2. Following bath application of ACh (0-5 ,tM), the cell current recordedat following

23 Pages·2005·2.74 MB·English

Journal ofPhysiology (1989), 419, pp. 665-687 665
With8text-figures
PrintedinGreatBritain
THE INITIATION OF CALCIUM RELEASE FOLLOWING MUSCARINIC
STIMULATION IN RAT LACRIMAL GLANDS
BYA. MARTY* AND Y. P. TANt
Fromthe *Laboratoire deNeurobiologie, EcoleNormaleSuperieure, 46 rued'Ulm,
75005Paris,Franceand tBogazifi Universitesi, BMMEPK2,Bebek,
Istanbul, Turkey
(Received 17March1989)
SUMMARY
1. Acinar cells were isolated from rat lacrimal glands, and the Ca2+ release
response of these cells was studied using two experimental approaches. In one
approach, changes in Ca2+ concentration, Ca2+, were monitored by measuring Ca2+_
dependent Cl- currents using tight-seal whole-cell recording. Alternatively, such
changeswere measured asafluorescence signal in cellsloadedwith Fura-2.
2. Following bath application of ACh (0-5,tM), the cell current recorded at
-60 mVwasunchangedforca0-8 s, thenroseinabiphasic manner. The initialphase
ofthe current rise ('hump') tookdifferent appearances dependingon the cell studied,
and it sometimes stood out from the main part ofthe response as a partially isolated
transient.
3. In cells which had been loaded with Fura-2, Ca?+ was found to rise abruptly
following a silent period. The delay was larger if ACh (0-2-0-5 /tM) was applied in
a depolarizing isotonic K+ saline than if it was applied in the normal saline. In
addition, the maximum of the Cai2 response was reduced with depolarizing
stimulating solutions. This indicates that membrane potential modulates the Ca1
response.
4. Responses to 5/iM-ACh, a saturating agonist concentration, were almost
identical in K+ saline and innormal saline.
5. Ifthe cell potential was hyperpolarized, the delay ofthe ACh-induced current
became shorter.
6. Breaking into an acinar cell with a pipette containing an elevated Ca2+
concentration (0-1-1 mM) led to a transient activation of Ca2+-induced currents
during the first secondsofwhole-cell recording. These transients were obtained more
reliably if the transition to the whole-cell mode was achieved by applying a sharp
pulse of potential ('zapping') rather than by applying suction to the pipette
compartment. At -60 mV, the transients elicited with the former method by
05 mM-Ca2+ had atime-to-peak near0-6 s andan amplitude varying between 10 and
600 pA. With 041 mM-Ca2+, similar transients were also observed, but a number of
cells failed to respond. Calcium-induced transients were blocked if cells were
previously loadedwith50/LM-Ruthenium Red.
IMS7589
666 A. MARTY AND Y. P. TAN
7. Performing the same experiments with inositol trisphosphate (InsP3, 20/tM) in
the pipette solutions also led to early transient Ca2+-induced currents. Amplitudes,
times-to-peak and 20-80% transition times were similar for05 mM-Ca2' and 20/tM-
InsP3 stimulations.
8. Calcium- or InsP3-induced transients were similar to 'humps' (point 2 above)
and torapid transients whichwere observed in certain cells upon application ofalow
ACh concentration (0-1 /LM). These results suggest a role of these transients in
shaping the ACh-induced currents.
9. The results indicate that Ca2+-induced Ca2+ release plays a key role in the
initiation oftheACh-inducedresponse. Itissuggested thatthesilentlagperiodisdue
to the accumulation ofactive phospholipase C molecules up to the pointwhere Ca2+_
induced Ca2+ release is initiated. It is finally proposed that the lag is determined by
the occupancy of the receptor, which can be modulated by changing either the
agonist concentration orthe membrane potential.
INTRODUCTION
Neurotransmitter-induced Ca2+ release is a multistep process involving a GTP-
binding protein and a phospholipase C which is responsible for the production of
inositol trisphosphate (Berridge, 1987; Cockcroft, 1987). Such responses have a
remarkably similar onset as measured in a number of preparations including rat
salivary and lacrimal gland, rat adrenal cortex, transfected neuroblastoma-glioma
cell line and guinea.-pig hepatocytes, consisting of a silent delay on the order of 1 s
followed by a steep Ca2+ rise (Merritt & Rink, 1987; Capiod, Ogden, Trentham &
Walker, 1988; Horn & Marty, 1988; Neher, Marty, Fukuda, Kubo & Numa, 1988;
Quinn, Williams & Tillotson, 1988). The exact nature of the events underlying the
delay andthesuddenCa2+risearenotunderstood. Theshapeoftheresponserequires
the existence of some positive feedback in the events leading to Ca2+ release. The
same conclusion can be reached from the finding that introduction of inositol
trisphosphate (InsP3) into the cells induces regenerative Ca2+-induced signals (Oron,
Dascal, Nadler & Lupu, 1985; Evans & Marty, 1986a; Capiod et al. 1988; Neher,
1988). In rat lacrimal cells, it was found that InsP3-induced signals gave a
characteristic frequency which is higher than that obtained with neurotransmitter
application, anditwasinferredthatonekind ofpositivefeedback atleasttakesplace
downstream of InsP3 production (Evans & Marty, 1986a). Furthermore, in two
preparations (rat mast cells and guinea-pig hepatocytes) the InsP3-induced signal
has a lower delay than that elicited by extracellularly acting agents, suggesting that
a large part ofthe delay corresponds to the production ofInsP3 (Capiod et al. 1988;
Neher, 1988).
In the present work, the onset ofthe Ca2+ response is analysed further. We have
first tried to identify the experimental parameters influencing the delay in order
to understand better the underlying cellular events. We have also studied responses
to sudden intracellular stimulation with Ca2+ and InsP3 in an attemptto identify the
positive feedbackresponsible forthe sudden onset oftheresponse. Experimentswere
performed in acinar cells ofrat lacrimal glands. In these cells, as in salivary secreting
cells, acetylcholine (ACh) induces the opening both of K+-selective and of Cl--
INITIATION OF Ca2+ RELEASE6 667
selective channels (Marty, Tan & Trautmann, 1984; Petersen & Maruyama, 1984;
Findlay & Petersen, 1985; Marty, Evans, Tan & Trautmann, 1986). The measure-
ment of Ca2+-dependent K+ and Cl- current responses allows the underlying
changes in internal Ca2+ concentration (Cai+) to be followed with a good temporal
resolution (in the millisecond range). Using this approach, a silent delay followed by
an abrupt rise ofCa2+-dependent current can be demonstrated at the onset ofACh
application (Horn & Marty, 1988).
An alternative approach is to measure CaF+ by use ofthe Ca2+-sensitive fluorescent
dye Fura-2 (Grynkiewicz, Poenie & Tsien, 1985). Direct measurements of Ca2+
increase induced by muscarinic agonists in exocrine glands have been performed
previously on cell suspensions (Takemura, 1985; Merritt & Rink, 1987) and in
individual cells (Gray, 1988; Foskett, Gunter-Smith, Melvin & Turner, 1989). This is
apreferable methodin thesensethat the Ca2+ signal can be more directlyinterpreted
than the current signal. However, the higher temporal resolution offered by current
recordings is essential to study the ratherfast events taking place at the onset ofthe
response. We have therefore combined the two approaches in the present work.
METHODS
Celldissociation
Manyoftheresultspresentedinthispaperonlyapplyifaminimumdoseofdissociatingenzymes
are employed. We used a dissociation procedure based on sequential treatment with trypsin and
collagenasewhichwasmodifiedfromthe methodofKanagasuntheram & Randle (1976) asfollows.
The exorbital lacrimal glands of Wistar rats (5 week old males) were rapidly removed under
pentobarbitone anaesthesia and placed in ice-cold divalent free mammalian saline solution
containing (mM): NaCl, 140; KCl, 5; HEPES-NaOH, 5 (pH 72). The capsulecontainingthegland
and the main secretory ducts was removed under a dissection microscope. The tissue was finely
mincedwithsurgical blades, andincubatedfor 10 minat37 °Cwith 2 mgtrypsin (type XI, Sigma)
in5mlofdivalent-freebicarbonate-buffered salinesolution, containing (mM): NaCl, 125; KCl, 2-5;
NaHCO3, 26; NaH2PO4, 1-25 (pH 7-4), supplemented with 10 mgglucoseand 25 mg bovine serum
albumin (BSA). Following trypsin treatment the cells were washed twice with the above solution
and incubated for 10min in the divalent-free bicarbonate-buffered saline solution supplemented
with glucose, BSA and EGTA (4mM). The cells were then washed twice and incubated for 25-
35min in 5 ml bicarbonate-buffered saline solution (BBS) containing (mM); NaCl, 125; KCl, 2-5;
MgCl2, 1; CaCl2, 1; NaHCO3, 26; Na2 HPO4, 1-25 (pH 74) supplemented with 10mg glucose and
07 mg collagenase (type II, Sigma). Throughout the dissociation procedure the solutions were
bubbled with a mixture of5% CO2 and 95% 02, and the vials (2-2 cm internal diameter) were
gently shaken (2 Hz, 5cm excursion). Trituration through a flame-polished plastic pipette gave a
suspension containing both single acinar cells and small aggregates. The cells were washed,
centrifuged twice at 20g and resuspended in culture medium (minimum essential medium,
GIBCO).
Isolated cells were placed on 35mm culture dishes (Greiner) and kept in culture medium
supplementedwithstreptomycinandpenicillininanincubatorat37 °Cinahumidifiedatmosphere
containing 5% CO2. Cells to be used for fluorescence measurements were plated on culture dishes
with acircular opening atthe bottom ofwhich microscope cover-slipsweregluedwithparaffin. In
general cellswereutilizedwithin 1-8hafterplating.
Electrophysiological measurements
Measurements ofmembrane currents were performed using the tight-seal whole-cell recording
method (Hamill, Marty, Neher, Sakmann & Sigworth, 1981). Before recording, the incubation
medium was replaced with an extracellular saline containing (mM): NaCl, 140; KCl, 5; CaCl2, 1;
MgCl2, 1; HEPES-NaOH, 5 (pH 7.2). Pipette resistance ranged from 2 to 3MQ when filled with
668 A. MARTY AND Y. P. TAN
standard intracellular solution. This solution contained (mM): KCl, 140; MgCl2, 2; EGTA-K, 0-5;
HEPES-KOH, 5 (pH 72). Insomeoftheexperimentsanelectronicperforator('zapper')wasused
to obtain fast rupture ofthe patch ofmembrane afterestablishing agigaohm seal. The procedure
consisted ofapplying repetitively a briefvoltage pulse (3-5V amplitude, 20,us duration, 0-1 Hz)
to the reference input of an EPC-7 amplifier's head stage (List Electronic, Darmstad, FRG). In
order to follow the pipette-cell conductance, repetitive voltage pulses (e.g. 5mV amplitude, 5 ms
duration, 5Hz) were given throughout the recording, and the resulting capacitive current was
displayedontheoscilloscope.Zappingdidnotalwayssucceed;inmanyinstances,thepipetteinput
conductance increased without any noticeable change in input capacitance, indicating that the
pipette-membrane seal gave way instead of the patch membrane. The percentage of successful
penetrationsvariedgreatlyfrom preparationtopreparation, withoutanyobviouscorrelationwith
othermeasurable cellparameters.
After establishing the whole-cell configuration, the cell potential was usually held at -60mV.
At this potential, Ca12+ increases elicited by muscarinic stimulation induce mainly an inward, Cl--
selective current (Marty et al. 1984). Under the ionic conditions used, this holding potential is
rather closetotheNernst potential for K+ (-85mV) butprovidesalargedrivingforceforinward
Cl- current (with a Cl- Nernst potential of0mV). In addition, Ca2+-dependent K+ channels are
voltagedependent (Trautmann&Marty, 1984)suchthatchoosingahyperpolarizedpotentialhelps
tominimizethe contribution ofthe K+conductance.
Calciummeasurements
Measurements ofintracellular Ca2+ concentration on single cells were done using the acetoxy-
methyl (AM) ester form of the Ca2+ indicator dye Fura-2 which is membrane permeant.
Fura-2 AM was added to the culture dish to give a05-1 pM concentration ofthedye and the cells
wereincubated forafurther 30 minat37 °C, afterwhichtheincubation mediumwasreplacedwith
therecordingsolution. Cellswerekeptfor 10minatroomtemperatureandwerewashedagainwith
the same solution in order to eliminate uncleaved Fura-2 AM molecules.
Fluorescence measurements were done on a Zeiss IM-35 inverted microscope equipped with
epifluorescence using a system similar to that ofAlmers & Neher (1985). The exact configuration
usedwasasdescribedbyGiaume, Randriamampita&Trautmann (1989)exceptthatthesignalwas
collected using a photomultiplier (Hamamatsu R647-D1) rather than a photodiode. Cell
autofluorescence wasfoundtobenegligibleandthereforenocorrectionneededtobe madeforit.
Conditionsforagonistapplication
In both the fluorescence measurements and electrical recordings drugs were applied with a fast
microperfusiontechnique (Krishtal& Pidoplichko, 1980;Fenwick,Marty&Neher, 1982).Thetime
constant ofsolution change atthe onset ofadrug application was estimated to be 30ms (Horn &
Marty, 1988).
Allexperimentswereperformedatroomtemperature (usually22-24°C).
RESULTS
'Humps' inACh-induced currents
Current responses induced by ACh and obtained under standard conditions are
shown in Fig. 1. As shown previously, the current response starts abruptly after a
silent period lasting about 1 s following agonist application (Trautmann & Marty,
1984; Horn & Marty, 1988). A careful examination of the rising phase of ACh-
induced Cl- currents measured at -60 mV revealed the presence of an inflexion
('hump')whichtookdiverseappearancesdepending onthe cell (Fig. 1). Thesehumps
were only observed ifa minimal dose ofdissociating enzymes had been employed as
described under Methods. Provided that this was done, however, the large majority
INITIATION OF Ca2+ RELEASE 669
of the cells displayed humps. In most cells a shoulder-shaped component was
obtained (Fig. 1A, B and E); in some cells, only a change of slope was visible
(Fig. ID); in others, a clear peak was leading the main part ofthe current response
(Fig. ICandF).
A D
t ~ ~
_____
B E
C F
I O5nA
1 s
Fig.1. EarlycomponentinACh-inducedcurrentresponses.Recordsobtainedat-60mVin
response tobath application of0-5,sM-ACh in various cells (barsabove records). In most
cells a shoulder was apparent as examplified in A, B and E. Occasionally a simple
inflexionwasvisible (asinD)orapeakwasapparent (asinCandE). Theamplituderatio
between early and late components varies from 0-14 (B) to 1-1 (F).
Thepossibility thathumps couldbeduetoacontaminationoftheCl- currentwith
aK+-selective currenthaving adifferenttime course could be safelyruledouton two
grounds. First, humps kept their characteristic features ifthe holding potential was
hyperpolarizedfrom -60 mVtotheequilibrium potentialforK+ (-85 mV) orbelow
(e.g. -100 mV). Secondly, thepresenceinthe cellinteriorof10 mM-decamethonium,
a potent blocker of Ca2+-dependent K+ channels of rat lacrimal glands (Llano,
Tanguy & Marty, 1987), did not alter the appearance ofthe humps (Fig. 2). Humps
therefore belong to the genuine time course of ACh-induced Cl- current responses.
Humps can actually also be observed at 0 mV in ACh-induced K+ current responses
(Marty, Horn, Tan & Zimmerberg, 1989). It was not possible to distinguish humps
in Fura-2 signals, however, due to the much lower signal-to-noise ratio of such
signals compared to that ofelectrical recordings.
Humps were present if the cells were stimulated with a Ca2+-free extracellular
solution. This observation rules out the possibility that humps could be related to
Ca2+ entry through the plasma membrane. In conclusion, the results of Fig. 1,
together with the various control experiments just described, indicate that Ca2+
670 A. MARTY AND Y. P. TAN
release proceeds in two phases, with a rapid, early transient immediately followed by
a longer, usually larger response.
TheCa2+ release response toACh
Internal Ca2+ responses to several applications of ACh (01-5#fM) in a Fura-2-
loaded lacrimal acinar cell are shown in Fig. 2. Successive applications were
separated by 10 min periods. Under such conditions, desensitization was minimal, as
indicated by the finding that a test of0 2 /tM-ACh at the end ofthe experiment gave
aCaW+ response very similar to that obtained initially (last and second low-speed
records in Fig. 2). Various qualitative features of the response are apparent. The
amplitude of the initial peak of the response saturates rapidly (at 0-2 ftM agonist
concentration) while the size of the sustained component only saturates near1I tM-
ACh concentration. At 0-1-0{5,tM-ACh, there are slow, damped oscillations of Ca?+
matching those previously described for Ca2+-dependent currents (Evans & Marty,
1986a; see Gray, 1988). The Ca2+ signal was found to rise after a certain lag. At a
concentration of0 5,tM-ACh, the mean time to the half-maximum Ca2+ response was
1-43+0-11 s (mean±s.D,, n = 3), close to the value of1-39+0-43 s (n = 18) found for
thetime-to-peak ofACh-inducedhumps (Table 1). Furthermore, thelagoftheFura-2
signals varied with the agonist concentration, as described previously (Merritt &
Rink, 1987; Horn & Marty, 1988). Thus in the experiment ofFig. 2 the time-to-half-
CaCi2+ changewas 2-0 s at01 /LM- and0-8 sat 2 /M-ACh (see the expanded traces in the
right lower panel of the figure).
The Fura-2 method was used to examine the effects ofmembrane potential on the
response of ACh. The cells were not voltage clamped, but the cell potential was
partially controlled during the early part ofthe ACh application by using either the
standard high-Na+ solution or an isotonic-K+ solution. If ACh was applied in the
standard solution, no potential change was of course expected up to the time when
CaF1 started to rise. If however, ACh was applied in the high-K+ solution, the cell
was expected to depolarize since the resting conductance is primarily K+ selective
(Trautmann & Marty, 1984). The speed of this depolarization was measured in
control experiments using whole-cell recording. The cells were found to depolarize
completely with a time for half-potential change of 65+29 ms (n = 3), close to the
exchange time of the apparatus. Thus, the cells could be considered to be entirely
depolarized by the high-K+ solution during most of the delay period. In the
experiments illustrated in Fig. 3A, individual cells were stimulated alternatively
with Na+- and K+-rich ACh solutions, with rest periods of 10 min in order to avoid
desensitization. The delay was clearly larger with the K+-rich ACh solution. These
experiments suggest that potential changes occurring simultaneously to the ACh
application areable tomodulate thedelay.
The delay ofthe ACh-induced current response is a linear function of the inverse
AChconcentration suchthatsaturatingagonistdosescorrespond toaminimum delay
value (Horn & Marty, 1988). The question therefore arose whether hyperpolarization
would be able to reduce this limiting value. To address this point, the experimental
protocol ofFig. 3A was repeated with a saturating ACh concentration (5 /tM). The
effect ofchanging Na+ for K+ in the stimulating solution was then hardly noticeable
(Fig. 3B and Table 1). Thus the limiting delay value obtained at saturating ACh
concentrations (Horn & Marty, 1988) is independent ofthe membrane potential.
INITIATION OF Ca2+ RELEASE 671
SensitivityofACh-induced current tomembranepotential
In cellswhereCa2+-dependent K+ channelswere blockedwithdecamethonium, the
sensitivity of ACh-induced Cl- currents to membrane potential could be examined
over a large potential range. Hyperpolarizing the cell membrane not only increased
500
(nM) 10 J
0 - 0.1 4M-ACh 0.2gM 0.5uMM
500 2MA
2+i,~~~~~~~~~~2
[Ca2~
(nM) ~~~~p~i~M~-~AC~h~~~~~~~~~~0.1
O - 2 ,uM5HM 0-2JUoM 2
100
~~ACh
______
0- 2M SumM 0-2 mM
Fig. 2. Ca2+responsestovariousAChconcentrations. AFura-2-loaded cellwassubjected
to ACh applications (bars under records) at various concentrations ranging from 01 to
5/SM. Rest intervals of 10min were given between successive ACh applications. Lower
right records: superimposed Ca2+ rises in response to 01 JtM-ACh (continuous line) and
2#M-ACh(dottedline).Notethedifferenceindelayandrisetime.Timestohalf-maximum
Ca2+ response were: 2-0 s (0-lIuM-ACh); 1 7 s (0-2 uM); 1-5s (0-5 mM); 08s (2/M); 08s
(5 uM); 1-9 s(0-2/M,lastlow-speedrecord).
theinward currentresponse, asexpectedfromtheaugmentation in drivingforce, but
it also shortened the delay. Itwas difficult to quantify this phenomenon because the
delay ofACh-induced currents increases with time in whole-cell recording as aresult
ofwash-out (Marty & Zimmerberg, 1989). Nevertheless, the effect ofpotential on the
delay was large enough to be clearly seen superimposed on the slow increase
associated with wash-out. In the experiment of Fig. 4, for example, the trace at
-20 mV in the first run had clearly a longer delay than that at -100 mV in the
second run (see Fig. 4B). Kinetic changes associated with cell potential can be seen
better by scaling the traces according to the driving force for Cl-. SinceEc1 = 0 mV,
the trace at -20 mV was multiplied by 5 and that at -60 mV by 5/3 in order to
match that recorded at -100 mV (Fig. 4A, lower records). It may be seen that the
scaled humps have similar amplitudes, the main difference between traces lying in
the delays (measured in Fig. 4B as the times-to-half-hump-amplitude). There was
672 A. MARTY AND Y. P. TAN
A
800r
600 Na+
[Ca2+i1
(nM)
400[
200F
1 s
ACh ACh ACh
0L
Na+ K+ Na+
B
400
ct.qv4p
.,
0* %%a-%
300 00 0
[Ca2+]; 9 0
011
(nM)
200 [
100,
1 s
0
o
ACh, Na+ ACh, K+ AACChh
Fig. 3. Ca2' responses to 0-5#M-ACh in Na+ and K+ salines. A, Ca2+ was measured in
response to ACh applications separated by 10min intervals. In the first and third
applications, ACh (0-5FM)wasdilutedinaNa+salinesimilartothestandardsalineplaced
in the bath solution, except that it contained no Ca2 , that it was supplemented with
0-2 mM-EGTA, and that it had 2mM-Mg2+ instead of1 mm. The second application was
performed with an isotonic K+ saline (containing no Ca2 , 02 mM-EGTA and 2 mr-
Mg2+). On the right, superimposed traces showing the response onsets for the three
stimulations. The [Ca2+]i rise is slower and smallerwith the K+ stimulation (continuous
line). B, same protocol as in A. ACh concentration, 5FM. In the first record, ACh was
diluted in Na+ saline; in the second record, ACh was diluted in K+ saline. The
corresponding rising phases (O, Na+; 0, K+) are displayed on the right. These rising
phases are almost exactly superimposed. (A third application with the Na+-rich ACh
solution later in the experiment again gave almost exactly the same response.)
also a systematic lengthening ofthe rise time with depolarization, as may be noted
in Fig. 4A.
The results of Fig. 4 confirm the effects observed with Fura-2 measurements
(Fig. 3). Theyfurthershowthattheincreaseddelayobservedindepolarizingsolution
(as in Fig. 3A) is linked to thepotential change ratherthan to the ionicnatureofthe
stimulating solution per se. Finally, it was found that delay changes observed either
INITIATION OF Ca2+ RELEASE 673
TABLE 1. Ca2+ response to ACh in resting and K+-depolarized cells
ACa2+ (K+) 4 (K+)
[ACh] ACa2+ (Na+) th(Nal)
c/sM) (nM) ACa2+ (Na+) (s) ti (Na4) n
02 264+27 074+022 2-57+0-41 P150+0-10 7
0.5 361+85 064+022 1-52+0-41 1-45+0-10 8
5 334+117 0-97+007 085+0-21 1P10+0-17 9
Individual experiments asinFig. 3. Foreach cell, the Ca2+responsewas measured sequentially
with Na+-, K+-, and again Na+-based ACh solutions. The control (Na+) values were taken as the
mean between the first and third measurement. ACa2+ isthe peak ACh-induced Ca2+ change. t. is
the timeelapsed between the onset ofthe ACh application and the half-Ca2+ response. Means and
S.D. ofn independent measurements are given.
A B
0.5-
0.0- -20mV 1.5 - ,
-0.5- -60 m-- 0
-10.,0-u _ 5
~~~~~~~~~~~~~~~1.0
-1.5
-2.0
0 1 2 3
0 ~~~~~~~--100~00~.5~ --0 mV-2
-1 .0-~~~ ~ ~ ~ ~~~~~~~.
-d0
-100 -20
~-2.~0~~~ ~ ~~IMembrane potential (mV)
Time (s)
Fig. 4. Voltage dependence ofACh-induced Cl- currents. Records were takenatvarious
holding potentials in response to bath applications of05,uM-ACh. The cell was dialysed
with 20mM-decamethonium in order to block Ca2+-dependent K+ channels and thus to
measure Ca2+-activated Cl- currents with little contamination from othercurrents. InA,
upperpanel,successiveresponsesrecordedat -100, -60and -20mVaresuperimposed.
The time origin corresponds tothe onset ofACh application. The lowerpanel shows the
same records after multiplication of the traces at -60 and -20mV by 5/3 and 5
respectively. ThegraphinBshowsthedelay(measuredasthetimeelapseduptothemid-
pointofthehump) asafunctionofmembranepotentialfortherunillustratedontheleft
part (0) as well as for the next run (0).
22 PHY419
674 A. MARTY AND Y. P. TAN
in Fura-2 or in whole-cell recording experiments were not influenced by the external
Ca2+ concentration, suggesting that Ca2+ entry, if present, did not modulate the
delay.
Local stimulation withanelevated Ca2+ solution
The rationale ofthese experiments was to stimulate the cell interior with a local
and rapid Ca2+ rise in orderto obtain Ca2+-induced Ca2+ release. Speedwas expected
tobe crucial since slowCa2+risesdesensitize theCa2+releaseprocesswithouteliciting
aresponse (Fabiato, 1985).
A C
L 0.5 mM-EGTA
0.2 mM-Ca2+
Ca2'-free saline
B 200 pA D
Em,-Ca2+
Fig. 5. Ca2+-induced transient current. Records start in the cell-attached configuration.
Holding potential, -60mV. InB, C andD, small-amplitude voltage pulses were given
repetitivelytotesttheaccessconductancetothecell. Verticalarrowsmarkthetransition
to the whole-cell recording mode induced by suction in the pipette compartment. InB,
the solution was maintained throughout; in the other cells, only a shortpulse ofsuction
was applied. The pipette contained the standard low-Ca21 internal solution (C) or else a
high-K+ solution containing 0-2 mm (A) or 1 mM-Ca2+ (B and D). The bath contained
standardsalineexceptfortherecordinD,whichwasperformedshortlyafterflushingthe
cellwith aCa2+-free saline containing0-2 mM-EGTA. Therepetitive artifactsinB, Cand
Dareduetofilteredcurrentresponsestothetestvoltagepulses.
Figure 5 illustrates the basic features ofthe responses which were obtained. The
pipette contained an intracellular solution without EGTA and with an added Ca2+
concentration of01-1 mM-Ca. In these experiments, a seal was established against
the cell membrane, and the transition to the whole-cell recording configuration was
obtained in a conventional manner using a sharp pulse of suction. The pipette
potential was held at -60 mV. Just following the break-in artifact, a small inward
current developed linearly with time. Superimposed on this signal, large inward
currenttransientsstartedtoriseafteralatencyof0-2-1-5 s (Fig. 5A andB). Thetime
between the break-in artifact and the peak ofthe transient ranged from 0-8 to 2-0 s,
and the transient amplitude varied from 100 to 600 pA. Acinar cells from exocrine