Excessive Calcium Release Research Articles

Preconditioned 70S30C bioactive glass foams promote osteogenesis in vivo

Bioactive glass scaffolds (70S30C; 70% SiO2 and 30% CaO) produced by a sol–gel foaming process are thought to be suitable matrices for bone tissue regeneration. Previous in vitro data showed bone matrix production and active remodelling in the presence of osteogenic cells. Here we report their ability to act as scaffolds for in vivo bone regeneration in a rat tibial defect model, but only when preconditioned. Pretreatment methods (dry, pre-wetted or preconditioned without blood) for the 70S30C scaffolds were compared against commercial synthetic bone grafts (NovaBone® and Actifuse®). Poor bone ingrowth was found for both dry and wetted sol–gel foams, associated with rapid increase in pH within the scaffolds. Bone ingrowth was quantified through histology and novel micro-CT image analysis. The percentage bone ingrowth into dry, wetted and preconditioned 70S30C scaffolds at 11weeks were 10±1%, 21±2% and 39±4%, respectively. Only the preconditioned sample showed above 60% material–bone contact, which was similar to that in NovaBone and Actifuse. Unlike the commercial products, preconditioned 70S30C scaffolds degraded and were replaced with new bone. The results suggest that bioactive glass compositions should be redesigned if sol–gel scaffolds are to be used without preconditioning to avoid excess calcium release.

Atividades de fosfolipase A2 secretada e glutationa peroxidase em filés PSE (pale, soft, exudative) de frango

The excessive release of calcium from the sarcoplasmic reticulum during the installation of PSE (Pale, Soft, Exudative) meat, leads to increase of phospholipase A2 (PLA2) activity and consequently causes a higher lipid oxidation. In contrast, glutathione peroxidase (GSH-Px) is a selenium-dependent antioxidant enzyme that prevents the oxidative damage. The aim of this work was to investigate the secreted PLA2 (sPLA2) and GSH-PX activities in PSE poultry meat. Breast meat samples (Pectoralis major m.) (n=24) were obtained from commercial slaughterhouse. Samples were classified as PSE and Control Meat based on pH and L* values, fillets with pH1h30? 6.0 and L24h* ?53.0 as PSE and fillets with pH1h30> 6.0 and L24h*<53.0 with Control. sPLA2 was analyzed using enzymatic assay kit and GSH-Px was measured a coupled assay procedure recording the NADPH (nicotinamide adenine dinucleotide phosphate reduced) oxidation. The sPLA2 average was 0.295 Umg-1 of protein for PSE meat and 0.380 Umg-1 of protein for Control meat, did not differ significantly (p >0,05). The GSH-Px activity was approximately 24,4% higher (p?0,05) for Control meat when compared to PSE meat. The sPLA2 activity of PSE fillets did not changed, however PSE fillets present the enzymatic system of antioxidant defense compromised with lower GSH-PX activity.

Atividades de fosfolipase A2 secretada e glutationa peroxidase em filés PSE (pale, soft, exudative) de frango

The excessive release of calcium from the sarcoplasmic reticulum during the installation of PSE (Pale, Soft, Exudative) meat, leads to increase of phospholipase A2 (PLA2) activity and consequently causes a higher lipid oxidation. In contrast, glutathione peroxidase (GSH-Px) is a selenium-dependent antioxidant enzyme that prevents the oxidative damage. The aim of this work was to investigate the secreted PLA2 (sPLA2) and GSH-PX activities in PSE poultry meat. Breast meat samples ( Pectoralis major m. ) (n=24) were obtained from commercial slaughterhouse. Samples were classified as PSE and Control Meat based on pH and L* values, fillets with pH1h30? 6.0 and L24h* ?53.0 as PSE and fillets with pH1h30> 6.0 and L24h* 0,05). The GSH-Px activity was approximately 24,4% higher (p?0,05) for Control meat when compared to PSE meat. The sPLA2 activity of PSE fillets did not changed, however PSE fillets present the enzymatic system of antioxidant defense compromised with lower GSH-PX activity.

The Bcl-2—IP3 Receptor Interaction As a Novel Target for Therapeutics in Myeloma

Abstract 4011The anti-apoptotic Bcl-2 (B-cell lymphoma 2) protein was first identified in follicular lymphoma, where the Bcl-2 gene was dysregulated as a result of chromosomal translocation. The anti-apoptotic activity of Bcl-2 can be attributed to two mechanisms conferred by structurally distinct domains. First, the BH domains 1–3 of Bcl-2 dimerize with pro-apoptotic members of the Bcl-2 family, such as Bax and Bak, and maintain mitochondrial integrity. Small molecules targeting these domains on Bcl-2 (e.g. ABT-737 and ABT-263/Navitoclax®) are under clinical evaluation. A less explored activity of Bcl-2 is that its BH4 domain functionally interacts with the IP3 receptor located on the endoplasmic reticulum (ER) membrane, the major organelle involved in regulating calcium homeostasis. Through this interaction, Bcl-2 inhibits IP3 receptor channel opening and prevents excessive calcium release from ER that can be harmful to cells. Our strategy is to disrupt the Bcl-2—IP3 receptor interaction to induce cell death in lymphoid malignant cells.Previously, our group has shown that an IP3 receptor derived peptide, or IDP, blocks the Bcl-2—IP3 receptor interaction and thereby induces elevation of cytosolic calcium in lymphoma cell lines. A modified version of the peptide—IDP-DD/AA, with an asparaginase cleavage site removed, is more potent in inducing calcium elevation as well as apoptosis in primary chronic lymphocytic leukemia (CLL) cells. Concentrations of the IDP-DD/AA peptide that killed CLL cells did not affect the viability of normal lymphocytes, suggesting a therapeutic window. We further exploited the cytotoxic activities in a panel of leukemia/lymphoma/myeloma cell lines and found that over 60% of myeloma cell lines tested were sensitive to the IDP-DD/AA peptide but not to the control peptide. Furthermore, in the sensitive myeloma cell lines treated with the IDP-DD/AA peptide, high amplitude cytosolic calcium elevation preceded caspase-3 activation and apoptotic morphology, suggesting the involvement of a calcium-mediated intrinsic apoptotic pathway.The in vivo anti-tumor activity of the IDP peptide was assessed in a myeloma xenograft model. Subcutaneous tumors were established in nude mice using the human myeloma cell line, NCI-H929. Randomly grouped mice treated by in-tumor injection of the IDP-DD/AA peptide showed prominent inhibition of tumor growth compared to saline-injected control. One of three mice treated with the IDP-DD/AA peptide showed no visible sign of tumor at the end of treatment. The data proffer promise of therapeutic use of the IDP peptide in myeloma disease.The effect of combinatory treatment of the IDP-DD/AA peptide and current anti-myeloma drugs (Bortezomib/Valcade®, doxorubicin/Adriamycin®, melphalan) were evaluated. Co-treatment of the myeloma cell line NCI-H929 cells with the IDP-DD/AA peptide and each one of the three anti-myeloma drugs induced synergistic cytotoxicity with combinatory indices (CI) less than 1. The data suggest that the IDP-DD/AA peptide augments cell killing activity of current chemotherapy drugs.In conclusion, the IDP-DD/AA peptide represents a novel anti-cancer agent that targets the anti-apoptotic Bcl-2 protein. The peptide exhibits both in vitro and in vivo anti-myeloma activity. Moreover, the IDP-DD/AA peptide can function as a single agent (in myeloma or CLL) as well as in combination with current anti-myeloma chemotherapy drugs. Disclosures:No relevant conflicts of interest to declare.

Rocuronium and malignant hyperthermia

Beggs et al.1 presented two interesting case reports postulating an association between rocuronium and malignant hyperthermia (MH). Although the observed fever in these patients declined after discontinuation of rocuronium, several points need further discussion. First, the definition of MH includes hypercapnia, lactic acidosis, and skeletal muscle hypermetabolism due to excessive sarcoplasmic calcium release via mutated ryanodine receptors after exposure to triggering agents.2 As both patients had complex medical conditions (e.g., acute respiratory distress syndrome, history of cardiopulmonary resuscitation, sepsis), disorders such as metabolic acidosis might have occurred for various reasons. Although MH certainly could have been one reason, this seems rather unlikely since the acidosis was not even temporally related to the use of rocuronium. Second, the body temperature in the reported cases remained elevated for several days, which is not typical of MH crisis. Even though there have been several reports of MH with hyperthermia as the only symptom,3 other findings, such as the modest creatine kinase elevation (351 units/L in patient 1), are inconsistent with a diagnosis of MH. Furthermore, unexplained fever among patients in the intensive care unit is very common and associated with a large variety of differential diagnoses.4

Multiple Defects in Energy Metabolism in Alzheimers Disease

Alzheimer's disease (AD) is the most common form of dementia in old age. Cognitive impairment in AD may be partially due to overall hypometabolism. Indeed, AD is characterized by an early region-specific decline in glucose utilization and by mitochondrial dysfunction, which have deleterious consequences for neurons through increased production of reactive oxygen species (ROS), ATP depletion and activation of cell death processes. In this article, we provide an overview of the alterations on energetic metabolism occurring in AD. First, we resume the evidences that link the 'metabolic syndrome' with increased risk for developing AD and revisit the major changes occurring on both extra-mitochondrial and mitochondrial metabolic pathways, as revealed by imaging studies and biochemical analysis of brain and peripheral samples obtained from AD patients. We also cover the recent findings on cellular and animal models that highlight mitochondrial dysfunction as a fundamental mechanism in AD pathogenesis. Recent evidence posits that mitochondrial abnormalities in this neurodegenerative disorder are associated with changes in mitochondrial dynamics and can be induced by amyloid-beta (Aβ) that progressively accumulates within this organelle, acting as a direct toxin. Furthermore, Aβ induces activation of glutamate N-methyl-D-aspartate receptors (NMDARs) and/or excessive release of calcium from endoplasmic reticulum (ER) that may underlie mitochondrial calcium dyshomeostasis thereby disturbing organelle functioning and, ultimately, damaging neurons. Throughout the review, we further discuss several therapeutic strategies aimed to restore neuronal metabolic function in cellular and animal models of AD, some of which have reached the stage of clinical trials.

Grape seed extract enhances eNOS expression and NO production through regulating calcium‐mediated AKT phosphorylation in H2O2‐treated endothelium

GSE (grape seed extract) has been shown to exhibit protective effects against cardiovascular events and atherosclerosis, although the underlying molecular mechanisms of action are unknown. Herein, we assessed the ability of GSE to enhance eNOS (endothelial nitric oxide synthase) expression and NO (nitric oxide) production in H2O2 (hydrogen peroxide)-treated HUVECs (human umbilical vein endothelial cells). GSE enhanced eNOS expression and NO release in H2O2-treated cells in a dose-dependent manner. GSE inhibited intracellular ROS (reactive oxygen species) and reduced intracellular calcium in a dose-dependent manner in H2O2-treated cells, as shown by confocal microscopy. ROS was inhibited in cells pretreated with 5.0 microM GSE, 2.0 microM TG (thapsigargin) and 20.0 microM 2-APB (2-aminoethoxydiphenyl borate) instead of 0.25 microM extracellular calcium. In addition, GSE enhanced eNOS expression and reduced ROS production via increasing p-AKT (AKT phosphorylation) with high extracellular calcium (13 mM). In conclusion, GSE protected against endothelial injury by up-regulation of eNOS and NO expression via inhibiting InsP3Rs (inositol 1,4,5-trisphosphate receptors)-mediated intracellular excessive calcium release and by activating p-AKT in endothelial cells.

The Cytoprotective Effects of Dantrolene

Calcium, as a second messenger, has an important role in a variety of cellular functions. However, disruption of intracellular calcium homeostasis leads to cytotoxicity and cell death. Excessive calcium release from intracellular stores, via the calcium channel ryanodine receptor, contributes to cell damage. Dysfunction of calcium homeostasis is established in tissue culture and animal models of ischemia, hypoxia, seizure, trauma, anesthesia, and neurodegenerative diseases. Dantrolene, the primary drug to treat malignant hyperthermia, is a ryanodine receptor antagonist. Dantrolene inhibits abnormal calcium release from the sarco-endoplasmic reticulum, which is the primary intracellular calcium store. Dantrolene has been investigated widely for its possible cytoprotective effects against cell damage in different tissue culture or animal models of diseases involving cytotoxicity induced by disruption of intracellular calcium homeostasis in pathogenesis. In this review, we summarize the role of the disruption of intracellular calcium homeostasis on cell death, the pharmacologic and pharmacokinetic features of dantrolene, and the cytoprotective effects and potential application of dantrolene for the inhibition of cell damage in a wide variety of models of stress and disease.

Exertional Rhabdomyolysis and Acute Kidney Injury

Skeletal muscle breakdown occurs normally with exercise, followed by muscle repair and physiologic adaption. Strenuous, unaccustomed, prolonged, and repetitive exercise, particularly when associated with other risk factors such as hot and humid climate or sickle cell trait can cause clinically significant exertional rhabdomyolysis (ER). Although most cases are asymptomatic and resolve without sequelae, ER is the most common cause of exercise-related myoglobinuric acute renal injury and acute renal failure in athletes. Exercise-related muscle pain, elevated serum creatine kinase (CK), and “cola-colored” urine have been described as a classic presentation of ER. The exact mechanism of ER has not been clearly elucidated. Most studies suggest a cascade of events that include depletion of adenosine triphosphate (ATP), impaired function of the Na+- K+ ATPase system, intracellular excess calcium accumulation, sarcolemma damage, and release of intracellular proteins and other substances into blood. Excess myoglobin that is filtered at the glomerulus can lead to myoglobinuric acute renal injury. Cessation of physical activity, relative rest during clinical recovery, and early aggressive fluid replacement are mainstays of treatment. Return to play after recovery from ER is influenced by associated risk factors that may predispose the athlete to recurrence and is guided by signs, symptoms, and CK levels. This article reviews the definitions, pathophysiology, diagnosis, and management of ER with specific relevance to acute kidney injury.

Ketamine: The best partner for isoflurane in neonatal anesthesia?

Isoflurane is one of the most commonly used inhalation anesthetic in neonatal anaesthesia. It has been suggested that isoflurane can induce caspase activation and apoptosis when applied in a clinically relevant concentration in the developing brain. Recent researches have indicated that a clinically relevant isoflurane treatment may induce neurodegeneration and apoptosis by activating the endoplasmic reticulum (ER) membrane inositol 1,4,5-trisphosphate (IP(3)) receptor, producing excessive calcium release from ER to the cytoplasm and triggering apoptosis. Although the exact mechanism by which isoflurane induces apoptosis still needs further study, it is generally accepted that the increase of cytosolic free calcium levels is the major risk factor. Previous studies have found that during early postnatal life, activation of gamma-aminobutyric acid (GABA(A)) receptor reduces the voltage-dependent Mg(2+) block of N-methyl-d-aspartate (NMDA) channels in neurons and increases cytosolic calcium levels by potentiated the Ca(2+) influx through NMDA channels; while in the adult, it may enhance the voltage-dependent Mg(2+) block of NMDA channels and decrease the Ca(2+) influx through NMDA channels. Since isoflurane acts at the GABA(A) receptor in an agonistic manner, here we presume that isoflurane increases intracellular calcium in neonatal neurons not only by activating IP(3) receptors in the endoplasmic reticulum (ER) membrane, but also by activating the GABA(A) receptor and depolarizing the postsynaptic membrane enough to facilitate NMDA receptor-mediated Ca(2+) influx. Meanwhile, we hypothesized that ketamine, a widely used pediatric anesthetic, acts as a noncompetitive antagonist of the NMDA type of glutamate receptors, which may be the best partner for isoflurane in neonatal anesthesia for it may attenuate isoflurane-induced caspase activation and apoptosis in the neonatal neurons by inhibiting the isoflurane-induced elevation in cytosolic calcium not only by blocking the NMDA receptors, but also by suppressing inositol triphosphate formation in the cytoplasm.

The Common Inhalational Anesthetic Isoflurane Induces Apoptosis via Activation of Inositol 1,4,5-Trisphosphate Receptors

Isoflurane induces cell apoptosis by an unknown mechanism. The authors hypothesized that isoflurane activates inositol 1,4,5-trisphosphate (IP3) receptors on the endoplasmic reticulum (ER) membrane, causing excessive calcium release, triggering apoptosis. The authors determined isoflurane-induced cytotoxicity by measuring caspase-3 activity, lactate dehydrogenase release, MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) reduction, and imaging analysis of cell damage markers (annexin V and propidium iodide staining) in different cell types. The authors used the chicken B lymphocyte with a total knock-out of IP3 receptors, PC12 cells with elevated IP3 receptor activity (transfected with L286V presenilin 1), striatal cells with a knock-in of Q111 Huntingtin, and each cell line's corresponding wild-type controls. The authors also measured the isoflurane-evoked changes of calcium concentration in cytosol and/or mitochondria in these cells. Isoflurane induced apoptosis concentration- and time-dependently, and sequentially elevated cytosolic and then mitochondrial calcium in the chicken B-lymphocyte wild-type but not the IP3 receptor total knock-out cells. Thapsigargin, a calcium adenosine triphosphatase inhibitor on ER membranes, induced apoptosis and elevations of calcium in cytosol and mitochondria in both chicken B-lymphocyte wild-type and IP3 receptor total knock-out cells. Isoflurane induced significantly more neurotoxicity and greater calcium release from the ER in L286V PC12 and Q111 Huntingtin striatal cells than in their corresponding wild-type controls, both of which were significantly inhibited by the IP3 receptor antagonist xestospongin C. These findings suggest that isoflurane activates the ER membrane IP3 receptor, producing excessive calcium release and triggering apoptosis. Neurons with enhanced IP3 receptor activity, as in certain cases of familial Alzheimer or Huntington disease, may be especially vulnerable to isoflurane cytotoxicity.

Maligne Hyperthermie

The term "malignant hyperthermia" (MH), regarded as the typical anaesthetic disease, refers to a clinical syndrome of varying intensity (from abortive courses to fulminant crises) and develops only under exposure of certain triggering substances or mechanisms. MH is caused by a defect in the ryanodine receptor subtype 1, which can often be proved genetically. Furthermore, it may also be generated by other mechanisms which disturb the membranous integrity of skeletal muscle cells (e.g. some myotonias, muscular dystrophies, malformation syndromes). Hyperthermia is only one of a number of life-threatening symptoms that may occur during a fulminant crisis, which ultimately results from an excessive release of calcium into the cytoplasm of muscle cells. Due to a current good knowledge about classical triggers, symptoms and therapeutic interventions, a clinical MH presentation may successfully be treated in the perioperative period. However, it appears to be likely that there are unreported cases outside hospitals since atypical courses or alternative MH triggers (e.g. alcohol, drugs, physical stress) may impair the correct diagnosis. In contrast severe hyperthermia can also arise from other drug-induced diseases, e.g. the neuroleptic malignant syndrome or the serotonin syndrome.

A Role for Reactive Oxygen/Nitrogen Species and Iron on Neuronal Synaptic Plasticity

A great body of experimental evidence collected over many years indicates that calcium has a central role in a variety of neuronal functions. In particular, calcium participates in synaptic plasticity, a neuronal process presumably correlated with cognitive brain functions such as learning and memory. In contrast, only recently, evidence has begun to emerge supporting a physiological role of reactive oxygen (ROS) and nitrogen (RNS) species in synaptic plasticity. This subject will be the central topic of this review. The authors also present recent results showing that, in hippocampal neurons, ROS/RNS, including ROS generated by iron through the Fenton reaction, stimulate ryanodine receptor-mediated calcium release, and how the resulting calcium signals activate the signaling cascades that lead to the transcription of genes known to participate in synaptic plasticity. They discuss the possible participation of ryanodine receptors jointly stimulated by calcium and ROS/RNS in the normal signaling cascades needed for synaptic plasticity, and how too much ROS production may contribute to neurodegeneration via excessive calcium release. In addition, the dual role of iron as a necessary, but potentially toxic, element for normal neuronal function is discussed.

Exciting, Radical, Suicidal

It is now widely accepted that not all brain cells die immediately after stroke. Surrounding a core of severe and rapid tissue injury, brain cell death evolves more slowly in a heterogeneous area that has been called the penumbra.1,2 In 1977, Astrup et al provided one of the first experimental demonstrations of the penumbra in a baboon model of cerebral artery occlusion.3 This region of brain in acute ischemic stroke was found to be electrically silent but sufficiently active metabolically to sustain membrane potentials. Neurons within the penumbra are functionally impaired but not yet dead. Without reperfusion, the penumbra collapses, brain cells die, and the lesion expands. Although the precise timing and cellular pathways involved are not fully understood, it is believed that mechanisms actively promoting cell death are triggered after stroke. Remarkable progress has been made in dissecting these mechanisms over the past 3 decades. Three major pathways have emerged: excitotoxicity, oxidative stress, and apoptosis, and they are inextricably linked. Here, we explore the notion that stroke is most fruitfully investigated by an integrative “systems biology” approach that encompasses cell death and survival signaling within all components of the neurovascular unit. How do brain cells die after stroke? A large body of data suggest that it may be exciting (glutamate and excitotoxicity), radical (oxidative stress and free radicals), and suicidal (apoptotic-like pathways).4–6 Simply put, when brain fails to generate sufficient ATP, such as after oxygen and glucose deprivation, energy failure occurs and ionic gradients are lost. Glutamate is released, reuptake processes are impaired, and this excitatory amino acid binds to its postsynaptic receptors and promotes excessive calcium entry and calcium release. Calcium-dependent synthases and proteases contribute to cell and tissue demise by degrading key cytoskeletal and enzymatic proteins, and generating nitric oxide and peroxynitrite. Mitochondrial functions …

Functional characterization of malignant hyperthermia-associated RyR1 mutations in exon 44, using the human myotube model

Malignant hyperthermia (MH) is a pharmacogenetic disorder with an autosomal dominant inheritance. During exposure to triggering agents as volatile anaesthetics, affected individuals may develop a potentially fatal hypermetabolic syndrome caused by excessive calcium release from the sarcoplasmic reticulum in skeletal muscle. More than 60 MH associated mutations were found in the gene of skeletal muscle ryanodine receptor (RyR1), but only some of them have been functionally characterized. Primary human myotubes were cultured from carriers of RyR1 mutations in exon 44 (Ala2350Thr, Arg2355Trp, Gly2375Ala) and from MH non-susceptible individuals. Investigation of calcium homeostasis with the calcium sensitive probe Fura 2 showed a higher sensitivity to the ryanodine receptor agonists 4-chloro- m-cresol, caffeine and halothane for the myotubes derived from the mutation carriers as compared to those of the control group. The presence of RyR1 mutations with impact on calcium homeostasis emphasizes the functional significance of exon 44.

Excitotoxic and excitoprotective mechanisms: abundant targets for the prevention and treatment of neurodegenerative disorders.

Activation of glutamate receptors can trigger the death of neurons and some types of glial cells, particularly when the cells are coincidentally subjected to adverse conditions such as reduced levels of oxygen or glucose, increased levels of oxidative stress, exposure to toxins or other pathogenic agents, or a disease-causing genetic mutation. Such excitotoxic cell death involves excessive calcium influx and release from internal organelles, oxyradical production, and engagement of programmed cell death (apoptosis) cascades. Apoptotic proteins such as p53, Bax, and Par-4 induce mitochondrial membrane permeability changes resulting in the release of cytochrome c and the activation of proteases, such as caspase-3. Events occurring at several subcellular sites, including the plasma membrane, endoplasmic reticulum, mitochondria and nucleus play important roles in excitotoxicity. Excitotoxic cascades are initiated in postsynaptic dendrites and may either cause local degeneration or plasticity of those synapses, or may propagate the signals to the cell body resulting in cell death. Cells possess an array of antiexcitotoxic mechanisms including neurotrophic signaling pathways, intrinsic stress-response pathways, and survival proteins such as protein chaperones, calcium-binding proteins, and inhibitor of apoptosis proteins. Considerable evidence supports roles for excitotoxicity in acute disorders such as epileptic seizures, stroke and traumatic brain and spinal cord injury, as well as in chronic age-related disorders such as Alzheimer's, Parkinson's, and Huntington's disease and amyotrophic lateral sclerosis. A better understanding of the excitotoxic process is not only leading to the development of novel therapeutic approaches for neurodegenerative disorders, but also to unexpected insight into mechanisms of synaptic plasticity.

Dantrolene, an inhibitor of intracellular calcium release, fails to increase survival in a rat model of intra-abdominal sepsis.

Increased release of intracellular calcium has been implicated in cell death and organ failure in endotoxemia and sepsis. We sought to test this hypothesis in a rat model of antibiotic-treated intraperitoneal sepsis with the use of dantrolene sodium, a specific inhibitor of intracellular calcium release. A prospective, randomized controlled trial. An experimental animal laboratory in a university hospital. Two hundred fourteen male Sprague-Dawley rats. Rats were rendered septic by intraperitoneal implantation of sterile feces mixed with live Escherichia coli and allocated to control, vehicle, or dantrolene treatment. A separate group of rats had arterial catheters implanted to allow blood sampling for determination of circulating tumor necrosis factor (TNF)-alpha and lactate concentrations. Additional rats were randomized to receive vehicle or dantrolene after intravenous injection of endotoxin. Over the 7-day study period, survival was significantly worse among rats that received dantrolene at a dose of 10 mg/kg, irrespective of whether treatment was started before or after induction of peritonitis. Mean whole blood lactate for each group peaked at 6 hrs after induction of infection. There were no significant differences in lactate concentration among the groups at any of the time points examined. Similarly, there were no differences among any of the groups for circulating concentrations of TNF-alpha. In rats challenged with endotoxin, dantrolene affected neither survival nor circulating concentrations of TNF-alpha. We conclude that dantrolene decreases survival in bacterial sepsis and has no effect on survival in endotoxemia in rats. The importance of excessive intracellular calcium release in sepsis remains to be elucidated.

Erythrocyte membrane fluidity in malignant hyperthermia

Malignant hyperthermia (MH) is a rare but serious complication of general anesthesia that potentially carries a high mortality and morbidity. It is associated with excessive release of calcium into skeletal muscle following exposure to certain drugs, including the volatile general anesthetics. Since these are recognized membrane fluidizing agents it has been speculated that this condition might represent a generalized defect in membrane physical properties either at rest or inducible by fluidizing agents. If this hypothesis were found to be correct, malignant hyperthermia might conveniently be detected by examining membrane physical properties of easily accessible cells rather than the cumbersome method of muscle biopsy currently employed. To test this hypothesis we identified patients proven to be susceptible to MH by muscle biopsy and a cohort of patients not susceptible to MH as defined by negative muscle biopsy testing. Erythrocytes were isolated from both groups and membrane physical properties examined using conventional, widely available, steady-state fluorescence polarization techniques. Erythrocyte membranes were evaluated with multiple probes both in the basal condition and following fluidization with either increasing temperature or two concentrations of a fluidizing alcohol. We report, contrary to previous publications, that no discernable differences were detectable between MH-positive or negative patients. Thus, we find no evidence for a generalized membrane defect in MH and conclude that the determination of erythrocyte membrane physical properties, by these techniques, are of no use in the preoperative screening for this disorder.

Calcitonin secretion in postmenopausal osteoporosis.

Calcitonin deficiency has been implicated in the pathogenesis of accelerated bone loss, especially in postmenopausal osteoporosis. To investigate this issue, we studied 25 patients with untreated postmenopausal osteoporosis, 14 age-matched and sex-matched healthy controls (spinal bone mineral density greater than or equal to age-specific and sex-specific mean), and 5 women who had undergone total thyroidectomy. Each subject received an intravenous infusion of 2 mg of elemental calcium per kilogram of body weight over 5 minutes, to test the C-cell secretory reserve. We measured calcitonin by radioimmunoassay in whole plasma and in silica-cartridge extracts of plasma, the latter method providing greatly improved sensitivity and specificity for monomeric calcitonin. Basal immunoreactive calcitonin concentrations, whether measured in whole plasma or in extracts, were significantly higher in the subjects with osteoporosis (P less than 0.01) than in the healthy controls. The calcitonin secretory reserve, as assessed by calcium stimulation, was normal in the osteoporotic group but virtually absent in the thyroidectomy group. We conclude that postmenopausal osteoporosis is not associated with and does not result from calcitonin deficiency. On the contrary, excessive skeletal calcium release may stimulate calcitonin secretion in patients with the disorder.

Calcium redistribution, calcification and stone formation in the parotid gland during experimental stimulation and hypercalcaemia. Cytochemical and X-ray microanalytical investigations.

Distribution and redistribution of intra- and pericellular calcium was investigated in the parotid gland of rats under secretory stimulation and hypercalcaemia. The effects of hypercalcaemia and secretory stimulation and of the combination of both were compared. Calcium content was determined by atomic absorption spectrometry. Calcium distribution within the tissue was demonstrated by light microscopical ( GBHA ) staining and electron microscopical (pyroantimonate method) cytochemistry in combination with X-ray microanalysis. Typical calcium depot sites were the basal and cellular membranes, the calcium buffer organelles (i.e. mitochondria) the secretory granules and the acinar lumina. After stimulation (by isoprenalin ) a decrease of calcium-enriched secretory granules and a depletion of intracellular calcium buffer organelles occurred. During hypercalcaemia (induced by dihydrotachysterol), a calcium overloading of the cell membrane and intracellular buffer organelles without calcification was observed. Combined stimulation and hypercalcaemia induced an excessive calcium overloading of all intra- and extracellular calcium depots with excessive calcium release into the acinar lumina resulting in calcium phosphate aggregates and stone formation. Secretory stimulation and simultaneous hypercalcaemia exert potentiating effects on intracellular and intraluminal calcification proposing an importance for pathogenesis of human sialolithiasis.