Downregulation of TRPC4 and TRPC5 Inhibits Smooth Muscle Cell Proliferation without Affecting Endothelial Cell Proliferation

Aims. The main treatment for coronary heart disease is percutaneous coronary intervention (PCI), and drug-eluting stents are designed to inhibit vascular smooth muscle cell (VSMCs) proliferation and migration causing restenosis by releasing pharmacological agents into the vessel wall. Once drug-eluting stents are deployed, these pharmacological agents exert many biological effects in the coronary circulation, not only inhibition of VSMCs but also extension to vascular endothelial cells (VECs). The purpose of this study was to explore target molecules that inhibit VSMCs proliferation without affecting VECs. Methods. mRNA and protein expressions of transient receptor potential channels (TRPCs) in cultured VSMCs and VECs were determined by western blotting and RT-qPCR. VSMCs and VECs proliferation was evaluated using CCK-8 assays and western blotting of proliferating cell nuclear antigen (PCNA). Calcium backfilling assays were performed to detect intracellular calcium ion concentration in cultured VSMCs and VECs. Results. The TRPC6 expression was more abundant in VECs than VSMCs, while TRPC4 and TRPC5 expressions were more abundant in VSMCs than VECs. Knockdown of TRPC4 or TRPC5 alone had no remarkable inhibitory effect on VSMC proliferation. Synergistic knockdown of TRPC4 and TRPC5 inhibited the proliferation of VSMCs, declined the expression of the PCNA, and reduced the intracellular calcium ion concentration but not VECs. Conclusion. These data suggest that concurrent inhibition of TRPC4 and TRPC5 inhibits VSMCs proliferation without affecting VECs, thus providing novel targets for developing pharmacological agents for drug-eluting stents.

Regulation of Dendrobium Polysaccharides on Proliferation and Oxidative Stress of Human Umbilical Vein Endothelial Cells in the High Glucose Environment

Backgrounds. Polysaccharides of Dendrobium candidum (PDC) showed a strong antioxidant effect on islet cells while the effects of PDC on human umbilical vein endothelial cells (HUVECs) under the high glucose condition remain unclear. Material and Method. HUVECs were incubated with high glucose (33.3 mmol/L) for 48 hours to induce injury, and cells were treated with PDC (100, 200, and 400 μg/mL) for 48 hours. The tetrazolium blue colorimetric (MTT) assay was used to detect cell proliferation, superoxide dismutase (SOD), and nitric oxide (NO) content in cell supernatants. Flow cytometry was used to detect reactive oxygen species (ROS) and calcium levels. Results. (1) Compared with the control group, the proliferation of HUVECs cells in the high glucose (33.3 mmol/L) group decreased ( P < 0.05 ). The intracellular calcium ion concentration and the intracellular ROS level increased ( P < 0.01 and P < 0.05 ). SOD activity and the level of NO in the culture medium were reduced (P <0.05). (2) Compared with the control group, PDC (50, 100, 200, 400, and 800 μg/mL) did not significantly affect the cell proliferation of HUVECs ( P > 0.05 ). (3) Compared with the high glucose group, the HUVEC cell viability of the high glucose + PDC (100, 200, and 400 μg/mL) group increased while the intracellular calcium ion concentration decreased in a concentration-dependent manner ( P < 0.05 ). Intracellular ROS levels were reduced, while SOD activity and the level of NO in culture fluids increased ( P < 0.05 ). Conclusion. PDC can promote the proliferation of HUVECs in the high glucose environment by reducing oxidative stress injury of HUVECs induced by high glucose.

Cav3.3 Down-Regulates CaMKII Beta to Ameliorate Neurotoxic Injury to the DRG Induced by Ropivacaine Hydrochloride

Background: The neurotoxicity of local anaesthetics is often reported. The present study aimed to investigate the relationship between Cav3.3 and CaMKⅡbeta in local anaesthetic neurotoxicity. Methods: An in vitro ropivacaine-induced model of neurotoxic injury to the dorsal root ganglion neurons was used. After specifically inhibiting the expression of Cav3.3 mRNA in DRG neurons by RNAi, cell viability, CaMKⅡbeta expression and the intracellular calcium ion level were detected in DRG neurons.Results: The results showed that the expression of CaMKⅡbeta decreased after the expression of Cav3.3 was inhibited. At the same time, in a model of ropivacaine hydrochloride-induced neurotoxic injury, inhibiting the expression of Cav3.3 mRNA improved cell viability and reduced the intracellular calcium level.Conclusions: These results suggest that Cav3.3 may be involved in neurotoxic injury induced by local anaesthetics by regulating the expression of CaMKⅡbeta. Both Cav3.3 and CaMKⅡbeta are therapeutic targets for neuronal injury induced by local anaesthetics.

Enhancement of DPP-IV Inhibitory Activity and GLP-1 Release Through RADA16-assisted Molecular Designed Rapeseed Peptide Nanogels

Objectives To produce and evaluate the microstructure and rheological behavior of a nanogel of pentapeptide IPQVS (RAP1) and octopeptide ELHQEEPL (RAP2), derived from rapeseed napin, through RADA16-assisted molecular design. In addition, to determine in vitro the dipeptidyl-peptidase IV (DPP-IV) inhibitory properties, glucagon-like peptide-1 (GLP-1) secretion through activation of calcium-sensing receptor (CaSR) and the intracellular calcium ion mobilization and receptor protein cAMP concentration. Methods An innovative nanogel, which loaded the RAP1 and RAP2 into RADA16 scaffold, respectively, was synthesized. The linker of double Gly was used in the connection of RADA16 and the functional oligopeptide region. DPP-IV inhibitory activity was evaluated in the Caco-2 cell monolayer. The microstructure and rheological behavior of RADA16-RAP1 and RADA16-RAP2 were characterized. The GLP-1 secretion through activation of CaSR receptor and the intracellular calcium ion mobilization and cAMP concentration were determined in STC-1 cells. Results DPP-IV inhibitory activity was reduced (more potent) by over 65% in the presence of 250 μM of the two formed nanogels RADA16–GG-IPQVS and RADA16- GG-ELHQEEPL (p &lt; 0.05). The two nanogel peptides had high stability at low temperature or body temperature and high dispersibility in water. Stable β-sheet structures increased by 5.6-fold and 5.2-fold, respectively, than the original oligopeptides, with a self-assembled fibrous morphology. The nanogels RADA16-RAP1 and RADA16-RAP2 might exhibit good rheological properties for potential injectable applications; storage modulus (G’) was 10 times higher than low modulus (G’’). Furthermore, the RAP2 and its RADA16-assisted nanogel peptide at the concentration of 250 μM significantly (p &lt; 0.05) increased the release of GLP-1 by 35.46% through calcium-sensing receptor (CaSR) pathway in the enteroendocrine STC-1 cells. Conclusions The innovated nanogel with the sequence of RADA16-GG-Xn has the possibility of oral and injection to management type 2 diabetes. DPP-IV inhibitory peptides IPQVS and ELHQEEPL can form a nanogel with high water content through the self-assembly gelation of RADA16. They may attach to the brush border of human intestinal epithelial cells to inhibit DPP-IV in the intestinal lumen. Funding Sources USDA-HATCH [grant number 1017440].

Isoprenoid Derivatives of Lysophosphatidylcholines Enhance Insulin and GLP-1 Secretion through Lipid-Binding GPCRs

Insulin plays a significant role in carbohydrate homeostasis as the blood glucose lowering hormone. Glucose-induced insulin secretion (GSIS) is augmented by glucagon-like peptide (GLP-1), a gastrointestinal peptide released in response to ingesting nutriments. The secretion of insulin and GLP-1 is mediated by the binding of nutrients to G protein-coupled receptors (GPCRs) expressed by pancreatic β-cells and enteroendocrine cells, respectively. Therefore, insulin secretagogues and incretin mimetics currently serve as antidiabetic treatments. This study demonstrates the potency of synthetic isoprenoid derivatives of lysophosphatidylcholines (LPCs) to stimulate GSIS and GLP-1 release. Murine insulinoma cell line (MIN6) and enteroendocrinal L cells (GLUTag) were incubated with LPCs bearing geranic acid (1-GA-LPC), citronellic acid (1-CA-LPC), 3,7-dimethyl-3-vinyloct-6-enoic acid (GERA-LPC), and (E)-3,7,11-trimethyl- 3-vinyldodeca-6,10-dienoic acid (1-FARA-LPC). Respective free terpene acids were also tested for comparison. Besides their insulin- and GLP-1-secreting capabilities, we also investigated the cytotoxicity of tested compounds, the ability to intracellular calcium ion mobilization, and targeted GPCRs involved in maintaining lipid and carbohydrate homeostasis. We observed the high cytotoxicity of 1-GERA-LPC and 1-FARA-LPC in contrast 1-CA-LPC and 1-GA-LPC. Moreover, 1-CA-LPC and 1-GA-LPC demonstrated the stimulatory effect on GSIS and 1-CA-LPC augmented GLP-1 secretion. Insulin and GLP-1 release appeared to be GPR40-, GPR55-, GPR119- and GPR120-dependent.

Mapping Co-regulatory Interactions among Ligand Binding sites in RyR1

Ryanodine receptor 1 (RyR1) is an intracellular calcium ion (Ca2+) release channel required for skeletal muscle contraction. Although cryo-electron microscopy identified binding sites of three coactivators Ca2+, ATP and caffeine (CFF), the mechanism of co-regulation and synergy of these activators is unknown. Here, we report allosteric connections among the three ligand binding sites and pore region in (i) Ca2+ bound-closed, (ii) ATP/CFF bound- closed, (iii) Ca2+/ATP/CFF bound-closed, and (iv) Ca2+/ATP/CFF bound-open RyR1 states. We identified two dominant interactions that mediate interactions between the Ca2+ binding site and pore region in Ca2+ bound-closed state, which partially overlapped with the pore communications in ATP/CFF bound-closed RyR1 state. In Ca2+/ATP/CFF bound-closed and -open RyR1 states, co-regulatory interactions were analogous to communications in the Ca2+ bound-closed and ATP/CFF bound- closed states. Both ATP- and CFF- binding sites mediate communication between the Ca2+ binding site and the pore region in Ca2+/ATP/CFF bound - open RyR1 structure. We conclude that Ca2+, ATP, and CFF propagate their effects to the pore region through a network of overlapping interactions that mediate allosteric control and molecular synergy in channel regulation.

Type I diabetes suppresses intracellular calcium ion increase normally evoked by heat stress in rat skeletal muscle

Heat stress, via its effects on muscle intracellular Ca2+ concentrations ([Ca2+]i), has been invoked as a putative therapeutic countermeasure to Type 1 diabetes-induced muscle atrophy. Using in vivo muscle preparation we tested the hypothesis that impaired muscle Ca2+ homeostasis in type I diabetic rats is due to attenuated heat stress tolerance mediated via TRPV1. Male Wistar rats were assigned to 1 of 4 groups: 1.control 30oC (CONT 30oC), 2.CONT 40oC, 3.diabetes 30oC (DIA 30oC), 4.DIA 40oC. 40oC was selected because it just exceeds the TRPV1 activation threshold. Spinotrapezius muscles were exteriorized in vivo and loaded with the fluorescent Ca2+ probe Fura-2AM. [Ca2+]i was estimated over 20min using fluorescence microscopy in quiescent muscle held at the required temperature using calibrated heat source applied to the ventral muscle surface. Western blotting was performed to determine the protein expression levels of TRPV1 in spinotrapezius muscle. After 20min of heat stress, the CONT 40oC condition induced a 12.3% [Ca2+]i elevation that was absent from the DIA 40oC or other conditions. Thus, no significant differences were found among DIA 40oC, DIA 30oC and CONT 30oC. TRPV1 protein expression was decreased by 42.0% in DIA compared with CONT (P<0.05) and, unlike CONT, heat stress did not increase TRPV1 phosphorylation. In conclusion, diabetes suppresses TRPV1 protein expression and function and inhibits the elevated myocyte [Ca2+]i evoked normally by heat stress. These results suggest that capsaicin or other therapeutic strategies to increase Ca2+ accumulation via TRPV1 might be more effective than hyperthermic therapy for Type I diabetic patients.

The investigation into neurotoxicity mechanisms of Nonylphenol: A narrative review

Background: Nonylphenol (NP), as a chemical compound that widely used in industry, is the result of the nonylphenol ethoxylate decomposition and it is known as an estrogen-like compound. Numerous studies and researches have shown that it has many destructive functions of various organs such as the brain. This toxicant causes oxidative stress in the cortex and hippocampus cells, which are two essential regions to preserve memory and learning in the brain. Methods: This review examines recent findings to better understand the mechanisms of NP neurotoxicity. We used Scopus, Google Scholar and PubMed databases to find articles with focus on the destructive effects of NP on the oxidative stress pathway and its defense mechanisms. Results: NP has potential human health hazard associated with gestational, peri- and postnatal exposure. NP can disrupt brain homeostasis in different ways, such as activation of inflammatory factors in brain especially in hippocampus and cortex, disruption of the cell cycle, changes in neuron, dendrites and synapses morphology, disruption of extra and intracellular calcium ion balance and also memory and learning disorders

In vivo Ca2+ dynamics during cooling after eccentric contractions in rat skeletal muscle

The effect of cooling on in vivo intracellular calcium ion concentration ([Ca2+]i) after eccentric contractions (ECs) remains to be determined. We tested the hypothesis that cryotherapy following ECs promotes an increased [Ca2+]i and induces greater muscle damage in two muscles with substantial IIb and IIx fiber populations. The thin spinotrapezius (SPINO) muscles of Wistar rats were used for in vivo [Ca2+]i imaging and tibialis anterior (TA) muscles provided greater fidelity and repeatability of contractile function measurements. SPINO [Ca2+]i was estimated using fura 2-AM and the magnitude, location and temporal profile of [Ca2+]i determined as the temperature near the muscle surface post-ECs was decreased from 30oC (control) to 20oC or 10oC. Subsequently, in the TA the effect of post-ECs cooling to 10oC on muscle contractile performance was determined at 1 and 2 days after ECs. TA muscle samples were examined by hematoxylin and eosin staining to assess damage. In SPINO reducing the muscle temperature from 30oC to 10oC post-ECs resulted in a 3.7-fold increase in the spread of high [Ca2+]i sites generated by ECs (P<0.05). These high [Ca2+]i sites demonstrated partial reversibility when rewarmed to 30oC. Dantrolene, a ryanodine receptor Ca2+ release inhibitor, reduced the presence of high [Ca2+] sites at 10oC. In the TA cooling exacerbated ECs-induced muscle strength deficits post-ECs via enhanced muscle fiber damage (P<0.05). By demonstrating that cooling post-ECs potentiates [Ca2+]i derangements, this in vivo approach supports a putative mechanistic basis for how post-exercise cryotherapy might augment muscle fiber damage and decrease subsequent exercise performance.