Browsing by Author "Morales, B"

Now showing 1 - 8 of 8
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    A physical chemistry theory of synergism on ciliated cell motility
    (BIOPHYSICAL SOCIETY, 2002) Barrera, N; Morales, B; Villalon, M
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    Cyclooxygenase-2/PGE(2) system mediates the effect of platelet activating factor in oviductal ciliated cells.
    (SOC STUDY REPRODUCTION, 1998) Villalon, M; Morales, B; Barrera, N; Oliva, C
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    Histidine 140 plays a key role in the inhibitory modulation of the P2X4 nucleotide receptor by copper but not zinc
    (2003) Coddou, C; Morales, B; González, J; Grauso, M; Gordillo, F; Bull, P; Rassendren, F; Huidobro-Toro, JP
    To elucidate the role of extracellular histidines in the modulation of the rat P2X(4) receptor by trace metals, we generated single, double, and triple histidine mutants for residues 140, 241, and 286, replacing them with alanines. cDNAs for the wild-type and receptor mutants were expressed in Xenopus laevis oocytes and in human embryonic kidney 293 cells and examined by the two electrode and patch clamp techniques, respectively. Whereas copper inhibited concentration-dependently the ATP-gated currents in the wild-type and in the single or double H241A and H286A receptor mutants, all receptors containing H140A were insensitive to copper in both cell systems. The characteristic bell-shaped concentration-response curve of zinc observed in the wildtype receptor became sigmoid in both oocytes and human embryonic kidney cells expressing the H140A mutant; in these mutants, the zinc potentiation was 2.5-4-fold larger than in the wild-type. Results with the H140T and H140R mutants further support the importance of a histidine residue at this position. We conclude that His-140 is critical for the action of copper, indicating that this histidine residue, but not His-241 or His-286, forms part of the inhibitory allosteric metal-binding site of the P2X4 receptor, which is distinct from the putative zinc facilitator binding site.
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    Plasma and intracellular membrane inositol 1,4,5-trisphosphate receptors mediate the Ca2+ increase associated with the ATP-induced increase in ciliary beat frequency
    (2004) Barrera, NP; Morales, B; Villalón, M
    An increase in intracellular free Ca2+ concentration ([Ca2+](i)) has been shown to be involved in the increase in ciliary beat frequency (CBF) in response to ATP; however, the signaling pathways associated with inositol 1,4,5-trisphosphate (IP3) receptor-dependent Ca2+ mobilization remain unresolved. Using radioimmunoassay techniques, we have demonstrated the appearance of two IP3 peaks occurring 10 and 60 s after ATP addition, which was strongly correlated with a release of intracellular Ca2+ from internal stores and an influx of extracellular Ca2+, respectively. In addition, ATP-dependent Ca2+ mobilization required protein kinase C (PKC) and Ca2+/ calmodulin-dependent protein kinase II activation. We found an increase in PKC activity in response to ATP, with a peak at 60 s after ATP addition. Xestospongin C, an IP3 receptor blocker, significantly diminished both the ATP-induced increase in CBF and the initial transient [Ca2+](i) component. ATP addition in the presence of xestospongin C or thapsigargin revealed that the Ca2+ influx is also dependent on IP3 receptor activation. Immunofluorescence and confocal microscopic studies showed the presence of IP3 receptor types 1 and 3 in cultured ciliated cells. Immunogold electron microscopy localized IP3 receptor type 3 to the nucleus, the endoplasmic reticulum, and, interestingly, the plasma membrane. In contrast, IP3 receptor type 1 was found exclusively in the nucleus and the endoplasmic reticulum. Our study demonstrates for the first time the presence of IP3 receptor type 3 in the plasma membrane in ciliated cells and leads us to postulate that the IP3 receptor can directly trigger Ca2+ influx in response to ATP.
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    Platelet activating factor increases ciliary activity in the hamster oviduct through epithelial production of prostaglandin E2
    (2001) Hermoso, M; Barrera, N; Morales, B; Pérez, S; Villalón, M
    We investigated the signal transduction mechanisms associated with an increase in ciliary beat frequency (CBF) produced by platelet activating factor (PAF) in oviductal ciliated cell cultures. In the range of concentrations similar to that produced by preimplantation embryos, PAF increased the CBF in a dose-dependent manner. The addition of PAF and prostaglandin E-2 (PGE,) to the cultures produced a synergic increase of ciliary beating, suggesting that PAF and PGE(2) signal transduction pathways may be associated. To demonstrate this hypothesis, cyclooxygenase-2 (COX-2) was selectively blocked by a specific inhibitor, NS-398, and the PAF-induced CBF increase was abolished. Moreover, a phospholipase A2 (PLA(2)) inhibitor, AACOCF3, blocks the PAF-induced CBF increase. PGE(2) production by oviductal epithelial cells is stimulated by PAF, and WEB-2086, a PAF-receptor blocker, specifically blocks the PAF-induced PGE(2) production. Using the fluorescent indicator fura-2, we measured the effect of PAF on intracellular Ca2+ concentration ([Ca2+](i)) in individual ciliated cells. PAF induced a transient increase of [Ca2+](i) that was blocked by WEB-2086 or by removal of extracellular Ca2+. We propose a mechanism for PAF-mediated signal transduction in the ciliated cells of the oviductal epithelium. Minimal doses of PAF trigger Ca2+ mobilization in tandem with increased PLA(2) activity and a COX-2-mediated increase in PGE(2). Local PGE(2) production by the oviductal mucosa suggests the presence of an autocrine loop controlling ciliary activity.
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    Zinc and copper modulate differentially the P2X4 receptor
    (2000) Acuña-Castillo, C; Morales, B; Huidobro-Toro, JP
    The rat ATP P2X(4) receptor was expressed in Xenopus laevis oocytes to assess the effect of zinc and copper as possible regulators of purinergic mechanisms. ATP applied for 20 s evoked an inward cationic current with a median effective concentration (EC50) of 21.4 +/- 2.8 mu M and a Hill coefficient (n(H)) of 1.5 +/- 0.1. Coapplication of ATP plus 10 mu M zinc displaced leftward, in a parallel fashion, the ATP concentration-response curve, reducing the EC50 to 8.4 +/- 1.8 mu M (p < 0.01) without altering the receptor n(H). The zinc potentiation was fast in onset, easily reversible, and voltage-independent and did not require metal preexposure. The zinc EC50 was 2-5 mu M, with a bell-shaped curve. At concentrations of 100-300 mu M, zinc produced less potentiation, and at 1 mM, it inhibited 50% the ATP current. The effect of zinc was mimicked by cadmium, in contrast, copper inhibited the ATP-evoked currents in a time- and concentration-dependent fashion, reducing the maximal current (I-max) without altering the EC50. The copper-induced inhibition was slow in onset, slowly reversible, and voltage-independent. Whereas coapplication of 300 mu M copper plus ATP reduced I-max to 36.2 +/- 5%, the coapplication of, or 60-s preexposure by, 10 mu M copper reduced I-max to 79 +/- 9.2% (p < 0.05) and 39.6 +/- 8.7% (p < 0.01), respectively. The inhibition was noncompetitive in nature and mimicked by mercury. Cobalt, barium, and manganese did not modify significantly the ATP-evoked current, demonstrating metal specificity. The simultaneous 1-min preapplication of both metals revealed that the 10 mu M zinc-induced potentiation was obliterated by 10 mu M copper, whereas 30 mu M copper not only reduced the potentiation, but inhibited the ATP response. Following coapplication of both metals for 20 s with ATP, at least 100 mu M copper was required to counteract the 10 mu M zinc-induced potentiation. The simultaneous preincubation with both metals provided evidence for a noncompetitive interaction. We hypothesize the existence of metal binding site(s), which are most likely localized in the extracellular domain of the P2X(4) receptor structure. These sites are selective and accessible to extracellular metal applications and bind micromolar concentrations of metals. The present results are compatible with the working hypothesis that trace metals, such as copper and zinc, are physiological modulators of the P2X(4) receptor. The modulation of brain purinergic transmission by physiologically and toxicologically relevant trace metal cations is highlighted.