©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Developmentally Expressed Ca-sensitive Adenylyl Cyclase Activity Is Disrupted in the Brains of Type I Adenylyl Cyclase Mutant Mice (*)

Enrique C. Villacres (1) (2), Zhiliang Wu (1)(§), Wenhui Hua (1), Mark D. Nielsen (1)(¶), Jyoti J. Watters (1)(¶), Chen Yan (1), Joseph Beavo (1), Daniel R. Storm (1)(**)

From the (1)Departments of Pharmacology and (2)Psychiatry & Behavioral Sciences, University of Washington School of Medicine, Seattle, Washington 98195

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The type I Ca-sensitive adenylyl cyclase has been implicated in several forms of synaptic plasticity in vertebrates. Mutant mice in which this enzyme was inactivated by targeted mutagenesis show deficient spatial memory and altered long term potentiation (Wu, Z. L., Thomas, S. A., Villacres, E. C., Xia, Z., Simmons, M. L., Chavkin, C., Palmiter, R. D., and Storm, D. R.(1995) Proc. Natl Acad Sci. U. S. A. 92, 220-224). Long term potentiation in the CA1 region of the rat hippocampus develops during the first 2 weeks after birth and reaches maximal expression at postnatal day 15 with a gradual decline at later stages of development. Here we report that Ca-stimulated adenylyl cyclase activity in rat hippocampus, cerebellum, and cortex increases significantly between postnatal days 1-16. This increase appears to be due to enhanced expression of type I adenylyl cyclase rather than type VIII adenylyl cyclase, the other adenylyl cyclase that is directly stimulated by Ca and calmodulin. Type I adenylyl cyclase mRNA in the hippocampus increased 7-fold during this developmental period. The developmental expression of Ca-stimulated adenylyl cyclase activity in mouse brain was attenuated in mutant mice lacking type I adenylyl cyclase. Changes in expression of the type I adenylyl cyclase during the period of long term potentiation development are consistent with the hypothesis that this enzyme is important for neuroplasticity and spatial memory in vertebrates.


INTRODUCTION

Intracellular cAMP regulates many important neuronal functions including ion channel activity (Nairn et al., 1985), transcription (Nigg et al., 1985; Hagiwara et al., 1993), and synaptic plasticity (for review, see Frank and Greenberg(1994)). For example, the late or long lasting form of long term potentiation (LTP)()in the CA1 region of the hippocampus is dependent upon the activity of cAMP-dependent protein kinases (Frey et al., 1993). Dopamine receptor antagonists block LTP in the CA1 as well as dopamine stimulation of adenylyl cyclase (Frey et al., 1991). Furthermore, cAMP elevating agents potentiate various forms of LTP in the hippocampus (Stanton and Sarvey, 1985; Hopkins and Johnston, 1988; Chavez-Noriega and Stevens, 1992, 1994; Weisskopf et al., 1994).

There is increasing evidence that Ca/calmodulin (CaM)-stimulated adenylyl cyclases may be important for some forms of neuroplasticity in vertebrates (Xia et al., 1991). The physiological function of the type I adenylyl cyclase (I-AC) and its role in neuroplasticity is of particular interest since it is neurospecific, Ca/CaM sensitive (Xia et al., 1993), and expressed in those areas of the brain that exhibit synaptic plasticity including the CA1, CA2, CA3 and dentate gyrus regions of the hippocampal formation (Xia et al., 1991). Furthermore, mutant mice deficient in I-AC are deficient in spatial memory and show depression of LTP in the hippocampus (Wu et al., 1995). However, I-AC mutant mouse brains still show residual Ca-stimulated adenylyl cyclase activity that is most likely due to VIII-AC, a Ca-sensitive adenylyl cyclase that is also expressed in the hippocampus (Cali et al., 1994). Approximately 50% of the Ca-stimulated adenylyl cyclase activity in the mouse hippocampus is contributed by I-AC (Wu et al., 1995).

Synaptogenesis and the expression of LTP in rodents occur during the first 3 weeks following birth (Pokorny and Yamamoto, 1981; Harris and Teyler, 1984). The objective of this study was to determine if Ca-stimulated adenylyl cyclase is expressed in the brain during this developmental period. Our data indicate that rat hippocampus, cerebellum, and cerebral cortex show a highly significant increase in Ca-stimulated adenylyl cyclase activity during the first 2 weeks of development, which is due primarily to increases in the expression of I-AC.


EXPERIMENTAL PROCEDURES

Cell Membrane Preparations

Membranes were prepared from HEK-293 cells as described previously (Choi et al., 1992b). Wild-type and I-AC mutant C57BL/6J mouse cerebellar membranes (Wu et al., 1995) were prepared from mice varying in age from postnatal day 2 (P2) to adulthood. Sprague-Dawley rat cerebellar, hippocampal, and cerebral cortical membranes were prepared from rats varying in age from P1 to P22. Brain tissue was resuspended in homogenization buffer (20 mM Tris-HCl, pH 7.4, 0.5 mM dithiothreitol, 1 mM EDTA, 1 mM EGTA, 5 µg/ml leupeptin, 2 mM MgCl, 0.5 mM phenylmethylsulfonyl fluoride) and homogenized at 4 °C. Unbroken cells and nuclei were removed by centrifugation at 600 g for 2 min, and the supernatants were subjected to centrifugation at 30,000 g for 20 min. The resulting membrane pellet was suspended in the homogenization buffer without EGTA and assayed for adenylyl cyclase activity.

Adenylyl Cyclase Assay

The enzyme assay was performed at 30 °C for 20 min by adding membrane fractions (10-75 µg of protein) to an assay solution containing 1 mM [-P]ATP (500 cpm/pmol), [H]cAMP (20,000 cpm/µmol), 5 mM MgCl, 1 mM EDTA, 2 mM cyclic AMP, 5 mM theophylline, 0.1% bovine serum albumin, 40 µg/ml CaM, 20 mM creatine phosphate, 60 units/ml creatine phosphokinase in 20 mM Tris-HCl, pH 7.4, in a final assay volume of 250 µl. The reaction was stopped by adding 750 µl of 1.5% sodium dodecyl sulfate. The reaction mixture was heated at 100 °C for 2 min, and the P- and H-labeled cyclic AMP generated was recovered using Dowex AG 50W-X4 and neutral alumina columns, as described by Salomon et al.(1974). The values of adenylyl cyclase activities represent the mean of triplicate determinations ± S.D. Protein concentrations were determined by using the Pierce BCA protein assay kit.

Quantitation of Free CaConcentrations

Free calcium concentrations in the adenylyl cyclase buffers were varied using a Ca/EGTA buffer system. Free Ca concentrations were estimated using the Bound and Determined computer program (Brooks and Storey, 1992).

RNA Analysis

Total RNA was isolated from frozen tissues by the acid guanidinium thiocyanate/phenol/chloroform extraction method (Chomczynski and Sacchi, 1987). Poly(A)-selected RNA was isolated from total RNA using type III oligo(dT) cellulose (Collaborative Research Inc., Bedford MA) and analyzed on a formaldehyde, 1.2% agarose gel in MOPS buffer (Ausubel et al., 1989). mRNA was transferred onto Nytran membranes (Schleicher & Schuell) in 10 SSC (saline sodium citrate) for 16-20 h, cross-linked at 80 °C for 1 h, and prehybridized at 42 °C in a hybridization buffer containing 50% formamide, 5 SSC, 1 Denhardt's solution, and 250 µg/ml denatured salmon sperm DNA for 4 h. [-P]dCTP random primed cDNA probes generated from the hypervariable region of a mouse I-AC clone (corresponding to amino acids 469-663) or from the first 771 nucleotides of the coding region and 329 nucleotides of the 5`-untranslated region of the VIII-AC were used for Northern analysis. These probes were hybridized to the immobilized RNA for 16-20 h at 42 °C in the hybridization buffer. The membrane was washed in 2 SSC, 0.5% SDS for 10 min at room temperature and once for 5 min at 65 °C. It was then washed 3 times for 20 min in 1 SSC, 0.5% SDS at 65 °C prior to autoradiography. The probe was stripped by boiling in 0.1 SSC for 10 min. The membrane was then hybridized with a -actin probe and then rehybridized again with an elongation factor 1 probe. PhosphorImager quantitation was performed by exposing the radioactive blot to a phosphor screen. After appropriate exposure, the screens were scanned using the PhosphorImager model 400S (Molecular Dynamics, Sunnyvale, CA).

In Situ Hybridization

Rats (postnatal days 7, 14, and adult) were decapitated, and the brains were rapidly isolated, frozen in OCT compound (Miles, Inc., Elkhart, IN) on dry ice, and stored at -70 °C until use. Sections (20 µm) were cut in a cryostat, thaw mounted on a poly-L-lysine-coated microscope slide, and stored at -70 °C. A template plasmid corresponding to nucleotides 1557-1981 of the bovine I-AC cDNA clone was used for the transcription of riboprobes (Xia et al., 1991). This sequence is found in a hypervariable region of the adenylyl cyclases and distinguishes I-AC from other adenylyl cyclases. The labeled antisense RNA probe was transcribed from the HindIII-linearized template with T3 RNA polymerase (Stratagene) in the presence of [S]UTP (DuPont NEN). The corresponding sense probe was transcribed from the SmaI-linearized template with T7 RNA polymerase. The specific activities of the probes were between 10 and 10 cpm/µg. In situ hybridizations were performed as described previously (Yan et al., 1994). Briefly, the sections were fixed in 4% (w/v) paraformaldehyde, pretreated with 0.2 M HCL, 2 µg/ml proteinase K, and 0.25% acetic anhydride in 0.1 M triethanolamine (pH 8), followed by graded dehydration for 2 min each in 30, 60, 80, 95, and 100% ethanol. Before hybridization, each section was prehybridized with 50 µl of hybridization buffer (50% deionized formamide, 10% dextran sulfate, 0.3 M NaCl, 1 Denhardt's solution, 10 mM dithiothreitol, 1 mM EDTA, 1 mg/ml yeast tRNA, and 100 mg/ml synthetic poly(A) RNA) in the absence of probe under parafilm coverslips in a humid chamber at 50 °C for 2-6 h. The prolonged prehybridization significantly reduces the background without affecting the specific hybridization signal intensity. The sections were then briefly rinsed in 2 SSC, dehydrated with ethanol, and air dried. Hybridization was performed in the hybridization buffer containing S-labeled antisense riboprobes (0.1 µg/ml/kilobase) at 50 °C for 16-20 h in a humid chamber under glass coverslips sealed by rubber cement. After hybridization, sections were washed twice with 2 SSC, 10 mM dithiothreitol for 30 min each at 55 °C. In order to remove nonspecifically bound single-stranded probe, the sections were treated with a solution containing 20 µg/ml RNase A, 100 units/ml RNase T1, 0.5 M NaCl, 10 mM Tris-HCl, pH 8, and 1 mM EDTA for 30 min at 37 °C. The sections were further washed in 2 SSC, 50% formamide, 10 mM dithiothreitol for 30 min at 55 °C and in 0.1 SSC, 10 mM dithiothreitol for 30 min at 55 °C. The sections were dried by graded dehydration with 30-100% ethanol and then coated with NTB2 emulsion (Eastman Kodak Co.) and exposed for 1-3 weeks at 4 °C. The emulsion-coated slides were developed, fixed, stained through the emulsion with hematoxylin or hematoxylin/eosin, and mounted with Permount (Fisher). Each comparative set of slides was dipped in the same batch of emulsion and exposed for the same period of time. Nonspecific hybridization was determined by parallel incubation of adjacent sections with -S-labeled sense riboprobes.


RESULTS

Developmental Expression of Ca-stimulated Adenylyl Cyclase Activity in Rat Hippocampus, Cerebellum, and Cerebral Cortex

I-AC mRNA is expressed in the hippocampal formation, cerebellum, and cerebral cortex of the adult rat brain, but it is absent from other areas of brain including the brain stem (Xia et al., 1991). We measured basal and Ca-stimulated adenylyl cyclase activity in rat brain membrane preparations during the first 3 weeks of postnatal development because this is the period of development in the hippocampus of the rat (Teyler et al., 1989). Ca-stimulated adenylyl cyclase activity in membranes from the rat hippocampus increased 5.5 ± 0.3-fold between P1 and P16 and declined somewhat after P16 (Fig. 1A). Although basal adenylyl cyclase activity also increased during this period, the relative increase in Ca-stimulated adenylyl cyclase activity was greater (5.5 ± 0.3- versus 3.5 ± 0.1-fold). Ca/CaM stimulation relative to basal activity was 3.6 ± 0.2-fold at P1 and 5.4 ± 0.3-fold at P16. In the cerebellum, both Ca-stimulated and basal adenylyl cyclase activities increased 7-10-fold between P5 and P18 (Fig. 1B). Basal and Ca-stimulated adenylyl cyclase activities in the cerebral cortex increased 3-4-fold during the same developmental period (Fig. 1C).


Figure 1: Effect of Ca on adenylyl cyclase activity in the developing postnatal rat hippocampus (A), cerebellum (B), and cerebral cortex (C). Membranes were prepared from Sprague-Dawley rat hippocampus (P1-P19), cerebellum (P3-P22), and cerebral cortex (P3-P18) and assayed for adenylyl cyclase activity in the presence () or absence () of 300 nM free Ca and 2.4 µM CaM as described under ``Experimental Procedures.'' Adenylyl cyclase activities are the mean of triplicate determinations ± S.D.



CaSensitivity of Adenylyl Cyclase in Rat Hippocampal Membranes

The developmental increase in Ca-stimulated adenylyl cyclase activity in the hippocampus was particularly interesting because of recent data implicating I-AC and other Ca-stimulated adenylyl cyclases in various forms of LTP in the hippocampus (Wu et al., 1995; Weisskopf et al., 1994). Two Ca-stimulated adenylyl cyclases are expressed in the hippocampus, I-AC (Xia etal., 1991) and VIII-AC (Cali etal., 1994). These two enzymes are the only known adenylyl cyclases that are directly stimulated by Ca and CaM in vitro. Therefore, the Ca sensitivity of adenylyl cyclase activity in the wild-type rat hippocampal membranes was compared with I-AC and VIII-AC expressed in HEK-293 cells to evaluate the contribution of these enzymes to Ca-stimulated adenylyl cyclase in this region of the brain (Fig. 2). Half-maximal stimulation of adenylyl cyclase activity in P10 rat hippocampal membranes was at 200 nM free Ca (Fig. 2A). I-AC and VIII-AC were half-maximally stimulated at 100 and 800 nM free Ca, respectively (Fig. 2, B and C). Furthermore, the Ca activation curves for I-AC and hippocampal adenylyl cyclase activities were both bell shaped, with a decrease in Ca-stimulated activity above 2.5 µM free Ca . These data suggest that I-AC makes a significant contribution to Ca-stimulated adenylyl cyclase activity in the rat hippocampus.


Figure 2: Comparison between the Ca sensitivity of hippocampal adenylyl cyclase activity and I-AC or VIII-AC. Membranes were isolated from 10-day-old Sprague-Dawley rat hippocampi and I-AC or VIII-AC stably transfected 293 cell and assayed for adenylyl cyclase in the presence of 2.4 µM CaM as described under ``Experimental Procedures.'' Free Ca concentrations were varied using EGTA/CaCl buffers as described under ``Experimental Procedures.'' Adenylyl cyclase activities are the mean of triplicate determinations ± S.D.



Developmental Expression of I-AC mRNA in Rat Brain

The developmental increase in Ca-stimulated adenylyl cyclase activity in rat brain could be due to changes in gene expression or modulation of adenylyl cyclase activity by other proteins, e.g. protein kinases. Consequently, we quantitated I-AC mRNA expression in brains from P2, P6, and P16 rats by Northern analysis using a probe specific for I-AC. The I-AC transcript was 11.5 kilobases, consistent with previous reports (Xia et al., 1993). In these experiments, poly(A)-selected RNA was isolated from the hippocampus, cerebellum, or whole rat brain, and the amount of I-AC mRNA present in each sample was normalized to elongation factor 1 mRNA (Fig. 3B). In the hippocampus, cerebellum, or whole brain, I-AC mRNA increased during the period from P2 to P16. For example, in the hippocampus, I-AC mRNA increased 7-fold between P2 and P16 (Fig. 3B), which is consistent with the increase in Ca-stimulated adenylyl cyclase activity in this tissue (Fig. 1). In contrast, VIII-AC mRNA increased 2-fold between P2 and P16 (data not shown). These data indicate that the developmental increase in Ca stimulated adenylyl cyclase activity was due primarily to expression of I-AC.


Figure 3: Northern analysis of I-AC mRNA in rat hippocampi, cerebella, and whole brains during postnatal development. A, Northern analysis for I-AC, -actin, and elongation factor 1 (EF-1) mRNA was carried out as described under ``Experimental Procedures.'' B, the relative levels of I-AC mRNA were normalized to elongation factor 1 mRNA by phosphorimaging analysis.



In Situ Hybridization Analysis for I-AC

The distribution of mRNA for I-AC in developing rat brains was also examined by in situ hybridization using a bovine I-AC-specific riproprobe corresponding to bases 1557-1981 (Fig. 4). The strongest hybridization signals were detected in the granule cell layers of the dentate gyrus, pyramidal cells of the CA2 region, and the granule cell layers of the cerebellum. Moderate levels of I-AC mRNA were observed in the pyramidal cells of the CA1 and CA3 fields. This expression pattern was evident in P7, P14, and adult rat brains. However, P14 rat brains showed stronger hybridization signals in the hippocampus, cortex, and cerebellum than did brains from P7. I-AC mRNA levels decreased somewhat from P14 to the adult stage, consistent with the decrease in Ca-stimulated adenylyl cyclase activity after 2 weeks of postnatal development in both rats and mice ( Fig. 1and 5A).


Figure 4: In situ hybridization analysis for I-AC in developing rat hippocampus and cerebellum. Sagittal sections of rat hippocampi (i) or cerebella (ii) were hybridized with -S-labeled antisense riboprobes directed to mRNA encoding I-AC and visualized using dark-field photomicroscopy during development. A, P7; B, P14; C, adult stage; D, hybridization of the adult stage with sense probe. CA1, CA2, and CA3, are the pyramidal cell layers of the hippocampus. DG, dentate gyrus; GL, granular layer.



Adenylyl Cyclase Activity in the Developing Cerebellum of the I-AC Mutant Mouse

The data discussed above indicate that increases in Ca-stimulated adenylyl cyclase activity during the first 2 weeks after birth may be primarily attributable to I-AC. Consequently, adenylyl cyclase activity was measured in the cerebellum from developing wild-type and I-AC mutant mice (Wu et al., 1995). Like rats, wild-type mice showed a significant increase in basal and Ca-stimulated adenylyl cyclase activity during the first 2 weeks of postnatal development (Fig. 5). The developmental increase in Ca-stimulated activity was greater than basal activity (6.5- versus 3.0-fold). I-AC mutant mice showed only a 2-fold increase in Ca-stimulated adenylyl cyclase activity between P2 and P16 and no significant increase in basal activity. Similarly, the large increase in forskolin-stimulated adenylyl cyclase activity between P2 and P16 in wild-type mice was due primarily to I-AC.


Figure 5: Developmental expression of Ca-stimulated adenylyl cyclase activity in cerebellar membranes from wild-type and I-AC mutant mice. Cerebellar membranes were isolated from wild-type () or I-AC mutant () mice (P2 through adulthood) and analyzed for adenylyl cyclase activity as described under ``Experimental Procedures.'' A, 300 nM free Ca and 2.4 µM CaM; B, basal activity; C, 10 µM forskolin. Adenylyl cyclase activities are the mean of triplicate determinations ± S.D.



Morphological Analysis of Brains from Wild-type and I-AC Mutant Mice

Because of the developmental expression pattern of I-AC, it was important to compare the structure of brains from wild-type and I-AC mutant mice. Analysis of 7-week-old mouse brain coronal sections stained with cresyl violet by light microscopy showed no detectable anatomical differences in the hippocampus, neocortex, or cerebellum (data not shown). There were also no apparent differences in the arrangement of cell body layers of the hippocampus or cerebellum when sections were examined at higher magnification. These data indicate that the type I adenylyl cyclase may not be crucial for hippocampal and cerebellar development and that the brains of mutant mice are anatomically indistinguishable from wild-type mice.


DISCUSSION

Several previous studies have reported that basal and forskolin-stimulated adenylyl cyclase activities in rat and mouse brains reach a maximum during the first 2-3 weeks following birth (Keshles and Levitzki, 1984; Broaddus and Bennet, 1990; Rius et al., 1994). However, the developmental expression of specific adenylyl cyclases including the Ca-stimulated adenylyl cyclase activity in brain had not been examined prior to this study. Of the 10 adenylyl cyclases cloned to date, only I-AC and VIII-AC are directly stimulated by Cain vitro and in vivo (Choi et al., 1992a; Cali et al., 1994), but neither is stimulated by -adrenergic agonists in vivo (Wayman et al., 1994; Cali et al., 1994). However, I-AC is synergistically stimulated by intracellular Ca and receptor activation in vivo (Wayman et al., 1994), whereas VIII-AC is not. It was the objective of this study to examine the developmental expression of Ca-stimulated adenylyl cyclases in rats and mice during P2-P16, a period during which various forms of synaptic plasticity develop in the rodent brain (Harris and Teyler, 1984).

Ca-stimulated adenylyl cyclase activity in mouse and rat brains increases significantly during the first 2 weeks of development, particularly in the hippocampus. Northern analysis suggests that this increase may be due primarily to elevations in I-AC mRNA. These developmental increases in I-AC mRNA were also evident when rat brain slices were analyzed by in situ hybridization. Although we did not determine whether the increase in I-AC mRNA was due to increased transcription or message stability, it is interesting that the I-AC transcript has a very large 3` noncoding region (approximately 7 kilobases) that may have specific sites for interaction with mRNA binding proteins. VIII-AC mRNA showed only minor increases during this developmental period. The increase in Ca-stimulated adenylyl cyclase activity was significantly disrupted in I-AC mutant mice confirming that I-AC is the major source for the developmental increases in activity.

The first 3 weeks following birth is an important period for the development of the mouse and rat brain. In the CA1 pyramidal cells, the number of basal dendrites and terminal branches of the apical dendrites are established by P5 (Pokorny and Yamamoto, 1981). The basal dendrites, the main shafts, and terminal fibers branching within the stratum moleculare of the apical dendrites develop by P10. Between P15 and P24, the lateral branches of the apical dendrites in the stratum radiatum and its preterminal fibers branching in the stratum lacunosum develop (Pokorny and Yamamoto, 1981). In a study of the postnatal development of the CA1 region of the rabbit hippocampus, cell branching and spine frequency reached near-adult levels by 3 weeks (Schwartzkroin, et al., 1982). In addition, it has been demonstrated that the CA1 region of the rat hippocampus shows little LTP prior to P5, maximal potentiated response around P15, and a subsequent decline to adult levels (Teyler et al., 1989). Therefore, synaptogenesis, dendritic development, and the development of LTP coincides with the developmental changes in I-AC expression and activity in the rat and mouse brain. However, anatomical analysis of brains from I-AC mutant and wild-type mice revealed no significant differences. The increased expression of I-AC in the hippocampus during the first 2 weeks following birth is, however, consistent with other evidence, suggesting that this enzyme contributes to signal transduction systems important for some forms of synaptic plasticity and memory (Xia et al., 1991; Impey et al., 1994; Wu et al., 1995).

In summary, I-AC mutant mice are deficient in developmental expression of Ca-stimulated adenylyl cyclase activity. These observations are consistent with the proposal that I-AC may play an important role in various forms of synaptic plasticity by coupling the Ca and cAMP signal transduction systems.


FOOTNOTES

*
This research was supported by National Institutes of Health Grants NS 01653, NS 20498, and DK 21723. 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.

§
Supported by a Keck Neuroscience postdoctoral fellowship.

Supported by National Institutes of Health Training Grant GM 07270.

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

The abbreviations used are: LTP, long term potentiation; CaM, calmodulin; Ca/CaM, the complex of four calcium ions bound to calmodulin; I-AC, type I adenylyl cyclase; VIII-AC, type VIII adenylyl cyclase; P, postnatal day; HEK-293 cells, human embryonic kidney-293 cells; MOPS, 4-morpholinepropanesulfonic acid.


ACKNOWLEDGEMENTS

We thank Dr. Jack Krupinski for providing the HEK-293 cells expressing VIII-AC. We also thank Dr. Z. Xia for providing the mouse type I adenylyl cyclase cDNA clone and Dr. Thomas R. Hinds for helpful discussion and advice.


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