QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

HOME  |   SEARCH  |   ARCHIVE  |   SUBSCRIBE  |   CONTACT  |   HELP

This Article Abstract Full Text (PDF) An erratum has been published An erratum has been published Submit an eLetter Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (56) Citing Articles via Google Scholar Google Scholar Articles by Antoni, F. A. Articles by Paterson, J. M. Search for Related Content PubMed PubMed Citation Articles by Antoni, F. A. Articles by Paterson, J. M.

 Previous Article  |  Next Article 

The Journal of Neuroscience, December 1, 1998, 18(23):9650-9661

Ca²⁺/Calcineurin-Inhibited Adenylyl Cyclase, Highly Abundant in Forebrain Regions, Is Important for Learning and Memory

F. A. Antoni¹, M. Palkovits², J. Simpson¹, S. M. Smith¹, A. L. Leitch¹, R. Rosie¹, G. Fink¹, and J. M. Paterson¹

¹ Medial Research Council Brain Metabolism Unit, University of Edinburgh, Edinburgh, EH8 9JZ, Scotland, United Kingdom, and ² Section on Genetics, National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland 20892

	ABSTRACT

Top Abstract Introduction Materials & Methods Results Discussion References

Activation of cAMP synthesis by intracellular Ca²⁺ is thought to be the main mode of cAMP generation in the brain. Accordingly, the Ca²⁺-activated adenylyl cyclases I and VIII are expressed prominently in forebrain neurons. The present study shows that the novel adenylyl cyclase type IX is inhibited by Ca²⁺ and that this effect is blocked selectively by inhibitors of calcineurin such as FK506 and cyclosporin A. Moreover, adenylyl cyclase IX is inhibited by the same range of intracellular free Ca²⁺ concentrations that stimulate adenylyl cyclase I. Adenylyl cyclase IX is expressed prominently in the forebrain. Substantial arrays of neurons positive for AC9 mRNA were found in the olfactory lobe, in limbic and neocortical areas, in the striatum, and in the cerebellar system. These data show that the initiation of the cAMP signal by adenylyl cyclase may be controlled by Ca²⁺/calcineurin and thus provide evidence for a novel mode of tuning the cAMP signal by protein phosphorylation/dephosphorylation cascades.

Key words: cAMP; protein phosphatase; hippocampus; neocortex; striatum; adenylyl cyclase I; calcium

	INTRODUCTION

Top Abstract Introduction Materials & Methods Results Discussion References

Molecular cloning of mammalian adenylyl cyclase (Krupinski et al., 1989) has led to the discovery of nine different isotypes. These can be classified broadly into Ca²⁺-stimulated cyclases, Ca²⁺-inhibited cyclases, and protein kinase C-activated cyclases on the basis of their distinct regulation by intracellular Ca²⁺ and protein phosphorylation (for review, see Sunahara et al., 1996; Antoni, 1997). Current evidence indicates that all three classes of adenylyl cyclase are expressed in the mammalian brain (for review, see Mons and Cooper, 1995; Antoni et al., 1998).

The Ca²⁺-stimulated enzymes adenylyl cyclase I (AC1) and VIII (AC8) have been assigned important roles in synaptic plasticity (Xia and Storm, 1997). In terms of neurotransmitter function, AC1 (Wayman et al., 1994) and AC8 (Cali et al., 1994) are both activated by Ca²⁺/calmodulin. Moreover, AC1 may generate cAMP as a coincidence detector for concerted signals from G_s-coupled receptors and ionotropic receptors that trigger changes in the membrane potential and an increase of intracellular free Ca²⁺.

Adenylyl cyclases II (AC2) and VII (AC7) are activated by protein kinase C (see summary by Ishikawa, 1998). Further, AC2 is also a coincidence detector stimulated by G subunits in the context of activation by G_s (Tang and Gilman, 1991; Chen et al., 1995). AC2 mRNA is abundant in the brain, including the neocortex and the limbic lobe (Mons and Cooper, 1995). AC7 has a more restricted distribution in the brain (Mons and Cooper, 1995), and its functional properties have not been analyzed in detail.

With respect to Ca²⁺-inhibited adenylyl cyclases, only the striatum and the mesolimbic dopaminergic system have been reported to express significant levels of AC5 (Glatt and Snyder, 1993; Mons and Cooper, 1995). Low levels of AC3 (Xia et al., 1992) and AC6 (Mons and Cooper, 1995) mRNA have been reported in whole brain.

By far, the most abundant cerebral adenylyl cyclase at the mRNA level appears to be AC9 (Paterson et al., 1995; Premont et al., 1996). The properties of AC9 are controversial, because studies from this laboratory (for review, see Antoni et al., 1998) suggested that receptor-induced synthesis of cAMP by AC9 is inhibited by calcineurin (protein phosphatase 2B). Others (Premont et al., 1996) have reported no effect of Ca²⁺ on AC9 overexpressed in Sf9 insect cells. Given the potential functional significance of a calcineurin-regulated adenylyl cyclase in the brain, we have analyzed the properties of AC9 overexpressed in human embryonic kidney (HEK-293) cells and compared the Ca²⁺ sensitivity of AC9 with that of AC1 in this system. Further, we have investigated the distribution of AC9 in mouse and rat forebrain.

The results showed that AC1 and AC9 are regulated reciprocally by intracellular free Ca²⁺. Moreover, the inhibition of AC9 by Ca²⁺ was blocked by the calcineurin inhibitors FK506 and cyclosporin A. Expression of AC9 mRNA was confined to the gray matter and appeared mainly neuronal. The limbic lobe, the neocortex, and the cerebellum all expressed AC9 mRNA. Expression of AC9 mRNA and protein was highest in the hippocampus. In sum, these studies reveal a novel mode of cAMP signaling in the brain in which the initiation of the cAMP signal is controlled by Ca²⁺/calcineurin.

	MATERIALS AND METHODS

Top Abstract Introduction Materials & Methods Results Discussion References

Animals. Adult (6-8 weeks old) male BALB/c mice and Wistar rats were housed under constant temperature and lighting (on, 5:00 A.M.; off, 7:00 P.M.) and had free access to pelleted food and tap water.

In situ hybridization histochemistry. Animals were decapitated, and the brains were removed rapidly from the skull and processed as previously described (Rosie et al., 1992; Paterson et al., 1995). ³⁵S-labeled riboprobes for AC9 were prepared by transcribing segments of the mouse (plasmid JP142) (Paterson et al., 1995) or rat AC9 cDNA (nucleotides 1107-1450) (J. M. Paterson, unpublished data) with the requisite RNA polymerases and used as previously described (Rosie et al., 1992; Paterson et al., 1995). Brain sections were exposed to x-ray film for 3-6 d or dipped in Kodak NTB-2 or Ilford K5 emulsion and exposed for 2-6 weeks. Cell nuclei were counterstained with pyronin. Measurements of optical densities and grain counts were performed according to published procedures (Rosie et al., 1992).

Immunodetection of AC9 protein. Antisera against the synthetic pentadecapeptide KQLSSNTHPKHCKYS (Severn Biotech, Kidderminster, UK) at the N-terminal region of AC9 were raised in rabbits against the synthetic peptide coupled to purified protein derivative (Lachmann et al., 1986). A similarly prepared Tyr-peptide-PPD conjugate derived from YEASELSKLNVSKSV at the C terminus of the protein also was used to immunize BCG-primed hens (courtesy of Professor Peter Sharp, The Roslin Institute, Edinburgh, Scotland).

For radioimmunoassay and immunoblot analysis the rats were decapitated, and brain regions were dissected rapidly while the brain was cooled on a glass plate on wet ice. The hippocampus, the cerebellum, the hypothalamus, and the adenohypophysis were homogenized in 50 mM Tris-HCl, 5 µg/ml leupeptin, 7 µg/ml pepstatin, 1 mM EGTA, 20 µl/ml Trasylol (Bayer, Wuppertal, Germany), and 1 mM MgSO₄, pH 7.4, at 4°C; crude membranes were prepared as previously described (Antoni et al., 1985). Similar membrane extracts were prepared from HEK-293 cell lines and mouse corticotrope tumor (AtT20) cells.

For immunoblots, membranes prepared from 10 mg of wet tissue or 10⁷ cells were resuspended in SDS-PAGE sample buffer (50 mM Tris, pH 7.2, 2% w/v SDS, and 5% v/v mercaptoethanol). After being heated to 95°C for 5 min and centrifuged at 8000 × g for 2 min, aliquots of the supernatant were separated by SDS-PAGE on 7.5% homogenous microgels. Electroblotting was performed on the same apparatus (Pharmacia PhastSystem, Uppsala, Sweden), and the blots were probed with rabbit anti-AC9 antiserum at 1:4000 dilution in conjunction with an ECL kit (Amersham, Aylesbury, UK).

For radioimmunoassay the membranes prepared from 10 mg of wet tissue or 10⁷ cells were solubilized in 0.5 ml of 154 mM NaCl, 50 mM Tris-HCl, pH 7.4, 1% NP40 (v/v), 0.25% deoxycholate (w/v), 1 mM EGTA, 1 mM PMSF, 1 µg/ml leupeptin, 1 µg/ml pepstatin, and 1.5 µl/ml Trasylol (Bayer) (RIPA buffer). After centrifugation for 30 min at 16,000 × g, the supernatant was collected and the aliquots were assayed for immunoreactive AC9 content. The C-terminally directed chicken serum was used at 1:30,000 final dilution, with radioiodinated peptide antigen prepared by the chloramine T method as the tracer. Bound and free ligand were separated by the double antibody precipitation method, using donkey anti-chicken IgY serum (courtesy of SAPU). The detection limit of the assay was 2 fmol of C-terminal antigen peptide per tube.

Production of stably transfected HEK-293 cells. HEK-293 cells were maintained as described previously (Paterson et al., 1995). The cells were transfected by electroporation with 10 µg of DNA per milliliter of the expression plasmid pcDNA3 (Invitrogen, De Schelp, The Netherlands) or pcDNA3 containing the cDNA of bovine AC1 (courtesy of D. R. Storm) or mouse AC9 (Paterson et al., 1995). Clonal cell lines were selected in 0.6 mg/ml of G418 and subsequently were propagated without antibiotic. Five different AC9-expressing clones that produced similar levels of cAMP were tested for the effects of Ca²⁺ on cAMP production, and all of these showed the inhibitory effect characterized in the present study. All four AC1-expressing clones that were tested showed stimulation by Ca²⁺; the cell line showing the highest cAMP response to Ca²⁺ was used in further experiments.

Assays of cAMP production. Incubations for the measurement of cAMP production were performed in cell suspensions as previously described (Paterson et al., 1995), with modifications as detailed below.

HEK-293 cells display complex intracellular Ca²⁺ handling mechanisms that may be controlled by cAMP and calcineurin (Querfurth and Selkoe, 1994; Lin et al., 1995; Seuwen and Boddeke, 1995). Therefore, intracellular stores of Ca²⁺ were depleted to reduce the contribution of intracellular Ca²⁺ pools to the intracellular free Ca²⁺ signal. All procedures were performed at 37°C; where required, the immunosuppressant drugs FK506 (courtesy of Fujisawa GmbH, Munich, Germany), cyclosporin A (courtesy of Novartis, UK), and L685,818 (courtesy of Merck, Rahway, NJ) or ethanol vehicle (0.1% v/v) were added to the medium at the onset of the Ca²⁺ depletion period. Two different Ca²⁺ depletion protocols were used.

In the first protocol the cells were incubated in a balanced salt solution containing (in mM) 133 NaCl, 5.4 KCl, 0.25 Na₂HPO₄, 0.44 KH₂PO₄, 1 MgSO₄, 2 EGTA, 5.6 D-glucose, 25 HEPES, pH 7.40, and 0.1% (w/v) bovine serum albumin (HBSS/EGTA) with 10 µM ryanodine and 1 µM thapsigargin (both from LC Labs, Boston, MA). These conditions deplete rapidly exchangeable Ca²⁺ pools and activate capacitative Ca²⁺ entry mechanisms (Cooper et al., 1994).

In the second protocol 4-Br-A23187 (5-15 µM) was used instead of ryanodine and thapsigargin and Ca/EGTA was added to obtain initial levels of [Ca²⁺]_i similar to those in the studies with ryanodine and thapsigargin. Under these conditions intracellular Ca²⁺ pools are depleted and, in addition to capacitative Ca²⁺ entry, Ca²⁺ influx may take place through the pores formed by the ionophore.

The cells were incubated in Ca²⁺-depleting medium for 20 min. Subsequently, HBSS/EGTA containing various amounts of CaCl₂ (Ca/EGTA) was added to achieve extracellular concentrations of Ca²⁺ ranging between 0.125 and 2 mM. The pH of this solution was 7.65 to minimize the change of pH caused by the displacement of protons from EGTAa pH shift from 7.40 to 7.29 occurred on achieving a free Ca²⁺ concentration of 2 mM. This protocol was modified when the effects of divalent cations were compared with those of Ca²⁺, because Ba²⁺ and Sr²⁺ potently displace Ca²⁺ from EGTA. Hence, cells were incubated for 20 min under Ca²⁺-depleting conditions and subsequently were pelleted by centrifugation at 200 × g for 5 min. The cells were resuspended in HBSS containing no added Ca²⁺, 0.2 mM EGTA, 10 µM ryanodine, and 1 µM thapsigargin. The additions of Ca²⁺ and other divalent cations were made assuming that 0.2 mM EGTA was present.

After Ca/EGTA was added, the cells were incubated for 5 min, after which the cyclic nucleotide phosphodiesterase (PDE) inhibitors isobutylmethylxanthine (IBMX) and rolipram, 1 and 0.1 mM, respectively, were introduced. Unless indicated otherwise, the cells were incubated for a further 10 min and then the incubation was terminated by the addition of 0.2 M HCl. The total cAMP content of the cells and the medium was determined after freeze-thawing and acetylation by radioimmunoassay (Antoni et al., 1995).

Membrane adenylyl cyclase assay. Crude membranes from HEK-293 cells were prepared as for immunodetection, except that 50 nM calyculin A also was included in the homogenization buffer to inhibit protein phosphatases. Membranes (6-10 µg of protein per tube) were incubated at 30°C in 50 mM Tris-HCl buffer, pH 7.1, containing (in mM) 0.3 ATP, 3 MgCl₂, 5 creatine phosphate, 1 EGTA, and 0.5 IBMX plus 0.5 mg/ml creatine phosphokinase, 0.25% (w/v) bovine serum albumin; the reaction was linear up to 20 min. cAMP content was measured by radioimmunoassay as above.

Estimation of intracellular free Ca²⁺. The measurement of intracellular free Ca²⁺ concentration ([Ca²⁺]_i) was performed in cells loaded with fura-2 AM (4 µM for 30 min at 37°C) in a Shimadzu model RF5000 spectrofluorometer. Approximately 3 × 10⁶ cells/ml were loaded into a cuvette; the solution was stirred and thermostatically heated to 37°C. The values for maximum and minimum fluorescence were obtained by recording the fluorescence intensities of a solution designed to mimic the intracellular environment (in mM): 25 Na-HEPES, pH 7.0, 10 NaCl, 120 KCl, 10 D-glucose, and 1 MgSO₄ at 340 and 380 nM excitation; the formula of Grynkiewicz et al. (Grynkiewicz et al., 1985) was applied. The K_D of fura-2 for Ca²⁺ was taken as 224 nM; maximum emission was determined at 4.1 mM CaCl₂, and minimum values were determined with the addition of 60 mM EGTA.

Data analysis. The production of cAMP is given as multiples of the cAMP content of those cells not treated with blockers of PDE; the range of these means was 35-80 fmol/well, depending on the cell line used and the passage number of the cells. Data were analyzed by one-way ANOVA, and Newman-Keuls test or Dunnett's test was used for multiple comparisons as appropriate.

	RESULTS

Top Abstract Introduction Materials & Methods Results Discussion References

Heterologous expression of AC9 in HEK-293 cells

Immunoreactive AC9 was undetectable in membrane extracts prepared from wild-type or pcDNA3-transfected HEK-293 cells (Fig. 1A). Cells stably transfected with AC9 cDNA contained a major ~164 kDa band on immunoblots, whereas in extracts of AtT20 cells, where the cyclase is expressed endogenously (Antoni et al., 1995), an immunoreactive band migrating at ~150 kDa was observed. The difference is plausibly attributable to cell-specific post-translational modifications such as glycosylation (Cali et al., 1994; Premont et al., 1996), phosphorylation (Kawabe et al., 1994; Antoni et al., 1998), or acylation (Mollner et al., 1995). Radioimmunoassay suggested that the stably transfected AC9 cell line expresses ~30-fold higher levels of AC9 protein than AtT20 cells (10.1 ± 0.9 vs 0.30 ± 0.02 pmol/mg protein; means ± SEM; n = 3) (Fig. 1B).

View larger version (16K): [in this window] [in a new window]   Figure 1.   Detection of AC9 protein by immunoblot and radioimmunoassay. A, Crude membrane fractions of AtT20 D16:16 mouse corticotrope tumor cells (AtT; 0.36 µg of protein/lane), wild-type HEK-293 cells (HEK; 0.36 µg of protein/lane), and HEK-293 cells stably transfected with AC9 protein (tHEK; 0.08 µg of protein/lane) were separated by SDS-PAGE on 7.5% homogenous microgels. Immunoblots were prepared and reacted (lanes marked with ) with rabbit antiserum 149 at 1:4000 directed against the N-terminal region of mouse AC9 protein or the same antiserum preabsorbed with the antigenic pentadecapeptide (1 µg/ml; lanes marked with +). Positions of molecular weight markers are shown on the left. B, Crude membrane fractions prepared from AtT20 D16:16 cells (filled squares), HEK-293 cells stably transfected with AC9 (open squares), and rat hippocampus (open triangles) were solubilized in RIPA buffer and analyzed by radioimmunoassay with a chicken antiserum directed against the C-terminal region of mouse AC9 and the antigen peptide (filled triangles) as a reference standard. The tracer was the radiolabeled antigen peptide; sample extracts were serially diluted twofold.

Selective monitoring of AC1- and AC9-derived cAMP

In wild-type or pcDNA3-transfected HEK-293 cells the PDE inhibitors caused no significant (p > 0.05 by one-way ANOVA) change of cAMP levels in the absence of receptor agonists (Fig. 2A), whereas forskolin and corticotropin-releasing factor markedly stimulated cAMP accumulation in these cells (Antoni et al., 1995, 1998).

View larger version (10K): [in this window] [in a new window]   Figure 2.   Effect of Ca2+ on cAMP production by HEK-293 cells stably transfected with adenylyl cyclase cDNAs. A, Time course of cAMP accumulation in cells transfected with AC9 (open triangles) or pcDNA3 vector alone (filled triangles). Cells were incubated under Ca2+-depleting conditions (2 mM EGTA and 5 µM 4Br-A23187). Blockers of phosphodiesterase (1 mM IBMX and 0.1 mM rolipram) were applied at time 0. Data are expressed as multiples of the amounts of cAMP found in cells not receiving PDE blockers at time 0. Means ± SEM; n = 4/group. B, Concentration-dependent inhibition of cAMP production by Ca2+ in cells stably transfected with AC9. HEK-293 cells overexpressing AC9 were incubated in medium containing no added calcium, 2 mM EGTA, 10 µM ryanodine, and 1 µM thapsigargin before the addition of various amounts of Ca/EGTA to the extracellular fluid. The abscissa shows the amount of free Ca2+ in the extracellular medium. Data are expressed as multiples of the amount of cAMP found in cells not receiving PDE blockers. Means ± SEM; n = 4/group. C, Concentration-dependent simulation of cAMP production by Ca2+ in cells stably transfected with AC1. Conditions are as in B except that HEK-293 cells overexpressing AC1 were used.

In contrast, large increases of cAMP were elicited by PDE inhibitors in cells transfected with AC9 (Fig. 2A) or AC1 (data not shown). The addition of forskolin or corticotropin-releasing factor with blockers of PDE produced further increases above the levels observed in the presence of PDE blockers alone. However, these were only partially attributable to the transfected enzymes once stimulated cAMP synthesis by the endogenous adenylyl cyclase system of the host cell line was taken into account (data not shown) (Antoni et al., 1998).

The high cAMP-synthesizing activity of cells expressing AC9 was not attributable to the "stripping" of G_s from heterotrimeric G_s. In membranes prepared from AC9-transfected cells, basal adenylyl cyclase activity was 532 ± 15 pmol/mg protein per 20 min, whereas in pcDNA3-transfected cells it was 26 ± 1 pmol/mg protein per 20 min (n = 4/group). Importantly, the application of GDP--S failed to alter basal adenylyl cyclase activity in AC9 transfected cells, whereas it blocked the stimulatory effect of GTP--S (Table 1).

View this table: [in this window] [in a new window]   Table 1.   Effect of GDP--S (100 µM) on membrane adenylyl cyclase activity of HEK-293 cells stably transfected with mouse AC9 in pcDNA3

In subsequent experiments PDE inhibitors alone were added to intact cells to elicit cAMP production, thus minimizing the contribution of the endogenous adenylyl cyclase system of the host cell line to cAMP biosynthesis. The addition of extracellular Ca²⁺ inhibited or stimulated cAMP production in cells transfected with AC9 (Fig. 2B) or AC1 (Fig. 2C), respectively. The inhibitory effect of Ca²⁺ on AC9 in this paradigm appeared specific because 2 mM SrCl₂, BaCl₂, or MgCl₂ had no significant influence on cAMP accumulation (Table 2).

View this table: [in this window] [in a new window]   Table 2.   Effect of divalent cations on cAMP production in HEK-293 cells stably transfected with AC9

Effect of Ca²⁺ on AC9 is mediated by calcineurin

The inhibitory effect of Ca²⁺ on AC9-derived cAMP formation in EGTA/ryanodine/thapsigargin-treated cells (Fig. 3A) was blocked by FK506, an inhibitor of calcineurin (Schreiber, 1992). L685,818, an analog of FK506 that does not affect calcineurin activity (Dumont et al., 1992), was without effect at 50 µM (Fig. 3B). Cyclosporin A, another blocker of calcineurin (Schreiber, 1992), also inhibited the effect of Ca²⁺ on cAMP formation (Fig. 3B). The pattern of [Ca²⁺]_i in AC9-transfected cells treated with EGTA/ryanodine/thapsigargin is shown in Figure 3C; identical results were obtained with AC1-transfected cells (data not shown). Overall, in AC9-transfected cells 10 µM FK506 had no significant effect on the initial levels of [Ca²⁺]_i (vehicle, 49.6 ± 8.5 nM vs FK506, 52 ± 6.5 nM; means ± SEM; n = 6) or the plateau levels achieved in the presence of 0.5 mM (94 ± 16 vs 85 ± 6; n = 3) or 2 mM extracellular Ca²⁺ (124.4 ± 15 vs 119 ± 13 in FK506; means ± SEM; n = 6).

View larger version (21K): [in this window] [in a new window]   Figure 3.   Effect of calcineurin blockers on the inhibitory effect of Ca2+ in HEK-293 cells overexpressing AC9. The cells were incubated in medium containing no added calcium, 2 mM EGTA, 10 µM ryanodine, and 1 µM thapsigargin before the addition of various amounts of Ca/EGTA to the extracellular fluid to produce the free Ca2+ concentrations indicated on the abscissa. A, Concentration-dependent reversal of the Ca2+ inhibitory effect on cAMP production by FK506. cAMP accumulation was evoked by the addition of 1 mM IBMX and 0.1 mM rolipram. Data are expressed as multiples of the amount of cAMP found in cells not receiving PDE. Means ± SEM; n = 4/group. B, Specificity of immunosuppressant action: 2 µM FK506 and 2 µM cyclosporin A (CsA) blocked the effect of 0.5 mM extracellular free Ca2+ on cAMP production, whereas L685,818 (50 µM) had no effect. Conditions are as in A. Open columns, 0 Ca2+ medium; striped columns, 0.5 mM Ca2+. *p < 0.05 when compared with respective 0 Ca2+ medium control; one-way ANOVA, followed by Newman-Keuls test. C, Measurement of [Ca2+]i in a suspension of HEK-293 cells transfected with AC9 and loaded with fura-2 AM. The lines indicate the start of the application of Ca/EGTA to the medium, and the numbers above the lines indicate the final concentrations (in mM) of free extracellular Ca2+ that are present; PDE-I indicates the application of 1 mM IBMX and 0.1 mM rolipram. The data are representative of six similar experiments.

In ionophore-treated cells, Ca²⁺ was also inhibitory to cAMP production by AC9 (Fig. 4A), whereas AC1 was stimulated robustly (data not shown). FK506 and cyclosporin A blocked the inhibitory effect of 0.25 mM CaCl₂ in the presence of 4-BrA23187, whereas L685,818 was without effect (Fig. 4B). The pattern of [Ca²⁺]_i under these conditions is shown in Figure 4C; note the lack of an effect of FK506.

View larger version (20K): [in this window] [in a new window]   Figure 4.   Analysis of the effect of Ca2+ and calcineurin blockers in ionophore-treated HEK-293 cells stably transfected with AC9. A, Cells were incubated in medium containing no added calcium, 2 mM EGTA, and 5 µM 4Br-A23187 before the addition of various amounts of Ca/EGTA to the extracellular fluid to produce the free Ca2+ concentrations that are indicated on the abscissa. Calcineurin inhibitors were applied 20 min before Ca/EGTA. cAMP accumulation was evoked by the addition of 1 mM IBMX and 0.1 mM rolipram; data are expressed as multiples of the amount of cAMP found in cells not receiving PDE blockers. Means ± SEM; n = 4/group. *p < 0.05 when compared with the respective 0 Ca2+ group. $p < 0.05 when compared with the respective groups receiving vehicle; one-way ANOVA, followed by Newman-Keuls test. B, Specificity of FK506 (10 µM) effect when compared with vehicle and L685,818 (50 µM). Open columns, 0 Ca2+ medium; striped columns, 0.5 mM Ca2+. Conditions are as in A. *p < 0.05 when compared with the respective 0 Ca2+ group; one-way ANOVA, followed by Newman-Keuls test. C, Measurement of [Ca2+]i in HEK-293 cells transfected with AC9, loaded with fura 2-AM, and pretreated with vehicle (filled symbols) or 10 µM FK506 (open symbols). The top line indicates the start of the application of Ca/EGTA yielding 0.25 mM free extracellular Ca2+ into the medium; the line below PDE-I indicates the application of 1 mM IBMX and 0.1 mM rolipram. The data are representative of six similar experiments.

Higher average levels of [Ca²⁺]_i (200-300 nM) were achieved by using 15 µM 4-BrA23187 and 0.25 mM CaCl₂. However, under these conditions the addition of PDE blockers to the cells dramatically enhanced [Ca²⁺]_i further to 1 µM. An almost complete suppression of cAMP synthesis by AC9 resulted that was not altered by FK506 or cyclosporin A (data not shown). In parallel experiments the stimulatory effect of Ca²⁺ on AC1 was observed no longer, suggesting the nonspecific toxicity of this ionophore concentration in the presence of Ca²⁺.

Distribution of AC9 mRNA in the forebrain

A summary of the overall distribution pattern of AC9 mRNA in mouse forebrain is presented in Table 3. The distribution of AC9 mRNA was identical in rat brain. The mRNA signal was restricted mainly to the gray matter and appeared to be associated with neuronal perikarya.

View this table: [in this window] [in a new window]   Table 3.   Relative abundance of AC9 mRNA in areas of the mouse brain

Olfactory lobe

A large number of neuronal perikarya in the anterior olfactory nucleus, the nucleus of the lateral olfactory tract, and the olfactory bulb were strongly positive for AC9 mRNA.

Limbic lobe and other limbic areas

The most intense mRNA signal was apparent in the limbic areas of the forebrain. In particular, neurons of the hippocampal complex, including the tenia tecta, the anterior hippocampus, indusium griseum, and hippocampus, were labeled very intensely for AC9 mRNA. In the hippocampus (Fig. 5A-E) the CA1-CA3 pyramidal cell layers and the dentate gyrus granule cells showed dense and specific labeling. Examination at higher magnification (Fig. 5E) showed that close to 70% of the perikarya in the pyramidal layer of CA3 and at least 50% of the granule cells in the dentate gyrus expressed AC9 mRNA. Strong specific hybridization signal also was detected in neurons of the subiculum (Fig. 5F,G) and the parasubiculum.

View larger version (101K): [in this window] [in a new window]   Figure 5.   Demonstration of AC9 mRNA in the hippocampus and the subiculum in coronal sections of mouse brain, using a 35S-labeled antisense riboprobe (except for B, G). A, Dark-field view of the anterior dorsal hippocampus. d, Dentate gyrus. Scale bar, 400 µm. B, Specificity control with 35S-labeled sense probe in coronal section of dorsal hippocampus; otherwise, the conditions are as in A. C, Dark-field view of the dorsal hippocampus at a more posterior level. Note the intense labeling of pyramidal neurons in the CA1 and CA3 regions as well as granule cells in the dentate gyrus (d). Scale bar, 100 µm. D, Dark-field view of the ventral hippocampus showing intense labeling of neuronal perikarya in the pyramidal layers of CA1-CA3 as well as the granule cells of the dentate gyrus (d). s, Subiculum. Scale bar, 200 µm. E, Bright-field view of dorsal hippocampus at higher magnification in coronal sections counterstained with pyronin to reveal cell nuclei. Scale bar, 50 µm. F, Bright-field view of the subiculum; intense labeling is evident in neurons. V, Dorsal third ventricle. Scale bar, 100 µm. G, Specificity control for F, using 35S-labeled sense riboprobe.

Neurons in the piriform, the entorhinal, and the cingulate cortices were also highly positive for AC9 mRNA. Labeled cell bodies in the cingulate (Fig. 6A,B) and the entorhinal cortices (data not shown) were most prominent in layers II and III.

View larger version (127K): [in this window] [in a new window]   Figure 6.   Distribution of AC9 mRNA in coronal sections of the cingulate cortex (A, C) and the parietal cortex (B, D) of the mouse brain. A, Cingulate cortex, dark-field view. Scale bar, 100 µm. Arrowhead points to layer II-III shown in C as a bright-field image at higher magnification, counterstained with pyronin. Scale bar, 50 µm. B, Parietal cortex, dark-field view. Scale bar, 200 µm. D, Bright-field view of layers II-III, counterstained with pyronin. Scale bar, 50 µm.

Other limbic areas showing distinct low-to-moderate levels of mRNA signal include the dorsal septal nucleus, the lateral, medial, and posterior amygdaloid nuclei, the medial habenular nucleus, and the interpeduncular nucleus.

Neocortex

In the neocortex AC9 mRNA was observed mainly in neurons of layers II, III, and VI and the large pyramidal cells of layer V (Fig. 6C,D). There were no apparent topographical variations in this pattern.

Striatonigral system

The caudate putamen contained a large number of cells expressing moderate levels of AC9 (Fig. 7A,B) mRNA, whereas the globus pallidus was essentially devoid of specific signal.

View larger version (62K): [in this window] [in a new window]   Figure 7.   Neurons positive for AC9 mRNA in the caudate nucleus (A, B). Scale bars: A, 100 µm; B, 25 µm.

Diencephalon

The anteroventral thalamic nucleus showed moderate mRNA signal; a lower level of signal was found in the paratenial and the centromedian thalamic nuclei. Both the supraoptic and the paraventricular nuclei contained low-to-moderate levels of mRNA. Low-level labeling also was detected in the median preoptic nucleus.

Cerebellar system

In the cerebellar cortex Purkinje cells were weakly positive for AC9 mRNA (Fig. 8). Other areas of the cerebellar system expressing AC9 mRNA included the cerebellar nuclei, the inferior olive, the pontine nuclei, and the red nucleus.

View larger version (109K): [in this window] [in a new window]   Figure 8.   AC9 mRNA labeling in coronal section of the cerebellum. Arrowheads, Purkinje cells; ic, interposed cerebellar nucleus; lc, lateral cerebellar nucleus. Scale bar, 200 µm.

Lower brainstem

Some reticular, serotonergic, and motor cells in the lower brainstem expressed moderate levels of AC9.

Detection of AC9 protein in brain

Immunoblot analysis of detergent extracts of membrane fractions from the hippocampus and hypothalamus showed the presence of a major ~155 kDa immunoreactive band (Fig. 9). Quantification by radioimmunoassay with a C-terminally directed antiserum suggested that crude membrane fractions prepared from the hippocampus have AC9 levels similar to those seen in AtT20 cells (Table 4). The cerebellum, the hypothalamus, and the anterior pituitary gland expressed lower levels of AC9 protein, which is consistent with the lower abundance of the mRNA signal in these tissues (Paterson et al., 1995; present study).

View larger version (28K): [in this window] [in a new window]   Figure 9.   Immunoblots of rat hippocampal (HIP; 2.5 µg protein/lane) and hypothalamic (HYP; 7.5 µg/lane) membranes reacted with anti-AC9 ( lanes) or the same antiserum preincubated with 1 µg/ml antigen peptide (+ lanes). Proteins were separated by SDS-PAGE on 7.5% homogenous microgels. The positions of molecular weight markers are indicated on the left of the graph.

View this table: [in this window] [in a new window]   Table 4.   The concentration of immunoreactive adenylyl cyclase 9 in selected regions of the rat brain and the anterior pituitary gland

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

These data show that the Ca²⁺/calcineurin-inhibited adenylyl cyclase (AC9) is present in areas of the brain previously thought to contain only Ca²⁺-activated (AC1 and AC8) or protein kinase C-activated (AC2 and AC7) adenylyl cyclases. The distribution of AC9 mRNA in the brain suggests close proximity to or possible colocalization in AC1- and AC2-expressing neurons in limbic and neocortical areas. Functionally, comparative analysis of AC1- and AC9-derived cAMP production in HEK-293 cells showed that AC1 and AC9 are regulated by the same range of intracellular free Ca²⁺ concentrations. Taken together, these data indicate that AC9 is a major cAMP-synthesizing enzyme in brain and is modulated by Ca²⁺ by way of the protein phosphatase calcineurin.

Properties of AC9

Previous work from our laboratory demonstrated calcineurin-mediated inhibition of agonist-induced cAMP responses in AtT20 cells that express relatively high levels of endogenous AC9 (Antoni et al., 1995) as well as HEK-293 cells transiently transfected with AC9 cDNA (Paterson et al., 1995). In these studies cAMP production was evoked by receptor agonists, and thus it remained to be demonstrated that the target of calcineurin-dependent control is AC9 itself rather than the efficiency of receptor-G_s-cyclase coupling. Meanwhile, others have reported that mouse AC9 is not influenced by Ca²⁺/calmodulin (Premont et al., 1996) and that a human AC9 variant recently cloned from the heart is not regulated by Ca²⁺-dependent processes (Hacker et al., 1998). The plausible explanations for these discrepancies are (1) Premont et al. (1996) used Sf9 insect cell membranes for which the profile of phosphorylation of AC9 may be different from that in mammalian systems, and (2) Hacker and coworkers (1998) reported an enzyme that has no significant homology in the C1b regulatory domain (see below) with mouse AC9 and hence may have different regulatory properties.

All mammalian adenylyl cyclases known so far conform to the "quasi-duplicated transporter" design (Taussig and Gilman, 1995) consisting of two membrane-spanning domains each, followed by substantial cytoplasmic loops that have been designated as C1 and C2, respectively. Current evidence indicates that the physical interaction of C1 and C2 underlies the catalytic activity of the enzyme. The interaction between C1 and C2 is facilitated by forskolin or G_s-GTP complexes (Tang and Gilman, 1995; Tesmer et al., 1997) that enhance the affinity of the interaction between C1 and C2 by ~50-fold (Whisnant et al., 1996). Therefore, overexpression of adenylyl cyclase in the region of 30-fold above normal, as in the present study, should produce substantial "basal" adenylyl cyclase activity, because the number of catalytically active C1-C2 complexes is increased by a similar factor as in the presence of forskolin or G_s-GTP. The adaptation of stably transfected cells to high levels of cAMP via increased gene expression and phosphorylation-dependent activation of PDEs is well documented (Conti et al., 1995; Houslay, 1998). Hence unstimulated levels of cAMP in cells stably transfected with adenylyl cyclase are similar to those in wild-type cells, whereas the pharmacological inhibition of PDE activity unmasks the enhanced production of cAMP by the transfected enzyme (Cali et al., 1994; Wayman et al., 1994; present study). With the use of GDP--S we have provided additional evidence that the increased adenylyl cyclase activity in the stably transfected cells is independent of G_s. The analysis of membrane adenylyl cyclase activity also confirmed previous reports (Premont et al., 1996; Antoni et al., 1998; Yan et al., 1998) that AC9 is stimulated only weakly by forskolin. Taken together, the characteristics of cAMP production in HEK-293 cells stably transfected with AC9 and treated with PDE blockers reflect the regulatory properties of the adenylyl cyclase catalytic moiety.

The present experiments show that intracellular free Ca²⁺ inhibits cAMP synthesis by AC9. The inhibitory effect of Ca²⁺ on AC9 was blocked by FK506 and cyclosporin A, but not by L685,818, which conforms with the current knowledge on the pharmacology of calcineurin (Schreiber, 1992; Sigal and Dumont, 1992). The degree of inhibition of AC9 by Ca²⁺/calcineurin was modest, i.e., 20-40%, which could be attributable to a number of factors, including the high levels of adenylyl cyclase relative to calcineurin expressed in the cells or the inactivation of calcineurin by [Ca²⁺]_i (Wang et al., 1996). Furthermore, depending on the rate of cAMP hydrolysis of the system under study, a relatively small reduction (~30%) of the rate of cAMP synthesis may lead to a marked fall (~80%) of intracellular cAMP levels (Chiono et al., 1995).

The concentration of intracellular Ca²⁺ sufficient for the inhibition of AC9 was in the region of 100 nM, a point at which calcineurin is already active (Perrino et al., 1995; Fruman et al., 1996). Further, this suggests that the fluctuations of [Ca²⁺]_i found under "basal" conditions are sufficient to modulate AC9 activity, i.e., constitutive phosphorylation/dephosphorylation may influence the level to which the enzyme may be activated. Indeed, in AtT20 cells, which display spontaneous [Ca²⁺]_i spikes with amplitudes up to 400 nM (Antoni et al., 1992), the inhibition of calcineurin with FK506 and cyclosporin A leads to enhanced activation of membrane adenylyl cyclase by corticotropin-releasing factor (Antoni et al., 1998).

Whether or not AC9 is a direct target for calcineurin or is regulated by a protein phosphatase downstream of calcineurin (Antaraki et al., 1997), i.e., a protein phosphatase cascade (Cohen, 1989), remains to be established. Moreover, the protein kinases that phosphorylate AC9 have not been identified.

Distribution of AC9 in the forebrain

The AC9 mRNA signal was prominent in neurons found in higher brain regions where substantial levels of mRNA for other Ca²⁺-inhibited adenylyl cyclases such as AC3, AC5, or AC6 have not been reported. Extraneuronal mRNA for AC9 was not observed. Overall, in the hippocampus, the neocortex, the striatum, and the cerebellum the same arrays of neurons that express significant levels of AC9 mRNA also contain relatively high levels of calcineurin (Kuno et al., 1992; Goto et al., 1993; Dawson et al., 1994) and FKBP12 (Dawson et al., 1994; Sabatini and Snyder, 1995). Within hippocampal pyramidal neurons (Sík et al., 1998) calcineurin is found in dendritic spines and the cell body, where adenylyl cyclase immunoreactivity also has been demonstrated with an antibody that recognizes all mammalian adenylyl cyclases (Mons et al., 1995). Thus, on the basis of current knowledge, the respective topographical distributions of AC9, calcineurin, and FKBP12 are consistent with the hypothesis that calcineurin is a regulator of AC9.

A comparison of the patterns of expression of AC9 and other adenylyl cyclases clearly indicates the close proximity of neurons expressing AC9 to those expressing the Ca²⁺-stimulated cyclases AC1/AC8 as well as AC2, an adenylyl cyclase stimulated by protein kinase C. Indeed, the distribution of AC9 mRNA in the hippocampal formation is identical to that of AC1, except in the pyramidal layer of CA1-CA3 where AC1 mRNA is relatively low (Xia et al., 1991), whereas AC9 mRNA is highly abundant. Thus mRNAs for three classes of adenylyl cyclase are expressed in the pyramidal cells of CA1-CA3 and in the granule cells of the dentate gyrus. Whether or not this is also the case at the level of protein expression and to what extent these cyclases are colocalized in the same neurons remain to be clarified.

The present study provides the first data on AC9 protein concentration in brain regions, which suggest that the level of AC9 in rat hippocampus is comparable to that in the clonal AtT20 cell line. In turn, AtT20 cells were found to express the expected level, i.e., ~0.05% of total membrane protein of immunoreactive AC9. Given the selective neuronal localization of AC9 mRNA, the concentration of AC9 protein in the hippocampus appears remarkably high. Similar data on other cyclase isotypes are not available at present.

Implications for synaptic function

Currently, any considerations of the physiological function of AC9 are speculative; however, two areas stand out as plausible and biologically significant.

First, in AtT20 cells AC9 is the target of an intracellular Ca²⁺ feedback loop that also involves BK-type K⁺ channels and Ca²⁺/calmodulin-activated PDE. This system regulates the membrane potential and the levels of cAMP to maintain cellular excitability (Antoni, 1996). AtT20 cells show the rhythmic firing of action potentials and intracellular free Ca²⁺ transients (Suprenant, 1982; Adler et al., 1983; Antoni et al., 1992). Prominent expression of AC9, Ca²⁺-activated K⁺ channels, and Ca²⁺/calmodulin PDE, i.e., the components of Ca²⁺ negative feedback on cAMP levels and the membrane potential, is evident in hippocampal principal neurons, which also exhibit various modes of rhythmic firing. Thus AC9 may be involved in the generation of rhythmic electrical activity in the hippocampus and possibly in other parts of the forebrain.

Second, hippocampal neurons have been analyzed extensively with respect to the role of cAMP in synaptic plasticity. The activation of cAMP synthesis by [Ca²⁺]_i has been attributed as a key role in various types of long-term potentiation (LTP) (for review, see Xia and Storm, 1997). However, the example of the Drosophila dunce mutant, which is deficient in a cyclic nucleotide PDE gene, indicates that the hydrolysis of cAMP is also a key process in memory formation and underscores the issue of a cAMP signal that is optimized in time as well as space (Dudai, 1997).

Calcineurin is a potent inhibitor of postsynaptic activity in hippocampal neurons (Raman et al., 1996; Wang and Kelly, 1997) and recently has been proposed as a "memory suppressor" gene (Abel et al., 1998). The overexpression of calcineurin in hippocampal neurons impaired a cAMP-dependent phase of tetanus-evoked LTP (Winder et al., 1998) as well as hippocampus-dependent memory formation (Mansuy et al., 1998). The prominent expression of AC9 in the hippocampus and the pivotal role of the tuning of the cAMP Ca²⁺ signal in synaptic plasticity (Blitzer et al., 1995) indicate that AC9 may be an important physiological target for protein kinase and phosphatase cascades that contribute to the long-term modulation of synaptic transmission.

	FOOTNOTES

Received Aug. 11, 1998; revised Sept. 14, 1998; accepted Sept. 14, 1998.

We thank J. Bennie, O. Grace, and S. Carroll for technical support. A generous supply of cAMP antiserum donated by K. J. Catt and A. Baukal (National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD) is gratefully acknowledged. We also thank D. R. Storm and W. L. Zhu (Seattle, WA) for kindly providing the plasmids used in this study and Peter Sharp (Roslin, Scotland, UK) for help in raising antisera in hens.

Correspondence should be addressed to Dr. F. Antoni, Medial Research Council Brain Metabolism Unit, Department of Neuroscience, University of Edinburgh, Edinburgh, EH8 9JZ, Scotland, UK.

	REFERENCES

Top Abstract Introduction Materials & Methods Results Discussion References

• Abel T, Martin KC, Bartsch D, Kandel ER (1998) Memory suppressor genes: inhibitory constraints on the storage of long-term memory. Science 279:338-341[Abstract/Free Full Text].
• Adler M, Wong BS, Sabol SL, Buis N, Jackson MB, Weight FF (1983) Action potentials and membrane ion channels in clonal anterior pituitary cells. Proc Natl Acad Sci USA 80:2086-2090[Abstract/Free Full Text].
• Antaraki A, Ang KL, Antoni FA (1997) Involvement of calyculin A inhibitable protein phosphatases in the cyclic AMP signal transduction pathway of mouse corticotroph tumour (AtT20) cells. Br J Pharmacol 121:991-999[Web of Science][Medline].
• Antoni FA (1996) Calcium checks cyclic AMP-corticosteroid feedback in adenohypophysial corticotrophs. J Neuroendocrinol 8:659-672[Web of Science][Medline].
• Antoni FA (1997) Calcium regulation of adenylyl cyclaserelevance for endocrine control. Trends Endocrinol Metab 8:7-13.
• Antoni FA, Holmes MC, Kiss JZ (1985) Pituitary binding of vasopressin is altered by experimental manipulations of the hypothalamo-pituitary-adrenocortical axis in normal as well as homozygous (di/di) Brattleboro rats. Endocrinology 117:1293-1299[Abstract/Free Full Text].
• Antoni FA, Hoyland J, Woods MD, Mason WT (1992) Glucocorticoid inhibition of stimulus-evoked adrenocorticotrophin release caused by suppression of intracellular calcium signals. J Endocrinol 133:R13-R16[Abstract/Free Full Text].
• Antoni FA, Barnard RJO, Shipston MJ, Smith SM, Simpson J, Paterson JM (1995) Calcineurin feedback inhibition of agonist-evoked cAMP formation. J Biol Chem 270:28055-28061[Abstract/Free Full Text].
• Antoni FA, Smith SM, Simpson J, Rosie R, Fink G, Paterson JM (1998) Calcium control of adenylyl cyclase: the calcineurin connection. Adv Second Messenger Phosphoprotein Res 32:153-172[Medline].
• Blitzer RD, Wong T, Nouranifar R, Iyengar R, Landau EM (1995) Postsynaptic cAMP pathway gates early LTP in hippocampal CA1 region. Neuron 15:1403-1414[Web of Science][Medline].
• Cali JJ, Zwaagstra JC, Mons N, Cooper DMF, Krupinski J (1994) Type VIII adenylyl cyclase. A Ca²⁺/calmodulin-stimulated enzyme expressed in discrete regions of rat brain. J Biol Chem 269:12190-12195[Abstract/Free Full Text].
• Chen J, DeVivo M, Dingus J, Harry A, Li J, Sui J, Carty DJ, Blank JL, Exton JH, Stoffel RH, Inglese J, Lefkowitz RJ, Logothetis DE, Hildebrandt JD, Iyengar R (1995) A region of adenylyl cyclase 2 critical for regulation by G-protein subunits. Science 268:1166-1169[Abstract/Free Full Text].
• Chiono M, Mahey R, Tate G, Cooper DMF (1995) Capacitive Ca²⁺ entry exclusively inhibits cAMP synthesis in C6-2B glioma cells. Evidence that physiologically evoked Ca²⁺ entry regulates Ca²⁺-inhibitable adenylyl cyclase in non-excitable cells. J Biol Chem 270:1149-1155[Abstract/Free Full Text].
• Cohen P (1989) Structure and regulation of protein phosphatases. Annu Rev Biochem 58:453-508[Web of Science][Medline].
• Conti M, Nemoz G, Sette C, Vicini E (1995) Recent progress in understanding the hormonal regulation of phosphodiesterases. Endocr Rev 16:370-389[Abstract/Free Full Text].
• Cooper DMF, Yoshimura M, Zhang Y, Chiono M, Mahey R (1994) Capacitative Ca²⁺ entry regulates Ca²⁺-sensitive adenylyl cyclases. Biochem J 297:437-440.
• Dawson TM, Steiner JP, Lyons WE, Fotuhi M, Blue M, Snyder SH (1994) The immunophilins, FK506 binding protein and cyclophilin, are discretely localized in the brain: relationship to calcineurin. Neuroscience 62:569-580[Web of Science][Medline].
• Dudai Y (1997) Time to remember. Neuron 18:179-182[Web of Science][Medline].
• Dumont FJ, Staruch MJ, Koprak SL, Siekierka JJ, Lin CS, Harrison R, Sewell T, Kindt VM, Beattie TR, Wyratt M, Sigal NH (1992) The immunosuppressive and toxic effects of FK506 are mechanistically related: pharmacology of a novel antagonist of FK506 and rapamycin. J Exp Med 176:751-760[Abstract/Free Full Text].
• Fruman DA, Pai SY, Klee CB, Burakoff SJ, Bierer BE (1996) Measurement of calcineurin phosphatase activity in cell extracts. Methods 9:146-154[Medline].
• Glatt CE, Snyder SH (1993) Cloning and expression of an adenylyl cyclase localized to the corpus striatum. Nature 361:536-538[Medline].
• Goto S, Nagahiro S, Korematsu K, Ushio Y, Fukunaga K, Miyamoto E, Hofer W (1993) Cellular colocalization of calcium calmodulin-dependent protein kinase-II and calcineurin in the rat cerebral cortex and hippocampus. Neurosci Lett 149:189-192[Web of Science][Medline].
• Grynkiewicz G, Poenie M, Tsien RY (1985) A new generation of Ca²⁺ indicators with greatly improved fluorescence properties. J Biol Chem 260:3440-3450[Abstract/Free Full Text].
• Hacker BM, Tomlinson JE, Wayman GA, Sultana R, Chan G, Villacres E, Disteche C, Storm DR (1998) Cloning, chromosomal mapping, and regulatory properties of the human type 9 adenylyl cyclase (ADCY9). Genomics 50:97-104[Web of Science][Medline].
• Houslay MD (1998) Adaptation in cyclic AMP signaling processes: a central role for cyclic AMP phosphodiesterases. Semin Cell Dev Biol 9:161-167[Web of Science][Medline].
• Ishikawa Y (1998) Regulation of cAMP signaling by phosphorylation. Adv Second Messenger Phosphoprotein Res 32:99-120[Medline].
• Kawabe JI, Iwami G, Ebine T, Ohno S, Katada T, Ueda Y, Homcy CJ, Ishikawa Y (1994) Differential activation of adenylyl cyclase by protein kinase C isozymes. J Biol Chem 269:16554-16558[Abstract/Free Full Text].
• Krupinski J, Coussen F, Bakalyar HA, Tang WJ, Feinstein PG, Orth K, Slaughter C, Reed RR, Gilman AG (1989) Adenylyl cyclase amino acid sequence: possible channel- or transporter-like structure. Science 244:1558-1564[Abstract/Free Full Text].
• Kuno T, Mukai H, Ito A, Chang CD, Kishima K, Saito N, Tanaka C (1992) Distinct cellular expression of calcineurin A- and A- in rat brain. J Neurochem 58:1643-1651[Web of Science][Medline].
• Lachmann P, Strangeways L, Vyakarnam A, Evan G (1986) Raising antibodies by coupling peptides to PPD and immunizing BCG-sensitized animals. Ciba Found Symp 119:25-57[Medline].
• Lin CW, Miller TR, Witte DG, Bianchi BR, Stashko M, Manelli AM, Frail DE (1995) Characterization of cloned human dopamine D₁ receptor-mediated calcium release in 293 cells. Mol Pharmacol 47:131-139[Abstract].
• Mansuy IM, Mayford M, Jacob B, Kandel ER, Bach ME (1998) Restricted and regulated overexpression reveals calcineurin as a key component in the transition from short-term to long-term memory. Cell 92:39-49[Web of Science][Medline].
• Mollner S, Beck K, Pfeuffer T (1995) Acylation of adenylyl cyclase catalyst is important for enzymic activity. FEBS Lett 371:241-244[Web of Science][Medline].
• Mons N, Cooper DMF (1995) Adenylate cyclasescritical foci in neuronal signaling. Trends Neurosci 18:536-542[Web of Science][Medline].
• Mons N, Harry A, Dubourg P, Premont RT, Iyengar R, Cooper DMF (1995) Immunohistochemical localization of adenylyl cyclase in rat brain indicates a highly selective concentration at synapses. Proc Natl Acad Sci USA 92:8473-8477[Abstract/Free Full Text].
• Paterson JM, Smith SM, Harmar AJ, Antoni FA (1995) Control of a novel adenylyl cyclase by calcineurin. Biochem Biophys Res Commun 214:1000-1008[Web of Science][Medline].
• Perrino BA, Ng LY, Soderling TR (1995) Calcium regulation of calcineurin phosphatase activity by its  subunit and calmodulin. Role of the autoinhibitory domain. J Biol Chem 270:7012[Abstract/Free Full Text].
• Premont RT, Matsuoka I, Mattei M-G, Pouille Y, Defer N, Hanoune J (1996) Identification and characterization of a widely expressed form of adenylyl cyclase. J Biol Chem 271:13900-13907[Abstract/Free Full Text].
• Querfurth HW, Selkoe DJ (1994) Calcium ionophore increases amyloid- peptide production by cultured cells. Biochemistry 33:4550-4561[Medline].
• Raman IM, Tong G, Jahr CE (1996) -Adrenergic regulation of synaptic NMDA receptors by cAMP-dependent protein kinase. Neuron 16:415-421[Web of Science][Medline].
• Rosie R, Thomson E, Blum M, Roberts JL, Fink G (1992) Oestrogen positive feedback reduces arcuate proopiomelanocortin messenger ribonucleic acid. J Neuroendocrinol 4:625-630[Web of Science].
• Sabatini DM, Snyder SH (1995) Immunophilins and the nervous system. Nat Med 1:32-37[Web of Science][Medline].
• Schreiber SL (1992) Immunophilin-sensitive protein phosphatase action in cell signaling pathways. Cell 70:365-368[Web of Science][Medline].
• Seuwen K, Boddeke H (1995) Heparin-insensitive calcium release from intracellular stores triggered by the recombinant human parathyroid hormone receptor. Br J Pharmacol 114:1613-1620[Web of Science][Medline].
• Sigal NH, Dumont FJ (1992) Cyclosporin A, FK-506, and rapamycin: pharmacologic probes of lymphocyte signal transduction. Annu Rev Immunol 10:519-560[Web of Science][Medline].
• Sík A, Hájos N, Gulácsi A, Mody I, Freund TF (1998) The absence of a major Ca²⁺ signaling pathway in GABAergic neurons of the hippocampus. Proc Natl Acad Sci USA 95:3245-3250[Abstract/Free Full Text].
• Sunahara RK, Dessauer CW, Gilman AG (1996) Complexity and diversity of mammalian adenylyl cyclases. Annu Rev Biochem 36:461-480.
• Suprenant A (1982) Correlation between electrical activity and ACTH/-endorphin secretion in mouse pituitary tumor cells. J Cell Biol 95:559-566[Abstract/Free Full Text].
• Tang WJ, Gilman AG (1991) Type-specific regulation of adenylyl cyclase by G-protein subunits. Science 254:1500-1503[Abstract/Free Full Text].
• Tang WJ, Gilman AG (1995) Construction of a soluble adenylyl cyclase activated by G_(s) and forskolin. Science 268:1769-1772[Abstract/Free Full Text].
• Taussig R, Gilman AG (1995) Mammalian membrane-bound adenylyl cyclases. J Biol Chem 270:1-4[Free Full Text].
• Tesmer JJ, Sunahara RK, Gilman AG, Sprang SR (1997) Crystal structure of the catalytic domains of adenylyl cyclase in a complex with G_s.GTPS. Science 278:1907-1916[Abstract/Free Full Text].
• Wang JH, Kelly PT (1997) Postsynaptic calcineurin activity downregulates synaptic transmission by weakening intracellular Ca²⁺ signaling mechanisms in hippocampal CA1 neurons. J Neurosci 17:4600-4611[Abstract/Free Full Text].
• Wang X, Culotta VC, Klee CB (1996) Superoxide dismutase protects calcineurin from inactivation. Nature 383:434-437[Medline].
• Wayman GA, Impey S, Wu Z, Kindsvogel W, Prichard L, Storm DR (1994) Synergistic activation of the type I adenylyl cyclase by Ca²⁺ and G_s-coupled receptors in vivo. J Biol Chem 269:25400-25405[Abstract/Free Full Text].
• Whisnant RE, Gilman AG, Dessauer CW (1996) Interaction of the two cytosolic domains of mammalian adenylyl cyclase. Proc Natl Acad Sci USA 93:6621-6625[Abstract/Free Full Text].
• Winder DG, Mansuy IM, Osman M, Moallem TM, Kandel ER (1998) Genetic and pharmacological evidence for a novel, intermediate phase of long-term potentiation suppressed by calcineurin. Cell 92:25-37[Web of Science][Medline].
• Xia ZG, Storm DR (1997) Calmodulin-regulated adenylyl cyclases and neuromodulation. Curr Opin Neurobiol 7:391-396[Web of Science][Medline].
• Xia ZG, Refsdal CD, Merchant KM, Dorsa DM, Storm DR (1991) Distribution of messenger RNA for the calmodulin-sensitive adenylate cyclase in rat brain expression in areas associated with learning and memory. Neuron 6:431-443[Web of Science][Medline].
• Xia ZG, Choi E, Wang F, Storm D (1992) The type III calcium calmodulin-sensitive adenylyl cyclase is not specific to olfactory sensory neurons. Neurosci Lett 144:169-173[Web of Science][Medline].
• Yan SZ, Huang ZH, Andrews RK, Tang WJ (1998) Conversion of forskolin-insensitive to forskolin-sensitive (mouse-type IX) adenylyl cyclase. Mol Pharmacol 53:182-187[Abstract/Free Full Text].

---

Copyright © 1998 Society for Neuroscience  0270-6474/98/18239650-12$05.00/0

This article has been cited by other articles:

				D. Willoughby and D. M. F. Cooper Organization and Ca2+ Regulation of Adenylyl Cyclases in cAMP Microdomains Physiol Rev, July 1, 2007; 87(3): 965 - 1010. [Abstract] [Full Text] [PDF]

				W. Cui, A. Smith, A. Darby-King, C. W. Harley, and J. H. McLean A temporal-specific and transient cAMP increase characterizes odorant classical conditioning Learn. Mem., March 2, 2007; 14(3): 126 - 133. [Abstract] [Full Text] [PDF]

				R. F. Watson, R. M. Abdel-Majid, M. W. Barnett, B. S. Willis, A. Katsnelson, T. H. Gillingwater, G. S. McKnight, P. C. Kind, and P. E. Neumann Involvement of protein kinase A in patterning of the mouse somatosensory cortex. J. Neurosci., May 17, 2006; 26(20): 5393 - 5401. [Abstract] [Full Text] [PDF]

				G. C.-K. Chan, S. Tonegawa, and D. R. Storm Hippocampal Neurons Express a Calcineurin-Activated Adenylyl Cyclase J. Neurosci., October 26, 2005; 25(43): 9913 - 9918. [Abstract] [Full Text] [PDF]

				X.-T. Hu, Y. Dong, X.-F. Zhang, and F. J. White Dopamine D2 Receptor-Activated Ca2+ Signaling Modulates Voltage-Sensitive Sodium Currents in Rat Nucleus Accumbens Neurons J Neurophysiol, March 1, 2005; 93(3): 1406 - 1417. [Abstract] [Full Text] [PDF]

				M. Townsend, Y. Liu, and M. Constantine-Paton Retina-Driven Dephosphorylation of the NR2A Subunit Correlates with Faster NMDA Receptor Kinetics at Developing Retinocollicular Synapses J. Neurosci., December 8, 2004; 24(49): 11098 - 11107. [Abstract] [Full Text] [PDF]

				M. G. Cumbay and V. J. Watts Novel Regulatory Properties of Human Type 9 Adenylate Cyclase J. Pharmacol. Exp. Ther., July 1, 2004; 310(1): 108 - 115. [Abstract] [Full Text] [PDF]

				F. A. Antoni, A. A. Sosunov, A. Haunso, J. M. Paterson, and J. Simpson Short-Term Plasticity of Cyclic Adenosine 3',5'-Monophosphate Signaling in Anterior Pituitary Corticotrope Cells: The Role of Adenylyl Cyclase Isotypes Mol. Endocrinol., April 1, 2003; 17(4): 692 - 703. [Abstract] [Full Text] [PDF]

				A. Haunso, J. Simpson, and F. A. Antoni Small Ligands Modulating the Activity of Mammalian Adenylyl Cyclases: A Novel Mode of Inhibition by Calmidazolium Mol. Pharmacol., March 1, 2003; 63(3): 624 - 631. [Abstract] [Full Text] [PDF]

				K.-W. Lee, J.-H. Hong, I. Y. Choi, Y. Che, J.-K. Lee, S.-D. Yang, C.-W. Song, H. S. Kang, J.-H. Lee, J. S. Noh, et al. Impaired D2 Dopamine Receptor Function in Mice Lacking Type 5 Adenylyl Cyclase J. Neurosci., September 15, 2002; 22(18): 7931 - 7940. [Abstract] [Full Text] [PDF]

				S.-T. Li, K. Kato, K. Tomizawa, M. Matsushita, A. Moriwaki, H. Matsui, and K. Mikoshiba Calcineurin Plays Different Roles in Group II Metabotropic Glutamate Receptor- and NMDA Receptor-Dependent Long-Term Depression J. Neurosci., June 15, 2002; 22(12): 5034 - 5041. [Abstract] [Full Text] [PDF]

				N. N Petrashevskaya, I. Bodi, M. Rubio, J. D Molkentin, and A. Schwartz Cardiac function and electrical remodeling of the calcineurin-overexpressed transgenic mouse Cardiovasc Res, April 1, 2002; 54(1): 117 - 132. [Abstract] [Full Text] [PDF]

				M. G. Cumbay and V. J. Watts Heterologous Sensitization of Recombinant Adenylate Cyclases by Activation of D2 Dopamine Receptors J. Pharmacol. Exp. Ther., June 1, 2001; 297(3): 1201 - 1209. [Abstract] [Full Text]

				J. E. Nash and J. M. Brotchie A Common Signaling Pathway for Striatal NMDA and Adenosine A2a Receptors: Implications for the Treatment of Parkinson's Disease J. Neurosci., October 15, 2000; 20(20): 7782 - 7789. [Abstract] [Full Text] [PDF]

				N. Defer, M. Best-Belpomme, and J. Hanoune Tissue specificity and physiological relevance of various isoforms of adenylyl cyclase Am J Physiol Renal Physiol, September 1, 2000; 279(3): F400 - F416. [Abstract] [Full Text] [PDF]

				J.-L. Guillou, G. M. Rose, and D. M. F. Cooper Differential Activation of Adenylyl Cyclases by Spatial and Procedural Learning J. Neurosci., July 15, 1999; 19(14): 6183 - 6190. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) An erratum has been published An erratum has been published Submit an eLetter Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (56) Citing Articles via Google Scholar Google Scholar Articles by Antoni, F. A. Articles by Paterson, J. M. Search for Related Content PubMed PubMed Citation Articles by Antoni, F. A. Articles by Paterson, J. M.

Home  |   Search  |   Archive  |   Subscribe  |   Contact  |   Help