Cellular impalements were used in combination with standard transepithelial electrical measurements to evaluate some of the determinants of the spontaneous lumen-positive voltage, Ve, which attends net Cl- absorption, JnetCl, and to assess how ADH might augment both JnetCl and Ve in the mouse medullary thick ascending limb of Henle microperfused in vitro. Substituting luminal 5 mM Ba++ for 5 mM K+ resulted in a tenfold increase in the apical-to-basal membrane resistance ratio, Ra/Rbl, and increasing luminal K+ from 5 to 50 mM in the presence of luminal 10(-4)M furosemide resulted in a 53-mV depolarization of apical membrane voltage, Va. Thus K+ accounted for at least 85% of apical membrane conductance. Either with or without ADH, 10(-4) M luminal furosemide reduced Ve and JnetCl to near zero values and hyperpolarized both Va and Vbl, the voltage across basolateral membranes; however, the depolarization of Vbl was greater in the presence than in the absence of hormone while the hormone had no significant effect on the depolarization of Va. Thus ADH-dependent increases in Ve were referable to greater depolarizations of Vbl in the presence of ADH than in the absence of ADH. 68% of the furosemide-induced hyperpolarization of Va was referable to a decrease in the K+ current across apical membranes, but, at a minimum, only 19% of the hyperpolarization of Vbl could be accounted for by a furosemide-induced reduction in basolateral membrane Cl- current. Thus an increase in intracellular Cl- activity may have contributed to the depolarization of Vbl during net Cl- absorption, and the intracellular Cl- activity was likely greater with ADH than without hormone. Since ADH increases apical K+ conductance and since the chemical driving force for electroneutral Na+, K+, 2Cl- cotransport from lumen to cell may have been less in the presence of ADH than in the absence of hormone, the cardinal effects of ADH may have been to increase the functional number of both Ba++-sensitive conductance K+ channels and electroneutral Na+, K+, 2Cl- cotransport units in apical plasma membranes.

Effects of antidiuretic hormone on cellular conductive pathways in mouse medullary thick ascending limbs of Henle: II. determinants of the ADH-mediated increases in transepithelial voltage and in net Cl-absorption.

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 vasopressin-induced 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 vasopressin-induced water permeability and not to changes in urea or sodium permeability.

https://dm5migu4zj3pb.cloudfront.net/manuscripts/108000/108673/JCI77108673.pdf

Relationship of aggregated intramembranous particles to water permeability in vasopressin-treated toad urinary bladder.

Examination of the toad urinary bladder by freeze-fracture electron microscopy reveals that the mitochondria-rich cells of the epithelium possess distinctive and characteristic membrane structural specialization. Unique rod-shaped intramembrane particles are found in luminal and basal membranes as well as certain intracellular vesicles of this cell type. The consistent finding of two discrete patterns of luminal membrane structural organization supports the possibility that two morphological forms of mitochondria-rich cell exist within the toad bladder epithelium.

Membrane structural specialization of the toad urinary bladder revealed by the freeze-fracture technique. II. The mitochondria-rich cell.

Examination of the toad urinary bladder by freeze-fracture electron microscopy reveals intramembrane particle arrays at a number of membrane sites. An array in which particles are aggregated into closely apposed parallel rows is found in the granular cell luminal membrane of dehydrated toads fixed in situ. These aggregates are structurally indistinguishable from those previously associated with vasopressin exposure in vitro. Aggregates are not found in granular cell luminal membrane in the case of hydrated toads fixed in situ. However, structurally similar arrays are found at low frequency in the membrane of cytoplasmic vacuoles in granular cells and in the plasma membrane of basal cells in both hydrated and dehydrated toads. Aggregates are also present at these sites in control and vasopressin-treated bladders from in vitro experiments. Particle arrays characteristic of gap junctions, desmosomes and hemidesmosomes also occur in the plasma membrane of basal cells. In addition, distinctive square arrays of particles exist in the plasma membrane of the bladder's mesothelium. Although a variety of intramembrane particle arrays exist in the toad urinary bladder, only the occurrence of organized particle aggregates in the luminal membrane of granular cells appears to be associated with vasopressin exposure.

Membrane structural specialization of the toad urinary bladder revealed by the freeze-fracture technique. III. Location, structure and vasopressin dependence of intramembrane particle arrays.

Freeze-fracture of granular cells of toad urinary bladder (Bufo marinus) reveals the presence, hitherto undescribed, of intramembranous particle aggregates in intracytoplasmic structures (tubules, vacuoles and vesicles) both in resting and vasopressin-stimulated epithelia.

Particle aggregates in plasma and intracellular membranes of toad bladder (granular cell).

Freeze-fracture electronmicroscopy demonstrates that vasopressin stimulation of isolated toad bladder results in a striking morphologic alteration of epithelial membrane structure. This alteration is characterized by the aggregation of intramembranous particles in orderly linear arrays at multiple sites in the luminal membranes of granular cells specifically. The size of these aggregates varies considerably, in terms of area, over a range from 0.5 to 70×10−3 μm2. The median aggregate size is about 10.5×10−3 μm2. Since the extent of vasopressin-associated particle aggregation, in terms of frequency of sites per area of membrane or cumulative area of membrane occupied by them, closely correlates with induced changes in transport function, as measured by osmotic water flow, the aggregates themselves appear to be of physiologic significance in the mechanism of action of vasopressin. This hypothesis is supported by the observations that sites of aggregation occur (a) in response to serosal exposure to hormone specifically, (b) independently of an osmotic gradient, and (c) following stimulation with cyclic adenosine monophosphate.

Carole C. Uiterwyk

Papers

Membrane structural and functional responses to vasopressin in toad bladder