Francisco Speroni

L, P-/Q- and T-type Ca2+ channels in smooth muscle cells from human umbilical artery.

The electrophysiological and pharmacological properties of Ca²⁺ current (I_Ca) were determined by the whole-cell configuration of the patch-clamp technique in smooth muscle cells from human umbilical artery. Using 5 mM extracellular Ca²⁺, depolarizing step pulses from -60 to 50 mV from a holding membrane potential of -80 mV evoked an I_Ca which activated at membrane potentials more positive than -50 mV and exhibited a maximum current density in a range of 10-20 mV. Steady-state inactivation protocols using a V_test of 10 mV gave a voltage at one-half inactivation and a slope factor of -35.6 mV and 9.5 mV, respectively. Nifedipine (1 µM), an L-type Ca²⁺ channels antagonist, completely inhibited I_Ca, while the L-type Ca²⁺ channels agonist Bay-K 8644 (1 µM) significantly increased I_Ca amplitude. Moreover, the selective blocker of P-/Q-type Ca²⁺ channels ω-agatoxin IVA partially blocked I_Ca (about 40 % inhibition at +20 mV by 20 nM). These pharmacological results suggest that L- and P-/Q-type Ca²⁺ channels, both nifedipine-sensitive, underlie the I_Ca registered using low extracellular Ca²⁺. The presence of the P-/Q-type Ca²⁺ channels was confirmed by immunoblot analysis. When I_Ca was recorded in a high concentration (30 mM) of extracellular Ca²⁺ or Ba²⁺ as current carrier, it was evident the presence of a nifedipine-insensitive component which completely inactivated during the course of the voltage-step (75 ms) at all potentials tested, and was blocked by the T-type Ca²⁺ channels blocker mibefradil (10 µM). Summarizing, this work shows for the first time the electrophysiological and pharmacological properties of voltage-activated Ca²⁺ currents in human umbilical artery smooth muscle cells.

Genistein effects on Ca2+ handling in human umbilical artery: inhibition of sarcoplasmic reticulum Ca2+ release and of voltage-operated Ca2+ channels

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.ResumenLas isoflavonas son un grupo de fitoestrógenos naturales que incluyen la genisteína. Al consumo de este compuesto se le han atribuido efectos beneficiosos para la salud, pero su actividad similar a los estrógenos permite pensar en efectos indeseados en niños o en el feto o la placenta durante el embarazo. En este trabajo se estudian los efectos de la genisteína sobre el manejo de Ca2+ por las células de músculo liso de la arteria umbilical humana (AUH). Mediante la utilización de técnicas fluorométricas se observó que la genisteína reduce el pico de Ca2+ intracelular producido por la serotonina en estas células incluso en ausencia de Ca2+ extracelular, lo que sugiere que la isoflavona reduce la liberación de Ca2+ a partir del retículo sarcoplásmico. Los experimentos de medida de fuerza refuerzan estos resultados, ya que la genisteína redujo la fuerza pico desarrollada por serotonina en anillos intactos de AUH en una solución libre de Ca2+. Además, la genisteína indujo la relajación de anillos de AUH precontraídos con serotonina o con una solución despolarizante de alto K+ extracelular, lo que apunta a una reducción de la entrada de Ca2+ desde el exterior de la célula. Con la técnica de “patch-clamp” en configuración de célula entera, los resultados confirmaron que la isoflavona inhibe corrientes iónicas a través de canales de Ca2+ operados por el voltaje. En resumen, mostramos que la genisteína inhibe dos mecanismos que incrementan el Ca2+ intracelular en células de músculo liso de AUH, comportándose de esta manera como un potencial vasorrelajante de los vasos fetales. Dado que la genisteína atraviesa la barrera placentaria, estos datos muestran que las isoflavonas podrían tener consecuencias en la regulación del flujo materno-fetal en mujeres embarazadas que incluyan productos derivados de la soja como parte de sus dietas.

Genistein inhibits contractile force, intracellular Ca2+ increase and Ca2+ oscillations induced by serotonin in rat aortic smooth muscle

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 μM) or a depolarizing high K+ solution produced the contraction of aortic rings, which were immediately relaxed by 20 μM genistein and by 20 μM 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 μM) 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.ResumenGenisteína y daidzeína, dos isoflavonas presentes en la soja, afectan el estado contráctil de diferentes tipos de músculo liso. Describimos aquí efectos agudos de estos compuestos sobre la fuerza contráctil y la concentración intracelular de Ca2+ ([Ca2+]i) en músculo liso aórtico de rata in situ. La serotonina (5-HT) (2 μM) o una solución despolarizante de alto K+ produjeron la contracción de anillos de aorta de rata, que fueron relajados inmediatamente por genisteína (20 μM) y daidzeína (20 μM). En concordancia con esto, tanto la 5-HT como el alto K+ incrementaron la [Ca2+]i en células de músculo liso aórtico in situ. La genisteína inhibió el aumento de [Ca2+]i producido por 5-HT (74,0±7,3%, n=11, p<0,05) y tuvo un efecto menor sobre aquel debido al alto K+ (19,9±4,0%, n=7, p<0,05). El bloqueante de canales de K+ tetraetilamonio (TEA) (0,5 mM) disminuyó los efectos de genisteína sobre el aumento de [Ca2+]i debido a 5-HT. Durante la aplicación prolongada de 5-HT, la [Ca2+]i comenzó a oscilar y una preincubación corta con genisteína (90 s) disminuyó significativamente la frecuencia de estas oscilaciones, siendo este efecto totalmente bloqueado por TEA. En conclusión, en el músculo liso aórtico de rata la genisteína es capaz de atenuar el aumento de [Ca2+]i y de la fuerza inducidos por 5-HT, así como de disminuir la frecuencia de las oscilaciones en la [Ca2+]i. El corto tiempo requerido por la genisteína, así como la relajación inducida por daidzeína sugieren que esto no se debe a inhibición de tirosín quinasas. El menor efecto de genisteína sobre el aumento de [Ca2+]i debido al alto K+, así como los efectos del TEA apuntan a la inhibición por parte de genisteína de canales de K+ activados por Ca2+.

Calcium Addition, pH, and High Hydrostatic Pressure Effects on Soybean Protein Isolates—Part 1: Colloidal Stability Improvement

Calcium addition to soybean protein dispersions increases nutritional value but harms functional properties, such as protein solubility and colloidal stability. The high hydrostatic pressure (HHP) treatment can reverse those effects. The aims of this work were to evaluate the influence of pH and protein and calcium concentration on HHP solubilizing/stabilizing effect and to characterize the physicochemical properties of HHP-stabilized species. Proteins without calcium addition were stabilized by HHP at both pHs. However, calcium-added proteins behaved differentially: at pHxa05.9, the effect was verified only at low protein concentration, whereas at pHxa07.0, the effect was verified under both assayed protein concentrations (5 and 10xa0gxa0L−1) and with a higher magnitude in calcium-added samples. Moreover, at pHxa07.0, the effect was independent of the order of calcium addition and HHP treatment, whereas at pHxa05.9, the effect was smaller when calcium was added after HHP treatment. At both pHs, the solubilizing/stabilizing effect of HHP on soybean proteins seemed to be largely dependent on the decrease in the size of protein species. The smaller the size, the greater the amount of protein that remained in dispersion after intense centrifugation (10,000g, 20xa0min, 4xa0°C). Although the effect of HHP consisted, at least in part, of stabilizing insoluble protein, turbidity decreased in all samples after HHP treatment. By combining different levels of pH, calcium, and protein concentrations, translucent or turbid colloidal-stable dispersions can be obtained by HHP treatment.

Calorimetric Study of Cowpea Protein Isolates. Effect of Calcium and High Hydrostatic Pressure

The thermal properties of cowpea protein isolates (CPI) were studied by differential scanning calorimetry under the influence of various conditions. An increase in the pH of protein extraction, from 8.0 to 10.0, during CPI preparation promoted a partial denaturation of cowpea proteins. Increases in enthalpy change of denaturation (ΔH) and temperature of denaturation (Td) were detected with increasing protein concentration from 7.5 to 10.5% (w/w). This behavior suggests that denaturation involves a first step of dissociation of protein aggregates. Calcium induced thermal stabilization in cowpea proteins, the increase in Td was ca. 0.3xa0°C/mM for protein dispersions of 7.5% (w/w) for 0 to 40xa0mM CaCl2. High hydrostatic pressure (HHP) induced denaturation in CPI in a pressure level dependent manner. The presence of calcium protected cowpea proteins towards HHP-induced denaturation when pressure level was 400xa0MPa, but not when it was 600xa0MPa. Thermal properties of cowpea protein isolates were very sensitive to processing conditions, these behaviors would have implications in processing of CPI-containing foodstuff.

Calcium Addition, pH and High Hydrostatic Pressure Effects on Soybean Protein Isolates—Part 2: Emulsifying Properties

Soybean protein isolates (SPI) represent an important source of proteins that are used to prepare oil-in-water (o/w) emulsions. The influence of an innovative treatment (high hydrostatic pressure, HHP) combined with calcium addition at different pH levels and protein concentrations on the formation and stability of o/w SPI emulsions was evaluated in this work. When applied separately, calcium addition or HHP treatment produced different effect at pHs 5.9 and 7.0. Calcium addition led to stable emulsions with decreased flocculation index (FI) at pHxa05.9 and low protein concentration (5xa0gxa0L−1), whereas at pHxa07.0, this effect was observed at high protein concentration (10xa0gxa0L−1). In these conditions, calcium would favor the arrival of big aggregates to interface, which would be modified and adsorbed during homogenization. Treatment with HHP decreased FI and stabilized emulsions during storage at pHxa07.0 (but not at pHxa05.9) when prepared from 10xa0gxa0L−1 protein dispersions. In these conditions, protein unfolding due to HHP-induced denaturation, and high ζ-potential would be responsible for emulsion improvement. Combination of calcium addition and HHP treatment impaired both formation and stabilization abilities of SPI at both pHs. Bridging flocculation was enhanced in these samples while interfacial protein concentration and percentage of adsorbed protein were increased. Thus, soybean proteins that were subjected to combined calcium addition and HHP treatment exhibited a great ability to associate each other, what can be useful to improve other functional properties such as gelation.