No abstract

Using the methods described in the preceding paper (Levine et al., 1984) for measuring the magnitude of the water-permeable barriers in series with the luminal membrane, we correct measured values of Pd(w) in bladders stimulated with low doses of antidiuretic hormone (ADH) or 8-bromo cyclic AMP to obtain their true values in the luminal membrane . Simultaneously, we also determine Pf . We thus are able to calculate Pf/Pd(w) for the hormone-induced water permeation pathway in the luminal membrane . Our finding is that Pf/Pd (w) = 17 . Two channel models consistent both with this value and the impermeability of the ADH-induced water permeation pathway to small nonelectrolytes are : (a) a long (=50 A), small-radius (=2 A) pore through which 17 water molecules pass in single-file array, and (b) a showerhead-like structure in which the stem is long and of large radius (=20 A) and the cap has numerous short, small-radius (=2 A) pores. A third possibility is that whereas the selective permeability to HQO results from small-radius ("-"2 A) pores, the large value of Pf/Pd(w) arises from their location in the walls of long tubular vesicles (^-2 ,um in length and 0

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-

Antidiuretic hormone (ADH) induces a large increase in the water permeability of the luminal membrane of toad urinary bladder. Measured

The interaction of vasopressin with prostaglandins were examined in the toad bladder by determining water flows, cAMP levels, and cAMP-dependent protein kinase activity. Both water flow and activation of cAMP-kinase in response to vasopressin were enhanced after prostaglandin inhibition, consistent with inhibition of vasopressin-induced cAMP generation by endogenous prostaglandins. On the other hand exogeneous PGE stimulated cAMP generation. PGE1 (10(-7) M) alone did not increase water flow but activated kinase more than vasopressin only. Addition of PGE1 (10(-7) M) and vasopressin inhibited water flow as compared with vasopressin along but increased the kinase ratio above that with vasopressin only. PGE2 (10(-5) M) increased the cAMP content and kinase ratio even more than vasopressin but again resulted in no water flow. Addition of vasopressin and PGE2 (10(-5) M) increased water flow but did not alter cAMP content or the kinase ratio compared with PGE2 alone. Similar results were obtained with PGE1. Accordingly, prostaglandin dissociates cAMP levels and kinase ratio from the hydroosmotic response, suggesting that PGE2 inhibits steps distal to cAMP. Consistent with this, in bladders pretreated with naproxen or meclofenamate, PGE2 (10(-8) to 10(-6) M) inhibited the response to submaximal doses of cAMP (5 mM) or 8-bromo-cAMP (0.03 mM). Furthermore, pretreatment with naproxen significantly enhanced the response to cAMP (5 mM). These studies provide evidence for vasopressin-PGE interaction at the site of cAMP generation and also at a step(s) unrelated to cAMP generation.