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Journal of Physiology (1991), 440, pp. 623-634 With 7 figures Printed in Great Britain
ATP-INDUCED Ca2+ RELEASE AND Cl- CURRENT IN CULTURED SMOOTH MUSCLE CELLS FROM PIG AORTA
BY G. DROOGMANS, G. CALLEWAERT, I. DECLERCK AND R. CASTEELS From the Laboratorium voor Fysiologie, KU Leuven, B-3000 Leuven, Belgium
(Received 26 October 1990) SUMMARY
1. The effect of exogenous ATP on transmembrane currents and on the cytoplasmic Ca2+ has been investigated in single cultured smooth muscle cells of pig aorta. 2. ATP applied to cells held at a potential of -50 mV evoked a transient inward current and a transient rise in [Ca2+]i. At a potential of + 20 mV the ATP-induced increase in [Ca2+]i was accompanied by an outward current. 3. At a potential of -50 mV, ATP evoked in Ca2+-free solution an inward current which was similar to that in the presence of external Ca2". A second application of ATP in Ca2+-free solution induced a much smaller current. 4. ATP induced in Ca2+-free solution a pronounced transient stimulation of the 45Ca2+ efflux from confluent smooth muscle monolayers. 5. The I-V curve of the ATP-activated current has a reversal potential close to 0 mV. A reduction of external Cl- shifts this reversal potential in accordance with the change of the Cl- equilibrium potential. 6. It is concluded that ATP causes a release of calcium from intracellular stores. The ensuing increase of [Ca2+]i activates a Cl- current, which can depolarize the cell membrane and thereby promote a voltage-gated Ca2+ entry. INTRODUCTION
ATP is released as an excitatory co-transmitter together with noradrenaline at sympathetic nerve terminals present in smooth muscle tissues. Using the patchclamp technique it has been demonstrated in a variety of smooth muscle cells that application of exogenous ATP activates a cation-selective channel which shows little discrimination between various monovalent cations (Benham, Bolton, Byrne & Large, 1987; Friel, 1988; Declerck, Droogmans & Casteels, 1989; Honore, Martin, Mironneau & Mironneau, 1989) and which is directly gated by ATP (Benham & Tsien, 1987; Nakazawa & Matsuki, 1987). In addition, this ATP-activated channel is permeable to divalent cations in rabbit ear artery (Benham et al. 1987; Declerck et al. 1989), and its activation increases the free intracellular calcium concentration ([Ca21]i) in cells which are exposed to normal physiological solutions (Benham, 1989b). It has also been demonstrated that in some smooth muscle cells ATP is able to release Ca2+ from inositol trisphosphate (1P3)-sensitive stores through activation of MS 8887
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G. DROOGMANS AND OTHERS
P2 purinergic receptors (Phaneuf, Berta, Casanova & Cavadore, 1987; Tawada, Furukawa & Shigekawa, 1987). This ATP-induced release of calcium has only been observed in cultured smooth muscle cells and also in a number of non-excitable cells. To our knowledge, the effect of ATP on membrane currents has not been measured in cultured smooth muscle cells. We have therefore investigated the effect of exogenous ATP on membrane currents and on [Ca21]i in primary cultured smooth muscle cells of pig aorta. METHODS
Cells Vascular smooth muscle cells from thoracic pig aorta, which were obtained from the local slaughterhouse, were cultured as described by Ross (1971), subcultured and used between the 5th and 12th passage. Solutions Cells were bathed in a modified Krebs solution containing (in mM): 1324 NaCl; 15 CaCl2; 5-9 KCl; 1-2 MgCl2; 11-5 HEPES-NaOH; 10 glucose; pH 7-3. Current and [Ca2+]i measurements Experiments were carried out on single cells 1 to 2 days after plating on cover-slips. Currents and [Ca2+]i were measured at room temperature (21-23 °C). External solutions were rapidly exchanged using a multibarrelled pipette with a common opening (Konnerth, Lux & Morad, 1987). The pipette (internal) solution was made up in Milli-Q water (Millipore) and contained (in mM): 120 CsCl; 5 NaCl; 1 MgCI2; 20 HEPES; 0-1 K5jndo-1 or 0-1 EGTA buffered at pH 7-2 with CsOH. Membrane currents were recorded with the standard patch-clamp technique (Hamill, Marty, Neher, Sakmann & Sigworth, 1981) using a List EPC-7 patch-clamp amplifier. Membrane currents were filtered with an 8-pole Bessel filter, digitized on line at sample intervals ranging between 1 and 2 ms and analysed by computer. [Ca2+]i was estimated from Indo-1 fluorescence by the ratio method using single-wavelength excitation and dual-wavelength emission (Grynkiewicz, Poenie & Tsien, 1985). Experiments were carried out on a Nikon Diaphot inverted microscope equipped for epifluorescence. The fluorescence probe was excited at 360 nm with a 75 W xenon arc lamp and fluorescence emitted from the cell was split by a dichroic mirror centred at 450 nm and detected by two parallel photomultipliers (Hamamatsu type R928) at 405 and 485 nm. Single cells were loaded with Indo-1 pentapotassium salt (100 /tM) by diffusion from the patch pipette. Loading of the dye was assessed by measuring changes in fluorescence intensity at 485 nm. Using pipette resistances of 2-3 MQ2, loading was complete within 5 min after formation of a whole-cell patch. Background fluorescence due to cell autofluorescence and Indo- 1 in the pipette was measured in cell-attached mode and subtracted from each record in whole-cell mode. The ratio of emitted fluorescence at 405 nm and at 485 nm (R) was then used to calculate [Ca2+] according to the relationship
[Ca2+]i = KdJ Rmax
min
where Rmin and Rmax are the fluorescence ratios obtained in the absence of Ca2+ and at saturating Ca2+ levels, Kd is the apparent dissociation constant for Indo- 1 for which a value of 213 nm was used (Benham, 1989 a), and ,? is the fluorescence ratio of the 485 nm signal in the absence of Ca2+ to that in the presence of saturating Ca2+ (Grynkiewicz et al. 1985). Rmax and Rmin were determined in vitro by placing a small drop of internal solution containing 2 mM-CaCl2 or 10 mM-EGTA between two cover-slips. No corrections were made for altered properties of Indo- 1 when loaded into the cytoplasm (Benham, 1989a).
45Ca2+ flux measurements Flux experiments were carried out on confluent monolayers of cells 4-5 days after plating in twelve well clusters (3-8 cm diameter). The cell density at the time of the experiments was 3 x 105
ATP-INDUCED Ca2+ RELEASE IN SMOOTH MUSCLE
625
cells well-'. The wells were fixed on a thermostated plate at 37 °C, which was placed on a mechanical shaker. After aspiration of the culture medium, the cells were washed twice with standard physiological solution. Cells were loaded with 45Ca2+ in a 0-2 mM-45Ca2+-containing solution (% 30 ,uCi ml-'). After 5 min the 45Ca2+ uptake was stopped by aspirating the radioactive medium and by washing the cells 4 times with an ice-cold Ca2+-free, 2 mM-EGTA-containing solution. One millilitre of Ca2+-free, 2 mM-EGTA-containing solution was added to the wells, and replaced every 2 min over a period of 30 min. At the end of this period the cells were solubilized in 2 % SDS (sodium dodecyl sulphate). The 45Ca2+ present in the efflux samples and the radioactivity remaining in the cells were determined in a liquid scintillation counter. The total activity in the cells at the beginning of each sampling period was obtained by adding the activity of the efflux samples in retrograde order to the activity in the cells at the end of the experiment. The tracer content of each efflux sample, divided by the total activity in the cells and by the duration of the sampling interval (2 min), gives the fractional 46Ca2+ loss (expressed in min-'). This quantity is represented as a function of efflux time. RESULTS
Effect of ATP on [Ca2+]i and on membrane current Figure 1 shows the effect of application of exogenous ATP (50#M) to a single smooth muscle cell held at a potential of -50 mV and which was dialysed with the Ca2+ indicator Indo-1 (100 /tM). After a latency of about 1 s ATP triggers a transient rise in [Ca2+]i from its resting value of about 85 nm to a peak value of 425 nm. This peak value is reached about 1 s after the onset of the response (lower trace). This transient increase in [Ca2+]i was accompanied by a transient inward current reaching a peak value of about 1P5 nA at the time when [Ca2+]i was maximal (upper trace). [Ca2+]i transients as shown in Fig. 1 were observed in all cells, and ranged from 215 to 580 nM, with a mean value of 390 + 45 nm (mean + standard error of the mean; n = 7 cells). The peak amplitude of the current varied from cell to cell, and ranged from 0-6 to 2-2 nA, with a mean value of 1-32 + 0-14 nA (n = 15 cells). After this transient increase of [Ca2+]i this parameter stabilized to a value near the resting [Ca2+]i. Whereas the increase of [Ca2+]i upon application of ATP was monophasic in all cells, we frequently observed that the rising phase of the concomitant transient current displayed several components. As illustrated in Fig. 2, an initial phase of the current can occur before any changes in [Ca2+]i could be detected. The time course of the ensuing second component is closely correlated with the changes in [Ca2+]i. It thus appears as if ATP exerts a dual action on the membrane current, i.e. an initial effect which is not related to changes in [Ca2+]i, and a second one which is clearly associated with changes in [Ca2+]i. The initial current component, which precedes the Ca2+ transient, was highly variable and did not occur in all cells. The ATP-activated Ca2+ transient, however, was present in all cells irrespective of the presence or absence of the initial current component. It is therefore unlikely that this initial ATP-activated current component causes an increase in [Ca2+]i, which then results in a subsequent activation of a Ca2+-dependent conductance. The long latency between the onset of this current, which occurs immediately after the application of ATP, and the increase in [Ca2+]i suggests an ATP-induced release of calcium rather than an ATP-activated
Ca2+-influx.
G. DROOGMANS AND OTHERS
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The increase in [Ca2+]i is not produced by the transient inward current The similarity of the time course of [Ca2+]i and the current evoked by ATP could be explained either by an inward Ca2+ current which causes a rise of [Ca2+]i, or by an increase of [Ca2+]i which itself activates an inward current. If the first hypothesis 50 /iM-ATP
0.5 nAJ
400
F
Ca2+ 200
(nM)
n v
L
2s 1. Effect of on Fig. 50,uM-ATP membrane current (in nA, top trace) and on [Ca2+]i (expressed in nm, bottom trace). The addition of ATP is indicated by the arrow. ATP was present for the rest of the record. The cell was held at a potential of -50 mV.
were true, then the rise in intracellular calcium would strongly depend on the transmembrane Ca2+ gradient. We have therefore applied ATP to cells which were held at various potentials. Figure 3 shows the results of such an experiment in which the effects of ATP were compared at two different holding potentials, i.e. at -50 mV (left panel) and at + 20 mV (right panel). At both potentials, application of ATP gives rise to a transient increase in [Ca21]i. At -50 mV this change in [Ca2+]i is accompanied by an inward current. The changes in [Ca2+]i at +20 mV are comparable to those at -50 mV, but they are accompanied by an outward current. Also the initial component of the ATP-activated current, which occurs before any change in [Ca2+]i, is reversed at + 20 mV.
ATP-INDUCED Ca2+ RELEASE IN SMOOTH MUSCLE
627
The ATP-evoked current does not depend on the presence of extracellular Ca2+ Although an outward current cannot directly result in an increase of [Ca2+]i, it cannot be excluded that this outward current masks an inward Ca2+ current which contributes to the increase in [Ca2+]i either directly or via a Ca2+-induced Ca2+ release mechanism. 50
,uM-ATP ~A
0.2 nA
400 L
Ca2+ 200 k
(nM)
nI
500 ms
Fig. 2. Effect of 50 /aM-ATP on the membrane current (in nA, top) and [Ca2"], (in nm, bottom). This recording shows on an expanded time scale the initial component of the current evoked by ATP. It is obvious that the initial current component is not associated with changes in [Ca2+]i. The holding potential was -50 mV.
We have therefore investigated the ATP-activated current in a Ca2+-free solution buffered with 2 mM-EGTA. The results of such an experiment are shown in Fig. 4. The top trace shows the current response evoked by ATP at a potential of -50 mV in a bathing solution containing 1.5 mM-Ca2+. The two subsequent responses were obtained in Ca2+-free solution at the same holding potential. The current induced by the application of ATP after about 30 s in Ca2+-free solution (middle trace) is not significantly different from that under control conditions. This observation is consistent with a minor contribution, if any at all, of Ca2+ influx to the ATPactivated current. A second application of ATP during the continued perfusion with Ca2+-free solution induces a much smaller transient inward current (bottom trace).
G. DROOGMANS AND OTHERS
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50 l~
uM-ATP l-
50 pM-ATP
1 nA
300 I 200
Ca2+ (nM)
100o 2s
Fig. 3. Effect of 50 /eM-ATP on membrane current (in nA, top) and [Ca2"], (in nm, bottom) at two different holding potentials. The left panel shows the data at -50 mV, the right panel those at a holding potential of + 20 mV.
50 yM-ATP O nA-
0 nA -
0 nA -
1 nA
5s
Fig. 4 Effect of extracellular Ca2+ on the current evoked by 50 /M-ATP at a holding potential of -50 mV. The upper trace was obtained in a bathing solution containing 1P5 mM-Ca2+. The middle and bottom trace show two consecutive applications of ATP in Ca2+-free solution. Note also the gradual increase of the leak current occurring in Ca2+-free solution.
ATP-INDUCED Ca2+ RELEASE IN SMOOTH MUSCLE
629
This residual current might represent a current component which is directly activated by ATP and is similar to the current component that occurs before any change in [Ca2+]i as described above. It is also possible that this current is activated by release of calcium from internal stores, which are not completely depleted of calcium during the preceding 'brief' exposure to ATP. 0.4
-
50 tM-ATP c
E
0
0O.2-
0
0.0
I
0
10
20
30
Time (min) Fig. 5. Effect of 50 ,tM-ATP on the fractional 45Ca2+ loss (min-') from confluent monolayers of smooth muscle cells exposed to a Ca2+-free solution. Data have been obtained from twelve wells.
Also the leak current gradually increases during exposure to Ca2+-free solution. This resulted in a loss of the seals in all experiments, except in one. Readmitting 1-5 mM-Ca2+ to the bathing solution resulted in a partial recovery of the ATP-evoked current in this cell (data now shown). ATP releases Ca2+ from intracellular stores The decline of the current upon repeated stimulation with ATP in Ca2+-free solution, and the partial recovery upon readmission of external Ca 2+ are consistent with a current that is activated by an ATP-induced release of intracellular calcium. In order to obtain more direct evidence for an ATP-induced release of calcium from intracellular stores, we have studied the effect of ATP on the 45Ca2+ efflux from confluent monolayers of smooth muscle cells. After loading the cells with 45Ca2+, the efflux was performed in a Ca2+-free solution in order to eliminate possible effects of ATP on the influx of Ca2+ (Fig. 5). The initial decline of the fractional 45Ca2+ loss mainly represents the wash-out of extracellular 'racer. After about 10 min of efflux, this fractional loss becomes almost constant, and it is assumed that it represents the 45Ca2+ efflux from the cells. ATP (50juM), applied to the cells after another 10 min of efflux, resulted in a 4-fold
G. DROOGMANS AND OTHERS
630
transient increase of the fractional loss. A second application of ATP did not evoke a second increase in efflux rate (not shown), suggesting that the transient effect is not due to desensitization, but to depletion of an intracellular Ca2+ store. Because the effect of ATP on these cultured cells seems to be different from its action in rabbit ear artery, we have performed a similar experiment on smooth
a O nA1
nAL
b
2s
I (nA)
Difference
~~-ATP
/ t
-50
/
V (mV)
50
-2
Fig. 6. Voltage dependence of the current evoked by 50 ,lM-ATP. The top trace shows the ATP-induced current, and the effect of applying a linear voltage ramp from -50 to + 50 mV before (a) and during (b) application of ATP. The lower panel shows the currents measured during the voltage ramp as a function of the instantaneous voltage. The ATPevoked current was calculated from the difference current between the control response and that measured in the presence of ATP. The reversal potential of the difference current was -7-5 mV in this cell.
muscle strips of the latter tissue. However, ATP did not stimulate the 45Ca2+ efflux in this tissue, whereas a subsequent stimulation with noradrenaline released a significant amount of 45Ca2+ (not shown). This observation confirms the different modes of action of ATP in these two smooth muscle types.
The ATP-activated current is a Cl- current The ionic nature of the ATP-activated current was investigated by applying linear voltage ramps between -50 and + 50 mV to the patch pipette. The difference between the current measured near the peak of the ATP-induced current and that measured in the absence of ATP is defined as the ATP-activated current. Figure 6 (top) shows a typical recording. At 'a' a linear ramp was applied in control conditions, at 'b' the same voltage ramp was applied in the presence of ATP. These currents, as well as the difference current, are represented as a function of the
ATP-INDUCED Ca2+ RELEASE IN SMOOTH MUSCLE
631
instantaneous voltage in the lower panel. The current-voltage relationships are approximately linear, and the difference current reverses at a potential around - 75 mV. The mean value of the reversal potential calculated from six experiments, amounts to -54 + 0-6 mV. This value is close to the equilibrium potential for Cl/ (nA)
1.0
100% Cl0.5
-
50% C-
-50
/50
V (mV)
-1.0
-
Fig. 7. Effect of substitution of 50% of external C1- ions by gluconate on the current-voltage relationship of the ATP-evoked current. The I-V curves of the ATPevoked current were obtained as described in Fig. 6. Substitution of half of the extracellular C1- ions by gluconate significantly shifts the reversal potential to more positive values.
ions (around 0 mV) and is therefore consistent with a current which is mainly carried by Cl- ions. However, these findings cannot exclude a current which would pass through a nonselective cation channel and would have a reversal potential near 0 mV. In order to further identify the ionic nature of the current we have measured the reversal potential of the ATP-evoked current in a solution in which half of the external Clions were replaced by gluconate ions. The I-V relationships of the ATP-activated current in 100 and 50 % extracellular Cl- are shown in Fig. 7. It can be observed that this substitution shifts the reversal potential to more positive potentials from a value of -5-5 mV in 100 % to + 1-2 mV in 50 % Cl-. The magnitude of this shift is close to the value of the shift of the equilibrium potential of Cl- ions by a 50 % reduction of [Cl-]0 (17-5 mV). DISCUSSION
These experimental results demonstrate an ATP-mediated Ca2+ release in primary cultures of pig aorta, which is accompanied by activation of a Ca2+-dependent Clcurrent. This action of ATP is different from that observed in rabbit ear artery cells, where the rise in [Ca2+]i could be associated with the activation of a non-selective
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G. DROOGMANS AND OTHERS
channel, which is also permeable to calcium (Benham, 1989b). A release of 45Ca2+ from internal stores could be clearly demonstrated in these cultured cells, but cation
ATP was unable to induce a release of calcium in rabbit ear artery cells. Calcium mobilization from internal Ca2+ stores by extracellular ATP via activation ofP2 purinergic receptors has been observed in a number of excitable and non-excitable cells (Hallam & Pearson, 1986; Gylfe & Helman, 1987; McMillian, Soltoff, Cantley & Talamo, 1987; Phaneuf et al. 1987; Tawada et al. 1987; Fine, Cole & Davidson, 1989; Pearce, Murphy, Jeremy, Morrow & Dandona, 1989; Sasakawa, Nakaki, Yamamoto & Kato, 1989). It has been shown that in several cell types these receptors are coupled to the activation of inositol phospholipid break-down, and that the generated 1P3 mediates the release of calcium. Such anIP3-induced Ca2+ release has also been observed in our cultured cells after, permeabilization with. saponin (L. Missiaen, unpublished observation). The current activated by ATP was biphasic in some cells: an initial current component, which occurred before any changes in [Ca2+]i, was followed by a second component which was associated with the ATP-induced changes in [Ca2+]i. We cannot completely rule out a contribution of an ATP-activated Ca2+ influx to the observed changes in [Ca2+]i. However, our experimental results indicate that such an influx of calcium is only marginal, and that the release of calcium from internal stores is the main mechanism which leads to the ATP-induced increase of [Ca2+]i. A reduction of the electrochemical Ca2+ gradient by changing the holding potential from -50 to + 20 mV does not significantly affect the ATP-induced change in [Ca2+]i. In addition, the current recorded during the first application of ATP in free solution is not significantly different from that of the preceding stimulation with ATP in the presence of external calcium. The ATP-induced increase of [Ca2+]i is closely correlated with the second component of the ATP-activated current. The finding that this current as well as the 45Ca2+ release disappear upon repeated stimulation with ATP in Ca2+-free solution indicates that there exists a causal relation between the increase in [Ca2+]i and the evoked current. The effect of changes of [Cl-]O on the reversal potential of the ATPactivated current is consistent with the change in Ec, (equilibrium potential for chloride), and supports the proposal that this current is due to activation of Clchannels by a rise in [Ca2+]i. Ca2+-activated Cl- currents could also be induced by caffeine in rat anococcygeus muscle (Byrne & Large, 1987) and in rabbit portal vein (Byrne & Large, 1988). It has also been reported that noradrenaline opens Ca2+activated Cl--selective channels in rabbit portal vein (Byrne & Large, 1988) and in rabbit ear artery (Amedee, Benham, Bolton, Byrne & Large, 1990). Such a Clcurrent will in cells, that are not held under voltage control, depolarize the cell membrane, and thereby cause further influx of Ca2+ through voltage-sensitive Ca2+_ channels. In summary, the mechanism of ATP action on these cultured smooth muscle cells of pig aorta differs from that observed in rabbit ear artery. Its main action is to release calcium in this tissue, and to activate a non-selective cation channel in rabbit ear artery (Benham et al. 1987; Declerck et al. 1988). The initial current component, which occurs before any change in might be due to activation of this nonselective cation channel, but this hypothesis needs further investigation. Although
Ca2+-
[Ca2+]i,
ATP-INDUCED Ca2+ RELEASE IN SMOOTH MUSCLE
633
Ca2+ influx during this initial current component cannot be excluded, its contribution to the observed changes in [Ca21]i is only marginal. It is not clear whether these different actions of ATP represent differences between types of smooth muscle cells, or whether they are due to a differentiation process occurring in the cultured cells. This work has been supported by research grant No. 3.0064.88 of the Fonds voor Wetenschappelijk Geneeskundig Onderzoek (Belgium). We thank Drs H. De Smedt and L. Missiaen for generously supplying us with the cultures of pig aorta cells used in this study. REFERENCES
AMEDE'E, T., BENHAM, C. D., BOLTON, T. B., BYRNE, N. G. & LARGE, W. A. (1990). Potassium, chloride and non-selective cation conductances opened by noradrenaline in rabbit ear artery cells. Journal of Physiology 423, 551-568. BENHAM, C. D. (1989a). Voltage-gated and agonist-mediated rises in intracellular Ca2+ in rat clonal pituitary cells (GH3) held under voltage clamp. Journal of Physiology 415, 143-158. BENHAM, C. D. (1989b). ATP-activated channels gate calcium entry in single smooth muscle cells dissociated from rabbit ear artery. Journal of Physiology 419, 689-701. BENHAM, C. D., BOLTON, T. B., BYRNE, N. G. & LARGE, W. A. (1987). Action of externally applied adenosine triphosphate on single smooth muscle cells dispersed from rabbit ear artery. Journal of Physiology 387, 473-488. BENHAM, C. D. & TSIEN, R. W. (1987). Receptor-operated, Ca2+-permeable channels activated by ATP in arterial smooth muscle. Nature 328, 275-278. BYRNE, N. G. & LARGE, W. A. (1987). Action of noradrenaline on single smooth muscle cells freshly dispersed from the rat anococcygeus muscle. Journal of Physiology 389, 513-525. BYRNE, N. G. & LARGE, W. A. (1988). Membrane ionic mechanisms activated by noradrenaline in cells isolated from the rabbit portal vein. Journal of Physiology 404, 557-573. DECLERCK, I., DROOGMANS, G. & CASTEELS, R. (1989). Excitatory agonists and Ca2+-permeable channels in arterial smooth muscle cells. In Essential Hypertension 2, ed. AOKI, K., pp. 45-56. Springer Verlag, Berlin. FINE, J., COLE, P. & DAVIDSON, J. S. (1989). Extracellular nucleotides stimulate receptor-mediated calcium mobilization and inositol phosphate production in human fibroblasts. Biochemical Journal 263, 371-376. FRIEL, D. D. (1988). An ATP-sensitive conductance in single smooth muscle cells from the rat vas deferens. Journal of Physiology 401, 361-380. GRYNKIEWICZ, G., POENIE, M. & TSIEN, R. Y. (1985). A new generation of Ca2+ indicators with greatly improved fluorescence properties. Journal of Biological Chemistry 260, 3440-3450. GYLFE, E. & HELMAN, B. (1987). External ATP mimics carbachol in initiating calcium mobilization from pancreatic f-cells conditioned by previous exposure to glucose. British Journal of Pharmacology 92, 281-289. HALLAM, T. J. & PEARSON, J. D. (1986). Exogenous ATP raises cytoplasmic free calcium in fura2 loaded piglet aortic endothelial cells. FEBS Letters 207, 95-99. HAMILL, 0. P., MARTY, A., NEHER, E., SAKMANN, B. & SIGWORTH, F. J. (1981). Improved patchclamp technique for high-resolution current recordings from cells and cell-free membrane patches. Pflugers Archiv 391, 85-100. HONORE', E., MARTIN, C., MIRONNEAU, C. & MIRONNEAU, J. (1989). An ATP-sensitive conductance in cultured smooth muscle cells from pregnant rat myometrium. American Journal of Physiology 257, C297-305. KONNERTH, A., Lux, H. D. & MORAD, M. (1987). Proton-induced transformation of calcium channel in chick dorsal root ganglion cells. Journal of Physiology 386, 603-633. MCMILLIAN, M. K., SOLTOFF, S. P., CANTLEY, L. C. & TALAMO, B. R. (1987). Extracellular ATP elevates intracellular free calcium in rat parotid acinar cells. Biochemical and Biophysical Research Communications 149, 523-530. NAKAZAWA, K. & MATSUKI, N. (1987). Adenosine triphosphate-activated inward current in isolated smooth muscle cells from rat vas deferens. Pflugers Archiv 409, 644-646.
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PEARCE, B., MUIRPHY, S.. JEREMY, J., MORROW, C. & DANDONA, P. (1989). ATP-evoked Ca2" mobilisation and prostainoid release from astrocytes: P2-purinergic receptors linked to phosphoinositide hydrolysis. Journal of Neurochemistry 52, 971-977. PHANEUF, S., BERTA, P., CASANOVA, J. & CAVADORE, J.-C. (1987). ATP stimulates iniositol phosphate accumulation and calcium mobilization in a primary culture of rat aortic myocytes. Biochemical and Biophysical Research Communications 143, 454-460. Ross, R. (1971). The smooth muscle cell. TT. Growth of smooth muscle in culture and formation of elastic fibers. Journal of Cell Biology 50, 172-186. SASAKAWA, N., NAKAKI, T., YAMAMOTO, S. & KATO, R. (1989). Stimulation by ATP of inositol trisphosphate accumulation and calcium mobilization in cultured adrenal chromaffin cells. Journal of Neurochemistry 52, 441-447. TAWADA. Y., FURUKAWA, K.-I. & SHIGEKAWA, M. (1987). ATP-induced calcium transient in cultured rat aortic smooth muscle cells. Journal of Biochemistry 102. 1499-1509.
Journal of Physiology (1991), 440, pp. 623-634 With 7 figures Printed in Great Britain
ATP-INDUCED Ca2+ RELEASE AND Cl- CURRENT IN CULTURED SMOOTH MUSCLE CELLS FROM PIG AORTA
BY G. DROOGMANS, G. CALLEWAERT, I. DECLERCK AND R. CASTEELS From the Laboratorium voor Fysiologie, KU Leuven, B-3000 Leuven, Belgium
(Received 26 October 1990) SUMMARY
1. The effect of exogenous ATP on transmembrane currents and on the cytoplasmic Ca2+ has been investigated in single cultured smooth muscle cells of pig aorta. 2. ATP applied to cells held at a potential of -50 mV evoked a transient inward current and a transient rise in [Ca2+]i. At a potential of + 20 mV the ATP-induced increase in [Ca2+]i was accompanied by an outward current. 3. At a potential of -50 mV, ATP evoked in Ca2+-free solution an inward current which was similar to that in the presence of external Ca2". A second application of ATP in Ca2+-free solution induced a much smaller current. 4. ATP induced in Ca2+-free solution a pronounced transient stimulation of the 45Ca2+ efflux from confluent smooth muscle monolayers. 5. The I-V curve of the ATP-activated current has a reversal potential close to 0 mV. A reduction of external Cl- shifts this reversal potential in accordance with the change of the Cl- equilibrium potential. 6. It is concluded that ATP causes a release of calcium from intracellular stores. The ensuing increase of [Ca2+]i activates a Cl- current, which can depolarize the cell membrane and thereby promote a voltage-gated Ca2+ entry. INTRODUCTION
ATP is released as an excitatory co-transmitter together with noradrenaline at sympathetic nerve terminals present in smooth muscle tissues. Using the patchclamp technique it has been demonstrated in a variety of smooth muscle cells that application of exogenous ATP activates a cation-selective channel which shows little discrimination between various monovalent cations (Benham, Bolton, Byrne & Large, 1987; Friel, 1988; Declerck, Droogmans & Casteels, 1989; Honore, Martin, Mironneau & Mironneau, 1989) and which is directly gated by ATP (Benham & Tsien, 1987; Nakazawa & Matsuki, 1987). In addition, this ATP-activated channel is permeable to divalent cations in rabbit ear artery (Benham et al. 1987; Declerck et al. 1989), and its activation increases the free intracellular calcium concentration ([Ca21]i) in cells which are exposed to normal physiological solutions (Benham, 1989b). It has also been demonstrated that in some smooth muscle cells ATP is able to release Ca2+ from inositol trisphosphate (1P3)-sensitive stores through activation of MS 8887
624
G. DROOGMANS AND OTHERS
P2 purinergic receptors (Phaneuf, Berta, Casanova & Cavadore, 1987; Tawada, Furukawa & Shigekawa, 1987). This ATP-induced release of calcium has only been observed in cultured smooth muscle cells and also in a number of non-excitable cells. To our knowledge, the effect of ATP on membrane currents has not been measured in cultured smooth muscle cells. We have therefore investigated the effect of exogenous ATP on membrane currents and on [Ca21]i in primary cultured smooth muscle cells of pig aorta. METHODS
Cells Vascular smooth muscle cells from thoracic pig aorta, which were obtained from the local slaughterhouse, were cultured as described by Ross (1971), subcultured and used between the 5th and 12th passage. Solutions Cells were bathed in a modified Krebs solution containing (in mM): 1324 NaCl; 15 CaCl2; 5-9 KCl; 1-2 MgCl2; 11-5 HEPES-NaOH; 10 glucose; pH 7-3. Current and [Ca2+]i measurements Experiments were carried out on single cells 1 to 2 days after plating on cover-slips. Currents and [Ca2+]i were measured at room temperature (21-23 °C). External solutions were rapidly exchanged using a multibarrelled pipette with a common opening (Konnerth, Lux & Morad, 1987). The pipette (internal) solution was made up in Milli-Q water (Millipore) and contained (in mM): 120 CsCl; 5 NaCl; 1 MgCI2; 20 HEPES; 0-1 K5jndo-1 or 0-1 EGTA buffered at pH 7-2 with CsOH. Membrane currents were recorded with the standard patch-clamp technique (Hamill, Marty, Neher, Sakmann & Sigworth, 1981) using a List EPC-7 patch-clamp amplifier. Membrane currents were filtered with an 8-pole Bessel filter, digitized on line at sample intervals ranging between 1 and 2 ms and analysed by computer. [Ca2+]i was estimated from Indo-1 fluorescence by the ratio method using single-wavelength excitation and dual-wavelength emission (Grynkiewicz, Poenie & Tsien, 1985). Experiments were carried out on a Nikon Diaphot inverted microscope equipped for epifluorescence. The fluorescence probe was excited at 360 nm with a 75 W xenon arc lamp and fluorescence emitted from the cell was split by a dichroic mirror centred at 450 nm and detected by two parallel photomultipliers (Hamamatsu type R928) at 405 and 485 nm. Single cells were loaded with Indo-1 pentapotassium salt (100 /tM) by diffusion from the patch pipette. Loading of the dye was assessed by measuring changes in fluorescence intensity at 485 nm. Using pipette resistances of 2-3 MQ2, loading was complete within 5 min after formation of a whole-cell patch. Background fluorescence due to cell autofluorescence and Indo- 1 in the pipette was measured in cell-attached mode and subtracted from each record in whole-cell mode. The ratio of emitted fluorescence at 405 nm and at 485 nm (R) was then used to calculate [Ca2+] according to the relationship
[Ca2+]i = KdJ Rmax
min
where Rmin and Rmax are the fluorescence ratios obtained in the absence of Ca2+ and at saturating Ca2+ levels, Kd is the apparent dissociation constant for Indo- 1 for which a value of 213 nm was used (Benham, 1989 a), and ,? is the fluorescence ratio of the 485 nm signal in the absence of Ca2+ to that in the presence of saturating Ca2+ (Grynkiewicz et al. 1985). Rmax and Rmin were determined in vitro by placing a small drop of internal solution containing 2 mM-CaCl2 or 10 mM-EGTA between two cover-slips. No corrections were made for altered properties of Indo- 1 when loaded into the cytoplasm (Benham, 1989a).
45Ca2+ flux measurements Flux experiments were carried out on confluent monolayers of cells 4-5 days after plating in twelve well clusters (3-8 cm diameter). The cell density at the time of the experiments was 3 x 105
ATP-INDUCED Ca2+ RELEASE IN SMOOTH MUSCLE
625
cells well-'. The wells were fixed on a thermostated plate at 37 °C, which was placed on a mechanical shaker. After aspiration of the culture medium, the cells were washed twice with standard physiological solution. Cells were loaded with 45Ca2+ in a 0-2 mM-45Ca2+-containing solution (% 30 ,uCi ml-'). After 5 min the 45Ca2+ uptake was stopped by aspirating the radioactive medium and by washing the cells 4 times with an ice-cold Ca2+-free, 2 mM-EGTA-containing solution. One millilitre of Ca2+-free, 2 mM-EGTA-containing solution was added to the wells, and replaced every 2 min over a period of 30 min. At the end of this period the cells were solubilized in 2 % SDS (sodium dodecyl sulphate). The 45Ca2+ present in the efflux samples and the radioactivity remaining in the cells were determined in a liquid scintillation counter. The total activity in the cells at the beginning of each sampling period was obtained by adding the activity of the efflux samples in retrograde order to the activity in the cells at the end of the experiment. The tracer content of each efflux sample, divided by the total activity in the cells and by the duration of the sampling interval (2 min), gives the fractional 46Ca2+ loss (expressed in min-'). This quantity is represented as a function of efflux time. RESULTS
Effect of ATP on [Ca2+]i and on membrane current Figure 1 shows the effect of application of exogenous ATP (50#M) to a single smooth muscle cell held at a potential of -50 mV and which was dialysed with the Ca2+ indicator Indo-1 (100 /tM). After a latency of about 1 s ATP triggers a transient rise in [Ca2+]i from its resting value of about 85 nm to a peak value of 425 nm. This peak value is reached about 1 s after the onset of the response (lower trace). This transient increase in [Ca2+]i was accompanied by a transient inward current reaching a peak value of about 1P5 nA at the time when [Ca2+]i was maximal (upper trace). [Ca2+]i transients as shown in Fig. 1 were observed in all cells, and ranged from 215 to 580 nM, with a mean value of 390 + 45 nm (mean + standard error of the mean; n = 7 cells). The peak amplitude of the current varied from cell to cell, and ranged from 0-6 to 2-2 nA, with a mean value of 1-32 + 0-14 nA (n = 15 cells). After this transient increase of [Ca2+]i this parameter stabilized to a value near the resting [Ca2+]i. Whereas the increase of [Ca2+]i upon application of ATP was monophasic in all cells, we frequently observed that the rising phase of the concomitant transient current displayed several components. As illustrated in Fig. 2, an initial phase of the current can occur before any changes in [Ca2+]i could be detected. The time course of the ensuing second component is closely correlated with the changes in [Ca2+]i. It thus appears as if ATP exerts a dual action on the membrane current, i.e. an initial effect which is not related to changes in [Ca2+]i, and a second one which is clearly associated with changes in [Ca2+]i. The initial current component, which precedes the Ca2+ transient, was highly variable and did not occur in all cells. The ATP-activated Ca2+ transient, however, was present in all cells irrespective of the presence or absence of the initial current component. It is therefore unlikely that this initial ATP-activated current component causes an increase in [Ca2+]i, which then results in a subsequent activation of a Ca2+-dependent conductance. The long latency between the onset of this current, which occurs immediately after the application of ATP, and the increase in [Ca2+]i suggests an ATP-induced release of calcium rather than an ATP-activated
Ca2+-influx.
G. DROOGMANS AND OTHERS
626
The increase in [Ca2+]i is not produced by the transient inward current The similarity of the time course of [Ca2+]i and the current evoked by ATP could be explained either by an inward Ca2+ current which causes a rise of [Ca2+]i, or by an increase of [Ca2+]i which itself activates an inward current. If the first hypothesis 50 /iM-ATP
0.5 nAJ
400
F
Ca2+ 200
(nM)
n v
L
2s 1. Effect of on Fig. 50,uM-ATP membrane current (in nA, top trace) and on [Ca2+]i (expressed in nm, bottom trace). The addition of ATP is indicated by the arrow. ATP was present for the rest of the record. The cell was held at a potential of -50 mV.
were true, then the rise in intracellular calcium would strongly depend on the transmembrane Ca2+ gradient. We have therefore applied ATP to cells which were held at various potentials. Figure 3 shows the results of such an experiment in which the effects of ATP were compared at two different holding potentials, i.e. at -50 mV (left panel) and at + 20 mV (right panel). At both potentials, application of ATP gives rise to a transient increase in [Ca21]i. At -50 mV this change in [Ca2+]i is accompanied by an inward current. The changes in [Ca2+]i at +20 mV are comparable to those at -50 mV, but they are accompanied by an outward current. Also the initial component of the ATP-activated current, which occurs before any change in [Ca2+]i, is reversed at + 20 mV.
ATP-INDUCED Ca2+ RELEASE IN SMOOTH MUSCLE
627
The ATP-evoked current does not depend on the presence of extracellular Ca2+ Although an outward current cannot directly result in an increase of [Ca2+]i, it cannot be excluded that this outward current masks an inward Ca2+ current which contributes to the increase in [Ca2+]i either directly or via a Ca2+-induced Ca2+ release mechanism. 50
,uM-ATP ~A
0.2 nA
400 L
Ca2+ 200 k
(nM)
nI
500 ms
Fig. 2. Effect of 50 /aM-ATP on the membrane current (in nA, top) and [Ca2"], (in nm, bottom). This recording shows on an expanded time scale the initial component of the current evoked by ATP. It is obvious that the initial current component is not associated with changes in [Ca2+]i. The holding potential was -50 mV.
We have therefore investigated the ATP-activated current in a Ca2+-free solution buffered with 2 mM-EGTA. The results of such an experiment are shown in Fig. 4. The top trace shows the current response evoked by ATP at a potential of -50 mV in a bathing solution containing 1.5 mM-Ca2+. The two subsequent responses were obtained in Ca2+-free solution at the same holding potential. The current induced by the application of ATP after about 30 s in Ca2+-free solution (middle trace) is not significantly different from that under control conditions. This observation is consistent with a minor contribution, if any at all, of Ca2+ influx to the ATPactivated current. A second application of ATP during the continued perfusion with Ca2+-free solution induces a much smaller transient inward current (bottom trace).
G. DROOGMANS AND OTHERS
628
50 l~
uM-ATP l-
50 pM-ATP
1 nA
300 I 200
Ca2+ (nM)
100o 2s
Fig. 3. Effect of 50 /eM-ATP on membrane current (in nA, top) and [Ca2"], (in nm, bottom) at two different holding potentials. The left panel shows the data at -50 mV, the right panel those at a holding potential of + 20 mV.
50 yM-ATP O nA-
0 nA -
0 nA -
1 nA
5s
Fig. 4 Effect of extracellular Ca2+ on the current evoked by 50 /M-ATP at a holding potential of -50 mV. The upper trace was obtained in a bathing solution containing 1P5 mM-Ca2+. The middle and bottom trace show two consecutive applications of ATP in Ca2+-free solution. Note also the gradual increase of the leak current occurring in Ca2+-free solution.
ATP-INDUCED Ca2+ RELEASE IN SMOOTH MUSCLE
629
This residual current might represent a current component which is directly activated by ATP and is similar to the current component that occurs before any change in [Ca2+]i as described above. It is also possible that this current is activated by release of calcium from internal stores, which are not completely depleted of calcium during the preceding 'brief' exposure to ATP. 0.4
-
50 tM-ATP c
E
0
0O.2-
0
0.0
I
0
10
20
30
Time (min) Fig. 5. Effect of 50 ,tM-ATP on the fractional 45Ca2+ loss (min-') from confluent monolayers of smooth muscle cells exposed to a Ca2+-free solution. Data have been obtained from twelve wells.
Also the leak current gradually increases during exposure to Ca2+-free solution. This resulted in a loss of the seals in all experiments, except in one. Readmitting 1-5 mM-Ca2+ to the bathing solution resulted in a partial recovery of the ATP-evoked current in this cell (data now shown). ATP releases Ca2+ from intracellular stores The decline of the current upon repeated stimulation with ATP in Ca2+-free solution, and the partial recovery upon readmission of external Ca 2+ are consistent with a current that is activated by an ATP-induced release of intracellular calcium. In order to obtain more direct evidence for an ATP-induced release of calcium from intracellular stores, we have studied the effect of ATP on the 45Ca2+ efflux from confluent monolayers of smooth muscle cells. After loading the cells with 45Ca2+, the efflux was performed in a Ca2+-free solution in order to eliminate possible effects of ATP on the influx of Ca2+ (Fig. 5). The initial decline of the fractional 45Ca2+ loss mainly represents the wash-out of extracellular 'racer. After about 10 min of efflux, this fractional loss becomes almost constant, and it is assumed that it represents the 45Ca2+ efflux from the cells. ATP (50juM), applied to the cells after another 10 min of efflux, resulted in a 4-fold
G. DROOGMANS AND OTHERS
630
transient increase of the fractional loss. A second application of ATP did not evoke a second increase in efflux rate (not shown), suggesting that the transient effect is not due to desensitization, but to depletion of an intracellular Ca2+ store. Because the effect of ATP on these cultured cells seems to be different from its action in rabbit ear artery, we have performed a similar experiment on smooth
a O nA1
nAL
b
2s
I (nA)
Difference
~~-ATP
/ t
-50
/
V (mV)
50
-2
Fig. 6. Voltage dependence of the current evoked by 50 ,lM-ATP. The top trace shows the ATP-induced current, and the effect of applying a linear voltage ramp from -50 to + 50 mV before (a) and during (b) application of ATP. The lower panel shows the currents measured during the voltage ramp as a function of the instantaneous voltage. The ATPevoked current was calculated from the difference current between the control response and that measured in the presence of ATP. The reversal potential of the difference current was -7-5 mV in this cell.
muscle strips of the latter tissue. However, ATP did not stimulate the 45Ca2+ efflux in this tissue, whereas a subsequent stimulation with noradrenaline released a significant amount of 45Ca2+ (not shown). This observation confirms the different modes of action of ATP in these two smooth muscle types.
The ATP-activated current is a Cl- current The ionic nature of the ATP-activated current was investigated by applying linear voltage ramps between -50 and + 50 mV to the patch pipette. The difference between the current measured near the peak of the ATP-induced current and that measured in the absence of ATP is defined as the ATP-activated current. Figure 6 (top) shows a typical recording. At 'a' a linear ramp was applied in control conditions, at 'b' the same voltage ramp was applied in the presence of ATP. These currents, as well as the difference current, are represented as a function of the
ATP-INDUCED Ca2+ RELEASE IN SMOOTH MUSCLE
631
instantaneous voltage in the lower panel. The current-voltage relationships are approximately linear, and the difference current reverses at a potential around - 75 mV. The mean value of the reversal potential calculated from six experiments, amounts to -54 + 0-6 mV. This value is close to the equilibrium potential for Cl/ (nA)
1.0
100% Cl0.5
-
50% C-
-50
/50
V (mV)
-1.0
-
Fig. 7. Effect of substitution of 50% of external C1- ions by gluconate on the current-voltage relationship of the ATP-evoked current. The I-V curves of the ATPevoked current were obtained as described in Fig. 6. Substitution of half of the extracellular C1- ions by gluconate significantly shifts the reversal potential to more positive values.
ions (around 0 mV) and is therefore consistent with a current which is mainly carried by Cl- ions. However, these findings cannot exclude a current which would pass through a nonselective cation channel and would have a reversal potential near 0 mV. In order to further identify the ionic nature of the current we have measured the reversal potential of the ATP-evoked current in a solution in which half of the external Clions were replaced by gluconate ions. The I-V relationships of the ATP-activated current in 100 and 50 % extracellular Cl- are shown in Fig. 7. It can be observed that this substitution shifts the reversal potential to more positive potentials from a value of -5-5 mV in 100 % to + 1-2 mV in 50 % Cl-. The magnitude of this shift is close to the value of the shift of the equilibrium potential of Cl- ions by a 50 % reduction of [Cl-]0 (17-5 mV). DISCUSSION
These experimental results demonstrate an ATP-mediated Ca2+ release in primary cultures of pig aorta, which is accompanied by activation of a Ca2+-dependent Clcurrent. This action of ATP is different from that observed in rabbit ear artery cells, where the rise in [Ca2+]i could be associated with the activation of a non-selective
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G. DROOGMANS AND OTHERS
channel, which is also permeable to calcium (Benham, 1989b). A release of 45Ca2+ from internal stores could be clearly demonstrated in these cultured cells, but cation
ATP was unable to induce a release of calcium in rabbit ear artery cells. Calcium mobilization from internal Ca2+ stores by extracellular ATP via activation ofP2 purinergic receptors has been observed in a number of excitable and non-excitable cells (Hallam & Pearson, 1986; Gylfe & Helman, 1987; McMillian, Soltoff, Cantley & Talamo, 1987; Phaneuf et al. 1987; Tawada et al. 1987; Fine, Cole & Davidson, 1989; Pearce, Murphy, Jeremy, Morrow & Dandona, 1989; Sasakawa, Nakaki, Yamamoto & Kato, 1989). It has been shown that in several cell types these receptors are coupled to the activation of inositol phospholipid break-down, and that the generated 1P3 mediates the release of calcium. Such anIP3-induced Ca2+ release has also been observed in our cultured cells after, permeabilization with. saponin (L. Missiaen, unpublished observation). The current activated by ATP was biphasic in some cells: an initial current component, which occurred before any changes in [Ca2+]i, was followed by a second component which was associated with the ATP-induced changes in [Ca2+]i. We cannot completely rule out a contribution of an ATP-activated Ca2+ influx to the observed changes in [Ca2+]i. However, our experimental results indicate that such an influx of calcium is only marginal, and that the release of calcium from internal stores is the main mechanism which leads to the ATP-induced increase of [Ca2+]i. A reduction of the electrochemical Ca2+ gradient by changing the holding potential from -50 to + 20 mV does not significantly affect the ATP-induced change in [Ca2+]i. In addition, the current recorded during the first application of ATP in free solution is not significantly different from that of the preceding stimulation with ATP in the presence of external calcium. The ATP-induced increase of [Ca2+]i is closely correlated with the second component of the ATP-activated current. The finding that this current as well as the 45Ca2+ release disappear upon repeated stimulation with ATP in Ca2+-free solution indicates that there exists a causal relation between the increase in [Ca2+]i and the evoked current. The effect of changes of [Cl-]O on the reversal potential of the ATPactivated current is consistent with the change in Ec, (equilibrium potential for chloride), and supports the proposal that this current is due to activation of Clchannels by a rise in [Ca2+]i. Ca2+-activated Cl- currents could also be induced by caffeine in rat anococcygeus muscle (Byrne & Large, 1987) and in rabbit portal vein (Byrne & Large, 1988). It has also been reported that noradrenaline opens Ca2+activated Cl--selective channels in rabbit portal vein (Byrne & Large, 1988) and in rabbit ear artery (Amedee, Benham, Bolton, Byrne & Large, 1990). Such a Clcurrent will in cells, that are not held under voltage control, depolarize the cell membrane, and thereby cause further influx of Ca2+ through voltage-sensitive Ca2+_ channels. In summary, the mechanism of ATP action on these cultured smooth muscle cells of pig aorta differs from that observed in rabbit ear artery. Its main action is to release calcium in this tissue, and to activate a non-selective cation channel in rabbit ear artery (Benham et al. 1987; Declerck et al. 1988). The initial current component, which occurs before any change in might be due to activation of this nonselective cation channel, but this hypothesis needs further investigation. Although
Ca2+-
[Ca2+]i,
ATP-INDUCED Ca2+ RELEASE IN SMOOTH MUSCLE
633
Ca2+ influx during this initial current component cannot be excluded, its contribution to the observed changes in [Ca21]i is only marginal. It is not clear whether these different actions of ATP represent differences between types of smooth muscle cells, or whether they are due to a differentiation process occurring in the cultured cells. This work has been supported by research grant No. 3.0064.88 of the Fonds voor Wetenschappelijk Geneeskundig Onderzoek (Belgium). We thank Drs H. De Smedt and L. Missiaen for generously supplying us with the cultures of pig aorta cells used in this study. REFERENCES
AMEDE'E, T., BENHAM, C. D., BOLTON, T. B., BYRNE, N. G. & LARGE, W. A. (1990). Potassium, chloride and non-selective cation conductances opened by noradrenaline in rabbit ear artery cells. Journal of Physiology 423, 551-568. BENHAM, C. D. (1989a). Voltage-gated and agonist-mediated rises in intracellular Ca2+ in rat clonal pituitary cells (GH3) held under voltage clamp. Journal of Physiology 415, 143-158. BENHAM, C. D. (1989b). ATP-activated channels gate calcium entry in single smooth muscle cells dissociated from rabbit ear artery. Journal of Physiology 419, 689-701. BENHAM, C. D., BOLTON, T. B., BYRNE, N. G. & LARGE, W. A. (1987). Action of externally applied adenosine triphosphate on single smooth muscle cells dispersed from rabbit ear artery. Journal of Physiology 387, 473-488. BENHAM, C. D. & TSIEN, R. W. (1987). Receptor-operated, Ca2+-permeable channels activated by ATP in arterial smooth muscle. Nature 328, 275-278. BYRNE, N. G. & LARGE, W. A. (1987). Action of noradrenaline on single smooth muscle cells freshly dispersed from the rat anococcygeus muscle. Journal of Physiology 389, 513-525. BYRNE, N. G. & LARGE, W. A. (1988). Membrane ionic mechanisms activated by noradrenaline in cells isolated from the rabbit portal vein. Journal of Physiology 404, 557-573. DECLERCK, I., DROOGMANS, G. & CASTEELS, R. (1989). Excitatory agonists and Ca2+-permeable channels in arterial smooth muscle cells. In Essential Hypertension 2, ed. AOKI, K., pp. 45-56. Springer Verlag, Berlin. FINE, J., COLE, P. & DAVIDSON, J. S. (1989). Extracellular nucleotides stimulate receptor-mediated calcium mobilization and inositol phosphate production in human fibroblasts. Biochemical Journal 263, 371-376. FRIEL, D. D. (1988). An ATP-sensitive conductance in single smooth muscle cells from the rat vas deferens. Journal of Physiology 401, 361-380. GRYNKIEWICZ, G., POENIE, M. & TSIEN, R. Y. (1985). A new generation of Ca2+ indicators with greatly improved fluorescence properties. Journal of Biological Chemistry 260, 3440-3450. GYLFE, E. & HELMAN, B. (1987). External ATP mimics carbachol in initiating calcium mobilization from pancreatic f-cells conditioned by previous exposure to glucose. British Journal of Pharmacology 92, 281-289. HALLAM, T. J. & PEARSON, J. D. (1986). Exogenous ATP raises cytoplasmic free calcium in fura2 loaded piglet aortic endothelial cells. FEBS Letters 207, 95-99. HAMILL, 0. P., MARTY, A., NEHER, E., SAKMANN, B. & SIGWORTH, F. J. (1981). Improved patchclamp technique for high-resolution current recordings from cells and cell-free membrane patches. Pflugers Archiv 391, 85-100. HONORE', E., MARTIN, C., MIRONNEAU, C. & MIRONNEAU, J. (1989). An ATP-sensitive conductance in cultured smooth muscle cells from pregnant rat myometrium. American Journal of Physiology 257, C297-305. KONNERTH, A., Lux, H. D. & MORAD, M. (1987). Proton-induced transformation of calcium channel in chick dorsal root ganglion cells. Journal of Physiology 386, 603-633. MCMILLIAN, M. K., SOLTOFF, S. P., CANTLEY, L. C. & TALAMO, B. R. (1987). Extracellular ATP elevates intracellular free calcium in rat parotid acinar cells. Biochemical and Biophysical Research Communications 149, 523-530. NAKAZAWA, K. & MATSUKI, N. (1987). Adenosine triphosphate-activated inward current in isolated smooth muscle cells from rat vas deferens. Pflugers Archiv 409, 644-646.
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PEARCE, B., MUIRPHY, S.. JEREMY, J., MORROW, C. & DANDONA, P. (1989). ATP-evoked Ca2" mobilisation and prostainoid release from astrocytes: P2-purinergic receptors linked to phosphoinositide hydrolysis. Journal of Neurochemistry 52, 971-977. PHANEUF, S., BERTA, P., CASANOVA, J. & CAVADORE, J.-C. (1987). ATP stimulates iniositol phosphate accumulation and calcium mobilization in a primary culture of rat aortic myocytes. Biochemical and Biophysical Research Communications 143, 454-460. Ross, R. (1971). The smooth muscle cell. TT. Growth of smooth muscle in culture and formation of elastic fibers. Journal of Cell Biology 50, 172-186. SASAKAWA, N., NAKAKI, T., YAMAMOTO, S. & KATO, R. (1989). Stimulation by ATP of inositol trisphosphate accumulation and calcium mobilization in cultured adrenal chromaffin cells. Journal of Neurochemistry 52, 441-447. TAWADA. Y., FURUKAWA, K.-I. & SHIGEKAWA, M. (1987). ATP-induced calcium transient in cultured rat aortic smooth muscle cells. Journal of Biochemistry 102. 1499-1509.
