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Predominant expression of type-VI adenylate cyclase in C6-2B rat glioma cells may account for inhibition of cyclic AMP accumulation by calcium.

ABSTRACT: In C6-2B cells, agonist-stimulated cyclic AMP accumulation is inhibited when the cytosolic Ca2+ concentration is increased. We now demonstrate that in C6-2B cells: (i) the early kinetics of the cyclic AMP inhibition by substance K (t1/2 = 35 s) and thapsigargin (t1/2 = 1.6 min) closely mimic the kinetics of the cytosolic Ca2+ increase evoked by either agent (t1/2 = 25 s and 1.5 min respectively); (ii) the Ca2+ rise and cyclic AMP inhibition by substance K or thapsigargin are similarly affected in EGTA-containing medium; (iii) PCR detects type-III and type-VI adenylate cyclase cDNAs, and RNAase protection assays show that the mRNA for type-VI adenylate cyclase, an isoform inhibitable by submicromolar Ca2+ concentrations, is the predominant species, strongly suggesting that type-VI adenylate cyclase is probably the target molecule for Ca(2+)-mediated inhibition of cyclic AMP accumulation.

SUBMITTER: Debernardi MA

PROVIDER: S-EPMC1134362 | biostudies-other | 1993 Jul

REPOSITORIES: biostudies-other

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Project description:Stable cell lines, derived from NG108-15 cells and transfected to express both the beta 2-adrenoceptor and adenylate cyclase type II, were produced and examined. The absence of adenylate cyclase type II in the parental cells and its presence in these clones was demonstrated by reverse transcriptase-PCR. Total cellular levels of adenylate cyclase were increased in a number of clones between 3- and 8-fold, as assessed by guanine nucleotide-stimulated specific high-affinity binding of [3H]forskolin to cellular membranes. Basal adenylate cyclase activity was markedly elevated compared with a clone expressing similar levels of the beta 2-adrenoceptor in the absence of adenylate cyclase type II. Each of NaF, forskolin and guanosine 5'-[beta, gamma-imido]triphosphate (a poorly hydrolysed analogue of GTP) produced substantially higher levels of adenylate cyclase activity in membranes of the clones positive for expression of adenylate cyclase type II than was achieved with the parental cells. Both isoprenaline, acting at the introduced beta 2-adrenoceptor, and iloprost, acting at the endogenously expressed IP prostanoid receptor, stimulated adenylate cyclase activity to much higher levels in the clones expressing adenylate cyclase type II compared with the clone lacking this adenylate cyclase; however, the concentration-effect curves for adenylate cyclase stimulation by these two agonists were not different between parental cells and clones over-expressing adenylate cyclase type II. A maximally effective concentration of the beta-adrenoceptor partial agonist ephedrine displayed similar intrinsic activity and potency to stimulate adenylate cyclase in membranes of clones both with and without adenylate cyclase type II. Both secretin and 5'-N-ethylcarbox-amidoadenosine (acting at an endogenous A2 adenosine receptor) were also able to produce substantially greater maximal activations of adenylate cyclase in the clones expressing excess adenylate cyclase type II, without alterations in agonist intrinsic activity or potency. These results demonstrate that the maximal output of the stimulatory arm of the adenylate cyclase cascade can be increased by increasing total levels of adenylate cyclase in the genetic background of NG108-15 cells.

Project description:A central feature of the immune synapse (IS) is the tight compartmentalization of membrane receptors and signaling mediators that is functional for its ability to coordinate T cell activation. Second messengers centrally implicated in this process, such as Ca2+ and diacyl glycerol, also undergo compartmentalization at the IS. Current evidence suggests a more complex scenario for cyclic AMP (cAMP), which acts both as positive and as negative regulator of T-cell antigen receptor (TCR) signaling and which, as such, must be subjected to a tight spatiotemporal control to allow for signaling at the IS. Here, we have used two bacterial adenylate cyclase toxins that produce cAMP at different subcellular localizations as the result of their distinct routes of cell invasion, namely Bordetella pertussis CyaA and Bacillus anthracis edema toxin (ET), to address the ability of the T cell to confine cAMP to the site of production and to address the impact of compartmentalized cAMP production on IS assembly and function. We show that CyaA, which produces cAMP close to the synaptic membrane, affects IS stability by modulating not only the distribution of LFA-1 and its partners talin and L-plastin, as previously partly reported but also by promoting the sustained synaptic accumulation of the A-kinase adaptor protein ezrin and protein kinase A while suppressing the ?-arrestin-mediated recruitment of phosphodiesterase 4B. These effects are dependent on the catalytic activity of the toxin and can be reproduced by treatment with a non-hydrolyzable cAMP analog. Remarkably, none of these effects are elicited by ET, which produces cAMP at a perinuclear localization, despite its ability to suppress TCR signaling and T cell activation through its cAMP-elevating activity. These results show that the IS responds solely to local elevations of cAMP and provide evidence that potent compartmentalization mechanisms are operational in T cells to contain cAMP close to the site of production, even when produced at supraphysiological levels.

Project description:Treatment of cultured tracheal smooth-muscle cells (TSM) with phorbol 12-myristate 13-acetate (PMA) (100 nM) or bradykinin (100 nM) elicited enhanced basal and guanosine 5'-[beta gamma-imido]-triphosphate-stimulated adenylate cyclase activities in subsequently isolated membranes. Combined stimulation of cells was non-additive, indicating that both agents activate adenylate cyclase via similar routes. Both PMA (100 nM) and bradykinin (100 nM) allowed the alpha subunit of Gs to act as a more favourable substrate for its cholera-toxin-catalysed ADP-ribosylation in vitro. PMA was without effect on intracellular cyclic AMP in control cells. However, constitutive activation of Gs by treatment in vivo with cholera toxin (0.5 ng/ml, 18 h) sensitized the cells to PMA stimulation, resulting in a concentration-dependent increase in intracellular cyclic AMP accumulation (EC50 = 7.3 +/- 2.5 nM, n = 5). Bradykinin also elicited a concentration-dependent increase in intracellular cyclic AMP (EC50 = 63.3 +/- 14.5 nM, n = 3). Constitutive activation of Gs resulted in an increased maximal response (10-fold) and potency (EC50 = 6.17 +/- 1.6 nM, n = 3) to bradykinin. This response was not affected by the B2-receptor antagonist, NPC567 [which selectively blocks bradykinin-stimulated phospholipase C (PLC), with minor activity against phospholipase D (PLD) activity]. Des-Arg9-bradykinin (a B1-receptor agonist) was without activity. These results suggest that the receptor sub-type capable of activating PLD may also be stimulatory for cyclic AMP accumulation. Furthermore, pre-treatment of the cells with butan-l-ol (0.3%, v/v), which traps phosphatidate derived from PLD reactions, blocked the bradykinin-stimulated increase in intracellular cyclic AMP. These studies suggest that there may be a causal link between PLD-derived phosphatidate and the positive modulation of adenylate cyclase activity. In support of this, the concentration-dependence for bradykinin-stimulated adenylate cyclase activity was identical with that of bradykinin-stimulated phospholipase D activity (EC50 = 5 nM). Bradykinin, but not PMA, was also capable of eliciting the inhibition of cyclic AMP phosphodiesterase activity in TSM cells (EC50 > 100 nM) via an unidentified mechanism. These studies indicate that cross-regulation between the cyclic AMP pathway and phospholipid-derived second messengers in TSM cells does not occur as a consequence of PLC-catalysed PtdIns(4,5)P2 hydrolysis, but may involve, in part, PLD-catalysed phosphatidylcholine hydrolysis.

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Predominant expression of type-VI adenylate cyclase in C6-2B rat glioma cells may account for inhibition of cyclic AMP accumulation by calcium.

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