Voltage-gated Calcium Channels Research Articles

Abstract TP379: Post-stroke whole body vibration therapy alters transcriptome and reduces ischemic brain damage in reproductively senescent female rats.

Introduction: Rehabilitative physical therapy is essential for reducing stroke-related functional deficits; however, comorbidities may limit patient participation. Whole body vibration (WBV; 40 Hz) offers an exercise-like alternative. Our studies show that one month of post-stroke WBV reduces ischemic damage and improves motor and cognitive function in middle-aged rats. Notably, WBV significantly increased circulating irisin, a muscle-derived hormone. We hypothesize that post-stroke WBV modifies the cerebral transcriptome and irisin treatment improves stroke outcome in middle-aged female rats. Methods: Middle-aged Sprague-Dawley rats were randomized to sham or transient middle cerebral artery occlusion (tMCAO; 90 min) surgery and divided into two cohorts. A cohort received either no-WBV (steady platform) or WBV (platform vibrating at 40 Hz) for 15 minutes twice a day for a week. Cortical tissue was then collected for RNA sequencing (RNAseq) and gene enrichment analysis. The second cohort received either saline or irisin (PeproTech, 0.2 µg/g BW) treatment at 4.5 hours post-tMCAO and then once a week for a month. At 21 days post-tMCAO, rats were assessed for cognitive deficits via the Morris water maze. At 1-month post-tMCAO, brains were collected for histological analysis. Results: RNAseq revealed significant (p<0.05) differential expression of 581 genes in the cortex due to WBV. Specifically, there was downregulation (log 2 (fold change) = -2.9; p= 0.03943) of Calcium Voltage-Gated Channel Auxiliary Subunit Gamma 7 ( Cacng7 ) and calcium-release channel activity (GO:0015278), which are both implicated in excitotoxicity following stroke. The expression of Vacuolar Protein Sorting-Associated Protein 37C ( Vps37c ), a component of an endosomal sorting complex that mediates apoptosis, was also downregulated (log 2 (fold change) = -7.1; p= 0.01888). In our second cohort, rats treated with irisin for 1-month post-tMCAO, as compared to saline, demonstrated significantly (p<0.05) reduced cognitive deficits in spatial learning (hidden platform) and memory (probe trial). Additionally, irisin treatment significantly (p<0.05) reduced infarct damage at 1-month post-tMCAO. Conclusion: Post-stroke WBV increases the expression of genes responsible for ischemic tolerance and irisin treatment improves cognitive outcomes in middle-aged rats. Future studies are needed to understand the underlying mechanism(s) responsible for irisin-conferred ischemic protection.

Inactivation of CaV1 and CaV2 channels.

Voltage-gated Ca2+ channels (VGCCs) are highly expressed throughout numerous biological systems and play critical roles in synaptic transmission, cardiac excitation, and muscle contraction. To perform these various functions, VGCCs are highly regulated. Inactivation comprises a critical mechanism controlling the entry of Ca2+ through these channels and constitutes an important means to regulate cellular excitability, shape action potentials, control intracellular Ca2+ levels, and contribute to long-term potentiation and depression. For CaV1 and CaV2 channel families, inactivation proceeds via two distinct processes. Voltage-dependent inactivation (VDI) reduces Ca2+ entry through the channel in response to sustained or repetitive depolarization, while Ca2+-dependent inactivation (CDI) occurs in response to elevations in intracellular Ca2+ levels. These processes are critical for physiological function and undergo exquisite fine-tuning through multiple mechanisms. Here, we review known determinants and modulatory features of these two critical forms of channel regulation and their role in normal physiology and pathophysiology.

P-128. Evaluating dopamine agonists to confer colonization resistance against bacterial enteric infection: population-based propensity-matched cohort study

Abstract Background Uncovering gut microbiota mechanisms can afford new preventive therapies against enteric infection amidst the antimicrobial resistance crisis. Recent groundbreaking work in mice models suggest microbial-mediated metabolism of diet-derived tryptophan helps confer colonization resistance against enteric pathogens via agonism of dopamine receptors. This study aims to assess the risk of bacterial enteric infection in restless leg syndrome patients using a dopamine agonist (ropinirole) compared to a voltage-gated calcium channel modulator (gabapentin).Table 1:Characteristics of the ropinirole and gabapentin cohorts before and after propensity score matching Methods The TriNetX Analytics Platform containing 89 million unique patients from 63 health care organizations was used. These data represent 27% of the US population from all 50 states, and includes persons from diverse geographic, age, race, ethnicity, income, and insurance groups. Persons >18 years of age diagnosed with restless leg syndrome and prescribed ropinirole or gabapentin between Jan 2018- Jan 2023 were included as the study and control cohort, respectively. Individuals who received both ropinirole and gabapentin were excluded. The two cohorts were propensity score matched for age, sex, race, ethnicity, social determinants of health, obesity, ischemic heart disease, type 2 diabetes mellitus, hypertension, hyperlipidemia, human immunodeficiency virus and organ transplant status. Using the matched cohorts, we calculated the relative risk of first-ever and recurrent bacterial enteric infection 1-year after index events in the respective cohorts. Results We identified 38,122 and 82,961 persons with restless leg syndrome receiving ropinirole or gabapentin, respectively. After 1:1 propensity matching, demographic and clinical characteristics were balanced (SD < 0.1) (Table 1). Ropinirole compared to gabapentin use among persons with restless leg syndrome was associated with decreased 1-year risk of both recurrent (RR 0.85, 95% CI 0.74-0.98) and new-onset bacterial enteric infection (RR 0.77, 95% CI 0.65-0.92). Conclusion This study found that persons with restless leg syndrome receiving dopamine-agonist (ropinirole) compared to voltage-gated calcium channel modulator therapy (gabapentin) was associated with reduced risk of recurrent and new-onset bacterial enteric infection. Disclosures David C. Kaelber, MD, PhD, MPH, FAAP, FACP, FACMI, FAMIA, Dynavax Technologies Corporation: Advisor/Consultant

Biophysical modeling and experimental analysis of the dynamics of C. elegans body-wall muscle cells.

This study combines experimental techniques and mathematical modeling to investigate the dynamics of C. elegans body-wall muscle cells. Specifically, by conducting voltage clamp and mutant experiments, we identify key ion channels, particularly the L-type voltage-gated calcium channel (EGL-19) and potassium channels (SHK-1, SLO-2), which are crucial for generating action potentials. We develop Hodgkin-Huxley-based models for these channels and integrate them to capture the cells' electrical activity. To ensure the model accurately reflects cellular responses under depolarizing currents, we develop a parallel simulation-based inference method for determining the model's free parameters. This method performs rapid parallel sampling across high-dimensional parameter spaces, fitting the model to the responses of muscle cells to specific stimuli and yielding accurate parameter estimates. We validate our model by comparing its predictions against cellular responses to various current stimuli in experiments and show that our approach effectively determines suitable parameters for accurately modeling the dynamics in mutant cases. Additionally, we discover an optimal response frequency in body-wall muscle cells, which corresponds to a burst firing mode rather than regular firing mode. Our work provides the first experimentally constrained and biophysically detailed muscle cell model of C. elegans, and our analytical framework combined with robust and efficient parametric estimation method can be extended to model construction in other species.

Development of a Triethylborane-Mediated Giese Cyclization/Aldol Reaction Cascade for the Total Synthesis of Ganoapplanin

AbstractWe present our synthetic endeavors towards the Ganoderma meroterpenoid ganoapplanin. This natural product was isolated from a Ganoderma fungus in 2016 and was found to be an inhibitor for T-type voltage-gated calcium channels. Our synthetic approach is based on a powerful intramolecular Giese cyclization/intermolecular aldol cascade to link the northern aromatic to the southern terpenoid fragment. This article highlights the synthetic studies that ultimately led to the successful development of the key cascade reaction, culminating in the first total synthesis of ganoapplanin.1 Introduction2 Synthesis of the Southern Terpenoid Fragment3 Synthesis of the Northern Terpenoid Fragment4 Triethylborane-Mediated Giese Cyclization/Aldol Reaction Cascades5 Completion of the Total Synthesis of Ganoapplanin6 Conclusion

Voltage-Gated Calcium Channels and the Parity-Dependent Differential Uterine Response to Oxytocin in Rats.

The experience of pregnancy affects uterine function well beyond delivery. We previously demonstrated that the response to oxytocin is more robust in the uteri of proven breeder rats. This study investigates the contribution of T-type calcium channels (TTCCs) and L-type calcium channels (LTCCs) to the distinct response of virgin (V) and proven breeder (PB) rat uteri to oxytocin. Dose-inhibition responses to mibefradil (TTCC inhibitor) and verapamil (LTCC inhibitor) were conducted on isolated V and PB uterine strips. These experiments were followed by dose-response curves to oxytocin (10-10 to 10-5M) in the presence of 10µM of each inhibitor. Area-under-the-curve (AUC), amplitude, frequency, and duration of contractions were measured. V uteri generally showed a greater dependence on VGCCs, especially TTCCs. However, PB uteri exhibited a stronger frequency response to oxytocin. Blocking TTCCs had a more pronounced impact on the differential oxytocin response, particularly affecting the frequency component of contractions. The stronger frequency response in PB uteri may be due to a higher concentration of TTCCs in their myometrial pacemaker cells. This study provides supporting evidence that pregnancy induces lasting changes in uterine calcium handling. Our findings suggest that TTCCs play a more important role than LTCC in the parity-dependent differential response to oxytocin. The impact of ORAI and TRP channels still needs to be evaluated, to gain a more comprehensive understanding of the relative impact of voltage-gated calcium channels vs. storage-operated calcium entry channels on this phenomenon.

Thymol and Carvacrol as Potential Tocolytic and Anti-inflammatory Agents in Pregnant Rat Uterus.

This work aimed to evaluate the anti-inflammatory and myorelaxant effect of thymol (TM) and carvacrol (CAR) in the pregnant rat uterus. Both compounds exhibit considerable antimicrobial, antispasmodic, and anti-inflammatory effects and due to these properties, they were studied in this in vitro model of premature birth induced by infection. All uterine tissues were studied in uterine contraction tests to determine the inhibitory effect of TM, CAR (10, 56, 100, 150, and 230 μM), and nifedipine (a calcium channel antagonist) on phasic and tonic contraction induced by electro- and pharmacomechanical stimuli. The quantitative determination of cyclic adenosine monophosphate (cAMP) induced by TM and CAR in the uterine lysate was carried out by ELISA. For the determination of the anti-inflammatory effect of TM, the pro-inflammatory cytokine, interleukin (IL)-1β, in uterine samples stimulated with lipopolysaccharide (LPS) was measured. Forskolin (FSK) was used as a positive control to evaluate the cAMP and cytokine levels. TM, CAR, and nifedipine inhibited the uterine contractions at the highest concentration level, however, nifedipine was the most equipotent (p<0.05). In addition, TM and CAR did not increase the intracellular cAMP production in comparison with FSK (p<0.05). However, both compounds were able to decrease the LPS-induced production in a concentration-dependent manner that was considered statistically significant (p>0.05). Finally, both the anti-inflammatory and uterine relaxing effects induced by TM and CAR were neither associated with the increase in cAMP levels nor with the production of IL-1β in pregnant rat uterine samples. Therefore, TM and CAR can be considered as alternative adjuvants for the treatment of infection-induced preterm labor. Before the in vitro experiments, an in-silico analysis was conducted using the Expaisy online server to evaluate the biological effects of thymol on uterine contraction. It is crucial to know the interaction and identification of genes encoding the Voltage-dependent L-type calcium channel subunit alpha-1C proteins, because of the functional relationship it may have in the inhibition of the uterine contraction. These properties place TM as a potentially safe and effective adjuvant agent in cases of preterm birth, an area of pharmacological treatment that requires urgent improvement.

Transcriptomic and Functional Landscape of Adult Human Spinal Cord NSPCs Compared to iPSC-Derived Neural Progenitor Cells.

The adult human spinal cord harbors diverse populations of neural stem/progenitor cells (NSPCs) essential for neuroregeneration and central nervous system repair. While induced pluripotent stem cell (iPSC)-derived NSPCs offer significant therapeutic potential, understanding their molecular and functional alignment with bona fide spinal cord NSPCs is crucial for developing autologous cell therapies that enhance spinal cord regeneration and minimize immune rejection. In this study, we present the first direct transcriptomic and functional comparison of syngeneic adult human NSPC populations, including bona fide spinal cord NSPCs and iPSC-derived NSPCs regionalized to the spinal cord (iPSC-SC) and forebrain (iPSC-Br). RNA sequencing analysis revealed distinct transcriptomic profiles and functional disparities among NSPC types. iPSC-Br NSPCs exhibited a close resemblance to bona fide spinal cord NSPCs, characterized by enriched expression of neurogenesis, axon guidance, synaptic signaling, and voltage-gated calcium channel activity pathways. Conversely, iPSC-SC NSPCs displayed significant heterogeneity, suboptimal regional specification, and elevated expression of neural crest and immune response-associated genes. Functional assays corroborated the transcriptomic findings, demonstrating superior neurogenic potential in iPSC-Br NSPCs. Additionally, we assessed donor-specific influences on NSPC behavior by analyzing gene expression and differentiation outcomes across syngeneic populations from multiple individuals. Donor-specific factors significantly modulated transcriptomic profiles, with notable variability in the alignment of iPSC-derived NSPCs to bona fide spinal cord NSPCs. Enrichment of pathways related to neurogenesis, axon guidance, and synaptic signaling varied across donors, highlighting the impact of genetic and epigenetic individuality on NSPC behavior.

Systematic review of the impact of calcium channel blockers on sperm function.

This study explores the effects of calcium channel blockers (CCBs) on sperm function, a critical aspect of male fertility. Male infertility, responsible for 30-50 % of infertility cases, often involves issues with sperm motility and capacitation, both of which are heavily influenced by calcium ions and specific ion channels like CatSper and voltage-dependent calcium channels (VDCCs). CCBs, which are commonly prescribed for cardiovascular conditions, inhibit these calcium channels, potentially disrupting sperm function. A comprehensive literature review showed varied results: some studies demonstrated that CCBs such as nifedipine reduce sperm motility and the acrosome reaction, causing reversible infertility, while others found no significant impact on fertilization rates in IVF treatments. Supporting these findings, animal studies indicated that CCBs impair spermatogenesis and sperm function without necessarily affecting hormonal levels. The research suggests that the impact of CCBs on male fertility might be reversible, emphasizing the need for more extensive in vivo studies to further understand these effects and their clinical significance for patients on CCB therapy.

GABAB Receptor Modulation of Membrane Excitability in Human Pluripotent Stem Cell-Derived Sensory Neurons by Baclofen and α-Conotoxin Vc1.1.

GABAB receptor (GABABR) activation is known to alleviate pain by reducing neuronal excitability, primarily through inhibition of high voltage-activated (HVA) calcium (CaV2.2) channels and potentiating G protein-coupled inwardly rectifying potassium (GIRK) channels. Although the analgesic properties of small molecules and peptides have been primarily tested on isolated murine dorsal root ganglion (DRG) neurons, emerging strategies to develop, study, and characterise human pluripotent stem cell (hPSC)-derived sensory neurons present a promising alternative. In this study, hPSCs were efficiently differentiated into peripheral DRG-induced sensory neurons (iSNs) using a combined chemical and transcription factor-driven approach via a neural crest cell intermediate. Molecular characterisation and transcriptomic analysis confirmed the expression of key DRG markers such as BRN3A, ISLET1, and PRPH, in addition to GABABR and ion channels including CaV2.2 and GIRK1 in iSNs. Functional characterisation of GABABR was conducted using whole-cell patch clamp electrophysiology, assessing neuronal excitability under current-clamp conditions in the absence and presence of GABABR agonists baclofen and α-conotoxin Vc1.1. Both baclofen (100 μM) and Vc1.1 (1 μM) significantly reduced membrane excitability by hyperpolarising the resting membrane potential and increasing the rheobase for action potential firing. In voltage-clamp mode, baclofen and Vc1.1 inhibited HVA Ca2+ channel currents, which were attenuated by the selective GABABR antagonist CGP 55845. However, modulation of GIRK channels by GABABRs was not observed in the presence of baclofen or Vc1.1, suggesting that functional GIRK1/2 channels were not coupled to GABABRs in hPSC-derived iSNs. This study is the first to report GABABR modulation of membrane excitability in iSNs by baclofen and Vc1.1, highlighting their potential as a future model for studying analgesic compounds.

Unexpected effects of old drugs – antihypoxic properties of lactulose

Faced with soaring costs of developing new drugs, researchers are looking for ways to repurpose old ones, that is «...the most fruitful basis for discovering a new drug is to start with an old drug». Lactulose is a well-known synthetic sugar used to treat constipation. It is not absorbed in the small intestine and is not broken down by human enzymes. Lactulose is metabolized in the colon by bacterial flora to short-chain fatty acids, monosaccharides, lactic and acetic acids, volatile fatty acids, hydrogen and methane. The problem is that all of this happens in the colon. Could something similar happen in Krebs solution under in vitro conditions?The aim of study – to test experimentally the hypothesis about the possible antihypoxic effect of the drug lactudose in vitro. However lactulose unexpectedly demonstrated a clear ability to restore spontaneous activity of the rat portal vein inhibited under hypoxia in vitro. Lactulose effectively restores the amplitude of vascular contractions caused by field electrical stimulation that was inhibited by hypoxia. When lactulose was added to the organ bath both before hypoxic exposure and electrical stimulation, stimulation caused a paradoxical increase in isometric tension. Preliminary data obtained suggest that the plasma membrane of vascular smooth muscle cells may primarily participate, and the mechanisms of energy supply to the contractile apparatus do not take such a large part in the response to hypoxia. The experiments showed, that one of the most vulnerablel inks in the excitation-contraction coupling chain in vascular tissues during hypoxia is the system of passive transmembrane Ca2+ transport. Probably, sugars that are not hydrolyzed by a living cell can have biological effects during hypoxia that are not related to their substrate influence. As an object of study, we focused on the disaccharide lactulose, which contains fructose and galactose. In the body, outside the intestines, there are no enzymes that could metabolize this bond and hydrolyze this compound. It seems that if lactulose has an antihypoxic effect, then this effect will depend exclusively on non-metabolic factors, which will make a significant contribution to the understanding of the fundamental mechanisms of the development of hypoxic conditions. The voltage-sensing domain of voltage-gated ion channels is a protein structure important for membrane voltage sensing that is characterized by specific, conserved, and charged amino acid residues. It is generally assumed that molecules are neutral and do not carry unpaired electrons. Charged molecules are called molecular ions, molecules with a multiplicity other than unity (i. e., with unpaired electrons and unsaturated valences). Thus, under certain changes in environmental conditions (for example, a change in pH), a previously neutral molecule can become electrically charged and thus acquire the ability to interact with a potential sensor in ion channels. Interestingly, even if a carbohydrate does not carry a formal charge, when bound it will change the dielectric environment, since the region previously filled with water (in the absence of significant conformational changes) will be occupied by an environment with a much lower dielectric constant. Thus, carbohydrates can act as universal electrostatic modulators of protein function, even if their binding causes little or no conformational change in the protein molecule. In conclusion, it should be noted, the fact that lactulose has antihypoxic properties is beyond doubt. From a clinician's point of view, this is already good. The compound is well known and completely harmless. But pharmacology is the science of the mechanisms of action of chemicals on living systems, and therefore we are obliged to propose such mechanisms. If not absolutely demonstrative, then at least theoretically acceptable. A possible metabolic shift towards glycolysis would be a good potential candidate for evidence, but is currently difficult to substantiate. It remains only to assume the possible interaction of the electrically modified (under conditions of hypoxia and associated acidosis) lactulose molecule with voltage-dependent calcium channels and the restoration of their activity, suppressed during hypoxia.

Voltage-dependent calcium channels in mammalian motor synapses – triggers and modulators of neuromuscular transmission

The initiation of fast synchronous quantal release of neurotransmitters in central and peripheral synapses is ensured by a local increase in the concentration of Ca2+ ions in the nerve terminals near the Ca2+ sensors of synaptic vesicles in response to depolarization of the presynaptic membrane by an action potential (AP) propagating along the axon. The Ca2+- entry from the outside through presynaptic voltage-dependent Ca2+ channels CaV2.1 or CaV2.2 (P/Q- or N-type) is the main way of forming a dynamic Ca2+ signal that initiates the process of exocytosis of synaptic vesicles in virtually all types of chemical synapses and is capable of inducing the development of certain Ca2+-dependent forms of synaptic plasticity. However, in recent years it has become obvious that the set of sources and the spectrum of presynaptic Ca2+ signals are very diverse. Identification of the ensemble of regulatory Ca2+-entries operating in combination with their corresponding targets, description of their contribution to the mechanisms controlling quantal release of neurotransmitter is a topical area of modern synaptic physiology. Among such additional to the trigger Ca2+-inputs, L-type Ca2+-channels are of particular interest. Their role and activation conditions in neuromuscular junctions (NMJs) are poorly studied and do not provide an unambiguous idea of the place of this Ca2+-entry in the regulation of acetylcholine (ACh) release in vertebrate motor synapses. This review systematizes the currently available research results on the diverse functional role of voltage-gated Ca2+-channels in mammalian NMJs and presynaptic signaling pathways that control these Ca2+-inputs and their participation in the processes of fine-tuning the ACh quantal release.

Association between CACNA1A and ATP1A2 Variants are Responsible for Severe Neurodevelopmental Disorder.

ATP1A2 and CACNA1A genes encode proteins forming transmembrane channels, Na+/K+/ATPase transporter, and voltage-gated calcium channels, respectively. Pathogenic variants in these genes are associated with hemiplegic migraines, movement disorders, and developmental and epileptic encephalopathy.We report a child presenting epileptic encephalopathy with cognitive and behavioral troubles. He carries a likely pathogenic variant in the ATP1A2 gene, inherited from his mother who presents hemiplegic migraines, and a variant of uncertain significance in the CACNA1A gene, inherited from his asymptomatic father and also found in his brother, who presents a milder neurodevelopmental disorder (NDD). No other significant copy number or single nucleotide variations were identified after an in-depth genetic study including whole exome sequencing, array comparative genomic hybridization, and screening for Fragile X and Prader-Willi/Angelman syndromes.We illustrate the synergetic impact of ATP1A2 and CACNA1A genes in NDDs.

Action Potential Firing Patterns Regulate Dopamine Release via Voltage-Sensitive Dopamine D2 Autoreceptors in Mouse Striatum In Vivo.

Dopamine (DA) in the striatum is vital for motor and cognitive behaviors. Midbrain dopaminergic neurons generate both tonic and phasic action potential (AP) firing patterns in behavior mice. Besides AP numbers, whether and how different AP firing patterns per se modulate DA release remain largely unknown. Here by using in vivo and ex vivo models, it is shown that the AP frequency per se modulates DA release through the D2 receptor (D2R), which contributes up to 50% of total DA release. D2R has a voltage-sensing site at D131 and can be deactivated in a frequency-dependent manner by membrane depolarization. This voltage-dependent D2R inhibition of DA release is mediated via the facilitation of voltage-gated Ca2+ channels (VGCCs). Collectively, this work establishes a novel mechanism that APs per se modulate DA overflow by disinhibiting the voltage-sensitive autoreceptor D2R and thus the facilitation of VGCCs, providing a pivotal pathway and insight into mammalian DA-dependent functions in vivo.

Evaluation of the anti-spasmodic activity of essential oils of Ammodaucus leucotrichus fruits and its main chemical component “perillaldehyde” on intestinal smooth muscle contractions of rodents: ex vivo and in silico approaches

Ethnopharmacological relevanceIn Moroccan traditional medicine, plants from the Apiaceae family are widely utilized in folk medicine to treat various diseases associated with the digestive system. Ammodaucus leucotrichus plays an important role as an antispasmodic that has been traditionally used, especially to treat digestive tract diseases in children.Aim of the studyThe aim of this research was to verify the traditional use by assessing the relaxant and spasmolytic activities of A. leucotrichus essential oil (ALEO) and then comparing them to the effects and potency of the major constituent of ALEO, which is perillaldehyde.Materials and methodsThe in vitro evaluation of ALEO’s relaxant and spasmolytic effects was carried out on isolated rats and rabbit jejunum in an organ bath setup. Intestinal contractility was recorded using an isotonic transducer connected to an amplifier. GC/MS analysis was conducted to identify components within ALEO. Subsequently, these compounds underwent in silico absorption, toxicity, and molecular docking studies.ResultsGC/MS analysis of this essential oil studied revealed seven compounds, which account for 98.67% of the oil, with the dominance of two compounds, namely, perillaldehyde (91.12%) and limonene (6.33%). ALEO and its main compound, perillaldehyde, reversibly relaxed the basal tone of rabbit jejunum, with the IC50 values 158.68 ± 13.89 and 95.03 ± 0.93 μg/mL, respectively. Moreover, ALEO caused a dose-dependent spasmolytic effect on Carbachol (CCh) and KCl provoked jejunum contraction in rats. Furthermore, the decrease in contractions of pre-contracted jejunum by CCh was more pronounced for perillaldehyde compared to ALEO, with an IC50 value of 68.59 ± 6.57 μg/mL, which was half compared to that of ALEO. The pre-treatment of the tissue with concentrations ranging from 30 to 100 μg/mL caused a rightward and downward shift in the concentration–response curves for CaCl2 and CCh. These results suggest that the spasmolytic effect of ALEO is mediated possibly through a non-competitive antagonist of calcium channel or muscarinic receptors. Our results are confirmed by the fact that perillaldehyde exhibited the highest docking scores on muscarinic acetylcholine receptors (M2 and M3) and voltage-gated calcium channels, with D-limonene showing lower binding energies in comparison. These remarks confirm that the activity of ALEO is attributed to the presence of perillaldehyde. In addition, perillaldehyde exhibits a low degree of in silico acute toxicity and high percent of intestinal absorption.ConclusionIn summary, ALEO exhibits myorelaxant and antispasmodic effects by inhibiting muscarinic receptors and calcium channels, which can be attributed to the presence of perillaldehyde. This provides a scientific foundation for the traditional use of A. leucotrichus in treating gastrointestinal disorders and opens up possibilities for developing a more effective and less toxic drug-utilizing perillaldehyde.

Dexmedetomidine suppresses glucose-stimulated insulin secretion in pancreatic β-cells.

Proper glycemic control is crucial for patient management in critical care, including perioperative care, and can influence patient prognosis. Blood glucose concentration determines insulin secretion and sensitivity and affects the intricate balance between the glucose metabolism. Human and other animal studies have demonstrated that perioperative drugs, including volatile anesthetics and intravenous anesthetics, affect glucose-stimulated insulin secretion (GSIS). Dexmedetomidine (DEX) decreases insulin release and affects glucose metabolism; however, the specific mechanism underlying this phenomenon remains largely unknown. Thus, we investigated the effect and mechanism of DEX on insulin secretion using mouse and rat pancreatic β-cell-derived MIN6 and INS-1 cell lines and primary pancreatic β-cells/islets extracted from mice. The amount of insulin secreted into the culture medium was determined using an enzyme-linked immunosorbent assay. Cell viability, cytotoxicity, and electrophysiological effects were investigated. Clinically relevant doses of DEX suppressed GSIS in MIN6 cells, INS-1 cells, and pancreatic β-cells/islets. Furthermore, DEX suppressed insulin secretion facilitated by insulinotropic factors. There was no significant difference in oxygen consumption rate, intracellular ATP levels, or caspase-3/7 activity. Electrophysiological evaluation using the patch-clamp method showed that DEX did not affect ATP-sensitive potassium (KATP) channels, voltage-dependent potassium channels, or voltage-gated calcium channels. We demonstrated that clinically relevant doses of DEX significantly suppressed GSIS. These findings suggest that DEX inhibits a signaling pathway via α2-adrenoceptor or insulin vesicle exocytosis, resulting in GSIS suppression. Our results support the hypothesis that DEX suppresses insulin secretion and reveal some underlying mechanisms.

CaV3.2 T-type calcium channels contribute to CGRP- induced allodynia in a rodent model of experimental migraine

BackgroundMigraine is a painful neurological syndrome characterized by attacks of throbbing headache, of moderate to severe intensity, which is associated with photo- and phono- sensitivity as well as nausea and vomiting. It affects about 15% of the world’s population being 2–3 times more prevalent in females. The calcitonin gene-related peptide (CGRP) is a key mediator in the pathophysiology of migraine, and a significant advance in the field has been the development of anti-CGRP therapies. The trigeminal ganglion (TG) is thought to be an important site of action for these drugs. Moreover, experimental migraine can be induced by CGRP injection in the TG. The signaling pathway induced by CGRP in the TG is not fully understood, but studies suggest that voltage-gated calcium channels contribute to CGRP effects relevant to migraine.ObjectiveWe hypothesised that CGRP injection in the TG enhances CaV3.2 T-type calcium channel currents to contribute to the development of periorbital mechanical allodynia.ResultsA Co-Immunoprecipitation assay in tsA-201 cells revealed that CaV3.2 channels form a complex with RAMP-1, a component of the CGRP receptor. Constitutive CGRPR activity was able to inhibit CaV3.2 channels and induce a depolarizing shift in both activation and inactivation curves. Incubation of TG neurons with CGRP increased T-type current density by ~ 3.6 fold, an effect that was not observed in TG neurons from CaV3.2 knockout mice. Incubation of TG neurons with Z944, a pan T-type channel blocker, resulted in an approximately 80% inhibition of T-type currents. In vivo, this treatment abolished the development of periorbital mechanical allodynia induced by CGRP in male and female mice. Likewise, CaV3.2 knockout mice did not develop periorbital mechanical allodynia after intraganglionic CGRP injection. Finally, we demonstrated that the CGRP effect depends on the activation of its canonical GPCR, followed by protein kinase A activation.ConclusionThe present study suggests that CGRP modulates CaV3.2 in the TG, an effect possibly mediated by the canonical CGRP receptor and PKA activation. The increase in T-type currents in the TG may represent a contributing factor for the initiation and maintenance of the headache pain during migraine.Graphical abstract

Peptide Lv and Angiogenesis: A Newly Discovered Angiogenic Peptide.

Peptide Lv is a small endogenous secretory peptide with ~40 amino acids and is highly conserved among certain several species. While it was first discovered that it augments L-type voltage-gated calcium channels (LTCCs) in neurons, thus it was named peptide "Lv", it can bind to vascular endothelial growth factor receptor 2 (VEGFR2) and has VEGF-like activities, including eliciting vasodilation and promoting angiogenesis. Not only does peptide Lv augment LTCCs in neurons and cardiomyocytes, but it also promotes the expression of intermediate-conductance KCa channels (KCa3.1) in vascular endothelial cells. Peptide Lv is upregulated in the retinas of patients with early proliferative diabetic retinopathy, a disease involving pathological angiogenesis. This review will provide an overview of peptide Lv, its known bioactivities in vitro and in vivo, and its clinical relevance, with a focus on its role in angiogenesis. As there is more about peptide Lv to be explored, this article serves as a foundation for possible future developments of peptide Lv-related therapeutics to treat or prevent diseases.

Estradiol elicits distinct firing patterns in arcuate nucleus kisspeptin neurons of females through altering ion channel conductances.

Hypothalamic kisspeptin (Kiss1) neurons are vital for pubertal development and reproduction. Arcuate nucleus Kiss1 (Kiss1ARH) neurons are responsible for the pulsatile release of gonadotropin-releasing hormone (GnRH). In females, the behavior of Kiss1ARH neurons, expressing Kiss1, neurokinin B (NKB), and dynorphin (Dyn), varies throughout the ovarian cycle. Studies indicate that 17β-estradiol (E2) reduces peptide expression but increases Slc17a6 (Vglut2) mRNA and glutamate neurotransmission in these neurons, suggesting a shift from peptidergic to glutamatergic signaling. To investigate this shift, we combined transcriptomics, electrophysiology, and mathematical modeling. Our results demonstrate that E2 treatment upregulates the mRNA expression of voltage-activated calcium channels, elevating the whole-cell calcium current that contributes to high-frequency burst firing. Additionally, E2 treatment decreased the mRNA levels of canonical transient receptor potential (TPRC) 5 and G protein-coupled K+ (GIRK) channels. When Trpc5 channels in Kiss1ARH neurons were deleted using CRISPR/SaCas9, the slow excitatory postsynaptic potential was eliminated. Our data enabled us to formulate a biophysically realistic mathematical model of Kiss1ARH neurons, suggesting that E2 modifies ionic conductances in these neurons, enabling the transition from high-frequency synchronous firing through NKB-driven activation of TRPC5 channels to a short bursting mode facilitating glutamate release. In a low E2 milieu, synchronous firing of Kiss1ARH neurons drives pulsatile release of GnRH, while the transition to burst firing with high, preovulatory levels of E2 would facilitate the GnRH surge through its glutamatergic synaptic connection to preoptic Kiss1 neurons.

Behavioral and genetic markers of susceptibility to escalate fentanyl intake.

The "loss of control" over drug consumption, present in opioid use disorder (OUD) and known as escalation of intake, is well-established in preclinical rodent models. However, little is known about how antecedent behavioral characteristics, such as valuation of hedonic reinforcers prior to drug use, may impact the trajectory of fentanyl intake over time. Moreover, it is unclear if distinct escalation phenotypes may be driven by genetic markers predictive of OUD susceptibility. Male and female Sprague-Dawley rats (n=63) were trained in a sucrose reinforcement task using a progressive ratio schedule. Individual differences in responsivity to sucrose were hypothesized to predict escalation of fentanyl intake. Rats underwent daily 1-h acquisition sessions for i.v. fentanyl self-administration (2.5 µg/kg; FR1) for 7 days, followed by 21 6-h escalation sessions, then tissue from prefrontal cortex was collected for RNA sequencing and qPCR. Latent growth curve and group-based trajectory modeling were used, respectively, to evaluate the association between sucrose reinforcement and fentanyl self-administration and to identify whether distinct escalation phenotypes can be linked to gene expression patterns. Sucrose breakpoints were not predictive of fentanyl acquisition nor change during escalation, but did predict fentanyl intake on the first day of extended access to fentanyl. Permutation analyses did not identify associations between behavior and single gene expression when evaluated overall, or between our ascertained phenotypes. However, weighted genome correlation network analysis (WGCNA) and gene set enrichment analysis (GSEA) determined several gene modules linked to escalated fentanyl intake, including genes coding for voltage-gated potassium channels, calcium channels, and genes involved in excitatory synaptic signaling. Transcription factor analyses identified EZH2 and JARID2 as potential transcriptional regulators associated with escalated fentanyl intake. Genome-wide association study (GWAS) term categories were also generated and positively associated with terms relating to substance use disorders. Escalation of opioid intake is largely distinct from motivation for natural reward, such as sucrose. Further, the gene networks associated with fentanyl escalation suggest that engagement of select molecular pathways distinguish individuals with "addiction prone" behavioral endophenotypes, potentially representing druggable targets for opioid use disorder. Our extended in silico identification of SNPs and transcription factors associated with the "addiction prone" high escalating rats highlights the importance of integrating findings from translational preclinical models. Through a precision medicine approach, our results may aid in the development of patient-centered treatment options for those with OUD.