The increased K+ leak of malaria-infected erythrocytes is not via a Ca(2+)-activated K+ channel.

Charybdotoxin and nitrendipine both inhibited K+(86Rb+) influx via the Ca(2+)-activated channel of uninfected erythrocytes but had no effect on K+(86Rb+) transport in malaria-infected cells. Activation of the channel in uninfected cells in which the cytoplasmic [Na+]/[K+] ratio was adjusted to be comparable with that of late-stage malaria-infected erythrocytes resulted in a large (nitrendipine-sensitive) increase in K+(86Rb+) influx. These results suggest that the endogenous Ca(2+)-activated K+ channel remains inactive in human red cells infected with late-stage parasites. The identity of the pathway which mediates the increased K(+)-leak in infected erythrocytes remains to be established.

Characteristics of 86Rb+ transport in human erythrocytes infected with Plasmodium falciparum.

Human red cells infected in vitro with Plasmodium falciparum showed a significant increase in the rate of both ouabain-sensitive and ouabain-insensitive 86Rb+ influx. The increase in ouabain-insensitive 86Rb+ influx was due, in part, to increased transport via a bumetanide-sensitive system and, in part to transport via a pathway that was absent (or at least inactive) in uninfected cells. The parasite-induced pathway was inhibited by piperine and had a dose response very similar to that of the Gardos channel of uninfected cells but was less sensitive than the Gardos channel to inhibition by quinine.

Glibenclamide and meglitinide block the transport of low molecular weight solutes into malaria-infected erythrocytes.

Following infection by the malaria parasite, human erythrocytes show increased uptake of a wide variety of low molecular weight solutes via pathways with functional characteristics different from those of the transporters of normal erythrocytes. In this study glibenclamide and meglitinide were shown to inhibit the induced transport of a sugar alcohol (sorbitol), an amino acid (threonine), an inorganic anion (Cl-) and an organic cation (choline) into human erythrocytes infected in vitro with Plasmodium falciparum. The results are consistent with the hypothesis that a diverse range of substrates enter malaria-infected cells via common pathways which have features in common with Cl- channels in other cell types. glibenclamide and meglitinide were also shown to inhibit the in vitro growth of the intracellular parasite which would suggest that these pathways may be a viable chemotherapeutic target.

Selective stage-specific changes in the permeability to small hydrophilic solutes of human erythrocytes infected with Plasmodium falciparum.

The permeability characteristics of Plasmodium falciparum-infected human erythrocytes to various 3H-labelled solutes were measured during the maturation of the parasites in sorbitol-synchronised cultures. Using [14C]inulin as the extracellular marker, estimates were made of the influx kinetics of [3H]amino acids into trichloroacetic acid (TCA)-soluble pools within the erythrocyte and concomitant incorporation into TCA-precipitable material. These measurements provided values of the rates of protein synthesis by the parasite and the initial influx rates for the transport of precursor amino acids into the erythrocyte. For about 12-15 h after parasitisation, the influx of L-[3H]glutamine remained at a low level comparable to that in the uninfected cell (2-9 nmol g-1 cells min-1). As pigment appeared in the trophozoite, the initial rate of influx of L-glutamine increased to a value up to 100-fold higher than in the uninfected erythrocyte. The increase in permeability affected only the parasitised cells in a culture of partially infected erythrocytes, and was selective with respect to substrate since the influx kinetics for both [3H]isoleucine and [3H]arginine were not affected by parasitisation. The permeability changes occurred mainly over a 4-8 h period in the development of the young trophozoite, during which time [3H]glycine influx was enhanced by a factor of 3-10, with a comparable increase in the uptake of myo-[3H]inositol. L-[3H]glutamate, which did not penetrate significantly into uninfected erythrocytes, entered red cells infected with mature trophozoites at a rate which was much less than 1% of the parasite-induced-L-glutamine influx. At the stages when the permeability to L-glutamine was markedly enhanced, parasitised cells remained impermeable to [3H]sucrose. An analysis of the relative 3H activities in glutathione and free amino acid pools indicated that, if L-glutamine permeation did not increase during parasite maturation beyond the ring stage, or was blocked by a potential antimalarial compound, an insufficient supply of L-glutamine would be available for the increased rates of parasite protein synthesis and glutathione turnover within the red cell.

Potentiation of the antimalarial activity of qinghaosu by methoxylated flavones.

Interaction between the flavones casticin and artemetin and the antimalarial activity of chloroquine and qinghaosu (QHS) was examined using an in vitro growth assay based on [3H]hypoxanthine incorporation in synchronized cultures of a cloned line of Plasmodium falciparum. Casticin, and to a lesser extent artemetin, selectively enhanced the inhibition of growth by QHS, but had little effect on the activity of chloroquine. The findings suggest that flavones indigenous to Artemisia annua, from which QHS is isolated, might significantly alter the clinical potential of this novel antimalarial drug in the treatment of chloroquine-resistant malaria.

Plasma glutamine levels and falciparum malaria.

Glutamine deficiency is associated with increased rates of sepsis and mortality, which can be prevented by glutamine supplementation. Changes in glutamine concentration were examined in Ghanaian children with acute falciparum malaria and control cases. The mean (SD) plasma glutamine concentration was lower in patients with acute malaria (401 (82) mumol/L, n = 50) than in control patients (623 (67) mumol/L, n = 7; P < 0.001). Plasma glutamine concentrations all rose in convalescence. The mean (SD) increase in plasma glutamine was 202 (123) mumol/L (n = 18; P < 0.001) compared with acute infection. We conclude that acute falciparum malaria is associated with large decreases in plasma glutamine and these falls may increase susceptibility to sepsis and dyserythropoeisis.

Effectiveness of the particle repositioning maneuver in subtypes of benign paroxysmal positional vertigo.

OBJECTIVES: To assess the efficacy of the particle repositioning maneuver (PRM) in patients presenting with idiopathic benign paroxysmal positional vertigo (BPPV) compared with those with evidence of additional peripheral vestibulopathies. METHODS: Retrospective administration of the Dizziness Handicap Inventory (DHI) to 41 patients with primary BPPV and 31 patients with secondary BPPV to subjectively evaluate their symptoms before and after the PRM. RESULTS: Both groups indicated a marked improvement in symptoms after the PRM. Only two patients reported an increase in their symptoms after the PRM and both had secondary BPPV. CONCLUSIONS: The PRM was found to be highly effective in all forms of BPPV, but careful history and judicious testing may identify patients requiring additional intervention to relieve their symptoms.

Cerebrospinal Fluid Studies in Kenyan Children with Severe Falciparum Malaria.

The pathogenesis of the neurological complications of Plasmodium falciparum malaria is unclear. We measured proteins and amino acids in paired plasma and cerebrospinal fluid (CSF) samples in children with severe falciparum malaria, to assess the integrity of the blood brain barrier (BBB), and look for evidence of intrathecal synthesis of immunoglobulins, excitotoxins and brain damage. METHODS: Proteins of different molecular sizes and immunoglobulins were measured in paired CSF and plasma samples in children with falciparum malaria and either impaired consciousness, prostrate, or seizures. RESULTS: The ratio of CSF to plasma albumin (Q(alb)) exceeded the reference values in 42 (51%) children. The CSF concentrations of the excitotoxic amino acid aspartate and many non-polar amino acids, except alanine, were above the reference value, despite normal plasma concentrations. IgM concentrations were elevated in 21 (46%) and the IgM index was raised in 22 (52%). Identical IgG oligoclonal bands were found in 9 (35%), but only one patient had an increase in the CSF IgG without a concomitant increase in plasma indicating intrathecal synthesis of IgG. CONCLUSIONS: This study indicates that the BBB is mildly impaired in some children with severe falciparum malaria, and this impairment is not confined to cerebral malaria, but also occurs in children with prostrate malaria and to a lesser extent the children with malaria and seizures. There is evidence of intrathecal synthesis of immunoglobulins in children with malaria, but this requires further investigation. This finding, together with raised level of excitotoxic amino acid aspartate could contribute to the pathogenesis of neurological complications in malaria.

L-Glutamine influx in malaria-infected erythrocytes: a target for antimalarials?

When malaria parasites enter red blood cells they precipitate on influx of substrates necessary for their development. For example, intraerythrocytic trophozoites of Plasmodium falciparum use exogenous t-glutamine in increasing amounts during maturation from the ring-stage. This is made possible by a marked and selective increase in the permeability of the host cell membrane. Several compounds have now been identified as inhibitors of the l-glutamine influx induced by P. falciparum; they are all natural products - either analogues of l-glutamine, or related to indigenous traditional remedies for malaria. In this article, Barry El ford shows that although these compounds may not be of immediate practical value as antimolarials, they can provide valuable insight into the mechanisms underlying the regulation of these parasite-mediated transport processes.

Permeation and distribution of deuterated and tritiated water in smooth and striated muscle.

1. The diffusion of deuterated water (HDO) into and out of isolated guinea-pig taenia coli and frog sartorius was followed by means of a recording electronic microbalance.2. A comparison was made of the rates of efflux of deuterated and tritiated (HTO) water using the microbalance and a standard tracer technique.3. The time course of the exchange of HDO in both smooth and striated muscle was fitted to the sum of two exponential functions; however, it was not possible to correlate sizes of the intra- and extracellular spaces in the muscles with the sizes of the two compartments in model systems based on compartmental theory of tracer kinetics.4. The efflux of HDO, which was measured by means of the microbalance under stagnant conditions, occurred at a rate approximately 3 times slower than the efflux of HTO which took place while muscles were agitated vigorously in their bathing media.5. There was a marked deviation at short times from a double exponential time course during the efflux of HTO.6. Values of the tracer water permeability, P(d), of smooth and striated muscle fibres, and the extracellular space, E, of the muscles were obtained using a computer to numerically evaluate the series solutions of the equations describing the permeation of labelled water in the muscles.7. It was necessary to assume a value for the diffusion coefficient of the tracer in extracellular fluid in order to compute a unique fit to an exchange curve.8. Values of P(d) and E given by the analysis depended upon the method used for monitoring the exchange, and an interpretation of this dependence was made difficult by the variation in the thickness of unstirred layers on the surface of the muscles under the different experimental conditions.

Diffusion and distribution of dimethyl sulphoxide in the isolated guinea-pig taenia coli.

1. The diffusion of the cryoprotective non-electrolyte dimethyl sulphoxide (DMSO) in the isolated guinea-pig taenia coli at 37, 25 and 0 degrees C has been studied using [(35)S]DMSO.2. Within 1 hr after immersion at 37 degrees C in Krebs solution containing 20% (w/v) DMSO and trace amounts of [(35)S]DMSO, the non-electrolyte was distributed uniformly throughout a volume equivalent to the total initial water content of the muscle.3. The kinetics of efflux of [(35)S]DMSO from muscles at constant volume were analysed on the basis of two models: one incorporated radial diffusion in extracellular fluid with simultaneous permeation into the cells, the other involved only radial diffusion in homogeneous cylinders of tissue having no internal barriers to diffusion; the former was found to give a better representation of the efflux kinetics.4. If it was assumed that the rate of diffusion of DMSO in the extracellular space of taenia coli was the same as that in the bathing medium, the values of the extracellular space and the permeability of smooth muscle to DMSO, obtained from the analysis of the efflux kinetics, were 454 +/- 19 ml./kg and 2.36 +/- 0.05 x 10(-6) cm sec(-1) at 37 degrees C.5. The activation energy for the transfer of DMSO across the surface of the cell was estimated to be 6.0 kcal/mole at 37 degrees C, 6.6 kcal/mole at 25 degrees C and 11.6 kcal/mole at 0 degrees C, indicating either that the equivalent pore radius of the cells decreased with temperature or that the cell permeability represented the sum of two fluxes, one through the aqueous pores of the cell and the other through the lipid phase of the cell membrane, each with a different energy of activation.6. A net flux of water across the surface of the cells, superimposed on the efflux of DMSO, markedly affected the rate of diffusion of the non-electrolyte out of the whole tissue; however, it was considered that an analysis of the efflux kinetics was not possible under these conditions.7. These results provide a basis for methods which will be used to investigate the possibility of preserving tissue in unfrozen aqueous media at sub-zero temperatures.

Interactions between temperature and tonicity on cation transport in dog red cells.

1. The temperature-dependence of the uptake of 24Na and 42K into dog red cells between 38 and 4 degrees C has been investigated. The effects on the cation fluxes of partial dehydration of the cells in hyperosmolar sucrose (50-125 mM) have also been studied. 2. A Hamilton gas-tight syringe was used to pipette accurately reproducible volumes of packed cells which contained in addition to 24Na or 42K either [131I]albumin or [51Cr]EDTA as extracellular markers. 3. At 38 degrees C Na flux (m-equiv/l. isosmolar cell volume. hr) increased from 2-8 +/- 0-1 (n = 8) in cells of normal volume to 226 +/- 8 (n = 8) when the cells were shrunken by 27-4 +/- 0-6% (n = 8) in media containing sucrose (100 mM). K influx remained relatively constant under these conditions. 4. The exchange of 24Na in shrunken cells followed a single exponential time course but about 9% of the intracellular Na apparently did not exchange with 24Na in the bathing medium. 5. The steady-state influx of Na in cells of normal volume was maximal at about 22 degrees C. The temperature dependence of the Na fluxes in shrunken cells was described by an Arrhenius relationship with a change in slope at about 22 degrees C. 6. The K influx in cells of normal volume decreased as the temperature was lowered from 38 degrees C, to about 12 degrees C, at which temperature the flux was at a well defined minimum. Above 12 degrees C, cell shrinkage had hardly any effect on K influx, but below 12 degrees C the influx in shrunken cells was significantly less than in cells of normal volume. 7. The selective increase in Na flux induced by cell shrinkage results from a Na:Na exchange process which cannot be explained in terms of Ussing's (1947) model of carrier-mediated exchange diffusion. 8. The lack of coupling between the effects of temperature and cell volume on the fluxes of Na and K indicates that localized structural changes of lipid-protein complexes specific for Na or K are responsible for the cation transport characteristics of dog red cells, and that phase transitions in the lipids of the cell membrane are unlikely to account for the temperature dependence of the fluxes.

Transport of diverse substrates into malaria-infected erythrocytes via a pathway showing functional characteristics of a chloride channel.

Following infection by the malaria parasite, Plasmodium falciparum, human erythrocytes show increased permeability to a variety of low molecular weight solutes. In this study a number of anion transport blockers were identified as potent inhibitors of the transport of a wide range of solutes into human erythrocytes infected in vitro with P. falciparum. 5-Nitro-2-(3-phenyl-propylamino)benzoic acid (NPPB), furosemide, and niflumate blocked the malaria-induced transport of monovalent cations, neutral amino acids, sugars, nucleosides, and monovalent anions. For all of the substrates tested the order of potency of these three inhibitors was the same (NPPB > furosemide > niflumate) and dose-response curves for the effect of these inhibitors on malaria-induced choline transport were similar to those for malaria-induced thymidine transport. The data suggest that much, if not all, of the high capacity (non-saturable) transport of low molecular weight solutes into P. falciparum-infected erythrocytes is via a single type of pathway. The broad specificity of the pathway, its non-saturability in the physiological concentration range, and its failure to distinguish between stereoisomers (L- and D-alanine) are consistent with its being a type of pore or channel. For those substrates for which quantitative influx measurements were made the magnitude of the malaria-induced (inhibitor-sensitive) transport was in the order: Cl- > lactate > thymidine, adenosine > carnitine > choline > K+. The pathway is therefore anion-selective. The pharmacological and substrate-selectivity properties of the pathway show marked similarities to those of chloride channels in other cell types; this raises the possibility that the high capacity transport of small organic solutes may be an important and, as yet, largely unrecognized role for such channels in other tissues.

Enhanced choline and Rb+ transport in human erythrocytes infected with the malaria parasite Plasmodium falciparum.

Human erythrocytes infected in vitro with the malaria parasite Plasmodium falciparum showed a markedly increased rate of choline influx compared with normal cells. Choline transport into uninfected cells (cultured in parallel with infected cells) obeyed Michaelis-Menten kinetics (Km approximately 11 microM). In malaria-parasite-infected cells there was an additional choline-transport component which failed to saturate at extracellular concentrations of up to 500 microM. This component was less sensitive than the endogenous transporter to inhibition by the Cinchona bark alkaloids quinine, quinidine, cinchonine and cinchonidine, but showed a much greater sensitivity than the native system to inhibition by piperine. The sensitivity of the induced choline transport to these reagents was similar to that of the malaria-induced (ouabain- and bumetanide-resistant) Rb(+)-transport pathway; however, the relative magnitudes of the piperine-sensitive choline and Rb+ fluxes in malaria-parasite-infected cells varied between cultures. This suggests either that the enhanced transport of the two cations was via functionally distinct (albeit pharmacologically similar) pathways, or that the transport was mediated by a pathway with variable substrate selectivity.

Potent antimalarial activity of clotrimazole in in vitro cultures of Plasmodium falciparum.

The increasing resistance of the malaria parasite Plasmodium falciparum to currently available drugs demands a continuous effort to develop new antimalarial agents. In this quest, the identification of antimalarial effects of drugs already in use for other therapies represents an attractive approach with potentially rapid clinical application. We have found that the extensively used antimycotic drug clotrimazole (CLT) effectively and rapidly inhibited parasite growth in five different strains of P. falciparum, in vitro, irrespective of their chloroquine sensitivity. The concentrations for 50% inhibition (IC(50)), assessed by parasite incorporation of [(3)H]hypoxanthine, were between 0.2 and 1.1 microM. CLT concentrations of 2 microM and above caused a sharp decline in parasitemia, complete inhibition of parasite replication, and destruction of parasites and host cells within a single intraerythrocytic asexual cycle (approximately 48 hr). These concentrations are within the plasma levels known to be attained in humans after oral administration of the drug. The effects were associated with distinct morphological changes. Transient exposure of ring-stage parasites to 2.5 microM CLT for a period of 12 hr caused a delay in development in a fraction of parasites that reverted to normal after drug removal; 24-hr exposure to the same concentration caused total destruction of parasites and parasitized cells. Chloroquine antagonized the effects of CLT whereas mefloquine was synergistic. The present study suggests that CLT holds much promise as an antimalarial agent and that it is suitable for a clinical study in P. falciparum malaria.

Antimalarial activity of Artemisia annua flavonoids from whole plants and cell cultures.

Cell suspension cultures developed from Artemisia annua exhibited antimalarial activity against Plasmodium faldparum in vitro both in the n-hexane extract of the plant cell culture medium and in the chloroform extract of the cells. Trace amounts of the antimalarial sesquiterpene lactone artemisinin may account for the activity of the n-hexane fraction but only the methoxylated flavonoids artemetin, chrysoplenetin, chrysosplenol-D and cirsilineol can account for the activity of the chloroform extract. These purified flavonoids were found to have IC50 values at 2.4 - 6.5 × 10(-5)M against P. falciparum in vitro compared with an IC50 value of about 3 × 10(-8)M for purified artimisinin. At concentrations of 5 × 10(-6)M these flavonoids were not active against P. falciparum but did have a marked and selective potentiating effect on the antiplasmodial activity of artemisinin.