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Enkephalin induces Ca2+ mobilization in single cells of bradykinin-sensitized differentiated neuroblastoma hybridoma (NG108-15) cells
Neuroscience Letters, 148 (1992) 93-96 © 1992 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/92/$ 05.00
93
NSL 09162
Enkephalin induces Ca 2+ mobilization in single cells of bradykininsensitized differentiated neuroblastoma hybridoma (NG 108-15) cells H i d e a k i T o m u r a , F u m i k a z u O k a j i m a a n d Yoichi K o n d o Department of Physical Biochemistry, Institute of Endocrinology, Gunma University, Maebashi (Japan) (Received 17 June 1992; Revised version received 18 September 1992; Accepted 21 September 1992)
Key words: Enkephalin; Bradykinin; Cytosolic Ca2+; Pertussis toxin: NG108-15 cell A study of the intracellular Ca ~+ ([Ca2+]) response of differentiated neuroblastoma x glioma hybrid cells (NG108-15 cell) to enkephalin (EK) was carried out by fura-2 video-imaging. EK alone did not influence [Ca:+]~ in single cells. The opioid did, however, induce a marked [Ca>I, rise, when the cells were incubated with bradykinin (BK) prior to the EK treatment. Such BK-assisted stimulation of the differentiated hybridoma cells by EK was completely abolished by pertussis toxin treatment. These results suggest that in single NG108-15 cells, EK induces Ca 2+ mobilization which is assisted by cross-talk between the EK and BK receptor systems via a pertussis toxin-sensitive G protein.
NG108-15 cells are neuroblastoma x glioma hybrid cells. When cAMP producing agents are added to the culture medium, the cells show some morphological and physiological features of neuronal cells. Among the neurotransmitter receptors found in cells [2, 8], bradykinin (BK) [14] and Pz-receptors [4] are known to be coupled to a phospholipase C (PLC) and are stimulatory. In contrast to these, opioid, az-adrenergic, somatostatin and muscarinic receptors are coupled to an adenylate cyclase and are inhibitory [8]. Enkephalin (EK), an opioid agonist, as well as somatostatin has also been shown to block Ca 2+ inward currents in differentiated NG108-15 cells [17]. On the other hand, we have found [13] that, in a suspension of undifferentiated NG108-15 cells, EK slightly but appreciably increases the intracellular Ca 2+ concentration ([Ca2+]i). Furthermore, this EK-dependent [Ca2+]i increase is markedly enhanced by prior stimulation of the cells with bradykinin (BK), a Ca 2+ mobilizing agent. These findings, however, raised two questions: first, whether the [Ca2+]i increase by EK without BK is due to the action of EK itself, or due to the EK action under the influence of an endogenous stimulant(s) which can act similarly to BK. A candidate for such a stimulant is ATP which might be liberated from damaged cells during harvesting. This seems reasonable since exogenously added Correspondence: H. Tomura, Department of Physical Biochemistry, Institute of Endocrinology, Gunma University, Maebashi 371, Japan. Fax: (81) (272) 34-1788.
ATP has been shown to act as a Ca z+ mobilizing agent and has been shown to mimic the action of BK by enhancing EK-dependent [Ca2+]~ increase. The second question is whether the enhanced EK-dependent [Ca2+]~ increase really occurs in the cell where the BK-induced [Ca2+]i transient has been observed. To answer these questions, in the present study, we analyzed EK-induced [Ca2+]~ changes by fura-2 video imaging of single NG 10815 cells grown on glass plates without harvesting. NG108-15 cells were provided by Dr. Higashida, Kanazawa University, Japan. The cells were grown on coverslips (Matsunami Glass, Tokyo, Japan) in an atmosphere of 5% C O 2 - 9 5 % air at 37°C, remaining undifferentiated in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS) (GIBCO) and HAT (100 p M hypoxanthine, 1 pM aminopterin and 16 p M thymidine) [4]. The cells were differentiatd by cultivating them for 7 days in D M E M supplemented with 2% FCS, 1 mM dibutyryl cyclide AMP and HAT [6]. To investigate the effect of pertussis toxin (PT), the cells were treated by the toxin (10 ng/ml) added to the medium 18 h before each experiment. For monitoring [Ca2+]~ changes, the cells were preincubated with 2 ,uM fura-2AM in a Ham's-10 medium containing 0.1% bovine serum albumin (fraction V, Sigma) for 20 rain at 37 ° C. After washing twice with a HEPES-buffered medium [10], the fura-2-1oaded cells were perfused at room temperature (about 25°C) at a 3 ml/min flow rate in a perfusion chamber (bath volume, 200 pl) mounted on the stage of an inverted epi-fluorescence microscope.
~4 HEPES-buffered medium containing BK (1 nM), EK (1 /aM) or BK (1 n M ) + E K (1/aM) was used for the perfusion and switched from one to the other at the lime indicated in the figures. The Cae+-induced fluorescence images of each single cell obtained by UV excitation at 340 and 380 nm, were analyzed by means of a digital image processor ( A R G U S 100, H a m a m a t s u Photonic) as previously described [9, 18]. Fig. I A shows time-dependent changes in [Ca>I, in
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Fig. 1. Effect of the prior application of BK on the EK-induced [Ca>]i rise in single differentiated cells. The fluorescence ratios (ratio of the emission intensity obtained by the excitation at 340 to that at 380 nm) of single cells were represented as [Ca2+]~and plotted against time. The cells were grown and differentiated on a coverslip, and were perfused with the medium supplemented with BK (1 nM) and that with BK (1 nM)+ EK (1/tM), successively.Both media contained 2 mM Ca2.. A: time courses of the cells treated with EK following BK treatment, a-g: time courses of [Ca2÷]i of cell (a) to cell (g). The cells were chosen at random from about 40 cells appearing in the same field, h: averaged time course of lCa2+l,of(a) to (g) +_S.D. B: an averaged time course of [Ca2+]~of 7 cells treated with EK without BK pretreatment. A representative of at least 4 separate experiments is shown.
differentiated NG108-15 cells. Undifferentiated cells showed essentially the same results (dala not shown~. The time course patterns (a) to (g) show [Ca:']~ changes in 7 arbitrarily chosen single cells and (h) shows the averaged [Ca2+]i change in these 7 cells. When the BK (1 nM)containing medium was switched to the BK (l nM) + EK (1/aM) medium, the average [Ca2'-]~ clearly increased in response to EK. Although the response levels of individual cells were different from each other, in most single cells a dual [Ca2+]i rise was observed (a d, 13. In contrast to the harvested cells in suspension, the cells on a glass slip showed no detectable [Ca>]~ increase following the application of E K (1/aM) alone (Fig. I B). Based on this result, we conclude that EK causes [Ca>]~ increase only under the influence of BK, and that the previously observed [Ca2+]i increase by E K alone [13] was probably due to E K cooperating with an endogenous stimulant such as released ATP as mentioned before. This idea is consistent with a preliminary result that the action of l /aM E K without BK was undetectable when a cell suspension was treated with apyrase, a hydrolytic enzyme mixture for ATP and its derivatives. Fig. 2 shows that pretreatment with PT 10 ng/ml) completely suppressed the E K (1/aM)-induced [Ca2+]i increase which occurred when 1 nM BK was also present in the medium but did not suppress the [Ca2+]~ increase caused by l nM BK alone. This suggests that a PT-sensitive G-protein is involved in the mechanism of the EKinduced [Ca2~]i rise, but not in the [Ca2']~ rise caused by BK alone. As shown in Fig. 3, the [Ca2+]~ increases brought about by BK (1 nM) alone and E K (1/aM) and BK (1 nM) in combination were basically unaffected by lowering the extracellular Ca 2+ concentration to such an extent that the K*-induced opening of a voltage-dependent Ca 2~ channel cannot occur. This result shows that both BKand EK-induced [Ca>]~ increases are mainly due to intracellular Ca 2+ mobilization. We have recently found a similar cross-talk between 2 receptor systems. Adenosine or Aj-purinergic agonists, signals for adenylate cyclase inhibition, induced intracellular Ca 2+ mobilization in the presence of known Ca 2~ mobilizing agents, including 0t-adrenergic and P2-purinergic agonists, and thyrotropin [7, 11, 12, 16]. Only the adenosine-dependent part of the [Ca>]~ increase was abolished by the PT treatment of the cells. Since a similar permissive stimulation by adenosine was found in phospholipase C (PLC) activation in a PT-sensitive manner, the adenosine-dependent Ca 2+ mobilization was explained as a result of the increased phosphoinositide hydrolysis [7, 11, 12, 16]. These results suggest an analogous mechanism for the EK-induced Ca 2+ mobilization. In fact, in our prelimi-
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Fig. 2. Effect of pertussis toxin treatment on the BK-assisted EK-induced [Ca>], rise. Cells were treated with pertussis toxin (10 ng/ml) 18 h before the experiments. Other experimental conditions and the expression of results were the same as described in the legend to Fig. 1.
nary experiment using undifferentiated NG108-15 cells, PT-sensitive and EK-dependent PLC stimulation was found in the presence of BK. Therefore, the cooperation between EK and BK could primarily induce a functional coupling between E K and PLC via a PT-sensitive G-protein. With respect to the mechanism of the cooperation, the primary [Ca>]i increase caused by BK may not be the key to the sensitization of the Ca 2+ signaling mechanism to EK, since as seen in the Figs. 1A and 3, EK-induced [Ca>]t increase occurred after the [Ca2+]i returned to the original level. On the other hand, BK must be continuously present in the medium in order to sensitize the cells to EK. This idea is not inconsistent with our preliminary result in which the addition of apyrase, the ATP hydrolytic enzyme, allowed 30 p M ATP which mimics the BK action, to induce a weak [Ca2+]i transient, but not the EK-dependent Ca `,+ mobilization. This is probably due to the exhaustion of ATP in the later period when EK (1 /tM) was applied. Further details of the cross-talk mechanism are under investigation. The physiological role of the EK-induced [Ca>]~ increase in the cells with neuronal activity is unknown.
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Fig. 3, Effect of low extracellular Ca-'* concentration. The medium for perfusion was supplemented with 2.2 m M EGTA. Other experimental conditions and the expression of results were the same as described m the legend 1o Fig. I.
Since this EK action is expressed even in the undifferentiated NG108-15 cells, this response is not likely to directly link to neuronal activities specific to differentiated cells. On the other hand, when we consider that the EK-induced [Ca2+]i increase only occurs in cooperation with BK, a pain mediator [1], this 'paradoxical effect' of EK may account for some aspects of opioid-induced "excitory effects', which have recently been reported [5, 15]. We thank Drs. H. Higashida (Kanazawa University) and M. Ui (University of Tokyo) for providing NG10815 cells and pertussis toxin, respectively. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan. 1 Baccaglini, P.I. and Hogan, RC., Some rat sensory neurons in culture express characteristics of differentiated pain sensory cclls., Proc. Natl. Acad. Sci. USA, 80 (1983) 594-598. 2 Brown, D.A., Higashida, H., Adams, P.R., Marrion, N.V. and Smart, T.G., Role of G-protein coupled phosphatidylinositide system in signal transduction in vertebrate neurons: experiments on neuroblastoma hybrid cells and ganglion cells, Cold Spring Harbor Syrup. Quant. Biol., 53 (1988) 375 384.
96 3 Gilman, A.G., G-protein: transducers of receptor-generated signals., Annu. Rev. Biochem., 56 (1987) 617 649. 4 Hirano, Y., Okajima, F., Tomura, H., Majid, M.A.. Takeuchi, T. and Kondo, Y., Change of intracellular calcium of neural cells reduced by extracellular ATR FEBS Lett., 284 (1991) 235-237. 5 Kayser, V., Besson, J.M. and Guilband, G., Paradoxical hyperalgesic effect of exceedingly low doses of systemic morphine in an animal model of persistent pain (Freund's adjuvant-induced arthritic rats), Brain Res., 414 (1987) 155 157. 6 Mullaney, I., Magee, A.I., Unson, C.G. and Milligan, G., Differential regulation of amounts of the guanine-nucleotide-binding proreins G~ and G o in neuroblastoma × glioma hybrid cells in response to dibutyryl cyclic AMP, Biochem. J., 256 (1988) 649-656. 7 Nazarea, M., Okajima, F. and Kondo, Y., P2-purinergic activation of phosphoinositide turnover is potentiated by A~-receptor stimulation in thyroid cells, Eur. J. Pharmacol., 206 (1991) 4% 52. 8 Nirenberg, M., Wilson, S., Higashida, H., Rotter, A., Krueger, K., Busis, N., Ray, R., Kenimer, J.G. and Adler, M., Modulation of synapse formation by cyclic adenosine monophosphate, Science, 222 (1983) 794-799. 9 0 g u r a , A., Myojo, Y. and Higashida, H., Bradykinin-evoked acetylcholine release via inositol trispbosphate-dependent elevation in free calcium in neuroblastoma x glioma hybrid NG108-15 cells, J. Biol. Chem., 265 (1990) 3577 3584. 10 Okajima, F., Sho, K. and Kondo, Y., Inhibition by islet-activating protein, pertussis toxin, of P2-purinergic receptor-mediated iodide efflux and phosphoinositide turnover in FRTL-5 cells, Endocrinology, 123 (1988) 1035 1043. 11 Okajima, F., Sato, K., Nazarea, M., Sho, K. and Kondo, Y., A permissive role of pertussis toxin substrate G-protein in P2-puriner-
gic stimulation of phosphoinositide turnover and arachidonate release in FRTL-5 thyroid cells, J. Biol. Chem., 264 (1989) 13029 13037. 12 Okajima, F., Sato, K., Sho, K. and Kondo, Y., Stimulation otadenosine receptor enhances ~-adrenergic receptor-mediated activation of phospholipase C and Ca > mobilization in a pertussis toxinsensitive manner in FRTL-5 thyroid cells, FEBS Lett., 248 (1989) 145 149. 13 Okajima, F. and Kondo, Y., Synergism in cytosolic Ca 2 mobilization between bradykinin and agonists for pertussis toxin-sensitive G-protein-coupled receptors in NG108-15 cells, FEBS Lett., 301 (1992) 223-226. 14 Osugi, T., Imaizumi, T., Misushima, A., Uchida, S. and Yoshida, H., Phorbol ester inhibits bradykinin-stimulated inositol trisphosphate formation and calcium mobilization in neurobtastoma × glioma hybrid NG 108-15 cells, J. Pharmacol. Exp. Ther., 240 (1987) 617-622. 15 Shen, K.-F. and Craia, S.M., Dual opioid modulation of the action potential duration of mouse dorsal root ganglion neurons in culture, Brain Res., 491 (1989) 227-242. 16 Sho, K., Okajima, F., Majid, M.A. and Kondo, Y., Reciprocal modulation of thyrotropin actions by P~-purinergic agonists in FRTL-5 thyroid ceils, J. Biol. Chem., 266 (1991) 12180-12184. 17 Tsunoo, A., Yoshii, M. and Narahashi, T., Block of calcium channels by enkephalin and somatostatin in neuroblastoma-glioma hybrid NGI08-15 cells, Proc. Natl. Acad. Sci. USA, 83 (1986) 9832 9836. 18 Tzuzuki, K., Iino, M. and Ozawa, S., Change in calcium permeability caused by quinolinic and in cultured rat hippocampal neurons, Neurosci. Lett., 105 (1989) 269-274.
93
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Enkephalin induces Ca 2+ mobilization in single cells of bradykininsensitized differentiated neuroblastoma hybridoma (NG 108-15) cells H i d e a k i T o m u r a , F u m i k a z u O k a j i m a a n d Yoichi K o n d o Department of Physical Biochemistry, Institute of Endocrinology, Gunma University, Maebashi (Japan) (Received 17 June 1992; Revised version received 18 September 1992; Accepted 21 September 1992)
Key words: Enkephalin; Bradykinin; Cytosolic Ca2+; Pertussis toxin: NG108-15 cell A study of the intracellular Ca ~+ ([Ca2+]) response of differentiated neuroblastoma x glioma hybrid cells (NG108-15 cell) to enkephalin (EK) was carried out by fura-2 video-imaging. EK alone did not influence [Ca:+]~ in single cells. The opioid did, however, induce a marked [Ca>I, rise, when the cells were incubated with bradykinin (BK) prior to the EK treatment. Such BK-assisted stimulation of the differentiated hybridoma cells by EK was completely abolished by pertussis toxin treatment. These results suggest that in single NG108-15 cells, EK induces Ca 2+ mobilization which is assisted by cross-talk between the EK and BK receptor systems via a pertussis toxin-sensitive G protein.
NG108-15 cells are neuroblastoma x glioma hybrid cells. When cAMP producing agents are added to the culture medium, the cells show some morphological and physiological features of neuronal cells. Among the neurotransmitter receptors found in cells [2, 8], bradykinin (BK) [14] and Pz-receptors [4] are known to be coupled to a phospholipase C (PLC) and are stimulatory. In contrast to these, opioid, az-adrenergic, somatostatin and muscarinic receptors are coupled to an adenylate cyclase and are inhibitory [8]. Enkephalin (EK), an opioid agonist, as well as somatostatin has also been shown to block Ca 2+ inward currents in differentiated NG108-15 cells [17]. On the other hand, we have found [13] that, in a suspension of undifferentiated NG108-15 cells, EK slightly but appreciably increases the intracellular Ca 2+ concentration ([Ca2+]i). Furthermore, this EK-dependent [Ca2+]i increase is markedly enhanced by prior stimulation of the cells with bradykinin (BK), a Ca 2+ mobilizing agent. These findings, however, raised two questions: first, whether the [Ca2+]i increase by EK without BK is due to the action of EK itself, or due to the EK action under the influence of an endogenous stimulant(s) which can act similarly to BK. A candidate for such a stimulant is ATP which might be liberated from damaged cells during harvesting. This seems reasonable since exogenously added Correspondence: H. Tomura, Department of Physical Biochemistry, Institute of Endocrinology, Gunma University, Maebashi 371, Japan. Fax: (81) (272) 34-1788.
ATP has been shown to act as a Ca z+ mobilizing agent and has been shown to mimic the action of BK by enhancing EK-dependent [Ca2+]~ increase. The second question is whether the enhanced EK-dependent [Ca2+]~ increase really occurs in the cell where the BK-induced [Ca2+]i transient has been observed. To answer these questions, in the present study, we analyzed EK-induced [Ca2+]~ changes by fura-2 video imaging of single NG 10815 cells grown on glass plates without harvesting. NG108-15 cells were provided by Dr. Higashida, Kanazawa University, Japan. The cells were grown on coverslips (Matsunami Glass, Tokyo, Japan) in an atmosphere of 5% C O 2 - 9 5 % air at 37°C, remaining undifferentiated in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS) (GIBCO) and HAT (100 p M hypoxanthine, 1 pM aminopterin and 16 p M thymidine) [4]. The cells were differentiatd by cultivating them for 7 days in D M E M supplemented with 2% FCS, 1 mM dibutyryl cyclide AMP and HAT [6]. To investigate the effect of pertussis toxin (PT), the cells were treated by the toxin (10 ng/ml) added to the medium 18 h before each experiment. For monitoring [Ca2+]~ changes, the cells were preincubated with 2 ,uM fura-2AM in a Ham's-10 medium containing 0.1% bovine serum albumin (fraction V, Sigma) for 20 rain at 37 ° C. After washing twice with a HEPES-buffered medium [10], the fura-2-1oaded cells were perfused at room temperature (about 25°C) at a 3 ml/min flow rate in a perfusion chamber (bath volume, 200 pl) mounted on the stage of an inverted epi-fluorescence microscope.
~4 HEPES-buffered medium containing BK (1 nM), EK (1 /aM) or BK (1 n M ) + E K (1/aM) was used for the perfusion and switched from one to the other at the lime indicated in the figures. The Cae+-induced fluorescence images of each single cell obtained by UV excitation at 340 and 380 nm, were analyzed by means of a digital image processor ( A R G U S 100, H a m a m a t s u Photonic) as previously described [9, 18]. Fig. I A shows time-dependent changes in [Ca>I, in
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Fig. 1. Effect of the prior application of BK on the EK-induced [Ca>]i rise in single differentiated cells. The fluorescence ratios (ratio of the emission intensity obtained by the excitation at 340 to that at 380 nm) of single cells were represented as [Ca2+]~and plotted against time. The cells were grown and differentiated on a coverslip, and were perfused with the medium supplemented with BK (1 nM) and that with BK (1 nM)+ EK (1/tM), successively.Both media contained 2 mM Ca2.. A: time courses of the cells treated with EK following BK treatment, a-g: time courses of [Ca2÷]i of cell (a) to cell (g). The cells were chosen at random from about 40 cells appearing in the same field, h: averaged time course of lCa2+l,of(a) to (g) +_S.D. B: an averaged time course of [Ca2+]~of 7 cells treated with EK without BK pretreatment. A representative of at least 4 separate experiments is shown.
differentiated NG108-15 cells. Undifferentiated cells showed essentially the same results (dala not shown~. The time course patterns (a) to (g) show [Ca:']~ changes in 7 arbitrarily chosen single cells and (h) shows the averaged [Ca2+]i change in these 7 cells. When the BK (1 nM)containing medium was switched to the BK (l nM) + EK (1/aM) medium, the average [Ca2'-]~ clearly increased in response to EK. Although the response levels of individual cells were different from each other, in most single cells a dual [Ca2+]i rise was observed (a d, 13. In contrast to the harvested cells in suspension, the cells on a glass slip showed no detectable [Ca>]~ increase following the application of E K (1/aM) alone (Fig. I B). Based on this result, we conclude that EK causes [Ca>]~ increase only under the influence of BK, and that the previously observed [Ca2+]i increase by E K alone [13] was probably due to E K cooperating with an endogenous stimulant such as released ATP as mentioned before. This idea is consistent with a preliminary result that the action of l /aM E K without BK was undetectable when a cell suspension was treated with apyrase, a hydrolytic enzyme mixture for ATP and its derivatives. Fig. 2 shows that pretreatment with PT 10 ng/ml) completely suppressed the E K (1/aM)-induced [Ca2+]i increase which occurred when 1 nM BK was also present in the medium but did not suppress the [Ca2+]~ increase caused by l nM BK alone. This suggests that a PT-sensitive G-protein is involved in the mechanism of the EKinduced [Ca2~]i rise, but not in the [Ca2']~ rise caused by BK alone. As shown in Fig. 3, the [Ca2+]~ increases brought about by BK (1 nM) alone and E K (1/aM) and BK (1 nM) in combination were basically unaffected by lowering the extracellular Ca 2+ concentration to such an extent that the K*-induced opening of a voltage-dependent Ca 2~ channel cannot occur. This result shows that both BKand EK-induced [Ca>]~ increases are mainly due to intracellular Ca 2+ mobilization. We have recently found a similar cross-talk between 2 receptor systems. Adenosine or Aj-purinergic agonists, signals for adenylate cyclase inhibition, induced intracellular Ca 2+ mobilization in the presence of known Ca 2~ mobilizing agents, including 0t-adrenergic and P2-purinergic agonists, and thyrotropin [7, 11, 12, 16]. Only the adenosine-dependent part of the [Ca>]~ increase was abolished by the PT treatment of the cells. Since a similar permissive stimulation by adenosine was found in phospholipase C (PLC) activation in a PT-sensitive manner, the adenosine-dependent Ca 2+ mobilization was explained as a result of the increased phosphoinositide hydrolysis [7, 11, 12, 16]. These results suggest an analogous mechanism for the EK-induced Ca 2+ mobilization. In fact, in our prelimi-
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Fig. 2. Effect of pertussis toxin treatment on the BK-assisted EK-induced [Ca>], rise. Cells were treated with pertussis toxin (10 ng/ml) 18 h before the experiments. Other experimental conditions and the expression of results were the same as described in the legend to Fig. 1.
nary experiment using undifferentiated NG108-15 cells, PT-sensitive and EK-dependent PLC stimulation was found in the presence of BK. Therefore, the cooperation between EK and BK could primarily induce a functional coupling between E K and PLC via a PT-sensitive G-protein. With respect to the mechanism of the cooperation, the primary [Ca>]i increase caused by BK may not be the key to the sensitization of the Ca 2+ signaling mechanism to EK, since as seen in the Figs. 1A and 3, EK-induced [Ca>]t increase occurred after the [Ca2+]i returned to the original level. On the other hand, BK must be continuously present in the medium in order to sensitize the cells to EK. This idea is not inconsistent with our preliminary result in which the addition of apyrase, the ATP hydrolytic enzyme, allowed 30 p M ATP which mimics the BK action, to induce a weak [Ca2+]i transient, but not the EK-dependent Ca `,+ mobilization. This is probably due to the exhaustion of ATP in the later period when EK (1 /tM) was applied. Further details of the cross-talk mechanism are under investigation. The physiological role of the EK-induced [Ca>]~ increase in the cells with neuronal activity is unknown.
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Fig. 3, Effect of low extracellular Ca-'* concentration. The medium for perfusion was supplemented with 2.2 m M EGTA. Other experimental conditions and the expression of results were the same as described m the legend 1o Fig. I.
Since this EK action is expressed even in the undifferentiated NG108-15 cells, this response is not likely to directly link to neuronal activities specific to differentiated cells. On the other hand, when we consider that the EK-induced [Ca2+]i increase only occurs in cooperation with BK, a pain mediator [1], this 'paradoxical effect' of EK may account for some aspects of opioid-induced "excitory effects', which have recently been reported [5, 15]. We thank Drs. H. Higashida (Kanazawa University) and M. Ui (University of Tokyo) for providing NG10815 cells and pertussis toxin, respectively. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan. 1 Baccaglini, P.I. and Hogan, RC., Some rat sensory neurons in culture express characteristics of differentiated pain sensory cclls., Proc. Natl. Acad. Sci. USA, 80 (1983) 594-598. 2 Brown, D.A., Higashida, H., Adams, P.R., Marrion, N.V. and Smart, T.G., Role of G-protein coupled phosphatidylinositide system in signal transduction in vertebrate neurons: experiments on neuroblastoma hybrid cells and ganglion cells, Cold Spring Harbor Syrup. Quant. Biol., 53 (1988) 375 384.
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