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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha -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 alpha -adrenergic receptors.

intracellular calcium store; signal transduction; acetylcholine; norepinephrine; mandibular gland; G protein; G protein-coupled receptor


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

IN SALIVARY ACINAR CELLS, stimulation of muscarinic, alpha -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 alpha -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 alpha -adrenergic, but not the SP, messenger pathways.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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).

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.

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.

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.

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.

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 alpha -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.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha 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-beta 1 (13, 21, 33). In addition, there have been several previous reports that stimulation of muscarinic, alpha -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, alpha -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 alpha -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 alpha -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 alpha -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, alpha 1-adrenergic, and NK1 receptors (32). The three receptors show an overall homology of 17-20%, due mostly to homology within highly conserved transmembrane alpha -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 alpha 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 alpha 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 alpha 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 alpha 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 alpha -adrenergic receptors but is not activated by the NK1 receptor. This is because the amino acid differences between the M3 and alpha -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 alpha -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 alpha -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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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