Dynamics of pavement cell-chloride cell interactions during abrupt salinity change in Fundulus heteroclitus

Freshwater-adapted killifish (Fundulus heteroclitus) opercular epithelia were dissected and subjected to blood-side hypertonic bathing solution in Ussing-style chambers to simulate the increase in blood osmolality during migration to sea water. Conversely, seawater-acclimated killifish opercular epithelia were subjected to hypotonic bathing solutions to simulate the initial stages of migration to fresh water. Freshwater-acclimation (hypertonic stress) induced a rapid (approximately 30 min) increase in membrane conductance (G(t)) from 3.10+/-0.56 to 7.52+/-1.15 mS x cm(-2) (P<0.01, N=27), whereas seawater-acclimation (hypotonic stress) induced a rapid decrease in G(t) from 8.22+/-1.15 to 4.41+/-1.00 mS x cm(-2) (P<0.01, N=27; means +/- S.E.M.). Control seawater-acclimated membranes had a density of apical crypts (where chloride cells are exposed to the environment; detected by scanning electron microscopy) of 1133+/-96.4 crypts x mm(-2) (N=12), whereas the hypotonically shocked specimens had a lower crypt density of 870+/-36.7 crypts x mm(-2) (P<0.01 N=10; means +/- S.E.M.). Hypertonic shock of freshwater membranes increased crypt density from 383.3+/-73.9 (N=12) to 630+/-102. 9 crypts x mm(-2) (P<0.05; N=11; means +/- S.E.M.). There was no change in density of chloride cells, as detected by fluorescence microscopy; hence, osmotic stress changes the degree of exposure, not the number of chloride cells. Cytochalasin D (5.0 micromol x l(-1)) completely blocked the conductance response to hypotonic shock and the reduction in apical crypt density measured by scanning electron microscopy, while phalloidin (33 micromol x l(-1)), colchicine (3x10(-4)mol x l(-1)) and griseofulvin (1.0 micromol x l(-1)) were ineffective. Actin imaging by phalloidin staining and confocal microscopy revealed extensive actin cords in pavement cell microridges and a ring of actin at the apex of chloride cells. We conclude that the actin cytoskeleton of chloride cells is required to maintain crypt opening and that osmotic shock causes chloride cells to adjust their apical crypt size.