The FASEB Journal article fj.14-268466. Published online April 30, 2015.

The FASEB Journal • Research Communication

Adenylyl cyclases 5 and 6 underlie PIP3-dependent regulation Gopireddy Raghavender Reddy,* Hariharan Subramanian,† Alexandra Birk,* Markus Milde,‡ Viacheslav O. Nikolaev,† and Moritz B¨unemann*,1 *Faculty of Pharmacy, Philipps University, Marburg, Marburg, Germany; †Institute of Experimental Cardiovascular Research, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; and ‡ Interfakult¨ares Institut f¨ur Biochemie, University of Tu¨ bingen, Tu¨ bingen, Germany Many different neurotransmitters and hormones control intracellular signaling by regulating the production of the second messenger cAMP. The function of the broadly expressed adenylyl cyclases (ACs) 5 and 6 is regulated by either stimulatory or inhibitory G proteins. By analyzing a well-known rebound stimulation phenomenon after withdrawal of Gi protein in atrial myocytes, we discovered that AC5 and -6 are tightly regulated by the second messenger PIP3. By monitoring cAMP levels in real time by means of F¨orster resonance energy transfer (FRET)–based biosensors, we reproduced the rebound stimulation in a heterologous expression system specifically for AC5 or -6. Strikingly, this cAMP rebound stimulation was completely blocked by the PI3K inhibitor wortmannin, both in atrial myocytes and in transfected human embryonic kidney cells. Similar effects were observed by heterologous expression of the PIP3 phosphatase and tensin homolog (PTEN). However, general kinase inhibitors or inhibitors of Akt had no effect, suggesting a PIP3-dependent mechanism. These findings demonstrate the existence of a novel general pathway for regulation of AC5 and -6 activity via PIP3 that leads to pronounced alterations of cytosolic cAMP levels.—Reddy, G. R., Subramanian, H., Birk, A., Milde, M., Nikolaev, V. O., B¨unemann, M. Adenylyl cyclases 5 and 6 underlie PIP3-dependent regulation. FASEB J. 29, 000–000 (2015). www.fasebj.org ABSTRACT

Key Words: cAMP • GPCR • PI3K • Gai • FRET CAMP IS A UBIQUITOUS SECOND

messenger that transmits signaling information from receptors to many different effector proteins within the cell. Because of its involvement in a wide range of cellular processes, such as cardiac contraction, insulin secretion, and modulation of neurotransmission (1), cAMP levels are highly regulated at the levels of both production and hydrolysis (2). cAMP is produced by adenylyl cyclases (ACs) which are under tight control of various modulators such as heterotrimeric G Abbreviations: AR, adrenoreceptor; AC, adenylyl cyclase; ACh, acetylcholine; AKAR, A kinase activity reporter; AKT-PH-YFP, pleckstrin homology domain of AKT protein kinase fused to yellow fluorescent protein; CFP, cyan fluorescent protein; Epac, exchange protein directly activated by cAMP; FRET, F¨orster resonance energy transfer; Gi/o, inhibitory/other G proteins (continued on next page)

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proteins, Ca2+, and protein kinases (3). Elevated cAMP levels activate a variety of different effectors, including cAMP-dependent protein kinase A (PKA) (4), cyclic nucleotide gated ion channels (5), and exchange protein directly activated by cAMP (Epac) (6). All membrane-bound AC isoforms are stimulated by stimulatory G proteins (Gs). The function of the broadly expressed AC5 and AC6 is attenuated by inhibitory Gi proteins and Ca2+ (7, 8). These 2 isoforms are widely expressed throughout the body and play important roles in mediating neurotransmission, including sympathetic and parasympathetic responses (9, 10). The importance of AC5 and -6 in the cardiovascular system is exemplified in the heart. Both AC5- and -6-knockout (KO) mice exhibit pronounced cardiac phenotypes, as both are involved in mediating the sympathetic regulation of the heart (11). Inhibition of these isoforms via M2-muscarinic acetylcholine receptors (M2AChR) results in the negative chronotropic and dromotropic response during vagal activity (12). Those isoforms are extremely sensitive toward activation of Gi/o-coupled receptors, most likely because of their prolonged interactions with Ga subunits (13). In addition to the direct inhibition of cAMP responses via inhibitory G (Gi) proteins, a second level of cAMP regulation via Gi proteins has been reported (14–16), for which the mechanism is not yet clear. In cardiac myocytes, most prominent in atrial myocytes, it has been observed that M2AChR activation not only inhibits b-adrenergic responses, it also facilitates b-adrenergic responses immediately after the termination of M2AChR stimulation (defined as a rebound response) (14–18). These rebound effects can explain physiologic phenomena such as postvagal tachycardia (19) and arrhythmogenic mechanisms (20). The underlying mechanism is disputed, but it has not been linked to the regulation of ACs (21). Based on initial observations that showed a cAMP rebound response specifically in AC5- and -6-expressing cells we sought to elucidate the underlying pathway. Signaling via Gi proteins could lead to the regulation of several different pathways, most of which are mediated by Gbg 1

Correspondence: Philipps-Universit¨at, Marburg, Karl-vonFrisch-Strasse 1, D-35032 Marburg, Germany. E-mail: moritz. [email protected] doi: 10.1096/fj.14-268466 This article includes supplemental data. Please visit http:// www.fasebj.org to obtain this information.

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subunits (22–24). A prominent example is the activation of PI3Kg via pertussis-toxin (PTX)-sensitive proteins. This activation of PI3Kg has been linked to a direct association of its catalytic domain with the bg-subunits of Gi proteins (25). It primarily leads to the generation of phosphatidylinositol (3,4,5)-triphosphate (PIP3), which represents an important signaling molecule of the inner leaflet of the plasma membrane. PIP3 binds to many (probably hundreds) proteins (26), such as kinases and ion channels (27–29). Many, but not all, of these proteins bind PIP3 via their pleckstrin homology (PH) domains, such as phosphoinositide-dependent kinase (PDK1) and Akt serine/threonine kinase (28, 30). PIP3 binding to proteins contributes to membrane targeting of cytosolic proteins and regulation of their activity. So far, it is unclear whether the Gi/o protein-activated PI3K signaling axis is connected to the cAMP rebound response described above. In the present study, we applied real-time cAMP imaging in cardiac myocytes and in heterologous expression systems and discovered a novel PIP3-dependent regulatory pathway that leads to robust regulation of AC5 and -6 and thereby to the cAMP rebound phenomenon. MATERIALS AND METHODS Plasmids All plasmids encoding for hAC5, hAC6, and hAC4 [wild-type (wt)] have been described elsewhere (31, 32). Phosphatase and tensin homolog (PTEN) was provided by Dr. Dominik Oliver (Philipps University, Marburg, Germany). FRET-based biosensors The Epac1-camps sensor has been described (33). AKAR4 was provided by Dr. Jin Zhang (Johns Hopkins University School of Medicine, Baltimore, MD, USA); Eevee-PKC was kindly provided by Dr. Kazuhiro Aoki (Kyoto University, Kyoto, Japan); and AKTPH-YFP (pleckstrin homology domain of AKT protein kinase fused to yellow fluorescent protein) was provided by Dr. Oliver. Reagents AS1949490 was purchased from Tocris Bioscience (Wiesbaden, Germany); platelet-derived growth factor (PDGF) BB from Miltenyi Biotec (Bergisch Gladbach, Germany); and staurosporine, rolipram, cilostamide, and SH-5 from Santa Cruz Biotechnology (Heidelberg, Germany). Unless otherwise noted, all other reagents were obtained from Sigma-Aldrich (Seelze, Germany).

(continued from previous page) Gs, stimulatory G proteins; HEK, human embryonic kidney (cell); Iso, isoprenaline; LED, light-emitting diode; M2AChR, M2muscarinic acetylcholine receptor; NE, norepinephrine; PDE, phosphodiesterase; PDGF, platelet derived growth factor; PDK1, phosphoinositide-dependent kinase; PH, pleckstrin homology; PIP3, phosphatidylinositol (3,4,5)-triphosphate; PTEN, phosphatase and tensin homolog; PTX, pertussis toxin; qRT-PCR, quantitative RT-PCR; SH, Src homology; SHIP2, SH2 domain containing inositol 5-phosphatase; wt, wild-type

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Cell culture and transfection HEK293T and HeLa cells were cultured in DMEM supplemented with fetal calf serum, L-glutamine, penicillin, and streptomycin (32) and were transfected with Effectene transfection reagent (Qiagen, Hilden, Germany). Transfections were stopped the next morning with normal culture medium or yohimbine-supplemented (100 nM) culture medium for Epac/a2A-adrenoreceptor (AR) experiments, to prevent serum activation of the a2A-AR. Approximately 24 h after transfection, the cells were split onto polyL-lysine-coated glass coverslips and analyzed the following day. For the cAMP experiments, 0.25 mg Epac1-camps (or AKAR4), 0.3 mg AC (type 4, 5, or 6), and 0.1 mg receptor (a2A-AR or M2AChR) were used. For the AKT-YFP translocation assay, we used 0.5 mg AKT-YFP sensor, 0.5 mg AC5, and 0.5 mg a2A-AR. Empty pcDNA3 was used to adjust the transfection mixture to the final amount of 1.35 mg DNA for cAMP measurements and 2.0 mg DNA for the AKT-YFP assay. For the F¨orster resonance energy transfer (FRET) biosensor activation measurements, including AKAR4, Eevee PKC, and AKT-YFP-PH, cells were transfected with only a 1 mg concentration of their respective cDNA.

FRET measurements of cAMP in intact cells FRET measurements were performed on an inverted fluorescence microscope (Axiovert 100; Zeiss, Jena Germany) equipped with a PLAN/Apo N 360/1.45 oil objective (Olympus, Hamburg, Germany) and 2 (440–500 nm) cooled light-emitting diodes (LEDs; pE-100; CoolLED, Andover, United Kingdom) (34). For FRET measurements, cyan fluorescent protein (CFP) was excited by using an ET 436/20 (Chroma, Olching, Germany) excitation filter in combination with an LP 458 beam splitter (Semrock, Rochester, NY, USA). For direct YFP excitation, the 500 nm LED was used in combination with the CFP/YFP dual-band HC filter set (Semrock). Simultaneous detection of CFP and YFP fluorescence was achieved by placing an Optosplit II (Cairn Research, Kent, United Kingdom) equipped with an LP 505 beamsplitter and HC 470/24 (CFP) and HC 525/39 (YFP) emission filters (all from Semrock) in front of the SPOT Pursuit Camera (SPOT Imaging Solutions, Sterling Heights, MI, USA). Normalization was performed by dividing the resulting FRET ratio by the initial values.

Translocation measurements Translocation experiments were performed on an inverted confocal microscope (IX 71; Olympus) with a 3100 oil immersion objective (UPlanSApo 3100/1.40 oil; Olympus), a CCD camera (EM-CCD Digital Camera; Hamamatsu, Herrsching am Ammersee, Germany), equipped with a confocal imaging system (VT-HAWK; VisiTech International, Sundeland, United Kingdom) and the following filters: T495lpxr, ET 470/403 and 535/30m (Chroma). The samples were illuminated with a 491 nm laser (VisiTech International). Fluorescence recordings were processed with VoxCell Scan (VisiTech International). The cells were superfused with buffer containing (in millimolar) 137 NaCl, 5.4 KCl, 2 CaCl2, 1 MgCl2, and 10 HEPES (pH 7.4) or agonist-containing buffer. For translocation experiments AKT-PH-YFP transfected cells were directly excited at 491 nm and YFP fluorescence recorded at 0.2 Hz. YFP emission images were collected every 5 s with a 250 ms integration time. To analyze membrane translocation of AKT-YFP, we defined 2 regions of interest: one in the cell cytosol (Fcytosol) and the other around the whole cell (Ftotal). Then the quotient Fcytosol/Ftotal was calculated. The 20 time points of Fcytosol/Ftotal values before the first stimulation were averaged and used as a reference. The ratio of raw Fcytosol/Ftotal to the reference value was defined as the normalized Fcytosol/Ftotal.

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Cardiac myocyte isolation and biosensor expression To monitor cAMP levels in cardiac myocytes, we used transgenic mice with ubiquitous expression of the cAMP FRET sensor Epac1camps (35). We isolated atrial myocytes from the adult transgenic mice by Langendorff perfusion. Isolated cells were seeded onto coverslips, mounted onto Ibidi (Madison, WI, USA) perfusion chambers, positioned on the stage of a Ti-S inverted fluorescence microscope (Nikon, Tokyo, Japan), and superfused with buffer (see Translocation measurements). Images were obtained with a 360 oil immersion objective (CFI P-Apo 60- Lambda, Nikon) and CCD camera (ORCA-03G; Hamamatsu Photonics). CFP excitation was achieved with a 440 nm CoolLED light source with an ET 436/30m excitation filter and a DCLP455 dichroic mirror. For ratiometric CFP and YFP FRET recordings, we used a DV2 Dual View equipped with the 05-EM filter set (both Photometrics, Huntington Beach, CA, USA) containing the 505 DCXR dichroic mirror plus ET 480/30m and ET 535/40m emission filter for CFP and YFP (all from Chroma), respectively. Cells were excited for 10–50 ms once every 5 s. FYFP was corrected for direct excitation and bleed-through. FRET ratios were calculated as the ratio of corrected YFP over CFP emissions (FYFP/FCFP). The ratio of FYFP/ FCFP value to the baseline value was defined as the normalized value (FYFP/FCFP).

and counted in a liquid scintillation counter (1600 TR; Packard; GMI, Inc., Ramsey, MN, USA). Software and statistics Data are presented as means 6 SEM. Statistics were obtained by 1-way ANOVA with the Bonferroni post hoc test to determine the significance between treatments. As an exception, the data shown in Fig. 1 were evaluated with the unpaired Student’s t test. Differences were considered significant at P , 0.05. To evaluate the data, we used Excel 2007 (Microsoft Corp., Redmond, WA, USA) and OriginPro 9.1 (OriginLabs, Northampton, MA, USA). We processed the confocal images with ImageJ (1.48; http://imagej. nih.gov/ij/; National Institutes of Health, Bethesda, MD, USA) and Photo Paint X4 (Corel, Ottawa, ON, Canada).

RESULTS Muscarinic receptor-mediated cAMP rebound response in cardiac myocytes

Total RNA was extracted from HEK and HeLa cells transfected with either ACs (AC5 or -6) or pcDNA3, together with Epac1camps and a2A-AR. RNA samples were treated with TURBO DNAse (Life Technologies-Ambion, Carlsbad, CA, USA) to remove DNA contamination. cDNA was prepared with the iScript cDNA synthesis kit (Bio-Rad, Munich, Germany). Real-time quantitative (q)RT-PCR was performed on a StepOnePlus RealTime PCR system (Life Technologies-Applied Biosystems, Forster City, CA, USA), using iTaq Universal SYBER Green Supermix (Bio-Rad). Samples were measured in triplicate. The qRT-PCR data were processed and analyzed by the 22DDCT method, where cycle threshold values were first normalized to internal control (GAPDH) and then to the mean of the control sample (defined as 1). Primers were used as follows: AC5, forward 59-GCAC AGGAGCACAACATCAG-39, reverse 59-CACGATGAGCACGTAGATGAG-39; AC6, forward 59-CAAACAATGAGGGTGTCGAGT-39, reverse 59-TGCTACCAATCGTCTTGATCTT-39 (transcripts of AC5 and AC6 are specific for human); and GAPDH, forward 59CCAGGCGCCCAATACG-39, reverse 59-CCACATCGCTCAGA CACCAT-39.

To measure real-time dynamics of the well-described cAMP rebound phenomenon after withdrawal of muscarinic stimulation (14, 16, 37), we chose atrial myocytes, because overall they show more responsiveness to muscarinic receptor stimulation than do ventricular myocytes (38). We therefore acutely isolated atrial myocytes from transgenic mice expressing the well-established cAMP biosensor Epac1-camps (35) and imaged cAMP levels by means of FRET. Upon exposure to low concentrations of the b-AR agonist isoprenaline (Iso), cAMP levels moderately increased, as indicated by a submaximal decrease in the FRET signal (Fig. 1Aa). Addition of ACh for 2 min reduced cAMP to basal levels (Fig.1Ab). Within 2 min after withdrawal of ACh, the FRET signal dropped significantly below the values reached before the exposure of ACh (Fig. 1A–C, c vs. b), indicating a robust increase in cAMP, which represents the cAMP rebound phenomenon. Based on the fact that AC5 and -6 are the dominant cardiac isoforms of ACs (39) and that at least AC5 is functionally expressed in atrial myocytes (40), we tested whether the observed cAMP rebound stimulation can be reproduced in HEK293T cells expressing Epac1-camps, AC5-wt, and M2AChR.

Ligand-binding assay

cAMP rebound response is specific for AC5 and -6

To determine a2A-AR expression levels, we performed a radioligand binding assay, with minor modifications to the published method (36). Cell membranes were prepared from HEK293T cells transfected with a2A-AR, together with Epac1-camps and AC5 or from nontransfected control cells. The cells were washed once with warm PBS and detached with buffer containing (in millimolar) 150 NaCl, 20 NaH2PO4 H2O, 3 MgCl2, 1 EDTA, and 1 tablet of protease inhibitor cocktail (Roche, Penzberg, Germany) per 10 ml lysis-buffer (pH 7.4). Membrane receptor–specific binding was determined by incubating saturating concentrations (700 nM) of [3H]-clonidine hydrochloride (Perkin Elmer, Billerica, MA, USA) overnight at 4°C, and 10 mM yohimbine hydrochloride was used to determine nonspecific binding. GF/C glass fiber filters (Merck Millipore, Darmstadt, Germany) were used to separate bound and unbound ligand by vacuum filtration. The filters were washed 4 times with ice-cold 50 mM Tris washing buffer (pH 7.4)

We adapted the stimulation protocol for transfected HEK293T cells by lowering the ACh concentrations, accounting for the huge spare receptor phenomenon observed for heterologously expressed Gi-coupled receptors (32). Similar to myocytes, cAMP levels increased after stimulation of endogenous b-ARs with 3 nM Iso, as reflected by a decrease in FRET. This rise in cAMP was inhibited to baseline by the addition of 0.3 nM ACh, attributable to Gi/o-mediated inhibition of ACs. However, the subsequent washout of ACh resulted in a rapid reversal of the inhibitory effect, leading to an increase in cAMP levels well beyond that observed in the presence of Iso before exposure to ACh (Fig. 1D–F) or Iso alone (Fig. 2A, blue trace) and close to the sensor saturation determined by exposure to 10 mM Iso (data not shown). This rebound induced

Total RNA extraction and quantitative real-time PCR

PIP3-MEDIATED RISE IN CAMP

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Figure 1. Acetylcholine-induced rebound stimulation of cAMP in murine adult atrial myocytes and HEK293T cells. A) FRETbased recordings of alterations in cAMP were conducted in single freshly isolated atrial cells from mice transgenically expressing the cAMP biosensor Epac1-camps, as depicted in this representative experiment. The fluorescence of the biosensor was excited at 436 6 15 nm, and emissions of CFP (480 6 15 nm) and YFP (535 6 20 nm) were detected with ratiometric imaging. The FYFP (corrected for bleed-through of CFP; yellow trace):FCFP ( blue trace) emission ratio (black trace) was normalized to initial values and plotted over time. Horizontal bars: exposure of cells to Iso and ACh. B) Pseudocolor FRET ratio images of mouse atrial cardiac myocytes expressing Epac1-camps are shown at the time points in (A). The calibration bar indicates FYFP/FCFP. C) Iso (3 nM)-evoked FRET changes were quantified as alterations in FYFP/FCFP (norm.) and are plotted for Iso before ACh (Aa), during costimulation with ACh (Ab), and after withdrawal of ACh (rebound stimulation, Ac) (n = 6). D) M2AChR-mediated cAMP responses in HEK293T cells expressing M2AChR, AC5, and Epac1-camps. E) FRET ratio images of HEK293T cells expressing Epac1-camps taken at the time points in (D), with calibration bar. F) Iso-evoked FRET changes were quantified as alterations in FYFP/FCFP (normalized) (n = 6–8). Scale bar, 10 mm.

a decrease in the Epac1-camps FRET by 10.2 6 1.4% compared to 6.1 6 1.6% (Fig. 1F, a–c). In principle, the use of Epac1-camps should allow the absolute quantification of cAMP concentrations in intact cells. We attempted to translate FRET values measured in HEK293T cells into cAMP concentrations by extrapolating Epac1-camps FRET calibrations performed in vitro [calibration procedure described in Nikolaev et al. (33)]. Thereby, we estimated an ACh rebound-attributable increase in cAMP from 0.9 to 3.1 mM. In cells where pcDNA-AC5 was replaced by empty vector, cAMP levels were significantly lower after withdrawal of ACh (Fig. 2A, B). We further confirmed that other Gi-coupled receptors, such as the a2A-AR, could also induce cAMP rebound 4

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stimulation in an AC5-dependent manner (Fig. 2C, D). The a2A-AR expression was measured by means of radioligand binding. The expression of a2A-AR was 10.98 6 0.9 pmol/mg membrane protein, and no specific binding of [3H]-clonidine hydrochloride was detected in the nontransfected control cells. Similar cAMP rebound responses were obtained in HEK293T cells transfected with cDNA for AC6 instead of AC5, but not in mock-transfected cells (Fig. 2E, F), which revealed an AC5 and -6 dependency of rebound stimulation. We also ruled out that a peculiarity of HEK293T cells was responsible for the rebound stimulation, by demonstrating a similar effect in HeLa cells (Fig. 3A, B). To confirm that AC5 and -6 transfection induces higher

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Figure 2. Gai-induced cAMP rebound stimulation is specific for AC5 and -6. FRET recordings were obtained from single HEK293T cells transfected with cDNA for Epac1-camps and the indicated receptors or ACs. Cells were subjected to the indicated agonist exposure protocol to elicit cAMP rebound stimulation. A) Summarized FRET recordings (n = 8–10) from cells that were transfected with M2AChR (all) and AC5 (red and blue) or empty pcDNA3 (black). B) Isoevoked alterations in FRET of the experiments shown in (A) were quantified as (FYFP/FCFP normalized at time point b) minus (FYFP/FCFP norm. at time point a) for all 3 conditions. C) Summarized FRET recordings showing alterations in cAMP induced by application and subsequent withdrawal of 0.3 nM NE (with the exception of experiments shown in black) in HEK cells transfected with Epac1camps or a2A-AR, with or without AC5 (n = 6–7). The FRET signal of Epac1-camps was normalized to initial values. D) Iso-evoked FRET changes between indicated time points (b–a) of the experiments shown in (C). E) Similar experiments were performed in cells transfected with M2AChR and with or without AC6 (n = 6–7). F) Iso-induced alterations in the normalized FRET signal were plotted as described in (C). *P , 0.05.

expression than endogenous levels, we measured AC5 and -6 mRNA expression levels in both HEK293T and HeLa cells by means of real-time PCR. The transfection efficiency of ;20–50% of AC5 and -6 mRNA was increased by at least 8-fold in both cell types relative to mock-transfected cells (Supplemental Fig. S1A, B). PIP3-MEDIATED RISE IN CAMP

Based on previously proposed mechanisms (16, 21) underlying ACh-induced cAMP rebound stimulation, we tested whether the observed rebound stimulation is inhibited by pretreatment with PTX. PTX-treated cells (30 ng/ml for 4–6 h) were effectively uncoupled from Gi proteins and exhibited a significantly attenuated cAMP 5

Figure 3. cAMP rebound stimulation is mediated via Gai and is not dependent on the cell type. A) Averaged FRET recordings showing cAMP alterations in HeLa cells. Cells transfected with M2AChR or Epac1-camps, with or without AC5 (n = 6–7), were subjected to the cAMP rebound stimulation protocol (n = 5–7). B) Iso-induced alterations in normalized (norm.) FYFP/FCFP (b–a) were determined after withdrawal of ACh and compared to that in cells transfected with or without AC5. C) Treatment of AC5- and a2A-AR-expressing cells with PTX (30 ng/ml; 4–6 h) attenuated both the initial NE-induced decline in cAMP and the subsequent rebound stimulation (n = 6–8). D) Quantification as in (B). E) Similar experiments were performed in cells transfected with the indicated AC isoforms (n = 7–8). *P , 0.05.

rebound phenomenon after withdrawal of a2A-AR stimulation (Fig. 3C, D). By comparing cAMP rebound responses in HEK293T cells transfected with AC4, -5, or -6, we found that a cAMP rebound response was induced in cells expressing AC5 or -6, but not in those expressing AC4. Functional and dominant expression of AC4 was indicated by the complete lack of Gi-mediated inhibition (Fig. 3E). Collectively, these data indicate that the Gai6

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induced cAMP rebound phenomenon was mediated via PTX-sensitive G proteins and AC5 and -6. Two studies reported that the phosphodiesterase (PDE) 3-dependent NO-cGMP pathway may be involved in the Giinduced rebound increase of cAMP (16, 41). Therefore, we examined whether PDE3- and -4-specific inhibitors might affect the Gi-induced cAMP rebound responses in HEK293T cells. We found that neither rolipram (10 mM)

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nor cilostamide (10 mM) attenuated the Gai-induced rebound response (Supplemental Fig. S1C–G). It has been reported that a selective PKA inhibitor diminished Gai-induced rebound responses via cAMPdependent PKA in atrial myocytes (16). We therefore pretreated cells with the specific PKA inhibitor KT5720 (1 mM) and found the cAMP rebound stimulation to be unaffected (Fig. 4A, B). Furthermore, we tested whether the cAMP rebound response is translated into altered PKA activity. We could confirm this effect by using the PKAspecific FRET biosensor AKAR4 (Fig. 4C–E). The AKAR4 sensor was also used to demonstrate effective inhibition of PKA by KT5720 (Fig. 4C) under conditions similar to those used for the experiments shown in Fig. 4A. Furthermore, the broad-spectrum protein kinase inhibitor staurosporine (1 mM) did not attenuate Gai-dependent cAMP rebound responses (Fig. 4F, G) under conditions where it virtually abrogated PKC activity (Fig. 4H). These results led us to conclude that Gi-induced rebound stimulation is not mediated by PKA, PKC, PKG, and other staurosporine sensitive kinases. Gai -induced cAMP rebound stimulation is dependent on PIP 3 Class1B PI3K (also known as PI3Kg) can be activated via PTX-sensitive Gi proteins (25). We therefore tested the involvement of PI3K on Gai-induced cAMP rebound responses by pretreatment with the PI3K inhibitor wortmannin. Application of 1 mM wortmannin significantly attenuated the AC5- and -6-mediated cAMP rebound stimulation (reduction of FRET decrease from 11.2 6 1.2 to 5.6 6 1.5% for AC5; 11.8 6 0.9 to 0.7 6 1.3% for AC6). Based on in vitro cAMP calibration of Epac1-camps, we estimated that wortmannin reduced cAMP concentrations from 4.1 mM to levels of ;0.7 mM for AC5 and 4.3–0.7 mM for AC6 (Fig. 5A–D). Indeed, exposure to wortmannin also significantly lowered b2-AR-mediated elevation of cAMP levels in cells expressing AC5 and -6 but not AC4 (Fig. 5E–H). We controlled for robust PIP3 depletion caused by wortmannin by demonstrating wortmannin-dependent translocation of AKT-PH-YFP from the plasma membrane to the cytosol (Supplemental Fig. S2A–C). To delineate which phosphoinositol species may be involved in the PI3K-dependent cAMP rebound stimulation, we induced expression of the PIP3-specific phosphatase PTEN, which is known to dephosphorylate PIP3, thereby lowering PIP3 levels (42). Overexpression of PTEN in cells that were cotransfected with AC5 significantly lowered cAMP rebound stimulation, as reflected by a reduction in the decline in FRET, from 12.1 6 0.9 to 7.8 6 1.4% (Fig. 6A, B). We estimated that cAMP concentrations after norepinephrine (NE) withdrawal were decreased from 6.0 (control) to 1.0 mM in the presence of PTEN. Having demonstrated that both PTEN and wortmannin exerted very similar effects on AC5-mediated cAMP production, we conclude that the generation of PIP3 is necessary for cAMP rebound stimulation. We next examined a possible involvement of PIP2 on Gai-mediated cAMP rebound responses by pretreatment with the Src homology (SH)2 domain containing inositol 5-phosphatase (SHIP2) inhibitor AS1949490. SHIP is a phosphatase that catalyzes the PIP3-MEDIATED RISE IN CAMP

conversion of PIP3 into PIP2 (43). Application of 1 mM AS1949490 did not attenuate Gai-dependent cAMP rebound stimulation (Fig. 6C, D), indicating that this phenomenon is more likely to be dependent on PIP3 than on PIP2. We further tested whether important downstream effectors of PIP3 are involved in the generation of the cAMP rebound stimulation (27, 30). We examined the involvement of Akt by pretreatment with the Akt-specific inhibitor SH-5. Application of 1 mM SH-5 did not affect Gai-mediated cAMP rebound stimulation (Fig. 6E, F). Indeed, the data collected earlier indicated that PDK1 also is not involved (Fig. 4F), as we did not observe any effect of the nonselective kinase inhibitor staurosporine (which also inhibits PDK1) on the rebound response. As a positive control for SH-5 we tested for Akt inhibition by using the AKT-PH-YFP sensor (Supplemental Fig. S2D). These data suggest that PIP3 itself either directly or indirectly mediates Gai-induced cAMP rebound responses. To test whether the moderate a2A-AR stimulation used in the cAMP rebound protocol actually leads to an increase in PIP3, we tested for NE-mediated translocation of AKTPH-YFP in HEK cells. Based on the above-mentioned finding that in the absence of wortmannin, AKT-PH-YFP is completely localized at the plasma membrane (Supplemental Fig. S2A), these experiments were performed in the presence of low (subsaturating) concentrations of wortmannin (50 nM; Supplemental Fig. S2B). Our results clearly demonstrate that a2A-AR-mediated Gi stimulation does indeed increase PIP3 levels as reflected by an increase in AKT-PH-YFP membrane localization (Fig. 7A). As expected, this increase in PIP3 levels induced via activation of Gi-coupled receptors was completely abolished by pretreatment with PTX (Fig. 7B, C). These findings suggest that PI3K mediates the Gai-induced cAMP rebound stimulation of AC5 and -6 expressing cells. G protein-independent elevations of PIP3 potentiate b-adrenergic stimulation of cAMP production We tested whether elevation of PIP3 induced by means other than Gi activation would sensitize AC5. Therefore, we stimulated PDGF receptors, which are well known to activate PI3K activity in many cells, including HEK293T cells (44). As expected, PDGF receptor stimulation elevated PIP3 levels, as determined by AKT-PH-YFP translocation (Supplemental Fig. S2C). Stimulation of Epac1-campsexpressing cells with 50 ng/ml PDGF-BB significantly increased Gas-induced cAMP levels compared with those in control conditions (Fig. 7D, E). These results strongly support the hypothesis that PIP3 mediates the Gai-induced cAMP rebound stimulation in AC5-expressing cells. PIP3-mediated Gi-induced cAMP rebound responses in cardiac myocytes Having demonstrated that the Gai-mediated cAMP rebound stimulation is induced via PIP3-dependent stimulation of AC5 and -6 activity by means of a heterologous expression system, we tested whether, in atrial cardiac myocytes, the ACh-induced cAMP rebound phenomenon is also mediated by PIP3. We therefore tested atrial 7

Figure 4. Protein kinases are not involved in Gai-induced cAMP rebound stimulation. A) Averaged FRET recordings of HEK293T cells expressing Epac1-camps, AC5, and a2A-AR. Cells were preincubated with 1 mM KT5720 or vehicle for 30 min (n = 6–8). B) Iso-evoked alterations in the FRET were measured after withdrawal of NE (b–a) and compared with those in cells treated with vehicle or KT5720. C) Iso-evoked changes in FRET of the PKA biosensor AKAR4 in cells that were pretreated with a low (submaximum) concentration of KT5720 (50 nM), to reduce high basal phosphorylation of this sensor in HEK293T cells. We then tested whether 1 mM KT5720 would attenuate the Iso response (C, n = 5). D) To test how the cAMP rebound stimulation is translated into alterations in PKA activity, we measured FRET in cells transfected with AKAR4 or a2A-AR, with or without AC5 (n = 9–10). E) Iso-evoked alterations in AKAR4-FRET measured after the withdrawal of NE (b–a). F) Summarized FRET recordings obtained in response to the cAMP rebound stimulation protocol of HEK293T cells transfected with a2A-AR, Epac1camps, and AC5. Before the measurement, cells were incubated with 1 mM staurosporine or vehicle (n = 8–10) for 30 min. G) Data shown in (F) were quantified in comparison with Iso-evoked alterations in FRET subsequent to withdrawal of NE. H) Inhibition of phorbol ester (tetracdeanoyl-phorbol-13-acetate; TPA)-mediated PKC activation by staurosporine was measured with the Eevee-PKC-FRET biosensor, with averaged FRET recordings normalized to initial values of cells preincubated with or without staurosporine (n = 5–6). *P , 0.05.

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Figure 5. cAMP rebound stimulation is mediated through PI3K. A) Epac1-camps-based measurement of cAMP rebound stimulation was performed in HEK293T cells transfected with a2A-AR, with or without AC5. Cells were preincubated with 1 mM PI3K-specific inhibitor wortmannin or vehicle for 30 min (n = 10–11). B) Iso-evoked alterations in FYFP/FCFP (normalized) were quantified as described in Fig. 2B and compared for the indicated conditions (b–a). a2A-AR-mediated cAMP rebound stimulation in AC6-expressing cells was (C) measured and (D) quantified , (n = 7–8). E–G) Wortmannin’s effects on Iso (3 nM)-mediated elevations in cAMP levels were compared in cells expressing Epac1-camps, as well as (E) AC4 , (F) AC5, or (G) AC6. FRET recordings of individual cells treated with or without 1 mM wortmannin were normalized (norm.) to initial values, averaged, and plotted over time (n = 7). H) Iso-evoked FRET changes of (E), (F), and (G) were quantified as alterations in FYFP/FCFP (norm.). *P , 0.05; ns, not significant.

myocytes from Epac1-camps-expressing transgenic mice for wortmannin sensitivity of the cAMP rebound stimulation. Wortmannin (1 mM) markedly and significantly reduced the ACh-induced cAMP rebound levels, suggesting that cAMP rebound stimulation in murine PIP3-MEDIATED RISE IN CAMP

cardiac myocytes is also largely dependent on PIP3-mediated AC sensitization (Fig. 8A, B). In an intriguing finding, similar to AC5-expressing HEK293T cells, wortmannin had a tendency to lower AC activity in response to b-AR stimulation (Fig. 5F, G). 9

Figure 6. cAMP rebound stimulation requires PIP3. a2A-AR-mediated cAMP rebound stimulation in AC5-, Epac1-camps-, or a2A-AR-expressing HEK293T cells was (A) measured and (B) the PTEN-dependent coexpression quantified (n = 7–8). Effects of (C, D) pharmacological SHIP2 or (E, F) Akt inhibition on cAMP rebound stimulation were studied. C, E) Averaged FRET recordings (normalized to initial values) obtained from HEK293T cells transfected with AC5-wt, a2A-AR, and Epac1-camps were plotted over time. Cells incubated with (C) 10 mM SHIP2 inhibitor AS1949490 or vehicle (n = 5–6) or (E) 10 mM Akt-specific inhibitor SH-5 or vehicle (n = 6–7) for 15 min were subjected to the cAMP rebound stimulation protocol. D, F) Iso-evoked FRET changes after withdrawal of NE of the experiments shown in (C) or (E). *P , 0.05; ns, not significant.

DISCUSSION When studying the mechanism of the well-known phenomenon of cAMP rebound stimulation after withdrawal of Gi stimulation in cardiac myocytes (Fig. 1), we discovered a novel regulatory pathway that requires PI3K activity and leads to sensitization of AC5 and -6, as illustrated in Fig. 8C. The Gi/o-mediated cAMP rebound stimulation was detected by real-time cAMP imaging with the FRET-based biosensor Epac1-camps in single intact cells. A robust cAMP rebound stimulation was reproducible after withdrawal of Gi/o stimulation in 2 different heterologous expression systems (HEK 293T and HeLa cells) (Figs. 2A and 3A), only if AC5 or -6, but not if AC4, was expressed (Fig. 3E). 10

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We observed a similar degree of cAMP rebound stimulation after stimulation with M2-AChRs compared to a2A-ARs and found the rebound stimulation to be PTX-sensitive (Fig. 3C), as described previously for cardiac myocytes (14, 37). Therefore, it can be concluded that Gi/o proteins mediate this effect. It has been demonstrated that HEK293T cells endogenously express AC5 or -6 to some extent (45). However, we did not observe a clear wortmannindependent effect on cAMP rebound levels in cells transfected with receptor alone (Fig. 5A), suggesting that contribution of endogenous AC5 and -6 expression was too low to be detected. In general, a rise in intracellular cAMP can be caused by the stimulation of AC activity or an inhibition of PDE

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Figure 7. a2A-AR receptor stimulation leads to elevation of PIP3 levels. A) HEK293T cells transfected with the PIP3-sensitive AKT-PH-YFP translocation sensor AC5 and a2A-AR were subjected to fast 2-dimenaionalconfocal imaging. Cells were preincubated with nonsaturating concentrations of wortmannin (50 nM) to relocate AKTPH-YFP to the cytosol (left). They were then superfused, with or without 1 mM NE, leading to membrane targeting of AKTPH-YFP (right). B) AKT-PH-YFP translocation to the plasma membrane summarized by plotting the ratio of cytosolic AKT-PHYFP relative to total cell fluorescence. To increase cytosolic AKTPH-YFP localization, cells were preincubated with 50 nM wortmannin and stimulated with 1 mM [red (n = 10) and green (n = 5)] or 0.3 nM NE (blue; n = 4). Green: results from cells pretreated with PTX (30 ng/ml for 4–6 h). C) NE-induced alterations in YFP(cytosol)/YFP(total). D) Effect of PDGF receptor-mediated PIP3 production on Isomediated cAMP responses were tested in AC5-expressing cells (n = 8–10). Cells expressing Epac1-camps were (red) or were not (black) incubated with 50 ng/ml PDGF-BB at the indicated time point during FRET imaging. (E) Alterations of FYFP/ FCFP (normalized) on superfusion with 3 nM Iso were analyzed and quantified. *P , 0.05.

activity. Our results clearly argue against a major contribution of PDEs in the cAMP rebound response. Inhibitors of the major PDE isoforms PDE3 and -4 did not attenuate the cAMP rebound response (Supplemental Fig. S1D, F). Also, inhibition of PKA, which is known to stimulate PDE4 activity, had no detectable effect on the observed rebound stimulation. Furthermore, the complete absence of the rebound stimulation when cAMP was produced via AC4 (Fig. 3E) argues against a major role of PDEs in the rebound response. These findings also indicate that PDE3dependent NO-cGMP signaling is not necessary for cAMP rebound stimulation. This finding is in agreement with those in other studies (21, 37), but is in contrast to 2 reports that identified the NO-cGMP signaling axis to contribute to Gai-induced rebound cAMP responses in feline atrial myocytes (16, 41). An important finding is that the cAMP rebound stimulation was observed not only on the level of cAMP, but also by using AKAR4, a FRET reporter of PKA activity (Fig. 4D). PIP3-MEDIATED RISE IN CAMP

The general, potent PI3K inhibitor wortmannin completely abolished cAMP rebound stimulation of AC5 and -6 without having major effects on cAMP production via AC4 (Fig. 5A–D). Using the well-established PIP3 sensor AKTPH-YFP, we were able to demonstrate that stimulation of a2A-AR under conditions very similar to those used to study rebound stimulation led to an increase in PIP3 production (Fig. 7B). The PIP3 levels needed for AC5- and -6-specific stimulation were higher than those needed for translocation of the AKT-PH domain. This difference was deducible from the observation that the overall PI3K activity in the absence of Gi/o stimulation had to be reduced to see Gi/o-mediated translocation of the AKT-sensor (Supplemental Fig. S2A). Wortmannin pretreatment not only fully inhibited cAMP rebound stimulation in AC5-expressing HEK293T cells but also completely abolished this response in adult mouse atrial myocytes (Fig. 8A). These findings suggest that Gai-mediated cAMP rebound stimulation was mediated via a similar PI3K-dependent pathway, in both 11

Figure 8. Wortmannin abolishes rebound stimulation of cAMP in atrial myocytes. A) Isolated murine atrial myocytes from Epac1-camps-expressing mice were preincubated with 1 mM wortmannin or vehicle for 30 min and subsequently subjected to the cAMP-rebound stimulation protocol, as described in Fig.1A (n = 6–9). B) Experimental data derived from (A) were quantified compared with Iso-mediated alterations of FYFP/FCFP (normalized) after withdrawal of ACh. *P , 0.05. C) The proposed PIP3 pathway responsible for Gai-induced rebound stimulation of cAMP.

cardiac myocytes and heterologous expression systems. Expression profiling of ACs in atrial myocytes from guinea pigs suggest that AC5 is expressed in atrial myocytes, together with AC1 (40). Functional evidence of AC5 and -6 expression in atrial myocytes comes from the observation of strong inhibition in response to Gi activation (Fig. 1) (46), which represents a hallmark of AC5 and -6 regulation. Overexpression of the PIP3-specific phosphatase PTEN resulted in an attenuation of the cAMP rebound stimulation similar to that attained with PI3K inhibition (Fig. 6A), whereas both specific Akt inhibitors (Fig. 6E) and the broad-spectrum kinase inhibitor staurosporine failed to affect cAMP rebound stimulation (Fig. 4F). Wortmannin treatment also significantly reduced b-adrenergic responses in cells expressing only AC5 and -6 (Fig. 5E–H). These findings led us to conclude that PIP3 regulates AC5 and -6 activity. This conclusion is strengthened by the observation that surpassing Gai-mediated PI3K activation through PDGF receptor stimulation resulted in a similar stimulation of AC5 (Fig. 7D, E). Wortmannin and other PI3K inhibitors have been implicated in several cellular or tissue models to stimulate basal cAMP levels or increase cardiac contractility (47, 48). This effect has been demonstrated in ventricular cardiac myocytes which express lower amounts of M2AChR as compared to atrial cells (49). These findings seem to be in 12

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contrast to the inhibition of broadly expressed AC5 and -6 reported herein. However, in most tissues or cell types, the determinant for basal cAMP levels is not clear, not even in cells that predominantly express AC5 and -6. Indeed, if we treated nontransfected cells or cells that express AC4 with wortmannin, we did not see a reduction in cAMP concentrations; in some cases we saw instead a tendency toward faster cAMP production in response to b2-adrenergic stimulation (Fig. 5E). Similarly, cardiac-specific PI3Kg-KO mice exhibit an increase in basal contractility, whereas functional impairment of PTEN leads to opposing effects (42). Those data also seem to contradict our findings based on the information that AC5 and -6 are the most abundant cAMP-producing isoforms in the heart. One explanation could be that PI3Kg not only regulates PIP3 levels, but also provides a scaffold for other important regulatory proteins including PDEs (42, 50). Therefore, the overall effect of PIP3 levels on cAMP homeostasis most likely depends on the cellular context. Because of the general importance of PI3K signaling in various tissues and cell types, we predict that PIP3dependent AC regulation represents an important novel regulatory mechanism to fine tune cAMP homeostasis in cells. Whether the PIP3 dependence of AC5 and -6 activity uncovered in the present study is a direct effect of PIP3 on the level of the cyclase or is mediated indirectly via

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unidentified PIP3-dependent regulatory proteins needs to be evaluated in future studies. Taken together, the results of the present study demonstrate that AC5 and -6 underlie a PIP3-dependent regulation and identify the well-known Gai-induced cAMP rebound stimulation initially described in the heart that depends on Gi-mediated PI3K activation.

19. 20.

21.

The authors thank Drs. Dominik Oliver, Jin Zhang, and Kazuhiro Aoki for providing FRET- and PIP3-dependent translocation biosensors. This work was funded in part by Deutsche Forschungsgemeinschaft SFB593. The authors declare no conflicts of interest.

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Received for publication December 4, 2014. Accepted for publication April 17, 2015.

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