-
Anesthesiology Aug 2019Randomised Trial of Fentanyl Anesthesia in Preterm Babies Undergoing Surgery: Effects on the Stress Response. By Anand KJ, Sippell WG, and Aynsley-Green A. Lancet 1987;... (Randomized Controlled Trial)
Randomized Controlled Trial
Randomised Trial of Fentanyl Anesthesia in Preterm Babies Undergoing Surgery: Effects on the Stress Response. By Anand KJ, Sippell WG, and Aynsley-Green A. Lancet 1987; 1:243-8. Reprinted with permission.In a randomised controlled trial, preterm babies undergoing ligation of a patent ductus arteriosus were given nitrous oxide and D-tubocurarine, with (n = 8) or without (n = 8) the addition of fentanyl (10 μg/kg intravenously) to the anesthetic regimen. Major hormonal responses to surgery, as indicated by changes in plasma adrenaline, noradrenaline, glucagon, aldosterone, corticosterone, 11-deoxycorticosterone, and 11-deoxycortisol levels, in the insulin/glucagon molar ratio, and in blood glucose, lactate, and pyruvate concentrations were significantly greater in the nonfentanyl than in the fentanyl group. The urinary 3-methylhistidine/creatinine ratios were significantly greater in the nonfentanyl group on the second and third postoperative days. Compared with the fentanyl group, the nonfentanyl group had circulatory and metabolic complications postoperatively. The findings indicate that preterm babies mount a substantial stress response to surgery under anesthesia with nitrous oxide and curare and that prevention of this response by fentanyl anesthesia may be associated with an improved postoperative outcome.
Topics: Anesthetics, Intravenous; Ductus Arteriosus, Patent; Fentanyl; Humans; Infant, Newborn; Pain; Postoperative Complications; Stress, Physiological
PubMed: 31233407
DOI: 10.1097/ALN.0000000000002810 -
PloS One 2021Denervation reduces the abundance of Na+,K+-ATPase (NKA) in skeletal muscle, while reinnervation increases it. Primary human skeletal muscle cells, the most widely used...
Effect of differentiation, de novo innervation, and electrical pulse stimulation on mRNA and protein expression of Na+,K+-ATPase, FXYD1, and FXYD5 in cultured human skeletal muscle cells.
Denervation reduces the abundance of Na+,K+-ATPase (NKA) in skeletal muscle, while reinnervation increases it. Primary human skeletal muscle cells, the most widely used model to study human skeletal muscle in vitro, are usually cultured as myoblasts or myotubes without neurons and typically do not contract spontaneously, which might affect their ability to express and regulate NKA. We determined how differentiation, de novo innervation, and electrical pulse stimulation affect expression of NKA (α and β) subunits and NKA regulators FXYD1 (phospholemman) and FXYD5 (dysadherin). Differentiation of myoblasts into myotubes under low serum conditions increased expression of myogenic markers CD56 (NCAM1), desmin, myosin heavy chains, dihydropyridine receptor subunit α1S, and SERCA2 as well as NKAα2 and FXYD1, while it decreased expression of FXYD5 mRNA. Myotubes, which were innervated de novo by motor neurons in co-culture with the embryonic rat spinal cord explants, started to contract spontaneously within 7-10 days. A short-term co-culture (10-11 days) promoted mRNA expression of myokines, such as IL-6, IL-7, IL-8, and IL-15, but did not affect mRNA expression of NKA, FXYDs, or myokines, such as musclin, cathepsin B, meteorin-like protein, or SPARC. A long-term co-culture (21 days) increased the protein abundance of NKAα1, NKAα2, FXYD1, and phospho-FXYD1Ser68 without attendant changes in mRNA levels. Suppression of neuromuscular transmission with α-bungarotoxin or tubocurarine for 24 h did not alter NKA or FXYD mRNA expression. Electrical pulse stimulation (48 h) of non-innervated myotubes promoted mRNA expression of NKAβ2, NKAβ3, FXYD1, and FXYD5. In conclusion, low serum concentration promotes NKAα2 and FXYD1 expression, while de novo innervation is not essential for upregulation of NKAα2 and FXYD1 mRNA in cultured myotubes. Finally, although innervation and EPS both stimulate contractions of myotubes, they exert distinct effects on the expression of NKA and FXYDs.
Topics: Animals; Cell Differentiation; Cell Line; Cells, Cultured; Coculture Techniques; Electric Stimulation; Gene Expression Regulation; Humans; Ion Channels; Membrane Proteins; Microfilament Proteins; Muscle Contraction; Muscle Fibers, Skeletal; Muscle, Skeletal; Phosphoproteins; Rats; Sodium-Potassium-Exchanging ATPase
PubMed: 33635930
DOI: 10.1371/journal.pone.0247377 -
Archives of Toxicology Jun 2021Neonicotinoid pesticides, originally developed to target the insect nervous system, have been reported to interact with human receptors and to activate rodent neurons.... (Comparative Study)
Comparative Study
Neonicotinoid pesticides, originally developed to target the insect nervous system, have been reported to interact with human receptors and to activate rodent neurons. Therefore, we evaluated in how far these compounds may trigger signaling in human neurons, and thus, affect the human adult or developing nervous system. We used SH-SY5Y neuroblastoma cells as established model of nicotinic acetylcholine receptor (nAChR) signaling. In parallel, we profiled dopaminergic neurons, generated from LUHMES neuronal precursor cells, as novel system to study nAChR activation in human post-mitotic neurons. Changes of the free intracellular Ca concentration ([Ca]) were used as readout, and key findings were confirmed by patch clamp recordings. Nicotine triggered typical neuronal signaling responses that were blocked by antagonists, such as tubocurarine and mecamylamine. Pharmacological approaches suggested a functional expression of α7 and non-α7 nAChRs on LUHMES cells. In this novel test system, the neonicotinoids acetamiprid, imidacloprid, clothianidin and thiacloprid, but not thiamethoxam and dinotefuran, triggered [Ca] signaling at 10-100 µM. Strong synergy of the active neonicotinoids (at low micromolar concentrations) with the α7 nAChR-positive allosteric modulator PNU-120596 was observed in LUHMES and SH-SY5Y cells, and specific antagonists fully inhibited such signaling. To provide a third line of evidence for neonicotinoid signaling via nAChR, we studied cross-desensitization: pretreatment of LUHMES and SH-SY5Y cells with active neonicotinoids (at 1-10 µM) blunted the signaling response of nicotine. The pesticides (at 3-30 µM) also blunted the response to the non-α7 agonist ABT 594 in LUHMES cells. These data show that human neuronal cells are functionally affected by low micromolar concentrations of several neonicotinoids. An effect of such signals on nervous system development is a toxicological concern.
Topics: Calcium; Cell Line; Cell Line, Tumor; Dopaminergic Neurons; Dose-Response Relationship, Drug; Humans; Neonicotinoids; Neuroblastoma; Patch-Clamp Techniques; Pesticides; Receptors, Nicotinic; Signal Transduction
PubMed: 33778899
DOI: 10.1007/s00204-021-03031-1 -
Molecular Biology of the Cell Apr 2022Brain-specific angiogenesis inhibitor 1-associated protein 2-like protein 2 (BAIAP2L2), a membrane-binding protein required for the maintenance of mechanotransduction in...
Brain-specific angiogenesis inhibitor 1-associated protein 2-like protein 2 (BAIAP2L2), a membrane-binding protein required for the maintenance of mechanotransduction in hair cells, is selectively retained at the tips of transducing stereocilia. BAIAP2L2 trafficked to stereocilia tips in the absence of EPS8, but EPS8 increased the efficiency of localization. A tripartite complex of BAIAP2L2, EPS8, and MYO15A formed efficiently in vitro, and these three proteins robustly targeted to filopodia tips when coexpressed in cultured cells. Mice lacking functional transduction channels no longer concentrated BAIAP2L2 at row 2 stereocilia tips, a result that was phenocopied by blocking channels with tubocurarine in cochlear explants. Transduction channels permit Ca entry into stereocilia, and we found that membrane localization of BAIAP2L2 was enhanced in the presence of Ca. Finally, reduction of intracellular Ca in hair cells using BAPTA-AM led to a loss of BAIAP2L2 at stereocilia tips. Taken together, our results show that a MYO15A-EPS8 complex transports BAIAP2L2 to stereocilia tips, and Ca entry through open channels at row 2 tips retains BAIAP2L2 there.
Topics: Animals; Calcium; Hair Cells, Auditory; Mechanotransduction, Cellular; Membrane Proteins; Mice; Stereocilia
PubMed: 35044843
DOI: 10.1091/mbc.E21-10-0491 -
Nature Structural & Molecular Biology Apr 2022Binding of the neurotransmitter acetylcholine to its receptors on muscle fibers depolarizes the membrane and thereby triggers muscle contraction. We sought to understand...
Binding of the neurotransmitter acetylcholine to its receptors on muscle fibers depolarizes the membrane and thereby triggers muscle contraction. We sought to understand at the level of three-dimensional structure how agonists and antagonists alter nicotinic acetylcholine receptor conformation. We used the muscle-type receptor from the Torpedo ray to first define the structure of the receptor in a resting, activatable state. We then determined the receptor structure bound to the agonist carbachol, which stabilizes an asymmetric, closed channel desensitized state. We find conformational changes in a peripheral membrane helix are tied to recovery from desensitization. To probe mechanisms of antagonism, we obtained receptor structures with the active component of curare, a poison arrow toxin and precursor to modern muscle relaxants. d-Tubocurarine stabilizes the receptor in a desensitized-like state in the presence and absence of agonist. These findings define the transitions between resting and desensitized states and reveal divergent means by which antagonists block channel activity of the muscle-type nicotinic receptor.
Topics: Animals; Binding Sites; Curare; Muscles; Receptors, Nicotinic; Torpedo
PubMed: 35301478
DOI: 10.1038/s41594-022-00737-3 -
Journal of Clinical Medicine Oct 2020In many neurodegenerative and muscular disorders, and loss of innervation in sarcopenia, improper reinnervation of muscle and dysfunction of the motor unit (MU) are key...
BACKGROUND
In many neurodegenerative and muscular disorders, and loss of innervation in sarcopenia, improper reinnervation of muscle and dysfunction of the motor unit (MU) are key pathogenic features. studies of MUs are constrained due to difficulties isolating and extracting functional MUs, so there is a need for a simplified and reproducible system of engineered MUs.
OBJECTIVE
to develop and characterise a functional MU model , permitting the analysis of MU development and function.
METHODS
an immortalised human myoblast cell line was co-cultured with rat embryo spinal cord explants in a serum-free/growth fact media. MUs developed and the morphology of their components (neuromuscular junction (NMJ), myotubes and motor neurons) were characterised using immunocytochemistry, phase contrast and confocal microscopy. The function of the MU was evaluated through live observations and videography of spontaneous myotube contractions after challenge with cholinergic antagonists and glutamatergic agonists.
RESULTS
blocking acetylcholine receptors with α-bungarotoxin resulted in complete, cessation of myotube contractions, which was reversible with tubocurarine. Furthermore, myotube activity was significantly higher with the application of L-glutamic acid. All these observations indicate the formed MU are functional.
CONCLUSION
a functional nerve-muscle co-culture model was established that has potential for drug screening and pathophysiological studies of neuromuscular interactions.
PubMed: 33050427
DOI: 10.3390/jcm9103238 -
Journal of Neurophysiology Feb 2021Somatosensory input strength can be modulated by primary afferent depolarization (PAD) generated predominantly via presynaptic GABA receptors on afferent terminals. We...
Somatosensory input strength can be modulated by primary afferent depolarization (PAD) generated predominantly via presynaptic GABA receptors on afferent terminals. We investigated whether ionotropic nicotinic acetylcholine receptors (nAChRs) also provide modulatory actions, focusing on myelinated afferent excitability in in vitro murine spinal cord nerve-attached models. Primary afferent stimulation-evoked synaptic transmission was recorded in the deep dorsal horn as extracellular field potentials (EFPs), whereas concurrently recorded dorsal root potentials (DRPs) were used as an indirect measure of PAD. Changes in afferent membrane excitability were simultaneously measured as direct current (DC)-shifts in membrane polarization recorded in dorsal roots or peripheral nerves. The broad nAChR antagonist d-tubocurarine (d-TC) selectively and strongly depressed Aδ-evoked synaptic EFPs (36% of control) coincident with similarly depressed A-fiber DRP (43% of control), whereas afferent electrical excitability remained unchanged. In comparison, acetylcholine (ACh) and the nAChR agonists, epibatidine and nicotine, reduced afferent excitability by generating coincident depolarizing DC-shifts in peripheral axons and intraspinally. Progressive depolarization corresponded temporally with the emergence of spontaneous axonal spiking and reductions in the DRP and all afferent-evoked synaptic actions (31%-37% of control). Loss of evoked response was long-lasting, independent of DC repolarization, and likely due to mechanisms initiated by spontaneous C-fiber activity. DC-shifts were blocked with d-TC but not GABA receptor blockers and retained after tetrodotoxin block of voltage-gated Na channels. Notably, actions tested were comparable between three mouse strains, in rat, and when performed in different labs. Thus, nAChRs can regulate afferent excitability via two distinct mechanisms: by central Aδ-afferent actions, and by transient extrasynaptic axonal activation of high-threshold primary afferents. Primary afferents express many nicotinic ACh receptor (nAChR) subtypes but whether activation is linked to presynaptic inhibition, facilitation, or more complex and selective activity modulation is unknown. Recordings of afferent-evoked responses in the lumbar spinal cord identified two nAChR-mediated modulatory actions: ) selective control of Aδ afferent transmission and ) robust changes in axonal excitability initiated via extrasynaptic shifts in DC polarization. This work broadens the diversity of presynaptic modulation of primary afferents by nAChRs.
Topics: Animals; Ganglia, Spinal; Mice; Mice, Inbred BALB C; Neurons, Afferent; Nicotinic Agonists; Nicotinic Antagonists; Rats; Rats, Sprague-Dawley; Receptors, Nicotinic; Synaptic Potentials
PubMed: 33326305
DOI: 10.1152/jn.00228.2020 -
Toxins Nov 2020Gymnodimines and spirolides are cyclic imine phycotoxins and known antagonists of nicotinic acetylcholine receptors (nAChRs). We investigated the effect of gymnodimine A...
Gymnodimines and spirolides are cyclic imine phycotoxins and known antagonists of nicotinic acetylcholine receptors (nAChRs). We investigated the effect of gymnodimine A (GYM A) and 13-desmethyl spirolide C (SPX 1) from on rat pheochromocytoma (PC12) cells by monitoring intracellular calcium levels ([Ca]). Using whole cells, the presence of 0.5 µM of GYM A or SPX 1 induced an increase in [Ca] mediated by acetylcholine receptors (AChRs) and inhibited further activation of AChRs by acetylcholine (ACh). To differentiate the effects of GYM A or SPX 1, the toxins were applied to cells with pharmacologically isolated nAChRs and muscarinic AChRs (mAChRs) as mediated by the addition of atropine and tubocurarine, respectively. GYM A and SPX 1 activated nAChRs and inhibited the further activation of nAChRs by ACh, indicating that both toxins mimicked the activity of ACh. Regarding mAChRs, a differential response was observed between the two toxins. Only GYM A activated mAChRs, resulting in elevated [Ca] but both toxins prevented a subsequent activation by ACh. The absence of the triketal ring system in GYM A may provide the basis for a selective activation of mAChRs. GYM A and SPX 1 induced no changes in [Ca] when nAChRs and mAChRs were inhibited simultaneously, indicating that both toxins target AChRs.
Topics: Animals; Calcium; Calcium Channels; Calcium Signaling; Cell Line; Dinoflagellida; Heterocyclic Compounds, 4 or More Rings; Imines; Marine Toxins; Muscarinic Antagonists; Nicotinic Agonists; PC12 Cells; Rats; Receptors, Muscarinic; Receptors, Nicotinic; Spiro Compounds
PubMed: 33261221
DOI: 10.3390/toxins12120751 -
Scientific Reports Oct 2023Drug designing is high-priced and time taking process with low success rate. To overcome this obligation, computational drug repositioning technique is being promptly...
Drug designing is high-priced and time taking process with low success rate. To overcome this obligation, computational drug repositioning technique is being promptly used to predict the possible therapeutic effects of FDA approved drugs against multiple diseases. In this computational study, protein modeling, shape-based screening, molecular docking, pharmacogenomics, and molecular dynamic simulation approaches have been utilized to retrieve the FDA approved drugs against AD. The predicted MADD protein structure was designed by homology modeling and characterized through different computational resources. Donepezil and galantamine were implanted as standard drugs and drugs were screened out based on structural similarities. Furthermore, these drugs were evaluated and based on binding energy (Kcal/mol) profiles against MADD through PyRx tool. Moreover, pharmacogenomics analysis showed good possible associations with AD mediated genes and confirmed through detail literature survey. The best 6 drug (darifenacin, astemizole, tubocurarine, elacridar, sertindole and tariquidar) further docked and analyzed their interaction behavior through hydrogen binding. Finally, MD simulation study were carried out on these drugs and evaluated their stability behavior by generating root mean square deviation and fluctuations (RMSD/F), radius of gyration (Rg) and soluble accessible surface area (SASA) graphs. Taken together, darifenacin, astemizole, tubocurarine, elacridar, sertindole and tariquidar displayed good lead like profile as compared with standard and can be used as possible therapeutic agent in the treatment of AD after in-vitro and in-vivo assessment.
Topics: Humans; Drug Repositioning; Molecular Docking Simulation; Alzheimer Disease; Prognosis; Astemizole; Tubocurarine; Molecular Dynamics Simulation
PubMed: 37865690
DOI: 10.1038/s41598-023-45347-1