Release Of Calcium Stores Research Articles

The ion channel TRPV5 regulates B-cell signaling and activation.

B-cell activation triggers the release of endoplasmic reticulum calcium stores through the store-operated calcium entry (SOCE) pathway resulting in calcium influx by calcium release-activated calcium (CRAC) channels on the plasma membrane. B-cell-specific murine knockouts of SOCE do not impact humoral immunity suggesting that alternative channels may be important. We identified a member of the calcium-permeable transient receptor potential (TRP) ion channel family, TRPV5, as a candidate channel expressed in B cells by a quantitative polymerase chain reaction (qPCR) screen. To further investigate the role of TRPV5 in B-cell responses, we generated a murine TRPV5 knockout (KO) by CRISPR-Cas9. We found TRPV5 polarized to B-cell receptor (BCR) clusters upon stimulation in a PI3K-RhoA-dependent manner. TRPV5 KO mice have normal B-cell development and mature B-cell numbers. Surprisingly, calcium influx upon BCR stimulation in primary TRPV5 KO B cells was not impaired; however, differential expression of other calcium-regulating proteins, such as ORAI1, may contribute to a compensatory mechanism for calcium signaling in these cells. We demonstrate that TRPV5 KO B cells have impaired spreading and contraction in response to membrane-bound antigen. Consistent with this, TRPV5 KO B cells have reduced BCR signaling measured through phospho-tyrosine residues. Lastly, we also found that TRPV5 is important for early T-dependent antigen specific responses post-immunization. Thus, our findings identify a role for TRPV5 in BCR signaling and B-cell activation.

The Xanthine Derivative KMUP-1 Inhibits Hypoxia-Induced TRPC1 Expression and Store-Operated Ca2+ Entry in Pulmonary Arterial Smooth Muscle Cells.

Exposure to hypoxia results in the development of pulmonary arterial hypertension (PAH). An increase in the intracellular Ca2+ concentration ([Ca2+]i) in pulmonary artery smooth muscle cells (PASMCs) is a major trigger for pulmonary vasoconstriction and proliferation. This study investigated the mechanism by which KMUP-1, a xanthine derivative with phosphodiesterase inhibitory activity, inhibits hypoxia-induced canonical transient receptor potential channel 1 (TRPC1) protein overexpression and regulates [Ca2+]i through store-operated calcium channels (SOCs). Ex vivo PASMCs were cultured from Sprague-Dawley rats in a modular incubator chamber under 1% O2/5% CO2 for 24 h to elucidate TRPC1 overexpression and observe the Ca2+ release and entry. KMUP-1 (1 μM) inhibited hypoxia-induced TRPC family protein encoded for SOC overexpression, particularly TRPC1. KMUP-1 inhibition of TRPC1 protein was restored by the protein kinase G (PKG) inhibitor KT5823 (1 μM) and the protein kinase A (PKA) inhibitor KT5720 (1 μM). KMUP-1 attenuated protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA, 1 μM)-upregulated TRPC1. We suggest that the effects of KMUP-1 on TRPC1 might involve activating the cyclic guanosine monophosphate (cGMP)/PKG and cyclic adenosine monophosphate (cAMP)/PKA pathways and inhibiting the PKC pathway. We also used Fura 2-acetoxymethyl ester (Fura 2-AM, 5 μM) to measure the stored calcium release from the sarcoplasmic reticulum (SR) and calcium entry through SOCs in hypoxic PASMCs under treatment with thapsigargin (1 μM) and nifedipine (5 μM). In hypoxic conditions, store-operated calcium entry (SOCE) activity was enhanced in PASMCs, and KMUP-1 diminished this activity. In conclusion, KMUP-1 inhibited the expression of TRPC1 protein and the activity of SOC-mediated Ca2+ entry upon SR Ca2+ depletion in hypoxic PASMCs.

Investigation of platelet activation and calcium store release by a topical hemostatic xerogel dressing for improved blood clotting

AbstractAdequate topical hemostatic dressings are required as first‐line approach for reduction of uncontrolled hemorrhage, a leading cause of mortalities. Platelets play a major role during blood clotting to prevent hemorrhage during injuries. Here, we demonstrate the mechanisms activated through protease activating receptor 1 (PAR1), a platelet membrane protein in response to a porous composite xerogel dressing incorporating SiNPs (size 122 ± 10 nm) and calcium (2.5 mM), characterized by scanning electron microscopy–energy dispersive x‐ray spectroscopy and Fourier transform infrared spectroscopy. The composite displayed 13.9‐fold improved blood clotting index in comparison to commercial dressing. The composite displayed increased platelet aggregation due to development of well‐formed pseudopodia as compared to bare xerogel, SFLLRN (a thrombin mimic), adenosine di‐phosphate (a platelet activator), and heparin (a thrombin inhibitor). Further, PAR1 gene was significantly upregulated in model A549 epithelial cell line (1.2‐fold) and human platelets (1.4‐fold). The composite enhanced calcium release and its extrusion in A549 cells. Upregulation of PAR1 on the platelet surface and calcium release are crucial for platelet shape change and aggregation. The data indicate that xerogel composite containing SiNPs and calcium enhanced blood clotting through activation of PAR1. Such dressings can provide a potential hemostatic solution to reduce blood loss, disability, and mortality during surgery and trauma care.

Anti-seizures therapeutics in patients with epilepsy: an approach to levetiracetam – review

Epilepsy is a chronic condition that consists of a transient disorder of brain function manifested by recurrent and unprovoked seizures affecting approximately 50 million people worldwide. Antiepileptic treatment is aimed at possible improvement the quality of life by reducing epilepsy seizures with the lowest possible side effects. One of the newest broad spectrum antiepileptic drug (AED) for the treatment of wide range of epilepsies – focal, partial, myoclonic, tonic-clonic and primary generalized seizures that can be used in both children and adults is levetiracetam. Levetiracetam is believed to bind to SV2A (synaptic vesicle protein 2A) in the brain, prevent the release of calcium stores from neurons and inhibit GABA and glycine activity. Due to the unique mechanism of action, high bioavailability (in both oral and intravenous administration), rapid absorption, metabolism based on the hydrolysis of acetamide groups in the blood (independent of the liver) and the lack of activity of the resulting metabolites, the drug is considered to be safe and effective in reducing and stopping seizures. In this review, we present the latest reports on the effectiveness of levetiracetam and its potential in the treatment of epilepsy in children and adults.

LPA suppresses T cell function by altering the cytoskeleton and disrupting immune synapse formation

Cancer and chronic infections often increase levels of the bioactive lipid, lysophosphatidic acid (LPA), that we have demonstrated acts as an inhibitory ligand upon binding LPAR5 on CD8 T cells, suppressing cytotoxic activity and tumor control. This study, using human and mouse primary T lymphocytes, reveals how LPA disrupts antigen-specific CD8 T cell:target cell immune synapse (IS) formation and T cell function via competing for cytoskeletal regulation. Specifically, we find upon antigen-specific T cell:target cell formation, IP3R1 localizes to the IS by a process dependent on mDia1 and actin and microtubule polymerization. LPA not only inhibited IP3R1 from reaching the IS but also altered T cell receptor (TCR)–induced localization of RhoA and mDia1 impairing F-actin accumulation and altering the tubulin code. Consequently, LPA impeded calcium store release and IS-directed cytokine secretion. Thus, targeting LPA signaling in chronic inflammatory conditions may rescue T cell function and promote antiviral and antitumor immunity.

Organelle calcium-derived voltage oscillations in pacemaker neurons drive the motor program for food-seeking behavior in Aplysia.

The expression of motivated behaviors depends on both external and internally arising neural stimuli, yet the intrinsic releasing mechanisms for such variably occurring behaviors remain elusive. In isolated nervous system preparations of Aplysia, we have found that irregularly expressed cycles of motor output underlying food-seeking behavior arise from regular membrane potential oscillations of varying magnitude in an identified pair of interneurons (B63) in the bilateral buccal ganglia. This rhythmic signal, which is specific to the B63 cells, is generated by organelle-derived intracellular calcium fluxes that activate voltage-independent plasma membrane channels. The resulting voltage oscillation spreads throughout a subset of gap junction-coupled buccal network neurons and by triggering plateau potential-mediated bursts in B63, can initiate motor output driving food-seeking action. Thus, an atypical neuronal pacemaker mechanism, based on rhythmic intracellular calcium store release and intercellular propagation, can act as an autonomous intrinsic releaser for the occurrence of a motivated behavior.

Exaggerated activities of TRPM7 underlie bupivacaine-induced neurotoxicity in the SH-SY5Y cells preconditioned with high glucose.

Hyperglycemia is considered a risk factor for the enhancement of local anesthetic-induced neurotoxicity. Transient receptor potential melastatin 7 (TRPM7), a kinase-coupled cation channel, has been implicated in a variety of neuropathological processes, including intracellular calcium disturbance and high glucose-induced neuropathy. In this study, we investigated whether TRPM7-related pathophysiology is involved in bupivacaine-induced neurotoxicity in SH-SY5Y cells and how hyperglycemia acts as a risk factor. For initial neurotoxicity evaluation, it was confirmed that cell damage and apoptosis induced by acute exposure to bupivacaine were dependent on its concentration and glucose preconditioning. High glucose preconditioning facilitated the bupivacaine-induced fast and temporary rise in intracellular free calcium concentration ([Ca2+ ]i ), which was attributed to both calcium influx through TRPM7 and calcium store release. Additionally, bupivacaine was shown to increase TRPM7-like currents, particularly in cells preconditioned with high glucose. Bupivacaine-induced neurotoxicity in hyperglycemia was correlated with extracellular signal-regulated kinase (ERK), but not protein kinase B (AKT) activation. Inhibition of TRPM7 and ERK activity alleviates bupivacaine neurotoxicity. These results suggest that therapeutically targeting TRPM7-related pathophysiological changes could be a potential strategy for treating local anesthetic-induced neurotoxicity exacerbated by hyperglycemia.

Role of Internal Ca2+ Stores in KCa Channel Feedback Control of Human Uterine Contractions

Background Minute-to-minute regulation of intracellular calcium concentrations is essential to allow a laboring uterus to phasically contract and relax in a manner required for delivery. Cytosolic calcium levels increase through influx of calcium through L-type calcium channels and are amplified by SR calcium release through inositol 1, 4, 5-triphosphate receptor (IP3R) channels and calcium induced calcium release. A specific class of small conductance calcium-activated potassium (KCa2.3) channels has been strongly implicated in the negative feedback control of membrane potential and intracellular calcium levels. Uterine contractions can be suppressed by amplifying this calcium-dependent feedback system. CyPPA, a positive modulator of KCa2.3, reduces contraction amplitudes in human and rodent tissue. Previous studies of endothelial and neuronal cells suggest focused Ca2+ release from internal stores in close proximity to the plasma membrane, may play an important role in KCa channel control. Indeed, close proximity and interaction of endoplasmic reticulum Ca2+ release channels (e.g. IP3Rs) and KCa channels may regulate intracellular calcium in a variety of tissues. This study aims to assess whether internal stores contribute to KCa2.3 dependent feedback regulation of contraction in the human myometrium. Methods Myograph studies were performed on human uterine tissues collected from non-pregnant patients undergoing hysterectomy. Uterine strips were sutured end-to-end to form rings, mounted onto myograph pins, and allowed to develop spontaneous phasic contractions. Concentration-dependent suppression of contractions with the positive KCa2.3 modulator CyPPA (3 μM, 10 μM, 30 μM) was assessed following depletion of internal calcium stores (using the SERCA inhibitor cyclopiazonic acid, CPA) or following treatment with the vehicle DMSO (controls). Results Utilizing myographical recordings and LabChart software, contraction area under curve (AUC) was calculated and normalized to time. Addition of 3 μM CyPPA resulted in a control group mean AUC of 1.287 mN/min (± 0.417) and 2.261 mN/min (± 1.338) for the treatment group [n=5]. The mean AUC with 10 μM CyPPA was 1.129 mN/min (± 0.571) for the control group and 2.164 mN/min (± 1.387) for the treatment group. After 30 μM CyPPA the mean AUC for the control group was 0.734 mN/min (± 0.471) and 1.863 mN/min (± 1.364) for the treatment group. Our group found that CPA pretreated uterine tissue had a significantly higher AUC than the control group (p<0.5), after addition of 30 μM CyPPA. Further analysis demonstrated no significant difference with CyPPA concentrations of 3 and 10 μM, but did reveal a trend of reduced AUC in the control group compared to the treatment group. These findings suggest blocking SERCA and inhibiting SR reuptake of calcium may decrease CyPPAs ability to diminish contractions, especially at higher concentrations. Conclusions Phasic contractions of isolated human uterine strips vary considerably with respect to amplitude and frequency. Overall, the findings suggest internal calcium store release may play an important role in moderating uterine contractions and augmenting KCa2.3 channel suppression of uterine contractility. Extended studies will assess whether this mechanism can be exploited as an effective tocolytic strategy.

IP3R Channels in Male Reproduction.

As a second messenger in cellular signal transduction, calcium signaling extensively participates in various physiological activities, including spermatogenesis and the regulation of sperm function. Abnormal calcium signaling is highly correlated with male infertility. Calcium signaling is mainly regulated by both extracellular calcium influx and the release of calcium stores. Inositol 1,4,5-trisphosphate receptor (IP3R) is a widely expressed channel for calcium stores. After being activated by inositol 1,4,5-trisphosphate (IP3) and calcium signaling at a lower concentration, IP3R can regulate the release of Ca2+ from stores into cytoplasm, and eventually trigger downstream events. The closure of the IP3R channel caused by a rise in intracellular calcium signals and the activation of the calcium pump jointly restores the calcium store to a normal level. In this review, we aim to discuss structural features of IP3R channels and the underlying mechanism of IP3R channel-mediated calcium signaling and further focus on the research progress of IP3R expression and function in the male reproductive system. Finally, we propose key directions and strategies for research of IP3R in spermatogenesis and the regulation of sperm function to provide more understanding of the function and mechanism of IP3R channel actions in male reproduction.

Intercellular Bridge Mediates Ca2+ Signals between Micropatterned Cells via IP3 and Ca2+ Diffusion

Intercellular bridges are plasma continuities formed at the end of the cytokinesis process that facilitate intercellular mass transport between the two daughter cells. However, it remains largely unknown how the intercellular bridge mediates Ca2+ communication between postmitotic cells. In this work, we utilize BV-2 microglial cells planted on dumbbell-shaped micropatterned assemblies to resolve spatiotemporal characteristics of Ca2+ signal transfer over the intercellular bridges. With the use of such micropatterns, considerably longer and more regular intercellular bridges can be obtained than in conventional cell cultures. The initial Ca2+ signal is evoked by mechanical stimulation of one of the daughter cells. A considerable time delay is observed between the arrivals of passive Ca2+ diffusion and endogenous Ca2+ response in the intercellular-bridge-connected cell, indicating two different pathways of the Ca2+ communication. Extracellular Ca2+ and the paracrine pathway have practically no effect on the endogenous Ca2+ response, demonstrated by application of Ca2+-free medium, exogenous ATP, and P2Y13 receptor antagonist. In contrast, the endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin and inositol trisphosphate (IP3) receptor blocker 2-aminoethyl diphenylborate significantly inhibit the endogenous Ca2+ increase, which signifies involvement of IP3-sensitive calcium store release. Notably, passive Ca2+ diffusion into the connected cell can clearly be detected when IP3-sensitive calcium store release is abolished by 2-aminoethyl diphenylborate. Those observations prove that both passive Ca2+ diffusion and IP3-mediated endogenous Ca2+ response contribute to the Ca2+ increase in intercellular-bridge-connected cells. Moreover, a simulation model agreed well with the experimental observations.

The Participation of Endocannabinoid Receptors in the Regulation of Spontaneous Synaptic Activity at Neuromuscular Junctions of Mice

The mechanisms underlying changes in spontaneous release of acetylcholine (ACh) induced by exogenous agonists and antagonists of endocannabinoid receptors were investigated by recording miniature endplate potentials (MEPPs) in motor synapses of mouse diaphragm preparations with intracellular microelectrodes. There have not been observed any statistically significant changes either in the membrane potential of muscle fibers or in the frequency, amplitude, and time parameters of the MEPPs under the action of AM-251 (1 μM), antagonist/inverse agonist of CB1 receptors, for 60 min. Activation of CB receptors by their exogenous agonist WIN 55,212-2 (WIN) (20 μM) for 1 h did not cause statistically significant changes in the amplitude of the MEPPs. However, MEPP frequency increased by more than 50% as compared to the control under the action of WIN for 30–60 min. Blocking of CB1 receptors by AM-251 (1 μM) prevented the WIN-induced increase in MEPP frequency. A protein kinase A inhibitor H-89 (1 μM) did not prevent the enhancement of the ACh secretion upon activation of CB receptors by WIN. WIN lost the ability to increase MEPP frequency in the presence of a phospholipase C inhibitor U73122 (5 μM), which did not affect by itself the amplitude and temporal characteristics or MEPP frequency. A protein kinase C inhibitor, chelerythrin (4 μM) or ryanodine, which blocks the ryanodine receptors (RyRs) at a concentration of 5 μM, also prevented the WIN-induced increase in MEPP frequency. Thus, a signaling cascade triggered by stimulation of presynaptic CB1 receptors with subsequent activation of phospholipase C, protein kinase C, and the release of stored calcium, leading to an increase in the frequency of spontaneous quantal release of ACh, has been detected for the first time in mouse motor synapses. This suggests the presence of CB1 receptors on the presynaptic membrane in mouse motor nerve terminals and the possibility of activation of these receptors by endocannabinoids for potentiating the spontaneous secretion of ACh.

Successful treatment of extensive calcifications and acute pulmonary involvement in dermatomyositis with the Janus-Kinase inhibitor tofacitinib – A report of two cases

IntroductionDermatomyositis (DM) can be complicated by calcinosis and interstitial lung disease (ILD). Calcinosis can be severely debilitating or life-threatening and to date there is no treatment with proven efficacy. In DM type I interferon contributes to pathophysiology by inducing the expression of proinflammatory cytokines and the JAK-STAT (signal transducer and activator of transcription) pathway may be involved in the regulation of mitochondrial calcium store release, a process potentially important for calcification in DM. JAK-inhibition may therefore be an attractive therapy in DM complicated by calcifications. Methods and resultsWe report on the fast and persistent response of extensive and rapidly progressive DM-associated calcifications in two patients treated with the JAK-inhibitor tofacitinib. During the 28-week observation period in both patients no new calcifications formed and existing calcifications were either regressive or stable. Furthermore, concomitant life-threatening DM-associated ILD (acute fibrinous and organizing pneumonia; AFOP) in one patient rapidly responded to tofacitinib monotherapy. Both patients were able to taper concomitant glucocorticoids. Tofacitinib was well tolerated and safe. ConclusionsThe results of our study support the role of JAK/STAT signaling in the development of calcinosis and ILD in DM. Tofacitinib may be an effective and safe treatment for calcinosis in DM and potentially for other connective tissue disease complicated by calcinosis.

The chemical cue tetrabromopyrrole induces rapid cellular stress and mortality in phytoplankton

Eukaryotic phytoplankton contribute to the flow of elements through marine food webs, biogeochemical cycles, and Earth’s climate. Therefore, how phytoplankton die is a critical determinate of the flow and fate of nutrients. While heterotroph grazing and viral infection contribute to phytoplankton mortality, recent evidence suggests that bacteria-derived cues also control phytoplankton lysis. Here, we report exposure to nanomolar concentrations of 2,3,4,5-tetrabromopyrrole (TBP), a brominated chemical cue synthesized by marine γ-proteobacteria, resulted in mortality of seven phylogenetically-diverse phytoplankton species. A comparison of nine compounds of marine-origin containing a range of cyclic moieties and halogenation indicated that both a single pyrrole ring and increased bromination were most lethal to the coccolithophore, Emiliania huxleyi. TBP also rapidly induced the production of reactive oxygen species and the release of intracellular calcium stores, both of which can trigger the activation of cellular death pathways. Mining of the Ocean Gene Atlas indicated that TBP biosynthetic machinery is globally distributed throughout the water column in coastal areas. These findings suggest that bacterial cues play multiple functions in regulating phytoplankton communities by inducing biochemical changes associated with cellular death. Chemically-induced lysis by bacterial infochemicals is yet another variable that must be considered when modeling oceanic nutrient dynamics.

The Role of Endogenous Calcitonin Gene-Related Peptide in the Neurotransmitter Quantal Size Increase in Mouse Neuromuscular Junctions

Changes in parameters of spontaneous acetylcholine (ACh) quantal secretion caused by prolonged high-frequency burst activity of neuromuscular junctions and possible involvement of endogenous calcitonin gene-related peptide (CGRP) and its receptors in these changes were studied. With this purpose, miniature endplate potentials (MEPPs) were recorded using standard microelectrode technique in isolated neuromuscular preparations of m. EDL–n. peroneus after a prolonged high-frequency nerve stimulation (30 Hz for 2 min). An increase in the MEPP amplitudes and time course was observed in the postactivation period that reached maximum 20–30 min after nerve stimulation and progressively faded in the following 30 min of recording. Inhibition of vesicular ACh transporter with vesamicol (1 μM) fully prevented this “wave” of the MEPP enhancement. This indicates the presynaptic origin of the MEPP amplitude increase, possibly mediated via intensification of synaptic vesicle loading with ACh and subsequent increase of the quantal size. Competitive antagonist of the CGRP receptor, truncated peptide isoform CGRP8–37 (1 μM), had no effect on spontaneous secretion parameters by itself but was able to prevent the appearance of enhanced MEPPs in the postactivation period. This suggests the involvement of endogenous CGRP and its receptors in the observed MEPP enhancement after an intensive nerve stimulation. Ryanodine in high concentration (1 μM) that blocks ryanodine receptors and stored calcium release did not influence spontaneous ACh secretion but prevented the increase of the MEPP parameters in the postactivation period. Altogether, the data indicate that an intensive nerve stimulation, which activates neuromuscular junctions and muscle contractions, leads to a release of endogenous CGRP into synaptic cleft and this release strongly depends on the efflux of stored calcium. The released endogenous CGRP is able to exert an acute presynaptic effect on nerve terminals, which involves its specific receptor action and intracellular cascades leading to intensification of ACh loading into synaptic vesicles and an increase in the ACh quantal size.

Alpha 7 nicotinic receptors attenuate neurite development through calcium activation of calpain at the growth cone.

The α7 nicotinic acetylcholine receptor (nAChR) is a ligand-gated ion channel that plays an important role in cellular calcium signaling contributing to synaptic development and plasticity, and is a key drug target for the treatment of neurodegenerative conditions such as Alzheimer’s disease. Here we show that α7 nAChR mediated calcium signals in differentiating PC12 cells activate the proteolytic enzyme calpain leading to spectrin breakdown, microtubule retraction, and attenuation in neurite growth. Imaging in growth cones confirms that α7 activation decreases EB3 comet motility in a calcium dependent manner as demonstrated by the ability of α7 nAChR, ryanodine, or IP3 receptor antagonists to block the effect of α7 nAChR on growth. α7 nAChR mediated EB3 comet motility, spectrin breakdown, and neurite growth was also inhibited by the addition of the selective calpain blocker calpeptin and attenuated by the expression of an α7 subunit unable to bind Gαq and activate calcium store release. The findings indicate that α7 nAChRs regulate cytoskeletal dynamics through local calcium signals for calpain protease activity.

Ionotropic and Metabotropic Mechanisms of Allosteric Modulation of α7 Nicotinic Receptor Intracellular Calcium.

The pharmacological targeting of the α7 nicotinic acetylcholine receptor (α7) is a promising strategy in the development of new drugs for neurologic diseases. Because α7 receptors regulate cellular calcium, we investigated how the prototypical type II-positive allosteric modulator PNU120596 affects α7-mediated calcium signaling. Live imaging experiments show that PNU120596 augments ryanodine receptor-driven calcium-induced calcium release (CICR), inositol-induced calcium release (IICR), and phospholipase C activation by the α7 receptor. Both influx of calcium through the α7 nicotinic acetylcholine receptor (nAChR) channel as well as the binding of intracellular G proteins were involved in the effect of PNU120596 on intracellular calcium. This is evidenced by the findings that chelation of extracellular calcium, expression of α7D44A or α7345-348A mutant subunits, or blockade of calcium store release compromised the ability of PNU120596 to increase intracellular calcium transients generated by α7 ligand activation. Spatiotemporal stochastic modeling of calcium transient responses corroborates these results and indicates that α7 receptor activation enables calcium microdomains locally and to lesser extent in the distant cytosol. From the model, allosteric modulation of the receptor activates CICR locally via ryanodine receptors and augments IICR through enhanced calcium influx due to prolonged α7 nAChR opening. These findings provide a new mechanistic framework for understanding the effect of α7 receptor allosteric modulation on both local and global calcium dynamics.

Temporal reprogramming of calcium signalling via crosstalk of gonadotrophin receptors that associate as functionally asymmetric heteromers

Signal crosstalk between distinct G protein-coupled receptors (GPCRs) is one mechanism that underlies pleiotropic signalling. Such crosstalk is also pertinent for GPCRs activated by gonadotrophic hormones; follicle-stimulating hormone (FSH) and luteinising hormone (LH), with specific relevance to female reproduction. Here, we demonstrate that gonadotrophin receptor crosstalk alters LH-induced Gαq/11-calcium profiles. LH-induced calcium signals in both heterologous and primary human granulosa cells were prolonged by FSHR coexpression via influx of extracellular calcium in a receptor specific manner. LHR/FSHR crosstalk involves Gαq/11 activation as a Gαq/11 inhibitor abolished calcium responses. Interestingly, the enhanced LH-mediated calcium signalling induced by FSHR co-expression was dependent on intracellular calcium store release and involved Gβγ. Biophysical analysis of receptor and Gαq interactions indicated that ligand-dependent association between LHR and Gαq was rearranged in the presence of FSHR, enabling FSHR to closely associate with Gαq following LHR activation. This suggests that crosstalk may occur via close associations as heteromers. Super-resolution imaging revealed that LHR and FSHR formed constitutive heteromers at the plasma membrane. Intriguingly, the ratio of LHR:FSHR in heterotetramers was specifically altered following LH treatment. We propose that functionally significant FSHR/LHR crosstalk reprograms LH-mediated calcium signalling at the interface of receptor-G protein via formation of asymmetric complexes.

Linking muscarinic receptor activation to UPS-mediated object memory destabilization: Implications for long-term memory modification and storage

Consolidated memories can become destabilized during reactivation, resulting in a transient state of instability, a process that has been hypothesized to underlie long-term memory updating. Consistent with this notion, relatively remote memories, which are resistant to standard destabilization procedures, are reliably destabilized when novel information (i.e., the opportunity for memory updating) is present during reactivation. We have also shown that cholinergic muscarinic receptor (mAChR) activation can similarly destabilize consolidated object memories. Synaptic protein degradation via the ubiquitin proteasome system (UPS) has previously been linked to destabilization of fear and object-location memories. Given the role of calcium in regulating proteasome activity, we hypothesized that activation of cholinergic receptors, specifically M1 mAChRs, stimulates the UPS via inositol triphosphate receptor (IP3R)-mediated release of intracellular calcium stores to facilitate object memory destabilization. We present converging evidence for this hypothesis, which we tested using a modified spontaneous object recognition task for rats and microinfusions into perirhinal cortex (PRh), a brain region strongly implicated in object memory. We extend our previous findings by demonstrating that M1 mAChRs are necessary for novelty-induced object memory destabilization. We also show that proteasome inhibition or IP3R antagonism in PRh prevents object memory destabilization induced by novelty or M1 mAChR stimulation. These results establish an intracellular pathway linking M1 receptors, IP3Rs, and UPS activity to object memory destabilization and suggest a previously unacknowledged role for cholinergic signaling in long-term memory modification and storage.

Rodent Models of Non-classical Progesterone Action Regulating Ovulation.

It is becoming clear that steroid hormones act not only by binding to nuclear receptors that associate with specific response elements in the nucleus but also by binding to receptors on the cell membrane. In this newly discovered manner, steroid hormones can initiate intracellular signaling cascades which elicit rapid effects such as release of internal calcium stores and activation of kinases. We have learned much about the translocation and signaling of steroid hormone receptors from investigations into estrogen receptor α, which can be trafficked to, and signal from, the cell membrane. It is now clear that progesterone (P4) can also elicit effects that cannot be exclusively explained by transcriptional changes. Similar to E2 and its receptors, P4 can initiate signaling at the cell membrane, both through progesterone receptor and via a host of newly discovered membrane receptors (e.g., membrane progesterone receptors, progesterone receptor membrane components). This review discusses the parallels between neurotransmitter-like E2 action and the more recently investigated non-classical P4 signaling, in the context of reproductive behaviors in the rodent.

231 Super resolution imaging unveiling the dyadic ultrastructure in atrial and ventricular cardiomyocytes

Calcium-induced calcium release drives contraction in cardiomyocytes. Located on the sarcoplasmic reticulum, ryanodine receptors (RyR) are responsible for the release of intracellular calcium stores. It has recently become apparent that...