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M T de la Fuente

Publications and source records attributed to M T de la Fuente.

10 recordsLinked to original sources
PubMed2001

The class II tumor-suppressor gene RARRES3 is expressed in B cell lymphocytic leukemias and down-regulated with disease progression.

The molecular pathogenesis of B cell chronic lymphocytic leukemia (B-CLL), the most common form of leukemia, remains unknown. We have used the mRNA differential display technique to analyze genes that may be involved in the development/progression of B-CLL. We have identified the tumor suppressor retinoic acid receptor responder 3 (RARRES3) as a B-CLL-related gene. RARRES3 maps to chromosome band 11q23, a region frequently deleted in lymphoproliferative disorders. To assess the potential involvement of RARRES3 in leukemogenesis, we examined 24 cases of B-CLL, 10 of acute lymphocytic leukemia (ALL) and five related cell lines by RT-PCR and sequence analyses. We report a correlation between RARRES3 down-regulation and B-CLL progression. We also found decreased RARRES3 gene levels in ALL cases and in the five cell lines studied. We did not find mutations in any of the leukemia samples assayed, including those with 11q23 deletion. These results indicate that RARRES3 may play a role in B-CLL progression.

Adolescent↗
PubMed1999

Fibronectin interaction with alpha4beta1 integrin prevents apoptosis in B cell chronic lymphocytic leukemia: correlation with Bcl-2 and Bax.

B cell chronic lymphocytic leukemia (B-CLL) consists of the accumulation of malignant cells that apparently escape normal apoptotic regulation. We have studied the role of alpha4beta1 integrin/fibronectin interaction in preventing apoptosis of these cells in vitro. B cells from 16 patients showed constant expression of alpha4beta1 and little or no alpha5beta1. B-CLL cells cultured on fibronectin or two previously described fibronectin recombinant fragments (H89 and H0) which contain the ligands for alpha4beta1, consistently showed higher viability than control cells cultured on poly-lysine. The H89 fragment, containing the high affinity ligand CS-1, was the most efficient substrate with mean cell viability values of 72, 60 and 35% at days 2, 5 and 8 of culture, respectively. For control cells these values were 40, 27 and 15%, respectively. Parallel cell cycle analysis confirmed these results. The anti-apoptotic effect required direct contact with immobilized substrata since it was not observed when using B-CLL conditioned media alone or when clustering alpha4beta1 with specific mAbs in suspension. Quantitation of the apoptosis regulatory proteins Bcl-2 and Bax revealed that cells cultured on the H89 fragment showed high/moderate levels of Bcl-2 (with some interpatient variation) and low levels of Bax resulting in an elevated Bcl-2/Bax ratio. These results indicate that adhesion of B-CLL cells to fibronectin upregulate the Bcl-2/Bax ratio and this may contribute to the anti-apoptotic effect induced via alpha4beta1 integrin.

Adult↗
PubMed1997

Drastic facilitation by alpha-latrotoxin of bovine chromaffin cell exocytosis without measurable enhancement of Ca2+ entry or [Ca2+]i.

1. Latrotoxin (LTX, 1-3 nM) caused a gradual increase of the spontaneous catecholamine release rate in bovine adrenal chromaffin cells superfused with normal Krebs-Hepes solution containing 2.5 mM Ca2+. Ca2+ removal abolished this effect. LTX enhanced also the secretory responses to high K+ (35 or 70 mM) and to acetylcholine (ACh, 30 microM). 2. The application of Ca2+ pulses to cells previously superfused with a 0 Ca2+ solution (Krebs-Hepes deprived of CaCl2) induced secretory responses that gradually reached 400-800 nA of catecholamines, provided that LTX was present. The responses to ACh or 35 mM K+ pulses (in the presence of Ca2+) were also enhanced by LTX, from around 100-200 nA to over 1000 nA. Though such enhancement remained in the presence of Ca2+ channel blockers, it disappeared upon the lowering of [Na+]o or in electroporated cells. 3. Using protocols similar to those of secretion, LTX did not enhance basal 45Ca2+ uptake, whole-cell Ca2+ currents or basal [Ca2+]i. In fact, LTX attenuated the K(+)- or ACh-evoked increases in 45Ca2+ uptake and [Ca2+]i. 4. It is proposed that the secretory response to brief periods of Ca2+ reintroductions is triggered by local subplasmalemmal Ca2+i transients, produced by the Na(+)-Ca2+ exchanger of the plasma membrane working in the reverse mode. This situation might be physiologically reproduced during ACh stimulation of chromaffin cells, which is followed by the firing of Na(+)-dependent action potentials.

Acetylcholine↗
PubMed1996

Effects of omega-conotoxin MVIIC on veratridine-induced cytotoxicity and cytosolic Ca(2+) oscillations.

External Ca(2+) entry through various Ca(2+)-channel subtypes is responsible for the large oscillations of the cytosolic Ca(2+) concentrations, [Ca(2+)](i), and cell death induced by veratridine in primary cultures of bovine chromaffin cells. Blockade by omega-conotoxin GVIA (GVIA) of N-type Ca(2+) channels, by omega-agatoxin IVA (IVA) of P-type Ca(2+) channels, or by furnidipine of L-type Ca(2+) channels did not afford cytoprotection. However, (omega-conotoxin MVIIC (MVIIC), a wide-spectrum blocker of N-, P- and Q-type Ca(2+) channels greatly protected the cells against the cytotoxic effects of veratridine. Furnidipine further enhanced the cytoprotecting effects of MVIIC. MVIIC but not furuidipine, markedly reduced the oscillations of [Ca(2+)](i) induced by veratridine in single fura-2-loaded chromaffin cells. The results suggest that Ca(2+) entry through any of the different Ca(2+) channel subtypes present in bovine chromaffin cells might be cytotoxic. They also support two ideas: (i) that wide-spectrum neuronal Ca(2+) channel blockers (i.e. MVIIC) might be better cytoprotecting agents than more specific neuronal Ca(2+) channel blockers (i.e., GVIA, IVA, furnidipine); and (ii) that combined Ca(2+) channel blockers may provide greater cytoprotection than single compounds.

Adrenal Glands↗
PubMed1995

Density of apamin-sensitive Ca(2+)-dependent K+ channels in bovine chromaffin cells: relevance to secretion.

Three objectives were defined when planning this study: (i) to identify binding sites for [125I]-apamin in intact bovine adrenal medulla chromaffin cells and to estimate their density and selectivity; (ii) to determine whether apamin modified the release of catecholamines evoked by brief pulses of dimethylphenylpiperazinium (DMPP, 1 or 5 microM for 10 sec), histamine (10 microM for 10 sec) or high K+ (20, 35 or 70 mM for 10 sec) applied to superfused cells; and (iii) to test whether apamin affected the profiles of the changes in cytosolic Ca2+ concentrations [Ca2+]i obtained in suspensions of cells loaded with fura-2 and stimulated with DMPP or histamine. At equilibrium, increasing concentrations of [125I]-apamin gave a saturation curve whose Scatchard transformation produced a Kd of 132 pM and a Bmax of 0.72 fmol/10(6) cells. Quinine, tetraethylammonium, charybdotoxin or glibenclamide (blockers of various subtypes of K+ channels) did not inhibit [125I]apamin binding. Binding was blocked by apamin and by d-tubocurarine, two blockers of small-conductance Ca(2+)-activated K+ channels (SK channels). The number of binding sites for [125I]apamin amounted to approx. 900 per single chromaffin cell, 0.72 sites per micron 2 surface area. Apamin (1 microM) enhanced the secretory response to histamine (10 microM), DMPP (1 or 5 microM) and high K+ (20 or 35 mM) by 2-3-fold. The response to 70 mM K+, however, was unaffected. Apamin also enhanced the peak [Ca2+]i increase produced by DMPP or histamine by approx. 30%. Overall, these results strongly support the hypothesis that under physiological conditions, SK channels control some of the electrical activity of chromaffin cells and indirectly, the opening of voltage-dependent Ca2+ channels, the access of Ca2+ to the secretory machinery and the rate of catecholamine release to the circulation from the intact adrenal gland.

Adrenal Medulla↗
PubMed1994

Interactions between Ca2+, PCA50941 and Bay K 8644 in bovine chromaffin cells.

We describe here the effects of PCA50941 (a novel 1,4-dihydropyridine derivative) comparatively with Bay K 8644 on various parameters in bovine adrenal chromaffin cells. The binding of [3H](+)-isradipine to bovine adrenal medulla plasma membranes was inhibited similarly by PCA50941 and Bay K 8644 at various [Ca2+]o suggesting a common binding site for both compounds on the dihydropyridine receptor. In voltage-clamped chromaffin cells PCA50941 (1 microM) and Bay K 8644 (1 microM) shifted the I-V relationship of whole-cell Ca2+ currents by about 5-10 mV towards more hyperpolarizing potentials. At -20 mV, PCA50941 enhanced ICa by 195 +/- 16% and Bay K 8644 by 288 +/- 51%. Stimulation of fura 2-loaded chromaffin cell suspensions with 17.7 K+/0.5 Ca2+ increased 3-fold the basal [Ca2+]i. PCA50941 increased further the K(+)-evoked peak to 655 nM, and Bay K 8644 to 1129 nM. In the presence of 5 mM Ca2+, PCA50941 or Bay K 8644 increased the [Ca2+] peaks to 427 and 350 nM, respectively. PCA50941 potentiated the release of catecholamines from perfused bovine adrenal glands evoked by 30 s pulses of 17.7 mM K+ in a manner dependent on the [Ca2+]o. Thus at 1, 2.5, 5 and 10 mM Ca2+, secretion was 2.3-, 3.8-, 5- and 4-fold greater than in control glands. Bay K 8644 enhanced the K(+)-induced response 3- and 9-fold at [Ca2+]o of 0.25 or 0.5 mM, respectively; at higher [Ca2+]o the potentiation was similar to that of PCA50941.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1994

Localized L-type calcium channels control exocytosis in cat chromaffin cells.

Depolarizing 1-s pulses to 0 mV from a holding potential of -70 mV, induced whole-cell currents through Ca2+ channels (ICa) in patch-clamped cat adrenal medulla chromaffin cells. The dihydropyridine (DHP) furnidipine (3 microM) reduced the peak current by 47% and the late current by 80%. omega-Conotoxin GVIA (CgTx, 1 microM) reduced the peak ICa by 42% and the late ICa by 55%. Pulses (10 s duration) with 70 mM K+/2.5 mM Ca2+ solution (70 K+/2.5 Ca2+), applied to single fura-2-loaded cat chromaffin cells increased the cytosolic Ca2+ concentration ([Ca2+]i) from 0.1 to 2.21 microM; this increase was reduced by 43.7% by furnidipine and by 42.5% by CgTx. In the perfused cat adrenal gland, secretion evoked by 10-s pulses of 70 K+/2.5 Ca2+ was reduced by 25% by CgTx and by 96% by furnidipine. Similar results were obtained when secretion from superfused isolated cat adrenal chromaffin cells was studied and when using a tenfold lower [Ca2+]o. The results are compatible with the existence of DHP-sensitive (L-type) as well as CgTx-sensitive (N-type) voltage-dependent Ca2+ channels in cat chromaffin cells. It seems, however, that though extracellular Ca2+ entry through both channel types leads to similar increments of averaged [Ca2+]i, the control of catecholamine release is dominated only by Ca2+ entering through L-type Ca2+ channels. This supports the idea of a preferential segregation of L-type Ca2+ channels to localized "hot spots" in the plasmalemma of chromaffin cells where exocytosis occurs.

Adrenal Medulla↗
PubMed1994

Modulation by L-type Ca2+ channels and apamin-sensitive K+ channels of muscarinic responses in cat chromaffin cells.

In the perfused cat adrenal gland stimulated with the muscarinic agonist methacholine chloride (100 microM for 3 min), two components were detected in the catecholamine secretory response: 1) an early phasic component that peaked at 300 ng/5 s catecholamine release and 2) a tonic component whose peak was transient and declined to a plateau of about 140 ng/5 s. Apamin (0.1 microM) increased the phasic component to 1,200 ng/5 s and the tonic component to approximately 350 ng/5 s. In single fura 2-loaded cat adrenal chromaffin cells, the cytosolic Ca2+ concentration ([Ca2+]i) also followed a biphasic pattern after stimulation with methacholine. Depletion of extracellular Ca2+ reduced the phasic [Ca2+]i peak by > 50% and the phasic secretory peak by approximately 90%; both the tonic components of [Ca2+]i and secretion were abolished. Depletion of intracellular Ca2+ pools decreased the phasic and tonic components of [Ca2+]i and secretion with respect to control values; however, the phasic components diminished more than the tonic components of [Ca2+]i and secretion. Although 3 microM furnidipine (a dihydropyridine L-type Ca2+ channel blocker) inhibited the phasic component of [Ca2+]i and secretion, its effects were more pronounced on the tonic component. omega-Conotoxin GVIA (1 microM, an N-type Ca2+ channel blocker) did not affect the [Ca2+]i or the methacholine secretory responses. The secretion peak seems to depend on both extracellular free Ca2+ (Cao2+) entry through L-type Ca2+ channels as well as on the mobilization of Ca2+ from intracellular stores; the plateau depends only on Cao2+ entry through L-type Ca2+ channels.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Medulla↗
PubMed1993

R56865 inhibits catecholamine release from bovine chromaffin cells by blocking calcium channels.

1. The effects of R56865 (a new class of cardioprotective agent which prevents Na+ and Ca2+ overload in cardiac myocytes) on catecholamine release, whole-cell current through Ca2+ channels (IBa) and cytosolic Ca2+ concentrations, [Ca2+]i, have been studied in bovine chromaffin cells. 2. R56865 caused a time- and concentration-dependent blockade of catecholamine release from superfused cells stimulated intermittently with 5 s pulses of 59 mM K+. After 5 min superfusion, a 3 microM concentration inhibited secretion by 20%; the blockade increased gradually with perfusion time, to reach 85% after 40 min. The IC50 to block secretion after 5 min periods of exposure to increasing concentrations of R56865 was around 3.1 microM. The blocking effects of R56865 were reversible after 5-15 min wash out. In high Ca2+ solution (10 mM Ca2+), the degree of blockade of secretion diminished by 20% in comparison with 1 mM Ca2+. 3. In electroporated cells, R56865 (10 microM) did not modify the secretory response induced by the application of 10 microM free Ca2+. 4. R56865 blocked the peak IBa current in a concentration- and time-dependent manner; its IC50 was very similar to that obtained for secretion (3 microM). The compound not only reduced the size of the peak current but also promoted its inactivation; when the effects of R56865 were measured at the most inactivated part of the current, its IC50 was 1 microM. Both the inactivation and the reduction of the peak currents were reversible upon washing out the drug. 5. In fura-2-loaded single chromaffin cells the basal [Ca2+]i of around 100 nM was elevated to a peak of1.5 microM by the application of a 5 s pulse of 59 mM K+. R56865 (10 microM) did not affect the basal [Ca2+]but blocked by 90% the K+-evoked increase. This effect was fully reversible after 5-10 min of wash out.6. The results are compatible with the idea that R56865 blocks Ca2+ entry into K+-depolarized chromaffin cells by promoting the inactivation of voltage-dependent Ca2+ channels; this would lead to the limitation of the rise in [Ca2+]i and of the release of catecholamines. The restriction of catecholamine release may favour indirectly the known direct beneficial cardioprotective actions of R56865.

Animals↗