BY KEN NAKAZAWA*, KANNOSUKE FUJIMORI, AKIRA TAKANAKA AND KAZUHIDEINOUE 2

16 Pages·2005·1.54 MB·English

Joulrnal ofPhysiology (1990). 428, pp. 257-272 257
W'ith 8figures
Printed in GreatBritain
AN ATP-ACTIVATED CONDUCTANCE IN PHEOCHROMOCYTOMA
CELLS AND ITS SUPPRESSION BY EXTRACELLULAR CALCIUM
BY KEN NAKAZAWA*, KANNOSUKE FUJIMORI, AKIRA TAKANAKA
AND KAZUHIDE INOUE
From the Division ofPharmacology, Biological Safety Research Center, National
Institute ofHygienic Sciences, 1-18-1 Kamiyoga, Setagaya, Tokyo 158, Japan
(Received 11 October 1989)
SUMMARY
1. ATP-activated inward current in PC12 pheochromocytoma cells was charac-
terized using the whole-cell voltage-clamp technique.
2. ATP (1004aM) applied extracellularly elicited an inward rectifying current with
areversal potential ofabout +7 mV. The current wasdesensitized in secondsin spite
of continued presence of ATP.
3. A comparison was made ofthe ability ofATP and its analogues. The order of
potency in activating the inward current was ATP > ATPyS > ADP; AMP,
adenosine and a,,-methylene ATP were inactive at concentrations up to 1 mM.
4. The ATP-activated current wasalso observed whenexternal Na+ andCa2+were
replaced with K+, TEA, Tris or glucosamine. The order of ion selectivity was
Na+ > K+ > TEA * Tris > glucosamine.
5. The ATP-activated current was also recorded in extracellular solutions
containing Ca2+ as the only external cation. The amplitude increased as the
concentration ofCa2+ was increased in the range between 1-8 and 16-2 mm. However,
the current amplitude decreased at higher Ca2+ concentrations and the current was
not recorded in 110 mM-Ca2+ solution.
6. In the presence of 140 mM-Na+ in the external solution, the current amplitude
also decreased as the external Ca2+ concentration was increased (from 1-8 to
16-2 mM).
7. The results indicate that Ca2+ as well as monovalent cations permeate through
the ATP-sensitive pathway and that Ca2+ blocks ion permeation, including its own
permeation through the pathway. This regulation by extracellular Ca2+ is different
to the ATP-activated current in smooth muscle cells.
INTRODUCTION
Extracellular adenosine 5'-triphosphate (ATP) has recently been regarded as a
regulator in various kinds oftissues (Burnstock, 1978; Burnstock & Kennedy, 1985;
Gordon, 1986). ATP released from nerve terminals exerts both excitatory and
inhibitory effects. A most typical example for excitatory effects may be smooth
* To whom all correspondence and reprint requests should be addressed.
MS8007
9 PHY428
258 K. NAKAZA "'A AND OTHERS
muscle contraction which is thought to be triggered by excitatory junctional
potentials due to activation of receptor operated non-specific cation channels
(Benham, Bolton, Byrne & Large, 1987; Benham & Tsien, 1987; Nakazawa &
Matsuki, 1987; Friel, 1988) while that for inhibitory effects be suppression of
may
neurotransmitter release which is thought to be due to inhibition of voltage-gated
Ca2" channels existing in nerve terminals (Tsien, Lipscombe, Madison, Bley & Fox,
1988).
Although ATP and its analogues are regarded rather as an inhibitory regulator of
neurotransmitter release via presynaptic adenosine receptors in neuronal cells, there
is some evidence indicating that ATP also exerts excitatory effects in these cells
(Jahr & Jessel, 1983; Krishtal, Marchenko, Obukhov & Volkova, 1988). We
previously showed such an excitatory effect ofATP in PC12 pheochromocytoma cells
where ATP stimulated noradrenaline secretion (Inoue, Nakazawa, Fujimori &
Takanaka, 1989). The stimulation of catecholamine secretion in these cells by ATP
seemed to result from activation of an inward current carried by cations. In the
present study, we further investigate the ionic basis ofthe ATP-activated current in
PC12 cells. Our results demonstrate that the current is regulated by extracellular
Ca2" in a different fashion from the ATP-activated current in smooth muscle cells.
METHODS
Rat pheochromocytoma-derived PC12 cells were cultured according to methods described by
Inoue & Kenimer (1988). The cells were cultured in culture flasks with Dulbecco's modified Eagle's
medium containing 7% fetal bovine serum, 7% heat-inactivated (560C. 30 min) horse serum.
2mM-L-glutamine and 50,ug/ml gentamicin in a humidified atmosphere of90% air and10% CO2
at 37 'C. The cells were removed and plated into collagen-coated 35 mm polystyrene dishes
(1x 106/dish). Theplated cells were cultured furtherunder thesame conditions asstated above and
used for experimentation 2 or 3 days after plating.
Membrane currents were measured usingthewhole-cell voltage-clamp technique (Hamill, Marty,
Neher, Sakmann & Sigworth, 1981) under the conditions described previously (Nakazawa, Saito &
Matsuki, 1988; Nakazawa, Fujimori, Takanaka & Inoue, 1989). The dish containing cultured cells
was placed on the stage ofan inverted microscope (OlympusIMT-2) and continuously superfused
with a standard external solution (solution1 ofTable 1). Heat-polished patch pipettes filled with
an internal solution containing 145 mM-CsCl, 5 mM-EGTA and 10 mM-HEPES (pH 7-4 with CsOH)
had tipresistance of2-5 MQ. Currents were measured usingapatch-clamp amplifier (Nihon Koden
CEZ2200). Series resistance compensation was employed. Electrical signals were filtered at 1 kHz
and stored on a magnetic tape-recorder (TEAC MR-10). The data was analysed on a personal
computer (NEC PC9801 RA2) using a data analysis program developed and provided by Dr
Matsuki of the University of Tokyo. All the experiments were performed at room temperature
(about 22 'C).
The ionic composition ofexternal solutions used in the present study was listed in Table 1. All
the solutions except solution 8 were adjusted to pH7-4. Solution 8 which contained 145 mm-
glucosamine as a main external cation was adjusted to pH6-4 so that glucosamine(pKa 86) was
> 99% ionized (Benham & Tsien, 1987).
Drugs used were adenosine 5'-triphosphate disodium salt (ATP, Sigma), adenosine 5'-
diphosphate sodium salt (ADP, Sigma), adenosine 5'-monophosphate sodium salt (AMP, Sigma),
adenosine (Sigma),a,,f-methyleneadenosine 5'-triphosphate lithium salt(a,fl-mATP, Sigma) and
adenosine-5'o-3-thiotriphosphate tetralithium salt (ATPyS, Boehringer Mannheim).
RESULTS
Figure1A shows a typical recording of ATP-activated current in PC12 cells
bathed in normal solution (solution 1). The current was activated upon superfusion
ATP-ACTIVATED CURRENT IN PC12 CELLS 259
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260 K. NAKAZAW1rA AsND OTHERS
with 100,tM-ATP. The current always desensitized during continuous superfusion
with ATP. At 5 min, the current was only 1 % of initial amplitude (09+00%,
mean+S.E.M.. n = 4).
Peak amplitude of the ATP-activated current was about 800 pA at -50 mV
(834+71 pA, n = 10), which was comparable to the amplitude ofthe ATP-activated
A
100AJM-ATP
B
0 5 10s
750-"*,
500-
r= 5.6s
250-
pA
Fig. 1. A. inward current activated by 100,/M-ATP recorded from a PC12 cell bathed in
normal solution (solution 1). Holding potential was -60 mV. The current trace digitized
at 125 Hz wasshown. The current wasinactivated inspite ofcontinued presenceofATP.
B, inactivation time course ofthe current shown inA. Amplitude ofthe current afterthe
periodofpeakamplitudewasplottedlogarithmically against time.Thetimeconstant was
calculated from the current 2s after the beginning ofthe inactivation.
current in isolated smooth muscle cells from rat vas deferens (Nakazawa & Matsuki,
1987). We calculated the surface area ofthe PC12 cells from the input capacitance
estimated in current-clamp recording by injecting a hyperpolarizing step of -10 pA.
The estimated membrane area of the PC12 cells (IV6+0-2 x 10-5 cm2, n = 10) was
3-fold smaller than that ofsmooth muscle cells from rat vas deferens (5-2 x 10-5 cm2;
Nakazawa, Matsuki, Shigenobu & Kasuya, 1987) (assuming specific membrane
capacitance of 1 aFcm-2). Based upon these values, thedensity ofthe ATP-activated
channels in PC12 cells is several fold larger than that in smooth muscle cells from rat
vas deferens.
Figure lB shows inactivation time course of the ATP-activated current.
Inactivation ofthe current followed a single exponential and the time constant was
5-6 s in this cell (mean: 65+15 s, n = 10). The result suggests that the ATP-
activated current is due to a homogeneous population of ion channels.
Although inactivation of the ATP-activated current developed in seconds,
ATP-ACTIVATED CURRENT IN PC12 CELLS 261
A
2 min 5 min
B - - 7400 pA
-a.0
Q0
E 0.5
a)
1 2 3 4 5 min
Interval
Fig. 2. Time course ofrecovery from inactivation. Cells were bathed in normal solution
(solution 1) andheldat -60 mV. First, thecellswereexposedto 100,uM-ATPasreference
applications, and were exposed to the same concentration of ATP as test applications
following various periods ofrinse with drug-free solution as shown inA. Recovery from
inactivation was assayed by measuring the peak amplitude elicited by the test
applicationsandnormalizedbythepeakcurrentelicitedbythereferenceapplications (B).
Each symbol denotes mean values ofthree to seven cells tested. Bars are S.E.M.
A
100 pM-ATP 1 mM-adenosine 1 mM-AMP 1 mM-ADP
B
100pM-ATP 1 mM-a,fr-mATP 300_gM-ATPYS
<K-f- 7200 pA
Fig. 3. Agonist selectivity ofthe ATP-activated current. A andB are obtained from two
different cells. Thecellswere bathed innormal solution (solution 1) and held at -50 mV.
A, effectsofadenosine nucleotides andadenosine. Tracesarefoursuccessiverecordings of
whole-cell currents on applications of 100,SM-ATP, 1 mM-adenosine, 1 mM-AMP and
1 mM-ADP (lefttoright). Eachapplication wasseparatedby5min.B,effectsofATPand
non-hydrolysable analogues ofATP. Current traces onsuccessive applications of 100/tM-
ATP, 1 mM-a,/-methylene ATP (mATP) and 3001uM-ATPyS (left to right) were shown.
Each application was separated by 5 min.
recovery from inactivation took much longer. Full recovery required about 5 min
after removal of ATP as shown in Fig. 2. The slow recovery from inactivation was
similar to that of ATP-activated current in smooth muscle cells (Benham & Tsien,
1987; Nakazawa&Matsuki, 1987; Friel, 1988). Inthefollowingsections, applications
262 K. NAKAZAWA AND OTHERS
of ATP or its analogues were separated by 5 min to allow complete recovery from
inactivation.
Agoni8t 8electivity
The sensitivity ofATP analogues in producing the inward current is illustrated in
Fig. 3. Figure 3A shows how the effectiveness ofATP depended on the presence of
A ATP
2s
400 pA
B
1.0 .n
-20 E
-3-0
m
G
4-0
Fig. 4. Current-voltage relationship fortheATP-activated current recorded withnormal
solution (solution 1). The cell was held at -50 mV, and 100ms hyperpolarizing or
depolarizing steps ranging from -120 to +20mV, in 1OmV increments were applied
onceinevery500ms.Whilesteppingthroughthisrangeofpotentialsrepetitively,thecell
was exposed to 100/SM-ATP as shown in A. The difference between the current in the
presenceandabsenceofATPforeachtestpotentialwasmeasuredandnormalizedbythe
difference attheholding potential (-50mV). The normalized amplitude plotted against
the potentials was shown inB.
the 5'-polyphosphate group. Neither adenosine nor AMP produced any obvious
changeincurrentupto 1 mM(n = 4). ADPdidnotexertanyeffectsat 100/SM (n = 3),
but induced a small inward current at 1 mm (19-6+5-0% of the current activated
by 100,uM-ATP, n = 5). Although 5'-polyphosphate group seemed indispensable for
activation of the current, hydrolysis of ATP was not related to the activation
because ATPyS, a non-hydrolysable analogue, elicited a similar inward current in
PC12 cells (Fig. 3B). The peak amplitude of the current induced by ATPyS was
10-0+1-7% at 100gUM (n = 3) and 44-3+6-8% at 300/uM (n = 4) when compared
with the amplitude of the current induced by 100gUM-ATP. The idea that the
activation is not related to hydrolysis was also supported by the result that ATP-
ATP-ACTIVATED CURRENT IN PC12 CELLS 263
activated currentwasnotaffected by500,tM-K-252b (95-5+8.0% ofcontrol, n = 4),
a drug which inhibits enzymatic hydrolysis of extracellular ATP (Kase, Iwahashi,
Nakanishi, Matsuda, Yamada, Takahashi, Murakata, Sato & Kaneko, 1987).
ATP (100 /M) activated the inward current which was unaffected by 100 /M-
hexamethonium (930±3-0%, n = 3), suggesting ATP acted on receptors different
from those recognized by nicotinic agents. Judging from the order of potency of
agonists, ATP-activated current seemed to be described as a P2 purinergic response
(Burnstock, 1978). However, a,,6-mATP, which is known as a P2 agonist in smooth
muscle tissues (Meldrum & Burnstock, 1983; Amobi & Smith, 1987), did not affect
membrane current up to 1 mm (Fig. 3B). It was reported that a,fl-mATP exerted a
desensitizing effect on P2purinoceptors in addition to itsagonisticeffect andblocked
an excitatory effect ofATP successively applied to smooth muscle tissues (Meldrum
& Burnstock, 1983; Amobi & Smith, 1987). A test was made for a possible
desensitizing effect ofa,,-mATP on ATP-activated current. PC12 cellswere exposed
to 1 mM-a,fl-mATP and then tested with 100,SM-ATP in the continued presence of
a,,f-mATP. Infour cells tested, x,,-mATP neitherelicited an inward current nordid
it cause clear inhibition of response to ATP. The results suggest that receptors
mediating the ATP-activated current in PC12 cells have certain pharmacological
differences from theP2 typepurinoceptors mediating contraction ofsmooth muscles.
Ionic selectivity
Figure 4 shows the current-voltage relationship for the ATP-activated current.
The cell was loaded with 150 mM-Cs' and bathed in normal external solution
(solution 1). The ATP-activated current exhibited inward rectification and reversed
near 0 mV (+6-7+3-6 mV, n = 6). The reversal potential of +6-7 mV suggests that
the ATP-sensitive channels are permeable to more than one ionic species.
Thepoorion selectivitywasalso supported bytheresultsshowninFig. 5A. Inthis
series of experiments, ion selectivity was assessed by replacing external Na+ with
various kinds ofcations (solution 5-9). The cells were first exposed to 100/LM-ATP
dissolved in a Ca2+-free solution containing 145 mM-NaCl (solution 2) and then
exposed to ATP dissolved in test solutions after a 5 min rinse with a drug-free
solution. In 145 mM-KCl solution, ATP induced an inward current ofhalfaslarge as
that in 145 mM-NaCl solution. ATP also induced a considerable inward current in
145 mM-TEA or Tris solution and induced a small inward current (about 30pA) in
145 mM-glucosamine solution. The order of permeability derived from the results
shown in Fig. 5A was Na+ > K+ > TEA. Tris > glucosamine, which accorded with
the order oftheir molecular weight.
In contrast to the case of monovalent cations, ATP did not induce the inward
current when external NaCl was replaced with 110 mM-CaCl2 (Fig. 5A). The result
was different from the characteristics of ATP-activated current in smooth muscle
cells from rabbit ear artery, where ATP activated an inward current in 110 mM-Ca2+
comparable to that activated in 135 mM-Na+ (Benham & Tsien, 1987). Lack of
detection of ATP-activated current in this study was not due to a possible cell
damage orsomefailureinrecordings, whichwasindicated bytheresultshown inFig.
5B. Inthis case, theexternal solution waschanged to 110 mM-Ca2+from 145 mM-Na+
while the ATP-activated current persisted. The ATP-induced current rapidly
264 K. NAKAZAWA AND OTHERS
disappeared upon switching to 110 mM-Ca2+ and reappeared on returning of the
solution to 145 mM-Na+.
The effects of Ca2+ on the ATP-activated current were investigated further and
described in the following section.
A
V 50- T,
E > E ELEo E
LnLTn~~~~~~~(
T
B
ATP
110 mM-CaCI2
4s
7 200 pA
Fig. 5. A, ATP-activated current recorded with various external cations as a charge
carrier. The cellswerebathed in 145 mM-Na+, Ca2+-freeexternal solution (solution 2) and
held at -80 mV. The cells were exposed to ATP (100#M) dissolved in the 145mM-Na+
solution, and the peak amplitude of the inward current evoked was measured as a
reference. Aftera 5minrinse with drug-free solution, theexternal solution was switched
toatestsolution containing adesired cation asa charge carrier (solutions 5-9), and, 30s
afterthe switching, the cellswereexposed toATP (100/sM) dissolved inthetestsolution.
The peak amplitude ofthe current activated in the test solutions was normalized by the
references and illustrated. Each column represents the mean value of four to six cells
tested. Bars are S.E.M. B, disappearance of ATP-activated current during a brief
treatment with 110mM-CaC12. The cell was bathed in 145 mM-Na+ solution (solution 2)
and held at -60 mV. The inward current was induced by 100,uM-ATP, the external
solution was replaced with 110mM-CaCl2 solution while the ATP current persisted, and
the Ca2+ solution was changed to the original one (100,uM-ATP continuously existed
throughout these procedures).
Effects ofCa2+ on ATP-activated current
As shown in Fig. 5, ATP induced a large inward current in an external solution
containing 145 mM-NaCl but did not induce any detectable inward current in
110 mM-Ca2+ solution. A simplest interpretation for the results was that the ATP-
sensitive pathway was monovalent cation selective and that Na+ but not Ca2+ could
permeate through the pathway. Ifthis is the case, thereversal potential ofthe ATP-
activated current obtained with only Na+ and Ca2+ asexternal cations would change
depending on solely on Na+ concentration, according to the Goldman's equation
(Goldman, 1943):
=RT1nPNa[Na+],
ErEvrv=F P~5CsI]C ' ( 1 (1)
where Erev is a reversal potential of the ATP-activated current, PNa and PCS are
permeability ofNa+ andCs+, respectively, [Na+]o isextracellular Na+ concentration,
[Cs+]i is intracellular Cs+ concentration, and R, T and F are their usual meanings.
ATP-ACTIVATED CURRENT IN PC12 CELLS 265
A test was made forthis hypothesis and theresults are shown in Fig. 6. As cations
oflarge molecular weight could permeate throughATP-sensitive pathway (Fig. 5A),
external NaCl was replaced with sucrose in this case. The reversal potential was
+70 mV in a solution containing 150 mM-Na. According to equation (1) with
A ATP
1200 pA
B
ATP
Y100
pA
0.5s
C [Nallo (mM)
, +20 1-9 5.5 16.7 50 -
c 0 I q
o150
X E 0 a
CO) 0-
a: -100 ,,
Fig. 6. External Na+dependency ofthereversal potential oftheATP-activated current.
External Na+ concentration was adjusted to 1P9, 55, 16-7 or50mm bymixing solution 3
and solution 10. A andB, measurement ofthereversal potentials. The cellswere held at
-80mV and exposed to a solution containing 50mm (A) or 5-5mM (B) of Na+. ATP
(100/M) was applied while four depolarizing steps (increments, 10mV) of 50ms were
applied every 100ms. The depolarizing steps applied to the cells ranged from -50 to
-20 mV (A) or from -60 to -30mV (B) in these cases. C, the reversal potentials
estimatedwiththemethodshowninAandB.Thereversalpotentialswereplottedagainst
external Na+ concentrations. The broken line was calculated from reversal potentials
asuming PNa/P.. = 1-3 andPc.=0 (see text). Each symbol represents the mean offour
to six cells tested. Bars are S.E.M.
[Na+]O = 150 mm and [Cs+]i = 150 mM, permeability ratio, PNa/cPs equals 1-3 in this
condition. Reversal potentials predicted from equation (1) with PNa/PCs = 1P3 were
indicated with abroken line in Fig. 6C. Ameasuredreversal potentialwaswellfitted
to this line when external Na+ was lowered to 50 mm. However, measured reversal
potentials didnot follow the predicted values at concentrations lower than 16-7 mm,
rather approached the reversal potentials predicted by the hypothesis that Ca2+ is a
major permeable cation (see below). The results indicate that the ATP-activated
current was, at least, not due to a simple pathway which only monovalent cations
permeate through depending on their concentrations.
Next we investigated effects of ATP under the condition that Na+ was omitted
from external solutions (solutions 9, 10 and their mixture were used). With
266 K. NIAKAZAWA AND OTHERS
A 48.6 mM-Ca2+, 5.4 mM-Ca2+,
Normal Na+-free Normal Na+-free
1200 pA
1 s
B C
30- > 1-0
.
20)
~~~~~~0*5-
CL
E 10-
1.8 5.4 16.2 48-6 110 1 8 5.4 16.2 48-6 110
[Ca+lo (mM) [Ca+lo (mM)
Fig. 7. ATP-activated current recorded with Ca2" as the only external cation. External
Ca2+ was adjusted to desired concentrations by mixing solution 9 and solution 10.A, the
currentrecordingsobtainedfromPC12cellswith normalsolution (solution 1),orsolutions
containing 48-6 or5-4 mM-Ca2' asthe only external cation. The current responses to four
successive applications of 100,tM-ATP were shown. Each application was separated by
5min. Horizontal bar represents ATP application. B, external Ca2' dependency ofATP
current carried by Ca2+. The peak amplitude ofthe current with the solutions containing
Ca2'astheonlyexternal cationwasnormalized againsttheamplitudeofthecurrent with
normal solution (solution 1), and plotted against external Ca2+ concentration. Each
symbol represents mean of four to five cells tested. Bars are S.E.M. C, concentration
dependency ofthe blocking effect ofexternal Ca2+ on its own permeation through the
ATP-activated pathway. The mean current amplitude with each concentration ofCa2+
shown inBwasdivided bythe concentration. Thevalueswere normalized and plotted as
relative permeability (P,.) against the Ca2+ concentrations. Curve fitting was made
assuming a model for the ATP-sensitive pathway:
nCa2+
unblocked < Ca2+-blocked.
According to conventional theory ofconcentration-response relation such as Tallarida &
Jacob (1979), the relation between PCa and [Ca2+]o was given by
(Pca=18mm-PCa)/PCa-1.8mm = 1/(1+(K/[Ca2+]0)n),
where K is a dissociation constant. The values used in the curve fitting were
K= 40x 10-5 M and n = 2-0.
1-8-48-6 mM-Ca2+ as an only external cation, ATP induced an inward current at
potentials more negative than -60 mV (Fig. 7C). ATP did not induce such an
inward current when the cells were bathed in Ca2+-free, 300 mM-sucrose (Ca2+ was
omitted from solution 10 and pH was adjusted with TEA-OH; final concentration of
TEA was about 3 mm). Therefore, we interpreted the inward current as inflow of
Ca2+. The amplitude ofthe ATP-activated Ca21 current increased whenexternal Ca2+
concentration wasraised in arange between 1-8 and 16-2 mm (Fig. 7B). However, the