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Kidney International, Vol. 9 (1976) p. 223—230

Antidiuretic hormone and water transfer

RICHARD M. HAYS

Department of Medicine, Division of Nephrology, Albert Einstein College of Medicine, Bronx, New York

There has been remarkable progress in recent years the rat collecting duct has also been shown to in- in our understanding of the physiology of antidiuretic crease following administration [11]; re- hormone (vasopressin), from its synthesis and release cent studies with the isolated papillary collecting duct in the central nervous system to its action on the renal of the rabbit, however, have been stated to show tubular cell. Several recent reviews [1—5] have consid- no effect of vasopressin on urea permeability [14]. ered the physiology of antidiuretic hormone in detail, and only a brief summary will be given of the steps Activation and control of cyclic AMP leading to the permeability response of the collecting duct. The question to be emphasized in this article is Attachment of vasopressin to its receptor activates the following: how does vasopressin increase the rate adenylate cyclase, a membrane-bound enzyme that of osmotic water flow across the cell membrane? converts ATP to adenosine 3',5'-monophosphate (cyclic AMP) [IS]. The rise in the intracellular cyclic Synthesis, release and binding AMP level ranges from ten-fold in the rat inner me- dulla [16] to three-fold in rat outer medulla and toad Vasopressin is synthesized in the supraoptic and bladder [16, 17]. Many factors control the paraventricular nuclei of the hypothalamus, probably intracellular level of cyclic AMP, including phospho- along with the neurophysin that acts as its binding diesterase, E1, calcium, magnesium, protein within the central nervous system [6, 7]. Once adrenal steroids and adrenergic agents (see [1, 4]). synthesized, vasopressin and neurophysin are trans- It has become increasingly evident that more than ported in the form of membrane-enclosed granules to one intracellular pool of cyclic AMP is responsible the median eminence and posterior lobe of the pi- for the control of water and solute permeability. tuitary, where they are stored, and then released Thus, in the toad bladder, an increased serosal cal- along with neurophysin. Release is by a process of cium concentration reduced osmotic water flow, but exocytosis [8], in which calcium appears to play a not active sodium transport in response to a sub- critical role [9, 10]. The major stimuli for release are maximal dose of vasopressin [18, 19]. There was no hyperosmolality and volume depletion, but a large effect of an elevated calcium concentration, however, number of additional stimuli, including pain, stress on cyclic AMP-stimulated water flow [19], indicating and a number of drugs can stimulate vasopressin that the site of inhibition by calcium was prior to the secretion [2, 5]. generation of cyclic AMP. It was concluded that two Once released, vasopressin moves in the plasma at functionally independent pools of cyclic AMP were concentrations in the range of 10-12 to l0"M, and is involved, one directed towards the control of water eventually bound by receptors at the basolateral sur- movement, the other towards active sodium trans- face of the collecting duct cells. Here, a sequence of port. A recent study by Flores et al [20] has provided steps is initiated which ends in the transformation of evidence for an adenylate cyclase in the toad bladder the luminal cell membrane, and an increase in the which does not control water flow, but which, under passive osmotic flow of water from tubular lumen to certain circumstances, can contribute cyclic AMP to cell of 7- to 50-fold [11, 12]. Urea reabsorption in the "water flow compartment". The concept of sepa- tissues such as the isolated urinary bladder of the rate cyclic AMP pools will be an important one in the toad is enhanced as well [13]. Urea reabsorption by subsequent discussion of water and urea transport. Not only may there be several pools of cyclic © 1976, by the International Society of Nephrology. AMP, but the possibility that vasopressin may stimu-

223 224 Hays late cyclic AMP production in one cell type (the the term "luminal membrane" is not necessarily re- mitochondria-rich cell of the toad bladder), with stricted to the membrane of a single cell type, either transfer of cycIicAMP to the granular cells via inter- in toad bladder or collecting duct. cellular channels, has been suggested by Goodman et Diffusion and solvent drag. What is the structural al [21]. change in the luminal membrane? It had originally Finally, there is the question of what steps lie be- been proposed that water and many solutes crossed yond cyclic AMP, ending in the transformation of the the cell membrane together, via aqueous channels or luminal membrane. Two views have emerged: the "pores" in the cell membrane [12, 35]. This view first, based on studies in the toad bladder, is that stemmed from two observations: 1) The rate at which cyclic AMP increases the activity of a protein individual labeled water molecules diffused across the phosphatase, leading to a net dephosphorylation of cell membrane following vasopressin appeared far the protein and a resultant change in membrane too slow to account for the large osmotic water flows permeability [22, 23]. Schwartz et al, however, using observed [35]. 2) The movement of small molecules carefully separated luminal membrane fragments such as urea and thiourea was accelerated in the from bovine collecting ducts, have reported a net direction of net water flow [27, 36], and this cyclic AMP-activated phosphorylation of membrane phenomenon of "solvent drag" could only take place protein, suggesting that a membrane-bound kinase is if solutes moved in a stream of water crossing the primarily involved in the permeability change [24]. membrane in aqueous channels. Vasopressin was There is now evidence for several fractions of kinase thought to dilate these channels, permitting Poiseuille activity in a given cell [25], and it is possible that flow of water rather than diffusion, and with it, the several kinases are activated by cyclic AMP, with solvent drag of small molecules (Fig. 1). each kinase governing some particular permeability The pore enlargement hypothesis, however, has property of the cell membrane. This important been brought into question by a number of problem is still unresolved. observations. First, the rate of diffusion of water molecules across the luminal cell membrane is far Water movement across the cell membrane faster than had been recognized in earlier experiments. The retarding effect of unstirred layers, The final step in the sequence initiated by cell cytoplasm and other tissue elements lying in vasopressin is a change in the structure of the luminal series with the luminal membrane had obscured the cell membrane, as yet undefined, which increases its fact that vasopressin may increase water diffusion by permeability to water. In tissues such as toad bladder as much as 20-fold in a tissue such as the toad blad- and frog skin, the permeability to sodium and to a der. Recognition of the importance of diffusion in the number of solutes, notably urea, is increased as well transfer of water across membranes has come from a [26—28]. The extent to which sodium and number of laboratories, using a variety of techniques nonelectrolyte movement is increased in the mamma- lian collecting duct is not completely resolved, as discussed earlier; there is one segment, the rabbit cortical collecting duct, in which there is good evi- dence that vasopressin increases water flow, but not -o urea movement [29]. The evidence that it is the luminal or outer-facing membrane that is transformed by vasopressin comes from a series of studies of frog skin [30], toad bladder [12, 13, 31] and rabbit and rat cortical collecting duct Vaso. __Water,__ [32—34]. These studies have shown that movement of water (or, in appropriate tissues, of urea) across the luminal permeability barrier, as demonstrated by cell swelling, shrinking or labeling of cell water by iso- topic urea, is accelerated by vasopressin. This can best be explained by an increase in the permeability of the luminal barrier. Estimates of the capacity of the intercellular pathway to transport water have Fig. 1. Pore enlargement hypothesis, in which vasopressin enlarges fallen far below the osmotic water flows actually ob- an aqueous channel in the cell membrane, permitting increased flow served [33]. It should be emphasized, however, that of water and solvent drag of small solutes. Antidiuretic hormone and water transfer 225

hypothesis is solvent drag. There is little doubt that solvent drag takes place across epithelia in the pres- 100 ence of net water flow, but most or all of it may take place in the intercellular channels, rather than through pores in the cell membrane itself [41, 45]. Thus, both the more recent estimates of the diffusion rate of water and the nature of the solvent drag effect

1.. have raised the possibility that water transfer across the cell membrane may not require aqueous channels. Perhaps the most decisive evidence has come from studies employing selective inhibitors of water or so- L lute transport. These will now be considered. a 1) Selective inhibitors. In 1970, Macey and Farmer [46] reported that phioretin, the aglucone of phiori- zen, blocked urea movement into the red blood cell, but had no effect on the entry of water. They

V concluded from this experiment that water and urea

V might cross the cell membrane via independent path- ways. Phloretin had the same effect when placed in the luminal bathing medium of the toad bladder [28]: inhibitionof the vasopressin-stimulated transport of urea (and all other amides tested), as well as thiourea and formaldehyde, but no effect on osmotic water flow, or the movement of ethanol or ethylene glycol Fig. 2. Effectof lOM phioretin on the permeability of the toad (Fig. 2). A number of agents are now known which, bladder to water and solutes. Data are shownas percentages of when placed in the luminal medium, block amide paired, untreated contro!s. There is a significant inhibition of the transport across the toad bladder, with no effect on movement of the amides urea and acetamide, but no effect of phioretin on water flow, or the movement of ethanol or ethylene osmotic water flow. These include tannic acid and g!yco! [28]. the oxidizing agents chromate and permanganate [47, 48]. Chromate and permanganate are of particular and membrane preparations [37—44]. A more detailed interest, since they block vasopressin-stimulated urea consideration of the role of diffusion can be found in transport irreversibly [47, 48]. They may act by oxi- [411. dizing one or more key components of the urea trans- A second problem in relation to the pore port pathway; if they do, their actions are relatively

Untreated 2 X 105M permanganate

400

300

200 j

Fig. 3. Effectof 2 >( 1O-5M permanganate on osmotic waler flow and urea (right- hand pane!), compared to untreated blad- 100 der (!eft-handpane!). Water flow and Ktra urea are shown in unstimu!ated contro! b!adders (C), and b!adders stimu- lated by !.5mvi8-bromo-cyc!ic AMP. Per- manganate significant!y depressed Ktrn C 8-Br- C 8-Br- C 8-Br- C 8-Br— urea, but not osmotic water flow. Vertical cAMP cAMP cAMP cAMP bars: +! servi 148]. WaLer Urea Water Urea 226 summarizes theinhibitoryeffectsofphloretinand confined totheerythrocyteandtoadbladder.Table1 membrane, asintheerythrocyte. for waterandsolutesdoexistintheepithelialcell [22—24], yond cyclicAMPisindeedintheluminalmembrane cyclic AMPanalogues([48];Fig.3).Ifthestepbe- pronounced inbladderstreatedwithcyclicAMPor AMP, sincetheirinhibitoryactionisjustas to actatapointbeyondthegenerationofcyclic fect onureatransport[481.Furthermore,theyappear (perchlorate, peroxideandferricyanide)havenoef- specific, sinceanumberofoxidizingagents K1 raflst SCC X / (1 Inhibition ofureatransportbytheseagentsisnot 0' 00 0 Chromate Chromate Tannic acid Tannic acid

I I Periodate Permanganate Phioretin Phloretin Phioretin Phloretin Phloretin Agent it Uric acid I .1 would appearthatindependentpathways

AdeUI I I

Fluorouracil I

Tetracycline -' I I

a o' 00 0 Concentration 05-I X 0 0 0 1-5 X

o 0 5 X1O- 2 >(10' 1-6 X I I I I 5 X 5 Xl0 l0- 10—s l0- 10-i l0- Cli10 rain p1 icti icol 10 l0- = l0

tSUflliuZ.id I I Table

Pheiiobarhital .- I 1. Toad bladder Toad bladder Toad bladder Toad bladder Toad bladder Amphibian, mammalian Dogfish kidney Human erythrocyte Dogfish kidney Gall bladder Human erythrocyte Diphenylbydantoin I I Agents inhibitingureatransport erythrocyte • IJ oIa °0 tn 0 °Ui

o a 0 0 Tissue Hays I I I I I

(;lnteth rnide II problem ofwatertransferisthefindingthatamole- the membrane.Ofmoredirectimportanceto itory agentslisted,maybeaproteincomponentof transport systemwhich,fromthenatureofinhib- has emergedfromthesestudiesisthatofafacilitated birds [49]. species (hagfish,dogfish,skates,boneyfish)orof tiles andmammals,butnotthoseofmoreprimitive of ureaacrosstheerythrocytesamphibians,rep- rabbit gallbladder.Phloretininhibitsthemovement sorption isbelievedtobeanactiveprocess)and tissues, includingdogfishkidney(whereureareab- oxidizing agentsonureatransportacrossavarietyof The pictureoftheamidetransportpathwaythat (y c I oh cx a no lie I -I

13u t a rid L-J Characteristics Partially reversible Irreversible Irreversible Irreversible Irreversible Irreversible Reversible Reversible Reversible — — compounds areontherightside ofthe ber ofdrugs.Themorehighlylipid-soluble including uricacid,antibioticsand anum- series ofcompoundsbiologicalinterest, Fig. 4. was statisticallysignificant [621. vasopressin. Inallcases,theincrement absent; clearportion:incrementfollowing figure. Hatchedportionofbar:vasopressin Permeability of thetoadbladdertoa 46, Reference 48 47 47 49 55 54 53 52 51 28 49, 50 Antidiuretic hormone and water transfer 227 cute as small as urea appears to be excluded from the pressin. A recent study in the toad bladder by Pietras water pathway. This suggests that water crosses the and Wright [61] has described a small but definite membrane either I) by "pure" diffusion (the entry of effect of vasopressin on lipophilic solutes, with com- single water molecules into the membrane phase); 2) pounds such as caffeine increasing their permeability throughchannels so small that urea is physically by 30% following administration of the hormone. excluded (i.e., channels approaching the molecular Levine et at [62] have found that vasopressin acceler- size of water); or 3) through larger aqueous channels ates the movement across the toad bladder of a large in which water is highly organized ("ice-like") and, series of lipophilic drugs, including glutethimide, for this reason, excludes solutes. There is evidence diphenylhydantoin and phenobarbital (Fig. 4). These both from nuclear magnetic resonance studies [56] compounds have decanol/water partition coefficients and studies of the activation energy for water diffu- as high as 160: 1, and may well penetrate membrane sion [57] that water in close association to cell mem- lipid directly. The increase in permeability was, seen branes is indeed highly structured. when cyclic AMP was substituted for vasopressin, It could still be argued, however, that urea and and was not inhibited by phloretin. Thus, the lipid water do penetrate the membrane via a common phase of the cell membrane appears to share in the pathway, and that phloretin and the oxidizing agents response to vasopressin, suggesting that the mem- distort the pathway slightly, preventing access of brane response is a broad one and may involve a urea, but not interfering with water flow. This now change in membrane fluidity (see [63]). Figure 5 sum- seems unlikely, in view of the recent observation that marizes, in diagrammatic form, our present view of three anesthetic agents (methohexital, methoxy- the effect of vasopressin on the luminal cell flurane and halothane) have an effect opposite to that membrane. Multiple pathways are shown, for so- of phloretin, namely, inhibition of vasopressin-stimu- dium, the am ides, water, solutes such as ethanol and lated water flow across the toad bladder with no effect on urea transport [58]. An inhibitory effect of anesthetic agents on water flow alone had already - — been reported [59j; the finding that urea, a larger - —0 molecule, was unimpeded is strong evidence against O—Sodium—O—0 distortion of a common pathway for water and urea. —- —0 In contrast to chromate and permanganate, the — —0 inhibitory effect of at least one of the anesthetic agents, methohexital, appears to take place at a step Blocked by: Phloretin prior to the generation of cyclic AMP, since these Tannic acid agents do not depress water flow generated by the Chromatefm1des cyclic AMP analogue 8-bromo cyclic AMP [58]. V These studies, therefore, have provided the first A indication that two separate vasopressin-sensitive, S adenylate cyclase-cyclic A MP systems may control Blocked by: 0 Methoxyflurane O—Watcr—--—O P water and urea transport. Whether this means that Halothane R there are two cyclic AMP pools within the same 0-— -—0 E granular cell, or whether two different cell types regu- S 0— —0 S late water and urea transport, is unclear at this point. 0-- --0 It is also possible that the "independent pathway" for 0— - N urea is really a paracellular one [60]. It is conceivable Ethanol, that the failure of the cortical collecting duct to in- Ethylene crease its permeability to urea following vasopressin 0-——0 administration, while its water permeability is greatly - —-0 enhanced, is due to the absence of an entire urea 0— — control system within the cell, or the absence of a 0— —-0 vasopressin-responsive urea transporting cell. 0— Lipophilic-0 2) The lipid phase of the membrane. Thus far, the o— molecules —0 discussion has centered on the effect of vasopressin 0— —-0 on the movement of water and hydrophilic solutes. 0— --0 There is now good evidence that the movement of Fig. 5. Multiple, selective pathways for water and solute transport highly lipophilic solutes is also accelerated by vaso- across the luminal cell membrane. 228 Hays

Fig. 6. Electron micrographs of freeze- fracture faces of granular cell luminal membrane from toad bladder, unstimu- lated (a and b)orstimulated (candd) withvasopressin. (a) Innerand (b) Outer fracture faces; without vasopres- sin stimulation, intramembranous par- ticles are not aggregated. (c) Inner fracture face after vasopressin stimu- lation; separate sites of aggregated in- tramembranous particles are empha- sized (singlearrows). (d)Outer fracture face which is complementary . to that shown in (c) after vasopressin

'"i stimulation; organized linear arrays of depressions corresponding to the ag- gregated intramembranous particles shown in (c) are emphasized (single arrows). Circled arrows indicateshad- owing direction (X80,000). (Reprinted

st'T with permission from [651.) ethylene glycol and, finally, the lipophilic molecules. methohexital,one of the anesthetics that inhibit wa- Some of the pathways may be predominantly protein, ter flow but not urea or sodium transport, others predominantly lipid in composition. significantly reduces the frequency and size of the particle aggregations. Thus, aggregation may be spe- cifically associated with water rather than solute Freeze fracture studies transport. Interpretation of these results will require more Until recently, attempts to demonstrate understanding of the freeze fracture technique itself, vasopressin-induced changes in the cell membrane by as well as its application to tissues such as the toad electron microscopy have not been successful. In bladder. There is evidence from studies in other tis- 1974, Chevalier, Bourguet and Hugon [64], using the sues [67—70] that the intramembranous particles are freeze fracture technique, found that there was a proteins. Their aggregation may be the result of a striking aggregation of intramembranous particles in change in membrane fluidity, as discussed earlier. the inner face of the luminal membrane of the frog It has also been suggested that the membrane particles urinary bladder following vasopressin adminis- themselves are the sites of water transfer [71], a view tration. This finding was confirmed by Kachadorian, that is supported by recent studies of red blood cell Wade and DiScala ([65], Fig. 6), who also noted a membrane polypeptides labeled with an inhibitor of close correlation between the frequency of water flow [72]. Clearly, there are new possibilities aggregation sites per area of toad bladder membrane worthy of being explored, and new techniques which and the extent of vasopressin-induced osmotic flow. may permit us to look directly at the membrane More recent studies [66] have shown that transformation induced by vasopressin. Antidiuretic hormone and water transfer 229

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