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  <cit> .
the latter culminates in inositol  <dig> ,5-trisphosphate -induced ca2+ release through insp <dig> receptor ca2+ channels  from the endoplasmic reticulum  and an elevation in intracellular ca2+ concentration .
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  <cit> .
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  <cit> .
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 insp <dig> generation  <cit>  and insp3-induced ca2+ release from the er.
camp exerts its physiological effects through downstream effector proteins, either protein kinase a  or camp-specific guanine nucleotide exchange factors  known as exchange proteins directly activated by camp   <cit> .
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  <cit> .
on the other hand, physiological evidence from pancreatic β cells indicates that epac sensitizes ca2+-induced ca2+ release  via insp3-r in a camp-dependent manner  <cit> .
although we are beginning to understand the functional consequences of insp <dig> 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  <cit> .
only a single insp3r isoform is expressed in drosophila melanogaster   <cit>  and caenorhabditis elegans .
insp3r in both species share the main functional properties with mammalian insp3r: insp <dig> sensitivity, single channel conductance, gating, and a bell-shaped ca2+ dependence  <cit> .
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-ht activates, in parallel, the camp and the phosphoinositide signaling cascade  <cit> .
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  <cit> .
the ca2+ elevation activates transepithelial cl- transport, whereas the increase in camp level stimulates transepithelial k+ transport  <cit> .
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  induced intracellular ca2+ oscillations, whereas saturating 5-ht concentrations  produced biphasic ca2+ responses that consisted of an initial transient followed by a plateau of elevated i .
to test whether these two types of response patterns were affected by camp, we increased the intracellular camp by bath application of  <dig> mm camp,  <dig> μm ibmx, or  <dig> μm forskolin.
these substances/concentrations had no effect on resting i  <cit> .
as shown in fig.
1a,  <dig> nm 5-ht induced intracellular ca2+ oscillations, as described previously.
application of forskolin to the bath in the continuous presence of  <dig> nm 5-ht converted the oscillatory i changes into a sustained increase .
treatment with camp or ibmx had the same effect as forskolin at all tested preparations .
forskolin did not affect the sustained ca2+ elevation produced by  <dig> nm 5-ht , a concentration that saturates the rate of fluid secretion  <cit> .
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  and applied forskolin in ca2+-free ps .
as seen in fig.
1c,  <dig> nm 5-ht was below the concentration that induced marked ca2+ oscillations , but application of  <dig> 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 .
blowfly salivary glands, however, seem to lack ryr  <cit> , 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- <dig>  this dye accumulates within the er and after β-escin permeabilization of the plasma membrane in an artificial "intracellular medium"  and loss of cytosolic dye, it monitors intraluminal ca2+   <cit> .
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- <dig> fluorescence ratios into a percentage scale, with 0% ca2+ release representing the intraluminal mag-fura- <dig> 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  <dig> 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+ .
treatment with  <dig> μm insp <dig>  on the other hand, caused a partial ca2+ release, and the subsequent addition of  <dig> μm camp resulted in a further ca2+ release , 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   was measured after camp addition to icm containing  <dig> μm insp <dig>  the sigmoidal dose-response curve fitted to the mean values of the insp3-induced ca2+ release gave a mean half maximal camp concentration  of  <dig>  μm .
in order to exclude that the augmentation of insp3-induced ca2+ release was not simply the result of the addition of fresh insp3-containing icm, we superfused several preparations with insp3-containing icm twice.
a second insp <dig> application never increased ca2+ release induced by a prior insp <dig> application .
moreover, mock stimulation with  <dig> μm  or  <dig> μm  8-br-rp-camps  had no significant effect on the insp3-induced ca2+ release .
to determine whether camp increased the affinity of the insp3r for insp <dig>  we examined ca2+ release induced by increasing insp3-concentrations in the absence  and presence of  <dig> μm camp .
the two resulting dose-response curves indicated that camp increased the affinity of the insp3r for insp <dig>  because camp shifted the dose-response curve to lower insp <dig> 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  <cit> .
to distinguish between these possibilities, camp-analogs that activate either pka or epac or both downstream effectors were used instead of camp  <cit> .
these camp analogs were applied at concentrations of  <dig> μm and  <dig> μm.
one problem in the quantitative evaluation of these experiments was, that the mag-fura- <dig> fluorescence ratio in the β-escin-permeabilized preparations continuously declined as ca2+ leaked out of the er , and this decline in fluorescence ratio varied between preparations.
therefore, we did not measure and compare the magnitude of ca2+ release from the er , but rather its rate as measured by the decline in the mag-fura- <dig> 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 .
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  <dig> μm none of the epac activators augmented insp3-induced ca2+ release .
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  <dig> μm, whereas the other two epac activators were ineffective at  <dig> μm.
since epac links camp to the activation of the small g protein rap <dig>  <cit>  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  <dig> mm gtp .
in contrast to the epac activators all tested pka-specific camp analogs augmented insp3-induced ca2+release significantly in a dose-dependent manner .
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  <cit> , and of the pka inhibitor h- <dig>  <cit>  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  <dig> μm insp <dig> .
these results provided further support for our conclusion that the camp-evoked augmentation of insp3-induced ca2+ release was mediated by pka.
does camp-mediated augmentation of insp3-induced ca2+ release affect transepithelial electrolyte transport?
the transepithelial potential  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+  <cit> .
we used tep measurements in order to examine whether camp was able to amplify transepithelial cl- transport induced  by 5-ht concentrations that were just sufficient to stimulate fluid secretion and  by saturating 5-ht concentrations.
because camp also stimulates transepithelial k+ transport by activating an apical vacuolar-type h+-atpase that energizes k+ transport  <cit> , 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  <dig>  mm of the k+ channel blocker ba2+ to block basolateral k+ entry  <cit> , as illustrated in fig.
5a.
a brief control stimulation with  <dig> 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  <cit> .
superfusion of the preparation with bacl2-containig ps caused the tep to become negative by about  <dig> mv, because the resting tep was slightly positive attributable to some transepithelial k+ transport in the unstimulated gland.
upon application of  <dig> nm 5-ht to the bacl2-containing ps, the tep became more negative , as a result of 5-ht-induced ca2+ release  <cit>  and a ca2+-induced activation of transepithelial cl- transport.
most significantly,  <dig> μm ibmx caused the tep to become even more negative in the presence of  <dig> 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  <dig> 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 .
the results of these tep measurements indicate that an increase in intracellular camp concentration  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 insp <dig>  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 insp <dig> by about a factor of  <dig>  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 insp <dig> 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  <cit> .
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 .
most reports suggest that insp3r phosphorylation augments insp3-induced ca2+ release   <cit> .
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 insp <dig> receptor.
we experienced that, at least for other proteins, results for putative phosphorylation sites vary by using different bioinformatic algorithms .
sequence information for calliphora insp3r is still lacking but the dipteran fly calliphora is closely related to drosophila.
thus, whether fly insp <dig> 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  <cit> .
the activity of these two kinases can be affected by pka  <cit> .
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 insp <dig> 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  <cit>  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 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  <cit> .
the enhanced ca2+ release is probably not the result of a camp-dependent stimulation of insp <dig> production  <cit> , although camp has been shown to potentiate insp <dig> production in hepatocytes and parotid acinar cells  <cit> .
this possibility can be excluded in calliphora salivary glands, as ibmx, although it potentiates 5-ht-induced fluid secretion , has no effect on 5-ht-induced inositol release from isolated glands  <cit> .
thus, in calliphora salivary glands, in parotid salivary glands, and in a number of other secretory cell types , the insp3r ca2+ channel obviously functions as a coincidence detector  <cit>  that monitors a simultaneous increase of insp <dig>  camp, and ca2+ concentrations, the last-mentioned because insp3r is also regulated by ca2+ .
recordings of the transepithelial potential  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  <dig> years.
in the early 1970s, berridge  <cit>  found that the phosphodiesterase inhibitor theophylline sensitized 5-ht-induced fluid secretion from calliphora salivary glands by a factor of about  <dig>
CONCLUSIONS
taking all these data together, we can now ascribe two physiological effects to camp in calliphora salivary glands:  the activation of an apical vacuolar-type h+-atpase  <cit>  that energizes the apical membrane for nh+/k+-antiporter-mediated k+ transport, and  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  <cit> .

 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. the latter culminates in inositol,5-trisphosphate -induced ca2+ release through insp receptor ca2+ channels from the endoplasmic reticulum and an elevation in intracellular ca2+ concentration. 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. 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. 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 insp generation and insp3-induced ca2+ release from the er. camp exerts its physiological effects through downstream effector proteins, either protein kinase a or camp-specific guanine nucleotide exchange factors known as exchange proteins directly activated by camp. 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. on the other hand, physiological evidence from pancreatic β cells indicates that epac sensitizes ca2+-induced ca2+ release via insp3-r in a camp-dependent manner. although we are beginning to understand the functional consequences of insp 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. only a single insp3r isoform is expressed in drosophila melanogaster and caenorhabditis elegans. insp3r in both species share the main functional properties with mammalian insp3r: insp sensitivity, single channel conductance, gating, and a bell-shaped ca2+ dependence. 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-ht activates, in parallel, the camp and the phosphoinositide signaling cascade. 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. the ca2+ elevation activates transepithelial cl- transport, whereas the increase in camp level stimulates transepithelial k+ transport. 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.  camp affects 5-ht-induced ca2+ signaling threshold concentrations of 5-ht induced intracellular ca2+ oscillations, whereas saturating 5-ht concentrations produced biphasic ca2+ responses that consisted of an initial transient followed by a plateau of elevated i. to test whether these two types of response patterns were affected by camp, we increased the intracellular camp by bath application of mm camp, μm ibmx, or μm forskolin. these substances/concentrations had no effect on resting i. as shown in fig. 1a, nm 5-ht induced intracellular ca2+ oscillations, as described previously. application of forskolin to the bath in the continuous presence of nm 5-ht converted the oscillatory i changes into a sustained increase. treatment with camp or ibmx had the same effect as forskolin at all tested preparations. forskolin did not affect the sustained ca2+ elevation produced by nm 5-ht, a concentration that saturates the rate of fluid secretion. 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 and applied forskolin in ca2+-free ps. as seen in fig. 1c, nm 5-ht was below the concentration that induced marked ca2+ oscillations, but application of 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. blowfly salivary glands, however, seem to lack ryr, 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- this dye accumulates within the er and after β-escin permeabilization of the plasma membrane in an artificial "intracellular medium" and loss of cytosolic dye, it monitors intraluminal ca2+. 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- fluorescence ratios into a percentage scale, with 0% ca2+ release representing the intraluminal mag-fura- 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 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+. treatment with μm insp on the other hand, caused a partial ca2+ release, and the subsequent addition of μm camp resulted in a further ca2+ release, 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 was measured after camp addition to icm containing μm insp the sigmoidal dose-response curve fitted to the mean values of the insp3-induced ca2+ release gave a mean half maximal camp concentration of μm. in order to exclude that the augmentation of insp3-induced ca2+ release was not simply the result of the addition of fresh insp3-containing icm, we superfused several preparations with insp3-containing icm twice. a second insp application never increased ca2+ release induced by a prior insp application. moreover, mock stimulation with μm or μm 8-br-rp-camps had no significant effect on the insp3-induced ca2+ release. to determine whether camp increased the affinity of the insp3r for insp we examined ca2+ release induced by increasing insp3-concentrations in the absence and presence of μm camp. the two resulting dose-response curves indicated that camp increased the affinity of the insp3r for insp because camp shifted the dose-response curve to lower insp 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. to distinguish between these possibilities, camp-analogs that activate either pka or epac or both downstream effectors were used instead of camp. these camp analogs were applied at concentrations of μm and μm. one problem in the quantitative evaluation of these experiments was, that the mag-fura- fluorescence ratio in the β-escin-permeabilized preparations continuously declined as ca2+ leaked out of the er, and this decline in fluorescence ratio varied between preparations. therefore, we did not measure and compare the magnitude of ca2+ release from the er, but rather its rate as measured by the decline in the mag-fura- 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. 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 μm none of the epac activators augmented insp3-induced ca2+ release. 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 μm, whereas the other two epac activators were ineffective at μm. since epac links camp to the activation of the small g protein rap 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 mm gtp. in contrast to the epac activators all tested pka-specific camp analogs augmented insp3-induced ca2+release significantly in a dose-dependent manner. 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, and of the pka inhibitor h- 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 μm insp. these results provided further support for our conclusion that the camp-evoked augmentation of insp3-induced ca2+ release was mediated by pka. does camp-mediated augmentation of insp3-induced ca2+ release affect transepithelial electrolyte transport? the transepithelial potential 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+. we used tep measurements in order to examine whether camp was able to amplify transepithelial cl- transport induced by 5-ht concentrations that were just sufficient to stimulate fluid secretion and by saturating 5-ht concentrations. because camp also stimulates transepithelial k+ transport by activating an apical vacuolar-type h+-atpase that energizes k+ transport, 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 mm of the k+ channel blocker ba2+ to block basolateral k+ entry, as illustrated in fig. 5a. a brief control stimulation with 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. superfusion of the preparation with bacl2-containig ps caused the tep to become negative by about mv, because the resting tep was slightly positive attributable to some transepithelial k+ transport in the unstimulated gland. upon application of nm 5-ht to the bacl2-containing ps, the tep became more negative, as a result of 5-ht-induced ca2+ release and a ca2+-induced activation of transepithelial cl- transport. most significantly, μm ibmx caused the tep to become even more negative in the presence of 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 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. the results of these tep measurements indicate that an increase in intracellular camp concentration 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 insp 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 insp by about a factor of 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 insp 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. 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. most reports suggest that insp3r phosphorylation augments insp3-induced ca2+ release. 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 insp receptor. we experienced that, at least for other proteins, results for putative phosphorylation sites vary by using different bioinformatic algorithms. sequence information for calliphora insp3r is still lacking but the dipteran fly calliphora is closely related to drosophila. thus, whether fly insp 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. the activity of these two kinases can be affected by pka. 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 insp 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 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 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. the enhanced ca2+ release is probably not the result of a camp-dependent stimulation of insp production, although camp has been shown to potentiate insp production in hepatocytes and parotid acinar cells. this possibility can be excluded in calliphora salivary glands, as ibmx, although it potentiates 5-ht-induced fluid secretion, has no effect on 5-ht-induced inositol release from isolated glands. thus, in calliphora salivary glands, in parotid salivary glands, and in a number of other secretory cell types, the insp3r ca2+ channel obviously functions as a coincidence detector that monitors a simultaneous increase of insp camp, and ca2+ concentrations, the last-mentioned because insp3r is also regulated by ca2+. recordings of the transepithelial potential 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 years. in the early 1970s, berridge found that the phosphodiesterase inhibitor theophylline sensitized 5-ht-induced fluid secretion from calliphora salivary glands by a factor of about  taking all these data together, we can now ascribe two physiological effects to camp in calliphora salivary glands: the activation of an apical vacuolar-type h+-atpase that energizes the apical membrane for nh+/k+-antiporter-mediated k+ transport, and 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. 
increasing intracellular camp level by bath application of forskolin, ibmx or camp in the continuous presence of threshold 5-ht concentrations converted oscillatory i changes into a sustained increase. 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. this indicated that camp sensitized the insp receptor ca2+ channel for insp by using camp analogs that activated either protein kinase a 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. we investigated whether camp affects 5-ht-induced ca2+ signaling and insp3-induced ca2+ release from the endoplasmic reticulum. serotonin induces fluid secretion from calliphora salivary glands by the parallel activation of the insp3/ca2+ and camp signaling pathways. intraluminal ca2+ measurements in the er of β-escin-permeabilized glands with mag-fura- revealed that camp augmented insp3-induced ca2+ release in a concentration-dependent manner. 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.
 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.  increasing intracellular camp level by bath application of forskolin, ibmx or camp in the continuous presence of threshold 5-ht concentrations converted oscillatory i changes into a sustained increase. intraluminal ca2+ measurements in the er of β-escin-permeabilized glands with mag-fura- revealed that camp augmented insp3-induced ca2+ release in a concentration-dependent manner. this indicated that camp sensitized the insp receptor ca2+ channel for insp by using camp analogs that activated either protein kinase a 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.  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. 