Hyperpolarization Of Membrane Potential Research Articles

Membrane potential hyperpolarization: a critical factor in acrosomal exocytosis and fertilization in sperm within the female reproductive tract.

Hyperpolarization of the membrane potential (Em), a phenomenon regulated by SLO3 channels, stands as a central feature in sperm capacitation-a crucial process conferring upon sperm the ability to fertilize the oocyte. In vitro studies demonstrated that Em hyperpolarization plays a pivotal role in facilitating the mechanisms necessary for the development of hyperactivated motility (HA) and acrosomal exocytosis (AE) occurrence. Nevertheless, the physiological significance of sperm Em within the female reproductive tract remains unexplored. As an approach to this question, we studied sperm migration and AE incidence within the oviduct in the absence of Em hyperpolarization using a novel mouse model established by crossbreeding of SLO3 knock-out (KO) mice with EGFP/DsRed2 mice. Sperm from this model displays impaired HA and AE in vitro. Interestingly, examination of the female reproductive tract shows that SLO3 KO sperm can reach the ampulla, mirroring the quantity of sperm observed in wild-type (WT) counterparts, supporting that the HA needed to reach the fertilization site is not affected. However, a noteworthy distinction emerges-unlike WT sperm, the majority of SLO3 KO sperm arrive at the ampulla with their acrosomes still intact. Of the few SLO3 KO sperm that do manage to reach the oocytes within this location, fertilization does not occur, as indicated by the absence of sperm pronuclei in the MII-oocytes recovered post-mating. In vitro, SLO3 KO sperm fail to penetrate the ZP and fuse with the oocytes. Collectively, these results underscore the vital role of Em hyperpolarization in AE and fertilization within their physiological context, while also revealing that Em is not a prerequisite for the development of the HA motility, essential for sperm migration through the female tract to the ampulla.

Contribution of Ih to Postnatal Maturation of Muscarinic Modulation of Inspiratory Bursting at Hypoglossal Motoneurons in Neonatal Mice

Activation of hypoglossal motoneurons (XII MNs) contributes to stiffening of the upper airway during inspiration. The magnitude of this excitation is decreased during sleep, likely due to a combination of withdrawal of noradrenaline and acetylcholine release at XII MNs. Acetylcholine acts at muscarinic acetylcholine receptors (mAChRs) and surprisingly mAChR activation has an excitatory effect on inspiratory bursting in neonatal preparations, which becomes inhibitory in adult preparations. mAChRs modulate several ion channels and characterizing muscarinic effect changes at the XII nucleus will help to explain the mechanism of the inhibitory shift over postnatal maturation. The hyperpolarization activated cyclic nucleotide gated (HCN) channel is positively modulated by mAChRs. HCN channels give rise to Ih, a mixed cation current activated at hyperpolarized membrane potentials that contribute to membrane depolarization. Ihincreases dramatically with postnatal maturation in XII MNs. Thus, we hypothesize that 1) the effects of muscarinic modulation of Ih increases with postnatal maturation, and 2) that muscarinic activation of Ih contributes to the net inhibitory component of muscarinic modulation of inspiratory bursting at XII motoneurons. To test our hypotheses, we used the rhythmic medullary slice preparation to test the functional effects of muscarinic modulation of Ih on inspiratory bursting in neonatal CD1 mice (postnatal day, P0-P4) in combination with pharmacological block of Ih with ZD7288 (25 μM, 150s). Local application of muscarine (100μM, 30s) in the XII motor nucleus increased inspiratory burst amplitude (189 ± 58% of baseline, n = 9). Contrary to our hypothesis, there was no statistically significant difference between inspiratory burst amplitude elicited by muscarine in the presence of ZD7288 (192 ± 47% of baseline, n = 9) versus with the aCSF vehicle control (174 ± 43% of baseline, p = 0.098). We next tested a higher ZD7288 concentration (100 μM) and longer application times (150s, 330s) in neonatal preparations. Preliminary data (n=2) indicate that ZD7288 may increase muscarinic potentiation of inspiratory burst amplitude at XII MNs (muscarinic potentiation (100μM, 30s): control — 112 ±65 % of baseline, aCSF vehicle control - 131 ± 38% of baseline; ZD7288 100μM, 150s - 148 ± 44% of baseline; ZD7288 100μM, 330s - 107 ± 72% of baseline). Future research will elucidate whether the Ih contribution to muscarinic modulation of inspiratory bursting increases with postnatal maturation. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Pulmonary Fibrosis Augments CGRP-Dependent Hyperpolarization and Vasodilation of Mouse Pulmonary Arteries

Calcitonin gene related peptide (CGRP) hyperpolarizes membrane potential (Vm) of pulmonary arterial smooth muscle cells (SMCs) and endothelial cells (ECs) through PKA-dependent activation of KATP channels. CGRP can diminish the severity of pulmonary fibrosis (PF), however, the effects on vascular signaling were poorly defined. We hypothesized that hyperpolarization to CGRP would be augmented in a mouse model of PF. PF was induced by intratracheal delivery of bleomycin (3 wk), and saline was used as a control (sham). Pulmonary arteries (PAs; 100-150 μm diameter) from male C57BL/6J mice (age, ~5 mo), were cannulated, pressurized to 16 cmH2O, and studied at 37°C. Alternatively, intact endothelial tubes were studied at 32°C following dissociation of SMCs. Vm was recorded continuously using intracellular microelectrodes. At rest, Vm was similar for SMCs (bleomycin = -43±3 mV, sham = -44±3 mV; n = 5/group) and ECs (bleomycin = -45±4 mV, sham = -44±3 mV; n=5/group). Increasing concentrations of CGRP (10−10-10−6 M) evoked hyperpolarization of Vm with greater potency in SMCs (EC50: bleomycin = 2 nM, sham = 15 nM) and ECs (EC50: bleomycin = 3 nM, sham = 8 nM) from PF mice, without changing effcacy (SMCs: bleomycin ΔVm = -24±4 mV, sham ΔVm = -21±3 mV; ECs: bleomycin ΔVm = -26±2 mV, sham ΔVm=23±4 mV). Consistently, vasodilation to CGRP in PAs preconstricted with uridine triphosphate (UTP, 5 μM; EC50) was enhanced in PF (92±6% maximum dilation;) vs. sham (83±4% maximum dilation) mice (n=5/group). Greater vasodilation and hyperpolarization of SMCs to CGRP persisted in endothelium-disrupted PAs and during treatment with L-NAME (10−4 M) indicating that ECs are not required for greater responsiveness to CGRP in SMCs. With no effect on resting Vm, inhibition of KATP channels with 1 μM glibenclamide or PKA with protein kinase inhibitor [PKI-(14-22)-amide, myristoylated; 1 μM] significantly attenuated (p<0.05) hyperpolarization of SMCs and ECs (ΔVm ≤5 mV for all groups). Both glibenclamide and PKI attenuated vasodilation to CGRP in PAs and eliminated differences between groups. In a mouse model of PF, CGRP-dependent hyperpolarization of pulmonary arterial SMCs and ECs is augmented through increased PKA-dependent activation of KATP channels leading to increased vasodilator sensitivity. AHA CDA#931652. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Characterization of HCN channel subtypes at hypoglossal motoneurons throughout postnatal maturation in mice

The inspiratory phase of breathing includes a stiffening of the upper airway to decrease airway resistance and is mediated by excitatory motor output from hypoglossal motoneurons (XII MNs). This excitation decreases from wakefulness to sleep. This decrease in excitation is in large part due to increasing acetylcholine levels, which activate muscarinic acetylcholine receptors (mAChRs) that have a net inhibitory effect in adult preparations. By contrast, activation of mAChRs in neonatal rodent preparations has a net effect of potentiating inspiratory burst amplitude. Therefore, characterizing changes in muscarinic effects at XII MNs across postnatal maturation will help elucidate the mechanism underlying the shift from muscarinic excitation to inhibition. mAChRs modulate several effector ion channels including the hyperpolarization activated cyclic nucleotide gated (HCN) channel. HCN channels give rise to I h, a mixed cation current activated at hyperpolarized membrane potentials. I h additionally shows upregulation at XII MNs with postnatal maturation. Since there are four HCN subtypes (HCN1-4), the reported changes in I h may be caused by changes in HCN channel subtype levels. HCN1 and HCN2 are most prominent in adult XII MNs, whereas the expression of HCN1-4 in neonates is unknown. Thus, the purpose of this research is to characterize changes in HCN gene and channel expression levels at XII MNs across postnatal maturation. We hypothesis that both HCN1 and HCN2 subtypes will increase with postnatal maturation, and that HCN2 has higher levels than HCN1. To test this hypothesis, we used neuroanatomical techniques to evaluate changes in expression patterns of the four HCN proteins at the XII nuclei across postnatal maturation in CD-1 mice. We performed double-labeled immunofluorescence experiments against HCN1 and HCN2 and co-labeled with choline acetyltransferase (ChAT) on 20μm transverse brainstem slices across six postnatal age groups under identical conditions: P0-P1, P4-P6, P9-P10, P12-P14, P17-P19, and adult. Images of the XII nuclei were collected using a confocal microscope. ImageJ was used to determine the average XII MN HCN subtype intensity across postnatal maturation. Preliminary data suggest that HCN1 (n=2) channels undergo a decrease in expression at P17-19 before a final increase in adulthood (P0-1= 97-100%, P4-6= 92-100%, P9-10= 85-95%, P12-14= 77-97%, P17-19= 61-67%, adult= 71-82%). By contrast, preliminary data suggest that HCN2 (n=2) expression intensity remained relatively consistent with a decrease in labeling intensity at P4-6 (P0-1= 97-100%, P4-6=62-84%, P9-10= 45-95%, P12-14= 67-90%, P17-19= 50-93%, adult= 75-100%). These modest changes in labeling intensity of HCN channel subtypes may not contribute to the observed shift of mAChR modulation at XII MNs from excitation to inhibition with postnatal maturation. Biomedical Sciences, College of Graduate Studies; Department of Physiology, College of Graduate Studies, Midwestern University; NIH/NHLBI Funding: R15HL148870. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Learning-induced bidirectional enhancement of inhibitory synaptic metaplasticity.

Training rodents in a particularly difficult olfactory-discrimination (OD) task results in the acquisition of the ability to perform the task well, termed 'rule learning'. In addition to enhanced intrinsic excitability and synaptic excitation in piriform cortex pyramidal neurons, rule learning results in increased synaptic inhibition across the whole cortical network to the point where it precisely maintains the balance between inhibition and excitation. The mechanism underlying such precise inhibitory enhancement remains to be explored. Here, we use brain slices from transgenic mice (VGAT-ChR2-EYFP), enabling optogenetic stimulation of single GABAergic neurons and recordings of unitary synaptic events in pyramidal neurons. Quantal analysis revealed that learning-induced enhanced inhibition is mediated by increased quantal size of the evoked inhibitory events. Next, we examined the plasticity of synaptic inhibition induced by long-lasting, intrinsically evoked spike firing in post-synaptic neurons. Repetitive depolarizing current pulses from depolarized (-70mV) or hyperpolarized (-90mV) membrane potentials induced long-term depression (LTD) and long-term potentiation (LTP) of synaptic inhibition, respectively. We found a profound bidirectional increase in the ability to induce both LTD, mediated by L-type calcium channels, and LTP, mediated by R-type calcium channels after rule learning. Blocking the GABAB receptor reversed the effect of intrinsic stimulation at -90mV from LTP to LTD. We suggest that learning greatly enhances the ability to modify the strength of synaptic inhibition of principal neurons in both directions. Such plasticity of synaptic plasticity allows fine-tuning of inhibition on each particular neuron, thereby stabilizing the network while maintaining the memory of the rule. KEY POINTS: Olfactory discrimination rule learning results in long-lasting enhancement of synaptic inhibition on piriform cortex pyramidal neurons. Quantal analysis of unitary inhibitory synaptic events, evoked by optogenetic minimal stimulation, revealed that enhanced synaptic inhibition is mediated by increased quantal size. Surprisingly, metaplasticity of synaptic inhibition, induced by intrinsically evoked repetitive spike firing, is increased bidirectionally. The susceptibility to both long-term depression (LTD) and long-term potentiation (LTP) of inhibition is enhanced after learning. LTD of synaptic inhibition is mediated by L-type calcium channels and LTP by R-type calcium channels. LTP is also dependent on activation of GABAB receptors. We suggest that learning-induced changes in the metaplasticity of synaptic inhibition enable the fine-tuning of inhibition on each particular neuron, thereby stabilizing the network while maintaining the memory of the rule.

Abstract 438: Inhibition of de novo lipogenesis with PUFA supplementation induces ferroptosis in advanced prostate cancer

Abstract Background: Metabolic alterations are recognized as a hallmark of cancer progression. In prostate cancer (PCa), in particular, a notable lipid reprogramming occurs with increased expression levels of multiple enzymes involved in the fatty acid synthesis pathway to fulfill the metabolic and structural demands of cancer cells. Fatty acids are vital for membrane building, ATP production through B-oxidation, and storage in lipid droplets for high-energy demands. In castration-resistant prostate cancer (CRPC), the aberrant overexpression of Fatty Acid Synthase (FASN), the rate-limiting enzyme in de novo lipogenesis (DNL), results in synthesis of saturated (palmitate) and monounsaturated fatty acids (SFA and MUFA). If DNL is inhibited, polyunsaturated fatty acids (PUFAs) are taken up from the environment. These could undergo lipid peroxidation generating reactive oxygen species (ROS) and contribute to ferroptosis. We hypothesize that combining the inhibition of DNL with the addition of exogenous PUFAs could increase cell death by ferroptosis. Methods: PCa cells (LNCaP, 22Rv1 and C4-2) and human organoids were exposed to pharmacologic FASN inhibition and PUFAs for 6 days. Cell viability, lipid peroxidation, superoxide production and, protein carbonylation as a measure of intracellular ROS as well as lipidomic profiling, were assessed. Results: FASN inhibition significantly increased lipid peroxidation and intracellular ROS, with subsequent membrane potential hyperpolarization in prostate cancer cells. These effects were partially rescued by palmitate, while FASNi supplemented with PUFA potentiated protein carbonylation. Expression levels of GPX4, the main regulator of ferroptosis, were reduced after DNL inhibition and rescued with palmitate addition. Suppression of DNL combined with supplemental addition of PUFAs resulted in significant cell growth inhibition in prostate cancer cells and organoids. Finally, lipid profiling demonstrated that the suppression of DNL triggers increased acyl chain length in phospholipid and several other lipid classes. Again, this effect was significantly potentiated when combining FASN inhibition with PUFA supplementation. Conclusion: FASN inhibition produces a cascade of intracellular events, including lipid remodeling, increased ROS production and ferroptosis, all potentiated by PUFAs. These findings provide compelling evidence for the use a combined approach involving DNL inhibition and PUFA supplementation as a novel therapeutic avenue to treat advanced prostate cancer. Citation Format: Silvia Daniele Rodrigues, Isadora Ferrari Teixeira, Pier Vitale Nuzzo, Hubert Pakula, David Nanus, Massimo Loda. Inhibition of de novo lipogenesis with PUFA supplementation induces ferroptosis in advanced prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 438.

Exploring Mechanisms of Antifungal Lipopeptide Iturin A from Bacillus against Aspergillus niger.

The control of Aspergillus niger (A. niger) is of great significance for the agricultural economy and food safety. In this study, the antifungal effect and mechanism of iturin A from Bacillus amyloliquefaciens (CGMCC No. 8473) against A. niger (ATCC 16404) were investigated using biochemical analyses and proteomics. Changes in a mycelium treated with iturin A were observed using scanning electron microscopy and transmission electron microscopy, including mycelial twisting and collapse, organelle disintegration, and intracellular vacuolization. The cytomembrane integrity of A. niger was affected by iturin A, as detected by propidium iodide staining. In addition, the generation of excess reactive oxygen species, the hyperpolarization of the mitochondrial membrane potential and malondialdehyde accumulation also indicated that iturin A induced apoptosis in A. niger through the oxidative stress pathway. Proteomics results showed that 310 proteins were differentially expressed in the A. niger mycelium exposed to iturin A, including 159 upregulated proteins and 151 downregulated proteins, which were mainly associated with energy metabolism of A. niger. We propose that iturin A might inhibit the growth of A. niger by disrupting cytomembrane integrity, via oxidative stress, and by interfering with glycolysis/gluconeogenesis and the tricarboxylic acid cycle. Overall, iturin A is a promising antifungal agent that provides a rationale for controlling A. niger contamination in food.

LRRC52 is likely a functional component of human KSper†.

Completion of fertilization is orchestrated by various ion channels in sperm membrane. Hyperpolarization of membrane potential, an indispensable event during the capacitation process, is dominated by sperm potassium channel (KSper). In addition to sperm-specific SLO3, which forms the channel pore, the auxiliary subunit leucine-rich-repeat-containing protein 52 (LRRC52) is required to form mKSper to function under physiological conditions. However, in human sperm, although most evidence supports that hSLO3 is the pore-forming subunit, whether hLRRC52 contributes to hKSper conductance and modulates sperm function remains to be understood. Here, using an extracellular segment that is homologous between mice and humans as an antigen, we developed a polyclonal antibody designed as LID1 that specifically detected mLRRC52 and performed co-immunoprecipitation with mSLO3. Additionally, patch-clamp recordings of mouse sperm showed that, physiological activation of mKSper and sperm functions were dramatically attenuated after treatment with LID1, indicating that LID1 functionally disrupted the regulation of mLRRC52 on mKSper. Next, LID1 was used to investigate the significance of hLRRC52 for hKSper activation. As a result, hLRRC52 was expressed in human sperm and might be assembled with hSLO3. More importantly, LID1 inhibited hKSper currents and depolarized sperm membrane potential, supporting essential modulation of hLRRC52 in hKSper. Ca2+ signaling of human sperm was also compromised in the presence of LID1, which impaired sperm motility and acrosome reaction. Because LID1 specifically inhibited both mKSper and hKSper but not mCatSper or hCatSper, our results suggest that hLRRC52 functions as an important component of hKSper and regulates sperm physiological functions.

Hyperpolarisation of Mitochondrial Membranes Is a Critical Component of the Antifungal Mechanism of the Plant Defensin, Ppdef1.

Plant defensins are a large family of small cationic proteins with diverse functions and mechanisms of action, most of which assert antifungal activity against a broad spectrum of fungi. The partial mechanism of action has been resolved for a small number of members of plant defensins, and studies have revealed that many act by more than one mechanism. The plant defensin Ppdef1 has a unique sequence and long loop 5 with fungicidal activity against a range of human fungal pathogens, but little is known about its mechanism of action. We screened the S. cerevisiae non-essential gene deletion library and identified the involvement of the mitochondria in the mechanism of action of Ppdef1. Further analysis revealed that the hyperpolarisation of the mitochondrial membrane potential (MMP) activates ROS production, vacuolar fusion and cell death and is an important step in the mechanism of action of Ppdef1, and it is likely that a similar mechanism acts in Trichophyton rubrum.

The cyclin-dependent kinase inhibitor abemaciclib-induced hepatotoxicity: Insight on the molecular mechanisms in HepG2/THP-1 co-culture model

Drug-induced liver injury (DILI) is one of the widespread causes of liver injury and immune system plays important role. Abemaciclib (ABE) is a cyclin-dependent kinase inhibitor used as monotherapy or combination therapy in the treatment of breast cancer. Like other kinase inhibitors, the underlying mechanisms of ABE-induced hepatotoxicity are not completely known yet. In the current study, hepatotoxicity of ABE was evaluated with HepG2/THP-1 co-culture model which has been developed in recent years for the evaluation of DILI potential. Following ABE treatment, oxidative stress, mitochondrial damage, cytokine secretion levels, apoptotic/necrotic cell death were determined. According to our results, ROS production along with GSH depletion was observed in HepG2 cells after ABE treatment. ABE promoted secretion of pro-inflammatory mediators (TNF-α and MCP-1) and declined anti-inflammatory cytokine IL-10 release. Besides, NFKβ and JNK1 protein expression levels increased following ABE treatment. ABE enhanced intracellular calcium levels, induced early apoptotic and necrotic cell deaths in HepG2 cells. Furthermore, the changes in some mitochondrial parameters including a reducement in intracellular ATP levels and complex V activity; hyperpolarized mitochondrial membrane potential and enhanced mitochondrial ROS levels were observed, whereas mitochondrial mass did not show any differences after ABE treatments. Therefore, ABE-induced hepatotoxic effects is probably via oxidative stress, inflammatory response and necrotic cell death rather than direct mitochondrial toxicity. In conclusion; the study makes a significant contribution to strengthening the infrastructure we have on in vitro toxicity mechanism evaluations, which are the basis of preclinical toxicity studies.

Plasticity occurs in a specific phenotype of neurons in the nucleus tractus solitarius of dystrophin gene-mutated rats.

Duchenne muscular dystrophy (DMD) is a severe progressive neuromuscular disorder that causes cardiac and respiratory failure. Patients with DMD have tachycardia and autonomic nervous dysfunction at a young age, which can potentially worsen cardiorespiratory function. Therefore, we hypothesised that plasticity occurs in neurons of the cardiorespiratory brainstem nucleus (nucleus tractus solitarius [NTS]) due to DMD, thus affecting neuronal regulation because afferent information from cardiorespiratory organs changes with disease progression. Patch-clamp experiments were performed on second-order NTS neurons from Dmd-mutated (Dm) rats that showed no functional dystrophin protein expression, as confirmed by immunohistochemistry. NTS neurons are classified into two electrophysiological phenotypes: one showing a delayed onset of spiking from hyperpolarised membrane potentials, namely, delayed-onset spiking (DS)-type neurons, and the other showing a rapid onset, namely, rapid-onset spiking-type neurons. Neuroplasticity mainly occurs in DS-type neurons in Dm rats and is characterised by blunted neuronal excitability accompanied by reduced outward currents and a facilitatory effect on synaptic transmission, that is, an increased frequency of spontaneous and miniature excitatory postsynaptic currents (EPSCs) without changes in the amplitude and an increased amplitude of tractus solitarius-evoked EPSCs without changes in the paired-pulse ratio. Although we cannot rule out the possibility that the neuroplastic changes observed in Dm rats were caused by dystrophin deficiency in the neurons themselves, the plasticity could be caused by cardiorespiratory deterioration and/or adaptation in DMD patients.

The roles of surround inhibition for the intrinsic function of the striatum, analyzed in silico.

The basal ganglia are important for action initiation, selection, and motor learning. The input level, the striatum, receives input preferentially from the cortex and thalamus and is to 95% composed of striatal projection neurons (SPNs) with sparse GABAergic collaterals targeting distal dendrites of neighboring SPNs, in a distance-dependent manner. The remaining 5% are GABAergic and cholinergic interneurons. Our aim here is to investigate the role of surround inhibition for the intrinsic function of the striatum. Large-scale striatal networks of 20 to 40 thousand neurons were simulated with detailed multicompartmental models of different cell types, corresponding to the size of a module of the dorsolateral striatum, like the forelimb area (mouse). The effect of surround inhibition on dendritic computation and network activity was investigated, while groups of SPNs were activated. The SPN-induced surround inhibition in distal dendrites shunted effectively the corticostriatal EPSPs. The size of dendritic plateau-like potentials within the specific dendritic segment was both reduced and enhanced by inhibition, due to the hyperpolarized membrane potential of SPNs and the reversal-potential of GABA. On a population level, the competition between two subpopulations of SPNs was found to depend on the distance between the two units, the size of each unit, the activity level in each subgroup and the dopaminergic modulation of the dSPNs and iSPNs. The SPNs provided the dominating source of inhibition within the striatum, while the fast-spiking interneuron mainly had an initial effect due to short-term synaptic plasticity as shown in with ablation of the synaptic interaction.

The Complex Effects of PKM2 and PKM2:IP3R Disruption on Intracellular Ca2+ Handling and Cellular Functions.

Pyruvate kinase M (PKM) 2 was described to interact with the inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) and suppress its activity. To further investigate the physiological importance of the PKM2:IP3R interaction, we developed and characterized HeLa PKM2 knockout (KO) cells. In the HeLa PKM2 KO cells, the release of Ca2+ to the cytosol appears to be more sensitive to low agonist concentrations than in HeLa wild-type (WT) cells. However, upon an identical IP3-induced Ca2+ release, Ca2+ uptake in the mitochondria is decreased in HeLa PKM2 KO cells, which may be explained by the smaller number of contact sites between the ER and the mitochondria. Furthermore, in HeLa PKM2 KO cells, mitochondria are more numerous, though they are smaller and less branched and have a hyperpolarized membrane potential. TAT-D5SD, a cell-permeable peptide representing a sequence derived from IP3R1 that can disrupt the PKM2:IP3R interaction, induces Ca2+ release into the cytosol and Ca2+ uptake into mitochondria in both HeLa WT and PKM2 KO cells. Moreover, TAT-D5SD induced apoptosis in HeLa WT and PKM2 KO cells but not in HeLa cells completely devoid of IP3Rs. These results indicate that PKM2 separately regulates cytosolic and mitochondrial Ca2+ handling and that the cytotoxic effect of TAT-D5SD depends on IP3R activity but not on PKM2. However, the tyrosine kinase Lck, which also interacts with the D5SD sequence, is expressed neither in HeLa WT nor PKM2 KO cells, and we can also exclude a role for PKM1, which is upregulated in HeLa PKM2 KO cells, indicating that the TAT-D5SD peptide has a more complex mode of action than anticipated.

Dopaminergic Dependency of Cholinergic Pallidal Neurons.

We employed the whole-cell patch-clamp method and ChAT-Cre mice to study the electrophysiological attributes of cholinergic neurons in the external globus pallidus. Most neurons were inactive, although approximately 20% displayed spontaneous firing, including burst firing. The resting membrane potential, the whole neuron input resistance, the membrane time constant and the total neuron membrane capacitance were also characterized. The current-voltage relationship showed time-independent inward rectification without a "sag". Firing induced by current injections had a brief initial fast adaptation followed by tonic firing with minimal accommodation. Intensity-frequency plots exhibited maximal average firing rates of about 10Hz. These traits are similar to those of some cholinergic neurons in the basal forebrain. Also, we examined their dopamine sensitivity by acutely blocking dopamine receptors. This action demonstrated that the membrane potential, excitability, and firing pattern of pallidal cholinergic neurons rely on the constitutive activity of dopamine receptors, primarily D2-class receptors. The blockade of these receptors induced a resting membrane potential hyperpolarization, a decrease in firing for the same stimulus, the disappearance of fast adaptation, and the emergence of a depolarization block. This shift in physiological characteristics was evident even when the hyperpolarization was corrected with D.C. current. Neither the currents that generate the action potentials nor those from synaptic inputs were responsible. Instead, our findings suggest, that subthreshold slow ion currents, that require further investigation, are the target of this novel dopaminergic signaling.

The aggressiveness of succinate dehydrogenase subunit B-deficient chromaffin cells is reduced when their bioelectrical properties are restored by glibenclamide.

Pheochromocytomas/paragangliomas (PPGLs) are neuroendocrine tumours, mostly resulting from mutations in predisposing genes. Mutations of succinate dehydrogenase (SDH) subunit B (SDHB) are associated with high probability of metastatic disease. Since bioelectrical properties and signalling in cancer are an emerging field, we investigated the metabolic, functional and electrophysiological characteristics in human succinate dehydrogenase subunit B (SDHB)-deficient pheochromocytoma cells. These cells exhibited reduced SDH function with elevated succinate-to-fumarate ratio and reduced intracellular ATP levels. The analysis of membrane passive properties revealed a more hyperpolarized membrane potential and a lower cell capacitance of SDHB-deficient cells compared to the parental ones. These bioelectrical changes were associated with reduced proliferation and adhesion capacity of SDHB-deficient cells. Only in SDHB-deficient cells, we also observed an increased amplitude of potassium currents suggesting an activation of ATP-sensitive potassium channels (KATP). Indeed, exposure of the SDHB-deficient cells to glibenclamide, a specific KATP inhibitor, or to ATP caused normalization of potassium current features and altered proliferation and adhesion. In this work, we show for the first time that reduced intracellular ATP levels in SDHB-deficient chromaffin cells impaired cell bioelectrical properties, which, in turn, are associated with an increased cell aggressiveness. Moreover, we first ever demonstrated that glibenclamide not only reduced the outward potassium currents in SDHB-deficient cells but increased their growth capacity, reduced their ability to migrate and shifted their phenotype towards one more similar to that of parental one.

Activation and polarization of striatal microglia and astrocytes are involved in bradykinesia and allodynia in early-stage parkinsonian mice

In addition to the cardinal motor symptoms, pain is a major non-motor symptom of Parkinson's disease (PD). Neuroinflammation in the substantia nigra pars compacta and dorsal striatum is involved in neurodegeneration in PD. But the polarization of microglia and astrocytes in the dorsal striatum and their contribution to motor deficits and hyperalgesia in PD have not been characterized. In the present study, we observed that hemiparkinsonian mice established by unilateral 6-OHDA injection in the medial forebrain bundle exhibited motor deficits and mechanical allodynia. In these mice, both microglia and astrocytes in the dorsal striatum were activated and polarized to M1 / M2 microglia and A1 / A2 astrocytes as genes specific to these cells were upregulated. These effects peaked 7 days after 6-OHDA injection. Meanwhile, striatal astrocytes in parkinsonian mice also displayed hyperpolarized membrane potentials, enhanced voltage-gated potassium currents, and dysfunction in inwardly rectifying potassium channels and glutamate transporters. Systemic administration of minocycline, a microglia inhibitor, attenuated the expression of genes specific to M1 microglia and A1 astrocytes in the dorsal striatum (but not those specific to M2 microglia and A2 astrocytes), attenuated the damage in the nigrostriatal dopaminergic system, and alleviated the motor deficits and mechanical allodynia in parkinsonian mice. By contrast, local administration of minocycline into the dorsal striatum of parkinsonian mice mitigated only hyperalgesia. This study suggests that M1 microglia and A1 astrocytes in the dorsal striatum may play important roles in the development of pathophysiology underlying hyperalgesia in the early stages of PD.

P-096 Prediction of fertilization success by voltage-dependent intracellular Ca2+ dynamics in non-normozoospermic human semen samples undergoing in vitro fertilization: preliminary results

Abstract Study question Does the change in intracellular calcium levels evoked by artificial membrane depolarizations predict fertilization success in a non-normozoospermic semen sample undergoing assisted reproductive techniques? Summary answer The increase in the intracellular calcium after artificial plasma membrane depolarization correlates negatively with fertilization rates in conventional IVF but not with ICSI What is known already Sperm capacitation-related features, like the regulation of the intracellular calcium concentration ([Ca2+]i) and the hyperpolarization of the plasma membrane potential (Vm), have been proposed as predictors of success during artificial reproduction techniques (ART). Intriguingly, upon Vm depolarization, sperm exhibit a rapid and transitory increase in the [Ca2+]i controlled, likely, by the activation of the sperm-specific calcium channel CatSper. The regulation of CatSper activity is essential for sperm function and is crucial for fertilization. Therefore we wondered if the Vm depolarization-dependent rise in the [Ca2+]i can be used to predict fertilization success rates in non-normozoospermic semen samples of men undergoing ART. Study design, size, duration This prospective study employed unidentified semen samples from 29 non-normozoospermic men (37.8 ± 5.6 years old) undergoing fertility treatment from March 2022 to the present at CITMER Reproductive Medicine, Mexico City. Frozen semen samples and procedures with less than 3 oocytes were excluded. Ten normozoospermic semen samples were analyzed as a control. The depolarization-dependent [Ca2+]i changes were evaluated by flow cytometry under four different Vm conditions: resting (variable among samples), –64, –46, and –32 mV. Participants/materials, setting, methods Semen samples were obtained on the same day as oocyte retrieval. Sperm cells were washed and incubated in capacitating HTF media for further use in conventional IVF or ICSI procedures. When available, the surplus cells were stained with vitality, Ca2+, and Vm-sensitive fluorescent dyes. Fluorescence changes were evaluated by AccuriC6 flow cytometer. The kinetical analysis of the transitory [Ca2+]i increase as well as the statistical comparisons were performed using custom-made R programming language code. Main results and the role of chance Analysis of resting Vm showed that normozoospermic semen samples presented more depolarized Vm than non-normozoospermic controls (–75.7 ± 6.9 vs –67.3 ± 10.5 mV, respectively, p = 0.02). The former group of semen samples was either used for conventional IVF (n = 11) or ICSI (n = 14). The semen samples in these last categories showed a tendency to present a more depolarized Vm than non-normozoospermic samples (–69.9 ± 8.8 and –66.6 ± 11.3 mV, respectively). No differences were seen in the resting Ca2+ levels. We then induced a Vm hyperpolarization by adding the K+ ionophore, valinomycin followed by increasing concentrations of extracellular K+. Each depolarizing stimulus evoked a fast and transitory increase in the [Ca2+]i. We then calculated seven different kinetic parameters of such responses: basal Ca2+ level, increase and decrease velocity, maximum Ca2+ reached, response durability, and area under the curve (AUC). The basal Ca2+ level reached after depolarizing stimuli correlated negatively (R = –0.64, p = 0.03) with the conventional IVF fertilization ratio (number of zygotes/total number of mature oocytes) when the Vm was clamped either to –64 and –46 mV. A negative and significant correlation (R = –0.76, p = 0.006) was seen for the AUC analysis at –64 mV. No relevant correlations were found in the ICSI samples. Limitations, reasons for caution This is an in vitro study, and caution must be taken when extrapolating these results in vivo. The preliminary nature of the results implies a necessity to increase the number of analyzed semen samples (in progress). Wider implications of the findings Further research into the molecular regulation of the CatSper channel could offer promising alternatives for clinicians to appropriately choose between IVF and ICSI for each couple. This protocol is fast and simple to perform in a laboratory equipped with a flow cytometer or any fluorescence-based reader. Trial registration number NA

Energy stress modulation of AMPK/FoxO3 signaling inhibits mitochondria-associated ferroptosis

Cancer cells and ischemic diseases exhibit unique metabolic responses and adaptations to energy stress. Forkhead box O 3a (FoxO3a) is a transcription factor that plays an important role in cell metabolism, mitochondrial dysfunction and oxidative stress response. Although the AMP-activated protein kinase (AMPK)/FoxO3a signaling pathway plays a pivotal role in maintaining energy homeostasis under conditions of energy stress, the role of AMPK/FoxO3a signaling in mitochondria-associated ferroptosis has not yet been fully elucidated. We show that glucose starvation induced AMPK/FoxO3a activation and inhibited ferroptosis induced by erastin. Inhibition of AMPK or loss of FoxO3a in cancer cells under the glucose starvation condition can sensitize these cells to ferroptosis. Glucose deprivation inhibited mitochondria-related gene expression, reduced mitochondrial DNA(mtDNA) copy number, decreased expression of mitochondrial proteins and lowered the levels of respiratory complexes by inducing FoxO3a. Loss of FoxO3a promoted mitochondrial membrane potential hyperpolarization, oxygen consumption, lipid peroxide accumulation and abolished the protective effects of energy stress on ferroptosis in vitro. In addition, we identified a FDA-approved antipsychotic agent, the potent FoxO3a agonist trifluoperazine, which largely reduced ferroptosis-associated cerebral ischemia-reperfusion (CIR) injuries in rats through AMPK/FoxO3a/HIF-1α signaling and mitochondria-dependent mechanisms. We found that FoxO3a binds to the promoters of SLC7A11 and reduces CIR-mediated glutamate excitotoxicity through inhibiting the expression of SLC7A11. Collectively, these results suggest that energy stress modulation of AMPK/FoxO3a signaling regulates mitochondrial activity and alters the ferroptosis response. The regulation of FoxO3a by AMPK may play a crucial role in mitochondrial gene expression that controls energy balance and confers resistance to mitochondria-associated ferroptosis and CIR injuries.

Acid-sensing ion channel 1a regulates mitochondrial membrane potential and pulmonary arterial smooth muscle cell apoptosis

Acid-sensing ion channel 1a (ASIC1a) is a proton-gated cation channel that contributes to the development of chronic hypoxia (CH)-induced pulmonary hypertension (PH). Our prior studies demonstrate that ASIC1a is expressed in pulmonary arterial smooth muscle cells (PASMCs) and contributes to plasmalemmal membrane potential ( E m ) depolarization and augmented intracellular Ca 2+ signaling leading to enhanced vasoconstrictor reactivity and vascular remodeling. During PH, mitochondrial dysfunction associated with mitochondrial membrane potential (Δψm) hyperpolarization can inhibit opening of the mitochondrial permeability transition pore (mPTP), thereby repressing apoptosis and promoting the hyperproliferative and apoptosis-resistant PASMC phenotype. Recent evidence suggests that ASIC1a is localized to mitochondria and regulates the mPTP. However, it is unknown if mitochondrial-localized ASIC1a (mtASIC1a) contributes to the pathogenesis of PH. Therefore, the overall objective of this study is to determine the functional role of mtASIC1a in the apoptosis-resistant PASMC phenotype associated with PH. We hypothesized that CH causes Δψm hyperpolarization and apoptosis-resistance in PASMCs due to loss of mtASIC1a localization. To test this hypothesis, we housed Asic1a knockout ( Asic1a -/- ) and wild-type ( Asic1a +/+ ) mice in a hypobaric chamber (barometric pressure: ~380 mmHg) for 4 weeks to induce PH. Following CH, we found increased expression of ASIC1a at the plasma membrane and decreased expression in mitochondria, which were associated with PASMC E m depolarization and Δψm hyperpolarization. Using tetramethylrhodamine (TMRE) fluorescence to measure Δψm, we found that TMRE fluorescence was significantly increased (p=0.02) in PASMCs from normoxic Asic1a -/- mice compared to Asic1a +/+ mice, indicating that deletion of Asic1a leads to Δψm hyperpolarization. We utilized a lentiviral vector encoding mitochondrial-targeted human ASIC1a (mtASIC1a LV) to overexpress mtASIC1a in PASMCs (p=0.003) and found that transduction with mtASIC1a LV in PASMCs from Asic1a -/- mice significantly decreased TMRE fluorescence (p=0.004), further supporting the functional role of mtASIC1a to regulate Δψm. To assess apoptosis, we measured levels of active caspase-3 by western blot analysis in intrapulmonary arteries (PAs) from Asic1a +/+ and Asic1a -/- mice following CH. CH decreased levels of active caspase-3 in PAs from Asic1a +/+ (p=0.032), but not Asic1a -/- mice. Under normoxic conditions, active caspase-3 was lower in PAs from Asic1a -/- compared to Asic1a +/+ mice (p=0.044). These data suggest mtASIC1a plays an important role in the regulation of Δψm and that the loss of mtASIC1a contributes to Δψm hyperpolarization and apoptosis resistance in PASMCs following CH. Future studies will examine the contributions of mtASIC1a to the pathogenesis of PH through the regulation of mitochondrial reactive oxygen species, mitochondrial Ca 2+ levels, mPTP sensitivity, and mitochondrial dynamics. NIH R01 HL111084 to N.L. Jernigan and NIH T32 HL007736 to T.C. Resta This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Compartment-specific dendritic information processing in striatal cholinergic interneurons is reconfigured by peptide neuromodulation

Cholinergic interneurons are central hubs of the striatal neuronal network, controlling information processing in a behavioral-state-dependent manner. It remains unknown, however, how such state transitions influence the integrative properties of these neurons. To address this, we made simultaneous somato-dendritic recordings from identified rodent cholinergic interneurons, revealing that action potentials are initiated at dendritic sites because of a dendritic axonal origin. Functionally, this anatomical arrangement ensured that the action potential initiation threshold was lowest at axon-bearing dendritic sites, a privilege efficacy powerfully accentuated at the hyperpolarized membrane potentials achieved in cholinergic interneurons following salient behavioral stimuli. Experimental analysis revealed the voltage-dependent attenuation of the efficacy of non-axon-bearing dendritic excitatory input was mediated by the recruitment of dendritic potassium channels, a regulatory mechanism that, in turn, was controlled by the pharmacological activation of neurokinin receptors. Together, these results indicate that the neuropeptide microenvironment dynamically controls state- and compartment-dependent dendritic information processing in striatal cholinergic interneurons.