©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Ca Inhibition of Type III Adenylyl Cyclase in Vivo(*)

(Received for publication, January 31, 1995; and in revised form, June 13, 1995)

Gary A. Wayman Soren Impey Daniel R. Storm (§)

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

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Type III adenylyl cyclase is stimulated by beta-adrenergic agonists and glucagon in vitro and in vivo, but not by Ca and calmodulin. However, the enzyme is stimulated by Ca and calmodulin in vitro when it is concomitantly activated by the guanyl nucleotide stimulatory protein G(s) (Choi, E. J., Xia, Z., and Storm, D. R. (1992a) Biochemistry 31, 6492-6498). Here, we examined regulation of type III adenylyl cyclase by G(s)-coupled receptors and intracellular Cain vivo. Surprisingly, intracellular Ca inhibited hormone-stimulated type III adenylyl cyclase activity. Submicromolar concentrations of intracellular free Ca, which stimulated type I adenylyl cyclase, inhibited glucagon- or isoproterenol-stimulated type III adenylyl cyclase. Inhibition of type III adenylyl cyclase by intracellular Ca was not mediated by G(i), cAMP-dependent protein kinase, or protein kinase C. However, an inhibitor of CaM kinases antagonized Ca inhibition of the enzyme, and coexpression of constitutively activated CaM kinase II completely inhibited isoproterenol-stimulated type III adenylyl cyclase activity. We propose that Ca inhibition of type III adenylyl cyclase may serve as a regulatory mechanism to attenuate hormone-stimulated cAMP levels in some tissues.


INTRODUCTION

Adenylyl cyclases are regulated by extracellular and intracellular signals including neurotransmitters, hormones, and intracellular Ca (reviewed in Tang and Gilman(1992) and Choi et al. (1993a). Each of the eight adenylyl cyclases that have been cloned (Krupinski et al., 1989; Feinstein et al., 1991; Bakalyar and Reed, 1990; Gao and Gilman, 1991; Ishikawa et al., 1992; Katsushika et al., 1992; Yoshimura and Cooper, 1992; Krupinski et al., 1992; Cali et al., 1994; Watson et al., 1994) has distinct regulatory properties. For example, I-AC (^1)(Tang et al., 1991; Choi et al., 1992a), III-AC (Choi et al., 1992a), and VIII-AC (Cali et al., 1994) are stimulated by Ca and calmodulin (CaM) in vitro but II-AC, IV-AC, V-AC, VI-AC, and VII-AC are not.

In contrast to I-AC and VIII-AC which are directly stimulated by Ca and CaM in vitro, III-AC is not stimulated by Ca and CaM unless it is also activated by GppNHp or forskolin (Choi et al., 1992a). Furthermore, the concentrations of free Ca for half-maximal stimulation of I-AC and III-AC are 150 nM and 5.0 µM Ca, respectively. These data suggested that III-AC might be synergistically stimulated by intracellular Ca- and G(s)-coupled receptors in vivo. To test this hypothesis, we examined the sensitivity of III-AC to G(s)-coupled receptor activation and intracellular Ca in HEK-293 cells. Contrary to our expectations, intracellular Ca inhibited glucagon- and isoproterenol-stimulated III-AC activities in vivo.


EXPERIMENTAL PROCEDURES

Cell Culture

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

Expression of 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), and the III-AC cDNA clone (Bakalyar and Reed, 1990) was generously provided by R. R. Reed (The John Hopkins University, Baltimore, MD). The coding sequence of III-AC and I-AC were ligated into CDM-8 for expression in HEK-293 cells. HEK-293 cells stably expressing I-AC (CDM(I-AC)) or III-AC (CDM(III-AC)) and neomycin resistance have been characterized previously (Choi et al., 1992a, 1992b, 1993a, 1993b; Wu et al., 1993) and were used for co-transfection with the rat glucagon receptor cDNA vector (Jelinek et al., 1993). Both I-AC and III-AC cell lines were stably transfected with a hygromycin resistance vector and either the pZCEP expression vector encoding the rat glucagon receptor (pLJ4) or pZCEP 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 DNA/plate) by the calcium 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 using the cAMP accumulation assay described below. After selection, cells were maintained in media containing 230 units/ml hygromycin B and 300 µg/ml G418. Multiple hygromycin/neomycin-resistant clones of each cell type, expressing the rat glucagon receptor (GluR) and III-AC or I-AC were isolated.

Adenylyl Cyclase Assay

Membrane preparations were isolated from HEK-293 cells, and the adenylyl cyclase assay was carried out as described previously (Choi et al., 1992a). Assay solutions contained 1 mM [alpha-P]ATP (500 cpm/pmol), ^3H-labeled cyclic AMP (20,000 cpm/µM), 5 mM MgCl(2), 0.2 mM EGTA, 1 mM EDTA, 2 mM cAMP, 5 mM theophylline, 0.5% bovine serum albumin, 20 mM creatine phosphate, and 100 units/ml creatine phosphokinase in 20 mM Tris-HCl, pH 7.4, unless otherwise specified. When CaCl(2) and EGTA were included in the assay, the concentration of free Ca was calculated by the method of Brooks and Storey(1992). Adenylyl cyclase activities are the mean of triplicate determinations.

cAMP Accumulation

Changes in intracellular cAMP were measured by determining the ratio of [^3H]cAMP to total ATP, ADP, and AMP pool in adenine-loaded cells (Wong et al., 1991). Absolute numbers for cAMP accumulation generally show some variation between experiments using different sets of cells (Federman et al., 1992; Dittman et al., 1994). However, relative changes in cAMP were highly consistent between experiments. Confluent cells in 6-well plates were initially incubated in DMEM 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 Dulbecco's modified Eagle's media (DMEM, Life Technologies, Inc.) containing 1.0 mM isobutylmethylxanthine and various effectors as indicated. Reactions were terminated by aspiration, washing cells once with 150 mM NaCl, and 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). Unless otherwise stated, cAMP accumulation data were corrected for endogenous adenylyl cyclase activity present in HEK-293 cells. This was accomplished by carrying out parallel experiments using the parental cell line under identical conditions. The cAMP values obtained from the parental line were subtracted from those obtained with I-AC or III-AC expressing cell lines to obtain the contribution from exogenously expressed adenylyl cyclases. Data are reported as the average of triplicate determinations ± S.D.

Coexpression of Constitutively Activated CaM Kinase II with III-AC in HEK-293 Cells

CaMKII-290 HEK-293 cells stably transformed with MT-CEV-CaMKII-290, a Zn-inducible expression vector containing the coding sequence of CaM kinase II-290, were transiently transfected with III-AC. For transfections, CaMKII-290 cells were plated at a density of 7 times 10^6 cells/100-mm plate and were maintained in DMEM, 10% fetal calf serum, 100 units/ml penicillin, and 100 µg/ml streptomycin 24 h prior to transfection and cells. On the day of transfection, the medium was aspirated and cells were rinsed with serum-free DMEM, and the media were replaced with 6.4 ml of serum-free DMEM. A mixture of 8 µg of DNA (either control CDM8 alone or CDM8 encoding type III adenylyl cyclase, CDM8-IIIAC) in 800 µl of Opti-MEM (Life Technologies, Inc.) and 64 µl of LipofectAMINE (Life Technologies, Inc.) in 800 µl of Opti-MEM were mixed, and the DNA-lipid complex was allowed to incubate for 40 min. The DNA-lipid mixture was added to each plate to be transfected, and cells were then incubated at 37 °C, 7% CO(2) for 8-10 h. The cells were then washed with serum-free DMEM, pooled, and split into 12-well plates containing DMEM, 10% fetal calf serum. On day 2, the medium was changed to serum-free DMEM with 50 µM ZnSO(4) for one-half the cells and to serum-free DMEM for the other half, for 12-18 h. Expression of CaMKII-290 was induced by the presence of Zn in the media. Cells were then assayed for cAMP accumulation on day 3 as described above.

Miscellaneous Procedures

Protein concentrations were determined by the method of Hill and Straka(1988). CaM was purified from bovine brain (Masure et al., 1984).


RESULTS

Synergistic Stimulation of III-AC by Caand Activated Gin Membranes

To examine the Ca sensitivity of III-AC in vitro and in vivo, III-AC and glucagon receptors were stably expressed in HEK-293 cells. The sensitivity of III-AC to Ca and hormones was examined in isolated membranes or intact cells. HEK-293 cells do not express I-AC or VIII-AC, and endogenous adenylyl cyclase activity is not stimulated by Ca and CaM. In the absence of other effectors, Ca and CaM did not significantly stimulate III-AC in isolated membranes (Fig. 1A). However, III-AC was stimulated by Ca and CaM when the enzyme was activated by GppNHp, a nonhydrolyzable GTP analogue that activates the guanyl nucleotide stimulatory protein G(s). In the presence of 100 µM GppNHp, Ca and CaM stimulated III-AC activity 2.1 ± 0.1-fold.


Figure 1: Synergistic stimulation of III-AC by Ca and activated G(s) in membrane preparations. A, stimulation of III-AC by Ca and CaM was assayed as a function of GppNHp concentration in membranes. B, stimulation of III-AC by Ca and CaM was assayed as a function of glucagon concentration in membranes. Membranes were prepared from HEK-293 cells stably expressing III-AC and the glucagon receptor (III-AC-G cells) and adenylyl cyclase was assayed as described under ``Experimental Procedures.'' When present, free Ca and CaM were 200 nM and 2.4 µM, respectively. Data were corrected for endogenous adenylyl cyclase activity by subtracting activity obtained from HEK-293 cells expressing only the glucagon receptor (293-G).The data are mean ± S.D. of triplicate assays.



To determine if Ca and receptor-activated G(s) will also synergistically stimulate III-AC in membranes, the sensitivity of the enzyme to CaM and Ca was analyzed in the presence of glucagon (Fig. 1B). In membrane preparations, III-AC was stimulated 4.1 ± 0.1-fold by glucagon with an EC of 7 nM. Glucagon-stimulated III-AC activity was enhanced 45 ± 6.1% by CaM and Ca; however, the EC for glucagon was not significantly affected by CaM. These data suggested that Ca stimulation of III-AC is conditional upon G(s) activation, and that Ca and hormones might synergistically activate the enzyme in vivo.

CaInhibition of Glucagon-stimulated III-AC Activity in Vivo

Glucagon stimulated III-AC 222 ± 16 fold in vivo, but I-AC was insensitive to glucagon (Fig. 2A), consistent with previous data reporting that I-AC is not stimulated by G(s)-coupled receptors in vivo (Wayman et al., 1994). The slight stimulation of cAMP levels seen with I-AC-G cells was due to glucagon stimulation of endogenous adenylyl cyclase activity. Although endogenous adenylyl cyclases in HEK-293 cells have not been fully characterized, these cells express low levels of III-AC (Xia at al., 1992) which may account for glucagon stimulation of cAMP levels in 293-G and I-AC-G cells.


Figure 2: Ca inhibits glucagon-stimulated III-AC activity in vivo. A, HEK-293 cells expressing no glucagon receptor (293), the glucagon receptor (293-G), I-AC and the glucagon receptor (I-AC-G), or III-AC and the glucagon receptor (III-AC-G) were treated with increasing concentrations of glucagon and assayed for cAMP accumulation as described under ``Experimental Procedures.'' cAMP accumulation assays were performed in triplicate, and the data are the mean ± S.D. of triplicate assays. B, HEK-293 cells expressing III-AC and the glucagon receptor (III-AC-G) were treated with increasing concentrations of glucagon in the presence or absence of 10 µM A23187 and 1.8 mM CaCl(2) and assayed for cAMP accumulation as described under ``Experimental Procedures.'' The data are the mean ± S.D. of triplicate assays.



From in vitro data using isolated membranes, we expected that glucagon and intracellular Ca would synergistically stimulate III-AC in intact cells. In fact, increases in intracellular Ca, generated by A23187 and extracellular Ca, inhibited glucagon-stimulated III-AC activity 60% (Fig. 2B). A23187 and extracellular Ca had no effect on the basal activity of III-AC or endogenous adenylyl cyclase activity (data not shown). Ca inhibited glucagon-stimulated III-AC activity in several different III-AC stable cell lines and was not due to clonal variation. With different cell lines, Ca inhibition of glucagon-stimulated III-AC activity varied from 40-60%.

The Ca dependences for inhibition of III-AC and stimulation of I-AC in vivo were compared using A23187 and varying amounts of extracellular Ca (Fig. 3). In this experiment, III-AC-G or I-AC-G cells were treated with 100 nM glucagon, but only III-AC was stimulated by glucagon. Glucagon-stimulated III-AC activity was inhibited by Ca concentrations which stimulated I-AC, and the curves were almost mirror images of each other. The concentration of free intracellular Ca for half-maximal inhibition of glucagon-stimulated III-AC activity was estimated at 150 to 200 nM using Fura-2 imagining.


Figure 3: Ca concentration dependence for inhibition of glucagon-stimulated III-AC activity in vivo. HEK-293 cells stably expressing I-AC (I-AC-G) or III-AC (III-AC-G) were treated with glucagon (100 nM), 10 µM A23187, and increasing concentrations of CaCl(2) as described under ``Experimental Procedures.'' Relative cAMP accumulations were determined as described under ``Experimental Procedures.'' The III-AC and I-AC data are presented as percentage of the ratio (cAMP/[ATP + ADP + AMP]) times 100 with no added CaCl(2) and are the mean ± S.D. of triplicate assays.



CaInhibition of Isoproterenol-stimulated III-AC Activity in Vivo

To address the generality of the phenomenon described above, we also examined the effect of intracellular Ca increases on isoproterenol stimulated III-AC activity. HEK-293 cells express endogenous beta-adrenergic receptors that are coupled to stimulation of III-AC in vivo (Fig. 4). Isoproterenol-stimulated III-AC activity was inhibited 41 ± 3% by A23187 indicating that Ca inhibition of III-AC activity was not a unique property of glucagon-stimulated activity.


Figure 4: Ca inhibits isoproterenol- stimulated III-AC activity in vivo. HEK-293 cells expressing III-AC were exposed to increasing concentrations of the beta-adrenergic agonist isoproterenol, in the absence or presence of 10 µM A23187 and 1.8 mM CaCl(2) as described under ``Experimental Procedures.''



In the experiments described above, intracellular Ca was elevated using A23187 and it was of interest to determine if Ca generated by physiologically relevant signals would also inhibit hormone stimulated III-AC. HEK-293 cells contain muscarinic receptors coupled to the mobilization of intracellular Ca. Treatment of HEK-293 cells with 10 µM carbachol elevates intracellular Ca to approximately 300 nM free Ca and stimulates I-AC (Choi et al., 1992b). Carbachol alone did not significantly affect III-AC activity but did inhibit isoproterenol-stimulated activity 43 ± 5% (Fig. 5). Inhibition of isoproterenol-stimulated III-AC activity by carbachol was insensitive to pertussis toxin and therefore not due to endogenous muscarinic receptors coupled to III-AC through G(i) (data not shown). These data indicate that physiologically relevant concentrations of intracellular Ca inhibit hormone-stimulated III-AC activity in vivo.


Figure 5: Inhibition of isoproterenol-stimulated type III adenylyl cyclase by carbachol. HEK-293 cells expressing III-AC (III-AC-G) were exposed to increasing concentrations of the beta-adrenergic agonist isoproterenol in the presence or absence of 10 µM carbachol. Under these conditions, carbachol increased intracellular free Ca from approximately 50 nM to 300 nM. cAMP accumulations were monitored as described under ``Experimental Procedures,'' and the data are the mean ± S.D. of triplicate assays.



CaInhibition of Forskolin-stimulated III-AC Activity in Vivo

Since inhibition of hormone-stimulated III-AC activity was not receptor-specific, the effect of Ca could occur through G(s), G(i), or the catalytic subunit. Therefore, we examined the influence of Ca on forskolin-stimulated III-AC activity since forskolin interacts directly with the catalytic subunit of adenylyl cyclases. Forskolin stimulation is not dependent upon the presence of G(s) or receptors for its actions (Seamon and Daly, 1981). Cells expressing III-AC were treated with increasing concentrations of forskolin in the presence or absence of 10 µM A23187 and 1.8 mM CaCl(2). In the absence of A23187, maximal forskolin stimulation of III-AC was 753 ± 20 fold with an EC of approximately 10 µM (Fig. 6). A23187 inhibited forskolin-stimulated III-AC activity 53 ± 5%. These data indicate that Ca inhibition of III-AC may be due to modifications of the catalytic subunit that affect its stimulation by forskolin or activated G(s).


Figure 6: Ca inhibits forskolin-stimulated III-AC activity in vivo. HEK-293 cells stably expressing III-AC (III-AC-G) were treated with increasing concentrations of forskolin in the presence or absence of 10 µM A23187 and 1.8 mM CaCl(2). cAMP accumulations were monitored as described under ``Experimental Procedures.'' The data are the mean ± S.D. of triplicate assays.



Inhibition of G(s)-stimulated III-AC Activity Is Not Due to GActivation

One of the major mechanisms for inhibition of adenylyl cyclases is by activation of G(i). For example, stimulation of G(i) by activation of M(4) muscarinic receptors inhibits III-AC activity in vivo (Dittman et al., 1994). To address the role of G(i) for Ca inhibition of III-AC, cells expressing III-AC were pretreated with pertussis toxin, an agent which ADP-ribosylates G(i)-alpha and blocks G(i)-mediated inhibition of adenylyl cyclases (Katada and Ui, 1982; Bokoch et al., 1983). M(4) muscarinic receptor inhibition of III-AC in HEK-293 cells is prevented by pertussis toxin treatment (Dittman et al., 1994). Pertussis toxin-treated cells were analyzed for isoproterenol-stimulated III-AC activity in the presence and absence of A23187 and CaCl(2). Although pertussis toxin treatment caused characteristic morphological changes in treated cells which are indicative of active toxin delivery, it had no significant effect on basal, glucagon, or isoproterenol-stimulated activity. Inhibition of isoproterenol-stimulated activity by Ca was also not affected by pertussis toxin, suggesting that G(i) does not contribute to Ca inhibition (data not shown). Similar results were obtained with glucagon and forskolin-stimulated III-AC activities; pertussis toxin did not block Ca inhibition of glucagon- or forskolin-stimulated activities.

CaM kinase II Inhibits Isoproterenol and Forskolin-stimulated III-AC Activity in Vivo

Ca inhibition of III-AC-stimulated activities might be due to the action of one of the Ca-sensitive protein kinases. This question was initially addressed by examining the effect of several protein kinase inhibitors on Ca inhibition of III-AC. The cAMP protein kinase inhibitors H89 and Rp-cAMP (Rothermel et al., 1988) as well as calphostin C, an inhibitor of protein kinase C, did not affect Ca inhibition of III-AC (data not shown). We are confident that H89 inhibits the activity of cAMP-dependent protein kinase in HEK-293 cells because this inhibitor blocked cAMP stimulation of CRE-mediated transcription in these cells (Impey et al., 1994). Furthermore, we have determined that calphostin C inhibits phorbol ester stimulation of adenylyl cyclase activity in HEK-293 cells. (^2)KN-62, a specific inhibitor of CaM kinases (Enslen et al., 1994), blocked Ca inhibition of glucagon-stimulated III-AC activity (Fig. 7). Ten µM KN-62 almost completely abolished Ca inhibition of glucagon-stimulated III-AC activity. Calmidazolium, a CaM antagonist, also blocked Ca inhibition of III-AC. These data suggest that Ca activation of CaM kinases may contribute to Ca inhibition of III-AC.


Figure 7: Effect of KN-62 on Ca inhibition of III-AC in vivo. HEK-293 cells expressing the rat glucagon receptor and III-AC were pretreated for 1 h with increasing doses of KN-62, an inhibitor of CaM kinases. The cells were then treated with 100 nM glucagon in the presence and absence of 10 µM A23187 and 1.8 mM CaCl(2) to quantitate Ca inhibition of glucagon-stimulated III-AC activity. cAMP accumulations were monitored as described under ``Experimental Procedures,'' and the data are presented as percentage inhibition of cAMP accumulation caused by A23187 and Ca. KN-62 blocked Ca inhibition of glucagon-stimulated III-AC activity. The data are the mean ± S.D. of triplicate assays.



To determine if CaM kinase II inhibits the activity of III-AC activity in vivo, we made stable transfectants in HEK-293 cells expressing CaM kinase II under the control of a metallothionein promoter. The CaM kinase II used in this experiment (KII-290) contains a point mutation that truncates the protein, removes its autoinhibitory domain, and makes it constitutively active (Matthews et al., 1994). These cells were then transiently transfected with a construct encoding III-AC, and the sensitivity of the adenylyl cyclase to CaM kinase II was evaluated by inducing the expression of the kinase with Zn. Zn treatment of cells not expressing KII-290 had no effect on basal, isoproterenol, or forskolin-stimulated III-AC activities. However, induction of CaM kinase II activity in KII-290 cells expressing III-AC completely inhibited isoproterenol (Fig. 8A) and forskolin-stimulated (Fig. 8B) III-AC activities. These data suggest that Ca inhibition of III-AC in vivo may be mediated by CaM kinase II. Thus far, we have been unable to inhibit hormone stimulation of III-AC in membrane preparations using purified CaM kinase II suggesting this kinase may not directly phosphorylate III-AC. However, further experimentation is required to elucidate the mechanism for CaM kinase II regulation of adenylyl cyclase activity.


Figure 8: Inhibition of isoproterenol and forskolin-stimulated type III adenylyl cyclase by CaM kinase II-290. KII-290 cells stably transfected with the inducible, constitutively active CaM kinase II-290 which were transiently transfected with III-AC, were exposed to either isoproterenol (A) or forskolin (B) ± induction of CaM kinase II-290 by Zn. cAMP accumulations were determined as described under ``Experimental Procedures.'' The data are corrected for endogenous adenylyl cyclase activity as described under ``Experimental Procedures.'' The data are the mean ± S.E. of triplicate assays.




DISCUSSION

The adenylyl cyclases exhibit diverse regulatory properties that provide a number of interesting mechanisms for regulation of intracellular cAMP by extracellular and intracellular signals. Several of the adenylyl cyclases are synergistically stimulated by signals arising from different pathways and therefore can generate enhanced cAMP signals in response to signal convergence. For example, the betabullet complex from G proteins stimulates G(s)-activated II-AC and IV-AC (Tang and Gilman, 1992) providing a mechanism for signal integration. I-AC is synergistically activated by Ca and neurotransmitters in vivo (Wayman et al., 1994), a regulatory property that may be important for some forms of synaptic plasticity and spatial memory in mice (Wu et al., 1995). Although synergistic stimulation of adenylyl cyclases by two or more signals may be important for some physiological process including cAMP-mediated transcription (Impey et al., 1994), mechanisms for inhibition of adenylyl cyclase activity and optimization of cAMP levels may be equally important. The data in this study identify a new mechanism for regulation of adenylyl cyclase activity; physiologically significant levels of intracellular Ca attenuate hormone stimulation of III-AC.

III-AC is stimulated by Ca and CaM when it is activated by G(s)in vitro, but hormone-stimulated III-AC is inhibited by Cain vivo. Glucagon, isoproterenol, and forskolin-stimulated III-AC activities were all partially inhibited by physiologically relevant concentrations of intracellular Ca (100 to 300 nM free Ca). The mechanism for Ca inhibition of III-AC activity was not dependent upon the activity of cAMP-dependent protein kinase, protein kinase C, or G(i). However, KN-62, an inhibitor of CaM kinases, blocked Ca inhibition suggesting the interesting possibility that Ca activation of CaM kinases may directly or indirectly inhibit III-AC activity in vivo. Furthermore, expression of constitutively active CaM kinase II completely blocked hormone stimulation of III-AC activity in vivo.

To date, five Ca-regulated adenylyl cyclases have been identified: I-AC, III-AC, V-AC, VI-AC, and VIII-AC. I-AC and VIII-AC are stimulated by intracellular Cain vivo (Choi et al., 1992b; Cali et al., 1994) and mutagenesis of the CaM binding domain of I-AC has established that Ca stimulation is mediated by CaM (Wu et al., 1993). Neither I-AC nor VIII-AC is stimulated by G(s)-coupled receptors in vivo (Wayman et al., 1994; Cali et al., 1994). Although I-AC is synergistically regulated by intracellular Ca and hormones in vivo, VIII-AC is not (Cali et al., 1994). In contrast, V-AC and VI-AC are directly inhibited by Ca in membranes (Yoshimura and Cooper, 1992; Katsushika et al., 1992), and VI-AC is inhibited by submicromolar Cain vivo (Cooper et al., 1994). Regulation of III-AC by Ca and hormones is distinct from all of the other adenylyl cyclases characterized thus far; it is stimulated by hormones in vivo, and increases in intracellular Ca inhibit this response.

Although in vitro studies using isolated membrane preparations or purified recombinant adenylyl cyclases and G proteins have provided valuable insight concerning mechanisms for regulation of adenylyl cyclases, it is becoming increasingly evident that conclusions drawn from in vitro data do not necessarily apply in vivo. For example, purified I-AC or I-AC in membranes is stimulated by addition of relatively high levels of activated recombinant G(s)-alpha, demonstrating that this enzyme has a G(s)-alpha interaction domain (Tang et al., 1991). However, I-AC is not stimulated by activation of G(s)-coupled receptors in HEK-293 cells (Wayman et al., 1994) or in cultured neurons. (^3)VIII-AC is synergistically stimulated by CaM and recombinant G(s)in vitro, but it is not synergistically stimulated by Ca and G(s) activation in vivo (Cali et al., 1994). Characterization of mechanisms for regulation of III-AC described in this study also demonstrates the importance of defining the regulatory properties of each adenylyl cyclase in vivo.

The physiological significance of Ca inhibition of hormone-stimulated III-AC activity remains to be established. Adenylyl cyclase activity in most tissues is inhibited by millimolar levels of Ca which has been attributed to formation of complexes between ATP and Ca, or binding of Ca to a Mg regulatory site on adenylyl cyclases (Steer and Levitzki, 1975). Several tissues including heart muscle (Potter et al., 1980) have been reported to contain adenylyl cyclase activity that is inhibited by submicromolar Ca. It is interesting that III-AC (Xia et al., 1992) and VI-AC (Yoshimura and Cooper, 1992; Katsushika et al., 1992) are both expressed in heart. The presence of III-AC activity in heart may provide a mechanism whereby the positive ionotropic and chronotropic effects of beta-adrenergic agonists are attenuated by increased intracellular Ca. The development of transgenic mice strains deficient in III-AC should provide valuable information concerning the physiological functions of the enzyme and the significance of this regulatory mechanism for specific physiological processes including heart muscle contractility and olfactory signal transduction.

In summary, this study describes a novel mechanism for regulation of adenylyl cyclase activity and is the first report showing that CaM kinases can regulate adenylyl cyclase activity in vivo. This regulatory mechanism may be important for a variety of physiological processes including heart muscle contractility and attenuation of neurotransmitter-stimulated cAMP levels in neurons.


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: I-AC, II-AC, etc., type I adenylyl cyclase, type II adenylyl cyclase, etc.; 293, HEK-293 kidney cells; 293-G, HEK-293 cells expressing the glucagon receptor; I-AC-G, HEK-293 cells expressing I-AC and the glucagon receptor; III-AC-G, HEK-293 cells expressing III-AC and the glucagon receptor; CaM, calmodulin; GppNHp, 5`-guanylyl-beta,-imidodiphosphate; DMEM, Dulbecco's modified Eagle's medium.

(^2)
S. Impey and D. R. Storm, unpublished data.

(^3)
R. Reddy and D. R. Storm, unpublished observations.


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. The construct encoding CaM kinase II was generously provided by R. P. Matthews and G. S. McKnight. 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.


REFERENCES

  1. Bacskai, B. J., Hochner, B., Mahaut-Smith, M., Adams, S. R., Kaang, B. K., Kandel, E. R., and Tsien, R. Y. (1983) Science 260,222-226
  2. Bakalyar, H. A., and Reed, R. R. (1990) Science 250,1403-1406 [Medline] [Order article via Infotrieve]
  3. Bokoch, G. M., Katada, T., Northup, J. K., Hewlett, E. L., and Gilman, A. G. (1983) J. Biol. Chem. 258,2072-2075 [Abstract/Free Full Text]
  4. Brooks, S. P. J., and Storey, K. B. (1992) Anal. Biochem. 201,119-126 [Medline] [Order article via Infotrieve]
  5. Cali, J. J., Zwaagstra, J. C., Mons, N., Cooper, D. M. F., and Krupinski, J. (1994) J. Biol. Chem. 269,12190-12195 [Abstract/Free Full Text]
  6. Chen, C., and Okayama, H. (1987) Mol. Cell. Biol. 7,2745-2752 [Medline] [Order article via Infotrieve]
  7. Choi, E. J., Xia, Z., and Storm, D. R. (1992a) Biochemistry 31,6492-6498 [Medline] [Order article via Infotrieve]
  8. Choi, E. J., Wong, S. T., Hinds, T. J., and Storm, D. R. (1992b) J. Biol. Chem. 267,12440-12442 [Abstract/Free Full Text]
  9. Choi E. J., Xia, Z., Villacres, E. C., and Storm, D. R. (1993a) Curr. Opin. Cell Biol. 5,269-273 [Medline] [Order article via Infotrieve]
  10. Choi, E. J., Wong, S. T., Dittman, A. H., and Storm, D. R. (1993b) Biochemistry 32,1891-1894 [Medline] [Order article via Infotrieve]
  11. Cooper, D. M., Yoshimura, M., Zhang, Y., Chiono, M., and Mahey, R. (1994) Biochem. J. 297,437-440 [CrossRef][Medline] [Order article via Infotrieve]
  12. Dittman, A. H., Weber, J. P., Hinds, T. J., Choi, E. J., Migeon, J. C., and Nathanson, N. M. and Storm, D. R. (1994) Biochemistry 33,943-951 [Medline] [Order article via Infotrieve]
  13. Enslen, H., Sun, P., Brickey, D., Soderling, S. H., Klamo, E., and Soderling, T. R. (1994) J. Biol. Chem. 269,15520-15527 [Abstract/Free Full Text]
  14. Federman, A. D., Conklin, B. R., Schrader, K. A., Reed, R. R., and Bourne, H. R. (1992) Nature 356,159-161 [CrossRef][Medline] [Order article via Infotrieve]
  15. Feinstein, P. G., Schrader, K. A., Bakalyar, H. A., Tang, W. J., Krupinski, J., Gilman, A. G., and Reed, R. R. (1991) Proc. Natl. Acad. Sci. U. S. A. 88,10173-10177 [Abstract]
  16. Gao, B., and Gilman A. (1991) Proc. Natl. Acad. Sci. U. S. A. 88,10178-10182 [Abstract]
  17. Hill, H. D., and Straka, J. G. (1988) Anal. Biochem. 170,203-208 [Medline] [Order article via Infotrieve]
  18. Impey, S., Wayman, G., Wu, Z., and Storm, D. R. (1994) Mol. Cell. Biol. 14,8272-8281 [Abstract]
  19. Ishikawa, Y., Katsushika, S., Chen, L., Halnon, N. J., Kawabe, J., amd Homcy, C. J. (1992) J. Biol. Chem. 267,13553-13557 [Abstract/Free Full Text]
  20. Jelinek, L. J., Lok, S. Rosenberg, G. B., Smith, R. A., Grant. F. J., Biggs, S., Bensch, P. A., Kuijper, J. L., Sheppard, P. O., Sprecher, C. A., and Kinsvogel, W. (1993) Science 259,1614-1616 [Medline] [Order article via Infotrieve]
  21. Katada, T., and Ui, M. (1982) Proc. Natl. Acad. Sci. U. S. A. 79,3129-3133 [Abstract]
  22. Katsushika, S., Chen, L., Kawabe, J., Nilakantan, R., Halnon, N. J. Homcy, C. J., and Ishikawa, Y. (1992) Proc. Natl. Acad. Sci. U. S. A. 89,8774-8778 [Abstract]
  23. Krupinski, J., Coussen, F., Bakalyar, H. A., Tang, W. J., Feinstein, P. G., Orth, K., Slaughter, C., Reed, R. R., and Gilman, A. G. (1989) Science 244,1558-1564 [Medline] [Order article via Infotrieve]
  24. Krupinski, J., Lehman, T. C., Frankenfield, C. D., Zwaagstra, J. C., and Watson, P.A. (1992) J. Biol. Chem. 267,24858-24862 [Abstract/Free Full Text]
  25. Masure, H. R., Head, J. R., and Tice, H. M. (1984) Biochem. J. 218,691-696 [Medline] [Order article via Infotrieve]
  26. Matthews, R. P., Guthrie, C. R., Wailes, L. M., Zhao, X., Means, A. R., and McKnight, G. S. (1994) Mol. Cell. Biol. 14,6107-6116 [Abstract]
  27. Potter, J. D., Piascik, M. T., Wisler, P. L., Robertson, S. P., and Johnson, C. L. (1980) Ann. N. Y. Acad. Sci. 356,220-231 [Abstract]
  28. Rothermel, J. D. (1988) J. Biochem. (Tokyo) 251,757-762
  29. Salomon, Y., Londos, C., and Rodbell, M. (1979) Anal. Biochem. 58,541-548
  30. Seamon, K., and Daly, J. W. (1981) J. Biol. Chem. 256,9799-9801 [Abstract/Free Full Text]
  31. Steer, M. L., and Levitzki, A. (1975) J. Biol. Chem. 250,2080-2084 [Abstract]
  32. Tang, W. J., and Gilman, A. G. (1992) Cell 70,869-872 [Medline] [Order article via Infotrieve]
  33. Tang, W. J., Krupinski, J., and Gilman, A. G. (1991) J. Biol. Chem. 266,8595-8603 [Abstract/Free Full Text]
  34. Watson, P. A., Krupinski, J., Kempinski, A. M., and Frankenfield, C. D. (1994) J. Biol. Chem. 269,28893-28898 [Abstract/Free Full Text]
  35. Wayman, G. A., Impey, S., Wu, Z., Kinsvogel, W., Prichard, L., and Storm, D. R. (1994) J. Biol. Chem. 269,25400-25405 [Abstract/Free Full Text]
  36. Wong, Y. H., Federman, A., Pace, A. M., Zachary, I., Evans, T., Pouyssegur, J., and Bourne, H. R. (1991) Nature 351,63-65 [CrossRef][Medline] [Order article via Infotrieve]
  37. Wu, Z., Wong, S. T., and Storm, D. R. (1993) J. Biol. Chem. 268,23766-23768 [Abstract/Free Full Text]
  38. Wu, Z., Thomas, S. A., Xia, Z., Villacres, E. C., Palmiter, R. D., and Storm, D. R. (1995) Proc. Natl. Acad. Sci. U. S. A. 92,220-224 [Abstract]
  39. Xia, Z., Cheryl, D. R., Merchant, K. M., Dorsa, D. M., and Storm, D. R. (1991) Neuron 6,431-443 [Medline] [Order article via Infotrieve]
  40. Xia, Z., Choi, E. J., Wang, F., and Storm, D. R. (1992) Neurosci. Lett. 144,169-173 [CrossRef][Medline] [Order article via Infotrieve]
  41. Yoshimura, M., and Cooper, D. M. (1992) Proc. Natl. Acad. Sci. U. S. A. 89,6716-6720 [Abstract]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.