349
Journal of Cell Science 101, 349-361 (1992)
Printed in Great Britain © The Company of Biologists Limited 1992
Transport of the cationic fluorochrome rhodamine 123 in an insect's
Malpighian tubule: indications of a reabsorptive function of the secondary
cell type
W. MEULEMANS and A. De LOOF
Zoological Institute of the University of Leuven, Naamsestraat 59, B-3000 Leuven, Belgium
Summary
The pathway of rhodamine 123 was examined after
injection into Sarcophaga flies and after in vitro labeling
of the Malpighian tubules. After in vitro labeling the
primary cells only retained this potential-sensitive dye
for a short period while all secondary cells accumulated
the dye from the tubule lumen. In vivo the secondary
cells also accumulated rhodamine 123 from the lumen,
but the primary cells in the distal parts of all four tubules
retained the dye for prolonged periods. This was most
pronounced in the distal part of the anterior Malpighian
tubules, where rhodamine 123 was eventually precipitated on the luminal concretions. Rhodamine 123
initially accumulated in the secondary cell mitochondria
and eventually in intensely fluorescing vesicles, probably
lysosomes. No evidence for endocytotic processes from
the lumen was found using Lucifer Yellow CH,
fluorescent dextrans and fluorescent albumin. Prior
incubation with the ionophores valinomycin, nigericin,
CCCP (all 1 pg/ml), dinitrophenol (1 mM) and NaN3
(10~ 2 M) inhibited the selective accumulation of rhodamine 123 to a large extent while monensin (1-5 /ig/ml)
showed little inhibitory effect. Furthermore, only cationic and no anionic or neutral dyes were accumulated
by the secondary cells. In the fleshfly Calliphora and the
fruitfly Drosophila, the dye rhodamine 123 also selectively accumulated in the secondary cells, as well in vitro
as in vivo.
Introduction
has been described (Lison, 1937; Maddrell et al., 1974;
Bresler et al., 1990). The excretion of basic dyes like
neutral red and methylene blue (Lison, 1938; Maddrell,
1977) in some insect Malpighian tubules has been linked
to the capability of these tubules to actively excrete
toxic alkaloids like nicotine, atropine and morphine,
which are also organic bases (Maddrell, 1977).
In this paper we present evidence that in vitro and in
vivo the secondary (S) cells of larval and adult
Malpighian tubules of some Diptera (Brachycera) are
accumulating membrane-permeant cationic dyes but
not anionic or neutral dyes from the luminal fluid.
Furthermore, in the distal part of the anterior Malpighian tubules rhodamine 123 was precipitated on the
luminal concretions.
Besides the Malpighian tubules of the fleshfly
Sarcophaga bullata, some comparative investigations
were also conducted with the Malpighian tubules of the
fruitfly Drosophila melanogaster and a related fleshfly,
Calliphora erythrocephala.
The Malpighian tubules are the so-called 'kidney
tubules' of the insects. Extensive research has been
devoted to the study of the mechanisms underlying the
primary urine secretion by Malpighian tubules (Maddrell, 1978a; Bradley, 1985; Maddrell and Overton,
1990). It has been shown that along with the large
amount of fluid passing through or in between the cells,
many small molecules also enter the primary urine
(Maddrell and Gardiner, 1974; Bradley, 1985). Small
essential molecules like amino acids or sugars leaking
into the lumen would then be reabsorbed along their
passage through the long Malpighian tubules or in parts
of the gut such as the rectum. Until now, only an active
reabsorption of glucose from the primary urine has
been described in the Malpighian tubules of some
insects (Knowles, 1975; Rafaeli-Bernstein and Mordue,
1979). Direct uptake of any molecule by a particular cell
type in the Malpighian tubules has not been described.
Coloured organic molecules have often been used to
characterize the excretory systems of insects. Acidic
organic molecules like fluorescein, indigocarmine and
amaranth are actively cleared from the haemolymph by
the Malpighian tubules, but no accumulation in the cells
Key words: Malpighian tubules, dye transport,
reabsorption, secondary cells, rhodamine 123.
Materials and methods
Animals
Sarcophaga bullata (Parker) was reared as described by
350
W. Meulemans and A. De Loof
Huybrechts and De Loof (1977). In our experiments, male
and female flies of day 3 were used, unless mentioned
otherwise. Drosophila melanogaster was reared as described
by Callaerts et al. (1988). The wild type of Calliphora
erythrocephala was caught in Leuven and reared as described
for Sarcophaga.
Supravital staining of the Malpighian tubules
In vitro staining of the tubules
The Malpighian tubules were carefully dissected in Ringer's
solution (adapted from Chan and Gehring (1971), in mM: 120
NaCl, 10 KC1, 15 MgSO4, 5 CaCl2, 10 Tricine, 20 glucose, 50
sucrose, pH 6.8). In this solution the tubules could actively
secrete fluid for at least 24 hours without oxygen supply, as
was measured with the in vitro bio-assay according to Ramsay
(1954). Some control experiments were conducted in a
glucose-free bicarbonate buffer (Sarcophaga Ringer's (in
mM): 121.5 NaCl, 10 KC1, 1 NaH2PO4, 10 NaHCO3, 0.7
MgCl2, 2.2 CaCl2, pH 6.8, aerated with 95% Oj/5% CO2),
according to Verachtert and De Loof (1988).
After staining and rinsing in Ringer's solution, the tubules
were transferred to a hollow glass slide in a few drops of
Ringer's fluid, and covered with a coverglass. In this covered
slide the tubule cells remained viable for many hours (cell
viability tested by Trypan blue exclusion; Ashburner, 1989).
For immediate photography of these whole-mounts, a flat
glass slide with two glass bridges was used.
The fluorescently stained tubules were observed under a
Leitz Laborlux S fluorescence microscope, using the rhodamine and fluorescein excitation filters. Rhodamine 123,
Lucifer Yellow CH, sulphorhodamine B, FTTC-labeled dextrans of 4.4 and 35 kDa and a TRTTC-labeled dextran of 10
kDa were purchased from Sigma. Bovine serum albumin
(Serva) was conjugated with FITC (Sigma) as described by
Haaijman (1983). Eosins Y and B and rhodamine 6G were
purchased from Merck; Pyronin Y(G) was purchased from
Serva; merocyanin 540 from Fluka; and fluorescein (sodium)
from UCB. Fluorescence was recorded using Kodak T MAX
400 ASA film.
observed under the fluorescence microscope. Larvae were
also injected with 100 /ig/ml.
In vitro application of drugs and antibiotics
The drugs and antibiotics were tested at different concentrations for 5-120 minutes on Malpighian tubules in vitro. The
following provided the best minimum incubation time and
lowest concentration for a clearly detectable effect: valinomycin (Janssen Chimica, Beerse, Belgium), 30 min in 1 ^g/ml;
nigericin (Sigma), 30 min in 1 jug/ml; monensin (Serva), up to
120 min in 5 ^g/ml; CCCP (Serva), 30 min in 1 ^M; 2,4-DNP
(Serva), 30 min in 1 mM; sodium azide (Merck), 30 min in
10~2 M. Whenever appropriate, the same concentration of
the primary solvent acetone or ethanol was included in the
Ringer's solution in the control conditions (max. 0.5%). Only
tubules with viable cells were used in the experiments.
Results
General morphology and terminology
The fleshfly Sarcophaga bullata (like most other
Diptera) contains 4 Malpighian tubules (Fig. 1), 2
oriented anteriorly (anterior Malpighian tubules or
AMT) and 2 oriented posteriorly (posterior Malpighian
tubules or PMT) in the abdomen. Each AMT contains a
morphologically distinct distal part (dAMT), while the
In vitro injection of fluorochromes into the lumen
The lumen of the proximal half of a freshly dissected tubule
was perfused with a short pulse of dye solution by means of
small glass capillaries with an outer tip diameter of 25-40 /zm.
The dye containing Ringer's solution was injected into the
lumen until the coloured fluid was seen to leave the other
open tubule end. After 1 to 5 minutes of incubation the lumen
was rinsed with Ringer's solution and the tubule was mounted
in fresh Ringer's fluid in a hollow slide, and covered with a
coverglass.
In vitro transport of fluorochromes in the lumen
For these experiments, whole Malpighian tubules were set up
between a fluorochrome solution and a dye-free Ringer's
solution in two separate bathing droplets under paraffin oil
(see Maddrell and Overton, 1990).
Intravital staining of the Malpighian tubules
In these experiments 2 )A of rhodamine 123 (100 ^g/ml) or
other dyes in Ringer's fluid (0.1-1 mg/ml) were injected into
the thorax of adult Sarcophaga bullata by means of small glass
capillaries with a tip diameter of about 100 /an. In each
experiment a control group, injected with Ringer's solution
alone, was kept under identical conditions. After the proper
incubation time, the tubules of experimental and control flies
were removed in Ringer's fluid, and were immediately
Fig. 1. General morphology and orientation of Malpighian
tubules in Sarcophaga bullata (after Wessing and
Eichelberg, 1969). Notice the swollen distal part (d) of the
anterior Malpighian tubule (A) and the morphologically
homogeneous posterior Malpighian tubule (P), which
resembles the proximal part (p) of the anterior tubule (M,
midgut, H, hindgut).
Dye transport in insect Malpighian tubules
PMT is morphologically homogeneous and similar to
the proximal part of the AMT (pAMT) (see Fig. 1).
Each tubule consists of two cell types, the primary (P)
and the secondary (S) cell type. Although in dipteran
Malpighian tubules most of the S cells have a stellate
cell shape, we prefer naming them secondary instead of
stellate cells, since similar cell types in other insect
groups are not stellate (Bradley, 1985). In comparison
to the P cells the smaller S cells in Diptera have shorter
microvilli, which contain little or no mitochondria, and
they have a smaller nucleus and a transparent cytoplasm containing little or no granular deposits.
Alkassis and Schoeller-Raccaud (1984) named the
cells in the distal part of the AMT of larval Calliphora
erythrocephala differently. We suggest that these cells
are also named S and P cells, since as well as some
differences the cells in distal and proximal tubule parts
have some apparent characteristics in common: a small
nucleus and a transparent cytoplasm in all S cells
compared to a granular cytoplasm containing a large
nucleus in all P cells. Furthermore, a very similar Factin cytoskeleton was found in the S and P cells in both
parts of the AMT (Meulemans and De Loof, 1990a,b).
We will however always indicate whether the cells are
found in the distal or proximal part of the AMT.
Supravital and intravital labeling of the Malpighian
tubules with rhodamine 123 and other fluorochromes
Rhodamine 123 (Rh 123) is a very fluorescent rhodamine derivative that is known to be non-toxic to
vertebrate cells after a short application of 10 ^g/ml
(Johnson et al., 1980, 1981; Chen, 1989). This fluorochrome is known to be attracted by the high potential
gradient over the mitochondrial membranes in living
cells. Experiments with the ion-selective ionophore
nigericin (K + /H + exchanger) showed that it is the
potential difference across the mitochondrial membranes rather than the pH gradient that attracts the dye
molecules (Johnson et al., 1981).
Supravital labeling of the Malpighian tubules
In vitro labeling
Posterior tubules. In a posterior Malpighian tubule
stained in vitro in 5 jig/ml Rh 123 for 10 minutes and
washed with the Ringer's solution for 5 minutes, the
mitochondria were well labeled. All P cells displayed an
intense labeling while the S cells appeared largely
unstained (Fig. 2A). Because of the granular cytoplasm
and the extensive labeling of the P cells, separate
mitochondria could not be detected. But the high
amount of mitochondria in the P cell microvilli was
clearly recognized as an intense apical zone upon these
cells. This was most obvious when looking at the P cell
brush border through the transparent S cells. Also, due
to the intensive staining of the underlying P cells the
mitochondria in the S cells, which were poorly labeled,
were hard to detect.
If such a living Rh 123-stained tubule was left in the
Ringer's solution for about two hours, an entirely
different staining pattern was found. Only the S cells
were labeled very intensely while the previously well
351
labeled P cells had lost most of the fluorochrome (Fig.
2B). Even the P cells in the most distal part of the PMT
lost their fluorescence after two hours in vitro. Separate
mitochondria were clearly recognized as tubular vesicles throughout the entire S cell. This pattern resembled that described for vertebrate cultured cells
(Johnson et al., 1980). At first this selective staining of
the S cells was thought to be an artefact caused by the
prolonged incubation of the tubule in the covered
hollow slide. However, when the same tubule was again
stained in a fluorochrome-containing Ringer's solution
the P cells were labeled as intensely as after the first
staining. So their mitochondria did not lose the ability
to accumulate Rh 123. The dye must have leaked from
the P cells to the lumen.
When such a tubule with brightly labeled S cells was
left in the Ringer's solution for another hour or longer,
large round vesicles were formed (Fig. 2D). Concomitantly the typical mitochondrial pattern disappeared.
Possibly these are enlarged, poisoned mitochondria or
lysosomal vesicles that finally accumulated the dye
molecules.
Anterior tubules. Immediately after in vitro labeling all
P cells in the AMT were brightly labeled, even those in
the distal part of the AMT. As in the PMT, the S cells
only displayed minor fluorescence. When a tubule was
left in the Ringer's solution for two hours, even the S
cells in the distal part of the AMT selectively accumulated the dye (Fig. 2C). After prolonged incubation in
Ringer's solution, the S cells also displayed bright
round vesicles in both distal and proximal parts of the
AMT. The results of the in vitro staining of both AMT
and PMT are summarised in Table 1.
In vitro injection into the Malpighian tubule lumen
Posterior tubules. To test whether the S cells had indeed
accumulated the dye from the luminal cavity, proximal
parts of posterior Malpighian tubules were luminally
injected with a 10 /Ug/ml solution of Rh 123 in Ringer's
fluid. After incubating for 1 or 2 minutes, it was obvious
that only the S cells had accumulated the fluorochrome;
the P cells were not labeled (Fig. 2E). It was even
unnecessary to rinse the lumen, since little dye
remained in the luminal cavity. If the same tubule was
then labeled at its haemolymph side, the P cells were
brightly stained. They did not lose their ability to take
up the Rh 123 but their apical membrane seemed to be
quite impermeable to this cationic fluorochrome. If Rh
123 (10 ^g/ml) was injected into the lumen of the most
distal part of the PMT, no selective accumulation in the
S cells was noticed. The P cells accumulated most of the
dye while the S cells only displayed minor labeling. So
only the more proximal S cells accumulated the dye
from the lumen in these posterior tubules. The apical
membranes of the P cells in the distal part of the PMT
seem to be permeable to this cationic dye, while the
proximally located P cells were largely impermeable.
Anterior tubules. Injection of the dye solution into the
lumen of the proximal part of the AMT resulted in the
352
W. Meulemans and A. De Loof
Fig. 2. Malpighian tubules were stained with Rh 123 in vitro. (A) Immediately after staining for 10 min in 5 |Ug/ml; the
secondary cells (S cells) remain largely unstained; (B and C) proximal (B) and distal (C) part of AMT, 120 min in Ringer's
fluid after staining; all S cells accumulated the dye while all P cells lost it; (D) S cell, 180 min in Ringer's solution after
staining; the mitochondnal pattern has disappeared and round vesicles are found; (E) Rh 123 (10 jig/ml) was injected into
the lumen of the proximal part of a tubule; only the S cells accumulated the dye from the lumen; (F-I) the following
fluorochromes were injected into the proximal part of a tubule: 10 /ig/ml rhodamine B (F), 20 j/g/ml merocyanin 540 (G),
20 jig/ml fluorescein (H) and 20 ng/m\ pyronine Y (I). Only the latter (cationic) dye is accumulated by the S cells. The
anionic dyes (in G and H) are found in high concentrations between the P cell microvilli (arrowheads). Bars, 10 jwn, A, B,
D, F-I; 20 ion, C and E.
Dye transport in insect Malpighian tubules
353
Table 1. Relativefluorescenceintensities after in vitro labeling of adult Malpighian tubules
Time after labeling
10 min
Prox.
PMT
AMT
S cell
Pcell
S cell
Pcell
T
180 min
120 min
Dist.
+
r
Prox.
I
Dist.
4+
Prox.
Dist.
*•*
*•*
• •*
**•
The tubules were stained for 10 min in 5 /<g/ml rhodamine 123 and were then incubated for the indicated period in Ringer's fluid (AMT,
anterior Malpighian tubule; Dist., distal part; P cell, primary cell; PMT, posterior Malpighian tubule; Prox., proximal part; S cell,
secondary cell).
***The S cell is diffusely labeled, but some very intensely fluorescing vesicles are spread throughout the cell. These vesicles contain a
higher concentration of label than the previously labeled mitochondria.
same pattern: the S cells accumulated the dye from the
lumen while the P cells remained unlabeled. Injection
into the distal part of the AMT was difficult due to the
high amount of granular material in the lumen, but in
the few tubules that were successfully injected only the
S cells accumulated the dye.
In vitro transport through the Malpighian tubule
lumen
To determine if there really was any transport of dye
through the tubule lumen, we set up living posterior
tubules as a connection between the fluorochome
solution (10 ng/m\ Rh 123) and the Ringer's solution
(Maddrell and Overton, 1990). It was obvious from
these experiments that the Rh 123 was indeed transported through the cells of the distal part (lying in the
fluorochrome solution) to the lumen and that the S cells
(but not the P cells), which were more proximally
located, had accumulated the dye from the lumen. The
dye was also found to have accumulated in the S cell
mitochondria.
Intravital labeling of the Malpighian tubules
Posterior tubules
About 10 minutes after injection of Rh 123 (100 /ig/ml)
in intact flies all P cells were heavily stained, while the S
cell mitochondria were poorly labeled (Fig. 3A). After
60 minutes or longer post injection the more proximally
located S cells were labeled intensely while the P cells
had lost their fluorescence in this part (Fig. 3B). Only
the P cells in the more distal part of the PMT (Fig. 3C)
kept the dye for prolonged periods. The S cells in the
distal regions accumulated much less fluorescence
compared to those parts in which the P cells had lost the
dye. Only about 40 hours after injection into the adult,
the dye had left the primary cells of all tubules. In
contrast to the AMT, the terms proximal and distal
parts of the PMT do not refer to certain clearly
recognizable zones. Even in electron microscopical
investigations, the PMT of the related fleshfly Calliphora could not be subdivided into different regions
(Berridge and Oschman, 1969; Alkassis and SchoellerRaccaud, 1984). The 'distal part' comprises from about
one third to one fifth of the tubule length. There is a
gradual transition of stained versus non-stained P cells.
After prolonged periods post injection the length of the
distal part of the PMT that displays brightly labeled P
cells decreases and eventually all P cells have lost the
fluorochrome. Concomitantly, the S cells in such parts
display an increase in fluorescence intensity.
Granular concretions were also found in the lumen of
the PMT, but unlike those in the distal part of the AMT
(see below) they did not display any detectable
fluorescence.
After about 40 hours post injection, the S cells
displayed diffuse labeling of the whole cell instead of
the mitochondrial pattern. As occurs after prolonged in
vitro incubation of a stained tubule, most S cells
contained some very fluorescent vesicles in their
cytoplasm (Fig. 3D). These vesicles had a round shape
and they exhibited an intense fluorescence after both
fluorescein and rhodamine excitation. In the intact fly
these vesicles were smaller and they displayed a
brighter fluorescence than the in vitro formed vesicles;
they might be lysosomes that finally removed the dye
from the cell.
An identical selective accumulation in the S cells was
found after injection of Rh 123 in older flies that were
fed liver (besides water and sugar).
After injection of 2 /A of 100 jig/ml Rh 123 into thirdinstar feeding larvae, different observations were made.
The S cells also accumulated the dye from the lumen
while the P cells lost the dye after about two hours; but
in larval tubules the P cells in the distal part of the PMT
also lost the dye rather quickly, while the S cells in this
part were also well labeled.
Anterior tubules
Also in the AMT, about 10 minutes after injection of
Rh 123 (100 jig/ml) into adults all P cells were heavily
labeled, while the S cell mitochondria were poorly
labeled. After 60 minutes or longer post injection the
more proximally located S cells were labeled intensely
while the P cells had lost their fluorescence in this part
(Fig. 3B).
Only the P cells in the distal part of the AMT kept the
dye for prolonged periods. This part showed the highest
fluorescence intensity of all the tubules. The S cells in
the distal regions accumulated much less fluorescence
than those of the proximal parts. But after prolonged
354
W. Meulemans and A. De Loof
Fig. 3. Injection of Rh 123 into intact flies: (A) 30 min after injection; (B) 24 hours after injection; proximal part of the
PMT showing selective accumulation in the S cell mitochondria; (C) same fly 24 hours after injection; more distal part of
the PMT showing P cells that retain dye longer while the S cells (S) appear largely unlabeled; (D) 30 hours after injection,
showing intensely fluorescing round vesicles in the S cells; (E) distal part of the AMT 30 hours after injection; the lumen
contains brightly labeled concretions while the cells appear unstained; (F) granules taken from the lumen of the distal
AMT 30 hours after injection, showing a rim of Rh 123 around most granules; (G) 90 hours after injection; Rh 123 is
found in the centre of most granules. Bars, 5 /m\, F and G; 10 ^m, B-D; 20 /.im, A and E.
Dye transport in insect Malpighian tubules
355
Table 2. Relative labeling intensities of the different cell types after Rh 123 injection
Time after injection
10 min
Prox.
PMT
AMT
Scell
P cell
Scell
Pcell
40h
60 min
Dist.
+
r
Prox.
Dist.
+
+
++
r
Prox.
Dist.
••**
***•
*•+•
2 jA of Rh 123 at 100 /ig/ml was injected into adults. See Table 1 legend for abbreviations.
"**S cells are diffusely labeled but contain some very fluorescently labeled vesicles. These vesicles are smaller but intenser as those
found 180 min after in vitro labeling.
periods post injection, even the mitochondria in some S
cells in the distal AMT were labeled more intensely
than neighbouring P cells. At only about 40 hours after
injection into the adult, the dye had left the primary
cells of all tubules.
At about the moment the S cells started losing their
mitochondrial pattern, the so-called concretions
(Brown, 1982; Sohal et al., 1976) in the lumen of the
distal part of the AMT were fluorescently labeled (Fig.
3E). When comparing the granular deposits in the distal
part of the AMT lumen with those of control flies, it was
obvious that the concretions in the dye-injected flies
contained the dye rhodamine 123 in a rim around their
periphery (Fig. 3F). After about 90 hours post
injection, the concretions were still labeled, but now the
fluorescence was found in the central part of the
concretions (Fig. 3G). These were preformed granules
upon which non-fluorescent material had been deposited in the lumen. The latter granules were clearly
larger than those in earlier stages. These granules have
also been seen to descend along the lumen of the
proximal part of the AMT.
After about 40 hours post injection, the S cells in the
AMT also displayed a rather diffuse staining of the
whole cell instead of the mitochondrial pattern. As in
the posterior adult tubules, most S cells contained some
very fluorescent vesicles in their cytoplasm (Fig. 3D).
All the different processes in intact flies could not be
accurately timed, since the injected flies showed great
variation in their speed of dye clearance. Some flies
cleared the dye from all their cells in 40 hours, while in
some other flies brightly labeled P cells could still be
found at this time. All Rh 123-injected flies survived as
well as the controls injected with Ringer's solution.
After injection of Rh 123 into third-instar feeding
larvae, the S cells of the proximal part of the AMT were
brightly labeled after about two hours. In contrast to
the adult, neither the P cells nor the S cells in the distal
part of the AMT accumulated any dye: two hours after
injection both S and P cells appeared unstained. So in
larvae, most P cells had lost the dye after some time
while all S cells (except those in the distal part of the
AMT) had accumulated the dye in their mitochondria.
No deposition of dye was found in concretions in the
lumen of the distal part of larval AMT. The results of
the intravital staining with Rh 123 are summarised in
Table 2 for adults and in Table 3 for larvae.
Table 3. Relative labeling intensities after injection of
2 [d rhodamine 123 (100 jJgJml) into third-instar
feeding larvae
Time after labeling
10 min
Prox.
PMT
AMT
Dist.
120 min
Prox.
Dist.
S cell
Pcell
S cell
Pcell
No long-term experiments were conducted.
Selective accumulation of other dyes?
In these experiments only the proximal part of adult
posterior Malpighian tubules was used. In experiments
in which 10 mg/ml Lucifer Yellow CH in Ringer's
solution was injected into the lumen of adult tubules no
evidence was found of endocytotic processes occurring
in the S cell type. Lucifer Yellow CH is a highly
fluorescent naphthalimide dye that normally does not
penetrate membranes of living cells (Stewart, 1978) and
can be employed as a marker for endocytosis (Swanson,
1989). After injection of FITC- or TRITC-labeled
dextrans with relative molecular masses of 4.4, 10 and
35X103 (10 mg/ml) or FITC-labeled bovine serum
albumin (10 mg/ml) in the lumen of the proximal part of
a Malpighian tubule no uptake of fluorescence was
noticed after 60 minutes of incubation. No endocytosis
was detected after incubation in the Ringer's solution or
in the complex Schneider's medium (Serva).
Injection of other fluorochromes indicated that only
positively charged molecules were accumulating in the
proximal S cells. The cationic dyes rhodamine 6G and
pyronin Y (Fig. 21) were also accumulating in the S cells
after injection into the tubule lumen (20 /xg/ml) and
after injection into adults (1 mg/ml). The acid (anionic)
dyes eosin Y and B, sodium fluorescein (Fig. 2H),
sulphorhodamine B and merocyanin 540 (Fig. 2G) were
not accumulating in the S cells after injection into the
tubule lumen (10-20 ^g/ml) or into adults (1 mg/ml).
Also, the neutral dye rhodamine B did not selectively
accumulate in the S cells after injection into the tubule
lumen (10 jig/ml) or into adults (100 /xg/ml; Fig. 2F).
This neutral and the acid dyes entered the P cells after
356
W. Meulemans and A. De Loof
the S cells to a great extent (Fig. 4C). At the low
concentrations (1-10 ng/ml) used on cell cultures, it was
ineffective on the Malpighian tubules.
Treatment with 10 fJM CCCP for 5 minutes prior to
injection of Rh 123 in the lumen inhibited the uptake of
Comparison with other Diptera (Brachycera)
Rh 123 in the S cells (Fig. 4D). After 15-60 minutes in 1
The Malpighian tubules of adult Drosophila melanogas- /zM CCCP some diffuse labeling was still found in the S
ter and adult Calliphora erythrocephala also showed an cells, but no mitochondria accumulated the dye. So
accumulation of Rh 123 in their S cells, after in vitro and when CCCP was applied prior to injection of Rh 123
in vivo labeling. Remarkably, the S cells in the distal
into the lumen little uptake was seen, but when CCCP
part of the AMT also, which are often described as a
was applied after staining a whole tubule the S cells
different cell type, accumulated Rh 123 from the
accumulated much more dye, although not in their
Malpighian tubule lumen.
mitochondria. In the latter condition most fluorescence
leaks from the P cells, so the S cells are surrounded by a
In vitro administration of drugs and antibiotics
high concentration of Rh 123.
In an effort to understand the process of selective
After treatment with 1 mM 2,4-DNP or 10~2 M NaN3
accumulation of Rh 123 and other cationic dyes by the
for 20 minutes before injection of Rh 123 into the tubule
secondary cells, some ion-selective ionophores (Presslumen, no accumulation of Rh 123 was found in the S
man, 1976) and drugs that interfere with the metabcell mitochondria (Fig. 4E), although some diffuse
olism were applied to posterior Malpighian tubules
intracellular accumulation could often be found.
before or after staining with Rh 123.
Drug administration after luminal dye-injection
If tubules were incubated for 15 minutes in nigericin (1
Drug administration after in vitro labeling
When tubules previously stained at their haemolymph
;Ug/ml, Fig. 4F) or monensin (1 jig/ml) after injection of
side with Rh 123 in vitro were treated with the
Rh 123 into the lumen, the S cell mitochondria were
protonophore 2,4-dinitrophenol (2,4-DNP, 1 mM) or
very brightly labeled, more then in control tubules
with the inhibitor of electron transport sodium azide
incubated in Ringer's solution (Fig. 4G). This differ(10~2 M), for 30 minutes, the S cells also accumulated
ence in mitochondrial membrane potential was most
the dye after about two hours of incubation in fresh
obvious after injection of lower concentrations of
Ringer's solution, but less than in the control. The
rhodamine 123 (0.5-1 jig/ml) into the tubule lumen.
staining was diffuse, no mitochondria were labeled. The
This increase in concentration of the dye can be
P cell mitochondria lost the dye more rapidly in these
ascribed to the expected increase in mitochondrial
tubules, while the S cells still accumulated some dye
potential, as described for nigericin (Johnson et al.,
1981).
diffusely throughout their cytoplasm. Administration of
the protonophore carbonyl cyanide 3-chlorophenyl
Treatment with valinomycin (1 ^g/ml), CCCP (1
hydrazone (CCCP) to tubules previously stained with
/iM), 2,4-DNP (10~3 M) and NaN3 (10~2 M) after
Rh 123 showed that after 60 minutes in 10 [M CCCP
injection of Rh 123 into the lumen caused the dye to
much dye was accumulated by the S cells, although no
leak from the S cell mitochondria. In the case of CCCP,
mitochondria were stained. The fluorescence was
much of the fluorescence was retained in large round
spread diffusely throughout the cell, and was also
vesicles in the S cells. All the latter treatments are
concentrated in some round vesicles in the S cell
known to abolish the mitochondrial membrane potencytoplasm. Although the mitochondrial membrane
tial (Johnson et al., 1981; Harold, 1986). Together with
potential, and thus after a while also the cell membrane
the stimulation of dye uptake by nigericin, it may be
potential, is inhibited in such cells, the dye still
concluded that the S cell mitochondria were the
accumulated in the S cells to a large extent (although
organelles accumulating Rh 123.
not in the mitochondria).
Influence of other parameters
Drug administration prior to luminal dye-injection
Injection of Rh 123 into tubules bathed for 60 minutes
Treatment of tubules with several ionophores prior to
in Ringer's solution with NaCl, or KC1 replaced by
injection of Rh 123 in the tubule lumen gave interesting
equimolar quantities of choline chloride (Serva), had
results: little loss of uptake was seen in S cells treated
no effect on the accumulation of Rh 123.
with monensin (1-5 /zg/ml for upto 120 minutes, Fig.
Incubating adult tubules in Sarcophaga Ringer's fluid
4B). Treatment with the ionophore nigericin (1 ^g/ml
(Verachtert and De Loof, 1988) for two hours after in
for 15-60 minutes, Fig. 4A) greatly reduced the amount
vitro labeling or dissection of tubules in this Ringer's
of Rh 123 accumulating in the S cells. Although they
solution from dye-injected flies did not result in any
can both transport Na + and K+ against H + , nigericin is
different
results. So whenever a possible change in
essentially a K + /H + ionophore, while monensin is
internal pH was caused by the bicarbonate-free
+
+
essentially a Na /H ionophore (Pressman, 1976).
Ringer's that we used (Thomas, 1989), this did not
Incubation for 15-60 minutes in 1 /ig/ml of the K result in an inhibition of dye uptake by the S cells. The S
selective ionophore valinomycin prior to luminal dyecell mitochondria were also brightly labeled in this
injection also inhibited the uptake of fluorochrome by
Ringer's fluid while the P cells were mostly unlabeled.
injection into the lumen, while the S cells were poorly
labeled.
No difference in response to the various dyes was
noticed between the sexes.
Dye transport in insect Malpighian tubules
357
Fig. 4. Injection of Rh 123 into the Malpighian tubule lumen after addition of drug or antibiotic: little or no dye is
accumulated by the S cells (S) after addition of nigericin (A, 1 /ig/ml for 20 min), valinomycin (C, 1 /ig/ml for 60 min),
CCCP (D, 10 fM for 30 min) and 2,4-DNP (E, 1 raM for 60 min), while the S cell mitochondria remain well labeled after
120 min in 1 /ig/ml monensin (B). Incubation in nigericin (1 /ig/ml for 15 min) after injection of Rh 123 (1 /ig/ml) into the
tubule lumen shows brightly labeled S cells (F), while another tubule of the same fly, incubated in Ringer's fluid, shows S
cells that accumulated much less dye (G). Bars, 10 fan A-E; 20 fan, F and G.
358
W. Meulemans and A. De hoof
Incubating a tubule for two hours in Ringer's solution
at 4°C prior to injection of cold Rh 123 into the lumen
did not decrease the accumulation of Rh 123 into the S
cell mitochondria. If a tubule stained in Rh 123 at its
haemolymph side was left for three hours in Ringer's
solution at 4°C, the P cells were still brightly labeled
while the S cells had not accumulated any dye. So the
low temperature inhibited the release of the dye
molecules from the P cell mitochondria. The S cells can
still accumulate the dye from the lumen under this
condition, but since little dye was leaking from the P
cells they appear largely unstained.
Injection of a pulse of the non-ionic detergent Triton
X-100 (0.1% in Ringer's solution) into the tubule lumen
prior to injection of Rh 123 (10 /ig/ml) decreased the
dye accumulation in the S cell mitochondria, but the
proximal P cells also were diffusely labeled. So
permeabilisation of the S and P cell apical membranes
decreases the mitochondrial staining, but the P cells
become permeable to the dye molecules.
Incubating a tubule for up to two hours in Ringer's
solution containing the cardiac glycoside ouabain (10~4
M, Serva) prior to injection of Rh 123 into the lumen
had no detectable effect on the accumulation of the dye
in the S cells.
Discussion
Our experiments show that secondary cells can accumulate some small organic molecules with positive charge
(at physiological pH) from the luminal fluid. The
primary cell apical membrane in the more proximal
parts of the tubules seems to be largely impermeable to
these cationic dyes. As seen in the experiments with
CCCP, 2,4-DNP and NaN3, the large fluid flow through
the P cells towards the lumen is not responsible for the
impermeability of the P cell apical membrane. These
drugs completely abolish fluid transport by the Malpighian tubules (Berridge, 1966). Furthermore, after
incubation in Ringer's solution without K+ no uptake of
luminal dye by the P cells was noticed. Fluid transport
through the P cells should be strongly inhibited in the
absence of K+ (Berridge, 1968). The experiments with
nigericin and some inhibitors of the mitochondrial
membrane potential indicate that Rh 123 was indeed
accumulating in the S cell mitochondria.
Experiments using the K + /H + ionophore nigericin
prior to injection of Rh 123 into the lumen showed a
strong inhibition of dye accumulation by the S cells,
although their mitochondrial membrane potential
should be increased by this treatment (Johnson et al.,
1981). In cultured vertebrate cells, no inhibition of dye
uptake by nigericin was found (Johnson et al., 1981).
Monensin (a Na + /H + ionophore) on the contrary does
not inhibit the uptake of Rh 123. These two ionophores
cause a non-electrogenic dissipation of Na + or K+
gradients, with a concomitant increase or decrease in
cellular pH. So altering intracellular K+ concentration
and/or pH with nigericin seems to inhibit the uptake of
rhodamine 123, although the mitochondrial membrane
potential is increased. The very specific (electrogenic)
potassium ionophore valinomycin also inhibits the dye
uptake, but this ionophore also dissipates the electrochemical gradient across the mitochondrial membranes
(Johnson et al., 1981).
No inhibition of accumulation of Rh 123 by the S cells
was found with ouabain (10~4 M). Atzbacher et al.
(1974) described inhibitory effects of ouabain on the
excretion of acidic dyes in Drosophila, and ouabainsensitive ATPases have been characterized in some
insect Malpighian tubules (Bradley, 1985). The uptake
of Rh 123 by the S cells was also not inhibited by the
absence of extracellular Na + or K + . Bresler et al. (1985,
1990) described Na+-dependent fluorescein transport in
the Malpighian tubules of several insects. RafaeliBernstein and Mordue (1979) found no inhibitory effect
of very low sodium concentrations on glucose reabsorption in Locusta Malpighian tubules, although according
to these authors this transport should be sodiumdependent.
The process of accumulation by the S cells is quite
fast. This might be an indication of facilitated diffusion
of Rh 123 through the apical S cell membrane. Possibly
the S cells contain carrier proteins in their apical
membrane that facilitate passage into the S cells.
Rather non-specific carriers have been described for the
transport of organic acids and bases in the vertebrate
kidney tubules (Ullrich, 1979) and for organic acids in
insect Malpighian tubules (Bresler et al., 1985, 1990).
This transport is believed to be carrier-mediated, since
it was completely inhibited by competitive inhibitors.
Strictly speaking, the transport of organic bases in the
mammalian proximal kidney tubules is not active, since
it represents facilitated diffusion that is largely dependent on the cell membrane potential and thus on
cellular metabolism (Ullrich, 1979).
The mechanism for selective accumulation by the S
cells remains unknown, but the impermeability of the
apical P cell membrane might play an important role in
this process. If the apical P cell membrane is largely
impermeable, then Rh 123 can only diffuse into the S
cell. In the distal parts of the posterior tubules, little
accumulation by the S cells is noted, while the P cells
take up the Rh 123 in these parts. When after some time
the P cells start loosing the dye in this part, the S cells
accumulate the dye from the lumen. The mitochondrial
membrane potential seems to be the major driving force
for this accumulation. Since Rh 123 only labels the S
cells well when presented at their apical membrane,
some process at this membrane must facilitate the entry
into the cell, or the basal cell membrane must be quite
impermeable to the dye. The specific inhibition of
uptake after incubation in nigericin also indicates some
process at the apical membrane that might facilitate
entry into the S cell. The lack of information concerning
ion transport phenomena in the secondary cell type
makes it difficult to explain the accumulation by the S
cells.
Also of interest is the apparent regionalisation of the
morphologically uniform PMT, at least in relation to
the release and uptake of Rh 123. Maddrell (1978b) also
Dye transport in insect Malpighian tubules
described physiological differences in the morphologically homogeneous lower Malpighian tubule of Rhodnius prolixus.
Johnson et al. (1981) indicated that cells with
differing mitochondrial membrane potentials can be
distinguished by means of the degree of their uptake of
Rh 123. This probably does not apply to the primary
cells, since after application of the dye to their basal cell
side the P cells are labeled intensely. We suggest,
rather, that there would be a difference in membrane
permeability for this cationic dye of apical versus basal
P cell membranes, and of proximal versus distal regions
of the tubules. Bernal et al. (1982) described an
accumulation of rhodamine 123 in all the cell types that
they examined, but the cell types differed in their ability
to retain the dye. Carcinoma cells retained the dye for
two to five days, while normal epithelial cells lost the
dye within one to 16 hours. They suggested that this
difference in retention of rhodamine 123 was caused by
the sensitivity of carcinoma cells to the dye, while noncarcinoma cells would possess mechanisms for inactivating rhodamine 123 or for excluding it from vulnerable cell compartments. Similarly, most P cells seem to
possess mechanisms for losing the dye rather quickly,
while the S cells lose the dye very slowly. The S cells
eventually accumulate the dye in small, round vesicles.
These vesicles might be of lysosomal origin, and may
destroy the dye molecules or any mitochondria poisoned by Rh 123. Lysosomal vesicles containing
mitochondria have been described (De Duve and
Wattiaux, 1966). Although after a short application this
dye is non-toxic, prolonged administration of the dye
has been found to disrupt mitochondrial functions in
some vertebrate cultured cells (Bernal et al., 1982).
It is also very interesting that Rh 123 is precipitated
on the luminal granules in the distal part of the AMT.
Much confusion still exists in the literature about the
origin and function of these extracellular concretions
(Sohal et al., 1976; Brown, 1982). After injection of Rh
123 in adults the dye was seen to precipitate on the
luminal granules, and the granules were seen to grow in
the tubule lumen by deposition of material upon
preformed granules. Wigglesworth (1965) had already
indicated that these luminal granules could be formed
by the precipitation of excreted materials within the
lumen. This precipitation of Rh 123 seems to be limited
to the distal part of the AMT, although similar
concretions can be found in the distal part of the PMT.
Brown (1982) described several types of such metalcontaining granules in invertebrate tissues. Periodic
precipitation seems to be responsible for the concentric
nature of the granules in some invertebrates. Whether
the extracellular granules originate from intracellular
ones or whether they are formed entirely extracellularly
remains unclear in many insects, but they may be
important in detoxification of metallic (cationic!) ions.
In our experiments the exogeneous molecule Rh 123
was precipitated on granules in a specialised part of the
Malpighian tubule lumen and then carried along the
tubule lumen towards the gut (where it was probably
excreted).
359
No evidence was found for endocytosis of the
fluorescently labeled dextrans, fluorescently labeled
bovine serum albumin or Lucifer Yellow CH from the
Malpighian tubule lumen. This of course does not
exclude any endocytosis of other molecules, but it
strongly suggests that there is no bulk pinocytosis of
luminal contents. The speed with which Rh 123
accumulates in the S cells also indicates a nonendocytotic process. Vigorous endocytosis of luminally
injected dextrans has been described in mammalian
kidney tubules (Schwartz and Al-Awqati, 1990).
Many potassium-transporting insect tissues like the
midgut and the Malpighian tubules contain (apical)
membrane-bound particles (Gupta and Berridge, 1966;
Berridge and Oschman, 1969). These 'portasomes'
were first described as apical K + - transporting pumps
(Harvey et al., 1983). Finally, the potassium transport
in lepidopteran goblet cells was found to depend upon
apical proton ATPases (Wieczorek et al., 1989, 1991).
These vacuolar-type proton ATPases were found to
provide the energy for potassium transport out of the
cell, by means of K + /hH + antiport. Although in most
animal cells Na + gradients are used as an energy source
for organic ion transport, several proton-gradientbased organic ion transport systems have been found in
animal cells. Ganapathy and Leibach (1991) described
proton-coupled solute transporter in different vertebrate cell types, among which is an organic cation
transporter. This transport system would be driven by a
proton gradient, using both the chemical and electrical
components of this proton gradient as an energy source.
In the brush border of renal proximal tubule cells,
Na + /H + exchange would provide the energy for the
transport of the cation N-methylnicotinamide (NMN)
by creating a H + gradient (Ross and Holohan, 1983).
Vacuolar-type proton ATPases generate the electrochemical gradient that permits the accumulation of
neutral or positively charged biogenic amines into
acidic vesicles (Mellman et al., 1986). Possibly the
organic cation Rh 123 could use similar routes for
entering the S cells, by a possible combination with H + ATPase. However, it would seem unlikely that the
positively charged (and permeant) dye molecule would
need such a system to enter the (negatively charged) S
cell's cytoplasm. More probably, any Rh 123 entering
the P cells could be returned to the lumen by means of
such a system. This system could eliminate cationic
molecules via the urine under normal circumstances.
However, no evidence of the short-circuiting of such a
mechanism by means of proton ionophores was found
in this study.
Recently, Bertram et al. (1991) described the inhibition of in vitro fluid secretion in Drosophila Malpighian tubules by bafilomycin A± and other inhibitors
of vacuolar-type ATPases. Leyssens et al. (1991) found
evidence for an electrogenic proton ATPase at the
apical membrane of Formica Malpighian tubules. The
same group described an alkalinisation of intracellular
pH upon stimulation of KCl and fluid secretion (Zhang
et al., 1991). So evidence is accumulating to suggest that
the Malpighian tubules of insects secrete primary urine
360
W. Meulemans and A. De Loof
by means of proton ATPase-dependent cation (K + )
transport. Recently, Klein et al. (1991) have even
localised the vacuolar proton ATPase in the apical
membranes of midgut and Malpighian tubules of
Manduca sexta moths. According to the authors, these
Malpighian tubules only comprise one type of cell. Such
plasma membrane proton ATPases were also detected
in the K+-transporting insect sensilla by means of
monoclonal antibodies (Klein and Zimmermann,
1991). Further studies will have to be focussed on the
presence and possible involvement of vacuolar-type
ATPases in the reabsorption and/or exclusion of organic
cations.
We have already indicated the suitability of the
fluorochrome rhodamine 123 as a selective supravital
label for the secondary cell type in the Malpighian
tubules of Sarcophaga bullata (Meulemans and De
Loof, 1991). This also seems to hold for the intravital
labeling of the other Diptera (Brachycera) examined.
Such selective vital labeling may facilitate future
research on these relatively unknown secondary cells.
This dye also clearly shows the polarity of the different
Malpighian tubule cells. In the P, as well as in the S
cells, differences in intracellular fluorescence intensity
are found after application of the dye to the apical or
basolateral cell membranes. As well as facilitated
transport processes, the possible influence of extracellular (charged) molecules of the cell's glycocalyx on the
differential permeability of membranes should not be
overlooked (Gupta, 1989).
Berridge and Oschman (1969) suggested that the S
cells in Calliphora could reabsorb sodium from the
lumen (hypothetically), while Taylor (1971) described
similar cells in Carausius and other insects as mucocytes
because of their positive reaction for acid mucopolysaccharides. In many insect Malpighian tubules cells with
similar morphology to the secondary cells of Diptera
are found (Martoja and Ballan-Dufrancais, 1984).
Whether they also exhibit similar characteristics remains to be investigated. Principal and intercalated cell
types have also been distinguished by means of a
mitochondria-selective (carbocyanine) dye in the mammalian kidney tubules (Schwartz and Al-Awqati, 1990).
This differential staining seemed to be based upon
differences in numbers of mitochondria rather then on
differences in membrane permeability or dye retention.
Cells with opposite polarities of H + secretion have been
described in the kidney collecting duct (Schwartz et al.,
1985).
The authors thank Prof. M. Quaghebeur for the use of the
fluorescence microscope. We also thank P. Callaerts for
helpful discussions, Prof. H. Wieczorek for providing recent
information on insect vacuolar-type ATPases and Mrs. J.
Puttemans for drawing. W.M. thanks the I.W.O.N.L. of
Belgium for financial support.
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(Received 15 July 1991 - Accepted, in revised form,
25 October 1991)