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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.
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,
Corresponding author: [email protected]
Received August 7, 2006.
Accepted May 8, 2007.
J. Anim. Sci. 2007. 85:2205–2213
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
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.
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
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.
Rate of Uptake of AA into Cultured Skin Strips
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
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
Na status
All AA
All AA
Ala, Cys, Lys
Ala, Cys, Lys
All AA
All AA
Ala, Cys
Ala, Cys
MeAIB (20 mM)
MeAIB (20 mM)
BCH (20 mM)
BCH (20 mM)
MeAIB (20 mM) + BCH (20 mM)
MeAIB (20 mM) + BCH (20 mM)
MeAIB = α-(methylamino)isobutyric acid; BCH = 2-amino-2-norbonanecarboxylic acid.
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.).
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
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
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.
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,
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
Km, mM
Vmax, nmolⴢg−1ⴢmin−1
Kd, nmolⴢg−1ⴢmin−1ⴢmM−1
0.04a ± 0.006
0.48b ± 0.051
1.01c ± 0.442
1.30a ± 0.033
0.07b ± 0.043
3.54c ± 0.384
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.
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.
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.
Beach, E. F., B. Munks, and A. Robinson. 1943. The amino acid
composition of animal tissue protein. J. Biol. Chem. 148:431–
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. Neutral amino acid transport
by isolated small intestinal cells from guinea pigs. Am. J. Physiol. 261:G1030–G1036.
Downes, A. M., A. G. Lyne, and W. H. Clarke. 1962. Radioautographic studies of the incorporation of [35S] cystine into wool.
Aust. J. Biol. Sci. 17:140–153.
Eddy, A. A. 1981. The amino acid pumps of living cells. Sci. Prog.
Oxf. 67:245–270.
Guidotti, G. G., A. F. Borghetti, and G. C. Gazzola. 1978. The regulation of amino acid transport in animal cells. Biochim. Biophys.
Acta 515:329–366.
Harvey, C. M., and J. C. Ellory. 1989. Identification of amino acid
transporters in the red blood cell. Methods Enzymol.
Hynd, P. I. 1989. Factors influencing the cellular events in the wool
follicle. Pages 169–184 in The Biology of Wool and Hair Growth.
G. E. Rogers, P. J. Reis, K. A. Ward, and R. C. Marshall, ed.
Chapman and Hall, London, UK.
Hynd, P. I. 2000. The nutritional biochemistry of wool and hair
follicles. Anim. Sci. 70:181–195.
Hynd, P. I., and B. K. Everett. 2000. Estimation of cell birth rate
in the wool follicle bulb using colchicine metaphase arrest or
DNA labelling with bromodeoxyuridine. Aust. J. Agric. Res.
Hynd, P. I., G. Nattrass, N. Wilson, and B. C. Powell. 1999. Amino
acid transport in wool and hair follicles. Exp. Dermatol.
Matheson, H. B., G. E. Westgate, P. P. Parmar, C. Riches, M. A.
Blount, and J. C. Ellory. 1999. Nutrition and metabolism in
isolated hair follicles. Exp. Dermatol. 8:319–320.
Orwin, D. F. G., R. W. Thomson, and N. E. Flower. 1973. Plasma
membrane differentiations of keratinizing cells of the wool follicle. III. Tight junctions. J. Ultrastruct. Res. 45:30–40.
Powell, B. C., A. Nesci, and G. E. Rogers. 1991. Regulation of keratin
gene expression in hair follicle differentiation. Ann. N. Y. Acad.
Sci. 642:1–20.
Powell, B. C., and G. E. Rogers. 1994. Differentiation in hard keratin
tissues: Hair and related structures. Pages 401–436 in The
Keratinocyte Handbook. I. Leigh, B. Lane, and F. Watt, ed.
Cambridge Univ. Press, UK.
Reis, P. J. 1979. Effects of amino acids on the growth and properties
of wool. Pages 223–242 in Physiological and Environmental
Limitations to Wool Growth. J. L. Black and P. J. Reis, ed. Univ.
N. Engl. Publ. Unit, Armidale New South Wales, Australia.
Rogers, G. E., M. J. Fietz, and A. Fratini. 1991. Trichohyalin and
matrix proteins. Ann. N. Y. Acad. Sci. 642:64–80.
Segel, I. H. 1975. Enzyme Kinetics. Wiley, New York, NY.
Ward, K. A., and R. L. N. Harris. 1976. Inhibition of wool follicle
DNA synthesis by mimosone and related 4(1H)-pyridones. Aust.
J. Biol. Sci. 29:189–196.
White, M. F., G. C. Gazzola, and H. N. Christensen. 1982. Cationic
amino acid transport into cultured animal cells. I. Influx into
cultured human fibroblasts. J. Biol. Chem. 257:4443–4449.
Williams, A. J. 1987. Physiological consequences of selection for
increased fleece weight. Pages 481–495 in Merino Improvement
Programs in Australia. Proc. Natl. Symp. Leura. B. J. McGuirk,
ed. Aust. Wool Corp., Sydney.
Wright, E. M., B. R. Stevens, and B. E. Peerce. 1986. Neutral amino
acid transport in rabbit intestinal brush-border membranes.
Fed. Proc. 45:2450–2454.