A novel rapid additive manufacturing concept for architectural composite shell construction inspired by the shell formation in land snails.

State-of-the-art rapid additive manufacturing (RAM)-specifically fused filament fabrication (FFF)-has gained popularity among architects, engineers and designers for the quick prototyping of technical devices, the rapid production of small series and even the construction scale fabrication of architectural elements. The spectrum of producible shapes and the resolution of detail, however, are determined and constrained by the layer-based nature of the fabrication process. These aspects significantly limit FFF-based approaches for the prefabrication and in situ fabrication of free-form shells at the architectural scale. Snails exhibit a shell building process that suggests ways to overcome these limits. They produce a soft, pliable proteinaceous film-the periostracum-which later hardens and serves, among other functions, as a form-giving surface for an inner calcium carbonate layer. Snail shell formation behavior is interpreted from a technical point of view to extract potentially useful aspects for a biomimetic transfer. A RAM concept for the continuous extrusion of thin free-form composite shells inspired by the snail shell formation is presented.

Trastuzumab therapy vs tetracycline controlled ERBB2 downregulation: influence on tumour development in an ERBB2-dependent mouse tumour model.

Trastuzumab (Herceptin) has improved therapy of breast cancer. Only patients overexpressing ERBB2 are treated with trastuzumab, whereas its use in tumours without ERBB2 expression is useless. This led to the concept that the subgroup of trastuzumab-sensitive tumours is 'ERBB2-dependent', meaning that ERBB2 signalling is indispensable for growth of these tumours. We used a mouse model that allows anhydrotetracycline (ATc)-controlled downregulation of ERBB2 in tumour tissue. ERBB2 mRNA and protein expression were downregulated below detection limit leading to a macroscopically complete tumour remission within 14 days. Tumour remission was accompanied by a strong decrease in proliferation, a moderate increase in apoptosis, as well as dephosphorylation of ERK1/2 and AKT/PKB. These data clearly indicate ERBB2 dependence. Therefore, a high sensitivity to trastuzumab may be suspected. Surprisingly, trastuzumab caused a much weaker effect compared to ATc-induced ERBB2 downregulation, although a decrease in ERBB2 membrane localisation was induced. Only a slight decrease in proliferation and a weak transient increase in apoptosis were observed. Interestingly, tumours responded to trastuzumab by a sharp fivefold increase in phosphorylated AKT/PKB as well as a 3.5- and 5.3-fold increase in AKT1 and AKT2 mRNA levels, respectively. In conclusion, 'ERBB2 dependence' is not sufficient to define trastuzumab-responsive tumours. The suboptimal effect of trastuzumab compared to the maximally possible effect induced by ATc demonstrates a high potential for improved ERBB2 blocking therapies.

P2 receptor expression in the dopaminergic system of the rat brain during development.

Extracellular ATP facilitates the release of dopamine via P2 receptor activation in parts of the mesolimbic system. To characterize P2X/Y receptor subtypes in the developing dopaminergic system, their expression in organotypic slice co-cultures including the ventral tegmental area/substantia nigra (VTA/SN) complex and the prefrontal cortex (PFC) was studied in comparison to the receptor expression in 3-5 day-old and adult rats. Reverse transcriptase-polymerase chain reaction (RT-PCR) with specific primers for the P2X(1,2,3,4,6,7) and P2Y(1) receptors in the tissue extracts of organotypic co-cultures revealed the presence of the P2X and P2Y receptor mRNAs investigated. Multiple immunofluorescence labeling of the P2X/Y receptor protein indicated differences in the regional expression in the organotypic co-cultures after 10 days of cultivation (VTA/SN, P2X(1,2,3,4,6,7), P2Y(1,6,12); PFC, P2X(1,3,4,6,7), P2Y(1,2,4,6,12)). At postnatal days 3-5, an immunofluorescence mostly comparable to that of adult rats was observed (VTA/SN and PFC: P2X(1,2,3,4,6,7), P2Y(1,2,4,6,12)). There was one important exception: the P2X(7) receptor immunocytochemistry was not found in adult tissue, suggesting a potential role of this receptor in the development. Only few P2 receptors (e.g. P2X(1), P2Y(1)) were expressed at fibers interconnecting the dopaminergic VTA/SN with the PFC in the organotypic co-cultures. The treatment of the cultures with the ATP analogues 2-methylthio-ATP and alpha,beta-methylene-ATP induced an increase in axonal outgrowth and fiber density, which could be inhibited by pre-treatment with the P2X/Y receptor antagonist pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid. The co-localization of the dopamine-(D1) receptor with the P2X(1) receptor in organotypic slice cultures was evident. In the PFC of the co-cultures, and that of young but not adult rats, a number of tyrosine hydroxylase (TH)-positive cells also possessed P2Y(1)-immunoreactivity (IR). Additionally, a strong P2Y(1)-IR was observed on astrocytes. The present results show a time-, region- and cell type-dependent in vitro and in vivo expression pattern of different P2 receptor subtypes in the dopaminergic system indicating the involvement of ATP and its receptors in neuronal development and growth.

Inhibition of cyclin-dependent kinases is neuroprotective in 1-methyl-4-phenylpyridinium-induced apoptosis in neurons.

The biochemical pathways involved in neuronal cell death in Parkinson's disease are not completely characterized. Mitochondrial dysfunction, specifically alteration of the mitochondrial complex I, is the primary target of the parkinsonian neurotoxin 1-methyl-4-phenylpyridinium (MPP+) induced apoptosis in neurons. In the present study, we examine the role of caspase-dependent and -independent routes in MPP+-induced apoptosis in rat cerebellar granule neurons (CGNs). We show a distinct increase in the expression of the cell cycle proteins cyclin D, cyclin E, cdk2, cdk4 and the transcription factor E2F-1 following a MPP+ treatment of CGNs. Flavopiridol (FLAV), a broad inhibitor of cyclin-dependent kinases (CDKs), attenuated the neurotoxic effects of MPP+ and significantly attenuates apoptosis mediated by MPP+ 200 microM. Likewise, the antioxidant vitamin E (vit E) increases neuronal cell viability and attenuates apoptosis induced by MPP+. Moreover, the expression levels of cyclin D and E2F-1 induced by this parkinsonian neurotoxin were also attenuated by vit E. Since, the broad-spectrum caspase inhibitor zVAD-fmk did not attenuate MPP+-induced apoptosis in CGNs, our data provide a caspase-independent mechanism mediated by neuronal reentry in the cell cycle and increased expression of the pro-apoptotic transcription factor E2F-1. Our results also suggest a potential role of oxidative stress in neuronal reentry in the cell cycle mediated by MPP+. Finally, our data further support the therapeutic potential of flavopiridol, for the treatment of Parkinson's disease.

Implication of cyclin-dependent kinase 5 in the neuroprotective properties of lithium.

Although numerous studies have demonstrated a neuroprotective and anti-apoptotic role of lithium in neuronal cell cultures, the precise mechanism by which this occurs, remains to be elucidated. In this study, we evaluated the lithium-mediated neuroprotection against colchicine-induced apoptosis in cultured cerebellar granule neurons. Previously, it has been demonstrated that colchicine mediates apoptosis in cerebellar granule neurons through cytoskeletal alteration and activation of an intrinsic pro-apoptotic pathway. Recently we also demonstrated a potential role of cyclin-dependent kinase 5 (cdk5) in this pathway. Here we report that colchicine induces dephosphorylation in Ser-9 and phosphorylation in Tyr-216, and thus activation, of glycogen synthase kinase-3beta in cerebellar granule neurons, and that this modification is inhibited by the presence of 5 mM lithium. However, the selective glycogen synthase kinase-3beta inhibitors SB-415286 and SB-216763 were unable to prevent colchicine-induced apoptosis in these cells, suggesting that the anti-apoptotic activity of lithium is not mediated by glycogen synthase kinase-3beta under these conditions. On the other hand, 5 mM lithium prevented the colchicine-induced increase in cdk5 expression and breakdown of cdk5/p35 to cdk5/p25. In addition, we show that up-regulation of cdk5/p25 is unrelated to inhibition of the activity of myocyte enhancer factor 2, a pro-survival transcription factor. These data suggest a previously undescribed neuroprotective mechanism of lithium associated with the modulation of cdk5/p35 or cdk5/p25 expression.

P2Y receptor expression on astrocytes in the nucleus accumbens of rats.

The expression of purinoceptor (P2)Y-subtypes on astrocytes in vivo under physiological conditions and after stab wound injury was investigated. Reverse transcriptase-polymerase chain reaction with specific primers for the receptor-subtypes P2Y1,2,4,6,12 in tissue extracts of the nucleus accumbens of untreated rats revealed the presence of all P2Y receptor mRNAs investigated. Double immunofluorescence visualized with laser scanning microscopy indicated the expression of the P2Y1,4 receptors on glial fibrillary acidic protein (GFAP)-labeled astrocytes under physiological conditions. After stab wound injury the additional expression of the P2Y2 and P2Y6 receptors, and an up-regulation of the P2Y1,4 receptor-labeling on astrocytic cell bodies and/or processes was observed. Astrocytes of cortical, in contrast to accumbal areas exhibited P2Y1,2,4,6 receptor-immunoreactivity (IR) under control conditions, which was up-regulated after stab would injury. Labeling for the P2Y12 receptor was not observed on GFAP-positive cortical and accumbal astrocytes under any of the conditions used. For the first time, the co-localization of different P2 receptor-subtypes (e.g. P2Y1 and P2X3) on the same astrocyte was shown immunocytochemically. The up-regulation of P2Y1 receptor-IR on astrocytes and non-glial cells after mechanical injury could be facilitated by microinfusion of the P2Y1,12,13 receptor agonist adenosine 5'-O-(2-thiodiphosphate) (ADPbetaS). Proliferative changes after ADPbetaS-microinjection were characterized by means of double-staining with antibodies against GFAP and 5-bromo-2'-deoxyuridine. The non-selective P2 receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid, the P2Y1 receptor antagonist N6-methyl-2'-deoxyadenosine 3',5'-bisphosphate and the P2Y1 receptor-antibody itself inhibited the agonist-induced effects. The data indicate the region-specific presence of P2Y receptors on astrocytes in vivo and their up-regulation after injury as well as the co-localization of P2X and P2Y receptor-subtypes on the same astrocyte. The dominant role of P2Y1 receptors in proliferation and the additional stimulation of non-P2Y1 receptors has been demonstrated in vivo suggesting the involvement of this receptor-type in the gliotic response under physiological and pathological conditions.

Somatic and axonal effects of ATP via P2X2 but not P2X7 receptors in rat thoracolumbar sympathetic neurones.

Excitatory ATP responses in rat cultured thoracolumbar sympathetic neurones are mediated by somatic P2X(2) receptors. The present study investigated a possible role of axonal P2X(2) as well as P2X(7) receptors on the same preparation. Confocal laser scanning microscopy demonstrated P2X(2) and P2X(7) immunoreactivity along the axons as well as P2X(7) immunoreactivity surrounding the cell nuclei. P2X(7) mRNA expression was detected in individual neurones using a single-cell RT-PCR approach. Adenosine triphosphate (ATP) caused a significant increase in axonal Ca(2+) concentration which was dependent on external Ca(2+) but insensitive to depletion of the cellular Ca(2+) pools by cyclopiazonic acid. Pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (PPADS; 30 micro m) virtually abolished the ATP response, whereas brilliant blue G (0.1 micro m), a selective P2X(7) receptor antagonist, had no effect. Dibenzoyl-ATP (BzATP; 100 micro m) induced a much smaller increase in axonal [Ca(2+)] concentration than ATP at equimolar concentrations. The response to BzATP was distinctly reduced by PPADS but not by brilliant blue G. The overall pharmacological profile of the axonal P2X receptors resembled closely that of the somatic P2X(2) receptors. In conclusion, the present data suggest the occurrence of axonal excitatory P2X(2) receptors in thoracolumbar sympathetic neurones. However, the functional significance of axonal and (peri)-nuclear P2X(7) receptors has still to be proven.

Formalin-induced changes of NMDA receptor subunit expression in the spinal cord of the rat.

Using RT-PCR, the present study investigated the effects of formalin administration on mRNA expression coding for NMDA receptor (NR) subunits and splice variants in rat lumbar spinal cord. Subsequent to formalin injection (5%; subcutaneously) into the hind paw of Sprague-Dawley rats, the animals exhibited the typical biphasic behavioural pain response. Spinal cord (L3-6) was prepared six hours after formalin injection. In controls, NR1-b predominated over NR1-a, and NR1-2 and NR1-4 exceeded over NR1-1 and NR1-3, respectively. Regarding the NR2 subunit expression in controls, NR2B exhibited the highest expression, followed by decreasing proportions of NR2C, NR2A, and NR2D. Formalin treatment did not affect NR1 splice variant expression but significantly increased and decreased the proportion of NR2A and NR2C, respectively. In summary, the present data demonstrate adaptive changes in the NR subunit expression pattern in rat spinal cord due to formalin injection.

Ethanol sensitivity of NMDA receptors.

NMDA receptors are ionotropic glutamate receptors assembled of subunits of the NR1 and of the NR2 family (NR2A-NR2D). The subunit diversity largely affects the pharmacological properties of NMDA receptors and, hence, gives rise to receptor heterogeneity. As an overall result of studies on recombinant and native NMDA receptors, ethanol inhibits the function of receptors containing the subunits NR2A and/or NR2B to a greater extent than those containing NR2C or NR2D. For example, in rat cultured mesencephalic neurons, NR2C expression was developmentally increased, whereas expression of NR2A and NR2B was decreased. These changes coincided with a developmental loss of sensitivity of NMDA responses to ethanol and ifenprodil, a non-competitive NMDA receptor antagonist that shows selectivity for NR2B-containing receptors. Also in rat locus coeruleus neurons, the low ethanol sensitivity of somatic NMDA receptors could be explained by a prominent expression of NR2C. The inhibitory site of action for ethanol on the NMDA receptor is not yet known. Patch-clamp studies suggest a target site exposed to or only accessible from the extracellular environment. Apparently, amino acid residue Phe(639), located in the TM3 domain of NR1, plays a crucial role in the inhibition of NMDA receptor function by ethanol. Since this phenylalanine site is common to all NMDA and non-NMDA receptor (AMPA/kainate receptor) subunits, this observation is consistent with accumulating evidence for a similar ethanol sensitivity of a variety of NMDA and non-NMDA receptors, but it cannot explain the differences in ethanol sensitivity observed with different NR2 subunits.

P2X receptor expression on astrocytes in the nucleus accumbens of rats.

Astrocytes express a variety of neurotransmitter receptors which render them capable of responding to extracellular stimuli, like ATP. Release of ATP, e.g. after brain injury, may initiate reactive gliosis via stimulation of purinergic P2X and P2Y receptors. In the present study, the expression and cellular localization of P2X receptor subtypes on astrocytes in the nucleus accumbens of rats under normal physiological conditions and after stab wound were investigated. Reverse transcription-polymerase chain reaction (RT-PCR) with specific P2X(1-7) primers, and double immunofluorescence with antibodies to glial fibrillary acidic protein (GFAP, a specific marker of fibrous astrocytes) and to different P2X receptor subtypes (P2X(1-4), P2X(7)) were used. The RT-PCR of tissue extracts of the nucleus accumbens of untreated rats revealed the presence of all seven currently known P2X receptor subtype mRNAs indicating the presence of these receptors in this region. A double immunofluorescence approach with confocal laser scanning microscopy showed the localization of P2X(2-4) receptor subtypes on GFAP-labelled astrocytes in untreated rats. Labelling for P2X(1) and P2X(7) receptor subtypes was not found. After mechanical damage all P2X receptor subtypes studied (P2X(1-4), P2X(7)) were observed on the GFAP-labelled reactive astrocytes. A characteristic distribution of the P2X receptors on astrocytic processes and cell bodies as well as an up-regulation of the P2X-immunofluorescence was found. In conclusion, the data show the presence of P2X receptors on rat nucleus accumbens astrocytes and suggest that astrogliosis in vivo is associated with an up-regulation of distinct P2X receptor subtypes.

P2X(2), P2X(2-2) and P2X(5) receptor subunit expression and function in rat thoracolumbar sympathetic neurons.

The present study investigated the pharmacological properties of excitatory P2X receptors and P2X(2) and P2X(5) receptor subunit expression in rat-cultured thoracolumbar sympathetic neurons. In patch-clamp recordings, ATP (3-1000 microM; applied for 1 s) induced inward currents in a concentration-dependent manner. Pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (PPADS; 30 microM) counteracted the ATP response. In contrast to ATP, alpha,beta-meATP (30 microM; for 1 s) was virtually ineffective. Prolonged application of ATP (100 microM; 10 s) induced receptor desensitization in a significant proportion of sympathetic neurons in a manner typical for P2X(2-2) splice variant-mediated responses. Using single-cell RT-PCR, P2X(2), P2X(2-2) and P2X(5) mRNA expression was detectable in individual tyrosine hydroxylase-positive neurons; coexpression of both P2X(2) isoforms was not observed. Laser scanning microscopy revealed both P2X(2) and P2X(5) immunoreactivity in virtually every TH-positive neuron. P2X(2) immunoreactivity was largely distributed over the cell body, whereas P2X(5) immunoreactivity was most distinctly located close to the nucleus. In summary, the present study demonstrates the expression of P2X(2), P2X(2-2) and P2X(5) receptor subunits in rat thoracolumbar neurons. The functional data in conjunction with a preferential membranous localization of P2X(2)/P2X(2-2) compared with P2X(5) suggest that the excitatory P2X responses are mediated by P2X(2) and P2X(2-2) receptors. Apparently there exist two types of P2X(2) receptor-bearing sympathetic neurons: one major population expressing the unspliced isoform and another minor population expressing the P2X(2-2) splice variant.

Neuroreceptors and ion channels as targets of alcohol.

This article represents the proceedings of a symposium at the 2000 ISBRA Meeting in Yokohama, Japan. The chairs were Toshio Narahashi and Kinya Kuriyama. The presentations were (1) Modulation of neuroreceptors and ion channels by alcohol, by T. Narahashi; (2) Inhibition by ethanol of NMDA and AMPA receptor-channels, by P. Illes, K. Wirkner, W. Fischer, K. Mühlberg, P. Scheibler, and C. Allgaier; (3) Effects of ethanol on metabotropic glutamate receptors, by K. Minami; (4) Acute alcohol actions on the 5-HT3 ligand-gated ion channel, by D. Lovinger; (5) Inhibition of NMDA receptors by MK801 attenuates ethanol-induced taurine release from the hippocampus, by F. Lallemand, R.J. Ward, and P. DeWitte; and (6) Effect of ethanol on voltage-operated Ca2+ channels in hepatic stellate cells, by T. Itatsu, Y. Takei, H. Oide, M. Hirose, X. E. Wang, S. Watanabe, M. Tateyama, R. Ochi, and N. Sato.

UTP evokes noradrenaline release from rat sympathetic neurons by activation of protein kinase C.

The pathway involved in UTP-evoked noradrenaline release was investigated in cultures of rat superior cervical ganglia. Northern blots revealed an age-related increase in levels of mRNA for P2Y6 receptors in cultures obtained at postnatal days 1 and 5, respectively, but no change in transcripts for P2Y1 and P2Y2. Likewise, UTP-evoked overflow of previously incorporated [(3)H]noradrenaline was six-fold higher in neurons obtained at postanatal day 5. Various protein kinase C inhibitors diminished UTP-, but not electrically, induced tritium overflow by > 70%, as did down-regulation of protein kinase C by 24 h exposure to phorbol ester. beta-Phorbol-12,13-dibutyrate and dioctanoylglycerol caused concentration-dependent increases in [(3)H] outflow of up to 6% of total radioactivity, and the secretagogue actions of these agents were reduced in the presence of protein kinase C inhibitors and in neurons pretreated with phorbol ester. Overflow evoked by dioctanoylglycerol was attenuated in the absence of extracellular Ca(2+) and in the presence of tetrodotoxin or Cd(2+). In addition to triggering tritium overflow, UTP reduced currents through muscarinic K(+) channels which, however, were not affected by phorbol esters. This action of UTP was not altered by protein kinase C inhibitors. These results indicate that P2Y6 receptors mediate UTP-evoked noradrenaline release from rat sympathetic neurons via activation of protein kinase C, but not inhibition of K(M) channels.

Effect of an adenosine A(1) receptor agonist and a novel pyrimidoindole on membrane properties and neurotransmitter release in rat cortical and hippocampal neurons.

Activation of adenosine A(1) receptors by endogenous adenosine plays a neuroprotective role under various pathophysiological conditions including hypoxia. Intracellular recordings were made in rat pyramidal cells of the somatosensory cortex. Hypoxia (5 min) induced a membrane depolarization and a decrease of input resistance. The A(1) receptor agonist N(6)-cyclopentyladenosine (CPA, 100 microM) reversibly inhibited the hypoxic depolarization. The inhibition was also present after blockade of the A(2A), A(2B) and A(3) receptor subtypes by selective antagonists. CPA had no effect on the hypoxic decrease of input resistance. 1,3-Dipropyl-8-cyclopentylxanthine (DPCPX), a selective A(1) receptor antagonist, which did not alter hypoxic depolarization when given alone abolished the inhibitory effect of CPA. Neither CPA nor DPCPX influenced membrane potential or apparent input resistance under normoxic conditions. The novel pyrimidoindole (R)-9-(1-methylbenzyl)-2-(4'-pyridyl)-9H-pyrimido[4,5-b]indole-4-amine (APPPI, 1 and 10 microM) reversibly diminished hypoxic depolarization but had no significant effect on input resistance. The effect of APPPI at a concentration of 1 microM, but not at 10 microM, was blocked by DPCPX (0.1 microM). CPA (100 microM) inhibited [(3)H]-noradrenaline ([(3)H]-NA) release from rat hippocampal brain slices significantly only in the presence of rauwolscine (0.1 microM), an alpha(2)-adrenoceptor antagonist. APPPI (1 and 10 microM) exhibited an inhibitory effect similar to that observed with CPA. The effects of both CPA and APPPI were antagonized by DPCPX (0.1 microM). The present data suggest that mainly presynaptic mechanisms prevent neurons from hypoxic changes by an inhibition of transmitter release. However, in contrast to CPA, APPPI exhibited additional effects, which require further investigation.

Single-cell RT-PCR analysis of N-methyl-D-aspartate receptor subunit expression in rat locus coeruleus neurones.

Whole-cell patch-clamp recordings were performed on 12- to 15-day-old rat locus coeruleus neurones in a midpontine slice preparation. Application of noradrenaline (100 microM) and N-methyl-D-aspartate (NMDA; 100 microM) induced a small outward current and a distinct inward current, respectively. Single-cell reverse transcriptase-polymerase chain reaction (scRT-PCR), used to analyse the expression pattern of NMDA receptor subunits 2A, 2B, and 2C (NR2A-C) subsequent to electrophysiological characterization, demonstrated differences in the capacity of individual locus coeruleus neurones to express NR2A-C mRNA. NR2C mRNA expression predominated over those of NR2A and NR2B mRNA in most neurones. In addition, in neurones containing NR2C mRNA NMDA induced significantly larger currents than in cells lacking expression of this gene. RT-PCR studies performed on tissue preparations of adult rats also revealed a distinct expression of NR2C mRNA. In conclusion, the present data demonstrate differences in the mRNA expression pattern of NR2A-C of individual locus coeruleus neurones with a predominant NR2C mRNA expression in the majority of the cells.

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.

P2X7 receptors in Müller glial cells from the human retina.

ATP has been shown to be an important extracellular signaling molecule. There are two subgroups of receptors for ATP (and other purines and pyrimidines): the ionotropic P2X and the G-protein-coupled P2Y receptors. Different subtypes of these receptors have been identified by molecular biology, but little is known about their functional properties in the nervous system. Here we present data for the existence of P2 receptors in Müller (glial) cells of the human retina. The cells were studied by immunocytochemistry, electrophysiology, Ca(2+)-microfluorimetry, and molecular biology. They displayed both P2Y and P2X receptors. Freshly enzymatically isolated cells were used throughout the study. Although the [Ca(2+)](i) response to ATP was dominated by release from intracellular stores, there is multiple evidence that the ATP-induced membrane currents were caused by an activation of P2X(7) receptors. Immunocytochemistry and single-cell RT-PCR revealed the expression of P2X(7) receptors by Müller cells. In patch-clamp studies, we found that (1) benzoyl-benzoyl ATP (BzATP) was the most effective agonist to evoke large inward currents and (2) the currents were abolished by P2X antagonists; however, (3) long-lasting application of BzATP did not cause an opening of large pores in addition to the cationic channels. By microfluorimetry it was shown that the P2X receptors mediated a Ca(2+) influx that contributed a small component to the total [Ca(2+)](i) response. Activation of P2X receptors may modulate the uptake of neurotransmitters from the extracellular space by Müller cells in the retina.

Inhibition by chloral hydrate and trichloroethanol of AMPA-induced Ca(2+) influx in rat cultured cortical neurones.

The effects of chloral hydrate and its main metabolite 2,2, 2-trichloroethanol were investigated on the (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-induced rise of intracellular Ca(2+) concentration ([Ca(2+)](i)) in cultured non-pyramidal cortical neurones of rats by using single-cell fura-2 microfluorimetry. AMPA elicited a concentration-dependent effect that peaked at 300 microM (EC(50), 7. 5 microM). Responses to AMPA (30 microM) were markedly inhibited by superfusion with chloral hydrate (IC(50), 4.5 mM) or trichloroethanol (IC(50), 0.9 mM). By contrast, ethanol (100 mM) caused only slight inhibition. In conclusion, the results demonstrate that chloral hydrate and especially its metabolite trichloroethanol, inhibit the AMPA-induced rise of [Ca(2+)](i) by depressing the entry of Ca(2) into cortical neurones via the AMPA receptor-channel.

P2Y receptors contribute to ATP-induced increases in intracellular calcium in differentiated but not undifferentiated PC12 cells.

ATP-induced Ca2+ transients were examined in individual PC12 cells of a well defined clone, before and after treatment with nerve growth factor (NGF) to induce a neurone-like phenotype. Using reverse transcriptase PCR these cells were found to express mRNA for several P2 receptors. In undifferentiated cells the ATP-induced Ca2+ response was entirely dependent on Ca2+ influx, could not be mimicked by UTP, alpha,beta-methylene ATP or dibenzoyl ATP or be blocked by pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS). ATP had no significant effect on levels of cyclic AMP or inositol 1,4,5-trisphosphate (InsP3). These results suggest that in undifferentiated PC12 cells ATP mainly acts on a P2X receptor, possibly the P2X4 subtype. After treatment with NGF for 7 days the ATP response was increased and partially sensitive to PPADS. A component of the ATP-induced Ca2+ increase was due to mobilisation of intracellular Ca2+ stores and another to capacitative Ca2+ entry. UTP caused an increase in intracellular Ca2+, and InsP3 formation could be stimulated by ATP and UTP. ATP also caused a small increase in cyclic AMP, but this was abolished in the presence of indomethacin. Thus, after NGF treatment ATP acts partially via a P2Y receptor, possibly the P2Y2 subtype.

Mechanism of inhibition by ethanol of NMDA and AMPA receptor channel functions in cultured rat cortical neurons.

The effect and mode of action of ethanol on N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors of rat cortical neurons in primary culture were compared by means of the patch-clamp technique. The maxima of the concentration-response curves for both NMDA and AMPA were markedly depressed by ethanol without an apparent shift of the curves to the right. Ethanol inhibited the effects of NMDA and AMPA concentration-dependently and equally well. Excitatory amino acid (EAA) currents were depressed to the largest extent when ethanol was continuously superfused during and between agonist applications; a smaller inhibitory effect was observed when ethanol was intermittently superfused during agonist applications only. There was no inhibition by ethanol, when its superfusion was between two agonist applications. According to expectations, the analysis of the ratios of plateau to peak currents failed to suggest a use-dependent blockade by ethanol. In addition, comparison of the voltage-current curves of NMDA and AMPA in the absence and presence of ethanol indicated a voltage-independent effect and no change in the reversal potential of the two agonists. Finally, the measurement of activation and deactivation time constants for the ethanol-induced inhibition of NMDA and AMPA responses confirms the failure of ethanol to cause an open-channel block. In conclusion, these findings as a whole indicate that ethanol inhibits NMDA and AMPA currents by a noncompetitive mechanism. The mode of action appears to be similar for both NMDA and AMPA; under the conditions of the present study, a selective interference with structural motifs of the NMDA receptor is unlikely.