Ca2+ inhibition of type III adenylyl cyclase in vivo.

Type III adenylyl cyclase is stimulated by β-adrenergic agonists and glucagon in vitro and in vivo, but not by Ca2+ and calmodulin. However, the enzyme is stimulated by Ca2+ and calmodulin in vitro when it is concomitantly activated by the guanyl nucleotide stimulatory protein Gs (Choi, E. J., Xia, Z., and Storm, D. R. (1992a) Biochemistry 31, 6492-6498). Here, we examined regulation of type III adenylyl cyclase by Gs-coupled receptors and intracellular Ca2+in vivo. Surprisingly, intracellular Ca2+ inhibited hormone-stimulated type III adenylyl cyclase activity. Submicromolar concentrations of intracellular free Ca2+, which stimulated type I adenylyl cyclase, inhibited glucagon- or isoproterenol-stimulated type III adenylyl cyclase. Inhibition of type III adenylyl cyclase by intracellular Ca2+ was not mediated by Gi, cAMP-dependent protein kinase, or protein kinase C. However, an inhibitor of CaM kinases antagonized Ca2+ inhibition of the enzyme, and coexpression of constitutively activated CaM kinase II completely inhibited isoproterenol-stimulated type III adenylyl cyclase activity. We propose that Ca2+ inhibition of type III adenylyl cyclase may serve as a regulatory mechanism to attenuate hormone-stimulated cAMP levels in some tissues.

Type III adenylyl cyclase is stimulated by ␤-adrenergic agonists and glucagon in vitro and in vivo, but not by Ca 2؉ and calmodulin. However, the enzyme is stimulated by Ca 2؉ 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 Ca 2؉ in vivo. Surprisingly, intracellular Ca 2؉ inhibited hormone-stimulated type III adenylyl cyclase activity. Submicromolar concentrations of intracellular free Ca 2؉ , which stimulated type I adenylyl cyclase, inhibited glucagon-or isoproterenol-stimulated type III adenylyl cyclase. Inhibition of type III adenylyl cyclase by intracellular Ca 2؉ was not mediated by G i , cAMP-dependent protein kinase, or protein kinase C. However, an inhibitor of CaM kinases antagonized Ca 2؉ 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 2؉ inhibition of type III adenylyl cyclase may serve as a regulatory mechanism to attenuate hormone-stimulated cAMP levels in some tissues.
In contrast to I-AC and VIII-AC which are directly stimulated by Ca 2ϩ and CaM in vitro, III-AC is not stimulated by Ca 2ϩ and CaM unless it is also activated by GppNHp or forskolin (Choi et al., 1992a). Furthermore, the concentrations of free Ca 2ϩ for half-maximal stimulation of I-AC and III-AC are 150 nM and 5.0 M Ca 2ϩ , respectively. These data suggested that III-AC might be synergistically stimulated by intracellular Ca 2ϩ -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 2ϩ in HEK-293 cells. Contrary to our expectations, intracellular Ca 2ϩ 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(Choi et al., , 1992b(Choi et al., , 1993a(Choi et al., , 1993bWu et al., 1993) and were used for cotransfection 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 ϫ 10 6 cells/plate, grown overnight, and transfected with the pZCEP control vector (1 g of DNA/plate) and a hygromycin resistance vector (1 g 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 g/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 g/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.
cAMP Accumulation-Changes in intracellular cAMP were measured by determining the ratio of [ 3 H]cAMP to total ATP, ADP, and AMP pool in [ 3 H]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 [ 3 H]adenine (2.0 Ci/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% trichlo-* 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 be 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; Gp-pNHp, 5Ј-guanylyl-␤,␥-imidodiphosphate; DMEM, Dulbecco's modified Eagle's medium. roacetic acid containing 1.0 M 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 Ϯ 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 2ϩ -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 ϫ 10 6 cells/100-mm plate and were maintained in DMEM, 10% fetal calf serum, 100 units/ml penicillin, and 100 g/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 g of DNA (either control CDM8 alone or CDM8 encoding type III adenylyl cyclase, CDM8-IIIAC) in 800 l of Opti-MEM (Life Technologies, Inc.) and 64 l of LipofectAMINE (Life Technologies, Inc.) in 800 l 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 M 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 2ϩ 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).

Synergistic Stimulation of III-AC by Ca 2ϩ and Activated G s in Membranes-
To examine the Ca 2ϩ 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 2ϩ 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 2ϩ and CaM. In the absence of other effectors, Ca 2ϩ and CaM did not significantly stimulate III-AC in isolated membranes (Fig.  1A). However, III-AC was stimulated by Ca 2ϩ 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 M GppNHp, Ca 2ϩ and CaM stimulated III-AC activity 2.1 Ϯ 0.1-fold.
To determine if Ca 2ϩ and receptor-activated G s will also synergistically stimulate III-AC in membranes, the sensitivity of the enzyme to CaM and Ca 2ϩ was analyzed in the presence of glucagon (Fig. 1B). In membrane preparations, III-AC was stimulated 4.1 Ϯ 0.1-fold by glucagon with an EC 50 of 7 nM. Glucagon-stimulated III-AC activity was enhanced 45 Ϯ 6.1% by CaM and Ca 2ϩ ; however, the EC 50 for glucagon was not significantly affected by CaM. These data suggested that Ca 2ϩ stimulation of III-AC is conditional upon G s activation, and that Ca 2ϩ and hormones might synergistically activate the enzyme in vivo.
Ca 2ϩ Inhibition of Glucagon-stimulated III-AC Activity in Vivo-Glucagon stimulated III-AC 222 Ϯ 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 scoupled receptors in vivo . 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.
From in vitro data using isolated membranes, we expected that glucagon and intracellular Ca 2ϩ would synergistically stimulate III-AC in intact cells. In fact, increases in intracellular Ca 2ϩ , generated by A23187 and extracellular Ca 2ϩ , inhibited glucagon-stimulated III-AC activity 60% (Fig. 2B). A23187 and extracellular Ca 2ϩ had no effect on the basal activity of III-AC or endogenous adenylyl cyclase activity (data not shown). Ca 2ϩ 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 2ϩ inhibition of glucagon-stimulated III-AC activity varied from 40 -60%.
The Ca 2ϩ dependences for inhibition of III-AC and stimulation of I-AC in vivo were compared using A23187 and varying amounts of extracellular Ca 2ϩ (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 2ϩ concentrations which stimulated I-AC, and the curves were almost mirror images of each other. The concentration of free intracellular Ca 2ϩ for half-maximal inhibition of glucagon-stimulated III-AC activity was estimated at 150 to 200 nM using Fura-2 imagining.
Ca 2ϩ Inhibition 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 2ϩ increases on isoproterenol stimulated III-AC activity. HEK-293 cells express endogenous ␤-adrenergic receptors that are coupled to stimulation of III-AC in vivo (Fig. 4). Isoproterenolstimulated III-AC activity was inhibited 41 Ϯ 3% by A23187 indicating that Ca 2ϩ inhibition of III-AC activity was not a unique property of glucagon-stimulated activity.
In the experiments described above, intracellular Ca 2ϩ was elevated using A23187 and it was of interest to determine if Ca 2ϩ 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 2ϩ . Treatment of HEK-293 cells with 10 M carbachol elevates intracellular Ca 2ϩ to approximately 300 nM free Ca 2ϩ and stimulates I-AC (Choi et al., 1992b). Carbachol alone did not significantly affect III-AC activity but did inhibit isoproterenol-stimulated activity 43 Ϯ 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 2ϩ inhibit hormone-stimulated III-AC activity in vivo.
Ca 2ϩ Inhibition of Forskolin-stimulated III-AC Activity in Vivo-Since inhibition of hormone-stimulated III-AC activity was not receptor-specific, the effect of Ca 2ϩ could occur through G s , G i , or the catalytic subunit. Therefore, we examined the influence of Ca 2ϩ 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 M A23187 and 1.8 mM CaCl 2 . In the absence of A23187, maximal forskolin stimulation of III-AC was 753 Ϯ 20 fold with an EC 50

FIG. 2. Ca 2؉ 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 Ϯ 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 M A23187 and 1.8 mM CaCl 2 and assayed for cAMP accumulation as described under "Experimental Procedures." The data are the mean Ϯ S.D. of triplicate assays. of approximately 10 M (Fig. 6). A23187 inhibited forskolinstimulated III-AC activity 53 Ϯ 5%. These data indicate that Ca 2ϩ inhibition of III-AC may be due to modifications of the catalytic subunit that affect its stimulation by forskolin or activated G s .
Inhibition of G s -stimulated III-AC Activity Is Not Due to G i Activation-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 2ϩ inhibition of III-AC, cells expressing III-AC were pretreated with pertussis toxin, an agent which ADPribosylates G i -␣ 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 isoproterenolstimulated 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 2ϩ was also not affected by pertussis toxin, suggesting that G i does not contribute to Ca 2ϩ inhibition (data not shown). Similar results were ob- tained with glucagon and forskolin-stimulated III-AC activities; pertussis toxin did not block Ca 2ϩ inhibition of glucagonor forskolin-stimulated activities.
CaM kinase II Inhibits Isoproterenol and Forskolin-stimulated III-AC Activity in Vivo-Ca 2ϩ inhibition of III-AC-stimulated activities might be due to the action of one of the Ca 2ϩsensitive protein kinases. This question was initially addressed by examining the effect of several protein kinase inhibitors on Ca 2ϩ 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 2ϩ 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 . 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 2ϩ inhibition of glucagon-stimulated III-AC activity (Fig. 7). Ten M KN-62 almost completely abolished Ca 2ϩ inhibition of glucagon-stimulated III-AC activity. Calmidazolium, a CaM antagonist, also blocked Ca 2ϩ inhibition of III-AC. These data suggest that Ca 2ϩ activation of CaM kinases may contribute to Ca 2ϩ inhibition of III-AC.
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 tran-siently 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 2ϩ . Zn 2ϩ 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 2ϩ 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.

DISCUSSION
The adenylyl cyclases exhibit diverse regulatory properties that provide a number of interesting mechanisms for regulation of intracellular cAMP by extracellular and intracellular 2 S. Impey and D. R. Storm, unpublished data.

FIG. 7. Effect of KN-62 on Ca 2؉ 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 M A23187 and 1.8 mM CaCl 2 to quantitate Ca 2ϩ 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 2ϩ . KN-62 blocked Ca 2ϩ inhibition of glucagon-stimulated III-AC activity. The data are the mean Ϯ S.D. of triplicate assays.
FIG. 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) Ϯ induction of CaM kinase II-290 by Zn 2ϩ . 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 Ϯ S.E. of triplicate assays. 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 ␤⅐␥ 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 2ϩ and neurotransmitters in vivo , 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 , 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 2ϩ attenuate hormone stimulation of III-AC.
III-AC is stimulated by Ca 2ϩ and CaM when it is activated by G s in vitro, but hormone-stimulated III-AC is inhibited by Ca 2ϩ in vivo. Glucagon, isoproterenol, and forskolin-stimulated III-AC activities were all partially inhibited by physiologically relevant concentrations of intracellular Ca 2ϩ (100 to 300 nM free Ca 2ϩ ). The mechanism for Ca 2ϩ 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 2ϩ inhibition suggesting the interesting possibility that Ca 2ϩ 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 2ϩ -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 Ca 2ϩ in vivo (Choi et al., 1992b;Cali et al., 1994) and mutagenesis of the CaM binding domain of I-AC has established that Ca 2ϩ stimulation is mediated by CaM (Wu et al., 1993). Neither I-AC nor VIII-AC is stimulated by G s -coupled receptors in vivo Cali et al., 1994). Although I-AC is synergistically regulated by intracellular Ca 2ϩ and hormones in vivo, VIII-AC is not (Cali et al., 1994). In contrast, V-AC and VI-AC are directly inhibited by Ca 2ϩ in membranes (Yoshimura and Cooper, 1992;Katsushika et al., 1992), and VI-AC is inhibited by submicromolar Ca 2ϩ in vivo . Regulation of III-AC by Ca 2ϩ 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 2ϩ 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 -␣, demonstrating that this enzyme has a G s -␣ interaction domain . However, I-AC is not stimulated by activation of G s -coupled receptors in HEK-293 cells  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 2ϩ 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 2ϩ inhibition of hormonestimulated III-AC activity remains to be established. Adenylyl cyclase activity in most tissues is inhibited by millimolar levels of Ca 2ϩ which has been attributed to formation of complexes between ATP and Ca 2ϩ , or binding of Ca 2ϩ to a Mg 2ϩ 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 2ϩ . 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 ␤-adrenergic agonists are attenuated by increased intracellular Ca 2ϩ . 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.