©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Heterologous Desensitization of Opioid-stimulated Ca Increase by Bradykinin or ATP in NG108-15 Cells (*)

Sheau-Huei Chueh (§) , Shu-Ling Song (1), Tsui-Ying Liu

From the (1)Department of Biochemistry and Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan, Republic of China

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Leucine-enkephalin (Leu-EK) dose-dependently elicited an increase in cytosolic Ca concentration ([Ca]) with an EC of 1.2 µM via the phosphoinositide cascade in NG108-15 cells. Chronic treatment of cells with [D-Ala,D-Leu]enkephalin caused time-dependent homologous desensitization. In the presence of extracellular Ca, ATP as well as bradykinin stimulated significantly higher increases in inositol 1,4,5-trisphosphate (IP) generation than did Leu-EK; however, the magnitude of intracellular Ca pools increased after ATP stimulation, whereas bradykinin depleted intracellular pools. Hence, cells lost their [Ca] response to Leu-EK if bradykinin was first added to induce a [Ca] increase, whereas the response was unchanged if Leu-EK was added after addition of ATP. When Leu-EK was added simultaneously with bradykinin or ATP, an additive response was observed in IP generation; however, the rise in [Ca] reached the same level as that induced by bradykinin or ATP alone. In the absence of extracellular Ca in which the replenishment of intracellular pools was not possible, ATP displayed an inhibitory effect similar to that of bradykinin on the Leu-EK-induced [Ca] increase. Prior treatment of cells with Leu-EK slightly heterologously desensitized the action of bradykinin, but had no effect on the ATP response. Our results suggest that a shared intracellular Ca pool is sensitive to the opioid, bradykinin and P-purinoceptor agonists; however, a defined pool of phosphatidylinositol 4,5-bisphosphate or a specific phospholipase C is responsible for each receptor.


INTRODUCTION

Neuroblastoma glioma hybrid NG108-15 cells have proven to be a favorable model system for the study of signal transduction processes in neuronal cells(1) . NG108-15 cells possess various receptors, including opioid(2) , -adrenergic(3) , muscarinic cholinergic(4) , bradykinin(5) , prostaglandin(6) , P-purinoceptor(7) , endothelin(8) , and angiotensin II (9) receptors. The NG108-15 cell line has been extensively used by opioid investigators, since it only possesses the -opioid receptor(10, 11) . -Opioid activation results in the inhibition of adenylate cyclase via a pertussis toxin-sensitive G in NG108-15 cells(12) . It has also been demonstrated that opioid agonists block voltage-sensitive Ca channels in the same cells and that a pertussis toxin-sensitive G is involved in the coupling process (13-15). In addition to its inhibitory action, opioids have very recently been found to elicit a direct excitatory effect in NG108-15 cells. Thus, opioid agonists stimulate an increase in the cytosolic Ca concentration ([Ca]).()Depending upon the differentiation status of the cells, the [Ca] increase results from either Ca influx or Ca release from the internal stores(16, 17) . Therefore, at least four distinct signaling pathways are evoked upon opioid activation within NG108-15 cells. Furthermore, chronic treatment of NG108-15 cells with opioid agonists results in a series of molecular processes resembling tolerance and dependence. These processes include desensitization and down-regulation of receptors and an increase in adenylate cyclase activity(18, 19) .

Using NG108-15 cells, we have previously reported that high extracellular K induced a greater rise in [Ca] in dibutyryl cAMP-treated cells than in control cells due to the development of both L- and N-type Ca channels during the cAMP-induced differentiation process(20) . In addition, ATP specifically elevated [Ca]. The ATP-induced rise in [Ca] was switched from a mechanism that originated from both Ca influx and IP-induced Ca release to one that predominantly resulted from Ca influx after differentiation(21, 22) . On the other hand, it was shown that bradykinin stimulated phospholipase C to generate IP and diacylglycerol and that IP then triggered Ca release from internal Ca stores in NG108-15 cells(5, 23) . We have shown that high extracellular K synergistically enhanced the bradykinin action on the [Ca] increase(24) .

Many studies have shown that cross-interaction between distinct receptor signaling systems modulates cellular functions. In the current study, we characterized the possible cross-interactions among Leu-EK, bradykinin, and ATP in the Ca signaling in undifferentiated NG108-15 cells. We found that cells lost their [Ca] response to Leu-EK following depletion of a shared Ca pool by bradykinin or ATP in the absence of extracellular Ca. In contrast, ATP did not affect the action of Leu-EK in the presence of extracellular Ca, since the Ca pools were replenished by ATP through the entry of extracellular Ca.


EXPERIMENTAL PROCEDURES

Culture of Neuroblastoma Glioma Cells

Clonal neuroblastoma glioma NG108-15 cells, obtained from Dr. M. Nirenberg, National Institutes of Health (Bethesda, MD), were used between passages 21 and 35. Cells were maintained at 37 °C in Dulbecco's modified Eagle's medium (with high glucose) supplemented with 10% fetal bovine serum, 100 µM hypoxanthine, 1 µM aminopterin, and 16 µM thymidine in a humidified atmosphere of 95% air and 5% CO. Cells were plated in 100 20-mm tissue culture dishes (Falcon). Where indicated, [D-Ala,D-Leu]enkephalin (DADLE) treatment of the cells was performed by adding 100 nM DADLE to the medium 4, 24, or 48 h before experiments.

Measurement of [Ca]

Elevation of [Ca] in populations of cells was calculated from the change of the fluorescence ratio of fura-2-loaded cells, as described(21, 24) . Briefly, after harvesting cells from the culture dishes, cells were suspended at a density of 1 10 cells/ml and incubated with 5 µM fura-2 acetoxymethyl ester in a buffer containing 150 mM NaCl, 5 mM KCl, 5 mM glucose, 1 mM MgCl, 2.2 mM CaCl, and 10 mM HEPES, pH 7.4 (designated loading buffer) at 37 °C for 30 min. Changes of the fluorescence ratio at 340 and 380 nm in response to various agonists were measured on line with an emission wavelength of 505 nm (Spex; CM System). The [Ca] was then calculated from the fluorescence ratio with a K of 224 nM for fura-2 and Ca equilibrium, as described previously(25) . All experiments were performed at least three times using different batches of cells. Results from one representative experiment are illustrated graphically, and mean values ± S.D. for [Ca] increase, calculated from n experiments, are shown. Statistical differences between mean values were assessed by Student's t test.

Determination of IPGeneration

IP generation within cells stimulated by Leu-EK, ATP, bradykinin, Leu-EK and bradykinin, or Leu-EK and ATP either in the presence or in the absence of extracellular Ca was quantified as described previously(22) . Briefly, aliquots of cells (about 0.5 mg of cell protein) were incubated with the indicated agonists at 37 °C for 15 s in loading buffer in a total volume of 100 µl and then IP generation was terminated by the addition of 3.33% (v/v) perchloric acid. After incubation for 20 min at 4 °C, the precipitated cells were removed by centrifugation at 12,000 g for 5 min. After neutralization to pH 7.5, IP production in extracts were determined by using the D-myo-[H]IP radioreceptor assay system (Amersham) according to the manufacturer's instructions. All experiments were carried out at least three times in triplicate with similar results, and the data presented are mean values ± S.D. from one representative triplicate experiment.

Estimation of cAMP Production

Cyclic AMP production within cells in response to prostaglandin E, bradykinin, or ATP either in the absence or in the presence of Leu-EK was determined as described(22) . Briefly, aliquots of cells (approximate 0.5 mg of cell protein) were incubated with the indicated agonists at 37 °C for 15 min in a total volume of 100 µl of loading buffer. Cells were then chilled on ice for 1 h after cAMP production was terminated by the addition of 10 µl of 1 N HCl. After boiling for 1 min, cells were centrifuged at 12,000 g for 10 min, and supernatants were neutralized to pH 7.4 before cAMP generation was quantified. Aliquots of extract were used for determination of cAMP by the [H]cAMP assay system (Amersham) according to the manufacturer's instructions. Typical triplicate experimental results are presented as mean values ± S.D. The same experiments were carried out three times with similar results.

Materials

Dulbecco's modified Eagle's medium, hypoxanthine/aminopterin/thymidine, and fetal bovine serum were purchased from Life Technologies, Inc. fura-2 acetoxymethyl ester was from Molecular Probes (Eugene, OR). Bradykinin, Leu-EK, ATP, DADLE, naloxone, and ionomycin were obtained from the Sigma. Thapsigargin was purchased from Research Biochemicals Inc. (Natick, MA). The [H]IP and [H]cAMP assay systems were obtained from the Amersham Corp. All other chemicals used were of analytical grade and were obtained from Merck.


RESULTS

Heterologous desensitization of the rise in [Ca] after -opioid and bradykinin receptor activation by sequential addition of these two receptor agonists is illustrated in Fig. 1. In the presence of extracellular Ca, 10 µM Leu-EK induced a small, but significant, increase in [Ca] (Fig. 1A, trace a). The [Ca] increase in response to 10 µM bradykinin was even higher (Fig. 1A, trace b). When Leu-EK was added after cells had been previously exposed to bradykinin, the [Ca] increase was significantly inhibited; [Ca] rose only marginally (Fig. 1A, trace b). In contrast, if bradykinin was added after a Leu-EK evoked [Ca] rise had occurred, only a slight inhibition was observed (Fig. 1A, trace a). Similar experiments were undertaken to examine possible cross interactions between -opioid receptor and P-purinoceptor. As shown in Fig. 1A, traces c and d, ATP (3 mM) did not change the subsequent effect of Leu-EK (10 µM) on [Ca], nor did Leu-EK change the response of [Ca] to ATP at the same concentration. The statistical data of the changes in [Ca]induced by Leu-EK, bradykinin, or ATP under the conditions described above are shown in Fig. 1B.


Figure 1: Heterologous desensitization of the [Ca] increase induced by sequential agonist stimulation. A, changes of [Ca] in fura-2-loaded NG108-15 cells (2 10 cells) were measured in the presence of extracellular Ca in response to sequential stimulation. Sequential stimulation was induced as follows: Leu-EK (10 µM) (EK) and then bradykinin (10 µM) (BK) (trace a) or vice versa (trace b), Leu-EK (10 µM) and then ATP (3 mM) (trace c) or vice versa (trace d). B, the statistical data of [Ca] changes induced by Leu-EK, bradykinin, or ATP under the conditions described above are summarized in a and b. The values are the mean ± S.D., n = 18.



The concentration dependence of the change of [Ca] upon bradykinin or ATP addition in cells with or without Leu-EK pretreatment is illustrated in Fig. 2, A and B, respectively. Pretreatment of cells with Leu-EK only lowered by approximately 25% the maximal response in the bradykinin-evoked [Ca] increase without affecting the EC value for bradykinin; the EC values for bradykinin were 1.5 and 2.5 µM for cells with or without preexposure to Leu-EK, respectively (Fig. 2A). On the other hand, the concentration dependence of ATP on the [Ca] increase displayed similar profiles in both sets of cells regardless of cell exposure to Leu-EK (Fig. 2B). The EC for ATP was about 1 mM, which is consistent with our previous results (21). It appears that Leu-EK transduces its signal through a pathway which is analogous to that of bradykinin but different from that of ATP. This notion is further supported by the experimental results of the Leu-EK dependence in the rise in [Ca]within cells in which the [Ca] increase had been initially induced by bradykinin or ATP. Thus, the response of the [Ca] increase stimulated by Leu-EK for all concentrations tested remained at only 15% if the cells had been preincubated with 10 µM bradykinin, whereas preincubation of cells with ATP had no effect on Leu-EK sensitivity (Fig. 2C). The EC of [Ca] increase for Leu-EK was about 1.2 µM.


Figure 2: Dose-dependent effect of bradykinin, ATP, or Leu-EK on [Ca] increase. The rise in [Ca] was measured for the indicated doses of bradykinin (A) or ATP (B) 120 s after cells were () or were not () initially stimulated with 10 µM Leu-EK. After prior treatment cells with buffer (), 10 µM bradykinin () or 3 mM ATP () for 120 s, the Leu-EK induced [Ca] increase was determined under various concentrations as indicated (C). The maximal [Ca] increases induced by 10 µM bradykinin (A), 3 mM ATP (B), or 10 µM Leu-EK (C) of 305 ± 46, 395 ± 54, and 96 ± 18 nM, respectively, were taken as 100%. Data are mean values ± S.D. from four independent experiments.



The effect of -opioid activation on the rise in [Ca] has been documented only recently. The action of Leu-EK in the current study was specific to the activation of the -opioid receptor. Thus, 10 µM naloxone antagonized the ability of Leu-EK to increase [Ca], but had no effect on the receptor activation induced by bradykinin or P-purinoceptor (Fig. 3). Furthermore, it has been shown previously that chronic treatment of NG108-15 cells with opioid agonists caused homologous desensitization in the adenylate cyclase signaling pathway (18, 19). Our results also indicate homologous desensitization in the rise in [Ca] induced by -receptor activation. As depicted in Fig. 4A, after preincubation of cells with 100 nM DADLE for 4, 24, or 48 h, the Leu-EK-mediated [Ca] increase was inhibited by approximately 53, 53, or 78%, respectively, compared with a [Ca] increase of 94 ± 11 nM (n = 18) in control cells. On the other hand, chronic treatment with DADLE only induced partial heterologous desensitization of the bradykinin receptor induced rise in [Ca] (Fig. 4B), whereas no significant cross-interaction was observed between P and receptors after similar treatment of cells (Fig. 4C).


Figure 3: Leu-EK-induced [Ca] increases mediated by the opioid receptor. fura-2-loaded cells were initially exposed to 10 µM naloxone (Nal) in all traces. After 35 s, 10 µM Leu-EK (EK) (trace a), 10 µM bradykinin (BK) (trace b), or 3 mM ATP (trace c) was added as indicated. The same experiments were carried out three times with similar results.




Figure 4: Effect of chronic DADLE treatment on the [Ca] increases evoked by Leu-EK, bradykinin, or ATP. Cells were incubated with 100 nM DADLE for 0, 4, 24, or 48 h in culture as indicated; then the [Ca] increase evoked by 10 µM Leu-EK (A), 10 µM bradykinin (B), or 3 mM ATP (C) was measured individually. Data are presented as mean values ± S.D., n = 15. The asterisk represents p < 0.001. The change of [Ca] induced by Leu-EK is significantly different from that in control cells without DADLE pretreatment; the number sign represents p < 0.001, significantly different from the bradykinin-induced [Ca] increase in control cells.



Based on the above data, it appears that Leu-EK may induce Ca release from intracellular Ca pools in a way similar to the well established bradykinin mechanism(22, 24) rather than by Ca influx as in the well known ATP mechanism(21, 22) . To study further whether internal Ca release is triggered by Leu-EK, we examined the relevance of intracellular Ca pools in the Leu-EK-induced Ca signaling event compared with that induced by bradykinin or ATP. As shown in Fig. 5A, in the absence of extracellular Ca and with 0.5 mM EGTA included, virtually all receptor activation still caused a [Ca] increase; the [Ca] increased from a basal level of 30 ± 6 nM (n = 19) to a maximal peak level of 65 ± 9 nM (n = 5) (trace b), 98 ± 10 nM (n = 5) (trace c), and 48 ± 6 nM (n = 4) (trace d) in response to 10 µM Leu-EK, 10 µM bradykinin, and 3 mM ATP, respectively. When Ca was subsequently reintroduced, [Ca] rose immediately in all sets of cells and remained at a sustained level of 120 ± 14 nM (n = 5) in control cells (trace a). The sustained level in Leu-EK- or bradykinin-stimulated cells was close to that of control cells. However, the sustained level of [Ca] was about twice that of control cells in ATP-stimulated cells. Thapsigargin inhibits the internal Ca pump and the subsequent refilling of the intracellular Ca pools, causing them to become depleted(26) . ATP was found to cause a significant increase in [Ca]in thapsigargin-treated cells, although the magnitude was decreased by about 60% on average (Fig. 5B, trace c). In contrast, the effects of Leu-EK or bradykinin on [Ca] was abolished almost completely by thapsigargin pretreatment (Fig. 5B, traces a and b).


Figure 5: Independence or dependence of [Ca] responses on extracellular Ca or intracellular Ca pools. A, fura-2-loaded cells were resuspended in Ca-free loading buffer containing 0.5 mM EGTA, and [Ca] changes were determined in response to the addition of buffer (trace a), 10 µM Leu-EK (EK) (trace b), 10 µM bradykinin (BK) (trace c), or 3 mM ATP (trace d) as indicated. After [Ca] returned to the basal level, 2.2 mM CaCl (Ca) was added in all traces. B, Leu-EK (10 µM) (EK) (trace a), bradykinin (10 µM) (BK) (trace b), or ATP (3 mM) (trace c) was added as indicated in the presence of extracellular Ca after intracellular Ca pools were depleted by treatment with 1 µM thapsigargin (TG).



Our results suggest that Leu-EK triggers Ca release from an intracellular Ca pool as did bradykinin; hence, bradykinin heterologously desensitized the activation of the -opioid receptor (Fig. 1A, trace b, Fig. 2C, and Fig. 5B). To further determine why Leu-EK only slightly inhibited the effect of bradykinin, we next measured IP generation within cells after induction by these agonists. Fig. 6shows that basal IP accumulation during a 15-s incubation period at 37 °C was 12.5 ± 1.6 pmol/mg, n = 3. Bradykinin (10 µM) or ATP (3 mM) significantly increased IP generation to 59.5 ± 1.7 or 51.3 ± 3.5 pmol/mg, respectively, whereas 10 µM Leu-EK only raised the level to 24.6 ± 1.1 pmol/mg. When cells were stimulated by Leu-EK plus bradykinin, or Leu-EK plus ATP, a further increase in IP production was observed; IP production was 71.1 ± 2.4 or 61.8 ± 4.0 pmol/mg after treatment with Leu-EK plus bradykinin or Leu-EK plus ATP, respectively. Removal of extracellular Ca reduced the basal IP production to 5.3 ± 0.2 pmol/mg. Similarly, IP generation upon bradykinin stimulation (39.2 ± 2.5 pmol/mg) was still much higher than after Leu-EK stimulation (8.1 ± 1.1 pmol/mg). On the other hand, ATP stimulation of IP production was severely inhibited in the absence of extracellular Ca (8.2 ± 1.2 pmol/mg). Simultaneous addition of Leu-EK with either bradykinin or ATP caused a further increase in IP production to 46.9 ± 3.6 or 13.5 ± 1.8 pmol/mg, respectively. After subtraction of basal IP generation, in the presence of extracellular Ca an additive effect on IP production was observed when Leu-EK was added simultaneously with bradykinin or ATP, and an effect greater than the additive effect was observed in the absence of extracellular Ca (Fig. 6). In contrast, when Leu-EK was included together with either bradykinin or ATP, the [Ca] increase reached the same level as that induced by bradykinin or ATP alone in the presence of extracellular Ca (Fig. 7).


Figure 6: Effect of extracellular Ca on IP generation induced by Leu-EK, bradykinin, or ATP. Aliquots of cells (about 0.5 mg of cell protein) were stimulated with 10 µM Leu-EK (EK), 10 µM bradykinin (BK), 3 mM ATP, 10 µM Leu-EK and 10 µM bradykinin, or 10 µM Leu-EK and 3 mM ATP as indicated at 37 °C for 15 s in loading buffer (A) or in nominally Ca-free loading buffer (B). IP was then extracted and determined by radioreceptor assay. The Leu-EK-induced IP generation is significantly different from the basal value, p < 0.001 and p < 0.02 in the presence and absence of extracellular Ca, respectively. Results are mean values ± S.D. of triplicate experiments.




Figure 7: [Ca] elevation in response to concomitant stimulation with Leu-EK and bradykinin or Leu-EK and ATP. [Ca] elevation in response to 10 µM Leu-EK (EK) (trace a), 10 µM bradykinin (BK) (trace b), 3 mM ATP (trace c), 10 µM Leu-EK and 10 µM bradykinin (trace d), or 10 µM Leu-EK and 3 mM ATP (trace e) was determined as described under ``Experimental Procedures.'' The same experiments were carried out three times with similar results.



Our results indicate that in the presence of extracellular Ca, bradykinin and ATP display similar effectiveness in activating phospholipase C and that these compounds were significantly more active than Leu-EK (Fig. 6). The reason that ATP did not heterologously desensitize the effect of Leu-EK as bradykinin did could be related to the ability of ATP to stimulate Ca influx (Fig. 5). It is possible that the Ca that entered the cells after exposure to ATP was used to fill the intracellular Ca pools and that the availability of Ca within intracellular Ca pools was a critical consequence of ATP pretreatment. We therefore examined the remaining size of the intracellular Ca pools after cells had been exposed to these agonists. The size of the [Ca] transient induced by ionomycin and EGTA was used as an index of the size of the intracellular Ca pools(27) . As shown in Fig. 8, in the presence of extracellular Ca, the [Ca] transient induced by ionomycin and EGTA displayed an increase in ATP-stimulated cells, but a decrease in bradykinin-, Leu-EK-, or thapsigargin-stimulated cells compared with control cells. The peak levels of [Ca] induced by ionomycin and EGTA were 745 ± 98 nM in control cells and 453 ± 68, 291 ± 53, 1038 ± 168, and 194 ± 44 nM (n = 6) in cells that had been treated with Leu-EK, bradykinin, ATP, and thapsigargin, respectively.


Figure 8: Changes in the magnitude of intracellular Ca pools after exposure to Leu-EK, bradykinin, ATP, or thapsigargin. fura-2-loaded cells were initially exposed to buffer (trace a), 10 µM Leu-EK (EK) (trace b), 10 µM bradykinin (BK) (trace c), 3 mM ATP (trace d), or 1 µM thapsigargin (TG) (trace e). After 120 s, 5 mM EGTA (indicated by a triangle) and 10 µM ionomycin (large arrowhead) was added to estimate the size of intracellular Ca pools.



The above results show that in the presence of extracellular Ca, the size of the intracellular Ca pools was indeed increased after exposure of cells to ATP. We next reassessed the effect of Leu-EK on [Ca] increase in bradykinin- or ATP-stimulated cells in the absence of extracellular Ca to eliminate the effect of ATP on the refilling of intracellular Ca pools. As shown in Fig. 9, stimulation of cells with bradykinin in nominally Ca-free medium caused a concentration-dependent transient in [Ca] that returned to the basal level within 1 min (Fig. 9A). A subsequent challenge with 10 µM Leu-EK caused an additional [Ca] increase, the magnitude of which was inversely proportional to the magnitude of the response of the first stimulation (Fig. 9A). A similar inverse relationship between the amount of Ca mobilized by a submaximal ATP concentration and a subsequent maximal Leu-EK stimulation is clearly demonstrated in Fig. 9B. These data suggest that in the absence of extracellular Ca, both bradykinin and ATP heterologously desensitized the subsequent action of Leu-EK.


Figure 9: Effect of Leu-EK on [Ca] increases in bradykinin- or ATP-stimulated cells in the absence of extracellular Ca. Cells suspended in nominally Ca-free loading buffer were first stimulated with submaximal concentrations of bradykinin (BK) (A) (30 nM (trace a), 3 nM (trace b), 0.3 nM (trace c), or 0.03 nM (trace d)) or submaximal concentrations of ATP (B) (300 µM (trace a), 30 µM (trace b), 3 µM (trace c), or 0.3 µM (trace d)) as indicated by the arrowhead. A maximal concentration of Leu-EK (10 µM) (EK) was added in all traces 50 s after the first stimulation as indicated by the arrow. The [Ca] increase was measured as described under ``Experimental Procedures.'' Similar responses were observed in at least three independent experiments.



It has been established that Leu-EK inhibited the cAMP generation within cells through the receptor activated G. To further determine the involvement of the cAMP signaling pathway in the cross-interaction of Leu-EK with bradykinin and ATP in the rise in [Ca], we measured cAMP production in NG108-15 cells in response to stimulation by a variety of agonists. As depicted in Fig. 10, the basal cAMP accumulation during a 15-min incubation period at 37 °C was 142 ± 20 pmol/mg (n = 3). Prostaglandin E at 1 µM induced an increase in cAMP generation to 412 ± 32 pmol/mg, whereas 10 µM bradykinin and 3 mM ATP did not significantly affect the cAMP signaling pathway; the cAMP values were 140 ± 24 and 135 ± 10 pmol/mg, respectively. When 10 µM Leu-EK was added either alone or in combination with prostaglandin E, bradykinin, or ATP, the cAMP content was 98 ± 36, 277 ± 42, 105 ± 10, and 97 ± 14 pmol/mg, respectively. Thus, cAMP generation revealed a similar approximately 30% decrease once Leu-EK was incorporated together with any agonists tested, although the reduction of the basal cAMP level induced by Leu-EK was not statistically significant.


Figure 10: Effect of various agonists on cellular cAMP accumulation. After incubation with 0.5 mM isobutylmethylxanthine for 30 min in culture, aliquots of cells (0.5 mg of cell protein) were stimulated at 37 °C for 15 min with buffer (Basal), 1 µM prostaglandin E (PGE), 10 µM bradykinin (BK), or 3 mM ATP in the presence or absence of 10 µM Leu-EK (EK) as indicated. Results are mean values ± S.D. of triplicate experiments. Asterisk and number sign, the cAMP levels are significant different from corresponding cells without Leu-EK, p < 0.01 and p < 0.05, respectively.




DISCUSSION

In addition to its inhibitory effect, we have demonstrated in the current study that activation of -opioid receptors in NG108-15 cells induced an increase in [Ca]. Furthermore, the source of the increased [Ca] evoked by Leu-EK was shown to be mainly due to Ca release from intracellular Ca pools (Fig. 5). Previously, we have demonstrated that bradykinin and ATP caused a [Ca] increase via distinct mechanisms in dibutyryl cAMP-induced differentiated NG108-15 cells. Bradykinin stimulated phospholipase C to generate IP, and IP in turn triggered Ca release from intracellular Ca pools(22, 24) . In contrast, extracellular Ca influx was activated by ATP, and two distinct Ca influx mechanisms were responsible for the [Ca] rise; a receptor-operated cation channel and pores formed by free acid ATP, ATP(21, 22) . In the current study, using nondifferentiated NG108-15 cells, we first demonstrated that ATP induced a considerable amount of Ca entry from the extracellular space in addition to its intracellular Ca releasing activity, but that the [Ca] increase of the bradykinin- and the Leu-EK-induced responses originated mainly from intracellular Ca pools (Fig. 5). Second, we observed that only ATP-stimulated IP generation exhibited remarkably high sensitivity to extracellular Ca (Fig. 6). Finally, although intracellular Ca could be partially released upon ATP stimulation in the absence of extracellular Ca, in the presence of extracellular Ca, the ATP-stimulated Ca influx which raised the [Ca]was even more relevant to the filling status of intracellular Ca pools (Fig. 8). Based on the current study and previously reported results(21, 22) , it appears that in the presence of extracellular Ca, ATP stimulates a Ca influx that raises [Ca], which in turn modulates IP generation and increases the size of intracellular Ca pools (Fig. 5, 6, and 8). Therefore, in the presence of extracellular Ca, no heterologous desensitization between Leu-EK and ATP was observed; however, bradykinin exhibited complete heterologous desensitization to the response elicited by Leu-EK, although Leu-EK only partially desensitized the response evoked by bradykinin ( Fig. 1and Fig. 2). On the other hand, in the absence of extracellular Ca, both bradykinin and ATP heterologously desensitized the action of Leu-EK in a dose-dependent manner by depleting the common stores that were accessible to Leu-EK (Fig. 9).

Stimulation of NG108-15 cells with 10 µM bradykinin resulted in a 4- or 11-fold higher rate of IP generation as compared with that evoked by 10 µM Leu-EK in the presence or absence of extracellular Ca, respectively (Fig. 6). However, the difference in the [Ca] rise was only 3-fold (Fig. 1). An explanation for this discrepancy is that the measurement of [Ca] reflects the final status of cytosolic Ca after Ca ``turnover'' in intact cells. In NG108-15 cells, bradykinin causes depolarization via protein kinase C activation(28) , whereas Leu-EK inhibits the voltage-dependent Ca channel via pertussis toxin-sensitive G-proteins(13, 14) . Alternatively, the intracellular Ca pools may have been limiting in the phospholipase C signaling pathway. A defined pool of phosphatidylinositol 4,5-bisphosphate or a specific phospholipase C may have been responsible for each receptor agonist; hence, an additive or a synergistic increase in IP generation was observed due to simultaneous addition of two agonists (Fig. 6). However, a further increase in [Ca] was not found when Leu-EK was also included with bradykinin or ATP (Fig. 7). Either the availability of Ca was reduced or the IP receptor was saturated if a shared intracellular Ca pool was acted on by all receptor agonists. Indeed, it has recently been shown that stimulation of SH-SY5Y human neuroblastoma cells with carbachol induced down-regulation of IP receptors, though half-maximal effects occurred after 1 h of treatment(29) .

Using a population of cells, the results of the current study demonstrate that Leu-EK stimulates phospholipase C to generate IP, probably via a pertussis toxin-sensitive G-protein (17, 30), followed by IP-triggered Ca release from intracellular Ca pools. Therefore, a common Ca pool was accessible to both Leu-EK, ATP and bradykinin. Similar results were reported by Jin et al.(17) using single NG108-15 cell. Thus, the bradykinin-induced [Ca] increase was lower in cells that had been previously treated with DADLE compared with control cells(17) . Because the magnitude of the [Ca] transients induced by Leu-EK was smaller than those induced by bradykinin and ATP, it is possible that only a subset of the cells that were activated by bradykinin and ATP were activated by Leu-EK. Findings contradictory to our results have also been reported. Prior treatment of NG108-15 cells with submaximal concentrations of bradykinin or ATP synergistically enhanced the [Ca] increase induced by Leu-EK, whereas Leu-EK alone had no effect on [Ca]. However, the potentiation of Leu-EK activity was reduced at higher bradykinin concentrations. At 10 nM, bradykinin did not affect Leu-EK induction of [Ca](30, 31) . The use of 10 µM bradykinin in the current study may explain the different results.

In previous studies, we found that undifferentiated NG108-15 cells lacked a significant depolarization-evoked increase in [Ca]; however, depolarization induced a greater rise in [Ca] after cells had been differentiated by elevating the cellular cAMP level(20) . Furthermore, ATP stimulated a [Ca] increase which predominantly resulted from Ca influx in differentiated cells but resulted from both intracellular Ca release and Ca influx in undifferentiated cells(21, 22) . Consistent with our results, it has recently been shown that removal of extracellular Ca reduced, but did not block, the opioid-induced [Ca] increase in undifferentiated NG108-15 cells, whereas the response was completely blocked by removal of extracellular Ca in differentiated cells(16) . In the current study, undifferentiated cells were utilized. Both ATP and Leu-EK induced IP generation. However, ATP exhibited high sensitivity to extracellular Ca (Fig. 6). In the presence of extracellular Ca, a large portion of IP generation in response to ATP might have been due to the indirect activation of phospholipase C by the ATP-stimulated influx of Ca(32) .

Even though ATP stimulated IP generation as effectively as did bradykinin (Fig. 6), the Ca pool which was accessible to Leu-EK became enlarged after ATP pretreatment (Fig. 8). Therefore, in contrast to bradykinin, prior treatment of cells with ATP did not heterologously desensitize the subsequent [Ca]response induced by Leu-EK in the presence of extracellular Ca (Fig. 1). ATP exerted a dual influence on the size of intracellular Ca pools. First, the Ca that entered cells after exposure to ATP was used to fill intracellular Ca pools. Second, we have previously shown that ATP can stimulate Na influx via a receptor-operated cation channel (22). Increased cytosolic Na may have enhanced the size of the intracellular Ca pool. It has been shown in vascular smooth muscle cells that elevation of intracellular Na increased the stored Ca in the sarcoplasmic reticulum; consequently, agonist-induced [Ca] increases were amplified(33) . On the other hand, ATP displayed effects similar to those of bradykinin on the Leu-EK-induced [Ca] increase in the absence of extracellular Ca in which replenishment of intracellular Ca pools was no longer possible (Fig. 9). Our data further indicate that an adenlylate cyclase cascade contributed very little to the cross-interaction between Leu-EK and bradykinin or ATP in Ca signaling in NG108-15 cells (Fig. 10).


FOOTNOTES

*
This work was supported by grants from the National Science Council (NSC83-0412-B016-123) and the Department of Health (DOH84-HR-402), Republic of China. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed: Dept. of Biochemistry, National Defense Medical Center, Taipei, Taiwan, ROC. Tel.: 886-2-365-4542; Fax: 886-2-368-1354.

The abbreviations used are: [Ca], cytosolic free Ca concentration; Leu-EK, leucine-enkephalin; DADLE, [D-Ala-D-Leu]enkephalin; IP, inositol 1,4,5-trisphosphate.


REFERENCES
  1. Hamprecht, B. (1977) Int. Rev. Cytol.49, 99-170 [Medline] [Order article via Infotrieve]
  2. Klee, W. A., and Nirenberg, M. (1974) Proc. Natl. Acad. Sci. U. S. A.71, 3474-3477 [Abstract]
  3. Sabol, S. L., and Nirenberg, M. (1979) J. Biol. Chem.254, 1913-1920 [Abstract]
  4. Green, D. A., and Clark, R. B. (1982) J. Neurochem.39, 1125-1131 [Medline] [Order article via Infotrieve]
  5. Yano, K., Higashida, H., Inoue, R., and Nozawa, Y. (1984) J. Biol. Chem.259, 10201-10207 [Abstract/Free Full Text]
  6. Miwa, N., Sugino, H., Ueno, R., and Hayaishi, O. (1988) J. Neurochem.50, 1418-1424 [Medline] [Order article via Infotrieve]
  7. Ehrlich, Y. H., Snider, R. M., Kornecki, E., Garfield, M. G., and Lenox, R. H. (1988) J. Neurochem.50, 295-301 [Medline] [Order article via Infotrieve]
  8. Chan, J., and Greenberg, D. A. (1991) J. Pharmacol. Exp. Ther.258, 524-530 [Abstract]
  9. Carrithers, M. D., Koide, K. A., Raman, V. K., Masuda, S., and Weyhenmeyer, J. A. (1990) Biochem. Biophys. Res. Commun.170, 1096-1101 [Medline] [Order article via Infotrieve]
  10. Low, P. Y., Hom, D. S., and Loh, H. H. (1983) Mol. Pharmacol.23, 26-35 [Abstract]
  11. Prather, P. L., Loh, H. H., and Law, P. Y. (1994) Mol. Pharmacol.45, 997-1003 [Abstract]
  12. Koski, G., and Klee, W. A. (1981) Proc. Natl. Acad. Sci. U. S. A.78, 4185-4189 [Abstract]
  13. Tsunoo, A., Yoshii, M., and Narahashi, T. (1986) Proc. Natl. Acad. Sci. U. S. A.83, 9832-9836 [Abstract]
  14. Hescheler, J., Rosenthal, W., Trautwein, W., and Schultz, G. (1987) Nature325, 445-447 [CrossRef][Medline] [Order article via Infotrieve]
  15. Jin, W., Lee, N. M., Loh, H. H., and Thayer, S. A. (1993) Brain Res.607, 17-22 [Medline] [Order article via Infotrieve]
  16. Jin, W., Lee, N. M., Loh, H. H., and Thayer, S. A. (1992) Mol. Pharmacol.42, 1083-1089 [Abstract]
  17. Jin, W., Lee, N. M., Loh, H. H., and Thayer, S. A. (1994) J. Neurosci.14, 1920-1929 [Abstract]
  18. Sharma, S. K., Klee, W. A., and Nirenberg, M. (1975) Proc. Natl. Acad. Sci. U. S. A.72, 3092-3096 [Abstract]
  19. Law, P. Y., Hom, D. S., and Loh, H. H. (1991) J. Pharmacol. Exp. Ther.256, 710-716 [Abstract]
  20. Chueh, S.-H., Kao, L.-S., and Liu, Y.-T. (1994) Brain Res.660, 81-87 [CrossRef][Medline] [Order article via Infotrieve]
  21. Chueh, S.-H., and Kao, L.-S. (1993) J. Neurochem.61, 1782-1788 [Medline] [Order article via Infotrieve]
  22. Chueh, S.-H., Hsu, L.-S., and Song, S.-L. (1994) Mol. Pharmacol.45, 532-539 [Abstract]
  23. Berridge, M. J. (1993) Nature361, 315-325 [CrossRef][Medline] [Order article via Infotrieve]
  24. Chueh, S.-H., and Kao, L.-S. (1994) Am. J. Physiol.266, C1006-C1012
  25. Grynkiewicz, G., Poenie, M., and Tsien, R. Y. (1985) J. Biol. Chem.260, 3440-3450 [Abstract]
  26. Thastrup, O., Cullen, P. J., Drobak, B. K., Hanley, M. R., and Dawson, A. P. (1990) Proc. Natl. Acad. Sci. U. S. A.87, 2466-2470 [Abstract]
  27. Montero, M., Alvarez, J., and Garcia-Sancho, J. (1990) Biochem. J.271, 535-540 [Medline] [Order article via Infotrieve]
  28. Shimahara, T., Icard-Liepkalins, C., Ohmori, H., and Shigemoto, T. (1990) Brain Res.524, 219-224 [Medline] [Order article via Infotrieve]
  29. Wojcikiewicz, R. J. H., Furuichi, T., Nakade, S., Mikoshiba, K., and Nahorski, S. R. (1994) J. Biol Chem.269, 7963-7969 [Abstract/Free Full Text]
  30. Okajima, F., Tomura, H., and Kondo, Y. (1993) Biochem. J.290, 241-247 [Medline] [Order article via Infotrieve]
  31. Okajima, F., and Kondo, Y. (1992) FEBS Lett.301, 223-226 [CrossRef][Medline] [Order article via Infotrieve]
  32. Eberhard, D. A., and Holz, R. W. (1988) Trends Neurosci.11, 517-520 [CrossRef][Medline] [Order article via Infotrieve]
  33. Borin, M. L., Tribe, R. M., and Blaustein, M. P. (1994) Am. J. Physiol.266, C311-C317

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