J. Exp. Biol. (1969), 50, 15-28
With 8 text-figwret
Printed in Great Britain
jr
URINE FORMATION BY THE MALPIGHIAN
TUBULES 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 transport of potassium (Ramsay, 1953, 1955; Berridge, 1967, 1968). However, potassium
transport 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 with
a non-transportable anion such as sulphate, fluid transport ceases. The mechanism
of transporting anions to accompany the active secretion of potassium has been
investigated.
MATERIAL AND METHODS
Three-day-old adult female Calliphora erythrocephala were used throughout this
study. The methods of culturing insects and setting up isolated Malpighian tubules
were similar to those described previously (Berridge, 1966).
Different anion media were obtained by adding the sodium salt of the anion to a
stock solution containing essential cations and metabolites (Table 1). The pH of all
media 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 phosphate is present in both the monobasic (20%) and dibasic (80%) forms. Since the pH
of 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.)
Compound
KOH
CaSO4.2H,O
MgSO4.7H,O
Trehalose
Glucose
Glutamine
a-Alanine
mg./ioo ml.
225
24
250
180
180
70
40
Compound
Glycine
Citric acid
Fumaric acid
Malic acid
Penicillin
Streptomycin
mg./ioo mL
SO
100
100
100
3
10
Unless indicated otherwise, rate of urine formation was measured 30—90 min. after
setting up the tubules and the urine collected over this period was used for chemical
analysis. Chloride and phosphate concentrations were determined by the Spinco/Beckman 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 determinations
and the vertical lines indicate ± twice the standard error of each mean.
In this study the abbreviation U\P has been borrowed from vertebrate physiology
to indicate the relationship between the concentration of a substance in the urine and
its concentration in the outside medium (Ramsay, 1958). In kidney physiology, the
outside medium is usually plasma, whereas, in the present study, it was an artificial
medium. Nevertheless, it will be less confusing to retain the convention of kidney
physiology 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 of
1*98 A. (Solomon, 1960a). When the effect of anion size on rate of urine formation was
tested, 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 secrete
urine when the chloride concentration of the bathing medium was varied. In order to
maintain a constant osmotic pressure, lower concentrations of chloride were obtained
by substitution with either sulphate or phosphate. In a full chloride medium urine
was 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 be
presented later (Fig. 5) which suggests that this inability of sulphate to support urine
formation is related to its large size.
In contrast to the above results, progressive replacement of chloride with phosphate
caused a considerable increase in rate of urine production (Fig. 1 a, open circles). These
changes in rate of urine formation depending on whether sulphate or phosphate
replaced chloride were reflected in marked differences in the chloride concentration
of the urine. When sulphate replaced chloride, the concentration of chloride in urine
was linearly related and slightly higher than its concentration in the bathing medium
(Fig. 1 b, closed circles). However, when chloride was replaced with phosphate, chloride
concentration in the urine was suppressed, especially at low external chloride concentrations (Fig. 1 b, open circles). The experiments to be described next indicate that the
ability of tubule cells to concentrate phosphate accounts for this displacement of
chloride from the urine.
Phosphate transport
Phosphate transport has been studied using the same experimental design as that
just described for chloride transport. A range of phosphate concentrations was obtained
by progressive substitution with either sulphate or chloride.
When sulphate replaced phosphate in the bathing medium, rate of urine formation
was 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 with
chloride had no effect on rate of urine production but thereafter the rate decreased
towards the level of tubules functioning in a full chloride medium (Fig. id).
10
140 equiv./
o
X
1/
120
/
8
in urme I
S
o
g
o
1
60
80
100
120
Chloride concentration in medium
(m-equiv./L)
80
i
§
o
40
c
o
2
40
100
60
8u
20
/
o
20
:
/{
l
A
Ay
- / /
—<!>T^"
1
1
1
1
1
20
40
60
80
100
120
Chloride concentration in medium
(m-equiv./l.)
(a)
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 the
medium. 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 concentration
in the urine did not differ depending on the presence of sulphate (closed circles) or
chloride (open circles). On the other hand, we have just seen that chloride concentration in the urine was suppressed to a greater extent in the presence of phosphate than
in the presence of sulphate (Fig. ib).
This difference between phosphate and chloride transport is shown more clearly if
the UjP ratios for these two anions are plotted against their concentration in the
bathing medium. Figure 3 illustrates that the mechanism concentrating phosphate in
the urine is not altered by the presence of chloride or sulphate. Although the U/P
ratio decreases with an increase in external phosphate concentration, the ratio remains
significantly above 1 throughout the range of phosphate concentration employed.
The presence of an impermeant anion (sulphate) as compared to a permeant anion
2
Exp. BioL 50, 1
i8
o
M. J. BERRDDGE
10
-
9
-
10 h
2
8
7
7
6
6
e 5
o
5
I
a
o
c
8
•a
fi
4
3
4
3
"C
u
2
I
2
a
1
I
0
1 2
3
4
5
6
7
0
1
2
3
4
5
6
7
8
Phosphate concentration in medium (fig. POJfiL)
Phosphate concentration in medium (jig.
(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 the
medium. In both cases, osmotic pressure of the bathing medium was adjusted with sulphate ( • )
or chloride (O).
1-2
26 -
.
.
2-4 "
2-2 -
o
o
iate
a,
b 20
18 -
8
1-fr _
1
OH
08
o
X
0-6.
(
6
0-4
/o
/
o
o
/
o
o
14 -
o
o
1-2 -
0-2
I
n.n
0
5
~*~~/^
/
_
10
•
1
2
3
4
I
5
6
I
1
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 bathing
medium. Data obtained from Fig. zb where phosphate was balanced against sulphate ( • ) or
chloride (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 concentration was varied by replacement with sulphate the U/P ratio for chloride remained
constant ( I - O - I - I 6 ) . In the presence of phosphate, however, chloride movement was
suppressed and the U/P ratio declined to a low level as phosphate progressively
replaced chloride in the bathing medium.
12 r-
10
NO,"'
Br-<
a• CIO33
ao4-
•a
2
a
o
o
<>HPO4*
' SCN"
-4
I
HCCV
CH,COOCH,CH 2 COO1
2
3
4
5
Citrate
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 chloride
and phosphate transport. The former probably occurs by passive diffusion down the
electrical gradient created by the active secretion of potassium. The simplest hypothesis
would be that chloride permeates through aqueous-filled channels or pores in the celJ
membranes. It seems unlikely that phosphate will move through such pores because
it has a much larger hydrated radius than chloride, or even sulphate, yet it is capable
of maintaining a high rate of urine formation. Consequently, it is necessary to postulate a carrier-mediated transport mechanism to account for this high level of phosphate
transport. Such a carrier mechanism is consistent with the observation that tubule
cells can concentrate phosphate in the urine. Further experiments were devised to test
the 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, rate
of urine formation should be related to the size of the permeating anions. In order to
test this, rate of urine formation was measured in the presence of anions with different
hydrated radii. Figure 5 shows that a wide variety of anions can support urine formation 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 in
artificial medium containing the anion to be studied; after a h., copper was added to six
tubules 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. Phosphate
was 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 was
higher than for any other anion (Fig. 5). The result obtained with the divalent form
must be accepted with reservation because the alkaline environment resulted in the
formation of a flocculent precipitate of calcium and/or magnesium phosphate which
could have reduced rate of urine formation (Berridge, 1968). Therefore, the ability of
tubules to secrete urine in the presence of divalent phosphate ions is even more remarkable because this anion has a hydrated radius considerably larger than sulphate
and propionate which could not support urine formation. These observations strengthen
the hypothesis that phosphate transport involves a specific carrier mechanism to facilitate 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 between
the two mechanisms by which anions move across the cell. Arsenate, which has a
molecular configuration similar to that of phosphate, will inhibit phosphate transport
but not chloride transport. And indeed, competitive inhibition of phosphate transport
by arsenate resulted in a considerable augmentation of chloride transport. When
arsenate (1 x io" 8 M) was added to tubules in the presence of both anions, the phosphate
UjP declined from 4-1 to 1-7, whereas the chloride U/P increased from 0-37 to 0-84.
2a
12
18
16
10
I 14
12
o
Is
10
a
u
50
100
150
200
250
300
40
80
120
Minutes
Minutes
Fig. 7
Fig. 8
160
200
240
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 in
the bathing medium for the period between the arrows (60-140 min.). At 140 min., six tubules
0).
were placed in a phosphate medium (O — O) and six tubules in a chloride medium (©
Fig. 8. Copper inhibition of chloride transport in the presence of phosphate. Six tubules were
set up in a phosphate medium containing 1 x io~* M copper ( O — O) and six tubules were set
up in a copper-free phosphate medium ( •
• ) . Both groups of tubules were placed in a
copper-free chloride medium at 90 min. (arrow).
The action of copper on rate of urine formation also suggests the possibility of two
separate pathways for anion transport. When tubules were set up in a phosphate
medium, copper (1 x io~* M) had no effect on rate of urine production (Fig. 6). On the
other hand, tubules utilizing anions such as chloride or nitrate were inhibited by
copper and rate of urine formation declined after addition of this heavymetal. Complete
inhibition 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 are
placed in a phosphate medium (Fig. 7). In this experiment twelve tubules were set up
in a chloride medium and allowed to secrete urine for 60 min., at which time copper
was introduced into the bathing medium resulting in a characteristic reduction of
urine flow. At 140 min. copper was removed and six tubules were placed in a chloride
medium and six tubules in a phosphate medium. Inhibition of urine formation
remained in those tubules placed in the chloride medium but was reversed in the
phosphate medium (Fig. 7). Urine formation was possible in the phosphate medium
because this anion could utilize its carrier mechanism, which is apparently unaffected
by 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 by
binding this heavy metal. The following experiment, however, clearly indicates that
copper inhibition of chloride transport can develop even in the presence of a high
concentration of phosphate (Fig. 8). Malpighian tubules were set up in a phosphate
medium containing copper and, as noted earlier (Fig. 6), rate of urine formation
resembled that of the control tubules functioning in a phosphate medium minus
copper. 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 had
previously been treated with copper, indicating that this heavy metal can inhibit the
chloride transport mechanism despite a high concentration of phosphate. Control
tubules showed a slight decrease in rate of urine formation in the chloride medium
because 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 of
chloride or phosphate therefore further suggest that transport of these two anions
involves separate mechanisms.
DISCUSSION
Rate of urine formation by the Malpighian tubules of CaUiphora depends on the
availability of anions to accompany the active secretion of potassium. Since the plasma
membrane of cells is lipoidal, special structural features must be present to allow
anions to pass through it. In CaUiphora, anions seem to permeate the membranes of
tubule 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 anion
transport, but also lead us to a better understanding of membrane structure and
function.
Since Boyle & Conway (1941) postulated the presence of pores in the surface membrane of muscle fibres, the sieve-like action of the cell membrane towards ions has
received considerable verification (Solomon, 19606). The exterior and interior media
of many cells seem to communicate through pores, and small ions pass through the
membrane, whereas those larger than the pore size are all non-penetrating. For
example, the inhibitory post-synaptic potential (I.P.S.P.) develops when the inhibitory
transmitter substance converts the post-synaptic membrane into a selective ionic
sieve. Coombs, Eccles & Fatt (1955) tested the ability of different anions to support
the 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 have
substantiated the 'pore-structure' hypothesis of the activated post-synaptic membrane (Araki et al. 1961; Ito, Kostyuk & Oshima, 1962; Kerkut & Thomas, 1964).
Araki et al. (1961) found that separation between penetrating and non-penetrating
anions occurred between chlorate and bromate and hence they estimated that the
effective pore radius of the activated inhibitory post-synaptic membrane of cat motoneurones lay between the hydrated sizes of these two anions, i.e. 2-85-3-25 A. respectively. 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 wide
variety of membranes. Despite the different techniques used, the effective pore sizes
Table 2. The effective radii of pores in different membranes
Cell or tissue
Human red blood cells
Beef red blood cells
Dog red blood cells
Human red blood cells
Human red blood cells
Necturus kidney slices
Nectwus kidney slices+ADH
Squid giant axon
Cat spinal motoneurone
Toad skin
Toad skin+ADH
Rat intestine
Frog muscle fibres
Frog gastric mucosa
Human ileum
Human jejunum
Radius of pore (A.)
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)
35
4i
7-4
3S~4-2
4-2
S-6
6-S
425
2-85-3-25
4-5
6-5
4-0
4-°
3-°-4-5
3-4
T5
are remarkably similar for all these different membranes (Table 2). The term' effective'
implies that these estimates apply to an idealized membrane containing 'uniform right
cylindrical' pores. Living membranes obviously do not conform to such rigid specifications. Instead of being considered as a fixed sieve-like structure, the cell membrane
should be regarded more as a dynamic structure in delicate equilibrium with its
surroundings. This dynamic state of the membrane is reflected in its ability to undergo
sudden and dramatic alterations in pore size, e.g. during synaptic transmission or
during the action of vasopressin on various excretory epithelia. Therefore, although
the term 'pore' has become rather a statistical concept, it is a convenient means of
describing the permeability characteristics of cell membranes.
Evidence for pores in the membranes of Malpighian tubule cells depends on the
observation that rate of urine formation is related to anion size (phosphate was the
only exception but the possible existence of a separate transport mechanism for this
substance will be discussed later). The ability of tubule cells to transport a variety of
anions with widely different chemical characteristics argues against the presence of
a specific carrier mechanism for chloride. Since the demarcation between penetrating
and non-penetrating anions occurred between acetate and sulphate, a rough estimate
of the effective pore radius would be about 36 A., which is well within the range of
pore 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 same
channels as do anions.
In contrast to the pore theory outlined above, a specific carrier-mediated mechanism
is probably involved in phosphate transport by Malpighian tubules. Despite its large
size, phosphate can support urine formation more effectively than any other anion
tested. Although the mechanism of phosphate transport across cell membranes is little
understood, present information suggests that carrier-mediated phosphate transport
may occur in a wide variety of cells.
Earlier studies on phosphate uptake by red blood cells (Gourley, 1952a, b), seaurchin eggs (Lindberg, 1950) and muscle (Causey & Harris, 1951) suggested that
phosphate may first accumulate on the cell surface and enter the cell only after partaking in a phosphorylative process. When 3SP was added to blood cells, label appeared
to a greater extent, and at an earner stage, in compounds such as ATP, ADP and
2,3-diphosphoglyceric acid than in intracellular phosphate ions (Vestergaard-Bogind,
1963). Some authors have even suggested that the carrier molecule might be 1,3-diphosphoglyceric acid (Bartlett, 1958; Prankerd & Airman, 1954) or ATP (Gourley,
1952a, b). The appearance of label in such molecules, however, does not necessarily
implicate them as the actual carriers, because if glycolytic processes take place near the
cell membrane, phosphate will rapidly enter the metabolic pool, especially since
phosphate turnover is rapid and the intracellular concentration of phosphate is low
(Vestergaard-Bogind, 1963).
Bacteria can accumulate phosphate against steep concentration gradients by means
of 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 absorption
of phosphate competitively (Rothstein, 1963; Borst Pauwels, 1964), the transport
process is probably very specific. As in red blood cells, addition of labelled phosphate
results in the rapid appearance of label in various organic molecules but the nature of
the actual carrier has not been determined. In the case of a flagellate, Euglena, it has
been suggested that an induced acid phosphatase may function in phosphate transport
(Blum, 1966). Rothstein & Meier (1949) and Borst Pauwels (1964), however, have
effectively excluded such a possibility for yeast cells because they were able to inhibit
the acid phosphatase without affecting phosphate uptake.
Considerable work has also been performed on mitochondria which are relatively
impermeable to chloride but allow phosphate to penetrate freely. This rapid translocation of phosphate is believed to occur through an exchange-diffusion carrier which
permits HgPO4~ ion to enter the mitochondria in exchange for OH~ (Chappel &
Crofts, 1966). As in bacteria, arsenate can also inhibit phosphate uptake by mitochondria (Chappel & Crofts, 1966; Hanson & Miller, 1967).
So far, the mode of phosphate transport has been considered only in simple systems
such as red blood cells, bacteria and isolated mitochondria. When epithelia are considered, the whole subject becomes far more complex, especially since phosphate may
become involved in cellular metabolism during its passage across the cell. In the
vertebrate kidney, phosphate excretion is achieved by glomenilar filtration and tubular
reabsorption (Smith, 1956). The reabsorptive process is considered to have a definite
maximal saturation value beyond which filtered phosphate is lost in the urine. Several
factors 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 reabsorption
in the proximal tubule (Samiy, Hirsch & Ramsay, 1965). Although it is well established
that 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 concerning
phosphate 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 those
described earlier.
The Malpighian tubules of Calliphora are capable of forming urine by secreting
potassium phosphate and it is postulated that the phosphate crosses the basal and apical
membranes by means of carriers (Berridge, 1967). Attachment of phosphate to the
carrier on one side of the membrane and its release on the opposite side may involve
phosphorylation and dephosphorylation respectively. This would explain how arsenate
inhibits phosphate excretion, because it may compete with phosphate in a similar way
to its competition in phosphorylative reactions of intermediate metabolism (Ter Welle
& Slater, 1967). The ability of arsenate to block the transport of phosphate but not
chloride provides circumstantial evidence for two different mechanisms of anion
transport.
The existence of separate pathways is further substantiated by the observation that
copper inhibits urine formation in the presence of chloride, but not in the presence of
phosphate. Although phosphate can reverse the potentiation of muscle contraction
induced by certain heavy metals (Sandow & Isaacson, 1966), it cannot protect tubule
cells against the inhibitory effect of copper. Koefoed-Johnsen & Ussing (1958) originally suggested that copper caused frog skin to hyperpolarize by making the cell
membranes impermeable to chloride. In molluscan neurones copper also inhibits the
passive permeability of the cell membrane to chloride (Chiarandini, Stefani &
Gerschenfeld, 1967). The present study indicates that copper blocks urine formation
only in the presence of those anions which are thought to pass through pores. Copper
has little effect on the potential difference across the skin of larval salamanders where
chloride permeability is low and this anion is probably transported by a carrier (Dietz,
Kirschner & Porter, 1967). Apparently, copper blocks chloride transport only when
this occurs passively through pores in the membrane. Copper may clog up these pores
by being bound to various ligands close to or within these apertures. However, if
anions and water pass through the same pores as suggested earlier, these pores cannot
be completely blocked because copper does not inhibit water transport. Perhaps when
copper is bound to various sites on the cell membrane the pores undergo subtle
alterations in conformation and charge distribution which do not impede the smaller
water molecules but severely restrict the passage of chloride ions.
It now becomes feasible to integrate the results of the present investigation with the
working hypothesis on the mechanism of urine formation outlined earlier (Berridge,
1967, 1968). This hypothesis was based on Ramsay's (1956) original suggestion that
potassium transport from the blood into the lumen is the 'prime mover' during urine
secretion. In Calliphora, both the apical and basal surfaces seem to be involved in
potassium transport. Urine formation occurs if this movement of potassium is accompanied 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 shows
characteristics which indicate that it might be active. This question of active versus
passive anion transport must await information concerning the electrochemical
gradients across tubule cells. Nevertheless, a net transport of electrolyte first from the
outside medium into the cell and then from the cell into the lumen could establish the
local osmotic gradients which are necessary to promote a passive flow of water. It is
further postulated that these local osmotic gradients are developed within the long
narrow channels formed by membrane infoldings on the basal side or by microviUi on
the luminal surface (Berridge, 1968; Berridge & Oschman, 1969).
SUMMARY
1. The ability of chloride and phosphate to support urine formation has been studied
under different conditions. When sulphate is used as a balancing anion, rate of urine
formation is linearly related to the external chloride or phosphate concentration.
2. Tubules can concentrate phosphate in the urine by a mechanism which is
independent of other anions. Phosphate U/P declines with an increase in external
phosphate 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 was
related to the size of the anions. Phosphate was the only exception because despite its
large size it supported the highest rate of urine production.
5. Arsenate competitively inhibits phosphate transport, resulting in an augmentation of chloride transport. Copper, however, blocks urine formation in the presence
of chloride but not in the presence of phosphate.
6. The experiments indicate two separate mechanisms for anion transport. Anions
such 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 critically
reviewing the manuscript. This study was supported by U.S.P.H.S. grant AM-0997503 awarded to Dr B. Schmidt-Nielsen.
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motoneurone membrane. J. PkysioL, Lond. 159, 410-35.
BARTLETT, G. R. (I 958). Organization of red cell glycolytic enzymes: cell coat phosphorus transfer. Aim.
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