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Volume regulation following hypotonic shock in isolated crypts of mouse distal colon
Author(s) -
Mignen Olivier,
Le Gall Christelle,
Harvey Brian J.,
Thomas Serge
Publication year - 1999
Publication title -
the journal of physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.802
H-Index - 240
eISSN - 1469-7793
pISSN - 0022-3751
DOI - 10.1111/j.1469-7793.1999.501ac.x
Subject(s) - charybdotoxin , chemistry , tonicity , bapta , extracellular , bumetanide , channel blocker , osmotic concentration , endocrinology , intracellular , biophysics , medicine , calcium , biochemistry , ion transporter , biology , membrane potential , organic chemistry , membrane
1 A video‐imaging technique of morphometry was used to measure the diameter as an index of cell volume in intact mouse distal colon crypts submitted to hypotonic shock. 2 Transition from isotonic (310 mosmol l −1 ) to hypotonic (240 mosmol l −1 ) saline caused a pronounced increase in crypt diameter immediately followed by regulatory volume decrease (RVD). 3 Exposure of crypts to Cl − ‐free hyposmotic medium increased the rapidity of both cell swelling and RVD. Exposure of crypts to Na + ‐free hyposmotic medium reduced the total duration of swelling. Return to initial diameter was followed by further shrinkage of the crypt cells. 4 The chloride channel inhibitor NPPB (50 μM) delayed the swelling phase and prevented the subsequent normal decrease in diameter. 5 The K + channel blockers barium (10 mM), charybdotoxin (10 nM) and TEA (5 mM) inhibited RVD by 51, 44 and 32%, respectively. 6 Intracellular [Ca 2+ ] rose from a baseline of 174 ± 17 nM ( n = 8 ) to 448 ± 45 nM ( n = 8 ) during the initial swelling phase 7 The Ca 2+ channel blockers verapamil (50 μM) and nifedipine (10 μM), the chelator of intracellular Ca 2+ BAPTA AM (30 μM), or the inhibitor of Ca 2+ release TMB‐8 (10 μM), dramatically reduced volume recovery, leading to 51% ( n = 9 ), 25% ( n = 7 ), 37% ( n = 6 ), 32% ( n = 8 ) inhibition of RVD, respectively. TFP (50 μM), an antagonist of the Ca 2+ ‐calmodulin complex, significantly slowed RVD. The Ca 2+ ionophore A23187 (2 μM) provoked a dramatic reduction of the duration and amplitude of cell swelling followed by extensive shrinkage. The release of Ca 2+ from intracellular stores using bradykinin (1 μM) or blockade of reabsorption with thapsigargin (1 μM) decreased the duration of RVD. 8 Prostaglandin E 2 (PGE 2 , 5 μM) slightly delayed RVD, whereas leukotriene D 4 (LTD 4 , 100 nM) and arachidonic acid (10 μM) reduced the duration of RVD. Blockade of phospholipase A 2 by quinacrine (10 μM) inhibited RVD by 53%. Common inhibition of PGE 2 and LTD 4 synthesis by ETYA (50 μM) or separate blockade of PGE 2 synthesis by 1 μM indomethacin reduced the duration of RVD. Blockade of LTD 4 synthesis by nordihydroguaiaretic acid (NDGA) did not produce any significant effect on cell swelling or subsequent RVD. 9 Staurosporine (1 μM), an inhibitor of protein kinases, inhibited RVD by 58%. Taken together the experiments demonstrate that the RVD process is under the control of conductive pathways, extra‐ and intracellular Ca 2+ ions, protein kinases, prostaglandins and leukotrienes.