Trichostatin A blocks aldosterone-induced Na+ transport and control of serum- and glucocorticoid-inducible kinase 1 in cortical collecting duct cells.

BACKGROUND AND PURPOSE: Aldosterone stimulates epithelial Na+ channel (ENaC)-dependent Na+ retention in the cortical collecting duct (CCD) of the kidney by activating mineralocorticoid receptors that promote expression of serum and glucocorticoid-inducible kinase 1 (SGK1). This response is critical to BP homeostasis. It has previously been suggested that inhibiting lysine deacetylases (KDACs) can post-transcriptionally disrupt this response by promoting acetylation of the mineralocorticoid receptor. The present study critically evaluates this hypothesis. EXPERIMENTAL APPROACH: Electrometric and molecular methods were used to define the effects of a pan-KDAC inhibitor, trichostatin A, on the responses to a physiologically relevant concentration of aldosterone (3 nM) in murine mCCDcl1 cells. KEY RESULTS: Aldosterone augmented ENaC-induced Na+ absorption and increased SGK1 activity and abundance, as expected. In the presence of trichostatin A, these responses were suppressed. Trichostatin A-induced inhibition of KDAC was confirmed by increased acetylation of histone H3, H4, and α-tubulin. Trichostatin A did not block the electrometric response to insulin, a hormone that activates SGK1 independently of increased transcription, indicating that trichostatin A has no direct effect upon the SGK1/ENaC pathway. CONCLUSIONS AND IMPLICATIONS: Inhibition of lysine de-acetylation suppresses aldosterone-dependent control over the SGK1-ENaC pathway but does not perturb post-transcriptional signalling, providing a physiological basis for the anti-hypertensive action of KDAC inhibition seen in vivo.

Dexamethasone and insulin activate serum and glucocorticoid-inducible kinase 1 (SGK1) via different molecular mechanisms in cortical collecting duct cells.

Serum and glucocorticoid-inducible kinase 1 (SGK1) is a protein kinase that contributes to the hormonal control of renal Na(+) retention by regulating the abundance of epithelial Na(+) channels (ENaC) at the apical surface of the principal cells of the cortical collecting duct (CCD). Although glucocorticoids and insulin stimulate Na(+) transport by activating SGK1, the responses follow different time courses suggesting that these hormones act by different mechanisms. We therefore explored the signaling pathways that allow dexamethasone and insulin to stimulate Na(+) transport in mouse CCD cells (mpkCCDcl4). Dexamethasone evoked a progressive augmentation of electrogenic Na(+) transport that became apparent after ~45 min latency and was associated with increases in SGK1 activity and abundance and with increased expression of SGK1 mRNA Although the catalytic activity of SGK1 is maintained by phosphatidylinositol-OH-3-kinase (PI3K), dexamethasone had no effect upon PI3K activity. Insulin also stimulated Na(+) transport but this response occurred with no discernible latency. Moreover, although insulin also activated SGK1, it had no effect upon SGK1 protein or mRNA abundance. Insulin did, however, evoke a clear increase in cellular PI3K activity. Our data are consistent with earlier work, which shows that glucocorticoids regulate Na(+) retention by inducing sgk1 gene expression, and also establish that this occurs independently of increased PI3K activity. Insulin, on the other hand, stimulates Na(+) transport via a mechanism independent of sgk1 gene expression that involves PI3K activation. Although both hormones act via SGK1, our data show that they activate this kinase by distinct physiological mechanisms.

Depolarization of sperm membrane potential is a common feature of men with subfertility and is associated with low fertilization rate at IVF.

STUDY QUESTION: Are significant abnormalities in outward (K(+)) conductance and resting membrane potential (Vm) present in the spermatozoa of patients undertaking IVF and ICSI and if so, what is their functional effect on fertilization success? SUMMARY ANSWER: Negligible outward conductance (≈5% of patients) or an enhanced inward conductance (≈4% of patients), both of which caused depolarization of Vm, were associated with a low rate of fertilization following IVF. WHAT IS KNOWN ALREADY: Sperm-specific potassium channel knockout mice are infertile with defects in sperm function, suggesting that these channels are essential for fertility. These observations suggest that malfunction of K(+) channels in human spermatozoa might contribute significantly to the occurrence of subfertility in men. However, remarkably little is known of the nature of K(+) channels in human spermatozoa or the incidence and functional consequences of K(+) channel defects. STUDY DESIGN, SIZE AND DURATION: Spermatozoa were obtained from healthy volunteer research donors and subfertile IVF and ICSI patients attending a hospital assisted reproductive techniques clinic between May 2013 and December 2015. In total, 40 IVF patients, 41 ICSI patients and 26 normozoospermic donors took part in the study. PARTICIPANTS/MATERIALS, SETTING, METHODS: Samples were examined using electrophysiology (whole-cell patch clamping). Where abnormal electrophysiological characteristics were identified, spermatozoa were further examined for Ca(2+) influx induced by progesterone and penetration into viscous media if sufficient sample was available. Full exome sequencing was performed to specifically evaluate potassium calcium-activated channel subfamily M α 1 (KCNMA1), potassium calcium-activated channel subfamily U member 1 (KCNU1) and leucine-rich repeat containing 52 (LRRC52) genes and others associated with K(+) signalling. In IVF patients, comparison with fertilization rates was done to assess the functional significance of the electrophysiological abnormalities. MAIN RESULTS AND THE ROLE OF CHANCE: Patch clamp electrophysiology was used to assess outward (K(+)) conductance and resting membrane potential (Vm) and signalling/motility assays were used to assess functional characteristics of sperm from IVF and ICSI patient samples. The mean Vm and outward membrane conductance in sperm from IVF and ICSI patients were not significantly different from those of control (donor) sperm prepared under the same conditions, but variation between individuals was significantly greater (P< 0.02) with a large number of outliers (>25%). In particular, in ≈10% of patients (7/81), we observed either a negligible outward conductance (4 patients) or an enhanced inward current (3 patients), both of which caused depolarization of Vm. Analysis of clinical data from the IVF patients showed significant association of depolarized Vm (≥0 mV) with low fertilization rate (P= 0.012). Spermatozoa with electrophysiological abnormities (conductance and Vm) responded normally to progesterone with elevation of [Ca(2+)]i and penetration of viscous medium, indicating retention of cation channel of sperm (CatSper) channel function. LIMITATIONS, REASONS FOR CAUTION: For practical, technical, ethical and logistical reasons, we could not obtain sufficient additional semen samples from men with conductance abnormalities to establish the cause of the conductance defects. Full exome sequencing was only available in two men with conductance defects. WIDER IMPLICATIONS OF THE FINDINGS: These data add significantly to the understanding of the role of ion channels in human sperm function and its impact on male fertility. Impaired potassium channel conductance (Gm) and/or Vm regulation is both common and complex in human spermatozoa and importantly is associated with impaired fertilization capacity when the Vm of cells is completely depolarized. STUDY FUNDING/COMPETING INTERESTS: The majority of the data were obtained using funding from MRC project grants (#MR/K013343/1, MR/012492/1). Additional funding was provided by NHS Tayside, TENOVUS, Chief Scientist Office NRS Fellowship and University of Abertay. The authors declare that there is no conflict of interest. TRIAL REGISTRATION NUMBER: Not applicable.

Specific loss of CatSper function is sufficient to compromise fertilizing capacity of human spermatozoa.

STUDY QUESTION: Are significant abnormalities of CatSper function present in IVF patients with normal sperm concentration and motility and if so what is their functional significance for fertilization success? SUMMARY ANSWER: Sperm with a near absence of CatSper current failed to respond to activation of CatSper by progesterone and there was fertilization failure at IVF. WHAT IS KNOWN ALREADY: In human spermatozoa, Ca(2+) influx induced by progesterone is mediated by CatSper, a sperm-specific Ca(2+) channel. A suboptimal Ca(2+) influx is significantly associated with, and more prevalent in, men with abnormal semen parameters, and is associated with reduced fertilizing capacity. However, abnormalities in CatSper current can only be assessed directly using electrophysiology. There is only one report of a CatSper-deficient man who showed no progesterone potentiated CatSper current. A CatSper 2 genetic abnormality was present but there was no information on the [Ca(2+)]i response to CatSper activation by progesterone. Additionally, the semen samples had indicating significant abnormalities (oligoasthenoteratozoospermia) multiple suboptimal functional responses in the spermatozoon. As such it cannot be concluded that impaired CatSper function alone causes infertility or that CatSper blockade is a potential safe target for contraception. STUDY DESIGN, SIZE, DURATION: Spermatozoa were obtained from donors and subfertile IVF patients attending a hospital assisted reproductive techniques clinic between January 2013 and December 2014. In total 134 IVF patients, 28 normozoospermic donors and 10 patients recalled due to a history of failed/low fertilization at IVF took part in the study. PARTICIPANTS/MATERIALS, SETTING, METHODS: Samples were primarily screened using the Ca(2+) influx induced by progesterone and, if cell number was sufficient, samples were also assessed by hyperactivation and penetration into viscous media. A defective Ca(2+) response to progesterone was defined using the 99% confidence interval from the distribution of response amplitudes in normozoospermic donors. Samples showing a defective Ca(2+) response were further examined in order to characterize the potential CatSper abnormalities. In men where there was a consistent and robust failure of calcium signalling, a direct assessment of CatSper function was performed using electrophysiology (patch clamping), and a blood sample was obtained for genetic analysis. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 101/102 (99%) IVF patients and 22/23 (96%) donors exhibited a normal Ca(2+) response. The mean (± SD) normalized peak response did not differ between donors and IVF patients (2.57 ± 0.68 [n = 34 ejaculates from 23 different donors] versus 2.66 ± 0.68 [n = 102 IVF patients], P = 0.63). In recall patients, 9/10 (90%) showed a normal Ca(2+) response. Three men were initially identified with a defective Ca(2+) influx. However, only one (Patient 1) had a defective response in repeat semen samples. Electrophysiology experiments on sperm from Patient 1 showed a near absence of CatSper current and exon screening demonstrated no mutations in the coding regions of the CatSper complex. There was no increase in penetration of viscous media when the spermatozoa were stimulated with progesterone and importantly there was failed fertilization at IVF. LIMITATIONS, REASONS FOR CAUTION: A key limitation relates to working with a specific functional parameter (Ca(2+) influx induced by progesterone) in fresh sperm samples from donors and patients that have limited viability. Therefore, for practical, technical and logistical reasons, some men (∼ 22% of IVF patients) could not be screened. As such the incidence of significant Ca(2+) abnormalities induced by progesterone may be higher than the ∼ 1% observed here. Additionally, we used a strict definition of a defective Ca(2+) influx such that only substantial abnormalities were selected for further study. Furthermore, electrophysiology was only performed on one patient with a robust and repeatable defective calcium response. This man had negligible CatSper current but more subtle abnormalities (e.g. currents present but significantly smaller) may have been present in men with either normal or below normal Ca(2+) influx. WIDER IMPLICATIONS OF THE FINDINGS: These data add significantly to the understanding of the role of CatSper in human sperm function and its impact on male fertility. Remarkably, these findings provide the first direct evidence that CatSper is a suitable and specific target for human male contraception.

The phosphorylation of endogenous Nedd4-2 In Na(+)-absorbing human airway epithelial cells.

Neural precursor cell expressed, developmentally down-regulated protein 4-2 (Nedd4-2) mediates the internalisation / degradation of epithelial Na(+) channel subunits (α-, ß- and γ-ENaC). Serum / glucocorticoid inducible kinase 1 (SGK1) and protein kinase A (PKA) both appear to inhibit this process by phosphorylating Nedd4-2-Ser(221), -Ser(327) and -Thr(246). This Nedd4-2 inactivation process is thought to be central to the hormonal control of Na(+) absorption. The present study of H441 human airway epithelial cells therefore explores the effects of SGK1 and / or PKA upon the phosphorylation / abundance of endogenous Nedd4-2; the surface expression of ENaC subunits, and electrogenic Na(+) transport. Effects on Nedd4-2 phosphorylation/abundance and the surface expression of ENaC were monitored by western analysis, whilst Na(+) absorption was quantified electrometrically. Acutely (20min) activating PKA in glucocorticoid-deprived (24h) cells increased the abundance of Ser(221)-phosphorylated, Ser(327)-phosphorylated and total Nedd4-2 without altering the abundance of Thr(246)-phosphorylated Nedd4-2. Activating PKA under these conditions did not cause a co-ordinated increase in the surface abundance of α-, ß- and γ-ENaC and had only a very small effect upon electrogenic Na(+) absorption. Activating PKA (20min) in glucocorticoid-treated (0.2µM dexamethasone, 24h) cells, on the other hand, increased the abundance of Ser(221)-, Ser(327)- and Thr(246)-phosphorylated and total Nedd4-2; increased the surface abundance of α-, ß- and γ-ENaC and evoked a clear stimulation of Na(+) transport. Chronic glucocorticoid stimulation therefore appears to allow cAMP-dependent control of Na(+) absorption by facilitating the effects of PKA upon the Nedd4-2 and ENaC subunits.

p,p'-DDE activates CatSper and compromises human sperm function at environmentally relevant concentrations.

STUDY QUESTION: Is the environmental endocrine disruptor p,p'-dichlorodiphenyldichloroethylene (p,p'-DDE) able to induce non-genomic changes in human sperm and consequently affect functional sperm parameters? SUMMARY ANSWER: p,p'-DDE promoted Ca(2+) flux into human sperm by activating CatSper channels even at doses found in human reproductive fluids, ultimately compromising sperm parameters important for fertilization. WHAT IS KNOWN ALREADY: p,p'-DDE may promote non-genomic actions and interact directly with pre-existing signaling pathways, as already observed in other cell types. However, although often found in both male and female reproductive fluids, its effects on human spermatozoa function are not known. STUDY DESIGN, SIZE, DURATION: Normozoospermic sperm samples from healthy individuals were included in this study. Samples were exposed to several p,p'-DDE concentrations for 3 days at 37°C and 5% CO2 in vitro to mimic the putative continuous exposure to this toxicant in the female reproductive tract in vivo. Shorter p,p'-DDE incubation periods were also performed in order to monitor sperm rapid Ca(2+) responses. All experiments were repeated on a minimum of five sperm samples from different individuals. PARTICIPANTS/MATERIALS, SETTING, METHODS: All healthy individuals were recruited at the Biosciences School, University of Birmingham, the Medical Research Institute, University of Dundee and in the Human Reproduction Service at University Hospitals of Coimbra. Intracellular Ca(2+) concentration ([Ca(2+)]i) was monitored by imaging single spermatozoa loaded with Oregon Green BAPTA-1AM and further whole-cell patch-clamp recordings were performed to validate our results. Sperm viability and acrosomal integrity were assessed using the LIVE/DEAD sperm vitality kit and the acrosomal content marker PSA-FITC, respectively. MAIN RESULTS AND THE ROLE OF CHANCE: p,p'-DDE rapidly increased [Ca(2+)]i (P < 0.05) even at extremely low doses (1 pM and 1 nM), with magnitudes of response up to 200%, without affecting sperm viability, except after 3 days of continuous exposure to the highest concentration tested (P < 0.05). Furthermore, experiments performed in a low Ca(2+) medium demonstrated that extracellular Ca(2+) influx was responsible for this Ca(2+) increase (P < 0.01). Mibefradil and NNC 55-0396, both inhibitors of the sperm-specific CatSper channel, reversed the p,p'-DDE-induced [Ca(2+)]i rise, suggesting the participation of CatSper in this process (P < 0.05). In fact, whole-cell patch-clamp recordings confirmed CatSper as a target of p,p'-DDE action by monitoring an increase in CatSper currents of >100% (P < 0.01). Finally, acrosomal integrity was adversely affected after 2 days of exposure to p,p'-DDE concentrations, suggesting that [Ca(2+)]i rise may cause premature acrosome reaction (P < 0.05). LIMITATIONS, REASONS FOR CAUTION: This is an in vitro study, and caution must be taken when extrapolating the results. WIDER IMPLICATIONS OF THE FINDINGS: A novel non-genomic p,p'-DDE mechanism specific to sperm is shown in this study. p,p'-DDE was able to induce [Ca(2+)]i rise in human sperm through the opening of CatSper consequently compromising male fertility. The promiscuous nature of CatSper activation may predispose human sperm to the action of some persistent endocrine disruptors. STUDY FUNDING/COMPETING INTEREST(S): The study was supported by both the Portuguese National Science Foundation (FCT; PEst-C/SAU/LA0001/2011) and the UK Wellcome Trust (Grant #86470). SM was supported by the Infertility Research Trust. RST is a recipient of a PhD fellowship from FCT (SFRH/BD/46002/2008). None of the authors has any conflict of interest to declare.

Increased vapor pressure deficit due to higher temperature leads to greater transpiration and faster mortality during drought for tree seedlings common to the forest-grassland ecotone.

Tree species growing along the forest-grassland ecotone are near the moisture limit of their range. Small increases in temperature can increase vapor pressure deficit (VPD) which may increase tree water use and potentially hasten mortality during severe drought. We tested a 40% increase in VPD due to an increase in growing temperature from 30 to 33°C (constant dewpoint 21°C) on seedlings of 10 tree species common to the forest-grassland ecotone in the southern Great Plains, USA. Measurement at 33 vs 30°C during reciprocal leaf gas exchange measurements, that is, measurement of all seedlings at both growing temperatures, increased transpiration for seedlings grown at 30°C by 40% and 20% for seedlings grown at 33°C. Higher initial transpiration of seedlings in the 33°C growing temperature treatment resulted in more negative xylem water potentials and fewer days until transpiration decreased after watering was withheld. The seedlings grown at 33°C died 13% (average 2 d) sooner than seedlings grown at 30°C during terminal drought. If temperature and severity of droughts increase in the future, the forest-grassland ecotone could shift because low seedling survival rate may not sufficiently support forest regeneration and migration.

2-APB-potentiated channels amplify CatSper-induced Ca(2+) signals in human sperm.

Ca2+i signalling is pivotal to sperm function. Progesterone, the best-characterized agonist of human sperm Ca2+i signalling, stimulates a biphasic [Ca2+]i rise, comprising a transient and subsequent sustained phase. In accordance with recent reports that progesterone directly activates CatSper, the [Ca2+]i transient was detectable in the anterior flagellum (where CatSper is expressed) 1-2 s before responses in the head and neck. Pre-treatment with 5 µM 2-APB (2-aminoethoxydiphenyl borate), which enhances activity of store-operated channel proteins (Orai) by facilitating interaction with their activator [STIM (stromal interaction molecule)] 'amplified' progesterone-induced [Ca2+]i transients at the sperm neck/midpiece without modifying kinetics. The flagellar [Ca2+]i response was unchanged. 2-APB (5 µM) also enhanced the sustained response in the midpiece, possibly reflecting mitochondrial Ca2+ accumulation downstream of the potentiated [Ca2+]i transient. Pre-treatment with 50-100 µM 2-APB failed to potentiate the transient and suppressed sustained [Ca2+]i elevation. When applied during the [Ca2+]i plateau, 50-100 µM 2-APB caused a transient fall in [Ca2+]i, which then recovered despite the continued presence of 2-APB. Loperamide (a chemically different store-operated channel agonist) enhanced the progesterone-induced [Ca2+]i signal and potentiated progesterone-induced hyperactivated motility. Neither 2-APB nor loperamide raised pHi (which would activate CatSper) and both compounds inhibited CatSper currents. STIM and Orai were detected and localized primarily to the neck/midpiece and acrosome where Ca2+ stores are present and the effects of 2-APB are focussed, but store-operated currents could not be detected in human sperm. We propose that 2-APB-sensitive channels amplify [Ca2+]i elevation induced by progesterone (and other CatSper agonists), amplifying, propagating and providing spatio-temporal complexity in [Ca2+]i signals of human sperm.

Epithelial Na⁺ channel activity in human airway epithelial cells: the role of serum and glucocorticoid-inducible kinase 1.

BACKGROUND AND PURPOSE: Glucocorticoids appear to control Na⁺ absorption in pulmonary epithelial cells via a mechanism dependent upon serum and glucocorticoid-inducible kinase 1 (SGK1), a kinase that allows control over the surface abundance of epithelial Na⁺ channel subunits (α-, ß- and γ-ENaC). However, not all data support this model and the present study re-evaluates this hypothesis in order to clarify the mechanism that allows glucocorticoids to control ENaC activity. EXPERIMENTAL APPROACH: Electrophysiological studies explored the effects of agents that suppress SGK1 activity upon glucocorticoid-induced ENaC activity in H441 human airway epithelial cells, whilst analyses of extracted proteins explored the associated changes to the activities of endogenous protein kinase substrates and the overall/surface expression of ENaC subunits. KEY RESULTS: Although dexamethasone-induced (24 h) ENaC activity was dependent upon SGK1, prolonged exposure to this glucocorticoid did not cause sustained activation of this kinase and neither did it induce a coordinated increase in the surface abundance of α-, ß- and γ-ENaC. Brief (3 h) exposure to dexamethasone, on the other hand, did not evoke Na⁺ current but did activate SGK1 and cause SGK1-dependent increases in the surface abundance of α-, ß- and γ-ENaC. CONCLUSIONS AND IMPLICATIONS: Although glucocorticoids activated SGK1 and increased the surface abundance of α-, ß- and γ-ENaC, these responses were transient and could not account for the sustained activation of ENaC. The maintenance of ENaC activity did, however, depend upon SGK1 and this protein kinase must therefore play an important but permissive role in glucocorticoid-induced ENaC activation.

Novel applications of PCR through the use of DNA substrates.

The ability to amplify genetic material using PCR has transformed the field of diagnostics. Now any organism can be detected by identifying the presence of specific nucleic acids. However, there still remain areas in which traditional PCR cannot easily be applied. In this chapter, we describe a different flavour of PCR in which an enzyme acts on a DNA substrate to convert it into a DNA product that can be detected by PCR. This opens up new areas for the application of the PCR technique and we will show how the technique can be used to detect immunoconjugates, bacterial drug resistance, and bacterial contamination.

Effects of nominally selective inhibitors of the kinases PI3K, SGK1 and PKB on the insulin-dependent control of epithelial Na+ absorption.

BACKGROUND AND PURPOSE: Insulin-induced Na(+) retention in the distal nephron may contribute to the development of oedema/hypertension in patients with type 2 diabetes. This response to insulin is usually attributed to phosphatidylinositol-3-kinase (PI3K)/serum and glucocorticoid-inducible kinase 1 (SGK1) but a role for protein kinase B (PKB) has been proposed. The present study therefore aimed to clarify the way in which insulin can evoke Na(+) retention. EXPERIMENTAL APPROACH: We examined the effects of nominally selective inhibitors of PI3K (wortmannin, PI103, GDC-0941), SGK1 (GSK650394A) and PKB (Akti-1/2) on Na(+) transport in hormone-deprived and insulin-stimulated cortical collecting duct (mpkCCD) cells, while PI3K, SGK1 and PKB activities were assayed by monitoring the phosphorylation of endogenous proteins. KEY RESULTS: Wortmannin substantially inhibited basal Na(+) transport whereas PI103 and GDC-0941 had only very small effects. However, these PI3K inhibitors all abolished insulin-induced Na(+) absorption and inactivated PI3K, SGK1 and PKB fully. GSK650394A and Akti-1/2 also inhibited insulin-evoked Na(+) absorption and while GSK650394A inhibited SGK1 without affecting PKB, Akti-1/2 inactivated both kinases. CONCLUSION AND IMPLICATIONS: While studies undertaken using PI103 and GDC-0941 show that hormone-deprived cells can absorb Na(+) independently of PI3K, PI3K seems to be essential for insulin induced Na(+) transport. Akti-1/2 does not act as a selective inhibitor of PKB and data obtained using this compound must therefore be treated with caution. GSK650394A, on the other hand, selectively inhibits SGK1 and the finding that GSK650394A suppressed insulin-induced Na(+) absorption suggests that this response is dependent upon signalling via PI3K/SGK1.

Dysregulation of epithelial Na+ absorption induced by inhibition of the kinases TORC1 and TORC2.

BACKGROUND AND PURPOSE: Although the serum and glucocorticoid-inducible protein kinase 1 (SGK1) appears to be involved in controlling epithelial Na(+) absorption, its role in this physiologically important ion transport process is undefined. As SGK1 activity is dependent upon target of rapamycin complex 2 (TORC2)-catalysed phosphorylation of SGK1-Ser(422) , we have explored the effects of inhibiting TORC2 and/or TORC1 upon the hormonal control of Na(+) absorption. EXPERIMENTAL APPROACH: Na(+) absorption was quantified electrometrically in mouse cortical collecting duct cells (mpkCCD) grown to confluence on permeable membranes. Kinase activities were assessed by monitoring endogenous protein phosphorylation, with or without TORC1/2 inhibitors (TORIN1 and PP242) and the TORC1 inhibitor: rapamycin. KEY RESULTS: Inhibition of TORC1/2 (TORIN1, PP242) suppressed basal SGK1 activity, prevented insulin- and dexamethasone-induced SGK1 activation, and caused modest (10-20%) inhibition of basal Na(+) absorption and substantial (∼80%) inhibition of insulin/dexamethasone-induced Na(+) transport. Inhibition of TORC1 did not impair SGK1 activation or insulin-induced Na(+) transport, but did inhibit (∼80%) dexamethasone-induced Na(+) absorption. Arginine vasopressin stimulated Na(+) absorption via a TORC1/2-independent mechanism. CONCLUSION AND IMPLICATIONS: Target of rapamycin complex 2, but not TORC1, is important to SGK1 activation. Signalling via phosphoinositide-3-kinase/TORC2/SGK1 can explain insulin-induced Na(+) absorption. TORC2, but not TORC1, is also involved in glucocorticoid-induced SGK1 activation but its role is permissive. Glucocorticoid-induced Na(+) transport displayed a requirement for TORC1 activity. Therefore, TORC1 and TORC2 contribute to the regulation of Na(+) absorption. Pharmacological manipulation of TORC1/2 signalling may provide novel therapies for Na(+)-sensitive hypertension.

Effects of peroxisome proliferator-activated receptor gamma agonists on Na+ transport and activity of the kinase SGK1 in epithelial cells from lung and kidney.

BACKGROUND AND PURPOSE: Peroxisome proliferator-activated receptor gamma (PPARgamma) agonists, such as rosiglitazone and pioglitazone, sensitize cells to insulin, and are therefore used to treat type 2 diabetes. However, in some patients, these drugs induce oedema, and the present study tests the hypothesis that this side effect reflects serum and glucocorticoid-inducible kinase 1 (SGK1)-dependent enhancement of epithelia Na(+) absorption. EXPERIMENTAL APPROACH: Na(+) absorbing epithelial cells (H441 cells, mpkCCD cells) on permeable membranes were mounted in Ussing chambers, and the effects of rosiglitazone (2 microM) and pioglitazone (10 microM) on transepithelial Na(+) absorption were quantified electrometrically. Changes in SGK1 activity were assessed by monitoring phosphorylation of residues within an endogenous protein. KEY RESULTS: Both cell types absorbed Na(+) via an electrogenic process that was enhanced by insulin. In mpkCCD cells, this stimulation of Na(+) transport was associated with increased activity of SGK1, whereas insulin regulated Na(+) transport in H441 cells through a mechanism that did not involve activation of this kinase. Rosiglitazone and pioglitazone had no discernible effect on transepithelial Na(+) absorption in unstimulated or insulin-stimulated cells and failed to alter cellular SGK1 activity. CONCLUSIONS AND IMPLICATIONS: Our results do not support the view that PPARgamma agonists stimulate epithelial Na(+) absorption or alter the control of cellular SGK1 activity. It is therefore likely that other mechanisms are involved in PPARgamma-mediated fluid retention, and a better understanding of these mechanisms may help with the identification of patients likely to develop oedema or heart failure when treated with these drugs.

Glucocorticoids can activate the alpha-ENaC gene promoter independently of SGK1.

The role of SGK1 (serum- and glucocorticoid-induced protein kinase 1) in the glucocorticoid induction of alpha-ENaC (epithelial Na+ channel alpha subunit) gene transcription was explored by monitoring the transcriptional activity of a luciferase-linked, alpha-ENaC reporter gene construct (pGL3-KR1) expressed in H441 airway epithelial cells. Dexamethasone evoked a concentration-dependent (EC50 approximately 4 microM) increase in transcriptional activity dependent upon a glucocorticoid response element in the alpha-ENaC sequence. Although dexamethasone also activated endogenous SGK1, artificially increasing cellular SGK1 activity by expressing a constitutively active SGK1 mutant (SGK1-S422D) in hormone-deprived cells did not activate pGL3-KR1. Moreover, expression of catalytically inactive SGK1 (SGK1-K127A) suppressed the activation of endogenous SGK1 without affecting the transcriptional response to dexamethasone. Increasing cellular PI3K (phosphoinositide 3-kinase) activity by expressing a membrane-anchored form of the catalytic PI3K-P110alpha subunit [CD2 (cluster of differentiation 2)-P110alpha] also activated endogenous SGK1 without affecting pGL3-KR1activity. A catalytically inactive form of CD2-P110alpha (R1130P), on the other hand, prevented the dexamethasone-induced activation of SGK1, but did not inhibit the activation of pGL3-KR1. However, expression of SGK1-S422D or CD2-P110alpha enhanced the transcriptional responses to maximally effective concentrations of dexamethasone and this effect occurred with no change in EC50. Dexamethasone-induced (0.3-300 nM) activation of pGL3-KR1 was unaffected by inhibitors of PI3K (PI-103 and wortmanin) and by rapamycin, a selective inhibitor of the TORC1 (target of rapamycin complex 1) signalling complex. Dexamethasone-induced activation of the alpha-ENaC gene promoter can thus occur independently of SGK1/PI3K, although this pathway does provide a mechanism that allows this transcriptional response to dexamethasone to be enhanced.

The regulation of selective and nonselective Na+ conductances in H441 human airway epithelial cells.

Analysis of membrane currents recorded from hormone-deprived H441 cells showed that the membrane potential (V(m)) in single cells (approximately -80 mV) was unaffected by lowering [Na+]o or [Cl(-)]o, indicating that cellular Na+ and Cl(-) conductances (GNa and GCl, respectively) are negligible. Although insulin (20 nM, approximately 24 h) and dexamethasone (0.2 microM, approximately 24 h) both depolarized Vm by approximately 20 mV, the response to insulin reflected a rise in GCl mediated via phosphatidylinositol 3-kinase (PI3K) whereas dexamethasone acted by inducing a serum- and glucocorticoid-regulated kinase 1 (SGK1)-dependent rise in GNa. Although insulin stimulation/PI3K-P110 alpha expression did not directly increase GNa, these maneuvers augmented the dexamethasone-induced conductance. The glucocorticoid/SGK1-induced GNa in single cells discriminated poorly between Na+ and K+ (PNa/PK approximately 0.6), was insensitive to amiloride (1 mM), but was partially blocked by LaCl3 (La3+; 1 mM, approximately 80%), pimozide (0.1 mM, approximately 40%), and dichlorobenzamil (15 microM, approximately 15%). Cells growing as small groups, on the other hand, expressed an amiloride-sensitive (10 microM), selective GNa that displayed the same pattern of hormonal regulation as the nonselective conductance in single cells. These data therefore 1) confirm that H441 cells can express selective or nonselective GNa (14, 48), 2) show that these conductances are both induced by glucocorticoids/SGK1 and subject to PI3K-dependent regulation, and 3) establish that cell-cell contact is vitally important to the development of Na+ selectivity and amiloride sensitivity.

Protein kinase CK2, cystic fibrosis transmembrane conductance regulator, and the deltaF508 mutation: F508 deletion disrupts a kinase-binding site.

Deletion of phenylalanine 508 (DeltaF508) from the first nucleotide-binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) is the most common mutation in cystic fibrosis. The F508 region lies within a surface-exposed loop that has not been assigned any interaction with associated proteins. Here we demonstrate that the pleiotropic protein kinase CK2 that controls protein trafficking, cell proliferation, and development binds wild-type CFTR near F508 and phosphorylates NBD1 at Ser-511 in vivo and that mutation of Ser-511 disrupts CFTR channel gating. Importantly, the interaction of CK2 with NBD1 is selectively abrogated by the DeltaF508 mutation without disrupting four established CFTR-associated kinases and two phosphatases. Loss of CK2 association is functionally corroborated by the insensitivity of DeltaF508-CFTR to CK2 inhibition, the absence of CK2 activity in DeltaF508 CFTR-expressing cell membranes, and inhibition of CFTR channel activity by a peptide that mimics the F508 region of CFTR (but not the equivalent DeltaF508 peptide). Disruption of this CK2-CFTR association is the first described DeltaF508-dependent protein-protein interaction that provides a new molecular paradigm in the most frequent form of cystic fibrosis.

A Ba2+-resistant, acid-sensitive K+ conductance in Na+-absorbing H441 human airway epithelial cells.

By analysis of whole cell membrane currents in Na(+)-absorbing H441 human airway epithelial cells, we have identified a K(+) conductance (G(K)) resistant to Ba(2+) but sensitive to bupivacaine or extracellular acidification. In polarized H441 monolayers, we have demonstrated that bupivacaine, lidocaine, and quinidine inhibit basolateral membrane K(+) current (I(Bl)) whereas Ba(2+) has only a weak inhibitory effect. I(Bl) was also inhibited by basolateral acidification, and, although subsequent addition of bupivacaine caused a further fall in I(Bl), acidification had no effect after bupivacaine, demonstrating that cells grown under these conditions express at least two different bupivacaine-sensitive K(+) channels, only one of which is acid sensitive. Basolateral acidification also inhibited short-circuit current (I(SC)), and basolateral bupivacaine, lidocaine, quinidine, and Ba(2+) inhibited I(SC) at concentrations similar to those needed to inhibit I(Bl), suggesting that the K(+) channels underlying I(Bl) are part of the absorptive mechanism. Analyses using RT-PCR showed that mRNA encoding several two-pore domain K(+) (K2P) channels was detected in cells grown under standard conditions (TWIK-1, TREK-1, TASK-2, TWIK-2, KCNK-7, TASK-3, TREK-2, THIK-1, and TALK-2). We therefore suggest that K2P channels underlie G(K) in unstimulated cells and so maintain the driving force for Na(+) absorption. Since this ion transport process is vital to lung function, K2P channels thus play an important but previously undocumented role in pulmonary physiology.