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The effects of changing the osmolarity of the ringer on acetylcholine release at the frog neuromuscular junction
rire sciences vol. io, Parc z, ~. 14as-14sa, 1971 . Printed in Great Britain
Pergamon Preen
TH8 EFFECP3 OF CHANGII~G THE OSl~OLARITY OF THE BINGER ON ACSTYLCHOLINE RELEASE AT THE FROG NE[TBQiQSCOLAR JQNCTION Hiroshi Rita, Ailliam Van der Rloot Departaant of Physiology b Biophysics, Health Sciences Center State IIniversity of New York, Stony Brook, New York 11790
(Received SO June 1971 ; in final form 22 November 1971) SIII~lARY Increases in the oemolarity of the Ringer are known to increase spontaneous acetylcholiae release from actor nerve endings while pafado~d.cally decreasing release from stiaulated nerves . Evidence ie presented suggesting the rise in osaolarity acts by increasing the ionic strength of the azoplason, altering electrostatic interactions between the acetylcholine-containing vesicles and the iffier face of the nerve terminal .
In the terainale of vertebrate motor nerves, the transmitter, acetylcholine (ACh), apparently is stored in vesicles (1,2) .
A resting nerve occasionally
releases a quanta of ACh into the gap between nerve and muscle ; the liberated ACh reacts with the auscle endplate =mbrane to produce a miniature endplate potential (IMP) .
Whnn an action potential invades the aeree terminal, approz-
iaately two-hundred quanta of ACh are released within e fraction of a milliseccad, generating as endplate potential (EPP) .
The aechaaisms by which an occa-
sional vesicle in a resting nerve terminal discharges its ACh or by which the action potential vastly accelerates ACh release are largely unknown. One clpe to the forces involved is that as increase in the tonicity of the Ringer, produced by adding NaCl or sucrose, causes a substantial increase in IMP frequency (3) .
Somewhat paradoxically, markedly hypertonic solutions also
block neurastuscular .tranemiseion, because when the action potential invades the terminal Iess ACh is released (4,5) .
Ae have analyzed the effects of increased
osmolarity on nauramuscular traneaissioa in the frog and suggnat that electrostatic forces are prominently involved in the interaction between the vesicles
14aß
1424
Osmolaríty and Acetylchollne Release
Vol . 10, No. 24
sad the inner face of the nerve membrane (6,7) . Methods The sciatic nerve-eartoriue muscle preparation from the frog was studied with conventional microelectrode methods .
In etudping EPP's the nerve was atim-
Mated supermaaimally once every 2 seconds and at least 200 responses were recorded .
MEPP's were recorded for at least one minute .
was kept inside the fibre throughout the experiment .
Usually the electrode Only in experiments in-
chiding temperature changes the electrode was withdrawn before the change, because the shift took same time and the muscle frequently changed its tension ; each run was limited to three temperature, to avoid drastic drops in resting potential .
The Ringer contained 100 mM NaCl, 2 mM KC1, 2 .5 mM CaC12, 3 mM
MgC12, and 8 mM tris maleate buffer (pH 7 .4) .
Solution osmolarity was checked
by measuring freezing point depression . Results MSPP freguency Bass and Moors (8) propose that increases in osmotic pressure increase MEPP frequency because the vesicles are surrounded by a shell of water moleculea that must be stripped away before the vesicles can fuse with the membrane sad release their ACh .
An increase in pressure decreases the energy required
to shed the layer of water and thereby accelerates spontaneous release .
This
mechanism does not account for the fact that solutes that can enter the nerve terminal do not produce these effects .
If glycerol, which is a permeant solute,
is added to the Ringer bathiag the eartoriue muscle, there is no change in MEPP frequency and we have not found any marked effect on the amplitude of the EPP . The model of Bass and Moor~ also fails to account for the effects of hypertonic solutions in decreasing the amount of ACh released from the stimulated nerve . Ona obvious possibility is that the increase in,MEPP frequency depletes the nerve terminal of the vesicles that are poised to release transmitter, so that when an action potential arrives, less ACh can be released .
This ezplanation is
Vol . 10, No . 24
Osmolarity and Acetylcholine Release
125
unlikely because as soon aB we can begin measurements after changing to a hypertonic solution, the amplitude of the EPP is decreased.
This suggests that there
is no need for an interval during which the stock of available quanta can be depleted before there is an effect on transmission .
furthermore,
in those ezperi-
mente in which frequeaciea are low enough for accurate measurements, the nuobera of quanta released both spontaneously and with nerve stimulation fit the Poisson distribution (Table 1) ; this fit implies thaç there are always a large number of available quanta (10,11,12) . TABLE I Fit of MEPP frequency and of number of quanta/EPP to the Poisson distribution for muscles in hypertonic solution in typical experiments .
The ezpected values were
calculated from the equation Nx ~ Nte~z/zl where Nt is the total number of samplea containing a number of MEPP's or z number of quanta/EPP and oc is the mean number of MEPP's or of quanta/EPP in the eemple .
The 0.71 sec time interval was
chosen because this was coroenient with our film viewing system . a MEPP 's/0 .71 sec in Binger + 100 mM sucrose Observed without added CaCh 0 38 1 42 2 32 3 15 2 4 1 5 P < 0.01 Integral [EPP amplitude/mean MBPP amplitude] in Binger + 220 mM sucrose at lOoC 0 5 1 20 2 33 3 40 4 23 32 5 6 15 7 8 8 5 9 3 10 0 11 1 P < 0.01
N=
Ez~ected 36 .8 46 .4 29 .3 12 .3 3 .9 1 .0
4 .6 17 .0 38 .4 38 .7 35 .7 26 .2 16 .3 8 .5 3 .9 1 .7 0.6 0.2
Since these effects are produced only by impermeant solutes, changes in the volume of the nerve terminal may be more significant than changes in the osmolarity of the solution .
In a hypertonic Ginger, the nerve terminals shrink,
so there is an increase ín the ionic strength of the aaoplasm .
Blioch et al (13)
suggested that the vesicles and the inner face of the resting nerve membrane
Oemolarity aid Acetyleholine Release
Y~188
Vol. 10, No. 24
have electrical surface charges of the smme sign produced by ionised groups on the membrane .
The surface charges produce a local accusiulation in the solution
of ions of the opposite sign, creating an electrical double layer .
The double
layers on the sesbrana s~ on the vesicles might have the ease sign, so they would repel one another and prevent aoet of the vesicles from approaching close ly to the m®brane.
Those vesicles that managed to get close to the sembrane
would be attracted by short-range, van der Waale forces ; if they collide into the proper Bite on the m®brave thn ACh from the vesicle night be released to the outside of the cell membrane . Increases in ionic strength decrease the thickness of the electrical double layers set up ae a result of the surface charges oa membranes, so perpapa the vesicles could. approach sore closely to the membrane .
The thickness
of the double layer is an iroerae function of the square root of the ionic strength of the solution .
As a simple model consider the effacte of ionic
strength on the rates of reaction bntwaen ions of different chemical species . If the ions have the same sign, a plot of log (reaction rate) ae a function of (ionic etrength)~ " 5 gives a straight line with a positive elope.
The reaction
rate gone up markedly ae the ionic strength increases, because the ions in solution screen the like charges on the reactants from one another . Plots of the log (MEPP/sec) as a function of points fall close to a straight line (Figure 1) .
(ommolarity)D " 5 show that the The fit to the plot suggests
that the vesicles and the inner face of the nerve membrane have surface charges of the same sign and that the increase in ionic strength produced by shrinking the nerve terminals is responsible for the increased release rate* .
However we
~bvioualy the interactions between a spherical vesicle and the inner face of a cylindrical membrane are more complicated than the interactions between single ions in solution . We have derived a general e:pression for the potential energy of interaction between vesicle and membrane using the Debye-Huckel approximation for low surface potentials . The length parameter enters this equation in several terms, so with substantial changes in ionic strength there are significant deviations from linearity in plots of predicted collision frequencies se a function of ~,1D .5 These details do not affect in any substantial way the arguments presented here and will be discussed further in a more detailed report .
Vol. 10, No . 24
Osmola~ity and Acetylcholine Release
127
are well aware that plots of logarithms as a function of square roots are apt to give spurious appearances of linearity, the model.
so we looked for another way to check
An increase in ionic strength ape~a the reaction rate between two
chemical species with charges of the same sign because there is a decrease in the free energy of activation,p ~ (14) . dF$
-
OH~
-
T~S'
100
u 0 a W â d ai
É
10
1
14
16
IS (mOsm)
20
°.d
22
24
FIG . 1. The relation between MEPP frequency and oemolarity at a frog neuromusin Dashed line : in a Ringer wade without CaCl2 . cular junction . Solid line : Straight liaea sre also oba Ringer made without CaC12 containing 1 mM PIgEDTA. tained when the muscles are kept in solutions with usual Ca++ concentrations .
The~ ~ has two components :
the activation heat, pH* and the product of
the absolute temperature, T and the activation entropy A Sam . ionic strength SIB is decreased because AS* ie increased . strength also produces a slight increase in
~R
With a higher A higher ionic
For instance, if there ie a
reaction between two univalesnt substances with charges of the same sign, an in crease in the ionic strength from 0 .1 to 0.2 will increase pH* by 0.004 e0. .
The
1428
Oemolarity and Acetylcholine Release
Yol. 10, No. 24
* -- becauaeA 1~ falls owreaction is speeded up -- despite the increase in ~H ing to an even greater increase in .,es - .
We ezperimentally estimated~t~ for
the release of ACh from nerve endings by measuring the !4EPP/sec at three different temperatures .
Arrhenius plots of log (I~PP/sec) va .
(1/T) show that the
points fall on a straight line ; the elope of the line is the activation energy EAct' EAct
Table IIA shove measured EAct's for spontaneous ACh release from nerve terminals in Ringer and in Ringer made hypersonic by adding sucrose .
Because
of the limited number of points, we cannot claim high accuracy, but clearly there ie not a sufficient change in EAct to account for the many-fold increase in the rate of release from the nerve in hypartonic solutions .
This increase
in rate cannot be owing to a fall in ~li~ so there moat be an increase in ASS. Ae described before, changes in ionic strength alter reaction rates by changing ~1S~, so we regard this result ae support for the idea that changes in the ionic strength of the a~plasm are responsible for the effects . EPP'e What can account for the effects of hypertonic solutions in decreasing ACh release from etiaulated nerve terminals?
Once again, plots of log (EPP) vs .
(oamolarity) O " 5 roughly approach straight lines ($ig . 2) but with negative slopes .
This suggests that in the stimulated terminal there is an interaction
between groups with opposite charges .
Osmolarity and Acetylcholine Release
vol, 10, No . â4
196
m0am
256
324
400
484
5T6
16
18
20
22
24
1429
10
É â ci
14
(mOsm) °`a Fig . 2 . Th~ relation between SPP amplitude and osmolarity in four typical eapnriments . When necessary, the amplitude of the EPP was decreased by increasing th~ MgCl2 concentration of the RLnger until a transaiseion block was obtained . Activation energies for ACh release from stimulated nerve terminals were calculated by determining the mean BPP from a series of 200 etimulations given once every two seconds at each of three different temperatures and using an Arrheaius plot .
As shown in Table IIB, there is no detectable change in $Act
with increasne in oamolarity . cause of a decrease in
eS,
Therefore, the decrease in release must occur be-
like that produced by an increase in ionic etreagth .
An alternative interpretation is the changes in ionic strength produce a conforoational change in the membranes, this could also lead to changes in release rate without changea in ,s8 ' .
1430
Oemolarlty a~ Acetylcholine Release
Vol. 10, No. 24
TABLE II Activation Energy for ACh Release A.
B.
Spontaneous release frani resting terminals Ringer
EAct(eV)
Numbei of ezperimente
+ 7 to 9 mT! M,g++
1.26 ± 0.24
13
+ 100 mM Sucrose
1.16 + 0.18
7
+ 200 mM Sucrose
1.70 + 0 .39
3
Release following nerve stimulation Ringer
EAct(eV)
Number of e:periaents
+ Curare
0 .83 + 0.16
4
+ 100 mM Sucrose
0 .82 + 0.13
7
+ 200 mM Sucrose
0.86 + 0.20
3
Summary and Conclusione We believe that in the resting nerve terminal the surface charges on the ACh-containing vesicles and on release sites on the inner face of the nerve terminal are the same sign (probably negative like known biological membranes) . They are electrostatically repelled from one another ; only an occasional vesicle has sufficient kinetic eaergy to pass through this electrostatic barrier so that it fuses with appropriate poiata on the membrane and releases its ACh .
Hyper-
tonic solutions cause an increase in the ionic strength in the nerve terminals; this decreases the electrostatic barrier and increases MEPP frequency.
After
the nerve is stimulated the surface charge on the inner face of the nerve terminal must decrease .
When the electrostatic barrier is reduced, eoany vesicles
release their ACh and an EPP is produced .
An increase in the ionic strength of
the azoplasm decreases the electrical attraction, thereby decreasing release rates . If this interpretation is correct,
the key event in transmission ie the
Vol. 10, No. 24
Osmolarity and Acetylcholine Release
1431
change in the magnitude of the surface charge on the inner face of the nerve terminal .
Ratz and Miledi
(15), hewn shown that Ca++ in the Ringer is essen-
tial for ACh release fron the stimulated nerve (though, as shown in Figure 1, Ca++ in the Ringer is not required for apontanaous release) . soma Ca++ enters the stimulated nerve terminal .
They suggest that
Perhaps this Ca++ combines
with negatively charged sites on the inner face of the menbrann to produce a decrease in surface charge, so the vesicles are more likely to reach the membraes and thereby release their ACh.
Increases in the ionic strength of the
azoplasm would decrease the interaction between Ca++ and the inner face of the m®braes,
eo there would be leas change in surface charge .
The result would be
a decrease in ACh release from the. stimulated nerve .
Acknowledgment Support by Grant 09096NS from the U .S .P .H .S .
Much of the study was conducted at
The New York University School of Medicine .
References 1.
B. Ratz, Proc . R. Soc.
1558 , 455 (1962) .
2.
D. F . Wilson and J . I. Hubhard, Fed. Proc . 29, 716 Abs . (1970) .
3.
P . Fett sad B . Ratz, J . Physiology i, 689 (1952) .
4.
S . Thaeleff, J. Physiol. 148, 659 (1959) .
5 ..
J. I . Hubhard, S. F. Jones, and 8. M . Landau, J. Physiol . 197, 639 (1968) .
6.
H. Rita and W. . Van der Rloot, Fed . Proc .
7.
H. &its sad W. Van der Rloot, Proc . 7CaV . Int.
~
457 (1969) . (}OIIII .
Phvaiol.
(in the Preen),
(1971) . 8.
L.~Baea and W. J . Moore, Proc . U . S. Nat . Aced .
9.
S. 7 . Furshpan, J. Physiol. 134, 689 (1956) .
Sçi ., 55, 1214 (1966) .
10 .
J. del Castillo and B. Ratz, J. Physiol. 124, 560 (1954) .
11 .
A. R. Martin, J. Physiol. 130, 144 (1955) .
12 .
A. R. Martin, Physiol . Rev. 46, 51 (1966) .
1432 13 .
Osmolarity and Acetylcholine Release
Z. L. Blioch, I . M . GLagoleva, B. A. Liberman, and V . A . Nenashev, J . Physiol. 199,
14 .
11 (1968) .
S. Glastone, R. J . Leider, and H. Eyring, The Theory of Rate Processes. (McGrav Rill, N .Y . 1941) pp . 427-442.
15 .
Vol . 10, No. 24
B . Rata and R. Miledi, J. Phyeiol. 207, 789,
(1970) .
Pergamon Preen
TH8 EFFECP3 OF CHANGII~G THE OSl~OLARITY OF THE BINGER ON ACSTYLCHOLINE RELEASE AT THE FROG NE[TBQiQSCOLAR JQNCTION Hiroshi Rita, Ailliam Van der Rloot Departaant of Physiology b Biophysics, Health Sciences Center State IIniversity of New York, Stony Brook, New York 11790
(Received SO June 1971 ; in final form 22 November 1971) SIII~lARY Increases in the oemolarity of the Ringer are known to increase spontaneous acetylcholiae release from actor nerve endings while pafado~d.cally decreasing release from stiaulated nerves . Evidence ie presented suggesting the rise in osaolarity acts by increasing the ionic strength of the azoplason, altering electrostatic interactions between the acetylcholine-containing vesicles and the iffier face of the nerve terminal .
In the terainale of vertebrate motor nerves, the transmitter, acetylcholine (ACh), apparently is stored in vesicles (1,2) .
A resting nerve occasionally
releases a quanta of ACh into the gap between nerve and muscle ; the liberated ACh reacts with the auscle endplate =mbrane to produce a miniature endplate potential (IMP) .
Whnn an action potential invades the aeree terminal, approz-
iaately two-hundred quanta of ACh are released within e fraction of a milliseccad, generating as endplate potential (EPP) .
The aechaaisms by which an occa-
sional vesicle in a resting nerve terminal discharges its ACh or by which the action potential vastly accelerates ACh release are largely unknown. One clpe to the forces involved is that as increase in the tonicity of the Ringer, produced by adding NaCl or sucrose, causes a substantial increase in IMP frequency (3) .
Somewhat paradoxically, markedly hypertonic solutions also
block neurastuscular .tranemiseion, because when the action potential invades the terminal Iess ACh is released (4,5) .
Ae have analyzed the effects of increased
osmolarity on nauramuscular traneaissioa in the frog and suggnat that electrostatic forces are prominently involved in the interaction between the vesicles
14aß
1424
Osmolaríty and Acetylchollne Release
Vol . 10, No. 24
sad the inner face of the nerve membrane (6,7) . Methods The sciatic nerve-eartoriue muscle preparation from the frog was studied with conventional microelectrode methods .
In etudping EPP's the nerve was atim-
Mated supermaaimally once every 2 seconds and at least 200 responses were recorded .
MEPP's were recorded for at least one minute .
was kept inside the fibre throughout the experiment .
Usually the electrode Only in experiments in-
chiding temperature changes the electrode was withdrawn before the change, because the shift took same time and the muscle frequently changed its tension ; each run was limited to three temperature, to avoid drastic drops in resting potential .
The Ringer contained 100 mM NaCl, 2 mM KC1, 2 .5 mM CaC12, 3 mM
MgC12, and 8 mM tris maleate buffer (pH 7 .4) .
Solution osmolarity was checked
by measuring freezing point depression . Results MSPP freguency Bass and Moors (8) propose that increases in osmotic pressure increase MEPP frequency because the vesicles are surrounded by a shell of water moleculea that must be stripped away before the vesicles can fuse with the membrane sad release their ACh .
An increase in pressure decreases the energy required
to shed the layer of water and thereby accelerates spontaneous release .
This
mechanism does not account for the fact that solutes that can enter the nerve terminal do not produce these effects .
If glycerol, which is a permeant solute,
is added to the Ringer bathiag the eartoriue muscle, there is no change in MEPP frequency and we have not found any marked effect on the amplitude of the EPP . The model of Bass and Moor~ also fails to account for the effects of hypertonic solutions in decreasing the amount of ACh released from the stimulated nerve . Ona obvious possibility is that the increase in,MEPP frequency depletes the nerve terminal of the vesicles that are poised to release transmitter, so that when an action potential arrives, less ACh can be released .
This ezplanation is
Vol . 10, No . 24
Osmolarity and Acetylcholine Release
125
unlikely because as soon aB we can begin measurements after changing to a hypertonic solution, the amplitude of the EPP is decreased.
This suggests that there
is no need for an interval during which the stock of available quanta can be depleted before there is an effect on transmission .
furthermore,
in those ezperi-
mente in which frequeaciea are low enough for accurate measurements, the nuobera of quanta released both spontaneously and with nerve stimulation fit the Poisson distribution (Table 1) ; this fit implies thaç there are always a large number of available quanta (10,11,12) . TABLE I Fit of MEPP frequency and of number of quanta/EPP to the Poisson distribution for muscles in hypertonic solution in typical experiments .
The ezpected values were
calculated from the equation Nx ~ Nte~z/zl where Nt is the total number of samplea containing a number of MEPP's or z number of quanta/EPP and oc is the mean number of MEPP's or of quanta/EPP in the eemple .
The 0.71 sec time interval was
chosen because this was coroenient with our film viewing system . a MEPP 's/0 .71 sec in Binger + 100 mM sucrose Observed without added CaCh 0 38 1 42 2 32 3 15 2 4 1 5 P < 0.01 Integral [EPP amplitude/mean MBPP amplitude] in Binger + 220 mM sucrose at lOoC 0 5 1 20 2 33 3 40 4 23 32 5 6 15 7 8 8 5 9 3 10 0 11 1 P < 0.01
N=
Ez~ected 36 .8 46 .4 29 .3 12 .3 3 .9 1 .0
4 .6 17 .0 38 .4 38 .7 35 .7 26 .2 16 .3 8 .5 3 .9 1 .7 0.6 0.2
Since these effects are produced only by impermeant solutes, changes in the volume of the nerve terminal may be more significant than changes in the osmolarity of the solution .
In a hypertonic Ginger, the nerve terminals shrink,
so there is an increase ín the ionic strength of the aaoplasm .
Blioch et al (13)
suggested that the vesicles and the inner face of the resting nerve membrane
Oemolarity aid Acetyleholine Release
Y~188
Vol. 10, No. 24
have electrical surface charges of the smme sign produced by ionised groups on the membrane .
The surface charges produce a local accusiulation in the solution
of ions of the opposite sign, creating an electrical double layer .
The double
layers on the sesbrana s~ on the vesicles might have the ease sign, so they would repel one another and prevent aoet of the vesicles from approaching close ly to the m®brane.
Those vesicles that managed to get close to the sembrane
would be attracted by short-range, van der Waale forces ; if they collide into the proper Bite on the m®brave thn ACh from the vesicle night be released to the outside of the cell membrane . Increases in ionic strength decrease the thickness of the electrical double layers set up ae a result of the surface charges oa membranes, so perpapa the vesicles could. approach sore closely to the membrane .
The thickness
of the double layer is an iroerae function of the square root of the ionic strength of the solution .
As a simple model consider the effacte of ionic
strength on the rates of reaction bntwaen ions of different chemical species . If the ions have the same sign, a plot of log (reaction rate) ae a function of (ionic etrength)~ " 5 gives a straight line with a positive elope.
The reaction
rate gone up markedly ae the ionic strength increases, because the ions in solution screen the like charges on the reactants from one another . Plots of the log (MEPP/sec) as a function of points fall close to a straight line (Figure 1) .
(ommolarity)D " 5 show that the The fit to the plot suggests
that the vesicles and the inner face of the nerve membrane have surface charges of the same sign and that the increase in ionic strength produced by shrinking the nerve terminals is responsible for the increased release rate* .
However we
~bvioualy the interactions between a spherical vesicle and the inner face of a cylindrical membrane are more complicated than the interactions between single ions in solution . We have derived a general e:pression for the potential energy of interaction between vesicle and membrane using the Debye-Huckel approximation for low surface potentials . The length parameter enters this equation in several terms, so with substantial changes in ionic strength there are significant deviations from linearity in plots of predicted collision frequencies se a function of ~,1D .5 These details do not affect in any substantial way the arguments presented here and will be discussed further in a more detailed report .
Vol. 10, No . 24
Osmola~ity and Acetylcholine Release
127
are well aware that plots of logarithms as a function of square roots are apt to give spurious appearances of linearity, the model.
so we looked for another way to check
An increase in ionic strength ape~a the reaction rate between two
chemical species with charges of the same sign because there is a decrease in the free energy of activation,p ~ (14) . dF$
-
OH~
-
T~S'
100
u 0 a W â d ai
É
10
1
14
16
IS (mOsm)
20
°.d
22
24
FIG . 1. The relation between MEPP frequency and oemolarity at a frog neuromusin Dashed line : in a Ringer wade without CaCl2 . cular junction . Solid line : Straight liaea sre also oba Ringer made without CaC12 containing 1 mM PIgEDTA. tained when the muscles are kept in solutions with usual Ca++ concentrations .
The~ ~ has two components :
the activation heat, pH* and the product of
the absolute temperature, T and the activation entropy A Sam . ionic strength SIB is decreased because AS* ie increased . strength also produces a slight increase in
~R
With a higher A higher ionic
For instance, if there ie a
reaction between two univalesnt substances with charges of the same sign, an in crease in the ionic strength from 0 .1 to 0.2 will increase pH* by 0.004 e0. .
The
1428
Oemolarity and Acetylcholine Release
Yol. 10, No. 24
* -- becauaeA 1~ falls owreaction is speeded up -- despite the increase in ~H ing to an even greater increase in .,es - .
We ezperimentally estimated~t~ for
the release of ACh from nerve endings by measuring the !4EPP/sec at three different temperatures .
Arrhenius plots of log (I~PP/sec) va .
(1/T) show that the
points fall on a straight line ; the elope of the line is the activation energy EAct' EAct
Table IIA shove measured EAct's for spontaneous ACh release from nerve terminals in Ringer and in Ringer made hypersonic by adding sucrose .
Because
of the limited number of points, we cannot claim high accuracy, but clearly there ie not a sufficient change in EAct to account for the many-fold increase in the rate of release from the nerve in hypartonic solutions .
This increase
in rate cannot be owing to a fall in ~li~ so there moat be an increase in ASS. Ae described before, changes in ionic strength alter reaction rates by changing ~1S~, so we regard this result ae support for the idea that changes in the ionic strength of the a~plasm are responsible for the effects . EPP'e What can account for the effects of hypertonic solutions in decreasing ACh release from etiaulated nerve terminals?
Once again, plots of log (EPP) vs .
(oamolarity) O " 5 roughly approach straight lines ($ig . 2) but with negative slopes .
This suggests that in the stimulated terminal there is an interaction
between groups with opposite charges .
Osmolarity and Acetylcholine Release
vol, 10, No . â4
196
m0am
256
324
400
484
5T6
16
18
20
22
24
1429
10
É â ci
14
(mOsm) °`a Fig . 2 . Th~ relation between SPP amplitude and osmolarity in four typical eapnriments . When necessary, the amplitude of the EPP was decreased by increasing th~ MgCl2 concentration of the RLnger until a transaiseion block was obtained . Activation energies for ACh release from stimulated nerve terminals were calculated by determining the mean BPP from a series of 200 etimulations given once every two seconds at each of three different temperatures and using an Arrheaius plot .
As shown in Table IIB, there is no detectable change in $Act
with increasne in oamolarity . cause of a decrease in
eS,
Therefore, the decrease in release must occur be-
like that produced by an increase in ionic etreagth .
An alternative interpretation is the changes in ionic strength produce a conforoational change in the membranes, this could also lead to changes in release rate without changea in ,s8 ' .
1430
Oemolarlty a~ Acetylcholine Release
Vol. 10, No. 24
TABLE II Activation Energy for ACh Release A.
B.
Spontaneous release frani resting terminals Ringer
EAct(eV)
Numbei of ezperimente
+ 7 to 9 mT! M,g++
1.26 ± 0.24
13
+ 100 mM Sucrose
1.16 + 0.18
7
+ 200 mM Sucrose
1.70 + 0 .39
3
Release following nerve stimulation Ringer
EAct(eV)
Number of e:periaents
+ Curare
0 .83 + 0.16
4
+ 100 mM Sucrose
0 .82 + 0.13
7
+ 200 mM Sucrose
0.86 + 0.20
3
Summary and Conclusione We believe that in the resting nerve terminal the surface charges on the ACh-containing vesicles and on release sites on the inner face of the nerve terminal are the same sign (probably negative like known biological membranes) . They are electrostatically repelled from one another ; only an occasional vesicle has sufficient kinetic eaergy to pass through this electrostatic barrier so that it fuses with appropriate poiata on the membrane and releases its ACh .
Hyper-
tonic solutions cause an increase in the ionic strength in the nerve terminals; this decreases the electrostatic barrier and increases MEPP frequency.
After
the nerve is stimulated the surface charge on the inner face of the nerve terminal must decrease .
When the electrostatic barrier is reduced, eoany vesicles
release their ACh and an EPP is produced .
An increase in the ionic strength of
the azoplasm decreases the electrical attraction, thereby decreasing release rates . If this interpretation is correct,
the key event in transmission ie the
Vol. 10, No. 24
Osmolarity and Acetylcholine Release
1431
change in the magnitude of the surface charge on the inner face of the nerve terminal .
Ratz and Miledi
(15), hewn shown that Ca++ in the Ringer is essen-
tial for ACh release fron the stimulated nerve (though, as shown in Figure 1, Ca++ in the Ringer is not required for apontanaous release) . soma Ca++ enters the stimulated nerve terminal .
They suggest that
Perhaps this Ca++ combines
with negatively charged sites on the inner face of the menbrann to produce a decrease in surface charge, so the vesicles are more likely to reach the membraes and thereby release their ACh.
Increases in the ionic strength of the
azoplasm would decrease the interaction between Ca++ and the inner face of the m®braes,
eo there would be leas change in surface charge .
The result would be
a decrease in ACh release from the. stimulated nerve .
Acknowledgment Support by Grant 09096NS from the U .S .P .H .S .
Much of the study was conducted at
The New York University School of Medicine .
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