Effects of mitochondria-associated Ca2+ transporters suppression on oocyte activation.

Oocyte activation deficiency leads to female infertility. [Ca2+ ]i oscillations are required for mitochondrial energy supplement transition from the resting to the excited state, but the underlying mechanisms are still very little known. Three mitochondrial Ca2+ channels, Mitochondria Calcium Uniporter (MCU), Na+ /Ca2+ Exchanger (NCLX) and Voltage-dependent Ca2+ Channel (VDAC), were deactivated by inhibitors RU360, CGP37157 and Erastin, respectively. Both Erastin and CGP37157 inhibited mitochondrial activity significantly while attenuating [Ca2+ ]i and [Ca2+ ]m oscillations, which caused developmental block of pronuclear formation. Thus, NCLX and VDAC are two mitochondria-associated Ca2+ transporter proteins regulating oocyte activation, which may be used as potential targets to treat female infertility. SIGNIFICANCE OF THE STUDY: NCLX and VDAC are two mitochondria-associated Ca2+ transporter proteins regulating oocyte activation.

Antinociceptive activity of 3ß-6ß-16ß-trihydroxylup-20 (29)-ene triterpene isolated from Combretum leprosum leaves in adult zebrafish (Danio rerio).

Drugs used to treat pain are associated with adverse effects, increasing the search for new drugs as an alternative treatment for pain. Therefore, we evaluated the antinociceptive behavior and possible neuromodulation mechanisms of triterpene 3ß, 6ß, 16ß-trihydroxylup-20(29)-ene (CLF-1) isolated from Combretum leprosum leaves in zebrafish. Zebrafish (n = 6/group) were pretreated with CLF-1 (0.1 or 0.3 or 1.0 mg/mL; i.p.) and underwent nociception behavior tests. The antinociceptive effect of CFL-1 was tested for modulation by opioid (naloxone), nitrergic (L-NAME), nitric oxide and guanylate cyclase synthesis inhibitor (methylene blue), NMDA (Ketamine), TRPV1 (ruthenium red), TRPA1 (camphor), or ASIC (amiloride) antagonists. The corneal antinociceptive effect of CFL-1 was tested for modulation by TRPV1 (capsazepine). The effect of CFL-1 on zebrafish locomotor behavior was evaluated with the open field test. The acute toxicity study was conducted. CLF-1 reduced nociceptive behavior and corneal in zebrafish without mortalities and without altering the animals' locomotion. Thus, CFL-1 presenting pharmacological potential for the treatment of acute pain and corneal pain, and this effect is modulated by the opioids, nitrergic system, NMDA receptors and TRP and ASIC channels.

Role of TRPV1/TRPV3 channels in olanzapine-induced metabolic alteration: Possible involvement in hypothalamic energy-sensing, appetite regulation, inflammation and mesolimbic pathway.

Atypical antipsychotics (AAPs) have the tendency of inducing severe metabolic alterations like obesity, diabetes mellitus, insulin resistance, dyslipidemia and cardiovascular complications. These alterations have been attributed to altered hypothalamic appetite regulation, energy sensing, insulin/leptin signaling, inflammatory reactions and active reward anticipation. Line of evidence suggests that transient receptor potential vanilloid type 1 and 3 (TRPV1 and TRPV3) channels are emerging targets in treatment of obesity, diabetes mellitus and could modulate feed intake. The present study was aimed to investigate the putative role TRPV1/TRPV3 in olanzapine-induced metabolic alterations in mice. Female BALB/c mice were treated with olanzapine for six weeks to induce metabolic alterations. Non-selective TRPV1/TRPV3 antagonist (ruthenium red) and selective TRPV1 (capsazepine) and TRPV3 antagonists (2,2-diphenyltetrahydrofuran or DPTHF) were used to investigate the involvement of TRPV1/TRPV3 in chronic olanzapine-induced metabolic alterations. These metabolic alterations were differentially reversed by ruthenium red and capsazepine, while DPTHF didn't show any significant effect. Olanzapine treatment also altered the mRNA expression of hypothalamic appetite-regulating and nutrient-sensing factors, inflammatory genes and TRPV1/TRPV3, which were reversed with ruthenium red and capsazepine treatment. Furthermore, olanzapine treatment also increased expression of TRPV1/TRPV3 in nucleus accumbens (NAc), TRPV3 expression in ventral tegmental area (VTA), which were reversed by the respective antagonists. However, DPTHF treatment showed reduced feed intake in olanzapine treated mice, which might be due to TRPV3 specific antagonism and reduced hedonic feed intake. In conclusion, our results suggested the putative role TRPV1 in hypothalamic dysregulations and TRPV3 in the mesolimbic pathway; both regulate feeding in olanzapine treated mice.

Cannabidiol Increases Proliferation, Migration, Tubulogenesis, and Integrity of Human Brain Endothelial Cells through TRPV2 Activation.

The effect of cannabidiol (CBD), a high-affinity agonist of the transient receptor potential vanilloid-2 (TRPV2) channel, has been poorly investigated in human brain microvessel endothelial cells (BMEC) forming the blood-brain barrier (BBB). TRPV2 expression and its role on Ca2+ cellular dynamics, trans-endothelial electrical resistance (TEER), cell viability and growth, migration, and tubulogenesis were evaluated in human primary cultures of BMEC (hPBMEC) or in the human cerebral microvessel endothelial hCMEC/D3 cell line. Abundant TRPV2 expression was measured in hCMEC/D3 and hPBMEC by qRT-PCR, Western blotting, nontargeted proteomics, and cellular immunofluorescence studies. Intracellular Ca2+ levels were increased by heat and CBD and blocked by the nonspecific TRP antagonist ruthenium red (RR) and the selective TRPV2 inhibitor tranilast (TNL) or by silencing cells with TRPV2 siRNA. CBD dose-dependently induced the hCMEC/D3 cell number (EC50 0.3 ± 0.1 µM), and this effect was fully abolished by TNL or TRPV2 siRNA. A wound healing assay showed that CBD induced cell migration, which was also inhibited by TNL or TRPV2 siRNA. Tubulogenesis of hCMEC/D3 cells in 3D matrigel cultures was significantly increased by 41 and 73% after a 7 or 24 h CBD treatment, respectively, and abolished by TNL. CBD also increased the TEER of hPBMEC monolayers cultured in transwell, and this was blocked by TNL. Our results show that CBD, at extracellular concentrations close to those observed in plasma of patients treated by CBD, induces proliferation, migration, tubulogenesis, and TEER increase in human brain endothelial cells, suggesting CBD might be a potent target for modulating the human BBB.

Calcium ion regulation by BAPTA-AM and ruthenium red improved the fertilisation capacity and developmental ability of vitrified bovine oocytes.

Vitrification reduces the fertilisation capacity and developmental ability of mammalian oocytes; this effect is closely associated with an abnormal increase of cytoplasmic free calcium ions ([Ca2+]i). However, little information about the mechanism by which vitrification increases [Ca2+]i levels or a procedure to regulate [Ca2+]i levels in these oocytes is available. Vitrified bovine oocytes were used to analyse the effect of vitrification on [Ca2+]i, endoplasmic reticulum Ca2+ (ER Ca2+), and mitochondrial Ca2+ (mCa2+) levels. Our results showed that vitrification, especially with dimethyl sulfoxide (DMSO), can induce ER Ca2+ release into the cytoplasm, consequently increasing the [Ca2+]i and mCa2+ levels. Supplementing the cells with 10 µM 1,2-bis (o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM or BAPTA) significantly decreased the [Ca2+]i level and maintained the normal distribution of cortical granules in the vitrified bovine oocytes, increasing their fertilisation ability and cleavage rate after in vitro fertilisation (IVF). Treating vitrified bovine oocytes with 1 µM ruthenium red (RR) significantly inhibited the Ca2+ flux from the cytoplasm into mitochondria; maintained normal mCa2+ levels, mitochondrial membrane potential, and ATP content; and inhibited apoptosis. Treating vitrified oocytes with a combination of BAPTA and RR significantly improved embryo development and quality after IVF.

Grip strength in mice with joint inflammation: A rheumatology function test sensitive to pain and analgesia.

Grip strength deficit is a measure of pain-induced functional disability in rheumatic disease. We tested whether this parameter and tactile allodynia, the standard pain measure in preclinical studies, show parallels in their response to analgesics and basic mechanisms. Mice with periarticular injections of complete Freund's adjuvant (CFA) in the ankles showed periarticular immune infiltration and synovial membrane alterations, together with pronounced grip strength deficits and tactile allodynia measured with von Frey hairs. However, inflammation-induced tactile allodynia lasted longer than grip strength alterations, and therefore did not drive the functional deficits. Oral administration of the opioid drugs oxycodone (1-8 mg/kg) and tramadol (10-80 mg/kg) induced a better recovery of grip strength than acetaminophen (40-320 mg/kg) or the nonsteroidal antiinflammatory drugs ibuprofen (10-80 mg/kg) or celecoxib (40-160 mg/kg); these results are consistent with their analgesic efficacy in humans. Functional impairment was generally a more sensitive indicator of drug-induced analgesia than tactile allodynia, as drug doses that attenuated grip strength deficits showed little or no effect on von Frey thresholds. Finally, ruthenium red (a nonselective TRP antagonist) or the in vivo ablation of TRPV1-expressing neurons with resiniferatoxin abolished tactile allodynia without altering grip strength deficits, indicating that the neurobiology of tactile allodynia and grip strength deficits differ. In conclusion, grip strength deficits are due to a distinct type of pain that reflects an important aspect of the human pain experience, and therefore merits further exploration in preclinical studies to improve the translation of new analgesics from bench to bedside.

Five hTRPA1 Agonists Found in Indigenous Korean Mint, Agastache rugosa.

Transient receptor potential ankyrin1 (TRPA1) and transient receptor potential vanilloid 1 (TRPV1) are members of the TRP superfamily of structurally related, nonselective cation channels and mediators of several signaling pathways. Previously, we identified methyl syringate as an hTRPA1 agonist with efficacy against gastric emptying. The aim of this study was to find hTRPA1 and/or hTRPV1 activators in Agastache rugosa (Fisch. et Meyer) O. Kuntze (A.rugosa), commonly known as Korean mint to improve hTRPA1-related phenomena. An extract of the stem and leaves of A.rugosa (Labiatae) selectively activated hTRPA1 and hTRPV1. We next investigated the effects of commercially available compounds found in A.rugosa (acacetin, 4-allylanisole, p-anisaldehyde, apigenin 7-glucoside, L-carveol, ß-caryophyllene, trans-p-methoxycinnamaldehyde, methyl eugenol, pachypodol, and rosmarinic acid) on cultured hTRPA1- and hTRPV1-expressing cells. Of the ten compounds, L-carveol, trans-p-methoxycinnamaldehyde, methyl eugenol, 4-allylanisole, and p-anisaldehyde selectively activated hTRPA1, with EC50 values of 189.1±26.8, 29.8±14.9, 160.2±21.9, 1535±315.7, and 546.5±73.0 µM, respectively. The activities of these compounds were effectively inhibited by the hTRPA1 antagonists, ruthenium red and HC-030031. Although the five active compounds showed weaker calcium responses than allyl isothiocyanate (EC50=7.2±1.4 µM), our results suggest that these compounds from the stem and leaves of A.rugosa are specific and selective agonists of hTRPA1.

A FluoPol-ABPP PAD2 high-throughput screen identifies the first calcium site inhibitor targeting the PADs.

The protein arginine deiminases (PADs) catalyze the post-translational hydrolysis of peptidyl-arginine to form peptidyl-citrulline in a process termed deimination or citrullination. PADs likely play a role in the progression of a range of disease states because dysregulated PAD activity is observed in a host of inflammatory diseases and cancer. For example, recent studies have shown that PAD2 activates ERα target gene expression in breast cancer cells by citrullinating histone H3 at ER target promoters. To date, all known PAD inhibitors bind directly to the enzyme active site. PADs, however, also require calcium ions to drive a conformational change between the inactive apo-state and the fully active calcium bound holoenzyme, suggesting that it would be possible to identify inhibitors that bind the apoenzyme and prevent this conformational change. As such, we set out to develop a screen that can identify PAD2 inhibitors that bind to either the apo or calcium bound form of PAD2. Herein, we provide definitive proof of concept for this approach and report the first PAD inhibitor, ruthenium red (Ki of 17 µM), to preferentially bind the apoenzyme.

Ca2+ signal transduction related to neutral lipid synthesis in an oil-producing green alga Chlorella sp. C2.

Changes in the cytosolic Ca(2+) levels and the role of Ca(2+) signal transduction in neutral lipid synthesis in Chlorella sp. C2 under nitrogen starvation conditions were investigated. The results detected by using the scanning ion-selective electrode technique demonstrate that nitrogen starvation induced significant Ca(2+) influx across the plasma membrane into cells. Ca(2+) fluorescence imaging and flow cytometry were used to estimate the effect of this Ca(2+) influx on the generation of the Ca(2+) signal, and the results showed that the cytosolic Ca(2+) concentration increased transiently and then remained at a stable, high level when the cells were exposed to nitrogen starvation. However, the increase could be inhibited by pre-treatment with the Ca(2+) channel blockers ruthenium red, verapamil and GdCl3, indicating that both the influx of Ca(2+) from the extracellular space via Ca(2+) channels that are localized in the plasma membrane and the release of Ca(2+) from intracellular calcium storage via the internal calcium store were required for the generation and transduction of the Ca(2+) signal. During nitrogen starvation, neutral lipid synthesis in Chlorella sp. C2 in response to stress conditions was also inhibited to differing degrees by pre-treatment with the three Ca(2+) channel blockers, demonstrating the regulation of Ca(2+) via these Ca(2+) channels in neutral lipid synthesis. The results suggested that by transduction of extracellular stress signals into the cell and the regulation of the Ca(2+) signal in neutral lipid synthesis, Ca(2+) signal transduction played important roles in the response mechanism of Chlorella sp. C2 to nitrogen starvation.

Nutrient deprivation increases vulnerability of endothelial cells to proinflammatory insults.

Nutrient deprivation is a stimulus for oxidative stress and is an established method for induction of cell autophagy and apoptosis. The aims of this study were to identify conditions that evoke superoxide production in cultured human umbilical vein endothelial cells (HUVECs), determine the mechanism of action for this response, and examine whether the stimulus might facilitate the adhesion of human isolated neutrophils to the HUVECs. HUVECs were incubated in M199 medium under conditions of serum starvation (serum-free M199 medium), low serum (medium containing 2% fetal calf serum), and high serum (medium containing 20% fetal calf serum). HUVECs were also incubated under proinflammatory conditions, in medium supplemented with 50ng/ml tumor necrosis factor-α (TNF-α) or neutrophils preactivated with 10nM phorbol 12-myristate 13-acetate (PMA). Superoxide production was increased fourfold in serum-starved HUVECs compared to cells incubated in 20% medium, and this was reduced by inhibitors of the mitochondrial electron transport chain and mitochondrial Ca(2+) uniporter. Superoxide production was 23.6% higher in HUVECs incubated with TNF-α in 2% medium compared to 2% medium alone, but unchanged with TNF-α in 20% medium. PMA-activated neutrophils adhered to morphologically aberrant HUVECs, which were mainly evident under the low-serum condition. The findings show a role of mitochondrial enzymes in superoxide production in response to nutrient deprivation and suggest that proinflammatory responses in HUVECs become manifest when HUVECs are in an already-compromised state.

Anti-inflammatory and anti-ulcer activities of carvacrol, a monoterpene present in the essential oil of oregano.

This study reports a pharmacological evaluation of anti-inflammatory and anti-ulcer activities of carvacrol, a phenolic monoterpene constituent of essential oils produced by oregano and other several aromatic plants and spices, in experimental models of edema induced by different phlogistic agents and gastric lesions induced by acetic acid. In models of paw edema induced by dextran or histamine, carvacrol was effective at 50 mg/kg (46% and 35%, respectively); in these models, cyproheptadine reduced edema formation (61% and 43%, respectively). In edema induced by substance P, carvacrol (100 mg/kg) and ruthenium red (3 mg/kg) also decreased the edema formation (46% and 40%, respectively). Carvacrol significantly reduced the ear edema induced by 12-O-tetradecanoylphorbol acetate and arachidonic acid at 0.1 mg per ear (43% and 33%, respectively), similar to indomethacin at 0.5 mg per ear or 2.0 mg per ear (55% and 57%, respectively). Carvacrol (at doses of 25, 50, and 100 mg/kg) showed a healing capacity on gastric lesions induced by acid acetic (60%, 91%, and 81%, respectively) after 14 days of treatment. These results suggest that carvacrol acts on different pharmacological targets, probably interfering in release and/or synthesis of inflammatory mediators, such as the prostanoids, and thus favoring the healing process for gastric ulcers.

Glucose starvation stimulates Zn2+ toxicity in cultures of cerebellar granule neurons.

Zinc chloride (0.02 mM, 3h) did not have any influence on the survival of cerebellar granule neurons (CGNs) incubated in balanced salt solution (BSS). However, in the absence of glucose ZnCl(2) caused severe neuronal damage, decreasing cell survival to 12±2%. Either the blockade of ionotropic glutamate NMDA-receptors with MK-801 or APV or supplementation the medium with ruthenium red (mitochondrial Ca(2+) uniporter blocker) almost entirely protected CGNs from the toxic effect of ZnCl(2) during glucose deprivation (GD). However, NBQX (AMPA/kainate glutamate receptor blocker) did not show protective effect. Measurements of intracellular calcium ions concentration using fluorescent probe (Fluo-4 AM) and zinc ions (FluoZin-3AM) demonstrated that 1.5h-exposure to GD induced intensive increase of Fluo-4 fluorescence and small increase of FluoZin-3 fluorescence in neurons. The supplementation of medium with ZnCl(2) caused equal increase of FluoZin-3 fluorescence at both GD and normoglycemia, whereas the potentiation of Fluo-4 fluorescence by zinc was observed only under GD and could be prevented by MK-801. However, neither MK-801 nor NBQX could influence [Zn(2+)](i) increase caused by zinc addition under GD, while ruthenium red did cause significant increase of [Zn(2+)](i). This data implies that zinc ions during GD induce an additional overload of CGNs with calcium ions that get transported through activated NMDA-channel. Zinc and calcium ions accumulate in mitochondria and amplify individual destructive action on these organelles leading to neuronal death.

Determinants of TRPV4 activity following selective activation by small molecule agonist GSK1016790A.

TRPV4 (Transient Receptor Potential Vanilloid 4) channels are activated by a wide range of stimuli, including hypotonic stress, non-noxious heat and mechanical stress and some small molecule agonists (e.g. phorbol ester 4α-PDD). GSK1016790A (GSK101) is a recently discovered specific small molecule agonist of TRPV4. Its effects on physical determinants of TRPV4 activity were evaluated in HeLa cells transiently transfected with TRPV4 (HeLa-TRPV4). GSK101 (10 nM) causes a TRPV4 specific Ca(2+) influx in HeLa-TRPV4 cells, but not in control transfected cells, which can be inhibited by ruthenium red and Ca(2+)-free medium more significantly at the early stage of the activation rather than the late stage, reflecting apparent partial desensitization. Western blot analysis showed that GSK101 activation did not induce an increase in TRPV4 expression at the plasma membrane, but caused an immediate and sustained downregulation of TRPV4 on the plasma membrane in HeLa-TRPV4 cells. Patch clamp analysis also revealed an early partial desensitization of the channel which was Ca(2+)-independent. FRET analysis of TRPV4 subunit assembly demonstrated that the GSK101-induced TRPV4 channel activation/desensitization was not due to alterations in homotetrameric channel formation on the plasma membrane. It is concluded that GSK101 specifically activates TRPV4 channels, leading to a rapid partial desensitization and downregulation of the channel expression on the plasma membrane. TRPV4 subunit assembly appears to occur during trafficking from the ER/Golgi to the plasma membrane and is not altered by agonist stimulation.

Laser modulation of heat and capsaicin receptor TRPV1 leads to thermal antinociception.

Er,Cr:YSGG lasers are used clinically in dentistry. The advantages of laser therapy include minimal thermal damage and the alleviation of pain. This study examined whether the Er,Cr:YSGG laser has in vivo and in vitro antinociceptive effects in itself. In capsaicin-evoked acute licking/shaking tests and Hargreaves tests, laser irradiation with an aerated water spray suppressed nociceptive behavior in mice. Laser irradiation attenuated TRPV1 activation by capsaicin in Ca(2+) imaging experiments with TRPV1-overexpressing cells and cultured trigeminal neurons. Therefore, the laser-induced behavioral changes are probably due to the loss of TRPV1 activity. TRPV4 activity was also attenuated, but limited mechanical antinociception by the laser was observed. The laser failed to alter the other receptor functions, which indicates that the antinociceptive effect of the laser is dependent on TRPV1. These results suggest that the Er,Cr:YSGG laser has analgesic effects via TRPV1 inhibition. Such mechanistic approaches may help define the laser-sensitive pain modality and increase its beneficial uses.

Effects of shear stress on intracellular calcium change and histamine release in rat basophilic leukemia (RBL-2H3) cells.

Massage, one form of physical therapy, is widely used for a large number of musculoskeletal disorders, but its exact mechanism still remains to be elucidated. One hypothesis is that the shear stress caused by massage may induce cutaneous mast cells to release histamine, thereby improving the local tissue microcirculation of blood. In the present work, a mast cell line (rat basophilic leukemia cells, RBL-2H3) was used in vitro to study cellular responses to the stimulus of shear stress generated by a rotating rotor in a cell dish. The intracellular calcium ([Ca2+]c) was studied by confocal fluorescence microscopy with Fluo-3/AM staining and the released histamine was measured with a fluorescence spectrometer using o-phthalaldehyde (OPA) staining. An elevation of [Ca2+]c occurred immediately after the shear stress, followed by histamine release. However, both [Ca2+]c increase and histamine release disappeared when a Ca2+-free saline was used, indicating that the rise in the [Ca2+]c is due to a Ca2+ influx from the extracellular buffer. Furthermore, Ruthenium red, a transient receptor potential vanilloid (TRPV) inhibitor, could effectively block the shear stressinduced histamine release, suggesting that TRPV membrane proteins are the likely targets of the shear stress. Because histamine is a well-known mediator of microvascular tissue dilation, these results may have an important impact on understanding the mechanism involved in massage therapy.

[Effects of ruthenium red on body temperature in rats with lipopolysaccharide-induced fever].

OBJECTIVE: To observe the effect of ruthenium red (RR) on the body temperature of rats with lipopolysaccharide (LPS)-induced fever and investigate the possible mechanism. METHODS: Rat models of fever were established with lipopolysaccharide and the effects of RR at different doses were observed on the body temperature of the rats and the content of TRPV4 in the hypothalamus. RESULTS: Compared with those in LPS group, the rats with LPS-induced fever receiving RR treatment showed a dose-dependent lowering of the body temperature. The rats with RR treatment had lower body temperature than those with saline injection. The content of TRPV4 in the saline group was significantly higher than that in RR+LPS and RR group. CONCLUSIONS: RR inhibits LPS-induced fever in rats and regulates the hypothalamal expression of TRPV4 channels, which may participate in the maintenance of normal body temperature.

Mechanisms of gastroprotective effect of eugenol in indomethacin-induced ulcer in rats.

Possible mechanisms underlying the gastroprotective effect of eugenol against indomethacin-induced ulcer in rats were investigated. Pyloric ligation was performed for collection of gastric juice, and gastric ulceration was induced by a single intraperitoneal (i.p.) injection of indomethacin (30 mg/kg). Pretreatment with a single dose of eugenol (100 mg/kg, orally), 1 h before indomethacin administration caused significant reductions in gastric mucosal lesions, gastric acid outputs and pepsin activity associated with a significant increase in mucin concentration. Additionally, eugenol significantly attenuated the elevations in gastric mucosal malondialdehyde and total nitrite, and the decrease in reduced glutathione observed with indomethacin. The protective effect afforded by eugenol was significantly inhibited by prior administration of glibenclamide, the ATP-sensitive potassium (K(ATP)) channel blocker, but not by prior use of ruthenium red, the transient receptor potential vanilloid 1 (TRPV1) antagonist. The results indicate that the anti-ulcer effect of eugenol is mediated by opening of K(ATP) channels, scavenging free radicals, decreasing acid-pepsin secretion, increasing mucin production, and preventing the deleterious rise in nitric oxide level.

Disruption of the intracellular Ca2+ homeostasis in the cardiac excitation-contraction coupling is a crucial mechanism of arrhythmic toxicity in aconitine-induced cardiomyocytes.

Aconitine is an effective ingredient in Aconite tuber, an important traditional Chinese medicine. Aconitine is also known to be a highly toxic diterpenoid alkaloid with arrhythmogenic effects. In the present study, we have characterized the properties of arrhythmic cytotoxicity and explored the possible mechanisms of aconitine-induced cardiomyocytes. Results show that aconitine induces significant abnormity in the spontaneous beating rate, amplitude of spontaneous oscillations and the relative intracellular Ca(2+) concentration. Also, mRNA transcription levels and protein expressions of SR Ca(2+) release channel RyR(2) and sarcolemmal NCX were elevated in aconitine-induced cardiomyocytes. However, co-treatment with ruthenium red (RR), a RyR channel inhibitor, could reverse the aconitine-induced abnormity in intracellular Ca(2+) signals. These results demonstrate that disruption of intracellular Ca(2+) homeostasis in the cardiac excitation-contraction coupling (EC coupling) is a crucial mechanism of arrhythmic cytotoxicity in aconitine-induced cardiomyocytes. Moreover, certain inhibitors appear to play an important role in the detoxification of aconitine-induced Ca(2+)-dependent arrhythmias.

Transient receptor potential vanilloid 1 is required for intrinsic osmoreception in organum vasculosum lamina terminalis neurons and for normal thirst responses to systemic hyperosmolality.

Recent studies have indicated that members of the transient receptor potential vanilloid (TRPV) family of cation channels are required for the generation of normal osmoregulatory responses, yet the mechanism of osmosensory transduction in primary osmoreceptor neurons of the CNS remains to be defined. Indeed, despite ample evidence suggesting that the organum vasculosum lamina terminalis (OVLT) serves as the primary locus of the brain for the detection of osmotic stimuli, evidence that neurons in the OVLT are intrinsically osmosensitive has remained elusive. Here we show that murine OVLT neurons are intrinsically sensitive to increases in the osmolality of the extracellular fluid. Hypertonic conditions provoked increases in membrane cation conductance that resulted in the generation of an inward current, depolarizing osmoreceptor potentials, and enhanced action potential discharge. Moreover, we found that this osmosensory signal transduction cascade was absent in OVLT neurons from TRPV1 knock-out (TRPV1-/-) mice and that responses of wild type (WT) OVLT neurons could be blocked by ruthenium red, an inhibitor of TRPV channels. Finally, TRPV1-/- mice showed significantly attenuated water intake in response to systemic hypertonicity compared with WT controls. These findings indicate that OVLT neurons act as primary osmoreceptors and that a product of the trpv1 gene is required for osmosensory transduction.

Attenuation of visceral nociception by alpha- and beta-amyrin, a triterpenoid mixture isolated from the resin of Protium heptaphyllum, in mice.

In the search for novel natural compounds effective against visceral nociception, the triterpenoid mixture alpha- and beta-amyrin, isolated from Protium heptaphyllum resin, was assessed in two established mouse models of visceral nociception. Mice were pretreated orally with alpha- and beta-amyrin (3, 10, 30, and 100 mg/kg) or vehicle, and the pain-related behavioral responses to intraperitoneal cyclophosphamide or to intracolonic mustard oil were analyzed. The triterpenoid mixture showed a dose-related significant antinociception against the cyclophosphamide-induced bladder pain, and at 100 mg/kg, the nociceptive behavioral expression was almost completely suppressed. Intracolonic mustard oil-induced nociceptive behaviors were maximally inhibited by 10 mg/kg alpha- and beta-amyrin mixture in a naloxone-reversible manner. While pretreatment with ruthenium red (3 mg/kg, s. c.), a non-specific transient receptor potential cation channel V1 (TRPV1) antagonist, also caused significant inhibition, the alpha (2)-adrenoceptor antagonist, yohimbine (2 mg/kg, s. c.), showed no significant effect. The triterpene mixture (10 mg/kg, p. o.) neither altered significantly the pentobarbital sleeping time, nor impaired the ambulation or motor coordination in open-field and rotarod tests, respectively, indicating the absence of sedative or motor abnormalities that could account for its antinociception. These results indicate that the antinociceptive potential of alpha- and beta-amyrin possibly involves the opioid and vanilloid (TRPV1) receptor mechanisms and further suggests that it could be useful to treat visceral pain of intestinal and pelvic origins.