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Distribution of glutamate dehydrogenase in the intestine of the earthworm (Lumbricus terrestris), and some physiological implications
Camp.
Biachem.
Physid.
Vol.
924
No.
2, pp.
229-233,
1989 Q
Printed in Great Britain
0300-9629189 $3.00 + 0.00 1989 Pergamon Press plc
DISTRIlBUTION OF GLUTAMATE DEHYDROGENASE IN THE INTESTINE OF THE EARTHWORM (LUMBRICUS TERRE,STRIS), AND SOME PHYSIOLOGICAL IMPLICATIONS POULPRENT~ Institute of Cell Biology and Anatomy, University of Copenhagen, Universitetspatken Copenhagen, 2100 Denmark
1.5,
(Received 5 J&y 1988) Abstract-l. Intestines of fresh and dehydrated-starved L. terresrris were compared to tissue and anterior-posterior distribution of glutamate dehydrogenase (GDH) and other mitochondrial or cytosol dehydrogenases. 2. For any dehydrogenase, including GDH, practically all the activity was in the gut epithelium. This distribution of GDH supports Tillinghast (1967, 1968) as to the excretory route for ammonia. 3. While the distributions of the marker dehydrogenases were reasonably uniform along the intestine, the GDH activity was predominantly @O-90% of the total activity) in the last third of the mid-intestine, indicating a true physiological differentiation of the midgut tube. The GDH activity of the typhlosole was about two times the activity in the peripheral epithelium. The GDH distribution was independent of the physiological state of the worm. 4. From the distribution of GDH it follows that the mid-intestine, imm~iately before the hindgut, is the main region both for amino acid uptake and catabolism. As regards amino acids, it typifies the primitive digestive tube by having both the absorptive and the liver functions.
INTRODUCDON
Prents (1987) found that glutamate dehydrogenase activity was high in the midgut epithelium of the earthworm Lumbricus terrestris L., but nearly absent from the chloragocytes, indicating that ammonia formation takes place predominantly in the intestinal epithelium, which is in accordance with Tillinghasts (1967) conclusion that ammonia is voided through the gut. Needham (1960) found that the earthworm midgut contained nearly all the omithine cycle enzymes and thus two tissues, the epithelium of the intestine and the chloragog tissue, contend for urea biogenesis. For many years the chloragog tissue has been considered analogous to vertebrate liver and thus the most probable site for urea biosynthesis. It was decided to investigate further the spatial relationships of these two aspects of nitrogen metabolism; ammonia production through oxidative deamination, and urea biosynthesis. The present paper deals with the location of oxidative deamination as revealed by the relative activity of glutamate dehydrogenase. The location of urea biosynthesis and its relation to ammonia formation is dealt with in another paper. The present paper concludes that while the distribution of most of the investigated dehydrogenases may indicate a longitudinal metabolic gradient, as proposed by O’Brien (1957), the distribution of glutamate dehydrogenase demonstrates a physiological, regional differentiation of the intestine, even though this differentiation is not morphologically visible, and that the posterior part of the midintestine is the major
site both for amino acid absorption acid catabolism.
and for amino
MATERIALS AND METHODS All reagents were analytical grade. Biochemical and histochemical reagents were from Sigma Chemical Company. Mature Lumbrinrs terrestris L. were collected on grass lawn by means of electric current. The worms were used inside 48 hr or kept without feeding in slightly moist oellulose granulate for l-2 months, leading to slight dehydration. Before preparation the worms were anaesthetized with cold 10% ethanol and rolled once on paper toweling. Fractionation For each fractionation 1-2 worms were used. In some cases the total intestine behind the gizzard was divided into eight regions of about the same length and fractionated separately. The fore-intestine with lateral “sacs” (Arthur, 1963) was fraction 1, and the hind-intestine (without typhlosole) + rectum was fractions 7 and 8 (in some cases a small part of the midintestine was present in fraction 7). Physiological solution (Drewes and Pax, 1974) was used for dissection. The homogeni~tion medium was 50% physiological solution and 50% 0.30 mol/l sucrose with 10 mmol/l of EDTA and of sodium bicarbonate. All homogenates were centrifuged at SOtlOg-min to remove larger fragments and chloragosomes and the sediment discarded. The final supernate volumes were for body wall about 4m1, and for the other tissues, at most, 1 ml per worm. Biochemicalassays The dehydrogenase reactions were as described by Bergmeyer (1974). Lactate dehydrogenase (LDH) was determined according to Bergmeyer and Bemt. Glutamate dehydrogenase (GDH) was assayed according to Schmidt, and 229
POUL
230
isocitrate dehydrogenase (ICDH) according to Bernt and Bergmeyer. Succinate dehydrogenase (SDH) and NADH diaphorase (NADH-Dp) were asayed as described by Prente (1987). Protein was determined by use of Bradford’s (1976) Coomassie brilliant blue G-250 (C.I. No. 42655) method with bovine serum albumin (BSA) as a standard. Photometric measurements were made by use of a Beckman acta CIII recording spectrophotometer.* Calculations As discussed by Prento (1987) the % distribution of an enzyme activity between the various tissue fractions gives a fair indication of the importance of the enzyme in the physiology of the tissues in question. Comparisons between the tissue may then be performed by use of % distributions and relative specific activities (RSAs). The activities of most enzymes are simply calculated as A = (dE/dr) x V/u. where t is the chosen unit time, V is the total fraction volume and tl is the fraction volume present in the measured assay. As each enzyme was determined on several worms, with variation in the physiological state, in the amount of tissue and probably in the preparative procedures, the % variation is also the most suitable for simple statistical treatment. An approximate determination of the actual GDH content was calculated from several experiments as the mean activity U = pmol NAD+ formed per min.
Regions of worms were quick-frozen at -70°C freezesectioned at 10pm and used the same day for enzyme histochemistry. Before incubation the sections were stabilized by immersion in acetone at -30°C for 15 min and air-dried. LDH and SDH reactions were performed essentially according to Kiernan (1981), and GDH according to Chayen et al. (1973). In all cases 30 ng Meldolas Blue (C.I. 5117.5; Basic Blue 6) was added per ml reaction medium (Kugler and Wrobal, 1978). For LDH the reaction medium contained 20% polyvinylpyrohdone (Sigma PVP-40). After incubation (1530 min at 30°C) the sections were rinsed. treated with 0.5% periodic acid for stabilization and
*The spectrophotometer is a grant from the Danish Science Council (Grant No. I l-0362). Table
1. Relative
Natural
PENT0
mounted with 50% PVP-40. Controls were neighboring sections incubated in the absence of the substrate. Cyfo-densitome~y. Optical density was measured on a Nikon Microphot-FX microscope by means of the spot measuring area and a 40X plan objective. The difference between exposure time values for enzyme incubated epithelium and control epithelium represents the optical density, from which the % absorbance was calculated. For the addition of the peripheral and the typhlosole epithelium it was assumed that the sectional areas of these two locations are similar, and for the comparison of the gut regions it was assumed that the sectional areas of the various regions have the same size. As neither assumption is true, the % distribution is only roughly approximate.
RESULTS Biochemical
assays
shows
Table 1 and specific
activities
enzyme distributions the intestine. SDH,
NADH-Dp, LDH, and ICDH specific and absolute activities exhibit a rather uniform distribution, with a slight fall in activity towards the posterior intestine (regions 7 and 8). The distribution of GDH is very different, having more than 80% of the activity concentrated in regions 5 + 6, and only 2-5% of the activity in any of the other regions. This is the case both for fresh and for starved, dehydrated animals. In some cases regions 14 were pooled and the GDH activity for this part of the intestine never then exceeded 5% of the total GDH activity, indicating that at the low activities present in the separate regions 14, the actual GDH activity tends to be overestimated. All the dehydrogenases, including GDH, are found in the intestinal epithelium. Except for a very low LDH activity, the chloragog fractions are virtually devoid of dehydrogenase activities. The intracellular distribution of GDH was investigated by sedimenting at 60,00Og-min 80% of the GDH activity was recovered in the sediment. The
enzyme distributions along the gut. Region I fore-intestine; regions hind-intestine (defined as the reaions without tvuhlosole) Regions
24
mid-intestine;
regions
7-X
alone the cut 6
7
8
44 (13) 4.28 (1.31)
2 (2) 0.60 (0.21)
0.27 (0.13)
7 (4) 0.96 (0.17)
8 (I) I .85 (0.27)
9 (3) 2.18 (0.78)
13 (1) I.11 (0.09)
15 (2)
2 (1)
I .24 (0.04)
0.71 (0.21)
3 (1) I.15 (0.21)
8 (1) 0.78 (0.05)
1 l (4) 0.96 (0.05)
12 (7) 0.99 (0.2 I)
6 (0.3) 0.97 (0.07)
7 (0.2) 0.98 (0.12)
10 (2) 0.69 (0.1 I)
7 (I) 0.73 (0.14)
6 (1) 0.72 (0.21)
7 (1) 0.96 (0.07)
7 (I) I.10 (0.03)
I
2
3
4
4 (2) 0.24 (0.14)
3 (2) 0.18 (0.14)
2 (1) 0.15 (0.04)
3 (2) 0.45 (0.21)
LDH (N = 5) % RSA SD
25 (6) I .03 (0.09)
18 (3) 0.79 (0.17)
15 (3) 0.93 (0.09)
9 (2) 0.86 (0.21)
0.78 (0.06)
ICDH (N = 3) % RSA SD
21 (2) 0.91 (0.04)
20 (1) 0.85 (0.14)
13 (2) 0.98 (0.08)
12 (2) 1.34 (0.12)
NADHDp % RSA SD
22 (2) I .06 (0.1 1)
21 (5) I.15 (0.23)
12 (3) I .07 (0.16)
26(9 I .32
24 (3) I .39 (0.26)
14 (2) 0.90 (0.15)
GDH’ (N = 9) % RSA SD
relative along
5 40 (9) 3.44 (0.75)
8 (1)
1 (1)
(N = 3)
SDH (N = 4) % RSA SD *For GDH N = Number
determinations
of worms. SD given in parentheses.
(0.22)
5 fresh and 4 starved
worms were used.
GDH in earthworm intestine su~rnat~nt had 20% GDH activity and a RSA of 0.26, while the sediment had 80% activity and a RSA of 3.64, so the GDH, as in other organisms, is localized to the mitochondrial matrix. From 10 worms the mean specific intestine GDH activity was found to be 25 pmol NAD+/min/g wet wt midgut (SD, 11) or about 50 U/g wet wt intestinal epithelium. For regions 5 and 6 alone, the epithelial activity must be about 200 p/g, which is reasonably comparable to mammalian liver.
Hktochemistry
(Fig.
1)
and LDH exhibit essentially simiiar distribution along the peripheral and typhlosole SDH
231
epithelium. GDH is very dissimilar, having very little activity in the intestine regions 14 and 7 and 8, but a very high activity in regions 5 and 6. The GDH activity is distinctly higher in the typhlosole than in the peripheral epithelium. The chloragog tissue exhibited no discernible SDH, LDH or GDH activity. The cyto-densitometric results are given in Table 2. As mentioned in the Methods and Materials section the relative distribution is only approximate. For GDH the actual distribution of course tends to minimize the % error. SDH and LDH exhibit slight maxima for the fore- and hind-intestine regions, and the distribution of activity between typhlosole and peripheral epithelium is about 1: 1. Regions 5 and 6
Fig. 1. Cross-section of intestinal epithelium of earthworm, x60. Left column; reaction for succinate dehydrogenase. Right column; reaction for glutamate dehydrogenase. First row, region 1; second row, region 3; third row, region 5; fourth row, region 7. Epithelium of typhlosole, te; peripheral epithelium, pe; chloragog tissue, ch.
POUL
232
PRENTB
Table 2. Densltometric measurements of cytoplasm of intestinal epithelium from dehydrogenaseincubated IO pm fresh-frozen sections of L. rerreslris. The measurements were converted to % of t”tal optlcal density. Periph = peripheral epithelium; typh = typhlosole epithelium. (A) Percent optical density of dehydrogenase
reaction product
measured on cross-sectioned
body regmns
Gut region
I
Ree. 3
Rec. 5
Reg. 6
3.5 (I) 3.5 (0.7)
2.5 (1.5) 2.5 (0.5)
14 (4) 35 (IO)
7 (I) 30 (II)
7 (20)
5 (1)
49 (14)
37 (I?)
3 (2)
18 (3) (2) I6
(2) 98 (2)
8 (2) 7 (1)
7 (1) (1) 6
II (3)
periph. + typh.
34 (5)
17 (4)
I5 (3)
I3 (2)
22 (5)
SDH
I3 13 (I) (1)
8 (1) 13 (I)
8 7 (2) (3)
8 8 (2) (3)
10.5 (3)
26 (2)
21 (3)
15 (5)
16 (5)
21 (5)
Ree.
Enzvme:trssue GDH
periph typh.
periph. + typh. LDH
perlph. typh.
periph. typh.
periph. + typh. GDH:
Typh.!periph.
ratto regions
Table 28. Percent optical
14:
R =
Rer. 7 1.5 (I)
I.14 (0.30), (n = 30): 5-6: R = 2.25 (1.1) (n = 25)
density of glutamate dehydrogenase sections of post-gizzard
reaction gut
product
measured
on longitudinal
Gut reeions Enzyme/tissue GDH
periph. twh.
periph. + typh
I 2 2 4 (1)
2
3
4
3 3
I.5 I.5
0.7 I.3
I4 20
20 28
0.5
I
6 (4)
3 (I)
2 (1)
34 (IO)
48 (9)
1 (1)
2 (1)
contain about 20% of the total intestinal SDH or LDH activity, or a little below the mean for the preceding regions. In contrast, GDH exhibits a very pronounced maximum for region 5 and 6, which amounts to more than 80% of the total activity of the intestine. Furthermore, the ratio of distribution of GDH activity between typhlosole and peripheral epithelium is about 2.2 : 1. As previously stated the agreement between the biochemical and the histochemical GDH distribution must be to some degree incidental, even though both types of measurement are based on length, but implies that the GDH activity is relatively constant along most of regions 5 and 6. DISCUSSION
Prents (1987) found that about 86% of the total GDH activity of the worm was present in the intestinal epithelium. This high relative activity of GDH in the intestinal epithehum supports Tillinghast’s (1967, 1968) conclusion, that ammonia is excreted into the intestinal lumen. As clearly demonstrated in the results section the GDH activity is very low in the anterior two-thirds and high in the last third of the typhlosolar intestine, and again low in the typhlosole-less hind-intestine. That this distribution is not a spurious result of differences in metabolic activity along the digestive tube can be ruled out as the SDH, LDH, ICDH and NADH-Dp activities run parallel to the protein contents of the various regions. Neither does the GDH distribution reflect the general metabolic gradient proposed by O’Brien (1957) essentially from the distribution of SDH and the distribution of lactate production. Along a length of the intestine corresponding to the last 40% of the mid-intestine proper, or about 25% of the post-gizzard gut, was found about 84% of the total GDH activity of the gut. The errors inherent in measuring low GDH activities entails that even this high value is probably too small.
5
6
7
8
This means that at least 75% of the total GDH of the worm is present in the epithehum of the hind-region of the mid-intestine. There can be little doubt that this localized high capacity for oxidative amino acid deamination must be functionally related to amino acid uptake and catabolism. The hind-mid-epithelium localization of most of the GDH activity is in contrast to Needham (1962) who found the maximum gut ammonia production more anteriorly, but is very logical if the last region of the mid-intestine is the one responsible for most of the absorption of amino acids. This discrepancy cannot be explained on the basis of the present data. Interestingly the GDH activity is also heterogeneously distributed inside the hind-midintestine, the typhlosole epithelium having approximately twice the activity of the peripheral epithelium (Fig. 1 and Table 2). No doubt the GDH distribution reflects a true physiological differentiation of the intestine. The paucity of dehydrogenases in the chloragog tissue implies that the catabolism of absorbed amino acids must take place in the intestinal epithelium, and bearing in mind the toxicity of ammonia it is reasonable to place its formation far back in the alimentary canal. The arginase distribution in fresh, well-fed and well-hydrated worms supports this (Prento, 1989). Even in worms starved for more than weeks, where arginase is present along the full length of the postgizzard gut, the distribution of GDH activity is unchanged. The mean specific GDH activity of the hind-midintestine of about 200 U/g wet wt is fairly comparable to the activity of mammalian liver and supports the fact that the hind-mid-intestine, as regards amino acid metabolism, is physiologically liver-like. Bishop and Campbell (1965) found that in contrast to the situation in vertebrates the ornithine cycle enzymes in L. terrestris are nearly exclusively extramitochondrial. The localization of GDH, however, is quite conventionally mitochondrial, as demonstrated
GDH in earthworm intestine by the distribution of the enzyme following sedimentation at 60,000 g-min. Two conclusions may be drawn from the above: (1) Even though morphologically undifferentiated along its length, the mid-intestine is none-the-less physiologically differentiated, the posterior 25% being the main absorptive region, at least for amino acids. This is reasonable and similar to the situation in for instance mammals, where the jejunum-ileum is most active in absorption. (2) In its absorptive function and in its function in intermediary amino acid metabolism the Lumbricus hind-mid-intestine exemplifies the region of primitive gut (Prents, 1987), which in vertebrates differentiated into a purely absorptive gut and a liver. Interestingly enough this region of the intestinal epithelium also exhibits considerable arginase activity (Prento, 1989) and may thus also carry out urea biogenesis. This further supports the concept (see Bishop and Campbell, 1963). The distribution of GDH should be of interest for the analysis of the functional differentiation of the intestine and the location of intermediary metabolism in other invertebrates. As the histochemical demonstration of GDH is very straightforward, this analysis may be performed even for animals too small or too complex to permit fractionation techniques. Acknowledgements-The
author wishes to thank Alice Kristiansen for her skilled technical assistance.
REFERENCES
Arthur D. R. (1963) The post-pharyngeal gut of the earthworm Lumbricus terrestris L. Proc. 2001. Sot. Lond. 141, 663-615.
Bergmeyer H. U. (1974) Methods of Enzymatic Analysis. Academic Press, New York.
c B.P. 92,2*--F
233
Bishop S. H. and Campbell J. W. (1963) Carbamyl phosphate synthesis in the earthworm Lumbricus terrestris. Science 142, 1583-1585. Bishop S. H. and Campbell J. W. (1965) Arginine and urea biosynthesis in the earthworm Lumbricus terrestris. Comp. Biochem. Physiol. 15, 51-71.
Bradford M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analyt. Biochem. 86, 142-146.
Chayen J., Bitensky L. and Butcher R. G. (1973) Practical Histochemistry. John Wiley & Sons, London. Drewes C. D. and Pax R. A. (1974) Neuromuscular physiology of the longitudinal muscle of the earthworm Lumbricus terrestris. I. Effects of different physiological salines. J. exp. Biol. 60, 445-452. Kiernan J. A. (188 1) Histological and Histochemical Meth odr: Theory and Practice. Pergamon Press, Oxford. Needham A. E. (1960) The arginase activity of the tissues of the earthworm Lumbricus terrestris L. and Eisenia foetida (Savignvl
J. exe. Biol. 7. 7755782.
Needham ‘A. k.‘(l962) Distribution of arginase activity along the body of earthworms. Comp. Biochem. Physiol. 5, 69-82.
O’Brien B. R. A. (1957) Evidence in support of an axial metabolic gradient in the earthworm. Aust. J. exp. Biol. 35, 83-92.
Prents P. (1987) Distribution of 20 enzymes in the midgut region of the earthworm, Lumbricus terrestris L., with particular emphasis on the physiological role of the chloragog tissue. Comp. Biochem. Physiol. 87A, 135-142.
Prente P. (1989) Distribution of arginase in the gut of the earthworm Lumbricus terrestris L. and some physiological and comparative implications. Comp. Biochem. Physiol. (to be submitted). Tillinghast E. K. (1967) Excretory pathways of ammonia and urea in the earthworm Lumbricus terrestris L. J. exp. Zool. 166, 295-300. Tillinghast E. K. (1968) Variations in blood and coelomic fluid ammonia and urea levels in the earthworm Lumbricus terrestris L. Comp. Biochem. Physiol. 24, 621-623.
Biachem.
Physid.
Vol.
924
No.
2, pp.
229-233,
1989 Q
Printed in Great Britain
0300-9629189 $3.00 + 0.00 1989 Pergamon Press plc
DISTRIlBUTION OF GLUTAMATE DEHYDROGENASE IN THE INTESTINE OF THE EARTHWORM (LUMBRICUS TERRE,STRIS), AND SOME PHYSIOLOGICAL IMPLICATIONS POULPRENT~ Institute of Cell Biology and Anatomy, University of Copenhagen, Universitetspatken Copenhagen, 2100 Denmark
1.5,
(Received 5 J&y 1988) Abstract-l. Intestines of fresh and dehydrated-starved L. terresrris were compared to tissue and anterior-posterior distribution of glutamate dehydrogenase (GDH) and other mitochondrial or cytosol dehydrogenases. 2. For any dehydrogenase, including GDH, practically all the activity was in the gut epithelium. This distribution of GDH supports Tillinghast (1967, 1968) as to the excretory route for ammonia. 3. While the distributions of the marker dehydrogenases were reasonably uniform along the intestine, the GDH activity was predominantly @O-90% of the total activity) in the last third of the mid-intestine, indicating a true physiological differentiation of the midgut tube. The GDH activity of the typhlosole was about two times the activity in the peripheral epithelium. The GDH distribution was independent of the physiological state of the worm. 4. From the distribution of GDH it follows that the mid-intestine, imm~iately before the hindgut, is the main region both for amino acid uptake and catabolism. As regards amino acids, it typifies the primitive digestive tube by having both the absorptive and the liver functions.
INTRODUCDON
Prents (1987) found that glutamate dehydrogenase activity was high in the midgut epithelium of the earthworm Lumbricus terrestris L., but nearly absent from the chloragocytes, indicating that ammonia formation takes place predominantly in the intestinal epithelium, which is in accordance with Tillinghasts (1967) conclusion that ammonia is voided through the gut. Needham (1960) found that the earthworm midgut contained nearly all the omithine cycle enzymes and thus two tissues, the epithelium of the intestine and the chloragog tissue, contend for urea biogenesis. For many years the chloragog tissue has been considered analogous to vertebrate liver and thus the most probable site for urea biosynthesis. It was decided to investigate further the spatial relationships of these two aspects of nitrogen metabolism; ammonia production through oxidative deamination, and urea biosynthesis. The present paper deals with the location of oxidative deamination as revealed by the relative activity of glutamate dehydrogenase. The location of urea biosynthesis and its relation to ammonia formation is dealt with in another paper. The present paper concludes that while the distribution of most of the investigated dehydrogenases may indicate a longitudinal metabolic gradient, as proposed by O’Brien (1957), the distribution of glutamate dehydrogenase demonstrates a physiological, regional differentiation of the intestine, even though this differentiation is not morphologically visible, and that the posterior part of the midintestine is the major
site both for amino acid absorption acid catabolism.
and for amino
MATERIALS AND METHODS All reagents were analytical grade. Biochemical and histochemical reagents were from Sigma Chemical Company. Mature Lumbrinrs terrestris L. were collected on grass lawn by means of electric current. The worms were used inside 48 hr or kept without feeding in slightly moist oellulose granulate for l-2 months, leading to slight dehydration. Before preparation the worms were anaesthetized with cold 10% ethanol and rolled once on paper toweling. Fractionation For each fractionation 1-2 worms were used. In some cases the total intestine behind the gizzard was divided into eight regions of about the same length and fractionated separately. The fore-intestine with lateral “sacs” (Arthur, 1963) was fraction 1, and the hind-intestine (without typhlosole) + rectum was fractions 7 and 8 (in some cases a small part of the midintestine was present in fraction 7). Physiological solution (Drewes and Pax, 1974) was used for dissection. The homogeni~tion medium was 50% physiological solution and 50% 0.30 mol/l sucrose with 10 mmol/l of EDTA and of sodium bicarbonate. All homogenates were centrifuged at SOtlOg-min to remove larger fragments and chloragosomes and the sediment discarded. The final supernate volumes were for body wall about 4m1, and for the other tissues, at most, 1 ml per worm. Biochemicalassays The dehydrogenase reactions were as described by Bergmeyer (1974). Lactate dehydrogenase (LDH) was determined according to Bergmeyer and Bemt. Glutamate dehydrogenase (GDH) was assayed according to Schmidt, and 229
POUL
230
isocitrate dehydrogenase (ICDH) according to Bernt and Bergmeyer. Succinate dehydrogenase (SDH) and NADH diaphorase (NADH-Dp) were asayed as described by Prente (1987). Protein was determined by use of Bradford’s (1976) Coomassie brilliant blue G-250 (C.I. No. 42655) method with bovine serum albumin (BSA) as a standard. Photometric measurements were made by use of a Beckman acta CIII recording spectrophotometer.* Calculations As discussed by Prento (1987) the % distribution of an enzyme activity between the various tissue fractions gives a fair indication of the importance of the enzyme in the physiology of the tissues in question. Comparisons between the tissue may then be performed by use of % distributions and relative specific activities (RSAs). The activities of most enzymes are simply calculated as A = (dE/dr) x V/u. where t is the chosen unit time, V is the total fraction volume and tl is the fraction volume present in the measured assay. As each enzyme was determined on several worms, with variation in the physiological state, in the amount of tissue and probably in the preparative procedures, the % variation is also the most suitable for simple statistical treatment. An approximate determination of the actual GDH content was calculated from several experiments as the mean activity U = pmol NAD+ formed per min.
Regions of worms were quick-frozen at -70°C freezesectioned at 10pm and used the same day for enzyme histochemistry. Before incubation the sections were stabilized by immersion in acetone at -30°C for 15 min and air-dried. LDH and SDH reactions were performed essentially according to Kiernan (1981), and GDH according to Chayen et al. (1973). In all cases 30 ng Meldolas Blue (C.I. 5117.5; Basic Blue 6) was added per ml reaction medium (Kugler and Wrobal, 1978). For LDH the reaction medium contained 20% polyvinylpyrohdone (Sigma PVP-40). After incubation (1530 min at 30°C) the sections were rinsed. treated with 0.5% periodic acid for stabilization and
*The spectrophotometer is a grant from the Danish Science Council (Grant No. I l-0362). Table
1. Relative
Natural
PENT0
mounted with 50% PVP-40. Controls were neighboring sections incubated in the absence of the substrate. Cyfo-densitome~y. Optical density was measured on a Nikon Microphot-FX microscope by means of the spot measuring area and a 40X plan objective. The difference between exposure time values for enzyme incubated epithelium and control epithelium represents the optical density, from which the % absorbance was calculated. For the addition of the peripheral and the typhlosole epithelium it was assumed that the sectional areas of these two locations are similar, and for the comparison of the gut regions it was assumed that the sectional areas of the various regions have the same size. As neither assumption is true, the % distribution is only roughly approximate.
RESULTS Biochemical
assays
shows
Table 1 and specific
activities
enzyme distributions the intestine. SDH,
NADH-Dp, LDH, and ICDH specific and absolute activities exhibit a rather uniform distribution, with a slight fall in activity towards the posterior intestine (regions 7 and 8). The distribution of GDH is very different, having more than 80% of the activity concentrated in regions 5 + 6, and only 2-5% of the activity in any of the other regions. This is the case both for fresh and for starved, dehydrated animals. In some cases regions 14 were pooled and the GDH activity for this part of the intestine never then exceeded 5% of the total GDH activity, indicating that at the low activities present in the separate regions 14, the actual GDH activity tends to be overestimated. All the dehydrogenases, including GDH, are found in the intestinal epithelium. Except for a very low LDH activity, the chloragog fractions are virtually devoid of dehydrogenase activities. The intracellular distribution of GDH was investigated by sedimenting at 60,00Og-min 80% of the GDH activity was recovered in the sediment. The
enzyme distributions along the gut. Region I fore-intestine; regions hind-intestine (defined as the reaions without tvuhlosole) Regions
24
mid-intestine;
regions
7-X
alone the cut 6
7
8
44 (13) 4.28 (1.31)
2 (2) 0.60 (0.21)
0.27 (0.13)
7 (4) 0.96 (0.17)
8 (I) I .85 (0.27)
9 (3) 2.18 (0.78)
13 (1) I.11 (0.09)
15 (2)
2 (1)
I .24 (0.04)
0.71 (0.21)
3 (1) I.15 (0.21)
8 (1) 0.78 (0.05)
1 l (4) 0.96 (0.05)
12 (7) 0.99 (0.2 I)
6 (0.3) 0.97 (0.07)
7 (0.2) 0.98 (0.12)
10 (2) 0.69 (0.1 I)
7 (I) 0.73 (0.14)
6 (1) 0.72 (0.21)
7 (1) 0.96 (0.07)
7 (I) I.10 (0.03)
I
2
3
4
4 (2) 0.24 (0.14)
3 (2) 0.18 (0.14)
2 (1) 0.15 (0.04)
3 (2) 0.45 (0.21)
LDH (N = 5) % RSA SD
25 (6) I .03 (0.09)
18 (3) 0.79 (0.17)
15 (3) 0.93 (0.09)
9 (2) 0.86 (0.21)
0.78 (0.06)
ICDH (N = 3) % RSA SD
21 (2) 0.91 (0.04)
20 (1) 0.85 (0.14)
13 (2) 0.98 (0.08)
12 (2) 1.34 (0.12)
NADHDp % RSA SD
22 (2) I .06 (0.1 1)
21 (5) I.15 (0.23)
12 (3) I .07 (0.16)
26(9 I .32
24 (3) I .39 (0.26)
14 (2) 0.90 (0.15)
GDH’ (N = 9) % RSA SD
relative along
5 40 (9) 3.44 (0.75)
8 (1)
1 (1)
(N = 3)
SDH (N = 4) % RSA SD *For GDH N = Number
determinations
of worms. SD given in parentheses.
(0.22)
5 fresh and 4 starved
worms were used.
GDH in earthworm intestine su~rnat~nt had 20% GDH activity and a RSA of 0.26, while the sediment had 80% activity and a RSA of 3.64, so the GDH, as in other organisms, is localized to the mitochondrial matrix. From 10 worms the mean specific intestine GDH activity was found to be 25 pmol NAD+/min/g wet wt midgut (SD, 11) or about 50 U/g wet wt intestinal epithelium. For regions 5 and 6 alone, the epithelial activity must be about 200 p/g, which is reasonably comparable to mammalian liver.
Hktochemistry
(Fig.
1)
and LDH exhibit essentially simiiar distribution along the peripheral and typhlosole SDH
231
epithelium. GDH is very dissimilar, having very little activity in the intestine regions 14 and 7 and 8, but a very high activity in regions 5 and 6. The GDH activity is distinctly higher in the typhlosole than in the peripheral epithelium. The chloragog tissue exhibited no discernible SDH, LDH or GDH activity. The cyto-densitometric results are given in Table 2. As mentioned in the Methods and Materials section the relative distribution is only approximate. For GDH the actual distribution of course tends to minimize the % error. SDH and LDH exhibit slight maxima for the fore- and hind-intestine regions, and the distribution of activity between typhlosole and peripheral epithelium is about 1: 1. Regions 5 and 6
Fig. 1. Cross-section of intestinal epithelium of earthworm, x60. Left column; reaction for succinate dehydrogenase. Right column; reaction for glutamate dehydrogenase. First row, region 1; second row, region 3; third row, region 5; fourth row, region 7. Epithelium of typhlosole, te; peripheral epithelium, pe; chloragog tissue, ch.
POUL
232
PRENTB
Table 2. Densltometric measurements of cytoplasm of intestinal epithelium from dehydrogenaseincubated IO pm fresh-frozen sections of L. rerreslris. The measurements were converted to % of t”tal optlcal density. Periph = peripheral epithelium; typh = typhlosole epithelium. (A) Percent optical density of dehydrogenase
reaction product
measured on cross-sectioned
body regmns
Gut region
I
Ree. 3
Rec. 5
Reg. 6
3.5 (I) 3.5 (0.7)
2.5 (1.5) 2.5 (0.5)
14 (4) 35 (IO)
7 (I) 30 (II)
7 (20)
5 (1)
49 (14)
37 (I?)
3 (2)
18 (3) (2) I6
(2) 98 (2)
8 (2) 7 (1)
7 (1) (1) 6
II (3)
periph. + typh.
34 (5)
17 (4)
I5 (3)
I3 (2)
22 (5)
SDH
I3 13 (I) (1)
8 (1) 13 (I)
8 7 (2) (3)
8 8 (2) (3)
10.5 (3)
26 (2)
21 (3)
15 (5)
16 (5)
21 (5)
Ree.
Enzvme:trssue GDH
periph typh.
periph. + typh. LDH
perlph. typh.
periph. typh.
periph. + typh. GDH:
Typh.!periph.
ratto regions
Table 28. Percent optical
14:
R =
Rer. 7 1.5 (I)
I.14 (0.30), (n = 30): 5-6: R = 2.25 (1.1) (n = 25)
density of glutamate dehydrogenase sections of post-gizzard
reaction gut
product
measured
on longitudinal
Gut reeions Enzyme/tissue GDH
periph. twh.
periph. + typh
I 2 2 4 (1)
2
3
4
3 3
I.5 I.5
0.7 I.3
I4 20
20 28
0.5
I
6 (4)
3 (I)
2 (1)
34 (IO)
48 (9)
1 (1)
2 (1)
contain about 20% of the total intestinal SDH or LDH activity, or a little below the mean for the preceding regions. In contrast, GDH exhibits a very pronounced maximum for region 5 and 6, which amounts to more than 80% of the total activity of the intestine. Furthermore, the ratio of distribution of GDH activity between typhlosole and peripheral epithelium is about 2.2 : 1. As previously stated the agreement between the biochemical and the histochemical GDH distribution must be to some degree incidental, even though both types of measurement are based on length, but implies that the GDH activity is relatively constant along most of regions 5 and 6. DISCUSSION
Prents (1987) found that about 86% of the total GDH activity of the worm was present in the intestinal epithelium. This high relative activity of GDH in the intestinal epithehum supports Tillinghast’s (1967, 1968) conclusion, that ammonia is excreted into the intestinal lumen. As clearly demonstrated in the results section the GDH activity is very low in the anterior two-thirds and high in the last third of the typhlosolar intestine, and again low in the typhlosole-less hind-intestine. That this distribution is not a spurious result of differences in metabolic activity along the digestive tube can be ruled out as the SDH, LDH, ICDH and NADH-Dp activities run parallel to the protein contents of the various regions. Neither does the GDH distribution reflect the general metabolic gradient proposed by O’Brien (1957) essentially from the distribution of SDH and the distribution of lactate production. Along a length of the intestine corresponding to the last 40% of the mid-intestine proper, or about 25% of the post-gizzard gut, was found about 84% of the total GDH activity of the gut. The errors inherent in measuring low GDH activities entails that even this high value is probably too small.
5
6
7
8
This means that at least 75% of the total GDH of the worm is present in the epithehum of the hind-region of the mid-intestine. There can be little doubt that this localized high capacity for oxidative amino acid deamination must be functionally related to amino acid uptake and catabolism. The hind-mid-epithelium localization of most of the GDH activity is in contrast to Needham (1962) who found the maximum gut ammonia production more anteriorly, but is very logical if the last region of the mid-intestine is the one responsible for most of the absorption of amino acids. This discrepancy cannot be explained on the basis of the present data. Interestingly the GDH activity is also heterogeneously distributed inside the hind-midintestine, the typhlosole epithelium having approximately twice the activity of the peripheral epithelium (Fig. 1 and Table 2). No doubt the GDH distribution reflects a true physiological differentiation of the intestine. The paucity of dehydrogenases in the chloragog tissue implies that the catabolism of absorbed amino acids must take place in the intestinal epithelium, and bearing in mind the toxicity of ammonia it is reasonable to place its formation far back in the alimentary canal. The arginase distribution in fresh, well-fed and well-hydrated worms supports this (Prento, 1989). Even in worms starved for more than weeks, where arginase is present along the full length of the postgizzard gut, the distribution of GDH activity is unchanged. The mean specific GDH activity of the hind-midintestine of about 200 U/g wet wt is fairly comparable to the activity of mammalian liver and supports the fact that the hind-mid-intestine, as regards amino acid metabolism, is physiologically liver-like. Bishop and Campbell (1965) found that in contrast to the situation in vertebrates the ornithine cycle enzymes in L. terrestris are nearly exclusively extramitochondrial. The localization of GDH, however, is quite conventionally mitochondrial, as demonstrated
GDH in earthworm intestine by the distribution of the enzyme following sedimentation at 60,000 g-min. Two conclusions may be drawn from the above: (1) Even though morphologically undifferentiated along its length, the mid-intestine is none-the-less physiologically differentiated, the posterior 25% being the main absorptive region, at least for amino acids. This is reasonable and similar to the situation in for instance mammals, where the jejunum-ileum is most active in absorption. (2) In its absorptive function and in its function in intermediary amino acid metabolism the Lumbricus hind-mid-intestine exemplifies the region of primitive gut (Prents, 1987), which in vertebrates differentiated into a purely absorptive gut and a liver. Interestingly enough this region of the intestinal epithelium also exhibits considerable arginase activity (Prento, 1989) and may thus also carry out urea biogenesis. This further supports the concept (see Bishop and Campbell, 1963). The distribution of GDH should be of interest for the analysis of the functional differentiation of the intestine and the location of intermediary metabolism in other invertebrates. As the histochemical demonstration of GDH is very straightforward, this analysis may be performed even for animals too small or too complex to permit fractionation techniques. Acknowledgements-The
author wishes to thank Alice Kristiansen for her skilled technical assistance.
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