Membrane effects of antidiuretic hormone

Membrane

Effects of Antidiuretic ALEXANDER Boston,

IdEAF,

M.D.

Massachusetts

hydrated toad the transparently thin bilobed bladder with its content of hypotonic urine may occupy from one third to OIX half of the entire abdominal cavity. Histologically this tissue consists of a single la)-er of specialized epithelial cells lining the mucosal or rlrinar) surface of the bladder. This frmctional epithelium is supported on a loose stroma of connective tissue in which are occasional bllndles of smooth muscle and capillaries. The contramucosal surface is a serosa. The total sohlte concentration of such urine Ina!- be as low as 50 mOsm. per kg. water, as dilllte as the hIllnan kidney can excrete during maximal \.\-ater diuresis. Such dilute urine may remain in the bladder for hours without apparent decrease in volume or increase in solute concentration. Although in the absence of antidiuretic hormone the bladder acts like a tight \-essel for the contained urine, Table I [,3] shows that the unidirectional permeabilit!to Icater measured isotopically with deuteriated or tritiated water is quite high. Measurements \vere made in vitro with the bladder wall separating two halves of a lucite chamber. Isotopically labeled water was added to the medium bathing one side of the bladder and its rate of appearance on the opposite side determined. Results arc expressed as microliters of \vater crossing 1 cm.2 of bladder wall per hour. It is seen that the rate of transport remains quite constant over three successive thirty minute periods. Furthermore, addition of vasopressin (‘mammalian antidiuretic hormone) to the medium bathing the serosal surface of the half-bladder after the first thirty minute control period produced a significant increase in the unidirectional diffusion permeability to water b\. some 70 per cent. Quite a different impression of the permeability of the bladder to water is obtained if

animals the ability to control the water content of their bodies is an important feature of their adaptation to a terrestrial existence. This is generally accomplished by the kidneys in which the tubular epithelium of the distal portions of the nephron is capable of reabsorbing solute, largely salts of sodium, from the luminal fluid without the accompanying water. This leaves a dilute urine from which water may- be reabsorbed facultatively, depending upon the presence or absence of the In the well hydrated antidiuretic hormone. animal, water is present in surfeit, no antidiuretic hormone circulates, and a dilute urine is excreted, ridding the animal of the excess water. During periods of water deprivation, antidittretic hormone is secreted and so modifies the epithelium in the distal portions of the nephron that water is reabsorbed and its content in the body is preserved. Because of technical difficulties in studying the processes whereby neurohypophysial hormones modify the permeability properties of the renal tubular epithelium in the myriads of microscopic nephrons comprising the mammalian kidney, investigators have sought more accessible responsive tissues. The skin and urinary bladder of frogs and toads have proved themselves invaluable in this context. The first major conceptual advance in our understanding of the mode of action of vasopressin came in 1953 from studies of Koefoed-Johnsen and Ussing [I]. In the toad the functions assigned in mammals to the epithelium of distal portions of the renal tubule are served as well by the urinary bladder. Ewer [Z’] observed that the urinar) bladder of the toad served as a reservoir of water which could be reabsorbed during periods of water deprivation or rapidly in response to In the injections of antidiuretic hormone.

I

Hormone*

N HIGHER

* From the Departments of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts. This study was supported in part by grants from the John A. Hartford Foundation, Inc. and the Cr. S. Public Health Service (No. HE-06664 from the National Heart Institute, and AM-04501 from the National Institute of Arthritis and Metabolic Diseases). VOL.

42,

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745

Membrane

Effects

of ADH-Leaf

TABLE I EFFECT 0~ v~s0nREs~In ON DIFFUSION PERMEABILITY (UNIDIRECTIONAL WATER FLUX) OF ISOLATEDTOAD BLADDER~0 WATER MEASUREDWITH DHO OR THO IN ABSENCE OF OSMOTIC GRADIENT [3,26] Permeability (~1. cmd2. hr-I.) Group

Period 1

Period 2

Period 3

Control Treated with hormone*

343 338

338 543

339 599

Mean Difference (period 2-l)

S.E. Mean Difference

P

z!z9 zt35

>0.5
,:05

* Hormone added at end of first period (2 units commercial vasopressin to medium bathing serosal surface). Control includes ten experiments; seven measured from mucosal to serosal surface and three in opposite direction. Hormonetreated group includes thirteen experiments; nine were mucosal to serosal fluxes and four were measured in reverse direction. 15 ml. frog Ringer’s solution bathing each surface; 3.14 cm2. area of chambers.

the net transfers of water occurring across this tissue is measured. This may be accomplished either gravimetrically by weighing the volume of solutions placed on either side of the bladder initially and again at the termination of an experiment or volumetrically by having one half of the chamber sealed and connected to a horizontal calibrated pipette in which the volume flow per unit time may be monitored continuously. Because of the distensibility of the bladder wall it is necessary to support it against a nylon or Dacron@ mesh during these observations so that the volume of the half chamber does not change due to billowing of the bladder to one side or the other. The results of a large number of measurements of net trans-

FIG. 1. Dependence of net water flux on osmotic gradient in presence or absence of neurohypophysial hormone. With hormone, the points at osmotic gradients of 59, 150 and 170 represent six, eight and fifteen experiments, respectively, and their mean values and standard error of means are 77.2 f 6.9, 186 % 8.8 and 209 f 7.9 ~1. per cm2. per hour, respectively. The regression equation relating water movement to osmotic gradient is y = 1.22 X f4.22. Twelve experiments without hormone but with an osmotic gradient of 160 mOsm. per kg. water gave a mean net flux of t4.3 f 1.3 1.41.per cm2. per hour [3,26].

port of water made in the absence and presence of antidiuretic hormone are shown in Figure 1 [3]. The abscissa is the osmotic gradient across the bladder wall obtained by diluting the medium bathing the mucosal surface to the desired concentration, but keeping constant that at the opposite serosal surface. The ordinate is the net water transport or flux. In the absence of an osmotic gradient, with or without antidiuretic hormone, essentially no net transfer of water occurs. We conclude, therefore, that the high isotopic permeability presented in Table I represents an equal permeability to water in the two directions across the tissue, from mucosal to serosal and serosal to mucosal surfaces. The lower line of Figure 1 indicates the small net transfers of water across the bladder which occur in the absence of the hormone, despite large osmotic gradients (160 mOsm. per kg. water on the abscissa is equivalent to some 4 atmospheres of hydrostatic pressure). This is consistent with the findings mentioned of dilute urine remaining in the bladder for long periods of time with little or no detectable change in concentration. The upper line shows the large net transfer of water which may occur in the presence of vasopressin. At the higher osmotic gradients studied, the volume of water crossing a unit area of bladder per minute is equal to the total water content of the same area of tissue and attains a magnitude of more than one third of the simultaneously determined unidirectional permeability to water. The finding that net movement of water is proportional to the transepithelial osmotic gradient and that no net water transfer occurs in the absence of such an external driving force indicates that water moves passively across the bladder. Furthermore, the hormonal effects on AMERICAN

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MEDICINE

Membrane

Effects

net water transport can be demonstrated in the complete absence of sodium ions [3] and, therefore, the active transport of sodium by this (issue contributes little dire&y to net water transport. The osmotjc gradients in VZOO,of course, arise from the active reabsorption of sodium, and this is the energy-requiring step in water reabsorption. The findings thus indicate that water moves passively across the toad bladder and that, despite a high isotopic permeability to water, little net movement occurs in the absence of the hormone. Vasopressin possesses the ability to induce large net transfers of water with only a moderate further increase in the unidirectional Before asking in what diffusion permeability. way the hormone modifies the bladder 10 produce these effects on the transport of water it may be useful to digress briefly and to consider some current views regarding water itself which make these observed effects possible. The umqueness and suitabi!ity of INater a~ a substrate for life has long been appreciated [4]. The structure of water which accounts for its unusual properties is the sllbject of extensive Although many questions study at present. about liquid water remain unsolved, it is the fact that water is a highly associated liquid which is pertinent to the present discussion. The crystallographic study of ice indicates it to consist of regular tetrahedrally placed molecules of water [5]. Each molecule of water is surrounded by four nearest neighbor molecules and the rescllcing tetrahedral configuration ir stabilized by hydrogen bonding of the water molecules. Each molecule is maximally bonded by two hydrogen bonds to its nearest neighbors. Pauling [5] has pointed out that when ice melts at zero degrees, the heat of melting is sufficient to break only some 15 per cent of the hydrogen bonds present in Ihe fully coordinated structure of ice. The residual hydrogen bonds in liquid water account for many of its unique properties. Obviously the physical properties of ice and water are so different that the latter cannot contain in a static state. 85 per tent oi the structure of the former. The association between a molecule and its neighbors can be only temporary, as the structure is continually broken down by thermal agitation. Such associations need persist over small regians or clusters for time5 long ertfi@ that the order be “seen” by x-ray or even infra-red radiation and longer than the VOL.

42.

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1967

of ADH--Leaf relaxation time for rotation of a single water molecule, some 1O-12 seconds. Thus water is continuously and raptdly making, breaking and reforming clusters. Frank IS] has picturesquely referred to the “flickering cluster” structure of water. From their statistical mechanical theory of water, Nemethy and Scheraga [7] have estimated the average size of clusters of water molecules in liquid water at 20”~. to be approxjmate+ 57 molecules. Below this temperature the size of the aggregates will be larger and at higher temperatures, smallerOnly in the vapor phase, however, does water behave like a monomer. Various factors are known to increase or stihiiize the struclture of water and others to break down its structure. It has long been appreciated that individual water molecules are permanent electrical dipoles because of the asymmetrical arrangement of the negative oxygen and positive hydrogen atoms in the V&aped moiec& of water. T&. interaction of these djpolcs with the strong electrical fields of ions results in ionic hydration shells. More recently it has been appreciated that water exists in an increased state of order in apposition to nonpolar molecules or surfaces [6]. Since the physica\ ptaperties of buik water and bulk ice are so difTerent we may expect the propettles of water to vary between these extremes at local sites. Since the coefficient of self diffusion of water in water is 2.4 X lo-%m?. set-‘. at 25”~. [8] and that of dater in ice is 1.0 X 10-1~cm2. see-‘. at -1.5 to -2.O”c. ISi, the transport properties of water may be expected to be strongly dependent upon the degree to which it is structured. In the measurements of the diffusion permeability of the bladder presented in Table I, labeled water was added to the medium bathing one surface of the bladder and its rate of appearance in the medium bathing the opposite surface determined in the absence of net transfers of water across the tissue to prevent effects from solvent drag. The individual tagged water molecule penetrates the barrier by diffusion, a process of isotopic exchange, in which the gradient of chemical potential of the isotopic species or a gradient of specific activity is the driving force and is equal to the entropy of mixing multiplied by the absolute temperature. As the isotopic water molecule penetrates the barrier it wilf he subject to Ihe frictional forces between water and water, f,,, and between

Membrane

748

Effects

water and membrane, f-, as it randomly jumps from one position to the next in response to thermal agitation. It may at one moment be associated with one cluster of water molecules and the next moment be part of another cluster. In the absence of net transfers of water across the bladder, the clusters will have no over-all direction and the progress of the labeled water molecules will depend upon the molecular friction between it and its neighboring water, fww, or membrane molecules, f,. In a finely porous membrane fw, > fww but in coarse, porous membranes, since they are mole friction terms, fVmdiminishes and f,, becomes the determining factor, as in self diffusion. From the self diffusion coefficient, D” , of water in water a “molecular radius” of the diffusing species may be computed from the Einstein-Stokes relation [ 701: Do = 6$

= F WV?

where k is Boltzmann’s constant, R is the gas constant, T is absolute temperature, a! is the radius of the diffusing species, and q is the viscosity of bulk water. The resultant value of (11is constant for measurements made over a considerable temperature range indicating that in the process of diffusion we are dealing with the transport of a single moving particle. Furthermore, the magnitude of a! (~1 8) indicates that the moving unit in diffusion is a single water molecule. The low value for OLas compared with the known molecular radius of water of 1.38 A from x-ray crystallography is probably due to the limitations of Stokes’ law when applied to the movement of particles which approximate in size those of the suspending medium. When the transport process involves movement of single water molecules, as in diffusion or movement through channels so small that dimensions approach that of a water molecule, the ratio of the two unidirectional fluxes, Jr2 and Jzr, across the barrier is proportional only to the ratio of the chemical activity of the water on the two sides of the barrier, al and at. Thus: J 12 -=-

al

JZI

a2

This condition is not favorable for net transport of water as can be quickly appreciated by substituting concentrations of water for activities in the experiments shown in Figure 1. With

of ADH--Leaf 160 mOsm. per kg. of water as the difference in the concentration of water across the bladder, J -=12 J 21

55.56 -

0.06

55.56 -

0.22 = “Oo3

If 512 is taken from Table I as 340 ~1. cm-*. l-n-‘., then J2i will be 339 ~1. cme2. hr-l. and the net flux expected by diffusion is only some 1 ~1. cme2. hr-l., whereas the observed net flux averaged some 200 ~1. cme2. hr-l. Diffusion is thus an ineffectual means of producing net transfer of water and can account for only a small fraction of the observed water transport across the bladder in the presence of vasopressin. In contrast to the process of diffusion, net water movement across a barrier in response to a gradient of hydrostatic or osmotic pressure will depend on the movement of individual water molecules only when the dimensions of the channels through which the water moves approximate the dimensions of individual water molecules. Through all pores of larger dimensions the associated nature of water will result in the movement of clusters of water molecules which thereby reduces the friction per molecule and allows larger net transfers of water for a given pressure difference than could occur by diffusion alone. To account for the observed net transfers of water across the bladder in the absence of vasopressin and again with the hormone the mean pore radius [1,11-131 must be some 8 A and 40 ,&, respectively. We may now ask in what way neurohypophysial hormones modify the bladder to produce the effects on water transport. Koefoed-Johnsen and Ussing [7] have proposed the most satisfactory explanation at the present time on the basis of the “pore hypothesis.” According to their hypothesis, net water movement occurs predominantly by bulk flow in aqueous pores through the bladder rather than by diffusion ; and vasopressin increases the net transfer of water by enlarging the radius of individual pores. Since diffusion is dependent upon the area (or pore radius squared) available for penetration by the diffusing species whereas laminar flow, according to the Poiseuille equation, is a function of the fourth power of the radius of the individual pores, a small increase in radius of individual pores, Ar, will affect diffusion only by (r + Ar)2 - r2 whereas bulk flow will be increased by (r + Ar)” - r4. They picture neurohypophysial hormones as altering the responsive AMERICAN

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Membrane

Effects of ADH-Leaf

Effect of neurohypoFIG. 2. physial hormone on permeability of toad bladder to urea, acetamide and thiourea. ‘The appropriately labeled molecule was added to the medium bathing one surface of the membrane and its rate of appearance on the opposite side was determined. two periods of thirty After minutes each, vasopressin was added to the serosal medium and its rate of appearance for an additional two periods was determined In every instance the permeability to urea and acetamide was enhanced by hormone, whereas permeability to thiourea was unaffected [ 76.261.

,NHp *

VASOPRESSIN

TRANSPORT

That neurohypophysial hormones may affect permeability to solute molecules as well as to water was first indicated by Andersen and Ussing 1741 who found that vasopressin produced an increase in permeability of the isolated toad skin to thiourea and acetamide. The striking feature of this effect on the permeability of the toad bladder to small molecules is its specificity. Figure 2 [75] contrasts the large increase in permeability to urea and acetamide that followed addition of vasopressin with the absence of an effect on thiourea. Of some 40 compounds whose rate of penetration has been tested, only in the case of certain small uncharged amides and certain small alcohols listed in Table II was the permeability of the toad bladder increased by vasopressin [ 761. The mode of penetration of the toad bladder by urea and the few similar small molecules appears to be passive, as was demonstrated ior VOL.

42,

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1967

acetamide

‘NH?

,N Hz Cc.5 *

TIYE

SOLUTE

urea

,CHn C=O

thioureo

‘NHz

1 120

60

membrane from one containing many small pores to one containing fewer larger pores. Little change in area or diffusion permeability would be consistent with large increases in bulk transfer of water with vasopressin according to this hypothesis of hormonal action. Examination of the permeability of the bladder to small solutes, however, shows the inapplicability of any explanation for the hormonal action on the transport of water, which requires the presence only of large pores.

749

IN

MINUTES

water. They penetrate the bladder at equal rate in both directions, as shown in Table III for urea [76]. The permeabilities from mucosal to serosal surface and from serosal to mucosal surface were simultaneously determined using V-urea and N’s-urea to measure the two unidirectional permeability coefficients, Although vasopressin produced approximately a tenfold average increase in the permeability coefficients, the values in the two directions remained equal. Furthermore, evidence for carrier-mediated or facilitated diffusion by either self-depression or competition studies was not elicited [75]. Thus these compounds appear to pass through the rate-limiting permeability barrier of the bladder by a process of free diffusion without interaction with the bladder. However, since the very fact of specificity necessitates interaction with the barrier, one suspects the presence of hydrogen bonding, which is the form of interaction most likely to escape detection by the criteria of self depression or competition. An effect of solvent flow through the bladder on the penetration of the bladder by urea has been demonstrated [75]. Imposing net transport of water across the bladder during simultaneous measurements of the two unidirectional fluxes of urea distorted the flux ratio from a value of unity by accelerating the urea flux in the direction of water movement and retarding the flux upstream. This finding indicates that urea and water probably occupy some common channel during a portion, at least, of their course

750

Membrane

Effects TABLE

COMPOUNDS

PENETRATING

TOAD

BLADDER

of ADH--Leaf II

MORE RAPIDLY

WITH

[ 75,361

VASOPRESSIN

Permeability Coefficients K trsna (1 O-km. set-l.) Molecular Weight

Vasopressin After Before

S.E. Mean Difference

n

Amides Urea (NHjINH Acetamide

2) 0

(CHs 4 NHZ)

60

26

274

zt5

37

59

44

196

zt26

73

97

215

87 42

132 127

180 282

f31

4 6

89

581

639

f18

6

73

174

259

fl0

6

122

26

40

f2

6

73

87

242

f16

6

18 :z

944 825

1580 913

f35 kl9

!

62

575 16

678 35

*20 ZtG

:

23

36

52

f2.7

14

9

0 Propionamide

Butyramide Cyanamide

Urethane

(CH&Hz

L‘NH21 0

(CHsCHzCHz bjNH1) (NHsCzN) 0 (NH2 b‘OCH~CHI) 0 CHB

Dimethylformamide

Nicotinamide

H&N’

(C 5H 4NiNH 0

Methylacetamide Water and alcohols

(CH&

\

CHs 2)

NHCHs)

Water Methanol(HOH) (CHsOH) Ethanol (CH&HZOH) Ethylene glycol (CH~OHCHTOH) Inorganic ions Sodium

across the bladder. Side1 and Hoffman [ 771 have observed solvent drag on urea using a synthetic liquid “membrane” of well stirred mesityl oxide separating two aqueous solutions in which transport of water probably occurs in droplets,

certainly not in continuous aqueous channels. These considerations make one interpret the evidence of a solvent drag effect on urea with some caution. TABLE REFLECTION

TABLE SIMULTANEOUS

PERMEABILITY

THE TWO DIRECTIONS

THROUGH

III

Hormone

Absent Present

17 13

M

-

26.0 251.3

S”

COEFFICIENTS

UREA

COEFFICIENTS

FOR

THE TOAD BLADDER

Permeability Coefficients (X 107 cm. xc-l.) No. of Periods

2

UREA

THROUGH

S-MM*

A

26.8 261.2

0.8 9.9

f2.3 zk7.9

TOAD

BLADDER

CHLORIDE

AND

[75,26]

IN

[ 76,26]

S.E. ofh

IV

FOR THIOUREA,

Compound

No. of Experiments

T&urea Chloride Urea

6 3 29

Mean AW (pl. cm? hr’.)

Mean

200 207 200

v

0.995 0.993 0.79

Range c

0.988-1.00 0.990-0.994 ...

Net solute flux * Mucosal to serosal and serosal to mucoaal permeabilities measured simultaneously with NIB- and C’~labeled urea.

NOTE:

Reelection coe5cient,

AMERICAN

q 4

1 -

JOURNAL

Net water flux Medium OF

concentration MEDlCINE

Membrane

Effects

Another means of testing the interaction of solute and solvent as they cross a permeability barrier is to evaluate the reflection coefficient, r. This expression has been introduced by Staverlnan [18] to indicate the penetration of mclubranes bb- solute relative to solvent. A truly- semipermeable membrane will act as a perfect sieve, the concentration of impermeant solute in the filtrate will be zero, and CJ = 1.0. On the other hand, the concentration of solute in the filtrate from a nonselective barrier will be the same as in the original medium, and (T = 0. Table IV [ 151 shows the reflection coefficients obtained for thiourea, chloride and urea in the presence of large net transfers of water averaging some 200 ~1. cmp2. hr-I. The reflection coefficient for thiourea and chloride was essentially 1.0, indicating that even in the presence of large net movements of water the membrane retains a high degree of impermeability to thiourea and chloride. Even urea is retarded some 80 per cent in its rate of penetration relative to that of water. Thus thiourea and chloride are excluded from access to the channels in which bulk flow of water occurs and the results, even with respect to urea, are not unambiguous in indicating that water and urea move in the same channels [ 791. SODIUM

t

PITRESSIN

0

1

90

120

150

I60

[ZO]. Energy derived from metabolism is Itsed to pump sodium ions uphill thermodynanlically from luminal fluid to body fluids. This transport is highly specific for sodium ions. It is of interest that this transport activity of the bladder can be stimulated by mammalian neurohypophl-sial hormones. This is shown in Figure 3 in which the active transport of sodiunl ions is the ordinate and time is the abscissa. Even a large amount of Pitressin’ added to the medium bathing the mucosal or urinary surface has no effect on sodium transport. The same ailloimt v

HORMONES

Hours Group

60

)

Demonstration of the unilateral stimulatory FIG. 3. effect of neurohypophysial hormone. When neurohypophysial hormone, Pitressin, was added to the Ringer’s solution bathing the mucosal surface no effect on shortcircuit current was noted. The same amount of hormonal preparation added to the solution bathing the serosal side resulted in a prompt and large stimulation [20,26].

TABLE NEUROHYPOPHYSIAL

30

SIDE

MINUTES

In contrast to water and the urea-like compounds which move passively, the toad bladder -like the renal tubular epithelium-actively transports sodium ions from urine to body fluids

Or

LUNITS

(SEROSAL

TRANSPORT

EFIEC’I‘

of ,41)H---l,m/

2

ON qo,

3

OF T04D

BLADDER

[26‘]

Mean Difference (hours 2-1)

S.L. Mean Difference

P

+0.02 +0.49

zto.03 Ito.

0 0
-0.04 0

..

Ten Paired Exfierimcnts A. Sodium Ringer’s Solution Control Treated with hormone*

1.20 1 .23

1.22 1 .72

1.06 1 .76

Nineteen Paired Experiments B. Sodium-Free Ringer’s Solution Control Treatrd with hormone*

1 .Ol 1.12

0.97 1.12

0.93 1 .lO

NOTE: Measurements of oxygen consumption were made for three consecutive hours on paired bladder halves in Warburg vessels by classic manometric technics. Hormone was added at the end of one hour to one bladder half while the other served as control. Measurements were made in sodium Ringer’s solution (.4) and in sodium-free Ringer’s solution (B) in which all the sodium had been replaced by magnesium or choline. * Hormone added at end of first hour. VOL.

42,

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Membrane

752

Effects of ADH-Lea]

of hormone added to the serosal medium produces a prompt and large stimulation of the current. It has been shown by simultaneous measurements of sodium flux across the bladder using two different radioactive isotopes of sodium that all the increase in short-circuit current after the administration of vasopressin results from an increase in the transport of sodium from mucosal to serosal surfaces [21]. The increase in sodium transport associated with exposure of the bladder to vasopressin is accompanied, as expected, by an increase in metabolism [21]. Table v (part A) shows the increase in oxygen consumption stimulated by vasopressin. The metabolic effects are, however, not a direct effect of the hormone but only secondary to its action on sodium transport. Thus, when sodium is removed from the bathing medium so that there is no sodium to be transported, the stimulation of oxygen consumption by the hormone is abolished (Table v, part B). In the absence of sodium, however, neurohypophysial hormones still exert their usual effects on the permeability of the bladder to water and urea [3]. A recent study by Civan and associates [22] further indicates that the action of vasopressin with respect to the permeability of the bladder to sodium is upon a passive process. Under conditions of zero net transport of sodium achieved by reducing the electrochemical gradient for chloride ions across the tissue to zero, the driving force for sodium would vanish across any “passive” permeability barrier. The addition of vasopressin in this special situation did not regularly increase the transepithelial electrical potential although it did cause the usual drop in its electrical resistance. The results suggest an action of vasopressin only on permeability barriers to sodium and not directly upon the active energy-requiring transport processes affecting this ion. Thus far, there is no direct evidence that vasopressin affects the sodium transport processes in the mammalian nephron and the evidence with respect to an effect on the transport of urea is equivocal. SITE OF ACTION

OF VASOPRESSIN

Having considered the penetration of the bladder wall by water, by solutes generally, by a special group of solutes typified by urea and by sodium ions, we should try to draw these

separate phenomena together into a unified concept of the permeability properties of this tissue and of the action of vasopressin thereon. Some unity can be achieved by examining the site at which the hormonal effect is mediated. Since such a site of action must coincide with a rate-limiting permeability barrier, identification of such sites will aid in the localization of the important permeability barriers within the tissue. The most direct approach to the site of the hormonal effect on permeability was that of Peachey and Rasmussen [23] and has been confirmed by Jard 1241. These workers exposed the mucosal surfaces of bladders to hypotonic fluid in the presence and absence of vasopressin. Following appropriate preparation they then examined the tissue histologically. In the absence of hormone there was no swelling of the epithelial layer of cells despite the hypotonic fluid bathing the mucosal surface. In the presence of vasopressin these cells were markedly swollen. When the same hypotonic solution was applied to the serosal surface of the tissue the cells were observed to swell promptly in the absence of the hormone. These observations, taken together with the knowledge that vasopressin induces large net transfers of water across the bladder when the mucosal bathing medium is hypotonic, clearly indicate that a permeability barrier at or near the mucosal surface normally excludes water from the cells. Neurohypophysial hormones affect this mucosal barrier to allow water to cross the tissue in the process of which osmotic swelling of the epithelial cells occurs. MacRobbie and Ussing [25] came to similar conclusions from direct measurements of the thickness of the epithelial layer of frog skin, which is functionally analogous to the toad bladder. This tissue only swells when its outside surface is bathed with hypotonic fluid in the presence of vasopressin. This finding implies that a barrier to penetration by water near the outside surface of the skin had been reduced, allowing net transport of water, as occurs in the toad bladder and renal tubule, with concomitant swelling of the epithelial cells. Although not so direct, similar evidence may be obtained from radioisotope experiments. Table VI [26] shows the effect of vasopressin on the permeability of the bladder to labeled water (THO), urea and thiourea and to the accumulation of the isotopic species within the tissue (per AMERICAN JO”“NAL

OF

MEDICINE

Membrane

EFFECT

OF

VASOPRESSIN ON PERMEABILITY AND

LABELING

Effects

of ADH-Leaf

753

TABLE VI OF TOADBLADDERBY THO,

TO THE MLICOSAL

MEDIUM

(Y-UREA AND CY-THIOITREAADDED

[26]

Permeability Coefficients (K trans X lO_km. set -I.)

% Labeling *

Vasopressin

Vasopressin

Compound

No. of Paired Experiments

Absent

Present

Absent

Present

THO (Y-urea CWthiourea

10 8 7

940 18 13

1,600 329 13

20.8 11 2.6

27.2 38 3.5

A

S.E. Mean Difference

P

6.4 27 0.9

12.3 rt3.9 f0.4

0.02
in tissue water (X 100). Since vasopressin increased the transepithelial concentration in mucosal medium permeabilitv coefficient. K+,,., ..-., and the tissue labeling for water, urea and thiourea, its site of action must be at or near the mkosal surface of the bladder. * 7’ labeling

=

average concentration

cent labeling) in the steady state following addition of the isotopic species to the medium bathing the mucosal surface. In the case of THO and C14-urea the increase in transport across the bladder is associated with an increased content of the radioactive species within the tissue. This finding is again consistent with an action of vasopressin to increase the permeability of a barrier in or near the apical surface of the mucosal layer of cells with respect to each species. By contrast, vasopressin has no significant effect on the low permeability of the bladder to thiourea and the tissue labeling was also not significantly affected by the hormone. Furthermore, the low values for tissue labeling averaging 2.6 and 3.5 per cent in seven paired experiments in the absence and the presence of vasopressin set an upper limit for tissue labeling since they include C14-thiourea adherent to the mucosal surface which was not removed by the blotting technic as well as molecules in transit across the tissue. This indicates that the selective barrier which effectively screens out most solute molecules must also be located near the mucosal surface. If we consider the relatively simple histology of the tissue, the apical surface of the mucosal cells is bounded by a unit plasma membrane and it seems likely that the hormonal effect is exerted upon this membrane. The only structure regularly present outside the unit plasma membrane is the fuzzy deposit seen by electron microscopy [23], but this appears to be much too porous a structure to constitute the permeability barriers we are discussing, unless VOL

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in life this layer is much more homogeneous and only in the process of fixation and histologic preparation becomes fibrillar and porous. In either case we are dealing with a superficial barrier at the mucosal surface of the tissue. Such a location for the major permeability barrier in these tissues is indeed fortunate as it protects the epithelial cells from being buffeted by the vicissitudes of urinary composition with respect to tonicity, pH, ammonium ions and other noxious factors which must be excreted in the urine at concentrations which would be toxic, if not lethal, to most cells. An interesting feature of the functional orientation of the mucosal cells in this tissue is that the response to neurohypophysial hormones occurs only when these are added to the medium bathing the serosal surface despite the effect of the hormone at the mucosal or apical surface of the cells. We have not been able to detect any hormonal effects with even very large amounts of the hormones added to the mucosal medium. This may simply mean that the polypeptide hormones cannot gain access to their site of action from the mucosal medium because of the general impermeability of the apical barrier to most solutes. The actual receptor sites for the hormones may be located at the basal surface of the mucosal layer of cells and this would be consistent with the evidence presented elsewhere in this Symposium that the action of vasopressin is mediated through a series of chemical events including the synthesis of 3 ‘, 5 ‘-cyclic adenylate, induced by the hormone in this responsive tissue.

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FIG. 4. Comparison of the effects of amphotericin B and vasopressin on short-circuit current, potential difference and net water movement across the toad bladder. Addition of amphotericin B to the mucosal medium produced a large increase in shortcurrent without affecting net movement of water. Subsequent addition of vasopressin failed to augment the short-circuit current, but produced its usual large effect on transport of water. When vasopressin was removed, the net transport of water was reduced. In this experiment sodium Ringer’s solution was as the serosal bathing used medium and sodium Ringer’s solution (diluted 1:4) as the mucosal bathing medium [28].

9-

MfNU

FIG. 5. Effect of amphotericin B and vasopressin on net water transfer and permeability to urea. Amphotericin B added to the mucosal bathing medium produced a large increase in the permeability of the bladder to urea and a slight increase in net water movement. Subsequently vasopressin had no further effect on the tissue permeability to urea but did cause the usual large increase in net water transfer. Removal of vasoprcssin reduced net water transfer to lower levels. In this experiment the initial shortcircuit current was 56 Mamp., the potential difference was 6 mv.; the final values were 58 ramp. and 6 mv., respectively. Sodium Ringer’s solution (diluted 1: 1) on the mucosal side provided the osmotic gradient. K,,,,, = permeability coefficient [28].

TES

SEROSAL MEDIUM

MUCOSAL MEDIUM

Fro. 6. Schematic representation of the mucosal permeability barricr. The urinary surface of the mucosal cells is represented as a dual barrier, a dense diffusion and a porous barrier, in series. All substances including water arc retarded at the diffusion barrier. Vasopressin enhances the permeability of this tissue to urea and sodium by an effect on the dense diffusion barrier, and to water by an effect on the porous barrier [28].

I I-

MUCOSAL Dense

BARRIER Porous

WATER

ii

UREA

I# I--+

OTHER SOLUTES SODIUM

8 Bl

K irons

MUCOSAL

K tram

BARRIER

( mnec-!x 10-T)

__) +

-.I 1

--ooo -30 __)

-2-20

-2-20

-35

I VASOPRESSIN

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l~inding the locus of action of vasopressin wilh respect to its effect on water, urea and sodiunl transport to a site in or near the apical surface of the mucosal layer of cells does not circllln\.ent the apparent paradox that the tissue can rvlnain selective to solute molecules of small dimensions at a time when large channels would seem necessary to accommodate the net transport of water across the tissue. This apparent difficulty is readily overcome theoretically by assuming that the permeability barrier is not a simple homogeneous structure but rather a complex system of at least two barriers with different properties in series [27]. Thus a dense diffusion barrier in series with an underlying porous barrier could theoretically account for the permeability properties of the bladder. The dense barrier might screen out most solutes but allow water, urea and sodium to pass. Modification of the porosity of the deeper barrier by. vasopressin could then provide the specific changes in permeability induced by the hormone. It seenncd that such a dual barrier hypothesis was likely to remain in the realm of purely probability until Dr. Norman speculative Lichtenstein made the observation that amphotericin B added to the mucosal medium bathing the toad bladder will separate functionally the two barriers [28]. Amphotericin B is a polyene antibiotic known to react with sterols [29J. For this reason it is toxic to fungi but not to bacteria which do not synthesize sterols and, therefore, contain none in their cell walls. By carefullv adjusting the amounts of amphotericin B used in experiments with the toad bladder it was possible to effect functional removal of the selective permeability barrier, leaving the second barrier relatively intact. Figure 4 [28] shows that amphotericin B added to the mucosal bathing medium resulted in a stimulation of sodium transport (short-circuit current) which was not further augmented by vasopressin. By contrast, net transport of water across the tissue was not significantly affected by the concentration of amphotericin B used in this experiment but increased in typical fashion in response to vasopressin. A similar dissociation of the effects of amphotericin B and of vasopressin on the permeability of the bladder to urea and to net transfer of water could also be made, as shown in Figure 5 [28]. Following application of amphotericin B to the mucosal surface of the bladder the permeability of the tissue to all

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of ADH--Leaf

7.5 5

small solutes is Inarkedly increased anti cellular constituents leak out into the medium, as has been shown in relation to the toxic effect of this antibiotic on molds [30,37]. Thus smphotericin functionally removes the selective perrrleability barrier in the bladder. By- quantitative comparisons of the changes in permeability induced by vasopressin and amphotericin B it was possible to ascribe the hormonal effects discussed to either of the two series barriers as summarized in Figure 6. Not? that it is still necessary for the hormone. to exert separate effects on each of the two hypothetical barriers. Further investigations into the structure of cell membranes and the molecular changes induced in responsive membranes by- neurohypophysial hormones will be necessary before we can say whether it will ever be possible to account for the hormonal effects by a silqle action exerted at a single site. REFERENCES 1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

KOEPOED-JOHNSEN, V. and USSING, H. H. ‘The contributions of diffusion and flow to the passage of D&l through living membranes. Acto ph_ysiol. scandinav., 28: 60, 1953. EWER, R. F. The effect of pituitrin on fluid distribution in Rujo regularis Reuss. J. Exper. Riol., 29: 173, 1952. HAYS, R. M. and LEAF, A. Studies on the movement of water through the isolated toad bladder and its modification by vasopressin. ./. &n. Physiol., 45: 905, 1962. IIENDERSON, L. .I. The Fitness of the Environment. 4n Inquiry into the Biological Significance of the Properties of Matter. New York, 1913. The Macmillan Co. PAIII.ING, L. The Nature of the Chemical Bond, 3rd ed., p. 465. Ithaca, New York, 1960. Cornell Univrrsity Press. FRANK, H. S. Covalenry in the hydrogen bond and the properties of water and ice. PAL. Roy. Sot. Med., 247: 481, 1958. NEMETHY, G. and SCIIERAGA, H. A. Structurr of water and hydrophobic bonding in proteins. I. A model for the thermodynamic properties of liquid water. J. Chem. Phys., 36: 3382, 1962. WANG, J. \V., ROBINSON, C. V. and EDELMAN, I. S. Self-diffusion and structure of liquid water. m. Measurements of the self-diffusion of liquid water with Ilz, H3 and 018 as tracers. .I. Am. Chm. Sot., 75 : 466, 1953. KUHN, W. and THURKAUF, M. lsotopentrennung b&n Gefrieren van Wasser und Diffusionskonstanten van D und ‘80 im Eis. H&et. chim. actn, 41: 938, 1958. EINSTEIN, A. ober die van der molekularkinetischen Theoric der WIrme geforderte Bcwegung van in ruhenden Fliissigkeiten suspendierten Teilchen. Ann. Phvs.. 17: 549. 1905. Enelish translation. In: ln&tigations on' the Thcoriof the Brownian Movement, p. 12. Edited by Fiirth, R. London,

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15.

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18. 19.

20.

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Effects of ADH--Leaf

1926. Methuen & Co. Ltd. Reprinted New York, 1956. Dover Publications. PAPPENHEIMER,J. R., RENKIN, E. M. and BORRERO, L. M. Filtration, diffusion and molecular sieving through peripheral capillary membranes. Am. J. Physiol., 167: 13, 1951. SOLOMON, A. K. Pores in the cell membrane. SC.Am., 203: 146, 1960. ROBBINS, E. and MAURO, A. Experimental study of the independence of diffusion and hydrodynamic permeability coefficients in collodion membranes. J. Gen. Physiol., 43: 523, 1960. ANDERSEN,B. and USSING, H. H. Solvent drag on non-electrolytes during osmotic flow through isolated toad skin and its response to antidiuretic hormone. Acta physiol. scandinav., 39: 228, 1957. LEAF, A. and HAYS, R. M. Permeability of the isolated toad bladder to solutes and its modification by vasopressin. J. Gen. Physiol., 45: 921, 1962. MAFFLY, R. H., HAYS, R. M., LAMDIN, E. and LEAF, A. The effect of neurohypophyseal hormones on the permeability of the toad bladder to urea. J. Clin. Invest., 39: 630, 1960. SIDEL,V. W. and HOFFMAN,J. F. Apparent “solventdrag” across a liquid membrane. In: Biophysical Society Abstracts, Chicago Meeting, 1963. STAVERMAN, A. J. The theory of measurement of osmotic pressure. Rec. trav. chim., 70: 344, 1951. KEDEM, 0. and KATCHALSKY, A. Thermodynamic analysis of the permeability of biological membranes to non-electrolytes. Biochim. et biophys. acta, 27: 229, 1958. LEAF, A., ANDERSON, J. and PAGE, L. B. Active sodium transport by the isolated toad bladder. J. Gen. Physiol., 41: 657, 1958.

21. LEAF, A. and DEMPSEY, E. F. Some effects of mammalian neurohypophyseal hormones on metabolism and active transport of sodium by the isolated toad bladder. J. Biol. Chem., 235: 2160, 1960. 22. CIVAN, M. M., KEDEM, 0. and LEAF, A. Effect of vasopressin on toad bladder under conditions of zero net sodium transport. Am. J. Physiol., 211: 569, 1966. 23. PEACNEY,L. D. and RASMUSSEN,H. Structure of the toad’s urinary bladder as related to its physiology. J. Biocham. d Biophys. Cytol., 10: 529, 1961. 24. JARD, S. Personal communication. 25. MACROBBIE, E. A. C. and USSINC, H. H. Osmotic behavior of the epithelial cells of frog skin. Acta physiol. scandinav., 53: 348, 1961. 26. LEAF, A. Transepithelial transport and its hormonal control in the toad bladder. Ergebn. Physiol., 56: 216, 1965. 27. KEDEM, 0. and KATCHALSKY, A. Permeability of composite membranes. Part 3. Series array of elements. Tr. Faraday SOG., 59: 1941, 1963. 28. LICHTENSTEIN, N. S. and LEAF, A. Effect of amphotericin B on the permeability of the toad bladder. J. Clin. Invest., 44: 1328, 1965. 29. KINSKY, S. C., LUSE, S. A. and VAN DEENEN, L. L. M. Interaction of polyene antibiotics and artificial membrane systems. Fed. Proc., 25: 1503, 1966. 30. KINSKY, S. C. The effect of polyene antibiotics in permeability in Neurospora crassa. Biochem. H Biophys. Res. Commun., 4: 353, 1961. 31. KINSKY, S. C. Alterations in the permeability of Newospora crassa due to polyene antibiotics. J. Back, 82: 889, 1961.

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