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Relation between Na+-K+ transport and the uptake of choline by brain slices
Vol . 6, pp . 293-295, 1967 . LIFE SCIENCES Printed in Great Britain .
Pergamon Press Ltd.
RELATION BETWEEN Na+ -S+ TRANSPORT AAD TriS IIYT~ OF CHCLIAE BY BRAIN SLICS9 J . Schuberth, A . Sandwall and B . SSrbo Research Institute of National Defenoe~ Department l~ Sundbyberg 4, Sweden
(Received 10 October 1966 ; in final form 21 November 1966) We have previously conoluded that oholiae is taken up by brain oort+ex elioee by an aotive prooeae (1), mace the compound is taken up against a oonoentration gradient by a prooese inhibited by dinitrophenol~ anozia and exolusion of glucose from the medium . 31noe Ha±â+ stimulated ionio movements of oatione across the oell membrane has been shown to be intimately oonneoted with other transport mechanisms (2) we have studied the effeote of oatione and inhibitors of Na+-B+ transport on the aotive uptake of oholine . As seen in Table 1 mA~ m,~m uptake was obtained with eodium~ whereas repleoement of this ion with other monovalent nations leads to a deorease of the choline uptake . Strong evidence eaists for the participation of a Na+ B+ stimulated ATP-ass in the unidirectional transport of Ha+ and g+ through osll membraves (3) end this ATP-ass is inhibited by oligoa~yoin sad ouabaia (4~5) . It oould in faot be demonstrated that oligomyoia and ouabain also inhibit the choline uptake and that mush less inhibition is given by ousbaia in a system where Na+ ie replaced by Li+ (Table 1) . That the inhibition of transport processes by ouabaia requires the presence of Na+ has previously been demonstrated (6) . It should b~ pointed out that the concentration ratios given in Table 1 represent the total uptake of oholine~ not oorreoted for the "passive" oompoaent desoribed is our pre-" vious oommunioation (1) . This does not affect the oonolusione arrived at is the present paper, as the passive oomponsnt is onlf abet 2S ~ of the total uptake found in the oontrol e:perimente . 293
294
CHOLINE UPTAKE
Yol . 6, No . 3
TABLE 1 Effeots of Na± Defiçiencr on Accumulation of Choline in Brain Slices . Mouse brain oortez slices were incubated at 37 0 for 45 min in a medium containing 3 " 10-5 M tritiated aholine . The choline uptake was determined as previously described (1) . The standard medium was a modified %rebe-Henseleit solution (1) in which NaCl was replaced by equivalent amounts of other salts as indicated below . As oligomycin had to be used in an ethanolio solutions the results obtained with this inhibifor were compared with those obtained in experiments where the same amount of ethanol was added to the standard medium . Values are given as means + standard error . Numbers of experiments are given within braokets .
Incubation medium
3 .32 ± o .os2 (77)
Standard medium "
Concentration ratiô
2 .14 + 0 .118 (15)~
+ ouabain 10 -4M
2 .65 + 0 .126 (29)~
Na+ replaced by L1+ Na+ Na +
"
"
Cs
1 .67 ± 0 .065 (3)
Na+
"
"
NR4+
1 .87 + 0 .197 (3)
"
"
L1 + + ouabain 10 -4M
2 .48 + 0 .152 (15)
~ ~x
Na+ Na+
3 .12 ± 0 .151 (9)
standarg medium + ethanol (2 ~)
n
"
"
"
"
+ oligoutiyoin
5 wg/~ 2 .24 + 0 .174 (3)
"
"
"
"
+ oligomycin 12 ~g/ml 2 .41 + 0 .162 (6)
~ ~
* The ratio of the concentration of radioactivity in the tissue per g initial wet weight to the radioactivity in the suspending medium per ml . Significantly different from the control value at 1 ~ level .
Vol . 6, No . 3
-K+
The Na+
295
CHOLINE UPTAKE
stimulated ATP-ase has been implicated to participate in
tha transport oY Na+ and K+ by a direct reaction with these cations (3) " Since choline is concentrated against a high intracellular concentration gradient in the same way as K+ , the possibility was considered that the active choline transport involved a direct reaction between choline and ATP-sae . In this case choline should be expected to activate the Na+-S+ stimulated ATP-sae in the same way as K+ ~ but no such stimulation of the brain ATP-ase ~-~ae found by Aldridge ('() . A more likely explanation ßor the "coupling" of the choline transport system with the PIs+-g+ pump may be that the energy required îor the choline uptake is derived from the 27s+ electrochemical gradient established across the cell membr :~ne, as previously suggested (8) in the case of the active transport of amino acids into cells .
Reßerencea 1.
J . SCHUBERTH, A . SUNDî~ALL, B . SORBO and J .-0 . LINDELL~ J . Neurochem. 1~, 347 (1966) .
2.
J .H . QUASTEL, Proc . Roy . Soc . B 16~, 169 (1965) .
3.
J .C . SKOU, Biochim . Biophys . Actes 42, 6 (1960) .
4.
P .J . G naReHeM snd J .M . GLYNN, Nature 20 = s7 , 1098 (1965) .
5.
R . IYHITTALi, K .P . Y7FD;ELER and A . BLAKE, Nature ~0~, 720 (1964) .
6.
Li, FOX, S . TfiIEß, L . ROSENBEi~G and S . SEG.'1L, Biochim . Biophys . Acts 79~ 167 (1964) .
7.
S7 .Pi . ALDRIDGE, Biochem . J . 8~, 527 (1962) .
8.
D .D~ . .KIPNIS in : Control of Energy Metabolism , edited by B . Chance, R .w . Estabrook and J . williameon, p 221, Academio Press, New York (1965) .
Pergamon Press Ltd.
RELATION BETWEEN Na+ -S+ TRANSPORT AAD TriS IIYT~ OF CHCLIAE BY BRAIN SLICS9 J . Schuberth, A . Sandwall and B . SSrbo Research Institute of National Defenoe~ Department l~ Sundbyberg 4, Sweden
(Received 10 October 1966 ; in final form 21 November 1966) We have previously conoluded that oholiae is taken up by brain oort+ex elioee by an aotive prooeae (1), mace the compound is taken up against a oonoentration gradient by a prooese inhibited by dinitrophenol~ anozia and exolusion of glucose from the medium . 31noe Ha±â+ stimulated ionio movements of oatione across the oell membrane has been shown to be intimately oonneoted with other transport mechanisms (2) we have studied the effeote of oatione and inhibitors of Na+-B+ transport on the aotive uptake of oholine . As seen in Table 1 mA~ m,~m uptake was obtained with eodium~ whereas repleoement of this ion with other monovalent nations leads to a deorease of the choline uptake . Strong evidence eaists for the participation of a Na+ B+ stimulated ATP-ass in the unidirectional transport of Ha+ and g+ through osll membraves (3) end this ATP-ass is inhibited by oligoa~yoin sad ouabaia (4~5) . It oould in faot be demonstrated that oligomyoia and ouabain also inhibit the choline uptake and that mush less inhibition is given by ousbaia in a system where Na+ ie replaced by Li+ (Table 1) . That the inhibition of transport processes by ouabaia requires the presence of Na+ has previously been demonstrated (6) . It should b~ pointed out that the concentration ratios given in Table 1 represent the total uptake of oholine~ not oorreoted for the "passive" oompoaent desoribed is our pre-" vious oommunioation (1) . This does not affect the oonolusione arrived at is the present paper, as the passive oomponsnt is onlf abet 2S ~ of the total uptake found in the oontrol e:perimente . 293
294
CHOLINE UPTAKE
Yol . 6, No . 3
TABLE 1 Effeots of Na± Defiçiencr on Accumulation of Choline in Brain Slices . Mouse brain oortez slices were incubated at 37 0 for 45 min in a medium containing 3 " 10-5 M tritiated aholine . The choline uptake was determined as previously described (1) . The standard medium was a modified %rebe-Henseleit solution (1) in which NaCl was replaced by equivalent amounts of other salts as indicated below . As oligomycin had to be used in an ethanolio solutions the results obtained with this inhibifor were compared with those obtained in experiments where the same amount of ethanol was added to the standard medium . Values are given as means + standard error . Numbers of experiments are given within braokets .
Incubation medium
3 .32 ± o .os2 (77)
Standard medium "
Concentration ratiô
2 .14 + 0 .118 (15)~
+ ouabain 10 -4M
2 .65 + 0 .126 (29)~
Na+ replaced by L1+ Na+ Na +
"
"
Cs
1 .67 ± 0 .065 (3)
Na+
"
"
NR4+
1 .87 + 0 .197 (3)
"
"
L1 + + ouabain 10 -4M
2 .48 + 0 .152 (15)
~ ~x
Na+ Na+
3 .12 ± 0 .151 (9)
standarg medium + ethanol (2 ~)
n
"
"
"
"
+ oligoutiyoin
5 wg/~ 2 .24 + 0 .174 (3)
"
"
"
"
+ oligomycin 12 ~g/ml 2 .41 + 0 .162 (6)
~ ~
* The ratio of the concentration of radioactivity in the tissue per g initial wet weight to the radioactivity in the suspending medium per ml . Significantly different from the control value at 1 ~ level .
Vol . 6, No . 3
-K+
The Na+
295
CHOLINE UPTAKE
stimulated ATP-ase has been implicated to participate in
tha transport oY Na+ and K+ by a direct reaction with these cations (3) " Since choline is concentrated against a high intracellular concentration gradient in the same way as K+ , the possibility was considered that the active choline transport involved a direct reaction between choline and ATP-sae . In this case choline should be expected to activate the Na+-S+ stimulated ATP-sae in the same way as K+ ~ but no such stimulation of the brain ATP-ase ~-~ae found by Aldridge ('() . A more likely explanation ßor the "coupling" of the choline transport system with the PIs+-g+ pump may be that the energy required îor the choline uptake is derived from the 27s+ electrochemical gradient established across the cell membr :~ne, as previously suggested (8) in the case of the active transport of amino acids into cells .
Reßerencea 1.
J . SCHUBERTH, A . SUNDî~ALL, B . SORBO and J .-0 . LINDELL~ J . Neurochem. 1~, 347 (1966) .
2.
J .H . QUASTEL, Proc . Roy . Soc . B 16~, 169 (1965) .
3.
J .C . SKOU, Biochim . Biophys . Actes 42, 6 (1960) .
4.
P .J . G naReHeM snd J .M . GLYNN, Nature 20 = s7 , 1098 (1965) .
5.
R . IYHITTALi, K .P . Y7FD;ELER and A . BLAKE, Nature ~0~, 720 (1964) .
6.
Li, FOX, S . TfiIEß, L . ROSENBEi~G and S . SEG.'1L, Biochim . Biophys . Acts 79~ 167 (1964) .
7.
S7 .Pi . ALDRIDGE, Biochem . J . 8~, 527 (1962) .
8.
D .D~ . .KIPNIS in : Control of Energy Metabolism , edited by B . Chance, R .w . Estabrook and J . williameon, p 221, Academio Press, New York (1965) .