Dermot M. F. Cooper

Capacitative Ca Entry Exclusively Inhibits cAMP Synthesis in C6-2B Glioma Cells EVIDENCE THAT PHYSIOLOGICALLY EVOKED Ca ENTRY REGULATES Ca-INHIBITABLE ADENYLYL CYCLASE IN NON-EXCITABLE CELLS

Elevation of cytosolic free Ca inhibits the type VI adenylyl cyclase that predominates in C6-2B cells. However, it is not known whether there is any selective requirement for Ca entry or release for inhibition of cAMP accumulation to occur. In the present study, the effectiveness of intracellular Ca release evoked by three independent methods (thapsigargin, ionomycin, and UTP) was compared with the capacitative Ca entry that was triggered by these treatments. In each situation, only Ca entry could inhibit cAMP accumulation (La ions blocked the effect); Ca release, which was substantial in some cases, was without effect. A moderate inhibition, as was elicited by a modest degree of Ca entry, could be rendered substantial in the absence of phosphodiesterase inhibitors. Such conditions more closely mimic the physiological situation of normal cells. These results are particularly significant, in demonstrating not only that Ca entry mediates the inhibitory effects of Ca on cAMP accumulation, but also that diffuse elevations in [Ca] are ineffective in modulating cAMP synthesis. This property suggests that, as with certain Ca-sensitive ion channels, Ca-sensitive adenylyl cyclases may be functionally colocalized with Ca entry channels.

Functional Co-localization of Transfected Ca2+-stimulable Adenylyl Cyclases with Capacitative Ca2+ Entry Sites

Three adenylyl cyclases (ACI, ACIII, and ACVIII) have been described, which are putatively Ca2+-stimulable, based on in vitro assays. However, it is not clear that these enzymes can be regulated by physiological rises in [Ca2+]i when expressed in intact cells. Furthermore, it is not known whether transfected adenylyl cyclases might display the strict requirement for capacitative Ca2+ entry that is shown by the Ca2+-inhibitable ACVI, which is indigenous to C6-2B glioma cells (Chiono, M., Mahey, R., Tate, G., and Cooper, D. M. F.(1995) J. Biol. Chem. 270, 1149-1155). In the present study, ACI, ACIII, and ACVIII were heterologously expressed in HEK 293 cells, and conditions were devised that distinguished capacitative Ca2+ entry from both internal release and nonspecific elevation in [Ca2+]i around the plasma membrane. Remarkably, not only were ACI and ACVIII largely insensitive to Ca2+ release from stores, but they were robustly stimulated only by capacitative Ca2+ entry and not at all by a substantial increase in [Ca2+]i at the plasma membrane elicited by ionophore. (ACIII, reflecting its feeble in vitro sensitivity to Ca2+, was unaffected by any [Ca2+]i rise.) These results suggest a quite unsuspected, essential association of Ca2+-sensitive adenylyl cyclases with capacitative Ca2+ entry sites, even when expressed heterologously.

Bimodal regulation of adenylate cyclase

The role of GTP in the stimulation of adenylate cyclase has received intense attention in the last 10 years. A number of excellent reviews of this area have appeared [1-3]. As a prelude to a discussion of inhibition of adenylate cyclase it seems appropriate to present a brief perspective on stimulation of adenylate cyclase. Many hormones interact with cell surface receptors to transmit a stimulatory signal to the catalytic unit of adenylate cyclase through the intervention of a GTP-regulatory protein (termed Ns) +. By a mechanism that remains unclear (due largely to a limited knowledge of the components of the system), GTP both decreases the affinity of hormones for their receptors and synergistically amplifies hormonal stimulation of activity. In general, the non-hydrolysable GTP analogue, Gpp(NH)p, promotes the latter action more effectively than the native compound. These and allied findings have led to the development of a general hypothesis, which proposes that hormone binding to receptors leads to the release of previously bound GDP (an ineffective stimulator), which allows occupancy by GTP and thus attainment of a more active R s • N s • C complex. On hydrolysis of GTP to GDP, the complex reverts to an inactive form, which coincides with the release of hormone [1-3]. Considerable gaps exist in our knowledge of stimulatory adenylate cyclase systems. Quantitative infor-

Adenylate cyclases: critical foci in neuronal signaling

Current findings show that adenylate cyclases comprise a heterogeneous multigene family, members of which are variously regulated by the alpha and beta gamma subunits of G proteins, by Ca2+ and by protein kinases. In the CNS, individual isoforms of adenylate cyclase are expressed discretely in select regions of the brain. At the subcellular level, adenylate cyclases can be concentrated into dendritic spines, thereby increasing their susceptibility to multiple regulatory influences. Altogether, such findings greatly expand knowledge of the potential role of this archetypical signaling system in the modulation of neuronal function.

Dependence of the Ca2+-inhibitable Adenylyl Cyclase of C6–2B Glioma Cells on Capacitative Ca2+ Entry

The ability of adenylyl cyclases to be regulated by physiological transitions in Ca2+ provides a key point for integration of cytosolic Ca2+ concentration ([Ca2+]i) and cAMP signaling. Ca2+-sensitive adenylyl cyclases, whether endogenously or heterologously expressed, require Ca2+ entry for their regulation, rather than Ca2+ release from intracellular stores (Chiono, M., Mahey, R., Tate, G., and Cooper, D. M. F. (1995) J. Biol. Chem. 270, 1149–1155; Fagan, K., Mahey, R., and Cooper, D. M. F. (1996) J. Biol. Chem. 271, 12438–12444). The present study compared the regulation by capacitative Ca2+ entry versusionophore-mediated Ca2+ entry of an endogenously expressed Ca2+-inhibitable adenylyl cyclase in C6–2B cells. Even in the face of a dramatic [Ca2+]i rise generated by ionophore, Ca2+ entry via capacitative Ca2+entry channels was solely responsible for the regulation of the adenylyl cyclase. Selective efficacy of BAPTA over equal concentrations of EGTA in blunting the regulation of the cyclase by capacitative Ca2+ entry defined the intimacy between the adenylyl cyclase and the capacitative Ca2+ entry sites. This association could not be impaired by disruption of the cytoskeleton by a variety of strategies. These results not only establish an intimate spatial relationship between an endogenously expressed Ca2+-inhibitable adenylyl cyclase with capacitative Ca2+ entry sites but also provide a physiological role for capacitative Ca2+ entry other than store refilling.

Inhibition by Calcium of Mammalian Adenylyl Cyclases

Ca2+ regulates mammalian adenylyl cyclases in a type-specific manner. Stimulatory regulation is moderately well understood. By contrast, even the concentration range over which Ca2+ inhibits adenylyl cyclases AC5 and AC6 is not unambiguously defined; even less so is the mechanism of inhibition. In the present study, we compared the regulation of Ca2+-stimulable and Ca2+-inhibitable adenylyl cyclases expressed in Sf9 cells with tissues that predominantly express these activities in the mouse brain. Soluble forms of AC5 containing either intact or truncated major cytosolic domains were also examined. All adenylyl cyclases, except AC2 and the soluble forms of AC5, displayed biphasic Ca2+ responses, suggesting the presence of two Ca2+ sites of high (∼0.2 μm) and low affinity (∼0.1 mm). With a high affinity, Ca2+ (i) stimulated AC1 and cerebellar adenylyl cyclases, (ii) inhibited AC6 and striatal adenylyl cyclase, and (iii) was without effect on AC2. With a low affinity, Ca2+inhibited all adenylyl cyclases, including AC1, AC2, AC6, and both soluble forms of AC5. The mechanism of both high and low affinity inhibition was revealed to be competition for a stimulatory Mg2+ site(s). A remarkable selectivity for Ca2+was displayed by the high affinity site, with a K i value of ∼0.2 μm, in the face of a 5000-fold excess of Mg2+. The present results show that high and low affinity inhibition by Ca2+ can be clearly distinguished and that the inhibition occurs type-specifically in discrete adenylyl cyclases. Distinction between these sites is essential, or quite spurious inferences may be drawn on the nature or location of high affinity binding sites in the Ca2+-inhibitable adenylyl cyclases.

Selective expression of one Ca2+-inhibitable adenylyl cyclase in dopaminergically innervated rat brain regions

Type I adenylyl cyclase, which can be stimulated by elevated cellular levels of Ca2+, has been proposed to provide a positive coincidence signal detection system, which can integrate signals arising via Gs- and Ca(2+)-mediated pathways. The occurrence of this adenylyl cyclase in brain regions implicated with associative learning in invertebrates and with the mammalian model of plasticity--hippocampal long-term potentiation, supports the notion that the ability of this species of adenylyl cyclase to detect two signals simultaneously may play a role in this neuronal function. In the present study, two recently cloned, closely-related adenylyl cyclases (Types V and VI), are shown to be inhibited by physiological elevation in [Ca2+]i. As a first step towards probing the neuronal significance of Ca(2+)-inhibitable adenylyl cyclases, their distribution was evaluated by in situ hybridization analysis of the rat brain. Strikingly distinct patterns of gene expression were found, ranging from a highly selective distribution of Type V mRNA within the striatum, nucleus accumbens and olfactory tubercle, to a weak and ubiquitous distribution of Type VI mRNA. Type V AC mRNA is expressed exclusively in medium-sized striatal neurons, which also express D1-dopaminergic (Gs-linked) and M1-muscarinic cholinergic (Ca(2+)-linked) receptors. Thus the adenylyl cyclase is primed for simultaneous detection of opposing regulatory influences. The utility of this novel mode of signal detection to dopaminergic function remains to be established.

Calmodulin-binding Sites on Adenylyl Cyclase Type VIII

Ca2+ stimulation of adenylyl cyclase type VIII (ACVIII) occurs through loosely bound calmodulin. However, where calmodulin binds in ACVIII and how the binding activates this cyclase have not yet been investigated. We have located two putative calmodulin-binding sites in ACVIII. One site is located at the N terminus as revealed by overlay assays; the other is located at the C terminus, as indicated by mutagenesis studies. Both of these calmodulin-binding sites were confirmed by synthetic peptide studies. The N-terminal site has the typical motif of a Ca2+-dependent calmodulin-binding domain, which is defined by a characteristic pattern of hydrophobic amino acids, basic and aromatic amino acids, and a tendency to form amphipathic α-helix structures. Functional, mutagenesis studies suggest that this binding makes a minor contribution to the Ca2+ stimulation of ACVIII activity, although it might be involved in calmodulin trapping by ACVIII. The primary structure of the C-terminal site resembles another calmodulin-binding motif, the so-called IQ motif, which is commonly Ca2+-independent. Mutagenesis and functional assays indicate that this latter site is a calcium-dependent calmodulin-binding site, which is largely responsible for the Ca2+ stimulation of ACVIII. Removal of this latter calmodulin-binding region from ACVIII results in a hyperactivated enzyme state and a loss of Ca2+ sensitivity. Thus, Ca2+/calmodulin regulation of ACVIII may be through a disinhibitory mechanism, as is the case for a number of other targets of Ca2+/calmodulin.

Adenosine as a Regulator of Adenylate Cyclase

The role of adenosine as a ‘local hormone’ has been firmly established in the past decade. Recently, several reviews have appeared on the numerous physiological effects of adenosine (Arch and Newsholme, 1978; Fox and Kelly, 1978) and on the various mechanisms of nucleoside metabolism, including phosphorylation, deamination, degradation (Arch and Newsholme, 1978; Fox and Kelly, 1978), and entry into the S-adenosyl homocysteine pathway (Usdin et al., 1979). Fain and Malbon (1979) have reviewed the evidence that links adenosine action at plasma membranes with cellular cyclic nucleotide metabolism. Burnstock (1979) has compared and contrasted the effects of adenosine and adenine nucleotides on various physiological systems. The conclusion derived from these reviews is that many, if not all, cells are subject to regulation by adenosine.

Ca2+/Calmodulin Distinguishes Between Guanyl-5′-yl-Imidodiphosphate-and Opiate-Mediated Inhibition of Rat Striatal Adenylate Cyclase

Abstract: The inhibition of adenylate cyclase from rat striatal plasma membranes by guanyl‐5′‐yl‐imidodiphos‐phate [Gpp(NH)p] and morphine was compared to determine whether Gpp(NH)p‐mediated inhibition accurately reflected hormone‐mediated inhibition in this system. Inhibition of adenylate cyclase activity by Gpp(NH)p and morphine was examined with respect to temperature, divalent cation concentration, and the presence of Ca2+/calmodulin (Ca2+/CaM). Gpp(NH)p‐mediated inhibition was dependent on the presence of Ca2+/CaM at 24°C; the inhibition was independent of Ca2+/CaM at 18°C; and inhibition could not be detected in the presence, or absence, of Ca2+/ CaM at 30°C. In contrast, naloxone‐reversible, morphine‐induced inhibition of adenylate cyclase was independent of both temperature and the presence of Ca2+/CaM. Mg2+dose‐response curves also reinforced the differences in the Ca2+/CaM requirement for Gpp(NH)p‐and morphine‐induced inhibition. Because Gpp(NH)p‐mediated inhibition was independent of Ca2+/CaM at low basal activities (i.e., 18°C, or below 1 mM Mg2+) and dependent on the presence of Ca2+/CaM at higher basal activities (24°C, or above 1 mM Mg2+), the inhibitory effects of Gpp(NH)p were examined at 1 mM Mg2+ in the presence of 100 nM forskolin. Under these conditions, both Gpp(NH)p‐and morphine‐induced inhibition of adenylate cyclase were independent of Ca2+/CaM. The results demonstrate that the requirement for Ca2+/CaM to observe Gpp(NH)p‐mediated inhibition depends on the basal activity of adenylate cyclase, whereas hormone‐mediated inhibition is Ca2+/CaM independent under all conditions. Further, this study shows that, under certain conditions, Gpp(NH)p‐mediated inhibition of adenylate cyclase does not mimic the inhibition of adenylate cyclase produced by a complete interaction between the inhibitory hormone receptor, the guanine nucleotide‐regulatory component that mediates inhibition, and the catalytic unit of the adenylate cyclase complex in striatal plasma membranes.