Human adrenal glomerulosa cells express K2P and GIRK potassium channels that are inhibited by ANG II and ACTH.

In whole cell patch clamp recordings, it was discovered that normal human adrenal zona glomerulosa (AZG) cells express members of the three major families of K+ channels. Among these are a two-pore (K2P) leak-type and a G protein-coupled, inwardly rectifying (GIRK) channel, both inhibited by peptide hormones that stimulate aldosterone secretion. The K2P current displayed properties identifying it as TREK-1 (KCNK2). This outwardly rectifying current was activated by arachidonic acid and inhibited by angiotensin II (ANG II), adrenocorticotrophic hormone (ACTH), and forskolin. The activation and inhibition of TREK-1 was coupled to AZG cell hyperpolarization and depolarization, respectively. A second K2P channel, TASK-1 (KCNK3), was expressed at a lower density in AZG cells. Human AZG cells also express inwardly rectifying K+ current(s) (KIR) that include quasi-instantaneous and time-dependent components. This is the first report demonstrating the presence of KIR in whole cell recordings from AZG cells of any species. The time-dependent current was selectively inhibited by ANG II, and ACTH, identifying it as a G protein-coupled (GIRK) channel, most likely KIR3.4 (KCNJ5). The quasi-instantaneous KIR current was not inhibited by ANG II or ACTH and may be a separate non-GIRK current. Finally, AZG cells express a voltage-gated, rapidly inactivating K+ current whose properties identified as KV1.4 (KCNA4), a conclusion confirmed by Northern blot. These findings demonstrate that human AZG cells express K2P and GIRK channels whose inhibition by ANG II and ACTH is likely coupled to depolarization-dependent secretion. They further demonstrate that human AZG K+ channels differ fundamentally from the widely adopted rodent models for human aldosterone secretion.

Adrenal fasciculata cells express T-type and rapidly and slowly activating L-type Ca2+ channels that regulate cortisol secretion.

In whole cell patch-clamp recordings, we characterized the L-type Ca(2+) currents in bovine adrenal zona fasciculata (AZF) cells and explored their role, along with the role of T-type channels, in ACTH- and angiotensin II (ANG II)-stimulated cortisol secretion. Two distinct dihydropyridine-sensitive L-type currents were identified, both of which were activated at relatively hyperpolarized potentials. One activated with rapid kinetics and, in conjunction with Northern blotting and PCR, was determined to be Cav1.3. The other, expressed in approximately one-half of AZF cells, activated with extremely slow voltage-dependent kinetics and combined properties not previously reported for an L-type Ca(2+) channel. The T-type Ca(2+) channel antagonist 3,5-dichloro-N-[1-(2,2-dimethyl-tetrahydro-pyran-4-ylmethyl)-4-fluoro-piperidin-4-ylmethyl]-benzamide (TTA-P2) inhibited Cav3.2 current in these cells, as well as ACTH- and ANG II-stimulated cortisol secretion, at concentrations that did not affect L-type currents. In contrast, nifedipine specifically inhibited L-type currents and cortisol secretion, but less effectively than TTA-P2. Diphenylbutylpiperidine Ca(2+) antagonists, including pimozide, penfluridol, and fluspirilene, and the dihydropyridine niguldipine blocked Cav3.2 and L-type currents and inhibited ACTH-stimulated cortisol secretion with similar potency. This study shows that bovine AZF cells express three Ca(2+) channels, the voltage-dependent gating and kinetics of which could orchestrate complex mechanisms linking peptide hormone receptors to cortisol secretion through action potentials or sustained depolarization. The function of the novel, slowly activating L-type channel is of particular interest in this respect. Regardless, the well-correlated selective inhibition of T- and L-type currents and ACTH- and ANG II-stimulated cortisol secretion by TTA-P2 and nifedipine establish the critical importance of these channels in AZF cell physiology.

Ca2+ and K+ channels of normal human adrenal zona fasciculata cells: properties and modulation by ACTH and AngII.

In whole cell patch clamp recordings, we found that normal human adrenal zona fasciculata (AZF) cells express voltage-gated, rapidly inactivating Ca(2+) and K(+) currents and a noninactivating, leak-type K(+) current. Characterization of these currents with respect to voltage-dependent gating and kinetic properties, pharmacology, and modulation by the peptide hormones adrenocorticotropic hormone (ACTH) and AngII, in conjunction with Northern blot analysis, identified these channels as Cav3.2 (encoded by CACNA1H), Kv1.4 (KCNA4), and TREK-1 (KCNK2). In particular, the low voltage-activated, rapidly inactivating and slowly deactivating Ca(2+) current (Cav3.2) was potently blocked by Ni(2+) with an IC50 of 3 µM. The voltage-gated, rapidly inactivating K(+) current (Kv1.4) was robustly expressed in nearly every cell, with a current density of 95.0 ± 7.2 pA/pF (n = 64). The noninactivating, outwardly rectifying K(+) current (TREK-1) grew to a stable maximum over a period of minutes when recording at a holding potential of -80 mV. This noninactivating K(+) current was markedly activated by cinnamyl 1-3,4-dihydroxy-α-cyanocinnamate (CDC) and arachidonic acid (AA) and inhibited almost completely by forskolin, properties which are specific to TREK-1 among the K2P family of K(+) channels. The activation of TREK-1 by AA and inhibition by forskolin were closely linked to membrane hyperpolarization and depolarization, respectively. ACTH and AngII selectively inhibited the noninactivating K(+) current in human AZF cells at concentrations that stimulated cortisol secretion. Accordingly, mibefradil and CDC at concentrations that, respectively, blocked Cav3.2 and activated TREK-1, each inhibited both ACTH- and AngII-stimulated cortisol secretion. These results characterize the major Ca(2+) and K(+) channels expressed by normal human AZF cells and identify TREK-1 as the primary leak-type channel involved in establishing the membrane potential. These findings also suggest a model for cortisol secretion in human AZF cells wherein ACTH and AngII receptor activation is coupled to membrane depolarization and the activation of Cav3.2 channels through inhibition of hTREK-1.

Evidence for cAMP-independent bTREK-1 inhibition by ACTH and NPS-ACTH in adrenocortical cells.

Bovine adrenal zona fasciculata (AZF) cells express bTREK-1 K(+) channels that are inhibited by ACTH through cAMP-dependent pathways. In whole cell patch clamp recordings from AZF cells, we found that ACTH may also inhibit bTREK-1 by a cAMP-independent mechanism. When the potent adenylyl cyclase (AC) antagonist 2,5-dideoxyadenosine-3'-triphosphate (2,5-dd-3'-ATP) was applied intracellularly through the patch pipette, bTREK-1 inhibition by the AC activator forskolin was blocked. In contrast, bTREK-1 inhibition by ACTH was unaltered. The selective G(Sα) antagonist NF449 also failed to blunt bTREK-1 inhibition by ACTH. At concentrations that produce little measurable increase in cAMP in bovine AZF cells, the O-nitrophenyl, sulfenyl-derivative of ACTH (NPS-ACTH) also inhibited bTREK-1 almost completely. Accordingly, 2,5-dd-3'-ATP at concentrations more than 1000× its reported IC(50) did not block bTREK-1 inhibition by NPS-ACTH. These results indicate that ACTH and NPS-ACTH can inhibit native bTREK-1 K(+) channels in AZF cells by a mechanism that does not involve activation of AC.

8-Phenylthio-adenines stimulate the expression of steroid hydroxylases, Cav3.2 Ca²âº channels, and cortisol synthesis by a cAMP-independent mechanism.

The regulation of cortisol synthesis and the expression of genes coding for steroidogenic proteins by 8-substituted cAMP and 8-substituted adenine derivatives were studied in bovine adrenal zona fasciculata (AZF) cells. At concentrations of 10-50 µM, 8-(4-chlorophenylthio)-cAMP (8CPT-cAMP), but not the poorly hydrolyzable Sp-8CPT-cAMP, stimulated large increases in cortisol synthesis and CYP17 mRNA expression. Of the three Epac (exchange protein activated by cAMP)-specific cAMP analogs, 8CPT-2'-OMe-cAMP, but not 8HPT-2'-OMe-cAMP or 8MeOPT-2'-OMe-cAMP, induced mRNAs for CYP17 and CYP11a1 steroid hydroxylases and stimulated cortisol synthesis. 8-Substituted adenine derivatives (10-200 µM), including 8PT-adenine, 8MeOPT-adenine, and 8CPT-adenine, stimulated similar large, concentration-dependent, and reversible increases in cortisol synthesis and steroid hydroxylase gene expression, whereas 8Br-adenine was ineffective. The phenylthio-adenine derivatives produced additive effects on cortisol synthesis when applied to AZF cells in combination with 8Br-cAMP. In contrast, no additivity was observed for these three compounds when used in combination with ACTH. 8PT-adenine did not activate PKA or inhibit DNA synthesis by AZF cells. 8PT-adenine-stimulated cortisol secretion and CYP17 steroid hydroxylase mRNA expression were potently inhibited by diphenyl-butylpiperidine T-type Ca(2+) antagonists. In AZF cells, 8PT-adenine and 8MeOPT-adenine induced the expression of both CACNA1H mRNA and associated Ca(v)3.2 Ca(2+) current. These results indicate that 8-chloro (but not 8-hydroxy- or 8-methoxy-)-phenylthio-cAMP analogs are converted to an active metabolite, 8CPT-adenine, that induces the expression of genes coding for steroidogenic proteins in bovine AZF cells. Other PT-adenine analogs also potently stimulate cortisol synthesis through the same unidentified signaling pathway that requires the expression of functional Ca(v)3.2 Ca(2+) channels. These phenylthio-adenine compounds and ACTH may stimulate cortisol synthesis through the same cAMP-independent mechanism.

Calcium-dependent inhibition of adrenal TREK-1 channels by angiotensin II and ionomycin.

Bovine adrenocortical cells express bTREK-1 K(+) (bovine KCNK2) channels that are inhibited by ANG II through a Gq-coupled receptor by separate Ca(2+) and ATP hydrolysis-dependent signaling pathways. Whole cell and single patch clamp recording from adrenal zona fasciculata (AZF) cells were used to characterize Ca(2+)-dependent inhibition of bTREK-1. In whole cell recordings with pipette solutions containing 0.5 mM EGTA and no ATP, the Ca(2+) ionophore ionomycin (1 µM) produced a transient inhibition of bTREK-1 that reversed spontaneously within minutes. At higher concentrations, ionomycin (5-10 µM) produced a sustained inhibition of bTREK-1 that was reversible upon washing, even in the absence of hydrolyzable [ATP](i). BAPTA was much more effective than EGTA at suppressing bTREK-1 inhibition by ANG II. When intracellular Ca(2+) concentration ([Ca(2+)](i)) was buffered to 20 nM with either 11 mM BAPTA or EGTA, ANG II (10 nM) inhibited bTREK-1 by 12.0 ± 4.5% (n=11) and 59.3 ± 8.4% (n=4), respectively. Inclusion of the water-soluble phosphatidylinositol 4,5-bisphosphate (PIP(2)) analog DiC(8)PI(4,5)P(2) in the pipette failed to increase bTREK-1 expression or reduce its inhibition by ANG II. The open probability (P(o)) of unitary bTREK-1 channels recorded from inside-out patches was reduced by Ca(2+) (10-35 µM) in a concentration-dependent manner. These results are consistent with a model in which ANG II inhibits bTREK-1 K(+) channels by a Ca(2+)-dependent mechanism that does not require the depletion of membrane-associated PIP(2). They further indicate that the Ca(2+) source is located in close proximity within a "Ca(2+) nanodomain" of bTREK-1 channels, where [Ca(2+)](i) may reach concentrations of >10 µM. bTREK-1 is the first two-pore K(+) channel shown to be inhibited by Ca(2+) through activation of a G protein-coupled receptor.

ACTH induces Cav3.2 current and mRNA by cAMP-dependent and cAMP-independent mechanisms.

Bovine adrenal zona fasciculata (AZF) cells express Ca(v)3.2 T-type Ca(2+) channels that function pivotally in adrenocorticotropic hormone (ACTH)-stimulated cortisol secretion. The regulation of Ca(v)3.2 expression in AZF cells by ACTH, cAMP analogs, and their metabolites was studied using Northern blot and patch clamp recording. Exposing AZF cells to ACTH for 3-6 days markedly enhanced the expression of Ca(v)3.2 current. The increase in Ca(v)3.2 current was preceded by an increase in corresponding CACNA1H mRNA. O-Nitrophenyl,sulfenyl-adrenocorticotropin, which produces a minimal increase in cAMP, also enhanced Ca(v)3.2 current. cAMP analogs, including 8-bromoadenosine cAMP (600 mum) and 6-benzoyladenosine cAMP (300 mum) induced CACNA1H mRNA, but not Ca(v)3.2 current. In contrast, 8-(4-chlorophenylthio) (8CPT)-cAMP (10-50 mum) enhanced CACNA1H mRNA and Ca(v)3.2 current, whereas nonhydrolyzable Sp-8CPT-cAMP failed to increase either Ca(v)3.2 current or mRNA. Metabolites of 8CPT-cAMP, including 8CPT-adenosine and 8CPT-adenine, increased Ca(v)3.2 current and mRNA with a potency and effectiveness similar to the parent compound. The Epac activator 8CPT-2'-O-methyl-cAMP and its metabolites 8CPT-2'-OMe-5'-AMP and 8CPT-2'-O-methyl-adenosine increased CACNA1H mRNA and Ca(v)3.2 current; Sp-8CPT-2'-O-methyl-cAMP increased neither Ca(v)3.2 current nor mRNA. These results reveal an interesting dichotomy between ACTH and cAMP with regard to regulation of CACNA1H mRNA and Ca(2+) current. Specifically, ACTH induces expression of CACNA1H mRNA and Ca(v)3.2 current in AZF cells by mechanisms that depend at most only partly on cAMP. In contrast, cAMP enhances expression of CACNA1H mRNA but not the corresponding Ca(2+) current. Surprisingly, chlorophenylthio-cAMP analogs stimulate the expression of Ca(v)3.2 current indirectly through metabolites. ACTH and the metabolites may induce Ca(v)3.2 expression by the same, unidentified mechanism.

cAMP analogs and their metabolites enhance TREK-1 mRNA and K+ current expression in adrenocortical cells.

bTREK-1 K(+) channels set the resting membrane potential of bovine adrenal zona fasciculata (AZF) cells and function pivotally in the physiology of cortisol secretion. Adrenocorticotropic hormone controls the function and expression of bTREK-1 channels through signaling mechanisms that may involve cAMP and downstream effectors including protein kinase A (PKA) and exchange protein 2 directly activated by cAMP (Epac2). Using patch-clamp and Northern blot analysis, we explored the regulation of bTREK-1 mRNA and K(+) current expression by cAMP analogs and several of their putative metabolites in bovine AZF cells. At concentrations sufficient to activate both PKA and Epac2, 8-bromoadenosine-cAMP enhanced the expression of both bTREK-1 mRNA and K(+) current. N(6)-Benzoyladenosine-cAMP, which activates PKA but not Epac, also enhanced the expression of bTREK-1 mRNA and K(+) current measured at times from 24 to 96 h. An Epac-selective cAMP analog, 8-(4-chlorophenylthio)-2'-O-methyl-cAMP (8CPT-2'-OMe-cAMP), potently stimulated bTREK-1 mRNA and K(+) current expression, whereas the nonhydrolyzable Epac activator 8-(4-chlorophenylthio)-2'-O-methyl-cAMP, Sp-isomer was ineffective. Metabolites of 8CPT-2'-OMe-cAMP, including 8-(4-chlorophenylthio)-2'-O-methyladenosine-5'-O-monophosphate and 8CPT-2'-OMe-adenosine, promoted the expression of bTREK-1 transcripts and ion current with a temporal pattern, potency, and effectiveness resembling that of the parent compound. Likewise, at low concentrations, 8-(4-chlorophenylthio)-cAMP (8CPT-cAMP; 30 microM) but not its nonhydrolyzable analog 8-(4-chlorophenylthio)-cAMP, Sp-isomer, enhanced the expression of bTREK-1 mRNA and current. 8CPT-cAMP metabolites, including 8CPT-adenosine and 8CPT-adenine, also increased bTREK-1 expression. These results indicate that cAMP increases the expression of bTREK-1 mRNA and K(+) current through a cAMP-dependent but Epac2-independent mechanism. They further demonstrate that one or more metabolites of 8-(4-chlorophenylthio)-cAMP analogs potently stimulate bTREK-1 expression by activation of a novel cAMP-independent mechanism. These findings raise significant questions regarding the specificity of 8-(4-chlorophenylthio)-cAMP analogs as cAMP mimetics.

N6-substituted cAMP analogs inhibit bTREK-1 K+ channels and stimulate cortisol secretion by a protein kinase A-independent mechanism.

Bovine adrenal zona fasciculata (AZF) cells express bTREK-1 K+ channels whose inhibition by cAMP is coupled to membrane depolarization and cortisol secretion through complex signaling mechanisms. cAMP analogs with substitutions in the 6 position of the adenine ring selectively activate cAMP-dependent protein kinase (PKA) but not exchange proteins activated by cAMP (Epacs). In whole-cell patch-clamp recordings from AZF cells, we found that 6-benzoyl-cAMP (6-Bnz-cAMP) and 6-monobutyryl-cAMP potently inhibit bTREK-1 K+ channels, even under conditions in which PKA activity was abolished. Specifically, when applied through the patch electrode, 6-Bnz-cAMP inhibited bTREK-1 with an IC(50) of less than 0.2 microM. Inhibition of bTREK-1 by 6-Bnz-cAMP was not diminished by PKA antagonists, including N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline (H-89), adenosine 3'-5'cyclic monophosphothiate, Rp-isomer, protein kinase inhibitor (PKI) (6-22) amide, and myristoylated PKI (14-22), applied alone or in combination, externally and intracellularly through the patch pipette. Under similar conditions, these same antagonists completely blocked PKA activation by 6-Bnz-cAMP. Inhibition of bTREK-1 by 6-Bnz-cAMP was voltage-independent and eliminated in the absence of ATP in the pipette solution. 6-Bnz-cAMP also produced delayed increases in cortisol synthesis and the expression of CYP11a1 mRNA that were only partially blocked by PKA antagonists. These results indicate that 6-Bnz-cAMP and other 6-substituted cAMP analogs can inhibit bTREK-1 K+ channels and stimulate delayed increases in cortisol synthesis by AZF cells through a PKA- and Epac-independent mechanism. They also suggest that adrenocorticotropin and cAMP function in these cells through a third cAMP-dependent protein. Finally, although 6-modified cAMP analogs exhibit high selectivity in activating PKA over Epac, they also may interact with other unidentified proteins expressed by eukaryotic cells.

Curcumin inhibits ACTH- and angiotensin II-stimulated cortisol secretion and Ca(v)3.2 current.

Adrenocorticotropic hormone and angiotensin II stimulate cortisol secretion from bovine adrenal zona fasciculata cells by the activation of adenylate cyclase and phospholipase C-coupled receptors. Curcumin (1- 20 muM), a compound found in the spice turmeric, inhibited cortisol secretion stimulated by ACTH, AngII, and 8CPT-cAMP. Curcumin also suppressed ACTH-stimulated increases in mRNAs coding for steroid acute regulatory protein and CYP11a1 steroid hydroxylase. In whole cell patch clamp recordings from AZF cells, curcumin at slightly higher concentrations also inhibited Ca(v)3.2 current. These results identify curcumin as an effective inhibitor of ACTH- and AngII-stimulated cortisol secretion. The inhibition of Ca(v)3.2 current by curcumin may contribute to its suppression of secretion.

Metabolites of an Epac-selective cAMP analog induce cortisol synthesis by adrenocortical cells through a cAMP-independent pathway.

Adrenal zona fasciculata (AZF) cells express a cAMP-activated guanine nucleotide exchange protein (Epac2) that may function in ACTH-stimulated cortisol synthesis. Experiments were done to determine whether cAMP analogs that selectively activate Epacs could induce cortisol synthesis and the expression of genes coding for steroidogenic proteins in bovine AZF cells. Treatment of AZF cells with the Epac-selective cAMP analog (ESCA) 8CPT-2'-OMe-cAMP induced large (>100 fold), concentration-dependent, delayed increases in cortisol synthesis and the expression of mRNAs coding for the steroid hydroxylases CYP11a1, CYP17, CYP21, and the steroid acute regulatory protein (StAR). However, a non-hydrolyzable analog of this ESCA, Sp-8CPT-2'-OMe-cAMP, failed to stimulate cortisol production even at concentrations that activated Rap1, a downstream effector of Epac2. Accordingly, putative metabolites of 8CPT-2'-OMe-cAMP, including 8CPT-2'-OMe-5'AMP, 8CPT-2'-OMe-adenosine, and 8CPT-adenine all induced cortisol synthesis and steroid hydroxylase mRNA expression with a temporal pattern, potency, and effectiveness similar to the parent compound. At concentrations that markedly stimulated cortisol production, none of these metabolites significantly activated cAMP-dependent protein kinase (PKA). These results show that one or more metabolites of the ESCA 8CPT-2'-OMe-cAMP induce cortico-steroidogenesis by activating a panel of genes that code for steroidogenic proteins. The remarkable increases in cortisol synthesis observed in this study appear to be mediated by a novel cAMP-, Epac- and PKA-independent signaling pathway.

ACTH inhibits bTREK-1 K+ channels through multiple cAMP-dependent signaling pathways.

Bovine adrenal zona fasciculata (AZF) cells express bTREK-1 K(+) channels that set the resting membrane potential and function pivotally in the physiology of cortisol secretion. Inhibition of these K(+) channels by adrenocorticotropic hormone (ACTH) or cAMP is coupled to depolarization and Ca(2+) entry. The mechanism of ACTH and cAMP-mediated inhibition of bTREK-1 was explored in whole cell patch clamp recordings from AZF cells. Inhibition of bTREK-1 by ACTH and forskolin was not affected by the addition of both H-89 and PKI (6-22) amide to the pipette solution at concentrations that completely blocked activation of cAMP-dependent protein kinase (PKA) in these cells. The ACTH derivative, O-nitrophenyl, sulfenyl-adrenocorticotropin (NPS-ACTH), at concentrations that produced little or no activation of PKA, inhibited bTREK-1 by a Ca(2+)-independent mechanism. Northern blot analysis showed that bovine AZF cells robustly express mRNA for Epac2, a guanine nucleotide exchange protein activated by cAMP. The selective Epac activator, 8-pCPT-2'-O-Me-cAMP, applied intracellularly through the patch pipette, inhibited bTREK-1 (IC(50) = 0.63 microM) at concentrations that did not activate PKA. Inhibition by this agent was unaffected by PKA inhibitors, including RpcAMPS, but was eliminated in the absence of hydrolyzable ATP. Culturing AZF cells in the presence of ACTH markedly reduced the expression of Epac2 mRNA. 8-pCPT-2'-O-Me-cAMP failed to inhibit bTREK-1 current in AZF cells that had been treated with ACTH for 3-4 d while inhibition by 8-br-cAMP was not affected. 8-pCPT-2'-O-Me-cAMP failed to inhibit bTREK-1 expressed in HEK293 cells, which express little or no Epac2. These findings demonstrate that, in addition to the well-described PKA-dependent TREK-1 inhibition, ACTH, NPS-ACTH, forskolin, and 8-pCPT-2'-O-Me-cAMP also inhibit these K(+) channels by a PKA-independent signaling pathway. The convergent inhibition of bTREK-1 through parallel PKA- and Epac-dependent mechanisms may provide for failsafe membrane depolarization by ACTH.

Curcumin inhibits bTREK-1 K+ channels and stimulates cortisol secretion from adrenocortical cells.

Bovine adrenal zona fasciculata (AZF) cells express bTREK-1 K(+) channels that set the resting membrane potential. Inhibition of these channels by adrenocorticotropic hormone (ACTH) is coupled to membrane depolarization and cortisol secretion. Curcumin, a phytochemical with medicinal properties extracted from the spice turmeric, was found to modulate both bTREK-1 K(+) currents and cortisol secretion from AZF cells. In whole-cell patch clamp experiments, curcumin inhibited bTREK-1 with an IC(50) of 0.93muM by a mechanism that was voltage-independent. bTREK-1 inhibition by curcumin occurred through interaction with an external binding site and was independent of ATP hydrolysis. Curcumin produced a concentration-dependent increase in cortisol secretion that persisted for up to 24h. At a maximally effective concentration of 50muM, curcumin increased secretion as much as 10-fold. These results demonstrate that curcumin potently inhibits bTREK-1 K(+) channels and stimulates cortisol secretion from bovine AZF cells. The inhibition of bTREK-1 by curcumin may be linked to cortisol secretion through membrane depolarization. Since TREK-1 is widely expressed in a variety of cells, it is likely that some of the biological actions of curcumin, including its therapeutic effects, may be mediated through inhibition of these K(+) channels.

Potent inhibition of native TREK-1 K+ channels by selected dihydropyridine Ca2+ channel antagonists.

Bovine adrenal zona fasciculata (AZF) cells express bTREK-1 background K+ channels that set the resting membrane potential. Whole-cell and single-channel patch-clamp recording were used to compare five Ca2+ channel antagonists with respect to their potency as inhibitors of native bTREK-1 K+ channels. The dihydropyridine (DHP) Ca2+ channel antagonists amlodipine and niguldipine potently and specifically inhibited bTREK-1 with IC50 values of 0.43 and 0.75 microM, respectively. The other Ca2+ channel antagonists, including the DHP nifedipine, the diphenyldiperazine flunarizine, and the cannabinoid anandamide were less potent, with IC50 values of 8.18, 2.48, and 5.07 microM, respectively. Additional studies with the highly prescribed antihypertensive amlodipine showed that inhibition of bTREK-1 by this agent was voltage-independent and specific. At concentrations that produced near complete block of bTREK-1, amlodipine inhibited voltage-gated Kv1.4 K+ and T-type Ca2+ currents in AZF cells by less than 10%. At the single-channel level, amlodipine reduced bTREK-1 open probability without altering the unitary conductance. The results demonstrate that selected DHP L-type Ca2+ channel antagonists potently inhibit native bTREK-1 K+ channels, whereas other Ca2+ channel antagonists also inhibit bTREK-1 at higher concentrations. Collectively, organic Ca2+ channel antagonists make up the most potent class of TREK-1 inhibitors yet described. Because TREK-1 K+ channels are widely expressed in the central nervous and cardiovascular systems, it is possible that some of the therapeutic or toxic effects of frequently prescribed drugs such as amlodipine may be due to their interaction with TREK-1 K+ rather L-type Ca2+ channels.

Angiotensin II inhibits native bTREK-1 K+ channels through a PLC-, kinase C-, and PIP2-independent pathway requiring ATP hydrolysis.

Angiotensin II (ANG II) inhibits bTREK-1 (bovine KCNK2) K(+) channels in bovine adrenocortical cells through a Gq-coupled AT(1) receptor by activation of separate Ca(2+)- and ATP hydrolysis-dependent signaling pathways. Whole cell patch-clamp recording from bovine adrenal zona fasciculata (AZF) cells was used to characterize the ATP-dependent signaling mechanism for inhibition of bTREK-1 by ANG II. We discovered that ATP-dependent inhibition of bTREK-1 by ANG II occurred through a novel mechanism that was independent of PLC and its established downstream effectors. The ATP-dependent inhibition of bTREK-1 by ANG II was not reduced by the PLC antagonists edelfosine and U73122, or by the PKC antagonists bisindolylmaleimide I (BIM) or calphostin C. bTREK-1 was partially inhibited ( approximately 25%) by the PKC activator phorbol 12,13 dibutyrate (PDBu) through an ATP-dependent mechanism that was blocked by BIM. Addition of Phosphatidylinositol(4,5) bisphosphate diC8 [DiC(8)PI(4,5)P(2)], a water-soluble derivative of phosphotidyl inositol 4,5 bisphosphate (PIP(2)) to the pipette solution failed to alter inhibition by ANG II. bTREK-1 inhibition by ANG II was also insensitive to antagonists of other protein kinases activated by ANG II in adrenocortical cells but was completely blocked by inorganic polytriphosphate PPPi. DiC(8)PI(4,5)P(2) was a weak activator of bTREK-1 channels, compared with the high-affinity ATP analog N(6)-(2-phenylethyl)adenosine-5'-O-triphosphate (6-PhEt-ATP). These results demonstrate that the modulation of bTREK-1 channels in bovine AZF cells is distinctive with respect to activation by phosphoinositides and nucleotides and inhibition by Gq-coupled receptors. Importantly, ANG II inhibits bTREK-1 channels through a novel pathway that is different from that described for inhibition of native TREK-1 channels in neurons, or cloned channels expressed in cell lines. They also indicate that, under physiological conditions, ANG II inhibits bTREK-1 and depolarizes AZF cells by two, novel, independent pathways that diverge proximal to the activation of PLC.

Angiotensin II inhibits bTREK-1 K+ channels in adrenocortical cells by separate Ca2+- and ATP hydrolysis-dependent mechanisms.

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.

Modulation of native T-type calcium channels by omega-3 fatty acids.

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.

TREK-1 K+ channels couple angiotensin II receptors to membrane depolarization and aldosterone secretion in bovine adrenal glomerulosa cells.

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.

Caffeic acid esters activate TREK-1 potassium channels and inhibit depolarization-dependent secretion.

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.

Corticotropin induces the expression of TREK-1 mRNA and K+ current in adrenocortical cells.

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.