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Reserpine and cocaine blocking of the uptake and storage mechanisms in adrenergic nerves
Life Sciences Vol . 3, pp. 703-708, 1984 . Pergamon Press, Inc . Printed in the ûnited States.
RE3ERPINE AND COCAINF BLOCKING OF THE UPTAKE AND STORAGE MECHANISMS IN ADRENERGIC NERVES Nils-eke Hillarp and Torbjörn Malmfore Department of Histology, Karolinska Institutet, Stockholm, Sweden
(Received 27 May 1964) It has been shown (1, 2,
3)
that the mechanisms for the uptake and
storage of catecholaminea in adrenergic nerves can be studied in an entirely new and direct way by means of a sensitive fluorescence method that permits, for instance,
the demonstration of the adrenergic transmitter at the cellular
level . The entire adrenergic neuron was found ~1, 2) to have the basic property of taking up noradrenaline (NA) and related amines by a mechanism that is different from that of the amine storage granules (cf .
4, 5, 6)
and
which is localized to the oell membrane . This mechanism is not only a transport mechanism to facilitate the entry of NA into the azoplasm, where the granules could then be responsible for the efficient accumulation . On the contrary, there is strong evidence that the membrane mechanism especially in the non-terminal azons and terminals - represents a highly efficient and important uptake-conoentration mechanism, which is capable of concentrating the amines against a very high gradient and giving a tremendous accumulation of the amines in the azoplasm without the cooperation of the storage granules . Since the non-terminal atone - in contrast to the terminals - have a very low granule content, the membrane mechanism can furthermore be studied in this part of the neuron without arty serious interference by the granule storage mechanism (1,
2) . The significance of this is obvious .
It offers new possibilities, for instance, of anlyzing the mode and site of
703
704
RESER.PINE AND COCAINE BLOCKII~iG
Vol . 3, No. 7
action of oertain drugs - such as reaerpine and cocaine - which are important tools in studies on âdrenergic transmission . The rat iris is very suitable for studies in this field (2 ) . Catecholamines injected in the anterior chamber of the eye are readly taken up by its adrenergic nerves and within 15 minutes réaah intraneuronal levels caloulated to be 1000 to 10,000 times higher than the initial ezternal amine levels both when low (5210
-8
M) ang high
(10 5
M) intraoaular concentrations are used .
The whole eateneive network of adrenergio avons and terminals and the localization of the amines taken up can furthermore be easily ezrnined (see microphotos in papers 1 and 2) . Methods The uptake of L-NA and L-a-rmethyl-AA was studied in the rat iris with the oonvenient technique described in detail in another paper (2 ) . The amines were administered intravenously in the lingual vein (slow injection of 0 .01-0 .25 mg~kg) or intraocularly (initial concentrations in the anterior chamber about 5s10~-10~ M) under light ether anesthesia . Heserpine (1-10 mg~kg) and - in the ezperiments with NA - nialamide (5-100 mg~kg) were administered intraperitoneally 3-24 and 2-6 hours respectively before the injeotions of the aateoholamines . Cocaine was administered to several groups of rate given reaerpine 24 hours earlier . It was injected in two equal doses of 25-50 mg~kg i .p . or 5-10 mg~kg i .v . 30 and 5 minutes before the administration of the catecholamines . The animals were killed by deaapitation at various intervals after administration of the amines (0 .1-8 hours) . The amine uptake can be studied in both the terminals and non-terminal anon after a reaerpine-induced depletion of the adrenergic transmitter . Only the non-terminal avons can be used in normal animals, however, since the terminals have a very high oontent of endogenous RA . aesults and Discussion The cateaholamines could be seen - even after the lowest doses - to
RESERPINE AND COCAII~iE BLOCKIIJG
Vol . 3, No . 7
705
accumulate within some minutes in both terminals and non-terminal azona 24 hours after the administration of reserpine . When higher doses (0 .25 mg~kg -6
i .v . or 10
~ intraocularly) were used the amines were concentrated to very
high levels, comparable to those of the endogenous AA in the terminals of untreated animals (probably in the order of 10 mg~kg ; cf . 7) . The amines showed a mazimal accumulation within 15-30 minutes and then slowly disappeared during the next
4-S
hours .
As shown in the previous papers, the amines taken up in the reaerpinized animals are not bound in storage sites of the amine granules or elsewhere within the axons . NA - in contrast to a-methyl-NA which is not attacked by monoamine azona (MAO) - therefore showed anly a low and very transient accumulation if 2;fA0 was not efficiently inhibited by a potent inhibitor (e .g . nialamide, 20 mg~kg or more) or anoxia (see 2) . The a-methylated amine, on the other hand, accumulated both in untreated and nialamide-treated animals in the same way as NA in nialamide-treated animals . On the basis of these findings it seems safe to conclude that this drug does not act by blocking the release of the amines but by inhibition of MAO . A correlate to this is that MAO is so active within or possibly just outside the adrenergic fibres
that it plays an important role for the uptake of NA
and other amines readily attacked by it when the storage function is blocked by reaerpine (cf . 8,
9,
10, 11 ) . This is strongly supported by the finding
that at the moat a low and very transient accumulation of NA could be obtained in the non-terminal azona of non-reeerpinized animals if MAO was not effectively inhibited . The most reasonable ezplanation is that the amines taken up can only to a low degree be inoorporated in storage granules (see above) and thus cannot be protected from MAO . It consequently seems improbable that the adrenergic transmitter can ezist intraneuronally outside the storage granules to arty great eztent . No certain differenaea in the uptake-oonoentration of the asteahol-
70 6
RESERPINE AND COCAINE BLOCKII~TG
Vol . 3, No. 7
amities were observed on testing with either low or high amine doses after different treatments with reaerpine . The amines accumulated readily both 3 hours after the administration of the highest reaerpine dose (10 mg~kg) and 24 hours after the lowest dose (1 mg~) " This was the case also in animals which had received two doses of 10 mg~kg each 24 hours and 1 hour before the injection of the catecholamines . The amines accumulated rapidly and then slowly dissappeared in both the non-terminal aeons and terminals of the reaerpinized rats and in the non-terminal axons of untreated animals . Since these parts of the axons both possess the cell membrane mechanism but entirely differ in respect of the number of storage granules they contain (see above) the observations show clearly that the reaerpine-induced amine depletion and blockade of the storage function of the adrenergic nerves are not brought about by a drug action on the cell membrane mechanism. It has often been suggested (see e .g . 12, 13, 14) that reaerpine acts primarily be interfering with a postulated cell membrane mechanism . The evidence for this view is entirely indirect, however, and will be discussed in forthcoming papers . In contrast to reaerpine, cocaine blocked the accumulation of the administered catecholaminea after intravenous amine doses up to 0 .05 mg~kg or intraocular concentrations up to 10 -7 ~. At 5 to 10 times larger doses or concentrations the block was partially overcome but the accumulation was markedly reduced . When the highest intraooular ooncentrations were used no certain inhibition was observed . If cocaine was given after the injection of the catecholamines the disappearance of the accumulated amines was not accelerated very markedly . It has been shown in several ways that reaerpine and cocaine block the uptake and~or storage of monoamines but views on their mode and site of action are contradiotory (cf . 5, 11, 12, 15, 16) . The experiments reported in this and previous papers (1, 2) have furnished the first direct evidence that the very efficient uptake and concentration of NA in adrenergic nerves -
Vol . 3, No . 7
RESERPINE AND COCAINE BLOCKING
707
shown to occur both in the non-terminal axons and along the entire longth of the terminals - are achieved primarily by a mechanism in the cell membrane and that this mechanism is strongly inhibited by cocaine but insensitive to reserpine . This strongly supports the view (6) that reserpine acts specifically and primarily through its blocking of the Lig-ATP dependent and
uptake-storage mechanism in the amine granules . Studies on depletion recovery of aatecholamines and on the formation of storage granules in
central monoamine neurons following the administration of reserpine have given further evidence for this view (17,
18) .
Acknowledgments This investigation was supported by a Public Health Service Research Grant
(NB 02854-04) from the National Institute of Neurological Diaaases and
Blindness and by a grant from the Swedish Medical Research Council . - For skilful technical assistance we are indebted to a~ira .Gun Matz . Reference 1 . B .HAI~®ERGER, T .?dALb4~ORS, K .-A .NORBERG and Sc .SACHS, Biochem . Pharmacol . i n press (1964) .
2 . T .MALt~`ORS, Z .Zellforsch . i n press (1964) . 3 . K .FUXE and G .SEDVALL, Acta physiol . stand . i n press (1964) . 4 . U .S . v . EDLER and F .LISHAJKO, Acta phyaiol . stand . ~, 468 (1963) . 5 . U .S . v . EDLER and F .LISHAJKO, Acta phyaiol . stand . 60, 217 (1964) . 6 . A .CARLSSON, N .-A .HILLARP and B .WALDECK, Acta physiol . stand . Suppl . 215 (1963) . 7 . A .CARLSSON, B .FALCK, K .Fü7CE and N .-A .HILLARP, Acta phyaiol . stand . 60, 112 (1964) . 8 . A .CARLSSON, Discussion in Ciba Found . S.ymp . on Adrenergia Mechanisms . Ed . : J .R .Vane, Q .E .W .Wolstenholme, and M.0'Connor . London J . and A .Churahill Ltd . (1960 ) .
9 " A .CARLSSON and N .-A .HILLARP, Aota physiol . stand . ~~ 95 (1962) .
708
10 .
REBERPINE AND COCAINE BLOCKIIJG
I .J .KOPIN and E .K .GORDON,
11 . A .CARLSSON,
12 . F .B .HUGHES and B .B .BRODIE,
J . Pharmacol . exp . Ther .
(1963) .
12 , 96 (1959) " Int .
J.
23 (1962) .
L .STJÄRNE, Acta physiol . stand .
62,
Suppl .
228 (1964) .
15 . J .A%ELROD, The Clinical Chemistry of 5~onoaminea p .
16 .
207
(1964) .
H .J .D~+iGLER, L A .MIC3AELSON, H .E .SPIEGEL and E .TITUS, Neuropharmacol . 1,
14 .
J . Pharmacol . exp . Ther . ~,
Hanâb . der Exp . Pharmakol . Ergängzungswerk . Vol . XIX .
Ed . s V .Erspamer . i n press
13 .
Vol . 3, No. 7
5.
~sevier Publ .
Co ., Amsterdam
(1963) .
R .F .FURCHGOTT,
S .M .KIRPEKAR s M .RIEKEH and A .SC~PAB, J . Pharmacol .
ezp . Ther . ~
39 (1963) "
17 . A .DAHLSTRÛ1d and K .F[J7CE, Acta Physiol . stand . in press
(1964) .
18 . A .DA?H,STRi~Pd, K .FVXE and N .-P~ .HILLARP, Aota pharmacol . e t to=icol . in press
(1964) .
RE3ERPINE AND COCAINF BLOCKING OF THE UPTAKE AND STORAGE MECHANISMS IN ADRENERGIC NERVES Nils-eke Hillarp and Torbjörn Malmfore Department of Histology, Karolinska Institutet, Stockholm, Sweden
(Received 27 May 1964) It has been shown (1, 2,
3)
that the mechanisms for the uptake and
storage of catecholaminea in adrenergic nerves can be studied in an entirely new and direct way by means of a sensitive fluorescence method that permits, for instance,
the demonstration of the adrenergic transmitter at the cellular
level . The entire adrenergic neuron was found ~1, 2) to have the basic property of taking up noradrenaline (NA) and related amines by a mechanism that is different from that of the amine storage granules (cf .
4, 5, 6)
and
which is localized to the oell membrane . This mechanism is not only a transport mechanism to facilitate the entry of NA into the azoplasm, where the granules could then be responsible for the efficient accumulation . On the contrary, there is strong evidence that the membrane mechanism especially in the non-terminal azons and terminals - represents a highly efficient and important uptake-conoentration mechanism, which is capable of concentrating the amines against a very high gradient and giving a tremendous accumulation of the amines in the azoplasm without the cooperation of the storage granules . Since the non-terminal atone - in contrast to the terminals - have a very low granule content, the membrane mechanism can furthermore be studied in this part of the neuron without arty serious interference by the granule storage mechanism (1,
2) . The significance of this is obvious .
It offers new possibilities, for instance, of anlyzing the mode and site of
703
704
RESER.PINE AND COCAINE BLOCKII~iG
Vol . 3, No. 7
action of oertain drugs - such as reaerpine and cocaine - which are important tools in studies on âdrenergic transmission . The rat iris is very suitable for studies in this field (2 ) . Catecholamines injected in the anterior chamber of the eye are readly taken up by its adrenergic nerves and within 15 minutes réaah intraneuronal levels caloulated to be 1000 to 10,000 times higher than the initial ezternal amine levels both when low (5210
-8
M) ang high
(10 5
M) intraoaular concentrations are used .
The whole eateneive network of adrenergio avons and terminals and the localization of the amines taken up can furthermore be easily ezrnined (see microphotos in papers 1 and 2) . Methods The uptake of L-NA and L-a-rmethyl-AA was studied in the rat iris with the oonvenient technique described in detail in another paper (2 ) . The amines were administered intravenously in the lingual vein (slow injection of 0 .01-0 .25 mg~kg) or intraocularly (initial concentrations in the anterior chamber about 5s10~-10~ M) under light ether anesthesia . Heserpine (1-10 mg~kg) and - in the ezperiments with NA - nialamide (5-100 mg~kg) were administered intraperitoneally 3-24 and 2-6 hours respectively before the injeotions of the aateoholamines . Cocaine was administered to several groups of rate given reaerpine 24 hours earlier . It was injected in two equal doses of 25-50 mg~kg i .p . or 5-10 mg~kg i .v . 30 and 5 minutes before the administration of the catecholamines . The animals were killed by deaapitation at various intervals after administration of the amines (0 .1-8 hours) . The amine uptake can be studied in both the terminals and non-terminal anon after a reaerpine-induced depletion of the adrenergic transmitter . Only the non-terminal avons can be used in normal animals, however, since the terminals have a very high oontent of endogenous RA . aesults and Discussion The cateaholamines could be seen - even after the lowest doses - to
RESERPINE AND COCAII~iE BLOCKIIJG
Vol . 3, No . 7
705
accumulate within some minutes in both terminals and non-terminal azona 24 hours after the administration of reserpine . When higher doses (0 .25 mg~kg -6
i .v . or 10
~ intraocularly) were used the amines were concentrated to very
high levels, comparable to those of the endogenous AA in the terminals of untreated animals (probably in the order of 10 mg~kg ; cf . 7) . The amines showed a mazimal accumulation within 15-30 minutes and then slowly disappeared during the next
4-S
hours .
As shown in the previous papers, the amines taken up in the reaerpinized animals are not bound in storage sites of the amine granules or elsewhere within the axons . NA - in contrast to a-methyl-NA which is not attacked by monoamine azona (MAO) - therefore showed anly a low and very transient accumulation if 2;fA0 was not efficiently inhibited by a potent inhibitor (e .g . nialamide, 20 mg~kg or more) or anoxia (see 2) . The a-methylated amine, on the other hand, accumulated both in untreated and nialamide-treated animals in the same way as NA in nialamide-treated animals . On the basis of these findings it seems safe to conclude that this drug does not act by blocking the release of the amines but by inhibition of MAO . A correlate to this is that MAO is so active within or possibly just outside the adrenergic fibres
that it plays an important role for the uptake of NA
and other amines readily attacked by it when the storage function is blocked by reaerpine (cf . 8,
9,
10, 11 ) . This is strongly supported by the finding
that at the moat a low and very transient accumulation of NA could be obtained in the non-terminal azona of non-reeerpinized animals if MAO was not effectively inhibited . The most reasonable ezplanation is that the amines taken up can only to a low degree be inoorporated in storage granules (see above) and thus cannot be protected from MAO . It consequently seems improbable that the adrenergic transmitter can ezist intraneuronally outside the storage granules to arty great eztent . No certain differenaea in the uptake-oonoentration of the asteahol-
70 6
RESERPINE AND COCAINE BLOCKII~TG
Vol . 3, No. 7
amities were observed on testing with either low or high amine doses after different treatments with reaerpine . The amines accumulated readily both 3 hours after the administration of the highest reaerpine dose (10 mg~kg) and 24 hours after the lowest dose (1 mg~) " This was the case also in animals which had received two doses of 10 mg~kg each 24 hours and 1 hour before the injection of the catecholamines . The amines accumulated rapidly and then slowly dissappeared in both the non-terminal aeons and terminals of the reaerpinized rats and in the non-terminal axons of untreated animals . Since these parts of the axons both possess the cell membrane mechanism but entirely differ in respect of the number of storage granules they contain (see above) the observations show clearly that the reaerpine-induced amine depletion and blockade of the storage function of the adrenergic nerves are not brought about by a drug action on the cell membrane mechanism. It has often been suggested (see e .g . 12, 13, 14) that reaerpine acts primarily be interfering with a postulated cell membrane mechanism . The evidence for this view is entirely indirect, however, and will be discussed in forthcoming papers . In contrast to reaerpine, cocaine blocked the accumulation of the administered catecholaminea after intravenous amine doses up to 0 .05 mg~kg or intraocular concentrations up to 10 -7 ~. At 5 to 10 times larger doses or concentrations the block was partially overcome but the accumulation was markedly reduced . When the highest intraooular ooncentrations were used no certain inhibition was observed . If cocaine was given after the injection of the catecholamines the disappearance of the accumulated amines was not accelerated very markedly . It has been shown in several ways that reaerpine and cocaine block the uptake and~or storage of monoamines but views on their mode and site of action are contradiotory (cf . 5, 11, 12, 15, 16) . The experiments reported in this and previous papers (1, 2) have furnished the first direct evidence that the very efficient uptake and concentration of NA in adrenergic nerves -
Vol . 3, No . 7
RESERPINE AND COCAINE BLOCKING
707
shown to occur both in the non-terminal axons and along the entire longth of the terminals - are achieved primarily by a mechanism in the cell membrane and that this mechanism is strongly inhibited by cocaine but insensitive to reserpine . This strongly supports the view (6) that reserpine acts specifically and primarily through its blocking of the Lig-ATP dependent and
uptake-storage mechanism in the amine granules . Studies on depletion recovery of aatecholamines and on the formation of storage granules in
central monoamine neurons following the administration of reserpine have given further evidence for this view (17,
18) .
Acknowledgments This investigation was supported by a Public Health Service Research Grant
(NB 02854-04) from the National Institute of Neurological Diaaases and
Blindness and by a grant from the Swedish Medical Research Council . - For skilful technical assistance we are indebted to a~ira .Gun Matz . Reference 1 . B .HAI~®ERGER, T .?dALb4~ORS, K .-A .NORBERG and Sc .SACHS, Biochem . Pharmacol . i n press (1964) .
2 . T .MALt~`ORS, Z .Zellforsch . i n press (1964) . 3 . K .FUXE and G .SEDVALL, Acta physiol . stand . i n press (1964) . 4 . U .S . v . EDLER and F .LISHAJKO, Acta phyaiol . stand . ~, 468 (1963) . 5 . U .S . v . EDLER and F .LISHAJKO, Acta phyaiol . stand . 60, 217 (1964) . 6 . A .CARLSSON, N .-A .HILLARP and B .WALDECK, Acta physiol . stand . Suppl . 215 (1963) . 7 . A .CARLSSON, B .FALCK, K .Fü7CE and N .-A .HILLARP, Acta phyaiol . stand . 60, 112 (1964) . 8 . A .CARLSSON, Discussion in Ciba Found . S.ymp . on Adrenergia Mechanisms . Ed . : J .R .Vane, Q .E .W .Wolstenholme, and M.0'Connor . London J . and A .Churahill Ltd . (1960 ) .
9 " A .CARLSSON and N .-A .HILLARP, Aota physiol . stand . ~~ 95 (1962) .
708
10 .
REBERPINE AND COCAINE BLOCKIIJG
I .J .KOPIN and E .K .GORDON,
11 . A .CARLSSON,
12 . F .B .HUGHES and B .B .BRODIE,
J . Pharmacol . exp . Ther .
(1963) .
12 , 96 (1959) " Int .
J.
23 (1962) .
L .STJÄRNE, Acta physiol . stand .
62,
Suppl .
228 (1964) .
15 . J .A%ELROD, The Clinical Chemistry of 5~onoaminea p .
16 .
207
(1964) .
H .J .D~+iGLER, L A .MIC3AELSON, H .E .SPIEGEL and E .TITUS, Neuropharmacol . 1,
14 .
J . Pharmacol . exp . Ther . ~,
Hanâb . der Exp . Pharmakol . Ergängzungswerk . Vol . XIX .
Ed . s V .Erspamer . i n press
13 .
Vol . 3, No. 7
5.
~sevier Publ .
Co ., Amsterdam
(1963) .
R .F .FURCHGOTT,
S .M .KIRPEKAR s M .RIEKEH and A .SC~PAB, J . Pharmacol .
ezp . Ther . ~
39 (1963) "
17 . A .DAHLSTRÛ1d and K .F[J7CE, Acta Physiol . stand . in press
(1964) .
18 . A .DA?H,STRi~Pd, K .FVXE and N .-P~ .HILLARP, Aota pharmacol . e t to=icol . in press
(1964) .