Rekha Chevli

High-affinity accumulation of chloroquine by mouse erythrocytes infected with Plasmodium berghei.

Washed erythrocytes infected with chloroquine-susceptible (CS) or with chloroquine-resistant (CR) P. berghei were used in model systems in vitro to study the accumulation of chloroquine with high affinity. The CS model could achieve distribution ratios (chloroquine in cells: chloroquine in medium) of 100 in the absence of substrate. 200-300 in the presence of 10 mM pyruvate or lactate, and over 600 in the presence of 1 mM glucose or glycerol. In comparable studies of the CR model, the distribution ratios were 100 in the absence of substrate and 300 or less in the presence of glucose or glycerol. The presence of lactate stimulated chloroquine accumulation in the CR model, whereas the presence of pyruvate did not. Lactate production from glucose and glycerol was undiminished in the CR model, and ATP concentrations were higher than in the CS model. Cold, iodoacetate, 2,4-dinitrophenol, or decreasing pH inhibited chloroquine accumulation in both models. These findings demonstrate substrate involvement in the accumulation of chloroquine with high affinity.In studies of the CS model, certain compounds competitively inhibited chloroquine accumulation, while others did not. This finding is attributable to a specific receptor that imposes structural constraints on the process of accumulation. For chloroquine analogues, the position and length of the side chain, the terminal nitrogen atom of the side chain, and the nitrogen atom in the quinoline ring are important determinants of binding to this receptor.

β-Guanidinopropionate and phosphorylated β-guanidinopropionate as substrates for creatine kinase

Abstract Rabbit muscle creatine kinase catalyzes transphosphorylation between ATP and β-guanidinopropionate at pH 9 and 23°C with a K m for β-guanidinopropionate of 50 m m and a V max of 0.21 μmole/min/mg of creatine kinase. The comparable values for the phosphorylation of creatine are 16.7 for K m and 75.1 for V max . In the reverse direction at pH 7.05 and 23°C, phosphorylated β-guanidinopropionate has a K m of 4.9 and a V max of 0.22 in comparison to a K m of 2.2 and a V max of 231 for phosphocreatine.

Chloroquine-Resistant Plasmodium falciparum: Effect of Substrate on Chloroquine and Amodiaquin Accumulation

Glucose stimulates the high-affinity processes of chloroquine and amodiaquin accumulation in owl monkey erythrocytes infected with a chloroquine-susceptible strain of Plasmodium falciparum. Although these erythrocytes have greater ability to accumulate amodiaquin than chloroquine, glucose has relatively less effect on amodiaquin accumulation than on chloroquine accumulation. In contrast to these findings with chloroquine-susceptible P. falciparum, glucose stimulates amodiaquin but not chloroquine accumulation in erythrocytes infected with chloroquine-resistant P. falciparum. This lack of function of a substrate-dependent component of chloroquine accumulation distinguishes chloroquine-resistant from chloroquine-susceptible P. falciparum.

Chloroquine Resistance in Malaria: Accessibility of Drug Receptors to Mefloquine

The process of mefloquine accumulation was studied in mouse erythrocytes infected with either Plasmodium berghei CS (chloroquine susceptible) or P. berghei CR (chloroquine resistant). In both cases, mefloquine was accumulated by a saturable process with an apparent dissociation constant of 2.5 × 10−6 M and an apparent maximal capacity of 700 μmol per kg of erythrocyte pellet; uninfected mouse erythrocytes accumulated more than half as much mefloquine as infected erythrocytes. The process of accumulation was not stimulated by providing glucose as a substrate, and it was not inhibited in infected erythrocytes by azide, iodoacetate, or incubation at 2°C. Although mefloquine was accumulated more effectively than chloroquine by uninfected erythrocytes and by erythrocytes infected with P. berghei CR, competition between chloroquine and mefloquine was observed, raising the possibility that the same process of accumulation serves both drugs. Chloroquine competitively inhibits mefloquine accumulation, with an apparent inhibitor constant of 1.7 × 10−3 M, and mefloquine competitively inhibits chloroquine accumulation, with an apparent inhibitor constant of 2 × 10−6 M. The same process of accumulation and the same group of receptors could serve both drugs if mefloquine has greater access than chloroquine to the receptors. Regardless of whether the same process serves both drugs, undiminished accumulation by erythrocytes infected with P. berghei CR provides an explanation for the superiority of mefloquine in treating chloroquine-resistant malaria.

Inhibition of creatine and phosphocreatine accumulation in skeletal muscle and heart

Abstract A series of creatine analogues was tested for ability to inhibit 14C-creatine accumulation in vivo and for ability to serve as substrates for creatine kinase in vitro. The most potent inhibitors of accumulation were 1-carboxymethyl-2-imino-hexahydropyrimidine, N-methylamidino-N-methylglycine, β-guanidinopropionate, and DL-β-guanidinobutyrate. The first two compounds are known to be inactive as substrates for creatine kinase, and DL-β-guanidinobutyrate was shown to be inactive in the present experiments, producing a Vmax less than 0.01% of that produced by creatine. The latter compound and β-guanidinopropionate were also found to be ineffective as inhibitors of creatine kinase in vitro. The best substrates for creatine kinase were N-ethyl-N-amidinoglycine and guanidinoacetate; these two compounds were inefficient as inhibitors of 14C-creatine accumulation. Moreover, adding a methyl group to β-guanidinopropionate to form N-methyl-N-amidino-β-alanine eliminated the compounds ability to inhibit 14C-creatine accumulation but had little effect on its ability to serve as a substrate for creatine kinase. Thus, we found no evidence that creatine kinase mediates creatine transport. In feeding trials with rats given DL-β-guanidinobutyric acid as 2% or 6% of their diet for 1 mo, this compound accumulated to concentrations of 14 μmole/g wet weight in skeletal muscle and of 4 μmole/g wet weight in heart. Simultaneously, in skeletal muscle there was a depletion of phosphocreatine to 50%, and of ATP to 80%, of control values. The total creatine content (creatine + phosphocreatine) of the heart also decreased, going from 12.8 to 2.8 μmole/g wet weight. Animals fed DL-β-guanidinobutyric acid should be suitable for studies of the long-term effects of creatine and phosphocreatine depletion in skeletal muscle and heart.

Biochemical and ultrastructural changes in skeletal muscle induced by a creatine antagonist

To evaluate the essentiality of creatine and phosphocreatine for the maintenance of the ultrastructure of skeletal muscle, chicks were fed a creatine antagonist, beta-guanidinobutyric acid (beta-GBA), as 2% of a Chow diet. Chicks fed beta-GBA exhibited growth retardation and weakness, and they accumulated large amounts of a monosubstituted guanidino compound, presumably beta-GBA, in their skeletal muscles. After 2 wk, there was a 74% decrease in the uptake of [14C]-1-creatine into pectoralis muscles of chicks fed beta-GBA. After 2 wk there as a significant decrease in phosphocreatine concentrations in pectoralis muscles from 20.1 +/- 2.8 mumoles per g wet weight (mean +/- S.D.) for 8 control chicks to 16.5 +/- 2.5 for 7 chicks fed beta-GBA. Selected fibers of the pectoralis and gastrocnemius muscles of chicks fed beta-GBA exhibited ultrastructural abnormalities including loss of thick and thin filaments, disruption of the Z band, dilated mitochondria, and dilated and displaced sarcoplasmic reticulum. The pectoralis muscles of chicks given 6% creatine in addition to 2% beta-GBA in the diet accumulated little beta-GBA, maintained normal phosphocreatine concentrations, and exhibited no significant ultrastructural abnormalities. These findings are the first experimental evidence that high concentrations of phosphocreatine are essential for the maintenance of the ultrastructural integrity of skeletal muscle.

Sustained isometric contraction of skeletal muscle depleted of phosphocreatine.

Abstract To evaluate the need for phosphocreatine as an energy reservoir to sustain isometric contraction of skeletal muscle, rats were depleted of phosphocreatine by feeding β-GPA (β-guanidinopropionate) as 1% of the diet. In the place of phosphocreatine, β-GPAP (phosphorylated β-GPA) accumulated to concentrations of 20–25 μmoles/g wet weight of muscle. Although the maximum isometric tension produced by the soleus was always less than that produced by the plantaris muscle, the maximum for either muscle was not significantly affected by feeding β-GPA. The endurance of experimental soleus muscles was prolonged, however. These muscles held 70% of their maximum isometric tension for 106 ± 40 seconds (mean ± SD, n = 4) whereas the value for five control muscles was 43 ± 18 seconds. With fatiguing, isometric contractions of control plantaris and soleus muscles, phosphocreatine concentrations decreased by 68–70%; in experimental muscles, the β-GPA concentration decreased less than 12%. This difference in phosphagen consumption demonstrates that skeletal muscle can sustain fatiguing, isometric contractions without using large amounts of phosphocreatine or a substitute phosphagen as an energy reservoir. Phosphocreatine hydrolysis during muscle contraction normally may serve some other purpose.

Measurement of β-guanidinopropionate and phosphorylated β-guanidinopropionate in tissues

Abstract The Sakaguchi color reaction for monosubstituted guanidino compounds was applied to the measurement of β-guanidinopropionate and phosphorylated β-guanidinopropionate. The phosphorylated derivative was measured as an increase in β-guanidinopropionate following incubation with 0.1 n HCl in a boiling-water bath for 10 min. After feeding rats 1% of β-guanidinopropionic acid in their diet for 69 days, skeletal muscle, heart, liver, kidney, and spleen contained 5–10 μmoles of a monosubstituted guanidino compound per gram wet weight of tissue. No β-guanidinopropionate was detected in brain or testes. Phosphorylated β-guanidinopropionate was found only in skeletal muscle (27 μmoles/g) and in heart (7 μmoles/g). Creatine hydrate (2%) added to the diet containing β-guanidinopropionic acid inhibited the accumulation of phosphorylated β-guanidinopropionate in the heart and partially inhibited its accumulation in skeletal muscle.

Chloroquine accumulation by erythrocytes: A latent capability

Abstract Treatment of normal mouse erythrocytes with a nonspecific protease from streptomyces griseus activates a saturable process for chloroquine accumulation. The apparent dissociation constant for the interaction of chloroquine with the saturable component of this latent process is 50 nM. This value is similar to that of a high-affinity process of chloroquine accumulation which hitherto has been observed only in erythrocytes infected with malaria parasites. In addition, the capacity of the latent process, approximately 30 μmoles per Kg of erythrocytes, is sufficient to account for the accumulation of chloroquine by erythrocytes infected with malaria parasites. These findings demonstrate the plausibility of attributing the ability to accumulate chloroquine with high affinity to the erythrocyte host rather than to the parasite.

Erythrocyte Surface: Novel Determinant of Drug Susceptibility in Rodent Malaria

To study the role of the erythrocyte membrane in the process of chloroquine accumulation, surface polypeptides were digested with a nonspecific protease from Streptomyces griseus. This treatment activated a saturable process of chloroquine accumulation with an affinity and a specificity similar to those of mouse erythrocytes infected with Plasmodium berghei CS (chloroquine susceptible). Studies of competitive inhibitors of chloroquine accumulation yielded the following approximate values for Ki: amodiaquine, 2 × 10−7 M; quinacrine, 5 × 10−7 M; quinine, 2 × 10−6 M; and mefloquine, 2 × 10−5 M. Lack of a substrate requirement distinguished this process from the one used by P. berghei and permitted the protease to be used in studies of infected erythrocytes. Protease treatment of erythrocytes infected with P. berghei CR (chloroquine resistant) produced a dramatic transformation. Instead of describing a sigmoid curve, the process of chloroquine accumulation became saturable and substrate dependent, with a Kdiss of approximately 10−8 M; i.e., protease-treated erythrocytes infected with P. berghei CR now behaved similarly to those infected with P. berghei CS. Coating the erythrocyte surface with albumin completely inhibited the protease-activated process of chloroquine accumulation. These findings are presented as evidence that erythrocyte surface components determine the affinity with which chloroquine is accumulated and thereby determine whether or not the malaria parasite will be susceptible to the drug.