IP3 Levels Research Articles

Impacts of exposure to and subsequent discontinuation of clozapine on tripartite synaptic transmission.

Clozapine is an effective antipsychotic for treatment-resistant schizophrenia, but its discontinuation leads to discontinuation syndrome/catatonia complicated by benzodiazepine-resistance and rhabdomyolysis. This study determined time-dependent effects of exposure and subsequent discontinuation of clozapine on expression of connexin43, 5-HT receptors, intracellular L-β-aminoisobutyrate (L-BAIBA) and 2nd-messengers and signalling of AMPK, PP2A and Akt in cultured astrocytes and rat frontal cortex. Intracellular L-BAIBA levels increased during clozapine exposure but immediately recovered after discontinuation. Both exposure to clozapine and L-BAIBA increased connexin43 and signalling of AMPK/Akt time-dependently, but reduced PP2A signalling, 5-HT receptor expression and IP3 level. These changes recovered within 2 weeks after discontinuation, while 5-HT receptors and IP3 transiently increased during the recovery process. L-BAIBA activated AMPK signalling, leading to attenuated PP2A signalling. Astroglial D-serine release was increased by clozapine exposure but continued to increase within 1 week after discontinuation via activation of IP3 receptor function. Clozapine discontinuation restored PP2A signalling due to decreased L-BAIBA, increased 5-HT receptor expression via probably enhanced 5-HT receptor recycling, but increased astroglial D-serine release persisted by transiently activated IP3 receptors via transiently increased IP3 level. Decreased L-BAIBA caused by clozapine discontinuation is, at least partially, involved in the transiently increased 5-HT receptor and astroglial D-serine release.

In vitro effect of diazoxon on cell signaling and second messengers in Nile tilapia (Oreochromis niloticus) leukocytes.

The physiological and molecular responses of leukocytes are altered by organophosphate pesticides (OPs). Some reports have shown that diazinon (DZN) causes immunotoxic effects; diazoxon (DZO), the oxon metabolite of DZN, is attributed to influence the immune response by affecting the leukocyte cholinergic system. In this study, the in vitro effects of DZO on molecules involved in cell signaling (cAMP, IP3, DAG, JAK1, and STAT3), which play a crucial role in the activation, differentiation, and survival of leukocytes, were evaluated. Data indicate that DZO leads to a decrease in cAMP concentration and an increase in basal IP3 levels. However, DZO does not affect basal levels of JAK1 and STAT3 phosphorylation. Instead, DZO inhibits leukocyte responsiveness to PMA and ionomycin, substances that, under normal conditions, enhance JAK/STAT signaling. These findings demonstrate that DZO significantly affects key molecular parameters related to cell signaling.

Computational model of interacting system dynamics of calcium, IP3 and β-amyloid in ischemic neuron cells

The study of single independent dynamics of calcium ([Ca2+]), IP3, and β-amyloid (Aβ) in neurons provide limited information. Some attempts are reported for the dynamics of two interacting systems of [Ca2+] and IP3, and calcium and Aβ, which gave some novel insights about the phenomena. However, the interactions of these three systems have not been analyzed till date in neurons. Therefore, a novel model is constructed to study the interactions of the spatiotemporal systems of [Ca2+], IP3, and Aβ in neurons. A two-way feedback mechanism between [Ca2+] and IP3, and [Ca2+] and Aβ has been incorporated into the model. The model is formulated by coupling three reaction-diffusion equations of [Ca2+], IP3 and Aβ, respectively. This coupling automatically takes care of the indirect two-way feedback process between IP3 and β-amyloid in neuron cells. The finite element method (FEM) with the Crank-Nicolson scheme (CNS) is utilized to study the contribution of various ER-associated processes like RyR, IP3R, SERCA pump, buffer approximation, etc on the neuronal interactions of [Ca2+], IP3, and β-amyloid during Ischemia. The numerical findings provide novel insights into alterations in ER handling during Ischemia, resulting in disturbances in the neuronal calcium, IP3, and Aβ levels, which may cause the advancement of Alzheimer’s illness and be responsible for neurotoxicity and cell death.

Insight into the increased risk associated with CRISPR-generated African G6PD variant (N126D) in rat model of sugen-5416-induced pulmonary hypertension

Pulmonary Hypertension (PH) is a cardiopulmonary disease associated with sustained increase in pulmonary arterial and right ventricular pressures, resulting in smooth muscle cell remodeling and the thickening of vasculature. Glucose-6-phosphate dehydrogenase (G6PD) is the rate limiting enzyme in the pentose phosphate pathway and is often associated with oxidative stress and the pulmonary vascular remodeling mechanism seen in PH. CRISPR-mediated single nucleotide polymorphism modeling was used to generate the African G6PD variant (N126D; G6PDN126D), which is an X-linked gene mutation resulting in a 20% decrease in G6PD activity and subsequent hypothesized increased risk of cardiovascular diseases for sub-Saharan individuals. The goal of this study was to determine the effects of the G6PDN126D variant using the Sugen-5416 (20 mg/kg in DMSO)/Normoxia (SU/NX)-induced PH rat model. Cardiac catheterization determined that the G6PDN126D variant-SU/Nx group had higher (G6PDN126D: 29.3 ± 5.2 vs WT: 19.5 ± 2.2; mmHg; p<0.05) mean right ventricle pressure and pulmonary artery remodeling as compared to the WT-SU/Nx group. Further, G6PDN126D variant-SU/Nx had lower nitric oxide levels (G6PDN126D: 10.6 vs WT: 13.6; mM/mg protein; p<0.05) and higher second messenger IP3 levels (G6PDN126D: 4.00 vs WT: 0.52; ng/mg protein; p<0.05) as compared to the WT-SU/Nx group. The data suggests that the African G6PD (N126D) mutation results in increased PH in rats and may be associated with increased risks of severe PH in the sub-Saharan population. These results imply more studies are required to develop personalized medicine for the treatment of PH. RO1HL132574 (SAG), RO1HL166546 (SAG), AHA Grant-in-Aid 17GRNT33670454 (SAG) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Electroacupuncture preconditioning relieves cutaneous passive anaphylaxis by down-regulating IP3 mediated ROS/TRPM2 signaling pathway and inhibiting mast cell degranulation in rats with urticaria

To observe the effect of electroacupuncture (EA) preconditioning of "Quchi" (LI11) and "Xuehai" (SP10) on mast cell (MC) degranulation, and expressions of inositol triphosphate(IP3), reactive oxygen species (ROS), transient receptor potential (TRP) M2, calmodulin (CaM) in rats with urticaria, so as to reveal its molecular mechanism under-lying improving urticaria. Thirty-two male SD rats were randomly divided into blank control, model, preconditioning of EA (Pre-EA) and medication groups (n=8 rats/group). The urticaria model was established by intradermal injection of dilute allogeneic antioalbumin serum at the spots of the bilateral symmetry of the spine on the back, and followed by tail venous injection of mixture solution of egg albumin diluent, plus 0.5% Evans blue and normal saline. Ten days before the end of modeling, rats of the pre-EA group received EA stimulation of LI11 and SP10 for 20 min, once a day for 10 consecutive days, and those of the medication group received gavage of loratadine tablets diluted solution (1 mg/kg) once a day for 10 days. The times of rat's scratching the sensitized skin were recorded, the diameter of the sensitized blue spots was measured and the degranulation rate of skin MCs was counted under microscope after toluidine blue staining. The expression levels of IP3, ROS, TRPM2 and CaM in the skin tissue were measured by immunohistochemistry and western blot, respectively. Compared with the blank control group, the scratching times, diameter of the sensitized blue spots, degranulation rate of MCs, and the expression levels of ion channel related proteins (IP3, ROS, TRPM2 and CaM) were significantly increased (P<0.01) in the model group. In comparison with the model group, the scratching times, diameter of sensitized blue spot, degranulation rate of MCs, and the expression levels of IP3, ROS, TRPM2 and CaM in both pre-EA and medication groups were significantly down-regulated (P<0.01, P<0.05). No significant differences were found between Pre-EA and medication groups in down-regulating the levels of the above-mentioned 7 indexes. EA-LI11 and SP10 preconditioning can reduce the cutaneous anaphylaxis in urticaria rats, which may be related to its effects in inhibiting the degranulation of MCs, and the expression of TRP channel related proteins.

Inositol hexaphosphate promotes intestinal adaptation in short bowel syndrome via an HDAC3-mediated epigenetic pathway.

Short bowel syndrome (SBS) has high morbidity and mortality rates, and promoting intestinal adaptation of the residual intestine is a critical treatment. Dietary inositol hexaphosphate (IP6) plays an important role in maintaining intestinal homeostasis, but its effect on SBS remains unclear. This study aimed at investigating the effect of IP6 on SBS and clarified its underlying mechanism. Forty male Sprague-Dawley rats (3-week-old) were randomly assigned into four groups (Sham, Sham + IP6, SBS, and SBS + IP6 groups). Rats were fed standard pelleted rat chow and underwent resection of 75% of the small intestine after 1 week of acclimation. They received 1 mL IP6 treatment (2 mg/g) or sterile water daily for 13 days by gavage. Intestinal length, levels of inositol 1,4,5-trisphosphate (IP3), histone deacetylase 3 (HDAC3) activity, and proliferation of intestinal epithelial cell-6 (IEC-6) were detected. IP6 treatment increased the length of the residual intestine in rats with SBS. Furthermore, IP6 treatment caused an increase in body weight, intestinal mucosal weight, and IEC proliferation, and a decrease in intestinal permeability. IP6 treatment led to higher levels of IP3 in feces and serum, and higher HDAC3 activity of the intestine. Interestingly, HDAC3 activity was positively correlated with the levels of IP3 in feces (r = 0.49, P = 0.01) and serum (r = 0.44, P = 0.03). Consistently, IP3 treatment promoted the proliferation of IEC-6 cells by increasing HDAC3 activity in vitro. IP3 regulated the Forkhead box O3 (FOXO3)/Cyclin D1 (CCND1) signaling pathway. IP6 treatment promotes intestinal adaptation in rats with SBS. IP6 is metabolized to IP3 to increase HDAC3 activity to regulate the FOXO3/CCND1 signaling pathway and may represent a potential therapeutic approach for patients with SBS.

Computational Model of Noradrenaline Modulation of Astrocyte Responses to Synaptic Activity

The mathematical modeling of synaptically connected neuronal networks is an established instrument for gaining insights into dynamics of neuronal ensembles and information processing in the nervous system. Recently, calcium signaling in astrocytes—glial cells controlling local tissue metabolism and synapse homeostasis—and its corresponding downstream effect on synaptic plasticity and neuromodulation appeared in the limelight of modeling studies. Here, we used mechanism-based mathematical modeling to disentangle signaling pathways and feedback loops in the astrocytic calcium response to noradrenaline, an important neuromodulator marking periods of heightened alertness and arousal. The proposed model is based on an experiment-based 2D representation of astrocyte morphology, discrete random glutamate synapses with placement probability defined by the morphology pattern, and spatially heterogeneous noradrenaline sources, reflecting axonal varicosities of the adrenergic axons. Both glutamate and noradrenaline drive Ca2+ dynamics in the astrocyte in an additive or synergistic manner. Our simulations replicate the global activation of astrocytes by noradrenaline and predict the generation of high-frequency Ca2+ waves in a dose-dependent manner and the preferred Ca2+ wave origination near noradrenaline release sites if they colocalise with high-density clusters of glutamate synapses. We tested positive feedback loops between noradrenaline release and glutamate spillover directly or mediated by gliotransmitter release from the activated astrocyte. The simulations suggest that the coupled stochastic drive by glutamate and noradrenaline release converges on the graded modulation of the IP3 level, which is translated into whole-cell Ca2+ waves of different frequencies. Thus, the proposed approach is supported by experimental data and can be used to address situations inaccessible directly by experiment, and is a starting point for a more detailed model that includes other signaling mechanisms providing negative feedback.

The Correlation between Serum Inositol 1,4,5 Triphosphate Level and Primary Hypothyroidism

Background: Most primary hypothyroidism patients also experience inefficiency and irregularity. It is possible to understand the significance of myo-inositol in treating the thyroid gland by relating it to the synthesis of thyroid hormones. Study aimed to estimate serum of inositol 1,4,5-triphosphate (IP3) in primary hypothyroidism disorder and through that level it can shed light on whether it is accused of inactivity of the thyroid gland and at the same time open the doors for the use as a treatment. Subject and Methods: The study was taken from the analytical cross-sectional design.120 subjects were divided into three groups, the first group included 40 healthy subjects, the second group included 25 patients with subclinical hypothyroidism, and the last group had 55 patients with primary hypothyroidism. with the subjects chosen from a teaching laboratory in the medical city. thyroid hormones and serum TSH was determined using Enzyme Immunoassay by Tosoh instrument assay, while serum inositol 1,4,5-triphosphate (IP3) using (ELISA) system. Results: primary hypothyroidism patients showed a significant (p≤0.05) decrease level of serum IP3 when compared with healthy subjects. There is significant positive correlation with serum inositol 1,4,5 triphosphate (IP3) and each triiodothyronine S.T3 (r = 0.581, p ≤0.05), thyroxine S.T4 (r = 0.597, p ≤0.05), and significant negative correlation thyroid-stimulating hormone S.TSH (r=-0.820, p≤0.05), in primary hypothyroidism Patients. Conclusions: inositol 1,4,5 triphosphate (IP3) deficiency in primary hypothyroidism disorder may be a cause of it happening, at the same time may be useful in its treatment even if it was not studied adequately in the study, but through its effect on a thyroid hormone.

Ginsenoside Rg1 treatment protects against cognitive dysfunction via inhibiting PLC–CN–NFAT1 signaling in T2DM mice

BackgroundAs a complication of Type II Diabetes Mellitus (T2DM), the etiology, pathogenesis, and treatment of cognitive dysfunction are still undefined. Recent studies demonstrated that Ginsenoside Rg1 (Rg1) has promising neuroprotective properties, but the effect and mechanism in diabetes-associated cognitive dysfunction (DACD) deserve further investigation. MethodsAfter establishing the T2DM model with a high-fat diet and STZ intraperitoneal injection, Rg1 was given for 8 weeks. The behavior alterations and neuronal lesions were judged using the open field test (OFT) and Morris water maze (MWM), as well as HE and Nissl staining. The protein or mRNA changes of NOX2, p-PLC, TRPC6, CN, NFAT1, APP, BACE1, NCSTN, and Aβ1-42 were investigated by immunoblot, immunofluorescence or qPCR. Commercial kits were used to evaluate the levels of IP3, DAG, and calcium ion (Ca2+) in brain tissues. ResultsRg1 therapy improved memory impairment and neuronal injury, decreased ROS, IP3, and DAG levels to revert Ca2+ overload, downregulated the expressions of p-PLC, TRPC6, CN, and NFAT1 nuclear translocation, and alleviated Aβ deposition in T2DM mice. In addition, Rg1 therapy elevated the expression of PSD95 and SYN in T2DM mice, which in turn improved synaptic dysfunction. ConclusionsRg1 therapy may improve neuronal injury and DACD via mediating PLC–CN–NFAT1 signal pathway to reduce Aβ generation in T2DM mice.

The Effect of A2E on the Ca2+-PKC Signaling Pathway in Human RPE Cells Exposed to Blue Light.

Aims In a model of blue light-induced damage in N-retinylidene-N-retinylethanolamine (A2E)-loaded human retinal pigment epithelial (RPE) cells, we examined the effect of A2E on the calcium (Ca2+)-protein kinase C (PKC) signaling pathway. Methods Primary human RPE cells were cultured, and the cells in the 4th–6th passages were used in this study. The cells were divided into 5 groups: control cells (no A2E, no blue light), blue light-treated cells, blue light + chloroquine-treated cells, blue light + A2E-treated cells, and blue light + A2E + chloroquine-treated cells. The cells were first treated with chloroquine (15 μM for 12 h) and then loaded with A2E (25 μM for 2 h).The blue light intensity was 2000 ± 500 lux, and the duration was 6 h. After blue light exposure, the cells were cultured for 24 h. Fluo-3/AM staining was used to determine the level of cytoplasmic Ca2+, and the cells were photographed using a laser scanning confocal microscope to analyze the fluorescence intensity. The intracellular levels of inositol triphosphate (IP3) and diacylglycerol (DAG) were measured by enzyme-linked immunosorbent assay (ELISA). Intracellular PKC activity was measured with a nonradioactive nuclide assay. Results Among all cell groups, the levels of Ca2+, DAG, and IP3 were lowest in the control cells (P < 0.05). The Ca2+, DAG, and IP3 levels in the blue light + A2E-treated cells and blue light + chloroquine-treated cells were higher than those in the blue light-treated cells (P < 0.05). The Ca2+, DAG, and IP3 levels were highest in the blue light + A2E + chloroquine-treated group (P < 0.05). PKC activity was lowest in the control cells (P < 0.05). The PKC activity of the blue light + A2E-treated cells and blue light + chloroquine-treated cells was higher than that of the blue light-treated cells (P < 0.05), and the PKC activity of the blue light + A2E + chloroquine-treated cells was the highest (P < 0.05). Conclusion Blue light and A2E increased the levels of Ca2+, IP3, and DAG in human RPE cells and enhanced PKC activity, and blue light and A2E had a synergistic effect. Chloroquine further increased the levels of Ca2+, IP3, and DAG and PKC activity in RPE cells or A2E-loaded RPE cells exposed to blue light.

A2A R mediated modulation in IP3 levels altering the [Ca2+]i through cAMP-dependent PKA signalling pathway

Stimulation of A2A receptors (A2A R) coupled to Gs/olf protein activates Adenylyl cyclase (AC) leading to the release of cAMP which activates the cAMP-dependent PKA phosphorylation. The possible role of A2A R in the modulation of free cytosolic Ca2+ concentration ([Ca2+]i) involving IP3, cAMP and PKA was investigated in HEK 293-A2A R. The levels of IP3 and cAMP were observed by enzyme immunoassay detection method and [Ca2+]i using Fluo-4 AM. Moreover, cAMP-dependent PKA was determined using the PKA Colorimetric Activity Kit. We observed that the cells pre-treated with A2A R agonist NECA showed increased levels of cAMP, PKA, IP3 and [Ca2+]i levels. However, the reverse effect was observed with A2A R antagonists (ZM241385 and caffeine). Blocking the Gαq/PLC/DAG/IP3 pathway with neomycin, a PLC inhibitor did not affect the modulation of IP3 and [Ca2+]i levels in HEK 293-A2A R cells. To investigate the Gαi/AC/cAMP/PKA, HEK 293-A2A R cells pre-treated with pertussis toxin followed by forskolin in the presence of A2A R agonist (NECA) showed no effect on cAMP levels. Further, Gαs/AC/cAMP/PKA pathway was investigated to elucidate the role of cAMP-dependent PKA in IP3 mediated [Ca2+]i modulation. In the HEK 293-A2A R cells pre-treated with PKA inhibitor KT5720 and treated with NECA led to inhibit the IP3 and [Ca2+]i levels. The study distinctly demonstrated that A2A R modulates IP3 levels to release the [Ca2+]i via cAMP-dependent PKA. The role of A2A R mediated Gαs pathway inducing IP3 mediated [Ca2+]i release may open new avenues in the therapy of neurodegenerative disorder.

KRAP is required for diffuse and punctate IP3-mediated Ca2+ liberation and determines the number of functional IP3R channels within clusters

KRas-induced actin-interacting protein (KRAP) has been identified as crucial for the appropriate localization and functioning of the inositol trisphosphate receptors (IP3Rs) that mediate Ca2+ release from the endoplasmic reticulum. Here, we used siRNA knockdown of KRAP expression in HeLa and HEK293 cells to examine the roles of KRAP in the generation of IP3-mediated local Ca2+ puffs and global, cell-wide Ca2+ signals. High resolution Ca2+ imaging revealed that the mean amplitude of puffs was strongly reduced by KRAP knockdown, whereas the Ca2+ flux during openings of individual IP3R channels was little affected. In both control and KRAP knockdown cells the numbers of functional channels in the clusters underlying puff sites were stochastically distributed following a Poisson relationship, but the mean number of functional channels per site was reduced by about two thirds by KRAP knockdown. We conclude that KRAP is required for activity of IP3R channels at puff sites and stochastically ‘licenses’ the function of individual channels on a one-to-one basis, rather than determining the functioning of the puff site as a whole. In addition to puff activity (‘punctate’ Ca2+ release), global, cell-wide Ca2+ signals evoked by higher levels of IP3 are further composed from a discrete ‘diffuse’ mode of Ca2+ release. By applying fluctuation analysis to isolate the punctate component during global Ca2+ signals, we find that KRAP knockdown suppresses to similar extents punctate and diffuse Ca2+ release in wild-type cells and in HEK293 cells exclusively expressing type 1 and type 3 IP3Rs. Thus, KRAP appears essential for the functioning of the IP3Rs involved in diffuse Ca2+ release as well as the clustered IP3Rs that generate local Ca2+ puffs.

Deletion Of G‐Protein‐Coupled Receptor 75 Prevents Hypoxia‐Induced Pulmonary Vasoconstriction and Hypertension

Pulmonary Hypertension (PH) is a cardiopulmonary disease estimated to affect between 20 million to 70 million individuals globally, with poor prognosis and inadequate treatment. Recent studies showed the orphan G Protein Coupled Receptor 75 (GPR75) is upregulated in the pulmonary vessels of PH patients, especially in females. Therefore, we hypothesized that Gpr75knock out (Gpr75KO) mice will be protected from developing PH. To test our hypothesis, first we determined the vasoreactivity of isolated intra‐lobar pulmonary arteries from wild‐type (WT) and GPR75KO mice induced by KCl (WT=6; Gpr75KO=8), U46619 (WT=8; Gpr75KO=8), ZD7288 (WT=7; Gpr75KO=7), and Endothelin (WT=7; Gpr75KO=8) to test their vasoreactivity. The Gpr75KO as compared with wild‐type pulmonary arteries contracted less to all the contractile agents. Next, we determine if Gpr75KO mice were protected from developing hypoxia‐induced PH. We placed 3‐month‐old female Gpr75KO (n=8) and control wild‐type (C57BL/C) mice (n=18) in a hypoxic chamber with 10% Oxygen or at atmospheric oxygen for 5 weeks. At the end of 5 weeks, we performed right heart catheterizations. Gpr75KO mice developed less (P&lt;0.05) right ventricle systolic pressure (RVSP: 24.3±1.2 mmHg) and right ventricle diastolic pressure (RVDP: 2.0±0.0 mmHg) than the wild‐type mice under hypoxia (RSVP and RDVP of 56.6±3.5 mmHg and 9.8±0.9 mmHg, respectively). Since GPR75 is a G‐protein coupled receptor to determine secondary messengers that potentially contribute to regulating contractile function of pulmonary arteries, we measured cAMP and IP3 in lungs of wild‐type and GPR75KO mice. While we found no difference in the IP3 levels between the WT and Gpr75KO groups, cAMP levels were significantly higher in Gpr75KO compared to WT mice. These results suggest GPR75 plays a major role in the development of PH by potentially attenuating cAMP‐dependent signaling and concomitantly augmenting pulmonary constriction in response to hypoxia.

Hydroxycarboxylic acid receptor 3 and GPR84 – Two metabolite-sensing G protein-coupled receptors with opposing functions in innate immune cells

G protein-coupled receptors (GPCRs) are key regulatory proteins of immune cell function inducing signaling in response to extracellular (pathogenic) stimuli. Although unrelated, hydroxycarboxylic acid receptor 3 (HCA3) and GPR84 share signaling via Gαi/o proteins and the agonist 3-hydroxydecanoic acid (3HDec). Both receptors are abundantly expressed in monocytes, macrophages and neutrophils but have opposing functions in these innate immune cells. Detailed insights into the molecular mechanisms and signaling components involved in immune cell regulation by GPR84 and HCA3 are still lacking. Here, we report that GPR84-mediated pro-inflammatory signaling depends on coupling to the hematopoietic cell-specific Gα15 protein in human macrophages, while HCA3 exclusively couples to Gαi protein. We show that activated GPR84 induces Gα15-dependent ERK activation, increases intracellular Ca2+ and IP3 levels as well as ROS production. In contrast, HCA3 activation shifts macrophage metabolism to a less glycolytic phenotype, which is associated with anti-inflammatory responses. This is supported by an increased release of anti-inflammatory IL-10 and a decreased secretion of pro-inflammatory IL-1β. In primary human neutrophils, stimulation with HCA3 agonists counteracts the GPR84-induced neutrophil activation. Our analyses reveal that 3HDec acts solely through GPR84 but not HCA3 activation in macrophages. In summary, this study shows that HCA3 mediates hyporesponsiveness in response to metabolites derived from dietary lactic acid bacteria and uncovers that GPR84, which is already targeted in clinical trials, promotes pro-inflammatory signaling via Gα15 protein in macrophages.

The role of calcium oscillations in the phenotype selection in endothelial cells

Angiogenesis is an important process in the formation and maintenance of tissues which is driven by a complex system of intracellular and intercellular signaling mechanisms. Endothelial cells taking part in early angiogenesis must select their phenotype as either a tip cells (leading, migratory) or a stalk cells (following). Recent experiments have demonstrated that rapid calcium oscillations within active cells characterize this phenotype selection process and that these oscillations play a necessary role in governing phenotype selection and eventual vessel architecture. In this work, we develop a mathematical model capable of describing these oscillations and their role in phenotype selection then use it to improve our understanding of the biological mechanisms at play. We developed a model based on two previously published and experimentally validated mathematical models of calcium and angiogenesis then use our resulting model to simulate various multi-cell scenarios. We are able to capture essential calcium oscillation dynamics and intercellular communication between neighboring cells. The results of our model show that although the late DLL4 (a transmembrane protein that activates Notch pathway) levels of a cell are connected with its initial IP3 (Inositol 1,4,5-trisphosphate) level, cell-to-cell communication determines its eventual phenotype.

Alpha2- Adrenergic blockers restore noise-induced biochemical and cognitive disorders

Noise-induced stress causes central nervous system dysfunction and neurotransmission impairment in the brain and changes hormone levels which potentially result in psychological and behavioral problems. Considering the significance of the potential negative outcome of the chronic stress, the aim of our investigation was to study the level of inositol triphosphate (IP3) in the mitochondrial fraction of the brain (MFB), behavioral characteristics and the effects of the α2-adrenoblockers beditin and mesedin to reveal their antioxidative effect during noise exposure. The open field tests were used in order to evaluate the behavioral states of the rats. Investigations were carried out on albino rats divided into 4 groups. The 1st group of rats served as a control. The 2nd, 3rd and 4th groups were exposed to 91 dBA of noise; the duration of exposure was 8 hours per day for 60 days. The 3rd group was injected with beditin and the 4th group with mesedin, both intraperitoneally and repeatedly. According to our results, the chronic exposure to high-level noise leads to the decreased level of IP3 resulting development of oxidative stress and metabolism imbalance. The open field test revealed that chronic noise exposure caused noise durationdependent delay in the locomotor and behavioral activity. Intensity of these changes were dependent on the duration of noise exposure. The administration of α2-adrenoblockers to the noise-exposed animals had a regulatory effect on the studied parameters under the conditions of chronic acoustic stress.

HIV infection of astrocytes compromises inter-organelle interactions and inositol phosphate metabolism: A potential mechanism of bystander damage and viral reservoir survival.

HIV-associated neurological dysfunction is observed in more than half of the HIV-infected population, even in the current antiretroviral era. The mechanisms by which HIV mediates CNS dysfunction are not well understood but have been associated with the presence of long-lasting HIV reservoirs. In the CNS, macrophage/microglia and a small population of astrocytes harbor the virus. However, the low number of HIV-infected cells does not correlate with the high degree of damage, suggesting that mechanisms of damage amplification may be involved. Here, we demonstrate that the survival mechanism of HIV-infected cells and the apoptosis of surrounding uninfected cells is regulated by inter-organelle interactions among the mitochondria/Golgi/endoplasmic reticulum system and the associated signaling mediated by IP3 and calcium. We identified that latently HIV-infected astrocytes had elevated intracellular levels of IP3, a master regulator second messenger, which diffuses via gap junctions into neighboring uninfected astrocytes resulting in their apoptosis. In addition, using laser capture microdissection, we confirmed that bystander apoptosis of uninfected astrocytes and the survival of HIV-infected astrocytes were dependent on mitochondrial function, intracellular calcium, and IP3 signaling. Blocking gap junction channels did not prevent an increase in IP3 or inter-organelle dysfunction in HIV-infected cells but reduced the amplification of apoptosis into uninfected neighboring cells. Our data provide a mechanistic explanation for bystander damage induced by surviving infected cells that serve as viral reservoirs and provide potential targets for interventions to reduce the devastating consequences of HIV within the brain.

Selective or Specific M1 Muscarinic Receptor Antagonists Exhibit Biased Agonism at the M1 Receptor via β‐arrestin Signaling

Introduction Selective (pirenzepine; PZ) and specific (muscarinic toxin 7; MT7) antagonists of the muscarinic acetylcholine type 1 receptor (M1R) reverse nerve degeneration in rodent models of peripheral neuropathy. These drugs drive axonal outgrowth of adult sensory neurons and signaling is mediated via extracellular-regulated protein kinase (ERK) and β-arrestin. In order to understand the pharmacological mechanism of action of PZ and MT7, we hypothesized that these drugs exhibited biased agonism at the M1R mediated via b-arrestin signaling. Methods Nano bioluminescence resonance energy transfer (NanoBRET) assay: Human embryonic kidney (HEK293) cells co-expressing human M1R-Nluc and Halo-tagged β-arrestin 2 were treated with different doses of M1R agonists (muscarine or carbachol) or antagonists (PZ and MT7)at different time points. NanoBRET assay was employed to measure ligand-induced β-arrestin 2 recruitment to the M1R. Assays were performed in triplicate with 3 independent experiments completed. Data are presented as corrected milli-BRET (mBRET) units. Substrate was added 2 min before BRET detection, and drugs were applied at various time points. Data are reported as the mean ± SD (statistics by 1-way ANOVA with Tukey's post-hoc test). Inositol-phosphate one (IP1) measurement: Intracellular IP1 levels were measured using IP-One assay (Cisbio Bioassays, USA). HEK293 cells co-expressing hM1R-Nluc and Halo-tagged β-arrestin 2 were plated onto 384-well white microplates (20000 cells per well) and were treated with stimulation buffer with or without 1) different doses of M1R agonist (muscarine), 2) M1R antagonists (PZ and MT7) or 3) both M1R antagonist (different doses of PZ) and agonist (muscarine, 1mM) for different time points at 37 °C. Cells were lysed by addition of the supplied buffer containing d2-labeled IP1, followed by addition of terbium cryptate-labeled anti-IP1 antibody, according to the manufacturer's instructions. Plates were incubated for 1 h at room temperature and fluorescence signals were measured at 620 and 665 nm using the Biotek Synergy Neo2 plate reader. HTRF ratio 665 nm/620 nm of each well were extrapolated on the standard curve to obtain IP1 concentrations. Results Both carbachol (Fig. 1A) and muscarine (Fig. 1B) induced Halo-tagged β-arrestin 2 recruitment to hM1R-Nluc in a dose-dependent manner at 5 min of treatment. pirenzepine (Fig. 1C) and MT7 (Fig. 1D) induced Halo-tagged β-arrestin 2 recruitment to hM1R-Nluc in a dose-dependent manner at longer time points (e.g. after 30 min). Interestingly, over short periods, e.g. 2-3 min, PZ and MT7 acted as classical antagonists with blockade of muscarine-induced b-arrestin 2 association with M1R. Unlike MT7 and PZ, muscarine increased IP1 levels in transfected cells (Fig. 2A). Also, PZ dose-dependently inhibited the formation of IP1 following muscarine (1mM) treatment (Fig. 2B). Conclusion Thisstudy for the first time provides molecular evidence to support a role for selective/specific muscarinic receptor antagonists acting as biased agonists at the M1R. This novel signaling pathway may contribute to driving axonal regeneration in adult sensory neurons and could be mobilized for nerve repair in neuropathic disease.

Allosteric modulation of adenosine A1 and cannabinoid 1 receptor signaling by G-peptides.

While allosteric modulation of GPCR signaling has gained prominence to address the need for receptor specificity, efforts have mainly focused on allosteric sites adjacent to the orthosteric ligand‐binding pocket and lipophilic molecules that target transmembrane helices. In this study, we examined the allosteric influence of native peptides derived from the C‐terminus of the Gα subunit (G‐peptides) on signaling from two Gi‐coupled receptors, adenosine A1 receptor (A1R) and cannabinoid receptor 1 (CB1). We expressed A1R and CB1 fusions with G‐peptides derived from Gαs, Gαi, and Gαq in HEK 293 cells using systematic protein affinity strength modulation (SPASM) and monitored the impact on downstream signaling in the cell compared to a construct lacking G‐peptides. We used agonists N6‐Cyclopentyladenosine (CPA) and 5’‐N‐Ethylcarboxamidoadenosine (NECA) for A1R and 2‐Arachidonoylglycerol (2‐AG) and WIN 55,212‐2 mesylate (WN) for CB1. G‐peptides derived from Gαi and Gαq enhance agonist‐dependent cAMP inhibition, demonstrating their effect as positive allosteric modulators of Gi‐coupled signaling. In contrast, both G‐peptides suppress agonist‐dependent IP1 levels suggesting that they differentially function as negative allosteric modulators of Gq‐coupled signaling. Taken together with our previous studies on Gs‐coupled receptors, this study provides an extended model for the allosteric effects of G‐peptides on GPCR signaling, and highlights their potential as probe molecules to enhance receptor specificity.

Differential signaling signatures evoked by DOI versus lisuride stimulation of the 5-HT2A receptor

The 5-HT2A receptor is a target for hallucinogenic and non-hallucinogenic ligands that evoke unique behavioral, electrophysiological and molecular consequences. Here, we explored the differential effects of distinct 5-HT2A receptor ligands on signaling pathways downstream to the 5-HT2A receptor. The hallucinogenic 5-HT2A receptor agonist DOI evoked an enhanced signaling response compared to the non-hallucinogenic 5-HT2A receptor agonist lisuride in human/rat 5-HT2AR-EGFP receptor expressing HEK293 cell lines and cortical neuronal cultures. We noted higher levels of phospho-PLC, pPKC, pERK, pCaMKII, pCREB, as well as higher levels of IP3 and DAG production following 5-HT2A receptor stimulation with DOI. Our study reveals distinct signaling signatures, differing in magnitude and kinetics at the 5-HT2A receptor in response to DOI versus lisuride.