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Effect of reserpine on monoamine oxidase activity in guinea pig heart
Vol . 6, pp, 2333-2343, 1967 . Life Sciences Printed in Great Britain .
Pergamon Press Ltd .
EFFECT OF R~ERPINE ON MONOAMINE OXIDASE ACTIVITY IN GUINEA PIG HEART F .Izumi, M .Oka, H.Yoshida and R.Imaizumi Department of Pharmacology, Faculty of Medicine, Osaka University, Kitaku, Osaka, Japan (Received 19 June 1967 ; in final form 8 August. 1967) It has been shown by several groups of workers (1-8) that there are at least two pools of bound norepinephrine (NE) in tissues ; one is a small "available" pool which car. be released by tyramine and by nerve stimulation (tyramine-sensitive pool) and the other is a larger, "firmly bound" pool which can be released by reserpine (tyramine-resistant pool),
Moreover, the
metabolic fate of the NE released from these two pools has been demonstrated. to be different (3,9) ; the NE released from the "available pool" is primarily 0-methylated by catechol-0-methyltransferase (COMT), whereas the NE released from the "firmly bound" pool is predominantly deaminated by monoamine oxidase (B~AO) .
This is supported by the experiments of Kopin and Gordon
(10-12) showing that NE released by tyramine mostly passes directly into the circulation, though some is 0-methylated in the tissues .
On the other hand, the NE released by reserpine is
predominantly deaminated in the tissues, and only a little is released in an active form . Recently, however, the presence of more than one bound pool of NE in the heart (the tyramine sensitive and tyramine resistant pools) has become doubtful .
Neff et al . (13) and Gutman and
Weil-Malherbe (14) found that, when a high concentration of
2334
EFFECT OF RESERPINE
Vol . 6, No . 21
tyramiae is maintained by continuous infusion, the NE in the heart decreases with a single exponential rate and NE stores are lowered by a maximum of 95 %.
Jonsson et al . (15) also
reported similar results with phenylethylamine .
From these
findings it seems questionable whether NE stores can be divided into tyramine sensitive and tyramine resistant pools . Furthermore, the finding that the NE released by reserpine is predominantly deaminated, while the NE released by tyramine is not, may also be simply explained as being the result of activation of mitochondrial MAO by reserpine, rather than representing a true difference in the metabolic fates of two actual NE pools released by reserpine and tyramine, respectively . Therefore, the present studies were undertaken to investigate the effect of reserpine on MAO activity is guinea pig heart . It has already been reported from our laboratory that MAO acti vity in the brain stem of guinea pigs is stimulated by reserpine administration (16) . Methods The methods used to estimate the activity of MAO in the heart, were based on measurements of radioactivity in deaminated products of 14C-labeled catecholamines, 14 C-NE and 14 C-Dopamine (DA), or 14 C-Tyramine . Guinea pigs (250-300g) were used throughout the study .
A
single intraperitoneal injection of 5 mB/KB of reserpine(sexL pasil, Ciba), was given and animals were killed by decapitation 2 .5 hr later.
Their hearts were pooled and chilled in ice cold
Ringer solution (154 mid NaC1, 5.6 mM KC1, 2 .2 mM CaC1 2 , 1 .3 MgS04 , 10 mM glucose, 40 mM Tris maleate buffer, pH 6 .8) . In experiments with slices, hearts were sliced with a
Vol . 6, No . 21
EFFECT OF RESERPINE
2335
Stadie-Rigg slicer and weighed immediately on a torsion balance . About 200 mg of slices (each slice weighed about 50 mg) were incubated is
3 ml of Ringer solution as described in the foot-
note of Table 1 .
The reaction urns stopped by addition of per-
chloric acid at a final concentration of 0.4 N .
Slices were
homogenized in a glass Potter homogenizer and 14C-deaminated amine compounds were extracted by standing the homogenate at 0°C for 1 hr .
Then the homogenate was centrifuged and the precipi-
tate was washed once with 2 ml of 0 .4 N perchloric acid .
The
washings and supernatant were pooled and charged onto a Dowex
50 column (H + form, 0 .4 x 4 cm) to absorb the 14 C-amines .
In
this vray, the radioactivity of 14 C-amine was absorbed onto the Dowex
50 column .
The radioactivity in the fractions of eluate
containing the deaminated products of 14 C-amine was measured in a Tricarb scintillation spectrophotometer. For experinenta in which heart homogenates were used for estimation of L1A0 activity, hearts were homogenized using a glass Potter homogenizer in Ringer solution containing 40 mM Tris maleate buffer, pH 6 .8 .
The homogenates were filtered
through gauze to remove coarse debris .
The homogenate was in-
cubated vrith 14 C-amine without shaking, as shown in the footnote of Table 2 .
Subsequent procedures were the same as those
used with slices . 'Nhen the r~itochondrial fraction of heart was used as the t7+A0 preparation, hearts were homogenized in ten volumes of 0.25 P,I sucrose containing 40 mf~ Tris maleate buffer, pH 6 .8 .
The
homogenate urns centrifuged at 600 x & for 10 min, to remove the debris and the supernatant was then centrifuged. at 8,000 x ~ for 20 min .
The resulting sediment was suspended in Ringer solution
2336
EFFECT OF RESERPINE
containing 40 mY Tris maleate buffer, pH 6 .8 .
Vol . 6, No . 21
Incubations of the
mitochondrial fraction with 14 C-amine were carried out without shaking as described in the footnote of Table 3 .
Subsequent
procedures for estimation of MAO activity were the same as is ezperüents with slices .
As the substrate for MAO, NE-7-14 C
(bitarturate), DA-~140 (HC1), Tyr-7-14C (gam) was used at final concentrations of 5 .3x10-6M (62104 CPM), 5 .3x10-85i (4,8x104 CPM), and 4.4x10-51I (10.9x104 CPèI), respectively . Results and Discussion Heart slices from normal guinea pigs and those pretreated 2 .5 hr previously with reserpine were incubated with l4 C-NE in Finger medium at 37oC for 1 hr .
As shown in Table 1, in the
latter slices, the amount of deaminated metabolites of 14 C-NE was more than in the controls . vious findings of Iversen et al .
This result agrees with the pre(17) that on perfusion of
hearts isolated after pretreatment with reserpine the 3HrNE taken up from the perfusion medium is mainly metabolized to deaminuted compounds . Table 2 shows the deamination of 14C-NE in heart homogenates of normal guinea pigs and those pretreated with reserpine . After pretreatment with reserpine the deaminated products of 14G~ were also significantly more than normal . This increase of deamination of 14 C-NE in slices or homogenates of reserpinized hearts is probably a consequence of activation of heart MAO activity by reserpine pretreatment .
How
ever, it is also possible that reserpine inhibits the binding of 14GRE into intracellular granules so that more free 14 C-NE is metabolized by MAO instead of being bound .
Vol . 6, No . 21
EFFECT OF RFSERPINE
2337
TABLE 1 Increase oî MAO Activity by Reaerpine Treatment (Guinea Pig Heart Slices) MAO Activity (CPM x10-2 /100 mg wet weight of tissue /hr) Control
Reserpiaized
12915
183 112
(15) (5) *CPM of Deaminated Metabolites
(96) Increase 40115
Guinea Pigs were treated with reserpine (5 mB/Sg, LP .) 2 .5 hours previously . The incubation medium coatain~d 200 mg of heart slices, 5 .3x1 6 M of dl- ~4 C-NE (6zl CP1t), 40 mM of tris maleate buffer (pH 6 .8), 154 mY of NaCl, 5 " 6 mM of gCl, 2 .2 mM of CaC12 , 1 .3 ~ of 1Lg.Sq~and 10 mY of glucose in a total volume of 3 ml . The incubation was carried out at 37° C for 1 hour with shaking.
TeHLE 2 Increase of MAO Activity by Reaerpine Treatment (Guinea Pig Heart Homogenate) 11A0 Activity (CPId*/mg proteia/hr) Control 237 f 11 (20)
Reaerpinized 425 t 84 (20)
(96) Increase 79
t
39
*CPM of Deaminated Metabolites Guinea Pigs were treated with reserpine (5 mg/gg, i.p .) 2 .5 hours previously . The 3~cubation medium contained 5 .3x10`6 M of dl-~4 C-NE (6xl0~CPM) 40 mM of tris maleate buffer (pH 6.8), 154 mM of NaC1, ~ .6 mM of gCl, 2.2 mY of CaCh , 1 .3 ~ of M~04, 10 mM of glucose and about 50 mg of protein 1n a total volume of 3 ml . The incubation was carried out at 37 °C for 1 hour without shaking.
Vol . (i, No . 21
EFFECT OF RESERPINE
2338
The major storage sites for endogenous and exogenous NE are kno~tn to be localized, not in the mitochondrial fraction, but in the microsomal fraction in the heart (18-21) .
There
fore, is the following experiments, the mitochondsial fractions of hearts from normal and reserpine-pretreated guinea pigs were used as the enzyme preparations of M&C .
As shown in Table 3,
an increased amount of deaminated products of 14 C-NE was also observed in the mitochondrial fraction from reserpinized guinea pig hearts as compared. to that in the mitochondrial fractions from normal guinea pig hearts . TABLE 3 Increase of MAO Activity by Reserpine Treatment (Guinea Pig Heart ll[itochondria)
MAO Activity (CPM* /mg protein /hr) Control
Reserpinized
451t19
643t68
(15)
(~) Increase 45t23
(15)
*CP1I of Deaminated metabolites Guinea Piggy were treated with reserpine (5 mg/Kg, i .p .) 2 .5 hours previously . The incubation medium contained 5 " 3x10'6 M of dl-~ 4C-NE (6x10 4CPM), 40 mM tris maleate buffer (pH 6.8) 154 mM of NaCl, 5.6 mM of KC1, 2 .2 mM of CaC12, 1 .3 ~ of MgSCq, 10 mè~ of ~r_lucose and about 10 mg of protein in a total volume of 3 ml . Incubation was carried out at 37 ° C for 1 hour without shaking. Therefore, these results show that reserpine treatment activates heart mitochondrial MAO activity, resulting in increased deamination of 14 C-NE in reserpinized guinea pig hearts .
EFFECT OF RESERPINE
Vol . 6, No . 21
2339
The mechanism by which reserpine activates heart mitochondrial MAO is not yet known .
However, as shown in Table 4, it
was observed that when 14C-DA or 14C-tyramine was used as a substrate for estimation of 31A0 activity, deamination of DA and tyramine was much more than that of NE .
However there was little
increase is the deamination of DA and tyramine in the reserpine pretreated hearts relative to the controls . TAHLE 4 Increase of MAO Activity by Reserpiae Treatment (Guinea Pig Heart Homogenate) MAO Activity (CPèt* /mg protein/hr) Substaate
Control
Noradrenaline
237 t 11 (20)
Dopamine
620
t
Reserpinized
50
( 5) Tyramine
1600 t 125 ( 5)
84
79
t 39
770 * 112
24
t
425
f
(96) Increase
(20) (5) 1605
f 117
4
0
(5)
*CPP~; of Deaminated Metabolites Guinea Pigs were treated with reserpine (5 mg/Kg, i .p .) 2 .5 hours previously . Incubation was carried out in normal Ringer solution at H 6 .8 (40 mho tris maleate buffer) with~4 C-NE (5 .3x10-6 è6, 6x10 1 CPN.~, ~4 C-DA (5 .3x10- ~ M, 4 .8x10aCPtd) or ~4 C-Tyramine (4 .4x lU'sM, 10 .9x10~CPP:i) as substrate . The final volume of the incubation medium was 3 ml and incubations were at 37°C for 1 hour ~r;ithout shaking . It was also found, as shown in Tables 5 and 6, that even when 14C-NE was used as a substrate for MAO, there was little or no increase of deamination of NE in reserpinized guinea pigs
2340
EFFECT OF RESERPINE
Vol . 6, No . 21
hearts in hypotonic medium which presumably caused swelling or structural changes of heart mitochondria leading to increased deamination of 14C-NE .
The increase of NE deamination
under hypotonic conditions seems to be is accordance with our previous results (22) and those of others (23) showing that the activity of mitochondrial MAO is related to the structural state of the mitochondria .
' TASLE 5
$ffect ôf Tonicity on the MAO Activity of a Guinea Pig Heart Homogenate YAO Activity (CPM*/mg protein/hr) Tonicity
Control
Reserpinized
(°~) Increase
Isotonic
194 t 15 C5)
350 t 27 (5)
80
Hypotonic
291 f 12 (5)
312 f 19 (5)
7
t 38 f
2
*CPY of Deaminated Metabolites Guinea Pigs were treated with reserpine (5 mg/Kg, i .p .) 2 .5 hours previously . Hearts were homogenized in isotonic (AaCl 154 mY, KC1 5 .6 mY) and hypotonic (NaCl-BCl-free) medium both containing 40 mM of tris maleate buffer (pH 6.8) and used as the enzyme preparations . dl-~ 4 C-NE (5 .3ac10 -6 M, 6x10 4C~PM) was used as substrate . Incubation urea carried out at 37°C for 1 hour without shaking . An interesting possibility based on these findings is that reserpine may act on the mitochondria) membrane or structure to increase the penetration of NE to mitochondria) MAO.
In this
way the deaminatioa of NE would be increased is reserpinized hearts .
The well known fact that the NE released by reserpine
is mainly metabolized to deaminated compounds has been explained
Yol. 6, No . 21
EFFECT OF RESERPINE
2341
TAHL$ 6 Sffect of Tonicity oa the léAO Activity of Guinea Pig Heart Mitochondria MAO Activity (CPiI*/mg protein/hr) Tonicity
Control
Isotonic
370 t (5)
18
537 t ~
Hypotonic
507 ~
29
5~
(5)
Eeserpinized
(5)
t 23
(~) Increase 45 t
19
0
(5)
*CPM of Deaminated Setabolites Guinea Pigs were treated with reaerpine (5 mg/%g, i.p .) 2.5 hours previousl . Hearts were homogenized in NaCl (154 mM)-BC1(5 .6 mll~ solution containing 40 mM of tris maleate buffer (pH 6.8) and mitochondria were isolated by the routine method . Mitochondria were suspended is isotonic (NaCl 154 mM, BC1 5.6 mM) and hypotonic (NaClSC1-free) solutions both containing 40 mM of trig maleate buffer (pH 6 .8) . d1-14 C-NF (5odp'6M 6x104CPM) was used as substrate . Incubation wen carried out at 37 ° C for 1 hour without shaking . by the attractive hypothesis proposed by gopin et al . (10,11,12) that reserpine can release the NE from the "firmly bound" NEpool and ao it is primarily deaminated .
However, this pheno-
menon can also be egplaiaed simply by the action of reserpine in facilitating the deamination of NE by mitochondrial MAO, as described above.
The finding that the NE released by tyramine
is primarily metabolized by COMT might also be considered to be because tyramine inhibits the deaminatioa of released NE by mitochondrial MAO competitively since it is known to be a better substrate of MAO than NE . vated by COMT than by MAO .
In this way much more NE is inacti-
2342
EFFECT OF RFSERPINE
Yol . 6, No . 21
Further studies are required on the relationship between the depletion of tissue RE and the increase of MAO activity induced by reserpine .
However, an interesting possibility is
that the action of reserpine is increasing deamiaation of NE by mitochoadrial MAO may play a significant role in the NE depletiag effect of reserpiae .
This possibility is supported
by the previous findings (24-28) that pretreatment with ~IAOinhibitors prevents the depletion of tissue NE caused by reserpine administration . Sumne~r~
8eserpine treatment activates mitochondrial MAO activity of guinea pig hearts .
Reßerpiae may act on the mitochondrial
membrane or structure to increase the penetration of norepine phriae to mitochondrial D~AO .
Ia this way the deaminatioa of
norepinephrine would be increased in reserpinized hearts . References 1 . U. Trendelenburg, J . Phar.aacol . , 134, 8 (1961) . 2 . L .T . Potter, J . Axelrod and LJ . Kopin, Biochem. Pharmacol . , 1~, 254 (1962) . 3, I .J . Kopin, G . Hertting and E .K . Gordon, J_ Pharmacol . , 34 (1962), 4, J . Axelrod, E.S : Gordon, G. Herttiag,I .J .Kopin and L .T . Potter, Br it . J . Pharmacol . , 12, 56 (1962) . 5 . T .R . Crout, A .J . Muskus and U . Trendelenburg, Brit . J . Pharmacol ., 18, 600 (1962) . 6. J .H . Burn and M.J . Rand, J . Physiol . , 144, 314 (1958) . ~. J .H . Burn and. M.J . Rand, J . Physio l-, 142, 135 (1959) . 8 . J .H . $urn and M.J . Rand, J . Physiol . , 1~0, 295 (1960) .
Vol . 6, No . 21
EFFECT OF RESERPINE
2343
9 . LJ . %opia and P.% . Cordon, Fed . Proc .,21, 332 (1962) . 10 . LJ . %opin and T.% . Cordon, J . Pharmacol . , ~, 351 (1962) . 11 . I.J . %opin and L.% . Cordon, J. Pharmacol . , 140, 207 (1963) . 12 . LJ . %opin, Pharmacol . Rev . , 16, 179 (1964) . 13 . N.H . Heff, T .N . Toter, W. Hammer and B.B . Bsodie, Life Sciences , 4, 1869 (1965) " 14 . Y. Guturan and H. Weil-Malherbe, Life Sciences , ~, 1293 (1966) . 15 . J . Jonsson, H. Grobecker and P. Holtz, Life Sciences, 2235 (1966) . 16 . T . Ithoh, M. Matauoka, %. Nakajima, B. Taßawa and R. Imaizumi, Jap . J . Pharmacol . , 12, 130 (1962) . 17 . L .L . Iversen, J . Glowinski and J . Axelrod, J . Pharmacol . , 173 (1965) " 18 . L .T . Potter and J . Axelrod, J . Pharmacol . , 142, 291 (1963) . 19 . L .T . Potter and J . Axelrod, Rature , ~, 581 (1963) . 20 . I .A . Michaelson, %.C . Richardaon, S .N . Snyder and E .O . Titus, Life Sciences ~, 971 (1964) . 21 . L .T . Potter, Pharmacol . Rev . ,18, 439 (1966) . 22 . T . Nukada, T . Sakurai and R . Imaizumi, Jap . J. Pharmacol . , 124 (1963) . 23 . H . Aebi, F . Stocker and M. Eberhardt, Biochem-Z ., 526 (1963) . 24 . A . Horita and 'sN.R . McGrath, Biochem. Pharmacol ., ~, 206 (1960) . 25 . S . Spector, R . %untzman, P .A . Shore and B.B . Brodie, J . Pharmacol . f ~, 256 (1960) . 26 . J . Axelrod, G. Hertting and R.W . Patrick, J . Pharmacol . , 325 (1961) . 27 . F. Clementi and G .P . Zocche, J, of Cell Biol~, ~, 28 . F. Clementi, Experientia , 21, 171 (1965)
587 (1963) .
Pergamon Press Ltd .
EFFECT OF R~ERPINE ON MONOAMINE OXIDASE ACTIVITY IN GUINEA PIG HEART F .Izumi, M .Oka, H.Yoshida and R.Imaizumi Department of Pharmacology, Faculty of Medicine, Osaka University, Kitaku, Osaka, Japan (Received 19 June 1967 ; in final form 8 August. 1967) It has been shown by several groups of workers (1-8) that there are at least two pools of bound norepinephrine (NE) in tissues ; one is a small "available" pool which car. be released by tyramine and by nerve stimulation (tyramine-sensitive pool) and the other is a larger, "firmly bound" pool which can be released by reserpine (tyramine-resistant pool),
Moreover, the
metabolic fate of the NE released from these two pools has been demonstrated. to be different (3,9) ; the NE released from the "available pool" is primarily 0-methylated by catechol-0-methyltransferase (COMT), whereas the NE released from the "firmly bound" pool is predominantly deaminated by monoamine oxidase (B~AO) .
This is supported by the experiments of Kopin and Gordon
(10-12) showing that NE released by tyramine mostly passes directly into the circulation, though some is 0-methylated in the tissues .
On the other hand, the NE released by reserpine is
predominantly deaminated in the tissues, and only a little is released in an active form . Recently, however, the presence of more than one bound pool of NE in the heart (the tyramine sensitive and tyramine resistant pools) has become doubtful .
Neff et al . (13) and Gutman and
Weil-Malherbe (14) found that, when a high concentration of
2334
EFFECT OF RESERPINE
Vol . 6, No . 21
tyramiae is maintained by continuous infusion, the NE in the heart decreases with a single exponential rate and NE stores are lowered by a maximum of 95 %.
Jonsson et al . (15) also
reported similar results with phenylethylamine .
From these
findings it seems questionable whether NE stores can be divided into tyramine sensitive and tyramine resistant pools . Furthermore, the finding that the NE released by reserpine is predominantly deaminated, while the NE released by tyramine is not, may also be simply explained as being the result of activation of mitochondrial MAO by reserpine, rather than representing a true difference in the metabolic fates of two actual NE pools released by reserpine and tyramine, respectively . Therefore, the present studies were undertaken to investigate the effect of reserpine on MAO activity is guinea pig heart . It has already been reported from our laboratory that MAO acti vity in the brain stem of guinea pigs is stimulated by reserpine administration (16) . Methods The methods used to estimate the activity of MAO in the heart, were based on measurements of radioactivity in deaminated products of 14C-labeled catecholamines, 14 C-NE and 14 C-Dopamine (DA), or 14 C-Tyramine . Guinea pigs (250-300g) were used throughout the study .
A
single intraperitoneal injection of 5 mB/KB of reserpine(sexL pasil, Ciba), was given and animals were killed by decapitation 2 .5 hr later.
Their hearts were pooled and chilled in ice cold
Ringer solution (154 mid NaC1, 5.6 mM KC1, 2 .2 mM CaC1 2 , 1 .3 MgS04 , 10 mM glucose, 40 mM Tris maleate buffer, pH 6 .8) . In experiments with slices, hearts were sliced with a
Vol . 6, No . 21
EFFECT OF RESERPINE
2335
Stadie-Rigg slicer and weighed immediately on a torsion balance . About 200 mg of slices (each slice weighed about 50 mg) were incubated is
3 ml of Ringer solution as described in the foot-
note of Table 1 .
The reaction urns stopped by addition of per-
chloric acid at a final concentration of 0.4 N .
Slices were
homogenized in a glass Potter homogenizer and 14C-deaminated amine compounds were extracted by standing the homogenate at 0°C for 1 hr .
Then the homogenate was centrifuged and the precipi-
tate was washed once with 2 ml of 0 .4 N perchloric acid .
The
washings and supernatant were pooled and charged onto a Dowex
50 column (H + form, 0 .4 x 4 cm) to absorb the 14 C-amines .
In
this vray, the radioactivity of 14 C-amine was absorbed onto the Dowex
50 column .
The radioactivity in the fractions of eluate
containing the deaminated products of 14 C-amine was measured in a Tricarb scintillation spectrophotometer. For experinenta in which heart homogenates were used for estimation of L1A0 activity, hearts were homogenized using a glass Potter homogenizer in Ringer solution containing 40 mM Tris maleate buffer, pH 6 .8 .
The homogenates were filtered
through gauze to remove coarse debris .
The homogenate was in-
cubated vrith 14 C-amine without shaking, as shown in the footnote of Table 2 .
Subsequent procedures were the same as those
used with slices . 'Nhen the r~itochondrial fraction of heart was used as the t7+A0 preparation, hearts were homogenized in ten volumes of 0.25 P,I sucrose containing 40 mf~ Tris maleate buffer, pH 6 .8 .
The
homogenate urns centrifuged at 600 x & for 10 min, to remove the debris and the supernatant was then centrifuged. at 8,000 x ~ for 20 min .
The resulting sediment was suspended in Ringer solution
2336
EFFECT OF RESERPINE
containing 40 mY Tris maleate buffer, pH 6 .8 .
Vol . 6, No . 21
Incubations of the
mitochondrial fraction with 14 C-amine were carried out without shaking as described in the footnote of Table 3 .
Subsequent
procedures for estimation of MAO activity were the same as is ezperüents with slices .
As the substrate for MAO, NE-7-14 C
(bitarturate), DA-~140 (HC1), Tyr-7-14C (gam) was used at final concentrations of 5 .3x10-6M (62104 CPM), 5 .3x10-85i (4,8x104 CPM), and 4.4x10-51I (10.9x104 CPèI), respectively . Results and Discussion Heart slices from normal guinea pigs and those pretreated 2 .5 hr previously with reserpine were incubated with l4 C-NE in Finger medium at 37oC for 1 hr .
As shown in Table 1, in the
latter slices, the amount of deaminated metabolites of 14 C-NE was more than in the controls . vious findings of Iversen et al .
This result agrees with the pre(17) that on perfusion of
hearts isolated after pretreatment with reserpine the 3HrNE taken up from the perfusion medium is mainly metabolized to deaminuted compounds . Table 2 shows the deamination of 14C-NE in heart homogenates of normal guinea pigs and those pretreated with reserpine . After pretreatment with reserpine the deaminated products of 14G~ were also significantly more than normal . This increase of deamination of 14 C-NE in slices or homogenates of reserpinized hearts is probably a consequence of activation of heart MAO activity by reserpine pretreatment .
How
ever, it is also possible that reserpine inhibits the binding of 14GRE into intracellular granules so that more free 14 C-NE is metabolized by MAO instead of being bound .
Vol . 6, No . 21
EFFECT OF RFSERPINE
2337
TABLE 1 Increase oî MAO Activity by Reaerpine Treatment (Guinea Pig Heart Slices) MAO Activity (CPM x10-2 /100 mg wet weight of tissue /hr) Control
Reserpiaized
12915
183 112
(15) (5) *CPM of Deaminated Metabolites
(96) Increase 40115
Guinea Pigs were treated with reserpine (5 mB/Sg, LP .) 2 .5 hours previously . The incubation medium coatain~d 200 mg of heart slices, 5 .3x1 6 M of dl- ~4 C-NE (6zl CP1t), 40 mM of tris maleate buffer (pH 6 .8), 154 mY of NaCl, 5 " 6 mM of gCl, 2 .2 mM of CaC12 , 1 .3 ~ of 1Lg.Sq~and 10 mY of glucose in a total volume of 3 ml . The incubation was carried out at 37° C for 1 hour with shaking.
TeHLE 2 Increase of MAO Activity by Reaerpine Treatment (Guinea Pig Heart Homogenate) 11A0 Activity (CPId*/mg proteia/hr) Control 237 f 11 (20)
Reaerpinized 425 t 84 (20)
(96) Increase 79
t
39
*CPM of Deaminated Metabolites Guinea Pigs were treated with reserpine (5 mg/gg, i.p .) 2 .5 hours previously . The 3~cubation medium contained 5 .3x10`6 M of dl-~4 C-NE (6xl0~CPM) 40 mM of tris maleate buffer (pH 6.8), 154 mM of NaC1, ~ .6 mM of gCl, 2.2 mY of CaCh , 1 .3 ~ of M~04, 10 mM of glucose and about 50 mg of protein 1n a total volume of 3 ml . The incubation was carried out at 37 °C for 1 hour without shaking.
Vol . (i, No . 21
EFFECT OF RESERPINE
2338
The major storage sites for endogenous and exogenous NE are kno~tn to be localized, not in the mitochondrial fraction, but in the microsomal fraction in the heart (18-21) .
There
fore, is the following experiments, the mitochondsial fractions of hearts from normal and reserpine-pretreated guinea pigs were used as the enzyme preparations of M&C .
As shown in Table 3,
an increased amount of deaminated products of 14 C-NE was also observed in the mitochondrial fraction from reserpinized guinea pig hearts as compared. to that in the mitochondrial fractions from normal guinea pig hearts . TABLE 3 Increase of MAO Activity by Reserpine Treatment (Guinea Pig Heart ll[itochondria)
MAO Activity (CPM* /mg protein /hr) Control
Reserpinized
451t19
643t68
(15)
(~) Increase 45t23
(15)
*CP1I of Deaminated metabolites Guinea Piggy were treated with reserpine (5 mg/Kg, i .p .) 2 .5 hours previously . The incubation medium contained 5 " 3x10'6 M of dl-~ 4C-NE (6x10 4CPM), 40 mM tris maleate buffer (pH 6.8) 154 mM of NaCl, 5.6 mM of KC1, 2 .2 mM of CaC12, 1 .3 ~ of MgSCq, 10 mè~ of ~r_lucose and about 10 mg of protein in a total volume of 3 ml . Incubation was carried out at 37 ° C for 1 hour without shaking. Therefore, these results show that reserpine treatment activates heart mitochondrial MAO activity, resulting in increased deamination of 14 C-NE in reserpinized guinea pig hearts .
EFFECT OF RESERPINE
Vol . 6, No . 21
2339
The mechanism by which reserpine activates heart mitochondrial MAO is not yet known .
However, as shown in Table 4, it
was observed that when 14C-DA or 14C-tyramine was used as a substrate for estimation of 31A0 activity, deamination of DA and tyramine was much more than that of NE .
However there was little
increase is the deamination of DA and tyramine in the reserpine pretreated hearts relative to the controls . TAHLE 4 Increase of MAO Activity by Reserpiae Treatment (Guinea Pig Heart Homogenate) MAO Activity (CPèt* /mg protein/hr) Substaate
Control
Noradrenaline
237 t 11 (20)
Dopamine
620
t
Reserpinized
50
( 5) Tyramine
1600 t 125 ( 5)
84
79
t 39
770 * 112
24
t
425
f
(96) Increase
(20) (5) 1605
f 117
4
0
(5)
*CPP~; of Deaminated Metabolites Guinea Pigs were treated with reserpine (5 mg/Kg, i .p .) 2 .5 hours previously . Incubation was carried out in normal Ringer solution at H 6 .8 (40 mho tris maleate buffer) with~4 C-NE (5 .3x10-6 è6, 6x10 1 CPN.~, ~4 C-DA (5 .3x10- ~ M, 4 .8x10aCPtd) or ~4 C-Tyramine (4 .4x lU'sM, 10 .9x10~CPP:i) as substrate . The final volume of the incubation medium was 3 ml and incubations were at 37°C for 1 hour ~r;ithout shaking . It was also found, as shown in Tables 5 and 6, that even when 14C-NE was used as a substrate for MAO, there was little or no increase of deamination of NE in reserpinized guinea pigs
2340
EFFECT OF RESERPINE
Vol . 6, No . 21
hearts in hypotonic medium which presumably caused swelling or structural changes of heart mitochondria leading to increased deamination of 14C-NE .
The increase of NE deamination
under hypotonic conditions seems to be is accordance with our previous results (22) and those of others (23) showing that the activity of mitochondrial MAO is related to the structural state of the mitochondria .
' TASLE 5
$ffect ôf Tonicity on the MAO Activity of a Guinea Pig Heart Homogenate YAO Activity (CPM*/mg protein/hr) Tonicity
Control
Reserpinized
(°~) Increase
Isotonic
194 t 15 C5)
350 t 27 (5)
80
Hypotonic
291 f 12 (5)
312 f 19 (5)
7
t 38 f
2
*CPY of Deaminated Metabolites Guinea Pigs were treated with reserpine (5 mg/Kg, i .p .) 2 .5 hours previously . Hearts were homogenized in isotonic (AaCl 154 mY, KC1 5 .6 mY) and hypotonic (NaCl-BCl-free) medium both containing 40 mM of tris maleate buffer (pH 6.8) and used as the enzyme preparations . dl-~ 4 C-NE (5 .3ac10 -6 M, 6x10 4C~PM) was used as substrate . Incubation urea carried out at 37°C for 1 hour without shaking . An interesting possibility based on these findings is that reserpine may act on the mitochondria) membrane or structure to increase the penetration of NE to mitochondria) MAO.
In this
way the deaminatioa of NE would be increased is reserpinized hearts .
The well known fact that the NE released by reserpine
is mainly metabolized to deaminated compounds has been explained
Yol. 6, No . 21
EFFECT OF RESERPINE
2341
TAHL$ 6 Sffect of Tonicity oa the léAO Activity of Guinea Pig Heart Mitochondria MAO Activity (CPiI*/mg protein/hr) Tonicity
Control
Isotonic
370 t (5)
18
537 t ~
Hypotonic
507 ~
29
5~
(5)
Eeserpinized
(5)
t 23
(~) Increase 45 t
19
0
(5)
*CPM of Deaminated Setabolites Guinea Pigs were treated with reaerpine (5 mg/%g, i.p .) 2.5 hours previousl . Hearts were homogenized in NaCl (154 mM)-BC1(5 .6 mll~ solution containing 40 mM of tris maleate buffer (pH 6.8) and mitochondria were isolated by the routine method . Mitochondria were suspended is isotonic (NaCl 154 mM, BC1 5.6 mM) and hypotonic (NaClSC1-free) solutions both containing 40 mM of trig maleate buffer (pH 6 .8) . d1-14 C-NF (5odp'6M 6x104CPM) was used as substrate . Incubation wen carried out at 37 ° C for 1 hour without shaking . by the attractive hypothesis proposed by gopin et al . (10,11,12) that reserpine can release the NE from the "firmly bound" NEpool and ao it is primarily deaminated .
However, this pheno-
menon can also be egplaiaed simply by the action of reserpine in facilitating the deamination of NE by mitochondrial MAO, as described above.
The finding that the NE released by tyramine
is primarily metabolized by COMT might also be considered to be because tyramine inhibits the deaminatioa of released NE by mitochondrial MAO competitively since it is known to be a better substrate of MAO than NE . vated by COMT than by MAO .
In this way much more NE is inacti-
2342
EFFECT OF RFSERPINE
Yol . 6, No . 21
Further studies are required on the relationship between the depletion of tissue RE and the increase of MAO activity induced by reserpine .
However, an interesting possibility is
that the action of reserpine is increasing deamiaation of NE by mitochoadrial MAO may play a significant role in the NE depletiag effect of reserpiae .
This possibility is supported
by the previous findings (24-28) that pretreatment with ~IAOinhibitors prevents the depletion of tissue NE caused by reserpine administration . Sumne~r~
8eserpine treatment activates mitochondrial MAO activity of guinea pig hearts .
Reßerpiae may act on the mitochondrial
membrane or structure to increase the penetration of norepine phriae to mitochondrial D~AO .
Ia this way the deaminatioa of
norepinephrine would be increased in reserpinized hearts . References 1 . U. Trendelenburg, J . Phar.aacol . , 134, 8 (1961) . 2 . L .T . Potter, J . Axelrod and LJ . Kopin, Biochem. Pharmacol . , 1~, 254 (1962) . 3, I .J . Kopin, G . Hertting and E .K . Gordon, J_ Pharmacol . , 34 (1962), 4, J . Axelrod, E.S : Gordon, G. Herttiag,I .J .Kopin and L .T . Potter, Br it . J . Pharmacol . , 12, 56 (1962) . 5 . T .R . Crout, A .J . Muskus and U . Trendelenburg, Brit . J . Pharmacol ., 18, 600 (1962) . 6. J .H . Burn and M.J . Rand, J . Physiol . , 144, 314 (1958) . ~. J .H . Burn and. M.J . Rand, J . Physio l-, 142, 135 (1959) . 8 . J .H . $urn and M.J . Rand, J . Physiol . , 1~0, 295 (1960) .
Vol . 6, No . 21
EFFECT OF RESERPINE
2343
9 . LJ . %opia and P.% . Cordon, Fed . Proc .,21, 332 (1962) . 10 . LJ . %opin and T.% . Cordon, J . Pharmacol . , ~, 351 (1962) . 11 . I.J . %opin and L.% . Cordon, J. Pharmacol . , 140, 207 (1963) . 12 . LJ . %opin, Pharmacol . Rev . , 16, 179 (1964) . 13 . N.H . Heff, T .N . Toter, W. Hammer and B.B . Bsodie, Life Sciences , 4, 1869 (1965) " 14 . Y. Guturan and H. Weil-Malherbe, Life Sciences , ~, 1293 (1966) . 15 . J . Jonsson, H. Grobecker and P. Holtz, Life Sciences, 2235 (1966) . 16 . T . Ithoh, M. Matauoka, %. Nakajima, B. Taßawa and R. Imaizumi, Jap . J . Pharmacol . , 12, 130 (1962) . 17 . L .L . Iversen, J . Glowinski and J . Axelrod, J . Pharmacol . , 173 (1965) " 18 . L .T . Potter and J . Axelrod, J . Pharmacol . , 142, 291 (1963) . 19 . L .T . Potter and J . Axelrod, Rature , ~, 581 (1963) . 20 . I .A . Michaelson, %.C . Richardaon, S .N . Snyder and E .O . Titus, Life Sciences ~, 971 (1964) . 21 . L .T . Potter, Pharmacol . Rev . ,18, 439 (1966) . 22 . T . Nukada, T . Sakurai and R . Imaizumi, Jap . J. Pharmacol . , 124 (1963) . 23 . H . Aebi, F . Stocker and M. Eberhardt, Biochem-Z ., 526 (1963) . 24 . A . Horita and 'sN.R . McGrath, Biochem. Pharmacol ., ~, 206 (1960) . 25 . S . Spector, R . %untzman, P .A . Shore and B.B . Brodie, J . Pharmacol . f ~, 256 (1960) . 26 . J . Axelrod, G. Hertting and R.W . Patrick, J . Pharmacol . , 325 (1961) . 27 . F. Clementi and G .P . Zocche, J, of Cell Biol~, ~, 28 . F. Clementi, Experientia , 21, 171 (1965)
587 (1963) .