- Email: [email protected]
Giardia lamblia: Expression of alkaline phosphatase activity in infected rat intestine
Experimental Parasitology 110 (2005) 91–95 www.elsevier.com/locate/yexpr
Giardia lamblia: Expression of alkaline phosphatase activity in infected rat intestine Safrun Mahmood a,b,¤, Kamaljit Kaur a, Nidhi Mittal a, Akhtar Mahmood a a
b
Department of Biochemistry, Panjab University, Chandigarh 160014, India Department of Experimental Medicine and Biotechnology, Postgraduate Institute of Medical Education and Research, Chandigarh 160012, India Received 30 April 2004; received in revised form 8 March 2005; accepted 9 March 2005 Available online 20 April 2005
Abstract Alkaline phosphatase (IAP) is a marker of intestinal microvillus membrane. Changes in IAP activity have been studied as a function of Giardia lamblia (G. lamblia) infection using rat as the experimental model. At day 11 and 15 post-infection, enzyme activity was reduced (p < 0.01) compared to controls. The enzyme levels were essentially similar to control values by day 30 post-infection. Analysis of the enzyme activity in cell fractions across crypt–villus axis revealed a marked decrease in enzyme activity in the villus tip and mid villus regions but a considerable increase (p < 0.01) in enzyme activity in the crypt base of 11 day post-infected animals compared to that in controls. The observed changes in IAP activity were conWrmed by assaying the enzyme activity in acrylamide gels using bromo-chloro-indolyl phosphate staining under non-denaturing conditions. These Wndings indicate diVerential changes across the crypt–villus axis, but impaired alkaline phosphatase levels in G. lamblia infected rat intestine. 2005 Elsevier Inc. All rights reserved. Keywords: Rat intestine; Giardia lamblia infection; Alkaline phosphatase expression
1. Introduction Giardia lamblia infection is a well-known cause of intestinal malabsorption and diarrhea in humans. However, the underlying mechanism of intestinal dysfunction in giardiasis remains largely unknown. Studies in both humans and experimental animals have shown that parasitic infection induces morphological and biochemical changes in intestine (Adam, 2001; Phillip and Smith, 1985; Vesy and Peterson, 1999). A marked decrease in the transport of nutrients and the activity of brush border enzymes has been reported (Anand et al., 1982; Mahmood et al., 2002). The observed decrease in brush border disaccharidases in giardial infection is a consequence of the downregulation of mRNA expression encoding sucrase and lactase (Mahmood et al., 2002). However, it is not known how
*
Corresponding author. E-mail address: [email protected] (S. Mahmood).
0014-4894/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2005.03.017
parasitic infection aVects the development of enzymes across crypt–villus axis in the intestine. Alkaline phosphatase (AP) is a marker enzyme of microvillus membranes (MVM) in intestine and exists both in the soluble and membrane bound forms (Young et al., 1981). The two isoforms of the enzyme are encoded by 2.7 and 3.0 kb mRNA transcripts (Eliakim et al., 1990; Lowe et al., 1990). The isoenzymes undergo considerable modiWcation during postnatal development (Sandhu and Mahmood, 1990). In view of these observations, in the present study, we studied the eVect of G. lamblia on the expression of AP in rat intestine. 2. Materials and methods 2.1. Animals and treatments Male Wistar rats, 6–8 weeks, old were used. G. lamblia cysts obtained from the human stool were puriWed on
92
S. Mahmood et al. / Experimental Parasitology 110 (2005) 91–95
sucrose gradient (Roberts-Thomson et al., 1976). An inoculum of 10,000 cysts/animal in 0.2 ml of normal saline was given orally by Ryle’s tube to each rat. Overnight fasted animals were sacriWced on day 0, 6, 11, 15, 30, and 40 post-infection under ether anesthesia. Starting from the ligament of Treitz, the entire intestine was removed and thoroughly washed with normal saline. Trophozoites were counted in a haemocytometer and the results were expressed as number of trophozoites per milliliter in the drained intestinal perfusate. 2.2. Preparation of brush border membranes Brush border membranes (BBM) were isolated and puriWed following the method of Schmitz et al. (1973). The membranes were suspended in 50 mM sodium maleate buVer, pH 6.8. The Wnal membrane preparation exhibited 10- to 14-fold enrichment of the marker enzymes, sucrase or AP compared to those in the homogenate. 2.3. Enzyme assay Alkaline phosphatase activity was determined according to the method of Bergmeyer (1963). Protein was estimated by the method of Lowry et al. (1951) using bovine serum albumin (BSA) as the standard. 2.4. Preparation of cell fractions Epithelial cells from intestine were isolated following the method of Weiser (1973). The intestine was rinsed thoroughly with solution containing 0.154 M NaCl and 1 mM dithiothreitol and incubated at 37 °C for 15 min. The Xuid was discarded, and the intestine was reWlled with 1.5 mM ethylene diamine tetra acetic acid (EDTA) and phosphate-buVered saline (PBS), pH 7.2. By a series of incubations and washings of intestinal loop at 37 °C, sequential fractions of epithelial cells were isolated. The cell fractions were suspended in 50 mM sodium maleate buVer, pH 6.8, and homogenized in the same buVer. The cell homogenate was centrifuged at 1000g for 10 min at 4 °C and the supernatant was used for biochemical analysis.
desired color intensity were obtained, the reaction was stopped by transferring the gels to 10% acetic acid. Finally the gel was washed with distilled water and dried under vacuum in a gel dryer at 80 °C for 2–3 h using cellophane membranes. The densitometric scan of the gels was obtained at a wavelength of 540 nm. 2.6. Statistical analysis Statistical analysis of the data were done using unpaired Student’s t test with p D 0.05 as the limit of statistical signiWcance.
3. Results The number of trophozoites in the intestine of G. lamblia infected animals was markedly enhanced from day 6 to 15 after infection (Fig. 1). However, by day 30–40 post-infection the trophozoite counts were appreciably reduced. As shown in Table 1, AP activity assayed in tissue homogenates as well as in puriWed microvillus membranes was signiWcantly reduced (p < 0.01) at day 11 and 15 post-infection. These Wndings were further conWrmed by assaying the enzyme activity in membrane proteins separated by SDS–PAGE under non-denaturing conditions (Fig. 2A). The enzyme activity was located in the gels by hydrolysis of BCIP, which appeared blue in color at the site of enzyme activity. The intensity of the color corresponded to enzyme levels in the membrane (Fig. 2B). The color intensity representing AP activity was low in membranes isolated from 11 and 15-day post-infected animals but the enzyme staining was essentially similar by day 30 of post-infection when compared to the equivalent controls.
2.5. 3⬘-Bromo-4-chloro indolyl phosphate (BCIP) staining Separation of brush border membrane proteins solubilized with 0.1% sodium dodecyl sulfate (SDS) was carried out by polyacrylamide gel electrophoresis (PAGE) under non-denaturing conditions (Lammeli, 1970). Hundred micrograms of the SDS solubilized membrane protein was run on 8% acrylamide gels. AP activity was stained in situ in presence of BCIP (1 mg/ml in 50 mM Tris–HCl, pH 7.6) solution at 37 °C. After bands of
Fig. 1. Trophozoite counts in G. lamblia infected rat intestine. Values are meana § SD of 6–8 animals.
S. Mahmood et al. / Experimental Parasitology 110 (2005) 91–95
93
Table 1 EVect of G. lamblia infection on alkaline phosphatase activity in rat intestine
Homogenate BBM
Control (0 day)
6th Day
11th Day
15th Day
30th Day
40th Day
0.05 § 0.01 0.28 § 0.07
0.05 § 0.003 0.25 § 0.030
0.04 § 0.01¤ 0.15 § 0.09¤
0.04 § 0.004¤ 0.11 § 0.02¤
0.07 § 0.02 0.29 § 0.03
0.05 § 0.01 0.34 § 0.09
Values are means § SD of 6–8 animals, as units/mg protein. ¤ p < 0.01 vs. control.
Fig. 2. (A) BCIP staining of acrylamide gels for alkaline phosphatase activity in G. lamblia infected rat intestine. 100 g membrane protein was applied to each well. (B) Densitometric scan of the data given in (A).
The eVect of G. lamblia infection on AP activity in epithelial cells across the crypt–villus axis was also analyzed. The serial cell fractions representing villus tip to crypt base from control and 11 day post-infected animals were isolated and enzyme activity was assayed. These results are shown in Fig. 3, AP activity was much higher in the upper villus cells and was almost absent in the crypt region of the control animals. However, in rats at 11 day, post-infection, AP activity was reduced (p < 0.01) in the villus tip and mid villus (p < 0.001) regions and was signiWcantly higher (p < 0.01) in the crypt base compared to corresponding villus and crypt cells in the control animals. These Wndings were further conWrmed by assaying the enzyme activity in the polyacrylamide gels using BCIP staining (Fig. 4). As expected, the staining of gels for IAP activity was quite high in the villus tip cells compared to the crypt cells in the control animals. However, the BCIP staining of enzyme activity was low in villus tip and mid villus regions but was relatively higher in crypt cells in G. lamblia infected rats compared to the corresponding cell fractions in the controls (Fig. 4B).
Fig. 3. Distribution of alkaline phosphatase activity in cell fractions across the crypt–villus axis in the control and G. lamblia infected rat intestine. Means § SD of 6–8 animals. *p < 0.05; **p < 0.01; and ***p < 0.001 vs. control.
4. Discussion The data presented here indicate that AP activity in the crypt base was signiWcantly higher in G. lamblia infected animals compared to controls but reverse was the case in the villus tip region under these conditions. The AP ratio in the villus tip to crypt base was around 12 in control animals, which was reduced to 4 in infected group. Although the parasitic infection was established as early as day 4 (results not shown), by day 6 post-infection, the animals exhibited considerable levels of Giardia trophozoites in intestinal perfusate. AP activity was considerably reduced in intestine of animals infected with G. lamblia by day 11 and 15 post-infection compared to controls. The observed decrease in AP activity at day 11 and 15 post-infection is similar to that reported by others for AP and disaccharidases in rat intestine (Anand et al., 1982; Mahmood et al., 2002). At day 11–15 post-infection, the enzyme levels in intestine were considerably lower than those in controls. This was associated with a signiWcant decrease in enzyme activity in the upper villus
94
S. Mahmood et al. / Experimental Parasitology 110 (2005) 91–95
AP activity in giardial infection. This is indeed corroborated by the fact that considerably low enzyme activity was detected in microvillus membrane by BCIP staining of membrane proteins resolved on acrylamide gels. Thus, the migration of epithelial cells across crypt–villus axis is altered by the parasitic infections. We previously reported a considerable decrease in the activity of brush border sucrase and lactase, which is associated with low levels of mRNA encoding sucrase and lactase in G. lamblia infected rat intestine (Nidhi et al., 1999). Recently, Mahmood et al. (2002) described low mRNA encoding sodium–glucose co-transporter (SGLT1) in Giardia infected rats. This suggested that downregulation of the expression of these proteins might be responsible for the observed decline in enzyme activity. Although similar studies have not been carried out with intestinal alkaline phosphatase, it is possible that a similar phenomenon may occur with this enzyme in G. lamblia infection.
Fig. 4. (A) BCIP staining of the acrylamide gel for alkaline phosphatase activity in villus tip (lanes 1, 4), mid villus (lanes 2, 5), and crypt region (lanes 3, 6) of control (lanes 1, 2, and 3) and G. lamblia infected (lanes 4, 5, and 6) rats. 100 g membrane protein was applied to each well. (B) Densitometric scan of the data given in (A).
and mid villus cells compared to that in the controls. However, in the crypt base, AP activity was elevated in infected animals compared to controls. Mc Donald and Ferguson (1978) showed that the turnover rate of enterocytes across the crypt–villus axis is considerably increased in chronic G. muris infected mice. This was associated with enhanced hyperplasia of crypt cells, such an increase in the number of enterocytes in the crypt base would also explain the increase in AP activity in the crypt region as seen in the present study. G. lamblia infection also induces an increase in epithelial cell mitosis (Smith, 1985), which could be responsible for the observed increase in IAP activity under these conditions. Xie et al. (1997) have demonstrated that mRNA encoding two IAP isoforms is diVerently distributed across crypt–villus axis in rat intestine. Alkaline phosphatase-I is mainly present in the villus tip but intestinal alkaline phosphatase-II mRNA is abundantly present in crypt as well as in villus tip cells. Thus diVerential expression of the two isoforms of IAP mRNAs across crypt–villus unit in the infected tissue may also be responsible for the observed changes in the enzyme level. However, studies on mRNA levels encoding IAP in G. lamblia infected animals are required to further explain these Wndings. Morphological studies have shown shortening of villi and distortion of the microvilli in giardiasis (Harloug et al., 1979). Ganguly et al. (1985) observed disruption of the microvilli under electron microscopy in G. lamblia infected intestine. This may also explain the low levels of
Acknowledgments Financial assistance from Indian Council of Medical Research, New Delhi for this study is gratefully acknowledged. Stool samples of patients suVering from giardiasis were obtained from the Department of Parasitology, PGIMER, Chandigarh.
References Adam, R.D., 2001. Biology of Giardia lamblia. Clinical Microbiology Reviews 14, 447–475. Anand, B.S., Mahmood, A., Ganguly, N.K., Rehani, M.H., Dilawari, J.B., Mahajan, R.C., 1982. Transport studies and enzyme assays in mice infected with human Giardia lamblia. Transactions of the Royal Society of Tropical Medicine and Hygiene 76, 616–619. Bergmeyer, M.V.C., 1963. Methods of Enzymatic Analysis, Academic Press, New York, vol. 40, pp. 783–785. Eliakim, R., Seetharam, S., Tietze, C.C., Alpers, D.H., 1990. DiVerential regulation of the mRNAs encoding for rat intestinal alkaline phosphatase. American Journal of Physiology 259, G93–G98. Ganguly, N.K., Mahajan, R.C., Radhakrishna, V., Bhagwat, A.G., 1985. Longitudinal electron microscopic studies in the jejunum of Giardia lamblia infected mice. Indian Journal of Medical Research 86, 102–107. Harloug, N.A., Gourley, W.K., Arvanitakis, C., 1979. Giardiasis: clinical spectrum and functional structural abnormalities of small intestinal mucosa. Gastroenterology 77, 161–169. Lammeli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of Bacteriophage T4. Nature 227, 680–685. Lowe, M., Strauss, A.W., Alpers, R., Seetharam, S., Alpers, D.H., 1990. Molecular cloning and expression of cDNA encoding the membrane associated rat intestinal alkaline phosphatase. Biochimica Biophysica Acta 1037, 170–177. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Ravdall, R.J., 1951. Protein measurement with Folin phenol reagent. Journal of Biological Chemistry 193, 265–275. Mahmood, S., Sodhi, C.P., Ganguly, N.K., 2002. Expression of sodium–glucose cotransporter and brush border disaccharidases in
S. Mahmood et al. / Experimental Parasitology 110 (2005) 91–95 Giardia lamblia infected rat intestine. Indian Journal of Biochemistry and Biophysics 39, 185–190. Mc Donald, T.T., Ferguson, A., 1978. Small intestinal epithelial cell kinetics and protozoan infection in mice. Gastroenterology 74, 496–500. Nidhi, M., Nagpaul, J.P.N., Mahmood, S., Mahmood, A., 1999. Ethanol eVects on intestinal functions in Giardia lamblia infected rats. Journal of Clinical Biochemistry and Nutrition 26, 17–25. Phillip, D., Smith, M.D., 1985. Pathophysiology and immunology of Giardiasis. Annual Review of Medicine 36, 295–307. Roberts-Thomson, J.C., Stevens, D.P., Mahmoud, A.F., Warren, K.S., 1976. Giardiasis in mice—an animal model. Gastroenterology 71, 571–576. Sandhu, M., Mahmood, A., 1990. Kinetic characteristics of soluble and brush border alkaline phosphatase and sucrase activities in developing rat intestine: eVect of hormones. Indian Journal of Biochemistry and Biophysics 27, 88–92.
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Schmitz, J., Presier, H., Maestracci, D., Ghosh, B.K., Crane, R.K., 1973. PuriWcation of human intestinal microvillus membranes. Biochimica Biophysica Acta 323, 98–112. Smith, P.D., 1985. Pathophysiology and immunology of Giardiasis. Annual Review of Medicine 36, 295–307. Vesy, C.J., Peterson, W.L., 1999. The management of Giardiasis. Alimentary Pharmacology and Therapeutics 13, 843–850. Weiser, M.M., 1973. Intestinal epithelial cell surface membrane glycoproteins synthesis: an indicator of cellular diVerentiation. Journal of Biological Chemistry 248, 2536–2541. Xie, Q.M., Zhang, Y., Mahmood, S., Alpers, D.H., 1997. Rat intestinal alkaline phosphatase II mRNA is present in duodenal crypt and villus cells. Gastroenterology 112, 376–387. Young, G.P., Yeldin, S.T., Alpers, D.H., 1981. Distribution of soluble and membranous forms of alkaline phosphatase in the small intestine of the rat. Biochimica Biophysica Acta 676, 257–265.
Giardia lamblia: Expression of alkaline phosphatase activity in infected rat intestine Safrun Mahmood a,b,¤, Kamaljit Kaur a, Nidhi Mittal a, Akhtar Mahmood a a
b
Department of Biochemistry, Panjab University, Chandigarh 160014, India Department of Experimental Medicine and Biotechnology, Postgraduate Institute of Medical Education and Research, Chandigarh 160012, India Received 30 April 2004; received in revised form 8 March 2005; accepted 9 March 2005 Available online 20 April 2005
Abstract Alkaline phosphatase (IAP) is a marker of intestinal microvillus membrane. Changes in IAP activity have been studied as a function of Giardia lamblia (G. lamblia) infection using rat as the experimental model. At day 11 and 15 post-infection, enzyme activity was reduced (p < 0.01) compared to controls. The enzyme levels were essentially similar to control values by day 30 post-infection. Analysis of the enzyme activity in cell fractions across crypt–villus axis revealed a marked decrease in enzyme activity in the villus tip and mid villus regions but a considerable increase (p < 0.01) in enzyme activity in the crypt base of 11 day post-infected animals compared to that in controls. The observed changes in IAP activity were conWrmed by assaying the enzyme activity in acrylamide gels using bromo-chloro-indolyl phosphate staining under non-denaturing conditions. These Wndings indicate diVerential changes across the crypt–villus axis, but impaired alkaline phosphatase levels in G. lamblia infected rat intestine. 2005 Elsevier Inc. All rights reserved. Keywords: Rat intestine; Giardia lamblia infection; Alkaline phosphatase expression
1. Introduction Giardia lamblia infection is a well-known cause of intestinal malabsorption and diarrhea in humans. However, the underlying mechanism of intestinal dysfunction in giardiasis remains largely unknown. Studies in both humans and experimental animals have shown that parasitic infection induces morphological and biochemical changes in intestine (Adam, 2001; Phillip and Smith, 1985; Vesy and Peterson, 1999). A marked decrease in the transport of nutrients and the activity of brush border enzymes has been reported (Anand et al., 1982; Mahmood et al., 2002). The observed decrease in brush border disaccharidases in giardial infection is a consequence of the downregulation of mRNA expression encoding sucrase and lactase (Mahmood et al., 2002). However, it is not known how
*
Corresponding author. E-mail address: [email protected] (S. Mahmood).
0014-4894/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2005.03.017
parasitic infection aVects the development of enzymes across crypt–villus axis in the intestine. Alkaline phosphatase (AP) is a marker enzyme of microvillus membranes (MVM) in intestine and exists both in the soluble and membrane bound forms (Young et al., 1981). The two isoforms of the enzyme are encoded by 2.7 and 3.0 kb mRNA transcripts (Eliakim et al., 1990; Lowe et al., 1990). The isoenzymes undergo considerable modiWcation during postnatal development (Sandhu and Mahmood, 1990). In view of these observations, in the present study, we studied the eVect of G. lamblia on the expression of AP in rat intestine. 2. Materials and methods 2.1. Animals and treatments Male Wistar rats, 6–8 weeks, old were used. G. lamblia cysts obtained from the human stool were puriWed on
92
S. Mahmood et al. / Experimental Parasitology 110 (2005) 91–95
sucrose gradient (Roberts-Thomson et al., 1976). An inoculum of 10,000 cysts/animal in 0.2 ml of normal saline was given orally by Ryle’s tube to each rat. Overnight fasted animals were sacriWced on day 0, 6, 11, 15, 30, and 40 post-infection under ether anesthesia. Starting from the ligament of Treitz, the entire intestine was removed and thoroughly washed with normal saline. Trophozoites were counted in a haemocytometer and the results were expressed as number of trophozoites per milliliter in the drained intestinal perfusate. 2.2. Preparation of brush border membranes Brush border membranes (BBM) were isolated and puriWed following the method of Schmitz et al. (1973). The membranes were suspended in 50 mM sodium maleate buVer, pH 6.8. The Wnal membrane preparation exhibited 10- to 14-fold enrichment of the marker enzymes, sucrase or AP compared to those in the homogenate. 2.3. Enzyme assay Alkaline phosphatase activity was determined according to the method of Bergmeyer (1963). Protein was estimated by the method of Lowry et al. (1951) using bovine serum albumin (BSA) as the standard. 2.4. Preparation of cell fractions Epithelial cells from intestine were isolated following the method of Weiser (1973). The intestine was rinsed thoroughly with solution containing 0.154 M NaCl and 1 mM dithiothreitol and incubated at 37 °C for 15 min. The Xuid was discarded, and the intestine was reWlled with 1.5 mM ethylene diamine tetra acetic acid (EDTA) and phosphate-buVered saline (PBS), pH 7.2. By a series of incubations and washings of intestinal loop at 37 °C, sequential fractions of epithelial cells were isolated. The cell fractions were suspended in 50 mM sodium maleate buVer, pH 6.8, and homogenized in the same buVer. The cell homogenate was centrifuged at 1000g for 10 min at 4 °C and the supernatant was used for biochemical analysis.
desired color intensity were obtained, the reaction was stopped by transferring the gels to 10% acetic acid. Finally the gel was washed with distilled water and dried under vacuum in a gel dryer at 80 °C for 2–3 h using cellophane membranes. The densitometric scan of the gels was obtained at a wavelength of 540 nm. 2.6. Statistical analysis Statistical analysis of the data were done using unpaired Student’s t test with p D 0.05 as the limit of statistical signiWcance.
3. Results The number of trophozoites in the intestine of G. lamblia infected animals was markedly enhanced from day 6 to 15 after infection (Fig. 1). However, by day 30–40 post-infection the trophozoite counts were appreciably reduced. As shown in Table 1, AP activity assayed in tissue homogenates as well as in puriWed microvillus membranes was signiWcantly reduced (p < 0.01) at day 11 and 15 post-infection. These Wndings were further conWrmed by assaying the enzyme activity in membrane proteins separated by SDS–PAGE under non-denaturing conditions (Fig. 2A). The enzyme activity was located in the gels by hydrolysis of BCIP, which appeared blue in color at the site of enzyme activity. The intensity of the color corresponded to enzyme levels in the membrane (Fig. 2B). The color intensity representing AP activity was low in membranes isolated from 11 and 15-day post-infected animals but the enzyme staining was essentially similar by day 30 of post-infection when compared to the equivalent controls.
2.5. 3⬘-Bromo-4-chloro indolyl phosphate (BCIP) staining Separation of brush border membrane proteins solubilized with 0.1% sodium dodecyl sulfate (SDS) was carried out by polyacrylamide gel electrophoresis (PAGE) under non-denaturing conditions (Lammeli, 1970). Hundred micrograms of the SDS solubilized membrane protein was run on 8% acrylamide gels. AP activity was stained in situ in presence of BCIP (1 mg/ml in 50 mM Tris–HCl, pH 7.6) solution at 37 °C. After bands of
Fig. 1. Trophozoite counts in G. lamblia infected rat intestine. Values are meana § SD of 6–8 animals.
S. Mahmood et al. / Experimental Parasitology 110 (2005) 91–95
93
Table 1 EVect of G. lamblia infection on alkaline phosphatase activity in rat intestine
Homogenate BBM
Control (0 day)
6th Day
11th Day
15th Day
30th Day
40th Day
0.05 § 0.01 0.28 § 0.07
0.05 § 0.003 0.25 § 0.030
0.04 § 0.01¤ 0.15 § 0.09¤
0.04 § 0.004¤ 0.11 § 0.02¤
0.07 § 0.02 0.29 § 0.03
0.05 § 0.01 0.34 § 0.09
Values are means § SD of 6–8 animals, as units/mg protein. ¤ p < 0.01 vs. control.
Fig. 2. (A) BCIP staining of acrylamide gels for alkaline phosphatase activity in G. lamblia infected rat intestine. 100 g membrane protein was applied to each well. (B) Densitometric scan of the data given in (A).
The eVect of G. lamblia infection on AP activity in epithelial cells across the crypt–villus axis was also analyzed. The serial cell fractions representing villus tip to crypt base from control and 11 day post-infected animals were isolated and enzyme activity was assayed. These results are shown in Fig. 3, AP activity was much higher in the upper villus cells and was almost absent in the crypt region of the control animals. However, in rats at 11 day, post-infection, AP activity was reduced (p < 0.01) in the villus tip and mid villus (p < 0.001) regions and was signiWcantly higher (p < 0.01) in the crypt base compared to corresponding villus and crypt cells in the control animals. These Wndings were further conWrmed by assaying the enzyme activity in the polyacrylamide gels using BCIP staining (Fig. 4). As expected, the staining of gels for IAP activity was quite high in the villus tip cells compared to the crypt cells in the control animals. However, the BCIP staining of enzyme activity was low in villus tip and mid villus regions but was relatively higher in crypt cells in G. lamblia infected rats compared to the corresponding cell fractions in the controls (Fig. 4B).
Fig. 3. Distribution of alkaline phosphatase activity in cell fractions across the crypt–villus axis in the control and G. lamblia infected rat intestine. Means § SD of 6–8 animals. *p < 0.05; **p < 0.01; and ***p < 0.001 vs. control.
4. Discussion The data presented here indicate that AP activity in the crypt base was signiWcantly higher in G. lamblia infected animals compared to controls but reverse was the case in the villus tip region under these conditions. The AP ratio in the villus tip to crypt base was around 12 in control animals, which was reduced to 4 in infected group. Although the parasitic infection was established as early as day 4 (results not shown), by day 6 post-infection, the animals exhibited considerable levels of Giardia trophozoites in intestinal perfusate. AP activity was considerably reduced in intestine of animals infected with G. lamblia by day 11 and 15 post-infection compared to controls. The observed decrease in AP activity at day 11 and 15 post-infection is similar to that reported by others for AP and disaccharidases in rat intestine (Anand et al., 1982; Mahmood et al., 2002). At day 11–15 post-infection, the enzyme levels in intestine were considerably lower than those in controls. This was associated with a signiWcant decrease in enzyme activity in the upper villus
94
S. Mahmood et al. / Experimental Parasitology 110 (2005) 91–95
AP activity in giardial infection. This is indeed corroborated by the fact that considerably low enzyme activity was detected in microvillus membrane by BCIP staining of membrane proteins resolved on acrylamide gels. Thus, the migration of epithelial cells across crypt–villus axis is altered by the parasitic infections. We previously reported a considerable decrease in the activity of brush border sucrase and lactase, which is associated with low levels of mRNA encoding sucrase and lactase in G. lamblia infected rat intestine (Nidhi et al., 1999). Recently, Mahmood et al. (2002) described low mRNA encoding sodium–glucose co-transporter (SGLT1) in Giardia infected rats. This suggested that downregulation of the expression of these proteins might be responsible for the observed decline in enzyme activity. Although similar studies have not been carried out with intestinal alkaline phosphatase, it is possible that a similar phenomenon may occur with this enzyme in G. lamblia infection.
Fig. 4. (A) BCIP staining of the acrylamide gel for alkaline phosphatase activity in villus tip (lanes 1, 4), mid villus (lanes 2, 5), and crypt region (lanes 3, 6) of control (lanes 1, 2, and 3) and G. lamblia infected (lanes 4, 5, and 6) rats. 100 g membrane protein was applied to each well. (B) Densitometric scan of the data given in (A).
and mid villus cells compared to that in the controls. However, in the crypt base, AP activity was elevated in infected animals compared to controls. Mc Donald and Ferguson (1978) showed that the turnover rate of enterocytes across the crypt–villus axis is considerably increased in chronic G. muris infected mice. This was associated with enhanced hyperplasia of crypt cells, such an increase in the number of enterocytes in the crypt base would also explain the increase in AP activity in the crypt region as seen in the present study. G. lamblia infection also induces an increase in epithelial cell mitosis (Smith, 1985), which could be responsible for the observed increase in IAP activity under these conditions. Xie et al. (1997) have demonstrated that mRNA encoding two IAP isoforms is diVerently distributed across crypt–villus axis in rat intestine. Alkaline phosphatase-I is mainly present in the villus tip but intestinal alkaline phosphatase-II mRNA is abundantly present in crypt as well as in villus tip cells. Thus diVerential expression of the two isoforms of IAP mRNAs across crypt–villus unit in the infected tissue may also be responsible for the observed changes in the enzyme level. However, studies on mRNA levels encoding IAP in G. lamblia infected animals are required to further explain these Wndings. Morphological studies have shown shortening of villi and distortion of the microvilli in giardiasis (Harloug et al., 1979). Ganguly et al. (1985) observed disruption of the microvilli under electron microscopy in G. lamblia infected intestine. This may also explain the low levels of
Acknowledgments Financial assistance from Indian Council of Medical Research, New Delhi for this study is gratefully acknowledged. Stool samples of patients suVering from giardiasis were obtained from the Department of Parasitology, PGIMER, Chandigarh.
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