Resveratrol reduces store-operated Ca2+ entry and enhances the apoptosis of fibroblast-like synoviocytes in adjuvant arthritis rats model via targeting ORAI1-STIM1 complex

BACKGROUND: Resveratrol was reported to trigger the apoptosis of fibroblast-like synoviocytes In adjuvant arthritis rats but the subcellular mechanism remains unclear. Since ER stress, mitochondrial dysfunction and oxidative stress were involved in the effects of resveratrol with imbalance of calcium bio-transmission, store operated calcium entry (SOCE), a novel intracellular calcium regulatory pathway, may also participate in this process. RESULTS: In the present study, Resveratrol was found to suppress ORAI1 expression of a dose dependent manner while have no evident effects on STIM1 expressive level. Besides, resveratrol had no effects on ATP or TG induced calcium depletion but present partly dose-dependent suppression of SOCE. On the one hand, microinjection of ORAI1 overexpressed vector in sick toe partly counteracted the therapeutic effects of resveratrol on adjuvant arthritis and serum inflammatory cytokine including IL-1, IL-6, IL-8, IL-10 and TNF-α. On the other hand, ORAI1 SiRNA injection provided slight relief to adjuvant arthritis in rats. In addition, ORAI1 overexpression partly diminished the alleviation of hemogram abnormality induced by adjuvant arthritis after resveratrol treatment while ORAI1 knockdown presented mild resveratrol-like effect on hemogram in rats model. CONCLUSION: These results indicated that resveratrol reduced store-operated Ca2+ entry and enhanced the apoptosis of fibroblast-like synoviocytes in adjuvant arthritis rats model via targeting ORAI1-STIM1 complex, providing a theoretical basis for ORAI1 targeted therapy in future treatment with resveratrol on rheumatoid arthritis.

Involvement of K+ATP and Ca2+ channels in hydrogen sulfide-suppressed ageing of porcine oocytes

BACKGROUND: Hydrogen sulfide has been shown to improve the quality of oocytes destined for in vitro fertilization. Although hydrogen sulfide is capable of modulating ion channel activity in somatic cells, the role of hydrogen sulfide in gametes and embryos remains unknown. Our observations confirmed the hypothesis that the KATP and L-type Ca2+ ion channels play roles in porcine oocyte ageing and revealed a plausible contribution of hydrogen sulfide to the modulation of ion channel activity. RESULTS: We confirmed the benefits of the activation and suppression of the KATP and L-type Ca2+ ion channels, respectively, for the preservation of oocyte quality. CONCLUSIONS: Our experiments identified hydrogen sulfide as promoting the desired ion channel activity, with the capacity to protect porcine oocytes against cell death. Further experiments are needed to determine the exact mechanism of hydrogen sulfide in gametes and embryos.

El Ca2+ y los esfingolípidos como moduladores de la apoptosis y el cáncer / Ca2+ and sphingolipids as modulators for apoptosis and cancer

El Ca2+ es un segundo mensajero que regula funciones directamente relacionadas con el cáncer como la proliferación, diferenciación y la apoptosis. La concentración intracelular de Ca2+ ([Ca2+]i) está altamente regulada por diversos mecanismos entre los que destacan canales iónicos, la Ca2+-ATPasa del retículo endoplasmático (SERCA) y de la membrana plasmática (PMCA), y el transporte de Ca2+ mitocondrial. En el cáncer, la célula tumoral prolifera sin control tras su incapacidad de reconocer señales apoptóticas. La apoptosis es mediada a través de cambios en la actividad de ciertas proteínas como las caspasas y miembros de la familia Bcl-2. Adicionalmente, el “estrés del retículo”, promovido por la acumulación y agregación de proteínas mal plegadas en el interior del retículo endoplasmático (RE), puede desencadenar la apoptosis. El “estrés del retículo” es inducido por una variedad de agentes, entre los que destaca la tapsigargina, inhibidor específico de la SERCA, la cual promueve un notable aumento en la [Ca2+]i, induciendo además apoptosis. En consecuencia, actualmente se están ensayando exitosamente derivados de la tapsigargina como agentes antineoplásicos. El Ca2+ es transferido a la mitocondria desencadenando señales apoptóticas. Por otra parte, los esfingolípidos, como la ceramida y la esfingosina, y sus derivados fosforilados, la ceramida-1-fosfato y la esfingosina-1-fosfato, los cuales regulan la [Ca2+]i, también están estrechamente vinculados con la señalización intracelular en procesos relacionados con el cáncer. Esta revisión discute nuevas evidencias sobre el efecto de estos esfingolípidos en la homeostasis de Ca+2 intracelular y su conexión con la apoptosis y el cáncer.

Ca2+ is a second messenger which regulates many functions directly related with cancer such as proliferation, differentiation and apoptosis. The intracellular Ca2+ concentration ([Ca2+]i) is finely regulated by several mechanisms, among them ionic channels, the endoplasmic reticulum Ca2+-ATPase (SERCA), the plasma membrane calcium pump (PMCA) and the mitochondrial Ca2+ transport. In cancer, the tumour cell proliferates without control since the capacity to recognize apoptotic signals has been lost. The apoptosis is regulated by changes in several proteins, as caspases and the Bcl-2 family members, among others. Additionally, the “reticulum stress”, promoted by the accumulation and aggregation of unfolded proteins in the interior of the endoplasmic reticulum (ER), ussually leads to apoptosis. The “reticulum stress” can be induced by several agents, remarkably with thapsigargin, a selective inhibitor of the SERCA, which in turn induces a large increment in [Ca2+]I, leading to apoptosis. As a consequence, currently, derivatives of thapsigargin are successfully been assayed as anti-neoplastic agents. Ca2+ is then transferred to the mitochondria, where it is known to constitute a main apoptotic signal. On the other hand, several sphingolipids, such as ceramide and sphingosine, and their phosphorylated derivatives ceramide-1-phosphate and sphingosine-1-phosphate, directly involved in the [Ca2+]I regulation, are also recognized as signal messengers related with cancer processes. In this review we discuss new evidences on the effect of several sphingolipids in the intracellular Ca2+ homeostasis and its relationship with apoptosis and cancer.

The Role of the Calcium and the Voltage Clocks in Sinoatrial Node Dysfunction

Recent evidence indicates that the voltage clock (cyclic activation and deactivation of membrane ion channels) and Ca2+ clocks (rhythmic spontaneous sarcoplasmic reticulum Ca2+ release) jointly regulate sinoatrial node (SAN) automaticity. However, the relative importance of the voltage clock and Ca2+ clock for pacemaking was not revealed in sick sinus syndrome. Previously, we mapped the intracellular calcium (Cai) and membrane potentials of the normal intact SAN simultaneously using optical mapping in Langendorff-perfused canine right atrium. We demonstrated that the sinus rate increased and the leading pacemaker shifted to the superior SAN with robust late diastolic Cai elevation (LDCAE) during beta-adrenergic stimulation. We also showed that the LDCAE was caused by spontaneous diastolic sarcoplasmic reticulum (SR) Ca2+ release and was closely related to heart rate changes. In contrast, in pacing induced canine atrial fibrillation and SAN dysfunction models, Ca2+ clock of SAN was unresponsiveness to beta-adrenergic stimulation and caffeine. Ryanodine receptor 2 (RyR2) in SAN was down-regulated. Using the prolonged low dose isoproterenol together with funny current block, we produced a tachybradycardia model. In this model, chronically elevated sympathetic tone results in abnormal pacemaking hierarchy in the right atrium, including suppression of the superior SAN and enhanced pacemaking from ectopic sites. Finally, if the LDCAE was too small to trigger an action potential, then it induced only delayed afterdepolarization (DAD)-like diastolic depolarization (DD). The failure of DAD-like DD to consistently trigger a sinus beat is a novel mechanism of atrial arrhythmogenesis. We conclude that dysfunction of both the Ca2+ clock and the voltage clock are important in sick sinus syndrome.

New nitric oxide donors based on ruthenium complexes

Nitric oxide (NO) donors produce NO-related activity when applied to biological systems. Among its diverse functions, NO has been implicated in vascular smooth muscle relaxation. Despite the great importance of NO in biological systems, its pharmacological and physiological studies have been limited due to its high reactivity and short half-life. In this review we will focus on our recent investigations of nitrosyl ruthenium complexes as NO-delivery agents and their effects on vascular smooth muscle cell relaxation. The high affinity of ruthenium for NO is a marked feature of its chemistry. The main signaling pathway responsible for the vascular relaxation induced by NO involves the activation of soluble guanylyl-cyclase, with subsequent accumulation of cGMP and activation of cGMP-dependent protein kinase. This in turn can activate several proteins such as K+ channels as well as induce vasodilatation by a decrease in cytosolic Ca2+. Oxidative stress and associated oxidative damage are mediators of vascular damage in several cardiovascular diseases, including hypertension. The increased production of the superoxide anion (O2-) by the vascular wall has been observed in different animal models of hypertension. Vascular relaxation to the endogenous NO-related response or to NO released from NO deliverers is impaired in vessels from renal hypertensive (2K-1C) rats. A growing amount of evidence supports the possibility that increased NO inactivation by excess O2- may account for the decreased NO bioavailability and vascular dysfunction in hypertension.

Aspectos fisiológicos do cálcio e sua disfunção em patologias neurodegenerativas / Physiologic aspects of calcium and your dysfunction in neurodegenerative pathologies

O íon cálcio funciona como um segundo mensageiro que regula um amplo espectro de processos celulares. A diminuição ou perda do controle dos mecanismos que regulam a concentração intracelular desse íon está associada, respectivamente, ao envelhecimento dos neurônios e a doenças neurodegenerativas. A gênese dessas modificações é desconhecida. Entretanto, estudos recentes apontam para uma provável correlação entre expressão gênica alterada, estresse do retículo endoplasmático e os processos patológicos associados à disfunção na concentração intracelular do cálcio. O esclarecimento dessas questões poderá trazer novos alvos terapêuticos capazes de frear ou reverter tais alterações, combatendo, dessa forma, tanto o envelhecimento neuronal quanto as doenças neurodegenerativas.

Calcium is a second messenger that regulates a lot of cellular functions. The following mechanisms regulate the intracellular concentrations of the ion: influx, release, extrusion and storage. Decrease or loss in control of these mechanisms is related to aging of neurons and neurodegenerative diseases, respectively. The genesis of these alterations is unknown. However, recent studies point to a correlation between calcium dysfunction and altered gene expression. There is also a correlation between endoplasmic reticulum stress and pathological processes. Further investigations may reveal new therapeutical targets that can block or revert these alterations.

Effect of some ions on sperm activation in Brycon henni(Eigenmann 1913)

Spermatozoa in Characid fish remain immobile in seminal plasma and are activated when freed into water where the ionic balance seems to be the main factor starting the activation process. This process was the target of the present study with emphasis on the activation of motility and on motility maintenance over time. The effect of isosmotic solutions was analyzed taking into account the possible combinations of the following ions, Ca2+, K+, Mg2+ and Na+ as well as the effect of channel blocking agents. The parameters measured were cells with motility (per cent), duration of motility (s), plasma membrane potential, and the effect of channel blockers on activation time and on motility. There was an increased motility when the semen was incubated in solutions containing K+ (p<0.05) compared with the control (CaNaMgK solution); the longest duration of motility was attained when the incubation was performed in solutions containing Na+ and Mg2+ (p<0.05). All solutions induced a change in membrane potential detected after 15 s of activation. Blocking K+, Ca2+ and Na+ channels did not alter motility but decreased the activation time (p<0.05). Potassium induced activation at all concentrations up to 105 mM, but motility was drastically decreased at concentrations higher than 140 mM (p<0.05). The conclusion is that interaction of the ionic environment with the cell membrane leads to changes in membrane potential and intracellular signalling that trigger sperm motility in Brycon henni.

Slow Ca2+ channels neither contribute to upstroke of action potential nor to pacemaker potential in spontaneously active freshly isolated three day embryonic chick ventricle.

Contribution of slow Ca2+ channels to the upstroke of action potential (AP) and pacemaker potential was studied by observing the effects of Ca2+ channel activators- high [Ca2+]0, Bay-K-8644, isoproterenol, forskolin and dibutyryl-cAMP on spontaneous AP of freshly isolated 3 day embryonic chick ventricle (3 day ECV). The spontaneous APs showed maximal upstroke velocity (+Vmax), maximum diastolic potential (MDP), overshoot (Eov) and AP duration at -20 mv (APD20) of 42.60 +/- 2.40 V/sec, -59.05 +/- 0.95 my, 16.30 +/- 0.53 mv and 70.32 +/- 4.60 msec, respectively (an average value of 35 preparations). Bay-K-8644 (0.1-0.8 microM), isoproterenol (5-10 pM) and forskolin (0.1-2.0 microM) induced a concentration-dependent increase in APD20 and Eov without affecting +Vmax. Dibutyryl-cAMP (1 microM) also enhanced the APD20 and Eov and had no effect on +Vmax. Elevation of [Ca2+]0 from 0.6 mM to 9.6 mM caused a concentration-dependent increase in APD20 and Eov leaving +Vmax unaltered. Elevated [Ca2+] and the other Ca2+ channel activators had no significant effect on MDP in above concentration range. Increase in APD20 and Eov could be explained at least by activation of slow Ca2+ channels but the lack of any change in +Vmax clearly suggests that the slow Ca2+ channels do not contribute to the upstroke of AP. All these interventions reduced the rate of spontaneous firing without any noticeable effect on MDP. This finding shows that the slow Ca2+ channels also do not contribute directly to the generation of pacemaker potential in spontaneously active freshly isolated 3 day ECV.

Calcium-transporters in myocardial cells.

This is a concise review of important calcium-transporters on the sarcolemma and organellar membranes of myocardial cells, and their functional roles in cell physiology. It briefly addresses L and T type calcium channels, store-operated calcium channel (SOC), sodium-calcium exchanger (NCX), and the plasma membrane calcium ATPase (PMCA) on the sarcolemma, ryanodine receptor (RyR), IP3 receptor (IP3R) and the sarcoplasmic reticulum (SR) calcium ATPase (SAERCA) on the SR membrane and their contributions to contraction and rhythm-generation. Several agonists and blockers for every transporter that are commonly used in research, and those with therapeutic applications have also been discussed.

Large conductance Ca2+-activated K+ (BK) channel: Activation by Ca2+ and voltage

Large conductance Ca2+-activated K+ (BK) channels belong to the S4 superfamily of K+ channels that include voltage-dependent K+ (Kv) channels characterized by having six (S1-S6) transmembrane domains and a positively charged S4 domain. As Kv channels, BK channels contain a S4 domain, but they have an extra (S0) transmembrane domain that leads to an external NH2-terminus. The BK channel is activated by internal Ca2+, and using chimeric channels and mutagenesis, three distinct Ca2+-dependent regulatory mechanisms with different divalent cation selectivity have been identified in its large COOH-terminus. Two of these putative Ca2+-binding domains activate the BK channel when cytoplasmic Ca2+ reaches micromolar concentrations, and a low Ca2+ affinity mechanism may be involved in the physiological regulation by Mg2+. The presence in the BK channel of multiple Ca2+-binding sites explains the huge Ca2+ concentration range (0.1 ìM-100 ìM) in which the divalent cation influences channel gating. BK channels are also voltage-dependent, and all the experimental evidence points toward the S4 domain as the domain in charge of sensing the voltage. Calcium can open BK channels when all the voltage sensors are in their resting configuration, and voltage is able to activate channels in the complete absence of Ca2+. Therefore, Ca2+ and voltage act independently to enhance channel opening, and this behavior can be explained using a two-tiered allosteric gating mechanism.

Altered calcium currents in cultured sensory neurons of normal and trisomy 16 mouse fetuses, an animal model for human trisomy 21 (Down Syndrome)

Down syndrome is determined by the presence of an extra copy of autosome 21 and is expressed by multiple abnormalities, with mental retardation being the most striking feature. The condition results in altered electrical membrane properties of fetal dorsal root ganglia (DRG) neurons, as in the trisomy 16 fetal mouse, an animal model of the human condition. Cultured trisomic DRG neurons from human and mouse fetuses present faster rates of depolarization and repolarization in the action potential compared to normal controls and a shorter spike duration. Also, trisomy 16 brain and spinal cord tissue exhibit reduced acetylcholine secretion. Therefore, we decided to study Ca2+ currents in cultured DRG neurons from trisomy 16 and age-matched control mice, using the whole-cell patch-clamp technique. Trisomic neurons exhibited a 62 percent reduction in Ca2+ current amplitude and reduced voltage dependence of current activation at -30 and -20 mV levels. Also, trisomic neurons showed slower activation kinetics for Ca2+ currents, with up to 80 percent increase in time constant values. Kinetics of the inactivation phase were similar in both conditions. The results indicate that murine trisomy 16 alter Ca2+ currents, which may contribute to impaired cell function, including neurotransmitter release. These abnormalities also may alter neural development.

Properties of voltage-gated Ca2+ currents measured from mouse pancreatic â-cells in situ

We used the single-microelectrode voltage-clamp technique to record ionic currents from pancreatic â-cells within intact mouse islets of Langerhans at 37C, the typical preparation for studies of glucose-induced "bursting" electrical activity. Cells were impaled with intracellular microelectrodes, and voltage pulses were applied in the presence of tetraethylammonium. Under these conditions, a voltage-dependent Ca2+ current (I Cav), containing L-type and non-L-type components, was observed. The current measured in situ was larger than that measured in single cells with whole-cell patch clamping, particularly at membrane potentials corresponding to the action potentials of â-cell electrical activity. The temperature dependence of I Cav was not sufficient to account for the difference in size of the currents recorded with the two methods. During prolonged pulses, the voltage-dependent Ca2+ current measured in situ displayed both rapid and slow components of inactivation. The rapid component was Ca2+-dependent and was inhibited by the membrane-permeable Ca2+ chelator, BAPTA-AM. The effect of BAPTA-AM on â-cell electrical activity then demonstrated that Ca2+-dependent inactivation of I Cav contributes to action potential repolarization and to control of burst frequency. Our results demonstrate the utility of voltage clamping â-cells in situ for determining the roles of ion channels in electrical activity and insulin secretion.

Acute hypercalcemia-induced hypertension: the roles of calcium channel and alpha-1 adrenergic receptor.

OBJECTIVE: To study the mechanism(s) of acute hypercalcemia-induced hypertension in dogs. MATERIAL AND METHOD: Adult male mongrel dogs were intravenously infused with: 1) normal saline solution, 2) CaCl2 solution, 3) CaCl2 + calcium channel blocker (verapamil), 4) CaCl2 + selective alpha-1 adrenergic receptor blocker (prazosin), or 5) CaCl2 + verapamil + prazosin. Either verapamil or prazosin treatment was started at forty minutes before CaCl2 infusion and then was co-administered throughout the three-hour experimental period. Systemic and renal hemodynamics parameters were determined. RESULTS: Infusion of CaCl2 caused increases in mean arterial blood pressure (p < 0.01), total peripheral resistance (p < 0.001), and renal vascular resistance (p < 0.001). Prior treatment with either verapamil or prazosin lowered baseline blood pressure (p < 0.01) and could prevent hypercalcemia-induced hypertension. This occurred accompanying regaining to near normal values of abnormal systemic hemodynamics parameters. Combination of both drugs showed more profound effects, particularly on lowering renal vascular resistance. CONCLUSION: Acute hypercalemic hypertension is caused by an increase in vascular resistance mediated via the direct effect of calcium on vascular smooth muscle as well as the indirect effect of calcium induced hypercatecholaminemia. The stimulatory effect of hypercalcemia on renal vascular resistance is more prominent than that on peripheral vascular resistance.

Mitochondrial Ca2+ homeostasis in health and disease

Although it has long been known that mitochondria possess a complex molecular repertoire for accumulating and releasing Ca2+, only in recent years has a large body of data demonstrated that these organelles promptly respond to Ca2+-mediated cell stimulations. In this contribution, we will review the principles of mitochondrial Ca2+ homeostasis and its signaling role in different physiological and pathological conditions.

Inositol 1, 4, 5-trisphosphate receptors in the heart

Inositol 1,4,5-trisphosphate (InsP3) is an established calcium-mobilizing messenger, which is well-known to activate Ca2+ signaling in many cell types. Contractile cardiomyocytes express hormone receptors that are coupled to the production of InsP3. Such cardioactive hormones, including endothelin, may have profound inotropic and arrhythmogenic actions, but it is unclear whether InsP3 underlies any of these effects. We have examined the expression and localization of InsP3 receptors (InsP3Rs), and the potential role of InsP3 in modulating cardiac excitation-contraction coupling (EC coupling). Stimulation of electrically-paced atrial and ventricular myocytes with a membrane-permeant InsP3 ester was found to evoke an increase in the amplitudes of action potential-evoked Ca2+ transients and to cause pro-arrhythmic diastolic Ca2+ transients. All the effects of the InsP3 ester could be blocked using a membrane-permeant antagonist of InsP3Rs (2-aminoethoxydiphenyl borate; 2-APB). Furthermore, 2-APB blocked arrhythmias evoked by endothelin and delayed the onset of positive inotropic responses. Our data indicate that atrial and ventricular cardiomyocytes express functional InsP3Rs, and these channels have the potential to influence EC coupling.

Acute and chronic effects of nitrendipine on naloxone precipitated morphine withdrawal in mice

There is growing evidence indicating that neuronal calcium channels play an important role in the mechanism of morphine dependence. In this study, the effects of acute and chronic administration of nitrendipine on naloxone precipitated morphine withdrawal signs were investigated. Mice were rendered dependent to morphine by subcutaneous injection of morphine over a period of 5 days. In chronic studies, nitrendipine [25 and 50 mg/kg, i.p.], or vehicle injections were given once a day during the morphine treatment, and the last injection of nitrendipine was given 24 h before the morphine withdrawal. For acute studies, nitrendipine [25 and 50 mg/kg, i.p.] was given 1 h after the last dose of morphine [1 h before naloxone]. A single injection of nitrendipine at 25 mg/kg was ineffective in blocking most signs of morphine withdrawal, however, at 50 mg/kg nitrendipine blocked signs such as hair raising, sniffing, diarrhea and number of jumping. The concurrent injections of nitrendipine with morphine prevented most signs of morphine withdrawal. In agreement with previous findings, these results suggest that alterations in voltage-sensitive calcium channels play a role in the adaptations that occur on chronic treatment with morphine

Voltage activated calcium channels in somatic muscle of filarial nematode Setaria cervi.

Acetylcholine (Ach), levamisole and pyrantel pamoate all cause stimulation of spontaneous rhythmic movements of whole worm and nerve muscle preparation of filarial nematode Setaria cervi. These stimulant effects are manifested only in the presence of available Ca2+ or extracellular Ca2+. Electrical stimulation of nerve muscle preparation of Setaria cervi elicited depolarization and increase in amplitude and tone of contractions. Electrical current stimulates Ca2+ entry leading to depolarization and during the phase of depolarization addition of any of the three stimulants viz. Ach, levamisole or pyrantel pamoate fails to elicit any response on nerve muscle preparation. The findings indicate that electrical stimulation, excitatory neurotransmitter Ach and stimulant anthelmintics levamisole and pyrantel pamoate all produce their stimulant effect by triggering entry of Ca2+ into the muscle cell. Further, blocking the calcium channels by nifedepine and thereby the entry of Ca2+ into the cells blocks the stimulant effect of Ach levamisole and pyrantel pamoate.

Involvement of calcium in pain and antinociception

Calcium ions are widely recognized to play a fundamental role in the regulation of several biological processes. Transient changes in cytoplasmic calcium ion concentration represent a key step for neurotransmitter release and the modulation of cell membrane excitability. Evidence has accumulated for the involvement of calcium ions also in nociception and antinociception, including the analgesic effects produced by opioids. The combination of opioids with drugs able to interfere with calcium ion functions in neurons has been pointed out as a useful alternative for safer clinical pain management. Alternatively, drugs that reduce the flux of calcium ions into neurons have been indicated as analgesic alternatives to opioids. This article reviews the manners by which calcium ions penetrate cell membranes and the changes in these mechanisms caused by opioids and calcium antagonists regarding nociceptive and antinociceptive events

Reaçäo acrossômica na exploraçäo de infertilidade masculina em fecundaçäo in vitro / Place of acrosomal reaction in the exploration of male infertility in in vitro fertilization

O acrossoma, vesícula de origem golgiana, tem um papel essencial no desenvolvimento da fecundaçäo, tanto in vivo com in vitro. Neste artigo estudaram diferentes métodos de induçäo da reaçäo cromossômica (RA). Os indutores como o líquido folicular, a progesterona e o cálcio iônico permitem a exploraçäo da dinâmica da RA e a análise dos espermatozóides aptos a realizar esta RA. Este trabalho mostra o interesse destas exploraçöes nos casos de infertilidade masculina em Reproduçäo Assistida.