Transient Hyperpolarization Research Articles

Interstitial cells of the sip syncytium regulate basal membrane potential in murine gastric corpus.

Smooth muscle cells (SMCs), Interstitial cells of Cajal (ICC) and Platelet-derived growth factor receptor α positive (PDGFRα+) cells form an integrated, electrical syncytium within the gastrointestinal (GI) muscular tissues known as the SIP syncytium. Immunohistochemical analysis of gastric corpus muscles showed that c-KIT+/ANO1+ ICC-IM and PDGFRα+ cells were closely apposed to one another in the same anatomical niches. We used intracellular microelectrode recording from corpus muscle bundles to characterize the roles of intramuscular ICC and PDGFRα+ cells in conditioning membrane potentials of gastric muscles. In muscle bundles, that have a relatively higher input impedance than larger muscle strips or sheets, we recorded an ongoing discharge of stochastic fluctuations in membrane potential, previously called unitary potentials or spontaneous transient depolarizations (STDs) and spontaneous transient hyperpolarizations (STHs). We reasoned that STDs should be blocked by antagonists of ANO1, the signature conductance of ICC. Activation of ANO1 has been shown to generate spontaneous transient inward currents (STICs), which are the basis for STDs. Ani9 reduced membrane noise and caused hyperpolarization, but this agent did not block the fluctuations in membrane potential quantitatively. Apamin, an antagonist of small conductance Ca2+-activated K+ channels (SK3), the signature conductance in PDGFRα+ cells, further reduced membrane noise and caused depolarization. Reversing the order of channel antagonists reversed the sequence of depolarization and hyperpolarization. These experiments show that the ongoing discharge of STDs and STHs by ICC and PDGFRα+ cells, respectively, exerts conditioning effects on membrane potentials in the SIP syncytium that would effectively regulate the excitability of SMCs.

Dipolar Order Induced Electron Spin Hyperpolarization.

The structure of coupled electron spin systems is of fundamental interest to many applications, including dynamic nuclear polarization (DNP), enhanced nuclear magnetic resonance (NMR), the generation of electron spin qubits for quantum information science (QIS), and quantitative studies of paramagnetic systems by electron paramagnetic resonance (EPR). However, the characterization of electron spin coupling networks is nontrivial, especially at high magnetic fields. This study focuses on a system containing high concentrations of trityl radicals that give rise to a DNP enhancement profile of 1H NMR characteristic of the presence of electron spin clusters. When this system is subject to selective microwave saturation through pump-probe ELectron DOuble Resonance (ELDOR) experiments, electron spin hyperpolarization is observed. We show that the generation of an out-of-equilibrium longitudinal dipolar order is responsible for the transient hyperpolarization of electron spins. Notably, the coupled electron spin system needs to form an AX-like system (where the difference in the Zeeman interactions of two spins is larger than their coupling interaction) such that selective microwave irradiation can generate signatures of electron spin hyperpolarization. We show that the extent of dipolar order, as manifested in the extent of electron spin hyperpolarization generated, can be altered by tuning the pump or probe pulse length, or the interpulse delay in ELDOR experiments that change the efficiency to generate or readout longitudinal dipolar order. Pump-probe ELDOR with selective saturation is an effective means for characterizing coupled electron spins forming AX-type spin systems that are foundational for DNP and quantum sensing.

Demystifying the Venus flytrap action potential.

All plants are electrically excitable, but only few are known to fire a well-defined, all-or-nothing action potential (AP). The Venus flytrap Dionaea muscipula displays APs with an extraordinarily high firing frequency and speed, enabling the capture organ of this carnivorous plant to catch small animals as fast as flies. The number of APs triggered by the prey is counted and serves as the basis for decisions within the flytrap's hunting cycle. The archetypical Dionaea AP lasts 1 s and consists of fivephases: Starting from the resting state, an initial cytosolic Ca2+ transient is followed by depolarization, repolarization and a transient hyperpolarization (overshoot) before the original membrane potential is finally recovered. When the flytrap matures and becomes excitable, a distinct set of ion channels, pumps and carriers is expressed, each mastering a distinct AP phase.

VDAC as a voltage-dependent mitochondrial gatekeeper under physiological conditions

Mitochondria, composed of two membranes, play a key role in energy production in eukaryotic cells. The main function of the inner membrane is oxidative phosphorylation, while the mitochondrial outer membrane (MOM) seems to control the energy flux and exchange of various charged metabolites between mitochondria and the cytosol. Metabolites cross MOM via the various isoforms of voltage-dependent anion channel (VDAC). In turn, VDACs interact with some enzymes, other proteins and molecules, including drugs. This work aimed to analyze various literature experimental data related to targeting mitochondrial VDACs and VDAC-kinase complexes on the basis of the hypothesis of generation of the outer membrane potential (OMP) and OMP-dependent reprogramming of cell energy metabolism. Our previous model of the VDAC-hexokinase-linked generation of OMP was further complemented in this study with an additional regulation of the MOM permeability by the OMP-dependent docking of cytosolic proteins like tubulin to VDACs. Computational analysis of the model suggests that OMP changes might be involved in the mechanisms of apoptosis promotion through the so-called transient hyperpolarization of mitochondria. The high concordance of the performed computational estimations with many published experimental data allows concluding that OMP generation under physiological conditions is highly probable and VDAC might function as an OMP-dependent gatekeeper of mitochondria, controlling cell life and death. The proposed model of OMP generation allows understanding in more detail the mechanisms of cancer death resistance and anticancer action of various drugs and treatments influencing VDAC voltage-gating properties, VDAC content, mitochondrial hexokinase activity and VDAC-kinase interactions in MOM.

Collective polarization dynamics in bacterial colonies signify the occurrence of distinct subpopulations.

Membrane potential in bacterial systems has been shown to be dynamic and tightly related to survivability at the single-cell level. However, little is known about spatiotemporal patterns of membrane potential in bacterial colonies and biofilms. Here, we discovered a transition from uncorrelated to collective dynamics within colonies formed by the human pathogen Neisseria gonorrhoeae. In freshly assembled colonies, polarization is heterogeneous with instances of transient and uncorrelated hyper- or depolarization of individual cells. As colonies reach a critical size, the polarization behavior transitions to collective dynamics: A hyperpolarized shell forms at the center, travels radially outward, and halts several micrometers from the colony periphery. Once the shell has passed, we detect an influx of potassium correlated with depolarization. Transient hyperpolarization also demarks the transition from volume to surface growth. By combining simulations and the use of an alternative electron acceptor for the respiratory chain, we provide strong evidence that local oxygen gradients shape the collective polarization dynamics. Finally, we show that within the hyperpolarized shell, tolerance against aminoglycoside antibiotics increases. These findings highlight that the polarization pattern can signify the differentiation into distinct subpopulations with different growth rates and antibiotic tolerance.

Zebrafish Embryos Display Characteristic Bioelectric Signals during Early Development.

Bioelectricity is defined as endogenous electrical signaling mediated by the dynamic distribution of charged molecules. Recently, increasing evidence has revealed that cellular bioelectric signaling is critical for regulating embryonic development, regeneration, and congenital diseases. However, systematic real-time in vivo dynamic electrical activity monitoring of whole organisms has been limited, mainly due to the lack of a suitable model system and voltage measurement tools for in vivo biology. Here, we addressed this gap by utilizing a genetically stable zebrafish line, Tg (ubiquitin: ASAP1), and ASAP1 (Accelerated sensor of action potentials 1), a genetically encoded voltage indicator (GEVI). With light-sheet microscopy, we systematically investigated cell membrane potential (Vm) signals during different embryonic stages. We found cells of zebrafish embryos showed local membrane hyperpolarization at the cleavage furrows during the cleavage period of embryogenesis. This signal appeared before cytokinesis and fluctuated as it progressed. In contrast, whole-cell transient hyperpolarization was observed during the blastula and gastrula stages. These signals were generally limited to the superficial blastomere, but they could be detected within the deeper cells during the gastrulation period. Moreover, the zebrafish embryos exhibit tissue-level cell Vm signals during the segmentation period. Middle-aged somites had strong and dynamic Vm fluctuations starting at about the 12-somite stage. These embryonic stage-specific characteristic cellular bioelectric signals suggest that they might play a diverse role in zebrafish embryogenesis that could underlie human congenital diseases.

Voltage imaging reveals the dynamic electrical signatures of human breast cancer cells

Cancer cells feature a resting membrane potential (Vm) that is depolarized compared to normal cells, and express active ionic conductances, which factor directly in their pathophysiological behavior. Despite similarities to ‘excitable’ tissues, relatively little is known about cancer cell Vm dynamics. Here high-throughput, cellular-resolution Vm imaging reveals that Vm fluctuates dynamically in several breast cancer cell lines compared to non-cancerous MCF-10A cells. We characterize Vm fluctuations of hundreds of human triple-negative breast cancer MDA-MB-231 cells. By quantifying their Dynamic Electrical Signatures (DESs) through an unsupervised machine-learning protocol, we identify four classes ranging from "noisy” to “blinking/waving“. The Vm of MDA-MB-231 cells exhibits spontaneous, transient hyperpolarizations inhibited by the voltage-gated sodium channel blocker tetrodotoxin, and by calcium-activated potassium channel inhibitors apamin and iberiotoxin. The Vm of MCF-10A cells is comparatively static, but fluctuations increase following treatment with transforming growth factor-β1, a canonical inducer of the epithelial-to-mesenchymal transition. These data suggest that the ability to generate Vm fluctuations may be a property of hybrid epithelial-mesenchymal cells or those originated from luminal progenitors.

Altered functional responses by PAR1 agonist in murine dextran sodium sulphate-treated colon

Protease-activated receptor-1 (PAR1) is highly expressed in murine colonic smooth muscles. Responses to PAR1 activation are complex and result from responses in multiple cell types. We investigated whether PAR1 responses are altered in inflamed colon induced by dextran sodium sulfate (DSS)-treatment. Colitis was induced in C57BL/6 mice by administration of 3% DSS in drinking water for 7 days. Measurements of isometric force, transmembrane potentials from impaled smooth muscle cells, quantitative PCR and Western blots were performed. Thrombin, an activator of PAR1, caused transient hyperpolarization and relaxation of untreated colons, but these responses decreased in DSS-treated colons. Apamin caused depolarization and increased contractions of muscles from untreated mice. This response was decreased in DSS-treated colons. Expression of Kcnn3 and Pdgfra also decreased in DSS-treated muscles. A second phase of thrombin responses is depolarization and increased contractions in untreated muscles. However, thrombin did cause depolarization in DSS-treated colon, yet it increased colonic contractions. The latter effect was associated with enhanced expression of MYPT1 and CPI-17. The propagation velocity and frequency of colonic migrating motor complexes in DSS-treated colon was significantly higher compared to control colons. In summary, DSS treatment causes loss of transient relaxations due to downregulation of SK3 channels in PDGFRα+ cells and may increase contractile responses due to increased Ca2+ sensitization of smooth muscle cells via PAR1 activation.

Effects of ANP and BNP on the generation of respiratory rhythms in brainstem-spinal cord preparation isolated from newborn rats.

Natriuretic peptides (NPs) are a family of peptide hormones produced in cardiac muscle cells and consist mainly of three types: atrial NP (ANP), B-type (or brain) NP (BNP), and C-type NP. We herein report the effects of ANP and BNP on central respiratory activity in brainstem-spinal cord preparation isolated from newborn rats. Bath application of these peptides (100 nM) induced a weak transient depression of the respiratory rhythm followed by recovery. Respiratory-related neurons in the rostral ventrolateral medulla showed a tendency for transient hyperpolarization followed by recovery during the application of ANP or BNP. The application of a membrane-permeable cGMP, 8-Br-cGMP (10 or 20 μM), did not induce significant effects on respiratory rhythm, suggesting no involvement of guanylyl cyclase in effects of ANP or BNP. We also examined effects of BNP on respiratory depression induced by the sedative dexmedetomidine, which exerts an inhibitory influence on respiratory rhythm. When pretreated with 50 nM BNP, the inhibitory effect of 100 nM dexmedetomidine was significantly reduced. Our findings suggest that ANP and BNP act as mild excitatory agents with sustained effects on respiratory rhythm after an initial transient depression.

Ent-kaurane diterpenes isolated from n-hexane extract of Baccharis sphenophylla by bioactivity-guided fractionation target the acidocalcisomes in Trypanosoma cruzi

BackgroundIn the present work the bioactivity-guided fractionation of n-hexane extract from aerial parts of Baccharis sphenophylla (Asteraceae) against trypomastigote forms of Trypanosoma cruzi was performed. PurposeTo evaluate the antitrypanosomal potential of diterpenes ent‑kaurenoic (1), grandifloric (2). and 15β-tiglinoyloxy‑ent-kaurenoic (3) acids, isolated from n-hexane extract from aerial parts of B. sphenophylla, and elucidate their mechanism of action against T. cruzi. Methods/Study designn-Hexane and MeOH extracts from aerial parts of B. sphenophylla were prepared and caused, respectively, 100% and 50% of death of trypomastigote forms of T. cruzi. Based on these results, the n-hexane extract was subjected to bioactivity-guided fractionation procedures to afford three related ent‑kaurane diterpenoids (1–3). Based on spectrofluorometric assays and flow cytometry analysis, the mechanism of action of compounds 1 and 3 was investigated. ResultsCompounds 1 and 3, isolated from n-hexane extract from aerial parts of B. sphenophylla, showed potent activity against parasites with EC50 values of 10.6 μM (SI > 18.8) and 2.4 μM (SI = 34.8), respectively. On the other hand, compound 2 was inactive against trypomastigotes. In mechanism of action studies using the fluorescent probe SYTOX Green, the plasma membrane permeability was unaltered after treatment with compounds 1 and 3, but compound 1 induced a depolarization of the plasma membrane electric potential (ΔΨp). No substantial alterations were observed in the mitochondria after treatment with compound 3, but a transient hyperpolarization of the mitochondrial membrane potential (ΔΨm) by compound 1. Despite the increased ATP levels induced by compounds 1 and 3, no alterations of ROS and Ca2+ levels were registered. However, both compounds promoted a time-dependent alkalinization of the acidocalcisomes, probably contributing to an osmotic imbalance of the cell. In silico physicochemical studies of compounds 1–3 suggested that lipophilicity and molecular complexity may play an important role in the antitrypanosomal activity. Moreover, no pan-assay interference compounds (PAINS) alerts were detected for compounds 1–3. ConclusionObtained data indicated that the isolated ent‑kaurane diterpenes from n-hexane extract from aerial parts of B. sphenophylla, especially compound 3, could be considered interesting prototypes for further modifications aiming the discovery of new hits against T. cruzi.

Fusicoccin (FC)-Induced Rapid Growth, Proton Extrusion and Membrane Potential Changes in Maize (Zea mays L.) Coleoptile Cells: Comparison to Auxin Responses

The fungal toxin fusicoccin (FC) induces rapid cell elongation, proton extrusion and plasma membrane hyperpolarization in maize coleoptile cells. Here, these three parameters were simultaneously measured using non-abraded and non-peeled segments with the incubation medium having access to their lumen. The dose–response curve for the FC-induced growth was sigmoidal shaped with the maximum at 10−6 M over 10 h. The amplitudes of the rapid growth and proton extrusion were significantly higher for FC than those for indole-3-acetic acid (IAA). The differences between the membrane potential changes that were observed in the presence of FC and IAA relate to the permanent membrane hyperpolarization for FC and transient hyperpolarization for IAA. It was also found that the lag times of the rapid growth, proton extrusion and membrane hyperpolarization were shorter for FC compared to IAA. At 30 °C, the biphasic kinetics of the IAA-induced growth rate could be changed into a monophasic (parabolic) one, which is characteristic for FC-induced rapid growth. It has been suggested that the rates of the initial phase of the FC- and IAA-induced growth involve two common mechanisms that consist of the proton pumps and potassium channels whose contribution to the action of both effectors on the rapid growth is different.

Structure and function of the gap junctional network of photoreceptive ganglion cells.

Intrinsically photosensitive retinal ganglion cells (ipRGCs) signal not only anterogradely to drive behavioral responses, but also retrogradely to some amacrine interneurons to modulate retinal physiology. We previously found that all displaced amacrine cells with spiking, tonic excitatory photoresponses receive gap-junction input from ipRGCs, but the connectivity patterns and functional roles of ipRGC-amacrine coupling remained largely unknown. Here, we injected PoPro1 fluorescent tracer into all six types of mouse ipRGCs to identify coupled amacrine cells, and analyzed the latter's morphological and electrophysiological properties. We also examined how genetically disrupting ipRGC-amacrine coupling affected ipRGC photoresponses. Results showed that ipRGCs couple with not just ON- and ON/OFF-stratified amacrine cells in the ganglion-cell layer as previously reported, but also OFF-stratified amacrine cells in both ganglion-cell and inner nuclear layers. M1- and M3-type ipRGCs couple mainly with ON/OFF-stratified amacrine cells, whereas the other ipRGC types couple almost exclusively with ON-stratified ones. ipRGCs transmit melanopsin-based light responses to at least 93% of the coupled amacrine cells. Some of the ON-stratifying ipRGC-coupled amacrine cells exhibit transient hyperpolarizing light responses. We detected bidirectional electrical transmission between an ipRGC and a coupled amacrine cell, although transmission was asymmetric for this particular cell pair, favoring the ipRGC-to-amacrine direction. We also observed electrical transmission between two amacrine cells coupled to the same ipRGC. In both scenarios of coupling, the coupled cells often spiked synchronously. While ipRGC-amacrine coupling somewhat reduces the peak firing rates of ipRGCs' intrinsic melanopsin-based photoresponses, it renders these responses more sustained and longer-lasting. In summary, ipRGCs' gap junctional network involves more amacrine cell types and plays more roles than previously appreciated.

Neuronal firing modulation by a membrane-targeted photoswitch

Optical technologies allowing modulation of neuronal activity at high spatio-temporal resolution are becoming paramount in neuroscience. In this respect, azobenzene-based photoswitches are promising nanoscale tools for neuronal photostimulation. Here we engineered a light-sensitive azobenzene compound (Ziapin2) that stably partitions into the plasma membrane and causes its thinning through trans-dimerization in the dark, resulting in an increased membrane capacitance at steady state. We demonstrated that in neurons loaded with the compound, millisecond pulses of visible light induce a transient hyperpolarization followed by a delayed depolarization that triggers action potential firing. These effects are persistent and can be evoked in vivo up to 7 days, proving the potential of Ziapin2 for the modulation of membrane capacitance in the millisecond timescale, without directly affecting ion channels or local temperature.

How Pulsatile Kisspeptin Stimulation and GnRH Autocrine Feedback Can Drive GnRH Secretion: A Modeling Investigation.

Pulsatile secretion of GnRH from hypothalamic GnRH neurons tightly regulates the release of mammalian reproductive hormones. Although key factors such as electrical activity and stimulation by kisspeptin have been extensively studied, the underlying mechanisms that regulate GnRH release are still not fully understood. Previously developed mathematical models studied hormonal release and electrical properties of GnRH neurons separately, but they never integrated both components. Herein, we present a more complete biophysical model to investigate how electrical activity and hormonal release interact. The model consists of two components: an electrical submodel comprised of a modified Izhikevich formalism incorporating several key ionic currents to reproduce GnRH neuronal bursting behavior, and a hormonal submodel that incorporates pulsatile kisspeptin stimulation and a GnRH autocrine feedback mechanism. Using the model, we examine the electrical activity of GnRH neurons and how kisspeptin affects GnRH pulsatility. The model reproduces the noise-driven bursting behavior of GnRH neurons as well as the experimentally observed electrophysiological effects induced by GnRH and kisspeptin. Specifically, the model reveals that external application of GnRH causes a transient hyperpolarization followed by an increase in firing frequency, whereas administration of kisspeptin leads to long-lasting depolarization of the neuron. The model also shows that GnRH release follows a pulsatile profile similar to that observed experimentally and that kisspeptin and GnRH exhibit ∼7-1 locking in their pulsatility. These results suggest that external kisspeptin stimulation with a period of ∼8 minutes drives the autocrine mechanism beyond a threshold to generate pronounced GnRH pulses every hour.

The anti-stress effects of therapy dogs on college students

The relationship between dysmyelination and the progression of neuropathy in Charcot–Marie–Tooth (CMT) hereditary polyneuropathy is unclear. Mice heterozygously deficient for the myelin protein P0 gene (P0+/−) are indistinguishable from wild-type (WT) at birth and then develop a slowly progressing demyelinating neuropathy reminiscent of CMT Type 1b. Accumulating evidence suggests that impulse conduction can become lethal to acutely demyelinated central and peripheral axons. Here we investigated the vulnerability of motor axons to long-lasting, high-frequency repetitive stimulation (RS) in P0+/− mice as compared to WT littermates at 7, 12, and 20 months of age. RS was carried out in interrupted trains of 200 Hz trains for 3 h. Tibial nerves were stimulated at the ankle while the evoked compound muscle action potentials (CMAPs) and the ascending compound nerve action potentials (CNAPs) were recorded from plantar muscles and the sciatic nerve, respectively. In 7-month old mice, there was recovery of CMAP and CNAP following RS. When mice were about one year old, electrophysiological recovery following RS was incomplete and in P0+/− also associated with morphological signs of partial Wallerian degeneration. The effect of RS was larger in P0+/− as compared to age-matched WT. When mice were about 2 years old, the effect was stronger and became similar between WT and P0+/−. RS was followed by a transient hyperpolarization, which decreased with age and was smaller in P0+/− than in WT. Our data suggest that both aging and the dysmyelinating disease process may contribute to the susceptibility to activity-induced axonal degeneration. It is possible that in aging mice and in P0+/− there is inadequate energy-dependent Na+/K+ pumping, as indicated by the reduced post-stimulation hyperpolarization, which may lead to a lethal Na+ overload in some axons.

The functional role of protease-activated receptors on contractile responses by activation of Ca2+ sensitization pathways in simian colonic muscles.

It has been known that activation of protease-activated receptors (PARs) affects gastrointestinal motility. In this study, we tested the effects of PAR agonists on electrical and contractile responses and Ca2+ sensitization pathways in simian colonic muscles. The Simian colonic muscle was initially hyperpolarized by PAR agonists. After the transient hyperpolarization, simian colonic muscle repolarized to the control resting membrane potential (RMP) without a delayed depolarization. Apamin significantly reduced the initial hyperpolarization, suggesting that activation of small conductance Ca2+-activated K+ (SK) channels is involved in the initial hyperpolarization. In contractile experiments, PAR agonists caused an initial relaxation followed by an increase in contractions. These delayed contractile responses were not matched with the electrical responses that showed no after depolarization of the RMP. To investigate the possible involvement of Rho-associated protein kinase 2 (ROCK) pathways in the PAR effects, muscle strips were treated with ROCK inhibitors, which significantly reduced the PAR agonist-induced contractions. Furthermore, PAR agonists increased MYPT1 phosphorylation, and ROCK inhibitors completely blocked MYPT1 phosphorylation. PAR agonists alone had no effect on CPI-17 phosphorylation. In the presence of apamin, PAR agonists significantly increased CPI-17 phosphorylation, which was blocked by protein kinase C (PKC) inhibitors suggesting that Ca2+ influx is increased by apamin and is activating PKC. In conclusion, these studies show that PAR activators induce biphasic responses in simian colonic muscles. The initial inhibitory responses by PAR agonists are mainly mediated by activation of SK channels and delayed contractile responses are mainly mediated by the CPI-17 and ROCK Ca2+ sensitization pathways in simian colonic muscles. NEW & NOTEWORTHY In the present study, we found that the contractile responses of simian colonic muscles to protease-activated receptor (PAR) agonists are different from the previously reported contractile responses of murine colonic muscles. Ca2+ sensitization pathways mediate the contractile responses of simian colonic muscles to PAR agonists without affecting the membrane potential. These findings emphasize novel mechanisms of PAR agonist-induced contractions possibly related to colonic dysmotility in inflammatory bowel disease.

The synthetic cannabinoid XLR-11 induces in vitro nephrotoxicity by impairment of endocannabinoid-mediated regulation of mitochondrial function homeostasis and triggering of apoptosis

Synthetic cannabinoids (SCBs)-related intoxications and deaths have been increasingly reported, turning its widespread recreational use into a major public health concern. Specifically, a direct link between SCBs and acute kidney injury (AKI) has been established. XLR-11 is an SCB commonly found in the toxicological analysis of patients with SCB-associated AKI. However, the pathophysiology of AKI among SCB consumers remains unknown. This work thus represents the first in vitro assessment of SCB nephrotoxicity, as a first approach to identify its cellular targets.We demonstrate that XLR-11, at biologically relevant concentrations (in the nanomolar range), primarily targets mitochondrial function in human proximal tubule (HK-2) cells, inducing a transient hyperpolarization of the mitochondrial membrane and increasing ATP production, accompanied by Bax translocation from cytosol into mitochondria. These phenomena further triggered energy-dependent apoptotic cell death pathways, indicated by increased caspase-3 activity and chromatin condensation. Experiments using SR141716A and SR144258, specific antagonists for CB1 and CB2 receptors, respectively, as well as HEK293T cells (which do not express CBRs) highlighted these processes’ dependence on CBR activation. Nevertheless, ATP formation seemed to follow a CBR-independent pathway. Our findings using specific inhibitors of endogenous cannabinoids biosynthesis (i.e. MAFP and THL) further evidenced the involvement of the endocannabinoid system in the regulation of these processes, as XLR-11 binding to CBRs seemed to compromise endocannabinoid-mediated preservation of mitochondrial function. Nevertheless, the exact mechanisms involved require further clarification.

Electrical interactions between photoreceptors in the compound eye of Periplaneta americana.

The compound eye of Periplaneta americana contains two spectral classes of photoreceptors: narrow-band UV-sensitive and broad-band green-sensitive. In intracellular recordings, stimulation of green-sensitive photoreceptors with flashes of relatively bright UV/violet light produced anomalous delayed depolarization after the end of the normal light response, whereas stimulation of UV-sensitive photoreceptors with green light elicited biphasic responses characterized by initial transient hyperpolarization followed by prolonged delayed depolarization. To explore the basis for these findings, we used RNA interference to selectively suppress expression of the genes encoding green opsin (GO1), UV opsin (UVO) or both. The hyperpolarizing component in UV-sensitive photoreceptors was eliminated and the delayed depolarization was reduced after GO1 knockdown, suggesting that the hyperpolarization represents fast inhibitory interactions between green- and UV-sensitive photoreceptors. Green-sensitive photoreceptor responses of GO1 knockdowns to flashes of UV/violet were almost exclusively biphasic, whereas residual responses to green had normal kinetics. Knockdown of UVO reduced the responses of UV-sensitive photoreceptors but had minor effects on delayed depolarization in green-sensitive photoreceptors. Angular sensitivity analysis indicated that delayed depolarization of green-sensitive photoreceptors by violet light originates from excitation of (an)other photoreceptor(s) in the same ommatidium. The angle at which the maximal delayed depolarization was observed in green-sensitive photoreceptors stimulated with violet light did not match the angle of the maximal transient depolarization. In contrast, no significant mismatch was observed for delayed depolarization elicited by green light. These results suggest that the cellular sources of the normal transient and additional delayed depolarization by violet light are separate and distinct.

Extracellular Cl- regulates electrical slow waves and setting of smooth muscle membrane potential by interstitial cells of Cajal in mouse jejunum.

What is the central question of this study? The aim was to investigate the roles of extracellular chloride in electrical slow waves and resting membrane potential of mouse jejunal smooth muscle by replacing chloride with the impermeant anions gluconate and isethionate. What is the main finding and its importance? The main finding was that in smooth muscle cells, the resting Cl- conductance is low, whereas transmembrane Cl- movement in interstitial cells of Cajal (ICCs) is a major contributor to the shape of electrical slow waves. Furthermore, the data confirm that ICCs set the smooth muscle membrane potential and that altering Cl- homeostasis in ICCs can alter the smooth muscle membrane potential. Intracellular Cl- homeostasis is regulated by anion-permeable channels and transporters and contributes to excitability of many cell types, including smooth muscle and interstitial cells of Cajal (ICCs). Our aims were to investigate the effects on electrical activity in mouse jejunal muscle strips of replacing extracellular Cl- (Cl-o ) with the impermeant anions gluconate and isethionate. On reducing Cl-o , effects were observed on electrical slow waves, with small effects on smooth muscle membrane voltage (Em ). Restoration of Cl- hyperpolarized smooth muscle Em proportional to the change in Cl-o concentration. Replacement of 90% of Cl-o with gluconate reversibly abolished slow waves in five of nine preparations. Slow waves were maintained in isethionate. Gluconate and isethionate substitution had similar concentration-dependent effects on peak amplitude, frequency, width at half peak amplitude, rise time and decay time of residual slow waves. Gluconate reduced free ionized Ca2+ in Krebs solutions to 0.13mm. In Krebs solutions containing normal Cl- and 0.13mm free Ca2+ , slow wave frequency was lower, width at half peak amplitude was smaller, and decay time was faster. The transient hyperpolarization following restoration of Cl-o was not observed in W/Wv mice, which lack pacemaker ICCs in the small intestine. We conclude that in smooth muscle cells, the resting Cl- conductance is low, whereas transmembrane Cl- movement in ICCs plays a major role in generation or propagation of slow waves. Furthermore, these data support a role for ICCs in setting smooth muscle Em and that altering Cl- homeostasis in ICCs can alter smooth muscle Em .

Early trans-plasma membrane responses to Tobacco mosaic virus infection

Early trans-plasma membrane behavior after in vivo mechanical inoculation of Nicotiana tabacum with Tobacco mosaic virus (TMV) was investigated and compared to virus quantification in leaf tissues. To identify early events related to virus/host interaction, the systemic virus TMV was used to infect lower leaves and tests were carried out on upper leaves which were not directly infected. Non-invasive microelectrodes were used to estimate trans-plasma membrane electron transport and membrane potential after artificial inoculum of virus, monitoring the plant for the following 15 days. Virus infection was assessed by ELISA and quantified by quantitative RT-PCR. Collected data showed that after 2-day post-inoculation (dpi), TMV was able to modify membrane parameters: transient hyperpolarization of trans-membrane potential was observed until 10 dpi, while redox activity in infected samples was higher compared to control until end of tests. Conversely, ELISA diagnostic test was not able to reveal the virus presence in tobacco leaves until 6 dpi, while leaf symptoms were manifested after 13 dpi.