Kidney International, Vol. 31(1987), pp. 530—537
Effects of antidiuretic hormone on the collecting duct RICHARD M. HAYS, NICHOLAS FRANKI, and GUOHUA DING Department of Medicine, Division of Nephrology, Albert Einstein College of Medicine, Bronx, New York, USA
One can imagine the consternation that swept the residents of cellular response to ADH; among them are prostaglandin E2 the Cambrian sea when it was announced that some of them [11, 12], calcium [13], protein kinase C [14], aldosterone [15], would be chosen to emerge on dry land. They had enjoyed their and certain amino acids [161. The final step in the sequence became clear when, as already easy equilibrium with their environment, without a thought about water balance. Now all this would change. noted, Chevalier, Bourguet and Hugon showed by freeze— Their features were groundless. Planning for this moment had fracture electron microscopy the appearance of the particle started long ago. New structures would be combined with old aggregates in the luminal membrane [4]. Wade [17] and Humbert hormones to create thirst. Skin and bladder would serve to and co-workers [18] then showed that the aggregates originated conserve water in amphibia, and a new, exquisitely designed from tubular structures in the cytoplasm which fused with the countercurrent system would come into being for mammals. luminal membrane. The essential features of the cellular reVasopressin, which had been transmitting primitive messages sponse to ADH are shown in Figure 1. Despite the progress that has been made, a great number of in prehistoric brains for millions of years, would be chosen to mediate the new signal for water conservation. The response of questions remain. Among them are how ADH initiates the the target organs would be prompt, making use of cyclic AMP process of fusion, how the cytoskeleton participates in this and vesicular fusion, old and reliable mechanisms. All of this process, and what happens to tubules after they fuse. This would be done without the help of the NIH, and the gifted group review will center on the structural changes that take place in of scientists, Dr. Berliner among them, who would later under- the ADH receptor cells of the toad bladder, particularly the changes in tubule position that may give clues to the relationstand and explain these principles of water conservation. ship of the cytoskeleton to fusion. We will attempt to integrate Collecting duct and amphibian bladder our work, and the work of others, into a scheme which we In the mammalian kidney, cells along the entire collecting concede is speculative, and probably no better than a number of duct respond to ADH, showing significant swelling in the alternative possibilities. The only virtue of such a presentation presence of an osmotic gradient [1]. In the amphibian bladder, is the questions it raises. The basis for the work to be presented is the large number of the granular cells, which are the predominant cells, show a similar response [21. Despite their existence in very different transmission electron microscopy (TEM) photographs we have tissues, the target cells for ADH in amphibian bladder and taken of bladders fixed by the tannic acid—glutaraldehyde mammalian collecting duct have much in common. The final method of Begg, Rodewald and Rebhun [19]. This method of event in both cells is the appearance of particle aggregates in the fixation reveals the filamentous structure of the apical region of urine—facing cell membrane [3, 4]; it is these particles that are the cell, Since tubules are not abundant in the cytoplasm, many believed to promote water flow across the membrane. The thin sections must be inspected before any pattern of tubular discovery of the particle aggregates came many years after the position becomes evident. proposal by Koefoed—Johnsen and Ussing that water flow might take place through pores or channels in the membrane [51, and
The resting cell the demonstration by Maffly et al [6], that the luminal memLet us first consider the appearance and position of the brane was the rate—limiting barrier that was transformed by tubules in cells unstimulated by ADH. Representative sections ADH. The membrane channels were found to be specific for are shown in Figures 2A and 2B, as well as in succeeding water, excluding solutes as small as urea [7]. It was proposed figures. A number of features occur regularly in tubules seen by that the water—conducting channels might be extremely narrow, transmission, scanning and freeze—fracture electron microsand that water molecules might move in single—file fashion copy. They are elongated, thin structures. Their mean diameter through them [8]. Cyclic AMP was shown to be the intracellular by TEM is 0.094 0.002 jxm, agreeing closely with data mediator of ADH [9], and the cytoskeleton was shown to be published by others [20]. TEM is not an ideal method for important in the intracellular response to the hormone [10]. A estimating tubule length, since it is unusual to visualize the large number of agents have now been shown to modify the entire tubule in a single section; in the few instances where this has been possible in a single or in serial sections, (for example, Fig. 2B), the maximum tubule length noted was 0.85 pm. Received for publication July 9, 1986 The tubules give an impression of rigidity as they lie within the cytoplasm; it is rare to see a bent tubule in control © 1987 by the International Society of Nephrology 530
531
ADH and the collecting duct
may be to anchor and position tubules -within the cytoskeleton; it is also possible that some of these filaments are responsible
for tubular motility, to be discussed in a later section. In addition to the filaments, there are narrow tubular elements that occasionally appear in close approximation to the tubules; their function is unclear. Finally, we come to the important question of tubule position with respect to the luminal membrane. Position, and its alter-
ation following stimulation by ADH, may give clues to the forces acting on the tubules that eventually lead to their fusion. We have, therefore, extended our earlier studies [22] to define
tubule angle in relation to distance of the tubule from the luminal membrane in control cells, and at several time points following ADH stimulation. We measured the distance from the luminal membrane to the lowest point of the tubule, and the angle of the tubule from a horizontal line parallel to the luminal membrane. Figure 3 shows the mean values for these measurements, at three ranges of depth from the luminal membrane: 0 to
0.19 m; 0.20 to 0.49 m, and 0.50 to 0.79 jim. In the absence of ADH, the tubules are virtually parallel to the luminal membrane at all depths (Fig. 3, Column 1). Soon
after stimulation by ADH, the angulation of the tubules Fig. 1. Diagrammatic view of the intracellular action of ADH. The hormone is bound to receptors on the basolateral membrane of the receptor cell and activates adenylate cyclase, increasing the intracellular concentration of cyclic AMP. A series of intermediate steps take place, including activation of a cyclic AMP-dependent protein kinase. Cytoplasmic tubular structures (A) are induced to fuse with the luminal membrane (B), and deliver aggregates of water—conducting particles to the membrane (C). Gr, subluminal granule.
changes, and the sequence of tubule fusion and particle delivery begins. ADH stimulation
The response to the toad bladder to the administration of ADH is rapid. By 2.5 minutes, Kachadorian, Casey and DiScala
have shown that some tubules have already fused with the luminal membrane and that particle aggregates have appeared
in the membrane [23]. Aggregate delivery and water flow accelerate to a maximum at approximately 20 minutes, and then decline. Figure 4 show some features of ADH-treated epithelial
preparations, while occasional bending is seen in fused tubules following ADH. The apparent rigidity may be created by the helical array of particles inserted into the tubule wall, or to supporting structures outside the tubule. The linearity of the tubules has enabled us to determine with some confidence the angulation of the tubules under a variety of conditions. Many tubules show a suggestion of segmentation (for example, Figs. 2A). One end of the tubule is often spherical with a constricted neck, a feature seen in several TEM figures in this review, and
cells. In Figure 4A, fixed 15 minutes after administration of
spherical heads, there are protuberances from the sides of the tubules [221. These are prominent in Figure 2B, and appear to be vesicles fusing with, or leaving the main body of the tubule. A coated vesicle is also seen. Do these vesicles represent stages
active Golgi complex, with a cascade of tubular structures below it; another Golgi complex is shown in Figure 4C, also with a tubular structure in its vicinity. Sections such as these
ADH, a tubule is touching the luminal membrane. The tubule is
approximately vertical, and shows slight segmentation of its body. A spherical head is again noted. There is a small dense
patch at the point of contact of the head with the luminal membrane, and there are filaments running across the base of the tubule. The tubule is probably undergoing fusion, rather
than detaching from the luminal membrane, since colloidal gold had been placed in the luminal bathing medium, and would have to an even more pronounced degree in earlier SEM and entered the tubule had it already fused (Figs. 5A-D). Deeper in freeze—fracture studies [21, 22], We have found that the spher- the cytoplasm, there is a well—developed Golgi complex. ical head is often coated with clathrin, suggesting that it is a Figure 4B, fixed 45 minutes after ADH administration, shows coated vesicle (see note added in proof). a fused tubule with an unusually square base, which may be Throughout our studies, we have noted that in addition to the formed by transversely—running filaments. Again, there is an
have raised the possibility that the tubules originate as vesicles
in tubule assembly, in which vesicles coalesce to form the in the Golgi complex, which then coalesce to form the elongated tubule, and/or do vesicles leave the tubule, bound for other sites tubules. However, while the Golgi is unquestionably a major within the cell? This question cannot be answered at present, site of origin of vesicles [24] we have no direct evidence for its but there is certainly evidence of activity in and around the role in the creation of tubules, and the origin of the tubules must tubule in addition to the major phenomenon of tubule fusion. Filaments can be seen running from the sides and ends of the tubules to the surrounding cytoskeleton (Fig. 2A). These were
also seen in earlier scanning studies in which the resinless embedding technique was used [22]. Their primary function
remain an open question.
How does the process of fusion begin? Can one detect a change in tubule position prior to actual fusion? Returning to our study of angulation (Fig. 3), the second column shows tubule angle five minutes after the administration of ADH.
532
Hays et a!
Fig. 2. Sections of two control bladders, showing tubules in approximately horizontal positions. A. Several tubules are present. The left arrow shows a series of filaments emerging from the base of the tubule; the arrow at the right indicates the spherical head. The lumen (L) and several microvilli (MV) are shown. B. A long tubule is shown, with several budding vesicles (arrows) and a nearby coated vesicle (arrowhead). (A x50,000; B x50,000.)
Isotonic Ringer's bathed both sides of the bladder at this time point, so that water flow and cell swelling were not factors in any change in tubule position. We wanted to make our measurements at this early time point to "catch" the tubules prior to fusion, and to minimize the percentage of tubules that had already fused and were returning. At a distance of 0 to 0.19 tm from the luminal surface, the mean angle of the tubules in the ADH-treated tissues was not significantly greater than in the controls. However, at 0.2 to 0.49 jsm from the surface, there was a significant increase in angulation of tubules compared to the control. It is unlikely that these tubules were already fused, with the point of fusion being out of the plane of the section; rather, we think that the increase in angulation may be the first step in the movement toward fusion, and that some element in the cytoskeleton may be responsible for the change in tubule position. At the deepest level surveyed, 0.50.t 0.79 m from the surface, the difference between control and ADH-treated tubules was not significant. Thus, the "signal", whatever it may be, appears to affect primarily the tubules at the middle range of depth in the cytoplasm. By 15 minutes (Fig. 3, third column), the angle of the mid-range tubing is steeper. There were not enough superficial tubules to include at this time point. Also, there was an osmotic gradient in the group of experiments
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3. Tubule angles as a function of distance from the luminal
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a significantly greater angle than the controls. Tubules undergoing fusion, and at points beyond fusion, contain colloidal gold. Angles SEM are indicated below each group of tubules.
angulation. Column 4 shows fused tubules, with a mean angle of 610.
After fusion takes place, the tubules may retain their basal carried out at this time point, which may have influenced anchorage. This observation was made in earlier freeze—crack
ADH and the collecting duct
533
Fig. 4. Tubule about to fuse with the luminal membrane after 15 mm of ADH stimulation. The spherical head and slightly segmented body are
apparent. Transverse filaments are seen at the base (arrow). A Golgi complex (G) lies deeper in the cytoplasm. B. Fused tubule (T) and Golgi complex 45 mm after ADH stimulation. A series of tubular structures is seen below the Golgi complex, between the two arrows. C. 5 mm of ADH-stimulatjon; Golgi complex, showing a tubular structure nearby (arrow). Several coated vesicles (multiple arrows) are shown in the upper right hand corner. (A x43,000; B x43,000; C x50,000.)
Hays et a!
534
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Fig. 5. Colloidal gold—containing tubules during continuous ADH stimulation. In B, a thin tubular structure runs below the tubule. C shows a highly angulated tubule (arrow), and a multivesicular body (M). In D, a spherical head is clearly seen. A-C: 15 mm of ADH stimulation; D: 45 mm of ADH stimulation. (A-D x43,000.)
studies [22]; Figure 4A also shows filaments at the base of the tubule which can serve as anchoring structures. The filaments are not invariably seen at the tubule bases, and the question of basal anchorage is still unresolved.
absence of an osmotic gradient may be important in determining
the rate of tubule detachment [27]. Our own studies [28] have been carried out in the presence of an osmotic gradient, with colloidal gold in the luminal membrane as a marker. As early as 15 minutes after the beginning of ADH stimulation, tubules were found in the cytoplasm containing colloidal gold, evidence Fusion and detachment of tubules that they had fused and then detached in the presence of ADH Because the methods used to study fusion have generally (Fig. 5A-D). Serial sections showed that the tubules had sepainvolved fixed tissues, it has been difficult to distinguish between two patterns of fusion: the first is one in which a given rated completely from the membrane. Thus, we favor the view tubule remains fixed throughout the period of hormonal stimu- that fusion and detachment may take place continuously, at lation; the second is one in which the tubules are continuously least in the presence of an osmotic gradient, and that the fusing and detaching from the luminal membrane. Studies by lifetime of a given fusion event is less than 15 minutes. After Gronowitz, Masur and Holtzman [25], Wade, Stetson and detaching, the tubules remained significantly angulated at both Lewis [261, and Masur, Cooper and Rubin [27], employing the middle and lowest level surveyed (Fig. 3, column 5). In horseradish peroxidase as a marker, favor the latter possibility. addition, virtually all of them had lost their spherical heads. What is the fate of the detached tubules? It appears possible Muller, Kachadorian and DiScala have concluded that detachment does not take place during the period of hormonal from the studies of Muller, Kachadorian and DiScala [20], and, stimulation [20]. It is important to point out that the presence or most recently, Harris, Wade and Handler [29] that many
ADH and the collecting duct
535
in regulating the entire system, and that linkages exist between tubules and the surrounding cytoskeleton.
However, it is abundantly clear that there are far more questions than answers at this point. We do not know the actual time that the tubules spend in the fused state, the percentage of their entire complement of aggregates that they deliver to the
luminal membrane in a single fusion, or the mechanism by which they fuse or detach. In addition, we do not have the information that would permit us to estimate the rate at which new tubules are generated and used tubules are destroyed or reutilized. This is important when one considers the receptor 'S
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"tubular reserve", and how does it provide a continuous supply of aggregates? We also have no clear idea of how the tubules move from one position to another within the cell. Cell biologists have proposed some interesting models for organelle movement, which we can briefly consider. The cytoskeleton and organelle movement
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cell during a period of sustained antidiuresis: what is its
The cytoskeleton is a highly cross—linked structure, capable of rapid assembly and breakdown. Small changes in intracellu-
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Fig. 6. Colloidal gold in vacuole (A) and multivesicular body (B), 45 and 135 mm after continuous ADH stimulation. (x43,000.)
lar pH and calcium are critical in determining the extent of cross—linking, which, in turn, determines the extent to which the cytoplasm exists as a gel (high degree of cross—linking), or a sol (breakdown of cross—linking). Several studies have reported that hormones may reduce the extent of cytoskeletal organization in target cells [30—32]. Thus, it is possible that
angulation or translation may take place when cytoskeletal barriers are removed in response to ADH. Ausiello and Hartwig [33] have proposed such a mechanism in which ADH increases cytoplasmic cyclic AMP, which activates protein kinase. Actin binding protein is phosphorylated, and thus inactivated, with a
tubules transfer their label to multivesicular bodies and other
vesicular structures when ADH is removed. It is entirely possible that transfer of tubular contents, and destruction of tubules themselves, can take place during and after ADH stimulation. Figures 6A and B show colloidal gold in large vacuoles and multivesicular bodies after 45 and 135 minutes of continuous vasopressin stimulation. However, some tubules, possibly a majority, may follow a different route by returning to their horizontal "resting" position upon withdrawal of ADH, becoming available for a second fusion (Fig. 3, 6th column). Indeed, the entire cycle from resting to fusion back to resting may simply involve angulation of the tubules, pivoting around a
resulting decrease in the cross—linking of the cortical actin network. If there were also to be an increase in calcium, villin would be activated as an actin—severing protein, diminishing the
actin network and permitting the tubules to approach the membrane. Alternative mechanisms for organelle movement have been proposed which involve the interaction of the organelles with microtubules, or with actin filaments. Several investigators [34,
35] have shown that microtubules play an active role in organelle transport. Microtubule—associated proteins appear to
link organelles to microtubules and to propel the organelles along the microtubule. The process is an ATP-dependent one. fixed base to a position of fusion, then back to a resting Alternatively, recent studies by Spudich, Kron and Sheetz [36] have shown that myosin—coated beads can move along pure position. actin filaments. This process, too, is ATP-dependent. Thus, in ADH-responsive cells it is possible that tubular angulation or Discussion movement may be guided by one of these mechanisms, rather The picture of intracellular events in ADH-stimulated cells than by an overall dissolution of the actin network. Microtuthat is beginning to emerge from the work of several laborato- bules are present in the terminal web area [20] (Figs. 7A and B), ries is one of a busy traffic pattern, in which tubules move from but there is no evidence yet, beyond the presence of microtutheir resting positions to angulation and fusion, then to detach- bules and of abundant actin filaments [37], for such a means of ment and either reuse or destruction. There may also be an organelle transport. What does emerge from the studies of toad important role for small vesicles engaged in endocytosis of bladder is the tight control of tubule position and movement, aggregates and intracellular transfer of aggregate—rich mem- which may require some type of "track" both for fusion and for branes from one site to another. Tubules may originate from the the events following detachment. Much more work will be fusion of vesicles, originating from sites such as the Golgi necessary on every phase of the ADH response before the complex. There is evidence that the cytoskeleton is important events surrounding fusion are understood.
Hays et a!
536
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Note added in proof Recently published data found in: FRANKI N, DING G, QUINTANA N,
HAYS RM: Evidence that the heads of ADH-sensitive aggrephores are
clathrin-coated vesicles: implications for aggrephore structure and function. Tiss Cell 18:803—807, 1986.
8. ROSENBERG PA, FINKELSTEIN A: Water permeability of gramicidin A-treated lipid bilayer membranes. J Gen Physiol 72:341—350, 1978
9. HANDLER JS, ORLOFF J: The mechanism of action of antidiuretic hormone, in Handbook of Physiology, Renal Physiology, edited by ORLOFF J, BERLINER RW. Washington, D.C., Am Physiol Soc, 1973, pp. 791—814
10. TAYLOR A: Role of microtubules and microfilaments in the action of
Acknowledgments
vasopressin, in Disturbances in Body Fluid Osmolality, edited by
This work was presented in part at the 100th annual meeting of the Association of American Physicians, Washington, May 1986. Sup-
ANDREOLI TE, GRANTHAM JJ, RECTOR FC, JR. Bethesda, Am Physiol Soc, 1977, pp. 97—124 11. HANDLER iS, ORLOFF J: Antidiuretic hormone. Annu Rev Physiol
lic of China. The careful preparation of the thin sections by Mr. Nelson Quintana is gratefully acknowledged.
13. PETERSEN Mi, EDELMAN IS: Calcium inhibition of the action of
ported by NIH Grant AM 03858, and with the help of a National 43:611—624, 1981 Science Foundation Instrumentation Grant. Dr. Ding is a visiting 12. ZUSMAN RM, KEI5ER JR, HANDLER iS: Vasopressin—stimulated scientist from Hubei Medical College, Wuhan, Hubei, People's Repubprostaglandin F biosynthesis in the toad urinary bladder. J Clin Invest 60:13.1339—1347, 1977
vasopressin on the urinary bladder of the toad. J Clin Invest 43: 583—592, 1964 Reprint requests to Richard M. Hays, Albert Einstein College of Medicine, Department of Medicine, Division of Nephrology, 1300 14. SCHLONDORFF D, LEVINE SD: Inhibition of vasopressin—stimulated water flow in toad bladder by phorbol myristate acetate, Morris Park Avenue, Bronx, New York 10461, USA. dioctanoyl glycerol, and RHC-80267. J Clin Invest 76:1071—1078,
References 1. GRANTHAM JJ, GANOTE CE, BURG M, ORLOFF J: Paths of trans-
tubular water flow in isolated renal collecting tubules. J Cell Biol 41:562—576, 1969
2, DIBONA DR, CIVAN MM, LEAF A: The cellular specificity of the effect of vasopressin on toad urinary bladder. J Memb Biol 1:79—91, 1969
3. HARMANCI MC, KACHADORIAN WA, VALTIN H, DISCALA VA:
Antidiuretic—hormone induced intramembranous alterations in mammalian collecting ducts. Am J Physiol 235:F440—F443, 1978 4. CHEVALIER J, BOURGUET J, HUGON JS: Membrane associated particles: Distribution in frog urinary bladder epithelium at rest and after oxytocin treatment. Cell Tissue Res 152:129-140, 1974 5. KOEFOED—JOHNSEN V, Us5ING HH: The contribution of diffusion
and flow to the passage of D20 through living membranes. Acta Physiol Scand 28:60—76, 1953
6. MAFFLY RH, HAYS RM, LAMDIN F, LEAF A: The effect of
neurohypophysical hormones on the permeability of the toad bladder to urea. J Clin Invest 39:630-641, 1960 7. LEVINE 5, FRANK! N, HAYS RM: Effect of phloretin on water and solute movement in the toad bladder. J Clin Invest 52:1435—1442, 1973
1985
15. STOFF iS, HANDLER JS, ORLOFF J: The effect of aldosterone on the
accumulation of adenosine 3', 5'-cyclic monophosphate in toad bladder epithelial cells in response to vasopressin and theophylline. Proc Nat! Acad Sci USA 69:805—808, 1972 16. CARVOUNIS CP, CARVOUNIS G, WILK BJ: Importance of amino
acids in vasopressin—stimulated water flow. J Clin Invest 76: 779—788, 1985
17. WADE JB: Membrane structural specialization of the toad urinary bladder revealed by the freeze—fracture technique. III Location, structure and vasopressin dependence of intramembranous particle array. J Membr Biol special issue: 281—296, 1978 18. HUMBERT F, MONTESANO R, GROSS A, DES0usA RC, ORcI L: Particle aggregates in plasma and intracellular membranes of toad bladder (granular cell). Experientia 33:1364—1367, 1977 19. BEGG DA, RODEWALD R, REBHUN L: The visualization of actin filament polarity in thin sections. Evidence for the uniform polarity of membrane—associated filaments. J Cell Biol 79:846—852, 1978 20. MULLER J, KACHADORIAN WA, DISCALA VA: Evidence that ADH-stimulated intramembrane particle aggregates are transferred from cytoplasmic to luminal membranes in toad bladder epithelial cells. J Cell Biol 85:83—95, 1980 21. HAYS RM, MEITELES L, FANT J, FRANK! N, SALISBURY J: A
ADH and the collecting duct scanning electron microscopic study of the cytoplasmic surface of the toad bladder luminal membrane. Scan Electron Microsc Part 11:789—795, 1982
22. SASAKI J, TILLES S, CONDEELIS J, CARB0NE J, MEITELES L, FRANKI N, BOLON R, ROBERTSON C, HAYS RM: Electron— microscopic study of the apical region of the toad bladder epithelial cells. Am J Physiol 247(Cell Physiol 16):C268—C281, 1984 23. KACHADORIAN WA, CASEY C, DISCALA VA: Time course of
ADH-induced intramembranous particle aggregation in toad urinary bladder. Am J Physiol 234:F461—F465, 1978 24. FARQUHAR MG: Progress in unraveling pathways of Golgi traffic. Ann Rev Cell Biol 1:447—488, 1985 25. GRONOWICZ G, MASUR SK, HOLTZMAN E: Quantitative analysis of
exocytosis and endocytosis in the hydroosmotic response of the toad bladder. J Memb Biol 52:221—225, 1980 26. WADE JB, STETSON DL, LEWIS SA: ADH action: Evidence for a membrane shuttle mechanism. Ann NYAcad Sci 372:106—117, 1981 27. MASUR SK, COOPERS, RuBIN M: Effect of an osmotic gradient on antidiuretic hormone—induced endocytosis and hydroosmosis in the toad urinary bladder. Am J Physiol 247:F370—F379, 1984
28. DING G, FRANKI N, HAYS RM: Evidence for cycling of aggregate—containing tubules in toad urinary bladder. Biol Cell 55: 213—218, 1985
29. HARRIS HW, WADE JB, HANDLER JS: Fluorescent markers to study membrane retrieval in ADH-treated toad urinary bladder. Am J Physiol (in press) 30. MILLER SS, WOLFF AM, ARNAUD CD: Bone cells in culture:
537
Morphologic transformation by hormones. Science 192:1340—1343, 1976
31. LAWRENCE TS, GINZBERG RD, GILULA NB, BEERS WH: Hormon-
ally induced cell shape change in cultured rat ovarian granulosa cells. J Cell Biol 80:21—36, 1979 32. WESTERMARK B, PORTER KR: Hormonally induced changes in the cytoskeleton of human thyroid cells in culture. J Cell Biol 94:42—50, 1982
33. AUSIELLO DA, HARTWIG JH: Microfilament organization and vasopressin action, in Vasopressin, edited by SCHRIER RW. New York, Raven Press, p. 89, 1985 34. ALLEN RD, WEiss DG, HAYDEN JH, BROWN DT, FUGIWAKE H, SIMPSON M: Gliding movement of and bidirectional transport along
single native microtubules from squid axoplasm: Evidence for an active role of microtubules in cytoplasmic transport. J Cell Biol 100:1736—1752, 1985
35. HIROKAWA N, BLOOM GS, VALLEE RB: Cytoskeletal architecture
and immunocytochemical localization of microtubule—associated proteins in regions of axons associated with rapid axonal transport: The B, B' iminodipropionitrile—intoxicated axon as a model system. J Cell Biol 101:227—239, 1985 36. SPUDICH JA, KRON Si, SHEETZ MP: Movement of myosin—coated
beads on oriented filaments reconstituted from purified actin. Nature 315:584—586, 1985 37. PEARL M, TAYLOR A: Actin filaments and vasopressin—stimulated
water flow in toad urinary bladder. Am J Physiol 245 (Cell Physiol 14):C28—C39, 1983