Very high water permeability in vasopressin-induced endocytic vesicles from toad urinary bladder.

Abstract

The regulation of transepithelial water permeability in toad urinary bladder is believed to involve a cycling of endocytic vesicles containing water transporters between an intracellular compartment and the cell luminal membrane. Endocytic vesicles arising from luminal membrane were labeled selectively in the intact toad bladder with the impermeant fluid-phase markers 6-carboxyfluorescein (6CF) or fluorescein-dextran. A microsomal preparation containing labeled endocytic vesicles was prepared by cell scraping, homogenization, and differential centrifugation. Osmotic water permeability was measured by a stopped-flow fluorescence technique in which microsomes containing 50 mM mannitol, 5 mM K phosphate, pH 8.5 were subject to a 60-mM inwardly directed gradient of sucrose; the time course of endosome volume, representing osmotic water transport, was inferred from the time course of fluorescence self-quenching. Endocytic vesicles were prepared from toad bladders with hypoosmotic lumen solution treated with (group A) or without (group B) serosal vasopressin at 23~ and bladders in which endocytosis was inhibited by treatment with vasopressin at 0-2~ (group C), or with vasopressin plus sodium azide at 23~ (group D). Stopped-flow results in all four groups showed a slow rate of 6CF fluorescence decrease (time constants 1.0-1.7 s for exponential fit) indicating a component of nonendocytic 6CF entrapment into sealed vesicles. However, in vesicles from group A only, there was a very rapid 6CF fluorescence decrease (time constant 9.6 _+ 0.2 ms, SEM, 18 separate preparations) with an osmotic water permeability coefficient (Pf) of >0.1 cm/s (18~ and activation energy of 3.9 -+ 0.8 kcal/mol (16 kJ/mol). Pr was inhibited reversibly by >60% by 1 mM HgC12. The rapid fluorescence decrease was absent in vesicles in groups B, C, and D. These results demonstrate the presence of functional water transporters in vasopressin-induced endocytic vesicles from toad bladder, supporting the hypothesis that water channels are cycled to and from the luminal membrane and providing a functional marker for the vasopressin-sensitive water channel. The calculated Pt in the vasopressin-induced endocytic vesicles is the highest Pt reported for any biological or artificial membrane.

Current understanding of the cellular biology and molecular structure of the antidiuretic hormone-stimulated water transport pathway.

“…1). These retrieved vesicles containing either entrapped carboxyfluorescein, fluorescein dextran (F dextran), or horseradish peroxidase (HRP) have been recovered from homogenates ofgranular cells (6,(24)(25)(26)(27)(28) and the renal papillas ofBrattleboro rats (29) and used to measure vesicle membrane Pf and proton permeability (PH+). The Pf of these water channel-containing vesicles (WCV) is extraordinarily high, demonstrating that functional water channels are removed from the apical membrane by vesicle endocytosis.…”

“…This is combined with the fact that the Pf exhibited by WCV is so large that it precludes accurate determinations ofvesicle PD (24,25). Together these considerations have made it difficult to relate the apparent PdPD ratios exhibited by ADH water flow to actual characteristics of ADH channel structure.…”

“…When a combination ofmercurials and sulfhydryl protection agents such as mercaptoethanol is added to either intact epithelia or WCV, no inhibition ofADH water flow is observed (16,25).…”

“…In contrast, ADH stimulation causes fusion of large numbers of subapical aggrephores with the apical membrane. Retrieval of water channel-containing membrane is dramatically increased by transepithelial water flow or removal of ADH (36) allowing selective entrapment of fluid phase markers within the lumens of WCV (24)(25)(26)(27) that possess large numbers of functional water channels.…”

Evidence from oocyte expression that the erythrocyte water channel is distinct from band 3 and the glucose transporter.

“…Additional evidence supporting the existence of an erythrocyte water channel is a low Arrhenius activation energy for Pf (Ea < 5 kcal/mol), a high ratio of osmotic-to-diffiusional water permeability (PdPd> 3) and strong inhibition of Pf by mercurial sulfhydryl reagents (1)(2)(3). Mercurial-sensitive water channels are also present on plasma and intracellular membranes of kidney proximal (4,5) and collecting tubules (6)(7)(8), and amphibian urinary bladder (6,9,10), but not in other cell types including intestine, trachea, placenta, and platelets (3).…”