Cytoplasmic Free Calcium Concentration Research Articles

Sex-dependent intra-islet structural rearrangements affecting alpha-to-beta cell interactions lead to adaptive enhancements of Ca2+ dynamics in prediabetic beta cells

Aims/hypothesisPrediabetic pancreatic beta cells can adapt their function to maintain normoglycaemia for a limited period of time, after which diabetes mellitus will manifest upon beta cell exhaustion. Understanding sex-specific beta cell compensatory mechanisms and their failure in prediabetes (impaired glucose tolerance) is crucial for early disease diagnosis and individualised treatment. Our aims were as follows: (1) to determine the key time points of the progression from beta cells’ functional adaptations to their failure in vivo; and (2) to mechanistically explain in vivo sex-specific beta cell compensatory mechanisms and their failure in prediabetes.MethodsIslets from male and female transgenic Ins1CreERT2-GCaMP3 mice were transplanted into the anterior chamber of the eye of 10- to 12-week-old sex-matched C57BL/6J mice. Recipient mice were fed either a control diet (CD) or western diet (WD) for a maximum of 4 months. Metabolic variables were evaluated monthly. Beta cell cytoplasmic free calcium concentration ([Ca2+]i) dynamics were monitored in vivo longitudinally by image fluorescence of the GCaMP3 reporter islets. Global islet beta cell [Ca2+]i dynamics in line with single beta cell [Ca2+]i analysis were used for beta cell coordination studies. The glucagon receptor antagonist L-168,049 (4 mmol/l) was applied topically to the transplanted eyes to evaluate in vivo the effect of glucagon on beta cell [Ca2+]idynamics. Human islets from non-diabetic women and men were cultured for 24 h in either a control medium or high-fat/high-glucose medium in the presence or absence of the glucagon receptor antagonist L-168,049. [Ca2+]i dynamics of human islets were evaluated in vitro after 1 h exposure to Fura-10.ResultsMice fed a WD for 1 month displayed increased beta cell [Ca2+]i dynamics linked to enhanced insulin secretion as a functional compensatory mechanism in prediabetes. Recruitment of inactive beta cells in WD-fed mice explained the improved beta cell function adaptation observed in vivo; this occurred in a sex-specific manner. Mechanistically, this was attributable to an intra-islet structural rearrangement involving alpha cells. These sex-dependent cytoarchitecture reorganisations, observed in both mice and humans, induced enhanced paracrine input from adjacent alpha cells, adjusting the glucose setpoint and amplifying the insulin secretion pathway. When WD feeding was prolonged, female mice maintained the adaptive mechanism due to their intrinsically high proportion of alpha cells. In males, [Ca2+]i dynamics progressively declined subsequent to glucose stimulation while insulin secretion continue to increase, suggesting uncoordinated beta cell function as an early sign of diabetes.Conclusions/interpretationWe identified increased coordination of [Ca2+]i dynamics as a beta cell functional adaptation mechanisms in prediabetes. Importantly, we uncovered the mechanisms by which sex-dependent beta cell [Ca2+]i dynamics coordination is orchestrated by an intra-islet structure reorganisation increasing the paracrine input from alpha cells on beta cell function. Moreover, we identified reduced [Ca2+]i dynamics coordination in response to glucose as an early sign of diabetes preceding beta cell secretory dysfunction, with males being more vulnerable. Alterations in coordination capacity of [Ca2+]i dynamics may thus serve as an early marker for beta cell failure in prediabetes.Graphical

Synergistic Interaction of 5-HT1B and 5-HT2B Receptors in Cytoplasmic Ca2+ Regulation in Human Umbilical Vein Endothelial Cells: Possible Involvement in Pathologies.

The aim of this work was to explore the involvement of 5-HT1B and 5-HT2B receptors (5-HT1BR and 5-HT2BR) in the regulation of free cytoplasmic calcium concentration ([Ca2+]i) in human umbilical vein endothelial cells (HUVEC). We have shown by quantitative PCR analysis, that 5-HT1BR and 5-HT2BR mRNAs levels are almost equal in HUVEC. Immunofluorescent staining demonstrated, that 5-HT1BR and 5-HT2BR are expressed both in plasma membrane and inside the cells. Intracellular 5-HT1BR are localized mainly in the nuclear region, whereas 5-HT2BR receptors are almost evenly distributed in HUVEC. 5-HT, 5-HT1BR agonist CGS12066B, or 5-HT2BR agonist BW723C86 added to HUVEC caused a slight increase in [Ca2+]i, which was much lower than that of histamine, ATP, or SFLLRN, an agonist of protease-activated receptors (PAR1). However, activation of 5-HT1BR with CGS12066B followed by activation of 5-HT2BR with BW723C86 manifested a synergism of response, since several-fold higher rise in [Ca2+]i occurred. CGS12066B caused more than a 5-fold increase in [Ca2+]i rise in HUVEC in response to 5-HT. This 5-HT induced [Ca2+]i rise was abolished by 5-HT2BR antagonist RS127445, indicating that extracellular 5-HT acts through 5-HT2BR. Synergistic [Ca2+]i rise in response to activation of 5-HT1BR and 5-HT2BR persisted in a calcium-free medium. It was suppressed by the phospholipase C inhibitor U73122 and was not inhibited by the ryanodine and NAADP receptors antagonists dantrolene and NED-19. [Ca2+]i measurements in single cells demonstrated that activation of 5-HT2BR alone by BW723C86 caused single asynchronous [Ca2+]i oscillations in 19.8 ± 4.2% (n = 3) of HUVEC that occur with a long delay (66.1 ± 4.3 s, n = 71). On the contrary, histamine causes a simultaneous and almost immediate increase in [Ca2+]i in all the cells. Pre-activation of 5-HT1BR by CGS12066B led to a 3-4 fold increase in the number of HUVEC responding to BW723C86, to synchronization of their responses with a delay shortening, and to the bursts of [Ca2+]i oscillations in addition to single oscillations. In conclusion, to get a full rise of [Ca2+]i in HUVEC in response to 5-HT, simultaneous activation of 5-HT1BR and 5-HT2BR is required. 5-HT causes an increase in [Ca2+]i via 5-HT2BR while 5-HT1BR could be activated by the membrane-permeable agonist CGS12066B. We hypothesized that CGS12066B acts via intracellular 5-HT1BR inaccessible to extracellular 5-HT. Intracellular 5-HT1BR might be activated by 5-HT which could be accumulated in EC under certain pathological conditions.

Effect of Buffers with Multiple Binding Sites on Calcium Waves

The existence and properties of intracellular waves of increased free cytoplasmic calcium concentration (calcium waves) are strongly affected by the binding and unbinding of calcium ions to a multitude of different buffers in the cell. These buffers can be mobile or immobile and, in general, have multiple binding sites that are not independent. Previous theoretical studies have focused on the case when each buffer molecule binds a single calcium ion. In this study, we analyze how calcium waves are affected by calcium buffers with two non-independent binding sites, and show that the interactions between the calcium binding sites can result in the emergence of new behaviors. In particular, for certain combinations of kinetic parameters, the profiles of buffer molecules with one calcium ion bound can be non-monotone.

Genome-Wide Identification and Expression Profile of OSCA Gene Family Members in Triticum aestivum L.

Hyperosmolality and various other stimuli can trigger an increase in cytoplasmic-free calcium concentration ([Ca2+]cyt). Members of the Arabidopsis thaliana (L.) reduced hyperosmolality-gated calcium-permeable channels (OSCA) gene family are reported to be involved in sensing extracellular changes to trigger hyperosmolality-induced [Ca2+]cyt increases and controlling stomatal closure during immune signaling. Wheat (Triticum aestivum L.) is a very important food crop, but there are few studies of its OSCA gene family members. In this study, 42 OSCA members were identified in the wheat genome, and phylogenetic analysis can divide them into four clades. The members of each clade have similar gene structures, conserved motifs, and domains. TaOSCA genes were predicted to be regulated by cis-acting elements such as STRE, MBS, DRE1, ABRE, etc. Quantitative PCR results showed that they have different expression patterns in different tissues. The expression profiles of 15 selected TaOSCAs were examined after PEG (polyethylene glycol), NaCl, and ABA (abscisic acid) treatment. All 15 TaOSCA members responded to PEG treatment, while TaOSCA12/-39 responded simultaneously to PEG and ABA. This study informs research into the biological function and evolution of TaOSCA and lays the foundation for the breeding and genetic improvement of wheat.

Effect of WSSV infection on cell cycle regulation, respiratory burst and cytoplasmic free calcium concentration in Penaeus vannamei

Effect of WSSV infection on cell cycle regulation, respiratory burst and cytoplasmic free calcium concentration in <i>Penaeus vannamei</i>

The Role of Two-Pore Channels in Norepinephrine-Induced [Ca2+]i Rise in Rat Aortic Smooth Muscle Cells and Aorta Contraction.

Second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) triggers Ca2+ release via two-pore channels (TPCs) localized in endolysosomal vesicles. The aim of the present work is to evaluate the role of TPCs in the action of norepinephrine (NE), angiotensin II (AngII), vasopressin (AVP), and 5-hydroxytriptamine (5-HT) on free cytoplasmic calcium concentration ([Ca2+]i) in smooth muscle cells (SMCs) isolated from rat aorta and on aorta contraction. To address this issue, the NAADP structural analogue and inhibitor of TPCs, NED 19, was applied. We have demonstrated a high degree of colocalization of the fluorescent signals of cis-NED 19 and endolysosmal probe LysoTracker in SMCs. Both cis- or trans-NED 19 inhibited the rise of [Ca2+]i in SMCs induced by 100 μM NE by 50–60%. IC50 for cis- and trans-NED 19 were 2.7 and 8.9 μM, respectively. The inhibition by NED 19 stereoisomers of the effects of AngII, AVP, and 5-HT was much weaker. Both forms of NED 19 caused relaxation of aortic rings preconstricted by NE, with relative potency of cis-NED 19 several times higher than that of trans-NED 19. Inhibition by cis-NED 19 of NE-induced contraction was maintained after intensive washing and slowly reversed within an hour of incubation. Cis- and trans-NED 19 did not cause decrease in the force of aorta contraction in response to Ang II and AVP, and only slightly relaxed aorta preconstricted by 5-HT and by KCl. Suppression of TPC1 in SMCs with siRNA caused a 40% decrease in [Ca2+]i in response to NE, whereas siRNA against TPC2 did not change NE calcium signaling. These data suggest that TPC1 is involved in the NE-stimulated [Ca2+]i rise in SMCs. Inhibition of TPC1 activity by NED 19 could be the reason for partial inhibition of aortic rings contraction in response to NE.

VAS2870 Inhibits Histamine-Induced Calcium Signaling and vWF Secretion in Human Umbilical Vein Endothelial Cells.

In this study, we investigated the effects of NAD(P)H oxidase (NOX) inhibitor VAS2870 (3-benzyl-7-(2-benzoxazolyl)thio-1,2,3-triazolo[4,5-d]pyrimidine) on the histamine-induced elevation of free cytoplasmic calcium concentration ([Ca2+]i) and the secretion of von Willebrand factor (vWF) in human umbilical vein endothelial cells (HUVECs) and on relaxation of rat aorta in response to histamine. At 10 μM concentration, VAS2870 suppressed the [Ca2+]i rise induced by histamine. Inhibition was not competitive, with IC50 3.64 and 3.22 μM at 1 and 100 μM concentrations of histamine, respectively. There was no inhibition of [Ca2+]i elevation by VAS2870 in HUVECs in response to the agonist of type 1 protease-activated receptor SFLLRN. VAS2870 attenuated histamine-induced secretion of vWF and did not inhibit basal secretion. VAS2870 did not change the degree of histamine-induced relaxation of rat aortic rings constricted by norepinephrine. We suggest that NOX inhibitors might be used as a tool for preventing thrombosis induced by histamine release from mast cells without affecting vasorelaxation.

The biphasic effect of extracellular glucose concentration on carbachol-induced fluid secretion from mouse submandibular glands.

Cholinergic agonists evoke elevations of the cytoplasmic free-calcium concentration ([Ca2+ ]i ) to stimulate fluid secretion in salivary glands. Salivary flow rates are significantly reduced in diabetic patients. However, it remains elusive how salivary secretion is impaired in diabetes. Here, we used an exvivo submandibular gland perfusion technique to characterize the dependency of salivary flow rates on extracellular glucose concentration and activities of glucose transporters expressed in the glands. The cholinergic agonist carbachol (CCh) induced sustained fluid secretion, the rates of which were modulated by the extracellular glucose concentration in a biphasic manner. Both lowering the extracellular glucose concentration to less than 2.5mM and elevating it to higher than 5mM resulted in decreased CCh-induced fluid secretion. The CCh-induced salivary flow was suppressed by phlorizin, an inhibitor of the sodium-glucose cotransporter 1 (SGLT1) located basolaterally in submandibular acinar cells, which is altered at the protein expression level in diabetic animal models. Our data suggest that SGLT1-mediated glucose uptake in acinar cells is required to maintain the fluid secretion by sustaining Cl- secretion in real-time. High extracellular glucose levels may suppress the CCh-induced secretion of salivary fluid by altering the activities of ion channels and transporters downstream of [Ca2+ ]i signals.

Silence of inositol 1,4,5-trisphosphate receptor expression decreases cyantraniliprole susceptibility in Bemisia tabaci

Cyantraniliprole is the second active ingredient of anthranilic diamide insecticide, and the first to control a cross-spectrum of chewing and sucking pests such as sweetpotato whitefly, Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae). The inositol 1,4,5-trisphosphate receptor (IP3R) and ryanodine receptor (RyR) are two families of Ca2+ release channels to raise the cytoplasmic free calcium concentration when it is activated by various extracellular stimuli. Previous study proved the over-expression of ryanodine receptor (RyR) was associated with the resistance to diamide insecticides, while the roles of IP3R in diamide resistance remain unknown. In this study, a full-length cDNA sequence of IP3R was cloned from B. tabaci through RT-PCR and rapid amplification of cDNA ends (RACE). The gene (named BtIP3R) is 9922bps long, with an open reading frame (ORF) of 8202bps, encoding a predicted IP3R of 2733 amino acids. The BtIP3R shares 47–78% identity with other insect IP3Rs. Quantitative real-time PCR (qRT-PCR) analysis showed that the BtIP3R was highly expressed in larva, pseudopupa, and female adult, while lowly expressed in egg and male adult. RNA interference (RNAi) by dietary introduction of double-stranded RNA (dsRNA) of BtIP3R significantly reduced the mRNA levels of the target gene in the adult, and dramatically decreased the susceptibility of adult B. tabaci to cyantraniliprole. The results shed light on further understanding of cyantraniliprole resistance mechanisms in B. tabaci as well as in other insects.

Suppression of histamine-induced relaxation of rat aorta and calcium signaling in endothelial cells by two-pore channel blocker trans-NED19 and hydrogen peroxide

The blocker of two-pore channels trans-NED 19 and hydrogen peroxide were found to inhibit histamine-induced relaxation of rat-aorta. The degree of inhibition depended on histamine concentration. The relaxation in response to I μM histamine of rat aorta preconstricted with 30 mM KCI, serotonin, or endothelin- 1, was completely abolished by 30 μM trans-NED 19. On the other hand, trans-NED 19 decreased the relaxation of the aorta in the presence of 10 μM histamine only by 2.1-fold to 2.4-fold, and there was almost no inhibition by trans-NED 19 of the relaxationinduced by 100 ptM histamine.) Relaxation of precontracted with serotonin aorta in response to 10 and 100 μM histamine was reduced by hydrogen peroxide (200 M) by 10- and 2.5-fold, respectively. Suppression of aorta relaxation by trans-NED 19 and H202 correlated with their inhibitory effect on the histamine-induced increase in the cytoplasmic free calcium concentration in human umbilical vein endothelial cells. With the use of a fluorescent probe LysoTracker, the cis-NED19 binding sites were demonstrated to be localized in endolysosomes of the endothelial cells. These data indicate that two-pore calcium channels participate in the histamine-induced endothelium-dependent relaxation of rat aorta. Furthermore, their functional role is exhibited much more clearly at low histamine concentrations. We suggest that hydrogen peroxide evokes depletion of intracellular calcium depots thereby suppressing the response to histamine.

The CRaZy Calcium Cycle.

Calcium is an essential cation for a cell. This cation participates in the regulation of numerous processes in either prokaryotes or eukaryotes, from bacteria to humans. Saccharomyces cerevisiae has served as a model organism to understand calcium homeostasis and calcium-dependent signaling in fungi. In this chapter it will be reviewed known and predicted transport mechanisms that mediate calcium homeostasis in the yeast. How and when calcium enters the cell, how and where it is stored, when is reutilized, and finally secreted to the environment to close the cycle. As a second messenger, maintenance of a controlled free intracellular calcium concentration is important for mediating transcriptional regulation. Many environmental stimuli modify the concentration of cytoplasmic free calcium generating the "calcium signal". This is sensed and transduced through the calmodulin/calcineurin pathway to a transcription factor, named calcineurin-responsive zinc finger, CRZ, also known as "crazy", to mediate transcriptional regulation of a large number of genes of diverse pathways including a negative feedback regulation of the calcium homeostasis system.

TRPV5/V6 Channels Mediate Ca(2+) Influx in Jurkat T Cells Under the Control of Extracellular pH.

Regulation of cytoplasmic free calcium concentration [Ca(2+)]i is a key factor for the maintenance of cellular homeostasis in different cell types, including lymphocytes. During T lymphocyte activation as well as production of cytokines, sustained Ca(2+) influx is essential, however, it remains unclear how this influx is regulated. Previously, we reported the expression and functional activity of calcium channels TRPV5 and TRPV6 (transient receptor potential vanilloid type 5 and 6) in human leukemia Jurkat T cells. In this study, using single channel recordings, we found that activity of calcium channels TRPV5/V6 in Jurkat T cells is subject to strong control of external stimuli such as a low- or high-pH stressor. We showed that extracellular acidic pH reduces the activity of TRPV5/V6 channels, whereas alkaline pH increases the activity of TRPV5/V6 channels in Jurkat T cells. Using calcium imaging, we found that Ca(2+) influx in Jurkat T cells displayed sensitivity to extracellular pH, similar to that shown for the calcium channels TRPV5/V6. Double immunostaining of Jurkat T cells revealed that TRPV5 and TRPV6 channels colocalize with clathrin and the early endocytosis marker, EEA1. Moreover, we demonstrated that a specific inhibitor of clathrin-dependent endocytosis, dynasore, blocked TRPV5/V6 activity, and Ca(2+) influx into Jurkat T cells. Overall, our findings indicate that strong environmental cues may affect the intracellular calcium level in Jurkat T cells by influencing the traffic of TRPV5/V6 channels in lymphocytes.

Enhanced Membrane Permeability of &lt;i&gt;Saccharomyces ce&lt;/i&gt;&lt;i&gt;revisiae&lt;/i&gt; Induced by Dielectric Barrier Discharge Plasma at Atmospheric Pressure

In this study, we investigated the effect of DBD plasma at atmospheric pressure on membrane permeability, potential membrane and cytoplasmic free calcium concentration inSaccharomyces cerevisiae. S. cerevisiaecells were treated with DBD plasma for 0, 1, 2, 3, 4 and 5 min and then subjected to various analyses before the commencement of fermentation or at the middle (9 h) and end (21 h) stages of fermentation. The results show that compared to non-treated cells, cells treated with plasma for 2-5 min show significant increase in membrane potential, while cells treated for 2-4 min show significant increase in membrane permeability prior to fermentation. Plasma-treated cells also show increased cytoplasmic free Ca2+that depends on plasma treatment time, with the highest increase, 35%, observed for 5-min treatment time. However, at the middle (9 h) and end (21 h) stages of fermentation, these changes become insignificant compared to the control.

Arachidonic acid activates release of calcium ions from reticulum via ryanodine receptor channels in C2C12 skeletal myotubes.

Arachidonic acid causes an increase in free cytoplasmic calcium concentration ([Ca2+]i) in differentiated skeletal multinucleated myotubes C2C12 and does not induce calcium response in C2C12 myoblasts. The same reaction of myotubes to arachidonic acid is observed in Ca2+-free medium. This indicates that arachidonic acid induces release of calcium ions from intracellular stores. The blocker of ryanodine receptor channels of sarcoplasmic reticulum dantrolene (20µM) inhibits this effect by 68.7 ± 6.3% (p < 0.001). The inhibitor of two-pore calcium channels of endolysosomal vesicles trans-NED19 (10µM) decreases the response to arachidonic acid by 35.8 ± 5.4% (p < 0.05). The phospholipase C inhibitor U73122 (10µM) has no effect. These data indicate the involvement of ryanodine receptor calcium channels of sarcoplasmic reticulum in [Ca2+]i elevation in skeletal myotubes caused by arachidonic acid and possible participation of two-pore calcium channels from endolysosomal vesicles in this process.

Stimulation of NOX2 in isolated hearts reversibly sensitizes RyR2 channels to activation by cytoplasmic calcium

The response of ryanodine receptor (RyR) channels to cytoplasmic free calcium concentration ([Ca2+]) is redox sensitive. Here, we report the effects of a mild oxidative stress on cardiac RyR (RyR2) channels in Langendorff perfused rat hearts. Single RyR2 channels from control ventricles displayed the same three responses to Ca2+ reported in other mammalian tissues, characterized by low, moderate, or high maximal activation. A single episode of 5min of global ischemia, followed by 1min of reperfusion, enhanced 2.3-fold the activity of NOX2 compared to controls and changed the frequency distribution of the different responses of RyR2 channels to calcium, favoring the more active ones: high activity response increased and low activity response decreased with respect to controls. This change was fully prevented by perfusion with apocynin or VAS 2870 before ischemia and totally reversed by the extension of the reperfusion period to 15min. In vitro activation of NOX2 in control SR vesicles mimicked the effect of the ischemia/reperfusion episode on the frequencies of emergence of single RyR2 channel responses to [Ca2+] and increased 2.2-fold the rate of calcium release in Ca2+-loaded SR vesicles. In vitro changes were reversed at the single channel level by DTT and in isolated SR vesicles by glutaredoxin. Our results indicate that in whole hearts a mild oxidative stress enhances the response of cardiac RyR2 channels to calcium via NOX2 activation, probably by S-glutathionylation of RyR2 protein. This change is transitory and fully reversible, suggesting a possible role of redox modification in the physiological response of cardiac RyR2 to cellular calcium influx.

Modulation of monosodium urate crystal-induced responses in neutrophils by the myeloid inhibitory C-type lectin-like receptor: potential therapeutic implications

IntroductionMonosodium urate crystals (MSU), the etiological agent of gout, are one of the most potent proinflammatory stimuli for neutrophils. The modulation of MSU-induced neutrophil activation by inhibitory receptors remains poorly characterized. The expression of the myeloid inhibitory C-type lectin-like receptor (MICL) in neutrophils is downregulated by several proinflammatory stimuli, suggestive of a role for this receptor in neutrophil function. We thus investigated the potential role of MICL in MSU-induced neutrophil activation.MethodsThe expression of MICL was monitored in human neutrophils by flow cytometry and Western blot analysis after stimulation with MSU. Protein tyrosine phosphorylation was also assessed by Western blot analysis and the production of IL-1 and IL-8 by enzyme-linked immunosorbent assay. Changes in the concentration of cytoplasmic free calcium were monitored with the Fura-2-acetoxymethyl ester calcium indicator. MICL expression was modulated with an anti-MICL antibody in neutrophils and siRNA in the PLB-985 neutrophil-like cell line.ResultsMSU induced the downregulation of MICL expression in neutrophils. A diminution in the expression of MICL induced by antibody cross-linking or siRNA enhanced the MSU-dependent increase in cytoplasmic calcium levels, protein tyrosine phosphorylation and IL-8 but not IL-1 production. Pretreatment of neutrophils with colchicine inhibited the MSU-induced downregulation of MICL expression.ConclusionsOur findings strongly suggest that MICL acts as an inhibitory receptor in human neutrophils since the downregulation of MICL expression enhances MSU-induced neutrophil activation. Since MSU downregulates the expression of MICL, MICL may play a pathogenic role in gout by enhancing neutrophil effector functions. In support of this notion, colchicine counteracts the MSU-induced loss of MICL expression. Our findings thus also provide further insight into the potential molecular mechanisms behind the anti-inflammatory properties of this drug.

Neurosteroids, trigger of the LH surge

Recent experiments from our laboratory are consistent with the idea that hypothalamic astrocytes are critical components of the central nervous system (CNS) mediated estrogen positive feedback mechanism. The “astrocrine hypothesis” maintains that ovarian estradiol rapidly increases free cytoplasmic calcium concentrations ([Ca2+]i) that facilitate progesterone synthesis in astrocytes. This hypothalamic neuroprogesterone along with the elevated estrogen from the ovaries allows for the surge release of gonadotropin-releasing hormone (GnRH) that triggers the pituitary luteinizing hormone (LH) surge. A narrow range of estradiol stimulated progesterone production supports an “off-on-off” mechanism regulating the transition from estrogen negative feedback to estrogen positive feedback, and back again. The rapidity of the [Ca2+]i response and progesterone synthesis support a non-genomic, membrane-initiated signaling mechanism. In hypothalamic astrocytes, membrane-associated estrogen receptors (mERs) signal through transactivation of the metabotropic glutamate receptor type 1a (mGluR1a), implying that astrocytic function is influenced by surrounding glutamatergic nerve terminals. Although other putative mERs, such as mERβ, STX-activated mER-Gαq, and G protein-coupled receptor 30 (GPR30), are present and participate in membrane-mediated signaling, their influence in reproduction is still obscure since female reproduction be it estrogen positive feedback or lordosis behavior requires mERα. The astrocrine hypothesis is also consistent with the well-known sexual dimorphism of estrogen positive feedback. In rodents, only post-pubertal females exhibit this positive feedback. Hypothalamic astrocytes cultured from females, but not males, responded to estradiol by increasing progesterone synthesis. Estrogen autoregulates its own signaling by regulating levels of mERα in the plasma membrane of female astrocytes. In male astrocytes, the estradiol-induced increase in mERα was attenuated, suggesting that membrane-initiated estradiol signaling (MIES) would also be blunted. Indeed, estradiol induced [Ca2+]i release in male astrocytes, but not to levels required to stimulate progesterone synthesis. Investigation of this sexual differentiation was performed using hypothalamic astrocytes from post-pubertal four core genotype (FCG) mice. In this model, genetic sex is uncoupled from gonadal sex. We demonstrated that animals that developed testes (XYM and XXM) lacked estrogen positive feedback, strongly suggesting that the sexual differentiation of progesterone synthesis is driven by the sex steroid environment during early development.This article is part of a Special Issue entitled ‘Neurosteroids’.

Acute stimulation of glucagon secretion by linoleic acid results from GPR40 activation and [Ca2+]i increase in pancreatic islet α-cells

The role of free fatty acids (FFAs) in glucagon secretion has not been well established, and the involvement of FFA receptor GPR40 and its downstream signaling pathways in regulating glucagon secretion are rarely demonstrated. In this study, it was found that linoleic acid (LA) acutely stimulated glucagon secretion from primary cultured rat pancreatic islets. LA at 20 and 40 μmol/l dose-dependently increased glucagon secretion both at 3 mmol/l glucose and at 15 mmol/l glucose, although 15 mmol/l glucose reduced basal glucagon levels. LA induced an increase in cytoplasmic free calcium concentrations ([Ca(2)(+)](i)) in identified rat α-cells, which is reflected by increased Fluo-3 intensity under confocal microscopy recording. The increase in [Ca(2)(+)](i) was partly inhibited by removal of extracellular Ca(2)(+) and eliminated overall by further exhaustion of intracellular Ca(2)(+) stores using thapsigargin treatment, suggesting that both Ca(2)(+) release and Ca(2)(+) influx contributed to the LA-stimulated increase in [Ca(2)(+)](i) in α-cells. Double immunocytochemical stainings showed that GPR40 was expressed in glucagon-positive α-cells. LA-stimulated increase in [Ca(2)(+)](i) was blocked by inhibition of GPR40 expression in α-cells after GPR40-specific antisense treatment. The inhibition of phospholipase C activity by U73122 also blocked the increase in [Ca(2)(+)](i) by LA. It is concluded that LA activates GPR40 and phospholipase C (and downstream signaling pathways) to increase Ca(2)(+) release and associated Ca(2)(+) influx through Ca(2)(+) channels, resulting in increase in [Ca(2)(+)](i) and glucagon secretion.

Activation of “silent” vasoconstrictive 5-HT1A receptors as a possible mechanism of synergism in angiotensin II and serotonin effect on vascular tone

It has been shown that the agonist of 5HT1A-receptors 8-OH-DPAT induces contraction of aortic rings in the presence of angiotensin II. This effect is not associated with activation of alpha1-adrenoceptors by 8-OH-DPAT as it is reproduced in the presence of prazosin which completely suppresses the nonspecific vasoconstrictive effect of 8-OH-DPAT via alpha1-adrenoceptors on the aorta incubated without angiotensin II. Synergism in the action of angiotensin II and 8-OH-DPAT is completely preserved after partial desensitization of the receptors of angiotensin II. It has been found that 8-OH-DPAT increases the free cytoplasmic calcium concentration in cultured smooth muscle cells from the rat aorta. The data obtained support the hypothesis about the existence of "silent" vasoconstrictive 5HT1A-receptors. It has been suggested that activation of these receptors underlies synergism in vasoconstrictive action of serotonin and angiotensin II.

Elevation of the expression of the inositol-1,4,5-trisphosphate receptors and increase of calcium signaling in response to ATP during differentiation of skeletal myoblasts C2C12 into myotubes

The expression levels of inositol-1,4,5-trisphosphate receptors (InsP3R) and cytoplasmic free calcium concentration ([Ca2+]c) changes in response to ATP in cultured skeletal myoblasts C2C12, in myoblasts at different stages of differentiation into myotubes, and in differentiated multinucleated myotubes have been determined. Using reverse transcription and real-time polymerase chain reaction we have shown that during differentiation of myoblasts into myotubes the expression of all three InsP3R increased 2–2.5-fold and at all stages of differentiation the expression levels of InsP3R2 and InsP3R3 were nearly equal to each other and 1.5–2 times higher than the expression level of InsP3R1. The kinetics of calcium signal in myotubes is characterized by higher maximal [Ca2+]c rise and more prolonged second phase in comparison with myoblasts. The data obtained provide evidence in favor of the increasing role of InsP3-dependent calcium mobilization at the first stages of skeletal muscles maturation.