Ca2+/calcineurin-inhibited adenylyl cyclase, highly abundant in forebrain regions, is important for learning and memory

Abstract

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.

Fig. 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.

Fig. 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 mmEGTA 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 time0. 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. Theabscissa 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.

Fig. 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 theabscissa. 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. Thelines indicate the start of the application of Ca/EGTA to the medium, and the numbers above thelines 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.

Fig. 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 theabscissa. 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 mmfree extracellular Ca²⁺ into the medium; theline below PDE-I indicates the application of 1 mm IBMX and 0.1 mm rolipram. The data are representative of six similar experiments.

Fig. 5.

Demonstration of AC9 mRNA in the hippocampus and the subiculum in coronal sections of mouse brain, using a³⁵S-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 andCA3 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 ofCA1–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 forF, using ³⁵S-labeled sense riboprobe.

Fig. 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. Arrowheadpoints 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.

Fig. 7.

Neurons positive for AC9 mRNA in the caudate nucleus (A, B). Scale bars:A, 100 μm; B, 25 μm.

Fig. 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.

Fig. 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.