©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Capacitative Ca Entry Exclusively Inhibits cAMP Synthesis in C6-2B Glioma Cells
EVIDENCE THAT PHYSIOLOGICALLY EVOKED Ca ENTRY REGULATES Ca-INHIBITABLE ADENYLYL CYCLASE IN NON-EXCITABLE CELLS (*)

(Received for publication, September 9, 1994; and in revised form, November 8, 1994)

Matthew Chiono (§) Rajesh Mahey Glenda Tate Dermot M. F. Cooper (¶)

From the Department of Pharmacology, University of Colorado Health Sciences Center, Denver, Colorado 80262

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Elevation of cytosolic free Ca inhibits the type VI adenylyl cyclase that predominates in C6-2B cells. However, it is not known whether there is any selective requirement for Ca entry or release for inhibition of cAMP accumulation to occur. In the present study, the effectiveness of intracellular Ca release evoked by three independent methods (thapsigargin, ionomycin, and UTP) was compared with the capacitative Ca entry that was triggered by these treatments. In each situation, only Ca entry could inhibit cAMP accumulation (La ions blocked the effect); Ca release, which was substantial in some cases, was without effect. A moderate inhibition, as was elicited by a modest degree of Ca entry, could be rendered substantial in the absence of phosphodiesterase inhibitors. Such conditions more closely mimic the physiological situation of normal cells. These results are particularly significant, in demonstrating not only that Ca entry mediates the inhibitory effects of Ca on cAMP accumulation, but also that diffuse elevations in [Ca] are ineffective in modulating cAMP synthesis. This property suggests that, as with certain Ca-sensitive ion channels, Ca-sensitive adenylyl cyclases may be functionally colocalized with Ca entry channels.


INTRODUCTION

A rise in cytosolic free calcium, [Ca], (^1)has been associated with the inhibition of cAMP accumulation in various tissues and cell lines(1, 2, 3, 4, 5, 6, 7, 8, 9, 10) . In a number of these situations, a Ca-dependent stimulation of phosphodiesterase (PDE) was the apparent mechanism for the inhibition of cAMP accumulation(1, 3) . However, in many of these cases, a Ca stimulation of PDE has been specifically precluded and a direct inhibitory effect of elevated [Ca] on adenylyl cyclase has been demonstrated to be the likely mechanism(2, 4, 5, 6, 7, 8, 9, 10, 11, 12) . A number of the sources displaying this behavior also express either adenylyl cyclase activity that is inhibited by submicromolar [Ca] in in vitro assays(2, 6, 7, 8, 9) , or mRNAs corresponding to either types V or VI adenylyl cyclase(13, 14, 15, 16, 17, 18, 19) ; the latter are recently cloned species that can be inhibited by Ca when their cDNAs are transfected into HEK 293 cells(13, 19, 20, 21) . Such negative influences of [Ca] on cAMP synthesis have been speculated to represent a useful feedback in systems where cAMP controls [Ca], such as cardiac myocytes(8, 22, 23) . However, an unresolved issue, which may cast light on the organization of Ca-sensitive adenylyl cyclases within cells, is whether there is any selective ability of Ca released from intracellular pools or Ca entry to inhibit adenylyl cyclase.

The C6-2B glioma cell line is a useful model system for evaluating the efficacy of Ca entry and internal release on the inhibition of cAMP synthesis. In these nonexcitable cells, agonist stimulation of [Ca] elevation involves an initial release of Ca from inositol 1,4,5-trisphosphate (IP(3))-sensitive stores, accompanied by Ca entry(7, 11, 24) . Strikingly, this cell line expresses almost exclusively Type VI adenylyl cyclase, along with trace amounts of type III(14) . Previous studies in this cell line have established an inhibition of cAMP accumulation that is compatible with direct effects of [Ca] on type VI adenylyl cyclase(7, 11, 14) . The focus of the present study was to determine whether there was a selective role for either Ca entry or release in the inhibition of cAMP accumulation in the C6-2B cell line. Three independent tools were utilized to manipulate [Ca]: the purinergic agonist UTP(25) , the Ca ionophore ionomycin (IM)(26, 27, 28) , and the microsomal Ca-ATPase inhibitor thapsigargin (TG)(29, 30) . These three agents provoke [Ca] rises and capacitative Ca entry by different mechanisms(31, 32) . Subsequent experiments with these agents clearly demonstrated that Ca entry, rather than a simple rise in [Ca], exclusively inhibits cAMP accumulation in C6-2B cells. The results suggest that, as with certain Ca-sensitive ion channels(33) , Ca-inhibitable adenylyl cyclase is functionally co-localized with sites of Ca entry.


EXPERIMENTAL PROCEDURES

Materials

Thapsigargin and ionomycin were from L C Services Corp. and Calbiochem, respectively. [2-^3H]Adenine (23-31 Ci/mmol) was obtained from Amersham Corp. Fura-2/AM and Pluronic F-127 were purchased from Molecular Probes, Inc. HEPES was purchased from Boehringer Mannheim. Other reagents were from Sigma

Cell Culture

Early phase C6-2B cells, kindly provided by Dr. G. L. Brooker (Georgetown University School of Medicine, Washington, DC) were grown in 75-cm^2 culture flasks in Ham's F-10 medium, containing 10% calf serum in an atmosphere of 95% air, 5% CO(2) at 37 °C, without antibiotics. Cells were used 4-7 days after passage.

Measurement of [Ca](i) in Cell Populations

[Ca](i) was measured by the Fura-2 technique using an H& series 300 spectrofluorimeter, as described previously(34) , with modifications. Briefly, cells were detached with phosphate-buffered saline (12.1 mM Na(2)HPO(4), 4 mM KH(2)PO(4), and 130 mM NaCl, pH 7.4) containing 0.01% EDTA and loaded with 2 µM fura-2/AM and 0.02% Pluronic F-127 for 20 min at room temperature. The cells were washed and kept at room temperature until use. [Ca](i) measurements were made in either Krebs buffer or a nominally Ca-free Krebs buffer. The Krebs buffer consisted of 120 mM NaCl, 4.75 mM KCl, 1 mM KH(2)PO(4), 5 mM NaHCO(3), 1.44 mM MgSO(4), 1.1 mM CaCl(2), 0.1 mM EGTA, 11 mM glucose, 25 mM HEPES, and 0.1% bovine serum albumin (fraction V) adjusted to pH 7.4 with 2 M Tris base. The nominally Ca-free Krebs buffer contained 120 mM NaCl, 4.75 mM KCl, 1.44 mM MgSO(4), 11 mM glucose, 25 mM HEPES, and 0.1% bovine serum albumin adjusted to pH 7.4 with 2 M Tris base. Approximately 4 times 10^6 cells were diluted with one of the above buffers, centrifuged, resuspended in the same buffer (3 ml), and transferred to a stirred cuvette at 29.5 °C. After a 1-min equilibration time, test substances were added from 100-fold concentrated stocks. Fluorescence ratios were converted to [Ca](i) values as described previously (34) based on the formula of Grynkiewicz et al.(35) .

Measurement of cAMP Accumulation

cAMP accumulation in intact cells was measured according to the method of Evans et al. (36) as described previously (34) with some modifications. C6-2B cells were incubated in Ham's F-10 medium (60 min, 37 °C) with 1 µCi of [^3H]adenine/1.5 times 10^6 cells to label the ATP pool. The cells were then detached, counted, centrifuged at 1,000 rpm for 6 min (IEC HN-SII centrifuge) and resuspended in either the Krebs buffer or the nominally Ca-free Krebs buffer, described above. Aliquots (900 µl) of the cell suspension were incubated at 37 °C for 10 min with PDE inhibitors, 500 µM 3-isobutyl-1-methylxanthine and 100 µM Ro 20-1724. Test agents (100 µl) were added for time periods indicated in the figure legends. The test substance addition also included isoproterenol or forskolin, as indicated, for stimulation of cAMP accumulation. The assay was terminated by addition of 5% (w/v, final) trichloroacetic acid. Unlabeled cAMP (1 mM final) was added to each sample. The samples were centrifuged, and the [^3H]ATP and [^3H]cAMP content of the supernatant was quantified according to the method of Salomon et al.(37) . The accumulation of cAMP is expressed as the percent conversion of [^3H]ATP into [^3H]cAMP.


RESULTS

Thapsigargin-induced [Ca](i) Rise and Inhibition of cAMP Synthesis

Compounds that elevate [Ca](i) independent of receptor activation and second messenger pathways are essential tools, when attempting to distinguish the role of Ca entry versus intracellular release on cAMP accumulation. The desire to elevate [Ca](i) independently of receptor occupation is particularly germane, given the selective susceptibility of certain adenylyl cyclase species to modulation by protein kinase C and beta subunits of G-proteins(38, 39) , whose activation are likely outcomes of receptor occupancy. TG, by inhibiting microsomal Ca-ATPases(29, 30) , causes a net elevation in [Ca](i) independent of receptor activation and IP(3)-induced release.

Resting [Ca](i) in C6-2B cell populations was approximately 90 nM. TG evoked a large and sustained elevation in [Ca](i) in Ca-containing Krebs buffer (Fig. 1A). The addition of EGTA rapidly reduced the [Ca](i) rise to below basal levels, suggesting that the sustained [Ca](i) rise was largely due to Ca entry. Adding Ca to the cells after EGTA caused a dramatic, concentration-dependent Ca entry (Fig. 1A). These data are consistent with the premise that the status of intracellular Ca stores dictates the degree of capacitative Ca entry, as initially proposed by Putney (31, 32) .


Figure 1: Effect of TG on [Ca] and cAMP accumulation in C6-2B cells in Krebs-HEPES buffer. A, [Ca] was determined as described under ``Experimental Procedures.'' TG (100 nM) was added at 60 s. Ca was chelated with 1.1 mM EGTA at 480 s and 3.0 mM (a), 1.0 mM (b), or 0.5 mM (c) CaCl(2) was added at 540 s. The results are representative of at least three experiments with similar results. B, cAMP production stimulated by isoproterenol (10 µM) and forskolin (10 µM) was measured for 1 min in the presence or absence of additional 1 mM CaCl(2), as indicated, following a 10-min preincubation in the presence of 100 nM TG alone (TG), or 1.1 mM EGTA + TG (EGTG). Control cAMP was determined in the absence of TG pretreatment and represented 9.4 ± 0.9% ATP converted (n = 11). The results shown represent mean ± S.E. from at least three separate experiments; *, significantly different from controls (p < 0.05);**, significantly different from TG preincubation (p < 0.05);***, significantly different from EGTA/TG preincubation (p < 0.05).



The consequences of the foregoing TG-induced [Ca](i) rises on cAMP accumulation are explored in the experiments represented in Fig. 1B. In Ca-containing Krebs buffer, TG pretreatment decreased cAMP production by 20% (Fig. 1B).)^2 The inhibitory effect of TG was eliminated by EGTA in excess of extracellular Ca (Fig. 1B). Reintroduction of CaCl(2), following a 10-min incubation with TG and EGTA, inhibited cAMP production by 36%. These results implicate a significant role for Ca entry in the inhibition that is evoked by TG; however, they do not evaluate the potential of the intracellular release that is evoked by TG to cause inhibition. That issue is more effectively addressed in a Ca-free buffer, as outlined in the following series of experiments.

The effect of TG on [Ca](i) in a nominally Ca-free Krebs buffer is shown in Fig. 2A. TG evokes a transient [Ca](i) elevation that slowly declines to basal values, which reflects the emptying and extrusion of intracellular Ca stores. Prominent Ca entry can be demonstrated upon introduction of CaCl(2) to the extracellular medium (3 mM, trace a; 1 mM, traceb). This Ca entry is blocked by LaCl(3) (tracesc and d, Fig. 2A).


Figure 2: Effect of TG on [Ca] and cAMP accumulation in C6-2B cells in Ca-free Krebs buffer. A, [Ca] was determined as described under ``Experimental Procedures.'' TG (100 nM) was added at 60 s and 3.0 mM (a) or 1.0 mM (b) CaCl(2) was added at 420 s. In tracesc and d, 50 µM LaCl(3) was added at 360 s, prior to the addition of 3.0 mM or 1.0 mM CaCl(2), respectively. The results are representative of at least three experiments with similar results. B, production of cAMP was determined for 2 min, in the presence of isoproterenol (80 µM), either acutely upon the addition of 100 nM TG (left) or in a parallel analysis, after preincubation with 100 nM TG for 15 min (right), as indicated. Where CaCl(2) (3.0 mM) or LaCl(3) (200 µM) were used, they were added acutely for the 2-min incubation, as indicated. The control cAMP levels were 3.38 ± 0.07% ATP converted. The data shown are means of triplicates and S.E. values from a representative of three similar experiments; *, significantly different from controls (p < 0.05);**, significantly different from CaCl(2) (p < 0.05);***, significantly different from LaCl(3) (p < 0.05).



In parallel experiments, the consequences of these [Ca](i) rises on cAMP accumulation are explored (Fig. 2B). In calcium-free Krebs buffer, acute incubation with TG causes no effect on cAMP accumulation (Fig. 2B, compare with Fig. 1B). However, upon the introduction of 3 mM CaCl(2) to the extracellular medium, a prominent inhibition of cAMP production is achieved. This inhibition is blocked by extracellular (200 µM) LaCl(3), which suggests that Ca entry exclusively mediates the inhibition of cAMP synthesis that is achieved by TG.

The acute consequences on cAMP synthesis of the various changes in [Ca](i) evoked by TG are explored in a time-course experiment, where all of the elements are added simultaneously to cells in a nominally Ca-free Krebs buffer (Fig. 3). The time courses were designed to measure the effects of these compounds prior to the initial [Ca](i) rise, during the Ca spike and during the sustained second phase of the [Ca](i) rise. TG, without extracellular Ca, did not affect cAMP accumulation at any time point examined (Fig. 3), even at early times e.g. 1 min, where (from Fig. 2A) a considerable elevation in [Ca](i) is achieved. However, a slow onset inhibition is apparent when CaCl(2) is added along with TG (Fig. 3). Presumably this reflects the TG-promoted emptying of pools and the entry that is associated with this process. These data strongly suggest that only the Ca entry promoted by TG, and none of the Ca release, can inhibit cAMP synthesis.


Figure 3: Time course of the effect of TG alone and in the presence of Ca on cAMP accumulation stimulated by isoproterenol (10 µM) and forskolin (10 µM) in C6-2B cells in nominally Ca-free Krebs buffer. TG (100 nM) and CaCl(2) (1 mM) were added simultaneously where indicated and incubated for the specified time period. The control cAMP accumulation was determined in the absence of TG and Ca. The results represent the mean ± S.E. from at least five experiments done in triplicate; *, significantly different from TG alone.



Ionomycin-induced [Ca](i) Rise and Inhibition of Adenylyl Cyclase

The Ca ionophore IM(26, 27, 28) also elevates [Ca](i) independent of receptor activation and IP(3)-induced release. At modest concentrations, it is somewhat selective for intracellular membranes (27, 28) , whereas at higher doses, it also renders the plasma membrane permeable to Ca(26) . In Ca-containing Krebs buffer, IM (400 nM) caused a dramatic Ca rise, followed by a substantial and sustained second phase (Fig. 4A). This initial spike and second phase corresponded to the release of intracellular Ca and a Ca entry phase, respectively. The addition of EGTA eliminated the Ca entry and decreased [Ca](i) to basal levels. A subsequent reintroduction of CaCl(2) in excess of the EGTA yielded concentration-dependent Ca entry (Fig. 4, a-c). The consequences of these IM-induced [Ca](i) rises on cAMP synthesis are explored in the following experiments.


Figure 4: Effect of ionomycin on [Ca] and cAMP accumulation in C6-2B cells in Krebs-HEPES buffer. A, [Ca] was determined as described under ``Experimental Procedures.'' Ionomycin (400 nM) was added at 60 s. Ca was chelated with 1.1 mM EGTA at 480 s and 3.0 mM (a), 1.0 mM (b), or 0.5 mM (c) CaCl(2) was added at 540 s. The data are representative of at least three experiments with similar results. B, cAMP production was measured in the presence of isoproterenol (10 µM) and forskolin (10 µM), along with 400 nM ionomycin alone (IM), 1.1 mM EGTA + IM (EGIM) or EGTA + IM + 1.0 mM CaCl(2) (EGIMCA). Where indicated, EGTA (EG) was present in the preincubation medium, i.e. for 10 min prior to initiation of cAMP production. The control cAMP was determined in the absence of IM and represented 9.4 ± 0.9% ATP converted (n = 11). The results shown represent mean ± S.E. from at least three separate experiments; *, significantly different from controls (p < 0.05);**, significantly different from IM (p < 0.05);***, significantly different from EGTA + IM (p < 0.05).



In Ca-containing Krebs buffer, a 3-min incubation with IM alone led to a modest but significant (12%) inhibition of cAMP production (Fig. 4B). No such inhibition was observed when EGTA was included with the IM (Fig. 4B). A subsequent addition of excess CaCl(2) (1 mM) in the presence of IM and EGTA caused a 25% inhibition of cAMP accumulation. These data again suggest an important role for Ca entry in cAMP inhibition, but do not exclude possible contributions from intracellular release.

In nominally calcium-free Krebs buffer, IM produced only half the [Ca](i) rise compared to that observed in normal Krebs buffer (cf. Fig. 5A and 4A). The elevation in [Ca](i) returned rather rapidly to base-line values. These responses reflect the preclusion of Ca entry in nominally Ca-free Krebs buffer. The introduction of extracellular CaCl(2) yielded concentration-dependent Ca entry (Fig. 5A, tracesa and b), which was effectively blocked by LaCl(3) (Fig. 5A, tracesc and d).


Figure 5: Effect of ionomycin on [Ca] and cAMP accumulation in C6-2B cells in Ca-free Krebs buffer. A, [Ca] was determined as described under ``Experimental Procedures.'' Ionomycin (400 nM) was added at 60 s and 3.0 mM (a) or 1.0 mM (b) CaCl(2) was added at 360 s. In tracesc and d, 50 µM LaCl(3) was added at 300 s, prior to the addition of 3.0 mM or 1.0 mM CaCl(2), respectively. The results are representative of at least three experiments with similar results. B, production of cAMP was determined in a 2-min assay in the presence of isoproterenol (80 µM), either acutely upon the addition of 400 nM IM (left) or in a parallel analysis, after preincubation with 400 nM IM for 10 min (right), as indicated. Where CaCl(2) (3.0 mM) or LaCl(3) (200 µM) were used, they were added acutely for the 2-min incubation, as indicated. The control cAMP levels were 3.12 ± 0.02% ATP converted. The data shown are means and S.E. values of triplicate determinations from a representative of three similar experiments; *, significantly different from controls (p < 0.05);**, significantly different from Ca (p < 0.05).



Acute incubation with IM in Ca-free Krebs buffer did not affect cAMP production (Fig. 5B). However, the addition of CaCl(2) following incubation with IM resulted in a 27% decrease in cAMP accumulation. This decrease was blocked by LaCl(3). These results confirm that Ca entry mediates all of the IM-induced inhibition of cAMP accumulation.

In a time-course study, analogous to that performed with TG, IM elicited no inhibition of cAMP synthesis at any time, in the nominal absence of extracellular Ca (Fig. 6), despite the rather substantial [Ca](i) rise that is evoked, particularly in the first minute of its action (cf. Fig. 5A). However, the presence of extracellular Ca permits substantial and significant inhibition of cAMP accumulation from 30 s onward (Fig. 6). These results are important in demonstrating, not only that Ca entry mediates the inhibitory effects of IM on cAMP accumulation, but also that the diffuse elevation in [Ca](i) caused by IM is ineffective in modulating cAMP synthesis.


Figure 6: Time course of the effect of ionomycin alone and in the presence of Ca on cAMP accumulation in C6-2B cells in nominally Ca-free Krebs buffer. IM (400 nM) and CaCl(2) (1 mM) were added simultaneously where indicated and incubated for the specified time period. cAMP accumulation stimulated by isoproterenol (10 µM) and forskolin (10 µM) was measured using the method described under ``Experimental Procedures.'' The control cAMP accumulation was determined in the absence of IM and Ca. The results represent the mean ± S.E. of triplicates from at least five experiments; *, significantly different from IM alone (p < 0.05).



UTP-induced [Ca](i) Rise and Inhibition of cAMP Synthesis

Earlier it was pointed out that there are additional mechanisms, other than direct effects of Ca on adenylyl cyclase, whereby agents that act via G-proteins to elevate [Ca](i) might also modulate cAMP synthesis. Being mindful of possible ambiguities arising from such effects, the purinergic P agonist, UTP, was investigated to determine whether it would yield results that would support the interpretation derived from studies with TG and IM. UTP acts through the phosphoinositide pathway to release Ca from intracellular stores via IP(3) receptors(25, 40) . The effect of UTP on [Ca](i) is shown in Fig. 7A. In calcium-containing Krebs buffer, UTP elicits a rapid [Ca](i) rise followed by a sustained second phase. The second phase is associated with Ca entry, since it can be eliminated with EGTA and reduced to below basal values. Addition of excess CaCl(2) (Fig. 7A, tracesa, b, and c) resulted in a concentration-dependent increase in [Ca](i), which reflects capacitative Ca entry.


Figure 7: Effect of UTP on [Ca] and cAMP accumulation in C6-2B cells in Ca-containing Krebs-HEPES buffer. A, [Ca] was determined as described under ``Experimental Procedures.'' UTP (100 µM) was added at 60 s. Ca was chelated with 1.1 mM EGTA at 240 s and 3.0 mM (a), 1.0 mM (b), or 0.5 mM (c) CaCl(2) was added at 300 s. The results are representative of at least three experiments with similar results. B, cAMP production, stimulated by isoproterenol (10 µM) and forskolin (10 µM), was measured for 1 min as described under ``Experimental Procedures,'' in the presence of 100 µM UTP (UTP), 1.1 mM EGTA + UTP (EGU), or EGTA + UTP + 1.0 mM CaCl(2) (EGUCA). Where indicated, EGTA (EG) was present in the preincubation medium for 10 min prior to the measurement of cAMP production. The control cAMP was determined in the absence of UTP and represented 9.4 ± 0.9% ATP converted (n = 11). The results shown represent mean ± S.E. from at least four separate experiments; *, significantly different from controls (p < 0.05);**, significantly different from EGTA + UTP (p < 0.05).



The effects of UTP on cAMP accumulation under analogous conditions to those used to manipulate [Ca](i) are presented in Fig. 7B. UTP, which causes release of intracellular Ca, along with Ca entry (see Fig. 7A) resulted in a significant (16%) inhibition of cAMP synthesis. This inhibition was abolished in the presence of EGTA. Readdition of excess CaCl(2) with UTP in the presence of EGTA restored the inhibition of cAMP production. Since EGTA eliminates the Ca influx contribution to the [Ca](i) rise elicited by UTP (Fig. 7A), these observations support a role of Ca entry in inhibition of cAMP accumulation caused by UTP.

In nominally Ca-free medium, stimulation with UTP resulted in an initial spike due to Ca released from intracellular stores, without a Ca entry phase (Fig. 8A). The initial spike due to UTP was smaller than in Fig. 7A. Capacitative Ca entry is evident upon the subsequent introduction of CaCl(2) (Fig. 2A, tracesa and b). This entry could be eliminated by LaCl(3) (tracesc and d).


Figure 8: Effect of UTP on [Ca] and cAMP accumulation in C6-2B cells in Ca-free Krebs buffer. A, [Ca] was determined as described under ``Experimental Procedures.'' UTP (100 µM) was added at 60 s and 3.0 mM (a) or 1.0 mM (b) CaCl(2) was added at 300 s. In traces c and d, 50 µM LaCl(3) was added at 240 s, prior to the addition of 3.0 mM or 1.0 mM CaCl(2), respectively. The results are representative of at least three experiments with similar results. B, production of cAMP stimulated by isoproterenol (80 µM) was determined for 90 s, either acutely upon the addition of 100 µM UTP (left) or in a parallel analysis, after preincubation with 100 µM UTP for 4 min (right), as indicated. Where CaCl(2) (3.0 mM) or LaCl(3) (200 µM) were used, they were added acutely for the 90-s incubation, as indicated. The control cAMP levels were 2.23 ± 0.12% ATP converted. The data shown are means and S.E. values from a representative of three similar experiments; *, significantly different from controls (p < 0.05);**, significantly different from Ca (p < 0.05).



The effects of UTP and UTP-induced Ca entry on cAMP accumulation in a nominally Ca-free Krebs buffer are explored in Fig. 8B. UTP in the absence of extracellular Ca did not affect cAMP production. However, the addition of extracellular Ca (3 mM CaCl(2)) subsequent to a UTP treatment decreased cAMP accumulation by 25%; this inhibition was fully blocked by LaCl(3). The essential contribution of extracellular Ca to the inhibition of cAMP production elicited by UTP, clearly demonstrates the required role of Ca entry in regulating adenylyl cyclase in C6-2B cells. (^3)

The Impact of Phosphodiesterase Activity on [Ca](i)-mediated Inhibition of Steady State cAMP Levels

The following experiments address the impact of a Ca inhibition of adenylyl cyclase in a normal physiological setting, i.e. in the absence of a persistent pharmacological inhibition of PDE. In experiments on Ca-inhibition of adenylyl cyclase in whole cells, (as in the preceding series) PDE is normally inhibited by methylxanthines and Ro 20-1784, for two reasons: (i) to increase the magnitude (and therefore, detectability) of the cAMP signal and (ii) to remove any confusion in interpretation caused by possible effects of Ca not only directly on adenylyl cyclase, but also directly on Ca/calmodulin-stimulated PDE(1, 3) . In a normal cell, however, steady state cAMP levels are the balance between synthetic rates (V(1)) and degradation rates (V(2)), as described by .

the concentration of cAMP relates to these synthetic and degradation rates and the K(m) of PDE, as follows:

where K(m) is the affinity of the PDE for cAMP; ATP cAMP can be considered to be a first order reaction, since the substrate, ATP, is in great excess(43) . Consider the outcome for steady state [cAMP] following a 33% inhibition of adenylyl cyclase (V(1)), where [cAMP] is calculated based on the previous formula: 1) at initial state, V(1) = 9, V(2) = 10, [cAMP] = 9 K(m) units (arbitrary units, adapted from (43) ); 2) at 33% inhibition of adenylyl cyclase, V(1) = 6; V(2) = 10; [cAMP] = 1.5 K(m) units.

This modest inhibition of cAMP synthesis leads to 83% inhibition in cAMP accumulation. Thus, theoretically, a modest inhibition of cAMP synthesis, even against an unchanging background of PDE, can lead to a drastic loss in cAMP accumulation; if this is combined with a stimulation of PDE, loss in cAMP can be profound. The following experiment explores the impact of PDE activity on the inhibition of cAMP levels caused by a rise in [Ca](i).

C6-2B cells were pretreated with either TG or UTP in the absence of extracellular Ca to deplete stores and prime the cells for a substantial or modest inhibition of cAMP accumulation (based on the results presented in Fig. 2and Fig. 8) in the absence or presence of PDE inhibitors (Table 1). After 10 min, cAMP accumulation, stimulated by isoproterenol, was measured in the absence or presence added extracellular CaCl(2) to provoke Ca entry as in Fig. 2. The absence of PDE inhibitors resulted overall in approximately 2-fold less cAMP accumulation (Table 1). However, the degree of inhibition was increased from 40 and 16% to almost 80 and 33%, respectively, in response to the substantial or modest degree of entry evoked by TG and UTP (Table 1). These data emphasize the significance of a background PDE activity in a normal physiological setting, i.e. a partial inhibition of adenylyl cyclase can result in almost total inhibition of cAMP accumulation.




DISCUSSION

In the present study, the efficacy of Ca entry versus release from intracellular stores at inhibiting cAMP accumulation was compared in C6-2B cells. Previous studies in the C6-2B glioma cell line had already established that an increase in [Ca](i) was accompanied by inhibition of cAMP accumulation(7, 11) . This inhibition was independent of PDE, protein kinase C, and Bordetella pertussis toxin (PTX) effects. However, it was not possible from the experiments performed to evaluate the efficacy of Ca entry versus release, at inhibiting cAMP synthesis. (^4)A selective requirement for Ca emanating from a particular source, would suggest a functional co-localization of the adenylyl cyclase with the source of the [Ca](i) rise. We have adopted strategies that elevate [Ca](i) independent of receptor activation and second messenger pathways, since certain adenylyl cyclase species are subject to modulation by protein kinase C and beta subunits of G-proteins, whose activation can arise from receptor occupancy(38, 39) . Each of the strategies applied provides convincing evidence that only Ca entry modulates cAMP synthesis. Thus, in the presence of extracellular Ca, TG, IM, and UTP inhibited cAMP synthesis to varying extents, but in the absence of extracellular Ca, the intracellular release induced by each of these treatments was incapable of eliciting inhibition. The capacitative Ca entry that was provoked by these various means of depleting Ca stores could inhibit cAMP synthesis and this effect was fully blocked by extracellular LaCl(3). The importance of entry versus release is further emphasized when the ineffective, but substantial, rise in [Ca](i) due to release that was elicited by TG, IM, or UTP (peak increments of 240, 400, and 90 nM, respectively; see Fig. 2, Fig. 5, and Fig. 8) is compared with the inhibitory action of the modest degree of entry (100 nM over basal; Fig. 8) evoked by UTP. In other words, even though the elevation in [Ca](i) arising from intracellular release in response to TG, IM, or UTP is substantially greater than, or equal to, the elevation in [Ca](i) arising from entry in response to UTP, only the [Ca](i) rise due to entry caused inhibition of cAMP synthesis. The time-course studies reinforce the fact that it is the onset of Ca entry, rather than the net elevation in [Ca](i), that correlates with inhibition of cAMP synthesis.

The increasing instances in which [Ca](i) elevation is associated with inhibition of cAMP synthesis, apparently as a result of a direct inhibition by Ca of a type V or VI adenylyl cyclase(2, 4, 5, 6, 7, 8, 9, 10, 11, 12) hints at potentially important physiological roles for Ca-inhibitable adenylyl cyclases. For instance, in the case of cardiac contractility, it has been proposed that feedback inhibition by Ca on the cAMP signal may provide a delicate mechanism for fine-tuning cAMP control of [Ca](i) elevation(8, 22, 23) . However, the magnitude of the inhibition often observed in studies such as those described presently may cast doubt on whether such effects can be sensed physiologically. A number of technical issues are relevant in this context. (i) for ease of measurement of cAMP levels, PDE is normally pharmacologically inhibited, unlike the situation in normal cells; consequently, a modest inhibition of cAMP synthesis, coupled with (possibly, a Ca-stimulated) PDE action, could result in dramatic inhibition of cAMP accumulation(1, 3) . Indeed, in the present studies, when PDE was not inhibited, a modest inhibition of adenylyl cyclase was converted into a dramatic loss in steady state cAMP levels. (ii) cAMP, rather than the activity state of its major cellular target, cAMP-dependent protein kinase, is measured in most studies; the kinase, or its targets, may be very sensitive to small changes in ambient cAMP concentrations, as is the case in adipocytes, where a 25% change in cAMP-dependent protein kinase activity is accompanied by a 90% reduction in lipolysis(45) . (iii) cAMP is measured on a very gross time scale, relative to some of the events that it regulates, such as the opening of ion channels(46) . Measurements of cAMP formation with high temporal resolution would significantly clarify the kinetic features of the regulation of its synthesis. Thus, our present limited ability to measure these phenomena should not imply that cellular elements cannot respond to and exploit such regulatory events.

In conclusion, the present results convincingly demonstrate a selective requirement for Ca entry, and not release or a diffuse elevation in [Ca](i), to inhibit adenylyl cyclase in C6-2B cells. The fact that inhibition is observed even when cytosolic Ca concentrations arising from release are greater than the concentrations deriving from entry, implies that the entry sites and adenylyl cyclase must be relatively close inside the cell, i.e. they must be sufficiently close that, regardless of the diffusion of Ca away from entry channels, Ca concentrations must remain high enough to inhibit adenylyl cyclase. This situation is directly analogous to the requirement for localized Ca entry for neurotransmitter and exocytotic secretion (47, 48) in which diffuse [Ca](i) rises fail to mimic the actions of Ca entry through discrete channels. An extension of this speculation is that the density of Ca entry channels relative to adenylyl cyclases will dictate whether adenylyl cyclases can respond to Ca. It will be interesting to determine whether any cell type can release sufficient Ca from intracellular stores so that efficacious concentrations reach the plasma membrane, or whether it is always necessary that Ca-sensitive adenylyl cyclases and Ca entry channels are functionally colocalized for cAMP synthesis to be regulated by [Ca](i). Given the limited range of Ca diffusion through cells because of mobile and immobile buffers(49) , it is possible that unless release sites can be very closely apposed to adenylyl cyclase, released Ca will never reach adenylyl cyclase in sufficient concentration. For the present, these studies establish that Ca-sensitive adenylyl cyclase in C6-2B cells is regulated only by the entry of Ca. Whether this functional colocalization is coincidental or directed will be an interesting avenue for future research.


FOOTNOTES

*
The studies were supported in part by National Institutes of Health Grant GM 32483 (to D. M. F. C.). 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.

§
Supported in part by Training Grant GM 07063 from the National Institutes of Health.

To whom correspondence should be addressed: Dept. of Pharmacology, Box C-236, University of Colorado Health Sciences Center, 4200 E. Ninth Ave., Denver, CO 80262. Tel.: 303-270-8964; Fax: 303-270-7097.

(^1)
The abbreviations used are: [Ca], cytosolic free Ca; G-protein, guanine nucleotide-binding regulatory protein; PDE, cAMP phosphodiesterase; TG, thapsigargin; IM, ionomycin; PTX, the toxin derived from B. pertussis, which ADP-ribosylates G-protein alpha subunits; IP(3), inositol 1,4,5-trisphosphate.

(^2)
In these studies, whether isoproterenol alone, forskolin alone, or the combination was used to stimulate cAMP accumulation, the degree of inhibition associated with Ca entry was not strikingly different (data not shown). Furthermore, agents that were used to elevate cAMP were without effect on either Ca entry or release and so were not included in the [Ca] measurements.

(^3)
The present effects of UTP are distinct from those of ATP, which has been reported to directly inhibit adenylyl cyclase in C6-2B cells, via a PTX-sensitive G(i) protein, without the involvement of Ca-influx(41, 42) . Whereas ATP can bind to all purinoceptors, regardless of the signal pathways that they utilize, UTP acts selectively through the P purinoceptor, which is coupled to phospholipase C via a PTX-insensitive G(q) protein in C6-2B cells(25, 40) .

(^4)
Quite curiously, in this same cell line, Charpentier et al. (44) observed a prominent Ca-dependent stimulation of cAMP accumulation, which appeared to be largely due to Ca influx. The fact that we, as well as others, have demonstrated inhibition of cAMP accumulation by calcium in C6-2B cells, rather than stimulation, may be due to divergence of the cell line over time. As noted earlier, the C6-2B cells used in the present study express predominantly type VI adenylyl cyclase mRNA along with small amounts of type III(14) ; it is conceivable that, with time, type III might have become the dominant isoform in these other cultures.


ACKNOWLEDGEMENTS

We thank Dr. R. A. Harris for continuing use of his spectrofluorimeter, Dr. G. Brooker for helpful discussions, and Dr. P. Mollard for useful comments on the manuscript.


REFERENCES

  1. Meeker, R. B., and Harden, T. K. (1982) Mol. Pharmacol. 22, 310-319 [Abstract]
  2. Dorflinger, L. J., Albert, P. J., Williams, A. T., and Behrman, H. R. (1984) Endocrinology 114, 1208-1215 [Abstract]
  3. Erneux, C., Sande, J. V., Miot, F., Cochaux, P., Decoster, C., and Dumont, J. E. (1985) Mol. Cell. Endocrinol. 43, 123-134 [Medline] [Order article via Infotrieve]
  4. Pereira, M. E., Segaloff, D. L., and Ascoli, M. (1988) Endocrinology 122, 2232-2239 [Abstract]
  5. Aakerlund, L., Gether, U., Fuhlendorff, J., Schwartz, T. W., and Thastrup, O. (1990) FEBS Lett. 260, 73-78 [CrossRef][Medline] [Order article via Infotrieve]
  6. Boyajian, C., Garritsen, A., and Cooper, D. M. F. (1991) J. Biol. Chem. 266, 4995-5003 [Abstract/Free Full Text]
  7. DeBernardi, M. A., Seki, T., and Brooker, G. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 9257-9261 [Abstract]
  8. Yu, H. J., Ma, H., and Green, R. D. (1993) Mol. Pharmacol. 44, 689-693 [Abstract]
  9. Altiok, N., and Fredholm, B. B. (1993) Cell. Signalling 5, 279-288 [CrossRef][Medline] [Order article via Infotrieve]
  10. Zgombick, J. M., Borden, L. A., Cochran, T. L., Kucharewicz, S. A., Weinshank, R. L., and Branchek, T. A. (1993) Mol. Pharmacol. 44, 575-582 [Abstract]
  11. DeBernardi, M. A., Munshi, R., and Brooker, G. (1993) Mol. Pharmacol. 43, 451-458 [Abstract]
  12. Garritsen, A., Zhang, Y., Firestone, J. A., Browning, M. D., and Cooper, D. M. F. (1992) J. Neurochem. 59, 1630-1639 [Medline] [Order article via Infotrieve]
  13. Yoshimura, M., and Cooper, D. M. F. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 6716-6720 [Abstract]
  14. DeBernardi, M. A., Munshi, R., Yoshimura, M., Cooper, D. M. F., and Brooker, G. (1993) Biochem. J. 293, 325-328 [Medline] [Order article via Infotrieve]
  15. Ishikawa, Y., Katsushika, S., Chen, L., Halnon, N. J., Kawabe, J., and Homcy, C. J. (1992) J. Biol. Chem. 267, 13553-13557 [Abstract/Free Full Text]
  16. Premont, R. T., Chen, J., Ma, H., Ponnapalli, M., and Iyengar, R. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 9809-9813 [Abstract]
  17. Katsushika, S., Chen, L., Kawabe, J., Nilakantan, R., Halnon, N. J., Homcy, C. J., and Ishikawa, Y. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 8774-8778 [Abstract]
  18. Krupinski, J., Lehman, T. C., Frankenfield, C. D., Zwaagstra, J. C., and Watson, P. A. (1992) J. Biol. Chem. 267, 24858-24862 [Abstract/Free Full Text]
  19. Wallach, J., Droste, M., Kluxen, F. W., Pfeuffer, T., and Frank, R. (1994) FEBS Lett. 338, 257-263 [CrossRef][Medline] [Order article via Infotrieve]
  20. Cooper, D. M. F., Yoshimura, M., Zhang, Y., Chiono, M., and Mahey, R. (1994) Biochem. J. 297, 437-440 [CrossRef][Medline] [Order article via Infotrieve]
  21. Mons, N., and Cooper, D. M. F. (1994) Mol. Brain Res. 22, 236-244 [Medline] [Order article via Infotrieve]
  22. Colvin, R. A., Oibo, J. A., and Allen, R. A. (1991) Cell Calcium 12, 19-27 [CrossRef][Medline] [Order article via Infotrieve]
  23. Cooper, D. M. F., and Brooker, G. (1993) Trends Pharmacol. Sci. 14, 34-36 [Medline] [Order article via Infotrieve]
  24. Lin, W.-W., Kiang, J. G., and Chuang, D.-M. (1992) J. Neurosci. 12, 1077-1085 [Abstract]
  25. Burnstock, G. (1990) Ann. N. Y. Acad. Sci. 603, 1-17
  26. Liu, C.-M., and Hermann, T. E. (1978) J. Biol. Chem. 253, 5892-5894 [Abstract]
  27. Albert, P. R., and Tashjian, A. H., Jr. (1986) Am. J. Physiol. 251, C887-C891
  28. Morgan, A. J., and Jacob, R. (1994) Biochem. J. 300, 665-672 [Medline] [Order article via Infotrieve]
  29. Thastrup, O., Cullen, P. J., Drøbak, B. K., Hanley, M. R., and Dawson, A. P. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 2466-2470 [Abstract]
  30. Thastrup, O., Dawson, A. P., Scharff, O., Foder, B., Cullen, P. J., Drøbak, B. K., Bjerrum, P. J., Christensen, S. B., and Hanley, M. R. (1989) Agents Actions 27, 17-23 [Medline] [Order article via Infotrieve]
  31. Putney, J. W., Jr. (1986) Cell Calcium 7, 1-12 [Medline] [Order article via Infotrieve]
  32. Putney, J. W., Jr. (1990) Cell Calcium 11, 611-624 [Medline] [Order article via Infotrieve]
  33. Robitaille, R., Garcia, M. L., Kaczorowski, G. J., and Charlton, M. P. (1993) Neuron 11, 645-655 [Medline] [Order article via Infotrieve]
  34. Garritsen, A., and Cooper, D. M. F. (1992) J. Neurochem. 59, 190-199 [Medline] [Order article via Infotrieve]
  35. Grynkiewicz, G., Poenie, M., and Tsien, R. Y. (1985) J. Biol. Chem. 260, 3440-3450 [Abstract]
  36. Evans, T., Smith, M. M., Tanner, L. I., and Harden, T. K. (1984) Mol. Pharmacol. 26, 395-404 [Abstract]
  37. Salomon, Y., Londos, C., and Rodbell, M. (1974) Anal. Biochem. 58, 541-548 [Medline] [Order article via Infotrieve]
  38. Tang, W.-J., and Gilman, A. G. (1992) Cell 70, 869-872 [Medline] [Order article via Infotrieve]
  39. Iyengar, R. (1993) FASEB J. 7, 768-775 [Abstract/Free Full Text]
  40. Munshi, R., DeBernardi, M. A., and Brooker, G. (1993) Mol. Pharmacol. 44, 1185-1191 [Abstract]
  41. Lin, W.-W., and Chuang, D.-M. (1993) Mol. Pharmacol. 44, 158-165 [Abstract]
  42. Pianet, I., Merle, M., and Labouesse, J. (1989) Biochem. Biophys. Res. Commun. 163, 1150-1157 [Medline] [Order article via Infotrieve]
  43. Newsholme, E. A., and Start, C. (1973) Regulation in Metabolism , Wiley Press, New York
  44. Charpentier, N., Prézeau, L., Carrette, J., Bertorelli, R., Le Cam, G., Manzoni, O., Bockaert, J., and Homburger, V. (1993) J. Biol. Chem. 268, 8980-8989 [Abstract/Free Full Text]
  45. Honnor, R. C., Dhillon, G. S., and Londos, C. (1985) J. Biol. Chem. 260, 15130-15138 [Abstract/Free Full Text]
  46. Zufall, F., Hatt, H., and Firestein, S. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 9335-9339 [Abstract]
  47. Sihra, T. S., Bogonez, E., and Nicholls, D. G. (1992) J. Biol. Chem. 267, 1983-1989 [Abstract/Free Full Text]
  48. Maruyama, Y., Inooka, G., Li, Y. X., Miyashita, Y., and Kasai, H. (1993) EMBO J. 12, 3017-3022 [Abstract]
  49. Allbritton, N. L., Meyer, T., and Stryer, L. (1992) Science 258, 1812-1815 [Medline] [Order article via Infotrieve]

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