Insulin resistance, obesity and the metabolic syndrome. Is there a therapeutic role for endothelin-1 antagonists?

There is increasing evidence to suggest that chronic activation of the endothelin-1 system can lead to heterologous desensitization of the glucose-regulatory and mitogenic actions of insulin with subsequent development of glucose intolerance, hyperinsulinemia, impaired endothelial function and exacerbation of cardiovascular disease. Effects are mediated through a variety of mechanisms that include attenuation of key insulin signalling pathways and decreased tyrosine phosphorylation of insulin receptor substrates IRS-1, SHC and G alpha q/11. Other actions involve hemodynamic changes leading to reduced delivery of insulin and glucose to peripheral tissues as well as enhanced hepatic glycogenolysis, decreased glucose-transporter translocation and modulation of various adipokines that regulate insulin action. Overall the data suggest that ET-1 antagonists may provide an effective means of improving cardiac dysfunction and favourably influencing glucose tolerance in obese humans and patients with early insulin sensitivity where there is clear evidence for activation of the ET-1 system. Although most effects of ET-1 that modulate mechanisms leading to glucose intolerance appear to involve the ETA receptor subtype recent data indicates that combined ETA/ETB receptor antagonists may function as effectively as selective ETA blockers. Prospective trials are needed to assess whether ET-1 antagonists, either alone or in combination, are superior to other more conventional therapies such as insulin sensitizers and to evaluate effects of combined treatments on the development of insulin resistance and the progression of diabetes. Early screening of patients at risk for evidence of ET-1 activation would help to identify subjects who may benefit most from such treatment.

Cholecystokinin dipeptoid antagonists: design, synthesis, and anxiolytic profile of some novel CCK-A and CCK-B selective and "mixed" CCK-A/CCK-B antagonists.

The design, synthesis, and structure-activity relationships (SAR) for the development of selective dipeptoid ligands for both of the cholecystokinin (CCK) receptor subtypes CCK-A and CCK-B are described. The SAR developed is used to design a ligand with equal nanomolar binding affinity for both the CCK-A and CCK-B receptors. Example compounds such as [1R-[1 alpha[R*(R*)],2 beta]]-4-[[2-[[3-(1H-indol-3-yl)- 2-methyl-2-[[[(2-methylcyclohexyl)oxy]carbonyl]amino]-1- oxopropyl]-amino]-1-phenylethyl]amino]-4-oxo-butanoic acid (24c), (1R-trans)-N-[alpha-methyl-N-[[(2-methylcyclohexyl)oxy] carbonyl]-D-tryptophyl]-L-3-(phenylmethyl)-beta-alanine (28i), and N-[alpha-methyl-N-[(tricyclo[3.3.1.1]dec-2-yloxy) carbonyl]-D-tryptophanyl]-L-3-(phenylmethyl)-beta-alanine (30m) are CCK-B selective compounds having CCK-B binding affinities of IC50 = 3.9, 0.34, and 0.15 nM with a CCK-A/CCK-B ratio of 464, 53, and 170, respectively. Other compounds such as (1R-trans)-N-[alpha-methyl-N-[[(2-methylcyclohexyl)oxy]carbonyl]- L-tryptophyl]-D-3-(phenylmethyl)-beta-alanine (281) and N-(alpha-methyl-N-[(tricyclo[3.3.1.1]dec-2-yloxy)carbonyl]-L - tryptophyl]-D-3-(phenylmethyl)-beta-alanine (30p) are CCK-A-selective compounds having CCK-A binding affinities of IC50 = 7.9 and 2.82 nM with a CCK-A/CCK-B ratio of 0.007 and 0.01, respectively. Further to these, (1S-trans)-N-[alpha-methyl-N-[[(2-methylcyclohexyl)oxy] carbonyl]-D-tryptophyl]-L-3-(phenylmethyl)-beta-alanine (28h) is a mixed CCK-A/CCK-B ligand with a CCK-A binding affinity of IC50 = 3.9 nM and a CCK-B binding affinity of IC50 = 4.2, producing a CCK-A/CCK-B ratio of unity. The CCK-B selective compounds are shown to be antagonists in electrophysiological tests on the rat ventromedial nucleus of the hypothalamus with an equilibrium constant (Ke) value of 2.8 nM for 30m and are also shown to be anxiolytic in the mouse ligh/dark box test with a minimum effective dose of 0.01 mg/kg, sc, for 30m. The CCK-A selective compounds are also shown to be competitive antagonists by the inhibition of CCK-8S-evoked amylase secretion from pancreatic acinar cells with a Ke value of 16 nM for 30p. In electrophysiological tests on the rat dorsal raphé (an area rich in CCK-A receptors) 30p had a Ke value of 12.8 nM. The mixed CCK-A/CCK-B compound 28h showed antagonistic properties in both CCK-A and CCK-B models; thus it inhibited CCK-8S-evoked amylase secretion from pancreatic acinar cells and is anxiolytic in the light/dark box paradigm.(ABSTRACT TRUNCATED AT 400 WORDS)

Use of a dipeptide chemical library in the development of non-peptide tachykinin NK3 receptor selective antagonists.

The use of a dipeptide library as the source of a micromolar chemical lead compound for the human tachykinin NK3 receptor is described. The screening of a dipeptide library through a cloned human NK3 receptor binding assay resulted in the identification of Boc(S)Phe(S)PheNH2 (1), which has subsequently been developed, following a 'peptoid' design strategy, into a series of high-affinity NK3 receptor selective antagonists. The structure-activity relationship of the C-terminal portion of this dipeptide lead was first explored and led to the identification of the urea derivative Boc(S)Phe(R)alphaMePheNH(CH2)7NHCONH2 (41, PD157672). This modified dipeptide has a Ke of 7 nM in blocking senktide-induced increases in intracellular calcium levels in human NK3 receptors stably expressed in CHO cells. Subsequent optimization of the N-terminal BocPhe group and the alphaMePhe residue side chain of 41 led to the identification of [S-(R*,S*)]-[2-(2,3-difluorophenyl)-1-methyl-1-[(7-ureidoheptyl)ca r bamoyl]ethyl]carbamic acid 2-methyl-1-phenylpropyl ester (60, PD161182), a non-peptide NK3 receptor selective antagonist. Compound 60 blocks the senktide-evoked increases in intracellular calcium levels in cloned human NK3 receptors stably expressed in CHO cells with Ke of 0.9 nM.

Second generation "peptoid" CCK-B receptor antagonists: identification and development of N-(adamantyloxycarbonyl)-alpha-methyl-(R)-tryptophan derivative (CI-1015) with an improved pharmacokinetic profile.

We have previously described the design and development of CI-988, a peptoid analogue of CCK-4 with excellent binding affinity and selectivity for the CCK-B receptor. Due to its anxiolytic profile in animal models of anxiety, this compound was developed as a clinical candidate. However, during its development, it was determined that CI-988 had low bioavailability in both rodent and nonrodent species. In the clinic, it was further established that CI-988 had poor bioavailability. Thus, there was a need to identify an analogue with an improved pharmacokinetic (PK) profile. The poor bioavailability was attributed to poor absorption and efficient hepatic extraction. We envisaged that reducing the molecular weight of the parent compound (5, MW = 614) would lead to better absorption. Thus, we synthesized a series of analogues in which the key alpha-methyltryptophan and adamantyloxycarbonyl moieties, required for receptor binding, were kept intact and the C-terminus was extensively modified. This SAR study led to the identification of tricyclo[3.3.1.1(3,7)]dec-2-yl [1S-[1 alpha(S*)2 beta]-[2-[(2-hydroxycyclohexyl)amino]-1-(1H-indol-3- ylmethyl)-1-methyl-2-oxoethyl]carbamate (CI-1015, 31) with binding affinities of 3.0 and 2900 nM for the CCK-B and CCK-A receptors, respectively. The compound showed CCK-B antagonist profile in the rat ventromedial hypothalamus assay with a Ke of 34 nM. It also showed an anxiolytic like profile orally in a standard anxiety paradigm (X-maze) with a minimum effective dose (MED) of 0.1 microgram/kg. Although the compound is less water soluble than CI-988, oral bioavailability in rat was improved nearly 10 times relative to CI-988 when dosed in HP beta CD. The blood-brain permeability of CI-1015 (31) was also enhanced relative to CI-988 (5). On the basis of the overall improved pharmacokinetic profile as well as enhanced brain penetration, CI-1015 (31) was chosen as a development candidate.

Presence of NK3-sensitive neurones in different proportions in the medial habenula of guinea-pig, rat and gerbil.

Electrophysiological recordings were made from neurones of the medial habenula (Mhb) in brain slices obtained from guinea-pig, rat and gerbil brain. The selective NK3 agonist, senktide, was used to determine the relative number of NK3-sensitive-neurones in the Mhb of each species. The proportion of neurones responding to NK1 (Sar9Met(O2)11SP) and NK2 (beta Ala8NKA(4-10) agonists was also assessed. All (65/65) of the guinea-pig Mhb neurones tested were excited by the NK3 agonist, but NK1 and NK2 agonists were without effect. NK3 responses in the guinea-pig were not altered by the presence of a selective NK1 antagonist, CP-99,994. NK1, NK2 and NK3 agonists were without effect on Mhb neurones from gerbil brain slices. In agreement with findings from previous studies, a population of rat Mhb neurones responded to NK1 or NK3 agonists alone or were excited by both. These data show that there is a difference in both the number of NK-sensitive neurones and the type of NK response found in the medial habenula of the three species. The high sensitivity to an NK3 agonist, combined with the apparent lack of NK1 and NK2 responses in the guinea-pig Mhb makes this preparation ideal for studies of central NK3-mediated events.

Benzodiazepine/cholecystokinin interactions at functional CCK receptors in rat brain.

1. The effects of benzodiazepines on cholecystokinin (CCK) responses produced following activation of CCKB receptors by pentagastrin in the ventromedial hypothalamus (VMH) or CCKA receptors by CCK-8S in the dorsal raphe of the rat brain in vitro have been investigated. 2. The benzodiazepine agonist, flurazepam, at high concentrations, blocked pentagastrin-induced excitations in the rat VMH yielding an equilibrium constant (Ke) value of 12.5 microM. 3. In the rat dorsal raphe, where activation of CCKA receptors leads to neuronal depolarization, flurazepam also produced a weak block of the CCK response. 4. Flurazepam blocked CCK responses but not carbachol-induced excitations of VMH neurones. The inhibition of CCK responses by flurazepam was not blocked by the benzodiazepine antagonist, flumazenil. 5. These data suggest that flurazepam is a weak antagonist at central CCKB receptors. 6. At central CCKA receptors, flurazepam blocked CCK-8S responses but the inhibition was not competitive, with a reduction in the peak CCK-8S obtainable in the presence of flurazepam. These results suggest that flurazepam acts at a site other than the CCKA receptor itself to block CCK responses in the dorsal raphe.

Effects of cholecystokinin and related peptides on neuronal activity in the ventromedial nucleus of the rat hypothalamus.

1. An investigation into the effects of cholecystokinin octapeptide (CCK-8S) and its pentapeptide analogue, pentagastrin, on neurones located in ventromedial nuclei of rat hypothalamic slices maintained in vitro has been undertaken. 2. CCK-8S (0.01-1.0 microM) applied in the perfusion medium produced a concentration-dependent increase in firing rate. This effect could be mimicked by pentagastrin and was selectively blocked by L-364,718, a potent peripheral CCK receptor antagonist that has been shown to possess micromolar affinity for central CCK receptors. 3. Intracellular recordings from ventromedial nucleus neurons revealed two distinct populations with comparable resting membrane parameters but differing neuronal activity. One group fired tetrodotoxin (TTX)-sensitive action potentials spontaneously at resting membrane potential whilst the second group fired action potentials only on injection of depolarizing current and were otherwise silent. 4. Application of CCK-8S or pentagastrin to spontaneously active neurones produced a small depolarization concomitant with an increase in action potential firing rate but the peptides had no effect on membrane properties of 'silent' neurones. 5. These data suggest the existence of at least two populations of neurones in the ventromedial hypothalamus, only one of which is excited by CCK-8S and pentagastrin.

Effect of chronic ethanol treatment in vivo on excitability in mouse cortical neurones in vitro.

1. The effects of cessation of chronic ethanol ingestion on seizure activity in vivo and on the characteristics of the evoked synaptic potentials in cortical neurones in vitro have been investigated in mice. Withdrawal from chronic ethanol treatment increased handling seizure ratings in mice between 4 and 16 h post-withdrawal. This ethanol-induced increase in seizure rating was unaffected by carbamazepine (30 mg kg(-1)) but significantly reduced at a higher concentration (130 mg kg(-1)). 2. Intracellular recordings were made from cortical layer II neurones in vitro from control mice and from mice following chronic ethanol ingestion. Evoked synaptic potentials were generated in these neurones through intralaminar stimulation. 3. Neurones from control mice displayed an evoked potential consisting of a fast excitatory postsynaptic potential (e.p.s.p.) mediated by AMPA-type glutamate receptors and an inhibitory postsynaptic potential (i.p.s.p.) mediated via GABA(A) receptors. Application of pentylenetetrazole (PTZ) or bicuculline onto these neurones inhibited the i.p.s.p., caused a large increase in both the amplitude and duration of the e.p.s.p. and initiated spontaneous excitatory activity. The resulting large evoked e.p.s.p. was mediated via both NMDA- and AMPA-type glutamate receptors. 4. Most neurones (77%) from ethanol treated mice displayed an evoked potential which comprised a large e.p.s.p. and no i.p.s.p. The e.p.s.p. consisted of several distinct components and in addition these neurones displayed spontaneous paroxysmal depolarizing shifts. This multi-component e.p.s.p. was mediated through both NMDA- and AMPA-type glutamate receptors. A population (23%) of neurones from ethanol treated mice exhibited evoked potentials which possessed both inhibitory and excitatory components and these neurones were effectively identical to those obtained from control mice. 5. Carbamazepine reduced the duration of the e.p.s.p. in neurones from ethanol treated mice and in PTZ-treated control neurones. 6. Prolonged ethanol ingestion is known to create a neurochemical imbalance in cortical neurones resulting in abnormal neurotransmission. The present study highlights the functional consequences that arise as a result of these neurochemical changes leading to over-excitation of neurones and pronounced epileptiform activity.

Tolbutamide excites rat glucoreceptive ventromedial hypothalamic neurones by indirect inhibition of ATP-K+ channels.

1. The sulphonylureas, tolbutamide (0.1-10 mM) and glibenclamide (0.1-100 microM) shown not to inhibit ATP-K+ channel currents when applied to inside-out membrane patches excised from rat cultured cerebral cortex or freshly-dispersed ventromedial hypothalamic nucleus (VMHN) neurones. 2. Saturable binding sites for [3H]-glibenclamide, with similar affinity constants are present in rat cerebral cortex and hypothalamic membranes. The density of binding sites was lower in the hypothalamus than cortex. 3. Intracellular recordings from glucoreceptive VMHN neurones in hypothalamic slices were obtained. In the absence of glucose, tolbutamide (0.1 mM) depolarized these cells, increased membrane resistance and elicited action potentials. 4. Tolbutamide (0.1 mM) inhibited ATP-K+ channel currents and induced action current activity in cell-attached recordings from glucoreceptive VMHN neurones. 5. Glibenclamide (10-500 nM) had no effect per se on glucoreceptive VMHN neurones but did antagonize the actions of tolbutamide. 6. It is concluded that the hypothalamic (and perhaps cortical) sulphonylurea receptors are not directly coupled to ATP-K+ channels.

Pharmacology of a cholecystokinin receptor on 5-hydroxytryptamine neurones in the dorsal raphe of the rat brain.

1. The effect of bath application of sulphated cholecystokinin octapeptide (CCK-8) was studied on neurones in slices containing rat raphe nucleus. 2. Intracellular recordings were made from neurones in the dorsal raphe nucleus. Some of the neurones with the characteristics of 5-hydroxytryptamine (5-HT)-containing cells which were inhibited by 5-HT and excited by noradrenaline were excited by cholecystokinin. The response to cholecystokinin was dose-dependent over the range 10 to 1000 nM. 3. The response to CCK-8 persisted in the presence of tetrodotoxin. Either reduction of extracellular calcium or addition of 25 mM magnesium did not block the CCK response, suggesting it was mediated by receptors located on the membrane of the raphe neurones. 4. The agonist and antagonist specificity of the CCK response was determined. The CCKB selective agonist, pentagastrin, was inactive when applied at concentrations up to 10 microM. the CCKA receptor antagonist L-364,718 (1 to 100 nM) blocked the response to cholecystokinin. Much higher (1-10 microM) concentrations of the CCKB receptor antagonist L-365,260 were required for inhibition of the CCK response. 5. These data support the existence of a CCK receptor, located on raphe neurones in the rat, with a pharmacological profile very similar to that described for the CCKA type.

Lack of effect of potassium channel openers on ATP-modulated potassium channels recorded from rat ventromedial hypothalamic neurones.

1. Single neuronal cells were freshly isolated from the ventromedial hypothalamic nuclei (VMHN) of the rat brain. Currents through ATP-modulated and large conductance (160 and 250 pS) calcium-activated potassium channels were recorded by the cell-attached and excised inside-out patch techniques. 2. BRL38227 (lemakalim; 30-90 microM) applied to the superfusing medium produced no change in firing rate of isolated glucose-receptive VMHN neurones in cell-attached recordings. 3. BRL38227, at concentrations of between 30-100 microM applied to the intracellular (cytoplasmic) aspect of inside-out patches, had no effect on the activity of ATP-sensitive K+ channels in the absence of ATP or in the presence of a sub-maximal inhibitory concentration (3 mM) of ATP. Cromakalim, pinacidil, minoxidil sulphate and diazoxide also produced no effect under these conditions. 4. The potassium channel openers (KCO's) were tested on ATP-activated potassium channels recorded from a further subpopulation of VMHN neurones. Application of BRL38227 (up to and including 100 microM) to this channel in inside-out patches either in the absence of ATP or when activated by 5 mM ATP had no effect on channel activity. Identical results were obtained with cromakalim and pinacidil. 5. BRL38227 had no effect on either of the large conductance (250 pS and 160 pS) calcium-activated potassium channels in VMHN neurones. 6. Intracellular recordings were made from glucose-receptive VMHN neurones in rat brain slices. Cromakalim (50 microM) or diazoxide (60 microM) did not alter the firing rate or passive membrane properties of these neurones demonstrated to be sensitive to tolbutamide (0.1 mM). 7. These results show that the KCO's tested in this study have no effect either on VMHN neurones contained in brain slices or on the activity of any of the ATP-modulated potassium channels under isolated patch conditions associated with these neurones.

Actions of the GABAB agonist, (-)-baclofen, on neurones in deep dorsal horn of the rat spinal cord in vitro.

1. The electrophysiological actions of the GABAB agonist, (-)-baclofen, on deep dorsal horn neurones were studied using an in vitro preparation of the spinal cord of 9-16 day old rat. 2. On all neurones tested, (-)-baclofen (100 nM-30 microM) had a hyperpolarizing action which was associated with a reduction in apparent membrane input resistance. The increase in membrane conductance was dose-dependent and had a Hill coefficient of 1.0. 3. The (-)-baclofen-activated hyperpolarization persisted in the presence of bicuculline (50 microM) and Mg2+ (20 mM). 4. The reversal potential of the hyperpolarizing event was estimated at 102 mV and was made less negative by increasing the external concentration of potassium ions. 5. Over the same concentration range, (-)-baclofen also depressed the polysynaptic composite excitatory postsynaptic potentials (e.p.s.ps) evoked in these neurones by electrical stimulation of the dorsal root entry zone. 6. The potassium channel blockers caesium, applied intracellularly, and barium, applied extracellularly, depressed the postsynaptic response to baclofen but not its effect on e.p.s.ps. 7. We propose that (-)-baclofen has more than one mechanism of action in spinal dorsal horn: a postsynaptic action mediated via an increase in potassium conductance and a presynaptic action that is not associated with potassium channels and may be mediated via calcium channels. Since previous studies have demonstrated little effect of (-)-baclofen on transmitter release in spinal cord, it is possible that the postsynaptic hyperpolarizing action of (-)-baclofen may account for its clinical potency as an anti-spastic agent.

Evaluation of a series of novel CCKB antagonists using a functional assay in the rat central nervous system.

1. Electrophysiological recordings from rat ventromedial hypothalamus (VMH) in vitro have been used to compare the effects of novel chemical entities on CCKB receptor activation in the rat central nervous system. 2. Twenty compounds from three different chemical series were evaluated for their ability to reduce pentagastrin-induced increases in action potential firing rate. 3. All twenty compounds studies were found to be CCKB antagonists, with equilibrium constants spanning a concentration-range of several orders of magnitude. The rank order for their ability to block pentagastrin responses correlated well with values obtained for their relative affinities for the mouse cortex CCKB binding site. 4. It is concluded that the VMH preparation provides a good functional correlate to binding assays in the rodent central nervous system for a structurally diverse series of CCKB antagonists.

Effects of cholecystokinin and pentagastrin on rat hippocampal neurones maintained in vitro.

Extracellular and intracellular recordings from CA1 neurones of rat hippocampal slices were undertaken to assess the relative potencies of cholecystokinin fragments. The CCK peptides displayed a large variability in their effects on extracellularly recorded population spikes. Intracellular recordings from CA1 neurones revealed a more consistent excitant action of these compounds. The C-terminal octapeptide CCK-8S, the tetrapeptide CCK-4 and pentagastrin were all found to be agonists when applied to hippocampal CA1 neurones maintained in vitro. Repeated application of the peptide fragments to the same cell resulted in a loss of activity. Neurones pre-treated with a CCK peptide showed no response to an application of a second, different, CCK fragment indicative of receptor cross-desensitization. Depolarisations induced by the excitatory amino acid L-glutamate remained unaffected by peptide application. These data suggest that the CCK fragments are agonists at rat CA1 neurones and share a common mode of action distinct from that of the excitatory amino acid L-glutamate.

Rat hippocampal slices 'in vitro' display spontaneous epileptiform activity following long-term organotypic culture.

Organotypic cultures of rat hippocampal slices were maintained for periods of up to 12 weeks in vitro. Cultures adopted a two-dimensional architecture whilst retaining the subfields characteristic of intact hippocampal slices. Coventional intracellular onset of spontaneous long-lasting epileptiform activity. Epileptiform activity characteristic of both interictal and ictal events (paroxysmal depolarising shifts, tonic/clonic phases and afterdischarges) was observed in the absence of pharmacological manipulation or of orthodromic stimulation. Epileptiform activity was abolished in the presence of high Mg2+ concentration or tetrodotoxin, agents known to block synaptic transmission. In addition, the frequency of epileptiform events was independent of membrane potential and the amplitude of the paroxysmal depolarising shift (PDS) displayed a near linear relationship with membrane potential. The PDS could be reversed at potentials approaching synaptic equilibrium potential. The N-methyl-D-aspartate (NMDA)-receptor antagonist DL-2-amino-5-phosphonovalerate (DL-APV) dose-dependently reduced both the amplitude and duration of the spontaneous paroxysmal shift, having no effect on the initiation of the event or the resting membrane parameters of the neurone. DL-APV also attenuated a late component of the synaptically evoked excitatory postsynaptic potentials (epsp) not observed in non-epileptiform neurones. Application of GABAA receptor antagonists bicuculline or picrotoxin converted interictal events to ictus. In the presence of these agents, ictal events were up to 90 s in duration. These results suggest that long-term culturing of hippocampal explants leads to an alteration in the balance of excitatory and inhibitory synaptic activity. This allows the expression of an excitatory amino acid depolarisation acting through NMDA receptors which contributes to the generation and maintenance of spontaneous epileptiform activity which is synaptic in origin.

Troglitazone inhibits type 2KATP channel activity and depolarises tolbutamide-sensitive neurones in the rat ventromedial hypothalamus.

Patch-clamp recording techniques were used to examine the effect of troglitazone upon visually identified neurones contained in slices from the rat ventromedial hypothalamus. Bath application of 1-50 microM troglitazone depolarised tolbutamide-sensitive neurones in a poorly reversible manner. In outside-out patches, troglitazone inhibited KATP channel activity when added to the bath solution. It is concluded that troglitazone is a novel modulator of the type 2 KATP channel.

Postsynaptic block of a glutamatergic synapse by low molecular weight fractions of spider venom.

Fractions of low molecular weight (ca. 600-1000 dalton) isolated by high performance liquid chromatography (HPLC) and thin layer chromatography (TLC) from the venoms of the New-World spiders, Argiope trifasciata and Araneus gemma block transmission at glutamatergic nerve-muscle junctions in the locust, Schistocerca gregaria. These fractions are probably small peptides containing phenolic or indolic residues. Their effects on the neurally evoked twitch contraction, the junctional potential to glutamate ionophoresis and the voltage-clamped excitatory postsynaptic current of locust muscle indicate uncompetitive antagonism of the glutamate receptor channel complex through open channel block. In view of their slow reversibility of action they should make useful tools for future biochemical studies of locust glutamate receptors.

The action of natural and synthetic isomers of quisqualic acid at a well-defined glutamatergic synapse.

L-, D- and DL-quisqualic acid have been synthesized and their activities at the glutamatergic locust nerve-muscle junction have been compared with those of natural quisqualic acid and with glutamic acid. Two well-characterised locust nerve-muscle preparations were used in these studies, the retractor unguis nerve-muscle system and the extensor tibiae nerve-muscle system. The amino acids were tested on the whole nerve-muscle system in the former, when reduction in neurally evoked twitch contraction amplitude was the measured parameter, and by ionophoretic application to single excitatory junctional sites in the latter, when amplitude of junctional depolarization was the measured parameter. Synthetic L-quisqualic acid exhibited identical potency to its natural counterpart. However, D-quisqualic acid and DL-quisqualic acid were more active than expected from the known stereospecificity of this glutamatergic system towards D- and L-glutamic acid. The hydantoin analogue of quisqualic acid was inactive. X-ray crystallographic analysis of L-quisqualic acid and the hydantoin analogue showed that the ring junction in the former is pyramidal whereas in the latter it is planar. This may account for the high potency of L-quisqualic acid on a receptor system which identifies a partially folded conformation of L-glutamic acid. A pyramidal configuration of D-quisqualic acid would allow either rapid interconversion between active and inactive configurations at its ring junction or adoption of a trigonal configuration in solution. Either interpretation could explain the unexpected potency of D-quisqualic acid.

Agonists selective for tachykinin NK1 and NK3 receptors excite subpopulations of neurons in the rat medial habenula nucleus in vitro.

The purpose of the present study was to determine the tachykinin receptor types present on neurones of the rat medial habenula nucleus. Extracellular recordings were made from spontaneously active medial habenula neurones in tissue slices of rat brain. The tachykinin analogues [Sar9,Met(O2)11]substance P, [beta-Ala8]neurokinin A-(4-10) and senktide were chosen for their potency and selectivity at NK1, NK2 and NK3 receptors, respectively. No neurone was observed to respond to [beta-Ala8]neurokinin A-(4-10). Neurones did respond to [Sar9,Met(O2)11]substance P and to senktide with a rapid and concentration-dependent increase in firing rate. Of the neurones where recordings were made, responses were obtained to one, both or neither of the agonists. These data suggest that receptors similar to NK1 and NK3, but not NK2 receptors, are differentially expressed on subpopulations of neurones of this nucleus.

Inhibition of KATP channel activity by troglitazone in CRI-G1 insulin-secreting cells.

Patch-clamp recording techniques were used to examine the effect of troglitazone on KATP channel activity in Cambridge rat insulinoma-G1 (CRI-G1) insulin-secreting cells. In both inside-out and outside-out patch recordings, bath application of troglitazone reduced KATP channel activity. This inhibition was independent of the membrane voltage and was poorly reversible. In whole-cell studies, troglitazone inhibited KATP channel currents with an IC50 of 697 +/- 92 nM and an associated Hill coefficient of 1.2 +/- 0.2. In current clamp recordings 10 microM troglitazone depolarised the CRI-G1 cell membrane by 36.8 +/- 3.9 mV with a concomitant decrease in membrane conductance. However, in contrast to the rapid depolarisation produced by tolbutamide, the effects of troglitazone developed more slowly, usually taking 15-20 min to develop.