ACTIVE TRANSPORT OF WATER BY INSECT MALPIGHIAN TUBULES
Cambridge University
shpm100{at}hermes.cam.ac.uk
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Simon Maddrell writes about J. A. Ramsay's 1954 publication `Active transport of water by the Malpighian tubules of the stick insect Dixippus morosus (Orthoptera; Phasmidae).' A pdf file of Ramsay's paper can be accessed as supplemental data at jeb.biologists.org
Through most of his career, Arthur Ramsay was fascinated by matters
osmotic. In the period after the Second World War, he worked on osmotic
relations of the earthworm (Ramsay,
1949a), developing typically novel methods for measuring the
melting-point and sodium content of minute quantities of fluids (Ramsay,
1949b
,
1950
). However, in the early
1950s, Ramsay started working on insect Malpighian tubules. He soon found that
the tubules secreted potassium ions into the lumen against an electrochemical
gradient, showing that this transport was an active one. This led Ramsay to
the attractive idea that "the secretion of potassium (together with
some anion) into the tubule will set up an osmotic pressure, which in its turn
will promote a passive inward diffusion of water"
(Ramsay, 1954
). According to
this idea "the secretion of potassium is the prime mover in
generating the flow of urine; and if the theory is true it follows that the
osmotic pressure of the urine should be equal to or greater than, but never
less than, the osmotic pressure of the haemolymph"
(Ramsay, 1954
). However, in
the classic paper that is the subject of the present comments, he provided
evidence that he thought destroyed this simple idea. Although we now believe
him to have been wrong about this, his paper
(Ramsay, 1954
) is nonetheless
held as a classic publication, as it describes his most novel and powerful
technique with which he will always be associated.
In the paper, Ramsay tested whether the establishment of hyper-osmotic conditions in the lumen of Malpighian tubules might cause osmotic entry of water. He found that the osmotic pressure of the fluid secreted by isolated Malpighian tubules of the stick insect Dixippus (now Carausius) morosus was, if anything, slightly but significantly hypo-osmotic to the experimental bathing fluid. He believed that this made his hypothesis untenable. He did, however, point out that his results could be explained if hyperosmotic fluid were to be transported into the tubule in one region and solutes reabsorbed in another, which we now know to be the case. He observed that this argument could not be refuted on the evidence then available, but argued that active transport of water was the simplest explanation. "The onus of disproof rests upon the opponents of this view", he concluded, rather characteristically!
In fact, 50 years on, we are confident that his earlier theory was entirely
correct and that fluid secretion does indeed depend on potassium transport,
albeit achieved by a complex of membrane proteins in which proton transport by
the ubiquitous V-ATPase is coupled with an antiporter that exchanges
H+ for K+ (Maddrell
and O'Donnell, 1992; Beyenbach,
2003
). As Ramsay supposed, this potassium transport leads to
anions flowing down their electrochemical potential gradient with water
movements being secondary to this transport of ions. The lowered osmotic
concentration of the fluid secreted by the tubules of Dixippus
(Carausius), which caused Ramsay to conclude that water movement
driven by potassium transport could not be correct, we now suppose to be
explained by active reabsorption of solutes, probably potassium plus anion.
Just such a system is found in the production of hypo-osmotic fluid by tubules
from the blood-sucking insect, Rhodnius prolixus
(Maddrell and Phillips, 1975
)
and tubules from several other insects behave similarly (for example, see
Spring and Hazelton, 1987
;
Marshall et al., 1993
;
O'Donnell and Maddrell,
1995
).
So how is it that Ramsay's paper is still so widely quoted, probably more often now even than in the first few years after it was written, even though its main conclusion is almost certainly wrong?
The answer is that the impact of the paper has been, not in its attempt to
find out how water movement is achieved, but in the technique Ramsay developed
to allow him to isolate the Malpighian tubules from the stick insect, keep
them alive for some hours, and observe them secreting fluid. This technique,
modified over the years but in essence the same as he devised, has been and
still is very widely used to study not only Malpighian tubules but other
fluid-secreting tubules, such as fly salivary glands
(Berridge and Patel, 1968). The
technique in its essentials is shown in
Fig. 1, taken from his classic
paper. The key points are the use of a container, a watch-glass in Ramsay's
original version, with a hydrophilic surface; he used `varnish'. On this was
poured a layer of liquid paraffin (mineral oil) deep enough to cover a drop of
fluid, originally haemolymph (blood) from the insect, in which a Malpighian
tubule could be placed. The cut end of the tubule could then be pulled out and
held outside the drop, in Ramsay's hands by a fine silk thread tied round the
cut end. He then would cut the tubule near the ligature (very likely with a
pair of ultrafine scissors that he used to make from tiny electrolytically
sharpened tungsten wires brazed to two steel plates, joined at their farther
ends) so that fluid secreted by the tubule would emerge from the cut and be
held there with no tendency to run back into the drop of fluid bathing the
main part of the tubule.
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Ramsay began the Summary of his paper by stating "1) Single
Malpighian tubules of the stick insect have been studied as preparations
isolated in drops of haemolymph under liquid paraffin." It is this
part of the paper that has so effectively survived and had such a major effect
on the world of insect fluid-secreting tubules. Indeed a very recent paper
(Beyenbach, 2003) contains as
part of its first figure a representation of technique instantly recognizable
from Ramsay's original. The technique is engagingly simple and elegant and,
using it, one can often see within seconds whether an experiment, say with a
suspected stimulant, has worked.
The technique has now been modified so as to allow easier observation and
handling of the secreted fluid. So the floor of the container is now usually
paraffin wax or silicone with a series of depressions in it so that many
tubules can be studied at once without the tendency of the droplets of a
bathing fluid to coalesce. For example up to 20 tubules from adult
Drosophila can be conveniently studied at once. The transparent floor
allows the tubules to be observed by transmitted light. The cut ends of the
tubules are today usually looped over small metal or glass pins stuck in the
wax or silicone to which they adhere. Secreted drops are either dislodged with
very fine glass rods or sucked off with a Gilson pipette and deposited on the
container floor. Their volumes are calculated just as Ramsay did by observing
their diameters with a micrometer eyepiece in the viewing binocular
microscope. He assumed the droplets resting on the floor of the container to
be spherical, as we do now, but he thought this was manifestly untrue.
However, tests of this, using radioactive counting of droplets of known
radioactive content, have in fact showed that the droplets are indeed almost
perfectly spherical, provided they are smaller than about 1 µl. This may be
due to the use of relatively high-density liquid paraffin, which means that
the droplets are more nearly neutrally buoyant and so made spherical by
surface tension, a powerful force when the droplets are small. Ramsay felt it
necessary to hold a small bubble of oxygen against the bathing drop so as to
supply the tubule with this gas. No-one now does this. It seems that the
surface area/volume ratio of a Malpighian tubule is so large that oxygen
dissolved in the bathing drop has easy access to the tubule and supports
secretion relatively unchecked. However, rather larger drops of fluid are used
to bathe an isolated tubule so that the oxygen demands of the tubule do not
exhaust the oxygen content of the drop. In any case, oxygen diffuses through
the liquid paraffin surrounding the bathing drop and the tubule in it. Ramsay
was impeded in his research by the need to include some haemolymph in the
fluid that bathed the isolated tubules. Other tubules, it turned out, required
no such special treatment; the tubules of Rhodnius would secrete for
hours in a simple saline containing only glucose as an energy supply, indeed
they would secrete at 35% of the normal rate in a solution of ammonium nitrate
plus glucose, containing no potassium, sodium or chloride
(Maddrell, 1969). Ironically,
it has emerged that many Malpighian tubules will only secrete normally when
bathed in a fluid containing amino acids, particularly glutamine, glutamate or
aspartate, possibly because they function as compatible intracellular
osmolytes that are necessary for sustained secretion at high rates by the
Malpighian tubules (Hazel et al.,
2003
). The irony derives from Ramsay's development of a dissecting
fluid which indeed contained glutamate, histidine and glycine, any of which,
it is now known, will support rapid fluid secretion when added to a simple
salt-based saline (Hazel et al.,
2003
). He supposed that the composition of his
"dissecting fluid should be put on record, though it has no special
merits to recommend it"!
The major effect of Ramsay's paper is that it has allowed a whole field of
investigation to be opened up, in which fluid-secreting tubules can be studied
in isolation. In early days, studies were made of the effects of stimulants,
such as hormones, on the rates of fluid secretion and the effects of salines
with different concentrations of the various physiologically important ions.
Later, it was possible to measure trans-epithelial potential
differences merely by placing electrodes in the bathing drop and the droplet
of secreted fluid (O'Donnell and Maddrell,
1984; Ianowski and O'Donnell,
2001
), though this method cannot be used to study tubules with too
narrow a lumen (Aneshansley et al.,
1988
). It has been used to study the effects of genetically
modifying the relative proportions of the different cell types
(Denholm et al., 2003
). It is
very pleasing that such a simple, elegant and powerful technique has survived
close to 50 years essentially without change.
References
Aneshansley, D. J., Marler, C. E. and Beyenbach, K. W. (1988). Transepithelial voltage measurements in isolated Malpighian tubules of Aedes egypti. J. Insect Physiol. 35, 41-52.
Beyenbach, K. W. (2003). Transport mechanisms
of diuresis in Malpighian tubules of insects. J. Exp.
Biol. 206,3845
-3856.
Berridge, M. J. and Patel, N. G. (1968). Insect salivary glands: stimulation of fluid secretion by 5-hydroxytryptaminre and adenosine 3',5'-monophosphate. Science 162,462 -463.[Medline]
Denholm, B., Sudarsan, V., Pasalodos Sanchez, S., Artero, R., Lawrence, P., Maddrell, S., Baylies, M. and Skaer, H. (2003). Dual origin of the renal tubules in Drosophila: mesodermal cells integrate and polarise to establish secretory function. Curr. Biol. 13,1052 -1057.[CrossRef][Medline]
Hazel, M. H., Ianowski, J. P., Christensen, R. J., Maddrell, S.
H. P. and O'Donnell, M. J. (2003). Amino acids modulate ion
transport and fluid secretion by insect Malpighian tubules. J. Exp.
Biol. 206,79
-91.
Ianowski, J. P. and O'Donnell, M. J. (2001). Transepithelial potential in Malpighian tubules of Rhodnius prolixus: lumen-negative voltages and the triphasic response to serotonin. J. Insect Physiol. 47,411 -421.[CrossRef][Medline]
Maddrell, S. H. P. (1969). Secretion by the Malpighian tubules of Rhodnius. The movements of ions and water. J. Exp. Biol. 52,71 -97.
Maddrell, S. H. P. and O'Donnell, M. J. (1992).
Insect Malpighian tubules: V-ATPase action in ion and fluid transport.
J. Exp. Biol. 172,417
-429.
Maddrell, S. H. P. and Phillips, J. E. (1975). Secretion of hypo-osmotic fluid by the lower Malpighian tubules of Rhodnius prolixus. J. Exp. Biol. 62,671 -683.
Marshall, A. T., Cooper, P., Rippon, G. D. and Patak, A. E.
(1993). Ion and fluid secretion by different segments of the
Malpighian tubules of the black filed cricket, Teleogryllus oceanicus.J. Exp. Biol. 177,1
-22.
O'Donnell, M. J. and Maddrell, S. H. P. (1984). Secretion by the Malpighian tubules of Rhodnius prolixus Stål: electrical events. J. Exp. Biol. 110,275 -290.[Abstract]
O'Donnell, M. J. and Maddrell, S. H. P. (1995). Fluid reabsorption and ion transport by the lower Malpighian tubules of adult female Drosophila. J. Exp. Biol. 198,1647 -1653.[Medline]
Spring, J. H. and Hazelton, S. R. (1987). Excretion in the house cricket (Acheta domesticus): stimulation of diuresis by tissue homogenates. J. Exp. Biol. 129, 63-81.
Ramsay, J. A. (1949a). The osmotic relations of the earthworm. J. Exp. Biol. 26, 46-56.
Ramsay, J. A. (1949b). A new method of freezing-point determination for small quantities. J. Exp. Biol. 26,57 -64.
Ramsay, J. A. (1950). The determination of sodium in small volumes of fluid by flame photometry J. Exp. Biol. 27,407 -419.
Ramsay, J. A. (1954). Active transport of water by the Malpighian tubules of the stick insect, Dixippus morosus (Orthoptera; Phasmidae). J. Exp. Biol. 31,104 -113.