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Published December 8, 2014 Characterization of transport systems for cysteine, lysine, alanine, and leucine in wool follicles of sheep N. Thomas,* D. R. Tivey,† N. M. Penno,† G. Nattrass,‡ and P. I. Hynd†1 *Discipline of Molecular and Biomedical Science, University of Adelaide, Adelaide, South Australia 5001; †Discipline of Agricultural and Animal Science, University of Adelaide, Roseworthy Campus, Roseworthy, South Australia 5371; and ‡South Australian Research and Development Institute Livestock Systems, Roseworthy Campus, Roseworthy, South Australia 5371 ABSTRACT: Aspects of the uptake of the AA Cys, Leu, Ala, and Lys into wool follicles were investigated using short-term culture of thin strips of sheep skin. Following verification of the reliability of the model system, the sites of uptake of the radiolabeled AA were shown to differ and to be consistent with their different roles in fiber production. Cysteine appeared in the zone of keratinization immediately distal to the follicle bulb. Lysine was incorporated into the germinative cells of the follicle bulb and the cells of the inner root sheath. Leucine and Ala were incorporated into the follicle bulb, inner root sheath, and keratinizing fiber. The incorporation of all AA into the dermal papilla was low. The relative rates of uptake of the AA into the wool follicle were as follows: L-Cys (100), L-Leu (5.5), L-Ala (2.5), and L-Lys (0.8). Uptake of Cys was saturable and followed Michaelis-Menten kinetics, suggesting a carrier-mediated system, with little or no diffusion. The majority (70%) of Cys uptake into follicles was via a Na-independent system that was not inhibited by α-(methyl- amino)isobutyric acid or 2-amino-2-norbonanecarboxylic acid and therefore is not via the normal Cys transport systems A, ASC, or L. Uptake of Cys appeared to be via a low-affinity, high-capacity transport system, which may be unique to the fiber-producing follicle. The majority of Ala transport had characteristics consistent with the functioning of system A (Na-dependent, inhibited by α-(methylamino)isobutyric acid, and low substrate affinity). Leucine uptake was inhibited by 2amino-2-norbonanecarboxylic acid but was Na-dependent, suggesting that a variant of system L operates in the follicle to transport Leu. Lysine uptake was consistent with the operation of the usual Lys transporter system y+. Diets designed to maximize wool growth should provide AA profiles reflecting the relative rates of uptake demonstrated in this study. Investigations of possible polymorphisms in genes encoding AA transport proteins in follicles may reveal a source of genetic differences in wool growth potential among genotypes. Key words: amino acid, follicle, kinetics, sheep, wool ©2007 American Society of Animal Science. All rights reserved. INTRODUCTION The production of wool and hair fibers in mammals has been a key determinant of their success, allowing greater control over thermoregulation, sexual communication, camouflage, and protection from the physical and chemical environment (Hynd, 2000). The production of fibers from animals also represents a small but economically important component of the global textile fiber market. A unique feature of animal fibers, in contrast to cellulosic fibers such as cotton, is that they largely consist of proteins. Wool fibers, for example, 1 Corresponding author: [email protected] Received August 7, 2006. Accepted May 8, 2007. J. Anim. Sci. 2007. 85:2205–2213 doi:10.2527/jas.2006-541 comprise from 50 to 100 spatially arranged keratins, which are encoded by discrete and exquisitely timed expression of genes within the wool follicle (Powell et al., 1991). The proteins of the fiber are unusual in that they contain from 15 to 37% mol Cys residues (Powell and Rogers, 1994), in contrast to most tissues, which contain from 0.6 to 1.5% mol Cys residues (Beach et al., 1943). Not surprisingly, the rate of fiber production is limited by the supply of Cys and its precursor sulfur AA, Met (Reis, 1979). Lysine appears to influence the rate of cell division in the follicle bulb (Hynd, 1989), but little is known of the role of other AA in wool growth processes. Despite the obvious importance of AA nutrition to fiber production, few attempts have been made to characterize the AA transport systems that operate in the wool follicle to supply the large quantity and unique 2205 2206 Thomas et al. pattern of AA required. It is feasible that the unusual AA demand may have elicited the development, in evolution, of specialized AA transport systems to ensure that the production of fiber, so important to the survival of the animal, is maintained. We examined the sites of uptake of several AA into cultured sheep skin and characterized the kinetics of uptake of these AA. The results support the hypothesis that the wool follicle has developed unique transporter systems for Cys and possibly Leu. MATERIALS AND METHODS The work reported in this paper was approved by the Animal Ethics Committee of the University of Adelaide, South Australia, in compliance with the Code of Practice for the Care and Use of Animals for Scientific Purposes, Sixth Edition, under the Prevention of Cruelty to Animals Act 1985. Collection and Culture of Skin Strips Adult Corriedale sheep (approximately 50 kg of BW) were housed in an animal house in individual pens and fed a maintenance ration of sheep pellets containing 9 MJ of ME/kg of DM and 160 g of CP/kg of DM. On the day of experiments, the midpoint of 1 side of the sheep was clipped closely with small animal clippers (Oster number 40 blade). Several dorsoventral rows of skin (approximately 200 mm long) were injected s.c. with local anesthetic (Lignocaine 2%, Troy Laboratories Pty Ltd., New South Wales, Australia). Five minutes after injection, incisions were made along the injected lines using a double-bladed scalpel, with the blades spaced at 1 mm. The incisions were made to a depth that allowed the skin to be released from the subcutis. Strips were removed using curved forceps to elevate the skin and a pair of surgical-grade iris scissors to remove the skin from the underlying fascia. The strips were blotted thoroughly to remove blood and were placed immediately in Krebs-Ringer phosphate (KRP) buffer (137 mM NaCl, 14.7 mM KCl, 1.2 mM MgSO4.7H2O, 0.3 mM NaH2PO4.2H2O, 14 mM Trizma base, 12 mM HCl, and 5.5 mM glucose; adjusted to pH 7.2) or Na-free phosphate buffer (containing 137 mM choline chloride or CaCl2 in place of NaCl, with all other ingredients as for KRP buffer). In all Na-free treatments, choline chloride replaced NaCl. The strips were transferred as quickly as possible to a humidified incubator at 37°C and 5% CO2. The strip wounds were treated with antiseptic spray [Cetrigen, Virbac (Aust.) Pty Ltd., New South Sales, Australia]. The excised strips were then prepared by cutting them into 10-mm pieces and dissecting immediately below the sebaceous glands and below the follicles (Figure 1). The upper (epidermis and sebaceous glands) and lower (s.c. adipose and muscle) regions were discarded. This created a region of skin rich in the metabolically active regions of the proximal follicle (the germinative bulb, the kera- Figure 1. Schematic diagram of the sheep skin strip preparation for investigations of AA transport. Note the removal of the sebaceous glands, epidermis, and hypodermis togenous zone, the inner root sheath, the outer root sheath, and the connective tissue sheath; approximately 400 follicles/strip) and devoid of other tissues. Cell cluster trays (Costar 24-well plates, Sigma, St. Louis, MO) were prepared with 0.5 mL of buffer, and at time 0, the skin strips were added to the wells. Various times of incubation, concentrations of substrates, buffer types, and inhibitors of specific transporter classes were used in the experiments described below. Viability of Skin Strips To determine the metabolic viability and health of the cultured skin strips, the uptake of [6-3H]thymidine (26 Ci/mmol) into the strips was measured. Skin strips collected as above were incubated in buffers containing 5 Ci/mL of 3H-thymidine for 0, 30, 60, 90, or 120 min. The strips were washed repeatedly as above, blotted dry, weighed, and placed in a scintillation vial containing 0.8 mL of Soluene-35D tissue solubilizer (Packard Instrument Co., Meriden, CT). The vials were incubated at 60°C until the tissue had dissolved completely. Five milliliters of biodegradable counting scintillant (Amersham Corp., Arlington Heights, IL) was added to each vial, and 1 mL of 1.0 M HCl was added to reduce chemiluminescence. The vials were stored overnight in the dark and counted for radioactivity in a liquid scintillation counter (1215 Rackbeta II, LKB Wallac, Turku, Finland). An aliquot of each incubation medium was counted to allow calculation of the specific activity of each labeled solute. The specific activities were then used to convert disintegrations per minute to nanomoles of solute taken up in each incubation period. All values were the averages of 5 replicates, and the amount of substrate taken up by the tissue was expressed as nanomoles per gram × minutes. Determination of the Sites of Uptake of AA into the Wool Follicle One 10-mm long skin strip was placed into each well with 0.5 mL of KRP buffer containing 2.5 Ci of either Amino acid transport in wool follicles 3 H-Ala, 3H-Leu, 3H-Lys, 3H-thymidine, 14C-inulin, or S-Cys. The radiolabeled AA were purchased from Amersham Corp. and were L-2,3-3H-Ala plus 2% ethanol (48 Ci/mmol), L-[4,5-3H]Leu plus 2% ethanol (85 Ci/ mmol), L-35S-Cys.HCl (56.74 mCi/mmol), L-[4,5-3H]Lys.HCl (85 Ci/mmol), and inulin-[14C]carboxylic acid (4.92 mCi/mmol). The incubation in inulin-[14C]carboxylic acid provided an estimation of the nonspecific extracellular entrapment of radioactive label after the washing procedure. All uptake estimates were corrected for this nonspecific entrapment (<0.1% of total). The strips were incubated for 60 min. The cultures were terminated by addition of 0.5 mL of 5% ice-cold trichloroacetic acid (TCA) to each well. The strips were then repeatedly washed (4 times) by transferring them to new wells containing fresh ice cold TCA with 15 min between each wash. Skin strips were then fixed in 10% buffered formalin for 48 h, paraffin-embedded, and sectioned at 8-m thickness in a plane longitudinal to the follicles. The sections were mounted onto TESPA-coated glass slides (Sigma). The sections were deparaffinized and hydrated through decreasing ethanol solutions. For autoradiographic detection of the sites of radiolabeled AA uptake, the slides were dipped in Ilford L4 gel emulsion, air dried, and exposed at 4°C in a lightproof box with silica gel desiccant. The length of exposure for 35S-Cys, 3H-Leu, 3H-Ala, and 3H-Lys was 9, 11, 12, and 21 d, respectively. The slides were then developed in Kodak D19 developer (Eastman Kodak Co., Rochester, NY) for 2.5 min, rinsed in distilled water for 10 to 20 s, and transferred to Kodak T-Fixer (diluted 1:4) for 2 min. The slides were then washed in distilled water for 5 min under a Ilford safelight F904 (Polysciences Inc., Warrington, PA) and then a further 25 min in daylight. After washing, the slides were airdried and stained using the SACPIC protocol (Auber, 1950). Coverslips were then placed on the sections using DePeX mountant (Sigma) and allowed to dry. The slides were viewed under both bright and dark field microscopy, and photographs were taken using Fujichrome 400 film (Fuji Sales, Edison, NJ) or Kodak Pan film. To quantify the relative uptake of labeled AA into various regions of the follicle, photographs of a skin section deemed to be representative of the particular treatment were scanned (Canonscan LiDE25, Canon Australia, Nth Ryde, New South Wales) at high resolution (600 dpi). Scans were made within 4 regions (follicle bulb, dermal papilla, inner root sheath, and keratogenous zone) of the follicle fiber. In addition, a background scan was taken outside of the follicles. The scanned areas were then analyzed for grayscale using an image analysis system (Analysis Five, Olympus Soft Imaging Solutions GmbH, Munster, Germany). The relative intensity of labeling in each region was determined as follows: (mean grayscale value − background grayscale value)/(specific activity of isotope × time of incubation). The units are arbitrary but reflect the relative extent of uptake of the AA into the various regions of the follicle. 2207 Rate of Uptake of AA into Cultured Skin Strips 35 To derive accurate kinetic parameters, it is essential that uptake is relatively constant over the period of measurement. To determine the period in which this assumption holds for the skin strip model, skin strips were incubated as described above in KRP medium for a minimum of 30 min before transfer to similar medium containing radiolabeled AA. The strips were then incubated for 0, 30, 60, 90, and 120 min and repeatedly washed in cold 5% TCA to remove extracellular AA. Radiolabel uptake was estimated as described previously. Michaelis-Menten Kinetics of AA Uptake into Cultured Skin Skin strips were cultured as described above but with addition of varying levels of unlabeled AA (from 0.005 to 15 mM). Addition of up to 15 mM had no effect on the tonicity of the media. The kinetic parameters were evaluated by analysis of the initial rate of uptake data using the program Enzfitter Version 1.0 (BioSoft, Cambridge, UK). The equations used were as follows: (a) for a single saturable system: V = (Vmax × [S])/(Km + [S]); and (b) for a saturable system plus diffusion: V = {(Vmax × [S]) + (Kd [S])}/(Km + [S]), where Vmax = the limiting velocity observed when the system is saturated with substrate; Km = the concentration of substrate at which the reaction rate is half-maximal; V = the estimated initial velocity; [S] = the substrate concentration; and Kd = the diffusion constant (Segel, 1975). The slope of the linear part of the curve approximates Kd (Del Castillo and Muniz, 1991), and the diffusive component at each substrate concentration is obtained as the product of Kd and [S]. The difference between total uptake and the diffusive component was derived as the saturable substrate uptake. Characterization of Transporter Class for L-Ala, L-Leu, L-Cys, and L-Lys Based on Na Dependence and Response to Specific Transporter Inhibitors Several of the AA transport systems can be distinguished by their dependence on Na, response to other competitive AA, and response to specific nonmetabolizable AA analogues. System A, for example, is inhibited by α-(methylamino)isobutyric acid (MeAIB), whereas system L is inhibited by 2-amino-2-norbonanecarboxylic acid (BCH). A combination of treatments was applied to incubated skin strips to define the AA transport systems operating for L-Ala, L-Leu, L-Cys, and L-Lys (Table 1). The concentrations of inhibitors were greater than 15 mM, so the tonicity of the media was kept constant by reducing the concentration of Na or choline chloride accordingly. The strips were incubated for 120 min in the presence or absence of Na, and the uptake of each AA was determined. Effects of other AA on uptake of specific individual AA were determined by adding them 2208 Thomas et al. Table 1. The combination of treatments used to discriminate among AA transport systems operating in the follicle cells of cultured sheep skin Inhibitor1 Na status Substrate2 +Na −Na +Na −Na +Na −Na +Na −Na All AA All AA Ala, Cys, Lys Ala, Cys, Lys All AA All AA Ala, Cys Ala, Cys None None MeAIB (20 mM) MeAIB (20 mM) BCH (20 mM) BCH (20 mM) MeAIB (20 mM) + BCH (20 mM) MeAIB (20 mM) + BCH (20 mM) 1 MeAIB = α-(methylamino)isobutyric acid; BCH = 2-amino-2-norbonanecarboxylic acid. 2 The saturating substrate concentrations used were 10 mM Ala, 3 mM Cys, 2 mM Leu, and 5 mM Lys. at saturating concentrations of 10 mM Ala, 3 mM Cys, 2 mM Leu, and 5 mM Lys. Statistical Analyses All comparisons were made using 1-way (treatment as the factor) or 2-way (treatment and time as the factors) ANOVA (GenStat for Windows, Version 6.1, VSN International Ltd.). RESULTS Cultured Skin Strips Incorporate Thymidine in a Linear Fashion for up to 120 min Figure 2 shows the accumulation of radiolabeled thymidine into cultured skin strips with time of incubation in media containing Na (KRP), Na-free medium with choline chloride as the Na substitute, and Na-free medium with CaCl2 as the Na substitute. The rate of uptake into skin strips was linear for up to 120 min of incubation. Choline chloride, but not CaCl2, was a suitable Na replacement in the medium as evaluated by rate of thymidine incorporation. Radiolabeled AA are Taken up by Different Cells and Regions of Cultured Follicles The sites and rates of uptake of the radiolabeled AA differed (Figure 3). L-Cysteine appeared almost exclusively in the zone extending from immediately above the germinative region of the follicle bulb to the distal end of the zone of keratinization (Figure 3b). Label was most intense in the migrating and keratinizing cortical cells (43 units). No L-Cys appeared in the germinative bulb, inner root sheath, or dermal papilla. L-Alanine was distributed (Figure 3c) throughout the bulb region (10 units), the keratogenous zone (6 units), and the inner root sheath (6 units). There was little LAla label in the dermal papilla. L-Lysine was localized to the germinative cells of the follicle bulb (3 units), the cells of the inner root sheath (3 units), and scattered Figure 2. Uptake of thymidine by cultured sheep skin. The skin strips incorporated thymidine in a linear fashion with time of incubation. Rate of uptake of thymidine was not affected by replacement of Na with choline chloride (䊏) but was significantly reduced by substitution with CaCl2 (▲) compared with controls (◆). Values are means ± SEM. a,bSlopes with different superscripts differ (P < 0.05). cells in the outer root sheath (Figure 3d). No Lys label appeared in the keratogenous zone of the follicle. LLeucine distribution was similar to that of Ala, with label apparent in the bulb (6 units), the keratogenous zone (3 units), and the inner root sheath (1 unit; Figure 3e). Tritiated thymidine uptake, as expected, was found almost exclusively in the germinative cells of the follicle bulb (13 units; Figure 3f). Radiolabeled AA Enter Cultured Skin in a Linear Fashion with Time of Incubation The extracellular entrapment of radioactive label (i.e., not associated with cellular uptake) was estimated as the quantity of 14C-inulin carboxylic acid remaining in the tissue after the washing procedure. Only 0.1% of the total label in the incubation medium remained trapped in the tissue. Uptake results were corrected for this small residue. For all AA, the rates of uptake were linear for at least 120 min of incubation (Figure 4). The rate of uptake of L-Cys into the follicles was approximately 20 times greater than that of L-Leu, 40 times greater than that of L-Ala, and 125 times greater than that of L-Lys. Response of AA Uptake to Na Depletion and to the Presence of Specific Inhibitors of Various Transport Classes Figure 5 shows the responses of L-Ala (a), and L-Cys (b) to the presence or absence of Na and to the inhibitors of system A (MeAIB) and system L (BCH). Uptake of L-Ala was reduced by 40% by removal of Na from the Amino acid transport in wool follicles 2209 Figure 4. Uptake of radiolabeled (a) L-Cys and (b) LLeu (䊉), L-Ala (䊏), and L-Lys (▲) by cultured sheep skin. All rates differed significantly at P < 0.05. Values are means ± SEM. Note that the scale on the L-Cys figure is greater than that for the other AA. Figure 3. Sites of uptake of radiolabeled AA by cultured sheep skin, with actual micrographs on the left and a schematic diagram of the pattern of uptake on the right of each panel. (a)A labeled diagram of the wool follicle, (b) L-Cys, (c) L-Ala, (d) L-Lys, (e) L-Leu, and (f) thymidine. Labels refer to the grayscale values of the scanned sections of the follicle (arbitrary units corrected for background grayscale, specific activity of the radiolabeled AA, and time of incubation). Bulb = germinative region of the follicle bulb; DP = dermal papilla; IRS = inner root sheath; KZ = keratogenous zone of the follicle. media (Figure 5a). The MeAIB reduced Ala uptake regardless of Na status. The BCH had no effect on Ala uptake regardless of Na status. Surprisingly, a combination of MeAIB and BCH appeared to block the inhibitory effect of MeAIB alone on Ala uptake. The majority of Cys uptake was Na-independent, with more than 70% of the uptake apparent in Nadeficient media (Figure 5b). The MeAIB and BCH produced small decreases in Cys uptake when Na was present but not in Na-free media. A combination of the inhibitors produced no further reduction in Cys uptake. The majority (60%) of Leu uptake was Na-dependent (Figure 6a). The BCH significantly reduced Leu uptake in both Na-containing and Na-free media. The uptake of L-Lys was not inhibited by the removal of Na from 2210 Thomas et al. Figure 5. Effects of Na and the specific transport inhibitors α-(methylamino)isobutyric acid (MeAIB) and 2-amino2-norbonanecarboxylic acid (BCH) on uptake of (a) L-Ala and (b) L-Cys by cultured sheep skin. Values are means ± SEM (n = 5). a–dMeans not bearing a common letter above the bar differ (P < 0.05). the incubation medium, nor did MeAIB or BCH affect Lys uptake (Figure 6b). Kinetics of AA Transport into Cultured Skin Figure 7 shows the relationship between uptake of and L-Lys and the concentration of cold or unlabeled AA in the medium. Diffusion contributed significantly to the uptake of Leu, Ala, and Lys by the cells in the skin strip at concentrations greater than 0.5, 5, and 1 mM, respectively. No diffusive component was evident for L-Cys at concentrations from 3 to 10 mM, as indicated by the apparent plateau in Cys uptake above 4 to 5 mM. The smooth rectangular hyperbolas fitted to the saturable components of uptake of Leu, Ala, and Cys are consistent with the function of a single transport system exhibiting Michaelis-Menten characteristics. Estimated kinetic variables are summarized in Table 2. The uptake of Cys was via a low- L-Leu, L-Ala, L-Cys, affinity, high-capacity system(s); Leu via a high-affinity, high-capacity system(s), and Ala via a low-affinity, low-capacity system. The curve for Lys uptake had a sigmoidal component indicative of the presence of more than 1 binding site mediating Lys uptake (Segel, 1975). Calculation of kinetic parameters in such situations is not simple, but through use of the velocity curve parameters rather than traditional kinetic methods (Wright et al., 1986), estimates of Lys uptake indicated a low-affinity (Km = 0.5 mM) and low-capacity [Vmax = 0.6 nmol/(g × min)] transporter. DISCUSSION Given the high rate of cell division and protein synthesis occurring in the fiber-producing follicles in the skin of animals (Hynd and Everett, 2000) and the unique AA composition of the fibers so produced (Reis, 2211 Amino acid transport in wool follicles Figure 6. Effects of Na and the inhibitor 2-amino-2norbonanecarboxylic acid (BCH) on the rate of uptake of (a) L-Leu and (b) L-Lys by wool follicles in cultured sheep skin. Values are means ± SEM (n = 5). a–cMeans not bearing a common letter above the bar differ (P < 0.05). 1979), it is surprising that aside from the preliminary report of the current study, there has been only 1 published report into the mechanisms of AA transport into the wool or hair follicle. Matheson et al. (1999) studied Cys uptake into isolated cultured human hair follicles and into subconfluent outer root sheath cells. Arginine was taken up predominantly by the cells of the outer root sheath in intact follicles, whereas Cys was taken up by the fiber-forming cells. The authors found that the Cys transporter, ASC, was present in the intact hair follicles and cells of the outer root sheath. The Arg transporter, y+, was also expressed by follicles and was functionally active in the cells of the outer root sheath. These studies demonstrate that cultured follicles and outer root sheath cells can be used to characterize the kinetics of AA transport. However, the availability of human hair follicles and follicle cells is limited given the large amounts of material needed to fully characterize the transport systems. The skin strip model used in our experiments provides a convenient model for such studies and appears to satisfy most of the criteria necessary for precise and accurate dissection of transport systems. First, the small strips of skin contain a high concentration of follicle material (approximately 400 follicles in each skin strip), so, provided most of the cell activity is in the lower follicle, the preparations will reflect uptake predominantly into follicle cells. Ward and Harris (1976) showed that even when the preparation contains epidermis and upper dermis, approximately 90% of the DNA synthetic activity takes place in the follicle bulb. The linear uptake of thymidine over the period of measurement indicates that the cells are metabolically active and continue to synthesize DNA and proteins. A reduction in thymidine uptake when choline replaced Na in the culture medium (Figure 2) and changes in AA uptake due to media depletion or inhibitor inclusion indicate the cells of the follicle are responsive to the culture conditions. However, results related to Na depletion of the media must be treated with some caution in that the tissue may retain some cellular and extracellular Na for sufficient time to influence the transporter studies. In most cases, Na depletion of the media reduced AA transport (Figures 5 and 6), suggesting a degree of Na dependence. The actual quantitative value of this dependence will be influenced by the extent of Na washout from the cell membranes during the culture period. Entrapment of chemicals in extracellular spaces was very low (<0.1%). Overall, the skin strip model provides a convenient, repeatable, and responsive means of studying AA transport into follicles and has the distinct advantage over cell or cell membrane systems that tissue and cell interactions (such as tight junctions) are maintained. Table 2. Michaelis-Menten kinetic parameters derived from the uptake data for L-Leu, LAla, L-Cys, and L-Lys into the cells of cultured sheep skin1 Substrate L-Leu L-Ala L-Cys L-Lys Km, mM Vmax, nmolⴢg−1ⴢmin−1 Kd, nmolⴢg−1ⴢmin−1ⴢmM−1 0.04a ± 0.006 0.48b ± 0.051 1.01c ± 0.442 Multisites 1.30a ± 0.033 0.07b ± 0.043 3.54c ± 0.384 Multisites 0.52 0.17 0.03 0.15 Km and Vmax differ among substrates (P < 0.05). Km = concentration of substrate at which the reaction rate is half of maximum rate; Vmax = maximal velocity of reaction observed when the system is saturated with substrate; Kd = the diffusion constant defined as the slope of the linear part of the uptake curve. a–c 1 2212 Thomas et al. Cysteine is required for the synthesis of the keratin proteins, which are produced in the keratogenous zone of the follicle, defined as the region from the end of the germinative portion of the follicle bulb to a point approximately one-third of the distance to the skin surface. Labeled Cys was found in precisely this region after skin strip culture (Figure 3b), confirming the result found after i.v. injection of radiolabeled Cys in live animals (Downes et al., 1962). Presumably, the Cys must leave the blood vessels surrounding the follicle, enter the extracellular space around the follicle, and move through the connective tissue sheath, through the cells of the outer root sheath, through the cells of the lower inner root sheath, and finally into the precortical cells of the fiber. This migration must be rapid, because radiolabeled Cys can be found in the suprabulbar region of the follicle only 2 min and in the fibrillary region only 15 min after i.v. injection (Downes et al., 1962). Although it is possible that Cys may traverse some cell layers of the follicle by moving between the cells, the presence of tight junctions between the cells of the inner root sheath in the keratogenous zone where Cys is taken up (Orwin et al., 1973) might suggest that the majority of Cys movement is transcellular. That is, the Cys probably crosses cell membranes by diffusion, active transport, or both. The fact that the uptake of Cys followed saturable Michaelis-Menten kinetics suggests that carrier-mediated transport systems are indeed operating in the cell membranes. For Cys, there appeared to be virtually no diffusion of the AA into the follicles (Figure 7). The most common mammalian Cys transporter, system ASC, is Na-dependent, has a high affinity for Cys (Km = 50 to 150 M), and is not inhibited by either BCH or MeAIB. Uptake of Cys into the sheep skin strips was only partially inhibited by Na depletion and appeared to be via a low-affinity, high-capacity transporter [Km = 1.01 mM, Vmax = 3.54 nmol/(g × min)]. Further, there was a slight but significant inhibition of uptake in the presence of BCH. Together, these results suggest that there may be more than 1 Cys transport mechanism operating in the wool or hair follicle or that the follicle has developed a unique Cys transport system with some, but not all, of the characteristics of system ASC. The isolation of ASC messenger RNA from hair follicles (Matheson et al., 1999) is consistent with our findings, but further functional studies of follicle-specific transporter genes are required to confirm or refute follicle specificity. Site-specific variations in Cys transport characteristics have been reported for other tissues such as the ovine erythrocyte, which has a system ASC with a very low (Km = 12 mM) affinity (Harvey and Ellory, 1989). The uptake of L-Lys into the cells of the lower germinative region of the follicle bulb (Figure 3d) is consistent with the role of Lys in histone synthesis in rapidly dividing cells (Alberts et al., 1994). Hynd (1989) showed that depletion of L-Lys in proteins entering the intestines of sheep results in a dramatic drop in the rate of division of follicle bulb cells, presumably Figure 7. Uptake of (a) L-Leu, (b) L-Ala, (c) L-Cys, (d) and L-Lys into sheep skin incubated in media containing different concentrations of unlabeled AA. Total uptake (solid line) and uptake by the saturable component (broken line) are indicated. Values are means ± SEM. Amino acid transport in wool follicles reflecting this deficit in histone and DNA synthesis. The presence of L-Lys in the cells of the inner root sheath is consistent with the finding that Lys-rich trichohyalin granules are present in these cells (Rogers et al., 1991). Lysine uptake was Na-independent and unaffected by MeAIB and BCH. This is consistent with the operation of transporter system y+ (Guidotti et al., 1978; White et al., 1982; Christensen, 1989). L-Alanine appeared uniformly distributed throughout the follicles after incubation (Figure 3c), although, as for all the AA, little label appeared in the cells of the dermal papilla. Presumably, Ala is required for general protein synthesis with no particular site-specific demand for unusual proteins in the follicle. The uptake was characteristic of the transporter system A (Na-dependent, saturable, low substrate affinity, low transport capacity, and inhibited by MeAIB; Eddy, 1981; Christensen, 1984). The distribution of L-Leu in the wool follicle was similar to that of L-Ala (Figure 3e). Little Leu label was found in the dermal papilla. System L, a common transport system for Leu, is Na-independent, but Leu incorporation into the skin strips was Na-dependent. This suggests that the follicles may utilize a different transporter for Leu than other tissues. Leucine uptake was, however, inhibited by the system L inhibitor BCH, suggesting a variant of system L, such as system T described for human erythrocytes (Christensen, 1989), may be operative in the follicle. Molecular approaches such as those described by Hynd et al. (1999) and Matheson et al. (1999) will be invaluable in dissecting the precise mechanisms of AA transport into cells of the wool and hair follicles of mammals. Elucidation of these mechanisms is important, because they may reveal one of the sources of genetic difference in fiber growth among animals. Genetically superior wool-producing sheep, for example, have lower concentrations of Cys in the circulating plasma (Williams, 1987) and remove almost 3 times more Cys from the plasma each day than low producers. Differences in the affinity of the membrane-bound Cys transporters or in the rate with which they transfer Cys across the plasma membrane may explain at least some of the genetic capacity for fiber production. Other applications of elucidation of AA transport systems in wool follicles include more precise formulation of diets designed to maximize wool growth rates and potential targets for biological defleecing agents or hair-growth modulation. LITERATURE CITED Alberts, B., D. Bray, J. Lewis, M. Raff, K. Roberts, and J. D. Watson. 1994. Molecular Biology of the Cell. Garland Publ., New York, NY. Auber, L. 1950. The anatomy of follicles producing wool fibers, with special reference to keratinisation. Trans. R. Soc. Edinb. 62:191–254. 2213 Beach, E. F., B. Munks, and A. Robinson. 1943. The amino acid composition of animal tissue protein. J. Biol. Chem. 148:431– 439. Christensen, H. N. 1984. Organic ion transport during seven decades: The amino acids. Biochim. Biophys. Acta 779:255–269. Christensen, H. N. 1989. Distinguishing amino acid transport systems of a given cell or tissue. Methods Enzymol. 173:576–616. Del Castillo, J. D., and R. Muniz. 1991. 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