John J. Enyeart

Block of current through T-type calcium channels by trivalent metal cations and nickel in neural rat and human cells.

1. The effects of the trivalent cations yttrium (Y3+), lanthanum (La3+), cerium (Ce3+), neodymium (Nd3+), gadolinium (Gd3+), holmium (Ho3+), erbium (Er3+), ytterbium (Yb3+) and the divalent cation nickel (Ni2+) on the T‐type voltage gated calcium channel (VGCC) were characterized by the whole‐cell patch clamp technique using rat and human thyroid C cell lines. 2. All the metal cations (M3+) studied, blocked current through T‐type VGCC (IT) in a concentration‐dependent manner. Smaller trivalents were the best T‐channel antagonists and potency varied inversely with ionic radii for the larger M3+ ions. Estimation of half‐maximal blocking concentrations (IC50s) for IT carried by 10 mM Ca2+ resulted in the following potency sequence: Ho3+ (IC50 = 0.107 microM) approximately Y3+ (0.117) approximately Yb3+ (0.124) > or = Er3+ (0.153) > Gd3+ (0.267) > Nd3+ (0.429) > Ce3+ (0.728) > La3+ (1.015) >> Ni2+ (5.65). 3. Tail current measurements and conditioning protocols were used to study the influence of membrane voltage on the potency of these antagonists. Block of IT by Ni2+, Y3+, La3+ and the lanthanides was voltage independent in the range from ‐200 to +80 mV. In addition, the antagonists did not affect macroscopic inactivation and deactivation of T‐type VGCC. 4. Increasing the extracellular Ca2+ concentration reduced the potency of IT block by Ho3+, indicative of competitive antagonism between this blocker and the permeant ion for a binding site. 5. The results suggest that the mechanism of metal cation block of T‐type VGCC is occlusion of the channel pore by the antagonist binding to a Ca2+/M3+ binding site, located out of the membrane electric field. 6. Block of T‐type VGCC by Y3+, lanthanides and La3+ differ from the inhibition of high voltage‐activated VGCC block in several respects: smaller cations are more potent IT antagonists; block is voltage independent and the antagonists do not permeate T‐type channels. These differences suggest corresponding structural dissimilarities in the permeation pathways of low and high voltage‐activated Ca2+ channels.

An ACTH- and ATP-regulated background K+ channel in adrenocortical cells is TREK-1.

Bovine adrenal zona fasciculata (AZF) cells express a background K+channel (IAC) 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 IAC. 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 IAC in AZF cells. Specifically, bTREK-1 currents were outwardly rectifying with a large instantaneous and smaller time-dependent component. Similar to IAC, bTREK-1 increased spontaneously in amplitude over many minutes of whole cell recording and was blocked potently by Ca2+ antagonists including penfluridol and mibefradil and by 8-(4-chlorophenylthio)-cAMP. Unitary TREK-1 and IAC currents were nearly identical in amplitude. The native IAC 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 IAC, 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 IAC 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 IAC 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 IAC 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.

ACTH AND AII DIFFERENTIALLY STIMULATE STEROID HORMONE ORPHAN RECEPTOR MRNAS IN ADRENAL CORTICAL CELLS

NGFI-B and Ad4BP are steroid hormone receptor-like transcription factor that may control steroidogenesis, growth and differentiation in the adrenal cortex. We have studied the induction of NGFI-B and Ad4BP and mRNAs by the peptide hormones, ACTH, AII, IGF, FGF, and by KCl depolarization in cultured bovine adrenocortical cells. The mRNAs for these two transcription factors were most effectively but differentially induced by ACTH and AII. mRNA for NGFI-B was typically undetectable in unstimulated cells, but rapidly (< 30 min) accumulated in response to ACTH and AII. Peak increases occurred within 2-3 h after which mRNA levels declined. At maximally effective concentrations, AII produced increases in NGFI-B mRNA 2.7-fold larger than those triggered by ACTH (n = 7). In contrast to NGFI-B, Ad4BP mRNA was readily detectable in unstimulated cells. ACTH and AII induced smaller, slower and more sustained increases in Ad4BP mRNA. Peak values were obtained in 6-8 h and Ad4BP mRNA remained elevated for at least 18 h. ACTH produced increases in Ad4BP that were 2.6-fold larger than those stimulated by AII (n = 8). Antagonists of major signaling pathways that couple ACTH and AII receptors to cortisol secretion, including T-type Ca2+ antagonist Ni2+ and penfluridol, the CaM kinase antagonist KN-62, the A-kinase antagonist H-89 and the non-selective kinase antagonist staurosporine, all failed to suppress increases in NGFI-B and Ad4BP mRNAs triggered by these two peptides. Each of these agents effectively inhibited cortisol production stimulated by the peptides. Further, arguing against their proposed role as transcription factors for steroidogenic enzymes, ACTH- and AII-stimulated increases in steroid orphan receptor mRNAs were not correlated with corresponding increases in cortisol production measured over 24 h. The results show that NGFI-B and Ad4BP mRNAs are differentially regulated by ACTH and AII. Only NGFI-B is rapidly and transiently increased with kinetics common to immediate early genes. The lack of correlation between peptide-stimulated increases in orphan receptor mRNAs and cortisol production in combination with the apparent divergence in the associated signaling pathways argue against a primary role for these transcription factors in ACTH- and AII-stimulated steroidogenesis. The dual function of these peptide hormones as mediators of development and corticosteroid synthesis could necessitate the presence of separate, parallel signaling pathways.

Modulation of native TREK-1 and Kv1.4 K+ channels by polyunsaturated fatty acids and lysophospholipids.

The modulation of TREK-1 leak and Kv1.4 voltage-gated K+ channels by fatty acids and lysophospholipids was studied in bovine adrenal zona fasciculata (AZF) cells. In whole-cell patch-clamp recordings, arachidonic acid (AA) (1–20 µM) dramatically and reversibly increased the activity of bTREK-1, while inhibiting bKv1.4 current by mechanisms that occurred with distinctly different kinetics. bTREK-1 was also activated by the polyunsaturated cis fatty acid linoleic acid but not by the trans polyunsaturated fatty acid linolelaidic acid or saturated fatty acids. Eicosatetraynoic acid (ETYA), which blocks formation of active AA metabolites, failed to inhibit AA activation of bTREK-1, indicating that AA acts directly. Compared to activation of bTREK-1, inhibition of bKv1.4 by AA was rapid and accompanied by a pronounced acceleration of inactivation kinetics. Cis polyunsaturated fatty acids were much more effective than trans or saturated fatty acids at inhibiting bKv1.4. ETYA also effectively inhibited bKv1.4, but less potently than AA. bTREK-1 current was markedly increased by lysophospholipids including lysophosphatidyl choline (LPC) and lysophosphatidyl inositol (LPI). At concentrations from 1–5 µM, LPC produced a rapid, transient increase in bTREK-1 that peaked within one minute and then rapidly desensitized. The transient lysophospholipid-induced increases in bTREK-1 did not require the presence of ATP or GTP in the pipette solution. These results indicate that the activity of native leak and voltage-gated K+ channels are directly modulated in reciprocal fashion by AA and other cis unsaturated fatty acids. They also show that lysophospholipids enhance bTREK-1, but with a strikingly different temporal pattern. The modulation of native K+ channels by these agents differs from their effects on the same channels expressed in heterologous cells, highlighting the critical importance of auxiliary subunits and signaling. Finally, these results reveal that AZF cells express thousands of bTREK-1 K+ channels that lie dormant until activated by metabolites including phospholipase A2 (PLA2)-generated fatty acids and lysophospholipids. These metabolites may alter the electrical and secretory properties of AZF cells by modulating bTREK-1 and bKv1.4 K+ channels.

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.

A Bovine Adrenocortical Kv1.4 K+ Channel Whose Expression Is Potently Inhibited by ACTH

We have cloned a bovine adrenal cortical (bKv1.4) K+ channel cDNA whose expression is rapidly inhibited by adrenocorticotropic hormone (ACTH). The 4386-nucleotide cDNA is homologous to other voltage-gated, rapidly inactivating Kv1.4 channels, and includes a 1986-nucleotide coding region and large 5′- and 3′-untranslated regions. Bovine Kv1.4-specific mRNA from adrenal zona fasciculata (AZF) cells was rapidly and potently reduced by ACTH, with a t 1 2 of approximately 1 h and an IC50 of 1.2 pm. The membrane-permeable cAMP analog 8-pcpt-cAMP also reduced bKv1.4 mRNA expression with kinetics similar to that observed with ACTH. Reduction of bKv1.4 mRNA expression by ACTH and 8-pcpt-cAMP was only partially inhibited by the selective protein kinase A antagonist H-89. Consistent with their effect on bKv1.4 mRNA, ACTH and 8-pcpt-cAMP both dramatically reduced the expression of bKv1.4-associated A-type current measured over 72 h. These results demonstrate that bovine AZF cells synthesize a Kv1.4-type channel whose expression is inhibited at the pretranslational level by ACTH and 8-pcpt-cAMP by a mechanism that is partially dependent on the activation of protein kinase A. The rapid, potent reduction of bKv1.4 mRNA produced by ACTH and 8-pcpt-cAMP indicates that the expression of this K+ channel is under tonic inhibitory control of the hypothalamic-pituitary-adrenal axis. The basic electrical properties of AZF cells might be tightly regulated at the transcriptional level by the normal diurnal pattern of ACTH secretion, and altered during bouts of stress by the enhanced release of this pituitary peptide. Under conditions of prolonged stress or adrenal insufficiency, persistent ACTH-induced changes in the electrical properties of AZF cells could be coupled to parallel changes in cortisol secretion.

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.

ACTH Induces Cav3.2 Current and mRNA by cAMP-dependent and cAMP-independent Mechanisms

Bovine adrenal zona fasciculata (AZF) cells express Cav3.2 T-type Ca2+ channels that function pivotally in adrenocorticotropic hormone (ACTH)-stimulated cortisol secretion. The regulation of Cav3.2 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 Cav3.2 current. The increase in Cav3.2 current was preceded by an increase in corresponding CACNA1H mRNA. O-Nitrophenyl,sulfenyl-adrenocorticotropin, which produces a minimal increase in cAMP, also enhanced Cav3.2 current. cAMP analogs, including 8-bromoadenosine cAMP (600 μm) and 6-benzoyladenosine cAMP (300 μm) induced CACNA1H mRNA, but not Cav3.2 current. In contrast, 8-(4-chlorophenylthio) (8CPT)-cAMP (10–50 μm) enhanced CACNA1H mRNA and Cav3.2 current, whereas nonhydrolyzable Sp-8CPT-cAMP failed to increase either Cav3.2 current or mRNA. Metabolites of 8CPT-cAMP, including 8CPT-adenosine and 8CPT-adenine, increased Cav3.2 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 Cav3.2 current; Sp-8CPT-2′-O-methyl-cAMP increased neither Cav3.2 current nor mRNA. These results reveal an interesting dichotomy between ACTH and cAMP with regard to regulation of CACNA1H mRNA and Ca2+ current. Specifically, ACTH induces expression of CACNA1H mRNA and Cav3.2 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 Ca2+ current. Surprisingly, chlorophenylthio-cAMP analogs stimulate the expression of Cav3.2 current indirectly through metabolites. ACTH and the metabolites may induce Cav3.2 expression by the same, unidentified mechanism.

Nucleotide sequence of the complementary DNA for turkey growth hormone

Summary Near-full length complementary DNA (cDNA) clones encoding turkey growth hormone (GH) have been isolated from a pituitary library. The longer of the two turkey GH cDNA clones that were sequenced is 803 base pairs (bp) in length and contains 41 nucleotides of the 5′-untranslated region (UTR), an open reading frame of 648 bp that encodes a 25 amino acid leader polypeptide segment as well as a 191 amino acid mature turkey GH protein, and a 3′-UTR that is 92 bp long followed by a 22 bp poly A tract. Comparison of the turkey GH nucleotide sequence to that of other avian GH clones shows the coding region to be greater than 98% homologous while the homology to mammalian GH sequences is between 68 and 78%. Northern blot analysis showed an approximate 800 bp turkey GH processed mRNA transcript that hybridized to the turkey GH cDNA probe. A large up-regulation of turkey GH transcription occurred when intact cultured pituitaries were treated with 1 nM human growth hormone releasing hormone but only modest changes were observed when cultures were treated with thyroid releasing hormone or somatostatin.

Adenosine inhibits a non-inactivating K+ current in bovine adrenal cortical cells by activation of multiple P1 receptors.

1 Bovine adrenal zona fasciculata (AZF) cells express a non‐inactivating K+ current (IAC) that sets the resting potential while it is activated by intracellular ATP. In whole‐cell patch clamp recordings from bovine AZF cells, we found that adenosine selectively inhibited IAC by a maximum of 78·4 ± 4·6% (n= 8) with an IC50 of 71 nM. The non‐selective adenosine receptor agonist NECA effectively inhibited IAC by 79·3 ± 2·9% (n= 24) at a concentration of 100 nM. 2 Inhibition of IAC was mediated through multiple P1 adenosine receptor subtypes. The A1‐selective agonist CCPA (10 nM), the A2A‐selective agonist CGS 21680 (100 nM) and the A3‐selective agonist IB‐MECA (10 nM) inhibited IAC by 64·8 ± 8·4, 78·4 ± 4·6 and 69·3 ± 6·9%, respectively. 3 Specific adenosine receptor subtype antagonists including DPCPX (A1), ZM 241385 (A2A) and MRS 1191 (A3) effectively blocked inhibition of IAC by adenosine receptor‐selective agonists. 4 A mixture of the three adenosine receptor antagonists completely suppressed inhibition of IAC by adenosine, but failed to alter inhibition by external ATP which acts through a separate P2 nucleotide receptor. 5 Inhibition of IAC by adenosine or NECA was eliminated by substituting GDP‐β‐S for GTP in the pipette, or by replacing ATP with AMP‐PNP or UTP. 6 In addition to inhibiting IAC, adenosine (10 μM) depolarized AZF cells by 46·2 ± 5·8 mV (n= 6). 7 These results show that bovine AZF cells express at least three adenosine receptor subtypes (A1, A2A, A3), each of which is coupled to the inhibition of IAC K+ channels through a G‐protein‐dependent mechanism requiring ATP hydrolysis. Adenosine‐mediated inhibition of IAC is associated with membrane depolarization. Adenosine and other purines may co‐ordinate the stress‐induced secretion of corticosteroids and catecholamines from the adrenal gland.