The terminal web of the intestinal brush border contains a spectrin-like protein, TW 260/240 (Glenney, J. R ., Jr., P. Glenney, M. Osborne, and K. Weber, 1982, Cell, 28:843-854 .) that interconnects the "rootlet" ends of microvillar filament bundles in the terminal web (Hirokawa, N ., R. E. Cheng, and M . Willard, 1983, Cell, 32 :953-965; Glenney J . R ., P. Glenney, and K. , /. Cell Biol., 96:1491-1496 . We have investigated further the structural properties of TW 260/240 and the interaction of this protein with actin . Salt extraction of TW 260/240 from isolated brush borders results in a loss of terminal web cross-linkers primarily from the apical zone directly beneath the plasma membrane. Morphological studies on purified TW 260/240 using the rotary shadowing technique confirm earlier results that this protein is spectrin-like and is in the tetrameric state in buffers of low ionic strength . However, examination of TW 260/240 tetramers by negative staining revealed a molecule much straighter and more uniform in diameter than rotary-shadowed molecules . At salt concentrations at (150 mM KCI) and above (300 mM KCI) the physiological range, we observed a partial dissociation of tetramers into dimers that occurred at both 0°and 37°C. We also observed (in the presence of 75 mM KCI) a concentration-dependent self-association of TW 260/240 into sedimentable aggregates .We have studied the interaction of TW 260/240 with actin using techniques of cosedimentation, viscometry, and both light and electron microscopy. We observed that TW 260/240 can bind and cross-link actin filaments and that this interaction is salt-and pHdependent . Under optimum conditions (25-75 mM KCI, at pH 7 .0) TW 260/240 cross-linked F-actin into long, large-diameter bundles. The filaments within these bundles were tightly packed but loosely ordered . At higher pH (7.5) such bundles were not observed, although binding and cross-linking were detectable by co-sedimentation and viscometry. At higher salt (>150 mM KCI), the binding of TW 260/240 to actin was inhibited . The presence of skeletal muscle tropomyosin had no significant effect on the salt-dependent binding of TW 260/240 to F-actin .The ubiquitous presence of spectrin-like proteins in the "cortical" cytoplasm of cells has been recently established by numerous biochemical and immunological studies on a wide variety of cell types and tissues (reviewed in references 1 and 2). By analogy with erythrocyte spectrin

No abstract

Vasopressin increases the water permeability of the apical membrane of the granular epithelial cells of the toad urinary bladder. Cytochalasin B inhibits this action of the hormone, indicating that microfilaments may play a role in the water permeability response. We have extended previous functional studies with cytochalasin B and have demonstrated that dihydrocytochalasin B, a more specific inhibitor of actin filament elongation, similarly diminishes the hydrosmotic response to vasopressin. Biochemical studies of isolated epithelial cells indicate that an actin-like protein accounts for about 10% of the soluble protein of the epithelium. Morphological studies of whole toad bladders incubated with heavy meromyosin conclusively demonstrate that actin is a component of the epithelial cells and that actin-containing filaments are associated with both plasma membranes and cytoplasmic organelle membranes. Taken together, these findings provide strong, albeit indirect, evidence that actin microfilaments play a functional role in the hormone-induced increase in water permeability in the toad urinary bladder.

No abstract

The effects of experimental procedures believed to increase cytosolic calcium on basal and vasopressin-stimulated osmotic water flow and transepithelial sodium transport were examined in the toad urinary bladder. Exposure of isolated toad bladders to quinidine, calcium ionophores (A23187, X537A), or low-sodium or potassium-free serosal solutions resulted in a dose-dependent decrease in the hydrosmotic response to vasopressin or exogenous adenosine 3',5'-cyclic monophosphate (cAMP). The degree of inhibition of cAMP-induced water flow induced by low-sodium or potassium-free serosal bathing media varied, and in a similar manner, with the serosal calcium concentration. The effects of quinidine sulfate (2 X 10-4 M), X537A (2 X 10(-5) M), and low serosal sodium (20 mM), but not that of A23187 (10(-5) M), were readily reversible. Exposure to quinidine (4 X 10(-4) M), A23187 (10(-5) M), X537A (5 X 10(-6) M), or low serosal sodium (2 mM) also inhibited the basal short-circuit current (SCC). Vasopressin, 4-20 mU/ml, completely overcame the inhibition of the SCC induced by quinidine, A23187, or low serosal sodium, but a submaximal dose of hormone (4 mU/ml) failed to fully reverse the inhibitory effect of X537A, 5 X 10(-6) M. These results are consistent with the view that 1) a Na-Ca exchange process operates across the basolateral surface of the granular epithelial cells of the toad urinary bladder in vivo, and 2) the level of free calcium in the granular cell cytosol plays a modulatory role in the control of apical membrane water and sodium permeability by vasopressin, and in the regulation of the basal rate of transepithelial sodium transport.