Voltar
Artigo Acesso aberto Revisado por pares
BIBTEX RIS

Long Lasting Inhibition of Adenylyl Cyclase Selectively Mediated by Inositol 1,4,5-Trisphosphate-evoked Calcium Release

2005; Elsevier BV; Volume: 280; Issue: 10 Linguagem: Inglês

10.1074/jbc.m410045200

ISSN

1083-351X

Autores

Jeanette L. Dyer, Yingjie Liu, Irene Pino de la Huerga, Colin W. Taylor,

Tópico(s)

Carbohydrate Chemistry and Synthesis

Resumo

In A7r5 smooth muscle cells, vasopressin stimulates release of Ca2+ from intracellular stores and Ca2+ entry, and it inhibits adenylyl cyclase (AC) activity. Inhibition of AC is prevented by inhibition of phospholipase C or when the increase in cytosolic [Ca2+] is prevented by the Ca2+ buffer, BAPTA. It is unaffected by pertussis toxin, inhibition of protein kinase C, or L-type Ca2+ channels or by removal of extracellular Ca2+. The independence of extracellular Ca2+ occurs despite inhibition of AC by vasopressin persisting for at least 15 min, whereas the cytosolic [Ca2+] returns to its basal level within 1–2 min in Ca2+-free medium. Although capacitative Ca2+ entry (CCE), activated by emptying stores with thapsigargin, inhibits AC, Ca2+ entry via CCE or L-type Ca2+ channels activated by vasopressin is ineffective. Temporally separating vasopressin-evoked Ca2+ release from the assessment of AC activity revealed that the transient Ca2+ signal resulting from Ca2+ mobilization causes a long lasting inhibition of AC. By contrast, inhibition of AC by thapsigargin-evoked CCE reverses rapidly after removal of extracellular Ca2+. Inhibition of AC by vasopressin is prevented by inhibition of Ca2+-calmodulin-dependent protein kinase II. We conclude that persistent inhibition of AC (probably AC-3) by vasopressin is mediated by inositol trisphosphate-evoked Ca2+ release causing activation of Ca2+-calmodulin-dependent protein kinase II. Our results establish that an important interaction between two ubiquitous signaling pathways is tuned selectively to Ca2+ release via inositol trisphosphate receptors and that the interaction transduces a transient Ca2+ signal into a long lasting inhibition of AC. In A7r5 smooth muscle cells, vasopressin stimulates release of Ca2+ from intracellular stores and Ca2+ entry, and it inhibits adenylyl cyclase (AC) activity. Inhibition of AC is prevented by inhibition of phospholipase C or when the increase in cytosolic [Ca2+] is prevented by the Ca2+ buffer, BAPTA. It is unaffected by pertussis toxin, inhibition of protein kinase C, or L-type Ca2+ channels or by removal of extracellular Ca2+. The independence of extracellular Ca2+ occurs despite inhibition of AC by vasopressin persisting for at least 15 min, whereas the cytosolic [Ca2+] returns to its basal level within 1–2 min in Ca2+-free medium. Although capacitative Ca2+ entry (CCE), activated by emptying stores with thapsigargin, inhibits AC, Ca2+ entry via CCE or L-type Ca2+ channels activated by vasopressin is ineffective. Temporally separating vasopressin-evoked Ca2+ release from the assessment of AC activity revealed that the transient Ca2+ signal resulting from Ca2+ mobilization causes a long lasting inhibition of AC. By contrast, inhibition of AC by thapsigargin-evoked CCE reverses rapidly after removal of extracellular Ca2+. Inhibition of AC by vasopressin is prevented by inhibition of Ca2+-calmodulin-dependent protein kinase II. We conclude that persistent inhibition of AC (probably AC-3) by vasopressin is mediated by inositol trisphosphate-evoked Ca2+ release causing activation of Ca2+-calmodulin-dependent protein kinase II. Our results establish that an important interaction between two ubiquitous signaling pathways is tuned selectively to Ca2+ release via inositol trisphosphate receptors and that the interaction transduces a transient Ca2+ signal into a long lasting inhibition of AC. Each of the nine membrane-bound isoforms of adenylyl cyclase (AC) 1The abbreviations used are: AC, adenylyl cyclase; AVP, Arg8-vasopressin; BHQ, 2,5-di-(tert-butyl)-1,4-benzohydroquinone; [Ca2+]i, intracellular free Ca2+ concentration; CaMKII, Ca2+-calmodulin-dependent protein kinase II; CCE, capacitative Ca2+ entry; CCh, carbamylcholine; HBS, hepes-buffered saline; IBMX, 3-isobutyl-1-methylxanthine; IP3, inositol 1,4,5-trisphosphate; NCCE, noncapacitative Ca2+ entry.1The abbreviations used are: AC, adenylyl cyclase; AVP, Arg8-vasopressin; BHQ, 2,5-di-(tert-butyl)-1,4-benzohydroquinone; [Ca2+]i, intracellular free Ca2+ concentration; CaMKII, Ca2+-calmodulin-dependent protein kinase II; CCE, capacitative Ca2+ entry; CCh, carbamylcholine; HBS, hepes-buffered saline; IBMX, 3-isobutyl-1-methylxanthine; IP3, inositol 1,4,5-trisphosphate; NCCE, noncapacitative Ca2+ entry. catalyzes formation of cAMP from ATP, and each can be stimulated by the G protein, Gs, or (with the exception of AC-9) by forskolin (1Hanoune J. Defer N. Annu. Rev. Pharmacol. Toxicol. 2001; 41: 145-174Crossref PubMed Scopus (545) Google Scholar, 2Hurley J.H. J. Biol. Chem. 1999; 274: 7599-7602Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). ACs are also regulated by many additional signals including protein kinases, other G protein subunits, nitric oxide, membrane potential, and Ca2+ (3Cooper D.M.F. Mons N. Karpen J.W. Nature. 1995; 374: 421-424Crossref PubMed Scopus (553) Google Scholar, 4Sunahara R.K. Dessauer C.W. Gilman A.G. Annu. Rev. Pharmacol. Toxicol. 1996; 36: 461-480Crossref PubMed Scopus (727) Google Scholar, 5Mons N. Decorte L. Jaffrad R. Cooper D.M. Life Sci. 1998; 62: 1647-1652Crossref PubMed Scopus (73) Google Scholar, 6Antoni F.A. Front. Endocrinol. 2000; 21: 103-132Google Scholar). Because the isoforms differ in their regulation by these additional signals and each cell type expresses a different complement of AC isoforms, there is enormous potential for tailoring the cross-talk between AC and other signaling pathways to meet the specific needs of particular cell types. The effects of Ca2+ on AC activity are particularly significant because they allow interplay between two of the most important and ubiquitously expressed intracellular signaling pathways (3Cooper D.M.F. Mons N. Karpen J.W. Nature. 1995; 374: 421-424Crossref PubMed Scopus (553) Google Scholar, 7Cooper D.M.F. Biochem. J. 2003; 375: 517-529Crossref PubMed Scopus (268) Google Scholar). Four isoforms of AC are regulated directly by Ca2+: AC-1 and AC-8 are stimulated by Ca2+-calmodulin, whereas AC-5 and AC-6 are inhibited by Ca2+. In addition, some isoforms are regulated by enzymes that are themselves Ca2+-regulated: AC-9 is inhibited by Ca2+-calcineurin (8Antoni F.A. Barnard R.J.O. Shipston M.J. Smith S.M. Simpson J. Paterson J.M. J. Biol. Chem. 1995; 270: 28055-28061Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar), and AC-1 and AC-3 are inhibited by Ca2+-calmodulin-dependent protein kinases (CaMK) IV and II, respectively (1Hanoune J. Defer N. Annu. Rev. Pharmacol. Toxicol. 2001; 41: 145-174Crossref PubMed Scopus (545) Google Scholar, 9Wei G. Wayman G. Storm D.R. J. Biol. Chem. 1996; 271: 24231-24235Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). For AC-3, regulation by CaMKII is probably more important than any direct effect of Ca2+, which, via calmodulin, potentiates stimulation of AC by forskolin or Gs but only at unphysiologically high [Ca2+] (9Wei G. Wayman G. Storm D.R. J. Biol. Chem. 1996; 271: 24231-24235Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 10Fagan K.A. Mahey R. Cooper D.M.F. J. Biol. Chem. 1996; 271: 12438-12444Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 11Wei J. Zhao A.Z. Chan G.C.K. Baker L.P. Impey S. Beavo J.A. Storm D.R. Neuron. 1998; 21: 495-504Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). The effects of Ca2+ on AC also depend upon the source of the Ca2+ signal. In most cells, depletion of intracellular Ca2+ stores stimulates, by mechanisms that remain contentious, Ca2+ entry across the plasma membrane via the so-called "capacitative Ca2+ entry" (CCE) pathway (12Putney Jr., J.W. Capacitative Calcium Entry. R. G. Landes Company, Austin, TX1997Crossref Google Scholar). The Ca2+ signals resulting from CCE have been shown to be most effective in inhibiting AC-5 and AC-6 (13Cooper D.M.F. Yoshimura M. Zhang Y. Chioni M. Mahey R. Biochem. J. 1994; 297: 437-440Crossref PubMed Scopus (77) Google Scholar, 14Chiono M. Mahey R. Tate G. Cooper D.M.F. J. Biol. Chem. 1995; 270: 1149-1155Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 15Fagan K.A. Mons N. Cooper D.M.F. J. Biol. Chem. 1998; 273: 9297-9305Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 16Wong M.P. Cooper D.M. Young K.W. Young J.M. Br. J. Pharmacol. 2000; 130: 1021-1030Crossref PubMed Scopus (11) Google Scholar) and in stimulating AC-1 and AC-8 (10Fagan K.A. Mahey R. Cooper D.M.F. J. Biol. Chem. 1996; 271: 12438-12444Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 13Cooper D.M.F. Yoshimura M. Zhang Y. Chioni M. Mahey R. Biochem. J. 1994; 297: 437-440Crossref PubMed Scopus (77) Google Scholar, 17Shuttleworth T.J. Thompson J.L. J. Biol. Chem. 1999; 274: 31174-31178Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 18Fagan K.A. Graf R.A. Tolman S. Schaak J. Cooper D.M.F. J. Biol. Chem. 2000; 275: 40187-40194Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). By contrast, release of Ca2+ from intracellular stores, whether mediated by IP3, ionophores, or inhibition of endoplasmic reticulum Ca2+-ATPases, is almost ineffective in regulating these isoforms of AC (10Fagan K.A. Mahey R. Cooper D.M.F. J. Biol. Chem. 1996; 271: 12438-12444Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 18Fagan K.A. Graf R.A. Tolman S. Schaak J. Cooper D.M.F. J. Biol. Chem. 2000; 275: 40187-40194Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). This selective regulation of AC by CCE probably reflects the colocalization of some isoforms of AC and the Ca2+ channels that mediate CCE in caveolae or lipid rafts (19Huang C. Hepler J.R. Chen L.T. Gilman A.G. Anderson R.G.W. Mumby S.M. Mol. Biol. Cell. 1997; 8: 2365-2378Crossref PubMed Scopus (188) Google Scholar, 20Fagan K.A. Smith K.E. Cooper D.M.F. J. Biol. Chem. 2000; 275: 26530-26537Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 21Smith K.E. Gu C. Fagan K.A. Hu B. Cooper D.M.F. J. Biol. Chem. 2002; 277: 6025-6031Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 22Ishikawa Y. Grabnt B.S. Okumura S. Schwencke C. Yamamoto M. Mol. Cell. Endocrinol. 2000; 162: 107-112Crossref PubMed Scopus (12) Google Scholar). CCE is not, however, universally associated with regulation of AC. Ca2+ entering via L-type Ca2+ channels effectively inhibits AC in cardiac myocytes (23Yu H.J. Ma H. Green R.D. Mol. Pharmacol. 1993; 44: 689-693PubMed Google Scholar) and stimulates it in cerebellar granule cells (24Cooper D.M.F. Schell M.J. Thorn P. Irvine R.F. J. Biol. Chem. 1998; 273: 27703-27707Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar), although in GH4C1 cells such Ca2+ signals are less effective than those evoked by CCE (18Fagan K.A. Graf R.A. Tolman S. Schaak J. Cooper D.M.F. J. Biol. Chem. 2000; 275: 40187-40194Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Interactions between Ca2+ and cyclic nucleotides are important in regulating the activities of many cell types (25Bruce J.I.E. Straub S.V. Yule D.I. Cell Calcium. 2003; 34: 431-444Crossref PubMed Scopus (101) Google Scholar, 26Werry T.D. Wilkinson G.F. Willars G.B. Biochem. J. 2003; 374: 281-296Crossref PubMed Scopus (141) Google Scholar). In smooth muscle, such interactions are major determinants of both contractile activity and proliferation (27Wong S.T. Baker L.P. Trinh K. Hetman M. Suzuki L.A. Storm D.R. Bornfeldt K.E. J. Biol. Chem. 2001; 276: 34206-34212Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Ca2+ promotes contraction by stimulating phosphorylation of myosin light chain, whereas cGMP and cAMP, via their respective kinases, usually cause relaxation by either attenuating Ca2+ signals or by more direct interactions with the contractile machinery (28Komalavilas P. Lincoln T.M. J. Biol. Chem. 1994; 269: 8701-8707Abstract Full Text PDF PubMed Google Scholar, 29Woodrum D.A. Brophy C.M. Mol. Cell. Endocrinol. 2001; 177: 135-143Crossref PubMed Scopus (32) Google Scholar, 30Ammendola A. Geiselhöringer A. Hofmann F. Schlossmann J. J. Biol. Chem. 2001; 276: 24153-24159Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 31Abdel-Latif A.A. Exp. Biol. Med. 2001; 226: 153-163Crossref PubMed Scopus (92) Google Scholar). The importance of interactions between cyclic nucleotides and Ca2+ in controlling vascular smooth muscle is clear from the many clinically useful drugs that target the Ca2+ channels, guanylyl cyclases, or cyclic nucleotide phosphodiesterases of the cardiovascular system. A7r5 cells, a cell line originally derived from rat aorta (32Kimes B.W. Brandt B.L. Exp. Cell Res. 1976; 98: 349-366Crossref PubMed Scopus (319) Google Scholar), express at least three isoforms of AC (isoforms 3, 5, and 6) (33Webb J.G. Yates P.W. Yang Q. Mukhin Y.V. Lanier S.M. Am. J. Physiol. 2001; 281: H1545-H1552PubMed Google Scholar), and all three are directly (types 5 and 6) or indirectly (type 3) inhibited by Ca2+. A similar pattern of expression is found in vascular smooth muscle, where types 2–7 AC have been identified in different species, with AC-3 perhaps being particularly important (27Wong S.T. Baker L.P. Trinh K. Hetman M. Suzuki L.A. Storm D.R. Bornfeldt K.E. J. Biol. Chem. 2001; 276: 34206-34212Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 34Xia Z. Choi E.-J. Wang F. Storm D.R. Neurosci. Lett. 1992; 144: 169-173Crossref PubMed Scopus (74) Google Scholar, 35Guldmeester A. Stenmark K.R. Brough G.H. Stevens T. Am. J. Physiol. 1999; 276: L1010-L1017PubMed Google Scholar, 36Zhang J. Sato M. Duzic E. Kubalak S.W. Lanier S.M. Webb J.G. Am. J. Physiol. 1997; 273: H971-HH980PubMed Google Scholar). In A7r5 cells, Arg8-vasopressin (AVP), via V1A receptors (37Thibonnier M. Bayer A.L. Simonson M.S. Kester M. Endocrinology. 1991; 129: 2845-2856Crossref PubMed Scopus (69) Google Scholar), stimulates phospholipase C and thereby both release of Ca2+ from intracellular stores and Ca2+ entry across the plasma membrane. The latter involves a complex interplay between different Ca2+ channels: AVP promotes opening of L-type voltage-gated Ca2+ channels (38Byron K.L. Lucchesi P.A. J. Biol. Chem. 2002; 277: 7298-7307Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar), and it can reciprocally regulate CCE and a noncapacitative Ca2+ entry (NCCE) pathway (39Moneer Z. Taylor C.W. Biochem. J. 2002; 362: 13-21Crossref PubMed Scopus (74) Google Scholar, 40Moneer Z. Dyer J.L. Taylor C.W. Biochem. J. 2003; 370: 439-448Crossref PubMed Scopus (54) Google Scholar). Given the importance of Ca2+-cAMP interactions in vascular smooth muscle, the evidence that not all Ca2+ signals are equally effective in regulating AC (7Cooper D.M.F. Biochem. J. 2003; 375: 517-529Crossref PubMed Scopus (268) Google Scholar), and evidence that AVP inhibits AC in A7r5 cells (33Webb J.G. Yates P.W. Yang Q. Mukhin Y.V. Lanier S.M. Am. J. Physiol. 2001; 281: H1545-H1552PubMed Google Scholar), we decided to examine whether inhibition of AC in these cells was regulated selectively by distinct Ca2+ entry pathways. We unexpectedly found that inhibition of AC by AVP is mediated by release of Ca2+ from intracellular stores and that the inhibition long outlasts the Ca2+ signal that initiates it. We suggest that the Ca2+ released through IP3 receptors selectively activates CaMKII leading to a relatively long lasting inhibition of AC activity. Our results demonstrate that an important interaction between two ubiquitous signaling pathways can be tuned selectively to release of Ca2+ from intracellular stores and that the interaction transduces a transient Ca2+ signal into a long lasting inhibition of AC. Materials—Alumina, ascorbic acid, AVP, carbamylcholine (CCh), GF 109203X (bisindoylmaleimide), 3-isobutyl-1-methylxanthine (IBMX), isoprenaline, pertussis toxin, and most routine reagents were from Sigma. Ascorbic acid (0.8 mm) was always included with isoprenaline. Forskolin and nimodipine were from Tocris (Avonmouth, UK). Ro-31-8220, U73122 (1-[6((17β-3-methoxyestra-1,3,5 (10)-trien-17-yl)amino)-hexyl]-1H-pyrrole-2,5-dione), U73122 (1-[6((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-2,5-pyrroledione), and cAMP were from Calbiochem (Nottingham, UK). Dowex AG 50W-X8 (100–200 mesh) was from Bio-Rad Laboratories. Fluo4 AM and BAPTA-AM were from Molecular Probes (Leiden, The Netherlands). [2,8-3H]Adenine (30 Ci/mmol) was from PerkinElmer Life Sciences. Cell culture materials were from Invitrogen or Sigma (serum). Cell Culture—A7r5 cells, originally from the American Type Culture Collection (Rockville, MD), were grown in 12-well plates in Dulbecco's modified Eagle's medium supplemented with fetal calf serum (1%) at 37 °C in a humidified atmosphere of 95% air and 5% CO2. Our initial intention had been to explore the relative effectiveness of Ca2+ entry via CCE and NCCE pathways in causing inhibition of AC, but we began our experiments (and then continued) using a variant of the cell line in which AVP neither inhibits CCE nor activates NCCE (39Moneer Z. Taylor C.W. Biochem. J. 2002; 362: 13-21Crossref PubMed Scopus (74) Google Scholar). In the cells used for this study, therefore, all AVP-evoked Ca2+ entry is mediated by CCE. Measurements of cAMP—For measurements of cAMP formation, cells were labeled with [3H]adenine. [3H]cAMP was then separated from [3H]ATP, [3H]ADP, and [3H]AMP using Dowex columns (to which cAMP adheres until eluted with water) and then alumina columns to which any residual [3H]ATP, [3H]ADP, and [3H]AMP adhere (41Johnson R.A. Salomon Y. Methods Enzymol. 1991; 195: 3-19Crossref PubMed Scopus (121) Google Scholar, 42Farndale R.W. Allan L.M. Martin B.R. Milligan G. Cell Signalling: A Practical Approach. 1st Ed. IRL Press, Oxford1992: 75-103Google Scholar). When the cells were confluent, they were incubated with 1 μCi/well [3H]adenine in the same medium for at least 2 h. Immediately before an experiment, the labeling medium was removed and replaced with Hepes-buffered saline (HBS: 135 mm NaCl, 5.9 mm KCl, 1.2 mm MgCl2, 1.5 mm CaCl2, 11.5 mm glucose, 11.6 mm Hepes, pH 7.3, at 20 °C). After two washes, the cells were incubated in HBS (or Ca2+-free HBS in which CaCl2 was replaced by 1 mm EGTA) supplemented with 1 mm IBMX and appropriate inhibitors. Further additions (details in figure legends) were made after 5 min. To allow direct comparison with measurements of [Ca2+]i, all assays were performed at 20 °C. After an appropriate interval, the incubations were terminated by addition of ice-cold trichloroacetic acid (5% final concentration). [3H]ATP, [3H]ADP, and [3H]AMP were separated from [3H]cAMP by sequential elution from anion exchange (Dowex 50 equilibrated with 1 m HCl) and alumina columns (equilibrated with 0.1 m imidazole, pH 7.5) (43Salomon Y. Londos C. Rodbell M. Anal. Biochem. 1974; 58: 541-548Crossref PubMed Scopus (3367) Google Scholar, 44Ho M.K.C. Wong Y.H. Milligan G. Signal Transduction: A Practical Approach. 2nd Ed. Oxford University Press, New York1992: 223-253Google Scholar). After 30 min on ice, the supernatants were loaded onto Dowex columns (1 ml): [3H]ATP (with [3H]ADP and [3H]AMP) was eluted in 3 ml of water, and after elution with a further 10 ml of water, the eluate was loaded onto a neutral alumina column from which [3H]cAMP was eluted with 100 mm imidazole-HCl, pH 7.5. Levels of [3H]cAMP are expressed as percentages of [3H]ATP. Measurements of [Ca2+]i—A7r5 cells grown (as above) in 96-well plates were loaded with fluo4 by incubation with fluo4 AM (2 μm in fresh dimethyl sulfoxide, final concentration 0.2%) and 0.2 mg/ml Pluronic F-127 in HBS. After 1 h at 20 °C, the cells were washed three times with HBS, and then after 1 h in HBS supplemented with 0.1% bovine serum albumin (to allow complete hydrolysis of the indicator) the cells were again washed and used for experiments. For fluorescence measurements, the 96-well plate was mounted in a FlexStation (Molecular Devices), which allows up to three automated additions to each well. A xenon lamp provided the excitation light, and a scanning dual monochromator allowed control of excitation (485 nm) and emission (525 nm) wavelengths. Fluorescence was captured at 1-s intervals using SoftMaxPro software (Molecular Devices) and then averaged (over periods of 5 s). At the end of each experiment, fluorescence of the Ca2+-saturated indicator (Fmax) was determined for each well by addition of 10 μm ionomycin and CaCl2 (final free concentration 10 mm). The fluorescence recorded in the absence of Ca2+ (equivalent to autofluorescence because Ca2+-free fluo4 is not fluorescent) was determined on each plate by prolonged incubation (>10 min) of cells in at least 6 wells with 10 μm ionomycin in Ca2+-free HBS supplemented with 10 mm BAPTA. Autofluorescence was subtracted from all values before analysis. Fluorescence was calibrated to [Ca2+]i using Equation 1[Ca2+] i=Kd⋅F/(Fmax−F)(Eq. 1) where the Kd of fluo4 for Ca2+ is 345 nm; F and Fmax are the autofluorescence-corrected fluorescence recorded from the sample and after saturating the indicator with Ca2+. All results are reported as means ± S.E. Inhibition of AC by AVP Requires Activation of Phospholipase C—In the presence of 1 mm IBMX to inhibit cyclic nucleotide phosphodiesterases, AVP, at concentrations (≤1 μm, 5 min) more than sufficient to stimulate release of Ca2+ from intracellular stores maximally, had no effect on cAMP levels (Fig. 1A). Under identical conditions, 5 min of incubation with forskolin caused a concentration-dependent (half-maximal effective concentration, EC50 ∼12 μm) stimulation of AC activity, and this was inhibited by AVP (Fig. 1A). A maximal concentration of AVP (1 μm) caused 33 ± 2% (n = 3) inhibition of the response evoked by 100 μm forskolin, and half-maximal inhibition (IC50) occurred with 0.89 ± 0.22 nm AVP (Fig. 1B). Similar results were obtained when 10 μm isoprenaline, an agonist of β-adrenoceptors, was used to stimulate AC (Fig. 1A): AVP again caused a concentration-dependent inhibition (IC50 = 0.90 ± 0.3 nm). We have used forskolin and isoprenaline interchangeably to stimulate AC activity with indistinguishable results. The experiments reported here were performed over a substantial period during which the amplitude of the Ca2+ signals evoked by AVP varied by as much as 2-fold. All comparisons of the effects of AVP on AC and cytosolic [Ca2+] ([Ca2+]i) were therefore performed in parallel experiments. AVP stimulated both release of Ca2+ from intracellular stores and Ca2+ entry (Fig. 1C). In the absence of extracellular Ca2+, both the duration of the response and the maximal amplitude of the initial increase in [Ca2+]i (1.69 ± 0.05 μmversus 1.25 ± 0.09 μm) were reduced (Fig. 1C). The EC50 for AVP-evoked Ca2+ signals was similar in the presence (EC50 = 14 ± 3nm) and absence (36 ± 25 nm) of extracellular Ca2+ (Fig. 1D), but significantly higher than the IC50 for inhibition of AC by AVP (Figs. 1B and 4A). Neither component of the Ca2+ response was affected by IBMX and forskolin (Fig. 1E) or isoprenaline (not shown). This is important because it allows the effects of Ca2+ on AC activity to be addressed without the complexity of cAMP affecting the Ca2+ signals. Treatment of cells with 100 ng/ml pertussis toxin for 24 h to uncouple the G protein, Gi, from cell surface receptors, had no effect on the ability of AVP to inhibit AC activity, although it substantially reduced the effectiveness of CCh, an agonist of muscarinic acetylcholine receptors (Fig. 2A). We conclude, consistent with a previous report (33Webb J.G. Yates P.W. Yang Q. Mukhin Y.V. Lanier S.M. Am. J. Physiol. 2001; 281: H1545-H1552PubMed Google Scholar), that inhibition of AC by AVP is not mediated by Gi or any other pertussis toxin-sensitive G protein. Inhibition of AC by AVP was completely inhibited by 10 μm U73122, a selective inhibitor of phospholipase C (45Bleasdale J.E. Thakur N.R. Gremban R.S. Bundy G.L. Fitzpatrick F.A. Smith R.J. Bunting S. J. Pharmacol. Exp. Ther. 1990; 255: 756-768PubMed Google Scholar), although not by its inactive analog, U73343 (Fig. 2B). Nor did inhibitors of protein kinase C, GF 109203X (1 μm, Fig. 2B), or Ro 31-8220 (10 μm, not shown) prevent AVP from inhibiting AC. Indeed, and consistent with earlier work suggesting that protein kinase C tonically represses signaling from the AVP receptor (46Broad L.M. Cannon T.R. Taylor C.W. J. Physiol. 1999; 517: 121-134Crossref PubMed Scopus (194) Google Scholar, 47Caramelo C. Tsai P. Okada K. Briner V.A. Schrier R.W. Am. J. Physiol. 1991; 260: F46-F52PubMed Google Scholar), inhibition of protein kinase C exaggerated the ability of AVP to inhibit AC. After preincubation with 10 μm Ro 81-3220 for 5 min, a maximal concentration of AVP inhibited forskolin-evoked AC activity by 57 ± 1% (n = 3, versus 35 ± 2% without inhibitor), although the sensitivity to AVP was unaltered (IC50 = 0.6 ± 0.2 nm). We conclude that inhibition of AC by AVP requires activation of phospholipase C but not protein kinase C, despite the presence in A7r5 cells of AC-6 (33Webb J.G. Yates P.W. Yang Q. Mukhin Y.V. Lanier S.M. Am. J. Physiol. 2001; 281: H1545-H1552PubMed Google Scholar), which is known to be inhibited by protein kinase C (48Lai H.-L. Yang T.-H. Messing R.O. Ching Y.-H. Lin S.-C. Chern Y. J. Biol. Chem. 1997; 272: 4970-4977Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). In subsequent experiments, we therefore examined the role of Ca2+ in mediating the effects of AVP. Sustained Inhibition of AC by AVP Requires an Increase in Cytosolic [Ca2+]—Fig. 3 shows the time course of cAMP accumulation in the presence of forskolin and IBMX alone or with 100 nm AVP. As expected, cAMP continued to accumulate for at least 15 min in the presence of forskolin. After 2–3 min, the inhibitory effect of AVP had fully developed (∼30% inhibition). Thereafter, and despite further stimulation of AC by forskolin, AVP maintained that 30% inhibition (Fig. 3). The mechanism that links AVP to inhibition of AC therefore appears to take 2–3 min to become maximally active but then remains effective for at least 15 min after the addition of AVP. Inhibition of forskolin-stimulated AC by AVP, measured during a 5-min incubation, was unaffected by removal of extracellular Ca2+. Neither the maximal effect of AVP (52 ± 10% versus 48 ± 8% inhibition in the presence and absence of Ca2+, respectively) nor the sensitivity to AVP (IC50 = 2.9 ± 1.9 nmversus 1.2 ± 0.8 nm) was affected by removal of extracellular Ca2+ (Fig. 4A). In cells loaded with 50 μm BAPTA-AM for 1 h in Ca2+-free HBS to buffer intracellular Ca2+, the Ca2+ signal evoked by AVP was abolished (Fig. 4C). Similar results were obtained when the intracellular stores were emptied by preincubation with 1 μm thapsigargin or 10 μm BHQ for 15 min in Ca2+-free HBS to inhibit the endoplasmic reticulum Ca2+ pumps. In each of these situations, AVP failed to inhibit AC activity, although inhibition of AC by CCh, mediated by Gi, was unaffected (Fig. 4B). Although the Ca2+ release evoked by AVP inhibited AC, thapsigargin and BHQ were ineffective despite evoking peak Ca2+ signals larger than, and sustained Ca2+ signals similar to, those evoked by a maximal concentration of AVP (Fig. 4, D and E). These results establish that an increase in [Ca2+]i is required for AVP to inhibit AC and that AVP-evoked, but not thapsigargin-evoked, release of Ca2+ from intracellular stores can mediate the inhibition. In subsequent experiments, we sought to establish whether release of Ca2+ from intracellular stores was uniquely capable of inhibiting AC or whether Ca2+ signals resulting from Ca2+ entry were also effective. Inhibition of AC by Ca2+Entry—Depolarization (by the addition of HBS containing 53 mm KCl in place of NaCl) stimulated an increase in [Ca2+]i which was prevented by 100 nm nimodipine or 10 μm verapamil and potentiated by 100 nm BAYK8644, confirming the involvement of L-type Ca2+ channels (39Moneer Z. Taylor C.W. Biochem. J. 2002; 362: 13-21Crossref PubMed Scopus (74) Google Scholar, 49Byron K.L. Taylor C.W. J. Biol. Chem. 1993; 268: 6945-6952Abstract Full Text PDF PubMed Google Scholar) (Fig. 5A). Although the peak [Ca2+]i evoked by L-type Ca2+ channels was significantly smaller than that evoked by a maximal concentration of AVP (0.31 ± 0.03 μmversus 1.7 ± 0.05 μm), it was comparable with the response evoked by ∼3 nm AVP, which did inhibit AC (Fig. 1, B and E). Nevertheless, depolarization had no significant effect on AC activity, whether stimulated by forskolin or isoprenaline (Fig. 5B). More importantly, neither nimodipine nor depolarization affected the inhibition of AC by AVP (Fig. 5B), despite modestly reducing (nimodipine) or increasing (depolarization) the amplitude of the initial AVP-evoked Ca2+ signals (Fig. 5C). These results establish that although AVP does stimulate Ca2+ entry via L-type Ca2+ channels, as reported by others (50Byron K.L. Circ. Res. 1996; 78: 813-820Crossref PubMed Scopus (41) Google Scholar), this component of the Ca2+ signal does not effectively inhibit AC activity. Ionomycin (1 μm), which we previously used to empty intracellular Ca2+ stores rapidly (39Moneer Z. Taylor C.W. Biochem. J. 2002; 362: 13-21Crossref PubMed Scopus (74) Google Scholar), directly inhibited AC independent of its effects on intracellular Ca2+ (not shown). We therefore used prolonged pretreatment (15 min) with either 1 μm thapsigargin or 10 μm BHQ in Ca2+-free HBS to empty intracellular Ca2+ stores and so activate CCE. Restoration of Ca2+ then evoked an increase in [Ca2+]i the amplitude of which increased (to a maximum of 871 ± 89 nm, with 10 mm extracellular Ca2+) as a function of the extracellular Ca2+ concentration (Fig. 6, A and B). Restoration of 1.5 mm Ca2+ to cells treated with thapsigargin caused [Ca2+]i to increase to 588 ±

Referência(s)