Increase In Intracellular Calcium Concentration Research Articles

Effect of Hypoxic Paracrine Media on Calcium-Regulatory Proteins in Infarcted Rat Myocardium

Background and ObjectivesAn increase in intracellular calcium concentration due to loss of Ca2+ homeostasis triggers arrhythmia or cardiac cell death in the heart. Paracrine factors released from stem cells have beneficial cardioprotective effects. However, the mechanism of modulation of Ca2+ homeostasis by paracrine factors in ischemic myocardium remains unclear.Materials and MethodsWe isolated rat bone marrow-derived mesenchymal stem cells (MSCs), and prepared paracrine media (PM) from MSCs under hypoxic or normoxic conditions (hypoxic PM and normoxic PM). We induced rat myocardial infarction by left anterior descending ligation for 1 hour, and treated PM into the border region of infarcted myocardium (n=6/group) to identify the alteration in calcium-regulated proteins. We isolated and stained the heart tissue with specific calcium-related antibodies after 11 days.ResultsThe hypoxic PM treatment increased Ca2+-related proteins such as L-type Ca2+ channel, sarcoplasmic reticulum Ca2+ ATPase, Na+/K+ ATPase, and calmodulin, whereas the normoxic PM treatment increased those proteins only slightly. The sodium-calcium exchanger was significantly reduced by hypoxic PM treatment, compared to moderate suppression by the normoxic PM treatment.ConclusionOur results suggest that hypoxic PM was significantly associated with the positive regulation of Ca2+ homeostasis in infarcted myocardium.

The expression and function of histamine H3 receptors in pancreatic beta cells

Histamine and its receptors in the CNS play important roles in energy homeostasis. Here, we have investigated the expression and role of histamine receptors in pancreatic beta cells, which secrete insulin. The expression of histamine receptors in pancreatic beta cells was examined by RT-PCR, Western blotting and immunostaining. Insulin secretion assay, ATP measurement and calcium imaging studies were performed to determine the function and signalling pathway of histamine H₃ receptors in glucose-induced insulin secretion (GIIS) from MIN6 cells, a mouse pancreatic beta cell line. The function and signalling pathway of H₃ receptors in MIN6 cell proliferation were examined using pharmacological assay and Western blotting. Histamine H₃ receptors were expressed in pancreatic beta cells. A selective H₃ receptor agonist, imetit, and a selective inverse H₃ receptor agonist, JNJ-5207852, had inhibitory and facilitatory effects, respectively, on GIIS in MIN6 cells. Neither imetit nor JNJ-5207852 altered intracellular ATP concentration, or intracellular calcium concentration stimulated by glucose and KCl, indicating that GIIS signalling was affected by H3 receptor signalling downstream of the increase in intracellular calcium concentration. Moreover, imetit attenuated bromodeoxyuridine incorporation in MIN6 cells. The phosphorylation of cAMP response element-binding protein (CREB), which facilitated beta cell proliferation, was inhibited, though not significantly, by imetit, indicating that activated H₃ receptors inhibited MIN6 cell proliferation, possibly by decreasing CREB phosphorylation. Histamine H₃ receptors were expressed in mouse beta cells and could play a role in insulin secretion and, possibly, beta cell proliferation.

20-Hydroxyeicosatetraenoic Acid Contributes to the Inhibition of K+ Channel Activity and Vasoconstrictor Response to Angiotensin II in Rat Renal Microvessels

The present study examined whether 20-hydroxyeicosatetraenoic acid (HETE) contributes to the vasoconstrictor effect of angiotensin II (ANG II) in renal microvessels by preventing activation of the large conductance Ca2+-activated K+ channel (KCa) in vascular smooth muscle (VSM) cells. ANG II increased the production of 20-HETE in rat renal microvessels. This response was attenuated by the 20-HETE synthesis inhibitors, 17-ODYA and HET0016, a phospholipase A2 inhibitor AACOF3, and the AT1 receptor blocker, Losartan, but not by the AT2 receptor blocker, PD123319. ANG II (10-11 to 10-6 M) dose-dependently decreased the diameter of renal microvessels by 41 ± 5%. This effect was blocked by 17-ODYA. ANG II (10-7 M) did not alter KCa channel activity recorded from cell-attached patches on renal VSM cells under control conditions. However, it did reduce the NPo of the KCa channel by 93.4 ± 3.1% after the channels were activated by increasing intracellular calcium levels with ionomycin. The inhibitory effect of ANG II on KCa channel activity in the presence of ionomycin was attenuated by 17-ODYA, AACOF3, and the phospholipase C (PLC) inhibitor U-73122. ANG II induced a peak followed by a steady-state increase in intracellular calcium concentration in renal VSM cells. 17-ODYA (10-5 M) had no effect on the peak response, but it blocked the steady-state increase. These results indicate that ANG II stimulates the formation of 20-HETE in rat renal microvessels via the AT1 receptor activation and that 20-HETE contributes to the vasoconstrictor response to ANG II by blocking activation of KCa channel and facilitating calcium entry.

Role of GRK4 in the Regulation of Arterial AT 1 Receptor in Hypertension

G-protein-coupled receptor kinase 4 (GRK4) gene variants, via impairment of renal dopamine receptor and enhancement of renin-angiotensin system functions, cause sodium retention and increase blood pressure. Whether GRK4 and the angiotensin type 1 receptor (AT(1)R) interact in the aorta is not known. We report that GRK4 is expressed in vascular smooth muscle cells of the aorta. Heterologous expression of the GRK4γ variant 142V in A10 cells increased AT(1)R protein expression and AT(1)R-mediated increase in intracellular calcium concentration. The increase in AT(1)R expression was related to an increase in AT(1)R mRNA expression via the NF-κB pathway. As compared with control, cells expressing GRK4γ 142V had greater NF-κB activity with more NF-κB bound to the AT(1)R promoter. The increased AT(1)R expression in cells expressing GRK4γ 142V was also associated with decreased AT(1)R degradation, which may be ascribed to lower AT(1)R phosphorylation. There was a direct interaction between GRK4γ and AT(1)R that was decreased by GRK4γ 142V. The regulation of AT(1)R expression by GRK4γ 142V in A10 cells was confirmed in GRK4γ 142V transgenic mice; AT(1)R expression was higher in the aorta of GRK4γ 142V transgenic mice than control GRK4γ wild-type mice. Angiotensin II-mediated vasoconstriction of the aorta was also higher in GRK4γ 142V than in wild-type transgenic mice. This study provides a mechanism by which GRK4, via regulation of arterial AT(1)R expression and function, participates in the pathogenesis of conduit vessel abnormalities in hypertension.

A novel method using Autographa californica multiple nucleopolyhedrovirus for increasing the sensitivity of insecticide through calcium influx in insect cell line

Due to an intensive use of chemical insecticides, resistance mechanisms to insecticides together with adverse effects on non-target organisms have been largely reported. Improvement in pest control strategy represents an urgent need to optimize efficiency in the control of pest insects. In this context, a novel method based on the use of insect specific virus applied in combination with chemical insecticide, which could lead to sensitization of the insect target to insecticides is described. Insect virus, the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV), applied onto Sf9 cells induces an increase of intracellular calcium concentration via extracellular calcium influx. Co-application of AcMNPV with chlorpyrifos-ethyl onto Sf9 cells expressing the key enzyme acetylcholinesterase (AChE), known to be targeted by organophosphate insecticides, increases 1.5-fold the sensitivity of AChE to the insecticide. This effect is correlated with intracellular calcium concentration rise since AcMNPV-induced potentiating insecticide effect is counteracted by pretreatment with the calcium channel blocker, cadmium chloride. Increasing insecticide target sensitivity through intracellular calcium modulation by using insect virus co-applied with a chemical insecticide is a very promising strategy allowing optimization of insecticide treatment while reducing the concentration of insecticides used.

Fluctuating vs. continuous exposure to H₂O₂: the effects on mitochondrial membrane potential, intracellular calcium, and NF-κB in astroglia.

The effects of H2O2 are widely studied in cell cultures and other in vitro systems. However, such investigations are performed with the assumption that H2O2 concentration is constant, which may not properly reflect in vivo settings, particularly in redox-turbulent microenvironments such as mitochondria. Here we introduced and tested a novel concept of fluctuating oxidative stress. We treated C6 astroglial cells and primary astrocytes with H2O2, using three regimes of exposure – continuous, as well as fluctuating at low or high rate, and evaluated mitochondrial membrane potential and other parameters of mitochondrial activity – respiration, reducing capacity, and superoxide production, as well as intracellular ATP, intracellular calcium, and NF-κB activation. When compared to continuous exposure, fluctuating H2O2 induced a pronounced hyperpolarization in mitochondria, whereas the activity of electron transport chain appears not to be significantly affected. H2O2 provoked a decrease of ATP level and an increase of intracellular calcium concentration, independently of the regime of treatment. However, fluctuating H2O2 induced a specific pattern of large-amplitude fluctuations of calcium concentration. An impact on NF-κB activation was observed for high rate fluctuations, whereas continuous and low rate fluctuating oxidative stress did not provoke significant effects. Presented results outline the (patho)physiological relevance of redox fluctuations.

Abstract 568: Regulation of GRK4 on Arterial AT 1 Receptor in Hypertension

G protein-coupled receptor kinase 4 (GRK4) gene variants or increased GRK4 expression, via impairment of renal dopamine receptor and enhancement of renin angiotensin system function, impairs renal sodium excretion, resulting in sodium retention and an increase in blood pressure. Increased aortic stiffness, a risk factor in cardiovascular disease, may be related to increased activity of the renin-angiotensin system Whether or not GRK4 and the angiotensin type 1 receptor (AT 1 R) interact in the aorta is not known. We now report that GRK4 is expressed in vascular smooth muscle cells (VSMCs) of the aorta. Exogenous expression of the GRK4 variant 142V in aortic A10 cells increased AT 1 R protein expression and AT 1 R-mediated increase in intracellular calcium concentration. The increased AT 1 R expression was caused by an increase in AT 1 R mRNA expression via NF-κB, because blockade of NF-κB abolished those effects of GRK4 A142V. As compared with control (vector-transfected) cells, cells expressing 142V had higher NF-kB activity and more NF-kB bound to AT 1 R promoter. The increased AT 1 R expression in cells expressing GRK4 142V was associated with decreased AT 1 R degradation, which was ascribed to the lower AT 1 R phosphorylation. There was direct interaction between GRK4 and AT 1 R in A10 cells which was decreased by GRK4 that could have caused the lower AT 1 R phosphorylation and degradation. The regulation of GRK4 of AT 1 R expression was confirmed in GRK4142V transgenic mice, the AT 1 R expression was higher, while AT 1 R phosphorylation was lower in aorta in GRK4 142V than control mice. Angiotensin II- mediated vasoconstriction was higher in A142V mice. This study provides a mechanism that GRK4, via regulation of arterial AT 1 R expression and function, engaged into the pathogenesis of hypertension.

Capsaicin mimics mechanical load-induced intracellular signaling events

Mechanical load-induced intracellular signaling events are important for subsequent skeletal muscle hypertrophy. We previously showed that load-induced activation of the cation channel TRPV1 caused an increase in intracellular calcium concentrations ([Ca2+]i) and that this activated mammalian target of rapamycin (mTOR) and promoted muscle hypertrophy. However, the link between mechanical load-induced intracellular signaling events, and the TRPV1-mediated increases in [Ca2+]i are not fully understood. Here we show that administration of the TRPV1 agonist, capsaicin, induces phosphorylation of mTOR, p70S6K, S6, Erk1/2 and p38 MAPK, but not Akt, AMPK or GSK3β. Furthermore, the TRPV1-induced phosphorylation patterns resembled those induced by mechanical load. Our results continue to highlight the importance of TRPV1-mediated calcium signaling in load-induced intracellular signaling pathways.

Calcium signaling mediates cold sensing in insect tissues.

The ability to rapidly respond to changes in temperature is a critical adaptation for insects and other ectotherms living in thermally variable environments. In a process called rapid cold hardening (RCH), insects significantly enhance cold tolerance following brief (i.e., minutes to hours) exposure to nonlethal chilling. Although the ecological relevance of RCH is well-established, the underlying physiological mechanisms that trigger RCH are poorly understood. RCH can be elicited in isolated tissues ex vivo, suggesting cold-sensing and downstream hardening pathways are governed by brain-independent signaling mechanisms. We previously provided preliminary evidence that calcium is involved in RCH, and here we firmly establish that calcium signaling mediates cold sensing in insect tissues. In tracheal cells of the freeze-tolerant goldenrod gall fly, Eurosta solidaginis, chilling to 0 °C evoked a 40% increase in intracellular calcium concentration as determined by live-cell confocal imaging. Downstream of calcium entry, RCH conditions significantly increased the activity of calcium/calmodulin-dependent protein kinase II (CaMKII) while reducing phosphorylation of the inhibitory Thr306 residue. Pharmacological inhibitors of calcium entry, calmodulin activation, and CaMKII activity all prevented ex vivo RCH in midgut and salivary gland tissues, indicating that calcium signaling is required for RCH to occur. Similar results were obtained for a freeze-intolerant species, adults of the flesh fly, Sarcophaga bullata, suggesting that calcium-mediated cold sensing is a general feature of insects. Our results imply that insect tissues use calcium signaling to instantly detect decreases in temperature and trigger downstream cold-hardening mechanisms.

Calcium pathways such as cAMP modulate clothianidin action through activation of α-bungarotoxin-sensitive and -insensitive nicotinic acetylcholine receptors

Clothianidin is a neonicotinoid insecticide developed in the early 2000s. We have recently demonstrated that it was a full agonist of α-bungarotoxin-sensitive and -insensitive nicotinic acetylcholine receptors expressed in the cockroach dorsal unpaired median neurons. Clothianidin was able to act as an agonist of imidacloprid-insensitive nAChR2 receptor and internal regulation of cAMP concentration modulated nAChR2 sensitivity to clothianidin. In the present study, we demonstrated that cAMP modulated the agonist action of clothianidin via α-bungarotoxin-sensitive and insensitive receptors. Clothianidin-induced current–voltage curves were dependent to clothianidin concentrations. At 10μM clothianidin, increasing cAMP concentration induced a linear current–voltage curve. Clothianidin effects were blocked by 0.5μM α-bungarotoxin suggesting that cAMP modulation occurred through α-bungarotoxin-sensitive receptors. At 1mM clothianidin, cAMP effects were associated to α-bungarotoxin-insensitive receptors because clothianidin-induced currents were blocked by 5μM mecamylamine and 20μM d-tubocurarine. In addition, we found that application of 1mM clothianidin induced a strong increase of intracellular calcium concentration. These data reinforced the finding that calcium pathways including cAMP modulated clothianidin action on insect nicotinic acetylcholine receptors. We proposed that intracellular calcium pathways such as cAMP could be a target to modulate the mode of action of neonicotinoid insecticides.

Characterization of new G protein-coupled adenine receptors in mouse and hamster.

The nucleobase adenine has previously been reported to activate G protein-coupled receptors in rat and mouse. Adenine receptors (AdeR) thus constitute a new family of purine receptors, for which the designation "P0-receptors" has been suggested. We now describe the cloning and characterization of two new members of the AdeR family from mouse (MrgA10, termed mAde1R) and hamster (cAdeR). Both receptors were expressed in Sf9 insect cells, and radioligand binding studies were performed using [(3)H]adenine. Specific binding of the radioligand was detected in transfected, but not in untransfected cells, and K D values of 286 nM (mAde1R, B max 1.18 pmol/mg protein) and 301 nM (cAdeR, B max 17.7 pmol/mg protein), respectively, were determined. A series of adenine derivatives was investigated in competition binding assays. Minor structural modifications generally led to a reduction or loss of affinity, with one exception: 2-fluoroadenine was at least as potent as adenine itself at the cAdeR. Structure-activity relationships at all AdeR orthologs and subtypes investigated so far were similar, but not identical. For functional analyses, the cAdeR was homologously expressed in Chinese hamster ovary (CHO) cells, while the mAde1R was heterologously expressed in 1321N1 astrocytoma cells. Like the previously described AdeRs from rat (rAdeR) and mouse (mAde2R), the mAde1R (EC50 9.77 nM) and the cAdeR (EC50 51.6 nM) were coupled to inhibition of adenylate cyclase. In addition, the cAdeR from hamster expressed in CHO cells produced an increase in intracellular calcium concentrations (EC50 6.24 nM) and was found to be additionally coupled to Gq proteins.

Insulin-Like Growth Factor Binding Proteins Increase Intracellular Calcium Levels in Two Different Cell Lines

BackgroundInsulin-like growth factor binding proteins (IGFBPs) are six related secreted proteins that share IGF-dependent and -independent functions. If the former functions begin to be well described, the latter are somewhat more difficult to investigate and to characterize. At the cellular level, IGFBPs were shown to modulate numerous processes including cell growth, differentiation and apoptosis. However, the molecular mechanisms implicated remain largely unknown. We previously demonstrated that IGFBP-3, but not IGFBP-1 or IGFBP-5, increase intracellular calcium concentration in MCF-7 cells (Ricort J-M et al. (2002) FEBS lett 527: 293–297).Methodology/Principal FindingsWe perform a global analysis in which we studied, by two different approaches, the binding of each IGFBP isoform (i.e., IGFBP-1 to -6) to the surface of two different cellular models, MCF-7 breast adenocarcinoma cells and C2 myoblast proliferative cells, as well as the IGFBP-induced increase of intracellular calcium concentration. Using both confocal fluorescence microscopy and flow cytometry analysis, we showed that all IGFBPs bind to MCF-7 cell surface. By contrast, only four IGFBPs can bind to C2 cell surface since neither IGFBP-2 nor IGFBP-4 were detected. Among the six IGFBPs tested, only IGFBP-1 did not increased intracellular calcium concentration whatever the cellular model studied. By contrast, IGFBP-2, -3, -4 and -6, in MCF-7 cells, and IGFBP-3, -5 and -6, in C2 proliferative cells, induce a rapid and transient increase in intracellular free calcium concentration. Moreover, IGFBP-2 and -3 (in MCF-7 cells) and IGFBP-5 (in C2 cells) increase intracellular free calcium concentration by a pertussis toxin sensitive signaling pathway.ConclusionsOur results demonstrate that IGFBPs are able to bind to cell surface and increase intracellular calcium concentration. By characterizing the IGFBPs-induced cell responses and intracellular couplings, we highlight the cellular specificity and complexity of the IGF-independent actions of these IGF binding proteins.

Rapid human melanoma cell death induced by sanguinarine through oxidative stress

Sanguinarine is a natural isoquinoline alkaloid derived from the root of Sanguinaria canadensis and from other poppy fumaria species, and is known to have a broad spectrum of pharmacological properties. Here we have found that sanguinarine, at low micromolar concentrations, showed a remarkably rapid killing activity against human melanoma cells. Time-lapse videomicroscopy showed rapid morphological changes compatible with an apoptotic cell death, which was further supported by biochemical markers, including caspase activation, poly(ADP-ribose) polymerase (PARP) cleavage and DNA breakdown. Pan-caspase inhibition blocked sanguinarine-induced cell death. Sanguinarine treatment also induced an increase in intracellular calcium concentration, which was inhibited by dantrolene, and promoted cleavage of BAP-31, thus suggesting a putative role for Ca2+ release from endoplasmic reticulum and a cross-talk between endoplasmic reticulum and mitochondria in the anti-melanoma action of sanguinarine. Sanguinarine disrupted the mitochondrial transmembrane potential (ΔΨm), released cytochrome c and Smac/DIABLO from mitochondria to cytosol, and induced oxidative stress. Overexpression of Bcl-XL by gene transfer did not prevent sanguinarine-mediated cell death, oxidative stress or release of mitochondrial apoptogenic proteins. However, preincubation with N-acetyl-l-cysteine (NAC) prevented sanguinarine-induced oxidative stress, PARP cleavage, release of apoptogenic mitochondrial proteins, and cell death. Pretreatment with glutathione (GSH) also inhibited the anti-melanoma activity of sanguinarine. Thus, pretreatment with the thiol antioxidants NAC and GSH abrogated the killing activity of sanguinarine. Taking together, our data indicate that sanguinarine is a very rapid inducer of human melanoma caspase-dependent cell death that is mediated by oxidative stress.

Distinct activity of BK channel β1-subunit in cerebral and pulmonary artery smooth muscle cells

This study was designed to test a hypothesis that the functional activity of big-conductance, Ca(2+)-activated K(+) (BK) channels is different in cerebral and pulmonary artery smooth muscle cells (CASMCs and PASMCs). Using patch-clamp recordings, we found that the activity of whole cell and single BK channels were significantly higher in CASMCs than in PASMCs. The voltage and Ca(2+) sensitivity of BK channels were greater in CASMCs than in PASMCs. Targeted gene knockout of β(1)-subunits significantly reduced BK currents in CASMCs but had no effect in PASMCs. Western blotting experiments revealed that BK channel α-subunit protein expression level was comparable in CASMCs and PASMCs; however, β(1)-subunit protein expression level was higher in CASMCs than in PASMCs. Inhibition of BK channels by the specific blocker iberiotoxin enhanced norepinephrine-induced increase in intracellular calcium concentration in CASMCs but not in PASMCs. Systemic artery blood pressure was elevated in β(1)(-/-) mice. In contrast, pulmonary artery blood pressure was normal in β(1)(-/-) mice. These findings provide the first evidence that the activity of BK channels is higher in cerebral than in PASMCs. This heterogeneity is primarily determined by the differential β(1)-subunit function and contributes to diverse cellular responses in these two distinct types of cells.

Neutrophil elastase induces MUC5AC secretion via protease-activated receptor 2

Mucus hypersecretion is a major manifestation in patients with chronic inflammatory airway diseases, and mucin5AC (MUC5AC) protein is a major component of airway mucus. Previous studies have demonstrated that neutrophil elastase (NE) stimulates the secretion of MUC5AC from airway epithelial cells, however, the mechanism is poorly understood. NE is a known ligand for protein active receptors (PARs), which have been confirmed to participate in releasing MUC5AC in the airways. However, the role of PARs in NE-induced MUC5AC secretion remains unclear. We demonstrated that airway goblet-like Calu-3 cells express PAR1, PAR2, and PAR3 with a predominant level of PAR2. NE can increase PAR2 expression and MUC5AC release. In our study, we showed that NE binding to PAR2 can increase the cytosolic calcium concentration and subsequently activate PKC, leading to MUC5AC secretion. In order to investigate the mechanism of increased cytosolic calcium in Calu-3 cells, thapsigargin was used to exhaust the endoplasmic reticulum (ER) calcium pools, and 2-aminoethoxydiphenyl borate was used to inhibit the function of the store-operated calcium entry (SOCE) channels in the plasma membrane. We found that the NE-induced increase in intracellular calcium concentration is derived from release of the ER calcium pool and its subsequent calcium internal flux from the extracellular space via SOCE channels, which is dependent on sufficient levels of extracellular calcium.

Focal adhesion kinase dependent activation of the PI3 kinase pathway by the functional soluble form of neurotensin receptor-3 in HT29 cells

The neurotensin (NT) receptor-3 (NTSR3), also called sortilin, is thought to display several functions including a role as a receptor or a co-receptor, in the sorting to plasma membrane and to lysosomes, and in the regulated secretion. The aim of this study was to investigate the function of the soluble form of NTSR3 (sNTSR3) released from several cell lines including colonic cancer cells. The human adenocarcinoma epithelial cell line HT29 has been used to monitor the release, the binding and internalization of sNTSR3 by radioreceptor assays and confocal microscopy. The modulation of the intracellular signaling pathways by the protein has been investigated by using Fura-2 fluorescence calcium imaging microscopy and Western blots analysis. We demonstrated that sNTSR3 specifically binds and internalizes into HT29 cells. This binding, independent from the transactivation of the epidermal growth factor receptor, leads to the increase of intracellular calcium concentration and to the activation of a FAK/Src-dependent activation of the PI3 kinase pathway. In conclusion, sNTSR3 released from the membrane bound NTSR3 is a functional protein able to activate intracellular pathways involved in cell survival but probably not in cell growth.

Rutin potentiates calcium uptake via voltage-dependent calcium channel associated with stimulation of glucose uptake in skeletal muscle

Rutin is a flavonoid with several pharmacological properties and it has been demonstrated that rutin can modulate glucose homeostasis. In skeletal muscle, an increase in intracellular calcium concentration may induce glucose transporter-4 (GLUT-4) translocation with consequent glucose uptake. The aim of this study was to investigate the effect of rutin and intracellular pathways on calcium uptake as well as the involvement of calcium in glucose uptake in skeletal muscle. The results show that rutin significantly stimulated calcium uptake through voltage-dependent calcium channels as well as mitogen-activated kinase (MEK) and protein kinase A (PKA) signaling pathways. Also, rutin stimulated glucose uptake in the soleus muscle and this effect was mediated by extracellular calcium and calcium–calmodulin-dependent protein kinase II (CaMKII) activation. In conclusion, rutin significantly stimulates calcium uptake in rat soleus muscles. Furthermore, the increase in intracellular calcium concentration is involved in DNA activation by rutin. Also, rutin-induced glucose uptake via CaMKII may result in GLUT-4 translocation to the plasma membrane, characterizing an insulin-independent pathway. These findings indicate that rutin is a potential drug candidate for diabetes therapy.

The role of calcium in intracellular pathways of rutin in rat pancreatic islets: potential insulin secretagogue effect

Rutin is a flavonol glycoside with multiple biological activities and it has been demonstrated that rutin modulates glucose homeostasis. In pancreatic β-cell, an increase in intracellular calcium concentration triggers exocytosis and thus insulin secretion. The aim of the study reported herein was to investigate the effect of rutin associated intracellular pathways on Ca2+ uptake in isolated rat pancreatic islets. We focused on the acute effects of rutin on in vivo insulin secretion and the in vitro cellular signaling of pancreatic islets related to this effect. The results show that rutin significantly increased glucose-induced insulin secretion in an in vivo treatment. Moreover, it was demonstrated that rutin stimulated Ca2+ uptake after 10min of incubation compared with the respective control group. The involvement of L-type voltage-dependent Ca2+ channels (L-VDCCs) was evidenced using nifedipine, while the use of glibenclamide and diazoxide demonstrated that the ATP-sensitive potassium (KATP) channels are not involved in the rutin action in pancreatic islets. In conclusion, rutin diminish glycemia, potentiate insulin secretion in vivo and significantly stimulates Ca2+ uptake in rat pancreatic islets. A novel cellular mechanism of action of rutin in Ca2+ uptake on pancreatic β-cells was elucidated. Rutin modulates Ca2+ uptake in pancreatic islets by opening L-VDCCs, alter intracellular Ca2+, PLC and PKC signaling pathways, characterizing KATP channel-independent pathways. These findings highlight rutin, a dietary adjuvant, as a potential insulin secretagogue contributing to glucose homeostasis.

Coxsackievirus B transmission and possible new roles for extracellular vesicles

Coxsackievirus B1, a member of the Picornaviridae family is a non-enveloped single-stranded RNA virus associated with human diseases including myocarditis and pancreatitis. Infection of the intestinal mucosa, lined by polarized epithelial cells, requires interaction of coxsackievirus with apically located DAF (decay-accelerating factor) before transport to the basolaterally located CAR (coxsackie and adenovirus receptor), where entry is mediated by endocytosis. As with many other non-enveloped viruses, coxsackievirus has to induce lysis of host cells in order to perpetuate infection. However, recent evidence indicates that virus spread to secondary sites is not only achieved by a lytic mechanism and a non-lytic cell-cell strategy has been suggested for coxsackievirus B3. A physical interaction between infected and non-infected cells has been shown to be an efficient mechanism for retroviral transmission and one type of extracellular vesicle, the exosome, has been implicated in HIV-1 transmission. HIV-1 also takes advantage of depolymerization of actin for spread between T-cells. Calpain-mediated depolymerization of the actin cytoskeleton, as a result of increases in intracellular calcium concentration during coxsackievirus infection, would result in a release of host cell-derived microvesicles. If so, we speculate that maybe such microvesicles, increasingly recognized as major vehicles mediating intercellular communication, could play a role in the intercellular transmission of non-enveloped viruses.

Rapid modulation of aromatase activity in the vertebrate brain.

Numerous steroid hormones, including 17β-estradiol (E2), activate rapid and transient cellular, physiological, and behavioral changes in addition to their well-described genomic effects. Aromatase is the key-limiting enzyme in the production of estrogens, and the rapid modulation of this enzymatic activity could produce rapid changes in local E2 concentrations. The mechanisms that might mediate such rapid enzymatic changes are not fully understood but are currently under intense scrutiny. Recent studies in our laboratory indicate that brain aromatase activity is rapidly inhibited by an increase in intracellular calcium concentration resulting from potassium-induced depolarization or from the activation of glutamatergic receptors. Phosphorylating conditions also reduce aromatase activity within minutes, and this inhibition is blocked by the addition of multiple protein kinase inhibitors. This rapid modulation of aromatase activity by phosphorylating conditions is a general mechanism observed in different cell types and tissues derived from a variety of species, including human aromatase expressed in various cell lines. Phosphorylation processes affect aromatase itself and do not involve changes in aromatase protein concentration. The control of aromatase activity by multiple kinases suggests that several amino acids must be concomitantly phosphorylated to modify enzymatic activity but site-directed mutagenesis of several amino acids alone or in combination has not to date revealed the identity of the targeted residue(s). Altogether, the phosphorylation processes affecting aromatase activity provide a new general mechanism by which the concentration of estrogens can be rapidly altered in the brain.