Premium
Calcium regulated chloride permeabilities in primary cultures of rabbit colonocytes
Author(s) -
Sahi Jasminder,
Wiggins Michael P.,
Gibori Gil B.,
Layden Thomas J.,
Rao Mrinalini C.
Publication year - 1996
Publication title -
journal of cellular physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.529
H-Index - 174
eISSN - 1097-4652
pISSN - 0021-9541
DOI - 10.1002/(sici)1097-4652(199608)168:2<276::aid-jcp6>3.0.co;2-m
Subject(s) - calcium , rabbit (cipher) , chemistry , chloride , microbiology and biotechnology , primary (astronomy) , biophysics , biochemistry , biology , organic chemistry , statistics , physics , mathematics , astronomy
To determine if calcium‐dependent secretagogues directly act on epithelial cells to elicit CI − secretion, their effects on CI − transport and intracellular Ca 2+ concentrations ([Ca 2+ ] i ) were determined in primary cultures of rabbit distal colonic crypt cells. The Cl − sensitive fluorescent probe, 6‐methoxyquinolyl acetoethyl ester, MQAE and the Ca 2+ ‐sensitive fluorescent probe, fura‐2AM were used to assess Cl − transport and [Ca 2+ ] i , respectively. Basal Cl − transport (0.274 ± 0.09 mM/sec) was inhibited significantly by the Cl − channel blocker diphenylamine‐2‐carboxylate (DPC, 50 μM, 0.068 ± 0.02 mM/sec; P < 0.001) and the Na + /K + /2Cl − cotransport inhibitor furosemide (1 μM, 0.137 ± 0.04 mM/sec; P < 0.01). Ion substitution studies using different halides revealed the basal influx to be I − > F − ≥ Cl − > Br − . DPC inhibited I − influx by ∼50%, F − influx by 80%, Cl − influx by 85%, and Br − influx by 90%. Furosemide significantly inhibited influx of Br − (84%) and Cl − (81%) but not of F − and I − . The effects of agents known to alter biological response by increasing [Ca 2+ ] i in other epithelial systems were used to stimulate Cl − transport. Cl − influx in mM/second was stimulated by 1 μM histamine (0.58 ± 0.05), 10 μM neurotensin (2.07 ± 0.32), 1 μM serotonin (1.63 ± 0.28), and 0.1 μM of the Ca 2+ ionophore A23187 (2.05 ± 0.40). The Cl − permeability stimulated by neurotensin, serotonin, and A23187 was partially blocked by DPC or furosemide added alone or in combination. Histamine‐induced Cl − influx was significantly inhibited by only furosemide. Indomethacin blocked histamine‐stimulated Cl − permeability but had no effect on the actions of the other agents. These studies, focusing on isolated colonocytes without the contribution of submucosal elements, reveal that (1) histamine stimulates Cl − transport by activating the Na + /K + /2Cl − cotransporter via a cyclooxygenase‐dependent pathway; (2) neurotensin, serotonin, and A23187 activate both Cl − channels and the cotransporter, and their actions are cyclooxygenase‐independent. © 1996 Wiley‐Liss, Inc.