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A method for determining nonelectrolyte uptake by adult Schistosoma mansoni using compartmental analysis of hexose fluxes
Molecular and Biochemical Parasitology, 5 (1982) 345- 352
345
Elsevier Biomedical Press
A METHOD F O R DETERMINING NONELECTROLYTE UPTAKE BY ADULT S C H I S T O S O M A M A N S O N ! USING COMPARTMENTAL ANALYSIS OF HEXOSE
FLUXES
RON B. PODESTA and LYN L. DEAN Department o f Zoology, University o f Western Ontario, London, Ontario, Canada N6A 5B7.
(Received 4 November 1981; accepted 8 February 1982)
A steady-state compartmental analysis of galactose, glucose and 3-O-methylglucose fluxes was conducted on male and female Schistosoma mansoni. The method has several advantages over previously used initial rate studies. A nonlinear log-linear plot of glucose exchange is discussed in terms of differentiating between metabolized and nonmetabolized substrates in transport studies. K t values and marker distribution volumes are estimated from the compartmental analysis and it is concluded that 3-O-methylglucose is not suited as a substrate for hexose transport studies in S. mansoni. Key words: Schistosoma mansoni, Compartmental analysis, Hexose transport, Distribution volumes.
INTRODUCTION Considerable effort has been expended endeavoring to examine fluxes o f organic solutes across the surface o f schistosomes [1]. These studies have been hampered by an inability to distinguish between radiolabeled solutes adhering to the surface o f the parasites, label entering the cecum o f the parasites, label being taken up across the cecal epithelium and label entering the parasites across the surface epithelial syncytium [ t ] . Another problem arises from the use in previous studies o f initial rate studies in which the diffusion barrier represented b y the surface hydrodynamic layers alters active and/or diffusive kinetics o f overall transport across the surface o f the parasites [1 ]. The above problems have been partially overcome in a promising method devised recently by Cornford and co-workers [2, 3 ] . These workers used a nonabsorbable marker which effectively eliminates from uptake determinations labeled substrates which are distributed in the surface unstirred layer, crypts and folds and label present in the lumen o f the cecum. However, the method o f normalization o f the data presented by these workers depends on the assumption that water uptake across the surface o f the parasites occurs by simple diffusion. This assumption has not been confirmed in a recent
Abbreviations: Gal, galactose; Glu, glucose; MG, 3-O-methylglucose; PEG, polyethylene glycol.
0166-6851/82/0000-0000/$02.75
© 1982 Elsevier Biomedical Press
346 study of the diffusive and osmotic components of water fluxes across the surface epithelial syncytium of Schistosoma mansoni [4]. Also, Cornford et al. [2, 3] used initial rate studies so that the derived kinetics will be subject to change pending the outcome of studies designed to examine the effect of unstirred layers. In the present study we develop a simple steady-state flux method using compartmental analysis of hexose fluxes in S. mansoni. MATERIALS AND METHODS
Schistosoma mansoni (Puerto Rican strain) were maintained in our laboratory through the infection of Syrian hamsters and snails (Biornphalaria glabrata). Adult parasites were obtained from the perfused hepatic portal veins of hamsters, 45 days post-infection, using conventional techniques. The composition of the balanced electrolyte solution used for perfusions and incubations in this study, has been described elsewhere [4, 5]. Groups of five male or female flukes were then incubated for 30 min in 5 ml of incubation fluid in a shaking water bath at 37°C. During this incubation period, the parasites were either exposed to 1 mM unlabeled sugar (glucose, galactose or 3-O-methylglucose) for inward exchange rate determinations, or radiolabeled sugar (1.0/aCi/ml) for outward exchange rate determinations. The non-permeable marker compound [3 HI polyethylene glycol 4000 (PEG; NEN) was used to quantify the amount of fluid and sugar adherent to the worm surface and distributed in the cecal lumen. The 30 min incubation was followed by a second incubation period of up to 60 min in a fluid containing 1 mM sugar in the efflux experiments and labeled sugar with [3H] PEG for the inward exchange group. Changes in levels of radiolabeled sugar and marker were determined at specific time intervals during the second incubation by solubilizing the worms in NCS (Amersham) and determining the radioactivity by liquid scintillation. Conventional double isotope counting techniques were used [5] and dpm determinations normalized according to tissue dry weight. Weight per worm values determined in preliminary experiments, were 29.6 +- 0.71 /ag (n = 100) for females and 75.0 + 1.29/ag (n = 100) for males. The data were analyzed by compartmental analysis using 'curve-peeling' techniques [6, 7] (see Results). All data are presented as means • standard errors of slopes and intercepts [81. RESULTS The data obtained in the present study are shown in Fig. 1 and 2 while the constants derived from these figures are shown in Table I. The curves all depicted first order kinetics in linear plots (not shown). In Fig. 1 and 2 the data are plotted semilogarithmically with the vertical axis a ratio of radiolabel at each time interval (Gait, Glut, MGt) over the initial tissue activity (Gait=o, Glut= 0, MGt=o ) for outward exchange studies, or the asymptote of first order kinetic curves obtained by iteration [5,9] for inward exchange studies (Galex, GlUex, MGex ). By convention it is necessary to subtract the positive in-
347
•
"2"..
tf~
~t " ""§'. i
.Ol
*galex
*gait= o ,u,
*a I
I
1
-
minutes 1601 r
_-
*R I
I
I
16ol
*T I
I
I
I
-
minutes
16C~
*T IIIIJ~
Fig. l. Rate curves for inward exchange (*R) and outward exchange (*T) of galactose in adult male and female S. mansoni. Curves 1 and 2 represent PEG retention where curve 1 is a smaller, more rapidly exchanging pool (PEG retained on surface) than curve 2, a larger more slowly exchanging pool (PEG accumulates in the cecum). Curve 5 is the total exchange curve for galactose while curve 3 is the difference between curve 5 and 1+2. Each point is the average of at least 6 experiments. See Table I for constants derived from this figure and see text for details. ward exchange values from unity to obtain exponentially decreasing functions [6, 10]. The slopes have the same numerical values as only the sign is changed by this convention. This procedure also allows for direct graphical comparisons o f outward and inward rates o f exchange of labeled substrates. The constants depicted in Table I were derived by a 'curve peeling' procedure [6, 10]. Considering first curves 1 and 2 (results for PEG expressed as proportions o f total uncorrected exchange o f sugars, curve 5), this procedure involves adding points from right to left o f each curve until a significant deviation from linearity occurs, i f the curve can be represented as a single log-linear function, then one assumes that the label is exchanging with a single compartment or pool. However, as in the case o f PEG results, a significant deviation from linearity indicates more than one pool. In this case, curve 2 was subtracted from curves 1 + 2 and, in Fig. 1, the difference is plotted as a second pool for PEG marked as the lower curve 1. Thus, PEG exchanges with two different pools; one which is larger and more slowly exchangeable and a second smaller, more rapidly exchanging pool. With all the results for PEG combined, the latter compartment was equivalent to 11.6% o f the total exchangeable sugar compartment with an exchange rate of 0.278/amol • min -~ . The larger PEG pool was equivalent Io 19% of the total exchangeable sugar pool and exchanged at a rate o f 0.007 /~mol • min -a . The smaller pool is termed the label in the surface unstirred layer, crypts and folds, while the larger compartment is considered to represent the label exchanging with the contents o f the cecum (see Discussion).
0.338 ± 0.014
I 0.084 -+ 0.011 O 0.145 ± 0.010
M
0.360 ± 0.031 0,252 ± 0.015
0.353 ± 0,025 0.234 ± 0.019
0.222±0.009
I 0.047 -+ 0.009 O 0.115 ± 0.008
0.352-+0.012
0.072-+0.009
O 0.116-+0.011
0.214 ± 0.019
1
0,344 ± 0.011
0.039 ± 0.008
O 0.101 ± 0.008
0.162 ± 0.012
1
0.319 ± 0,015
0.168 +- 0.013
0.188 -+ 0.010
O 0 . t 7 5 -+ 0.010
0.175 ± 0,011
O
I
0.155 ± 0.016
1
F
M
IT
M
F
0.122 ± 0.008 0.185 ± 0.014
0.120 ± 0.010 0.165 ±- 0.011
0.172-+0.009
0.116±0.008
0.151 ± 0.013
0.104 +- 0.013
0.214 -+ 0.014
0.351 ± 0.015
0.254 ± 0.012
0.326 -+ 0.018
0.006 ± 0,001 0.002 -+ 0.001
0.012 ± 0,001 0.003 ± 0.001
0.003 -+ 0~001
0.00850.001
0.004 ± 0.001
0.011 ± 0.002
0.009 -+ 0.001
0.009 ÷ 0 . 0 0 l
0.008 ± 0.001
0.010 + 0,002
X2
0.790 ± 0.021 0.670 ± 0.028
0.831 ± 0.029 0.721 ± 0.024
0.571±0,010
0.663-+0.014
0.591 ± 0.015
0.659 -+ 0~021
0.637 -+ 0,012
0.515 ± 0.018
0.570 ± 0.014
0.520 -+ 0.021
a3
Tissue
0.061 -+ 0.004 0.033 ± 0,001
0.048 ± 0.004 0.031 ± 0.003
0.041+-0.003
0.072±0.002
0.036 ± 0.002
0.064 ± 0.003
0,040 ± 0.002
0.030 -+ 0.003
0.039 -+ 0.003
0.027 ÷ 0.002
X3
0.146±0.011
0.151 -+0.011
0.162 ± 0.015
0 A 9 1 -+ 0.007
a4
0.t18±0.006
0.141 -+0.002
0,166 ± 0.011
0.128 + 0.002
X4
Metabolic c o m p o n e n t
x I - x 4 are the slopes ± S.E. of lines 1 - 4 in Figs. 1 and 2. The slopes are in units of p m o l • min -1 and the intercepts are d ecimal fractions (%) of total exchangeable sugar pools,
The data are presented in the form; total exchangeable pool ( y ) = a~ e x~ t = a2e-.X~ t + a3e-X~ t + a4e-X~ t where a~-o a 4 are the intercepts ± S,E, and
Gal
Gtu
MG
aS
al
XI
Cecum
Surface volume
in adult male (M) and female [F) Schistosoma mansoni
Steady-state inward (/) and outward (O) rates of exchange and c o m p a r t m e n t s of radiolabeled glucose (Glu), galactose (Gal) and 3-O-methylglucose (MG)
TABLE I
349
However, the curve peeling procedures used in this study are usually restricted to the separation o f 2 to 3 compartments [6, 10]. For example, the total uncorrected exchange o f sugar (curve 5 in Figs. 1 and 2) is a complex curve which we could not analyze by 'curve peeling' since no more than approximately four or five points yielded a straight line. Hence, more compartments could be distinguished than could be considered realistically or statistically significant using 'curve peeling'. However, since the sugars will exchange with the same pools delineated b y the extracellular marker (PEG) [5, 9, 10], it is possible to exclude these pools from the sugar data b y subtracting curves 1 and 2 from curve 5. In the present study, this procedure was used to derive curve 3 in Figs. 1 and 2, which is labeled as the tissue compartment (Table I ) . The results for glucose indicated the presence o f a second tissue compartment represented as curve 4 (metabolic component in Table I; see Discussion). Therefore, the steady-state hexose fluxes examined in the present study could be described by a rate equation o f the following form:
y =ale-X~ t+a2e-X2t+a3e-X3t+a4e-X4t
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~
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,
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~O
Fig. 2. Rate curves for inward exchange (A,B) and outward exchange (C,D) of 3-O-methylglucose (MG) and glucose (Glu) into adult male and female S. mansoni. Curves 1 and 2 represent PEG retention where curve t represents a small, rapidly exchanging pool (PEG retained on surface of flukes) and curve 2 represents a larger, more slowly exchanging pool of PEG (PEG accumulation in gut of flukes). Curve 5 is total exchange of sugar while curve 3 is the difference between curves 5 and 1+2. For glucose results, alinear portion at top of curve 3 is labeled as curve 4. Each point is the average of at least 6 experiments, each consisting of incubations of 5 worms. See text for details and Table I for constants derived from these curves.
350 where y is the total exchangeable pool of hexose, a represents the relative size of each compartment; x the rate constant for exchange of label with each compartment; t is time; and subscripts 1 - 4 represent different compartments where 1 is the surface unstirred layer; 2 the cecal volume, 3 the tissue compartment and, 4 the metabolic component in the case of glucose fluxes. With all of the results for the three sugars combined, the tissue pool represented 64.5% of the total and the exchange rate across the surface was 0.044 /amol • rain -1 . There were no significant differences in relative pool sizes or exchange rates for any of the compartments between male and female parasites although a~ and a2 were generally larger in males (Table I). Also, a~ was larger (p<0.05, Student's t-test) in the outward exchange studies than in the inward exchange studies but x~ was smaller (p<0.05) in the outward direction. For the tissue compartment, a 3 and x3 were larger (p<0.05) when the net exchange of sugar occurred from outside to inside than vice versa. For both male and female parasites, x3 was greatest for glucose (p<0.05) followed by galactose and 3-0methylglucose when inward exchange rates were considered. No significant differences occurred in x3 when outward exchange rates were considered. DISCUSSION The steady-state compartmental analysis of hexose fluxes presented in this study for male and female S. m a n s o n i has several advantages over methods used previously to examine sugar fluxes in schistosomes and other helminths. The kinetics derived from steady-state fluxes are not altered by unstirred layer effects as in short-term initial-rate studies [1]. The use of a non-absorbable marker (PEG) excludes from the flux determination all labeled substrate in surface unstirred layers, label trapped in surface pits and crypts, and label in the gut of the parasites. Compartmental analysis yields both the rate constants for the fluxes and the relative sizes of the radiolabeled substrate pools. Since long-term incubations are used, then fewer parasites and less labeled substrate are required to load tissues with significant amounts of labeled substrate. The rate constants can be compared with previous studies by converting the rate constants to K t values in units of mM [11]. Using the steady-state definition of K t [11], we derived values for galactose of 0.65 mM (9) and 0.54 mM (d), for glucose 0.56 mM (9) and 0.57 mM (d) and for methylglucose, 1.44 mM (9) and 1.33 mM (d). These K t values are lower than those determined elsewhere [2, 3, 12], the reason being that previous studies used initial-rate kinetics which, due to unstirred layer effects, yield artifactually high K t values [9]. The method used in the present study yields sizes and rate constants for radiotracer compartments but does not indicate the morphological or functional aspects of the pools. The first pool (a~, x~ ) is considered in this study to be equivalent to the surface unstirred layers since the rate constant is close to that obtained for similar hydrodynamic surface layers in H y m e n o l e p i s d i m i n u t a [9]. Since the second compartment is also labeled by PEG and is therefore extracellular, it must represent the cecal compartment.
351 This leaves the third compartment which must therefore represent sugar fluxes across the surface epithelial syncytium into the tissue compartment of S. mansoni. However, we have not determined in the present study the rate constant for uptake across the cecal epithelium, estimates of which will appear in a separate study. Nevertheless, the latter cannot exceed the rate constants obtained for entry into or out of the cecum (x2), indicating that this route of uptake will be slow relative to transport across the surface epithelial syncytium (on average x2 was approximately 16% of x3). The major problem with the method used in this study, as in all long-term uptake studies, is metabolism of the substrate. Of the three substrates used in this study only glucose is readily metabolized, although galactose may be utilized to a smaller extent [9, 12]. The major difference in the exchange kinetics of the three sugars was the alinear portion observed in the glucose curves but not those for galactose and methylglucose. Two pools were therefore distinguished for glucose fluxes, a larger, more slowly exchanging pool and a smaller, more rapidly exchanging pool. Although further studies will be required to identify these glucose pools, we report in the present study that the smaller, more rapidly exchanging pool (aa, x4) probably represents that component in which glucose is being removed from or added to the transport pool of glucose through metabolic processes. Our basis for this conclusion is that during steady-state exchange the tissue pools of glucose should be relatively constant and high so that the inward and outward exchange rates (aa) will be similar relative to the more rapid metabolic compartment (a4). The flux of glucose through metabolic pathways should be rapid relative to the rate of transport of glucose into or out of a tissue pool where glucose must traverse a long diffusion path and cross a series of membranes. Regardless of the outcome of further studies to identify the glucose pools, the difference between metabolized and nonmetabolized substrates may lie in the linearity of the steady-state compartmental fluxes. Although the inward and outward exchange rates were smaller than the metabolic component, the inward rate constants were greater than the outward rate constants for glucose (I/0: 9 1.78; 6 1.76) and galactose (I/0: ~? 1.55; d 1.85). This asymmetry is typical of transporting epithelia [1, 4]. However, the ratios obtained for methylglucose were less than unity (9 0.69; c~0.75). The difference between methylglucose and the other two sugars was not in the outward exchange rates, which were similar for the three sugars, but in the inward rate constants. This would be expected if, as indicated in the K t calculations above, the surface transport agency has a lower affinity for methylglucose. Also, the surface (al) and cecal (a2) compartments were generally larger when the worms were exposed to methylglucose. Although the reasons for this difference are not clear, these results indicate that methylglucose may not be an ideal substrate for hexose transport studies in S. mansoni. The results of the present study also give information relating to the size of the unstirred layers and cecal compartments from the distribution volumes of the marker substance (PEG) [13]. In the present study, we calculated the size of the unstirred layer on female S. mansoni (29.6 ~tg dry weight) of 0.64 ~1 and 1.01 /.d for males (75.0
352
/ag dry weight). The female c e c u m was 0 . 3 4 / A while the male c e c u m was 1.28 tal, averaged for the PEG distribution volumes in all o f the experiments. ACKNOWLEDGEMENTS This study was supported by grants to R.B.P. f r o m the Natural Sciences and Engineering Research Council o f Canada and the J.P. Bickell F o u n d a t i o n . Technical assistance was supplied b y S.F. T i m m e r s in some o f these studies. REFERENCES 1
2 3 4 5
6 7 8 9 10 11 12 13
Podesta, R.B. (1980) Concepts of membrane biology in Hymenolepis diminuta. In: The Biology of the Tapeworm Hymenolepis diminuta, (Ariai, H.P. ed.), pp. 505-549, Academic Press, Inc. New York. Cornford, E.M. and Oldendorf, W.H. (1979) Transintegumental uptake of metabolic substrates in male and female Schistosoma mansoni. J. Parasitol. 65,375-383. Cornford, E.M., Bocash, W.D. and Oldendorf, W.H. (1981) Transintegumental glucose uptake in Schistosornatium douthitti. J. Parasitol. 67, 2 4 - 3 0 . Brodie, D.A. and Podesta, R.B. (1981) 3HOH-osmotic water fluxes and ultrastructure of an epithelial syncytium. J. Membrane Biol. 61,107-114. Podesta, R.B., Stallard, H.E., Evans, W.S., Lussier, P.E., Jackson, D.J. and Mettrick, D.F. (1977) Hymenolepis diminuta: Determination of unidirectional uptake rates for nonelectrolytes across the surface epithelial membrane. Exp. Parasitol. 4 2 , 3 0 0 - 3 1 7 . Jacquez, J.A. (1972) Compartmental Analysis in Biology and Medicine, Elsevier Publishing Co., Amsterdam. Brown, R.F. (1980) Compartmental system analysis: State of the art. Trans. Biomed. Engineering 27, 1-11. Sokal, R.R. and Rohlf, F.J. (1969) Biometry, W.H. Freeman and Co., San Francisco. Podesta, R.B. (1977) Hymenolepis diminuta: Unstirred layer thickness and effects on active and passive transport kinetics. Exp. Parasitol. 43, 12- 24. Kotyk, A. and Janacek, K. (1975) Cell Membrane Transport. Principles and Techniques, Plenum Press, New York and London. Bergmeyer, H.U. (1978) Principles of Enzymatic Analysis, Verlag Chemie, Weinheim, New York. Pappas, P.W. and Read, C.P. (1975) Membrane transport in helminth parasites. Exp. Parasitol. 37,469-530. Podesta, R.B. (1977) Hymenolepis diminuta: Marker distribution volumes of tissues and mucosal extracellular space. Exp. Parasitol. 41,289 299.
345
Elsevier Biomedical Press
A METHOD F O R DETERMINING NONELECTROLYTE UPTAKE BY ADULT S C H I S T O S O M A M A N S O N ! USING COMPARTMENTAL ANALYSIS OF HEXOSE
FLUXES
RON B. PODESTA and LYN L. DEAN Department o f Zoology, University o f Western Ontario, London, Ontario, Canada N6A 5B7.
(Received 4 November 1981; accepted 8 February 1982)
A steady-state compartmental analysis of galactose, glucose and 3-O-methylglucose fluxes was conducted on male and female Schistosoma mansoni. The method has several advantages over previously used initial rate studies. A nonlinear log-linear plot of glucose exchange is discussed in terms of differentiating between metabolized and nonmetabolized substrates in transport studies. K t values and marker distribution volumes are estimated from the compartmental analysis and it is concluded that 3-O-methylglucose is not suited as a substrate for hexose transport studies in S. mansoni. Key words: Schistosoma mansoni, Compartmental analysis, Hexose transport, Distribution volumes.
INTRODUCTION Considerable effort has been expended endeavoring to examine fluxes o f organic solutes across the surface o f schistosomes [1]. These studies have been hampered by an inability to distinguish between radiolabeled solutes adhering to the surface o f the parasites, label entering the cecum o f the parasites, label being taken up across the cecal epithelium and label entering the parasites across the surface epithelial syncytium [ t ] . Another problem arises from the use in previous studies o f initial rate studies in which the diffusion barrier represented b y the surface hydrodynamic layers alters active and/or diffusive kinetics o f overall transport across the surface o f the parasites [1 ]. The above problems have been partially overcome in a promising method devised recently by Cornford and co-workers [2, 3 ] . These workers used a nonabsorbable marker which effectively eliminates from uptake determinations labeled substrates which are distributed in the surface unstirred layer, crypts and folds and label present in the lumen o f the cecum. However, the method o f normalization o f the data presented by these workers depends on the assumption that water uptake across the surface o f the parasites occurs by simple diffusion. This assumption has not been confirmed in a recent
Abbreviations: Gal, galactose; Glu, glucose; MG, 3-O-methylglucose; PEG, polyethylene glycol.
0166-6851/82/0000-0000/$02.75
© 1982 Elsevier Biomedical Press
346 study of the diffusive and osmotic components of water fluxes across the surface epithelial syncytium of Schistosoma mansoni [4]. Also, Cornford et al. [2, 3] used initial rate studies so that the derived kinetics will be subject to change pending the outcome of studies designed to examine the effect of unstirred layers. In the present study we develop a simple steady-state flux method using compartmental analysis of hexose fluxes in S. mansoni. MATERIALS AND METHODS
Schistosoma mansoni (Puerto Rican strain) were maintained in our laboratory through the infection of Syrian hamsters and snails (Biornphalaria glabrata). Adult parasites were obtained from the perfused hepatic portal veins of hamsters, 45 days post-infection, using conventional techniques. The composition of the balanced electrolyte solution used for perfusions and incubations in this study, has been described elsewhere [4, 5]. Groups of five male or female flukes were then incubated for 30 min in 5 ml of incubation fluid in a shaking water bath at 37°C. During this incubation period, the parasites were either exposed to 1 mM unlabeled sugar (glucose, galactose or 3-O-methylglucose) for inward exchange rate determinations, or radiolabeled sugar (1.0/aCi/ml) for outward exchange rate determinations. The non-permeable marker compound [3 HI polyethylene glycol 4000 (PEG; NEN) was used to quantify the amount of fluid and sugar adherent to the worm surface and distributed in the cecal lumen. The 30 min incubation was followed by a second incubation period of up to 60 min in a fluid containing 1 mM sugar in the efflux experiments and labeled sugar with [3H] PEG for the inward exchange group. Changes in levels of radiolabeled sugar and marker were determined at specific time intervals during the second incubation by solubilizing the worms in NCS (Amersham) and determining the radioactivity by liquid scintillation. Conventional double isotope counting techniques were used [5] and dpm determinations normalized according to tissue dry weight. Weight per worm values determined in preliminary experiments, were 29.6 +- 0.71 /ag (n = 100) for females and 75.0 + 1.29/ag (n = 100) for males. The data were analyzed by compartmental analysis using 'curve-peeling' techniques [6, 7] (see Results). All data are presented as means • standard errors of slopes and intercepts [81. RESULTS The data obtained in the present study are shown in Fig. 1 and 2 while the constants derived from these figures are shown in Table I. The curves all depicted first order kinetics in linear plots (not shown). In Fig. 1 and 2 the data are plotted semilogarithmically with the vertical axis a ratio of radiolabel at each time interval (Gait, Glut, MGt) over the initial tissue activity (Gait=o, Glut= 0, MGt=o ) for outward exchange studies, or the asymptote of first order kinetic curves obtained by iteration [5,9] for inward exchange studies (Galex, GlUex, MGex ). By convention it is necessary to subtract the positive in-
347
•
"2"..
tf~
~t " ""§'. i
.Ol
*galex
*gait= o ,u,
*a I
I
1
-
minutes 1601 r
_-
*R I
I
I
16ol
*T I
I
I
I
-
minutes
16C~
*T IIIIJ~
Fig. l. Rate curves for inward exchange (*R) and outward exchange (*T) of galactose in adult male and female S. mansoni. Curves 1 and 2 represent PEG retention where curve 1 is a smaller, more rapidly exchanging pool (PEG retained on surface) than curve 2, a larger more slowly exchanging pool (PEG accumulates in the cecum). Curve 5 is the total exchange curve for galactose while curve 3 is the difference between curve 5 and 1+2. Each point is the average of at least 6 experiments. See Table I for constants derived from this figure and see text for details. ward exchange values from unity to obtain exponentially decreasing functions [6, 10]. The slopes have the same numerical values as only the sign is changed by this convention. This procedure also allows for direct graphical comparisons o f outward and inward rates o f exchange of labeled substrates. The constants depicted in Table I were derived by a 'curve peeling' procedure [6, 10]. Considering first curves 1 and 2 (results for PEG expressed as proportions o f total uncorrected exchange o f sugars, curve 5), this procedure involves adding points from right to left o f each curve until a significant deviation from linearity occurs, i f the curve can be represented as a single log-linear function, then one assumes that the label is exchanging with a single compartment or pool. However, as in the case o f PEG results, a significant deviation from linearity indicates more than one pool. In this case, curve 2 was subtracted from curves 1 + 2 and, in Fig. 1, the difference is plotted as a second pool for PEG marked as the lower curve 1. Thus, PEG exchanges with two different pools; one which is larger and more slowly exchangeable and a second smaller, more rapidly exchanging pool. With all the results for PEG combined, the latter compartment was equivalent to 11.6% o f the total exchangeable sugar compartment with an exchange rate of 0.278/amol • min -~ . The larger PEG pool was equivalent Io 19% of the total exchangeable sugar pool and exchanged at a rate o f 0.007 /~mol • min -a . The smaller pool is termed the label in the surface unstirred layer, crypts and folds, while the larger compartment is considered to represent the label exchanging with the contents o f the cecum (see Discussion).
0.338 ± 0.014
I 0.084 -+ 0.011 O 0.145 ± 0.010
M
0.360 ± 0.031 0,252 ± 0.015
0.353 ± 0,025 0.234 ± 0.019
0.222±0.009
I 0.047 -+ 0.009 O 0.115 ± 0.008
0.352-+0.012
0.072-+0.009
O 0.116-+0.011
0.214 ± 0.019
1
0,344 ± 0.011
0.039 ± 0.008
O 0.101 ± 0.008
0.162 ± 0.012
1
0.319 ± 0,015
0.168 +- 0.013
0.188 -+ 0.010
O 0 . t 7 5 -+ 0.010
0.175 ± 0,011
O
I
0.155 ± 0.016
1
F
M
IT
M
F
0.122 ± 0.008 0.185 ± 0.014
0.120 ± 0.010 0.165 ±- 0.011
0.172-+0.009
0.116±0.008
0.151 ± 0.013
0.104 +- 0.013
0.214 -+ 0.014
0.351 ± 0.015
0.254 ± 0.012
0.326 -+ 0.018
0.006 ± 0,001 0.002 -+ 0.001
0.012 ± 0,001 0.003 ± 0.001
0.003 -+ 0~001
0.00850.001
0.004 ± 0.001
0.011 ± 0.002
0.009 -+ 0.001
0.009 ÷ 0 . 0 0 l
0.008 ± 0.001
0.010 + 0,002
X2
0.790 ± 0.021 0.670 ± 0.028
0.831 ± 0.029 0.721 ± 0.024
0.571±0,010
0.663-+0.014
0.591 ± 0.015
0.659 -+ 0~021
0.637 -+ 0,012
0.515 ± 0.018
0.570 ± 0.014
0.520 -+ 0.021
a3
Tissue
0.061 -+ 0.004 0.033 ± 0,001
0.048 ± 0.004 0.031 ± 0.003
0.041+-0.003
0.072±0.002
0.036 ± 0.002
0.064 ± 0.003
0,040 ± 0.002
0.030 -+ 0.003
0.039 -+ 0.003
0.027 ÷ 0.002
X3
0.146±0.011
0.151 -+0.011
0.162 ± 0.015
0 A 9 1 -+ 0.007
a4
0.t18±0.006
0.141 -+0.002
0,166 ± 0.011
0.128 + 0.002
X4
Metabolic c o m p o n e n t
x I - x 4 are the slopes ± S.E. of lines 1 - 4 in Figs. 1 and 2. The slopes are in units of p m o l • min -1 and the intercepts are d ecimal fractions (%) of total exchangeable sugar pools,
The data are presented in the form; total exchangeable pool ( y ) = a~ e x~ t = a2e-.X~ t + a3e-X~ t + a4e-X~ t where a~-o a 4 are the intercepts ± S,E, and
Gal
Gtu
MG
aS
al
XI
Cecum
Surface volume
in adult male (M) and female [F) Schistosoma mansoni
Steady-state inward (/) and outward (O) rates of exchange and c o m p a r t m e n t s of radiolabeled glucose (Glu), galactose (Gal) and 3-O-methylglucose (MG)
TABLE I
349
However, the curve peeling procedures used in this study are usually restricted to the separation o f 2 to 3 compartments [6, 10]. For example, the total uncorrected exchange o f sugar (curve 5 in Figs. 1 and 2) is a complex curve which we could not analyze by 'curve peeling' since no more than approximately four or five points yielded a straight line. Hence, more compartments could be distinguished than could be considered realistically or statistically significant using 'curve peeling'. However, since the sugars will exchange with the same pools delineated b y the extracellular marker (PEG) [5, 9, 10], it is possible to exclude these pools from the sugar data b y subtracting curves 1 and 2 from curve 5. In the present study, this procedure was used to derive curve 3 in Figs. 1 and 2, which is labeled as the tissue compartment (Table I ) . The results for glucose indicated the presence o f a second tissue compartment represented as curve 4 (metabolic component in Table I; see Discussion). Therefore, the steady-state hexose fluxes examined in the present study could be described by a rate equation o f the following form:
y =ale-X~ t+a2e-X2t+a3e-X3t+a4e-X4t
d
~
".oo
" -5 "~.2.
\
• .2:
o,"
"
"5
"
"\
of
\.
AI
I
""
-5.
\ "','.2.
~ l l l l
~
.". •.
"~'.A • •.
o
..o \
\'.
,,.5 01
'
~ "
0.'
.
"5 ' \ "2"
,\ \
3-
D
I
r,Ominutes
6o
,
\,3
,
m~nOutes
I
\
~O
Fig. 2. Rate curves for inward exchange (A,B) and outward exchange (C,D) of 3-O-methylglucose (MG) and glucose (Glu) into adult male and female S. mansoni. Curves 1 and 2 represent PEG retention where curve t represents a small, rapidly exchanging pool (PEG retained on surface of flukes) and curve 2 represents a larger, more slowly exchanging pool of PEG (PEG accumulation in gut of flukes). Curve 5 is total exchange of sugar while curve 3 is the difference between curves 5 and 1+2. For glucose results, alinear portion at top of curve 3 is labeled as curve 4. Each point is the average of at least 6 experiments, each consisting of incubations of 5 worms. See text for details and Table I for constants derived from these curves.
350 where y is the total exchangeable pool of hexose, a represents the relative size of each compartment; x the rate constant for exchange of label with each compartment; t is time; and subscripts 1 - 4 represent different compartments where 1 is the surface unstirred layer; 2 the cecal volume, 3 the tissue compartment and, 4 the metabolic component in the case of glucose fluxes. With all of the results for the three sugars combined, the tissue pool represented 64.5% of the total and the exchange rate across the surface was 0.044 /amol • rain -1 . There were no significant differences in relative pool sizes or exchange rates for any of the compartments between male and female parasites although a~ and a2 were generally larger in males (Table I). Also, a~ was larger (p<0.05, Student's t-test) in the outward exchange studies than in the inward exchange studies but x~ was smaller (p<0.05) in the outward direction. For the tissue compartment, a 3 and x3 were larger (p<0.05) when the net exchange of sugar occurred from outside to inside than vice versa. For both male and female parasites, x3 was greatest for glucose (p<0.05) followed by galactose and 3-0methylglucose when inward exchange rates were considered. No significant differences occurred in x3 when outward exchange rates were considered. DISCUSSION The steady-state compartmental analysis of hexose fluxes presented in this study for male and female S. m a n s o n i has several advantages over methods used previously to examine sugar fluxes in schistosomes and other helminths. The kinetics derived from steady-state fluxes are not altered by unstirred layer effects as in short-term initial-rate studies [1]. The use of a non-absorbable marker (PEG) excludes from the flux determination all labeled substrate in surface unstirred layers, label trapped in surface pits and crypts, and label in the gut of the parasites. Compartmental analysis yields both the rate constants for the fluxes and the relative sizes of the radiolabeled substrate pools. Since long-term incubations are used, then fewer parasites and less labeled substrate are required to load tissues with significant amounts of labeled substrate. The rate constants can be compared with previous studies by converting the rate constants to K t values in units of mM [11]. Using the steady-state definition of K t [11], we derived values for galactose of 0.65 mM (9) and 0.54 mM (d), for glucose 0.56 mM (9) and 0.57 mM (d) and for methylglucose, 1.44 mM (9) and 1.33 mM (d). These K t values are lower than those determined elsewhere [2, 3, 12], the reason being that previous studies used initial-rate kinetics which, due to unstirred layer effects, yield artifactually high K t values [9]. The method used in the present study yields sizes and rate constants for radiotracer compartments but does not indicate the morphological or functional aspects of the pools. The first pool (a~, x~ ) is considered in this study to be equivalent to the surface unstirred layers since the rate constant is close to that obtained for similar hydrodynamic surface layers in H y m e n o l e p i s d i m i n u t a [9]. Since the second compartment is also labeled by PEG and is therefore extracellular, it must represent the cecal compartment.
351 This leaves the third compartment which must therefore represent sugar fluxes across the surface epithelial syncytium into the tissue compartment of S. mansoni. However, we have not determined in the present study the rate constant for uptake across the cecal epithelium, estimates of which will appear in a separate study. Nevertheless, the latter cannot exceed the rate constants obtained for entry into or out of the cecum (x2), indicating that this route of uptake will be slow relative to transport across the surface epithelial syncytium (on average x2 was approximately 16% of x3). The major problem with the method used in this study, as in all long-term uptake studies, is metabolism of the substrate. Of the three substrates used in this study only glucose is readily metabolized, although galactose may be utilized to a smaller extent [9, 12]. The major difference in the exchange kinetics of the three sugars was the alinear portion observed in the glucose curves but not those for galactose and methylglucose. Two pools were therefore distinguished for glucose fluxes, a larger, more slowly exchanging pool and a smaller, more rapidly exchanging pool. Although further studies will be required to identify these glucose pools, we report in the present study that the smaller, more rapidly exchanging pool (aa, x4) probably represents that component in which glucose is being removed from or added to the transport pool of glucose through metabolic processes. Our basis for this conclusion is that during steady-state exchange the tissue pools of glucose should be relatively constant and high so that the inward and outward exchange rates (aa) will be similar relative to the more rapid metabolic compartment (a4). The flux of glucose through metabolic pathways should be rapid relative to the rate of transport of glucose into or out of a tissue pool where glucose must traverse a long diffusion path and cross a series of membranes. Regardless of the outcome of further studies to identify the glucose pools, the difference between metabolized and nonmetabolized substrates may lie in the linearity of the steady-state compartmental fluxes. Although the inward and outward exchange rates were smaller than the metabolic component, the inward rate constants were greater than the outward rate constants for glucose (I/0: 9 1.78; 6 1.76) and galactose (I/0: ~? 1.55; d 1.85). This asymmetry is typical of transporting epithelia [1, 4]. However, the ratios obtained for methylglucose were less than unity (9 0.69; c~0.75). The difference between methylglucose and the other two sugars was not in the outward exchange rates, which were similar for the three sugars, but in the inward rate constants. This would be expected if, as indicated in the K t calculations above, the surface transport agency has a lower affinity for methylglucose. Also, the surface (al) and cecal (a2) compartments were generally larger when the worms were exposed to methylglucose. Although the reasons for this difference are not clear, these results indicate that methylglucose may not be an ideal substrate for hexose transport studies in S. mansoni. The results of the present study also give information relating to the size of the unstirred layers and cecal compartments from the distribution volumes of the marker substance (PEG) [13]. In the present study, we calculated the size of the unstirred layer on female S. mansoni (29.6 ~tg dry weight) of 0.64 ~1 and 1.01 /.d for males (75.0
352
/ag dry weight). The female c e c u m was 0 . 3 4 / A while the male c e c u m was 1.28 tal, averaged for the PEG distribution volumes in all o f the experiments. ACKNOWLEDGEMENTS This study was supported by grants to R.B.P. f r o m the Natural Sciences and Engineering Research Council o f Canada and the J.P. Bickell F o u n d a t i o n . Technical assistance was supplied b y S.F. T i m m e r s in some o f these studies. REFERENCES 1
2 3 4 5
6 7 8 9 10 11 12 13
Podesta, R.B. (1980) Concepts of membrane biology in Hymenolepis diminuta. In: The Biology of the Tapeworm Hymenolepis diminuta, (Ariai, H.P. ed.), pp. 505-549, Academic Press, Inc. New York. Cornford, E.M. and Oldendorf, W.H. (1979) Transintegumental uptake of metabolic substrates in male and female Schistosoma mansoni. J. Parasitol. 65,375-383. Cornford, E.M., Bocash, W.D. and Oldendorf, W.H. (1981) Transintegumental glucose uptake in Schistosornatium douthitti. J. Parasitol. 67, 2 4 - 3 0 . Brodie, D.A. and Podesta, R.B. (1981) 3HOH-osmotic water fluxes and ultrastructure of an epithelial syncytium. J. Membrane Biol. 61,107-114. Podesta, R.B., Stallard, H.E., Evans, W.S., Lussier, P.E., Jackson, D.J. and Mettrick, D.F. (1977) Hymenolepis diminuta: Determination of unidirectional uptake rates for nonelectrolytes across the surface epithelial membrane. Exp. Parasitol. 4 2 , 3 0 0 - 3 1 7 . Jacquez, J.A. (1972) Compartmental Analysis in Biology and Medicine, Elsevier Publishing Co., Amsterdam. Brown, R.F. (1980) Compartmental system analysis: State of the art. Trans. Biomed. Engineering 27, 1-11. Sokal, R.R. and Rohlf, F.J. (1969) Biometry, W.H. Freeman and Co., San Francisco. Podesta, R.B. (1977) Hymenolepis diminuta: Unstirred layer thickness and effects on active and passive transport kinetics. Exp. Parasitol. 43, 12- 24. Kotyk, A. and Janacek, K. (1975) Cell Membrane Transport. Principles and Techniques, Plenum Press, New York and London. Bergmeyer, H.U. (1978) Principles of Enzymatic Analysis, Verlag Chemie, Weinheim, New York. Pappas, P.W. and Read, C.P. (1975) Membrane transport in helminth parasites. Exp. Parasitol. 37,469-530. Podesta, R.B. (1977) Hymenolepis diminuta: Marker distribution volumes of tissues and mucosal extracellular space. Exp. Parasitol. 41,289 299.