Phosphoinositide substrates of myotubularin affect voltage-activated Ca2+ release in skeletal muscle

Skeletal muscle excitation–contraction (E–C) coupling is altered in several models of phosphatidylinositol phosphate (PtdInsP) phosphatase deficiency and ryanodine receptor activity measured in vitro was reported to be affected by certain PtdInsPs, thus prompting investigation of the physiological role of PtdInsPs in E–C coupling. We measured intracellular Ca²⁺ transients in voltage-clamped mouse muscle fibres microinjected with a solution containing a PtdInsP substrate (PtdIns(3,5)P ₂ or PtdIns(3)P) or product (PtdIns(5)P or PtdIns) of the myotubularin phosphatase MTM1. No significant change was observed in the presence of either PtdIns(5)P or PtdIns but peak SR Ca²⁺ release was depressed by ~30% and 50% in fibres injected with PtdIns(3,5)P ₂ and PtdIns(3)P, respectively, with no concurrent alteration in the membrane current signals associated with the DHPR function as well as in the voltage dependence of Ca²⁺ release inactivation. In permeabilized muscle fibres, the frequency of spontaneous Ca²⁺ release events was depressed in the presence of the three tested phosphorylated forms of PtdInsP with PtdIns(3,5)P ₂ being the most effective, leading to an almost complete disappearance of Ca²⁺ release events. Results support the possibility that pathological accumulation of MTM1 substrates may acutely depress ryanodine receptor-mediated Ca²⁺ release. Overexpression of a mCherry-tagged form of MTM1 in muscle fibres revealed a striated pattern consistent with the triadic area. Ca²⁺ release remained although unaffected by MTM1 overexpression and was also unaffected by the PtdIns-3-kinase inhibitor LY2940002, suggesting that the 3-phosphorylated PtdIns lipids active on voltage-activated Ca²⁺ release are inherently maintained at a low level, inefficient on Ca²⁺ release in normal conditions.     

Application of difference circular dichroism (CD) spectroscopy for determination of pharmaceutical compounds. Analysis of stereoisomers by the HPLC-CD/UV method

Circular dichroism (CD) spectroscopy is gaining an increasing importance in pharmaceutical analysis. One of the main advantages of the CD spectroscopic method is its inherent selectivity, which provides the selective analysis of chiral molecules possessing optically active absorbance bands. In addition to direct CD spectroscopic methods, several new procedures have been developed based on CD spectroscopy for the analysis and determination of pharmaceutical compounds in the past few years. One of the most progressive fields of chiroptical analysis is the combination of high performance liquid chromatographic separation and CD spectroscopy and the application of simultaneous dual CD/UV detection. The aim of our research work was to work out new procedures based on CD and CD related techniques and to investigate the application of these methods in pharmaceutical analysis. This work presents a new, selective and sensitive difference CD spectroscopic method based on oxime formation of the keto group for the determination of delta 4-3-ketosteroids and 6-keto-morphinans. Molar CD and UV spectral parameters of E and Z oxime isomers, produced in the course of oxime formation reaction, have been determined by HPLC separation and simultaneous dual CD and UV detection, without isolation of the isomers. The stereochemistry of spirocyclic pyridopyridazine compounds has been investigated by simultaneous dual CD and UV spectroscopy following HPLC separation. The enantiomeric relationship of the pyridopyridazine stereoisomers has been proved by the application of ellipticity-absorbance ratio spectra in a novel way.     

Colocalization and nonrandom distribution of Kv1.3 potassium channels and CD3 molecules in the plasma membrane of human T lymphocytes

Distribution and lateral organization of Kv1.3 potassium channels and CD3 molecules were studied by using electron microscopy, confocal laser scanning microscopy, and fluorescence resonance energy transfer. Immunogold labeling and electron microscopy showed that the distribution of FLAG epitope-tagged Kv1.3 channels (Kv1.3/FLAG) significantly differs from the stochastic Poisson distribution in the plasma membrane of human T lymphoma cells. Confocal laser scanning microscopy images showed that Kv1.3/FLAG channels and CD3 molecules accumulated in largely overlapping membrane areas. The numerical analysis of crosscorrelation of the spatial intensity distributions yielded a high correlation coefficient (C = 0.64). A different hierarchical level of molecular proximity between Kv1.3/FLAG and CD3 proteins was reported by a high fluorescence resonance energy transfer efficiency (E = 51%). These findings implicate that reciprocal regulation of ion-channel activity, membrane potential, and the function of receptor complexes may contribute to the proper functioning of the immunological synapse.     

Sarcoplasmic reticulum Ca2+ refilling controls recovery from Ca2+-induced Ca2+ release refractoriness in heart muscle

In cardiac muscle Ca2+-induced Ca2+ release (CICR) from the sarcoplasmic reticulum (SR) is initiated by Ca2+ influx via L-type Ca2+ channels. At present, the mechanisms underlying termination of SR Ca2+ release, which are required to ensure stable excitation-contraction coupling cycles, are not precisely known. However, the same mechanism leading to refractoriness of SR Ca2+ release could also be responsible for the termination of CICR. To examine the refractoriness of SR Ca2+ release, we analyzed Na+-Ca2+ exchange currents reflecting cytosolic Ca2+ signals induced by UV-laser flash-photolysis of caged Ca2+. Pairs of UV flashes were applied at various intervals to examine the time course of recovery from CICR refractoriness. In cardiomyocytes isolated from guinea-pigs and mice, beta-adrenergic stimulation with isoproterenol-accelerated recovery from refractoriness by approximately 2-fold. Application of cyclopiazonic acid at moderate concentrations (<10 micromol/L) slowed down recovery from refractoriness in a dose-dependent manner. Compared with cells from wild-type littermates, those from phospholamban knockout (PLB-KO) mice exhibited almost 5-fold accelerated recovery from refractoriness. Our results suggest that SR Ca2+ refilling mediated by the SR Ca2+-pump corresponds to the rate-limiting step for recovery from CICR refractoriness. Thus, the Ca2+ sensitivity of CICR appears to be regulated by SR Ca2+ content, possibly resulting from a change in the steady-state Ca2+ sensitivity and in the gating kinetics of the SR Ca2+ release channels (ryanodine receptors). During Ca2+ release, the concomitant reduction in Ca2+ sensitivity of the ryanodine receptors might also underlie Ca2+ spark termination by deactivation.     

Regulation of the rat sarcoplasmic reticulum calcium release channel by calcium

The regulation by calcium of the ryanodine receptor/SR calcium release channel (RyR) from rat skeletal muscle was studied under isolated conditions and in situ. RyRs were either solubilized and incorporated into lipid bilayers or single fibres were mounted into a Vaseline gap voltage clamp. Single channel data were compared to parameters determined from the calculated calcium release flux. With K⁺ (250 mM) being the charge carrier the single channel conductance was 529 pS at 50 M Ca²⁺ cis and trans, and decreased with increasing cis [Ca²⁺]. Open probability showed a bell shaped calcium dependence revealing an activatory and an inhibitory Ca²⁺ binding site (Hill coefficients of 1.18 and 1.28, respectively) with half activatory and inhibitory concentrations of 9.4 and 298 M. The parameters of the inhibitory site agreed with the calcium dependence of channel inactivation deduced from the decline in SR calcium release in isolated fibres. Mean open time showed slight [Ca²⁺] dependence following a single exponential at every Ca²⁺ concentration tested. Closed time histograms, at high [Ca²⁺], were fitted with three exponentials, from which the longest was calcium independent, and resembled the recovery time constant of SR inactivation (115 ± 15 ms) obtained in isolated fibres. The data are in agreement with a model where calcium binding to the inhibitory site on RyR would be responsible for the calcium dependent inactivation in situ.     

In-Hospital Patient Education Markedly Reduces Alcohol Consumption after Alcohol-Induced Acute Pancreatitis

Although excessive alcohol consumption is by far the most frequent cause of recurrent acute pancreatitis (AP) cases, specific therapy is still not well established to prevent recurrence. Generally, psychological therapy (e.g., brief intervention (BI)) is the cornerstone of cessation programs; however, it is not yet widely used in everyday practice. We conducted a post-hoc analysis of a prospectively collected database. Patients suffering from alcohol-induced AP between 2016 and 2021 received 30 min BI by a physician. Patient-reported alcohol consumption, serum gamma-glutamyl-transferase (GGT) level, and mean corpuscular volume (MCV) of red blood cells were collected on admission and at the 1-month follow-up visit to monitor patients’ drinking habits. Ninety-nine patients with alcohol-induced AP were enrolled in the study (mean age: 50 ± 11, 89% male). A significant decrease was detected both in mean GGT value (294 ± 251 U/L vs. 103 ± 113 U/L, p < 0.001) and in MCV level (93.7 ± 5.3 U/L vs. 92.1 ± 5.1 U/L, p < 0.001) in patients with elevated on-admission GGT levels. Notably, 79% of the patients (78/99) reported alcohol abstinence at the 1-month control visit. Brief intervention is an effective tool to reduce alcohol consumption and to prevent recurrent AP. Longitudinal randomized clinical studies are needed to identify the adequate structure and frequency of BIs in alcohol-induced AP.     

ERCP is more challenging in cases of acute biliary pancreatitis than in acute cholangitis – Analysis of the Hungarian ERCP registry data

BackgroundEndoscopic retrograde cholangiopancreatography (ERCP) is an important therapeutic modality in acute biliary pancreatitis (ABP) cases with cholangitis or ongoing common bile duct obstruction. Theoretically, inflammation of the surrounding tissues would result in a more difficult procedure. No previous studies examined this hypothesis.ObjectivesABP and acute cholangitis (AC) without ABP cases were compared to assess difficulty of ERCP.MethodsThe rate of successful biliary access, advanced cannulation method, adverse events, cannulation and fluoroscopy time were compared in 240 ABP cases and 250 AC cases without ABP. Previous papillotomy, altered gastroduodenal anatomy, and cases with biliary stricture were excluded.ResultsSignificantly more pancreatic guidewire manipulation (adjusted odds ratio (aOR) 1.921 [1.241–2.974]) and prophylactic pancreatic stent use (aOR 4.687 [2.415–9.098]) were seen in the ABP than in AC group. Average cannulation time in the ABP patients (248 vs. 185 s; p = 0.043) were longer than in AC cases. No difference was found between biliary cannulation and adverse events rates.ConclusionERCP in ABP cases seem to be more challenging than in AC. Difficult biliary access is more frequent in the ABP cases which warrants the involvement of an experienced endoscopist.