©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Hormone Stimulation of Type III Adenylyl Cyclase Induces Ca Oscillations in HEK-293 Cells (*)

(Received for publication, July 17, 1995)

Gary A. Wayman Thomas R. Hinds Daniel R. Storm (§)

From the Department of Pharmacology, University of Washington, Seattle, Washington 98195

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Various forms of cross-talk between the Ca and cAMP signal transduction systems can occur in animal cells depending upon the types of adenylyl cyclases present. Here, we report that Ca oscillations can be generated by hormone stimulation of type III adenylyl cyclase expressed in HEK-293 cells. These Ca oscillations are apparently due to the unique regulatory features of type III adenylyl cyclase, which is stimulated by hormones and inhibited by elevated Cain vivo. Ca oscillations were generated by glucagon, isoproterenol, or forskolin stimulation of type III adenylyl cyclase and were dependent upon the activity of cAMP- and calmodulin-dependent protein kinases. Ca oscillations were not solely dependent upon cAMP increases since dibutyryl cAMP or (S(p))-cAMP did not stimulate Ca oscillations. We hypothesize that stimulation of type III adenylyl cyclase leads to increased cAMP, activation of inositol 1,4,5-trisphosphate receptors, and elevation of intracellular Ca. As free Ca increases, type III adenylyl cyclase activity is attenuated by CaM kinase(s) and intracellular cAMP levels decrease. When cAMP levels drop below a threshold level, the inositol 1,4,5-trisphosphate receptor is dephosphorylated and Ca is resequestered. This cycle is repeated if type III adenylyl cyclase is chronically exposed to an activator. This unique mechanism for generation of Ca oscillations in cells is distinct from others documented in the literature.


INTRODUCTION

In most mammalian tissues the Ca and cAMP signal transduction systems are tightly coupled, and cross-talk between these two regulatory systems may play an important role for various physiological phenomena including synaptic plasticity (Xia et al., 1991; Choi et al., 1993b). Intracellular free Ca (Ca) (^1)can affect cAMP levels by modulation of adenylyl cyclase or phosphodiesterase activities (reviewed by Choi et al. (1993a) and Beavo and Reifsnyder(1990)). On the other hand, cAMP-dependent protein kinase (PKA) or cAMP can affect Ca by regulating Ca ion channel activity (reviewed by Hell et al.(1994)). Because of the regulatory diversity of adenylyl cyclases, phosphodiesterases, and protein kinases, different patterns of cross-talk between the Ca and cAMP regulatory systems may be established in specific cell types.

cDNA clones for eight adenylyl cyclases have been isolated, and each of these enzymes has distinct regulatory properties (Krupinski et al., 1989, 1992; Bakalyar and Reed, 1990; Feinstein et al., 1991; Gao and Gilman, 1991; Ishikawa et al., 1992; Katsushika et al., 1992; Yoshimura and Cooper, 1992; Cali et al., 1994; Watson et al., 1994). Five of these enzymes: I-AC (Tang et al., 1991; Choi et al., 1992b), III-AC (Choi et al., 1992a; Wayman et al., 1994), V-AC and VI-AC (Yoshimura and Cooper, 1992; Katsushika et al., 1992), and VIII-AC (Cali et al., 1994), are regulated by Ca. I-AC and VIII-AC are stimulated by intracellular Cain vivo (Choi, 1992b; Cali et al., 1994), whereas V-AC and VI-AC are inhibited by Ca.

III-AC is stimulated by Ca and calmodulin (CaM) in isolated membranes when the enzyme is also activated by G(s) (Choi et al., 1992). However, we recently discovered that Ca inhibits hormone-stimulated III-AC activity in vivo (Wayman et al., 1995). Ca inhibition of III-AC is not due to activation of G(i) or protein kinase C and is apparently mediated by one of the CaM kinases. For example, Ca inhibition of III-AC is blocked by KN-62, which is an inhibitor of CaM kinases. Furthermore, III-AC is inhibited by coexpression of III-AC and constitutively activated CaM kinase-II in HEK-293 cells. The CaM kinase construct used was under the control of the metallothionein promoter, which allowed the induction CaM kinase-II expression with Zn. Ca inhibition of III-AC in vivo provides a feedback mechanism for attenuation of hormone-stimulated adenylyl cyclase activity. Since activation of PKA can increase Ca and hormone stimulation of III-AC is inhibited by Ca, one might expect Ca oscillations to be generated by hormone stimulation of III-AC. Here, we report glucagon and isoproterenol stimulation of Ca oscillations in HEK-293 cells expressing III-AC and the glucagon receptor.


EXPERIMENTAL PROCEDURES

Cell Culture

Human embryonic kidney 293 (HEK-293) cells were grown at 37 °C in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum in a humidified 95% air, 5% CO(2) incubator. Cell culture materials were from Life Technologies, Inc. unless otherwise noted.

Expression of I-AC, III-AC, and the Glucagon Receptor in HEK-293 Cells

The I-AC cDNA clone was isolated from a bovine brain cDNA library as described by Xia et al.(1991). The III-AC cDNA clone in pBluescript SK- (Bakalyar and Reed, 1990) was obtained from R. R. Reed (John Hopkins University, Baltimore, MD). The coding sequence of III-AC was ligated to CDM-8 (CDM-8(III-AC)) for expression in HEK-293 cells. Neomycin-resistant HEK-293 cells stably transfected with an expression vector CDM8 that contained cDNA for I-AC (CDM8(I-AC)), III-AC (CDM8(III-AC)), or no exogenous DNA were used for this study. These clones have been characterized previously (Choi et al., 1992a, 1992b, 1993a, 1993b; Wu et al., 1993) and were used for subsequent cotransfection with the rat glucagon receptor (Jelinek et al., 1993). Each of these cell lines was stably transfected with either the pZCEP expression vector encoding the rat glucagon receptor (pLJ4) or vector alone. For DNA transfections, cells were plated on 100-mm dishes at a density of 2 times 10^6 cells/plate, grown overnight, and transfected with the pZCEP control vector (1 µg of DNA/plate) and a hygromycin resistance vector (1 µg of DNA/plate) by the Ca phosphate method (Chen and Okayama, 1987). Hygromycin-resistant cells were selected in culture medium containing hygromycin B (Sigma, 460 units/ml) and 300 µg/ml G418. Hygromycin/neomycin-resistant cells were assayed for glucagon-stimulated adenylyl cyclase activity by use of a cAMP accumulation assay. After selection, cells were maintained in medium containing 230 units/ml hygromycin B and 300 µg/ml G418. Multiple hygromycin/neomycin-resistant clones of each type, expressing the rat glucagon receptor (GluR) and III-AC were isolated. Cells were grown for imaging as follows. Day 1 cells were plated on poly-L-lysine- or poly-D-lysine-coated Lab-Tek four-chambered coverglass slides (60,000 cells/well) and were Ca imaged on day 6.

cAMP Accumulation

Changes in intracellular cAMP levels were measured by determining the ratio of [^3H] cAMP to total ATP, ADP, and AMP pool in [^3H]adenine-loaded cells as described by Wong et al.(1991). This assay system allows rapid and extremely sensitive measurements of relative changes in intracellular cAMP levels in response to various effects. Absolute ratios for cAMP accumulation generally show some variation between experiments using different sets of cells (Federman et al., 1992; Dittman et al., 1993). It is important to emphasize, however, that relative changes in cAMP were highly consistent between experiments. Confluent cells in six-well plates were initially incubated in Dulbecco's modified Eagle's medium containing [^3H]adenine (2.0 µCi/ml, ICN) for 16-20 h, washed once with 150 mM NaCl, and incubated at 37 °C for 30 min in incubation buffer (118 mM NaCl, 4.7 mM KCl, 2.0 mM CaCl(2), 1.2 mM MgSO(4), 1.2 mM KH(2)PO(4), 0.5 mM EDTA, 10.0 mM glucose, 20.0 mM HEPES, pH 7.4) containing 1.0 mM IBMX, and various effectors as indicated. Reactions were terminated by aspiration, washing cells once with 150 mM NaCl, and the addition of 1.0 ml of ice-cold 5% trichloroacetic acid containing 1.0 µM cAMP. Culture dishes were maintained at 4 °C for 1-4 h, and acid-soluble nucleotides were separated by ion-exchange chromatography as described (Salomon et al., 1979). Reported data are the average of triplicate determinations.

Determination of Phosphoinositide Production

Cells were plated in six-well (35 mm diameter) plates and allowed to grow until nearly confluent. Cells were then assayed for phosphoinositide turnover in response to forskolin, isoproterenol, glucagon, and carbachol by the method of Masters et al.(1985) and Subers et al. (1988). Reported data are the average of triplicate determinations.

Calcium Imaging

A 75-watt xenon lamp and a Metaltek (NC) filter wheel and shutter were separated from the Nikon microscope to prevent vibrations from affecting the optical recordings. A G. W. Ellis fiberoptic light scrambler (Technical Video Ltd.) was used to transmit the light to the microscope. Excitation, emission, and neutral density filters were from Omega Optical. The objectives used were a Nikon Fluor 20/1.3 and a Nikon Fluor 40/0.85 NA. The images were intensified with a GenIIsys image intensifier (Dage-MTI) and acquired with a Dage-MTI CCD-72 series camera. All image acquisition was computer-controlled with the Universal Imaging Corporation's Image-1/FL program. Images were viewed on a Sony Trinitron color video monitor (PVM-1343MD) and printer (UP-5000) and a Javelin Electronics video monitor (model BWM9x) and stored via a Panasonic optical disk drive (LF-7010). Images were acquired at 2-3-s intervals to reduce photobleaching. Preliminary experiments verified the linearity of the Fura-2 response at the camera settings utilized in these experiments. In vivo calibrations were performed by incubating Fura-2-loaded HEK-293 cells with 20 µM A23187 for 30 min in either (a) Ca-free imaging buffer containing 1 mM EGTA to determine R(min) or (b) imaging buffer containing 30 mM CaCl(2) to determine R(max) (S380 is the ratio of the signal at 380 in Ca-free buffer divided by the 380 signal in the presence of Ca). Cells were loaded with 100 nM Fura-2-AM in Dulbecco's modified Eagle's medium at 26 C for 30 min and allowed to rest in imaging buffer at 26 C for 1 h before imaging. The Ca imaging buffer contained 140 mM NaCl, 10 mM HEPES, 5 mM KCl, 0.5 mM MgCl(2), 1.5 mM CaCl(2), 10 mM glucose in distilled H(2)O at pH 7.4. The buffers were filter sterilized through a nalge 0.2 filter. Ca-free buffer was identical to imaging buffer except that Ca was omitted and 1 mM EGTA was included. All images were corrected for background fluorescence and shading across the field of view. Conversion of the ratio of the fluorescent intensities at each excitation wavelength (340 nm and 380 nm) to intracellular free Ca was determined through standard equations (Grynkiewicz et al., 1985). The Fura-2 K(d) of 224 nM was utilized in this equation.


RESULTS

Glucagon Stimulates CaOscillations in HEK-293 Cells Expressing III-AC

In these experiments we used several types of transformed HEK-293 cell lines to analyze the contribution of III-AC to Ca transients. Glucagon does not increase intracellular Ca or cAMP in the control HEK-293 cells because they do not express glucagon receptors (Wayman et al., 1994). I-AC-expressing cells were also used as a control. I-AC is stimulated by concentrations of intracellular Ca that inhibit the activity of glucagon-stimulated III-AC activity, and it is not stimulated by glucagon or beta-adrenergic agonists in vivo (Wayman et al., 1994).

HEK-293 cells stably expressing the glucagon receptor (293-G), the glucagon receptor with I-AC (I-AC-G), or III-AC (III-AC-G) were treated with 100 nM glucagon and individual cells were Ca imaged using Fura-2 (Fig. 1). In agreement with the observations of Jelinek et al.(1993), treatment of 293-G cells with glucagon caused a single spike of intracellular Ca (Fig. 1A). Additional exposures to glucagon resulted in no further increase in Ca. Cells expressing I-AC and the glucagon receptor gave a similar response; a single peak of Ca with no additional increase with subsequent exposures to glucagon (Fig. 1B). In contrast, Ca oscillations were generated when III-AC-G cells were treated with glucagon (Fig. 1C). These oscillations were dependent upon the continued presence of glucagon and were not generated by transient exposure to the hormone.


Figure 1: Glucagon stimulation of Ca oscillations in HEK-293 cells expressing the glucagon receptor and III-AC. HEK-293 cells expressing the rat glucagon receptor (293-G), or the glucagon receptor with I-AC (I-AC-G) or III-AC (III-AC-G) were treated with 100 nM glucagon and Ca imaged using Fura-2 as described under ``Experimental Procedures.'' Representative traces from individual cells are presented.



Glucagon elicited three general types of Ca response in these cells (Fig. 2, Table 1). Only 7% of III-AC-G cells gave a single Ca spike (Fig. 2A), 9% showed an intermediate response best described as spike-plateau (Fig. 2B), and 84% exhibited Ca oscillations (Fig. 1C). In contrast, only 4% of the control 293-G cells responded with Ca oscillations (Table 1). The initial Ca spike in III-AC-G cells averaged 500 ± 136 nM and was followed by multiple Ca transients (336 ± 128 nM), which continued for at least 60 min. These oscillations were at an average frequency of 4.3 peaks/15 min. In 293-G cells, the single Ca spike averaged 332 ± 75 nM (Table 1). Several different stable cell lines expressing III-AC and the glucagon receptor were examined with analogous results.


Figure 2: Representative Ca responses to glucagon in III-AC-G cells. Typical examples of Ca responses in III-AC-G cells treated with 100 nM glucagon are presented. A, 7% of the cells showed one Ca spike; B, 8% showed a spike plateau; C, 85% showed Ca oscillations. Intracellular Ca in individual cells was measured with Fura-2 as described under ``Experimental Procedures.'' Representative traces from individual cells are presented.





Isoproterenol Stimulates CaOscillations in HEK-293 Cells Expressing III-AC

To determine if initiation of Ca oscillations was strictly dependent on glucagon, III-AC-G cells were treated with the beta-adrenergic agonist isoproterenol. HEK-293 cells express endogenous beta-adrenergic receptors that are coupled to the stimulation of III-AC in vivo (Wayman et al., 1994). Incubation of HEK-293, I-AC-G (data not shown), or 293-G cells with 10 µM isoproterenol caused a single transient Ca peak (Fig. 3A). This response was similar in amplitude and duration to that elicited by glucagon. Exposure of III-AC-G cells to isoproterenol caused Ca oscillations that strongly resembled those induced by glucagon (Fig. 3B). Therefore, the phenomenon under consideration is not specific to glucagon stimulation of III-AC.


Figure 3: Isoproterenol stimulation of Ca oscillations in HEK-293 cells expressing III-AC. A, HEK-293 cells expressing the glucagon receptor (293-G) or the glucagon receptor with III-AC (III-AC-G) were treated with 10 µM isoproterenol and Ca imaged using Fura-2 as described under ``Experimental Procedures.'' Representative traces from individual cells are presented.



Forskolin Stimulates CaOscillations in HEK-293 Cells Expressing III-AC

To determine if other adenylyl cyclase activators stimulate Ca oscillations, III-AC-G cells were treated with forskolin and Ca imaged. Forskolin stimulates adenylyl cyclases through direct interactions with the catalytic subunit and does not require G proteins (Seamon and Daly, 1981). Forskolin, but not its inactive analogue 1,9-dideoxy-forskolin, induced Ca oscillations in III-AC-G cells (Fig. 4). These oscillations were of comparable amplitude and frequency to those stimulated by either glucagon or isoproterenol. Treatment of 293-G cells with forskolin caused a single Ca peak (Fig. 7C). These results indicate that Ca oscillations in III-AC-G cells can be initiated by several different activators of III-AC and are not dependent upon hormone stimulation of the enzyme.


Figure 4: Forskolin stimulation of Ca oscillations in HEK-293 cells expressing III-AC. HEK-293 cells expressing type III adenylyl cyclase (III-AC-G) were treated with 100 µM forskolin and Ca imaged using Fura-2 as described under ``Experimental Procedures.'' A representative trace from an individual cell is presented.




Figure 7: Stimulation of Ca oscillations in 293-G cells by combinations of glucagon and IBMX. 293-G cells were exposed to either 100 nM glucagon (A and B) or 100 µM forskolin (C and D) in the presence (B and D) or absence (A and C) of 300 µM IBMX. Changes in intracellular Ca are represented by the relative change in fluorescence ratio (340 nm/380 nm), which is proportional to intracellular free Ca as described under ``Experimental Procedures.'' When present, IBMX (300 µM) was present through the duration of the experiment. Representative traces from individual cells are presented.



Comparison of Intracellular cAMP Increases Stimulated by Hormones in III-AC-G and 293-G Cells

The data described thus far suggest that elevations in cAMP may stimulate Ca oscillations in III-AC-G cells. The absence of hormone-stimulated Ca oscillations in 293-G cells, which express low levels of endogenous III-AC, might be due to insufficient cAMP increases. Therefore, 293-G, I-AC-G, and III-AC-G cells were treated with 100 nM glucagon (Fig. 5A) or 10 µM isoproterenol (Fig. 5B) and intracellular cAMP accumulations were measured. Glucagon- or isoproterenol-stimulated cAMP increases were 2-3-fold greater in III-AC-G cells than in 293-G or I-AC-G cells (Fig. 5, A and B). Forskolin-stimulated increases in cAMP were also significantly greater in III-AC-G cells compared to 293-G cells (Fig. 6). These data suggest that Ca oscillations in HEK-293 cells may require a threshold cAMP increase that is generated in III-AC-G cells, but not in 293-G or I-AC-G cells. However, other data discussed below suggest that increases in cAMP may be necessary but not sufficient for generation of Ca oscillations.


Figure 5: Hormone-stimulated cAMP increases in 293-G, I-AC-G, and III-AC-G cells. HEK-293 cells expressing the glucagon receptor alone (293-G, box) or the glucagon receptor with I-AC (I-AC-G, &cjs2112;) or III-AC (III-AC-G, ) were treated with 100 nM glucagon (A) or 10 µM isoproterenol (B). Relative cAMP accumulations were determined as described under ``Experimental Procedures.'' The data are the mean ± S.D. of triplicate assays.




Figure 6: Forskolin-stimulated cAMP increases in 293-G and III-AC-G cells. HEK-293 cells expressing the glucagon receptor alone (293-G) or the glucagon receptor and III-AC (III-AC-G) were treated with increasing concentrations of forskolin. Relative cAMP accumulations were determined as described under ``Experimental Procedures.'' The data are the mean ± S.D. of triplicate assays.



If Ca oscillations in HEK-293 cells require a threshold cAMP increase, then it might be possible to stimulate Ca oscillations in 293-G cells using glucagon or forskolin in combination with cAMP phosphodiesterase inhibitors, which increase cAMP signals. The cAMP increases produced in III-AC-G cells by 100 nM glucagon or 10 µM isoproterenol are comparable to those produced in 293-G cells by a combination of IBMX and glucagon. Exposure of 293-G cells to either glucagon or forskolin in the absence of IBMX, a phosphodiesterase inhibitor, produced a single Ca peak (Fig. 7, A and C). IBMX alone had no effect on intracellular Ca (data not shown); however, combinations of glucagon and IBMX (Fig. 7B) or forskolin and IBMX (Fig. 7D) resulted in Ca oscillations. The predominant form(s) of endogenous adenylyl cyclase in HEK-293 cells is Ca-inhibitable. For example, isoproterenol stimulated adenyly cyclase activity in HEK-293 control cells approximately 6.0-fold, and this stimulation was inhibited 60% by increasing intracellular free Ca. These data are consistent with the proposal that a minimal cAMP increase is necessary for Ca oscillations.

cAMP Analogues Alone Do Not Produce CaOscillations in HEK-293 Cells

If Ca oscillations are solely dependent upon minimal cAMP increases, then it should be possible to generate oscillations with high concentrations of membrane-permeable cAMP analogues such as dibutyryl cAMP or (S(p))-cAMP. Incubation of 293-G cells with either 1 mM dibutyryl cAMP (Fig. 8A) or 400 µM (S(p))-cAMP (Fig. 8B) resulted in a single Ca transient. Secondary challenges with greater concentrations of cAMP analogues (e.g. 5 mM dibutyryl cAMP) caused no further increase in Ca. These levels of (S(p))-cAMP or dibutyryl cAMP are sufficient to fully activate PKA in 293 cells (Impey et al., 1994). We conclude that elevated cAMP may be necessary but not sufficient for Ca oscillations. Other regulatory properties of III-AC, for example its sensitivity to Ca inhibition, may contribute to this phenomenon.


Figure 8: Effect of dibutyryl cAMP and (S(p))-cAMP on intracellular free Ca in HEK-293 cells. The effect of 1 mM dibutyryl cAMP (A) or 400 µM (S(p))-cAMP (B) on intracellular free Ca in III-AC-G cells was determined as described under ``Experimental Procedures.'' Representative traces from individual cells are presented.



Activation of PKA Is Required for Generation of CaOscillations

cAMP can regulate intracellular Ca by several mechanisms including direct interactions with Ca channels or indirectly by activation of PKA, which phosphorylates IP(3) (Nakade et al., 1994) and ryanodine receptors channels (Suko et al., 1993; Hohenegger, 1993). To determine if PKA activation is required for generation of Ca oscillations in III-AC-G cells, the effects of two PKA inhibitors, H-89 and (R(p))-cAMP (Rothermel et al., 1988), were examined. Preincubation of III-AC-G cells for 30 min with 20 µM H-89 did not block the initial Ca rise stimulated by glucagon, but it did inhibit Ca oscillations (Fig. 9A). Similarly, treatment of these cells with (R(p))-cAMP, prior to addition of isoproterenol, also blocked Ca oscillations but not the initial Ca response (Fig. 9B). These PKA inhibitors had no significant effect on basal or hormone-stimulated intracellular cAMP levels (Wayman et al., 1994). Furthermore, basal Ca levels and the magnitude of the first Ca spike were also unaffected by (R(p))-cAMP or H-89. The inability of H-89 or (R(p))-cAMP to block the initial cAMP induced Ca transients may be due to incomplete inhibition PKA or to a PKA-independent mechanism for mobilization of Ca by cAMP. These data indicate that PKA activity is required for both glucagon- and isoproterenol-stimulated Ca oscillations.


Figure 9: Inhibitors of cAMP-dependent protein kinase block hormone-stimulated Ca oscillations in III-AC-G cells. Intracellular Ca responses to 100 nM glucagon or 10 µM isoproterenol were monitored in the presence of 20 µM H-89 (A) or 300 µM (R(p))-cAMP (B). The protein kinase inhibitors were added 30 min prior to imaging. Intracellular free Ca was measured and determined as described under ``Experimental Procedures.'' Representative traces from individual cells are presented.



The CaM Kinase Inhibitor KN-62 Blocks CaOscillations

It is our working hypothesis that CaM kinase activity may contribute to Ca oscillations by inhibiting hormone or forskolin stimulation of III-AC as intracellular Ca increases. III-AC is inhibited by elevations in intracellular Ca in HEK-293 cells, and this inhibition is blocked by KN-62, a specific inhibitor of CaM kinases (Wayman et al., 1995). Consequently, Ca oscillations may arise from a cycle that includes hormone activation of III-AC, inhibition of III-AC by CaM kinases as Ca increases, and subsequent decreases in cAMP, followed by sequestration of Ca and reinitiation of the cycle when Ca drops. If this hypothesis is valid, then KN-62 should inhibit glucagon and forskolin stimulation of Ca oscillations.

KN-62 had no effect on either basal or carbachol-stimulated intracellular free Ca (data not shown). Although KN-62 did not block the initial Ca peak stimulated by glucagon and forskolin, Ca oscillations were inhibited by KN-62 (Fig. 10). However, Ca(i) did increase approximately 2-fold over the base line and stayed at this level for at least 30 min. These data are consistent with the hypothesis that Ca inhibition of III-AC, by CaM kinases, may contribute to Ca oscillations. Because CaM kinases regulate a number of proteins involved in the regulation of intracellular Ca, they may also be important for the resequestration of intracellular Ca. For example, phospholambin (Xu et al., 1993) and a sarcoplasmic reticulum Ca pump (Hawkins et al., 1994) are both phosphorylated by CaM kinases. Phosphorylation of the sarcoplasmic reticulum Ca ATPase results in a 2-fold increase in catalytic activity. Therefore, inhibition of CaM kinase activity in HEK-293 cells may inhibit the cell's ability to return intracellular free Ca to basal levels.


Figure 10: Hormone-stimulated Ca oscillations in III-AC-G cells are blocked by the CaM kinase inhibitor KN-62. Intracellular Ca responses to 100 nM glucagon or 10 µM isoproterenol were monitored in the presence of 10 µM KN-62, an inhibitor of CaM kinases. KN-62 was added 30 min prior to Ca imaging. Intracellular free Ca was measured and determined as described under ``Experimental Procedures.'' Representative traces from individual cells are presented.



CaOscillations Are Not Due to Hormone Stimulation of IP(3)Turnover

One of the major mechanisms for coupling of hormone receptors to mobilization of intracellular Ca is through stimulation of phospholipase C and activation of the inositol trisphosphate cascade (reviewed by Berridge(1993)). Inositol 1,4,5-trisphosphate (IP(3)) stimulates release of Ca from nonmitochondrial intracellular stores and, in some systems, activation of the IP(3) pathway stimulates Ca oscillations (reviewed by Berridge(1990) and Fewtrell(1993)). Intracellular cAMP has been reported to regulate IP(3) production in either a positive (Horn et al., 1991) or negative fashion (Campbell et al., 1990). To address the role of this mechanism for hormone-stimulated Ca oscillations in III-AC-G cells, we examined the effect of glucagon, isoproterenol, and forskolin on IP(3) turnover (Fig. 11). Although the muscarinic agonist carbachol increased phosphoinositide turnover 80%, glucagon, isoproterenol, and forskolin had no significant effect. These data suggest that Ca oscillations induced by hormone or forskolin stimulation of III-AC were not due to stimulation of the IP(3) pathway.


Figure 11: Effect of adenylyl cyclase activators on phosphoinositol turnover in III-AC-G cells. III-AC-G or 293-G cells were incubated with 10 µM isoproterenol, 100 µM forskolin, 100 nM glucagon, or 1 mM carbachol for 30 min. Total intracellular phosphoinositol was then determined as described under ``Experimental Procedures.'' The data are the mean ± S.D. of triplicate assays.



The IP(3)-regulated CaPool Is the Primary Source For CaOscillations

Elevations in Ca(i) can occur by several mechanisms including the opening of plasma membrane Ca channels or the release of Ca from intracellular stores. To identify the Ca pool that contributes to glucagon-stimulated Ca oscillations in III-AC-G cells, we examined the effect of glucagon and forskolin on Ca oscillations in the absence of extracellular Ca. Glucagon stimulated Ca oscillation in the absence of extracellular Ca (Fig. 12A). The amplitude of the initial Ca peak was comparable in the presence and absence of extracellular Ca. However, the amplitude of subsequent Ca peaks decayed relatively rapidly suggesting that internal pools were depleted. Therefore, extracellular Ca is not required for the initiation of oscillations but may be required for maintenance of Ca oscillations over an extended period of time.


Figure 12: Extracellular Ca is not required for hormone-stimulated Ca oscillations in III-AC-G cells. A, the effect of glucagon on intracellular free Ca in the absence of extracellular Ca (no extracellular Ca, 1 mM EGTA) was monitored. B, the effect of thapsigargin on glucagon-stimulated Ca oscillations was examined. III-AC-G cells were pretreated with 100 nM thapsigargin in the absence of extracellular Ca (Ca-free, 1 mM EGTA), followed by 100 nM glucagon as indicated. Intracellular free Ca was measured and determined as described under ``Experimental Procedures.'' Representative traces from individual cells are presented.



If the primary source of Ca for oscillations is an internal Ca pool, then thapsigargin should inhibit glucagon-stimulated Ca oscillations since this drug is an inhibitor of the intracellular sarcoenodplasmic reticulum Ca ATPases (Thastrup et al., 1990; Lytton et al., 1991). Treatment of III-AC-G cells with thapsigargin in Ca-free medium caused a rapid release and depletion of intracellular Ca stores, and intracellular free Ca returned to basal levels within 15 min (Fig. 12B). Glucagon-stimulated Ca oscillations were completely blocked by pretreatment with thapsigargin, suggesting that Ca oscillations were dependent upon intracellular Ca pools.

Two of the major intracellular Ca pools are the ryanodine- and IP(3)- sensitive pools, both of which are regulated by cAMP through PKA (Bird et al. 1993; Nakade et al., 1994; Yoshida et al., 1992). High concentrations of ryanodine inhibits the release of Ca from the ryanodine-sensitive Ca pool. Treatment of III-AC-G cells with ryanodine (1-50 µM) had no effect on glucagon-stimulated Ca oscillations in III-AC-G cells, indicating that the ryanodine-sensitive Ca pool does not contribute to this phenomenon (Fig. 13).


Figure 13: Ryanodine does not affect glucagon-stimulated Ca oscillations in III-AC-G cells. III-AC-G cells were pretreated with 40 µM ryanodine followed by 100 nM glucagon as indicated, and intracellular free Ca was measured and determined as described under ``Experimental Procedures.'' A representative trace from an individual cell is presented.



HEK-293 cells express muscarinic receptors, which are coupled to mobilization of intracellular free Ca through the phospholipase C/IP(3) pathway. The muscarinic agonist carbachol increases IP(3) turnover and intracellular Ca in these cells. Furthermore, PKA phosphorylation of IP(3) receptors stimulates Ca release from intracellular stores (Burgess et al., 1991; Bird et al., 1993; Joseph and Ryan, 1993; Nakade et al., 1994) and could account for the cAMP-generated Ca transients caused by forskolin, glucagon, or isoproterenol in III-AC-G cells. If the IP(3)-sensitive Ca pool contributes to the Ca oscillations stimulated by forskolin, then pretreatment of III-AC-G cells with carbachol in the absence of extracellular Ca should exhaust the IP(3)-sensitive pool and inhibit forskolin-stimulated Ca oscillations. Incubation of III-AC-G cells with carbachol in Ca-free media gave a single Ca transient, and subsequent addition of forskolin did not stimulate Ca oscillations (Fig. 14). Furthermore, pretreatment of these cells with forskolin for 5 min, in the absence of external Ca, diminished the Ca increase caused by subsequent application of carbachol, indicating that both reagents stimulated Ca release from a common pool. Collectively, these data suggest that the major Ca pool contributing to glucagon and forskolin-stimulated Ca oscillations was the IP(3)-sensitive pool.


Figure 14: Carbachol pretreatment inhibits forskolin-stimulated Ca oscillations in III-AC-G cells. A, III-AC-G cells were pretreated with 1 mM carbachol and no extracellular Ca followed by 100 µM forskolin as indicated, and intracellular free Ca was measured. B, III-AC-G cells were pretreated with 100 µM forskolin followed by 1 mM carbachol as indicated, and intracellular free Ca was measured. Intracellular free Ca was measured and determined as described under ``Experimental Procedures.'' Representative traces from individual cells are presented.




DISCUSSION

There is increasing interest in molecular mechanisms for generation of Ca oscillations in non-excitable cells (reviewed by Fewtrell(1993) and Berridge(1990, 1992)). Presumably Ca oscillations provide enhanced Ca signals averaged over an extended period of time without the toxicity associated with persistently elevated Ca(i). One of the most extensively characterized mechanism for generation of Ca oscillations is through stimulation of the phospholipase C/IP(3) pathway. For example, Bird et al.(1993) have proposed that IP(3)-generated sinusoidal oscillations in intracellular Ca require negative feedback regulation of phospholipase C by protein kinase C. In this study we describe a new mechanism for generation of Ca oscillations that is based upon the unique regulatory features of III-AC, an enzyme that is stimulated by G(s)-coupled receptors in vivo but inhibited by elevated Ca(i).

Forskolin, glucagon, and isoproterenol stimulated Ca oscillations in HEK-293 cells that were stably transfected with III-AC. Control HEK-293 cells did not show Ca oscillations unless glucagon or forskolin were applied with IBMX. Since HEK-293 cells express III-AC activity (Xia et al., 1993) and hormone stimulation of endogenous adenylyl cyclase activity is also Ca-inhibitable, it seems likely that this enzyme contributed to Ca oscillations in 293-G cells when endogenous cAMP phosphodiesterase activity was inhibited by IBMX. Hormone-stimulated Ca oscillations in III-AC-G cells were dependent upon PKA activity, the IP(3)-sensitive Ca pool, and they were inhibited by KN-62, a CaM kinase inhibitor. High levels of cAMP analogues, that were sufficient to generation single Ca peaks, did not stimulate Ca oscillations suggesting that elevated cAMP was necessary but not sufficient to account for Ca oscillations. Glucagon, forskolin and isoproterenol did not generate Ca oscillations by stimulating IP(3) turnover.

What is the mechanism for hormone-stimulated Ca oscillations in III-AC-G cells? Our data are most consistent with the following model (Fig. 15). When III-AC is activated by hormones, cAMP stimulates PKA, which phosphorylates and activates IP(3) receptors. As intracellular Ca rises, III-AC activity is attenuated by CaM kinase(s) and intracellular cAMP levels decrease because of cAMP phosphodiesterases. When cAMP levels drops below a threshold point and the IP(3) receptor is dephosphorylated, Ca is resequestered and the cycle can be repeated if III-AC is chronically exposed to an activator such as forskolin or glucagon. In fact, Ca oscillations do not occur with a single exposure to the hormone or forskolin; the adenylyl cyclase activator has to be constantly present for the Ca oscillations to persist. Interestingly, it has been hypothesized that feedback inhibition of adenylyl cyclase activity by intracellular Ca may lead to Ca oscillations (Rapp and Berridge, 1977; Cooper et al., 1995). The data described in this report are the first evidence that Ca inhibition of adenylyl cyclase activity can lead to Ca oscillations in animal cells.


Figure 15: Mechanism for hormone-stimulated Ca oscillations in III-AC-G cells. It is hypothesized that stimulation of III-AC-G by hormones or forskolin leads to activation of PKA, stimulation of IP(3) receptors, and increases in intracellular Ca. As intracellular Ca increases, III-AC activity is inhibited and cAMP levels are decreased by cAMP phosphodiesterases. When cAMP drops below a threshold level, Ca is resequestered and the cycle is repeated as long as activators of III-AC are present. R(s), adenylyl cyclase stimulatory receptor; III-AC, type III adenylyl cyclase; CaM, calmodulin; PKC, protein kinase C; PKA, cAMP-dependent protein kinase; PLC, phospholipase C; DAG, diacylglycerol; IP(3), inositol 1,4,5-trisphosphate; IP(3)R, IP(3) receptor/channel; CaMK II/IV, CaM kinase type II or IV; PDE, cAMP phosphodiesterase.




FOOTNOTES

*
This work was supported by National Institutes of Health Grant HL 44948. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 206-543-9280; Fax: 206-685-3822.

(^1)
The abbreviations used are: Ca, intracellular free Ca; PKA, cAMP-dependent protein kinase; CaM, calmodulin; I-AC, type I adenylyl cyclase; III-AC, type III adenylyl cyclase; 293-G, HEK-293 cells expressing the rat glucagon receptor; I-AC-G, HEK-293 cells expressing the rat glucagon receptor and I-AC; III-AC-G, HEK-293 cells expressing the rat glucagon receptor and III-AC; IBMX, isobutylmethylxanthine; IP(3), inositol 1,4,5-trisphosphate.


ACKNOWLEDGEMENTS

We thank Dr. Randy Reed for providing the III-AC cDNA clone, Dr. Zhengui Xia for providing the I-AC clone, and Dr. E. J. Choi and Dr. Andy Dittman for providing HEK-293 cells stably expressing I-AC and III-AC. We also thank Dr. Enrique Villacres, Dr. Lauren Baker, Dr. Wenhui Hua, Dr. Guy Chan, Mark Nielsen, Soren Impey, and Scott Wong for critical reading of this manuscript.


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