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Fluid secretion by isolated Malpighian tubules of the housefly Musca domestica
FLUID SECRETION BY ISOLATED MALPIGHIAN TUBULES OF THE HOUSEFLY MUSCA DOMES7’1CA TERENCE DALTON and DAVID M. WINDMILL
Department of Zoology, Westfield College, Kidderpore Avenue, London, NW3 7ST. U.K (Received
5 November 1979)
Abstract--The Malpighian tubules of Musca domeska secrete a fluid with a high concentration 01 potassium and low concentrations of sodium. calcium. magnesium and chloridecompared with the isolating medium. Low secretion rates are produced by low medium potassium concentrations ( c 7 mM). with low sodium concentrations (up to 5 mM) increasing secretion; higher potassium concentrations produce higher secretion rates whilst higher sodium concentrations have no further effect. Calcium and magnesium are essential for secretion. The rate of tubule secretion is inversely proportional to the osmotic pressure of the isolating medium and the osmotic pressure of the secreted fluid is slightly hyper-osmotic to the medium over a range of medium osmotic pressures. The metabolic inhibitors cyanide. iodoacetate and 2.4-dinitrophenol inhibit secretion; CUE’ ions. arsenate and ouabain have no effect whereas ethacrynic acid abolishes secretion. 5-hydroxytryptamine. qchc AMP and theophylline have no effect on secretion. Sodium ~h~c)anatu ~umula~sa fluid secretion and Increases the osmotic pressure and the concentration of sodium and chloride, but not potassium. in the secreted fluid.
INTRODUCTION
the bathing medium was investigated by incubating tubules in a series of solutions with Na:K compositions ranging from 132 mM Na:O mM K to 102 mM Na:30 mM K. A similar series of experiments was performed to determine the dependence of secretion on sodium ions. In this case tubules were incubated in a series of solutions with compositions ranging from 0 mM Na:lOmM K to 122 mM Na:lO mM K; the osmotic pressure and chloride concentration of these solutions being maintained by the addition of the appropriate concentration of choline chloride. In investigating the effect of the osmotic pressure of the bathing medium on secretion the osmotic pressure changes were achieved by dilution of the standard medium with distilled water or the addition of sucrose. Sodium, potassium, calcium and magnesium concentrations were determined using a Perkin-Elmer 300s Atomic Absorption Spectrophotometer with Heated Graphite Atomiser attachment; chloride concentrations were measured using the electrometric method of RAMSAYet al. (1955) and osmotic pressures determined by the cryoscopic method of RAMSAY and
THE FUNCTION of insect Malpighian tubules has been described for only a few species (see MADDRELL, 1971 for review).- This paper describes work carried out on the isolated Malpighian tubules of the housefly Musca domestica and compares their function in an artificial medium with the tubules of other insects. MATERIALS
AND METHODS
A breeding stock of Musca domestica, obtained from the World Health Organisation, Geneva, was maintained at a temperature of 26”C+ 1°C. The Malpighian tubules of 3-day-old female flies were set up for the measurement of fluid secretion using the method of BERRIDGEand PATEL (1968). Each tubule was isolated in a 10 pl droplet of medium under liquid paraffin maintained in a constant temperature bath at 22°C; tubules were incubated for I hr before the beginning of an experiment to allow the secretion rate to stabilise. Secretion rate was measured by removal of secreted droplet at frequent intervals, the measurement of the droplet diameter and its volume obtained by calculation. The standard incubation medium used throughout the study contained (g/l) NaCl: 5.50; KCI: 1.50; NaH,PO,: 0.265; Na,HPO,: 0.540; NaOH: 0.10; MgC1,.6Hz0: 2.00; CaCl,.2H,O: 0.44; trehalose: 0.80; glucose: 0.70; sodium pyruvate: 0.50; sodium malate: 0.50; sodium-a-ketoglutarate: 0.25; di-sodium succinate: 0.05; sodium fumarate; 0.05. This gives the following concentrations of the major ions (mM) Na: 112: K: 20; Ca: 3; Mg: 10; Cl: 114; Phosphate: 6. The dependence of secretion on potassium ions in
BROWN (1955).
RESULTS Sodium, calcium, magnesium and chloride are present at lower concentrations in the secreted fluid than in the bathing medium whilst potassium concentrations are elevated (Table 1). Secretion is critically dependent upon the presence of potassium and sodium in the bathing medium; in the absence of potassium (Fig. 1) sodium is able. to maintain a very 281
282
TERENCE
DALTON
AND
DAVID M. WINDMILL
Table 1. The concentration of ions in the secreted fluid of Musca Malpighian tubules incubated in standard bathing medium and in medium containing 10-2M sodium thiocyanate (NaSCN)
Bathing Na+ K+ CaZ + Mg2 + Cl-
Normal
medium
115.2+_3.7 18.7* 1.1 9.8kO.7 9.7kO.8 117.1+3.3
medium are included
low rate of secretion and is secreted at a high concentration (Fig. 2). Replacement of 7 mM of sodium with an equivalent amount of potassium produces a large increase in fluid secretion and a change in the sodium and potassium concentrations in the secreted fluid, potassium almost completely replacing sodium.
Concentrotlon
200
39.7+_ 1.8 140.3_+4.2
121.6k2.8 for comparison.
of potossum In bothlng medum.
Concentrations
are
mM
concentration on the rate of secretion; the osmotic medium was kept constant with sodium.
I50
NaSCN
Replacement of further sodium with potassium produces much smaller increments in fluid secretion and ion transfer. Sodium is necessary for normal secretion however, since potassium alone can only maintain a low rate of secretion (Fig. 3), addition of 5 mM sodium restores maximal secretion. The divalent cations calcium and magnesium are
loo
Concentratton in bothlng medwm. Fig. 2. The concentration
Secreted fluid Medium+ 10-2M
16.2kO.8 142.6* 3.9 5.3kl.4 4.6 f 0.9 95.3 + 3.1
Values obtained for the analysis of the bathing expressed as mM + S.E.M., n = 8.
Fig. 1. The effect of potassium
medium
50
pressure
[No]
of the bathing
’
mM
of sodium (-O----O-) and potassium C-M-) in the secreted function of their concentration in the bathing medium.
lluid as a
Fluid secretion by malpighian tubules of
I
housefly
I
I
I
50
loo
150
mM
Concentration of sodium in bothkng medium.
Ftg. 3. The effect of sodium concentration
on the rate of fluid secretion. The bathing medium contained 10 mM potassium for each sodium concentration tested and the osmotic pressure was maintained by the addition of choline chloride.
Concentratlcn in bathing medium, Fig. 4. The effect of calcium
(-O----O-)and magnesium (-u-j
also necessary for fluid secretion; in their absence (Fig. 4) fluid secretion ceases but increases upon the addition of either calcium or magnesium to the maximal secretion rates arc bathing medium; produced by 3 mM Ca and IO mM Mg. The rate of fluid secretion is inversely related to the osmotic pressure of the bathing medium (Fig. 5) and the osmotic pressure of the secreted fluid is slightly. but consistently. hypertonic to the bathing medium (average A”C _tiauid-AYCm~ium = 0.016”C + O.Ol”C) (Fig. 6). Table 2 summarises the effects of inhibitors and drugs upon tubule secretion. All the metabolic inhibitors tested abolished secretion; ouabain. Cu’+ ions and arsenate had no effect whilst ethacrynic acid abolished and sodium thiocvanate stimulated secretion. 5_hydroxytryptamine,* dibutyryl cyclic AMP and theoohvlline had no effect. The sodium thidcyanate stimulated fluid secretion is accompanied by increased concentrations of sodium and chloride in the secreted fluid (Table 1); the concentration of potassium remains unchanged and the osmotic pressure of the secreted fluid is significantly increased. Figure 7 shows that the
mM
on the rate of fluid secretion.
osmotic pressure of the fluid secreted by stimulated tubules is consistently hyper-osmotic to the external medium over a wide range of external osmotic pressures (average A CEecreredauld -A Cmedlum = 0.103 CiO.027.C).
DlSCUSSION The secretion of cations by the tubules of Musca shows great similarities to the situation described for Calliphora (BERRIDGE, 1967. 1968). Caruusius (RAMSAY, 1955, 1956). Tip& (COAST, 1969) and Schistocvrca (MADDRELL and KLUNSUWAN, 1973) but contrasts with the tubules of Rhodnius (MADDRELL, 1969) and Glossirza (GEE, 1976a, b). Sodium, calcium and magnesium are present at lower concentrations in the tubule fluid than in the bathing medium whilst potassium is secreted at a higher concentration. Secretion is dependent upon the presence of potassium although in its absence sodium is able to maintain a very low rate of secretion and is secreted at a high concentration. The preferential secretion of potassium by the tubules is indicated by the fact that when small
TERENCEDALTONAND
284
Medwmosmotic pessure.
A-C
@.mOfiCpressure of medium, A”c
Fig. 5. The effect of varying the osmotic concentration of the bathing medium on the rate of fluid secretion. Higher osmotic concentrations were produced by the addition of sucrose and lower concentrations by dilution of the medium with distilled water. The line has been drawn by eye.
of potassium (< 10 mM) are added to the medium the secretion rate increases sharply, the concentration of potassium increases and the concentration of sodium decreases in the secreted fluid. Sodium secretion is not slowed by the presence of potassium but the potassium stimulates a very large increase in the rate of fluid secretion so that sodium appears at a much lower concentration. Sodium is required for normal secretion rates however, since potassium alone can maintain only a low rate of secretion. Models for ion and fluid movement across Malpighian tubules have been proposed (MADDRELL, amounts
Table 2. The effect of inhibitors
Test Compound Cyanide
DNP lodoacetate cuz+ Arsenate Cuz + + arsenate Ouabain Ethacrynic acid Sodium
thiocyanate
Shydroxytryptamine Adrenaline Dibutyryl
cyclic AMP
Theophylline
DAVIDM. WINDMILL
Fig. 6. The relationship between the osmotic pressure of the medium and that of the secreted fluid. The line indicates where the two solutions are isosmotic.
197 1;GEE, 1976a) and with the exceptions of Rhodnius and Glossina, potassium is actively transported across the tubule wall with water following down the generated osmotic gradient. BERRIDGE and OSCHMAN (1969) suggested that the luminal membrane possesses an electrogenic potassium pump and the basal membrane a Na/K exchange pump. The use of inhibitors has not proven very useful in establishing the presence of these pumps; ouabain, a specific inhibitor of Na + - K +-activated, Mg2 + dependent ATP-ase-an enzyme closely linked with (if not identical with) Na/K pumps (SKOI!, 1965)-is reported to have no effect on fluid secretion by the tubules of Calliphora (BERRIDGE, 1968), Cavausius
and drugs on the rate of fluid secretion tubules of Musca
Concentration (MI 10-J 5 X lo-” 10-3 10-4 10-4 10-3 10-3 10-a lo-3:10-a 10-3 10-4 10-a 10-3 10-z 10-s 10-s 10-6 10-a lo-’ 10-z 10-s 10-z
Rates are expressed as the Mean +_S.E.M.
by Malpighian
Rate of secretion 30 min after administration of drug as 7; original rate 0, 1’0
n
96.8 + 4.2 40.5& 3.8 0 0 42.8k5.3 0 102.612.9 103.3* 3.5 97. I + 4.2 91.4 & 3.7 18.8+6.1 0 95.6 + 4.6 158.6k6.8 91.1 * 5.3 97.2k2.9 101.6+ I.1 98.6+ 3.5 lOl.4+2.4 97.2k3.8 106.3k5.9 96.7k4.1
5 5 4 4 6 4 5 6 5 6 6 4 6 8 5 6 5 5 6 6 6 6
Fluid secretion by malpighian tubules of housefly
Osmotic pressure of medum.
AT
Fig. 7. The relationship between the osmotic pressure of the medium and that of the secreted fluid in sodium thiocyanate treated tubules. lsosmoticity is indicated by the straight line.
(PILCHER, 1970b). Rhodnius (MADDRELL, 1969) and Glossina (GEE, 1976b), whereas fluid secretion (and a Na + - K + -activated ATP-ase) is inhibited by ouabain in the tubules of Locusta (ANSTEEand BELL, 1975) and Schistocerc,a (PEACOCK et al.. 1972). Ouabain has no effect on fluid secretion by Musca tubules when they are incubated in normal medium (20 mM K+). This does not preclude the presence of a Na/K-ATP-ase however. since it has been shown that potassium at concentrations of the order of 20 mM can block ouabain inhibition (WILLIS, 1968; JUNGREIS, 1977). Furthermore, ethacrynic acid, which appears to inhibit the same.pump enzymes as ouabain (PODEVIN and BOUMENDIL-PODEVIN,1972; JAIRALA, et al., 1972) does inhibit secretion so that it seems likely that a Na/K-ATP-ase is essential for transport. The dependence of secretion upon the presence of Mg2+ ions in the incubation medium may reflect the dependence of this enzyme upon Mg* + ions as in other tissues. In a high chloride:low phosphate medium (114 mM:6 mM) the concentration of chloride in Musca tubule fluid is lower than that of the medium, a situation described for Calliphora ( BERRIDGE, 1969) and Carausius (RAMSAY. 1956; PILCHER, 1970b) but in contrast to Rhodnius (MADDRELL, 1969) and Schistocerca (MADDRELL and KLUNSUWAN. 1973). BERRIDGE (1969) concluded that anions were transported across Calliphora tubules via two independent routes: (I ) anions such as chloride pass through pores in the membrane at a rate dependent upon their hydrated ionic radius-a route that is blocked by Cu’* ions, and (2) phosphate is actively transported by a carrier and is blocked by arsenate. Phosphate will not support fluid secretion in Rhodnius (MADDRELL. 1969) but maintains a higher rate of secretion in Calliphora (BERRIDGE, 1969). Carausius (RAMSAY. 1956) and Schistocerca (MADDRELL and KLUNSIIWAN. 1973) and is more concentrated in the secreted fluid than in the medium. Cuz+ ions inhibit fluid secretion by Calliphora tubules in a chloride medium but have no effect in a high phosphate medium; arsenate sharply reduces the rate of secretion of tubules in a phosphate medium. Phosphate
2x5
secretion in Carausius appears to be different since CuL+ ions inhibit secretion in a phosphate medium (PILCHER. 1970b). The observations in Musca of reduced chloride concentration and a positive luminal potential difference (approx. 4 mV: unpublished data) strongly suggest that chloride movement across the tubule is passive. The lack of effect of Cu2 ’ ions on secretion was at first thought to be due to phosphate being actively transported across the tissue and maintaining secretion. however, the lack of any effect of arsenate. either alone or in combination with CuL + ions. would negate this possibility. It is possible that all anions share a common Cu2+ and arsenateinsensitive route. The effect of varying the osmotic pressure of the bathing medium on tubule secretion in Musca is similar to that observed in all other species: decreasing the osmotic pressure increases the rate of secretion and the secreted fluid is slightly hyper-osmotic to the medium. The effects of metabolic inhibitors are similar to those described on tubules of Calliphora (BERRIDGE. 1966) and Rhodnius ( MADDRELL, 1969): iodoacetate, which blocks the conversion of fructose-6-phosphate to pyruvate; cyanide, which inhibits cytochrome oxidase and 2,4-dinitrophenol, which uncouples oxidative phosphorylation all abolish secretion. Thus fluid secretion is dependent upon both glycolysis and oxidative metabolism. Low concentrations of 5-hydroxytryptamine and cyclic AMP stimulate fluid secretion by the Malpighian tubules of Rhodnius and Carausius (MADDRELL, ef al. 1971). 5-hydroxytryptamine appears to mimic the action of the insect diuretic hormone and cyclic AMP is thought to act as secondary messenger in mediating the response. The tubules of Schistocerca are stimulated by cyclic AMP but not by 5-hydroxytryptamine (MADDRELL and KLUNSUWAN. 1973). The absence of any effect of 5_hydroxytryptamine, cyclic AMP or theophylline (a methyl xanthine which elevates intracellular levels of cyclic AMP by inhibition of phosphodiesterase, the enzyme responsible for cyclic AMP breakdown) on Musca tubules implies the absence of a hormonal mechanism for controlling fluid secretion by the Malpighian tubules similar to that found in Rhodnius (MADDRELL, 1963). Carausius (PILTHIR. 1970a) and Srhistocercu (MORDUE, 1969). The observed effects of sodium thiocyanate on Musca tubules can be explained in terms of the tubules secreting a hyper-osmotic fluid by the active transport of potassium and sodium (with potassium being the ‘prime-mover’ in fluid formation) with chloride moving passively down the electrochemical gradient as the major anion and sodium and chloride being actively resorbed from the luminal fluid via a Na +linked chloride pump thus reducing the osmolarity of the tubule fluid to near that of the bathing medium. Sodium thiocyanate inhibits Na-dependent active chloride transport in fish gills (EPSTEIN. rt ul.. 1973) and amphibian cornea (ZADUNAISKY et al.. 1971) so that in Musca it is postulated that it abolishes the NaCl resorption pathway thus raising the concentrations of sodium and chloride in the tubule fluid: if chloride movement into the lumen during secretion is passive as suggested. then he observed equilibration with the medium chloride concentration is to be expected. The
286
TERENCE DALTON ANI) DAVID M. WINDMILI
increase in fluid secretion probably reflects flow of water down the increased osmotic gradient but the observation that the potassium concentration in the secreted fluid remains constant suggests that water movement is intimately linked with, and limited by. potassium transport and that the potassium transport pathway, and hence fluid movement normally operates close to maximal capacity.
REFERENCES
ANSTEE J. H. and BELL D. M. (1975) Relationship of Na+-K+-activated ATP-ase to fluid production by Malpighian tubules of Locusra migratoria. J. Insect Physiol. 21, 1779-I 784. BERRIDGE M. J. (1966) Metabolic pathways of the isolated Malpighian tubules of the blowfly functioning in an artificial medium. J. Insect Physiol. 12, 1523-1538. BERRIDGE M. J. (1967) Ion and water transport across epithelia. In Znsecrs and Physiology (Ed. by BEAMENT J. W. L. and TREHERNE J. H.). Oliver and Boyd. Edinburgh. BERRIDGE M. J. (1968) Urine formation by the Malpighian tubules of Calliphora I Cations. J. e-up. Biol. 48, 159-l 74. BERRIDGE M. J. (1969) Urine formation by the Malpighian tubules of Calliphora II Anions. J. exp. Biol. 50, 15-28. BERRIDGE M. J. and OSCHMAN J. L. (1969) A structural basis for fluid secretion by Malpighian tubules. Tissue and Cell 1, 247-272.
BERRIDGE M. J. and PATEL N. G. (1968) Insect salivary stimulation of fluid secretion by glands: S-hydroxytryptamine and adenosine 3’-5’. monophosphate. Science, Wash. 162, 462-463. COAST G. M. (1969) Formation of urinary fluid by Malpighian tubules of an insect. J. Physiol. (LO&J 202, 102P-lO3P. EPSTEIN F. H., MAETZ J. and RENZIS G. (1973) Active transport of chloride by the teleost gill: inhibition by thiocyanate. Am. J. Physiol. 224, 1295-1299. GEE J. D. (1976a) Active transport of sodium by the Malpighian tubules of the tsetse fly Glossina morsirans. J. exp. Biol. 64, 357-368.
GEE J. D. (1976b) Fluid secretion by the Malpighian tubules of the tsetse fly Glossina morsitans: The effects of ouabain, ethacrynic acid and amiloride. J. exp. Biol. 65, 323-332. JAIRALA S. W.. VIEYRA A. and MACLAUGHLIN M. (1973) Influence of ethacrynic acid and ouabain on the oxygen consumption and potassium and sodium content of the kidney medulla of the dog. Biochim. biophys. Acta 279. 320-330.
JUNGREIS A. M. (1977) Comparative aspects of invertebrate epithelial transport. In Water Relations in Membrane Transport in Planrs and Animals (Ed. by JUNGREIS A. M.. HODGES T. K.. KLEINZELLER A. and SCHULTZ S. G.). Academic Press, New York
MADDRELL S. H. P. (1963) Excretion in the blood suckmghug Rhodnius prolr.ws Stal. I The control of diurests. J. e\p. Biol. 40, 247-256.
MAI~DR~LL S. H. P. (1969) Secretion tubules of Rhodnius. The movements
by the Malpighian of ions and water.
J. e-x-p. Biol. 51, 71-97.
MADURELL S. H. P. (1971) The mechanisms of Insect Excretory Systems. Adv. Insect Phswl. 8, 199-33 I. MAUI~RELL S. H. P. and KLUXSUWAIU S. (1973) Fluid secretion by it1 vitro preparations of the Malpighian tubules of the desert locust Schi.vtoc,ewa grqaria. J Insecr Ph.vsiol. 19, 1369-l 376. MADDRELL S. H. P.. PIL~HER D. E. M. and GARIXN~R B. 0. C. (197 I) Pharmacology of the Malpighian tubules of Rhodnius and Carausius: the structure-activity of tryptamine analogues and the role of cyclic AMP. J. c_xp. Biol. 54, 779-804.
MORDLI~W. (1969) Hormonalcontrol of Malpighian tubules and rectal function in the desert locust Schistowrco gregaria.
J. Insecr Ph_vsiol. 15, 273-285.
PEACOCK A. J., BOWLER K. and ANSTEE J. H. (1972) Demonstration of a Na’-K’-Mg2+ dependent ATP-ase in a preparation from hindgut and Malpighian tubules of two species of insect. Esperierllia 28, 901-902. PILCHER D. E. M. (1970a) Hormonal control of the Malpighian tubules of the stick insect Curuusius morosus. J. erp.
Biol. 52, 653-665.
PILCHER D. E. M. (1970b) The influence of the diuretic hormone on the process of urine secretion in the Malpighian tubules of Curausius morosus. J. e.vp. Biol. 53, 465-484.
PODEVIN R. A. and BOUMENDIL-PODEVINE. F. (1972) Effects of temperature. medium K+. ouabain and ethacynic acid on transport of electrolytes and water by separated renal tubules. Biochim. biophys. Ac,ta 282, 234-249. RAMSAYJ. A. (1955) The excretion of sodium. potassium and water by the Malpighian tubules of the stick insect Di.uippu.r morosu.r (Orthoptera, Phasmidae). J. exp. Biol. 32, ZOO-2 16. RAMSAYJ. A. (1956) Excretion by the Malpighian tubules of insect Dl.uippus morosus (Orthoptera, the stick Phasmidae): calcium. magnesium, chloride. phosphate and hydrogen ions. J. e.xp. Biol. 33, 696708. RAMSAY J. A. and BROWN R. H. J. (1955) Simplitied and procedure for freezing-point deapparatus terminations upon small volumes of fluid. J. vcient. Instrum.
32, 372-375.
RAMSAY J. A., BROWN R. H. J. and CROC~HANP. C. (1955) Electrometric titration ofchloride in small volumes. J. c.~p. Biol. 32, 822-829.
SKOU J. C. (1965) Enzymatic basis for active transport of Na’ and K+ across cell membranes. PII~N~/. Rev. 45. 5966
17.
WILLIS J. S. (1968) The interactton of K +, ouabain and Na ’ on the cation transport and respiration of renal cortical cells of hamsters and ground squirrels. Biochim. bioph~s. Arta.
163. 516-530.
ZADLINAIXY J. A., LANDE M. A. and HAFNER J. (1971) Further studies in chloride transport in the frog cornea. .4m. J. Phxsiol. 221, 1832-1836.
Department of Zoology, Westfield College, Kidderpore Avenue, London, NW3 7ST. U.K (Received
5 November 1979)
Abstract--The Malpighian tubules of Musca domeska secrete a fluid with a high concentration 01 potassium and low concentrations of sodium. calcium. magnesium and chloridecompared with the isolating medium. Low secretion rates are produced by low medium potassium concentrations ( c 7 mM). with low sodium concentrations (up to 5 mM) increasing secretion; higher potassium concentrations produce higher secretion rates whilst higher sodium concentrations have no further effect. Calcium and magnesium are essential for secretion. The rate of tubule secretion is inversely proportional to the osmotic pressure of the isolating medium and the osmotic pressure of the secreted fluid is slightly hyper-osmotic to the medium over a range of medium osmotic pressures. The metabolic inhibitors cyanide. iodoacetate and 2.4-dinitrophenol inhibit secretion; CUE’ ions. arsenate and ouabain have no effect whereas ethacrynic acid abolishes secretion. 5-hydroxytryptamine. qchc AMP and theophylline have no effect on secretion. Sodium ~h~c)anatu ~umula~sa fluid secretion and Increases the osmotic pressure and the concentration of sodium and chloride, but not potassium. in the secreted fluid.
INTRODUCTION
the bathing medium was investigated by incubating tubules in a series of solutions with Na:K compositions ranging from 132 mM Na:O mM K to 102 mM Na:30 mM K. A similar series of experiments was performed to determine the dependence of secretion on sodium ions. In this case tubules were incubated in a series of solutions with compositions ranging from 0 mM Na:lOmM K to 122 mM Na:lO mM K; the osmotic pressure and chloride concentration of these solutions being maintained by the addition of the appropriate concentration of choline chloride. In investigating the effect of the osmotic pressure of the bathing medium on secretion the osmotic pressure changes were achieved by dilution of the standard medium with distilled water or the addition of sucrose. Sodium, potassium, calcium and magnesium concentrations were determined using a Perkin-Elmer 300s Atomic Absorption Spectrophotometer with Heated Graphite Atomiser attachment; chloride concentrations were measured using the electrometric method of RAMSAYet al. (1955) and osmotic pressures determined by the cryoscopic method of RAMSAY and
THE FUNCTION of insect Malpighian tubules has been described for only a few species (see MADDRELL, 1971 for review).- This paper describes work carried out on the isolated Malpighian tubules of the housefly Musca domestica and compares their function in an artificial medium with the tubules of other insects. MATERIALS
AND METHODS
A breeding stock of Musca domestica, obtained from the World Health Organisation, Geneva, was maintained at a temperature of 26”C+ 1°C. The Malpighian tubules of 3-day-old female flies were set up for the measurement of fluid secretion using the method of BERRIDGEand PATEL (1968). Each tubule was isolated in a 10 pl droplet of medium under liquid paraffin maintained in a constant temperature bath at 22°C; tubules were incubated for I hr before the beginning of an experiment to allow the secretion rate to stabilise. Secretion rate was measured by removal of secreted droplet at frequent intervals, the measurement of the droplet diameter and its volume obtained by calculation. The standard incubation medium used throughout the study contained (g/l) NaCl: 5.50; KCI: 1.50; NaH,PO,: 0.265; Na,HPO,: 0.540; NaOH: 0.10; MgC1,.6Hz0: 2.00; CaCl,.2H,O: 0.44; trehalose: 0.80; glucose: 0.70; sodium pyruvate: 0.50; sodium malate: 0.50; sodium-a-ketoglutarate: 0.25; di-sodium succinate: 0.05; sodium fumarate; 0.05. This gives the following concentrations of the major ions (mM) Na: 112: K: 20; Ca: 3; Mg: 10; Cl: 114; Phosphate: 6. The dependence of secretion on potassium ions in
BROWN (1955).
RESULTS Sodium, calcium, magnesium and chloride are present at lower concentrations in the secreted fluid than in the bathing medium whilst potassium concentrations are elevated (Table 1). Secretion is critically dependent upon the presence of potassium and sodium in the bathing medium; in the absence of potassium (Fig. 1) sodium is able. to maintain a very 281
282
TERENCE
DALTON
AND
DAVID M. WINDMILL
Table 1. The concentration of ions in the secreted fluid of Musca Malpighian tubules incubated in standard bathing medium and in medium containing 10-2M sodium thiocyanate (NaSCN)
Bathing Na+ K+ CaZ + Mg2 + Cl-
Normal
medium
115.2+_3.7 18.7* 1.1 9.8kO.7 9.7kO.8 117.1+3.3
medium are included
low rate of secretion and is secreted at a high concentration (Fig. 2). Replacement of 7 mM of sodium with an equivalent amount of potassium produces a large increase in fluid secretion and a change in the sodium and potassium concentrations in the secreted fluid, potassium almost completely replacing sodium.
Concentrotlon
200
39.7+_ 1.8 140.3_+4.2
121.6k2.8 for comparison.
of potossum In bothlng medum.
Concentrations
are
mM
concentration on the rate of secretion; the osmotic medium was kept constant with sodium.
I50
NaSCN
Replacement of further sodium with potassium produces much smaller increments in fluid secretion and ion transfer. Sodium is necessary for normal secretion however, since potassium alone can only maintain a low rate of secretion (Fig. 3), addition of 5 mM sodium restores maximal secretion. The divalent cations calcium and magnesium are
loo
Concentratton in bothlng medwm. Fig. 2. The concentration
Secreted fluid Medium+ 10-2M
16.2kO.8 142.6* 3.9 5.3kl.4 4.6 f 0.9 95.3 + 3.1
Values obtained for the analysis of the bathing expressed as mM + S.E.M., n = 8.
Fig. 1. The effect of potassium
medium
50
pressure
[No]
of the bathing
’
mM
of sodium (-O----O-) and potassium C-M-) in the secreted function of their concentration in the bathing medium.
lluid as a
Fluid secretion by malpighian tubules of
I
housefly
I
I
I
50
loo
150
mM
Concentration of sodium in bothkng medium.
Ftg. 3. The effect of sodium concentration
on the rate of fluid secretion. The bathing medium contained 10 mM potassium for each sodium concentration tested and the osmotic pressure was maintained by the addition of choline chloride.
Concentratlcn in bathing medium, Fig. 4. The effect of calcium
(-O----O-)and magnesium (-u-j
also necessary for fluid secretion; in their absence (Fig. 4) fluid secretion ceases but increases upon the addition of either calcium or magnesium to the maximal secretion rates arc bathing medium; produced by 3 mM Ca and IO mM Mg. The rate of fluid secretion is inversely related to the osmotic pressure of the bathing medium (Fig. 5) and the osmotic pressure of the secreted fluid is slightly. but consistently. hypertonic to the bathing medium (average A”C _tiauid-AYCm~ium = 0.016”C + O.Ol”C) (Fig. 6). Table 2 summarises the effects of inhibitors and drugs upon tubule secretion. All the metabolic inhibitors tested abolished secretion; ouabain. Cu’+ ions and arsenate had no effect whilst ethacrynic acid abolished and sodium thiocvanate stimulated secretion. 5_hydroxytryptamine,* dibutyryl cyclic AMP and theoohvlline had no effect. The sodium thidcyanate stimulated fluid secretion is accompanied by increased concentrations of sodium and chloride in the secreted fluid (Table 1); the concentration of potassium remains unchanged and the osmotic pressure of the secreted fluid is significantly increased. Figure 7 shows that the
mM
on the rate of fluid secretion.
osmotic pressure of the fluid secreted by stimulated tubules is consistently hyper-osmotic to the external medium over a wide range of external osmotic pressures (average A CEecreredauld -A Cmedlum = 0.103 CiO.027.C).
DlSCUSSION The secretion of cations by the tubules of Musca shows great similarities to the situation described for Calliphora (BERRIDGE, 1967. 1968). Caruusius (RAMSAY, 1955, 1956). Tip& (COAST, 1969) and Schistocvrca (MADDRELL and KLUNSUWAN, 1973) but contrasts with the tubules of Rhodnius (MADDRELL, 1969) and Glossirza (GEE, 1976a, b). Sodium, calcium and magnesium are present at lower concentrations in the tubule fluid than in the bathing medium whilst potassium is secreted at a higher concentration. Secretion is dependent upon the presence of potassium although in its absence sodium is able to maintain a very low rate of secretion and is secreted at a high concentration. The preferential secretion of potassium by the tubules is indicated by the fact that when small
TERENCEDALTONAND
284
Medwmosmotic pessure.
A-C
@.mOfiCpressure of medium, A”c
Fig. 5. The effect of varying the osmotic concentration of the bathing medium on the rate of fluid secretion. Higher osmotic concentrations were produced by the addition of sucrose and lower concentrations by dilution of the medium with distilled water. The line has been drawn by eye.
of potassium (< 10 mM) are added to the medium the secretion rate increases sharply, the concentration of potassium increases and the concentration of sodium decreases in the secreted fluid. Sodium secretion is not slowed by the presence of potassium but the potassium stimulates a very large increase in the rate of fluid secretion so that sodium appears at a much lower concentration. Sodium is required for normal secretion rates however, since potassium alone can maintain only a low rate of secretion. Models for ion and fluid movement across Malpighian tubules have been proposed (MADDRELL, amounts
Table 2. The effect of inhibitors
Test Compound Cyanide
DNP lodoacetate cuz+ Arsenate Cuz + + arsenate Ouabain Ethacrynic acid Sodium
thiocyanate
Shydroxytryptamine Adrenaline Dibutyryl
cyclic AMP
Theophylline
DAVIDM. WINDMILL
Fig. 6. The relationship between the osmotic pressure of the medium and that of the secreted fluid. The line indicates where the two solutions are isosmotic.
197 1;GEE, 1976a) and with the exceptions of Rhodnius and Glossina, potassium is actively transported across the tubule wall with water following down the generated osmotic gradient. BERRIDGE and OSCHMAN (1969) suggested that the luminal membrane possesses an electrogenic potassium pump and the basal membrane a Na/K exchange pump. The use of inhibitors has not proven very useful in establishing the presence of these pumps; ouabain, a specific inhibitor of Na + - K +-activated, Mg2 + dependent ATP-ase-an enzyme closely linked with (if not identical with) Na/K pumps (SKOI!, 1965)-is reported to have no effect on fluid secretion by the tubules of Calliphora (BERRIDGE, 1968), Cavausius
and drugs on the rate of fluid secretion tubules of Musca
Concentration (MI 10-J 5 X lo-” 10-3 10-4 10-4 10-3 10-3 10-a lo-3:10-a 10-3 10-4 10-a 10-3 10-z 10-s 10-s 10-6 10-a lo-’ 10-z 10-s 10-z
Rates are expressed as the Mean +_S.E.M.
by Malpighian
Rate of secretion 30 min after administration of drug as 7; original rate 0, 1’0
n
96.8 + 4.2 40.5& 3.8 0 0 42.8k5.3 0 102.612.9 103.3* 3.5 97. I + 4.2 91.4 & 3.7 18.8+6.1 0 95.6 + 4.6 158.6k6.8 91.1 * 5.3 97.2k2.9 101.6+ I.1 98.6+ 3.5 lOl.4+2.4 97.2k3.8 106.3k5.9 96.7k4.1
5 5 4 4 6 4 5 6 5 6 6 4 6 8 5 6 5 5 6 6 6 6
Fluid secretion by malpighian tubules of housefly
Osmotic pressure of medum.
AT
Fig. 7. The relationship between the osmotic pressure of the medium and that of the secreted fluid in sodium thiocyanate treated tubules. lsosmoticity is indicated by the straight line.
(PILCHER, 1970b). Rhodnius (MADDRELL, 1969) and Glossina (GEE, 1976b), whereas fluid secretion (and a Na + - K + -activated ATP-ase) is inhibited by ouabain in the tubules of Locusta (ANSTEEand BELL, 1975) and Schistocerc,a (PEACOCK et al.. 1972). Ouabain has no effect on fluid secretion by Musca tubules when they are incubated in normal medium (20 mM K+). This does not preclude the presence of a Na/K-ATP-ase however. since it has been shown that potassium at concentrations of the order of 20 mM can block ouabain inhibition (WILLIS, 1968; JUNGREIS, 1977). Furthermore, ethacrynic acid, which appears to inhibit the same.pump enzymes as ouabain (PODEVIN and BOUMENDIL-PODEVIN,1972; JAIRALA, et al., 1972) does inhibit secretion so that it seems likely that a Na/K-ATP-ase is essential for transport. The dependence of secretion upon the presence of Mg2+ ions in the incubation medium may reflect the dependence of this enzyme upon Mg* + ions as in other tissues. In a high chloride:low phosphate medium (114 mM:6 mM) the concentration of chloride in Musca tubule fluid is lower than that of the medium, a situation described for Calliphora ( BERRIDGE, 1969) and Carausius (RAMSAY. 1956; PILCHER, 1970b) but in contrast to Rhodnius (MADDRELL, 1969) and Schistocerca (MADDRELL and KLUNSUWAN. 1973). BERRIDGE (1969) concluded that anions were transported across Calliphora tubules via two independent routes: (I ) anions such as chloride pass through pores in the membrane at a rate dependent upon their hydrated ionic radius-a route that is blocked by Cu’* ions, and (2) phosphate is actively transported by a carrier and is blocked by arsenate. Phosphate will not support fluid secretion in Rhodnius (MADDRELL. 1969) but maintains a higher rate of secretion in Calliphora (BERRIDGE, 1969). Carausius (RAMSAY. 1956) and Schistocerca (MADDRELL and KLUNSIIWAN. 1973) and is more concentrated in the secreted fluid than in the medium. Cuz+ ions inhibit fluid secretion by Calliphora tubules in a chloride medium but have no effect in a high phosphate medium; arsenate sharply reduces the rate of secretion of tubules in a phosphate medium. Phosphate
2x5
secretion in Carausius appears to be different since CuL+ ions inhibit secretion in a phosphate medium (PILCHER. 1970b). The observations in Musca of reduced chloride concentration and a positive luminal potential difference (approx. 4 mV: unpublished data) strongly suggest that chloride movement across the tubule is passive. The lack of effect of Cu2 ’ ions on secretion was at first thought to be due to phosphate being actively transported across the tissue and maintaining secretion. however, the lack of any effect of arsenate. either alone or in combination with CuL + ions. would negate this possibility. It is possible that all anions share a common Cu2+ and arsenateinsensitive route. The effect of varying the osmotic pressure of the bathing medium on tubule secretion in Musca is similar to that observed in all other species: decreasing the osmotic pressure increases the rate of secretion and the secreted fluid is slightly hyper-osmotic to the medium. The effects of metabolic inhibitors are similar to those described on tubules of Calliphora (BERRIDGE. 1966) and Rhodnius ( MADDRELL, 1969): iodoacetate, which blocks the conversion of fructose-6-phosphate to pyruvate; cyanide, which inhibits cytochrome oxidase and 2,4-dinitrophenol, which uncouples oxidative phosphorylation all abolish secretion. Thus fluid secretion is dependent upon both glycolysis and oxidative metabolism. Low concentrations of 5-hydroxytryptamine and cyclic AMP stimulate fluid secretion by the Malpighian tubules of Rhodnius and Carausius (MADDRELL, ef al. 1971). 5-hydroxytryptamine appears to mimic the action of the insect diuretic hormone and cyclic AMP is thought to act as secondary messenger in mediating the response. The tubules of Schistocerca are stimulated by cyclic AMP but not by 5-hydroxytryptamine (MADDRELL and KLUNSUWAN. 1973). The absence of any effect of 5_hydroxytryptamine, cyclic AMP or theophylline (a methyl xanthine which elevates intracellular levels of cyclic AMP by inhibition of phosphodiesterase, the enzyme responsible for cyclic AMP breakdown) on Musca tubules implies the absence of a hormonal mechanism for controlling fluid secretion by the Malpighian tubules similar to that found in Rhodnius (MADDRELL, 1963). Carausius (PILTHIR. 1970a) and Srhistocercu (MORDUE, 1969). The observed effects of sodium thiocyanate on Musca tubules can be explained in terms of the tubules secreting a hyper-osmotic fluid by the active transport of potassium and sodium (with potassium being the ‘prime-mover’ in fluid formation) with chloride moving passively down the electrochemical gradient as the major anion and sodium and chloride being actively resorbed from the luminal fluid via a Na +linked chloride pump thus reducing the osmolarity of the tubule fluid to near that of the bathing medium. Sodium thiocyanate inhibits Na-dependent active chloride transport in fish gills (EPSTEIN. rt ul.. 1973) and amphibian cornea (ZADUNAISKY et al.. 1971) so that in Musca it is postulated that it abolishes the NaCl resorption pathway thus raising the concentrations of sodium and chloride in the tubule fluid: if chloride movement into the lumen during secretion is passive as suggested. then he observed equilibration with the medium chloride concentration is to be expected. The
286
TERENCE DALTON ANI) DAVID M. WINDMILI
increase in fluid secretion probably reflects flow of water down the increased osmotic gradient but the observation that the potassium concentration in the secreted fluid remains constant suggests that water movement is intimately linked with, and limited by. potassium transport and that the potassium transport pathway, and hence fluid movement normally operates close to maximal capacity.
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