Biochimica et Biophysica Acta, 1071 (1991) 255-271

255

© 1991 Elsevier Science Publishers B.V. All rights reserved 0304-4157/91/$03.50

Review

BBAREV 85385

Structure-function relationships in intestinal brush border membranes P. P r o u k Department of Biochemistry, Faculty of Medicine, University of Ottawa, Ottawa (Canada) (Received 18 June 1991)

Contents I.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

255

II.

Preparation of brush border membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

256

I11.

Proteins of brush border membrane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

257

IV.

Lipids of brush border membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Transbilayer distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

258 258 259

V.

Structural studies on brush border membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Phase transitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Fluidity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I. Polarity of the membranes and differentiation along the length of the intestine . . . . . . . . . 2. Differentiation along the crypt-villus axis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Postnatal development and ageing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

259 259 260 261 261 262

VI.

Effects of lipid uptake in vitro on structure and function of brush border membranes . . . . . . . . . A. Fatty acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Cholesterol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

262 262 263

Vll.

Effects of dietary supplementation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

264

VIII. Modulation of calcium transport by vitamin D . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

265

IX.

267

Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

267

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

267

L Introduction

The enterocyte is highly polarized displaying at maturity, a well developed brush border at the luminal surface of the cell which is separated from the basolateral region of the plasmalemma by junctional corn-

Correspondence: P. Prouix, Dept. Biochemistry, Faculty of Medicine, University of Ottawa, 451 Smyth Road, Ottawa, Ontario, Canada KIH 8M5.

plexes that consist of tight junctions, belt desmosomes and spot desmosomes. The tight junctions appose lateral membranes of adjacent cells and play an important role in the control of epithelial permeability. The brush border consists of an array of several thousand microviUi per cell each consisting of a finger like structure comprising a plasma membrane and an elaborate supportive cytoskeleton. The anatomical features of the small intestinal epithelium [1-5] as well as those of the microvilli with particular reference to the cytoskeletal elements and their organization [6-15] have been extensively reviewed in the last decade.

256 Intestinal brush border (microvillus, luminal, apical) membranes continue to be the object of numerous investigations. The reasons for their wide experimental use stem from their varied enzymatic and transport activities particularly in regards to digestion, absorption and secretion as well as from the ease with which they can be isolated in relatively pure form. Since a sufficiently high proportion of the membranes are isolated as sealed vesicles they have been extensively used as models for transport measurements. Many of the studies on brush border membranes have been aimed at elucidating structure-function relationships. Particular interest has been given to the influence of lipid composition and fluidity on enzyme and transport activities. Such experimentation has led to detailed knowledge of the lipid compositional and fluidity variations resulting from cell differentiation along the villus-crypt axis and aborally along the intestinal tract, or resulting from dietary manipulation, the administration of hormones or other agents or again, resulting from the exposure of isolated membranes to exogenous lipids in vitro. In some cases attempts to correlate such alterations with functional changes have been made. The present article summarizes recent findings in these areas and together with other recent reviews [16,17] addresses the question of structure-function relationships in intestinal brush border membranes. There is evidence that not only is the enterocyte membrane polarized into apical and basolaterial regions but that these regions are themselves polarized into subdomains of different composition and function [18]. The mechanisms whereby such polarization is generated and maintained is presently an area of considerable interest [19-34]. The structural and functional polarization of the enterocyte plasmalemma into brush border and basolateral regions is explained by very distinct protein, enzyme and lipid compositions characteristic of each membrane segment. However, it is not the purpose of this review to compare the biochemical properties of these membrane segments but rather to bring out those features characteristic of the brush border membrane. Consequently discussions that follow will be centered mainly on the isolation and the compositional, structural and functional aspects of this latter membrane. II. Preparation of brush border membranes Studies on the structure and function of the brush border membranes have been greatly aided by the development of rapid methods for their isolation (see reviews in Refs. 5, 35, 36, 48). These membranes are routinely prepared by homogenizing mucosal scrapings and precipitating most of the undesired organelles and basolateral membranes with

10 mM CaC! z or MgCI 2 [37-47]. In some procedures, the divalent cation precipitation method is combined with density gradient centrifugation [43,49] or with passage through a column of Sepharose 4B [50] as additional purification steps. Brush border memb~aiics and basolateral membranes can be isolated simultaneously from small intestine by a method exploiting Ca 2÷ precipitation and Percoll gradient centrifugation [44]. Basolateral membranes from isolated enterocytes, mucosal scrapings, colonocytes as well as from other types of cells are obtained by methods based on density gradient centrifugation as are also, the brush border membranes from colonocytes [51-58]. Intestinal brush border membranes purified by the Ca2+precipitation method have a diameter range between 0.05 to 0.3 g M and display a right-side out orientation as evidenced by transmission and freeze fracture electron microscopic examination, by the activity of sucrase in the presence and absence of detergent and by immunological methods [36,39,59]. A similar right side out orientation was found for renal brush border membranes as revealed by the use of monoclonal antibodies recognizing separately either the cytoplasmic or the extracytoplasmic domain of neutral endopeptidase [60]. Vesculation follows dissociation of the cytoskeletal core fibres and it has been suggested that the right side out orientation depends on the adherence of cytoskeletal elements to the membrane [611. Although brush border membrane vesicles prepared by the divalent cation precipitation method have been widely used for transport studies, the rates obtained are probably underestimated since there is strong evidence that a large portion of the vesicles are leaky to molecules of low molecular weight. Accordingly, it has been shown that actin, considered to be a marker of the cytoplasmic side can be labelled by a number of reagents of molecular weight < 700 [62-66]. Also Gains and Hauser [67] comparing the compartmentation of radiolabelled glucose, raffinose, inulin and inulin carboxylic acid estimatcd that only 1 in 4 to 6 of the vesicles were sealed to small molecules. However, both intestinal and renal brush border vesicles are sealed to large molecules exceeding 5000 in molecular weight [39,60,68]. The use of Mg 2÷ has been advocated, since when rabbit brush border membranes are isolated by Ca 2÷ precipitation, unusually high levels of lysophosphatidylcholine and lysophosphatidylethanolamine, corresponding to 16-26% of the total lipid phosphorus, can arise [41]. The breakdown of phospholipids was attributed to an activation of phospholipase A by Ca 2÷. There are at this time however not very much grounds for preferring the use of Mg 2÷. Membranes prepared with this cation are less pure [37,69] and several other reports have indicated the presence of quite normal

257 amounts of lysophospholipids in luminal membranes prepared with C a 2 + [70-72]. Furthermore, recent studies have clearly characterized phospholipase A of brush border membranes as a Ca2+-independent enzyme [45,73]. It seems, therefore, that the accumulation of iyso derivatives and the formation of free fatty acids are not related to the type of divalent cation used but t,~_,the treatment and manner of storage of the isolated membranes or of the tissue from which these membranes are isolated [45,72]. Breakdown of phospholipids by phospholipase A of brush borders has been shown to persist even during storage at -20°C [45]. Treatment of isolated microvilli membranes with KSCN removes much of their interior dense cytoskeletal material and yields a membrane fraction in which the alkaline phosphatase and sucrase activities have been enriched by up to 40- and 100-fold, respectively, as compared to the original homogenate [74]. Detailed examination of brush border membranes by transmission and freeze fracture electron microscopy, by quasielastic light scattering and by SDS-PAGE analysis of their proteins brought Bjorkman et al. [75] to the conclusion that vesicles prepared by Ca 2+ o r M g 2+ precipitation with or without additional treatment with KSCN differ in size, shape, intramembranous particle distribution and protein composition. Recent studies by Bjorkman and Brigham [76] have also indicated that these different methods of membrane preparation affect phospholipid composition and both the static and dynamic components of fluidity. As pointed out by the authors, the structural heterogeneity of such preparations should be considered in the interpretation of functional studies. However, a recent report by Johnston et al. [77] indicated no differences in the o-~lucose

transport ability of brush border membrane vesicles prepared by these four procedures. III. Proteins of brush border membrane

Brush border membranes contain a number of characteristic hydrolytic enzymes and transport systems. The hydrolases are important for the terminal digestion of oligosaccharides and peptides as well as for the activation of pancreatic enzymes. The transport systems are required for the mediated entry of digestion products into the cell and are often Na÷-coupled [5,78-82]. Except for the Na ~-dependent D-glucose transporter (see reviews by Semenza et al. [35] and Semenza and Corceili [79]) not much is known about the properties of the proteins involved in transport. The Na÷-glucose cotransporter has been isolated as a 72 kDa protein [83] and has been cloned and sequenced [84]. Candidates for transport of bile salt [85], proline [86], fatty acid [87] and phosphate [88,251] have been identified along with receptors involved in the uptake of factorbound cobalamin [89,90], nerve growth factor [91], epidermal growth factor [92] and IgG [93]. Because of their relative abundance, the hydrolases have been more easily purified and characterized. These enzymes form a distinct class of membrane proteins termed ectoenzymes. The structural and enzymatic properties of these proteins [78-81,94-96] as well as their biosynthesis and intracellular transport [97] have been reviewed. In general, the ectoenzymes are integral proteins that project their catalytic sites on the outside of the cell. They are heavily glycosylated proteins and possess three distinct domains. More than

TABLE I

Lipid composition of brush-border menzbranesfrom different species Numbers in parentheses represent the references from which these values were taken. Chick

Total lipid/protein Phospholipid/protein ( / z g / m g protein)

Pig

Rabbit (72)

(100)

(101)

(102)

(40)

(42)

(45)

0.49

0.54

0.41

150 *

270 *

205

Neutral lipid (/zg/mg protein)

97 *

Glycosphingolipid ( # g / r a g protein)

(98)

(99)

(44)

0.6

0.56

0.50

0.43

0.65

0.63

0.62

150

133

191 *

190

168

131

128

160

I l0

119

195 *

160 *

104

110

93

105

351 *

168 *

209 *

403

207

290

80 *

50

60

77

89

0.5

0.74

1.14

1.39

(70)

165 *

Cholesterol ( ~ g / ' m g protein)

121

Cholesterol/phospholipid (mol/mol)

1.5 *

Mouse

Rat

~ 66 * 0.5

* Calculated from the original references.

85

90

85

1."2

1.2

1.26

0.87

91 *

258 mouse [102] and rat [70,104] but not of young rabbit [105]. Further characteristic of the luminal membrane is the higher sphingomyelin/phosphatidylcholine ratio compared to that of the contraluminal membranes. The renal brush border and basolateral membranes of rat display the same differences in amounts of choline and ethanolamine lipids as those of their intestinal counterparts [106]. The phospholipids of rabbit small intestine brush border membranes comprise besides the diacyl derivatives, plasmalogen and alkylacyl subclasses. The phosphatidylcholine and phosphatidylethanolamine fractions contain 10% and 33% ether lipid, respectively [41]. Although the fatff acid composition of each lipid class of small intestine brush border membranes differs appreciably [40,45] there is a common high abundance of certain fatty acids displayed. Palmitic and stearic acids account for up to 40-80% of the total and 18: l(n - 9), 18: 2(n - 6) and 20: 4(n - 6) account for most of the remainder [40,41,45,101,107,108]. For rabbit and rat the degree of saturation is higher in microvilli membranes than in basolateral membranes [17,104,109,159]; however, for mice the same degree of saturation is seen in both membranes [101]. Morita et al. [110] gave qualitative evidence that the protein, glycoprotein and glycolipid compositions of microvilli membranes were quite different from those of the basolateral membrane and reflected the functional polarity of the intestinal epithelial cell. The microvillus membrane is very rich in glycosphingolipids and may contain up to 1.5- to 2-fold the amount found in the basolateral membrane. In mouse, rat and rabbit microvilli membranes [40,41,101,102,111,112], the major glycosphingolipid consists of monohexosylceramide but in pig, digalactosylceramide and pentahexosylceramide are present as the major components [42]. Other glycosphingolipids such as trihexosylceramide, various oligosaccharide-containing lipids and ganglio-

90% of the mass of the protein is accounted for by a large globular glycosylated domain containing the catalytic site. A second domain anchors the protein to the bilayer by hydrophobic interactions and may involve its N-terminus or, less frequently, its C-terminus or a phosphatidylinositol-glycan moiety. The third domain is a polypeptide stalk that joins the other two domains. IV. Lipids of brush border membranes

IV.-A. Composition The lipid composition of small intestinal brush border membranes was reported for several species by a number of laboratories using different isolation methods. Part of the variation found may reflect the influence of the nutritional state of the animals which is not always clearly specified. Results summarized in Table I reveal nonetheless, certain consistencies. The apical membranes in general display relatively high cholesterol/ phospholipid ratios, low lipid/protein ratios and a high glycosphiagolipid content (Table I) when compared to basoiaterai membranes [99,104]. Cholesterol accounts for more than half of the neutral lipids. The levels of free fatty acids (not shown) usually exceed the trace amounts found in tissues [41,42,72] and may arise in part from phospholipase A activity during the membrane isolation procedure [103] but they probably represent as well, exogenously-derived lipid accumulated during absorption. The phospholipid composition of brush border membranes for these same species is shown in Table II. Phosphatidylcholine and phosphatidylethanolamine account for 60-70% of the total. In most instances, particularly in mouse and rabbit, phosphatidylethanolamine was found to be more abundant than its choline analogue. By contrast phosphatidylcholine is the predominant phospholipid of basolateral membranes of TABLE !I

Phospholipid composition of brush-border membranes from different mammalian species Numbers in parentheses represent the references from which these values were taken. Phospholipid

Phosphatidylethanolamine Phosphatidylcholine Phosphatidylserine Phosphatidylinositol Sphingomyelin Lysophosphatidylethanolamine Lysophosphatidylcholine Phosphatidic acid Cardiolipin Phosphatidylglycerol

Percent of total phospholipid pig

rat

(42)

(41)

(45)

(70)

(98)

(104)

(41)

(72)

(101)

(102)

34.0 34.0 12.0 10.0 10.0

38.3 32.6 13.0 8.7 6.4

37.4 26.8 11.2 6.4 7.0 6.9 4.3

11.9 26.7 4.4 22.3 24.1

39.5 20.7 14.0

47.2 20.6 13.3 5.6 7.3 2.8 3.4

49.1 25.1 6.0 11.6 8.3

1.7

30.3 35.0 7.5 8.3 10.5 3.6 3.1 1.2

61.7 16.2 14.2 7.9

8.7 0.9

28.4 36.4 6.8 7.4 8.0 2.8 4.0 2.5 2.6 1.4

1.0

rabbit

6.7

0.5

mouse

259 sides have also been tentatively identified or reported to occur in different species [42,101,111-114]. In rat microvilli membranes, the monohexosylceramide fraction contains glucose as the sugar constituent [l l 1], whereas in pig membranes, both glucosyl- and galactosylceramide are present [42]. The levels of glycolipid increase with differentiation and development. Accordingly as brush border membrane surface enlargement occurs during maturation of crypt cells into villus cells, there is in rat, a sizeable increase in glycolipid content [115,116]. Presumably, much of this glycolipid is incorporated into the luminal membranes. Studies with rodent small intestine have also indicated alterations in glycosphingolipid content and composition following administration of steroids [117,118] or during development with age [119,120]. It is not known, however, at which membrane level these changes occur or what particular role these lipids play in the maturation and developmental processes. II/-B. Transbilayer distribution Studies by Barsukov et al. [121] revealed that only about 25% of the two most abundant phospholipids (phosphatidyicholine and phosphatidylethadolamine) of intestinal microvilli membranes were readily accessible to phospholipase A 2 and phosphatidylcholine exchange protein. The major pool of these lipids appeared to be located on the cytoplasmic side of the bilayer and could exchange rapidly with the outer pool [121,122]. This topical orientation of the major phospholipids is probably linked to the high glycosphingolipid content of these membranes and the usual outer leaflet distribution of this latter lipid [123]. For renal brush border membranes, recent reports have indicated a similar inner leaflet oriented distribution of phosphatidylcholine and phosphatidylethanolamine together with phosphatidylserine and phosphatidylinositol. The only major lipid of the exterior leaflet detected by sphingomyelinase, phospho!ipase C and trinitrobenzene suifonate used as probes, was sphingomeylin accounting for 75% of the phospholipids [124,125]. Both, cytoskeletal elements linked to brush border membranes and sphingomyelin having a low transbilayer migration rate, were suggested to play important roles in maintaining phospholipid asymmetry in these membranes [125]. The transbilayer distribution of amino phospholipids in trout intestinal brush border membrane was examined by Pelletier et al. [126] using trinitrobenzene sulfonate as probe. A symmetrical distribution of phosphatidylethanolamine between the two leaflets was reported for mid intestine membrane whereas in posterior intestine membrane, 64% of the ethanolamine lipid was located in the inner membrane leaflet. Close to 70% of the phosphatidylserine had an inner leaflet

orientation in membranes from both regions of the intestine. Attempts to probe the distribution of cholesterol in rabbit small intestine brush border membranes with cholesterol oxidase were unsuccessful. The enzyme was active only in the presence of detergent at concentrations which disrupt the membrane [127]. V. Structural studies on brush border membranes

V.-A. Phase transitions Thermotropic transitions in intestinal cell membranes have been assessed with the help of fluorescence techniques, differential scanning calorimetry (DSC), electron spin resonance as well as by examining the temperature dependence of enzyme activities. Except in the case of DSC analysis, phase transition temperatures are revealed by discontinuities in Arrhenius plots of the data. It is known, however, that such discontinuities can represent several phenomena, such as a lipid phase transition from gel to liquid crystalline state [128] a lateral lipid phase separation [129] or the interaction between the boundary lipid phase associated with membrane protein and the bulk lipid phase [130]. It is not usually clear which of these phenomena is involved when discontinuities are reported. DSC studies of rat enterocyte microvilli membranes revealed relatively broad transitions with a peak of approx. 30°C and a lower critical temperature similar to that measured by steady state fluorescence polarization [131]. The upper critical temperatures (39-40°C) were just above the physiological temperature which means that for rat at least, some of the membrane functions could be influenced by the physical state of the lipids at body temperature. Rather low transition enthalpies are observed in these membranes which was explained by the relatively high content of cholesterol in the membrane and the significant involvement of protein-lipid interactions. Accordingly, the transition enthalpies of the rehydrated extracted lipids from enterocyte membranes are 2-5-fold higher than those of the intact membranes. Protein-lipid interactions in the antipodal membranes were further indicated by diphenylhexatriene (DPH), 2-(9-anthroyloxy)-stearate (2-AS) and 12-AS steady state fluorescence anisotropy measurements which revealed for rat, significantly higher values in the intact membranes than in their extracted lipids [108,131]. However, these interactions as indicated by fluorescense anisotropy differences between membranes and extracted lipid have been shown to be greatly dependent on the age of the animal [109]. Thermotropic transitions were also examined in rabbit microvilli membranes by Hauser et al. [132] using 5-doxyl-, 12-doxyl- and 16-doxylstearic acid as well as 5-doxyl phosphatidylcholine. With the first three

260 probes, Arrhenius plots of the order parameter or the rotational correlation time indicated respective discontinuities at 30, 20 and 13°C. Since the apparent transition temperature depended on the location of the spin probe along the bilayer normal, the authors suggested that chain melting is progressive and spreads with increasing temperature from the center of the membrane outward. Subsequent results of Miitsch et al. [133] obtained by DSC analysis of the same membranes revealed a broad reversible lipid phase transition in the range 10 to 30°C with a peak at 25°C in both intact microvilli membranes and their extracted lipids. Removal of exteriorly oriented proteins with papain or the inner cytoskeleton by alkaline treatment had no significant effect on the transition. It appeared that lipid was entirely responsible for the transition and protein-lipid interactions did not play a predominant role although extracted lipids did display somewhat lower packing and slightly higher fluidity than the brush border membranes [132]. In another study involving rabbit, a comparison of DPH-steady state fluorescence anisotropy values obtained with the intact microvilli membranes and liposomes generated from their lipid extracts was made. The anisotropy values were only slightly lower in the liposomes at temperatures below 30°C indicating minor influence of protein on fluidity. It appears from these different results that the extent of lipid-protein interactions in the enterocyte membranes may be found to vary not only with age but also with species and the techniques used for their study [134]. An important difference in the lipid phase transitions of rabbit membranes compared to those of rat membranes is noticeable in that the latter occur in a temperature range approximately 10°C higher. The reason for this is probably related to differences in lipid composition: however, the acyl chain compositions of adult animals of both species are similar [41,80] and the too large variations in the phospholipid and glycolipid content sported by different groups for each of the species do not allow any definite conclusions at this time. Ohyashiki et al. [135] assessed the thermotropic transitions of porcine microvilli plasmalemma by labelling their protein constituents with a fluorgenic thiol reagent, N-[7-dimethylamino-4-methyl-coumarinyl] maleimide and obtained a transition point at 35°C when the effect of temperature on fluorescence parameters was examined. On the basis of this and other evidence, the authors concluded that a phase transition of the lipid from gel to liquid crystalline state occurs over a temperature range from 30 to 35°C. Phase transitions in small intestinal and cotonocyte membranes have also been examined by assessing the temperature dependence of enzymatic and transport activities [58,99,136-139]. In general, enzymes the cat-

alytic functions of which operate outside the lipid environment, did not display breakpoint temperatures in Arrhenius plots of their activities. Among such enymes were dissacharidases, iactase, leucine aminopeptidase, glutamyl transpeptidase and phospholipid methyltransferase II [131,137]. Most of the other enzymes and transport systems studied displayed discontinuities in the Arrhenius plots of their activities in a temperature range of 20-30°C. On the basis of studies involving delipidation and relipidation of alkaline phosphatase Brasitus et al. [139] suggested that the breakpoint temperature obtained by examining the temperature dependence of membrane catalytic and transport proteins is determined by the lipid immediately surrounding the protein rather than by the bulk lipid of the membrane. However, experiments clearly demonstrating the role of the surrounding lipid on the conformation of these proteins have not been performed. Furthermore, while increases in fluidity brought about by benzyl alcohol result in an augmentation of the phosphatase activity of human brush border membranes [141] such fluidity increases do not affect this activity in rat membranes [140]. Consequently, it is difficult to relate causally, and in a generalized manner, temperature-sensitive alterations in protein activity with changes in lipid structure. V.-B. Fluidity Fluidity, or the relative motional freedom of lipid molecules in biomembranes, has been conveniently assessed by measuring steady state fluorescence anisotropy using appropriate fluorescent probes [142]. In earlier work (reviewed by Shinitzky and Barenholz [143]) fluidity was expressed in terms of microviscosity as calculated from ~b, the rotational relaxation time, obtained by applying the Perrin equation: r0 r s _ - - ~

1 + "r/~b

where r s is the steady state of fluorescence, r 0 is the maximal anisotropy value and ~- is the fluorescence lifetime. Recent time resolved fluorescence anisotropy decay measurements have indicated however that the rotation of certain probes such as DPH is restricted in both natural and artifical membranes by lipid anisotropy [144-146]. The following modified Perrin equation takes this hindrance into account [144]: rs=

r o - r~ +r~ ! + r/~

in which r~ is the limiting hindered anisotropy. The first term of the equation represents a kinetic component and the second, a structural component. For

261 probes such as DPH which are rod shaped, r~ values are high and largely account for r~ [144]. For spherical probes such as 12-(9-antroyloxy) stearate, r= values are small and r~ in this case reflects mainly the rotational relaxation time [144]. Accordingly, probes of the first category have been used to report on the static component of fluidity; whereas, probes of the second, on the dynamic component of fluidity [17,144].

V.-B. 1. Polarity of the membranes and differentiation along the length of the intestine Studies of membrane labelled with fluorescent probes such as DPH, AS derivatives, retinol and dansyl phosphatidylethanolamine have yielded higher steady state anisotropy values for microvillus membranes compared to basolateral membranes. The lesser fluidity in the luminal membranes suggested by the anisotropy measurements were noticed in small intestine as well as in the colon [16,17,58,99,104,147]. Furthermore in both the small and large intestine, for both types of membranes, fluorescence anisotropy values were higher in the distal than in the proximal segments. The fluidity of the antipodal membranes was also estimated by assessing short-range lateral diffusior, as revealed by quantification of the intramolecular excimer fluorescence of dipyrenylpropane [16]. Indicative of greater fluidity, the excimer/monimer intensity ratio was appreciably higher in the basolaterai membranes for the temperature range studied. Differences in fluidity between renal microvillus and basolateral membranes were reported to be similar to those characterizing the antipodal membranes of the intestine [148,149]. The lower fluidity displayed by microvilli membranes compared to basolateral membranes can be explained by the higher cholesterol/phospholipid ratio, the lower lipid/protein ratio and the greater degree of acyl chain saturation in luminal membranes [17]. Also to be considered is the fact that much of the phosphatidylcholine of brush border membranes has been replaced by glycosphingolipids which are more highly ordered as a result of hydrogen binding [123,150]. Accordingly, desialylation of intestinal of porcine intestinal brush border membrane with neuraminidase results in perturbations in the proteins of the membrane [151] and an increased fluidity as revealed by the fluorescence parameters of pyrene and DPH-labelled membranes [152]. lllsley and co-workers [153] have recently questioned the adequacy of steady state fluorescence anisotropy measurements alone for the assessment of fluidity. In their case, different conclusions regarding the fluidity of renal antipodal membranes were arrived at on the basis of lifetime and tryptophan-parinaric acid fluorescence energy transfer measurements using cis- and trans-parinaric acid as probes. While the application of additional methods such as for example, fluorescence

anisotropy decay measurements, for assessing fluidity in biomembranes can be recommended, steady state fluorescence anisotropy measurements have been conveniently and reliably used for assessing average fluidity of membrane lipids. Results from such determinations should concorde with those obtained by estimating the mole fractions of probes present as long lifetime components (that fraction in the solid phase) of the bilayer. The accuracy of the latter estimations depends however on the proper evaluation of the preexponential factors which may be difficult to achieve when made with only three measurements by phase and modulation methods [153]. The data obtained with tryptophan-parinaric acid fluorescence energy transfer measurements do not necessarily contradict those resulting from steady state anisotropy determinations. The former report on the lipid phase in the close vicinity of those hydrophobic anchor peptides containing tryptophan whereas the latter report on the average fluidity throughout the entire bilayer. The lipid composition of microvilli membranes changes along the length of the small intestine and the colon such that when compared to the proximal segments, the distal portion displays an increased content of cholesterol, a greater cholesterol/phospholipid ratio and a higher degree of saturation in the phospholipid acyl chains [108,109,147]. Similar differences in lipid composition are displayed by the basolaterai membrane, in pig, a very large increase in the cholesterol/phospholipid ratio is seen in the microvilli membranes of jejunum when compared to those of duodenum [38]. These regional changes in lipid composition do result in significant changes in fluidity as was mentioned previously.

V-B. 2. Differentiation along the crypt-villus axis Decreases in fluidity as reported by fluorescence anisotropy measurements of fluorophor-labelled luminal plasmalemma, have also been noted when membranes of cells originating from the crypt are compared to those which have migrated to the villus tip [154]. This change in order was noticed however, only in small intestinal membranes and not in colon membranes [155]. The physical changes resulting from migration to the villus tip were matched by increases in the protein/lipid, choiesteroi/phospholipid and cholesterol/protein ratios as well as in the degree of acyl chain saturation [154]. Similar changes in the cholesterol/phospholipid ratio and an increase in relative amounts of phosphatidylethanolamine were also noted for rabbit microvilli membranes as cells matured along the crypt-villus axis [156]. These compositional changes were accompanied by changes in fluidity and in glucose transport kinetics. Quite surprisingly, however, results from a more recent study performed with Lewis rats by Dudeja et

262 al. [157] revealed a gradient in fluidity and corresponding changes in lipid composition opposite to those noted earlier with Sherman rats [154]. It is evident from these last results that generalizations regarding fluidity gradients in brush border membranes accompanying maturation along the crypt-villus axis cannot be made because of important biological variations even within strains of the same species.

V-B. 3. Postnatal decelopment and ageing Besides those describing changes during cell maturation several studies have revealed decreases in microvilli and basolaterai membrane fluidity during postnatal development up to adulthood [105,108,118,158, 159]. Accompanying these differentiation and developmental changes are a number Gf functional changes [160-166]. In one study, Hiibner et al. measured the steady state fluorescence anisotropy to assess fluidity in rat small intestine brush border membranes that had been labelled with a series of fluorophores monitoring different depths of the bilayer. With all probes, the fluorescence anisotropy increased with age from the newborn to the adult stage [167]. Increases with age, of fluorescence anisotropy and transition temperature of DPH-labelled microvilli membranes were also observed in rats for a period extending between 6 to 17 weeks but not beyond [108]. Pang et al. [100] also examined rabbit microvilli membranes by electron spin resonance using 5-doxylstearic acid as probe. When the hyperfine splitting parameter was plotted as a function of temperature, a discontinuity was noticed at 39.6°C for membranes of mature animals, a value considerably higher than that reported by Hauser et al. [132]. No discontinuity was noticed for membranes of newborn rabbits. Schwarz et al. [109] also failed to see a phase transition in microviili membranes from suckling rats but did observe one for membranes of weaned animals. It is evident from these results that membranes from the very young are less ordered than those from mature animals and this may be an important factor accounting for the more facile import of macromolecules in younger animals. This import which is pinocytosis dependent would probably be enhanced by the easier attachment of the macromolecules to the surface of the more fluid membranes of newborns [100]. Maturational increases in order were also found to occur in rabbit basolateral membranes when examined by DPH steady state fluorescence anisotropy as a function of temperature [105]. The physical changes in the enterocyte membranes accompanying postnatal development up to adulthood are coincidental with alterations in lipid composition [108,109,147,154,168]. Microvilli membranes from jejunum and ileum of suckling rats were reported to display a higher sphingomyelin/phosphatidyicholine ratio, and were richer in total lipid, cholesterol and

phospholipid per mg prot~in than their counterparts from postweaning rats (6 weeks). Hiibner et al. [167] also working with rat small intestinal brush border membranes found the molar ratio of saturated/cis unsaturated fatty acids to increase abruptly from the natal to the suckling period and remained relatively constant thereafter. The cholesteroi/phospholipid ratio increased quite steadily from the natal to the adult stage and was due to a rise in cholesterol paralleled by a fall in phospholipid content throughout most of the period examined. Similar increases of the cholesterol/ phospholipid ratio with age were reported by Engelhardt et ai. [169] who also showed a parallel decrease in phosphocholine transferase activity. A study by Brasitus et al. [108] with post weaned rats, the microvilli membranes of which were examined over a 6 to 117 week period revealed important increases in the cholesterol/phospholipid ratio and a fall in the lipid/ protein and sphingomyelin/phosphatidylcholine ratios as a function of age up to adulthood in both proximal and distal segments of the small intestine. These changes were accompanied by an increase in the degree of saturation of the acyl chains in the proximal segment and a decrease in arachidonic acid. VI. Effects of lipid uptake in vitro on structure and function of brush border membranes

Uptake studies [170-181] have indicated that the lipid composition of brush border membranes can he altered directly by interacting isolated microvilli membranes with fatty acids of different types, with choiesterol or with complex lipids containing difierent polar headgroups. Alternatively the composition can be changed by dietary manipulation however, in the latter case, changes are less pronounced becat~e, of compensatory mechanisms which affect the acy! chain composition or cholesterol content. These in vitro modifications could possibly reflect the transie~ zhanges in composition that occur in the membrane during absorption since there is at present no evidence that at least part of the absorptive process involves passive diffusion through the bilayer.

VI-A. FatO, acids Uptake of methyl esters of vaccinic acid were reported to increase the fluidity ~f chick brush border membranes as assessed by DPH fluorescence polarization measurements, As a consequence, the rate of Ca -'+ uptake increased [182,183]. In another study, it was shown that Ca -'+ uptake was enhanced in rabbit brush border membranes treated with low concentrations of unsaturated fatty acids or with various concentrations of caprylic acid [184]. The uptake was inhibited

263 however when the membranes were treated with higher concentrations of unsaturated fatty acids (0.2 to 0.60 raM). Saturated fatty acids had no marked effects on Ca 2+ uptake. Quite predictably the uptake of oleic acid, linoleic acid and methyl oleate decreased the fluorescence anisotropy of membranes labelled with DPH in a concentration-dependent manner, in contrast, paimitic acid had little or no effect on DPH-reportable order. These findings were similar to those of Klausner et al. [185] who, making use of DPH and anilinonaphthyisulfonate as fluorescent probes, showed that the membranes of mouse lymphocytes and baby hamster kidney cells contained lipid domains of different fluidities. Saturated and trans-unsaturated fatty acids were taken up by the less fluid domains without apparent change in order of the interior apolar regions and consequently DPH fluorescence polarization was unaffected. cis-Unsaturated fatty acids on the other hand were taken up by more fluid domains where they caused further disruption of acyi chain packing and a lowering of DPH fluorescence polarization. Incorporation of cis-unsaturated fatty acids in domains harboring the Ca 2+ uptake process increases entry of Ca 2+ in concert with the increased fluidity. However, when concentrations of such fatty acids in these domains become sufficiently great, the presence of a largely increased number of free carboxyl groups at the membrane surfaces is likely to result. The binding of Ca 2+ to these carboxyl groups at the exterior of the membrane would compete with the Ca 2+ transport process. Such superficially bound divalent cation is removed by the wash procedure, used in the method used for studying Ca 2÷ uptake and consequently a decrease in Ca 2÷ uptake values is seen. Excess fluidization in response to a large incorporation of cis-unsaturated fatty acids cannot be in itself the cause of inhibition of Ca 2 ÷ uptake at high concentrations since methyl oleate would have caused a similar inhibitory effect. The effect of caprylate on membrane structure appears to be different than that of cis-unsaturated fatty acids. This agent does cause perturbations in the structure of the luminal plasmalemma as assessed by decreases in the fluorescence polarization of dansylchloride, fluorescein isothiocynate and eosin maleimide convalently linked to the membrane; however, its action appears to be exerted mainly on the conformation of proteins rather than on the lipid bilayer [186]. Consequently the enhancing effects of this fatty acid on Ca 2+ uptake could not be correlated with changes in the order within the bilayer structure. Uptake of fatty acids as 'their CoA esters by chick brush border membranes was also studied [187]. Of the derivatives tried, only oleic and linoleic acids were effective in increasing the rate of Ca 2+ uptake. Satu-

rated derivatives and arachidonic acid were without effect. The precise mechanism whereby unsaturated fatty acids stimulate Ca 2÷ uptake remains unknown. Maenz and Forsyth [188] suggested from their results that ricinoleate, like deoxycholate, may stimulate the uptake of Ca 2+ by acting as a calcium ionophore. However such a mechanism would imply the functionality of the carboxyl group and since methyl esters of unsaturated fatty acids enhance Ca 2÷ uptake whereas free saturated fatty acids do not, it would seem that effects on order in the lipids surrounding the Ca ,-÷ transport system are more important. Tiruppathi et al. [189] showed recently that treatment of rabbit small intestinal brush border membranes with free fatty acids resulted in the inhibition of Na+-H + exchange. Unsaturated fatty acids were the most inhibitory, whereas saturated fatty acids of intermediate chain length showed moderate inhibitory effects. Na+-gradient dependent D-glucose and L-alanine transport and H+-dependent dipeptide transport systems were also affected by unsaturated fatty acid treatment. The inhibitory effects of oleic acid on glucose and alanine transport were attributed to an enhanced collapse of the Na+-gradient; however, inhibition of the Na+-H + exchanger was not due to an accelerated dissipation of the H + gradient since dimethylamiloride-sensitive Na + uptake measured in the absence of an H + gradient was also inhibited by oleic acid. The role that fluidity may play in the regulation of certain activities of brush border membranes was further investigated by Fernandez et ai. [190] who studied the effects of uptake of aliphatic alcohols (Cl to C8) on Na+coupled D-glucose influx, Mg2+-ATPase and suerase. Glucose uptake was 80% inhibited by concentrations of the eight alcohols which increased the fluidity of the membrane by no more than 3%. Only the first alcohols of the series produced any inhibition of Mg2+-ATPase and sucrase. Effects of these agents on the glucose transport system may be at least partly hydrophooic in nature causing a perturbation of lipidcarrier interactions [190]. However, there is good experimental basis for concluding in this case that the inhibition is largely due to indirect effects resulting from a decreased interior size of the brush border membrane vesicles and the formation of channels favoring passage of sodium and a faster collapse of the sodium gradient [191-103]. It should be appreciated, therefore, that the use of alcohols to assess the role of fluidity on transport activities may be misleading.

VI-B. Cholesterol Increasing the cholesterol of chick brush border membranes [194] was accomplished in vitro by incubat-

264 ing everted sacs of jejunum with liposomes of different cholesterol/phospholipid ratios. The brush border membranes of such preparations were isolated and found to display respectively, a 3-fold increase and a 2-fold decrease in molar cholesterol/phospholipid ratio when incubations were performed with iiposomes of high and low cholesterol/phospholipid ratios. No changes in fatty acid and phosphatidylcholine composition accompanied the incorporation of cholesterol. The rate of L-ieucine uptake of enterocytes prepared from lipid-treated jejunum segments was however unaffected by the changes in cholesterol content. While effects on fluidity in this case were not assessed, it is possible that cholesterol intercalated in regions of the bilayer other than those constituting the environment of the leucine transport system. In a recent study [140] rat microvilli membranes were enriched in or depleted of their cholesterol content by incubation with non specific lipid transfer protein and phosphatidylcholine liposomes containing or lacking cholesterol. These modifications were accompanied by corresponding changes in fluidity as assessed by steady-state fluorescence polarization .measurements of plasmalemma labelled with DPH. A fall in sterol/phosphatidylcholine ratio increased alkaline phosphatase activity by up to 30%, whereas a rise had an opposite effect. Sucrase, maltase, and lactase were unaffected by changes in cholesterol content. The decrease in alkaline phosphatase activity which is seen with higher ratios was not related directly to a change in fluidity since benzyi alcohol restored the fluidity of cholesterol enriched membranes without altering alkaline phosphatase activity. It appears that alkaline phosphatase but not disaccharidase can be modulated by changes in the cholesterol/ phospholipid ratio and that this modulation is not secondary to changes in fluidity. The precise manner in which cholesterol influences alkaline phosphatase activity is unknown but it was suggested that its effects might involve changes in protein-protein interactions, in the tightly bound annular lipid or in the bulk lipid properties of the microvillus membrane [140]. Although the possibility of other means of its attachment have not been precluded [195] it appears that intestinal alkaline phosphatase is secured to the brush border membrane by a phosphatidylinositol glycan anchor [94,196]. This being the case, rather than exertitig its effect by modifying the lipid anulus, cholesterol could serve to dilute and make less available phospholipid choline groups, the vicinity of which are required to protect the bound form of the enzyme from inhibition by endogenous phosphate groups [197]. Alternatively, packing of the phosphatidylinositol moiety within the bilayer, likely to be affected by cholesterol, could possibly influence the conformation of the phosphatase with resultant effects on its catalytic activity.

On the other hand, the sucrase-isomaltase complex is anchored in the membranes by a single segment of the isomalt,~se subunit that spans the bilayer near the cytoplasm-oriented N-terminus [198,199]. In this case, it is quite clear that the lipid environment of this anchor [140,200] in whichever manner it may possibly influence structure of the dissacharidase complex, does not affect catalytic activity. VII. Effects of dietary supplementation A number of studies have explored the effects of dietary manipulation on the lipid composition and structure-function relationships of intestinal membranes [201-209]. In rats maintained on diets supplemented with unsaturated fats the enterocyte antipodal membranes displayed small but significant increases in fluidity as compared to membranes of rats receiving a diet supplemented with saturated fat [201]. The increased fluidity was due to increased linoleic acid in microvilli membranes and both linoleic and arachidonic acids in basolateral membranes. The fluidisation process triggered a compensatory response which caused an increase in the cholesterol/phospholipid ratio but was nevertheless accompanied by an enhanced p-nitrophenylphosphatase activity in microvilli membranes and augmented levels of Na+-K + ATPase activity in the basolateral membrane. However, on the basis of later evidence obtained by Brasitus and coworkers it is unlikely that these enhancements were directly attributable to changes in fluidity [140]. In piglets fed semipurified diets supplemented with hydrogenated coconut oil as compared to those receiving similar diets supplemented with corn oil, the jejunum microvilli membranes displayed decreased overall unsaturation of their lipids [202]. Linoleic acid and arachidonic acid levels were markedly depressed and partly replaced by 5,8,11-cicosatrienoic acid [203]. The cholesterol/phospholipid and the sphingomyelin/PC ratio remained unchanged. Electron spin resonance measurements of membranes labelled for both lipids and proteins indicated decreased fluidity resulting from essential fatty acid deficiency and notable changes in the membrane protein behaviour which could be interpreted in terms of reduced lipid-protein interactions [203]. Evidence was also given that the degree of disclosure of reactant groups of glycoconjugates at the membrane surface was affected by saturated fatty supplementation in the diet. Rats, fed with fish oil-enriched diets compared to rats fed with normal diets were characterized by microvilli membranes containing higher levels of palmitoleic acid and eicosapentaenoic acid and lower levels of stearic acid and linoleic acid [204]. Fluidity as measured by steady state fluorescence anisotmpy of membranes labelled with lipid-soluble probes, was unal-

265 tered whereas the activity of alkaline phosphatase was enhanced by the fish oil intake. This enhancement was ascribed to local changes in the lipid environment of the enzyme rather than to general changes affecting the overall fluidity of the membrane [204]. On the other hand when brush border membranes obtained from rats fed fish oil- or butter fat-enriched diets were compared, significant differences in the fatty acid composition of membrane lipids occurred as well as in the molecular species of phosphatidylcholine. The fish oilenriched membranes displayed greater fluidity and Na+-dependent D-glucose transport with a higher maximum velocity [205]. Fish oil supplementation in the diet was also reported to prevent the enhanced active transport of galactose that occurs in small intestine of animals with high cholesterol intake [206]. Treatment of rats with HCG-917 results in the replacement of 87-90% of the cholesterol with 7-dihydrocholesterol in jejunal and ileal microvilli membranes. This change was accompanied by a decreased fluidity of the outer region of the bilayer of jejunum but no changes in the fluidity of ileal membrane were noted. The increased rigidity in the jejunum was accompanied by a decreased rate of fatty acid absorption [207]. The effect of feeding high cholesterol diets on the hydrolase and transport activities of guinea pig small intestinal brush border membranes was also studied [208]. Cholesterol-rich diets caused an inhibition of Na÷dependent glucose transport, as well as of Ca 2÷ transport and dissacharidase activities. Although the noted functional changes were attributed to enrichment of cholesterol and increases in microviscosity of the membranes, these parameters were not measured. Intestinal absorption of many nutrients is increased in diabetic rats. When rats with streptozotocin induced diabetes were fed a polyunsaturated diet, the enhanced jejunal and ileal uptake of glucose was reduced or normalized :~nd these effects could not be explained by alterations ~, intestinal morphology or brush-border content of cholesterol or phospholipids [209]. Polyunsaturated diets also increased alkaline phosphatase activity in diabetic jejunum and ileum. The results as a whole supported the contention that dietary lipid manipulation can influence important intestinal functions including transport processes, possibly by altering the fluidity of brush border membranes. But in fact, the mechanisms mediating these effects remain largely unknown. It can be added at this point that brush border membranes of diabetic rats prepared from enterocytes lining the entire crypt-villus axis have been found to be less fluid than their control counterparts [210]. Feeding a polyunsaturated diet would be expected to increase the fluidity in the membranes of the experimental animals and one might propose this alteration as the basis for decreased transport activity. However, a recent study by Dudeja et al. [157], in which a detailed

assessment was made of fluidity and D-glucose transport along different parts of the crypt-villus axis in both diabetic and control animals, failed to show any direct or inverse r,~iationship between average membrane fluidity and transport ability. Although these last results do not preclude the possibility that glucose carriers are sensitive to the fluidity of their immediate environment, they invite other explanations for the dietary effects. For example the carrier density could be altered or specific fatty acids of the diet may have direct effects on the carrier activity. VIIL Modulation of transport by vitamin D It is currently well known that Ca 2+ transport across the intestine proceeds via an overall saturable, vitamin D-regulated transcellular process and a seco-steroid independent paracellular mechanism which is non saturable [211-213]. The first step in the transcellular process involves translocation across the luminal membrane by a mechanism which has been reported to comprise both a saturable, facilitated diffusion process and a non saturable component [214,215]. The entry into the cell is followed by transcytosolic diffusion and an extrusion across the contraluminal membrane [211]. The mechanisms whereby vitamin D regulation occurs are not completely understood and aspects of the mode of action of this agent are still presently debated. Besides genomic effects which increase levels of cytosolic calcium binding protein (CaBP) [216,217], a protein proposed to function in the transcytosolic diffusion step [211,218], there are reported, protein synthesis-independent mechanisms which begin at a very early stage following secosteroid administration [219-222]. In this regard, however, it may be added that some of the more recent results obtained with embryonic chick duodenal cultures [223] and with isolated chick duodenal cells [224] have indicated the appearance of CaBP prior to Ca 2÷ uptake responses and an inhibition by cycloheximide of 1,25(OH)zD3 effects. To explain non genomic regulation by vitamin D, Rasmussen and coworkers proposed the liponomic control hypothesis [225,226]. Liponomic control is a process whereby 1,25-dihydroxyvitamin D 3 (1,25(OH)2D~) increases brush border membrane fluidity by promoting changes in the lipid composition and causes either unmasking of cryptic calcium transport components or an increase in their turnover number. In support of this hypothesis is the fact that Ca 2÷ uptake by isolated brush border membrane vesicles is enhanced when the acyl chain composition of their lipids is modified in vitro to increase fluidity [135,182,183,226]. Administration of vitamin D to animals destabilizes the brush border membrane [224] by causing, in the phosphatidylcholine component of its lipids, an increase in the degree of unsaturation [40] while enhancing the activity of the

266 phospholipid deacylation-reacylation cycle [228] as well as de novo synthesis of phosphatidylcholine [229]. These protein synthesis-independent changes which favor greater fluidity also closely correlate with the time course of protein synthesis-independent increases in calcium uptake following administration of 1,25(OH)~ D 3 [219,222,227-233]. The liponomic hypothesis is based to a large extent on a correlation between increased fluidity and increased Ca 2÷ transport which was demonstrated in vitro [182,183]. However, not all changes in lipid composition of brush border membranes brought about by incorporation of exogenous lipids result in changes in fluidity that correlate well with Ca 2+ uptake [234]. Furthermore, dietary supplementation of vitamin D [227] or acute administration of 1,25(OH)2D a does not necessarily change the fluidity of the luminal membrane [183,235] but this may depend on which component of fluidity measurements are made [236]. In vivo, changes in fluidity along the length of the small intestine correlate well with C a 2 + accumulating ability, duodenal brush border membranes displaying the highest fluidity and Ca 2+ uptake values [108,109,183]. However, other factors could come into play which were not precluded, such as for example, the existence of an aborai concentration gradient of the calcium channel proteins. Moreover, along the crypt-villus axis, rather than a direct correlation, some of the data reported suggest the existence of an inverse relationship between fluidity and the C a 2 + uptake ability of the brush border membranes although in this regard, the parameters involved were measured, each in a different species [154,253]. Summarizing the evidence at hand, Wasserman and Fullmer [237] concluded that vitamin D performs dual effects on the enterocyte, one dependent on protein synthesis and the other, on changes in lipid microenvironment in the brush border membrane. However, the issue as to whether the early events resulting in increased Ca 2+ uptake following vitamin D administration are the result of mechanisms dependent or not on protein synthesis is still controversial. Furthermore, the evidence that the e trly effects of vitamin D administration are mediated by mechanisms leading to increased fluidity is not compelling. In this regard, results suggest that fluidity increases may play a supportive rather than a determinant role in the early mediation process. A novel mechanism has recently been proposed, in which the receptor for calmodulin in the microvilli membrane (a 105 kDa protein) is a calcium channel. Following entry through this channel, Ca 2+ would bind to calmodulin and the complex would then diffuse to a coated pit area at the base of the microviUi where the divalent ion would be internalized by endocytosis. This model and the evidence in its supp¢~rt were recently presented by Bikle [238]. In accord with this concept is

the fact that administration of 1,25(OH)2D 3 to vitamin D-deficient chicks rapidly increases the calmodulin content of microvilli membranes in parallel with Ca 2+ u~dke ~ctivity in a protein synthesis-independent manner. Th~ increase in calmodulin is due to an elevated bind!ag to the 105 kDa protein present in the brush border membrane. The secosteroid-increased Ca 2+ transport ~ctivity is blocked by calmodulin antagonists. The distribvtion of caimodulin and its receptor protein parallels that of Ca 2+ transport activity in the crypt-villus axis. Of capital importance for the eventual acceptance of the model would be a clear demonstration that the 105 kDa protein is indeed involved as a channel for Ca 2+ transport. In this regard, reconstitution studies with isolated complexes containing calmodulin and its receptor [239,240] or inhibition of Ca 2÷ transport by antibodies against the 105 kDa protein could be useful. However, present evidence indicates that the calmodulin receptor protein is not an integral protein and may be linked to the brush border membrane via a glycoprotein [240,242]. Such findings cannot be accommodated by the model in its present form. The transcytosolic pathway for the movement of Ca 2÷ is not yet clearly known. The implication of organelles has been suggested [243-247] but the alleged, ferrying role of CaBP [217] in these processes is not clear. Possibly an organelle-bound form of CaBP is involved [247]. Recently endocytotic vesicles, the formation of which may be enhanced by increases in luminal membrane fluidity have been proposed as carriers of Ca 2+ from the plasmalemma to the ly~osomes [245-247]. The mechanisms would follow the endocytotic pathway recently described for epithehal cells [249]. These organeUes would in turn transport Ca 2+ to the basolateral membranes. It appears, however, that effects on other elements of the transport pathway are required to explain the action of 1,25(OH)2D 3 [250]. Indeed, recent reports have indicated that the secosteroid hormone action may be exerted at the level of the basolateral membrane calcium channels. BAYK 8644, a Ca 2+ channel agonist, acting only on the basolateral membrane in vascularly perfused duodenal loops of vitamin D replete chicks, mimicks 1,25(OH)2D 3 by rapidly enhancing Ca 2÷ transport [251]. It was suggested on this basis that early effects of vitamin D may be to transiently increase influx of Ca 2+ at the basolateral membrane level, a process which would in turn activate exocytosis of Ca2+-containing vesicles as well as efflux by the Ca2+-pump and Na+-Ca 2+ exchanger resulting in a net increase in Ca 2+ transport. Interestir.gly, Wali et al. [252] have just recently shown with rat colonic epithelium "'-mat one very rapid action of the secosteroid hormone is to stimulate phosphoinositide hydrolysis and cause the translocation and activation of protein kinase C. Such effects are accom-

267 I,UMINAL MEMBRANE

HAS( ) L A T E R : ~,I MEMBRANE I'C" SYNTIIESIS AC'YL ('IIAIN RI'ITAII,t)RI Nt ;

" ~ ' © Ca q II-'~N~I..I qII'ENH

Na.~ T " @ .

~ C a

Oa

~ORGANELI.ES

~ 3 ExOCYTOSIS Fig. 1. Effect of 1,25-dihydroxy ,,itamin D s on transcellular transport of Ca 2+. Non genomic effects of the secosteroid are pictured as occurring partly at the level of the basolateral membrane where opening of the calcium channels possibly coupled with or additive to vitamin D-induced hydrolysis of phosphoinositides and the activation of protein kinase C would result in a transient increase in cytosolic Ca 2+ concentration. This rise would activate the Ca 2+ pump, the Na+-Ca 2+ exchanger and exocytosis processes and increase net Ca 2+ efflux. Other non genomic effects include an increase in the synthesis of phosphatidylcholine (PC) and the level of unsaturation in this lipid by removal and replacement of acyl groups which would in turn cause fluidisation of the luminal membrane. This fluidisation would then enhance diffusion ~hrough Ca 2+ channels, a process coupled to transcytosolic ferrying by free or organelle-bound calcium binding protein (CaBP) or by organelles directly. Transiocation through the cytoplasm would be favoured by increased CaBP production occurring as a genomic effect of the secosteroid. Alternatively, diffusion through the luminal membrane and the cytosol would be linked to the formation of endocytotic vesicles and their eventual fusion to iysosomes. Endocytotic activity would increa:e as a result of a vitamin D-enhanced binding of caimodulin (CAM) to its receptor, a 105 kDa component believed to function as a Ca 2÷ channel protein. Once loaded with Ca 2+, "he CAM-105 kDa protein complex wculd move to a coated pit area at the base of the microvilli where endocytosis of the Ca 2 + would occur. • Processes stimulated by 1,25-dihydroxy vntamin D s.

panied by an equally rapid rise in cytosolic C a 2+ a s measured in isolated colonocytes, part of which is probably due to Ca 2÷ influx. It is not presently known however whether these responses can occur in both antipodal membranes of the cell or whether they are polarized. Some of the present concepts involving transcellular Ca 2÷ transport and the influence of vitamin D on this process are summarized in Fig. 1.

IX. Concluding remarks Studies on structure-function relationships in brush border membranes have been largely concerned with revealing the effect of lipid composition and fluidity on the activity of enzymes and transport systems. Results in general have indicated that fluidity in this, as in

other membranes, is controlled to a great extent by the degree of unsaturation of the acyl chains and the relative proportion of cholesterol. Although effects of proteins on fluidity have been reported, their magnitude seems to vary with such factors as the technique used for assessing fluidity as well as the age and species of animals examined. With regards to the brush border membrane at least, because of biological variation, generalizations concerning relationships between fluidity and function cannot be made: a direct relationship in one strain of rat for example may become an inverse relationship in another strain. Modification of bulk lipid composition and fluidity does modulate certain enzyme and transport activities as well as processes involved in lipid absorption and Ca 2+ uptake. In many cases, however, it remains to be determined whether the modulation of activity is due to change in fluidity itself, to a specific interaction of lipid with protein, to changes in levels of proteins associated with the activity or to some other indirect effect. Clearly more detailed investigations are required to establish what role lipids and fluidity may play on the structure of the proteins specifying the biological activity. In this regard, membrane modification or reconstitution studies with purified enzyme or transporter components, designed not only to examine changes in the function but also in the conformation of the protein may prove very valuable.

Acknowledgements The author is gratefid to the many colleagues who have responded to his requests for reprints, to Dr. A.G. Szabo for his advice in the evaluation of some of the published fluorescence spectrophotometry data and to Ms. J. Normand and Ms. G. Villeneuve for their skillful typing of the manuscript. Work in the author's laboratory was supported by the Medical Research Council of Canada.

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