Stimulation of Type 1 and Type 8 Ca2+/Calmodulin-sensitive Adenylyl Cyclases by the Gs-coupled 5-Hydroxytryptamine Subtype 5-HT7A Receptor*

Lauren P. BakerDagger , Mark D. NielsenDagger , Soren Impey, Mark A. Metcalf§, Steven W. Poser, Guy Chan, Karl Obrietan, Mark W. Hamblin§, and Daniel R. Storm

From the Department of Pharmacology, University of Washington, Seattle, Washington 98195, the § Department of Psychiatry and Behavioral Sciences, University of Washington, Seattle, Washington 98195 and Veterans Affairs Medical Center, Seattle, Washington 98108

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

The neurotransmitter serotonin (5-hydroxytryptamine, 5-HT) plays an important regulatory role in developing and adult nervous systems. With the exception of the 5-HT3 receptor, all of the cloned serotonin receptors belong to the G protein-coupled receptor superfamily. Subtypes 5-HT6 and 5-HT7 couple to stimulation of adenylyl cyclases through Gs and display high affinities for antipsychotic and antidepressant drugs. In the brain, mRNA for 5-HT6 is found at high levels in the hippocampus, striatum, and nucleus accumbens. 5-HT7 mRNA is most abundant in the hippocampus, neocortex, and hypothalamus. To better understand how serotonin might control cAMP levels in the brain, we coexpressed 5-HT6 or 5-HT7A receptors with specific isoforms of adenylyl cyclase in HEK 293 cells. The 5-HT6 receptor functioned as a typical Gs-coupled receptor in that it stimulated AC5, a Gs-sensitive adenylyl cyclase, but not AC1 or AC8, calmodulin (CaM)-stimulated adenylyl cyclases that are not activated by Gs-coupled receptors in vivo. Surprisingly, serotonin activation of 5-HT7A stimulated AC1 and AC8 by increasing intracellular Ca2+. 5-HT also increased intracellular Ca2+ in primary neuron cultures. These data define a novel mechanism for the regulation of intracellular cAMP by serotonin.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Serotonin (5-hydroxytryptamine, 5-HT)1 is a ubiquitous neurotransmitter that elicits a variety of physiological effects peripherally and centrally (1-3). A growing family of plasma membrane receptors bind 5-HT and mediate its cellular effects (4). All of the 5-HT receptors except 5-HT3 belong to the superfamily of G protein-coupled receptors. The recently cloned 5-HT6 (5, 6) and 5-HT7 (7-10) receptors activate adenylyl cyclase(s) through the heterotrimeric G protein Gs, and expression of these receptors in cultured cells couples serotonin to increases in cAMP (6, 8). 5-HT6 and 5-HT7 display high affinities for antipsychotic and antidepressant drugs including clozapine, amoxapine, and amitryptiline (5-8), suggesting a role for these receptors in affective function. In the brain, the 5-HT6 receptor is most highly expressed in the hippocampus, nucleus accumbens, striatum, and limbic regions, and the 5-HT7 receptor is found in the hypothalamus, hippocampus, and cortex. The distribution of these receptors in brain is consistent with the hypothesis that they play a role in mood and affect (8, 11-13). In addition, the expression of the 5-HT7 receptor in the suprachiasmatic nucleus (SCN) and the ability of serotonin and cAMP to advance the mammalian circadian rhythm indicates that 5-HT7 plays an important role in circadian physiology (10, 14, 15). 5-HT7 receptors are also expressed in glial cells (16, 17) and at lower levels in peripheral tissues including the spleen, intestine, and vascular smooth muscle (7-9, 18).

Many psychotherapeutic agents modulate the cAMP signaling pathway (19) and perturbation of this signal transduction system may contribute to some affective disorders. To date, nine distinct cDNA clones for mammalian adenylyl cyclases have been described (20-29), each with a unique tissue distribution and regulatory properties (30-32). All of the known adenylyl cyclase isoforms are sensitive to Gs stimulation in vitro (30-32). However, the Ca2+/calmodulin-stimulated isoforms AC1 and AC8 are insensitive to Gs in vivo (33-35). In addition, AC1 and AC8 are neural-specific (21, 36) and are expressed in areas of the brain where 5-HT6 and 5-HT7 are localized. For example, 5-HT6 and AC1 are expressed in the hippocampus and cerebellum (13, 37), 5-HT7 and AC1 are found in the hippocampus (8-10, 37), and 5-HT7 and AC8 are expressed in the hypothalamus and hippocampus (8-10, 21). In addition, 5-HT6 and AC5 are localized to the striatum and nucleus accumbens (13, 38). Recent studies have described rat and human 5-HT7 splice variants (39-41), adding a layer of complexity to the study of 5-HT7 coupling in vivo.

To explore mechanisms for regulation of cAMP in brain by serotonin, we examined the coupling of 5-HT6 and 5-HT7A to specific adenylyl cyclases expressed in HEK 293 cells. Our data indicate that 5-HT6 acts as a typical Gs-coupled receptor, stimulating the Gs-sensitive AC5 isoform, but not the Gs-insensitive AC1 or AC8 isoforms (35). Unexpectedly, 5-HT7A stimulated AC1 and AC8 by increasing intracellular Ca2+. These data suggest that serotonin may regulate cAMP in certain areas of the brain by mobilization of intracellular Ca2+ through activation of 5-HT7A receptors.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Materials-- 3-Isobutylmethylxanthine (IBMX), carbachol, and 5-HT were purchased from Sigma (St. Louis, MO). Clozapine and methiothepin were purchased from Research Biochemicals (Natick, MA). Thapsigargin, pertussis toxin, cholera toxin, bisindolylmaleimide, and Fura-2/AM were from Calbiochem (La Jolla, CA). Restriction endonucleases and T4 DNA ligase were purchased from New England Biolabs (Beverly, MA). The Klenow fragment of DNA polymerase was obtained from Boehringer Mannheim.

Cell Culture-- Human embryonic kidney 293 (HEK 293) cells were grown at 37 °C in HEPES-buffered Dulbecco's modified Eagle's medium (H-DMEM) supplemented with 10% bovine calf serum (BCS) and 1% penicillin/streptomycin in a humidified 95% O2, 5% CO2 incubator. Cell culture materials were obtained from Life Technologies, Inc. unless otherwise indicated.

Hypothalamic cultures were prepared according to Obrietan and van den Pol (42). Briefly, the mediobasal hypothalamus extending from the mamillary bodies to the preoptic area was removed from postnatal day 1 rats. The tissue was placed in dissociation medium (DM; 75 mM Na2SO4, 27 mM K2SO4, 15 mM MgCl2, 0.25 mM CaCl2, 1.85 mM Hepes, 18 mM glucose, and 1 mM kyneurenic acid, pH 7.4) containing papain (10 units/ml) and L-cysteine (0.2 mg/ml) for 30 min. The tissue was pelleted, and the protease solution was removed by aspiration. The tissue was triturated into a single cell suspension in standard tissue culture medium (glutamate- and glutamine-free DMEM supplemented with 10% fetal bovine serum, 100 units/ml penicillin/streptomycin, and 6 gm/l glucose). The suspended cells were plated onto poly-D-lysine-coated glass coverslips. Cytosine arabinofuranoside (8 µM) was added to the tissue culture medium on the second day in culture to inhibit glial cell proliferation and was removed on the sixth day in culture. Cell cultures were maintained at 37 °C and 5% CO2.

For hippocampal cultures, hippocampi were dissected from postnatal day 1 rats. Dissociation medium plus papain (10 units/ml) was added and incubated with occasional agitation at 37 °C for 45 min. After rinsing twice with neurobasal medium (Life Technologies, Inc.), the tissue was triturated 25 times in DM with a 5-ml plastic pipette. After 5 min, the supernatant was transferred to a fresh tube, and the remaining tissue was resuspended in fresh DM and triturated once more. The supernatants were pooled, diluted in neurobasal medium to the appropriate cell density and plated onto poly-D-lysine-coated glass coverslips. After incubating in 5% CO2, 37 °C for 2 h, the medium was changed to fresh Neurobasal medium containing 1× B-27 supplement (Life Technologies, Inc.), 0.5 mM glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin. Twenty four h later, 5 µM cytosine D-arabinofuranoside (Sigma) was added.

Subcloning of Expression Vectors-- The human 5-HT6 cDNA clone in pBluescript II KS (-) (Stratagene, La Jolla, CA) was digested with XbaI, and the 5' overhang was filled in with Klenow fragment. Subsequently, the 5-HT6 cDNA was released from pBluescript II KS (-) by digestion with XhoI. The isolated cDNA fragment was subcloned into pCDNAIII (Invitrogen, San Diego, CA) using EcoRV and XhoI restriction sites. The human 5-HT7A cDNA clone in pCDSRalpha was released by digestion with EcoRI and XbaI and subcloned into pCDNAIII that was previously digested with the same two enzymes. The correct constructs were confirmed by restriction endonuclease digestion and agarose gel electrophoresis. The AC1 cDNA clone was isolated from a bovine brain cDNA library as described previously (37). Dr. Ravi Iyengar (Mount Sinai School of Medicine, New York, NY) generously provided the cDNA clone for AC5. The cDNA clone for AC8 was a gift from Dr. John Krupinski (Weiss Center for Research, Geisinger Clinic, Danville, PA). All adenylyl cyclases were cloned into the same parent vector pCEP4 (Invitrogen, Carlsbad, CA).

Coexpression of 5-HT6 or 5-HT7A with Adenylyl Cyclases in HEK 293 Cells-- Polyclonal populations of G418- and hygromycin-resistant HEK 293 cells (Calbiochem, 500 µg/ml and 500 units/ml, respectively) were obtained by stable transfection using the calcium phosphate method (43). 5-HT6 or 5-HT7A were cotransfected with the parent pCEP4 expression vector or one of the aforementioned adenylyl cyclases cloned into pCEP4. Expression of adenylyl cyclases and 5-HT6 and 5-HT7A receptors was confirmed by cAMP accumulation assays as described below and by receptor binding assays.

cAMP Accumulation Assay-- Changes in intracellular cAMP were measured by determining the ratio of [3H]cAMP to the total ATP, ADP, and AMP pool in [3H]adenine-loaded cells as described previously (44). This assay system allows for rapid and sensitive determination of relative changes in intracellular cAMP levels. Whereas the ratios measured between assays showed some variation, the relative changes in cAMP levels between assays were reproducible. Briefly, cells in 12-well plates (approximately 90% confluent) were incubated in H-DMEM + 10% BCS containing 1-2 µCi/well [3H]adenine (ICN, Costa Mesa, CA) for 16-20 h. The next day, cells were washed once with 150 mM NaCl and incubated in H-DMEM plus 1% penicillin/streptomycin containing the indicated effectors (e.g. serotonin) plus 1 mM IBMX for 30 min. For assays in which receptor antagonists were used, cells were pretreated with the indicated antagonists for 15 min in H-DMEM. Assays were initiated by addition of stimulators and IBMX in the continued presence or absence of antagonists. Ca2+-free (-Ca2+) experiments were performed in DMEM/F-12 (1:1) lacking CaCl2 and containing 0.5 mM EGTA. The same medium was used during thapsigargin pretreatment and the assay. For assays in the presence of Ca2+ (+Ca2+), medium containing 1.8 mM CaCl2 was used. Reactions were terminated by aspiration and addition of 1 ml of ice-cold 5% trichloroacetic acid and 1 µM cAMP. Culture dishes were maintained at 4 °C for 1-4 h and acid-soluble nucleotides were separated by Dowex AG50WX-4 followed by neutral alumina chromatography as described previously (45). The data are presented as the average of triplicate determinations.

Ca2+ Imaging by Fura-2 Fluorescence Ratio Determination-- Intracellular calcium levels in HEK 293 cells stably expressing 5-HT6, 5-HT7A, 5-HT7A/AC1, or 5-HT7A/AC8 were analyzed according to Wayman et al., 1995 (46). Cells were plated on poly-D-lysine-coated 4-well Lab-TEK cover glass chambers (Nalge Nunc, Naperville, IL) at a density of approximately 50,000 cells per well and imaged after 4 days. Cells were loaded with 100 nM Fura-2/AM (Molecular Probes, Eugene, OR) in imaging buffer (140 mM NaCl, 5 mM KCl, 0.5 mM MgCl2, 1.5 mM CaCl2, 10 mM glucose, 10 mM HEPES, pH 7.4) for 30 min, rinsed, and allowed to rest in 250 µl of imaging buffer for 1 h before imaging. Cells were imaged using a Nikon inverted microscope, a 75-watt xenon lamp, a Metaltek filter wheel, and Image I software (Universal Imaging, West Chester, PA). The intracellular calcium concentration was calibrated according to Wayman et al., 1995 (46). For agonist treatments, 2 µM 5-HT or 200 µM ATP in 250 µl of imaging buffer was added for a final concentration of 1 µM 5-HT or 100 µM ATP, respectively.

Primary neuronal cultures were imaged in a similar manner except that they were imaged on glass coverslips mounted in a custom-built chamber, and cells were loaded with 3-5 µM fura-2-AM.

Determination of Phosphatidyl Inositol Turnover-- Cells were plated onto 6-well tissue culture dishes and allowed to reach approximately 80% confluency. Cells were then labeled with 1 µCi/well myo-[3H]inositol (Amersham Pharmacia Biotech, 115 Ci/mmol) for 16 h in H-DMEM containing 10% BCS. For the assay, the medium was aspirated, and the cells were washed once and incubated for 30 min at 37 °C with PSS/LiCl (118 mM NaCl, 4.7 mM KCl, 3 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 10 mM glucose, 0.5 mM EDTA, 10 mM LiCl, 20 mM HEPES, pH 7.4). Effectors dissolved in PSS/LiCl were then added for 15 min at 37 °C. The reactions were terminated by washing once with 4 °C PSS/LiCl and then adding 1 ml of 4 °C methanol. After 2 h at 4 °C, the cells were scraped from the dishes and added to 15-ml conical tubes. One-half-ml of CHCl3 and 0.4 ml of dH2O were added to each tube and the mixture was sonicated for 15 s on ice. An additional 0.5 ml CHCl3 and 0.5 ml dH2O were then added to the tubes with vortexing, and the samples were centrifuged at 1300 × g for 10 min. The upper aqueous phase was collected, loaded onto freshly prepared ion-exchange columns (AG-1 × 8, formate form, 100-200 mesh), and total inositol phosphates were measured as described previously (47, 48).

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Expression of 5-HT6 and 5-HT7A Receptors in HEK 293 Cells-- Cell lines stably expressing 5-HT6 or 5-HT7A were generated using G418 and hygromycin as selectable markers. Expression of functional receptors was confirmed by radioligand binding (data not shown) and intracellular cAMP accumulation assays. HEK 293 cells do not express endogenous 5-HT receptors, and addition of 5-HT did not increase intracellular cAMP (data not shown). However, cells expressing 5-HT6 or 5-HT7A displayed substantial increases in intracellular cAMP in response to 5-HT (Fig. 1, A and B). To examine the pharmacology of these receptors, cAMP accumulation assays were carried out over a range of 5-HT concentrations in the presence or absence of several antagonists. In 5-HT6-expressing cells, half-maximal stimulation of cAMP occurred at approximately 30 nM 5-HT (Fig. 1A), with a maximal stimulation of 40-fold over basal at 50 µM 5-HT. Clozapine and methiothepin, antagonists of the 5-HT6 receptor, shifted the 5-HT activation curve to the right, with Ki values of 35.7 and 7.1 nM, respectively. In HEK 293 cells expressing 5-HT7A, half-maximal stimulation of cAMP levels occurred at approximately 15 nM 5-HT (Fig. 1B), with a maximal stimulation of 7-fold at 250 nM 5-HT. Clozapine, chlorpromazine, and amitryptiline, antagonists of the 5-HT7A receptor, shifted the 5-HT activation curve to the right, with Ki values of 11.8 nM, 137 nM, and 250 nM, respectively, in agreement with published values (7, 8, 49). These antagonists appear to be mixed competitive/noncompetitive at the 5-HT6 and 5-HT7A receptors.


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Fig. 1.   Expression of 5-HT6 and 5-HT7 receptors in HEK 293 cells. A, HEK 293 cells expressing 5-HT6 were pretreated for 15 min with 0.5 µM clozapine, 0.1 µM methiothepin, or vehicle (Me2SO) for 5-HT alone. Assays were initiated by the addition of the indicated concentrations of 5-HT and IBMX in the continued presence or absence of antagonist. B, HEK 293 cells expressing 5-HT7 were pretreated for 15 min with 0.5 µM clozapine, 1 µM chlorpromazine, 1 µM amitryptiline, or vehicle (Me2SO) for 5-HT alone. Assays were initiated by the addition of the indicated concentrations of 5-HT and IBMX in the continued presence or absence of antagonist. Relative cAMP accumulation was determined as described under "Experimental Procedures." The data are the mean ± S.D. of triplicate assays for a single experiment (n >=  7 experiments for 5-HT6 and 5-HT7 dose-response curves in the absence of antagonists). Approximate Ki values were calculated according to the Schild equation.

The 5-HT6 and 5-HT7A Receptors Stimulate AC5-- Coupling of 5-HT6 and 5-HT7A to adenylyl cyclase isoforms was assessed by measuring intracellular cAMP accumulation in polyclonal populations of HEK 293 cells coexpressing 5-HT6 or 5-HT7A with specific adenylyl cyclases. Cells coexpressing 5-HT6 and AC5, a Gs-sensitive adenylyl cyclase, demonstrated robust increases in cAMP in response to 5-HT (Fig. 2A). AC5 was stimulated approximately 7-fold by 10 µM 5-HT. Similarly, in cells coexpressing 5-HT7A and AC5, cAMP increased approximately 7-fold in response to 10 µM 5-HT (Fig. 2B). The response of AC5 to 5-HT in both 5-HT6/AC5 and 5-HT7A/AC5 cells was blocked by clozapine and methiothepin (Fig. 3, A and B). These results indicate that the 5-HT6 and 5-HT7A receptors couple to AC5 via stimulation of Gs.


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Fig. 2.   Stimulation of AC5 by 5-HT6 and 5-HT7 receptors in HEK 293 cells. HEK 293 cells coexpressing 5-HT6 and AC5 (A) or 5-HT7 and AC5 (B) were treated with the indicated concentrations of 5-HT. The data are the mean ± S.D. of triplicate assays for a single representative experiment (panel A, n = four experiments; panel B, n = four experiments). The contribution of the endogenous HEK 293 cell adenylyl cyclases has been subtracted from AC5-transfected cell cAMP values.


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Fig. 3.   Stimulation of AC5 by 5-HT6 and 5-HT7 is blocked by clozapine and methiothepin. HEK 293 cells coexpressing 5-HT6 and AC5 (A) or 5-HT7 and AC5 (B) were pretreated with 10 µM clozapine, 1 µM methiothepin, or Me2SO vehicle for 15 min. cAMP assays were initiated by addition of vehicle or 1 µM 5-HT in the presence or absence of the indicated antagonists. The data are the mean ± S.D. of triplicate assays for a single experiment. The contribution of the endogenous HEK 293 cell adenylyl cyclases has been subtracted from AC5-transfected cell cAMP values.

The 5-HT6 Receptor Does Not Couple to AC1 or AC8-- AC1 is not stimulated by Gs-coupled receptors in vivo unless intracellular Ca2+ is simultaneously elevated (33, 34) or beta gamma subunits released from Gsalpha are scavenged (35). AC8 is not stimulated by Gs-coupled receptors in vivo even in the presence of Ca2+ (35). To determine whether 5-HT6 is capable of stimulating AC1 or AC8, cells expressing 5-HT6 with AC1 or AC8 were treated with increasing concentrations of 5-HT and intracellular cAMP was measured. 5-HT6 did not mediate significant stimulation of AC1 or AC8 at any concentration of 5-HT tested (Fig. 4, A and B), whereas the Ca2+ ionophore A23187 stimulated AC1 and AC8 robustly (data not shown; Ref. 35). These data indicate that 5-HT6 acts as a representative Gs-coupled receptor.


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Fig. 4.   AC1 and AC8 are not stimulated by 5-HT6 receptors. HEK 293 cells coexpressing 5-HT6 and AC1 (A) or 5-HT6 and AC8 (B) were treated with the indicated concentrations of 5-HT. The data are the mean ± S.D. of triplicate assays for a single representative experiment (panel A, n = four experiments; panel B, n = three experiments).

The 5-HT7A Receptor Couples Serotonin to Stimulation of AC1 and AC8-- To determine whether 5-HT7A can couple to activation of AC1 or AC8, cells expressing 5-HT7A with these adenylyl cyclases were treated with increasing concentrations of 5-HT, and cAMP accumulation was measured. Unexpectedly, 5-HT7A stimulated both AC1 and AC8 (Fig. 5, A and B). One µM 5-HT stimulated AC1 approximately 6-fold and AC8 approximately 4-fold. Both clozapine and methiothepin blocked activation of AC1 and AC8 by 5-HT7A (Fig. 5, A and B).


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Fig. 5.   Stimulation of AC1 and AC8 by 5-HT7 receptors. HEK 293 cells coexpressing 5-HT7 and AC1 (A) or 5-HT7 and AC8 (B) were treated with the indicated concentrations of 5-HT in the presence or absence of 10 µM clozapine or 1 µM methiothepin. Antagonists were added to cells 15 min prior to initiation of assays. The data are the mean ± S.D. of triplicate assays from a single experiment (n = two experiments for 5-HT7/AC1 and for 5-HT7/AC8 dose-responses in the absence of antagonists). The contribution of the endogenous HEK 293 cell adenylyl cyclases has been subtracted from the AC1- or AC8-expressing cell cAMP values.

5-HT7A Stimulation of AC1 and AC8 Is Mediated by Increases in Intracellular Ca2+-- Because AC1 and AC8 are Ca2+/CaM-stimulated adenylyl cyclases, the role of Ca2+ in 5-HT7A receptor activation was evaluated. Intracellular cAMP accumulation assays were carried out in the presence of 1.8 mM extracellular CaCl2 (+Ca2+), or under Ca2+-free conditions (-Ca2+). In the latter case, cells were pretreated for 30 min with 250 nM thapsigargin, which depletes intracellular Ca2+ stores (46), in Ca2+-free medium containing 0.5 mM EGTA. Following pretreatment, cells were assayed in the same medium (minus thapsigargin) in the presence of 0.5 mM EGTA. In the presence of Ca2+, 5-HT7A stimulated AC1 robustly and AC8 moderately (Fig. 6, A and B). Under conditions in which internal Ca2+ stores were depleted and extracellular Ca2+ was absent, 5-HT7A stimulation of AC1 or AC8 was greatly attenuated. The role of intracellular Ca2+ increases was also evaluated using BAPTA-AM to chelate intracellular Ca2+. BAPTA-AM pretreatment attenuated 5-HT7A stimulation of AC1 by 50 to 75% and blocked stimulation of AC8 completely (Fig. 7, A and B). The incomplete block of 5-HT stimulation of AC1 by BAPTA-AM, may be due to incomplete chelation of intracellular Ca2+. These data suggest that the 5-HT7A stimulation of AC1 and AC8 may occur via increases in intracellular Ca2+.


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Fig. 6.   Intracellular Ca2+ depletion attenuates 5-HT7 stimulation of AC1 and AC8. HEK 293 cells coexpressing 5-HT7 and AC1 (A), or 5-HT7 and AC8 (B) were pretreated for 30 min with Me2SO vehicle in normal DMEM/F-12 (1:1) (+Ca2+) or 250 nM thapsigargin in Ca2+-free DMEM/F-12 (1:1) supplemented with 0.5 mM EGTA (-Ca2+). Following pretreatment, the medium was aspirated, and cells were treated with the indicated concentrations of 5-HT and assayed for cAMP accumulation under the same conditions as above (± Ca2+), except that thapsigargin or Me2SO was omitted. The data are the mean ± S.D. of triplicate assays for a single experiment (panel A, n = three experiments; panel B, n = three experiments). The contribution of the endogenous HEK 293 cell adenylyl cyclases has been subtracted from the AC1- or AC8-expressing cell cAMP values.


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Fig. 7.   Intracellular Ca2+ chelation inhibits 5-HT7 stimulation of AC1 and AC8. HEK 293 cells co-expressing 5-HT7 and AC1 (A), or 5-HT7 and AC8 (B) were pretreated for 15 min with 80 µM BAPTA/AM or Me2SO vehicle in H-DMEM + 1% penicillin/streptomycin + 10% BCS. Following the preincubation period, cells were treated with the indicated concentrations of 5-HT in the presence or absence of 80 µM BAPTA-AM. The data are the mean ± S.D. of triplicate assays for a single experiment. The contribution of the endogenous HEK 293 cell adenylyl cyclases has been subtracted from the AC1- or AC8-expressing cell cAMP values.

To determine whether activation of 5-HT7A increases intracellular Ca2+, cells expressing 5-HT6 and AC1, or 5-HT7A alone or with AC1 or AC8 were Ca2+-imaged using Fura-2 fluorescence. Treatment of 5-HT6/AC1 cells with 1 µM 5-HT did not increase intracellular Ca2+ (Fig. 8A), whereas treatment of 5-HT7A cells with 1 µM 5-HT elicited increases in intracellular Ca2+ from a resting level of approximately 40 nM to 250-500 nM (Fig. 8B). In cells expressing 5-HT7A with AC1 or AC8, 1 µM 5-HT also increased intracellular Ca2+ (Fig. 8, C and D).


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Fig. 8.   Cells expressing 5-HT7 increase intracellular calcium in response to 5-HT. The intracellular calcium concentration was measured in HEK 293 cells expressing 5-HT6/AC1 (A), 5-HT7 (B), 5-HT7/AC1 (C), and 5-HT7/AC8 (D) as described under "Experimental Procedures." In each case, 1 µM 5-HT was added at the beginning of the experiment. As a control for the ability of the cells to increase intracellular calcium, 100 µM ATP was added at the end of the experiment. None of the 5-HT6/AC1 cells increased intracellular calcium in response to 5-HT. However, 5-HT induced increased intracellular calcium in cells expressing 5-HT7 (17% 5-HT7, 67% 5-HT7/AC1, 6% 5-HT7/AC8). Shown are representative responses from single cells (n = 287 cells for 5-HT6/AC1; n = 875 cells for 5-HT7; n = 438 cells for 5-HT7/AC1; n = 424 cells for 5-HT7/AC8).

Activation of 5-HT7A Does Not Increase Phosphatidyl Inositol Turnover-- A possible mechanism for activation of AC1 and AC8 by 5-HT7A receptors is through phosphatidylinositol (PI) turnover and the generation of intracellular Ca2+ release by IP3. Therefore, total inositol phosphates were measured in 5-HT-stimulated cells labeled with myo-[3H]-inositol using ion-exchange chromatography. Whereas 0.5 mM carbachol increased PI turnover in HEK 293 cells and in cell lines expressing 5-HT7A, 5-HT7A/AC1, and 5-HT7A/AC8 (Fig. 9), no increase in turnover was observed in response to 1 µM 5-HT. In addition, pretreatment of the same cell lines with bisindolylmaleimide, a PKC inhibitor, had no effect on 5-HT-induced stimulation of cAMP (data not shown).


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Fig. 9.   5-HT does not induce PI turnover in cells expressing 5-HT7 and AC1 or AC8. Phosphatidylinositol turnover was measured as described under "Experimental Procedures." Carbachol (0.5 mM) induced PI turnover in HEK 293 cells as well as cells expressing 5-HT7, 5-HT7/AC1, or 5-HT7/AC8. 5-HT (1 µM) had no effect on PI turnover in any of the cell lines. The data are the mean ± S.D. for a single experiment (n = two experiments for HEK 293; n = seven experiments for 5-HT7; n = six experiments for 5-HT7/AC1; n = three experiments for 5-HT7/AC8).

5-HT7A Does Not Modulate Adenylyl Cyclase Activity Through Gi-- To determine whether the 5-HT7A receptor couples to adenylyl cyclases through Gi, e.g. by release of beta gamma subunits, HEK 293 cells expressing 5-HT7A were pretreated with 50 ng/ml pertussis toxin for 18 h. Although the cells underwent a reproducible morphological change characteristic of pertussis toxin treatment, pertussis toxin had no effect on the ability of 5-HT7A to couple to endogenous adenylyl cyclases (Fig. 10). Experiments were also carried out to examine the effect of cholera toxin. Pretreatment of cells expressing 5-HT7A with 0.5 µg/ml cholera toxin for 18 h increased the basal level of cAMP. However, 5-HT did not increase cAMP above the levels obtained with cholera toxin alone (Fig. 10).


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Fig. 10.   5-HT7A does not couple to adenylyl cyclase via Gi. HEK 293 cells expressing 5-HT7A were pretreated with 50 ng/ml pertussis toxin or with 0.5 µg/ml cholera toxin for 18 h. Following the preincubation period, cells were treated with the indicated concentrations of 5-HT. The data are the mean ± S.D. of triplicate assays for a single experiment (n = 2 experiments).

Serotonin Increases Intracellular Ca2+ in Cultured Neurons-- To determine whether 5-HT can induce a Ca2+ increase in neurons, primary cultures were prepared from the hypothalamus and hippocampus of postnatal day 1 rats. After 10 to 14 days in culture, cells were subjected to Ca2+ imaging using Fura-2 fluorescence. As shown in Fig. 11, serotonin and 8-OH-DEPAT, a 5-HT7 (and 5-HT1A) agonist, both increased intracellular Ca2+ in hypothalamic neurons. In hypothalamic neuron cultures, 38% of the fields contained cells that responded to 5-HT. Of those fields, 26% of the cells responded to 10 µM 5-HT with an increase in Ca2+. In addition, 14% of the fields contained cells that responded to 8-OH-DEPAT. Of those fields, 11% of the cells responded to 50 µM 8-OH-DEPAT with an increase in Ca2+ (n = 543 cells over five experiments). A response to 50 µM NMDA was included to verify that the cells imaged were neurons. Although a smaller percentage of hippocampal neurons responded, similar results were obtained (data not shown).


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Fig. 11.   5-HT increases intracellular 5-HT7 in cultured neurons. Hypothalamic neurons were cultured according procedures under "Experimental Procedures." An increase in intracellular calcium was observed in response to application of 50 µM 8-OH-DEPAT or 10 µM 5-HT. At approximately 10 min, the imaging buffer was altered to exclude Mg2+ and to include 20 µM glycine, in order to facilitate activation of NMDA receptors. The basal Ca2+ level was slightly increased in response to this buffer change and increased further in response to application of exogenous NMDA (50 µM), indicating the cells were healthy neurons. The trace shown is from a single representative neuron (n = 543 cells over 5 experiments). Although the percentage of responding cells was lower in hippocampal cultures, similar results were observed.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Serotonin modulates a variety of physiological functions, including sleep (50), appetite (3), vascular tone (2), sexual behavior (51), and mood (1). The family of serotonin receptors and splice variants probably evolved to mediate the diverse roles of serotonin in the developing and the adult nervous system (4). Several of the recently cloned members of the serotonin receptor family (e.g. 5-HT6 and 5-HT7) couple to stimulation of adenylyl cyclase, and in the brain, their mRNA expression patterns overlap with those of several adenylyl cyclases.

To better understand how serotonin might control cAMP, we have coexpressed 5-HT6 or 5-HT7A with either AC1, AC5, or AC8 in HEK 293 cells and evaluated coupling in vivo by intracellular cAMP assay. Our results demonstrate that 5-HT6 acts as a typical Gs-linked receptor, in that it stimulated AC5, but not AC1 or AC8 (21, 33, 34). Unexpectedly, 5-HT7A not only stimulated AC5, but also AC1 and AC8. Because AC1 and AC8 are insensitive to Gs-coupled receptors, but are sensitive to Ca2+ and/or Ca2+/calmodulin, we examined the role of Ca2+ in 5-HT7A stimulation of AC1 and AC8 and found that activation of 5-HT7A increased intracellular Ca2+. In addition, depletion and/or chelation of Ca2+ attenuated 5-HT7A stimulation of AC1 and AC8. Therefore, 5-HT7A stimulation of the Ca2+/CaM-stimulated adenylyl cyclases AC1 and AC8 is most likely because of elevated intracellular Ca2+.

The ability of 5-HT7A activation to increase intracellular Ca2+ has not previously been reported. Serotonin- and 8-OH-DEPAT-induced increases in Ca2+ in primary neuronal cultures indicates that this coupling to Ca2+-stimulated adenylyl cyclases may occur in neurons. 8-OH-DEPAT has also been reported to increase intracellular Ca2+ in suprachiasmatic nucleus cultures (52). In addition, Mork and Geisler (53) have reported that 5-HT receptor agonists regulate Ca2+-stimulated adenylyl cyclase activity in rat hippocampus and cortex. Whether these effects in neurons are specific for 5-HT7A receptor signaling is not yet known because specific agonists and antagonists for the 5-HT7 receptor have not been identified.

Many G protein-linked receptors have multifunctional signaling potential when expressed in a given cell type, either through activation of more than one type of G protein or through pleiotropic effects of a single G protein (61). The ability of the 5-HT7A to increase intracellular Ca2+ suggests that this receptor may signal via dual coupling to Gs and another G protein. However, our data indicate that the Ca2+ increase occurs independent of phosphoinositide turnover and PKC. Furthermore, 5-HT7A coupling to adenylyl cyclases occurs independent of Gi. Several other G protein-linked members of the 5-HT receptor family, including 5-HT1A, 5-HT1D, 5-HT1F, 5-HT2C, and 5-HT4 couple to increases in calcium (54-58), and endogenously expressed 5-HT1B and 5-HT2B receptors increase intracellular calcium independent of phosphoinositide turnover (59, 60).

The coupling between 5-HT6 and AC5 is interesting given that 5-HT6 and AC5 are expressed in the striatum and nucleus accumbens, important loci for antipsychotic drug effects (62). This suggests that antagonism of 5-HT6 stimulation of AC5 in striatum and nucleus accumbens may contribute, in part, to the therapeutic effects of clozapine and other psychotherapeutic agents. The coupling of 5-HT6 and AC5 also indicates that AC5 may mediate serotonergic effects on striatal function in general.

The coupling of 5-HT7A to AC1 and AC8 was surprising and not predicted from the known regulatory properties of these two adenylyl cyclases. Coexpression of mRNA for 5-HT6, 5-HT7, AC1, and AC8 occurs in the hippocampus (8, 13, 21, 37). The role of 5-HT in the hippocampus is unclear, in part because almost all of the 5-HT receptor subtypes are expressed in the hippocampus, and subtype-specific drugs are not yet available. When 5-HT7A-selective agents become available, it will be of interest to determine whether activation of this receptor in neurons increases intracellular Ca2+, and if 5-HT7A modulates specific forms of synaptic plasticity. The coupling of 5-HT7A to AC8 in the hypothalamus may also play an important role in endocrine or circadian regulation.

In summary, these data demonstrate that 5-HT6 acts as a typical Gs-coupled receptor by stimulating AC5, but not AC1 or AC8. The discovery that 5-HT7A stimulates AC1 and AC8 through increases in intracellular Ca2+ provides a novel mechanism for serotonergic regulation of intracellular cAMP in the brain and other tissues.

    ACKNOWLEDGEMENTS

We thank Dr. Ravi Iyengar for the cDNA clone for AC5, and Dr. John Krupinski for the cDNA clone for AC8. We thank Drs. Ulrika Lernmark, Beth Hacker, and Lisa Prichard and Scott Wong for critical reading of the manuscript.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant 20498.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger The first two authors contributed equally to this work.

To whom correspondence should be addressed: Dept. of Pharmacology, Box 357280, University of Washington, Seattle, WA 98195-7280. Tel.: 206-543-7028; Fax: 206-685-3822; E-mail: dstorm{at}u.washington.edu.

1 The abbreviations used are: 5-HT, 5-hydroxytryptamine; AC1, AC5, and AC8, adenylyl cyclase types 1, 5, and 8, respectively; BAPTA-AM, 1,2-bis(2-amino-5,5'-difluorophenoxy)ethane-tetrakis (acetoxymethyl) ester; BCS, bovine calf serum; CaM, calmodulin; H-DMEM, HEPES-buffered Dulbecco's modified Eagle's medium; IBMX, isobutylmethylxanthine; PI, phosphatidylinositol; PKC, protein kinase C; NMDA, N-methyl-D-aspartate.

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Abstract
Introduction
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Results
Discussion
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