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 2627). 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.

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.

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) 323536. 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.

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.

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.

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 937 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.

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 3940, 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.

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⁺ 3438. 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 334243, 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).

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.

All three mammalian InsP₃R subtypes have the potential to undergo phosphorylation by PKA and by some other kinases including PKG, PKC and CaM-kinase 2245. 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 146). Most reports suggest that InsP₃R phosphorylation augments InsP₃-induced Ca²⁺ release (e.g. 121517474849], whereas others indicate that Ca²⁺ release is attenuated [e.g. 1450].

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) 192122. 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 17515253. Thus, cAMP might affect DmInsP₃R via other kinases or unknown accessory proteins that are phosphorylated by PKA.

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 5455. 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 5758 found that the phosphodiesterase inhibitor theophylline sensitized 5-HT-induced fluid secretion from Calliphora salivary glands by a factor of about 10.

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.

Because the transepithelial potential (TEP) is a sensitive indicator of the transepithelial K⁺ and Cl^- transport 283438, 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.

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.

[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.

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.