©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Voltage-sensitive Adenylyl Cyclase Activity in Cultured Neurons
A CALCIUM-INDEPENDENT PHENOMENON (*)

Raghava Reddy , Dave Smith , Gary Wayman , Zhiliang Wu , Enrique C. Villacres , Daniel R. Storm (§)

From the (1)Department of Pharmacology, University of Washington, Seattle, Washington 98195

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Catalytic subunits of mammalian adenylyl cyclases have been proposed to contain 12 transmembrane domains, a property shared with some voltage-sensitive ion channels. Here we report that adenylyl cyclase activity in cerebellar neurons is synergistically stimulated by depolarizing agents and -adrenergic receptor activation. This phenomenon is Ca-independent and not attributable to Ca-stimulated adenylyl cyclase activity. Cholera toxin and forskolin also synergistically stimulate adenylyl cyclase activity in combination with depolarizing agents. We hypothesize that conformational changes in the catalytic subunit of the enzymes caused by changes in the membrane potential may enhance stimulation of adenylyl cyclases by the guanylyl nucleotide stimulatory protein. This novel mechanism for regulation of adenylyl cyclases generates robust cAMP signals that may contribute to various neuromodulatory events including some forms of neuroplasticity.


INTRODUCTION

Molecular mechanisms underlying synaptic plasticity have been intensely studied because of their potential importance for learning and memory (reviewed in Frank and Greenberg(1994) and Stevens(1994)). Several regulatory systems have been implicated in neuroplasticity including the cAMP signal transduction system (Kandel and Schwartz, 1982; Chavez-Noriega and Stevens, 1992, 1994; Chetkovich and Sweatt, 1993; Frey et al., 1993; Weisskopf et al., 1994; Wu et al., 1995). cAMP-dependent protein kinases regulate several neuronal functions including ion channel activity, neurotransmitter synthesis, synaptic transmission, and gene expression (reviewed in Krebs and Beavo(1979), Nestler and Greengard(1983), and Nairn et al.(1985)). Evidence from invertebrates (Kandel and Schwartz, 1982; Dudai, 1988; Livingston, 1985; Levin et al., 1992) and mammalian brain (Xia et al., 1991, 1993; Wu et al., 1995) indicates that adenylyl cyclases may be important for some forms of synaptic plasticity. For example, transgenic mice lacking the Ca-stimulated type I adenylyl cyclase are deficient in spatial memory and show altered LTP in the CA1 region of the hippocampus (Wu et al., 1995).

Brain adenylyl cyclases are regulated by neurotransmitter receptors coupled to the enzymes through the G regulatory proteins, G and G (reviewed in Ross and Gilman(1980)) and by intracellular Ca (reviewed in Cheung and Storm (1982)). Clones for eight distinct adenylyl cyclases have been published (Krupinski et al., 1989, 1992; Feinstein et al., 1991; Bakalyar and Reed, 1990; Gao & Gilman, 1991; Ishikawa et al., 1992; Katsushika et al., 1992; Yoshimura and Cooper, 1992; Cali et al., 1994), all of which are expressed in mammalian brain. Although these enzymes share sequence homology, they contain hypervariable regions and exhibit different regulatory properties. For example, type I (Tang et al., 1991; Choi et al., 1992b), type III (Choi et al., 1992a), and type VIII adenylyl cyclases (Cali et al., 1994) are stimulated by Ca and calmodulin whereas type II, IV, V, VI, and VII are not. Synergistic regulation of adenylyl cyclase activity in neurons by neurotransmitters and Ca may play an important role in synaptic plasticity by coupling of the Ca and cAMP regulatory systems (Xia et al., 1991; Choi et al., 1993; Wayman et al., 1994; Impey et al., 1994; Wu et al., 1995).

On the basis of hydropathy plots, the adenylyl cyclases are proposed to contain 12 transmembrane sequences and two large cytoplasmic domains (Krupinski et al., 1989; Gao and Gilman, 1991). Although topographical similarities between mammalian adenylyl cyclases and voltage-sensitive ion channels raised the possibility that they may have ion channel activity, none of these enzymes have been reported to function as ion channels or membrane transport systems. In this study, we report that adenylyl cyclase activity in primary cultured neurons is synergistically stimulated by membrane depolarization and various activators of adenylyl cyclase including -adrenergic agonists.


EXPERIMENTAL PROCEDURES

Primary Neuron Cultures

Rat or mouse pups (postnatal day 5-8) were used for cerebellar neuron cultures, and hippocampal neurons were obtained from 2-day-old pups. The cerebellum or hippocampus was removed and placed into high glucose Dulbecco's modified Eagle's medium in a 100-mm culture dish at room temperature. The tissue was transferred to a 15-ml conical tube containing 5 ml of prewarmed trypsin/EDTA solution (0.25% trypsin and 1 mM EDTA) and incubated at 37 °C for 20 min with agitation. At the end of incubation, the issue was allowed to settle, the supernatant was discarded, and the trypsin treatment was repeated. The trypsinized tissue was then resuspended in 10 ml of Dulbecco's modified Eagle's medium containing 10% bovine calf serum and 100 units/ml penicillin G and 100 µg/ml streptomycin sulfate. The tissue was triturated by gentle pipetting. The tissue debris was allowed to settle, the supernatant containing dissociated neurons was recovered, and cell viability was examined using trypan blue exclusion. The cells were plated onto poly-L-lysine-coated 35-mm wells in a 6-well plate at a density of 6 million cells/well. 2 days after plating, the growth medium was supplemented with cytosine -arabinofuroside to a final concentration of 10 µM to inhibit the growth of non- neuronal cells. On the 8th day after plating, neurons were treated with various adenylyl cyclase activators and used for cAMP accumulation assays.

cAMP Accumulation

Changes in intracellular cAMP levels were measured by determining the ratio of [H]cAMP to total ATP, ADP, and AMP pool in [H]adenine-loaded cells as described by Wong et al.(1991). The growth medium of the cells was supplemented with [H]adenine (5 µCi/ml), and cells were incubated for 2 h. Growth medium was removed, and cells were washed twice with Krebs-Ringer-Hepes (KRH)()buffer (128 mM NaCl, 5 mM KCl, 1 mM NaHPO 10 mM glucose, 20 mM Hepes, pH 7.40, 1.2 mM MgSO, and 2.7 mM CaCl). The cells were preincubated with KRH buffer for 30 min, and effectors were added to KRH buffer and incubated an additional 30 min. When cAMP accumulations were examined in the absence of Ca, CaCl was omitted in the KRH buffer, and it was supplemented with 2.0 mM EGTA and 15 uM BAPTA/AM. Veratridine depolarizations were carried out in Ca-free buffer in which the NaCl was increased to 150 mM. Antagonism of veratridine-stimulated Na channel activity was accomplished by pretreatment with 1 µM tetrodotoxin overnight in culture medium. All cAMP accumulation assays were done in the presence of 1.0 mM isobutylmethylxanthine, an inhibitor of cAMP phosphodiesterase activity. After treating neurons with various effectors, the buffer was removed, and the reaction was stopped by adding 1 ml of 5% trichloroacetic acid containing 1 µM cAMP. Following 30 min of incubation at room temperature, acid-soluble nucleotides were separated by ion-exchange chromatography as described (Salomon et al., 1974).

CaImaging Using Fura-2

Neurons were subcultured onto poly-L-lysine-coated, 4-chambered NUNC dishes. Within 72 h after subculturing, cells were rinsed once with KRH or Ca-free KRH buffer and then loaded with 4 µM Fura-2 at 37 °C in the dark. After 40 min of loading with Fura-2, cells were rinsed twice with KRH or Ca-free KRH buffer and allowed to sit for 30 min. Ca imaging was carried out in either a Ca-containing KRH buffer or a Ca-free KRH buffer supplemented with 2 mM EGTA and 15 uM BAPTA/AM using a Nikon Diaphote inverted microscope. The four-chambered coverglass (Lab-Tek, Nunc) was epi-illuminated through a 20 objective at 340 and 380 nm using a filter wheel and a 75-W xenon lamp at 25 °C. Emitted fluorescence was collected by the 20 objective and filtered through a 510-nm band pass filter. Fluorescence was subsequently magnified with a 2 lens, and an image was obtained with an intensified CCD camera. The ratios of 340/380 were obtained every 8 s up to 20 min with no observable photo-bleaching. Control of the camera and filter wheel and the rate of sampling, data collection, data display, and analysis was done with the software Image-1/FL (Universal Imaging Corp.).


RESULTS

Synergistic Stimulation of Intracellular cAMP Levels in Cerebellar Neurons by KCl and Isoproterenol Is Ca-independent

Our objective was to determine if adenylyl cyclase activity in neurons is affected by changes in the membrane potential. This question was initially addressed by treatment of cultured neurons with KCl, which depolarizes neuronal membranes (Di Virgilio et al., 1987). Neurons were chosen for this study because they have excitable membranes that can be depolarized by several agents. Depolarization of primary cultured neurons from rat cerebellum with 60 mM KCl in the presence of 2.7 mM extracellular CaCl caused a 2.0 ± 0.1-fold increase in intracellular cAMP (Fig. 1A), presumably because of the presence of type I adenylyl cyclase or other Ca-sensitive adenylyl cyclases (Xia et al., 1991; Wu et al., 1995). Isoproterenol stimulated cAMP accumulation 3.1 ± 0.2-fold, and the combination of isoproterenol and KCl was synergistic, elevating cAMP 11.2 ± 1.1-fold. Because these experiments were carried out in the presence of cyclic nucleotide phosphodiesterase inhibitors, the increases in intracellular cAMP were due to stimulation of adenylyl cyclase activity rather than inhibition of phosphodiesterases. Nifedipine, an L-type voltage-sensitive Ca channel antagonist, completely inhibited cAMP increases stimulated by KCl alone. However, synergistic stimulation of adenylyl cyclase activity by KCl and isoproterenol was not affected by nifedipine, suggesting that activation of voltage-sensitive Ca channels may not be required for this phenomenon and that it may be Ca-independent (Fig. 1B).


Figure 1: Effect of KCl and isoproterenol on cAMP production in primary cultured neurons from rat cerebellum. Rat cerebellar neurons were isolated and treated with 60 mM KCl, 10 µM isoproterenol (Iso), or both, for 30 min without (A) or with (B) nifedipine as described under ``Experimental Procedures.'' When present, nifedipine was at 5 µM. Relative cAMP accumulations were determined as described under ``Experimental Procedures.'' Reported values are the averages of triplicate determinations ± S.D.



To address the role of intracellular Ca for synergistic stimulation of adenylyl cyclase by KCl and isoproterenol, the experiments described above were repeated under conditions that inhibited intracellular Ca increases (no extracellular Ca, 2.0 mM EGTA, and 15 µM BAPTA/AM). Under Ca-free conditions, KCl alone did not stimulate intracellular cAMP in cerebellar neurons, but isoproterenol did (Fig. 2A). Synergistic stimulation of cAMP formation by KCl and isoproterenol was consistently seen in eight independent experiments using different preparations of cerebellar neurons. Under Ca-free conditions, isoproterenol stimulation was 3.3 ± 0.5-fold, whereas the combination of isoproterenol and 60 mM KCl stimulated 12.1 ± 2.0-fold. Similar results were obtained with cultured hippocampal neurons; isoproterenol stimulated 6.6 ± 0.3-fold, KCl did not increase cAMP, and the combination of isoproterenol and 60 mM KCl stimulated cAMP accumulation 18.1 ± 0.6-fold (Fig. 2B). Substitution of 60 mM KCl with 60 mM of NaCl (total NaCl increased from 128 mM to 188 mM) did not increase isoproterenol stimulation of cAMP levels.


Figure 2: Effect of KCl and isoproterenol on cAMP production in primary cultured neurons from rat cerebellum and hippocampus in the absence of Ca. Rat cerebellar (A) or hippocampal (B) neurons were isolated and treated with 60 mM KCl, 10 µM isoproterenol, or both, KCl for 30 min under Ca-free conditions as described under ``Experimental Procedures.'' Increases in intracellular Ca were inhibited by treatment of neurons in Ca-free KRH buffer containing 2.0 mM EGTA and 15 µM BAPTA/AM. Intracellular cAMP was determined as described under ``Experimental Procedures.'' Reported values are the averages of triplicate determinations ± S.D.



To verify that KCl did not actually increase intracellular Ca under Ca-free conditions, cultured cerebellar neurons were treated with 60 mM KCl in the presence or the absence of extracellular Ca and imaged for changes in intracellular Ca using Fura-2 (Fig. 3). In the presence of extracellular Ca, 60 mM KCl and 10 µM isoproterenol caused a significant increase in intracellular Ca that persisted for greater than 15 min. Under Ca-free conditions (no extracellular Ca, 2.0 mM EGTA, and 15 µM BAPTA/AM), no increase in intracellular Ca was detectable when neurons were treated with 60 mM KCl and 10 µM isoproterenol. These data strongly support the conclusion that synergistic stimulation of adenylyl cyclase activity by KCl and isoproterenol was not dependent upon increases in intracellular Ca. Under Ca-free conditions, KCl or combinations of KCl and isoproterenol did not increase intracellular Ca.


Figure 3: KCl depolarization of cultured neurons does not in-crease intracellular Ca under Ca-free conditions. Rat cerebellar neurons were isolated and treated with 60 mM KCl and 10 µM isoproterenol in the presence or the absence of Ca as described under ``Experimental Procedures.'' Relative increases in intracellular Ca (relative fluorescence ratio, 340/380) were monitored using Fura-2-loaded cells.



The synergism between isoproterenol and KCl was dependent upon the concentrations of both reagents (Fig. 4). In the presence of 60 mM KCl, half-maximal stimulation of intracellular cAMP occurred between 0.1 and 1.0 µM isoproterenol and was completely blocked by the -adrenergic antagonist propranolol. KCl as low as 15 mM significantly enhanced isoproterenol stimulation of cAMP accumulation. KSO, a depolarizing agent that does not cause cell swelling, also synergistically increased intracellular cAMP with isoproterenol (Fig. 4C). These data indicate that adenylyl cyclase activity in cultured neurons is synergistically activated by stimulation of -adrenergic receptors and KCl without increases in intracellular Ca.


Figure 4: Isoproterenol, KCl, and KSO concentration dependence for synergistic stimulation of adenylyl cyclase in cerebellar neurons. Rat cerebellar neurons were isolated and treated with 60 mM KCl and varying concentrations of isoproterenol (A), 10 µM isoproterenol and varying concentrations of KCl (B), or 10 µM isoproterenol and varying concentrations of KSO (C) for 30 min under Ca-free conditions, and intracellular cAMP was determined as described under ``Experimental Procedures.'' When present, propranolol was at 10 µM. Data are the averages of triplicate determinations ± S.D.



Increases in cAMP stimulated by KCl plus isoproterenol were reversibly dependent upon the presence of KCl (). When neurons were pretreated with KCl for 30 min, washed, and then assayed for cAMP accumulation in the presence of isoproterenol, cAMP levels were identical to those seen without pretreatment with KCl. Furthermore, isoproterenol stimulation of adenylyl cyclase activity in membranes prepared from neurons was not increased when the neurons were pretreated with 60 mM KCl prior to membrane isolation (data not shown). Changes in the adenylyl cyclase system caused by depolarization or isoproterenol alone were readily reversible, and synergistic stimulation required the simultaneous presence of both agents. Apparently, membrane depolarization does not cause stable covalent modifications of the enzyme that enhance stimulation by -adrenergic receptors.

Because changes in osmolarity have been reported to affect adenylyl cyclase activity in other types of cells (Watson, 1990), we evaluated the effect of increased osmolarity on adenylyl cyclase activity in cultured neurons using 60 or 120 mM sucrose (). Sucrose at 120 mM did not affect basal adenylyl cyclase activity or its sensitivity to isoproterenol in the absence of Ca. Similar results were obtained with 120 mM glucose when NaCl in the growth media was increased by 60 mM or when the growth media were diluted to decrease osmolarity (data not shown). Furthermore, KSO, a depolarizing agent that does not cause cell swelling, also gave synergistic stimulation of cAMP levels when applied with isoproterenol (Fig. 4C). Therefore, it is unlikely that changes in osmolarity or cell swelling contributed to the changes in adenylyl cyclase activity caused by combinations of KCl and isoproterenol.

Synergistic Stimulation of Intracellular cAMP Levels in Cerebellar Neurons by KCl and Isoproterenol Is Not Due to Type I Adenylyl Cyclase

The major Ca-stimulated adenylyl cyclase in rat and mouse cerebellum is type I adenylyl cyclase (Xia et al., 1991; Wu et al., 1995). Because type I adenylyl cyclase responds synergistically to combinations of activators (Wayman et al., 1994) and is one of the major forms of adenylyl cyclase present in cerebellar neurons, the increases in intracellular cAMP caused by KCl and -adrenergic receptor stimulation might be due to this enzyme. Recently, we disrupted the gene for type I adenylyl cyclase in mice and reported that Ca-stimulated adenylyl cyclase activity in the cerebellar neurons from the mutant mice is significantly reduced (Wu et al., 1995). Cultured cerebellar neurons from wild type and mutant mice lacking type I adenylyl cyclase were analyzed for sensitivity to KCl and isoproterenol. In the presence of Ca, cerebellar neurons from wild type and type I adenylyl cyclase mutant mice showed synergistic stimulation of adenylyl cyclase activity by KCl and isoproterenol, but neurons from mutant mice showed little cAMP increase in response to KCl alone (Fig. 5A). Isoproterenol-stimulated cAMP increases were not depressed in type I adenylyl cyclase mutant neurons because type I adenylyl cyclase is not stimulated by G-coupled receptors in vivo (Wayman et al., 1994). In the absence of Ca, KCl and isoproterenol synergistically stimulated cAMP in neurons from mutant mice, indicating that type I adenylyl cyclase did not contribute to this process (Fig. 5B).


Figure 5: KCl and isoproterenol synergistically stimulate cAMP in neurons from type I adenylyl cyclase mutant mice. Cerebellar neurons from wild type and mutant mice lacking type I adenylyl cyclase were treated with 60 mM KCl, 10 uM isoproterenol, or both, for 30 min in the presence (A) or the absence (B) of Ca as described under ``Experimental Procedures.'' Data are the averages of triplicate determinations ± S.D.



Adenylyl Cyclase Activity in Cultured Neurons Is Synergistically Stimulated by Veratridine and Isoproterenol

The data described above strongly suggested that one or more adenylyl cyclases in cerebellar neurons may be sensitive to the membrane potential and that the phenomenon is Ca-independent. We hypothesize that membrane depolarization may cause conformational changes in the adenylyl cyclase system that enhance stimulation by activated G or other effectors. If this hypothesis is valid, treatment of neurons with other depolarizing agents and isoproterenol in the absence of Ca should synergistically elevate cAMP. Veratridine is a sodium channel agonist that depolarizes neuronal membranes in the presence of extracellular Na by promoting the influx of Na (Catterall, 1974). In the absence of Ca, veratridine at concentrations up to 200 µM had no effect on intracellular cAMP in cerebellar neurons (Fig. 6A). Combinations of veratridine and isoproterenol, however, synergistically stimulated intracellular cAMP. Half-maximal stimulation was at 100 µM veratridine, consistent with the K of this drug for Na channels (Catterall, 1974). Stimulation of intracellular cAMP by veratridine and isoproterenol was partially inhibited by tetrodotoxin, a sodium channel antagonist (Narahashi et al., 1964).


Figure 6: Depolarization of membranes with veratridine or TEA/4AP synergistically stimulates adenylyl cyclase activity in cerebellar neurons. A, stimulation of cAMP accumulation in cerebellar neurons in the presence of increasing concentrations of veratridine. When present, tetrodotoxin was at 1.0 µM. B, stimulation of cAMP accumulations by combinations of isoproterenol and depolarization using TEA/4AP. When present, KCl was at 60 mM, TEA was at 10 mM, 4AP was at 10 mM, and isoproterenol was at 10 µM. Intracellular cAMP was determined as described under ``Experimental Procedures.'' Data are the averages of triplicate determinations ± S.D.



Excitable cells can also be depolarized using tetraethylammonium (TEA) and 4-aminopyridine (4AP), agents that depolarize neurons and prolong the action potential (Barrett et al., 1988). Like KCl and veratridine, TEA/4AP had no effect on cAMP in the absence of Ca; however, synergistic stimulation of cAMP was seen in combination with isoproterenol (Fig. 6B). Intracellular cAMP levels stimulated by TEA/4AP plus isoproterenol were comparable with those caused by KCl and isoproterenol. Thus, three different depolarizing agents enhanced isoproterenol stimulation of adenylyl cyclase activity under Ca-free conditions consistent with the proposal that the membrane potential may regulate sensitivity of adenylyl cyclases to -adrenergic agonists.

KCl Enhances Cholera Toxin- and Forskolin-stimulated Adenylyl Cyclase Activities

-Adrenergic stimulation of adenylyl cyclases requires three protein components; the catalytic subunit, the guanylyl nucleotide stimulatory complex G, and the -adrenergic receptor (May et al., 1985). Each of these proteins is associated with the cytoplasmic membrane and could, in principle, be a voltage-sensitive subunit of the adenylyl cyclase system. In an attempt to identify the voltage-sensitive component of the enzymes, we examined the cholera toxin and forskolin sensitivity of adenylyl cyclase activity with 60 mM KCl. Cholera toxin stimulates adenylyl cyclases by catalyzing the ADP-ribosylation of the subunit of G, thereby inhibiting its intrinsic GTPase activity (Cassel and Selinger, 1977; Moss and Vaughan, 1977). Treatment of cerebellar neurons with cholera toxin alone in the absence of Ca increased intracellular cAMP 5.0 ± 0.2-fold (Fig. 7A). A combination of cholera toxin treatment and KCl depolarization enhanced cAMP levels 12.1 ± 0.5-fold. Similar results were obtained when neurons were treated with cholera toxin and TEA/4AP (data not shown). These data suggested that stimulation of the catalytic activity by cholera toxin-activated G was sensitive to the membrane potential.


Figure 7: Cholera toxin or forskolin synergistically stimulate adenylyl cyclase activity with KCl depolarization. A, cerebellar neurons were pretreated with cholera toxin (1.0 µg/ml) for 24 h and then assayed for cAMP accumulation at varying concentrations of KCl in Ca-free KRH buffer as described under ``Experimental Procedures.'' B, cerebellar neurons were treated with 60 mM KCl with varying concentrations of forskolin and were assayed for cAMP in free Ca-free KRH buffer as described under ``Experimental Procedures.'' Data are the averages of triplicate determinations ± S.D.



Forskolin activates adenylyl cyclases by direct interaction with the catalytic subunit, and purified adenylyl cyclase catalytic subunits are stimulated by forskolin in the absence of G-coupling proteins or receptors (Seamon and Daly, 1981). Forskolin at 50 µM increased intracellular cAMP approximately 10-fold relative to untreated controls (Fig. 7B). In Ca-free buffer, combinations of forskolin and KCl synergistically stimulated adenylyl cyclase activities. For example, 50 µM forskolin and 60 mM KCl stimulated cAMP 73.5-fold ± 4.2-fold, demonstrating that forskolin activation of the enzyme is voltage-sensitive. The effect of depolarization on forskolin-stimulated adenylyl cyclase activity strongly suggests that the voltage-sensitive component of the adenylyl cyclase system is the catalytic subunit.


DISCUSSION

Adenylyl cyclases are regulated by a number of physiologically important messengers, including neurotransmitters, intracellular Ca, and hormones (reviewed in Tang and Gilman(1992), Choi et al.(1993), and Pieroni et al.(1993)). Hydropathy analysis of the amino acid sequence of mammalian adenylyl cyclases indicates that these enzymes have general structural similarity to voltage-sensitive ion channels. Therefore, it was of interest to determine whether adenylyl cyclase activity in cultured neurons is voltage-sensitive. Our data indicate that cerebellar and hippocampal neurons contain voltage-sensitive adenylyl cyclase activity that responds synergistically to depolarization and various effectors including -adrenergic agonists.

In the presence of extracellular Ca, adenylyl cyclase activity in cerebellar and hippocampal neurons was stimulated by KCl depolarization, consistent with the presence of type I adenylyl cyclase in rat cerebellum and hippocampus (Xia et al., 1991; Wu et al., 1995) and type VIII adenylyl cyclase in hippocampus (Cali et al., 1994). Furthermore, Ca-dependent KCl stimulation of adenylyl cyclase activity in mouse cerebellar neurons was greatly diminished in neurons from type I adenylyl cyclase mutant mice. In the absence of Ca, adenylyl cyclase activity in neurons was not directly stimulated by depolarizing agents. However, combinations of depolarizing agents with isoproterenol, cholera toxin, or forskolin synergistically stimulated adenylyl cyclase in the absence of increased intracellular Ca. In addition, neurons from mutant mice lacking type I adenylyl cyclase also showed synergistic stimulation of cAMP by KCl and isoproterenol. The fact that neurons from type I adenylyl cyclase still showed synergistic stimulation of adenylyl cyclase activity by KCl and isoproterenol supports the general conclusion that this phenomenon is not due to a Ca-stimulated adenylyl cyclase.

Veratridine and other depolarizing agents have been reported to increase the formation of cAMP in brain slices by elevation of intracellular Ca and stimulation of neurotransmitter release (Shimizu et al., 1970). It is unlikely that the phenomenon described in this study was due to the release of neurotransmitters because Ca is generally critical for neurotransmitter release. If veratridine and KCl stimulated neurotransmitter release under Ca-free conditions, one would expect stimulation of adenylyl cyclases activity by depolarizing agents alone. KCl, KSO, veratridine, or TEA/4AP had no effect on intracellular cAMP unless paired with other activators of adenylyl cyclase. There are four possible mechanisms that might explain the effect of KCl on adenylyl cyclase activity: increases in intracellular Ca, cell swelling, specific chemical effects of KCl on adenylyl cyclases, or changes in membrane depolarization. The phenomenon was Ca-independent, and it was not due to cell swelling. Several distinct depolarizing agents with different chemical properties synergistically stimulated adenylyl cyclase activity with isoproterenol. Therefore, the phenomenon described in this paper is most likely due to synergistic stimulation of adenylyl cyclase activity by changes in membrane potential coupled with adenylyl cyclase activators.

The voltage-sensitive protein subunit of the adenylyl cyclase system was not identified, although the evidence most strongly implicated the catalytic subunit. Since cholera toxin activation was synergistic with depolarizing agents, it seems likely that either G and/or the catalytic subunit may be sensitive to the membrane potential. Although the subunit of G is membrane-associated, membrane attachment is through palmitylation of Cys-3 (Degtyarev et al., 1993; Wedegaertner et al., 1993) and is less likely to be sensitive to the membrane potential than the catalytic subunit(s). Although forskolin and / synergistically stimulate some adenylyl cyclases, there is no evidence that membrane depolarization releases /. The fact that depolarizing agents and forskolin synergistically stimulated adenylyl cyclase activity is consistent with the conclusion that the catalytic subunit is probably the voltage-sensitive component of the adenylyl cyclase system in neurons.

The voltage-sensitive adenylyl cyclase(s) present in neurons were not identified; however, type I adenylyl cyclase was eliminated from consideration using neurons from type I adenylyl cyclase mutant mice. Although individual adenylyl cyclases can be expressed in several cell lines including HEK-293 cells, these non-neuronal cell lines cannot be used to study the voltage sensitivity of individual adenylyl cyclases because it is not possible to vary the membrane potential over a range comparable with that of neurons. Analysis of the amino acid sequences of the cloned mammalian adenylyl cyclases suggests that they all may contain two tandem arrays of six transmembrane helices separated by a cytoplasmic loop (Krupinski et al., 1989). The principle subunits of voltage-gated ion channels contain four groups of six probable transmembrane -helices surrounding a central pore (reviewed in Catterall(1994)). The S4 transmembrane domain of voltage-sensitive ion channels, which is thought be responsible for the voltage sensitivity of these proteins, has charged amino acids at every third residue. Adenylyl cyclases do contain charged amino acids within putative transmembrane domains that may impart voltage sensitivity. For example, several of the adenylyl cyclases contain charged amino acids within the 1st, 6th, 7th, and 12th transmembrane domains. The membrane topology of mammalian adenylyl cyclases has not been experimentally defined, and identification of transmembrane domains and voltage-sensitive elements will require more extensive analysis.

There are several mechanisms that may account for the phenomenon described in this study. Voltage-dependent conformational changes in the catalytic subunit of the enzyme may enhance stimulation by activated G or forskolin. It is also possible that conformational changes caused by membrane depolarization may allow phosphorylation of the enzyme by specific protein kinases, which increases sensitivity to activated G. Although our data does not distinguish between these two mechanisms, stimulation by combinations of KCl and isoproterenol required the simultaneous presence of both agents. Pretreatment of neurons with KCl followed by washout did not increase stimulation by subsequent addition of isoproterenol. Thus it seems unlikely that membrane depolarization led to stable covalent modifications of the catalytic subunit that enhanced stimulation by various effectors. Alternatively, specific adenylyl cyclases may be in close proximity with voltage-sensitive proteins that influence their sensitivity to activated G. Regardless of the exact mechanism, the data presented in this study describe a new mechanism for the regulation of adenylyl cyclase activity in neurons that is distinct from all others documented in the literature.

It is becoming increasingly evident that specific adenylyl cyclases in the brain are synergistically regulated by combinations of signals. For example, type I adenylyl cyclase is not stimulated by G-coupled receptors in vivo unless the enzyme is also activated by Ca and calmodulin (Wayman et al., 1994). The / complex from G proteins stimulates G-activated type II adenylyl cyclase and type IV adenylyl cyclase (Tang and Gilman, 1991), providing another mechanism by which adenylyl cyclases can function as coincidence detectors (Bourne and Nicoll, 1993). The data in this report identify another mechanism for cross-talk and integration of signals by adenylyl cyclases. Prolonged or robust cAMP signals are particularly important for cAMP-mediated stimulated transcription, which may be crucial for some forms of synaptic plasticity in vertebrates, or for positive feedback regulation of Ca ion channels (Chetkovich et al., 1991; Frey et al., 1993; Bacskai et al., 1993; Impey et al., 1994; Weisskopf et al., 1994).

To the best of our knowledge, this is the first report describing a voltage-sensitive enzyme. The discovery that adenylyl cyclase activity in neurons may be sensitive to membrane potential is a new regulatory mechanism that has important implications for neuron function. Depolarization of neuronal membranes caused by various physiological stimuli coupled with receptor activation can synergistically stimulate adenylyl cyclase activity and generate exceptionally high intracellular cAMP. This regulatory mechanism may be important for some forms of neuroplasticity and other neuromodulatory events.

  
Table: Synergistic stimulation of adenylyl cyclase activity by KCl and isoproterenol is reversibly dependent upon KCl depolarization


  
Table: Increased osmolarity does not affect adenylyl cyclase activity in primary cultured neurons from the cerebellum



FOOTNOTES

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

§
To whom correspondence should be addressed. Tel.: 206-543-7028; Fax: 206-685-3822.

The abbreviations used are: KRH, Krebs-Ringer-Hepes; TEA, tetraethylammonium; 4AP, 4-aminopyridine; BAPTA/AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N`,N`-tetra(acetoxymethyl)ester.


ACKNOWLEDGEMENTS

We thank Dr. William A. Catterall, Dr. Niel Nathanson, and Scott Wong for advice and discussions.


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