Life Sciences Vol. 3, pp . 1117-1122, 1964 . Pergamon Press, Inc . Printed in the United States .
THE EFFECT OF INDOLEPYRUVIC ACID, PHENYL PYRUVIC ACID AND PARAHYDROXY PHENYLPYRUVIC ACID ON ISOLATED RAT UTERUS Tor Norset.h and Ragnar Haavaldsen Institute of Occupational Health, Oslo 3, Norway
(Received 22 June 1964 ; in final form 15 July 1964) In a recent publication (1) it has been reported that, all aromatic amino acids except histidine are transaminated by rat, brain extract, and three enzymes mediating these reactions are separated (2) .
The possibility therefore exists
that the corresponding keto-acids are normal metabolites of the brain, and in fact,, phenylpyruvic acid and indolpyruvic acid have been identified (3 ) .
The
central position of these keto-acids in some forms of mental anomalies (4) initiated the investigation of a possible pharmacological effect of phenylpyruvic acid (PPA), indolpyruvic acid (IPA) parahydroxy phenylpyruvic acid (pHPPA), Materials and Methods All chemicals were of purest obtainable quality and deion_ized water was used throughout for preparation of solutions . Tests for biological activity were performed on isolated uterus from virgin female rats injected intraperitoneally with 0 .5 mg diethylstilboestrol 21~ hours prior t .o extirpat.ing the muscle . tion at about I~
0
The uterine horns were kept in Dejalons solu-
for 12 t,o 36 hours, and then tested in an organ bath with 0
Dejalons solution at 30 .
With the test substance added in doses of 0 .1 to 0,5
ml the bath in total contained 5 ml . concentrations added t.o the bath,
All substances used are expressed in molar
Serotonin, at the concentration 1 x 10
served as a standard stimulation of the muscles . otherwise not. stated .
Contact . time was ßt5 seconds if
Usually 5 minutes passed betyreen tests,
All solutions
were made fresh daily and used within 6 hours . The isotonic contractions of the muscles were recorded on a smoked drum
1117
1118
EFFECT OF IPA, PPA, AND pHPPA
using horrizontal levels magnifying about peaks .
5
Vol . 3, No. 10
times and loaded to give suitable
In some tests serot.onin and the keno-acid were acting together in the
bath, and a possible chemical reaction between the substances was excluded by recording the spectrum of the mixtures, and by chromatography on paper (5) . Results Figure I shows that PPA induced a contraction of the muscle if concentrations of
5
x 10 ~ or 1 x 10 -3 M are used .
The concentration 1 x 10 -4M, however,
did not give any contraction, nor did it affect the response to serotonin stimulation . It is seen from figure II that the serotonin-induced contraction was antagonized by IPA in the concentrations 1 x 10 -3 M and 5 x 10 -3M .
5mn .
UUUU~JUUUUUU`
1
2
3
4
5
6
7
B
9
10
11
'2
13
FIG . I 4,5,8 indicate 1,2,3 ,,7,9,11,13 indicate standard serot.onin stimulation . PPA 1 x 10 - M, 5 x 10 -1=M and I x 10' M respectively . 10 and 12 indicate PPA 1 x 10'~M, contact time 30 seconds, and standard stimulation with PPA in the bath . Further tests have shown a slight antagonism when using the concentration
5
x 10 -4M, but . n o effect with 1 x 10 - ~M .
Also figure II shows that the IPA
antagonism is present for several standard serotonin stimulations subsequent to the test where IPA is added to the bath .
This inhibitory effect gradually de-
creases with repeated serotonin stimulations . As it appears from figure III the effect is also found when IPA is added to the bath between the serotonin stimulations, and consequently is not acting together with serot .onin in the bath,
but the concentrations and contact time
EFFECT OF IPA, PPA, AND pHPPA
Vol. 3, No . 10
1119
AA
J~ILiu~uu~~~u
1
2
3
5
4
6
7
8
9
10 11
12 ~
Y+
FIG . II All numbers indicate s+andard serotonin stimulation . 3 and 5 indicate IPA 1 x 10 -3 M and 5 x 10 -3 M respectively, contact time 15 seconds with washing before standard stimulation . 7 and 10 indicate IPA 1 x 10 -3 M and 5 x 10 -3 M respectively, contact time 30 seconds without washing before standard stimulation . 5min . -,
w
Jv~UU~U~ 5 6 1 2 3 4 7 FIG . III Between 2 and 3 IPA All numbers indicate standard serotonin stimulation . 5 x 10 - M is added 3 times with a total contact time of 3 minutes with washing before serotonin is added . then have to be increased . Figure IV and V show that. pHPPA has almost identical effect as IPA, figure II and III . All results were reproduced with identical peaks at least. twice on different uterine muscles, and all tests have given the same pattern . Discussion Y~ittle has been published concerning the biological effects of aromatic
EFFECT OF IPA, PPA, AND pHPPA
1120
Vol . 3, No. 10
5min .
1
2
3
4
5
6
7
8
9
10
FIG . IV All numb rs indicate standard serotonin stimulation . 3 and ~ indicate pHPPA 1 x 10 -~M and 5 x 10 -3 M respectively, contact time 15 seconds with washing before standard stimulation . 5 and 8 indicate pHPPA 1 x 10-3 M and 5 x 10 -3 M, contact, time 30 seconds without washing before standard stimulation .
A
'VUUUUU~J~1
2
3
4
5
6
~
8
FIG, V All numbers indicate standard serotonin stimulation . Between 3 and ~ pHPPA 5 x 10 -3M is added 3 times with a total contact time of 3 minutes with washing before serotonin is added . keto-acids and related compounds .
Recently the aldehydes of serotonin and
trypt.amine were reported to be without pharmacological effect. on the isolated rat uterus and guinea-pig ileum (6) .
Howell and Lee (7) have found that phenyl-
pyruvic acid causes a decrease in oxygen utilization of rat. brain slices .
No
conclusion is given, and the possibility of a connection t.o the mental symptons of some diseases of the amino acid metabolism is outlined .
Pogrund, Drell and
Olark (8) have reported that met.ahydroxyphenylpyruvic acid and 3,L~-dihydro-
EFFECT OF IPA, PPA, AND pHPPA
Vol . 3, No. 10
1121
xyphenylpyruvic acid increase the blood pressure of cats and rats when injected intravenously .
Metatyramine and dopamine were present in the urine from the
animals, and it is suggested that these substances mediated the pressor response .
No effect was found after parahydroxyphenylpyruvic acid, nor UTas t .yramine
found in the urine . As IPA is found as a normal metabolite in brain tissue, the possible antagonistic effect to serotonin is of interest due to the pharmacological effect observed from serotonin antagonists and substances containing indole st.ruct .ure . irlhether this antagonistic effect is due to inhibition of some enzymic process in the production of serotonin, uncertain .
or inflicts some transmission mechanism, is
The lack of a lag time in the effect of PPA on the uterine muscle
indicates that some transmission mechanism is involved .
This is also a possible
explanation why the effects on the three keto acids are not identical, as different effects of related substances in this mechanism would not be surprising .
Some effect of these keto acids has also been found in the guinea-pig and
rat ileum, but the results are not so easily interpreted . more detailed report elsewhere .
GTe hope to give a
Concerning the effect. of these keto acids on
other organs not much can be stated, heart, blood vessels and bronchical tree are perhaps the most possible physiological target organs in addition t.o the central nervous system . The present publication gives some very interesting aspects, as the amino acid precursor of the amines can also give rise to other pharmacological active substances, i .e . aromatic
-keto acids . References
196, 577 (1962) .
1.
R . HAAVALDSEN, Nature (Lund .)
2.
F . FGNNUM, R . HAAVALDSEN and 0 . TANGEN, J . Neurochem . , 11,
3.
R . HAAVALDSEN,
4.
A.
5.
K . SCHtyARZ, Arch . Biochem . biophys . ,
Biochem . J,
109 (1964) .
in press .
FELLING, Hoppe-Seglers Z, physiol . Chem . ,
227, 169 (1934) .
92, 168 (1961) .
EFFECT OF IPA, PPA, AND pHPPA
1122 6.
Vol . 3, No. 10
J, RENSGN, H . GTEISSBACH and S . UDENFRIEND, J, Pharmacol . exp, Ther . ,
113,
326 (19610 , 113, 660 (1963) .
7.
R .K, HOGIELL and M, LEE, Proc, Soc, exp, Biol, and Med . ,
8,
R, S, POGRTJND, W . DRELL and~~i .G, CLARK, J . Pharmachol, exp . Ther . ,
(1961) .
131, 29~