319

J. Phyeiol. (1977), 271, pp. 319-336 With I plate and 6 text-figurew Printed in Great Britain

KINETICS OF GLUCOSE TRANSPORT BY THE PERFUSED MID-GUT OF THE FRESHWATER PRAWN MACROBRACHIUM ROSENBERGII* BY GREGORY A. AHEARN AND LEIGH A. MAGINNISSt From the Department of Zoology, University of Hawaii at Manoa, Honolulu, Hawaii 96822, U.S.A.

(Received 23 August 1976) SUMMARY

1. Mucosal influx of [3H]glucose was examined in the mid-gut of a freshwater prawn, Macrobrachium rosenbergii, using an in vitro perfusion technique. 2. [3H]glucose transfer across the apical cell membrane of the epithelium exhibited Michaelis-Menten kinetics (Jmi, * = 0- 15 ,mole glucose equiv/g. min Kt = 0417 mM). Under Na-free conditions, glucose influx was significantly reduced and a linear function of substrate concentration, indicative of either slow cellular diffusion (KD = 7-6 x 10-3 mole glucose equiv/g. min. mM) or a facilitated process with a low carrier affinity for the sugar. 3. Phlorizin was a potent competitive inhibitor of glucose influx (K1 = 3-6 x 10-3 mM), galactose and 3-O-methylglucose (3-0-MG) were weak inhibitors, and fructose had no evident effect on glucose uptake. Azide, but not iodoacetate (IAA), significantly depressed influx. 4. Absorbed [3H]glucose was rapidly metabolized by the mid-gut. The majority of accumulated activity within the tissue was in the form of phosphorylated compounds and tritiated water (THO), while only 0-3 % was recovered as free-glucose. 5. Preliminary studies examining transmural [3H]glucose transport, however, demonstrated a significant net mucosal to serosal free-glucose flux across the prawn mid-gut which was Na-dependent and IAA- and phlorizin-sensitive. Two alternative interpretations of the data are advanced as possible mechanisms for transepithelial glucose transport: (1) group translocation, or (2) the operation of an energized, high affinity, baso-lateral sugar transport carrier. * Contribution no. 522 from the Hawaii Institute of Marine Biology, University of Hawaii, Honolulu. t Present address: Department of Physiology, School of Medicine, State University of New York, Buffalo, N.Y. 14214, U.S.A. 11-2

320

G. A. AHEARN AND L. A. MAGINNISS INTRODUCTION

Among decapod crustaceans, considerable research in nutritional physiology has been directed toward the identification and localization of digestive enzymes, and the determination of pH in various alimentary compartments (Vonk, 1960; van Weel, 1970), while little attention has been focused on elucidating the sites and capacity for absorption of organic molecules. Based on histological studies, Yonge (1924) and van Weel (1955) attributed a primary absorptive role to the digestive gland, a secondary role to the mid-gut (intestine), and little or no nutrient uptake to the chitinized foregut and hind gut. Speck & Urich (1970) confirmed the absorptive importance of the digestive gland, but also demonstrated that all segments of the gastro-intestinal tract (stomach, digestive gland, mid-gut, and hind gut) were capable of concentrating radioactively labelled glucose and amino acids. Until recently, no detailed analysis of membrane transfer mechanisms for organic molecules by the crustacean gut had been undertaken. Ahearn (1974, 1976) has since characterized the processes of glycine absorption by the isolated mid-gut of the marine shrimp Penaeus marginatus, and Brick (1975), using an in vitro perfusion technique, has examined lysine transport across the mid-gut of the freshwater Malaysian prawn Macrobrachium ro8enbergii. These studies demonstrated the presence of carriermediated, energy- and Na-dependent transfer mechanisms at the apical membrane of the mid-gut epithelial cells in both crustacean species. These authors propose that amino acid absorption in these animals consists of accumulative processes into the intestinal tissue followed by passive flow into the haemolymph. In the present report, the physiological mechanisms involved in intestinal absorption of the metabolically important substrate, D-glucose, by the Malaysian prawn are investigated. Preliminary accounts of these findings have been published (Maginniss, 1974, 1976). METHODS

Experimental animals Adult Macrobrachium rosenbergii were purchased from a commercial prawn farm in Punaluu, Oahu, Hawaii and were maintained in laboratory holding tanks supplied with circulating fresh water (23-26 'C). The animals were fed a prepared diet consisting of tuna, shrimp, soybean, corn, high gluten wheat flour, and a vitamin supplement (Balazs, Ross & Brooks, 1973).

GLUCOSE TRANSPORT BY PRAWN MID-GUT

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Incubation media Formulation of a physiological saline was based primarily on the ionic composition and osmotic pressure of the shrimp haemolymph serum (Ahearn, Maginniss, Song & Tornquist, 1977). The incubation medium consisted of the following ion concentrations (mM): Na, 221K1; K, 8-1; Ca, 12-0; Mg, 4-8; Cl, 211-3; S04, 25K1; HCO3, 0 7; H2P04, 0-2; HPO, 0-2. A sodium-free medium was prepared by substituting choline chloride and choline bicarbonate for the respective sodium salts, in addition to making minor alterations in chloride and sulphate concentrations. Both solutions had the same osmotic pressure (430 m-osmole/kg) and were adjusted to a pH of 7-5. Perfusion chamber ntesti ne v ~~~~I

Perf usate

P[~~~~tTeperatueiPUM Asantte Experimental medium

Text-fig. 1. Diagrammatic representation of perfusion apparatus used to measure [3H]glucose transport by the mid-gut of the freshwater prawn

Macrobrachium rosenbergii. Perfusion apparatus In vitro mucosal influx of glucose by the prawn mid-gut was examined using a perfusion apparatus illustrated diagrammatically in Text-fig. 1. The lucite chamber consisted of a V-shaped trough submerged in a water-bath. The gut was mounted and secured on two epoxy-coated stainless steel needles, and perfused with a peristaltic pump (Buchler Instruments). The serosal bathing medium (10 ml. vol.) was maintained at 24-25 'C and was aerated and circulated by three polyethylene (Polythene) tubes extending into the chamber. Mucosal influx The intestinal contents of the excised mid-gut were flushed with incubation medium using a peristaltic pump. The cleansed mid-gut was then mounted in the incubation chamber and perfused with glucose-free medium at a flow rate of 0-20 ml./min for approx. 5 min prior to experimentation. A preliminary study revealed little variation in mucosal glucose influx over a range of flow rates varying from

a. A. AHEARN AND L. A. MAGINNISS 0-10 to 0-25 ml./min. Following pre-incubation, the mid-gut was perfused with

322

medium containing trace amounts of [3H]glucose (6-3H) (New England Nuclear), together with unlabelled glucose to final sugar concentrations between 0-01-1-0 mm. No glucose was added to the serosal solution. Following the experimental influx period, the mid-gut was rinsed for 20 sec with unlabelled glucose-free medium at an increased flow rate, cut from the needle mounts and rinsed for an additional 1-2 sec in ice-cold medium. The mid-gut was then blotted with absorbent tissue, weighed to the nearest 0-02 mg on an analytical balance and extracted for 48 hr in 5 ml. of 70 % ethanol. Samples of extract and experimental medium were counted -by liquid scintillation spectrometry (Beckman LS 230) using a toluene-based cocktail and corrected for quench by the external standard method. Less than 1 % of the total extractable activity was recovered through a second 48 hr exposure to 70 % ethanol. In many experiments, a second aliquot portion of extract was evaporated to dryness in an empty counting vial, redissolved in an equal volume of 70 % ethanol, and counted as above. The difference in activity between the two samples was assumed to represent a volatile fraction, probably tritiated water (THO). Accumulated radioactivity, not extracted in 70 % ethanol (bound fraction), was measured by dissolving the mid-gut in 0-25 ml. Protocol tissue solubilizer (New England Nuclear) and counting by liquid scintillation as described. For several experiments, total accumulated activity was determined by digesting the mid-gut in Protosol immediately following [3H]glucose exposure, eliminating the ethanol extraction procedure. An extracellular space (adherent and intercellular volume) was measured for the mid-gut mucosal surface using [3H]inulin (New England Nuclear). The experimental procedure was identical to that described above with the exception that [3H]inulin was counted in Aquasol scintillation cocktail (New England Nuclear). The calculated space, expressed as % wet tissue weight, was used to correct all glucose influx measurements.

Transmural flux Preliminary results of transmural glucose transport across the prawn mid-gut (future publication) are also presented in this report. Unidirectional transepithelial flux from mucosa to serosa (M -+S) was determined by perfusing the mid-gut for 60-90 min with 0-10 mM [3H]glucose. Small aliquot portions of the serosal bath were withdrawn at 10 min intervals and transferred to empty scintillation vials. The samples were then evaporated to dryness, redissolved in equal volumes of distilled water, and counted by liquid scintillation techniques. The data were converted to units of non-volatile glucose equiv/g, plotted as a function of time, and the transmural flux calculated by linear regression. Serosa to mucosa (S -+ M) transepithelial transport was determined in a like manner, with the exceptions that the labelled glucose solution was added to the serosal compartment and the perfusate effluent was sampled periodically for non-volatile glucose activity.

Chromatography The chemical state of tritiated activity accumulated during several mucosal influx studies was examined by one-dimensional ascending thin-layer chromatography. Concentrated samples of extract were spotted on cellulose chromagrams (Eastman Kodak Co.) and run for 10 cm at room temperature (24-25 "C) using n-butanoll pyridine/water (6:4: 3 v/v). The serosal medium, following several M -+ S transport experiments, was also analysed chromatographically using two additional solvent systems: acetone/water (9:1, v/v) and n-butanol/acetic acid/water (2:1 :1, v/v).

GLUCOSE TRANSPORT BY PRAWN MID-GUT

323

Following separation, the plates were cut into 0 5 x 16 cm segments along the separation path and counted by liquid scintillation. The Rf values for tritiated and unlabeled glucose standards were resolved by liquid scintillation and aniline/ phthalate spray reagent, respectively.

RESULTS

Mid-gut morphology The prawn intestine is a straight narrow tube extending from the stomach in the cephalothorax to a short hind gut in the posterior portion of the abdomen. Histologically, the mid-gut consists of a luminal layer of epithelial cells surrounded by connective tissue, and both circular and longitudinal muscle. The mucosal epithelium is composed of long narrow cells with basally located nuclei. The apical membrane is highly folded, forming tightly packed microvilli which provide the cell with an extensive surface area for transport processes (PI. 1). A brush border of this type is characteristic of absorptive epithelia in general (Berridge & Oschman, 1972). An abundance of mitochondria appear concentrated in the apical region (between the nucleus and brush border membrane). This distribution may be indicative of the location of cellular transport sites requiring energy from oxidative metabolism. Time course of [3H]glucose uptake A time course of [3H]glucose accumulation (0.10 mm glucose) was determined in Na and choline medium to establish appropriate mucosal influx exposure periods (Text-fig. 2). The plotted data represent ethanol extracted tritiated activity, and are therefore labelled as glucose equivalents, since free-glucose concentrations were not determined. In Na medium, accumulation of tritiated activity was hyperbolic, approaching steady state by 60 min. At steady state, the mid-gut had accumulated activity to a level ten times that in the perfusion medium. These data, however, do not demonstrate the tissue capable of concentrating free-glucose, since the proportion of [3H]glucose in the extracted activity is unknown. In Na medium, the accumulation remained linear for the first 5 min. An exposure period of 2-5 min was assumed, therefore, to be adequate for the measurement of mucosal influx. Tissue accumulation of [3H]glucose activity in choline medium was quite slow, and remained linear over the entire uptake period (Text-fig. 2). At 30 min, the mid-gut had accumulated tritiated activity to a level less than half that in the external medium. All mucosal influx measurements conducted in choline medium were determined using 20 min exposure

periods.

3. A. AHEARN AND L. A. MAGINNISS

324

1/00 [Na]=221

mM

-) 0-75

so0n

70

0-50

XI

0

EI 30-25

El

0

20

[Nal=0 mM 60 Time (min)

40

80

100

Text-fig. 2. Time course of [3H]glucose accumulation by the mucosal surface of the perfused mid-gut in Na and Na-free bathing media. The plotted points are means (five or more mid-guts/mean) and the vertical lines are + 1 s.E. of means. Absence of the S.E. of mean verticals indicates that the statistical variability lies within the area of the plotted points. The dashed horizontal line represents the glucose concentration of the perfusate throughout the uptake period (0.10 mm glucose).

Extracellular space Mucosal influx experiments were corrected for a calculated extracellular space (ECS) using [3H]inulin as follows: Cellular influx = total influx - [ECS (% wet wt.) x [glucose].edijm]. The time course of [3H]inulin accumulation appeared to be initially hyperbolic, followed by a slower linear phase of uptake (Text-fig. 3). These two components were considered to represent rapid filling of the extracellular compartment and diffusional penetration of the cells, respectively. Similar results have been reported using [3H]mannitol, [3H]inulin, and [3H]raffinose (Ahearn, 1974; Ahearn & Townsley, 1975; Brick, 1975; Esposito & Csaky, 1974). The latter authors speculate that the tritiated inulin (also purchased from NEN) may have contained small labelled monomers which could have penetrated easily into the cellular compartment. An ECS was estimated by extrapolating the linear component of the [3H]inulin curve to time zero, and using the vertical intercept (3-54 %

GLUCOSE TRANSPORT BY PRAWN MID-GUT 325 wet wt.) as an ECS correction for the 20 min influx exposures in choline medium. Since the space marker had not attained this level (3-54 %) by 2-5 min, the mean of the [3H]inulin space determinations at 2-5 min (1.90 %) was employed for all influx experiments conducted in Na medium. These calculated values are not intended to represent the true mucosal ECS, but rather the apparent space remaining after the normal rinsing procedure used for all influx determinations.

15

10 _

|

c1o

c , )/ -5 C

0

10

l I 20

~

30

~~~~~~~~~~~~~~~~~~~~ I

40

Time (min)

Text-fig. 3. Time course of [3H]inulin uptake by the mucosal surface of the perfused mid-gut. Symbols and sample size as described in Text-fig. 2.

Effect of external glucose concentration on mucosal influx Mucosal glucose influx (1umole glucose equiv/g. min) plotted as a function of substrate concentration (0-01-1-0 mM) exhibits a hyperbolic relationship characteristic of carrier-mediated transport (Text-fig. 4). This type of saturation kinetics can be described by the Michaelis-Menten equation: Jrnax.[G] Jin -Ktin +[G]' where Jj, represents the rate of mucosal influx, Jmax. is the maximal influx rate, Kt the glucose concentration resulting in half maximal influx, and [G] the glucose concentration in the perfusion medium. The kinetic constants (JmaX. = 0-15 mole glucose equiv/g.min, Kt = 0-17 mM) were derived by the double reciprocal method of Lineweaver & Burk (1934),

3. A. AHEARN AND L. A. MAGINNISS 326 as illustrated in Text-fig. 5. The hyperbolic curve in Text-fig. 4 was calculated from these constants and describes a satisfactory fit to the data. No significant diffusional entry of glucose was evident, although high variability in the data may preclude any definitive statement on this point. -

016

o012

T

C,,

o

,0.08_

008I T il

[Na]=221 mm

/

en

0.040

[Na=- 0mM 0

0 25

0 50

0

0-

0 75

1 00

[Glucose] (mM)

Text-fig. 4. Influx of [3H]glucose as a function of mucosal glucose concentration in Na and Na-free bathing media. Symbols and sample size as described in Text-fig. 2. The hyperbolic curve fitted to the influx data under normal Na conditions (221 mM) was calculated from the MichaelisMenten equation using the kinetic constants derived from the LineweaverBurk plot, illustrated in Text-fig. 5.

Effect of Na-free medium on glucose influx Total replacement of mucosal and serosal Na with choline significantly reduced glucose influx (Text-fig. 4), suggesting a near absolute dependence on this cation for transport across the mucosal membrane. In the absence of Na, glucose influx was linear over the concentration range examined (0-025-- 0 mM), indicative of either a facilitated process with a high Kt or cellular diffusion (KD = 7-6 x 10-3 ,umole/g. min. mM). Effects of phlorizin, hexoses and metabolic inhibitors The glycoside phlorizin was a potent inhibitor of glucose influx. At 0-10 mm glucose, an equimolar concentration of phlorizin reduced mucosal entry by 93 %. At a fixed inhibitor level of 0-01 mm, glucose influx was measured over a broad substrate concentration range (0-025-10 mM). The results are presented in double reciprocal fashion along with the

GLUCOSE TRANSPORT BY PRAWN MID-GUT 327 uninhibited glucose influx data in Text-fig. 5. The similar vertical intercepts (common JjmnaX) and dissimilar horizontal intercepts (different Kt values) indicate competitive inhibition. An inhibitor constant (Ki) of 150 0

Glucose

120

+

0-01 mM phiorizin 0

90 a

0

60

0

Glucose

30

-5

0

5

10

15

20

1/[GI Text-fig. 5. Lineweaver- Burk double reciprocal plot of mean glucose influx values under normal Na conditions, and in the presence of 0-01 mmphlorizin.

2-8 x 10-3 mm was derived by the method of Dixon the equation: K [i (Ka/Kt)-1'

&

Webb (1964) using

where Ka is the apparent Kt in the presence of phlorizin, [i] the phlorizin concentration, and Kt as previously described. At constant glucose (0-10 mM) and varying phlorizin concentrations (00-010 mM) (Table 1), a K1 of 4-3 x 10-3 mm was calculated by the method of Dixon (1953). A mean phlorizin K1 of 3-6 x 10-3 mm was assumed, therefore, to represent the inhibitor constant for glucose influx across the brush border. Galactose and 3-0-methylglucose (3-0-MG) were both weak inhibitors

328 G. A. AHEARN AND L. A. MAGINNISS of 0.10 mm glucose influx (Table 1). Although 0 10 mm galactose had no effect on glucose entry, galactose and 3-0-MG, at concentrations of 5 mM and above, significantly reduced influx. These data, however, do not reveal the type of inhibition induced (competitive or non-competitive). Fructose, at concentrations up to 25 mm, had no significant effect on 0-10 mM glucose influx. TABLE 1. Effect of phlorizin, sugars, and metabolic inhibitors 04I0 mm glucose influx Control No.* Influx (smole/g. min) Inhibitor 6 100 0 057± 0010a Control 32 6 0-01 mM phlorizin 0*018± 0003 23 6 0-013+ 0002 0 03 mm phlorizin 6 7 0 004± 0 001 04 0 mM phlorizin

Control 04 0 mM galactose 5 mm galactose 25 mm galactose 04I0 mm fructose 5 mm fructose 25 mm fructose Control 5 mM-3-O-MG 10 mM-3-0-MG 25 mM-3-O-MG Control 1 mM-IAAM

1 mM azidefl 10 mm azidefl *

0-048± 0007 0050+ 0010 0*030 0-006 0*012 + 0*003 0-056 ± 0*016 0-051 0-007 0-046 ± 0*006

0*051 0-006 0*028 ± 0*005 0-023± 0004 0*020 ± 0004 0*047 ± 0*010 0*046 0-009 0*023 ± 0003 0*005± 0001

12 4 13 8 4 10 8

100 104 63 25 117 106 96

5 8 6 6

100 55 45 39

6 6 12 6

100

98 49 11

on

Pt P < 0*01 P < 0.01 P < 0*01

N.S. P 0-05 P < 0*01

N.S. N.S. N.S. P < 0*025 P < 0*01 P < 0*01

N.S. P < 0*01 P < 0.01

No, of experiments.

t t test (Snedecor & Cochran, 1968),

a Mean + I s.E. of mean. 8 Mid-guts pre-incubated on both mucosal and serosal surfaces with inhibitor for 20 min prior to influx determination.

The effect of metabolic inhibitors on 0-10 mm glucose influx is presented in Table 1. Mid-guts were pre-incubated with either 1 mm iodoacetate (JAA) or azide (1 and 10 mM) on both surfaces (mucosal and serosal) for 20 min prior to influx determination. While IAA had no evident effect, azide significantly inhibited glucose uptake.

GLUCOSE TRANSPORT BY PRAWN MID-GUT

329

Chemical state of tritiated activity following ntucosal [3H]glucose influx Three forms of tritiated activity accumulated during mucosal uptake were detected at ten substrate concentrations ranging between 0 011.0 mM-glucose. The bound fraction (non-extractable in 70 % ethanol) accounted for 5-0 + 0*1 % (mean + 1 S.E. of mean) of the total accumulated X

30

I

I ll

002 mm

20

l

l

l

006 mm

., 0

0~~~02m

lit:~ Ditac 'ro /YA\An(mm)

10

-~~~~~~~~~~~~~~~~~~~~~

1 Distance from origin (cm) Text-fig. 6. Distribution of radioactivity on thin-layer chromagrams of mid-gut extract from four mucosal [3H]glucose influx experiments. The dashed verticals encompass the migration zone occupied by [3H]glucose and 'cold' glucose. The concentrations quoted refer to glucose.

activity and may represent a cellular storage product such as glycogen. The volatile fraction (19-5 + 1-7 %) was probably THO, a by-product of glucose metabolism. The remaining fraction of accumulated radioactivity (75-5 + 1-7 /) was extractable and non-volatile. Thin-layer chromatographic separation of this non-volatile component

3. A. AHEARN AND L. A. MAGINNISS 330 from several influx experiments revealed that only a small fraction of activity (0-3 %) represented free-glucose, the majority of tritium label being concentrated in two peaks nearer the origin (Text-fig. 6). A similar chromatographic distribution of non-volatile activity was observed for mid-guts perfused with 1 mm unlabelled glucose for 1 hr prior to 1 mM[3H]glucose influx determinations. Thus, the metabolism of absorbed [3H]glucose observed during normal influx determinations was not due to a dissipation of endogenous energy reserves incurred during the tissue preparation phase preceding glucose exposure. Rather, this high level of cellular metabolism may represent normal steady-state processes in the presence of mucosal glucose. TABLE 2. Transmural flux of 0-10 mM-[3H]-glucose

No.t n-mole/g. min* Experimental conditions M -+ S flux 24 21-32 ± 2-86 Normal conditions 6 0-53±0-22 Mucosal[Na] = 0mM 5 0-80 ± 0-24 Mucosal phlorizin (0.10 mM) 3-43±0-64 6 1 mM-IAA (M+S) S -+ M flux 5 0-93 ± 0-18 Normal conditions Net flux 20-39 Normal conditions * Transmural flux + 1 S.E. of mean of non-volatile tritiated activity. t No. of experiments.

Treatment of non-volatile activity from several mucosal influx studies with alkaline phosphatase (Tris-HCl, pH = 8-9 for 60 min at 37 'C) evoked a near complete loss of the origin peak, with a significant increase of activity in the free-glucose zone (Rf = 42) and an area beyond the normal glucose range (Rr = 58). These results indicate that a large proportion of this non-volatile activity was phosphorylated, and a substantial fraction was probably in the form of hexose phosphatess.

Transmural [3H]glucose transport A preliminary investigation of transmural glucose transport demonstrated the prawn mid-gut capable of maintaining a significant net M -+- S flux of non-volatile tritiated activity (Table 2). At an external concentration of 0-10 mm glucose, the M- S flux was 0-021 #tmole/g.min (41 % of mucosal accumulation rate), while the S -o- M flux was only 0-00 1 /Imole/ g. min. Chromatographic analysis of the serosal medium following several M -a S transport experiments using three solvent systems indicated that

331 GLUCOSE TRANSPORT BY PRAWN MID-GUT 50-90 % of the serosal activity migrated in a manner indistinguishable from free-glucose. These results were confirmed by enzymatic assay for glucose using the hexokinase/glucose-6-phosphate dehydrogenase/fluorometric procedure modified from Lowry & Passonneau (1972). The M- S flux was Na-dependent and inhibited by phlorizin and IAA which suggests that transepithelial glucose transport is a cellular rather than paracellular process. DISCUSSION

Functional comparisons with mammalian intestine Na-dependent, carrier-mediated glucose influx A Na-dependent, carrier-mediated transport mechanism for glucose influx was clearly demonstrated for the mid-gut epithelium of Macrobrachium rosenbergii, as has been reported extensively for mammalian intestine (Schultz & Curran, 1970). In the absence of Na, glucose absorption was significantly reduced, the kinetics of influx being characteristic of either slow cellular diffusion or a facilitated system with a low affinity for the sugar. Although the functional role of Na in intestinal transport is still contested (Schultz & Curran, 1970; Kimmich, 1973; Baker, Lo & Nunn, 1974), dependence upon this cation for active absorption of organic non-electrolytes is widely reported in the literature. Under normal Na conditions, the Michaelis constant (Kt) derived for mucosal influx in the prawn mid-gut was an order of magnitude below values determined for in vitro mammalian intestine (Fisher & Parsons, 1953a; Crane, 1960a; Goldner, Schultz & Curran, 1969). This high affinity transport system may be indicative of normal physiological glucose concentrations encountered by the mid-gut epithelium, since the digestive gland is considered a primary site for nutrient absorption in decapod crustaceans (Yonge, 1924; van Weel, 1970; Speck & Urich, 1970). Food matter entering the prawn intestine is probably already substantially depleted of its initial glucose load; the presence of a high affinity carrier mechanism would appear, therefore, physiologically advantageous for more complete recovery of the metabolically important solute. Highaffinity transport systems for glycine and lysine have also been reported for shrimp mid-gut (Ahearn, 1974; Brick, 1975).

Inhibition of glucose influx Phlorizin was a strong competitive inhibitor of glucose transport in the prawn mid-gut, the inhibitor constant (Ki = 3-6 x 10-3 mM) being within the same range reported for mammalian intestine (Alvarado & Crane, 1962, 1964). This high affinity for the sugar carrier has been

332 3. A. AHEARN AND L. A. MAGINNISS attributed to the synergistic effect of two different attachment sites for phlorizin, a sugar and a phenol binding site (Alvarado, 1967). The influences of galactose, 3-0-MG, and fructose on glucose influx in the prawn and vertebrate intestines were similar; the former two hexoses exerting an inhibitory effect, while interactions between fructose and glucose (if present) had no apparent influence on influx. However, the inhibitory potency of galactose and 3-0-MG does appear to vary between the two animal groups. Glucose, galactose, and 3-0-MG all share a common transport mechanism in the mammalian intestine and the carrier specificity for the three sugars is relatively similar (within an order of magnitude) (Fisher & Parsons, 1953b; Crane 1960a; Goldner et al. 1969). In Macrobrachium, competitive inhibition of glucose influx by galactose and 3-0-MG has not as yet been demonstrated. If interaction between the monosaccharides does occur at the binding site level, then the carrier specificity is indeed much greater for glucose than galactose or 3-0-MG. Azide, but not IAA, inhibited mucosal glucose influx in the prawn midgut, suggesting the uptake mechanism may be dependent on aerobic metabolism. Similar results were reported for 3-0-MG absorption by rabbit ileum, influx being reduced by an inhibitor of oxidative metabolism (cyanide in this case), while IAA was without significant effect (Goldner, Hajjar & Curran, 1972). These authors, however, indicate that IAA inhibition might have been observed had the pre-incubation period been longer than 30 min.

Transcellular glucose transport As indicated by the above discussion, the mucosal influx mechanism of the prawn mid-gut exhibits several basic characteristics similar to the vertebrate absorption process. The chemical state of intracellular tritiated activity recovered following [3H]glucose influx in Macrobrachium, however, does not appear consistent with the expected function of intestinal tissue-luminal absorption and cellular concentration of free-glucose, followed by passive or facilitated diffusion into the blood for general body distribution. Only a negligible quantity of labelled sugar was recovered from the mid-gut as free-glucose, the majority of activity being in various states of metabolic alteration. An initial investigation of transepithelial glucose transport, however, revealed a significant net Mu- S flux of non-volatile activity (Table 2), of which the majority was identified as free-glucose using both chromatographic and enzymatic procedures. The sensitivity to phlorizin and IAA, and dependence upon Na suggests that the net transmural flux was a cellular rather than paracellular process. In addition, while IAA had no apparent effect on the influx mechanism (inhibited by azide), the glyco-

GLUCOSE TRANSPORT BY PRAWN MID-GUT 333 lytic poison depressed 0i10 mm glucose M-* S flux by 84%. These results provide strong suggestive evidence for, the existence of a distinct basolateral carrier mechanism involved with transcellular glucose transport. IAA may influence this transport process metabolically (inhibition of glycolysis) and/or through reactivity with carrier protein sulphydryl groups. Although a definitive statement regarding the mechanisms) of glucose transport across the prawn mid-gut cannot be advanced at this time, two possible interpretations of the data are proposed. One hypothesis (group translocation) suggests that glucose absorption may involve a phosphorylation-linked influx process, followed by a baso-lateral dephosphorylation efflux mechanism. Consistent with this interpretation is the observed translocation of free-glucose from lumen to blood, while intracellular activity levels resulting from mucosal influx reveal high concentrations of phosphorylated compounds and an apparent absence of free-hexose. This type of transport mechanism was first proposed by Wilbrandt & Laszt (1933) to describe sugar absorption by mammalian intestine. Much of the evidence upon which this theory was originally founded, though, has since been refuted (Crane, 1960b). A phosphorylation-linked influx step has been demonstrated essential, however, for accumulation of sugars in yeast (van Steveninck, 1968, 1969, 1970, 1972) and bacteria (Kundig, Ghosh & Roseman, 1964; Kaback, 1968). A second possible interpretation proposes the existence of a high affinity, baso-lateral glucose pump which is functionally accessible to the intracellular release point of the mucosal carrier. This transport system would tend also to maintain a low intracellular free-glucose concentration and facilitate the transmural movement of the sugar. A similar glucose transport mechanism has been reported for the perfused rat intestine (Esposito, Faelli & Capraro, 1973). Resolution of the mechanisms) responsible for transepithelial glucose transport is presently the subject of on-going research. The authors wish to extend thanks to Messrs William J. Cooke and Melvin Lum (University of Hawaii) for making available electron micrographs of the prawn mid-gut epithelium and to Dr Richard J. Guillory (University of Hawaii) for providing materials and assistance with the enzymatic assay for glucose. Appreciation is also extended to Drs John E. Bardach and Robert C. May (Hawaii Institute of Marine Biology) and Robert W. Brick (Texas A & M University) for their comments and suggestions on the manuscript during its preparation. This investigation was supported by the University of Hawaii Sea Grant Program under grant numbers 04-3-158-29 and 04-5-158-17 from the National Oceanic and Atmospheric Administration and the National Science Foundation under grant number BMS 74-20663.

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REFERENCES AHEARN, G. A. (1974). Kinetic characteristics of glycine transport by the isolated midgut of the marine shrimp, Penaeue8 marginatou. J. exp. Biol. 61, 677- 696. AHEARN, G. A. (1976). Co-transport of glycine and sodium across the mucosal border of the midgut epithelium in the marine shrimp, Penaeu8s marginatu*. J. Phygiol. 258, 499-520. AHEARN, G. A. & TowNsLEY, S. J. (1975). Integumentary amino acid transport and metabolism in the apodous sea cucumber, Chiridota rigida. J. exp. Biol. 62, 733-752. AHEARN, G. A., MAGINNISS, L. A., SONG, Y. K. & TORNQUIST, A. (1977). Intestinal water and ion transport in freshwater malacostracan prawns (Crustacea). In Water Relations in Membrane Transport in Plants and Animals, ed. JTNGOREIs, A. M., HODGES, T., KLEINZELLER, A. M. & SCHULTZ, S. G., pp. 129-142. New York: Academic. ALvARADo, F. & CRANE, R. K. (1962). Phlorizin as a competitive inhibitor of the active transport of sugars by hamster small intestine, in vitro. Biochim. biophy8.

Acta 56, 170-172. ALvARADo, F. & CRANE, R. K. (1964). Studies on the mechanism of intestinal absorption of sugars. VII. Phenylglycoside transport and its possible relationship to phlorizin inhibition of the active transport of sugars by the small intestine. Biochim. biophy8. Acta 93, 116-135. ALVARADO, F. (1967). Hypothesis for the interaction of phlorizin and phloretin with membrane carriers for sugars. Biochim. biophy8. Acta 135, 483-495. BAKER, R. D., Lo, C. & NUNN, A. S. (1974). Galactose fluxes across brush border of hamster jejunal epithelium: effects of mucosal anaerobiosis. J. Membrane Biol. 19, 55-78. BAI~zs, G. H., Ross, E. & BROOKS, C. C. (1973). Preliminary studies on the preparation and feeding of crustacean diets. Aquaculture 2, 369-377. BERRIDGE, M. J. & OscH1Aw, J. L. (1972). Transporting Epithelia. New York: Academic. BRICK, R. W. (1975). Transport of lysine across the intestine of the freshwater prawn, Macrobrachium ro8enbergii. Ph.D. Thesis, University of Hawaii. CRANE, R. K. (1960a). Studies on the mechanism of the intestinal absorption of sugars. III. Mutual inhibition in vitro, between some actively transported sugars. Biochim. biophys. Acta 45, 477--482. CRPANE, R. K. (1960b). Intestinal absorption of sugars. Phyeiol. Rev. 40, 789-825. DIXON, M. (1953). The determination of enzyme inhibitor constants. Biochem. J. 55, 170-171. DIXON, M. & WEBB, E. C. (1964). Enzymes, 2nd edn. New York: Academic. Esposrtro, G., FAELLI, A. & CAPRARo, V. (1973). Sugar and electrolyte absorption in the rat intestine perfused 'in vivo'. Pflugere Arch. gee. Phyeiol. 340, 335-348. EsPosrro, G. & CSAKY, T. Z. (1974). Extracellular space in the epithelium of rat's small intestine. Am. J. Phyaiol. 226, 50-55. FISHER, R. B. & PARSONs, D. S. (1953a). Glucose movements across the wall of the rat small intestine. J. Phyeiol. 119, 210-223. FISHER, R. B. & PARSONS, D. S. (1953b). Galactose absorption from the surviving small intestine of the rat. J. Phyeiol. 119, 224-232. GOLDNER, A. M., SCHULTZ, S. G. & CuIRRAN, P. F. (1969). Sodium and sugar fluxes across the mucosal border of rabbit ileum. J. gen. Phyeiol. 53, 362-383. GOLDNER, A. M., HAJJAR, J. J. & CuRRAN, P. F. (1972). Effects of inhibitors on 3-O-methylglucose transport in rabbit ileum. J. Membrane Biol. 10, 267-278.

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KABACK, H. R. (1968). The role of the phosphoenolpyruvate phosphotransferase system in the transport of sugars by isolated membrane preparations of Ekcherichia coli. J. biol. Chem. 243, 3711-3724. KIMMtcH, G. A. (1973). Coupling between Na+ and sugar transport in small intestine. Biochim. biophy8. Acta 300, 31-78. KUNDIG, W., GiEOSH, S. & ROSEMAN, S. (1964). Phosphate bound to histidine in a protein as an intermediate in a novel phosphotransferase system. Proc. natn. Acad. Sci. U.S.A. 52, 1067-1074. LINEwEAvER, H. & BURK, D. (1934). The determination of enzyme dissociation constants. J. chem. Soc. 56, 658-666. LowRy, 0. H. & PAsso;NrEAu, J. V. (1972). A Flexible Sy8tem of Enzymatic Analysi. New York: Academic. MAGiINNISS, L. A. (1974). Glucose transport by the isolated midgut of the Malaysian prawn, Macrobrachium rosenbergii. Am. Zool. 14, 1292. MAGINNISs, L. A. (1976). Transcellular glucose transport by the perfused midgut of the freshwater prawn, Macrobrachium roeenbergii. Am. Zool. 16, 237. SCHuLTZ, S. G. & CUXRAN, P. F. (1970). Coupled transport of sodium and organic solutes. Phyeiol. Rev. 50, 637-718. SNEDECOR, G. W. & CocmuAN, W. G. (1968). Statistical Methods, 6th edn. Ames, Iowa: The Iowa State University Press. SPECK, U. & URICH, K. (1970). The metabolic fate of nutrients in the crayfish (Orconecte8 limosu8). Z. vergl. Phyeiol. 68, 318-333. VAN STEVENINCK, J. (1968). Transport and transport-associated phosphorylation of 2-deoxy-D-glucose in yeast. Biochim. biophys. Acta 163, 386-394. VAN STEVENINCK, J. (1969). The mechanism of transmembrane glucose transport in yeast: evidence for phosphorylation, associated with transport. Arch8 Biochem. Biophys. 130, 244-252. vAN STEVENINCK, J. (1970). The transport mechanism of ac-methylglucoside in yeast. Evidence for transport-associated phosphorylation. Biochim. biophys. Acta 203, 376-384. VAN STEVENTNCK, J. (1972). Transport and transport-associated phosphorylation of galactose in Saccharomyces cerevisiae. Biochim. biophys. Acta 274, 575-583. VAN WEEL, P. B. (1955). Processes of secretion, restitution, and resorption in gland of midgut (glandula media intestine) of Atya spinipee Newport (DecapodaBrachyura). Physiol. Zool. 28, 40-54. VAN WEEL, P. B. (1970). Digestion in crustacea. In Chemical Zoology, vol. v, ed. FLORKIN, M. & SCHEER, B. T., pp. 97-115. New York: Academic. VoNK, H. J. (1960). Digestion and metabolism. In The Physiology of Crustacea, vol. i, ed. WATERMAN, T. H., pp. 291-316. New York: Academic. WILBRANDT, W. & LASZT, L. (1933). Causes of the selective absorption of sugars from the intestine. Biochem. Z. 259, 398-417. YONGE, C. M. (1924). Studies on the comparative physiology of digestion. II. The mechanism of feeding, digestion, and assimilation in Nephrops norvegicus. J. exp. Biol. 1, 343-390.

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L. A. MAGINNISS AND G. A. AHEARN EXPLANATION OF PLATE

PLATE 1 Electron micrographs of mucosal aspect of Macrobrachiutn mid-gut epithelium. Mid-gut tissue was fixed by perfusion with 2 % glutaraldehyde, post-fixed in I % osmium tetroxide, stained with uranyl acetate and lead citrate, and examined on a Hitachi HS-8 electron microscope. A, Luminal epithelium exhibits a tightly packed brush border which provides the cell with an extensive surface area for transport processes. Mitochondria are concentrated in the apical region and this distribution may be indicative of the location of cellular transport sites requiring energy from oxidative metabolism. Arrows indicate lateral spaces between epithelial cells. B. In comparison with the mammalian intestinal brush border, the prawn mid-gut microvilli are similar in diameter (0I flm) and approx. twice the length (3 5 j#m). The relative density of microvilli, however, appears much higher in the vertebrate epithelium (Berridge & Oscbman, 1972). C, Septate desmosome at the luminal surface joining two adjacent epithelial cells. In contrast, vertebrate epithelium exhibits a tight junction.

The Journal of Physiology, Vol. 271, No. 2

Plate 1

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L. A. AIAGINNISS AND G. A. AHEARN

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Kinetics of glucose transport by the perfused mid-gut of the freshwater prawn Macrobrachium rosenberg ii.

319 J. Phyeiol. (1977), 271, pp. 319-336 With I plate and 6 text-figurew Printed in Great Britain KINETICS OF GLUCOSE TRANSPORT BY THE PERFUSED MID-...
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