Download Carbohydrate and amino acid metabolism in the A10 vascular

633rd MEETING, LONDON
66 1
Carbohydrate and amino acid metabolism in the A,, vascular smooth muscle cell line
PAUL R. KEMP, GEORGE K. RADDA and
ANNE-MARIE L. SEYMOUR
Department of Biochemistry, Oxford Universiry, South Parks
Road, Oxford OX1 3QU, U.K.
Littlc is known about the metabolism of the A,,, vascular
smooth muscle cell line, which has been used as a model for
vascular smooth muscle. The metabolism of these cells was
investigated to provide a basis for comparison with freshly
isolated vascular smooth muscle, the metabolism of which
has been studied previously [ 11. Vascular smooth muscle has
been shown to have a high rate of aerobic lactate production,
which accounts for less than 30% of the energy yield of the
cells, but over 90% of the glucose entering the cell [2].
The method chosen to follow substrate utilization and the
release of the end-products of metabolism in this study was
' H n.m.r. These findings were subsequently substantiated by
assaying the maximal rates of key enzymes in the pathways of
glycogenolysis, glycolysis and the citric acid cycle isolated
from the cells.
A,,, cells were grown in Dulbecco's modified Eagles
medium (DMEM)+ 10"/0 (v/v) fetal calf serum (FCS) in 75
cm? flasks until confluence was reached. The medium was
then replaced with 10 ml of fresh DMEM + 10% (v/v) FCS.
This medium was removed after 24 or 48 h, freeze-dried,
and redissolved in 3 ml of ?H,O in the presence of Chelex.
The pH was adjusted to 7.2 before the sample was filtered
through non-absorbent cotton wool into an n.m.r. tube. A
capillary containing a trisilylpentane standard was placed
into the centre of the tube, IH n.m.r. spectra were collected at
300 MHz over a 13 p.p.m. sweep width using a pulse angle of
70" and an interpulse delay of 10 s. The water resonance was
suppressed by irradiation during the interpulse delay.
Maximal enzyme activities were assayed on homogenates
of cells grown to confluence in 6 cm Petri dishes.
The major substrate used by the Al,, cells was glucose, in
the first 24 h period, SO f 1.3 pmol of glucose was consumed
compared with 9.8 f 0.8 pmol of glutamine and 4.3 k 0.1
y mol o f branched-chain amino acids. In this period,
14.7 f 0.6 pmol of lactate and 1.7 f 0.1 pmol of alanine werc
released into the medium. In the second 24 h only 31.1 f 1
pmol of glucose, 4.32 f 0 . 1 pmol of glutamine and
0.07 f 0.05 pmol of the branched-chain amino acids werc
consumed. The rate of alanine release was reduced to
1.2 f 0.2 pmol, but the rate of lactate release was constant.
Therc was no evidence of the production of aspartate or
citrate, potential end products of glutamine metabolism.
Table 1. Maximal catalytic rates of enzymes of glycogenoly.si.s,
glycolysis and the citric acid cycle in A , , vascular smooth muscle
cell lines
n = 4 in all cases; values are mean f s.D.,,
Enzyme
Phosphorylase
Hexokinase
P hosphofructokinase
Lactate dehydrogenase
Citrate synthase
Glutamate dehydrogenase
I
Max. rate
I mg protein
(nmol min
I)
I .7 f0.03
18.2 f 3.8
31.6f 1.3
262 f 32
139k 14
1 30 k 7
These results suggest that unlike the freshly isolated tissue,
exogenously supplied glucose is the major substrate in these
cells. This glucose may, predominantly, be converted into
COz, since only 15% of the glucose carbon is converted into
lactate in the first 24 h. During the second 24 h period, the
proportion of glucose converted into lactate is slightly
increased (24%).This result is again a deviation from results
of studies on freshly isolated tissue where over 90% of the
glucose transported into the cell is converted into lactate.
The data also suggest that glutamine may be oxidized to COz
rather than partially oxidized to lactate, citrate or aspartate.
The maximum catalytic rates of phosphorylase, hcxokinase, phosphofructokinase, lactate dehydrogenase, glutamate dehydrogenase and citrate synthase in vitro are given in
Table 1. These data show that citric acid cycle enzymes arc
present in the A,,, cells at activities in excess of some glycogenolytic and glycolytic enzymes. Thus it is reasonable to
conclude that the citric acid cycle can oxidize most or all of
the pyruvate produced by glycolysis.
In conclusion it would appear that the A,,, cell line, like
freshly isolated vascular smooth muscle generates most of its
energy via oxidative phosphorylation. Unlike the freshly
isolated tissue most of the glucose taken into the A,,, cell is
oxidized completely.
I . Paul, R. J. ( I 980) in Hundbook oJ Physiology (Bohr, D. F.,
Sparks, H. V. & Somlyo, A. R., eds.), pp. 201-235, American
Physiological Society. MD
2. Paul, R. J., Lynch, R. M. & Krisanda, J. M. ( 1986) Adv. Exp.
MU'. Biol. 194, 1 17- 1 2 1
Received 23 November I989
Nerve growth factor receptor-positivefibre pathways in the human neocortex
JANET M. KERWIN, ELAINE K. PERRY,
CHRISTOPHER M. MORRIS and ROBERT H. PERRY
M.R. C'. Neurochemical Pathology Unil, Newcastle General
Iiospital, Westgate Road, Newcastle Upon Tyne, NE4 6RE,
U.K.
/?-Nerve growth factor (NGF)is a 27 kDa protein, composed
of two identical subunits 118 amino acids in length, which
promotes the survival of peripheral sensory and sympathetic
neurons, and has also been shown to be associated with the
cells of the basal forebrain cholinergic system of the central
Abbreviations used: NGF, nerve growth factor; PBS, phosphatebuffered saline; AChE, acetylcholinesterase.
Vol. 18
nervous system. By interacting with specific membrane
receptors on nerve terminals, NGF is transported retrogradely to the cell bodies where it exerts physiological
actions increasing the transcription rate of specific gcncs
governing cell growth, membrane adhesion, and transmitter
levels.
Previous reports have demonstrated localization of NGF
receptor in the cholinergic neurons o f the basal forebrain in
rat 11, 21 and human 131, but have failed to demonstrate
axonal immunoreactivity. We have investigated, using NGF
receptor immunocytochemistry, the human temporal cortex
and adjacent forebrain nuclei in post-mortem brain tissue
from normal individuals, with a view to mapping the distribution of the receptor in axonal processes projecting from cell