Robert Reinhardt

Characterization of P2X3, P2Y1 and P2Y4 receptors in cultured HEK293‐hP2X3 cells and their inhibition by ethanol and trichloroethanol

Membrane currents and changes in the intracellular Ca2+ concentration ([Ca2+]i) were measured in HEK293 cells transfected with the human P2X3 receptor (HEK293‐hP2X3). RT‐PCR and immunocytochemistry indicated the additional presence of endogenous P2Y1 and to some extent P2Y4 receptors. P2 receptor agonists induced inward currents in HEK293‐hP2X3 cells with the rank order of potency α,β‐meATPu2003≈u2003ATPu2003>u2003ADP‐β‐Su2003>u2003UTP. A comparable rise in [Ca2+]i was observed after the slow superfusion of ATP, ADP‐β‐S and UTP; α,β‐meATP was ineffective. These data, in conjunction with results obtained by using the P2 receptor antagonists TNP‐ATP, PPADS and MRS2179 indicate that the current response to α,β‐meATP is due to P2X3 receptor activation, while the ATP‐induced rise in [Ca2+]i is evoked by P2Y1 and P2Y4 receptor activation. TCE depressed the α,β‐meATP current in a manner compatible with a non‐competitive antagonism. The ATP‐induced increase of [Ca2+]i was much less sensitive to the inhibitory effect of TCE than the current response to α,β‐meATP. The present study indicates that in HEK293‐hP2X3 cells, TCE, but not ethanol, potently inhibits ligand‐gated P2X3 receptors and, in addition, moderately interferes with G protein‐coupled P2Y1 and P2Y4 receptors. Such an effect may be relevant for the interruption of pain transmission in dorsal root ganglion neurons following ingestion of chloral hydrate or trichloroethylene.

P2X2 and P2Y1 immunofluorescence in rat neostriatal medium‐spiny projection neurones and cholinergic interneurones is not linked to respective purinergic receptor function

The presence of ionotropic P2X receptors, targets of ATP in fast synaptic transmission, as well as metabotropic P2Y receptors, known to activate K+ currents in cultured neostriatal neurones, was investigated in medium‐spiny neurones and cholinergic interneurones contained in neostriatal brain slices from 5–26‐day‐old rats. In these cells, adenosine‐5′‐triphosphate (ATP) (100–1000 μM), 2‐methylthioadenosine‐5′‐triphosphate (2MeSATP), α,β‐methyleneadenosine‐5′‐triphosphate (α,βmeATP, 30–300 μM, each) and adenosine‐5′‐O‐(3‐thiotriphosphate (ATPγS) (100 μM) failed to evoke P2X receptor currents even when 8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX, 0.1 μM), apyrase (10 U ml−1) or intracellular Cs+ was used to prevent occluding effects of the ATP breakdown product adenosine, desensitisation of P2X receptors by endogenous ATP and an interference with the activation of K+ channels, respectively. P2X receptor agonists were also ineffective in outside‐out patches withdrawn from the brain slice tissue. Muscimol (10 μM) evoked GABAA receptor‐mediated currents under all these conditions. When used as a control, locus coeruleus neurones responded with P2X receptor‐mediated currents to ATP (300 μM), 2MeSATP and α,βmeATP (100 μM, each). ATP and adenosine‐5′‐diphosphate (ADP) (100 μM, each) did not activate K+ currents in the neostriatal neurones. Despite the observed lack of function, P2X2 and P2Y1 immunofluorescence was found in roughly 50% of the medium‐spiny neurones and cholinergic interneurones. A role of ATP in synaptic transmission to striatal medium‐spiny neurones and cholinergic interneurones appears unlikely, however, the otherwise silent P2X and P2Y receptors may gain functionality under certain yet unknown conditions.

Molecular simulation applied to 2-(N′-alkylidenehydrazino)- and 2-(N′-aralkylidenehydrazino)adenosine A2 agonists

Summary The planar double ring system of adenine is conformationally rigid. Rotations of the ribose ring of adenosine around the N -glycosidic bond are hindered by nonbonded electrostatic and steric forces. The flexibility of the sugar ring and the changed conformations of the substituents of 2-( N′ -alkylidenehydrazino)-and 2-( N′ -aralkylidenehydrazino)adenosine A 2 agonists may be observed under changing solvent conditions and by energetic activation. Four hydrogen-bonding forces between the glutarimide rings of a recently designed pseudoreceptor and the ‘pharmacophoric groups’ of the adenosine derivatives may be hypothesized. Structure-activity relationships show that the A 2 agonist potency is mainly determined by the molar refraction of the substituents, the point-charge dipole moment of the molecule, and the type of substitution (aliphatic or aromatic groups).

Hyperbaric and normobaric reoxygenation of hypoxic rat brain slices – impact on purine nucleotides and cell viability

Hyperbaric oxygen treatment has been suggested as able to reduce hypoxia induced neuronal damage. The aim of the study was to compare the impact of different reoxygenation strategies on early metabolical (purine nucleotide content determined by HPLC) and morphological changes (index of cell injury after celestine blue/acid fuchsin staining) of hypoxically damaged rat neocortical brain slices. For this purpose slices (300 microm and 900 microm) were subjected to either 5 or 30 min of hypoxia by gassing the incubation medium with nitrogen. During the following reoxygenation period treatment groups were administered either 100% oxygen (O) or room air (A) at normobaric (1 atm absolute, NB-O; NB-A) or hyperbaric (2.5 atm absolute, HB-O; HB-A) conditions. After 5 min of hypoxia, both HB-O and NB-O led to a complete nucleotide status restoration (ATP/ADP; GTP/GDP) in 300 microm slices. However, reoxygenation after 30 min of hypoxia was less effective, irrespective of the oxygen pressure. Furthermore, administering hyperbaric room air resulted in no significant posthypoxic nucleotide recovery. In 900 microm slices, both control incubation as well as 30 min of hypoxia resulted in significantly lower trinucleotide and higher dinucleotide levels compared to 300 microm slices. While there was no significant difference between HB-O and NB-O on the nucleotide status, morphological evaluation revealed a better recovery of the index of cell injury (profoundly injured/intact cell-ratio) in the HB-O group. Conclusively, the posthypoxic recovery of metabolical characteristics was dependent on the duration of hypoxia and slice thickness, but not on the reoxygenation pressure. A clear restorative effect on purine nucleotides was found only in early-administered HB-O as well as NB-O in contrast to room air treated slices. However, these pressure independent metabolic changes were morphologically accompanied by a significantly improved index of cell injury, indicating a possible neuroprotective role of HB-O in early posthypoxic reoxygenation.

Investigation of the conformational behaviour of permethylated cyclodextrins by molecular modelling

Conformations of manually built native and permethylated alpha-, beta-, and gamma-cyclodextrins (CD) were investigated using various computer assisted molecular modelling methods. Calculations were carried by applying the MM+ and the Tripos force field. The influences of atomic charges on the macrocyclic conformations during the optimization procedure were analyzed. The permethylation of hydroxyl groups of cyclodextrins changes bond and torsion angles between the glucose monomers and of the primary substituents. A method to determine the diameters of the cyclodextrin cavity by a modelling approach is described. It is shown that due to permethylation the larger cavity opening is increased and the primary substituents are canted outwards. As a consequence, the torus shape of the molecule changes, which is an important feature for docking and fitting studies.

Chapter 18 P2 receptor-mediated activation of noradrenergic and dopaminergic neurons in the rat brain

Publisher Summary Focal electrical stimulation evokes in the rat locus coeruleus (LC) biphasic synaptic potentials consisting of early depolarizing (p.s.p.) and late hyperpolarizing (i.p.s.p.) components. It has been found that the p.s.p. is because of the release of glutamate from afferent fibres predominantly onto non-N-methyl-D- aspartate (non-NMDA) receptors and of γ-aminobutyric acid (GABA) onto GABA A receptors. In the chapter, the investigations demonstrate the presence of release stimulatory P2 receptors in the central noradrenergic and dopaminergic systems. Hence, exogenously applied or endogenously released ATP may excite noradrenergic neurons of the LC and dopaminergic neurons of the ventral tegmental area (VTA). ATP and its structural analog 2-MeSATP depolarized LC neurons in a slice preparation. In addition, there is strong evidence for the contribution of ATP to excitatory synaptic potentials recorded from the neurons themselves; this ATP appears to be coreleased with noradrenaline from recurrent axon collaterals or dendrites. A combined methodological approach was used to prove the presence of P2 receptors in the VTA and its main projection target in the NAc. Although electrophysiological recordings from VTA neurons in a slice preparation failed to show a depolarizing response to 2-MeSATP, the application of 2-MeSATP into the VTA via a microdialysis probe was a powerful stimulus of dopamine release in vivo .

Rapid assay for one-run determination of purine and pyrimidine nucleotide contents in neocortical slices and cell cultures.

The present study describes the measurement of endogenous nucleoside di- and triphosphate contents (ATP, GTP, UTP, CTP, ADP, GDP and UDP) in rat neocortical brain slices and mixed neuronal/astrocytic corticoencephalic cultures. Determination was by means of anion-exchange HPLC using a binary gradient of 0.3 M ammonium carbonate and water. In addition, a new method is described for the identification of nucleoside triphosphates, using digestion of the nucleotides by phosphoglycerate kinase and partial splitting of nucleoside diphosphates to shift the equilibrium of the phosphoglycerate kinase reaction in direction of breakdown of nucleoside triphosphates. Finally, the determination of the sum of creatine and creatine phosphate is suggested as an alternative reference value instead of protein under conditions when cells are cultured in protein-containing medium.

Early biochemical and histological changes during hyperbaric or normobaric reoxygenation after in vitro ischaemia in primary corticoencephalic cell cultures of rats.

In a first series of experiments, the morphological changes of corticoencephalic cells by ischaemia were determined by staining with celestine blue-acid fuchsin in order to classify cells as intact, dark basophilic (supposedly reversibly injured) and preacidophilic or acidophilic (profoundly injured). Hypoxia and glucose-deprivation (in vitro ischaemia) markedly decreased the number of intact cells and correspondingly increased the number of both reversibly and profoundly damaged cells. The morphological characteristics indicated a partial recovery during reoxygenation either in the absence or presence of glucose and irrespective of whether normobaric or hyperbaric oxygen was used. In a second series of experiments, nucleoside triphosphate and diphosphate levels were determined in corticoencephalic cultures by high-performance liquid chromatography. Hypoxia in combination with glucose-deficiency markedly decreased the ATP:ADP, GTP:GDP and UTP:UDP ratios. A still larger fall of these ratios was observed both after normobaric and hyperbaric reoxygenation. In contrast, both normobaric and hyperbaric reoxygenation in the presence of glucose led to an almost complete recovery near the control normoxic values. In conclusion, the histological changes were not adequately reflected by changes in the nucleoside triphosphate:diphosphate ratios and, in addition, hyperbaric oxygen had neither favourable nor unfavourable effects on the early morphological and functional restitution of ischaemically damaged cells under the conditions of the present study.

Investigation of gas chromatographic interaction mechanism on permethylated cyclodextrins by molecular modelling

SummaryThe capillary gas chromatographic retention behavior of α-pinene and tricyclene has been investigated on stationary phases of different polarities. On all but one of the columns employed, tricyclene eluted before α-pinene; only permethylated β-cyclodextrins dissolved in moderately polar polisiloxanes gave a reversed elution order. The intermolecular interactions which caused the unexpected retention behavior were investigated in detail, applying methods of computer simulation. To achieve this, we have developed a calculation algorithm on the basis of molecular mechanical optimizations and programmed it in a macro. This makes it possible to systematically investigate a given configuration space in which all the possible interactions can take place. It was shown that permethylated β-cyclodextrin as host molecule for both guest molecules offers an optimum cavity size. As a result the number of energetically favorable contacts between host and guest molecules as well as the strength of the interactions in this stationary phase were larger. As a consequence the elution order, normally only influenced by the vapor pressure of the compounds at a given temperature, was changed. Nonspecific interactions played an especially important role for these kinds of substances.

Alterations of purine and pyrimidine nucleotide contents in rat corticoencephalic cell cultures following metabolic damage and treatment with openers and blockers of ATP-sensitive potassium channels

Rat corticoencephalic cell cultures were investigated by high performance liquid chromatography for changes in the levels of adenosine 5-triphosphate (ATP), guanosine 5-triphosphate (GTP), uridine 5-triphosphate (UTP), cytidine 5-triphosphate (CTP), and the respective nucleoside diphosphates. Hypoxia was induced by gassing the incubation medium for 30 min with 100% argon. Removal of glucose was caused by washing the cultures in glucose-free medium at the beginning of the 30 min incubation period. Whereas hypoxia or glucose-deficiency alone failed to alter the nucleotide levels, the combination of these two manipulations was clearly inhibitory. Diazoxide (300 microM) an opener of ATP-dependent potassium channels (K(ATP)) did not alter the nucleotide contents either in a normoxic and glucose-containing medium, or a hypoxic and glucose-free medium. By contrast, the K(ATP) channel antagonist tolbutamide (300 microM) aggravated the hypoxic decrease of nucleotide levels in a glucose-free medium, although it was ineffective in a normoxic and glucose-containing medium. Hypoxia and glucose-deficiency decreased the ATP/ADP and UTP/UDP ratios, but failed to change the GTP/GDP ratio. Diazoxide and tolbutamide (300 microM each) had no effect on the nucleoside triphosphate/diphosphate ratios either during normoxic or during hypoxic conditions. In conclusion, corticoencephalic cultures are rather resistant to in vitro ischemia. Although they clearly respond to the blockade of plasmalemmal K(ATP) channels (plasmaK(ATP)) by tolbutamide, these channels appear to be maximally open as a consequence of the fall in intracellular nucleotides and, therefore, diazoxide has no further effect.