Total Receptor Number Research Articles

Mathematical Modeling Unveils Optimization Strategies for Targeted Radionuclide Therapy of Blood Cancers.

Targeted radionuclide therapy is based on injections of cancer-specific molecules conjugated with radioactive nuclides. Despite the specificity of this treatment, it is not devoid of side-effects limiting its use and is especially harmful for rapidly proliferating organs well perfused by blood, like bone marrow. Optimization of radioconjugates administration accounting for toxicity constraints can increase treatment efficacy. Based on our experiments on disseminated multiple myeloma mouse model treated by 225Ac-DOTA-daratumumab, we developed a mathematical model which investigation highlighted the following principles for optimization of targeted radionuclide therapy. 1) Nuclide to antibody ratio importance. The density of radioconjugates on cancer cells determines the density of radiation energy deposited in them. Low labeling ratio as well as accumulation of unlabeled antibodies and antibodies attached to decay products in the bloodstream can mitigate cancer radiation damage due to excessive occupation of specific receptors by antibodies devoid of radioactive nuclides. 2) Cancer binding capacity-based dosing. The total number of specific receptors on cancer cells is a critical factor for treatment optimization, which estimation may allow increasing treatment efficacy close to its theoretical limit. Injection of doses significantly exceeding cancer binding capacity should be avoided since radioconjugates remaining in the bloodstream have negligible efficacy to toxicity ratio. 3) Particle range-guided multi-dosing. The use of short-range particle emitters and high-affinity antibodies can allow for robust treatment optimization via initial saturation of cancer binding capacity, enabling redistribution of further injected radioconjugates and deposited dose towards still viable cells that continue expressing specific receptors.

Riding a VeSPha in the classroom: A Versatile Simulation tool for effective didactic illustration of Pharmacodynamic concepts

A solid mastery of Pharmacodynamics concepts is a cornerstone of effective pharmacology education. Understanding the meaning and appropriate use of pharmacodynamics terms such as potency, affinity, intrinsic efficacy, or receptor reserve is extremely valuable but challenging for most trainees. The onus, therefore, is on the educators to devise effective strategies and tools to facilitate the acquisition of this critical competency.When approaching pharmacodynamics, the ability of learners to explore the influence of driving parameters on the magnitude and position of concentration response curves is a powerful way to visualize and make concrete the basic concepts of pharmacodynamics. To this end, I have developed VeSPha, a Versatile Simulation tool for Pharmacodynamics based on a rapid equilibrium generalization of Furchgott’s views on agonism. Through multiple sliders, the user can vary the parameters of the system, such as the concentrations of orthosteric and allosteric ligands, their respective affinities, the allosteric coupling constant, the intrinsic efficacies of each of the receptor species, the total number of receptors and the sensitivity of the system to the stimulus. A graph panel allows the display of the resulting response curves as a function of the concentration of ligand as well as orthosteric and allosteric ligand occupancy curves. Dropdown lines with corresponding EC50 as well as apparent Kd values for each ligand are also displayed. VeSPha also includes bar graphs as well as an intuitive graphic representation of the model displaying the values of the parameters, the distribution of the receptor species at the chosen state, the total stimulus as well as the resulting responses.The tool allows easy exploration of a wide gamut of possibilities that illustrate basic concepts of affinity, intrinsic efficacy, partial agonism, constitutive activity, inverse agonism, competitive antagonism, as well as allosteric modulation of both affinity and efficacy in any combination. The tool is implemented as an easy‐to‐use Excel file. This allows for flexible standalone use and for straightforward embedding into PowerPoint presentations for real‐time illustrations during lectures. The tool also readily generates simulated data including sampling errors for composing realistic, internally consistent assessment items such as quiz, problem set and exam questions.Implementation of this didactic tool is straightforward and effective in the author’s experience across a wide range of learners spanning from undergraduates, Master’s, Ph.D. M.D. Pharm D and DDS students. The tool, including a tutorial video can be made available to the Pharmacology teaching community upon request and an online version is under development.

ALPHA(1)-ADRENOCEPTORS ACTIVATION DECREASES MYOCARDIAL CONTRACTILITY IN NEWBORN RATS

Alpha(1)-adrenergic receptors (α1-AR) are found in cardiomyocytes, endothelial cells, and smooth muscle cells of humans and animals. Despite the fact that α1-AR make up 10% of the total number of adrenergic receptors, these receptors also involved in the regulation of inotropic and chronotropic functions of the heart. According to some scientists, the effects of α1-AR activation are not required for the basal contractile function of the heart while other group of researchers believe that α1-AR can be considered as cardioprotective targets; in particular, it is postulated that the α1A-subtype of adrenergic receptors can provide significant inotropic support in cardiac pathologies. This study was carried out on 6-7-day-old outbred newborn rat pups to evaluate the effect of alpha(1)-adrenoceptors activation on the myocardial contractility in newborn rats. For this, Alpha1-adrenergic receptors were stimulated by the pharmacological drug methoxamine at concentrations of 10-9-10-6 mol and the reaction of the contractile force of the strips of myocardium ventricles and heart atria in response to the agonist was investigated. Results of study revealed that stimulation of alpha1-adrenergic receptors, regardless of the methoxamine concentration, led to a negative inotropic reaction of the myocardium of atria and ventricles of newborn rat pups. This study showed unidirectional inotropic responses on rat atrial and ventricular myocardium in response to α1-adrenergic receptors stimulation. Methoxamine smoothly reduces the contractile force of the strips of myocardium atria and ventricles. At the same time, the concentration dependence on the inotropic reaction of the myocardium was observed. Results of study suggested that probably α1-adrenergic receptors along with the main regulators β-adrenergic receptors carry out fine tuning of the heart activity.

Mechanisms of Linear Growth Acceleration in Childhood Obesity

The obesity epidemic is alarming. Nearly one‐third of children in the Unites States are overweight or obese by the time they reach their pre‐teen years. Tall stature and accelerated bone elongation are distinctive clinical features of childhood obesity, which tend to co‐occur with painful conditions such as limb bowing, slipped epiphyses, fractures, and early onset osteoarthritis. Although linear growth rate is markedly increased, obese children paradoxically have normal circulating levels of IGF‐I, the major growth‐stimulating hormone. The causes of obesity‐induced growth acceleration remain elusive. Our lab has used a well‐established mouse model of excess dietary fat in order to examine bone elongation changes that occur with the development of juvenile obesity in young growing mice. We found, surprisingly, that brief exposure to a high‐fat diet accelerates bone elongation rate without increasing body mass or circulating IGF‐I levels. Interestingly, when we used in vivo multiphoton microscopy to measure how much systemic IGF‐I localized to growth plates in real time, we found significantly more (nearly twice as much) IGF‐I in the vasculature surrounding the growth plates of mice on a high‐fat diet relative to controls. Post‐mortem immunostaining confirmed that a high‐fat diet increased the number of activated IGF‐I receptors in the growth plate, but not total receptor number. Together, these results suggest that a high‐fat diet increases local bioavailability of IGF‐I in growth plate cartilage without altering systemic IGF‐I levels, and before changes in body mass become evident. This unique method of imaging growth factor transport in cartilage is an important advancement for addressing why linear growth is accelerated in obese children who have normal levels of systemic growth‐promoting hormones. While diets high in fat are well‐recognized for cardiovascular and metabolic complications, the role of dietary fat in bone lengthening is a novel and timely area of research. These studies may provide essential data for testing whether impeding IGF‐I actions in growth plates could be a beneficial approach for mitigating some of the deleterious skeletal consequences of childhood obesity.Support or Funding InformationSupported by the National Institute of General Medical Sciences (1P20GM121299‐01) and National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health (1R15AR067451‐01).

Modeling and predictions of biphasic mechanosensitive cell migration altered by cell-intrinsic properties and matrix confinement

Motile cells sense the stiffness of their extracellular matrix through adhesions and respond by modulating the generated forces, which in turn lead to varying mechanosensitive migration phenotypes. Through modeling and experiments, cell migration speed is known to vary with matrix stiffness in a biphasic manner, with optimal motility at an intermediate stiffness. Here, we present a 2D cell model defined by nodes and elements, integrated with subcellular modeling components corresponding to mechanotransductive adhesion formation, force generation, protrusions and node displacement. On 2D matrices, our calculations reproduce the classic biphasic dependence of migration speed on matrix stiffness and predict that cell types with higher force-generating ability do not slow down on very stiff matrices, thus disabling the biphasic response. We also predict that cell types defined by a lower number of total receptors require stiffer matrices for optimal motility, which also limits the biphasic response. For a cell type with robust biphasic migration on a 2D surface, simulations in channel-like confined environments of varying width and height predict faster migration in more confined matrices. Simulations performed in shallower channels predict that the biphasic mechanosensitive cell migration response is more robust on 2D micro-patterns compared to the channel-like 3D confinement. Thus, variations in the dimensionality of matrix confinement alters the way migratory cells sense and respond to the matrix stiffness. Our calculations reveal new phenotypes of stiffness- and topography-sensitive cell migration that critically depend on both cell-intrinsic and matrix properties. These predictions may inform our understanding of various mechanosensitive modes of cell motility that could enable tumor invasion through topographically heterogeneous microenvironments.

Identification and Evolution of TGF-β Signaling Pathway Members in Twenty-Four Animal Species and Expression in Tilapia.

Transforming growth factor β (TGF-β) signaling controls diverse cellular processes during embryogenesis as well as in mature tissues of multicellular animals. Here we carried out a comprehensive analysis of TGF-β pathway members in 24 representative animal species. The appearance of the TGF-β pathway was intrinsically linked to the emergence of metazoan. The total number of TGF-β ligands, receptors, and smads changed slightly in all invertebrates and jawless vertebrates analyzed. In contrast, expansion of the pathway members, especially ligands, was observed in jawed vertebrates most likely due to the second round of whole genome duplication (2R) and additional rounds in teleosts. Duplications of TGFB2, TGFBR2, ACVR1, SMAD4 and SMAD6, which were resulted from 2R, were first isolated. Type II receptors may be originated from the ACVR2-like ancestor. Interestingly, AMHR2 was not identified in Chimaeriformes and Cypriniformes even though they had the ligand AMH. Based on transcriptome data, TGF-β ligands exhibited a tissue-specific expression especially in the heart and gonads. However, most receptors and smads were expressed in multiple tissues indicating they were shared by different ligands. Spatial and temporal expression profiles of 8 genes in gonads of different developmental stages provided a fundamental clue for understanding their important roles in sex determination and reproduction. Taken together, our findings provided a global insight into the phylogeny and expression patterns of the TGF-β pathway genes, and hence contribute to the greater understanding of their biological roles in the organism especially in teleosts.

MicroRNA‐135a Regulates NHE9 To Inhibit Proliferation and Migration of Glioblastoma Cells

Glioblastoma multiformae (GBM) is the most aggressive type of malignant brain tumor with complex molecular profile. Overexpression of Na+/H+ Exchanger isoform 9 (NHE9) promotes tumor progression and correlates positively with insensitivity to radiochemotherapy and poor prognosis. However, molecular mechanisms responsible for increase in NHE9 levels beyond a critical threshold have not been identified.We show that microRNA 135a (miR‐135a) targets NHE9 to downregulate its expression in U87 cells. MiR‐135a levels are significantly lower in glioblastoma cells compared to normal brain tissue. Downregulation of NHE9 expression by miR‐135a reduced proliferative and migratory capacity of U87 cells. Selectively increasing NHE9 expression in these cells restored their ability to proliferate and migrate. We demonstrate that miR‐135a takes a two‐pronged approach affecting epidermal growth factor receptors (EGFRs) to suppress tumor cell growth and migration. EGFR activity is a potent stimulator of oncogenic signaling. While miR‐135a targets EGFR transcripts to decrease the total number of receptors made, by targeting NHE9 it routes the few EGFRs made away from the plasma membrane to dampen oncogenic signaling. NHE9 is localized to sorting endosomes in glioblastoma cells where it alkalinizes the endosome lumen by leaking protons. Downregulation of NHE9 expression by miR‐135a acidifies sorting endosomes limiting EGFR trafficking to the glioblastoma cell membrane. We propose downregulation of miR‐135a as a potential mechanism underlying the high NHE9 expression observed in subset of glioblastomas. Future studies should explore miR‐135a as a potential therapeutic for glioblastomas with NHE9 overexpression.Support or Funding InformationFunds from the Department of Natural Sciences and the Office of Research and Sponsored Programs at the University of Michigan DearbornThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

MicroRNA-135a regulates NHE9 to inhibit proliferation and migration of glioblastoma cells

BackgroundGlioblastoma multiformae (GBM) is the most aggressive type of malignant brain tumor with complex molecular profile. Overexpression of Na+/H+ Exchanger isoform 9 (NHE9) promotes tumor progression and correlates positively with insensitivity to radiochemotherapy and poor prognosis. However, molecular mechanisms responsible for increase in NHE9 levels beyond a critical threshold have not been identified.MethodsBioinformatics analysis, luciferase reporter assays, real-time PCR and western blotting were conducted to examine the expression profiles and identify microRNAs (miRNA) that target NHE9. Cell proliferation and migration assays were conducted in U87 glioblastoma cells to determine the consequence of miRNA mediated targeting of NHE9. Endosomal pH measurements, immunofluorescence microscopy and surface biotinylation experiments were conducted to characterize the mechanistic basis of regulation.ResultsWe show that microRNA 135a (miR-135a) targets NHE9 to downregulate its expression in U87 cells. MiR-135a levels are significantly lower in glioblastoma cells compared to normal brain tissue. Downregulation of NHE9 expression by miR-135a affects proliferative and migratory capacity of U87 cells. Selectively increasing NHE9 expression in these cells restored their ability to proliferate and migrate. We demonstrate that miR-135a takes a two-pronged approach affecting epidermal growth factor receptors (EGFRs) to suppress tumor cell growth and migration. EGFR activity is a potent stimulator of oncogenic signaling. While miR-135a targets EGFR transcripts to decrease the total number of receptors made, by targeting NHE9 it routes the few EGFRs made away from the plasma membrane to dampen oncogenic signaling. NHE9 is localized to sorting endosomes in glioblastoma cells where it alkalinizes the endosome lumen by leaking protons. Downregulation of NHE9 expression by miR-135a acidifies sorting endosomes limiting EGFR trafficking to the glioblastoma cell membrane.ConclusionsWe propose downregulation of miR-135a as a potential mechanism underlying the high NHE9 expression observed in subset of glioblastomas. Future studies should explore miR-135a as a potential therapeutic for glioblastomas with NHE9 overexpression.

Status Epilepticus Impairs Synaptic Plasticity in Rat Hippocampus and Is Followed by Changes in Expression of NMDA Receptors.

Cognitive deficits and memory loss are frequent in patients with temporal lobe epilepsy. Persistent changes in synaptic efficacy are considered as a cellular substrate underlying memory processes. Electrophysiological studies have shown that the properties of short-term and long-term synaptic plasticity in the cortex and hippocampus may undergo substantial changes after seizures. However, the neural mechanisms responsible for these changes are not clear. In this study, we investigated the properties of short-term and long-term synaptic plasticity in rat hippocampal slices 24h after pentylenetetrazole (PTZ)-induced status epilepticus. We found that the induction of long-term potentiation (LTP) in CA1 pyramidal cells is reduced compared to the control, while short-term facilitation is increased. The experimental results do not support the hypothesis that status epilepticus leads to background potentiation of hippocampal synapses and further LTP induction becomes weaker due to occlusion, as the dependence of synaptic responses on the strength of input stimulation was not different in the control and experimental animals. The decrease in LTP can be caused by impairment of molecular mechanisms of neuronal plasticity, including those associated with NMDA receptors and/or changes in their subunit composition. Real-time PCR demonstrated significant increases in the expression of GluN1 and GluN2A subunits 3h after PTZ-induced status epilepticus. The overexpression of obligate GluN1 subunit suggests an increase in the total number of NMDA receptors in the hippocampus. A 3-fold increase in the expression of the GluN2B subunit observed 24h after PTZ-induced status epilepticus might be indicative of an increase in the proportion of GluN2B-containing NMDA receptors. Increased expression of the GluN2B subunit may be a cause for reducing the magnitude of LTP at hippocampal synapses after status epilepticus.

Evaluation of plasma PCSK9 concentrations, transcript of LDL receptor, as well as the total number of monocyte LDL receptors in acute coronary syndrome patients

Before our study, there were no data concerning complex evaluation of: plasma PCSK9 concentrations, transcript LDL receptor (LDLR), as well as the total amount of monocytes' LDLR in acute coronary syndrome (ACS) patients. PCSK9 levels in a few cohort studies were found to correlate with the number of white blood cells (WBC) or platelets (PLT). The study aims to evaluate PCSK9-LDLR concentrations, as well as to find any association between PCSK9 and WBC or PLT. The study group included 95 consecutive patients with acute myocardial infarction, in whom angiography/angioplasty of the culprit vessel was performed. The control group consisted of 10 healthy young volunteers. Thirty patients from the studied group were qualified for further percutaneous revascularization after 3 months. Laboratory tests were performed using commercially available kits. LDLR expression on monocyte surface was measured by flow cytometry, but the mRNA level for LDLR was established by real time polymerase chain reaction. The PCSK9 plasma concentration was measured by ELISA kits. Higher concentration of PCSK9 and amount of LDLR on monocytes surface were observed in patients with ACS compared with healthy young volunteers (number of LDLRs on monocytes [reaction units] 10.8 ± 9.6 vs. 41.8 ± 11.8, p < 0.001, PCSK9 [ng/mL] 295.4 ± 76.4 vs. 213 ± 63.2, p < 0.001). A similar relationship was observed after application of 3-month intensive lipid-lowering therapy in patients with ACS (n = 30, PCSK9 [ng/mL] 281.1 ± 59.5 vs. 358.5 ± 74.7, p < 0.001, LDLR transcript [reaction units] 0.6 ± 0.32 vs. 1.87 ± 0.24, p < 0.001, number of LDLRs on monocytes [reaction units] 5.9 ± 3.1 vs. 22.3 ± 3.8, p < 0.001). There were no significant differences in levels of PCSK9, LDLR between patients with ST-segment elevation myocardial infarction (STEMI) and non-ST-segment elevation myocardial infarction (NSTEMI). There was no relation of the PCSK9 with WBC as well as with PLT. We observed significantly higher concentration of PCSK9, and significantly higher levels of mRNA LDLR transcript in patients with ACS compared with healthy young volunteers. A similar pattern was observed after 3 months of intensive statin therapy among patients with ACS. There were no differences in these parameters between patients with STEMI vs. NSTEMI. The results of the study require confirmation in a larger population of patients.

Staphylococcus Aureus Targets the Duffy Antigen Receptor for Chemokines (DARC) to Lyse Erythrocytes

Introduction: Staphylococcus aureus is a major human pathogen. Infections by this bacterium range from minor skin and soft tissue infections, to more invasive and life threating infections like sepsis, osteomyelitis, and pneumonia. In order to successfully infect the mammalian host, S. aureus has to overcome iron scarcity within the host. As such, S. aureus is thought to produce toxins that lyse erythrocytes, releasing hemoglobin, a critical iron source for S. aureus in mammals. The bi-component β-barrel pore-forming leukocidins kill human neutrophils and were mostly considered as virulence factors fighting the host innate immune system. We show here thatHlgAB and LukED are two potent hemolytic leukocidins against human erythrocytes. Furthermore, we describe the identification of the Duffy antigen receptor for chemokines (DARC) as the red blood cell receptor for both LukED and HlgAB leukocidins.Results: We took advantage of erythrocyte samples from different Duffy (Fy) genotyped individuals from the French National Blood Transfusion Institute. We evaluated the receptor expression on the surface of erythrocytes (Figure 1). DARC-negative erythrocytes (phenotype Fya-/b-) were fully resistant to HlgAB and LukED. Compared to individuals expressing normal levels of DARC (phenotypes Fya+/b+, Fya+/b+weak, Fya+/a+, and Fyb+/b+), individuals expressing intermediate (phenotype Fyb+weak/b+weak) or very low levels of DARC (phenotype Fyb-/b+weak) showed intermediate susceptibility to both HlgAB and LukED (Figure 1). These data demonstrate that the hemolytic activity of HlgAB and LukED is dependent on the expression of DARC at the red blood cell surface. To better understand the interaction of HlgAB and LukED with DARC, we screened HEK293T cells transfected with plasmids encoding DARC mutants (Tournamille et al, 2003). We demonstrate that while both HlgAB and LukED bind to DARC with nanomolar affinities, they do so by recognizing different domains of the receptor. To directly evaluate whether LukED and HlgAB can promote bacterial replication as a result of erythrocyte lysis, S. aureus was grown in iron-starved medium supplemented with cell-free extracts of erythrocytes treated with LukED or HlgAB. We observed that HlgAB and LukED were each capable of promoting S. aureus growth in a DARC and hemoglobin scavenging system (IsdBH)-dependent manner. These in vitro studies were supported by a murine bacteremia model.Discussion: By combining human studies of DARC polymorphisms with gain and loss of function experiments and biochemical analyses, we demonstrate that DARC is necessary and sufficient to render host cells susceptible to LukED and HlgAB. By targeting DARC, HlgAB and LukED support S. aureus growth in a hemoglobin-acquisition dependent-manner. Thus, these findings provide the missing link of how S. aureus targets and lyses erythrocytes to release one of the scarcest nutrient within the mammalian host. Human epidemiological studies comparing the severity of S. aureus infection in patients with DARC positive or DARC negative erythrocytes are now required to evaluate the contribution of DARC-mediated hemolysis in human staphylococcal diseases. Given the resistance of DARC negative erythrocytes to the parasites Plasmodium vivax and P. knowlesi, and now to the hemolytic activity of the bacterium S. aureus, our findings suggest the possibility of a positive selection event in response to these important human pathogens.(A) Susceptibility of human erythrocytes to S. aureus β-barrel pore forming toxins. The dashed line indicates 50% hemolysis. n = 6.(B) Levels of DARC and CD55 on erythrocytes of donors with different Fy phenotypes. The dashed line indicates the detection threshold. n = 2-7 ± SEM.(C,E) Susceptibility of human erythrocytes with different Fy phenotypes to HlgAB (C) and LukED (E). The dashed line indicates 50% hemolysis. n = 2-7 ± SEM.(D, F) Correlation of half-maximal effective concentrations (EC50) of HlgAB (D) and LukED (F) with the total number of receptors expressed on the erythrocyte surface.For Fyb+weak/Fyb- donors, LukED EC50 could not be calculated. n = 2-7 ± SEM. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Epileptogenic effects of NMDAR antibodies in a passive transfer mouse model.

Most patients with N-methyl D-aspartate-receptor antibody encephalitis develop seizures but the epileptogenicity of the antibodies has not been investigated in vivo. Wireless electroencephalogram transmitters were implanted into 23 C57BL/6 mice before left lateral ventricle injection of antibody-positive (test) or healthy (control) immunoglobulin G. Mice were challenged 48 h later with a subthreshold dose (40 mg/kg) of the chemo-convulsant pentylenetetrazol and events recorded over 1 h. Seizures were assessed by video observation of each animal and the electroencephalogram by an automated seizure detection programme. No spontaneous seizures were seen with the antibody injections. However, after the pro-convulsant, the test mice (n = 9) had increased numbers of observed convulsive seizures (P = 0.004), a higher total seizure score (P = 0.003), and a higher number of epileptic 'spike' events (P = 0.023) than the control mice (n = 6). At post-mortem, surprisingly, the total number of N-methyl D-aspartate receptors did not differ between test and control mice, but in test mice the levels of immunoglobulin G bound to the left hippocampus were higher (P < 0.0001) and the level of bound immunoglobulin G correlated with the seizure scores (R(2) = 0.8, P = 0.04, n = 5). Our findings demonstrate the epileptogenicity of N-methyl D-aspartate receptor antibodies in vivo, and suggest that binding of immunoglobulin G either reduced synaptic localization of N-methyl D-aspartate receptors, or had a direct effect on receptor function, which could be responsible for seizure susceptibility in this acute short-term model.

CHARACTERISTICS OF A SENSOR BASED ON POLYANILINE IN STOICHIOMETRY (1 ↔ 4)

A model of interaction of an analyte with a chemoresistive sensor is offered. According to the model, a receptor molecule can conduct a current after binding four molecules of analyte. It is taken into account the process of penetration of the analyte into the sensor and noninstantaneous filling of a chamber containing the sensor with analyte, which is characteristic of the experimental configuration used. A system of nonlinear differential equations is derived that describes the kinetics of processes in the investigated analyte–chemosensor system. Using the proposed theoretical model and the experimental data for the acetone–chemosensor system based on polyaniline, wedetermine the values of structural-functional characteristics of the chemosensor which were not estimated before (the total number of receptors in the polymer, the current through a single conducting receptor, association–dissociation rate constants of acetone molecule with receptor molecule, and rate constants of exchange with acetone between the polymer and the environment). The developed theoretical model satisfactory reproduces the experimental kinetic curve of the current through the sensor for the time range for which we have reliable experimental data.

Homeostatic Competition between Phasic and Tonic Inhibition

The GABAA receptors are the major inhibitory receptors in the brain and are localized at both synaptic and extrasynaptic membranes. Synaptic GABAA receptors mediate phasic inhibition, whereas extrasynaptic GABAA receptors mediate tonic inhibition. Both phasic and tonic inhibitions regulate neuronal activity, but whether they regulate each other is not very clear. Here, we investigated the functional interaction between synaptic and extrasynaptic GABAA receptors through various molecular manipulations. Overexpression of extrasynaptic α6β3δ-GABAA receptors in mouse hippocampal pyramidal neurons significantly increased tonic currents. Surprisingly, the increase of tonic inhibition was accompanied by a dramatic reduction of the phasic inhibition, suggesting a possible homeostatic regulation of the total inhibition. Overexpressing the α6 subunit alone induced an up-regulation of δ subunit expression and suppressed phasic inhibition similar to overexpressing the α6β3δ subunits. Interestingly, blocking all GABAA receptors after overexpressing α6β3δ receptors could not restore the synaptic GABAergic transmission, suggesting that receptor activation is not required for the homeostatic interplay. Furthermore, insertion of a gephyrin-binding-site (GBS) into the α6 and δ subunits recruited α6(GBS)β3δ(GBS) receptors to postsynaptic sites but failed to rescue synaptic GABAergic transmission. Thus, it is not the positional effect of extrasynaptic α6β3δ receptors that causes the down-regulation of phasic inhibition. Overexpressing α5β3γ2 subunits similarly reduced synaptic GABAergic transmission. We propose a working model that both synaptic and extrasynaptic GABAA receptors may compete for limited receptor slots on the plasma membrane to maintain a homeostatic range of the total inhibition.

Higher pretreatment 5-HT1A receptor binding potential in bipolar disorder depression is associated with treatment remission: a naturalistic treatment pilot PET study.

Bipolar disorder is a major cause of disability and a high risk for suicide. The pathophysiology of the disorder remains largely unknown. Medication choice for bipolar depression patients involves trial and error. Our group reported previously that brain serotonin 1A (5-HT(1A)) receptor binding measured by positron emission tomography (PET) is higher in bipolar depression. We now investigated whether pretreatment 5-HT(1A) levels correlates with antidepressant medication outcome. Forty-one medication-free DSM-IV diagnosed, bipolar patients in a major depressive episode had brain PET scans performed using [(11)C]WAY-100635 and a metabolite corrected arterial input function. The patients then received naturalistic psychopharmacologic treatment as outpatients and a follow up Hamilton Depression Rating Scale (HDRS) after 3 months of treatment. Patients with 24 item HDRS scores less than 10 were considered to have remitted. A linear mixed effects model was used to compare BP(F) (binding potential, proportional to the total number of available receptors) in 13 brain regions of interest between remitters and nonremitters. Thirty-four patients completed 3 months of treatment and ratings; 9 had remitted. Remitters and nonremitters did not differ in age, sex, or recent medication history with serotonergic medications. Remitters had higher [(11)C]WAY-100635 BP(F) across all brain regions compared with nonremitters (P = 0.02). Higher pretreatment brain 5-HT(1A) receptor binding was associated with remission after 3 months of pharmacological treatment in bipolar depression. Prospective treatment studies are warranted to determine whether this test predicts outcome of specific types of treatment.

NMDA Receptors in GABAergic Synapses during Postnatal Development

GABA (gamma-aminobutyric-acid), the main inhibitory neurotransmitter in the adult brain, exerts depolarizing (excitatory) actions during development and this GABAergic depolarization cooperates with NMDARs (N-methyl-D-aspartate receptors) to drive spontaneous synchronous activity (SSA) that is fundamentally important for developing neuronal networks. Although GABAergic depolarization is known to assist in the activation of NMDARs during development, the subcellular localization of NMDARs relative to GABAergic synapses is still unknown. Here, we investigated the subcellular distribution of NMDARs in association with GABAergic synapses at the developmental stage when SSA is most prominent in mice. Using multiple immunofluorescent labeling and confocal laser-scanning microscopy in the developing mouse hippocampus, we found that NMDARs were associated with both glutamatergic and GABAergic synapses at postnatal day 6–7 and we observed a direct colocalization of GABAA- and NMDA-receptor labeling in GABAergic synapses. Electron microscopy of pre-embedding immunogold-immunoperoxidase reactions confirmed that GluN1, GluN2A and GluN2B NMDAR subunits were all expressed in glutamatergic and GABAergic synapses postsynaptically. Finally, quantitative post-embedding immunogold labeling revealed that the density of NMDARs was 3 times higher in glutamatergic than in GABAergic synapses. Since GABAergic synapses were larger, there was little difference in the total number of NMDA receptors in the two types of synapses. In addition, receptor density in synapses was substantially higher than extrasynaptically. These data can provide the neuroanatomical basis of a new interpretation of previous physiological data regarding the GABAAR-NMDAR cooperation during early development. We suggest that during SSA, synaptic GABAAR-mediated depolarization assists NMDAR activation right inside GABAergic synapses and this effective spatial cooperation of receptors and local change of membrane potential will reach developing glutamatergic synapses with a higher probability and efficiency even further away on the dendrites. This additional level of cooperation that operates within the depolarizing GABAergic synapse, may also allow its own modification triggered by Ca2+-influx through the NMDA receptors.

Mean Platelet Volume and Integrin Alleles Strongly Correlate with Increased Levels of Integrins αIIbβ3 and α2β1: Clinical Implications for Platelet Reactivity in Acute Coronary Syndome

Abstract 32 Background- Differences in the level of integrins expressed on the surface membranes of platelets can have a significant effect on platelet reactivity and adverse outcomes in a variety of thrombotic and hematologic dyscrasias. Among normal individuals, the basal level of platelet integrin aIIbb3 varies at least two-fold, while that of the integrin a2b1 exhibits a greater range of variation. The total number of integrin molecules and other receptors on platelets is influenced to a large extent by the total surface area of plasma membrane and consequently the mean platelet volume (MPV), which itself has a very strong genetic component. We sought to determine the relative contribution of mean platelet volume (MPV) and integrin gene alleles to expression levels of these integrins in patients with acute coronary syndrome (ACS) and normal subjects. Methods and Results- Monoclonal antibodies AP2 and 8C12 (or 12F1) were used in flow cytometry to measure platelet aIIbb3 and a2b1, respectively, in whole blood from 341 patients with ACS and 128 normal subjects. Comparisons were made with MPV, platelet count (PC) and in the normal subjects ITGA2 rs1126643 and ITGB3 rs5918 alleles. In both ACS patients and controls, there was a strong direct correlation between MPV and aIIbb3 level (p < 0.001). In control subjects, MPV and aIIbb3 level did not correlate with ITGB3 rs5918 alleles. For a2b1, MPV contributed modestly in either patients or controls, while ITGA2 rs1126643 alleles exerted the greatest effect. Moreover, we observed an inverse correlation between MPV and the rs1126643 minor allele. Conclusions- Our results indicate that MPV is the major effector of platelet membrane levels of aIIbb3, in both ACS patients and normal subjects, while ITGA2 rs1126643 contributes most strongly to a2b1 levels. Hyperactivity of larger platelets results from increased levels of key platelet receptors, notably integrin aIIbb3, in support of the contention that MPV is an accurate marker of risk for adverse outcome in ACS. This is consistent with published findings that MPV, the most common metric of platelet size, correlates very well with platelet reactivity, such that larger platelets have greater prothombotic potential, and elevated MPV is associated with increased platelet aggregation, thromboxane synthesis, b-thromboglobulin release, and expression of adhesion molecules. The most intriguing finding of our study is the inverse association of the ITGA2 rs1126643 minor allele T with MPV. The rs1126643 minor allele likely exerts this effect via the documented influence of a2b1 on megakaryocyte (MK) maturation. Disclosures:No relevant conflicts of interest to declare.

Amelioration of hypoxia-induced striatal 5-HT2A receptor, 5-HT transporter and HIF1 alterations by glucose, oxygen and epinephrine in neonatal rats

Alterations in neurotransmitters and its receptors expression induce brain injury during neonatal hypoxic insult. Molecular processes regulating the serotonergic receptors play an important role in the control of respiration under hypoxic insult. The present study focused on the serotonergic regulation of neonatal hypoxia and its resuscitation methods. Receptor binding assays and gene expression studies were done to evaluate the changes in 5HT2A receptors and its transporter in the corpus striatum of hypoxic neonatal rats and hypoxic rats resuscitated with glucose, oxygen and epinephrine. Total 5HT and 5HT2A receptor number was increased in hypoxic neonates along with an up regulation of 5HT2A receptor and 5HT transporter gene. The enhanced striatal 5HT2A receptors modulate the ventilatory response to hypoxia. Immediate glucose resuscitation was found to ameliorate the receptor and transporter alterations. Hypoxia induced ATP depletion mediated reduction in blood glucose levels can be encountered by glucose administration and oxygenation helps in overcoming the anaerobic condition. The adverse effect of immediate oxygenation and epinephrine supplementation was also reported. This has immense clinical significance in establishing a proper resuscitation for the management of neonatal hypoxia.

Physical Basis behind Achondroplasia, the Most Common Form of Human Dwarfism

Fibroblast growth factor receptor 3 (FGFR3) is a receptor tyrosine kinase that plays an important role in long bone development. The G380R mutation in FGFR3 transmembrane domain is known as the genetic cause for achondroplasia, the most common form of human dwarfism. Despite many studies, there is no consensus about the exact mechanism underlying the pathology. To gain further understanding into the physical basis behind the disorder, here we measure the activation of wild-type and mutant FGFR3 in mammalian cells using Western blots, and we analyze the activation within the frame of a physical-chemical model describing dimerization, ligand binding, and phosphorylation probabilities within the dimers. The data analysis presented here suggests that the mutation does not increase FGFR3 dimerization, as proposed previously. Instead, FGFR3 activity in achondroplasia is increased due to increased probability for phosphorylation of the unliganded mutant dimers. This finding has implications for the design of targeted molecular treatments for achondroplasia.

Adenosine A2A Receptor Signaling and Golf Assembly Show a Specific Requirement for the γ7 Subtype in the Striatum

The adenosine A(2A) receptor (A(2A)R) is increasingly recognized as a novel therapeutic target in Parkinson disease. In striatopallidal neurons, the G-protein α(olf) subtype is required to couple this receptor to adenylyl cyclase activation. It is now well established that the βγ dimer also performs an active role in this signal transduction process. In principal, sixty distinct βγ dimers could arise from combinatorial association of the five known β and 12 γ subunit genes. However, key questions regarding which βγ subunit combinations exist and whether they perform specific signaling roles in the context of the organism remain to be answered. To explore these questions, we used a gene targeting approach to specifically ablate the G-protein γ(7) subtype. Revealing a potentially new signaling paradigm, we show that the level of the γ(7) protein controls the hierarchial assembly of a specific G-protein α(olf)β(2)γ(7) heterotrimer in the striatum. Providing a probable basis for the selectivity of receptor signaling, we further demonstrate that loss of this specific G-protein heterotrimer leads to reduced A(2A)R activation of adenylyl cyclase. Finally, substantiating an important role for this signaling pathway in pyschostimulant responsiveness, we show that mice lacking the G-protein γ(7) subtype exhibit an attenuated behavioral response to caffeine. Collectively, these results further support the A(2A)R G-protein α(olf)β(2)γ(7) interface as a possible therapeutic target for Parkinson disease.