Angiotensin II,vasopressin and GTP[γ-S] inhibit inward-rectifying K+ channels in porcine cerebral capillary endothelial cells

Summary Cerebral capillaries from porcine brain were isolated. and endothelial cells were grown in primary culture. The whole-cell tight seal patch-clamp method was applied to freshly isolated single endothelial cells, and cells which were held in culture up to one week. With high K⁺ solution in the patch pipette and in the bath we observed inward-rectifying K⁺ currents, showing a time-dependent decay in part of the experiments. Ba²⁺ (1–10mm) in the bath blocked this current, whereas outside tetraethylammonium (10mm) decreased the peak current but increased the steady-state current. Addition of 1 m of angiotensin II or of arginine-vasopressin to the extracellular side caused a time-dependent inhibition of the inward-rectifying K⁺ current in part of the experiments. Addition of 100 m GTP[-S] to the patch pipette blocked the K⁺ inward rectifier. In cell-attached membrane patches two types of single inward-rectifying K⁺ channels were observed, with single channel conductances of 7 and 35 pS. Cell-attached patches were also obtained at the antiluminal membrane of intact isolated cerebral capillaries. Only one type of K⁺ channel withg=30 pS was recorded. In conclusion, inwardly rectifying K⁺ channels, which can be inhibited by extracellular angiotensin II and arginine-vasopressin, are present in cerebral capillary endothelial cells. The inhibition of this K⁺ conductance by GTP[-S] indicates that G-proteins are involved in channel regulation. It is suggested that angiotensin II and vasopressin regulate K⁺ transport across the blood-brain barrier, mediating their effects via G-proteins.     

The Susceptibility of Cerebral Endothelial Cells to Astroglial Induction of Blood-Brain Barrier Enzymes Depends on Their Proliferative State

Primary cultures of brain capillary endothelial cells (BCECs) were used to investigate the induction of blood-brain barrier (BBB) characteristics in vitro. Enzymatic activities of gamma-glutamyltranspeptidase (gamma-GT) and alkaline phosphatase (ALP) were taken as indicators for the expression of the BBB phenotype. We were able to show that a coculture system with a direct cell-cell contact between astroglial cells and BCECs is the necessary precondition for an increase of these enzyme activities that are lost in pure BCEC cultures. Coculture with both astrocytes and C6-glioma cells reestablishes the BBB phenotype whereas conditioned media as well as an astrocyte-derived extracellular matrix were ineffective. The susceptibility of the BCECs to an astroglial stimulus depends on the proliferative state of the BCECs. Cells in an early highly proliferative culture phase were stimulated to express an enzymatic activity level similar to the in vivo situation. Confluent BCEC monolayers were not induced at all. With the ALP we observed a spatial induction within a BCEC colony. Astrocyte-induced ALP activity was first observed at an outer belt of BCEC colonies in direct contact with the astrocyte layer. However, this signal is transferred to the center of the colony with time in culture. We conclude that direct contact of BCECs with astroglial cells is necessary for the induction of the BBB phenotype in cultured BCECs and that this signal may be transferred from induced to noninduced BCECs.     

Metabolization of 3,5-dichlorocatechol by Alcaligenes eutrophus JMP 134

2,4-Dichloro-cis,cis-muconate is established as ringcleavage product in the degradation of 3,5-dichlorocatechol by Alcaligenes eutrophus JMP 134. The formerly described isomerization of 2-chloro-trans- to 2-chlorocis-4-carboxymethylenebut-2-en-4-olide as an essential catabolic step could not be certified.     

A Two One-Sided Parametric Tolerance Interval Test for Control of Delivered Dose Uniformity—Part 2—Effect of Changing Parameters

This article examines the effects of changing parameters in the test which was proposed by the FDA at the October 2005 Advisory Committee meeting for confirming delivered dose uniformity in orally inhaled and nasal drug products. This article is an extension of the characterization study presented in an accompanying article (Part 1). The goal of this study is to understand how parameters of the test affect the test performance. The effects of changing test parameters such as target interval, maximum allowable proportion in the tail area, and sample size are examined. The results show that changing the maximum allowable tail area and/or the target interval have the largest impact on the test outcomes, i.e., probability of acceptance for a given batch mean and standard deviation. The presented information may provide potential users of the test with a set of tools for optimizing the test characteristics for a particular product.     

The Polarity of Choroid Plexus Epithelial Cells In Vitro Is Improved in Serum-Free Medium

Abstract: The influence of culture conditions on the development of normal characteristics of the choroid plexus epithelium has been investigated in vitro with respect to polarity, barrier properties, transport, and secretory activity. Withdrawal of serum supplement in the culture medium of cells grown on filters caused morphologically visible changes by an increased trimming of microvilli at the apical membrane side, which is accompanied by an increased expression of the Na⁺,K⁺-ATPase. Moreover cells under serum-free conditions exhibit structural changes in tight junctional zonula occludens protein-1 (ZO-1) organization, a reduced permeability, and a drastically increased electrical resistance from 150 Ω· cm² in the presence of serum to 1,500 Ω· cm² after serum withdrawal. Under these conditions, cell monolayers are able to build up a transcellular proton gradient and to secrete fluid into the upper (apical) filter compartment, which is accompanied by a polarized secretion of proteins like transthyretin. Active transport of the dyes fluorescein and phenol red by the organic anion transporter is found to be driven by the Na⁺,K⁺-ATPase. We come to the conclusion that removal of serum favors the differentiation process of the plexus epithelium in vitro, which brings the cell culture model closer to the physiological situation in vivo. We present preliminary evidence that epidermal growth factor may be one component in serum preventing the proper in vitro differentiation.     

Meisen(Parus) erkennen den Grad der Gef?hrdung durch einen Feind

Zusammenfassung In Klappfallen gefangene Kohl-, Blau- und Weidenmeisen(Parus major, P. caeruleus, P. montanus) haßten um so häufiger, je näher ihnen ein Mensch kam (Situation 1–2). Beim Herausgreifen haßte keine (Situation 3), sondern erst, wenn sie anschließend weider wegfliegen konnten (Situation 4). Das Hassen freier Umgebungsvögel derselben Arten nahm mit dem Grad der Feindbedrohung des Fänglings zu und konnte auch allein durch den Anblick eines menschenbedrohten Fänglings ausgelöst werden (Tab.).

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Tits(Parus) recognize the degree of threat imposed by an enemy

Summary Trapped Great, Blue, and Willow Tits (Parus major, P. caeruleus, P. montanus) mobbed more strongly as they were approached by a human being (situation 1–2). While being removed from the trap a tit did not mob (situation 3), but it did so again while flying away (situation 4). The incidence of mobbing of free birds nearby, belonging to these three species, increased with growing threat to the trapped tit. Mobbing of these observing birds can be released merely by the view of a trapped tit threatened by a human being (Tab.).

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Mit Unterstützung der DFG, Az. Cu 4/18.     

LigPath: a module for predictive calculation of a ligand’s pathway into a receptor-application to the gpH<Subscript>1</Subscript> - receptor

Until now, the access of ligands into the binding pocket of a G-protein coupled receptor has scarcely been studied using molecular-modeling techniques because of the lack of sufficient algorithms. Neither with Monte-Carlo- nor with Molecular Dynamics Simulations can the penetration of a ligand into the binding pocket of a receptor be calculated because of the excessive amount of computing time needed. Therefore, a new algorithm LigPath for approximate calculation of a ligand’s pathway into the binding pocket has been developed. This new algorithm is based on a linkage of directional guiding of the ligand, Monte-Carlo-Search and minimization. In order to evaluate the performance of the algorithm, the guinea-pig histamine H₁ receptor was investigated in combination with one of its potent agonists, histaprodifen, which is proposed to bind in a pocket deep between the transmembrane helices of the receptor. Our calculations show that the amino acids Tyr194, Phe193, Phe436 and Phe433 guide the positively charged histaprodifen from the extracellular part of the receptor into the binding pocket.