Metabolic Biomarkers Differentiate a Surgical Intervertebral Disc from a Nonsurgical Intervertebral Disc.

BACKGROUND: Degeneration of the intervertebral disc (IVD) is caused by disturbances in metabolic processes, which lead to structural disorders. The aim of this report is to analyze metabolic disorders in the degeneration process by comparing control discs with degenerated discs. In our research on the nucleus pulposus (NP), we used NMR spectroscopy of extracts of hydrophilic and hydrophobic compounds of the tissue. METHODS: Nuclear magnetic resonance (NMR) spectroscopy allows the study of biochemistry and cellular metabolism in vitro. Hydrophilic and hydrophobic compounds were extracted from the NP of the intervertebral disc. In the NMR spectra, metabolites were identified and quantitatively analyzed. The results of our research indicate disturbances in the biosynthesis and metabolism of cholesterol, the biosynthesis and degradation of various fatty acid groups, ketone bodies, or lysine, and the metabolism of glycerophospholipids, purines, glycine, inositol, galactose, alanine, glutamate, and pyruvate in the biosynthesis of valine and isoleucine, leucine. All these disorders indicate pathomechanisms related to oxidative stress, energy, neurotransmission disturbances, and disturbances in the structure and functioning of cell membranes, inflammation, or chronic pain generators. CONCLUSIONS: NMR spectroscopy allows the identification of metabolites differentiating surgical from nonsurgical discs. These data may provide guidance in in vivo MRS studies in assessing the severity of lesions of the disc.

Forced Oscillation Measurements in Patients with Idiopathic Interstitial Pneumonia Subjected to Pulmonary Rehabilitation.

(1) Background: Pulmonary rehabilitation (PR) plays a significant therapeutic role for patients with idiopathic interstitial pneumonia (IIP). The study assessed the impact of physical activity on lung function measured by forced oscillation technique (FOT). (2) Methods: The study involved 48 patients with IIP subjected to a 3-week inpatient PR. The control group included IIP patients (n = 44) on a 3-week interval without PR. All patients were assessed at baseline and after 3 weeks of PR by FOT, spirometry, plethysmography, grip strength measurement and the 6-minute walk test. (3) Results: There were no significant changes in FOT measurements in the PR group, except for reduced reactance at 11 Hz, observed in both groups (p < 0.05). Patients who completed PR significantly improved their 6-min walk distance (6MWD) and forced vital capacity (FVC). The change in 6MWD was better in patients with higher baseline reactance (p = 0.045). (4) Conclusions: Patients with IIP benefit from PR by an increased FVC and 6MWD; however, no improvement in FOT values was noticed. Slow disease progression was observed in the study and control groups, as measured by reduced reactance at 11 Hz. Patients with lower baseline reactance limitations achieve better 6MWD improvement.

Serum metabolic profiles and metal levels of patients with multiple sclerosis and patients with neuromyelitis optica spectrum disorders - NMR spectroscopy and ICP-MS studies.

BACKGROUND AND OBJECTIVES: Neuromyelitis optica spectrum disorder (NMOSD) is a disease misdiagnosed with multiple sclerosis (MS). We hypothesized that the serum metabolic profile could be helpful in the differentiation of both diseases in an early stage. METHODS: We included controls, patients with MS diagnosed according to the McDonald criteria of 2010, and patients with NMOSD diagnosed according to the criteria from 2015. Blood samples were collected on clots from all participants after overnight overfasting. We obtained metabolic profiles using proton magnetic resonance spectroscopy (1HNMR) of serum hydrophilic and hydrophobic compounds. Serum metal levels were measured using isotope-specific detection mass spectrometry (ICP-MS). For statistical analyzes, we used ANOVA tests and multivariate analysis (MVA) - orthogonal partial least square discriminant analysis (OPLS-DA). RESULTS: We analyzed metabolite levels in patient groups compared to controls. We observed significantly different levels of ten metabolite signals in patients with MS vs controls and eighteen metabolite signals in patients with NMSOD vs controls. We observed significantly different levels of five signals in patients with MS vs NMOSD. In the MVA analysis of patient groups, we indicated compounds that most differentiated the groups. All of these compounds are involved in cycles connected to the inflammation process and/or oxidative stress. The results of metallomics studies confirmed metal participation in these processes. DISCUSSION: It is possible to distinguish patients with MS and NMOSD from controls using ANOVA and MVA tests. The chosen metabolite profile analyzes might possibly be helpful in distinguishing the two diseases from each other in some seronegative and radiologically negative cases.

Zinc and Copper Brain Levels and Expression of Neurotransmitter Receptors in Two Rat ASD Models.

Zinc and copper are important trace elements necessary for the proper functioning of neurons. Impaired zinc and/or copper metabolism and signaling are implicated in many brain diseases, including autism (ASD). In our studies, autistic-like behavior in rat offsprings was induced by application to pregnant mothers valproic acid or thalidomide. Zinc and copper contents were measured in serum and brain structures: hippocampus, cerebral cortex, and cerebellum. Our research shows no interconnections in the particular metal concentrations measured in autistic animal brains and their sera. Based on patient researches, we studied 26 genes belonging to disturbed neurotransmitter pathways. In the same brain regions, we examined the expression of genes encoding proteins of cholinergic, adrenergic, serotonin, and dopamine receptors. In both rats' ASD models, 17 out of the tested gene expression were decreased. In the cerebellum and cerebral cortex, expression of genes encoding cholinergic, adrenergic, and dopaminergic receptors decreased, whereas in the hippocampus only expression of serotoninergic receptors genes was downregulated. The changes in metals content observed in the rat brain can be secondary phenomena, perhaps elements of mechanisms that compensate for neurotransmission dysfunctions.

The effects of exercise training on lipid profile in patients with sarcoidosis.

This study aimed to determine the use of lipid profiling to assess the effects of moderate intensity exercise training (ET) on patients with sarcoidosis. Fourteen patients with sarcoidosis (mean age, 46.0 ± 9.6 years) were examined before and after 3-week of ET programme in hospital settings. Symptoms (fatigue: FAS, dyspnoea: MRC), lung function tests and physical function tests (6 MWT, muscle force) were measured before and after ET. Proton nuclear magnetic resonance (NMR) spectroscopy combined with orthogonal partial least squares-discriminant analysis (OPLS-DA) was used to determine lipid profile before and after ET. Twenty-five NMR signals from lipid compounds were selected for further analysis as well as serum lipid and inflammatory markers. Three weeks of ET results in improvement of symptoms (FAS: 27.5 vs. 21.0; p < 0.001, MRC: 0.86 vs. 0.14; p = 0.002) and physical function (6MWT: 508.43 vs. 547.29; p = 0.039). OPLS-DA analysis of the lipid profiles of patients with sarcoidosis revealed differences among the samples before and after ET, including decreases in fatty acids (p < 0.017), triglycerides (p < 0.022) and total cholesterol (p < 0.020). Other changes included shifts in fatty acids oxidation products and triacylglycerol esters. A short-time, in-hospital exercise training benefits patients with sarcoidosis by enhancing their physical function. Additionally, positive effect on lipid profile was observed also in this study. It is suggested that lipid profiling could become a new prognostic method to assess effects of pulmonary rehabilitation in patients with sarcoidosis.

Changes in the metabolic profiles of the serum and putamen in Parkinson's disease patients - In vitro and in vivo NMR spectroscopy studies.

The aim of this study was to investigate the relationship between serum metabolomic biomarkers and brain in vivo magnetic resonance spectroscopy (MRS) biomarkers in patients with Parkinson's disease (PD) as well as to investigate compound concentration changes by comparing the results with healthy control subjects. Univariate statistical analysis of the serum showed significant differences in the levels of phenylalanine, tyrosine, lysine, glutamine, glutamate, acetone, acetate, 3-hydroxybutyrate, and 1-monoacylglycerol (1-MAG) between the PD patient group and the control group. Orthogonal partial least squares discriminant analysis showed significantly different compound concentrations of acetate, 3-hydroxybutyrate, glutamine, tyrosine, 1-MAG and testosterone. In vivo MRS of the putamen showed significantly higher concentrations of glutamine/glutamate complex and glutamine in patients with PD in comparison to control subjects. Following disrupted metabolic pathways in patients with PD were identified: dopamine synthesis, steroid hormone biosynthesis, fatty acid biosynthesis, the synthesis and degradation of ketone bodies, the metabolism of pyruvate, arginine, proline, alanine, aspartate, glutamate, tyrosine and phenylalanine. The obtained results may indicate changes in neurotransmission, disturbances in energy production and an altered cell membrane structure.

Assessment of the brain ischemia during orthostatic stress and lower body negative pressure in air force pilots by near-infrared spectroscopy.

A methodology for the assessment of the cerebral hemodynamic reaction to normotensive hypovolemia, reduction in cerebral perfusion and orthostatic stress leading to ischemic hypoxia and reduced muscular tension is presented. Most frequently, the pilots of highly maneuverable aircraft are exposed to these phenomena. Studies were carried out using the system consisting of a chamber that generates low pressure around the lower part of the body - LBNP (lower body negative pressure) placed on the tilt table. An in-house developed 6-channel NIRS system operating at 735 and 850 nm was used in order to assess the oxygenation of the cerebral cortex, based on measurements of diffusely reflected light in reflectance geometry. The measurements were carried out on a group of 12 active pilots and cadets of the Polish Air Force Academy and 12 healthy volunteers. The dynamics of changes in cerebral oxygenation was evaluated as a response to LBNP stimuli with a simultaneous rapid change of the tilt table angle. Parameters based on calculated changes of total hemoglobin concentration were proposed allowing to evaluate differences in reactions observed in control subjects and pilots/cadets. The results of orthogonal partial least squares-discriminant analysis based on these parameters show that the subjects can be classified into their groups with 100% accuracy.

Gene polymorphisms and motor levodopa-induced complications in Parkinson's disease.

OBJECTIVE: The aim of the study was to evaluate the association of individual and combined single-nucleotide polymorphisms in brain-derived neurotrophic factor (BDNF), dopamine transporter (DAT), and catechol-O-methyltransferase (COMT) genes with the occurrence of motor levodopa-induced complications (MLIC) in Parkinson's disease (PD). MATERIALS AND METHODS: We studied 76 patients with PD (MLIC occurred in 56.6%) and 60 controls. Allelic discrimination of rs6265 BDNF (Val66Met), rs397595 DAT (SLC6A3), and rs4680 COMT (Val158Met) genes were genotyped. Odds ratios (OR) and 95% confidence intervals (95% CI) were calculated using multinominal logistic regression. Orthogonal partial least squares (OPLS) analysis and OPLS discriminant analysis (OPLS-DA) were used to analyze qualitative genetic data. RESULTS: The risk of PD in subjects with the AG BDNF genotype was increased sixfold (OR = 6.12, 95% CI = 2.88-13.02, p < .0001), and AG BDNF and AG DAT genotypes were correlated with PD in OPLS-DA (VIP > 1). There were no differences in distributions of BDNF, DAT and COMT genotypes between PD groups with and without MLIC, while OPLS model showed that genotype combination of AG BDNF, AG DAT, and GG COMT was correlated with MLIC and genotypes combination of GG BDNF, AA DAT, and AA COMT with lack of MLIC in PD patients (VIP > 1). CONCLUSIONS: Our results confirmed the association of rs6265 BDNF (Val66Met) with the risk of PD and suggest a synergic effect of rs6265 BDNF (Val66Met), rs397595 DAT (SLC6A3), and rs4680 COMT (Val158Met) polymorphisms on the occurrence of MLIC.

Glutamate, Glutamine and GABA Levels in Rat Brain Measured Using MRS, HPLC and NMR Methods in Study of Two Models of Autism.

The disorders of the glutamatergic neurotransmission have been associated with pathogenesis of autism. In this study we evaluated the impact of the in vivo and ex vivo test methodology on measurements of levels of neurotransmitter amino acids in hippocampus of rats for valproic acid- (VPA) and thalidomide- (THAL) induced models of autism. The main goal was to compare the changes in concentrations of glutamate (Glu), glutamine (Gln) and GABA between both autistic groups and the control, measured in vivo and ex vivo in homogenates. The rat pups underwent three in vivo tests: ultrasonic vocalization (USV), magnetic resonance spectroscopy (MRS) and unilateral microdialysis of the hippocampus. Analyses of homogenates of rat hippocampus were performed using high-performance liquid chromatography (HPLC) and nuclear magnetic resonance (NMR) spectroscopy. For the statistical analysis, we performed univariate and multivariate tests. USV test, which is considered in rodents as an indicator of pathology similar to autism, showed decreased USV in VPA and THAL groups. In vivo MRS studies demonstrated increases of Glu content in male rat's hippocampus in VPA and THAL groups, while the microdialysis, which allows examination of the contents in the extracellular space, detected decreases in the basal level of Gln concentrations in VPA and THAL groups. Ex vivo HPLC studies showed that levels of Glu, Gln and GABA significantly increased in male rat's hippocampus in the VPA and THAL groups, while NMR studies showed increased levels of Gln and GABA in the VPA group. Collectively, these results are consistent with the hypothesis suggesting the role of the glutamatergic disturbances on the pathogenesis of autism. For all methods used, the values of measured changes were in the same direction. The orthogonal partial least square discriminant analysis confirmed that both animal models of autism tested here can be used to trace neurochemical changes in the brain.

Confirmation of brain death using optical methods based on tracking of an optical contrast agent: assessment of diagnostic feasibility.

We aimed to determine whether optical methods based on bolus tracking of an optical contrast agent are useful for the confirmation of cerebral circulation cessation in patients being evaluated for brain death. Different stages of cerebral perfusion disturbance were compared in three groups of subjects: controls, patients with posttraumatic cerebral edema, and patients with brain death. We used a time-resolved near-infrared spectroscopy setup and indocyanine green (ICG) as an intravascular flow tracer. Orthogonal partial least squares-discriminant analysis (OPLS-DA) was carried out to build statistical models allowing for group separation. Thirty of 37 subjects (81.1%) were classified correctly (8 of 9 control subjects, 88.9%; 13 of 15 patients with edema, 86.7%; and 9 of 13 patients with brain death, 69.2%; p < 0.0001). Depending on the combination of variables used in the OPLS-DA model, sensitivity, specificity, and accuracy were 66.7-92.9%, 81.8-92.9%, and 77.3-89.3%, respectively. The method was feasible and promising in the demanding intensive care unit environment. However, its accuracy did not reach the level required for brain death confirmation. The potential usefulness of the method may be improved by increasing the depth of light penetration, confirming its accuracy against other methods evaluating cerebral flow cessation, and developing absolute parameters for cerebral perfusion.

Serum lipidomics in diagnostics of sarcoidosis.

The aim of this study was to determine the use of the lipid profile of patients with sarcoidosis and compare it with healthy subjects. We assume that lipid profile of serum in sarcoidosis differs from the lipid profile of control subjects. Serum was collected from 14 patients with II stage of sarcoidosis and 14 control subjects (healthy volunteers). Proton NMR spectroscopy combined with discriminant analyses, OPLS-DA (orthogonal partial least squares projections to latent structures discriminant analysis), was used. Thirty four NMR signals of lipid compounds were selected. OPLS-DA model consisted of three components and very good explain the data and also predict the data. Discriminant analysis correctly classified patients according to their groups for 92.9% of sarcoidose and 100% of control. From multivariate discriminant analysis we obtain a list of potentialbiomarkers which are statistically significant and which separate one class from another. These biomarkers are statistically significant, but not necessarily biochemically significant. They may have biochemical significance and they may be the biomarkers we are interested in, however, this must be established through extensive testing. Presented method allows distinguishing between healthy subject and sarcoidosis patients. (Sarcoidosis Vasc Diffuse Lung Dis 2018; 35: 150-153).

Role of Ryanodine and NMDA Receptors in Tetrabromobisphenol A-Induced Calcium Imbalance and Cytotoxicity in Primary Cultures of Rat Cerebellar Granule Cells.

The study assessed the role of ryanodine receptors (RyRs) and NMDA receptors (NMDARs) in the Ca(2+) transients and cytotoxicity induced in neurons by the brominated flame retardant tetrabromobisphenol A (TBBPA). Primary cultures of rat cerebellar granule cells (CGC) were exposed to 7.5, 10, or 25 µM TBBPA for 30 min, and cell viability was assessed after 24 h. Moreover, (45)Ca uptake was measured, and changes in the intracellular Ca(2+) concentration ([Ca(2+)]i) were studied using the fluo-3 probe. The involvement of NMDARs and RyRs was verified using the pertinent receptor antagonists, 0.5 µM MK-801 and 2.5 µM bastadin 12, which was co-applied with 200 µM ryanodine, respectively. The results show that TBBPA concentration-dependently induces an increase in [Ca(2+)]i. This effect was partly suppressed by the inhibitors of RyRs and NMDARs when administered separately, and completely abrogated by their combined application. A concentration-dependent activation of (45)Ca uptake by TBBPA was prevented by MK-801 but not by RyR inhibitors. Application of ≥ 10 µM TBBPA concentration-dependently reduced neuronal viability, and this effect was only partially and to an equal degree reduced by NMDAR and RyR antagonists given either separately or in combination. Our results directly demonstrate that both the RyR-mediated release of intracellular Ca(2+) and the NMDAR-mediated influx of Ca(2+) into neurons participate in the mechanism of TBBPA-induced Ca(2+) imbalance in CGC and play a significant, albeit not exclusive, role in the mechanisms of TBBPA cytotoxicity.

Neurotoxic effects of indocyanine green -cerebellar granule cell culture viability study.

The aim of this study was to examine neurotoxicity indocyanine green (ICG). We assessed viability of primary cerebellar granule cell culture (CGC) exposed to ICG to test two mechanisms that could be the first triggers causing neuronal toxicity: imbalance in calcium homeostasis and the degree of oligomerization of ICG molecules. We have observed this imbalance in CGC after exposure to 75-125µΜ ICG and dose and application sequence dependent protective effect of Gadovist on surviving neurons in vitro when used with ICG. Spectroscopic studies suggest the major cause of toxicity of the ICG is connected with oligomers formation. ICG at concentration of 25 µM (which is about 4 times higher than the highest concentration of ICG in the brain applied in in-vivo human studies) is not neurotoxic in the cell culture.

Synergistic neurotoxicity of oxygen-glucose deprivation and tetrabromobisphenol A in vitro: role of oxidative stress.

BACKGROUND: Tetrabromobisphenol A (TBBPA) is a toxic brominated flame retardant. Previous studies have demonstrated that exposure of primary cultures of rat cerebellar granule cells (CGC) to ≥ 10 µM TBBPA induces toxicity and excitotoxicity, and the underlying mechanism may involve calcium imbalance and oxidative stress. Here we examined whether the application of TBBPA at subtoxic concentrations may exacerbate acute damage of CGC challenged with oxygen-glucose deprivation (OGD), and evaluated with fluorescent indicators the involvement of calcium imbalance, mitochondrial depolarization and oxidative stress. METHODS: Survival of CGC was assessed 24 h after OGD/TBBPA using fluorescent dyes. An OGD challenge lasting for 45, 60 or 75 min induced a duration-dependent injury to the neurons. RESULTS: Application of 2.5, 5 or 7.5 µM TBBPA for 45 min to normoxic and glucose-containing incubation medium did not reduce the viability of cultured CGC, but this compound exacerbated the toxic effects of OGD in a concentration-dependent way. Moreover, TBBPA had a slight effect on calcium homeostasis and mitochondrial membrane potential, but significantly activated the production of reactive oxygen species in CGC. The application of H(2)O(2) at 5, 10 and 25 µM mimicked the effects of TBBPA on OGD toxicity, while 0.1 mM ascorbic acid or 1 mM glutathione ameliorated this toxicity. CONCLUSION: These results suggest the involvement of oxidative stress in the synergistic neurotoxic effects of TBBPA and OGD.

Multiwavelength time-resolved detection of fluorescence during the inflow of indocyanine green into the adult's brain.

Optical technique based on diffuse reflectance measurement combined with indocyanine green (ICG) bolus tracking is extensively tested as a method for clinical assessment of brain perfusion in adults at the bedside. Methodology of multiwavelength and time-resolved detection of fluorescence light excited in the ICG is presented and advantages of measurements at multiple wavelengths are discussed. Measurements were carried out: 1. on a physical homogeneous phantom to study the concentration dependence of the fluorescence signal, 2. on the phantom to simulate the dynamic inflow of ICG at different depths, and 3. in vivo on surface of the human head. Pattern of inflow and washout of ICG in the head of healthy volunteers after intravenous injection of the dye was observed for the first time with time-resolved instrumentation at multiple emission wavelengths. The multiwavelength detection of fluorescence signal confirms that at longer emission wavelengths, probability of reabsorption of the fluorescence light by the dye itself is reduced. Considering different light penetration depths at different wavelengths, and the pronounced reabsorption at longer wavelengths, the time-resolved multiwavelength technique may be useful in signal decomposition, leading to evaluation of extra- and intracerebral components of the measured signals.

Wavelength-resolved measurements of fluorescence lifetime of indocyanine green.

We study fluorescence lifetime of indocyanine green (ICG) using femtosecond laser and sensitive detection based on time-correlated single-photon counting. A time-resolved multichannel spectral system is constructed and applied for determination of the fluorescence lifetime of the ICG in different solvents. Emission properties of ICG in water, milk, and 1% intralipid solution are investigated. Fluorescence of the fluorophore of different concentrations (in a range of 1.7-160 µM) dissolved in different solutions is excited by femtosecond pulses generated with the use of Ti:Sa laser tuned within the range of 740-790 nm. It is observed that fluorescence lifetime of ICG in water is 0.166 ± 0.02 ns and does not depend on excitation and emission wavelengths. We also show that for the diffusely scattering solvents (milk and intralipid), the lifetime may depend on the dye concentration (especially for large concentrations of ICG). This effect should be taken into account when analyzing changes in the mean time of arrival of fluorescence photons excited in ICG dissolved in such optically turbid media.

Serum interleukin (IL-2, IL-10, IL-6, IL-4), TNFalpha, and INFgamma concentrations are elevated in patients with atypical and idiopathic parkinsonism.

We investigated serum levels of interleukin (IL)-2, IL-10, IL-6, IL-4, TNFalpha, INFgamma in 7 patients with atypical parkinsonism (AP), 31 idiopathic PD (iPD) patients, 17 idiopathic PD with cardiovascular risk factor (iPD-CVRF) patients, and 20 age-matched controls (healthy, non-parkinsonian patients). Cytokine concentrations were measured using the Becton Dickinson (BD) human Th1/Th2 Cytokine kit II with a flow cytometry system. The concentrations of IL-2, IL-10, IL-4, IL-6, TNFalpha, and INFgamma were detectable in the serum from all groups, including the control. Increased serum IL-2, IL-10, IL-4, IL-6, TNFalpha, and INFgamma concentrations were found in all groups of parkinsonian patients, as compared to the control group. The highest elevations of serum IL-2, IL-4, IL-6, TNFalpha, and INFgamma concentrations were observed in AP patients, as compared to the iPD and iPD-CVRF groups. However, the serum IL-6 concentration was higher in the iPD-CVRF group than in the iPD group. The IL-10 level was significantly higher in all groups of PD patients relative to the control group, but was the lowest in the serum from the AP patients. Moreover, the serum levels of lipid peroxidation products were enhanced 2.1- and 1.5-fold in AP and both iPD groups, respectively. These results argue in favor of the involvement of immunological events in the process of neurodegeneration in AP and PD.

Changes in concentration of cerebrospinal fluid components in patients with traumatic brain injury.

Brain injury, like other central nervous system pathologies, causes changes in the composition of the cerebrospinal fluid (CSF). In this study, changes in the concentration of small molecules of the CSF, which are in the minimal micromolar concentration, were observed and monitored using high-resolution proton (NMR) spectroscopy. Twenty-two patients with isolated traumatic brain injuries (TBI) and 15 patients making up the control group were recruited for the study. CSF samples were collected by lumbar puncture from the lumbar subarachnoid space in the patients just before commencement of therapy and on the first, third, seventh and fourteenth days of therapy at the ICU. Forty-four signals of the NMR spectra and NO concentration of the CSF samples were analyzed. The analysis shows that the amino acid and organic acid concentrations change during the therapy and mostly are higher than in the control group. Significant differences in concentration of the analyzed CSF components between the TBI patients and the control group have been noted. The rate of the lactate to pyruvate conversion increased because the L/P ratio showed no significant differences between the TBI group and the control group, while the concentrations of both components were significantly higher in the TBI patients than in the control group. Citrulline, arginine and nitric oxide concentrations were the focus of the analysis. Citrulline concentration changes overlapped NO changes from 0 until 3rd day of therapy, while for the remaining days of observation the NO concentration stabilized at the control level, whereas citrulline concentration significantly decreased.

Synthetic bastadins modify the activity of ryanodine receptors in cultured cerebellar granule cells.

Although the interactions of several natural bastadins with the RyR1 isoform of the ryanodine receptor in sarcoplasmic reticulum has been described, their structure-dependent interference with the RyR2 isoform, mainly expressed in cardiac muscle and brain neurons, has not been studied. In this work, we examined calcium transients induced by natural bastadin 10 and several synthetic bastadins in cultured cerebellar granule cells known to contain RyR2. The fluorescent calcium indicator fluo-3 and confocal microscopy were used to evaluate changes in the intracellular Ca(2+) concentration (Ca(i)), and the involvement of ryanodine receptors was assessed using pharmacological tools. Our results demonstrate that apart from the inactive BAST218F6 (a bisdebromo analogue of bastadin 10), synthetic bastadin 5, and synthetic analogues BAST217B, BAST240 and BAST268 (at concentrations >20 microM) increased Ca(i) in a concentration-dependent, ryanodine- and FK-506-sensitive way, with a potency significantly exceeding that of 20 mM caffeine. Moreover, the same active bastadins at a concentration of 5 muM in the presence of ryanodine prevented a thapsigargin-induced increase in Ca(i). These results indicate that bastadins, acting in a structure-dependent manner, modify the activity of RyR2 in primary neuronal culture and provide new information about structure-related pharmacological properties of bastadins.