RESUMEN
Cardiovascular diseases are the most important cause of morbidity and mortality in the civilized world. Stenosis or occlusion of blood vessels leads not only to events that are directly life-threatening, such as myocardial infarction or stroke, but also to a significant reduction in quality of life, for example in lower limb ischemia as a consequence of metabolic diseases. The first synthetic polymeric vascular replacements were used clinically in the early 1950s. However, they proved to be suitable only for larger-diameter vessels, where the blood flow prevents the attachment of platelets, pro-inflammatory cells and smooth muscle cells on their inner surface, whereas in smaller-diameter grafts (6 mm or less), these phenomena lead to stenosis and failure of the graft. Moreover, these polymeric vascular replacements, like biological grafts (decellularized or devitalized), are cell-free, i.e. there are no reconstructed physiological layers of the blood vessel wall, i.e. an inner layer of endothelial cells to prevent thrombosis, a middle layer of smooth muscle cells to perform the contractile function, and an outer layer to provide innervation and vascularization of the vessel wall. Vascular substitutes with these cellular components can be constructed by tissue engineering methods. However, it has to be admitted that even about 70 years after the first polymeric vascular prostheses were implanted into human patients, there are still no functional small-diameter vascular grafts on the market. The damage to small-diameter blood vessels has to be addressed by endovascular approaches or by autologous vascular substitutes, which leads to some skepticism about the potential of tissue engineering. However, new possibilities of this approach lie in the use of modern technologies such as 3D bioprinting and/or electrospinning in combination with stem cells and pre-vascularization of tissue-engineered vascular grafts. In this endeavor, sex-related differences in the removal of degradable biomaterials by the cells and in the behavior of stem cells and pre-differentiated vascular cells need to be taken into account. Key words: Blood vessel prosthesis, Regenerative medicine, Stem cells, Footprint-free iPSCs, sr-RNA, Dynamic bioreactor, Sex-related differences.
Asunto(s)
Prótesis Vascular , Ingeniería de Tejidos , Humanos , Ingeniería de Tejidos/métodos , AnimalesRESUMEN
Autologous and allogenic human pericardia used as biomaterials for cardiovascular surgery are traditionally crosslinked with glutaraldehyde. In this work, we have evaluated the resistivity to collagenase digestion and the cytotoxicity of human pericardium crosslinked with various concentrations of glutaraldehyde in comparison with pericardium crosslinked by genipin, nordihydroguaiaretic acid, tannic acid, and in comparison with unmodified pericardium. Crosslinking retained the wavy-like morphology of native pericardium visualized by second harmonic generation microscopy. The collagenase digestion products were analyzed using SDS-PAGE, capillary electrophoresis, and a hydroxyproline assay. Glutaraldehyde and genipin crosslinking protected the native pericardium efficiently against digestion with collagenase III. Only low protection was provided by the other crosslinking agents. The cytotoxicity of crosslinked pericardium was evaluated using xCELLigence by monitoring the viability of porcine valve interstitial cells cultured in eluates from crosslinked pericardium. The highest cell index, reflecting both the number and the shape of the monitored cells was observed in eluates from genipin. Crosslinking pericardium grafts with genipin therefore seems to be a promising alternative procedure to the traditional crosslinking with glutaraldehyde, because it provides similarly high protection against degradation with collagenase, without cytotoxic effects.
Asunto(s)
Reactivos de Enlaces Cruzados , Pericardio/química , Trasplantes/química , Materiales Biocompatibles , Glutaral , Humanos , Iridoides , Masoprocol , TaninosRESUMEN
The intestinal transport of nutrients exhibits distinct diurnal rhythmicity, and the enterocytes harbor a circadian clock. However, temporal regulation of the genes involved in colonic ion transport, i.e., ion transporters and channels operating in absorption and secretion, remains poorly understood. To address this issue, we assessed the 24-h profiles of expression of genes encoding the sodium pump (subunits Atp1a1 and Atp1b1), channels (α-, ß-, and γ-subunits of Enac and Cftr), transporters (Dra, Ae1, Nkcc1, Kcc1, and Nhe3), and the Na(+)/H(+) exchanger (NHE) regulatory factor (Nherf1) in rat colonic mucosa. Furthermore, we investigated temporal changes in the spatial localization of the clock genes Per1, Per2, and Bmal1 and the genes encoding ion transporters and channels along the crypt axis. In rats fed ad libitum, the expression of Atp1a1, γEnac, Dra, Ae1, Nhe3, and Nherf1 showed circadian variation with maximal expression at circadian time 12, i.e., at the beginning of the subjective night. The peak γEnac expression coincided with the rise in plasma aldosterone. Restricted feeding phase advanced the expression of Dra, Ae1, Nherf, and γEnac and decreased expression of Atp1a1. The genes Atp1b1, Cftr, αEnac, ßEnac, Nkcc1, and Kcc1 did not show any diurnal variations in mRNA levels. A low-salt diet upregulated the expression of ßEnac and γEnac during the subjective night but did not affect expression of αEnac. Similarly, colonic electrogenic Na(+) transport was much higher during the subjective night than the subjective day. These findings indicate that the transporters and channels operating in NaCl absorption undergo diurnal regulation and suggest a role of an intestinal clock in the coordination of colonic NaCl absorption.
Asunto(s)
Ritmo Circadiano/genética , Colon/fisiología , Electrólitos/farmacocinética , Perfilación de la Expresión Génica , Absorción Intestinal/genética , Aldosterona/sangre , Animales , Proteínas Portadoras/genética , Colon/citología , Ingestión de Alimentos/genética , Enterocitos/metabolismo , Mucosa Intestinal/metabolismo , Canales Iónicos/genética , Masculino , Proteínas Circadianas Period/genética , Ratas , Ratas Wistar , Cloruro de Sodio Dietético/farmacocinéticaRESUMEN
We studied the effects of Ca(2+) and K(+) on non-transferrin iron uptake from ferric citrate complex by HeLa and K562 cells. Uptake experiments in Na-HEPES buffer (137 mM NaCl, 4 mM KCl) showed that extracellular Ca(2+) stimulated the iron uptake. The rate of iron uptake in 4 mM Ca(2+) was about 3-5 times higher than without Ca(2+). The iron uptake in K-HEPES buffer (68 mM NaCl, 75 mM KCl) with a high K(+) level was transiently stimulated during the first 10 min. The rate of iron uptake for 0.4 mM Ca(2+) was approximately 3 times higher in K-HEPES buffer than in Na-HEPES buffer. The calcium channel blockers verapamil (50 microM) and nifedipine (5 microM) had no effect on the uptake either in control Na-HEPES buffer or after K(+) stimulation in K-HEPES buffer. The sodium channel blocker lidocaine (50 microM) also had no effect on the uptake of iron in Na-HEPES buffer as well as after K(+) stimulation. Furthermore, the iron uptake was not significantly affected when Na(+) in the Na-HEPES and K-HEPES buffers was replaced by isotonic saccharose. We conclude that extracellular calcium per se, and not intracellular calcium or Ca(2+) transport, stimulates ferric iron uptake by both HeLa and K562 cells. A high level of extracellular K(+) also stimulates the uptake, probably via cell membrane depolarization. Na(+) is not involved in these stimulations of iron uptake. The transient K(+) effect and continuous Ca(2+) effect seem to be additive.
Asunto(s)
Calcio/farmacología , Células HeLa/efectos de los fármacos , Hierro/metabolismo , Potasio/farmacología , Transporte Biológico , Tampones (Química) , Calcimicina/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Membrana Celular/efectos de los fármacos , Compuestos Férricos/metabolismo , Células HeLa/metabolismo , Humanos , Células K562/efectos de los fármacos , Células K562/metabolismo , Lidocaína/farmacología , Cloruro de Litio/farmacología , Factores de Tiempo , Regulación hacia ArribaRESUMEN
We studied transport of non-transferrin iron into HeLa cells adapted for growth in defined medium, containing either 5 micrograms/ml of iron-saturated transferrin (HeLa/Tf cells) or 5 microM ferric citrate (HeLa/Fe5 cells) as a source of iron. Employing 55Fe-ferric citrate, iron uptake by intact cells was compared with iron binding to isolated membranes. Uptake characteristics of both HeLa/Tf and HeLa/Fe5 cells seemed to be similar: Km = 14 microM and Vmax = 135 pmol Fe/min/10(5) cells for HeLa/Tf, Km = 22 microM and Vmax = 165 pmol Fe/min/10(5) cells for HeLa/Fe5. Increasing concentrations (0.3-1.2 microM) of 55Fe-ferric citrate, producing levels of free 55Fe which were independent of total Fe under the experimental conditions used, led to increased binding of 55Fe for both HeLa/Tf and HeLa/Fe5 cells (1.08-8.03 nmol Fe/h/10(5) cells). This corresponds with the suggestion that iron was bound in the form of ferric citrate rather than in the form of free iron. Dissociation constants of Fe binding, KD = 0.61 microM for HeLa/Tf and KD = 1.53 microM for HeLa/Fe5, were obtained from competition experiments. We conclude that specific binding sites for ferric citrate are constitutively expressed in plasma membrane and that their expression does not require the induction by the presence of ferric citrate. The uptake of non-transferrin iron is realized in at least two steps. The first step is iron binding to the specific binding sites in plasma membrane. The binding does not represent a limiting step of the uptake.
Asunto(s)
Membrana Celular/metabolismo , Hierro/metabolismo , Sitios de Unión , Transporte Biológico , Células Clonales , Células HeLa , Humanos , Hierro/farmacocinética , Transferrina/metabolismoRESUMEN
We studied the factors that determine the differing growth requirements of low-iron-tolerant (LIT) versus high-iron-dependent (HID) cells for extracellular nontransferrin iron. The growth of LIT cells HeLa and THP-1, when transferred from transferrin (5 micrograms/ml) medium into low-iron (5 microM ferric citrate) medium, was not significantly affected while HID cells Jiyoye and K562 showed nearly no growth. HeLa and THP-1 cells, as well as Jiyoye and K562 cells, do not produce transferrin in sufficient amounts to support their growth in low-iron medium. Surprisingly, similar rates of iron uptake in low-iron medium (0.033 and 0.032 nmol Fe/min and 10(6) cells) were found for LIT cells HeLa and HID cells K562. Furthermore, the intracellular iron level (4.64 nmol/10(6) cells) of HeLa cells grown in low-iron medium was much higher than iron levels (0.15 or 0.20 nmol/10(6) cells) of HeLa or K562 cells grown in transferrin medium. We demonstrated that the activity (ratio activated/total) of the iron regulatory protein (IRP) in HID cells Jiyoye and K562 increased more than twofold (from 0.32 to 0.79 and from 0.47 to 1.12, respectively) within 48 h after their transfer into low-iron medium. In the case of LIT cells HeLa and THP-1, IRP activity stayed at similar or slightly decreased levels (0.86-0.73 and 0.58-0.55, respectively). Addition of iron chelator deferoxamine (50 microM, i.e., about half-maximal growth-inhibitory dose) resulted in significantly increased activity of IRP also in HeLa and THP-1 cells. We hypothesize that the relatively higher bioavailability of nontransferrin iron in LIT cells, over that in HID cells, determines the differing responses observed under low-iron conditions.
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División Celular , Proteínas Hierro-Azufre/metabolismo , Hierro/metabolismo , Proteínas de Unión al ARN/metabolismo , Transporte Biológico , Linfoma de Burkitt , Medios de Cultivo , Deferoxamina/farmacología , Células HeLa , Humanos , Proteínas Reguladoras del Hierro , Cinética , Leucemia Eritroblástica Aguda , Leucemia Monocítica Aguda , Transferrina/farmacología , Células Tumorales CultivadasRESUMEN
Alcuronium was found earlier to increase the binding of (3H)methyl-N-scopolamine [(3H)NMS] to muscarinic receptors in membranes of rat heart atria by increasing the receptors' affinity for (3H)NMS, without altering the number of (3H)NMS binding sites. We have now investigated how the interaction of alcuronium with muscarinic receptors is affected by solubilization. In experiments on pig heart atria, alcuronium had a positive allosteric effect on (3H)NMS binding both to unsolubilized receptors and to receptors solubilized by digitonin and deoxycholate. The cooperativity coefficient alpha was 0.39 +/- 0.01 for unsolubilized and 0.57 +/- 0.01 for solubilized receptors. Under the conditions used for solubilization, muscarinic receptors did not interact with G proteins, as indicated by experiments with the binding of carbachol in the absence and presence of a stable GTP analogue. Alcuronium slowed down the rates of (3H)NMS association with, and dissociation from, solubilized receptors; the dissociation rate constant was diminished more than 300 times by 30 microM alcuronium. The results show that the positive allosteric action of alcuronium on cardiac muscarinic receptors and its effect on the kinetics of radioligand binding are preserved after receptor solubilization and do not depend on the interaction of receptors with G proteins.
Asunto(s)
Alcuronio/farmacología , Miocardio/metabolismo , Receptores Muscarínicos/efectos de los fármacos , Regulación Alostérica , Animales , Unión Competitiva , Carbacol/metabolismo , Femenino , Guanilil Imidodifosfato/farmacología , Masculino , N-Metilescopolamina , Concentración Osmolar , Receptores Muscarínicos/metabolismo , Derivados de Escopolamina/metabolismo , Solubilidad , PorcinosRESUMEN
Cholinesterase inhibitor tacrine (1,2,3,4-tetrahydro-9-aminoacridine) is known to interfere with the binding of specific ligands to muscarinic receptors with unusually steep binding inhibition curves. We investigated whether the concentration dependence of the inhibition of binding is associated with the inhibitory effect of tacrine on the activity of cholinesterases, and compared the effect of tacrine with that of 7-methoxytacrine. Tacrine was found to inhibit the specific binding of [3H]quinuclidinyl benzilate (QNB) in rat brain cortex with IC50 values of 11 microM both in the absence and in the presence of 100 microM eserine, which had been added to ensure complete inhibition of cholinesterases at all concentrations of tacrine; in the cerebellum, the IC50 value was 10 microM in the absence and 14 microM in the presence of eserine; Hill slope factors (nH) were in the range of 1.55-1.79 and were not significantly affected by the presence of eserine. 7-Methoxytacrine inhibited the binding of [3H]QNB with an IC50 value of 2.3 microM in the cortex and of 2.6 microM in the cerebellum. The results indicate that the degree and the steep course of the inhibition of [3H]QNB binding to M1 and M2 muscarinic receptors by tacrine do not depend on its inhibitory effect on cholinesterases, and that 7-methoxytacrine is likely to interfere with the function of muscarinic receptors 4-5 times more strongly than tacrine.
Asunto(s)
Cerebelo/metabolismo , Corteza Cerebral/metabolismo , Inhibidores de la Colinesterasa/farmacología , Fisostigmina/farmacología , Receptores Muscarínicos/metabolismo , Tacrina/análogos & derivados , Tacrina/farmacología , Animales , Unión Competitiva , Femenino , Cinética , Quinuclidinil Bencilato/metabolismo , Ratas , Ratas Endogámicas , Receptores Muscarínicos/efectos de los fármacosRESUMEN
On isolated rat heart atria, atracurium competitively antagonized the negative chronotropic effect of methylfurmethide, shifting the concentration-response curve to the right without diminishing the agonist's maximal effect; Kd calculated from dose ratios was 3.0 mumol/l. On the longitudinal muscle of rat ileum, atracurium antagonized the effect of methylfurmethide in a non-competitive manner; at 50 mumol/l atracurium, the maximum response to methylfurmethide was diminished by about 50%. Atracurium antagonized the binding of (3H)quinuclidinyl benzilate [3H)QNB) to muscarinic binding sites in the atria, ileal longitudinal muscle and cerebellum with IC50 values of 5-8 mumol/l, and in brain cortex of 25 mumol/l. Atracurium was little efficient, however, in antagonizing the binding of N-(3H-methyl) scopolamine [3H)NMS) to muscarinic binding sites. Complete blockade was not achieved at concentrations up to 1 mmol/l. Concentrations required to diminish the binding by 50% were 10 - 1000 times higher for (3H)NMS than for (3H)QNB. Atracurium brought about the dissociation of (3H)QNB-receptor complexes, but its effect was considerably stronger at a concentration of 30 mumol/l than at 1 mmol/l. Atracurium slowed down the dissociation of (3H)QNB-receptor complexes observed after the addition of atropine. The effects of atracurium on the dissociation of (3H)NMS-receptor complexes were similar to those on (3H)QNB-receptor complexes, but a high concentration of atracurium (1 mmol/l) produced a transient increase in (3H)NMS binding preceding its subsequent dissociation. Although the observations of the antagonism by atracurium of the effect of methylfurmethide on the heart atria, and of the inhibition of the specific binding of (3H)QNB to the atria, ileal smooth muscle, cerebellum and brain cortex are compatible with the assumption of a competitive interaction, the discrepancy between the effects of atracurium on the binding of (3H)QNB and (3H)NMS indicates that atracurium does not bind to the same binding site as (3H)QNB and (3H)NMS. It appears that most effects of atracurium on muscarinic receptors are allosteric and that both negative and positive cooperatives play a role in interactions between atracurium and muscarinic ligands.
Asunto(s)
Atracurio/metabolismo , Receptores Colinérgicos/metabolismo , Animales , Atropina/farmacología , Corteza Cerebral/metabolismo , Corteza Cerebral/ultraestructura , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Femenino , Atrios Cardíacos/metabolismo , Masculino , Muscarina/análogos & derivados , Muscarina/farmacología , Músculo Liso/metabolismo , Músculo Liso/ultraestructura , N-Metilescopolamina , Parasimpatolíticos/metabolismo , Parasimpaticomiméticos/farmacología , Quinuclidinil Bencilato/metabolismo , Ratas , Ratas Endogámicas , Derivados de Escopolamina/metabolismo , TritioRESUMEN
The effect of the neuromuscular blocker alcuronium on the binding of N-[3H]-methylscopolamine [( 3H]NMS) and l-[3H]quinuclidinylbenzilate ([3H]QNB) to muscarinic binding sites in rat heart atria, longitudinal smooth muscle of the ileum, cerebral cortex, cerebellum, and submaxillary glands was measured using filtration techniques. In the presence of 10(-5) M alcuronium, the binding of [3H]NMS (which was present at a subsaturating concentration of 2 x 10(-10) M) was increased 5.3-fold in the atria and smooth muscle and 3-fold in the cerebellum; no increase was observed in the brain cortex and salivary glands. The binding of [3H]NMS was inhibited at 10(-3) M and higher concentrations of alcuronium. The rates of [3H]NMS association to and dissociation from muscarinic binding sites in the atria were diminished by 10(-5) M alcuronium. Scatchard plots of [3H]NMS binding data obtained with and without 10(-5) M alcuronium indicated that the maximum number of binding sites was not altered by the drug, whereas the apparent Kd for [3H]NMS was diminished. In contrast to [3H] NMS, the effects of alcuronium on the binding of [3H]QNB were only inhibitory. The concentration of alcuronium required to diminish the binding of [3H]QNB by 50% (IC50) was 4-7 microM in the atria, ileal smooth muscle, and the cerebellum, 140 microM in the brain cortex, and 1200 microM in the parotid gland. The results suggest that the binding of low concentrations of alcuronium to muscarinic receptors in the heart, ileal smooth muscle, and cerebellum allosterically increases the affinity of muscarinic receptors towards [3H]NMS, although not [3H]QNB. At high concentrations, alcuronium inhibits the binding of muscarinic ligands, presumably by competition for the classical muscarinic binding site. Positive cooperativity induced by alcuronium appears to be specific for the m2 (cardiac) subtype of muscarinic receptors.