ATPase Pump Activity Research Articles

FRI550 Case Of Thyrotoxic Periodic Paralysis In African American Man With Cannabis Use

Abstract Disclosure: H. Al Jumaili: None. A. Mahaldar: None. S. Campbell: None. A. Gosmanova: None. Introduction: Recent changes in cannabis use (CU) laws led to growing interest in its use for recreational and therapeutic purposes. may have serious health problems such as hypokalemia, paralysis and rhabdomyolysis. Thyrotoxic Periodic Paralysis (TPP) is a rare and life-threatening complication of the thyrotoxicosis characterized by acute reversable painless muscle weakness or paralysis and hypokalemia due to massive intracellular shift of potassium. This condition is mainly seen males of Asian descent. Case: A 31-year-old African American man with Graves’ disease admitted to the hospital for upper and lower extremities weakness after Thanksgiving Day. He woke up with sudden inability to move any of his extremities with. He had high carbohydrate meal, on the evening. Patient reported having similar periodic episodes of paralysis, usually worse in the lower extremities for the last 2 years. This episode lasted longer than usual. On exam, he had enlarged thyroid gland and tachycardia. motor weakness, worse on lower extremities, hyporeflexia and intact sensation. Potassium was 1.8 mmol/l, magnesium 1.5 mg/dl, TSH <0.01 mIU/ml, FT4 4.3 ng/dl, FT3 18.3 ng/dl, anti-thyroid antibodies were elevated at 18 IU/L, and urine screen was positive for cannabinoid. Potassium was replaced, he was given propranolol and continued on methimazole. Patient's paralysis had improved in all four limbs, with serum potassium of 4.1 mmol/L. Discussion: TPP is a rarely seen condition in US but is known in an eastern Asian population, and in US the incidence rate was reported to be around 0.1 to 0.2%. American Indian are thought to be the higher risk. Underlying mechanism is imbalance in potassium homeostasis due to increased cellular Na-K ATPase pump activity with shift of potassium to the intracellular space. There are many theories regarding the cause of exaggerated intracellular potassium influx in TPP. Na-K ATPase activity is directly stimulated by thyroid hormone and insulin response to carbohydrate ingestion which promotes potassium uptake into muscle correlating with clinical observation that paralysis seen after a high carbohydrate meal. KCNJ18 gene mutations which alter the function of an inwardly rectifying potassium channel named Kir2.6 have been detected in some patients. In our patient, additional factor such as cannabis use potentiated intracellular potassium shift and hypokalemia. Recent evidence suggests that the CB1 receptor is expressed not only in the brain, but also found in lungs, heart, and skeletal muscle. Stimulation of (CB1) and activation of G protein-coupled inwardly rectifying potassium (GIRK) channels through intra-cellular signal cascades promotes hypokalemia and paralysis. Conclusion: This case raises awareness of TPP in non-Asian population and highlights the importance to consider cannabis as additional trigger for hypokalemia and paralysis. Presentation: Friday, June 16, 2023

A comparative study between angiotensin receptor neprilysin inhibitor (thiorphan/irbesartan) with each of nitrate and carvedilol in a rat model of myocardial ischemic reperfusion injury.

The combined angiotensin receptor neprilysin inhibitor is a promising cardioprotective pharmacological agent. This study investigated the beneficial effects of thiorphan (TH)/irbesartan (IRB), in myocardial ischemia-reperfusion (IR) injury, compared to each of nitroglycerin and carvedilol. Male Wistar rats were divided into five groups (10 rats/group): Sham, untreated I/R, TH/IRB + IR (0.1/10mg/kg), nitroglycerin + IR (0.2mg/kg), and carvedilol + IR (10mg/kg). Mean arterial blood pressure, cardiac functions and arrhythmia incidence, duration and score were assessed. Cardiac levels of creatine kinase-MB (CK-MB), oxidative stress, endothelin-1, ATP, Na+ /K+ ATPase pump activity and mitochondria complexes activities were measured. Histopathological examination, Bcl/Bax immunohistochemistry studies and electron microscopy examination of left ventricle were performed. TH/IRB preserved the cardiac functions and mitochondrial complexes activities, mitigated cardiac damage, reduced oxidative stress and arrhythmia severity, improved the histopathological changes and decreased cardiac apoptosis. TH/IRB showed a comparable effect to each of nitroglycerin and carvedilol in alleviating the IR injury consequences. TH/IRB showed significant preservation of mitochondrial complexes activity I and II compared to nitroglycerin. TH/IRB significantly increased LVdP/dtmax and decreased oxidative stress, cardiac damage and endothelin-1 along with increasing the ATP content, Na+ /K+ ATPase pump activity and mitochondrial complexes activity when compared to carvedilol. TH/IRB showed a cardioprotective effect in reducing IR injury that is comparable to each of nitroglycerin and carvedilol that could be explained in part by its ability to preserve mitochondrial function, increase ATP, decrease oxidative stress as well as endothelin 1.

Impairment of adrenergically-regulated thermogenesis in brown fat of obesity-resistant mice is compensated by non-shivering thermogenesis in skeletal muscle

ObjectiveNon-shivering thermogenesis (NST) mediated by uncoupling protein 1 (UCP1) in brown adipose tissue (BAT) can be activated via the adrenergic system in response to cold or diet, contributing to both thermal and energy homeostasis. Other mechanisms, including metabolism of skeletal muscle, may also be involved in NST. However, relative contribution of these energy dissipating pathways and their adaptability remain a matter of long-standing controversy. MethodsWe used warm-acclimated (30 °C) mice to characterize the effect of an up to 7-day cold acclimation (6 °C; CA) on thermoregulatory thermogenesis, comparing inbred mice with a genetic background conferring resistance (A/J) or susceptibility (C57BL/6 J) to obesity. ResultsBoth warm-acclimated C57BL/6 J and A/J mice exhibited similar cold endurance, assessed as a capability to maintain core body temperature during acute exposure to cold, which improved in response to CA, resulting in comparable cold endurance and similar induction of UCP1 protein in BAT of mice of both genotypes. Despite this, adrenergic NST in BAT was induced only in C57BL/6 J, not in A/J mice subjected to CA. Cold tolerance phenotype of A/J mice subjected to CA was not based on increased shivering, improved insulation, or changes in physical activity. On the contrary, lipidomic, proteomic and gene expression analyses along with palmitoyl carnitine oxidation and cytochrome c oxidase activity revealed induction of lipid oxidation exclusively in skeletal muscle of A/J mice subjected to CA. These changes appear to be related to skeletal muscle NST, mediated by sarcolipin-induced uncoupling of sarco(endo)plasmic reticulum calcium ATPase pump activity and accentuated by changes in mitochondrial respiratory chain supercomplexes assembly. ConclusionsOur results suggest that NST in skeletal muscle could be adaptively augmented in the face of insufficient adrenergic NST in BAT, depending on the genetic background of the mice. It may provide both protection from cold and resistance to obesity, more effectively than BAT.

Luminal H2 O2 promotes ER Ca2+ dysregulation and toxicity of palmitate in insulin-secreting INS-1E cells.

The endoplasmic reticulum (ER) lumen is not only the major site for the assembly and folding of newly synthesized proteins but also the main intracellular Ca2+ store. Ca2+ ions are involved in versatile biochemical processes, including posttranslational processing and folding of nascent proteins. Disruption of ER Ca2+ homeostasis is usually accompanied by an ER stress response that can ultimately lead to apoptosis if unresolved. Abnormal ER Ca2+ depletion has been linked to pancreatic β-cell dysfunction and death under lipotoxic conditions. However, the underlying mechanisms how the β-cell toxic saturated free fatty acid palmitate perturbs ER Ca2+ homeostasis and its interplay with other organelles are not fully understood. In the present study, we demonstrate that treatment of insulin-secreting INS-1E cells with palmitate diminished ER Ca2+ levels, elevated cytosolic/mitochondrial Ca2+ content, lowered the mitochondrial membrane potential, and ATP content. In addition, palmitate-pretreated β-cells contained significantly less luminal Ca2+ , revealed a severely impaired ER Ca2+ reuptake rate, and substantially lower insulin content. Importantly, detoxification of luminal H2 O2 by expression of the ER-resident glutathione peroxidase 8 (GPx8) abrogated the lipotoxic effects of palmitate. Moreover, GPx8 supported oxidative protein folding and preserved insulin content under lipotoxic conditions. A direct involvement of luminal H2 O2 in palmitate-mediated ER Ca2+ depletion could be corroborated by the ectopic expression of an ER-luminal active catalase. Our data point to the critical role of luminal H2 O2 in palmitate-mediated depletion of ER Ca2+ through redox-dependent impairment of Ca2+ ATPase pump activity upstream of mitochondrial dysfunction in insulin-secreting INS-1E cells.

ODP535 Thyrotoxic Periodic Paralysis (TPP) in A Young African American Male: Post Exercise

Abstract Thyrotoxic periodic paralysis (TPP) is a rare fatal disorder with hypokalemia as the hallmark feature associated with thyrotoxicosis. Compared with existing studies that TPP is prevalent among younger men of Asian descent, this study intends to complement this area of study by addressing aspects currently not fully covered involving the existence of TPP among men of African descent. This study documents the case of a 37year old African American Man with no previous history of hyperthyroidism who presented with sudden onset lower extremity paralysis and hypokalemia. It is particularly important to note that while majority of patients with TPP have preexisting hyperthyroidism, our patient presented with no history of overactive thyroid. The finding of this work fills a gap in literature highlighting the importance of separating Familial Hypokalemic periodic paralysis (FPP) and TPP at its earliest detection. We find strong and robust evidence suggesting that patients may be easily termed FPP in view of its similarity to TPP thereby leading to a delay in the diagnosis and treatment. TPP has been linked with genetic mutations in specific ion channels (calcium channels and KIR) and increased Na-K ATPase pump activity. This leads to a massive intracellular shift in potassium causing hypokalemia under the influence of excess thyroid hormone. We find that both TPP and FPP have similar clinical presentations and same precipitants hence the need for a high index of suspicion in patients with hypokalemia and sudden onset paralysis with no history of hyperthyroidism to prevent lethal complications. Our results suggest that prompt correction of hypokalemia and treatment of hyperthyroidism is key in resolving attacks and preventing lethal complications. Presentation: No date and time listed

High doses of eugenol cause structural and functional damage to the rat liver

Eugenol is a phenolic compound found in clove extract and extensively used in traditional medicine. It is unclear whether its intake can cause positive or negative effects on liver morphology and physiology in healthy individuals. Thus, we aimed to evaluate liver parameters of rats treated with 10, 20, and 40 mg kg−1 eugenol. After 60 days of treatment, liver samples were collected and analyzed by biometric, histological, biochemical, and oxidative analyses. Our results showed that 10, 20, and 40 mg kg−1 eugenol did not alter body and liver weights, serum and hepatic ALT levels and catalase, glutathione-s-transferase, total, Ca2+, and Mg2+ ATPases activities in treated animals. However, 20 and 40 mg kg−1 eugenol reduced Na+/K+ ATPase pump activity and blood glucose levels. They also increased hepatic glycogen content, superoxide dismutase activity, ferric reducing antioxidant power, and nitric oxide and malondialdehyde levels. Still, 20 and 40 mg kg−1 eugenol caused structural and functional damage to the liver tissue of eugenol-treated rats. We concluded that 10 mg kg−1 eugenol is a safe dose for consumption in long-term treatment for rats. Doses higher than 20 mg kg−1 lead to hepatic damage that can impair vital processes of liver functionality.

Calcium cycling as a mediator of thermogenic metabolism in adipose tissue.

Canonical non-shivering thermogenesis (NST) in brown and beige fat relies on uncoupling protein 1 (UCP1)-mediated heat generation, although alternative mechanisms of NST have been identified, including sarcoplasmic reticulum (SR)-calcium cycling. Intracellular calcium is a crucial cell signaling molecule for which compartmentalization is tightly regulated, and the sarco-endoplasmic calcium ATPase (SERCA) actively pumps calcium from the cytosol into the SR. In this review, we discuss the capacity of SERCA-mediated calcium cycling as a significant mediator of thermogenesis in both brown and beige adipocytes. Here, we suggest two primary mechanisms of SR calcium mediated thermogenesis. The first mechanism is through direct uncoupling of the ATPase and calcium pump activity of SERCA, resulting in the energy of ATP catalysis being expended as heat in the absence of calcium transport. Regulins, a class of SR membrane proteins, act to decrease the calcium affinity of SERCA and uncouple the calcium transport function from ATPase activity, but remain largely unexplored in adipose tissue thermogenesis. A second mechanism is through futile cycling of SR calcium whereby SERCA-mediated SR calcium influx is equally offset by SR calcium efflux, resulting in ATP consumption without a net change in calcium compartmentalization. A fuller understanding of the functional and mechanistic role of calcium cycling as a mediator of adipose tissue thermogenesis and how manipulation of these pathways can be harnessed for therapeutic gain remains unexplored. Significance Statement Enhancing thermogenic metabolism in brown or beige adipose tissue may be of broad therapeutic utility to reduce obesity and metabolic syndrome. Canonical BAT-mediated thermogenesis occurs via uncoupling protein 1 (UCP1). However, UCP1-independent pathways of thermogenesis, such as sarcoplasmic (SR) calcium cycling, have also been identified, but the regulatory mechanisms and functional significance of these pathways remain largely unexplored. Thus, this mini-review discusses the state of the field with regard to calcium cycling as a thermogenic mediator in adipose tissue.

Na+/K+-ATPase Activation by cAMP in the Midgut of Lutzomyia longipalpis (Lutz & Neiva, 1912; Diptera: Psychodidae).

Lutzomyia longipalpis (Lutz & Neiva, 1912) females have been intensively studied regarding the regulation of midgut pH. The mechanisms involved in pH regulation are complex, and some aspects remain to be clarified. Here, we investigated the role of the Na+/K+-ATPase pump as an electrochemical potential generator and its modulation by the second messenger cAMP in the midgut of female L. longipalpis. Our results suggest that not only may Na+/K+-ATPase be the main generator of an electrochemical potential across membranes in the midgut of female L. longipalpis, but also its activity is positively regulated by cAMP. cAMP-mediated Na+/K+-ATPase pump activity might be necessary to maintain the transport of the nutrients produced during blood digestion.

Microanatomy of the antennal glands and their Na+/K+-ATPase activity in three true crab species (Brachyura), Portunus pelagicus (Linnaeus, 1758) (Portunidae), Macrophthalmus dentipes Lucas in Guérin, 1836 (Macrophthalmidae) and Eriocheir hepuensis Dai, 1991 (Varunidae)

Abstract There are differences between various crab species in the function and structure of organs involved in ionic and osmotic regulation processes. The antennal glands together constitute one of the most important organs involved in the osmoregulation in crabs. The present investigation aimed to study the tissue structure of the antennal glands and their Na+/K+-ATPase (NKA) pump activity in three true crab species from three different habitats, including the marine (Portunus pelagicus (Linnaeus, 1758)), the estuarine (Macrophthalmus dentipes Lucas in Guérin, 1836) and the freshwater habitat (Eriocheir hepuensis Dai, 1991). In this regard, the tissue structure of the antennal glands and the activity of the Na+/K+-ATPase (NKA) pump were assessed in these three selected species. The results showed that the antennal glands in all studied species consisted of two anterior parts and a posterior part. The anterior parts are composed of the proximal tubular region (PT) and the distal tubular region (DT). The PT and DT parts comprised the coelomosac and labyrinths in the anterior portion, and the bladder located in the posterior portion. However, despite the similarity in the general tissue structure of the antennal gland in marine, estuarine and freshwater crab species, some structural differences were observed between those species. Labyrinth cells, coelomosac podocytes, and bladder cells in the estuarine crab M. dentipes contained large vacuoles especially on the top (i.e., near the lumen) of the cells. The highest amount of NKA pump activity was measured in the antennal glands of M. dentipes (). The NKA pump plays a more important role in the estuary and seawater adaptation of crabs, but freshwater species are not highly dependent on the NKA enzyme for osmoregulation.

Increasing SERCA function promotes initiation of calcium sparks and breakup of calcium waves.

Increasing sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) pump activity enhances sarcoplasmic reticulum calcium (Ca) load, which increases both ryanodine receptor opening and driving force of Ca release flux. Both of these effects promote Ca spark formation and wave propagation. However, increasing SERCA activity also accelerates local cytosolic Ca decay as the wave front travels to the next cluster, which limits wave propagation. As a result, increasing SERCA pump activity has a biphasic effect on the propensity of arrhythmogenic Ca waves, but a monotonic effect to increase Ca spark frequency and amplitude. Waves of sarcoplasmic reticulum (SR) calcium (Ca) release can cause arrhythmogenic afterdepolarizations in cardiac myocytes. Ca waves propagate when Ca sparks at one Ca release unit (CRU) recruit new Ca sparks in neighbouring CRUs. Under normal conditions, Ca sparks are too small to recruit neighbouring Ca sparks where Ca sensitivity is also low. However, under pathological conditions such as a Ca overload or ryanodine receptor (RyR) sensitization, Ca sparks can be larger and propagate more readily as macro-sparks or full Ca waves. Increasing SERCA pump activity promotes SR Ca load, which promotes RyR opening and increases driving force of the Ca release flux from SR to cytosol, promoting Ca waves. However, high sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) activity can also decrease local cytosolic [Ca] as it approaches the next CRU, thereby reducing wave appearance and propagation. In this study, we use a physiologically detailed model of subcellular Ca cycling and experiments in phospholamban-knockout mice, to show how Ca waves are initiated and propagate and how different conditions contribute to the generation and propagation of Ca waves. We show that reducing diffusive coupling between Ca sparks by increasing SERCA activity prevents Ca waves by reducing [Ca] at the next CRU, as do Ca buffers, low intra-SR Ca diffusion and distance between CRUs. Increasing SR Ca uptake rate has a biphasic effect on Ca wave propagation; initially it enhances Ca spark probability and amplitude and CRU coupling, thereby promoting arrhythmogenic Ca wave propagation, but at higher levels SR Ca uptake can abort those arrhythmogenic Ca waves.

Hypoxia-induced increase in GABA content is essential for restoration of membrane potential and preventing ROS-induced disturbance to ion homeostasis

When plants are exposed to hypoxic conditions, the level of γ-aminobutyric acid (GABA) in plant tissues increases by several orders of magnitude. The physiological rationale behind this elevation remains largely unanswered. By combining genetic and electrophysiological approach, in this work we show that hypoxia-induced increase in GABA content is essential for restoration of membrane potential and preventing ROS-induced disturbance to cytosolic K+ homeostasis and Ca2+ signaling. We show that reduced O2 availability affects H+-ATPase pumping activity, leading to membrane depolarization and K+ loss via outward-rectifying GORK channels. Hypoxia stress also results in H2O2 accumulation in the cell that activates ROS-inducible Ca2+ uptake channels and triggers self-amplifying “ROS-Ca hub,” further exacerbating K+ loss via non-selective cation channels that results in the loss of the cell's viability. Hypoxia-induced elevation in the GABA level may restore membrane potential by pH-dependent regulation of H+-ATPase and/or by generating more energy through the activation of the GABA shunt pathway and TCA cycle. Elevated GABA can also provide better control of the ROS-Ca2+ hub by transcriptional control of RBOH genes thus preventing over-excessive H2O2 accumulation. Finally, GABA can operate as a ligand directly controlling the open probability and conductance of K+ efflux GORK channels, thus enabling plants adaptation to hypoxic conditions.

Cardiac Glycosides as Immune System Modulators.

Cardiac glycosides (CGs) are natural steroid compounds occurring both in plants and animals. They are known for long as cardiotonic agents commonly used for various cardiac diseases due to inhibition of Na+/K+-ATPase (NKA) pumping activity and modulating heart muscle contractility. However, recent studies show that the portfolio of diseases potentially treatable with CGs is much broader. Currently, CGs are mostly studied as anticancer agents. Their antiproliferative properties are based on the induction of multiple signaling pathways in an NKA signalosome complex. In addition, they are strongly connected to immunogenic cell death, a complex mechanism of induction of anticancer immune response. Moreover, CGs exert various immunomodulatory effects, the foremost of which are connected with suppressing the activity of T-helper cells or modulating transcription of many immune response genes by inhibiting nuclear factor kappa B. The resulting modulations of cytokine and chemokine levels and changes in immune cell ratios could be potentially useful in treating sundry autoimmune and inflammatory diseases. This review aims to summarize current knowledge in the field of immunomodulatory properties of CGs and emphasize the large area of potential clinical use of these compounds.

GENES ASSOCIATED WITH BLOOD PRESSURE TRAITS SHOW CELL TYPE-SPECIFIC EXPRESSION AND CONTROL MULTIPLE CAUSAL LINKS TO BLOOD PRESSURE REGULATION IN THE HUMAN KIDNEY

Objective: Hypertension affects 35% of the global adult population. It is a major risk factor for stroke, heart failure and coronary artery disease and is the leading preventable risk factor for premature death globally. Here we describe our approach to determine the causal pathways between genetic variants and blood pressure through the expression of kidney genes. Design and method: We used a catalogue of all known blood pressure associated genetic variants, gene expression profiles from 430 human kidney tissue samples and a single-cell gene expression data set to identify causal relationships with blood pressure using a colocalisation analysis and an ensemble of Mendelian randomisation methods. Results: By combining genome-wide genotypes and gene expression profiles from RNA-sequencing in an expression quantitative trait loci (eQTL) analysis we identify 418 genes where expression is modified by genetic variants previously associated with blood pressure traits. To investigate how these genes are expressed in the kidney we used gene expression profiles from 41,778 human renal cells and identified 390 (93%) of these genes to be expressed in these cells with 69 (18%) showing expression specific to individual renal cell types. There was a statistically significant enrichment for expression of blood pressure associated genes in juxtaglomerular cells and cells from the collecting tubule. We identified 111 genes where the eQTL and blood pressure association colocalised and then applied an ensemble of Mendelian randomisation (MR) methods to determine which genes also have a causal effect on blood pressure in the kidney. Our MR analysis identified 53 (13%) genes where expression is causally linked to blood pressure, these included: guanylate cyclase 1 (GUCY1A3), the main receptor for nitric oxide and the target of riociguat and nuclear receptor coactivator 7 (NCOA7) which regulates vacuolar proton pumping ATPase (V-ATPase) activity in the intercalated cells of the collecting duct. Conclusions: We have identified 53 kidney genes where expression is causally linked to blood pressure. This not only highlights the importance of the kidney in blood pressure regulation, but also identifies putative targets for drug development or repurposing.

Cardioprotective action of chia (Salvia hispanica L.) in ovariectomized rats fed a high fat diet.

The reduction in estrogen levels is associated with the increased risk factors for cardiovascular disease development. The present study aimed to evaluate the effect of chia consumption in a standard diet (SD) or high fat diet (HFD) on ovariectomized (OVX) and non-ovariectomized (SHAM) rats, in relation to biometric measurements, oxidative stress, mineral content and ATPase enzymes in the heart. The study was conducted with 80 female Wistar rats, which received a SD or HFD for 18 weeks. During the first 7 weeks, the animals received the SD or HFD. Then, 40 rats were ovariectomized and 40 rats were SHAM operated. After recovery from surgery, the animals were allocated to 8 groups (n = 10) and they received one of the following diets for 8 weeks: SD, SD + chia, HFD and HFD + chia. In the OVX group, HFD increased weight gain, adiposity, cardiac hypertrophy, and nitric oxide (NO) and K concentration and decreased the Na+/K+ATPase activity. In combination with HFD, ovariectomy decreased the catalase activity, Mg, Cu and Zn concentration, total ATPase activity, and Na+/K+ATPase and Mg2 + ATPase activities; this group also presented higher NO, Ca, K, Fe and Mn concentration in the heart. The SHAM group fed chia presented a lower fat content in the heart. In the OVX group fed HFD, chia increased the activity of superoxide dismutase, decreased NO and maintained the content of minerals and ATPase enzymes. Thus, chia improved the biometric parameters of the heart, the antioxidant activity and maintained the content of minerals and ATPase enzymes, showing a cardioprotective action, but without reversing the deleterious effects of ovariectomy.

Angiotensin receptor-neprilysin inhibitior (thiorphan/irbesartan) decreased ischemia-reperfusion induced ventricular arrhythmias in rat; in vivo study

Ventricular arrhythmias are considered as a major risk of sudden cardiac death. This study was designed to investigate the potential effects of angiotensin receptor neprilysin inhibitor; thiorphan/irbesartan (TH/IRB) combination therapy on myocardial ischemic-reperfusion (I/R)-induced arrhythmia. Fifty male Wistar rats were divided into 5 groups; (I, II): Sham, I/R both received DMSO intraperitoneally before the procedure. (III, IV, V): TH/IRB + IR (0.1/5 mg/kg, 0.1/10 mg/kg and 0.1/15 mg/kg). The drugs were injected intraperitoneally 15 min before I/R induction. Electrocardiograms changes, mean arterial blood pressure, incidence of ventricular tachycardia (VT), incidence of ventricular fibrillation (VF) and arrhythmia score were assessed. Cardiac levels of creatinine kinase–MB (CK-MB), Malondialdehyde (MDA), superoxide dismutase (SOD), endothelin-1 (ET-1), ATP content, and Na+/K+-ATPase pump activity were measured. TH (0.1 mg/kg) in combination with IRB (5, 10 and 15 mg/kg) produced significant decrease in QTc interval duration, ST height, incidence of VT and VF, duration of VT + VF, and arrhythmia score compared to I/R group. All treated groups showed significant decrease in the cardiac levels of: CK-MB, MDA and ET-1 and significant increase in SOD, ATP content, and Na+/K+-ATPase pump activity compared to I/R. TH/IRB + IR (0.1/10 mg/kg) group produced significant decrease in CK-MB, MDA and ET-1 and a significant increase in SOD, ATP content, and Na+/K+-ATPase pump activity compared to other treated groups. In conclusion, angiotensin receptor neprilysin inhibitor (thiorphan/irbesartan) decreased arrhythmia score and decreased cardiac damage. These could be explained in part by its ability to decrease oxidative stress and ET-1, increase ATP, and Na+/K+-ATPase pump activity in this rat model of I/R-induced arrhythmia.

Effect of 5-HT2C receptor agonist and antagonist on chronic unpredictable stress (CUS) - Mediated anxiety and depression in adolescent Wistar albino rat: Implicating serotonin and mitochondrial ETC-I function in serotonergic neurotransmission

Anxiety and depression are among the major neuropsychiatric disorders worldwide, and yet the etiologies of these disorders remain unclear to date. Chronic unpredictable stress (CUS) procedure mimics several behavioral characteristics such as anxiety and depression in rodents. Using this animal model, we have attempted to understand the serotonergic system in the hippocampus and prefrontal cortex, while using the 5-HT2CR agonist and antagonist in evaluating 5-HT2C receptor neurotransmission. A decrease in serotonin (5-HT) level, tryptophan hydroxylase-2 activity and, 5-HT2CR receptor protein down-regulation in the CUS exposed group, explains the involvement of 5-HT and 5-HT2CR neurotransmission in the genesis of anxiety and depression. Besides, the oxidative stress - attenuated electrolyte imbalance via decrease ATPase pump activity, and compromised oxidative phosphorylation via decrease ETC-I activity are some of the underlying factors affecting neuronal cell survival and serotonergic neurotransmission. To complement our finding, altered behavioral performance scored in the open field test, elevated plus maze test, and the forced swim test, when exposed to CUS is indicative or consistent with anxiety, depression, emotional and locomotor status of the animals. Keeping these findings in mind, treatment with 5-HT2CR agonist (1-Methylpsilocin at 0.7 mg/kg), and 5-HT2CR antagonist (RS-102221 hydrochloride at 1 mg/kg) displayed varying results. One prominent finding was the anxiolytic ability of the 5-HT2CR agonist and the anti-depressive ability of the 5-HT2CR antagonist on the 7th-day treatment. Though the exact mechanism of action is not clear, their ability to equilibrate brain redox status, restoring Ca2+ level via Ca2+ATPase pump activity, and sustaining the mitochondrial bioenergetics can all be accounted for facilitating neurogenesis and the serotonergic system.

SUN-LB88 Thyrotoxic Periodic Paralysis in Hispanic Patients

BACKGROUND: Thyrotoxic periodic paralysis (TPP) presents as acute intermittent attacks of weakness related to hypokalemia, commonly reported in Asians and rare in Hispanics(1). Patients with TPP will have triiodothyronine (T3) triggered increased Na+/K+ ATPase pump activity and transcription of the KCNJ18 gene that encodes for the Kir2.6 channel(2). This permits insulin, catecholamines, stress and alcohol(3) to increase cellular intake of potassium, which causes depolarization and leads to weakness and paralysis. We report a case of TPP in a young Hispanic man who presented with lower extremity weakness and falls. CASE PRESENTATION: A 34-year-old Hispanic man with Graves’ disease, non-adherent to medications presented with generalized weakness, more pronounced in legs, and recurrent falls. Physical examination was unremarkable except for mild enlargement of thyroid gland and abnormal gait due to weakness. Laboratory data showed hypokalemia of 1.8 mmol/L (3.7-5.1 mmol/L) and a TSH level of <0.004 mIU/L (0.34-5.6 mIU/L). Free T4 3.74 ng/dL (0.6-1.6 ng/dL), free T3 597 pg/dL (230-420 Pg/dL), thyroid stimulating Ig 148 (<130). Electrocardiogram did not show U waves. Radio iodine 123 scan of thyroid revealed diffusely increased 24-hour radioactive uptake of 66.5% (10-30%). The patient was diagnosed with TPP and supplemented with three doses of potassium 40 mEq IV infusion. Methimazole and metoprolol were started. He made a good clinical recovery within days. After discharge, he was treated with I-131 (13 mci) and developed postablative hypothyroidism on long term. He was euthyroid on levothyroxine. He did not have any recurrence of weakness at 7-year follow-up. CONCLUSION: TPP is uncommonly seen in Hispanics patients as opposed to Asians(3). Physicians should consider TPP as part of the differential diagnosis in young hyperthyroid Hispanic men presenting with weakness or paralysis, as early recognition and treatment can reduce recovery time and potentially prevent tachyarrhythmia or death. REFERENCES: 1. Matta A, Koppala J, Gossman W. Thyrotoxic hypokalaemic periodic paralysis: a rare presentation of Graves’ disease in a Hispanic patient. BMJ Case Rep. 2014;2014. 2. Ryan DP, Ptacek LJ. Mutations in Potassium Channel Kir2.6 Cause Susceptibility to Thyrotoxic Hypokalemic Periodic Paralysis. Cell, 140(1), pp.88-98. 3. Amblee, A. and Gulati, S. (2016). Thyrotoxic Periodic Paralysis: Eight Cases in Males of Hispanic Origin from a Single Hospital. AACE Clinical Case Reports, 2(1), pp.e58-e64.

Changes in Ca2+ release in human red blood cells under pulsed magnetic field

Since Ca2+ is related to a highly versatile intracellular signal to regulate many different cellular processes, the role of Ca2+ and its concentration in red blood cells (RBCs) are very important. Accordingly, in this study, we have elucidated the relationship between pulsed magnetic field (PMF) and intracellular Ca2+ level to understand how PMF affects oxidative stressed RBCs. In addition, the effect of verapamil on RBCs in Ca2+ level, compared to the effect of PMF was investigated because verapamil, as a reducing agent, blocks Ca2+ influx into RBCs. Tert-butyl hydroperoxide (tBHP) was used to induce oxidative stress in RBCs with various concentrations of 0.1 to 400 mM. Our PMF stimulator has the maximum intensity of 0.27 T at a transition time of 102 μs with pulse intervals of 1 Hz. Our result showed that Ca2+ level is remarkably decreased in RBCs treated verapamil, RBCs exposed to PMF after tBHP(0.1 mM) treatment, and RBCs treated tBHP(0.1 mM) after PMF exposure, respectively. This means that PMF not only reduces Ca2+ level of inner RBC effectively but also blocks inhibiting Ca2+ pump ATPase activity. Also it seems that PMF appears to act similar to reducing agent. For hemolysis change in oxidative stressed RBCs with or without PMF, using western blot and gel analysis, it is observed that hemolysis declines in control RBCs and tBHP(0.1 mM)-treated RBCs exposed to PMF, and in verapamil treated sample. The RBC deformability (RBCD) was also improved after PMF exposure. Therefore, the results of the present study support that PMF gives RBCs positive effect consistently in Ca2+ level and plays a role in preventing RBC hemolysis from oxidative stress and improving RBCD. In conclusion, our study suggests a possibility to use PMF as a new modality in improving circulatory regulation in hemodynamic derangements in disease. We need to optimize PMF intensity and stimulated duration for clinical use.

Uncoupling of sarcoendoplasmic reticulum calcium ATPase pump activity by sarcolipin as the basis for muscle non-shivering thermogenesis.

Thermogenesis in endotherms relies on both shivering and non-shivering thermogenesis (NST). The role of brown adipose tissue (BAT) in NST is well recognized, but the role of muscle-based NST has been contested. However, recent studies have provided substantial evidence for the importance of muscle-based NST in mammals. This review focuses primarily on the role of sarcoplasmic reticulum (SR) Ca2+-cycling in muscle NST; specifically, it will discuss recent data showing how uncoupling of sarcoendoplasmic reticulum calcium ATPase (SERCA) (inhibition of Ca2+ transport but not ATP hydrolysis) by sarcolipin (SLN) results in futile SERCA pump activity, increased ATP hydrolysis and heat production contributing to muscle NST. It will also critically examine how activation of muscle NST can be an important factor in regulating metabolic rate and whole-body energy homeostasis. In this regard, SLN has emerged as a powerful signalling molecule to promote mitochondrial biogenesis and oxidative metabolism in muscle. Furthermore, we will discuss the functional interplay between BAT and muscle, especially with respect to how reduced BAT function in mammals could be compensated by muscle-based NST. Based on the existing data, we argue that SLN-mediated thermogenesis is an integral part of muscle NST and that muscle NST potentially contributed to the evolution of endothermy within the vertebrate clade. This article is part of the theme issue 'Vertebrate palaeophysiology'.

Ascleposide, a natural cardenolide, induces anticancer signaling in human castration-resistant prostatic cancer through Na+ /K+ -ATPase internalization and tubulin acetylation.

Cardiac glycosides, which inhibit Na+ /K+ -ATPase, display inotropic effects for the treatment of congestive heart failure and cardiac arrhythmia. Recent studies have suggested signaling downstream of Na+ /K+ -ATPase action in the regulation of cell proliferation and apoptosis and have revealed the anticancer activity of cardiac glycosides. The study aims to characterize the anticancer potential of ascleposide, a natural cardenolide, and to uncover its primary target and underlying mechanism against human castration-resistant prostate cancer (CRPC). Cell proliferation was examined in CRPC PC-3 and DU-145 cells using sulforhodamine B assay, carboxyfluorescein succinimidyl ester staining assay and clonogenic examination. Flow cytometric analysis was used to detect the distribution of cell cycle phase, mitochondrial membrane potential, intracellular Na+ and Ca2+ levels, and reactive oxygen species production. Protein expression was examined using Western blot analysis. Endocytosis of Na+ /K+ -ATPase was determined using confocal immunofluorescence microscopic examination. Ascleposide induced an increase of intracellular Na+ and a potent antiproliferative effect. It also induced a decrease of G1 phase distribution while an increase in both G2/M and apoptotic sub-G1 phases, and downregulated several cell cycle regulator proteins, including cyclins, Cdk, p21, and p27 Cip/Kip proteins, Rb and c-Myc. Ascleposide decreased the expression of antiapoptotic Bcl-2 members (eg, Bcl-2 and Mcl-1) but upregulated proapoptotic member (eg, Bak), leading to a significant loss of mitochondrial membrane potential and activation of both caspase-9 and caspase-3. Ascleposide also dramatically induced tubulin acetylation, leading to inhibition of the catalytic activity of Na+ /K+ -ATPase. Notably, extracellular high K+ (16 mM) significantly blunted ascleposide-mediated effects. Furthermore, ascleposide induced a p38 MAPK-dependent endocytosis of Na+ /K+ -ATPase and downregulated the protein expression of Na+ /K+ -ATPase α1 subunit. Ascleposide displays antiproliferative and apoptotic activities dependent on the inhibition of Na+ /K+ -ATPase pumping activity through p38 MAPK-mediated endocytosis of Na+ /K+ -ATPase and downregulation of α1 subunit, which in turn cause tubulin acetylation and cell cycle arrest. Cell apoptosis is ultimately triggered by the activation of caspase cascade attributed to mitochondrial damage through the downregulation of Bcl-2 and Mcl-1 protein expressions while upregulation of Bak protein levels. The data also suggest the potential of ascleposide in anti-CRPC development.