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1.
Skelet Muscle ; 12(1): 3, 2022 01 29.
Article in English | MEDLINE | ID: mdl-35093178

ABSTRACT

Motor unit remodelling involving repeated denervation and re-innervation occurs throughout life. The efficiency of this process declines with age contributing to neuromuscular deficits. This study investigated differentially expressed genes (DEG) in muscle following peroneal nerve crush to model motor unit remodelling in C57BL/6 J mice. Muscle RNA was isolated at 3 days post-crush, RNA libraries were generated using poly-A selection, sequenced and analysed using gene ontology and pathway tools. Three hundred thirty-four DEG were found in quiescent muscle from (26mnth) old compared with (4-6mnth) adult mice and these same DEG were present in muscle from adult mice following nerve crush. Peroneal crush induced 7133 DEG in muscles of adult and 699 DEG in muscles from old mice, although only one DEG (ZCCHC17) was found when directly comparing nerve-crushed muscles from old and adult mice. This analysis revealed key differences in muscle responses which may underlie the diminished ability of old mice to repair following nerve injury.


Subject(s)
Crush Injuries , Muscle Denervation , Aging/genetics , Animals , Mice , Mice, Inbred C57BL , Muscle, Skeletal/innervation , Nerve Crush , Nerve Regeneration/physiology , RNA , Transcriptome
2.
Sci Rep ; 9(1): 14461, 2019 10 08.
Article in English | MEDLINE | ID: mdl-31595023

ABSTRACT

To determine the role of denervation and motor unit turnover in the age-related increase in skeletal muscle oxidative stress, the hydrogen peroxide (H2O2) specific, genetically-encoded, fluorescent cyto-HyPer2 probe was expressed in mouse anterior tibialis (AT) muscle and compared with ex vivo measurements of mitochondrial oxidant generation. Crush of the peroneal nerve induced increased mitochondrial peroxide generation, measured in permeabilised AT fibers ex vivo and intra vital confocal microscopy of cyto-HyPer2 fluorescence showed increased cytosolic H2O2 in a sub-set (~24%) of individual fibers associated with onset of fiber atrophy. In comparison, mitochondrial peroxide generation was also increased in resting muscle from old (26 month) mice compared with adult (6-8 month) mice, but no age effect on fiber cytosolic H2O2 in vivo was seen. Thus ageing is associated with an increased ability of muscle fibers to maintain cytosolic redox homeostasis in the presence of denervation-induced increase in mitochondrial peroxide generation.


Subject(s)
Aging/metabolism , Hydrogen Peroxide/metabolism , Molecular Probes/metabolism , Muscle Fibers, Skeletal/metabolism , Animals , Fluorescent Dyes , Male , Mice , Mitochondria/metabolism , Muscular Atrophy/metabolism , Nerve Crush , Neuromuscular Junction/metabolism , Oxidants/metabolism , Oxidative Stress , Sarcopenia/metabolism
3.
Exp Physiol ; 103(4): 535-544, 2018 04 01.
Article in English | MEDLINE | ID: mdl-29441689

ABSTRACT

NEW FINDINGS: What is the central question of this study? Can modulation of inducible NO synthase reduce pain behaviour and pro-inflammatory cytokine signalling in a rat model of neuropathic pain? What is the main finding and its importance? Nitric oxide synthase-based therapies could be effective for the treatment of peripheral neuropathic pain. ABSTRACT: Peripheral neuropathic pain (PNP), resulting from injury to or dysfunction of a peripheral nerve, is a major health problem that affects 7-8% of the population. It is inadequately controlled by current drugs and is characterized by pain hypersensitivity, which is believed to be attributable to sensitization of peripheral and CNS neurons by various inflammatory mediators. Here we examined, in a rat model of PNP: (i) whether reducing levels of nitric oxide (NO) with 1400W, a highly selective inhibitor of inducible NO synthase (iNOS), would prevent or attenuate pain hypersensitivity; and (ii) the effects of 1400W on plasma concentrations of several cytokines that are secreted after iNOS upregulation during chronic pain states. The L5 spinal nerve axotomy (SNA) model of PNP was used, and 1400W (20 mg kg-1 ) was administered i.p. at 8 h intervals for 3 days starting at 18 h post-SNA. Changes in plasma concentrations of 12 cytokines in SNA rats treated with 1400W were examined using multiplex enzyme-linked immunosorbent assay. The SNA rats developed behavioural signs of mechanical and heat hypersensitivity. Compared with the vehicle/control, 1400W significantly: (i) limited development of mechanical hypersensitivity at 66 h post-SNA and of heat hypersensitivity at 42 h and at several time points tested thereafter; and (ii) increased the plasma concentrations of interleukin (IL)-1α, IL-1ß and IL-10 in the SNA rats. The findings suggest that 1400W might exert its analgesic effects by reducing iNOS and altering the balance between the pro-inflammatory (IL-1ß and IL-1α) and anti-inflammatory (IL-10) cytokines and that therapies targeting NO or its enzymes might be effective for the treatment of PNP.


Subject(s)
Amidines/pharmacology , Benzylamines/pharmacology , Neuralgia/drug therapy , Neuralgia/metabolism , Nitric Oxide Synthase Type II/antagonists & inhibitors , Animals , Cytokines/metabolism , Disease Models, Animal , Male , Nitric Oxide/metabolism , Rats , Rats, Wistar
4.
Osteoarthritis Cartilage ; 25(8): 1199-1209, 2017 08.
Article in English | MEDLINE | ID: mdl-28232143

ABSTRACT

The extracellular matrix (ECM) of articular cartilage is comprised of complex networks of proteins and glycoproteins, all of which are expressed by its resident cell, the chondrocyte. Cartilage is a unique tissue given its complexity and ability to resist repeated load and deformation. The mechanisms by which articular cartilage maintains its integrity throughout our lifetime is not fully understood, however there are numerous regulatory pathways known to govern ECM turnover in response to mechanical stimuli. To further our understanding of this field, we envision that proteomic analysis of the secretome will provide information on how the chondrocyte remodels the surrounding ECM in response to load, in addition to providing information on the metabolic state of the cell. In this review, we attempt to summarize the recent mass spectrometry-based proteomic discoveries in healthy and diseased cartilage and chondrocytes, to facilitate the discovery of novel biomarkers linked to degenerative pathologies, such as osteoarthritis (OA).


Subject(s)
Osteoarthritis/diagnosis , Biomarkers/metabolism , Cartilage, Articular/metabolism , Chondrocytes/metabolism , Cytokines/metabolism , Enzymes/metabolism , Extracellular Matrix/metabolism , Humans , Mass Spectrometry , Proteins/metabolism , Proteomics/methods
5.
Osteoarthritis Cartilage ; 24(10): 1786-1794, 2016 10.
Article in English | MEDLINE | ID: mdl-27266646

ABSTRACT

OBJECTIVE: The anterior cruciate ligament transection (ACLT) rabbit osteoarthritis (OA) model confers permanent knee instability and induces joint degeneration. The degeneration process is complex, but includes chondrocyte apoptosis and OA-like loss of cartilage integrity. Previously, we reported that activation of a volume-sensitive Cl(-) current (ICl,vol) can mediate cell shrinkage and apoptosis in rabbit articular chondrocytes. Our objective was therefore to investigate whether ICl,vol was activated in the early stages of the rabbit ACLT OA model. DESIGN: Adult Rabbits underwent unilateral ACLT and contralateral arthrotomy (sham) surgery. Rabbits were euthanized at 2 or 4 weeks. Samples were analyzed histologically and with assays of cell volume, apoptosis and electrophysiological characterization of ICl,vol. RESULTS: At 2 and 4 weeks post ACLT cartilage appeared histologically normal, nevertheless cell swelling and caspase 3/7 activity were both significantly increased compared to sham controls. In cell-volume experiments, exposure of chondrocytes to hypotonic solution led to a greater increase in cell size in ACLT compared to controls. Caspase-3/7 activity, an indicator of apoptosis, was elevated in both ACLT 2wk and 4wk. Whole-cell currents were recorded with patch clamp of chondrocytes in iso-osmotic and hypo-osmotic external solutions under conditions where Na(+), K(+) and Ca(2+) currents were minimized. ACLT treatment resulted in a large increase in hypotonic-activated chloride conductance. CONCLUSION: Changes in chondrocyte ion channels take place prior to the onset of apparent cartilage loss in the ACLT rabbit model of OA. Further studies are needed to investigate if pharmacological inhibition of ICl,vol decreases progression of OA in animal models.


Subject(s)
Chondrocytes , Animals , Anterior Cruciate Ligament , Anterior Cruciate Ligament Injuries , Cartilage, Articular , Disease Models, Animal , Osteoarthritis , Osteoarthritis, Knee , Rabbits
6.
Organogenesis ; 12(2): 94-107, 2016 04 02.
Article in English | MEDLINE | ID: mdl-27116676

ABSTRACT

Chondrocytes are the uniquely resident cells found in all types of cartilage and key to their function is the ability to respond to mechanical loads with changes of metabolic activity. This mechanotransduction property is, in part, mediated through the activity of a range of expressed transmembrane channels; ion channels, gap junction proteins, and porins. Appropriate expression of ion channels has been shown essential for production of extracellular matrix and differential expression of transmembrane channels is correlated to musculoskeletal diseases such as osteoarthritis and Albers-Schönberg. In this study we analyzed the consistency of gene expression between channelomes of chondrocytes from human articular and costal (teenage and fetal origin) cartilages. Notably, we found 14 ion channel genes commonly expressed between articular and both types of costal cartilage chondrocytes. There were several other ion channel genes expressed only in articular (6 genes) or costal chondrocytes (5 genes). Significant differences in expression of BEST1 and KCNJ2 (Kir2.1) were observed between fetal and teenage costal cartilage. Interestingly, the large Ca(2+) activated potassium channel (BKα, or KCNMA1) was very highly expressed in all chondrocytes examined. Expression of the gap junction genes for Panx1, GJA1 (Cx43) and GJC1 (Cx45) was also observed in chondrocytes from all cartilage samples. Together, this data highlights similarities between chondrocyte membrane channel gene expressions in cells derived from different anatomical sites, and may imply that common electrophysiological signaling pathways underlie cellular control. The high expression of a range of mechanically and metabolically sensitive membrane channels suggest that chondrocyte mechanotransduction may be more complex than previously thought.


Subject(s)
Cartilage, Articular/cytology , Chondrocytes/metabolism , Gene Expression Regulation , Ion Channels/genetics , Membrane Proteins/genetics , Adolescent , Child , Chondrocytes/cytology , Female , Gap Junctions/genetics , Humans , Ion Channels/metabolism , Male , Membrane Proteins/metabolism , Middle Aged , Phenotype
7.
Br J Pharmacol ; 172(7): 1753-68, 2015 Apr.
Article in English | MEDLINE | ID: mdl-25421636

ABSTRACT

BACKGROUND AND PURPOSE: Transient receptor potential vanilloid type 4 (TRPV4) and calcium-activated potassium channels (KCa ) mediate osmosensing in many tissues. Both TRPV4 and KCa channels are found in the paraventricular nucleus (PVN) of the hypothalamus, an area critical for sympathetic control of cardiovascular and renal function. Here, we have investigated whether TRPV4 channels functionally couple to KCa channels to mediate osmosensing in PVN parvocellular neurones and have characterized, pharmacologically, the subtype of KCa channel involved. EXPERIMENTAL APPROACH: We investigated osmosensing roles for TRPV4 and KCa channels in parvocellular PVN neurones using cell-attached and whole-cell electrophysiology in mouse brain slices and rat isolated PVN neurons. Intracellular Ca(2+) was recorded using Fura-2AM. The system was modelled in the NEURON simulation environment. KEY RESULTS: Hypotonic saline reduced action current frequency in hypothalamic slices; a response mimicked by TRPV4 channel agonists 4αPDD (1 µM) and GSK1016790A (100 nM), and blocked by inhibitors of either TRPV4 channels (RN1734 (5 µM) and HC067047 (300 nM) or the low-conductance calcium-activated potassium (SK) channel (UCL-1684 30 nM); iberiotoxin and TRAM-34 had no effect. Our model was compatible with coupling between TRPV4 and KCa channels, predicting the presence of positive and negative feedback loops. These predictions were verified using isolated PVN neurons. Both hypotonic challenge and 4αPDD increased intracellular Ca(2+) and UCL-1684 reduced the action of hypotonic challenge. CONCLUSIONS AND IMPLICATIONS: There was functional coupling between TRPV4 and SK channels in parvocellular neurones. This mechanism contributes to osmosensing in the PVN and may provide a novel pharmacological target for the cardiovascular or renal systems.


Subject(s)
Paraventricular Hypothalamic Nucleus/physiology , Potassium Channels, Calcium-Activated/physiology , TRPV Cation Channels/physiology , Animals , Female , In Vitro Techniques , Male , Membrane Potentials/physiology , Mice , Models, Biological , Neurons/physiology , Rats, Wistar
8.
Curr Pain Headache Rep ; 17(12): 378, 2013 Dec.
Article in English | MEDLINE | ID: mdl-24198035

ABSTRACT

Osteoarthritis (OA) is a debilitating chronic condition widely prevalent in ageing populations. Because the pathology of the disease includes cartilage erosion and joint remodelling, OA patients experience a great deal of pain. Despite numerous studies, details of OA are frequently inseparable from other types of chronic pain, and its causes are unknown. In most circumstances in OA, the cartilage lacks afferent innervation, although other joint tissues contain nociceptive neurones. In addition to physical joint damage, there is a strong element of joint inflammation. Genetic studies have identified several associations between ion channels and OA pain, including NaV1.7, P2X7, and TRPV1, but several other channels have also been implicated. Many ion channels involved with OA pain are common to those seen in inflammatory pain. This review considers causes of OA pain and discusses three possible pain-reducing strategies involving ion channel modulation: chondroprotection, innate afferent nerve inhibition, and inhibition of inflammatory hyperalgesia. Future targets for OA pain analgesia could involve a number of ion channels.


Subject(s)
Analgesics/therapeutic use , Anti-Inflammatory Agents, Non-Steroidal/therapeutic use , Chronic Pain/drug therapy , Ion Channels/drug effects , Nociceptors/drug effects , Osteoarthritis/drug therapy , Pain/drug therapy , Aged , Aged, 80 and over , Chronic Pain/etiology , Chronic Pain/physiopathology , Female , Humans , Male , Middle Aged , Osteoarthritis/complications , Osteoarthritis/physiopathology , Pain/etiology , Pain/physiopathology , Pain Measurement , Treatment Outcome
9.
Br J Pharmacol ; 168(7): 1584-96, 2013 Apr.
Article in English | MEDLINE | ID: mdl-22928819

ABSTRACT

BACKGROUND AND PURPOSE: Chondrocytes exist within cartilage and serve to maintain the extracellular matrix. It has been postulated that osteoarthritic (OA) chondrocytes lose the ability to regulate their volume, affecting extracellular matrix production. In previous studies, we identified expression of epithelial sodium channels (ENaC) in human chondrocytes, but their function remained unknown. Although ENaC typically has Na(+) transport roles, it is also involved in the cell volume regulation of rat hepatocytes. ENaC is a member of the degenerin (Deg) family, and ENaC/Deg-like channels have a low conductance and high sensitivity to benzamil. In this study, we investigated whether canine chondrocytes express functional ENaC/Deg-like ion channels and, if so, what their function may be. EXPERIMENTAL APPROACH: Canine chondrocytes were harvested from dogs killed for unassociated welfare reasons. We used immunohistochemistry and patch-clamp electrophysiology to investigate ENaC expression and video microscopy to analyse the effects of pharmacological inhibition of ENaC/Deg on cell volume regulation. KEY RESULTS: Immunofluorescence showed that canine chondrocytes expressed ENaC protein. Single-channel recordings demonstrated expression of a benzamil-sensitive Na(+) conductance (9 pS), and whole-cell experiments show this to be approximately 1.5 nS per cell with high selectivity for Na(+) . Benzamil hyperpolarized chondrocytes by approximately 8 mV with a pD2 8.4. Chondrocyte regulatory volume decrease (RVI) was inhibited by benzamil (pD2 7.5) but persisted when extracellular Na(+) ions were replaced by Li(+) . CONCLUSION AND IMPLICATIONS: Our data suggest that benzamil inhibits RVI by reducing the influx of Na(+) ions through ENaC/Deg-like ion channels and present ENaC/Deg as a possible target for pharmacological modulation of chondrocyte volume.


Subject(s)
Amiloride/analogs & derivatives , Chondrocytes/drug effects , Degenerin Sodium Channels/metabolism , Epithelial Sodium Channels/metabolism , Amiloride/pharmacology , Animals , Cell Size/drug effects , Chondrocytes/cytology , Chondrocytes/metabolism , Dogs , Fluorescent Antibody Technique , In Vitro Techniques , Patch-Clamp Techniques
10.
Osteoarthritis Cartilage ; 15(1): 1-8, 2007 Jan.
Article in English | MEDLINE | ID: mdl-16891130

ABSTRACT

OBJECTIVE: Chondrocytes are highly sensitive to variations in extracellular glucose and oxygen levels in the extracellular matrix. As such, they must possess a number of mechanisms to detect and respond to alterations in the metabolic state of cartilage. In other organs such as the pancreas, heart and brain, such detection is partly mediated by a family of potassium channels known as K(ATP) (adenosine 5'-triphosphate-sensitive potassium) channels. Here we investigate whether chondrocytes too express functional K(ATP) channels, which might, potentially, serve to couple metabolic state with cell activity. METHODS: Immunohistochemistry was used to explore K(ATP) channel expression in equine and human chondrocytes. Biophysical properties of equine chondrocyte K(ATP) channels were investigated with patch-clamp electrophysiology. RESULTS: Polyclonal antibodies directed against the K(ATP) Kir6.1 subunit revealed high levels of expression in human and equine chondrocytes mainly in superficial and middle zones of normal cartilage. Kir6.1 was also detected in superficial chondrocytes in osteoarthritic (OA) cartilage. In single-channel electrophysiological studies of equine chondrocytes, we found K(ATP) channels to have a maximum unitary conductance of 47 +/- 9 pS (n=5) and a density of expression comparable to that seen in excitable cells. CONCLUSION: We have shown, for the first time, functional K(ATP) channels in chondrocytes. This suggests that K(ATP) channels are involved in coupling metabolic and electrical activities in chondrocytes through sensing of extracellular glucose and intracellular adenosine triphosphate (ATP) levels. Altered K(ATP) channel expression in OA chondrocytes may result in impaired intracellular ATP sensing and optimal metabolic regulation.


Subject(s)
Cartilage, Articular/metabolism , Chondrocytes/metabolism , Osteoarthritis/physiopathology , Potassium Channels, Inwardly Rectifying/metabolism , Adenosine Triphosphate/metabolism , Animals , Horses , Humans , Immunohistochemistry , Membrane Potentials/physiology , Patch-Clamp Techniques , Potassium Channels, Inwardly Rectifying/physiology
11.
Br J Pharmacol ; 149(5): 600-7, 2006 Nov.
Article in English | MEDLINE | ID: mdl-17001301

ABSTRACT

BACKGROUND AND PURPOSE: alpha-tetrahydrodeoxycorticosterone (THDOC) is an endogenous neuroactive steroid which increases in plasma and brain concentration during stress. It has both positive and negative modulatory effects on GABA activated GABAA currents, dependent upon the dose. We investigated the effects of THDOC on spinally-projecting "pre-sympathetic" neurones in the parvocellular subnucleus of the hypothalamic paraventricular nucleus (PVN), to determine whether it activates or inhibits these neurones, and by what mechanism. EXPERIMENTAL APPROACH: Rat spinally-projecting (parvocellular) PVN neurones were identified by retrograde labelling and the action of THDOC investigated with three modes of patch-clamp: cell-attached action current, whole-cell voltage-clamp and cell-attached single-channel recording. KEY RESULTS: In cell-attached patch mode, parvocellular neurones fired action potentials spontaneously with an average frequency of 3.6 +/- 1.1 Hz. Bath application of THDOC reduced this with an EC50 of 67 nM (95% confidence limits: 54 to 84 nM), Hill coefficient 0.8 +/- 0.04, n = 5. In whole-cell patch-clamp mode, pressure ejection of GABA evoked inward currents. These were clearly GABAA currents, since they were inhibited by the GABAA receptor antagonist bicuculline, and reversed near the chloride equilibrium potential. THDOC significantly potentiated GABAA currents (1 microM THDOC: 148 +/- 15% of control, n = 5, p < or = 0.05, ANOVA). Single-channel analysis showed no differences in conductance or corrected mean open times in the presence of 1 microM THDOC. CONCLUSIONS AND IMPLICATIONS: THDOC inhibited parvocellular neuronal activity without showing any evidence of the bidirectional activity demonstrated previously with cultured hypothalamic neurones. Our data are consistent with the hypothesis that THDOC acts by potentiating the post-synaptic activity of endogenously released GABA.


Subject(s)
Action Potentials/drug effects , Desoxycorticosterone/analogs & derivatives , Neurons/drug effects , Animals , Bicuculline/pharmacology , Desoxycorticosterone/pharmacology , Dose-Response Relationship, Drug , Drug Synergism , Female , GABA Antagonists/pharmacology , Hypothalamus/cytology , Hypothalamus/drug effects , Hypothalamus/physiology , Ion Channel Gating/drug effects , Ion Channel Gating/physiology , Kinetics , Male , Neurons/cytology , Neurons/physiology , Paraventricular Hypothalamic Nucleus/cytology , Paraventricular Hypothalamic Nucleus/drug effects , Paraventricular Hypothalamic Nucleus/physiology , Patch-Clamp Techniques/methods , Rats , Rats, Wistar , Receptors, GABA-A/physiology , Sympathetic Nervous System/cytology , Sympathetic Nervous System/drug effects , Sympathetic Nervous System/physiology , Synaptic Transmission/drug effects , gamma-Aminobutyric Acid/metabolism , gamma-Aminobutyric Acid/pharmacology
12.
Am J Physiol Regul Integr Comp Physiol ; 289(1): R172-80, 2005 Jul.
Article in English | MEDLINE | ID: mdl-15802557

ABSTRACT

In this comparative study, we have established in vitro models of equine and elephant articular chondrocytes, examined their basic morphology, and characterized the biophysical properties of their primary voltage-gated potassium channel (Kv) currents. Using whole cell patch-clamp electrophysiological recording from first-expansion and first-passage cells, we measured a maximum Kv conductance of 0.15 +/- 0.04 pS/pF (n = 10) in equine chondrocytes, whereas that in elephant chondrocytes was significantly larger (0.8 +/- 0.4 pS/pF, n = 4, P

Subject(s)
Cartilage, Articular/metabolism , Chondrocytes/metabolism , Elephants/metabolism , Horses/metabolism , Potassium Channels, Voltage-Gated/physiology , 4-Aminopyridine/pharmacology , Animals , Cartilage, Articular/cytology , Elapid Venoms/pharmacology , Electrophysiology , Immunohistochemistry , Kinetics , Models, Biological , Potassium Channel Blockers/pharmacology , Potassium Channels, Voltage-Gated/drug effects , Potassium Channels, Voltage-Gated/metabolism , Tetraethylammonium/pharmacology
13.
Br J Pharmacol ; 137(1): 87-97, 2002 Sep.
Article in English | MEDLINE | ID: mdl-12183334

ABSTRACT

1. We have used a range of in vitro electrophysiological techniques to investigate the mechanism of rapid cortisol neuromodulation of parvocellular neurones in the rat paraventricular nucleus. 2. In our study, we found that cortisol (10 microM) increased spontaneous action-current firing frequency to 193%. This effect was insensitive to the glucocorticoid intracellular-receptor antagonist mifepristone. 3. Cortisol (0.1-10 microM) had no detectable effects on whole-cell GABA current amplitudes, or GABA(A) single-channel kinetics. 4. Cortisol (10 microM) inhibited whole-cell potassium currents in parvocellular neurones by shifting the steady-state activation curve by 14 mV to the right. 5. Additionally, in a cell line expressing both the glucocorticoid intracellular receptor and recombinant, fast inactivating potassium channels (hKv1.3), cortisol (1 and 10 microM) inhibited potassium currents by shifting their steady-state activation curves to the right by 12 mV (10 microM cortisol). This effect was also insensitive to the cortisol antagonist, mifepristone. 6. These data suggest that inhibition of voltage-gated potassium channels may contribute to the rapid neuromodulatory effects of cortisol, possibly by direct interaction with the ion channel itself.


Subject(s)
Hydrocortisone/pharmacology , Paraventricular Hypothalamic Nucleus/drug effects , Animals , Dose-Response Relationship, Drug , Electric Conductivity , Hormone Antagonists/pharmacology , In Vitro Techniques , Kv1.3 Potassium Channel , Mifepristone/pharmacology , Neurons/metabolism , Neurons/physiology , Paraventricular Hypothalamic Nucleus/metabolism , Paraventricular Hypothalamic Nucleus/physiology , Patch-Clamp Techniques , Potassium Channel Blockers/pharmacology , Potassium Channels/drug effects , Potassium Channels/physiology , Potassium Channels, Voltage-Gated/drug effects , Potassium Channels, Voltage-Gated/physiology , Rats , Receptors, GABA/drug effects , Receptors, GABA/physiology , Receptors, Glucocorticoid/metabolism
14.
Br J Pharmacol ; 133(5): 673-8, 2001 Jul.
Article in English | MEDLINE | ID: mdl-11429391

ABSTRACT

The GABA-related compound nipecotic acid is commonly used to inhibit GABA uptake. This report shows that nipecotic acid can also directly activate GABA(A)-like chloride channels. When applied to outside-out patches of paraventricular neurones, nipecotic acid (1 mM) activated inward unitary currents (approximately 3 pA at a holding potential of -60 mV, E(Cl)+44 mV). The EC(50) for ion channel activation was approximately 300 microM, 3 fold greater than that found for GABA itself in this preparation. The nipecotic acid activated channels had similar conductance and kinetic properties to those of GABA activated channels in the same patches, reversed near E(Cl) and were inhibited by bicuculline (3 microM). This study indicates that for experiments in which relatively high concentrations of nipecotic acid are used, possible direct GABA(A) receptor agonist properties should be considered.


Subject(s)
Ion Channels/drug effects , Nipecotic Acids/pharmacology , Receptors, GABA-A/drug effects , Animals , Bicuculline/pharmacology , Chloride Channels/drug effects , Chloride Channels/physiology , Dose-Response Relationship, Drug , GABA Agonists/pharmacology , GABA Antagonists/pharmacology , Ion Channels/physiology , Membrane Potentials/drug effects , Neurons/drug effects , Neurons/physiology , Paraventricular Hypothalamic Nucleus/cytology , Paraventricular Hypothalamic Nucleus/drug effects , Paraventricular Hypothalamic Nucleus/physiology , Rats , Receptors, GABA-A/physiology , gamma-Aminobutyric Acid/pharmacology
15.
J Neurosci Methods ; 102(1): 25-33, 2000 Oct 15.
Article in English | MEDLINE | ID: mdl-11000408

ABSTRACT

The paraventricular nucleus (PVN) of the hypothalamus modulates cardiovascular function via a sub-population of neurones which project directly to sympathetic centres of the spinal cord. Identification and patch-clamp recording from these neurones is difficult, however, because of the complex organisation and neuronal heterogeneity of the PVN. We report here on methods for the in vitro recording of voltage-gated potassium channel (K(V)) currents from those neurones within the PVN which project to the intermediolateral column of the rat spinal cord, and are believed to directly modulate cardiovascular function. We show K(V) channel currents of spinally projecting neurones to be slowly inactivating (tau >> 100 ms) and weakly sensitive to TEA (K(d)>10 mM). These methods will be useful for the study of K(V) and other ion channel modulation in spinally projecting neurones of the PVN.


Subject(s)
Adrenergic Fibers/metabolism , Efferent Pathways/metabolism , Neurons/metabolism , Paraventricular Hypothalamic Nucleus/metabolism , Potassium Channels/metabolism , Spinal Cord/metabolism , Stilbamidines , Adrenergic Fibers/drug effects , Adrenergic Fibers/ultrastructure , Animals , Efferent Pathways/cytology , Efferent Pathways/drug effects , Fluorescent Dyes/pharmacology , Neurons/cytology , Neurons/drug effects , Paraventricular Hypothalamic Nucleus/cytology , Paraventricular Hypothalamic Nucleus/drug effects , Patch-Clamp Techniques , Potassium Channels/drug effects , Rats , Rats, Wistar , Spinal Cord/cytology , Spinal Cord/drug effects , Tetraethylammonium/pharmacology
16.
Br J Pharmacol ; 128(4): 909-16, 1999 Oct.
Article in English | MEDLINE | ID: mdl-10556925

ABSTRACT

1 The aim of this study was to investigate the selectivity of the ATP-sensitive potassium (K(ATP)) channel inhibitor U-37883A (4-morpholinecarboximidine-N-1-adamantyl-N'-1-cyclohexyl). Membrane currents through K(ATP) channels were recorded in single muscle cells enzymatically isolated from rat mesenteric artery, cardiac ventricle and skeletal muscle (flexor digitorum brevis). K(ATP) currents were induced either by cell dialysis with 0.1 mM ATP and 0.1 mM ADP, or by application of synthetic potassium channel openers (levcromakalim or pinacidil). 2 U-37883A inhibited K(ATP) currents in smooth muscle cells from rat mesenteric artery. Half inhibition of 10 microM levcromakalim-induced currents occurred at a concentration of 3.5 microM. 3 Relaxations of rat mesenteric vessels caused by levcromakalim were reversed by U-37883A. 1 microM levcromakalim-induced relaxations were inhibited at a similar concentration of U-37883A (half inhibition, 1.1 microM) to levcromakalim-induced KATP currents. 4 K(ATP) currents activated by 100 microM pinacidil were also studied in single myocytes from rat mesenteric artery, skeletal muscle and cardiac ventricle. 10 microM U-37883A substantially inhibited K(ATP) currents in vascular cells, but had little effect in skeletal or cardiac myocytes. Higher concentrations of U-37883A (100 microM) caused a modest decrease in K(ATP) currents in skeletal and cardiac muscle. The sulphonylurea K(ATP) channel antagonist glibenclamide (10 microM) abolished currents in all muscle types. 5 The effect of U-37883A on vascular inward rectifier (KIR) and voltage-dependent potassium (KV) currents was also examined. While 10 microM U-37883A had little effect on these currents, some inhibition was apparent at higher concentrations (100 microM) of the compound. 6 We conclude that U-37883A inhibits K(ATP) channels in arterial smooth muscle more effectively than in cardiac and skeletal muscle. Furthermore, this compound is selective for K(ATP) channels over KV and KIR channels in smooth muscle cells.


Subject(s)
Adamantane/analogs & derivatives , Adenosine Triphosphate/metabolism , Morpholines/pharmacology , Muscle, Skeletal/drug effects , Myocardium/metabolism , Potassium Channel Blockers , Potassium Channels, Inwardly Rectifying , Adamantane/pharmacology , Animals , Cromakalim/pharmacology , In Vitro Techniques , Male , Mesenteric Arteries/drug effects , Mesenteric Arteries/physiology , Muscle Relaxation/drug effects , Muscle, Skeletal/metabolism , Rats , Rats, Wistar
17.
Br J Pharmacol ; 128(3): 760-6, 1999 Oct.
Article in English | MEDLINE | ID: mdl-10516659

ABSTRACT

1. We have investigated the inhibition of inwardly rectifying potassium channels by the alpha-adrenergic agonist/antagonist chloroethylclonidine (CEC). We used two preparations; two-electrode voltage-clamp of rat isolated flexor digitorum brevis muscle and whole-cell patch-clamp of cell lines transfected with Kir2.1 (IRK1). 2. In skeletal muscle and at a membrane potential of -50 mV, chloroethylclonidine (CEC), an agonist at alpha2-adrenergic receptors and an antagonist at alpha1x-receptors, was found to inhibit the inward rectifier current with a Ki of 30 microM. 3. The inhibition of skeletal muscle inward rectifier current by CEC was not mimicked by clonidine, adrenaline or noradrenaline and was not sensitive to high concentrations of alpha1-(prazosin) or alpha2-(rauwolscine) antagonists. 4. The degree of current inhibition by CEC was found to vary with the membrane potential (approximately 70% block at -50 mV c.f. approximately 10% block at -190 mV). The kinetics of this voltage dependence were further investigated using recombinant inward rectifier K+ channels (Kir2.1) expressed in the MEL cell line. Using a two pulse protocol, we calculated the time constant for block to be approximately 8 s at 0 mV, and the rate of unblock was described by the relationship tau=exp((Vm+149)/22) s. 5. This block was effective when CEC was applied to either the inside or the outside of patch clamped cells, but ineffective when a polyamine binding site (aspartate 172) was mutated to asparagine. 6. The data suggest that the clonidine-like imidazoline compound, CEC, inhibits inward rectifier K+ channels independently of alpha-receptors by directly blocking the channel pore, possibly at an intracellular polyamine binding site.


Subject(s)
Clonidine/analogs & derivatives , Potassium Channel Blockers , Potassium Channels, Inwardly Rectifying , Animals , Clonidine/pharmacology , Kinetics , Rats , Receptors, Adrenergic, alpha/physiology , Recombinant Proteins/antagonists & inhibitors
18.
J Physiol ; 511 ( Pt 1): 15-24, 1998 Aug 15.
Article in English | MEDLINE | ID: mdl-9679159

ABSTRACT

Externally applied Ag+ (100-200 nM) irreversibly blocked the strong inwardly rectifying K+ channel, Kir2.1. Mutation to serine of a cysteine residue at position 149 in the pore-forming H5 region of Kir2.1 abolished Ag+ blockage. To determine how many of the binding sites must be occupied by Ag+ before the channel is blocked, we measured the rate of channel block and found that our results were best fitted assuming that only one Ag+ ion need bind to eliminate channel current. We tested our hypothesis further by constructing covalently linked dimers and tetramers of Kir2.1 in which cysteine had been replaced by serine in one (dimer) or three (tetramer) of the linked subunits. When expressed, these constructs yielded functional channels with either two (dimer) or one (tetramer) cysteines per channel at position 149. Blockage in the tetramer was complete after sufficient exposure to 200 nM Ag+, a result that is also consistent with only one Ag+ being required to bind to Cys149 to block fully. The rate of development of blockage was 16 times slower than in wild-type channels; the rate was 4 times slower in channels formed from dimers.


Subject(s)
Cysteine , Potassium Channels, Inwardly Rectifying , Potassium Channels/chemistry , Potassium Channels/physiology , Silver/pharmacology , Amino Acid Sequence , Amino Acid Substitution , Animals , Base Sequence , Binding Sites , CHO Cells , Conserved Sequence , Cricetinae , Dimerization , Kinetics , Macromolecular Substances , Mice , Models, Chemical , Molecular Sequence Data , Mutagenesis, Site-Directed , Oligodeoxyribonucleotides , Potassium Channel Blockers , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Serine , Transfection
19.
Br J Pharmacol ; 123(6): 1103-10, 1998 Mar.
Article in English | MEDLINE | ID: mdl-9559893

ABSTRACT

1. The aim of this study was to characterize the K(ATP) channel of intact rat skeletal muscle (rat flexor digitorum brevis muscle). Changes in membrane currents were recorded with two-electrode voltage-clamp of whole fibres. 2. The K(ATP) channel openers, levcromakalim and pinacidil (10-400 microM), caused a concentration-dependent increase in whole-cell chord conductance (up to approximately 1.5 mScm(-2)). The activated current had a weak inwardly rectifying current-voltage relation, a reversal potential near E(K) and nanomolar sensitivity to glibenclamide--characteristic of a K(ATP) channel current. Concentration-effect analysis revealed that levcromakalim and pinacidil were not particularly potent (EC50 approximately 186 microM, approximately 30 microM, respectively), but diazoxide was completely inactive. 3. The ability of both classical K(ATP) channel inhibitors (glibenclamide, tolbutamide, glipizide and 5-hydroxydecanoic acid) and a number of structurally related glibenclamide analogues to antagonize the levcromakalim-induced current was determined. Glibenclamide was the most potent compound with an IC50 of approximately 5 nM. However, the non-sulphonylurea (but cardioactive) compound 5-hydroxydecanoic acid was inactive in this preparation. 4. Regression analysis showed that the glibenclamide analogues used have a similar rank order of potency to that observed previously in vascular smooth muscle and cerebral tissue. However, two compounds (glipizide and DK13) were found to have unexpectedly low potency in skeletal muscle. 5. These experiments revealed K(ATP) channels of skeletal muscle to be at least 10x more sensitive to glibenclamide than previously found; this may be because of the requirement for an intact intracellular environment for the full effect of sulphonylureas to be realised. Pharmacologically, K(ATP) channels of mammalian skeletal muscle appear to resemble most closely K(ATP) channels of cardiac myocytes.


Subject(s)
Muscle Fibers, Skeletal/metabolism , Muscle, Skeletal/metabolism , Potassium Channels, Inwardly Rectifying , Potassium Channels/metabolism , Animals , Cromakalim/pharmacology , Glyburide/pharmacology , In Vitro Techniques , Potassium Channel Blockers , Potassium Channels/agonists , Rats
20.
J Membr Biol ; 155(3): 257-62, 1997 Feb 01.
Article in English | MEDLINE | ID: mdl-9050449

ABSTRACT

We investigated the block of KATP channels by glibenclamide in inside-out membrane patches of rat flexor digitorum brevis muscle. (1) We found that glibenclamide inhibited KATP channels with an apparent Ki of 63 nm and a Hill coefficient of 0.85. The inhibition of KATP channels by glibenclamide was unaffected by internal Mg2+. (2) Glibenclamide altered all kinetic parameters measured; mean open time and burst length were reduced, whereas mean closed time was increased.(3) By making the assumption that binding of glibenclamide to the sulphonylurea receptor (SUR) leads to channel closure, we have used the relation between mean open time, glibenclamide concentration and KD to estimate binding and unbinding rate constants. We found an apparent rate constant for glibenclamide binding of 9.9 x 10(7) m-1 sec-1 and an unbinding rate of 6.26 sec-1. (4) Glibenclamide is a lipophilic molecule and is likely to act on sulfonylurea receptors from within the hydrophobic phase of the cell membrane. The glibenclamide concentration within this phase will be greater than that in the aqueous solution and we have taken this into account to estimate a true binding rate constant of 1.66 x 10(6) m-1 sec-1.


Subject(s)
Adenosine Triphosphate/physiology , Glyburide/pharmacology , Hypoglycemic Agents/pharmacology , Muscle, Skeletal/drug effects , Potassium Channels/physiology , Animals , Binding Sites , Glyburide/metabolism , Hypoglycemic Agents/metabolism , Ion Channel Gating/drug effects , Iontophoresis , Muscle, Skeletal/metabolism , Patch-Clamp Techniques , Potassium Channel Blockers , Potassium Channels/drug effects , Rats
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