From the
Leucine-enkephalin (Leu-EK) dose-dependently elicited an
increase in cytosolic Ca
Neuroblastoma
Using NG108-15 cells, we have
previously reported that high extracellular K
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
Heterologous desensitization of the rise in
[Ca
In addition to its inhibitory effect, we have demonstrated in
the current study that activation of
Stimulation of NG108-15 cells with 10
µM bradykinin resulted in a 4- or 11-fold higher rate of
IP
Using a
population of cells, the results of the current study demonstrate that
Leu-EK stimulates phospholipase C to generate IP
In previous studies, we found that
undifferentiated NG108-15 cells lacked a significant
depolarization-evoked increase in
[Ca
Even though ATP stimulated
IP
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.
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) .
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) .
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
.
Culture of Neuroblastoma
Clonal neuroblastoma Glioma
Cells
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 IP
IPGeneration
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.
]
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.
-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).
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) .
, 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.
]
; 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) .
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).
], cytosolic free Ca
concentration; Leu-EK, leucine-enkephalin; DADLE,
[D-Ala
-D-Leu
]enkephalin;
IP
, inositol 1,4,5-trisphosphate.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.