Impact of sympathetic nervous system activity on post‐exercise flow‐mediated dilatation in humans

AbstractTransient reduction in vascular function following systemic large muscle group exercise has previously been reported in humans. The mechanisms responsible are currently unknown. We hypothesised that sympathetic nervous system activation, induced by cycle ergometer exercise, would contribute to post‐exercise reductions in flow‐mediated dilatation (FMD). Ten healthy male subjects (28 ± 5 years) undertook two 30 min sessions of cycle exercise at 75% HR_max. Prior to exercise, individuals ingested either a placebo or an α₁‐adrenoreceptor blocker (prazosin; 0.05 mg kg⁻¹). Central haemodynamics, brachial artery shear rate (SR) and blood flow profiles were assessed throughout each exercise bout and in response to brachial artery FMD, measured prior to, immediately after and 60 min after exercise. Cycle exercise increased both mean and antegrade SR (P < 0.001) with retrograde SR also elevated under both conditions (P < 0.001). Pre‐exercise FMD was similar on both occasions, and was significantly reduced (27%) immediately following exercise in the placebo condition (t‐test, P = 0.03). In contrast, FMD increased (37%) immediately following exercise in the prazosin condition (t‐test, P = 0.004, interaction effect P = 0.01). Post‐exercise FMD remained different between conditions after correction for baseline diameters preceding cuff deflation and also post‐deflation SR. No differences in FMD or other variables were evident 60 min following recovery. Our results indicate that sympathetic vasoconstriction competes with endothelium‐dependent dilator activity to determine post‐exercise arterial function. These findings have implications for understanding the chronic impacts of interventions, such as exercise training, which affect both sympathetic activity and arterial shear stress.

Abbreviations

BF

blood flow

CO

cardiac output

FMD

flow‐mediated dilatation

HR

heart rate

LBNP

lower body negative pressure

MAP

mean arterial pressure

MSNA

muscle sympathetic nerve activity

SNS

sympathetic nervous system

SR

shear rate

SR_AUC

shear rate area under curve

SV

stroke volume

TPRi

total peripheral resistance index

     

Signalling beyond photon absorption: extracellular retinoids and growth factors modulate rod photoreceptor sensitivity

Key points

• We propose that the end product of chromophore bleaching in rod photoreceptors, all‐trans retinol, is part of a feedback loop that increases the sensitivity of the phototransduction cascade in rods.
• A previously described light‐induced hypersensitivity in rods, termed adaptive potentiation, is reduced by exogenously applied all‐trans retinol but not all‐trans retinal.
• This potentiation is produced by insulin‐like growth factor‐1, whose binding proteins are located in the extracellular matrix, even in our isolated retina preparation after removal of the retinal pigmented epithelium.
• Simple modelling suggests that the light stimuli used in the present study will produce sufficient all‐trans retinol within the interphotoreceptor matrix to explain the potentiation effect.

Abstract

Photoreceptors translate the absorption of photons into electrical signals for propagation through the visual system. Mammalian photoreceptor signalling has largely been studied in isolated cells, and such studies have necessarily avoided the complex environment of supportive proteins that surround the photoreceptors. The interphotoreceptor matrix (IPM) contains an array of proteins that aid in both structural maintenance and cellular homeostasis, including chromophore turnover. In signalling photon absorption, the chromophore 11‐cis retinal is first isomerized to all‐trans retinal, followed by conversion to all‐trans retinol (ROL) for removal from the photoreceptor. Interphotoreceptor retinoid‐binding protein (IRBP) is the most abundant protein in the IPM, and it promotes the removal of bleached chromophores and recycling in the nearby retinal pigment epithelium. By studying the light responses of isolated mouse retinas, we demonstrate that ROL can act as a feedback signal onto photoreceptors that influences the sensitivity of phototransduction. In addition to IRBP, the IPM also contains insulin‐like growth factor‐1 (IGF‐1) and its associated binding proteins, although their functions have not yet been described. We demonstrate that extracellular application of physiological concentrations of IGF‐1 can increase rod photoreceptor sensitivity in mammalian retinas. We also determine that chromophores and growth factors can limit the range of a newly described form of photoreceptor light adaptation. Finally, fluorescent antibodies demonstrate the presence of IRBP and IGFBP‐3 in isolated retinas. A simple model of the formation and release of ROL into the extracellular space quantitatively describes this novel feedback loop.

Abbreviations

AP

adaptive potentiation

AP‐4

(±)‐2‐amino‐4‐phosphonobutyric acid

CNG

cyclic nucleotide gated

DAPI

4′,6‐diamidino‐2‐phenylindole

ERG

electroretinography

FGF

fibroblast growth factor

IGF‐1

insulin‐like growth factor‐1

IGFBP

insulin‐like growth factor binding protein

IGF‐1R

insulin‐like growth factor‐1 receptor

IPM

interphotoreceptor matrix

IR

insulin receptor

IRBP

interphotoreceptor retinoid‐binding protein

RAL

all‐trans retinal

ROL

all‐trans retinol

ROS

rod outer segment

RPE

retinal pigmented epithelium

     

Endogenous casein kinase‐1 modulates NMDA receptor activity of hypothalamic presympathetic neurons and sympathetic outflow in hypertension

Key points

• Increased NMDA receptor activity and excitability of presympathetic neurons in the hypothalamus can increase sympathetic nerve discharges leading to hypertension.
• In this study, we determined how protein kinases and phosphatases are involved in regulating NMDA receptor activity and firing activity of presympathetic neurons in the hypothalamus in normotensive and hypertensive rats.
• We show that casein kinase‐1 inhibition increases NMDA receptor activity and excitability of presympathetic neurons in the hypothalamus and augments sympathetic nerve discharges in normotensive, but not in hypertensive, rats.
• Our data indicate that casein kinase‐1 tonically regulates NMDA receptor activity by interacting with casein kinase‐2 and protein phosphatases in the hypothalamus and that imbalance of NMDA receptor phosphorylation can augment the excitability of hypothalamic presympathetic neurons and sympathetic nerve discharges in hypertension.
• These findings help us understand the neuronal mechanism of hypertension, and reducing the NMDA receptor phosphorylation level may be effective for treating neurogenic hypertension.

Abstract

Increased N‐methyl‐d‐aspartate receptor (NMDAR) activity in the paraventricular nucleus (PVN) of the hypothalamus is involved in elevated sympathetic outflow in hypertension. However, the molecular mechanisms underlying augmented NMDAR activity in hypertension remain unclear. In this study, we determined the role of casein kinase‐1 (CK1) in regulating NMDAR activity in the PVN. NMDAR‐mediated excitatory postsynaptic currents (EPSCs) and puff NMDA‐elicited currents were recorded in spinally projecting PVN neurons in spontaneously hypertensive rats (SHRs) and Wistar–Kyoto (WKY) rats. The basal amplitudes of evoked NMDAR‐EPSCs and puff NMDA currents were significantly higher in SHRs than in WKY rats. The CK1 inhibitor PF4800567 or PF670462 significantly increased the amplitude of NMDAR‐EPSCs and puff NMDA currents in PVN neurons in WKY rats but not in SHRs. PF4800567 caused an NMDAR‐dependent increase in the excitability of PVN neurons only in WKY rats. Also, the CK1ε protein level in the PVN was significantly lower in SHRs than in WKY rats. Furthermore, intracerebroventricular infusion of PF4800567 increased blood pressure and lumbar sympathetic nerve activity in WKY rats, and this effect was eliminated by microinjection of the NMDAR antagonist into the PVN. In addition, PF4800567 failed to increase NMDAR activity in brain slices of WKY rats pretreated with the protein phosphatase 1/2A, calcineurin, or casein kinase‐2 inhibitor. Our findings suggest that CK1 tonically suppresses NMDAR activity in the PVN by reducing the NMDAR phosphorylation level. Diminished CK1 activity may contribute to potentiated glutamatergic synaptic input to PVN presympathetic neurons and elevated sympathetic vasomotor tone in neurogenic hypertension.

Abbreviations

ABP

arterial blood pressure

aCSF

artificial cerebrospinal fluid

AMPA

α‐amino‐3‐hydroxy‐4‐isoxazoleproprionic acid

AMPAR

AMPA receptor

CK1

casein kinase‐1

CK2

casein kinase‐2

CNQX

6‐cyano‐7‐nitroquinoxaline‐2,3‐dione

EPSC

excitatory postsynaptic current

GABA

γ‐aminobutyric acid

HR

heart rate

LSNA

lumbar sympathetic nerve activity

NMDA

N‐methyl‐d‐aspartate

NMDAR

NMDA receptor

PVN

paraventricular nucleus

PP1

protein phosphatase 1

PP2A

protein phosphatase 2A

PP2B

protein phosphatase 2B

PKC

protein kinase C

RVLM

rostral ventrolateral medulla

SHR

spontaneously hypertensive rat

WKY

Wistar–Kyoto

     

Hypoxic induction of T‐type Ca2+ channels in rat cardiac myocytes: role of HIF‐1α and RhoA/ROCK signalling

Key points

• T‐type Ca²⁺ channels are expressed in the ventricular myocytes of the fetal and perinatal heart, but are downregulated as development progresses. However, these channels are re‐expressed in adult cardiomyocytes under pathological conditions.
• Hypoxia induces the upregulation of the T‐type Ca²⁺ channel Ca_v3.2 mRNA in cardiac myocytes, whereas Ca_v3.1 mRNA is not significantly altered.
• The effect of hypoxia on Ca_v3.2 mRNA requires hypoxia inducible factor‐1α (HIF‐1α) stabilization and involves the small monomeric G‐protein RhoA and its effector ROCKI.
• Our results suggest that the hypoxic regulation of the Ca_v3.2 channels may be involved in the increased probability of developing arrhythmias observed in ischemic situations, and in the pathogenesis of diseases associated with hypoxic Ca²⁺ overload.

Abstract

T‐type Ca²⁺ channels are expressed in the ventricular myocytes of the fetal and perinatal heart, but are normally downregulated as development progresses. Interestingly, however, these channels are re‐expressed in adult cardiomyocytes under pathological conditions. We investigated low voltage‐activated T‐type Ca²⁺ channel regulation in hypoxia in rat cardiomyocytes. Molecular studies revealed that hypoxia induces the upregulation of Ca_v3.2 mRNA, whereas Ca_v3.1 mRNA is not significantly altered. The effect of hypoxia on Ca_v3.2 mRNA was time‐ and dose‐dependent, and required hypoxia inducible factor‐1α (HIF‐1α) stabilization. Patch‐clamp recordings confirmed that T‐type Ca²⁺ channel currents were upregulated in hypoxic conditions, and the addition of 50 μm NiCl₂ (a T‐type channel blocker) demonstrated that the Ca_v3.2 channel is responsible for this upregulation. This increase in current density was not accompanied by significant changes in the Ca_v3.2 channel electrophysiological properties. The small monomeric G‐protein RhoA and its effector Rho‐associated kinase I (ROCKI), which are known to play important roles in cardiovascular physiology, were also upregulated in neonatal rat ventricular myocytes subjected to hypoxia. Pharmacological experiments indicated that both proteins were involved in the observed upregulation of the Ca_v3.2 channel and the stabilization of HIF‐1α that occurred in response to hypoxia. These results suggest a possible role for Ca_v3.2 channels in the increased probability of developing arrhythmias observed in ischaemic situations, and in the pathogenesis of diseases associated with hypoxic Ca²⁺ overload.

Abbreviations

Ca_V channels

voltage‐gated Ca²⁺ channels

DRB

5,6‐dichloro‐1‐β‐d‐ribofuranosylbenzimidazole

DMOG

dimethyloxalylglycine

DPI

diphenyliodonium

HIF

hypoxia inducible factor

NMDG

N‐methyl‐d‐glucamine

NRVMs

neonatal rat ventricular myocytes

P_O2

partial pressure of oxygen

ROCK

Rho‐associated kinase

ROS

reactive oxygen species

siRNA

small interfering RNA

     

Murine startle mutant Nmf11 affects the structural stability of the glycine receptor and increases deactivation

AbstractDysfunctional glycinergic inhibitory transmission underlies the debilitating neurological condition, hyperekplexia, which is characterised by exaggerated startle reflexes, muscle hypertonia and apnoea. Here we investigated the N46K missense mutation in the GlyR α1 subunit gene found in the ethylnitrosourea (ENU) murine mutant, Nmf11, which causes reduced body size, evoked tremor, seizures, muscle stiffness, and morbidity by postnatal day 21. Introducing the N46K mutation into recombinant GlyR α1 homomeric receptors, expressed in HEK cells, reduced the potencies of glycine, β‐alanine and taurine by 9‐, 6‐ and 3‐fold respectively, and that of the competitive antagonist strychnine by 15‐fold. Replacing N46 with hydrophobic, charged or polar residues revealed that the amide moiety of asparagine was crucial for GlyR activation. Co‐mutating N61, located on a neighbouring β loop to N46, rescued the wild‐type phenotype depending on the amino acid charge. Single‐channel recording identified that burst length for the N46K mutant was reduced and fast agonist application revealed faster glycine deactivation times for the N46K mutant compared with the WT receptor. Overall, these data are consistent with N46 ensuring correct alignment of the α1 subunit interface by interaction with juxtaposed residues to preserve the structural integrity of the glycine binding site. This represents a new mechanism by which GlyR dysfunction induces startle disease.

Abbreviations

DMEM

Dulbecco''s modified Eagle medium

DR

dose‐ratio

ENU

ethylnitrosourea

GFP

green fluorescent protein

GLRA1

GlyR α1 subunit gene

GLRB

GlyR β subunit gene

GluCl

glutamate‐activated Cl^˗ channel

GlyR

glycine receptor

HEK‐293

human embryonic kidney 293 cells

K_B

equilibrium dissociation constant

V_patch

trans‐patch potential

WT

wild type

     

Nitrergic signalling via interstitial cells of Cajal regulates motor activity in murine colon

Key points

• Dysregulation of nitric oxide (NO) signalling is associated with GI motility dysfunctions like chronic constipation, achalasia or Hirschsprung''s disease. The inhibitory effect of NO is mainly exerted via NO‐sensitive guanylyl cyclase (NO‐GC) which is found in different gastrointestinal (GI) cell types including smooth muscle cells (SMCs) and interstitial cells of Cajal (ICC).
• Here, we focus on the investigation of NO‐GC function in murine colon. Using cell‐specific knock‐out mice, we demonstrate that NO‐GC is expressed in myenteric ICC of murine colon and participates in regulation of colonic spontaneous contractions in longitudinal smooth muscle.
• We report a novel finding that basal enteric NO release acts via myenteric ICC to influence the generation of spontaneous contractions whereas the effects of elevated endogenous NO are mediated by SMCS in the murine proximal colon.
• These results help in understanding possible pathological mechanisms involved in slowed colonic action and colonic inertia.

Abstract

In the enteric nervous systems, NO is released from nitrergic neurons as a major inhibitory neurotransmitter. NO acts via NO‐sensitive guanylyl cyclase (NO‐GC), which is found in different gastrointestinal (GI) cell types including smooth muscle cells (SMCs) and interstitial cells of Cajal (ICC). The precise mechanism of nitrergic signalling through these two cell types to regulate colonic spontaneous contractions is not fully understood yet. In the present study we investigated the impact of endogenous and exogenous NO on colonic contractile motor activity using mice lacking nitric oxide‐sensitive guanylyl cyclase (NO‐GC) globally and specifically in SMCs and ICC. Longitudinal smooth muscle of proximal colon from wild‐type (WT) and knockout (KO) mouse strains exhibited spontaneous contractile activity ex vivo. WT and smooth muscle‐specific guanylyl cyclase knockout (SMC‐GCKO) colon showed an arrhythmic contractile activity with varying amplitudes and frequencies. In contrast, colon from global and ICC‐specific guanylyl cyclase knockout (ICC‐GCKO) animals showed a regular contractile rhythm with constant duration and amplitude of the rhythmic contractions. Nerve blockade (tetrodotoxin) or specific blockade of NO signalling (l‐NAME, ODQ) did not significantly affect contractions of GCKO and ICC‐GCKO colon whereas the arrhythmic contractile patterns of WT and SMC‐GCKO colon were transformed into uniform motor patterns. In contrast, the response to electric field‐stimulated neuronal NO release was similar in SMC‐GCKO and global GCKO. In conclusion, our results indicate that basal enteric NO release acts via myenteric ICC to influence the generation of spontaneous contractions whereas the effects of elevated endogenous NO are mediated by SMCs in the murine proximal colon.

Abbreviations

DEA‐NO

2‐(N,N‐diethylamino)‐diazenolate‐2‐oxide diethylammonium salt

EFS

electrical field stimulation

GCKO

guanylyl cyclase knockout

ICC

interstitial cells of Cajal

ICC‐GCKO

ICC‐specific guanylyl cyclase knockout

KO

knockout

NO

nitric oxide

NO‐GC

nitric oxide‐sensitive guanylyl cyclase

l‐NAME

N ^G‐nitro‐l‐arginine methyl ester

nNOS

neuronal nitric oxide synthase

ODQ

1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one

PKG

cGMP‐dependent protein kinase

SMC

smooth muscle cell

SMC‐GCKO

smooth muscle‐specific guanylyl cyclase knockout

TTX

tetrodotoxin

WT

wild‐type

     

GABAB receptor‐mediated feed‐forward circuit dysfunction in the mouse model of fragile X syndrome

Key points

• Cortico‐hippocampal feed‐forward circuits formed by the temporoammonic (TA) pathway exhibit a marked increase in excitation/inhibition ratio and abnormal spike modulation functions in Fmr1 knock‐out (KO) mice.
• Inhibitory, but not excitatory, synapse dysfunction underlies cortico‐hippocampal feed‐forward circuit abnormalities in Fmr1 KO mice.
• GABA release is reduced in TA‐associated inhibitory synapses of Fmr1 KO mice in a GABA_B receptor‐dependent manner.
• Inhibitory synapse and feed‐forward circuit defects are mediated predominately by presynaptic GABA_B receptor signalling in the TA pathway of Fmr1 KO mice.
• GABA_B receptor‐mediated inhibitory synapse defects are circuit‐specific and are not observed in the Schaffer collateral pathway‐associated inhibitory synapses in stratum radiatum.

Abstract

Circuit hyperexcitability has been implicated in neuropathology of fragile X syndrome, the most common inheritable cause of intellectual disability. Yet, how canonical unitary circuits are affected in this disorder remains poorly understood. Here, we examined this question in the context of the canonical feed‐forward inhibitory circuit formed by the temporoammonic (TA) branch of the perforant path, the major cortical input to the hippocampus. TA feed‐forward circuits exhibited a marked increase in excitation/inhibition ratio and major functional defects in spike modulation tasks in Fmr1 knock‐out (KO) mice, a fragile X mouse model. Changes in feed‐forward circuits were caused specifically by inhibitory, but not excitatory, synapse defects. TA‐associated inhibitory synapses exhibited increase in paired‐pulse ratio and in the coefficient of variation of IPSPs, consistent with decreased GABA release probability. TA‐associated inhibitory synaptic transmission in Fmr1 KO mice was also more sensitive to inhibition of GABA_B receptors, suggesting an increase in presynaptic GABA_B receptor (GABA_BR) signalling. Indeed, the differences in inhibitory synaptic transmission between Fmr1 KO and wild‐type (WT) mice were eliminated by a GABA_BR antagonist. Inhibition of GABA_BRs or selective activation of presynaptic GABA_BRs also abolished the differences in the TA feed‐forward circuit properties between Fmr1 KO and WT mice. These GABA_BR‐mediated defects were circuit‐specific and were not observed in the Schaffer collateral pathway‐associated inhibitory synapses. Our results suggest that the inhibitory synapse dysfunction in the cortico‐hippocampal pathway of Fmr1 KO mice causes hyperexcitability and feed‐forward circuit defects, which are mediated in part by a presynaptic GABA_BR‐dependent reduction in GABA release.

Abbreviations

AP

action potential

APV

2‐amino‐5‐phosphonopentanoic acid

CV

coefficient of variation

DNQX

6,7‐dinitroquinoxaline‐2,3‐dione

E/I

excitatory/inhibitory

EPSP

excitatory postsynaptic potential

FFI

feed‐forward inhibitory

FMRP

fragile X mental retardation protein

Fmr1

fragile X mental retardation 1

FWHM

full‐width half‐maximum

FXS

fragile X syndrome

GABA_AR

γ‐aminobutyric acid type A receptor

GABA_BR

γ‐aminobutyric acid type B receptor

IPSP

inhibitory postsynaptic potential

KO

knock‐out

PPR

paired‐pulse ratio

RMS

root mean square

SC

Schaffer collateral

SLM

stratum lacunosum moleculare

SR

stratum radiatum

TA

temporoammonic

WT

wild‐type

     

Perivascular tissue inhibits rho‐kinase‐dependent smooth muscle Ca2+ sensitivity and endothelium‐dependent H2S signalling in rat coronary arteries

Key points

• Local regulation of vascular resistance adjusts coronary blood flow to metabolic demand, although the mechanisms involved are not comprehensively understood
• We show that heart tissue surrounding rat coronary arteries releases diffusible factors that regulate vasoconstriction and relaxation
• Perivascular tissue reduces rho‐kinase‐dependent smooth muscle Ca²⁺ sensitivity and constriction of coronary arteries to serotonin, the thromboxane analogue U46619 and the α₁‐adrenergic agonist phenylephrine
• Endothelium‐dependent relaxation of coronary arteries in response to cholinergic stimulation is inhibited by perivascular tissue as a result of reduced endothelial Ca²⁺ responses and attenuated H₂S‐dependent signalling
• These results establish cellular mechanisms by which perivascular heart tissue can modify local vascular tone and coronary blood flow

Abstract

Interactions between perivascular tissue (PVT) and the vascular wall modify artery tone and contribute to local blood flow regulation. Using isometric myography, fluorescence microscopy, membrane potential recordings and phosphospecific immunoblotting, we investigated the cellular mechanisms by which PVT affects constriction and relaxation of rat coronary septal arteries. PVT inhibited vasoconstriction to thromboxane, serotonin and α₁‐adrenergic stimulation but not to depolarization with elevated extracellular [K⁺]. When PVT was wrapped around isolated arteries or placed at the bottom of the myograph chamber, a smaller yet significant inhibition of vasoconstriction was observed. Resting membrane potential, depolarization to serotonin or thromboxane stimulation, and resting and serotonin‐stimulated vascular smooth muscle [Ca²⁺]‐levels were unaffected by PVT. Serotonin‐induced vasoconstriction was almost abolished by rho‐kinase inhibitor Y‐27632 and modestly reduced by protein kinase C inhibitor bisindolylmaleimide X. PVT reduced phosphorylation of myosin phosphatase targeting subunit (MYPT) at Thr850 by ∼40% in serotonin‐stimulated arteries but had no effect on MYPT‐phosphorylation in arteries depolarized with elevated extracellular [K⁺]. The net anti‐contractile effect of PVT was accentuated after endothelial denudation. PVT also impaired vasorelaxation and endothelial Ca²⁺ responses to cholinergic stimulation. Methacholine‐induced vasorelaxation was mediated by NO and H₂S, and particularly the H₂S‐dependent (dl‐propargylglycine‐ and XE991‐sensitive) component was attenuated by PVT. Vasorelaxation to NO‐ and H₂S‐donors was maintained in arteries with PVT. In conclusion, cardiomyocyte‐rich PVT surrounding coronary arteries releases diffusible factors that reduce rho‐kinase‐dependent smooth muscle Ca²⁺ sensitivity and endothelial Ca²⁺ responses. These mechanisms inhibit agonist‐induced vasoconstriction and endothelium‐dependent vasorelaxation and suggest new signalling pathways for metabolic regulation of blood flow.

Abbreviations

8‐SPT

8‐(p‐sulphophenyl)theophylline

ACh

acetylcholine

AUC

area under the curve

Bis‐10

bisindolylmaleimide X

CSE

cystathionine γ‐lyase

EC

endothelial cell

K‐PSS

physiological saline solution with elevated [K⁺]

l‐NAME

N‐nitro‐l‐arginine methyl ester

MYPT

myosin phosphatase targeting subunit

PKC

protein kinase C

PPG

dl‐propargylglycine

PSS

physiological saline solution

PVT

perivascular tissue

SNAP

S‐nitroso‐N‐acetyl‐d,l‐penicillamine

SNP

sodium nitroprusside

VSMC

vascular smooth muscle cell

     

Contrasting actions of a convulsant barbiturate and its anticonvulsant enantiomer on the α1β3γ2L GABAA receptor account for their in vivo effects

Key Points

• Most barbiturates are anaesthetics but unexpectedly a few are convulsants whose mechanism of action is poorly understood.
• We synthesized and characterized a novel pair of chiral barbiturates that are capable of photolabelling their binding sites on GABA_A receptors. In mice the S‐enantiomer is a convulsant, but the R‐enantiomer is an anticonvulsant.
• The convulsant S‐enantiomer binds solely at an inhibitory site. It is both an open state inhibitor and a resting state inhibitor. Its action is pH independent, suggesting the pyrimidine ring plays little part in binding. The inhibitory site is not enantioselective because the R‐enantiomer inhibits with equal affinity.
• In contrast, only the anticonvulsant R‐enantiomer binds to the enhancing site on open channels, causing them to stay open longer. The enhancing site is enantioselective.
• The in vivo actions of the convulsant S‐enantiomer are accounted for by its interactions with GABA_A receptors.

Abstract

Most barbiturates are anaesthetics but a few unexpectedly are convulsants. We recently located the anaesthetic sites on GABA_A receptors (GABA_ARs) by photolabelling with an anaesthetic barbiturate. To apply the same strategy to locate the convulsant sites requires the creation and mechanistic characterization of a suitable agent. We synthesized enantiomers of a novel, photoactivable barbiturate, 1‐methyl‐5‐propyly‐5‐(m‐trifluoromethyldiazirinyl) phenyl barbituric acid (mTFD‐MPPB). In mice, S‐mTFD‐MPPB acted as a convulsant, whereas R‐mTFD‐MPPB acted as an anticonvulsant. Using patch clamp electrophysiology and fast solution exchange on recombinant human α₁β₃γ_2L GABA_ARs expressed in HEK cells, we found that S‐mTFD‐MPPB inhibited GABA‐induced currents, whereas R‐mTFD‐MPPB enhanced them. S‐mTFD‐MPPB caused inhibition by binding to either of two inhibitory sites on open channels with bimolecular kinetics. It also inhibited closed, resting state receptors at similar concentrations, decreasing the channel opening rate and shifting the GABA concentration–response curve to the right. R‐mTFD‐MPPB, like most anaesthetics, enhanced receptor gating by rapidly binding to allosteric sites on open channels, initiating a rate‐limiting conformation change to stabilized open channel states. These states had slower closing rates, thus shifting the GABA concentration–response curve to the left. Under conditions when most GABA_ARs were open, an inhibitory action of R‐mTFD‐MPPB was revealed that had a similar IC₅₀ to that of S‐mTFD‐MPPB. Thus, the inhibitory sites are not enantioselective, and the convulsant action of S‐mTFD‐MPPB results from its negligible affinity for the enhancing, anaesthetic sites. Interactions with these two classes of barbiturate binding sites on GABA_ARs underlie the enantiomers’ different pharmacological activities in mice.

Abbreviations

C

channel closed, resting state

CD₅₀

median clonic siezure dose

CI

confidence interval

G₂O

open channel state bound with two GABA molecules

GABA_AR

GABA receptor Type A

HEK

human embryonic kidney

I

peak current amplitude

I_max

maximal peak current amplitude

k₋₁

dissociation rate constant

k₊₁

binding rate constant

k_act

activation energy

LoRR

loss of righting reflex

MPPB

1‐methyl‐5‐phenyl‐5‐propyl‐barbituric acid

nAChR

nicotinic acetylcholine receptor

O

open channel state

O’

stabilized open channel state

pK

acid dissociation constant

PTZ

pentylenetetrazol

R‐mTFD‐MPAB

R‐5‐allyl‐1‐methyl‐5‐(m‐trifluoromethyl‐diazirynylphenyl) barbituric acid

R‐mTFD‐MPPB

R‐1‐methyl‐5‐propyly‐5‐(m‐trifluoromethyldiazirinyl) phenyl barbituric acid

S‐mTFD‐MPAB

S‐5‐allyl‐1‐methyl‐5‐(m‐trifluoromethyl‐diazirynylphenyl) barbituric acid

S‐mTFD‐MPPB

S‐1‐methyl‐5‐propyly‐5‐(m‐trifluoromethyldiazirinyl) phenyl barbituric acid

TID

3‐(trifluoromethyl)‐3‐(m‐iodophenyl) diazirine

α

channel closing rate

β

channel opening rate

     

Heart failure induces changes in acid‐sensing ion channels in sensory neurons innervating skeletal muscle

Key points

• Heart failure is characterized by an elevated sympathetic state and exercise intolerance, which is partially driven by exaggerated autonomic reflexes triggered by skeletal muscle afferents.
• Acid‐sensing ion channels (ASICs) are highly expressed in skeletal muscle afferents and contribute to exercise mediated reflexes.
• Here we show that ASIC currents recorded from isolated skeletal muscle sensory neurons display diminished pH sensitivity, altered desensitization kinetics, and faster recovery from desensitization in a mouse model of heart failure.
• These results indicate ASICs in muscle afferents are altered in heart failure, and may contribute to the associated sympathoexcitation and exercise intolerance.

Abstract

Heart failure is associated with diminished exercise capacity, which is driven, in part, by alterations in exercise‐induced autonomic reflexes triggered by skeletal muscle sensory neurons (afferents). These overactive reflexes may also contribute to the chronic state of sympathetic excitation, which is a major contributor to the morbidity and mortality of heart failure. Acid‐sensing ion channels (ASICs) are highly expressed in muscle afferents where they sense metabolic changes associated with ischaemia and exercise, and contribute to the metabolic component of these reflexes. Therefore, we tested if ASICs within muscle afferents are altered in heart failure. We used whole‐cell patch clamp to study the electrophysiological properties of acid‐evoked currents in isolated, labelled muscle afferent neurons from control and heart failure (induced by myocardial infarction) mice. We found that the percentage of muscle afferents that displayed ASIC‐like currents, the current amplitudes, and the pH dose–response relationships were not altered in mice with heart failure. On the other hand, the biophysical properties of ASIC‐like currents were significantly different in a subpopulation of cells (40%) from heart failure mice. This population displayed diminished pH sensitivity, altered desensitization kinetics, and very fast recovery from desensitization. These unique properties define these channels within this subpopulation of muscle afferents as being heteromeric channels composed of ASIC2a and ‐3 subunits. Heart failure induced a shift in the subunit composition of ASICs within muscle afferents, which significantly altered their pH sensing characteristics. These results might, in part, contribute to the changes in exercise‐mediated reflexes that are associated with heart failure.

Abbreviations

ASICs

acid‐sensing ion channels

BP

blood pressure

DiI

1,1‐dioctadecyl‐3,3,3,3 tetramethylindocarbocyanine perchlorate

DRG

dorsal root ganglion

EPR

exercise pressor reflex

HF

heart failure

HR

heart rate

     

Sodium nitrate alleviates functional muscle ischaemia in patients with Becker muscular dystrophy

AbstractBecker muscular dystrophy (BMD) is a progressive X‐linked muscle wasting disease for which there is no treatment. BMD is caused by in‐frame mutations in the gene encoding dystrophin, a structural cytoskeletal protein that also targets other proteins to the sarcolemma. Among these is neuronal nitric oxide synthase mu (nNOSμ), which requires specific spectrin‐like repeats (SR16/17) in dystrophin''s rod domain and the adaptor protein α‐syntrophin for sarcolemmal targeting. When healthy skeletal muscle is exercised, sarcolemmal nNOSμ‐derived nitric oxide (NO) attenuates α‐adrenergic vasoconstriction, thus optimizing perfusion. In the mdx mouse model of dystrophinopathy, this protective mechanism (functional sympatholysis) is defective, resulting in functional muscle ischaemia. Treatment with a NO‐donating non‐steroidal anti‐inflammatory drug (NSAID) alleviates this ischaemia and improves the murine dystrophic phenotype. In the present study, we report that, in 13 men with BMD, sympatholysis is defective mainly in patients whose mutations disrupt sarcolemmal targeting of nNOSμ, with the vasoconstrictor response measured as a decrease in muscle oxygenation (near infrared spectroscopy) to reflex sympathetic activation. Then, in a single‐arm, open‐label trial in 11 BMD patients and a double‐blind, placebo‐controlled cross‐over trial in six patients, we show that acute treatment with oral sodium nitrate, an inorganic NO donor without a NSIAD moiety, restores sympatholysis and improves post‐exercise hyperaemia (Doppler ultrasound). By contrast, sodium nitrate improves neither sympatholysis, nor hyperaemia in healthy controls. Thus, a simple NO donor recapitulates the vasoregulatory actions of sarcolemmal nNOS in BMD patients, and constitutes a putative novel therapy for this disease.

Abbreviations

BMD

Becker muscular dystrophy

deoxyHb

deoxyhaemoglobin

deoxyMb

deoxymyoglobin

DMD

Duchenne muscular dystrophy

LBNP

lower body negative pressure

MVC

maximal voluntary contraction

NIRS

near infrared spectroscopy

NO

nitric oxide

NO₂⁻

nitrite

NO₃⁻

nitrate

nNOSμ

neuronal nitric oxide synthase mu

NSAID

non‐steroidal anti‐inflammatory drug

PDE5

phosphodiesterase 5

TLS

total labile signal

     

μ opioid receptor activation hyperpolarizes respiratory‐controlling K?lliker–Fuse neurons and suppresses post‐inspiratory drive

Key points

• In addition to reductions in respiratory rate, opioids also cause aspiration and difficulty swallowing, indicating impairment of the upper airways. The Kölliker–Fuse (KF) maintains upper airway patency and a normal respiratory pattern.
• In this study, activation of μ opioid receptors in the KF reduced respiratory frequency and tidal volume in anaesthetized rats.
• Nerve recordings in an in situ preparation showed that activation of μ opioid receptors in the KF eliminated the post‐inspiration phase of the respiratory cycle.
• In brain slices, μ opioid agonists hyperpolarized a distinct population (61%) of KF neurons by activation of an inwardly rectifying potassium conductance.
• These results suggest that KF neurons that are hyperpolarized by opioids could contribute to opioid‐induced respiratory disturbances, particularly the impairment of upper airways.

Abstract

Opioid‐induced respiratory effects include aspiration and difficulty swallowing, suggesting impairment of the upper airways. The pontine Kölliker–Fuse nucleus (KF) controls upper airway patency and regulates respiration, in particular the inspiratory/expiratory phase transition. Given the importance of the KF in coordinating respiratory pattern, the mechanisms of μ opioid receptor activation in this nucleus were investigated at the systems and cellular level. In anaesthetized, vagi‐intact rats, injection of opioid agonists DAMGO or [Met⁵]enkephalin (ME) into the KF reduced respiratory frequency and amplitude. The μ opioid agonist DAMGO applied directly into the KF of the in situ arterially perfused working heart–brainstem preparation of rat resulted in robust apneusis (lengthened low amplitude inspiration due to loss of post‐inspiratory drive) that was rapidly reversed by the opioid antagonist naloxone. In brain slice preparations, activation of μ opioid receptors on KF neurons hyperpolarized a distinct population (61%) of neurons. As expected, the opioid‐induced hyperpolarization reduced the excitability of the neuron in response to either current injection or local application of glutamate. In voltage‐clamp recordings the outward current produced by the opioid agonist ME was concentration dependent, reversed at the potassium equilibrium potential and was blocked by BaCl₂, characteristics of a G protein‐coupled inwardly rectifying potassium (GIRK) conductance. The clinically used drug morphine produced an outward current in KF neurons with similar potency to morphine‐mediated currents in locus coeruleus brain slice preparations. Thus, the population of KF neurons that are hyperpolarized by μ opioid agonists are likely mediators of the opioid‐induced loss of post‐inspiration and induction of apneusis.

Abbreviations

ACSF

artificial cerebrospinal fluid

ANOVA

analysis of variance

AP

action potential

CTAP

d‐Phe‐Cys‐Tyr‐d‐Trp‐Arg‐Thr‐Pen‐Thr‐NH₂

CV

coefficient of variation

cVN

central vagus nerve

DAMGO

([d‐Ala², N‐Me‐Phe⁴, Gly⁵‐ol]‐enkephalin

DNQX

6,7‐dinitroquinoxaline‐2,3‐dione

E2

stage 2 expiration

GIRK

G protein‐coupled inwardly rectifying potassium

KF

Kölliker–Fuse

LC

locus coeruleus

ME

[Met]⁵enkephalin

NLX

naloxone

PB

parabrachial

PN

phrenic nerve; post‐I, post‐inspiration; preBötC, preBötzinger complex; scp, superior cerebellar peduncle; T _E, expiratory time; T _I, inspiratory time

     

The exercise‐induced biochemical milieu enhances collagen content and tensile strength of engineered ligaments

Key points

• Exercise acutely increases the concentrations of metabolites and hormones such as growth hormone (GH) and, to a lesser extent, insulin‐like growth factor 1 (IGF‐1); however, the biological function of this response is unclear.
• Pharmacological administration of these hormones stimulates collagen synthesis in muscle and tendon; however, whether the post‐exercise biochemical milieu has a similar action is unknown.
• Treating engineered ligaments with serum obtained from young healthy men after exercise resulted in more collagen and improved tensile strength over those treated with serum from resting men.
• Further, we show that the increase in collagen induced by post‐exercise serum (i) is not reproduced by treatment with recombinant GH or IGF‐1, and (ii) is associated with the activation of PI3 kinase/mTORC1 and ERK1/2 signalling.

Abstract

Exercise stimulates a dramatic change in the concentration of circulating hormones, such as growth hormone (GH), but the biological functions of this response are unclear. Pharmacological GH administration stimulates collagen synthesis; however, whether the post‐exercise systemic milieu has a similar action is unknown. We aimed to determine whether the collagen content and tensile strength of tissue‐engineered ligaments is enhanced by serum obtained post‐exercise. Primary cells from a human anterior cruciate ligament (ACL) were used to engineer ligament constructs in vitro. Blood obtained from 12 healthy young men 15 min after resistance exercise contained GH concentrations that were ∼7‐fold greater than resting serum (P < 0.001), whereas IGF‐1 was not elevated at this time point (P = 0.21 vs. rest). Ligament constructs were treated for 7 days with medium supplemented with serum obtained at rest (RestTx) or 15 min post‐exercise (ExTx), before tensile testing and collagen content analysis. Compared with RestTx, ExTx enhanced collagen content (+19%; 181 ± 33 vs. 215 ± 40 μg per construct P = 0.001) and ligament mechanical properties – maximal tensile load (+17%, P = 0.03 vs. RestTx) and ultimate tensile strength (+10%, P = 0.15 vs. RestTx). In a separate set of engineered ligaments, recombinant IGF‐1, but not GH, enhanced collagen content and mechanics. Bioassays in 2D culture revealed that acute treatment with post‐exercise serum activated mTORC1 and ERK1/2. In conclusion, the post‐exercise biochemical milieu, but not recombinant GH, enhances collagen content and tensile strength of engineered ligaments, in association with mTORC1 and ERK1/2 activation.

Abbreviations

ACL

anterior cruciate ligament

ERK

extracellular signal‐regulated kinase

GH

growth hormone

IGF‐1

insulin‐like growth factor 1

mTORC1

mechanistic/mammalian target of rapamycin complex 1

TGF‐β1

transforming growth factor‐β1

     

Kainate receptor pore‐forming and auxiliary subunits regulate channel block by a novel mechanism

Key points

• Kainate receptor heteromerization and auxiliary subunits, Neto1 and Neto2, attenuate polyamine ion‐channel block by facilitating blocker permeation.
• Relief of polyamine block in GluK2/GluK5 heteromers results from a key proline residue that produces architectural changes in the channel pore α‐helical region.
• Auxiliary subunits exert an additive effect to heteromerization, and thus relief of polyamine block is due to a different mechanism.
• Our findings have broad implications for work on polyamine block of other cation‐selective ion channels.

Abstract

Channel block and permeation by cytoplasmic polyamines is a common feature of many cation‐selective ion channels. Although the channel block mechanism has been studied extensively, polyamine permeation has been considered less significant as it occurs at extreme positive membrane potentials. Here, we show that kainate receptor (KAR) heteromerization and association with auxiliary proteins, Neto1 and Neto2, attenuate polyamine block by enhancing blocker permeation. Consequently, polyamine permeation and unblock occur at more negative and physiologically relevant membrane potentials. In GluK2/GluK5 heteromers, enhanced permeation is due to a single proline residue in GluK5 that alters the dynamics of the α‐helical region of the selectivity filter. The effect of auxiliary proteins is additive, and therefore the structural basis of polyamine permeation and unblock is through a different mechanism. As native receptors are thought to assemble as heteromers in complex with auxiliary proteins, our data identify an unappreciated impact of polyamine permeation in shaping the signalling properties of neuronal KARs and point to a structural mechanism that may be shared amongst other cation‐selective ion channels.

Abbreviations

iGluR

ionotropic glutamate receptor

KAR

kainate receptor

l‐Glu

l‐glutamate

MD

molecular dynamics

POPC

1‐palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐phosphocholine

Put

putrescine

RMSD

root mean square deviation

SMD

steered molecular dynamics

Spm

spermine

Spd

spermidine

     

Increased susceptibility of spontaneously hypertensive rats to ventricular tachyarrhythmias in early hypertension

Key points

• Hypertension is a risk factor for sudden cardiac death caused by ventricular tachycardia and fibrillation.
• Whether hypertension in its early stage is associated with an increased risk of ventricular tachyarrhythmias is not known.
• Based on experiments performed at the cellular and whole heart levels, we show that, even early in chronic hypertension, the hypertrophied and fibrotic ventricles of spontaneously hypertensive rats aged 5 to 6 months have already developed increased stress‐induced arrhythmogenicity, and this increased susceptibility to ventricular arrhythmias is primarily a result of tissue remodelling rather than cellular electrophysiological changes.
• Our findings highlight the need for early hypertension treatment to minimize myocardial fibrosis, ventricular hypertrophy, and arrhythmias.

Abstract

Hypertension is a risk factor for sudden cardiac death caused by ventricular tachycardia and fibrillation (VT/VF). We hypothesized that, in early hypertension, the susceptibility to stress‐induced VT/VF increases. We compared the susceptibility of 5‐ to 6‐month‐old male spontaneously hypertensive rats (SHR) and age/sex‐matched normotensive rats (NR) to VT/VF during challenge with oxidative stress (H₂O₂; 0.15 mmol l⁻¹). We found that only SHR hearts exhibited left ventricular fibrosis and hypertrophy. H₂O₂ promoted VT in all 30 SHR but none of the NR hearts. In 33% of SHR cases, focal VT degenerated to VF within 3 s. Simultaneous voltage‐calcium optical mapping of Langendorff‐perfused SHR hearts revealed that H₂O₂‐induced VT/VF arose spontaneously from focal activations at the base and mid left ventricular epicardium. Microelectrode recording of SHR hearts showed that VT was initiated by early afterdepolarization (EAD)‐mediated triggered activity. However, despite the increased susceptibility of SHR hearts to VT/VF, patch clamped isolated SHR ventricular myocytes developed EADs and triggered activity to the same extent as NR ventricular myocytes, except with larger EAD amplitude. During the early stages of hypertension, when challenged with oxidative stress, SHR hearts showed an increased ventricular arrhythmogenicity that stems primarily from tissue remodelling (hypertrophy, fibrosis) rather than cellular electrophysiological changes. Our findings highlight the need for early hypertension treatment to minimize myocardial fibrosis, ventricular hypertrophy, and arrhythmias.

Abbreviations

AP

action potential

APD

action potential duration

APD₉₀

action potential at 90% duration

CaMKII

calcium/calmodulin‐dependent protein kinase II

CaT

calcium transient

CaTD₉₀

calcium transient at 90% duration

CI

confidence interval

DBP

diastolic blood pressure

EAD/DAD

early/delayed after‐depolarization

HR

heart rate

ICC

interclass correlation

I_Ca,L

L‐type calcium current

I_Ks

slow delayed rectifier potassium current

I_Na

sodium current

I_to

transient outward potassium current

IVS(d,s) interventricular septum thickness (during diastole

during systole)

LV

left ventricle

LVEF

left ventricular ejection fraction

LVFS

left ventricular fractional shortening

LVH

left ventricular hypertrophy

LVID(d,s) left ventricular internal diameter (during diastole

during systole)

MV

mitral valve

NR

normotensive rats

PA peak vel

pulmonary artery peak velocity

(P)CL

(pacing) cycle length

PW

posterior wall

P‐ECG

pseudo‐electrocardiogram

RV

right ventricle

RWT

relative wall thickness

SHR

spontaneously hypertensive rats

SHHF

spontaneously hypertensive heart failure

SBP

systolic blood pressure

VT/VF

ventricular tachycardia and fibrillation

     

Dysferlin deficiency blunts β‐adrenergic‐dependent lusitropic function of mouse heart

AbstractDysferlin is a cell membrane bound protein with a role in the repair of skeletal and cardiac muscle cells. Deficiency of dysferlin leads to limb‐girdle muscular dystrophy 2B (LGMD2B) and Miyoshi myopathy. In cardiac muscle, dysferlin is located at the intercalated disc and transverse tubule membranes. Loss of dysferlin causes death of cardiomyocytes, notably in ageing hearts, leading to dilated cardiomyopathy and heart failure in LGM2B patients. To understand the primary pathogenesis and pathophysiology of dysferlin cardiomyopathy, we studied cardiac phenotypes of young adult dysferlin knockout mice and found early myocardial hypertrophy with largely compensated baseline cardiac function. Cardiomyocytes isolated from dysferlin‐deficient mice showed normal shortening and re‐lengthening velocities in the absence of external load with normal peak systolic Ca²⁺ but slower Ca²⁺ re‐sequestration than wild‐type controls. The effects of isoproterenol on relaxation velocity, left ventricular systolic pressure and stroke volume were blunted in dysferlin‐deficient mouse hearts compared with that in wild‐type hearts. Young dysferlin‐deficient mouse hearts expressed normal isoforms of myofilament proteins whereas the phosphorylation of ventricular myosin light chain 2 was significantly increased, implying a molecular response to the impaired lusitropic function. These early phenotypes of diastolic cardiac dysfunction and blunted lusitropic response of cardiac muscle to β‐adrenergic stimulation indicate a novel pathogenic mechanism of dysferlin cardiomyopathy.

Abbreviations

CaMKII

calmodulin kinase II

cMyBP‐C

cardiac myosin binding protein‐C

LGMD2B

limb‐girdle muscular dystrophy 2B

DTT

dithiothreitol

LVP_max

left ventricular systolic peak pressure

LVP_min

left ventricular end diastolic pressure

mAb

monoclonal antibody

MHC

myosin heavy chain

MLC2v

ventricular myosin light chain 2

MM

Myoshimyopathy

TnI

troponin I

TnT

troponin T

T_P

time for reaching peak cytosolic calcium

TR₂₅

time for 25% calcium re‐sequestration

TR₇₅

time for 75% calcium re‐sequestration

     

Large conductance Ca2+‐activated K+ (BK) channels promote secretagogue‐induced transition from spiking to bursting in murine anterior pituitary corticotrophs

Key points

• Corticotroph cells of the anterior pituitary are electrically excitable and are an integral component of the hypothalamic‐pituitary‐adrenal axis which governs the neuroendocrine response to stress.
• Corticotrophs display predominantly single spike activity under basal conditions that transition to complex bursting behaviours upon stimulation by the hypothalamic secretagogues corticotrophin‐releasing hormone (CRH) and arginine vasopressin (AVP); however, the underlying mechanisms controlling bursting are unknown.
• In this study, we show that CRH and AVP induce different patterns of corticotroph electrical activity, and we use an electrophysiological approach combined with mathematical modelling to show the ionic mechanisms for these differential effects.
• The data reveal that secretagogue‐induced bursting is dependent on large conductance Ca²⁺‐activated K⁺ (BK) channels and is driven primarily by CRH whereas AVP promotes an increase in single‐spike frequency through BK‐independent pathways involving activation of non‐selective cation conductances.
• As corticotroph excitability is differentially regulated by CRH and AVP this may allow corticotrophs to respond appropriately to different stressors.

Abstract

Anterior pituitary corticotroph cells are a central component of the hypothalamic‐pituitary‐adrenal (HPA) axis essential for the neuroendocrine response to stress. Corticotrophs are excitable cells that receive input from two hypothalamic secretagogues, corticotrophin‐releasing hormone (CRH) and arginine vasopressin (AVP) to control the release of adrenocorticotrophic hormone (ACTH). Although corticotrophs are spontaneously active and increase in excitability in response to CRH and AVP the patterns of electrical excitability and underlying ionic conductances are poorly understood. In this study, we have used electrophysiological, pharmacological and genetic approaches coupled with mathematical modelling to investigate whether CRH and AVP promote distinct patterns of electrical excitability and to interrogate the role of large conductance calcium‐ and voltage‐activated potassium (BK) channels in spontaneous and secretagogue‐induced activity. We reveal that BK channels do not play a significant role in the generation of spontaneous activity but are critical for the transition to bursting in response to CRH. In contrast, AVP promotes an increase in single spike frequency, a mechanism independent of BK channels but dependent on background non‐selective conductances. Co‐stimulation with CRH and AVP results in complex patterns of excitability including increases in both single spike frequency and bursting. The ability of corticotroph excitability to be differentially regulated by hypothalamic secretagogues provides a mechanism for differential control of corticotroph excitability in response to different stressors.

Abbreviations

ACTH

adrenocorticotrophic hormone

AVP

arginine vasopressin

BF

burstiness factor

BK channel

large conductance Ca²⁺‐ and voltage‐activated K⁺ channel

BK‐far

BK channels located distantly from voltage‐gated Ca²⁺ channels

BK‐near

BK channels in close proximity to voltage‐activated Ca²⁺ channels

CRH

corticotrophin‐releasing hormone

GFP

green fluorescent protein

HPA

hypothalamic‐pituitary‐adrenal

IK channel

intermediate conductance Ca²⁺‐activated K⁺ channel

IP₃

inositol trisphosphate

NMDG

N‐methyl‐d‐glucamine

PKA

protein kinase A

PKC

protein kinase C

POMC

proopiomelanocortin

STREX

stress regulated exon

ZERO

BK channels lacking STREX insert