J. Physiol. (1982), 333, pp.

125-139 With 11 text-Jigure8 Printed in Great Britain

125

MUSCARINIC AGONISTS INACTIVATE POTASSIUM CONDUCTANCE OF GUINEA-PIG MYENTERIC NEURONES BY K. MORITA, R. A. NORTH AND T. TOKIMASA From the Neuropharmacology Laboratory, Department of Nutrition and Food Science, Massachu8etts In8titute of Technology, Cambridge, MA 02193, U.S.A.

(Received 22 January 1982)

SUMMARY

1. The effects of muscarinic agonists applied both by perfusion and ionophoresis to myenteric neurones of the guinea-pig ileum were investigated by intracellular recording methods. 2. Perfusion with muscarinic agonists (acetylcholine, oxotremorine, methacholine, bethanechol) in concentrations of 100 nm to 10 ,UM caused membrane depolarizations. Brief ionophoretic applications of oxotremorine, or acetylcholine in the presence of hexamethonium, evoked depolarizations with a latency of 100 ms to 1 s and a duration of 5-60 s. 3. The depolarizations were completely antagonized by low concentrations (1-10 nM) of the muscarinic antagonists hyoscine or atropine. 4. The latency and time course of the muscarinic depolarizations were about one thousand times longer than those of nicotinic responses evoked in the same cell by acetylcholine applied from the same ionophoresis electrode. 5. The muscarinic depolarization was associated with a conductance decrease and reversed polarity at a membrane potential close to the potassium equilibrium

potential. 6. The muscarinic depolarization became smaller but did not disappear completely during prolonged (up to 60 min) perfusion or repeated (more than 0O02 Hz) ionophoretic applications of muscarinic agonists. 7. Lower concentrations (3-30 nM) of oxotremorine, which did not change membrane potential, reduced the amplitude and duration of the calcium-dependent increase in potassium conductance which follows a burst ofaction potentials. 8. It is concluded that the muscarinic depolarization of myenteric neurones is due to potassium inactivation.
INTRODUCTION

Acetylcholine (ACh) can cause both muscarinic and nicotinic responses in autonomic neurones. The nicotinic depolarization is responsible for the generation of the fast excitatory post-synaptic potential (e.p.s.p.) (Nishi & Koketsu, 1960), whereas in some ganglia the muscarinic receptor mediates much more prolonged synaptic potentials of both polarities (Libet, 1964; Nishi & Koketsu, 1968; Hartzell, Kuffler, Stickgold & Yoshikami, 1977; Horn & Dodd, 1981).
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K. MORITA, R. A; NORTH AND T. TOKIMASA Myenteric neurones of the guinea-pig ileum also have nicotinic fast e.p.s.p.s (Nishi & North, 1973) but the action of muscarinic agonists has not been studied. The purpose of the present experiments was to determine the nature and ionic mechanism of the action of muscarinic agonists on membrane properties of myenteric neurones. A preliminary report has appeared (Tokimasa, Morita & North, 1981).
126
METHODS

Adult guinea-pigs (250-350 g) were heavily stunned and bled from the neck. The ileum was rapidly removed and placed in Krebs solution of the following composition (mM): NaCl, 117; KCl, 4-7; CaCl2, 2-5;,;MgCl2, 1-2; NaH2PO4, 1-2; NaHCO3, 25; glucose, 11; gassed with 5% CO2 and 95 % 02. The myenteric plexus-longitudinal muscle preparation used was prepared as described by Nishi & North (1973). Single myenteric ganglia were immobilized by means of pinning through the closely adjacent muscle using stainless steel pins of 10 Aim in diameter. The ganglion was visualized by differential interference contrast optics (total magnification x 500). Intracellular recordings were made with glass micro-electrodes containing 2 M-potassium chloride (d.c. resistance 60-100 MCI), using a pre-amplifier (WP instruments, M701) which permitted current injection. Values for resting potential were determined by sudden withdrawal of the micro-electrode. A glass micro-electrode (tip diameter 10-.30 ,um) filled with Krebs solution was used to stimulate neuronal elements within the ganglion. The tip of this electrode was positioned on the surface of the ganglion and moved to different positions around the impaled neurone under visual control. Single or repeated cathodal current pulses (100-500 us duration) were passed through this electrode. Myenteric neurones were classed according to the criteria originally proposed by Nishi & North (1973) and Hirst, Holman & Spence (1974). If the neurone responded to focal stimulation with a fast excitatory post-synaptic potential (e.p.s.p.) it was called a Type 1 (S) cell. Those neurones which had no fast e.p.s.p. and which showed a prolonged after hyperpolarization following a single soma action potential were called Type 2 (AH) cells. The preparation was perfused (1-4 ml/min) with a pre-heated Krebs solution so that the temperature at the recording site was kept at 36-37 TC. Different solutions were applied to the preparation by means of a tap which changed the inflow to the tissue. The bath volume was 1-2 ml. Solutions of differing ionic composition were maintained iso-osmotic by adjusting the sodium chloride concentration. Acetylcholine and oxotremorine were applied by ionophoresis. The ionophoresis electrodes contained ACh chloride (100 mM) or oxotremorine sesquifumarate (30 mM). The tip of such an electrode was positioned within 3-10 sum of the soma of the neurone from which the intracellular recording was made, and outward current pulses were used to eject the drugs. A small (3 nA) inward current was routinely applied to reduce diffusion. Drugs used were acetylcholine chloride (Sigma), oxotremorine sesquifumarate (Aldrich), hexamethonium bromide (Sigma), atropine sulphate (MSD), hyoscine hydrochloride (Sigma), methacholinebromide (Calbiochem), bethanechol chloride (Sigma), and tetrodotoxin (Sankyo).
RESULTS

Muscarinic depolarization Application by perfusion. Methacholine, oxotremorine, acetylcholine (ACh) and bethanechol gave essentially similar results, but in the majority of experiments methacholine or oxotremorine were used. These muscarinic agonists caused a membrane depolarization which readily reached threshold for action potential generation (Fig. 1). The depolarization began within 10-30 s of the drug-containing solution reaching the tissue, and the membrane potential returned to its original value within 5-10 min of washing with drug-free solution. For a given agonist, the amplitude of the depolarization was dependent on the concentration applied (Fig. 2). Oxotremorine was approximately ten times more potent than methacholine.
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MUSCARINIC EFFECTS ON OK
A

127

a-T
a

~~~b

da

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Fig. 1. Methacholine depolarization. A, during the period indicated by the filled bar the perfusing solution contained methacholine (10 /SM). Action potentials (full height not recorded) were evoked by stimulating presynaptic nerves, except during methacholine application when the cell fired spontaneously. Downward deflections are anelectrotonic potentials caused by passing a constant current pulse across the cell membrane. B, oscilloscope records at different times during A. a, e.p.s.p. and action potential; b, during methacholine depolarization the e.p.s.p. is much reduced; c, washout; d, anelectrotonic potential; e, during methacholine depolarization the resistance was increased and anode break excitation occurred; f, washout. Resting potential was -60 mV.

E
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._ N

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0.

a
/

/
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Agonist concentration (log M) Fig. 2. Depolarization induced by perfusion with muscarinic agonists as a function of agonist concentration. 0, oxotremorine; *, methacholine. Bars indicate S.E. of mean for number of observations (Type 1 neurones) indicated.

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K. MORITA, R. A. NORTH AND T. TOKIMASA

Depolarizations were less often observed in Type 2 (AH) neurones and effective concentrations were generally higher than those depolarizing Type 1 (S) neurones. Application by ionophoresis. Passage of small currents (5-30 nA, 01-40 ms, 2-100 pC) through the ACh-containing pipette induced depolarizations in Type 1 (S) neurones which had amplitudes and time courses similar to the fast e.p.s.p. These
A

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10,50

10,100

15,1000

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0

15 10 MV 10S

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Fig. 3. Ionophoretic application of (A) acetylcholine and (B) oxotremorine. A, increasing the strength (nA) or duration (ms) of the ionophoretic current increased the amplitude ofthe slow depolarization (hexamethonium (200 /M) present). B, a similar experiment with oxotremorine. In both cases the depolarization was associated with an increase in input resistance. C, time course of potential change evoked by ionophoretic application of ACh. Amplitudes are scaled to a peak equal to 1: actual peak amplitude was 1I 7 + 26 mV (mean +s.E. of mean, n = 10). Continuous line is y = 1-69 (exp (-t/7 5)-exp (-t/P12)).

depolarizations, like the fast e.p.s.p., were reversibly abolished by hexamethonium (200 LM) and are termed nicotinic. Type 2 (AH) cells were not affected by these low ionophoretic currents of ACh, and never showed any depolarizations which were blocked by hexamethonium. ACh evoked a depolarization of much slower time course in both Type 1 (S) and Type 2 (AH) cells. In Type 1 (S) cells, this was occasionally observed with application of as little as 2 pC, and occurred after the subsidence of the initial nicotinic depolarization (see North & Tokimasa, 1982). However, in most Type 1 (S) cells and in all Type 2 (AH) cells it was necessary to eject at least ten times more ionophoretic
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129

charge to evoke the slow depolarization. Both long duration single pulses (5-20 nA, 10 ms to 3 s, typical charge 10 nC) and repeated passage of smaller pulses were equally effective. The slow ACh depolarization began after a minimum latency of 100 ms from the beginning of passage ofthe ionophoretic current (latency was 170 + 22 ms (mean + S.E. of mean) in thirty-one cells) and reached its peak amplitude within 1-5 s, the rise time being faster for larger ejection charges. This latency was increased at least
A
B

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Fig. 4. Hyoscine reversibly prevents the depolarizations induced by oxotremorine and ACh. A, oxotremorine perfusion (1/SM). Top, control. The arrow down and the arrow up indicate the switching on and off of d.c. polarizing current in order that the resistance could be measured at the resting level. Middle, hyoscine (10 nM). Bottom, wash. B, ACh ionophoresis (15 nA/5 ins; in the presence of hexamethonium (200 4M); thirty pulses at 10 Hz).Top, control. Middle, hyoscine (3 nM). Bottom, wash. C, oxotremorine ionophoresis (5 nA/2 s). Top, control. Middle, hyoscine (30 nM). Bottom, wash.

two-fold when the temperature of the perfusing solution was reduced from 37 to 30 0C. The total duration of the slow ACh depolarization was from 5 to 60 s. When the muscarinic response from ten different cells was averaged, its time course was found to be well fitted by a double exponential function with time constants for the rising and falling phases of 1P2 and 7-5 s (Fig. 30). In a given neurone, the amplitude of the slow depolarization was dependent on the ionophoretic current strength (Fig. 3). Single current pulses passed through ionophoresis pipettes containing oxotremorineevoked depolarizations similar in all respects to these caused by ACh in the presence of hexamethonium (Fig. 3A, B). Neuronal sensitivity to ACh and oxotremorine appeared to be similar, ranging from 01 to 500 mV/nC, with Type 1 (S) neurones being significantly more sensitive to oxotremorine' than type 2 (AR) neurones. Tetrodotoxin (100 nM) did not affect the depolarization induced by ACh or oxotremorine, although it abolished the action potential of Type 1 (5) neurones, and all synaptic potentials. This suggests that the effects of the muscarinic agonists are probably not mediated by the release of other transmitter substances from neighbouring neurones. Antaqonism by hyoscine and atropine. Hyoscine (10 nM) prevented the depolarization
5

~~~~~~~~~~~~~~~~~~~~~P HY 333

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K. MORITA, R. A. NORTH AND T. TOKIMASA

induced by perfusion with oxotremorine, ACh, methacholine or bethanechol (Fig. 4). This antagonism reversed 30-60 min after washout of the antagonist. Atropine and hyoscine had no effect on the resting membrane potential, input resistance or the action potential. The effects of ionophoresis of ACh or oxotremorine were also completely prevented by perfusion with hyoscine (1-30 nM) (Fig. 4). When the
A

a-/<~~b J

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10 Ms

los

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Fig. 5. A, effect of oxotremorine on action potential and input resistance. In each record, top trace is transmembrane current and bottom trace is membrane potential. a, control; t, oxotremorine (1 jSM) caused a membrane depolarization and a prolongation of the action potential; c, wash; d, control; e, oxotremorine (1 /SM) depolarized cell;f, the increase in input resistance was apparent when the potential was restored to the resting level by passing a steady hyperpolarizing current; g, wash. B, ACh depolarization induced by ionophoresis (15 nA/5 ms, thirty pulses at 10 Hz) in presence of hexamethonium (200 FM). C, oxotremorine (Oxo) depolarization induced by ionophoresis (20 nA/2 s). In both B and C, manual clamping of the slow potential change (traces marked b) revealed the increase in input resistance.

response to ACh was completely abolished by hyoscine, it could be restored by increasing either the amplitude or the duration of the ACh ejection current. The antagonism by hyoscine reversed after 10-30 min of washing. Ionic mechanism. Depolarizations evoked by perfusion with muscarinic agonists were usually associated with an increase in the amplitude of hyperpolarizing electrotonic potentials, reflecting an increased neuronal input resistance (Figs. 1, 4
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131

& 5). When the agonist-induced depolarization was prevented by passing current through the recording electrode (manual clamp), an increase in amplitude of electrotonic potentials was observed in all neurones (eg. Figs. 4 & 5 ). Action potentials evoked by passing depolarizing currents had an increased duration in the presence of muscarinic agonists (Fig. 5), and it was noticed that action potentials were
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lins Fig. 6. Reversal of slow ACh potential. A, response to ionophoresis of ACh (10 nA/ I00 ms) recorded at the membrane potential shown beside each trace. Electrotonic potentials were removed from all but two traces. Hexamethonium (200,uM) present throughout. B, amplitude of ACh potential as a function of membrane potential. Reversal occurs near -90 mV.

readily generated at the termination of hyperpolarizing current pulses (anode break excitation). The depolarizing effect of perfused muscarinic agonists was decreased when they were applied during passage of a constant hyperpolarizing current. A more complete study of the ionic mechanism was made with ionophoresis of ACh and oxotremorine. The membrane resistance was increased during the depolarization (Figs. 5 & 6) and this was still observed with the manual-clamp technique (Fig. 5). The amplitude of the ACh depolarization became smaller when it was evoked during membrane hyperpolarization and larger during membrane depolarization. Application of ACh to neurones hyperpolarized beyond about -90 mV caused a hyperpolarizing response which became larger with further membrane hyperpolarization (Fig. 6). The reversal potential in thirteen cells was - 88-0 + 1P5 mV (mean S.E. of mean). Effects on afterhyperpolarization. An afterhyperpolarization lasting up to 20 s follows a single action potential in Type 2 (AH) neurones. This was reduced in amplitude and duration by oxotremorine, even in concentrations (3-30 nM) which had no effect on membrane potential. The afterhyperpolarization which followed a
5-2

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132

K. MORITA, R. A. NORTH AND T. TOKIMASA

A

I AL

20I

C
a Control

Oxotremorine

c Wash

d Hyoscine

e Hyoscine and

oxotrenopine

f Hyoscine

gControl

h Oxotremorine

i Wah

lOos
Fig. 7. Muscarinic inhibition of the afterhyperpolarization which follows the action potential. A, the triangle indicates the time of ionophoretic application of ACh (80 nA/3 8). The upward deflections are depolarizing pulses of sufficient intensity to evoke a single action potential (monitored by oscilloscope). Type 2 (AH) cell, resting potential -60 mV, in hexamethonium (200 pM). B, an experiment similar to that in A, except that repeated ACh ionophoretic pulses were used (15 nA/5 ms; 10 Hz, 3 s). The afterhyperpolarization was evoked by thirty action potentials (10 Hz, 3 s). Type 1 (S) cell, resting resting potential -45 mV, in hexamethonium (200 ,M). Calibrations in B apply to A. C, afterhyperpolarization evoked by thirty action potentials. Perfusion with oxotremorine (30 nM) reduced the amplitude and duration of the afterhyperpolarization without changing resting potential (panel b). This effect washed out (c). Hyoscine (10 nM) alone did not change the afterhyperpolarization (d) but completely prevented the effect of oxotremorine (30 nM) (e). Following washout of hyoscine, oxotremorine (30 nM) had its usual effect (g-i).

burst of action potentials (post-tetanic hyperpolarization; p.t.h.) in both Type 1 (S) and Type 2 (AH) neurones was also reduced by oxotremorine (Fig. 7). In those cells in which resting potential was unchanged, oxotremorine (30 nM) reduced the peak amplitude of the p.t.h. to 76.2 + 6-2 %, the total duration to 62-6 + 8-7 %, and the half-decay time to 71-1+ 12-6 % of their respective control values (mean+s.E. of mean, n = 7). These effects of oxotremorine were reversibly antagonized by hyoscine (1 nM) (Fig. 7). Essentially similar findings were made when either oxotremorine or
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133

ACh were applied by ionophoresis. During the muscarinic depolarization, the action potential afterhyperpolarization or p.t.h. were greatly shortened, and often reduced in amplitude despite the increased driving force for their generation (Fig. 7). Desensitization Prolonged applications. The depolarization caused by perfusion with oxotremorine persisted throughout periods of application up to 60 min. However, both the potential change and the underlying conductance decrease progressively diminished in amplitude, reaching half their initial value after approximately 20 min (Fig. 8).
A

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0 30

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Time (min) Fig. 8. Desensitization during prolonged application of oxotremorine. A, sample of pen recordings taken during a 60 min application of oxotremorine (1 M). Action potentials occurred throughout the depolarization. On occasions the membrane potential was restored to the resting level (manual clamp) and input resistance measured by passing a constant current pulse. B, input resistance (0; control is 100%) and resting potential (mV, 0) as functions of time.

Repeated applications. Muscarinic depolarizations induced by ionophoretic application of ACh were identical when evoked at intervals exceeding 1 min. Application at intervals of between 30 and 60 s was associated with a progressive decline in the response amplitude (Fig. 11); shorter intervals could not be studied because of the long time course of the response. Muscarinic ACh depolarizations evoked at intervals of 30 s declined to half their initial amplitude in 3-8 min. When the ACh response was desensitized by repeated applications, it recovered its control amplitude when
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K. MORITA, R. A. NORTH AND T. TOKIMASA

the interval between applications was allowed to exceed 1 min. The response to ionophoretic application of ACh was also depressed by perfusion with concentrations ofoxotremorine ( < 30 nM) or ACh (< 100 nm) which were too low to produce depolarizations (Fig. 9). This depression rapidly recovered following washout of the perfusing agonists. These results indicate that the muscarinic response desensitizes to half its initial amplitude in the order of several minutes, and that recovery from the desensitized condition occurs within about 1 min.

A_
-

Y~~~~~~~~~~~~~

20 s Fig. 9. Desensitization of muscarinic depolarization. ACh was applied (15 nA/5 ms, thirty pulses at 10 Hz) at intervals of 1 0 s (triangles) and caused reproducible slow depolarizations. Perfusion with oxotremorine (30 nM) greatly reduced the response to ACh, even though this concentration is too low to cause any depolarization. Hexamethonium (200 #M)

present throughout.

Comparison of nicotinic and muscarinic responses Depolarization. In many neurones, the nicotinic and muscarinic actions of ACh were compared before and after addition of hexamethonium. Fig. 10 shows such a result. The ACh-containing pipette was placed close to the soma membrane, so that application of 45 pC of ACh evoked a depolarization quite similar in time course to the fast e.p.s.p. The latency of the response was 3 ms, the threshold ejection charge was 15 pC and the sensitivity was 230 mV/nC. This response reversed its polarity at a membrane potential close to -15 mV. After addition of hexamethonium (200 /M), a depolarization could only be evoked by applying several pulses through the ionophoretic pipette. This depolarization had a latency of 400 ms, and the sensitivity was 3-1 mV/nC. This depolarization reversed its polarity at -91 mV, and was completely eliminated by hyoscine (10 nM). Thus, the same neurone bears both nicotinic and muscarinic receptors linked to different ionic conductances. Desensitization. Nicotinic responses evoked at frequencies exceeding 5 Hz underwent a progressive decline in amplitude, and this was more marked at higher frequencies (Fig. 11). The rate of desensitization was greater with a larger initial response amplitudes or higher frequencies of application. When the initial response was 15 mV, repeated applications at 5 Hz evoked declining depolarizations which reached an amplitude of 7-5 mV after 5-6 + I 1 s (mean + S.E. of mean, n = 10). The time required
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MUSCARINIC EFFECTS ON 9K
A

135

5 ms

]20mV

20 ms 20mV

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A

ACh
4ns

ACh
C

AK

10 mV
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A

-40
A

-7
-85

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CA

20 ms

i 20 mV

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mV

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Fig. 10. Comparison of fast and slow effects of ACh on membrane potential of one myenteric neurone. A, fast ACh effects. Left, fast e.p.s.p. evoked by single stimulus (150 ,ss, 40 V) (ns). Right, depolarizations induced by ionophoretic application of ACh (15 nA, durations were, from left to right, 3, 8 and 10 ms) (triangles indicate termination of application). B, slow ACh effects after addition of hexamethonium (100jC/M). ACh, ionophoresis (10 nA/40 ms; 5 Hz/3 s). Left, control. Middle, 5 min after introducing hyoscine (3 nM). Right, after washout of hyoscine. C, reversal of fast ACh responses. ACh ionophoresis (15 nA/7 ms) (triangles). The membrane potential at which the response was evoked is indicated beside each recording. D, Reversal of slow ACh responses. ACh was applied repeatedly (as in B) in the presence of hexamethonium. Resting potential was -55 mV.

for the response to recover half its original amplitude from the fully desensitized steady state was 25 + 01 (n = 10). This recovery time constant was independent of the magnitude of the desensitization (Katz & Thesleff, 1957). The nature of the muscarinic desensitization was described above; this was compared with the nicotinic response desensitization in the same neurones, using initial response amplitudes which were approximately equal (Fig. 11). The desensitization time constant for the muscarinic response was 50-150 times greater than that
s

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K. MORITA, R. A. NORTH AND T. TOKIMASA

for the nicotinic response, and the time constants of recovery from desensitization also differed by a factor of about 50.
DISCUSSION

Ionic mechani m. Our results indicate that the muscarinic response is generated by a reduction in the membrane potassium conductance. The major evidence for this is the reduction in input conductance which accompanies the potential change, and
A

%J
20s

20 mV

~~~~~~~

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OmA

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ACh

l
Is

---,-

...* ...................... * i a,jlOmV

I.II II lI i AIlII.1ILit L 1iII.,1 I , i

I L

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Fig. 1 1. Comparison of muscarinic and nicotinic depolarizations with repeated applications. A, in hexamethonium (200 FM). ACh ionophoresis (triangles; 15 nA/5 ms pulses; 10 Hz, 3 8). No desensitization was apparent with applications separated by 50 s, but when this interval was reduced to approximately 30 s, the amplitude of the response progressively declined. B, upper trace, membrane potential. Lower trace, ionophoretic current monitor. ACh was applied by single pulses (10 nA, 7 ms). At intervals of about 28, no desensitization is apparent, but when the interval was reduced to 200 ms, the nicotinic response progressively declined.

the linear reduction in response amplitude with membrane hyperpolarization. The reversal of the muscarinic response at a membrane potential close to the potassium equilibrium potential would also be predicted for such a potassium inactivation, if one assumes that those channels which are closing are the ones through which the polarizing current passes. Inactivation of the potassium conductance has been proposed as the principal effect of muscarinic receptor occupation in both central (Krnjevic, Pumain & Renaud,
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137 MUSCARINIC EFFECTS ON 1K 1971; Dodd, Dingledine & Kelly, 1981) and peripheral (Kobayashi & Libet, 1970; Weight & Votava, 1970; Brown & Constanti, 1980) synapses. However, clear reversals of the response have generally not been demonstrated. The difficulty in reversing the muscarinic depolarization has been attributed by Brown & Adams (1980) to the rectifying properties of the membrane, since the potassium conductance which was closed by muscarinic agonists in bullfrog ganglion cells was open only at membrane potentials less negative than about -60 mV. Such a voltage-sensitive potassium conductance seems not to predominate in myenteric neurones, since little rectification occurs in the potential range of -60 to -90 mV (Nishi & North, 1973; present observations, eg. Fig. 6). Furthermore, the clear reversal of the muscarinic depolarization implies that the potassium channels remain open and sensitive to muscarinic agonists at even more negative potentials. Further insight into the ionic mechanism of the muscarinic action is afforded by our finding that oxQtremorine reduced the amplitude and duration of the calciumdependent potassium conductance which follows the action potential (Morita, North & Tokimasa, 1982a). This may indicate that the potassium channels closed by the muscarinic agonists are also sensitive to the intracellular calcium concentration. It is possible that the reduction in the afterhyperpolarization results from a direct decrease in calcium ion entry during the action potential; however, this seems a little unlikely because the action potential duration was always increased and never shortened by muscarinic agonists (eg. Fig. 5A). The minimum latency of 100 ms which we observed is similar to that reported for several other muscarinic responses to ACh including activation of potassium conductance in frog parasympathetic neurones (Hartzell et at. 1977) and cardiac muscle (Hill-Smith & Purves, 1978) or the depolarization of frog sympathetic ganglion cells (Koketsu, Nishi & Soeda, 1968) and smooth muscle (Bolton, 1976) (see also Hartzell, 1981). Because the muscarinic response is due to potassium channel closure, it seems necessary to invoke a closure of channels over the entire (electrotonically close) cell membrane. This contrasts with an agonist induced conductance increase in which the potential change is generated by passive flow across non-synaptic membrane which originates in a few specialized areas where channels are opened. It is possible that the latency arises because of the need for ACh to diffuse over and affect the entire cell surface. Calculations based on the diffusion of ACh in free solution suggest that this might not account for such a long delay (Krnjevic' & Mitchell, 1960; Hill-Smith & Purves, 1978). An alternative reason for the latency and slow time course may be the requirement for an intracellular intermediate to be generated and affect potassium channels throughout the cell. Further experiments are required to distinguish these possibilities. It seems unlikely that the kinetics of the potassium channel contribute to the delay since in various preparations the latency of the response to muscarinic agonists is similar whether the end result is opening or closing of potassium channels (Hartzell, 1981). It might be asked whether ACh released under physiological conditions could produce the muscarinic effects which we observed with exogenous application. In favourable experimental conditions, muscarinic depolarizations were observed in response to amounts of ACh about ten times more than those needed to mimic the
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K. MORITA, R. A. NORTH AND T. TOKIMASA

fast e.p.s.p. (500 mV/nC compared with 5 mV/pC; see North & Tokimasa, 1982). However, the resulting muscarinic response is a hundred to a thousand times longer than the nicotinic depolarization and is additionally excitatory by virtue of the increased input resistance and the reduction in the afterhyperpolarization. The following papers describe experiments which show that ACh released by' a single stimulus to nerves can indeed have significant effects on both the presynaptic (Morita, North & Tokimasa, 1982b) and the post-synaptic (North & Tokimasa, 1982) membranes.
This work supported by U.S. Department of Health and Human Services Grants NIH NS181 11 and. ADAMHA DA03160.
REFERENCES

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