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Departments of Gastroenterology and Clinical Research (H.-P.B., B.G.), University of Berne, CH-3010 Berne, Switzerland; Department of Pharmacology (B.M.), Medical School, Philipps University, D-35033 Marburg, Germany; and Clinical Research Unit for Gastrointestinal Endocrinology (R.D.), Department of Medicine, Philipps University, D-35033 Marburg, Germany
Address all correspondence and requests for reprints to: Dr. H.-P. Bode, Departments of Clinical Research and Gastroenterology, University of Berne, Murtenstrasse 35, CH-3010 Berne, Switzerland. E-mail: bode{at}dkf4.unibe.ch
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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GLP-1 signal transduction still is not fully understood. It seems that GLP-1 mediates its effects mainly by stimulation of adenylate cyclase and subsequent elevation of cAMP. In contrast, the ensuing steps mostly are not known or are subject to controversial discussion. Effects of elevated cAMP are often mediated by protein kinase A (PKA). At least one important exception, however, is the modulation of ion channels. Several types of channels can be modulated by binding of cAMP, independently of phosphorylation by PKA (6).
Recently, it was proposed that also the GLP-1-induced [Ca2+]cyt elevation is associated with direct ion channel modulation by cAMP. In a study that was mainly performed with insulinoma cells, but included some measurements with native rat ß-cells, GLP-1 was found to activate an inward current with properties typical for nonselective cation channels (7). Earlier, in excised patches from a less frequently used insulinoma cell line (CRI-G1), a nonselective cation channel had been described with direct modulation by cAMP, independent of PKA (8, 9). It was therefore suggested (7) that GLP-1 elevates [Ca2+]cyt via cAMP-induced opening of a nonselective cation channel, leading to depolarization by sodium influx and subsequent opening of voltage-dependent calcium channels (VDCCs). Surprisingly, Rp-cAMPS, a PKA inhibitor that acts by blocking the cAMP binding site (10), mediated similar effects as GLP-1 (7). This was viewed as further support for the cAMP-regulated cation channel thesis. It was proposed that a less specific cAMP binding site on the channel (than in PKA) could have been activated by Rp-cAMPS, reflecting the findings in excised patches from CRI-G1 cells (9). However, no other, structurally distinct PKA inhibitors were examined, and the effect of Rp-cAMPS pretreatment was not tested. Thus, the participation of PKA was not assessed conclusively.
Other studies, however, have reached the opposite conclusion: mediation of the GLP-1-induced [Ca2+]cyt rise by PKA activation. In a study with ßTC3 insulinoma cells (11), the elevating action of GLP-1 on [Ca2+]cyt was completely blocked by a low concentration of Rp-cAMPS (10 µM), suggesting PKA involvement. In another study, preincubation with the PKA inhibitor H-89 (12) abolished the calcium elevations induced by the cAMP agonist dibutyryladenosine 3',5'-cyclic monophosphate in rat ß-cells (13). Although a high concentration of H-89 (40 µM) was required, partly inhibiting the glucose-induced calcium response, these results were taken as evidence that cAMP-mediated calcium elevations depend on PKA. Finally, an earlier study reported that Rp-cAMPS inhibited the facilitating effect of GLP-1 on glucose-induced depolarization in a rat ß-cell preparation that displayed low glucose sensitivity (14).
These conflicting observations prompted us to readdress this issue, employing both a suitable ß-cell model and adequate tools for PKA inhibition. For this purpose, we chose a combination of mouse islets on the one hand and the differentiated, glucose-sensitive ß-cell line INS-1 (15) on the other. We considered regular islets as most useful because they allow us to assess ß-cell behavior within a natural environment (16, 17). The risk of preparation-induced artefacts is much lower than in single, dispersed ß-cells, where it is an obvious problem. On the other hand, ß-cells in isolated islets may be influenced by the adjacent endocrine cell types in an uncontrolled manner. Therefore, we used INS-1 cells, in addition.
We employed a new, highly specific PKA inhibitor, the staurosporine derivative KT5720 (18), as well as the unselective, but very potent, kinase inhibitor staurosporine itself (19) to avoid interpretation problems caused by the obvious limitations of Rp-cAMPS and H-89. Both, if solely used, do not allow a sufficient clarification of the role of PKA for [Ca2+]cyt under GLP-1 stimulation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The cell culture chemicals RPMI 1640, FCS, and solutions containing penicillin, streptomycin, EDTA, trypsin, or pyruvate, were obtained from Life Technologies, Inc. Fura-2 acetoxymethylester and fura-2 potassium salt were from Molecular Probes, Inc. (Eugene, OR). GLP-1 (7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36)amide was obtained from Bachem Biochemica GmbH (Heidelberg, Germany). Forskolin and H-89 were from BIOMOL Research Laboratories, Inc. (Hamburg, Germany). Staurosporine was purchased from Sigma Chemical Co. (Deisenhofen, Germany). KT5720 was from Calbiochem (Bad Soden, Germany). Rp-cAMPS was from Biolog (Bremen, Germany). Collagenase, from Clostridium histolyticum, 0.9 U/mg, was from Serva (Heidelberg, Germany).
For insulin determination in the perifusion experiments, a commercially available RIA was used, the SRI-13K Sensitive Rat Insulin RIA Kit, with rat insulin as standard and 100% cross-reactivity for mouse insulin, from Linco Research, Inc. (St. Charles, MO), purchased through Labodia, (Chanta-Merloz, Yens, Switzerland).
Measurements of cytosolic calcium,
[Ca2+]cyt, in the rat ß-cell
line INS-1
INS-1 cells, passages 80–100 (kindly donated by C. B.
Wollheim, University of Geneva, Switzerland) were grown in RPMI 1640
medium with 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, 1
mM sodium pyruvate, 10 mM HEPES, and 50
µM mercaptoethanol, as described (15). After 6–8 days,
before confluency, the cells were detached with EDTA/trypsin, then
maintained in spinner culture for 2–3 h, at 37 C, in the same medium,
but with 25 mM HEPES and 5% FCS. The cells were then
loaded with fura-2, by spinner culture in the presence of 2
µM fura-2 acetoxymethylester (fura-2/AM) for 30 min, at
37 C. After washing, the cells were kept in spinner culture at room
temperature and were used subsequently for calcium measurements.
Measurements of [Ca2+]cyt were performed with 106 cells/ml in stirred, thermostated (37 C) cuvettes in a spectrofluorimeter (Perkin-Elmer GmbH, Langen, Germany, LS 50 B). Excitation and emission wavelengths were set to 340 and 505 nm, respectively. Calibration and compensation for extracellular fura-2 were done as described before (20).
Preparation of mouse islets for measurements of islet cytosolic
calcium
Mouse islets were prepared with collagenase treatment of
pancreata from DBA/2 mice (male, 6–8 weeks old, fed ad
libitum, obtained from Charles River Deutschland GmbH, Sulzfeld, Germany). After preparation, the
isolated islets were cultured for 3–6 days in RPMI 1640 (11
mM glucose) supplemented with 10% FCS, 100 µg
streptomycin/ml, 100 U penicillin/ml, at 37 C, gassed with 95%
O2-5% CO2. For calcium measurements, islets
were then loaded with fura-2 in this medium, by 40 min incubation with
5 µM fura-2/AM, at 37 C.
Measurements of islet cytosolic calcium,
[Ca2+]cyt, with digital imaging
fluorescence microscopy
Single fura-2-loaded islets were transferred to a coverslip,
which formed the bottom of an open superfusion chamber. The chamber was
mounted onto the stage of an inverted microscope (Zeiss Axiovert 135
TV, Carl Zeiss, Oberkochen, Germany), held by an aperture
in a thermostated metal block on the stage. The islets in the chamber
were superfused continuously at 1 ml/min, using a peristaltic pump. The
chamber vol was 700 µl. Solution changes were accomplished rapidly by
means of a valve attached to an 8-chambered superfusion reservoir. The
reservoir and the metal block on the microscope stage were thermostated
to 37 C. Measurements of [Ca2+]cyt were
performed using a Zeiss/Attofluor RatioVision digital imaging system,
with alternating excitation of the cells at 334 and 380 nm, monitoring
of the resultant emission at 520 nm by an intensified CCD camera
(512 x 512 pixels), and subsequent digitizing of the signal. For
determination of [Ca2+]cyt, the ratio (R) of
the emissions at the two excitation wavelengths was formed, and
[Ca2+]cyt was calculated according to the
published equation: [Ca2+]cyt =
(R-Rmin)/(Rmax-R) x dissociation
constant (Kd) x
(Sf2/Sb2) (21). Calibration was done by
measuring two external standards, containing calcium-saturated and
calcium-free fura-2, respectively. This yielded Rmax,
Rmin, Sf2, and Sb2. As
Kd, 224 nM was used (21).
Measurement of insulin secretion from mouse islets
The same islet preparation, with islets 3–4 days after
isolation (as for the calcium measurements) was also used for
measurements of insulin secretion. For each experiment, 50 islets were
kept in a perifusion chamber, as described previously (22), at 37 C,
and perifused at a rate of 1 ml/min with Krebs-Ringer buffer
supplemented with 10 mM HEPES buffer (adjusted to pH 7.4
with NaOH) and 1 mg BSA/ml. A valve allowed switching of perifusion
buffers, for perifusion with different glucose concentrations and other
added substances. All perifusion buffers were saturated with carbogen
gas, 95% O2-5% CO2. The perifusate was
collected over 1 or 3 min, as demonstrated by the spacing of data
points (see Fig. 3C
). The experiments started with 20 min perifusion
with 3 mM ambient glucose, before sampling. Insulin was
determined with the SRI-13 K RIA from Linco Research, Inc.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In addition to GLP-1, the effect of the adenylate cyclase activator forskolin was examined. The use of forskolin enabled us to examine, selectively, GLP-1 signal transduction downstream of receptor and G protein. This seemed useful, in light of possible kinase inhibitor side actions on receptor activation and coupling to adenylate cyclase.
In INS-1 cells, GLP-1 (Fig. 1A) or forskolin (Fig. 1C) induced a
biphasic [Ca2+]cyt elevation. The effects of
GLP-1 and forskolin were distinguished only by an
indentation in the GLP-1 traces between initial peak and
plateau, and by a lower plateau in the case of GLP-1.
These differences may reflect desensitization of the GLP-1
receptor. A maximal effect on [Ca2+]cyt
required 10-8 M GLP-1
(10-9, 5 x 10-9, 10-8, and
2 x 10-8 M examined) or 1
µM forskolin (0.1, 0.5, 2, and 5 µM
examined), respectively. These concentrations were thus employed
throughout the study. The height of the initial calcium peak, after
GLP-1 or forskolin addition, was used to assess the action
of the inhibitors.
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); 1 mM Rp-cAMPS mediated a very small calcium elevation
during the first 2 min after its addition, peaking at 9 ± 4
nM (n = 5). The PKA inhibitor H-89 reduced the
[Ca2+]cyt elevations at higher concentrations
(10–40 µM, Fig. 1). However, 5 µM H-89, in
many studies (23, 24, 25, 26, 27) mediating a strong antagonistic effect on
PKA-dependent processes, did not produce a significant inhibition (Fig. 1).
In light of the strong inhibitory effect of higher H-89 concentrations,
it was of interest to examine whether H-89 can have actions on
cytosolic calcium possibly not mediated by PKA inhibition. For this
purpose, we investigated the effect of H-89 on calcium release from
intracellular stores. Calcium transport by intracellular stores in
ß-cells, so far, does not seem to be influenced by the cAMP pathway
and PKA, although this issue is still under investigation. To examine
calcium stores, 1 µM thapsigargin was added to INS-1
cells under the same conditions as GLP-1 or forskolin in
the experiments shown in Fig. 1. Thapsigargin inhibits the calcium
pumps of intracellular stores, which empties the stores rapidly, via a
leak conductance of the store membrane (28). This effect is used to
assess the filling state of the stores. H-89 had a pronounced
inhibitory effect on calcium store filling. Thapsigargin elevated
[Ca2+]cyt in INS-1 cells by 249 ± 23
nM (mean ± SD) in the absence of H-89.
This calcium rise was decreased to 110 ± 20 nM,
48 ± 5 nM, or 19 ± 4 nM in the
presence of 10, 20, or 40 µM H-89, respectively (n =
4).
After H-89, we investigated the effect of staurosporine. Staurosporine
is a very potent, albeit unselective, protein kinase inhibitor (19).
Because of its high potency, it is especially suited to reveal a
modulatory influence of protein kinases, including PKA. As H-89,
staurosporine inhibited the [Ca2+]cyt
elevations induced by GLP-1 or forskolin only at higher
concentrations (Fig. 2).
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, A and B. Increasing the glucose
concentration produced a calcium peak that was followed by oscillations
at 8, 11, and 15 mM and by a plateau-like calcium elevation
at 20 mM glucose. Additional superfusion of the islets with
10-8 M GLP-1 induced a further
calcium elevation, with attenuation or abolition of calcium
oscillations (Fig. 3A). For a better resolution of the
GLP-1 effect, superfusion with the peptide was kept short
and limited to complete evolution of the initial peak in most
experiments, to allow for return to the prestimulatory
[Ca2+]cyt values during the experiment.
Examining glucose concentrations of 8, 11, 15, and 20 mM,
the effect of GLP-1 was highest at 8 and 11 mM
glucose. Because the glucose-induced calcium changes were less variable
at 11 than at 8 mM glucose, all further experiments were
conducted at 11 mM glucose. The effect of 1
µM forskolin (Fig. 3B) again, as in INS-1 cells, was
similar to that of 10-8 M GLP-1.
Some variability between islets was encountered in the absolute calcium
levels (e.g. see Fig. 3, A and B). However, the effects of
GLP-1 or forskolin were remarkably constant in relation to
other calcium elevations in the experiments, such as the
glucose-induced effects.
To verify the relevance of our data, we examined cAMP-induced effects
on insulin secretion in our islet preparation (Fig. 3C). Our
measurements showed that the preparation responded to glucose or cAMP
elevations in a typical way. Elevation of ambient glucose from 3–11
mM produced a short decrease followed by a pronounced
peak-like increase in insulin secretion. The intermittent secretion
decrease is reminiscent of similar calcium decreases in some of our
measurements (e.g. see Fig. 6A) or in experiments by others
(31). A glucose secretion peak, followed by return to (or nearly to)
prestimulatory levels, has often been observed in secretion experiments
with mouse islets (32, 33).
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None of the three PKA inhibitors under study abolished the GLP-1-induced [Ca2+]cyt elevation in islets, even when used in concentrations in excess of those that have mediated a complete inhibition of PKA-dependent processes in other studies.
In the presence of 1 mM Rp-cAMPS (Fig. 4A), GLP-1 elevated
[Ca2+]cyt in the same manner as in the
absence of this inhibitor (Fig. 3A). Rp-cAMPS reduced amplitude and
increased frequency of the glucose-induced calcium oscillations (Fig. 4A). The mechanism of this effect remains unclear. It could be
attributed to inhibition of residual PKA activity generated, for
example, by glucagon from islet 
-cells (35). However, similar
effects were observed also after stimulation with GLP-1 or
forskolin. The oscillation amplitude was further reduced by increasing
the Rp-cAMPS concentration to 3 mM (not shown). This,
however, did not alter the GLP-1-induced calcium
elevation. At 3 mM Rp-cAMPS, added to the superfusion
medium 10 min before GLP-1, 10-8
M GLP-1 raised
[Ca2+]cyt to 291 nM (mean of two
experiments). The effect of GLP-1, furthermore, was not
affected by prolonged pretreatment with Rp-cAMPS. When 1 mM
Rp-cAMPS was added to the superfusion medium 25 min before
GLP-1, 10-8 M GLP-1
raised [Ca2+]cyt to 293 nM (mean
of two experiments). Even the highest Rp-cAMPS concentration examined
(3 mM) did not produce a
[Ca2+]cyt elevation.
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). KT5720 influenced the glucose-induced calcium oscillations in a
fashion similar to that of Rp-cAMPS, lowering amplitude and increasing
frequency. However, KT5720 also mediated a slight decrease of the mean
[Ca2+]cyt during glucose- induced calcium
oscillations (Fig. 4B), in contrast to Rp-cAMPS. The attenuation of the
GLP-1-induced calcium elevation by KT5720 may partly be
caused by the preceding decreased mean
[Ca2+]cyt.
H-89, at high concentrations (20 µM, Fig. 5A; or 40 µM, Fig. 5B),
produced a considerable inhibition of the GLP-1-
induced [Ca2+]cyt elevation in islets.
This inhibition, however, was paralleled by a proportional inhibition
of the underlying effects of glucose on
[Ca2+]cyt (Fig. 5). This action comprised two
separate effects. As with Rp-cAMPS and KT5720, H-89 decreased amplitude
and increased frequency of the glucose-dependent
[Ca2+]cyt oscillations. Still, it also
mediated a very pronounced decrease of the mean cytosolic calcium level
during glucose-dependent calcium oscillations.
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). The effect of forskolin disappeared
in parallel to the inhibition of glucose-dependent
[Ca2+]cyt changes by staurosporine (Fig. 6, C
and D). The same was found for the GLP-1-induced
[Ca2+]cyt elevation in islets (not
shown). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Rp-cAMPS (10, 36, 37) is selective for PKA, because it competes for the cAMP binding site of the enzyme. Rp-cAMPS has been used successfully to identify a number of PKA-dependent processes in diverse cell types. In most cases, 10-4 M or lower concentrations of Rp-cAMPS, with a preincubation time not longer than 15 min, have been sufficient for complete inhibition of PKA-mediated effects (36, 37). In our study, a 10-fold higher concentration of the inhibitor did not inhibit GLP-1- or forskolin-induced [Ca2+]cyt elevations, either in INS-1 cells or in islets. This observation clearly argued against involvement of PKA. However, a potential disadvantage of Rp-cAMPS is its slow penetration into cells (36, 37). Keeping the presently-used high concentrations in mind (up to 3 mM in islets), this was not a likely cause for the absence of an inhibitory action. Still, we nevertheless examined the effects of the more lipophilic, and thus more permeant, inhibitors KT5720 (18) and H-89 (12).
In the majority of studies with KT5720, concentrations below 10 µM have been used for complete inhibition of PKA-dependent processes (38, 39, 40). In our study, 10 µM KT5720 mediated only a slight inhibition of the GLP-1-induced [Ca2+]cyt elevation in islets. Lack of a relevant inhibitory action of KT5720 was furthermore confirmed by its insignificant effect in INS-1 cells.
Unlike the other two inhibitors, H-89 produced a considerable inhibition of GLP-1- or forskolin-induced [Ca2+]cyt elevations, albeit at rather high concentrations. At these concentrations, H-89 also inhibited glucose-dependent [Ca2+]cyt elevations in islets, and calcium transport by intracellular stores in INS-1 cells. These processes probably do not depend on PKA to such a large extent, although (particularly) the involvement of PKA in calcium storage is still under investigation. The glucose-induced [Ca2+]cyt elevation involves closure of ATP-sensitive potassium channels and opening of L-type VDCCs (41). PKA has no central role, if any at all, in the nutrient-mediated closure of ATP-sensitive potassium channels (42). Furthermore, in contrast to other cell types, PKA has only a very small activating effect on L-type VDCCs in ß-cells (5, 42). Calcium transport by intracellular stores in ß-cells, so far, does not seem to be under the influence of PKA (43, 44). In conclusion, the results revealed pronounced unspecific inhibitory actions of higher H-89 concentrations against several calcium transport processes. This obviously demonstrates a limited usefulness of H-89 as PKA inhibitor in studies on [Ca2+]cyt regulation.
In contrast to our study, a number of cAMP-induced processes were reported to be inhibited by concentrations of H-89 below 1 or 10 µM (45, 23, 24, 25, 26, 27).
In the present study, H-89 at concentrations below 10 µM had only small inhibitory effects. Considering the high concentration of H-89 required for inhibition of the GLP-1- induced [Ca2+]cyt elevation, its effects on glucose-induced [Ca2+]cyt elevations and calcium storage, and the lacking inhibitory action of the other inhibitors, it has to be assumed that the effect of H-89 on GLP-1 calcium signaling is of an unspecific nature, different from PKA inhibition.
The results we obtained with staurosporine, a potent (but unselective) protein kinase inhibitor, also did not suggest involvement of PKA. Again, high concentrations were required for inhibitory effects, and these concentrations also inhibited glucose-induced [Ca2+]cyt elevations. Remarkably, already 20 nM staurosporine abolished the GLP-1-induced augmentation of insulin secretion in perifused rat islets (46). This effect can be attributed to a PKA-regulated step in insulin secretion that is downstream of GLP-1-induced depolarization and [Ca2+]cyt elevation (4, 5). With regard to [Ca2+]cyt, we observed inhibitory effects only at staurosporine concentrations approximately 2 orders of magnitude higher. Staurosporine, like H-89, may thus have acted unspecificly. Both staurosporine (19) and H-89 (12) inhibit protein kinases by competing with ATP for its binding site. In ß-cells, ATP binding sites exist with regulatory function for [Ca2+]cyt and insulin secretion; for example, on ATP-sensitive potassium channels (41). Higher concentrations of H-89 and staurosporine may mediate inhibitory actions by binding to these sites.
Other investigators have reached different conclusions about PKA involvement in the GLP-1-induced depolarization and [Ca2+]cyt elevation. In a study with rat ß-cells, the cAMP-mediated [Ca2+]cyt elevation was abolished by pretreatment with 40 µM H-89 for 20 min (13). However, similar to our observations, this also led to a partial inhibition of glucose-mediated [Ca2+]cyt elevations. As we have shown, these results, obtained with a high concentration of H-89, do not allow us to reach conclusions on a mediation of the GLP-1-induced [Ca2+]cyt rise by PKA.
In studies with isolated rat (14) or mouse (5) ß-cells, Rp-cAMPS inhibited the GLP-1-induced depolarization. This effect was attributed to a decreased inhibition of ATP- sensitive potassium channels by GLP-1. Because the results have been obtained with the selective PKA inhibitor Rp-cAMPS, the observations most likely reflect involvement of PKA. However, several points argue against central relevance of these observations. An earlier investigation, with mouse islets, has provided substantial evidence against an involvement of ATP-sensitive potassium channels in this process (34). Elevation of cAMP did not decrease ß-cell potassium conductance. These results are now complemented by our data arguing against PKA involvement. Furthermore, the recent molecular characterization of the ß-cell ATP-sensitive potassium channel has not yielded evidence for an inhibitory modulation by PKA (47, 48).
The inhibitory effects of Rp-cAMPS in isolated ß-cells have been observed either at a low glucose concentration, 5 mM (5), or with cells that displayed a considerably reduced responsiveness to glucose (14). Interestingly, a very small forskolin-mediated decrease of rubidium efflux at 3 mM glucose, pointing to a cAMP-mediated decrease of ß-cell potassium conductance, was furthermore found in mouse islets (34). It is possible that PKA has a slight inhibitory action on ATP-sensitive potassium channels that is manifest at substimulatory glucose concentrations. On the other hand, it cannot be ruled out that the observations reflect altered properties of ß-cells, because of the isolation procedure. In any case, the present study provides results with general relevance for the mechanisms of GLP-1 action on [Ca2+]cyt, because glucose concentrations were used that enable maximal GLP-1 effects, and the examination of islets reflects ß-cell activity in a more physiological environment.
Some indirect evidence against PKA involvement in the GLP-1-induced [Ca2+]cyt rise has already been presented by others. In one study, the PKA inhibitor Rp-cAMPS surprisingly mimicked the depolarization and [Ca2+]cyt rise that was induced in ßTC6 insulinoma cells by GLP-1, the cAMP analog 8-bromo-cAMP, or the PKA agonist Sp-cAMPS (7). It was concluded that the effects were mediated by a cAMP-binding site with lower specificity than that in PKA. For example, the results seemed compatible with direct activation of an ion channel by cAMP. No further use of Rp-cAMPS was made in that study, and no other PKA inhibitors were tested. The observation of prominent agonistic properties of Rp-cAMPS is not confirmed by our findings. We only found a comparatively small and protracted [Ca2+]cyt rise by Rp-cAMPS in INS-1 cells, and none at all in islets. It is conceivable that these differences are attributable to the rapid, focal application of substances by a micropipette in the mentioned study (7). This may have led to an extraordinarily rapid concentration increase of Rp-cAMPS within the cells, which may have unmasked a weak partial agonistic activity of the inhibitor (9). In summary, care should be taken in the interpretation of such results, with regard to PKA involvement.
In CRI-G1 insulinoma cells, a nonselective cation channel has been characterized in excised membrane patches that is activated by addition of cAMP, probably reflecting PKA-independent regulation by cAMP (8, 9). Our results encourage an intensified search for such channels in ß-cells.
In summary, PKA-independent [Ca2+]cyt elevation seems to be one of two branches of GLP-1 signal transduction in ß-cells, the other branch being the already-known PKA- dependent stimulation of the secretory machinery, resulting in increased recruitment of secretory granules to the plasma membrane. The divergent features of these two branches could be associated with similarly divergent functional roles, in the normal as well as in the pathological state.
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Acknowledgments |
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Footnotes |
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Received October 26, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 G. Kang, O. G Chepurny, M. J Rindler, L. Collis, Z. Chepurny, W.-h. Li, M. Harbeck, M. W Roe, and G. G Holz A cAMP and Ca2+ coincidence detector in support of Ca2+-induced Ca2+ release in mouse pancreatic {beta} cells J. Physiol., July 1, 2005; 566(1): 173 - 188. [Abstract] [Full Text] [PDF]
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 D. A. D'Alessio and T. P. Vahl Glucagon-like peptide 1: evolution of an incretin into a treatment for diabetes Am J Physiol Endocrinol Metab, June 1, 2004; 286(6): E882 - E890. [Abstract] [Full Text] [PDF]
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G. G. Holz Epac: A New cAMP-Binding Protein in Support of Glucagon-Like Peptide-1 Receptor-Mediated Signal Transduction in the Pancreatic {beta}-Cell Diabetes, January 1, 2004; 53(1): 5 - 13. [Abstract] [Full Text] [PDF]
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Y. Miura and H. Matsui Glucagon-like peptide-1 induces a cAMP-dependent increase of [Na+]i associated with insulin secretion in pancreatic {beta}-cells Am J Physiol Endocrinol Metab, November 1, 2003; 285(5): E1001 - E1009. [Abstract] [Full Text] [PDF]
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 D. Arnette, T. B. Gibson, M. C. Lawrence, B. January, S. Khoo, K. McGlynn, C. A. Vanderbilt, and M. H. Cobb Regulation of ERK1 and ERK2 by Glucose and Peptide Hormones in Pancreatic {beta} Cells J. Biol. Chem., August 29, 2003; 278(35): 32517 - 32525. [Abstract] [Full Text] [PDF]
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 K. E. Mayo, L. J. Miller, D. Bataille, S. Dalle, B. Goke, B. Thorens, and D. J. Drucker International Union of Pharmacology. XXXV. The Glucagon Receptor Family Pharmacol. Rev., March 1, 2003; 55(1): 167 - 194. [Abstract] [Full Text] [PDF]
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G. Kang, J. W. Joseph, O. G. Chepurny, M. Monaco, M. B. Wheeler, J. L. Bos, F. Schwede, H.-G. Genieser, and G. G. Holz Epac-selective cAMP Analog 8-pCPT-2'-O-Me-cAMP as a Stimulus for Ca2+-induced Ca2+ Release and Exocytosis in Pancreatic beta -Cells J. Biol. Chem., February 28, 2003; 278(10): 8279 - 8285. [Abstract] [Full Text] [PDF]
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G. Kang and G. G Holz Amplification of exocytosis by Ca2+-induced Ca2+ release in INS-1 pancreatic {beta} cells J. Physiol., January 1, 2003; 546(1): 175 - 189. [Abstract] [Full Text] [PDF]
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E. Gomez, C. Pritchard, and T. P. Herbert cAMP-dependent Protein Kinase and Ca2+ Influx through L-type Voltage-gated Calcium Channels Mediate Raf-independent Activation of Extracellular Regulated Kinase in Response to Glucagon-like Peptide-1 in Pancreatic beta -Cells J. Biol. Chem., December 6, 2002; 277(50): 48146 - 48151. [Abstract] [Full Text] [PDF]
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M. Nakazaki, A. Crane, M. Hu, V. Seghers, S. Ullrich, L. Aguilar-Bryan, and J. Bryan cAMP-Activated Protein Kinase-Independent Potentiation of Insulin Secretion by cAMP Is Impaired in SUR1 Null Islets Diabetes, December 1, 2002; 51(12): 3440 - 3449. [Abstract] [Full Text] [PDF]
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P. E. MacDonald, W. El-kholy, M. J. Riedel, A. M. F. Salapatek, P. E. Light, and M. B. Wheeler The Multiple Actions of GLP-1 on the Process of Glucose-Stimulated Insulin Secretion Diabetes, December 1, 2002; 51(90003): S434 - 442. [Abstract] [Full Text] [PDF]
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Y. Kashima, T. Miki, T. Shibasaki, N. Ozaki, M. Miyazaki, H. Yano, and S. Seino Critical Role of cAMP-GEFII{middle dot}Rim2 Complex in Incretin-potentiated Insulin Secretion J. Biol. Chem., November 30, 2001; 276(49): 46046 - 46053. [Abstract] [Full Text] [PDF]
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G. Kang, O. G Chepurny, and G. G Holz cAMP-regulated guanine nucleotide exchange factor II (Epac2) mediates Ca2+-induced Ca2+ release in INS-1 pancreatic {beta}-cells J. Physiol., October 15, 2001; 536(2): 375 - 385. [Abstract] [Full Text] [PDF]
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X. Wang, J. Zhou, M. E. Doyle, and J. M. Egan Glucagon-Like Peptide-1 Causes Pancreatic Duodenal Homeobox-1 Protein Translocation from the Cytoplasm to the Nucleus of Pancreatic {beta}-Cells by a Cyclic Adenosine Monophosphate/Protein Kinase A-Dependent Mechanism Endocrinology, May 1, 2001; 142(5): 1820 - 1827. [Abstract] [Full Text]
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D. J. Drucker Minireview: The Glucagon-Like Peptides Endocrinology, February 1, 2001; 142(2): 521 - 527. [Abstract] [Full Text] [PDF]
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[Ca2+]cyt, as displayed in A and C) was used
to quantify the effect of GLP-1 (10-8
M) and forskolin (1 µM). B and D, The
indicated concentrations of the PKA inhibitors Rp-cAMPS, KT5720, or
H-89 were added 15 min before GLP-1 (B) or forskolin (D).
Data are mean ± SD of five experiments. *,
Significant differences (unpaired t test,
P < 0.01) to the control.
