J. Exp. Biol. (1969), 50, 15-28 j rWith 8 text-figwretPrinted in Great Britain

URINE FORMATION BY THE MALPIGHIANTUBULES OF CALLIPHORA

II. ANIONS

BY M. J. BERRIDGE

Department of Biology, Case Western Reserve University, Cleveland, Ohio 44106

{Received 8 May 1968)

INTRODUCTION

Urine formation by the Malpighian tubules of insects depends on an active trans-port of potassium (Ramsay, 1953, 1955; Berridge, 1967, 1968). However, potassiumtransport alone will not induce water transport unless it is accompanied by an anion.For example, if the chloride bathing the isolated tubules of Calliphora is replaced witha non-transportable anion such as sulphate, fluid transport ceases. The mechanismof transporting anions to accompany the active secretion of potassium has beeninvestigated.

MATERIAL AND METHODS

Three-day-old adult female Calliphora erythrocephala were used throughout thisstudy. The methods of culturing insects and setting up isolated Malpighian tubuleswere similar to those described previously (Berridge, 1966).

Different anion media were obtained by adding the sodium salt of the anion to astock solution containing essential cations and metabolites (Table 1). The pH of allmedia was adjusted to 7-4. The final concentration of the test anions was n o m-equiv./l.for monovalent anions and 146 m-equiv./l. for the divalent anions. At pH 7-4 phos-phate is present in both the monobasic (20%) and dibasic (80%) forms. Since the pHof urine was not measured, phosphate concentration has been expressed as fig. PO^/A.

Table 1. Composition of the stock solution of essential cations and metabolites

(The sodium salt of the anion to be tested was dissolved in this stock solution.)

CompoundKOHCaSO4.2H,OMgSO4.7H,OTrehaloseGlucoseGlutaminea-Alanine

mg./ioo ml.225

24250180180

704 0

CompoundGlycineCitric acidFumaric acidMalic acidPenicillinStreptomycin

mg./ioo mLSO

1 0 0

1 0 0

1 0 0

31 0

Unless indicated otherwise, rate of urine formation was measured 30—90 min. aftersetting up the tubules and the urine collected over this period was used for chemicalanalysis. Chloride and phosphate concentrations were determined by the Spinco/Beck-man adaptations of the methods of Schales & Schales (1941) and Fiske & Subbarow

16 M. J. BERRIDGE

(1925) respectively. The points on the graphs represent the mean of six determinationsand the vertical lines indicate ± twice the standard error of each mean.

In this study the abbreviation U\P has been borrowed from vertebrate physiologyto indicate the relationship between the concentration of a substance in the urine andits concentration in the outside medium (Ramsay, 1958). In kidney physiology, theoutside medium is usually plasma, whereas, in the present study, it was an artificialmedium. Nevertheless, it will be less confusing to retain the convention of kidneyphysiology than to introduce a new term for studies on Malpighian tubules.

The hydrated radii of anions was estimated from their relative hydrated sizes(Araki, Ito & Oscarsson, 1961) assuming that potassium has a hydrated radius of1*98 A. (Solomon, 1960a). When the effect of anion size on rate of urine formation wastested, the pH of the phosphate and bicarbonate solutions was not adjusted.

RESULTS

Urine formation in the presence of chloride or phosphate

Chloride transport

The first group of experiments tested the ability of Malpighian tubules to secreteurine when the chloride concentration of the bathing medium was varied. In order tomaintain a constant osmotic pressure, lower concentrations of chloride were obtainedby substitution with either sulphate or phosphate. In a full chloride medium urinewas secreted at a rate of 4-6 mm.8 x io^/min. Replacement of chloride with sulphate(Fig. 1 a, closed circles) resulted in a progressive decrease in rate of urine production.When the chloride concentration was reduced below 30 m-equiv./l. urine flow ceased,indicating that sulphate alone cannot support urine formation. Evidence will bepresented later (Fig. 5) which suggests that this inability of sulphate to support urineformation is related to its large size.

In contrast to the above results, progressive replacement of chloride with phosphatecaused a considerable increase in rate of urine production (Fig. 1 a, open circles). Thesechanges in rate of urine formation depending on whether sulphate or phosphatereplaced chloride were reflected in marked differences in the chloride concentrationof the urine. When sulphate replaced chloride, the concentration of chloride in urinewas linearly related and slightly higher than its concentration in the bathing medium(Fig. 1 b, closed circles). However, when chloride was replaced with phosphate, chlorideconcentration in the urine was suppressed, especially at low external chloride concen-trations (Fig. 1 b, open circles). The experiments to be described next indicate that theability of tubule cells to concentrate phosphate accounts for this displacement ofchloride from the urine.

Phosphate transport

Phosphate transport has been studied using the same experimental design as thatjust described for chloride transport. A range of phosphate concentrations was obtainedby progressive substitution with either sulphate or chloride.

When sulphate replaced phosphate in the bathing medium, rate of urine formationwas linearly related to the external phosphate concentration (Fig. za, closed circles).The effect of varying chloride and phosphate in the bathing medium on rate of urine

Urine formation by the Malptgkian tubules of Calliphora. / / . 17

formation has already been described (Fig. 1 a, open circles); to facilitate comparison,the values have been replotted on Fig. 2a. A 50% replacement of phosphate withchloride had no effect on rate of urine production but thereafter the rate decreasedtowards the level of tubules functioning in a full chloride medium (Fig. id).

10

oX

8o

o

1

20 40 60 80 100 120

Chloride concentration in medium(m-equiv./L)

(a)

equi

v./

Sin

urm

e I

gi§o8uco2o

140

120

100

80

60

40

20

-

: /{A l

- A y- / /

—<!>T^" 1 1 1 1

1 /

//

1

20 40 60 80 100 120

Chloride concentration in medium(m-equiv./l.)

Fig. i. (a) Effect of chloride concentration in the bathing medium on rate of urine production.(6) Chloride concentration in the urine as a function of the chloride concentration in themedium. In both cases, osmotic pressure of the bathing medium was adjusted with sulphate ( • )or phosphate (O).

The Malpighian tubules of Calliphora resemble those of Dixippus (Ramsay, 1956)in that they can concentrate phosphate in the urine (Fig. zb). Phosphate concentrationin the urine did not differ depending on the presence of sulphate (closed circles) orchloride (open circles). On the other hand, we have just seen that chloride concentra-tion in the urine was suppressed to a greater extent in the presence of phosphate thanin the presence of sulphate (Fig. ib).

This difference between phosphate and chloride transport is shown more clearly ifthe UjP ratios for these two anions are plotted against their concentration in thebathing medium. Figure 3 illustrates that the mechanism concentrating phosphate inthe urine is not altered by the presence of chloride or sulphate. Although the U/Pratio decreases with an increase in external phosphate concentration, the ratio remainssignificantly above 1 throughout the range of phosphate concentration employed.

The presence of an impermeant anion (sulphate) as compared to a permeant anion2 Exp. BioL 50, 1

i 8 M. J. BERRDDGE

o

Iaoc

"C

I

10 -

9 -

8

7

6

5

4

3

2

1

2

eo•a

fi

u

a

10 h

8

7

6

5

4

3

2

I0 1 2 3 4 5 6 7

Phosphate concentration in medium (jig.

0 1 2 3 4 5 6 7 8

Phosphate concentration in medium (fig. POJfiL)

(a) (ft)

Fig. 2. (a) Effect of phosphate concentration in the bathing medium on rate of urine production.(b) Phosphate concentration in the urine as a function of the phosphate concentration in themedium. In both cases, osmotic pressure of the bathing medium was adjusted with sulphate ( • )or chloride (O).

a,b

i at e

81OH

26

2-4

2-2

20

18

1-fr

14

1-2

n.n

- . .

" o

-

-

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-

-

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o

o

I I I 1

1-2

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5 08

X 0-6.

6 0-4

0-2

~*~~/^_ /

/ o

o /

/

0 1 2 3 4 5 6 7 8

Phosphate concentration in medium (jig. POJfA.)

Fig. 3

0 20 40 60 80 100 120

Chloride concentration in medium (m-equiv./l.)

Fig- 4

Fig. 3. The relation between phosphate UjP and phosphate concentration in the bathingmedium. Data obtained from Fig. zb where phosphate was balanced against sulphate ( • ) orchloride (O).

Fig. 4. The relation between chloride UjP and chloride concentration in the bathing medium.Data taken from Fig. 1 b where chloride was balanced against sulphate ( • ) or phosphate (O).

Urine formation by the Malpighian tubules of Calliphora. / / . 19

(phosphate) has a marked effect on chloride transport (Fig. 4). When chloride con-centration was varied by replacement with sulphate the U/P ratio for chloride remainedconstant ( I -O-I - I6) . In the presence of phosphate, however, chloride movement wassuppressed and the U/P ratio declined to a low level as phosphate progressivelyreplaced chloride in the bathing medium.

12 r-

10

3•a2ao

o -4

I

NO,"'

Br-<a-

ao4-• CIO3-

' SCN" <>HPO4*

HCCV

CH,COO-

CH,CH2COO- Citrate

1 2 3 4 5 6 7

Hydrated radius (A.)

Fig. 5. The ability of anions with different hydrated radii to support urine formation.

The experiments described so far suggest a qualitative difference between chlorideand phosphate transport. The former probably occurs by passive diffusion down theelectrical gradient created by the active secretion of potassium. The simplest hypothesiswould be that chloride permeates through aqueous-filled channels or pores in the celJmembranes. It seems unlikely that phosphate will move through such pores becauseit has a much larger hydrated radius than chloride, or even sulphate, yet it is capableof maintaining a high rate of urine formation. Consequently, it is necessary to postu-late a carrier-mediated transport mechanism to account for this high level of phosphatetransport. Such a carrier mechanism is consistent with the observation that tubulecells can concentrate phosphate in the urine. Further experiments were devised to testthe hypothesis of two separate pathways for anion transport.

2O M. J. BERRIDGE

Urine formation in the presence of onions with different hydrated radii

If one of the transport mechanisms involves a passage of anions through pores, rateof urine formation should be related to the size of the permeating anions. In order totest this, rate of urine formation was measured in the presence of anions with differenthydrated radii. Figure 5 shows that a wide variety of anions can support urine forma-tion and that a relationship exists between rate of urine formation and the size of the

11 T -

Time (hr.)

Fig. 6. Effect of copper (1 x io~* M) on rate of urine formation. Twelve tubules were set up inartificial medium containing the anion to be studied; after a h., copper was added to sixtubules and the remainder served as controls. Phosphate control ( • ) , phosphate+ copper ( O);nitrate control (A), nitrate+ copper (A); chloride control ( • ) , chloride+copper ( • ) .

different anions. Anions larger than acetate cannot support urine formation. Phosphatewas the only anion tested which did not conform to this pattern. Despite its large size,the monovalent form of phosphate maintained a rate of urine formation which washigher than for any other anion (Fig. 5). The result obtained with the divalent formmust be accepted with reservation because the alkaline environment resulted in theformation of a flocculent precipitate of calcium and/or magnesium phosphate whichcould have reduced rate of urine formation (Berridge, 1968). Therefore, the ability oftubules to secrete urine in the presence of divalent phosphate ions is even more re-markable because this anion has a hydrated radius considerably larger than sulphateand propionate which could not support urine formation. These observations strengthenthe hypothesis that phosphate transport involves a specific carrier mechanism to facili-tate the passage of this large anion through the cell membranes.

Urine formation by the Malpighian tubules of Calliphora. / / . 21

Effect of arsenate and copper on avion transport

A specific competitor and inhibitor have been employed to differentiate betweenthe two mechanisms by which anions move across the cell. Arsenate, which has amolecular configuration similar to that of phosphate, will inhibit phosphate transportbut not chloride transport. And indeed, competitive inhibition of phosphate transportby arsenate resulted in a considerable augmentation of chloride transport. Whenarsenate (1 x io"8 M) was added to tubules in the presence of both anions, the phosphateUjP declined from 4-1 to 1-7, whereas the chloride U/P increased from 0-37 to 0-84.

12

10

Is

50 100 150 200 250 300

Minutes

Fig. 7

2a

18

16

I 14

12

o 10

au

40 80 120 160 200 240

Minutes

Fig. 8

Fig. 7. Ability of phosphate to reverse the inhibitory effect of copper on urine formation.Initially twelve tubules were set up in a chloride medium; copper (1 x io~* M) was present inthe bathing medium for the period between the arrows (60-140 min.). At 140 min., six tubuleswere placed in a phosphate medium (O — O) and six tubules in a chloride medium (© 0 ) .

Fig. 8. Copper inhibition of chloride transport in the presence of phosphate. Six tubules wereset up in a phosphate medium containing 1 x io~* M copper ( O — O) and six tubules were setup in a copper-free phosphate medium ( • • ) . Both groups of tubules were placed in acopper-free chloride medium at 90 min. (arrow).

The action of copper on rate of urine formation also suggests the possibility of twoseparate pathways for anion transport. When tubules were set up in a phosphatemedium, copper (1 x io~* M) had no effect on rate of urine production (Fig. 6). On theother hand, tubules utilizing anions such as chloride or nitrate were inhibited bycopper and rate of urine formation declined after addition of this heavymetal. Completeinhibition of urine formation occurred after 2 hr. (Fig. 6).

22 M. J. BERRIDGE

Inhibition of urine formation by copper can be reversed if Malpighian tubules areplaced in a phosphate medium (Fig. 7). In this experiment twelve tubules were set upin a chloride medium and allowed to secrete urine for 60 min., at which time copperwas introduced into the bathing medium resulting in a characteristic reduction ofurine flow. At 140 min. copper was removed and six tubules were placed in a chloridemedium and six tubules in a phosphate medium. Inhibition of urine formationremained in those tubules placed in the chloride medium but was reversed in thephosphate medium (Fig. 7). Urine formation was possible in the phosphate mediumbecause this anion could utilize its carrier mechanism, which is apparently unaffectedby copper.

Before these observations can be accepted as evidence for two separate pathways,it is necessary to exclude the possibility that phosphate reverses copper inhibition bybinding this heavy metal. The following experiment, however, clearly indicates thatcopper inhibition of chloride transport can develop even in the presence of a highconcentration of phosphate (Fig. 8). Malpighian tubules were set up in a phosphatemedium containing copper and, as noted earlier (Fig. 6), rate of urine formationresembled that of the control tubules functioning in a phosphate medium minuscopper. After 90 minutes all tubules were placed in a copper-free chloride medium(Fig. 8). Inhibition of urine production occurred in only those tubules which hadpreviously been treated with copper, indicating that this heavy metal can inhibit thechloride transport mechanism despite a high concentration of phosphate. Controltubules showed a slight decrease in rate of urine formation in the chloride mediumbecause this anion is less effective than phosphate in promoting urine formation(Fig. 1 a). These studies on copper inhibition of urine formation in the presence ofchloride or phosphate therefore further suggest that transport of these two anionsinvolves separate mechanisms.

DISCUSSION

Rate of urine formation by the Malpighian tubules of CaUiphora depends on theavailability of anions to accompany the active secretion of potassium. Since the plasmamembrane of cells is lipoidal, special structural features must be present to allowanions to pass through it. In CaUiphora, anions seem to permeate the membranes oftubule cells by two independent routes, namely through pores in the case of chloride(and a number of other anions), or by means of carriers in the case of phosphate.Therefore the present observations not only contribute to our knowledge of aniontransport, but also lead us to a better understanding of membrane structure andfunction.

Since Boyle & Conway (1941) postulated the presence of pores in the surface mem-brane of muscle fibres, the sieve-like action of the cell membrane towards ions hasreceived considerable verification (Solomon, 19606). The exterior and interior mediaof many cells seem to communicate through pores, and small ions pass through themembrane, whereas those larger than the pore size are all non-penetrating. Forexample, the inhibitory post-synaptic potential (I.P.S.P.) develops when the inhibitorytransmitter substance converts the post-synaptic membrane into a selective ionicsieve. Coombs, Eccles & Fatt (1955) tested the ability of different anions to supportthe I.P.S.P. and found that discrimination between anions was closely related to their

Urine formation by the Malpighian tubules of Calliphora. / / . 23

relative hydrated sizes. Further studies utilizing a more extensive anion series havesubstantiated the 'pore-structure' hypothesis of the activated post-synaptic mem-brane (Araki et al. 1961; Ito, Kostyuk & Oshima, 1962; Kerkut & Thomas, 1964).Araki et al. (1961) found that separation between penetrating and non-penetratinganions occurred between chlorate and bromate and hence they estimated that theeffective pore radius of the activated inhibitory post-synaptic membrane of cat moto-neurones lay between the hydrated sizes of these two anions, i.e. 2-85-3-25 A. respec-tively. Other techniques employing a comparison of diffusion and bulk flow of water,or reflexion coefficients, have been used to calculate the effective pore sizes of a widevariety of membranes. Despite the different techniques used, the effective pore sizes

Table 2. The effective radii of pores in different membranes

Reference

Paganelli & Solomon (1957)Villegas, Barton & Solomon (1958)Villegas, Barton & Solomon (1958)Solomon (1960 a)Goldstein & Solomon (i960)Whittembury, Sugino & Solomon (i960)Whittembury, Sugino & Solomon (i960)Villegas & Bamola (1961)Araki et al. (1961)Whittembury (1962)Whittembury (1962)Lindemann & Solomon (1962)Zadunaisky, Parisi & Montoreano (1963)Villegas (1963)Fordtran & Dietschy (1966)Fordtran & Dietschy (1966)

Cell or tissueHuman red blood cellsBeef red blood cellsDog red blood cellsHuman red blood cellsHuman red blood cellsNecturus kidney slicesNectwus kidney slices+ADHSquid giant axonCat spinal motoneuroneToad skinToad skin+ADHRat intestineFrog muscle fibresFrog gastric mucosaHuman ileumHuman jejunum

Radius of pore (A.)

3-54 i7-4

3S~4-24-2S-66-S425

2-85-3-254-56-54-0

4-°3-°-4-5

3-4T5

are remarkably similar for all these different membranes (Table 2). The term' effective'implies that these estimates apply to an idealized membrane containing 'uniform rightcylindrical' pores. Living membranes obviously do not conform to such rigid specifica-tions. Instead of being considered as a fixed sieve-like structure, the cell membraneshould be regarded more as a dynamic structure in delicate equilibrium with itssurroundings. This dynamic state of the membrane is reflected in its ability to undergosudden and dramatic alterations in pore size, e.g. during synaptic transmission orduring the action of vasopressin on various excretory epithelia. Therefore, althoughthe term 'pore' has become rather a statistical concept, it is a convenient means ofdescribing the permeability characteristics of cell membranes.

Evidence for pores in the membranes of Malpighian tubule cells depends on theobservation that rate of urine formation is related to anion size (phosphate was theonly exception but the possible existence of a separate transport mechanism for thissubstance will be discussed later). The ability of tubule cells to transport a variety ofanions with widely different chemical characteristics argues against the presence ofa specific carrier mechanism for chloride. Since the demarcation between penetratingand non-penetrating anions occurred between acetate and sulphate, a rough estimateof the effective pore radius would be about 36 A., which is well within the range ofpore sizes found in other cells (Table 2). Since the radius of water molecules (1*5 A.) is

24 M. J . BERRTOGE

considerably less than that of the pores, water presumably passes through the samechannels as do anions.

In contrast to the pore theory outlined above, a specific carrier-mediated mechanismis probably involved in phosphate transport by Malpighian tubules. Despite its largesize, phosphate can support urine formation more effectively than any other aniontested. Although the mechanism of phosphate transport across cell membranes is littleunderstood, present information suggests that carrier-mediated phosphate transportmay occur in a wide variety of cells.

Earlier studies on phosphate uptake by red blood cells (Gourley, 1952a, b), sea-urchin eggs (Lindberg, 1950) and muscle (Causey & Harris, 1951) suggested thatphosphate may first accumulate on the cell surface and enter the cell only after par-taking in a phosphorylative process. When 3SP was added to blood cells, label appearedto a greater extent, and at an earner stage, in compounds such as ATP, ADP and2,3-diphosphoglyceric acid than in intracellular phosphate ions (Vestergaard-Bogind,1963). Some authors have even suggested that the carrier molecule might be 1,3-di-phosphoglyceric acid (Bartlett, 1958; Prankerd & Airman, 1954) or ATP (Gourley,1952a, b). The appearance of label in such molecules, however, does not necessarilyimplicate them as the actual carriers, because if glycolytic processes take place near thecell membrane, phosphate will rapidly enter the metabolic pool, especially sincephosphate turnover is rapid and the intracellular concentration of phosphate is low(Vestergaard-Bogind, 1963).

Bacteria can accumulate phosphate against steep concentration gradients by meansof a carrier mechanism which seems to be coupled to glycolysis (Goodman & Rothstein,1957). Since arsenate is the only compound which was found to inhibit the absorptionof phosphate competitively (Rothstein, 1963; Borst Pauwels, 1964), the transportprocess is probably very specific. As in red blood cells, addition of labelled phosphateresults in the rapid appearance of label in various organic molecules but the nature ofthe actual carrier has not been determined. In the case of a flagellate, Euglena, it hasbeen suggested that an induced acid phosphatase may function in phosphate transport(Blum, 1966). Rothstein & Meier (1949) and Borst Pauwels (1964), however, haveeffectively excluded such a possibility for yeast cells because they were able to inhibitthe acid phosphatase without affecting phosphate uptake.

Considerable work has also been performed on mitochondria which are relativelyimpermeable to chloride but allow phosphate to penetrate freely. This rapid trans-location of phosphate is believed to occur through an exchange-diffusion carrier whichpermits HgPO4~ ion to enter the mitochondria in exchange for OH~ (Chappel &Crofts, 1966). As in bacteria, arsenate can also inhibit phosphate uptake by mito-chondria (Chappel & Crofts, 1966; Hanson & Miller, 1967).

So far, the mode of phosphate transport has been considered only in simple systemssuch as red blood cells, bacteria and isolated mitochondria. When epithelia are con-sidered, the whole subject becomes far more complex, especially since phosphate maybecome involved in cellular metabolism during its passage across the cell. In thevertebrate kidney, phosphate excretion is achieved by glomenilar filtration and tubularreabsorption (Smith, 1956). The reabsorptive process is considered to have a definitemaximal saturation value beyond which filtered phosphate is lost in the urine. Severalfactors can modify this reabsorptive process, most notable of which is the parathyroid

Urine formation by the Malpigtuan tubules of Calliphora. / / . 25

hormone, which exerts its phosphaturic action by inhibiting phosphate reabsorptionin the proximal tubule (Samiy, Hirsch & Ramsay, 1965). Although it is well establishedthat phosphate is secreted by the renal tubules of certain fish (Grafflin, 1936; Smith,1939) and of the chicken (Levinsky & Davidson, 1957), there is some doubt concerningphosphate secretion by mammalian kidney tubules (Handler, 1962; Webster, Mann &Hills, 1967). The renal transport mechanism is inhibited by arsenate (Ginsburg &Lotspeich, 1963), which might indicate a transport mechanism similar to thosedescribed earlier.

The Malpighian tubules of Calliphora are capable of forming urine by secretingpotassium phosphate and it is postulated that the phosphate crosses the basal and apicalmembranes by means of carriers (Berridge, 1967). Attachment of phosphate to thecarrier on one side of the membrane and its release on the opposite side may involvephosphorylation and dephosphorylation respectively. This would explain how arsenateinhibits phosphate excretion, because it may compete with phosphate in a similar wayto its competition in phosphorylative reactions of intermediate metabolism (Ter Welle& Slater, 1967). The ability of arsenate to block the transport of phosphate but notchloride provides circumstantial evidence for two different mechanisms of aniontransport.

The existence of separate pathways is further substantiated by the observation thatcopper inhibits urine formation in the presence of chloride, but not in the presence ofphosphate. Although phosphate can reverse the potentiation of muscle contractioninduced by certain heavy metals (Sandow & Isaacson, 1966), it cannot protect tubulecells against the inhibitory effect of copper. Koefoed-Johnsen & Ussing (1958) origi-nally suggested that copper caused frog skin to hyperpolarize by making the cellmembranes impermeable to chloride. In molluscan neurones copper also inhibits thepassive permeability of the cell membrane to chloride (Chiarandini, Stefani &Gerschenfeld, 1967). The present study indicates that copper blocks urine formationonly in the presence of those anions which are thought to pass through pores. Copperhas little effect on the potential difference across the skin of larval salamanders wherechloride permeability is low and this anion is probably transported by a carrier (Dietz,Kirschner & Porter, 1967). Apparently, copper blocks chloride transport only whenthis occurs passively through pores in the membrane. Copper may clog up these poresby being bound to various ligands close to or within these apertures. However, ifanions and water pass through the same pores as suggested earlier, these pores cannotbe completely blocked because copper does not inhibit water transport. Perhaps whencopper is bound to various sites on the cell membrane the pores undergo subtlealterations in conformation and charge distribution which do not impede the smallerwater molecules but severely restrict the passage of chloride ions.

It now becomes feasible to integrate the results of the present investigation with theworking hypothesis on the mechanism of urine formation outlined earlier (Berridge,1967, 1968). This hypothesis was based on Ramsay's (1956) original suggestion thatpotassium transport from the blood into the lumen is the 'prime mover' during urinesecretion. In Calliphora, both the apical and basal surfaces seem to be involved inpotassium transport. Urine formation occurs if this movement of potassium is accom-panied by a movement of anions by either of the mechanisms mentioned above.Although chloride transport probably occurs passively down the electrochemical

26 M. J. BERRIDGE

gradient due to potassium transport (Ramsay, 1956), phosphate transport showscharacteristics which indicate that it might be active. This question of active versuspassive anion transport must await information concerning the electrochemicalgradients across tubule cells. Nevertheless, a net transport of electrolyte first from theoutside medium into the cell and then from the cell into the lumen could establish thelocal osmotic gradients which are necessary to promote a passive flow of water. It isfurther postulated that these local osmotic gradients are developed within the longnarrow channels formed by membrane infoldings on the basal side or by microviUi onthe luminal surface (Berridge, 1968; Berridge & Oschman, 1969).

SUMMARY

1. The ability of chloride and phosphate to support urine formation has been studiedunder different conditions. When sulphate is used as a balancing anion, rate of urineformation is linearly related to the external chloride or phosphate concentration.

2. Tubules can concentrate phosphate in the urine by a mechanism which isindependent of other anions. Phosphate U/P declines with an increase in externalphosphate concentration.

3. In the absence of phosphate, chloride is slightly concentrated in the urine.Chloride U/P is reduced by phosphate but unaffected by sulphate.

4. When an extensive series of anions was tested, rate of urine production wasrelated to the size of the anions. Phosphate was the only exception because despite itslarge size it supported the highest rate of urine production.

5. Arsenate competitively inhibits phosphate transport, resulting in an augmenta-tion of chloride transport. Copper, however, blocks urine formation in the presenceof chloride but not in the presence of phosphate.

6. The experiments indicate two separate mechanisms for anion transport. Anionssuch as chloride probably pass through pores with an estimated radius of 3-6 A.,whereas phosphate is transported by a carrier.

I would like to thank Dr B. Schmidt-Nielsen and Dr R. K. Josephson for criticallyreviewing the manuscript. This study was supported by U.S.P.H.S. grant AM-09975-03 awarded to Dr B. Schmidt-Nielsen.

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BARTLETT, G. R. (I 958). Organization of red cell glycolytic enzymes: cell coat phosphorus transfer. Aim.N.Y. Acad. Sri. U.S.A. 75, 110-14.

BERRIDGE, M. J. (1966). Metabolic pathways of isolated Malpighian tubules of the blowfly functioningin an artificial medium. J. Insect Pkytiol. 13, 1523-38.

BERRIDGE, M. J. (1967). Ion and water transport across epithelia. In Insects and Physiology, pp. 329—47.Ed. J. W. L. Beament and J. E. Treheme. Edinburgh and London: Oliver and Boyd.

BERRIDGE, M. J. (1968). Urine formation by the Malpighian tubules of CaUiphora. I. Cations. J'. exp. Biol.48, 159-74-

BERRIDGE, M. J. & OSCHMAN, J. L. (1969). A structural basis for fluid secretion by Malpighian tubules.Tissue and Cell (in the Press).

BLUM, J. J. (1966). Phosphate uptake by phosphate-starved Euglena. J. gen. Pkysiol. 49, 1125-37.

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activity of yeast and phosphate uptake. Biochim. biophys. Acta 93, 659-61.BOYLE, P. J. & CONWAY, E. J. (1941). Potassium accumulation in muscle and associated changes.

J. Pkytiol., Land. 100, 1-63.CAUSEY, G. & HARRIS, E. J. (1951). The uptake and loss of phosphate by frog muscle. Biochem. J. 49,

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chondria. In Regulation ofMetabolic Processes in Mitochondria, pp. 293-316. Ed. J. M. Tager, S. Papa,E. Quagliariello and E. C. Slater. Amsterdam: Elsevier Publishing Company.

CHIARANDINI, D. J., STEFANI, E. & GERSCHENFELD, H. M. (1967). Inhibition of membrane permeabilityto chloride by copper in molluscan neurones. Nature, Land. 313, 97—9.

COOMBS, J. S., ECCLES, J. C. & FATT, P. (1955). The specific ion conductances and the ionic movementsacross the motoneuronal membrane that produce the inhibitory post-synaptic potential. J. Physiol.,Land. 130, 326-73.

DIETZ, T. H., KIRSCHNER, L. B. & PORTER, D. (1967). The roles of sodium transport and anion permea-bility in generating transepithelial potential differences in larval salamanders. J. exp. Biol. 46, 85-96.

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