RESUMO
The Membrane Attack Complex-Perforin (MACPF) family is ubiquitously found in all kingdoms. They have diverse cellular roles, however MACPFs with pore-forming toxic function in venoms and poisons are very rare in animals. Here we present the structure of PmPV2, a MACPF toxin from the poisonous apple snail eggs, that can affect the digestive and nervous systems of potential predators. We report the three-dimensional structure of PmPV2, at 17.2 Å resolution determined by negative-stain electron microscopy and its solution structure by small angle X-ray scattering (SAXS). We found that PV2s differ from nearly all MACPFs in two respects: it is a dimer in solution and protomers combine two immune proteins into an AB toxin. The MACPF chain is linked by a single disulfide bond to a tachylectin chain, and two heterodimers are arranged head-to-tail by non-covalent forces in the native protein. MACPF domain is fused with a putative new Ct-accessory domain exclusive to invertebrates. The tachylectin is a six-bladed ß-propeller, similar to animal tectonins. We experimentally validated the predicted functions of both subunits and demonstrated for the first time that PV2s are true pore-forming toxins. The tachylectin "B" delivery subunit would bind to target membranes, and then the MACPF "A" toxic subunit would disrupt lipid bilayers forming large pores altering the plasma membrane conductance. These results indicate that PV2s toxicity evolved by linking two immune proteins where their combined preexisting functions gave rise to a new toxic entity with a novel role in defense against predation. This structure is an unparalleled example of protein exaptation.
Assuntos
Complexo de Ataque à Membrana do Sistema Complemento/ultraestrutura , Lectinas/ultraestrutura , Perforina/ultraestrutura , Conformação Proteica , Sequência de Aminoácidos/genética , Animais , Membrana Celular/química , Membrana Celular/ultraestrutura , Complexo de Ataque à Membrana do Sistema Complemento/química , Complexo de Ataque à Membrana do Sistema Complemento/imunologia , Cristalografia por Raios X , Dimerização , Lectinas/química , Lectinas/imunologia , Modelos Moleculares , Perforina/química , Perforina/imunologia , Subunidades Proteicas/genética , Espalhamento a Baixo Ângulo , Caramujos/ultraestrutura , Difração de Raios XRESUMO
Ionotropic purinergic receptors (P2X) are expressed in endothelial and smooth muscle cells of blood vessels. ATP acting on smooth muscle P2X receptors is able to induce vasoconstriction in different kind of vessels. However, to our knowledge, there are no reports that directly show the activity of these purinergic receptors in native human vascular smooth muscle cells. In this work, we describe for the first time an ATP-induced current in freshly isolated human umbilical artery (HUA) smooth muscle cells. The current was measured by patch-clamp technique in whole-cell condition on cells clamped at -50 mV. At 100 µM of ATP the current showed a rapid activation and desensitization, and was carried by both Na(+) and Ca(2+). The current was completely blocked by suramin (300 µM) and partially blocked by 100 µM of Zn(2+) without affecting the kinetic of desensitization. All these properties suggest that the ATP-induced ionic currents are mediated through P2X(1)-like receptors. Moreover, we show that ATP transiently increased cytosolic Ca(2+) in "in situ" smooth muscle cells of intact HUA segments and that this response is dependent of extracellular and intracellular Ca(2+). These data expand the knowledge of purinergic receptors properties in vascular smooth muscle cells and the probable role of ATP as a paracrine modulator of contractile tone in a human artery which is fundamental for feto-placental blood flow.
Assuntos
Trifosfato de Adenosina/fisiologia , Cálcio/metabolismo , Citosol/metabolismo , Líquido Extracelular/fisiologia , Miócitos de Músculo Liso/metabolismo , Artérias Umbilicais/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Citosol/efeitos dos fármacos , Líquido Extracelular/efeitos dos fármacos , Feminino , Humanos , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacos , Gravidez , Suramina/farmacologia , Fatores de Tempo , Artérias Umbilicais/efeitos dos fármacosRESUMO
Isoflavones are a group of natural phytoestrogens including the compound genistein. Health beneficial effects have been attributed to the consumption of this compound, but the fact that it has estrogen-like activity has raised doubts regarding its potential risk in infants, newborns, or in the fetus and placenta during pregnancy. This work is aimed at studying genistein effects on Ca2+ handling by smooth muscle cells of the human umbilical artery (HUA). Using fluorometric techniques, we found that in these cells genistein reduces the intracellular Ca2+ peak produced by serotonin. The same result could be demonstrated in absence of extracellular Ca2+, suggesting that the isoflavone reduces Ca2+ release from the sarcoplasmic reticulum. Force measurement experiments strengthen these results, since genistein reduced the peak force attained by intact HUA rings stimulated by serotonin in a Ca2+-free solution. Moreover, genistein induced the relaxation of HUA rings precontracted either with serotonin or a depolarizing high-extracellular K+ solution, hinting at a reduction of extracellular Ca2+ entry to the cell. This was confirmed by whole-cell patch-clamp experiments where it was shown that the isoflavone inhibits ionic currents through voltage-operated Ca2+ channels. In summary, we show that genistein inhibits two mechanisms that could increase intracellular Ca2+ in human umbilical smooth muscle cells, behaving in this way as a potential vasorelaxing substance of fetal vessels. Taking into account that genistein is able to cross the placental barrier, these data show that isoflavones may have important implications in the regulation of feto-maternal blood flow in pregnant women who consume soy-derived products as part of their meals.
Assuntos
Cálcio/metabolismo , Genisteína/farmacologia , Canais de Cálcio/efeitos dos fármacos , Feminino , Humanos , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Técnicas de Patch-Clamp , Gravidez , Retículo Sarcoplasmático/efeitos dos fármacos , Retículo Sarcoplasmático/metabolismo , Artérias Umbilicais/metabolismoRESUMO
The soy-derived isoflavones genistein and daidzein affect the contractile state of different kinds of smooth muscle. We describe acute effects of genistein and daidzein on contractile force and intracellular Ca2+ concentration ([Ca2+]i) in in situ smooth muscle of rat aorta. Serotonin (5-HT) (2 microM) or a depolarizing high K+ solution produced the contraction of aortic rings, which were immediately relaxed by 20 microM genistein and by 20 microM daidzein. Accordingly, both 5-HT and a high K+ solution increased the [Ca2+]i in in situ smooth muscle cells. Genistein strongly inhibited the [Ca2+]i increase evoked by 5-HT (74.0 +/- 7.3%, n = 11, p < 0.05), and had a smaller effect on high K+ induced [Ca2+]i increase (19.9 +/- 4.0%, n = 7, p < 0.05). The K+ channels blocker tetraethylammonium (TEA) (0.5 mM) diminished genistein effects on 5-HT-induced [Ca2+]i increase. Interestingly, during prolonged application of 5-HT, the [Ca2+]i oscillated and a short (90 s) preincubation with genistein (20 microM) significantly diminished the frequency of the oscillations. This effect was totally abolished by TEA. In conclusion, in rat aortic smooth muscle, genistein is capable of diminishing the increase in [Ca2+]i and in force evoked by 5-HT and high K+ solution, and of decreasing the frequency of [Ca2+]i oscillations induced by 5-HT. The short time required by genistein, and the relaxing effect of daidzein suggest that tyrosine kinases inhibition is not involved. The small inhibiting effect of genistein on the [Ca2+]i increase evoked by high K+ and the effect of TEA point to the activation by genistein of calcium-activated K+ channels.
Assuntos
Aorta Torácica/citologia , Cálcio/antagonistas & inibidores , Genisteína/farmacologia , Contração Muscular/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Serotonina/farmacologia , Animais , Citofotometria/métodos , Contração Isométrica/efeitos dos fármacos , Masculino , Oscilometria/métodos , Ratos , Ratos WistarRESUMO
The data presented in this work suggest that in human umbilical artery (HUA) smooth muscle cells, the Na(+)/Ca(2+) exchanger (NCX) is active and working in the reverse mode. This supposition is based on the following results: (i) microfluorimetry in HUA smooth muscle cells in situ showed that a Ca(2+)-free extracellular solution diminished intracellular Ca(2+) ([Ca(2+)](i)), and KB-R7943 (5microM), a specific inhibitor of the Ca(2+) entry mode of the exchanger, also decreased [Ca(2+)](i) (40.6+/-4.5% of Ca(2+)-free effect); (ii) KB-R7943 produced the relaxation of HUA rings (-24.7+/-7.3gF/gW, n=8, p<0.05); (iii) stimulation of the NCX by lowering extracellular Na(+) increases basal [Ca(2+)](i) proportionally to Na(+) reduction (Delta fluorescence ratio=0.593+/-0.141 for Na(+)-free solution, n=8) and HUA rings' contraction (peak force=181.5+/-39.7 for 130mM reduction, n=8), both inhibited by KB-R7943 and a Ca(2+)-free extracellular solution. In conclusion, the NCX represents an important Ca(2+) entry route in HUA smooth muscle cells.
Assuntos
Cálcio/metabolismo , Contração Muscular/fisiologia , Miócitos de Músculo Liso/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Sódio/metabolismo , Artérias Umbilicais/enzimologia , Células Cultivadas , Humanos , Contração Muscular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Sódio/farmacologia , Artérias Umbilicais/efeitos dos fármacosRESUMO
The aim of our work was to investigate the presence of non-selective cation channels (NSCC) in freshly isolated smooth muscle cells from the human umbilical artery (HUA), one of the vessels involved in fetal-placental circulation. We studied the electrophysiological properties of NSCC using the patch-clamp technique in whole-cell configuration, and their possible role in the contractile state of intact vessels' rings. Recording with a high intracellular Cs(+) solution and a near physiological extracellular saline solution, we found a Gd(3+)-sensitive current (IC(50) = 1.05 microM) with a linear current-voltage relationship showing a reversal potential (E(rev)) of -2.1 +/- 1.2 mV (n =15 cells). La(3+) (100 microM) and Mg(2+) (5 mM) also blocked this current. In such conditions, inward currents were carried by Na(+) and Ca(2+); hence, a Na(+)-free solution inhibited only inward current (-67.3 +/- 11.4%, at -40 mV, n = 7, p < 0.05) and a Ca(2+)-Na(+)-free solution decreased the current even further with respect to values obtained in Na(+)-free solution (-69.8 +/- 8.8% at -40 mV, n = 9, p < 0.05). The permeability ratios (P(X)/P(Cs(+))) for monovalent and divalent cations were 1, 0.9, 0.7, 0.7, 0.7, and 0.5 where X = Cs(+), Na(+), Li(+), Ca(2+), Ba(2+) and Tris(+), respectively. In intact tissue, a 0 Ca(2+) extracellular solution, Gd(3+) (100-250 microM), La(3+) (200 microM) and Mg(2+) (5 mM) induced vasorelaxation in non-stimulated HUA rings.
Assuntos
Cátions/metabolismo , Canais Iônicos/fisiologia , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Artérias Umbilicais/metabolismo , Cordão Umbilical/irrigação sanguínea , Relação Dose-Resposta a Droga , Eletrofisiologia , Gadolínio/metabolismo , Gadolínio/farmacologia , Humanos , Lantânio/metabolismo , Lantânio/farmacologia , Magnésio/metabolismo , Magnésio/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Técnicas de Patch-Clamp , Artérias Umbilicais/efeitos dos fármacos , Vasodilatação/efeitos dos fármacosRESUMO
Hemodynamic care during postoperative management of myocardial revascularization should include vasorelaxing drugs to insure adequate graft and coronary flow, and stimulation of stroke volume to maintain vascular perfusion pressure. We tested the cardiac (inotropic and lusitropic) and vascular (relaxant) effects of diltiazem (0.1 nM to 0.1 mM), dobutamine (10 microM to 10 mM) and amrinone (10 microM to 1 mM) on isolated rat atria and thoracic aorta, and also on isolated human saphenous vein (HSV) and human mammary artery (HMA). Dobutamine produced a maximal positive inotropic effect (+dF/dt max = 29 +/- 7%) at its ED50 for aortic relaxation (88 +/- 7 microM). Conversely, at their ED50 for aortic relaxation diltiazem depressed myocardial contractility and amrinone did not exhibit myocardial effects. In HSV and HMA contracted with 80 mM potassium, diltiazem and dobutamine (but not amrinone) had a vasorelaxant activity similar to that in rat aorta. Norepinephrine-contracted human vessels were significantly more sensitive than potassium-contracted vessels to the relaxant effect of amrinone (ED50 HMA = 15 +/- 5 microM, ED50 HSV = 72 +/- 31 microM, P < 0.05). We conclude that at concentrations still devoid of myocardial effects dobutamine and amrinone are effective dilators in graft segment vessels and rat aorta contracted by membrane depolarization. If the difference between aortic and myocardial tissue still holds in human tissues, at the appropriate concentrations these drugs should be expected to improve cardiac performance while still contributing to the maintenance of graft patency.
Assuntos
Amrinona/farmacologia , Cardiotônicos/farmacologia , Diltiazem/farmacologia , Dobutamina/farmacologia , Revascularização Miocárdica , Vasodilatadores/farmacologia , Animais , Aorta/efeitos dos fármacos , Aorta/fisiologia , Feminino , Átrios do Coração/efeitos dos fármacos , Humanos , Masculino , Artéria Torácica Interna/efeitos dos fármacos , Artéria Torácica Interna/fisiologia , Ratos , Ratos Sprague-Dawley , Veia Safena/efeitos dos fármacos , Veia Safena/fisiologiaRESUMO
Hemodynamic care during postoperative management of myocardial revascularization should include vasorelaxing drugs to insure adequate graft and coronary flow, and stimulation of stroke volume to maintain vascular perfusion pressure. We tested the cardiac (inotropic and lusitropic) and vascular (relaxant) effects of diltiazem (0.1 nM to 0.1 mM), dobutamine (10 æM to 10 mM) and amrinone (10 æM to 1 mM) on isolated rat atria and thoracic aorta, and also on isolated human saphenous vein (HSV) and human mammary artery (HMA). Dobutamine produced a maximal positive inotropic effect (+dF/dt max = 29 ñ 7 percent) at its ED50 for aortic relaxation (88 ñ 7 æM). Conversely, at their ED50 for aortic relaxation diltiazem depressed myocardial contractility and amrinone did not exhibit myocardial effects. In HSV and HMA contracted with 80 mM potassium, diltiazem and dobutamine (but not amrinone) had a vasorelaxant activity similar to that in rat aorta. Norepinephrine-contracted human vessels were significantly more sensitive than potassium-contracted vessels to the relaxant effect of amrinone (ED50 HMA = 15 ñ 5 æM, ED50 HSV = 72 ñ 31 æM, P < 0.05). We conclude that at concentrations still devoid of myocardial effects dobutamine and amrinone are effective dilators in graft segment vessels and rat aorta contracted by membrane depolarization. If the difference between aortic and myocardial tissue still holds in human tissues, at the appropriate concentrations these drugs should be expected to improve cardiac performance while still contributing to the maintenance of graft patency.
Assuntos
Animais , Masculino , Feminino , Ratos , Aorta , Cardiotônicos , Átrios do Coração , Revascularização Miocárdica , Vasodilatadores , Amrinona , Diltiazem , Dobutamina , Ratos Sprague-DawleyRESUMO
Rat atria is richly innervated by sensory nerve fibers that release CGRP when stimulated either by capsaicin or acid pH. We studied the physiological relevance of acid pH-induced CGRP release on changes in atrial contractility and relaxation produced by lowering the pH. Isolated atria electrically paced at 2.77 Hz were exposed to a 10-minute period of metabolic acidosis (pH=6.73+/-0.01, n=28) after: 1) CGRP release induced by capsaicin 0.5 microM; 2) blockage of CGRP release with ruthenium red (RR) 5 microM; 3) no pretreatment; and 4) CGRP receptor blockage with CGRP(8-37) 1 microM. Contractility and relaxation were significantly less depressed by acid pH when CGRP release was prevented by RR or CGRP receptor activation was blocked by CGRP(8-37). The results suggest that CGRP release and the activation of CGRP receptors may be physiologically involved in contributing to the depression of contractility and relaxation induced by acid pH in rat atria.
Assuntos
Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Contração Miocárdica/fisiologia , Ácidos , Animais , Função Atrial , Antagonistas do Receptor do Peptídeo Relacionado ao Gene de Calcitonina , Capsaicina/farmacologia , Átrios do Coração/inervação , Átrios do Coração/metabolismo , Frequência Cardíaca/fisiologia , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Precondicionamento Isquêmico Miocárdico , Masculino , Miocárdio/metabolismo , Fibras Nervosas/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/fisiologia , Rutênio VermelhoRESUMO
The human saphenous vein (HSV) is currently used as a graft in coronary revascularization as well as in some other vascular beds, namely those of the inferior limbs. Since a significant proportion of HSV grafts develop stenosis, many studies have focused on the factors that could promote graft failure. This article reviews the results on structural and functional features that might be concurrent in the production of saphenous vein graft stenosis. The reactivity of HSV to several physiological agonists is analyzed, including those derived from the endothelium with contractile or relaxing properties, since these are relevant inducers of graft spasm and/or modifiers of the expression of graft factors involved in either tissue growth or thrombotic-atherosclerotic processes. Mechanisms that regulate vascular smooth muscle contractile state, in particular the activity of K+ channels of the plasma membrane, are described.