14C-Desferrioxamine B: Uptake into erythrocytes infected withPlasmodium falciparum

Plasmodium falciparum-infected human erythrocytes (early trophozoite stages) and non-infected erythrocytes were incubated in 1.7 mM ¹⁴C-desferrioxamine B (specific activity 1 Ci/2.6 mg desferrioxamine B). After 270 min the cells were washed and the radioactivity was measured in the cell pellet and, after lysis, in cytoplasm and membranes. The results indicate that Desferrioxamine B can the red blood cell and pass through the parasite membrane and that the parasites are killed by the intracellular action of the chelator.     

Ebselen is a specific inhibitor of LTB4-mediated migration of human neutrophils

Ebselen is a seleno-organic anti-inflammatory compound with glutathione peroxidase-like activity that has the unique characteristic of mediating the isomerization of 5-HETE and LTB₄ to their biologically inactive trans isomers, both directly in fluid phase and indirectly through metabolic pathways in stimulated peripheral blood leukocytes. LTB₄ is an inflammatory mediator with potent chemotactic activity for neutrophilic leukocytes. We studied the effects of ebselen on the chemotactic and chemokinetic responses with human-blood-derived neutrophils. With the use of 120-m-thick 5-m-pore durapore filters and low BSA concentrations (0.05%) in the chemotaxis buffers, ebselen was evaluated for its effect on both chemotactic and chemokinetic responses to LTB₄, C5a, and fMLP. Ebselen at 3–20 M concentrations inhibited both chemotactic and chemokinetic responses to optimal concentrations of LTB₄ without altering chemotactic responses to C5a or fMLP. Likewise, ebselen at 20 M specifically inhibited LTB₄-stimulated transendothelial migration of neutrophils, while not altering responses to C5a nor fMLP.     

Naegleria fowleri andN. lovaniensis: Differences in sensitivity to trimethoprim and other antifolates

The growth ofNaegleria fowleri cultures in a BCS medium was not affected either by trimethoprim at 400 g/ml or by aminopterine, 3,5-diaminopterine and methotrexate at 500 g/ml.N. lovaniensis propagation in the same medium was inhibited with 10 g/ml of trimethoprim, 50 g/ml methotrexate and 100 g/ml 3,5-diaminopteridine. Aminopterine was ineffective at a concentration of 500 g/ml. The inhibitory effect of trimethoprim onN. lovaniensis cultures depended on the medium composition and could be neutralized by an addition of folic or tetrahydrofolic acids and a suspension of heat-killedEnterobacter aerogenes. Thymine, thymidine, hypoxantine and 2-amino-4-hydroxy-6-(tatrahydroxybutyl)-pteridine did not have an adverse effect. Trimethoprim activity inN. fowleri cultures could not be enhanced by the addition of Triton X-100 and Polymyxine B. Cryolyzate ofN. fowleri amoebae did not influence the trimethoprim inhibition ofN. lovaniensis cultures. Deviation in dihydrofolatereductase chemical structure or thymine dependency seems to be the probable explanation forN. fowleri antifolate resistance.     

Glutathione and peroxide metabolism in malaria-parasitized erythrocytes

The glutathione metabolism of Plasmodium falciparum, P. vinckei and P. berghei has been investigated. Human erythrocytes with low glutathione reductase and synthetase activity are still capable of harbouring P. falciparum. Both enzymes have been demonstrated in Plasmodium spp. Moreover, evidence is given for a selenium-independent glutathione peroxidase in malaria parasites.     

Phenotypic differences between induced and spontaneous 2μ-plasmid-free segregants of Saccharomyces cerevisiae

Summary The maximum specific growth rates (_max) of 2 -plasmid-free ([cir°]) segregants of three haploid and one diploid strain of Saccharomyces cerevisiae have been determined and compared with the _max of their 2 -plasmid-containing ([cir ⁺]) progenitors. Two classes of [cir°] strains have been examined: those induced by transformation with a 2 -based recombinant plasmid according to the method of Dobson et al. (1980) and those isolated as spontaneous [cir°] segregants from glucose-limited continuous cultures. The _max of the spontaneous [cir°] segregants was not found to differ significantly from that of their [cir ⁺] parents. In all cases, however, the induced [cir°] strains had a _max which was significantly less than that of their [cir ⁺] counterparts. This effect was particularly marked in the case of the diploid strain where a 34% reduction in _max was observed. The implications of these results are discussed in terms of the effect of the transformation process on host yeast cells.     

Susceptibility testing quality control studies with fosfomycin tromethamine

Multiple studies were performed to define the quality control ranges for susceptibility tests of fosfomycin tromethamine. For disk diffusion tests, the limits proposed are 22 to 30 mm forEscherichia coli ATCC 25922 and 25 to 33 mm forStaphylococcus aureus ATCC 25923. Broth microdilution tests were not reproducible. For agar dilution tests, ranges proposed are 0.5 to 2.0 g/ml forEscherichia coli ATCC 25922, 0.5 to 4.0 g/ml forStaphylococcus aureus ATCC 29213; 2.0 to 8.0 g/ml forPseudomonas aeruginosa ATCC 27853, and 32 to 128 g/ml forEnterococcus faecalis ATCC 29212.     

On the regulation of the expressed L-type calcium channel by cAMP-dependent phosphorylation

The Ca²⁺ channel subunits _1C-a and _1C-b were stably expressed in Chinese hamster ovary (CHO) and human embryonic kidney (HEK) 293 cells. The peak Ba²⁺ current (I _Ba) of these cells was not affected significantly by internal dialysis with 0.1 mM cAMP-dependent protein kinase inhibitor peptide (mPKI), 25 M cAMP-dependent protein kinase catalytic subunit (PKA), or a combination of 25 M PKA and 1 M okadaic acid. The activity of the _1C-b channel subunit expressed stably in HEK 293 cells was depressed by 1 M H 89 and was not increased by superfusion with 5 M forskolin plus 20 M isobutylmethylxanthine (IBMX). The _1C-a·₂·₂/ complex was transiently expressed in HEK 293 cells; it was inhibited by internal dialysis of the cells with 1 M H 89, but was not affected by internal dialysis with mPKI, PKA or microcystin. Internal dialysis of cells expressing the _1C-a·₂·₂/ channel with 10 M PKA did not induce facilitation after a 150-ms prepulse to +50 mV. The Ca²⁺ current (I _Ca) of cardiac myocytes increased threefold during internal dialysis with 5 M PKA or 25 M microcystin and during external superfusion with 0.1 M isoproterenol or 5 M forskolin plus 50 M IBMX. These results indicate that the L-type Ca²⁺ channel expressed is not modulated by cAMP-dependent phosphorylation to the same extent as in native cardiac myocytes.     

Control of pulmonary vascular smooth muscle tone by sarcoplasmic reticulum ca2+ pump blockers: thapsigargin and cyclopiazonic acid

The effect of thapsigargin (TG) and cyclopiazonic acid (CPA) on the mechanical activity of the rat pulmonary artery were investigated. In chemically (-escin)-skinned arterial strips, application of TG (0.1–1 M) or CPA (0.5–10 M) prior and throughout the loading procedure of the internal Ca²⁺ stores (0.3 M free Ca²⁺ ions for 8–10 min) concentration dependently inhibited the subsequent contractile response induced by noradrenaline (NA, 10 M) or caffeine (25 mM). In intact strips repeatedly incubated in a Ca²⁺-containing solution (2.5 mM for 10 min), followed by incubation in a Ca²⁺-free solution 12 min before NA-stimulation, TG and CPA not only inhibited the NA-induced contraction but also increased the tension which appeared during the exposure time to Ca²⁺. The two phenomena developed with similar time courses. The increase in tension during the readmission of Ca²⁺ ions was not antagonized by verapamil (10 M) or nifedipine (1 M) but was blocked by La³⁺ (50 M) and Co²⁺ (1 mM) ions. The amplitude of the verapamil-insensitive TG (or CPA)-induced contraction was dependent on the external [Ca²⁺] [0.1–10 mM, concentration for half maximal effect (EC₅₀) =0.85 mM], not modified by the reduction of the external [Na⁺] (from 130 to 10 mM) and decreased by depolarization of the strip using K⁺-rich (30–120 mM) solutions. Under the latter condition, 38±9 and 83±4% reduction (n=5) was observed in the presence of 60 and 120 mM K⁺ respectively. This contraction was also concentration dependently inhibited by the tyrosine kinase inhibitors genistein (0.5–50 M) and tyrphostin (2–50 M). Sr²⁺ ions, which contracted both depolarized intact and skinned strips, failed to replace Ca²⁺ ions in the verapamil-insensitive contraction induced by TG or CPA (n=4). Finally, TG (1 M) and CPA (10 M) did not modify the pCa tension relationship in skinned strips (n=5). These results show that the main action of TG and CPA in rat pulmonary artery is to prevent the refilling of the internal Ca²⁺ store. TG and CPA also seem to facilitate a Ca²⁺ influx through a specific verapamil-insensitive pathway. The biophysical and molecular characteristics of this pathway remain to be elucitated, although it appears to involve a tyrosine kinase activity.     

Expression of surface membrane IgG on pokeweed mitogen-reactive anti-tetanus toxoid antibody-producing cells

Anti-tetanus toxoid antibody-producing cells, differentially expressing surface membrane IgM, were analyzed for the additional expression of surface membrane IgG. ⁺ and ^– cells were rosetted with anti--ox red blood cells and separated by density centrifugation into fractions enriched or depleted or ⁺ cells. These B-cell subsets were assayed for the production of IgM and IgG anti-tetanus toxoid antibody and total IgM and IgG. The results indicated that the majority of anti-tetanus toxoid antibody synthesis in the ^– fraction was by ⁺ cells. In the ⁺ fraction, however, both IgM and IgG anti-tetanus toxoid antibody production was detected in the ^+– and ⁺⁺ fraction. The inclusion of isotype-specific antisera during the first 2 days of culture further established that was expressed on the surface of the majority of the precursors for IgG anti-tetanus antibody productionin vitro. Studies performed to determine the culture requirements of ^– and ⁺ cells revealed that production of IgG anti-tetanus toxoid antibody by both cell subsets was dependent on T cells and pokeweed mitogen. However, some ^– cells could produce IgG in the presence of T cells alone.     

Growth-inhibitory effect of a fucoidan from brown seaweed <Emphasis Type="Italic">Undaria pinnatifida</Emphasis> on <Emphasis Type="Italic">Plasmodium</Emphasis> parasites

The present study was undertaken to investigate the inhibitory effects of fucoidan, a sulfated polysaccharide isolated from the edible brown seaweed Undaria pinnatifida, on the growth of Plasmodium parasites. In order to assess the anti-malarial activity of fucoidan, growth inhibition activities were evaluated using cultured Plasmodium falciparum parasites in vitro and on Plasmodium berghei-infected mice in vivo. Fucoidan significantly inhibited the invasion of erythrocytes by P. falciparum merozoites, and its 50% inhibition concentration was similar to those for the chloroquine-sensitive P. falciparum 3D7 strain and the chloroquine-resistant K1 strain. Four-day suppressive testing in P. berghei-infected mice with fucoidan resulted in a 37% suppressive effect versus the control group and a delay in death associated with anemia (P < 0.05). In addition, fucoidans had no toxic effect on RAW 264.7 cells. These findings indicate that fucoidans from the Korean brown algae U. pinnatifida inhibits the invasion of Plasmodium merozoites into erythrocytes in vitro and in vivo. J.-H. Chen and J.-D. Lim contributed equally to this work.     

The effects of TMB-8 on the shape changes of vascular endothelial cells resulting from exposure to various inflammatory agents

Pre-treatment of the endothelial cells lining the guinea pig inferior vena cava with 8-(diethylamino) octyl 3,4,5-trimethoxybenzoate (TMB-8) (10 M)in vitro significantly reduced the shape changes resulting from subsequent exposure to platelet activating factor (PAF) (0.1 M), calcium ionophore A23187 (10 M), histamine (300 M), bradykinin (2 M), prostaglandin E₂ (PGE₂) (30 M), or leukotrienes (LT) C₄, D₄ or E₄ (1 M). Since TMB-8 is an intracellular calcium antagonist, this provides evidence to support the suggestion that these inflammatory agents increase the concentration of intracellular calcium which brings about a contraction of the actin-myosin complex resulting in endothelial cell shape changes, and the formation of interendothelial cell gaps.     

The effect of praziquantel on the pseudophyllidean cestodeBothriocephalus acheilognathi in vitro

AdultBothriocephalus acheilognathi were incubated in solutions containing 0 (control), 0.1, 1.0, 10.0 and 100 g praziquantel per ml (0, 10², 10³, 10⁴ and 10⁵ gl^–1) of 0.9% saline for 5, 15 and 60 min at a temperature of 18°C. The worms contracted immediately upon being placed in the drug. Scanning and transmission electron microscopy revealed considerable tegumental damage particularly in the neck region. Vacuolization and bubbling to the tegument occurred in all of the drug solutions tested. Exposure to drug concentrations of more than 1.0 gml^–1 (10³ gl^–1) praziquantel for 15 min or greater resulted in many of the bubbles bursting and releasing their contents to the exterior. Mature proglottides were distorted and had occasional large swellings resulting in the mass expulsion of eggs. Praziquantel had no ovicidal activity. Exposure to drug concentrations of 100 g (10⁵ gl^–1) praziquantel per ml saline for 24 h was not lethal to the worms.     

Functional expression and properties of the human skeletal muscle sodium channel

Full-length deoxyribonucleic acid, complementary (cDNA) constructs encoding the-subunit of the adult human skeletal muscle Na⁺ channel, hSkM1, were prepared. Functional expression was studied by electrophysiological recordings from cRNA-injectedXenopus oocytes and from transiently transfected tsA201 cells. The Na⁺ currents of hSkM1 had abnormally slow inactivation kinetics in oocytes, but relatively normal kinetics when expressed in the mammalian cell line. The inactivation kinetics of Na⁺ currents in oocytes, during a depolarization, were fitted by a weighted sum of two decaying exponentials. The time constant of the fast component was comparable to that of the single component observed in mammalian cells. The block of hSkM1 Na⁺ currents by the extracellular toxins tetrodotoxin (TTX) and -conotoxin (CTX) was measured. The IC₅₀ values were 25 nM (TTX) and 1.2 M (CTX) in oocytes. The potency of TTX is similar to that observed for the rat homolog rSkM1, but the potency of CTX is 22-fold lower in hSkM1, primarily due to a higher rate of toxin dissociation in hSkM1. Single-channel recordings were obtained from outside-out patches of oocytes expressing hSkM1. The single-channel conductance, 24.9 pS, is similar to that observed for rSkM1 expressed in oocytes.     

Tubular transport processes in proximal tubules of hypothyroid rats. Lack of relationship between thyroidal dependent rise of isotonic fluid reabsorption and Na+−K+-ATPase activity

Hypothyroid rats reconstituted with 10 g/kg b.w. per day of tri-iodothironine (T₃) for 4 days resulted in normal free T₃ and TSH levels. FT₃ levels were: 0.53±0.3 pg/ml in hypothyroid rats; 2.78±1.21 pg/ml in hormone reconstituted rats and 2.90±0.90 pg/ml in euthyroid rats. TSH levels were 3,508±513 g/ml in hypothyroid rats; 1,008±204 g/ml in reconstituted rats and 270±184 ng/ml in euthyroid rats.When hypothyroid rats were reconstituted with 50 g T₃/kg b.w. per day, TSH levels were nearly normal after 4 days (1,157±621 ng/ml). However FT₃ levels after 1–4 days were always higher than in euthyroid rats.Hypothyroid rats show a decrease in isotonic fluid reabsorption (J _v) in the proximal tubule (1.50±0.08 versus 4.96±0.23 10^–2 nl·mm^–1·s^–1 in euthyroid animals). 1 day after T₃ (10 g/kg b.w./day) injectionJ _v was increased significantly to 2.05±0.20 10^–2 nl·mm^–1·s^–1 and continued to increase during 4 days of T₃ reconstitution.When 50 g T₃/kg b.w./day was used,J _v increased to 2.75±0.07 after 1 day and to 3.10±0.42 10^–2 nl·mm^–1·s^–1 after 4 days.J _v was never reaching a value close to that of euthyroid rats because the tubular radius in hypothyroid rats (14.7±1.8 m) is less than that of euthyroid rats (19.2±0.5 m). The radius in hypothyroid rats treated with T₃ was unchanged over a 4 day course with either high or low doses of T₃.Na⁺–K⁺-ATPase activity was found to be 2.91±0.16 M P_i/h×mg protein in homogenates of kidney cortex from hypothyroid rats. Treatment of hypothyroid rats with 10 g or 50 g of T₃ resulted in an initial decrease in ATPase activity, followed by an increase to base level in hypothyroid rats with 10 g and a significantly higher level with 50 g. This decrease in ATPase activity was contrasted to the increase inJ _v.These data indicate that there is a dissociation between the effects of physiological doses of thyroid hormones on proximal tubular reabsorption and the effects of T₃ on Na⁺–K⁺-ATPase activity of kidney cortex. This leads to question the relationship between sodium transport and ATPase activity under physiological doses of thyroid hormones. An early effect of physiological doses of thyroid hormones on brush border Na⁺ permeability is suggested.     

Interaction of the novel penem CGP 31 608 and Its enantiomer with type Id beta-lactamase and penicillin-binding proteins

The novel penem CGP 31 608 (5R, 6S, 8R) and its enantiomer CGP 32 879 (5S, 6R, 8S) were shown to be essentially stable against hydrolysis by type Id -lactamase isolated from Pseudomonas aeruginosa 18S/H. CGP 31 608 was a potent progressive inhibitor of this enzyme (I50=32 M), which was only weakly inhibited by CGP 32 879 (I50=460 M). CGP 31 608 had the highest affinity for penicillin-binding protein (PBP) 4 from Escherichia coli K-12 (I50= 1 g/ml), followed by PBPs 2 (10 g/ml) and 1A/1Bs (100 g/ml); CGP 32 879 did not inhibit binding of ¹⁴ C-benzylpenicillin to the PBPs. The steric configuration of the -lactam nucleus of penems appears to strongly influence their affinity for -Iactamases and target PBPs. The balanced spectrum of CGP 31 608 may be explained by its -lactamase stability and affinity for several vital PBPs.     

Effect of thermal stress on glutathione metabolism in human erythrocytes

This is the first experiment to investigate the effect of heat and cold stress on glutathione metabolism in human erythrocytes. We immersed men at three different water temperatures for 10 min. At 39°C, no remarkable changes were observed. Levels of glutathione (GSH) decreased from 2.44 (0.14) to 1.80 (0.10) mol·ml red blood cells^–1 [mol·ml RBC^–1; mean (SEM);P<0.0005] and those of lipid peroxides increased from 1.87 (0.03) to 2.06 (0.04) nmol · ml RBC^–1 (P<0.01) after the immersion at 42°C. In contrast, levels of GSH increased from 2.46 (0.17) to 2.91 (0.17) mol · ml RBC^–1 (P<0.05) and those of lipid peroxides did not change after the immersion at 25°C. The activities of glutathione peroxidase decreased from 35.90 (1.83) to 34.33 (1.66) IU · g Hb^–1 (P < 0.01) after the immersion at 42°C; however, these activities did not change after the immersion at 25°C. The activities of glutathione reductase (both active and inactive forms) showed no changes at any temperatures. These changes indicate that heat stress causes oxidative stress in the human body; however, cold stress is thought to augment the activity of the antioxidative defence system. It is suggested that body exposure to hot environmental conditions should not be recommended for patients suffering from a damaged antioxidative defence system.     

Effects of ketamine on GABA-evoked excitability of peripheral nerve

Summary The effects of the dissociative anaesthetic, ketamine on GABA-evoked changes in the excitability of myelinated fibers of dorsal and ventral roots of isolated bullfrog sciatic nerves were examined. Ketamine alone (0.01–1000 M) evoked small increases (<5%) in dorsal root fiber excitability, as reflected in the half-maximal A-fiber compound action potential when concentrations were >10 M; with 0.1 M even larger increases (10%) were elicited in the ventral root fibers. As well, the increases evoked by 10 M ketamine were followed by graded decreases. 0.1 and 10 M ketamine, concentrations which by themselves had small or no effect, produced a dose-dependent depression of the large increases in excitability which are induced by activation of GABA receptors. In the presence of ketamine GABA concentration-response curves of the dorsal root fibers showed depression of the maximal response, while those of the ventral root fibers were shifted to the right. This apparent antagonism of GABA responses by ketamine may arise from blockade of receptor-mediated effects (e.g. K⁺/Cl^- currents and/or secondary depolarization from K⁺ accumulation), but could also be caused by a selective potentiation of hyperpolarizing receptors.     

Force–velocity and unloaded shortening velocity during graded potassium contractures in frog skeletal muscle fibres

Steady-state conditions of contraction, at maximal and submaximal forces, were produced in intact single muscle fibres, from Rana esculenta, using full tetani and graded K⁺-contractures. The uniformity in radial direction, of spreading of activation produced in K⁺-contractures, was checked in relation to the fibre diameters. The absolute isometric force was similar in tetani and maximal contractures, for fibres with diameters between 40 and 60 m, but not for fibres with diameters greater than about 70 m in which contracture force never reached tetanic force. The force–[K⁺]_o relation was similar for fibres with diameters between 40 and 60 m, but it was right shifted and it had a minor slope for fibres with diameters greater than 65–70 m. This suggests that only in the small diameter fibres (40–60 m) the activation does not fail to penetrate uniformly from the surface towards the fibre core. For fibres selected in the diameter range between 40 and 60 m, force–velocity relations and unloaded shortening velocities were determined in tetani and maximal and submaximal contractures. Data were obtained across a force range of 0.3 to 1 P ₀ (tetanic plateau force). Controlled velocity method was used to obtain force–velocity relations, and slack test to determine the unloaded shortening velocity (V _U). The values of the parameters characterising the force–velocity relation (V ₀ and a/P ₀) and V _U as determined by the slack test did not differ significantly in tetani and contractures, independent of the activation level or absolute force developed by the fibre. These results show that, at least within the range of forces tested, crossbridge kinetics is independent of the number of cycling crossbridges, in agreement with the prediction of the recruitment model of myofilament activation.     

Giant excised cardiac sarcolemmal membrane patches: sodium and sodium-calcium exchange currents

Summary A tightly-sealed cardiac sarcolemmal patch preparation of large diameter (10–16 m with 5–25 G seals) has been developed to study regulation of selected electrogenic mechanisms. Formation of a large readily accessible membrane surface area is achieved by incubating freshly isolated myocytes in a high KCl/zero calcium solution, which promotes separation of sarcolemma from myofilaments. The formation of large-diameter, high resistance seals is facilitated by depositing a neutral hydrocarbon film on electrode tips. Sodium currents in excised patches are stable for patch life-times of 15–40 min with peak current densities of 120 to 350 A/cm². Outward sodium-calcium exchange current is identified by its specific dependence on external calcium and internal sodium, inhibition by cobalt and dichlorobenzamil, and activation by <0.1M internal free, calcium. Maximum exchange current density is >20 A/cm².Supported by a Grant-in-Aid and an Established Investigatorship of the American Heart Association.     

The hypocalciuric effect of thiazides: subcellular localization of the action

The acute administration of thiazides results in a decrease in the urinary Ca²⁺/Na⁺ ratio, whereas chronic administration of these diuretics decreases calciuria. In both situations, Ca²⁺ transport is enhanced in the early part of the distal tubule. The purpose of our study was to determine whether the hypocalciuric action of thiazides was due to a change in the active transport of Ca²⁺ through the basolateral membrane of the nephron or to an effect (direct or indirect) on the permeability of the distal tubule luminal membrane to calcium. In order to detect intrinsic differences between membranes of the proximal and distal tubules, the effect of the diuretic was examined in proximal and distal tubule preparations, and in basolateral and luminal membranes from the two segments separately.Preincubation of microdissected distal tubules in hypotonic solution containing 500 M hydrochlorothiazide (HCTZ) did not influence the Ca²⁺-dependent ATP hydrolysis (Ca²⁺=1 M) nor the Mg²⁺-dependent ATP hydrolysis (Mg²⁺=100 M). Similarly 100 M HCTZ did not change the Ca²⁺ ATPase activity in intact proximal and distal tubule suspensions, at Ca²⁺ concentrations ranging from 0.05 M to 1 M.ATP-dependent Ca²⁺ transport was present in basolateral membrane vesicles from proximal and distal tubule suspensions. Preincubation of the membranes with 100 M HCTZ did not influence this transport. A Na⁺/ Ca²⁺ exchanger, present in the basolateral membranes from the distal tubule, was also insensitive to HCTZ. In contrast, preincubation of luminal membranes from the distal tubules (but not proximal tubules) with 500 M HCTZ significantly increased the Ca²⁺ uptake by these membranes. This increase in Ca²⁺ uptake, in the presence of Na⁺, was dose-dependent; the minimal and the maximal effects of the diuretic were observed at concentrations of 25 M and 100 M respectively. HCTZ increased the V _maxCa²⁺ from2.5±0.3 pmol g^–1 (10 s^–1) to 3.7±0.6 pmol g^–1 (10 s^–1) (P<0.01), but did not influence the K _m (1.43±0.25 mM and 1.37±0.1 mM Ca²⁺ in experimental and control membranes, respectively). Na⁺ was necessary for this effect. Na⁺ per se decreased Ca²⁺ uptake in a concentration-dependent manner and HCTZ partially reestablished Ca²⁺ uptake to the levels observed in a Na⁺-free medium. The anion of the Na⁺ salt also modulated the effect of HCTZ on Ca²⁺ transport. While Cl^– and SCN^– permitted HCTZ to enhance Ca²⁺ uptake, the SO ₄ ^2– anion did not. It is therefore concluded that (a) the hypocalciuric effect of thiazides is primarily due to an increase in the Ca²⁺ uptake of the luminal membrane from the distal tubule, (b) Na⁺ and Ca²⁺ transports are tightly related in the distal luminal membrane, (c) HCTZ modulates this interrelationship by decreasing the inhibitory effect of Na⁺ on Ca²⁺ uptake. Whether the Ca²⁺ and Na⁺ carriers are the same molecule or different entities needs further investigation.