Channel Agonist Research Articles

ERK Phosphorylation Regulates the Aml1/Runx1 Splice Variants and the TRP Channels Expression during the Differentiation of Glioma Stem Cell Lines.

The identification of cancer stem cells in brain tumors paved the way for new therapeutic approaches. Recently, a role for the transcriptional factor Runx1/Aml1 and the downstream ion channel genes in brain cancer development and progression has been suggested. This study aimed to explore the expression and the role of Runx1/Aml1, its Aml1b and Aml1c splice variants and the downstream TRPA1 and TRPV1 ion channels in undifferentiated and day-14 differentiated neural stem cells (NSCs and D-NSCs) and glioblastoma stem cells (GSCs and D-GSCs) lines with different proneural (PN) or mesenchymal (MES) phenotype. Gene and protein expression were evaluated by qRT-PCR, cytofluorimetric, western blot and confocal microscopy analyses. Moreover, by western blot, we observed that ERK phosphorylation enhances the Aml1b and Aml1c protein expression during glioma differentiation. Furthermore, the agonists of TRPA1 and TRPV1 channels stimulated apoptosis/necrosis in GSCs and D-GSCs as evaluated by Annexin V and PI staining and cytofluorimetric analysis. Finally, by qRT-PCR, the modulation of Wnt/β catenin, FGF, and TGFβ/SMAD signaling pathways in PN- and MES-GSCs was reported. Overall, our results provide new evidence regarding Runx1/Aml1 isoform overexpression and modulation in TRP channel expression during gliomagenesis, thus offering new directions for glioblastoma therapy.

Effects of pharmacological agents for neurogenic oropharyngeal dysphagia: A systematic review and meta-analysis.

BackgroundThis systematic review and meta‐analysis aimed to evaluate the effects of pharmacological agents for neurogenic oropharyngeal dysphagia based on evidence from randomized controlled trials (RCTs).MethodsElectronic databases were systematically searched between January 1970 and March 2021. Two reviewers independently extracted and synthesized the data. The outcome measure was changed in (any) relevant clinical swallowing‐related characteristics.Key resultsData from 2186 dysphagic patients were collected from 14 RCT studies across a range of pharmacotherapies. The pooled effect size of transient receptor potential (TRP) channel agonists was large compared to placebo interventions (SMD[95%CI] =1.27[0.74,1.80], p < 0.001; I 2 = 79%). Data were limited for other pharmacological agents and the overall pooled effect size of these agents was non‐significant (SMD [95% CI] =0.25 [−0.24, 0.73]; p = 0.31; I 2 = 85%). When analyzed separately, large effect sizes were observed with Nifedipine (SMD[95%CI] =1.13[0.09,2.18]; p = 0.03) and Metoclopramide (SMD[95%CI] =1.68[1.08,2.27]; p < 0.001). By contrast, the effects of angiotensin‐converting enzyme (ACE) inhibitors (SMD[95%CI] = −0.67[−2.32,0.99]; p = 0.43; I 2 = 61%), Physostigmine (SMD[95%CI] = −0.05[−1.03,0.93]; p = 0.92) and Glyceryl Trinitrate (GTN) (SMD [95% CI] = −0.01 [−0.11, 0.08]; p = 0.78) were non‐significant. Within stroke patients, subgroup analysis showed that TRP channel agonists had a moderate pooled effect size (SMD[95%CI] =0.74[0.10,1.39]; p = 0.02; I 2 = 82%) whereas the effects of other agents were non‐significant (SMD[95%CI] =0.40[−0.04,0.84]; p = 0.07; I 2 = 87%).Conclusions & InferencesOur results showed that TRP channel agonists, Nifedipine and Metoclopromide may be beneficial for neurogenic dysphagic patients. Large scale, multicenter clinical trials are warranted to fully explore their therapeutic effects on swallowing.

Activation of SK3 channel plays a pivotal role in modulation of trigeminal neuralgia

ABSTRACT Objective: To investigate whether small conductance Ca2+ activatedK+ channels; Trigeminal ganglion; Trigeminal neuralgia. (SK3) exists in normal rats’ trigeminal ganglions (TG) and its effect on their pain thresholds. Methods: In total, 110 healthy adult male Sprague–Dawley (SD) rats were involved in this study. Ten rats were dissected to collect their liver tissues, TG and DRG. The rest of the rats were randomly assigned to either the experimental group or the control group. The animal model of trigeminal neuralgia (TN) was established by infraorbital nerve ligation. The expression of SK3 channels in their livers, TG and dorsal root ganglions (DRG) were detected. And different doses of SK3 channel activator and inhibitor were administered to the rats in both groups 15 days after the operation; meanwhile, their pain thresholds were also measured. Results: The expression of SK3 channel was found in TG. In the experimental group, the pain threshold was significantly decreased and there was a decreased level of SK3 than that in the control group at 15 days after operation. The administration of SK3 channel agonist (CyPPA) could significantly improve the pain threshold, while, the pain threshold decreased after administration of SK3 channel antagonist (Apamin). Conclusion: The SK3 channel may play a pivotal role in the pathogenesis of trigeminal neuralgia, and it may be one of the potential targets for the treatment of trigeminal neuralgia.

INVESTIGATION OF THE THERAPEUTIC AND PROTECTIVE EFFECTS OF ATP SENSITIVE POTASSIUM CHANNEL (KATP) AGONISTS AND ANTAGONISTS ON PENICILLIN INDUCED EXPERIMENTAL EPILEPSY MODELS IN RATS

Epilepsy is a disease occurring because of extreme activation of nervous system. Increased glutamate and Ca++ in brain is another reason of epilepsy. In this study, we investigated the effects of ATP sensitive potassium channel (KATP) agonists and antagonists on penicillin induced epileptiform activity in male wistar albino rats. Rats were divided three experimental main groups; (1) Control, (2) Before seizures (BS) groups, (3) During seizure (DS) groups. DS and BS groups are divided into four subgroups; (a)5HD, (b)HMR1098, (c)Bepridil (d)P 1075. Bepridil and p1075, reduce the number of spike-waves, while the effect of Bepridil appears to have a similar effect when administered both during and before the seizure. HMR1098 and 5-HD both increased the seizures both when administered before or during the seizure, different from the other studies. When K ATP channel agonists are administered before and during the seizure, they reduce the seizures, while antagonists increase the seizure.

TRP Channel Agonists Activate Different Afferent Neuromodulatory Mechanisms in Guinea Pig Urinary Bladder.

Activation of TRP channels expressed in urinary bladder afferent nerves and urothelium releases neurotransmitters that influence bladder function. Experiments were undertaken to examine the mechanisms underlying effects of TRPA1 (allyl isothiocyanate, AITC), TRPV1 (capsaicin, CAPS), and TRPC (oleoyl-2-acetyl-sn-glycerol, OAG) agonists on guinea pig bladder activity. Effects of these agonists were compared with effects of nitro-oleic acid (OA-NO2), an electrophilic nitro-fatty acid, known to activate TRPV1, TRPA1 or TRPC channels in sensory neurons. AITC (100 μM) increased (231%) area of spontaneous bladder contractions (SBCs) an effect reduced by a TRPA1 antagonist (HC3-03001, HC3, 10 μM) and reversed to inhibition by indomethacin (INDO, 500 nM) a cyclooxygenase inhibitor. The post-INDO inhibitory effect of AITC was mimicked (39% depression) by calcitonin gene-related peptide (CGRP, 100 nM) and blocked by a CGRP antagonist (BIBN, 25 μM). CAPS (1 μM) suppressed SBCs by 30% in 81% of strips, an effect blocked by a TRPV1 antagonist (diarylpiperazine, 1 μM) or BIBN. SBCs were suppressed by OA-NO2 (30 μM, 21% in 77% of strips) or by OAG (50 μM, 30%) an effect blocked by BIBN. OA-NO2 effects were not altered by HC3 or diarylpiperazine. OA-NO2 also induced excitation in 23% of bladder strips. These observations raise the possibility that guinea pig bladder is innervated by at least two types of afferent nerves: [1] Type A express TRPA1 receptors that induce the release of prostaglandins and excite the detrusor, [2] Type B express TRPV1, TRPA1 and TRPC receptors and release CGRP that inhibits the detrusor.

"Time window" effect of Yoda1-evoked Piezo1 channel activity during mouse skeletal muscle differentiation.

AimMechanosensitive Piezo1 ion channels emerged recently as important contributors to various vital functions including modulation of the blood supply to skeletal muscles. The specific Piezo1 channel agonist Yoda1 was shown to regulate the tone of blood vessels similarly to physical exercise. However, the direct role of Piezo1 channels in muscle function has been little studied so far. We therefore investigated the action of Yoda1 on the functional state of skeletal muscle precursors (satellite cells and myotubes) and on adult muscle fibres.MethodsImmunostaining, electrophysiological intracellular recordings and Ca2+ imaging experiments were performed to localize and assess the effect of the chemical activation of Piezo1 channels with Yoda1, on myogenic precursors, adult myofibres and at the adult neuromuscular junction.ResultsPiezo1 channels were detected by immunostaining in satellite cells (SCs) and myotubes as well as in adult myofibres. In the skeletal muscle precursors, Yoda1 treatment stimulated the differentiation and cell fusion rather than the proliferation of SCs. Moreover, in myotubes, Yoda1 induced significant [Ca2+] i transients, without detectable [Ca2+] i response in adult myofibres. Furthermore, although expression of Piezo1 channels was detected around the muscle endplate region, Yoda1 application did not alter either the nerve‐evoked or spontaneous synaptic activity or muscle contractions in adult myofibres.ConclusionOur data indicate that the chemical activation of Piezo1 channels specifically enhances the differentiation of skeletal muscle precursors, suggesting a possible new strategy to promote muscle regeneration.

Activation of Transient Receptor Potential Ankyrin 1 and Vanilloid 1 Channels Promotes Odontogenic Differentiation of Human Dental Pulp Cells

IntroductionTransient receptor potential ankyrin 1 (TRPA1) and vanilloid 1 (TRPV1) are thermosensitive channels that play an important role in thermal sensation or tooth pain by regulating intracellular Ca2+ concentration that is essential for pulp tissue repair. The aim of this study was to evaluate the role of TRPA1 and TRPV1 channels in the odontogenic differentiation of human dental pulp cells (HDPCs). MethodsHDPCs were isolated from healthy human intact third molars and cultured in odontogenic differentiation medium. Gene and protein expression levels of TRPA1 and TRPV1 channels during the odontogenic differentiation of HDPCs were evaluated by real-time quantitative polymerase chain reaction and Western blot analysis. HDPCs were then treated with channel agonists or antagonists, and the expression levels of odontogenic markers dentin sialophosphoprotein (DSPP) and osteopontin (OPN) were examined. Alkaline phosphatase activity and alizarin red staining were also conducted to detect mineralization levels. ResultsConsistent with the mineralization degree and DSPP and OPN expression, messenger RNA and protein expression of TRPA1 and TRPV1 channels was up-regulated during the odontogenic differentiation of HDPCs. The application of TRPA1 or TRPV1 agonists increased the mineralized nodules of alizarin red staining and alkaline phosphatase activity and up-regulated the messenger RNA and protein expression of DSPP and OPN, respectively, with the highest values reached on the seventh day (P < .05). On the contrary, the mineralization level and DSPP and OPN expression could be suppressed by using the antagonists of these 2 channels. ConclusionsTRPA1 and TRPV1 channels not only showed up-regulated expression along with the odontogenic differentiation of HDPCs but also could affect the odontogenic differentiation by regulating intracellular Ca2+ concentration.

Protective effect of HCN2-induced SON sensitization on chronic visceral hypersensitivity in neonatal-CRD rat model

Abnormal brain-gut interactions contribute to the development of chronic visceral hypersensitivity (CVH), which is the pivotal feature of irritable bowel syndrome (IBS). Despite the consensus with respect to the vital role of hyperpolarization-activated cyclic nucleotide-gated 2 (HCN2) channels in promoting painful symptoms in the peripheral nervous system, we identified that the upregulation of HCN2 in supraoptic nucleus (SON) was involved in the modulation of CVH in rat model of neonatal colorectal distention (n-CRD). Specifically, colorectal distention (CRD) upregulated the expression of c-Fos in SON in adult CVH rats, indicating the involvement of SON sensitazation in visceral sensation. Moreover, the administration of ZD7288 (the pan-HCN channel inhibitor) rather than 8-Br-cAMP (the non-specific HCN channel agonist) aggravated the CVH symptoms and reduced the phosphorylation level of CaMKII-CREB cascade. Together, the findings indicated that the upregulation of supraoptic HCN2 contributed to the sensitization of SON, which had protective effects on the modulation of CVH with the involvement of CaMKII-CREB cascade in n-CRD rat model.

Regulation of TRPML1 channel activity and inflammatory exosome release by endogenously produced reactive oxygen species in mouse podocytes

The nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome in podocytes has been implicated in the initiation of glomerular inflammation during hyperhomocysteinemia (hHcy). However, the mechanism by which NLRP3 inflammasome products are released from podocytes remains unknown. The present study tested whether exosome secretion from podocytes is enhanced by NADPH oxidase-produced reactive oxygen species (ROS), which may serve as a pathogenic mechanism mediating the release of inflammatory cytokines produced by the NLRP3 inflammasome in podocytes after Hcy stimulation. We first demonstrated the remarkable elevation of endogenously produced ROS in podocytes treated with Hcy compared with control podocytes, which was abolished by pre-treatment with the NADPH oxidase inhibitors, gp91 ds-tat peptide and diphenyleneiodonium (DPI). In addition, Hcy induced activation in podocytes of NLRP3 inflammasomes and the formation of multivesicular bodies (MVBs) containing inflammatory cytokines, which were prevented by treatment with gp91 ds-tat or the ROS scavenger, catalase. Given the importance of the transient receptor potential mucolipin 1 (TRPML1) channel in Ca2+-dependent lysosome trafficking and consequent lysosome-MVB interaction, we tested whether lysosomal Ca2+ release through TRPML1 channels is inhibited by endogenously produced ROS in podocytes after Hcy stimulation. By GCaMP3 Ca2+ imaging, we confirmed the inhibition of TRPML1 channel activity by Hcy which was remarkably ameliorated by catalase and gp91 ds-tat peptide. By structured illumination microscopy (SIM) and nanoparticle tracking analysis (NTA), we found that ML-SA1, a TRPML1 channel agonist, significantly enhanced lysosome-MVB interaction and reduced exosome release in podocytes, which were attenuated by Hcy. Pre-treatment of podocytes with catalase or gp91 ds-tat peptide restored ML-SA1-induced changes in lysosome-MVB interaction and exosome secretion. Moreover, we found that hydrogen peroxide (H2O2) mimicked the effect of Hcy on TRPML1 channel activity, lysosome-MVB interaction, and exosome secretion in podocytes. Based on these results, we conclude that endogenously produced ROS importantly contributes to inflammatory exosome secretion from podocytes through inhibition of TRPML1 channel activity, which may contribute to the initiation of glomerular inflammation during hHcy.

The TRPA1 Agonist Cinnamaldehyde Induces the Secretion of HCO3- by the Porcine Colon.

A therapeutic potential of the TRPA1 channel agonist cinnamaldehyde for use in inflammatory bowel disease is emerging, but the mechanisms are unclear. Semi-quantitative qPCR of various parts of the porcine gastrointestinal tract showed that mRNA for TRPA1 was highest in the colonic mucosa. In Ussing chambers, 1 mmol·L−1 cinnamaldehyde induced increases in short circuit current (ΔIsc) and conductance (ΔGt) across the colon that were higher than those across the jejunum or after 1 mmol·L−1 thymol. Lidocaine, amiloride or bumetanide did not change the response. The application of 1 mmol·L−1 quinidine or the bilateral replacement of 120 Na+, 120 Cl− or 25 HCO3− reduced ΔGt, while the removal of Ca2+ enhanced ΔGt with ΔIsc numerically higher. ΔIsc decreased after 0.5 NPPB, 0.01 indometacin and the bilateral replacement of 120 Na+ or 25 HCO3−. The removal of 120 Cl− had no effect. Cinnamaldehyde also activates TRPV3, but comparative measurements involving patch clamp experiments on overexpressing cells demonstrated that much higher concentrations are required. We suggest that cinnamaldehyde stimulates the secretion of HCO3− via apical CFTR and basolateral Na+-HCO3− cotransport, preventing acidosis and damage to the epithelium and the colonic microbiome. Signaling may involve the opening of TRPA1, depolarization of the epithelium and a rise in PGE2 following a lower uptake of prostaglandins via OATP2A1.

Redox Regulation of Lysosome‐MVB Interaction and Inflammatory Exosome Release during NLRP3 Inflammasome Activation by High Homocysteine in Mouse Podocytes

The nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome in podocytes has been implicated in the initiation of glomerular inflammation during hyperhomocysteinemia (hHcy). However, the mechanism by which NLRP3 inflammasome products are released from podocytes remains unknown. The present study tested whether exosome secretion from podocytes is enhanced by NADPH oxidase-produced reactive oxygen species (ROS), which may serve as a pathogenic mechanism mediating the release of products by NLRP3 inflammasome activation in podocytes. We first demonstrated the remarkable elevation of endogenously produced ROS in podocytes treated with homocysteine (Hcy) compared with control podocytes, which was abolished by pre-treatment with catalase, a hydrogen peroxide (H2O2) scavenger or gp91 ds-tat peptide, a NADPH oxidase inhibitor. Hcy also induced activation of NLRP3 inflammasome and formation of MVBs containing the products of NLRP3 inflammasome activation such as IL-1β in podocytes, which were prevented by catalase and gp91 ds-tat peptide. By super-resolution microscopy and nanoparticle tracking analysis (NTA), we found that ML-SA1, a TRPML1 channel agonist that increases lysosome trafficking by calcium release, significantly enhanced lysosome-MVB interaction and reduced exosome release in podocytes, which attenuated Hcy effects. In the presence of catalase or gp91 ds-tat peptide, moreover, Hcy-induced reduction of lysosome-MVB interaction and exosome secretion was restored. We also tested whether exogenous ROS can mimic the effects of Hcy on exosome release in podocytes. It was found that H2O2 activated NLRP3 inflammasome and induced formation of MVBs containing inflammatory cytokines in a dose-dependent manner. Furthermore, we confirmed that H2O2 decreased lysosome-MVB interaction and increased exosome release in podocytes. Based on these results, we conclude that endogenously produced ROS importantly contributes to inflammatory exosome secretion from podocytes during activation of NLRP3 inflammasome in response to high Hcy due to inhibition of lysosome-MVB interaction. This redox regulation of Hcy-induced inflammatory exosome release may be a critical molecular mechanism triggering glomerular inflammatory response during hHcy (supported by NIH grant DK120491).

Contribution of Enhancer of Zeste Homolog 2 gene in Calcification Nidus Formation and Exosome Release in Arterial Smooth Muscle Cells

Enhancer of zeste homolog 2 (Ezh2) as a histone methyltransferasehas been reported to epigenetically control gene expression, and it is known to be involved in phenotypic transition and osteogenesis, playing a critical role in multiple inflammatory diseases. Lysosomal transient receptor potential mucolipin 1 (TRPML1) channels regulating lysosome interaction with multivesicular bodies (MVBs) and consequent exosome release play a crucial role in the SMC phenotype transition to osteogenic during arterial medial calcification (AMC). However, there is lack of evidence to show whether Ezh2 participates in the phenotypic transition and vascular calcification process. The aim of this study was to determine the role of Ezh2 in high phosphate (Pi)-induced calcification and to explore its contribution to the phenotype change and exosome secretion in vascular smooth muscle cells (SMCs) that is essential for AMC. Using aortic SMCs isolated from SMC-specific Ezh2 knockout (Ezh2fl/fl/SMCre) mice and wild-type littermates, we demonstrated that high Pi treatment increased more calcium deposition in aortic SMCs as shown by the presence of Alizarin Red-stained nodules when Ezh2 gene is deleted or its activity is inhibited by GSK126. Further, this calcium deposition was significantly decreased by MLSA-1, a TRPML-1 channel agonist in these cells. Correspondingly, Ezh2 gene deletion induced phenotypic change in Pi-treated SMCs, as depicted by decreased SM22-α (SMC marker) and increased expression of osteogenic gene osteopontin in Western blot analysis, indicating that Ezh2 contributes to phenotype change in SMCs leading to calcification nidus formation. Using nanoparticle tracking analysis, Pi-induced secretion of exosomes (100-150 nm in size) were found significantly increased in SMCs, which was augmented by Ezh2 gene deletion or GSK126, or attenuated by MLSA-1. Similarly, GSK126 significantly increased calcification and exosome secretion in Pi-treated and human aortic SMCs (HASMCs), while MLSA-1 markedly decreased calcium deposition and exosome release in these cells. These results suggest that a normal expression of Ezh2 genecritically controls phenotype transition and exosome secretion in arterial SMCs and its defect or deficiency may be an important molecular mechanism leading to AMC.

Control of TRPML1 Channel Activity and Lysosome Trafficking by Acid Ceramidase in Mouse Coronary Arterial Endothelial Cells

Previous studies have demonstrated that lysosomal acid ceramidase (AC) regulates lysosome-multivesicular body (MVB) interaction and exosome release in mouse coronary arterial endothelial cells (CAECs). However, it remains unknown how exosome secretion is controlled by AC. In this study, we tested a hypothesis that enhancement of lysosome trafficking by AC activity increases lysosome-MVB interaction determining MVB fate to promote exosome release, which may be attributed to its action on lysosomal TRPML1 channel-mediated Ca2+ release in CAECs. By GCaMP3 Ca2+ imaging, we found that ML-SA1 as a TRPML1 channel agonist remarkably increased lysosomal Ca2+ release through TRPML1 channel in WT/WT CAECs. In Asah1fl/fl/ECcre CAECs, however, ML-SA1 failed to influence TRPML1 channel-mediated Ca2+ release. As an AC product, sphingosine remarkably increased lysosomal Ca2+ release through TRPML1 channel in both WT/WT and Asah1fl/fl/ECcre CAECs. With whole-lysosome patch clamp recording, we detected ML-SA1-induced Ca2+ currents in a dose-dependent manner through the membrane of lysosomes isolated from WT/WT CAECs, while intact lysosomes isolated from Asah1fl/fl/ECcre CAECs had evidently blunted opening of TRPML1 channels in response to ML-SA1. Different effects of AC associated sphingolipids on TRPML1 channel activity were also examined in lysosomes isolated from WT/WT CAECs. It was found that sphingomyelin inhibited, but sphingosine enhanced lysosomal TRPML1 channel activity. Ceramide did not alter this lysosomal channel activity. In lysosomes isolated from Asah1fl/fl/ECcre CAECs, only sphingosine increased ML-SA1-induced TRPML1 channel-mediated Ca2+ release. Using confocal microscopy for measurement of lysosomes movement, we demonstrated that lysosomal TRPML1 channel activation by ML-SA1 in CAECs markedly enhanced lysosome trafficking toward nucleus, which was blocked by genetic deletion of AC. These results suggest that AC-mediated sphingolipid metabolism controls lysosomal TRPML1 channel-mediated Ca2+ release and lysosome trafficking in CAECs, which may contribute to the regulation of lysosome-MVB interaction and exosome release by AC (supported by NIH grant DK120491).

Lacidipine Prevents Scopolamine-Induced Memory Impairment by Reducing Brain Oxido-nitrosative Stress in Mice.

Cholinergic deficits and oxido-nitrosative stress are consistently associated with Alzheimer's disease (AD). Previous findings indicate that acetylcholine subdues Ca2+ current in the brain. Cholinergic antagonists (e.g., scopolamine) can instigate Ca2+-induced redox imbalance, inflammation, and cell-death pathways leading to AD-type memory impairment. Earlier, several Ca2+-channel blockers (CCB, e.g., dihydropyridine type) or cholinergic enhancers showed promising results in animal models of AD. In the present research, pretreatment effects of lacidipine (L-type CCB) on learning and memory functions were investigated using the scopolamine mouse model of AD. Swiss albino mice (20-25g) were administered lacidipine (1 and 3mg/kg) for 14days. Scopolamine, an anti-muscarinic drug, was given (1mg/kg) from days 8 to 14. The mice were subjected to elevated plus maze (EPM) and passive-avoidance (PA) paradigms. Bay-K8644 (a Ca2+-channel agonist) was administered before behavioral studies on days 13 and 14. Biochemical parameters of oxidative stress and acetylcholinesterase (AChE) activity were quantified using the whole brain. Behavioral studies showed an increase in transfer latency (TL) in the EPM test and a decrease in step-through latency (STL) in the PA test in scopolamine-administered mice. Scopolamine enhanced the AChE activity and oxidative stress in the brain of mice which resulted in memory impairment. Lacidipine prevented the amnesia against scopolamine and reduced the oxidative stress and AChE activity in the brain of mice. Bay-K8644 attenuated the lacidipine-induced improvement in memory and redox balance in scopolamine-administered mice. Lacidipine can prevent the oxidative stress and improve the cholinergic function in the brain. These properties of lacidipine can mitigate the pathogenesis of AD-type dementia.

Hypoxic Conditions Promote Rhythmic Contractile Oscillations Mediated by Voltage-Gated Sodium Channels Activation in Human Arteries.

Arterial smooth muscle exhibits rhythmic oscillatory contractions called vasomotion and believed to be a protective mechanism against tissue hypoperfusion or hypoxia. Oscillations of vascular tone depend on voltage and follow oscillations of the membrane potential. Voltage-gated sodium channels (Nav), responsible for the initiation and propagation of action potentials in excitable cells, have also been evidenced both in animal and human vascular smooth muscle cells (SMCs). For example, they contribute to arterial contraction in rats, but their physiopathological relevance has not been established in human vessels. In the present study, we investigated the functional role of Nav in the human artery. Experiments were performed on human uterine arteries obtained after hysterectomy and on SMCs dissociated from these arteries. In SMCs, we recorded a tetrodotoxin (TTX)-sensitive and fast inactivating voltage-dependent INa current. Various Nav genes, encoding α-subunit isoforms sensitive (Nav 1.2; 1.3; 1.7) and resistant (Nav 1.5) to TTX, were detected both in arterial tissue and in SMCs. Nav channels immunostaining showed uniform distribution in SMCs and endothelial cells. On arterial tissue, we recorded variations of isometric tension, ex vivo, in response to various agonists and antagonists. In arterial rings placed under hypoxic conditions, the depolarizing agent KCl and veratridine, a specific Nav channels agonist, both induced a sustained contraction overlaid with rhythmic oscillations of tension. After suppression of sympathetic control either by blocking the release of catecholamine or by antagonizing the target adrenergic response, rhythmic activity persisted while the sustained contraction was abolished. This rhythmic activity of the arteries was suppressed by TTX but, in contrast, only attenuated by antagonists of calcium channels, Na+/Ca2+ exchanger, Na+/K+-ATPase and the cardiac Nav channel. These results highlight the role of Nav as a novel key element in the vasomotion of human arteries. Hypoxia promotes activation of Nav channels involved in the initiation of rhythmic oscillatory contractile activity.

15727 KATP channel prodrugs as therapeutics for chronic pain and substance abuse disorders

ABSTRACT IMPACT: Pharmacological activation of KATP channels may provide analgesia and attenuate opioid tolerance and withdrawal OBJECTIVES/GOALS: Our long term goal is to develop therapeutics for the treatment of the overuse of opioids. The objective of this application is to test novel KATP channel-targeting prodrugs in rodent models of neuropathic and inflammatory pain in addition to opioid tolerance after chronic morphine administration. METHODS/STUDY POPULATION: In one study, two different measures for chronic pain were implemented in mice. Male and female mice (n=10) were subjected to spinal nerve ligation (SNL) or intraplantar injection of Complete Freund’s Adjuvant (CFA) to induce neuropathic and inflammatory pain, respectively. Administration of KATP channel prodrugs (60ug, it) attenuated mechanical hypersensitivity after SNL or CFA compared to vehicle (saline). In a separate study, changes in mechanical hypersensitivity were tested while mice undergo chronic morphine treatment (15mg/kg, 2x, 5 days) with administration of the prodrugs. Tolerance was measured as the loss of antinociception, and withdrawal is measured ˜24 hours after the final morphine injection. RESULTS/ANTICIPATED RESULTS: Intrathecal administration of either KATP channel prodrugs significantly attenuated mechanical hypersensitivity after SNL and significantly attenuated mechanical hypersensitivity after CFA in mice. We predict that intrathecal administration of these prodrugs will also attenuate morphine tolerance and withdrawal in mice. This hypothesis is based off our previous data indicating non-water soluble KATP channel agonists produce analgesia and attenuate morphine tolerance in mice. DISCUSSION/SIGNIFICANCE OF FINDINGS: Pharmaceutical strategies to utilize KATP channels for therapeutics have been hindered due to the low solubility and low ability to cross the neurovascular unit. Newly developed, water-soluble KATP channel openers could be useful pharmaceutical strategy to reduce chronic pain, opioid tolerance, and withdrawal in human populations.

KATP and TRPM2-like channels couple metabolic status to resting membrane potential of octopus neurons in the mouse ventral cochlear nucleus

ATP-sensitive potassium (KATP) channels and transient receptor potential melastatin 2 (TRPM2) channels are commonly expressed both pre- and postsynaptically in the central nervous system (CNS). We hypothesized that KATP and TRPM2 may couple metabolic status to the resting membrane potential of octopus neurons of the mouse ventral cochlear nucleus (VCN).Therefore, we studied the expression of KATP channels and TRPM2 channels in octopus cells by immunohistochemical techniques and their contribution to neuronal electrical properties by the electrophysiological patch clamp technique.In immunohistochemical staining of octopus cells, labelling with Kir6.2 and SUR1 antibodies was strong, and labelling with the SUR2 antibody was moderate, but labelling with Kir6.1 was very weak. Octopus cells had intense staining with TRPM2 antibodies.In patch clamp recordings, bath application of KATP channel agonists H2O2 (880 μM), ATZ (1 mM), cromakalim (50 μM), diazoxide (200 μM), NNC 55-0118 and NN 414 separately resulted in hyperpolarizations of resting potential to different extents. Application of 8-Bro-cADPR (50 μM), a specific antagonist of TRPM2 channels, in the presence of H2O2 (880 μM) resulted in further hyperpolarization by approximately 1 mV. The amplitudes of H2O2-induced outward KATP currents and ADPR-induced inward currents were 206.1 ± 31.5 pA (n = 4) and 136.8 ± 22.4 pA, respectively, at rest. Their respective reversal potentials were -77 ± 2.6 mV (n = 3) and -6.3 ± 2.9 (n = 3) and -6.3 ± 2.9 (n = 3).In conclusion, octopus cells appear to possess both KATP channels and TRPM2-like channels. KATP might largely be constituted by SUR1–Kir6.2 subunits and SUR2–Kir6.2 subunits. Both KATP and TRPM2-like channels might have a modulatory action in setting the membrane potential.

Genetically Engineered Addition of a Ca2+ Buffering Capacity in Mouse Cardiac Muscle Thin Filament using a Glu-Rich Segment of Insect Troponin T Alters Cardiac Performance

In addition to a core function in Ca2+ regulation of muscle contraction, insect troponin T (TnT) has a unique glutamic acid-rich long C-terminal extension. With sequence similarity to this diverged structure, the N-terminal segment of avian flight muscle TnT binds Ca2+ with a physiologically relevant affinity (Zhang et al., Biochemistry 43:2645-55, 2004). Deletion of the C-terminal extension of Drosophila TnT significantly decreased muscle and heart functions (Cao et al., JBC 295:3794-3807, 2020). We propose a hypothesis that this structure serves as a myofilament Ca2+ reservoir to reduce the amount of Ca2+ cycled by Ca2+ pumps during muscle contraction and relaxation. A CRISP/Cas9 knock-in (KI) mouse model was constructed to replace the N-terminal variable region of cardiac TnT with 51 Glu residues derived from the C-terminal extension of bee TnT. Cardiac function is studied using ex vivo working hearts and tested for responses to beta-adrenergic stimulation or L-Ca2+ channel agonist Bay-K8644. At baseline, cTnT-51E hearts showed lower systolic velocity than that of WT control. Upon isoproterenol or Bay-K8644 treatment, cTnT-51E and wild type hearts both had positive responses in performance. However, cTnT-51E hearts exhibited significantly more increases in ventricular peak systolic pressure and diastolic velocity but still lower stroke volume as compared to that of WT controls. This phenotype is likely due to an altered response to the increase in intracellular calcium. Further analysis of Ca2+ responses of skinned muscle sections, cardiac energetic efficiency and intracellular Ca2+ transient are underway to establish the novel Ca2+ buffering capacity of TnT and its function in contractile kinetics of striated muscle.

Isoliquiritigenin, an active ingredient of Glycyrrhiza, elicits antinociceptive effects via inhibition of Nav channels.

Glycyrrhiza extract has been used for the treatment of oral and gastric ulcers, but the analgesic mechanism remains unknown. In the present study, we investigated the effects of isoliquiritigenin, an active ingredient of Glycyrrhiza, on Nav channels in vitro and nociceptive behaviors in vivo. In an autopatch-clamp study, isoliquiritigenin inhibited the currents of Nav1.1, Nav1.3, Nav1.6, Nav1.7, and Nav1.8 in a channel expression system. In small- and medium-sized cultured trigeminal ganglion neurons, the compound suppressed Nav currents in many neurons (78%) and Kv currents in all neurons, dose-dependently. In current-clamp mode, isoliquiritigenin blocked action potential generation in many neurons (64%), but it conversely accelerated action potential generation in the remaining neurons. The opposing effects on action potentials were reproduced in a computational simulation of a modified Hodgkin-Huxley-based model, based on the electrophysiological data. In behavioral experiments, local treatment with isoliquiritigenin suppressed nociceptive behaviors in response to oral ulcer development or nociceptive TRP channel agonists in the oral mucosa and hind paw. These results suggest that isoliquiritigenin exerts an analgesic effect predominantly via inhibitory action on Nav channels on sensory nociceptive fibers. This pharmacological mechanism indicates that isoliquiritigenin is useful for pain relief and provides scientific evidence for Glycyrrhiza at the ingredient level.

Inhibition of CRAC channel current by TRPV1 channel agonist and antagonist in RBL cells

Inhibition of CRAC channel current by TRPV1 channel agonist and antagonist in RBL cells