J . Bruirr Physrol. 106. 3668-3673. MATSLMOTO M.. KOBAYASHI K.. KISHILAWA H . & MORI A. (1976) IRCS bled. Sci. 4. 65 LOWRV0. H.. ROSEBROLGHN.H.. FARRA. L. & RA\DALL R. J. (1951) J . h i d Clirm. 193. 26.5-275 MARTINJ. B. & DOTYD. M. (1949) ,411uh.r C l i t ~ .21. 965-967. MIZUNOA,. MLKAWAJ.. KOBAYASHI K. & MORi A (1975) IRCS Mrd. Sci. 3. 385.
NOILVJINnMIN03 LXOHS
SHORT COMMUNICATION
Effects of guanidino compounds on rabbit brain microsomal Na’-K’ ATPase activity (Received 31 December 1975. Accepted 16 March 1976)
SOMCG U A N I U I N O compounds induced convulsions in rabbits when administered intracisternally; among them are taurocyamine. glycocyamine. N-acetylarginine. y-guanidinobutyric acid. guanidine and methylguanidine (JINNAI C’I d . 1966. 1969; MATSLMOTO t’f ul., 1976; Mizcho ei u l , 1975). The mechanism by which these compounds induce conv~lsioiihas not yet been clarified. It may he possible that guanidino compounds cause convulsion in the same manner as ouabain. a specific inhibitor of Na’-K’ activated ATPase activity. causing convulsion in rats through the disruption of the membrane potential (BIGNAMI & PALLANDI. 1966; CORNOGet ul.. 1967). It was the purpose of this study to examine whether convulsions induced by guanidino compounds are associated with an inhibition of rabbit brain microsomal ATPase activity. An adult white rabbit (body weight 2.0 kg) was killed with a strong blow to the head without use of anesthesia and the whole hrain except for the cerebellum taken out. Thc brain tissues were homogenized with 9 vol. of 0.25 M-sucrose. I mM-EDTA (pH 7.0) using a Teflon-glass homogenizer. The whole procedure was carried out at 0 -4‘C. The homogenate was centrifuged at 1O.oOOy for 70 min to precipitate unbroken cells, nuclei and mitochondrial fractions. The supernatant was centrifuged at 105.000y for 60 min to obtain a microsomal fraction. The precipitate was washed twice with 0.2.5 M-sucrose-l mhi-
TAFIL~ I EFFKTS
EDTA and repeating the centrifugation at 105.000g for 60 min. The final sediment was suspended in the same sucrose-EDTA medium to give a protein concentration of 4 5 rng/ml and kept at -2O’C until further. Protein concentration was assayed by the method of LOWRY et ol. (1951). ATPase assay was carried out as follows: the complete reaction mixture contained 50 mM-imidazole buffer (pH 7.0). 5 mM-MgCl,, 100 mM-NaCI. 20 mM-KCI and 5&100 pg enzyme protein in a total volume of 1.0 ml. Ouabain concentration was, where used 0.2 mM. The mixture was preincubated at 37’C for 5 min. The reaction was started with the addition of 0.1 ml of a 40mM-ATP solution. After 10 min at 37°C the reaction was terminated by the addition of 1.0 ml of cold 16% perchloric acid. The precipitated protein was removed by centrifugation. The liberated inorganic phosphate content was determined by the method of MARTIN& D ~ T (1949). Y Enzyme activity was expressed as pmol P, released in I h per mg protein. All assays were carried out in triplicate. The average specific activity of total ATPase (14 experiments) in the preparation was found to be 47 5pmol/mg protein/h and 17 3 for Mg2+ ATPase. 30 7 for Na’-K+ ATPase respectively. ERects of guanidino compounds o n ATPase were examined by adding each compound to the reaction mixture
+
OF GUANIDINO COMPOLINDSou RABBIT BRAIN MICROSOMAL
5.0 S'O SO S'O
S'O
s
S'O
5'0
i
05 5'0 s '0 5'0 S'O
E
E P El 8
* Slgnificantly differenc from control ( P < 0 I ” J 635
+
ATPASEA C T I ~ I T I
616
Short communication
to produce a final concentration of 0.5 mM. The examined gusnidino compounds %ere taurocyamine. glycocyamine. guanidinosuccinic acid. ,V-acetylarginine. ,6-guanidinopropionic acid, ;-guanidino-B-hydroxybutyric acid, 7-guanidinobutyric acid. arginine. homoarginine. guanidine and methylguan idine. Out of the eleven guanidino compounds. only methylguanidine reduced Na+-K' ATPase activity (20 2 7 pmol, mg protein:h) to 67"" of control values ( P < 0.1"" for the difference between the control and the methylguanidrne added group). None of the other ten guanidino compounds, including taurocyamine. glycocyamine, N-acetylarginine, 7-guanidinobutyric acid and guanidine, which have convulsive effects in rabbits, inhibited Na+-K+ ATPase activity. None of the eleven compounds tested showed any effect on Mg" ATPase activity (Table I ) . Methylguanidine behaved as a noncompetitive inhibitor. Its inhibitory activity was much lower than that of ouabain Our results suggest that the basic mechanism involved in convulsive eRects of guanidino compounds is not connected with brain microsomal Na'-K + ATPase activity.
1,lirirurr /or
Nuiirohiolog?. Okuyuim L:iiirrrsily A f r4icul School. Sltikoro-clro 2-5-1. Okajarriu. Jupurr
BIGNAMI
A. &
REFERENCES G . (1966)
PALLAND1
M MATS~'MUTO A Mo~i
A"1ttri-t..
Lnrid. 209.
4 13-414.
COKNOCJ. L.. JR.. CONATAS N. K. & Fi IrKMAl J. R . (19671 . 4 1 i i w . J . Parhol. 51. 573-590. JlNNAl D . SAWAl A. & MORIA. (1966) ~Vofiirc.. Loiitl. 212. 61 7 JINNAI D.. MOW A,. MCKAWA3 . OHKLISL H.. HOSOTASI M.. MIZCWO A. & TYEL. C. (1969) Jap J . Bruirr Physrol. 106. 3668-3673. MATSLMOTO M.. KOBAYASHI K.. KISHILAWA H . & MORI A. (1976) IRCS bled. Sci. 4. 65 LOWRV0. H.. ROSEBROLGHN.H.. FARRA. L. & RA\DALL R. J. (1951) J . h i d Clirm. 193. 26.5-275 MARTINJ. B. & DOTYD. M. (1949) ,411uh.r C l i t ~ .21. 965-967. MIZUNOA,. MLKAWAJ.. KOBAYASHI K. & MORi A (1975) IRCS Mrd. Sci. 3. 385.
