Biomedical subjects

Hubert H Kerschbaum

Publications and source records attributed to Hubert H Kerschbaum.

14 recordsLinked to original sources

PubMed2007

Nitric oxide decreases ammonium release in tadpoles of the clawed frog, Xenopus laevis, Daudin.

In the present study, we quantified the physiological consequences of nitric oxide (NO) on ammonium release in tadpoles of Xenopus laevis. Tadpoles exposed to S-nitro-N-acetylpenicillamine (SNAP), an NO-donor, or L: -arginine, the substrate of NO synthase (NOS), showed a reversible decrease, whereas animals exposed to the NOS inhibitor Nomega-methyl-L: -arginine (L: -NMMA) exhibited an increase in ammonium release. Release of ammonium may be of physiological relevance during stress response of the animal. Handling of tadpoles as well as exposure to hyposmotic environments increased ammonium release. To localize NO synthesizing cells, we used diaminofluorescein-diacetate (DAF-2DA), an NO-sensitive fluorescent dye, and NADPH-diaphorase histochemistry, an indicator for NOS activity. We observed a fluorescence signal as well as NADPH-diaphorase activity in small, solitary cells in the epidermis. Similarly to NADPH-diaphorase histochemistry, silver nitrate staining and rhodamine labelling, markers for mitochondria-rich cells, showed a strong reaction in these cells. These observations indicate that NO (1) inhibits ammonium release, and (2) is endogenously synthesized in mitochondria-rich cells in Xenopus tadpoles. Based on our histochemical results, we speculate that gill epithelium and epidermis work in parallel to release ammonium as epidermal tissue contains mitochondria-rich and NADPH-diaphorase positive cells.

Animals↗

PubMed2006

An artificial three-dimensional matrix promotes ramification in the microglial cell-line, BV-2.

Three-dimensional (3D) cell culture yields strikingly different cell phenotypes compared to two-dimensional (2D) cell culture. Since microglia, monocyte derived immune cells in the brain, exist in a variety of cell shapes ranging from amoeboid to ramified, we evaluated the impact of 2D versus 3D culture conditions on cell shape. The microglial cell-line, BV-2, was either cultured on poly-D-lysine coated dishes (2D culture conditions) or in a BD Pura Matrix Peptide Hydrogel (3D culture conditions) in the absence or presence of the extracellular matrix proteins, fibronectin and collagen type I, respectively. We identified five distinct morphological phenotypes (amoeboid, bipolar, tripolar, multipolar, ramified) and compared the frequency distribution of these phenotypes under different culture conditions using a chi(2) test. Culture of BV-2 cells in an inert 3D matrix shifted the frequency distribution from an amoeboid dominated population, which is typical for BV-2 cells cultured under conventional 2D conditions, to a population dominated by multipolar phenotypes. Fibronectin or collagen type I significantly suppressed matrix-induced ramification. These cell culture experiments illustrate the dependency of cell shape on spatial distribution of potential adhesion sites.

Animals↗

PubMed2006

Ultraviolet irradiation-induced apoptosis does not trigger nuclear fragmentation but translocation of chromatin from nucleus into cytoplasm in the microglial cell-line, BV-2.

Chromatin condensation, decrease of nuclear volume, and nuclear fragmentation are key features of apoptosis (programmed cell death) in many eukaryotic cells. How chromatin is redistributed in a continuously shrinking nucleus is an intriguing question. To evaluate this interesting spatial problem, we studied the ultrastructural distribution of chromatin in distinct stages of apoptosis using the microglial cell-line, BV-2, as a model and UV irradiation as a trigger of apoptosis. During apoptosis, condensed chromatin accumulated initially at the nuclear periphery and, subsequently, occupied almost the entire nucleus. Surprisingly, nuclei did not fragmentize, but apoptotic cells showed condensed chromatin in the nucleus as well as in the nucleus-attached cytoplasm. During apoptosis, the nuclear envelope dilated and decreased in extension by formation of numerous electron lucent vesicles, which accumulated in the cytoplasm. Furthermore, we observed in BV-2 cells well-known apoptotic features, like increased caspase-3/7 activity and annexin V labeling, as well as a sequence of cell morphological alterations, including cell shrinkage, zeiosis, and formation of apoptotic bodies. Thus, our findings suggest that UV-induced chromatin degradation is not restricted to the nucleus but may also take place in the cytoplasm in BV-2 cells.

Animals↗

PubMed2006

Gender-dependent differences in sensation seeking and social interaction are correlated with saliva testosterone titre in adolescents.

OBJECTIVES: In the present study, we tested the hypothesis that gender - dependent differences in novelty seeking, leadership, and sympathy might correlate with testosterone titre. Since several studies report that the impact of testosterone on personality traits is more visible under emotional challenging situations, we harvested saliva testosterone upon an anticipated stressor (academic examination) and under basal conditions. SETTING AND DESIGN: 19 female and 23 male adolescents (17 to 19 years of age) completed standardized questionnaires on sensation seeking, anxiety, and social interaction. Two weeks later, they had to write an anticipated, rigorous examination in mathematics in their school. Before and after the examination, saliva had been harvested from each subject and testosterone titre has been estimated. METHODS: Saliva testosterone was quantified using a luminescence immunoassay (LIA). Each subject completed questionnaires on sensations seeking according to Zuckerman (SSS - V), anxiety (STAI), and social interactions. RESULTS: Both genders showed an increase in their testosterone titre shortly after examination or announcement of test scores. A Spearman correlation coefficient indicated a significant correlation between testosterone titre and sensation seeking subscales in female but not in male subjects. Analysis of social interactions revealed that peers regarded male subjects, who had high testosterone titres, as leaders but not as likeable individuals, whereas they regarded female subjects, who had high testosterone titres, not as leaders but as highly likeable individuals. CONCLUSION: Theses findings strongly suggest that testosterone has gender specific effects on novelty seeking, dominance, and sympathy.

Adolescent↗

PubMed2006

Progesterone and its metabolites 5-dihydroprogesterone and 5-3-tetrahydroprogesterone decrease LPS-induced NO release in the murine microglial cell line, BV-2.

OBJECTIVE: Microglial cells, important immunosurveillance cells in the nervous system, respond to pathogens with an increase in inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) release. Because excessive release of NO may be neurotoxic, tight regulation of nitrosative stress is required. DESIGN AND SETTING: We cultured the murine microglial cell-line, BV-2, with bacterial lipopolysaccharide (LPS) to induce NO synthesis and quantified the impact of progesterone and its metabolites, 5alpha-dihydroprogesterone (DHP) and 5alpha-3alpha-tetrahydroprogesterone (THP), on NO release. RESULTS: Our in vitro experiments showed that in BV-2 cells LPS-induced NO release is suppressed by progesterone and THP. Both neurosteroids decreased NO release by about 40% when used at a concentration of 10 microM. NO release was less sensitive to DHP. This neurosteroid decreased NO release only by 20% when used at a concentration of 10 microM. NO release was sensitive to N(G)-methyl-L-arginine (L-NMMA), a completive inhibitor of NOS, indicating that LPS-induced NO release is mediated by NOS activity. Trypan blue exclusion experiments showed that the ratio of viable to dead cells did not vary using different concentrations of progesterone. Furthermore, progesterone did not increase apoptosis or necrosis when estimated by the distribution of DAPI-labelled condensed chromatin. CONCLUSION: These experiments indicate that the decline in NO release is mainly due to modulation of NOS activity or expression. Because progesterone, DHP, and THP are synthesized in astrocytes and oligodendrocytes, these neurosteroids may locally suppress an immune response.

5-alpha-Dihydroprogesterone↗

PubMed2006

Cortisol titre increases with novelty of academic oral examinations.

OBJECTIVE: In the present study we evaluated whether intensity and novelty of oral academic examinations are reflected in saliva cortisol titre. DESIGN AND SETTING: 91 pupils completed questionnaires on sensation seeking according to Zuckerman as well as on stress coping strategies and rated their individual stress intensity upon minor oral examinations. 26 of these students donated saliva samples before as well as ten and 30 minutes after the examination to quantify cortisol using an immuno assay. Oral examinations during the school year, where students may re-adjust their grade on subsequent examinations, were regarded as minor oral examinations, whereas school exit examinations, where students do not have the chance to correct their grade anymore and may even risk a delay of several months in their academic career when they fail, were regarded as major examinations. RESULTS: Minor oral examinations revealed either a moderate, but significant, increase in saliva cortisol titre by 1.1 fold or did not have a significant impact on cortisol titre. In contrast, school exit exams elevated cortisol titre 5.2 fold. In minor oral examinations, moderate correlations between cortisol titre and subsets in sensation seeking and coping strategies, respectively, were identified. CONCLUSION: Intensity and novelty of an academic examination is significantly correlated to cortisol titre. Minor oral examinations, with little consequences on academic career, only have a minor impact on cortisol titre, whereas school exit examinations, which are novel to students and may have major consequences on the academic career, elicit a major increase in cortisol titre.

Adolescent↗

PubMed2005

Blockade of chloride conductance antagonizes PMA-induced ramification in the murine microglial cell line, BV-2.

In microglial cells, activation of ion channels and ion transporters is associated with the transformation from an amoeboid to a ramified phenotype and vice versa. In the present study, we evaluated the contributions of protein kinase C (PKC) activity and ion conductance to the phorbol 12-myristate 13-acetate (PMA)-dependent ramification in the murine microglial cell line, BV-2. In a first set of experiments, we showed that PMA, a commonly used activator of PKC, but not the bioinactive analog 4 alpha-phorbol 12,13-didecanoate (4 alpha-PDD), induces ramification in BV-2 cells. Surprisingly, the PKC inhibitors calphostin C, chelerythrine, or bisindolylmaleimide II did not antagonize PMA-induced ramification. In a further set of experiments, we found that 4,4'-diisocyanatostilbene-2,2' disulfonic acid (DIDS), 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS), which block chloride channels and K-Cl cotransporters, and SKF 96365, a non-selective ion channel blocker, consistently suppressed PMA-induced ramification in BV-2 cells. Additional ion channel blockers, including lanthanides, amiloride, Ba2+, 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), and flufenamic acid did not affect PMA-induced ramification in BV-2 cells. Cs+ accentuated the PMA-dependent ramification in BV-2 cells. Thus, our results indicate (1) that a PMA-binding protein, excluding PKC isoforms, is critical in structural remodeling of microglial cells and (2) that chloride conductance plays a pivotal role in induction of ramification in microglial cells.

Animals↗

PubMed2005

A cGMP-dependent cascade enhances an L-type-like Ca2+ current in identified snail neurons.

We studied the impact of an NO-cGMP dependent signalling pathway on the high-voltage-activated (HVA) Ca(2+) current in identified neurons of the pulmonate snail, Helix pomatia, using Ba(2+) as charge carrier. The 3',5'-cyclic guanosine monophosphate (cGMP) analogues, dibutyryl-cGMP and 8-bromo-cGMP, consistently induced a biphasic response, consisting of an increase superseded by a decline of the Ba(2+) current. The NO donor, sodium nitroprusside (SNP), modulated only in a minority of neurons the Ba(2+) current. Blockade of protein kinase activity with 1-[5-isoquinolinesulfonyl]-2 methyl piperazine (H 7), a nonselective protein kinase inhibitor, or Rp-8-pCPT-cGMP, a selective protein kinase G (PKG) inhibitor, decreased, whereas Rp-cAMP, a selective protein kinase A (PKA) inhibitor, increased the Ba(2+) current upon application of cGMP analogues or SNP. Okadaic acid or calyculin, inhibitors of protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A), augmented the Ba(2+) current. Under these conditions, cGMP analogues or SNP had an additive-enhancing effect on the Ba(2+) current. When neurons were exposed to the nonselective phosphodiesterase (PDE) inhibitor 3-isobutyl-1-methylxanthine (IBMX), cGMP analogues induced a persistent increase of the Ba(2+) current, whereas SNP induced a biphasic response. These data suggest coexistence of cGMP-PKG and cGMP-PDE pathways as well as crosstalk between cGMP and 3',5'-cyclic adenosine monophosphate (cAMP) pathways, which converge on HVA Ca channels in Helix neurons. In this model, augmentation of the Ba(2+) current through HVA Ca channels is accomplished by PKA and PKG, whereas attenuation is mediated by PDEs, which prevent activation of protein kinases via hydrolysis of cyclic nucleotides.

Animals↗

PubMed2004

Nitric oxide decreases a calcium-activated potassium current via activation of phosphodiesterase 2 in Helix U-cells.

In the present study, we investigated the underlaying mechanism of nitric oxide (NO) and cGMP on the decline of a Ca2+-activated potassium (KCa) current in U-cells of the right parietal ganglion of the pulmonate snail, Helix pomatia. Using a two-electrode voltage-clamp technique, we activated a KCa-current either by opening of endogenous voltage-gated Ca2+-channels during depolarizing voltage steps or by ionophoretic injection of Ca2+ via a third electrode containing 100 mM Ca2+. KCa-current amplitude in U-cells was sensitive to Ba2+, TEA, iberiotoxin, kaliotoxin and charybdotoxin (ChTX), but not to 4-aminopyridine (4-AP) (up to 30 mM) and apamin (up to 300 nM). Thus, the biophysical and pharmacological profile of the KCa-current in U-cells shares similarities with the large-conductance KCa channel (BKCa). The NO-donor sodium nitroprusside (SNP) or S-nitro-N-acetylpenicillamine (SNAP) as well as NO-gas decreased the KCa-current amplitude and decreased the rate of KCa-current activation elicited by Ca2+-injection. Decline of the current amplitude and decrease of activation of KCa-current were qualitatively mimicked by the membrane-permeable cGMP analogue dibutyryl-cGMP (db-cGMP). NO-induced decrease of KCa-current was blocked by methylene blue (50 microM), an inhibitor of the guanylyl-cyclase, and by erytho-9-(2-hydroxyl-3-nonyl) adenine (EHNA) (100 microM), an inhibitor of the cGMP-stimulated phosphodiesterase 2 (PDE2). These experiments suggest that the NO-mediated decrease of KCa-current in U-cells results from synthesis of cGMP by activation of a guanylyl-cyclase and subsequent activation of PDE2.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed2003

Polyvalent cations as permeant probes of MIC and TRPM7 pores.

Recent studies in Jurkat T cells and in rat basophilic leukemia cells revealed an Mg(2+)-inhibited cation (MIC) channel that has electrophysiological properties similar to TRPM7 Eyring rate model expressed exogenously in mammalian cells. Here we compare the characteristics of several polyvalent cations and Mg(2+) to block monovalent MIC current from the outside. Putrescine, spermidine, spermine, PhTX-343 (a derivative of the naturally occurring polyamine toxin philanthotoxin), and Mg(2+) each blocked in a dose- and voltage-dependent manner, indicating a blocking site within the electric field of the ion channel. Spermine and the relatively bulky PhTX-343 exhibited voltage dependence steeper than that expected for the number of charges on the molecule. Polyamines and Mg(2+) are permeant blockers, as judged by relief of block at strongly negative membrane potentials. Intracellular dialysis with spermine (300 microM) had no effect, indicating an asymmetrical pore. At the single-channel level, spermine and Mg(2+) induced flickery block of 40-pS single channels. I/V characteristics and polyamine block are similar in expressed TRPM7 and in native MIC currents, consistent with the conclusion that native MIC channels are composed of TRPM7 subunits. An Eyring rate model is developed to account for I/V characteristics and block of MIC channels by polyvalent cations from the outside.

Animals↗

PubMed2003

Anticipated academic examinations induce distinct cortisol responses in adolescent pupils.

OBJECTIVES: Although it is widely accepted that academic examinations are accompanied by a cortisol increase, some recent studies reported either no effect or even a decline of the cortisol level. To reevaluate these discrepancies, we investigated whether personality traits predict the cortisol response upon academic examinations. SETTING AND DESIGN: Nineteen male and female adolescent pupils (17 to 19 years) participated in the study. Two anticipated, mandatory, routine written examinations were used as familiar stressful conditions, whereas an anticipated, mandatory oral examination in front of a board of known and unknown examiners was used as a novel stressful situation. METHODS: Baseline, pre- and postexamination salivary cortisol were quantified and correlated with psychometric measures, including self-estimated stress level, obtained from a five-point scale, and sensation seeking subscales according to Zuckerman. RESULTS: Salivary cortisol response, taken as an average of all subjects, showed a transient increase upon examinations. However, comparing individual cortisol responses revealed three distinct cortisol profiles, including a transient increase (Type 1), a transient decline (Type 2), or no response (Type 3). Type 1 predominates in examinations combined with novelty. A moderate negative association was noted between saliva cortisol concentrations on some sensation seeking subscales. Self-reported stress levels did not significantly correlate with salivary cortisol concentration. CONCLUSION: Our findings show that upon academic examinations the cortisol response varies among subjects. A moderate negative association was unveiled by correlating individual cortisol responses with sensation seeking subscales.

Adolescent↗

PubMed2002

Complementary distribution of NADPH-diaphorase and l-arginine in the snail nervous system.

Since the interneuronal messenger nitric oxide (NO) can not be stored in neurones, the regulation of the NO-producing enzyme nitric oxide synthase (NOS) is crucial. Neuronal NOS metabolises L-arginine to nitric oxide (NO) and L-citrulline in a Ca(2+)-dependent manner. Thus, availability of L-arginine to NOS may modulate NO production. In this study, we examined the cellular distribution of reduced nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase, L-arginine and L-citrulline. Using NADPH-diaphorase histochemistry to visualise putative NO-producing cells and immunocytochemistry to localise L-arginine, we showed that the distribution of L-arginine-immunoreactive neurones correlates well with those of NADPH-diaphorase-positive neurones in cerebral ganglia of the pulmonate Helix pomatia. However, substrate and enzyme were visualised in separate but adjacent neurones. We further examined whether NADPH-diaphorase-labelled cells contain the L-citrulline. Following elevation of intracellular Ca(2+) by the Ca(2+) ionophore, ionomycin, or by a high-K(+) solution, the number of L-citrulline-immunoreactive neurones in mesocerebrum and pedal lobe increased up to tenfold. Preincubation of ganglia with the NOS inhibitor N(G)-nitro-L-arginine prevented ionomycin or high-K(+) solution-induced L-citrulline synthesis. Most L-citrulline-immunoreactive neurones contain NADPH-diaphorase activity. In conclusion, these experiments indicate a complementary distribution of NOS and L-arginine and suggest an unknown signalling pathway between neurones to maintain L-arginine and NO homeostasis.

Animals↗

PubMed2002

A Shaker homologue encodes an A-type current in Xenopus laevis.

In Xenopus laevis, several distinct K(+)-channels (xKv1.1, xKv1.2, xKv2,1, xKv2.2, xKv3.1) have been cloned, sequenced, and electrophysiologically characterized. K(+)-channels significantly shape neuronal excitability by setting the membrane potential, and latency and duration of action potentials. We identified a further Shaker homologue, xKv1.4, in X. laevis. The open reading frame encodes a K(+)-channel that shares 72% of its 698 amino acids with the human Shaker homologue, hKv1.4. Northern blot analysis revealed xKv1.4 in the brain, muscle, and spleen but not in the ovary, intestine, heart, liver, kidney, lung, and skin. Whole-cell patch clamp recording from rat basophilic leukaemia (RBL) cells transfected with xKv1.4 revealed a voltage-gated, outward rectifying, transient A-type, K(+) selective current. xKv1.4 was strongly dependent on extracellular K(+). Exposure of cells to K(+) free bath solution almost completely abolished the current, whereas in the presence of high K(+), inactivation in response to a maintained depolarizing step and the frequency-dependent cumulative inactivation decreased. Ion channels encoded by xKv1.4 are sensitive to 4-aminopyridine and quinidine but insensitive to tetraethylammonium and the peptide toxins, charybdotoxin, margatoxin, and dendrotoxin. In conclusion, our results indicate that the biophysical and pharmacological signature of xKv1.4 closely resemble those of the A-current described in Xenopus embryonic neurons and is similar to the human Shaker homologue, hKv1.4.

Animals↗

PubMed2002

Distinct properties of CRAC and MIC channels in RBL cells.

In rat basophilic leukemia (RBL) cells and Jurkat T cells, Ca(2+) release-activated Ca(2+) (CRAC) channels open in response to passive Ca(2+) store depletion. Inwardly rectifying CRAC channels admit monovalent cations when external divalent ions are removed. Removal of internal Mg(2+) exposes an outwardly rectifying current (Mg(2+)-inhibited cation [MIC]) that also admits monovalent cations when external divalent ions are removed. Here we demonstrate that CRAC and MIC currents are separable by ion selectivity and rectification properties: by kinetics of activation and susceptibility to run-down and by pharmacological sensitivity to external Mg(2+), spermine, and SKF-96365. Importantly, selective run-down of MIC current allowed CRAC and MIC current to be characterized under identical ionic conditions with low internal Mg(2+). Removal of internal Mg(2+) induced MIC current despite widely varying Ca(2+) and EGTA levels, suggesting that Ca(2+)-store depletion is not involved in activation of MIC channels. Increasing internal Mg(2+) from submicromolar to millimolar levels decreased MIC currents without affecting rectification but did not alter CRAC current rectification or amplitudes. External Mg(2+) and Cs(+) carried current through MIC but not CRAC channels. SKF-96365 blocked CRAC current reversibly but inhibited MIC current irreversibly. At micromolar concentrations, both spermine and extracellular Mg(2+) blocked monovalent MIC current reversibly but not monovalent CRAC current. The biophysical characteristics of MIC current match well with cloned and expressed TRPM7 channels. Previous results are reevaluated in terms of separate CRAC and MIC channels.

Animals↗