No abstract

1 The pharmacological properties of CGP 37849 (DL-(E)-2-amino-4-methyl-5-phosphono-3-pentenoic acid; 4-methyl-APPA) and its carboxyethylester, CGP 39551, novel unsaturated analogues of the Nmethyl-D-aspartate (NMDA) receptor antagonist, 2-amino-5-phosphonopentanoate (AP5), were evaluated in rodent brain in vitro and in vivo. 2 Radioligand binding experiments demonstrated that CGP 37849 potently (K1 220 nM) and competitively inhibited NMDA-sensitive L-[3H]-glutamate binding to postsynaptic density (PSD) fractions from rat brain. It inhibited the binding of the selective NMDA receptor antagonist, [3H]-( ±)-342-carboxypiperazin-4-yl)propyl-1-phosphonate (CPP), with a Ki of 35 nm, and was 4, 5 and 7 fold more potent than the antagonists ((±)-cis-4-phosphonomethylpiperidine-2-carboxylic acid) (CGS 19755), CPP and D-AP5, respectively. Inhibitory activity was associated exclusively with the trans configuration of the APPA molecule and with the D-stereoisomer. CGP 39551 showed weaker activity at NMDA receptor recognition sites and both compounds were weak or inactive at 18 other receptor binding sites. 3 CGP 37849 and CGP 39551 were inactive as inhibitors of L-[3H]-glutamate uptake into rat brain synaptosomes and had no effect on the release of endogenous glutamate from rat hippocampal slices evoked by electrical field stimulation. 4 In the hippocampal slice in vitro, CGP 37849 selectively and reversibly antagonized NMDA-evoked increases in CAI pyramidal cell firing rate. In slices bathed in medium containing low Mg2+ levels, concentrations of CGP 37849 up to 10pM suppressed burst firing evoked in CAl neurones by stimulation of Schaffer collateral-commissural fibres without affecting the magnitude of the initial population spike; CGP 39551 exerted the same effect but was weaker. In vivo, oral administration to rats of either CGP 37849 or CGP 39551 selectively blocked firing in hippocampal neurones induced by ionophoreticallyapplied NMDA, without affecting the responses to quisqualate or kainate. al., 1988). Many studies have demonstrated that activation of the NMDA receptor is involved in the generation of epileptiform activity and in hypoxic-ischaemic neuronal damage, and conversely that NMDA receptor antagonists are anticonvulsant and cerebroprotective in animal models of epilepsy and stroke (see reviews by Meldrum, 1985;Rothman & Olney, 1986;Choi, 1988;Patel et al., 1988;Iversen et al., 1989;Albers et al., 1989 (Meldrum, 1985;Rothman & Olney, 1986;Albers et al., 1989;.Antagonism of NMDA receptor mechanisms potentially may be achieved by a number of different approaches. In addition to the transmitter recognition site, the NMDA receptor complex comprises an allosteric regulatory site and a channel binding domain (Foster & Fagg, 1987b), and possibly also sites defined by the actions of Zn2", polyamines, tricyclic antidepressants, ifenprodil and CGP 31358 (see Lodge, 1989;Baud et al., 1989). At present, however, the two most well characterized sites are (1) the transmitter recognition site, at which substances such as ...

No abstract

A key to enhance the low translatability of preclinical drug discovery are in vitro human three-dimensional (3D) microphysiological systems (MPS). Here, we show a new method for automated engineering of 3D human skeletal muscle models in microplates and functional compound screening to address the lack of muscle wasting disease medication. To this end, we adapted our recently described 24-well plate 3D bioprinting platform with a printhead cooling system to allow microvalve-based drop-on-demand printing of cell-laden Matrigel containing primary human muscle precursor cells. Mini skeletal muscle models develop within a week exhibiting contractile, striated myofibers aligned between two attachment posts. As an in vitro exercise model, repeated high impact stimulation of contractions for 3 h by a custom-made electrical pulse stimulation (EPS) system for 24-well plates induced interleukin-6 myokine expression and Akt hypertrophy pathway activation. Furthermore, the known muscle stimulators caffeine and Tirasemtiv acutely increase EPS-induced contractile force of the models. This validated new human muscle MPS will benefit development of drugs against muscle wasting diseases. Moreover, our Matrigel 3D bioprinting platform will allow engineering of non-self-organizing complex human 3D MPS.

1 Synaptic activation of g-aminobutyric acid (GABA) B receptors at GABA synapses causes (a) postsynaptic hyperpolarization mediating a slow inhibitory postsynaptic potential/current (IPSP/C) and (b) presynaptic inhibition of GABA release which depresses IPSPs and leads to paired-pulse widening of excitatory postsynaptic potentials (EPSPs). To address whether these e ects are mediated by pharmacologically identical receptors the e ects of six GABA B receptor antagonists of widely ranging potencies were tested against each response. 2 Monosynaptic IPSP B s were recorded in the presence of GABA A , AMPA/kainate and NMDA receptor antagonists. All GABA B receptor antagonists tested depressed the IPSP B with an IC 50 based rank order of potency of CGP556795CGP56433=CGP55845A=CGP524324CGP511764 CGP36742. 3 Paired-pulse EPSP widening was recorded as an index of paired-pulse depression of GABAmediated IPSP/Cs. A similar rank order of potency of antagonism of paired-pulse widening was observed to that for IPSP B inhibition. 4 Comparison of the IC 50 values for IPSP B inhibition and paired-pulse EPSP widening revealed a close correlation between the two e ects in that their IC 50 s lay within the 95% con®dence limits of a correlation line that described IC 50 values for inhibition of paired-pulse EPSP widening that were 7.3 times higher than those for IPSP B inhibition. 5 Using the compounds tested here it is not possible to assign di erent subtypes of GABA B receptor to pre-and post-synaptic loci at GABAergic synapses. However, 5 ± 10 fold higher concentrations of antagonist are required to block presynaptic as opposed to postsynaptic receptors when these are activated by synaptically released GABA.

No abstract

The goal of this study was to investigate whether locus coeruleus neurons of the rat are sensitive to agonists of the different excitatory amino acid receptors. All experiments were performed on a midpontine rat slice preparation. Bath-applied L-glutamate, kainate, N-methyl-D-aspartate (NMDA) and quisqualate induced concentration-dependent activations of all neurons which were reflected in an increase of the neurons' mean discharge rate. The rank order of cell activation was kainate approximately quisqualate greater than NMDA greater than L-glutamate. None of the agonists induced a bursting-type of discharge. The NMDA-receptor blocker DL-2-amino-5-phosphonovaleric acid (APV, 30 microM) selectively antagonized the NMDA-induced increase in cell firing. Kynurenic acid (100 microM) non-selectively attenuated the response to NMDA, kainate and quisqualate. Neither APV nor kynurenic acid per se had any effect on the spontaneous firing rate. If the Mg2+ concentration in the superfusion medium was lowered from 2 mM to nominally zero the response to NMDA was selectively increased. In conclusion, locus coeruleus neurons share with other neurons their sensitivity to agonists of all three types of excitatory amino acid receptors. However, in contrast to other neurons, they do not respond with a bursting type of discharge.

No abstract