Biochemical Society Transactions ( 1999) Volume 27, part 5
21
A Gene Transfer Strategy to Manipulate Nitric Oxide in
the Vasculature of a Hypertensive Rat Model.
M. Yvonne Alexander, M. J. Brosnan, J. Fennel], A. M. Devlin,
C.A. Hamilton, A.F. Dominiczak. Dept. of Medicine and
Therapeutics, University of Glasgow, Glasgow, GI 1 6NT, UK.
Nitric oxide plays an important role in vasorelaxation and studies
in our laboratory have shown a deficiency in nitric oxide and an
excess superoxide anion production in the stroke-prone
spontaneously hypertensive rat (SHRSP) model compared with its
normotensive control Wistar Kyoto (WKY). We are investigating
whether gene transfer of replication-deficient adenoviruses
containing endothelial nitric oxide synthase (eNOS) and
superoxide dismutase (SOD) cDNAs into rat carotid arteries in
vivo, and human veins from hypertensive patients ex vivo, will alter
the function of the vascular endothelium. Segments of rat arteries
have been exposed to AdCMVeNOS and AdCMVP-galactosidase,
(IX1Oi0 pfu/ml) in vivo and ex vivo. Western blot and
immunocytochemical analyses have demonstrated an increase in
expression of both bovine and human eNOS compared to control
vessels. Isometric tension recordings of the rat arteries infused with
the bovine eNOS virus ex vivo have shown restoration of nitric
oxide availability when compared to vessels treated with vehicle
alone. Current studies involve adenoviral-mediated delivery of
human NOS and human SOD cDNAs to determine the effect on
arterial function. Our findings to date demonstrate that
AdCMVeNOS gene transfer can i), restore nitric oxide availability
to that seen in the normal WKY strain; ii), is an effective means of
gene transfer into an intact endothelium and may have important
implications for the alteration of vascular tone in diseased vessels.
22 Comparative study of purine transporters in drug sensitive
and drug resistant Trypanosoma equiperdum.
Jane E. Leadsham and Simon M. Jarvis
Research School of Biosciences, University of Kent at
Canterbury, Canterbury, Kent, CT2 MJ.
23
A I49
Transport of purine nucleobases by COS-I cells occurs
via a nitrobenzylthioinosine-insensitive (ei) nucleoside
transporter
Daniel Johnson and Simon M Jarvis, Department of Biosciences,
University of Kent at Canterbury, Canterbury, Kent CT2 M J
The uptake of physiological nucleobases, and their cytotoxic
analogues by mammalian cells are mediated by several distinct
transporters. These transporters differ in their specificity,
sensitivity to inhibitors and their requirement for extracellular
sodium. However, the genes encoding these carrier proteins have
yet to be identified. As part of our ongoing studies to clone
nucleobase transporters, we have characterized hypoxanthine
transport in COS-I cells, as a possible expression system for
candidate nucleobase transporter cDNAs. Hypoxanthine uptake
into monolayer cultures of confluent COS-1 cells was shown to
be mediated by a saturable Na'hdependent process (K,
42+6 M; V, 300i18pmo1/1O6cell per min(n=3)). Initial rates
of [ Hlhypoxanthine influx were inhibited by purine and
?
pyrimidine nucleosides (ICsO of 55*2 and 424*77 pM for
adenosine and uridine respectively), but purine nucleobases were
less potent inhibitors of hypoxanthine uptake (adenine ICsO
800+55 pM). In addition, nitrobenzylthioinosine (NBMPR) was
able to inhibit hypoxanthine influx, but only at concentrations
that block the NBMPR-insensitive (ei) nucleoside transporter. In
conclusion, hypoxanthine enters COS-1 cells via the facilitateddiffusion NBMPR-insensitive nucleoside transporter. Thus,
COS-1 cells should be a suitable cell line for the transient
expression of cDNAs encoding Na+-dependent and nucleobase
specific transporters.
Supported by a grant from the MRC. Daniel Johnson is in receipt
of a BBSRC postgraduate studentship.
24
Characterisation of purine transporters in L6 rat skeletal
muscle cells.
Helen L. Maddock, Sarah Vine, ?Jeremy D. Pearson and Simon
M. Jarvis. Research School of Biosciences, University of Kent,
Canterbury, Kent CT2 M J , ?Vascular Biology Research Centre,
Biomedical Sciences Division, King's College, London W8 7AH.
Protozoa of the family Trypanosomatidae cause many severe
and widespread diseases in humans, cattle and horses. Like
all known parasitic protozoa, Trypanosoma are auxotrophic
for purines and therefore need to salvage purines from their
environment. The first step in purine salvage, the transport of
nucleosides across the cell membrane is achieved in
Trypanosoma brucei brucei by at least two high affinity
transporters (PI and P2) w t h overlapping permeant
specificities. Furthermore, the P2 transporter has also been
implicated in the transport of trypanocidal drugs. The aim of
the present study was to compare the nucleoside transport
properties of bloodstream forms of wild type and drug
resistant Tequiperdurn. Wild type Tequiperdum were shown
to possess both P1 and P2 like transport activity with
properties similar to those found in T b. brucei (K, and V,,
values of 0.3f0.05 pM, 2.4f0.4 pmol/107cells/s, 0.3f0.007
pM and 1.1k0.4 pmol/107cells/s (n=3) for P1 and P2
respectively). However, in drug resistant T. equiperdum the
P2 transporter was apparently not detected at an extracellular
adenosine concentration of 0.1pM. Further studies revealed
that the P2 transporter was present in the drug resistant strain
but the affinity for adenosine uptake was decreased by 100fold. The permeant specificity of the P2 transporter in drug
resistant T equiperdurn is currently under investigation.
The properties of purine transporters in confluent L6 rat skeletal
muscle cells were investigated. Kinetic analysis demonstrated that
adenosine uptake was saturable with an apparent K, 11 If: 1.5pM
and V,,
0.14 If: 0.02pmo1/106 cells/s (n=5). Uptake was by
facilitated diffusion and independent of extracellular sodium.
Nitrobenzylthioinosine (NBMPR) and dilazep (10pM) failed to
inhibit adenosine uptake, demonstrating adenosine influx via a
NBMPR-insensitive (ei) nucleoside transporter. Hypoxanthine
was a weak inhibitor of adenosine influx (apparent K, 880 5 34
pM, n=4). In contrast to the finding that hypoxanthine was a poor
inhibitor of adenosine uptake, direct [3H]hypoxanthine influx was
shown to be via a sodium-independent saturable system that
exhibited a medium affinity for the purine base (apparent K, 48 5
16pM and V,
0.38 5 0.07pmo1/106cells/s, n=5). Surprisingly,
hypoxanthine uptake was also sensitive to inhibition by
nucleosides. Deoxycoformycin (I pM, adenosine deaminase
inhibitor) had no effect on the potency of adenosine to block
hypoxanthine uptake (adenosine K, 58 If: 15pM, n=3). The
properties of this hypoxanthine transport are different from those
described in other cell types, thus suggesting the presence of a
previously uncharacterized hypoxanthine carrier in mammalian
cells.
JL is in receipt of a BBSRC postgraduate studentship
Supported by a grant from the British Heart Foundation.
0 I999 Biochemical Society