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.     

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.     

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 IC₅₀ of 0.93 μM 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 24 h. At a maximally effective concentration of 50 μM, 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.     

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.     

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.     

Modulation of Native TREK-1 and Kv1.4 K<Superscript>+</Superscript> 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.     

sn-1,2 dioctanoylglycerol mimics the effects of angiotensin II on aldosterone production and potassium permeability in isolated bovine glomerulosa cells

In order to elucidate the possible role in glomerulosa cells of diacylglycerol released by angiotensin II we have studied the action of a synthetic diacylglycerol, sn-1,2-dioctanoylglycerol (DiC8), on aldosterone production and potassium permeability in bovine adrenal cells. DiC8 elicited an increase in 86Rb efflux from cells previously equilibrated with the isotope. The action of DiC8 on the rate coefficient for 86Rb efflux was similar to that previously described for angiotensin II (Am. J. Physiol. 254 (1988) E144-149), i.e. DiC8 induced an immediate increase in 86Rb efflux followed by a sustained decrease in potassium permeability. This DiC8 induced inhibition was observed even in the presence of depolarizing concentrations of potassium. The effect of DiC8 on aldosterone secretion from adrenal glomerulosa cells was measured using a perifusion system. DiC8 (300 microM) caused a significant increase of aldosterone production, comparable to that seen with angiotensin II (100 nM). These results indicate that DiC8 has similar effects to angiotensin II on both potassium permeability and steroidogenesis, which suggests that activation of protein kinase C is involved in the changes of ionic permeability induced by this hormone in bovine adrenal glomerulosa cells.     

Properties of ATP-dependent K(+) channels in adrenocortical cells

Bovine adrenocortical zona fasciculata (AZF)cells express a novel ATP-dependent K⁺-permeable channel(I_AC). Whole cell and single-channel recordings were used to characterize I_AC channels withrespect to ionic selectivity, conductance, and modulation bynucleotides, inorganic phosphates, and angiotensin II (ANG II). Inoutside-out patch recordings, the activity of unitaryI_AC channels is enhanced by ATP in the patchpipette. These channels were K⁺ selective with nomeasurable Na⁺ or Ca²⁺ conductance. Insymmetrical K⁺ solutions with physiological concentrationsof divalent cations (M²⁺), I_ACchannels were outwardly rectifying with outward and inward chordconductances of 94.5 and 27.0 pS, respectively. In the absence ofM²⁺, conductance was nearly ohmic. Hydrolysis-resistantnucleotides including AMP-PNP and NaUTP were more potent than MgATP asactivators of whole cell I_AC currents. Inorganicpolytriphosphate (PPP_i) dramatically enhancedI_AC activity. In current-clamp recordings, nucleotides and PPP_i produced resting potentials in AZFcells that correlated with their effectiveness in activatingI_AC. ANG II (10 nM) inhibited whole cellI_AC currents when patch pipettes contained 5 mMMgATP but was ineffective in the presence of 5 mM NaUTP and 1 mM MgATP.Inhibition by ANG II was not reduced by selective kinase antagonists.These results demonstrate that I_AC is adistinctive K⁺-selective channel whose activity isincreased by nucleotide triphosphates and PPP_i.Furthermore, they suggest a model for I_AC gatingthat is controlled through a cycle of ATP binding and hydrolysis.

     

ANG II-stimulated DNA synthesis is mediated by ANG II receptor-dependent Ca(2+)/PKC as well as EGF receptor-dependent PI3K/Akt/mTOR/p70S6K1 signal pathways in mouse embryonic stem cells

Effect of angiotensin II (ANG II) on mouse embryonic stem (ES) cell proliferation was examined. ANG II increased [(3)H] thymidine incorporation in a time- (>4 h) and dose- (>10(-9) M) dependent manner. The ANG II-induced increase in [(3)H] thymidine incorporation was blocked by inhibition of ANG II type 1 (AT(1)) receptor but not by ANG II type 2 (AT(2)) receptor, and AT(1) receptor was expressed. ANG II increased inositol phosphates formation and [Ca(2+)](i), and translocated PKC alpha, delta, and zeta to the membrane fraction. Consequently, the inhibition of PLC/PKC suppressed ANG II-induced increase in [(3)H] thymidine incorporation. The inhibition of EGF receptor kinase or tyrosine kinase prevented ANG II-induced increase in [(3)H] thymidine incorporation. ANG II phosphorylated EGF receptor and increased Akt, mTOR, and p70S6K1 phosphorylation blocked by AG 1478 (EGF receptor kinase blocker). ANG II-induced increase in [(3)H] thymidine incorporation was blocked by the inhibition of p44/42 MAPKs but not by p38 MAPK inhibition. Indeed, ANG II phosphorylated p44/42 MAPKs, which was prevented by the inhibition of the PKC and AT(1) receptor. ANG II increased c-fos, c-jun, and c-myc levels. ANG II also increased the protein levels of cyclin D1, cyclin E, cyclin-dependent kinase (CDK) 2, and CDK4 but decreased the p21(cip1/waf1) and p27(kip1), CDK inhibitory proteins. These proteins were blocked by the inhibition of AT(1) receptor, PLC/PKC, p44/42 MAPKs, EGF receptor, or tyrosine kinase. In conclusion, ANG II-stimulated DNA synthesis is mediated by ANG II receptor-dependent Ca(2+)/PKC and EGF receptor-dependent PI3K/Akt/mTOR/p70S6K1 signal pathways in mouse ES cells.     

Protein kinase A modulates PLC-dependent regulation and PIP2-sensitivity of K+ channels

Neurotransmitter and hormone regulation of cellular function can result from a concomitant stimulation of different signaling pathways. Signaling cascades are strongly regulated during disease and are often targeted by commonly used drugs. Crosstalk of different signaling pathways can have profound effects on the regulation of cell excitability. Members of all the three main structural families of potassium channels: inward-rectifiers, voltage-gated and 2-P domain, have been shown to be regulated by direct phosphorylation and Gq-coupled receptor activation. Here we test members of each of the three families, Kir3.1/Kir3.4, KCNQ1/KCNE1 and TREK-1 channels, all of which have been shown to be regulated directly by phosphatidylinositol bisphosphate (PIP2). The three channels are inhibited by activation of Gq-coupled receptors and are differentially regulated by protein kinase A (PKA). We show that Gq-coupled receptor regulation can be physiologically modulated directly through specific channel phosphorylation sites. Our results suggest that PKA phosphorylation of these channels affects Gq-coupled receptor inhibition through modulation of the channel sensitivity to PIP2.     

Differential inhibition of the TRPM8 ion channel by Gαq and Gα11

Cold temperature is encoded by the cold-sensitive ion channel TRPM8 in somatosensory neurons. It has been unclear how TRPM8 is modulated so that it can mediate distinct type of cold signaling. We have recently reported that activated Gα_q directly inhibits TRPM8 after activation of Gq-coupled receptors. Here, we further show that activation of the muscarinic receptor M1R, which is known to inhibit M currents through PLCβ-mediated hydrolysis of PtdIns(4,5)P₂, similarly inhibited TRPM8 potently, but inhibition was not prevented by the PLC inhibitor U73122. Interestingly, although Gα_q and Gα₁₁ are indistinguishable in activating PLCβ and hydrolysing PtdIns(4,5)P₂, activated Gα₁₁ inhibited TRPM8 to a lesser extent than activated Gα_q. The differential TRPM8 inhibition is determined by a specific residue E197 on Gα₁₁, because mutating this residue to the corresponding residue on Gα_q restored TRPM8 inhibition to a similar degree as mediated by Gα_q. These results reinforce the idea that activated Gα_q directly inhibits TRPM8 independently from PtdIns(4,5)P₂ hydrolysis-mediated inhibition of TRPM8.     

Receptor-mediated inhibition of G protein-coupled inwardly rectifying potassium channels involves G(alpha)q family subunits, phospholipase C, and a readily diffusible messenger

G protein-coupled inwardly rectifying K+ (GIRK) channels can be activated or inhibited by distinct classes of receptor (G(alpha)i/o- and G(alpha)q-coupled), providing dynamic regulation of cellular excitability. Receptor-mediated activation involves direct effects of G(beta)gamma subunits on GIRK channels, but mechanisms involved in GIRK channel inhibition have not been fully elucidated. An HEK293 cell line that stably expresses GIRK1/4 channels was used to test G protein mechanisms that mediate GIRK channel inhibition. In cells transiently or stably cotransfected with 5-HT1A (G(alpha)i/o-coupled) and TRH-R1 (G(alpha)q-coupled) receptors, 5-HT (5-hydroxytryptamine; serotonin) enhanced GIRK channel currents, whereas thyrotropin-releasing hormone (TRH) inhibited both basal and 5-HT-activated GIRK channel currents. Inhibition of GIRK channel currents by TRH primarily involved signaling by G(alpha)q family subunits, rather than G(beta)gamma dimers: GIRK channel current inhibition was diminished by Pasteurella multocida toxin, mimicked by constitutively active members of the G(alpha)q family, and reduced by minigene constructs that disrupt G(alpha)q signaling, but was completely preserved in cells expressing constructs that interfere with signaling by G(beta)gamma subunits. Inhibition of GIRK channel currents by TRH and constitutively active G(alpha)q was reduced by, an inhibitor of phospholipase C (PLC). Moreover, TRH- R1-mediated GIRK channel inhibition was diminished by minigene constructs that reduce membrane levels of the PLC substrate phosphatidylinositol bisphosphate, further implicating PLC. However, we found no evidence for involvement of protein kinase C, inositol trisphosphate, or intracellular calcium. Although these downstream signaling intermediaries did not contribute to receptor-mediated GIRK channel inhibition, bath application of TRH decreased GIRK channel activity in cell-attached patches. Together, these data indicate that receptor-mediated inhibition of GIRK channels involves PLC activation by G(alpha) subunits of the G(alpha)q family and suggest that inhibition may be communicated at a distance to GIRK channels via unbinding and diffusion of phosphatidylinositol bisphosphate away from the channel.     

ANG II AT1 and AT2 receptors both inhibit bFGF-induced proliferation of bovine adrenocortical cells

Angiotensin II(ANG II) has long been known for its pressor and growth-promotingeffects, which are both mediated by theAT₁ receptor. By contrast, theAT₂ receptor has recently beenreported to mediate inhibition of proliferation through as yetundefined mechanisms. We report here that in bovine adrenal fasciculata cells ANG II by itself does not affect growth but inhibits basic fibroblast growth factor (bFGF)-induced DNA synthesis and blocks thecells in G₁ phase. Consistent withthis, ANG II inhibits cyclin D₁ expression and cyclinD₁-associated kinase activity. Theantimitogenic effect of ANG II is partly mimicked by theAT₂-selective agonist CGP-42112.It is also blocked partly and in an additive fashion by theAT₁- andAT₂-selective antagonists losartanand PD-123319, indicating the contribution of both receptor subtypes tothis response. AT₁-dependentantiproliferation is selectively blocked by the cyclooxygenaseinhibitor indomethacin and restored by prostaglandin E₂, whereasAT₂-receptor-mediated inhibitionof growth is suppressed by the tyrosine phosphatase inhibitorsorthovanadate and bpV(pic). Both pathways are, however,pertussis toxin sensitive. We hypothesize that, in fasciculatacells, the AT₁ receptor inhibitsbFGF-induced proliferation by stimulating prostaglandin synthesis,whereas the AT₂ receptor mediatesits effect through a pathway that requires protein tyrosine phosphataseactivation.

     

Distinct subunit contributions to the activation of M-type potassium channels by PI(4,5)P2

Low-threshold voltage-gated M-type potassium channels (M channels) are tetraheteromers, commonly of two Kv7.2 and two Kv7.3 subunits. Though gated by voltage, the channels have an absolute requirement for binding of the membrane phospholipid phosphatidylinositol-4,5-bisphosphate (PI(4,5)P(2)) to open. We have investigated the quantitative relation between the concentration of a water-soluble PI(4,5)P(2) analog, dioctanoyl-PI(4,5)P(2) (DiC(8)-PI(4,5)P(2)), and channel open probability (P(open)) by fast application of increasing concentrations of DiC(8)-PI(4,5)P(2) to the inside face of membrane patches excised from Chinese hamster ovary cells expressing M channels as heteromeric Kv7.2/7.3 subunits. The rationale for the experiments is that this will mimic the effect of changes in membrane PI(4,5)P(2) concentration. Single-channel conductances from channel current-voltage relations in cell-attached mode were 9.2 ± 0.1 pS with a 2.5-mM pipette [K(+)]. Plots of P(open) against DiC(8)-PI(4,5)P(2) concentration were best fitted using a two-component concentration-P(open) relationship with high and low affinity, half-maximal effective concentration (EC(50)) values of 1.3 ± 0.14 and 75.5 ± 2.5 μM, respectively, and Hill slopes of 1.4 ± 0.06. In contrast, homomeric channels from cells expressing only Kv7.2 or Kv7.3 constructs yielded single-component curves with EC(50) values of 76.2 ± 19.9 or 3.6 ± 1.0 μM, respectively. When wild-type (WT) Kv7.2 was coexpressed with a mutated Kv7.3 subunit with >100-fold reduced sensitivity to PI(4,5)P(2), the high-affinity component of the activation curve was lost. Fitting the data for WT and mutant channels to an activation mechanism with independent PI(4,5)P(2) binding to two Kv7.2 and two Kv7.3 subunits suggests that the two components of the M-channel activation curve correspond to the interaction of PI(4,5)P(2) with the Kv7.3 and Kv7.2 subunits, respectively, that channels can open when only the two Kv7.3 subunits have bound DiC(8)-PI(4,5)P(2), and that maximum channel opening requires binding to all four subunits.     

Inhibition of bovine brain calmodulin-dependent cGMP phosphodiesterase by peptide and non-peptide angiotensin receptor ligands.

Inhibition of a purified 60 KDa bovine brain calmodulin-dependent cGMP phosphodiesterase (PDE) was investigated for a number of peptides and non-peptides which are known to bind to angiotensin (ANG) receptors. The peptide antagonists sarilesin and sarmesin had KI = 120 and greater than 200 microM respectively, and the peptide agonists ANG II and ANG III had KI = greater than 200 and 45 microM respectively. Non-peptide ANG receptor antagonists related to DuP 753 exhibited KI values in the same range. For both peptide and non-peptide antagonists, inhibitory activities in the PDE assay reflected the order of antagonist potencies at ANG receptors in the rat isolated uterus assay and binding affinities at ANG receptors in rat uterine membranes, suggesting that molecular recognition factors are similar for both ANG receptors and cGMP PDE. The vasodilatory and blood pressure lowering effects of compounds related to DuP 753 may be due in part to inhibition of cGMP PDE. The differential effects of ANG II and ANG III at target tissues may relate in part to the marked differences in cGMP PDE inhibition associated with these two peptides hormones.     

Exposure of luminal membranes of LLC-PK1 cells to ANG II induces dimerization of AT1/AT2 receptors to activate SERCA and to promote Ca2+ mobilization

ANG II is secreted into the lumens of proximal tubules where it is also synthesized, thus increasing the local concentration of the peptide to levels of potential physiological relevance. In the present work, we studied the effect of ANG II via the luminal membranes of LLC-PK(1) cells on Ca(2+)-ATPase of the sarco(endo)plasmic reticulum (SERCA) and plasma membrane (PMCA). ANG II (at concentrations found in the lumen) stimulated rapid (30 s) and persistent (30 min) SERCA activity by more than 100% and increased Ca(2+) mobilization. Pretreatment with ANG II for 30 min enhanced the ANG II-induced Ca(2+) spark, demonstrating a positively self-sustained stimulus of Ca(2+) mobilization by ANG II. ANG II in the medium facing the luminal side of the cells decreased with time with no formation of metabolites, indicating peptide internalization. ANG II increased heterodimerization of AT(1) and AT(2) receptors by 140%, and either losartan or PD123319 completely blocked the stimulation of SERCA by ANG II. Using the PLC inhibitor U73122, PMA, and calphostin C, it was possible to demonstrate the involvement of a PLC→DAG(PMA)→PKC pathway in the stimulation of SERCA by ANG II with no effect on PMCA. We conclude that ANG II triggers SERCA activation via the luminal membrane, increasing the Ca(2+) stock in the reticulum to ensure a more efficient subsequent mobilization of Ca(2+). This first report on the regulation of SERCA activity by ANG II shows a new mechanism for Ca(2+) homeostasis in renal cells and also for regulation of Ca(2+)-modulated fluid reabsorption in proximal tubules.     

Angiotensin receptor subtype AT(1) mediates alveolar epithelial cell apoptosis in response to ANG II

Previous work from this laboratory demonstrated induction of apoptosis in lung alveolar epithelial cells (AEC) by purified angiotensin II (ANG II) and expression of mRNAs for both ANG II receptor subtypes AT(1) and AT(2) (Wang R, Zagariya A, Ibarra-Sunga O, Gidea C, Ang E, Deshmukh S, Chaudhary G, Baraboutis J, Filippatos G, and Uhal BD. Am J Physiol Lung Cell Mol Physiol 276: L885-L889, 1999.). The present study was designed to determine the ANG II receptor subtype mediating AEC apoptosis in response to ANG II. Apoptosis was induced with purified ANG II applied to the human lung AEC-derived carcinoma cell line A549 or to primary AEC isolated from Wistar rats. In both cell types, the AT(1)-selective receptor antagonists L-158809 or losartan inhibited ANG II-induced apoptosis by 90% at concentrations of 10(-8) M and 10(-7) M, respectively. The inhibition was concentration dependent with IC(50) of 10(-12) M and 10(-11) M on the primary rat AEC. In contrast, the AT(2)-selective antagonists PD-123319 or PD-126055 could not block ANG II-induced apoptosis in either cell type. In primary rat AEC, apoptosis in response to ANG II was blunted in a dose-dependent manner by the protein kinase C inhibitor chelerythrine but not by the tyrosine phosphatase inhibitor sodium orthovanadate. Together, these data indicate that AEC apoptosis in response to ANG II is mediated by receptor subtype AT(1), despite the expression of mRNAs for both AT(1) and AT(2).     

The protein kinase C inhibitor, bisindolylmaleimide (I), inhibits voltage-dependent K+ channels in coronary arterial smooth muscle cells

We examined the effects of the protein kinase C (PKC) inhibitor, bisindolylmaleimide (BIM) (I), on voltage-dependent K+ (K(V)) channels in rabbit coronary arterial smooth muscle cells using whole-cell patch clamp technique. BIM (I) reversibly and dose-dependently inhibited the K(V) currents with an apparent Kd value of 0.27 microM. The inhibition of the K(V) current by BIM (I) was highly voltage-dependent between -30 and +10 mV (voltage range of channel activation), and the additive inhibition of the K(V) current by BIM (I) was voltage-dependence in the full activation voltage range. The rate constants of association and dissociation for BIM (I) were 18.4 microM(-1) s(-1) and 4.7 s(-1), respectively. BIM (I) had no effect on the steady-state activation and inactivation of K(V) channels. BIM (I) caused use-dependent inhibition of K(V) current, which was consistent with the slow recovery from inactivation in the presence of BIM (I) (recovery time constants were 856.95 +/- 282.6 ms for control, and 1806.38 +/- 110.0 ms for 300 nM BIM (I)). ATP-sensitive K+ (K(ATP)), inward rectifier K+ (K(IR)), Ca2+-activated K+ (BK(Ca)) channels, which regulate the membrane potential and arterial tone, were not affected by BIM (I). The PKC inhibitor, chelerythrine, and protein kinase A (PKA) inhibitor, PKA-IP, had little effect on the K(V) current and did not significantly alter the inhibitory effects of BIM (I) on the K(V) current. These results suggest that BIM (I) inhibits K(V) channels in a phosphorylation-independent, and voltage-, time- and use-dependent manner.     

CRAC channel is inhibited by neomycin in a Ptdlns(4,5)P2‐independent manner

Depletion of intracellular Ca²⁺ stores evokes store‐operated Ca²⁺ entry through the Ca²⁺ release‐activated Ca²⁺ (CRAC) channels. In this study, we found that the store‐operated Ca²⁺ entry was inhibited by neomycin, an aminoglycoside that strongly binds phosphatidylinositol 4,5‐bisphosphate (PtdIns(4,5)P2). Patch clamp recordings revealed that neomycin blocked the CRAC currents reconstituted by co‐expression of Orai1 and Stim1 in HEK293 cells. Using a rapamycin‐inducible PtdIns(4,5)P2‐specific phosphatase (Inp54p) system to manipulate the PtdIns(4,5)P2 in the plasma membrane, we found that the CRAC current was not altered by PtdIns(4,5)P2 depletion. This result suggests that PtdIns(4,5)P2 is not required for CRAC channel activity, and thereby, neomycin inhibits CRAC channels in a manner that is independent of neomycin–PtdIns(4,5)P2 binding. Copyright © 2015 John Wiley & Sons, Ltd.