J. exp. Biol. 128, 349-369 (1987)
349
Printed in Great Britain © The Company of Biologists Limited 1987
INTRACELLULAR FREE POTASSIUM, SODIUM AND
CHLORIDE MEASURED WITH ION-SELECTIVE
MICROELECTRODES FROM SALIVARY GLAND CELLS OF
THE SNAIL PLANORBIS CORNEUS
BY ANDREW BARBER*
Physiological Institute, University of Munich, Pettenkoferstrasse 12,
8000 Munich 2, West Germany
Accepted 10 November 1986
SUMMARY
Double-barrelled, ion-selective microelectrodes (ISMEs) have been used to
measure the intracellular free concentrations of K + ([K + ],), Na + ([Na + ]i) and C P
([CP],), together with membrane potentials (E M ), from single salivary gland acinar
cells of the pond snail Planorbis corneus. After adjustments had been made for
the cross-sensitivities of the ion-exchangers to other intracellular ions, the mean
concentrations were estimated to be: [K + ],, 42-9mmolF 1 ; [Na + ],, 2-4mmolP 1 ;
and [Cl~]j, 10-3 mmoll" 1 . The mean Nernstian equilibrium potentials for K + , Na +
and C P were calculated to be — 88 mV, +74-4 mV and — 41 mV, respectively. The
basolateral membrane of Planorbis salivary cells appears to be permeable to K + and
Na + under resting conditions, because blocking the electrogenic N a + / K + pump
with K+-free saline or ouabain revealed the presence of a large passive efflux of K +
and an influx of Na + . Salivary gland cells also lose intracellular C P rapidly in C P free saline (extracellular C P replaced by sulphate) which, along with other evidence,
indicates a substantial resting permeability of the salivary cell membrane to C P .
Stimulating gland cells with 10~4 mol P 1 acetylcholine (ACh) led to a depolarization
of E M , a rise in [Na + ], and a fall in [K + ],. This was followed by a transient
hyperpolarization of EM and a recovery of [Na + ], and [K + ], to their original levels.
There was no evidence that [CP], changes after stimulation with ACh. The
mechanism of action of ACh on Planorbis salivary gland cells and its relevance for
secretion are discussed.
INTRODUCTION
The ionic mechanisms underlying the secretion of salt and water by exocrine
glands are only partially understood. It is generally agreed that the interaction of
agonists with secretory cells leads to an increase in intracellular free Ca 2+ ([Ca ];) in
•Present address: Abteilung Pharmakologie, E. Merck AG, Frankfurter Strasse 250, 6100
Darmstadt 1, West Germany.
Key words: salivary gland cells, intracellular K + , intracellular Na + , intracellular C P , ion-selective
microelectrodes, Planorbis corneus, acetylcholine.
350
A. BARBER
the cytosol of the gland cells and also to an increase in the conductance of the
basolateral membrane to one or more species of ions (Ginsborg & House, 1980).
These processes produce fluxes of ions which are believed to stimulate secretion by
activating ion pumps in the gland cell membranes. However, the ions involved in the
responses of gland cells to agonists have not always been identified unambiguously
(Marty, Tan & Trautmann, 1984) and the exact nature of the pumps or transport
mechanisms responsible for secretion are not entirely clear (Martinez & Cassity,
1984).
It is apparent that the measurement of intracellular ions in gland cells could offer
some insight into the process of secretion. In the present study, double-barrelled,
ion-selective microelectrodes (ISMEs) have therefore been used to make direct
measurements of the intracellular free concentrations of K + , Na+ and C P ([K + ],,
[Na + ]i and [CP];, respectively) in the cytosol of salivary acinar cells of the pond
snail Planorbis corneus. In addition, estimates of the permeability of the resting
membrane to these ions were made. The ionic mechanisms underlying the response
to acetylcholine (ACh) were also examined. Some of the results given below have
been presented in a preliminary form to a meeting of the German Physiological
Society (Barber & ten Bruggencate, 1985*3).
MATERIALS AND METHODS
Preparation and solutions
Pairs of salivary glands were isolated from specimens of Planorbis corneus as
described previously (Barber, 1985) and transferred to a recording chamber which
had a volume of 0-2 ml. The chamber was perfused continuously at a rate of
7-5 mlmin" 1 with snail salines at room temperature (20-28°C).
Standard physiological saline contained (in mmoll"'): NaCl, 39; KC1, 1*3;
CaCl2, 4-5; MgCl2, 1-5; NaHCO3, 7-0; and was bubbled to a pH between 7-5 and
7-2 with a CO2/O2 gas mixture. K+-free saline was made by replacing KC1 with an
equimolar amount of NaCl, while Ca +-free saline was made by substituting MgCl2
for CaCl2 and adding 1 mmoll" 1 EGTA. High-K+ saline, in which 25mmolP' of
the NaCl content of standard saline was replaced by an equimolar concentration of
KC1, was also Ca +-free. CP-free saline was made by using sulphate salts of Na + ,
K + , Ca2+ and Mg2"1". It should be noted that this type of saline had a different ionic
strength compared to standard snail saline. Similar results were, however, obtained
with low-CP saline, which contained 39mmolP 1 sodium glucuronate instead of
NaCl. Nominally bicarbonate-free saline was made by substituting Hepes buffer for
sodium bicarbonate (pH adjusted with NaOH) and was gassed with 100% O 2 . The
production of metabolic CO2 means that this solution is unlikely to have been
completely free of bicarbonate ions. Acetylcholine bromide (Sigma) and ouabain
(Serva) were dissolved in these salines and perfused through the chamber as
required.
Free ions in snail gland cells
351
Micrvelectrodes and electrical recording
Intracellular recordings were made from salivary gland acinar cells with doublebarrelled ISMEs which had very fine tip diameters (<0-5 fim as measured under a
light microscope). These electrodes were constructed as described by Grafe, Rimpel,
Reddy & ten Bruggencate (1982; see also Barber & ten Bruggencate, 19856; Barber,
1986). The liquid ion-exchanger resins used in the tips of the K + -, Na + - and
CP-selective electrodes were Corning477317, ETH227 (Fluka) and IE-170 (W-P
Instruments), respectively. The shank and barrel of the ion-selective side of the
electrode were filled with 200mmoir' KC1, NaCl or KC1 for K + -, Na + - and Cl~selective electrodes, respectively.
The reference barrels were filled with 1 molP 1 magnesium acetate in the case of
+
K - and Na + -ISMEs and l m o l P 1 magnesium sulphate for Cl~-ISMEs and had
resistances of 50—100 MQ measured in physiological saline. Both barrels of the
electrodes were connected via chlorided silver wires to the input stage of a high
impedance amplifier.
The membrane potential (E^) of the impaled cell was registered by the reference
barrel of the ISME (Vref). Vref was also subtracted electronically from the potential
registered by the ion-selective barrel in order to monitor a pure ion signal (Vlon).
Current pulses could be applied between the bath and ground in order to check that
this subtraction was being performed accurately (see Fig. 7). The experimental
chamber was normally grounded through an agar bridge, though broken singlebarrelled microelectrodes filled with 3 moll" 1 KC1 (resistance 1-2MQ) were sometimes used in an attempt to reduce bath ground artefacts during the exchange of
different salines.
Electrodes were calibrated immediately after being withdrawn from the gland cells
by running calibration solutions through the experimental chamber. K+-selective
electrodes were calibrated in solutions containing SmmolF 1 NaCl together with
different concentrations of KC1. The Vlon for these electrodes (V^) increased by
an average of 53 mV per 10-fold increase in K + concentration, while their mean
K + :Na + selectivity ratio was 1:0-013 (N =30). Calibration solutions for Na + electrodes contained SOmmoll"1 KC1, l m m o l F 1 EGTA and different concentrations of NaCl. Vion for Na + -ISMEs (VNa) changed by a mean of 53 mV per 10-fold
change in Na + concentration, and the mean Na + :K + selectivity of these electrodes
was 1:0-06 (N = 9). C\~-electrodes were calibrated in pure KC1 solutions (Thomas,
1978) and Vion for CP-ISMEs (VCi) changed by a mean of 51 mV per 10-fold change
in Cl~ concentration (N = 15). Values of [K + ],, [Na + ], and [Cl~], are given in terms
of free ion concentrations since conversions to ion activities were not performed [see
Thomas (1978) and Tsien (1983) for a discussion of this point].
The K + ion-exchanger used in the present study is known to be sensitive to ACh
(Kuramoto & Haber, 1981), its response consisting of a positive shift in V^. It was
assumed, however, that the fall in intracellular VK produced by a brief (10 s)
extracellular application of ACh (e.g. Fig. 6A) was not influenced by any direct
response of the ISME to ACh.
352
A. BARBER
Intracellular measurements were also made with conventional single-barrelled
microelectrodes which were filled with S m o l F 1 potassium acetate and had resistances of 10-50 MQ measured in physiological saline. The input resistances of
impaled salivary gland acinar cells were measured by injecting pulses of direct
current through a balanced bridge circuit.
Ion fluxes and permeability coefficients of the resting gland cell membrane
Estimates of ion fluxes and permeability coefficients were made from changes in
[K + ];, [Na + ]j and [CP]; measured from glands bathed in either K + -free or CP-free
saline. These calculations were based on the following assumptions.
1. Planorbis salivary gland cells are cuboidal in shape with sides of 15 /J.m (see
Barber, 1985) and that only the basolateral membrane is exposed to changes in
solutions.
2. Changes in [K + ];, [Na + ], and [CP]; reflect net movements of ions across the
cell membrane, and changes occur uniformly throughout the entire cell volume.
3. No significant intracellular binding or buffering of K + , Na + or C P takes place
(Thomas, 1978).
4. The operation of the electrogenic N a + / K + pump is the only process responsible for the active transport of Na + and K + and any C P pumps are inactive in C P free saline (but see Aickin & Brading, 1985).
5. Changes in extracellular ion concentrations have no effect on the permeability
of the cell membrane (see Discussion).
6. Measurements of [K + ],, [Na + ] ; and [ C P ] ; in these experiments are uninfluenced by elicited action potential activity (but see Edman, Gestrelius & Grampp,
1983; Hodgkin & Horowicz, 1959) or any basal or stimulated secretion.
The net flux of any given ion, M lon , was calculated from the relationship
Mlon
=
A io
[ "].
V
~^T'A'
(1)
where M is measured in molcrrP s~1, A [ion]; is the net change of the intracellular
ion concentration in mol P 1 , At is time measured in seconds, V is cell volume in litres
and A is the surface area of the cell in cm2.
The K + and Na + permeability coefficients (PK and PNa» respectively) were
calculated as
R T
exp(EMF/RT)-l
P =M
{L)
*-,„„ M,on j ^ p [ i o n ] . . e x p ( E M F / R T ) - [ion]e
(Hodgkin & Horowicz, 1959; Hodgkin & Katz, 1949), where P lon is measured in
cms" 1 , [ion]e represents extracellular K + or Na + and [ion]; represents intracellular
K + or Na + (all concentrations being in molP 1 ). R, T and F have their usual
thermodynamic meanings ( R T / F = 25 mV).
The C P permeability coefficient (PCi) was calculated from the relationship
P
= M
rci
1Vlci
. ^ 1 - .
l-exp(-EMF/RT)
EMF [ C r ] e - [ C r ] , - e x p ( - E M F / R T )
{i
>
Free ions in snail gland cells
353
(Hodgkin & Horowicz, 1959), where [Cl ] e represents extracellular Cl and both
[Cl~] e and [Cl~]; are measured in mol I"1.
The theoretical value of EM was then calculated from the normally employed form
of the Goldman (1943) equation,
EM =
RT
In
F
(4)
RESULTS
Resting concentrations offree intracellular ions
An example of a successful impalement of a Planorbis salivary acinar cell with an
ISME, in this case a K+-selective electrode, is shown in Fig. 1. It was found that
1 min
-20-
-40
W
-60-
\
-80
50
30
di
30/
60-
20
o
50
80-
or
2 /
40-
E
20-
1-3 J —>
n .
Fig. 1. Measurement of [K + ], from a Planorbis salivary gland acinar cell. (A) A typical
intracellular recording from a gland cell with a double-barrelled K+-selective microelectrode. The membrane potential (EM) of the impaled cell (top trace) is measured as the
potential from the reference barrel of the microelectrode (Vref), while [K + ], (bottom
trace) is the potential from the ion-selective barrel minus Vref (= V K ; see Materials and
Methods). Spontaneous action potentials were recorded from this gland cell, but the
amplitudes of these spikes are not shown at their full size. (B) The calibration of the ionselective microelectrode used in A following its withdrawal from the cell. The electrode,
which had a tip diameter of less than 0-5/im, was calibrated by running solutions with
known K + concentrations through the experimental chamber.
354
A. BARBER
stable intracellular recordings with ISMEs were usually not possible with electrodes
which had tip diameters greater than around 0-5 fim. Another general observation
was that even fine electrodes required periods of 5-20 min after impalement to 'seal
into' the gland cell membrane, as judged by the time necessary to measure stable
values of E^ and Vlon. The relationship between the measured values of EM and
[K+];, [Na+]i and [CP], are shown in Fig. 2.
Data concerning the resting levels of free intracellular ions in Planorbis salivary
gland cells are brought together in Table 1. Where appropriate these values of
[ion], have been corrected for inaccuracies introduced by cross-sensitivity of the
ion-exchangers to other intracellular ions ([Na + ], and [CP], measurements; see
= 0-7
60 -i
A
50403020/• = 0-5
10-
-52
-56
[cr],
30-
-60
•
•
20-
•
-64
•
-68
-72
-76
-80
r=-0-3
•
•
•
10-
0J
-52
-56
-60
-64
-68
-72
-76
-80
EM
Fig. 2. Relationship between [ion], and membrane potential (EM) for acinar cells impaled with double-barrelled ion-selective microelectrodes. The lines of best fit for each
ion were calculated by linear regression, and the correlation coefficient (r) is also
indicated.
355
Free ions in snail gland cells
Discussion). The labelling of the [ion]; ordinates of the figures, however, has not
been adjusted for interference.
Resting permeability of the gland cell membrane and mechanisms for maintaining
The high resting level of [K + ] ; and the very low resting value of [Na+][ in
Planorbis salivary gland cells (Table 1) imply the presence of active mechanisms
for accumulating K + and extruding Na + against their respective electrochemical
gradients. It is generally accepted that the resting distribution of these ions across
cell membranes is due chiefly to the operation of an electrogenic Na + /K + pump
(Petersen, 1980). The role of the Na + /K + pump in maintaining [K + ]; and [Na + ] ; in
Planorbis salivary glands was confirmed by reversibly inhibiting the activity of this
pump with K+-free saline. After a delay of 4-10 min, the cells began to lose K + at
-i
an initial and maximum rate of 2-6 mmoll min (s.D. ±0-53mmoll min
+
1
1
N = 4) and to gain Na at an initial and maximum rate of 0-15 mmoll" min" (s.D.
±0-03mmoir 1 min" 1 ,YV=5) (Fig. 3).
Table 1. A summary of data obtained from Planorbis salivary gland cells zuith ionselective microelectrodes
K+
Basal values
Mean [ion], ± S.D. (mmoll" 1 )
Mean membrane potential
(EM±s.D.;mV)
[ion] e (mmoll ')
Mean equilibrium potential
(E, o n ;mV)
Number
Apparent net flux
(M ion ; molcm~ 2 s~')
Apparent permeability
coefficient (P; on ; cms )
Na +
cr
Ca2+'
42-9 ±10-3
-66-5 ±5-7
2-4 ± 1-1
-68-6 ±5-8
10-3 ± 2-9
-63-3 ± 5-2
124±84nmoll" 1
1-3
-88-0
460
+74-4
52-3
-41
4-5
+ 132
32
6-5X10" 11
15
7-5X10" 12
19
2-5x10 - l i
9
—
7-6X10" 6
5-4X10" 8
8-9x10 - 7
—
Following stimulation with 10~ 4 moll~' acetylcholine
Mean A[ion],± s.D. (mmoll" 1 )
-4-0±2-0
+3-2 ± 1 0 none detected
Mean A E M ± S.D. (mV)
55-8 ±7-5
53-3 ±4-9
44-6 ±7-5
Number
17
23
12
Mean A[ion] e ± S.D.
+0-6 ± 0-3
—0-6 ± 0 1 none detected
-67-1 ±4-1
+ 290 ± lOOnmoll" 1
49-7 ±6-3
12
none detected
(mmoir')t
Number
47
12
10
15
Information from Barber (1986; •) and Barber & ten Bruggencate (19856; f) is also included.
N.B. Measurements of acetylcholine-induced changes in [K + ] e were made using valinomycin ionexchanger ion-selective microelectrodes, which are insensitive to acetylcholine (see Materials and
Methods).
Values of basal [Na + ]j and [Cl~], have been adjusted for the effects of cross sensitivity, as described in
the Discussion.
356
A. BARBER
Time lags in changes in [K + ], and [Na + ] ; were not observed when the N a + / K +
pump was blocked irreversibly by ouabain (e.g. Fig. 6B), which probably indicates
that some time is required in K+-free saline to deplete extracellular K + at the surface
of the gland cells. The rate of decrease of [K + ]; and increase of [Na + ]; were,
however, similar in glands treated with K+-free saline or ouabain (the rate of increase
of [Na + ], in Fig. 6B is approximately 0-13 mmolP 1 min" 1 ), which presumably
indicates that the N a + / K + pump is fully blocked in K+-free saline once K + is
washed from the experimental chamber.
Measurements of changes in [K + ], are unlikely to have been influenced to any
great extent by the parallel increase in [Na + ],. However, the relatively large fall in
[K + ]| and the comparatively poor Na + :K + selectivity ratio of the Na + -ISMEs (see
Materials and Methods) mean that the rise in [Na + ] ; is likely to have been underestimated by around 0-15 mmol I"1 min" 1 (i.e. [Na + ] ; actually rises by 0-3 mmol P 1
min~ in K+-free saline). These values were inserted into equation 1 and the net
efflux of K + (MK) and net influx of Na + (MNa) were calculated (Table 1). The
permeability coefficients for K + (PK) and Na + (PN E ) were calculated (Table 1) using
equation 2.
Readmitting normal K+-containing saline into the experimental chamber resulted
in a transient membrane hyperpolarization, presumably caused by the reactivation
of the electrogenic Na + /K + pump (see Poulsen & Oakley, 1979) and the rapid
restoration of the original levels of [K + ], and [Na + ], (Fig. 3).
C P was lost from salivary gland cells in CP-free saline (Fig. 4) at a mean initial
and maximum rate of 1-0 mmol P 1 min" 1 (s.D. ±0-47mmolP 1 min^1, N=7),
without the delay characteristic of measurements of [K + ], and [Na + ]; in K+-free
saline (Fig. 3). This rate of C P loss slowed gradually until [CP], stabilized after
10—20min at a mean value of 15-OmmolP1 (s.D. ±4 mmol P 1 , N = 5). This value of
rate of change of [CP], was inserted into equation 1 to calculate the net CP flux in
]2mV
K+-free saline
K+-free saline
Fig. 3. Effects of inhibiting the electrogenic N a + / K + pump on [ion]j. Reversibly
blocking the N a + / K + pump with K+-free saline led, after a delay, to (A) a fall in [K + ]j
and (B) a rise in [Na + ],. Readmitting normal saline to the experimental chamber elicited
a hyperpolarization of Eji, caused by the reactivation of the N a + / K + pump, and the
recovery of [K + ], and [Na + ], to their original values. Note the different time scales in A
and B.
357
Free ions in snail gland cells
Cl -free saline, Mci, and the Cl permeability coefficient, P Q , was then estimated
from equation 3 (Table 1).
Additional evidence in favour of a substantial resting permeability to C P was
obtained from measurements of changes in EM and input resistance (RM) in CP-free
saline. The introduction of CP-free saline into the experimental chamber produced a
rapid transient depolarization of Planorbis gland cells accompanied by a powerful
discharge of action-potential-like activity (Figs 4, 5), followed by a return of EM to its
original value. Similar transient depolarizations upon contact with CP-free saline
have been reported previously in other preparations known to have a high resting CP
permeability, such as frog skeletal muscle cells (Hodgkin & Horowicz, 1959). CPfree saline also produced a large increase in the RM of Planorbis salivary glands
(Fig. 5), as in other cells, such as mouse liver cells (Graf & Petersen, 1978), where
resting C P permeability is high.
The transient depolarization, the increase in R^ and the loss of CP in CP-free
saline were also observed after pre-incubating glands for lOmin in Ca +-free saline.
This indicates that these effects did not arise from an indirect depolarizing action of
1 min
-40 i
j
-60
u
-80
\r \Ay
J
28
3mV
U
14
Cl -free saline
Fig. 4. Effects of removing extracellular C P on [CP],. The introduction of CP-free
saline (CP replaced by sulphate) into the experimental chamber produced a rapid fall in
[CP]j to a lower stable value. CP-free saline also elicited a transient depolarization of
the salivary acinar cell and an increase in spontaneous action potential activity. The
reintroduction of extracellular C P led to an uptake of C P and a transient depolarization
of E M .
358
A. BARBER
Cl -free saline on presynaptic elements (Ascher, Kunze & Nield, 1976) or the low
Ca 2+ activity in CP-free saline made with sulphate salts (Hodgkin & Horowicz,
1959). These changes in E M , RM and [Cl~]; were also observed in low-CP solution
in which glucuronate was substituted for part of the C P . This means that these
effects were not artefacts caused by replacing C P with sulphate.
Transferring the glands back to normal saline led to the rapid recovery of the
original level of [CP] ; (Fig. 4), a transient depolarization and a transient increase in
RM (Fig. 5). The subject of the resting CP permeability of the gland cell membrane
will be considered further in the Discussion.
Changes in [ion]',- following stimulation with acetylcholine
The application of ACh or nicotinic agonists onto Planorbis salivary glands is
known to produce a dose-dependent biphasic electrical response from acinar cells
(Barber, 1985). In the present study it was found that the ACh-induced depolarization occurs in parallel with a fall in [K + ], (Fig. 6A) and a rise in [Na + ] ; (Fig. 6B;
Table 1). These data thus demonstrate that at least part of the K + released into the
bathing medium by ACh (Barber & ten Bruggencate, 19856) originates from acinar
gland cells rather than, or in addition to, presynaptic structures, and also confirm the
occurrence of a Na + influx during the ACh-induced depolarization (Barber, 1985).
In contrast to the results obtained for [K + ], and [Na + ],, no significant change in
[CP]; was detected following the application of ACh (Fig. 7). Any increase in C P
permeability would, however, be difficult to detect because the net driving force on
C P is comparatively weak (Table 1), and indeed reverses during the ACh-induced
depolarization.
-30
2min
-50
6
-70
-90
a
-free saline
Fig. 5. Changes in membrane potential (EM) and membrane resistance ( R M ) of
Planorbis salivary gland cells in Cl~-free saline. Membrane resistance was measured
with a conventional single-barrelled microelectrode by injecting 2nA pulses of hyperpolarizing current, 600 ms in duration, through a balanced bridge circuit at intervals of
20 s. CP-free saline produced an increase in the RM of salivary acinar cells, while the reintroduction of normal saline led to a transient increase in RM followed by a gradual
recovery to its original value. The changes in EM are similar to those documented in
Fig. 4.
Free ions in snail gland cells
359
The possibility that ACh elicits an undetected increase in membrane permeability
to C P was investigated by applying ACh repeatedly onto glands bathed in CP-free
saline (Fig. 8). CP-free saline produced an increase in the size of the depolarizing
phase of the response to ACh and this enhanced response was maintained for as long
as the preparation was bathed in CP-free medium (i.e. up to 10 applications over the
course of 2h). This last finding is not a result which would be expected if ACh made
the cell membrane more permeable to C P (see Adams & Brown, 1975; Iwatsuki &
Petersen, 1977). The increase in the size of the ACh depolarization is presumably a
consequence of the increase in RM in CP-free saline (see previous section) rather
than the result of an increase in the outward driving force on CP following the
removal of extracellular C P .
The mechanism of action of ACh on Planorbis gland cells was further examined by
comparing the effects of ACh on [K + ], and [Na + ] ; with those of high-K+ saline
(Fig. 9). High-K + saline was applied only very briefly (30 s) to salivary glands in
order to avoid the influx of KCl and water which can occur over time in these salines
(see Ascher et al. 1976; Iwasa, 1982). These experiments were also carried out in the
absence of extracellular Ca2+ so that the depolarizing action of high-K+ saline on
gland cells would not be influenced by any presynaptic excitatory effect of this saline.
It was found, though, that good impalements with ISMEs were more difficult to
obtain in Ca2+-free saline than in normal saline, with the result that recordings of
[K + ]j and [Na + ]; were not optimal in these experiments.
Depolarizing salivary cells with high-K+ saline led to an influx of K + (Fig. 9A),
which is in accordance with the reversed driving force on K + under these conditions.
2 min
-40-,
-20
10 mV %
-40
-60-
UJ
s
W
-60
1 min
-80
J
r^L 7-0o
E
]2mV
3mV
5010 4 moll 'ouabain
Fig. 6. Effects of stimulation with acetylcholine (ACM on membrane potential (E M ) and
[ion]; in salivary acinar cells. A 10s application of 10 mol P 1 ACh ( • ) led to a biphasic
(depolarizing-hyperpolanzing) change in E M and (A) a fall in [K + ], and (B) a rise in
[Na + ]i. The inset in (A) reveals that the ACh-induced depolarization is also accompanied
by a discharge of gland cell action potentials. The hyperpolarizing phase of the response
to ACh and the recovery of [Na + 1, to its original resting value were both blocked
(B) when the electrogenic Na + /K pump was inhibited with 10~ 4 molP' ouabain.
Inhibiting the N a + / K + pump also revealed the presence of a Na + influx.
360
A. BARBER
Although the extent to which K + may have leaked into salivary cells through a
poor seal between the ISME and cell membrane in these experiments remains
unknown, this result implies that a good part of the K + lost from gland cells during
-20-
UJ
-40
I
-60
bl * ]
— I
2mV
20 J
d.c.
Fig. 7. Effects of stimulation with acetylcholine (ACh) on gland cell [Cl ];. A 10 s
application of 10~ 4 moll~ 1 ACh ( • ) to the saline flowing over the gland produced no
significant change in [Cl~],. A calibrated pulse of 50 mV direct current (d.c.) was also
applied between the bath and ground in order to check that Vci was being recorded
accurately (see Materials and Methods).
-20
-40
-60
tu
-80
-100
-free saline
Fig. 8. Action of Cl -free saline on the gland cell response to acetylcholine (ACh). This
recording was made with a conventional single-barTelled microelectrode, and 10~4 mol 1~'
ACh (•) was applied for 10s to the saline flowing over the glands. Cl~-free saline
produced an increase in the size of the ACh-induced depolarization (size of the original
response in normal saline marked by dotted line) and hyperpolarization. This effect
persisted for as long as the gland remained in contact with Cl~-free saline.
Free ions in snail gland cells
361
1 min
J2mV
High-K+ salme
High-K+ saline
Fig. 9. Comparison of the effects of acetylcholine (ACh) and high-K+ saline on [K + ],
and [Na+]j in acinar cells. (A) The application of 10~4moll~' ACh (•) for 10s to the
salineflowingover the glands elicited a fall in [K + ],, while depolarizing the cell for 30s
with high-K+ saline produced arisein [K + ],. (B) ACh (#) caused an increase in [Na+],
whereas high-K+ saline had no apparent influence on [Na + ],. These recordings were
made in Ca2+-free saline so as to prevent presynaptic effects of high-K+ saline from
influencing these measurements. The slope of the [ion]; trace is due to the difficulty of
obtaining good recordings in the absence of [Ca2+]e.
ACh-induced depolarization leaves through voltage-dependent K + channels and/or
leakage pathways.
The brief application of high-K+ saline produced no change in [Na + ], (Fig. 9B),
though it should be noted that the inward driving force on Na + is reduced in this
saline. Despite this difference it seems likely that the majority of Na + which enters
Planorbis gland cells after the application of ACh does so through channels activated
directly by the ACh receptor.
The hyperpolarizing phase of the ACh response is associated with the gradual
recovery of [K + ], and [Na + ]; to their original levels (Fig. 6). The part played by the
N a + / K + pump in this recovery was confirmed in the present study by inhibiting the
activity of this pump irreversibly with 10~4moll~1 ouabain (Fig. 6B). Gland cells in
which the N a + / K + pump was blocked were unable to restore [Na + ] t (Fig. 6B) or
[K + ], to their original resting levels. These experiments therefore indicate that the
transient undershoot of [K + ] c and overshoot of [Na + ] e which occur during the
hyperpolarization (Barber & ten Bruggencate, 19856) are produced at least in part by
the electrogenic accumulation of K + and expulsion of Na + from the gland cells. The
N a + / K + pump is presumably activated by the increase in [Na + ], which occurs
during the depolarization, rather than by a direct action of ACh on the pump.
DISCUSSION
Measurements of gland cell [ion],
ISMEs have been used to measure [ion]; in a broad range of tissues (Walker &
Brown, 1977) and probably represent the most useful technique currently available
for determining the activity of K + , Na + and C\~ directly and continuously in living
362
A. BARBER
cells. One of the first studies to make use of ISMEs to measure intracellular ions in
gland cells was made by Palmer & Civan (1977), who measured [K + ],, [Na + ], and
[CP]; in the cytoplasm and nucleus of giant salivary cells of the insect Chironomus.
The smaller salivary gland cells of other insects (e.g. Berridge & Schlue, 1978) and
mammals (e.g. Poulson & Oakley, 1979) have since proved amenable to study with
ISMEs. However, the results of the present investigation, together with those of
Barber (1986), represent the first direct determinations of [ion], in molluscan gland
cells.
In the present study, no striking dependence was found between the presumed
degree of injury to the cells, as measured by E M , and the magnitude of [Na + ], or
[CP], (Fig. 2A,B). This implies that estimates of [Na+]j and [Cl~]; are unlikely to
have been influenced greatly by impalement artefacts. In the case of [K + ]; it was
found that cells with higher E^ values tended to have a higher [K + ]; and vice versa
(Fig. 2A). This probably reflects genuine variation in [K + ], and the dependence
of resting E^ on the transmembrane K + concentration gradient rather than a
correlation between the degree of cell damage and the size of [K + ];.
Another potential difficulty in the interpretation of these measurements is that the
ion-exchangers used in the tips of ISMEs are not perfectly sensitive to only one kind
of ion. Na + is the most likely interfering intracellular cation for the K + ionexchanger used in the present study (Meier et al. 1982). However, the very low levels
of [Na+]i in Planorbis salivary gland cells, together with the relatively good
selectivity of the K + -ISMEs against Na + (see Materials and Methods), mean that no
allowance for Na + need be applied to measurements of [K + ] ; .
With Na + -ISMEs the largest errors are likely to be caused by K + and Ca2+ (Meier
et al. 1982). The relatively high levels of [K + ] ; imply that intracellular K + may make
a significant contribution to the VNS signal. Calculations based upon the selectivity
ratios of the Na + -ISMEs (see Materials and Methods) and the mean value of [K + ],
(Table 1) indicate that [Na + ], may be overestimated by around 2-6 mmolP 1 . Levels
of intracellular free Ca 2+ ([Ca2+]j) in Planorbis salivary gland cells are very low
(Table 1) which suggests that Ca2+ probably does not interfere significantly with
measurements of [Na + ]j.
In contrast to recordings with K+- and Na + -ISMEs, the identity and concentrations of ions interfering with measurements with CP-ISMEs are usually not
known (Thomas, 1978). It was found, however, that apparent [CP]; in gland cells
soaked in CP-free saline falls to stabilize around ISmmoll" 1 . On the assumption
that all intracellular C P is eventually lost from cells bathed in CP-free saline
(Thomas, 1978), this would mean that true [CP]; lies around 10-3mmolP'
(Table 1) while the total interference in these cells is equivalent to ISmmolP 1
[CP]i.
Bicarbonate ions certainly contribute to this interference since exchanging standard snail Ringer in the experimental chamber with nominally bicarbonate-free
saline produced a rapid (complete within 2-3 min) and fully reversible reduction in
apparent [CP]; by around 4-7mmolP 1 (s.D. ± l - 5 m m o l P ' ; yV=3; A. Barber,
Free ions in snail gland cells
363
unpublished observations). Interference from intracellular bicarbonate is generally
assumed to be insignificant in bicarbonate-free saline (Bolton & Vaughan-Jones,
1977).
The sulphate ions used as a substitute for Cl~ would also be a source of
interference if they entered the cells (Saunders & Brown, 1977), as would sulphate
which diffused from the reference barrel of the ISME. Estimating the contribution
made by cross-sensitivity to these measurements is further complicated by the fact
that the selectivity of the CP-ISME deteriorates when [Cl~], is low (Vaughan-Jones,
1979; Aickin & Brading, 1983). Finally, it can also not be ruled out that salivary cells
in CP-free saline lose water and shrink (MacKnight, 1985), thus influencing
measurements of [Cl~],.
Tip potential artefacts recorded by the reference barrel of the ISMEs were another
possible reason for inaccurate measurements of [ion];. Although no systematic
investigation of such artefacts was carried out, it was observed that EM values
measured with the acetate-filled reference barrels of K + -, Na + - and Ca 2+ -ISMEs
were generally higher than those measured with the sulphate-filled barrels of the Cl~ISMEs (Table 1). Similar problems with reference barrels filled with sulphate salts
have been reported by a number of other workers (e.g. Bolton & Vaughan-Jones,
1977; Berridge & Schlue, 1978; Gardner & Moreton, 1985). Any underestimation of
EM would lead to an overestimation of V a (see Materials and Methods) which would
contribute to the interference encountered with measurements of [Cl~],.
The mean basal [K + ]j in Planorbis salivary gland cells was determined as
42-9mmoll~1 (Table 1), which is comparable to the [K + ]j values of 53-4mmoll~ 1
(Kostyuk, Sorokina & Kholodova, 1969) and 51-1 mmoU"1 (s.D. ±10-3 mmoU"1,
7V= 15; A. Barber, unpublished observations) measured in unidentified giant
neurones of Planorbis central ganglia. This value is also comparable to the
64mmoll~' estimated indirectly to be the [K + ], in salivary cells of the related snail
Helisoma (Hadley, Murphy & Kater, 1980), when allowance is made for the
osmolalities of the different Ringer salines (in Helisoma 64/130 = 0-49; in Planorbis
42-9/112 = 0-38).
The [Na + ], of Planorbis salivary gland cells (2-4mmolP'; Table 1), on the other
hand, is considerably lower than the n-SmmolF 1 measured from Planorbis
neurones with Na + -ISMEs of the protruding tip type (Kostyuk et al. 1969). This
relatively high value of neuronal [Na + ], may be due to the difficulties of properly
inserting ISMEs of this kind intracellularly (Thomas, 1972) or could represent a
genuine difference between [Na +]j in Planorbis neurones and salivary gland cells.
The [CP]; and equilibrium potential for C\~ found in the present study (Table 1)
are within the range previously measured in a variety of molluscan preparations
(Gardner & Moreton, 1985).
Apart from K + , Na+ and Cl~, the net cellular charge balance and osmolality are
probably made up mainly by amino acids, phosphorus compounds and the fixed
negative charges of proteins (Burton, 1983).
364
A. BARBER
Permeability of the gland cell membrane
The high basal value of [K + ]; and very low level of [Na+]i in Planorbis salivary
gland cells were found to be maintained by the activity of an electrogenic N a + / K +
pump. Inhibiting the activity of this pump reversibly with K+-free saline blocked the
active accumulation of K + and extrusion of Na + and revealed the presence of a
passive K + efflux and Na + influx (Fig. 3). The P K calculated from the K + efflux
(7-6X 10~ 6 cms~') is consistent with a high resting permeability to K + , as for
example in leech neurones (8xlO" 6 cms~ 1 ; Deitmer & Schlue, 1981). The P Na
estimated from the Na + influx (5-4X 10~ 8 cms~') is very low and a P N , / P K ratio of
0-007, if correct, would indicate that the resting basolateral membrane is very
selective against Na + as compared to K + .
Planorbis salivary gland cells also have a relatively high resting [CP], (10-3
mmol P ' ; Table 1) which implies the presence of some process, at present unknown,
for actively accumulating C P against its electrochemical gradient. At the same time
the resting C P permeability of the salivary cell membrane would appear to be quite
high. Thus when glands were bathed in CP-free saline, the result was a rapid fall in
[CP]i (Fig. 4), from which a P a of 8-9X lO^cms" 1 was calculated (Table 1). It
should be noted, however, that cells bathed in CP-free saline do not always lose C P
only as a result of passive leakage through the cell membrane. It has been demonstrated recently that the Cl~/bicarbonate exchange mechanism which normally
keeps [CP]i high in mammalian Purkinje fibres (Vaughan-Jones, 1979, 1982) and
smooth muscle cells (Aickin & Brading, 1985) reverses in CP-free saline to transport
C P out of these cells. If such an active extrusion of C P in CP-free medium also
takes place in Planorbis salivary cells, then this would clearly lead to a spuriously
high estimation of PCiThe increase in RM and the transient decrease in E^ in salivary gland cells
observed after transferring the glands to CP-free saline (Fig. 5) also appear consistent with a high resting Pci in these cells (Hodgkin & Horowicz, 1959; Graf &
Petersen, 1978). But an increase in leak resistance due to the lower solution
conductivity of CP-free saline (Adams & Brown, 1975), a decrease in electrical
coupling between neighbouring acinar cells (Barber, 1985) caused by the removal
of extracellular C P (Asada & Bennett, 1971), or a decrease in membrane K +
conductance in CP-free medium (Carmeliet & Verdonck, 1977) may also contribute
to the increased RM in CP-free saline.
The transient depolarization and increase in R^ observed after transferring the
glands back to normal saline (Fig. 5) were unexpected observations. If Pci were high
then the return to normal saline should have elicited a transient EM hvperpolarization
(Hodgkin & Horowicz, 1959; Iwatsuki & Petersen, 1977) and a gradual decrease in
R M . These effects of returning CP-depleted cells to normal saline are not readily
explained, and may be secondary to changes in intracellular pH (Thomas, 1982) or
cell shrinkage (MacKnight, 1985).
Given the reservations mentioned above, it was reassuring that a theoretical EM of
— 71-2 mV was calculated by inserting data from Table 1 into the Goldman equation
Free ions in snail gland cells
365
(equation 4). This estimate of EM is very close to the values measured with doublebarrelled ISMEs (Table 1) or single-barrelled conventional electrodes ( — 72 mV;
Barber, 1985). It would seem, then, that EM in Planorbis salivary gland cells can be
explained in terms of the measured concentration gradients of K + , Na + and C P
and their estimated resting permeabilities through the gland cell membrane. This
conclusion does not exclude the possibility that electrogenic ion pumps also make a
small contribution to the resting EM- Interestingly, the ionic basis of EM in salivary
gland cells of the related snail Helisoma appears to be different from that in
Planorbis. Helisoma salivary cell membranes are less selective against Na + ( P N 8 / P K
ratio, 0-04) and C P permeability makes no contribution to their EM (Hadley et al.
1980).
Stimulus-induced changes in [ion]; and the role of acetylcholine in secretion
It has been demonstrated in the present study that the stimulation of Planorbis
salivary gland cells with ACh leads to a fall in [K + ]; and a rise in [Na + ]; (Fig. 6). In
contrast, stimulation with ACh produced no sign of any change in [Cl~]; (Fig. 7),
though some passive changes in [Cl~], following EM might have been expected,
given the relatively high resting PC1. While agonist-induced changes in [ion], have
been recorded previously from gland cells in insects and mammals (e.g. Berridge &
Schlue, 1978; Poulsen & Oakley, 1979), this study, together with a preceding
investigation (Barber, 1986), represents the first measurements of such changes from
molluscan gland cells.
These observations are probably relevant for salivary secretion in intact specimens
of Planorbis. ACh is at present the most likely candidate for the transmitter between
the central nervous system and the salivary glands (Barber, 1985), as it is in several
other species of mollusc (Barber, 1983). As such it is suspected to mediate the large
(approx. 30 mV) excitatory postsynaptic potentials (EPSPs) which trigger action
potentials in these salivary cells (Barber, 1985). Although ACh-induced depolarizations are larger than individual EPSPs a volley of EPSPs (Barber, 1985) can
produce a depolarization not dissimilar to the ACh response in size and duration.
Finally, while it has not yet been shown whether Planorbis salivary gland cells
actually secrete saliva following the application of ACh, it is known that gland cells
which are electrically excitable do secrete when depolarized either by agonists (e.g.
WadaeZ al. 1984) or by high-K+ saline (e.g. Suchard, Lattanzio, Rubin & Pressman,
1982).
As far as the mechanism of action of ACh is concerned, the absence of any change
in [Na+]i when Planorbis salivary glands were bathed in high-K+ saline (Fig. 9B)
suggests that detectable amounts of Na + do not enter gland cells through voltagedependent Na + channels during the ACh-induced depolarization. Like Ca +
(Barber, 1986), the majority of Na + probably enters these cells through channels
opened directly by ACh. An ACh-induced depolarization produced by an exclusive
increase in membrane permeability to Na + and Ca2+ would explain the finding that
even large depolarizations of EM are accompanied by only relatively small decreases
in membrane resistance (Barber, 1985). The large inward driving force on Na + and
366
A. BARBER
Ca 2+ would mean that even a small increase in permeability to these ions would
produce a large depolarization (see Biihrle & Sonnhof, 1983).
Depolarizing salivary cells with high-K+ saline produced substantial changes in
the level of [K + ]; (Fig. 9A). This means that large numbers of potassium ions are
probably able to leave salivary cells during the ACh-induced depolarization either
through leakage pathways, as a result of the increased outward driving force on K + ,
or through voltage-dependent K + channels. Large amounts of K + probably do not
leave Planorbis salivary gland cells via either Ca2+-activated K + channels or Ca 2+ activated non-selective cation channels (Marty et al. 1984; Petersen & Maruyama,
1984) because the ACh-induced K+ efflux is also observed in Ca2+-free saline
(Fig. 9A; Barber & ten Bruggencate, 19856). The possibility that ACh-activated
channels in Planorbis salivary cells are also permeable to a small degree to K + has not
been ruled out by the present experiments, however. Whatever its mechanism, it
appears likely that this efflux of K + plays an important role in repolarizing the gland
cells at the end of the depolarizing phase of the ACh-response.
The electrogenic N a + / K + pump seems to have no part in this repolarization of the
gland cell membrane, at least during the short exposures to ACh used in the present
experiments. Salivary cells in which the Na + /K + pump was inhibited by ouabain
repolarized quite normally (Fig. 6B), though the repeated application of ACh under
these conditions does lead to a gradual depolarization of EM (Barber, 1985). The
function of the N a + / K + pump appears to be to maintain the basal values of [K + ] ;
and [Na + ], both in resting glands and following stimulation.
The suggested mechanism of action of agonist on Planorbis salivary gland cells,
namely an increase in membrane permeability to Na + and Ca 2+ , the subsequent
release of K + and the activation of an electrogenic Na + /K + pump, is different from
those proposed in a number of other exocrine glands (for reviews see Ginsborg
& House, 1980; Petersen, 1980). In mammalian salivary glands, for example,
stimulation with ACh leads to an efflux of K + through Ca2+-activated channels,
followed by a re-uptake of K + , along with Na + and C P , by a K + / N a + / C r cotransport system (Petersen & Maruyama, 1984; see also Martinez & Cassity, 1984).
This net uptake of NaCl appears to be sufficient to account for the secretion of NaCl
in the primary saliva (see also Marty et al. 1984).
The ion fluxes produced by ACh in Planorbis salivary gland cells, though, do not
seem to represent a balanced uptake of ions suitable for incorporation into a profuse,
watery salivary secretion. It may be that since Planorbis is an aquatic animal the
secretion of enzymes and mucus is a much more important function of these glands
than the secretion of salt and water. The salivary glands may not be required to
secrete very much fluid.
Previous accounts of the electrophysiology of molluscan gland cells have tended to
emphasize the role of action potentials in secretion. Salivary cells in Helisoma
(Hadley et al. 1980; Senseman, Horwitz, Cleeman & Orkand, 1985) and Ariolimax
(Goldring, Kater & Kater, 1983) and cells of the pedal gland of Ariolimax (Kater,
1977) all display action potentials whose inward currents are carried by Ca2+ and
Na + . The fact that action potentials and ACh produce similar changes in ion
Free ions in snail gland cells
367
permeability suggests, however, that EPSPs and action potentials may complement
one another in producing the ion fluxes which stimulate secretion.
Financial support for this work was provided by the University of Munich.
I express my thanks to Professor G. ten Bruggencate for having made my stay in
Munich possible.
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