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Reserpine-induced release of drugs from sympathetic nerve endings
Life Sciences Vo1 .3, pp. 839-844, 1964 . Pergamon Press, Inc . Printed in the United States.
RSSSxPïNS-INDUCED RâLSA3B OF DRUGS FRCS Si~ATfiBTIC NSRVR St~INGS C . C . Chang,* B . Conta and B . B . Brodie Laboratory of Chemical Pharmacology National Heart Institute National Institutes of Health Bethesda, Maryland
(Received 10 July 1964) STUDIES by Mataumoto and Horita (1) indicate that pretreatment of rabbits with metamphetamine not only prevents the depletion of heart aorepinephrine (NE) induced by guanethidine but antagonizes the uptake of guanethidine by this tissue .
This finding stimulated our interest in the nature of guanethidine
binding sites .
Recently we demonstrated that guanethidine is localized in rat
tissues by two kinds of binding sites :
unapecific sites, present in all tissues
and analagoua to those which reversibly bind most drugs with tissue components, and specific sites, present mainly in tissues containing NB in high amounts . The two kinds of binding were distinguished by the action of amphetamine or ephedrine which selectively prevent the uptake of guanethidine by specific sites ; in addition, amphetamine releases guanethidine from these sites (2) . The specific sites in the heart are readily saturable and take up a maximum of about 2 .7 molecules of guanethidine (3 .2 ;tg/g of heart) for each molecule of endogenous NS .
In contrast, the unspecific sites show no signs of
saturation at levels up to 15 Wg/g .
These results suggest that the drug might
be taken up into heart by a specialised transport process .
In accord with this
view are the results of Schanker and Morrison (3), which show that guanethidine is taken up into heart slices by two processes, only one of which is readily *Prasant Addrasa :
National Taiwan University, Taipei, Taiwan, China .
839
840
RESERPINE-II~TDUCED RELEASE OF DRUGS
Vol . 3, No. 8
saturated and is inhibited by anaerobic conditions and by amphetamine .
Again,
the maximal uptake of guanethidine by this process amounts to about 2 .7 molecules of drug to each molecule of endogenous NE . The present paper describes the effects of reaerpine on the uptake and release of guanethidine, tyramine and metaraminol from various tissues . Methods The rata were male, Sprague-Dawley, weighing about 200 grams .
Guanethi-
dine (8 mg/kg as free base) or H3 -guanethidine (25 ug/kg) was injected intravenously via e tail vein .
Five hours later, the animals were killed by
cervical fracture and the hearts rapidly removed, rinsed, blotted, and weighed . For the assay of guanethidine, HC1 .
tissues were homogenized in 2 volumes of O .O1N
The homogenates were adjusted to a pH of 10 and the guanethidine was
extracted into chloroform, returned to O .1N HC1, and assayed chemically (Chang et al ., this laboratory, unpublished) .
H3 -Guanethidine in the aqueous phase
wen assayed by scintillation counting .
The radioactivity was subjected to
paper chromatography (isopropanol, concentrated ammonia, water 200 :20 :30) and yielded only a single spot with an Rf value identical with that of H3 -guanethidine . The labeled
~-H3 -(octahydro-l-azocinyl)-ethy, -guanidine sulfate J (H 3 -guanethidine - 17 .3 etc/~M) was kindly donated b y Dr . H . Sheppard of CIBA Pharmaceutical Company, Summit, New Jersey . ßeaulta In a preliminary report, we described experiments showing that amphetamine not only blocks the sympetholytic effects of guanethidine but antagonizes the uptake of the drug by various tissues and prevents its NB-depleting action (2) . In addition, amphetamine releases a considerable portion of guanethidine from tissues and reverses its sympatholytic action .
Theee results suggest that
amphetamine prevents and reverses the pharmacological action of guanethidine by displacing it from sites which have a selective affinity for the drug .
Vol . 3, No. 8
RESERPII~TE-II~TDUCED RELEASE OF DRUGS
841
After the administration of the drug in small doses, the uptake by the heart, mainly confined to specific sites, is too low for accurate measurement . For this reason, we have carried out experiments with labelled ae well as unlabelled guanethidine .
The results in Table 1 show that amphetamine blocked
by 70x the uptake of H3 -guanethidine by heart and that tyramine and metaraminol blocked the uptake of H3 -guanethidine to about the same extent . TABLE 1 Bffect of Various Drugs on Uptake of H3 -Guanethidine by Heart Rata were treated with various drugs and 30 minutes later were given H 3 -guaaethidine (25 Fig/kg, i .v .) ; controls were given H3-guanethidine only . Animals were killed 5 hr after injection of H3 -guanethidine . Figures in parentheses indicate number of experiments .
Treatment
H -Guanethidine Level F~g/8
Guanethidine
0 .188 t 0 .008 S .B .
Amphetamine (0 .75 mg/kg, i .p .) + Guanethidine
0 .068 ; 0 .050 ; 0 .051
70
Tyramine (25 mg/kg, i .p .) + Guanethidine
0 .058 ; 0 .048 ; 0 .055
71
Metaraminol (5 mg/kg, + Guanethidine
0 .048 ; 0 .059 ; 0 .051
72
i .p .)
It must be noted, however,
Inhibition of Uptake
x
(10)
that .we have not yet established whether or not
tyramine and metaraminol prevent the uptake of guanethidine given in pharmacological doses . ile have considered the possibility that the Npecific sites of guanethidine uptake might be the NS storage depots and that guanethidine might actually be "stored" in the catecholamine compartments of sympathetic nerve endings .
If
thin postulate were correct, then reaerpine should not only prevent the specific uptake of guanethidine but also release the drug already taken up .
Vol . 3, No . 8
RESERPINE-II~TDUCED RELEASE OF DRUGS
842
Table 2 shows the effects of reaerpine on the uptake of H3-guanethidine (25 hg/kg, i .v .) .
The uptake of R 3 -guanethidine by heart was blocked by about
75X but the uptake by skeletal muscle, a tissue that contains relatively little NE, was relatively unaffected .
Furthermore, as shown in Table 3,
reaerpine given to the rata, pretreated with H 3 -guanethidine, released a considerable proportion of the drug from the heart . TABLE 2 Effect of Reaerpine on the Uptake of R3 -Guanethidine by Heart and Skeletal Muscle Rata were treated with reaerpine (1 mg/kg, i .p .) and 3 hr later with H3 -guanControl rata were given H3-guanethidine only . Rata ethidine (25 4~g/kg, i .v .) . were killed 5 hr after injection of H3-guanethidine . H~j Guanet idine Levels Skeletal Muscle F~8/8 WS/g
Treatment
Heart
Guanethidine
0 .161 ;
0 .196 ;
0 .210
Reaerpine + Guanethidine
0 .062 ; 0 .079 ; 0 .080 ; 0 .063
0 .006 ;
0 .012 ; 0 .013
0 .010 ; 0 .013 ; 0 .010 0 .010
TABLE 3 Depletion of H 3 -Guanethidine from Reart by Reaerpine Rata were given H 3 -guanethidine (25 I~g/kg, i .v .) 1 hr before reaerpine (2 mg/kg, i .p .) . Control rats were given H3-guanethidine only . Animals were killed 5 hr Figures in parentheses indicate number of after injection of H3 -guanethidine . experimeata .
H Guanethidine
Treatment
Guanethidine Guanethidine + Reaerpine
-
Level
~
X Release
0 .175 ± 0 .010 (4)
--
0 .095 t 0 007
45
4
The effects of reaerpine on the tissue binding of guanethidine were also determined after giving the drug in pharmacologically active doses (8 mg/kg, i .v .) .
Vol . 3, No. 8
RESERPINE-II~TDUCED RELEASE OF DRUGS
843
TABLE 4 Effect of Reaerpine on Uptake of Guanethidine by Rat Heart Rats were given reaerpine (2 mg/kg, i .p .) 5 hr before guanethidine (8 mg/kg, i .v .) ; controls were given guanethidine only . Animals were killed 5 hr after injection of guanethidine . Figures in parentheses indicate number of experiments .
Treatment
I
Heart 1~8/8 ± S .E .
Guanethidine Levels Skeletal Muscle F~g/g ± S .E .
Guanethidine
6 .3 ± 0 .7 (4)
2 .6 ± 0 .5
(4)
Reserpine + Guanethidine Z Blockade of Uptake
3 .3 ± 0 .3 (4) 47
3 .0 ± 0 .3 (9)
The results in Table 4 show that reaerpine lowered the uptake of guanethidine into heart by about 45% but did not affect the uptake by skeletal muscle .
It
may be inferred that reaerpine had completely blocked the uptake of guanethidine into adrenergic neurons since amphetamine (0 .75 mg/kg, i .p .) produced no additional inhibition of guanethidine uptake .
The uptake of tyramine was also
shown to be almost completely prevented by pretreatment of rats with reserpine (4) . al .
Moreover, preliminary results, which agree with those of Shore et
(5), show that reaerpine prevents the uptake of metaraminol . Discussion The present studies support the view that guanethidine is accumulated into
various tissues by two types of binding :
unspecific binding analogous to that
which reversibly binds most drugs onto tissue components, and specific binding by sympathetic nerve endings . which store endogenous NE .
The specific sites may be identical with those
In accord with this view, reaerpine releases con-
siderable amounts of guanethidine from tissues containing high levels of NE but releases little or no guanethidine from tissues having only small amounts of catecholamine . Other drugs that release DiS, including tyramine and metaraminol, are also taken up by adrenergic neurone .
The uptake of these drugs is also prevented
by pretreatment with reaerpine (4,5) .
844
Vol. 3, No. 8
RESERPINE-IIIDUCED RELEASE OF DRUGS
Studies in this Laboratory have shown that guanethidine (2,3) and tyramine (4) do not release NS bq a simple one-to-one displacement .
In fact,
guanethidine must first be taken up before appreciable amounts of catecholamines are released; initially, the drug actually shares occupancy of the nerve endings with the endogenous amine .
After entering the sympathetic neurone,
guaaethidine releases NS perhaps by enhancing the permeability of the terminal membranes .
At first, guanethidine, like tyramine, releases at a rapid rata
the more available pool of NS; in fact, large doses of guanethidine can elicit s pronounced sympathomimetic effect . mainly in their duration of action.
Guanethidine sad tyramine may differ Thus, guanethidine remains localized in
adrenergic neurone for a considerable period of time and causes a steady loss in catecholamines after the period of rapid release .
In contrast,
rapidly releases about 30x of the heart 103 and than disappears .
tyramine
Although
metaramiaol and cb-methyl noradranaline are localized in adrenergic neurons for a considerable period of time (6), these drugs do not elicit adrenergic blockade, perhaps because they exert a rather potent sympathomimetic affect of their own (7,8,9) . References
s,
1.
C. MATSIIMOTO and A . HORITA, Biochem. P'harmacol .
2.
B. COSTA, C, C .
3.
A . MORRISON and L. SCHAI~t,
4.
C . ?lATSÜl~07.~0, B . C08TA and B . B. BRODIE, The Pharmacolo~ist , in press,
CHAIiG and B . B .
295 (1963) .
BRODIB, The PharmacoloRist , in press,
(1964) .
to be published (1964) .
(1964) . 5.
P. A. SHORE and H. S . ALPSRS, Fad .
Proc . 1 350 (1964) .
6.
A. Cl1RLSSON and !1 . LIHDQVIST, Acta Ph9siol . Scared. ~, 87
7.
M. D . DAY and M. J . RAH J . Pharm. Pharmacol . ~, 221 (1963) .
(1962) .
~,
8.
li. S . Aè1DSH and T. M6G1iQ8801i, Biochem. Pharmacol . ~Ls
66 (1963) .
9.
M. D . DAY and li. J . RAl®, Iat . J . xeuroaharmacol . ~, 173 (1964) .
RSSSxPïNS-INDUCED RâLSA3B OF DRUGS FRCS Si~ATfiBTIC NSRVR St~INGS C . C . Chang,* B . Conta and B . B . Brodie Laboratory of Chemical Pharmacology National Heart Institute National Institutes of Health Bethesda, Maryland
(Received 10 July 1964) STUDIES by Mataumoto and Horita (1) indicate that pretreatment of rabbits with metamphetamine not only prevents the depletion of heart aorepinephrine (NE) induced by guanethidine but antagonizes the uptake of guanethidine by this tissue .
This finding stimulated our interest in the nature of guanethidine
binding sites .
Recently we demonstrated that guanethidine is localized in rat
tissues by two kinds of binding sites :
unapecific sites, present in all tissues
and analagoua to those which reversibly bind most drugs with tissue components, and specific sites, present mainly in tissues containing NB in high amounts . The two kinds of binding were distinguished by the action of amphetamine or ephedrine which selectively prevent the uptake of guanethidine by specific sites ; in addition, amphetamine releases guanethidine from these sites (2) . The specific sites in the heart are readily saturable and take up a maximum of about 2 .7 molecules of guanethidine (3 .2 ;tg/g of heart) for each molecule of endogenous NS .
In contrast, the unspecific sites show no signs of
saturation at levels up to 15 Wg/g .
These results suggest that the drug might
be taken up into heart by a specialised transport process .
In accord with this
view are the results of Schanker and Morrison (3), which show that guanethidine is taken up into heart slices by two processes, only one of which is readily *Prasant Addrasa :
National Taiwan University, Taipei, Taiwan, China .
839
840
RESERPINE-II~TDUCED RELEASE OF DRUGS
Vol . 3, No. 8
saturated and is inhibited by anaerobic conditions and by amphetamine .
Again,
the maximal uptake of guanethidine by this process amounts to about 2 .7 molecules of drug to each molecule of endogenous NE . The present paper describes the effects of reaerpine on the uptake and release of guanethidine, tyramine and metaraminol from various tissues . Methods The rata were male, Sprague-Dawley, weighing about 200 grams .
Guanethi-
dine (8 mg/kg as free base) or H3 -guanethidine (25 ug/kg) was injected intravenously via e tail vein .
Five hours later, the animals were killed by
cervical fracture and the hearts rapidly removed, rinsed, blotted, and weighed . For the assay of guanethidine, HC1 .
tissues were homogenized in 2 volumes of O .O1N
The homogenates were adjusted to a pH of 10 and the guanethidine was
extracted into chloroform, returned to O .1N HC1, and assayed chemically (Chang et al ., this laboratory, unpublished) .
H3 -Guanethidine in the aqueous phase
wen assayed by scintillation counting .
The radioactivity was subjected to
paper chromatography (isopropanol, concentrated ammonia, water 200 :20 :30) and yielded only a single spot with an Rf value identical with that of H3 -guanethidine . The labeled
~-H3 -(octahydro-l-azocinyl)-ethy, -guanidine sulfate J (H 3 -guanethidine - 17 .3 etc/~M) was kindly donated b y Dr . H . Sheppard of CIBA Pharmaceutical Company, Summit, New Jersey . ßeaulta In a preliminary report, we described experiments showing that amphetamine not only blocks the sympetholytic effects of guanethidine but antagonizes the uptake of the drug by various tissues and prevents its NB-depleting action (2) . In addition, amphetamine releases a considerable portion of guanethidine from tissues and reverses its sympatholytic action .
Theee results suggest that
amphetamine prevents and reverses the pharmacological action of guanethidine by displacing it from sites which have a selective affinity for the drug .
Vol . 3, No. 8
RESERPII~TE-II~TDUCED RELEASE OF DRUGS
841
After the administration of the drug in small doses, the uptake by the heart, mainly confined to specific sites, is too low for accurate measurement . For this reason, we have carried out experiments with labelled ae well as unlabelled guanethidine .
The results in Table 1 show that amphetamine blocked
by 70x the uptake of H3 -guanethidine by heart and that tyramine and metaraminol blocked the uptake of H3 -guanethidine to about the same extent . TABLE 1 Bffect of Various Drugs on Uptake of H3 -Guanethidine by Heart Rata were treated with various drugs and 30 minutes later were given H 3 -guaaethidine (25 Fig/kg, i .v .) ; controls were given H3-guanethidine only . Animals were killed 5 hr after injection of H3 -guanethidine . Figures in parentheses indicate number of experiments .
Treatment
H -Guanethidine Level F~g/8
Guanethidine
0 .188 t 0 .008 S .B .
Amphetamine (0 .75 mg/kg, i .p .) + Guanethidine
0 .068 ; 0 .050 ; 0 .051
70
Tyramine (25 mg/kg, i .p .) + Guanethidine
0 .058 ; 0 .048 ; 0 .055
71
Metaraminol (5 mg/kg, + Guanethidine
0 .048 ; 0 .059 ; 0 .051
72
i .p .)
It must be noted, however,
Inhibition of Uptake
x
(10)
that .we have not yet established whether or not
tyramine and metaraminol prevent the uptake of guanethidine given in pharmacological doses . ile have considered the possibility that the Npecific sites of guanethidine uptake might be the NS storage depots and that guanethidine might actually be "stored" in the catecholamine compartments of sympathetic nerve endings .
If
thin postulate were correct, then reaerpine should not only prevent the specific uptake of guanethidine but also release the drug already taken up .
Vol . 3, No . 8
RESERPINE-II~TDUCED RELEASE OF DRUGS
842
Table 2 shows the effects of reaerpine on the uptake of H3-guanethidine (25 hg/kg, i .v .) .
The uptake of R 3 -guanethidine by heart was blocked by about
75X but the uptake by skeletal muscle, a tissue that contains relatively little NE, was relatively unaffected .
Furthermore, as shown in Table 3,
reaerpine given to the rata, pretreated with H 3 -guanethidine, released a considerable proportion of the drug from the heart . TABLE 2 Effect of Reaerpine on the Uptake of R3 -Guanethidine by Heart and Skeletal Muscle Rata were treated with reaerpine (1 mg/kg, i .p .) and 3 hr later with H3 -guanControl rata were given H3-guanethidine only . Rata ethidine (25 4~g/kg, i .v .) . were killed 5 hr after injection of H3-guanethidine . H~j Guanet idine Levels Skeletal Muscle F~8/8 WS/g
Treatment
Heart
Guanethidine
0 .161 ;
0 .196 ;
0 .210
Reaerpine + Guanethidine
0 .062 ; 0 .079 ; 0 .080 ; 0 .063
0 .006 ;
0 .012 ; 0 .013
0 .010 ; 0 .013 ; 0 .010 0 .010
TABLE 3 Depletion of H 3 -Guanethidine from Reart by Reaerpine Rata were given H 3 -guanethidine (25 I~g/kg, i .v .) 1 hr before reaerpine (2 mg/kg, i .p .) . Control rats were given H3-guanethidine only . Animals were killed 5 hr Figures in parentheses indicate number of after injection of H3 -guanethidine . experimeata .
H Guanethidine
Treatment
Guanethidine Guanethidine + Reaerpine
-
Level
~
X Release
0 .175 ± 0 .010 (4)
--
0 .095 t 0 007
45
4
The effects of reaerpine on the tissue binding of guanethidine were also determined after giving the drug in pharmacologically active doses (8 mg/kg, i .v .) .
Vol . 3, No. 8
RESERPINE-II~TDUCED RELEASE OF DRUGS
843
TABLE 4 Effect of Reaerpine on Uptake of Guanethidine by Rat Heart Rats were given reaerpine (2 mg/kg, i .p .) 5 hr before guanethidine (8 mg/kg, i .v .) ; controls were given guanethidine only . Animals were killed 5 hr after injection of guanethidine . Figures in parentheses indicate number of experiments .
Treatment
I
Heart 1~8/8 ± S .E .
Guanethidine Levels Skeletal Muscle F~g/g ± S .E .
Guanethidine
6 .3 ± 0 .7 (4)
2 .6 ± 0 .5
(4)
Reserpine + Guanethidine Z Blockade of Uptake
3 .3 ± 0 .3 (4) 47
3 .0 ± 0 .3 (9)
The results in Table 4 show that reaerpine lowered the uptake of guanethidine into heart by about 45% but did not affect the uptake by skeletal muscle .
It
may be inferred that reaerpine had completely blocked the uptake of guanethidine into adrenergic neurons since amphetamine (0 .75 mg/kg, i .p .) produced no additional inhibition of guanethidine uptake .
The uptake of tyramine was also
shown to be almost completely prevented by pretreatment of rats with reserpine (4) . al .
Moreover, preliminary results, which agree with those of Shore et
(5), show that reaerpine prevents the uptake of metaraminol . Discussion The present studies support the view that guanethidine is accumulated into
various tissues by two types of binding :
unspecific binding analogous to that
which reversibly binds most drugs onto tissue components, and specific binding by sympathetic nerve endings . which store endogenous NE .
The specific sites may be identical with those
In accord with this view, reaerpine releases con-
siderable amounts of guanethidine from tissues containing high levels of NE but releases little or no guanethidine from tissues having only small amounts of catecholamine . Other drugs that release DiS, including tyramine and metaraminol, are also taken up by adrenergic neurone .
The uptake of these drugs is also prevented
by pretreatment with reaerpine (4,5) .
844
Vol. 3, No. 8
RESERPINE-IIIDUCED RELEASE OF DRUGS
Studies in this Laboratory have shown that guanethidine (2,3) and tyramine (4) do not release NS bq a simple one-to-one displacement .
In fact,
guanethidine must first be taken up before appreciable amounts of catecholamines are released; initially, the drug actually shares occupancy of the nerve endings with the endogenous amine .
After entering the sympathetic neurone,
guaaethidine releases NS perhaps by enhancing the permeability of the terminal membranes .
At first, guanethidine, like tyramine, releases at a rapid rata
the more available pool of NS; in fact, large doses of guanethidine can elicit s pronounced sympathomimetic effect . mainly in their duration of action.
Guanethidine sad tyramine may differ Thus, guanethidine remains localized in
adrenergic neurone for a considerable period of time and causes a steady loss in catecholamines after the period of rapid release .
In contrast,
rapidly releases about 30x of the heart 103 and than disappears .
tyramine
Although
metaramiaol and cb-methyl noradranaline are localized in adrenergic neurons for a considerable period of time (6), these drugs do not elicit adrenergic blockade, perhaps because they exert a rather potent sympathomimetic affect of their own (7,8,9) . References
s,
1.
C. MATSIIMOTO and A . HORITA, Biochem. P'harmacol .
2.
B. COSTA, C, C .
3.
A . MORRISON and L. SCHAI~t,
4.
C . ?lATSÜl~07.~0, B . C08TA and B . B. BRODIE, The Pharmacolo~ist , in press,
CHAIiG and B . B .
295 (1963) .
BRODIB, The PharmacoloRist , in press,
(1964) .
to be published (1964) .
(1964) . 5.
P. A. SHORE and H. S . ALPSRS, Fad .
Proc . 1 350 (1964) .
6.
A. Cl1RLSSON and !1 . LIHDQVIST, Acta Ph9siol . Scared. ~, 87
7.
M. D . DAY and M. J . RAH J . Pharm. Pharmacol . ~, 221 (1963) .
(1962) .
~,
8.
li. S . Aè1DSH and T. M6G1iQ8801i, Biochem. Pharmacol . ~Ls
66 (1963) .
9.
M. D . DAY and li. J . RAl®, Iat . J . xeuroaharmacol . ~, 173 (1964) .