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Proximal to distal variations in enzymes of the rat intestine
432
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 26636
P R O X I M A L TO D I S T A L V A R I A T I O N S IN ENZYMES OF T H E RAT INTESTINE
D. D. H A R R I S O N AND H. L. W E B S T E R
The Department of Biochemistry, Institute of Pathology, Royal dlexandra Hospital for Children, Sydney (Australia) ( R e c e i v e d M a r c h 5th, 1971)
SUMMARY
Studies have been made of the distribution of four enzymes, invertase, alkaline phosphatase, leucine aminopeptidase and esterase from the proximal to the distal ends of the rat intestine. The enzyme activities and protein content of ten segments were assayed on epithelial cells harvested from the everted gut by a vibrational technique previously elaborated by the authors. Invertase activity doubles its extreme proximal level to reach a distinct peak at the mid-jejunum and then rapidly falls to zero distal values. Leucine aminopeptidase and esterase are relatively constant in activity up to the mid-jejunum, then slowly decrease distally. Alkaline phosphatase distribution is quite different, showing a m a x i m u m at the most proximal point, falling very sharply in the near-proximal area to reach zero at the distal end. A critical evaluation of the possible explanations of these results is presented.
INTRODUCTION In recent years there has been a trend towards studies of the gut of experimental animals, particularly the rat, on a widening enzymological front. As a background to such investigations it is essential that information be available on the biochemical characteristics of the gut at different points from proximal to distal regions. It has been reported by FISHER AND PARSONS1 that the absorptive and digestive potential of the gut varies according to position along the tract. Studies on human intestinal biopsy material by NEWCOMER AND McGILL2 have also indicated regional variations in the activity of disaccharidases. In an earlier paper a, the authors reported on the activities of certain enzymes during the transformation of crypt to columnar epithelial cells. These results referred to combined cell collections from the proximal and near-distal regions. In the present paper, the object has been to provide further enzymic data with particular emphasis on specific regions of the intestinal tract. Biochim. Biophys. Acta, 244 (1971) 432-436
433
ENZYMIC ACTIVITIES ALONG THE INTESTINE MATERIALS
Experimental animals Male Wistar rats weighing about 25 ° g were used. The animals were fed and provided with water up to the time of killing, then stunned and bled via the severed thoracic vessels. The small intestine was removed, separated from mesenteric tissue and washed with isotonic salt solution.
Columnar intestinal cell preparation The small intestine was cut into ten equal lengths (about 15 cm), representing a progression from the proximal to the distal end of the gut. Each portion was everted onto rods, immersed in IOO ml o.14 M NaC1-5 mM EDTA solution and vibrated at 2 mm amplitude for 20 rain in the cold as previously described 3. Examination of the gut after these procedures showed that the crypts were still predominantly intact.
Chemicals All chemicals used were of Analytical Reagent grade. METHODS
Enzymic assays All enzymes were assayed colorimetrically. The unit of activity was taken as the transformation of I /,mole of substrate/min. Assays were conducted at 20 ° for esterase and 37 ° for the other enzymes. Leucine aminopeptidase was estimated using L-leucyl /5-naphthylamine as substrate according to the method of GREEN et al. 4. Invertase was assayed by utilising a glucose oxidase method to follow the rate of hydrolysis of sucrose. The conditions of assay were similar to those of DAHLQVIST 5. Alkaline phosphatase activity was measured by a modification of the method of BESSEY et al2. Zn ~+ (I mM) and Mg ~+ (5 raM) were included in the assay incubation, which was carried out at pH 9.2. p-Nitrophenyl acetate hydrolase activity was measured according to the method of HUGGINSAND L A P I D E S 7. Correction was made for the rate of spontaneous hydrolysis of the substrate. Although the substrate employed is an unphysiological ester, the assumption has been made that the cellular enzyme catalysing hydrolysis of esters of aliphatic alcohols and lower fatty acids has some affinity for this substrate. The enzyme is subsequently in this paper referred to as "esterase". Protein was estimated by the method of Lowry using bovine serum albumen or Hyland freeze-dried human serum as standards. RESULTS
Aliquots of villous cell suspensions in EDTA-saline from each of the gut segments, proximal to distal, were immediately taken for analysis. For protein, esterase, alkaline phosphatase and invertase assays these aliquots were used directly, after appropriate dilutions. These final dilutions were made with water to effect cell lysis and so remove limits on the access of substrates to the intracellular enzymes. For Biochim. Biophys. Acta, 244 (1971) 4 3 2 - 4 3 6
434
D.D.
H A R R I S O N , H . L. W E B S T E R
leucine aminopeptidase, the cellular material in an aliquot was centrifuged, drained and resuspended in water, from which a portion was taken for enzyme assay. The latter procedure was necessary to reduce the concentration of E D T A to levels which did not interfere with leucine aminopeptidase activity.
Protein The amount of villous cell protein recovered from each segment is shown in Table I. I t is seen that the cellular material is most concentrated in the proximal end of the gut with a suggestion of a m a x i m u m in Segments 2-5. Thereafter, more distally, the cellular protein decreases to a distal level of approximately half the peak value. TABLE
I
VILLOUS CELL PROTEIN
IN SEQUENTIAL
Segment (No.)
Protein (mg/segment) *
i (proximal) 2 3 4 5 6 7 8 9 IO ( d i s t a l )
49 54 61 60 60 52 43 37 32 31
GUT SEGMENTS
• Mean values for 8 experiments.
Enzymes The data for specific activities of four enzymes along the length of the gut is illustrated in Figs. IA to ID. For invertase (Fig. IC) the level of activity at the extreme proximal end increases approximately 2-fold to reach a sharp m a x i m u m at Segment 5 (the mid-j eiunum), then falls to a far-distal level of virtually zero. The distribution of alkaline phosphatase (Fig. IB), is in marked contrast. The m a x i m u m specific activity coincides with the most proximal area of the gut, falling very steeply in the second segment to half this value, thereafter being steadily reduced to very low far-distal levels. Leucine aminopeptidase activity (Fig. IA), appears to be fairly constant proximally (up to Segment 5) then falls to about half this level at the distal end of the gut. Esterase activity (Fig. ID), appears to be uniform throughout the gut length, tending possibly to become slightly less distally. DISCUSSION
The observed topographical variations in enzyme activity of villous ceils along the gut could be explained in a number of ways. It is possible that the crypt cells at various levels have an intrinsically different biochemical potential to produce digestive and other enzymes. Definitive evidence for this view is lacking; it is known for example that the absorption of vitamin BI2 in the rat is confined to the ileum s, nevertheless, other explanations for such functional foci could be advanced. Biochim. Biophys. Acta, 2 4 4 (1971) 4 3 2 - 4 3 6
ENZYMIC ACTIVITIES ALONG THE INTESTINE 40 t
435
n, 2
20
""-'--
A O]
°__°
"°
1000
i
{:::
ol k.
n=4
"\'\._.\
500
c E 0 o E v >.l
80.
.>
-
-
./_.7
/\
40
\.
.u
0
°°
\
C
"~.
•~
n =5 °~°~o~.~o~-~-°~o~.~o
O'
D 1
i 2
3
i
4 5 6 7 8 9 Segment no from most p r o x i m a l (1) to most distol (10)
i 10
Fig. I. Mean specific activities of e n z y m e s at various distances from the proximal end of the rat gut. A, B, C and D refer, respectively, to leucine aminopeptidase, alkaline phosphatase, invertase and esterase, n = number of experiments.
It m a y be argued that the potential of the crypts is the same and that differences in the production of certain enzymes during subsequent maturation depends on environmental luminal or humoral factors. Strong support for this view has been provided by DOWLING AND BOOTH 9, who observed adaptive ileal changes following proximal intestinal resection. The present data for invertase distribution are not inconsistent with this argument; the prominent peak of specific activity corresponds with the mid-jejunum m a x i m u m for glucose absorption reported by FISHER AND PARSONS1. A similar distribution has been found for maltase, sucrase and lactase in human intestinal biopsies by NEWCOMER AND McGILL2; however, the fall in sucrase activity toward the distal end of the human gut was not so marked as that observed in the present report on the rat intestine. Another explanation for variation is the possibility that the villi are more developed in concentration per unit area, or in height, at different points of the gut. The authors have described profiles of increasing activity for various enzymes as epithelial cells escalate toward the tip of the villus 3. Where the profile is steep, commencing from very low crypt values (e.g. invertase and alkaline phosphatase), it is conceivable that the greater maturation time involved in escalation of tall villi would be associated Bi oc hi m . Biophys. Acta, 244 (1971) 432-436
43b
D. D. HARRISON, H. L. WEBSTER
with higher enzyme activities. In this context, the present results are equivocal. While the invertase m a x i m u m coincides with the gut area of greatest villous cell protein concentration, the alkaline phosphatase data shows no such correlation. Leucine aminopeptidase and esterase have a less dramatic profile of increase from the crypt level during maturation a, so that the effect of variation in villus height would be expected to be less. This is consistent with the finding that up to the mid-jejunal region their activity is relatively constant, after which the levels are correlated with a decline in cellular protein concentration as tile distal end is approached. Correlation of enzyme activities with maximal development of brush border structures is not convincing. Invertase 1°, glycylglycine dipeptidase 11 and alkaline phosphatase 12, are claimed to be localised specifically in the brush borders, yet the distributions of these enzymes apparently have little in common. The distinctly different distribution of alkaline phosphatase is notable. Despite considerable investigation, the intestinal function of this enzyme is still not clear. If the present observations reflect this function, they draw attention to the immediate proximal region of the gut. It is tempting to speculate that membrane-bound alkaline phosphatase is an integral part of an ion and water transport system which could be functioning maximally in the making of osmotic adjustments immediately post pylorus. ACKNOWLEDGEMENTS
This work was supported by a research project grant from the National Health and Medical Research Council of Australia. REFERENCES R. ]3. FISHER AND D. S. PARSONS,J. Physiol., I1O (1949) 36. A. D. NEWCOMER AND D. B. McGILL, Gastroenlerology, 51 (1966) 481. H. L. WEBSTER AND D. D. HARRISON,Exptl. Cell Res., 56 (1969) 245. M. N. GREEN, K. T s o u , R. BRESSLER AND A. M. SELIGMAN, Arch. Biochem. Biophys., 57 (I955} 458 • 5 A. DAHLQVIST, Anal. Biochem., 7 (1964) 18. 6 0 . A. BESSEY, O. H. LOWRY AND M. J. BROCK, J. Biol. Chem., 164 (1946 ) 321. 7 C. HUGGINS AND J. LAPIDES, J. Biol. Chem., 17o (1947) 467 • 8 P. C. REYNELL, G. H. SPRAY AND K. B. TAYLOR, Clin. Sci., 16 (1957) 663. 9 R. H. DOWLIXG AND C. C. BOOTH, Clin. Sci., 32 (1967) 139. IO D. MILLER AND R. K. CRANE, Biochim. Biophys. Acta, 52 (1961) 293. i i J. H. HOLT AND D. MILLER, Biochim. Biophys. Acta, 58 (1962) 239. 12 F. R. JOHNSON AND J. H. KUGLER, J. Anat., 87 (1953) 247. I 2 3 4
Biochim. Biophys. Acta, 244 (I97I) 4 3 2 - 4 3 6
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 26636
P R O X I M A L TO D I S T A L V A R I A T I O N S IN ENZYMES OF T H E RAT INTESTINE
D. D. H A R R I S O N AND H. L. W E B S T E R
The Department of Biochemistry, Institute of Pathology, Royal dlexandra Hospital for Children, Sydney (Australia) ( R e c e i v e d M a r c h 5th, 1971)
SUMMARY
Studies have been made of the distribution of four enzymes, invertase, alkaline phosphatase, leucine aminopeptidase and esterase from the proximal to the distal ends of the rat intestine. The enzyme activities and protein content of ten segments were assayed on epithelial cells harvested from the everted gut by a vibrational technique previously elaborated by the authors. Invertase activity doubles its extreme proximal level to reach a distinct peak at the mid-jejunum and then rapidly falls to zero distal values. Leucine aminopeptidase and esterase are relatively constant in activity up to the mid-jejunum, then slowly decrease distally. Alkaline phosphatase distribution is quite different, showing a m a x i m u m at the most proximal point, falling very sharply in the near-proximal area to reach zero at the distal end. A critical evaluation of the possible explanations of these results is presented.
INTRODUCTION In recent years there has been a trend towards studies of the gut of experimental animals, particularly the rat, on a widening enzymological front. As a background to such investigations it is essential that information be available on the biochemical characteristics of the gut at different points from proximal to distal regions. It has been reported by FISHER AND PARSONS1 that the absorptive and digestive potential of the gut varies according to position along the tract. Studies on human intestinal biopsy material by NEWCOMER AND McGILL2 have also indicated regional variations in the activity of disaccharidases. In an earlier paper a, the authors reported on the activities of certain enzymes during the transformation of crypt to columnar epithelial cells. These results referred to combined cell collections from the proximal and near-distal regions. In the present paper, the object has been to provide further enzymic data with particular emphasis on specific regions of the intestinal tract. Biochim. Biophys. Acta, 244 (1971) 432-436
433
ENZYMIC ACTIVITIES ALONG THE INTESTINE MATERIALS
Experimental animals Male Wistar rats weighing about 25 ° g were used. The animals were fed and provided with water up to the time of killing, then stunned and bled via the severed thoracic vessels. The small intestine was removed, separated from mesenteric tissue and washed with isotonic salt solution.
Columnar intestinal cell preparation The small intestine was cut into ten equal lengths (about 15 cm), representing a progression from the proximal to the distal end of the gut. Each portion was everted onto rods, immersed in IOO ml o.14 M NaC1-5 mM EDTA solution and vibrated at 2 mm amplitude for 20 rain in the cold as previously described 3. Examination of the gut after these procedures showed that the crypts were still predominantly intact.
Chemicals All chemicals used were of Analytical Reagent grade. METHODS
Enzymic assays All enzymes were assayed colorimetrically. The unit of activity was taken as the transformation of I /,mole of substrate/min. Assays were conducted at 20 ° for esterase and 37 ° for the other enzymes. Leucine aminopeptidase was estimated using L-leucyl /5-naphthylamine as substrate according to the method of GREEN et al. 4. Invertase was assayed by utilising a glucose oxidase method to follow the rate of hydrolysis of sucrose. The conditions of assay were similar to those of DAHLQVIST 5. Alkaline phosphatase activity was measured by a modification of the method of BESSEY et al2. Zn ~+ (I mM) and Mg ~+ (5 raM) were included in the assay incubation, which was carried out at pH 9.2. p-Nitrophenyl acetate hydrolase activity was measured according to the method of HUGGINSAND L A P I D E S 7. Correction was made for the rate of spontaneous hydrolysis of the substrate. Although the substrate employed is an unphysiological ester, the assumption has been made that the cellular enzyme catalysing hydrolysis of esters of aliphatic alcohols and lower fatty acids has some affinity for this substrate. The enzyme is subsequently in this paper referred to as "esterase". Protein was estimated by the method of Lowry using bovine serum albumen or Hyland freeze-dried human serum as standards. RESULTS
Aliquots of villous cell suspensions in EDTA-saline from each of the gut segments, proximal to distal, were immediately taken for analysis. For protein, esterase, alkaline phosphatase and invertase assays these aliquots were used directly, after appropriate dilutions. These final dilutions were made with water to effect cell lysis and so remove limits on the access of substrates to the intracellular enzymes. For Biochim. Biophys. Acta, 244 (1971) 4 3 2 - 4 3 6
434
D.D.
H A R R I S O N , H . L. W E B S T E R
leucine aminopeptidase, the cellular material in an aliquot was centrifuged, drained and resuspended in water, from which a portion was taken for enzyme assay. The latter procedure was necessary to reduce the concentration of E D T A to levels which did not interfere with leucine aminopeptidase activity.
Protein The amount of villous cell protein recovered from each segment is shown in Table I. I t is seen that the cellular material is most concentrated in the proximal end of the gut with a suggestion of a m a x i m u m in Segments 2-5. Thereafter, more distally, the cellular protein decreases to a distal level of approximately half the peak value. TABLE
I
VILLOUS CELL PROTEIN
IN SEQUENTIAL
Segment (No.)
Protein (mg/segment) *
i (proximal) 2 3 4 5 6 7 8 9 IO ( d i s t a l )
49 54 61 60 60 52 43 37 32 31
GUT SEGMENTS
• Mean values for 8 experiments.
Enzymes The data for specific activities of four enzymes along the length of the gut is illustrated in Figs. IA to ID. For invertase (Fig. IC) the level of activity at the extreme proximal end increases approximately 2-fold to reach a sharp m a x i m u m at Segment 5 (the mid-j eiunum), then falls to a far-distal level of virtually zero. The distribution of alkaline phosphatase (Fig. IB), is in marked contrast. The m a x i m u m specific activity coincides with the most proximal area of the gut, falling very steeply in the second segment to half this value, thereafter being steadily reduced to very low far-distal levels. Leucine aminopeptidase activity (Fig. IA), appears to be fairly constant proximally (up to Segment 5) then falls to about half this level at the distal end of the gut. Esterase activity (Fig. ID), appears to be uniform throughout the gut length, tending possibly to become slightly less distally. DISCUSSION
The observed topographical variations in enzyme activity of villous ceils along the gut could be explained in a number of ways. It is possible that the crypt cells at various levels have an intrinsically different biochemical potential to produce digestive and other enzymes. Definitive evidence for this view is lacking; it is known for example that the absorption of vitamin BI2 in the rat is confined to the ileum s, nevertheless, other explanations for such functional foci could be advanced. Biochim. Biophys. Acta, 2 4 4 (1971) 4 3 2 - 4 3 6
ENZYMIC ACTIVITIES ALONG THE INTESTINE 40 t
435
n, 2
20
""-'--
A O]
°__°
"°
1000
i
{:::
ol k.
n=4
"\'\._.\
500
c E 0 o E v >.l
80.
.>
-
-
./_.7
/\
40
\.
.u
0
°°
\
C
"~.
•~
n =5 °~°~o~.~o~-~-°~o~.~o
O'
D 1
i 2
3
i
4 5 6 7 8 9 Segment no from most p r o x i m a l (1) to most distol (10)
i 10
Fig. I. Mean specific activities of e n z y m e s at various distances from the proximal end of the rat gut. A, B, C and D refer, respectively, to leucine aminopeptidase, alkaline phosphatase, invertase and esterase, n = number of experiments.
It m a y be argued that the potential of the crypts is the same and that differences in the production of certain enzymes during subsequent maturation depends on environmental luminal or humoral factors. Strong support for this view has been provided by DOWLING AND BOOTH 9, who observed adaptive ileal changes following proximal intestinal resection. The present data for invertase distribution are not inconsistent with this argument; the prominent peak of specific activity corresponds with the mid-jejunum m a x i m u m for glucose absorption reported by FISHER AND PARSONS1. A similar distribution has been found for maltase, sucrase and lactase in human intestinal biopsies by NEWCOMER AND McGILL2; however, the fall in sucrase activity toward the distal end of the human gut was not so marked as that observed in the present report on the rat intestine. Another explanation for variation is the possibility that the villi are more developed in concentration per unit area, or in height, at different points of the gut. The authors have described profiles of increasing activity for various enzymes as epithelial cells escalate toward the tip of the villus 3. Where the profile is steep, commencing from very low crypt values (e.g. invertase and alkaline phosphatase), it is conceivable that the greater maturation time involved in escalation of tall villi would be associated Bi oc hi m . Biophys. Acta, 244 (1971) 432-436
43b
D. D. HARRISON, H. L. WEBSTER
with higher enzyme activities. In this context, the present results are equivocal. While the invertase m a x i m u m coincides with the gut area of greatest villous cell protein concentration, the alkaline phosphatase data shows no such correlation. Leucine aminopeptidase and esterase have a less dramatic profile of increase from the crypt level during maturation a, so that the effect of variation in villus height would be expected to be less. This is consistent with the finding that up to the mid-jejunal region their activity is relatively constant, after which the levels are correlated with a decline in cellular protein concentration as tile distal end is approached. Correlation of enzyme activities with maximal development of brush border structures is not convincing. Invertase 1°, glycylglycine dipeptidase 11 and alkaline phosphatase 12, are claimed to be localised specifically in the brush borders, yet the distributions of these enzymes apparently have little in common. The distinctly different distribution of alkaline phosphatase is notable. Despite considerable investigation, the intestinal function of this enzyme is still not clear. If the present observations reflect this function, they draw attention to the immediate proximal region of the gut. It is tempting to speculate that membrane-bound alkaline phosphatase is an integral part of an ion and water transport system which could be functioning maximally in the making of osmotic adjustments immediately post pylorus. ACKNOWLEDGEMENTS
This work was supported by a research project grant from the National Health and Medical Research Council of Australia. REFERENCES R. ]3. FISHER AND D. S. PARSONS,J. Physiol., I1O (1949) 36. A. D. NEWCOMER AND D. B. McGILL, Gastroenlerology, 51 (1966) 481. H. L. WEBSTER AND D. D. HARRISON,Exptl. Cell Res., 56 (1969) 245. M. N. GREEN, K. T s o u , R. BRESSLER AND A. M. SELIGMAN, Arch. Biochem. Biophys., 57 (I955} 458 • 5 A. DAHLQVIST, Anal. Biochem., 7 (1964) 18. 6 0 . A. BESSEY, O. H. LOWRY AND M. J. BROCK, J. Biol. Chem., 164 (1946 ) 321. 7 C. HUGGINS AND J. LAPIDES, J. Biol. Chem., 17o (1947) 467 • 8 P. C. REYNELL, G. H. SPRAY AND K. B. TAYLOR, Clin. Sci., 16 (1957) 663. 9 R. H. DOWLIXG AND C. C. BOOTH, Clin. Sci., 32 (1967) 139. IO D. MILLER AND R. K. CRANE, Biochim. Biophys. Acta, 52 (1961) 293. i i J. H. HOLT AND D. MILLER, Biochim. Biophys. Acta, 58 (1962) 239. 12 F. R. JOHNSON AND J. H. KUGLER, J. Anat., 87 (1953) 247. I 2 3 4
Biochim. Biophys. Acta, 244 (I97I) 4 3 2 - 4 3 6