Lukas Weigl

Dihydropyridine‐induced Ca2+ release from ryanodine‐sensitive Ca2+ pools in human skeletal muscle cells

1 Dihydropyridines (DHPs) are widely used antihypertensive drugs and inhibit excitation‐contraction (E–C) coupling in vascular smooth muscle and in myocardial cells by antagonizing L‐type Ca2+ channels (DHP receptors). However, contradictory reports exist about the interaction of the DHP with the skeletal muscle isoform of the DHP receptor and E–C coupling in skeletal muscle cells. 2 Using the intracellular fluorescent Ca2+ indicator fura‐2, an increase in [Ca2+]i was observed after extracellular application of nifedipine to cultured human skeletal muscle cells. The rise in [Ca2+]i was dose dependent with a calculated EC50 of 614 ± 96 nm nifedipine and a maximum increment in [Ca2+]i of 80 ± 3.2 nm. Similar values were obtained with nitrendipine. 3 This effect of DHPs was restricted to differentiated skeletal muscle cells and was not seen in non‐differentiated cells or in PC12 cells. In spite of the observed increase in [Ca2+]i, whole‐cell patch clamp experiments revealed that 10 μm nifedipine abolished inward Ba2+ currents through L‐type Ca2+ channels completely. 4 Similar to nifedipine, (±)Bay K 8644, an agonist of the L‐type Ca2+ channel, also increased [Ca2+]i. This effect could not be enhanced by further addition of nifedipine, suggesting that both DHPs act via a common signalling pathway. 5 Based on the specific mechanism of the skeletal muscle E‐C coupling, we propose the stabilization of a conformational state of the DHP receptor by DHPs, which is sufficient to activate the ryanodine receptor.

No evidence for direct modulatory effects of Δ9-Tetrahydrocannabinol on human polymorphonuclear leukocytes

The effects of cannabinoids (CB) that have been reported in various leukocyte populations were mainly immunosuppressive or immunomodulatory. Almost nothing is known, however, about direct interactions of cannabinoids with human polymorphonuclear cells (PMN), although m-RNA for the cannabinoid receptor-2 (CB(2)) was found in human PMN. In order to investigate a potential influence of cannabinoids on human PMN, the migration and phagocytosis of PMN were studied in the presence of Delta(9)-Tetrahydrocannabinol (Delta(9)-THC) at final concentrations between 10(-10) and 10(-5) M. No effect was detectable on these essential PMN functions; and besides, no CB(2)-receptor expression could be detected using the Western blotting technique. Thus, circulating human PMN from healthy individuals remain unaffected by Delta(9)-THC due to the absence of functional CB(2)-receptor expression.

The mechanical properties of continuous spinal small-bore catheters.

Continuous spinal anesthesia (CSA) has a nearly 100-yr history. In situations of difficult removal of a CSA small-bore catheter, mechanical properties of the different catheters might be important, because breakage could occur. We compared 5 different CSA small-bore catheters, 22- to 28-gauge from 3 manufacturers, for tensile strength, tensile stress, distension, and yield strength. Maximal tensile strength is the force applied before breakage of the catheter. The material characteristics of different CSA small-bore catheters for maximal tensile strength were: 22-gauge = 29.56 ± 1.56 (mean ± sd) Newton (N), 24-gauge = 16.77 ± 1.61 N, 25-gauge = 9.20 ± 0.48 N, 27-gauge = 4.61 ± 0.25 N, 28-gauge = 5.07 ± 0.59 N at room temperature. A strong correlation between maximal tensile strength and the outer diameter (r = 0.957, P < 0.001) and maximal tensile strength and the wall thickness (r = 0.9, P < 0.001) was observed. Although extrapolation from experimental studies to clinical routine should be made with care, our data suggest that catheters with higher-strength characteristics may reduce the risk of catheter breakage in patients, although clinical correlations are lacking.

The disease modifying osteoarthritis drug diacerein is able to antagonize pro inflammatory state of chondrocytes under mild mechanical stimuli

OBJECTIVE To investigate the combination of mild mechanical stimuli and a disease modifying osteoarthritis drug (DMOAD) in inflammatory activated chondrocytes and to study the combination of drug and mechanical tension on the cellular level as a model for an integrated biophysical approach for osteoarthritis (OA) treatments. METHODS Interleukin-1beta (IL-1β) stimulated C28/I2 cells underwent mild mechanically treatment while cultured in the presence of the DMOAD diacerein. The pharmacological input of diacerein was evaluated by cell viability and cell proliferation measurements. Inflammation and treatment induced changes in key regulatory proteins and components of the extracellular matrix (ECM) were characterized by quantitative real-time PCR (qPCR). The effects on metalloproteinase-1 (MMP-1) activity and glycosaminoglycan (GAG) concentration in cell supernatants of treated cells were investigated. RESULTS C28/I2 cells demonstrated significant changes in expression of inflammatory and cartilage destructive proteins in response to IL-1β stimulation. The chondroprotective action of diacerein in mechanically stimulated cells was mediated by a decrease in interleukin-8 (IL-8), fibronectin-1 (FN-1), collagen type I (Col 1) and MMP-1 expression levels, respectively. Augmented expression of interleukin-6 receptor (IL-6R) and the fibroblast growth factor receptors (FGFRs) by diacerein was not abolished by mechanical treatment. The observed effects were accompanied by a reduced cell proliferation rate, attenuated cell viability and extenuated MMP-1 activity. CONCLUSION Diacerein diversely regulates the expression of main regulatory proteins as well as components important to regenerate and set up ECM. Mechanical stimulation does not negatively influence the chondroprotective effect induced by diacerein treatment in immortalized human C28/I2 chondrocytes.

Novel double and single ryanodine receptor 1 variants in two Austrian malignant hyperthermia families.

BACKGROUND: Malignant hyperthermia (MH) is a potentially lethal genetic disorder in response to volatile anesthetics and depolarizing muscle relaxants. To support the claim that a novel genetic variant causes MH, it is necessary to demonstrate that it has significant effects on the sensitivity of the ryanodine receptor (RYR1) calcium channel. In this study we focused on 2 Austrian families with strong MH disposition and new RYR1 variants. METHODS: We sequenced the entire coding region of the RYR1 from 2 Austrian MH individuals. Genotype–phenotype segregation and evolutionary conservation of the variants were considered. On a functional level, Ca2+ release experiments with fura-2-acetoxymethyl ester were performed in cultured skeletal muscle cells derived from individuals carrying the new variants and compared with control cells from nonsusceptible individuals. Caffeine, 4-chloro-m-cresole (4-CmC), and halothane were used as specific Ca2+ releasing agents. RESULTS: The variant p.A612P in family A segregated with an MH-susceptible phenotype and cells showed an increased sensitivity for all Ca2+-releasing substances tested. In family B, 2 variants (p.R2458H/p.R3348C) were identified. While p.R2458H and p.R2458H/p.R3348C segregated with an MH-susceptible diagnosis, p.R3348C alone showed an MH equivocal diagnosis. Ca2+-release experiments showed that exchanges of these highly conserved amino acids increased the sensitivities for the substances tested (except 4-CmC with p.R2458H and p.R3348C) when compared with the MH-negative control group. CONCLUSIONS: Our results suggest that these variants are new causative MH variants.

Activation of RhoB in simvastatin-induced apoptosis of differentiated human skeletal muscle cells

Statins are well-tolerated cholesterol lowering drugs. However, in rare cases myotoxicity or even rhabdomyolysis occur. We have recently shown that in vitro statins are able to induce apoptosis in differentiated human skeletal muscle cells. In particular, simvastatin triggers the mitochondrial pathway of apoptosis via translocation of Bax from the cytosol to the mitochondrial membrane. Accordingly, caspase 9 and caspase 3 were consecutively activated. Here we show that an early event in simvastatin action is up-regulation of RhoB on mRNA level which is paralleled by the downstream Kruppel-like factor-2 (KLF2) and KLF-6. Moreover, the level of active, GTP-bound RhoB was increased in simvastatin-treated cells in a concentration-dependent manner as determined by Rhotekin pull-down assay. Up-regulation of RhoB on mRNA and protein level was dependent on HMG-CoA reductase inhibition. Interestingly, the up-regulation of RhoB was paralleled by caspase 2 activation, which might be directly associated with RhoB and therefore be required for the cytotoxic effect of simvastatin.

3,5-Di-t-butyl catechol is a potent human ryanodine receptor 1 activator, not suitable for the diagnosis of malignant hyperthermia susceptibility

3,5-Di-t-butyl catechol (DTCAT) releases Ca(2+) from rat skeletal muscle sarcoplasmic reticulum (SR) vesicles. Hence, it is a candidate for use as a substitute for halothane or caffeine in the in vitro contracture test for the diagnosis of susceptibility to malignant hyperthermia (MH). To characterize the effect of DTCAT at cell level, Ca(2+) release experiments were performed on cultured, human skeletal muscle myotubes using the fluorescent Ca(2+) indicator fura2-AM. DTCAT was also assayed in the in vitro contracture test on human skeletal muscle bundles obtained from individuals diagnosed susceptible (MHS), normal (MHN) or equivocal for halothane (MHEH) and compared to the standard test substances caffeine and halothane. DTCAT increased, in a concentration-dependent manner and with a higher efficacy as compared to caffeine, the free, intracellular Ca(2+) levels of cultured MHN and MHS skeletal muscle myotubes. This effect was similar in both types of myotubes and involved the release of Ca(2+) from SR stores as well as Ca(2+)-influx from the extracellular space. Inhibition of ryanodine receptors either with ryanodine or with ruthenium red markedly reduced DTCAT-induced increase in intracellular Ca(2+) concentration while abolishing that induced by caffeine. In MHN skeletal muscle bundles, DTCAT induced contractures with an EC(50) value of 160 ± 91 μM. However, the sensitivity of MHS or MHEH muscles to DTCAT was similar to that of MHN muscles. In conclusion, DTCAT is not suitable for the diagnosis of MH susceptibility due to its failure to discriminate between MHN and MHS muscles.

3,5-Di-t-butyl catechol (DTCAT) as an activator of the human skeletal muscle ryanodine receptor Ca2+ channel and its evaluation as a test substance for the assessment of susceptibility to malignant hyperthermia

3,5-Di-t-butyl catechol (DTCAT) has been shown to release Ca2+ from rat skeletal muscle sarcoplasmic reticulum (SR) vesicles, which makes it a possible candidate for use as a substitute for halothane or caffeine in the in vitro contracture test (IVCT) for the assessment of susceptibility to malignant hyperthermia (MHS).