Freeze-fracture electron microscopy demonstrates that vasopressin stimulation of isolated toad bladder alters the structure of the luminal membrane of granular cells. This alteration consists of an ordered aggression of intramembranous particles, and appears to be of functional significance, since the frequency of aggregation sites per area of membrane is closely correlated with vasopressin-induced osmotic water flow.

In freeze-fracture (FF) preparations of ADH-stimulated toad urinary bladder, characteristic intramembrane particle (IMP) aggregates are seen on the protoplasmic (P) face of the luminal membrane of granular cells while complementary parallel grooves are found on the exoplasmic (E) face . These IMP aggregates specifically correlate with ADH-induced changes in water permeability . Tubular cytoplasmic structures whose membranes contain IMP aggregates which look identical to the IMP aggregates in the luminal membrane have also been described in granular cells from unstimulated and ADH-stimulated bladders . The diameter of these cytoplasmic structures (0.11 ± 0.004 ,um) corresponds to that of tubular invaginations of the luminal membrane seen in thin sections of ADH-treated bladders (0.13 ± 0.005 pm). Continuity between the membranes of these cytoplasmic structures (which are not granules) and the luminal membrane has been directly observed in favorable cross-fractures . In FF preparations of the luminal membrane, these apparent fusion events are seen as round, ice-filled invaginations (0.13 ± 0.01 ,um Diam), of which about half have the characteristic ADHassociated aggregates near the point of membrane fusion . They are less numerous than, but linearly related to, the number of aggregates counted in the same preparations (n = 78, r = 0.71, P < 0.01). These observations suggest that the IMP aggregates seen in luminal membrane after ADH stimulation are transferred preformed by fusion of cytoplasmic with luminal membrane .Freeze-fracture electron microscopy has shown that stimulation of the isolated amphibian urinary bladder with antidiuretic hormone (ADH) results in a structural change in the luminal membrane of granular epithelial cells (1,3,11,12) . Whereas in the unstimulated bladder intramembrane particles (IMP) are randomly distributed in an apparently J. CELL BIOLOGY0 The Rockefeller University Press -0021-9525/80/04/0083/13 $1 .00Volume 85 April 1980 83-95 homogeneous matrix, after ADH stimulation IMP aggregates of various sizes are seen at discrete membrane sites on the protoplasmic half-membrane (fracture face P) . Complementary areas of the apposed exoplasmic half-membrane (fracture face E) exhibit parallel rows of depressions (grooves), reflecting the linear organization of par-

Intramembranous particle aggregates in the luminal membrane of toad bladder granular cells after vasopressin stimulation have been found to correlate closely and specifically with induced alterations of water permeability. Roles for microtubules and microfilaments in mediating the latter response have been proposed on the basis of studies involving colchicine and cytochalasin B, respectively. In the present investigation the effects of these agents on both initiating and sustaining vasopressin-induced osmotic water flow and the particle aggregation phenomenon were studied. The results indicate that during initiation the aggregation and water flow responses to vasopressin are each colchicine- and cytochalasin B-sensitive and that these sensitivities can be wholly additive. However, after full vasopressin stimulation is established, the same responses demonstrate sensitivity only to cytochalasin B, not to colchicine. The findings, therefore, suggest that microtubules and microfilaments may be independently necessary for the initiation of the aggregation and water flow responses to vasopressin, and that microfilaments, but not microtubules, are required for their maintenance.

A B S T R A C T It has been previously demonstrated with freeze-fracture electron microscopy that vasopressin induces specific structural alterations of the luminal membrane of granular cells from toad urinary bladder in a dose-dependent fashion. These alterations consist of aggregated intramembranous particles and are observed both in the presence and absence of an osmotic gradient. We examined the effect of methohexital, a selective inhibitor of vasopressin-stimulated water flow, and the effect of phloretin, a selective inhibitor of urea permeability, on the structure of the granular cell luminal membrane. Methohexital treatment of the vasopressin-stimulated toad bladder reduced both the osmotic water flow and vasopressininduced alterations of membrane structure to the same extent. Phloretin reduced urea permeability but not water flow or particle aggregation. Since neither agent affects vasopressin-stimulated sodium movement, these findings indicate that the phenomenon of particle aggregation is specifically related to vaso-

Freeze-fracture electron microscopy had previously revealed antidiuretic hormone-induced aggregates of intramembranous particles in amphibian urinary bladder. To investigate the effects of antidiuretic hormone (ADH) in another ADH-sensitive epithelium, namely, mammalian renal collecting ducts, freeze-fracture studies were carried out in Brattleboro homozygous rats. Collecting duct luminal membranes of ADH-treated homozygotes showed intramembranous particle clusters (117 ± 17/100 μm2) that were loosely packed and that occurred on both exoplasmic (E) and protoplasmic (P) faces. Untreated, control homozygous rats had significantly less (3 ± 1/100 μm2) clusters. Changes similar to those seen in ADH-treated rats were observed in water-deprived Wistar rats. The clustered particles differed from those seen in ADH-treated amphibian urinary bladder in that the latter occurred only on the P face and were more densely packed. Nevertheless, our observations suggest a common membrane effect for ADH action that may apply in mammals and amphibia alike. freeze-fracture; Brattleboro homozygous rats; membrane particle clusters Submitted on March 6, 1978 Accepted on July 14, 1978