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Effect of vitamin a on in vitro alanine transport in isolated rabbit ileum
Camp. Biochem. Physiol.,
1977, Vol. %A, pp. 281 to 284. Pergsmon Press. Printed in Great Briton
EFFECT OF VITAMIN A ON IN VITRO ALANINE TRANSPORT IN ISOLATED RABBIT ILEUM JEAN-JACQUESHAJJAR, KAMIL NASSAR AND ANWAR BIKHAZI Department of Physiology, School of Medicine, American University of Beirut, Beirut. Lebanon (Received I January 1977) Abstract-l.
Retinol and retinal in relatively large pharmacological doses inhibit the transmural fluxes of r_-alanine in the isolated rabbit ileum. 2. Unidirectional alanine influx across the mucosal epithelial layer was also inhibited by vitamin A. 3. Cell alanine accumulation increases in the presence of vitamin A due in part to the relative shrinkage of the ileal cells and to the incorporation of alanine in cell proteins.
INTRODUCTION
One of the general metabolic functions of vitamin A is its role in maintaining the normal structure and function of cell membranes and epithelia. It is reported to cause chemical (Roels & Shah, 1969; Lucy, 1964; Mack et al., 1972) and structural (Glick & Kerr, 1968) changes in these membranes that lead to alterations in their transport functions. The effect of vitamin A on intestinal transport has not however been clearly defined. It is generally reported to cause an increase in permeability of cell membranes (Fell, 1964; Dingle & Lucy, 1965) but in vitamin A-deficient rats the intestinal absorption of amino acids increases (Varnell, 1972). This report describes the effect of vitamin A on amino acid absorption in the rabbit ileum in vitro. MATERIALS AND METHODS General procedure The distal part of the ileum of rabbit (Oryctolagus cunniculus) intestine was utilized in all these studies. The animal was killed by intravenous injection of sodium pentobarbital and its abdominal cavity was opened by a mid-line incision, and a segment of the distal ileum resected out and placed in Ringer’s solution which contained 140 mM NaCI, 10 mM KHCO,, 1.2 mM K,HP04, 0.2 mM KH,PO,. l.2mM MgCI,, 1.2mM CaCl, (pH7.2 when bubbled with a gas mixture of 95% 0, and 5% CO,). The intestinal segment was washed free of its luminal contents, cut open along its mesenteric line and dealt with in different ways as described below. In all studies the intestine was kept in solutions containing 0.2% Tween 80 (Fluka A.G., Switzerland) which solubilized all the tested concentrations ofretinol and retinal (Fluka A.G., Switzerland). All studies were done at 37°C. Transmural ,jlux measurements
The rabbit ileum was mounted as a flat sheet on a Lucite chamber and bathed on both of its sides by Ringer’s solution containing appropriate concentrations of vitamin A. The mucosal-to-serosal (J,,) and serosal-to mucosal (J,) fluxes were measured by a similar apparatus and procedure to that described by Schultz & Zalusky (1964). L-alanine
(Sigma Chemical Co.) was added at a concentration of 5 mM in the mucosal and serosal solutions. L-alanine-‘H (Radiochemical Center, Amersham) was placed on one side and after a period of equilibration of 40 min the steadystate rate of appearance of isotope on the opposite side was determined. The samples were counted in a liquid scintillation spectrometer using Bray’s (1960) solution. Measurement border
of alanine injux
across the mucosal brush
The flux of alanine from the luminal solution into the intestinal cell across the brush-border membrane was measured by a method similar to that described by Schultz et al. (1967). The intestine was mounted as a sheet on a Lucite chamber with its mucosal surface in contact with a compartment into which solutions could be instilled and withdrawn rapidly. The intestine was preincubated with Ringer’s solution for a period of 20min following which the solution was replaced by a test solution containing 5 mM L-alanine, L-alanine-3H and “C-inulin. The test solution was maintained in contact with the mucosal surface for about 60sec then withdrawn and replaced by chilled isotonic mannitol solution. The exposed intestine was then cut out and extracted in O.lN HNO, for 4 hr. The influx of alanine was estimated from the amount of tritiated alanine recovered in the tissue extract after correction for the inulin space. Vitamin A was added to both or either the test or preincubation solutions in different concentrations from stock solutions.
Measurement of intracellular alanine concentration
Mucosal strips of the ileum were obtained for these studies by scraping the mucosa with a microscope slide and were examined as described previously in detail (Hajjar et al., 1970). The strips were incubated in Ringer’s solution to which was added L-alanine, L-alanine-3H and 14C-inuEn. The incubation was maintained for 60 min following which they were removed, blotted with Whatman No. 1 filter paper, weighed and extracted in O.IN HNO,. Samples of the extracts and incubation media were counted in a three-channel liquid scintillation spectrometer. The dry weights of the extracted tissues were determined after drvina at 90°C for 12 hr. From these data. the extracellular spa&, cell volume, cell alanine concentration and cell alanine content were determined by similar methods as described previously (Hajjar et al., 1970).
281
JEAN-JACQIJES HAJJAR,KAMIL NASSAR AND ANWAKBIWIAXI
282
Table I. Effect of vitamin A on L-alanine transmural fluxes across rabbit ileum
Ringer + Retinol
(1 pgg/ml)
(5 &mU
+ Retinal
(1 pgg/ml) (5 fig/ml)
(20WmU (40 pgg/ml)
0.751 0.3 I8 0.201 0.501 0.350 0.146 0.128
+ + f + + + +
0.057 0.035* 0.022’ 0.031t 0.036* 0.026* 0.014*
0.160) 0.112 + 0.100 f 0. I64 i 0.142 + 0.100 * 0.040 f
0.018 0.011t 0.004* 0.018 0.019 0.0 I o* o.ooi*
0.591 0.106 0. IO1 0.516 0 3OK 0.046 0.08’
I-1
I3 I1 17
I? II I?
Fluxes are in pmol/hr per cm’; errors are standard errors of the mean J ne, = J,, - J,,. N is flux measurements from at least 4 rabbits. All solutions contained 0.2% Tween 80. and 5 mM alanine. t P < o.di * P < 0.001 Effect of vitamin A OII alanine in/lu.u
RESULTS Effect of vitamin A on the transmural ,ju.xes of alanine
The results of these experiments are summarized in Table I. The transmural fluxes in the control experiments were measured in the presence of Tween 80 and J,, was found to be slightly lower than that reported previously in comparable experiments where a Ringer’s solution without Tween was utilized (Hajjar et al., 1972). It is possible that Twecn may have a mild inhibitory action on alanine transport but we have justified its use in this study since in three preliminary experiments J, was measured in the presence and absence of Tween and was 0.74 f 0.06 p-equiv/hr per cm’ and 0.84 + 0.05 p-equiv/hr per cm2 respectively. J, however, was not altered by Tween. The effect of vitamin A on intestinal transmural alanine fluxes is summarized in Table 1. It inhibits both J,,,, and J, fluxes of the amino acid, with the J, inhibition being more pronounced, and observed with smaller doses of the vitamin analogues. Both retinol and retinal in concentrations above 1 /.&ml were inhibitory, with the alcohol being perhaps more effective on an equal dose level. We have initially tried smaller doses of retinol (0.1 and 0.5 s/ml) and they produced no significant inhibition of alanine fluxes. Thus, it appears from a consideration of the usual vitamin A daily intake that pharmacological rather than physiological doses of the vitamin are required to produce this effect.
In defining further the action of vitamin A on alanine transport we have examined its effect on the initial rate of uptake of alanine across the brushborder membrane. The results of these studies are summarized in Table 2. It is obvious that the vitamin. again in its alcohol or aldehyde forms, inhibits alanine influx into the mucosal brush-border membrane. This effect however is not instantaneous but occurs after a period of contact between the vitamin and the intestinal epithelium. As described in the methods section, in these experiments the preincubation period lasted for about 20 min and the test period for only 1 min. The vitamin inhibited alanine influx only when it was present during the 20min of preincubation and was relatively ineffective when in contact with the tissue for only I min. This suggests that the vitamin’s action is not specific but occurs after a period of interaction between the vitamin and the membrane. As a matter of fact the presence of retinol in the test solution was not necessary. since as shown at the bottom of Table 2, the mere preincubation with the vitamin was sufficient to inhibit alanine flux across the intestinal mucosa. EfJect of vitamin A on cell alanine uccumulatiorl
One other aspect of the effect of vitamin A on alanine transport is its effect on alanine accumulation in intestinal cells. The results of these studies are shown in Table 3. There are three points that arc
Table 2. Effect of vitamin A on alanine influx across the mucosal brush border of rabbit intestine
Control Retinal
(1 @ml)
Retinal
(1 tig/ml) (5 i&ml)
(15a/ml)
Preincubation solution
Test solution
Ringer Ringer Retinol Ringer Retinal Retinal dinger Retinal Retinal
Ringer Retinol Retinol Retinal Retinal Retinal Retinal Retinal Ringer
All errors are standard errors of the mean. N is number of determinations from at least 4 rabbits. All solutions contained 0.2% Tween 80 and 5 mM alanine. *P < 0.001 t P < 0.05
Influx (p-equiv/hr per cm2) 2.32 + 2.30 k 1.90 * 2.212 + 2.131 * 1.87 + 2.03 + 1.23 f 1.62 +
0.08 0.10 0.127 0.08 0.10 0.12t 0.08 0.07* 0.08’
I5 14 14 12 I2 12 12 12 12
283
Alanine transport Table 3. Effect of vitamin A on alanine accumulation in intestinal cells of rabbit ileum
Ringer + Retinol t fig/ml 5 pg/ml + Retinal 5 ng/mf 15 pg/ml 30 pg/m)
Extracellular space (pl/mg wet wt)
Cell volume Wmg dry wt)
0.22 f 0.02
4.8 + 0.2
20 * I
0.070 * 0.003
0.24 + 0.02 0.29 f 0.03
4.5 & 0.2 3.1 * 0.2
24 _+ l(21) 29 k 2 (26)t
0.099 f 0.004t 0.138 f 0.003*
0.24 k 0.02 0.27 f 0.03 0.34 + 0.03
4.4 * 0.2 4.1 f 0.3 3.5 * 0.4
27 f I (22) 34 If: 3 (23); 51 f 3 (27)*
0.121 f 0.002* 0.134 + 0.005* 0.150 + 0.005*
Cell alanine concentration (mW*
Cell alanine content (W/mg dry v-9
All values are the mean f SE. of 2&30 determinations from 4 rabbits. The values in parentheses are the calculated concentrations (the control concentration times the control over experimental cell
volumes). *P < 0.01
t P < 0.05 All solutions contained 0.2% Tween 80 and 5 mM alanine.
brought out by an examination of these findings. First, the extracellular space that is measured with 14C-inulin increases in the presence of vitamin A, the increase being more obvious when the concentration of the vitamin is raised. The vitamin may conceivably open some compartments of this space that are ordinarily inaccessible to inulin. In favour of this explanation are the findings of Lucy & Dingle (1964) and Glick & Kerr (1968) who report vacuolization, bleb formation and vesiculation of the cell membranes that are exposed to vitamin A in uitro. It is possible alternatively to explain this increase in extracellular space as being due to the possible penetration of inulin into the cells. This is unlikely, however, since as noted above the vitamin causes a reduction rather than an increase in alanine permeability across the intestinal epithelium. The second point is the observed decrease in cell volume due to the presence of vitamin A in the incubation solution. The reason for this shrinkage in epithelial cells is not clear, it may however be related to an electrolyte shift between the intestinal cell and its bathing medium. We have no experimental justification for this explanation but in isolated red cells Dingle & Lucy (1965) observed a rapid loss of cell K with the addition of vitamin A to the incubation medium. The third main observation in Table 3 is the increase in cell alanine concentration in the presence of vitamin A. This was observed both in the presence of retinol and reginal and was more obvious with increasing concentrations of the vitamin. The shrinkage of the cells can undoubtedly explain a good part of this increase in cell alanine concentration. There are nevertheless two findings in the results in Table 3, that suggest that amino acid accumulation does actually increase due to vitamin A. If we calculate the expected concentrations due to the cell volume change by multiplying the control concentration by the ratio of the control over experimental cell volume, we obtain values that are presented in Table 3 in parentheses. These calculated concentrations which represent the change due to cell shrinkage are smaller than the observed concentrations. Secondly, the content of alanine in intestinal cells increases in the presence of vitamin A. The cell alanine content is calculated per milligram dry weight of mucosal tissue, and is accord-
ingly not influenced by the cell volume changes. These two findings therefore, make us suspect that vitamin A causes an actual net alanine accumulation at steady state. DISCUSSION The results presented point out the nature of the action of vitamin A on alanine transport across the intestine. In concentrations below 0.5 pg/ml, the vitamin does not influence the transmural fluxes of alanine, but in larger concentrations it reduces the permeability of this epithelium as evidenced by a reduction in both the mucosal-to-serosal and serosalto-mucosal fluxes. The decrease in mucosal-to-serosal alanine flux is quite remarkable, which tends to suggest that active alanine transport is inhibited by vitamin A. This point is supported by the observed inhibition of alanine influx by vitamin A. The influx of alanine from the mucosal solution into the intestinal cell across the brush-border membrane is primarily responsible for the overall active transport of this amino acid across this epithelium (Schultz et al., 1967). The vitamin therefore must inhibit the active step in alanine transport. This inhibition however, does not appear immediately upon application of the vitamin to the mucosal surface but a lag period of time being required before it manifests. This indicates that the vitamin’s action is not a direct one but occurs after an interaction between the vitamin and the cell membrane. We have no experimental justification here to prove that such an interaction does occur, but vitamin A has been reported to cause alterations in stability and structure of artificial membranes, and to cause a decrease in thickness of the hydrophobic regions of the lipid bilayers of these membranes (Dreher et al., 1967; Anderson et al., 1967). Assuming that a similar change occurs in the intestinal mucosal membrane and the hydrophobic regions of the carrier sites are reduced or restricted by vitamin A, then this may account for the reduced affinity of the membrane carrier to amino acids. In a previous publication (Hajjar & Curran, 1970) the affinity of the mucosal carrier site to amino acids was observed to decrease with the decrease in the hydrophobic radicals of these amino acids. It is reasonable therefore to assume that
284
JEAN-JACQUESHAJJAR, KAMIL NAS~ARAND ANWAR BIKHAZI
the affinity of alanine to the carrier sites decreases as the hydrophobic sites of these carrier are restricted. Additional experiments are needed however to justify this proposed action of vitamin A. Another interesting aspect of the results presented is the effect of vitamin A on alanine accumulation in the intestinal cell. In spite of reducing the permeation of this amino acid across the mucosal membrane and decreasing its net transmural transfer it also results in an increase in its cellular concentration. Although the vitamin produces also a decrease in cell volume, this does not account for all the observed elevation in cell alanine concentration. nor the increase in cell alanine content. There are two possible explanations to account for this increased accumulation. One, would be to assume that the vitamin diminishes the exit of alanine from the cell. This reduction in efflux must however be greater than the reduction in alanine influx that was observed in Table 2, since a net accumulation of the amino acid would not otherwise be possible. At the moment, we do not have any experimental justification to support or reject this explanation. An alternative hypothesis would be to consider that the vitamin may induce an increased incorporation of amino acids into the cell proteins. The findings of De Luca et al. (1970) favour this proposition. They found that in vitamin A deficiency, there was a decreased incorporation of 3H-scrine into a glycopeptide from the rat intestine. We have actually tested the amount of 14C-alanine that gets incorporated in protein precipitates of the intestinal mucosa, using a method that has been described previously (Hajjar et al., 1972). In five pieces of intestinal mucosa that have been preincubated in vitamin A (5 pg/ml) the amount of the alanine incorporated was 0.48 + 0.03 PM/g dry weight of intestine, while in comparable control tissues it was 0.39 + 0.04. It is evident therefore that the vitamin increases the utilization of amino acids in the formation of proteins, which may account at least in part for the observed increase in cell alanine concentration. Ack,lowledgt,me,lts-This work was supported by the American University of Beirut Research Grants (No. 18-5203) and (No. 18-5163). We are indebted to Miss H. Hatzakordzian and Mr A. Hajj for valuable technical assistance.
REFERENCES ANDER~CIN 0. R.. ROELS0. A., DREHEKK. D. & SCHULMA~ J. H. (1967) The effect of retinol and a-tocopherol on the structure and stability of lipid bilayers. J. Ultra.srr~~ct Res. 19, 60&610. BRAY G. A. (1960) A simple etticient hquid scintillator for counting aqueous solutions in a liquid scintillation counter. Anal. Biochrm. 1, 279-2X5. DE LUCA L., SCHUMACHER M.. WOLF G. & NEWHERN~ P. M. (1970) Biosynthesis of a fucose-containing glycopeptide from rat small intestine in normal and vitamin A-deficient conditions. J. hiol. Chrm. 245, 455 I-4558. DINGLE J. T. & LUCY J. A. (1965) Vitamin A. carotenoids and cell function. Biol. Reo. 40, 42146 I. DREHER K. D.. SCHULMAN J. H.. ANIIERSON 0. R. B ROELS 0. A. (1967) Properties of lipid and lipoprotein monolayers on 0,04 solutions and the role of cholesterol. retinol, and tochopherol in stabilizing lecithin monolayers. J. Ultrastruct. Res. 19, 586~599. FELL H. B. (1964) In Vitumins and Hormotws (Edited by HARRIS R. S.. WOOL G. I. & LORAINF J. A.). Vol. 22. p. 81. Academic Press. New York. GLICK D. & KERR W. J. (1968) Effect of vitamin A 111 vitro on histamine release and structure of mast cells. Eupl. Cell Rrs. 53, 120 134. HAJJAR J. J. & CUKRA~ P. F. (1970) Characteristics of the amino acid transport system in the mucosal border of rabbit ileum. J. gets. Phwiol. 56, 673 691. HAJJAR J. J.. LAMONT A. S. & CCJRRAU P. F. (lY70) The Na-alanine interaction in rabbit Ileum: effect of Na on alanine fluxes. J. yen. Physiol. 55. 277~~296. HAJJAR J. J.. HAWRANI M. & KIIURI R. N. (1972) Alanine Rux across rabbit Peyer’s patches. Camp. Bioclwm. Physiol. 43A, 723--732. LUCY J. A. (1964) Globular hpid mlcelles and cell membranes. J. Theoret. Eiol. 7, 36@ 373 LUCY J. A. & DINGL~ J. T. (1964) Fat-soluble vitamins and biological membranes. Vatuw. Loud. 204, 156 I 60. MACK J. P.. Lu! N. S. T.. Roe~s 0. A. & ANDORRA 0. R. (1972) The occurrence of vitamin A in biological membranes. Biochim. hiopllys. Actu 288, 203 219. ROELS 0. A. & SHAH D. 0. (1969) Molecular interactlona in lecithin-retinol monolayers. .I Colloiil %i. 29, 279.-287. Sctiti~rz S. G. & ZALLISKYR. (1964) Ion transport In ISOlated rabbit ileum. J. pa. Physiol. 47. 567-584. SCHULTZ S. G.. C~JRRAY P. F.. CHEZ R. A. Jt F~ISZ R. E. (1967) Alanine and sodium fluxes across mucosal border of rabbit ileum. J. gw. Phvsiol. 50, 1731 1160 VARN~LL T. R. (1972) Effect of avitat&osis A and dletarq regime on intestinal transfer and hepatic concentration of free amino acids. III! J I’ir. Nurr. Rrs. 42. 271 ‘77.
1977, Vol. %A, pp. 281 to 284. Pergsmon Press. Printed in Great Briton
EFFECT OF VITAMIN A ON IN VITRO ALANINE TRANSPORT IN ISOLATED RABBIT ILEUM JEAN-JACQUESHAJJAR, KAMIL NASSAR AND ANWAR BIKHAZI Department of Physiology, School of Medicine, American University of Beirut, Beirut. Lebanon (Received I January 1977) Abstract-l.
Retinol and retinal in relatively large pharmacological doses inhibit the transmural fluxes of r_-alanine in the isolated rabbit ileum. 2. Unidirectional alanine influx across the mucosal epithelial layer was also inhibited by vitamin A. 3. Cell alanine accumulation increases in the presence of vitamin A due in part to the relative shrinkage of the ileal cells and to the incorporation of alanine in cell proteins.
INTRODUCTION
One of the general metabolic functions of vitamin A is its role in maintaining the normal structure and function of cell membranes and epithelia. It is reported to cause chemical (Roels & Shah, 1969; Lucy, 1964; Mack et al., 1972) and structural (Glick & Kerr, 1968) changes in these membranes that lead to alterations in their transport functions. The effect of vitamin A on intestinal transport has not however been clearly defined. It is generally reported to cause an increase in permeability of cell membranes (Fell, 1964; Dingle & Lucy, 1965) but in vitamin A-deficient rats the intestinal absorption of amino acids increases (Varnell, 1972). This report describes the effect of vitamin A on amino acid absorption in the rabbit ileum in vitro. MATERIALS AND METHODS General procedure The distal part of the ileum of rabbit (Oryctolagus cunniculus) intestine was utilized in all these studies. The animal was killed by intravenous injection of sodium pentobarbital and its abdominal cavity was opened by a mid-line incision, and a segment of the distal ileum resected out and placed in Ringer’s solution which contained 140 mM NaCI, 10 mM KHCO,, 1.2 mM K,HP04, 0.2 mM KH,PO,. l.2mM MgCI,, 1.2mM CaCl, (pH7.2 when bubbled with a gas mixture of 95% 0, and 5% CO,). The intestinal segment was washed free of its luminal contents, cut open along its mesenteric line and dealt with in different ways as described below. In all studies the intestine was kept in solutions containing 0.2% Tween 80 (Fluka A.G., Switzerland) which solubilized all the tested concentrations ofretinol and retinal (Fluka A.G., Switzerland). All studies were done at 37°C. Transmural ,jlux measurements
The rabbit ileum was mounted as a flat sheet on a Lucite chamber and bathed on both of its sides by Ringer’s solution containing appropriate concentrations of vitamin A. The mucosal-to-serosal (J,,) and serosal-to mucosal (J,) fluxes were measured by a similar apparatus and procedure to that described by Schultz & Zalusky (1964). L-alanine
(Sigma Chemical Co.) was added at a concentration of 5 mM in the mucosal and serosal solutions. L-alanine-‘H (Radiochemical Center, Amersham) was placed on one side and after a period of equilibration of 40 min the steadystate rate of appearance of isotope on the opposite side was determined. The samples were counted in a liquid scintillation spectrometer using Bray’s (1960) solution. Measurement border
of alanine injux
across the mucosal brush
The flux of alanine from the luminal solution into the intestinal cell across the brush-border membrane was measured by a method similar to that described by Schultz et al. (1967). The intestine was mounted as a sheet on a Lucite chamber with its mucosal surface in contact with a compartment into which solutions could be instilled and withdrawn rapidly. The intestine was preincubated with Ringer’s solution for a period of 20min following which the solution was replaced by a test solution containing 5 mM L-alanine, L-alanine-3H and “C-inulin. The test solution was maintained in contact with the mucosal surface for about 60sec then withdrawn and replaced by chilled isotonic mannitol solution. The exposed intestine was then cut out and extracted in O.lN HNO, for 4 hr. The influx of alanine was estimated from the amount of tritiated alanine recovered in the tissue extract after correction for the inulin space. Vitamin A was added to both or either the test or preincubation solutions in different concentrations from stock solutions.
Measurement of intracellular alanine concentration
Mucosal strips of the ileum were obtained for these studies by scraping the mucosa with a microscope slide and were examined as described previously in detail (Hajjar et al., 1970). The strips were incubated in Ringer’s solution to which was added L-alanine, L-alanine-3H and 14C-inuEn. The incubation was maintained for 60 min following which they were removed, blotted with Whatman No. 1 filter paper, weighed and extracted in O.IN HNO,. Samples of the extracts and incubation media were counted in a three-channel liquid scintillation spectrometer. The dry weights of the extracted tissues were determined after drvina at 90°C for 12 hr. From these data. the extracellular spa&, cell volume, cell alanine concentration and cell alanine content were determined by similar methods as described previously (Hajjar et al., 1970).
281
JEAN-JACQIJES HAJJAR,KAMIL NASSAR AND ANWAKBIWIAXI
282
Table I. Effect of vitamin A on L-alanine transmural fluxes across rabbit ileum
Ringer + Retinol
(1 pgg/ml)
(5 &mU
+ Retinal
(1 pgg/ml) (5 fig/ml)
(20WmU (40 pgg/ml)
0.751 0.3 I8 0.201 0.501 0.350 0.146 0.128
+ + f + + + +
0.057 0.035* 0.022’ 0.031t 0.036* 0.026* 0.014*
0.160) 0.112 + 0.100 f 0. I64 i 0.142 + 0.100 * 0.040 f
0.018 0.011t 0.004* 0.018 0.019 0.0 I o* o.ooi*
0.591 0.106 0. IO1 0.516 0 3OK 0.046 0.08’
I-1
I3 I1 17
I? II I?
Fluxes are in pmol/hr per cm’; errors are standard errors of the mean J ne, = J,, - J,,. N is flux measurements from at least 4 rabbits. All solutions contained 0.2% Tween 80. and 5 mM alanine. t P < o.di * P < 0.001 Effect of vitamin A OII alanine in/lu.u
RESULTS Effect of vitamin A on the transmural ,ju.xes of alanine
The results of these experiments are summarized in Table I. The transmural fluxes in the control experiments were measured in the presence of Tween 80 and J,, was found to be slightly lower than that reported previously in comparable experiments where a Ringer’s solution without Tween was utilized (Hajjar et al., 1972). It is possible that Twecn may have a mild inhibitory action on alanine transport but we have justified its use in this study since in three preliminary experiments J, was measured in the presence and absence of Tween and was 0.74 f 0.06 p-equiv/hr per cm’ and 0.84 + 0.05 p-equiv/hr per cm2 respectively. J, however, was not altered by Tween. The effect of vitamin A on intestinal transmural alanine fluxes is summarized in Table 1. It inhibits both J,,,, and J, fluxes of the amino acid, with the J, inhibition being more pronounced, and observed with smaller doses of the vitamin analogues. Both retinol and retinal in concentrations above 1 /.&ml were inhibitory, with the alcohol being perhaps more effective on an equal dose level. We have initially tried smaller doses of retinol (0.1 and 0.5 s/ml) and they produced no significant inhibition of alanine fluxes. Thus, it appears from a consideration of the usual vitamin A daily intake that pharmacological rather than physiological doses of the vitamin are required to produce this effect.
In defining further the action of vitamin A on alanine transport we have examined its effect on the initial rate of uptake of alanine across the brushborder membrane. The results of these studies are summarized in Table 2. It is obvious that the vitamin. again in its alcohol or aldehyde forms, inhibits alanine influx into the mucosal brush-border membrane. This effect however is not instantaneous but occurs after a period of contact between the vitamin and the intestinal epithelium. As described in the methods section, in these experiments the preincubation period lasted for about 20 min and the test period for only 1 min. The vitamin inhibited alanine influx only when it was present during the 20min of preincubation and was relatively ineffective when in contact with the tissue for only I min. This suggests that the vitamin’s action is not specific but occurs after a period of interaction between the vitamin and the membrane. As a matter of fact the presence of retinol in the test solution was not necessary. since as shown at the bottom of Table 2, the mere preincubation with the vitamin was sufficient to inhibit alanine flux across the intestinal mucosa. EfJect of vitamin A on cell alanine uccumulatiorl
One other aspect of the effect of vitamin A on alanine transport is its effect on alanine accumulation in intestinal cells. The results of these studies are shown in Table 3. There are three points that arc
Table 2. Effect of vitamin A on alanine influx across the mucosal brush border of rabbit intestine
Control Retinal
(1 @ml)
Retinal
(1 tig/ml) (5 i&ml)
(15a/ml)
Preincubation solution
Test solution
Ringer Ringer Retinol Ringer Retinal Retinal dinger Retinal Retinal
Ringer Retinol Retinol Retinal Retinal Retinal Retinal Retinal Ringer
All errors are standard errors of the mean. N is number of determinations from at least 4 rabbits. All solutions contained 0.2% Tween 80 and 5 mM alanine. *P < 0.001 t P < 0.05
Influx (p-equiv/hr per cm2) 2.32 + 2.30 k 1.90 * 2.212 + 2.131 * 1.87 + 2.03 + 1.23 f 1.62 +
0.08 0.10 0.127 0.08 0.10 0.12t 0.08 0.07* 0.08’
I5 14 14 12 I2 12 12 12 12
283
Alanine transport Table 3. Effect of vitamin A on alanine accumulation in intestinal cells of rabbit ileum
Ringer + Retinol t fig/ml 5 pg/ml + Retinal 5 ng/mf 15 pg/ml 30 pg/m)
Extracellular space (pl/mg wet wt)
Cell volume Wmg dry wt)
0.22 f 0.02
4.8 + 0.2
20 * I
0.070 * 0.003
0.24 + 0.02 0.29 f 0.03
4.5 & 0.2 3.1 * 0.2
24 _+ l(21) 29 k 2 (26)t
0.099 f 0.004t 0.138 f 0.003*
0.24 k 0.02 0.27 f 0.03 0.34 + 0.03
4.4 * 0.2 4.1 f 0.3 3.5 * 0.4
27 f I (22) 34 If: 3 (23); 51 f 3 (27)*
0.121 f 0.002* 0.134 + 0.005* 0.150 + 0.005*
Cell alanine concentration (mW*
Cell alanine content (W/mg dry v-9
All values are the mean f SE. of 2&30 determinations from 4 rabbits. The values in parentheses are the calculated concentrations (the control concentration times the control over experimental cell
volumes). *P < 0.01
t P < 0.05 All solutions contained 0.2% Tween 80 and 5 mM alanine.
brought out by an examination of these findings. First, the extracellular space that is measured with 14C-inulin increases in the presence of vitamin A, the increase being more obvious when the concentration of the vitamin is raised. The vitamin may conceivably open some compartments of this space that are ordinarily inaccessible to inulin. In favour of this explanation are the findings of Lucy & Dingle (1964) and Glick & Kerr (1968) who report vacuolization, bleb formation and vesiculation of the cell membranes that are exposed to vitamin A in uitro. It is possible alternatively to explain this increase in extracellular space as being due to the possible penetration of inulin into the cells. This is unlikely, however, since as noted above the vitamin causes a reduction rather than an increase in alanine permeability across the intestinal epithelium. The second point is the observed decrease in cell volume due to the presence of vitamin A in the incubation solution. The reason for this shrinkage in epithelial cells is not clear, it may however be related to an electrolyte shift between the intestinal cell and its bathing medium. We have no experimental justification for this explanation but in isolated red cells Dingle & Lucy (1965) observed a rapid loss of cell K with the addition of vitamin A to the incubation medium. The third main observation in Table 3 is the increase in cell alanine concentration in the presence of vitamin A. This was observed both in the presence of retinol and reginal and was more obvious with increasing concentrations of the vitamin. The shrinkage of the cells can undoubtedly explain a good part of this increase in cell alanine concentration. There are nevertheless two findings in the results in Table 3, that suggest that amino acid accumulation does actually increase due to vitamin A. If we calculate the expected concentrations due to the cell volume change by multiplying the control concentration by the ratio of the control over experimental cell volume, we obtain values that are presented in Table 3 in parentheses. These calculated concentrations which represent the change due to cell shrinkage are smaller than the observed concentrations. Secondly, the content of alanine in intestinal cells increases in the presence of vitamin A. The cell alanine content is calculated per milligram dry weight of mucosal tissue, and is accord-
ingly not influenced by the cell volume changes. These two findings therefore, make us suspect that vitamin A causes an actual net alanine accumulation at steady state. DISCUSSION The results presented point out the nature of the action of vitamin A on alanine transport across the intestine. In concentrations below 0.5 pg/ml, the vitamin does not influence the transmural fluxes of alanine, but in larger concentrations it reduces the permeability of this epithelium as evidenced by a reduction in both the mucosal-to-serosal and serosalto-mucosal fluxes. The decrease in mucosal-to-serosal alanine flux is quite remarkable, which tends to suggest that active alanine transport is inhibited by vitamin A. This point is supported by the observed inhibition of alanine influx by vitamin A. The influx of alanine from the mucosal solution into the intestinal cell across the brush-border membrane is primarily responsible for the overall active transport of this amino acid across this epithelium (Schultz et al., 1967). The vitamin therefore must inhibit the active step in alanine transport. This inhibition however, does not appear immediately upon application of the vitamin to the mucosal surface but a lag period of time being required before it manifests. This indicates that the vitamin’s action is not a direct one but occurs after an interaction between the vitamin and the cell membrane. We have no experimental justification here to prove that such an interaction does occur, but vitamin A has been reported to cause alterations in stability and structure of artificial membranes, and to cause a decrease in thickness of the hydrophobic regions of the lipid bilayers of these membranes (Dreher et al., 1967; Anderson et al., 1967). Assuming that a similar change occurs in the intestinal mucosal membrane and the hydrophobic regions of the carrier sites are reduced or restricted by vitamin A, then this may account for the reduced affinity of the membrane carrier to amino acids. In a previous publication (Hajjar & Curran, 1970) the affinity of the mucosal carrier site to amino acids was observed to decrease with the decrease in the hydrophobic radicals of these amino acids. It is reasonable therefore to assume that
284
JEAN-JACQUESHAJJAR, KAMIL NAS~ARAND ANWAR BIKHAZI
the affinity of alanine to the carrier sites decreases as the hydrophobic sites of these carrier are restricted. Additional experiments are needed however to justify this proposed action of vitamin A. Another interesting aspect of the results presented is the effect of vitamin A on alanine accumulation in the intestinal cell. In spite of reducing the permeation of this amino acid across the mucosal membrane and decreasing its net transmural transfer it also results in an increase in its cellular concentration. Although the vitamin produces also a decrease in cell volume, this does not account for all the observed elevation in cell alanine concentration. nor the increase in cell alanine content. There are two possible explanations to account for this increased accumulation. One, would be to assume that the vitamin diminishes the exit of alanine from the cell. This reduction in efflux must however be greater than the reduction in alanine influx that was observed in Table 2, since a net accumulation of the amino acid would not otherwise be possible. At the moment, we do not have any experimental justification to support or reject this explanation. An alternative hypothesis would be to consider that the vitamin may induce an increased incorporation of amino acids into the cell proteins. The findings of De Luca et al. (1970) favour this proposition. They found that in vitamin A deficiency, there was a decreased incorporation of 3H-scrine into a glycopeptide from the rat intestine. We have actually tested the amount of 14C-alanine that gets incorporated in protein precipitates of the intestinal mucosa, using a method that has been described previously (Hajjar et al., 1972). In five pieces of intestinal mucosa that have been preincubated in vitamin A (5 pg/ml) the amount of the alanine incorporated was 0.48 + 0.03 PM/g dry weight of intestine, while in comparable control tissues it was 0.39 + 0.04. It is evident therefore that the vitamin increases the utilization of amino acids in the formation of proteins, which may account at least in part for the observed increase in cell alanine concentration. Ack,lowledgt,me,lts-This work was supported by the American University of Beirut Research Grants (No. 18-5203) and (No. 18-5163). We are indebted to Miss H. Hatzakordzian and Mr A. Hajj for valuable technical assistance.
REFERENCES ANDER~CIN 0. R.. ROELS0. A., DREHEKK. D. & SCHULMA~ J. H. (1967) The effect of retinol and a-tocopherol on the structure and stability of lipid bilayers. J. Ultra.srr~~ct Res. 19, 60&610. BRAY G. A. (1960) A simple etticient hquid scintillator for counting aqueous solutions in a liquid scintillation counter. Anal. Biochrm. 1, 279-2X5. DE LUCA L., SCHUMACHER M.. WOLF G. & NEWHERN~ P. M. (1970) Biosynthesis of a fucose-containing glycopeptide from rat small intestine in normal and vitamin A-deficient conditions. J. hiol. Chrm. 245, 455 I-4558. DINGLE J. T. & LUCY J. A. (1965) Vitamin A. carotenoids and cell function. Biol. Reo. 40, 42146 I. DREHER K. D.. SCHULMAN J. H.. ANIIERSON 0. R. B ROELS 0. A. (1967) Properties of lipid and lipoprotein monolayers on 0,04 solutions and the role of cholesterol. retinol, and tochopherol in stabilizing lecithin monolayers. J. Ultrastruct. Res. 19, 586~599. FELL H. B. (1964) In Vitumins and Hormotws (Edited by HARRIS R. S.. WOOL G. I. & LORAINF J. A.). Vol. 22. p. 81. Academic Press. New York. GLICK D. & KERR W. J. (1968) Effect of vitamin A 111 vitro on histamine release and structure of mast cells. Eupl. Cell Rrs. 53, 120 134. HAJJAR J. J. & CUKRA~ P. F. (1970) Characteristics of the amino acid transport system in the mucosal border of rabbit ileum. J. gets. Phwiol. 56, 673 691. HAJJAR J. J.. LAMONT A. S. & CCJRRAU P. F. (lY70) The Na-alanine interaction in rabbit Ileum: effect of Na on alanine fluxes. J. yen. Physiol. 55. 277~~296. HAJJAR J. J.. HAWRANI M. & KIIURI R. N. (1972) Alanine Rux across rabbit Peyer’s patches. Camp. Bioclwm. Physiol. 43A, 723--732. LUCY J. A. (1964) Globular hpid mlcelles and cell membranes. J. Theoret. Eiol. 7, 36@ 373 LUCY J. A. & DINGL~ J. T. (1964) Fat-soluble vitamins and biological membranes. Vatuw. Loud. 204, 156 I 60. MACK J. P.. Lu! N. S. T.. Roe~s 0. A. & ANDORRA 0. R. (1972) The occurrence of vitamin A in biological membranes. Biochim. hiopllys. Actu 288, 203 219. ROELS 0. A. & SHAH D. 0. (1969) Molecular interactlona in lecithin-retinol monolayers. .I Colloiil %i. 29, 279.-287. Sctiti~rz S. G. & ZALLISKYR. (1964) Ion transport In ISOlated rabbit ileum. J. pa. Physiol. 47. 567-584. SCHULTZ S. G.. C~JRRAY P. F.. CHEZ R. A. Jt F~ISZ R. E. (1967) Alanine and sodium fluxes across mucosal border of rabbit ileum. J. gw. Phvsiol. 50, 1731 1160 VARN~LL T. R. (1972) Effect of avitat&osis A and dletarq regime on intestinal transfer and hepatic concentration of free amino acids. III! J I’ir. Nurr. Rrs. 42. 271 ‘77.