Life Sciences Vol . 20, pp . 1607-1612, 1977 . Printed in the U .S .A .
Pergamon Preae
TAUROCHOLATE UPTAKE BY MEMBRANE VESICLES PREPARED FROM ILEAL BRUSH BORDERS Leon Lack, James T . Walker, and Chi-Yin H . Hsu Department of Physiology and Pharmacology Duke University Medical Center Durham, North Carolina 27710 (Received in final form April 7, 1977)
Summary Taurocholate uptake by vesicles prepared from brush borders obtained from the small intestines of guinea pigs was studied . Vesicles obtained from the brush borders of ileums demonstrated an enhanced initial uptake in those incubations where a sodium ion gradient (extravesicular sodium concentration greater than intravesicular) was present at the outset . With the dissipation of this sodium gradient the intravesicular concentration of taurocholate declined . This overshoot phenomenon was absent in parallel incubations of vesicles made from jejunal tissue . When the sodium chloride was replaced by isosmotic amounts of mannitol no overshoot was observed in incubations of ileal vesicles until subsequent addition of sodium chloride to these incubations, These observations are in accord with the idea that those subcellular structural elements operating in the ileal bile salt transport system are associated with the brush border membranes of the ileal mucosal cells . Absorption of bile salts from the intestine involves an active transport system which, in all species studied, is located exclusively in the ileum (1) . This system is physiolo gically dependent on the presence of sodium ions in the luminal fluid (2,3,4) . Structure activity studies indicate that the initial recognition site for transport involves three components : an interaction between the steroid moiety of the bile salt substrate ; a coulombic interaction between the anionic charge of the bile salt and a cationic site on the transport system ; and, finally, an interaction between sodium ions present in the fluid bathing the mucosal surface of the ileal tissue and a closely positioned anionic site on the transport system . These three factors would appear to operate cooperatively (4,5) . Prior to initiating intensive efforts to isolate the macromolecules present in the ileal tissue which function in the active transport process, it was necessary to obtain more specific information on the location of this specialized system within the ileal mucosal cells . The demonstration by others of intact glucose transport in isolated brush border membranes (6,7) 1607
1608
Taurocholate Uptake by Membrane Vesicles
Vol . 20, No . 9, 1977
suggested the feasibility of utilizing such preparations for the study of bile salt transport . Thus specific uptake, with demonstrated sodium requirements, by such vesicles prepared from ileal brush borders would locate the presence of the transport system. This communication reports the results of such investigations . Methods Guinea pigs of the Hartley strain, 400-600 grams were used for the preparation of brush border membrane vesicles . Vesicles were made from the most proximal and most distal quarters of the small intestine . The procedure for preparing brush borders and their conversion to closed vesicles was essentially that described by Hopfer et al (6) . Certain modifications of the procedure were required in order to obtain adequate amounts of biological material from the ileal region of the small bowel . The mucosal scrapings were suspended in 75 volumes of 5mM EDTA, 1mM Hepes Tris (pH 7 .4) and homogenized for 25 seconds in a Sorvall dannimixer with the speed control set at position 4 . The homogenates were then filtered through #25 bolting cloth (200 mesh) and through glass wool . The rest of the procedure for the preparation of brush borders was that described by the original workers except that the filtration through glass wool in the presence of 90mM NaCl was omitted . The homogenization of brush borders (in a glass Teflon Homogenizer at 1000 rpm) was done in 100mM mannitol containing 1mM Tris Hepes buffer (pH 7 .4) . These procedures were all performed in the cold (0-4°C) . Aliquots of the final preparations were allowed to react at room temperature with 38 glutaraldehyde, .1M Cacodylate (pH 7 .2) for periodic monitoring by electron microscopy . These examinations were made by Dr Joachim R . Sommer of the Department of Pathology of the Duke Medical Center . The electron microscopic appearance of all the preparations was similar to those highly purified vesicular membrane fractions originally described by Hopfer et al (6) . A single preparation of the two types of vesicles (proximal and distal) involved the tissue from three guinea pigs . Incubations were carried out immediately following their preparation . 24[ 14C] sodium taurocholate was obtained from New England Nuclear Corporation and checked for radiopurity by thin layer chromatography using solvent system #2 of Hofmann (8) . This material could be used without subsequent purification . Reagent chemicals were obtained from Sigma Corporation . Incubation procedures were carried out at 37°C in air . Vesicles suspended in 100mM mannitol, Tris Hepes buffer, were preincubated for 5 minutes in the constant temperature bath . After 5 minutes an equal volume of SOmM NaCl containing the [14C] taurocholate (also preincubated at 37°C) was added . The final volume was 1 ml . The concentration of taurocholate (specific activity 52 mCi/mmole) was 15 nanomoles per ml and the concentration of vesicular protein was 2 mg per ml . Five .1 ml aliquots were removed at specified times and suspended in 2 ml of solution containing equal amounts of the mannitol and NaCl solutions maintained at 0°C . These were filtered through .45 micron Millipore filters . The filters were then washed 2 times with 2 ml portions of the same cold solution . The amounts of labelled taurocholate reuaaining on the filter were ascertained
Vol . 20, No . 9, 1977
Taurocholate Uptake by Membrane Vesicles
1609
by scintillation counting . Protein was determined by the method of Lowry et al (9) . Results Figure 1 gives the results of 4 separate experiments . The uptake of [ 14C] sodium taurocholate by vesicles made from proximal (jejunal) intestinal brush borders is compared with uptake by vesicles prepared from tissue from the distal small bowel (ileum) . It is apparent that distal vesicles are capable of initially taking up significantly more taurocholate than the proximal vesicles . With time the amount of material present in the distal vesicles decreases and at 10 minutes is indistinguishable from that measured in proximal vesicles . All data are presented as ~-ho maan
+ CF!M .
40
o Distal Vesicles . ProRimol Vesicles o c
~a E 0 0 .20
L0
2.0
Minutes
4.0
~ -JIO
FIG . 1 Uptake of 14C taurocholate by brush border membrane vesicles . The reactions were initiated by adding an equal volume of the taurocholate in 50 mmolar NaCl to a suspension of vesicles in 100mM mannitol (0 time) . Both solutions were buffered at pH 7 .4 in 1mM Tris Hepes . Final concentration taurocholate 15 nanomoles per ml, vesicle protein 2 mg per ml . Figure 2 gives the results of 5 experiments which compare the ability of these two types of membrane preparations to take up sodium taurocholate in the absence of a sodium ion gradient and then in its presence .
1610
Taurocholate Uptake by Membrane Vesicles
Proximal
---~-°Distol
Vol . 20, No . 9,
1977
.. "\. .
FIG . 2 Uptake of taurocholate by vesicles in the absence and presence of NaCl . Between 0 time and 4 minutes the vesicles were incubated with 100mM mannitol ; 1mM Tris Hepes, pH 7 .4 ; sodium taurocholate,concentration 15 nanomoles per ml . At point indicated by the arrow an equal volume of 50mM NaCl ; 1mM Tris Hepes, pH 7 .4y containing taurocholate,l5 nanomoles per ml, was added . When radiolabelled taurocholate in mannitol solution was added to the membrane vesicles suspended in mannitol there was minimal uptake and both types of preparations were indistinguishable . After four minutes an equal volume of NaCl solution was added . This is indicated by the arrow in Figure 2 . This solution had the same osmolality as the mannitol and contained 14C taurocholate in the same concentration as that already present in the incubation mixtures . Uptake by distal vesicles then ,proceded in a manner similar to that observed in Figure 1 . In incubations containing proximal Vesicles the uptake increased modestly and approached that seen previously (Figure 1 for this type of vesicle) . In this case the small incremental uptake between 4 .5 and 5 minutes probably reflects the fact that NaCl is entering the vesicles together with osmolar quantities
Vol .
20, No . 9, 1977
Taurocholate Uptake by Membrane Vesicles
1611
of water and thus increasing the intravesicular volume . Discussion The purpose of these studies was to ascertain if the relatively simple brush border vesicle system, demonstrated by others to serve as an intact in vitro glucose transport system, could be used to demonstrate comparable activity for taurocholate transport . Since uphill bile salt transport is now recognized as residing exclusively in the ileum, specialized uptake could be expected only in the membrane preparation derived from ileal tissue . In addition, this uptake should be expected to depend on the presence of sodium and should be manifest during the period of time that the activity of the external sodium exceeds that within the vesicle . With the dissipation of the sodium gradient, the taurocholate within the vesicular compartment would equilibrate and approach the levels of that outside the compartment . This is the basis of the overshoot phenomenon described by Murer and Hopfer in the case of sugars (7) . Figure 1 demonstrates that the initial overshoot occurs only in incubations of vesicles prepared from the ileal region . Furthermore, this uptake pattern is manifested only when sodium chloride is added to the extravesicular area (Figure 2) . It is concluded, therefore, that the initial interaction of the bile salt substrate with its ileal transport system occurs in the brush border membrane . Acknowledgements This work was supported by research grant AM-09582 from the National Institutes of Health, and in part by a grant (RR-30) from the General Clinical Research Centers Program of the Division of Research Sources, National Institutes of Health . The authors wish to express their deepest appreciation to Dr J .R . Sommer for his evaluation of the vesicle samples by electron microscopy . References 1. 2. 3. 4. 5. 6. 7. 8. 9.
L . Lack and I .M . Weiner, The Bile Acids, Vol 2, pp .33-54, P .P . Nair and D . Kritchevsky, editors, Plenum Press, New York (1973) . P . Holt, Amer . J . Ph siol . 207 1-7 (1964) . M .R . Playoust an R .J . Isselracher, J . Clin . Invest . 43, 467-476 (1964) . K . Gallagher, J . Marskopf, J .T . Walker, and L . Lack, J .' Li 'id Res . 17, 572-577 (1976) . R . un y, J . Marskopf, J .T . Walker, and L . Lack, J . Lipid Res . in press (1977) . U . Hopf er, K . Nelson, J . Perrotto, and K .J . Isselbacher, J .' Biol . Chem . _248, 25-32 (1973) . H . Murer an U . Hopf er, Proc . Nat . Acad . Sci . USA . 71, 484-488 (1974) . A .F . Hofmann, J . Lipid Res . 3, 127-128 (1962) . O .H . Lowry, N.J . Rosenbrough, and A .L . Farr, J . Biol . Chem . 193, 262-275 (1951) .
Pergamon Preae
TAUROCHOLATE UPTAKE BY MEMBRANE VESICLES PREPARED FROM ILEAL BRUSH BORDERS Leon Lack, James T . Walker, and Chi-Yin H . Hsu Department of Physiology and Pharmacology Duke University Medical Center Durham, North Carolina 27710 (Received in final form April 7, 1977)
Summary Taurocholate uptake by vesicles prepared from brush borders obtained from the small intestines of guinea pigs was studied . Vesicles obtained from the brush borders of ileums demonstrated an enhanced initial uptake in those incubations where a sodium ion gradient (extravesicular sodium concentration greater than intravesicular) was present at the outset . With the dissipation of this sodium gradient the intravesicular concentration of taurocholate declined . This overshoot phenomenon was absent in parallel incubations of vesicles made from jejunal tissue . When the sodium chloride was replaced by isosmotic amounts of mannitol no overshoot was observed in incubations of ileal vesicles until subsequent addition of sodium chloride to these incubations, These observations are in accord with the idea that those subcellular structural elements operating in the ileal bile salt transport system are associated with the brush border membranes of the ileal mucosal cells . Absorption of bile salts from the intestine involves an active transport system which, in all species studied, is located exclusively in the ileum (1) . This system is physiolo gically dependent on the presence of sodium ions in the luminal fluid (2,3,4) . Structure activity studies indicate that the initial recognition site for transport involves three components : an interaction between the steroid moiety of the bile salt substrate ; a coulombic interaction between the anionic charge of the bile salt and a cationic site on the transport system ; and, finally, an interaction between sodium ions present in the fluid bathing the mucosal surface of the ileal tissue and a closely positioned anionic site on the transport system . These three factors would appear to operate cooperatively (4,5) . Prior to initiating intensive efforts to isolate the macromolecules present in the ileal tissue which function in the active transport process, it was necessary to obtain more specific information on the location of this specialized system within the ileal mucosal cells . The demonstration by others of intact glucose transport in isolated brush border membranes (6,7) 1607
1608
Taurocholate Uptake by Membrane Vesicles
Vol . 20, No . 9, 1977
suggested the feasibility of utilizing such preparations for the study of bile salt transport . Thus specific uptake, with demonstrated sodium requirements, by such vesicles prepared from ileal brush borders would locate the presence of the transport system. This communication reports the results of such investigations . Methods Guinea pigs of the Hartley strain, 400-600 grams were used for the preparation of brush border membrane vesicles . Vesicles were made from the most proximal and most distal quarters of the small intestine . The procedure for preparing brush borders and their conversion to closed vesicles was essentially that described by Hopfer et al (6) . Certain modifications of the procedure were required in order to obtain adequate amounts of biological material from the ileal region of the small bowel . The mucosal scrapings were suspended in 75 volumes of 5mM EDTA, 1mM Hepes Tris (pH 7 .4) and homogenized for 25 seconds in a Sorvall dannimixer with the speed control set at position 4 . The homogenates were then filtered through #25 bolting cloth (200 mesh) and through glass wool . The rest of the procedure for the preparation of brush borders was that described by the original workers except that the filtration through glass wool in the presence of 90mM NaCl was omitted . The homogenization of brush borders (in a glass Teflon Homogenizer at 1000 rpm) was done in 100mM mannitol containing 1mM Tris Hepes buffer (pH 7 .4) . These procedures were all performed in the cold (0-4°C) . Aliquots of the final preparations were allowed to react at room temperature with 38 glutaraldehyde, .1M Cacodylate (pH 7 .2) for periodic monitoring by electron microscopy . These examinations were made by Dr Joachim R . Sommer of the Department of Pathology of the Duke Medical Center . The electron microscopic appearance of all the preparations was similar to those highly purified vesicular membrane fractions originally described by Hopfer et al (6) . A single preparation of the two types of vesicles (proximal and distal) involved the tissue from three guinea pigs . Incubations were carried out immediately following their preparation . 24[ 14C] sodium taurocholate was obtained from New England Nuclear Corporation and checked for radiopurity by thin layer chromatography using solvent system #2 of Hofmann (8) . This material could be used without subsequent purification . Reagent chemicals were obtained from Sigma Corporation . Incubation procedures were carried out at 37°C in air . Vesicles suspended in 100mM mannitol, Tris Hepes buffer, were preincubated for 5 minutes in the constant temperature bath . After 5 minutes an equal volume of SOmM NaCl containing the [14C] taurocholate (also preincubated at 37°C) was added . The final volume was 1 ml . The concentration of taurocholate (specific activity 52 mCi/mmole) was 15 nanomoles per ml and the concentration of vesicular protein was 2 mg per ml . Five .1 ml aliquots were removed at specified times and suspended in 2 ml of solution containing equal amounts of the mannitol and NaCl solutions maintained at 0°C . These were filtered through .45 micron Millipore filters . The filters were then washed 2 times with 2 ml portions of the same cold solution . The amounts of labelled taurocholate reuaaining on the filter were ascertained
Vol . 20, No . 9, 1977
Taurocholate Uptake by Membrane Vesicles
1609
by scintillation counting . Protein was determined by the method of Lowry et al (9) . Results Figure 1 gives the results of 4 separate experiments . The uptake of [ 14C] sodium taurocholate by vesicles made from proximal (jejunal) intestinal brush borders is compared with uptake by vesicles prepared from tissue from the distal small bowel (ileum) . It is apparent that distal vesicles are capable of initially taking up significantly more taurocholate than the proximal vesicles . With time the amount of material present in the distal vesicles decreases and at 10 minutes is indistinguishable from that measured in proximal vesicles . All data are presented as ~-ho maan
+ CF!M .
40
o Distal Vesicles . ProRimol Vesicles o c
~a E 0 0 .20
L0
2.0
Minutes
4.0
~ -JIO
FIG . 1 Uptake of 14C taurocholate by brush border membrane vesicles . The reactions were initiated by adding an equal volume of the taurocholate in 50 mmolar NaCl to a suspension of vesicles in 100mM mannitol (0 time) . Both solutions were buffered at pH 7 .4 in 1mM Tris Hepes . Final concentration taurocholate 15 nanomoles per ml, vesicle protein 2 mg per ml . Figure 2 gives the results of 5 experiments which compare the ability of these two types of membrane preparations to take up sodium taurocholate in the absence of a sodium ion gradient and then in its presence .
1610
Taurocholate Uptake by Membrane Vesicles
Proximal
---~-°Distol
Vol . 20, No . 9,
1977
.. "\. .
FIG . 2 Uptake of taurocholate by vesicles in the absence and presence of NaCl . Between 0 time and 4 minutes the vesicles were incubated with 100mM mannitol ; 1mM Tris Hepes, pH 7 .4 ; sodium taurocholate,concentration 15 nanomoles per ml . At point indicated by the arrow an equal volume of 50mM NaCl ; 1mM Tris Hepes, pH 7 .4y containing taurocholate,l5 nanomoles per ml, was added . When radiolabelled taurocholate in mannitol solution was added to the membrane vesicles suspended in mannitol there was minimal uptake and both types of preparations were indistinguishable . After four minutes an equal volume of NaCl solution was added . This is indicated by the arrow in Figure 2 . This solution had the same osmolality as the mannitol and contained 14C taurocholate in the same concentration as that already present in the incubation mixtures . Uptake by distal vesicles then ,proceded in a manner similar to that observed in Figure 1 . In incubations containing proximal Vesicles the uptake increased modestly and approached that seen previously (Figure 1 for this type of vesicle) . In this case the small incremental uptake between 4 .5 and 5 minutes probably reflects the fact that NaCl is entering the vesicles together with osmolar quantities
Vol .
20, No . 9, 1977
Taurocholate Uptake by Membrane Vesicles
1611
of water and thus increasing the intravesicular volume . Discussion The purpose of these studies was to ascertain if the relatively simple brush border vesicle system, demonstrated by others to serve as an intact in vitro glucose transport system, could be used to demonstrate comparable activity for taurocholate transport . Since uphill bile salt transport is now recognized as residing exclusively in the ileum, specialized uptake could be expected only in the membrane preparation derived from ileal tissue . In addition, this uptake should be expected to depend on the presence of sodium and should be manifest during the period of time that the activity of the external sodium exceeds that within the vesicle . With the dissipation of the sodium gradient, the taurocholate within the vesicular compartment would equilibrate and approach the levels of that outside the compartment . This is the basis of the overshoot phenomenon described by Murer and Hopfer in the case of sugars (7) . Figure 1 demonstrates that the initial overshoot occurs only in incubations of vesicles prepared from the ileal region . Furthermore, this uptake pattern is manifested only when sodium chloride is added to the extravesicular area (Figure 2) . It is concluded, therefore, that the initial interaction of the bile salt substrate with its ileal transport system occurs in the brush border membrane . Acknowledgements This work was supported by research grant AM-09582 from the National Institutes of Health, and in part by a grant (RR-30) from the General Clinical Research Centers Program of the Division of Research Sources, National Institutes of Health . The authors wish to express their deepest appreciation to Dr J .R . Sommer for his evaluation of the vesicle samples by electron microscopy . References 1. 2. 3. 4. 5. 6. 7. 8. 9.
L . Lack and I .M . Weiner, The Bile Acids, Vol 2, pp .33-54, P .P . Nair and D . Kritchevsky, editors, Plenum Press, New York (1973) . P . Holt, Amer . J . Ph siol . 207 1-7 (1964) . M .R . Playoust an R .J . Isselracher, J . Clin . Invest . 43, 467-476 (1964) . K . Gallagher, J . Marskopf, J .T . Walker, and L . Lack, J .' Li 'id Res . 17, 572-577 (1976) . R . un y, J . Marskopf, J .T . Walker, and L . Lack, J . Lipid Res . in press (1977) . U . Hopf er, K . Nelson, J . Perrotto, and K .J . Isselbacher, J .' Biol . Chem . _248, 25-32 (1973) . H . Murer an U . Hopf er, Proc . Nat . Acad . Sci . USA . 71, 484-488 (1974) . A .F . Hofmann, J . Lipid Res . 3, 127-128 (1962) . O .H . Lowry, N.J . Rosenbrough, and A .L . Farr, J . Biol . Chem . 193, 262-275 (1951) .
