The neuronal and synaptic dynamics underlying post-inhibitory rebound burst related to major depressive disorder in the lateral habenula neuron model.

A burst behavior observed in the lateral habenula (LHb) neuron related to major depressive disorder has attracted much attention. The burst is induced from silence by the excitatory N-methyl-D-aspartate (NMDA) synapse or by the inhibitory stimulation, i.e., a post-inhibitory rebound (PIR) burst, which has not been explained clearly. In the present paper, the neuronal and synaptic dynamics for the PIR burst are acquired in a theoretical neuron model. At first, dynamic cooperations between the fast rise of inhibitory γ-aminobutyric acid (GABA) synapse, slow rise of NMDA synapse, and T-type calcium current to evoke the PIR burst are obtained. Similar to the inhibitory pulse stimulation, fast rising GABA current can reduce the membrane potential to a level low enough to de-inactivate the low threshold T-type calcium current to evoke a PIR spike, which can enhance the slow rising NMDA current activated at a time before or after the PIR spike. The NMDA current following the PIR spike exhibits slow decay to induce multiple spikes to form the PIR burst. Such results present a theoretical explanation and a candidate for the PIR burst in real LHb neurons. Then, the dynamical mechanism for the PIR spike mediated by the T-type calcium channel is obtained. At large conductance of T-type calcium channel, the resting state corresponds to a stable focus near Hopf bifurcation and exhibits an "uncommon" threshold curve with membrane potential much lower than the resting membrane potential. Inhibitory modulation induces membrane potential decreased to run across the threshold curve to evoke the PIR spike. At small conductance of the T-type calcium channel, a stable node appears and manifests a common threshold curve with higher membrane potential, resulting in non-PIR phenomenon. The results present the dynamic cooperations between neuronal dynamics and fast/slow dynamics of different synapses for the PIR burst observed in the LHb neuron, which is helpful for the modulations to major depressive disorder.

Impact of quetiapine on ion channels and contractile dynamics in rat ventricular myocyte.

Antipsychotic drugs often lead to adverse effects, including those related to the cardiovascular system. Of these, quetiapine is known to cause significant changes in the QT interval although the underlying mechanism remains mysterious, prompting us to examine its effects on cardiac electrophysiological properties. Therefore, we investigated the effect of quetiapine on contraction, action potential (AP), and the associated membrane currents such as L-type Ca2+ and K+ using the whole-cell patch clamp method to examine its impacts on isolated rat ventricular myocytes. Our results showed that (1) quetiapine reduces cell contractility in a concentration-dependent manner and (2) leads to a significant prolongation in the duration of AP in isolated ventricular myocytes. This effect was both concentration and frequency-dependent; (3) quetiapine significantly decreased the Ca2+, transient outward K+, and steady-state K+ currents. However, only high concentration of quetiapine (100 µM) could significantly change the activation and reactivation kinetics of L-type Ca2+ channels. This study demonstrates that QT extension induced by quetiapine is mainly associated with the prolongation of AP. Moreover, quetiapine caused a significant decrease in contractile force and excitability of ventricular myocytes by suppressing Ca2+ and K+ currents.

Role of the CaV1.2 distal carboxy terminus in the regulation of L-type current.

L-type calcium channels are essential for the excitation-contraction coupling in cardiac muscle. The CaV1.2 channel is the most predominant isoform in the ventricle which consists of a multi-subunit membrane complex that includes the CaV1.2 pore-forming subunit and auxiliary subunits like CaVα2δ and CaVß2b. The CaV1.2 channel's C-terminus undergoes proteolytic cleavage, and the distal C-terminal domain (DCtermD) associates with the channel core through two domains known as proximal and distal C-terminal regulatory domain (PCRD and DCRD, respectively). The interaction between the DCtermD and the remaining C-terminus reduces the channel activity and modifies voltage- and calcium-dependent inactivation mechanisms, leading to an autoinhibitory effect. In this study, we investigate how the interaction between DCRD and PCRD affects the inactivation processes and CaV1.2 activity. We expressed a 14-amino acid peptide miming the DCRD-PCRD interaction sequence in both heterologous systems and cardiomyocytes. Our results show that overexpression of this small peptide can displace the DCtermD and replicate the effects of the entire DCtermD on voltage-dependent inactivation and channel inhibition. However, the effect on calcium-dependent inactivation requires the full DCtermD and is prevented by overexpression of calmodulin. In conclusion, our results suggest that the interaction between DCRD and PCRD is sufficient to bring about the current inhibition and alter the voltage-dependent inactivation, possibly in an allosteric manner. Additionally, our data suggest that the DCtermD competitively modifies the calcium-dependent mechanism. The identified peptide sequence provides a valuable tool for further dissecting the molecular mechanisms that regulate L-type calcium channels' basal activity in cardiomyocytes.

Bupleurum exerts antiarrhythmic effects by inhibiting L-type calcium channels in mouse ventricular myocytes.

BACKGROUND: Bupleurum (Bup), is a traditional effective medicine to treat colds and fevers in clinics. Multiple studies have demonstrated that Bup exhibites various biological activities, including cardioprotective effects, anti-inflammatory, anticancer, antipyretic, antimicrobial, and antiviral effects, etc. Currently, the effects of Bup on cardiac electrophysiology have not been reported yet. METHODS: Electrocardiogram recordings were used to investigate the effects of Bup on aconitine-induced arrhythmias. Patch-clamp techniques were used to explore the effects of Bup on APs and ion currents. RESULTS: Bup reduced the incidence of ventricular fibrillation (VF) and delayed the onset time of ventricular tachycardia (VT) in mice. Additionally, Bup (40 mg/mL) suppressed DADs induced by high-Ca2+ and shortened action potential duration at 50 % completion of repolarization (APD50) and action potential duration at 90 % completion of repolarization (APD90) to 60.89 % ± 8.40 % and 68.94 % ± 3.24 % of the control, respectively. Moreover, Bup inhibited L-type calcium currents (ICa.L) in a dose-dependent manner, with an IC50 value of 25.36 mg/mL. Furthermore, Bup affected the gated kinetics of L-type calcium channels by slowing down steady-state activation, accelerating the steady-state inactivation, and delaying the inactivation-recovery process. However, Bup had no effects on the Transient sodium current (INa.T), ATX II-increased late sodium current (INa.L), transient outward current (Ito), delayed rectifier potassium current (IK), or inward rectifier potassium current (IK1). CONCLUSION: Bup is an antiarrhythmic agent that may exert its antiarrhythmic effects by inhibiting L-type calcium channels.

Diabetes-Induced Amplification of Nociceptive DRG Neuron Output by Upregulation of Somatic T-Type Ca2+ Channels.

The development of pain symptoms in peripheral diabetic neuropathy (PDN) is associated with the upregulation of T-type Ca2+ channels (T-channels) in the soma of nociceptive DRG neurons. Moreover, a block of these channels in DRG neurons effectively reversed mechanical and thermal hyperalgesia in animal diabetic models, indicating that T-channel functioning in these neurons is causally linked to PDN. However, no particular mechanisms relating the upregulation of T-channels in the soma of nociceptive DRG neurons to the pathological pain processing in PDN have been suggested. Here we have electrophysiologically identified voltage-gated currents expressed in nociceptive DRG neurons and developed a computation model of the neurons, including peripheral and central axons. Simulations showed substantially stronger sensitivity of neuronal excitability to diabetes-induced T-channel upregulation at the normal body temperature compared to the ambient one. We also found that upregulation of somatic T-channels, observed in these neurons under diabetic conditions, amplifies a single action potential invading the soma from the periphery into a burst of multiple action potentials further propagated to the end of the central axon. We have concluded that the somatic T-channel-dependent amplification of the peripheral nociceptive input to the spinal cord demonstrated in this work may underlie abnormal nociception at different stages of diabetes development.

Patient-specific iPSC-derived cardiomyocytes reveal variable phenotypic severity of Brugada syndrome.

BACKGROUND: Brugada syndrome (BrS) is a cardiac channelopathy that can result in sudden cardiac death (SCD). SCN5A is the most frequent gene linked to BrS, but the genotype-phenotype correlations are not completely matched. Clinical phenotypes of a particular SCN5A variant may range from asymptomatic to SCD. Here, we used comparison of induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) derived from a SCN5A mutation-positive (D356Y) BrS family with severely affected proband, asymptomatic mutation carriers (AMCs) and healthy controls to investigate this variation. METHODS: 26 iPSC lines were generated from skin fibroblasts using nonintegrated Sendai virus. The generated iPSCs were differentiated into cardiomyocytes using a monolayer-based differentiation protocol. FINDINGS: D356Y iPSC-CMs exhibited increased beat interval variability, slower depolarization, cardiac arrhythmias, defects of Na+ channel function and irregular Ca2+ signaling, when compared to controls. Importantly, the phenotype severity observed in AMC iPSC-CMs was milder than that of proband iPSC-CMs, an observation exacerbated by flecainide. Interestingly, the iPSC-CMs of the proband exhibited markedly decreased Ca2+ currents in comparison with control and AMC iPSC-CMs. CRISPR/Cas9-mediated genome editing to correct D356Y in proband iPSC-CMs effectively rescued the arrhythmic phenotype and restored Na+ and Ca2+ currents. Moreover, drug screening using established BrS iPSC-CM models demonstrated that quinidine and sotalol possessed antiarrhythmic effects in an individual-dependent manner. Clinically, venous and oral administration of calcium partially reduced the malignant arrhythmic events of the proband in mid-term follow-up. INTERPRETATION: Patient-specific and genome-edited iPSC-CMs can recapitulate the varying phenotypic severity of BrS. Our findings suggest that preservation of the Ca2+ currents might be a compensatory mechanism to resist arrhythmogenesis in BrS AMCs. FUNDING: National Key R&D Program of China (2017YFA0103700), National Natural Science Foundation of China (81922006, 81870175), Natural Science Foundation of Zhejiang Province (LD21H020001, LR15H020001), National Natural Science Foundation of China (81970269), Key Research and Development Program of Zhejiang Province (2019C03022) and Natural Science Foundation of Zhejiang Province (LY16H020002).

Disruption of mitochondrial and lysosomal functions by human CACNA1C variants expressed in HEK 293 and CHO cells.

Objective: To investigate the pathogenesis of three novel de novo CACNA1C variants (p.E411D, p.V622G, and p.A272V) in causing neurodevelopmental disorders and arrhythmia. Methods: Several molecular experiments were carried out on transfected human embryonic kidney 293 (HEK 293) and Chinese hamster ovary (CHO) cells to explore the effects of p.E411D, p.V622G, and p.A272V variants on electrophysiology, mitochondrial and lysosomal functions. Electrophysiological studies, RT-qPCR, western blot, apoptosis assay, mito-tracker fluorescence intensity, lyso-tracker fluorescence intensity, mitochondrial calcium concentration test, and cell viability assay were performed. Besides, reactive oxygen species (ROS) levels, ATP levels, mitochondrial copy numbers, mitochondrial complex I, II, and cytochrome c functions were measured. Results: The p.E411D variant was found in a patient with attention deficit-hyperactive disorder (ADHD), and moderate intellectual disability (ID). This mutant demonstrated reduced calcium current density, mRNA, and protein expression, and it was localized in the nucleus, cytoplasm, lysosome, and mitochondria. It exhibited an accelerated apoptosis rate, impaired autophagy, and mitophagy. It also demonstrated compromised mitochondrial cytochrome c oxidase, complex I, and II enzymes, abnormal mitochondrial copy numbers, low ATP levels, abnormal mitochondria fluorescence intensity, impaired mitochondrial fusion and fission, and elevated mitochondrial calcium ions. The p.V622G variant was identified in a patient who presented with West syndrome and moderate global developmental delay. The p.A272V variant was found in a patient who presented with epilepsy and mild ID. Both mutants (p.V622G and p.A272V) exhibited reduced calcium current densities, decreased mRNA and protein expressions, and they were localized in the nucleus, cytoplasm, lysosome, and mitochondria. They exhibited accelerated apoptosis and proliferation rates, impaired autophagy, and mitophagy. They also exhibited abnormal mitochondrial cytochrome c oxidase, complex I and II enzymes, abnormal mitochondrial copy numbers, low ATP, high ROS levels, abnormal mitochondria fluorescence intensity, impaired mitochondrial fusion and fission, as well as elevated mitochondrial calcium ions. Conclusion: The p.E411D, p.V622G and p.A272V mutations of human CACNA1C reduce the expression level of CACNA1C proteins, and impair mitochondrial and lysosomal functions. These effects induced by CACNA1C variants may contribute to the pathogenesis of CACNA1C-related disorders.

Lack of the transcription factor Nfix causes tachycardia in mice sinus node and rats neonatal cardiomyocytes.

AIMS: Nfix is a transcription factor belonging to the Nuclear Factor I (NFI) family comprising four members (Nfia, b, c, x). Nfix plays important roles in the development and function of several organs. In muscle development, Nfix controls the switch from embryonic to fetal myogenesis by promoting fast twitching fibres. In the adult muscle, following injury, lack of Nfix impairs regeneration, inducing higher content of slow-twitching fibres. Nfix is expressed also in the heart, but its function has been never investigated before. We studied Nfix role in this organ. METHODS: Using Nfix-null and wild type (WT) mice we analyzed: (1) the expression pattern of Nfix during development by qPCR and (2) the functional alterations caused by its absence, by in vivo telemetry and in vitro patch clamp analysis. RESULTS AND CONCLUSIONS: Nfix expression start in the heart from E12.5. Adult hearts of Nfix-null mice show a hearts morphology and sarcomeric proteins expression similar to WT. However, Nfix-null animals show tachycardia that derives form an intrinsic higher beating rate of the sinus node (SAN). Molecular and functional analysis revealed that sinoatrial cells of Nfix-null mice express a significantly larger L-type calcium current (Cacna1d + Cacna1c). Interestingly, downregulation of Nfix by sh-RNA in primary cultures of neonatal rat ventricular cardiomyocytes induced a similar increase in their spontaneous beating rate and in ICaL current. In conclusion, our data provide the first demonstration of a role of Nfix that, increasing the L-type calcium current, modulates heart rate.

Expression and Impact of Adenosine A3 Receptors on Calcium Homeostasis in Human Right Atrium.

Increased adenosine A2A receptor (A2AR) expression and activation underlies a higher incidence of spontaneous calcium release in atrial fibrillation (AF). Adenosine A3 receptors (A3R) could counteract excessive A2AR activation, but their functional role in the atrium remains elusive, and we therefore aimed to address the impact of A3Rs on intracellular calcium homeostasis. For this purpose, we analyzed right atrial samples or myocytes from 53 patients without AF, using quantitative PCR, patch-clamp technique, immunofluorescent labeling or confocal calcium imaging. A3R mRNA accounted for 9% and A2AR mRNA for 32%. At baseline, A3R inhibition increased the transient inward current (ITI) frequency from 0.28 to 0.81 events/min (p < 0.05). Simultaneous stimulation of A2ARs and A3Rs increased the calcium spark frequency seven-fold (p < 0.001) and the ITI frequency from 0.14 to 0.64 events/min (p < 0.05). Subsequent A3R inhibition caused a strong additional increase in the ITI frequency (to 2.04 events/min; p < 0.01) and increased phosphorylation at s2808 1.7-fold (p < 0.001). These pharmacological treatments had no significant effects on L-type calcium current density or sarcoplasmic reticulum calcium load. In conclusion, A3Rs are expressed and blunt spontaneous calcium release at baseline and upon A2AR-stimulation in human atrial myocytes, pointing to A3R activation as a means to attenuate physiological and pathological elevations of spontaneous calcium release events.

Spatial Distribution of Calcium Sparks Determines Their Ability to Induce Afterdepolarizations in Human Atrial Myocytes.

Analysis of the spatio-temporal distribution of calcium sparks showed a preferential increase in sparks near the sarcolemma in atrial myocytes from patients with atrial fibrillation (AF), linked to higher ryanodine receptor (RyR2) phosphorylation at s2808 and lower calsequestrin-2 levels. Mathematical modeling, incorporating modulation of RyR2 gating, showed that only the observed combinations of RyR2 phosphorylation and calsequestrin-2 levels can account for the spatio-temporal distribution of sparks in patients with and without AF. Furthermore, we demonstrate that preferential calcium release near the sarcolemma is key to a higher incidence and amplitude of afterdepolarizations in atrial myocytes from patients with AF.

Models of the cardiac L-type calcium current: A quantitative review.

The L-type calcium current ( I CaL ) plays a critical role in cardiac electrophysiology, and models of I CaL are vital tools to predict arrhythmogenicity of drugs and mutations. Five decades of measuring and modeling I CaL have resulted in several competing theories (encoded in mathematical equations). However, the introduction of new models has not typically been accompanied by a data-driven critical comparison with previous work, so that it is unclear which model is best suited for any particular application. In this review, we describe and compare 73 published mammalian I CaL models and use simulated experiments to show that there is a large variability in their predictions, which is not substantially diminished when grouping by species or other categories. We provide model code for 60 models, list major data sources, and discuss experimental and modeling work that will be required to reduce this huge list of competing theories and ultimately develop a community consensus model of I CaL . This article is categorized under: Cardiovascular Diseases > Computational Models Cardiovascular Diseases > Molecular and Cellular Physiology.

[Changes of myocardial calcium currents in rats with myocardial injury induced by running exercise during acute hypoxia].

OBJECTIVE: To investigate the changes in myocardial calcium currents in rats subjected to forced running exercise during acute hypoxia and their association with myocardial injury. METHODS: Forty SD rats were randomized into quiescent group and running group either in normal oxygen (NQ and NR groups, respectively) or in acute hypoxia (HQ and HR groups, respectively). Hypoxia was induced by keeping the rats in a hypobaric oxygen chamber (PaO2=61.6kpa) for 4 h a day; the rats in the two running groups were forced to run on running wheels for 4 h each day. Rat ventricular myocytes was isolated by enzymatic digestion for recording action potentials and currents using patch clamp technique, and confocal Ca2+ imaging was used to monitor intracellular Ca2+ levels. The expressions of Cav1.2 channel and the cardiac ryanodine receptor (RyR2) were determined using Western blotting. RESULTS: Compared with those in NQ group, the rats in HR group showed significantly decreased SOD activity (P < 0.01), increased h-FABP, hs-CRP and IMA levels (P < 0.05 or 0.01), obvious myocardial pathology, and prolonged APD50 and APD90 (P < 0.05). Of the different stress conditions, forced running in acute hypoxia resulted in the most prominent increase of the densities of ICa, L currents, causing also a significant left shift of the steady state activation curve and a significant right shift of the steady state inactivation curve. Compared with those in NQ group, the rats in NR, HQ and HR groups all exhibited higher rates of spontaneous calcium wave events in the cardiac myocytes, increased frequency of calcium sparks with lowered amplitude, enhanced calcium release amplitude in the ventricular myocytes, and delayed calcium ion reabsorption; in particular, these changes were the most conspicuous in HR group (P < 0.05 or 0.01). There was also a significant increase in the protein levels of Cav1.2 channel and RyR2 receptor in HR group (P < 0.05 or 0.01). CONCLUSIONS: The mechanism of myocardial injury in rats subjected to forced running in acute hypoxia may involve the increase of oxidative stress and calcium current and intracellular calcium overload.

Modeling analysis of subthreshold voltage signaling along hippocampal mossy fiber axons.

Axons are classically thought of as electrically well isolated from other parts of the neurons due to the shape of a long cable-like structure. In contrast to this classical view on axonal compartmentalization, recent studies revealed that subthreshold depolarization of soma and dendrite passively propagates to the axons for a substantial distance, as demonstrated in some experimentally accessible axons including hippocampal mossy fibers and cortical pyramidal cell axons. Passive propagation of subthreshold dendritic EPSPs to the axons, defined as EPreSPs (excitatory presynaptic potentials), has been demonstrated to affect transmitter release from the axon terminals. To further characterize and explore the functional significance of passive subthreshold voltage signaling along the axons, the model of EPreSPs along hippocampal mossy fibers, proposed by Alle and Geiger, was reconstructed on the NEURON simulator. To test the effect of EPreSPs on action potentials and transmitter release from the axon terminals, additional conductances were incorporated into the previous passive propagation model. These include the axonal sodium, potassium, and leak channels as well as presynaptic calcium channels composed of P/Q-, N-, and R-types, which are reconstructed from the properties of those recorded from mossy fiber boutons experimentally. In this revised model, the preceding subthreshold EPreSPs slightly reduced the action potential-evoked presynaptic calcium currents by a decrease in the amplitude of action potentials due to the slow depolarization. It should be mentioned that EPreSPs by themselves elicited small calcium currents during subthreshold depolarization through these high-voltage activated calcium channels. Since the previous experimental study by simultaneous pre and postsynaptic recordings demonstrated that EPreSPs enhanced action potential-evoked transmitter release from the mossy fiber terminals, it has been suggested that different mechanisms from the enhancement of action potential-evoked presynaptic calcium entry may involve enhanced transmitter release by EPreSP. Small calcium entry by subthreshold EPreSPs may enhance transmitter release from the mossy fiber terminals by acting as high-affinity calcium sensors for enhancing transmitter release. Another form of axonal subthreshold voltage signaling, GABA-EPreSPs elicited by a spillover of GABA from surrounding interneurons, was also explored. Functional consequences of the two modes of axonal subthreshold voltage signaling were discussed with the simulation results.

Adrenoceptor sub-type involvement in Ca2+ current stimulation by noradrenaline in human and rabbit atrial myocytes.

Atrial fibrillation (AF) from elevated adrenergic activity may involve increased atrial L-type Ca2+ current (ICaL) by noradrenaline (NA). However, the contribution of the adrenoceptor (AR) sub-types to such ICaL-increase is poorly understood, particularly in human. We therefore investigated effects of various broad-action and sub-type-specific α- and ß-AR antagonists on NA-stimulated atrial ICaL. ICaL was recorded by whole-cell-patch clamp at 37 °C in myocytes isolated enzymatically from atrial tissues from consenting patients undergoing elective cardiac surgery and from rabbits. NA markedly increased human atrial ICaL, maximally by ~ 2.5-fold, with EC75 310 nM. Propranolol (ß1 + ß2-AR antagonist, 0.2 microM) substantially decreased NA (310 nM)-stimulated ICaL, in human and rabbit. Phentolamine (α1 + α2-AR antagonist, 1 microM) also decreased NA-stimulated ICaL. CGP20712A (ß1-AR antagonist, 0.3 microM) and prazosin (α1-AR antagonist, 0.5 microM) each decreased NA-stimulated ICaL in both species. ICI118551 (ß2-AR antagonist, 0.1 microM), in the presence of NA + CGP20712A, had no significant effect on ICaL in human atrial myocytes, but increased it in rabbit. Yohimbine (α2-AR antagonist, 10 microM), with NA + prazosin, had no significant effect on human or rabbit ICaL. Stimulation of atrial ICaL by NA is mediated, based on AR sub-type antagonist responses, mainly by activating ß1- and α1-ARs in both human and rabbit, with a ß2-inhibitory contribution evident in rabbit, and negligible α2 involvement in either species. This improved understanding of AR sub-type contributions to noradrenergic activation of atrial ICaL could help inform future potential optimisation of pharmacological AR-antagonism strategies for inhibiting adrenergic AF.

Synaptotagmin-7 Enhances Facilitation of Cav2.1 Calcium Channels.

Voltage-gated calcium channel Cav2.1 undergoes Ca2+-dependent facilitation and inactivation, which are important in short-term synaptic plasticity. In presynaptic terminals, Cav2.1 forms large protein complexes that include synaptotagmins. Synaptotagmin-7 (Syt-7) is essential to mediate short-term synaptic plasticity in many synapses. Here, based on evidence that Cav2.1 and Syt-7 are both required for short-term synaptic facilitation, we investigated the direct interaction of Syt-7 with Cav2.1 and probed its regulation of Cav2.1 function. We found that Syt-7 binds specifically to the α1A subunit of Cav2.1 through interaction with the synaptic-protein interaction (synprint) site. Surprisingly, this interaction enhances facilitation in paired-pulse protocols and accelerates the onset of facilitation. Syt-7α induces a depolarizing shift in the voltage dependence of activation of Cav2.1 and slows Ca2+-dependent inactivation, whereas Syt-7ß and Syt-7γ have smaller effects. Our results identify an unexpected, isoform-specific interaction between Cav2.1 and Syt-7 through the synprint site, which enhances Cav2.1 facilitation and modulates its inactivation.

[Discovery of exogenous ligands for orphan receptor BRS-3 from Chinese herbs].

Bombesin receptor subtype-3(BRS-3) is an orphan receptor in the bombesin receptor family. Its signal transduction mechanism and biological function have attracted much attention. Seeking the ligand for BRS-3 is of great significance for exploring its function. Considering the fact that the activation of BRS-3 receptor can induce the change in intracellular Ca~(2+) concentration, the fluo-rometric imaging plate reader(FLIPR) was utilized for ligand screening at the cellular level. Among more than 400 monomeric compounds isolated from Chinese herbs, yuanhunine from Corydalis Rhizoma and sophoraisoflavanone A and licoriphenone from Glycyrrhizae Radix et Rhizoma antagonized BRS-3 to varying degrees. It was confirmed in HEK293 cells expressing BRS-3 that yuanhunine, sophoraisoflavanone A, and licoriphenone inhibited the calcium current response after the activation of BRS-3 by [D-Phe~6,ß-Ala~(11),Phe~(13),Nle~(14)]bombesin-(6-14) in a dose-dependent manner with the IC_(50) values being 8.58, 4.10, and 2.04 µmol·L~(-1), respectively. Further study indicated that yuanhunine and sophoraisoflavanone A exhibited good selectivity for BRS-3. In this study, it was found for the first time that monomers derived from Chinese herbs had antagonistic activity against orphan receptor BRS-3, which has provided a tool for further study of BRS-3 and also the potential lead compounds for new drug discovery. At the same time, it provides reference for the research and development of innovative drugs based on the active ingredients of Chinese herbs.

Impact of IFN-γ Deficiency on the Cardiomyocyte Function in the First Stage of Experimental Chagas Disease.

Chagas disease (CD) is caused by the parasitic protozoan T. cruzi. The progression of CD in ~30% of patients results in Chagasic Cardiomyopathy (CCM). Currently, it is known that the inflammatory system plays a significant role in the CCM. Interferon-gamma (IFN-γ) is the major cytokine involved in parasitemia control but has also been linked to CCM. The L-type calcium current (ICa,L) is crucial in the excitation/contraction coupling in cardiomyocytes. Thus, we compared ICa,L and the mechanical properties of cardiomyocytes isolated from infected wild type (WT) and IFN-γ(-/-) mice in the first stage of T. cruzi infection. Using the patch clamp technique, we demonstrated that the infection attenuated ICa,L in isolated cardiomyocytes from the right and left ventricles of WT mice at 15 days post-infection (dpi), which was not observed in the IFN-γ(-/-) cardiomyocytes. However, ICa,L was attenuated between 26 and 30 dpi in both experimental groups. Interestingly, the same profile was observed in the context of the mechanical properties of isolated cardiomyocytes from both experimental groups. Simultaneously, we tracked the mortality and MCP-1, TNF-α, IL-12, IL-6, and IL-10 serum levels in the infected groups. Importantly, the IFN-γ(-/-) and WT mice presented similar parasitemia and serum inflammatory markers at 10 dpi, indicating that the modifications in the cardiomyocyte functions observed at 15 dpi were directly associated with IFN-γ(-/-) deficiency. Thus, we showed that IFN-γ plays a crucial role in the electromechanical remodeling of cardiomyocytes during experimental T. cruzi infection in mice.

The role of M3 receptors in regulation of electrical activity deteriorates in the rat heart during ageing.

Ageing is a complex process which affects all systems of the organism and therefore changes the environment where the heart is working. In this study we demonstrate the ageing-related changes in the mechanisms of parasympathetic regulation of mammalian heart. Electrophysiological effects produced by selective activation of M3-cholinoreceptors were compared in isolated cardiac preparations from young adult (4 months), adult (1 year) and ageing (2 years) rats using sharp glass microelectrode technique. M3-receptors were activated with muscarinic agonist pilocarpine (10-5M) in the presence of selective M2 antagonist AQ-RA741 (10-7M). In atrial and ventricular myocardium from young rats M3 stimulation induced shortening of action potentials(APs), while no significant effect was observed in both elder groups. The main mechanism of M3-induced AP shortening is inhibition of L-type Ca2+ current, estimated using whole-cell patch-clamp. It was negligible in atrial myocytes from ageing animals in comparison with young rats. The loss of sensitivity to stimulation of M3-receptors is due to decrease in M3 gene expression, shown by RT-PCR both in atrial and ventricular samples from ageing rats. Thus, in ageing rat heart M3-receptors are down-regulated and not involved in regulation of electrical activity.

Role of primary sensory neurone cannabinoid type-1 receptors in pain and the analgesic effects of the peripherally acting agonist CB-13 in mice.

BACKGROUND: Cannabinoid type-1 receptors (CB1Rs) are expressed in primary sensory neurones, but their role in pain modulation remains unclear. METHODS: We produced Pirt-CB1R conditional knockout (cKO) mice to delete CB1Rs in primary sensory neurones selectively, and used behavioural, pharmacological, and electrophysiological approaches to examine the influence of peripheral CB1R signalling on nociceptive and inflammatory pain. RESULTS: Conditional knockout of Pirt-CB1R did not alter mechanical or heat nociceptive thresholds, complete Freund adjuvant-induced inflammation, or heat hyperalgesia in vivo. The intrinsic membrane properties of small-diameter dorsal root ganglion neurones were also comparable between cKO and wild-type mice. Systemic administration of CB-13, a peripherally restricted CB1/CB2R dual agonist (5 mg kg-1), inhibited nociceptive pain and complete Freund adjuvant-induced inflammatory pain. These effects of CB-13 were diminished in Pirt-CB1R cKO mice. In small-diameter neurones from wild-type mice, CB-13 concentration-dependently inhibited high-voltage activated calcium current (HVA-ICa) and induced a rightward shift of the channel open probability curve. The effects of CB-13 were significantly attenuated by AM6545 (a CB1R antagonist) and Pirt-CB1R cKO. CONCLUSION: CB1R signalling in primary sensory neurones did not inhibit nociceptive or inflammatory pain, or the intrinsic excitability of nociceptive neurones. However, peripheral CB1Rs are important for the analgesic effects of systemically administered CB-13. In addition, HVA-ICa inhibition appears to be a key ionic mechanism for CB-13-induced pain inhibition. Thus, peripherally restricted CB1R agonists could have utility for pain treatment.