European Journal of Pharmacology, 215 (1992) 321-324

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© 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.00

EJP 21052 Short communication

Dopamine receptor agonists and antagonists enhance ATP-activated currents K a z u h i d e I n o u e , K e n N a k a z a w a , T o m o k a z u W a t a n o , Mica O h a r a - I m a i z u m i 1, K a n n o s u k e F u j i m o r i and Akira Takanaka Dil'ision of Pharmacology, National Institute of Hygienic Sciences, 1-18-1 Kamiyoga, Setagaya, Tokyo 158, Japan

Received 14 February 1992, accepted 10 March 1992

The effects of dopamine and related compounds on ATP-activated channels were investigated in pheochromocytoma PC12 cells. Dopamine (10 p~M) enhanced an inward current activated by 100 txM ATP. A similar enhancement of the ATP-activated current was observed with apomorphine (10 txM), a non-selective dopamine receptor agonist, with (+)-SKF-38393 (10 p~M), a selective dopamine D l receptor agonist, and with (-)-quinpirole (10 #M), a selective dopamine D 2 receptor agonist. Moreover, (+)-SCH-23390 (30/xM), a dopamine D I receptor antagonist, and (-)-sulpiride (30 txM), a dopamine D 2 receptor antagonist, also enhanced the ATP-activated current. The results suggest that ATP-activated channels are modulated by dopaminergic mechanisms, and that this modulation cannot be attributed to any single class of dopamine receptors. ATP; Ion channels; Dopamine; Dopamine receptor agonists; Dopamine receptor antagonists; Pheochromocytoma PC12 cells

1. Introduction

A family of extracellular ATP-activated channels has recently been characterized in neuronal and muscle cells (see review, Bean and Friel, 1990). These channels are coupled to receptors which are classified as P2-type on the basis of their agonist selectivity according to the conventional classification (e.g., Burnstock and Kennedy, 1985). Physiological roles for ATP-activated channels in neurotransmission, either by depolarizing postsynaptic membranes or by supplying Ca 2+ directly through the channels themselves, have been suggested (Bean and Friel, 1990). However, modulation of these channels by endogenous substances including neurotransimitters has not been reported. We previously reported that PC12 cells, a cell line derived from a rat pheochromocytoma, also possess ATP-activated channels (Nakazawa et al., 1990, 1991). As PC12 cells release endogenous dopamine upon various types of stimulation, including that by extracellular A T P (e.g., Inoue et al., 1991), some modulation of the ATP-activated channels by dopamine was expected. The present study examined the contribution of dopaminergic mechanisms to ATP-activated chan-

Correspondence to: K. Nakazawa, Department of Cellar and Molecular Physiology,Harvard Medical School, 25 Shattuck Street, Boston, MA 02115, U.S.A. Tel. 1.617.432 1012, fax 1.617.432 1144. i Present address: Life Science Institute, Sophia University, 7-1 Kioicho, Chiyoda-ku,Tokyo 102, Japan.

nels. We found that the inward current activated by extracellular ATP was enhanced not only by compounds known as agonists to both dopamine D~ and D 2 receptors but also by those known as antagonists.

2. Material and methods

PC12 cells were prepared as described by Inoue et al. (1991). Briefly, PC12 cells were cultured in Dulbecco's Modified Eagle's Medium containing 7% fetal bovine serum, 7% heat-inactivated horse serum, 2 mM L-glutamine, and 50 txg/ml gentamicin sulfate at 37°C under an atmosphere of 10% COz, and then plated onto collagen-coated polystyrene dishes at 10 6 cells per dish. After an additional 2 - 3 days of culture, the cells were used for experiments. Membrane currents were measured with whole-cell voltage-clamp techniques (Hamill et al., 1981). The cells were continuously superfused with an extracellular solution containing (in raM) 140 NaCI, 5.4 KC1, 1.8 CaCl 2, 1.0 MgCl 2, 11.1 glucose and 10.0 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) (adjusted with N a O H to pH 7.4). Heat-polished patch pipettes were used with a tip resistances of about 5 m ~ when filled with an intracellular solution containing (in mM) 150 CsC1, 10.0 H E P E S and 5.0 glycoletherdiamine-N,N,N',N'-tetraacetic acid (EGTA). Membrane currents were monitored on an memory oscilloscope (Nihon Kohden VC-11)via a patch-clamp ampli-

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tier (Nihon Kohden CEZ-2200). Electrical signals were filtered at 1 kHz and stored on magnetic tape for later analysis. Drugs used were ATP (adenosine 5'-triphosphate disodium salt, Sigma, St. Louis, MO, U.S.A.), dopamine (dopamine hydrochloride, Sigma), apomorphine (apomorphine hydrochloride, Wako Pure, Osaka, Japan). ( - )-Quinpirole, ( + )- and (-)-SKF-38393 [R( + )- and S(-)-SKF-38393 hydrochloride], ( - ) - and ( + ) sulpiride [S(-)- and R(+)-sulpiride] and SCH-23390 [R( + )-SCH-23390 hydrochloride] were purchased from Research Biochemicals Inc. (Natick, MA, U.S.A.). All the drugs were dissolved in the extracellular solution and applied to the ceils by superfusion. Cells were exposed to ATP for a short period (about 5 s) to avoid desensitization of ATP-activated channels (Nakazawa et al., 1990).

ATP 100 ~,M A.

=oo] T-

+ dopamine

T"

T"

1 S'i"

T*

7~

T*

o"

T ~'

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Fig. 2. Summarized data of the enhancement of the ATP-activated current by dopamine and related compounds. Cells were held at - 60 mV and exposed to 100 #,M ATP with or without each drug as in fig. 1A. The currents recorded in the presence of drugs were normalized to control currents for each cell. Each column indicates the mean from four to seven cells tested. Bars are S.E.M. Asterisks indicate statistically significant differences from the control by Student's paired t-test (P < 0.05) (comparisons were made using the initial values, i.e., before normalization).

Figure 1A shows an inward current from a PC12 cell that was activated by bath-application of ATP (100 /xM). This current could be enhanced by bath application of dopamine (10 # M ) along with ATP (100 /zM) (fig. 1A and 2A). Apomorphine (10 #M), another dopamine receptor agonist, was equipotent with dopamine in enhancing the ATP-activated current (fig. 2A). The inward current activated by 100/xM ATP and these agonists returned to the initial (i.e., 'before enhancement') level (mean + S.E.M.: dopamine 96.2_+

control

C.

Tt

150

3. R e s u l t s

A.

B.

5.4%, n = 6; apomorphine 99.9 _+ 10.4%, n = 6) after a 1-min washout period. Thus, the enhancement was reversible. Neither dopamine nor apomorphine alone could activate this inward current. Unlike the current activated by 100 M ATP (fig. 1A), the inward current activated by 1 mM ATP, a concentration which produces maximal inward currents as well as cate-

C.

-+250

ATP 100 gM

I

7 B,

control

I

I

I

+20 mV

+ dopamine

ATP 1 mM

7

-5O

+ dopamine

-500 pA

- - 7 2 0 0 pA

Fig. 1. Effects of dopamine on ATP-activated currents. A, B and C were obtained from three different cells. (A) Typical recordings of the enhancement by dopamine (10 p,M) of an inward current activated by 100/xM ATP. The cell was held at - 60 mV. Each application of ATP was separated by I min. Dopamine was present 30 s before and during application of ATP. (B) Lack of enhancement by dopamine (10 ttM) of an inward current activated by 1 mM ATP. The data were obtained as in (A). (C) Current-voltage relationship for the ATP-aetivated current in the absence or presence of dopamine. The cell was held at - 6 0 mV and a ramp pulse from - 6 0 to + 20 mV (100 m V / s ) was applied to the cell every 2 s. The cell was exposed to 100 ttM ATP during repetitive application of the ramp pulse. The difference between the current before and during ATP application was calculated every 5 mV. Each symbol represents the ATP-activated current in the absence (control, filled circles) or presence of 10 ttM dopamine ( + dopamine, open circles).

3~3

cholamine release in these cells (Nakazawa et al., 1991), was not increased by 10/~M dopamine (fig. 1B; mean, 92.6 _+ 1.2% of control, n = 4). The voltage dependence of the ATP-activated current was not affected by dopamine in any of the four cells tested (fig. 1C). Figure 2 shows a summary of the effects of dopamine and related compounds on the ATP-activated current. (+)-SKF-38393 (10 /~M), a selective dopamine D1 receptor agonist, and ( - ) - q u i n p i r o l e (10/~M), a selective dopamine D2 receptor agonist, enhanced the ATP-activated current (fig. 2B). (+)-SCH-23390 (30 ~M), a selective dopamine D1 receptor antagonist, and ( - ) - s u l p i r i d e (30 p.M), a selective dopamine D2 receptor antagonist, also enhanced the ATP-activated current (fig. 2C). All of these compounds affected the membrane current only in the presence of extracellular ATP. We also tested the effects of (-)-SKF-38393 (10 /zM), ( - ) - S C H - 2 3 3 9 0 (30 p.M) and (+)-sulpiride (30 /~M), which are inactive enantiomers at dopamine D l and D 2 receptors (fig. 2C), on the ATP-activated current. Of these enantiomers, only (-)-SKF-38393 could also enhance the ATP-activated current.

4. Discussion We have shown that dopamine enhances the current amplitude of ATP-activated channels in PC12 cells. Dopamine is the first endogenous substance identified as a modulator of ATP-activated currents. The enhancement of ATP-activated currents by dopamine and related compounds is not due to their agonist potency at Pz-purinoceptors but is due to some allosteric effect because none of these compounds produced an inward current in the absence of ATP. An increase in the number of available channels does not appear to be involved because a maximally activated inward current was not increased by dopamine (fig. 1B). Dopamine is a negative feedback regulator of catecholamine release and acts by inhibiting voltagegated CA 2+ channels and nicotinic acetylcholine receptor channels (Sontag et al., 1990) via dopamine D 2 receptors in dopamine-secreting cells. Artalejo et al. (1991) recently reported that dopamine, by acting via dopamine D~ receptor-mediated mechanisms, acts as a positive feedback regulator by enhancing the activity of voltage-gated Ca 2+ channels in bovine chromaffin cells. The enhancement of the ATP-activated channels in this report also suggests such a positive feedback regulatory role of dopamine. The enhancement of the ATP-activated current is not due to a single population of dopamine D 1 o r O 2 receptors, or a mixture of them because selective dopamine D 1 and D 2 receptor antagonists [(+)-SCH23390 and (-)-sulpiride] and an inactive compound (-)-SKF-38393 also enhanced the ATP-activated cur-

rent (fig. 2B,C). Inhibition by 'dopamine receptor agonists' of nicotine-stimulated dopamine release from bovine chromaffin cells is also not mediated via classical types of dopamine receptors (Huettl et al., 1991). The receptors involved in the enhancement of the ATP-activated currents may be different from the receptors in bovine chromaffin cells judging by the selectivity of agonists. For example, apomorphine but not dopamine or ( - ) - q u i n p i r o l e inhibits nicotine-stimulated dopamine release from bovine chromaffin cells (Huettl et al., 1991), whereas all of these compounds enhanced the ATP-activated current in PC12 cells (fig. 2A,B). Modulation by specific activation of the dopamine D 3 receptor, which was recently characterized by molecular cloning (Sokoloff et al., 1991), is also insufficient to account for our results because the agonist potency of (+)-SKF-38393 compared to that of the other compounds tested was much higher than that expected from its building to dopamine D 3 receptors (Sokoloff et al., 1991). We do not know whether the enhancement by dopamine and related compounds is mediated by one specific receptor or by multiple types of receptors. If the enhancement was mediated through one type of 'dopamine' receptor, that receptor must have a structural peculiarity that allows antagonists (and some inactive enantiomers) to induce the same conformational change in receptor molecules as agonists do. Another possibility that cannot be excluded is that these antagonists interact with binding sites distinct from those for the agonists.

Acknowledgments We thank Ms. T. Obama for culturing the cells. We also thank Dr. A.R. Rittenhouse for improving our manuscript.

References Artalejo, C.R., M.A. Ariano, R.L. Perlman and A.P. Fox, 1991, Activation of facilitation calcium channels in chromaffin cells by D 1 dopamine receptors through a cAMP/protein kinase A-dependent mechanism, Nature 348, 239. Bean, B.P. and D.D. Friel, 1990, ATP-activated channels in excitable cells, in: Ion Channels, Vol. 2, ed. T. Narahashi (Plenum Press, New York) p. 169. Burnstock, G. and C. Kennedy, 1985, A dual function for adenosine 5'-triphosphate in the regulation of vascular tone: excitatory cotransmitter with noradrenaline from perivascular nerves and locally released inhibitory intravascular agent, Circ. Res. 58, 319. Hamill, O.P., A. Marty, E. Neher, B. Sakmann and F.J. Sigworth, 1981, Improved patch clamp techniques for high-resolution current recordings from cells and cell-free patches, Pflfigers Arch. 391, 85. Huettl, P., G.A. Gerhardt, M.D. Browning and J.M. Masserano, 1991, Effects of dopamine receptor agonists and antagonists and

324 catecholamine release in bovine chromaffin cells, J. Pharmacol. Exp. Ther. 257, 567. Inoue, K., K. Nakazawa, M. Ohara-lmaizumi, T. Obama, K. Fujimori and A. Takanaka, 1991, Selective and competitive antagonism by suramin of ATP-stimulated catecholamine-secretion from PC12 phaeochromocytoma cells, Br. J. Pharmacol. 102, 581. Nakazawa, K., K, Fujimori, A. Takanaka and K. Inoue, 1990, ATPactivated conductance in pheochromocytoma cells and its suppression by extracellular calcium, J. Physiol. (London) 428, 257. Nakazawa, K., K. Fujimori, A. Takanaka and K. Inoue, 1991, Comparison of adenosine triphosphate- and nicotine-activated inward

currents in rat phaeochromocytoma cells, J. Physiol. (London) 434, 647. Sokoloff, P., B. Giros, M.-P. Martres, M.-L. Bouthenet and J.-C. Schwartz, 199l, Molecular cloning and characterization of a novel dopamine receptor (Dfl as a target for neuroleptics, Nature 347, 146. Sontag, J.-M,, P. Sanderson, M. Klepper, D. Aunis, K. Takeda and M.-F, Bader, 1990, Modulation of secretion by dopamine involves decrease in calcium and nicotinic currents in bovine chromaffin cells, J. Physiol. (London) 427, 495.

Dopamine receptor agonists and antagonists enhance ATP-activated currents.

The effects of dopamine and related compounds on ATP-activated channels were investigated in pheochromocytoma PC12 cells. Dopamine (10 microM) enhance...
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