Verónica Milesi

A BK (Slo1) channel journey from molecule to physiology

Calcium and voltage-activated potassium (BK) channels are key actors in cell physiology, both in neuronal and non-neuronal cells and tissues. Through negative feedback between intracellular Ca2+ and membrane voltage, BK channels provide a damping mechanism for excitatory signals. Molecular modulation of these channels by alternative splicing, auxiliary subunits and post-translational modifications showed that these channels are subjected to many mechanisms that add diversity to the BK channel α subunit gene. This complexity of interactions modulates BK channel gating, modifying the energetic barrier of voltage sensor domain activation and channel opening. Regions for voltage as well as Ca2+ sensitivity have been identified, and the crystal structure generated by the 2 RCK domains contained in the C-terminal of the channel has been described. The linkage of these channels to many intracellular metabolites and pathways, as well as their modulation by extracellular natural agents, has been found to be relevant in many physiological processes. This review includes the hallmarks of BK channel biophysics and its physiological impact on specific cells and tissues, highlighting its relationship with auxiliary subunit expression.

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.

Mechanical properties of human saphenous veins from normotensive and hypertensive patients.

BACKGROUND Different reactivities of saphenous vein grafts in hypertensive and normotensive patients could lead to differences in the postoperative patency of the grafts. METHODS In saphenous vein rings isolated from remnants of aorta-coronary grafts obtained from hypertensive and normotensive patients we studied the length-tension relationship; response to high levels of potassium, norepinephrine, and epinephrine; and relaxation in response to calcium deprivation. RESULTS The rings from hypertensive patients were stiffer and developed more force (grams force/grams weight) than the rings from normotensive subjects to 80 mmol/L potassium (59+/-16 versus 25+/-5, p < 0.05) and to 1 micromol/L norepinephrine (61+/-8 versus 36+/-7, p < 0.05), but not to 10 micromol/L epinephrine (57+/-11 and 54+/-11; not significant). The rings from hypertensive patients relaxed more slowly than those of the normotensive subjects in a calcium-free medium (time to half-relaxation of 976+/-180 versus 548+/-81 seconds; p < 0.05). CONCLUSIONS The saphenous vein from hypertensive patients is less distensible, slower to relax, and more reactive to at least two agonists. These differences could influence the grafts patency and the clinical outcome.

Effects of light ethanol consumption during pregnancy: increased frequency of minor anomalies in the newborn and altered contractility of umbilical cord artery.

This study explores the effects of light maternal ethanol consumption during pregnancy on the appearance of minor malformations in neonates as well as on the contractile properties of their umbilical cord arteries (UCAs). Clinical external findings of newborns of women declaring light ethanol consumption during any period of their pregnancies [ethanol-exposed group (E group), n = 79] were compared with those of nonexposed mothers [nonexposed to ethanol group (NE group), n = 100]. Women who smoked or had any associated pathology were excluded. E group mothers consumed, on average, 200–250 mL ethanol/trimester (upper limit 700 mL/trimester). Sixty-six percent of the neonates in the E group presented at least one minor malformation (retromicrognathia and minor anomalies of the auricular/preauricular area were the more common), whereas only 16% of the NE group did (p = 0.0000). The percentage of children exhibiting Apgar scores <7 was significantly greater in the E group (11% versus 2%, p = 0.0119). UCAs from the E group developed significantly less contractile force (p < 0.05) than those of the NE group when exposed to 1 μM serotonin (5-HT) or to a high K+ depolarizing solution. This difference persisted after inhibition of endothelial release of nitric oxide (NO) and prostacyclin. In conclusion, even light drinking should be considered a risk during pregnancy.

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.

Diversity of Potassium Channels in Human Umbilical Artery Smooth Muscle Cells A Review of Their Roles in Human Umbilical Artery Contraction

Through their control of cell membrane potential, potassium (K+) channels are among the best known regulators of vascular tone. This article discusses the expression and function of K+ channels in human umbilical artery smooth muscle cells (HUASMCs). We review the bibliographic reports and also present single-channel data recorded in freshly isolated cells. Electrophysiological properties of big conductance, voltage- and Ca2+-sensitive K+ channel and voltage-dependent K+ channels are clearly established in this vessel, where they are involved in contractile state regulation. Their role in the maintenance of membrane potential is an important control mechanism in the determination of the vessel diameter. Additionally, small conductance Ca2+-sensitive K+ channels, 2-pore domains K+ channels and inward rectifier K+ channels also appear to be present in HUASMCs, while intermediate conductance Ca2+-sensitive K+ channels and ATP-sensitive K+ channels could not be identified. In both cases, additional investigation is necessary to reach conclusive evidence of their expression and/or functional role in HUASMCs. Finally, we discuss the role of K+ channels in pregnancy-related pathologies like gestational diabetes and preeclampsia.

Bupivacaine inhibits large conductance, voltage- and Ca2+- activated K+ channels in human umbilical artery smooth muscle cells

Bupivacaine is a local anesthetic compound belonging to the amino amide group. Its anesthetic effect is commonly related to its inhibitory effect on voltage-gated sodium channels. However, several studies have shown that this drug can also inhibit voltage-operated K+ channels by a different blocking mechanism. This could explain the observed contractile effects of bupivacaine on blood vessels. Up to now, there were no previous reports in the literature about bupivacaine effects on large conductance voltage- and Ca2+-activated K+ channels (BKCa). Using the patch-clamp technique, it is shown that bupivacaine inhibits single-channel and whole-cell K+ currents carried by BKCa channels in smooth muscle cells isolated from human umbilical artery (HUA). At the single-channel level bupivacaine produced, in a concentration- and voltage-dependent manner (IC50 324 µM at +80 mV), a reduction of single-channel current amplitude and induced a flickery mode of the open channel state. Bupivacaine (300 µM) can also block whole-cell K+ currents (~45% blockage) in which, under our working conditions, BKCa is the main component. This study presents a new inhibitory effect of bupivacaine on an ion channel involved in different cell functions. Hence, the inhibitory effect of bupivacaine on BKCa channel activity could affect different physiological functions where these channels are involved. Since bupivacaine is commonly used during labor and delivery, its effects on umbilical arteries, where this channel is highly expressed, should be taken into account.

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+.

The Antineoplastic Agent Paclitaxel (Taxol®) Increases Contractile Activity in Human Saphenous Veins and Human Mammary Arteries

Abstract Effects of the antineoplastic agent paclitaxel (Taxol®) were studied on contractions of isolated human saphenom vein (HSV) and mammary artery (HMA). Peak force developed by vascular segments with cumulative concentrations of physiologic agonists was enhanced by paclitaxel, producing a shift to the left of dose-response curves. Paclitaxel 1 μM decreased ED50 (in μM) for norepinephrine from 1.01 ± 0.24 to 0.20 ± 0.06 (n = 16, p < 0.05) in HSV and from 1.30 ± 0.30 to 0.51 ± 0.21 (n = 15, p < 0.05) in HMA and for 5–hydroxytriptamine from 0.64 ± 0.19 to 0.21 ± 0.07 (n = 20, p < 0.05) in HSV. Paclitaxel 1 μM also significantly increased the peak force of contractions elicited by endothelin-1 0.01 μM in HSV. In contrast, it did not affect contractions evoked by KCl 80 mM. These results show that paclitaxel produces a hyperreactivity in human vessels challenged by physiologic agonists, which suggests that administration of paclitaxel to patients could augment peripheral resistance and increase blood pressure.

Role of insulin preincubation in the contractile reactivity of rat aortic rings

Preincubation with physiological concentrations of insulin affects contractile reactivity of isolated smooth muscle cells. We studied the effects of insulin on intact aortic rings of Wistar rats preincubated 1-2 h with 240 pM (I1) and 960 pM (I2) insulin with and without NO synthesis inhibition by N(omega)-nitro-L-arginine methyl ester (L-NAME). Resting force was tripled by 0.1 mM L-NAME in control (C) and I1 groups, but not in I2 groups. I1 treatment decreased the tachyphylaxis to two successive 1 microM arginine vasopressin (AVP) stimulations. Single contractions elicited by 1 microM AVP, 1 microM angiotensin II (AngII), or 0.01 microM endothelin (ET1) were not affected by insulin preincubation in either maximal force (Fmax) or relaxation times. L-NAME enhanced Fmax of AngII contractions by about 75% in C, 120% in I1, and 74% in I2 groups; accordingly, it augmented the final steady-state force in C and I1 but not in I2. Similarly, L-NAME increased Fmax (30-40%) of AVP and ET1 contractions in C and I1 groups but failed to do so in contractions of I2 group. Results obtained with 10 microM indomethacin suggest that this is due to insulin stimulation of prostacyclin effects.