Download Transamination and asymmetry in glutamate transport across the

Bioscience Reports i, 851-856 (1981)
Printed in Great Britain
gSl
T r a n s a m i n a t i o n and a s y m m e t r y in g l u t a m a t e t r a n s p o r t
across the b a s o l a t e r a l m e m b r a n e of f r o g s m a l l i n t e s t i n e
C. A. R. 5OYD and V. S. PERRING
Department of Human Anatomy, University of Oxford,
South Parks Road, Oxford OXI 3QX, U.K.
(Received 20 October 1981)
Transport
of L - g l u t a m a t e across the b a s o I a t e r a l
m e m b r a n e of frog small-intestinal epithelium, unlike
that of L-alanine, is highly asymmetric; thus the rate
c o n s t a n t (.Kentry) describing the entry of glutamate
into the eplthenum from the vascular bed across this
membrane is one order of magnitude greater than the
r a t e c o n s t a n t ( K e x i t ) d e s c r i b i n g its exit.
This
asymmetry, which appears not to depend upon the Na
g r a d i e n t , may be i m p o r t a n t in maintaining a high
i n t r a c e l l u l a r c o n c e n t r a t i o n of glutamate relative to
a l a n i n e t h e r e b y favouring the production of alanine
from glutamate by transamination.
It has long been recognized that the small intestine is a major site
of metabolism of e-amino N (Dent & Schilling, 1949; Wiseman, 1953;
Windmueller & Spaeth, 1974, 1975; Hanson & Parsons, 1977). Thus for
e x a m p l e t r a n s a m i n a t i o n of g l u t a m a t e , c a t a l y s e d by the enzyme
giutamic-alanine transaminase (EC 2.6.1.2), results i n the production
of alanine through the reaction
Ki
glutamic acid + pyruvate ,
9 alanine + e-ketoglutarate.
(1)
K2
This reaction is readily reversible ( K I / K 2 = 1.6; Boyer, 1973) and the
forward reaction will be favoured by a high cellular concentration of
g l u t a m a t e r e l a t i v e to alanine.
As the intraepithelial amino acid
concentrations will be determined, in part, by their cellular entry and
exit mechanisms, we have investigated the transport systems available
f o r g l u t a m a t e and a l a n i n e e n t r y and exit across the basolateral
membrane of frog (Rana ridibunda) intact small intestine.
Methods
Evidence that frog small
in vitro
intestine converts glutamate to alanine
Everted rings of R. ridibunda small intestine were incubated for
60 min at 20~
in Frog Ringer solution containing t0 mM L-glutamate, 1 mM D-glucose, and [l#C]inulin (an extracellular marker); as a
c o n t r o l , rings were i n c u b a t e d in a s i m i l a r medium from which
9
The Biochemical
Society
852
BOYD
& PERRING
glutamate had been omitted. After incubation, the tissue was removed
from the solutions and extracted in 0.04 M HNO 3 overnight at g0~
Samples of the extract were analysed by thin-layer chromatography
(Brenner et aJ., 1962).
Extracts of the control tissue gave rise to
two barely visible Ninhydrin-positive spots (Rf values 0.19 and 0.27);
in contrast, extracts of the tissue incubated with glutamate produced
seven spots after develpment with Ninhydrin, including two prominent,
purple-staining spots with Rf values 0.19 and 0.37, which were shown
by comparison with amino acid 'standards' to be glutamate and alanine
respectively.
Measurement
alan•
of
basolateral
membrane
transport
of glutamate
and
The vascularly perfused preparation of R. ridibunda small intestine
(Boyd et al., 1975) is ideally suited to the study of basolateralmembrane solute transport.
Washout of solute from the luminally or
vascularly preloaded epithelium into the vascular bed is c h a r a c t e r i z e d
by a rate constant, Kexit.
This describes exit from the epithelium
into the vascular bed across the basolateral membrane.
Wash-in to
the tissue from the vascular bed (Boyd & Parsons, 1979) enables a
f u r t h e r r a t e c o n s t a n t , Ken.try , to be calculated.
Rat e constants
describing glutamate and alanme entry and exit across the basolateral
membrane are presented in Table 1.
Results
and Discussion
Exit from the luminally preloaded epithelium
Fig. l shows the vascular appearance of alanine and glutamate
during washout from the luminally preloaded epithelium. It is clear
that alanine washout is biphasic (as is that of leucine; cf. Cheeseman,
1979), t h e initial phase being c h a r a c t e r i z e d by Kexit (fast,
Table I. Rate constants describing the transport of alanine
and glutamate across the basolateral face of the vascularly
perfused epithelium of R. ridibunda small intestine
Values are means • S.E.M.~ with the number of samples shown in parentheses.
Kentry (min -I )
for wash-in to epithelium
from vascular bed
Kexit (min -I) for washout from
epithelium into vascular bed
following loading from
Lumen
Alanine
Glutamic acid
0.196 • 0.019 (ii)
0.327 • 0.116 (i0)
Vascular bed
0.150 • 0.008 (14)
fast (unstripped)
0.181 • 0.051 (8)
fast (unstripped)
0.044 • 0.004 (14)
slow
0.031 • 0.007 (8)
slow
0.027 • 0.003 (12)
0.155 • 0.032 (i0)
fast (unstripped)
0.026 • 0.005 (I0)
slow
GLUTAMATE TRANSPORT AND TRANSAMINATION
100-
g53
Washout:Alanine, Glutamate
(after loadingfrom lumen)
Alanine
Kexit (unstripped)-0.165rain-1
50-
T
c~
E
ti0. 058 rain-1
| 10'
~
Kexti=O
O.3mi
6 nq ~
_
o
,r
o
'
'
zb
'
Minsofwashout
Fig. i.
Vascular appearance of glutamate and
alanine during washout from the luminally preloaded
epithelium of R. ridihunda small intestine.
Both
experiments were carried out on the same animal~ and
in each the epithelium was loaded from the lumen
with 1 mM amino acid until the rate of vascular
appearance reached a steady state; after the removal
of solute from the lumen~ washout was followed for
the subsequent 30 (alanine) or 35 (glutamate) min.
unstripped) = 0,165 rain-I; glutamate exit is monophasic and slow~ with
Kexit = 0.036 rain -I,
Wash-in to the epithelium from the vascular bed
The rate constants for amino acid entry into the epithelium from
the vascular bed across the basolatera] membrane (Table 1) indicate
that alanine entry and exit are remarkably similar~ there being no
significant difference between Kexit (fas% unstripped) and Kentry. In
contrast~ Ke l.i.l .l y. for glutamate is one order of magnitude greater than
Kexit ~ indicating that glutamate transport is highly asymmetric.
85#
BOYD & PERRING
Exit from the vascularly preloaded epithelium
Alanine washout from the vascularly preloaded epithelium (mean
Kexit [fast, unstripped] = 0.181 + 0.051 min -t) is similar to exit from
t h e e p i t h e l i u m a f t e r loading from the lumen (mean Kexit [fas%
unstripped] = 0.150 + 0.008 min-t). However~ glutamate washout from
t h e v a s c u l a r l y preloaded epitheJium is described by an initial fast
phase of vascular appearance (mean Kexit [fas% unstripped] = 0 . 1 5 5 +
0.032 min -t) and a subsequent slower phase of washout described by
rate constant, Kexit (slow) = 0.026 + 0.005 min -t, similar to that
describing the slow monoexponential washout of glutamate from the
luminally preloaded epithelium.
This suggests that the initial rapid phase of washout represents
v a s c u l a r a p p e a r a n c e of radioactive alanine (shown to be a major
p r o d u c t of g l u t a m a t e m e t a b o l i s m in e v e r t e d rings) derived by
t r a n s a m i n a t i o n from glutamate that was originally loaded into the
epithelium from the vascular side. The second phase of washout may
represent vascular appearance of unmetabolized glutamate.
Asymmetric glutamate transport
Glutamate exit across the basolateral membrane is extremely slow,
while entry is rapid; the biochemical basis for this asymmetry remains
unclear.
The rates of glutamate (and alanine) entry and exit across
the basotateral membrane are not significantly changed by lowering the
external Na concentration from 123 mM to 5 mM (Table 2); thus this
marked asymmetry in glutamate transport is maintained in the absence
of a sodium concentration gradient.
Burckhardt et al. (1980) and
Sacktor et al. (1981) have demonstrated that potassium is invoJved in
energizing glutamate uptake into vesicles prepared from rat-kidney
proximal-tubule basolateral and brush-border membranes.
Our findings
with intact tissue would fit nicely with such a mechanism. Mircheff
et al. (1980) have identified both Na-dependent and Na-independent
pathways for alanine in basolateral membrane vesicles from rat small
i n t e s t i n e ; our e v i d e n c e suggests that the Na-independent pathway
predominates in the frog small intestine.
The relation between basolateral transport and metabolism
The a s y m m e t r y in glutamate basolateral transport is likely to
maintain a relatively low plasma concentration of the potentially toxic
amino acid glutamate (the concentration of glutamate in the plasma
of Rana c a t e s b e i a n a is about 90 pM; Boyd, 1976), while the high
intraceilular level of glutamate will favour transamination to alanine.
Since the basolateral membrane transport of alanine appears to be
symmetrical, both exit and entry being fas% any alanine produced from
glutamate will be removed rapidly from the cells of the epithelium,
thereby favouring the forward reaction in equation t.
Acknowledgement
V. S. P. thanks the M.R.C. for a research studentship.
GLUTAMATE TRANSPORT AND TRANSAMINATION
855
Table 2. The effect of removing vascular Na (choline
substitution) upon rate constants describing glutamate
and alan•
movements between the epithelium and the
vascular bed of R. ridibunda small intestine expressed
as a ratio of the rate constant measured in the absence of
vascular Na to that measured in the presence of vascular Na
Values are means • S.E.M., with
parentheses.
the number of samples shown in
Kentry (-Na+)
Kexit (-Na+)
Kentry (+Na+)
Kexit (+Na+)
Alanine
0.8•
1.1•
Glutamic a c i d
0.7 • 0.i (3)
1.2•
References
Boyd CAR (1976) D. Phil. thesis, University of Oxford.
Boyd CAR & Parsons DS (1979) Movements of monosaccharides between
blood and tissues of vascularly perfused small intestine. J.
Physiol. 2879 371-391.
Boyd CAR, Cheeseman CI & Parsons DS (1975) Amino acid movements
across the wall of anuran small intestine perfused through the
vascular bed. J. Physiol. 250, 409-429.
Boyer PD (1973) The Enzymes, Vol. IX, Part B, p 464, Academic
Press, New York.
Brenner M, Niederwieser A & Pataki G (1962) Aminosauren und
derivate, in D u n n s c h i c h t - C h r o m a t o g r a p h i e (Stahl E, ed), pp
403-445, Springer-Verlag, Berlin.
Burckhardt G, Kinne R, Stange G & Murer H (1980) The effects of
potassium and membrane potential on sodium-dependent glutamic
acid uptake. Biochim. Biophys. Acta 5999 191-201.
Cheeseman CI (1979) Factors affecting the movement of amino acids
and small peptides across the vascularly perfused anuran small
intestine. J. Physiol. 293, 457-468.
Dent CE & Schilling JA (1949) Studies on the absorption of
proteins: the amino-acid pattern in the portal blood. Biochem.
J. 44~ 318-333.
Hanson PJ & Parsons DS (1977) Metabolism and transport of
glutamine and glucose in vascularly perfused small intestine of
the rat. Biochem. J. 166, 509-519.
Mircheff AK, van Os CH & Wright EM (1980) Pathways for alanine
transport in intestinal basal lateral membrane vesicles. J.
Memh. Biol. 52, 83-92.
856
BOYD & PERRING
Sacktor B, Rosenbloom IL, Liang T & Cheng L (1981) Sodium
gradient- and sodium plus potassium gradient-dependent
L-glutamate uptake in renal basolateral membrane vesicles. J.
Memb. Biol. 60, 63-71.
Windmueller HG & Spaeth AE (1974) Uptake and metabolism of plasma
glutamate by the small intestine. J. Biol. Chem. 249~ 50705079.
Windmueller HG & Spaeth AE (1975) Intestinal metabolism of glutamine and glutamate from the lumen as compared to glutamine from
the blood. Arch. Biochem. Biophys. 171, 662-672.
Wiseman G (1953) Absorption of amino acids using an in vitro
technique. J. Physiol. 120, 63-72.