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cAMP potentiates InsP3-induced Ca2+ release from the endoplasmic reticulum in blowfly salivary glands

BMC Physiology volume 8, Article number: 10 (2008) Cite this article

Abstract

Background

Serotonin induces fluid secretion from Calliphora salivary glands by the parallel activation of the InsP3/Ca2+ and cAMP signaling pathways. We investigated whether cAMP affects 5-HT-induced Ca2+ signaling and InsP3-induced Ca2+ release from the endoplasmic reticulum (ER).

Results

Increasing intracellular cAMP level by bath application of forskolin, IBMX or cAMP in the continuous presence of threshold 5-HT concentrations converted oscillatory [Ca2+]i changes into a sustained increase. Intraluminal Ca2+ measurements in the ER of β-escin-permeabilized glands with mag-fura-2 revealed that cAMP augmented InsP3-induced Ca2+ release in a concentration-dependent manner. This indicated that cAMP sensitized the InsP3 receptor Ca2+ channel for InsP3. By using cAMP analogs that activated either protein kinase A (PKA) or Epac and the application of PKA-inhibitors, we found that cAMP-induced augmentation of InsP3-induced Ca2+ release was mediated by PKA not by Epac. Recordings of the transepithelial potential of the glands suggested that cAMP sensitized the InsP3/Ca2+ signaling pathway for 5-HT, because IBMX potentiated Ca2+-dependent Cl- transport activated by a threshold 5-HT concentration.

Conclusion

This report shows, for the first time for an insect system, that cAMP can potentiate InsP3-induced Ca2+ release from the ER in a PKA-dependent manner, and that this crosstalk between cAMP and InsP3/Ca2+ signaling pathways enhances transepithelial electrolyte transport.

Background

Calcium ions and cyclic AMP are ubiquitous intracellular messengers that regulate a plethora of cellular processes. Indeed, the stimulation of many non-excitable cells by neurotransmitters or hormones causes the parallel activation of the cAMP and the phosphoinositide signaling pathways [1, 2]. The latter culminates in inositol 1,4,5-trisphosphate (InsP3)-induced Ca2+ release through InsP3 receptor Ca2+ channels (InsP3R) from the endoplasmic reticulum (ER) and an elevation in intracellular Ca2+ concentration ([Ca2+]i). InsP3-induced Ca2+ release with or without Ca2+ entry from the extracellular space generates temporally and spatially coordinated Ca2+ signals leading, in many cells, to intracellular Ca2+ oscillations and waves [3–5]. Thus, Ca2+ signals can be spatially compartmentalized and coded by amplitude, frequency, and/or shape: these parameters are important for the specificity of stimulus response coupling [5].

One way of controlling Ca2+ signals can be achieved by cAMP, which has been shown to affect Ca2+ signaling at multiple sites, e.g., at the level of InsP3 generation [6–8] and InsP3-induced Ca2+ release from the ER. cAMP exerts its physiological effects through downstream effector proteins, either protein kinase A (PKA) or cAMP-specific guanine nucleotide exchange factors (cAMP-GEF) known as e xchange p roteins directly a ctivated by c AMP (Epac) [9, 10]. Upon activation by cAMP, PKA is able to phosphorylate all three subtypes of vertebrate InsP3R and thus to modulate InsP3-induced Ca2+ release from the ER [1, 11–17]. On the other hand, physiological evidence from pancreatic β cells indicates that Epac sensitizes Ca2+-induced Ca2+ release (CICR) via InsP3-R in a cAMP-dependent manner [18].

Although we are beginning to understand the functional consequences of InsP3 receptor phosphorylation and its effects on InsP3-induced Ca2+ release in some mammalian cell types, little knowledge is currently available about whether cAMP affects InsP3-induced Ca2+ release in invertebrates [19]. Only a single InsP3R isoform is expressed in Drosophila melanogaster (DmInsP3R) [20, 21] and Caenorhabditis elegans (CeInsP3R). InsP3R in both species share the main functional properties with mammalian InsP3R: InsP3 sensitivity, single channel conductance, gating, and a bell-shaped Ca2+ dependence [22–24]. However, InsP3R phosphorylation has not been investigated in these species.

Since almost nothing is known regarding whether cAMP affects InsP3R function in invertebrates or its possible mode of action, we have studied this interaction in isolated salivary glands of the blowfly Calliphora vicina, a dipteran species closely related to Drosophila. Calliphora salivary glands secrete a KCl-rich saliva when stimulated with the neurohormone serotonin (5-hydroxytryptamine, 5-HT). 5-HT activates, in parallel, the cAMP and the phosphoinositide signaling cascade [25]. The latter leads to InsP3-induced Ca2+ release from the ER and, at low 5-HT concentrations, to intracellular Ca2+ oscillations through cyclical Ca2+ release from and reuptake into the ER [26, 27]. The Ca2+ elevation activates transepithelial Cl- transport, whereas the increase in cAMP level stimulates transepithelial K+ transport [28–31]. The aim of the present study has been to investigate whether cAMP affects 5-HT-induced Ca2+ signaling and InsP3-induced Ca2+ release from the ER. We provide evidence that cAMP sensitizes the InsP3-sensitivity of InsP3-induced Ca2+ release in a PKA-dependent manner.

Results

cAMP affects 5-HT-induced Ca2+ signaling

Threshold concentrations of 5-HT (1–3 nM) induced intracellular Ca2+ oscillations, whereas saturating 5-HT concentrations (> 30 nM) produced biphasic Ca2+ responses that consisted of an initial transient followed by a plateau of elevated [Ca2+]i (Figs. 1A, B, and [26, 27]). To test whether these two types of response patterns were affected by cAMP, we increased the intracellular cAMP by bath application of 10 mM cAMP, 100 μM IBMX, or 100 μM forskolin. These substances/concentrations had no effect on resting [Ca2+]i [33]. As shown in Fig. 1A, 3 nM 5-HT induced intracellular Ca2+ oscillations, as described previously. Application of forskolin to the bath in the continuous presence of 3 nM 5-HT converted the oscillatory [Ca2+]i changes into a sustained increase (n = 8). Treatment with cAMP or IBMX had the same effect as forskolin at all tested preparations (cAMP, n = 7; IBMX, n = 5). Forskolin did not affect the sustained Ca2+ elevation produced by 30 nM 5-HT (Fig. 1B), a concentration that saturates the rate of fluid secretion [34].

Figure 1
figure 1

Forskolin augments [Ca2+] i changes induced by low 5-HT concentrations in Calliphora salivary gland cells. (A) Stimulation with 3 nM 5-HT produces intracellular Ca2+ oscillations. Application of 100 μM forskolin converts oscillatory [Ca2+]i changes into a sustained increase (n = 8). (B) Stimulation of the gland with 30 nM 5-HT, a concentration that saturates fluid secretion, produces a biphasic Ca2+ response consisting of an initial transient followed by a plateau of elevated [Ca2+]i. The sustained phase of elevated [Ca2+]i is not effected by forskolin (n = 4). (C) Application of a threshold concentration of 5-HT (1 nM) in Ca2+-free PS (0-Ca, 2 mM EGTA) increases [Ca2+]i just measurably without triggering Ca2+ spikes. Additional application of 100 μM forskolin induces a transient Ca2+ elevation, showing that forskolin augments 5-HT-induced Ca2+ release, not Ca2+ entry (n = 8).

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To determine whether the extra Ca2+ increase produced by forskolin at low 5-HT concentrations was attributable to Ca2+ influx from the extracellular space, we stimulated glands with a sub-threshold concentration of 5-HT (in order to prevent fast Ca2+ store depletion [26]) and applied forskolin in Ca2+-free PS (no added Ca2+, 2 mM EGTA). As seen in Fig. 1C, 1 nM 5-HT was below the concentration that induced marked Ca2+ oscillations (in Ca2+-containing PS), but application of 100 μM forskolin stimulated a transient Ca2+ elevation even in the absence of extracellular Ca2+. Taken together, these results suggested that cAMP did not induce Ca2+ influx but rather augmented Ca2+ release from the ER produced by low 5-HT concentrations.

cAMP augments InsP3-induced Ca2+ release from the ER

Theoretically, there are two mechanisms for the release of Ca2+ from the ER: the InsP3R and the ryanodine receptor Ca2+ channel (RyR). Blowfly salivary glands, however, seem to lack RyR [26], leaving only the InsP3R as potential target for the cAMP pathway in order to enhance Ca2+ release.

To examine directly whether cAMP augmented InsP3-induced Ca2+ release we studied Ca2+ release from the ER by intraluminal Ca2+ measurements with the low-affinity Ca2+-indicator dye Mag-fura-2. This dye accumulates within the ER and after β-escin permeabilization of the plasma membrane in an artificial "intracellular medium" (ICM) and loss of cytosolic dye, it monitors intraluminal Ca2+ ([Ca2+]L) [32, 35, 36]. Figures 2A and 2B show two representative original recordings of intraluminal Ca2+ measurements. In order to facilitate the quantitative evaluation of this type of measurements, we converted Mag-fura-2 fluorescence ratios into a percentage scale, with 0% Ca2+ release representing the intraluminal Mag-fura-2 ratio at time zero of the recording, and 100% Ca2+ release representing the fluorescence ratio after the loss of intraluminal Ca2+ following ionomycin application.

Figure 2
figure 2

cAMP augments InsP 3 -induced Ca2+ release from β-escin permeabilized cells, as shown by intraluminal Ca2+ measurements with Mag-Fura-2. (A) cAMP does not induce Ca2+ release from the ER (n = 4). (B) Application of 5 μM InsP3 induces Ca2+ release from the ER and is augmented by 100 μM cAMP. (C, D) Ca2+ release induced by 5 μM InsP3 is neither enhanced by application of fresh InsP3 solution (C) nor by mock stimulation with Rp-cAMPS (D). (E) Quantification of the cAMP-dependent augmentation of Ca2+ release induced by 5 μM InsP3 from experiments as shown in B. 0% Ca2+ release is the intraluminal Mag-fura-2 ratio at time zero of the recording; 100% Ca2+ release is the fluorescence ratio after complete loss of intraluminal Ca2+ following ionomycin application. A sigmoidal dose-response curve fitted to mean values (R2 = 0.4) of the InsP3(+cAMP)-induced Ca2+ release gives an EC50, cAMP of 2.6 μM. (F) Dose-response relationship for InsP3-induced Ca2+ release in the presence (triangles) and absence (squares) of 100 μM cAMP. The leftward shift of the dose-response relationship indicates sensitization of InsP3-induced Ca2+ release for InsP3 by cAMP. (E, F) The number of measurements for every data point is given in brackets. Means ± S.D.

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Application of 100 μM cAMP to the permeabilized gland tubules did not induce Ca2+ release from the ER, whereas the Ca2+-ionophore ionomycin led to a dramatic loss in intraluminal Ca2+ (Fig. 2A). Treatment with 5 μM InsP3, on the other hand, caused a partial Ca2+ release, and the subsequent addition of 100 μM cAMP resulted in a further Ca2+ release (Fig. 2B), indicating that cAMP had augmented InsP3-induced Ca2+ release. In order to obtain the dose-response relationship for the effect of cAMP on InsP3-induced Ca2+ release, the cAMP concentration was systematically varied, and Ca2+ release (%) (Fig. 2E, squares) was measured after cAMP addition to ICM containing 5 μM InsP3. The sigmoidal dose-response curve fitted to the mean values of the InsP3(+cAMP)-induced Ca2+ release gave a mean half maximal cAMP concentration (EC50) of 2.5 μM (Fig. 2E).

In order to exclude that the augmentation of InsP3-induced Ca2+ release was not simply the result of the addition of fresh InsP3(+cAMP)-containing ICM, we superfused several preparations with InsP3(no cAMP)-containing ICM twice. A second InsP3 application never increased Ca2+ release induced by a prior InsP3 application (Fig. 2C; n = 5). Moreover, mock stimulation with 10 μM (n = 5) or 100 μM (n = 5) 8-Br-Rp-cAMPS (a competitive antagonist of cAMP binding to PKA) had no significant effect on the InsP3-induced Ca2+ release (Fig. 2D displays a representative original recording with 10 μM 8-Br-Rp-cAMP).

To determine whether cAMP increased the affinity of the InsP3R for InsP3, we examined Ca2+ release induced by increasing InsP3-concentrations in the absence (Fig. 2F, squares) and presence of 100 μM cAMP (Fig. 2F, triangles). The two resulting dose-response curves indicated that cAMP increased the affinity of the InsP3R for InsP3, because cAMP shifted the dose-response curve to lower InsP3 concentrations by about one order of magnitude.

Is the cAMP-dependent augmentation of InsP3-induced Ca2+ release mediated by PKA or EPAC?

The effect of cAMP on InsP3-induced Ca2+ release could be mediated by either PKA or Epac. Both target proteins are expressed in blowfly salivary glands [59]. To distinguish between these possibilities, cAMP-analogs that activate either PKA or Epac or both downstream effectors were used instead of cAMP [39]. These cAMP analogs were applied at concentrations of 10 μM and 100 μM. One problem in the quantitative evaluation of these experiments was, that the Mag-fura-2 fluorescence ratio in the β-escin-permeabilized preparations continuously declined as Ca2+ leaked out of the ER (see, for example, Figs. 2A, B; 3A, C, D), and this decline in fluorescence ratio varied between preparations. Therefore, we did not measure and compare the magnitude of Ca2+ release from the ER (as above), but rather its rate as measured by the decline in the Mag-fura-2 fluorescence ratio per minute. The rates were obtained from regression lines fitted to the fluorescence traces over a one minute period before and after application of the cAMP analog (see Fig. 3A, dotted lines). As shown in Figs. 3A and 3B, 8-CPT-cAMP, activating both PKA and Epac, augmented InsP3-induced Ca2+ release significantly and in a dose-dependent manner.

Figure 3
figure 3

InsP 3 -induced Ca2+ release is augmented by PKA activators and not by Epac activators. (A, C, D) Representative original recordings showing the effects of three cAMP analogs on InsP3-induced Ca2+ release as recorded by intraluminal Ca2+ measurements with Mag-Fura-2 in β-escin-permeabilized glands. (B, D, F) Summary of results obtained from experiments as illustrated in A, C and D. Ca2+ release is displayed as the change in the rate of the Mag-Fura-2 fluorescence ratio (ΔF340/F380·min-1) before and after addition of a cAMP analog as shown in (A), dotted lines. (A, B) The PKA and Epac activator 8-CPT-cAMP augments InsP3-induced Ca2+ release significantly in a concentration-dependent manner. (C, E) Neither 8-pMeOPT-2'-O-Me-cAMP nor the two other Epac activators (8-pHPT-2'-O-Me-cAMP and 8-pCPT-2'-O-Me-cAMP) has an effect on InsP3-induced Ca2+ release. 8-pCPT-2'-O-Me-cAMP was also ineffective in GTP-containing ICM (lowest two bars). (D, F) All three tested PKA activators (6-Phe-cAMP, 6-BNZ-cAMP, 6-MBC-cAMP) augment InsP3-induced Ca2+ release in a concentration-dependent manner. (B, E, F) Means ± S.D., paired t-test, *P < 0.05, **P < 0.01, ***P < 0.001.

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Figures 3C–F summarize the effect of three Epac-specific cAMP-analogs and of three PKA-specific analogs on InsP3-induced Ca2+ release. At a concentration of 10 μM none of the Epac activators augmented InsP3-induced Ca2+ release (Figs. 3C, E). The Epac-activator 8-pHPT-2'-O-Me-cAMP produced a slight but significant increase in the rate of Ca2+ release when applied at a concentration of 100 μM, whereas the other two Epac activators were ineffective at 100 μM. Since Epac links cAMP to the activation of the small G protein Rap1 [9, 37] and since our ICM did not contain GTP, we tested whether the above Epac activators were ineffective because of the lack of GTP. However, 8-CPT-O-2'-Me-cAMP had also no significant effect on InsP3-induced Ca2+ release when applied in ICM supplemented with 3 mM GTP (Fig. 3E).

In contrast to the Epac activators all tested PKA-specific cAMP analogs augmented InsP3-induced Ca2+release significantly in a dose-dependent manner (Figs. 3E, F). These findings indicated that the cAMP-dependent augmentation of InsP3-induced Ca2+ release was mediated by PKA rather than Epac.

PKA inhibitors block the augmentation of InsP3-induced Ca2+ release by cAMP

To examine by an alternative approach whether the cAMP evoked augmentation of the InsP3-induced Ca2+ release was mediated by PKA, we tested the effect of the competitive antagonist of cAMP-binding to PKA, 8-Br-Rp-cAMPS [39, 40], and of the PKA inhibitor H-89 [41] on 8-CPT-cAMP-augmented InsP3-induced Ca2+ release. Both substances reversed the extra-Ca2+ release produced by 8-CPT-cAMP on a background of 5 μM InsP3 (Figs. 4A–D). These results provided further support for our conclusion that the cAMP-evoked augmentation of InsP3-induced Ca2+ release was mediated by PKA.

Figure 4
figure 4

The competitive antagonist of cAMP-binding to PKA, 8-Br-Rp-cAMPS (A, B), and the PKA inhibitor H-89 (C, D) reverse augmentation of InsP 3 -induced Ca2+ release caused by 8-CPT-cAMP. Graphs constructed as described for Fig. 3.

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Does cAMP-mediated augmentation of InsP3-induced Ca2+ release affect transepithelial electrolyte transport?

The transepithelial potential (TEP) is a sensitive indicator of the transepithelial K+ and Cl- transport that results from 5-HT-induced activation of the InsP3/Ca2+ and cAMP signaling pathways, because K+ transport is activated by cAMP and Cl- transport is activated by Ca2+ [34, 38]. We used TEP measurements in order to examine whether cAMP was able to amplify transepithelial Cl- transport induced (1) by 5-HT concentrations that were just sufficient to stimulate fluid secretion and (2) by saturating 5-HT concentrations. Because cAMP also stimulates transepithelial K+ transport by activating an apical vacuolar-type H+-ATPase that energizes K+ transport [33, 42, 43], we had to minimize the contribution of transepithelial K+ transport to 5-HT-induced TEP changes. This was accomplished by using a K+-free PS containing 7.5 mM of the K+ channel blocker Ba2+ to block basolateral K+ entry [44], as illustrated in Fig. 5A. A brief control stimulation with 30 nM 5-HT produced a biphasic change of the TEP. The negative-going phase of the TEP change was attributable to transepithelial Cl- transport, and the positive-going phase was caused by the somewhat delayed transepithelial K+ transport [34]. Superfusion of the preparation with BaCl2-containig PS caused the TEP to become negative by about 10 mV, because the resting TEP was slightly positive attributable to some transepithelial K+ transport in the unstimulated gland. Upon application of 1 nM 5-HT to the BaCl2-containing PS, the TEP became more negative (Fig. 5A), as a result of 5-HT-induced Ca2+ release [26] and a Ca2+-induced activation of transepithelial Cl- transport. Most significantly, 500 μM IBMX caused the TEP to become even more negative in the presence of 1 nM 5-HT. The effects of IBMX, 5-HT, and Ba2+ were reversible. Fig. 5B summarizes the results of several experiments of this kind and displays the TEP recorded at four selected time points indicated in Fig. 5A. The experiment illustrated in Fig. 5C is identical, except that the preparation was stimulated with 30 nM 5-HT, a concentration that saturates the rate of fluid transport. At this high 5-HT concentration, IBMX caused no further change of the TEP (Fig. 5D).

Figure 5
figure 5

Effects of IBMX on 5-HT-induced changes in transepithelial potential (TEP) in Ba2+-containing PS. (A, B) Original recordings. The bar graphs (B, D) display and summarize the TEPs recorded at the time points (1–4) as indicated in A and C; means ± S.D. In both groups of experiments (A, C), an initial control stimulation with 30 nM 5-HT produces a biphasic TEP change. The TEP goes negative after superfusion of the preparation with Ba2+-containing PS. Addition of 1 nM and 30 nM 5-HT cause the TEP to go further negative. The TEP recorded in the presence of 1 nM 5-HT (A, B) but not 30 nM 5-HT (C, D) goes further negative by application of 500 μM IBMX in the presence of 5-HT.

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The results of these TEP measurements indicate that an increase in intracellular cAMP concentration (by application of the phosphodiesterase inhibitor IBMX) augments the effect of a threshold concentration of 5-HT on transepithelial Cl- transport. This result is in agreement with above finding that cAMP sensitizes the InsP3R Ca2+ channel for InsP3. The physiological consequence of InsP3R sensitization is measurable only when the glands are stimulated by low 5-HT concentrations.

Discussion

The results of this study provide physiological evidence that cAMP augments InsP3-induced Ca2+ release from the ER in the salivary glands of Calliphora vicina, a dipteran fly closely related to Drosophila melanogaster. Our intraluminal Ca2+ measurements in the ER of permeabilized cells in isolated glands show, in addition, that cAMP increases the affinity of the InsP3R for InsP3 by about a factor of 10. Using cAMP analogs that activate either PKA or Epac and PKA inhibitors we show further that this cAMP effect is mediated by PKA rather than Epac. Finally, intracellular Ca2+ measurements and electrophysiological recordings indicate that the cAMP-induced and PKA-mediated sensitization of the InsP3R for InsP3 affects Ca2+ signaling and transepithelial electrolyte transport.

cAMP-induced and PKA-mediated augmentation of InsP3-induced Ca2+ release

All three mammalian InsP3R subtypes have the potential to undergo phosphorylation by PKA and by some other kinases including PKG, PKC and CaM-kinase [22, 45]. The resulting phosphoregulation of Ca2+ release is thought to have profound effects on the spatio-temporal characteristics of Ca2+ signals and to provide a potential mechanism of crosstalk between different signaling pathways. Nevertheless, data on the effects of InsP3R phosphorylation on InsP3-induced Ca2+ release are contradictory (reviewed in [1, 46]). Most reports suggest that InsP3R phosphorylation augments InsP3-induced Ca2+ release (e.g. [12, 15, 17, 47–49]], whereas others indicate that Ca2+ release is attenuated [e.g. [14, 50]].

Here, we show that cAMP augments InsP3-induced Ca2+ release in permeabilized salivary glands of Calliphora, and that the effect of cAMP is mediated by PKA. The cAMP-dependent leftward shift in the dose-response relationship for InsP3 suggests that the augmentation of Ca2+ release is attributable to an increase of about 10-fold in the affinity of the InsP3R Ca2+ channel for InsP3. We can exclude the possibility that the cAMP-induced augmentation of Ca2+ release results from a stimulation of Ca2+ loading of the ER via SERCA, because the intraluminal Ca2+ concentration is not affected by cAMP-containing ICM in the permeabilized glands.

The involvement of PKA suggests that the cAMP effect is mediated by phosphorylation of InsP3R. However, although six potential PKA phosphorylation sites have been detected in the sequence of Caenorhabditis elegans InsP3R, no such sites have been identified in Drosophila melanogaster InsP3R (DmInsP3R) [19, 21, 22]. It must be noted, however, that only a single algorithm had been used to search for putative sites for PKA-mediated phosphorylation in the Drosophila InsP3 receptor. We experienced that, at least for other proteins, results for putative phosphorylation sites vary by using different bioinformatic algorithms [Voss et al., 2007]. Sequence information for Calliphora InsP3R is still lacking but the dipteran fly Calliphora is closely related to Drosophila. Thus, whether fly InsP3 receptor Ca2+ channels can be phosphorylated, or whether the InsP3R in Calliphora differs from that in Drosophila with respect to consensus sites for PKA-mediated phosphorylation remains unknown. Therefore, we cannot yet explain the molecular basis of the cAMP-induced and PKA-mediated sensitization of Ca2+ release in this species. DmInsP3R seems to have consensus sequences for phosphorylation by PKC and CaM-kinase II [21]. The activity of these two kinases can be affected by PKA [17, 51–53]. Thus, cAMP might affect DmInsP3R via other kinases or unknown accessory proteins that are phosphorylated by PKA.

Physiological consequences of cAMP-mediated sensitization of the InsP3R for InsP3R for InsP3

The cAMP-mediated sensitization of the InsP3R for InsP3 has measurable effects on Ca2+ signaling in Calliphora salivary glands. We have shown that increasing the intracellular cAMP concentration converts baseline Ca2+ spiking induced by threshold concentrations of 5-HT [26] into a sustained Ca2+ elevation. This effect of cAMP on Ca2+ spiking is remarkably similar to that reported for the parotid acinar cell. Here, forskolin potentiates carbachol-induced [Ca2+]i changes, and this potentiation also results from enhanced Ca2+ release attributable to cAMP-dependent and PKA-mediated potentiation of InsP3-induced Ca2+ release from the ER [17]. The enhanced Ca2+ release is probably not the result of a cAMP-dependent stimulation of InsP3 production [17], although cAMP has been shown to potentiate InsP3 production in hepatocytes and parotid acinar cells [54, 55]. This possibility can be excluded in Calliphora salivary glands, as IBMX, although it potentiates 5-HT-induced fluid secretion (see below), has no effect on 5-HT-induced [3H]inositol release from isolated glands [56]. Thus, in Calliphora salivary glands, in parotid salivary glands, and in a number of other secretory cell types (such as pancreatic β cells), the InsP3R Ca2+ channel obviously functions as a coincidence detector [18] that monitors a simultaneous increase of InsP3, cAMP, and Ca2+ concentrations, the last-mentioned because InsP3R is also regulated by Ca2+ [reviewed in [22]].

Recordings of the transepithelial potential (TEP) in Calliphora salivary glands indicate that cAMP also augments the Ca2+-dependent transepithelial Cl- transport induced by low 5-HT concentrations, an observation suggesting that the cAMP-dependent enhanced Ca2+ release additionally affects fluid secretion. This notion is supported by experiments dating back more than 30 years. In the early 1970s, Berridge [57, 58] found that the phosphodiesterase inhibitor theophylline sensitized 5-HT-induced fluid secretion from Calliphora salivary glands by a factor of about 10.

Conclusion

Taking all these data together, we can now ascribe two physiological effects to cAMP in Calliphora salivary glands: (1) the activation of an apical vacuolar-type H+-ATPase [33, 59] that energizes the apical membrane for n H+/K+-antiporter-mediated K+ transport, and (2) the augmentation of InsP3-induced Ca2+ release from the ER resulting in enhanced Ca2+ signaling and enhanced transepithelial Cl- transport and fluid secretion. Both actions of cAMP are mediated by PKA, which is present at the sites of these effector proteins, the ER, and the apical membrane [59].

Methods

Animals, preparation and solutions

The blowfly, Calliphora vicina, was reared at our Institute. Flies were kept at 24–26°C under a 12 h light: 12 h dark cycle. The abdominal region of the tubular salivary glands of adult flies was dissected under physiological solution (PS).

Normal PS contained (mM): 128 NaCl, 10 KCl, 2 CaCl2, 2 MgCl2, 2.8 maleic acid, 3 sodium glutamate, 10 TRIS-HCl, 10 D-Glucose, pH 7.2. Ca2+-free PS was prepared by omitting CaCl2 and adding 2 mM EGTA. "Intracellular-like" medium (ICM) was used for experiments with β-escin-permeabilized preparations and contained (mM): 125 KCl, 20 NaCl, 2 MgCl2, 3 Na2ATP, 0.1 EGTA, 0.06 CaCl2, 10 HEPES at pH 7.3. The free Ca2+ concentration in this medium was determined to be ~250 nM, as noted previously [32]. GTP-ICM contained (mM): 125 KCl, 20 NaCl, 2 MgCl2, 3 Na2ATP, 3 GTP, 0.1 EGTA, 0.06 CaCl2, 10 HEPES at pH 7.3.

Transepithelial potential recordings

Because the transepithelial potential (TEP) is a sensitive indicator of the transepithelial K+ and Cl- transport [28, 34, 38], we used TEP recordings to obtain information about the effects of cAMP on transepithelial Cl- transport that is activated by an increase in intracellular Ca2+ concentration. Isolated salivary gland tubules (ca. 10 mm long) were placed across a narrow paraffin oil gap into a two-well perfusion chamber that was modified according to [28]. One well contained the closed end of the gland tubule and was continuously perfused with PS. The cut end of the salivary gland opened into the other well. Both wells were connected via 3 M KCl agar-bridges and AgAgCl-pellets in microelectrode holders (WPI Int., Berlin, Germany) to a differential amplifier (npi-electronics, Tamm, Germany). Data were sampled and digitized at 2 Hz (A/D-board: DAS-1600; Keithley, Germering, Germany). The software EASYEST (Asyst Software Technologies Inc., Rochester, NY) was used for data acquisition and storage, and SigmaPlot 8.0 software for offline data analysis.

Dye loading and cell permeabilization

For intracellular Ca2+ measurements the dissected glands were loaded with fura-2 by incubation with 5 μM fura-2 acetoxymethylester in PS for 40–60 min at room temperature. After dye loading, the gland tubules were mounted on cover slips coated with VectaBond™ (Axxora, Grünberg, Germany) and placed in a superfusion chamber on the stage of a Zeiss Axiovert 135TV epifluorescence microscope. In all experiments, the preparations were continuously superfused with PS (or with Ca2+-free PS) at a rate of ~1 ml/min.

For intraluminal Ca2+ measurements in the ER the glands were loaded with mag-fura-2 by a 20 min incubation with 1 μM mag-fura-2 AM in PS and subsequently mounted in glass-bottomed perfusion chambers as described above. The glands were then permeabilized for 4–8 min in ICM containing 200 μg ml-1 (w/v) β-escin. After permeabilization, excessive β-escin was washed out with ICM. The progress of permeabilization was monitored by following the decrease in mag-fura-2 fluorescence until the signal had reached a stable level attributable to the loss of cytosolic dye.

Measurements of [Ca2+]i

[Ca2+]i was measured as described previously [26]. In brief, pairs of fluorescence images, excited at wavelengths of 340 nm and 380 nm (VisiChrome High Speed Polychromator System; Visitron Systems, Puchheim, Germany) via a 450 nm dichroic mirror and a Zeiss Fluar 20/0.75 objective, were captured at a rate of 1 Hz with a cooled frame transfer CCD camera (TE/CCD-512EFT; Princeton Instruments Corp., Trenton, NJ) via a 515–565 nm bandpass filter. Raw images were processed on a PC by using the software MetaFluor (Universal Imaging Corp., West Chester, PA). Fluorescence ratios (340 nm/380 nm) were calculated after subtraction of background fluorescence and cell autofluorescence both of which were determined at the end of every experiment by quenching fura-2 fluorescence by application of 20 mM MnCl2.

Statistical analysis

Signal processing and curve fitting were performed by using GraphPad Prism 4 (Version 4.01, GraphPad Software Inc.). Data are expressed as means ± S.D. Statistical comparisons were made by a Student's paired t-test, and P values < 0.05 were considered significant.

References

  1. Bruce JIE, Straub SV, Yule DI: Crosstalk between cAMP and Ca2+ signaling in non-excitable cells. Cell Calcium. 2003, 34: 431-444. 10.1016/S0143-4160(03)00150-7.

    Article  CAS  PubMed  Google Scholar 

  2. Zaccolo M, Pozzan T: cAMP and Ca2+ interplay: a matter of oscillation. TINS. 2003, 26: 53-55.

    CAS  PubMed  Google Scholar 

  3. Berridge MJ, Lipp P, Bootman MD: The versatility and universality of calcium signalling. Nature Rev Molec Cell Biol. 2000, 1: 11-21. 10.1038/35036035.

    Article  CAS  Google Scholar 

  4. Fewtrell C: Ca2+ oscillations in non-excitable cells. Annu Rev Physiol. 1993, 55: 427-454. 10.1146/annurev.ph.55.030193.002235.

    Article  CAS  PubMed  Google Scholar 

  5. Petersen OH, Michalak M, Verkhratsky A: Calcium signalling: past, present and future. Cell Calcium. 2005, 38: 161-169. 10.1016/j.ceca.2005.06.023.

    Article  CAS  PubMed  Google Scholar 

  6. Misaki N, Imaizumi TY, Watanabe Y: Cyclic AMP-dependent protein kinase interferes with GTP gamma S stimulated IP3 formation in differentiated HL-60 cell membranes. Life Sci. 1989, 45: 1671-1678. 10.1016/0024-3205(89)90277-4.

    Article  CAS  PubMed  Google Scholar 

  7. Wu D, Katz A, Simon MI: Activation of phospholipase C β2 by the α and βγ subunits of trimeric GTP-binding protein. Proc Natl Acad Sci USA. 2001, 90: 13049-13054.

    Google Scholar 

  8. Kennedy CR, Proulx PR, Hebert RL: Regulation of bradykinin-stimulated phospholipase C and arachidonic acid release by protein kinase A in MDCK-D1 cells. Biochim Biophys Acta. 1995, 1258: 206-214.

    Article  PubMed  Google Scholar 

  9. de Rooij J, Zwartkruis FJ, Verheijen MH, Cool RH, Nijman SM, Wittinhofer A, Bos JL: Epac is a Rap1 guanone-mucleotide-exchange factor directly activated by cyclic AMP. Nature. 1998, 396: 474-477. 10.1038/24884.

    Article  CAS  PubMed  Google Scholar 

  10. Bos JL: Epac: a new cAMP target and new avenues in cAMP research. Nat Rev Mol Cell Biol. 2003, 4: 733-738. 10.1038/nrm1197.

    Article  CAS  PubMed  Google Scholar 

  11. Ferris CD, Cameron AM, Bredt DS, Huganit RL, Snyder SH: Inositol 1,4,5-trisphosphate receptor is phosphorylated by cyclic AMP-dependent protein kinase at serins 1755 and 1589. Biochem Biophys Res Commun. 1991, 175: 192-198. 10.1016/S0006-291X(05)81219-7.

    Article  CAS  PubMed  Google Scholar 

  12. Hajnoczky G, Gao E, Nomura T, Hoek JB, Thomas AP: Multiple mechanisms by which protein kinase A potentiates inositol 1,4,5-trisphosphate-induced Ca2+ mobilization in permeabilized hepatocytes. Biochem J. 1993, 293: 413-422.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  13. Nakade S, Rhee SK, Hamanaka H, Mikoshiba K: Cyclic AMP-dependent phosphorylation of an immunoaffinity-purified homotetrameric inositol 1,4,5-trisphosphate receptor (type I) increases Ca2+ flux in reconstituted lipid vesicles. J Biol Chem. 1994, 269: 6735-6742.

    CAS  PubMed  Google Scholar 

  14. Tertyshnikova S, Fein A: Inhibition of inositol 1,4,5-trisphosphate-induced Ca2+ release by cAMP-dependent protein kinase in a living cell. Proc Natl Acad Sci USA. 1998, 95: 1613-1617. 10.1073/pnas.95.4.1613.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  15. Wojcikiewicz RJ, Luo SG: Phosphorylation of inositol 1,4,5-trisphosphate receptors by cAMP-dependent protein kinase. Type I, II, and III receptors are differentially susceptible to phosphorylation and are phosphorylated in intact cells. J Biol Chem. 1998, 273: 5670-5677. 10.1074/jbc.273.10.5670.

    Article  CAS  PubMed  Google Scholar 

  16. Giovannucci DR, Groblewski GE, Sneyd J, Yule DI: Target phosphorylation of inositol 1,4,5-trisphosphate receptors selectively inhibit localized Ca2+ release and shapes oscillatory Ca2+ signals. J Biol Chem. 2000, 275: 33704-33711. 10.1074/jbc.M004278200.

    Article  CAS  PubMed  Google Scholar 

  17. Bruce JIE, Shuttleworth TJ, Giovannucci DR, Yule DI: Phosphorylation of inositol 1,4,5-trisphosphate receptors in parotid acinar cells. J Biol Chem. 2002, 277: 1340-1348. 10.1074/jbc.M106609200.

    Article  CAS  PubMed  Google Scholar 

  18. Kang G, Chepurny OG, Rindler MJ, Collis L, Chepurny Z, Li WH, Harbeck M, Roe MW, Holz GG: A cAMP and Ca2+ coincidence detector in support of Ca2+-induced Ca2+ release in mouse pancreatic β cells. J Physiol (Lond). 2005, 566: 173-188. 10.1113/jphysiol.2005.087510.

    Article  CAS  Google Scholar 

  19. Venkatesh K, Siddharta G, Joshi R, Pate SL, Hasan G: Interactions between inositol 1,4,5-trisphosphate and cAMP signaling pathways regulate larval molting in Drosophila. Genetics. 2001, 158: 309-318.

    PubMed Central  CAS  PubMed  Google Scholar 

  20. Hasan G, Rosbash M: Drosophila homologs of two mammalian intracellular Ca2+-release channels: identification and expression patterns of inositol 1,4,5-trisphosphate and the ryanodine receptor genes. Development. 1992, 116: 967-975.

    CAS  PubMed  Google Scholar 

  21. Yoshikawa S, Tanimura T, Miyawaki A, Nakamura M, Yuzaki M, Furuichi T, Mikoshiba K: Molecular cloning and characterization of the inositol 1,4,5-trisphosphate receptor in Drosophila melanogaster. J Biol Chem. 1992, 267: 16613-16619.

    CAS  PubMed  Google Scholar 

  22. Bezprozvanny I: The inositol 1,4,5-trisphosphate receptors. Cell Calcium. 2005, 38: 261-272. 10.1016/j.ceca.2005.06.030.

    Article  CAS  PubMed  Google Scholar 

  23. Srikanth S, Wang Z, Tu H, Nair S, Mathew MK, Hasan G, Bezprozvanny I: Functional properties of the Drosophila melanogaster inositol 1,4,5-trisphosphate receptor mutants. Biophys J. 2004, 86: 3634-3646. 10.1529/biophysj.104.040121.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  24. Swatton JE, Morris SA, Wissing F, Taylor CW: Functional properties of Drosophila inositol trisphosphate receptors. Biochem J. 2001, 359: 435-441. 10.1042/0264-6021:3590435.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  25. Berridge MJ: the secrets of cell signaling. Annu Rev Physiol. 2005, 67: 1-21. 10.1146/annurev.physiol.67.040103.152647.

    Article  CAS  PubMed  Google Scholar 

  26. Zimmermann B, Walz B: Serotonin-induced intercellular calcium waves in salivary glands of the blowfly Calliphora erythrocephala. J Physiol (Lond). 1997, 500: 17-28.

    Article  CAS  Google Scholar 

  27. Zimmermann B, Walz B: The mechanism mediating regenerative intercellular Ca2+ waves in the blowfly salivary gland. EMBO J. 1999, 18: 3222-3231. 10.1093/emboj/18.12.3222.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  28. Berridge MJ, Prince WT: Transepithelial potential changes during stimulation of isolated salivary glands with 5-hydroxytryptamine and cyclic AMP. J Exp Biol. 1972, 56: 139-153.

    CAS  PubMed  Google Scholar 

  29. Prince WT, Berridge MJ: The effects of 5-hydroxytryptamine and cyclic AMP on the potential profile across isolated salivary glands. J Exp Biol. 1972, 56: 323-333.

    CAS  PubMed  Google Scholar 

  30. Berridge MJ, Lindley BD, Prince WT: Membrane permeability changes during stimulation of isolated salivary glands of Calliphora by 5-hydroxytryptamine. J Physiol (Lond). 1975, 244: 549-567.

    Article  CAS  Google Scholar 

  31. Berridge MJ, Lindley BD, Prince WT: Studies on the mechanisms of fluid secretion by isolated salivary glands of Calliphora. J Exp Biol. 1976, 64: 311-322.

    CAS  PubMed  Google Scholar 

  32. Zimmermann B: Control of InsP3-induced Ca2+ oscillations in permeabilized blowfly salivary gland cells: contribution of mitochondria. J Physiol (Lond). 2000, 525: 707-719. 10.1111/j.1469-7793.2000.t01-1-00707.x.

    Article  CAS  Google Scholar 

  33. Dames P, Zimmermann B, Schmidt R, Rein J, Voss M, Schewe B, Walz B, Baumann O: cAMP regulates plasma membrane vacuolar-type H+-ATPase assembly and activity in blowfly salivary glands. Proc Natl Acad Sci USA. 2006, 103: 3926-3931. 10.1073/pnas.0600011103.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  34. Berridge MJ: The role of 5-hydroxytryptamine and cyclic AMP in the control of fluid secretion by isolated salivary glands. J Exp Biol. 1970, 53: 171-186.

    CAS  PubMed  Google Scholar 

  35. Hofer AM, Machen TE: Direct measurements of free Ca2+ in organelles of gastric epithelial cells. Am J Physiol. 1994, 267: G442-G451.

    CAS  PubMed  Google Scholar 

  36. Chatton JY, Liu H, Stucki J: Simultaneous measurements of Ca2+ in the intracellular stores and the cytosol of hepatocytes during hormone-induced Ca2+ oscillations. FEBS Lett. 1995, 368: 165-168. 10.1016/0014-5793(95)00632-J.

    Article  CAS  PubMed  Google Scholar 

  37. Kawasaki H, Springett GMN, Mochizuki N, Toki S, Nakaya M, Matsuda M, Housman DE, Graybiel AM: A family of cAMP-binding proteins that directly activate Rap1. Science. 1998, 282: 2275-2279. 10.1126/science.282.5397.2275.

    Article  CAS  PubMed  Google Scholar 

  38. Berridge MJ, Patel NG: Insect salivary glands: stimulation of fluid secretion by 5-hydroxytryptamine and adenosine-3',5'-monophosphate. Science. 1968, 162: 462-463. 10.1126/science.162.3852.462.

    Article  CAS  PubMed  Google Scholar 

  39. Christensen AE, Selheim F, de Rooij J, Dremier S, Schwede F, Dao KK, Martinez A, Maenhaut C, Bos JL, Genieser HG, Doskeland SO: cAMP analog mapping of Epac1 and cAMP kinase. Discriminating analogs demonstrate that Epac and cAMP kinase act synergistically to promote PC-12 cell neurite extension. J Biol Chem. 2003, 278: 35394-35402. 10.1074/jbc.M302179200.

    Article  CAS  PubMed  Google Scholar 

  40. Gjertsen BT, Mellgren G, Otten A, Maronde E, Genieser HG, Jastorff B, Vintermyr OK, McKnight GS, Doskeland SO: Novel (Rp)-cAMPS analogs as tools for inhibition of cAMP-kinase in cell culture. Basal cAMP-kinase activity modulates interleukin-1 beta action. J Biol Chem. 1995, 270: 20599-20607. 10.1074/jbc.270.35.20599.

    Article  CAS  PubMed  Google Scholar 

  41. Chijiwa T, Mishima A, Hagiwara M, Sano M, Hayashi K, Inoue T, Nairo K, Toshioka T, Hidaka H: Inhibition of forskolin-induced neurite outgrowth and protein phosphorylation by a newly synthesized selective inhibitor of cyclic AMP-dependent protein kinase, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), of PC12D pheochromocytoma cells. J Biol Chem. 1990, 265: 5267-5272.

    CAS  PubMed  Google Scholar 

  42. Zimmermann BP, Dames PB, Walz BO, Baumann O: Distribution and serotonin-induced activation of vacuolar-type H+-ATPase in the salivary glands of the blowfly Calliphora vicina. J Exp Biol. 2003, 206: 1867-1876. 10.1242/jeb.00376.

    Article  CAS  PubMed  Google Scholar 

  43. Rein J, Zimmermann B, Hille C, Lang I, Walz B, Baumann O: Fluorescence measurements of serotonin-induced V-ATPase-dependent pH changes at the luminal surface in salivary glands of the blowfly Calliphora vicina. J Exp Biol. 2006, 209: 1716-1724. 10.1242/jeb.02187.

    Article  CAS  PubMed  Google Scholar 

  44. Ianowski JP, O'Donnell MJ: Basolateral ion transport mechanisms during fluid secretion by Drosophila Malpighian tubules: Na+:K+:2Cl- cotransport and Cl- conductance. J Exp Biol. 2004, 207: 2599-2609. 10.1242/jeb.01058.

    Article  CAS  PubMed  Google Scholar 

  45. Yule DI, Straub SV, Bruce JIE: Modulation of Ca2+ oscillations by phosphorylation of Ins(1,4,5)P3 receptors. Biochem Soc Trans. 2003, 31: 954-957.

    Article  CAS  PubMed  Google Scholar 

  46. Straub SV, Wagner LE, Bruce JIE, Yule DI: Modulation of cytosolic calcium signaling by protein kinase A-mediated phosphorylation of inositol 1,4,5-trisphosphate receptors. Biol Res. 2004, 37: 593-602.

    Article  PubMed  Google Scholar 

  47. Brown DA, Bruce JIE, Straub SV, Yule DI: cAMP potentiates ATP-evoked calcium signaling in human parotid acinar cells. J Biol Chem. 2004, 279: 39485-39494. 10.1074/jbc.M406201200.

    Article  CAS  PubMed  Google Scholar 

  48. Dyachok O, Gylfe E: Ca2+-induced Ca2+ release via inositol 1,4,5-trisphosphate receptors is amplified by protein kinase A and triggers exocytosis in pancreatic beta-cells. J Biol Chem. 2004, 279: 45455-45461. 10.1074/jbc.M407673200.

    Article  CAS  PubMed  Google Scholar 

  49. Joseph SK, Ryan SV: Phosphorylation of the inositol trisphosphate receptor in isolated rat hepatocytes. J Biol Chem. 1993, 268: 23059-23065.

    CAS  PubMed  Google Scholar 

  50. Volpe P, Alderson-Lang BH: Regulation of inositol 1,4,5-trisphosphate-induced Ca2+ release. II. Effect of cAMP-dependent protein kinase. Am J Physiol. 1990, 258: C1086-C1091.

    CAS  PubMed  Google Scholar 

  51. Daaka Y, Luttrell LM, Lefkowitz RJ: Switching of the coupling of the beta2-adrenergic receptor to different G-proteins by protein kinase A. Nature. 1997, 390: 88-91. 10.1038/36362.

    Article  CAS  PubMed  Google Scholar 

  52. Wayman GA, Tokumitsu H, Soderling TR: Inhibitory crosstalk by cAMP kinase on the calmodulin-dependent protein kinase cascade. J Biol Chem. 1997, 272: 16073-16076. 10.1074/jbc.272.26.16073.

    Article  CAS  PubMed  Google Scholar 

  53. Valverde RH, Tortelote GG, Lemos T, Mintz E, Vieyra A: Ca2+/calmodulin-dependent protein kinase II is an essential mediator in the coordinated regulation of electrocyte Ca2+-ATPase by calmodulin and protein kinase A. J Biol Chem. 2005, 280: 30611-30618. 10.1074/jbc.M501880200.

    Article  CAS  PubMed  Google Scholar 

  54. Pittner RA, Fain JN: Exposure of cultured hepatocytes to cyclic AMP enhances the vasopressin-mediated stimulation of inositol phosphate production. Biochem J. 1989, 257: 455-460.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  55. Horn VJ, Baum BJ, Ambudkar IS: Beta-adrenergic receptor stimulation induces inositol trisphosphate production and Ca2+ mobilization in rat parotid acinar cells. J Biol Chem. 1988, 263: 12454-12460.

    CAS  PubMed  Google Scholar 

  56. Fain JN, Berridge MJ: Relationship between hormonal activation of phosphatidyl hydrolysis, fluid secretion and calcium flux in the blowfly salivary gland. Biochem J. 1979, 178: 45-58.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  57. Berridge MJ: The role of 5-hydroxytryptamine and cyclic AMP in the control of fluid secretion by isolated salivary glands. J Exp Biol. 1970, 53: 171-186.

    CAS  PubMed  Google Scholar 

  58. Berridge MJ: Transepithelial potential changes during stimulation of isolated salivary glands with 5-hydroxytryptamine and cyclic AMP. J Exp Biol. 1972, 56: 139-153.

    CAS  PubMed  Google Scholar 

  59. Rein J, Voss M, Blenau W, Walz B, Baumann O: Hormone-induced assembly and activation of V-ATPase in blowfly salivary glands is mediated by protein kinase A. Am J Physiol Cell Physiol. 2008, 294: C56-C65. 10.1152/ajpcell.00041.2007.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

This work was supported by the Deutsche Forschungsgemeinschaft (Wa 463/9 to B.W. and O.B. and GRK 837 "Functional Insect Science" to B.W.).

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  1. Institute of Biochemistry and Biology, Department of Animal Physiology, University of Potsdam, Karl-Liebknecht-Str. 24-25, 14476, Potsdam-Golm, Germany

    Ruth Schmidt, Otto Baumann & Bernd Walz

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Correspondence to Bernd Walz.

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RS carried out all experiments and drafted the manuscript. BW and OB participated in the conception of the project and the design of the experiments, and they helped to write the manuscript. All authors approved the final manuscript.

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Schmidt, R., Baumann, O. & Walz, B. cAMP potentiates InsP3-induced Ca2+ release from the endoplasmic reticulum in blowfly salivary glands. BMC Physiol 8, 10 (2008). https://doi.org/10.1186/1472-6793-8-10

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