NG108-15 Cells Research Articles

Recombinant Spider Silk and Collagen-Based Nerve Guidance Conduits Support Neuronal Cell Differentiation and Functionality in Vitro.

Biomaterial scaffolds are under investigation as therapeutic tools to bridge nerve endings following traumatic peripheral nerve injury. The goal is to develop biocompatible nerve guidance conduits (NGCs) with internal guiding structures that promote longitudinally oriented cell migration and regeneration. In the present study, a nonwoven mesh (NWM) made of a recombinant spider silk protein was processed into a tubular structure, ensuring structural integrity of enclosed microfluidics-produced collagen fibers for cell and neurite guidance. The differentiated type of the neuroblastoma X glioma hybrid cell line NG108-15 was used as a model for studying neuronal differentiation on the individual components and on the complete NGC. Differentiated NG108-15 cells grown on recombinant spider silk NWM and collagen fibers formed neuronal networks and synapses. Additionally, whole-cell patch clamp recordings confirmed that all components supported the differentiation of NG108-15 cells into functional neurons. Our NGC demonstrated that tubes made of recombinant spider silk NWM filled with microfluidics-produced collagen fibers are well suited for peripheral nerve repair.

AIE Triggers the Circularly Polarized Luminescence of Atomically Precise Enantiomeric Copper(I) Alkynyl Clusters.

Atomically precise enantiomeric metal clusters are scarce, and copper(I) alkynyl clusters with intense circularly polarized luminescence (CPL) responses have not been reported. A pair of chiral alkynyl ligands, (R/S)-2-diphenyl-2-hydroxylmethylpyrrolidine-1-propyne (abbreviated as R/S-DPM) we successfully prepared and single crystals were characterized of optically pure enantiomeric pair of atomically-precise copper(I) clusters, [Cu14 (R/S-DPM)8 ](PF6 )6 (denoted as R/S-Cu14 ), which feature bright red luminescence and CPL with a high luminescence anisotropy factor (glum ). A dilute solution containing R/S-Cu14 was nonluminescent and CPL inactive at room temperature. Crystallization- and aggregation-induced emission (CIE and AIE, respectively) contribute to the triggering of the CPL of R/S-Cu14 in the crystalline and aggregated states. Their AIE behavior and good biocompatibility indicated applications of these copper(I) clusters in cell imaging in HeLa and NG108-15 cells.

Chronic Treatment With Imipramine Inhibits Cell Growth and Enhances Serotonin 2C Receptor mRNA Expression in NG 108-15 Cells

We previously reported that NG108-15 cells contain intrinsic serotonin 2C receptor (5-HT2CR). The effects of imipramine, a 5-HT2CR antagonist, on cell growth, cell viability, and the 5-HT2CR mRNA level were investigated in this study. Repeated treatment with imipramine at concentrations of 1 – 10 μM for 5 days inhibited cell growth in a concentration-dependent manner without affecting cell viability. In addition, the level of 5-HT2CR mRNA was elevated. At 30 μM, imipramine significantly reduced cell viability. Our findings suggest that the effect of imipramine on neuronal growth may be related to its effects on 5-HT2CR.

Gene expression profile of the hippocampus of rats subjected to traumatic brain injury.

Traumatic brain injury (TBI) is a serious public health problem as well as a leading cause of severe posttraumatic disability. Numerous studies indicate that the differentially expressed genes (DEGs) of neural signaling pathways are strongly correlated with brain injury. To further analyze the roles of the DGEs in the central nervous system, here we systematically investigated TBI on the hippocampus and its injury mechanism at the whole genome level. On the basis of Gene Ontology and Kyoto Encyclopedia of Genes and Genomes Analyses, we revealed that the DEGs were involved in many signaling pathways related to the nervous system, especially neuronal survival-related pathways. Finally, we verified the microarray results and detected the gene expression of neuronal survival-related genes in the hippocampus by using real-time quantitative polymerase chain reaction. With Western blot and axon growth assay, the expression of P2rx3 was upregulated in rats subjected to TBI, and overexpression of P2rx3 promoted neurite growth of NG108 cells. Our results suggested that the DEGs (especially P2rx3) and several signaling pathways might play a pivotal role in TBI. We also provided several targeted genes related to TBI for future investigation.

Producing Neurospheroids and Hydrogels to Create a Three-dimensional in Vitro Model for the Use of Conduits in Peripheral Nerve Regeneration

Abstract INTRODUCTION Peripheral nerve regeneration is a complex challenge that requires suitable nerve guidance systems to bridge the severed ends of 2 nerves back together. Current polymeric conduits on the market provide good cellular growth but are limited by the length of gap defect they can repair, and complete functional recovery is rare. This project focused on creating a three-dimensional (3D) in Vitro spheroidal sprouting assay for peripheral nerve regeneration, as well as producing and testing different polymeric hydrogels as potential scaffold materials for the conduit. METHODS Different concentrations of chitosan, methylcellulose (MC) and sodium alginate were produced, as well as blends of these materials. These hydrogels were seeded with 3D neurospheroids, along with NG108-15 (neuronal) cells and Schwann cells to test their biocompatibility. RESULTS MTT assays showed the mean absorbance of chitosan gels with NG108-15 cells at 24 hr (P < .001) and 72 hr (P > .05) was similar/slightly higher than the negative control. Live-Dead data showed 93.4% of live cells at DIV7 on MC: Ch blends, compared to 72% with chitosan alone. CONCLUSION Overall, both chitosan and MC were nontoxic and biocompatible with NG108-15 and Schwann cells. Blending chitosan with MC improved its chemical and physical properties. The cells formed spheroids that well on a gel; this pseudo-3D structure is excellent for research purposes compared to 2D as it mimics the body's internal environment.

Biosynthesis and characterization of a novel, biocompatible medium chain length polyhydroxyalkanoate by Pseudomonas mendocina CH50 using coconut oil as the carbon source.

This study validated the utilization of triacylglycerides (TAGs) by Pseudomonas mendocina CH50, a wild type strain, resulting in the production of novel mcl-PHAs with unique physical properties. A PHA yield of 58% dcw was obtained using 20 g/L of coconut oil. Chemical and structural characterisation confirmed that the mcl-PHA produced was a terpolymer comprising of three different repeating monomer units, 3-hydroxyoctanoate, 3-hydroxydecanoate and 3-hydroxydodecanoate or P(3HO-3HD-3HDD). Bearing in mind the potential of P(3HO-3HD-3HDD) in biomedical research, especially in neural tissue engineering, in vitro biocompatibility studies were carried out using NG108-15 (neuronal) cells. Cell viability data confirmed that P(3HO-3HD-3HDD) supported the attachment and proliferation of NG108-15 and was therefore confirmed to be biocompatible in nature and suitable for neural regeneration.

PEDOT Coated Thick Film Electrodes for In Situ Detection of Cell Adhesion in Cell Cultures.

Low temperature cofired ceramics (LTCC) provide a technology for the 3-dimensional integration of sensor arrays into bioreactors covering dimensions of several hundred micrometers. Since optical control in such assemblies is not possible, the in situ detection of cell adhesion on impedance electrodes with high spatial resolution would deliver crucial information. A current limitation is the increasing impedance of microelectrodes with decreasing diameter. This study evaluates the suitability of thick film gold electrodes, pristine and coated with electropolymerized poly(3,4-ethylenedioxythiophene) (PEDOT), for the detection of cell adhesion on the electrode surface. The impedance as criterion for cell attachment is measured with a recording system for electroactive cells with the aim of improving usability. Two cell cultures with different adhesion characteristic are used for adhesion assessment on planar test chips. The impedance increase measured on individual PEDOT coated electrodes due to tight contact of cells reaches a factor of 6.8 in cultures of well-adherent HepG2 cells. Less adhered NG108-15 cells produce a maximum impedance increase by a factor of 2.6. Since the electrode impedance is significantly reduced by PEDOT coating, a reduction of the electrode diameter to values below 100 µm and spatially resolved detection is possible. The results encourage further studies using PEDOT coated thick film electrodes as bio-electronic-interfaces. We presume that such miniaturized electrodes are suitable for 3-dimensional recordings in electroactive cell cultures, providing information of local cell adhesion at the same time.

An Optimized Collagen-Fibrin Blend Engineered Neural Tissue Promotes Peripheral Nerve Repair.

Tissue engineering approaches in nerve regeneration often aim to improve results by bridging nerve defects with conduits that mimic key features of the nerve autograft. One such approach uses Schwann cell self-alignment and stabilization within collagen gels to generate engineered neural tissue (EngNT). In this study, we investigated whether a novel blend of fibrin and collagen could be used to form EngNT, as before EngNT design a beneficial effect of fibrin on Schwann cell proliferation was observed. A range of blend formulations was tested in terms of mechanical behavior (gel formation, stabilization, swelling, tensile strength, and stiffness), and lead formulations were assessed in vitro. A 90% collagen 10% fibrin blend was found to promote SCL4.1/F7 Schwann cell viability and supported the formation of aligned EngNT, which enhanced neurite outgrowth in vitro (NG108 cells) compared to formulations with higher and lower fibrin content. Initial in vivo tests in an 8 mm rat sciatic nerve model using rolled collagen-fibrin EngNT rods revealed a significantly enhanced axonal count in the midsection of the repair, as well as in the distal part of the nerve after 4 weeks. This optimized collagen-fibrin blend therefore provides a novel way to improve the capacity of EngNT to promote regeneration following peripheral nerve injury.

Paclitaxel-induced HMGB1 release from macrophages and its implication for peripheral neuropathy in mice: Evidence for a neuroimmune crosstalk

Given our recent evidence for the role of high mobility group box 1 (HMGB1) in chemotherapy-induced peripheral neuropathy (CIPN) in rats, we examined the origin of HMGB1 and the upstream and downstream mechanisms of HMGB1 release involved in paclitaxel-induced neuropathy in mice. Paclitaxel treatment developed mechanical allodynia in mice, as assessed by von Frey test, which was prevented by an anti-HMGB1-neutralizing antibody or thrombomodulin alfa capable of inactivating HMGB1. RAGE or CXCR4 antagonists, ethyl pyruvate or minocycline, known to inhibit HMGB1 release from macrophages, and liposomal clodronate, a macrophage depletor, prevented the paclitaxel-induced allodynia. Paclitaxel caused upregulation of RAGE and CXCR4 in the dorsal root ganglia and macrophage accumulation in the sciatic nerve. In macrophage-like RAW264.7 cells, paclitaxel evoked cytoplasmic translocation of nuclear HMGB1 followed by its extracellular release, and overexpression of CBP and PCAF, histone acetyltransferases (HATs), known to cause acetylation and cytoplasmic translocation of HMGB1, which were suppressed by ethyl pyruvate, N-acetyl-l-cysteine, an anti-oxidant, and SB203580 and PDTC, inhibitors of p38 MAP kinase (p38MAPK) and NF-κB, respectively. Paclitaxel increased accumulation of reactive oxygen species (ROS) and phosphorylation of p38MAPK, NF-κB p65 and I-κB in RAW264.7 cells. In mice, N-acetyl-l-cysteine or PDTC prevented the paclitaxel-induced allodynia. Co-culture of neuron-like NG108-15 cells or stimulation with their conditioned medium promoted paclitaxel-induced HMGB1 release from RAW264.7 cells. Our data indicate that HMGB1 released from macrophages through the ROS/p38MAPK/NF-κB/HAT pathway participates in the paclitaxel-induced peripheral neuropathy in mice, and unveils an emerging therapeutic avenue targeting a neuroimmune crosstalk in CIPN.

Simulated Microgravity Culture Enhances the Neuroprotective Effects of Human Cranial Bone-Derived Mesenchymal Stem Cells in Traumatic Brain Injury.

Fundamental cures of central nervous system (CNS) diseases are rarely achieved due to the low regenerative ability of the CNS. Recently, cell-based therapy using mesenchymal stem cells (MSCs) has been explored as an effective treatment for CNS diseases. Among the various tissue-derived MSCs, we have isolated human cranial bone-derived MSCs (cMSCs) in our laboratory. In addition, we have focused on simulated microgravity (MG) as a valuable culture environment of MSCs. However, detailed mechanisms underlying functional recovery from transplantation of MSCs cultured under MG conditions remain unclear. In this study, we investigated the therapeutic mechanisms of transplantation of cMSCs cultured under MG conditions in traumatic brain injury (TBI) model mice. Human cMSCs were cultured under 1G and MG conditions, and cMSCs cultured under MG conditions expressed significantly higher messenger RNA (mRNA) levels of hepatocyte growth factor (HGF) and transforming growth factor beta (TGF-β). In TBI model mice, the transplantation of cMSCs cultured under MG conditions (group MG) showed greater motor functional improvement compared with only phosphate-buffered saline administration (group PBS). Moreover, the protein expression levels of tumor necrosis factor alpha (TNF-α) and the Bcl-2-associated X protein (Bax)/b cell leukemia/lymphoma 2 protein (Bcl-2) ratio were significantly lower at brain injury sites in mice of group MG than those of group PBS. In addition, an in vitro study showed that the conditioned medium of cMSCs cultured under MG conditions significantly suppressed the cell death of NG108-15 cells exposed to oxidative or inflammatory stress through anti-inflammatory and antiapoptosis effects. These findings demonstrate that culturing cMSCs under simulated MG increases the neuroprotective effects, suggesting that simulated MG cultures may be a useful method for cell-based therapy strategies for CNS diseases.

Blockade of T-type calcium channels by 6-prenylnaringenin, a hop component, alleviates neuropathic and visceral pain in mice

Since Cav3.2 T-type Ca2+ channels (T-channels) expressed in the primary afferents and CNS contribute to intractable pain, we explored T-channel-blocking components in distinct herbal extracts using a whole-cell patch-clamp technique in HEK293 cells stably expressing Cav3.2 or Cav3.1, and purified and identified sophoraflavanone G (SG) as an active compound from SOPHORAE RADIX (SR). Interestingly, hop-derived SG analogues, (2S)-6-prenylnaringenin (6-PNG) and (2S)-8-PNG, but not naringenin, also blocked T-channels; IC50 (μM) of SG, (2S)-6-PNG and (2S)-8-PNG was 0.68–0.75 for Cav3.2 and 0.99–1.41 for Cav3.1. (2S)-6-PNG and (2S)-8-PNG, but not SG, exhibited reversible inhibition. The racemic (2R/S)-6-PNG as well as (2S)-6-PNG potently blocked Cav3.2, but exhibited minor effect on high-voltage-activated Ca2+ channels and voltage-gated Na+ channels in differentiated NG108-15 cells. In mice, the mechanical allodynia following intraplantar (i.pl.) administration of an H2S donor was abolished by oral or i.p. SR extract and by i.pl. SG, (2S)-6-PNG or (2S)-8-PNG, but not naringenin. Intraperitoneal (2R/S)-6-PNG strongly suppressed visceral pain and spinal ERK phosphorylation following intracolonic administration of an H2S donor in mice. (2R/S)-6-PNG, administered i.pl. or i.p., suppressed the neuropathic allodynia induced by partial sciatic nerve ligation or oxaliplatin, an anti-cancer agent, in mice. (2R/S)-6-PNG had little or no effect on open-field behavior, motor performance or cardiovascular function in mice, and on the contractility of isolated rat aorta. (2R/S)-6-PNG, but not SG, was detectable in the brain after their i.p. administration in mice. Our data suggest that 6-PNG, a hop component, blocks T-channels, and alleviates neuropathic and visceral pain with little side effects.

Cell Mechanotransduction With Piconewton Forces Applied by Optical Tweezers.

Mechanical stresses are always present in the cellular environment and mechanotransduction occurs in all cells. Although many experimental approaches have been developed to investigate mechanotransduction, the physical properties of the mechanical stimulus have yet to be accurately characterized. Here, we propose a mechanical stimulation method employing an oscillatory optical trap to apply piconewton forces perpendicularly to the cell membrane, for short instants. We show that this stimulation produces membrane indentation and induces cellular calcium transients in mouse neuroblastoma NG108-15 cells dependent of the stimulus strength and the number of force pulses.

Confocal micrographs: automated segmentation and quantitative shape analysis of neuronal cells treated with ostreolysin A/pleurotolysin B pore-forming complex.

Detailed shape analysis of cells is important to better understand the physiological mechanisms of toxins and determine their effects on cell morphology. This study aimed to develop a procedure for accurate morphological analysis of cell shape and use it as a tool to estimate toxin activity. With the aim of optimizing the method of cell morphology analysis, we determined the influence of ostreolysin A and pleurotolysin B complex (OlyA/PlyB) on the morphology of murine neuronal NG108-15 cells. A computational method was introduced and successfully applied to quantify morphological attributes of the NG108-15 cell line before and after 30 and 60min exposure to OlyA/PlyB using confocal microscopy. The modified circularity measure [Formula: see text] for shape analysis was applied, which defines the degree to which the shape of the neuron differs from a perfect circle. It enables better detection of small changes in the shape of cells, making the outcome easily detectable numerically. Additionally, we analyzed the influence of OlyA/PlyB on the cell area, allowing us to detect the cells with blebs. This is important because the formation of plasma membrane protrusions such as blebs often reflects cell injury that leads to necrotic cell death. In summary, we offer a novel analytical method of neuronal cell shape analysis and its correlation with the toxic effects of the pore-forming OlyA/PlyB toxin in situ.

Simultaneous recording of electrical activity and the underlying ionic currents in NG108-15 cells cultured on gold substrate

This paper shows the simultaneous recording of electrical activity and the underlying ionic currents by using a gold substrate to culture NG108-15 cells. Cells grown on two different substrates (plastic Petri dishes and gold substrates) were characterized quantitatively through scanning electron microscopy (SEM) as well as qualitatively by optical and atomic force microscopy (AFM). No significant differences were observed between the surface area of cells cultured on gold substrates and Petri dishes, as indicated by measurements performed on SEM images. We also evaluated the electrophysiological compatibility of the cells through standard patch-clamp experiments by analyzing features such as the resting potential, membrane resistance, ionic currents, etc. Cells grown on both substrates showed no significant differences in their dependency on voltage, as well as in the magnitude of the Na+ and K+ current density; however, cells cultured on the gold substrate showed a lower membrane capacitance when compared to those grown on Petri dishes. By using two separate patch-clamp amplifiers, we were able to record the membrane current with the conventional patch-clamp technique and through the gold substrate simultaneously. Furthermore, the proposed technique allowed us to obtain simultaneous recordings of the electrical activity (such as action potentials firing) and the underlying membrane ionic currents. The excellent conductivity of gold makes it possible to overcome important difficulties found in conventional electrophysiological experiments such as those presented by the resistance of the electrolytic bath solution. We conclude that the technique here presented constitutes a solution to the problem of the simultaneous recording of electrical activity and the underlying ionic currents, which for decades, had been solved only partially.

Electropermeabilization of cells by closely spaced paired nanosecond-range pulses

Decreasing the time gap between two identical electric pulses is expected to render bioeffects similar to those of a single pulse of equivalent total duration. In this study, we show that it is not necessarily true, and that the effects vary for different permeabilization markers. We exposed individual CHO or NG108 cells to one 300-ns pulse (3.7–11.6 kV/cm), or a pair of such pulses (0.4–1000 μs interval), or to a single 600-ns pulse of the same amplitude. Electropermeabilization was evaluated (a) by the uptake of YO-PRO-1 (YP) dye; (b) by the amplitude of elicited Ca2+ transients, and (c) by the entry of Tl+ ions. For YP uptake, applying a 600-ns pulse or a pair of 300-ns pulses doubled the effect of a single 300-ns pulse; this additive effect did not depend on the time interval between pulses or the electric field, indicating that already permeabilized cells are as susceptible to electropermeabilization as naïve cells. In contrast, Ca2+ transients and Tl+ uptake increased in a supra-additive fashion when two pulses were delivered instead of one. Paired pulses at 3.7 kV/cm with minimal separation (0.4 and 1 μs) elicited 50–100% larger Ca2+ transients than either a single 600-ns pulse or paired pulses with longer separation (10–1000 μs). This paradoxically high efficiency of the closest spaced pulses was emphasized when Ca2+ transients were elicited in a Ca2+-free solution (when the endoplasmic reticulum (ER) was the sole significant source of Ca2+), but was eliminated by Ca2+ depletion from the ER and was not observed for Tl+ entry through the electropermeabilized membrane. We conclude that closely spaced paired pulses specifically target ER, by either permeabilizing it to a greater extent than a single double-duration pulse thus causing more Ca2+ leak, or by amplifying Ca2+-induced Ca2+ release by an unknown mechanism.

Surfactant-exfoliated graphene as a near-infrared photothermal ablation agent

The near-infrared photothermal ability of pristine surfactant-exfoliated graphene was studied and demonstrated through the thermal ablation of mammalian NG108-15 cells. The graphene microsheets were prepared with polyethylene glycol triblock surfactants to improve biocompatibility while achieving high yields using a scalable and low-cost production method without the need for costly and time-consuming purification steps. The cytotoxicity of the surfactant-coated graphene was studied and it showed moderate toxicity. The ability of the exfoliated graphene to withstand extreme temperature cycling was demonstrated, highlighting the suitability of this material for multiple thermal ablation exposures. The pristine nature of the graphene sheets results in greater absorption in the near-infrared compared with graphene oxide or reduced graphene oxide. The photothermal transduction efficiency was also enhanced and determined to be ≈80% at 808 nm leading to the high efficacy of pristine graphene as an ablation agent.

Simulated Cholinergic Reinnervation of β (INS-1) Cells: Antidiabetic Utility of Heterotypic Pseudoislets Containing β Cell and Cholinergic Cell.

Cholinergic neurons can functionally support pancreatic islets in controlling blood sugar levels. However, in islet transplantation, the level of cholinergic reinnervation is significantly lower compared to orthotopic pancreatic islets. This abnormal reinnervation affects the survival and function of islet grafts. In this study, the cholinergic reinnervation of beta cells was simulated by 2D and 3D coculture of INS-1 and NG108-15 cells. In 2D culture conditions, 20 mM glucose induced a 1.24-fold increase (p < 0.0001) in insulin secretion from the coculture group, while in the 3D culture condition, a 1.78-fold increase (p < 0.0001) in insulin secretion from heterotypic pseudoislet group was observed. Glucose-stimulated insulin secretion (GSIS) from 2D INS-1 cells showed minimal changes when compared to 3D structures. E-cadherin expressed in INS-1 and NG108-15 cells was the key adhesion molecule for the formation of heterotypic pseudoislets. NG108-15 cells hardly affected the proliferation of INS-1 cells in vitro. Heterotypic pseudoislet transplantation recipient mice reverted to normoglycemic levels faster and had a greater blood glucose clearance compared to INS-1 pseudoislet recipient mice. In conclusion, cholinergic cells can promote insulin-secreting cells to function better in vitro and in vivo and E-cadherin plays an important role in the formation of heterotypic pseudoislets.

SNC80 and naltrindole modulate voltage-dependent sodium, potassium and calcium channels via a putatively delta opioid receptor-independent mechanism.

SNC80 was designed as a highly selective nonpeptide delta opioid receptor (DOR) agonist. Antidepressant-like and antinociceptive effects of this compound were demonstrated in animal models. Naltrindole was synthetized as a highly selective DOR antagonist. Its antitussive and antinociceptive effects were reported. Observed effects of SNC80 and naltrindole may be accompanied by changes in neuronal excitability including modulation of voltage-dependent ion channels. We investigated possible DOR-independent modulation of neuronal sodium, calcium and potassium currents by both agents. NG108-15 cells lacking expression of DOR protein were used as model of neuronal cells. Cells were differentiated into neuronal phenotype by exposure to dibutyryl cyclic-AMP (dbcAMP). Lack of DORs expression in NG108-15 cells and the presence of DOR expression in brain and neuronal cultures were demonstrated by Western blot analysis. Both SNC80 and naltrindole exerted low to moderate modulatory effects on voltage-dependent ion currents. SNC80 weakly inhibited sodium current, potentiated calcium current, and did not act on potassium channels. Naltrindole inhibited sodium current, did not act on calcium current and inhibited potassium current at a high concentration. Such effects should be taken into account when these compounds are used for investigation of DOR-mediated signaling pathways.

Disruption of dopamine D1/D2 receptor complex is involved in the function of haloperidol in cardiac H9c2 cells

AimsHaloperidol is an antipsychotic agent and acts as dopamine D2 receptor (D2R) antagonist, as a prototypical ligand of sigma1 receptors (Sig1R) and it increases expression of type 1 IP3 receptors (IP3R1). However, precise mechanism of haloperidol action on cardiomyocytes through dopaminergic signaling was not described yet. This study investigated a role of dopamine receptors in haloperidol-induced increase in IP3R1 and Sig1R, and compared physiological effect of melperone and haloperidol on basic heart parameters in rats. Materials and methodsWe used differentiated NG-108 cells and H9c2 cells. Gene expression, Western blot and immunofluorescence were used to evaluate haloperidol-induced differences; proximity ligation assay (PLA) and immunoprecipitation to determine interactions of D1/D2 receptors. To evaluate cardiac parameters, Wistar albino male rats were used. Key findingsWe have shown that antagonism of D2R with either haloperidol or melperone results in upregulation of both, IP3R1 and Sig1R, which is associated with increased D2R, but reduced D1R expression. Immunofluorescence, immunoprecipitation and PLA support formation of heteromeric D1/D2 complexes in H9c2 cells. Treatment with haloperidol (but not melperone) caused decrease in systolic and diastolic blood pressure and significant increase in heart rate. SignificanceBecause D1R/D2R complexes can engage Gq-like signaling in other experimental systems, these results are consistent with the possibility that disruption of D1R/D2R complex in H9c2 cells might cause a decrease in IP3R1 activity, which in turn may account for the increase expression of IP3R and Sig1R. D2R is probably not responsible for changes in cardiac parameters, since melperone did not have any effect.

Recovery of 3-Iodothyronamine and Derivatives in Biological Matrixes: Problems and Pitfalls.

Difficulties have been reported in quantitating 3-iodothyronamine (T1AM) in blood or serum, and tentatively attributed to problems in extraction or other pre-analytical steps. For this reason, even cell culture experiments have often be performed with unphysiological protein-free media. The aim of this study was to evaluate the recovery of exogenous T1AM added to a standard cell culture medium, namely Dulbecco's minimum essential medium (DMEM) supplemented with fetal bovine serum (FBS), and to other biological matrixes. Cell culture media (Krebs-Ringer buffer, DMEM, FBS, DMEM + FBS, used either in the absence or in the presence of NG108-15 cells) and other biological matrixes (rat brain and liver homogenates, human plasma, and blood) were spiked with T1AM and/or deuterated T1AM (d4-T1AM) and incubated for times ranging from 0 to 240 minutes. Samples were then extracted using a liquid/liquid method and analyzed using liquid chromatography coupled to mass spectrometry in order to assay T1AM and its metabolites, namely 3-iodothyroacetic acid (TA1), thyronamine, thyroacetic acid, N-acetyl-T1AM, and T1AM esters. In FBS-containing buffers, T1AM decreased exponentially over time, with a half-life of 6-17 minutes, depending on FBS content, and after 60 minutes, it averaged 0-10% of the baseline. T1AM metabolites were not detected, except for minimum amounts of TA1. Notably, d4-T1AM decreased over time at a much lower rate, reaching 50-70% of the baseline at 60 minutes. These effects were completely abolished by protein denaturation and partly reduced by semicarbazide. In the presence of cells, T1AM concentration decreased virtually to 0 within 60 minutes, but TA1 accumulated in the incubation medium, with quantitative recovery. Spontaneous decrease in T1AM concentration with isotopic difference was confirmed in rat organ homogenates and human blood. These results suggest binding and sequestration of T1AM and/or its aldehyde derivative by blood and tissue proteins, with significant isotope effects. These issues might account for the technical problems complicating the analytical assays of endogenous T1AM.