Functional characteristics of calcium-sensitive adenylyl cyclase of the infusorian <Emphasis Type="Italic">Tetrahymena pyriformis</Emphasis>

The calcium-sensitive forms of adenylyl cyclases (AC) have been revealed in the majority of vertebrate and invertebrate animals, as well as in several representatives of unicellular organisms, including infusoria. We have found for the first time that the AC activity in the infusorian Tetrahymena pyriformis changes in the presence of calcium ions. Calcium ions at concentrations of 0.2–20 μM stimulated the activity of this enzyme, with the maximum of the stimulatory effect being observed at 2 μM Ca²⁺. At a concentration of 100 μM and higher, the calcium cations inhibited the AC activity. Antagonists of calmodulin W-5 and W-7 at concentrations of 20–100 μM decreased the stimulatory effect of 5 μM Ca²⁺, while at the higher concentrations inhibited it completely. Another calmodulin antagonist, chloropromazine, decreased the Ca²⁺-stimulated AC activity only at concentrations of 200–1000 μM. The stimulatory effect of serotonin, EGF, and cAMP on AC activity was enhanced in the presence of 5 μM Ca²⁺. The stimulatory effect of EGF, cAMP, and insulin on AC was decreased in the presence of 100 μM Ca²⁺, while the effect of cAMP was also observed in the presence of calmodulin antagonists (500 μM). At the same time, stimulatory effect of D-glucose did not change in the presence of Ca²⁺ and calmodulin antagonists. The obtained data indicate that, in the infusorian T. pyriformis, there are calcium-sensitive forms of AC that can be stimulated by EGF, cAMP, insulin, and serotonin.     

Regulation of multiple forms of cyclic nucleotide phosphodiesterase from bovine hypothalamus: New factors modulating enzyme activity

Studies of bovine hypothalamic cyclic nucleotide phosphodiesterase (PDE) indicate the presence of several peaks of PDE activity, distinguishable by DEAE-cellulose column chromatography, displaying different substrate specificities, kinetic behavior, and regulatory properties. Evidence is presented that chromatographically separated forms of PDE activity are subject to control by Ca²⁺-calmodulin, cyclic nucleotides, limited proteolysis, reagents affecting sulfhydryl groups, and neurohormone “C”—one of several new cardioactive compounds isolated from hypothalamic magnocellular nuclei of animals—in a complex substrate-specific and concentration-dependent manner. Of particular interest is the finding that each of the forms of cGMP PDE, being Ca²⁺/calmodulin-dependent, possesses sensitivity to activation by cAMP, especially under conditions favoring the oxidation of thiol groups of PDE, resulting in a loss in responsiveness of the enzyme to the activation by calmodulin. This effect appears to be relatively stable but readily reversible by sulfhydryl reducing reagents, which restore both the cGMP PDE sensitivity to competitive inhibition by cAMP and the responsiveness of the enzyme to activation by calmodulin. A reinterpretation of the regulatory properties of multiple forms of PDE is proposed. Special Issue dedicated to Dr. Eugene Kreps.     

Characterization of the cAMP phosphodiesterase domain in plant adenylyl cyclase/cAMP phosphodiesterase CAPE from the liverwort Marchantia polymorpha

Cyclic AMP (cAMP) acts as a second messenger and is involved in the regulation of various physiological responses. Recently, we identified the cAMP-synthesis/hydrolysis enzyme CAPE, which contains the two catalytic domains adenylyl cyclase (AC) and cAMP phosphodiesterase (PDE) from the liverwort Marchantia polymorpha. Here we characterize the PDE domain of M. polymorpha CAPE (MpCAPE-PDE) using the purified protein expressed in E. coli. The K_m and V_max of MpCAPE-PDE were 30 µM and 5.8 nmol min^?1 mg^?1, respectively. Further, we investigated the effect of divalent cations on PDE activity and found that Ca²⁺ enhanced PDE activity, suggesting that Ca²⁺ may be involved in cAMP signaling through the regulation of PDE activity of CAPE. Among the PDE inhibitors tested, only dipyridamole moderately inhibited PDE activity by approximately 40% at high concentrations. Conversely, 3-isobutyl-1-methylxanthine (IBMX) did not inhibit PDE activity.

     

Modulation of dihydropyridine‐sensitive calcium channels in drosophila by a cAMP‐mediated pathway

Drosophila has proved to be a valuable system for studying the structure and function of ion channels. However, relatively little is known about the regulation of ion channels, particularly that of Ca²⁺ channels, in Drosophila. Physiological and pharmacological differences between invertebrate and mammalian L‐type Ca²⁺ channels raise questions on the extent of conservation of Ca²⁺ channel modulatory pathways. We have examined the role of cyclic adenosine monophosphate (cAMP) cascade in modulating the dihydropyridine (DHP)‐sensitive Ca²⁺ channels in the larval muscles of Drosophila, using mutations and drugs that disrupt specific steps in this pathway. The L‐type (DHP‐sensitive) Ca²⁺ channel current was increased in the dunce mutants, which have high cAMP concentration owing to cAMP‐specific phosphodiesterase (PDE) disruption. The current was decreased in the rutabaga mutants, where adenylyl cyclase (AC) activity is altered thereby decreasing the cAMP concentration. The dunce effect was mimicked by 8‐Br‐cAMP, a cAMP analog, and IBMX, a PDE inhibitor. The rutabaga effect was rescued by forskolin, an AC activator. H‐89, an inhibitor of protein kinase‐A (PKA), reduced the current and inhibited the effect of 8‐Br‐cAMP. The data suggest modulation of L‐type Ca²⁺ channels of Drosophila via a cAMP‐PKA mediated pathway. While there are differences in L‐type channels, as well as in components of cAMP cascade, between Drosophila and vertebrates, main features of the modulatory pathway have been conserved. The data also raise questions on the likely role of DHP‐sensitive Ca²⁺ channel modulation in synaptic plasticity, and learning and memory, processes disrupted by the dnc and the rut mutations. © 1999 John Wiley & Sons, Inc. J Neurobiol 39: 491–500, 1999     

Effect of lipid peroxidation conditions on calcium-dependent activity of phosphodiesterase 3′,5′-cAMP in the rat brain

3′,5′-cAMP plays an important role as a second messenger molecule controlling multiple cellular processes in the brain. Its levels are decreased by phosphodiesterases (PDEs), responsible for hydrolysis of intracellular cAMP. A part of the PDE activity is dependent on the effect of calcium, mediated by its binding to calmodulin. During oxidative stress, precisely these changes in calcium concentration are responsible for cell damage. We have examined the effects of oxidative stress conditions on the activity of PDE in rat brain homogenates. We found a different influence of activated lipid peroxidation conditions (Fe²⁺ with ascorbate and increased temperature) on the calcium-dependent and calcium-independent PDE activity. The inhibition of Ca²⁺-dependent PDE was observed, while Ca²⁺-independent PDE was not influenced. We assume that it might be the impact of lipid peroxidation products or any mechanism activated by the higher temperature on the interaction of the Ca²⁺-dependent isoform of PDE with the complex calcium-calmodulin. Another explanation might be that the formation of the functioning calcium-calmodulin complex is impossible in these conditions.     

Regulation of cAMP dynamics by Ca2+ and G protein-coupled receptors in the pancreatic -cell: a computational approach

In this report we describe a mathematical model for the regulation of cAMP dynamics in pancreatic β-cells. Incretin hormones such as glucagon-like peptide 1 (GLP-1) increase cAMP and augment insulin secretion in pancreatic β-cells. Imaging experiments performed in MIN6 insulinoma cells expressing a genetically encoded cAMP biosensor and loaded with fura-2, a calcium indicator, showed that cAMP oscillations are differentially regulated by periodic changes in membrane potential and GLP-1. We modeled the interplay of intracellular calcium (Ca²⁺) and its interaction with calmodulin, G protein-coupled receptor activation, adenylyl cyclases (AC), and phosphodiesterases (PDE). Simulations with the model demonstrate that cAMP oscillations are coupled to cytoplasmic Ca²⁺ oscillations in the β-cell. Slow Ca²⁺ oscillations (<1 min^–1) produce low-frequency cAMP oscillations, and faster Ca²⁺ oscillations (>3–4 min^–1) entrain high-frequency, low-amplitude cAMP oscillations. The model predicts that GLP-1 receptor agonists induce cAMP oscillations in phase with cytoplasmic Ca²⁺ oscillations. In contrast, observed antiphasic Ca²⁺ and cAMP oscillations can be simulated following combined glucose and tetraethylammonium-induced changes in membrane potential. The model provides additional evidence for a pivotal role for Ca²⁺-dependent AC and PDE activation in coupling of Ca²⁺ and cAMP signals. Our results reveal important differences in the effects of glucose/TEA and GLP-1 on cAMP dynamics in MIN6 β-cells. adenylyl cyclase; calcium ion; glucagon-like peptide 1; modeling; oscillations     

Effect of streptozotocin-induced diabetes on phosphodiesterase activity in rat luteal cells

The present studies were carried out to characterize the cAMP-phosphodiesterase enzyme (PDE) in luteal cells recovered from pseudopregnant rats with streptozotocin-induced diabetes. A significant increase in the specific activity of the enzyme was detected in luteal cells from diabetic rats (Group D) with respect to control rats (Group C). This increase could not be prevented by insulin therapy (Group I). Luteal cells from Groups C and D rats responded in vitro to insulin by increasing their PDE activity (% of stimulus of specific activity: C = 75%, D = 110%). However, in cells isolated from Group I, the hormone caused an inhibition of PDE activity (% of inhibition of specific activity: 48%). When cytosolic fractions from Groups C, D, and I were submitted to ion exchange chromatography, two PDE activity peaks could be observed and the activity of the different fractions was increased in the presence of Ca²⁺ and calmodulin. Nevertheless, the Ca²⁺—calmodulin effect was much lower in the extracts from Groups D and I than for controls. Kinetic studies of luteal PDE showed nonlinear Lineweaver-Burk graphs with two apparent ATP hydrolysis sites. Similar K_m values were found for PDE from groups C, D, and I, whereas the V_max2 for the enzyme was higher in Groups D and I. The endogenous concentration of cAMP, measured by RIA, showed no significant differences among Groups C, D, and I. On the basis of these results, we conclude that the specific activity of PDE is significantly increased in luteal cells from streptozotocin-induced diabetic animals, which could explain the previously described reduction in LH-stimulated progesterone production by luteal cells in diabetic rats. © 1996 Wiley-Liss, Inc.     

Light-Induced Pigment Aggregation in Xanthophores of the Medaka,Oryzias latipes

The response mechanism of medaka xanthophores to light was examined at the cellular level. Innervated and denervated xanthophores of adult medakas responded to light (9,000 lux) within 30 sec by pigment aggregation, and this aggregation was not mediated through α-adrenoceptors on the cell membrane. Maximum sensitivity to light was at wavelengths of 410–420 nm, and the direct effect of light was reversible. Xanthophore responsiveness to light in summer was higher than that in winter. Ca²⁺ and calmodulin were not involved in the response, but rather, an important role for cAMP and phos-phodiesterase (PDE) was suggested. It seems likely that photoreception by visual pigment which is sensitive to light at wavelengths of 410–420 nm increases PDE activity, probably via a G-protein, such as occurs with visual cells in the retina, which causes a decrease in levels of cytosolic cAMP, in turn leading to pigment aggregation within medaka xanthophores.     

Isoforms of cyclic nucleotide phosphodiesterase PDE3 and their contribution to cAMP hydrolytic activity in subcellular fractions of human myocardium

Three isoforms of PDE3 (cGMP-inhibited) cyclic nucleotide phosphodiesterase regulate cAMP content in different intracellular compartments of cardiac myocytes in response to different signals. We characterized the catalytic activity and inhibitor sensitivity of these isoforms by using recombinant proteins. We determined their contribution to cAMP hydrolysis in cytosolic and microsomal fractions of human myocardium at 0.1 and 1.0 microm cAMP in the absence and presence of Ca(2+)/calmodulin. We examined the effects of cGMP on cAMP hydrolysis in these fractions. PDE3A-136, PDE3A-118, and PDE3A-94 have similar K(m) and k(cat) values for cAMP and are equal in their sensitivities to inhibition by cGMP and cilostazol. In microsomes, PDE3A-136, PDE3A-118, and PDE3A-94 comprise the majority of cAMP hydrolytic activity under all conditions. In cytosolic fractions, PDE3A-118 and PDE3A-94 comprise >50% of the cAMP hydrolytic activity at 0.1 microm cAMP, in the absence of Ca(2+)/calmodulin. At 1.0 microm cAMP, in the presence of Ca(2+)/calmodulin, activation of Ca(2+)/calmodulin-activated (PDE1) and other non-PDE3 phosphodiesterases reduces their contribution to <20% of cAMP hydrolytic activity. cGMP inhibits cAMP hydrolysis in microsomal fractions by inhibiting PDE3 and in cytosolic fractions by inhibiting both PDE3 and PDE1. These findings indicate that the contribution of PDE3 isoforms to the regulation of cAMP hydrolysis in intracellular compartments of human myocardium and the effects of PDE3 inhibition on cAMP hydrolysis in these compartments are highly dependent on intracellular [Ca(2+)] and [cAMP], which are lower in failing hearts than in normal hearts. cGMP may amplify cAMP-mediated signaling in intracellular compartments of human myocardium by PDE3-dependent and PDE3-independent mechanisms.     

Role of calcium in adult onset polycystic kidney disease

Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in genes encoding the polycystin (PC) 1 and 2 proteins. The goal of this study was to determine the role of calcium in regulating cyst growth. Stromal interaction molecule 1 (STIM1) protein expression was 15-fold higher in PC1-null proximal tubule cells (PN) than in heterozygote (PH) controls and 2-fold higher in an inducible, PC1 knockout, mouse model of ADPKD compared to a non-cystic match control. IP3 receptor protein expression was also higher in the cystic mice. Knocking down STIM1 with siRNA reduced cyst growth and lowered cAMP levels in PN cells. Fura2 measurements of intracellular Ca²⁺ showed higher levels of intracellular Ca²⁺, SOCE and thaspigargin-stimulated ER Ca²⁺ release in PN vs. PH cells. There was a dramatic reduction in thapsigargin-stimulated release of ER Ca²⁺ following STIM1 silencing or application of 2-APB, consistent with altered ER Ca²⁺ movement; the protein expression of the Ca²⁺-dependent adenylyl cyclases (AC) AC3 and AC6 was up- and down-regulated, respectively. Like STIM1 knockdown, application of the calmodulin inhibitor W7 lowered cAMP levels, further indicating that STIM1 regulates AC3 via Ca²⁺ We conclude that the high levels of STIM1 in ADPKD cells play a role in supporting cyst growth and promoting high cAMP levels and an increased release of Ca²⁺ from the ER. Thus, our results provide novel therapeutic targets for treating ADPKD.     

Cell type specific inhibition of cAMP phosphodiesterase activity during terminal differential in Dictyostelium discoideum

Cyclic AMP phosphodiesterase (PDE) activity reaches a peak during the aggregation stage of development where it functions to regulate extracellular levels of cAMP. During the subsequent differentiation of the two cell types at the culmination stage, the activity reappears but only in stalk cells. We found that extracts from the culmination stage contained PDE which could be activated by preincubation with Mg2+ and dithiothreitol (DTT), a treatment which is known to release an endogenous inhibitor from the aggregation stage enzyme. When the culmination stage extracts were subjected to chromatography on Biogel P300, two peaks of activity were eluted, PDE-I (Mr greater than 260,000) and PDE-II (Mr 100,000). Treatment of the fractions with Mg-DTT did not affect the low-molecular-weight enzyme but caused activation of the high-molecular-weight enzyme and the appearance of a third, intermediate form. Kinetic analysis of the two peaks revealed Km values for cAMP of 2 mM and 10 microM for PDE-I and PDE-II, respectively. We tested the possibility that these forms of the enzyme might be distributed differently in the two cell types by measuring the Km for cAMP and the effect of Mg-DTT treatment on isolated sections of stalk and spore cells. The spore sections contained a high Km form of the enzyme (0.3 mM) which was activated by preincubation with Mg . DTT whereas stalk sections contained a low Km form (3 microM) which was not affected by the activation treatment. We conclude that both cell types contain enzyme protein and that the apparent localization of PDE activity in stalk cells is due to the inhibition of activity in spore cells.     

Roles of Calcium/Calmodulin-Dependent Kinase II in Long-Term Memory Formation in Crickets

Ca²⁺/calmodulin (CaM)-dependent protein kinase II (CaMKII) is a key molecule in many systems of learning and memory in vertebrates, but roles of CaMKII in invertebrates have not been characterized in detail. We have suggested that serial activation of NO/cGMP signaling, cyclic nucleotide-gated channel, Ca²⁺/CaM and cAMP signaling participates in long-term memory (LTM) formation in olfactory conditioning in crickets, and here we show participation of CaMKII in LTM formation and propose its site of action in the biochemical cascades. Crickets subjected to 3-trial conditioning to associate an odor with reward exhibited memory that lasts for a few days, which is characterized as protein synthesis-dependent LTM. In contrast, animals subjected to 1-trial conditioning exhibited memory that lasts for only several hours (mid-term memory, MTM). Injection of a CaMKII inhibitor prior to 3-trial conditioning impaired 1-day memory retention but not 1-hour memory retention, suggesting that CaMKII participates in LTM formation but not in MTM formation. Animals injected with a cGMP analogue, calcium ionophore or cAMP analogue prior to 1-trial conditioning exhibited 1-day retention, and co-injection of a CaMKII inhibitor impaired induction of LTM by the cGMP analogue or that by the calcium ionophore but not that by the cAMP analogue, suggesting that CaMKII is downstream of cGMP production and Ca²⁺ influx and upstream of cAMP production in biochemical cascades for LTM formation. Animals injected with an adenylyl cyclase (AC) activator prior to 1-trial conditioning exhibited 1-day retention. Interestingly, a CaMKII inhibitor impaired LTM induction by the AC activator, although AC is expected to be a downstream target of CaMKII. The results suggest that CaMKII interacts with AC to facilitate cAMP production for LTM formation. We propose that CaMKII serves as a key molecule for interplay between Ca²⁺ signaling and cAMP signaling for LTM formation, a new role of CaMKII in learning and memory.     

cAMP controls rod photoreceptor sensitivity via multiple targets in the phototransduction cascade

In early studies, both cyclic AMP (cAMP) and cGMP were considered as potential secondary messengers regulating the conductivity of the vertebrate photoreceptor plasma membrane. Later discovery of the cGMP specificity of cyclic nucleotide–gated channels has shifted attention to cGMP as the only secondary messenger in the phototransduction cascade, and cAMP is not considered in modern schemes of phototransduction. Here, we report evidence that cAMP may also be involved in regulation of the phototransduction cascade. Using a suction pipette technique, we recorded light responses of isolated solitary rods from the frog retina in normal solution and in the medium containing 2 µM of adenylate cyclase activator forskolin. Under forskolin action, flash sensitivity rose more than twofold because of a retarded photoresponse turn-off. The same concentration of forskolin lead to a 2.5-fold increase in the rod outer segment cAMP, which is close to earlier reported natural day/night cAMP variations. Detailed analysis of cAMP action on the phototransduction cascade suggests that several targets are affected by cAMP increase: (a) basal dark phosphodiesterase (PDE) activity decreases; (b) at the same intensity of light background, steady background-induced PDE activity increases; (c) at light backgrounds, guanylate cyclase activity at a given fraction of open channels is reduced; and (d) the magnitude of the Ca²⁺ exchanger current rises 1.6-fold, which would correspond to a 1.6-fold elevation of [Ca²⁺]_in. Analysis by a complete model of rod phototransduction suggests that an increase of [Ca²⁺]_in might also explain effects (b) and (c). The mechanism(s) by which cAMP could regulate [Ca²⁺]_in and PDE basal activity is unclear. We suggest that these regulations may have adaptive significance and improve the performance of the visual system when it switches between day and night light conditions.     

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.     

Purification and characterization of calmodulin from porcine renal medulla

A calcium sensitive phosphodiesterase (PDE) activated by an endogenous calmodulin was identified in the cytosolic fraction of porcine renal medulla. The PDE and calmodulin were separated from each other by DEAE-cellulose column chromatography. Calmodulin was purified from a heat-treated supernatant by column chromatography with DEAE-cellulose and hydroxylapatite. The purified renal calmodulin has a molecular weight of 17,500, is heatstable, and has a pI of 4.2. Activation of the renal PDE by calmodulin was immediate and stoichiometric. The renal calmodulin and PDE cross react with bovine brain calmodulin and PDE, indicating a lack of tissue and species specificity. Thus, renal calmodulin is very similar to bovine brain calmodulin. However, renal calmodulin did not affect detergent-solubilized or membrane-bound renal adenylate cyclase or the antidiuretic hormone-stimulated activity of the enzyme. These results suggest that calmodulin may function in the renal medulla to regulate cAMP levels by stimulation of PDE but not adenylate cyclase. However, the ubiquitous distribution of calmodulin in eukaryotic cells and its effects on a number of other enzymes allow the possibility that calmodulin may have a role in renal function other than cAMP metabolism.     

Effects of 39 Compounds on Calmodulin-Regulated Adenylyl Cyclases AC1 and Bacillus anthracis Edema Factor

Adenylyl cyclases (ACs) catalyze the conversion of ATP into the second messenger cAMP. Membranous AC1 (AC1) is involved in processes of memory and learning and in muscle pain. The AC toxin edema factor (EF) of Bacillus anthracis is involved in the development of anthrax. Both ACs are stimulated by the eukaryotic Ca²⁺-sensor calmodulin (CaM). The CaM-AC interaction could constitute a potential target to enhance or impair the AC activity of AC1 and EF to intervene in above (patho)physiological mechanisms. Thus, we analyzed the impact of 39 compounds including typical CaM-inhibitors, an anticonvulsant, an anticholinergic, antidepressants, antipsychotics and Ca²⁺-antagonists on CaM-stimulated catalytic activity of AC1 and EF. Compounds were tested at 10 μM, i.e., a concentration that can be reached therapeutically for certain antidepressants and antipsychotics. Calmidazolium chloride decreased CaM-stimulated AC1 activity moderately by about 30%. In contrast, CaM-stimulated EF activity was abrogated by calmidazolium chloride and additionally decreased by chlorpromazine, felodipine, penfluridol and trifluoperazine by about 20–40%. The activity of both ACs was decreased by calmidazolium chloride in the presence and absence of CaM. Thus, CaM-stimulated AC1 activity is more insensitive to inhibition by small molecules than CaM-stimulated EF activity. Inhibition of AC1 and EF by calmidazolium chloride is largely mediated via a CaM-independent allosteric mechanism.     

EP2 receptor mediated cAMP release is augmented by PGF2α activation of the FP receptor via the calcium-calmodulin pathway

Prostaglandins exert their effects on target cells by coupling to specific G protein-coupled receptors (GPCRs) that are often co-expressed in the same cells and use alternate and in some cases opposing intracellular signaling pathways. This study investigated the cross-talk that influences intracellular signaling and gene expression profiling in response to co-activation of the EP2 and FP prostanoid receptors in Ishikawa cells stably expressing both receptors (FPEP2 cells). In this study we show that in FPEP2 cells, PGF alone does not alter adenosine 3′,5′-cyclic monophosphate (cAMP) production, but in combination with Butaprost enhances EP2 receptor mediated cAMP release compared to treatment with Butaprost alone. PGF-mediated potentiation of cAMP release was abolished by antagonism of the FP receptor, inhibition of phospholipase C (PLC) and inositol phosphate receptor (IP3R) whereas inhibition of protein kinase C (PKC) had no effect. Moreover, inhibition of calcium effectors using calmodulin antagonist (W7) or Ca²⁺/calmodulin-dependent kinase II (CaMK-II) inhibitor (KN-93) abolished PGF potentiation of Butaprost-mediated cAMP release. Using siRNA molecules targeted against the adenylyl cyclase 3 (AC3) isoform, we show that AC3 is responsible for the cross-talk between the FP and EP2 receptors. Using gene array studies we have identified a candidate gene, Spermidine/N1-acetyltransferase (SAT1), which is regulated by this cAMP mediated cross-talk. In conclusion, this study demonstrates that co-activation of the FP and EP2 receptors results in enhanced release of cAMP via FP receptor-Gα_q-Ca²⁺-calmodulin pathway by activating calcium sensitive AC3 isoform.     

Signal tranduction: regulation of cAMP concentration in cardiac muscle by calmodulin-dependent cyclic nucleotide phosphodiesterase

The bovine heart calmodulin-dependent phosphodiesterase can be phosphorylated by cAMP-dependent protein kinase, resulting in a decrease in the enzyme's affinity for calmodulin. The phosphorylation of calmodulin-dependent phosphodiesterase is blocked by Ca²⁺ and calmodulin and reversed by the calmodulin-dependent phosphatase. The dephosphorylation is accompanied by an increase in the affinity of the phosphodiesterase for calmodulin. The CaM-dependent phosphodiesterase isozymes of heart and brain are regulated by calmodulin, but the affinity for calmodulin are different. Furthermore, the bovine heart CaM-dependent phosphodiesterase isozyme in stimulated at much lower Ca²⁺ concentration than the bovine brain isozymes. Results from this study suggest that the activity of this phosphodiesterase is precisely regulated by cross-talk between Ca²⁺ and cAMP signalling pathways.     

Defective regulation of apical membrane chloride transport and exocytosis in cystic fibrosis

A biochemical link is proposed between recent observations on defective regulation of Cl^– transport in CF respiratory epithelial cells and studies showing altered biological activity of calmodulin in exocrine glands from CF patients. A consensus is emerging that defective -adrenergic secretory responsiveness in CF cells is caused by a defect in a regulator protein at a site distal to cyclic AMP formation. Our results indicate that this protein might be a specific calmodulin acceptor protein which modifies the activity of calmodulin in epithelial cells. Alteration in Ca²⁺/calmodulin dependent regulation of Cl^– transport and protein secretion could explain (i) alterations in Ca²⁺ homeostasis seen in CF, (ii) defective -adrenergic responses of CF cells, and (iii) the observed inability of cyclic AMP (acting via its specific protein kinase, A-kinase) to open apical membrane Cl^– channels in CF epithelial cells. Most of the physiological abnormalities of CF including elevated sweat electrolytes and hyperviscous mucus can be explained on this basis.Abbreviations -adrenergic agonist acting at its receptor cAMP cyclic AMP - PDE phosphodiesterase - CaM calmodulin - Pase phosphatase     

CFTR-Adenylyl Cyclase I Association Responsible for UTP Activation of CFTR in Well-Differentiated Primary Human Bronchial Cell Cultures

Chloride secretion by airway epithelial cells is defective in cystic fibrosis (CF). The conventional paradigm is that CFTR is activated through cAMP and protein kinase A (PKA), whereas the Ca²⁺-activated chloride channel (CaCC) is activated by Ca²⁺ agonists like UTP. We found that most chloride current elicited by Ca²⁺ agonists in primary cultures of human bronchial epithelial cells is mediated by CFTR by a mechanism involving Ca²⁺ activation of adenylyl cyclase I (AC1) and cAMP/PKA signaling. Use of selective inhibitors showed that Ca²⁺ agonists produced more chloride secretion from CFTR than from CaCC. CFTR-dependent chloride secretion was reduced by PKA inhibition and was absent in CF cell cultures. Ca²⁺ agonists produced cAMP elevation, which was blocked by adenylyl cyclase inhibition. AC1, a Ca²⁺/calmodulin-stimulated adenylyl cyclase, colocalized with CFTR in the cell apical membrane. RNAi knockdown of AC1 selectively reduced UTP-induced cAMP elevation and chloride secretion. These results, together with correlations between cAMP and chloride current, suggest that compartmentalized AC1–CFTR association is responsible for Ca²⁺/cAMP cross-talk. We further conclude that CFTR is the principal chloride secretory pathway in non-CF airways for both cAMP and Ca²⁺ agonists, providing a novel mechanism to link CFTR dysfunction to CF lung disease.