Cellular Localisation of Adenylyl Cyclase: A Post-genome Perspective

The intracellular messenger cAMP is essential for vital processes ranging from ovulation to cognition. There are 10 genes for adenylyl cyclase (AC), the biosynthetic enzyme of cAMP. Nine of these encode membrane-bound proteins and one gives rise to soluble AC. The understanding of the biological significance of this molecular diversity is incomplete. Membrane-bound ACs conform to the same structural blueprint but have markedly different regulatory characteristics. AC mRNAs are differentially distributed in the body suggesting non-redundant physiological functions. The subcellular localisation of AC isoforms has not been examined in detail. Here we discuss the current knowledge on the intracellular targeting of AC isoforms, and highlight the technical problems of AC detection, some of which appear to be caused by the poor quality-control of commercially supplied antibodies. The principal message is that intracellular targeting of ACs may be isoform-specific and also dependent on the cellular context of expression.Invocation: This paper was written to honour one of the founders of chemical neuroanatomy—Professor Miklós Palkovits on his 70th birthday.     

Organization of cAMP signalling microdomains for optimal regulation by Ca2+ entry

Cross-talk between cAMP and Ca2+ signalling pathways plays a critical role in cellular homoeostasis. Several AC (adenylate cyclase) isoforms, catalysing the production of cAMP from ATP, display sensitivity to submicromolar changes in intracellular Ca2+ and, as a consequence, are key sites for Ca2+ and cAMP interplay. Interestingly, these Ca2+-regulated ACs are not equally responsive to equivalent Ca2+ rises within the cell, but display a remarkable selectivity for regulation by SOCE (store-operated Ca2+ entry). Over the years, considerable efforts at investigating this phenomenon have provided indirect evidence of an intimate association between Ca2+-sensitive AC isoforms and sites of SOCE. Now, recent identification of the molecular components of SOCE [namely STIM1 (stromal interaction molecule 1) and Orai1], coupled with significant advances in the generation of high-resolution targeted biosensors for Ca2+ and cAMP, have provided the first detailed insight into the organization of the cellular microdomains associated with Ca2+-regulated ACs. In the present review, I summarize the findings that have helped to provide our most definitive understanding of the selective regulation of cAMP signalling by SOCE.     

Hydroxamate based inhibitors of adenylyl cyclase. Part 1: the effect of acyclic linkers on P-site binding

The adenylyl cyclases (ACs) are a family of enzymes that are key elements of signal transduction by virtue of their ability to convert ATP to cAMP. The catalytic mechanism of this transformation proceeds through initial binding of ATP to the purine binding site (P-site) followed by metal mediated cyclization with loss of pyrophosphate. Crystallographic analysis of ACs with known inhibitors reveals the presence of two metals in the active site. Presently, nine isoforms of adenylyl cyclase are known and unique isoform combinations are expressed in a tissue specific manner. The development of isoform specific inhibitors of adenylyl cyclase may prove to be a useful strategy toward the design of novel therapeutic agents. In order to develop novel AC inhibitors, we have chosen a design approach utilizing molecules with the adenine ring system joined to a metal-coordinating hydroxamic acid via flexible acyclic linkers. The designed inhibitors were assayed against type V AC with the size and heteroatom content of the linkers varied to probe the interaction of the nucleotide and metal binding sites within the enzyme.     

Adenylyl cyclase isoform expression in non-diabetic and diabetic Goto-Kakizaki (GK) rat pancreas. Evidence for distinct overexpression of type-8 adenylyl cyclase in diabetic GK rat islets

Glucose-induced insulin release is markedly decreased in the spontaneously diabetic Goto-Kakizaki (GK) rat pancreas. This defect was recently shown to be reversed by forskolin which markedly enhances cAMP generation in GK islets. These effects of forskolin were associated with overexpression of type-3 adenylyl cyclase (AC) mRNA due to the presence of two functional point mutations in the promoter region of AC3 gene in GK rat. Nine AC isoforms have been described, but their expression pattern in relation to the main pancreatic islet cell types, as well as their involvement in the diabetic state, is still unknown. Using antibodies raised against AC1–8, we have studied by double immunofluorescence the localisation of these AC isoforms in different endocrine cell types in both normal and diabetic GK rat pancreas. Our results demonstrated a clear immunoreaction (IR) to AC1–4 and 6 in normal and GK islet β-cells, while a smaller number of ACs were expressed in α- and δ-cells. No AC-IR was observed in pancreatic polypeptide cells. Moreover, we have found an increased IR of the Ca²⁺-stimulated AC1, AC3 and AC8 in diabetic β- and α-cells, compared with the corresponding IR in control pancreas. Most noticeable was the eliciting of a markedly enhanced AC8-IR in GK rat β- and α-cells, in contrast to a barely discernible AC8-IR in corresponding normal cells. In conclusion, AC expression exhibits a complex pattern in the endocrine pancreas, with specific differences between the normal and diabetic state. Accepted: 25 November 1999     

Particulate and soluble adenylyl cyclases participate in the sperm acrosome reaction

cAMP is important in sea urchin sperm signaling, yet the molecular nature of the adenylyl cyclases (ACs) involved remained unknown. These cells were recently shown to contain an ortholog of the mammalian soluble adenylyl cyclase (sAC). Here, we show that sAC is present in the sperm head and as in mammals is stimulated by bicarbonate. The acrosome reaction (AR), a process essential for fertilization, is influenced by the bicarbonate concentration in seawater. By using functional assays and immunofluorescence techniques we document that sea urchin sperm also express orthologs of multiple isoforms of transmembrane ACs (tmACs). Our findings employing selective inhibitors for each class of AC indicate that both sAC and tmACs participate in the sperm acrosome reaction.     

Altered cAMP signaling induced by lysophosphatidic acid in senescent human diploid fibroblasts

Lysophosphatidic acid (LPA) is a lipid mitogen that acts through G-protein-coupled receptors. LPA responsiveness has been reported to be dependent on the senescent state of the cells. To solve the mechanism underlying, we observed LPA-dependent cAMP status and found its age-dependent contrasting profile such as high level of cAMP in the senescent cells vs its low level in the young cells. In order to clarify the molecular mechanism of the ageing effect, we examined various molecular species involved in the cAMP signaling pathway by semi-quantitative RT-PCR. EDG-1 and EDG-4 were unchanged, but EDG-2 and EDG-7 were reduced with age. Senescent cells showed a partial reduction of Gi1, Gi2, and Gi3, but no change in the level of Gq. Decreased Gis and Gi-coupled LPA receptors may reduce the inhibitory effect of Gi alpha on adenylyl cyclases (ACs), resulting in cAMP accumulation via activation of adenylyl cyclase in senescent fibroblasts. We also observed an age-dependent increase in some of AC isoforms: II, IV, and VI. In conclusion, multiple changes in the cAMP signaling pathway of the senescent cells might explain the altered responsiveness to the mitogenic stimuli.     

AKAP79/150 Interacts with AC8 and Regulates Ca2+-dependent cAMP Synthesis in Pancreatic and Neuronal Systems

Protein kinase A anchoring proteins (AKAPs) provide the backbone for targeted multimolecular signaling complexes that serve to localize the activities of cAMP. Evidence is accumulating of direct associations between AKAPs and specific adenylyl cyclase (AC) isoforms to facilitate the actions of protein kinase A on cAMP production. It happens that some of the AC isoforms (AC1 and AC5/6) that bind specific AKAPs are regulated by submicromolar shifts in intracellular Ca²⁺. However, whether AKAPs play a role in the control of AC activity by Ca²⁺ is unknown. Using a combination of co-immunoprecipitation and high resolution live cell imaging techniques, we reveal an association of the Ca²⁺-stimulable AC8 with AKAP79/150 that limits the sensitivity of AC8 to intracellular Ca²⁺ events. This functional interaction between AKAP79/150 and AC8 was observed in HEK293 cells overexpressing the two signaling molecules. Similar findings were made in pancreatic insulin-secreting cells and cultured hippocampal neurons that endogenously express AKAP79/150 and AC8, which suggests important physiological implications for this protein-protein interaction with respect to Ca²⁺-stimulated cAMP production.     

Dependence of electrical activity and calcium influx-controlled prolactin release on adenylyl cyclase signaling pathway in pituitary lactotrophs

Pituitary lactotrophs in vitro fire extracellular Ca2+-dependent action potentials spontaneously through still unidentified pacemaking channels, and the associated voltage-gated Ca2+influx (VGCI) is sufficient to maintain basal prolactin (PRL) secretion high and steady. Numerous plasma membrane channels have been characterized in these cells, but the mechanism underlying their pacemaking activity is still not known. Here we studied the relevance of cyclic nucleotide signaling pathways in control of pacemaking, VGCI, and PRL release. In mixed anterior pituitary cells, both VGCI-inhibitable and -insensitive adenylyl cyclase (AC) subtypes contributed to the basal cAMP production, and soluble guanylyl cyclase was exclusively responsible for basal cGMP production. Inhibition of basal AC activity, but not soluble guanylyl cyclase activity, reduced PRL release. In contrast, forskolin stimulated cAMP and cGMP production as well as pacemaking, VGCI, and PRL secretion. Elevation in cAMP and cGMP levels by inhibition of phosphodiesterase activity was also accompanied with increased PRL release. The AC inhibitors attenuated forskolin-stimulated cyclic nucleotide production, VGCI, and PRL release. The cell-permeable 8-bromo-cAMP stimulated firing of action potentials and PRL release and rescued hormone secretion in cells with inhibited ACs in an extracellular Ca2+-dependent manner, whereas 8-bromo-cGMP and 8-(4-chlorophenylthio)-2'-O-methyl-cAMP were ineffective. Protein kinase A inhibitors did not stop spontaneous and forskolin-stimulated pacemaking, VGCI, and PRL release. These results indicate that cAMP facilitates pacemaking, VGCI, and PRL release in lactotrophs predominantly in a protein kinase A- and Epac cAMP receptor-independent manner.     

Genetic evidence for adenylyl cyclase 1 as a target for preventing neuronal excitotoxicity mediated by N-methyl-D-aspartate receptors

The excessive activation of N-methyl-D-aspartate (NMDA) receptors by glutamate results in neuronal excitotoxicity. cAMP is a key second messenger and contributes to NMDA receptor-dependent synaptic plasticity. Adenylyl cyclases 1 (AC1) and 8 (AC8) are the two major calcium-stimulated ACs in the central nervous system. Previous studies demonstrate AC1 and AC8 play important roles in synaptic plasticity, memory, and persistent pain. However, little is known about the possible roles of these two ACs in glutamate-induced neuronal excitotoxicity. Here, we report that genetic deletion of AC1 significantly attenuated neuronal death induced by glutamate in primary cultures of cortical neurons, whereas AC8 deletion did not produce a significant effect. AC1, but not AC8, contributes to intracellular cAMP production following NMDA receptor activation by glutamate in cultured cortical neurons. AC1 is involved in the dynamic modulation of cAMP-response element-binding protein activity in neuronal excitotoxicity. To explore the possible roles of AC1 in cell death in vivo, we studied neuronal excitotoxicity induced by an intracortical injection of NMDA. Cortical lesions induced by NMDA were significantly reduced in AC1 but not in AC8 knock-out mice. Our findings provide direct evidence that AC1 plays an important role in neuronal excitotoxicity and may serve as a therapeutic target for preventing excitotoxicity in stroke and neurodegenerative diseases.     

Physiological roles for G protein-regulated adenylyl cyclase isoforms: insights from knockout and overexpression studies

Cyclic AMP is a universal second messenger, produced by a family of adenylyl cyclase (AC) enzymes. The last three decades have brought a wealth of new information about the regulation of cyclic AMP production by ACs. Nine hormone-sensitive, membrane-bound AC isoforms have been identified in addition to a tenth isoform that lacks membrane spans and more closely resembles the cyanobacterial AC enzymes. New model systems for purifying and characterizing the catalytic domains of AC have led to the crystal structure of these domains and the mapping of numerous interaction sites. However, big hurdles remain in unraveling the roles of individual AC isoforms and their regulation in physiological systems. In this review we explore the latest on AC knockout and overexpression studies to better understand the roles of G protein regulation of ACs in the brain, olfactory bulb, and heart.     

Comparative electrostatic analysis of adenylyl cyclase for isoform dependent regulation properties

The enzyme adenylyl cyclase (AC) plays a pivotal role in a variety of signal transduction pathways inside the cell, where it catalyzes the cyclization of adenosine triphosphate (ATP) into the second‐messenger cyclic adenosine monophosphate (cAMP). Among other roles, AC regulates processes involved in neural plasticity, innervation of smooth muscles of the heart and the endocrine system of the pancreas. The functional diversity of AC is manifested in its different isoforms, each having a specific regulation pattern. There is an increasing amount of data available concerning the regulatory properties of AC isoforms, however little is known about the interactions on a structural level. Here, we conducted a comparative electrostatic analysis of the catalytic domains of all nine transmembrane AC isoforms with the aim of detecting, verifying and predicting the binding sites of molecular regulators on AC. The results provide support for the positioning of the binding site of the inhibitory protein G_iα at a pseudo‐symmetric position to the stimulatory G_sα binding site. They also provide a structural interpretation of the Gβγ interaction with ACs 2, 4, and 7 and suggest a new binding site for RGS2. Comparison of the small molecule binding sites on AC shows that overall they have high electrostatic similarity, but regions of electrostatic differences are identified. These could provide a basis for the development of novel compounds with isoform‐specific modulatory effects on AC. Proteins 2016; 84:1844–1858. © 2016 Wiley Periodicals, Inc.     

Differential inhibition of adenylyl cyclase isoforms and soluble guanylyl cyclase by purine and pyrimidine nucleotides

Mammals express nine membranous adenylyl cyclase isoforms (ACs 1-9), a structurally related soluble guanylyl cyclase (sGC) and a soluble AC (sAC). Moreover, Bacillus anthracis and Bacillus pertussis produce the AC toxins, edema factor (EF), and adenylyl cyclase toxin (ACT), respectively. 2'(3')-O-(N-methylanthraniloyl)-guanosine 5'-[gamma-thio]triphosphate is a potent competitive inhibitor of AC in S49 lymphoma cell membranes. These data prompted us to study systematically the effects of 24 nucleotides on AC in S49 and Sf9 insect cell membranes, ACs 1, 2, 5, and 6, expressed in Sf9 membranes and purified catalytic subunits of membranous ACs (C1 of AC5 and C2 of AC2), sAC, sGC, EF, and ACT in the presence of MnCl(2). N-Methylanthraniloyl (MANT)-GTP inhibited C1.C2 with a K(i) of 4.2 nm. Phe-889 and Ile-940 of C2 mediate hydrophobic interactions with the MANT group. MANT-inosine 5'-[gamma-thio]triphosphate potently inhibited C1.C2 and ACs 1, 5, and 6 but exhibited only low affinity for sGC, EF, ACT, and G-proteins. Inosine 5'-[gamma-thio]triphosphate and uridine 5'-[gamma-thio]triphosphate were mixed G-protein activators and AC inhibitors. AC5 was up to 15-fold more sensitive to inhibitors than AC2. EF and ACT exhibited unique inhibitor profiles. At sAC, 2',5'-dideoxyadenosine 3'-triphosphate was the most potent compound (IC(50), 690 nm). Several MANT-adenine and MANT-guanine nucleotides inhibited sGC with K(i) values in the 200-400 nm range. UTP and ATP exhibited similar affinities for sGC as GTP and were mixed sGC substrates and inhibitors. The exchange of MnCl(2) against MgCl(2) reduced inhibitor potencies at ACs and sGC 1.5-250-fold, depending on the nucleotide and cyclase studied. The omission of the NTP-regenerating system from cyclase reactions strongly reduced the potencies of MANT-ADP, indicative for phosphorylation to MANT-ATP by pyruvate kinase. Collectively, AC isoforms and sGC are differentially inhibited by purine and pyrimidine nucleotides.     

A fission yeast platform for heterologous expression of mammalian adenylyl cyclases and high throughput screening

The fission yeast Schizosaccharomyces pombe uses a cAMP signaling pathway to link glucose-sensing to Protein Kinase A activity in order to regulate cell growth, sexual development, gluconeogenesis, and exit from stationary phase. We previously used a PKA-repressed fbp1-ura4 reporter to conduct high throughput screens (HTSs) for inhibitors of heterologously-expressed mammalian cyclic nucleotide phosphodiesterases (PDEs). Here, we describe the successful expression of all ten mammalian adenylyl cyclase (AC) genes, along with the human GNAS Gα_s gene. By measuring expression of an fbp1-GFP reporter together with direct measurements of intracellular cAMP levels, we can detect both basal AC activity from all ten AC genes as well as GNAS-stimulated activity from eight of the nine transmembrane ACs (tmACs; AC2-AC9). The ability to use this platform to conduct HTS for novel chemical probes that reduce PKA activity was demonstrated by a pilot screen of the LOPAC®¹²⁸⁰ library, leading to the identification of diphenyleneiodonium chloride (DPI) as an inhibitor of basal AC activity. This screening technology could open the door to the development of therapeutic compounds that target GNAS or the ACs, an area in which there is significant unmet need.     

Genetic selection in<Emphasis Type="Italic"> Saccharomyces</Emphasis> of mutant mammalian adenylyl cyclases with elevated basal activities

We show that co-expression of rat Galphas together with type I, II, IV, or VI mammalian adenylyl cyclase (AC) can suppress the growth defect of cyr1 strains of Saccharomyces cerevisiae, which lack a functional endogenous AC. Complemention of cvr1 is not observed in the absence of Galphas, indicating that the mammalian ACs retain their normal regulatory behavior in yeast. Selection for Galphas-independent growth of (cyr1 strains expressing type IV AC yielded several ACIV mutants with enhanced basal activity, each of which had a single amino acid substitution in the conserved C1a or C2a region of the protein. Expression of two of the mutant ACs in HEK293 cells resulted in increased levels of cAMP and elevated adenylyl cyclase activity. Further selection for reverting mutations in one of these constitutively active AC mutants yielded three independent intragenic suppressor mutations. The distribution of the activating and suppressor mutations throughout both C1a and C2a is consistent with a model in which the enhanced basal activity results from an increase in the affinity between C1a and C2a. These results demonstrate the utility of Saccharomyces as a tool for the identification of informative mutant forms of mammalian ACs.     

Calcium-Dependent Increases in Protein Kinase-A Activity in Mouse Retinal Ganglion Cells Are Mediated by Multiple Adenylate Cyclases

Neurons undergo long term, activity dependent changes that are mediated by activation of second messenger cascades. In particular, calcium-dependent activation of the cyclic-AMP/Protein kinase A signaling cascade has been implicated in several developmental processes including cell survival, axonal outgrowth, and axonal refinement. The biochemical link between calcium influx and the activation of the cAMP/PKA pathway is primarily mediated through adenylate cyclases. Here, dual imaging of intracellular calcium concentration and PKA activity was used to assay the role of different classes of calcium-dependent adenylate cyclases (ACs) in the activation of the cAMP/PKA pathway in retinal ganglion cells (RGCs). Surprisingly, depolarization-induced calcium-dependent PKA transients persist in barrelless mice lacking AC1, the predominant calcium-dependent adenylate cyclase in RGCs, as well as in double knockout mice lacking both AC1 and AC8. Furthermore, in a subset of RGCs, depolarization-induced PKA transients persist during the inhibition of all transmembrane adenylate cyclases. These results are consistent with the existence of a soluble adenylate cyclase that plays a role in calcium-dependent activation of the cAMP/PKA cascade in neurons.     

Filamin A is a phosphorylation target of membrane but not cytosolic adenylyl cyclase activity

Transmembrane adenylyl cyclase (AC) generates a cAMP pool within the subplasma membrane compartment that strengthens the endothelial cell barrier. This cAMP signal is steered toward effectors that promote junctional integrity and is inactivated before it accesses microtubules, where the cAMP signal causes phosphorylation of tau, leading to microtubule disassembly and barrier disruption. During infection, Pseudomonas aeruginosa uses a type III secretion system to inject a soluble AC, ExoY, into the cytosol of pulmonary microvascular endothelial cells. ExoY generates a cAMP signal that disrupts the endothelial cell barrier. We tested the hypothesis that this ExoY-dependent cAMP signal causes phosphorylation of tau, without inducing phosphorylation of membrane effectors that strengthen endothelial barrier function. To approach this hypothesis, we first discerned the membrane compartment in which endogenous transmembrane AC6 resides. AC6 was resolved in caveolin-rich lipid raft fractions with calcium channel proteins and the cell adhesion molecules N-cadherin, E-cadherin, and activated leukocyte adhesion molecule. VE-cadherin was excluded from the caveolin-rich fractions and was detected in the bulk plasma membrane fractions. The actin binding protein, filamin A, was detected in all membrane fractions. Isoproterenol activation of ACs promoted filamin phosphorylation, whereas thrombin inhibition of AC6 reduced filamin phosphorylation within the membrane fraction. In contrast, ExoY produced a cAMP signal that did not cause filamin phosphorylation yet induced tau phosphorylation. Hence, our data indicate that cAMP signals are strictly compartmentalized; whereas cAMP emanating from transmembrane ACs activates barrier-enhancing targets, such as filamin, cAMP emanating from soluble ACs activates barrier-disrupting targets, such as tau.     

Evidence for multiple distinctly localized adenylyl cyclase isoforms in mammalian spermatozoa

In addition to a bicarbonate-regulated soluble adenylyl cyclase (sAC), mammalian spermatozoa, like somatic cells, appear to contain receptor/G protein-regulated AC activity that contributes to the modulation of specialized cell processes. This study provides evidence that agents, known to influence somatic membrane-associated AC (mAC) but apparently not germ cell sAC, can modulate cAMP production and functional state in mouse spermatozoa. Specifically, forskolin significantly enhanced cAMP production and capacitation, while inclusion of 2',5'-dideoxyadenosine significantly blocked these responses. Furthermore, GTPgammaS and NaF stimulated cAMP, but GDPbetaS and mastoparan had no apparent effect, consistent with recent evidence that G(s), but not G(i), contributes to AC/cAMP regulation in uncapacitated cells. In addition, intact mouse spermatozoa were screened for all known mAC isoforms by immunolocalization, using commercially available specific antibodies. The most abundant isoforms appeared to be AC2, AC3, and AC8, each with distinct distributions in the acrosomal and flagellar regions; AC1 and AC4 also appeared to be present, although less abundantly, in the midpiece and acrosomal cap regions, respectively. Intriguingly, however, Western blotting revealed that the major immunoreactive proteins in mouse sperm lysates were considerably smaller (approximately 50-60 kDa) than their somatic cell counterparts, suggesting that mature spermatozoa contain multiple mACs which may function in a shortened form. Of particular interest were AC3 and AC8, located in the same regions as, and hence possibly directly associated with, specific cell surface receptors and G proteins that are able to regulate the spermatozoon's acquisition and maintenance of fertilizing ability via changes in AC/cAMP.     

Kinetic properties of Ca2+/calmodulin-dependent phosphodiesterase isoforms dictate intracellular cAMP dynamics in response to elevation of cytosolic Ca2+

Multiply regulated adenylyl cyclases (AC) and phosphodiesterases (PDE) can yield complex intracellular cAMP signals. Ca2+-sensitive ACs have received far greater attention than the Ca2+/calmodulin-dependent PDE (PDE1) family in governing intracellular cAMP dynamics in response to changes in the cytosolic Ca2+ concentration ([Ca2+]i). Here, we have stably expressed two isoforms of PDE1, PDE1A2 and PDE1C4, in HEK-293 cells to determine whether they exert different impacts on cellular cAMP. Fractionation and imaging showed that both PDEs occurred mainly in the cytosol. However, PDE1A2 and PDE1C4 differed considerably in their ability to hydrolyze cAMP and in their susceptibility to inhibition by the non-selective PDE inhibitor, IBMX and the PDE1-selective inhibitor, MMX. PDE1A2 had an approximately 30-fold greater Km for cAMP than PDE1C4 and yet was more susceptible to inhibition by IBMX and MMX than was PDE1C4. These differences were mirrored in intact cells when thapsigargin-induced capacitative Ca2+ entry (CCE) activated the PDEs. Mirroring their kinetic properties, PDE1C4 was active at near basal cAMP levels, whereas PDE1A2 required agonist-triggered levels of cAMP, produced in response to stimulation of ACs. The effectiveness of IBMX and MMX to inhibit PDE1A2 and PDE1C4 in functional studies was inversely related to their respective affinities for cAMP. To assess the impact of the two isoforms on cAMP dynamics, real-time cAMP measurements were performed in single cells expressing the two PDE isoforms and a fluorescent Epac-1 cAMP biosensor, in response to CCE. These measurements showed that prostaglandin E1-mediated cAMP production was markedly attenuated in PDE1C4-expressing cells upon induction of CCE and cAMP hydrolysis occurred at a faster rate than in cells expressing PDE1A2 under similar conditions. These results prove that the kinetic properties of PDE isoforms play a major role in determining intracellular cAMP signals in response to physiological elevation of [Ca2+]i and thereby provide a rationale for the utility of diverse PDE1 species.