Cav3.2 T-Type Calcium Channel Mediates Acute Itch and Contributes to Chronic Itch and Inflammation in Experimental Atopic Dermatitis.

Voltage-gated calcium channels regulate neuronal excitability. The Cav3.2 isoform of the T-type voltage-activated calcium channel is expressed in sensory neurons and is implicated in pain transmission. However, its role in itch remains unclear. In this study, we demonstrated that Cav3.2 is expressed by mechanosensory and peptidergic subsets of mouse dorsal root ganglion neurons and colocalized with TRPV1 and receptors for type 2 cytokines. Cav3.2-positive neurons innervate human skin. A deficiency of Cav3.2 reduces histamine, IL-4/IL-13, and TSLP-induced itch in mice. Cav3.2 channels were upregulated in the dorsal root ganglia of an atopic dermatitis (AD)-like mouse model and mediated neuronal excitability. Genetic knockout of Cav3.2 or T-type calcium channel blocker mibefradil treatment reduced spontaneous and mechanically induced scratching behaviors and skin inflammation in an AD-like mouse model. Substance P and vasoactive intestinal polypeptide levels were increased in the trigeminal ganglia from AD-like mouse model, and genetic ablation or pharmacological inhibition of Cav3.2 reduced their gene expression. Cav3.2 knockout also attenuated the pathologic changes in ex vivo skin explants cocultured with trigeminal ganglia neurons from AD-induced mice. Our study identifies the role of Cav3.2 in both histaminergic and nonhistaminergic acute itch. Cav3.2 channel also contributes to AD-related chronic itch and neuroinflammation.

The Effect of Evogliptin Tartrate on Controlling Inflammatory Pain.

Background: Evogliptin tartrate inhibits dipeptidyl peptidase-4 (DPP-4), boosting glucagon-like peptide 1 (GLP-1) secretion and improving insulin release and glucose tolerance, while also exerting anti-inflammatory effects. We investigated its anti-inflammatory and analgesic effects. Methods: Forty male Sprague Dawley rats were divided into (N = 10 in each): (1) naïve, (2) complete Freund's adjuvant (CFA) inflammation + evogliptin tartrate (once for 10 mg/kg) (CFAE), (3) CFA + vehicle (same volume with normal saline with evogliptin tartrate/once) (CFAV), and (4) CFA + indomethacin (5 mg/mL/kg/1 time) (CFAI) groups. CFA was injected subcutaneously into rat plantar regions, and medications (evogliptin tartrate, vehicle, and indomethacin) were administered orally for 5 days. Post treatment, blood from the heart and plantar inflammatory tissue were collected to assess inflammatory cytokines. Evogliptin tartrate effects on controlling inflammation and pain were evaluated by measuring rat plantar paw thickness, paw withdrawal threshold, dorsal root ganglion (DRG) resting membrane potential, DRG action potential firing, and cytokine (TNF-α and IL-1ß) levels. Results: Compared with the naïve group, plantar paw thickness, cytokine (TNF-α and IL-1ß) levels, DRG resting membrane potential, and DRG action potential firing increased, whereas the paw withdrawal threshold decreased in all CFA groups. However, CFAE and CFAI rats showed recovery. The degree of CFAE recovery resembled that observed in the CFAI group. Conclusions: Evogliptin tartrate mirrored the anti-inflammatory pain relief of indomethacin. We aim to broaden its use as an anti-inflammatory drug or pain relief drug.

Long-lasting restoration of memory function and hippocampal synaptic plasticity by focused ultrasound in Alzheimer's disease.

BACKGROUND: Focused ultrasound (FUS) is a medical technology that non-invasively stimulates the brain and has been applied in thermal ablation, blood-brain barrier (BBB) opening, and neuromodulation. In recent years, numerous experiences and indications for the use of FUS in clinical and preclinical studies have rapidly expanded. Focused ultrasound-mediated BBB opening induces cognitive enhancement and neurogenesis; however, the underlying mechanisms have not been elucidated. METHODS: Here, we investigate the effects of FUS-mediated BBB opening on hippocampal long-term potentiation (LTP) and cognitive function in a 5xFAD mouse model of Alzheimer's disease (AD). We applied FUS with microbubble to the hippocampus and LTP was measured 6 weeks after BBB opening using FUS. Field recordings were made with a concentric bipolar electrode positioned in the CA1 region using an extracellular glass pipette filled with artificial cerebrospinal fluid. Morris water maze and Y-maze was performed to test cognitive function. RESULTS: Our results demonstrated that FUS-mediated BBB opening has a significant impact on increasing LTP at Schaffer collateral - CA1 synapses and rescues cognitive dysfunction and working memory. These effects persisted for up to 7 weeks post-treatment. Also, FUS-mediated BBB opening in the hippocampus increased PKA phosphorylation. CONCLUSION: Therefore, it could be a promising treatment for neurodegenerative diseases as it remarkably increases LTP, thereby improving working memory.

Modified (-)-gallocatechin gallate-enriched green tea extract rescues age-related cognitive deficits by restoring hippocampal synaptic plasticity.

Aging leads to cognitive impairments characterized by reduced hippocampal functions that are associated with impairment of long-term potentiation of CA1 synapses. Here, we assessed the safety and efficacy of modified (-)-gallocatechin gallate (GCG)-enriched green tea extract (HTP-GTE) in ameliorating the cognitive dysfunctions in late middle-aged murine model. We developed a novel HTP-GTE that was enriched with GCG via epimerization that involved heating. We compared the effects of oral administrations of conventional green tea and HTP-GTE in young and aged male C57/BL6 mice, and examined the changes in the hippocampal functions related to aging process. The functional outcome was assessed by the electrophysiological experiments to measure the long-term potentiation (LTP). HTP-GTE improved the age-related cognitive impairments via restoring long-term synaptic plasticity. We also identified that GCG was the main active component responsible for the HTP-GTE effect. The main molecular pathway in ameliorating the age-related cognitive dysfunctions involved protein kinase A (PKA) which was shown to be modulated by HTP-GTE. Thus, HTP-GTE has a therapeutic potential as a dietary supplement which may aid to rescue the impaired cognitive functions at the early phase of aging process through the modulation of LTP threshold.

Stem cell restores thalamocortical plasticity to rescue cognitive deficit in neonatal intraventricular hemorrhage.

Severe neonatal intraventricular hemorrhage (IVH) patients incur long-term neurologic deficits such as cognitive disabilities. Recently, the intraventricular transplantation of allogeneic human umbilical cord blood-derived mesenchymal stem cells (MSCs) has drawn attention as a therapeutic potential to treat severe IVH. However, its pathological synaptic mechanism is still elusive. We here demonstrated that the integration of the somatosensory input was significantly distorted by suppressing feed-forward inhibition (FFI) at the thalamocortical (TC) inputs in the barrel cortices of neonatal rats with IVH by using BOLD-fMRI signal and brain slice patch-clamp technique. This is induced by the suppression of Hebbian plasticity via an increase in tumor necrosis factor-α expression during the critical period, which can be effectively reversed by the transplantation of MSCs. Furthermore, we showed that MSC transplantation successfully rescued IVH-induced learning deficits in the sensory-guided decision-making in correlation with TC FFI in the layer 4 barrel cortex.

TFEB-GDF15 axis protects against obesity and insulin resistance as a lysosomal stress response.

TFEB, a key regulator of lysosomal biogenesis and autophagy, is induced not only by nutritional deficiency but also by organelle stress. Here, we find that Tfeb and its downstream genes are upregulated together with lipofuscin accumulation in adipose tissue macrophages (ATMs) of obese mice or humans, suggestive of obesity-associated lysosomal dysfunction/stress in ATMs. Macrophage-specific TFEB-overexpressing mice display complete abrogation of diet-induced obesity, adipose tissue inflammation and insulin resistance, which is independent of autophagy, but dependent on TFEB-induced GDF15 expression. Palmitic acid induces Gdf15 expression through lysosomal Ca2+-mediated TFEB nuclear translocation in response to lysosomal stress. In contrast, mice fed a high-fat diet with macrophage-specific Tfeb deletion show aggravated adipose tissue inflammation and insulin resistance, accompanied by reduced GDF15 level. Finally, we observe activation of TFEB-GDF15 in ATMs of obese humans as a consequence of lysosomal stress. These findings highlight the importance of the TFEB-GDF15 axis as a lysosomal stress response in obesity or metabolic syndrome and as a promising therapeutic target for treatment of these conditions.

(-)-Gallocatechin gallate from green tea rescues cognitive impairment through restoring hippocampal silent synapses in post-menopausal depression.

Post-menopausal depression (PMD) is a common psychological disorder accompanied by a cognitive deficit, which is caused by a series of uncontrolled emotional disruptions by strong environmental stressors during menopause. To overcome PMD-induced cognitive deficit, Green tea has been suggested as a dietary supplement because of its ameliorating effect on cognitive dysfunction induced by normal aging or neurodegenerative syndromes; however, its clinical use to improve PMD-accompanied cognitive deficit is still limited due to the controversy for the active ingredients and ambiguous mechanism of its action. Here, we developed modified high-temperature-processed green tea extract (HTP-GTE), which showed lower neuronal toxicity than the conventional green tea extract (GTE). We also demonstrated that HTP-GTE administration prevented the development of learned helplessness (LH) in a rat post-menopausal model. Additionally, HTP-GTE improved LH-induced cognitive impairments simultaneously with rescued the long-term synaptic plasticity. This occurred via the restoration of silent synapse formation by increasing the hippocampal BDNF-tyrosine receptor kinase B pathway in the helpless ovariectomized (OVX) rats. Likewise, we also identified that (-)-gallocatechin gallate was the main contributor of the HTP-GTE effect. Our findings suggested that HTP-GTE has a potential as a preventive nutritional supplement to ameliorate cognitive dysfunctions associated with PMD.

Enhanced Thermal Sensitivity of TRPV3 in Keratinocytes Underlies Heat-Induced Pruritogen Release and Pruritus in Atopic Dermatitis.

Itch in atopic dermatitis (AD) is aggravated under warm conditions. Transient receptor potential vanilloid (TRPV) 3, a member of the thermosensitive transient receptor potential channels, is activated by innocuous heat and is abundantly expressed in keratinocytes. The potential role of TRPV3 in itch is illustrated in TRPV3 channelopathies of humans and mice. However, the role of TRPV3 in heat-induced itch in AD and the underlying mechanisms are unclear. Here we showed that keratinocytes isolated from patients with AD exhibit enhanced expression and heat sensitivity with hyperactive channel function of TRPV3. Heat stimulus induced enhanced secretion of thymic stromal lymphopoietin, nerve growth factor, and prostaglandin E2 by keratinocytes from patients with AD through TRPV3 activation. TRPV3 agonists increased thymic stromal lymphopoietin, nerve growth factor, prostaglandin E2, and IL-33 production in human keratinocytes and induced scratching behavior upon intradermal injection in mice. TRPV3 was upregulated in the skin of MC903-induced AD mouse model. Heat stimulation to MC903-treated mice increased scratching behavior and produced higher levels of thymic stromal lymphopoietin, nerve growth factor, prostaglandin E2, and IL-33 from the epidermis, which were attenuated by pharmacologic inhibition of TRPV3. Moreover, neutralization of thymic stromal lymphopoietin reduced heat-evoked scratching in MC903-challenged mice. These results suggest that TRPV3 is a potential therapeutic target for heat-induced itch in AD.

Nanoelectrode-mediated single neuron activation.

Elucidating cellular dynamics at the level of a single neuron and its associated role within neuronal circuits is essential for interpreting the complex nature of the brain. To investigate the operation of neural activity within its network, it is necessary to precisely manipulate the activation of each neuron and verify its propagation path via the synaptic connection. In this study, by exploiting the intrinsic physical and electrical advantages of a nanoelectrode, a vertical nanowire multi electrode array (VNMEA) is developed as a neuronal activation platform presenting the spatially confined effect on the intracellular space of individual cells. VNMEA makes a distinct difference between the interior and exterior cell potential and the current density, deriving the superior effects on activating Ca2+ responses compared to extracellular methods under the same conditions, with about 2.9-fold higher amplitude of Ca2+ elevation and a 2.6-fold faster recovery rate. Moreover, the synchronized propagation of evoked activities is shown in connected neurons implying cell-to-cell communications following the intracellular stimulation. The simulation and experimental consequences prove the outstanding property of temporal/spatial confinement of VNMEA-mediated intracellular stimulation to activate a single neuron and show its potential in localizing spiking neurons within neuronal populations, which may be utilized to reveal the connection and activation modalities of neural networks.

Dynamic causal modeling for calcium imaging: Exploration of differential effective connectivity for sensory processing in a barrel cortical column.

Multi-photon calcium imaging (CaI) is an important tool to assess activities of neural populations within a column in the sensory cortex. However, the complex asymmetrical interactions among neural populations, termed effective connectivity, cannot be directly assessed by measuring the activity of each neuron or neural population using CaI but calls for computational modeling. To estimate effective connectivity among neural populations, we proposed a dynamic causal model (DCM) for CaI by combining a convolution-based dynamic neural state model and a dynamic calcium ion concentration model for CaI signals. After conducting a simulation study to evaluate DCM for CaI, we applied it to an experimental CaI signals measured at the layer 2/3 of a barrel cortical column that differentially responds to hit and error whisking trials in mice. We first identified neural populations and constructed computational models with intrinsic connectivity of neural populations within the layer 2/3 of the barrel cortex and extrinsic connectivity with latent external modes. Bayesian model inversion and comparison shows that interactions with latent inhibitory and excitatory external modes explain the observed CaI signals within the barrel cortical column better than any other tested models, with a single external mode or without any latent modes. The best model also showed differential intrinsic and extrinsic effective connectivity between hit and error trials in the functional hierarchy. Both simulation and experimental results suggest the usefulness of DCM for CaI in terms of exploration of hierarchical interactions among neural populations observed in CaI.

Physical exercise enhances adult cortical plasticity in a neonatal rat model of hypoxic-ischemic injury: Evidence from BOLD-fMRI and electrophysiological recordings.

Neuroplasticity is considered essential for recovery from brain injury in developing brains. Recent studies indicate that it is especially effective during early postnatal development and during the critical period. The current study used functional magnetic resonance imaging (fMRI) and local field potential (LFP) electrophysiological recordings in rats that experienced neonatal hypoxic-ischemic (HI) injury during the critical period to demonstrate that physical exercise (PE) can improve cortical plasticity even when performed during adulthood, after the critical period. We investigated to what extent the blood oxygen level-dependent (BOLD)-fMRI responses were increased in the contralesional spared cortex, and how these increases were related to the LFP electrophysiological measurements and the functional outcome. The balance of excitation and inhibition was assessed by measuring excitatory and inhibitory postsynaptic currents in stellate cells in the primary somatosensory (S1) cortex, which was compared with the BOLD-fMRI responses in the contralesional S1 cortex. The ratio of inhibitory postsynaptic current (IPSC) to excitatory postsynaptic current (EPSC) at the thalamocortical (TC) input to the spared S1 cortex was significantly increased by PE, which is consistent with the increased BOLD-fMRI responses and improved functional outcome. Our data clearly demonstrate in an experimental rat model of HI injury during the critical period that PE in adulthood enhances neuroplasticity and suggest that enhanced feed-forward inhibition at the TC input to the S1 cortex might underlie the PE-induced amelioration of the somatosensory deficits caused by the HI injury. In summary, the results of the current study indicate that PE, even if performed beyond the critical period or during adulthood, can be an effective therapy to treat neonatal brain injuries, providing a potential mechanism for the development of a potent rehabilitation strategy to alleviate HI-induced neurological impairments.

Erythropoietin Attenuates Postoperative Cognitive Dysfunction by Shifting Macrophage Activation toward the M2 Phenotype.

Postoperative cognitive dysfunction (POCD) may be driven by transference of the innate immune response to the brain after aseptic surgical damage. Macrophages are key mediators of innate immunity that can display a pro-inflammatory M1 phenotype or an anti-inflammatory M2 phenotype. Erythropoietin (EPO) is a hematopoietic hormone that exerts anti-inflammatory effects by influencing macrophage function. We hypothesized that EPO would prevent POCD by promoting macrophage phenotype switching to the M2 phenotype post-surgery. To evaluate the effects of EPO on POCD and macrophage polarization post-surgery, we administered EPO (5,000 U/kg) with or without an arginase inhibitor (amino-6-boronohexanoic acid, 10 mg/kg) to ICR mice before and after abdominal surgery. Forty-eight hours post-surgery, we assessed memory, synapse function, and macrophage/microglial phenotypes in the spleen and hippocampus. We also investigated M1/M2 phenotypes in RAW264.7 and BV2 cells stimulated with lipopolysaccharide and interferon-γ (M1 inducers) in the presence or absence of EPO. EPO prevented POCD, decreased surgery-related synaptic dysfunction, and attenuated pro-inflammatory cytokine generation in the hippocampus. Moreover, EPO suppressed M1-related genes expression and promoted M2 genes expression in the spleen and hippocampus post-surgery. Furthermore, EPO decreased the proportions of macrophages/microglia expressing an M1 surface marker (CD40) and increased those expressing an M2 surface marker (CD206). Arginase inhibition abolished the beneficial effects of EPO on POCD. In vitro, EPO treatment promoted switching of RAW264.7 and BV2 cells stimulated with M1 inducers to an M2 phenotype. In conclusion, EPO prevents POCD by promoting macrophage phenotype switching toward the M2 phenotype.

Cell-penetrating interactomic inhibition of nuclear factor-kappa B in a mouse model of postoperative cognitive dysfunction.

Some patients experience impaired cognitive functioning after surgery, a phenomenon referred to as postoperative cognitive dysfunction (POCD). Signs of POCD are closely associated with the development of systemic or hippocampal inflammation. However, the precise pathophysiological mechanisms of prevention/treatment options for POCD still remain unclear. After injury, the transcriptional factor nuclear factor-kappa B (NF-κB) is thought to regulate or stimulate inflammation amplification. Therefore, we designed a cell-penetrating fusion protein called nt-p65-TMD, which inhibits NF-κB p65 activation by translocating into the nucleus. In the present study, we discovered that nt-p65-TMD exerted effects on surgery-induced cognitive impairment in mice. Specifically, nt-p65-TMD exhibited strong immunoregulatory properties that were able to reduce surgery-induced elevations in cerebrovascular integrity impairment, subsequent peripheral immune-cell recruitment, and inflammation amplification, which ultimately lead to cognitive decline. The nt-p65-TMD has the unique ability to regulate and reduce systemic inflammation and inflammation amplification, suggesting a new strategy for preventing development of cognitive decline that occurs in POCD.

Trafficking patterns of NMDA and GABAA receptors in a Mg2+-free cultured hippocampal neuron model of status epilepticus.

An altered pattern of receptor trafficking is one of the pathophysiologic mechanisms of status epilepticus (SE). The gradual internalization of GABAA receptors (GABARs) occurs in both in vitro and in vivo models of SE and is thought to be a cause of decreased GABAergic inhibition. Unlike GABARs, little is known about alterations in NMDA receptor (NMDAR) trafficking during SE, even though increased activity of NMDARs is indispensable for the induction and maintenance of SE. Therefore, we aimed to simultaneously investigate the changes in the trafficking patterns of GABARs and NMDARs in an in vitro cultured hippocampal neuron model of SE. For induction of epileptiform discharges, hippocampal neurons were exposed to external medium without Mg2+. Biotinylation assay and immunofluorescence staining for GABAR ß2,3 and NMDAR NR1 subunits were performed to quantify and visualize surface GABARs and NMDARs, respectively. The frequency of spontaneous action potentials increased more than 4-fold after Mg2+-free induction. The level of surface GABARs decreased over time after Mg2+-free induction, dropping to approximately 50% of control levels an hour after Mg2+-free induction. By contrast, the trafficking of NMDARs to the surface was enhanced after a slight time lag, increasing by 30% of control levels an hour after Mg2+-free induction. Our data showed the changes of both NMDAR and GABAR trafficking during prolonged SE induced by a Mg2+-free extracellular environment and confirmed that this in vitro SE model is suitable for examining alterations in the receptor trafficking pattern by prolonged seizure activity. These results suggest that targeting of surface NMDAR could be a promising method in controlling benzodiazepine-resistant SE.

Peripheral Sensory Deprivation Restores Critical-Period-like Plasticity to Adult Somatosensory Thalamocortical Inputs.

Recent work has shown that thalamocortical (TC) inputs can be plastic after the developmental critical period has closed, but the mechanism that enables re-establishment of plasticity is unclear. Here, we find that long-term potentiation (LTP) at TC inputs is transiently restored in spared barrel cortex following either a unilateral infra-orbital nerve (ION) lesion, unilateral whisker trimming, or unilateral ablation of the rodent barrel cortex. Restoration of LTP is associated with increased potency at TC input and reactivates anatomical map plasticity induced by whisker follicle ablation. The reactivation of TC LTP is accompanied by reappearance of silent synapses. Both LTP and silent synapse formation are preceded by transient re-expression of synaptic GluN2B-containing N-methyl-D-aspartate (NMDA) receptors, which are required for the reappearance of TC plasticity. These results clearly demonstrate that peripheral sensory deprivation reactivates synaptic plasticity in the mature layer 4 barrel cortex with features similar to the developmental critical period.

Phospholipase C-dependent hydrolysis of phosphatidylinositol 4,5-bisphosphate underlies agmatine-induced suppression of N-type Ca2+ channel in rat celiac ganglion neurons.

Agmatine suppresses peripheral sympathetic tone by modulating Cav2.2 channels in peripheral sympathetic neurons. However, the detailed cellular signaling mechanism underlying the agmatine-induced Cav2.2 inhibition remains unclear. Therefore, in the present study, we investigated the electrophysiological mechanism for the agmatine-induced inhibition of Cav2.2 current (ICav2.2) in rat celiac ganglion (CG) neurons. Consistent with previous reports, agmatine inhibited ICav2.2 in a VI manner. The agmatine-induced inhibition of the ICav2.2 current was also almost completely hindered by the blockade of the imidazoline I2 receptor (IR2), and an IR2 agonist mimicked the inhibitory effect of agmatine on ICav2.2, implying involvement of IR2. The agmatine-induced ICav2.2 inhibition was significantly hampered by the blockade of G protein or phospholipase C (PLC), but not by the pretreatment with pertussis toxin. In addition, diC8-phosphatidylinositol 4,5-bisphosphate (PIP2) dialysis nearly completely hampered agmatine-induced inhibition, which became irreversible when PIP2 resynthesis was blocked. These results suggest that in rat peripheral sympathetic neurons, agmatine-induced IR2 activation suppresses Cav2.2 channel voltage-independently, and that the PLC-dependent PIP2 hydrolysis is responsible for the agmatine-induced suppression of the Cav2.2 channel.

Agmatine suppresses peripheral sympathetic tone by inhibiting N-type Ca(2+) channel activity via imidazoline I2 receptor activation.

Agmatine, a putative endogenous ligand of imidazoline receptors, suppresses cardiovascular function by inhibiting peripheral sympathetic tone. However, the molecular identity of imidazoline receptor subtypes and its cellular mechanism underlying the agmatine-induced sympathetic suppression remains unknown. Meanwhile, N-type Ca(2+) channels are important for the regulation of NA release in the peripheral sympathetic nervous system. Therefore, it is possible that agmatine suppresses NA release in peripheral sympathetic nerve terminals by inhibiting Ca(2+) influx through N-type Ca(2+) channels. We tested this hypothesis by investigating agmatine effect on electrical field stimulation (EFS)-evoked contraction and NA release in endothelium-denuded rat superior mesenteric arterial strips. We also investigated the effect of agmatine on the N-type Ca(2+) current in superior cervical ganglion (SCG) neurons in rats. Our study demonstrates that agmatine suppresses peripheral sympathetic outflow via the imidazoline I2 receptor in rat mesenteric arteries. In addition, the agmatine-induced suppression of peripheral vascular sympathetic tone is mediated by modulating voltage-dependent N-type Ca(2+) channels in sympathetic nerve terminals. These results suggest a potential cellular mechanism for the agmatine-induced suppression of peripheral sympathetic tone. Furthermore, they provide basic and theoretical information regarding the development of new agents to treat hypertension.

Protease-activated receptor 2 activation inhibits N-type Ca2+ currents in rat peripheral sympathetic neurons.

The protease-activated receptor (PAR)-2 is highly expressed in endothelial cells and vascular smooth muscle cells. It plays a crucial role in regulating blood pressure via the modulation of peripheral vascular tone. Although several mechanisms have been suggested to explain PAR-2-induced hypotension, the precise mechanism remains to be elucidated. To investigate this possibility, we investigated the effects of PAR-2 activation on N-type Ca(2+) currents (I(Ca-N)) in isolated neurons of the celiac ganglion (CG), which is involved in the sympathetic regulation of mesenteric artery vascular tone. PAR-2 agonists irreversibly diminished voltage-gated Ca(2+) currents (I(Ca)), measured using the patch-clamp method, in rat CG neurons, whereas thrombin had little effect on I(Ca). This PAR-2-induced inhibition was almost completely prevented by ω-CgTx, a potent N-type Ca(2+) channel blocker, suggesting the involvement of N-type Ca(2+) channels in PAR-2-induced inhibition. In addition, PAR-2 agonists inhibited I(Ca-N) in a voltage-independent manner in rat CG neurons. Moreover, PAR-2 agonists reduced action potential (AP) firing frequency as measured using the current-clamp method in rat CG neurons. This inhibition of AP firing induced by PAR-2 agonists was almost completely prevented by ω-CgTx, indicating that PAR-2 activation may regulate the membrane excitability of peripheral sympathetic neurons through modulation of N-type Ca(2+) channels. In conclusion, the present findings demonstrate that the activation of PAR-2 suppresses peripheral sympathetic outflow by modulating N-type Ca(2+) channel activity, which appears to be involved in PAR-2-induced hypotension, in peripheral sympathetic nerve terminals.

Suppression of peripheral sympathetic activity underlies protease-activated receptor 2-mediated hypotension.

Protease-activated receptor (PAR)-2 is expressed in endothelial cells and vascular smooth muscle cells. It plays a crucial role in regulating blood pressure via the modulation of peripheral vascular tone. Although some reports have suggested involvement of a neurogenic mechanism in PAR-2-induced hypotension, the accurate mechanism remains to be elucidated. To examine this possibility, we investigated the effect of PAR-2 activation on smooth muscle contraction evoked by electrical field stimulation (EFS) in the superior mesenteric artery. In the present study, PAR-2 agonists suppressed neurogenic contractions evoked by EFS in endothelium-denuded superior mesenteric arterial strips but did not affect contraction elicited by the external application of noradrenaline (NA). However, thrombin, a potent PAR-1 agonist, had no effect on EFS-evoked contraction. Additionally, ω-conotoxin GVIA (CgTx), a selective N-type Ca(2+) channel (ICa-N) blocker, significantly inhibited EFS-evoked contraction, and this blockade almost completely occluded the suppression of EFS-evoked contraction by PAR-2 agonists. Finally, PAR-2 agonists suppressed the EFS-evoked overflow of NA in endothelium-denuded rat superior mesenteric arterial strips and this suppression was nearly completely occluded by ω-CgTx. These results suggest that activation of PAR-2 may suppress peripheral sympathetic outflow by modulating activity of ICa-N which are located in peripheral sympathetic nerve terminals, which results in PAR-2-induced hypotension.

Transient receptor potential c4/5 like channel is involved in stretch-induced spontaneous uterine contraction of pregnant rat.

Spontaneous myometrial contraction (SMC) in pregnant uterus is greatly related with gestational age and growing in frequency and amplitude toward the end of gestation to initiate labor. But, an accurate mechanism has not been elucidated. In human and rat uterus, all TRPCs except TRPC2 are expressed in pregnant myometrium and among them, TRPC4 are predominant throughout gestation, suggesting a possible role in regulation of SMC. Therefore, we investigated whether the TRP channel may be involved SMC evoked by mechanical stretch in pregnant myometrial strips of rat using isometric tension measurement and patch-clamp technique. In the present results, hypoosmotic cell swelling activated a potent outward rectifying current in G protein-dependent manner in rat pregnant myocyte. The current was significantly potentiated by 1µM lanthanides (a potent TRPC4/5 stimulator) and suppressed by 10µM 2-APB (TRPC4-7 inhibitor). In addition, in isometric tension experiment, SMC which was evoked by passive stretch was greatly potentiated by lanthanide (1µM) and suppressed by 2-APB (10µM), suggesting a possible involvement of TRPC4/5 channel in regulation of SMC in pregnant myometrium. These results provide a possible cellular mechanism for regulation of SMC during pregnancy and provide basic information for developing a new agent for treatment of premature labor.