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• W2064576105 abstract "Elevation of cytosolic free Ca 2+ 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 2+ entry or release for inhibition of cAMP accumulation to occur. In the present study, the effectiveness of intracellular Ca 2+ release evoked by three independent methods (thapsigargin, ionomycin, and UTP) was compared with the capacitative Ca 2+ entry that was triggered by these treatments. In each situation, only Ca 2+ entry could inhibit cAMP accumulation (La 3+ ions blocked the effect); Ca 2+ release, which was substantial in some cases, was without effect. A moderate inhibition, as was elicited by a modest degree of Ca 2+ 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 2+ entry mediates the inhibitory effects of Ca 2+ on cAMP accumulation, but also that diffuse elevations in [Ca 2+] i are ineffective in modulating cAMP synthesis. This property suggests that, as with certain Ca 2+-sensitive ion channels, Ca 2+-sensitive adenylyl cyclases may be functionally colocalized with Ca 2+ entry channels. Elevation of cytosolic free Ca 2+ 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 2+ entry or release for inhibition of cAMP accumulation to occur. In the present study, the effectiveness of intracellular Ca 2+ release evoked by three independent methods (thapsigargin, ionomycin, and UTP) was compared with the capacitative Ca 2+ entry that was triggered by these treatments. In each situation, only Ca 2+ entry could inhibit cAMP accumulation (La 3+ ions blocked the effect); Ca 2+ release, which was substantial in some cases, was without effect. A moderate inhibition, as was elicited by a modest degree of Ca 2+ 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 2+ entry mediates the inhibitory effects of Ca 2+ on cAMP accumulation, but also that diffuse elevations in [Ca 2+] i are ineffective in modulating cAMP synthesis. This property suggests that, as with certain Ca 2+-sensitive ion channels, Ca 2+-sensitive adenylyl cyclases may be functionally colocalized with Ca 2+ entry channels. A rise in cytosolic free calcium, [Ca 2+] i, 1The abbreviations used are: [Ca 2+]cytosolic free Ca 2+G-proteinguanine nucleotide-binding regulatory proteinPDEcAMP phosphodiesteraseTGthapsigarginIMionomycinPTXthe toxin derived from B. pertussis, which ADP-ribosylates G-protein α, subunitsIP3inositol 1,4,5-trisphosphate. has been associated with the inhibition of cAMP accumulation in various tissues and cell lines(1Meeker R.B. Harden T.K. Mol. Pharmacol. 1982; 22: 310-319PubMed Google Scholar, 2Dorflinger L.J. Albert P.J. Williams A.T. Behrman H.R. Endocrinology. 1984; 114: 1208-1215Crossref PubMed Scopus (77) Google Scholar, 3Erneux C. Sande J.V. Miot F. Cochaux P. Decoster C. Dumont J.E. Mol. Cell. Endocrinol. 1985; 43: 123-134Crossref PubMed Scopus (39) Google Scholar, 4Pereira M.E. Segaloff D.L. Ascoli M. Endocrinology. 1988; 122: 2232-2239Crossref PubMed Scopus (21) Google Scholar, 5Aakerlund L. Gether U. Fuhlendorff J. Schwartz T.W. Thastrup O. FEBS Lett. 1990; 260: 73-78Crossref PubMed Scopus (130) Google Scholar, 6Boyajian C. Garritsen A. Cooper D.M.F. J. Biol. Chem. 1991; 266: 4995-5003Abstract Full Text PDF PubMed Google Scholar, 7DeBernardi M.A. Seki T. Brooker G. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9257-9261Crossref PubMed Scopus (45) Google Scholar, 8Yu H.J. Ma H. Green R.D. Mol. Pharmacol. 1993; 44: 689-693PubMed Google Scholar, 9Altiok N. Fredholm B.B. Cell. Signalling. 1993; 5: 279-288Crossref PubMed Scopus (17) Google Scholar, 10Zgombick J.M. Borden L.A. Cochran T.L. Kucharewicz S.A. Weinshank R.L. Branchek T.A. Mol. Pharmacol. 1993; 44: 575-582PubMed Google Scholar). In a number of these situations, a Ca 2+-dependent stimulation of phosphodiesterase (PDE) was the apparent mechanism for the inhibition of cAMP accumulation(1Meeker R.B. Harden T.K. Mol. Pharmacol. 1982; 22: 310-319PubMed Google Scholar, 3Erneux C. Sande J.V. Miot F. Cochaux P. Decoster C. Dumont J.E. Mol. Cell. Endocrinol. 1985; 43: 123-134Crossref PubMed Scopus (39) Google Scholar). However, in many of these cases, a Ca 2+ stimulation of PDE has been specifically precluded and a direct inhibitory effect of elevated [Ca 2+] i on adenylyl cyclase has been demonstrated to be the likely mechanism(2Dorflinger L.J. Albert P.J. Williams A.T. Behrman H.R. Endocrinology. 1984; 114: 1208-1215Crossref PubMed Scopus (77) Google Scholar, 4Pereira M.E. Segaloff D.L. Ascoli M. Endocrinology. 1988; 122: 2232-2239Crossref PubMed Scopus (21) Google Scholar, 5Aakerlund L. Gether U. Fuhlendorff J. Schwartz T.W. Thastrup O. FEBS Lett. 1990; 260: 73-78Crossref PubMed Scopus (130) Google Scholar, 6Boyajian C. Garritsen A. Cooper D.M.F. J. Biol. Chem. 1991; 266: 4995-5003Abstract Full Text PDF PubMed Google Scholar, 7DeBernardi M.A. Seki T. Brooker G. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9257-9261Crossref PubMed Scopus (45) Google Scholar, 8Yu H.J. Ma H. Green R.D. Mol. Pharmacol. 1993; 44: 689-693PubMed Google Scholar, 9Altiok N. Fredholm B.B. Cell. Signalling. 1993; 5: 279-288Crossref PubMed Scopus (17) Google Scholar, 10Zgombick J.M. Borden L.A. Cochran T.L. Kucharewicz S.A. Weinshank R.L. Branchek T.A. Mol. Pharmacol. 1993; 44: 575-582PubMed Google Scholar, 11DeBernardi M.A. Munshi R. Brooker G. Mol. Pharmacol. 1993; 43: 451-458PubMed Google Scholar, 12Garritsen A. Zhang Y. Firestone J.A. Browning M.D. Cooper D.M.F. J. Neurochem. 1992; 59: 1630-1639Crossref PubMed Scopus (18) Google Scholar). A number of the sources displaying this behavior also express either adenylyl cyclase activity that is inhibited by submicromolar [Ca 2+] in in vitro assays(2Dorflinger L.J. Albert P.J. Williams A.T. Behrman H.R. Endocrinology. 1984; 114: 1208-1215Crossref PubMed Scopus (77) Google Scholar, 6Boyajian C. Garritsen A. Cooper D.M.F. J. Biol. Chem. 1991; 266: 4995-5003Abstract Full Text PDF PubMed Google Scholar, 7DeBernardi M.A. Seki T. Brooker G. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9257-9261Crossref PubMed Scopus (45) Google Scholar, 8Yu H.J. Ma H. Green R.D. Mol. Pharmacol. 1993; 44: 689-693PubMed Google Scholar, 9Altiok N. Fredholm B.B. Cell. Signalling. 1993; 5: 279-288Crossref PubMed Scopus (17) Google Scholar), or mRNAs corresponding to either types V or VI adenylyl cyclase(13Yoshimura M. Cooper D.M.F. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6716-6720Crossref PubMed Scopus (204) Google Scholar, 14DeBernardi M.A. Munshi R. Yoshimura M. Cooper D.M.F. Brooker G. Biochem. J. 1993; 293: 325-328Crossref PubMed Scopus (42) Google Scholar, 15Ishikawa Y. Katsushika S. Chen L. Halnon N.J. Kawabe J. Homcy C.J. J. Biol. Chem. 1992; 267: 13553-13557Abstract Full Text PDF PubMed Google Scholar, 16Premont R.T. Chen J. Ma H. Ponnapalli M. Iyengar R. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9809-9813Crossref PubMed Scopus (170) Google Scholar, 17Katsushika S. Chen L. Kawabe J. Nilakantan R. Halnon N.J. Homcy C.J. Ishikawa Y. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8774-8778Crossref PubMed Scopus (195) Google Scholar, 18Krupinski J. Lehman T.C. Frankenfield C.D. Zwaagstra J.C. Watson P.A. J. Biol. Chem. 1992; 267: 24858-24862Abstract Full Text PDF PubMed Google Scholar, 19Wallach J. Droste M. Kluxen F.W. Pfeuffer T. Frank R. FEBS Lett. 1994; 338: 257-263Crossref PubMed Scopus (47) Google Scholar); the latter are recently cloned species that can be inhibited by Ca 2+ when their cDNAs are transfected into HEK 293 cells(13Yoshimura M. Cooper D.M.F. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6716-6720Crossref PubMed Scopus (204) Google Scholar, 19Wallach J. Droste M. Kluxen F.W. Pfeuffer T. Frank R. FEBS Lett. 1994; 338: 257-263Crossref PubMed Scopus (47) Google Scholar, 20Cooper D.M.F. Yoshimura M. Zhang Y. Chiono M. Mahey R. Biochem. J. 1994; 297: 437-440Crossref PubMed Scopus (78) Google Scholar, 21Mons N. Cooper D.M.F. Mol. Brain Res. 1994; 22: 236-244Crossref PubMed Scopus (95) Google Scholar). Such negative influences of [Ca 2+] i on cAMP synthesis have been speculated to represent a useful feedback in systems where cAMP controls [Ca 2+] i, such as cardiac myocytes(8Yu H.J. Ma H. Green R.D. Mol. Pharmacol. 1993; 44: 689-693PubMed Google Scholar, 22Colvin R.A. Oibo J.A. Allen R.A. Cell Calcium. 1991; 12: 19-27Crossref PubMed Scopus (50) Google Scholar, 23Cooper D.M.F. Brooker G. Trends Pharmacol. Sci. 1993; 14: 34-36Abstract Full Text PDF PubMed Scopus (35) Google Scholar). However, an unresolved issue, which may cast light on the organization of Ca 2+-sensitive adenylyl cyclases within cells, is whether there is any selective ability of Ca 2+ released from intracellular pools or Ca 2+ entry to inhibit adenylyl cyclase. cytosolic free Ca 2+ guanine nucleotide-binding regulatory protein cAMP phosphodiesterase thapsigargin ionomycin the toxin derived from B. pertussis, which ADP-ribosylates G-protein α, subunits inositol 1,4,5-trisphosphate. The C6-2B glioma cell line is a useful model system for evaluating the efficacy of Ca 2+ entry and internal release on the inhibition of cAMP synthesis. In these nonexcitable cells, agonist stimulation of [Ca 2+] i elevation involves an initial release of Ca 2+ from inositol 1,4,5-trisphosphate (IP3)-sensitive stores, accompanied by Ca 2+ entry(7DeBernardi M.A. Seki T. Brooker G. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9257-9261Crossref PubMed Scopus (45) Google Scholar, 11DeBernardi M.A. Munshi R. Brooker G. Mol. Pharmacol. 1993; 43: 451-458PubMed Google Scholar, 24Lin W.-W. Kiang J.G. Chuang D.-M. J. Neurosci. 1992; 12: 1077-1085Crossref PubMed Google Scholar). Strikingly, this cell line expresses almost exclusively Type VI adenylyl cyclase, along with trace amounts of type III(14DeBernardi M.A. Munshi R. Yoshimura M. Cooper D.M.F. Brooker G. Biochem. J. 1993; 293: 325-328Crossref PubMed Scopus (42) Google Scholar). Previous studies in this cell line have established an inhibition of cAMP accumulation that is compatible with direct effects of [Ca 2+] i on type VI adenylyl cyclase(7DeBernardi M.A. Seki T. Brooker G. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9257-9261Crossref PubMed Scopus (45) Google Scholar, 11DeBernardi M.A. Munshi R. Brooker G. Mol. Pharmacol. 1993; 43: 451-458PubMed Google Scholar, 14DeBernardi M.A. Munshi R. Yoshimura M. Cooper D.M.F. Brooker G. Biochem. J. 1993; 293: 325-328Crossref PubMed Scopus (42) Google Scholar). The focus of the present study was to determine whether there was a selective role for either Ca 2+ entry or release in the inhibition of cAMP accumulation in the C6-2B cell line. Three independent tools were utilized to manipulate [Ca 2+] i: the purinergic agonist UTP(25Burnstock G. Ann. N. Y. Acad. Sci. 1990; 603: 1-17Crossref PubMed Scopus (363) Google Scholar), the Ca 2+ ionophore ionomycin (IM)(26Liu C.-M. Hermann T.E. J. Biol. Chem. 1978; 253: 5892-5894Abstract Full Text PDF PubMed Google Scholar, 27Albert P.R. Tashjian Jr., A.H. Am. J. Physiol. 1986; 251: C887-C891Crossref PubMed Google Scholar, 28Morgan A.J. Jacob R. Biochem. J. 1994; 300: 665-672Crossref PubMed Scopus (256) Google Scholar), and the microsomal Ca 2+-ATPase inhibitor thapsigargin (TG)(29Thastrup O. Cullen P.J. Dr⊘bak B.K. Hanley M.R. Dawson A.P. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2466-2470Crossref PubMed Scopus (3009) Google Scholar, 30Thastrup O. Dawson A.P. Scharff O. Foder B. Cullen P.J. Dr⊘bak B.K. Bjerrum P.J. Christensen S.B. Hanley M.R. Agents Actions. 1989; 27: 17-23Crossref PubMed Scopus (435) Google Scholar). These three agents provoke [Ca 2+] i rises and capacitative Ca 2+ entry by different mechanisms(31Putney Jr., J.W. Cell Calcium. 1986; 7: 1-12Crossref PubMed Scopus (2115) Google Scholar, 32Putney Jr., J.W. Cell Calcium. 1990; 11: 611-624Crossref PubMed Scopus (1264) Google Scholar). Subsequent experiments with these agents clearly demonstrated that Ca 2+ entry, rather than a simple rise in [Ca 2+] i, exclusively inhibits cAMP accumulation in C6-2B cells. The results suggest that, as with certain Ca 2+-sensitive ion channels(33Robitaille R. Garcia M.L. Kaczorowski G.J. Charlton M.P. Neuron. 1993; 11: 645-655Abstract Full Text PDF PubMed Scopus (378) Google Scholar), Ca 2+-inhibitable adenylyl cyclase is functionally co-localized with sites of Ca 2+ entry. 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 Early phase C6-2B cells, kindly provided by Dr. G. L. Brooker (Georgetown University School of Medicine, Washington, DC) were grown in 75-cm2 culture flasks in Ham's F-10 medium, containing 10% calf serum in an atmosphere of 95% air, 5% CO2 at 37°C, without antibiotics. Cells were used 4-7 days after passage. [Ca 2+]i was measured by the Fura-2 technique using an H& series 300 spectrofluorimeter, as described previously(34Garritsen A. Cooper D.M.F. J. Neurochem. 1992; 59: 190-199Crossref PubMed Scopus (13) Google Scholar), with modifications. Briefly, cells were detached with phosphate-buffered saline (12.1 mM Na2HPO4, 4 mM KH2PO4, 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 2+]i measurements were made in either Krebs buffer or a nominally Ca 2+-free Krebs buffer. The Krebs buffer consisted of 120 mM NaCl, 4.75 mM KCl, 1 mM KH2PO4, 5 mM NaHCO3, 1.44 mM MgSO4, 1.1 mM CaCl2, 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 2+-free Krebs buffer contained 120 mM NaCl, 4.75 mM KCl, 1.44 mM MgSO4, 11 mM glucose, 25 mM HEPES, and 0.1% bovine serum albumin adjusted to pH 7.4 with 2 M Tris base. Approximately 4 × 106 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 2+]i values as described previously (34Garritsen A. Cooper D.M.F. J. Neurochem. 1992; 59: 190-199Crossref PubMed Scopus (13) Google Scholar) based on the formula of Grynkiewicz et al.(35Grynkiewicz G. Poenie M. Tsien R.Y. J. Biol. Chem. 1985; 260: 3440-3450Abstract Full Text PDF PubMed Scopus (80) Google Scholar). cAMP accumulation in intact cells was measured according to the method of Evans et al. (36Evans T. Smith M.M. Tanner L.I. Harden T.K. Mol. Pharmacol. 1984; 26: 395-404PubMed Google Scholar) as described previously (34Garritsen A. Cooper D.M.F. J. Neurochem. 1992; 59: 190-199Crossref PubMed Scopus (13) Google Scholar) 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 × 106 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 2+-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.(37Salomon Y. Londos C. Rodbell M. Anal. Biochem. 1974; 58: 541-548Crossref PubMed Scopus (3374) Google Scholar). The accumulation of cAMP is expressed as the percent conversion of [3H]ATP into [3H]cAMP. Compounds that elevate [Ca 2+]i independent of receptor activation and second messenger pathways are essential tools, when attempting to distinguish the role of Ca 2+ entry versus intracellular release on cAMP accumulation. The desire to elevate [Ca 2+]i independently of receptor occupation is particularly germane, given the selective susceptibility of certain adenylyl cyclase species to modulation by protein kinase C and β γ subunits of G-proteins(38Tang W.-J. Gilman A.G. Cell. 1992; 70: 869-872Abstract Full Text PDF PubMed Scopus (376) Google Scholar, 39Iyengar R. FASEB J. 1993; 7: 768-775Crossref PubMed Scopus (266) Google Scholar), whose activation are likely outcomes of receptor occupancy. TG, by inhibiting microsomal Ca 2+-ATPases(29Thastrup O. Cullen P.J. Dr⊘bak B.K. Hanley M.R. Dawson A.P. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2466-2470Crossref PubMed Scopus (3009) Google Scholar, 30Thastrup O. Dawson A.P. Scharff O. Foder B. Cullen P.J. Dr⊘bak B.K. Bjerrum P.J. Christensen S.B. Hanley M.R. Agents Actions. 1989; 27: 17-23Crossref PubMed Scopus (435) Google Scholar), causes a net elevation in [Ca 2+]i independent of receptor activation and IP3-induced release. Resting [Ca 2+]i in C6-2B cell populations was approximately 90 nM. TG evoked a large and sustained elevation in [Ca 2+]i in Ca 2+-containing Krebs buffer (Fig. 1A). The addition of EGTA rapidly reduced the [Ca 2+]i rise to below basal levels, suggesting that the sustained [Ca 2+]i rise was largely due to Ca 2+ entry. Adding Ca 2+ to the cells after EGTA caused a dramatic, concentration-dependent Ca 2+ entry (Fig. 1A). These data are consistent with the premise that the status of intracellular Ca 2+ stores dictates the degree of capacitative Ca 2+ entry, as initially proposed by Putney (31Putney Jr., J.W. Cell Calcium. 1986; 7: 1-12Crossref PubMed Scopus (2115) Google Scholar, 32Putney Jr., J.W. Cell Calcium. 1990; 11: 611-624Crossref PubMed Scopus (1264) Google Scholar). The consequences of the foregoing TG-induced [Ca 2+]i rises on cAMP accumulation are explored in the experiments represented in Fig. 1B. In Ca 2+-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 2+ (Fig. 1B). Reintroduction of CaCl2, following a 10-min incubation with TG and EGTA, inhibited cAMP production by 36%. These results implicate a significant role for Ca 2+ 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 2+-free buffer, as outlined in the following series of experiments. The effect of TG on [Ca 2+]i in a nominally Ca 2+-free Krebs buffer is shown in Fig. 2A. TG evokes a transient [Ca 2+]i elevation that slowly declines to basal values, which reflects the emptying and extrusion of intracellular Ca 2+ stores. Prominent Ca 2+ entry can be demonstrated upon introduction of CaCl2 to the extracellular medium (3 mM, trace a; 1 mM, traceb). This Ca 2+ entry is blocked by LaCl3 (tracesc and d, Fig. 2A). In parallel experiments, the consequences of these [Ca 2+]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 CaCl2 to the extracellular medium, a prominent inhibition of cAMP production is achieved. This inhibition is blocked by extracellular (200 μM) LaCl3, which suggests that Ca 2+ 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 2+]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 2+-free Krebs buffer (Fig. 3). The time courses were designed to measure the effects of these compounds prior to the initial [Ca 2+]i rise, during the Ca 2+ spike and during the sustained second phase of the [Ca 2+]i rise. TG, without extracellular Ca 2+, 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 2+]i is achieved. However, a slow onset inhibition is apparent when CaCl2 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 2+ entry promoted by TG, and none of the Ca 2+ release, can inhibit cAMP synthesis. The Ca 2+ ionophore IM(26Liu C.-M. Hermann T.E. J. Biol. Chem. 1978; 253: 5892-5894Abstract Full Text PDF PubMed Google Scholar, 27Albert P.R. Tashjian Jr., A.H. Am. J. Physiol. 1986; 251: C887-C891Crossref PubMed Google Scholar, 28Morgan A.J. Jacob R. Biochem. J. 1994; 300: 665-672Crossref PubMed Scopus (256) Google Scholar) also elevates [Ca 2+]i independent of receptor activation and IP3-induced release. At modest concentrations, it is somewhat selective for intracellular membranes (27Albert P.R. Tashjian Jr., A.H. Am. J. Physiol. 1986; 251: C887-C891Crossref PubMed Google Scholar, 28Morgan A.J. Jacob R. Biochem. J. 1994; 300: 665-672Crossref PubMed Scopus (256) Google Scholar), whereas at higher doses, it also renders the plasma membrane permeable to Ca 2+(26Liu C.-M. Hermann T.E. J. Biol. Chem. 1978; 253: 5892-5894Abstract Full Text PDF PubMed Google Scholar). In Ca 2+-containing Krebs buffer, IM (400 nM) caused a dramatic Ca 2+ rise, followed by a substantial and sustained second phase (Fig. 4A). This initial spike and second phase corresponded to the release of intracellular Ca 2+ and a Ca 2+ entry phase, respectively. The addition of EGTA eliminated the Ca 2+ entry and decreased [Ca 2+]i to basal levels. A subsequent reintroduction of CaCl2 in excess of the EGTA yielded concentration-dependent Ca 2+ entry (Fig. 4, a-c). The consequences of these IM-induced [Ca 2+]i rises on cAMP synthesis are explored in the following experiments. In Ca 2+-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 CaCl2 (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 2+ 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 2+]i rise compared to that observed in normal Krebs buffer (cf. Fig. 5A and 4A). The elevation in [Ca 2+]i returned rather rapidly to base-line values. These responses reflect the preclusion of Ca 2+ entry in nominally Ca 2+-free Krebs buffer. The introduction of extracellular CaCl2 yielded concentration-dependent Ca 2+ entry (Fig. 5A, tracesa and b), which was effectively blocked by LaCl3 (Fig. 5A, tracesc and d). Acute incubation with IM in Ca 2+-free Krebs buffer did not affect cAMP production (Fig. 5B). However, the addition of CaCl2 following incubation with IM resulted in a 27% decrease in cAMP accumulation. This decrease was blocked by LaCl3. These results confirm that Ca 2+ 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 2+ (Fig. 6), despite the rather substantial [Ca 2+]i rise that is evoked, particularly in the first minute of its action (cf. Fig. 5A). However, the presence of extracellular Ca 2+ permits substantial and significant inhibition of cAMP accumulation from 30 s onward (Fig. 6). These results are important in demonstrating, not only that Ca 2+ entry mediates the inhibitory effects of IM on cAMP accumulation, but also that the diffuse elevation in [Ca 2+]i caused by IM is ineffective in modulating cAMP synthesis. Earlier it was pointed out that there are additional mechanisms, other than direct effects of Ca 2+ on adenylyl cyclase, whereby agents that act via G-proteins to elevate [Ca 2+]i might also modulate cAMP synthesis. Being mindful of possible ambiguities arising from such effects, the purinergic P 2u 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 2+ from intracellular stores via IP3 receptors(25Burnstock G. Ann. N. Y. Acad. Sci. 1990; 603: 1-17Crossref PubMed Scopus (363) Google Scholar, 40Munshi R. DeBernardi M.A. Brooker G. Mol. Pharmacol. 1993; 44: 1185-1191PubMed Google Scholar). The effect of UTP on [Ca 2+]i is shown in Fig. 7A. In calcium-containing Krebs buffer, UTP elicits a rapid [Ca 2+]i rise followed by a sustained second phase. The second phase is associated with Ca 2+ entry, since it can be eliminated with EGTA and reduced to below basal values. Addition of excess CaCl2 (Fig. 7A, tracesa, b, and c) resulted in a concentration-dependent increase in [Ca 2+]i, which reflects capacitative Ca 2+ entry. The effects of UTP on cAMP accumulation under analogous conditions to those used to manipulate [Ca 2+]i are presented in Fig. 7B. UTP, which causes release of intracellular Ca 2+, along with Ca 2+ 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 CaCl2 with UTP in the presence of EGTA restored the inhibition of cAMP production. Since EGTA eliminates the Ca 2+ influx contribution to the [Ca 2+]i rise elicited by UTP (Fig. 7A), these observations support a role of Ca 2+ entry in inhibition of cAMP accumulation caused by UTP. In nominally Ca 2+-free medium, stimulation with UTP resulted in an initial spike due to Ca 2+ released from intracellular stores, without a Ca 2+ entry phase (Fig. 8A). The initial spike due to UTP was smaller than in Fig. 7A. Capacitative Ca 2+ entry is evident upon the subsequent introduction of CaCl2 (Fig. 2A, tracesa and b). This entry could be eliminated by LaCl3 (tracesc and d). The effects of UTP and UTP-induced Ca 2+ entry on cAMP accumulation in a nominally Ca 2+-free Krebs buffer are explored in Fig. 8B. UTP in the absence of extracellular Ca 2+ did not affect cAMP production. However, the addition of extracellular Ca 2+ (3 mM CaCl2) subsequent to a UTP treatment decreased cAMP accumulation by 25%; this inhibition was fully blocked by LaCl3. The essential contribution of extracellular Ca 2+ to the inhibition of cAMP production elicited by UTP, clearly demonstrates the required role of Ca 2+ entry in regulating adenylyl cyclase in C6-2B cells. 3The 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 Gi protein, without the involvement of Ca 2+-influx(41Lin W.-W. Chuang D.-M. Mol. Pharmacol. 1993; 44: 158-165PubMed Google Scholar, 42Pianet I. Merle M. Labouesse J. Biochem. Biophys. Res. Commun. 1989; 163: 1150-1157Crossref PubMed Scopus (44) Google Scholar). Whereas ATP can bind to all purinoceptors, regardless of the signal pathways that they utilize, UTP acts selectively through the P 2u purinoceptor, which is coupled to phospholipase C via a PTX-insensitive Gq protein in C6-2B cells(25Burnstock G. Ann. N. Y. Acad. Sci. 1990; 603: 1-17Crossref PubMed Scopus (363) Google Scholar, 40Munshi R. DeBernardi M.A. Brooker G. Mol. Pharmacol. 1993; 44: 1185-1191PubMed Google Scholar). The following experiments address the impact of a Ca 2+ 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 2+-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 2+ not only directly on adenylyl cyclase, but also directly on Ca 2+/calmodulin-stimulated PDE(1Meeker R.B. Harden T.K. Mol. Pharmacol. 1982; 22: 310-319PubMed Google Scholar, 3Erneux C. Sande J.V. Miot F. Cochaux P. Decoster C. Dumont J.E. Mol. Cell. Endocrinol. 1985; 43: 123-134Crossref PubMed Scopus (39) Google Scholar). In a normal cell, however, steady state cAMP levels are the balance between synthetic rates (V1) and degradation rates (V2), as described by. ATP⇀V1cAMP⇀V2AMP REACTION 1 the concentration of cAMP relates to these synthetic and degradation rates and the Km of PDE, as follows: [cAMP]-V1×Km(V2-VL)(Eq. 1) where Km 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(43Newsholme E.A. Start C. Regulation in Metabolism. Wiley Press, New York1973Google Scholar). Consider the outcome for steady state [cAMP] following a 33% inhibition of adenylyl cyclase (V1), where [cAMP] is calculated based on the previous formula: 1) at initial state, V1 = 9, V2 = 10, [cAMP] = 9 Km units (arbitrary unit" @default.
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