Measurement of intracellular chloride ion concentration in ICC in situ and in explant culture

Yaohui Zhu, S. P. Parsons, J. D. Huizinga

Research output: Contribution to journalArticle

Abstract

Background Chloride channels are proposed to play a central role in the electrical pacemaking mechanism of interstitial cells of Cajal (ICC). A key unknown factor in the consideration of this role is the chloride equilibrium potential (ECl), as determined by the relative concentrations of intra- ([Cl-]i) and extracellular ([Cl-] o) chloride ions. Methods To calculate the ECl of ICC, [Cl-]i was measured with the fluorescent chloride indicator N-(6-methoxyquinolyl) acetoethyl ester (MQAE). Pacemaker ICC in explant cultures or in situ, i.e. ICC associated with the myenteric plexus of the small intestine, were loaded with MQAE and fluorescence was measured by laser scanning confocal microscopy. The dye fluorescence was then calibrated against known [Cl-]i by treating the explants or in situ preparations with chloride ionophore and varying bath chloride concentrations. Key Results In explants, ICC [Cl-]i was measured as 13 mmol L-1 with [Cl-]o of 100 mmol L -1, giving an ECl of -23 mV. With [Cl-] o at 166 mmol L-1, [Cl-]i was 26 mmol L-1, giving an ECl of -21 mV. In situ, ICC [Cl -]i was measured as 26 mmol L-1 with [Cl -]o of 130 mmol L-1, giving an ECl of -18 mV. Importantly ICC compensate for changes in extracellular chloride by changing [Cl-]i and thus maintain ECl. In ICC explant clusters, [Cl-]i was seen to fluctuate, possibly evoked by rhythmic changes in intracellular calcium. Conclusions & Inferences The intracellular chloride concentration in ICC fluctuates to keep its equilibrium potential constant. The identification of ECl as positive to the resting membrane potential of ICC indicates that opening of chloride channels will depolarize ICC.

Original languageEnglish (US)
Pages (from-to)704-709
Number of pages6
JournalNeurogastroenterology and Motility
Volume22
Issue number6
DOIs
StatePublished - Jun 2010
Externally publishedYes

Fingerprint

Interstitial Cells of Cajal
Chlorides
Ions
Chloride Channels
Fluorescence
Myenteric Plexus
Ionophores
Baths
Confocal Microscopy
Membrane Potentials
Small Intestine
Coloring Agents

Keywords

  • Chloride
  • Chloride ion channels
  • Equilibrium potential
  • Interstitial cells of Cajal
  • Intestinal motility
  • Intestinal pacemaking
  • Reversal potential

ASJC Scopus subject areas

  • Endocrine and Autonomic Systems
  • Gastroenterology
  • Physiology

Cite this

Measurement of intracellular chloride ion concentration in ICC in situ and in explant culture. / Zhu, Yaohui; Parsons, S. P.; Huizinga, J. D.

In: Neurogastroenterology and Motility, Vol. 22, No. 6, 06.2010, p. 704-709.

Research output: Contribution to journalArticle

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abstract = "Background Chloride channels are proposed to play a central role in the electrical pacemaking mechanism of interstitial cells of Cajal (ICC). A key unknown factor in the consideration of this role is the chloride equilibrium potential (ECl), as determined by the relative concentrations of intra- ([Cl-]i) and extracellular ([Cl-] o) chloride ions. Methods To calculate the ECl of ICC, [Cl-]i was measured with the fluorescent chloride indicator N-(6-methoxyquinolyl) acetoethyl ester (MQAE). Pacemaker ICC in explant cultures or in situ, i.e. ICC associated with the myenteric plexus of the small intestine, were loaded with MQAE and fluorescence was measured by laser scanning confocal microscopy. The dye fluorescence was then calibrated against known [Cl-]i by treating the explants or in situ preparations with chloride ionophore and varying bath chloride concentrations. Key Results In explants, ICC [Cl-]i was measured as 13 mmol L-1 with [Cl-]o of 100 mmol L -1, giving an ECl of -23 mV. With [Cl-] o at 166 mmol L-1, [Cl-]i was 26 mmol L-1, giving an ECl of -21 mV. In situ, ICC [Cl -]i was measured as 26 mmol L-1 with [Cl -]o of 130 mmol L-1, giving an ECl of -18 mV. Importantly ICC compensate for changes in extracellular chloride by changing [Cl-]i and thus maintain ECl. In ICC explant clusters, [Cl-]i was seen to fluctuate, possibly evoked by rhythmic changes in intracellular calcium. Conclusions & Inferences The intracellular chloride concentration in ICC fluctuates to keep its equilibrium potential constant. The identification of ECl as positive to the resting membrane potential of ICC indicates that opening of chloride channels will depolarize ICC.",
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AU - Parsons, S. P.

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N2 - Background Chloride channels are proposed to play a central role in the electrical pacemaking mechanism of interstitial cells of Cajal (ICC). A key unknown factor in the consideration of this role is the chloride equilibrium potential (ECl), as determined by the relative concentrations of intra- ([Cl-]i) and extracellular ([Cl-] o) chloride ions. Methods To calculate the ECl of ICC, [Cl-]i was measured with the fluorescent chloride indicator N-(6-methoxyquinolyl) acetoethyl ester (MQAE). Pacemaker ICC in explant cultures or in situ, i.e. ICC associated with the myenteric plexus of the small intestine, were loaded with MQAE and fluorescence was measured by laser scanning confocal microscopy. The dye fluorescence was then calibrated against known [Cl-]i by treating the explants or in situ preparations with chloride ionophore and varying bath chloride concentrations. Key Results In explants, ICC [Cl-]i was measured as 13 mmol L-1 with [Cl-]o of 100 mmol L -1, giving an ECl of -23 mV. With [Cl-] o at 166 mmol L-1, [Cl-]i was 26 mmol L-1, giving an ECl of -21 mV. In situ, ICC [Cl -]i was measured as 26 mmol L-1 with [Cl -]o of 130 mmol L-1, giving an ECl of -18 mV. Importantly ICC compensate for changes in extracellular chloride by changing [Cl-]i and thus maintain ECl. In ICC explant clusters, [Cl-]i was seen to fluctuate, possibly evoked by rhythmic changes in intracellular calcium. Conclusions & Inferences The intracellular chloride concentration in ICC fluctuates to keep its equilibrium potential constant. The identification of ECl as positive to the resting membrane potential of ICC indicates that opening of chloride channels will depolarize ICC.

AB - Background Chloride channels are proposed to play a central role in the electrical pacemaking mechanism of interstitial cells of Cajal (ICC). A key unknown factor in the consideration of this role is the chloride equilibrium potential (ECl), as determined by the relative concentrations of intra- ([Cl-]i) and extracellular ([Cl-] o) chloride ions. Methods To calculate the ECl of ICC, [Cl-]i was measured with the fluorescent chloride indicator N-(6-methoxyquinolyl) acetoethyl ester (MQAE). Pacemaker ICC in explant cultures or in situ, i.e. ICC associated with the myenteric plexus of the small intestine, were loaded with MQAE and fluorescence was measured by laser scanning confocal microscopy. The dye fluorescence was then calibrated against known [Cl-]i by treating the explants or in situ preparations with chloride ionophore and varying bath chloride concentrations. Key Results In explants, ICC [Cl-]i was measured as 13 mmol L-1 with [Cl-]o of 100 mmol L -1, giving an ECl of -23 mV. With [Cl-] o at 166 mmol L-1, [Cl-]i was 26 mmol L-1, giving an ECl of -21 mV. In situ, ICC [Cl -]i was measured as 26 mmol L-1 with [Cl -]o of 130 mmol L-1, giving an ECl of -18 mV. Importantly ICC compensate for changes in extracellular chloride by changing [Cl-]i and thus maintain ECl. In ICC explant clusters, [Cl-]i was seen to fluctuate, possibly evoked by rhythmic changes in intracellular calcium. Conclusions & Inferences The intracellular chloride concentration in ICC fluctuates to keep its equilibrium potential constant. The identification of ECl as positive to the resting membrane potential of ICC indicates that opening of chloride channels will depolarize ICC.

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KW - Intestinal pacemaking

KW - Reversal potential

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