Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 14 de 14
Filter
Add more filters










Publication year range
1.
J Pharmacol Exp Ther ; 383(1): 11-24, 2022 10.
Article in English | MEDLINE | ID: mdl-35926869

ABSTRACT

Pharmacological inhibition of prolyl-4-hydroxylase domain (PHD) enzymes stabilizes hypoxia-inducible factors (HIFs), transcription factors that activate target genes that, among others, increase erythropoietin (EPO) synthesis, resulting in the production of new red blood cells (RBCs). Herein, we summarize the preclinical characteristics of the small molecule HIF prolyl-4-hydroxylase inhibitor vadadustat (AKB-6548), which is in development for the treatment of anemia in patients with chronic kidney disease (CKD). Vadadustat inhibits the enzyme activity of all three human PHD isozymes, PHD1, PHD2, and PHD3, with similar low nanomolar inhibitory constant values. PHD enzyme inhibition by vadadustat is competitive with endogenous cofactor 2-oxoglutarate and is insensitive to free iron concentration. In the human hepatocellular carcinoma cell line (Hep 3B) and human umbilical vein endothelial cells, PHD inhibition by vadadustat leads to the time- and concentration-dependent stabilization of HIF-1α and HIF-2α In Hep 3B cells, this in turn results in the synthesis and secretion of EPO; vascular endothelial growth factor is not measured at detectable levels. A single oral dose of vadadustat in rats potently increases circulating levels of EPO, and daily oral dosing for 14 days increases RBC indices in healthy rats and in the 5/6 nephrectomy model of CKD. In mice and dogs, once-daily repeat oral dosing increases hemoglobin and hematocrit. Vadadustat has a relatively short half-life in all nonclinical species evaluated and does not accumulate when administered as a single bolus dose (oral or intravenous) or upon repeat oral dosing. The pharmacological profile of vadadustat supports continued development for treatment of renal anemia. SIGNIFICANCE STATEMENT: Vadadustat (AKB-6548) is an orally bioavailable small molecule prolyl-4-hydroxylase inhibitor in development for anemia of chronic kidney disease. It is an equipotent inhibitor of the three human prolyl-4-hydroxylase domain isoforms, which activates erythropoiesis through stabilization of hypoxia-inducible factor (HIF)-1α and HIF-2α, increasing production of erythropoietin, without detectable stimulation of vascular endothelial growth factor.


Subject(s)
Anemia , Erythropoietin , Hypoxia-Inducible Factor-Proline Dioxygenases , Renal Insufficiency, Chronic , Animals , Dogs , Humans , Mice , Rats , Anemia/drug therapy , Anemia/etiology , Anemia/metabolism , Basic Helix-Loop-Helix Transcription Factors/metabolism , Endothelial Cells/metabolism , Erythropoietin/metabolism , Hypoxia-Inducible Factor-Proline Dioxygenases/antagonists & inhibitors , Prolyl Hydroxylases , Renal Insufficiency, Chronic/complications , Renal Insufficiency, Chronic/drug therapy , Vascular Endothelial Growth Factor A
2.
SLAS Discov ; 24(2): 111-120, 2019 02.
Article in English | MEDLINE | ID: mdl-30589598

ABSTRACT

SLC6A19 (B0AT1) is a neutral amino acid transporter, the loss of function of which results in Hartnup disease. SLC6A19 is also believed to have an important role in amino acid homeostasis, diabetes, and weight control. A small-molecule inhibitor of human SLC6A19 (hSLC6A19) was identified using two functional cell-based assays: a fluorescence imaging plate reader (FLIPR) membrane potential (FMP) assay and a stable isotope-labeled neutral amino acid uptake assay. A diverse collection of 3440 pharmacologically active compounds from the Microsource Spectrum and Tocriscreen collections were tested at 10 µM in both assays using MDCK cells stably expressing hSLC6A19 and its obligatory subunit, TMEM27. Compounds that inhibited SLC6A19 activity in both assays were further confirmed for activity and selectivity and characterized for potency in functional assays against hSLC6A19 and related transporters. A single compound, cinromide, was found to robustly, selectively, and reproducibly inhibit SLC6A19 in all functional assays. Structurally related analogs of cinromide were tested to demonstrate structure-activity relationship (SAR). The assays described here are suitable for carrying out high-throughput screening campaigns to identify modulators of SLC6A19.


Subject(s)
Amino Acid Transport Systems, Neutral/antagonists & inhibitors , Biological Assay/methods , Amino Acid Transport Systems, Neutral/metabolism , Animals , Cell Line , Fluorescence , Humans , Isotope Labeling , Membrane Potentials , Xenopus laevis
3.
ACS Med Chem Lett ; 3(3): 216-21, 2012 Mar 08.
Article in English | MEDLINE | ID: mdl-24900457

ABSTRACT

A series of CCR5 antagonists representing the thiophene-3-yl-methyl ureas were designed that met the pharmacological criteria for HIV-1 inhibition and mitigated a human ether-a-go-go related gene (hERG) inhibition liability. Reducing lipophilicity was the main design criteria used to identify compounds that did not inhibit the hERG channel, but subtle structural modifications were also important. Interestingly, within this series, compounds with low hERG inhibition prolonged the action potential duration (APD) in dog Purkinje fibers, suggesting a mixed effect on cardiac ion channels.

4.
J Am Chem Soc ; 133(41): 16477-85, 2011 Oct 19.
Article in English | MEDLINE | ID: mdl-21942640

ABSTRACT

The viral resistance of marketed antiviral drugs including the emergence of new viral resistance of the only marketed CCR5 entry inhibitor, maraviroc, makes it necessary to develop new CCR5 allosteric inhibitors. A mutagenesis/modeling approach was used (a) to remove the potential hERG liability in an otherwise very promising series of compounds and (b) to design a new class of compounds with an unique mutant fingerprint profile depending on residues in the N-terminus and the extracellular loop 2. On the basis of residues, which were identified by mutagenesis as key interaction sites, binding modes of compounds were derived and utilized for compound design in a prospective manner. The compounds were then synthesized, and in vitro evaluation not only showed that they had good antiviral potency but also fulfilled the requirement of low hERG inhibition, a criterion necessary because a potential approved drug would be administered chronically. This work utilized an interdisciplinary approach including medicinal chemistry, molecular biology, and computational chemistry merging the structural requirements for potency with the requirements of an acceptable in vitro profile for allosteric CCR5 inhibitors. The obtained mutant fingerprint profiles of CCR5 inhibitors were used to translate the CCR5 allosteric binding site into a general pharmacophore, which can be used for discovering new inhibitors.


Subject(s)
Anti-HIV Agents/pharmacology , Drug Design , Ether-A-Go-Go Potassium Channels/antagonists & inhibitors , HIV-1/drug effects , Leukocytes, Mononuclear/drug effects , Urea/pharmacology , Anti-HIV Agents/chemical synthesis , Anti-HIV Agents/chemistry , CCR5 Receptor Antagonists , Dose-Response Relationship, Drug , Humans , Microbial Sensitivity Tests , Models, Molecular , Molecular Structure , Molecular Weight , Mutagenesis , Stereoisomerism , Structure-Activity Relationship , Urea/analogs & derivatives , Urea/chemistry
5.
Bioorg Med Chem Lett ; 20(1): 228-31, 2010 Jan 01.
Article in English | MEDLINE | ID: mdl-19914064

ABSTRACT

Two sets of diaminopyrimidines, totalling 45 compounds, were synthesized and assayed against Plasmodium falciparum. The SAR was relatively shallow, with only the presence of a 2-(pyrrolidin-1-yl)ethyl group at R(2) significantly affecting activity. A subsequent series addressed high LogD values by introducing more polar side groups, with the most active compounds possessing diazepine and N-benzyl-4-aminopiperidyl groups at R(1)/R(2). A final series attempted to address high in vitro microsomal clearance by replacing the C6-Me group with CF(3), however antiplasmodial activity decreased without any improvement in clearance. The C6-CF(3) group decreased hERG inhibition, probably as a result of decreased amine basicity at C2/C4.


Subject(s)
Antimalarials/chemical synthesis , Pyrimidines/chemistry , Animals , Antimalarials/chemistry , Antimalarials/pharmacology , Ether-A-Go-Go Potassium Channels/metabolism , Humans , Microsomes, Liver/metabolism , Plasmodium falciparum/drug effects , Pyrimidines/chemical synthesis , Pyrimidines/pharmacology , Rats , Structure-Activity Relationship
6.
J Pharmacol Exp Ther ; 324(2): 427-33, 2008 Feb.
Article in English | MEDLINE | ID: mdl-17986649

ABSTRACT

The United States Food and Drug Administration (FDA) uses alfuzosin as an example of a drug having QT risk in humans that was not detected in nonclinical studies. FDA approval required a thorough clinical QT study (TCQS) that was weakly positive at high doses. The FDA has used the clinical/nonclinical discordance as a basis for mandatory TCQS, and this requirement has serious consequences for drug development. For this reason, we re-examined whether nonclinical signals of QT risk for alfuzosin were truly absent. Alfuzosin significantly prolonged action potential duration (APD)(60) in rabbit Purkinje fibers (p < 0.05) and QT in isolated rabbit hearts (p < 0.05) at the clinically relevant concentration of 300 nM. In man, the QT interval corrected with Fridericia's formula increased 7.7 ms, which exceeds the 5.0-ms threshold for a positive TCQS. Effects on hK(v)11.1, hK(v)4.3, and hK(v)7.1/hKCNE1 potassium currents and calcium current were not involved. At 300 nM, approximately 30x C(max), alfuzosin significantly increased whole-cell peak sodium (hNa(v)1.5) current (p < 0.05), increased the probability of late hNa(v)1.5 single-channel openings, and significantly shortened the slow time constant for recovery from inactivation. Alfuzosin also increased hNa(v)1.5 burst duration and number of openings per burst between 2- and 3-fold. Alfuzosin is a rare example of a non-antiarrhythmic drug that delays cardiac repolarization not by blocking hK(v)11.1 potassium current, but by increasing sodium current. Nonclinical studies clearly show that alfuzosin increases plateau potential and prolongs APD and QT, consistent with QT prolongation in man. The results challenge the FDA grounds for the absolute primacy of TCQS based on the claim of a false-negative, nonclinical study on alfuzosin.


Subject(s)
Action Potentials/drug effects , Myocytes, Cardiac/drug effects , Quinazolines/pharmacology , Action Potentials/physiology , Animals , Cell Line , Female , Guinea Pigs , Humans , Long QT Syndrome/drug therapy , Long QT Syndrome/physiopathology , Male , Myocytes, Cardiac/physiology , Purkinje Fibers/drug effects , Purkinje Fibers/physiology , Quinazolines/therapeutic use , Rabbits
7.
Biochem Biophys Res Commun ; 344(4): 1161-5, 2006 Jun 16.
Article in English | MEDLINE | ID: mdl-16647042

ABSTRACT

Curcumin, a major constituent of the spice turmeric, is a nutriceutical compound reported to possess therapeutic properties against a variety of diseases ranging from cancer to cystic fibrosis. In whole-cell patch-clamp experiments on bovine adrenal zona fasciculata (AZF) cells, curcumin reversibly inhibited the Kv1.4K+ current with an IC50 of 4.4 microM and a Hill coefficient of 2.32. Inhibition by curcumin was significantly enhanced by repeated depolarization; however, this agent did not alter the voltage-dependence of steady-state inactivation. Kv1.4 is the first voltage-gated ion channel demonstrated to be inhibited by curcumin. Furthermore, these results identify curcumin as one of the most potent antagonists of these K+ channels identified thus far. It remains to be seen whether any of the therapeutic actions of curcumin might originate with its ability to inhibit Kv1.4 or other voltage-gated K+ channel.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Antineoplastic Agents/pharmacology , Curcumin/pharmacology , Kv1.4 Potassium Channel/antagonists & inhibitors , Adrenal Glands/drug effects , Adrenal Glands/metabolism , Animals , Cattle , Cells, Cultured , Inhibitory Concentration 50 , Patch-Clamp Techniques
8.
Neurosci Lett ; 400(1-2): 63-8, 2006 May 29.
Article in English | MEDLINE | ID: mdl-16546320

ABSTRACT

Neuronal nAChRs are pentameric transmembrane proteins which function as ligand-gated ion channels and are composed of multiple alpha and beta subunits. Nine neuronal nAChR alpha subunit genes (alpha2-alpha10) and three nAChR beta subunit genes (beta2-beta4) have been identified. nAChR subtypes are heteromers, composed of various combinations of nAChR subunits or homomers composed of alpha7, alpha8, or alpha9 subunits. nAChR subtypes are widely expressed in the nervous system, yet each subunit has a distinct and unique pattern of expression. This report focuses on the expression of the nAChR alpha7 gene since homomeric nAChRs can be formed from this one subunit, simplifying a study of the expression of a specific nAChR subtype. Alpha7 nAChRs are involved in several important biological activities in addition to synaptic transmission including mediating neurite outgrowth, neuronal development and cell death, and in presynaptic control of neurotransmitter release. Transcriptional regulation of alpha7 gene expression may be important to control the location and timing of these events. We previously isolated a rat alpha7 nAChR promoter and studied expression in PC12 cells. In this study we examined the expression of the alpha7 promoter in PC12, HEK293, L6, SN17 and Neuro-2A cells in order to define elements necessary for cell-specific expression. Elements promoting expression of alpha7 in muscle and fibroblasts were identified. We also demonstrated that several other nAChR genes are also expressed in SN 17 and Neuro-2A cells, supporting use of these cell lines as models to study transcriptional control of nAChR genes.


Subject(s)
Gene Expression/physiology , Promoter Regions, Genetic/genetics , Receptors, Nicotinic/genetics , Animals , Blotting, Northern/methods , Cell Line , Humans , Mice , Protein Subunits/genetics , Protein Subunits/metabolism , RNA, Messenger/biosynthesis , Rats , Reverse Transcriptase Polymerase Chain Reaction/methods , Transfection/methods , alpha7 Nicotinic Acetylcholine Receptor
9.
J Biol Chem ; 280(35): 30814-28, 2005 Sep 02.
Article in English | MEDLINE | ID: mdl-15994319

ABSTRACT

Bovine adrenocortical cells express bTREK-1 K+ channels that set the resting membrane potential (V(m)) 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 microM) 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.


Subject(s)
Adenosine Triphosphate/metabolism , Adrenal Cortex/cytology , Angiotensin II/metabolism , Calcium/metabolism , Potassium Channels, Tandem Pore Domain/antagonists & inhibitors , Potassium Channels, Tandem Pore Domain/metabolism , Signal Transduction/physiology , Adrenal Cortex/metabolism , Adrenocorticotropic Hormone/metabolism , Animals , CHO Cells , Cattle , Cells, Cultured , Cricetinae , Cyclic AMP/analogs & derivatives , Cyclic AMP/metabolism , Hormone Antagonists/metabolism , Ionomycin/metabolism , Ionophores/metabolism , Male , Patch-Clamp Techniques , Peptide Fragments/metabolism , Type C Phospholipases/metabolism
10.
Biochem Biophys Res Commun ; 327(2): 485-93, 2005 Feb 11.
Article in English | MEDLINE | ID: mdl-15629140

ABSTRACT

Low voltage-activated, rapidly inactivating T-type Ca(2+) channels are found in a variety of cells where they regulate electrical activity and Ca(2+) entry. In whole-cell patch clamp recordings from bovine adrenal zona fasciculata cells, cis-polyunsaturated omega-3 fatty acids including docosahexaenoic acid (DHA), eicosapentaenoic acid, and alpha-linolenic acid inhibited T-type Ca(2+) current (I(T-Ca)) with IC(50)s of 2.4, 6.1, and 14.4microM, respectively. Inhibition of I(T-Ca) by DHA was partially use-dependent. In the absence of stimulation, DHA (5microM) inhibited I(T-Ca) by 59.7+/-8.1% (n=5). When voltage steps to -10mV were applied at 12s intervals, block increased to 80.5+/-7.2%. Inhibition of I(T-Ca) by DHA was accompanied by a shift of -11.7mV in the voltage dependence of steady-state inactivation, and a smaller -3.3mV shift in the voltage dependence of activation. omega-3 fatty acids also selectively altered the gating kinetics of T-type Ca(2+) channels. DHA accelerated T channel recovery from inactivation by approximately 3-fold, but did not affect the kinetics of T channel activation or deactivation. Arachidonic acid, an omega-6 polyunsaturated fatty acid, also inhibited T-type Ca(2+) current at micromolar concentrations, while the trans polyunsaturated fatty acid linolelaidic acid was ineffective. These results identify cis polyunsaturated fatty acids as relatively potent, new T-type Ca(2+) channel antagonists. omega-3 fatty acids are essential dietary components that have been shown to possess remarkable neuroprotective and cardioprotective properties that are likely mediated through suppression of electrical activity and associated Ca(2+) entry. Inhibition of T-type Ca(2+) channels in neurons and cardiac myocytes could contribute significantly to their protective actions.


Subject(s)
Calcium Channels, T-Type/metabolism , Fatty Acids, Omega-3/pharmacology , Ion Channel Gating/drug effects , Adrenal Glands/cytology , Animals , Calcium/metabolism , Calcium Channels, T-Type/chemistry , Cattle , Cells, Cultured , Electric Conductivity , Kinetics , Patch-Clamp Techniques
11.
Am J Physiol Endocrinol Metab ; 287(6): E1154-65, 2004 Dec.
Article in English | MEDLINE | ID: mdl-15315905

ABSTRACT

Bovine adrenal glomerulosa (AZG) cells were shown to express bTREK-1 background K(+) channels that set the resting membrane potential and couple angiotensin II (ANG II) receptor activation to membrane depolarization and aldosterone secretion. Northern blot and in situ hybridization studies demonstrated that bTREK-1 mRNA is uniformly distributed in the bovine adrenal cortex, including zona fasciculata and zona glomerulosa, but is absent from the medulla. TASK-3 mRNA, which codes for the predominant background K(+) channel in rat AZG cells, is undetectable in the bovine adrenal cortex. In whole cell voltage clamp recordings, bovine AZG cells express a rapidly inactivating voltage-gated K(+) current and a noninactivating background K(+) current with properties that collectively identify it as bTREK-1. The outwardly rectifying K(+) current was activated by intracellular acidification, ATP, and superfusion of bTREK-1 openers, including arachidonic acid (AA) and cinnamyl 1-3,4-dihydroxy-alpha-cyanocinnamate (CDC). Bovine chromaffin cells did not express this current. In voltage and current clamp recordings, ANG II (10 nM) selectively inhibited the noninactivating K(+) current by 82.1 +/- 6.1% and depolarized AZG cells by 31.6 +/- 2.3 mV. CDC and AA overwhelmed ANG II-mediated inhibition of bTREK-1 and restored the resting membrane potential to its control value even in the continued presence of ANG II. Vasopressin (50 nM), which also physiologically stimulates aldosterone secretion, inhibited the background K(+) current by 73.8 +/- 9.4%. In contrast to its potent inhibition of bTREK-1, ANG II failed to alter the T-type Ca(2+) current measured over a wide range of test potentials by using pipette solutions of identical nucleotide and Ca(2+)-buffering compositions. ANG II also failed to alter the voltage dependence of T channel activation under these same conditions. Overall, these results identify bTREK-1 K(+) channels as a pivotal control point where ANG II receptor activation is transduced to depolarization-dependent Ca(2+) entry and aldosterone secretion.


Subject(s)
Aldosterone/metabolism , Potassium Channels, Tandem Pore Domain/physiology , Receptors, Angiotensin/metabolism , Zona Glomerulosa/metabolism , Adrenal Glands/cytology , Adrenal Glands/metabolism , Angiotensin II/pharmacology , Animals , Arachidonic Acid/pharmacology , Caffeic Acids/pharmacology , Calcium Channels, T-Type/drug effects , Calcium Channels, T-Type/metabolism , Cattle , Cells, Cultured , Electric Conductivity , Electrophysiology , Membranes/physiology , Patch-Clamp Techniques , Potassium Channels, Tandem Pore Domain/antagonists & inhibitors , Potassium Channels, Tandem Pore Domain/drug effects , Potassium Channels, Tandem Pore Domain/genetics , RNA, Messenger/metabolism , Vasopressins/pharmacology , Zona Glomerulosa/cytology , Zona Glomerulosa/drug effects , Zona Glomerulosa/physiology
12.
Mol Pharmacol ; 65(3): 599-610, 2004 Mar.
Article in English | MEDLINE | ID: mdl-14978238

ABSTRACT

In whole-cell and single-channel patch-clamp recordings from bovine adrenal fasciculata cells, it was discovered that selected caffeic acid derivatives dramatically enhanced the activity of background TREK-1 K+ channels. Cinnamyl 1-3,4-dihydroxy-alpha-cyanocinnamate (CDC), activated TREK-1 when this agent was applied externally to cells or outside-out patches at concentrations of 5 to 10 microM. Structure/activity studies showed that native bTREK-1 channels were also activated by other caffeic acid esters, including caffeic acid phenethyl ester (CAPE), which contain a benzene or furan ring in the ester side chain. The activation of bTREK-1 by caffeic acid derivatives did not occur through inhibition of lipoxygenases because other potent lipoxygenase inhibitors failed to activate bTREK-1. In bovine adrenal zona fasciculata (AZF) cells, bTREK-1 K+ channels set the resting membrane potential. Inhibition of these channels by corticotropin leads to depolarization-dependent Ca2+ entry and cortisol secretion. CDC, which activates up to thousands of dormant bTREK-1 channels in AZF cells, was found to overwhelm the inhibition of bTREK-1 by corticotropin, reverse the membrane depolarization, and inhibit corticotropin-stimulated cortisol secretion. These results identify selected caffeic acid derivatives as novel K+ channel openers that activate TREK-1 background K+ channels. Because of their ability to stabilize the resting membrane potential and oppose electrical activity and depolarization-dependent Ca2+ entry, these compounds may have therapeutic potential as neuroprotective or cardioprotective agents.


Subject(s)
Caffeic Acids/chemistry , Caffeic Acids/pharmacology , Potassium Channels, Tandem Pore Domain , Potassium Channels/metabolism , Zona Fasciculata/drug effects , Animals , Cattle , Cells, Cultured , Hydrocortisone/metabolism , Membrane Potentials/drug effects , Membrane Potentials/physiology , Structure-Activity Relationship , Zona Fasciculata/cytology , Zona Fasciculata/metabolism
13.
Mol Pharmacol ; 64(1): 132-42, 2003 Jul.
Article in English | MEDLINE | ID: mdl-12815169

ABSTRACT

Bovine adrenal zona fasciculata (AZF) cells express a two-pore/four-transmembrane segment bTREK-1 K+ channel that sets the resting potential and couples hormonal signals to depolarization-dependent Ca2+ entry and cortisol secretion. It was discovered that corticotropin (1-2000 pM) enhances the expression of bTREK-1 mRNA and membrane current in cultured AZF cells. Forskolin and 8-pcpt-cAMP mimicked corticotropin induction of bTREK-1 mRNA, but angiotensin II (AII) was ineffective. The induction of bTREK-1 mRNA by corticotropin was partially blocked by the A-kinase antagonist H-89. 8-(4-Chloro-phenylthio)-2-O-methyladenosine-3'-5'-cyclic monophosphate, a cAMP analog that activates cAMP-regulated guanine nucleotide exchange factors (Epac), failed to increase bTREK-1 mRNA. Corticotropin-stimulated increases in bTREK-1 mRNA were eliminated by inhibitors of protein synthesis or gene transcription. bTREK-1 current disappeared after 24 h in serum-supplemented medium, but in the presence of corticotropin, bTREK-1 expression was maintained for at least 48 h. The enhancement of bTREK-1 mRNA and ionic current contrasts with the corticotropin-induced down-regulation of the Kv1.4 voltage-gated K+ current and associated mRNA in AZF cells. These results demonstrate that corticotropin rapidly and potently induces the expression of bTREK-1 in AZF cells at the pretranslational level by a cAMP-dependent mechanism that is partially dependent on A-kinase but independent of Epac and Ca2+. They further indicate that prolonged stimulation of AZF cells by corticotropin, as occurs during long-term stress or disease, may produce pronounced changes in the expression of genes encoding ion channels, thereby reshaping the electrical properties of these cells to enhance or limit cortisol secretion.


Subject(s)
Adrenocorticotropic Hormone/pharmacology , Gene Expression/drug effects , Potassium Channels, Tandem Pore Domain , Potassium Channels/biosynthesis , Zona Fasciculata/cytology , Animals , Calcium/metabolism , Cattle , Cells, Cultured , Cyclic AMP/metabolism , Electrophysiology , Male , Potassium Channels/genetics , Potassium Channels/physiology , RNA, Messenger/biosynthesis , RNA, Messenger/drug effects
14.
J Biol Chem ; 277(51): 49186-99, 2002 Dec 20.
Article in English | MEDLINE | ID: mdl-12368289

ABSTRACT

Bovine adrenal zona fasciculata (AZF) cells express a background K(+) channel (I(AC)) that sets the resting potential and acts pivotally in ACTH-stimulated cortisol secretion. We have cloned a bTREK-1 (KCNK2) tandem-pore K(+) channel cDNA from AZF cells with properties that identify it as the native I(AC). The bTREK-1 cDNA is expressed robustly in AZF cells and includes transcripts of 4.9, 3.6, and 2.8 kb. In patch clamp recordings made from transiently transfected cells, bTREK-1 displayed distinctive properties of I(AC) in AZF cells. Specifically, bTREK-1 currents were outwardly rectifying with a large instantaneous and smaller time-dependent component. Similar to I(AC), bTREK-1 increased spontaneously in amplitude over many minutes of whole cell recording and was blocked potently by Ca(2+) antagonists including penfluridol and mibefradil and by 8-(4-chlorophenylthio)-cAMP. Unitary TREK-1 and I(AC) currents were nearly identical in amplitude. The native I(AC) current, in turn, displayed properties that together are specific to TREK-1 K(+) channels. These include activation by intracellular acidification, enhancement by the neuroprotective agent riluzole, and outward rectification. bTREK-1 current differed from native K(+) current only in its lack of ATP dependence. In contrast to I(AC), the current density of bTREK-1 in human embryonic kidney-293 cells was not increased by raising pipette ATP from 0.1 to 5 mm. Further, the enhancement of I(AC) current in AZF cells by low pH and riluzole was facilitated by, and dependent on, ATP at millimolar concentrations in the pipette solution. Overall, these results establish the identity of I(AC) K(+) channels, demonstrate the expression of bTREK-1 in a specific endocrine cell, identify potent new TREK-1 antagonists, and assign a pivotal role for these tandem-pore channels in the physiology of cortisol secretion. The activation of I(AC) by ATP indicates that native bTREK-1 channels may function as sensors that couple the metabolic state of the cell to membrane potential, perhaps through an associated ATP-binding protein.


Subject(s)
Adenosine Triphosphate/metabolism , Adrenal Cortex/cytology , Adrenocorticotropic Hormone/metabolism , Potassium Channels, Tandem Pore Domain , Potassium Channels/metabolism , Adenosine Triphosphate/pharmacology , Amino Acid Sequence , Animals , CHO Cells , Cattle , Cell Line , Cells, Cultured , Cloning, Molecular , Cricetinae , Cyclic AMP/metabolism , DNA, Complementary/metabolism , Dose-Response Relationship, Drug , Humans , Hydrogen-Ion Concentration , Kinetics , Molecular Sequence Data , Patch-Clamp Techniques , Penfluridol/pharmacology , Protein Binding , RNA, Messenger/metabolism , Rats , Riluzole/pharmacology , Time Factors , Transfection
SELECTION OF CITATIONS
SEARCH DETAIL
...