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-

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A B S T R A C T The exaggerated natriuresis of hypothyroid rats receiving a 5% saline infusion was studied to determine the mechanism and the site within the nephron responsible for this increase in sodium excretion. Sodium clearance (CNa) and fractional sodium excretion were both demonstrated to be greater in hypothyroid rats for any amount of sodium infused. The rate of increase in fractional sodium excretion in response to saline loading was 3.4 times greater in hypothyroid animals. At the conclusion of the diuresis some of the hypothyroid animals excreted greater than 45% of the filtered sodium load, while no control animal excreted more than 12% of the filtered sodium load.The mean clearance of insulin during the saline diuresis was 36.6% lower (P < 0.001) in the hypothyroid rats. D-Aldosterone given to hypothyroid animals 3 hr before the experiment did not alter the magnitude or rate of increase in fractional sodium excretion. Inulin space determinations in nephrectomized rats revealed that extracellular fluid volume was contracted by 17.1% in the hypothyroid rats (P < 0.01). Plasma sodium was not significantly different in hypothyroid and control animals.A limit on solute free water reabsorption (T'Huo) per osmolar clearance (Co..) was demonstrated in the hypothyroid rats when these animals excreted greater than 12% of the filtered osmotic load. The limit on TCH20 formation was associated with an acceleration in the rate of sodium excretion and a decline in the rate of potassium excretion. Early in the diuresis when Co.., CNa, and TCH20 were comparable in hypothyroid and control Received for publication 5 January 1970.rats, the filtered sodium load was 31% lower (P < 0.01) in the hypothyroid animals. These findings indicate that diminished thyroid hormone activity decreases renal sodium reabsorptive capacity. Indirect evidence suggests that the distal and possibly the proximal tubules are the sites of this diminished sodium reabsorption in hypothyroid animals. INTRODUCTIONHypothyroidism has profound effects on salt and water metabolism in the rat. These are manifested by an increase in water consumption and excretion (1), inability to elaborate a maximally concentrated urine (2), exaggerated natriuresis during a water or saline diuresis (3,4), and impaired ability to conserve sodium, resulting in a negative sodium balance and death when sodium intake is restricted (5). Fregly, Cade, Waters, Straw, and Taylor (4,6) have demonstrated a decreased secretory rate and tubular response to aldosterone in hypothyroid rats, and they have proposed that thyroid insufficiency influences sodium reabsorption through this indirect mechanism. Reville and Stephan (7), however, have recently presented evidence in adrenalectomized hypothyroid rats which suggested that the alteration of sodium metabolism observed in hypothyroid animals was independent of adrenal function. In the present study several of the factors which regulate sodium reabsorption were evaluated with the hope of determining what mechanism might be responsi...

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