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.Genisteína 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⁺.Facultad de Ciencias Exacta

The mechanism of injury-induced [Ca2+]i oscillations in the endothelium of excised rat aorta

Endothelial injury is the primary event that leads to a variety of severe vascular disorders. The signal transduction pathway which drives the subsequent healing process is far from being fully elucidated. Mechanical injury elicits a Ca2+ response in the endothelium of intact rat aorta, which comprises an initial Ca2+ release from inositol-1,4,5-trisphosphate-sensitive (IP3Rs) stores followed by a long-lasting decay phase due to Ca2+ entry through uncoupled connexons. In a minor fraction of cells, the Ca2+ signal adopts an oscillatory pattern whose molecular underpinnings are yet to be elucidated. In the light of the role played by repetitive Ca2+ spikes in regulating tissue regeneration, the present study aims at elucidating the mechanisms underlying injury-induced Ca2+ oscillations. The repetitive Ca2+ signal reversibly ceased upon removal of extracellular Ca2+ or addition of the inorganic cations, La3+ and Ni2+. Moreover, the spiking response was abolished by the gap-junction blockers, heptanol and 18 beta-glycyrrhetinic acid and by interfering with the Ca2+ entry-mode mode of the Na+/Ca2+ exchanger (NCX). The InsP3-producing agonist, ATP, resumed Ca2+ oscillations in silent cells, while the phospholipase C inhibitor, U73122, inhibited the oscillatory signal. The latter was also prevented by the SERCA inhibitors, thapsigargin and cyclopiazonic acid acid. These data show that injury-induced [Ca2+]i oscillations require the coordinated interplay between NCX-mediated Ca2+ entry and InsP3-dependent Ca2+ release. Besides directly gating Ca2+ inflow, uncoupled connexons might let Na+ into the cells and stimulate Ca2+ entry through NCX by increasing submembranal Na+ levels

Cardiac microvascular endothelial cells express a functional Ca 2+-sensing receptor

The mechanism whereby extracellular Ca2+ exerts the endothelium-dependent control of vascular tone is still unclear. In this study, we assessed whether cardiac microvascular endothelial cells (CMEC) express a functional extracellular Ca2+-sensing receptor (CaSR) using a variety of techniques. CaSR mRNA was detected using RT-PCR, and CaSR protein was identified by immunocytochemical analysis. In order to assess the functionality of the receptor, CMEC were loaded with the Ca2+-sensitive fluorochrome, Fura-2/AM. A number of CaSR agonists, such as spermine, Gd 3+, La3+ and neomycin, elicited a heterogeneous intracellular Ca2+ signal, which was abolished by disruption of inositol 1,4,5-trisphosphate (InsP3) signaling and by depletion of intracellular stores with cyclopiazonic acid. The inhibition of the Na +/Ca2+ exchanger upon substitution of extracellular Na+ unmasked the Ca2+ signal triggered by an increase in extracellular Ca2+ levels. Finally, aromatic amino acids, which function as allosteric activators of CaSR, potentiated the Ca2+ response to the CaSR agonist La3+. These data provide evidence that CMEC express CaSR, which is able to respond to physiological agonists by mobilizing Ca2+ from intracellular InsP3-sensitive stores.Facultad de Ciencias Exacta

Cardiac microvascular endothelial cells express a functional Ca 2+-sensing receptor

The mechanism whereby extracellular Ca2+ exerts the endothelium-dependent control of vascular tone is still unclear. In this study, we assessed whether cardiac microvascular endothelial cells (CMEC) express a functional extracellular Ca2+-sensing receptor (CaSR) using a variety of techniques. CaSR mRNA was detected using RT-PCR, and CaSR protein was identified by immunocytochemical analysis. In order to assess the functionality of the receptor, CMEC were loaded with the Ca2+-sensitive fluorochrome, Fura-2/AM. A number of CaSR agonists, such as spermine, Gd 3+, La3+ and neomycin, elicited a heterogeneous intracellular Ca2+ signal, which was abolished by disruption of inositol 1,4,5-trisphosphate (InsP3) signaling and by depletion of intracellular stores with cyclopiazonic acid. The inhibition of the Na +/Ca2+ exchanger upon substitution of extracellular Na+ unmasked the Ca2+ signal triggered by an increase in extracellular Ca2+ levels. Finally, aromatic amino acids, which function as allosteric activators of CaSR, potentiated the Ca2+ response to the CaSR agonist La3+. These data provide evidence that CMEC express CaSR, which is able to respond to physiological agonists by mobilizing Ca2+ from intracellular InsP3-sensitive stores.Facultad de Ciencias Exacta

“VEGF induces human endothelial progenitor cells proliferations by eliciting oscillations in intracellular Ca2+ concentration”

Endothelial progenitor cells (EPCs) traffic from the bone marrow to the site of tissue regeneration and sustain neo-vascularization after acute vascular injury and upon the angiogenic switch in solid tumors. Therefore, they represent a suitable tool for cell-based therapy in regenerative medicine and provide a novel promising target in the fight against cancer. The main stimulus responsible for EPC egression from the bone marrow and engraftment within neovessels is vascular endothelial growth factor (VEGF). Intracellular Ca2+ signals regulate numerous endothelial functions, such as proliferation, migration, and differentiation, and underpin VEGF effect on mature endothelium. We have recently shown that EPC growth is governed by a store-dependent Ca2+ entry (SOCE) pathway on the plasma membrane, which is activated by depletion of the inositol-1,4,5-trisphosphate (InsP3)-sensitive Ca2+ pools1. The present study aimed at investigating the nature and the role of VEGF-elicited Ca2+ signals in EPCs. All the putative SOCE mediators (i.e. TRPC1, TRPC4, Orai1 and Stim1) were present in EPCs. VEGF induced long lasting Ca2+ oscillations, however, removal of external Ca2+ (0Ca2+) and SOCE inhibition with BTP-2 reduced the number of Ca2+ spikes. Blockade of phospholipase C-? (PLC-?) with U73122 and emptying the InsP3-sensitive Ca2+ pools with cyclopiazonic acid (CPA) prevented the Ca2+ response to VEGF. Accordingly, the Ca2+ response to VEGF was inhibited by superfusing CPA during the ongoing oscillations. Notably, VEGF induced EPC was abrogated by SOCE inhibition with BTP-2. Similarly, VEGF promoted NF-kB translocation into the nucleus in a BTP-2-sensitive manner. Thus, VEGF causes an initial InsP3-dependent Ca2+ discharge followed by SOCE-mediated Ca2+ entry in cEPCs. SOCE, in turn, controls store refilling and induces cell proliferation by recruiting NF-kB

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.Genisteína 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⁺.Facultad de Ciencias Exacta

Phenotypic and functional characterization of endothelial progenitor cells isolated from peripheral blood of renal cell carcinoma patients

Endothelial progenitor cells (EPCs) are mobilized from either bone marrow or arterial walls to restore blood perfusion to ischemic organs and establish the vascular network within growing tumors [1]. The Ca2+ machinery plays a key role in EPC activation and might serve a molecular target for novel therapies of highly angiogenic tumors, such as renal cell carcinoma (RCC) [1]. The Ca2+ toolkit is remodelled in EPCs isolated from RCC patients (RCC-EPCs) as respect to healthy donors [2]. The present study was undertaken to evaluate for the first time the functional properties of EPCs isolated from tumor patients by focusing on RCC-EPCs. We extended our analysis at microscopic level by monitoring the sub-cellular structure of RCC-EPCs relative to their Ca2+ signalling fingerprint. Our results showed a striking functional and ultrastructural difference between RCC-EPCs and their normal counterparts, which might be the basis for designing novel, more specific anti-angiogenic treatments

Cardiac microvascular endothelial cells express a functional Ca 2+-sensing receptor

The mechanism whereby extracellular Ca2+ exerts the endothelium-dependent control of vascular tone is still unclear. In this study, we assessed whether cardiac microvascular endothelial cells (CMEC) express a functional extracellular Ca2+-sensing receptor (CaSR) using a variety of techniques. CaSR mRNA was detected using RT-PCR, and CaSR protein was identified by immunocytochemical analysis. In order to assess the functionality of the receptor, CMEC were loaded with the Ca2+-sensitive fluorochrome, Fura-2/AM. A number of CaSR agonists, such as spermine, Gd 3+, La3+ and neomycin, elicited a heterogeneous intracellular Ca2+ signal, which was abolished by disruption of inositol 1,4,5-trisphosphate (InsP3) signaling and by depletion of intracellular stores with cyclopiazonic acid. The inhibition of the Na +/Ca2+ exchanger upon substitution of extracellular Na+ unmasked the Ca2+ signal triggered by an increase in extracellular Ca2+ levels. Finally, aromatic amino acids, which function as allosteric activators of CaSR, potentiated the Ca2+ response to the CaSR agonist La3+. These data provide evidence that CMEC express CaSR, which is able to respond to physiological agonists by mobilizing Ca2+ from intracellular InsP3-sensitive stores.Facultad de Ciencias Exacta