(Received for publication, August 1, 1995; and in revised form, September 14, 1995)
From the
The African trypanosome, Trypanosoma brucei brucei,
possesses at least two nucleoside transporter systems designated P1 and
P2, the latter being implicated in the selective uptake of
melaminophenyl arsenical drugs. Since arsenical-resistant trypanosomes
show cross-resistance in vivo to aromatic diamidines, we have
investigated whether these drugs are also substrates for the P2
nucleoside transporter. In melarsen-sensitive T. b. brucei,
the diamidines, including the commonly used trypanocides, pentamidine
and berenil, were found to abrogate lysis induced by the P2 transport
of melarsen oxide in vitro. Measurement of
[ring-H]pentamidine transport in
melarsen-sensitive T. b. brucei, demonstrated that uptake is
carrier-mediated, with a K
of 0.84
µM and a V
of 9.35 pmol
s
(10
cells)
.
Pentamidine transport appears to be P2-mediated in these cells, as
pentamidine strongly inhibited uptake of
[2`,5`,8-
H]adenosine by the P2 transporter, with
a K
of 0.56 µM. Furthermore,
[ring-
H]pentamidine transport was
blocked by a number of P2 transporter substrates and inhibitors, as
well as by other diamidine drugs. Analysis of the uptake of pentamidine
and other diamidines in melarsen-resistant trypanosomes in vitro and in vivo, which also show differential levels of
resistance to these compounds in vivo, indicated that P2
transport was altered in these cells and that accumulation of these
drugs was markedly reduced.
African trypanosomiasis continues to be a major public health and veterinary problem in many parts of Africa(1, 2) . Treatment of the disease in humans and animals is confounded by the limited repertoire of drugs and by the emerging threat of drug resistance in the field(3, 4) . One approach to the development of new drugs for the treatment of African sleeping sickness is the elucidation of the mode of action of existing drugs and the underlying mechanisms of drug resistance. The aromatic diamidine, pentamidine, is one of the most frequently administered drugs in the treatment of the early stage of the disease (5) . However, despite more than 50 years of its use in the field, little is known about pentamidine's mode of action or the mechanisms which mediate pentamidine resistance(6, 7) . Cross-resistance to pentamidine and other diamidines and to the melaminophenyl arsenicals has been observed frequently in laboratory strains of African trypanosomes(8, 9, 10, 11, 12, 13, 14) , but how this resistance is conferred remains elusive.
Recently, we identified a novel P2 nucleoside transporter in T. b. brucei, which appears to mediate the uptake of trivalent melaminophenyl arsenical drugs(15) . P2 transport was observed to be altered in a melarsen-resistant T. b. brucei clone, suggesting that resistance to these drugs may be conferred by a decreased accumulation of the drug due to alterations in P2 transport. The melarsen-resistant clone also displayed differential levels of resistance to various diamidines in vivo(10) . In this report, we show that pentamidine and other diamidines are P2 transport substrates, and, furthermore, that the differential levels of resistance to the diamidine drugs displayed in vivo may be due to alterations in P2 transport.
Both melarsen oxide and melarsoprol were generously provided by Specia, Rhône-Poulenc (Paris, France), and the diisethionate salts of stilbamidine, hydroxystilbamidine, propamidine, dibromopentamidine, and pentamidine by Rhône-Poulenc (formerly May & Baker; Dagenham, Essex, UK).
Uptake over longer time courses was
measured according to (20) . At zero time, trypanosomes (2
10
cells ml
), prewarmed to 25
°C, were mixed in an flask with an equal volume of CBSS/BSA (25
°C) containing [
H]pentamidine. At various
times, triplicate 0.1-ml aliquots were withdrawn and pipetted into
sequencing tubes (0.4 ml capacity, BDH/Merck Ltd.), containing 0.1 ml
12% (v/v) PCA overlaid with 0.1 ml of silicone oil. Uptake was
terminated by centrifuging the cells into the PCA layer in a Beckman
Microcentrifuge E fitted with a horizontal rotor. Cell pellets were
left to extract in PCA overnight at 4 °C. Duplicate samples (40
µl) of the PCA layer were counted in 3.0 ml of Picofluor 40
scintillation fluid in a Beckman LS6000LL liquid scintillation counter.
Figure 1: Effect of pentamidine on melarsen oxide-induced lysis in T. b. brucei. Cell lysis by 0.5 µM melarsen oxide was measured in the absence (0) or presence of pentamidine at a 1-, 10-, 25-, and 50-fold excess over the arsenical at 37 °C as described under ``Experimental Procedures.'' A control sample containing no drug is represented by the broken line.
Figure 2:
Pentamidine transport kinetics in T.
b. brucei. Transport of [H]pentamidine was
measured at 25 °C according to ``Experimental
Procedures.'' The inset represents the data transformed
by the Hanes-Woolf equation where S represents pentamidine
concentration (µM) and v represents the initial
rate of uptake (pmol s
(10
cells)
).
Figure 3:
Inhibition of uptake of
[H]adenosine on the P2 transporter by pentamidine
in T. b. brucei. The effect of pentamidine on transport of
[
H]adenosine (1 µM, open
circles. and 10 µM, closed circles) was
measured with 1 mM inosine included to saturate P1 transport.
Uptake was determined at 25 °C as described under
``Experimental Procedures.'' Results are expressed as (pmol
s
(10
cells)
)
versus pentamidine concentration.
Figure 4:
Uptake of
[H]pentamidine by melarsen-sensitive (c118) and
-resistant (cRU15) T. b. brucei in vitro. Uptake was measured
in T. b. brucei c118 (open circles) and cRU15 (closed circles) as described under ``Experimental
Procedures.'' a, 0.5 µM pentamidine; b, 10 µM pentamidine.
The initial rates of
uptake of pentamidine by cRU15 can be estimated from Fig. 4to
be 0.069 and 0.370 pmol s (10
cells)
for 0.5 µM and 10
µM pentamidine, respectively. Although initial rates
cannot be accurately determined for c118 from Fig. 4, they can
be calculated using the Michaelis-Menten equation to be 3.49 and 8.63
pmol s
(10
cells)
,
respectively. Thus, cRU15 appears to take up pentamidine at an initial
rate that is 20- to 50-fold slower than c118.
Our previous work suggested that uptake of the melaminophenyl
arsenical drugs into T. b. brucei is mediated by a P2
nucleoside transporter(15) . The present study demonstrates
that uptake of pentamidine is also carrier-mediated with several lines
of evidence suggesting that this involves the same P2 transporter.
First, uptake of pentamidine exhibits saturation kinetics consistent
with a carrier-mediated mechanism. The kinetic parameters determined
here for T. b. brucei S427 c118 (K = 0.84 µM and V
= 9.35 pmol s
(10
cells)
at 25 °C) are broadly in agreement with
previous estimates for T. b. brucei EATRO 110 (K
= 2.7 µM; V
= 6.1 pmol s
(10
cells)
at 37 °C) (24) .
Second, pentamidine is a potent inhibitor of adenosine uptake on the P2
transporter with a K
(0.56 µM)
similar to its K
(0.84 µM). Although
these data could be compatible with simple competitive inhibition,
where pentamidine competes with adenosine for uptake on the P2 carrier
and vice versa, the converse experiment did not yield the predicted
reciprocal kinetic parameters: adenosine only inhibits pentamidine
transport at concentrations >10 µM indicating that the K
must be significantly greater than its K
(0.59 µM) for the P2
transporter(15) . The reason for this discrepancy is not known.
Third, with this notable exception, all other inhibitors of
arsenical-induced lysis, which are also inhibitors (or competitive
substrates) of adenosine uptake on the P2 transporter (15) ,
strongly inhibit transport of pentamidine. In the case of the
diamidines, these findings are consistent with a previous report that
propamidine, stilbamidine, and hydroxystilbamidine are competitive
inhibitors of pentamidine transport with K
values
of the same order as the K
determined for
pentamidine(24) . Fourth, melarsen-resistant cells exhibit
profound alterations in their ability to transport both adenosine (15) and pentamidine (this study) via the P2 system in the
short term (seconds), which is reflected in their decreased ability to
accumulate both pentamidine and other diamidines in the longer term
(minutes or hours).
Our current results show that pentamidine can be rapidly accumulated and concentrated by several orders of magnitude within the cell. Others have shown that pentamidine transport can be partially inhibited by a number of metabolic inhibitors, suggesting involvement of an energy-dependent process(24) . However, it would be premature to discard the possibility that the high intracellular levels observed may be due to binding to intracellular sites, such as kinetoplast DNA(25, 26) . HPLC analysis of trypanosomal extracts reported here and elsewhere (16) indicates that there is no significant metabolism of diamidine drugs in the cell; thus, the maintenance of a concentration gradient by metabolic conversion of these drugs is unlikely.
Loss or alteration in the kinetic properties of the P2 transporter offers an attractive explanation for the well documented nonreciprocal cross-resistance between the melaminophenyl arsenical and diamidine classes of trypanocidal drugs(8, 9, 10, 11, 12, 13, 14) . Although the intracellular targets for these drugs are not known with any certainty, it is likely that they are different. Thus, the frequently observed nonreciprocal nature of this cross-resistance could depend on the relative contributions made either by alterations in the intracellular targets or by alterations in the P2 transporter to the overall resistance of the cell. However, this hypothesis does not account entirely for the differential levels of resistance of trivalent melaminophenyl arsenicals and diamidines in our melarsen-resistant clone(14) . Possibly, the wide range of resistance factors observed could depend on the extent to which these drugs can be accumulated to achieve lethal intracellular concentrations in cRU15, which, in turn, would depend on their pharmacokinetic profile. Precise information on the plasma pharmacokinetics of the biologically active forms of many of these drugs is lacking in rodents. However, prophylactic activity could provide some indication of gross pharmacological properties. Significantly, none of the melaminophenyl arsenicals have any prophylactic activity in rodents when subsequently challenged with trypanosomes(7) , correlating with cRU15 showing moderate to high resistance against these compounds (26-, 69-, and 121-fold for melarsen oxide, melarsoprol, and trimelarsen, respectively)(14) . In contrast, with the notable exception of berenil(27) , all of the diamidines possess considerable prophylactic activity (7, 28) with cRU15 showing low resistance to pentamidine, stilbamidine, and propamidine (1.6-, 5.7-, and 7.7-fold, respectively) versus moderate resistance to berenil (24-fold)(14) . Although comparative studies failed to distinguish between the prophylactic activity of pentamidine and stilbamidine(29) , radiotracer studies in mice suggest that stilbamidine is excreted more rapidly than pentamidine (50% elimination in 2 and 5-6 days, respectively)(30, 31, 32) . Unfortunately, most of the drug that is retained in the body is sequestered in tissues, especially liver and kidney, and the residual amounts in plasma were too low to measure. Nonetheless, these data support a correlation between pharmacological properties and resistance for these drugs. Our failure to find any striking difference between the intracellular concentrations of stilbamidine and pentamidine in cRU15 following 4-h exposure in vivo does not necessarily invalidate this hypothesis, since this interval may not be sufficient to reveal marked differences in diamidine concentrations necessary to achieve cell killing. It is not possible to extend this time course much beyond 4 h since pentamidine clears the parasitemia shortly thereafter(16) . Further work is required to test this hypothesis.
In conclusion, it appears that, like the melaminophenyl
arsenicals, the diamidines are also accumulated in T. b. brucei by a P2 nucleoside transporter and that resistance to the
diamidine drugs may be partly conferred by alterations in P2 transport
and the pharmacokinetic properties of the drug. The precise mechanism
by which P2 transport is altered is unclear at present, but it may be
envisaged that it arises from either point mutations within the gene
thereby altering K, V
, or
both of the transporter, or, alternatively, the expression of the P2
transporter may be down-regulated. Elucidation of the precise mechanism
of P2-mediated resistance awaits study by genetic analysis.