Searching for calcium antagonists for hypertension disease therapy from Moutan Cortex, using bioactivity integrated UHPLC-QTOF-MS.

INTRODUCTION: Calcium channel blockers (CCBs) are currently the most commonly used drugs for the treatment of hypertension. Moutan Cortex (MC), a traditional Chinese herb, has been found to have an anti-hypertensive effect. However, its potential mechanisms in the regulation of intracellular calcium concentration ([Ca2+ ]i ) remain poorly understood. OBJECTIVE: The main objective of this work was to identify the potential calcium antagonists from MC and study their molecular mechanisms. METHODS: Ultra-high performance liquid chromatography-quadrupole-time-of-fight-mass spectrometry (UHPLC-QTOF-MS) analysis combined with a dual-luciferase reporter assay was utilised to systematically screen the calcium antagonistic active ingredients in the methanol extract of MC. Additionally, the molecular mechanism of these compounds was further studied using live-cell imaging analysis with the calcium ion (Ca2+ ) probe dye fluo-4/AM to monitor changes in [Ca2+ ]i . RESULTS: Three monoterpenoids (paeoniflorin, benzoylpaeoniflorin and mudanpioside C), one phenolic acid (paeonol) and one gallotannin (1,2,3,4,6-O-pentagalloylglucose) were screened out as potential calcium antagonists in MC. Among them, the calcium antagonistic activity of benzoylpaeoniflorin, mudanpioside C and 1,2,3,4,6-O-pentagalloylglucose is first reported. Additionally, paeoniflorin, benzoylpaeoniflorin, mudanpioside C and paeonol can effectively block voltage-operated Ca2+ channels (VOCCs) to exert calcium antagonism, while 1,2,3,4,6-O-pentagalloylglucose plays a role in blocking inositol 1,4,5-trisphosphate receptors (IP3Rs). CONCLUSION: This work indicated that the anti-hypertensive efficacy of MC acted through multiple components selectively antagonising multiple cell signalling pathways to regulate [Ca2+ ]i . Furthermore, they could be considered as a reference standard for controlling the quality of Chinese medicinal materials.

Utilizing the planarian voltage-gated ion channel transcriptome to resolve a role for a Ca2+ channel in neuromuscular function and regeneration.

The robust regenerative capacity of planarian flatworms depends on the orchestration of signaling events from early wounding responses through the stem cell enacted differentiative outcomes that restore appropriate tissue types. Acute signaling events in excitable cells play an important role in determining regenerative polarity, rationalized by the discovery that sub-epidermal muscle cells express critical patterning genes known to control regenerative outcomes. These data imply a dual conductive (neuromuscular signaling) and instructive (anterior-posterior patterning) role for Ca2+ signaling in planarian regeneration. Here, to facilitate study of acute signaling events in the excitable cell niche, we provide a de novo transcriptome assembly from the planarian Dugesia japonica allowing characterization of the diverse ionotropic portfolio of this model organism. We demonstrate the utility of this resource by proceeding to characterize the individual role of each of the planarian voltage-operated Ca2+ channels during regeneration, and demonstrate that knockdown of a specific voltage operated Ca2+ channel (Cav1B) that impairs muscle function uniquely creates an environment permissive for anteriorization. Provision of the full transcriptomic dataset should facilitate further investigations of molecules within the planarian voltage-gated channel portfolio to explore the role of excitable cell physiology on regenerative outcomes. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.

Conditional Deletion of the L-Type Calcium Channel Cav1.2 in NG2-Positive Cells Impairs Remyelination in Mice.

Exploring the molecular mechanisms that drive the maturation of oligodendrocyte progenitor cells (OPCs) during the remyelination process is essential to developing new therapeutic tools to intervene in demyelinating diseases such as multiple sclerosis. To determine whether L-type voltage-gated calcium channels (L-VGCCs) are required for OPC development during remyelination, we generated an inducible conditional knock-out mouse in which the L-VGCC isoform Cav1.2 was deleted in NG2-positive OPCs (Cav1.2KO). Using the cuprizone (CPZ) model of demyelination and mice of either sex, we establish that Cav1.2 deletion in OPCs leads to less efficient remyelination of the adult brain. Specifically, Cav1.2KO OPCs mature slower and produce less myelin than control oligodendrocytes during the recovery period after CPZ intoxication. This reduced remyelination was accompanied by an important decline in the number of myelinating oligodendrocytes and in the rate of OPC proliferation. Furthermore, during the remyelination phase of the CPZ model, the corpus callosum of Cav1.2KO animals presented a significant decrease in the percentage of myelinated axons and a substantial increase in the mean g-ratio of myelinated axons compared with controls. In addition, in a mouse line in which the Cav1.2KO OPCs were identified by a Cre reporter, we establish that Cav1.2KO OPCs display a reduced maturational rate through the entire remyelination process. These results suggest that Ca2+ influx mediated by L-VGCCs in oligodendroglial cells is necessary for normal remyelination and is an essential Ca2+ channel for OPC maturation during the remyelination of the adult brain.SIGNIFICANCE STATEMENT Ion channels implicated in oligodendrocyte differentiation and maturation may induce positive signals for myelin recovery. Voltage-gated Ca2+ channels (VGCCs) are important for normal myelination by acting at several critical steps during oligodendrocyte progenitor cell (OPC) development. To determine whether voltage Ca2+ entry is involved in oligodendrocyte differentiation and remyelination, we used a conditional knockout mouse for VGCCs in OPCs. Our results indicate that VGCCs can modulate oligodendrocyte maturation in the demyelinated brain and suggest that voltage-gated Ca2+ influx in OPCs is critical for remyelination. These findings could lead to novel approaches for obtaining a better understanding of the factors that control OPC maturation in order to stimulate this pool of progenitors to replace myelin in demyelinating diseases.

Conditional Deletion of the L-Type Calcium Channel Cav1.2 in Oligodendrocyte Progenitor Cells Affects Postnatal Myelination in Mice.

To determine whether L-type voltage-operated Ca2+ channels (L-VOCCs) are required for oligodendrocyte progenitor cell (OPC) development, we generated an inducible conditional knock-out mouse in which the L-VOCC isoform Cav1.2 was postnatally deleted in NG2-positive OPCs. A significant hypomyelination was found in the brains of the Cav1.2 conditional knock-out (Cav1.2KO) mice specifically when the Cav1.2 deletion was induced in OPCs during the first 2 postnatal weeks. A decrease in myelin proteins expression was visible in several brain structures, including the corpus callosum, cortex, and striatum, and the corpus callosum of Cav1.2KO animals showed an important decrease in the percentage of myelinated axons and a substantial increase in the mean g-ratio of myelinated axons. The reduced myelination was accompanied by an important decline in the number of myelinating oligodendrocytes and in the rate of OPC proliferation. Furthermore, using a triple transgenic mouse in which all of the Cav1.2KO OPCs were tracked by a Cre reporter, we found that Cav1.2KO OPCs produce less mature oligodendrocytes than control cells. Finally, live-cell imaging in early postnatal brain slices revealed that the migration and proliferation of subventricular zone OPCs is decreased in the Cav1.2KO mice. These results indicate that the L-VOCC isoform Cav1.2 modulates oligodendrocyte development and suggest that Ca2+ influx mediated by L-VOCCs in OPCs is necessary for normal myelination. SIGNIFICANCE STATEMENT: Overall, it is clear that cells in the oligodendrocyte lineage exhibit remarkable plasticity with regard to the expression of Ca2+ channels and that perturbation of Ca2+ homeostasis likely plays an important role in the pathogenesis underlying demyelinating diseases. To determine whether voltage-gated Ca2+ entry is involved in oligodendrocyte maturation and myelination, we used a conditional knock-out mouse for voltage-operated Ca2+ channels in oligodendrocyte progenitor cells. Our results indicate that voltage-operated Ca2+ channels can modulate oligodendrocyte development in the postnatal brain and suggest that voltage-gated Ca2+ influx in oligodendroglial cells is critical for normal myelination. These findings could lead to novel approaches to intervene in neurodegenerative diseases in which myelin is lost or damaged.

Airway hyperresponsiveness induced by repeated esophageal infusion of HCl in guinea pigs.

Gastroesophageal reflux is a common disorder closely related to chronic airway diseases, such as chronic cough, asthma, chronic bronchitis, and chronic obstructive disease. Indeed, gastroesophageal acid reflux into the respiratory tract causes bronchoconstriction, but the underlying mechanisms have still not been clarified. This study aimed to elucidate functional changes of bronchial smooth muscles (BSMs) isolated from guinea pigs in an animal model of gastroesophageal reflux. The marked airway inflammation, hyperresponsiveness and remodeling were observed after guinea pigs were exposed to intraesophageal HCl infusion for 14 days. In addition, contractile responses to acetylcholine (ACh), KCl, electrical field stimulation, and extracellular Ca(2+) were greater in guinea pigs infused with HCl compared with control groups. The L-type voltage-dependent Ca(2+) channels (L-VDCC) blocker, nicardipine, significantly inhibited ACh- and Ca(2+)-enhanced BSM contractions in guinea pigs infused with HCl. The Rho-kinase inhibitor, Y27632, attenuated ACh-enhanced BSM contractions in guinea pigs infused with HCl. Moreover, mRNA and protein expressions for muscarinic M2 and M3 receptors, RhoA, and L-VDCC in BSM were detected by real-time PCR and Western blot. Expressions of mRNA and protein for muscarinic M3 receptors, RhoA, and L-VDCC were greater than in BSM of HCl-infused guinea pigs, whereas levels of muscarinic M2 receptors were unchanged. We demonstrate that acid infusion to the lower esophagus and, subsequently, microaspiration into the respiratory tract in guinea pigs leads to airway hyperresponsiveness and overactive BSM. Functional and molecular results indicate that overactive BSM is the reason for enhancement of extracellular Ca(2+) influx via L-VDCC and Ca(2+) sensitization through Rho-kinase signaling.

Role of STIM1 in neurodegeneration.

STIM1 is an endoplasmic reticulum (ER) protein with a key role in Ca2+ mobilization. Due to its ability to act as an ER-intraluminal Ca2+ sensor, it regulates store-operated Ca2+ entry (SOCE), which is a Ca2+ influx pathway involved in a wide variety of signalling pathways in eukaryotic cells. Despite its important role in Ca2+ transport, current knowledge about the role of STIM1 in neurons is much more limited. Growing evidence supports a role for STIM1 and SOCE in the preservation of dendritic spines required for long-term potentiation and the formation of memory. In this regard, recent studies have demonstrated that the loss of STIM1, which impairs Ca2+ mobilization in neurons, risks cell viability and could be the cause of neurodegenerative diseases. The role of STIM1 in neurodegeneration and the molecular basis of cell death triggered by low levels of STIM1 are discussed in this review.

Participation of the TRP channel in the cardiovascular effects induced by carvacrol in normotensive rat.

Carvacrol has been described as an agonist/antagonist of different transient receptor potential (TRP) channels and voltage-dependent calcium channels (Cavs). The aim of this study was to evaluate the role of Cav and TRP channels following carvacrol stimulation. Initially, in mesenteric artery rings carvacrol relaxed phenylephrine-induced contractions. Furthermore, carvacrol inhibited contraction elicited by CaCl2 in depolarizing nominally without Ca2+ medium and antagonized the contractions induced by S(-)-Bay K 8644 and inhibited Ca2+ currents indicating the inhibition of Ca2+ influx through L-type Cav. Additionally, carvacrol antagonized the contractions induced by CaCl2 in the presence of nifedipine/Cyclopiazonic acid/phenylephrine or nifedipine/Cyclopiazonic acid/KCl 60, suggesting a possible inhibition of calcium influx by store operated channels (SOCs), receptor operated channels (ROCs) and/or TRP channels. Interestingly, among the TRP channel blockers used, the effect induced by carvacrol was attenuated by Mg2+ and potentiated by La3+ and Gd3+, suggesting that TRP channels are involved in relaxation induced by carvacrol. Monoterpene also induced hypotension and bradycardia in non-anesthetized normotensive rats and negative inotropic and chronotropic effects. In conclusion, these results suggest that the hypotensive effect of carvacrol is probably due to bradycardia and a peripheral vasodilatation that involves, at least, the inhibition of the Ca2+ influx through Cav and TRP channels.

Phytochemical study guided by the myorelaxant activity of the crude extract, fractions and constituent from stem bark of Hymenaea courbaril L.

ETHNOPHARMACOLOGICAL RELEVANCE: Hymenaea courbaril L. (Caesalpinoideae) is used in Brazilian folk medicine to treat anemia, kidney problems, sore throat and other dysfunctions of the respiratory system, such as bronchitis and asthma, although such properties are yet to be scientifically validated. AIM OF THE STUDY: In order to give a scientific basis to support the traditional use of Hymenaea courbaril, this study was designed to evaluate antioxidant, myorelaxant and anti-inflammatory properties of the ethanol extract from stem bark and its fractions. The myorelaxant effect of astilbin, a flavonoid isolated from the bioactive ethyl acetate fraction (EAF), has also been evaluated. MATERIAL AND METHODS: In the present study ethanol extract from stem bark (EEHC) and fractions were analyzed using bioassay-guided fractionation. The following activities were investigated: antioxidant by 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, myorelaxant on rat tracheal smooth muscle, and anti-inflammatory using ovalbumin-induced leukocytosis and airway hyperresponsiveness in rats. RESULTS: The results of the present investigation show that the whole extract of Hymenaea courbaril and some of its fractions strongly scavenged DPPH radical. The extract showed myorelaxant activity on rat trachea, being EAF its highest efficient fraction. Bio-guided study allowed the isolation of astilbin, a well-known flavonoid. The activity induced by this compound indicates that it may be partly responsible for the myorelaxant effect of EAF. EAF reduced contractions that depended on divalent cation inflow through voltage-operated Ca(2+) channels (VOCCs) or receptor-operated Ca(2+) channels (ROCCs), but it was more potent to inhibit VOCC- than ROCC-dependent contraction induced by Ca(2+) addition in ACh-enriched Ca(2+)-free medium. Oral pretreatment of antigen-challenged animals with EAF prevented airway hyperresponsiveness on KCl-induced contraction and reduced the number of total white cells, particularly eosinophils and neutrophils in bronchoalveolar lavage. CONCLUSIONS: This study provided scientific basis that Hymenaea courbaril presents potential antioxidant, myorelaxant and anti-inflammatory actions, which support its use in folk medicine to treat inflammatory airway diseases.