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P J Romero

Publications and source records attributed to P J Romero.

At least 19 recordsLinked to original sources
PubMed2002

Caffeine activates a mechanosensitive Ca(2+) channel in human red cells.

Caffeine is known to activate influx of both mono- and divalent cations in various cell types, suggesting that this xanthine opens non-selective cation channels at the plasma membrane. This possibility was investigated in human erythrocytes, studying the caffeine action on net Ca(2+), Na(+) and K(+) movements in ATP-depleted cells. Whole populations and subpopulations of young and old erythrocytes were employed. Caffeine was tested in the presence of known mechanosensitive channel blockers (Gd(3+), neomycin and amiloride) and ruthenium red as a possible inhibitor. Caffeine enhanced net cation fluxes in a concentration-dependent way. In whole populations, the Ca(2+) entry elicited by 20 mM caffeine was fully suppressed by Gd(3+) (5 microM), amiloride (250 microM) and ruthenium red (100 microM) and partially blocked by neomycin (100 microM). The above blockers also inhibited caffeine-dependent Na(+) entry whilst showing antagonistic effects on the corresponding K(+) efflux. These compounds fully suppressed hypotonically-induced (-35 mOsm/kg) Ca(2+) influx at nearly the same concentrations completely blocking caffeine-stimulated Ca(2+) entry. The effect of inhibitors on Ca(2+) influx in young cells exceeded that in old cells at similar concentrations. The results clearly show that caffeine stimulates a stretch-activated Ca(2+) channel in human red cells and that aged cells are less susceptible to mechanosensitive channel blockers.

Adenosine Triphosphate↗
PubMed1999

The role of calcium metabolism in human red blood cell ageing: a proposal.

The mechanisms of recognition, trapping and destruction of senescent human erythrocytes are not completely understood. Here we discuss some evidences supporting the idea that red cell ageing is closely related to a progressive increase in intracellular ionic Ca. This in turn, is the determining factor of a series of events such as Ca pump proteolysis and opening of the Ca-dependent K channel, that finally lead to the removal of old cells by macrophages at spleen level.

Calcium↗
PubMed1997

Ionic calcium content of light dense human red cells separated by Percoll density gradients.

In this paper we have compared the adequacy of two methods using Percoll density gradients to separate light and dense erythrocytes from fresh human blood. After measuring the distribution of some classical age-markers such as haemoglobin, potassium and creatine contents, it was found that preformed gradients generated more stringent conditions for age-related density separations. Employing such gradients the free Ca2+ content of above sub-populations was assessed with Fura-2, under conditions where the viscosity effect was abolished. In five experiments, the free Ca2+ content (mean value +/- 1 S.D.) was 8.4 +/- 2.82 nM and 31.2 +/- 13.0 nM for the 7-10% lightest and densest cells, respectively. These results are discussed in connection to red cell senescence.

Acetylcholinesterase↗
PubMed1997

Differences in Ca2+ pumping activity between sub-populations of human red cells.

It has been shown recently that the free Ca2+ content of human red cells rises during ageing in vivo. With the aim of determining the mechanisms involved in such a change, we have investigated some aspects of Ca2+ homeostasis. Both the initial rate of Ca2+ influx and some kinetic parameters of the Ca2+ pump of human red cells were studied in light and dense sub-populations obtained through stringent, self-formed Percoll gradients. At 37 degrees C and pH 7.4, no differences in Ca2+ entry were found. By contrast, either at pH 7.0 or 7.4, the maximal Ca2+ extrusion rate of the approximately 10% heaviest cells was one-half of the corresponding lighter ones. The results demonstrate that the elevated free Ca2+ concentration distinctive of senescent cells, arises from a reduction in Ca2+ extrusion capacity during ageing. The possible physiological significance of this finding is discussed.

Calcium↗
PubMed1996

Action of protein kinase A activators on the caudal neuromuscular junction of toad tadpoles, recorded on synaptic spots.

Miniature end-plate currents (MEPCS), from synaptic spots on the caudal muscles of Bufo marinus tadpoles, were analyzed in both pre- and postsynaptic domains, when protein kinase A (PKA) activity modificators were used. Sp-cAMPS diasteromer induced an increase in MEPC frequency, which was completely reversed by Rp-cAMPS. However, changes in the decay time of MEPCS were not detected. Dibutyryl-cAMP produced a similar presynaptic action, but its postsynaptic action was similar to butyrate. Presynaptic effect of forskolin (FSK), if any, is masked by the increase of MEPC frequency produced by dimethylsulfoxide (DMSO), the solvent used.

Administration, Topical↗
PubMed1995

Alteration by EGTA of the human red cell Ca(2+)-ATPase.

The effect of EGTA on Ca(2+)-ATPase activity was studied in fragmented membranes and solubilized preparations from human red cells. A dual action was found. At low concentrations (0.1-1 mM), EGTA increased Ca2+ affinity without affecting Vmax. By contrast, at high concentrations (5-10 mM), EGTA was inhibitory. Both effects were partially reversible.

Calcium-Transporting ATPases↗
PubMed1995

Effects of magnesium plus vanadate on partial reactions of the Ca(2+)-ATPase from human red cell membranes.

Under conditions in which pretreatment with Mg2+ plus vanadate activate the Ca(2+)-ATPase, the initial rate of phosphorylation of the enzyme increased from 141 to 259 pmol/mg protein per s while the steady-state level of phosphoenzyme lowered from 1.9 to 1.1 pmol/mg protein. The drop in phosphoenzyme level was caused by incubation and washing during treatment rather than by vanadate. The data allowed to estimate a turnover number for the enzyme that raised by 170% after pretreatment. The results show that the activation of the Ca(2+)-ATPase by Mg2+ plus vanadate is due to changes in the kinetic properties of the enzyme.

Calcium-Transporting ATPases↗
PubMed1995

Characterization of the plasma-membrane calcium pump from Trypanosoma cruzi.

Despite previous reports [McLaughlin (1985) Mol. Biochem. Parasitol. 15, 189-201; Ghosh, Ray, Sarkar and Bhaduri (1990) J. Biol. Chem. 265, 11345-11351; Mazumder, Mukherjee, Ghosh, Ray and Bhaduri (1992) J. Biol. Chem. 267, 18440-18446] suggesting that the plasma-membrane Ca(2+)-ATPases of different trypanosomatids differ from the Ca2+ pumps present in mammalian cells, Trypanosoma cruzi plasma-membrane Ca(2+)-ATPase shares several characteristics with the Ca2+ pumps present in other systems. This enzyme could be partially purified from epimastigote plasma-membrane vesicles using calmodulin-agarose affinity chromatography. The activity of the partially purified enzyme was stimulated by T. cruzi or bovine brain calmodulin. In addition, the enzyme cross-reacted with antiserum and monoclonal antibody 5F10 raised against human red-blood-cell Ca(2+)-ATPase, has a molecular mass of 140 kDa and forms Ca(2+)-dependent hydroxylamine-sensitive phosphorylated intermediates. These results, together with its high sensitivity to vanadate, indicate that this enzyme belongs to the P-type class of ionic pumps.

Animals↗
PubMed1994

Activation of the human red cell calcium ATPase by calcium pretreatment.

Some kinetic parameters of the human red cell Ca(2+)-ATPase were studied on calmodulin-free membrane fragments following preincubation at 37 degrees C. After 30 min treatment with EGTA (1 mM) plus dithioerythritol (1 mM), a Vmax of about 0.4 mumol Pi/mg x hr and a Ks of 0.3 microM Ca2+ were found. When Mg2+ (10 mM) or Ca2+ (10 microM) were also added during preincubation, Vmax, but not Ks was altered. Ca2+ was more effective than Mg2+, thus increasing Vmax to about 1.3 mumol P/mg x hr. The presence of both Ca2+ and Mg2+ during pretreatment decreased Ks to 0.15 microM, while having no apparent effect on Vmax. Conversely, addition of ATP (2 mM) with either Ca2+ or Ca2+ plus Mg2+ increased Vmax without affecting Ks. Preincubation with Ca2+ for periods longer than 30 min further increased Vmax and reduced Ks to levels as low as found with calmodulin treatment. The Ca2+ activation was not prevented by adding proteinase inhibitors (iodoacetamide, 10 mM; leupeptin, 200 microM; pepstatin A, 100 microM; phenylmethanesulfonyl fluoride, 100 microM). The electrophoretic pattern of membranes preincubated with or without Mg2+, Ca2+ or Ca2+ plus Mg2+ did not differ significantly from each other. Moreover, immunodetection of Ca(2+)-ATPase by means of polyclonal antibodies revealed no mobility change after the various treatments. The above stimulation was not altered by neomycin (200 microM), washing with EGTA (5 mM) or by both incubating and washing with delipidized serum albumin (1 mg/ml), or omitting dithioerythritol from the preincubation medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1993

Synergistic activation of the human red cell calcium ATPase by magnesium and vanadate.

The Ca(2+)-ATPase activity of human red cells was studied on calmodulin-free membrane fragments after previous incubation with Mg2+ and vanadate. In the presence of EGTA (5 mM), the activity was slightly affected by either ion alone. However, when added together, both Ca2+ affinity and Vmax were increased up to levels found with calmodulin (0.3 microM). This synergistic activation was not abolished by proteinase inhibitors (iodoacetamide, 10 mM; leupeptin, 200 microM; pepstatin A, 100 microM; phenylmethanesulfonyl fluoride, 100 microM), neomycin (200 microM), washing with EDTA (5 mM) or by both incubating and washing with delipidized serum albumin (1 mg/ml). During preincubation under optimal Mg2+ and vanadate conditions, the replacement of K+ by Na+ or Li+ was without effect. Co2+ or Zn2+ (10 mM) could not substitute for Mg2+, whereas Mn2+ almost replaced it at equimolar amounts. By contrast, addition of ATPMg (2 mM) decreased the activation by about one-half. Like calmodulin, pretreatment with Mg2+ plus vanadate also increased the affinity for ATP and elicited appearance of a second (low) affinity site (apparent Km = 120 microM). The fluorescence depolarization of 1,6-diphenyl- and 1-(4-trimethylammonium phenyl)-6-phenyl 1,3,5-hexatriene incorporated into membrane fragments was not affected after preincubating with Mg2+, vanadate or Mg2+ plus vanadate. The results show that Mg2+ and vanadate are acting neither via proteolysis or fatty acid production nor by facilitating phospholipid metabolism or altering membrane fluidity. They may be enhancing the Ca(2+)-ATPase activity by stabilizing the E1 conformer or promoting an enzyme conformation which facilitates the E2-E1 transition.

Adenosine Triphosphate↗
PubMed1993

[Possible new modulators of the calcium pump of human erythrocytes].

A review on the human erythrocyte Ca pump is presented, discussing general aspects of the catalytic cycle and the effect of some naturally occurring modulators such as calmodulin, acidic phospholipids and polyunsaturated fatty acids, controlled calpain proteolysis and protein kinases. The effect of both ATP, mono and divalent cations and recent experiments by the author and coworkers are also discussed in the same context, pinpointing Ca and Mg as new Ca ATPase modulators.

Adenosine Triphosphate↗
PubMed1992

Inhibition of the human erythrocyte calcium pump by dimethyl sulfoxide.

The action of dimethyl sulfoxide on the human red cell Ca2+ pump was studied in inside-out vesicles. In a high-K+ medium at pH 7.6, the organic solvent inhibited both Ca2+ transport and ATP hydrolysis. Half-maximal effect was obtained with about 2% (v/v). At or below 10% dimethyl sulfoxide, the inhibition was overcome by adding inorganic phosphate or oxalate. In the absence of organic solvent, Ca2+ efflux from Ca(2+)-loaded vesicles consisted of a slow and a fast component whilst in its presence, there appears additionally a leakage component. The size of the latter depended markedly on dimethyl sulfoxide concentration, being about 3% at that level where Ca2+ uptake was half-maximally inhibited. ATP hydrolysis was more sensitive to dimethyl sulfoxide (10%) when free Ca2+ was increased within the millimolar level than when it was raised within the micromolar range. On the other hand, raising Ca2+ with organic solvent greatly stimulated ATP synthesis through ATP-Pi exchange, without reaching saturation. The results suggest that dimethyl sulfoxide blocks the red cell Ca2+ pump by increasing the affinity of the Ca2+ translocating site at the releasing step. They also show that at high concentrations, this solvent increases Ca2+ permeability.

Adenosine Triphosphate↗
PubMed1992

The effect of ATP on Ca(2+)-dependent K+ channels of human red cells.

The action of ATP on Ca(2+)-dependent K+ channels was studied in fresh human erythrocytes using patch-clamp techniques. Single-channel current was recorded at pH 6.5 from inside-out patches in the presence of symmetrical K+ gluconate solutions, containing both 1 microM free Ca2+ in the bath and 0.5 mM LaCl3 on the pipette side. With no ATP, the electrical activity revealed low-conductance K+ channels (25 pS), which showed inward rectification and an opening kinetics dependent on membrane potential. When ATP (1 mM) and Mg2+ (2 mM) were added together and a depolarizing potential was simultaneously applied, only a high-conductance channel (about 75 pS) was observed. This channel showed no rectifying properties and it was not found if ATP was added in the absence of Mg2+. Channel activity was enhanced by adding fluoride (10 mM) or trifluoperazine (50 microM) whilst it was reduced after incubating with dibutyryl cAMP (50 microM) or alkaline phosphatase (250 U/ml). On the other hand, when fragmented membranes from inside-out vesicles were incubated with gamma-32 P-ATP and 1 microM free Ca2+ under above conditions, only two high-molecular weight polypeptides (235 and 320 kDa) were labelled with 32P. The results suggest that ATP-mediated phosphorylation of Ca(2+)-dependent K+ channels leads to a high-conductance state.

Adenosine Triphosphate↗
PubMed1991

The effect of cholera toxin on human red cell Ca-ATPase.

The effect of cholera toxin on transport Ca-ATPase was studied in membrane fragments from human red cells. A consistently moderate inhibition was found when fragments were previously incubated with toxin in the presence of beta-NAD but not in its absence of after treatment with non-activated toxin. In calmodulin-free preparations, both Ca affinity and maximal rate of hydrolysis were affected whereas only affinity was altered in calmodulin-deficient membranes.

Ca(2+) Mg(2+)-ATPase↗
PubMed1990

A calcium pump in plasma membrane vesicles from Leishmania braziliensis.

A subcellular fraction highly enriched in plasma membrane vesicles was prepared from Leishmania promastigotes. This fraction showed (Ca2+ + Mg2+)-ATPase activity. This, however, represented a small fraction (about 25%) of the overall ATPase activity. The Ca2(+)-ATPase showed general characteristics common to plasma membrane ATPases involved in Ca2+ transport. Thus, the Ca2(+)-ATPase was activated by Ca2+ with a high affinity (Km about 0.7 microM), saturating at about 5 microM Ca2+. Furthermore, it was stimulated by calmodulin (about 70-80% with 5 micrograms/ml) and almost fully inhibited by trifluoperazine (100 microM). The above vesicles accumulated Ca2+ against a concentration gradient and released it after the addition of A23187, as shown independently by 45Ca2+ and Arsenazo III studies. The transport mechanism showed the same kinetics parameters as described for the enzyme, indicating a single molecular entity. In addition, Ca2(+)-ATPase activity and Ca2+ uptake were completely inhibited by vanadate (20 microM), indicating that an E1-E2 type mechanism is involved. The results clearly demonstrate the presence of a Ca2+ pump in the plasma membrane of Leishmania which is capable of maintaining a low cytoplasmic Ca2+ concentration.

Adenosine Triphosphate↗
PubMed1990

Metabolic control of the K+ channel of human red cells.

The effects of cAMP, ATP and GTP on the Ca2(+)-dependent K+ channel of fresh (1-2 days) or cold-stored (28-36 days) human red cells were studied using atomic absorption flame photometry of Ca2(+)-EGTA loaded ghosts which had been resealed to monovalent cations in dextran solution. When high-K+ ghosts were incubated in an isotonic Na+ medium, the rate constant of Ca2(+)-dependent K+ efflux was reduced by a half on increasing the theophylline concentration to 40 mM. This effect was observed in ghosts from both fresh and stored cells, but only if they were previously loaded with ATP. The inhibition was more marked when Mg2+ was added together with ATP, and it was abolished by raising free Ca2+ to the micromolar level. Like theophylline, isobutyl methylxanthine (10 mM) also affected K+ efflux. cAMP (0.2-0.5 mM), added both internally and externally (as free salt, dibutyryl or bromide derivatives), had no significant effect on K+ loss when the ghost free-Ca2+ level was below 1 microM, but it was slightly inhibitory at higher concentrations. The combined presence of cAMP (0.2 mM) plus either theophylline (10 mM), or isobutyl methylxanthine (0.5 mM), was more effective than cAMP alone. This inhibition showed a strict requirement for ATP plus Mg2+ and it was not overcome by raising internal Ca2+. Ghosts from stored cells seemed more sensitive than those from fresh cells, to the combined action of cAMP and methylxanthines. Loading ATP into ghosts from fresh or stored cells markedly decreased K+ loss. Although this effect was observed in the absence of added Mg2+ (0.5 mM EDTA present), it was potentiated upon adding 2 mM Mg2+. The K+ efflux from ATP-loaded ghosts was not altered by dithio-bis-nitrobenzoic acid (10 mM) or acridine orange (100 microM), while it was increased two- to fourfold by incubating with MgF2 (10 mM), or MgF2 (10 mM) + theophylline (40 mM), respectively. By contrast, a marked efflux reduction was obtained by incorporating 0.5 mM GTP into ATP-containing ghosts. The degree of phosphorylation obtained by incubating membranes with (gamma-32P)ATP under various conditions affecting K+ channel activity, was in direct correspondence to their effect on K+ efflux. The results suggest that the K+ channel of red cells is under complex metabolic control, via cAMP-mediated and nonmediated mechanisms, some which require ATP and presumably, involve phosphorylation of the channel proteins.

1-Methyl-3-isobutylxanthine↗