Volume regulation following hypotonic shock in isolated crypts of mouse distal colon

1. A video-imaging technique of morphometry was used to measure the diamete r 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 follo wed 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 me dium 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 mu M) 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 [Ca2+] rose from a baseline of 174 +/- 17 nM (n = 8) to 44 8 +/- 45 nM (n = 8) during the initial swelling phase 7. The Ca2+ channel blockers verapamil (50 mu M) and nifedipine (10 mu M), the chelator of intracellular Ca2+ BAPTA AM (30 mu M), or the inhibitor of Ca2+ release TMB-8 (10 mu M), dramatically reduced volume recovery, leading to 51% (n = 9), 25% (n = 7), 37 % (n = 6), 32% (n = 8) inhibition of RVD, respectively TFP (50 mu M), an antagonist of the Ca2+-calmodulin complex, s ignificantly slowed RVD. The Ca2+ ionophore A23187 (2 mu M) provoked a dram atic reduction of the duration and amplitude of cell swelling followed by e xtensive shrinkage. The release of Ca2+ from intracellular stores using bra dykinin (1 mu M) or blockade of reabsorption with thapsigargin (1 mu M) dec reased the duration of RVD. 8. Prostaglandin E-2 (PGE(2), 5 mu M) slightly delayed RVD, whereas leukotr iene D-4 (LTD4, 100 nM) and arachidonic acid (10 mu M) reduced the duration of RVD. Blockade of phospholipase A, by quinacrine (10 mu M) inhibited RVD by 53%. Common inhibition of PGE(2) and LTD4 synthesis by ETYA (50 mu M) o r separate blockade of PGE(2) synthesis by 1 mu M indomethacin reduced the duration of RVD. Blockade of LTD, synthesis by nordihydroguaiaretic acid (N DGA) did not produce any significant effect on cell swelling or subsequent RVD. 9. Staurosporine (1 mu M), an inhibitor of protein kinases, inhibited RVD b y 58%. Taken together the experiments demonstrate that the RVD process is u nder the control of conductive pathways, extra- and intracellular Ca2+ ions , protei:n kinases, prostaglandins and leukotrienes.