Tony Priestley

Antagonism of responses to excitatory amino acids on rat cortical neurones by the spider toxin, argiotoxin636

1 Several low molecular weight spider toxins have recently been shown to block potently glutama‐tergic neuromuscular transmission at the invertebrate neuromuscular junction. The aim of the present investigation was to evaluate the effects of one such toxin, argiotoxin636, on excitatory amino acid receptor‐mediated responses in mammalian neurones. 2 Membrane currents were recorded from rat cortical neurones after 2–6 weeks in cell culture, by the whole‐cell variant of the patch‐clamp technique. N‐methyl‐D‐aspartate (NMDA) and kainate were used as selective agonists for their respective receptor subtypes. α‐Amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole‐propionic acid (AMPA) was used as a selective agonist for the quisqualate receptor subtype. 3 Responses to these agonists were characterised with respect to their concentration and voltage‐dependence. Argiotoxin636 (3–30 μm) was found to attenuate markedly responses to NMDA in an agonist‐ and voltage‐dependent manner. Thus, argiotoxin636 progressively reduced successive responses to NMDA when membrane potentials were voltage clamped between −40 mV to −100 mV. The more negative the membrane potential the more rapid the development of the block of inward current. 4 The antagonism of NMDA‐induced currents by argiotoxin636 could be reversed by clamping the membrane at positive potentials (+ 20 to + 60 mV) and reapplying NMDA. 5 Responses to AMPA and kainate were less affected by argiotoxin636, with an antagonist action only becoming evident at a concentration of 100 μm 6 These results suggest that argiotoxin636 is an open‐channel blocker of the NMDA activated ion‐channel in mammalian neurones. Furthermore, our results indicate at least a 30 fold selectivity for NMDA over the quisqualate‐ and kainate‐activated ion‐channels.

The effect of NMDA receptor glycine site antagonists on hypoxia-induced neurodegeneration of rat cortical cell cultures

The neuroprotective potential of an antagonist (7-chlorokynurenic acid (7-CIKYNA)) and a low efficacy partial agonist (HA-966) for the glycine modulatory site on the N-methyl-D-aspartate (NMDA) receptor complex has been examined using a neuronal cell culture/hypoxia model of neurodegeneration. Their effects were compared to those of the potent uncompetitive NMDA antagonist, MK-801. Hypoxic cell injury was assessed visually and quantified by measuring the appearance of two cytosolic enzymes, lactate dehydrogenase (LDH) and neurone specific enolase (NSE), in the culture medium. MK-801 prevented the hypoxia-induced cell mortality in a concentration-related manner with an IC50 of 15 nM against increases in LDH levels. HA-966 and 7-CIKYNA also produced concentration-related protective effects with IC50s of 175 and 18 microM, respectively. Although both glycine antagonists were considerably weaker than MK-801 their maximum neuroprotective effects were comparable to that produced by MK-801, i.e. complete protection. This indicates that the level of NMDA receptor activation which can take place in the presence of the partial agonist HA-966 is insufficient to cause permanent neuronal damage. Concentration-effect curves were similar when NSE was used as the marker enzyme, supporting previous observations that the increases in LDH levels accurately and specifically reflect neuronal cell death. These results provide further evidence that hypoxia-induced injury to cortical neuronal cultures is mediated by an excessive stimulation of NMDA receptors and that glycine-site antagonists and partial agonists may have therapeutic potential in conditions where pathologically high levels of NMDA receptor activation are thought to occur.

Pharmacological differences between two muscarinic responses of the rat superior cervical ganglion in vitro

1 Pharmacological differences have been observed between the muscarinic agonist‐induced depolarizing and hyperpolarizing responses of the rat isolated superior cervical ganglion. 2 Pirenzepine (0.3 μm) selectively reduced the depolarizing response and unmasked the hyperpolarizing response. No such selectivity was observed with a concentration of N‐methylatropine which was equipotent with pirenzepine in antagonizing the depolarizing response. 3 The neuromuscular blocking agents gallamine (10 μm) and pancuronium (3 μm) exhibited the oppositive selectivity to pirenzepine, both dramatically reduced the hyperpolarization but only slightly antagonized the depolarization. 4 The potencies of a range of agonists in evoking the depolarizing and hyperpolarizing responses, the latter in the presence of 0.3 μm pirenzepine, have been determined. Methylfurmethide failed to hyperpolarize the ganglion at concentrations which evoked maximal depolarizations. 5 The muscarinic hyperpolarization did not appear to be mediated by the secondary release of catecholamines. 6 It was concluded that the two muscarinic responses on the rat superior cervical ganglion, the slow depolarization and faster hyperpolarization, are mediated by different muscarinic receptor subtypes.

Subtypes of NMDA receptor in neurones cultured from rat brain

The non-competitive N-methyl-D-aspartate (NMDA) antagonist, ifenprodil, discriminates two receptor populations, each of which shows a reciprocal abundance in cultured cortical and cerebellar granule cells. Thus approximately 70% of NMDA-gated membrane current was antagonized with high affinity (IC50 = 1.4 +/- 0.9 microM) in cortical neurones whereas only approximately 20% was antagonized with high affinity (IC50 = 1.3 +/- 0.3 microM) in granule cells. Inhibition curves for CGS 19755 appeared relatively monophasic: this competitive NMDA antagonist had a significantly higher affinity for the granule cell receptor (Ki = 0.8 +/- 0.2 microM) compared with that on cortical neurones (Ki = 2 +/- 0.6 microM). The data suggest that these two antagonists may be of value in identifying the expression of subpopulations of native NMDA receptors in other brain regions.

The effects of insulin-like growth factor analogues on survival of cultured cerebral cortex and cerebellar granule neurones.

Insulin and insulin-like growth factors (IGF-I, IGF-II) are closely related polypeptides which are found in the CNS and which promote neuronal survival and neurite outgrowth. They are each associated with specific cell surface receptors and several soluble binding proteins (IGFBPs) which are involved in regulating function and availability. Two analogues of IGF-I were produced by site directed mutagenesis: (Gln3, Ala4, Tyr15, (Leu16)IGF-1 (QAYL-IGF) and a B-chain mutant in which the first 16 amino acids of IGF-1 were replaced by the first 17 amino acids of insulin. These analogues have significantly reduced binding affinity for IGFBPs. Using glucose deprivation as a damaging stimulus and assaying lactate dehydrogenase released from cultures as a marker for cell death, we have investigated the effect of IGF analogues on cell death of cerebrocortical and cerebellar granule cell cultures. In the presence of IGF-I, QAYL-IGF or B-chain mutant, the amount of LDH released from cortical and cerebellar granule cell cultures was significantly reduced compared to control (no glucose), indicating that these molecules promote survival. Both QAYL and B-chain mutants, which have reduced affinity for IGFBPs, are as effective as IGF-I in promoting cell survival in conditions of glucose deprivation and their reduced affinity for IGFBPs has no apparent deleterious effect on their neuroprotective function. We also show that the neuroprotective effect of the IGF analogues is due to a direct effect on the neurones in these cultures and is independent of the presence of glia.

Derivatives of 1-hydroxy-3-aminopyrrolidin-2-one (HA-966). Partial agonists at the glycine site of the NMDA receptor

The in vitro activies of 4-substituted and bicyclic analogues of the glycine-site NMDA partial agonist HA-966 (1) reveal strict structure-activity requirements reflecting subtle conformational and steric requirements for receptor binding. The most active compounds have cis-4-methyl or hydroxyl substituents and it is suggested that the in vivo anticonvulsant activity and good brain penetration of the optimal compound (+) 2 (L-687,414) result from the high fraction of (+) 2 which is not ionised at physiological pH.

The effect of baclofen and somatostatin on neuronal activity in the rat ventromedial hypothalamic nucleus in vitro

The electrical properties of neurones within the ventromedial hypothalamic nucleus of the rat were studied in an in vitro slice preparation, using conventional intracellular recording techniques. A detailed analysis of 36 intracellular recordings appeared to suggest 3 cell types, based on membrane capacitance and resistance characteristics, confirming previous reports of a diversity of cell types within this nucleus. The responsiveness of each cell type to exogenously-applied baclofen and somatostatin was also investigated. The inhibitory responses to both of these drugs were concentration-related (over the range 100 nM to 1 microM), tetrodotoxin-resistant and consisted of a membrane hyperpolarization (mean +/- SEM = 6.7 +/- 1 and 10.7 +/- 1 mV for 1 microM somatostatin and baclofen, respectively) and an associated reduction in the firing frequency of spontaneously active cells. These agonist-evoked responses probably represented direct postsynaptic actions but they were not restricted to any single type of cell. Evidence for an additional presynaptic effect of baclofen was also obtained. Responses to baclofen were extremely robust and readily quantifiable, whereas those to somatostatin showed pronounced long-lasting desensitization, which was particularly marked a larger concentrations. These data support previous contentions, based on in vivo studies, that somatostatin and GABA are likely to participate in the control of complex functions by the ventromedial hypothalamic nucleus.

T-type calcium channel blockers: spiro-piperidine azetidines and azetidinones-optimization, design and synthesis.

A series of spiro-azetidines and azetidinones has been evaluated as novel blockers of the T-type calcium channel (Ca(V)3.2) which is a new therapeutic target for the potential treatment of both inflammatory and neuropathic pain. Confirmation and optimization of the potency, selectivity and DMPK properties of leads will be described.

Time-dependent changes in NMDA receptor expression in neurones cultured from rat brain

Several studies have shown marked changes in the regional expression pattern of N-methyl-D-aspartate (NMDA) receptor subunit composition in vivo in the developing brain. Similar developmental changes may also occur in vitro. Thus, displacement of [3H]MK-801 binding by the subunit-selective NMDA antagonist, ifenprodil, in membrane homogenates prepared from cultured neurones, has been shown to comprise two components, the relative proportion of which changed with time in culture [11]. In the present experiments we have used electrophysiological methods to determine the influence of time in culture on NMDA receptor subtype expression pattern in individual neurones. Shortly after plating (13 days in vitro (DIV)), approximately 70% of total NMDA-induced inward current in voltage-clamped rat cortical neurones is antagonised by ifenprodil with relatively high affinity (approximately 1 microM). By 65 DIV, however, the high-affinity component contributed to only approximately 20% of the overall antagonism. Cerebellar granule cells also appear to undergo a similar change in their NMDA receptor expression with the exception that, in general, they appear initially to show considerably less of the ifenprodil high-affinity component and this disappears completely by 15 DIV. These experiments suggest that individual cortical and granule cells express at least two different NMDA receptor subtypes and that their relative proportion changes with time in culture.

A 5‐HT1‐like receptor mediates a pertussis toxin‐sensitive inhibition of rat ventromedial hypothalamic neurones in vitro

5‐ Hydroxytryptamine (5‐HT), 5‐carboxamidotryptamine and 8‐hydroxy‐2‐(di‐n‐propylamino)‐tetralin inhibited rat ventromedial hypothalamic neurones in vitro in a concentration‐dependent manner. The agonist‐induced inhibition was reduced by spiperone (1 μM) and by pertussis toxin, but not by MDL 72222 (10 μM) or ketanserin (1 μM). The inhibition appeared to be mediated via 5‐HT1A‐receptors and a pertussis toxin‐sensitive pathway.