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Browsing by Author "Huidobro-Toro, JP"

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A_2B adenosine receptor mediates human chorionic vasoconstriction and signals through arachidonic acid cascade
(2005) Donoso, MV; López, RL; Miranda, R; Briones, R; Huidobro-Toro, JP
Because adenosine is a vascular tone modulator, we examined the effect of adenosine and congeners in the vascular reactivity of isolated human placental vessels and in perfused cotyledons. We characterized its vasomotor action and tentatively identified the receptor subtypes and their intracellular signaling mechanisms. We recorded isometric tension from the circular layer of chorionic vessel rings maintained under 1.5 g of basal tension or precontracted with KCl. The relative order of potency of adenosine and structural analogs is consistent with the expression of A2B receptors, 5'-(N-ethylcarboxamido) adenosine (NECA) being the most potent. The maximal contraction ranged from 45% to 60% of the KCl standard response, except for an A(2A) receptor agonist that did not exceed 15%. Consistently, NECA was 100-fold more potent than adenosine to raise the perfusion pressure of ex vivo perfused cotyledons. In contrast, a selective A(3) receptor agonist relaxed precontracted rings of chorionic vessels. Whereas a selective A3 receptor antagonist was ineffective to antagonize adenosine-induced contraction, A(2) or A(1) receptor antagonists reduced adenosine-induced vasoconstriction concentration-dependently. Denudation of the endothelial layer reduced adenosine- and NECA-induced contractions by 50-70%. Furthermore, indomethacin reduced adenosine- or NECA-induced contractions concentration-dependently in intact and endothelium-denuded rings. A thromboxane receptor antagonist blocked adenosine- and NECA-induced contractions in intact and endothelium-denuded rings, suggesting the involvement of an arachidonic acid metabolite as the mediator of the vasoconstriction. We propose that adenosine A(2B) receptors mediate the adenosine- induced contraction vasomotor effect in human chorionic vessels and that this involves synthesis of a thromboxane receptor activator or a related prostanoid.
Adenosine 5′-triphosphate and neuropeptide Y are co-transmitters in conjunction with noradrenaline in the human saphenous vein
(1999) Racchi, H; Irarrázabal, MJ; Howard, M; Morán, S; Zalaquett, R; Huidobro-Toro, JP
1 Human saphenous veins were used to assess the cooperative participation of adenosine 5-triphosphate (ATP), neuropeptide Y (NPY), and noradrenaline (NA) in the vasomotor responses elicited following electrical depolarization of the perivascular nerve terminals. Rings from recently dissected human biopsies were mounted to record isometric muscular contractions; the motor activity elicited in the circular muscle layer following electrical depolarization (2.5-20 Hz, 50 V, 0.5 msec) were recorded.
Aleph2, a Suspected Anxiolytic and Putative Hallucinogenic Phenylisopropylamine Derivative, Is a 5-Ht2a and 5-Ht2c Receptor Agonist
(2000) Acuña-Castillo, C; Scorza, C; Reyes-Parada, M; Cassels, BK; Huidobro-Toro, JP
To assess the pharmacodynamic profile of ALEPH-2, a phenylisopropylamine derivative with alleged anxiolytic and hallucinogenic properties,Xenopus laevis oocytes were microinjected with either of the rat cRNA for the 5-HT2A or the 5-HT2C receptor. Concentration-response curves were obtained following the exposure of the oocytes to varying concentrations of either ALEPH-2 or 5-hydroxy-tryptamine (5-HT) for 10 s. ALEPH-2 is a partial agonist on the 5-HT,,receptor with a similar potency to 5-HT. In contrast, ALEPH-2 is a full 5-HT2C receptor agonist and is about 15-fold less potent than 5-HT. Pre-application of 1 muM ritanserin antagonized the responses induced by 5-HT and ALEPH-2 to the same extent; however, the 5-HT2A receptor is more sensitive to ritanserin blockade than the 5-HT2C receptor. (C) 2000 Elsevier Science Inc. All rights reserved.
Clonidine-induced nitric oxide-dependent vasorelaxation mediated by endothelial α₂-adrenoceptor activation
(2001) Figueroa, XF; Poblete, MI; Boric, MP; Mendizábal, VE; Adler-Graschinsky, E; Huidobro-Toro, JP
1 To assess the involvement of endothelial alpha (2)-adrenoceptors in the clonidine-induced vasodilatation, the mesenteric artery of Sprague Dawley rats was cannulated and perfused with Tyrode solution (2 ml min(-1)). We measured perfusion pressure, nitric oxide (NO) in the perfusate using chemiluminescence, and tissue cyclic GMP by RIA.
Extracellular histidine residues identify common structural determinants in the copper/zinc P2X₂ receptor modulation
(2005) Lorca, RA; Coddou, C; Gazitúa, MC; Bull, P; Arredondo, C; Huidobro-Toro, JP
To assess the mechanism of P2X(2) receptor modulation by transition metals, the cDNA for the wild-type receptor was injected to Xenopus laevis oocytes and examined 48-72 h later by the two-electrode voltage-clamp technique. Copper was the most potent of the trace metals examined; at 10 mu M it evoked a 25-fold potentiation of the 10 mu M ATP-gated currents. Zinc, nickel or mercury required 10-fold larger concentrations to cause comparable potentiations, while palladium, cobalt or cadmium averaged only 12- and 3-fold potentiations, respectively. Platinum was inactive. The non-additive effect of copper and zinc at 10-100 mu M suggests a common site of action; these metals also shifted to the left the ATP concentration-response curves. To define residues necessary for trace metal modulation, alanines were singly substituted for each of the nine histidines in the extracellular domain of the rat P2X(2) receptor. The H120A and H213A mutants were resistant to the modulator action of copper, zinc and other metals with the exception of mercury. Mutant H192A showed a reduction but not an abrogation of the copper or zinc potentiation. H245A showed less affinity for copper while this mutant flattened the zinc-induced potentiation. Mutant H319A reduced the copper but not the zinc-induced potentiation. In contrast, mutants H125A, H146A, H152A and H174A conserved the wild-type receptor sensitivity to trace metal modulation. We propose that His120, His192, His213 and His245 form part of a common allosteric metal-binding site of the P2X(2) receptor, which for the specific coordination of copper, but not zinc, additionally involves His319.
Heavy metals modulate the activity of the purinergic P2X₄ receptor
(2005) Coddou, C; Lorca, RA; Acuña-Castillo, C; Grauso, M; Rassendren, F; Huidobro-Toro, JP
To further characterize the nature of the regulatory metal-binding sites of the rat P2X(4) receptor, several transition heavy metals were tested to examine their ability to mimic the facilitator action of zinc or the inhibitory action of copper. cDNA coding for the rat P2X(4) receptor was injected into Xenopus laevis oocytes; the two-electrode voltage-clamp technique was used to measure and quantify the ATP-evoked currents in the absence or presence of the metals. Cadmium facilitated the ATP-gated currents in a reversible and voltage-independent manner; maximal potentiation occurred within less than 1 min.
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.
Neuropeptide Y is released from human mammary and radial vascular biopsies and is a functional modulator of sympathetic cotransmission
(2004) Donoso, MV; Miranda, R; Irarrázaval, MJ; Huidobro-Toro, JP
The role of neuropeptide Y (NPY) as a modulator of the vasomotor responses mediated by sympathetic cotransmitters was examined by electrically evoking its release from the perivascular nerve terminals of second- to third-order human blood vessel biopsies and by studying the peptide-induced potentiation of the vasomotor responses evoked by exogenous adenosine 5' triphosphate (ATP) and noradrenaline (NA). Electrical depolarization of nerve terminals in mammary vessels and radial artery biopsies elicited a rise in superfusate immunoreactive NPY (ir-NPY), which was chromatographically identical to a standard of human NPY (hNPY); a second peak was identified as oxidized hNPY. The amount released corresponds to 4-6% of the total NPY content in these vessels. Tissue extracts also revealed two peaks; hNPY accounted for 68-85% of the ir-NPY, while oxidized hNPY corresponded to 7-15%. The release process depended on extracellular calcium and on the frequency and duration of the electrical stimuli; guanethidine blocked the release, confirming the peptide's sympathetic origin. Assessment of the functional activity of the oxidized product demonstrated that while it did not change basal tension, the NA-evoked contractions were potentiated to the same extent as with native hNPY. Moreover, NPY potentiated both the vasomotor action of ATP or NA alone and the vasoconstriction elicited by the simultaneous application of both cotransmitters. RT-PCR detected the mRNA coding for the NPY Y-1 receptor. In summary, the release of hNPY or its oxidized species, elicited by nerve terminal depolarization, coupled to the potentiation of the sympathetic cotransmitter vasomotor responses, highlights the modulator role of NPY in both arteries and veins, strongly suggesting its involvement in human vascular sympathetic reflexes. Copyright (C) 2004 S. Karger AG, Basel.
Nitric oxide synthase-independent release of nitric oxide induced by KCl in the perfused mesenteric bed of the rat
(2000) Mendizabal, VE; Poblete, I; Lomniczi, A; Rettori, V; Huidobro-Toro, JP; Adler-Graschinsky, E
The aim of the present study was to test whether the contractile responses elicited by KCI in the rat mesenteric bed are coupled to the release of nitric oxide (NO). Contractions induced by 70 mM KCl were coincident with the release of NO to the perfusate. The in vitro exposure to the nitric oxide synthase (NOS) inhibitor L-N-omega-nitro-L-arginine methyl ester, L-NAME (1-100 muM) potentiated the vascular responses to 70 mM KCI and, unexpectedly, increased the KCl-stimulated release of NO. Moreover, even after the chronic treatment with L-NAME (70 mg/kg/day during 4 weeks), the KCl-induced release of NO was not reduced, whereas the potentiation of contractile responses was indeed achieved. The possibility that NOS had not been completely inhibited under our experimental conditions can be precluded because NOS activity was significantly inhibited after both L-NAME treatments. After the in vitro treatment with 1 to 100 muM L-NAME, the inhibition of NOS was concentration-dependent (from 50% to 90%). With regard to the basal release of NO, the inhibition caused by L-NAME was not concentration-dependent and reached a maximum of 40%, suggesting that bas al NO outflow is only partially dependent on NOS activity. An eventual enhancement of NOS activity caused by KCI was disregarded because the activity of this enzyme measured in homogenates from mesenteric beds perfused with 70 mM KCl was significantly reduced. On the other hand, endothelium removal, employed as a negative control, almost abolished NOS activity, whereas the incubation with the Ca2+ ionophore A23187, employed as a positive control, induced an increase in NOS activity. It is concluded that in the mesenteric arterial bed of the rat. the contractile responses elicited by depolarization through KCl an coincident with a NOS-independent release of NO. This observation, which differs from the results obtained with noradrenaline, do not support the use of KCl as an alternative contractile agent whenever the participation of NO is under study. (C) 2000 Elsevier Science B.V. All rights reserved.
P2Y₁ and P2Y₂ receptors are coupled to the NO/cGMP pathway to vasodilate the rat arterial mesenteric bed
(2002) Buvinic, S; Briones, R; Huidobro-Toro, JP
1 To assess the role of nucleotide receptors in endothelial-smooth muscle signalling, changes in perfusion pressure of the rat arterial mesenteric bed, the luminal output of nitric oxide (NO) and guanosine 3',5' cyclic monophosphate (cGMP) accumulation were measured after the perfusion of nucleotides.
Pharmacological identification of P2X₁, P2X₄ and P2X₇ nucleotide receptors in the smooth muscles of human umbilical cord and chorionic blood vessels
(2003) Valdecantos, P; Briones, R; Moya, P; Germain, A; Huidobro-Toro, JP
To ascertain the role of extracellular adenosine 5'-triphosphate (ATP) receptors in human placenta circulation, we identified and pharmacologically characterized the P2X receptor population in its superficial vessels. Total RNA was extracted from segments of chorionic and umbilical arteries and veins of terminal placentae delivered by vaginal or Caesarian births. Polymerase chain reaction (PCR), followed by sequencing of the products, identified the presence of P2X 1, 4, 5, 6, and 7 mRNAs in smooth muscle from chorionic and umbilical arteries and veins. Umbilical vessels proximal to the fetus expressed the same population of P2X subtypes, except for the P2X(5), but additionally expressed the P2X(2). Rings of chorionic vessels contracted upon addition of nucleotides and analogs with the following relative rank order of potencies in arteries and veins: alpha,beta-methyleneATP>beta,gamma-methyleneATP>PNP>ATP=diBzATP>2-MeSATP>ADP>AMP; in umbilical vessels alpha,beta-methyleneATP was at least 100-fold more potent than ATP. Nucleotide potency was less than that of PGF(2alpha) or endothelin-2, but had the same magnitude as serotonin. ATP-desensitized receptors evidenced cross desensitization to alpha,beta-methyleneATP, 2-MeSATP and diBzATP, effect not observed when desensitization was elicited by alpha,beta-methyleneATP, confirming the presence of various P2X receptor subtypes in the smooth muscles of these vessels. The vasocontractile efficacy of alpha,beta-methyleneATP was unaltered by endothelium removal, while that of ATP was significantly attenuated and those elicited by 2-MeSATP were blunted, indicating the presence of additional endothelial nucleotide receptors. These results suggest that P2X receptors participate in the humoral regulation of placental blood flow. (C) 2002 Elsevier Science Ltd. All rights reserved.
Synergism between neuropeptide Y and norepinephrine highlights sympathetic cotransmission
(1999) Cortés, V; Donoso, MV; Brown, N; Fanjul, R; López, C; Fournier, A; Huidobro-Toro, JP
Although abundant literature supports the notion that neuropeptide Y (NPY) synergizes in vivo and in vitro, the vasomotor activity elicited by norepinephrine (NE), the converse interaction (i.e,, the adrenergic modulation of the NPY vasomotor response) has been less characterized. To assess whether NE synergizes the vasomotor effect of NPY, the rat arterial mesenteric bed was chosen as a model experimental system. Mesenteries were precontracted with NE and few minutes later were pelf used with exogenous NPY. Under these conditions, NPY contracted the arterial mesenteric bed with an EC50 value of 0.72 +/- 0.06 nM. NPY was unable to contract this vascular territory without an agonist-induced precontraction, Other agonists, such as endothelin-1, a synthetic analog of prostaglandin F-2 alpha, or 5-hydroxytryptamine, also were effective primers because in their presence, NPY was a potent vasoconstrictor. In contrast, mesenteries precontracted with KCI failed to evidence the NPY-induced rise in perfusion pressure. Two structural analogs of NPY, PW and [Leu(31),Pro(34)]NPY, mimicked the activity of NPY. The NPY fragment 13-36 did not elicit such a response. All NPY analogs exhibited less efficacy and potency relative to NPY. The NPY- and related structural analog-induced vasoconstriction was competitively and reversibly antagonized by BIBP 3226; the pA(2) of the NPY interaction was 7.0. The application of 0.1 to 1 mu M BIBP 3226 or 0.1 to 10 nM prazosin at the peak of the NPY vasomotor response elicited a gradual blockade of the vasoconstriction. Although BIBP 3226 blocked the increase in perfusion pressure elicited by NPY, leaving unaffected the NE-induced tone, 10 nM prazosin blocked the full response, including the NE-induced component. Tissue preincubation with 200 nM nifedipine abolished the NPY-induced vasoconstriction; likewise, the acute application of 10 to 100 nM nifedipine blocked gradually the maximal NPY-induced contraction. Removal of the mesenteric endothelial layer increased the potency of NPY by 2-fold; it also slightly potentiated the antagonist activity of BIBP 3226, The synergism between NPY and NE backs the principle of sympathetic cotransmission.
The human prion octarepeat fragment prevents and reverses the inhibitory action of copper in the P2X₄ receptor without modifying the zinc action
(2003) Lorca, RA; Chacón, M; Barría, MI; Inestrosa, NC; Huidobro-Toro, JP
Human prion protein fragments (PrP60-67 or PrP59-91) prevented and reversed the inhibition elicited by 5 mum copper on the P2X(4) receptor expressed in Xenopus laevis oocytes. A 60-s pre-application of 5 muM copper caused a 69.2 +/- 2.6% inhibition of the 10 muM adenosine triphosphate (ATP)-evoked currents, an effect that was prevented by mixing 5 muM copper with 0.01-10 muM of the PrP fragments 1-min prior to application. This interaction was selective, as PrP59-91 did not alter the facilitatory action of zinc. The EC50 of PrP60-67 and PrP59-91 for the reduction of the copper inhibition were 4.6 +/- 1 and 1.3 +/- 0.4 muM, respectively. A synthetic PrP59-91 variant in which all four His were replaced by Ala was inactive. However, the replacement of Trp in each of the four putative copper-binding domains by Ala slightly decreased its potency. Furthermore, the application of 10 muM PrP59-91 reversed the copper-evoked inhibition, restoring the ATP concentration curve to the same level as the non-inhibited state. Fragment 139-157 of betaA4 amyloid precursor protein also prevented the action of copper; its EC50 was 1.6 +/- 0.1 muM; the metal chelator penicillamine was equipotent with PrP60-67, but carnosine was significantly less potent. Our findings highlight the role of PrP in copper homeostasis and hint at its possible role as a modulator of synapses regulated by this trace metal.
The hypolipidemic drug metabolites nafenopin-CoA and ciprofibroyl-CoA are competitive P2Y₁ receptor antagonists
(2003) Coddou, C; Loyola, G; Boyer, JL; Bronfman, M; Huidobro-Toro, JP
Coenzyme A (CoA-SH), endogenous and drug-derived CoA-derivatives were tested as putative antagonists of P2Y receptors expressed in Xenopus laevis oocytes, a method used to determine calcium-activated chloride current, an indicator of the activation of these receptors. CoA-SH antagonized reversibly and in a concentration-dependent manner the ATP-gated currents evoked by the human P2Y(1) but not the P2Y(2) receptor. Palmitoyl-CoA was four-fold more potent than CoASH as an antagonist while palmitoyl-carnitine was inactive, highlighting the role of the CoA-SH moiety in the antagonism. The CoA derivatives of nafenopin and ciprofibrate, two clinically relevant hypolipidemic drugs, increased 13 and three-fold the potency of CoA-SH, respectively. The K(B)s of nafenopin-CoA and ciprofibroyl-CoA were 58 and 148 nM, respectively; the slopes of the Schild plots were unitary. Neither 100 muM nafenopin nor ciprofibrate alone altered the P2Y, receptor activity. Neither CoA-SH nor ciprofibroyl-CoA antagonized the rat P2X(2) or the P2X(4) nucleotide receptors nor interacted with the 5-HT2A/C receptors. The bulky drug CoA-SH derivatives identify a hydrophobic pocket, which may serve as a potential target for novel selective P2Y(1) antagonists. (C) 2003 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.
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.
α₂-adrenoceptors control the release of noradrenaline but not neuropeptide Y from perivascular nerve terminals
(2002) Donoso, MV; Carvajal, A; Paredes, A; Tomic, A; Koenig, CS; Huidobro-Toro, JP
Neuropeptide Y (NPY) and noradrenaline (NA) are co-transmitters at many sympathetic synapses, but it is not yet clear if their release is independently regulated. To address this question, we quantified the electrically evoked release of these co-transmitters from perivascular nerve terminals to the mesenteric circulation in control and drug-treated rats. 6-Hydroxydopamine reduced the tissue content and the electrically evoked release of ir-NPY and NA as well as the rise in perfusion pressure. A 0.001 mg/kg reserpine reduced the content of ir-NPY and NA, but did not modify their release nor altered the rise in perfusion pressure elicited by the electrical stimuli. However, 0.1 mg/kg reserpine reduced both the content and release of NA but decreased only the content but not the release of ir-NPY; the rise in perfusion pressure was halved. Clonidine did not affect the release of ir-NPY while it lowered the outflow of NA, not altering the rise in perfusion pressure elicited by the electrical stimuli. Yohimbine, did not modify the release of ir-NPY but increased the NA outflow, it antagonized the clonidine effect. Therefore, presynaptic alpha(2)-adrenoceptors modulate the release of NA but not NPY, implying separate regulatory mechanisms. (C) 2002 Elsevier Science Inc. All rights reserved.

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