Amiloride-sensitive channels are a major contributor to mechanotransduction in mammalian muscle spindles

Anna Simon, Fiona Shenton, Irene Hunter, Robert W Banks, Guy Smith Bewick

Research output: Contribution to journalArticle

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Abstract

We investigated whether channels of the epithelial sodium/amiloride-sensitive degenerin (ENaC/DEG) family are a major contributor to mechanosensory transduction in primary mechanosensory afferents, using adult rat muscle spindles as a model system. Stretch-evoked afferent discharge was reduced in a dose-dependent manner by amiloride and three analogues - benzamil, 5-(N-ethyl-N-isopropyl) amiloride (EIPA) and hexamethyleneamiloride (HMA), reaching > or = 85% inhibition at 1 mm. Moreover, firing was slightly but significantly increased by ENaC delta subunit agonists (icilin and capsazepine). HMA's profile of effects was distinct from that of the other drugs. Amiloride, benzamil and EIPA significantly decreased firing (P <0.01 each) at 1 microm, while 10 microm HMA was required for highly significant inhibition (P <0.0001). Conversely, amiloride, benzamil and EIPA rarely blocked firing entirely at 1 mm, whereas 1 mm HMA blocked 12 of 16 preparations. This pharmacology suggests low-affinity ENaCs are the important spindle mechanotransducer. In agreement with this, immunoreactivity to ENaC alpha, beta and gamma subunits was detected both by Western blot and immunocytochemistry. Immunofluorescence intensity ratios for ENaC alpha, beta or gamma relative to the vesicle marker synaptophysin in the same spindle all significantly exceeded controls (P <0.001). Ratios for the related brain sodium channel ASIC2 (BNaC1alpha) were also highly significantly greater (P <0.005). Analysis of confocal images showed strong colocalisation within the terminal of ENaC/ASIC2 subunits and synaptophysin. This study implicates ENaC and ASIC2 in mammalian mechanotransduction. Moreover, within the terminals they colocalise with synaptophysin, a marker for the synaptic-like vesicles which regulate afferent excitability in these mechanosensitive endings.
Original languageEnglish
Pages (from-to)171-185
Number of pages15
JournalThe Journal of Physiology
Volume588
Issue number1
DOIs
Publication statusPublished - 2010

Fingerprint

Epithelial Sodium Channels
Muscle Spindles
Amiloride
Synaptophysin
Degenerin Sodium Channels
Cellular Mechanotransduction
Sodium Channels
Synaptic Vesicles
Fluorescent Antibody Technique
Western Blotting
Immunohistochemistry
Pharmacology
Brain

Keywords

  • Action Potentials
  • Afferent Pathways
  • Animals
  • Cells, Cultured
  • Epithelial Sodium Channel
  • Ion Channel Gating
  • Male
  • Mechanotransduction, Cellular
  • Muscle Spindles
  • Muscle, Skeletal
  • Rats
  • Reflex, Stretch

Cite this

Amiloride-sensitive channels are a major contributor to mechanotransduction in mammalian muscle spindles. / Simon, Anna; Shenton, Fiona; Hunter, Irene; Banks, Robert W; Bewick, Guy Smith.

In: The Journal of Physiology, Vol. 588, No. 1, 2010, p. 171-185.

Research output: Contribution to journalArticle

Simon, Anna ; Shenton, Fiona ; Hunter, Irene ; Banks, Robert W ; Bewick, Guy Smith. / Amiloride-sensitive channels are a major contributor to mechanotransduction in mammalian muscle spindles. In: The Journal of Physiology. 2010 ; Vol. 588, No. 1. pp. 171-185.
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abstract = "We investigated whether channels of the epithelial sodium/amiloride-sensitive degenerin (ENaC/DEG) family are a major contributor to mechanosensory transduction in primary mechanosensory afferents, using adult rat muscle spindles as a model system. Stretch-evoked afferent discharge was reduced in a dose-dependent manner by amiloride and three analogues - benzamil, 5-(N-ethyl-N-isopropyl) amiloride (EIPA) and hexamethyleneamiloride (HMA), reaching > or = 85{\%} inhibition at 1 mm. Moreover, firing was slightly but significantly increased by ENaC delta subunit agonists (icilin and capsazepine). HMA's profile of effects was distinct from that of the other drugs. Amiloride, benzamil and EIPA significantly decreased firing (P <0.01 each) at 1 microm, while 10 microm HMA was required for highly significant inhibition (P <0.0001). Conversely, amiloride, benzamil and EIPA rarely blocked firing entirely at 1 mm, whereas 1 mm HMA blocked 12 of 16 preparations. This pharmacology suggests low-affinity ENaCs are the important spindle mechanotransducer. In agreement with this, immunoreactivity to ENaC alpha, beta and gamma subunits was detected both by Western blot and immunocytochemistry. Immunofluorescence intensity ratios for ENaC alpha, beta or gamma relative to the vesicle marker synaptophysin in the same spindle all significantly exceeded controls (P <0.001). Ratios for the related brain sodium channel ASIC2 (BNaC1alpha) were also highly significantly greater (P <0.005). Analysis of confocal images showed strong colocalisation within the terminal of ENaC/ASIC2 subunits and synaptophysin. This study implicates ENaC and ASIC2 in mammalian mechanotransduction. Moreover, within the terminals they colocalise with synaptophysin, a marker for the synaptic-like vesicles which regulate afferent excitability in these mechanosensitive endings.",
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T1 - Amiloride-sensitive channels are a major contributor to mechanotransduction in mammalian muscle spindles

AU - Simon, Anna

AU - Shenton, Fiona

AU - Hunter, Irene

AU - Banks, Robert W

AU - Bewick, Guy Smith

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N2 - We investigated whether channels of the epithelial sodium/amiloride-sensitive degenerin (ENaC/DEG) family are a major contributor to mechanosensory transduction in primary mechanosensory afferents, using adult rat muscle spindles as a model system. Stretch-evoked afferent discharge was reduced in a dose-dependent manner by amiloride and three analogues - benzamil, 5-(N-ethyl-N-isopropyl) amiloride (EIPA) and hexamethyleneamiloride (HMA), reaching > or = 85% inhibition at 1 mm. Moreover, firing was slightly but significantly increased by ENaC delta subunit agonists (icilin and capsazepine). HMA's profile of effects was distinct from that of the other drugs. Amiloride, benzamil and EIPA significantly decreased firing (P <0.01 each) at 1 microm, while 10 microm HMA was required for highly significant inhibition (P <0.0001). Conversely, amiloride, benzamil and EIPA rarely blocked firing entirely at 1 mm, whereas 1 mm HMA blocked 12 of 16 preparations. This pharmacology suggests low-affinity ENaCs are the important spindle mechanotransducer. In agreement with this, immunoreactivity to ENaC alpha, beta and gamma subunits was detected both by Western blot and immunocytochemistry. Immunofluorescence intensity ratios for ENaC alpha, beta or gamma relative to the vesicle marker synaptophysin in the same spindle all significantly exceeded controls (P <0.001). Ratios for the related brain sodium channel ASIC2 (BNaC1alpha) were also highly significantly greater (P <0.005). Analysis of confocal images showed strong colocalisation within the terminal of ENaC/ASIC2 subunits and synaptophysin. This study implicates ENaC and ASIC2 in mammalian mechanotransduction. Moreover, within the terminals they colocalise with synaptophysin, a marker for the synaptic-like vesicles which regulate afferent excitability in these mechanosensitive endings.

AB - We investigated whether channels of the epithelial sodium/amiloride-sensitive degenerin (ENaC/DEG) family are a major contributor to mechanosensory transduction in primary mechanosensory afferents, using adult rat muscle spindles as a model system. Stretch-evoked afferent discharge was reduced in a dose-dependent manner by amiloride and three analogues - benzamil, 5-(N-ethyl-N-isopropyl) amiloride (EIPA) and hexamethyleneamiloride (HMA), reaching > or = 85% inhibition at 1 mm. Moreover, firing was slightly but significantly increased by ENaC delta subunit agonists (icilin and capsazepine). HMA's profile of effects was distinct from that of the other drugs. Amiloride, benzamil and EIPA significantly decreased firing (P <0.01 each) at 1 microm, while 10 microm HMA was required for highly significant inhibition (P <0.0001). Conversely, amiloride, benzamil and EIPA rarely blocked firing entirely at 1 mm, whereas 1 mm HMA blocked 12 of 16 preparations. This pharmacology suggests low-affinity ENaCs are the important spindle mechanotransducer. In agreement with this, immunoreactivity to ENaC alpha, beta and gamma subunits was detected both by Western blot and immunocytochemistry. Immunofluorescence intensity ratios for ENaC alpha, beta or gamma relative to the vesicle marker synaptophysin in the same spindle all significantly exceeded controls (P <0.001). Ratios for the related brain sodium channel ASIC2 (BNaC1alpha) were also highly significantly greater (P <0.005). Analysis of confocal images showed strong colocalisation within the terminal of ENaC/ASIC2 subunits and synaptophysin. This study implicates ENaC and ASIC2 in mammalian mechanotransduction. Moreover, within the terminals they colocalise with synaptophysin, a marker for the synaptic-like vesicles which regulate afferent excitability in these mechanosensitive endings.

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KW - Cells, Cultured

KW - Epithelial Sodium Channel

KW - Ion Channel Gating

KW - Male

KW - Mechanotransduction, Cellular

KW - Muscle Spindles

KW - Muscle, Skeletal

KW - Rats

KW - Reflex, Stretch

U2 - 10.1113/jphysiol.2009.182683

DO - 10.1113/jphysiol.2009.182683

M3 - Article

VL - 588

SP - 171

EP - 185

JO - The Journal of Physiology

JF - The Journal of Physiology

SN - 0022-3751

IS - 1

ER -