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R DiPolo

Publications and source records attributed to R DiPolo.

At least 19 recordsLinked to original sources
PubMed2006

Depolymerisation and rearrangement of actin filaments during exocytosis in rat peritoneal mast cells: involvement of ryanodine-sensitive calcium stores.

Cytoskeletal F-actin associated with synaptic vesicles and granules plays an important role during Ca(2+)-mediated exocytosis. In the present work, we have used amperometry and confocal fluorescence to study the role of internal Ca(2+) in the rearrangement of F-actin (visualised with phalloidin-Alexa 546) during exocytosis in rat mast cells. The F-actin-depolymerising drug, latrunculin A, and the ryanodine receptor agonists ryanodine and caffeine that, per se did not induce exocytosis, enhanced the exocytotic responses elicited by compound 48/80 (C48/80). They also induced cortical actin depolymerisation in the presence or absence of external Ca(2+). Degranulation induced by C48/80 was accompanied by the formation of a cytoplasmic F-actin network. Depletion of internal Ca(2+) with cyclopiazonic acid inhibited latrunculin potentiation of C48/80-stimulated exocytosis and completely blocked the formation of the cytoplasmic F-actin network. This indicates that the mobilisation of Ca(2+) from ryanodine-sensitive intracellular stores plays an important role in the depolymerisation of the cortical F-actin barrier and possibly in the formation of the internal F-actin network during exocytotic activation of peritoneal mast cells.

Actin Cytoskeleton↗
PubMed2004

Evaluation of the presence of a thapsigargin-sensitive calcium store in trypanosomatids using Trypanosoma evansi as a model.

Ca2+ plays an important role in the regulation of several important activities in different trypanosomatids. These parasites possess a Ca2+ transport system in the endoplasmic reticulum (ER) involved in Ca2+ homeostasis, which has been reported to be insensitive to thapsigargin, a classical inhibitor of the sarcoplasmic-ER Ca2+ adenosine triphosphatase (ATPase) (SERCA) in most eukaryotic cells. However, currently there is a controversy regarding the existence of a thapsigargin-sensitive ER Ca2+ store in these parasites. Therefore, we decided to explore the effect of this inhibitor using different methodological approaches. First, we selected Trypanosoma evansi as a parasite model to warrant the homogeneity of the population because this parasite has only a single life cycle, i.e., bloodstream-form trypomastigotes. Second, we compared the thapsigargin effect on Ca2+ homeostasis by spectrophotometrical Ca2+ measurements using 3 different approaches: whole-cell populations, cells that have been permeabilized by treatment with digitonin, and intact single cells. Our results demonstrate that a low concentration of thapsigargin induces Ca2+ release from intracellular Ca2+ stores in this parasite, which can be observed independently of the method used. Furthermore, the addition of thapsigargin before or after nigericin did not abolish its effect, showing that thapsigargin acts specifically on the ER. In conclusion, our results indicate the presence of a nonmitochondrial thapsigargin-sensitive Ca2+ store in T. evansi.

Animals↗
PubMed2001

Trypanosoma evansi: a convenient model for studying intracellular Ca(2+) homeostasis using fluorometric ratio imaging from single parasites.

The aim of this work was to measure, for the first time, the basal cytosolic Ca(2+) levels of Trypanosoma evansi and to explore the possibility of observing changes in the intracellular Ca(2+) concentration ([Ca(2+)](i)) using fluorescence ratio imaging techniques in single isolated parasites of this species. Under appropriate loading conditions, the high intracellular levels of the Ca(2+) fluorescence probe Fura-2 permits resolution, in real time, of single parasite [Ca(2+)](i) signals. Measurements of the basal [Ca(2+)](i) indicate that homeostatic mechanisms maintain [Ca(2+)](i) at 106 +/- 38 (n = 32) nM in the presence of 2 mM extracellular calcium. The resting [Ca(2+)](i) was unaffected by changes in extracellular Ca(2+) in the range from 0 to 10 mM. The Ca(2+) ionophore A23187 induced a large increase in [Ca(2+)](i) which (i) reached a steady state value even in the simultaneous presence of both external calcium and ionophore and (ii) returned to base line upon removal of extracellular Ca(2+). A dose-response curve of the protonophore nigericin shows that T. evansi contains an important pH-sensitive intracellular pool which may be released by this drug with a K(1/2) of 8 microM. These data demonstrate that this parasite contains highly efficient systems to control [Ca(2+)](i). Finally, our results, with the use of sera as source of an antibody-complement to induce Ca(2+) entry, demonstrate that it is possible to resolve fast [Ca(2+)](i) signals in single parasites from T. evansi.

Animals↗
PubMed2000

In squid nerves intracellular Mg(2+) promotes deactivation of the ATP-upregulated Na(+)/Ca(2+) exchanger.

We investigated the role of intracellular Mg(2+) (Mg(i)(2+)) on the ATP regulation of Na(+)/Ca(2+) exchanger in squid axons and bovine heart. In squid axons and nerve vesicles, the ATP-upregulated exchanger remains activated after removal of cytoplasmic Mg(2+), even in the absence of ATP. Rapid and complete deactivation of the ATP-stimulated exchange occurs upon readmission of Mg(i)(2+). At constant ATP concentration, the effect of intracellular Mg(2+) concentration ([Mg(2+)](i)) on the ATP regulation of exchanger is biphasic: activation at low [Mg(2+)](i), followed by deactivation as [Mg(2+)](i) is increased. No correlation was found between the above results and the levels of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] measured in nerve membrane vesicles. Incorporation of PtdIns(4,5)P(2) into membrane vesicles activates Na(+)/Ca(2+) exchange in mammalian heart but not in squid nerve. Moreover, an exogenous phosphatase prevents MgATP activation in squid nerves but not in mammalian heart. It is concluded that 1) Mg(i)(2+) is an essential cofactor for the deactivation part of ATP regulation of the exchanger and 2) the metabolic pathway of ATP upregulation of the Na(+)/Ca(2+) exchanger is different in mammalian heart and squid nerves.

Adenosine Triphosphate↗
PubMed1998

Intracellular calcium clearance in Purkinje cell somata from rat cerebellar slices.

1. The mechanisms governing the return of intracellular calcium (Cai2+) to baseline levels following depolarization-evoked [Ca2+]i rises were investigated in Purkinje cell somata using tight-seal whole-cell recordings and fura-2 microfluorometry, for peak [Ca2+]i ranging from 50 nm to 2 microM. 2. Cai2+ decay was well fitted by a double exponential with time constants of O.6 and 3 s. Both time constants were independent of peak [Ca2+]i but the contribution of the faster component increased with [Ca2+]i. 3. Thapsigargin (10 microM) and cyclopiazonic acid (50 microM) prolonged Cai2+ decay indicating that sarco-endoplasmic reticulum Ca2+ (SERCA) pumps contribute to Purkinje cell Cai2+ clearance. 4. A modest participation in clearance was found for the plasma membrane Ca2+ (PMCA) pumps using 5,6-succinimidyl carboxyeosin (40 microM). 5. The Na(+)-Ca2+ exchanger also contributed to the clearance process, since replacement of extracellular Na+ by Li+ slowed Cai2+ decay. 6. Carbonyl cyanide m-chlorophenylhydrazone (CCCP, 2 microM) and rotenone (10 microM) increased [Ca2+]i and elicited large inward currents at -60 mV. Both effects were also obtained with CCCP in the absence of external Ca2+, suggesting that mitochondrial Ca2+ uptake uncouplers release Ca2+ from intracellular stores and may alter the membrane permeability to Ca2+. These effects were irreversible and impeded tests on the role of mitochondria in Cai2+ clearance. 7. The relative contribution of the clearance systems characterized in this study varied as a function of [Ca2+]i. At 0.5 microM Cai2+, SERCA pumps and the Na(+)-Ca2+ exchanger contribute equally to removal and account for 78% of the process. Only 45% of the removal at 2 microM Cai2+ can be explained by these systems. In this high [Ca2+]i range the major contribution is that of SERCA pumps (21%) and of the Na(+)-Ca2+ exchanger (18%), whereas the contribution of PMCA pumps is only 6%.

Animals↗
PubMed1998

Differential up-regulation of Na+-Ca2+ exchange by phosphoarginine and ATP in dialysed squid axons.

1. The aim of this study was to characterize further the two main metabolic pathways of regulation of the Na+-Ca2+ exchanger in squid axons induced by its two naturally ocurring high-energy compounds: ATP and phosphoarginine (Pa). [Na+]o-dependent Ca2+ efflux (forward Na+o-Ca2+i exchange) and [Ca2+]o-dependent Ca2+ efflux (Ca2+o-Ca2+i exchange) were measured in internally dialysed squid axons at 16-17 C. 2. Measurements of changes in the apparent affinity of the Na+-Ca2+ exchanger for transporting (Na+o, Na+i, Ca2+o, Ca2+i) and regulatory (Ca2+i) ions induced by ATP and Pa show marked differences for the two substrates: (i) ATP strongly alters the affinity for Na+o and Na+i, while Pa does not, and (ii) in the absence of Na+i, ATP has no stimulatiory effect; on the other hand, Pa causes a dramatic increase in Na+o-Ca2+i exchange with little activation of Ca2+o-Ca2+i exchange. 3. The MgATP analogue chromium-ATP (CrATP) completely inhibits MgATP stimulation of the Na+-Ca2+ exchanger. Nevertheless, even with the effects of the nucleotide blocked, Pa exhibits its usual activation of the [Na+]o-dependent Ca2+ efflux. 4. None of the classical serine-threonine-tyrosine kinase inhibitors, nor the PP1 and PP2 phosphatase inhibitors, affects either the ATP or the Pa effect. However, intracellular microinjections of an exogenous phosphatase (alkaline phosphatase) completely reverses the stimulation of the Na+-Ca2+ exchange induced by ATP and Pa. 5. Prolonged intracellular dialysis with highly permeable porous capillaries (18 kDa molecular weight cut-off), which normally induces a complete run-down of the MgATP effect, does not alter the Pa stimulation of the exchanger, even after 6 h of continuous dialysis. 6. We conclude that the ATP and Pa modulation of Na+-Ca2+ exchange in an invertebrate nerve fibre are two genuinely different mechanisms, which affect the carrier properties in very different ways. An interesting similarity between ATP and Pa is that a phosphorylation-dephosphorylation process seems to be a common feature of these two regulation modes.

Adenosine Triphosphate↗
PubMed1998

ATP stimulation of Na+/Ca2+ exchange in cardiac sarcolemmal vesicles.

In cardiac sarcolemmal vesicles, MgATP stimulates Na+/Ca2+ exchange with the following characteristics: 1) increases 10-fold the apparent affinity for cytosolic Ca2+; 2) a Michaelis constant for ATP of approximately 500 microM; 3) requires micromolar vanadate while millimolar concentrations are inhibitory; 4) not observed in the presence of 20 microM eosin alone but reinstated when vanadate is added; 5) mimicked by adenosine 5'-O-(3-thiotriphosphate), without the need for vanadate, but not by beta,gamma-methyleneadenosine 5'-triphosphate; and 6) not affected by unspecific protein alkaline phosphatase but abolished by a phosphatidylinositol-specific phospholipase C (PI-PLC). The PI-PLC effect is counteracted by phosphatidylinositol. In addition, in the absence of ATP, L-alpha-phosphatidylinositol 4,5-bisphosphate (PIP2) was able to stimulate the exchanger activity in vesicles pretreated with PI-PLC. This MgATP stimulation is not related to phosphorylation of the carrier, whereas phosphorylation appeared in the phosphoinositides, mainly PIP2, that coimmunoprecipitate with the exchanger. Vesicles incubated with MgATP and no Ca2+ show a marked synthesis of L-alpha-phosphatidylinositol 4-monophosphate (PIP) with little production of PIP2; in the presence of 1 microM Ca2+, the net synthesis of PIP is smaller, whereas that of PIP2 increases ninefold. These results indicate that PIP2 is involved in the MgATP stimulation of the cardiac Na+/Ca2+ exchanger through a fast phosphorylation chain: a Ca(2+)-independent PIP formation followed by a Ca(2+)-dependent synthesis of PIP2.

Adenosine Triphosphate↗
PubMed1997

A novel 13 kDa cytoplasmic soluble protein is required for the nucleotide (MgATP) modulation of the Na/Ca exchange in squid nerve fibers.

The Na/Ca exchange is a highly regulated transport mechanism in which MgATP, a powerful modulatory intracellular substrate, has important implications for its function. As occurs with some preparations, in squid axons, nucleotide regulation is lost after membrane vesicle isolation. This has been a significant obstacle in the biochemical characterization of the MgATP effect. An important clue in solving this long-standing puzzle is presented in this work by showing that prolonged intracellular dialysis of squid axons produces a complete run down of the MgATP effect. Here we report that a soluble cytoplasmic factor isolated from fresh squid axoplasm and brain reconstitutes the MgATP stimulation of the Na-gradient-dependent 45Ca uptake in squid optic nerve membrane vesicles. Partial purification of this factor uncovers the presence of a novel 13 kDa soluble cytoplasmic protein (SCPr) which, when microinjected in ATP de-regulated dialyzed squid axons, completely restores the MgATP stimulation of Na(o)-dependent Ca efflux. We propose that in the squid preparation this SCPr constitutes the link between the nucleotide and target effector: the Na/Ca exchanger itself, or other plasma membrane structures which may secondarily interact with the exchanger.

Adenosine Triphosphate↗
PubMed1995

Phosphoarginine stimulation of Na(+)-Ca2+ exchange in squid axons--a new pathway for metabolic regulation?

1. [Na+]o-dependent Ca2+ efflux (forward Na(+)-Ca2+ exchange), [32P]ATP wash-out curves and [ATP] were measured in internally dialysed squid giant axons at 17-18 degrees C. 2. We found that dialysing squid axons without ATP and with [Ca2+]i around 1 microM the basal levels of the [Na+]o-dependent Ca2+ efflux were significantly higher in the presence of N omega-phosphoarginine (PA). Phosphocreatine, a related phosphagen, is without effect. 3. PA stimulation of the Na(+)-Ca2+ exchange occurs in the complete absence of ATP (< 1 microM), being independent of, and additive to, the ATP-stimulated [Na+]o-dependent Ca2+ efflux. PA stimulation of [Na+]o-dependent Ca2+ efflux is fully and rapidly reversible with a Km around 7.7 mM. Activation by saturating [PA] is equivalent in magnitude to that of ATP. 4. PA stimulation of Na(+)-Ca2+ exchange is markedly dependent on intracellular Ca2+ and Mg2+ ions. Below 0.5 microM Ca2+i PA effect is negligible, becoming noticeable between 0.8 and 2 microM. In addition, Ca2+i considerably increases the rate at which PA activates the Na(+)-Ca2+ exchange. Although there is no absolute requirement of the PA effect for Mg2+ ions, this divalent cation largely stimulates the PA effect. 5. This work demonstrates, for the first time, the presence in squid axons of a new form of metabolic regulation of the Na(+)-Ca2+ exchange directly and solely related to PA and different from that of MgATP. This novel mechanism is likely to play a physiological role in Ca2+ extrusion through the Na(+)-Ca2+ exchanger, particularly at micromolar [Ca2+]i.

Animals↗
PubMed1994

Calcium-induced calcium release in cerebellar Purkinje cells.

Depolarization-induced intracellular Ca2+ rises were measured in fura-2-loaded, voltage-clamped Purkinje cells. The peak Ca2+ rise increased more than linearly with voltage step duration, suggesting the presence of Ca(2+)-induced Ca2+ release. In cells from young animals, in which Ca2+ currents could be satisfactorily recorded, a supralinear relation was also found between peak Ca2+ rise and Ca2+ current integral. Responses to long pulses were inhibited in cells dialyzed with 20 microM ruthenium red and potentiated in cells bathed in the presence of 20 microM ryanodine. Upon repetitive depolarization, increasing Ca2+ rises were elicited by successive voltage pulses, probably because of a potentiating effect of residual Ca2+. Altogether, the results indicate an important contribution of Ca(2+)-induced Ca2+ release to Ca2+ signals of Purkinje cells.

Aging↗
PubMed1994

Effects of vanadate on MgATP stimulation of Na-Ca exchange support kinase-phosphatase modulation in squid axons.

We have proposed that in squid axons MgATP stimulation of Na-Ca exchange involves a phosphorylation-dephosphorylation process catalyzed by a kinase-phosphatase system. In the present work, we used vanadate as a tool to gather further evidence about the mechanism of metabolic control of the Na-Ca exchanger in internally dialyzed and voltage-clamped squid axons. Vanadate, at concentrations up to 100 microM, stimulated extracellular Na (Nao)-dependent Ca efflux only in the presence of MgATP but failed to do so when the axons were dialyzed with the nonhydrolyzable ATP analogue beta, gamma-methyleneadenosine 5'-triphosphate or with CrATP, a MgATP analogue that completely abolishes MgATP stimulation of the Na-Ca exchange. In axons fully activated by Mg-adenosine 5'-O-(3-thiotriphosphate), vanadate had no effect on Na-Ca exchange. The dose-response curve for vanadate stimulation followed Michaelian kinetics with a Km of 5.6 +/- 0.4 microM and a maximum velocity of 216 +/- 10 fmol.cm-2.s-1 (intracellular Ca concentration = 0.8 microM). This coincides with the high affinity of vanadate in inhibiting the in vitro phosphatase activity of an alkaline phosphatase extracted from rat liver. In addition, vanadate increased fivefold the apparent affinity for MgATP (Km from 220 +/- 14 to 40 +/- 4 microM). Concentrations of vanadate in the millimolar range inhibited the MgATP-stimulated Na-Ca exchange (apparent Ki of 5.7 +/- 0.3 mM) and the in vitro phosphorylation by the catalytic subunit of a adenosine 3',5'-cyclic monophosphate protein kinase (apparent Ki 2.64 +/- 0.04 mM). We conclude that MgATP stimulation of Na-Ca exchange is proportional to the levels of phosphorylation that result from the balance of the activity of a kinase and a phosphatase activity.

Adenosine Triphosphate↗
PubMed1994

Cardiac sarcolemmal Na/Ca-inhibiting peptides XIP and FMRF-amide also inhibit Na/Ca exchange in squid axons.

The effect of two cardiac sarcolemmal inhibitory peptides, the 20-amino acid exchange inhibitory peptide (XIP) and the molluscan cardioexcitatory tetrapeptide amide Phe-Met-Arg-Phe-NH2 (FMRFa), were tested in dialyzed squid giant axons. XIP injected into axons causes a maximal inhibition of 52 +/- 8% (n = 6) in the external Na (Nao)-dependent Ca efflux. The inhibitory effect was the same in axons dialyzed with saturating intracellular Ca (Cai) concentration (100 microM) and no MgATP or in axons containing submicromolar Cai concentrations (0.7 microM) and 2 mM MgATP. FMRFa, a peptide that shows no obvious homology with XIP, also causes a marked inhibition in Nao-dependent Ca efflux. As in cardiac sarcolemmal vesicles, the peptide inhibits with low apparent affinity (Ki = 1.9 microM; n = 5). Like XIP, FMRFa has the same effect in axons dialyzed with or without MgATP. The data indicate that XIP, which resembles an endogenous calmodulin binding site that may have an autoregulatory function, and the tetrapeptide FM-RFa, which binds to a putative opiate site, both inhibit Na/Ca exchange in squid axons. The sites at which these peptides bind are not related to the nucleotide (MgATP) regulation of Na/Ca exchange. We therefore suggest that these two sites in the vertebrate cardiac Na/Ca exchange are conserved in the invertebrate axon exchanger.

Animals↗
PubMed1994

[Determination of micro and macronutrients in the cattle of the Venezuelan plains and their influence on the origin of bovine paraplegic syndrome].

We report a study carried out in three livestock-producing regions of Venezuela to determine the mineral status of grazing cattle and its relationship to the Síndrome Parpléjico del Bovino (SPB). Animal tissue samples from blood and liver were collected from a total of 17 farms within three regions: southwest (Apure), central (Guárico) and southeast (Bolívar) both during the dry and rainy seasons. In SPB free animals, the serum levels of sodium, potassium, chloride, magnesium, total and ionized calcium, phosphorus, and creatinine, were within the normal range. Glucose was found to be deficient in cattle from Bolívar and Guárico states and normal in Apure. With the exception of liver copper and serum zinc, all the other microelements analyzed (liver cobalt, and molybdenum, and serum iron) were found to be normal. Copper was found to be low in all regions studied with a mean value of 74.8 ppm indicating a moderate deficiency of this element. Similarly, in the central and southwest regions, zinc was found to be close to 0.34 ppm, significantly lower than the critical level of 0.7 ppm. In order to determine the effect of the dry and rainy seasons on the content of macro and microelements, controlled group of cattle from the three regions were followed in their contents of magnesium, calcium, copper and iron. In the dry season all of these elements tended to be much lower, showing a significant increase in the rainy season. This increase was much greater in cattle that received mineral supplementation and sanitary treatment. Bovines with diagnosis of SPB showed: low liver copper content, low serum magnesium and phosphorus levels significantly higher that control cattle.

Animals↗
PubMed1993

In squid axons the Ca2+i regulatory site of the Na+/Ca2+ exchanger is drastically modified by sulfhydryl blocking agents. Evidences that intracellular Ca2+i regulatory and transport sites are different.

We have explored the effect of the sulfhydryl group blocker p-chloromercuryphenylsulfonic acid (PCMBS) on Ca2+ and Na+ interactions with the Na+/Ca2+ exchanger in squid giant nerve fibers. Steady-state Na+o-dependent Ca2+ efflux (forward) and Na+i-dependent Ca2+ influx (reverse) were measured in internally dialyzed, voltage clamped squid axons. External PCMBS (0.5 mM, for 25-35 min) has no effect on the activation of Ca2+ efflux by Na+o, and Ca2+o or on the activatory external monovalent cation site. In contrast, when applied internally it drastically reduces the affinity of the Na+/Ca2+ exchanger towards Ca2+i ions without affecting its maximal rate of transport; in the presence of MgATP the K0.5 for Ca2+i activation of forward Na+/Ca2+ exchange increases from 1.5 microM to 95 microM; likewise the apparent affinity of the Ca2+i stimulation of the reversal exchange decreases 100-fold. Interestingly, no effect of PCMBS was found on the interactions between Na+i and Ca2+i ions with the internal transport site(s) (inhibition of Na+2o and Ca2+o-dependent Ca2+ efflux by Na+i). On the other hand, Na+i ions do not modify the interactions of Ca2+i with that site. Two important characteristics of the Ca2+i regulatory site are uncover in this work: (i) sulfhydryl groups are important in maintaining the integrity of the Ca2+ binding domain of the Ca2+i regulatory site and (ii) Na+i and Ca2+i regulatory, or Na+i and Ca2+i transporting sites, are different entities.

4-Chloromercuribenzenesulfonate↗
PubMed1993

Effects of some metal-ATP complexes on Na(+)-Ca2+ exchange in internally dialysed squid axons.

1. Na(+)o-dependent Ca2+ efflux (forward Na(+)-Ca2+ exchange), and in some cases the Na(+)i-dependent Ca2+ influx (reverse Na(+)-Ca2+ exchange) were measured in internally dialysed squid axons under membrane potential control. 2. We tested the effect on the Na(+)-Ca2+ exchange of the MgATP analogue bidentate chromium adenosine-5'-triphosphate (CrATP), substrate of several kinases, and cobalt tetrammine ATP (Co(NH3)4ATP), a poor substrate of most kinases. 3. CrATP completely blocked the MgATP and MgATP-gamma-S (ATP-gamma-S) stimulation of the Na(+)o-dependent Ca2+ efflux (forward exchange) and the Na+i-dependent Ca2+ influx (reverse exchange). The analogue only blocked the nucleotide-dependent fraction of the Na(+)-Ca2+ exchange without modifying any kinetic parameters of the exchange reactions. 4. The effects of CrATP were fully reversible with a very slow time constant (t 1/2 about 30 min). 5. The MgATP stimulation of the Na(+)-Ca2+ exchange was completely saturated at 1 mM. Higher MgATP concentrations (up to 15 mM) had no additional effects. Pentalysine (internal or external), the protein kinase C inhibitor H-7 (1-(5-isoquinolinylsulphonyl)-2-methylpiperazine) and several calmodulin inhibitors did not inhibit Na(+)-Ca2+ exchange either in the absence or presence of MgATP. 6. Our results do not agree with the idea of an aminophospholipid translocase being responsible for the ATP stimulation of the Na(+)-Ca2+ exchange in squid axons; they suggest that this is due to the action of a kinase system.

Adenosine Triphosphate↗