Regulation of Cx45 hemichannels mediated by extracellular and intracellular calcium

Connexin45 (Cx45) hemichannels (HCs) open in the absence of Ca(2+) and close in its presence. To elucidate the underlying mechanisms, we examined the role of extra- and intracellular Ca(2+) on the electrical properties of HCs. Experiments were performed on HeLa cells expressing Cx45 using electrical (voltage clamp) and optical (Ca(2+) imaging) methods. HCs exhibit a time- and voltage-dependent current (I(hc)), activating with depolarization and inactivating with hyperpolarization. Elevation of [Ca(2+)](o) from 20 nM to 2 μM reversibly decreases I(hc), decelerates its rate of activation, and accelerates its deactivation. Our data suggest that [Ca(2+)](o) modifies the channel properties by adhering to anionic sites in the channel lumen and/or its outer vestibule. In this way, it blocks the channel pore and reversibly lowers I(hc) and modifies its kinetics. Rapid lowering of [Ca(2+)](o) from 2 mM to 20 nM, achieved early during a depolarizing pulse, led to an outward I(hc) that developed with virtually no delay and grew exponentially in time paralleled by unaffected [Ca(2+)](i). A step increase of [Ca(2+)](i) evoked by photorelease of Ca(2+) early during a depolarizing pulse led to a transient decrease of I(hc) superimposed on a growing outward I(hc); a step decrease of [Ca(2+)](i) elicited by photoactivation of a Ca(2+) scavenger provoked a transient increase in I(hc). Hence, it is tempting to assume that Ca(2+) exerts a direct effect on Cx45 hemichannels.

Structure-activity relationships from in vitro efficacies of the thiazolide series against the intracellular apicomplexan protozoan Neospora caninum

Nitazoxanide (2-acetolyloxy-N-(5-nitro 2-thiazolyl) benzamide; NTZ) represents the parent compound of a novel class of broad-spectrum anti-parasitic compounds named thiazolides. NTZ is active against a wide variety of intestinal and tissue-dwelling helminths, protozoa, enteric bacteria and a number of viruses infecting animals and humans. While potent, this poses a problem in practice, since this obvious non-selectivity can lead to undesired side effects in both humans and animals. In this study, we used real time PCR to determine the in vitro activities of 29 different thiazolides (NTZ-derivatives), which carry distinct modifications on both the thiazole- and the benzene moieties, against the tachyzoite stage of the intracellular protozoan Neospora caninum. The goal was to identify a highly active compound lacking the undesirable nitro group, which would have a more specific applicability, such as in food animals. By applying self-organizing molecular field analysis (SOMFA), these data were used to develop a predictive model for future drug design. SOMFA performs self-alignment of the molecules, and takes into account the steric and electrostatic properties, in order to determine 3D-quantitative structure activity relationship models. The best model was obtained by overlay of the thiazole moieties. Plotting of predicted versus experimentally determined activity produced an r2 value of 0.8052 and cross-validation using the "leave one out" methodology resulted in a q2 value of 0.7987. A master grid map showed that large steric groups at the R2 position, the nitrogen of the amide bond and position Y could greatly reduce activity, and the presence of large steric groups placed at positions X, R4 and surrounding the oxygen atom of the amide bond, may increase the activity of thiazolides against Neospora caninum tachyzoites. The model obtained here will be an important predictive tool for future development of this important class of drugs.

Impact of del32-71-GH (exon 3 skipped GH) on intracellular GH distribution, secretion and cell viability: a quantitative confocal microscopy analysis

BACKGROUND: Familial isolated growth hormone deficiency (IGHD) is a disorder with about 5-30% of patients having affected relatives. Among those familial types, IGHD type II is an autosomal dominant form of short stature, associated in some families with mutations that result in missplicing to produce del32-71-GH, a GH peptide which cannot fold properly. The mechanism by which this mutant GH may alter the controlled secretory pathway and therefore suppress the secretion of the normal 22-kDa GH product of the normal allele is not known in detail. Previous studies have shown variance depending on cell type, transfection technique used, as well as on the method of analysis performed. AIM: The aim of our study was to analyse and compare the subcellular distribution/localization of del32-71-GH or wild-type (wt)-GH (22-kDa GH), each stably transfected into AtT-20, a mouse pituitary cell line endogenously producing ACTH, employed as the internal control for secretion assessment. METHODS: Colocalization of wt- and del32-71 mutant GH form was studied by quantitative confocal microscopy analysis. Using the immunofluorescent technique, cells were double stained for GH plus one of the following organelles: endoplasmic reticulum (ER anti-Grp94), Golgi (anti-betaCOP) or secretory granules (anti-Rab3a). In addition, GH secretion and cell viability were analysed in detail. RESULTS/CONCLUSIONS: Our results show that in AtT-20 neuroendocrine cells, in comparison to the wt-GH, the del32-71-GH has a major impact on the secretory pathway not only affecting GH but also other peptides such as ACTH. The del32-71-GH is still present at the secretory vesicles' level, albeit in reduced quantity when compared to wt-GH but, importantly, was secretion-deficient. Furthermore, while focusing on cell viability an additional finding presented that the various splice site mutations, even though leading eventually to the same end product, namely del32-71-GH, have different and specific consequences on cell viability and proliferation rate.

Annexins as intracellular calcium sensors

The annexins are a multigene family of Ca(2+)- and charged phospholipid-binding proteins. Although they have been ascribed with diverse functions, there is no consensus about the role played by this family as a whole. We have mapped the Ca(2+)-induced translocations of four members of the annexin family and of two truncated annexins in live cells, and demonstrated that these proteins interact with the plasma membrane as well as with internal membrane systems in a highly coordinated manner. Annexin 2 was the most Ca(2+) sensitive of the studied proteins, followed by annexins 6, 4 and 1. The calcium sensitivity of annexin 2 increased further following co-expression with S100A10. Upon elevation of [Ca(2+)](i), annexins 2 and 6 translocated to the plasma membrane, whereas annexins 4 and 1 also became associated with intracellular membranes and the nuclear envelope. The NH(2)-terminus had a modulatory effect on plasma membrane binding: its truncation increased the Ca(2+) sensitivity of annexin 1, and decreased that of annexin 2. Given the fact that several annexins are present within any one cell, it is likely that they form a sophisticated [Ca(2+)] sensing system, with a regulatory influence on other signaling pathways.

Activation of the orphan endothelial receptor Tie1 modifies Tie2-mediated intracellular signaling and cell survival

A critical role for Tie1, an orphan endothelial receptor, in blood vessel morphogenesis has emerged from mutant mouse studies. Moreover, it was recently demonstrated that certain angiopoietin (Ang) family members can activate Tie1. We report here that Ang1 induces Tie1 phosphorylation in endothelial cells. Tie1 phosphorylation was, however, Tie2 dependent because 1) Ang1 failed to induce Tie1 phosphorylation when Tie2 was down-regulated in endothelial cells; 2) Tie1 phosphorylation was induced in the absence of Ang1 by either a constitutively active form of Tie2 or a Tie2 agonistic antibody; 3) in HEK 293 cells Ang1 phosphorylated a form of Tie1 without kinase activity when coexpressed with Tie2, and Ang1 failed to phosphorylate Tie1 when coexpressed with kinase-defective Tie2. Ang1-mediated AKT and 42/44MAPK phosphorylation is predominantly Tie2 mediated, and Tie1 down-regulates this pathway. Finally, based on a battery of in vitro and in vivo data, we show that a main role for Tie1 is to modulate blood vessel morphogenesis by virtue of its ability to down-regulate Tie2-driven signaling and endothelial survival. Our new observations help to explain why Tie1 null embryos have increased capillary densities in several organ systems. The experiments also constitute a paradigm for how endothelial integrity is fine-tuned by the interplay between closely related receptors by a single growth factor.

A novel quantitative method for analyzing the distributions of nanoparticles between different tissue and intracellular compartments

The penetration, translocation, and distribution of ultrafine and nanoparticles in tissues and cells are challenging issues in aerosol research. This article describes a set of novel quantitative microscopic methods for evaluating particle distributions within sectional images of tissues and cells by addressing the following questions: (1) is the observed distribution of particles between spatial compartments random? (2) Which compartments are preferentially targeted by particles? and (3) Does the observed particle distribution shift between different experimental groups? Each of these questions can be addressed by testing an appropriate null hypothesis. The methods all require observed particle distributions to be estimated by counting the number of particles associated with each defined compartment. For studying preferential labeling of compartments, the size of each of the compartments must also be estimated by counting the number of points of a randomly superimposed test grid that hit the different compartments. The latter provides information about the particle distribution that would be expected if the particles were randomly distributed, that is, the expected number of particles. From these data, we can calculate a relative deposition index (RDI) by dividing the observed number of particles by the expected number of particles. The RDI indicates whether the observed number of particles corresponds to that predicted solely by compartment size (for which RDI = 1). Within one group, the observed and expected particle distributions are compared by chi-squared analysis. The total chi-squared value indicates whether an observed distribution is random. If not, the partial chi-squared values help to identify those compartments that are preferential targets of the particles (RDI > 1). Particle distributions between different groups can be compared in a similar way by contingency table analysis. We first describe the preconditions and the way to implement these methods, then provide three worked examples, and finally discuss the advantages, pitfalls, and limitations of this method.

Annexins sense changes in intracellular pH during hypoxia

The pH(i) (intracellular pH) is an important physiological parameter which is altered during hypoxia and ischaemia, pathological conditions accompanied by a dramatic decrease in pH(i). Sensors of pH(i) include ion transport systems which control intracellular Ca2+ gradients and link changes in pH(i) to functions as diverse as proliferation and apoptosis. The annexins are a protein family characterized by Ca2+-dependent interactions with cellular membranes. Additionally, in vitro evidence points to the existence of pH-dependent, Ca(2+)-independent membrane association of several annexins. We show that hypoxia promotes the interaction of the recombinant annexin A2-S100A10 (p11) and annexin A6 with the plasma membrane. We have investigated in vivo the influence of the pH(i) on the membrane association of human annexins A1, A2, A4, A5 and A6 tagged with fluorescent proteins, and characterized this interaction for endogenous annexins present in smooth muscle and HEK (human embryonic kidney)-293 cells biochemically and by immunofluorescence microscopy. Our results show that annexin A6 and the heterotetramer A2-S100A10 (but not annexins A1, A4 and A5) interact independently of Ca2+ with the plasma membrane at pH 6.2 and 6.6. The dimerization of annexin A2 within the annexin A2-S100A10 complex is essential for the pH-dependent membrane interaction at this pH range. The pH-induced membrane binding of annexins A6 and A2-S100A10 might have consequences for their functions as membrane organizers and channel modulators.

Distribution of intracellular and secreted surfactant during postnatal rat lung development

Pulmonary surfactant prevents alveolar collapse via reduction of surface tension. In contrast to human neonates, rats are born with saccular lungs. Therefore, rat lungs serve as a model for investigation of the surfactant system during postnatal alveolar formation. We hypothesized that this process is associated with characteristic structural and biochemical surfactant alterations. We aimed to discriminate changes related to alveolarization from those being either invariable or follow continuous patterns of postnatal changes. Secreted active (mainly tubular myelin (tm)) and inactive (unilamellar vesicles (ulv)) surfactant subtypes as well as intracellular surfactant (lamellar bodies (lb)) in type II pneumocytes (PNII) were quantified before (day (d) 1), during (d 7), at the end of alveolarization (d 14), and after completion of lung maturation (d 42) using electron microscopic methods supplemented by biochemical analyses (phospholipid quantification, immunoblotting for SP-A). Immunoelectron microscopy determined the localization of surfactant protein A (SP-A). (1) At d 1 secreted surfactant was increased relative to d 7-42 and then decreased significantly. (2) Air spaces of neonatal lungs comprised lower fractions of tm and increased ulv, which correlated with low SP-A concentrations in lung lavage fluid (LLF) and increased respiratory rates, respectively. (3) Alveolarization (d 7-14) was associated with decreasing PNII size although volume and sizes of Lb continuously increased. (4) The volume fractions of Lb correlated well with the pool sizes of phospholipids in lavaged lungs. Our study emphasizes differential patterns of developmental changes of the surfactant system relative to postnatal alveolarization.

Fructose-1,6-bisphosphate preserves intracellular glutathione and protects cortical neurons against oxidative stress

Fructose-1,6-bisphosphate (FBP), an endogenous intermediate of glycolysis, protects the brain against ischemia-reperfusion injury. The mechanisms of FBP protection after cerebral ischemia are not well understood. The current study was undertaken to determine whether FBP protects primary neurons against hypoxia and oxidative stress by preserving reduced glutathione (GSH). Cultures of pure cortical neurons were subjected to oxygen deprivation, a donor of nitric oxide and superoxide radicals (3-morpholinosydnonimine), an inhibitor of glutathione synthesis (L-buthionine-sulfoximine) or glutathione reductase (1,3-bis(2-chloroethyl)-1-nitrosourea) in the presence or absence of FBP (3.5 mM). Neuronal viability was determined using an 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay. FBP protected neurons against hypoxia-reoxygenation and oxidative stress under conditions of compromised GSH metabolism. The efficacy of FBP depended on duration of hypoxia and was associated with higher intracellular GSH concentration, an effect partly mediated via increased glutathione reductase activity.

Influence of CYP2B6 polymorphism on plasma and intracellular concentrations and toxicity of efavirenz and nevirapine in HIV-infected patients

BACKGROUND: Efavirenz (EFV) and nevirapine (NVP) are metabolized by cytochrome P450 2B6 (CYP2B6). Allele 516 G>T (Gln172His) is associated with diminished activity of this isoenzyme, and may lead to differences in drug exposure. METHODS: We evaluated this allele as a pharmacogenetic marker of EFV and NVP pharmacokinetics and EFV toxicity in 167 participants receiving EFV and 59 receiving NVP recruited within the genetics project of the Swiss HIV Cohort Study. Drug concentrations were measured in plasma and in peripheral blood mononuclear cells (PBMCs) from the same sample. Neuropsychological toxicity of EFV (sleep disorders, mood disorders, fatigue) was assessed using a standardized questionnaire. RESULTS AND CONCLUSIONS: CYP2B6 516TT was associated with greater plasma and intracellular exposure to EFV, and greater plasma exposure to NVP. Intracellular drug concentration, and CYP2B6 genotype were predictors of EFV neuropsychological toxicity. CYP2B6 genotyping may be useful to complement an individualization strategy based on plasma drug determinations to increase the safety and tolerability of EFV.

Vancomycin acts synergistically with gentamicin against penicillin-resistant pneumococci by increasing the intracellular penetration of gentamicin

Vancomycin and gentamicin act synergistically against penicillin-resistant pneumococci in vitro and in experimental rabbit meningitis. The aim of the present study was to investigate the underlying mechanism of this synergism. The intracellular concentration of gentamicin was measured by using the following experimental setting. Bacterial cultures were incubated with either gentamicin alone or gentamicin plus vancomycin for a short period (15 min). The gentamicin concentration was determined before and after grinding of the cultures by using the COBAS INTEGRA fluorescence polarization system (Roche). The grinding efficacies ranged between 44 and 54%, as determined by viable cell counts. In the combination regimen the intracellular concentration of gentamicin increased to 186% compared to that achieved with gentamicin monotherapy. These data suggest that the synergy observed in vivo and in vitro is based on an increased intracellular penetration of the aminoglycoside, probably due to the effect of vancomycin on the permeability of the cell wall.

Monomerization of dimeric IgG of intravenous immunoglobulin (IVIg) increases the antibody reactivity against intracellular antigens

Intravenous immunoglobulin (IVIg) preparations are derived from pooled plasma from up to 60,000 healthy human donors and reflect the immunologic experience of the donor population. IVIg contains monomeric and dimeric IgG populations which are in a dynamic equilibrium depending on concentration, pH, temperature, donor pool size, time and stabilizers added in order to keep the portion of dimeric IgG below a certain level. In the present study, monomeric and dimeric fractions were isolated by size exclusion chromatography. The dimeric fractions, however, showed a dynamic instability and tended to dissociate. Both dimeric and monomeric IgG fractions were acid treated (pH 4) in order to dissociate the dimeric IgG. Western-blot analysis identified a sub-population of SDS resistant IgG dimers. Furthermore, the reactivities of the fractions were tested against a panel of self- and exo-antigens. There was a marked increase in activity of the dimeric compared to the monomeric IgG fraction against various intracellular self-antigens. Our data indicates that the increased reactivities of pH 4-treated fractions can mainly be attributed to dimer dissociation, as pH 4-treated monomers do not show significantly increased activities against a range of antigens.