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Study Design: Preclinical pilot study., Objectives: To explore peripheral and central nociceptive mechanisms that contribute to muscle stretch-induced locomotor deficits following spinal cord injury., Setting: Kentucky Spinal Cord Injury Research Center, Louisville, KY, USA., Methods: Ten female Sprague-Dawley rats received moderate, 25 g/cm T10 contusion injuries and recovered for 4 weeks. Rats were divided into three groups: Morphine/Ibuprofen-treated, Acetaminophen/Baclofen-treated, and saline control. Each group received daily hindlimb muscle stretching during weeks 4, 5, 9, and 10 post-injury and drugs were administered with stretching during weeks 4 and 9 only. Locomotor function was assessed throughout the experiment using the BBB Open Field Locomotor Scale. Hindlimb responses including spasticity, writhing, and clonic-like vibrations during muscle stretching were classified and scored., Results: Consistent with our previous studies, hindlimb muscle stretching caused significant deficits in locomotor recovery following spinal cord injury. Baclofen and Ibuprofen partially mitigated the stretching effect, but none of the drugs significantly prevented the drop in locomotor function during stretching. Interestingly, treatment with Baclofen or Ibuprofen significantly reduced hindlimb responses such as spasticity and writhing during stretching, while Morphine exacerbated clonic-like vibrations in response to stretching maneuvers., Conclusions: These findings suggest that stretching may inhibit locomotor recovery through combined mechanisms of peripheral inflammation and sensitization of nociceptive afferents. When combined with central sprouting and loss of descending controls after SCI, this results in exaggerated nociceptive input during stretching. The inability of the applied clinical drugs to mitigate the detrimental effects of stretching highlights the complexity of the stretching phenomenon and emphasizes the need for further investigation., (© 2024. The Author(s), under exclusive licence to International Spinal Cord Society.)

Competing Interests: Disclosures The authors have no conflicts of interest to disclose.

Traumatic brain injury (TBI) triggers a bevy of changes including mitochondrial dysfunction, apoptosis, oxidative stress, neurobehavioural impairment, and neuroinflammation, among others. Dantrolene (DNT), a muscle relaxant which inhibits intracellular Ca 2+ signaling from the ER, has been repurposed as a potential neuroprotective agent in various neurological diseases. However, there have been limited studies on whether it can mitigate TBI-induced deficits and restore impaired mitochondrial dynamics. This study sought to evaluate whether Dantrolene can potentially provide neuroprotection in an in vivo model of TBI. Male wistar rats subjected to TBI were treated with DNT (10 mg/kg) 1 h and 12 h post surgery. Animals were assessed 24 h post-TBI to evaluate neurobehavioural deficits and cerebral edema. We evaluated the protein expressions of apoptotic, autophagic, and neuroinflammatory markers by immunoblotting, as well as Mitochondrial Membrane Potential (MMP) and Reactive Oxygen Species (ROS) via Flow Cytometry to ascertain the effects of DNT on TBI. We further analysed immunofluorescence staining with Glial Fibrillary Acidic Protein (GFAP) and immunohistochemistry with NF-κβ to investigate neuroinflammation. H&E staining was also performed post-TBI. Our findings revealed DNT administration inhibits mitochondria-mediated apoptotis and reduces heightened oxidative stress. DNT treatment was also found to reverse neurobehavioural impairments and offer neuroprotection by preserving neuronal architechture. We also demonstrated that DNT inhibits neuronal autophagy and alleviates neuroinflammation following TBI by modulating the NF-κβ/Akt signaling pathway. Thus, our results suggest a novel application of DNT in ameliorating the multitude of deficits induced by TBI, thereby conferring neuroprotection., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Objective: To study the histological changes in lung of rats, evaluate their dynamics and determine the Bax and Bcl-2 genes expression in bronchial epithelium, alveolar type 1 cells and neutrophils at different times after the administration of baclofen., Material and Methods: The experiment was conducted on 20 mature (at the age of 20 weeks) male Wistar rats with a mass of 290-350 g, distributed in 4 groups (5 rats in each). Animals in the control group did not receive baclofen. Rats in experimental groups received baclofen at a dose of 85 mg/kg: in the 1st group, the experiment duration was 3 hours (time to maximum observed blood drug concentration); in the 2nd group - 4.5 h (drug half-life and time to maximum observed concentration of the main drug's metabolite - beta-[p-chlorophenyl])-gamma-hydroxybutyric acid in the blood); in the 3rd group - 24 h., Results: A complex of pathological reactions developed in lungs of experimental animals when baclofen muscle relaxant was administered, namely circulatory disturbances at all levels of the microvasculature (venular and capillary congestion, hemorrhages in the interalveolar septums, alveoli, sludge), emphysema, sites of which punctuated with atelectases and dystelectases. The complex of pathological changes in the lungs had a certain dynamics and reached its highest severity by the 24th hour. Bax expression was strong, while Bcl-2 expression was moderate in the immunohistochemical (IHC) study of bronchical epithelium and alveolar type 1 cells, Bax and Bcl-2 expression in neutrophils was moderate in rats of the 1st group. The expression of Bax and Bcl-2 was strong in the bronchial epithelium and alveolar type 1 cells, the expression of Bax in neutrophils was moderate, and BCL-2 - strong in animals of the 2nd group. The expression of Bax in bronchial epithelium and alveolar type 1 cells was moderate, expression of Bcl-2 in bronchial epithelium and alveolar type 1 cells - strong, expression of Bax in neutrophils - weak and expression of Bcl-2 - strong among the rats of the 3rd group., Conclusion: The complex of pathological changes in lungs had a certain dynamic. Data on the histological changes in lungs in combination with the results of the chemical study, can be used to diagnose the poisoning by baclofen and to establish the time since the drug was administered. The results obtained during the IHC study suggest the involvement of apoptosis in the development of lesion of bronchial epithelium and alveolar type 1 cells. In addition, the expression of Bcl-2 in epithelial cells may play a role in the process of their regeneration.

Eperisone Hydrochloride was launched in Japan in 1983 and has been used to improve muscle tone and treat spastic paralysis (Originator: Eisai Co., Ltd.). However, its biochemical mechanism of action is unknown. SB Drug Discovery was used to evaluate purinergic P2X (P2X) receptor antagonism using fluorescence. In this study, we discovered that its target protein is the P2X7 receptor. Also, P2X receptor subtype selectivity was high. This finding demonstrates the (Eperisone-P2X7-pain linkage), the validity of P2X7 as a drug target, and the possibility of drug repositioning of Eperisone Hydrochloride.

Pharmacology graduates require an understanding of both in vitro and in vivo drug responses but there has been a decline in animal use in pharmacology education over the last 30 years. To address this, we present the novel invertebrate model, Lumbriculus variegatus, for in vivo testing of drugs in a teaching environment. We have developed two novel behavioral assays: the stereotypical movement assay, which measures the effect of drugs on the ability of L. variegatus to perform stereotypical movements following tactile stimulation, and the free locomotion assay, which measures drug effects on unstimulated movement. We report the effects of compounds with diverse pharmacodynamic properties on L. variegatus using these assays. The ryanodine receptor antagonist, dantrolene, altered the unstimulated movement of L. variegatus at 5 μM, whereas stimulated movement was inhibited at ≥25 μM. Lidocaine, a voltage-gated sodium channel blocker, and quinine, a nonselective sodium and potassium channel blocker, reduced both stimulated and unstimulated L. variegatus movement at ≥0.5 mM. Inhibitory effects of quinine persisted for up to 24 h after drug removal, whereas lidocaine effects were reduced 10 min after drug removal. Herein, we provide proof-of-concept utilization of L. variegatus as an organism for use in in vivo pharmacology education but without regulatory constraints or the need for specialized equipment and training., (© 2021 The Authors. Pharmacology Research & Perspectives published by British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics and John Wiley & Sons Ltd.)

Background: Rho/ROCK signaling has been demonstrated to be involved in the vascular reactivity of many arterial networks. However, RhoA expression and the contribution of Rho/ROCK pathway to the control of perfusion pressure have not been investigated in the rat hind limb vascular bed as a skeletal muscle vascular network., Aims: To investigate the contribution of the Rho/ROCK pathway in the control of perfusion pressure in the isolated-perfused rat hind limb vascular bed., Study Design: Animal experimentation., Methods: Two Rho inhibitors (atorvastatin and C3 exoenzyme) and ROCK inhibitors (Y-27632 and fasudil) were tested on the phenylephrine-elevated perfusion pressure in the isolated-perfused rat hind limb vascular bed. Furthermore, we sought the expression of RhoA protein in the femoral, popliteal and saphenous arteries as well as quadriceps and gastrocnemius muscles by Western blotting., Results: The ROCK inhibitors Y-27632 and fasudil (both 10-8 to 10-5 M) induced substantial vasodilatations. The maximum vasodilatations induced by Y-27632 and fasudil (both at 10-5 M) were 84.0 ± 6.9% and 76.9 ± 6.9%, respectively (P = .091). Y-27632 was not more potent than fasudil, as the EC50 values for Y-27632 and fasudil were 0.7 ± 2.1 μM and 2.5 ± 2.4 μM, respectively (P = .177). Atorvastatin (10-7 to 10-4 M) and C3 exoenzyme (3 × 10-8 M) also produced vasodilatation (maximum vasodilatation; 20.3 ± 1.7% and 13.7 ± 3.6%, respectively). The EC50 value for atorvastatin was 94.9 ± 1.2 μM. The western blot analysis showed that the femoral, saphenous, and popliteal arteries, as well as the gastrocnemius and quadriceps muscles, express RhoA protein., Conclusion: The Rho/ROCK pathway contributes significantly to the control of perfusion pressure in the rat hind limb vascular bed.

Moxifloxacin and gemifloxacin were tested on isolated rabbits' jejunal preparations as little is known about its effects on gastrointestinal tissues. Moxifloxacin and gemifloxacin were tested in concentrations 0.01-10μg/mL for possible effect(s) on isolated rabbits' jejunal preparations. The drugs were applied on spontaneous, on low K + (20mM)-induced contractions and on high K + (80mM)-induced contractions. Response was plotted as % of its respective controls. EC50 for Moxifloxacin and Gemifloxacin on spontaneous (without Glibenclamide) contractions are 2.83±0.5μg/mL and 1.11±0.2μg/mL, respectively. Moxifloxacin and Gemifloxacin relaxed the low K+ (20mM) -induced contractions, which were inhibited in presence of Glibenclamide (3μM). Our result indicates that the relaxant activity of Moxifloxacin and Gemifloxacin is mediated possibly through activation of ATP-sensitive potassium channels (KATP). The relaxant effect of Moxifloxacin and Gemifloxacin is predominantly mediated by activation of ATP-Sensitive potassium channels (K ATP ), which could be cause of one of relaxing mechanisms.

Intracellular Ca2+ level is under strict regulation through calcium channels and storage pools including the endoplasmic reticulum (ER). Mutations in certain ion channel subunits, which cause mis-regulated Ca2+ influx, induce the excitotoxic necrosis of neurons. In the nematode Caenorhabditis elegans, dominant mutations in the DEG/ENaC sodium channel subunit MEC-4 induce six mechanosensory (touch) neurons to undergo excitotoxic necrosis. These necrotic neurons are subsequently engulfed and digested by neighboring hypodermal cells. We previously reported that necrotic touch neurons actively expose phosphatidylserine (PS), an "eat-me" signal, to attract engulfing cells. However, the upstream signal that triggers PS externalization remained elusive. Here we report that a robust and transient increase of cytoplasmic Ca2+ level occurs prior to the exposure of PS on necrotic touch neurons. Inhibiting the release of Ca2+ from the ER, either pharmacologically or genetically, specifically impairs PS exposure on necrotic but not apoptotic cells. On the contrary, inhibiting the reuptake of cytoplasmic Ca2+ into the ER induces ectopic necrosis and PS exposure. Remarkably, PS exposure occurs independently of other necrosis events. Furthermore, unlike in mutants of DEG/ENaC channels, in dominant mutants of deg-3 and trp-4, which encode Ca2+ channels, PS exposure on necrotic neurons does not rely on the ER Ca2+ pool. Our findings indicate that high levels of cytoplasmic Ca2+ are necessary and sufficient for PS exposure. They further reveal two Ca2+-dependent, necrosis-specific pathways that promote PS exposure, a "two-step" pathway initiated by a modest influx of Ca2+ and further boosted by the release of Ca2+ from the ER, and another, ER-independent, pathway. Moreover, we found that ANOH-1, the worm homolog of mammalian phospholipid scramblase TMEM16F, is necessary for efficient PS exposure in thapsgargin-treated worms and trp-4 mutants, like in mec-4 mutants. We propose that both the ER-mediated and ER-independent Ca2+ pathways promote PS externalization through activating ANOH-1., Competing Interests: The authors have declared that no competing interests exist.

Patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) offer an attractive experimental platform to investigate cardiac diseases and therapeutic outcome. In this study, iPSC-CMs were utilized to study their calcium transient signals and drug effects by means of machine learning, a central part of artificial intelligence. Drug effects were assessed in six iPSC-lines carrying different mutations causing catecholaminergic polymorphic ventricular tachycardia (CPVT), a highly malignant inherited arrhythmogenic disorder. The antiarrhythmic effect of dantrolene, an inhibitor of sarcoplasmic calcium release, was studied in iPSC-CMs after adrenaline, an adrenergic agonist, stimulation by machine learning analysis of calcium transient signals. First, beats of transient signals were identified with our peak recognition algorithm previously developed. Then 12 peak variables were computed for every identified peak of a signal and by means of this data signals were classified into different classes corresponding to those affected by adrenaline or, thereafter, affected by a drug, dantrolene. The best classification accuracy was approximately 79% indicating that machine learning methods can be utilized in analysis of iPSC-CM drug effects. In the future, data analysis of iPSC-CM drug effects together with machine learning methods can create a very valuable and efficient platform to individualize medication in addition to drug screening and cardiotoxicity studies.

General anesthesia is obtained by administration of potent hypnotics, analgesics and muscle relaxants. Apart from their intended effects (loss of consciousness, pain relief and muscle relaxation), these agents profoundly affect the control of breathing, in part by an effect within the peripheral chemoreflex loop that originates at the carotid bodies. This review assesses the role of cholinergic chemotransmission in the peripheral chemoreflex loop and the mechanisms through which muscle relaxants and hypnotics interfere with peripheral chemosensitivity. Additionally, consequences for clinical practice are discussed.

Aromatic L-amino acid decarboxylase (AADC) deficiency is a rare, autosomal recessive inborn error of metabolism in which several neurotransmitters including serotonin, dopamine, norepinephrine and epinephrine are deficient. Symptoms typically appear in the first year of life and include oculogyric crises and dystonia, hypotonia, and global developmental delay. Dystonia is of particular concern as a dystonic storm can ensue leading to rhabdomyolysis. Rhabdomyolysis can become life-threating and therefore its recognition and prompt management is of significant importance. Here we present two cases of patients with AADC deficiency and a history of dystonic crisis causing rhabdomyolysis. We hypothesize that in addition to the hypodopaminergic, a hypercholinergic state is contributing to the pathophysiology of dystonia in AADC deficiency, as well as to the associated rhabdomyolysis. We were able to prevent rhabdomyolysis in both patients with using Dantrolene and we suggest using a trial of this medication in cases of sustained dystonic crisis in AADC deficiency patients., Competing Interests: None declared, (Georg Thieme Verlag KG Stuttgart · New York.)

Background: Overactivation of ryanodine receptors and the resulting impaired calcium homeostasis contribute to Alzheimer's disease-related pathophysiology. This study hypothesized that exposing neuronal progenitors derived from induced pluripotent stems cells of patients with Alzheimer's disease to dantrolene will increase survival, proliferation, neurogenesis, and synaptogenesis., Methods: Induced pluripotent stem cells obtained from skin fibroblast of healthy subjects and patients with familial and sporadic Alzheimer's disease were used. Biochemical and immunohistochemical methods were applied to determine the effects of dantrolene on the viability, proliferation, differentiation, and calcium dynamics of these cells., Results: Dantrolene promoted cell viability and proliferation in these two cell lines. Compared with the control, differentiation into basal forebrain cholinergic neurons significantly decreased by 10.7% (32.9 ± 3.6% vs. 22.2 ± 2.6%, N = 5, P = 0.004) and 9.2% (32.9 ± 3.6% vs. 23.7 ± 3.1%, N = 5, P = 0.017) in cell lines from sporadic and familial Alzheimer's patients, respectively, which were abolished by dantrolene. Synapse density was significantly decreased in cortical neurons generated from stem cells of sporadic Alzheimer's disease by 58.2% (237.0 ± 28.4 vs. 99.0 ± 16.6 arbitrary units, N = 4, P = 0.001) or familial Alzheimer's disease by 52.3% (237.0 ± 28.4 vs.113.0 ± 34.9 vs. arbitrary units, N = 5, P = 0.001), which was inhibited by dantrolene in the familial cell line. Compared with the control, adenosine triphosphate (30 µM) significantly increased higher peak elevation of cytosolic calcium concentrations in the cell line from sporadic Alzheimer's patients (84.1 ± 27.0% vs. 140.4 ± 40.2%, N = 5, P = 0.049), which was abolished by the pretreatment of dantrolene. Dantrolene inhibited the decrease of lysosomal vacuolar-type H-ATPase and the impairment of autophagy activity in these two cell lines from Alzheimer's disease patients., Conclusions: Dantrolene ameliorated the impairment of neurogenesis and synaptogenesis, in association with restoring intracellular Ca homeostasis and physiologic autophagy, cell survival, and proliferation in induced pluripotent stem cells and their derived neurons from sporadic and familial Alzheimer's disease patients.

Dicranopteris linearis leaf has been reported to exert antinociceptive activity. The present study elucidates the possible mechanisms of antinociception modulated by the methanol extract of D. linearis leaves (MEDL) using various mouse models. The extract (25, 150, and 300 mg/kg) was administered orally to mice for 30 min priot to subjection to the acetic acid-induced writhing-, hot plate- or formalin-test to establish the antinociceptive profile of MEDL. The most effective dose was then used in the elucidation of possible mechanisms of action stage. The extract was also subjected to the phytochemical analyses. The results confirmed that MEDL exerted significant ( p < 0.05) antinociceptive activity in those pain models as well as the capsaicin-, glutamate-, bradykinin- and phorbol 12-myristate 13-acetate (PMA)-induced paw licking model. Pretreatment with naloxone (a non-selective opioid antagonist) significantly ( p < 0.05) reversed MEDL effect on thermal nociception. Only l-arginine (a nitric oxide (NO) donor) but not N(ω)-nitro-l-arginine methyl ester (l-NAME; a NO inhibitor) or 1 H -[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; a specific soluble guanylyl cyclase inhibitor) significantly ( p < 0.05) modified MEDL effect on the writhing test. Several polyphenolics and volatile antinociceptive compounds were detected in MEDL. In conclusion, MEDL exerted the opioid/NO-mediated antinociceptive activity, thus, justify D. linearis as a potential source for new analgesic agents development.

Aberrant Ca 2+ release from cardiac ryanodine receptors (RyR2) has been shown to be one of the most important causes of lethal arrhythmia in various types of failing hearts. We previously showed that dantrolene, a specific agent for the treatment of malignant hyperthermia, inhibits Ca 2+ leakage from the RyR2 by correcting the defective inter-domain interaction between the N-terminal (1-619 amino acids) and central (2000-2500 amino acids) domains of the RyR2 and allosterically enhancing the binding affinity of calmodulin to the RyR2 in diseased hearts. In this study, we examined whether dantrolene inhibits this Ca 2+ leakage, thereby preventing the pharmacologically inducible ventricular tachycardia in ventricular pressure-overloaded failing hearts. Ventricular tachycardia (VT) was easily induced after an injection of epinephrine in mice after 8 weeks of transverse aortic constriction-induced pressure-overload. Pretreatment with dantrolene almost completely inhibited the pharmacologically inducible VT. In the presence of dantrolene, the occurrence of both Ca 2+ sparks and spontaneous Ca 2+ transients was inhibited, which was associated with enhanced calmodulin binding affinity to the RyR2. These results suggest that dantrolene could be a new potent agent in the treatment of lethal arrhythmia in cases of acquired heart failure., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background and Aim: Simaba ferruginea A.St.-Hil. Popularly known as "calunga," is a typical Brazilian cerrado plant whose rhizomes are popular for treating diarrhea., Aims: The aim of this study was to evaluate the spasmolytic activity and the antidiarrheal effect of the ethanolic extract obtained from S. ferruginea (Sf-EtOH)., Methods: Ileal segments (1-2 cm) from male Wistar rats were mounted in isolated organ baths and connected to a force transducer, and then to an amplifier which was connected to a computer (AVS Projetos/São Paulo-SP). After stabilization for 60 min, under tension (1 gf), two submaximal contractions were induced with KCl 40 mM or carbachol 10 -6  M on ileal segments. During the third tonic and sustained contraction, Sf-EtOH was added in cumulative concentrations to the organ bath. Incubations with L-NAME (10 -4  M), ODQ (10 -5  M), TEA + (5 or 1 mM), glibenclamide (10 -5  M), or apamine (100 nM) were prepared (n = 5), separately and used to verify the involvement of the nitric oxide synthase, guanylate cyclase, and potassium channels in the relaxing effect. The results were expressed as mean ± standard error of the mean and were statistically evaluated using one-way ANOVA followed by Bonferroni test, when necessary *p < 0.05., Results: Sf-EtOH promotes relaxation on rat isolated ileum pre-contracted with CCh and KCl in a concentration-dependent manner. Sf-EtOH also inhibited ileum contractions against cumulative concentrations of carbachol (CCh), KCl, and CaCl 2 , shifting the curves to the right in a non-parallel manner with an E max reduction. In the presence of potassium channel blockers, Sf-EtOH shifted the curves to the right with a reduction of E max , suggesting the involvement of BK Ca , K ATP , and SK Ca in its spasmolytic effect. In the presence of L-NAME or ODQ, the relaxation curves were shifted to the right, suggesting the involvement of this pathway in Sf-EtOH spasmolytic effect., Conclusions: Sf-EtOH acts in a concentration-dependent manner, involving the positive modulation of K + channels and NO pathway.

Deficiency of deleted in liver cancer 2 (DLC2), a novel domain to inhibit RhoA activity, plays an important role in inflammatory pain. However, the underlying mechanisms remain unclear. This study investigated the role of DLC2 and its downstream cascade of RhoA/ROCK in formalin-induced inflammatory pain using DLC2-knockout (DLC2 -/- ) mice and compared them with DLC2 wild-type (DLC2 +/+ ) mice. Mechanical allodynia and thermal hyperalgesia were evaluated using von Frey filament aesthesiometer and Hargreaves test, respectively. The spinal cord dorsal horn (L3-L5) was selected for molecular and cellular identification by Western blot and immunofluorescence. DLC2 -/- mice showed increased mechanical allodynia and thermal hyperalgesia. Expression of ROCK1, ROCK2 and IL-1β was significantly higher in DLC2 -/- mice. Intrathecal administration of RhoA inhibitor (C3 exoenzyme) or ROCK inhibitor (Y27632) significantly attenuated formalin-induced inflammatory hyperalgesia in DLC2 -/- mice. ROCK2 and IL-1β expression were reduced by C3 exoenzyme or Y27632. Spinal p38 activation was also inhibited by C3 exoenzyme or Y27632. Double-labelling immunofluorescence demonstrated co-localization of DLC2 with spinal dorsal microglia. The number of activated microglia in the spinal dorsal horn was significantly higher in DLC2 -/- mice, but was reduced by Y27632. These findings indicate that DLC2 deficiency increased formalin-induced inflammatory hyperalgesia through regulating RhoA/ROCK2, and IL-1β may be a downstream effector. Our results also suggest that RhoA/ROCK enhanced p38 activation plays an important role in formalin-induced inflammatory pain. The finding that DLC2 attenuated inflammatory pain through inhibiting RhoA/ROCK2 suggests that the DLC2/RhoA/ROCK2/p38/IL-β pathway may be a potential therapeutic target to reduce inflammatory pain., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: 2,4-Dinitrophenol (DNP) is a known uncoupler of oxidative phosphorylation that clinically results in hyperthermia, tachycardia, tachypnea, and metabolic acidosis. Overdoses of DNP are often fatal and there is no specific reversal therapy. Dantrolene interferes with calcium release in skeletal muscle and is traditionally used to treat malignant hyperthermia. There has been limited published data on its use in DNP toxicity. We present two cases of DNP toxicity that were treated with dantrolene. CASE 1: A 22-year-old male presented following an overdose of his bodybuilding supplements including DNP. He became altered, tachycardic, and hyperthermic to 40.0C. He required intubation and aggressive cooling. He received multiple doses of dantrolene over the initial 36 h with resolution of his hyperthermia. He was extubated and discharged home on hospital day 6. CASE 2: A 20-year-old male presented following a staggered ingestion of DNP. He was tachypneic and tachycardic on arrival. He became hyperthermic to 40.2C and required intubation. He underwent aggressive cooling and received 200 mg of IV dantrolene. His temperature normalized, however, he expired 4 h after ED arrival., Conclusion: DNP toxicity has limited treatment options. Dantrolene may ameliorate the hypermetabolic state in DNP toxicity by lessening excitation-contraction coupling in muscle cells and improving the associated hyperthermia. Our cases demonstrate the hyperthermia reducing effects of dantrolene in DNP toxicity and contribute to the existing literature on this topic. Being aware of the possible use of dantrolene to treat the associated hyperthermia could assist emergency physicians in the treatment of DNP toxicity., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Objectives: To explore and characterize the disposition and dynamics of micromotions in the wall of the intact resting teradotoxinized urinary bladder of the rabbit before and after the administration of adrenergic and cholinergic pharmaceutical agents., Methods: Spatiotemporal maps and related intravesical pressure were used to analyse propagating patches of contractions (PPCs) and their component individual myogenic contractions [propagating individual contractions (PICs)] in the wall of the tetradotoxinized urinary bladder., Results: The bladder wall exhibited two contractile states that were of similar frequencies to those of the two types of electrophysiological discharge described in previous studies; the first, in which cyclic PPCs predominated, the second in which small irregular PICs predominated. The addition of carbachol increased the size, frequency, speed and distance of propagation of PPCs, whereas the addition of isoprenaline temporarily halted the incorporation of PICs into PPCs, and reduced patch size and total area undergoing contraction. The RhoA kinase (ROCK) inhibitor Y-27632 reduced both largest patch index and mean patch size. Both carbenoxolone and ROCK inhibition decreased the duration of PPCs. Carbenoxolone also prolonged duration and accelerated PPC propagation velocity. The authors postulate that these differences arise from differing effects of these agents on myocytes and interstitial cells within the stress environment of the bladder, influencing the development, coordination and propagation of PPCs., Conclusions: The timings and structure of spontaneous micromotions in the wall of the isolated bladder change when it is treated with sympathetic/parasympathetic agonists and with myogenically active agents. Correspondingly, disorders of bladder wall contraction may result from disorders of either neurogenic or myogenic signalling and may be amenable to treatment with combinations of agents that influence both., (© 2019 The Authors BJU International © 2019 BJU International Published by John Wiley & Sons Ltd.)

Background: Approach for green chemistry for chemical synthesis is found to be very efficient as it makes the reaction more easily, less tedious, maximize desired products and minimize by-products., Materials & Methods: Utilizing this approach 1, 5-benzodiazepines and its derivatives have been synthesized and evaluated for skeletal muscle and antianxiety activity. 1, 5-benzodiazepine derivatives have attracted great attention due to its diversity of pharmacological activities and its application in heterocyclic synthesis and medicines. The target compounds were synthesized by first reacting o-phenylenediamine with acetophenone to yield 1, 5-benzodiazepines. In the next step the NH of 1, 5-benzodiazepines were chloroacetylated and then the chloro group was substituted with different anilines. The structures were confirmed on the basis of their TLC, IR, 1H NMR and CHN elemental studies. The physicochemical parameters were determined for BBB penetration through online software., Results: The Log P values of the compounds tested showed that compounds have the potential to be CNS active. The compounds were evaluated for the skeletal muscle relaxant activity and antianxiety activity. It was investigated that 1, 5-benzodiazepines derivatives possess significant differences between control group and treated group., Conclusion: Among these derivatives, the compound bearing chloro group possesses the highest skeletal muscle relaxant and antianxiety activity., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

There has been a resurgence in the use of 2,4-dinitrophenol, C 6 H 4 N 2 O 5 (DNP) recently as an illegal weight loss drug. We present a case of a healthy 25-year-old girl who took two tablets of DNP, purchased from an overseas online retailer. She was managed with aggressive, invasive cooling measures and 2.5 mg kg -1 dantrolene. Despite this, her temperature continued to rise exponentially to 41.5°C. Cardiac arrest occurred and resuscitation was unsuccessful. To our knowledge, this is the first reported case of the ineffective use of dantrolene in acute DNP poisoning. We review the pathophysiology of DNP toxicity and argue that the use of dantrolene therapy is biochemically implausible, based on poor evidence and likely to be futile. We have contacted the UK National Poisons Information Service (NPIS/TOXBASE) to propose changes to the management of acute DNP toxicity., Competing Interests: Competing interests: JVS is a ST4 Registrar in Anaesthetics and Intensive Care Medicine, North Central School of Anaesthetics, London and an Anaesthetic Academic Clinical Fellow who holds an Honorary Research Assistant post in the Division of Surgery and Interventional Science University College London. EK is a CT2 in General Surgery in the North West Thames Deanery. She has undertaken a rotation in Intensive Care Medicine as part of her surgical training. AT is a consultant in Anaesthetics and Intensive Care Medicine at The Hillingdon Hospitals NHS Trust., (© BMJ Publishing Group Limited 2018. No commercial re-use. See rights and permissions. Published by BMJ.)

Benzodiazepines (BDZ), which potentiate the action of GABA at four subtypes of GABA A receptors (α1, α2, α3, and α5GABA A Rs), are highly effective against anxiety disorders, but also cause severe side effects greatly limiting their clinical application. Both, preclinical studies in genetically engineered mice, and preclinical and clinical trials with subtype-selective compounds indicate that undesired effects can in principle be avoided by targeting specific GABA A R subtypes. While there is general consensus that activity at α1GABA A Rs should be avoided, controversy exists as to whether α2 or α3GABA A Rs need to be targeted for anxiolysis. While previous experiments in GABA A R point-mutated mice demonstrated a critical role of α2GABA A Rs, studies solely relying on pharmacological approaches suggested a dominant contribution of α3GABA A Rs. As most α1GABA A R-sparing BDZ site agonists discriminate little between α2 and α3GABA A Rs, these claims rest almost exclusively on a single compound, TP003, that has been reported to be a selective α3GABA A R modulator. Here, we have revisited the in vitro pharmacological profile of TP003 and, in addition, tested TP003 in GABA A R triple point-mutated mice, in which only either α1, α2, or α3GABA A Rs were left BDZ sensitive. These experiments revealed that TP003 behaves as a partial, rather non-selective BDZ site agonist in vitro that acts in vivo through α1, α2, and α3GABA A Rs (α5GABA A R-mediated effects were not tested). With respect to anxiolysis, our results support a critical contribution of α2GABA A Rs, but not of α3GABA A Rs. TP003 should therefore not be considered an α3GABA A R selective agent. Previously published studies using TP003 should be interpreted with caution., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Interstitial cystitis is a syndrome characterized by detrusor overactivity and chronic inflammation of the bladder. The mechanisms responsible for the altered smooth muscle contractility remain poorly understood. The aim of the study was to investigate the role of intracellular signalling pathways in carbachol-induced detrusor contraction in a rat model of interstitial cystitis. Cyclophosphamide (150 mg/kg, dissolved in saline) was injected to rats (Sprague-Dawley, female, 200-250 g) intraperitoneally once a day on days 1, 4 and 7 to induce interstitial cystitis. Control groups were injected with saline (0.9% NaCl). Detrusor smooth muscle strips were mounted in 1-ml organ baths containing HEPES-buffered modified Krebs' solution and permeabilized with 40 μM β-escin for 30 min. Carbachol-induced contractions were significantly increased from 21.2 ± 1.6% (saline-treated) to 44 ± 4.4% in cyclophosphamide-treated group. The Rho kinase inhibitor Y-27632 (8.8 ± 2%) and the protein kinase C inhibitor GF-109203X (11.7 ± 2.8%) inhibited the increased contractile response (44 ± 4.4%) in rats with cystitis. The increased carbachol-induced contraction (44 ± 4.4%) was also significantly inhibited by the sarcoplasmic reticulum ryanodine channel blocker ryanodine (25.8 ± 3.2%) and the sarcoplasmic reticulum IP 3 receptor blocker heparin (17.2 ± 2.2%) in cystitis. RhoA protein levels in the bladder of cyclophosphamide-treated rats were significantly increased while pan-protein kinase C (α, β and γ isoforms) protein expression was unaltered between experimental groups. Carbachol-induced calcium sensitization at constant and clamped calcium (pCa 6) was also increased in cystitis (from 15.8 ± 2.2% to 24.7 ± 2.8%). This increased response (24.7 ± 2.8%) was significantly inhibited by both Y-27632 (7.9 ± 0.7%) and GF-109203X (4.4 ± 1.5%). We conclude that interstitial cystitis is characterized by an enhanced carbachol contractile response as well as by calcium sensitization of the detrusor smooth muscle. Activation of Rho kinase and protein kinase C pathways may be the molecular culprits responsible for the augmented muscarinic response observed in cystitis., (© 2018 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).)

Background: Both vagal (VS) and sympathetic (SS) stimulations can increase atrial fibrillation (AF) inducibility, with VS being known as more arrhythmogenic in normal hearts. Heart failure (HF) results in autonomic dysfunction (characterized by sympathetic activation and vagal withdrawal) and is associated with an increased AF incidence. This study investigated whether failing hearts, compared with normal control hearts, respond differently to autonomic stimulation-induced AF arrhythmogenesis and the effect of dantrolene on SS-enhanced AF in HF., Methods and Results: A rat myocardial infarction (MI) HF model was used. In experiment 1, AF inducibility was compared in 9 MI-HF rats versus 10 sham-control animals at baseline, during VS, and during SS with isoproterenol infusion. In experiment 2, dantrolene treatment (n = 8) was compared with placebo-control (n = 9) on SS-induced AF inducibility in HF. Compared with the sham-control, baseline AF inducibility was higher in the MI-HF group. AF inducibility was augmented in both groups by autonomic stimulation. However, under VS the increased magnitude was less in the MI-HF group (49% ± 11% vs 80% ± 10%; P = .029), but under SS was significantly more (53% ± 8% vs 6% ± 7%; P < .001), compared with sham-control. Dantrolene significantly attenuated SS-enhanced AF in HF (69% ± 6% vs 29% ± 9%; P = .006)., Conclusions: Failing hearts are less sensitive to VS, but more vulnerable to SS-induced AF compared with normal-control hearts. Dantrolene can significantly attenuate SS-enhanced AF in HF, indicating that cardiac ryanodine receptor dysfunction may play a critical role in SS-enhanced AF in HF, and stabilizing leaky ryanodine receptor with the use of dantrolene may be a new treatment option in this condition., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Objectives: To investigate the effect of metabolic syndrome on the bladder cannabinoid receptors 1 and 2 (CB1/CB2) expression and function in the fructose-fed rats (FR)., Methods: Adult male Sprague-Dawley rats were divided into two groups: (i) Control rats fed with normal chow; and (ii) Rats fed with high-fructose diet (FR) for 9 weeks. The body weight, blood pressure, plasma sugar, insulin, triglyceride and cholesterol were measured. Bladder muscle strips were prepared in organ bath and pre-contracted with 1 µM/L acetylcholine (ACh) or 50 mM/L KCl. The relaxation responses to CB1/CB2 agonist Bay59-3047 (0.01-1 µM/L) were recorded. The effects of CB1 antagonist AM251, CB2 antagonist AM630, protein kinase A (PKA) inhibitor H-89 and ATP-sensitive potassium channel (K ATP ) inhibitor glibenclamide on the Bay59-3047-induced response were tested. Western blotting and real-time polymerase chain reaction (RT-PCR) analyses were performed for bladder CB1/CB2 receptors., Results: Significant increases of body weight, blood pressure, plasma glucose, insulin, cholesterol and triglyceride levels were found in the FR. Bay59-3047 reduced ACh and KCl pre-contracted bladder strip tension in a dose-dependent fashion. The relaxation responses were significantly decreased in the FR. The Bay59-3047-induced relaxation was attenuated by AM251, glibenclamide and H-89. Western blotting and RT-PCR showed decreased expressions of FR bladder CB1 and CB2 receptor protein and mRNA., Conclusion: CB1/CB2 receptors mediate rat bladder relaxation through the PKA and K ATP pathway. The CB1 receptor may play a more prominent role. The response is decreased in the FR bladder due to reduced expressions of the cannabinoid receptors., (© 2017 John Wiley & Sons Australia, Ltd.)

The objective of this in vitro study was to evaluate and compare the effects of scopolamine butylbromide (Spasmolax ® ) on xylazine-sensitized bovine pregnant uterine strips, at different stages of pregnancy. The procedures were carried out in isolated organ bath. Uterine motility, expressed with amplitude, frequency of contractions as well as the area under the curve, was recorded in different stages of pregnancy and data were collected at 5-min intervals starting 5 min before treatment until 10 min after treatment (5-min after xylazine administration and 5-min after scopolamine butylbromide addition). The results suggest that scopolamine butylbromide might decrease the tonic effect induced by xylazine on bovine pregnant uteri from 0-30 days to 240-270 days., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Acute RyR2 activation by exchange protein directly activated by cAMP (Epac) reversibly perturbs myocyte Ca 2+ homeostasis, slows myocardial action potential conduction, and exerts pro-arrhythmic effects. Loose patch-clamp studies, preserving in vivo extracellular and intracellular conditions, investigated Na + current in intact cardiomyocytes in murine atrial and ventricular preparations following Epac activation. Depolarising steps to varying test voltages activated typical voltage-dependent Na + currents. Plots of peak current against depolarisation from resting potential gave pretreatment maximum atrial and ventricular currents of -20.23 ± 1.48 (17) and -29.8 ± 2.4 (10) pA/μm 2 (mean ± SEM [n]). Challenge by 8-CPT (1 μmol/L) reduced these currents to -11.21 ± 0.91 (12) (P < .004) and -19.3 ± 1.6 (11) pA/μm 2 (P < .04) respectively. Currents following further addition of the RyR2 inhibitor dantrolene (10 μmol/L) (-19.91 ± 2.84 (13) and -26.6 ± 1.7 (17)), and dantrolene whether alone (-19.53 ± 1.97 (8) and -27.6 ± 1.9 (14)) or combined with 8-CPT (-19.93 ± 2.59 (12) and -29.9 ± 2.5(11)), were indistinguishable from pretreatment values (all P >> .05). Assessment of the inactivation that followed by applying subsequent steps to a fixed voltage 100 mV positive to resting potential gave concordant results. Half-maximal inactivation voltages and steepness factors, and time constants for Na + current recovery from inactivation in double-pulse experiments, were similar through all the pharmacological conditions. Intracellular sharp microelectrode membrane potential recordings in intact Langendorff-perfused preparations demonstrated concordant variations in maximum rates of atrial and ventricular action potential upstroke, (dV/dt) max . We thus demonstrate an acute, reversible, Na + channel inhibition offering a possible mechanism for previously reported pro-arrhythmic slowing of AP propagation following modifications of Ca 2+ homeostasis, complementing earlier findings from chronic alterations in Ca 2+ homeostasis in genetically-modified RyR2-P2328S hearts., (© 2017 The Authors. Clinical and Experimental Pharmacology and Physiology Published by John Wiley & Sons Australia, Ltd.)

Background: The mechanisms underlying spontaneous atrial fibrillation (AF) associated with atrial ischemia/infarction are incompletely elucidated. Here, we investigate the mechanisms underlying spontaneous AF in an ovine model of left atrial myocardial infarction (LAMI)., Methods and Results: LAMI was created by ligating the atrial branch of the left anterior descending coronary artery. ECG loop recorders were implanted to monitor AF episodes. In 7 sheep, dantrolene-a ryanodine receptor blocker-was administered in vivo during the 8-day observation period (LAMI-D, 2.5 mg/kg, IV, BID). LAMI animals experienced numerous spontaneous AF episodes during the 8-day monitoring period that were suppressed by dantrolene (LAMI, 26.1±5.1; sham, 4.3±1.1; LAMI-D, 2.8±0.8; mean±SEM episodes per sheep, P <0.01). Optical mapping showed spontaneous focal discharges (SFDs) originating from the ischemic/normal-zone border. SFDs were calcium driven, rate dependent, and enhanced by isoproterenol (0.03 µmol/L, from 210±87 to 3816±1450, SFDs per sheep) but suppressed by dantrolene (to 55.8±32.8, SFDs per sheep, mean±SEM). SFDs initiated AF-maintaining reentrant rotors anchored by marked conduction delays at the ischemic/normal-zone border. NOS1 (NO synthase-1) protein expression decreased in ischemic zone myocytes, whereas NADPH (nicotinamide adenine dinucleotide phosphate, reduced form) oxidase and xanthine oxidase enzyme activities and reactive oxygen species (DCF [6-carboxy-2',7'-dichlorodihydrofluorescein diacetate]-fluorescence) increased. CaM (calmodulin) aberrantly increased [ 3 H]ryanodine binding to cardiac RyR2 (ryanodine receptors) in the ischemic zone. Dantrolene restored the physiological binding of CaM to RyR2., Conclusions: Atrial ischemia causes spontaneous AF episodes in sheep, caused by SFDs that initiate reentry. Nitroso-redox imbalance in the ischemic zone is associated with intense reactive oxygen species production and altered RyR2 responses to CaM. Dantrolene administration normalizes the CaM response, prevents LAMI-related SFDs, and AF initiation. These findings provide novel insights into the mechanisms underlying ischemia-related atrial arrhythmias., (© 2018 American Heart Association, Inc.)

Aim: To investigate the effect of intrathecal baclofen (ITB) on function and activity in dyskinetic cerebral palsy (CP)., Method: A retrospective cohort study of records from 25 children (15 males, 10 females; mean age 10y 11mo, SD 4y 9mo). Five were classified in Gross Motor Function Classification level IV and 20 in level V. Parents were interviewed about activities in daily life, sitting, communication, pain, sleep, and gross and fine motor function. Differences before and 1 year after ITB were graded as positive, no change, or negative. Assessments of dystonia (using the Barry-Albright Dystonia Scale) and muscle tone (Ashworth Scale) were made. Joint range of motion (ROM) was measured., Results: Both dystonia and increased muscle tone, present in all participants before ITB, decreased after (p<0.001). Passive ROM was restricted, with no difference after. Parents reported improvements in activities in daily life (p<0.001), sitting (p<0.001), communication (p<0.001), and fine motor function (p=0.013), but no change in gross motor function. Before ITB, pain and disturbed sleep were reported. There was a reduction in pain (p=0.002) and sleep improved (p=0.004) after ITB., Interpretation: After ITB in individuals with dyskinetic CP, improvements were found in sitting, communication, and fine motor skills. There was a reduction in dystonia and muscle tone, and pain and sleep improved., What This Paper Adds: Intrathecal baclofen can affect specific aspects of functioning in dyskinetic cerebral palsy. Sitting, communication, and fine motor function improved. Dystonia and spasticity were reduced. Pain was reduced and sleep improved., (© 2017 Mac Keith Press.)

1. The purpose of the present study was to investigate the effect of piperine (PIP) on CYP2E1 enzyme activity and pharmacokinetics of chlorzoxazone (CHZ) in healthy volunteers. 2. An open-label, two period, sequential study was conducted in 12 healthy volunteers. A single dose of PIP 20 mg was administered daily for 10 days during treatment phase. A single dose of CHZ 250 mg was administered during control and after treatment phases under fasting conditions. The blood samples were collected at predetermined time intervals after CHZ dosing and analyzed by HPLC. 3. Treatment with PIP significantly enhanced maximum plasma concentration (C max ) (3.14-4.96 μg/mL) , area under the curve (AUC) (10.46-17.78 μg h/mL), half life (T 1/2 ) (1.26-1.82 h) and significantly decreased elimination rate constant (K el ) (0.57-0.41 h  -   1 ), apparent oral clearance (CL/F) (24.76-13.65 L/h) of CHZ when compared to control. In addition, treatment with PIP significantly decreased C max (0.22-0.15 μg/mL), AUC (0.94-0.68 μg h/mL), T 1/2 (2.54-1.68 h) and significantly increased K el (0.32-0.43 h  -   1 ) of 6-hydroxychlorzoxazone (6-OHCHZ) as compared to control. Furthermore, treatment with PIP significantly decreased metabolite to parent (6-OHCHZ/CHZ) ratios of C max , AUC, T 1/2 and significantly increased K el ratio of 6-OHCHZ/CHZ, which indicate the decreased formation of CHZ to 6-OHCHZ. 4. The results suggest that altered pharmacokinetics of CHZ might be attributed to PIP mediated inhibition of CYP2E1 enzyme, which indicate significant pharmacokinetic interaction present between PIP and CHZ. The inhibition of CYP2E1 by PIP may represent a novel therapeutic benefit for minimizing ethanol induced CYP2E1 enzyme activity and results in reduced hepatotoxicity of ethanol.

After spinal cord injury (SCI), reflexes become hyperexcitable, leading to debilitating muscle spasms and compromised motor function. Previous work has described adaptations in spinal systems that might underlie this hyperexcitability, including an increase in constitutively active 5-HT 2C receptors in spinal motoneurons. That work, however, examined adaptations following complete transection SCI, whereas SCI in humans is usually anatomically and functionally incomplete. We therefore evaluated whether constitutive activity of 5-HT 2C receptors contributes to reflex hyperexcitability in an incomplete compression model of SCI and to spasms in vitro and in vivo. Our results confirm that 5-HT 2C receptor constitutive activity contributes to reflex excitability after incomplete SCI. We also evaluated whether constitutive activity could be altered by manipulation of neural activity levels after SCI, testing the hypothesis that it reflects homeostatic processes acting to maintain spinal excitability. We decreased neural activity after SCI by administering baclofen and increased activity by administering the selective serotonin reuptake inhibitor (SSRI) fluoxetine. We found that drug administration produced minimal alterations in in vivo locomotor function or reflex excitability. Similarly, we found that neither baclofen nor fluoxetine altered the contribution of constitutively active 5-HT 2C receptors to reflexes after SCI, although the contribution of 5-HT 2C receptors to reflex activity was altered after SSRIs. These results confirm the importance of constitutive activity in 5-HT 2C receptors to spinal hyperexcitability following SCI in the clinically relevant case of incomplete SCI but suggest that this activity is not driven by homeostatic processes that act to maintain overall levels of spinal excitability. NEW & NOTEWORTHY After spinal cord injury (SCI), most people will develop muscle spasms below their level of injury that can severely impact function. In this work, we examine the adaptations that occur within the spinal cord after SCI that contribute to these motor dysfunctions. We also evaluate one hypothesis about how these adaptations develop, which will potentially lead to intervention strategies to improve functional outcomes in persons with SCI., (Copyright © 2017 the American Physiological Society.)

Extracts of Desmodium adscendens (Sw) DC are used for the treatment of various diseases but limited toxicological evaluations have been done on the medicinal plant. This study investigates toxicity effects of the leave extract of D adscendens, and the possibility of drug-drug interaction of the plant extract when co-administered with other drugs. Oral administrations of leaf extract of D adscendens to white Wistar rats in an acute toxicity studies allowed the estimation of an LD 50 (median lethal dose) value of 1122 mg/kg body weight. In a subchronic toxicity studies, the plant extract caused a decrease in zoxazolamine paralysis time and prevented thiopentone from causing sleep in test animals compared to controls. Overall, the results are consistent with the plant extract being safe at the doses administered in humans. However, the induction of the CYP enzymes is an indication of a possible drug interaction when the plant extract is co-administered with other drugs.

The muscle relaxant carisoprodol has recently been controlled at the federal level as a Schedule IV drug due to its high abuse potential and consequences of misuse, such as withdrawal syndrome, delusions, seizures, and even death. Recent work has shown that carisoprodol can directly gate and allosterically modulate the type A GABA (GABA A ) receptor. These actions are subunit-dependent; compared with other GABA A receptors, carisoprodol has nominal direct gating effects in α 3 β 2 γ 2 receptors. Here, using site-directed mutagenesis and whole-cell patch-clamp electrophysiology in transiently transfected human embryonic kidney 293 cells, we examined the role of GABA A receptor α subunit transmembrane domain 4 (TM4) amino acids in direct gating and allosteric modulatory actions of carisoprodol. Mutation of α 3 valine at position 440 to leucine (present in the equivalent position in the α 1 subunit) significantly increased the direct gating effects of carisoprodol without affecting its allosteric modulatory effects. The corresponding reverse mutation, α 1(L415V), decreased carisoprodol direct gating potency and efficacy. Analysis of a series of amino acid mutations at the 415 position demonstrated that amino acid volume correlated positively with carisoprodol efficacy, whereas polarity inversely correlated with carisoprodol efficacy. We conclude that α 1(415) of TM4 is involved in the direct gating, but not allosteric modulatory, actions of carisoprodol. In addition, the orientation of alkyl or hydroxyl groups at this position influences direct gating effects. These findings support the likelihood that the direct gating and allosteric modulatory effects of carisoprodol are mediated via distinct binding sites., (Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.)

Mutations in the skeletal muscle ryanodine receptor (RyR1) cause malignant hyperthermia (MH) and central core disease (CCD), whereas mutations in the cardiac ryanodine receptor (RyR2) lead to catecholaminergic polymorphic ventricular tachycardia (CPVT). Most disease-associated RyR1 and RyR2 mutations are located in the N-terminal, central, and C-terminal regions of the corresponding ryanodine receptor (RyR) isoform. An increasing body of evidence demonstrates that CPVT-associated RyR2 mutations enhance the propensity for spontaneous Ca 2+ release during store Ca 2+ overload, a process known as store overload-induced Ca 2+ release (SOICR). Considering the similar locations of disease-associated RyR1 and RyR2 mutations in the RyR structure, we hypothesize that like CPVT-associated RyR2 mutations, MH/CCD-associated RyR1 mutations also enhance SOICR. To test this hypothesis, we determined the impact on SOICR of 12 MH/CCD-associated RyR1 mutations E2347-del, R2163H, G2434R, R2435L, R2435H, and R2454H located in the central region, and Y4796C, T4826I, L4838V, A4940T, G4943V, and P4973L located in the C-terminal region of the channel. We found that all these RyR1 mutations reduced the threshold for SOICR. Dantrolene, an acute treatment for MH, suppressed SOICR in HEK293 cells expressing the RyR1 mutants R164C, Y523S, R2136H, R2435H, and Y4796C. Interestingly, carvedilol, a commonly used β-blocker that suppresses RyR2-mediated SOICR, also inhibits SOICR in these RyR1 mutant HEK293 cells. Therefore, these results indicate that a reduced SOICR threshold is a common defect of MH/CCD-associated RyR1 mutations, and that carvedilol, like dantrolene, can suppress RyR1-mediated SOICR. Clinical studies of the effectiveness of carvedilol as a long-term treatment for MH/CCD or other RyR1-associated disorders may be warranted., (© 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

The neurosteroid allopregnanolone (3α, 5α-tetra-hydroprogesterone: ALLO) elicits anxiolytic, anticonvulsant, and hypnotic anesthetic effects in vivo similar to those induced by other positive allosteric modulators of the GABA A receptor. Endogenous ALLO has been shown to be rapidly elevated in the brain by acute stress paradigms, such as immobilization, in animal models. The present study was designed to ascertain the role of neurosteroid biosynthesis in the anxiety-like behavior induced by immobilization stress. Mice were exposed to an immobilization stressor for 2 h. After 24 h, the mice that had been immobilized did not behave significantly differently in the elevated plus maze (EPM) test and in the elevated open platform (EOP) test than the mice that had not been immobilized. In contrast, finasteride-pretreated immobilization stressed mice did behave significantly differently in the EPM and EOP tests. These findings suggest that ALLO biosynthesis contributes to stress resistance. Furthermore, the ALLO mimetic drug alfaxalone appeared to antagonize the effects of finasteride by significantly changing the behavior in the EPM test or in the EOP test in finasteride (10 mg kg -1 )-pretreated immobilized mice. In addition, alfaxalone, unlike diazepam, did not affect the muscle tone of the mice, as measured by the grip strength test. These results suggest that alfaxalone is a promising anxiolytic candidate lacking benzodiazepine-like muscle-relaxant effects., (© 2017 Wiley Periodicals, Inc.)

Tissue oxygenation, extracellular acidity, and tissue reducing capacity are among crucial parameters of tumor microenvironment (TME) of significant importance for tumor pathophysiology. In this paper, we demonstrate the complementary application of particulate lithium octa-n-butoxy-naphthalocyanine and soluble nitroxide paramagnetic probes for monitoring of these TME parameters using electron paramagnetic resonance (EPR) technique. Two different types of therapeutic interventions were studied: hypothermia and systemic administration of metabolically active drug. In summary, the results demonstrate the utility of EPR technique for non-invasive concurrent longitudinal monitoring of physiologically relevant chemical parameters of TME in mouse xenograft tumor models, including that under therapeutic intervention.

Currently available anxiolytics cause numerous adverse effects and show craving and tolerance during long term treatment. Currently traditional medicines have been re-evaluated widely through work on various plant species. Numerous plants in traditional system show pharmacological activity with unlimited prospective for therapeutic use. Hence we planned to evaluate the effect of methanol extract of T. foenum-graecum L. seeds on anxiety, sedation and motor coordination in mice at different doses following 15 days of oral feeding. Effect on anxiety was assessed by Hole board test and Light and Dark transition models.Phenobarbitone induced sleeping time and Rota rod test were performed to assess effect on sedation and motor coordination. In Hole board test, T. foenum-graecum L. seeds decreased the number of head dips in mice at all the three doses. In Light and Dark transition model, T. foenum-graecum L. seeds increased the period spent in the light box and the number of moves among the two compartments at 100 and 200 mg/kg as compared to control animals. In phenobarbitone induced sleeping time, T. foenum-graecum L. seeds did not reveal any sedative effect. In Rota rod test, extract exhibited significant skeletal muscle relaxant effect at 200 mg/kg (at 90 min) as compared to the control animals. Results of our study shows significant antianxiety effects of T. foenum-graecum L. seeds and may also recommend improved adverse effect profile as compared to diazepam.

Objective: To assess changes in body composition, body weight and resting metabolic rate in patients who received intrathecal baclofen therapy for spasticity., Design: Prospective, longitudinal, quasi-experimental, with a pre/post design., Patients: Twelve patients with spasticity, fulfilling study criteria, and due for pump implantation for intrathecal baclofen therapy, completed the study., Methods: Data were obtained before, 6 months and 12 months after commencement of intrathecal baclofen therapy as regards body composition (by skinfold calliper), body weight, and resting metabolic rate (by resting oxygen consumption). Spasticity was assessed according to the Modified Ashworth Scale (MAS) and Penn Spasm Frequency Scale (PSFS)., Results: A reduction in spasticity according to MAS occurred. Mean fat body mass increased and mean lean body mass decreased. Mean body weight showed a non-significant increase and resting metabolic rate a non-significant decrease., Conclusion: This explorative study indicates that unfavourable changes in body composition might occur after intrathecal baclofen therapy. Since obesity and increased fat body mass contribute to an increased cardiovascular risk, these findings may indicate a need for initiation of countermeasures, e.g. increased physical activity and/or dietary measures, in conjunction with intrathecal baclofen therapy. Further studies, including larger study samples and control groups, are needed to corroborate these findings.

Dantrolene was introduced for treatment of malignant hyperthermia. It also has antiarrhythmic properties and may thus be an alternative to amiodarone for the treatment of ventricular fibrillation (VF). Aim of this study was to compare the return of spontaneous circulation (ROSC) with dantrolene and amiodarone in a pig model of cardiac arrest. VF was induced in anesthetized pigs. After 8 min of untreated VF, chest compressions and ventilation were started and one of the drugs (amiodarone 5 mg kg -1 , dantrolene 2.5 mg kg -1 or saline) was applied. After 4 min of initial CPR, defibrillation was attempted. ROSC rates, hemodynamics and cerebral perfusion measurements were measured. Initial ROSC rates were 7 of 14 animals in the dantrolene group vs. 5 of 14 for amiodarone, and 3 of 10 for saline). ROSC persisted for the 120 min follow-up in 6 animals in the dantrolene group, 4 after amiodarone and 2 in the saline group (n.s.). Hemodynamics were comparable in both dantrolene group amiodarone group after obtaining ROSC. Dantrolene and amiodarone had similar outcomes in our model of prolonged cardiac arrest, However, hemodynamic stability was not significantly improved using dantrolene. Dantrolene might be an alternative drug for resuscitation and should be further investigated., Competing Interests: TW is involved with B. Braun (advisory board) HW is involved with Teleflex, Vygon an B. Braun (speaker fees), TS is involved with Teleflex, Vygon and B.Braun (advisory board). None of the other authors declared any competing interests.

Background: A series of new N-(2-benzoyl-4-chlorophenyl)-2-(4-(substituted phenyl) piperazin-1-yl) acetamides (3a-j) have been synthesized by the chloroacetylation of 2-amino-5- chlorbenzophenone which was further reacted with substituted phenylpiperazine., Material: The chemical structures of the compounds were confirmed on the basis of their TLC, IR, 1HNMR, 13CNMR and by elemental analysis. The physicochemical similarity of the target compounds with respect to standard drug diazepam was assessed by calculating from a set of physicochemical properties using software programs., Conclusion: Molecular docking studies revealed that the target compounds correctly dock into the binding pocket of the GABAA receptor, while their bioavailability/drug-likeness was predicted to be acceptable but requires future optimization. The anxiolytic and skeletal muscle relaxant activity of the target compounds (3a-j) were evaluated in albino mice. Among them, compound 3h showed potent anxiolytic and skeletal muscle relaxant activity., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Dystrophin, an important protein of the dystrophin-glycoprotein complex, has been implicated in the pathogenesis of experimental Chagas disease. It is important for the maintenance of cell shape and contraction force transmission. Dystrophin loss has been related to end-stage cardiac myopathies and proposed as a common route for myocardial dysfunction and progression to advanced heart failure. Evidence suggests that calpains, calcium-dependent proteases, digest dystrophin when the calcium concentration is compatible with their activation. The objective of this in vitro study was to test the hypothesis that dantrolene, a calcium channel blocker, improves structural changes induced by serum from Trypanosoma cruzi-infected mice. Cultured neonatal cardiac myocytes were incubated with serum from T. cruzi-infected mice and treated with dantrolene for 24 h. Immunofluorescence and immunoblotting were performed to evaluate dystrophin and calpain-1 protein expression. The levels of dystrophin decreased 13 % and calpain increased 17 % after incubation of cultured neonatal cardiac myocytes with serum from T. cruzi-infected mice. The treatment with dantrolene restored the dystrophin and calpain levels near control levels. Our results demonstrate that alterations in calcium homeostasis in cardiac myocytes are responsible, in part, for cardiac structural changes in experimentally induced T. cruzi myocarditis and that calpain inhibitors may be beneficial in Chagasic heart disease.

Dantrolene is the only available drug for the treatment of malignant hyperthermia, a life-threatening inborn sensitivity of the ryanodine receptor (RyR1) in skeletal muscles to volatile anesthetics. Dantrolene is metabolized in the liver to 5-OH dantrolene. Both compounds are zwitterions or net negatively charged. Here, we investigated interactions of dantrolene and 5-OH dantrolene with solute carrier (SLC) family members occurring in skeletal muscle cells, hepatocytes, and renal proximal tubule cells. SLC22A8 (organic anion transporter 3, OAT3) was very sensitive to both compounds exhibiting IC 50 values of 0.35 ± 0.03 and 1.84 ± 0.34 μM, respectively. These IC 50 concentrations are well below the plasma concentration in patients treated with dantrolene (3-28 μM). SLC22A7 (OAT2) was less sensitive to dantrolene and 5-OH dantrolene with IC 50 values of 15.6 ± 2.1 and 15.8 ± 3.2 μM, respectively. SLCO1B1 (OATP1B1), SLCO1B3 (OATP1B3), and SLCO2B1 (OATP2B1) mainly interacted with 5-OH dantrolene albeit with higher IC 50 values than those observed for OAT2 and OAT3. Dantrolene and 5-OH dantrolene failed to inhibit uptake of 1-methyl-4-phenylpyrimidinium (MPP) by OCT1 and of carnitine by OCTN2. In counter-flow experiments on OAT3, dantrolene and 5-OH dantrolene decreased pre-equilibrated cellular [ 3 H]estrone-3-sulfate (ES) content as did the transported substrates glutarate, furosemide, and bumetanide. With OAT2, dantrolene and 5-OH dantrolene slightly decreased the pre-equilibrated [ 3 H]cGMP content. If no other transporter markedly contributes to uptake or release of ES or cGMP, respectively, these data suggest that OAT3 and OAT2 may be involved in absorption, metabolism, and excretion of dantrolene and its metabolite 5-OH dantrolene.

We and others have demonstrated a protective effect of pacing postconditioning (PPC) against ischemia/reperfusion (I/R) injury. However, the mechanisms underlying this protection are not completely clear. In the present study, we evaluated the effects of calcium release from the sarcoplasmic reticulum (SR) and the novel intracellular acidic stores (AS). Isolated rat hearts (n = 6 per group) were subjected to coronary occlusion followed by reperfusion using a modified Langendorff system. Cardiac hemodynamics and contractility were assessed using a data acquisition program, and cardiac injury was evaluated by creatine kinase (CK) and lactate dehydrogenase (LDH) levels. Hearts were subjected to 30 min of regional ischemia, produced by ligation of the left anterior descending (LAD) coronary artery, followed by 30 min of reperfusion. The hearts were also subjected to PPC (3 cycles of 30 s of left ventricle (LV) pacing alternated with 30 s of right atrium (RA) pacing) and/or were treated during reperfusion with agonists or antagonists of release of calcium from SR or AS. PPC significantly (P < 0.05) normalized LV, contractility, and coronary vascular dynamics and significantly (P < 0.001) decreased heart enzyme levels compared to the control treatments. The blockade of SR calcium release resulted in a significant (P < 0.01) recovery in LV function and contractility and a significant reduction in CK and LDH levels (P < 0.01) when applied alone or in combination with PPC. Interestingly, the release of calcium from AS alone or in combination with PPC significantly improved LV function and contractility (P < 0.05) and significantly decreased the CK and LDH levels (P < 0.01) compared to the control treatments. An additive effect was produced when agonism of calcium release from AS or blockade of calcium release from the SR was combined with PPC. Calcium release from AS and blockade of calcium release from the SR protect the heart against I/R. Combining calcium release from acidic stores or blockade of calcium release from the SR with PPC produced a synergistic protective effect.

Chronic constriction injury to the sciatic nerve was used as an animal model of neuropathic pain. Instead of frequently used reflex-based tests we used an operant thermal place preference test to evaluate signs of neuropathic pain and the effect of baclofen administration in rats with neuropathy. Chronic constriction injury was induced by four loose ligations of the sciatic nerve. Thermal place preference (45 °C vs. 22 °C and 45 °C vs. 11 °C) was measured after the ligation and after the administration of baclofen in sham and experimental rats. Rats with the chronic constriction injury spent significantly less time on the colder plate compared to sham operated animals at the combination 45 °C vs. 11 °C. After administration of baclofen (10 mg/kg s.c.), the aversion to the colder plate in rats with chronic constriction injury disappeared. At the combination 45 °C vs. 22 °C, no difference in time spent on colder and/or warmer plate was found between sham and experimental animals. These findings show the importance of cold allodynia evaluation in rats with chronic constriction injury and the effectiveness of baclofen in this neuropathic pain model.

It has been reported that several aminoglycoside antibiotics have a potential of prolonging the action of non-depolarizing muscle relaxants by drug interactions acting pre-synaptically to inhibit acetylcholine release, but antibiotics itself also have a strong effect on relaxing the smooth muscle. In this study, four antibiotics of aminoglycosides such as gentamicin, streptomycin, kanamycin and neomycin were compared with skeletal muscle relaxants baclofen, tubocurarine, pancuronium and succinylcholine, and a smooth muscle relaxant, papaverine. The muscle strips isolated from the rat bladder were stimulated with pulse trains of 40 V in amplitude and 10 s in duration, with pulse duration of 1 ms at the frequency of 1-8 Hz, at 1, 2, 4, 6, 8 Hz respectively. To test the effect of four antibiotics on bladder smooth muscle relaxation, each of them was treated cumulatively from 1 μM to 0.1 mM with an interval of 5 min. Among the four antibiotics, gentamicin and neomycin inhibited the EFS response. The skeletal muscle relaxants (baclofen, tubocurarine, pancuronium and succinylcholine) and inhibitory neurotransmitters (GABA and glycine) did not show any significant effect. However, papaverine, had a significant effect in the relaxation of the smooth muscle. It was suggested that the aminoglycoside antibiotics have inhibitory effect on the bladder smooth muscle.

Meprobamate is a schedule IV anxiolytic and the primary metabolite of the muscle relaxant carisoprodol. Meprobamate modulates GABAA (γ-aminobutyric acid Type A) receptors, and has barbiturate-like activity. To gain insight into its actions, we have conducted a series of studies using recombinant GABAA receptors. In αxβzγ2 GABAA receptors (where x=1-6 and z=1-3), the ability to enhance GABA-mediated current was evident for all α subunit isoforms, with the largest effect observed in α5-expressing receptors. Direct gating was present with all α subunits, although attenuated in α3-expressing receptors. Allosteric and direct effects were comparable in α1β1γ2 and α1β2γ2 receptors, whereas allosteric effects were enhanced in α1β2 compared to α1β2γ2 receptors. In "extrasynaptic" (α1β3δ and α4β3δ) receptors, meprobamate enhanced EC20 and saturating GABA currents, and directly activated these receptors. The barbiturate antagonist bemegride attenuated direct effects of meprobamate. Whereas pentobarbital directly gated homomeric β3 receptors, meprobamate did not, and instead blocked the spontaneously open current present in these receptors. In wild type homomeric ρ1 receptors, pentobarbital and meprobamate were ineffective in direct gating; a mutation known to confer sensitivity to pentobarbital did not confer sensitivity to meprobamate. Our results provide insight into the actions of meprobamate and parent therapeutic agents such as carisoprodol. Whereas in general actions of meprobamate were comparable to those of carisoprodol, differential effects of meprobamate at some receptor subtypes suggest potential advantages of meprobamate may be exploited. A re-assessment of previously synthesized meprobamate-related carbamate molecules for myorelaxant and other therapeutic indications is warranted., (Copyright © 2016 Elsevier B.V. All rights reserved.)

This case report describes a female with p.Lys4876Arg amino acid change in the ryanodine receptor type 1 (RYR1) and a sibling who died of malignant hyperthermia (MH) during anesthesia. After her diagnosis as MH susceptible, this patient was administered low-dose dantrolene daily for greater than 25 years for treatment of chronic muscle spasm and pain in her lower extremities and back limiting sleep. Her creatine phosphokinase (CPK) was as high as 2390 IU/L during labor and 900 IU at rest. With 25 mg dantrolene daily, muscle cramps were eliminated, and sleep was improved. Gait instability was noted with dantrolene in the morning, but not when taken at bedtime. There was no evidence of liver injury. This case suggests that low dose dantrolene by mouth could be considered for the treatment of chronic muscle pain in individuals with MH susceptibility.

In this study, methamphetamine (Meth)- and glutamate (Glu)-mediated intracellular Ca(++) (Ca(++) i) signals were examined in real time in primary cortical neurons overexpressing an intracellular Ca(++) probe, GCaMP5, by adeno-associated viral (AAV) serotype 1. Binding of Ca(++) to GCaMP increased green fluorescence intensity in cells. Both Meth and Glu induced a rapid increase in Ca(++) i, which was blocked by MK801, suggesting that Meth enhanced Ca(++) i through Glu receptor in neurons. The Meth-mediated Ca(++) signal was also blocked by Mg(++) , low Ca(++) or the L-type Ca(++) channel inhibitor nifedipine. The ryanodine receptor inhibitor dantrolene did not alter the initial Ca(++) influx but partially reduced the peak of Ca(++) i. These data suggest that Meth enhanced Ca(++) influx through membrane Ca(++) channels, which then triggered the release of Ca(++) from the endoplasmic reticulum in the cytosol. AAV-GCaMP5 was also injected to the parietal cortex of adult rats. Administration of Meth enhanced fluorescence in the ipsilateral cortex. Using immunohistochemistry, Meth-induced green fluorescence was found in the NeuN-containing cells in the cortex, suggesting that Meth increased Ca(++) in neurons in vivo. In conclusion, we have used in vitro and in vivo techniques to demonstrate a rapid increase of Ca(++) i by Meth in cortical neurons through overexpression of GCaMP5. As Meth induces behavioral responses and neurotoxicity through Ca(++) i, modulation of Ca(++) i may be useful to reduce Meth-related reactions., (© 2014 Society for the Study of Addiction.)

Background: Intracellular calcium overload is a major contributing factor to diaphragmatic dysfunction triggered by sepsis. In this study, the possible role of dantrolene, a ryanodine receptor inhibitor, in preventing the release of calcium from the sarcoplasmic reticulum in diaphragmatic dysfunction and weakness was explored., Methods: A middle-grade severity sepsis rat model was established for the effects of treatment with dantrolene, on diaphragm harvested 24 h after cecal ligation and puncture (CLP), and analyzed using functional, histologic, and biomarker assays., Results: It was found that in septic rats, treatment with dantrolene significantly improved the contractility, relaxation, and fatigue index of the diaphragm in a dose-dependent manner. The benefits are associated with improvement in ultrastructural changes of Z band integrity and myofilament arrangements along with increases both in the ratio of slow-twitch type composition. Moreover, dantrolene effectively inhibits the overexpression of high-mobility group box 1 and reduces the calpain-1-caspase-3 proteolytic activity., Conclusions: Dantrolene can effectively attenuate the dysfunction of diaphragm in septic rats; Furthermore, the beneficial effects were associated with downregulation of high-mobility group box 1 and calpain-1-caspase-3 proteolytic activity., (Copyright © 2016 Elsevier Inc. All rights reserved.)