Tanshinone II A sulfonate, but not tanshinone II A, acts as potent negative allosteric modulator of the human purinergic receptor P2X7.

Tanshinone II A sulfonate (TIIAS) was identified as a potent, selective blocker of purinergic receptor P2X7 in a compound library screen. In this study, a detailed characterization of the pharmacologic effects of TIIAS on P2X7 is provided. Because TIIAS is a derivative of tanshinone II A (TIIA) and both compounds have been used interchangeably, TIIA was included in some assays. Fluorometric and electrophysiologic assays were used to characterize effects of TIIAS and TIIA on recombinantly expressed human, rat, and mouse P2X7. Results were confirmed in human monocyte-derived macrophages expressing native P2X7. In all experiments, involvement of P2X7 was verified using established P2X7 antagonists. TIIAS, but not TIIA, reduces Ca(2+) influx via human P2X7 (hP2X7) with an IC50 of 4.3 µM. TIIAS was less potent at mouse P2X7 and poorly inhibited rat P2X7. Monitoring of YO-PRO-1 uptake confirmed these findings, indicating that formation of the hP2X7 pore is also suppressed by TIIAS. Electrophysiologic experiments revealed a noncompetitive mode of action. TIIAS time-dependently inhibits hP2X7 gating, possibly by binding to the intracellular domain of the receptor. Inhibition of native P2X7 in macrophages by TIIAS was confirmed by monitoring Ca(2+) influx, YO-PRO-1 uptake, and release of the proinflammatory cytokine interleukin-1ß. Fluorometric experiments involving recombinantly expressed rat P2X2 and human P2X4 were conducted and verified the compound's selectivity. Our data suggest that hP2X7 is a molecular target of TIIAS, but not of TIIA, a compound with different pharmacologic properties.

Positive allosteric modulation by ivermectin of human but not murine P2X7 receptors.

BACKGROUND AND PURPOSE: In mammalian cells, the anti-parasitic drug ivermectin is known as a positive allosteric modulator of the ATP-activated ion channel P2X4 and is used to discriminate between P2X4- and P2X7-mediated cellular responses. In this paper we provide evidence that the reported isoform selectivity of ivermectin is a species-specific phenomenon. EXPERIMENTAL APPROACH: Complementary electrophysiological and fluorometric methods were applied to evaluate the effect of ivermectin on recombinantly expressed and on native P2X7 receptors. A biophysical characterization of ionic currents and of the pore dilation properties is provided. KEY RESULTS: Unexpectedly, ivermectin potentiated currents in human monocyte-derived macrophages that endogenously express hP2X7 receptors. Likewise, currents and [Ca(2+) ](i) influx through recombinant human (hP2X7) receptors were potently enhanced by ivermectin at submaximal or saturating ATP concentrations. Since intracellular ivermectin did not mimic or prevent its activity when applied to the bath solution, the binding site of ivermectin on hP2X7 receptors appears to be accessible from the extracellular side. In contrast to currents through P2X4 receptors, ivermectin did not cause a delay in hP2X7 current decay upon ATP removal. Interestingly, NMDG(+) permeability and Yo-Pro-1 uptake were not affected by ivermectin. On rat or mouse P2X7 receptors, ivermectin was only poorly effective, suggesting a species-specific mode of action. CONCLUSIONS AND IMPLICATIONS: The data indicate a previously unrecognized species-specific modulation of human P2X7 receptors by ivermectin that should be considered when using this cell-biological tool in human cells and tissues.

Electrophysiological classification of P2X7 receptors in rat cultured neocortical astroglia.

BACKGROUND AND PURPOSE: P2X7 receptors are ATP-gated cation channels mediating important functions in microglial cells, such as the release of cytokines and phagocytosis. Electrophysiological evidence that these receptors also occur in CNS astroglia is rare and rather incomplete. EXPERIMENTAL APPROACH: We used whole-cell patch-clamp recordings to search for P2X7 receptors in astroglial-neuronal co-cultures prepared from the cerebral cortex of rats. KEY RESULTS: All the astroglial cells investigated responded to ATP with membrane currents, reversing around 0 mV. These currents could be also detected in isolated outside-out patch vesicles. The results of the experiments with the P2X [alpha,beta-methylene ATP and 2'-3'-O-(4-benzoyl) ATP] and P2Y receptor agonists [adenosine 5'-O-(2-thiodiphosphate), uridine 5'-diphosphate, uridine 5'-triphosphate (UTP) and UDP-glucose] suggested the involvement of P2X receptors in this response. The potentiation of ATP responses in a low divalent cation or alkaline bath, but not by ivermectin, made it likely that a P2X7 receptor is operational. Blockade of the ATP effect by the P2X7 antagonists Brilliant Blue G, calmidazolium and oxidized ATP corroborated this assumption. CONCLUSIONS AND IMPLICATIONS: Rat cultured cortical astroglia possesses functional P2X7 receptors. It is suggested that astrocytic P2X7 receptors respond to high local ATP concentrations during neuronal injury.

Acamprosate inhibits Ca2+ influx mediated by NMDA receptors and voltage-sensitive Ca2+ channels in cultured rat mesencephalic neurones.

Acamprosate has recently been introduced in relapse prophylaxis in weaned alcoholics. Using fura-2 microfluorimetry, the present study investigates whether acamprosate affects N-methyl-D-aspartate (NMDA) or K+-induced changes in free intracellular Ca2+ concentration ([Ca2+]i) in rat cultured mesencephalic neurones. Both application of NMDA (plus glycine) and elevation of extracellular K+ induced rapid increases in [Ca2+]i which respectively were insensitive and sensitive to omega-conotoxin (omega-CTX) MVIIC, a blocker of voltage-dependent Ca2+ channels (VDCCs). Acamprosate (100 microM and 300 microM) significantly attenuated the response induced by NMDA as well as that induced by K+ in a concentration-dependent manner. Concurrent application of omega-CTX MVIIC and acamprosate impaired the K+-induced increase in [Ca2+]i to the same extent as omega-CTX MVIIC alone. The present data suggest that acamprosate inhibits Ca2+ influx through both NMDA receptors and VDCCs.

Experiences with DHE: an order entry and result reporting case study.

Organization of hospital information systems that are currently in use is characterized by a multitude of simple, unfortunately less integrated components. For the integration of the already available as well as new components into a hospital information system, the "Committee European de Normalisation" (CEN) proposed a basic "Conceptual Architectural Framework". Based on the CEN proposal, in this paper we present first results of a case study in order to investigate the usability of the Distributed Healthcare Environment (DHE), as a standard architecture for hospital information systems. Another aim of this case study was to design an Order Entry and Result Reporting System and implement it by using DHE.

[Model for a computer-assisted quality assurance system in burn intensive care]. / Modell eines computergestützten Qualitätssicherungssystems in der Verbrennungsintensivtherapie.

The prerequisites for an integrated quality management system for hospitals were developed as a result of considerations regarding the necessity of an appropriate quality-assurance system for the intensive care sector. The basis for a dynamic quality-assurance system on the documentational level is a comprehensive data base containing patient files unlimited in terms of time and data technology. The necessary hardware and software structure will be provided by consistent application of a client-server architecture. Permanent surveillance of existing quality objectives within the scope of an economic close-loop system requires a system with flexible query mechanisms. Informational data access from all hierarchical level within the hospital organization is facilitated by the data warehouse concept, a data base system with subject-oriented, integrated, time-variable, and persistent data.