Inhibitors Of Transient Receptor Potential Channels Research Articles

ZBTB20 mediates stress-induced visceral hypersensitivity via activating the NF-κB/transient receptor potential channel pathway.

Psychological stress is a major trigger for visceral hypersensitivity (VH) in irritable bowel syndrome. The zinc finger protein ZBTB20 (ZBTB20) is implicated in somatic nociception via modulating transient receptor potential (TRP) channels, but its role in the development of VH is unclear. This study aimed to investigate the role of ZBTB20/TRP channel axis in stress-induced VH. Rats were subjected to water avoidance stress (WAS) for 10 consecutive days. Small interfering RNA (siRNA) targeting ZBTB20 was intrathecally administered. Inhibitors of TRP channels, stress hormone receptors, and nuclear factor kappa-B (NF-κB) were administered. Visceromotor response to colorectal distension was recorded. Dorsal root ganglia (DRGs) were dissected for Western blot, coimmunoprecipitation, and chromatin immunoprecipitation. The DRG-derived neuron cell line was applied for specific research. WAS-induced VH was suppressed by the inhibitor of TRPV1, TRPA1, or TRPM8, with enhanced expression of these channels in L6-S2 DRGs. The inhibitor of glucocorticoid receptor or β2-adrenergic receptor counteracted WAS-induced VH and TRP channel expression. Concurrently, WAS-induced stress hormone-dependent ZBTB20 expression and NF-κB activation in DRGs. Intrathecally injected ZBTB20 siRNA or an NF-κB inhibitor repressed WAS-caused effect. In cultured DRG-derived neurons, stress hormones promoted nuclear translocation of ZBTB20, which preceded p65 nuclear translocation. And, ZBTB20 siRNA suppressed stress hormone-caused NF-κB activation. Finally, WAS enhanced p65 binding to the promoter of TRPV1, TRPA1, or TRPM8 in rat DRGs. ZBTB20 mediates stress-induced VH via activating NF-κB/TRP channel pathway in nociceptive sensory neurons.

TRP Channels in Cancer: Signaling Mechanisms and Translational Approaches.

Ion channels play a crucial role in a wide range of biological processes, including cell cycle regulation and cancer progression. In particular, the transient receptor potential (TRP) family of channels has emerged as a promising therapeutic target due to its involvement in several stages of cancer development and dissemination. TRP channels are expressed in a large variety of cells and tissues, and by increasing cation intracellular concentration, they monitor mechanical, thermal, and chemical stimuli under physiological and pathological conditions. Some members of the TRP superfamily, namely vanilloid (TRPV), canonical (TRPC), melastatin (TRPM), and ankyrin (TRPA), have been investigated in different types of cancer, including breast, prostate, lung, and colorectal cancer. TRP channels are involved in processes such as cell proliferation, migration, invasion, angiogenesis, and drug resistance, all related to cancer progression. Some TRP channels have been mechanistically associated with the signaling of cancer pain. Understanding the cellular and molecular mechanisms by which TRP channels influence cancer provides new opportunities for the development of targeted therapeutic strategies. Selective inhibitors of TRP channels are under initial scrutiny in experimental animals as potential anti-cancer agents. In-depth knowledge of these channels and their regulatory mechanisms may lead to new therapeutic strategies for cancer treatment, providing new perspectives for the development of effective targeted therapies.

Mechanisms of the PD-1/PD-L1 pathway in itch: From acute itch model establishment to the role in chronic itch in mouse

Programmed cell death receptor/ligand 1 (PD-1/PD-L1) blockade therapy for various cancers induces itch. However, few studies have evaluated the mechanism underlying PD-1/PD-L1 inhibitor-induced itch. This study aimed to establish and evaluate a mouse model of acute itch induced by PD-1/PD-L1 inhibitors and to explore the role of the PD-1/PD-L1 pathway in chronic itch. The intradermal injection of the PD-1/PD-L1 small molecule inhibitors, or anti-PD-1/PD-L1 antibodies in the nape of the neck in the mice elicited intense spontaneous scratches. The model was evaluated using pharmacological methods. The number of scratches was reduced by naloxone but not by antihistamines or the transient receptor potential (TRP) channel inhibitor. Moreover, the PD-1 receptor was detected in the spinal cord of the mouse models of chronic itch that exhibited acetone, diethyl ether, and water (AEW)-induced dry skin, imiquimod-induced psoriasis, and 1-fluoro-2,4-dinitrobenzene (DNFB)-induced allergic contact dermatitis. Intrathecal PD-L1 (1 μg, 4 times a week for 1 week) suppressed the activation of the microglia in the spinal dorsal horn to relieve the chronic itch that was elicited by imiquimod-induced psoriasis and DNFB-induced allergic contact dermatitis. Although the activation of the microglia in the spinal dorsal horn was not detected in the AEW-treated mice, intrathecal PD-L1 still reduced the number of scratches that were elicited by AEW. Our findings suggest that histamine receptor inhibitors or TRP channel inhibitors have limited effects on PD-1/PD-L1 inhibitor-induced itch and that spinal PD-1 is important for the spinal activation of the microglia, which may underlie chronic itch.

Investigations into hydrogen sulfide-induced suppression of neuronal activity in vivo and calcium dysregulation in vitro.

Acute exposure to high concentrations of hydrogen sulfide (H2S) leads to sudden death and, if survived, lingering neurological disorders. Clinical signs include seizures, loss of consciousness, and dyspnea. The proximate mechanisms underlying H2S-induced acute toxicity and death have not been clearly elucidated. We investigated electrocerebral, cardiac and respiratory activity during H2S exposure using electroencephalogram (EEG), electrocardiogram (EKG) and plethysmography. H2S suppressed electrocerebral activity and disrupted breathing. Cardiac activity was comparatively less affected. To test whether Ca2+ dysregulation contributes to H2S-induced EEG suppression, we developed an in vitro real-time rapid throughput assay measuring patterns of spontaneous synchronized Ca2+ oscillations in cultured primary cortical neuronal networks loaded with the indicator Fluo-4 using the fluorescent imaging plate reader (FLIPR-Tetra®). Sulfide >5 ppm dysregulated synchronous calcium oscillation (SCO) patterns in a dose-dependent manner. Inhibitors of NMDA and AMPA receptors magnified H2S-induced SCO suppression. Inhibitors of L-type voltage gated Ca2+ channels and transient receptor potential channels prevented H2S-induced SCO suppression. Inhibitors of T-type voltage gated Ca2+ channels, ryanodine receptors, and sodium channels had no measurable influence on H2S-induced SCO suppression. Exposures to > 5 ppm sulfide also suppressed neuronal electrical activity in primary cortical neurons measured by multi-electrode array (MEA), an effect alleviated by pretreatment with the nonselective transient receptor potential channel inhibitor, 2-APB. 2-APB also reduced primary cortical neuronal cell death from sulfide exposure. These results improve our understanding of the role of different Ca2+ channels in acute H2S-induced neurotoxicity and identify transient receptor potential channel modulators as novel structures with potential therapeutic benefits.

Ultrashort nanosecond electric pulses activate a conductance in bovine adrenal chromaffin cells that involves cation entry through TRPC and NALCN channels

In whole-cell voltage clamped bovine adrenal chromaffin cells maintained at a holding potential of −70 mV, a single 5 ns, 5 MV/m pulse elicited an inward current carried mainly by Na+ that displayed inward rectification and a reversal potential near −3 mV, a voltage consistent with a non-selective cation current. The broad-spectrum inhibitors of transient receptor potential (TRP) channels, La3+ (10 μM), Gd3+ (10 μM), SKF-96365 (50 μM) and 2-aminoethoxydiphenyl borane (2-APB; 100 μM), inhibited the current similarly by ∼72%, ∼83%, ∼68% and ∼76%, respectively. Depleting membrane cholesterol with methyl-β-cyclodextrin (MβCD; 1–6 mg/ml) or inhibiting phosphatidylinositol 4,5-bisphosphate (PIP2) synthesis with wortmannin (20 and 40 μM) produced a similar level of inhibition on the NEP-induced conductance as the broad spectrum TRP channel inhibitors. Moreover, no additive inhibitory effect was detected by combining MβCD (3 mg/ml), wortmannin (20 μM) and La3+ (10 μM), suggesting that each agent targeted different levels of the same pathway to exert a full effect. RT-PCR experiments revealed robust expression at the mRNA level of TRPC4, TRPC5 and TRPM7 channels for which specific blockers were available. Whereas the TRPM7 blocker FTY720 had no effect, the TRPC4/5 channel inhibitor M084 (20 μM) blocked the conductance by ∼50%, indicating that TRPC4 and/or TRPC5 channel(s) may be partially involved in mediating the NEP-induced current. CP-96345 (20 μM), a specific blocker of the sodium leak current channel (NALCN), also reduced the NEP-induced current. The inhibition was ∼30% and additive to that caused by the TRPC4/5 blocker M084. RT-PCR experiments confirmed the expression of this channel at the mRNA level. Taken as a whole, these data provide evidence that a large fraction of the current evoked by a 5 ns pulse in adrenal chromaffin cells may be carried by both TRPC4/5 channels and the NALCN channel. Understanding the biophysical properties of the NEP-elicited conductance in a neural-type cell will be extremely valuable for the future development of NEP stimulation approaches for neuromodulation.

Functional Characterization of TRPV-Like Ion Channels Involved in Nematocyst Discharge from the Sea Anemone Diadumene lineata.

AbstractCnidarians require mechanical stimuli to trigger nematocyst discharge and initiate feeding behaviors. The interval from triggering stimulus to response is tens of microseconds, making it likely that mechanically gated ion channels trigger nematocyst discharge. Because many transient receptor potential channels are mechanically gated, we hypothesized that nematocyst discharge involves transient receptor potential channels. We therefore tested various transient receptor potential channel inhibitors to determine whether they inhibit nematocyst discharge and prey killing in the acontiate sea anemone (Actinaria) Diadumene lineata (a.k.a. Haliplanella luciae). Three types of cnidocyte supporting cell complexes regulate nematocyst discharge in anemones: Types C, B, and A. Discharge from Type Cs is directly triggered by stimulation of contact-sensitive mechanoreceptors, while Type Bs require activation of chemoreceptors from prey-derived N-acetylated sugars to sensitize contact-sensitive mechanoreceptors. In Type As, activated chemoreceptors tune vibration-sensitive mechanoreceptors that predispose contact-sensitive mechanoreceptors for triggering. The non-selective transient receptor potential channel blockers lanthanum and gadolinium dose-dependently inhibited about 80% of prey killing and all nematocyst discharge from Type Bs and Type Cs, but not Type As. The selective transient receptor potential vanilloid 4 (TRPV4) blocker GSK2193874 inhibited Type As and Type Bs. However, the selective TRPV4 blockers HC-067047 and RN-1734 inhibited only Type As. Thus, three TRPV4-selective blockers implicate TRPV-like involvement in discharge from Type As, whereas GSK2193874 also affected Type Bs. Our results suggest that a TRPV-like homolog plays an essential role in nematocyst-mediated prey killing from Type As, whereas other transient receptor potential channels are likely involved in discharge from Type B and C cnidocyte supporting cell complexes.

Transient Receptor Potential Channels and Botulinum Neurotoxins in Chronic Pain.

Pain afflicts more than 1.5 billion people worldwide, with hundreds of millions suffering from unrelieved chronic pain. Despite widespread recognition of the importance of developing better interventions for the relief of chronic pain, little is known about the mechanisms underlying this condition. However, transient receptor potential (TRP) ion channels in nociceptors have been shown to be essential players in the generation and progression of pain and have attracted the attention of several pharmaceutical companies as therapeutic targets. Unfortunately, TRP channel inhibitors have failed in clinical trials, at least in part due to their thermoregulatory function. Botulinum neurotoxins (BoNTs) have emerged as novel and safe pain therapeutics because of their regulation of exocytosis and pro-nociceptive neurotransmitters. However, it is becoming evident that BoNTs also regulate the expression and function of TRP channels, which may explain their analgesic effects. Here, we summarize the roles of TRP channels in pain, with a particular focus on TRPV1 and TRPA1, their regulation by BoNTs, and briefly discuss the use of BoNTs for the treatment of chronic pain.

When the Efferent Becomes Afferent ‐ the Cellular Response of Sympathetic Neurons to Sensory Stimuli

Responding to the so called “inflammatory soup” has often been regarded as the sole domain of sensory afferent neurons. We tested the hypothesis that efferent sympathetic neurons can also directly respond to traditionally afferent stimuli. We used single cell RNA‐sequencing (scRNAseq) to explore transcript expression of membrane G protein and ion channel coupled receptors in the sympathetic neuronal population within the stellate ganglia of 5–6‐week‐old Wistar rats. Agonist responses were then evaluated using Fura‐2 AM calcium imaging and perforated patch clamp recordings in isolated cultured neurons.ScRNAseq identified transcript expression of transient receptor potential (TRP) channels (TRPV2>TRM7>TRPC1>>TRM4). Certain TRP channel activators (10 mM camphor n=18, 10 mM cannabidiol n=16, and 100 mM 2‐APB n=15) caused large baseline calcium influxes, which were blocked by the TRP channel inhibitor ruthenium red (RuR, 10 mM; Camphor n=7; Cannabidiol n=24; 2‐APB n=11). An accompanying depolarization of the resting membrane potential and spontaneous firing was also observed, which was reduced by RuR. Other TRP channel activators (100 mM menthol n=15, 1 mM capsaicin n=16, 500 mM AITC n=9, 1 mM vanillin n=15, 300 nM JT010 n=15, 100 mM cavracol n=13, 10 mM icillin n=10, 50 mM naltriben n=15, pH=6.5 n=16 and 100 ng/ml IGF‐1 n=12) failed to produce significant responses.ScRNAseq also identified transcript expression of receptors for inflammatory mediators, for example histamine (HRH3) and prostaglandin E2 (PTGER2 and PTGER3). Stimulation of these receptors (100 mM histamine n=11 and 1 mM prostaglandin E2 n=8) had no effect on baseline [Ca2+]I, but were able to reduce Ca2+ influx in response to KCl depolarization (50 mM) or nicotine (100 mM).Overall, these data are important when interpreting stellate ganglia sympathetic neurons as purely efferent, with evidence that neuronal excitability can be directly influenced by sensory signaling pathways.Support or Funding InformationThis project was supported by the Wellcome trust and the British Heart Foundation

Group I metabotropic glutamate receptor activation induces TRPC6-dependent calcium influx and RhoA activation in cultured human kidney podocytes

Researches have shown that mice lacking the metabotropic glutamate receptor 1 (mGluR) showed albuminuria, remodeling of F-actin, with loss of stress fibers. Selective group I mGluRs agonist (S)-3,5-dihydroxyphenylglycine (DHPG) attenuated albuminuria in several rodent models of nephrotic syndrome. However, the molecular mechanism is obscure. Using a human podocyte cell line, we here investigated the molecular mechanisms of group I mGluRs-induced calcium influx and the formation of stress fibers. Our data showed that group I mGluRs activation by DHPG induced a significant calcium influx, and promoted cytoskeletal stress fiber formation and focal adhesions in podocytes. Pre-incubating podocytes with non-selective inhibitor of transient receptor potential channels (TRPC), or the knockdown of TRPC6 attenuated the calcium influx and the stress fiber formation induced by DHPG. Further, DHPG resulted in an increase of active RhoA expression. However, the knockdown of RhoA by siRNA abolished the DHPG-induced increase in stress fibers. Additionally, nonselective inhibitors of TRPC or TRPC6 knockdown clearly inhibited RhoA activation induced by DHPG, as assessed by Glutathione-S-transferase pull-down assay followed by Western blotting. Taken together, our findings suggest TRPC6 regulates actin stress fiber formation and focal adhesions via the RhoA pathway in response to group I mGluRs activation. Our data can potentially explain the mechanism of protective action of group I mGluRs in glomerular podocyte injury.

Pharmacological Disruption of Sea Urchin Tube Foot Motility and Behavior.

The understanding of the molecular basis of sea urchin behavior and sensory and motor systems lags far behind that of many other animal species. To investigate whole-animal behavior pharmacologically, we first demonstrated that immersion in drug solution is an effective drug administration route for sea urchins, whereas oral drug administration was found to be ineffective. Although intracoelomic injection was found to be effective at administering drugs, it was also found that injection itself can disrupt normal sea urchin behavior. Using the drug immersion procedure, we demonstrate that sea urchin locomotion and the sea urchin righting response are inhibited in a dose-dependent manner by the phosphodiesterase inhibitor theophylline and the transient receptor potential channel inhibitor 2-aminoethoxydiphenyl borate. The sea urchin righting response was also inhibited by the nitric oxide synthase inhibitor N(G)-nitro-l-arginine methyl ester and the Ca2+ channel inhibitor diltiazem, which, along with theophylline and 2-aminoethoxydiphenyl borate, would all be expected to disrupt smooth muscle function, based on studies in other animals. In addition, the removal of extracellular Ca2+ also inhibited the righting response, whereas an inhibitor of intracellular Ca2+ release, thapsigargin, did not affect the righting response, indicating that extracellular Ca2+ rather than intracellular Ca2+ stores are required for righting.

TRPV2 channel inhibitors attenuate fibroblast differentiation and contraction mediated by keratinocyte-derived TGF-β1 in an in vitro wound healing model of rats

BackgroundKeratinocytes release several factors that are involved in wound contracture and scar formation. We previously reported that a three-dimensional reconstruction model derived from rat skin represents a good wound healing model. ObjectiveWe characterized the role of transient receptor potential (TRP) channels in the release of transforming growth factor (TGF)-β1 from keratinocytes and the differentiation of fibroblasts to identify possible promising pharmacological approaches to prevent scar formation and contractures. MethodsThe three-dimensional culture model was made from rat keratinocytes seeded on a collagen gel in which dermal fibroblasts had been embedded. ResultsAmong the TRP channel inhibitors tested, the TRPV2 inhibitors SKF96365 and tranilast attenuated most potently keratinocyte-dependent and — independent collagen gel contraction due to TGF-β signaling as well as TGF-β1 release from keratinocytes and α-smooth muscle actin production in myofibroblasts. Besides the low amounts detected in normal dermis, TRPV2 mRNA and protein levels were increased after fibroblasts were embedded in the gel. TRPV2 was also expressed in the epidermis and keratinocyte layers of the model. Both inhibitors and TRPV2 siRNA attenuated the intracellular increase of Ca2+ induced by the TRPV agonist 2-aminoethoxydiphenyl borate in TGF-β1-pretreated fibroblasts. ConclusionThis is the first study to show that compounds targeting TRPV2 channels ameliorate wound contraction through the inhibition of TGF-β1 release and the differentiation of dermal fibroblasts in a culture model.

Intracellular stored calcium plays a minor role in Gq-coupled receptor-mediated contraction in rat airway smooth muscle

The general notion of activation of Gq-protein coupled receptors involves the mobilisation of stored and extracellular calcium and leads to smooth muscle tissue contraction. The aim of this study was to investigate the involvement of calcium mediated contractions in vascular and airway smooth muscles. Using the standard organ bath procedures, aortic and tracheal rings were obtained from 6 to 8 week-old male Sprague Dawley rats. To activate the Gq protein receptors, phenylephrine (PE), an α1-adrenoceptor agonist, and carbachol, a M3 cholinoceptor agonist, was added to baths containing the aortic and tracheal rings, respectively. The maximum response (Emax) to PE was reduced from 158.8 ± 11.8% (n=6) to 62.5 ± 12.4 % (n=8) upon removal of extracellular calcium in Krebs-Ringer solution. Maximal response to PE was also suppressed in the presence of nifedipine, a L-type Ca2+ channel inhibitor, (70.3 ± 11 %, n=8) and SKF96365, a canonical transient receptor potential channel inhibitor, (26.7 ± 13.2 %, n=5) when the influx of extracellular calcium was blocked. Removal of stored calcium also attenuated the PE contraction (p 0.05). From these observations, we conclude that the role of stored and extracellular calcium in Gq protein activation is not the same across different type of smooth muscle tissues.

Copper-induced early responses involve the activation of Transient Receptor Potential (TRP) channels, release of amino acids, serotonin and adrenalin, and activation of homologs of glutamate, adrenalin and serotonin receptors in the marine alga Ulva compressa

It has been previously shown that U. compressa cultivated with 10μM copper showed the activation of Transient Receptor Potential (TRP) channels at 4, 8 and 13min allowing copper ions entry and transient depolarizations. Here, the alga was incubated with copper and with inhibitors of human TRP channels that were incorporated just after copper addition (time 0), and after 1 and 2min of copper exposure, and membrane depolarizations were detected at 4, 8 and 13min. Copper induced the activation of a TRP A1/C5/M8 at time 0, a TRP A1/C4/M8/V1 after 1min, and a TRP A1/C5/M8, after 2min of copper exposure. In addition, EGTA, a specific calcium chelator, did not inhibit depolarization events when added at time 0, and after 1 and 2min, whereas bathocuproine, a specific copper chelator, inhibited transient depolarizations at 4, 8 and 13min. In addition, inhibitors of human GluR of NMDA type, added at time 0, and GluR of AMPA/KA types, added after 1 and 2min as well as inhibitors of serotonin and adrenalin receptors added after 2min inhibited depolarizations at 4, 8 and 13min. Furthermore, amino acids, serotonin and adrenalin were release to culture medium in response to copper excess. Thus, copper ions induces the activation of TRP channels allowing extracellular copper ions entry leading to transient depolarizations, release of aminoacids, serotonin, and adrenalin, and activation of homologs of glutamate, adrenalin and serotonin receptors which are involved in the activation of TRPs and depolarizations observed at 4, 8 and 12min of copper exposure.

Inhibitory effects of Tyrphostin AG-related compounds on oxidative stress-sensitive transient receptor potential channel activation

Some transient receptor potential (TRP) proteins including TRPA1, TPRM2 and TRPV1 are oxidative stress-sensitive Ca2+-permeable channels. Ca2+ signaling via these TRP channels activated by oxidative stress has been implicated in the aggravation of various inflammatory diseases and pain sensation. We recently reported that Tyrphostin AG490 exerted inhibitory effects on H2O2-induced TRPM2 activation by scavenging the hydroxyl radical. In order to identify stronger inhibitors of oxidative stress-sensitive TRP channels than AG490, we examined the inhibitory effects of Tyrphostin AG-related compounds on H2O2-induced TRP channel activation in human embryonic kidney 293 cells expressing TRP channels. AG555 and AG556 blocked the activation of TRPM2 by H2O2 more strongly than AG490. Regarding TRPV1 and TRPA1, none of the three compounds tested affected H2O2-induced TRPV1 activation; however, AG555 and AG556 reduced H2O2-induced TRPA1 activation more than AG490. Thus, we herein identified AG555 and AG556 as new compounds that exert stronger inhibitory effects on H2O2-induced TRPM2 and TRPA1 activation than AG490. Edaravone, a hydroxyl radical scavenger used in the treatment of cerebral hemorrhage and cerebral infarction, did not affect H2O2-induced TRPM2 or TRPA1 activation. AG555 and AG556 may be useful seed compounds as therapeutic agents for several TRP-related diseases associated with oxidative stress.

Estrogen Receptor Alpha Utilizes TRP Channel 6 in Cancer Cell Growth

The rapid signaling effects of the hormone estrogen (E2) are mediated through estrogen receptor alpha (ERα) that activates a Ca2+/calmodulin signaling pathway involving CaM KK, CaM KI, and ERK. Additionally, expression of transient receptor potential (TRP) channels, which are Ca2+ channels, may correlate with breast cancer cell growth. In this family of channels, TRPC6 has been shown to be expressed in MCF‐7 breast cancer cells and involved in ERK activation. Since both ERα and TRPC6 increase intracellular calcium levels, ERK activation, and cell growth our goal was to examine if ERα cooperates with TRPC6 to increase CaM Kinase and ERK phosphorylation as well as MCF‐7 cell growth. E2 treatment of MCF‐7 cells led to a significant rise in phosphorylation of CaM KI and its downstream target, ERK, and this effect was blocked by pretreatment with the ERa antagonist MPP. Treatment of MCF‐7 cells with E2 stimulated an increase in MCF‐7 cell growth that was blocked by pretreatment with MPP, but not by pretreatment with the ERb antagonist PHTPP. Interestingly, treatment of cells with the TRP channel inhibitor APB blocked E2 activation of ERK and cell growth. Immunoprecipitation (IP) of endogenous ERa pulled down TRPC6 as did the reciprocal IP from MCF‐7 cells. Confocal microscopy also revealed co‐localization of the proteins in MCF‐7 cells. Our data suggest E2 rapidly activates CaM KI and ERK via ERa and TRP channels and ERa may associate with TRPC6 to regulate breast cancer cell growth.Support or Funding InformationThe Paul K. and Evalyn E. C. Richter Memorial Fund.This work was supported through a Life Science grant from the M.J. Murdock Charitable Trust to J.M.S. grant #2011267.

Faint electric treatment-induced rapid and efficient delivery of extraneous hydrophilic molecules into the cytoplasm

Effective delivery of extraneous molecules into the cytoplasm of the target cells is important for several drug therapies. Previously, we showed effective in vivo transdermal delivery of naked siRNA into skin cells induced by faint electric treatment (ET) iontophoresis, and significant suppression of target mRNA levels (Kigasawa et al., Int. J. Pharm., 2010). This result indicates that electricity promoted the delivery of siRNA into cytoplasm. In the present study, we analyzed the intracellular delivery of naked anti-luciferase siRNA by faint ET, and found that the luciferase activity of cells expressing luciferase was reduced by in vitro ET like in vivo iontophoresis. Cellular uptake of fluorescent-label siRNA was increased by ET, while low temperature exposure, macropinocytosis inhibitor amiloride and caveolae-mediated endocytosis inhibitor filipin significantly prevented siRNA uptake. These results indicate that the cellular uptake mechanism involved endocytosis. In addition, voltage sensitive fluorescent dye DiBAC4 (3) penetration was increased by ET, and the transient receptor potential channel inhibitor SKF96365 reduced siRNA uptake, suggesting that faint ET reduced membrane potentials by changing intracellular ion levels. Moreover, to analyze cytoplasmic delivery, we used in-stem molecular beacon (ISMB), which fluoresces upon binding to target mRNA in the cytoplasm. Surprisingly, cytoplasmic ISMB fluorescence appeared rapidly and homogeneously after ET, indicating that cytoplasmic delivery is markedly enhanced by ET. In conclusion, we demonstrated for the first time that faint ET can enhance cellular uptake and cytoplasmic delivery of extraneous molecules.

Broad-range TRP channel inhibitors (2-APB, flufenamic acid, SKF-96365) affect differently contraction of resistance and conduit femoral arteries of rat

Transient receptor potential (TRP) channels are proposed to contribute to membrane depolarization and Ca2+ influx into vascular smooth muscle (VSM) cells. Our aim was to study the effects of widely used broad-range TRP channel inhibitors – 2-aminoethoxydiphenyl borate (2-APB), flufenamic acid (FFA) and SKF-96365 – on the contraction of freshly isolated small and large arteries.Endothelium-denuded resistance (≈250µm) and conduit (≈1000µm) femoral arteries were isolated from adult Wistar rats and mounted in wire myograph. The effects of the above mentioned TRP channel inhibitors and voltage-dependent calcium channel inhibitor nifedipine were studied on arterial contractions induced by phenylephrine, U-46619 or K+. Phenylephrine-induced contractions were also studied in the absence of extracellular Na+. mRNA expression of particular canonical and melastatin TRP channel subunits in femoral vascular bed was determined.TRP channel inhibitors attenuated K+-induced contraction less than nifedipine. Phenylephrine-induced contraction was more influenced by 2-APB in resistance arteries, while FFA completely prevented U-46619-induced contraction in both sizes of arteries. The absence of extracellular Na+ prevented the inhibitory effects of 2-APB, but not those of FFA.The observed effects of broad-range TRP channel inhibitors, which were dependent on the size of the artery, confirmed the involvement of TRP channels in agonist-induced contractions. The inhibitory effects of 2-APB (but not those of FFA or SKF-96365) were dependent on the presence of extracellular Na+.

TRP Channels Localize to Subdomains of the Apical Plasma Membrane in Human Fetal Retinal Pigment Epithelium.

Calcium regulates many functions of the RPE. Its concentration in the subretinal space and RPE cytoplasm is closely regulated. Transient receptor potential (TRP) channels are a superfamily of ion channels that are moderately calcium-selective. This study investigates the subcellular localization and potential functions of TRP channels in a first-passage culture model of human fetal RPE (hfRPE). The RPE isolated from 15- to 16-week gestation fetuses were maintained in serum-free media. Cultures were treated with barium chloride (BaCl2) in the absence and presence of TRP channel inhibitors and monitored by the transepithelial electrical resistance (TER). The expression of TRP channels was determined using quantitative RT-PCR, immunoblotting, and immunofluorescence confocal microscopy. Barium chloride substantially decreased TER and disrupted cell-cell contacts when added to the apical surface of RPE, but not when added to the basolateral surface. The effect could be partially blocked by the general TRP inhibitor, lanthanum chloride (LaCl3, ~75%), or an inhibitor of calpain (~25%). Family member-specific inhibitors, ML204 (TRPC4) and HC-067047 (TRPV4), had no effect on basal channel activity. Expression of TRPC4, TRPM1, TRPM3, TRPM7, and TRPV4 was detected by RT-PCR and immunoblotting. The TRPM3 localized to the base of the primary cilium, and TRPC4 and TRPM3 localized to apical tight junctions. The TRPV4 localized to apical microvilli in a small subset of cells. The TRP channels localized to subdomains of the apical membrane, and BaCl2 was only able to dissociate tight junctions when presented to the apical membrane. The data suggest a potential role for TRP channels as sensors of [Ca(2+)] in the subretinal space.

Inhibition of endothelial cell Ca²⁺ entry and transient receptor potential channels by Sigma-1 receptor ligands.

Background and PurposeThe Sigma-1 receptor (Sig1R) impacts on calcium ion signalling and has a plethora of ligands. This study investigated Sig1R and its ligands in relation to endogenous calcium events of endothelial cells and transient receptor potential (TRP) channels.Experimental ApproachIntracellular calcium and patch clamp measurements were made from human saphenous vein endothelial cells and HEK 293 cells expressing exogenous human TRPC5, TRPM2 or TRPM3. Sig1R ligands were applied and short interfering RNA was used to deplete Sig1R. TRP channels tagged with fluorescent proteins were used for subcellular localization studies.Key ResultsIn endothelial cells, 10–100 μM of the Sig1R antagonist BD1063 inhibited sustained but not transient calcium responses evoked by histamine. The Sig1R agonist 4-IBP and related antagonist BD1047 were also inhibitory. The Sig1R agonist SKF10047 had no effect. Sustained calcium entry evoked by VEGF or hydrogen peroxide was also inhibited by BD1063, BD1047 or 4-IBP, but not SKF10047. 4-IBP, BD1047 and BD1063 inhibited TRPC5 or TRPM3, but not TRPM2. Inhibitory effects of BD1047 were rapid in onset and readily reversed on washout. SKF10047 inhibited TRPC5 but not TRPM3 or TRPM2. Depletion of Sig1R did not prevent the inhibitory actions of BD1063 or BD1047 and Sig1R did not co-localize with TRPC5 or TRPM3.Conclusions and ImplicationsThe data suggest that two types of Sig1R ligand (BD1047/BD1063 and 4-IBP) are inhibitors of receptor- or chemically activated calcium entry channels, acting relatively directly and independently of the Sig1R. Chemical foundations for TRP channel inhibitors are suggested.

A cell-based impedance assay for monitoring transient receptor potential (TRP) ion channel activity

Transient receptor potential (TRP) channels are non-selective ion channels permeable to cations including Na +, Ca 2+ and Mg 2+. They play a unique role as cellular sensors and are involved in many Ca 2+-mediated cell functions. Failure in channel gating can contribute to complex pathophysiological mechanisms. Dysfunctions of TRP channels cause diseases but are also involved in the progress of diseases. We present a novel method to analyse chemical compounds as potential activators or inhibitors of TRP channels to provide pharmaceutical tools to regulate channel activity for disease control. Compared to common methods such as patch clamp or Ca 2+ imaging, the presented impedance assay is automatable, experimental less demanding and not restricted to Ca 2+ flow. We have chosen TRPA1 from the TRPA (‘ankyrin’) family as a model channel which was stimulated by allyl isothiocyanate (AITC). HEK293 cells stably transfected with human TRPA1 cDNA were grown on microelectrode arrays. Confluent cell layers of high density were analysed. Impedance spectra of cell-covered and non-covered electrodes yielded a cell-specific signal at frequencies between 70 and 120 kHz. Therefore, 100 kHz was chosen to monitor TRPA1 activity thereupon. An average impedance decrease to about 70% of its original value was observed after application of 10 μM AITC indicating an increased conductance of the cell layer mediated by TRPA1. Transfected cells pretreated with 10 μM of inhibitor ruthenium red to prevent channel conductance, as well as control cells lacking TRPA1, showed no impedance changes upon AITC stimuli demonstrating the specificity of the novel impedance assay.