J. A. Jeevendra Martyn

Up-and-down Regulation of Skeletal Muscle Acetylcholine Receptors Effects on Neuromuscular Blockers

Multiple factors alter the interaction of muscle relaxants with the NMJ. This review has focused on the aberrant responses caused principally by alterations in AChRs (table 1). Many pathologic states increase (up-regulate) AChR number. These include upper and lower motor neuron lesions, muscle trauma, burns, and immobilization. Pre- or postjunctional inhibition of neurotransmission by drugs or toxins also up-regulate AChRs. These include alpha- and beta-BT, NDMR, anticonvulsants, and clostridial toxins. We speculate that other bacterial toxins also up-regulate AChR. With proliferation of AChRs, agonist drug dose-response curves are shifted to the left. The exaggerated release of potassium when depolarization occurs with the use of agonists such as SCh and decamethonium can be attributed to the increased number of AChR. Thus, SCh should be avoided in patients who are in the susceptible phase (see section V). In the presence of increased AChR, the requirement for NDMR is markedly increased. Thus, the response to NDMR may be used as an indirect estimator of increased sensitivity to SCh (table 1). The most extensively studied pathologic state in which there is a decrease in AChRs is myasthenia gravis; there is immunologically mediated destruction and/or functional blockade of AChRs. The pathophysiologic and pharmacologic changes in LEMS are quite distinct from those of myasthenia gravis. Decreased AChRs in myasthenia gravis result in resistance to agonists and increased sensitivity to competitive antagonists. In conditioning exercise, the perturbed muscles show sensitivity to NDMR that may be due to decreased AChRs. Chronic elevations of ACh observed with organophosphorus poisoning or chronic use of reversible cholinesterase inhibitors results in down-regulation of AChRs. In this condition, SCh should be avoided because its metabolic breakdown would be impaired; the requirement for NDMR may be decreased. All of the varied responses to SCh and NDMR, which are associated with concomitant changes in AChRs, are analogous to drug-receptor interactions observed in other biologic systems.

Succinylcholine -induced hyperkalemia in acquired pathologic states : Etiologic factors and molecular mechanisms

Lethal hyperkalemic response to succinylcholine continues to be reported, but the molecular mechanisms for the hyperkalemia have not been completely elucidated. In the normal innervated mature muscle, the acetylcholine receptors (AChRs) are located only in the junctional area. In certain pathologic states, including upper or lower motor denervation, chemical denervation by muscle relaxants, drugs, or toxins, immobilization, infection, direct muscle trauma, muscle tumor, or muscle inflammation, and/or burn injury, there is up-regulation (increase) of AChRs spreading throughout the muscle membrane, with the additional expression of two new isoforms of AChRs. The depolarization of these AChRs that are spread throughout the muscle membrane by succinylcholine and its metabolites leads to potassium efflux from the muscle, leading to hyperkalemia. The nicotinic (neuronal) α7 acetylcholine receptors, recently described to be expressed in muscle also, can be depolarized not only by acetylcholine and succinylcholine but also by choline, persistently, and possibly play a critical role in the hyperkalemic response to succinylcholine in patients with up-regulated AChRs.

Comparison of Comet Assay, Electron Microscopy, and Flow Cytometry for Detection of Apoptosis

Differentiating apoptosis from necrosis is a challenge in single cells and in parenchymal tissues. The techniques available, including in situ TUNEL (Terminal deoxyribonucleotide transferase-mediated dUTP-X Nick End-Labeling) staining, DNA ladder assay, and flow cytometry, suffer from low sensitivity or from a high false-positive rate. This study, using a Jurkat cell model, initially evaluated the specificity of the neutral comet assay and flow cytometry compared to the gold standard, electron microscopy, for detection of apoptosis and necrosis. Neutral comet assay distinguished apoptosis from necrosis in Jurkat cells, as evidenced by the increased comet score in apoptotic cells and the almost zero comet score in necrotic cells. These findings were consistent with those of electron microscopy and flow cytometry. Furthermore, using rats with burn or ischemia/reperfusion injury, well-established models of skeletal and cardiac muscle tissue apoptosis, respectively, we applied the comet assay to detect apoptosis in these muscles. Neutral comet assay was able to detect apoptotic changes in both models. In the muscle samples from rats with burn or ischemia-reperfusion injury, the comet score was higher than that of muscle samples from their respective controls. These studies confirm the consistency of the comet assay for detection of apoptosis in single cells and provide evidence for its applicability as an additional method to detect apoptosis in parenchymal cells.

Obesity-induced insulin resistance and hyperglycemia: etiologic factors and molecular mechanisms.

Obesity is a major cause of type 2 diabetes, clinically evidenced as hyperglycemia. The altered glucose homeostasis is caused by faulty signal transduction via the insulin signaling proteins, which results in decreased glucose uptake by the muscle, altered lipogenesis, and increased glucose output by the liver. The etiology of this derangement in insulin signaling is related to a chronic inflammatory state, leading to the induction of inducible nitric oxide synthase and release of high levels of nitric oxide and reactive nitrogen species, which together cause posttranslational modifications in the signaling proteins. There are substantial differences in the molecular mechanisms of insulin resistance in muscle versus liver. Hormones and cytokines from adipocytes can enhance or inhibit both glycemic sensing and insulin signaling. The role of the central nervous system in glucose homeostasis also has been established. Multipronged therapies aimed at rectifying obesity-induced anomalies in both central nervous system and peripheral tissues may prove to be beneficial.

Primary Role of Functional Ischemia, Quantitative Evidence for the Two-Hit Mechanism, and Phosphodiesterase-5 Inhibitor Therapy in Mouse Muscular Dystrophy

Background Duchenne Muscular Dystrophy (DMD) is characterized by increased muscle damage and an abnormal blood flow after muscle contraction: the state of functional ischemia. Until now, however, the cause-effect relationship between the pathogenesis of DMD and functional ischemia was unclear. We examined (i) whether functional ischemia is necessary to cause contraction-induced myofiber damage and (ii) whether functional ischemia alone is sufficient to induce the damage. Methodology/Principal Findings In vivo microscopy was used to document assays developed to measure intramuscular red blood cell flux, to quantify the amount of vasodilatory molecules produced from myofibers, and to determine the extent of myofiber damage. Reversal of functional ischemia via pharmacological manipulation prevented contraction-induced myofiber damage in mdx mice, the murine equivalent of DMD. This result indicates that functional ischemia is required for, and thus an essential cause of, muscle damage in mdx mice. Next, to determine whether functional ischemia alone is enough to explain the disease, the extent of ischemia and the amount of myofiber damage were compared both in control and mdx mice. In control mice, functional ischemia alone was found insufficient to cause a similar degree of myofiber damage observed in mdx mice. Additional mechanisms are likely contributing to cause more severe myofiber damage in mdx mice, suggestive of the existence of a “two-hit” mechanism in the pathogenesis of this disease. Conclusions/Significance Evidence was provided supporting the essential role of functional ischemia in contraction-induced myofiber damage in mdx mice. Furthermore, the first quantitative evidence for the “two-hit” mechanism in this disease was documented. Significantly, the vasoactive drug tadalafil, a phosphodiesterase 5 inhibitor, administered to mdx mice ameliorated muscle damage.

Inducible Nitric-oxide Synthase and NO Donor Induce Insulin Receptor Substrate-1 Degradation in Skeletal Muscle Cells

Chronic inflammation plays an important role in insulin resistance. Inducible nitric-oxide synthase (iNOS), a mediator of inflammation, has been implicated in many human diseases including insulin resistance. However, the molecular mechanisms by which iNOS mediates insulin resistance remain largely unknown. Here we demonstrate that exposure to NO donor or iNOS transfection reduced insulin receptor substrate (IRS)-1 protein expression without altering the mRNA level in cultured skeletal muscle cells. NO donor increased IRS-1 ubiquitination, and proteasome inhibitors blocked NO donor-induced reduction in IRS-1 expression in cultured skeletal muscle cells. The effect of NO donor on IRS-1 expression was cGMP-independent and accentuated by concomitant oxidative stress, suggesting an involvement of nitrosative stress. Inhibitors for phosphatidylinositol-3 kinase, mammalian target of rapamycin, and c-Jun amino-terminal kinase failed to block NO donor-induced IRS-1 reduction, whereas these inhibitors prevented insulin-stimulated IRS-1 decrease. Moreover iNOS expression was increased in skeletal muscle of diabetic (ob/ob) mice compared with lean wild-type mice. iNOS gene disruption or treatment with iNOS inhibitor ameliorated depressed IRS-1 expression in skeletal muscle of diabetic (ob/ob) mice. These findings indicate that iNOS reduces IRS-1 expression in skeletal muscle via proteasome-mediated degradation and thereby may contribute to obesity-related insulin resistance.

Treatment of Pain in Acutely Burned Children

The child with burns suffers severe pain at the time of the burn and during subsequent treatment and rehabilitation. Pain has adverse physiological and emotional effects, and research suggests that pain management is an important factor in better outcomes. There is increasing understanding of the private experience of pain, and how children benefit from honest preparation for procedures. Developmentally appropriate and culturally sensitive pain assessment, pain relief, and reevaluation have improved, becoming essential in treatment. Pharmacological treatment is primary, strengthened by new concepts from neurobiology, clinical science, and the introduction of more effective drugs with fewer adverse side effects and less toxicity. Empirical evaluation of various hypnotic, cognitive, behavioral, and sensory treatment methods is advancing. Multidisciplinary assessment helps to integrate psychological and pharmacological pain-relieving interventions to reduce emotional and mental stress, and family stress as well. Optimal care encourages burn teams to integrate pain guidelines into protocols and critical pathways for improved care.

Thermodilution right ventricular volume: a novel and better predictor of volume replacement in acute thermal injury.

Management of acutely burned patients requires intense but meticulous fluid therapy. Indicators of satisfactory resuscitation include: intravascular and arterial pressures and urine output. The usefulness of these parameters as a predictor of cardiac index (CI) has not been tested. Compared to central venous pressure, right ventricular end-diastolic volume (RVEDV) may give a closer approximation of the preload of the right ventricle. Modifying the pulmonary artery (PA) catheter and using the thermodilution technique we measured RVEDV and right ventricular ejection fraction (RVEF) as well as the classical parameters in 16 patients of mean age 39.3 years and of mean body surface area burn 75.2%. CI best correlated with RVEDV (r = 0.75). Mean PA pressure, wedge pressure, RV end-diastolic pressure, and urine output correlated poorly with CI (r = 0.36, 0.32, 0.27, and 0.26, respectively). Unlike atrial pressures the RVEDV and RVEF are unaffected by malpositioning of transducers, airway pressure, and compliance changes of the ventricle. The measurement of RVEDV is a useful clinical tool for the assessment of preload and when used in combination with RVEF may indicate the need for inotropy.

Analysis of Thermal Injury-induced Insulin Resistance in Rodents IMPLICATION OF POSTRECEPTOR MECHANISMS

Burn injury is associated with insulin resistance. The molecular basis of this resistance was investigated by examining insulin receptor signaling in rats after thermal injury. The impaired insulin-stimulated transport of [3H]2-deoxyglucose into soleus muscle strips confirmed the insulin resistance following burns. In vivoinsulin-stimulated phosphoinositide 3-kinase activity, pivotal in translocation of GLUT4, was decreased in burns when assessed by its insulin receptor substrate-1 (IRS-1)-associated activity. Insulin-induced tyrosine kinase activity of insulin receptor (IR) and tyrosine phosphorylation of IRS-1 were also attenuated. Immunoprecipitated IR, however, appeared to have normal insulin-responsive kinase activity. Finally, immunoprecipitated IRS-1 was tested for its effect on partially purified recombinant IR and was found to inhibit its kinase activity. This inhibitory effect of IRS-1 was abolished by prior treatment of IRS-1 with alkaline phosphatase, indicating that burn injury-related hyperphosphorylation of IRS-1 is similar to that observed in TNFα-induced inhibition of IR signaling. All of these changes were observed in the absence of quantitative changes in IR, IRS-1, and phosphoinositide 3-kinase. Alterations in postreceptor insulin signaling, therefore, may be responsible for the insulin resistance after thermal injury.

Plasma protein binding of drugs after severe burn injury.

Plasma protein binding in seven severely burned (35% to 85% of body) patients 1 to 25 days after burn injury was examined for two drugs: diazepam (DZ), which binds mainly to serum albumin (ALB), and imipramine (IMI), which binds primarily to α1‐acid glycoprotein (AAG). Protein binding was determined by equilibrium dialysis, and AAG concentrations were measured by radial immunodiffusion. AAG concentrations increased from 36 to 99 mg/dl (day 1) to 221 to 268 mg/dl (days 5 to 20). The IMI free fraction values decreased from 11.2%‐19.7% to 5.5%‐7.8% and correlated well with AAG concentrations. IMI free fraction values were lower in burned patients (10.8% ±0.8%) than in healthy controls (15.3% ± 0.7%). In contrast, DZ free fraction did not correlate with AAG or ALB concentrations. The DZ free fraction ranged from 1.5% to 8.04%, and changes in a given individual did not relate to time after injury. Free fraction of DZ was higher in the burned population (3.5% ± 0.37%) than in healthy controls (1.25% ± 0.05%). Basic drugs that are highly bound to AAG may show progressive, increased binding after burn injury, whereas drugs binding to ALB may decrease after burn injury. Such binding changes can alter the interpretation of total serum or plasma drug concentrations.