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Hypothetical model of catecholamine uptake into adrenal medullary storage vesicles
Pergamon Press
Lüe Sciences Vol. 13, pp. 875-883 , 1973 . Printed in Great Britain
HYPOTHETICAL MODEL OF CATECHOLAMINE UPTAKE INTO ADRENAL MEDULLARY STORAGE VESICLES Theodore A . Slotkin Department of Physiology and Pharmacology Duke University Medical Center Durham, North Carolina 27710
(Received 18 June, 1973; in final form 31 July, 1973) SUMMARY Recent studies from several laboratories suggest that the uptake of catecholamines into adrenal medullary vesicles is mediated by a mobile carrier in the vesicle membrane . A transport model is proposed in which catecholamines attach to the carrier on the outside of the membrane and are detached on the inside surface by the action of ATP ; probable loci of action of uptake inhibitors (reserpine and N-ethylmaleimide) are identified .
In 1962, independent studies by Kirshner (1) and Carlsson et al . (2) demonstrated the existence of a catecholamine incorooration system in isolated adrenal medullary vesicles which was stimulated by ATP and magnesium and inhib ited by reserpine .
Since then, a vast number of reports have appeared concern-
ing the nature of the system and the effects of nucleotides, divalent cations and various drugs on the uptake, storage and release of vesicular amines .
The
present discussion attempts to unify studies from several laboratories in order to produce a schematic model of the uptake system . 1.
Uptake and storage are separable processes and uptake is mediated by a
membrane-bound carrier .
Prior to the discovery of the ATP and magnesium stim-
ulated incorporation mechanism, Hillary (3, 4, 5) suggested that catecholamines and ATP could form a stable storage complex within the vesicle, in a molar ratio of 4 catecholamines / 1 ATP .
The suggestion has been confirmed in part
by in vitro studies which further implicate the involvement of divalent cations (6, 7) and possibly other vesicle constituents (3, 8, 9) .
This raised the
question whether amine incorporation might in fact represent storage only ;
875
878
Vol. 13, No . 8
Model of Catecholamine Uptake
catecholamines and ATP could diffuse through the membrane (4, 10) and then become bound in a non-diffusable form .
It therefore became important to ask
whether uptake and storage represented the same or different processes .
fiat
they are different was demonstrated by studies from our laboratory utilizing simultaneous measurements of incorporation and effl ux of radioactively-labeled amines (11, 12) ; the latter parameter gives a measure of the stability with which a given amine is stored within the vesicle .
In comparing the incorpora-
tions and effl uxes of serotonin and epinephrine, it was found that serotonin was incorporated to a greater extent than epinephrine despite the fact that it was not stored as stably as epinephrine ; if the incorporation process had refl ected passive diffusion followed by binding, then the more stably bound amine would have been incorporated to a greater extent .
Since this was not
the case, a second process, namely uptake, must be implicated to account for the higher incorporation of serotonin .
Additionally, B-phenylethylamine,
which was not itself incorporated into the vesicles, was able to inhibit epinephrine incorporation by about 50~ in equimolar concentrations, indicating an interference with epinephrine incorporation at a point prior to storage . A series of studies from Taugner's laboratory (13, 14, 15) and more recently by Pletscher et al . (16) have examined the properties of storage vesicles which have reformed after hypo-osmotic shock .
The "emptied" vesicles
were able to incorporate exogenous amines against a Gradient by utilizing the magnesium-activated ATPase in the membrane ; however, the amines incorporated in this system demonstrated a rapid rate of efflux, indicating that they were not stored within the empty vesicles .
This confirms that uptake and storage
are different processes and further suggests that the uptake is mediated by a carrier within the membrane .
Additional evidence for carrier-mediated trans-
port is provided by the following studies : a.
The incorporation of exogenous amines in intact vesicles requires energy in the form of ATP and is linked to the activity of the magnesium-activâted ATPase in the membrane ; both the
877
Model o~f Catecholamine Uptake
Vol . 13, No. 8
ATPase and uptake are inhibited by sulfhydryl reagents, such as N-ethylmaleimide (17) . b.
The uptake is saturable, with an estimated ~ of 4 to 8 x 10' 4 M in cow adrenal vesicles (18, 19) and 3 x 10 -5 M in rat adrenal
vesicles (H .O . green and T.A . Slotkin, submitted for publication) ; competition occurs with related amines and competitive potency is unrelated to subsequent storage stability (11, 12) . c.
The uptake process displays specificity, since catecholamines are preferred over related phenethylamines (such as metaraminol) and serotonin is preferred over catecholamines ; the uptake preference differs from the stability of storage (epinephrine > serotonin > metaraminol) .
d.
Uptake is temperature dependent, with a Q10 of 3 to 6, which is typical of transport processes (1) .
e.
Catecholamines are bound firmly to purified vesicle membranes, and the binding is inhibited by N-ethylmaleimide or reserpine in the presence of magnesium; the binding of epinephrine has a Kdiss
°f about 10 -4 M, which is close to the uptake !~, and the
binding has the same specificity as uptake into intact vesicles, viz . serotonin > epinephrine > metaraminol (20) .
These data in-
dicate that the binding of amines to the membranes may involve binding to the putative carrier. f.
Reserpine, which inhibits the ATP-magnesium stimulated incorporation of catecholamines, is persistently bound to the vesicle membrane (21, 22) and the degree of binding of reserpine to the membranes correlates with the effect of the drug .
These data all indicate that the first step in amine incorporation into adrenal storage vesicles is mediated by a membrane-bound carrier which utilizes ATP to transport amines . 2.
ATP is utilized on the inside of the membrane to cause detachment of
678
Model od Catecholamine Uptake
amines from the carrier ; transphos hor lation ma
Vol. 13, No. 8
be involved . In intact
vesicles as well as re-fornied "empty" vesicles, the uptake of amines is dependent on the membrane ATPase (13, 14, 16, 17) .
However, the binding of amines
to purified vesicle membranes is substantially reduced by ATP ; ADP is considerably less effective and AMP totally ineffective (20) .
This suggests that ATP
is utilized to detach the amine from the membrane-bound carrier and that the utilization of ATP may occur on the inside surface on the membrane .
Thus, the
ATP would have to undergo translocation in order to activate uptake .
Studies
by Hillary (4), Carlsson et al . (23), Kirchner et al . (10 ), Stevens et al . (24) and Stitzel et al . (25) all indicate that adenine nucleotides do indeed pass through the vesicle membrane to the interior .
However, in order to facilitate
uptake, the ATP would have to exist in a form other than the catecholaminecontainina storage complex ; since the complex is presumably non-diffusable, it cannot serve as an energy source for uptake, and the ATP utilized must therefore come from a labile pool .
Does a labile pool exist?
The efflux of ATP
from isolated vesicles is biphasic with an initially rapid rate of loss followed by a much slower rate (26), and the half-lives of the two components correspond to those of loosely and stably stored catecholamine, indicating that there is indeed a labile ATP pool within the vesicle .
Furthermore, studies in
perfused glands indicate that newly-formed ATP is incorporated in a labile manner and is not immediately stored stably (25) .
The concept of utilization of
ATP on the inside of the membrane is not unique, and is commonly observed in mitochondria (27) ; it should not be overlooked that the catecholamine storage vesicles themselves are potentially the richest source of ATP available. It is noteworthy that reserpine, which competitively inhibits catecholamine uptake (18, 23) as well as catecholamine binding to vesicle membranes (20) does not affect ATPase activity (28) ; this provides further evidence that the utilization of ATP is involved in the detachment rather than attachment of catecholamines to the membrane-bound carrier, and that it acts at a site different frorom that of reserpine.
Model ad Catecholamine Uptake
Vol . 13, No. 8
87 9
If ATP is indeed utilized in the detachment, the question remains as to the mechanism by which this occurs . a.
There are two likely possibilities :
Detachment occurs by binding of ATP to a site on the carrier resulting in a non-competitive reduction in amine binding capacity; this is observed in purified vesicle membranes and does not re quire magnesium (20) .
Since energy is not utilized, this alone
cannot be the mechanism, but the effect may precede the second step : b.
The carrier may be transphosphorylated, releasing the amine by a reduction either in affinity or capacity .
Trifaro (29, 30)
reports that transphosphorylation of membrane components (and indeed of the ATPase itself) occurs, requiring the same conditions as ATPase activity and stimulated uptake, and all three processes are inhibited by N-ethylmaleimide .
The degree of
transphosphorylation is far lower than the ATPase activity, suggesting a rapid turnover of phosphorylated intermediates, although some components appear to be phosphorylated to form stable products .
Transphosphorylation has also been implicated
in other carrier-mediated transport systems (31) . From these data, the following model of catecholamine uptake in storage vesicles can be constructed (Fig . 1) :
a catecholamine molecule (epinephrine,
E) attaches to a carrier (C) at the outside surface of the membrane .
This does
not involve the participation of ATP or magnesiun, but is inhibited by reserpine or N-ethylmaleimide in the presence of magnesium (20) .
The amine-carrier
complex (EC) is dissociated on the inside surface by a direct decrease in binding capacity caused by combination of the carrier with ATP simultaneously with (or followed by) transphosphorylation of the carrier to form CtiP, utilizing the conversion of ATP to ADP by the magnesium-activated ATPase . available for storage .
E then becomes
Just as ATP can penetrate the vesicle membrane, the ADP
can diffuse to the exterior ; in any case, the net effect on the inside surface
680
Model of Catecholamine Uptake
Vol . 13, No. 6
is the splitting of one molecule of ATP to make available for storage one molecule of E.
At the outer surface, CtiP is dephosphorylated to release C for com-
bination with another amine molecule .
The net effect on the outside .is the
disappearance of one molecule of ATP and the appearance of ADP and Pi with the inward transport of one molecule of E .
Assuming this one-for-one relationship,
the ATPase activity of the storage vesicles is at least 6 times that necessary for maximal catecholamine transport (29, 30), so that the energy requirement of the model is certainly feasible .
FI~ . 1 Hypothetical model of catecholamine transport in storage vesicles, showing probable sites of action of reserpine and N-ethylmaleimide (NEM) . E is epinephrine, C is carrier, EC is epinephrine-carrier complex, CtiP is phosphorylated carrier, and Pi is inorganic phosphate . The following observable effects are all accounted for by a system of this type : a.
In intact vesicles and empty vesicles, amines would be pumped in and ATP would be hydrolyzed ; uptake would be stimulated by ATP and magnesium.
b . N-Ethylmaleimide would block transport by inhibiting the ATPase (at either or both the transphosphorylation and dephosphorylation steps) as well as by blocking attachment of catechplamines to the carrier ; the latter effect would explain why concentrations of N-ethylmaleimide which totally block uptake produce only 50~ inhibition of the ATPase (11, 12, 29, 37) . c.
Reserpine would block transport by inhibiting attachment of the
Vol. 13, No. 8
Model ad Catecholamine Uptake
88 1
catecholamines, but would not inhibit the ATPase . d.
In isolated membranes, the binding of catecholamines would be inhibited by the addition of ATP or by reserpine or N-ethylmaleimide + magnesium.
Of course, in any such system, prediction of observed phenomena does not constitute proof of the model, and such an interpretation should be treated as solely a schematic representation with which to compare additional results when available .
For example, the coupling of ATP to a phosphorylated carrier may be
more complex than the simple reaction indicated, and other vesicle proteins, phospholipids, etc . may be involved as well . Although another vesicular uptake system has been identified which is reserpine insensitive and is not stimulated by ATP and magnesium, this second system operates only at high amine concentration and does not possess specificity (11, 12) ; the proposed model applies solely to the reserpine sensitive system . It is hoped that the formulation of this working hypothesis can help in the design of specific experiments to examine the catecholamine uptake system on the molecular level . ACKNOWLEDGMENTS Work reported from this laboratory was supported by grants from the Council for Tobacco Research - USA, Inc ., the North Carolina Heart Association, the Pharmaceutical Manufacturers Association Foundation, and USPHS DA-00465-~1 .
REFERENCES 1.
N . KIRSHNER, J . Biol . Chem . _237 2311-2317 (1962) .
2.
A. CARLSSON, N .-A. HILLARP and B . WALDECK, Med. Exp. 6 47-53 (1962) .
3.
N .-~ . HILLARP, Acta Physiol . Scan d . 42 321-332 (1958) .
4.
N .-ß . HILLARP, Acta Physiol . Scan d . 47 271-279 (1959) .
0
5.
0 N .-A .
6.
K.H . BERNEIS, A. PLETSCHER and M . DAPRADA, Nature 224 281 (1969) .
HILLARP, Acta Physiol . Scand . 50 8-22 (1960) .
882
Vol . 13, No . 8
Model ad Catecholamine Uptake
7.
K .H . BERNEIS, A . PLETSCHER and M DAPRADA, Brit . J . Pharmacol . 39 382-389 (1970) .
8.
A . PHILIPPU and H .J . SCHÜMANN, Naunyn- Schmiedebergs Arch . Pharmacol . 252 339-358 (1966) .
9.
A .D . SMITH, in The Interaction of Dru s and Subcellular C Animal Cells (P . . mp e , e . a. ur , on n
onents in
10 . N . KIRSHNER, C . HOLLOWAY, W .J . SMITH and A .G . KIRSHNER, in Mechanisms of Release of Bio enic Amines (U .S . von Euler, S . Rosell an . vnas, eds.) p . u9 Nergamon, x or y 11 . T .A . SLOTKIN, R .M . FERRIS and N . KIRSHNER, Mol . Pharmacol . 7 308-316 (1971} 12 . T .A . SLOTKIN and N . KIRSHNER, Mol . Pharmacol . 7 581-592 (1971) . 13 . G . TAUGNER, Naunyn- Schmiedebergs Arch . Pharmacol . 270 392-406 (1971) . 14 . G . TAUGNER, Naunyn-Schmiedebergs Arch . Pharmacol . 274 299-314 (1972) . 15 . B . AGOSTINI and G . TAUGNER, Histochemie 30 255-272 (1973) . 16 . A . PLETSCHER, M . DAPRADA, H . STEFFEN, K .H . BERNEIS and B . LUTOLD, 3rd Intl . Catecholamine Sym~ Abs . (1973) . 17 . N . KIRSHNER, in Pharniacolo of Choline is and Adrene (G .B . Koelle, W . . u as an . ar sson, e . p . Medical Press, Prague 1965) .
is Transmission ze os ova
18 . J . JONASSON, E . ROSENGREN and B . WALDECK, Acta Physiol . Scand . _60 136-140 (1964) . 19 . P . LUNDBORG, Acta Physiol . Scand . 67 423-429 (1966) . 20 . T .A . SLOTKIN and N . KIRSHNER, Biochem . Pharmacol . i n press . 21 . S . NORN and P .A . SHORE, Biochem . Pharmacol . 20 1291-1295 (1971) . 22 . S . NORM and P .A . SHORE, Biochem . Pharmacol . 20 2133-2135 (1971) . 23 . A . CARLSSON, N .-~ . HILLARP and B . WALDECK, Acta Physiol . Scand . _59 Suppl . 215, 5-38 (1963) . 24 . P . STEVENS, R .L . ROBINSON . K . VAN DYKE and R . STITZEL, J . Pharmacol . Exp . Ther . 18 1 463-471 (1972) . 25 . R .E . STITZEL, R .L . ROBINSON and P . STEVENS, Fed-Proç. 32 784Abs (1973) . 26 . F . LISHAJKO, Acta Physiol . Scan d . 76 159-171 (1969) . 27 . M . KLINGENBERG, in Re ulation of Metabolic Processes in Mitochondria (J .M . , p . 80 Tager, S . Papa, E . Quag a e o an . . a~~r, e . vo . American Elsevier, New York (1966) . 28 . R .M . FERRIS, O .H . VIVEROS and N . KIRSHNER, Biochem . Pharmacol . _19 505-514 (1970) . 29 . J .M . TRIFARO and J . DWORKIND, Mol . Pharm a col . 7 52-65 (1971) .
Vol. 13, No . 8
Madel of Catecholamine Uptake
30 . J .M . TRIFARO and M . WARNER, Mol . Pharmacol . 8 159-169 (1972) . 31 . D .L . OXENDER, Ann . Rev ._ Biochem . 41 777-814 (1972) .
883
Lüe Sciences Vol. 13, pp. 875-883 , 1973 . Printed in Great Britain
HYPOTHETICAL MODEL OF CATECHOLAMINE UPTAKE INTO ADRENAL MEDULLARY STORAGE VESICLES Theodore A . Slotkin Department of Physiology and Pharmacology Duke University Medical Center Durham, North Carolina 27710
(Received 18 June, 1973; in final form 31 July, 1973) SUMMARY Recent studies from several laboratories suggest that the uptake of catecholamines into adrenal medullary vesicles is mediated by a mobile carrier in the vesicle membrane . A transport model is proposed in which catecholamines attach to the carrier on the outside of the membrane and are detached on the inside surface by the action of ATP ; probable loci of action of uptake inhibitors (reserpine and N-ethylmaleimide) are identified .
In 1962, independent studies by Kirshner (1) and Carlsson et al . (2) demonstrated the existence of a catecholamine incorooration system in isolated adrenal medullary vesicles which was stimulated by ATP and magnesium and inhib ited by reserpine .
Since then, a vast number of reports have appeared concern-
ing the nature of the system and the effects of nucleotides, divalent cations and various drugs on the uptake, storage and release of vesicular amines .
The
present discussion attempts to unify studies from several laboratories in order to produce a schematic model of the uptake system . 1.
Uptake and storage are separable processes and uptake is mediated by a
membrane-bound carrier .
Prior to the discovery of the ATP and magnesium stim-
ulated incorporation mechanism, Hillary (3, 4, 5) suggested that catecholamines and ATP could form a stable storage complex within the vesicle, in a molar ratio of 4 catecholamines / 1 ATP .
The suggestion has been confirmed in part
by in vitro studies which further implicate the involvement of divalent cations (6, 7) and possibly other vesicle constituents (3, 8, 9) .
This raised the
question whether amine incorporation might in fact represent storage only ;
875
878
Vol. 13, No . 8
Model of Catecholamine Uptake
catecholamines and ATP could diffuse through the membrane (4, 10) and then become bound in a non-diffusable form .
It therefore became important to ask
whether uptake and storage represented the same or different processes .
fiat
they are different was demonstrated by studies from our laboratory utilizing simultaneous measurements of incorporation and effl ux of radioactively-labeled amines (11, 12) ; the latter parameter gives a measure of the stability with which a given amine is stored within the vesicle .
In comparing the incorpora-
tions and effl uxes of serotonin and epinephrine, it was found that serotonin was incorporated to a greater extent than epinephrine despite the fact that it was not stored as stably as epinephrine ; if the incorporation process had refl ected passive diffusion followed by binding, then the more stably bound amine would have been incorporated to a greater extent .
Since this was not
the case, a second process, namely uptake, must be implicated to account for the higher incorporation of serotonin .
Additionally, B-phenylethylamine,
which was not itself incorporated into the vesicles, was able to inhibit epinephrine incorporation by about 50~ in equimolar concentrations, indicating an interference with epinephrine incorporation at a point prior to storage . A series of studies from Taugner's laboratory (13, 14, 15) and more recently by Pletscher et al . (16) have examined the properties of storage vesicles which have reformed after hypo-osmotic shock .
The "emptied" vesicles
were able to incorporate exogenous amines against a Gradient by utilizing the magnesium-activated ATPase in the membrane ; however, the amines incorporated in this system demonstrated a rapid rate of efflux, indicating that they were not stored within the empty vesicles .
This confirms that uptake and storage
are different processes and further suggests that the uptake is mediated by a carrier within the membrane .
Additional evidence for carrier-mediated trans-
port is provided by the following studies : a.
The incorporation of exogenous amines in intact vesicles requires energy in the form of ATP and is linked to the activity of the magnesium-activâted ATPase in the membrane ; both the
877
Model o~f Catecholamine Uptake
Vol . 13, No. 8
ATPase and uptake are inhibited by sulfhydryl reagents, such as N-ethylmaleimide (17) . b.
The uptake is saturable, with an estimated ~ of 4 to 8 x 10' 4 M in cow adrenal vesicles (18, 19) and 3 x 10 -5 M in rat adrenal
vesicles (H .O . green and T.A . Slotkin, submitted for publication) ; competition occurs with related amines and competitive potency is unrelated to subsequent storage stability (11, 12) . c.
The uptake process displays specificity, since catecholamines are preferred over related phenethylamines (such as metaraminol) and serotonin is preferred over catecholamines ; the uptake preference differs from the stability of storage (epinephrine > serotonin > metaraminol) .
d.
Uptake is temperature dependent, with a Q10 of 3 to 6, which is typical of transport processes (1) .
e.
Catecholamines are bound firmly to purified vesicle membranes, and the binding is inhibited by N-ethylmaleimide or reserpine in the presence of magnesium; the binding of epinephrine has a Kdiss
°f about 10 -4 M, which is close to the uptake !~, and the
binding has the same specificity as uptake into intact vesicles, viz . serotonin > epinephrine > metaraminol (20) .
These data in-
dicate that the binding of amines to the membranes may involve binding to the putative carrier. f.
Reserpine, which inhibits the ATP-magnesium stimulated incorporation of catecholamines, is persistently bound to the vesicle membrane (21, 22) and the degree of binding of reserpine to the membranes correlates with the effect of the drug .
These data all indicate that the first step in amine incorporation into adrenal storage vesicles is mediated by a membrane-bound carrier which utilizes ATP to transport amines . 2.
ATP is utilized on the inside of the membrane to cause detachment of
678
Model od Catecholamine Uptake
amines from the carrier ; transphos hor lation ma
Vol. 13, No. 8
be involved . In intact
vesicles as well as re-fornied "empty" vesicles, the uptake of amines is dependent on the membrane ATPase (13, 14, 16, 17) .
However, the binding of amines
to purified vesicle membranes is substantially reduced by ATP ; ADP is considerably less effective and AMP totally ineffective (20) .
This suggests that ATP
is utilized to detach the amine from the membrane-bound carrier and that the utilization of ATP may occur on the inside surface on the membrane .
Thus, the
ATP would have to undergo translocation in order to activate uptake .
Studies
by Hillary (4), Carlsson et al . (23), Kirchner et al . (10 ), Stevens et al . (24) and Stitzel et al . (25) all indicate that adenine nucleotides do indeed pass through the vesicle membrane to the interior .
However, in order to facilitate
uptake, the ATP would have to exist in a form other than the catecholaminecontainina storage complex ; since the complex is presumably non-diffusable, it cannot serve as an energy source for uptake, and the ATP utilized must therefore come from a labile pool .
Does a labile pool exist?
The efflux of ATP
from isolated vesicles is biphasic with an initially rapid rate of loss followed by a much slower rate (26), and the half-lives of the two components correspond to those of loosely and stably stored catecholamine, indicating that there is indeed a labile ATP pool within the vesicle .
Furthermore, studies in
perfused glands indicate that newly-formed ATP is incorporated in a labile manner and is not immediately stored stably (25) .
The concept of utilization of
ATP on the inside of the membrane is not unique, and is commonly observed in mitochondria (27) ; it should not be overlooked that the catecholamine storage vesicles themselves are potentially the richest source of ATP available. It is noteworthy that reserpine, which competitively inhibits catecholamine uptake (18, 23) as well as catecholamine binding to vesicle membranes (20) does not affect ATPase activity (28) ; this provides further evidence that the utilization of ATP is involved in the detachment rather than attachment of catecholamines to the membrane-bound carrier, and that it acts at a site different frorom that of reserpine.
Model ad Catecholamine Uptake
Vol . 13, No. 8
87 9
If ATP is indeed utilized in the detachment, the question remains as to the mechanism by which this occurs . a.
There are two likely possibilities :
Detachment occurs by binding of ATP to a site on the carrier resulting in a non-competitive reduction in amine binding capacity; this is observed in purified vesicle membranes and does not re quire magnesium (20) .
Since energy is not utilized, this alone
cannot be the mechanism, but the effect may precede the second step : b.
The carrier may be transphosphorylated, releasing the amine by a reduction either in affinity or capacity .
Trifaro (29, 30)
reports that transphosphorylation of membrane components (and indeed of the ATPase itself) occurs, requiring the same conditions as ATPase activity and stimulated uptake, and all three processes are inhibited by N-ethylmaleimide .
The degree of
transphosphorylation is far lower than the ATPase activity, suggesting a rapid turnover of phosphorylated intermediates, although some components appear to be phosphorylated to form stable products .
Transphosphorylation has also been implicated
in other carrier-mediated transport systems (31) . From these data, the following model of catecholamine uptake in storage vesicles can be constructed (Fig . 1) :
a catecholamine molecule (epinephrine,
E) attaches to a carrier (C) at the outside surface of the membrane .
This does
not involve the participation of ATP or magnesiun, but is inhibited by reserpine or N-ethylmaleimide in the presence of magnesium (20) .
The amine-carrier
complex (EC) is dissociated on the inside surface by a direct decrease in binding capacity caused by combination of the carrier with ATP simultaneously with (or followed by) transphosphorylation of the carrier to form CtiP, utilizing the conversion of ATP to ADP by the magnesium-activated ATPase . available for storage .
E then becomes
Just as ATP can penetrate the vesicle membrane, the ADP
can diffuse to the exterior ; in any case, the net effect on the inside surface
680
Model of Catecholamine Uptake
Vol . 13, No. 6
is the splitting of one molecule of ATP to make available for storage one molecule of E.
At the outer surface, CtiP is dephosphorylated to release C for com-
bination with another amine molecule .
The net effect on the outside .is the
disappearance of one molecule of ATP and the appearance of ADP and Pi with the inward transport of one molecule of E .
Assuming this one-for-one relationship,
the ATPase activity of the storage vesicles is at least 6 times that necessary for maximal catecholamine transport (29, 30), so that the energy requirement of the model is certainly feasible .
FI~ . 1 Hypothetical model of catecholamine transport in storage vesicles, showing probable sites of action of reserpine and N-ethylmaleimide (NEM) . E is epinephrine, C is carrier, EC is epinephrine-carrier complex, CtiP is phosphorylated carrier, and Pi is inorganic phosphate . The following observable effects are all accounted for by a system of this type : a.
In intact vesicles and empty vesicles, amines would be pumped in and ATP would be hydrolyzed ; uptake would be stimulated by ATP and magnesium.
b . N-Ethylmaleimide would block transport by inhibiting the ATPase (at either or both the transphosphorylation and dephosphorylation steps) as well as by blocking attachment of catechplamines to the carrier ; the latter effect would explain why concentrations of N-ethylmaleimide which totally block uptake produce only 50~ inhibition of the ATPase (11, 12, 29, 37) . c.
Reserpine would block transport by inhibiting attachment of the
Vol. 13, No. 8
Model ad Catecholamine Uptake
88 1
catecholamines, but would not inhibit the ATPase . d.
In isolated membranes, the binding of catecholamines would be inhibited by the addition of ATP or by reserpine or N-ethylmaleimide + magnesium.
Of course, in any such system, prediction of observed phenomena does not constitute proof of the model, and such an interpretation should be treated as solely a schematic representation with which to compare additional results when available .
For example, the coupling of ATP to a phosphorylated carrier may be
more complex than the simple reaction indicated, and other vesicle proteins, phospholipids, etc . may be involved as well . Although another vesicular uptake system has been identified which is reserpine insensitive and is not stimulated by ATP and magnesium, this second system operates only at high amine concentration and does not possess specificity (11, 12) ; the proposed model applies solely to the reserpine sensitive system . It is hoped that the formulation of this working hypothesis can help in the design of specific experiments to examine the catecholamine uptake system on the molecular level . ACKNOWLEDGMENTS Work reported from this laboratory was supported by grants from the Council for Tobacco Research - USA, Inc ., the North Carolina Heart Association, the Pharmaceutical Manufacturers Association Foundation, and USPHS DA-00465-~1 .
REFERENCES 1.
N . KIRSHNER, J . Biol . Chem . _237 2311-2317 (1962) .
2.
A. CARLSSON, N .-A. HILLARP and B . WALDECK, Med. Exp. 6 47-53 (1962) .
3.
N .-~ . HILLARP, Acta Physiol . Scan d . 42 321-332 (1958) .
4.
N .-ß . HILLARP, Acta Physiol . Scan d . 47 271-279 (1959) .
0
5.
0 N .-A .
6.
K.H . BERNEIS, A. PLETSCHER and M . DAPRADA, Nature 224 281 (1969) .
HILLARP, Acta Physiol . Scand . 50 8-22 (1960) .
882
Vol . 13, No . 8
Model ad Catecholamine Uptake
7.
K .H . BERNEIS, A . PLETSCHER and M DAPRADA, Brit . J . Pharmacol . 39 382-389 (1970) .
8.
A . PHILIPPU and H .J . SCHÜMANN, Naunyn- Schmiedebergs Arch . Pharmacol . 252 339-358 (1966) .
9.
A .D . SMITH, in The Interaction of Dru s and Subcellular C Animal Cells (P . . mp e , e . a. ur , on n
onents in
10 . N . KIRSHNER, C . HOLLOWAY, W .J . SMITH and A .G . KIRSHNER, in Mechanisms of Release of Bio enic Amines (U .S . von Euler, S . Rosell an . vnas, eds.) p . u9 Nergamon, x or y 11 . T .A . SLOTKIN, R .M . FERRIS and N . KIRSHNER, Mol . Pharmacol . 7 308-316 (1971} 12 . T .A . SLOTKIN and N . KIRSHNER, Mol . Pharmacol . 7 581-592 (1971) . 13 . G . TAUGNER, Naunyn- Schmiedebergs Arch . Pharmacol . 270 392-406 (1971) . 14 . G . TAUGNER, Naunyn-Schmiedebergs Arch . Pharmacol . 274 299-314 (1972) . 15 . B . AGOSTINI and G . TAUGNER, Histochemie 30 255-272 (1973) . 16 . A . PLETSCHER, M . DAPRADA, H . STEFFEN, K .H . BERNEIS and B . LUTOLD, 3rd Intl . Catecholamine Sym~ Abs . (1973) . 17 . N . KIRSHNER, in Pharniacolo of Choline is and Adrene (G .B . Koelle, W . . u as an . ar sson, e . p . Medical Press, Prague 1965) .
is Transmission ze os ova
18 . J . JONASSON, E . ROSENGREN and B . WALDECK, Acta Physiol . Scand . _60 136-140 (1964) . 19 . P . LUNDBORG, Acta Physiol . Scand . 67 423-429 (1966) . 20 . T .A . SLOTKIN and N . KIRSHNER, Biochem . Pharmacol . i n press . 21 . S . NORN and P .A . SHORE, Biochem . Pharmacol . 20 1291-1295 (1971) . 22 . S . NORM and P .A . SHORE, Biochem . Pharmacol . 20 2133-2135 (1971) . 23 . A . CARLSSON, N .-~ . HILLARP and B . WALDECK, Acta Physiol . Scand . _59 Suppl . 215, 5-38 (1963) . 24 . P . STEVENS, R .L . ROBINSON . K . VAN DYKE and R . STITZEL, J . Pharmacol . Exp . Ther . 18 1 463-471 (1972) . 25 . R .E . STITZEL, R .L . ROBINSON and P . STEVENS, Fed-Proç. 32 784Abs (1973) . 26 . F . LISHAJKO, Acta Physiol . Scan d . 76 159-171 (1969) . 27 . M . KLINGENBERG, in Re ulation of Metabolic Processes in Mitochondria (J .M . , p . 80 Tager, S . Papa, E . Quag a e o an . . a~~r, e . vo . American Elsevier, New York (1966) . 28 . R .M . FERRIS, O .H . VIVEROS and N . KIRSHNER, Biochem . Pharmacol . _19 505-514 (1970) . 29 . J .M . TRIFARO and J . DWORKIND, Mol . Pharm a col . 7 52-65 (1971) .
Vol. 13, No . 8
Madel of Catecholamine Uptake
30 . J .M . TRIFARO and M . WARNER, Mol . Pharmacol . 8 159-169 (1972) . 31 . D .L . OXENDER, Ann . Rev ._ Biochem . 41 777-814 (1972) .
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