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Journal of Phyuiology (1991), 442, pp. 691-710 With 12 figures Printed in Great Britain

PROPERTIES OF IONIC CURRENTS INDUCED BY EXTERNAL ATP IN A MOUSE MESODERMAL STEM CELL LINE

BY YOSHIHIRO KUBO From the Department of Neurophysiology, Tokyo Metropolitan Institute for Neurosciences, Fuchu-city, Tokyo 183, Japan (Received 3 January 1991) SUMMARY

1. ATP was puff applied to cells of a mesodermal stem cell line, C3H1OT1/2, and the responses were studied by whole-cell patch clamp recording. 2. In 91 % of the cells (90/99), K+ current lasting for tens of seconds was observed after several seconds latency. The current showed outward rectification. In 10% of the cells (9/99), ATP induced Cl- current which also lasted for tens of seconds after several seconds latency, but showed little rectification. In 6% of these cells (5/99), both K+ and Cl- currents were induced by ATP. 3. The K+ current induced by ATP was dose dependent, with a Kd of 0 4 [zM. The effects of ATP analogues were tested at a concentration of 20 [UM. ADP and ATP-yS induced the K+ current, while AMP and adenosine did not. a,f-Methylene ATP produced a diminished K+ current. 4. The ATP-induced K+ current was not observed when EGTA in the internal solution was raised from 0-1 to 5 mm. In Fluo-3-loaded cells, an increase in intracellular Ca2l concentration induced by the application of ATP was observed, and the time course was similar to the induced K+ current. Both the increase in intracellular Ca2+ and the K+ current were induced by ATP even in Ca2+-free external solution. Ryanodine (50 ,sM) in the external solution did not affect the ATP response, and application of 10 mM-caffeine alone to the external solution did not induce any response. 5. The variance of the steady-state fluctuations in the course of the ATP-induced slow K+ current was analysed. The single-channel conductance was estimated as 2-7 pS at 0 mV with external and internal K+ concentrations of 5 and 140 mM respectively. The K+ current was not affected by apamin at concentrations of up to 1 ,UM but was reduced to one-third by 140 mM-tetraethylammonium (TEA). 6. It was concluded that puff-applied ATP has two main effects in the mesodermal stem cells: an increase in the intracellular Ca2+ concentration and a succeeding hyperpolarization due to the Ca2+-activated K+ conductance which is present in this cell. The significance of the increase in intracellular Ca2+ caused by ATP is discussed. INTRODUCTION

The concept of purinergic neurotransmission, first proposed by (Burnstock, 1978, 1981), has been confirmed in various nerve cells, including spinal cord neurones (Jahr MS 9029

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& Jessell, 1983), dorsal root neurones (Bean, 1990; Bean, Williams & Ceelen, 1990), cochlear hair cells (Shigemoto & Ohmori, 1990) and adrenal chromaffin cells (Sasakawa, Nakaki, Yamamoto & Kato, 1989). In addition to nerve cells, ATP-activated responses have been reported in various cells of mesodermal origin. It has been reported that ATP causes multiple responses in chick skeletal muscle cells (Kolb & Wakelam, 1983; Haggblad, Eriksson & Heilbronn, 1985; Hume & Honig, 1986; Hume & Thomas, 1988), and that it activates nicotinic ACh receptors in Xenopus skeletal muscle cells (Igusa, 1988). ATP-activated currents have also been observed in smooth muscle cells of blood vessels (Benham & Tsien, 1987; Benham, Bolton, Byrne & Large, 1987) and vas deferens (Friel, 1988) and in cardiac muscle cells (Friel & Bean, 1988). In addition, ATP induces slow hyperpolarizing responses in a mouse fibroblast cell line, L6 (Okada, Yada, Ohno-Shosaku, Oiki, Ueda & Machida, 1984). C3H1OT1/2 is a cell line established from mouse whole embryo (Reznikoff, Brankow & Heidelberger, 1973). It can be induced to differentiate to various cells of mesodermal origin, such as muscles, lipocytes and chondrocytes by adding 5-aza-2'deoxy-cytidine to the medium (Constantinides, Jones & Gevers, 1977; Taylor & Jones, 1979), and is generally considered to be a mesodermal stem cell line. In this paper, the existence of ATP responses in this cell line was examined, and it was found that slow K+ current is elicited by ATP application. Reported ATP-induced currents are variable, especially in the following three ways. (1) Dose-response relation. The dose-response relation shows marked differences. The response was observed only at concentrations of more than 200 fiM (Okada et al. 1984), while in other preparations the half-maximally activating concentration (Kd) was as low as 2-7 /LM (Bean, 1990). (2) Pharmacological type of the receptor. The ATP receptors are categorized pharmacologically into P1 and P2 types (Burnstock, 1978) and the P2 receptors are further categorized into two subtypes (Burnstock & Kennedy, 1985). In some preparations, receptors are activated by the ATP analogue ADP (Okada et al. 1984), while in others they are activated only by ATP (Hume & Honig, 1986; Friel & Bean, 1988; Igusa, 1988). (3) Whether it is receptor coupled (short latency) or second messenger mediated (long latency). Most ATP responses are observed with short latency (Igusa, 1988; Bean, 1990) which suggests the direct coupling of the receptors to ionic channels, but in some preparations responses with long latencies are observed (Okada et al. 1984; Hume & Thomas, 1988; Shigemoto & Ohmori, 1990) suggesting mediation by a second messenger system. In adrenal chromaffin cells, inositol trisphosphate accumulation and Ca21 mobilization by ATP have been reported (Sasakawa et al. 1989). The properties of the ATP response in C3H1OT1/2 cells were investigated with respect to these three points. By monitoring changes in intracellular Ca21 concentration of Fluo-3-loaded cells during ATP responses, it was shown that the current is mediated by an increase in the intracellular Ca2 . In addition, properties of the activated K+ channels such as single-channel conductance and the effect of K+ channel blockers were investigated.

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METHODS

Material8 The mesodermal stem cell line C3H1OT1/2 established by Reznikoff et al. (1973) was used. C3HlOT1/2 clone 8 at a passage number of 9 was obtained from the Japanese Cancer Research Resources Bank (CCL 226, JCRB, Japan), and cells of up to a passage number of 15 were used for experiments. The culture medium was Basal Medium for Eagle's (BME; GIBCO), supplemented with 10 % heat-inactivated (56 °C, 30 min) fetal calf serum (FCS; GIBCO), 100 units/ml penicillin and 100 mg/ml streptomycin. The cells were seeded at a concentration of 104 cells/ml every 10 days. For electrophysiological experiments, the cells were seeded on poly-L-lysine- (PLL; P1274, Sigma) coated (100 ug/ml, 2 h) or uncoated cover-slips (104-105 cells/ml). As it was difficult to record from very well-attached flat cells, suitably rounded cells, obtained by leaving at room temperature for up to 1 h, were used for experiments. Electrophysiological recording Electrophysiological measurements were carried out on an inverted microscope (IMT-2, Olympus, Tokyo, Japan) with Nomarsky optics using a x 40 objective lens. Cells were voltage clamped using the whole-cell variation of the patch clamp technique by a patch clamp amplifier (CEZ-2200, Nihon Kohden, Tokyo, Japan) with a feedback resistor of 1 GQ. Patch electrodes were made of Corning hard glass micropipettes and fire-polished at the tips and their resistance ranged from 3 to 4 MQ. The voltage errors due to series resistance were not compensated during recording. In most cases the ATP response was less than 500 pA, and voltage errors due to series resistance were estimated to be less than 4 mV, assuming the series resistance to be twice that of the open pipette (Marty & Neher, 1983). The reference electrode was 3 M-KCl-Agar/Ag-AgCl. Voltage steps for voltage clamp experiments and pulses to control the 'puffer' were delivered from a 16-bit digital-analog converter (DAC98, Canopus, Osaka, Japan) controlled by a microcomputer (PC 9801 VX21, NEC, Tokyo, Japan). Data were filtered through an 8-pole Bessel low-pass filter with a cut-off frequency of 1 or 3 kHz and conveyed simultaneously to the same computer used for stimulation using the Direct Memory Access mode of a 12-bit analog-digital converter (ADX 98E, Canopus, Osaka, Japan). All electrophysiological experiments were carried out at room temperature (22-25 °C). Fluctuation analysis Cells whose ATP-induced currents were long lasting with a slow decline were chosen for steadystate fluctuation analysis around the mean. Data low-pass filtered at 3 kHz were acquired at a sampling frequency of 20 kHz for 800 ms (16000 points) with intervals of 1200 ms, sixteen times before and during the response. Steady parts of the response lasting for 400 ms (8000 points) at various current levels were chosen and the mean current level (I) and current variance (82) were measured. After subtraction of baseline current and variance, the mean current-variance relationship was fitted with the quadratic relationship: 42 = iI-P/N (Ehrenstein, Lecar & Nossal, 1970) by the simplex method using least squares as a criterion, to yield an estimate of i, the singlechannel current amplitude and N, the number of independent channels. To decrease baseline noise, the level of external solution was minimized. Although the pipettes were not Sylgard coated, the baseline noise variance was about 2 pA2. Drug application The following drugs were used in the experiment: ATP (A-0770, Sigma), ADP (A-6521, Sigma), AMP (A-1877, Sigma), adenosine (A-7636, Sigma), a,fl-methylene ATP (M-6517, Sigma) and ATP-y-S (102342, Boeringer Manheim). Drugs were applied with the 'puffer' method (Choi, 1978). Pipettes with a diameter of 2-4,sm were filled with the drug dissolved in the external solution and located about 50,um from the cell. For dose-response experiments, two puff pipettes containing drugs at standard and test concentrations separated by about 400 1am were advanced to the cell in turn. The gas pressure used to 'puff' was 0-4 kg/cm2. The electrical valve was operated by pulses delivered from a D-A converter. Solutions The compositions of external solutions used are listed in Table 1. External solution was changed by perfusing with a peristaltic pump (P-3, Pharmacia). In the experiments of Fig. 10 C, sufficiently

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quick (about 1 s) application of the drug was achieved by reducing the bath volume as much as possible, and applying more than ten times the bath volume of the solution directly to the bath by syringe. The internal solution had the following composition (in mM): KCI, 145; KOH, 7; HEPES, 10; and EGTA, 0-1. This solution was supplemented with 2 mM-ATP-Mg (A-0770, Sigma) shortly before use, and the pH adjusted to 7-4 by adding a small amount of HCl. The liquid junction potential between the internal and external solutions was about -2 mV, and was not compensated. TABLE 1. Composition of external solutions TMA TEA Cl- Glutamate Na+ Ca2+ Mg2+ K+ Na+-2 Ca2+-1 Mg2+ 140 2 1 5 146 Na+-0 Ca2+ 140 3 5 146 Na+-50 K+ 95 2 1 50 146 TMA-2 Ca2+-1 Mg2+ 2 1 140 5 146 1 TEA-2 Ca2+-1 Mg2+ 2 140 5 146 2 1 140 6 Na+-glutamate 5 140 All solutions contained 10 mM-HEPES and 17 mM-glucose. pH was adjusted to 7-4. The concentrations of ions are all millimolar. The Na+-2 Ca2+-1 Mg2+ solution is termed 'normal' in this paper.

Observation of Ca2+ increase Cells were incubated with the membrane-permeable Ca2+-sensitive dye Fluo-3 AM (342-05731, Dojindo, Kumamoto, Japan) at a concentration of 20 /tM in serum-free Dulbecco's Modified Eagle's Medium buffered by 20 mM-HEPES at 23 °C for 1 h. Observation was carried out by an Olympus objective lens UVFL x 20 attached to an inverted microscope (IMT-2, Olympus, Tokyo, Japan), with illumination by a 100 W mercury lamp. Fluorescence was excited and observed through a dichroic mirror unit for excitation by blue light (IMT2-DMB, Olympus). The images before and during the ATP response were acquired by CCD camera (WV-CD50, National, Tokyo, Japan) and recorded on videotape. After the experiment, the data were replayed and paused on a CRT video-monitor, and photos were taken with a fixed exposure time. Using a microcomputer equipped with a frame grabber (C2000, Hamamatsu Photonics, Hamamatsu, Japan), thirty continual images (1 s) were summated and the brightness of areas including responding cells was calculated. The values were obtained every 1-17 s. RESULTS

Membrane currents induced by ATP application to C3H1OT1/2 cells ATP was puff applied to C3HlOT1/2 cells under voltage clamp. After 3-10 s latency, outward current lasting for several tens of seconds was observed at the holding potential of -20 mV, in 91 % of cells (90/99). An example is shown in Fig. 1A. The short bar shows the timing of the puff application of ATP. In this case, the latency is about 3 s (at +20 mV). The response was outward even at -30 mV. After repetitive application of ATP, the response desensitized, and responses at potentials more hyperpolarized than -60 mV were not observed. Another example is shown in Fig. lB. In this case, the latency is about 8 s (at -20 mV). The response was outward at -40 mV, but reversed to an inward response at -100 mV. The reversal potential was estimated to be around -80 mV. Under current clamp condition, the resting membrane potential (-5 mV) hyperpolarized to -70 mV (Fig. 1 C). The hyperpolarized potential varied from -78 to -45 mV (n = 10), probably depending on the contamination of leak conductance. These results suggest that this current may be carried by K+.

ATP-INDUCED CURRENTS IN C3HlOTl/2 CELLS

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As the amplitude of the ATP-induced current had a tendency to decrease after several puff applications of ATP, a correct I-V relation for this current could not be obtained simply by plotting the peak amplitude at various holding potentials. To ascertain an accurate I-V relation, slow ramp potential commands were applied

A

B 50 pA

+20 mV 1_0 sl -20 mV

v

0

-60

-10 -20

-100 w

-30 ~

~

~

C

i

25 mV

10 s Fig. 1. ATP-induced K+ currents under voltage clamp at various holding potentials shown to the left of the traces (A and B). A and B were obtained from different cells; 10 sM- (A) and 20 /SM- (B) ATP were puff applied. The small bars near the traces show the timing of puff applications for 1 s. C, ATP-induced hyperpolarization under current clamp; 20 /SMATP was puff applied.

during the steady peak of the ATP response (Fig. 2B and F) and in the absence (Fig. 2A and E) of ATP. Records were taken with a holding potential of -20 mV, and ramps from - 120 to +80 mV, and back to - 120 mV, were applied at a rate of 01 mV/ms. The membrane currents thus obtained in the presence and absence of ATP (Fig. 2 C and G) and the difference currents (Fig. 2D and H) were plotted as a function of membrane potential. In standard external solution containing 5 mM-K+ (Fig. 2A, B, C and D), the I-V relation of ATP response (Fig. 2D) reversed at around -80 mV, suggesting that the current is carried by K+. The I-V relation showed a slight outward rectification. In external solution containing 50 mM-K+ (Fig. 2E, F, G and H), the I-V relation reversed at around -30 mV (Fig. 2H), with less obvious outward rectification. These results show that the ATP-induced current is carried by K+. In 10 % of C3HlOTL/2 cells (9/99), the ATP-induced response (Fig. 3A) displayed a different I-V relation (Fig. 3D), almost linear in standard external solution, with

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a reversal potential around 0 mV. Judging from the reversal potential, there were two main possibilities for the selectivity of the ionic channels underlying this current: (1) non-specific cation selective, or (2) Cl- selective. To clarify this point, the external solution was changed. When external Na+ was replaced with TMA, the

E A

50 mM-K+

Normal A

250 pA

B

F

5s

A

.pA G

-

40

40

mV I

H D I

Al

Fig. 2. Analysis of the I-V relation of the ATP-induced K+ response in external solution containing 5 mM-K+ (A, B, C and D) and 50 mM-K+ (E, F, G and H). Cells were voltage clamped at -20 mV and ramp pulse stimulation of + 100 mV (0-1 mV/ms) was applied to the cells before (A and E) and at the steady peak of the ATP responses (B and F). The current during the rising phases of the ramp stimulation was plotted as a function of voltage in C (for A and B) and in G (for E and F). The difference currents before and during the responses are shown as an I-V plot in D (for A and B) and in H (for C and D). ATP was puff applied at 20 /IM.

response at -20 mV was almost unchanged from that in standard external solution (Fig. 3B). When external C1- was replaced with glutamate, the response at + 20 mV became inward (Fig. 3C). These data suggest that the ATP-induced current which reversed at 0 mV is carried by Cl-. A similar experiment to that in Fig. 2 was carried

ATP-INDUCED CURRENTS IN C3HlOT1/2 CELLS A

E

\

+20 mV

2.5 nA

697

A.

0.5

0 -20

-/

2.5

F

500 pA 10 s

-80

5.0 10 s

B -20

Na+ TMA - L

Cl-

c

2.5

glutamate

'

+20

2*5

D H

-80 mV

. 0

4.

.I

0

Fig. 3. ATP-induced C1- currents under voltage clamp at various holding potentials shown to the left of the traces (A) and their I-V relation (D). B, the response at -20 mV when external Na+ was changed to TMA; C, the response at +20 mV when external C1was changed to glutamate. The current values are plotted as a function of voltage in D. 0, normal; *, TMA; *, glutamate. E, F, G and H, analysis of the I-V relation of the ATPinduced Cl- response. Cells were voltage clamped at -20 mV and ramp pulse stimulations of +100 mV (01 mV/ms) were applied to the cells before (E) and during (F) the responses. The rising phases of the ramp stimulation were plotted as an I-V relation in G and the difference current was plotted in H. Puff-applied ATP was 1 mm (A, B and C) and 20 uM (E and F).

out on the C1- response (Fig. 3E, F, G and H), which confirmed that the conductance had little voltage dependence (Fig. 3H). In contrast to the ATP-induced K+ response, this Cl- response did not show marked desensitization, and in some cases responses were observed more than fifteen times. Thus, it is possible that the receptors or internal messengers for these two responses are different.

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Both ATP-induced K+ and Cl- currents were observed to co-exist in 6 % of cells (5/99) (Fig. 4). The traces at -10 mV (Fig. 4B) and -20 mV (Fig. 4C) were clearly composed of two components: an outward component carried by K+ and an inward component carried by Cl-. At -60 mV (Fig. 4D), the tiny outward component is lost A

250 pA 0 mV B 125

-10

C

-20

250

U

D -60

500 20 s

Fig. 4. ATP-induced K+ and C1- currents observed in a cell at various potentials shown to the left of the traces. Puff-applied ATP was 1 mm.

in the large inward Cl- component. At 0 mV (Fig. 4A), the Cl- component is expected to be very small, and an outward current carried by K+ is observed to predominate. As cells which showed only ATP-induced Cl- current (4/99), or both K+ and Clcurrents (5/99), were rare, only the properties of the ATP-induced slow K+ current were investigated in further detail. To distinguish the K+ current from the rare ATPinduced Cl- current, responses were observed routinely at -20 mV. At this potential, K+ current is large and outward, and is clearly distinguishable from an inward Cl- current. Furthermore, responses at -80 mV were also routinely examined to make sure that ATP-induced inward Cl- current did not exist in the cell.

Dose response of ATP-induced K+ current Although the dose dependence of short-latency ATP-induced currents has been reported (Friel & Bean, 1988; Bean, 1990), that of long-latency ATP-induced K+ currents is not well studied. Thus, the ATP dose dependence of the slow K+ current

ATP-INDUCED CURRENTS IN C3HJOTJ/2 CELLS

699

20 gM 20

s

200

A

pUM

200 pA

2

B

400

N

C

200

D 100

E

0.02

2

50

F 1.0

I

a)

cnCl) CoQ) 0

0.5

a)

cc

0 0.02

2

200

ATP concentration (pM)

Fig. 5. The relationship between the dose of ATP and induced K+ current. Each of the two traces was obtained from the same cell (A, B, C, D and E). The responses induced by control ATP (20 M) (left traces) and test ATP (right traces) are shown. The concentrations of test ATP, ranging from 200 to 0-02 /M, are shown near the traces. Responses to control ATP were normalized to the same size. The holding potential was 0 mV. The mean values of the test/control responses at each test concentration are plotted in F. The standard deviation of each point is shown by a bar in F. The number of data at each test concentration are as follows. n = 3 at 200 #M; n = 7 at 2/M; n = 7 at 0-6 uM; n = 9 at 0-2 ,M; n = 3 at 0-02 1SM. The Kd and Hill coefficient of the best-fit curve were 0-412 ,M and 1-765, respectively.

was investigated. Responses were measured at various ATP concentrations and the amplitude of response relative to that observed in the same cell to ATP at a control concentration was calculated. As preliminary studies showed that 20 /,M-ATP causes

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a full response, this concentration was used as a control. As described in the Methods, control ATP solution and test ATP solution were loaded in pipettes separated by more than 400 ,sm. They were advanced to the cell in turn and the ATP of each pipette was puff applied. Only when the control response size before and after the application of the test solution did not change were the results adopted. Examples of responses to 200, 2, 0-6, 0-2 and 0-02 ,uM-ATP (right) and control 20 ,tM-ATP (left) are shown in Fig. 5A, B, C, D and E, respectively. The relative amplitudes were measured at the peak, and their means at each concentrations were plotted in Fig. 5F. Their standard deviations were also shown by bars. These points could be fitted with a dose-response curve with a Hill coefficient of 1-76 and a Kd of 0-412 /M. This Kd value is very low in comparison with the ATP response in other systems. The Kd of the short-latency ATP-induced currents in bull-frog atrial cells and in sensory neurones were reported to be 56 /tM (Friel & Bean, 1988) and 2-7 /M (Bean, 1990) respectively. Okada et al. (1984) reported that the hyperpolarizing responses in L fibroblastic cells, whose time course is similar to the K+ current in this report, could be induced only by an ATP concentration of more than 200 ftM. Thus, the 'machinery' that gives rise to ATP-induced K+ current in C2HlOTL/2 cells is very sensitive to external ATP. However, this does not necessarily imply a high sensitivity of the ATP receptors, because the value assayed is not ATP bound to the receptor, but a final output current. Similarly, the Hill coefficient may not necessarily reflect the properties of the receptor itself, but could, for example, result from positive feedback in the second or third messenger release system following receptor activation. In the following experiment, ATP was used at a concentration of 20 /m, since this was shown to be sufficient for a full response.

The effects of ATP analogues ATP responses have been categorized pharmacologically into two types, P1 and P2 (Burnstock, 1978). To ascertain the type of ATP receptors in C3H10T1/2 cells, several analogues of ATP were applied. All drugs were tested at a concentration of 20 /M, which gave a full response in the case of ATP. ADP gave rise to a clear slow K+ current (n = 5), although it was less potent than ATP (Fig. 6A). In contrast, AMP (n = 7) and adenosine (n = 6) never caused slow K+ current (Fig. 6B and C). These data suggest that the ATP-induced slow K+ current is induced by the activation of P2 receptors. Consistent with this, ATP-y-S (n = 2), which is an agonist of P2 receptors, also caused slow K+ current (Fig. 6D). a,,f-Methylene ATP, which is known to be more potent than ATP at P2x receptors (Burnstock & Kennedy, 1985), gave only a small response compared to ATP (Fig. 6E). The effect was investigated in six cells, and a clear slow K+ current was observed in only one cell. This result implies that the P2 receptors involved in this response are of the P2Y subtype rather than P2X. Increase in intracellular Ca2+ concentration upon ATP application The long latency of the ATP-induced K+ current, ranging from 3 s (Fig. 1 A) to 10 s (Fig. 5D), suggests that the ionic channels activated are not receptor coupled but are activated by second messengers. With the hypothesis that this K+ channel is

ATP-INDUCED CURRENTS IN C3HlOTl/2 CELLS

701

activated by Ca2" released from internal stores via some kind of second messengers, the EGTA concentration in the internal solution was raised to 5 mm to see if the ATP-induced response was blocked. The usual internal solution used contained EGTA at only 01 mm, so that the Ca2+ concentration could fluctuate easily with stimulation (Kubo & Kidokoro, 1989). ATP

ADP 200 pA

A -

--.

10 s

AMP B

Adenosine

ATP-y-S

D

a,j3-Methylene ATP E-

Fig. 6. K+ responses to ATP analogues. Each of the two traces was obtained from the same cell (A, B, C, D and E). The responses induced by ATP (left traces) and ATP analogues (right traces) are shown. The names of the analogues are shown near the traces. Each drug was applied at 20 uM, and the holding potentials were 0 mV.

The data in Fig. 7 are successive recordings from ten cells in the same dish. Internal solutions with 01 mm- (A, C, E, G, I) or 5 mm- (B, D, F, H, J) EGTA were used alternately. It is clear that the ATP-induced K+ currents observed in all cells with 041 mM-EGTA internal solution did not appear in cells with 5 mM-EGTA. In D, although a tiny response was elicited, it appeared to be blocked during the course of the rising phase. This result suggests that intracellular Ca2+ plays an important role in the ATP-induced K+ current.

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As a next step, changes in the intracellular Ca2+ concentration before and during the ATP response were measured. As described in the Methods, C310T1/2 cells were loaded with Fluo-3 AM, a membrane-permeable fluorescent indicator of Ca21 concentration (Kao, Harootunian & Tsien, 1989). The images were recorded by CCD 5 mM-EGTA

0.1 mM-EGTA

A

B

/

C

0 l_ D

F

f

E

G

J

H

J

200 pA 20 s

Fig. 7. The effect of intracellular EGTA on the ATP-induced K+ current. A-J are successive recordings from ten different cells in the same dish. Internal solutions with 0 1 mM- (A, C, E, G, I) and 5 mm- (B, D, F, H, J) EGTA were used alternately. Puff-applied ATP was 20 /zM, and holding potentials were 0 mV.

camera, stored on videotape, and analysed after experiments. Photographs before and after 3, 6, 9, 12, 15, 25, 35, 45 and 55 s of ATP application in normal external solution are shown in Fig. 8A a-j. The brightness of the area including the cell in Fig. 8A was calculated by summating thirty consecutive images (for 1 s), and plotted in Fig. 9A. The background brightness from a region without a cell was subtracted, and responses normalized to the peak value. A clear increase of the signal was observed upon application of ATP with a latency of about 3 s (Figs 8A c and 9A). In twentyfive of thirty-two cells investigated, an increase with similar time course to Fig. 8A was observed by monitoring the real time images under a microscope and the recorded images on a CRT. As the time course of the rising phase of intracellular Ca2+ concentration was similar to the electrophysiologically recorded ATP-induced K+

ATP-INDUCED CURRENTS IN C3HlOTl/2 CELLS A

703

B Normal

0

a

f

b

g

b

Ca2+

g

h

d

i

d

I

e

I

Fig. 8. The increase of intracellular Ca2+ monitored by Fluo-3 AM when 20 /LM-ATP was puff applied in normal (2 mM-Ca2+) (A) and in Ca2+-free normal (B) external solutions. All photographs are CCD images on the monitor TV. Cells observed under Nomarski optics are shown at the top of A and B. Photographs are before (a) and after 3 (b), 6 (c), 9 (d), 12 (e), 15 (f), 25 (g), 35 (h), 45 (i) and 55 s (j) of 20 IuM-ATP application. Scale bar is 20 /sm.

current, the K+ current was considered to be activated by intracellular Ca2+. The decay phase of intracellular Ca21 (Fig. 9A) was slower than that of ATP-induced K+ current. The reason for this is possibly the absence of intracellular EGTA which was used (0'1 mM) in the recording of ATP-induced K+ current.

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There are two candidates for the source of the increased Ca2+: release from intracellular Ca2+ stores or influx of extracellular Ca2+. To determine which is important in the present case, a similar experiment to measure intracellular Ca2+ was carried out in Ca2+-free external solution. Even in Ca2+-free external solution where A

Normal 1.0-1

0@*

*

* I.

0.

_-.

_

, ,,,,, 0~~~~~5~~ 0.0.v

0.5

a

B

*0

0

b c d e f

, g

. i

, h

j

s

o Ca2+

1.0

-

0*

s 0

* 000

*

.

_ ~~~~~~* ."00 ...".

___~~~~~~~~~~10*004t

n.s v

;.p

..

I

.

g h i i Fig. 9. Quantitative measurement of the intracellular Ca2+ increase monitored by Fluo3 AM when 20 /M-ATP was puff applied in normal (2 mM-Ca2+) (A) and in Ca2+-free normal (B) external solutions. A and B were obtained from the same responses as in Fig. 8A and B, respectively. The brightness of the area including the cell in Fig. 8 was calculated by summating thirty images (for 1 s), and plotted as a function of time. The background brightness where there was no cell observed was subtracted, and values normalized to a peak of 10. Small bars near the plots show the timing of 1 s puff applications of ATP. Small characters (a-j) on the time axis indicate the times when the photographs of Fig. 8 were taken. a b c de f

Ca21 was substituted with Mg2+, an increase in intracellular Ca2+ upon ATP application was clearly observed (Fig. 8B and 9B). In fifteen of nineteen cells investigated, an increase with similar time course to Fig. 8B was observed by monitoring the real time images under a microscope and the recorded images on a CRT. Furthermore, ATP-induced K+ currents of similar amplitudes were recorded in a cell in normal and in Ca2+-free external solution (Fig. IOA). Thus, the main source of the increased Ca2+ during the response appears to be not influx from the extracellular solution, but release from internal Ca2+ stores. This result suggests that the mechanism of the ATP-induced K+ current is as follows. Puff-applied ATP activates the receptor, and causes Ca2+ release from internal Ca2+ stores, probably via some second messenger system. The released Ca2+ activates the Ca2+-induced K+ channels. The extracellular Ca2+ might have an important role in replenishing the internal Ca2+ stores as observed in rat chromaffin cells (Kubo & Kidokoro, 1989).

ATP-INDUCED CURRENTS IN C3HlOT1/2 CELLS 705 As a next step, the effects of ryanodine, which is known to deplete internal Ca2" stores, and caffeine, which is known to cause Ca2+ release from internal Ca2+ stores, were tested (Fig. lOB and C). Surprisingly, even when cells were incubated in 50 ,UMryanodine, the ATP response was observed (Fig. lOB) (3/3). Furthermore, 1 mMA

0, Ca2ts

Normal

B

50OgM-ryanodine

100 pA C

10 mM-caffeine

10 s

Fig. 10. The ATP-induced K+ response in normal (left) and in Ca2+-free external solution (right) of one cell (A). The ATP-induced K+ response in a cell incubated in 50 /Mryanodine solution is shown in B. The external solution also contained 50 ,uM-ryanodine at the time of recording. In C, 10 mM-caffeine was applied in the external solution at the beginning of the trace. The holding potential in A, B and C was 0 mV. Puff-applied ATP in A and B was 20 uM.

caffeine applied to the external solution did not cause any membrane currents (Fig. lOC) (0/3). The upward deflection at the beginning of the trace is considered to be due to the capacitative disturbance of the quick application of caffeine solution to the bath by syringe. Thus, the internal Ca2+ stores, which are apparently involved in the ATP response, were shown to be unaffected by ryanodine and caffeine.

Properties of the K+ channel underlying ATP-induced K+ current To ascertain the properties of the K+ channel, steady-state fluctuation analysis of the ATP-induced K+ current was carried out. Cells whose ATP response was rather long lasting, with only slow decline, were selected for this analysis. The current was sampled at 20 kHz for 800 ms, discontinuously with 1200 ms intervals between periods of sampling, before and during the response. Seven example 800 ms segments of noise are shown in Fig. I1 B, and a typical response of the same cell is shown in Fig. 1 1A. From Fig. 11 A and B, it is clear that the declining phase is rather slow, and some parts can be considered to be stationary over 400 ms. These steady 400 ms domains at various current levels of the records were used for analysis. 23

PHY 442

Y. KUBO

706

The mean current and variance after subtraction of baseline levels were plotted as in Fig. 11 C, and the relationship fitted as described in the Methods. From the bestfitted curve, the single-channel current and total number of channels were estimated to be 0X21 pA and n = 2100, respectively. As the K+ equilibrium potential with 5 mMA 200 pA 10 s

C

B

20 _

_

0

Cu

~~~~>U10_0

= 100 pA

200 100 Mean current (pA) Fig. 11. Steady-state fluctuation analysis of the ATP-induced K+ current. 20 ,pM-ATP was puff applied at 0 mV, and sampled continuously for 40 s at 50 Hz (A) and discontinuously for 32 s at 20 kHz (B). A and B are different responses of the same cell. The filters used were 8-pole Bessel at 1 kHz (A) and at 3 kHz (B). Steady regions of the traces at various levels in B were used for fluctuation analysis. The baseline current and variance subtracted before plotting in C were 5 9 pA and 241 pA2, respectively. The relationship of current and variance was fitted as described in the Methods.

400 ms

0

internal and 140 mM-external K+ is - 83-9 mV, the estimated single-channel chord conductance was 2-4 pS. The value was almost constant in the six cells investigated (mean = 2-7 pS, standard deviation = 015 pS). The Ca2+-induced K+ channels involved in the ATP response in this cell are therefore much smaller than the several types of Ca2+-induced K+ channels previously reported (Blatz & Magleby, 1987). The effects of the K+ channel blockers TEA and apamin on this channel were studied. Although the ATP-induced response was not clearly affected when 5 mMTEA was added to the external solution, it was reduced to about one-third by replacing Na+ in the external solution with TEA (140 mM) (Fig. 12A). This decrease was not due to desensitization, because the response fully recovered when the bath was replaced with normal solution again (Fig. 12A, bottom). In contrast, apamin up to 1000 nmin the external solution did not affect the ATP response (Fig. 12B). The prolonged latency of the trace with 1000 nM-apamin is more likely to be due to desensitization by repetitive puff application of ATP, and not to an effect of apamin.

ATP-INDUCED CURRENTS IN C3HlOT1/2 CELLS A

707

B

Normal

Normal

TEA

100 nM-apamin

Normal 500 nM-apamin

1000 nM-apamin

200 pA 100 pA 10 s

Fig. 12. The effect of K+ channel blockers TEA (A) and apamin (B) on the K+ current induced by application of 20 1uM-ATP. The holding potential was 0 mV. A, all three traces were obtained from one cell. In the middle trace, all Na+ (140 mM) in the external solution was replaced with TEA (140 mM). B, all four traces were obtained from one cell. The prolonged latency of the fourth trace might be due to desensitization, which was often observed when ATP was applied repetitively.

DISCUSSION

ATP was puff applied to cells of a mesodermal stem cell line, C2HlOT1/2, and the responses were studied by the whole-cell variation of the patch clamp technique. The mechanism of the ATP-induced response From the results shown above, the mechanism of the ATP-induced K+ current in C3HlOT1/2 cells is believed to be as follows. (1) Extracellular ATP binds to P2Y receptors. (2) Activated P2y receptors induce Ca2+ release from intracellular Ca2+ stores, which are insensitive to caffeine and ryanodine, probably via some kind of second messenger, and cause an increase in the intracellular Ca2+ concentration. (3) The increased Ca2+ activates 2-7 pS Ca2+-induced K+ channels, which are blocked by TEA but not by apamin. 23-2

Y. KUBO It was not investigated -whether the ATP-induced C1- current, observed in some of these cells, is also activated via Ca2+. As the Cl- response showed only weak desensitization, in contrast to the K+ response which exhibited much stronger desensitization, it is possible that the intracellular mechanisms of these two responses are different. 708

The physiological significance of the ATP-induced respone In this report, it was found that C3H1OT1/2 mesodermal stem cells show ATPinduced K+ current with several seconds latency. The most characteristic property of this response was that it was mediated by Ca2' released from internal stores, as revealed by monitoring the intracellular Ca21 concentration. This fact means that ATP gives rise to two effects in C3HlOT1/2 cells: hyperpolarization of the membrane potential and an increase in the intracellular Ca2+ concentration. Although ATP responses have been reported in many preparations such as neurones and in various cells of the mesoderm, release of Ca2+ from internal stores elicited by ATP has only been reported in some preparations, such as cochlear hair cells (Shigemoto & Ohmori, 1990) and adrenal chromaffin cells (Sasakawa et al. 1989). It is possible that transient increase in intracellular Ca2+ play important roles at the turning points of various cells in differentiation by activating various kinds of intracellular processes. The ATP response found in C3H1OT1/2 cells is also expected to play an important role in the later mesodermal differentiation, by increasing the intracellular Ca2+ concentration. The ATP response was also observed even in cells of C3H1OTL/2-derived myogenic subelones and of C2C12 myoblasts which are at the turning point for muscle differentiation, and lost as muscle differentiation proceeds (Kubo, 1991 a, b). It is an important problem whether or not the ATP response really occurs in mesodermal stem cells in vivo, because innervation by purinergic neurones to mesodermal stem cells is not likely, and thus there would be no marked local increases in ATP concentration around mesodermal stem cells. From the doseresponse study, the Kd value of the ATP response was found to be as low as 0 4 #M. The very low Kd value allows the possibility that mesodermal stem cells show responses even to very diluted ATP in the extracellular space. Thus, it is possible that increases in the intracellular Ca2+ concentration of mesodermal stem cells produced by ATP actually occur in vivo. The author would like to thank Dr H. Koike for his support and constant encouragement throughout this research, and Professor K. Takahashi for his invaluable comments on the manuscript. The author would also like to thank Drs M. Shidara and Y. Okamura for their help in computer programming and Dr H. Robinson for improving the English. The mesodermal stem cell line C3H1OT1/2 was supplied by the Japanese Cancer Research Resources Bank (JCRB). This research was supported by grants in aid for scientific research from the Ministry of Education, Science and Culture of Japan. REFERENCES

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