Hymenolepis diminuta: The non-saturable component of methionine uptake

Internalionsl Journalfor Printed in Great Britain.

Pamsrlology Vo/. 12, No. 4, pp. 265-270,

1982.

0020-7519/82/040265-06$03.00/0 Pergomon Press Ltd. 0 1982Auslralian Society for Parasitology

HYMENOLEPIS DIMINUTA: THE NON-SATURABLE METHIONINE UPTAKE P. E. LUSSIER, R. B. PODESTA*

COMPONENT

OF

and D. F. METTRICK

Department of Zoology, University of Toronto, Toronto, Ontario M5S IAl, Canada (Received 25 February

1981)

Abstract-LussrER P. E., PODESTA R. B. and METTRICKD. F. 1982. Hymenolepis diminuta: the nonsaturable component of methionine uptake. International Journal for Parasitology 12: 265-270. The concentration dependence of in vitro unidirectional methionine influx by Hymenolepis diminuta was analysed by the relation: J = (J,C,)/(K,+ C,) + KAC,), where J,,, is the maximum uptake rate, K1 is the the apparent affinity constant and C, is the medium substrate concentration. The linear component was separated using an asymptotic least squares curve fitting procedure and the resulting constant, Kd, is thought to be an apparent permeability coefficient. Kd may be a reflection of a simple diffusive component, a second mediated component or a combination of a passive and mediated influx. The low Qlo value of the Kd’s for methionine uptake (Q,,,= 1.31) indicated that this component is probably a reflection of diffusion within the membrane. However, the decrease in the Kd component in the presence of leucine and glycine, implies that there is also a small, second, mediated component in addition to the diffusive component. Kd derived from the asymptotic portion of the concentration-flux relation was compared with the residual flux of methionine after near complete inhibition of the mediated component with leucine and glycine. The Kd component was found to be pH-sensitive, increasing as the pH decreased and was not affected by external sodium. Results indicate that the mediated component of methionine influx was accelerated by increasing external Na + and H f concentrations. INDEX KEY WORDS: Methionine; coefficient; Hymenolepis diminuta.

amino acid transport;

INTRODUCTION EXTENSIVE studies

on

amino acid uptake by Hymenolepis diminuta and other cestodes have indicated that there are six membrane components that mediate amino acid transport: (1) a serine system; (2) a leucine system; (3) a phenylalanine system; (4) a glycine system; (5) a dibasic system; and (6) a dicarboxylic system (see review by Pappas & Read, 1975). In contrast to transport studies done on other cells and tissues, amino acid competition has been the only criterion used in defining cestode transport systems and the techniques employed in most previous helminth studies, derived by Read, Rothman & Simmons (1963), must now be reconsidered (Podesta, Stallard, Evans, Lussier, Jackson & Mettrick, 1977). The saline solutions used ubiquitously in tapeworm transport studies contained maleate, a metabolic enzyme inhibitor (Webb, 1966; Podesta & Mettrick, 1974a) and tris, an Na+ ion competitor (Schultz & Curran, 1970). Secondly, extraction procedures of radiolabelled absorbates of widely different polarities have been shown to

*Present Address: Department of Zoology, University of Western Ontario, London, Ontario, N6A 3K7.

inhibitors;

Kd; apparent

permeability

increase error in flux determinations and in the case of helminths, there is an added error in uptake determination due to the increased unstirred layer effect when multiple worm incubations are used in calculating a simple flux rate (Podesta, Stallard, Evans, Lussier, Jackson & Mettrick, 1977). Thirdly, previous techniques fail to take into account the complexity of the transport process, as demonstrated by more recent theoretical and methodological advances for determining unidirectional flux rates in a variety of systems (see Podesta, Stallard, Evans, Lussier, Jackson & Mettrick, 1977). Attempts have been made to separate the passive component of amino acid uptake processes from saturated components (Woodward & Read, 1969; Uglem & Read, 1973; Pappas & Read, 1975), but the methods used can give rise to considerable error in the magnitude of the non-saturated uptake component (see Atkins & Gardner, 1977; Podesta, 1980). Quite apart from the methodological criticisms that can be made of previous studies concerning amino acid uptake by helminth parasites, the previous studies can also be criticised for using linear transformations of the data in determining kinetic parameters (Dowd & Riggs, 1965; Markus, Hess, Ottaway & Cornish-Bowden, 1976; Atkins & 265

P. E. LUSSIER,R. B. PODESTAUI~D. F. METTRICK

266

Gardner, 1977). A least square curvilinear procedure is now available which is considered to offer many over previous analytical methods advantages (Markus et al., 1976) and in addition, a diffusive component, &, is generated by this curve fitting procedure. Since virtually no work has been done on the latter component, experiments were designed to find out whether the passive permeability coefficient, Kd, was actually accounted for by simple passive diffusion, as the constant implies, or whether it represents a mediated component. The pH sensitivity and Na+ dependency for both passive and mediated components were also investigated.

I.J.P. VOL.12. 1982

where J, = flux of solute in n moles per g per h; J,,, = maximum saturable uptake rate in p moles per g per h; K, = apparent affinity constant in mM; K, = apparent permeability coefficient in g moles per g per h per mM; C, = bulk fluid substrate concentration. From this equation, the parameters 1,. Kt and Kd were calculated with the use of a non-linear least squares curve fitting procedure written in Fortran IV, obtained from Dr. J. A. Jacquez, University of Michigan. The asymptotic portion of the fitting procedure (J,,,Cb)/(K, + Cb) gives rise to the Kt and J,,, values. In the equation, Kd(Cb) is the linear component. From this, Kd, the apparent permeability coefficient, can be derived (see Atkins B Gardner, 1977, for a discussion of their method). RESULTS

MATERIALS AND METHODS Male Wistar (Woodlyn Breeding Farms, Guelph) strain rats (120 g) were infected with 30 cysticercoids of H. di~inu~a obtained from Tribo~~u~confuted. Rats were killed 9 days post-infection and the worms were flushed from the small intestine with a prewarmed balanced salt solution. The composition of the balanced salt solution (pH7 BSS) was 130 rnhl NaCl, 5.0 mM KCI, 1.2 rnM MgClz, 1.2 mM CaCI,, I.2 rnM NazHPO,, 0.78 mM NaHzPO,, 30 mM mannitol, 1 mM NaHCO, and ImM polyethylene glycol (PEG). The absorbate-containing balanced salt solutions, the wash and incubation solutions were prepared with the same ingredients as the BSS; however, in the case of the wash solution 5 mM glucose and 25 mM mannitol were added, while all amino arid-containing incubation solutions were prepared so that the combined concentration of amino acid and mannitol was 30 IIIM.The concentration of methionine ranged from 0.1 mM to 160 mr.r and the inhibitor concentrations of glycine and leucine were 4 mM. All balanced salt solutions were gassed with 95% N,: 5% CO, and kept at 37°C except when the effect of temperature dependency was being determined. In this case, the temperature was lowered to 27°C or raised to 47°C and maintained there throughout the experiment. The constituents of the Na + -free preincubation and incubation fluids were the same as for the BSS but instead of NaCI, choline chloride (130 mM) was employed in the basic medium and K + -phosphate buffers (K f increase of <3 mM) were used instead of the Na+-phosphate buffers. Following Bushing from the rat intestine, a11worms were kept for a maximum of 30 min in BSS (wash solution) prior to experimental incubation. Individual tapeworms were blotted on filter paper moistened with wash solution, weighed and placed in a 5 mM preincubation solution for 10 min. Worms were transferred for 2 min to an amino acid incubation solution containing radiolabelled substrate and marker, polyethylene glycoi (PEG) (Podesta, Stailard, Evans, Lussier, Jackson & Mettrick, 1977). Worms were then rinsed in 2 washes of BSS, blotted on moist filter paper and digested in 1 ml N.C.S. (Amersham/Searle) tissue solubilizer at 40°C for I2 h. To the worm digests I5 ml toluene containing PPO (6.0 g/l) and POPOP (0.075 g/l) was added and samnles were counted on a Packard Tricarb Liquid Scintillatjon Spectrometer. After determining the disintegrations per minute (d.p.m.) of 1% and 3H for the amino acids and the marker, the transport rate, J,. was caiculated (see Podesta, Stallard, Evans, Lussier, Jackson & Mettrick, 1977, for equations). The concentration dependence of amino acid uptake can be described by the equation: J,z__

Jmcb K, + cb

f

x;I(Cb)

The curve fitting

procedure, being an iterative approach for finding the best fit for a rectangular hyperbola with a linear portion, fits the relationship shown in Fig. 1 to eqn. I. In Fig. 1, ‘A’, depicting total methionine transport and ‘C’ were both fitted by the iterative curve fitting procedure; K(, J,,,, and I(d values were derived from these two curves. Earlier techniques (Woodward & Read, 1969) depended on maximally inhibiting uptake of the amino acid, giving rise to curve ‘D’. Glycine and leucine are both good inhibitors of methionine uptake (Fig. 2). Previous studies (Woodward & Read, 1969; Uglem & Read, 1973; Pappas & Read, 1975) have assumed that the mediated component in total uptake curve ‘A’ (Fig. 1) is completely inhibited so that the remaining transport, represented by curve ‘D’, is passive. However, since it was not known how to account for the curved shoulder present in the maximally inhibited curve ‘D’, the linear portion of ‘D’ was taken (which in the present case is from 4 mM to 16 mM) and drawn as a straight line with the same slope through the origin, giving rise to ‘E’. This represented a diffusive component. Subtracting ‘E’ from curve ‘A’ gave curve ‘B’, which was then assumed to be the mediated component from which K, and .I,,, values were calculated, However, two serious errors are involved in the estimation of the Kd component by maximally inhibiting the uptake curve. First, the linear portion of ‘D’, assumed to be equivalent to the straight line ‘E’, contains a linear component as well as a non-linear component. This will result in an overestimation of the Kd component of methionine uptake (Fig. 1, D) and an underestimation of the saturable component obtained from the total methionine uptake curve (Fig. 1, A). The kinetic parameters of these data are shown in Fig. 3. The Q,, of the Kd component was found to be 1.31 (Fig. 4). This is consistent with diffusive kinetics, indicating that &, is primarily a passive permeability component. However, a passive component would not be expected to decrease in the presence of other amino acids. As seen in Fig. 3, the inhibited uptake of methionine resulted in an increase in the affinity constant while J,,, decreased. The concomitant

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Methionine

uptake

4

by H. diminuta

8

267

12

16

FIG. 1. Methionine uptake by Hymenolepis diminuta (A) and inhibition by leucine and glycine (D). E represents the slope of the linear portion of D (4-16mM); B = (A-E); C = [A - Kd(Cb - Ci)] where C, is the inhibitor substrate concentration; see text for explanation.

‘“1 2

4

6

8

10

12

14

16

FIG. 2. Uptake of methionine (4.0 mM) by Hymenolepis diminufa in the presence of the competitive inhibitors leucine (W) and glycine (0). The inhibitor concentrations ranged from 0.0 mM to 16.0 mM. Coefficient of variation for each point was 4.9% based on an average sample of 9 worms for each mean.

Met[C] mM FIG. 3. Competitive inhibition of methionine leucine and glycine in Hymenolepis diminufa. concentrations 4 mM. Lineweaver-Burk plots curves shown in Figs. 1 and 2, were obtained kinetic parameters derived from the curve fitting

uptake by Inhibitor of uptake using the procedure.

decrease in Kd suggested that the apparent permeability coefficient contained a slowly saturating mediated component and a diffusive component. The Kd for methionine was found to be pHsensitive (Fig. S), increasing from 0.72 at pH 8-O to 1.33 at pH 6.0 (p
Methionine

268

uptake by H. diminuta

i

A

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12

16

Met [C]mM

FIG. 4. Effect of temperature

6

2

Met

FIG. 5.

Effect

of

and substrate concentration

14

10 [C]

mM

pH on uptake of methionine by

Hymenolepis diminuta.

DISCUSSION

Podesta (1977) has shown that Kd is a function of transfer across the tegumental membrane and not of diffusion across the membrane-bound water, for as

2

on uptake of methionine

the thickness of the unstirred layer was decreased from 251-377 pm to 9-35 pm, the resistance to solute flux offered by the unstirred layer was lowered and Kd increased. Also, because the Qlo of the Kd’s for methionine uptake at 37°C and 27°C was 1.31 (Fig. 4) it indicated that Kd was primarily a passive permeability component. However, since a passive component in the presence of other amino acids would not be expected to decrease (Fig. 3), it can be concluded that Kd is a combination of both passive and mediated uptake. Similar studies by Christensen & Liang (1966), using an asymptotic curve fitting procedure to study non-saturable migration of amino acids by tumor cells, revealed a temperature sensitivity for Kd so high for some amino acids as to exclude simple diffusion as the rate limiting step. Furthermore, the component demonstrated a considerable pH sensitivity. These workers concluded that Kd may represent a passive component, as indicated by low QIO’s, or a slowly saturable active component (Qtc1.3*0), or a combination of both passive and mediated uptake.

6 Met[C]

by Hymenolepis diminuta.

10

14

mM

FIG. 6. Effect of Na+-replacement on uptake of methionine by Hymenolepis diminuta. Choline chloride was used as the substitute for NaCl.

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In the present study, even though the Kd component was not found to be Na+ -dependent, a large mediated component was dependent on this cation. Further studies revealed that uptake of alanine, valine and glycine were also dependent on extracellular Na + (Lussier, Podesta & Mettrick, 1979). Read and co-workers considered that cestodes were unique in that they lacked Na + ion dependency in amino acid transport (Read et al., 1963; Pappas, Uglem & Read, 1974). However, evidence showing that amino acid transport was indeed dependent on extracellular Na + ion has been overlooked and a reexamination of the published data of both Read et al. (1963) and of Isseroff, Ertel & Levy (1976) shows that absorption of certain amino acids by platyhelminths are dependent on extracellular Na+ concentrations. Similarly, Podesta, Evans & Stallard (1977) have shown that alanine influx for both H. diminuta and H. microstoma exhibited both primary and secondary active transport characteristics where Na+ was the major co-ion transported. Another important finding of the present study was that both the passive and active components of methionine influx were accelerated at pH 6.0. In glucose transport, an acidic pH was believed to alter the tightness or mobility of membrane lipids increasing the permeability and solvent drag of glucose flow, while the active component of glucose uptake by H. diminuta was not affected (Podesta & Mettrick, 1974b). If increased H + concentration does alter membrane permeability, the passive component of methionine transport should similarly have increased, as was confirmed in the present study. With respect to the mediated component of methionine influx, the increased flux seems to represent the protonation of the substrate itself to a form which is more reactive with the transport loci. The estimated isoelectric point, where the zwitterionic species of neutral amino acids predominates, corresponds to pH 6.0. Below and above this value, the reactivity of the mediated component decreased (Lussier et al., 1979). In addition, it was found that the Na+ -independent component was responsible for the pH sensitivity while the Na+ dependent component was not significantly altered (Lussier et al., 1979). This is interesting in view of the acidic conditions prevailing in the infected gut (Podesta & Mettrick, 1974a; Mettrick, 1980). The development of new experimental procedures has allowed accurate estimates of unidirectional flux rates. In addition, the use of a curve fitting procedure allowed the determination, by means of a least squares approach, of the kinetic parameters J, and Kt as well as a second uptake component, Kd, overlooked by other methods. It is a more precise measure of the apparent passive permeability component than that which is obtained by inhibition experiments. The additional precision offered with the curve fitting procedure

269

Methionine uptake by H. diminuta

and other methodological

improvements (Podesta, 1977) give added confidence that the results are real and not an artifact of the methods used. It is clear that further experimentation is required to fully define the Kd coefficient of amino acid transport in helminths since virtually no work has been done on this transport parameter. work was supported by the Natural Sciences and Engineering Research Council of

Acknowledgements-This

Canada through grant No. A4667 to DFM and a Postdoctoral Fellowship to RBP.

REFERENCES

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