Cytoplasmic transport of lipids: Role of binding proteins

ISSN 0305-0491/96/$15.00 PII S0305-0491(96)00179-4

Comp. Biochem. Physiol. Vol. ll5B, No. 3, pp. 319-331, 1996 Copyright © 1996 Elsevier Science Inc. ELSEVIER

Cytoplasmic Transport of Lipids: Role of Binding Proteins Richard A. Weisiger UNIVERSITY OF CALIFORNIA, DIVISION OF GASTROENTEROLOGY, DEPARTMENT OF MEDICINE,

SAN FRANCISCO,CA 94143, U.S.A. ABSTRACT. After entering the cell, small molecules must penetrate the cytoplasm before they are metabolized, excreted or can convey information to the cell nucleus. Without efficient cytoplasmic transport, most such molecules would efflux back from the cell before they could reach their targets. Conversely, intracellular lipids generated by hydrolysis of triglycerides, phospholipids and other esters must be transported away from their site of formation to prevent toxic accumulation, lntracellular movement of all molecules is slowed by molecular crowding, tortuosity, and the greater viscosity of the cytosol relative to water. However, lipids and other amphipathic molecules are further slowed by their tendency to bind to cytoplasmic membranes. Cytoplasmic binding proteins reduce membrane binding by increasing the aqueous solubility of their ligands. These aqueous carriers catalyze the transport of lipid molecules across hydrophilic water layers just as plasma membrane carriers catalyze the transport of hydrophilic molecules across the hydrophobic membrane core. They even display the principal features of carrier-mediated transport, including saturation, mutual competition, and countertransport. Higher concentrations of cytoplasmic binding proteins are associated with more rapid cytoplasmic transport of longchain fatty acids. Available data suggest that substantial intracellular concentration gradients of fatty acids should exist, and that these gradients may help determine which metabolic pathway the fatty acid enters. Thus, cytoplasmic carrier proteins may help regulate the uptake and metabolism of fatty acids and other lipid molecules. Copyright © 1996 Elsevier Science Inc. COMe BIOCHEMPHYSIOL115B;3:319-331, 1996. KEY WORDS. Diffusion, amphipathic molecules, organic anions, fatty acids, fatty acid binding protein, ligandins, bile acid bindin proteins, carrier mediated transport, saturation, competition, countertransport

INTRODUCTION

T h e selective transport of small molecules is among the most fundamental functions of living cells. Selectivity usually reflects specific carrier proteins or channels in the plasma membrane that regulate the membrane's permeability for their substrates. Not all molecules require carriers to cross the plasma membrane, however. Sufficiently hydrophobic molecules can penetrate the membrane core without help, but have much greater difficulty crossing the aqueous layers on either side of the plasma membrane. For many such molecules, transport across these water layers may restrict the transport rate under physiologic circumstances. In response to this limitation, organisms have evolved aqueous carrier systems (the soluble binding proteins) that catalyze the transport of poorly soluble molecules across these water layers. This paper will discuss these transport processes and how soluble carrier systems may influence the observed uptake kinetics. I will show that plasma and cytosolic binding proteins represent true carrier systems, exAddress reprint requests to: Dr. Weisiger, Department of Medicine and the Liver (;enter, University of California, San Francisco, CA 94143-0538 U.S.A. Tel. 415-476-6422; Fax 415-476-0659; E-mail: [email protected] Received 26 May 1996; accepted 31 May 1996.

hibiting all the features of carrier-mediated kinetics. Accordingly, cytoplasmic carriers may regulate certain metabolic pathways for lipids and other amphipathic molecules.

DISCUSSION

Small molecules fall into three basic categories: hydrophilic compounds (e.g. carbohydrates and inorganic ions), hydrophobic compounds (e.g. triglycerides and cholesterol esters) and amphipathic compounds (e.g. molecules with both hydrophilic and hydrophobic portions such as fatty acids, bile acids, phospholipids, bilirubin, thyroid and steroid hormones). Most cellular lipids are amphipathic.

Barriers to Uptake Movement of molecules into and out of cells typically occurs by diffusion, a type of random motion driven by thermal vibrations. For hydrophilic molecules, the lipid core of the membrane represents a free energy barrier (Fig. 1). In order to cross the membrane, enough energy must be available to get across the free-energy barrier to the other side. By providing aqueous channels through membranes, mere-

320

R.A. Weisiger

Biological Compounds with Limited Solubility

Plasma Membrane O,--,v----,..4D Aqueous cytoplasm

Hydrophobic molecule

Amphipathic molecule ~0 Hydrophilic molecule ..A

Uptake FIG. 1. Barriers to uptake of small molecules by cells. Mole. cules are most soluble when their free energy of solution is low (vertical axis). Free energy barriers occur when the molecule must move into an area of low solubility where the free energy is high. Uptake of molecules into the cell is prevented whenever the molecule has insufficient thermal energy to climb the free energy barrier (portions of curves with rising slope). The membrane core represents a free en. ergy barrier to uptake of hydrophilic molecules (bottom curve), but not to hydrophobic molecules (top curve), which are much more soluble in lipid. Amphipathic molecules prefer to bind to the membrane surface, where their lipid portion can interact with the membrane core and their hydrophilic portion can interact with solvent water. The major barrier to uptake of amphipathic and hydrophobic molecules is often dissociation from membrane or protein binding sites and diffusion across aqueous layers.

brane carrier proteins reduce the free energy needed to cross the membrane (113,114), thus catalyzing the uptake rate (97). In some cases, the thermal energy may be augmented by other energy sources (active transport). In contrast, many lipid molecules are more soluble in the core of the membrane than in water (Fig. 1). For these molecules, transport across water layers may be much slower than transport through the plasma membrane (4,11,110, 113). The height of the free energy barrier generally reflects the number of hydrogen bonds in the solvent water that must be broken when the lipid molecule is dissolved. In other words, the surface tension of water tends to exclude amphipathic molecules on a molecular scale just as oil drops are excluded from water on a visible scale. By reducing aqueous solubility, these energy barriers restrict movement of amphipathic molecules through water layers in the cytoplasm.

Many biologically important compounds are only sparingly soluble in water. For example, the solubility of long chain fatty acids at physiologic pH is <10/aM (74), while that of bilirubin is < 1 nM (14). Other amphipathic molecules with low solubilities include phospholipids (118), hydrophobic bile acids (85), retinoids (9), CoA-esters (15), cholesterol (58), thyroid and steroid hormones (12), and a wide variety of exogenous drugs and toxins collectively referred to as "organic anions." The liver maintains efficient fluxes of these compounds despite their low aqueous solubilities. The input of energy required for an amphipathic or hydrophobic molecule to dissolve in water can be reduced by soluble binding proteins. These aqueous carriers provide mobile hydrophobic binding sites that allow arnphipathic molecules to penetrate into and across aqueous layers that would otherwise block transport (110). This function has been compared to "boats" (the binding proteins) carrying non-swimming passengers (the amphipaths) across a river (the water layer) (72). Cytoplasm contains a large number of soluble binding proteins. These include fatty acid binding protein (FABP) (10), bile acid binding proteins (101), retinol binding proteins (9,75), phospholipid binding proteins (118), thyroid hormone binding proteins (7,36), cholesterol and sterol binding proteins (58,91), heine binding protein (57), tocopherol binding protein (105,117), and a diverse group of glutathione S-transferases with overlapping specificities (48). is THE CELL "WELL-STIRRED"?The living cell is often viewed as a tiny bag of water containing dissolved molecules and suspended organelles. As such, the cytosol is commonly treated as a homogeneous pool, or compartment. Thus, we speak of "the" cytoplasmic pH and "the" cytosolic concentration of calcium, glucose or ATP as if these values must necessarily be identical everywhere within the cell. This ideal case may be termed the "compartmental cell" model. If one accepts this view, then the function of cytoplasmic binding proteins is unclear. They can have no role in cytoplasmic transport because the concentration of their ligands is already uniform (by definition). Proponents of this view have proposed that binding proteins act as buffers to absorb sudden influxes of their ligands, or that they help prevent toxicity caused by binding of organic anions to membranes. However, binding proteins can provide at best only shortterm protection until their binding capacity becomes saturated. For example, the normal uptake rate of fatty acids is sufficient to saturate all of the fatty acid binding protein in the liver in about 1 minute. Thus, the buffering capacity of soluble binding proteins is limited. If, however, the cytosolic concentration of small molecules is not uniform, then another function of these proteins is evident: They ferry molecules through the cytoplasm that would otherwise become immobilized by binding to cellular organelles.

Cytoplasmic Transport of Lipids

321

Mechanisms of Intracellular Transport Small molecules move through cells by three basic mechanisms: passive self-diffusion, convection, and active pumping. Convection, also known as bulk flow, is important only in certain cell types. These include cells with very active cytoplasmic streaming (e.g. amoebas and other cells that move by amoeboid motion), and cells with a substantial flow of cell water from one pole to another. A n example of the latter is the renal tubular cell (53). Because this flow is not turbulent, it does little to mix the contents of the cell. Instead, bulk water flow displaces any existing concentration gradients and mobile organelles in the direction of the flow (53). Active transport may occur when a "molecular motor" (kinesin) attached to a membrane vesicle or organelle moves through the cytoplasm following a cytoskeletal microfilament using a ratchet mechanism driven by hydrolysis of A T P (21,23). The latter mechanism is well developed in neurons, whose axons are far too long to rely on simple diffusion for transport (2), and is also found in liver where it is used to deliver certain membrane vesicles to the cell surface (69).

Cytoplasmic Diffusion Rates Diffusion is the primary mechanism of cytoplasmic transport in most cells. It occurs by random motion of molecules (i.e. Brownian motion), which is driven by thermal vibrations. According to Fick's law of diffusion, the diffusional flux J is dependent on the concentration difference AC, the diffusion constant D, and the distance x. J -

/ICD

(1)

X

T h e diffusion constant D is a measure of the mobility of molecules in solution. It is determined by properties of both the molecule and the medium. Thus, the diffusion "constant" for a molecule will be different in different media or for different conditions. According to the Stokes-Einstein equationh 1. D is inversely related to molecular diameter 2. D is proportional to the absolute temperature (°K) 3. D is inversely proportional to the viscosity of the medium

appears to display if one assumes that the cytoplasm is homogenous. While this approach is useful, Fick's law greatly oversimplifies the situation in cytoplasm. Most importantly, the high solute concentrations and small volumes invalidate certain statistical assumptions upon which it is based (1). Until a more complete theory of cytoplasmic diffusion is developed, Fick's law will continue to be used by most investigators with the knowledge that it is only an approximation.

Relative Rates of Cytoplasmic Diffusion If cytoplasm were merely a dilute aqueous solution, cytoplasmic diffusion rates would be similar to those in water. In fact, cytoplasmic diffusion rates for lipids are typically one to four orders of magnitude slower than in water (Table 1). This means that a liver cell with a diameter of 20/am will behave (in diffusional terms) as if it were a sphere of water from 200 mm to 2 cm in diameter. Aqueous diffusion barriers of this size commonly produce significant concentration gradients (8). Thus, we should not be surprised to find cytoplasmic concentration gradients for rapidly transported or metabolized compounds. Another way to characterize cytoplasmic transport rates is by how long it takes, on average, for a molecule to diffuse across the cell. This characteristic "relaxation time" is approximately x"/D~,, where x is the cell diameter. For a 20/am liver cell, the relaxation time varies from less than l s for water (31 ), to around 200 s for palmitate (38). Of course, pahnitate does not need to cross the entire liver cell to reach its site of initial metabolism on the endoplasmic reticuhun. Thus relaxation times, ahhough more intuitive than effective diffusion rates, may be misleading.

Permeability of Cytoplasm Like membranes, cytoplasm has a permeability P~, which reflects how rapidly a given molecule can penetrate it. The permeability of the cytoplasm is inversely related to the distance x that the transported molecule must cross to reach its target. Dde, p, -

(2) X

T h e diffusion constant of a molecule in cytoplasm is always lower than the comparable value in water due to greater viscosity, tortuosity and binding to cytoplasmic organelles. Because these properties are poorly defined in most cases, we lump them into the effective diffusion constant, D,.,. Thus, D~,~ is the diffusion constant that the molecule /The Stokes-Einstein equation is D = RT/6a'rr/A. It defines the diffusion constant D of a molecule as a function of the Boltzman constant R, the absolute temperature T, its r;adius of gyration r, the viscosityof the solvent I/and Aw~gadro'snumber A. For globular molecules, r is proportional to the cube ro.t ~,f the molecular weight.

We are used to thinking of the plasma membrane as the primary barrier to transport of molecules into and across cells. This is true, however, only when the permeability of the plasma membrane (P=,) is less than the permeability of the cytoplasm (Pro < Pc). On the other hand, if P, << Pro, then the plasma membrane is not a significant barrier to uptake. Instead, the uptake rate is determined by the rate of m o v e m e n t through the cytoplasm. This possibility needs to be considered when interpreting uptake data. As an example, consider a liver cell transporting bile

322

R.A. Weisiger

TABLE 1. Effective diflhsion rates for s o m e molecules in water and tissues* Molecule

Actin Actin Albumin Albumin Albumin Albumin Albumin Albumin Albumin Albumin Albumin Albumin Albumin Apoferritin Carboxyfluorescein Carboxyfluorescein Dextran (68 kDa) Fluorescein Hemoglobin Insulin Myoglobin Ovalbumin Palmitate Protons (hydronium) Protons (hydronium) Protons (hydronium) Stearate-NBD Tri-iodothyronine (T3) Tubulin Water (self-diffusion) Water (self-diffusion)

Deft cm2/s x 10 -s

10-50 0.3 1 29 40 51 60 63 66 88 0.6 1.7 9.2 1.6 1 2 2 0.33 6

0.9 50 3.5 0.2-0.6 100-200 140 9500 0.3-0.5 3 7.5 570 1940

Tissue

amoeba cytoplasm chicken gizzard cell fibroblast 15% albumin solution amoeba cytoplasm 5% albumin solution rabbit ear interstitium dilute water solution dilute water solution dilute water solution chicken gizzard cell fibroblast sea urchin oocyte fibroblast fibroblast liver liver liver erythrocyte fibroblast myocyte human macrophage liver muscle neuron dilute water solution liver liver sea urchin oocyte duck embryo dilute water solution

Method

Ref.

FRAP FRAP FRAP modeling FRAP modeling FRAP modeling lag time light scattering

(107) (51 ) (116) (22) (107) (22) (16) (22) (59) (28)

FRAP

(51 )

FRAP FRAP FRAP FRAP FRAP FRAP FRAP NMR FRAP spectroscopy FRAP MID modeling pH lag time lag time FRAP MID FRAP NMR NMR

(43) (90) (43) (43) (56) (81) (56) (52) (43) (46) (107) (38) (41) (3) (59) (67) (66) (90) (17) (17)

*Most studies were done at 25°C, but range from 18-37°C. Consult original referencesfor details. Table reprinted from (111).

acids from the plasma membrane on one side to the bile canaliculus on the other. If the permeability of the plasma membrane is much less than that of the cytoplasm, then transport across the plasma membrane will be rate limiting and no cytoplasmic concentration gradient will form (Fig. 2, top). If, however, Pm >'> Pc, then a large concentration gradient will form at steady state in the presence of an effective biliary secretion mechanism (Fig. 2, bottom). In the latter case, the concentration of the transported molecule in the layer of cytoplasm adjacent to the plasma membrane will increase until the rate of uptake into the cell (influx) approaches the back flux from the cell (efflux). By raising concentrations in the surface cytoplasm, slow cytoplasmic transport causes most molecules to efflux from the cell before they can be metabolized or excreted (arrows). VARIETIES OF CYTOPLASMICDIFFUSION. Depending on their physical and chemical properties, small molecules may diffuse through the cytoplasm while dissolved in the aqueous phase, while bound to soluble proteins, or while partially or completely bound to membranes. Diffusion of the

membrane-bound form may occur by lateral diffusion of the molecule within the membrane (94) or by diffusion of membrane vesicles with their bound ligands (88). Vesicles may also be actively transported through the cytoplasm by molecular motors that are driven by hydrolysis of ATP (23). The effective cytoplasmic diffusion constant, Doe, is the sum of the contributions of all of these pathways after weighing for the fraction of the molecule in each phase) Table 2 lists the major mechanisms of cytoplasmic transport with their approximate rates.

Factors That Influence Cytoplasmic Diffusion A number of factors modulate the diffusional flux of molecules within cells. These include tortuosity of the diffusional path, cytosolic viscosity, binding of the molecule to

2Mathematically, D~ = Y_,i'X,D,where Xi is the fraction of the molecule in that phase (e.g. bound to membranes)and D~is the diffusion constant of that form of the molecule within the cytoplasm(67).

Cytoplasmic Transport of Lipids

Blood

323

Bile

the composition of the cytoplasm. This approach is used because D~u is an experimentally measurable value, while other features of cytoplasm such as tortuosity and viscosity are difficult to quantitate.

Restricted Mobility Due To Cytoskeleton Cytoplasm contains a lattice of cytoskeletal filaments that significantly alters the solvent properties of cytoplasmic water (18,61,63). These filaments make up 16-21% of the volume of a typical cell and have a surface area of -19,000 cm z per ml (29). This network further restricts the diffusional mobility of large molecules by molecular sieving (60,61) and of many soluble molecules by reversible (e.g. Van der Waals) binding interactions (29,43). In addition, sizedependent molecular sieving is important for larger molecules,completely immobilizing molecules larger than about 520 A in diameter (60,61).

Viscosity and Molecular Crowding

FIG. 2. Effect of cytoplasmic transport rate on bile acid secre. tion. When membrane transport is rate limiting (top panel), efflux from the cell is minimal (small arrow) and most molecules reach the bile canaliculus (right) before they efflux from the cell. When cytoplasmic transport is slow, however, efflux nearly equals influx and less bile acid reaches the canaliculus before effluxing. Biliary excretion removes bile acid near the canaliculus faster than it can be replenished by transport, causing cytoplasmic concentration gradients. Pm = permeability of membrane. P~ = permeability of cytoplasm.

membranes and proteins, molecular crowding, and sizedependent sieving by cytoskeletal filaments. TORTUOSITY. Cells contain many membranous and fibrillar obstructions that increase the diffusional path and thus reduce the diffusional flux. For example, liver cytoplasm contains ~10,000 cm 2 of membranes (1 square meter) per ml of cytoplasm (108). Most cytosolic molecules are unable to pass through these obstructions but must follow aqueous channels around them. It has been estimated that this increases the length of the diffusional path by a factor of 5 or more (17,81,87). The effect of tortuosity can be incorporated into Eq. (1) as a larger value of the distance x, but is more commonly used to reduce the value of D, which thus becomes the effective diffusion constant D~-. This value is thus not a constant at all, but varies according to

Overall, cytoplasm has the consistency of a viscoelastic gel with bulk viscosity several thousand times that of water (61,64,70). The gel is created by cytoskeletal fibers, which make up a microtrabecular lattice (93). The viscosity of the cytosolic solution between these fibers is not much greater than water (47,62). The bulk viscosity of the gel appears regulated in part by pH and intracellular Ca ++ concentrations with maximum viscosity at physiologic values (61), although measurements in purified cytosol may not be representative of living tissue (18,62). A major portion of cell water is tightly bound to cytoplasmic membranes and proteins, thus limiting its mobility and further reducing cytoplasmic diffusion rates (17,61,63). The cytosolic protein concentration is 15-26% (27), suggesting there should be significant hydrodynamic, stearic and electrostatic interactions among dissolved proteins that should further limit their mobility (63). Because little water is free, small changes in cytoplasmic water content can produce dramatic changes in cytoplasmic viscosity and D,:,:f values (40,43, 47,70,80). These data suggest that diffusion of aqueous molecules should be dramatically slower in cytoplasm than in free solution. Using fluorescent dextrans, Peters estimated that cytoplasmic diffusion of hydrophilic molecules in liver cells is approximately 20 times slower than in water (81). Because amphipaths often bind extensively to intracellular membranes (20,58,67,101) their diffusion through cytoplasm should be slower than for comparably sized dextrans. Although energy-dependent vesicular transport pathways also exist (5,24,77), vesicular transport is relatively slow. For amphipathic molecules with cytoplasmic binding proteins, this mechanism may become important only at higher amphipath concentrations when the binding capacity of soluble proteins becomes saturated (25).

324

R.A. Weisiger

TABLE 2. Types of cytoplasmic transport*

Type

Mechanism

Rate

Ro~ ~r bmdmg proems

I

Aqueous diffusion of unbound form

Very fast D~ ~10 6 cm2/s

II

Aqueous diffusion of protein-bound form

Fast D~, <10 7 cmZ/s td((¢>10 s

Yes

III

Vesicular transport

--

IV

Lateral diffusion of membrane-bound form

V

Convection

Slow D~f~~ 10 -9 cmZ/s tdi, ~ 1000 s Very slow D~ ~ 10 lo Cln2/s td,ff ~10,000 s Variable

Effect of cytoskeletal

inhibitors

--

tdi(~0.1 - 1 s

-Yes

Inhibition if driven by kinesin --

Examples

Glucose ATP Potassium Bilirubin Fatty acids Calcium Bile acids ICG High conc. bile acids ICG plus colchicine

Inhibition

*The five major forms of cytoplasmic transport are listed along with order-of-magnitude estimates of their diffusion rates (D~u)and the approximate time required to diffuse across a liver cell. Table modified from (111).

Driving Forces for Cytoplasmic Diffusion Concentration gradients are inherently unstable) In the absence of energy input, they decay toward equilibrium. Thus, diffusive fluxes must in all cases reflect input of free energy. Driving forces may be generated within the same cell, such as by active transport of a molecule across the cell plasma membrane. For example, bile acids are actively pumped into liver cells across the basolateral membrane (89) while many organic anions and cations are actively pumped out of liver cells across the bile canalicular membrane (119). In each case, cytoplasmic concentration gradients may be created and sustained by the pumping mechanism if it is sufficiently rapid. In addition, gradients may be created within a cell by metabolic utilization of a substrate within the same cell (35) or in a different cell that may be adjacent or located remotely. Thus, rapid utilization of long chain fatty acids by heart muscle may generate diffusion gradients across the endothelial cells that line the cardiac capillaries (32).

Cytoplasmic Diffusion of Amphipathic Molecules Amphipaths are molecules with detergent properties, containing both hydrophobic and hydrophilic domains. As such, they tend to bind to membranes (104) and to various soluble binding proteins and have only limited solubility in water. Amphipaths are a very broad class that includes not only physiologic molecules such as long chain fatty acids, cholesterol, bilirubin, thyroid and steroid hormones, but ~More precisely, it is activity gradients that are unstable. The activity of a molecule in solution is equal to its thermodynamic free energy. In must cases, this value is proportional to the concentration. However, binding of the molecule to membranes or proteins may alter activity, as discussed in (110).

also exogenous drugs and toxins collectively referred to as organic anions and organic cations. Although convection and vesicular transport may contribute significantly to cytoplasmic transport of some amphipathic molecules (5,19, 24,37), most cytoplasmic transport is believed to occur by diffusion of the molecule while it is bound to cytoplasmic binding proteins such as ligandin and fatty acid binding protein (30,73,104). A role for soluble binding proteins in transport has long been postulated (71,73,96,104). Although these basic ideas were first proposed more than 15 years ago (73) and have gained wide support (95,96,101), no adequate methods were available to investigate cytoplasmic transport in living cells until recently. T h e idea that binding of a small molecule to a larger one can increase the diffusion rate in cytoplasm is counterintuitive. Normally, increasing the size of the diffusing species should decrease diffusion. However, amphipathic molecules spend only a small part of the time in the cytosol, often binding extensively to intracellular membranes, which are themselves essentially immobile (104). Because lateral diffusion within membranes is very slow (42), membranebound organic anions contribute relatively little to the diffusional flux in most cases. Diffusion rates should thus be increased by cytoplasmic binding proteins, which reduce the fraction of the organic anion in the relatively immobile membrane-bound pool (104). These predictions have been confirmed by recent studies showing that organic anions and other amphipaths diffuse through hepatic cytoplasm very slowly. Effective cytoplasmic diffusion constants range from - 3 × 10 s cmes 1 for tri-iodothyronine (66) to - 3 × 10 9 cme.s-i for a stearate analog (67) and for fluorescein (56). These values are

Cytoplasmic Transport of Lipids

325

TABLE 3. Effect of binding protein on distribution and mobility of fatty acids Relative amount of binding protein

Cytosolic fatty acid (percent)

D~ (cm 2 s -1)

100% 200%

18.2 +- 2.7 35.1 _+ 7

3.05 +_ 0.21 x 10 '~ 5.03 _+ 0.37 x 10 ~

Male Female Data are from reference(67).

2-3 orders of magnitude smaller than for the unbound anion in free water, and correspond to half-times for cytoplasmic equilibration of more than one minute. Hydrophobic molecules take longer to reach the bile than their more hydrophilic brethren (6), presumably reflecting more extensive binding to cytoplasmic membranes. Higher levels of binding proteins help prevent membrane binding. As shown in Table 3, diffusion constants for a fluorescent fatty acid were nearly twice as fast in female than in male hepatocytes (67) as expected from the fact that female cells contain substantially more fatty acid binding protein than male cells (76). Indeed, a plot of the intracellular mobility as a function of the cytosolic fraction is essentially linear (Fig. 3). This is the pattern expected if most intracellular transport is due to diffusion of protein-bound molecules.

Kinetics of Cytoplasmic Transport Cytoplasmic transport of amphipathic molecules displays saturation, mutual competition between similar substrates, and apparent countertransport (Fig. 4). These are the principal features of carrier-mediated transport, indicating that cytoplasmic binding proteins form a true carrier system Relative Mobility 6

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Cytoplasmic Concentration Gradients Caused by Metabolism Concentration gradients caused by transport of a molecule across cells were discussed earlier (see Fig. 2). However, concentration gradients may also occur due to rapid metabolism of the transported molecule. These gradients are produced when a metabolic pathway consumes the fatty acid more rapidly than it can be replaced by cytoplasmic transport. Examples of molecules with low aqueous solubility and rapid metabolic rates include long-chain fatty acids (67) and oxygen (35,45). In each case, cytoplasmic transport appears to depend on the carrier function of binding proteins: fatty acid binding protein (FABP) in the former case (112), and myoglobin in the latter (35). Figure 5 depicts the shape of the intracellular concentration gradient expected for palmitate within a liver cell. Note that fatty acid concentrations are approximately 3fold higher in the periphery of the cell than near the center. If the enzymes responsible for fatty acid metabolism are unevenly distributed within the cell, the shape of these concentration gradients may help determine the fate of the fatty acid.

Methods for Observing Cytoplasmic Transport

| |

. ,

i I

t

i~. - I J ~r . . . . . . . . . . . .

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(112). The Michaelis-Menten V ...... of the cytoplasmic carrier system is proportional to the concentration of the cytoplasmic binding protein, just as the V ...... of a membrane carrier is proportional to the number of carriers. The Km of the system reflects the relative affinity of the transported molecule for the binding protein and for membranes, but is not a simple function of these parameters due to regional variation of substrate concentration across the cell.

.l

0.35

S o l u b l e F r a c t i o n in C y t o s o l

FIG. 3. Cytoplasmic mobility is proportional to the soluble fraction. Cytoplasmic binding proteins increase the soluble fraction of amphipathic molecules in cytoplasm by reducing their binding to membranes. Because the soluble fraction is more mobile, binding proteins also increase cytoplasmic mobility. Data are for a fluorescent stearate analog and have been replotted from their original sources (65,67).

DIRECT METHODS. Molecules with intrinsic fluorescence or molecules that alter the fluorescence of other probes may be observed by several techniques. All are based on following the relaxation of an experimentally created concentration gradient with time. Stock and coworkers studied transport of fluorescent dextrans (18-157 kDa) across perfused liver cells by intravital microscopy (100). They found that the major resistance was not entry into the liver cell, but rather intracellular transport. Although not specifically commented on by the authors, this transport pathway presumably involved vesicles containing the probe that had

326

R.A. Weisiger

1.0

Saturation

~,2.5

cO

=m

E

¢¢

E 2

L_ 4.1

c-

1.5

g •e

O t0.5 O Ca

1

o

>

>

~0.5

=m

t~

Go 0

,,•

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....

I ....

10 15 20 25 30 35 40 Concentration (mM)

.

No inhibitor

?, 1 F F "-

O0O

- vit. iohibito 2 raM)

. 0.2 Il , , , - 0.4 , i , , , ,0.6 t,., 18. , , 1 1/Concentration (mM -1)

Added unlabeled substrateh~

o.6 0.4

•-

gOo -~-

>

°2I -0.4

-0.6 -0.8

0

5 from

cell s u r f a c e

10 (pm)

FIG. 5. Concentration gradients of palmitate within a liver ceil. Rapid hepatic metabolism of palmitate combined with relatively slow cytoplasmic transport should produce sub. stantial fatty acid concentration gradients within liver ceils. The shape of these gradients may direct fatty acids into dif. ferent metabolic pathways. Thus, metabolic systems near the cell periphery should be exposed to the highest concen. trations of fatty acid, while those more centrally located would see lower concentrations. For this computer simulation the rate of metabolism (0.7 min -l) was taken from reference (67) while De~ (4 x 10-9 c m Z / s ) w a s taken from (38). These values are assumed to be the same everywhere in the ceil. A spherical geometry was assumed such as would be expected for a suspended hepatocyte. Because not all exterior surfaces are exposed to plasma in the intact liver, a more complex geometry would be expected/n vivo. Non-saturat. ing concentrations of palmitate were assumed.

Countertransport

0.8

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-500

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500

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1000

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1500

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2000

Time (ms) FIG. 4. Cytoplasmic diffusion of amphipaths is carrier.

mediated. Diffusion of an amphipathic molecule across the plasma membrane and a 10.~m layer of cytoplasm was sim. ulated using a computer (112). The model shows three of the principal features of a carrier system, saturation, compe. tition and apparent countertransport. Concentrations and velocities shown are arbitrary, resulting from the choice of conditions. Saturation occurs when the capacity of the solu. ble binding proteins is exceeded, causing subsequently added molecules to bind primarily to immobile membranes.

entered by fluid-phase endocytosis (54,92). Direct microinjection of fluorescent probes into cells can be used (116), although this may create local anomalies in cytoplasmic composition. Jiirgens and coworkers used heine absorbance to show that the diffusion constant of myoglobin in rat diaphragm is - 5 × 10 -7 cme/s (46). However, the most commonly used method of direct observation is fluorescence repolarization after photobleaching (FRAP). FRAP uses a highly collimated laser beam to generate transient concentration gradients within cells. A highintensity beam is first used to irreversibly bleach the dye in one region of the cell (Fig. 6), and then low-intensity illumination is used to determine how rapidly the gradients dissipate due to diffusion of unbleached dye from the periphery (13,43,56,60,67,81,116). In most cases, cells have been previously loaded with the fluorescent molecule by preincubation or microinjection. With use of appropriate filters on the laser and photomultiplier, most common fluorescent molecules are suitable. Temperature and CO2 levels are typically physiologic. FRAP has numerous advantages for studying intracellular transport:

Cytoplasmic Transport of Lipids

327

1. It is direct and model-independent, requiring few assumptions about the transport system. 2. The portion of the cell cytoplasm to be studied can be selected. 3. Studies are performed in a single cell rather than averaging results over many cells, allowing cell heterogeneity to be investigated. 4. Multiple studies can be performed on a single cell or coverslip of cells. 5. Convection can be distinguished from diffusion. 6. Multiple intracellular pools can be detected (if present), providing their diffusion rate constants are sufficiently different and they do not rapidly interconvert.

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FRAP has been used to measure the intracellular mobility of proteins (43,44,50,116), calcium (13), dextrans (81), ficoils (60,63), fatty acids (44,65,67), fluorescein (56), carboxyfluorescein (56) and other molecules (43,47). In all of these studies, it was found that cytoplasmic diffusion is much slower than comparable diffusion rates in free solution, and that amphipathic and hydrophobic molecules diffuse through cytoplasm less rapidly than hydrophilic molecules. INDIRECT METHODS. Indirect methods infer cytoplasmic transport rates by measuring rates in vitro and then using

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1. Molecules to be studied must have suitable fluorescence properties. 2. A method for getting the molecule into the cytoplasm must exist. 3. The probe must not be metabolized or excreted by the cell before the photobleaching study is complete. 4. Addition of the fluorescent side group may alter the properties of the molecule.

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FIG. 7. Effect of slow cytoplasmic transport on efflux curves. The curves show the concentration in the effluent of a tissue perfused single.pass with buffer after being pre-loaded with a radioactive indicator. Left panel: Fast cytoplasmic trans. port. A single exponential is seen that reflects the permeability of the plasma membrane. Right panel: slow cytoplasmic transport. Initial efflux rates are limited by the plasma membrane, but the efflux rate slows later as cytoplasmic gradients develop. The final efflux rate reflects both membrane a n d cytoplasmic permeability. The rate of cytoplasmic transport c a n be c a l c u l a t e d from fitting an appropriate model to t h e data.

models to extrapolate to expected rates in vivo (26,39,82, 87,99). Alternatively, the rate of intracellular transport can be estimated from analysis of overall rates of uptake and/or efflux for an experimental system (33,34,45,98,106). As an example of the latter, we consider efflux from a tissue that has been uniformly loaded with a radioactive tracer. If cytoplasmic transport is rapid and perfusion even, the concentration in the effluent will be a simple exponential with time (Fig. 7, left panel). In contrast, if cytoplasmic transport is slow, 4 the curve will be nonlinear (Fig. 7, right panel). The nonlinearity develops when the rate of efflux from surface layers of cytoplasm exceeds the rate at which the radioactive molecule can be replenished by diffusion from deeper layer of cytoplasm. Unfortunately, this approach is sensitive to uneven perfusion of the tissue: Poorly perfused regions can simulate slow cytoplasmic transport by releasing their radioactivity back into the perfusate slowly, thus invalidating the approach. This problem can be largely overcome by using the multiple indicator dilution (MID) technique. This method is similar to that shown in Fig. 7, except that the tissue is not uniformly loaded at the start of the experiment but is instead perfused with a bolus of the radioactive tracer. Effluent radioactivity is expressed relative to a non-transported indi4In this context, "slow" means that the characteristic time for equilibration (x:/D) is longer than or comparable to the characteristic time fi~r removal by all other transport processes (membrane transport, plasma flow, etc.). These times are inversely related to the rate constant fi)r each step as is raore fully discussed in (109).

328

cator, which is used as a reference. Use of a reference makes the method insensitive to uneven tissue perfusion. Although it is less direct, the MID method (68) has the distinct advantage that it can be used with any molecule that can be obtained in radioactive form. Unlike FRAP, radioactive labeling of the molecule does not usually change its physical properties. Using this approach, the values for D~ffof tri-iodothyronine (66) and palmitate (38) were found to be 3 x 10 -s cm2/s and 2-6 x 10 -9 cm2/s, respectively. Importantly, the values obtained for palmitate by the MID method closely match the values obtained for NBD-stearate by the FRAP approach, thus helping to validate both methods. Luxon has further demonstrated that D~f~can be markedly reduced by displacing fatty acids from their cytosolic binding proteins (65). A reduction in D~ may explain why indomethacin, which blocks binding of taurocholate to its cytoplasmic binding protein, both delays and reduces biliary bile acid excretion in the perfused rat liver (103). The MID approach has also been used by Rivory and coworkers to estimate the cytoplasmic diffusion rate of water and certain lipophilic drugs in perfused liver (83,84). Cytoplasmic transport can also be inferred from fixation of tissues after brief exposure to labeled molecules (102). Cytoplasmic transport of hydronium ions (hydrated protons) has been measured from the lag between change in pH at two different sites in a giant neuron (3), while transport of water has been estimated by NMR techniques (49,55,87,115).

SUMMARY

Diseases Caused by Disordered Cytoplasmic Transport Various diseases have been shown to be caused by abnormal function of membrane carriers (e.g. cystic fibrosis). Thus, we would expect to find diseases associated with abnormal levels or functions of cytoplasmic carriers as well. NiemannPick type C is a disease in which cholesterol accumulates in lysosomes apparently due to lack of effective cytoplasmic transport (79). Although sterol carrier protein 2 (SCP2) levels are reduced in this disorder (86), it is not known if this is the cause of the disorder, or if some other cytoplasmic transport defect is responsible. A recent paper describes vitamin E deficiency due to deficient levels of cytoplasmic tocopherol binding protein (78). Other examples likely exist, but have not been looked for. In the liver, defective cytoplasmic transport might manifest as intrahepatic cholestasis or decreased hepatocellular function. It is tempting to speculate that the hepatic dysfunction seen in steatosis and other diseases associated with accumulation of intracellular debris (e.g. "storage" diseases) might in part reflect the greater size of affected liver cells and the extra impediment to diffusion caused by the cytoplasmic inclusions. Such changes might interfere with cytoplasmic transport not only for amphipaths, but also for essential energy substrates and information-carrying molecules. Further research to test this hypothesis is needed.

R.A. Weisigcr

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