Dénes Budai

CD59 blocks not only the insertion of C9 into MAC but inhibits ion channel formation by homologous C5b-8 as well as C5b-9

Activation of the complement system on the cell surface results in the insertion of pore forming membrane attack complexes (MAC, C5b‐9). In order to protect themselves from the complement attack, the cells express several regulatory molecules, including the terminal complex regulator CD59 that inhibits assembly of the large MACs by inhibiting the insertion of additional C9 molecules into the C5b‐9 complex. Using the whole cell patch clamp method, we were able to measure accumulation of homologous MACs in the membrane of CD59− human B‐cells, which formed non‐selective ion channels with a total conductance of 360 ± 24 pS as measured at the beginning of the steady‐state phase of the inward currents. C5b‐8 and small‐size MAC (MAC containing only a single C9) can also form ion channels. Nevertheless, in CD59+ human B‐cells in spite of small‐size MAC formation, an ion current could not be detected. In addition, restoring CD59 to the membrane of the CD59− cells inhibited the serum‐evoked inward current. The ion channels formed by the small‐size MAC were therefore sealed, indicating that CD59 directly interfered with the pore formation of C5b‐8 as well as that of small‐size C5b‐9. These results offer an explanation as to why CD59‐expressing cells are not leaky in spite of a buildup of homologous C5b‐8 and small‐size MAC. Our experiments also confirmed that ion channel inhibition by CD59 is subject to homologous restriction and that CD59 cannot block the conductivity of MAC when generated by xenogenic (rabbit) serum.

Divergent effects of Aβ1–42 on ionotropic glutamate receptor-mediated responses in CA1 neurons in vivo

Aggregated Abeta1-42 is hypothesized to be the central cause of Alzheimers disease. However, early changes in synaptic activity may be detected in the disease long before a significant cell loss is manifested. Despite the fact that Abeta1-42 interference with long-term potentiation (LTP) and the field excitatory postsynaptic potential (fEPSP) is well documented, the exact mechanism of these events remains to be clarified. Here we studied the effects of iontophoretically applied Abeta1-42 on the neuronal firing evoked in vivo on the CA1 hippocampal neurons of Wistar rats by different agonists of the ionotropic glutamate receptors: N-methyl-d-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and kainic acid (KA). NMDA elicited firing enhanced in all of the measured cells; in contrast, the AMPA-mediated responses decreased significantly after Abeta1-42 ejection. The changes in KA-evoked responses to Abeta1-42 revealed two types of cells. In the first type, the KA-mediated firing remained at the control level, while in the second type, Abeta1-42 attenuated the KA-evoked responses. A protective pentapeptide, Leu-Pro-Tyr-Phe-Asp-amide, was used to verify the specificity of these beta-amyloid-elicited effects. The pentapeptide protected against the modulatory effects of Abeta1-42 on the NMDA and AMPA responses. In conclusion, we have shown that Abeta1-42 exerts divergent effects on the activity of the ionotropic glutamate receptors in vivo. These results suggest that the LTP disruption and fEPSP attenuation seen after Abeta1-42 application are in part due to the altered function of these receptors.

Stimulation intensity as critical determinant of presynaptic receptor effectiveness

A number of reports in the literature suggest that there is an inverse correlation between the intensity of nerve activation and the effectiveness of presynaptic receptors that inhibit transmitter release. This common feature of many presynaptic inhibitory receptors may provide important insights into the mechanism by which transmitter release is controlled. Sue Duckles and Dénes Budai discuss the implications such a relationship between the pattern of nerve activation and effectiveness of presynaptic receptors has for understanding the physiological role of neuromodulators in general.

Endomorphin-2, an endogenous tetrapeptide, protects against Aβ1–42 in vitro and in vivo

The underlying cause of Alzheimers disease (AD) is thought to be the β‐amyloid aggregates formed mainly by Aβ1–42 peptide. Protective pentapeptides [e.g., Leu‐Pro‐Phe‐Phe‐Asp (LPFFD)] have been shown to prevent neuronal toxicity of Aβ1–42 by arresting and reversing fibril formation. Here we report that an endogenous tetrapeptide, endomorphin‐2 (End‐2, amino acid sequence: YPFF), defends against Aβ 1–42 induced neuromodulatory effects at the cellular level. Although End‐2 does not interfere with the kinetics of Aβ fibrillogenesis according to transmission electron microscopic studies and quasielastic light scattering measurements, it binds to Aβ1–42 during aggregation, as revealed by tritium‐labeled End‐2 binding assay and circular dichroism measurements. The tetrapeptide attenuates the inhibitory effect on cellular redox activity of Aβ1–42 in a dose‐dependent manner, as measured by 3‐(4,5‐dimethylthiazolyl‐2)‐2,‐5‐diphenyltetrazolium bromide (MIT) assay. In vitro and in vivo electrophysiological experiments show that End‐2 also protects against the field excitatory postsynaptic potential attenuating and the NMDA‐evoked responseenhancing effect of Aβ1–42. Studies using [d‐Ala (2), N‐Me‐Phe (4), Gly (5)‐ol]‐enkephalin (DAMGO), a µ‐opioid receptor agonist, show that the protective effects of the tetrapeptide are not µ‐receptor modulated. The endogenous tetrapeptide End‐2 mayserve as a lead compound for the drug development in the treatment of AD.—Szegedi, V., Juhász, G., Rózsa, E., Juhász‐Vedres, G., Datki, Z., Fülöp, L., Bozsó, Z., Lakatos, A., Laczkó, I., Farkas, T., Kis, Z., Tóth, G., Soós, K., Zarándi, M., Budai, D., Toldi, J., Penke, B. Endomorphin‐2, an endogenous tetrapeptide, protects against Aβ1–42 in vitro and in vivo. FASEB J. 20, E324–E333 (2006)

Anti-calmodulins and Tricyclic Adjuvants in Pain Therapy Block the TRPV1 Channel

Ca2+-loaded calmodulin normally inhibits multiple Ca2+-channels upon dangerous elevation of intracellular Ca2+ and protects cells from Ca2+-cytotoxicity, so blocking of calmodulin should theoretically lead to uncontrolled elevation of intracellular Ca2+. Paradoxically, classical anti-psychotic, anti-calmodulin drugs were noted here to inhibit Ca2+-uptake via the vanilloid inducible Ca2+-channel/inflamatory pain receptor 1 (TRPV1), which suggests that calmodulin inhibitors may block pore formation and Ca2+ entry. Functional assays on TRPV1 expressing cells support direct, dose-dependent inhibition of vanilloid-induced 45Ca2+-uptake at µM concentrations: calmidazolium (broad range)≥trifluoperazine (narrow range)>chlorpromazine/amitriptyline>fluphenazine>>W-7 and W-13 (only partially). Most likely a short acidic domain at the pore loop of the channel orifice functions as binding site either for Ca2+ or anti-calmodulin drugs. Camstatin, a selective peptide blocker of calmodulin, inhibits vanilloid-induced Ca2+-uptake in intact TRPV1+ cells, and suggests an extracellular site of inhibition. TRPV1+, inflammatory pain-conferring nociceptive neurons from sensory ganglia, were blocked by various anti-psychotic and anti-calmodulin drugs. Among them, calmidazolium, the most effective calmodulin agonist, blocked Ca2+-entry by a non-competitive kinetics, affecting the TRPV1 at a different site than the vanilloid binding pocket. Data suggest that various calmodulin antagonists dock to an extracellular site, not found in other Ca2+-channels. Calmodulin antagonist-evoked inhibition of TRPV1 and NMDA receptors/Ca2+-channels was validated by microiontophoresis of calmidazolium to laminectomised rat monitored with extracellular single unit recordings in vivo. These unexpected findings may explain empirically noted efficacy of clinical pain adjuvant therapy that justify efforts to develop hits into painkillers, selective to sensory Ca2+-channels but not affecting motoneurons.

GYKI 52466 inhibits AMPA/kainate and peripheral mechanical sensory activity

The selectivity of the 2,3-benzodiazepine compound, GYKI 52466, was tested on wide dynamic range (WDR) dorsal horn neurons of the rat spinal cord. Using extracellular recordings, neurons were characterized by stimulation with noxious and innocuous intensities of the receptive field. In most cells, responses to iontophoretically applied (R,S)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) or kainic acid (KA), but not N-methyl-D-aspartate (NMDA), were profoundly reduced by iontophoretic ejection of GYKI 52466. The inhibition usually lasted for 5-30 min following application of GYKI 52466. In a few neurons, responses to NMDA were also decreased by GYKI 52466. Responses to both noxious and innocuous mechanical stimulation were reduced in the presence of GYKI 52466. The results provide evidence for the selective inhibition by GYKI 52466 of AMPA/KA receptor-mediated functions and support the involvement of these receptors in spinal mechanical nociception.

An intraperitoneally administered pentapeptide protects against Aβ1-42 induced neuronal excitation in vivo

The underlying cause of Alzheimers disease (AD) is thought to be the accumulation and aggregation of a misfolded protein, amyloid-beta (Abeta). A promising strategy against AD is the application of protective, peptide-based neuroprotective agents that selectively bind to Abeta. We recently described a pentapeptide, LPYFDa, which recognizes Abeta (1-42) and protects neurons against the toxic effects of aggregated Abeta (1-42) both in vitro and in vivo. Our previous work indicated that the in vivo ejection of fibrillar Abeta (1-42) into the hippocampal CA1 region resulted in a massive increase in the NMDA-evoked neuronal firing rate. Our current aim was to study whether intraperitoneally administered LPYFDa is capable of protecting against the synaptotoxic action of fibrillar Abeta (1-42) administered by iontophoresis. Our investigations of the in vivo biodistribution of tritium-labelled LPYFDa and single-unit electrophysiology revealed that LPYFDa readily crosses the blood-brain barrier, and protects the synapses against the excitatory action of fibrillar Abeta (1-42) in a relatively wide temporal window in rat. This pentapeptide may serve as a lead compound for the design of novel drug candidates for the prevention of AD.

Changes in acetylcholine content, release and muscarinic receptors in rat hippocampus under cold stress.

The aim was to study the mechanism of the previously established decrease in acetylcholine (ACh) concentration in the rat hippocampus under cold stress. Male rats were exposed for 14 days to cold (5 degrees C) or kept (controls) at room temperature (24 degrees C). Acetylcholine content, release and muscarinic receptor binding were investigated in the hippocampus. Cold exposure resulted in a decrease of ACh concentration in the dorsal hippocampus. Moreover, the potassium-evoked release of ACh from hippocampal slices was increased and an increase of maximal binding capacity of [3H] (-) quinuclidinyl benzilate in the dorsal hippocampus of cold exposed animals was also observed. Thus the decrease of hippocampal ACh concentration under cold exposure is probably due to its increased release. On balance then, our results demonstrate that cold stress in the rat induces significant activation of the hippocampal cholinergic system.

Fibrillar Aβ1-42 Enhances NMDA Receptor Sensitivity via the Integrin Signaling Pathway

The aggregated form of amyloid-beta (Abeta) (1-42) has been shown to increase N-methyl-D-aspartic acid (NMDA) evoked neuronal activity in vivo. Here we further characterized this phenomenon by investigating the role of integrin activation and downstream Src kinase activity using in vivo electrophysiology and in vitro intracellular Ca (2+) measurements. Pretreatment of differentiated SH-SY5Y cells with fibrillar Abeta (1-42) markedly enhanced the intracellular calcium increases caused by NMDA receptor (NMDA-R) stimulation. Function blocking antibody against beta1 integrin depressed the facilitatory effects of Abeta (1-42). Similarly, Abeta (1-42) facilitated NMDA-R driven firing of hippocampal neurons in vivo, and this effect was reduced by neutralizing antibody against beta1 integrins. The positive action of Abeta (1-42) on NMDA-R dependent responses was also depressed by an inhibitor known to block Src kinase. These results support the hypothesis that aggregated Abeta (1-42) is recognized by the beta1 subunit containing integrins and may induce a Src kinase dependent NMDA receptor phosphorylation.

The modulatory effect of estrogen on the neuronal activity in the barrel cortex of the rat. An electrophysiological study

In acute experiments, the effects of iontophoretically applied 17β-estradiol hemisuccinate on the activity of the primary somatosensory cortical neurons were studied in ovariectomized rats by extracellular single-unit recording. 17β-Estradiol increased both the spontaneous and the vibrissa deflection- evoked responses, with an average latency of 24 min. It is suggested that this relatively long latency of the 17β-estradiol effect is based not so much on membrane mechanisms as on genomic mechanisms.