Caffeine does not inhibit substance P-evoked intracellular
Ca2+ mobilization in rat salivary
acinar cells
J. T.
Seo1,2,
H.
Sugiya3,
S. I.
Lee2,
M. C.
Steward1, and
A. C.
Elliott1
1 School of Biological
Sciences, University of Manchester, Manchester M13 9PT, United Kingdom;
2 Department of Oral Biology,
Yonsei University College of Dentistry, Shinchon-Dong 134, Seodaemoon-Gu, Seoul, South Korea; and
3 Department of Physiology,
Nihon University School of Dentistry at Matsudo, Matsudo, Chiba 271, Japan
 |
ABSTRACT |
We used the
Ca2+-sensitive fluorescent dye
fura 2, together with measurements of intracellular
D-myo-inositol
1,4,5-trisphosphate [Ins(1,4,5)P3],
to assess the inhibitory effects of caffeine on signal transduction via
G protein-coupled receptor pathways in isolated rat mandibular salivary
acinar cells. ACh, norepinephrine (NE), and substance P (SP) all evoked
substantial increases in the intracellular free
Ca2+ concentration
([Ca2+]i).
Responses to ACh and NE were markedly inhibited by prior application of
20 mM caffeine. The inhibitory effect of caffeine was not reproduced by
phosphodiesterase inhibition with IBMX or addition of cell-permeant
dibutyryl cAMP. In contrast to the ACh and NE responses, the
[Ca2+]i
response to SP was unaffected by caffeine. Despite this, SP and ACh
appeared to mobilize Ca2+ from a
common intracellular pool. Measurements of agonist-induced changes in
Ins(1,4,5)P3
levels confirmed that caffeine inhibited the stimulus-response coupling
pathway at a point before
Ins(1,4,5)P3 generation. Caffeine did not, however, inhibit
[Ca2+]i
responses evoked by direct activation of G proteins with 40 mM
F
. These data show that
caffeine inhibits G protein-coupled signal transduction in these cells
at some element that is common to the muscarinic and
-adrenergic
signaling pathways but is not shared by the SP signaling pathway. We
suggest that this element might be a specific structural motif on the G
protein-coupled muscarinic and
-adrenergic receptors.
intracellular calcium store; signal transduction; acetylcholine; norepinephrine; mandibular gland; G protein; G protein-coupled receptor
 |
INTRODUCTION |
IN SALIVARY ACINAR CELLS, stimulation of muscarinic,
-adrenergic, or substance P (SP) receptors induces an increase in
the intracellular free Ca2+
concentration
([Ca2+]i;
see, for example, Refs. 18 and 19). All of these receptors are coupled
via G proteins to phospholipase C (PLC) and hence promote the
production of
D-myo-inositol
1,4,5-trisphosphate
[Ins(1,4,5)P3] and diacylglycerol.
Ins(1,4,5)P3 in
turn induces an increase in [Ca2+]i
by mobilization of Ca2+ from
internal stores, with entry of extracellular
Ca2+ being activated by mechanisms
that remain the subject of controversy (26).
Evidence obtained in a variety of nonexcitable cell types indicates
that high (10-20 mM) concentrations of caffeine inhibit [Ca2+]i
responses evoked by
Ins(1,4,5)P3-generating
agonists (2, 23, 25, 31). The mechanism of this inhibitory action of caffeine is not known precisely, and a number of possibilities have
been suggested, including inhibition of
Ins(1,4,5)P3
generation, inhibition of
Ins(1,4,5)P3
binding to its receptor, and inhibition of the
Ca2+ channel activity of the
Ins(1,4,5)P3
receptor. In pancreatic acinar cells, where
[Ca2+]i
responses evoked by ACh, CCK, and ATP (which act via different receptors) are all inhibited by caffeine, it appears that caffeine acts
by blocking
Ins(1,4,5)P3
production (30). This last result suggests that caffeine would be
expected to block
[Ca2+]i
increases evoked by any
Ins(1,4,5)P3-generating
agonist in other exocrine acinar cell types, such as those of the
salivary glands. In the present study, however, we have found that
neither the increase in
Ins(1,4,5)P3 nor
the elevation of
[Ca2+]i
induced by stimulating SP receptors in rat mandibular acinar cells is
blocked by caffeine. This contrasts with the marked inhibition by
caffeine of responses to muscarinic or
-adrenergic stimulation. These data suggest, first, that caffeine does not act on the
Ins(1,4,5)P3 receptor or on PLC in this tissue and, second, that caffeine acts at a
site that is common to the muscarinic and
-adrenergic, but not the
SP, messenger pathways.
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MATERIALS AND METHODS |
Mandibular salivary gland acini were isolated from male Sprague-Dawley
rats by a slight modification of the method described in Ref. 14. In
brief, rats were killed by cervical dislocation, and both mandibular
salivary glands were isolated and dissected free from the surrounding
connective tissue. Sublingual glands, which are closely associated with
the mandibular glands, were also removed. The glands were then minced
and digested with collagenase (type IV from Sigma; 60 U/ml) for 1 h in
a HEPES-buffered physiological saline containing (in mM) 130 NaCl, 4.5 KCl, 1.0 NaH2PO4,
1.0 MgSO4, 1.5 CaCl2, 10 HEPES-Na, 10 HEPES free
acid, 10 D-glucose, and 2 glutamine, as well as 1× MEM amino acids and 1% BSA
(equilibrated with 100% O2; pH
adjusted to 7.4 with NaOH). During digestion, the cells were
mechanically dissociated by gentle pipetting at 20-min intervals. After
several washes with HEPES-buffered physiological saline, the cells were
again dissociated mechanically by pipetting. This method produced
primarily cell clusters and small acini containing 4-10 cells.
Fura 2 loading and
[Ca2+]i
measurements.
Acini were loaded with fura 2 by incubation with 2 µM fura 2-AM in
the HEPES-buffered solution for 30 min at room temperature under 100%
O2. They were then washed twice
and resuspended in a HCO
3-buffered
physiological saline containing (in mM) 110 NaCl, 4.5 KCl, 1.0 NaH2PO4,
1.0 MgSO4, 1.5 CaCl2, 5 HEPES-Na, 5 HEPES free
acid, 25 NaHCO3, and 10 D-glucose (equilibrated with
95% O2-5%
CO2 to give a pH of 7.4). Small
portions of this cell suspension were transferred to a chamber (volume
~0.3 ml) on the stage of a Nikon Diaphot inverted microscope for
[Ca2+]i
measurements. The remaining acini were kept on ice. Once the cells had
adhered to the coverslip that formed the base of the experimental
chamber, they were continuously superfused with the HCO
3-buffered solution at a flow rate
of 2 ml/min. In experiments in which caffeine was used, 10 mM NaCl was
replaced by 20 mM caffeine, whereas in experiments with
F
, NaF replaced NaCl on an
equimolar basis. In Ca2+-free
solutions, CaCl2 was omitted and 1 mM EGTA was added. All experiments were carried out at 37°C.
[Ca2+]i
was measured in individual acini or cell clusters by dual-wavelength
spectrofluorometry. Cells were exposed to excitation wavelengths of 340 and 380 nm at a frequency of 1 Hz using a Sutter Instruments (Novato,
CA) Lambda-ten filter wheel. The emitted fluorescence was measured at
510 nm with a photomultiplier tube (Thorn EMI, Ruislip, United Kingdom)
attached to a spectrofluorometer system (Cairn Research, Faversham,
Kent, United Kingdom). The ratio of light emitted at 340 and 380 nm
excitation was used as an index of
[Ca2+]i.
Measurements of cellular
Ins(1,4,5)P3.
For measurements of
Ins(1,4,5)P3,
cells were suspended in HCO
3-buffered
saline for 5 min at 37°C. Agonists were then added from
concentrated stock solutions to give the required final concentration.
Ten seconds later, the incubation was terminated by addition of 0.2 volumes of ice-cold 20% perchloric acid, after which the samples were
kept on ice for 30 min. After sedimentation of cellular proteins and
neutralization of the resulting supernatant, the amount of
Ins(1,4,5)P3 in
the samples was measured with a commercially available assay kit
(Amersham Life Science, Arlington Heights, IL) that was used according
to the manufacturer's instructions.
All chemicals were obtained from Sigma (Poole, United Kingdom), except
fura 2-AM, which was obtained from Molecular Probes (Eugene, OR) and
was made up as a 3 mM stock solution in DMSO. Data are presented as
means ± SE. Statistical analysis was carried out using unpaired
Student's t-tests.
 |
RESULTS |
Ca2+
responses to ACh and norepinephrine.
Pronounced desensitization of the SP response has been reported in a
variety of cell types (7, 29) and was also observed in our own
experiments when we stimulated cells with the peptide two or more times
(data not shown). We therefore chose to avoid repeated applications of
SP. Instead, we first stimulated acini with ACh, followed, after a
recovery period, either by a second stimulation with ACh or by
stimulation with norepinephrine (NE) or SP. The data were then
normalized by setting the peak value of the first ACh-evoked
[Ca2+]i
increase to 100%. The acini were stimulated with agonists for 1 min
and were allowed to recover for 6 min between stimulations. We chose 1 µM ACh as a standard concentration, since it is close to a maximum
dose for
[Ca2+]i
mobilization in rat mandibular cells (4) but evokes only a relatively
modest desensitization of the ACh response (see Fig. 1). Experiments in which cells
were pretreated with 20 mM caffeine were performed and analyzed in the
same fashion. Caffeine was introduced 4 min after the initial ACh
stimulation, so that the second agonist stimulation took place after 2 min of caffeine treatment. The period of time between the first and the
second agonist treatments thus remained constant at 6 min regardless of
whether or not caffeine was applied. Exposures to ACh and other agonists were kept brief to minimize any contribution to the
[Ca2+]i
signal from entry of extracellular
Ca2+ rather than from release of
stored Ca2+. Previous work on rat
mandibular (4) and parotid (18, 19) acinar cells has shown that the
initial peak of the
[Ca2+]i
response is derived overwhelmingly from
Ca2+ release from intracellular
stores.

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Fig. 1.
Caffeine (caff), but not IBMX or dibutyryl cAMP (DBcAMP), inhibits
intracellular free Ca2+
concentration
([Ca2+]i)
response to ACh stimulation in rat mandibular acinar cells.
A and
B: records of ratios of fluorescence
at 340 nm to fluorescence at 380 nm
(F340/F380)
from fura 2-loaded acinar cells, each representative of 7 separate
experiments on different individual cell preparations.
C: averaged results on 2nd (test)
ACh-evoked
[Ca2+]i
increase, expressed as a percentage of 1st (control) response to ACh.
Number of separate observations was 7 (ACh alone and ACh + caffeine) or 4 (ACh + IBMX or ACh + DBcAMP).
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As shown in Fig. 1, the peak value of the increase in
[Ca2+]i
caused by a second stimulation with 1 µM ACh was reduced to 79.4 ± 3.4% (n = 7) compared with the
first ACh response. This was probably due to slight desensitization of
the muscarinic
[Ca2+]i
response, as observed previously (4). In the presence of 20 mM
caffeine, the
[Ca2+]i
increase caused by the second stimulation with ACh was dramatically reduced, to 23.4 ± 3.0% (n = 7)
of the initial ACh response. By comparison of the second ACh responses
with or without caffeine, it could be calculated that 20 mM caffeine
reduced the ACh response to 29.5 ± 3.8%
(P < 0.001) of the control value.
Application of caffeine was generally, although not invariably,
associated with a small decrease in the fura 2 fluorescence ratio. This
is not a quenching effect, since caffeine does not quench fura 2 fluorescence (22), and thus it almost certainly represents a small
decrease in
[Ca2+]i
on application of caffeine. This effect of caffeine has previously been
attributed to enhanced uptake of
Ca2+ into intracellular stores
(10), although the present study does not provide any evidence on this point.
Camello et al. (8) recently suggested that the inhibitory actions of
caffeine in pancreatic acinar cells may result from inhibition of
phosphodiesterases. We investigated whether this was true for
mandibular cells by examining whether the inhibitory effect of caffeine
could be mimicked by the well-known phosphodiesterase inhibitor IBMX
(0.5 mM) or by the membrane-permeant cAMP dibutyryl cAMP (DBcAMP; 1 mM). Both compounds were tested in the same experimental protocol that
was used for caffeine. Compounds were applied to cells 2 min before
stimulation with ACh. Fig. 1C shows
averaged data, which clearly demonstrate that neither compound mimicked the inhibitory effect of caffeine (ACh response in presence of IBMX or
DBcAMP was 93.4 ± 4.3 and 85.3 ± 2.9%, respectively, of initial control response to ACh; both
n = 4).
The increase in
[Ca2+]i
caused by 3 µM NE was also greatly inhibited by pretreatment with
caffeine (Fig. 2). The acini were stimulated with 1 µM ACh followed by 3 µM NE. The size of the peak
[Ca2+]i
increase induced by 3 µM NE was 77.3 ± 6.8%
(n = 4) compared with the 1 µM
ACh-induced
[Ca2+]i
increase. This value was reduced to 28.2 ± 4.2%
(n = 6) by pretreatment with 20 mM
caffeine, and this change was statistically significant
(P < 0.001).

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Fig. 2.
Caffeine inhibits
[Ca2+]i
response to norepinephrine (NE) stimulation.
A and
B: records of
F340/F380
from fura 2-loaded cells, representative of 4 (A) and 6 (B) separate experiments.
C: averaged results. Format is similar
to that of Fig. 1.
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Ca2+
responses to SP.
Mandibular cells also possess SP receptors linked to
PLC activation. We therefore tested whether caffeine inhibited the
response to SP. In marked contrast to ACh and NE, the SP-induced
[Ca2+]i
increase was not significantly affected by pretreatment with caffeine
(Fig. 3). The
[Ca2+]i
increase caused by 5 nM SP was 103.8 ± 7.2%
(n = 8) compared with the first
stimulation with ACh. In caffeine-treated cells, the corresponding
value was 87.2 ± 4.6% (n = 10).
The difference between the two values was not significant. Caffeine
similarly had no effect on the
[Ca2+]i
response evoked by a 10-fold lower dose of SP (0.5 nM; no significant effect, n = 4; data not shown).

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Fig. 3.
Caffeine does not inhibit
[Ca2+]i
response to substance P (SP) stimulation.
A and
B: records of
F340/F380
in fura 2-loaded cells, representative of 8 (A) and 10 (B) separate experiments.
C: averaged results. Format is
identical to that of Fig. 2.
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Figure 4,
A and
B, shows a further difference between
the effects of caffeine on ACh- and SP-induced
[Ca2+]i
increases. In the presence of caffeine, ACh evoked a relatively small
increase in
[Ca2+]i,
as expected. When caffeine was subsequently removed, a substantial further rise in
[Ca2+]i
was observed, as previously reported by Toescu et al. (30) in
pancreatic acinar cells. In contrast, when cells were stimulated with
SP in the presence of caffeine (Fig.
4B), there was almost no further
increase in
[Ca2+]i
on withdrawal of caffeine (n = 5).

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Fig. 4.
ACh and SP release Ca2+ from same
intracellular pool. All records are of
F340/F380
in fura 2-loaded cells. A: caffeine
pretreatment reduces response to ACh.
B: caffeine pretreatment does not
affect SP response. C: SP-evoked
Ca2+ release is reduced when
intracellular Ca2+ stores have
previously been emptied by stimulation with ACh.
D: intracellular
Ca2+ stores remain loaded after 6 min of superfusion with Ca2+-free
medium. Traces in A,
C, and
D are representative of 3 separate
experiments; trace in B is
representative of 5 separate experiments.
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To investigate whether ACh and SP mobilize
Ca2+ from the same intracellular
Ca2+ pool, acini were stimulated
with ACh and then SP in the absence of superfusate
Ca2+ (Fig.
4C). Exposure to the
Ca2+-free superfusate induced a
slow decrease in
[Ca2+]i.
The cells were then stimulated with ACh for ~2 min. During this
period,
[Ca2+]i
increased and then returned to the basal level. Subsequent stimulation
with 5 nM SP caused no changes in
[Ca2+]i
(n = 3). The absence of an
[Ca2+]i
increase in response to SP was not due to the depletion of the
Ca2+ pool caused by the 6-min
exposure to the Ca2+-free
superfusate. As shown in Fig. 4D, the
peak value of the SP-induced
[Ca2+]i
increase, measured 6 min after
Ca2+ removal from the superfusate,
was 60.2 ± 10.6% (n = 3) compared with the ACh-induced
[Ca2+]i
response obtained before Ca2+
withdrawal. Because previous exposure to ACh does not desensitize the
SP response in this cell type (Fig. 3), the lack of an SP response in
Fig. 4C implies that the SP-sensitive
Ca2+ pool is emptied by ACh. This
strongly implies that ACh and SP mobilize
Ca2+ from the same intracellular
Ca2+ pool.
Ins(1,4,5)P3 responses
to ACh, NE, and SP.
We next went on to examine agonist-evoked
Ins(1,4,5)P3
production. In pancreatic acinar cells, caffeine inhibits responses to
PLC-coupled agonists at a point in the stimulus-response coupling pathway before the generation of
Ins(1,4,5)P3
(30). This is also the case in rat mandibular acinar cells stimulated
with either ACh or NE, since caffeine significantly reduced the
Ins(1,4,5)P3 production evoked by these two agonists (Fig.
5). However, the increase in
Ins(1,4,5)P3
evoked by SP was unaffected by caffeine, in keeping with the results on
[Ca2+]i
described above. The agonist-evoked increases in
Ins(1,4,5)P3 were
broadly similar among the different agonists when either supramaximal
doses of each agonist (10 µM ACh, 30 µM NE, 5 nM SP) or 10-fold
lower concentrations (1 µM ACh, 3 µM NE, and 0.5 nM SP) were
compared. The lack of effect of caffeine on the SP-evoked increase in
Ins(1,4,5)P3 was
apparent at both concentrations of SP. This confirms that the
insensitivity of the SP response to caffeine is not dependent on
agonist dose and represents a genuine difference between the ACh and NE
signal pathways and the SP signal pathway.

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Fig. 5.
Cellular
D-myo-inositol
1,4,5-trisphosphate
(InsP3) levels
in rat mandibular cells that were unstimulated (Unstim) or were treated
with ACh, NE, SP, or caffeine (means ± SE of 3 experiments on
separate cell preparations). Open bars, no caffeine; hatched bars, 20 mM caffeine. * Statistically significant difference (at 5%
level) from corresponding result in absence of caffeine.
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Direct activation of G proteins with
F
.
Because the inhibitory action of caffeine on signal transduction lies
upstream of
Ins(1,4,5)P3
generation, the site of inhibition must presumably be the receptor or
the G protein. To try to distinguish these two sites, we used
F
to activate the G
proteins directly. It has been reported that [Ca2+]i
responses can be elicited with comparatively low concentrations of
F
(5-10 mM) in some
exocrine cell types (20, 34). However, we found that 5, 10, or 20 mM
F
evoked only a very small
and gradual increase in
[Ca2+]i
in mandibular acinar cells. This was true regardless of whether 50 µM
AlCl3 was included to enhance
formation of the G protein-activating species
AlF
4. To produce a rapid increase
in [Ca2+]i,
we found that it was necessary to use 30 or 40 mM
F
. These experiments were
also carried out in Ca2+-free
medium to avoid problems with precipitation. Figure
6 shows that under these conditions 40 mM
F
evoked a large and rapid
increase in
[Ca2+]i
(Fig. 6A) that was unaffected by 20 mM caffeine (Fig. 6B; averaged results; response to 40 mM
F
without caffeine, 86.4 ± 8.7% of initial ACh response; with caffeine, 75.1 ± 11.6%;
values not significantly different). This result shows that caffeine
does not inhibit the response triggered by direct activation of G
protein(s) in acinar cells and therefore suggests that the inhibitory
action of caffeine may be mediated by a site on the muscarinic (and
presumably also the
-adrenergic) receptor.

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Fig. 6.
Caffeine does not inhibit
[Ca2+]i
response to 40 mM F .
A and
B: records of
F340/F380
in fura 2-loaded cells, representative of 4 experiments in each case.
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 |
DISCUSSION |
In this paper, we demonstrate that caffeine, which inhibits the
[Ca2+]i
increases induced by a range of
Ins(1,4,5)P3-generating
agonists in various nonexcitable cell types, does not block the
increases in
Ins(1,4,5)P3 and
[Ca2+]i
evoked by SP in rat mandibular acinar cells. Caffeine does, however,
block both
Ins(1,4,5)P3
production and the
[Ca2+]i
signals evoked by ACh and NE. This result raises several interrelated questions. The main questions concern the mechanism of caffeine inhibition of ACh and NE responses and hence the possible reasons for
the lack of inhibition of SP responses. In addition, there is the
question of whether caffeine acts directly on the receptor-G protein-PLC stimulus-response coupling machinery or whether the effects
of caffeine are mediated by another intracellular mechanism, for
instance, phosphodiesterase inhibition.
To take the second question first, our results clearly show that the
action of caffeine in mandibular cells is not related to inhibition of
phosphodiesterases and a subsequent increase in intracellular cAMP,
since neither IBMX nor DBcAMP mimicked the effects of caffeine. In
addition, it is extremely unlikely that the inhibitory effect of
caffeine is related to any
Ca2+-releasing action of caffeine
similar to that observed in excitable cells (see, for example, Ref.
22). This is because caffeine clearly did not cause any increase in
[Ca2+]i
in our experiments. In fact, application of 20 mM caffeine caused a
modest decrease in
[Ca2+]i,
as previously reported in mandibular cells (10), by an unknown mechanism. Overall, we think that the simplest hypothesis is that caffeine inhibits ACh and NE responses by acting directly on the stimulus-response coupling machinery. This is in accordance with the
rapid onset and, especially, the reversibility of the action of
caffeine (see Fig. 4 and Ref. 30). It is also supported by the
observation in pancreatic acinar cells that the inhibitory effect of
caffeine directly reflects the intracellular caffeine concentration on
a second-to-second basis (30).
Regardless of whether caffeine acts directly or indirectly, the lack of
inhibitory effect of caffeine on the SP-evoked
[Ca2+]i
increase could be most simply explained on the basis that SP stimulates
an intracellular signaling pathway quite distinct from that used by
both ACh and NE. However, SP, adrenergic, and muscarinic receptors are
all members of the same superfamily of G protein-coupled receptors (3,
6, 11, 32) and are all thought to activate the
Ins(1,4,5)P3/Ca2+
intracellular messenger pathway. More specifically, salivary gland
muscarinic and adrenergic receptors are thought to be of the
M3 and
1-adrenergic subtypes (9, 18,
19, 27), and these, together with the SP receptor (more commonly known
as the NK1 tachykinin receptor),
all couple to G proteins of the
Gq/11 subclass that stimulate
PLC-
1 (13, 21, 33). In
addition, there have been several previous reports that stimulation of
muscarinic,
-adrenergic, and
NK1 receptors increases
[Ca2+]i
via a common intracellular signaling pathway in salivary acinar cells
(18, 19, 27). Finally, our results confirm that ACh and SP mobilize
Ca2+ from the same intracellular
Ca2+ pool.
Given the strong evidence that most or all of the signaling pathway is
common to muscarinic,
-adrenergic, and
NK1 (SP) receptors in this tissue,
two related questions arise. 1) Why
does caffeine not block the
[Ca2+]i
increase evoked by SP? 2) Where is
the site of caffeine action? Our data suggest that the site of caffeine
action is not the
Ins(1,4,5)P3 receptor/Ca2+ release channel, as
inhibition of this system would clearly imply that the SP-induced
[Ca2+]i
increase should be inhibited by caffeine. Toescu et al. (30) suggested
that the site of caffeine action in pancreatic acinar cells was at or
before the level of
Ins(1,4,5)P3
production. The present study indicates either that this site is not
involved in the SP-mediated signaling pathway or that structural
differences exist in the proximal components of the signaling pathway
that may affect the susceptibility to caffeine inhibition. The two most
probable sites of caffeine action would seem to be the receptors or the
G protein(s). One attractive possibility is that muscarinic and
-adrenergic receptors in mandibular cells might operate through a
common, caffeine-sensitive, G protein to stimulate PLC, whereas NK1 (SP) receptors might couple to
a different, caffeine-insensitive one. Although receptors show a
preference for particular G proteins, studies in simplified expression
systems clearly demonstrate that receptors can couple to a number of
different G proteins. For instance, in the Sf 9 insect cell
expression system, the NK1
receptor can be shown to couple not only to
Gq but also to several G proteins of other subclasses (G12,
G13, and
Gz) (1), some of whose functions are unknown (32). Hitherto, the available evidence suggests that
M3 and
-adrenergic receptors in
the salivary glands couple to PLC primarily through G proteins of the
Gq/11 subclass (27, 28). However,
this subclass is now known to consist of at least five distinct G
proteins (32), and evidence in some cell types suggests that different
G proteins within this subclass may mediate distinct functions (17).
Alternatively, the site of caffeine action might be the receptor(s).
This is more consistent with the lack of inhibitory effect of caffeine
when the G proteins are activated directly using
F
. If the actions of
caffeine are indeed direct, as the evidence discussed above suggests,
then one might perhaps envisage caffeine binding to a structural motif
common to muscarinic and
-adrenergic receptors but not present on
the NK1 (SP) receptor. It is
tempting to speculate that such a caffeine-binding site might exist
within the second and third cytoplasmic loops of the G protein-coupled receptors, which are the major regions where sequence divergence is
thought to determine the specificity of receptor-G protein coupling
(24). This would be consistent with the proposed intracellular site of
action of caffeine (30).
Some indirect support for both of the above hypotheses can be derived
from a consideration of the published sequences of the M3,
1-adrenergic, and
NK1 receptors (32). The three
receptors show an overall homology of 17-20%, due mostly to
homology within highly conserved transmembrane
-helical regions
(amino acid sequence comparisons were made on protein sequences from
the SwissProt database using Geneworks version 2.5 software from Oxford
Molecular Group, Oxford, United Kingdom). However, the
M3 and
1-adrenergic receptors are also
notably homologous in the key regions for G protein coupling, namely
the 20-30 amino acids of the second intracellular loop and the
10-12 amino acids at each end of the third intracellular loop (5,
12, 15, 16, 24). For these 45-50 amino acids, homology between the
M3 receptors and
1-receptors is nearly 55%, whereas the homology of either receptor to the
NK1 receptor is much lower
(~20%). Even more strikingly, of 12 amino acid residues in the these
3 regions that have been mapped by the elegant site-directed mutagenesis experiments of Wess and co-workers (5, 12, 15, 16) as being
critical for coupling of the M3
receptor to Gq, 9 are identical in
the
1-adrenergic receptors, but
only 1 is identical in the NK1
receptor. The sequence information would thus be consistent with
caffeine binding to a site associated with G protein coupling and
present on the M3 and
1-receptors but not on the
NK1 receptor. However, this does
not eliminate the possibility that caffeine acts on a G protein that is
downstream of the M3 and
-adrenergic receptors but is not activated by the NK1 receptor. This is because the
amino acid differences between the
M3 and
-adrenergic receptors
and the NK1 receptor discussed above are equally suggestive of the coupling of these receptors to a
different G protein(s).
In conclusion, we found that the
NK1 (SP) receptor in salivary
glands triggers an
Ins(1,4,5)P3/Ca2+
response that is not susceptible to caffeine inhibition. This contrasts
with the inhibition by caffeine of responses to muscarinic or
-adrenergic receptor stimulation in the same cells. Our results show
that caffeine almost certainly acts at the level of receptor-G protein
coupling and therefore imply that the G protein activated by the
NK1 receptor in this cell type may
be distinct from the G protein(s) activated by muscarinic or
-adrenergic stimulation. Our results also suggest that caffeine
action may be a useful pharmacological tool to distinguish subtly
different G protein pathways.
 |
ACKNOWLEDGEMENTS |
This work was supported by Programme Grant 1.27 from the Wellcome
Trust and by a British Council Anglo-Korean Collaborative Research
Programme Grant. J. T. Seo was supported in Manchester by an Overseas
Research Student Award and by a University of Manchester Frederick
Craven Moore Scholarship. A. C. Elliott acknowledges the support of a
British Digestive Foundation/Schering-Plough Travel Fellowship.
 |
FOOTNOTES |
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: A. C. Elliott,
School of Biological Sciences, University of Manchester, G.38 Stopford
Bldg., Manchester M13 9PT, United Kingdom (E-mail:
acelliot{at}mh1.mcc.ac.uk).
Received 2 April 1998; accepted in final form 13 January 1999.
 |
REFERENCES |
1.
Barr, A. J.,
L. F. Brass,
and
D. R. Manning.
Reconstitution of receptors and GTP-binding regulatory proteins (G proteins) in Sf9 cells.
J. Biol. Chem.
272:
2223-2229,
1997[Abstract/Free Full Text].
2.
Berridge, M. J.
Caffeine inhibits inositol-trisphosphate-induced membrane potential oscillations in Xenopus oocytes.
Proc. R. Soc. Lond. B Biol. Sci.
244:
57-62,
1991[Medline].
3.
Berridge, M. J.
Inositol trisphosphate and calcium signalling.
Nature
361:
315-325,
1993[Medline].
4.
Berrie, C. P.,
and
A. C. Elliott.
Activation of protein kinase C does not cause desensitization in rat and rabbit mandibular acinar cells.
Pflügers Arch.
428:
163-172,
1994[Medline].
5.
Blin, N.,
J. Yun,
and
J. Wess.
Mapping of single amino acid residues required for selective activation of Gq/11 by the m3 muscarinic acetylcholine receptor.
J. Biol. Chem.
270:
17741-17748,
1995[Abstract/Free Full Text].
6.
Bonner, T. I.,
N. J. Buckley,
A. C. Young,
and
M. R. Brann.
Identification of a family of muscarinic acetylcholine receptor genes.
Science
237:
527-532,
1987[Medline].
7.
Bordey, A.,
P. Feltz,
and
J. Trouslard.
Mobilization of intracellular calcium by substance P in a human astrocytoma cell line (U-373 MG).
Glia
11:
277-283,
1994[Medline].
8.
Camello, P. J.,
O. H. Petersen,
and
E. C. Toescu.
Simultaneous presence of cAMP and cGMP exert a co-ordinated inhibitory effect on the agonist-evoked Ca2+ signal in pancreatic acinar cells.
Pflügers Arch.
432:
775-781,
1996[Medline].
9.
Dai, Y.,
I. S. Ambudkar,
V. J. Horn,
C.-K. Yeh,
E. E. Kousvelari,
S. J. Wall,
M. Li,
R. P. Yasuda,
B. B. Wolfe,
and
B. J. Baum.
Evidence that M3 muscarinic receptors in rat parotid gland couple to two second messenger systems.
Am. J. Physiol.
261 (Cell Physiol. 30):
C1063-C1073,
1991[Abstract/Free Full Text].
10.
Fukushi, Y.,
T. Ozawa,
A. Nishiyama,
H. Kase,
and
M. Wakui.
Depletion of ryanodine-sensitive Ca2+ stores activates Ca2+ entry in rat submandibular gland acinar cells.
Tohoku J. Exp. Med.
178:
399-411,
1996[Medline].
11.
Hershey, A. D.,
and
J. E. Krause.
Molecular characterization of a functional cDNA encoding the rat substance P receptor.
Science
247:
958-962,
1990[Medline].
12.
Kostenis, E.,
J. Gomeza,
C. Lerche,
and
J. Wess.
Genetic analysis of receptor-G coupling selectivity.
J. Biol. Chem.
272:
23675-23681,
1997[Abstract/Free Full Text].
13.
Kwatra, M. M.,
D. A. Schwinn,
J. Schreurs,
J. L. Blank,
C. M. Kim,
J. L. Benovic,
J. E. Krause,
M. G. Caron,
and
R. J. Lefkowitz.
The substance P receptor, which couples to Gq/11, is a substrate of beta-adrenergic receptor kinase 1 and 2.
J. Biol. Chem.
268:
9161-9164,
1993[Abstract/Free Full Text].
14.
Lau, K. R.,
A. C. Elliott,
and
P. D. Brown.
Acetylcholine-induced intracellular acidosis in rabbit salivary gland acinar cells.
Am. J. Physiol.
256 (Cell Physiol. 25):
C288-C295,
1989[Abstract/Free Full Text].
15.
Liu, J.,
N. Blin,
B. R. Conklin,
and
J. Wess.
Molecular mechanisms involved in muscarinic acetylcholine receptor-mediated G protein activation studied by insertion mutagenesis.
J. Biol. Chem.
271:
6172-6178,
1996[Abstract/Free Full Text].
16.
Liu, J.,
B. R. Conklin,
N. Blin,
J. Yun,
and
J. Wess.
Identification of a receptor/G-protein contact site critical for signalling specificity and G-protein activation.
Proc. Natl. Acad. Sci. USA
92:
11642-11646,
1995[Abstract].
17.
Macrez-Lepretre, N.,
F. Kalkbrenner,
G. Schultz,
and
J. Mironneau.
Distinct functions of Gq and G11 proteins in coupling
1-adrenoreceptors to Ca2+ release and Ca2+ entry in rat portal vein myocytes.
J. Biol. Chem.
272:
5261-5268,
1997[Abstract/Free Full Text].
18.
McMillian, M. K.,
S. P. Soltoff,
J. D. Lechleiter,
L. W. Cantley,
and
B. R. Talamo.
Extracellular ATP increases free cytosolic calcium in rat parotid acinar cells.
Biochem. J.
255:
291-300,
1988[Medline].
19.
Merritt, J. E.,
and
T. J. Rink.
The effects of substance P and carbachol on inositol tris- and tetrakisphosphate formation and cytosolic free calcium in rat parotid acinar cells. A correlation between inositol phosphate levels and calcium entry.
J. Biol. Chem.
262:
14912-14916,
1987[Abstract/Free Full Text].
20.
Mertz, L. M.,
V. J. Horn,
B. J. Baum,
and
I. S. Ambudkar.
Calcium entry in rat parotid acini: activation by carbachol and aluminum fluoride.
Am. J. Physiol.
258 (Cell Physiol. 27):
C654-C661,
1990[Abstract/Free Full Text].
21.
Offermanns, S.,
T. Wieland,
D. Homann,
J. Sandmann,
E. Bombien,
K. Spicher,
G. Schultz,
and
K. H. Jakobs.
Transfected muscarinic acetylcholine receptors selectively couple to Gi-type G proteins and Gq/11.
Mol. Pharmacol.
45:
890-898,
1994[Abstract].
22.
O'Neill, S. C.,
P. Donoso,
and
D. A. Eisner.
The role of [Ca2+]i and [Ca2+] sensitization in the caffeine contracture of rat myocytes: measurement of [Ca2+]i and [caffeine]i.
J. Physiol. (Lond.)
425:
55-70,
1990[Abstract].
23.
Osipchuk, Y. V.,
M. Wakui,
D. I. Yule,
D. V. Gallacher,
and
O. H. Petersen.
Cytoplasmic Ca2+ oscillations evoked by receptor stimulation, G-protein activation, internal application of inositol trisphosphate or Ca2+: simultaneous microfluorimetry and Ca2+-dependent Cl
current recording in single pancreatic acinar cells.
EMBO J.
9:
697-704,
1990[Abstract].
24.
Ostrowski, J.,
M. A. Kjelsberg,
M. G. Caron,
and
R. J. Lefkowitz.
Mutagenesis of the beta 2-adrenergic receptor: how structure elucidates function.
Annu. Rev. Pharmacol. Toxicol.
32:
167-183,
1992[Medline].
25.
Otun, H.,
J. I. Gillespie,
J. R. Greenwell,
and
W. Dunlop.
Inhibition of Ca2+ mobilization by caffeine in a cultured vascular smooth muscle cell line (A7r5).
Exp. Physiol.
76:
811-814,
1991[Abstract].
26.
Putney, J. W., Jr.,
and
G. S. Bird.
The signal for capacitative calcium entry.
Cell
75:
199-201,
1993[Medline].
27.
Sawaki, K.,
B. J. Baum,
and
I. S. Ambudkar.
1-Adrenergic and m3-muscarinic receptor stimulation of phosphatidylinositol 4,5-bisphosphate-specific phospholipase C are independently mediated by G
q/11 in rat parotid gland membranes.
Arch. Biochem. Biophys.
316:
535-540,
1995[Medline].
28.
Sawaki, K.,
Y. Hiramatsu,
B. J. Baum,
and
I. S. Ambudkar.
Involvement of G
q/11 in m3-muscarinic receptor stimulation of phosphatidylinositol 4,5-bisphosphate-specific phospholipase C in rat parotid gland membranes.
Arch. Biochem. Biophys.
305:
546-550,
1993[Medline].
29.
Sjodin, L.,
E. Viitanen,
and
E. Gylfe.
Rapid down-regulation of substance P binding to guinea-pig pancreatic acinar cells during homologous desensitization.
J. Physiol. (Lond.)
476:
69-77,
1994[Abstract].
30.
Toescu, E. C.,
S. C. O'Neill,
O. H. Petersen,
and
D. A. Eisner.
Caffeine inhibits the agonist-evoked cytosolic Ca2+ signal in mouse pancreatic acinar cells by blocking inositol trisphosphate production.
J. Biol. Chem.
267:
23467-23470,
1992[Abstract/Free Full Text].
31.
Wakui, M.,
H. Kase,
and
O. H. Petersen.
Cytoplasmic Ca2+ signals evoked by activation of cholecystokinin receptors: Ca2+-dependent current recording in internally perfused pancreatic acinar cells.
J. Membr. Biol.
124:
179-187,
1991[Medline].
32.
Watson, S. P.,
and
S. Arkinstall.
The G Protein-Linked Receptor Factsbook. San Diego, CA: Academic, 1994.
33.
Wu, D.,
A. Katz,
C. H. Lee,
and
M. I. Simon.
Activation of phospholipase C by alpha-1 adrenergic receptors is mediated by the alpha subunits of Gq family.
J. Biol. Chem.
267:
25798-25802,
1992[Abstract/Free Full Text].
34.
Yule, D. I.,
and
J. A. Williams.
Mastoparan induces oscillations of cytosolic Ca2+ in rat pancreatic acinar cells.
Biochem. Biophys. Res. Commun.
177:
159-165,
1991[Medline].
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