Andrea Blöchl

Redox hydrogel-based bienzyme microelectrodes for amperometric monitoring of L-glutamate

Fabrication and characterization of amperometric bienzyme L-glutamate sensitive microelectrodes are the prerequisite for monitoring changes Of L-glutamate concentration at glutamate-secreting cell cultures. The design of the glutamate microelectrodes is based on incorporating L-glutamate oxidase and horseradish peroxidase into a redox-hydrogel containing PVI19-dmeOs as the redox mediator and immobilizing this system onto the surface of platinum microdisk electrodes using, a dip-coating procedure. For amperometric measurements Of L-glutamate, these redox hydrogel-based bienzyme microelectrodes can be operated at low working potentials (-50 mV vs. Ag/AgCl) decreasing the influence of electroactive interferants possibly present in biological samples. The L-glutamate microsensors are characterized by a good operation stability and sensitivity (0.038+/-0.005 mAM(-1)), a low detection limit (0.5 muM in a conventional amperometric set-up and 0.03 muM in a Faraday cage, defined as three times the signal-to-noise ratio), a linear range up to 50 muM and a response time of about 35 s. The glutamate biosensors have been applied for the direct measurement Of L-glutamate release (upon chemical stimulation) from a population of immortalized hippocampal neurons (HN10 cells) demonstrating the possibility to amperometrically monitor in-Situ L-glutamate secretion from these cells. (Less)

Nerve growth factor stimulates MAPK via the low affinity receptor p75LNTR

Apart from its high affinity receptor TrkA, nerve growth factor (NGF) can also stimulate the low affinity receptor p75LNTR and induce a Trk‐independent signaling cascade. We examined the possible involvement of mitogen‐activated protein kinase (MAPK) in this signaling pathway in neuronal cultures of the cerebellum of P2‐aged rats and PCNA cells; both cell types express p75LNTR but not TrkA. We found a fast and transient phosphorylation of p42‐ and p44‐MAPK after stimulation with NGF or C2‐ceramide which proved to be sensitive to inhibition of MAPK kinase and protein kinase A (PKA). As stimulation with NGF also activated p21Ras it can be concluded that at least part of the observed MAPK activation was effected via p21Ras and via PKA.

Electrochemical High-Content Screening of Nitric Oxide Release from Endothelial Cells

Release of nitric oxide (NO) is of high importance for regulating endothelial cell functions during vasodilatation, vascular remodeling, and angiogenesis. Thus, a direct and reliable real‐time method for NO detection that takes into account time‐dependent variations of the NO concentration in the complex reaction within the diffusion zone above the cells is vital for obtaining information about the role of NO in intracellular endothelial signal transduction and its impact on the surrounding cells. In this study, the time course of vascular endothelial growth factor E (VEGF‐E) stimulated NO release from transformed human umbilical vein endothelial cells (T‐HUVEC) was investigated by means of metalloporphyrin‐based NO sensors employed in an electrochemical robotic system. The NO sensor was obtained by electrochemically induced deposition of NiII tetrakis(p‐nitrophenylporphyrin) on a 50‐μm diameter platinum disk electrode which was integrated, together with a 25‐μm diameter platinum disk, in a double‐barrel electrode arrangement. The second electrode was used as a guidance sensor for the automatic and highly reproducible positioning of the NO sensor at a known distance from a layer of adherently growing cells by using z‐approach curves in the negative feedback mode of scanning electrochemical microscopy (SECM). The electrochemical robotic system allows the fully automated detection of NO with high sensitivity and selectivity to be performed in real time within 96‐well microtiter plates. A functional cell assay was established to allow the standardized detection of NO released upon stimulation from T‐HUVEC with a sensor positioned at a known distance above the endothelial cells. The overall system was evaluated by automatic detection of NO release from T‐HUVEC upon stimulation with VEGF‐E after incubation with a variety of drugs that are known to act on different sites in the complex signal‐transduction pathway that finally invokes NO release.

Pyrrole functionalised metalloporphyrins as electrocatalysts for the oxidation of nitric oxide

Pyrrole-functionalised tetracarboxyphenyl porphyrin and trimethoxyphenylcarboxy-phenyl porphyrin containing Ni, Mn and Pd as the central metal ion were used to modify Pt-disk microelectrodes (slashed circle 50 mum) (by repetitive cyclic voltammetry, dip-dry and pulse-amperometry methods) for the detection of nitric oxide (NO). Electrodes modified with Mn(II) trimethoxyphenylcarboxyphenyl porphyrin using the pulse amperomery approach, were found to be sensitive, stable and fast in response towards the oxidation of NO. Thus, they were used for the detection of NO release from a population of transformed human umbilical vein endothelial cells (T-HUVEC) into a droplet of electrolyte solution following stimulation with vascular endothelial growth factor (VEGF). The electrode surface was covered with an additional layer of Nafion(R) to prevent interference from anionic molecules such as nitrite.

Inactivation and activation of Ras by the neurotrophin receptor p75

The neurotrophin receptor p75 induces neurotrophic and/or apoptotic signalling pathways and can also cooperate with the neurotrophic Trk receptor tyrosine kinases. Its intracellular part encloses a so‐called ‘death domain’ with a segment similar to the wasp venom mastoparan which binds small GTPases such as Rho. To study possible interactions of p75 and Ras (and Rho) we used wild‐type and mutant genes of p75 stably expressed by MDCK cells which normally have neither Trk nor p75. We found that p75 can directly bind the GTPases Ras and Rho and that the unstimulated p75 inactivates total cellular Ras through a differential influence on the dissociation of GDP and GTP from Ras and an exchange of bound Ras·GDP for free Ras·GTP. These properties of p75 could also be demonstrated in vitro and should therefore be cell type‐independent. Stimulation of p75 with nerve growth factor causes Ras activation via adapter proteins known from Trk signalling and induces rapid outgrowth of cellular processes. Both inactivation and activation of Ras by p75 are controlled by the phosphorylation state of the receptors two intracellular tyrosines. p75 also influences Rho activation and inactivation, and the combined interactions of the receptor with the two GTPases Ras and Rho can regulate neurite formation in an efficient, synergistic way.

Photic inhibition of TrkB/Ras activity in the pigeon's tectum during development: impact on brain asymmetry formation

Asymmetric photic stimulation during embryonic or post‐hatch development induces a functional lateralization of the pigeons visual system, which is accompanied by left–right differences in tectal cell sizes. The intracellular membrane‐anchored GTPase Ras can be activated by a number of upstream mechanisms including binding of brain‐derived neurotrophic factor to its specific TrkB receptor. Ras activity plays an important morphogenetic role in neurons and therefore might also be involved in the asymmetric differentiation of tectal cells. To investigate the role of Ras, we determined the relative levels of activated Ras and of signalling active phospho‐TrkB in tecta of light‐ and dark‐incubated pigeons and combined this with an immunohistochemical detection of Ras‐GTP and TrkB receptors. While Ras activation levels did not differ between light‐ and dark‐incubated pigeons during embryonic development, directly after hatching Ras activity was significantly decreased in the stronger stimulated left tectum of light‐incubated animals. This was accompanied by lower levels of TrkB phosphorylation. Immunohistochemical staining revealed Ras‐GTP‐positive cell bodies within the efferent cell layer. These cells were TrkB‐positive and developed enlarged soma sizes within the right tectum during the first week after hatching. This association suggests asymmetric Ras activation to be involved in the asymmetric differentiation of the efferent cells as a result of asymmetric TrkB signalling. Because asymmetric light exposure occurs only during embryonic development, the observed transient asymmetric inhibition of TrkB/Ras activity after hatching may reflect differential embryonic maturation of tectal inhibitory circuits leading to a functional superiority of the right eye in the adult organism.