Angiotensin II Inhibits bTREK-1 K+ Channels in Adrenocortical Cells by Separate Ca2+- and ATP Hydrolysis-dependent Mechanisms
2005; Elsevier BV; Volume: 280; Issue: 35 Linguagem: Inglês
10.1074/jbc.m504283200
ISSN1083-351X
AutoresJohn J. Enyeart, Sanjay Danthi, Haiyan Liu, Judith A. Enyeart,
Tópico(s)Neuroscience and Neuropharmacology Research
ResumoBovine adrenocortical cells express bTREK-1 K+ channels that set the resting membrane potential (Vm) and couple angiotensin II (AngII) and adrenocorticotropic hormone (ACTH) receptors to membrane depolarization and corticosteroid secretion. In this study, it was discovered that AngII inhibits bTREK-1 by separate Ca2+- and ATP hydrolysis-dependent signaling pathways. When whole cell patch clamp recordings were made with pipette solutions that support activation of both Ca2+- and ATP-dependent pathways, AngII was significantly more potent and effective at inhibiting bTREK-1 and depolarizing adrenal zona fasciculata cells, than when either pathway is activated separately. External ATP also inhibited bTREK-1 through these two pathways, but ACTH displayed no Ca2+-dependent inhibition. AngII-mediated inhibition of bTREK-1 through the novel Ca2+-dependent pathway was blocked by the AT1 receptor antagonist losartan, or by including guanosine-5′-O-(2-thiodiphosphate) in the pipette solution. The Ca2+-dependent inhibition of bTREK-1 by AngII was blunted in the absence of external Ca2+ or by including the phospholipase C antagonist U73122, the inositol 1,4,5-trisphosphate receptor antagonist 2-amino-ethoxydiphenyl borate, or a calmodulin inhibitory peptide in the pipette solution. The activity of unitary bTREK-1 channels in inside-out patches from adrenal zona fasciculata cells was inhibited by application of Ca2+ (5 or 10 μm) to the cytoplasmic membrane surface. The Ca2+ ionophore ionomycin also inhibited bTREK-1 currents through channels expressed in CHO-K1 cells. These results demonstrate that AngII and selected paracrine factors that act through phospholipase C inhibit bTREK-1 in adrenocortical cells through simultaneous activation of separate Ca2+- and ATP hydrolysis-dependent signaling pathways, providing for efficient membrane depolarization. The novel Ca2+-dependent pathway is distinctive in its lack of ATP dependence, and is clearly different from the calmodulin kinase-dependent mechanism by which AngII modulates T-type Ca2+ channels in these cells. Bovine adrenocortical cells express bTREK-1 K+ channels that set the resting membrane potential (Vm) and couple angiotensin II (AngII) and adrenocorticotropic hormone (ACTH) receptors to membrane depolarization and corticosteroid secretion. In this study, it was discovered that AngII inhibits bTREK-1 by separate Ca2+- and ATP hydrolysis-dependent signaling pathways. When whole cell patch clamp recordings were made with pipette solutions that support activation of both Ca2+- and ATP-dependent pathways, AngII was significantly more potent and effective at inhibiting bTREK-1 and depolarizing adrenal zona fasciculata cells, than when either pathway is activated separately. External ATP also inhibited bTREK-1 through these two pathways, but ACTH displayed no Ca2+-dependent inhibition. AngII-mediated inhibition of bTREK-1 through the novel Ca2+-dependent pathway was blocked by the AT1 receptor antagonist losartan, or by including guanosine-5′-O-(2-thiodiphosphate) in the pipette solution. The Ca2+-dependent inhibition of bTREK-1 by AngII was blunted in the absence of external Ca2+ or by including the phospholipase C antagonist U73122, the inositol 1,4,5-trisphosphate receptor antagonist 2-amino-ethoxydiphenyl borate, or a calmodulin inhibitory peptide in the pipette solution. The activity of unitary bTREK-1 channels in inside-out patches from adrenal zona fasciculata cells was inhibited by application of Ca2+ (5 or 10 μm) to the cytoplasmic membrane surface. The Ca2+ ionophore ionomycin also inhibited bTREK-1 currents through channels expressed in CHO-K1 cells. These results demonstrate that AngII and selected paracrine factors that act through phospholipase C inhibit bTREK-1 in adrenocortical cells through simultaneous activation of separate Ca2+- and ATP hydrolysis-dependent signaling pathways, providing for efficient membrane depolarization. The novel Ca2+-dependent pathway is distinctive in its lack of ATP dependence, and is clearly different from the calmodulin kinase-dependent mechanism by which AngII modulates T-type Ca2+ channels in these cells. Bovine adrenocortical cells, including cortisol-secreting AZF 1The abbreviations used are: AZF, bovine adrenal fasciculata; AZG, bovine adrenal glomerulosa; Vm, resting membrane potential; AngII, angiotensin II; ACTH, adrenocorticotropic hormone; PLC, phospholipase C; 2-APB, 2-aminoethoxydiphenyl borate; CIP, calmodulin inhibitory peptide; CHO, Chinese hamster ovary; BAPTA, 1,2-bis-(2-aminophenoxy)ethane-N,N,N′,N″-tetraacetic acid; IP3, inositol 1,4,5-trisphosphate; AMP-PNP, 5′-adenylyl-β,γ-imidodiphosphate; GDPβS, guanosine-5′-O-(2-thiodiphosphate); 8-pcpt-cAMP, 8-(4-chlorophenylthio) adenosine 3′,5′-cyclic monophosphate. 1The abbreviations used are: AZF, bovine adrenal fasciculata; AZG, bovine adrenal glomerulosa; Vm, resting membrane potential; AngII, angiotensin II; ACTH, adrenocorticotropic hormone; PLC, phospholipase C; 2-APB, 2-aminoethoxydiphenyl borate; CIP, calmodulin inhibitory peptide; CHO, Chinese hamster ovary; BAPTA, 1,2-bis-(2-aminophenoxy)ethane-N,N,N′,N″-tetraacetic acid; IP3, inositol 1,4,5-trisphosphate; AMP-PNP, 5′-adenylyl-β,γ-imidodiphosphate; GDPβS, guanosine-5′-O-(2-thiodiphosphate); 8-pcpt-cAMP, 8-(4-chlorophenylthio) adenosine 3′,5′-cyclic monophosphate. cells and aldosterone-secreting AZG cells express bTREK-1 leak-type K+ channels that function pivotally in the physiology of corticosteroid secretion (1Mlinar B. Biagi B.A. Enyeart J.J. J. Biol. Chem. 1993; 268: 8640-8644Abstract Full Text PDF PubMed Google Scholar, 2Enyeart J.J. Xu L. Danthi S. Enyeart J.A. J. Biol. Chem. 2002; 277: 49186-49199Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). bTREK-1 belongs to the mechanogated, thermo- and fatty acid-sensitive subgroup of two-pore/four-transmembrane fragment family of K+ channels (3Goldstein S.A. Bockenhauer D. O'Kelly I. Zilberberg N. Nat. Rev. Neurosci. 2001; 2: 175-184Crossref PubMed Scopus (562) Google Scholar, 4Maingret F. Lauritzen I. Patel A.J. Heurteaux C. Reyes R. Lesage F. Lazdunski M. Honore E. EMBO J. 2000; 19: 2483-2491Crossref PubMed Scopus (412) Google Scholar, 5Patel A.J. Honore E. Trends Neurosci. 2001; 24: 339-346Abstract Full Text Full Text PDF PubMed Scopus (374) Google Scholar, 6Maingret F. Patel A.J. Lesage F. Lazdunski M. Honore E. J. Biol. Chem. 2000; 275: 10128-10133Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar, 7Bang H. Kim Y. Kim D. J. Biol. Chem. 2000; 275: 17412-17419Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar, 8Kang D. Choe C. Kim D. J. Physiol. (Lond.). 2005; 564: 103-116Crossref Scopus (196) Google Scholar, 9Kim D. Trends Pharmacol. Sci. 2003; 24: 648-654Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). In the adrenal cortex, bTREK-1 channels couple hormonal signals originating at the cell membrane to depolarization-dependent Ca2+ entry (1Mlinar B. Biagi B.A. Enyeart J.J. J. Biol. Chem. 1993; 268: 8640-8644Abstract Full Text PDF PubMed Google Scholar, 2Enyeart J.J. Xu L. Danthi S. Enyeart J.A. J. Biol. Chem. 2002; 277: 49186-49199Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 10Enyeart J.J. Mlinar B. Enyeart J.A. Mol. Endocrinol. 1993; 7: 1031-1040Crossref PubMed Scopus (112) Google Scholar, 11Enyeart J.A. Danthi S.J. Enyeart J.J. Am. J. Physiol. 2004; 287: E1154-E1165Crossref PubMed Scopus (47) Google Scholar). bTREK-1 channels are inhibited by AngII and ACTH at concentrations identical to those that trigger membrane depolarization and corticosteroid secretion (1Mlinar B. Biagi B.A. Enyeart J.J. J. Biol. Chem. 1993; 268: 8640-8644Abstract Full Text PDF PubMed Google Scholar, 12Mlinar B. Biagi B.A. Enyeart J.J. J. Biol. Chem. 1995; 270: 20942-20951Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Other paracrine factors, including ATP, which stimulates cortisol secretion through a G protein-coupled P2Y3 receptor, also inhibit bTREK-1 and depolarize AZF cells with similar potency (13Hoey E.D. Nicol M. Williams B.C. Walker S.W. Endocrinology. 1994; 134: 1553-1560Crossref PubMed Scopus (18) Google Scholar, 14Xu L. Enyeart J.J. Mol. Pharmacol. 1999; 55: 364-376Crossref PubMed Scopus (21) Google Scholar). The signaling pathways that link the peptide hormones and paracrine factors to bTREK-1 inhibition are only partially understood. In particular, adrenocortical cells express two pharmacologically distinct types of AngII receptors (15Dudley D.T. Panek R.L. Major T.C. Lu G.H. Bruns R.F. Klinkefus B.A. Hodges J.C. Weishaar R.E. Mol. Pharmacol. 1990; 38: 370-377PubMed Google Scholar, 16Smith R.D. Chiu A.T. Wong P.C. Herblin W.F. Timmermans P.B. Annu. Rev. Pharmacol. Toxicol. 1992; 32: 135-165Crossref PubMed Scopus (249) Google Scholar, 17De Gasparo M. Catt K.J. Inagami T. Wright J.W. Unger Th. Pharmacol. Rev. 2000; 52: 415-472PubMed Google Scholar). Losartan-sensitive AT1 receptors are coupled to multiple signaling pathways (17De Gasparo M. Catt K.J. Inagami T. Wright J.W. Unger Th. Pharmacol. Rev. 2000; 52: 415-472PubMed Google Scholar, 18Smith R.D. Baukal A.J. Dent P. Catt K.J. Endocrinology. 1999; 140: 1385-1391Crossref PubMed Scopus (40) Google Scholar, 19Nadler J.L. Natarajan R. Stern N. J. Clin. Investig. 1987; 80: 1763-1769Crossref PubMed Scopus (119) Google Scholar). Most physiological responses, including AngII-stimulated corticosteroid secretion, are mediated through AT1 receptor-dependent activation of PLC, which catalyzes the synthesis of inositol trisphosphates (IP3) and diacylglycerol from phosphatidylinositol 4,5-bisphosphate (17De Gasparo M. Catt K.J. Inagami T. Wright J.W. Unger Th. Pharmacol. Rev. 2000; 52: 415-472PubMed Google Scholar, 20Spat A. Hunyady L. Physiol. Rev. 2004; 84: 489-539Crossref PubMed Scopus (374) Google Scholar). Adrenocortical cells also express losartan-insensitive AT2 receptors, which comprise about 20% of the AngII receptors in these cells (15Dudley D.T. Panek R.L. Major T.C. Lu G.H. Bruns R.F. Klinkefus B.A. Hodges J.C. Weishaar R.E. Mol. Pharmacol. 1990; 38: 370-377PubMed Google Scholar, 17De Gasparo M. Catt K.J. Inagami T. Wright J.W. Unger Th. Pharmacol. Rev. 2000; 52: 415-472PubMed Google Scholar). The signaling pathways and function of these receptors in the physiology of corticosteroid-secreting cells is unknown. In whole cell patch clamp recordings from bovine AZF and AZG cells, AngII maximally inhibited bTREK-1 by 72–77% with an IC50 of ∼145 pm, provided that ATP was present in the recording pipette at millimolar concentrations (1Mlinar B. Biagi B.A. Enyeart J.J. J. Biol. Chem. 1993; 268: 8640-8644Abstract Full Text PDF PubMed Google Scholar, 11Enyeart J.A. Danthi S.J. Enyeart J.J. Am. J. Physiol. 2004; 287: E1154-E1165Crossref PubMed Scopus (47) Google Scholar, 12Mlinar B. Biagi B.A. Enyeart J.J. J. Biol. Chem. 1995; 270: 20942-20951Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). AngII-mediated inhibition of bTREK-1 was eliminated when ATP in the pipette solution was replaced by the non-hydrolyzable ATP analog AMP-PNP, or UTP (12Mlinar B. Biagi B.A. Enyeart J.J. J. Biol. Chem. 1995; 270: 20942-20951Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 21Xu L. Enyeart J.J. Am. J. Physiol. 2001; 280: C199-C215Crossref Google Scholar). The kinase or ATPase that mediates AngII inhibition through this ATP hydrolysis-dependent pathway has not been identified. In these whole cell recording studies, [Ca2+]i was buffered to 22 nm with 11 mm BAPTA. In a separate study, we found that bTREK-1 in AZF cells was inhibited by raising the [Ca2+]i in the pipette solution from 22 nm to 2 μm (22Gomora J.C. Enyeart J.J. Am. J. Physiol. 1998; 275: C1526-C1537Crossref PubMed Google Scholar). bTREK-1 was also inhibited by superfusing cells with the Ca2+ ionophore ionomycin, provided that [Ca2+]i was buffered by 2 mm, rather than 11 mm BAPTA. At the single channel level, in excised inside-out patch recordings, the application of saline containing 35 μm Ca2+ to the cytoplasmic face of the membrane markedly inhibited bTREK-1 channel activity. In these experiments, 5 mm ATP was present at the cytoplasmic face of the membrane. Taken together, the results of these studies could suggest that AngII inhibits bTREK-1 through a single Ca2+- and ATP hydrolysis-dependent signaling pathway that requires the activation of a Ca2+-dependent kinase. In bovine AZG cells, AngII enhances the activity of T-type Ca2+ channels through the activation of Ca2+/calmodulin-dependent kinase II (23Lu H.K. Fern R.J. Nee J.J. Barrett P.Q. Am. J. Physiol. 1994; 267: F183-F189PubMed Google Scholar, 24Chen X.L. Bayliss D.A. Fern R.J. Barrett P.Q. Am. J. Physiol. 1999; 276: F674-F683Crossref PubMed Google Scholar, 25Wolfe J.T. Wang H. Perez-Reyes E. Barrett P.Q. J. Physiol. (Lond.). 2002; 538: 343-355Crossref Scopus (68) Google Scholar). Alternatively, the effective inhibition of bTREK-1 by AngII in whole cell recordings with [Ca2+]i strongly buffered to 22 nm using 11 mm BAPTA suggests that AngII may inhibit bTREK-1 by multiple signaling pathways, only one of which is Ca2+-dependent. In whole cell and single channel patch clamp recordings from bovine adrenocortical cells, we discovered that AngII does inhibit bTREK-1 through separate Ca2+ and ATP hydrolysis-dependent signaling pathways. Simultaneous activation of both pathways by AngII leads to near complete inhibition of bTREK-1 current and pronounced depolarization of bovine adrenocortical cells. Molecular features of the new Ca2+-dependent mechanism were identified. Tissue culture media, antibiotics, fibronectin, and fetal bovine sera were obtained from Invitrogen. Coverslips were from Bellco (Vineland, NJ). Enzymes, BAPTA, MgATP, Na2ATP, GDPβS, ACTH-(1–24), AngII, 8-pcpt-cAMP, ionomycin, and EGTA were obtained from Sigma. U73122, U73343, and 2-aminoethoxydiphenyl borate (2-APB) were purchased from Biomol (Plymouth Meeting, PA). The calmodulin inhibitory peptide (CIP) was obtained from Calbiochem (La Jolla, CA). AngII receptor antagonists, losartan and PD123319, were kindly provided by Dr. Ronald Smith (Merck Pharmaceutical Co.) and Dr. Joan Keiser (Parke-Davis), respectively. Isolation and Culture of AZF Cells—Bovine adrenal glands were obtained from steers (age 2–3 years) at a local slaughterhouse. Isolated AZF and AZG cells were obtained and prepared as previously described (26Enyeart J.J. Gomora J.C. Xu L. Enyeart J.A. J. Gen. Physiol. 1997; 110: 679-692Crossref PubMed Scopus (40) Google Scholar). After isolation, cells were either resuspended in Dulbecco's modified Eagle's medium/F-12 (1:1) with 10% fetal bovine serum, 100 units/ml penicillin, 0.1 mg/ml streptomycin, and the antioxidants 1 μm tocopherol, 20 nm selenite, and 100 μm ascorbic acid (Dulbecco's modified Eagle's medium/F-12+) and plated for immediate use, or resuspended in fetal bovine serum, 5% Me2SO, divided into 1-ml aliquots, and stored in liquid nitrogen for future use. For patch clamp experiments, cells were plated in Dulbecco's modified Eagle's medium/F-12+ in 35-mm dishes containing 9-mm2 glass coverslips. Coverslips were treated with fibronectin (10 μg/ml) at 37 °C for 30 min then rinsed with warm, sterile phosphate-buffered saline immediately before adding cells. Cells were maintained at 37 °C in a humidified atmosphere of 95% air, 5% CO2. Transient Transfection and Visual Identification of CHO-K1 or COS-7 Cells Expressing bTREK-1—For whole cell patch clamp recording of cloned bTREK-1 K+ currents, CHO-K1 or COS-7 cells were co-transfected with a mixture of pCR3.1 Uni-bTREK-1 and an expression plasmid (p3-CD8) for the α-subunit of the human CD8 lymphocyte surface antigen at a 5:1 ratio using Lipofectamine (Invitrogen). Cells were visualized 1–2 days post-transfection after a 15-min incubation with anti-CD8 antibody-coated beads (Dynal Biotech Inc., Lake Success, NY) as described (27Jurman M.E. Boland L.M. Liu Y. Yellen G. BioTechniques. 1994; 17: 876-881PubMed Google Scholar). Cells were used for patch clamping 24–48 h after transfection with bTREK-1. Transfected cells were plated on 9-mm glass coverslips as described above. Fifteen min before initiating a patch clamp experiment, anti-CD8 antibody-coated beads were added to the culture dish. Upon transferring coverslips to the recording chamber, transfected cells were identified based on decoration with the beads. Whole cell and single channel bTREK-1 currents were recorded from transfected cells as described below for AZF cells. Patch Clamp Experiments—Patch clamp recordings of K+ channel currents were made in the whole cell and inside-out patch configuration from bovine AZF cells. Although the results reported in this study were obtained by recording currents from AZF cells, preliminary recordings from AZG cells showed no difference in bTREK-1 currents with respect to modulation by AngII. For whole cell recordings, the standard external solution consisted of 140 mm NaCl, 5 mm KCl, 2 mm CaCl2, 2 mm MgCl2, 10mm HEPES, and 5 mm glucose, with pH adjusted to 7.3 using NaOH. The standard pipette solution consisted of (in mm): 120 KCl, 2 MgCl2, 10 HEPES, and 0.2 GTP, with pH titrated to 6.8 using KOH. The buffering capacity of pipette solutions was varied by adding combinations of Ca2+ and BAPTA or EGTA using the Bound and Determined software program (28Brooks S.P. Storey K.B. Anal. Biochem. 1992; 201: 119-126Crossref PubMed Scopus (324) Google Scholar). Low and high capacity Ca2+ buffering solutions contained 0.5 mm EGTA and 11 mm BAPTA, respectively. The low capacity Ca2+ buffering solution was normally Ca2+-free. [Ca2+]i was buffered to 22 nm in the high capacity buffering solution. Nucleotides, including MgATP, NaUTP, and AMP-PNP were added to pipette or bath solutions as noted in the text. For inside-out patch recordings, the standard external and pipette solutions used for whole cell recordings were switched. Patch clamp recordings of IT-Ca were made in the whole cell configuration. The standard pipette solution was in mm: 120 CsCl, 2 MgCl2, 2 NaUTP, 0.5 EGTA, 0.2 GTP, 10 HEPES with pH titrated to 7.2 using CsOH. The external solution contained (in mm): 117 tetraethylammonium, 5 CsCl, 20 CaCl2, 2 MgCl2, 5 HEPES, with pH adjusted to 7.3 using tetraethylammonium-OH. All solutions were filtered through 0.22-μm cellulose acetate filters. Recording Conditions and Electronics—AZF cells were used for patch clamp experiments 2–12 h after plating. Typically, cells with diameters <15 μm and capacitances of 10–15 pF were selected. Coverslips were transferred from 35-mm culture dishes to the recording chamber (volume: 1.5 ml) that was continuously perfused by gravity at a rate of 3–5 ml/min. For whole cell recordings, patch electrodes with resistances of 1.0–2.0 MΩ were fabricated from Corning 0010 glass (World Precision Instruments, Sarasota, FL). These electrodes routinely yielded access resistances of 1.5–4.0 MΩ and voltage-clamp time constants of <100 μs. For single channel recordings, patch electrodes with higher resistances (3–5 MΩ) were used. K+ currents were recorded at room temperature (22–25 °C) according to the procedure of Hamill et al. (29Hamill O.P. Marty A. Neher E. Sakmann B. Sigworth F.J. Pflügers Arch. 1981; 391: 85-100Crossref PubMed Scopus (15138) Google Scholar) using a List EPC-7 patch clamp amplifier. Pulse generation and data acquisition were done using a personal computer and PCLAMP software with TL-1 interface (Axon Instruments, Inc., Burlingame, CA). Currents were digitized at 2–10 KHz after filtering with an 8-pole Bessel filter (Frequency Devices, Haverhill, MA). Linear leak and capacity currents were subtracted from current records using summed scaled hyperpolarizing steps of ½ to ¼ pulse amplitude. Data were analyzed using PCLAMP (CLAMPFIT 9.2, FETCHAN 6.04, and PSAT 6.04) and SigmaPlot (version 8.0) software. Drugs were applied by bath perfusion, controlled manually by a six-way rotary valve. p values were calculated using Student's t test. AngII Inhibits bTREK-1 by Separate Ca2+ and ATP-dependent Mechanisms—Bovine AZF cells express two types of K+ channels, voltage gated, rapidly inactivating Kv1.4 channels, and leak-type bTREK-1 channels (2Enyeart J.J. Xu L. Danthi S. Enyeart J.A. J. Biol. Chem. 2002; 277: 49186-49199Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 30Mlinar B. Enyeart J.J. J. Gen. Physiol. 1993; 102: 239-255Crossref PubMed Scopus (36) Google Scholar, 31Enyeart J.A. Xu L. Enyeart J.J. J. Biol. Chem. 2000; 275: 34640-34649Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). In whole cell recordings, bTREK-1 amplitude often increases spontaneously over a period of minutes, provided that the recording pipette contains ATP or other nucleotide triphosphates at millimolar concentrations (1Mlinar B. Biagi B.A. Enyeart J.J. J. Biol. Chem. 1993; 268: 8640-8644Abstract Full Text PDF PubMed Google Scholar, 2Enyeart J.J. Xu L. Danthi S. Enyeart J.A. J. Biol. Chem. 2002; 277: 49186-49199Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 26Enyeart J.J. Gomora J.C. Xu L. Enyeart J.A. J. Gen. Physiol. 1997; 110: 679-692Crossref PubMed Scopus (40) Google Scholar). The absence of time- and voltage-dependent bTREK-1 inactivation allows the corresponding membrane current to be easily isolated in whole cell recordings, using either of two voltage clamp protocols. When voltage steps of 300 ms duration are applied from a holding potential of –80 mV, bTREK-1 can be measured near the end of a voltage step when the transient Kv1.4 current has inactivated (Fig. 1, A–C, left traces). Alternatively, bTREK-1 can be selectively activated by an identical voltage step, after a 10-s prepulse to –20 mV has fully inactivated Kv1.4 channels (Fig. 1, A–C, right traces). Measurement of bTREK-1 by either method yielded nearly identical results. In previous whole cell recording studies on AZF cells using patch electrodes containing 2–5 mm MgATP and [Ca2+]i strongly buffered to 22 nm with 11 mm BAPTA, AngII inhibited bTREK-1 by a maximum of 77–82% with an IC50 of 145 pm. However, when MgATP in the pipette was replaced with UTP, which is not a substrate for kinases or ATPases, AngII was ineffective (1Mlinar B. Biagi B.A. Enyeart J.J. J. Biol. Chem. 1993; 268: 8640-8644Abstract Full Text PDF PubMed Google Scholar, 12Mlinar B. Biagi B.A. Enyeart J.J. J. Biol. Chem. 1995; 270: 20942-20951Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 21Xu L. Enyeart J.J. Am. J. Physiol. 2001; 280: C199-C215Crossref Google Scholar). In the present study, AngII at a maximally effective concentration (10 nm), inhibited bTREK-1 by only 12.0 ± 4.5% (n = 11) when pipette solutions contained 2 mm UTP and 11 mm BAPTA (Fig. 1, A and D). However, when [Ca2+]i was weakly buffered by substituting 0.5 mm EGTA for 11 mm BAPTA, inhibition of bTREK-1 by AngII was restored, even in the presence of 2 mm UTP. With [Ca2+]i weakly buffered by 0.5 mm EGTA, AngII (2 or 10 nm) inhibited bTREK-1 by 67.4 ± 2.5% (n = 33) (Fig. 1, B and D). With weak [Ca2+]i buffering, AngII also inhibited bTREK-1 by 58.0 ± 7.5% (n = 8) when the pipette contained the non-hydrolyzable ATP analog AMP-PNP (2 mm) (Fig. 1D). These results suggest that AngII-mediated inhibition of bTREK-1 may occur through separate Ca2+ and ATP hydrolysis-dependent signaling pathways. If AngII modulates bTREK-1 by parallel mechanisms, each of which produces only partial inhibition, it is likely that simultaneous activation of both pathways would more effectively suppress this current. Accordingly, when bTREK-1 currents were recorded with a pipette solution whose composition supported activation of both ATP- and Ca2+-dependent pathways (0.5 mm EGTA, 5 mm MgATP), AngII (2 nm) was significantly more effective, inhibiting bTREK-1 by 92.9 ± 0.7% (n = 8) (Fig. 1, C and D). Not only was AngII more effective at inhibiting bTREK-1 when both the Ca2+ and ATP pathways were available, it was also more potent. To compare the potency of AngII as an inhibitor of bTREK-1 through Ca2+ and combined pathways, AZF cells were superfused with AngII at various concentrations and bTREK-1 currents were recorded with pipette solutions containing 0.5 mm EGTA and either 2 nm NaUTP or 5 mm MgATP. Inhibition curves constructed from data obtained in these experiments clearly showed that AngII was more potent, as well as more effective, at inhibiting bTREK-1 when Ca2+- and ATP-dependent pathways were available (Fig. 2A, left). With both pathways available for activation, AngII inhibited bTREK-1 almost completely, with an IC50 of 63 pm. By comparison, with only the Ca2+ pathway available for activation, AngII inhibited bTREK-1 by a maximum of ∼70%, with an IC50 of 149 pm (Fig. 2A). The marked enhancement of bTREK-1 inhibition by AngII when both Ca2+- and ATP-dependent pathways were activated was most pronounced at lower AngII concentrations. At low AngII concentrations, bTREK-1 inhibition was partially reversible with washing (Fig. 2A). Activation of Ca2+- and ATP-dependent Pathways Correlate with Membrane Depolarization—bTREK-1 channels are largely responsible for setting the resting potential of bovine adrenocortical cells (1Mlinar B. Biagi B.A. Enyeart J.J. J. Biol. Chem. 1993; 268: 8640-8644Abstract Full Text PDF PubMed Google Scholar, 12Mlinar B. Biagi B.A. Enyeart J.J. J. Biol. Chem. 1995; 270: 20942-20951Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). In the absence of a significant inward current, the Goldman-Hodgkin-Katz voltage equation predicts that a large fraction of the bTREK-1 channels would have to be inhibited to effectively depolarize these cells. The simultaneous activation of both the Ca2+- and ATP-dependent pathways should therefore provide a more efficient means of depolarizing adrenocortical cells. Accordingly, in combined current and voltage clamp experiments, AngII inhibited bTREK-1 and strongly depolarized AZF cells only when both the Ca2+- and ATP-dependent pathways were available for activation. When recordings were made with pipette solutions that sustained only activation of the Ca2+-dependent pathway, AngII (2 nm) inhibited bTREK-1 current by 62.2 ± 5.7%, whereas these cells were depolarized by an average of 11.9 ± 2.4 mV (n = 5) from their resting potential of –68.8 ± 4.4 mV (Fig. 2B). By comparison, when recordings were made with pipette solutions that allowed activation of both Ca2+- and ATP-dependent pathways, AngII (2 nm) inhibited bTREK-1 almost completely by 96.8 ± 1.0%, and depolarized AZF cells by an average of 44.3 ± 4.3 mV (n = 4) from their resting potential of –63.3 ± 3.2 mV (Fig. 2C). ATP, but Not ACTH or cAMP, Inhibits bTREK-1 through Both Ca2+- and ATP-dependent Pathways—Previously, we demonstrated that externally applied ATP and UTP inhibited bTREK-1 channels through activation of a G protein-coupled purinergic receptor with a P2Y3 agonist profile (32Xu L. Enyeart J.J. J. Physiol. (Lond.). 1999; 521: 81-97Crossref Scopus (15) Google Scholar). In that study where [Ca2+]i was strongly buffered with 11 mm BAPTA, ATP (100 μm) inhibited bTREK-1 by a maximum of 71.3 ± 3.2% and this inhibition was eliminated by substituting AMP-PNP for MgATP in the pipette solution. Thus, similar to AngII, externally applied ATP inhibits bTREK-1 through a ATP hydrolysis-dependent mechanism. P2Y3 receptors are coupled to PLC activation and the release of [Ca2+]i (33Webb T.E. Henderson D. King B.F. Wang S. Simon J. Bateson A.N. Burnstock G. Barnard E.A. Mol. Pharmacol. 1996; 50: 258-265PubMed Google Scholar, 34Williams M. Burnstock G. Jacobson K.A. Jarvis M.F. Purinergic Approaches in Experimental Therapeutics. Wiley-Liss, Inc., New York1997: 3-26Google Scholar). To determine whether externally applied ATP also inhibited bTREK-1 through a Ca2+-dependent mechanism, inhibition was studied with a pipette solution designed to permit activation of the Ca2+, but not the ATP-dependent pathway. With [Ca2+]i weakly buffered by 0.5 mm EGTA, ATP at 10 and 100 μm inhibited bTREK-1 by 26.2 ± 9.3 (n = 7) and 42.8 ± 4.4% (n = 6), respectively (Fig. 3, A and D). When considered in conjunction with the previously mentioned study (32Xu L. Enyeart J.J. J. Physiol. (Lond.). 1999; 521: 81-97Crossref Scopus (15) Google Scholar), these results indicate that similar to AngII, external ATP inhibits bTREK-1 through separate Ca2+- and ATP hydrolysis-dependent mechanisms. Accordingly, when AZF cells were superfused with ATP under conditions where Ca2+- and ATP-dependent pathways could be activated, bTREK-1 was inhibited by 85.6 ± 5.8% (n = 4) (Fig. 3D). ACTH also potently inhibits bTREK-1 in bovine AZF cells with an IC50 of ∼5pm (1Mlinar B. Biagi B.A. Enyeart J.J. J. Biol. Chem. 1993; 268: 8640-8644Abstract Full Text PDF PubMed Google Scholar). Although the signaling pathways are only partially understood, inhibition likely involves both protein kinase A-dependent and -independent actions of cAMP (5Patel A.J. Honore E. Trends Neurosci. 2001; 24: 339-346Abstract Full Text Full Text PDF PubMed Scopus (374) Google Scholar, 35Enyeart J.J. Mlinar B. Enyeart J.A. J. Gen. Physiol. 1996; 108: 251-264Crossref PubMed Scopus (49) Google Scholar). Inhibition by ACTH or cAMP is eliminated when AMP-PNP or UTP is substituted for ATP in the pipette solution with [Ca2+]i strongly buffered by 11 mm BAPTA (26Enyeart J.J. Gomora J.C. Xu L. Enyeart J.A. J. Gen. Physiol. 1997; 110: 679-692Crossref PubMed Scopus (40) Google Scholar, 35Enyeart J.J. Mlinar B. Enyeart J.A. J. Gen. Physiol. 1996; 108: 251-264Crossref PubMed Scopus (49) Google Scholar). Experiments were done to determine whether, in addition to the ATP-dependent mechanism, ACTH or cAMP could also inhibit bTREK-1 by a Ca2+-dependent process. In contrast to AngII, ACTH failed to inhibit bTREK-1 in whole cell recordings made with pipette soluti
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