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Circulation Research. 1997;80:297-304

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(Circulation Research. 1997;80:297-304.)
© 1997 American Heart Association, Inc.

Articles

The Adenylyl Cyclases as Integrators of Transmembrane Signal Transduction

Yoshihiro Ishikawa, Charles J. Homcy

the Department of Medicine (Y.I.), Brigham & Women's Hospital, Harvard Medical School, Boston, Mass, and COR Therapeutics Inc (C.J.H.), San Francisco, Calif.

Correspondence to Yoshihiro Ishikawa, MD, PhD, Brigham & Women's Hospital, 221 Longwood Ave, Boston, MA 02115, or Charles J. Homcy, MD, COR Therapeutics Inc, 256 E Grand Ave, South San Francisco, CA 94080.


Key Words: adenylyl cyclase • heart • regulation • transmembrane signaling


*    cAMP Generation in the Heart
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Activation of the sympathetic nerves initiates the most potent stimulus for enhancing cardiac output, both acutely and chronically (Fig 1Down). Norepinephrine released into the synaptic cleft at sympathetic nerve terminals binds to ß-adrenergic receptors (ßARs) on the cardiac sarcolemma and activates the stimulatory guanine nucleotide binding protein Gs by promoting the exchange of GDP for GTP. This reaction catalyzes the dissociation of the GTP-bound Gs{alpha} subunit from Gß{gamma}. GTP-bound Gs{alpha} then binds to and stimulates adenylyl cyclase. Adenylyl cyclase is a membrane-bound enzyme that catalyzes the conversion of ATP to cAMP.1 cAMP, an intracellular second messenger, activates protein kinase A by dissociating its regulatory subunit from the catalytic subunit.2 The free catalytic subunit thereupon initiates a series of enzymatic reactions leading to a phosphorylation cascade, activating multiple proteins that regulate both the rate and force of cardiac contraction. Phosphorylation of the L-type calcium channel, for example, enhances calcium entry into cardiocytes, leading to increased contractility.3 On phosphorylation of phospholamban, the inhibition exerted by the nonphosphorylated form of phospholamban on the sarcoplasmic reticulum calcium pump is removed, and its rate of calcium uptake is increased, thereby leading to a more rapid decrease of the cytosolic calcium concentration during diastole.4 Dissociation of the troponin C–calcium complex is also enhanced when troponin I is phosphorylated, which leads to an accelerated relaxation rate. These latter events underlie the lusitropic effects of ßAR stimulation.5 Thus, a series of reactions occurs within cardiocytes that is initiated at the level of the cell surface ßAR. The integrated effect is an enhancement in cardiac output. This process rapidly reverses when agonist occupancy of the receptor ceases, ie, at the removal of norepinephrine from the synaptic cleft. cAMP is eventually degraded to 5' AMP by phosphodiesterase.6 Protein kinase A is then inactivated by reassociation of the catalytic subunit with the regulatory subunit.2 Phosphorylated proteins are rapidly dephosphorylated by specific phosphatases, returning their conformation to a less active form. These changes are rapid, occurring in the millisecond-to-second time frame.



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  		Figure 1. cAMP signaling in the heart. ST indicates synaptic terminal; Nor, norepinephrine; SL, sarcolemma; AC, adenylyl cyclase; PKA, protein kinase A; and SR, sarcoplasmic reticulum.

In various pathophysiological conditions, such as heart failure, major changes develop within the catecholamine signaling pathway.7 Using various animal models and cardiac tissues obtained from failing human hearts, investigators have demonstrated that "malfunction" of multiple components within the cAMP signaling pathway occurs. Changes in the content and function of the receptor, G proteins, and adenylyl cyclase may thus constitute fundamental defects underlying certain cardiac diseases. Thus, it is important to understand the properties of each component of the ßAR–Gs–adenylyl cyclase signaling pathway. We will focus this review article on adenylyl cyclase in particular, which has been characterized for many years biochemically but cloned only recently.


*    The Structure of Adenylyl Cyclase
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Molecular cloning studies in the past decade have demonstrated that many proteins are actually members of large families, consisting of multiple subtypes that differ in tissue distribution and biochemical properties. Although this is the case for certain of the enzymes regulating the ßAR–Gs–adenylyl cyclase pathway, such as the G protein–coupled receptor kinases,8 there are, in contrast, only three ßAR subtypes and four splice variants of Gs{alpha}, the latter all derived from a single gene product. Except for the ß3AR, whose expression is generally restricted to adipose tissue, these components are all expressed rather ubiquitously, and their coupling to adenylyl cyclase shows no major differences.

Among the three major components of the ßAR–Gs–adenylyl cyclase signaling pathway, the last was cloned most recently. It is interesting to note, however, that a cAMP-synthesizing activity was identified within the membrane fraction of cells more than three decades ago. In the 1970s, adenylyl cyclase was successfully solubilized with nonionic detergents and partially purified, and its molecular mass (>100 kD) was calculated on the basis of its hydrodynamic properties.9 Biochemical characterization of the enzyme obtained from various tissues demonstrated that there were two distinct subtypes: calmodulin-sensitive and -insensitive isoforms.10 However, the cloning of the first adenylyl cyclase cDNA, followed by the identification of multiple other isoforms, was not achieved until the 1990s. This delay was due to the fact that adenylyl cyclase is a rare component of the cell membrane, constituting only {approx}0.001% of the total membrane protein. Furthermore, its enzymatic activity is fragile; incubation at 37°C for 0.5 hour readily inactivates the enzyme when it is uncoupled from Gs{alpha}.11

The first adenylyl cyclase isoform was cloned from the brain.12 The amino acid sequence deduced from the cDNA clone showed several interesting characteristics. It was known that adenylyl cyclase was a membrane-bound enzyme. However, the predicted structure from the brain isoform was unexpected: hydropathy analysis predicted a molecule consisting of a module of six transmembrane spans linked to a large cytoplasmic domain that was tandemly repeated (Fig 2Down). The amino acid sequence within the transmembrane domains (M1 and M2) did not show sequence homology to any other proteins. However, the sequences within the two cytoplasmic domains (C1a and C2a) showed significant homology to each other and to the sequences within both bacterial and yeast adenylyl cyclases and even within guanylyl cyclase. Because of highly conserved amino acid sequence homology, the two domains (C1a and C2a) are considered to be catalytic domains. These findings also suggest that both eukaryotic and prokaryotic adenylyl cyclases, as well as guanylyl cyclase, share the same ancestral origin.



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  		Figure 2. Structure of adenylyl cyclase. The entire molecule is subdivided into seven domains: N indicates the N-terminal cytoplasmic domain; M1, the first six transmembrane–spanning domain; C1a, the first cytoplasmic catalytic domain; C1b, the first cytoplasmic linker domain; M2, the second six transmembrane–spanning domain; C2a, the second cytoplasmic catalytic domain; and C2b, the second cytoplasmic noncatalytic domain. {alpha} represents the front half of the molecule; ß, the back half.

The complex structure of adenylyl cyclase is not typical for a membrane-bound enzyme; it is rather similar to that of transporters or ion channels.13 In particular, membrane transporters, such as P-glycoprotein, share the same membrane topology, ie, two modules of six transmembrane-spanning regions followed by a large cytoplasmic domain. The adenylyl cyclases may seem to be related to this larger family of membrane transporters, which are also referred to as the ABC (ATP-binding cassette) family of proteins. However, the adenylyl cyclases do not possess an ATP-binding or "Walker" motif. Whether a mammalian adenylyl cyclase might also possess transporter-like activity has also not been demonstrated, although an adenylyl cyclase from Paramecium possesses potassium channel activity.14

Why did the adenylyl cyclases evolve with such an intricate design? It seems unlikely that this exists simply to concentrate the catalyst in a membrane-delimited manner near the G protein so as to ensure rapid amplification of the agonist-triggered signal. As discussed below, the two cytoplasmic domains of adenylyl cyclase likely interact to initiate efficient catalytic activity. The enzyme's configuration might promote a proper interaction of the two catalytic domains upon its activation. Nevertheless, it remains unclear as to why such a complex membrane tethering evolved to support this enzyme's activity.


*    Diversity in Adenylyl Cyclase Isoforms
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The adenylyl cyclase isoform originally isolated from the brain (designated as type I) is calmodulin sensitive and expressed only in the brain. Subsequently, several groups, including our own, isolated additional adenylyl cyclase isoforms (types II through IX).15 16 17 18 19 20 21 22 23 24 So far, at least nine isoforms are known. They all share the same membrane topology, ie, a tandem repetition of a six-transmembrane domain and a large cytoplasmic domain. The amino acid sequence within the membrane domain is not conserved among these isoforms; however, that of the cytoplasmic domain is relatively well conserved. Interestingly, the degree of amino acid sequence homology is not similar among all isoforms within the adenylyl cyclase family; certain isoform subgroups, such as types II/IV/VII or types V/VI, exhibit greater amino acid sequence homology within the subgroup than within other isoforms. Subsequent biochemical studies also revealed that certain isoforms not only share a similar amino acid sequence but also display similar biochemical properties. Based on amino acid sequence homology, biochemical properties, and tissue distribution, the nine isoforms can be subdivided into at least five subgroups (TableDown). Importantly, the diversity in their biochemical properties, in particular, regulation by calmodulin and Gß{gamma} subunits, and in their tissue distribution may explain the conflicting findings of earlier studies in which membranes prepared from a variety of different tissues were used.


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  		Table 1. Adenylyl Cyclase Subgroups

Types I and VIII form the neuronal subgroup, which is expressed only in the brain. They are both stimulated by calmodulin. Sequence homology between these two isoforms is the least conserved among all the subgroups. Gß{gamma} subunits inhibit the catalytic activity of type I. There are at least three splice variants of the type VIII isoform that differ in calmodulin sensitivity25 ; in contrast, two potential splice variants of the type I isoform do not show any functional differences.26

Types II, IV, and VII form the ubiquitous subgroup, which is expressed in multiple tissues, including the heart. They are insensitive to calmodulin. In contrast to the neuronal isoforms, the ubiquitous isoforms are stimulated by Gß{gamma} subunits. The type IV and VII isoforms can be detected in most tissues, although one variant of type VII is expressed only in the retina.27 The ubiquitous isoforms are potently stimulated by protein kinase C,28 29 30 although other isoforms can also be stimulated by this kinase.31 The type II isoform seems to be the major isoform in the lung; this isoform is expressed in airway smooth muscle cells32 and in vascular endothelial cells from a variety of tissues.33 Since this isoform responds to a variety of signals emanating from different receptors and G proteins,34 including chemotactic35 and growth factor receptors, its output represents an integrated response to multiple extracellular stimuli, placing it at a key position to modulate airway resistance.32 Whether changes in the expression of this isoform occur under pathophysiological conditions has not been examined.

Originally isolated from olfactory tissue, the type III isoform is calmodulin sensitive.15 36 Although its expression is highest in olfactory tissue, its mRNA can also be detected in other tissues, such as the atria and brown fat.37

Types V and VI are the most closely related isoforms within the mammalian adenylyl cyclase family. Although these isoforms were cloned from several tissue sources, including the heart,18 19 liver,20 and neuronal cells,21 these represent the major adenylyl cyclase isoforms in the heart, as described below in detail, and constitute the cardiac subgroup. There are also splice variants of these isoforms.38 39

Type IX is the newest member of the mammalian adenylyl cyclase family. This isoform, isolated from a pituitary tumor cell line, shows a unique interaction with calcineurin,24 40 a calcium-sensitive serine-threonine phosphatase widely expressed in mammalian cells. This isoform can be stimulated by FK506, a calcineurin inhibitor, suggesting that the activity of this isoform is maintained through phosphorylation.

The distribution of the various isoforms within the brain is heterogeneous, suggesting that each isoform is involved in a distinct aspect of neuronal signaling.41 For example, the type V isoform is restricted to the striatum, implicating its involvement in motor regulation.39 The hippocampus is rich in the type I isoform, and since this isoform is activated by calcium-calmodulin, it has been speculated that it plays a role in long-term potentiation mediated by the glutamate receptor. Indeed, knockout mice lacking the type I isoform show an impaired learning capacity.42 The involvement of a particular isoform in the neuronal control of blood pressure or the circulation has not been investigated.

Each of the various isoforms has a unique chromosomal distribution. Unlike G-protein subtypes,43 all the adenylyl cyclase isoforms are found on different chromosomes, even within the same subgroup,44 45 indicating that the adenylyl cyclase gene family expanded by chromosomal duplication rather than by gene duplication. Since distinct promoter/enhancer elements control the transcription of these different adenylyl cyclase genes, their unique patterns of developmental- and tissue-specific expression would be predicted.

Thus, there is significant heterogeneity in the distribution and biochemical properties of the various adenylyl cyclase isoforms. It is also apparent that a single tissue or cell expresses multiple adenylyl cyclase isoforms; no cell type has been found thus far that expresses only one isoform of adenylyl cyclase. Each tissue and cell type, however, likely possesses a unique "mixture" of adenylyl cyclase isoforms. The heterogeneity among adenylyl cyclase isoforms distinguishes this enzyme family from the other components of the ßAR signaling pathway, specifically, the ßAR itself and Gs{alpha}, which lack this diversity either in the number of isoforms expressed or in their pattern of tissue-specific expression.


*    Mechanisms of Catalytic Activation
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Since the soluble forms of guanylyl cyclase share homology in their catalytic regions with adenylyl cyclase and since guanylyl cyclase subunits must heterodimerize to generate catalytic activity, it was speculated that the two putative catalytic domains of adenylyl cyclase (C1a and C2a) must dimerize to initiate catalysis (Fig 2Up). For guanylyl cyclase, it has been demonstrated that the two subunits, {alpha} and ß, which are distinct gene products, must be coexpressed to catalyze cGMP synthesis.46 An analogous situation appears to exist for the adenylyl cyclases. For the type I, II, and V isoforms,47 48 expression of either the {alpha} half (M1/C1) or ß half (M2/C2) of the molecule alone is insufficient to generate enzymatic activity. The type V isoform is particularly intriguing in that the single type V adenylyl cyclase gene can generate, in addition to its full-length product, an {alpha}-half molecule (M1/C1a) containing a single catalytic domain through the use of an alternative polyadenylation signal located within an intron in the middle of the gene.48 When either half of the molecule, {alpha} (M1C1) or ß (M2C2), is expressed alone, the product is inactive. Catalytic activity is rescued when the two halves are coexpressed. Additional evidence supporting this mechanism comes from the finding that a 20–amino acid residue peptide, whose sequence was taken from a highly conserved region in the first cytoplasmic domain of type V adenylyl cyclase, can directly inhibit the catalytic activity of all isoforms against which it was tested.49 It is hypothesized that this region of C1a directly contacts C2a and that the peptide, by way of competition, blocks the interaction and thereby inhibits catalytic activity. Interestingly, the two catalytic domains (or halves) need not originate from the same isoform; a chimeric molecule termed I-{alpha}/II-ß containing the front half (M1C1) of type I and the back half (M2C2) of type II is also active. However, active chimeric molecules cannot be randomly assembled from different isoform "halves." The rules that determine a successful mating have not been worked out.50

More recent studies have demonstrated that the two cytosolic domains alone (C1/C2), in the absence of their transmembrane regions, can generate enzymatic activity. A type I-C1/II-C2 chimera, consisting of only the cytoplasmic catalytic domain of each isoform connected by a short linker and devoid of the transmembrane spans, shows a high level of enzymatic activity. This soluble enzyme was produced using a bacterial expression system.51 52 53 The purified product could be stimulated by Gs{alpha} and forskolin as well as inhibited by Gß{gamma} and P-site inhibitors (adenosine and its analogues), indicating that the cytoplasmic domains alone can recapitulate the major biochemical properties of the wild-type enzyme. Other studies have shown that noncatalytic domains, such as the amino-terminal region, contribute to the catalytic activity of one isoform (type I)47 but not of another (type V),20 54 suggesting that certain regions may modify enzymatic activity but in an isoform-dependent manner. Unexpected properties may also exist within other noncatalytic domains, such as a voltage-sensing activity in the transmembrane regions.55 Depolarization of cultured neuronal cells with KCl was synergistic with a ßAR agonist in stimulating adenylyl cyclase activity. The concept that the transmembrane spans of adenylyl cyclase, like those of voltage-gated ion channels, can detect changes in membrane potential and thereby signal a conformational change in the enzyme is intriguing. This mechanism has particular implications for excitable cells such as neurons and cardiocytes, where, if it were operational, adenylyl cyclase itself might play a primary role in both sensing and modulating the cell's overall response to stimuli that otherwise could not directly activate a G protein–linked signaling pathway. Although the two catalytic domains (C1/C2) are necessary to generate enzymatic activity, the above studies suggest that other domains contribute to the complete biochemical and regulatory profile of the intact molecule.

Regions important for the interaction with various regulatory components of adenylyl cyclase have been demonstrated using synthetic peptides. A 27–amino acid residue peptide whose sequence was taken from the C2a region of type II adenylyl cyclase blocked Gß{gamma} stimulation of this isoform.56 This peptide also inhibited other enzymes that are known to be stimulated by Gß{gamma}, such as phospholipase ß, certain potassium channels, and ß-adrenergic receptor kinase. Interestingly, this peptide contained a motif QXXER that is shared by these Gß{gamma}-regulated enzymes, suggesting that this motif is important for Gß{gamma} interaction and that the region containing this motif within type II adenylyl cyclase is a binding domain for Gß{gamma}. A region important for calmodulin interaction has also been identified. A 28–amino acid residue peptide whose sequence was taken from the C1b region of type I adenylyl cyclase inhibited the calmodulin-stimulated activity of adenylyl cyclase purified from brain.57 Another study showed that the mutation, F503 to R503, in type I adenylyl cyclase within the region corresponding to this peptide abolished calmodulin stimulation of type I adenylyl cyclase,58 suggesting that this is the calmodulin binding domain of the type I isoform.


*    Diversity in Regulation by G-Protein Subunits
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Complementary DNA cloning of adenylyl cyclase isoforms has enabled investigators to examine each isoform independently. Numerous studies have indicated that no two isoforms are regulated in the same manner, even within the same subgroup. Potential regulators vary from simple cation concentration59 to various kinases.29 60 The pattern of regulation may differ, even for the same isoform, when expressed in different cell types, between splice variants of adenylyl cyclase isoforms or in response to various regulatory components. A striking finding that underscores the diversity in the regulation of adenylyl cyclase isoforms is their response to various G-protein subunits: the same G-protein subunit may stimulate certain adenylyl cyclase isoforms, inhibit other isoforms, and have no effect on the remaining isoforms. Gß{gamma} inhibits type I adenylyl cyclase while it stimulates type II adenylyl cyclase. The stimulation of type II adenylyl cyclase is augmented in the presence of Gs{alpha} stimulation of this isoform.61 62 Type IV adenylyl cyclase, an isoform closely related to type II, is also stimulated by Gß{gamma}. In contrast, type V and VI adenylyl cyclase isoforms are not directly responsive to Gß{gamma}. Inhibition of catalytic activity by Gi{alpha} is also not universal among the isoforms, and variable degrees of inhibition have been reported.61 63 In studies using membranes prepared from insect cells expressing a particular adenylyl cyclase isoform, all Gi{alpha} subtypes (Gi{alpha}1 to Gi{alpha}3) elicited a similar degree of inhibition of the type V and VI isoforms. Type I adenylyl cyclase was inhibited, but weakly, compared with types V and VI.61 Protein kinase C influences the Gi{alpha}-mediated inhibition of adenylyl cyclase.64 In studies using COS cell membranes coexpressing the type II adenylyl cyclase isoform and a constitutively active Gi{alpha}, inhibition of type II adenylyl cyclase by Gi{alpha} was abolished upon treatment of the cells with phorbol ester. These data suggest that Gi{alpha} inhibits adenylyl cyclase in an isoform-dependent manner and that this inhibition is influenced by other factors, such as the phosphorylation state of the enzyme. Inhibition by other G{alpha} subunits also occurs in an isoform-specific way. Go{alpha} inhibits type I adenylyl cyclase while minimally affecting type VI. G{alpha}12 does not affect a variety of adenylyl cyclase isoforms, whereas Gz{alpha} inhibits type I and V adenylyl cyclase.65 Finally, although all isoforms are stimulated by Gs{alpha}, the extent of this effect varies among the isoforms and is influenced by other factors as well.66 The outcome of these findings is that the combination of G-protein subtypes and adenylyl cyclase isoforms expressed by a cell is important in determining cAMP production in response to external stimuli. In certain cases, the same stimulus may result in the opposite effect on cAMP production within different cell types, such as stimulation of adenylyl cyclase through an inhibitory G protein–linked receptor. Stimulation of the {alpha}2-adrenergic receptor, which is coupled in an inhibitory manner to adenylyl cyclase through Gi, usually leads to decreased cAMP production. However, coexpression of this receptor with the type II adenylyl cyclase isoform results in increased cAMP production, probably through the stimulation of the enzyme by Gß{gamma} subunits.67 These results underscore the two major determinants of cAMP production within a cell: which adenylyl cyclase isoforms are expressed and what are their relative amounts? These factors, in addition to the repertoire of G protein–linked receptor subtypes expressed by the cell, are important in determining the direction and degree of cAMP signaling (Fig 3Down).



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  		Figure 3. Old and new paradigms for the regulation of adenylyl cyclases. cAMP production within the cell is not determined by the bulk amount of an adenylyl cyclase (old paradigm) but rather by the content of each adenylyl cyclase isoform that the cell expresses (new paradigm). Importantly, the content and mixture of adenylyl cyclase isoforms vary among different cell types. Note that the same regulatory component may stimulate certain adenylyl cyclase isoforms, inhibit other isoforms, and have no effect on the remaining isoforms. AC through AC''' represent different adenylyl cyclase isoforms expressed by the same cell type. The size variation of the symbol AC is meant to reflect the difference in content of each isoform, thereby contributing to the overall profile of the cAMP response to various stimuli. Gs indicates the stimulatory G protein; Gi, the inhibitory G protein; Ca, calcium; CaM, calmodulin; PKC, protein kinase C; ß{gamma}, G-protein ß{gamma} subunits; and Gz, G alpha z.


*    Calcium Sensitivity of Adenylyl Cyclase Isoforms
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In the heart, the ambient calcium concentration is continuously being regulated and, in turn, modulates a variety of signaling pathways, including cAMP production. The cardiac isoforms, types V and VI, are insensitive to both calmodulin and Gß{gamma} subunits. However, these isoforms have been shown to be directly inhibited by submicromolar concentrations of calcium.21 NCB-20 cells, which express mostly the type VI isoform, increase intracellular calcium concentration in response to bradykinin. This results from an initial release of calcium from inositol 1,4,5-tris-phosphate–sensitive stores, which, in turn, inhibits adenylyl cyclase catalytic activity.68 Interestingly, more generalized elevations of intracellular calcium produced by calcium ionophores do not necessarily inhibit cAMP production in these same cells; this suggests that calcium-sensitive adenylyl cyclase isoforms may be functionally colocalized with certain calcium entry channels.69

The type III isoform is stimulated by calcium/calmodulin in vitro when concomitantly stimulated with Gs{alpha}.36 However, when this isoform is hormonally stimulated in intact cells, it is inhibited by an increase in intracellular calcium. Since this inhibition is blocked by a calmodulin kinase inhibitor, it has been proposed that calmodulin kinase directly inhibits the type III isoform in vivo.70 Therefore, this calmodulin-sensitive isoform appears to be under dual regulation: stimulation by calcium/calmodulin and inhibition by calmodulin kinase.

The most recently proposed mechanism by which calcium can regulate adenylyl cyclase activity is through calcineurin, a calcium-sensitive phosphatase. The type IX isoform is activated by FK506, an inhibitor of this particular phosphatase, suggesting that calcium-dependent dephosphorylation (not phosphorylation) is a potentially important mechanism regulating the activity of this isoform.24

Thus, a variety of mechanisms involving calcium can impinge on the activity of various adenylyl cyclase isoforms; similarly, cAMP-triggered mechanisms can directly gate the strength and duration of the calcium signal. For example, ßAR-mediated activation of adenylyl cyclase and, thereby, protein kinase A leads to increased intracellular calcium concentrations by enhancing external calcium entry and the release of internal stores. This, in return, can regulate various adenylyl cyclases, either directly or indirectly, through the activation of various calcium-dependent kinases and phosphatases. Thus, cAMP and calcium concentrations may oscillate, providing both temporal and spatial information to the cell. Although dynamic interplay between these two second messenger pathways has been demonstrated,71 its significance for excitable cell types, such as the cardiocyte, has not been defined.


*    Adenylyl Cyclase Isoforms in the Heart
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Although the mRNAs of the ubiquitous isoforms, in particular, types IV and VII, are detectable in the heart, the steady state mRNA levels for the type V and VI isoforms are much higher than those of the other isoforms. Furthermore, these two isoforms are most dominantly expressed in the heart. Expression of the type V isoform has been reported in only two tissues in both dogs and humans,18 72 whereas type VI is expressed in most tissues at a low level.19 Species differences in the pattern of expression are also apparent. In rodents, type V is expressed in other tissues, including the kidney.20 The high level of expression of these two isoforms has also been demonstrated in isolated and purified cardiocyte preparations.72 73 Since specific antibodies with the requisite sensitivity to detect these isoforms are not available, expression at the protein level has not been quantified. Nevertheless, it is reasonable to suggest that these two isoforms importantly determine both the properties and the efficiency of ßAR signaling in the heart. Certainly the biochemical signature of cardiac adenylyl cyclase is most consistent with the known properties of the type V and VI isoforms.

An interesting question arises: why does the heart express two isoforms that are quite similar in their biochemical properties? Type V appears to be an adult isoform, whereas type VI is more highly expressed in the neonate.74 75 Expression of the type VI isoform is most abundant in the fetus, gradually declines with age, and reaches its lowest level in mature adults. The expression of type V follows an opposite pattern. The expression is lowest in the fetus, gradually increases with age, and reaches a maximum in the mature adult. Thus, two major isoforms in the heart appear to be developmentally regulated in an opposite manner, a pattern analogous to the expression of the {alpha}- and ß-myosin heavy chains.76 Nevertheless, by mRNA analysis, these two isoforms are the most abundant in the heart at each stage of development.

It has been suggested that a decreased capacity to generate cAMP may be a fundamental defect in cardiac disease, particularly heart failure.77 The expression of the cardiac adenylyl cyclase isoforms V and VI is significantly decreased in the dog after the development of heart failure generated by ventricular overdrive pacing. After 4 weeks of pacing, left ventricular end-diastolic pressure and heart rate increase significantly, coincident with the appearance of the signs and symptoms of heart failure, ie, edema, ascites, and exercise intolerance. Basal and forskolin-stimulated adenylyl cyclase activities decrease significantly. These changes are accompanied by a reduction in the steady state mRNA levels of both the type V and VI isoforms. These findings suggest that decreased adenylyl cyclase mRNA levels underlie the downregulation of catalytic activity. Whether this plays a causal role in the contractile dysfunction of heart failure remains unclear. Changes in adenylyl cyclase expression in other forms of cardiac disease have not been extensively investigated. In cardiac hypertrophy, for example, it would be interesting to determine whether the expression of the neonatal type VI isoform is increased.

Cardiac adenylyl cyclase may be regulated by protein kinase C under ischemic conditions and thereby play a role in maintaining cardiac contractility.78 79 Protein kinase C, which has classically been defined as a calcium-activated serine-threonine kinase, actually comprises at least 11 isozymes. These can be divided into three classes according to their cofactor requirements for activation: classic ({alpha}, ßI, ßII, and {gamma}, which are sensitive to both calcium and diacyl glycerol), novel ({delta}, {epsilon}, {eta}, {theta}, and µ, which are insensitive to calcium and sensitive to diacyl glycerol), and atypical ({zeta} and {lambda}, which are insensitive to both calcium and diacyl glycerol).80 The heart expresses at least the {alpha}, ß, {delta}, and {zeta} isozymes.81 The {alpha} and {zeta} isozymes directly phosphorylate and activate the type V cardiac adenylyl cyclase isoform in vitro60 ; the {zeta} isozyme activates type V adenylyl cyclase in a calcium-independent manner, and the {alpha} isozyme requires calcium, affording yet another mechanism for calcium-mediated regulation of adenylyl cyclase activity.

It is also known that growth factors, including insulin, can regulate cardiac cAMP production and contractility. Insulin receptor stimulation activates phosphatidylinositol-3-OH kinase, leading to the formation of specific lipid products, such as phosphatidylinositol-3,4,5 triphosphate,82 that activate protein kinase C{zeta}.83 In cells overexpressing the type V isoform, insulin augments cAMP production; this augmentation can be further enhanced by coexpressing the protein kinase C{zeta} isozyme.84 These data imply that growth factors regulate cAMP signaling via an atypical protein kinase C, such as protein kinase C{zeta}, although further studies are required to confirm this mechanism in vivo. Such regulation may occur in an isoform-specific manner; epidermal growth factor–mediated augmentation of cAMP production is also observed with the type V isoform, but not with other isoforms.85 More recent data, however, suggest that this stimulation is due, at least in part, to epidermal growth factor–stimulated phosphorylation of Gs{alpha}.86

In contrast to the enhancement of type V activity by protein kinase C–mediated phosphorylation, phosphorylation by protein kinase A directly inhibits its activity, by decreasing the Vmax of the enzyme without changing its Km for the substrate ATP.87 The phosphorylation and inhibition of type V adenylyl cyclase by protein kinase A suggest the presence of a negative-feedback loop at the level of adenylyl cyclase itself as well as a mechanism for heterologous desensitization of the cAMP signaling pathway.


*    Other Adenylyl Cyclases
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 up arrowThe Structure of Adenylyl...
 up arrowDiversity in Adenylyl Cyclase...
 up arrowMechanisms of Catalytic...
 up arrowDiversity in Regulation by...
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 up arrowAdenylyl Cyclase Isoforms in...
 *Other Adenylyl Cyclases
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Thus far, we have described the diversity that exists within the membrane-bound forms of mammalian adenylyl cyclases. However, another type of adenylyl cyclase that is not membrane bound exists in reproductive tissues.88 This soluble adenylyl cyclase(s) is not regulated by hormones, including gonadotrophins. Further characterization of this adenylyl cyclase type will require the cloning of its cDNA.


*    Future Studies
up arrowTop
 up arrowcAMP Generation in the...
 up arrowThe Structure of Adenylyl...
 up arrowDiversity in Adenylyl Cyclase...
 up arrowMechanisms of Catalytic...
 up arrowDiversity in Regulation by...
 up arrowCalcium Sensitivity of Adenylyl...
 up arrowAdenylyl Cyclase Isoforms in...
 up arrowOther Adenylyl Cyclases
 *Future Studies
down arrowReferences
 
It should be apparent that sympathetic regulation of the heart is importantly determined by the unique adenylyl cyclase isoforms expressed in this tissue. Although much data in vitro have been obtained to support this contention, the physiological relevance of this unique pattern of adenylyl cyclase isoform expression remains to be determined. The biochemical diversity afforded by the multiplicity of adenylyl cyclase isoforms enables a cell, by the nature of the isoforms it expresses, to integrate and modulate uniquely its responsiveness to both internal signals (eg, calcium) and to external stimuli (eg, hormones and neurotransmitters) (Fig 3Up). The type V isoform is the dominant isoform in the adult heart and thereby plays a key role in determining the cardiac response to a variety of stimuli, in particular, stimulation by the sympathetic nerves. Changes in the level of expression of type V and that of other isoforms in pathophysiological states may contribute to cardiac dysfunction. Certain of the aforementioned hypotheses can be approached using transgenic animal models.

In addition to the known isoforms, an additional cardiac adenylyl cyclase isoform may also exist. Although a calmodulin-sensitive adenylyl cyclase activity in the heart has been reported,89 no known calmodulin-sensitive isoforms are expressed in the heart, except for type III, which is found in the atria37 but not in the ventricle. Future cloning studies may isolate a new isoform(s) and further expand the repertoire of cardiac adenylyl cyclases.


*    Acknowledgments
 
This study was supported by grants from the US Public Health Service (HL-38070) and the American Heart Association (No. 13533945). Y.I. thanks Dr M. Ishii (Yokohama University) for his advice and encouragement. The authors also acknowledge profound contributions by many investigators whose work could not be explicitly cited here.


*    Footnotes
 
This manuscript was sent to Leslie A. Leinwand, Consulting Editor, for review by expert referees, editorial decision, and final disposition.

Received September 18, 1996; accepted November 8, 1996.


*    References
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 up arrowcAMP Generation in the...
 up arrowThe Structure of Adenylyl...
 up arrowDiversity in Adenylyl Cyclase...
 up arrowMechanisms of Catalytic...
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 up arrowAdenylyl Cyclase Isoforms in...
 up arrowOther Adenylyl Cyclases
 up arrowFuture Studies
 *References
 

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