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Electrophysiological characteristics of giant axons of the lobster Homarus Americanus and their response to cholinergic compounds
Comp. gen. Pharmac., 1971 , 2, 99-xo 5. [Scientechnica (Publishers) Ltd.]
99
E L E C T R O P H Y S I O L O G I C A L C H A R A C T E R I S T I C S OF GIANT AXONS OF T H E LOBSTER HOMARUS AMERICANUS AND T H E I R RESPONSE TO C H O L I N E R G I C C O M P O U N D S THEODORE
B. H O E K M A N
AND W-D. D E T T B A R N
Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee 372o3, U.S.A.
(Received28 Aug., 197o) ABSTRACT i. Using polarization tests, five apparently distinct categories of electrical response are observed in the population of 7-Io giant axons of the lobster connective. 2. Differences in the response to externally applied acetylcholine (I × i o -3 M) correlate with these electrical categories. 3. The depolarization response to ACh in this preparation is blocked by cholinesterase (ChE) inhibitors or by strongly buffered bathing media (o. i M Tris or glyeylglycine, at pH 7), while a similar depolarization and spike decrement is produced by bathing solutions acidified to pH 4-o--5.o. 4. The possibility that the acetylcholine depolarization results from local pH alteration induced by the hydrolysis products of the demonstrated high cholinesterase levels of these axons is discussed. IT has been known for some time that the giant axons of the lobster circumoesophageal connective respond to the external application ofacetylcholine (ACh) with a m e m b r a n e depolarization (Dettbarn and Bartels, 1968; Bartels, Dettbarn, and Brzin, i969). This p h e n o m e n o n has been of particular interest in view of a proposed role for acetylcholine in axonal conduction (Nachmansohn, 1959). At least two categories of response to A C h have been indicated in this population of axons, depolarization with spike decrement and depolarization initiating repetitive or spontaneous discharge. A full or partial block of depolarization by cholinesterase inhibitors has been reported (Bartels, Dettbarn, and Brzin, I969). T o m i t a a n d W r i g h t (I965) have demonstrated that the capability of certain crustacean giant axons for repetitive firing in response to a long depolarization is related to m e m b r a n e resistance and capacitance. O n this basis we have used the response of axons to long cathodal and anodal stimulating pulses as a screening criterion to distinguish differences in m e m b r a n e characteristics that m a y be related to differences in
their A C h sensitivity. W e have further investigated the relationship between cholinesterase activity a n d the A C h depolarization. MATERIALS AND METHODS Circumoesophageal connectives were excised from 2.5-3-1b. lobsters Homarus americanus and prepared for examination using methods very similar to those described by Tobias and Bryant (1955) and by Dalton (I958). A connective which was ligated at either end before excision was desheathed under a dissecting microscope and then mounted in a perfusion chamber that permits continuous flow of the bathing medium (2-4 ml. per minute) and rapid exchange of solutions without disturbing the micro-electrode. The resting potential and action potentials were monitored using an intracellular glass micro-electrode (3-10 megohms) coupled to a conventional high input impedance pre-amplifier (Bioelectric NF- I). The output of the pre-amplifier was displayed on an oscilloscope (Tektronix RM-564) and the resting potential recorded continuously on a strip-chart recorder (Varian G-iooo) connected to the cathode follower output of the oscilloscope. The nerve-bundle was stimulated electrically through a bipolar electrode driven by a conventional stimulator and stimulus isolation unit (Grass S-4 and SIU-5). To elicit a single conducted action potential a cathodal pulse of o'l-o.2msecond duration was used, while for the polarization test a
IO0
Comp. gen. Pharmac.
HOEKMAN AND DETTBARN
pulse 3o-5 ° mseconds in duration was applied. Immediately after each successful impalement of an axon, its response to long hyperpolarizing and depolarizing pulses was checked. Stimulus intensity was increased to a value of twice the threshold for a single spike if a repetitive response was not obtained using a slightly supra-threshold stimulus. An axon responding with a single spike at this level of stimulus strength was judged to be ' nonrepetitive' to the stimulus polarity being considered. Artificial sea-water (Dettbarn and Bartels, ]968 ) was the basic perfusion medium. Test substances were dissolved in it and the pH adjusted to pH 7"7. RESULTS Application of the test concentration of x × Io - 2 M A C h produced differing responses in the population of 7-xo of the larger giant axons which we have studied in this connective. Using the polarization screening test, five apparently distinct categories of response have been distinguished (Fig. x). E a c h class of axons (arbitrarily designated types I - V ) appears to have a characteristic response to A C h (Table I). T y p e I axons, which give single spike or no response u p o n hyperpolarization and a single spike on depolarization, are usually insensitive to ACh. T y p e I I axons, which show no response or anodal break-firing with hyperpolarization and repetitive discharge during depolarization, are also insensitive to ACh. T y p e I I I axons fire repetitively during hyperpolarization, discharge a single spike u p o n depolarization, a n d are very sensitive to ACh. T h e rapid depolarization which occurs in the presence of A C h (I × I o -2 M ) often leads to spontaneous repetitive firing. T y p e I V axons fire repetitively on the ' anodal break ' from hyperpolarization and with a single spike occurring during depolarization. A C h induces a slow depolarization leading to spike decrement and conduction block (Fig. 2). T y p e V axons fire repetitively u p o n hyperpolarization and depolarization but are so infrequently observed that no characterization of their A C h response has been made. Competitive inhibitors (atropine and curare) are either ineffective or produce distinct p r i m a r y effects in concentrations that interfere with A C h action. However, it has been shown (Dettbarn and Bartels, I968 ) that
cholinesterase inhibitors quite specifically block the depolarization response to A C h . O u r experiments demonstrate that physostigmine, a so-called reversible inhibitor,
anodal
cathodal
II
III
IV
. L _ _ ~
.....
V
FIG. I.--Photographic records of the polarization test response of lobster giant axons. Calibration pulse in all records is 20 mV., 0"5 msecond. The figure shows the response of axon types I - V (described in text) to 3o-5omsecond-anodal (hyperpolarizing) and cathodal (depolarizing) pulses. Only types I I I and IV were notable sensitive to ACh. completely blocked the A C h depolarization (Fig. 3A). Likewise paraoxon, an irreversible inhibitor, completely blocked the response, b u t when it was displaced from the enzyme by pyridine-2-aldoxime methiodide (2-PAM) the A C h depolarization is restored (Fig. 3B).
I97I,
IOI
CHARACTERISTICS OF LOBSTER GIANT AXONS
=
P a r a o x o n as well as other alkylphosphates inactivate by phosphorylating the esteratic site of the ChE. This reaction is only slowly or not at all reversible in vitro. In vivo experiments Table L ~ T H E
the more effective nucleophilic agents is 2-PAM. T o characterize the depolarization response further, acetylcholine analogues with
RESPONSE OF GIANT AXONS OF THE LOBSTER CIRCUMOESOPHAGEAL CONNECTIVE TO LONG ANODAL AND CATHODAL PULSES AND TO ACETYLCHOLINE
AXON
R E S P O N S ETO S
RESPONSE TO
RESPONSE TO
TYPE
ANODAL PULSE
CATHODAL PULSE
A C H (IO -~ M)
I
Single spike or no response
Single spike at onset
Usually no response; if present very slow, often irreversible conduction block
II
No response or single anodal break spike
Repetitive firing during depolarization
No response
III
Repetitive firing during hyperpolarization
Single spike at onset
Sensitive to ACh, rapid depolarization often leading to spontaneous repetitive firing; rapid recovery from depolarization.
IV
Repetitive firing upon ' anodal break'
Single spike at onset
Slow response to ACh, eventual spike block
V
Repetitive firing during hyperpolarization
Repetitive firing during depolarization
Infrequently observed, insufficient data to characterize
Time (minutes) 12 18
24
30
-40 SW A
ACh
-60
~
~
E W
-so
t
FIG. 2.--Strip-chart recording of resting potential (Era) showing depolarization and photographic records of changes in the action potential when the preparation is perfused with ACh I × 10 -2 M. Calibration pulse is 20 mV., o. 5 msecond. The time of each photograph is indicated by the markers below the strip-chart tracing. The time when ACh is introduced (ACh) and when wash-out begins (SW) are shown above the tracing. with the that even the e n z y m e nucleophilic
axons used here indicated after 2 4 hours o f washing remained inhibited unless a reagent was added. O n e of
demonstrated activity at the n e u r o m u s c u l a r j u n c t i o n were tested. Carbamylcholine, decam e t h o n i u m , succinyl choline, a n d butyryl choline which are not hydrolysed or only
I02
Comp. gen. Pharmac.
HOEKMANN AND DETTBARN
elicited depolarization, a n d its response characteristics were nearly identical to A C h (Fig. 4). Most of the tests w i t h this c o m p o u n d
very slowly b y A C h E proved ineffective. Acetyl-13-methylcholine (mecholyl) was the only c o m p o u n d other t h a n A C h which
(minutes)
Time 6
-40
I:?
A
39
SW
A C h - t - Physo.
/.
-6O
~"
51
4.'5
/ f ~' ,
-80
, 2 / / 5 , 60 6 6 / 3 2 ,3//5od62" ,68 ,,, E
- 50
IdJ
,80
,86
B SW
-70
ACh
~PX
S,W
ACh
PAM ACh
-90 FIG. 3.--Strip-chart records of the resting membrane potential of lobster giant axons. A, Perfusion with i × io -2 M ACh leads to depolarization and spike decrement. Washing with artificial sea-water (SW) restores E m and conduction. Perfusion with ACh (x x Io -2 M) in the presence of physostigmine (Physo) (z × IO-~ M) had no effect. B, Control perfusion with ACh (i × IO-2 M) produces the usual depolarization andspike decrement. Treatment with paraoxon (PX) (2"5 × Io -8) M produced a transient effect upon the membrane potential but after recovery the axon was insensitive to ACh (ACh). Treatment with the paraoxon antidote 2-PAM (PAM) for a few minutes restored axonal sensitivity to ACh ( I × IO - I m ) ,
/ / m Time (minutes) 12 18
6 !
,
i
i
i
24
30
i
,
-40 SW E -60
DL-Mech
"3
1
W
-80
~-
"'
t
t
Fxo. 4.--Strip-chart recording of the resting potential (E ,) and photographic records of changes in the action potential showing a depolarization and spike decrement in response to DL-mecholyl (DLMech) 2 × zo-2 M. The calibration pulse is 2o rnV, o. 5 msecond. The time of each photograph is indicated by the markers below the tracing, and the time when the drug is introduced and when wash-out begins (SW) above the tracing.
x97I,
CHARACTERISTIGS
OF
were m a d e with a DL-mixture, but a few experiments were done with the purified L-isomer. It has been reported (Hoskin, I963) that only the D-acetyl-[3-methylcholine as determined optically is hydrolysed by the cholinesterase and in our experiments only the
LOBSTER
GIANT
xo3
AXONS
and/or acetic acid? W e have investigated the latter possibility in some detail. Perfusion of choline bromide in concentrations as high as 2 × Io -3 M h a d no effect u p o n the resting or action potentials. Estimates of local acetic acid concentration were not
Time (minutes) 6
12 ,--9, 4 2
v
i
E
'~
48
,
54
i
|
|
SW
-4O
A
,
DL-Mech
'J"
L - Mech
SW
-60
E
W
-80
FIG. 5.--Strip-chart recording of the resting membrane potential (Em). Perfusion with a DL-mixture of mecholyl (DL-Mech) 2"5 × zo-2 M elicits a depolarization and spike decrement similar to that seen with ACh. L-Mecholyl (L-Mech) is not hydrolysed, and is not effective.
gg Time (minutes) 12 18
6
24 !
*
30 |
,
-40 $W
-60
-eo
L
pH5.0
t
t
FIG. @--Strip-chart recording of resting potential (E m) and photographic records of changes in the action potential during perfusion with artificial sea-water acidified to pH 5'o. A depolarization arid spike decrement occurs showing many similarities to the ACh response. The markers above the trace indicate time when acid sea-water was introduced (pH 5.o) and when normal sea-water at pH 7"7 was returned (SW). The markers below the tracing indicate the times of the photographs. Calibration pulse is 20 mV, 0. 5 msecond. D-isomer was effective in depolarization (Fig. 5). Thus, it is seen that only c o m p o u n d s which are hydrolysed by the C h E are active in depolarization. This raises a critical question as to the mechanism of action: Is A C h interacting in p r i m a r y fashion with a receptor integral with the ChE, or is this a secondary effect of the hydrolysis products, choline
satisfactory, but depolarization and a spike decrement similar to that seen in presence o f A C h was elicited by reducing the p H of the perfusion m e d i u m to p H 4.0-5-0 (Fig. 6). Unfortunately, with this procedure the depolarization was not completely reversible. I f the A C h effect is directly related to the increase in local concentration of acetic acid
IO4
HOEKMAN
Comp. gen. Pharmac.
AND DETTBARN
as a hydrolysis product, the presence of a high concentration of a suitable buffer should reduce or block the depolarization. This was seen to be the case for the slow, decrementing spike depolarization of type I and type I V axons (Fig. 7A) as well as for the repetitive firing response of type I I I axons (Fig. 7B). When ACh (I × I o -2 M) was perfused in a
(Dettbarn and Bartels, i968; Bartels, Dettbarn, and Brzin, 1969). The splitting of ACh generates hydrogen ions within or close to the membrane since the major activity of the enzyme is limited to the axolemma (De Lorenzo, Dettbarn, and Brzin, I969). The morphological localization of the enzyme, the high levels of activity reported in this
Time (minutes) 6
-40
-60
12 ,
A
,//
57 .
.
.
63
.
69
SW
~...../A C h + Tri$
ACh
E -80 v
W
6
E -40
B
-60
A,~ Ch
12
Sp. act.
J,
./.1
39
45
51
SW
~1~,L ACh'l" Tri=
SW
-80
FIG. 7.--Strip records of the resting membrane potential of lobster giant axons showing the effect of strong buffer solutions on the ACh response. A, A control perfusion of ACh (I × IO-8 M) elicits depolarization and spike decrement in a type IV axon: while the same concentration is ineffective in the presence of o.I M Tris (Tris). B, ACh, I × io -2 M, produces depolarization leading to spontaneous activity (sp. act.) in a type III axon. Subsequent application of I × 1o-= M ACh in the presence ofo. x M Tris is ineffective. bathing solution buffered with o.i M Trishydroxymethyl-aminomethane (Tris), neither of these axon types was responsive in the usual fashion. I f these same axons were washed for 15-3o minutes in normally buffered bathing solution the characteristic response to ACh was restored. Equivalent results have been obtained using o.I M glycylglycine buffer. DISCUSSION The findings reported here support the suggestion that the ACh action is due to localized acidification of the membrane, caused by the hydrolysing activity of AChE
tissue, and the fact that the effective concentration approximates to that necessary for a maximal hydrolysis rate would seem to favour an accumulation of hydrogen ions. Such a shift in p H may be sufficient to cause the observed electrophysiological changes. The likelihood of this postulate is further demonstrated by the effect of cholinesterase inhibitors such as physostigmine and paraoxon in blocking the ACh action. The absence of an ACh effect in well-buffered seawater and the fact that part of the ACh response can be mimicked by changes in the p H of the bathing solution also support this conclusion. The experiments demonstrating
x97I, z
CHARACTERISTICS OF LOBSTER GIANT AXONS
the lack of action when the non-hydrolysable isomer of mecholyl, L-mecholyl, or other non-hydrolysable agonists of ACh, such as carbachol and decamethonium, were used, give additional support to the role of the hydrolysing activity of A C h E in causing the a p p a r e n t effect of A C h on m e m b r a n e and action potential of the circumoesophageal giant axon of the lobster. O n the basis of these experiments it appears that : - I. T h e r e are distinct differences in sensitivity to the application of A C h that m a y be related to the electrical characteristics of a particular axon. 2. T h a t the A C h depolarization responses are dependent u p o n cholinesterase activity, in particular the accumulation of acetic acid as a hydrolysis product. It is possible that the mechanism is similar to that demonstrated recently by Walker and Brown (i97o) for certain cells of Aplysia ganglion. Decreases in the extracellular p H were shown to increase the m e m b r a n e conductance to chloride. Hyperpolarization, depolarization, or no response was elicited, depending u p o n the value of the chloride equilibrium potential relative to the resting potential for the cell. T h e changes in resting potential and the conducted response of lobster axons m a y very well be related to such a change in conductance, although it is not possible on the basis of the available data to designate the ion involved. T h e observed differences in capability for repetitive response and a corresponding variability from axon to axon of resting potential (Tomita and Wright, I965) seem to support the possibility that the equilibrium potentials for some o f the critical ion species are different for the different axon types. This would
xo5
a c c o u n t for the variety of response observed in these studies. ACKNOWLEDGEMENT
This work was supported by Health Science Advancement Award Number 5-SO4-FRo6o67 (W-D. D.) and Graduate Training Grant Number G.M. ooo58 (T. B. H.). REFERENCES BARTELS, E., DETTBARN, W-D., and BRZIN, M.
(1969), ' Action of acetylcholine in the presence of organo phosphates on single axon of the lobster ', Biochem. Pharmac., x8, 2591-2596. DALTON, J. C. (1958), ' Effects of ions on membrane potentials of a lobster giant axon ', J. gen. Physiol., 4 x, 529-542. DELoRENZO, A. J. D., DETTBARN, W-D., and BRZIN, M. (I969) , ' Fine structural organization of acetylcholinesterase in single axons ', a7. Ultrastruct. Res., 28~ 27-4 o. DETTBARN, W-D., and BARTELS,E. (I968), ' Tile action of acetylcholine and cholinesterase inhibitors on single axons of the lobster ', Biochem. Pharmac., x7, I833-x844. HOSKIN, F. C. G. (x963), ' Stereospecificity in the reactions of acetylcholinesterase ', Proc. Soc. exp. Biol. Med., Ix3, 32o-32x. NAeHMANSOHN, D. (1959), Chemical and Molecular Basis of Nerve Activity, Chapter 6. New York: Academic Press. TOBIAS, J. M., and BRYANT, S. H. (I955), ' A n isolated giant axon preparation from the lobster nerve-cord ', 07. cell. comp. Physiol., 46, I63-I82. TO~nTA, T., and WRIGHT, E. B. (I965), ' A study of the crustacean axon repetitive response. I. The effect of membrane potential and resistance', Ibid., 65, I95-2Io. WALKER, J. L., jun., and BROWN, A. M. (I97O), 'Unified account of the variable effects of carbon dioxide on nerve cells ', Science, N.Y., x67, 15OO--I504.
Key Word Index: Lobster giant axon, Homarus americanus, acetylcholine, cholinesterase inhibitor, effect ofpH alteration.
99
E L E C T R O P H Y S I O L O G I C A L C H A R A C T E R I S T I C S OF GIANT AXONS OF T H E LOBSTER HOMARUS AMERICANUS AND T H E I R RESPONSE TO C H O L I N E R G I C C O M P O U N D S THEODORE
B. H O E K M A N
AND W-D. D E T T B A R N
Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee 372o3, U.S.A.
(Received28 Aug., 197o) ABSTRACT i. Using polarization tests, five apparently distinct categories of electrical response are observed in the population of 7-Io giant axons of the lobster connective. 2. Differences in the response to externally applied acetylcholine (I × i o -3 M) correlate with these electrical categories. 3. The depolarization response to ACh in this preparation is blocked by cholinesterase (ChE) inhibitors or by strongly buffered bathing media (o. i M Tris or glyeylglycine, at pH 7), while a similar depolarization and spike decrement is produced by bathing solutions acidified to pH 4-o--5.o. 4. The possibility that the acetylcholine depolarization results from local pH alteration induced by the hydrolysis products of the demonstrated high cholinesterase levels of these axons is discussed. IT has been known for some time that the giant axons of the lobster circumoesophageal connective respond to the external application ofacetylcholine (ACh) with a m e m b r a n e depolarization (Dettbarn and Bartels, 1968; Bartels, Dettbarn, and Brzin, i969). This p h e n o m e n o n has been of particular interest in view of a proposed role for acetylcholine in axonal conduction (Nachmansohn, 1959). At least two categories of response to A C h have been indicated in this population of axons, depolarization with spike decrement and depolarization initiating repetitive or spontaneous discharge. A full or partial block of depolarization by cholinesterase inhibitors has been reported (Bartels, Dettbarn, and Brzin, I969). T o m i t a a n d W r i g h t (I965) have demonstrated that the capability of certain crustacean giant axons for repetitive firing in response to a long depolarization is related to m e m b r a n e resistance and capacitance. O n this basis we have used the response of axons to long cathodal and anodal stimulating pulses as a screening criterion to distinguish differences in m e m b r a n e characteristics that m a y be related to differences in
their A C h sensitivity. W e have further investigated the relationship between cholinesterase activity a n d the A C h depolarization. MATERIALS AND METHODS Circumoesophageal connectives were excised from 2.5-3-1b. lobsters Homarus americanus and prepared for examination using methods very similar to those described by Tobias and Bryant (1955) and by Dalton (I958). A connective which was ligated at either end before excision was desheathed under a dissecting microscope and then mounted in a perfusion chamber that permits continuous flow of the bathing medium (2-4 ml. per minute) and rapid exchange of solutions without disturbing the micro-electrode. The resting potential and action potentials were monitored using an intracellular glass micro-electrode (3-10 megohms) coupled to a conventional high input impedance pre-amplifier (Bioelectric NF- I). The output of the pre-amplifier was displayed on an oscilloscope (Tektronix RM-564) and the resting potential recorded continuously on a strip-chart recorder (Varian G-iooo) connected to the cathode follower output of the oscilloscope. The nerve-bundle was stimulated electrically through a bipolar electrode driven by a conventional stimulator and stimulus isolation unit (Grass S-4 and SIU-5). To elicit a single conducted action potential a cathodal pulse of o'l-o.2msecond duration was used, while for the polarization test a
IO0
Comp. gen. Pharmac.
HOEKMAN AND DETTBARN
pulse 3o-5 ° mseconds in duration was applied. Immediately after each successful impalement of an axon, its response to long hyperpolarizing and depolarizing pulses was checked. Stimulus intensity was increased to a value of twice the threshold for a single spike if a repetitive response was not obtained using a slightly supra-threshold stimulus. An axon responding with a single spike at this level of stimulus strength was judged to be ' nonrepetitive' to the stimulus polarity being considered. Artificial sea-water (Dettbarn and Bartels, ]968 ) was the basic perfusion medium. Test substances were dissolved in it and the pH adjusted to pH 7"7. RESULTS Application of the test concentration of x × Io - 2 M A C h produced differing responses in the population of 7-xo of the larger giant axons which we have studied in this connective. Using the polarization screening test, five apparently distinct categories of response have been distinguished (Fig. x). E a c h class of axons (arbitrarily designated types I - V ) appears to have a characteristic response to A C h (Table I). T y p e I axons, which give single spike or no response u p o n hyperpolarization and a single spike on depolarization, are usually insensitive to ACh. T y p e I I axons, which show no response or anodal break-firing with hyperpolarization and repetitive discharge during depolarization, are also insensitive to ACh. T y p e I I I axons fire repetitively during hyperpolarization, discharge a single spike u p o n depolarization, a n d are very sensitive to ACh. T h e rapid depolarization which occurs in the presence of A C h (I × I o -2 M ) often leads to spontaneous repetitive firing. T y p e I V axons fire repetitively on the ' anodal break ' from hyperpolarization and with a single spike occurring during depolarization. A C h induces a slow depolarization leading to spike decrement and conduction block (Fig. 2). T y p e V axons fire repetitively u p o n hyperpolarization and depolarization but are so infrequently observed that no characterization of their A C h response has been made. Competitive inhibitors (atropine and curare) are either ineffective or produce distinct p r i m a r y effects in concentrations that interfere with A C h action. However, it has been shown (Dettbarn and Bartels, I968 ) that
cholinesterase inhibitors quite specifically block the depolarization response to A C h . O u r experiments demonstrate that physostigmine, a so-called reversible inhibitor,
anodal
cathodal
II
III
IV
. L _ _ ~
.....
V
FIG. I.--Photographic records of the polarization test response of lobster giant axons. Calibration pulse in all records is 20 mV., 0"5 msecond. The figure shows the response of axon types I - V (described in text) to 3o-5omsecond-anodal (hyperpolarizing) and cathodal (depolarizing) pulses. Only types I I I and IV were notable sensitive to ACh. completely blocked the A C h depolarization (Fig. 3A). Likewise paraoxon, an irreversible inhibitor, completely blocked the response, b u t when it was displaced from the enzyme by pyridine-2-aldoxime methiodide (2-PAM) the A C h depolarization is restored (Fig. 3B).
I97I,
IOI
CHARACTERISTICS OF LOBSTER GIANT AXONS
=
P a r a o x o n as well as other alkylphosphates inactivate by phosphorylating the esteratic site of the ChE. This reaction is only slowly or not at all reversible in vitro. In vivo experiments Table L ~ T H E
the more effective nucleophilic agents is 2-PAM. T o characterize the depolarization response further, acetylcholine analogues with
RESPONSE OF GIANT AXONS OF THE LOBSTER CIRCUMOESOPHAGEAL CONNECTIVE TO LONG ANODAL AND CATHODAL PULSES AND TO ACETYLCHOLINE
AXON
R E S P O N S ETO S
RESPONSE TO
RESPONSE TO
TYPE
ANODAL PULSE
CATHODAL PULSE
A C H (IO -~ M)
I
Single spike or no response
Single spike at onset
Usually no response; if present very slow, often irreversible conduction block
II
No response or single anodal break spike
Repetitive firing during depolarization
No response
III
Repetitive firing during hyperpolarization
Single spike at onset
Sensitive to ACh, rapid depolarization often leading to spontaneous repetitive firing; rapid recovery from depolarization.
IV
Repetitive firing upon ' anodal break'
Single spike at onset
Slow response to ACh, eventual spike block
V
Repetitive firing during hyperpolarization
Repetitive firing during depolarization
Infrequently observed, insufficient data to characterize
Time (minutes) 12 18
24
30
-40 SW A
ACh
-60
~
~
E W
-so
t
FIG. 2.--Strip-chart recording of resting potential (Era) showing depolarization and photographic records of changes in the action potential when the preparation is perfused with ACh I × 10 -2 M. Calibration pulse is 20 mV., o. 5 msecond. The time of each photograph is indicated by the markers below the strip-chart tracing. The time when ACh is introduced (ACh) and when wash-out begins (SW) are shown above the tracing. with the that even the e n z y m e nucleophilic
axons used here indicated after 2 4 hours o f washing remained inhibited unless a reagent was added. O n e of
demonstrated activity at the n e u r o m u s c u l a r j u n c t i o n were tested. Carbamylcholine, decam e t h o n i u m , succinyl choline, a n d butyryl choline which are not hydrolysed or only
I02
Comp. gen. Pharmac.
HOEKMANN AND DETTBARN
elicited depolarization, a n d its response characteristics were nearly identical to A C h (Fig. 4). Most of the tests w i t h this c o m p o u n d
very slowly b y A C h E proved ineffective. Acetyl-13-methylcholine (mecholyl) was the only c o m p o u n d other t h a n A C h which
(minutes)
Time 6
-40
I:?
A
39
SW
A C h - t - Physo.
/.
-6O
~"
51
4.'5
/ f ~' ,
-80
, 2 / / 5 , 60 6 6 / 3 2 ,3//5od62" ,68 ,,, E
- 50
IdJ
,80
,86
B SW
-70
ACh
~PX
S,W
ACh
PAM ACh
-90 FIG. 3.--Strip-chart records of the resting membrane potential of lobster giant axons. A, Perfusion with i × io -2 M ACh leads to depolarization and spike decrement. Washing with artificial sea-water (SW) restores E m and conduction. Perfusion with ACh (x x Io -2 M) in the presence of physostigmine (Physo) (z × IO-~ M) had no effect. B, Control perfusion with ACh (i × IO-2 M) produces the usual depolarization andspike decrement. Treatment with paraoxon (PX) (2"5 × Io -8) M produced a transient effect upon the membrane potential but after recovery the axon was insensitive to ACh (ACh). Treatment with the paraoxon antidote 2-PAM (PAM) for a few minutes restored axonal sensitivity to ACh ( I × IO - I m ) ,
/ / m Time (minutes) 12 18
6 !
,
i
i
i
24
30
i
,
-40 SW E -60
DL-Mech
"3
1
W
-80
~-
"'
t
t
Fxo. 4.--Strip-chart recording of the resting potential (E ,) and photographic records of changes in the action potential showing a depolarization and spike decrement in response to DL-mecholyl (DLMech) 2 × zo-2 M. The calibration pulse is 2o rnV, o. 5 msecond. The time of each photograph is indicated by the markers below the tracing, and the time when the drug is introduced and when wash-out begins (SW) above the tracing.
x97I,
CHARACTERISTIGS
OF
were m a d e with a DL-mixture, but a few experiments were done with the purified L-isomer. It has been reported (Hoskin, I963) that only the D-acetyl-[3-methylcholine as determined optically is hydrolysed by the cholinesterase and in our experiments only the
LOBSTER
GIANT
xo3
AXONS
and/or acetic acid? W e have investigated the latter possibility in some detail. Perfusion of choline bromide in concentrations as high as 2 × Io -3 M h a d no effect u p o n the resting or action potentials. Estimates of local acetic acid concentration were not
Time (minutes) 6
12 ,--9, 4 2
v
i
E
'~
48
,
54
i
|
|
SW
-4O
A
,
DL-Mech
'J"
L - Mech
SW
-60
E
W
-80
FIG. 5.--Strip-chart recording of the resting membrane potential (Em). Perfusion with a DL-mixture of mecholyl (DL-Mech) 2"5 × zo-2 M elicits a depolarization and spike decrement similar to that seen with ACh. L-Mecholyl (L-Mech) is not hydrolysed, and is not effective.
gg Time (minutes) 12 18
6
24 !
*
30 |
,
-40 $W
-60
-eo
L
pH5.0
t
t
FIG. @--Strip-chart recording of resting potential (E m) and photographic records of changes in the action potential during perfusion with artificial sea-water acidified to pH 5'o. A depolarization arid spike decrement occurs showing many similarities to the ACh response. The markers above the trace indicate time when acid sea-water was introduced (pH 5.o) and when normal sea-water at pH 7"7 was returned (SW). The markers below the tracing indicate the times of the photographs. Calibration pulse is 20 mV, 0. 5 msecond. D-isomer was effective in depolarization (Fig. 5). Thus, it is seen that only c o m p o u n d s which are hydrolysed by the C h E are active in depolarization. This raises a critical question as to the mechanism of action: Is A C h interacting in p r i m a r y fashion with a receptor integral with the ChE, or is this a secondary effect of the hydrolysis products, choline
satisfactory, but depolarization and a spike decrement similar to that seen in presence o f A C h was elicited by reducing the p H of the perfusion m e d i u m to p H 4.0-5-0 (Fig. 6). Unfortunately, with this procedure the depolarization was not completely reversible. I f the A C h effect is directly related to the increase in local concentration of acetic acid
IO4
HOEKMAN
Comp. gen. Pharmac.
AND DETTBARN
as a hydrolysis product, the presence of a high concentration of a suitable buffer should reduce or block the depolarization. This was seen to be the case for the slow, decrementing spike depolarization of type I and type I V axons (Fig. 7A) as well as for the repetitive firing response of type I I I axons (Fig. 7B). When ACh (I × I o -2 M) was perfused in a
(Dettbarn and Bartels, i968; Bartels, Dettbarn, and Brzin, 1969). The splitting of ACh generates hydrogen ions within or close to the membrane since the major activity of the enzyme is limited to the axolemma (De Lorenzo, Dettbarn, and Brzin, I969). The morphological localization of the enzyme, the high levels of activity reported in this
Time (minutes) 6
-40
-60
12 ,
A
,//
57 .
.
.
63
.
69
SW
~...../A C h + Tri$
ACh
E -80 v
W
6
E -40
B
-60
A,~ Ch
12
Sp. act.
J,
./.1
39
45
51
SW
~1~,L ACh'l" Tri=
SW
-80
FIG. 7.--Strip records of the resting membrane potential of lobster giant axons showing the effect of strong buffer solutions on the ACh response. A, A control perfusion of ACh (I × IO-8 M) elicits depolarization and spike decrement in a type IV axon: while the same concentration is ineffective in the presence of o.I M Tris (Tris). B, ACh, I × io -2 M, produces depolarization leading to spontaneous activity (sp. act.) in a type III axon. Subsequent application of I × 1o-= M ACh in the presence ofo. x M Tris is ineffective. bathing solution buffered with o.i M Trishydroxymethyl-aminomethane (Tris), neither of these axon types was responsive in the usual fashion. I f these same axons were washed for 15-3o minutes in normally buffered bathing solution the characteristic response to ACh was restored. Equivalent results have been obtained using o.I M glycylglycine buffer. DISCUSSION The findings reported here support the suggestion that the ACh action is due to localized acidification of the membrane, caused by the hydrolysing activity of AChE
tissue, and the fact that the effective concentration approximates to that necessary for a maximal hydrolysis rate would seem to favour an accumulation of hydrogen ions. Such a shift in p H may be sufficient to cause the observed electrophysiological changes. The likelihood of this postulate is further demonstrated by the effect of cholinesterase inhibitors such as physostigmine and paraoxon in blocking the ACh action. The absence of an ACh effect in well-buffered seawater and the fact that part of the ACh response can be mimicked by changes in the p H of the bathing solution also support this conclusion. The experiments demonstrating
x97I, z
CHARACTERISTICS OF LOBSTER GIANT AXONS
the lack of action when the non-hydrolysable isomer of mecholyl, L-mecholyl, or other non-hydrolysable agonists of ACh, such as carbachol and decamethonium, were used, give additional support to the role of the hydrolysing activity of A C h E in causing the a p p a r e n t effect of A C h on m e m b r a n e and action potential of the circumoesophageal giant axon of the lobster. O n the basis of these experiments it appears that : - I. T h e r e are distinct differences in sensitivity to the application of A C h that m a y be related to the electrical characteristics of a particular axon. 2. T h a t the A C h depolarization responses are dependent u p o n cholinesterase activity, in particular the accumulation of acetic acid as a hydrolysis product. It is possible that the mechanism is similar to that demonstrated recently by Walker and Brown (i97o) for certain cells of Aplysia ganglion. Decreases in the extracellular p H were shown to increase the m e m b r a n e conductance to chloride. Hyperpolarization, depolarization, or no response was elicited, depending u p o n the value of the chloride equilibrium potential relative to the resting potential for the cell. T h e changes in resting potential and the conducted response of lobster axons m a y very well be related to such a change in conductance, although it is not possible on the basis of the available data to designate the ion involved. T h e observed differences in capability for repetitive response and a corresponding variability from axon to axon of resting potential (Tomita and Wright, I965) seem to support the possibility that the equilibrium potentials for some o f the critical ion species are different for the different axon types. This would
xo5
a c c o u n t for the variety of response observed in these studies. ACKNOWLEDGEMENT
This work was supported by Health Science Advancement Award Number 5-SO4-FRo6o67 (W-D. D.) and Graduate Training Grant Number G.M. ooo58 (T. B. H.). REFERENCES BARTELS, E., DETTBARN, W-D., and BRZIN, M.
(1969), ' Action of acetylcholine in the presence of organo phosphates on single axon of the lobster ', Biochem. Pharmac., x8, 2591-2596. DALTON, J. C. (1958), ' Effects of ions on membrane potentials of a lobster giant axon ', J. gen. Physiol., 4 x, 529-542. DELoRENZO, A. J. D., DETTBARN, W-D., and BRZIN, M. (I969) , ' Fine structural organization of acetylcholinesterase in single axons ', a7. Ultrastruct. Res., 28~ 27-4 o. DETTBARN, W-D., and BARTELS,E. (I968), ' Tile action of acetylcholine and cholinesterase inhibitors on single axons of the lobster ', Biochem. Pharmac., x7, I833-x844. HOSKIN, F. C. G. (x963), ' Stereospecificity in the reactions of acetylcholinesterase ', Proc. Soc. exp. Biol. Med., Ix3, 32o-32x. NAeHMANSOHN, D. (1959), Chemical and Molecular Basis of Nerve Activity, Chapter 6. New York: Academic Press. TOBIAS, J. M., and BRYANT, S. H. (I955), ' A n isolated giant axon preparation from the lobster nerve-cord ', 07. cell. comp. Physiol., 46, I63-I82. TO~nTA, T., and WRIGHT, E. B. (I965), ' A study of the crustacean axon repetitive response. I. The effect of membrane potential and resistance', Ibid., 65, I95-2Io. WALKER, J. L., jun., and BROWN, A. M. (I97O), 'Unified account of the variable effects of carbon dioxide on nerve cells ', Science, N.Y., x67, 15OO--I504.
Key Word Index: Lobster giant axon, Homarus americanus, acetylcholine, cholinesterase inhibitor, effect ofpH alteration.