help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2007-0319
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Brunton, P. J.
Right arrow Articles by Shipston, M. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Brunton, P. J.
Right arrow Articles by Shipston, M. J.
Right arrowPubmed/NCBI databases
*Gene*GEO Profiles
*HomoloGene*UniGene
*Compound via MeSH
*Substance via MeSH
Hazardous Substances DB
*HYDROCORTISONE
Endocrinology Vol. 148, No. 11 5496-5506
Copyright © 2007 by The Endocrine Society

Hypothalamic-Pituitary-Adrenal Axis Hyporesponsiveness to Restraint Stress in Mice Deficient for Large-Conductance Calcium- and Voltage-Activated Potassium (BK) Channels

Paula J. Brunton, Matthias Sausbier, Georg Wietzorrek, Ulrike Sausbier, Hans-Guenther Knaus, John A. Russell, Peter Ruth and Michael J. Shipston

Centre for Integrative Physiology (P.J.B., J.A.R., M.J.S.), School of Biomedical Science, Hugh Robson Building, University of Edinburgh, Edinburgh EH8 9XD, Scotland, United Kingdom; Pharmacology and Toxicology (M.S., U.S., P.R.), University Tübingen, Institute of Pharmacy, 72076 Tübingen, Germany; and Division of Molecular and Cellular Pharmacology (G.W., H.-G.K.), Medical University Innsbruck, Innsbruck A-6020, Austria

Address all correspondence and requests for reprints to: Michael J. Shipston, Centre for Integrative Physiology, School of Biomedical Science, Hugh Robson Building, University of Edinburgh, Edinburgh EH8 9XD, Scotland, United Kingdom. E-mail: mike.shipston{at}ed.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Stress activates the hypothalamic-pituitary-adrenal (HPA) axis, releasing ACTH from the anterior pituitary gland and glucocorticoids from the adrenal cortex. Stress also activates the sympathetic nervous system, evoking adrenaline release from the adrenal medulla. Large-conductance calcium- and voltage-activated potassium (BK) channels have been implicated in regulation of cellular excitability in these systems. Here, we examine the functional role of BK channels in HPA axis regulation in vivo using female mice genetically deficient (BK–/–) for the pore-forming subunits of BK channels. BK–/– phenotype in the HPA was confirmed by immunohistochemistry, Western blot analysis, and corticotrope patch-clamp recording. Restraint stress-induced plasma concentrations of ACTH and corticosterone were significantly blunted in BK–/– mice compared with wild type (WT) controls. This stress hyporesponsiveness was associated with reduced activation of hypothalamic paraventricular nucleus (PVN) neurons. Basal expression of CRH, but not arginine vasopressin mRNA in the PVN was significantly lower in BK–/– mice compared with WT controls. Total anterior pituitary ACTH peptide content, but not proopiomelanocortin mRNA expression or corticotrope number, was significantly reduced in BK–/– mice compared with WT. However, anterior pituitary corticotropes from BK–/– mice fully supported ACTH output, releasing a significantly greater proportion of stored ACTH in response to secretagogue in vitro compared with WT. These results support an important role for BK channels in both the neural circuitry and endocrine output of the HPA axis and indicate that the stress hyporesponsiveness in BK–/– mice primarily results from reduced activation of hypothalamic PVN neurosecretory neurons.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
STRESSFUL STIMULI ACTIVATE the hypothalamic-pituitary-adrenal (HPA) axis, resulting in secretion of ACTH from anterior pituitary corticotropes and glucocorticoids from the adrenal cortex as well as activating the fight-or-flight neural stress axis, resulting in release of adrenaline from the adrenal medulla (1, 2, 3). Increasing evidence suggests that the function of both the endocrine and neural stress axes is dependent upon cellular excitability controlled by large-conductance calcium- and voltage-activated potassium (BK) channels. In turn, the expression and function of BK channels are potently regulated by the endocrine outputs of the HPA axis (ACTH and glucocorticoids), suggesting that BK channel and stress-axis function are intimately linked.

Both ACTH and glucocorticoids regulate alternative pre-mRNA splicing of the BK channel pore-forming {alpha}-subunit in the adrenal gland (4, 5, 6), and glucocorticoids regulate splicing in the anterior pituitary (5). In rats, hypophysectomy results in changes in alternative pre-mRNA splicing of the BK channel pore-forming {alpha}-subunit in the medulla of the adrenal gland (6), which can be reversed by ACTH treatment. The functional consequence of this hypophysectomy-induced splicing decision is to modify the firing frequency of adrenal medullary chromaffin cells that release adrenaline (7). Furthermore, regulation of this same splicing decision is manifested in both the adrenals and anterior pituitary during the postnatal stress hyporesponsive period in rats (5) and in the adrenals after chronic social stress in the tree shrew (8). In vitro experiments provide direct evidence for glucocorticoid-mediated regulation of BK channel alternative pre-mRNA splicing in the adrenals and anterior pituitary (4, 5). Nanomolar concentrations of corticosterone (CORT) increase the expression of the stress axis-regulated exon (STREX) BK channel splice variant in cultured anterior pituitary cells via activation of the mineralocorticoid receptor (5). In contrast, micromolar concentrations of CORT decrease STREX inclusion in both anterior pituitary cells and adrenal chromaffin cells, an effect that is dependent upon activation of the glucocorticoid receptor (5).

Second, as well as these long-term effects on BK channel pre-mRNA splicing, glucocorticoids can modulate functional BK channel activity over the timescale of seconds to hours. For example, micromolar concentrations of glucocorticoid rapidly (within seconds to minutes) activate BK channels in endocrine tissues, including murine AtT20 corticotropes, via a nongenomic mechanism (9, 10, 11) that is likely to be dependent upon BK channel {alpha}-subunit assembly with regulatory β-subunits, at least in some systems (11). In contrast, glucocorticoids, in the timescale of minutes to hours, act via the intracellular glucocorticoid receptor to promote de novo mRNA and protein synthesis and antagonize phosphorylation-dependent inhibition of BK channels in murine AtT20 D16:16 corticotrope cells (12, 13). In this system CRH, via activation of protein kinase A, inhibits BK channels, and glucocorticoids prevent CRH-mediated inhibition of the BK channel. Both protein kinase A-mediated inhibition of BK channels (14) and glucocorticoid antagonism of this regulation (15) can be reconstituted by heterologous expression of the STREX BK channel splice variant in HEK293 cells. The functional effect of this regulation in AtT20 corticotropes is to allow a robust secretion of ACTH in response to CRH through inhibition of BK channels. In turn, glucocorticoids block the CRH-mediated inhibition of BK channels and hence blunt CRH-stimulated ACTH release (12).

However, the functional role of BK channels in the central components of the HPA axis, i.e. native CRH paraventricular nucleus (PVN) neurons or corticotropes, is essentially not known. Indeed, our understanding of the electrophysiological properties of native CRH neurons (16, 17) or corticotropes (18) is, rather surprisingly, rudimentary in any species. BK channels are expressed in the native murine hypothalamus and pituitary (19, 20), and based on the data presented above, we hypothesized that genetic deletion of the single gene encoding the pore-forming {alpha}-subunit of the BK channel, KCNMA1 (21), in mice would alter HPA axis function under basal conditions and in response to acute stress.

To investigate the functional role of BK channels in the HPA axis in vivo we 1) further validated the BK–/– phenotype by examining BK channel protein expression in the HPA axis, 2) measured ACTH and CORT secretion in response to restraint stress in vivo and stimulated ACTH release in vitro, 3) analyzed proopiomelanocortin (POMC) mRNA expression and ACTH content in the anterior pituitary gland (22), and 4) determined expression in the PVN of the mRNAs for arginine vasopressin (AVP) and CRH, the principal ACTH secretagogues (1, 2, 3), and nur77, an immediate-early gene that is a marker for neuronal activation (23, 24). Here, we demonstrate that mice lacking BK channels display a HPA axis hyporesponsiveness primarily resulting from reduced hypothalamic drive.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Mice lacking the pore exon of the BK channel {alpha}-subunit (BK–/–) were generated as described (21). Wild-type (WT) and BK–/– mice on the hybrid SV129/C57BL6 background (always F2 generation) were used. Either litter- or age-matched adult female mice were randomly assigned to each experimental condition. Mice were caged in groups of two to four under standard laboratory conditions (lights on at 0700 h, lights off at 1900 h, 21 C, with chow and tap water available ad libitum). All experiments and tissue collection were performed between 0800 and 1100 h and performed in accordance with accepted standards of humane animal care, United Kingdom Home Office requirements, and the German legislation on protection of animals.

Immunohistochemistry
Fixation and immunohistochemistry of coronal brain slices was performed as described previously (19). For immunohistochemistry analysis of pituitary and adrenal glands, BK–/– and WT mice were euthanized by cervical dislocation, and adrenal and pituitary glands removed, rinsed in 50 mM PBS, embedded in TissueTek OCT Compound (Sakura Finetek Europe BU, Zoeterwoude, The Netherlands), rapidly frozen over liquid nitrogen, and stored in airtight vials at –80 C until further processing. Cryostat sections (5–20 µm) were thaw-mounted onto poly-L-lysine-coated slides, rinsed in Tris-buffered saline (TBS) (150 mM NaCl; 100 mM Tris, pH 7.4) and fixed in either 4% paraformaldehyde in PBS (pH 7.4) or methanol for 5 min. Sections were washed twice in TBS and permeabilized in 0.2% Triton X-100 in TBS (TBS-T). Endogenous peroxidase was blocked in 0.6% H2O2/25% methanol in TBS for 25 min, followed by washes with TBS and TBS-T. Slides were preincubated in 2% nonimmune goat serum/2% BSA/0.2% skimmed milk powder in TBS-T, and incubated overnight with anti-BK{alpha}(674:1115) (1:1000 in 2% BSA in TBS-T) (19). This antibody is directed against the C terminus of the BK channel {alpha}-subunit. The specificity of this antibody has been extensively characterized for both immunohistochemistry and Western blotting, displaying similar characteristics compared with other rabbit polyclonal and mouse monoclonal antibodies in mouse and recognizes a single immunoreactive band in murine brain that is absent in BK–/– mice (19, 25) (see also Fig. 1Go). Sections were washed for 1 h with TBS-T and incubated with a horseradish peroxidase-conjugated goat antirabbit IgG. After washing with TBS, immunoreactivity was visualized using the diaminobenzidine method. Sections were dehydrated in an ascending series of ethanol (70/96/100%), cleared in xylene, and coverslipped with Entellan (Merck, Darmstadt, Germany).


Figure 1
View larger version (45K):
[in this window]
[in a new window]

  		FIG. 1. BK channel expression in the HPA axis. Representative sections from WT murine brain (A), pituitary (C), and adrenal gland (E) immunostained for the BK channel pore-forming {alpha}-subunit with corresponding sections from BK–/– mouse brain (B), pituitary (D), and adrenal (F) processed in parallel. Insets show low-magnification views of brain, pituitary, and adrenal sections with boxes indicating regions used for higher-magnification views. In A and B, the box is overlaid on the anterior hypothalamus. C and D, Posterior (P), intermediate (I), and anterior (A) pituitary; E and F, medulla (M), reticular (R), fascicular (F), and glomerular (G) layers of the adrenal gland. Sections were immunostained using 1:1000 dilution of anti-BK{alpha}(674:1115) and immunoreactivity visualized using the diaminobenzidine method. G, Representative Western blot of plasma membranes (10 µg) from adrenal gland (Adr), anterior pituitary (Pit), total brain, and hypothalamus (Hypo) from WT and BK–/– mice. Blots were probed for the BK channel {alpha}-subunit using anti-BK{alpha}(674:1115) and visualized by enhanced chemiluminescence. H, Representative whole-cell outward-current traces determined at +40 mV, from a holding potential of –70 mV, with intracellular free calcium buffered to 0.5 µM to activate calcium-dependent currents in WT and BK–/– isolated pituitary corticotropes. The selective BK channel blocker paxilline (10 µM) was applied for 5 min and reduced outward current in WT but not BK–/– corticotropes. I, Summary data of paxilline sensitive outward current in WT and BK–/– cells normalized to cell capacitance (nA/pF). Data are means ± SEM. **, P < 0.01 vs. WT by Student’s t test. WT, n = 5; BK–/–, n = 4.

 
Western blotting
For Western blot analysis, 10 µg membranes from the respective murine tissues were separated by 10% SDS-PAGE and transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 5% low-fat milk (Marvel) in washing buffer (10 mM PBS, 0.05% Tween 20) for 2 h at 20 C and incubated with the rabbit polyclonal antibody BK{alpha}(674:1115) (19) in washing buffer at 4 C overnight. Blots were rinsed for 30 min with washing buffer and thereafter incubated with peroxidase-conjugated goat antirabbit IgG (Sigma-Aldrich, St. Louis, MO) for 1 h at 20 C. After several washes with washing buffer, immunoreactive bands were detected by enhanced chemiluminescence.

Restraint stress paradigm and tissue collection
Restraint stress was used as a mixed physical and psychological stressor (26). Mice were randomly assigned to one of three experimental groups assayed in parallel: 1) control, nonstressed (basal); 2) restraint for 30 min (restraint); and 3) restraint for 30 min followed by 15 min solo recovery in the home cage (restraint + recovery). For the restraint groups, mice were placed individually in a clear plastic restraint tube [CH Technologies (USA) Inc., Westwood, NJ] of internal diameter 31 mm with a variable pusher, adjusted based on animal size. Mice were decapitated and trunk blood collected in chilled Eppendorf tubes containing 5 µl 5% EDTA. Brains and pituitaries were rapidly removed, frozen on dry ice, and stored at –70 C until tissue processing and in situ hybridization. Blood samples were chilled on ice and centrifuged at 13,000 rpm for 5 min, and plasma was separated and stored at –20 C until subsequent RIA for ACTH and CORT.

Plasma ACTH and CORT measurements
Plasma ACTH and CORT concentrations were determined in single RIAs using commercially available kits (IDS Ltd., Boldon, Tyne and Wear, UK). ACTH was measured in unextracted plasma samples using a two-site immunoradiometric assay (27). CORT was measured in unextracted plasma (diluted 1:25 in assay buffer) using a double-antibody RIA with [125I]CORT as the tracer (28). Sensitivities were 5 pg/ml and 0.5 ng/ml and the intraassay variation less than 7% and 8% for the ACTH and CORT assays, respectively.

In situ hybridization
Tissue was sectioned coronally on a cryostat at 15 µm and thaw-mounted onto Polysine slides. Sections were fixed with 4% (wt/vol) paraformaldehyde in 0.1 M PBS, washed in 0.1 M PBS, acetylated in triethanolamine/acetic anhydride solution, dehydrated in an ascending ethanol series (70–100%), delipidated in chloroform, and partially rehydrated in 95% (vol/vol) ethanol. Synthetic oligonucleotide probes were used (MWG-Biotech, Ebersberg, Germany), and for each probe, all sections were processed in the same hybridization reaction. To detect AVP mRNA expression, a 36-mer probe (5'-GAC CCG GGG CTT GGC AGA ATC CAC GGA CTC CCG TGT-3'), complementary to bases 466–501 of mouse AVP mRNA was used (GenBank NM009732). The CRH mRNA probe was 42-mer (5'-GCC TGT TGC TGT GAG CTT GCT GAG CTA ACT GCT CTG CCC GGG-3') complementary to bases 673–714 of mouse CRH mRNA (GenBank NM205769). The POMC probe was 42-mer (5'-GAA GCC ACC GTA ACG CTT GTC CTT GGG CGG GTT GCT CCA GCG-3') complementary to bases 734–775 of mouse POMC mRNA (GenBank NM008895). The nur77 mRNA probe was 36-mer (5'-GTC TCG GGG CTG GCC AGG TCC ATG GTA GGC TTG CCG-3'), complementary to bases 201–236 of mouse nur77 mRNA (GenBank NM010444).

Probes were 3'-labeled with [35S]dATP using terminal deoxynucleotidyl transferase and purified using spin columns (QIAquick nucleotide removal kit; QIAGEN, Crawley, West Sussex, UK). Sections were hybridized with 35S-radiolabeled probe (105 cpm labeled probe per section) in hybridization buffer (BSA, 1% wt/vol; dextran sulfate, 5% wt/vol; dithiothreitol, 15 mM; EDTA, 2 mM; Ficoll, 1% wt/vol; formamide, 50% vol/vol; PolyA, 0.1 mg/ml; polyvinylpyrrolidone, 1% (wt/vol); salmon testes DNA, 0.2 mg/ml; sodium chloride, 1.2 M; sodium pyrophosphate, 2.5% wt/vol; Tris, pH 7.6, 20 mM; yeast tRNA, 0.1 mg/ml; yeast total RNA, 0.1 mg/ml) overnight at 37 C in humidified chambers. Sections were rinsed three times in 1x saline sodium citrate at room temperature, washed four times for 15 min each in 1x saline sodium citrate at 59 C for AVP and 60 C for CRH, POMC and nur77 mRNA probes, followed by two 30-min washes at room temperature. Sections were air dried, dipped in liquid autoradiographic emulsion (Ilford K-5; Knutsford, Cheshire, UK), and exposed at 4 C. Exposure times for each mRNA probe were as follows: AVP, 7 d; CRH, 70 d; POMC, 28 d; and nur77, 63 d. Slides were developed (Kodak D-19 developer; Sigma), fixed (Ilford rapid fixer), counterstained with hematoxylin and eosin, and coverslipped with DePeX.

All in situ hybridization analysis was performed on coded slides. AVP mRNA in the PVN and POMC mRNA in the anterior pituitary were quantified from emulsion-dipped sections using a computerized image analysis system (objective magnification x4; NIH Image, version 1.62). For AVP and POMC mRNA analysis, grain area was measured over six PVN (magnocellular and parvocellular neurons) or eight anterior pituitary sections, respectively, rather than analysis of single cells. The area of each PVN or anterior pituitary profile was measured to calculate grain area (square millimeters) per PVN (square millimeters) or anterior pituitary (square millimeters). Background measurements were made over areas adjacent to the region of interest, converted to grain area per square millimeter and subtracted. There was no significant difference in the size of either the PVN or the anterior pituitary sections. The mean area of a single PVN section in control animals was 0.107 ± 0.002 mm2 and 0.107 ± 0.005 mm2 for WT (n = 6) and BK–/– (n = 7) mice, respectively. The mean area of an anterior pituitary section was 0.99 ± 0.14 mm2 and 0.98 ± 0.15 mm2 for WT (n = 20) and BK–/– (n = 20) mice, respectively. The number of cells expressing POMC mRNA in the anterior pituitary was manually counted in emulsion-dipped sections (at x40 magnification, in two pituitary profiles per mouse). A positive cell was defined as one with more overlying silver grains than the average background + 3 SD. Data are presented as the number of positive cells per anterior pituitary profile. CRH and nur77 mRNA hybridization was quantified using two methods. The number of cells expressing CRH or nur77 mRNA in the BK–/– parvocellular PVN (pPVN) was manually counted in emulsion-dipped sections (at x40 magnification, in six PVN profiles per mouse). A positive cell was defined as one with more overlying silver grains than the mean over 10 cells lateral to the PVN (background) + 3 SD. Data are presented as the number of positive cells per PVN profile. In addition, silver grain area was measured in the PVN over 120 individual neurons per mouse that were positively hybridized with the CRH or nur77 mRNA probe. Background measurements were made over the equivalent of 20 cells in neuropil adjacent to the PVN, divided by 20, and subtracted. Data are presented as grain density per cell (square micrometers). For all in situ hybridization measurements, the mean values for each mouse were used to calculate group means ± SEM.

ACTH secretion in vitro
ACTH secretion in vitro was evaluated using anterior pituitary segments in static incubation assays. Mice were killed as above and the anterior pituitary gland separated from the posterior pituitary using fine forceps under a stereomicroscope. The anterior pituitary from each animal was placed in 2 ml DMEM (Invitrogen, Carlsbad, CA) containing 25 mM HEPES (Sigma) and 0.1% BSA (Sigma). Anterior pituitaries were cut with a scalpel blade into four segments under a stereomicroscope at room temperature. The segments were incubated individually, each in a single well of a 24-well plate with 0.5 ml DMEM-BSA at room temperature for 1 h, gently washed with DMEM-BSA, and incubated for another hour at room temperature before another wash and incubation in 0.5 ml DMEM-BSA at 37 C in a water bath with gentle rocking. For secretion experiments, pituitary segments were transferred to fresh 24-well plates and incubated in 0.5 ml DMEM-BSA containing the appropriate secretagogue for 30 min at 37 C in a shaking water bath. Each pituitary segment from an individual mouse was randomly assigned to one of four treatments: basal (vehicle control), 10 nM CRH, 0.1 nM CRH plus 2 nM AVP, or 50 mM KCl. CRH and AVP were from Bachem AG (Bubendorf, Switzerland). Plates were then placed on ice and the supernatant removed, spun briefly at 100 x g, snap frozen on dry ice, and stored at –70 C until RIA.

ACTH content in pituitary segments was determined by rapidly rinsing segments three times in Hanks’ buffered salt solution, which were then snap frozen on dry ice in 1 ml 0.1 N HCl containing 0.01 N ascorbate and stored at –70 C. Pituitaries were rapidly thawed, homogenized through a 23-gauge needle attached to a 1-ml syringe, using 20 strokes, and briefly sonicated. Samples were diluted in RIA buffer and assayed for ACTH as below. Total protein content in each pituitary segment was determined using the BCA protein assay kit essentially as described by the manufacturer (Pierce Biotechnologies Inc., Rockford, IL). To estimate the initial ACTH content in each pituitary segment before exposure to secretagogues, in release experiments, the ACTH content remaining in each segment at the end of the 30-min incubation period was determined as above and added to the measured ACTH release into the medium during the 30-min incubation (i.e. initial total ACTH in segment = remaining ACTH in segment + ACTH secreted into the medium).

ACTH RIA.
ACTH in in vitro samples was determined, in duplicate, using a commercial RIA kit (ACTH kit RK-001-21; Phoenix Pharmaceuticals Inc., Belmont, CA) after appropriate dilution in RIA buffer. Assay sensitivity IC50 was 15.6 pg ACTH/tube with a sensitivity limit of 1 pg ACTH/tube. Intra- and interassay variability was less than 8 and 5%, respectively.

ACTH release was calculated either as amount of ACTH released relative to total protein in each pituitary segment (nanograms ACTH per milligram protein) or as a percentage of total pituitary ACTH content. Percent release was calculated by dividing the ACTH released into the medium by the initial total ACTH content of the pituitary segment, measured as described above. To calculate the fold stimulation of secretion by each secretagogue, the stimulated ACTH secretion value was divided by the corresponding basal release.

Patch clamp electrophysiology
For electrophysiology, pituitary cells were mechanically dispersed with 0.15% trypsin (Worthington Biochemical, Lakewood, NJ), plated on glass coverslips, and maintained in culture for 1–3 d in DMEM with 10% fetal calf serum before analysis. Cell identity was confirmed post hoc by immunostaining fixed cells for ACTH using a rabbit polyclonal antibody (Sigma-Aldrich). Conventional whole-cell patch clamp electrophysiology was used to determine outward potassium currents in physiological potassium gradients. The extracellular solution contained (mM) 140 NaCl, 5 KCl, 2 MgCl2, 1 CaCl2, 10 HEPES, and 20 glucose (pH 7.4), and 0.002 tetrodotoxin. Intracellular solution contained (mM) 140 KCl, 2 MgCl2, 10 HEPES, 10 glucose, 1 BAPTA, and 1 ATP (pH 7.3) with intracellular free calcium buffered to 0.5 µM to ensure robust activation of calcium-activated potassium currents in WT or BK–/– cells. Cells were voltage clamped at –70 mV and depolarized to the respective potentials for 400 msec. Steady-state outward current was determined 350 msec into the pulse. All data acquisition and voltage protocols were controlled by an Axopatch 200B amplifier with pCLAMP 9 software (Molecular Devices, Sunnyvale, CA). All data were sampled at 10 kHz and filtered at 2 kHz. Pipettes were manufactured from Harvard GC-150F or Garner EN-1 glass. The BK channel inhibitor paxilline (Sigma-Aldrich) was bath applied either by gravity-driven perfusion or direct application to the bath at a concentration of 10 µM.

Statistical analysis
Statistical analyses of the data were performed using Sigmastat software (Systat Software UK Ltd., Hounslow, London, UK). ACTH, CORT, and nur77 mRNA data were analyzed using a two-way ANOVA with Student-Newman-Keuls multiple comparison test. The AVP, CRH, POMC mRNA, and ACTH content and electrophysiology data were analyzed using a Student’s t test. Data are presented as group means ± SEM where n represents the number of experiments or mice. P values < 0.05 were considered statistically significant in each case.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Immunohistochemical and Western blot analysis revealed that the pore-forming {alpha}-subunit of the BK channel is expressed at multiple levels of the WT murine HPA axis including the hypothalamus, anterior pituitary, and adrenal gland (Fig. 1Go, A, C, E, and G). BK channel {alpha}-subunit protein is robustly expressed in anterior pituitary and adrenal glands. In the hypothalamus, BK channel expression is relatively low compared with other brain regions (Fig. 1Go, A and G) such as hippocampus as previously reported (19). Western blotting revealed an approximately 125-kDa immunoreactive band corresponding to the BK channel {alpha}-subunit in each of these tissues using the same antibody (Fig. 1GGo). In mice lacking the BK channel (BK–/–), no specific expression of the {alpha}-subunit protein was detectable in the brain, anterior pituitary, or adrenal gland (Fig. 1Go) as confirmed by both immunohistochemistry (Fig. 1Go, B, D, and F) and Western blotting (Fig. 1GGo). Similar data were also obtained by RT-PCR analysis for BK channel {alpha}-subunit mRNA expression (data not shown). In addition, whole-cell patch-clamp electrophysiological analysis of isolated corticotropes demonstrated functional BK potassium currents in WT corticotropes that were significantly reduced by the selective BK channel inhibitor paxilline (Fig. 1Go, H and I). In contrast, paxilline had no significant effect on outward potassium currents in BK–/– corticotropes as expected (Fig. 1Go, H and I).

Attenuated stress-induced plasma ACTH and CORT levels in BK–/– mice
To address whether HPA axis function was disrupted in mice deficient for the BK channel, WT and homozygous knockout (BK–/–) female mice were untreated (basal) or subjected to 30 min restraint stress with or without 15 min of recovery. No significant differences in basal plasma concentrations of ACTH (Fig. 2AGo) were observed between WT and BK–/– mice with 52.5 ± 14.4 pg/ml and 57.2 ± 20.7 pg/ml for WT (n = 6) and BK–/– (n = 6), respectively. Thirty minutes restraint resulted in robust stimulation of ACTH secretion in both WT and BK–/– mice; however, the ACTH response was significantly (P < 0.001, two-way ANOVA with Student-Newman-Keuls multiple comparison test) blunted in BK–/– mice to 56% of WT ACTH concentrations (Fig. 2AGo). Thus, although the increase in ACTH secretion above basal in WT was 6.1-fold, it was only 3.1-fold in BK–/– mice (Fig. 2AGo). After 15 min recovery after the restraint stress, plasma ACTH concentrations returned toward basal levels with no significant difference between WT and BK–/– mice.


Figure 2
View larger version (18K):
[in this window]
[in a new window]

  		FIG. 2. Effects of 30 min restraint on stress hormone secretion in WT and BK–/– mice. Mice were killed by decapitation either immediately after 30 min restraint or 15 min after the end of a 30-min period of restraint (restraint + recovery). Control (basal) mice were removed from their home cages and killed immediately. Trunk blood was collected in each case. Plasma was assayed for ACTH (A) and CORT (B). Values are plotted as group means ± SEM (n = 6 mice per group). Two-way ANOVA followed by Student-Newman-Keuls multiple comparison test was used for statistical analysis of the data: *, P < 0.02 vs. respective control group; #, P < 0.001 vs. BK–/–/restraint group.

 
Stress-induced ACTH concentrations in vivo were paralleled by plasma CORT levels (Fig. 2BGo). No significant difference in basal CORT (Fig. 2BGo) concentration was observed between WT (73.5 ± 12.4 ng/ml, n = 7) and BK–/– (49.8 ± 7.9 ng/ml, n = 7). Thirty minutes restraint resulted in a significant (P < 0.01, two-way ANOVA with Student-Newman-Keuls multiple comparison test) increase in CORT secretion in both WT and BK–/– mice; however, the response of BK–/– mice was again significantly (P < 0.05, two-way ANOVA with Student-Newman-Keuls multiple comparison test) blunted to 51% of WT CORT concentrations (Fig. 2BGo). Thus, although the increase in CORT secretion above basal in WT was 6.6-fold, it was only 4.9-fold in BK–/– mice (Fig. 2BGo). CORT levels remained elevated in both WT and BK–/– after 15 min recovery after the restraint stress. Taken together, these data demonstrate that both the ACTH and CORT response to restraint stress is markedly attenuated in mice lacking the BK channel.

Enhanced ACTH release from the anterior pituitary in vitro in BK–/– mice
We asked whether the attenuated plasma ACTH and CORT levels in response to restraint stress may result from a reduced capacity of anterior pituitary corticotropes to release ACTH. Quantitative in situ hybridization showed that POMC mRNA expression in the anterior pituitary was not significantly different between WT and BK–/– mice (Fig. 3Go, A–C). Furthermore, the total number of cells expressing POMC mRNA in the anterior pituitary was not altered between WT and BK–/– mice, demonstrating that the number of corticotropes is not modified by genetic deletion of BK channels (Fig. 3DGo) However, total anterior pituitary ACTH peptide content was significantly (P < 0.01 Student’s t test) reduced in pituitaries from BK–/– mice to 40% of the ACTH content in WT anterior pituitary (Fig. 3EGo). Taken together, these findings suggest that the reduced ACTH peptide content results from a reduction in ACTH content per corticotrope, and this results from mechanisms downstream of transcriptional regulation of the POMC gene.


Figure 3
View larger version (41K):
[in this window]
[in a new window]

  		FIG. 3. Basal POMC mRNA expression and ACTH content in the pituitary. Representative bright-field autoradiographs of coronal pituitary sections hybridized with a 35S-labeled oligonucleotide probe complementary to POMC mRNA from an untreated WT (A) or BK–/– (B) mouse: AP, Anterior pituitary; IL, intermediate lobe; PP, posterior pituitary. Scale bar, 500 µm. C–E, Quantification of POMC mRNA expression in the anterior pituitary. C, Total area of the anterior pituitary section and the overlying silver grain area were measured from autoradiographs, and the data presented as grain density (mm2/mm2). D, Number of cells expressing POMC mRNA was counted in anterior pituitary sections. In both C and D, values are group means ± SEM (WT, n = 7; BK–/–, n = 7 mice per group). Student’s t test revealed no significant differences between the groups. E, ACTH content in the anterior pituitary gland expressed as nanograms ACTH per milligram of total pituitary protein. Data are means ± SEM (WT, n = 8; BK–/–, n = 7). **, P < 0.01, Student’s t test vs. WT.

 
To address whether the reduced ACTH content resulted in a reduced ACTH secretion we measured both basal and stimulated ACTH release in vitro from anterior pituitary segments. Pituitary segments were used in an attempt to retain pituitary architecture as close as possible to that in vivo. Previous studies in mice demonstrated that restraint stress increases ACTH secretion within 2 min of onset of the stressor and that these elevated levels of ACTH remain essentially constant for the duration of the stressor (29, 30). To parallel the in vivo duration of the restraint stress paradigm, we stimulated the pituitary for 30 min in vitro with various secretagogues to ask whether a deficit in the ability of BK–/– pituitaries to support sustained ACTH release might underlie the hyporesponsiveness in vivo.

Basal ACTH release, expressed either as per milligram protein (Fig. 4AGo) or as a percentage of total ACTH content (Fig 4BGo), was not significantly different between WT and BK–/– pituitary segments in vitro, although fractional release tended to be greater in BK–/– pituitaries. Basal ACTH release in WT and BK–/– anterior pituitary segments was 7.1 ± 1.4% (n = 8) and 14.8 ± 2.0% (n = 7) (P = 0.06, ANOVA) of total ACTH content, respectively (Fig. 4BGo). Thus, basal ACTH release in vitro was not significantly different between WT and BK–/– as we also observed for plasma ACTH levels in vivo.


Figure 4
View larger version (22K):
[in this window]
[in a new window]

  		FIG. 4. ACTH release from the anterior pituitary gland in vitro. ACTH release was determined from WT or BK–/– anterior pituitary segments in vitro after a 30-min incubation at 37 C with vehicle control (basal), 10 nM CRH, 0.1 nM CRH plus 2 nM AVP, or 50 mM KCl. A, ACTH release data are expressed as relative to total pituitary protein (nanograms ACTH per milligram protein); B, ACTH release is expressed as a percentage of total initial ACTH stored in the pituitary. Data are means ± SEM. WT, n = 8; BK–/–, n = 7. Two-way ANOVA followed by Student-Newman-Keuls multiple comparison test was used for statistical analysis of the data: *, P < 0.05 vs. WT.

 
Furthermore, even though BK–/– pituitaries have reduced pituitary ACTH stores, there was no deficit in the ability of BK–/– pituitaries to maintain ACTH output in vitro compared with WT (Fig. 4AGo) when expressed as ACTH released per milligram of pituitary protein. Moreover, with a combined CRH and AVP stimulus that mimics the levels of CRH and AVP (0.1 nM CRH and 2 nM AVP) reported in portal blood (31, 32, 33), BK–/– pituitaries released significantly (P < 0.05, ANOVA with post hoc Student-Newman-Keuls test) more ACTH per milligram pituitary protein compared with WT (Fig. 4AGo). The ability of BK–/– pituitaries to support high-level ACTH output resulted from a significantly (P < 0.05, ANOVA with post hoc Student-Newman-Keuls test) enhanced fractional release of stored ACTH in response to the combined CRH and AVP stimulus (Fig. 4BGo). A similar significant enhancement of fractional ACTH release from BK–/– pituitaries was also observed using either 10 nM CRH or 50 mM KCl as secretagogue. This enhanced ACTH secretory response in vitro was also evident (P < 0.05, ANOVA with post hoc Student-Newman-Keuls test) when expressed as fold increase in percentage ACTH release above basal using either CRH alone or in combination with AVP, but not for KCl. Fold stimulation for CRH in WT and BK–/– was 1.7 ± 0.3 (n = 8) and 2.6 ± 0.3 (n = 7), respectively (P < 0.05, ANOVA with post hoc Student-Newman-Keuls test). Fold stimulation for the combined CRH/AVP stimulus was 2.6 ± 0.3 (n = 8) and 3.8 ± 0.6 (n = 7) (P < 0.05, ANOVA with post hoc Student-Newman-Keuls test) for WT and BK–/–, respectively. Taken together, these data demonstrate that absence of the BK channel does not attenuate the ability of the pituitary corticotrope to release ACTH during acute (30 min) exposure to secretagogues and thus argues against a pituitary defect as the primary cause of the stress hyporesponsiveness observed in vivo. Rather, BK channel deletion results in exaggerated secretagogue-stimulated ACTH release from the available store in the anterior pituitary gland.

Reduced basal expression of CRH mRNA in the PVN of BK–/– mice
The above data do not support a primary defect at the level of the anterior pituitary corticotrope that would explain the reduced ACTH and CORT responses to stress in vivo but instead indicate reduced hypothalamic drive during stress. Thus, we examined whether the stress hyporesponsiveness may be a result of reduced activation of the hypothalamic PVN CRH/AVP neurons.

We first examined basal levels of CRH and AVP mRNA expression in the PVN to address whether there is reduced hypothalamic drive to the pituitary in the BK–/– mice that may underlie the reduced ACTH content in the anterior pituitary of these animals. Quantitative in situ hybridization revealed a small but significant (P < 0.05, Student’s t test) 20% reduction in basal CRH mRNA expression in the pPVN of BK–/– mice compared with WT controls (Fig. 5Go, A and C). If this modest decrease in CRH mRNA expression is also reflected in significantly reduced CRH production and release, it might be predicted to contribute to the reduced pituitary ACTH content and plasma levels observed in BK–/– mice. In contrast, no significant differences in total PVN AVP mRNA expression (Fig. 5Go, B and D) were observed, suggesting that diminished AVP production in the hypothalamus is not responsible for the diminished ACTH response in vivo. However, the inability to unambiguously separate the parvocellular and magnocellular divisions of the PVN in mouse, and the high levels of AVP expression in magnocellular neurons, precludes definitively addressing whether AVP mRNA expression in pPVN neurons is diminished.


Figure 5
View larger version (92K):
[in this window]
[in a new window]

  		FIG. 5. Basal CRH and AVP mRNA expression in the PVN. Representative autoradiographs of coronal brain sections cut through the PVN (Bregma –0.75 mm) (62 ) and hybridized with a 35S-labeled oligonucleotide probe complementary to either CRH mRNA (A) (dark-field) or AVP mRNA (B) (bright-field) from an untreated WT and BK–/– mouse. 3V, Third ventricle. Scale bar, 100 µm. C, Quantification of CRH mRNA expression in the PVN. The expression per PVN was calculated by multiplying the number of neurons positively expressing CRH mRNA by the grain density per neuron. a.u., Arbitrary units. WT, n = 7; BK–/–, n = 7. *, P < 0.05, Student’s t test. Inset in C shows a representative bright-field autoradiograph of CRH mRNA-labeled neurons from a WT mouse. Black arrows indicate cells defined as positively expressing CRH mRNA and white arrows indicate cells not expressing CRH mRNA. Scale bar, 10 µm. D, Quantification of AVP mRNA expression in the PVN. Data are presented as grain density (square millimeter) per PVN (square millimeter). WT, n = 6; BK, n = 7 mice per group. In both cases, data are group means ± SEM. Student’s t test revealed no significant differences between the groups.

 
Reduced activation of neurons in the PVN in response to restraint stress in BK–/– mice
We next sought to examine whether the blunted ACTH and CORT responses to restraint stress in BK–/– mice could be explained by reduced central activation of the neurons in the PVN. Quantitative in situ mRNA analysis for the immediate-early gene nur77 (NGFI-B, nerve growth factor inducible gene-B; a reporter of neuronal activation) (23, 24, 34, 35) was performed to ask whether the number of neurons activated by restraint stress in the PVN is reduced in BK–/– mice and/or whether the extent of activation of individual neurons may be diminished. The number of cells expressing nur77 mRNA was not significantly different under basal conditions between WT (57.8 ± 6.0 nur77-positive cells, n = 7) and BK–/– mice (56.1 ± 4.8 nur77-positive cells, n = 6) (Fig. 6Go, A and B). Furthermore, nur77 mRNA grain density per neuron was 19.9 ± 1.3 µm2 (n = 7) and 17.7 ± 0.5 µm2 (n = 6) for WT and BK–/– mice, respectively (Fig. 6Go, A and C). Exposure to 30 min restraint significantly (P < 0.003, two-way ANOVA with Student-Newman-Keuls post hoc test) increased both the number of nur77-positive cells in the PVN and the nur77 grain density per neuron in WT and BK–/– mice (Fig. 6Go, A–C). However, in BK–/– mice, the number of nur77 mRNA-positive cells, as well as nur77 mRNA expression per cell, was significantly attenuated (P < 0.04 after restraint and P < 0.001 after restraint + recovery; two-way ANOVA with Student-Newman-Keuls multiple comparison test) compared with WT mice (Fig. 6Go, A–C). Thus, activation of the PVN neurons was significantly attenuated in BK–/– mice compared with WT, and this reduced activation of the neuroendocrine system most likely makes a significant contribution to the attenuated ACTH and CORT response to restraint in BK–/– animals.


Figure 6
View larger version (58K):
[in this window]
[in a new window]

  		FIG. 6. The effect of 30 min restraint on nur77 mRNA expression in the PVN. A, Representative dark-field autoradiographs of coronal brain sections cut through the PVN (Bregma –0.75 mm) (62 ) and hybridized with a 35S-labeled oligonucleotide probe complementary to nur77 mRNA from WT basal, WT restraint, BK–/– basal, and BK–/– restraint. 3V, Third ventricle. Scale bar, 100 µm. Quantification of nur77 mRNA expression in the PVN is presented as group means + SEM for number of positive cells/PVN (B) and grain density/neuron (square micrometers) (C). In both instances, numbers of mice per group were as follows: WT basal, n = 7; BK–/– basal, n = 6; WT restraint, n = 6; BK–/– restraint, n = 7; WT restraint + recovery, n = 7; BK–/– restraint + recovery, n = 6. *, P < 0.003 vs. respective basal group; #, P < 0.04; +, P < 0.001 vs. BK–/– mice for the same treatment group, two-way ANOVA with Student-Newman-Keuls multiple comparison test.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The regulation of cellular excitability in the HPA axis is essential for the maintenance of body homeostasis in response to stress. Here we demonstrate stress hyporesponsiveness in mice deficient for the pore-forming {alpha}-subunit encoding large-conductance calcium- and voltage-activated potassium (BK) channels. Genetic deletion of BK channels resulted in diverse effects on cellular function at multiple levels of the HPA axis. Although basal HPA axis function appears essentially normal, with no significant difference in basal plasma ACTH or CORT levels, BK–/– mice display blunted ACTH and CORT responses to restraint stress. This stress hyporesponsiveness primarily results from the reduced activation of hypothalamic PVN neurons in response to stress. In contrast, at the level of the anterior pituitary corticotrope, although ACTH content is diminished, secretagogue-stimulated ACTH release was enhanced from the anterior pituitary of BK–/– mice. Taken together, these data support the hypothesis that BK channels play an important, and multifunctional, role in regulating cellular function in the HPA axis.

Reduced activation of PVN neurons in response to stress
In these studies, we used restraint stress, a mixed physical and psychological stressor, to explore HPA axis responses in BK–/– mice (26). Maximal responses to restraint stress are commonly observed 30 min after the beginning of the restraint, as used here, and result in activation of multiple higher brain regions including the thalamus, cortex, and limbic system (1, 26). The pPVN neurosecretory neurons that express CRH and/or AVP are regulated by multiple inhibitory and excitatory inputs. Because BK channels are widely expressed in the murine brain (19), including regions important in HPA axis regulation (1, 19, 26), altered function in any of the brain regions activated by restraint stress, and regulating the PVN, might contribute to the reduced PVN neuron activation in BK–/– mice. However, we saw no significant difference in the number of PVN neurons activated under basal conditions between BK–/– mice and WT controls using the early-immediate gene nur77 as a marker of cellular activation (23, 24). This suggests lack of gross imbalance of basal inputs to CRH neurons in BK–/– mice; furthermore, it suggests that BK channels do not play a central role in determining the basal electrical excitability of PVN neurons per se. In contrast, in response to restraint stress, as evaluated by changes in nur77 mRNA expression, in BK–/– mice, a significantly reduced number of PVN neurons were activated, and the extent of activation in each neuron was also diminished.

The reduced stress-induced activation of PVN neurons in BK–/– mice may result from two distinct mechanisms: either 1) changes in afferent input to the CRH and/or AVP neurons or 2) changes in intrinsic PVN neuron excitability per se. In support of the former mechanism, BK channels control excitatory neurotransmission in other systems (21, 36, 37, 38). Thus, during stress, reduced activation of excitatory afferents and/or reduced excitatory synaptic input to PVN neurons may underlie the reduced activation of PVN neurons in BK–/– mice, as has been demonstrated during the stress hyporesponsive period in late pregnancy in rats (39). Alternatively, the reduced activation may be a consequence of reduced intrinsic excitability in CRH neurons in the PVN in response to stimulatory input. In support of such a mechanism, in BK–/– mice, a significant reduction in intrinsic excitability of cerebellar Purkinje neurons results from a depolarization block (21). Although PVN CRH neurons are pivotal to the physiological function of the HPA axis, very little is known about their electrophysiological properties or ion channel expression (16, 17). The precise functional role of BK channels in a particular system is not simple to predict because the role of BK channels in controlling cellular electrical excitability is dependent on both the properties of endogenous BK channels and the cellular context in which they are expressed. For example, in rat somatotropes, but not gonadotropes, BK channels have been proposed to paradoxically promote generation of spontaneous plateau-bursting activity and facilitated calcium entry that may be important for control of basal hormone secretion (40).

Clearly, more work is warranted, including use of alternative stressors, to address the functional role of BK channels in regulating both the central neural circuits regulating CRH neurons and the intrinsic electrical excitability of pPVN neurosecretory neurons.

Reduced CRH mRNA expression in the PVN of BK–/– mice
Although the stress hyporesponsiveness in BK–/– mice is likely to result from the reduced activation of PVN neurons, BK–/– mice also displayed a modest reduction in basal CRH mRNA expression in the pPVN. If this translates into a significant reduction in CRH peptide expression, this would also be predicted to contribute to the stress hyporesponsiveness. CRH expression in the PVN is under combinatorial control by protein kinase A and glucocorticoid signaling pathways as well as by cellular depolarization (34, 35, 41), although differences between species are apparent (42). Intriguingly, voltage-dependent calcium influx and activation of calmodulin-dependent protein kinase IV regulates both CRH (41) and BK channel expression in neurons (43, 44). Thus, excitability of PVN CRH neurons, determined by the afferent inputs (1, 26), glucocorticoid negative feedback (3, 35), and intrinsic electrical excitability may be important in regulating both CRH and BK channel expression in the PVN. CRH gene expression (45) as well as ACTH and CORT (3, 35) release also display a diurnal rhythm driven by the circadian oscillator in the suprachiasmatic nucleus. BK–/– mice have been shown to have a modified circadian output (46) during the dark phase. In this study, we analyzed CRH mRNA expression during the early light phase; thus, whether changes in circadian rhythms underlie the reduced CRH mRNA expression observed in BK–/– mice in our studies remains to be determined.

CRH release from the median eminence into the portal blood circulation is calcium dependent (2), and whether stimulus-secretion coupling is disrupted in hypothalamic neurons in BK–/– mice remains to be examined. In this regard, BK channels have been suggested to play an important role in neuropeptide secretion from the terminals of magnocellular neurons projecting to the posterior pituitary (47).

Changes in corticotrope function in BK–/– mice
The mechanisms underlying the reduced ACTH peptide content in the BK–/– pituitary are unknown but may arise from several distinct mechanisms including effects on POMC translation (22), POMC processing (48, 49), or depletion of the available secretory pool. However, even though pituitary ACTH stores were significantly reduced, the release of ACTH from BK–/– pituitaries in vitro in response to CRH and AVP was not compromised compared with WT under conditions of maximal secretagogue stimulation over the time course (30 min) in which stress hyporesponsiveness was observed in vivo. In fact, the proportion of total available ACTH that was released in response to CRH and AVP is significantly greater in BK–/– mice compared with WT controls with more than 50% of ACTH stores secreted in response to CRH/AVP in the 30-min exposure used here. Importantly, exhaustion of the releasable ACTH stores was not manifest within the 30-min maximal stimulation in vitro, paralleling the time course of the in vivo restraint stress paradigm used here. Thus, our data do not support a role for reduced pituitary ACTH content or release being a primary contributing factor to the stress hyporesponsiveness observed in response to acute restraint in vivo.

Because anterior pituitary basal POMC mRNA levels were not significantly different between BK–/– and WT mice, sustained activation of the HPA axis would be expected to result in a much faster depletion of ACTH stores and thus a potentially diminished ACTH response to repeated or chronic stress. Clearly, more work is required to address whether repeated, or chronic, stress paradigms also result in HPA axis hyporesponsiveness, in which depletion of pituitary ACTH stores may then be a significant contributing factor.

The enhanced secretagogue-stimulated ACTH release observed in vitro in BK–/– pituitaries is in general agreement with the role of BK channels in murine AtT20 D16:16 corticotropes. In this system, CRH inhibits BK channels through the cAMP-protein kinase A signaling pathway, thus removing the inhibitory effects of BK channels on voltage-dependent calcium influx required for sustained secretion (12). Inhibition of BK channels in AtT20 D16:16 corticotropes thus results in a more robust ACTH secretory response. Although AtT20 D16:16 corticotropes lack functional AVP receptors, the protein kinase C pathway, which is activated by AVP, also inhibits BK channels (13). Although our data demonstrate, for the first time, functional BK potassium currents in WT murine corticotropes, the contribution of BK channels to native murine corticotrope electrical excitability is essentially not known.

Physiological relevance
Although these studies clearly demonstrate an important role for BK channels in HPA function during acute stress, our data may have considerably wider implications for changes in HPA function seen during development and both physiological and pathophysiological challenges to homeostasis.

For example, the BK–/– mice also display a mild hypertensive phenotype (50); thus, interactions between cardiovascular changes and HPA axis disruption may compromise allostasis (51). Moreover, during the postnatal stress hyporesponsive period in rats, a switch in alternative splicing of BK {alpha}-subunits in the anterior pituitary parallels the fall in plasma glucocorticoid levels (5). Because the splice variant properties of BK channels are important for both CRH and glucocorticoid regulation of corticotrope function (12, 14, 15), changes in BK channel expression may underlie the stress hyporesponsive period.

In rats and mice, HPA axis hyporesponsiveness is also observed during late pregnancy and involves reduced activation of pPVN CRH neurons in response to a range of stressors as well as decreased basal CRH mRNA expression (24, 39, 52, 53, 54, 55, 56). Intriguingly, in rats, the HPA hyporesponsiveness in pregnancy also involves a marked enhancement of CRH- and AVP-stimulated ACTH secretion from the anterior pituitary as we observed here in the BK–/– mice (54). Thus, the HPA hyporesponsiveness in late pregnancy and in BK–/– mice share several common features including hyporesponsiveness of PVN neurons and hyperreponsiveness of anterior pituitary corticotropes. Although it is not known whether changes in BK channel expression, or activity, occur in the HPA, or any other, neural axis during pregnancy, significant changes in BK channel expression do occur in the myometrium (57, 58, 59, 60, 61). This suggests that changes in BK channel expression in the HPA axis may underlie, at least in part, changes in HPA function during pregnancy.

In conclusion, our data support the hypothesis that large-conductance calcium- and voltage-activated BK channels play important roles in regulating the HPA axis at multiple levels.


   Acknowledgments
 
We are grateful to Helen Cameron for expert assistance with analysis of in situ hybridization studies.


   Footnotes
 
This work was supported by The Wellcome Trust and Biotechnology and Biological Sciences Research Council.

Disclosure Summary: The authors have nothing to disclose.

First Published Online July 26, 2007

Abbreviations: AVP, Arginine vasopressin; BK, large-conductance calcium- and voltage-activated potassium; CORT, corticosterone; HPA, hypothalamic-pituitary-adrenal; POMC, proopiomelanocortin; pPVN, parvocellular paraventricular nucleus; STREX, stress axis-regulated exon; TBS, Tris-buffered saline; TBS-T, Triton X-100 in TBS; WT, wild type.

Received March 7, 2007.

Accepted for publication July 17, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Herman JP, Figueiredo H, Mueller NK, Ulrich-Lai Y, Ostrander MM, Choi DC, Cullinan WE 2003 Central mechanisms of stress integration: hierarchical circuitry controlling hypothalamo-pituitary-adrenocortical responsiveness. Front Neuroendocrinol 24:151–180[CrossRef][Medline]
  2. Antoni FA 1986 Hypothalamic control of adrenocorticotropin secretion: advances since the discovery of 41-residue corticotropin-releasing factor. Endocr Rev 7:351–378[Abstract/Free Full Text]
  3. Dallman MF, Akana SF, Cascio CS, Darlington DN, Jacobson L, Levin N 1987 Regulation of ACTH secretion: variations on a theme of B. Recent Prog Horm Res 43:113–173[Medline]
  4. Lai GJ, McCobb DP 2002 Opposing actions of adrenal androgens and glucocorticoids on alternative splicing of Slo potassium channels in bovine chromaffin cells. Proc Natl Acad Sci USA 99:7722–7727[Abstract/Free Full Text]
  5. Lai GJ, McCobb DP 2006 Regulation of alternative splicing of Slo K+ channels in adrenal and pituitary during the stress hyporesponsive period of rat development. Endocrinology 147:3961–3967[Abstract/Free Full Text]
  6. Xie J, McCobb DP 1998 Control of alternative splicing of potassium channels by stress hormones. Science 280:443–446[Abstract/Free Full Text]
  7. Lovell PV, McCobb DP 2001 Pituitary control of BK potassium channel function and intrinsic firing properties of adrenal chromaffin cells. J Neurosci 21:3429–3442[Abstract/Free Full Text]
  8. McCobb DP, Hara Y, Lai GJ, Mahmoud SF, Flugge G 2003 Subordination stress alters alternative splicing of the Slo gene in tree shrew adrenals. Horm Behav 43:180–186[CrossRef][Medline]
  9. Lovell PV, King JT, McCobb DP 2004 Acute modulation of adrenal chromaffin cell BK channel gating and cell excitability by glucocorticoids. J Neurophysiol 91:561–570[Abstract/Free Full Text]
  10. Huang MH, So E, Liu YC, Wu SN 2006 Glucocorticoids stimulate the activity of large-conductance Ca2+-activated K+ channels in pituitary GH3 and AtT-20 cells via a non-genomic mechanism. Steroids 71:129–140[CrossRef][Medline]
  11. King JT, Lovell PV, Rishniw M, Kotlikoff MI, Zeeman ML, McCobb DP 2006 β2 and β4 subunits of BK channels confer differential sensitivity to acute modulation by steroid hormones. J Neurophysiol 95:2878–2888[Abstract/Free Full Text]
  12. Shipston MJ, Kelly JS, Antoni FA 1996 Glucocorticoids block protein kinase A inhibition of calcium-activated potassium channels. J Biol Chem 271:9197–9200[Abstract/Free Full Text]
  13. Tian L, Philp JA, Shipston MJ 1999 Glucocorticoid block of protein kinase C signalling in mouse pituitary corticotroph AtT20 D16:16 cells. J Physiol 516:757–768[Abstract/Free Full Text]
  14. Tian L, Duncan RR, Hammond MSL, Coghill, LS, Wen H, Rusinova R, Clark AG, Levitan IB, Shipston MJ 2001 Alternative splicing switches potassium channel sensitivity to protein phosphorylation. J Biol Chem 276:7717–7720[Abstract/Free Full Text]
  15. Tian L, Hammond MS, Florance H, Antoni FA, Shipston MJ 2001 Alternative splicing determines sensitivity of murine calcium-activated potassium channels to glucocorticoids. J Physiol 537:57–68[Abstract/Free Full Text]
  16. Latchford KJ, Ferguson AV 2005 Angiotensin depolarizes parvocellular neurons in paraventricular nucleus through modulation of putative nonselective cationic and potassium conductances. Am J Physiol 289:R52–R58
  17. Luther JA, Daftary SS, Boudaba C, Gould GC, Halmos KC, Tasker JG 2002 Neurosecretory and non-neurosecretory parvocellular neurones of the hypothalamic paraventricular nucleus express distinct electrophysiological properties. J Neuroendocrinol 14:929–932[CrossRef][Medline]
  18. Ritchie AK, Kuryshev YA, Childs GV 1996 Corticotropin-releasing hormone and calcium signalling. Trends Endocrinol Metab 10:365–369
  19. Sausbier U, Sausbier M, Sailer CA, Arntz C, Knaus HG, Neuhuber W, Ruth P 2005 Ca2+-activated K+ channels of the BK-type in the mouse brain. Histochem Cell Biol 125:725–741[CrossRef][Medline]
  20. Chen L, Tian L, MacDonald SHF, McClafferty H, Hammond MSL, Huibant JM, Ruth P, Knaus HG, Shipston MJ 2005 Functionally diverse complement of large conductance calcium- and voltage-activated potassium channel (BK) {alpha}-subunits generated from a single site of splicing. J Biol Chem 280:33599–33609[Abstract/Free Full Text]
  21. Sausbier M, Hu H, Arntz C, Feil S, Kamm S, Adelsberger H, Sausbier U, Sailer CA, Feil R, Hofmann F, Korth M, Shipston MJ, Knaus HG, Wolfer DP, Pedroarena CM, Storm JF, Ruth P 2004 Cerebellar ataxia and Purkinje cell dysfunction caused by Ca2+-activated K+ channel deficiency. Proc Natl Acad Sci USA 101:9474–9478[Abstract/Free Full Text]
  22. Lundblad JR, Roberts JL 1988 Regulation of proopiomelanocortin gene expression in pituitary. Endocr Rev 9:135–157[Abstract/Free Full Text]
  23. Chan RKW, Brown ER, Ericsson A, Kovacs KJ, Sawchenko PE 1993 A comparison of 2 immediate-early genes, c-Fos and NGFI-B, as markers for functional activation in stress-related neuroendocrine circuitry. J Neurosci 13:5126–5138[Abstract]
  24. Douglas AJ, Brunton PJ, Bosch OJ, Russell JA, Neumann ID 2003 Neuroendocrine responses to stress in mice: hyporesponsiveness in pregnancy and parturition. Endocrinology 144:5268–5276[Abstract/Free Full Text]
  25. Sailer CA, Kaufmann WA, Kogler M, Chen L, Sausbier U, Ottersen OP, Ruth P, Shipston MJ, Knaus HG 2006 Immunolocalization of BK channels in hippocampal pyramidal neurons. Eur J Neurosci 24:442–454[CrossRef][Medline]
  26. Pacak K, Palkovits M 2001 Stressor specificity of central neuroendocrine responses: implications for stress-related disorders. Endocr Rev 22:502–548[Abstract/Free Full Text]
  27. Hodgkinson SC, Allolio B, Landon J, Lowry PJ 1984 Development of a non-extracted 2-site immunoradiometric assay for corticotropin utilizing extreme amino-terminally and carboxy-terminally directed antibodies. Biochem J 218:703–711[Medline]
  28. Keith LD, Winslow JR, Reynolds RW 1978 A general procedure for estimation of corticosteroid response in individual rats. Steroids 31:523–531[CrossRef][Medline]
  29. Muller MB, Zimmermann S, Sillaber I, Hagemeyer TP, Deussing JM, Timpl P, Kormann MSD, Droste SK, Kuhn R, Reul JM, Holsboer F, Wurst W 2003 Limbic corticotropin-releasing hormone receptor 1 mediates anxiety-related behavior and hormonal adaptation to stress. Nat Neurosci 6:1100–1107[CrossRef][Medline]
  30. Preil J, Muller MB, Gesing A, Reul JM, Sillaber I, van Gaalen MM, Landgrebe J, Holsboer F, Stenzel-Poore M, Wurst W 2001 Regulation of the hypothalamic-pituitary-adrenocortical system in mice deficient for CRH receptors 1 and 2. Endocrinology 142:4946–4955[Abstract/Free Full Text]
  31. Gibbs DM, Vale WW 1982 Presence of corticotropin releasing factor-like immunoreactivity in hypophysial portal blood. Endocrinology 111:1418–1420[Abstract/Free Full Text]
  32. Koenig JI, Meltzer HY, Devane GD, Gudelsky GA 1986 The concentration of arginine vasopressin in pituitary stalk plasma of the rat after adrenalectomy or morphine. Endocrinology 118:2534–2539[Abstract/Free Full Text]
  33. Recht LD, Hoffman DL, Haldar J, Silverman AJ, Zimmerman EA 1981 Vasopressin concentrations in hypophysial portal plasma: insignificant reduction following removal of the posterior pituitary gland. Neuroendocrinology 33:88–90[CrossRef][Medline]
  34. Nicholson RC, King BR, Smith R 2004 Complex regulatory interactions control CRH gene expression. Front Biosci 9:32–39[Medline]
  35. Watts AG 2005 Glucocorticoid regulation of peptide genes in neuroendocrine CRH neurons: a complexity beyond negative feedback. Front Neuroendocrinol 26:109–130[CrossRef][Medline]
  36. Shao LR, Halvorsrud R, Borg-Graham L, Storm JF 1999 The role of BK-type Ca2+-dependent K+ channels in spike broadening during repetitive firing in rat hippocampal pyramidal cells. J Physiol 521:135–146[Abstract/Free Full Text]
  37. Raffaelli G, Saviane C, Mohajerani MH, Pedarzani P, Cherubini E 2004 BK potassium channels control transmitter release at CA3-CA3 synapses in the rat hippocampus. J Physiol 557:147–157[Abstract/Free Full Text]
  38. Hu H, Shao L-R, Chavoshy S, Gu N, Trieb M, Behrens R, Laake P, Pongs O, Knaus HG, Ottersen OP, Storm JF 2001 Presynaptic Ca2+-activated K+ channels in glutamatergic hippocampal terminals and their role in spike repolarization and regulation of transmitter release. J Neurosci 21:9585–9597[Abstract/Free Full Text]
  39. Brunton PJ, Meddle SL, Ma S, Ochedalski T, Douglas AJ, Russell JA 2005 Endogenous opioids and attenuated hypothalamic-pituitary-adrenal axis responses to immune challenge in pregnant rats. J Neurosci 25:5117–5126[Abstract/Free Full Text]
  40. van Goor F, Li YX, Stojilkovic SS 2001 Paradoxical role of large-conductance calcium-activated K+ (BK) channels in controlling action potential-driven Ca2+ entry in anterior pituitary cells. J Neurosci 21:5902–5915[Abstract/Free Full Text]
  41. Yamamori E, Asai M, Yoshida M, Takano K, Itoi K, Oiso Y, Iwasaki Y 2004 Calcium/calmodulin kinase IV pathway is involved in the transcriptional regulation of the corticotropin-releasing hormone gene promoter in neuronal cells. J Mol Endocrinol 33:639–649[Abstract/Free Full Text]
  42. Imaki T, Katsumata H, Konishi SI, Kasagi Y, Minami S 2003 Corticotropin-releasing factor type-1 receptor mRNA is not induced in mouse hypothalamus by either stress or osmotic stimulation. J Neuroendocrinol 15:916–924[CrossRef][Medline]
  43. Xie J, Jan C, Stoilov P, Park J, Black DL 2005 A consensus CaMK IV-responsive RNA sequence mediates regulation of alternative exons in neurons. RNA 11:1825–1834[Abstract/Free Full Text]
  44. Xie J, Black DL 2001 A CaMK IV responsive RNA element mediates depolarization- induced alternative splicing of ion channels. Nature 410:936–939[CrossRef][Medline]
  45. Watts AG, Tanimura S, Sanchez-Watts G 2004 Corticotropin-releasing hormone and arginine vasopressin gene transcription in the hypothalamic paraventricular nucleus of unstressed rats: daily rhythms and their interactions with corticosterone. Endocrinology 145:529–540[Abstract/Free Full Text]
  46. Meredith AL, Wiler SW, Miller BH, Takahashi JS, Fodor AA, Ruby NF, Aldrich RW 2006 BK calcium-activated potassium channels regulate circadian behavioural rhythms and pacemaker output. Nat Neurosci 9:1041–1047[CrossRef][Medline]
  47. Dopico AM, Widmer H, Wang G, Lemos JR, Treistman SN 1999 Rat supraoptic magnocellular neurones show distinct large conductance, Ca2+-activated K+ channel subtypes in cell bodies versus nerve endings. J Physiol 519:101–114[Abstract/Free Full Text]
  48. Benjannet S, Rondeau N, Day R, Chretien M, Seidah NG 1991 Pc1 and Pc2 are proprotein convertases capable of cleaving proopiomelanocortin at distinct pairs of basic residues. Proc Natl Acad Sci USA 88:3564–3568[Abstract/Free Full Text]
  49. Zhu XR, Zhou A, Dey A, Norrbom C, Carroll R, Zhang CL, Laurent V, Lindberg I, Ugleholdt R, Holst JJ, Steiner DF 2002 Disruption of PC1/3 expression in mice causes dwarfism and multiple neuroendocrine peptide processing defects. Proc Natl Acad Sci USA 99:10293–10298[Abstract/Free Full Text]
  50. Sausbier M, Arntz C, Bucurenciu I, Zhao H, Zhou XB, Sausbier U, Feil S, Kamm S, Essin K, Sailer CA, Abdullah U, Krippeit-Drews P, Feil R, Hofmann F, Knaus HG, Kenyon C, Shipston MJ, Storm JF, Neuhuber W, Korth M, Schubert R, Gollasch M, Ruth P 2005 Elevated blood pressure linked to primary hyperaldosteronism and impaired vasodilation in BK channel-deficient mice. Circulation 112:60–68[Abstract/Free Full Text]
  51. McEwen BS, Wingfield JC 2003 The concept of allostasis in biology and biomedicine. Horm Behav 43:2–15[CrossRef][Medline]
  52. Brunton PJ, Bales J, Russell JA 2006 Neuroendocrine stress but not feeding responses to centrally administered neuropeptide Y are suppressed in pregnant rats. Endocrinology 147:3737–3745[Abstract/Free Full Text]
  53. Brunton PJ, Russell JA 2003 Hypothalamic-pituitary-adrenal responses to centrally administered orexin-A are suppressed in pregnant rats. J Neuroendocrinol 15:633–637[CrossRef][Medline]
  54. Ma S, Shipston MJ, Morilak D, Russell JA 2005 Reduced hypothalamic vasopressin secretion underlies attenuated adrenocorticotropin stress responses in pregnant rats. Endocrinology 146:1626–1637[Abstract/Free Full Text]
  55. Johnstone HA, Wigger A, Douglas AJ, Neumann ID, Landgraf R, Seckl JR, Russell JA 2000 Attenuation of hypothalamic-pituitary-adrenal axis stress responses in late pregnancy: changes in feedforward and feedback mechanisms. J Neuroendocrinol 12:811–822[CrossRef][Medline]
  56. Neumann ID, Johnstone HA, Hatzinger M, Liebsch G, Shipston MJ, Russell JA, Landgraf R, Douglas AJ 1998 Attenuated neuroendocrine responses to emotional and physical stressors in pregnant rats involve adenohypophysial changes. J Physiol 508:289–300[Abstract/Free Full Text]
  57. Song M, Zhu N, Olcese R, Barila B, Toro L, Stefani E 1999 Hormonal control of protein expression and mRNA levels of the MaxiK channel {alpha}-subunit in myometrium. FEBS Lett 460:427–432[CrossRef][Medline]
  58. Zhu N, Eghbali M, Helguera G, Song M, Stefani E, Toro L 2005 Alternative splicing of Slo channel gene programmed by estrogen, progesterone and pregnancy. FEBS Lett 579:4856–4860[Medline]
  59. Matharoo-Ball B, Ashford MLJ, Arulkumaran S, Khan RN 2003 Down-regulation of the {alpha}- and β-subunits of the calcium- activated potassium channel in human myometrium with parturition. Biol Reprod 68:2135–2141[Abstract/Free Full Text]
  60. Benkusky NA, Fergus DJ, Zucchero TM, England SK 2000 Regulation of the Ca2+-sensitive domains of the maxi-K channel in the mouse myometrium during gestation. J Biol Chem 275:27712–27719[Abstract/Free Full Text]
  61. Eghbali M, Toro L, Stefani E 2003 Diminished surface clustering and increased perinuclear accumulation of large conductance Ca2+-activated K+ channel in mouse myometrium with pregnancy. J Biol Chem 278:45311–45317[Abstract/Free Full Text]
  62. Franklin KBJ, Paxinos G 1997 The mouse brain in stereotaxic coordinates. San Diego: Academic Press



This article has been cited by other articles:


Home page
 Proc. Natl. Acad. Sci. USAHome page
P. R. Grimm, D. L. Irsik, D. C. Settles, J. D. Holtzclaw, and Steven. C. Sansom
Hypertension of Kcnmb1-/- is linked to deficient K secretion and aldosteronism
PNAS, July 14, 2009; 106(28): 11800 - 11805.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Brunton, P. J.
Right arrow Articles by Shipston, M. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Brunton, P. J.
Right arrow Articles by Shipston, M. J.
Right arrowPubmed/NCBI databases
*Gene*GEO Profiles
*HomoloGene*UniGene
*Compound via MeSH
*Substance via MeSH
Hazardous Substances DB
*HYDROCORTISONE

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals