scholarly journals Calcium transport of Plasmodium chabaudi-infected erythrocytes.

1982 ◽  
Vol 93 (3) ◽  
pp. 680-684 ◽  
Author(s):  
K Tanabe ◽  
R B Mikkelsen ◽  
D F Wallach
Keyword(s):  
Infected Erythrocyte ◽  
Calcium Content ◽  
Transport Processes ◽  
Metabolic Inhibitors ◽  
Parasite Development ◽  
Ionophore A23187 ◽  
Plasmodium Chabaudi ◽  
Carbonyl Cyanide ◽  
Flux Measurements ◽  
Infected Cells

The calcium content and transport processes of Plasmodium chabaudi-infected rat erythrocytes were analyzed by atomic absorption spectroscopy and 45Ca2+ flux measurements. Infected erythrocytes, after fractionation on metrizamide gradients according to stage of parasite development, exhibited progressively increasing levels of Ca2+ with schizont and gametocytes containing 10- to 20-fold greater calcium levels than normal cells (0.54 +/- 0.25 nmol/10(8) cells). 45Ca2+ flux experiments showed both increased influx and decreased efflux in infected erythrocytes. Tris/NH4Cl lysis of normal erythrocytes preloaded with 45Ca2+ with the Ca2+ ionophore A23187 released less than 90% of cell calcium after incubation in ethyleneglycol bis(aminoethylether) N,N'-tetraacetic acid containing buffer, whereas lysis of the infected erythrocyte membrane resulted in release of 10-20% cell Ca2+, with the remaining portion associated with the isolated parasite fraction. This information together with the effects of various metabolic inhibitors indicates the presence of a parasite Ca2+ compartment in P. chabaudi-infected erythrocytes. Dicyclohexylcarbodiimide (DCCD) an inhibitor of proton ATPases of chloroplasts, bacteria, yeast, and mitochondria, and the proton ionophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP), inhibited Ca2+ influx and stimulated efflux from infected cells. These results combined with evidence for a DCCD- and CCCP-sensitive membrane potential in P. chabaudi-infected cells (Mikkelsen et al., accompanying manuscript) suggest that Ca2+ transport of intraerythrocytic parasites is coupled to a proton-motive force across the Plasmodia plasma membrane.

Transport processes of 2-deoxy-D-glucose in erythrocytes infected withPlasmodium yoelii, a rodent malaria parasite

Parasitology ◽  
1989 ◽  
Vol 98 (3) ◽  
pp. 371-379 ◽  
Author(s):  
A. Izumo ◽  
K. Tanabe ◽  
M. Kato ◽  
S. Doi ◽  
K. Maekawa ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Glucose Transporter ◽  
Plasmodium Yoelii ◽  
Transport Processes ◽  
Diffusion System ◽  
Host Cells ◽  
Metabolic Inhibitors ◽  
Dependent Manner ◽  
Simple Diffusion ◽  
Infected Cells

SUMMARYThe transport processes of D-glucose inPlasmodium yoelii-infected mouse erythrocytes were investigated using 2-deoxy-D-glucose (2DOG), a non-metabolizable analogue of D-glucose. Infected cells showed an increase in the uptake of 2DOG compared to uninfected controls, and an effect which was more prominent in cells with mature-stage parasites. Kinetic studies measuring the initial rates of 2DOG uptake revealed two components in infected cells with late trophozoite and schizont-stage parasites: a simple diffusion system and a carrier (transporter)-mediated system. The transporter was common for D-glucose and 2DOG and had a kinetic constant indicating a high affinity for 2DOG (theKm= 0·18 mM and theVmax= 0·61 mmol/1010cells/min), as compared to the constant of the mouse erythrocyte carrier (theKm= 10 mM and theVmax= 1·8 mmol/1010cells/min). Determination of the distribution of [3H]2DOG in infected cells and experiments with metabolic inhibitors indicated that the simple diffusion system localizes in the membrane of host cells and the transporter in the parasite plasma membrane. The parasite glucose transporter was much less sensitive to cytochalasin B than that of the host cells and the uptake of 2DOG via the transporter was dependent on energy. Based on these findings, the following features emerge: D-glucose first gains access to the cytosol of infected erythrocytes via the simple diffusion system, which appears after infection by the parasite, and an active uptake against the concentration gradient takes place at the parasite plasma membrane via the parasite glucose transporter in an energy dependent manner. Finally, an energy transduction mechanism for the transport of glucose across the parasite plasma membrane is discussed.

scholarly journals Calcium metabolism in rat hepatocytes

1978 ◽  
Vol 170 (3) ◽  
pp. 615-625 ◽  
Author(s):  
S Foden ◽  
P J Randle
Keyword(s):  
Cyclic Amp ◽  
Time Course ◽  
Rat Hepatocytes ◽  
Ionophore A23187 ◽  
Total Calcium ◽  
Free Medium ◽  
Adrenergic Agonists ◽  
Calcium Efflux ◽  
Similar Time ◽  
Carbonyl Cyanide

1. The total calcium concentration in rat hepatocytes was 7.9 microgram-atoms/g dry wt.; 77% of this was mitochondrial. Approx. 20% of cell calcium exchanged with 45Ca within 2 min. Thereafter incorporation proceeded at a low rate to reach 28% of total calcium after 60 min. Incorporation into mitochondria showed a similar time course and accounted for 20% of mitochondrial total calcium after 60 min. 2. The alpha-adrenergic agonists phenylephrine and adrenaline + propranolol stimulated incorporation of 45Ca into hepatocytes. Phenylephrine was shown to increase total calcium in hepatocytes. Phenylephrine inhibited efflux fo 45Ca from hepatocytes perifused with calcium-free medium. 3. Glucagon, dibutryl cyclic AMP and beta-adrenergic agonists adrenaline and 3-isobutyl-1-methyl-xanthine stimulated calcium efflux from hepatocytes perifused with calcium-free medium. The effect of glucagon was blocked by insulin. Insulin itself had no effect on calcium efflux and it did not affect the response to dibutyryl cyclic AMP. 4. Incorporation of 45Ca into mitochondria in hepatocytes was stimulated by phenylephrine and inhibited by glucagon and by carbonyl cyanide p-trifluoromethoxyphenylhydrazone. The effect of glucagon was blocked by insulin. 5. Ionophore A23187 stimulated hepatocyte uptake of 45Ca, uptake of 45Ca into mitochondria in hepatocytes and efflux of 45Ca into a calcium-free medium.

scholarly journals Basolateral transport of tetraethylammonium by a clone of LLC-PK1 cells.

1992 ◽  
Vol 2 (10) ◽  
pp. 1507-1515
Author(s):  
T D McKinney ◽  
M B Scheller ◽  
M Hosford ◽  
M E Lesniak ◽  
T S Haseley
Keyword(s):  
Organic Cation ◽  
Basolateral Membrane ◽  
Transport Processes ◽  
Proton Exchange ◽  
Metabolic Inhibitors ◽  
Organic Cations ◽  
Dependent Manner ◽  
Electrogenic Transport ◽  
The Media ◽  
Uptake Medium

In these studies, a clone of cells derived from the porcine renal epithelial line LLC-PK1 grown on porous filters was used to evaluate basolateral uptake of the organic cation tetraethylammonium (TEA). (3H) TEA (1 microM) entered cells in a saturable and time-dependent manner achieving a steady-state value at 2 to 2.5 h. Uptake was reduced by hypothermia and the metabolic inhibitors sodium azide and iodoacetate. Several other organic cations in 1 mM concentrations inhibited the majority of TEA uptake. In lower concentrations, the inhibitory potency of these was: verapamil greater than cimetidine approximately amiloride approximately quinidine greater than procainamide approximately N1-methylnicotinamide. When sodium was replaced with potassium in the uptake medium, TEA uptake was also reduced consistent with electrogenic transport. However, uptake was reduced further by 1 mM cimetidine in the presence of both NaCl and KCl buffers. TEA uptake was not significantly different when the media pH was varied from 6.0 to 8.0. In addition, results of experiments in which intracellular pH was altered with NH4Cl were not consistent with the presence of organic cation/proton exchange. TEA/TEA exchange could not be demonstrated in experiments in which cells were preloaded with 1 mM nonradioactive TEA and uptake of (3H)TEA was measured or in which nonradioactive TEA in the external medium failed to enhance efflux from cells preloaded with (3H)TEA. These results indicate that the basolateral membrane of LLC-PKc10 cells has one or more transport processes for the mediated uptake of organic cations. However, the precise mechanism(s) involved in this transport remains to be elucidated.

Human High Molecular Weight Kininogen - A Secreted Platelet Coagulant Protein

1981 ◽  
Author(s):  
A H Schmaier ◽  
J Kuchibhotla ◽  
R W Colman
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Gel Filtration ◽  
Human Platelets ◽  
Metabolic Inhibitors ◽  
Ionophore A23187 ◽  
High Molecular Weight Kininogen ◽  
Contact Phase ◽  
Platelet Factor ◽  
Coagulant Activity

Platelets have been shown to contain a number of secret- able coagulant proteins, which participate as substrates or cofactors in plasma coagulation reactions. Since we have previously demonstrated that high molecular weight kininogen (HMWK) is immunochemically present in platelet extracts, we posited that HMWK is secreted during activation of platelets. Fresh normal platelets were washed by a combination of albumin-gradient and gel-filtration procedures. In 11 experiments the supernates of freeze-thaw lysates of normal human platelets contained a mean of 5.7 Units (range 3.16 to 8.14) of HMWK coagulant activity/3 × 1011 platelets. This coagulant activity was neutralized by a goat antiki- ninogen antibody. Using a 125I-HMWK tracer in PRP, the supernate of washed activated platelets contained 0.082% radioactivity as the starting PRP, suggesting that 14% of the total HMWK coagulant activity could be accounted for by plasma contamination. In four experiments, ionophore A23187 (15μM) induced a net secretion of 39% of the total platelet HMWK (range 16 to 49%). Platelet HMWK secretion by A23187 was concentration dependent (1 to 15 μM) . At A23187 (15μM) platelets released 75% 14C-5HT (range 61 to 99%) and 81% low affinity platelet Factor 4 (range 60 to 99%). Ninety-five percent of A23187-induced secretion of HMWK could be blocked by platelet pretreatment with metabolic inhibitors. LDH determinations indicated that only 5% (range 0 to 10%) of total secreted platelet HMWK could be attributed to lysis. Collagen and PGH2 also caused secretion of platelet HMWK coagulant activity. This study indicates that human platelets contain functional HMWK which may be secreted locally to modulate the reactions of the contact phase of plasma proteolysis.

scholarly journals Membrane potential of Plasmodium-infected erythrocytes.

1982 ◽  
Vol 93 (3) ◽  
pp. 685-689 ◽  
Author(s):  
R B Mikkelsen ◽  
K Tanabe ◽  
D F Wallach
Keyword(s):  
Membrane Potential ◽  
Plasma Membranes ◽  
Metabolic Inhibitors ◽  
Erythrocyte Membranes ◽  
Weak Acids ◽  
Atpase Inhibitor ◽  
Lipophilic Anion ◽  
Parasite Stage ◽  
Infected Cells ◽  
Electrogenic Proton Pump

The membrane potential (Em) of normal and Plasmodium chabaudi-infected rat erythrocytes was determined from the transmembrane distributions of the lipophilic anion, thiocyanate (SCN), and cation, triphenylmethylphosphonium (TPMP). The SCN- and TPMP-measured Em of normal erythrocytes are -6.5 +/- 3 mV and -10 +/- 4 mV, respectively. The TPMP-measured Em of infected cells depended on parasite developmental stage; "late" stages (schizonts and gametocytes) were characterized by a Em = -35 mV "early stages (ring and copurifying noninfected) by a low Em (-16 mV). The SCN-determined Em of infected cells was -7 mV regardless of parasite stage. Studies with different metabolic inhibitors including antimycin A, a proton ionophore (carbonylcyanide m-chlorophenylhydrazone [CCCP] ), and a H+ -ATPase inhibitor (N,N'-dicyclohexylcarbodiimide, [DCCD] ) indicate that SCN monitors the Em across the erythrocyte membrane of infected and normal cells whereas TPMP accumulation reflects the Em across the plasma membranes of both erythrocyte and parasite. These inhibitor studies also implicated proton fluxes in Em-generation of parasitized cells. Experiments with weak acids and bases to measure intracellular pH further support this proposal. Methylamine distribution and direct pH measurement after saponin lysis of erythrocyte membranes demonstrated an acidic pH for the erythrocyte matrix of infected cells. The transmembrane distributions of weak acids (acetate and 5,5-dimethyloxazolidine-2,4-dione) indicated a DCCD-sensitive alkaline compartment. The combined results suggest that the intraerythrocyte parasite Em and delta pH are in part the consequence of an electrogenic proton pump localized to the parasite plasma membrane.

scholarly journals A Comparison of the Effects of Metabolic Inhibitors on Chloride Uptake and Photosynthesis In Chara Oorallina

1969 ◽  
Vol 22 (2) ◽  
pp. 351 ◽  
Author(s):  
FA Smith ◽  
KR West
Keyword(s):  
Comparative Study ◽  
Electron Flow ◽  
External Solution ◽  
Metabolic Inhibitors ◽  
Chloride Uptake ◽  
Low Concentrations ◽  
Carbonyl Cyanide ◽  
Chloride Influx

A comparative study has been made of the effects of four metabolic inhibitors on chloride uptake and photosynthetic 14C02 fixation by cells of O. corallina, and on oxygen evolution by chloropl<1sts isolated from the cells. Low concentrations of phlorizin and Dio-9 inhibited chloride uptake, but this was not accompanied by an inhibition of photosynthesis in vivo, and could not be correlated with the measured inhibition of electron flow in vitro. Low concentrations of imidazole stimulated the chloride influx in light, but there was again no effect on photosynthetic 14C02 fixation, although imidazole did uncouple electron flow in vitro. The effect of imidazole was dependent on the pH of the external solution. Increasing concentrations of carbonyl cyanide m-chlorophenylhydrazone progressively reduced the chloride influx and 14C02 fixation, and uncoupled electron flow in vitro. The work provides no evidence to support the view that chloride uptake is directly linked to electron flow rather than phosphorylation.

Perturbation of the pump-leak balance for Na+ and K+ in malaria-infected erythrocytes

2001 ◽  
Vol 280 (6) ◽  
pp. C1576-C1587 ◽  
Author(s):  
Henry M. Staines ◽  
J. Clive Ellory ◽  
Kiaran Kirk
Keyword(s):  
Erythrocyte Membrane ◽  
Infected Erythrocyte ◽  
Ion Permeability ◽  
Permeability Ratio ◽  
Parasite Plasmodium ◽  
Initial Decrease ◽  
Parasite Plasmodium Falciparum ◽  
Flux Measurements ◽  
Cell Cytosol ◽  
Host Cell Cytosol

In human erythrocytes infected with the mature form of the malaria parasite Plasmodium falciparum, the cytosolic concentration of Na+ is increased and that of K+ is decreased. In this study, the membrane transport changes underlying this perturbation were investigated using a combination of86Rb+, 43K+, and22Na+ flux measurements and a semiquantitative hemolysis technique. From >15 h postinvasion, there appeared in the infected erythrocyte membrane new permeation pathways (NPP) that caused a significant increase in the basal ion permeability of the erythrocyte membrane and that were inhibited by furosemide (0.1 mM). The NPP showed the selectivity sequence Cs+ > Rb+ > K+ > Na+, with the K+-to-Na+permeability ratio estimated as 2.3. From 18 to 36 h postinvasion, the activity of the erythrocyte Na+/K+ pump increased in response to increased cytosolic Na+ (a consequence of the increased leakage of Na+ via the NPP) but underwent a progressive decrease in the latter 12 h of the parasite's occupancy of the erythrocyte (36–48 h postinvasion). Incorporation of the measured ion transport rates into a mathematical model of the human erythrocyte indicates that the induction of the NPP, together with the impairment of the Na+/K+pump, accounts for the altered Na+ and K+levels in the host cell cytosol, as well as predicting an initial decrease, followed by a lytic increase in the volume of the host erythrocyte.

Prostaglandin-induced pepsinogen secretion from dispersed gastric glands from guinea pig stomach

1985 ◽  
Vol 249 (5) ◽  
pp. G592-G598 ◽  
Author(s):  
S. Berger ◽  
J. P. Raufman
Keyword(s):  
Guinea Pig ◽  
Incubation Temperature ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Gastric Glands ◽  
Cellular Calcium ◽  
Pepsinogen Secretion ◽  
Carbonyl Cyanide ◽  
Guinea Pig Stomach

We examined the actions of several prostaglandins (PG) on pepsinogen secretion from dispersed gastric glands prepared from guinea pig stomach. Pepsinogen secretion was stimulated by PGA1, PGA2, PGB2, PGE1, and PGE2. PGE1 and PGE2 were 10 times more potent than PGA1 and PGA2. Reducing the incubation temperature from 37 degrees to 4 degrees C or adding carbonyl cyanide m-chlorophenylhydrazone reduced PG-stimulated pepsinogen secretion. PGE2-induced pepsinogen secretion was not altered by atropine or dibutyryl cGMP. Potentiation of pepsinogen secretion occurred with PGE2 plus carbachol or the calcium ionophore A23187 but not with PGE2 plus secretin or 8-bromo-cAMP. Isobutylmethylxanthine increased the potency and the efficacy of the action of PGE2 on pepsinogen secretion. These results indicate that PGE1, PGA2, PGB2, PGE1, and PGE2 can modulate pepsinogen secretion from dispersed gastric glands from guinea pig stomach. Moreover, potentiation of pepsinogen secretion occurs when PGE2 is combined with secretagogues whose actions appear to be mediated by changes in cellular calcium (carbachol and A23187) but not with secretagogues whose actions appear to be mediated by changes in cellular cAMP (secretin and 8-bromo-cAMP). These data suggest that PG-induced pepsinogen secretion may be mediated by changes in cellular cAMP.

The alteration of mitotic events by ionophore A23187 and carbonyl cyanide n-chlorophenylhydrazone

1985 ◽  
Vol 75 (1) ◽  
pp. 347-355
Author(s):  
M.L. Ziegler ◽  
J.E. Sisken ◽  
S. Vedbrat
Keyword(s):  
Oxidative Phosphorylation ◽  
Intracellular Calcium ◽  
Calcium Ions ◽  
Hela Cells ◽  
Cytosolic Calcium ◽  
Ionophore A23187 ◽  
Chromosome Movement ◽  
Low Concentrations ◽  
Carbonyl Cyanide ◽  
Stimulation Of

A large quantity of published work indicates that calcium ions may be involved in the regulation of mitotic events and recent reports suggest that the onset of chromosome movement is dependent upon a transient increase in free cytosolic calcium ions. In this paper we examine the effects of two agents known to perturb intracellular calcium pools on mitosis in HeLa cells. These were the calcium-selective ionophore A23187 and carbonyl cyanide n-chlorophenylhydrazone (CCCP), which is a protonophoric inhibitor of oxidative phosphorylation. Owing to a stimulation of glycolysis, the latter agent does not decrease intracellular ATP in HeLa but does cause mitochondria to release calcium ions. Our data show that, at low concentrations, both agents prolong metaphase but differ in their effects on anaphase and cytokinesis. Studies with chlorotetracycline, a commonly used probe for membrane-associated calcium, verify that these agents do affect calcium pools under the conditions of our experiments. The data presented are consistent with the idea that increased cytosolic calcium levels can directly or indirectly affect mitotic events but, contrary to other suggestions, cause a prolongation of metaphase, i.e. they delay the onset of chromosome movement.

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