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.

Involvement of the central tachykinin NK1 receptor during maintenance of mechanical hypersensitivity induced by diabetes in the rat.

Our study examines the role of central and peripheral neurokinin1 (NK1) receptors in diabetes-induced mechanical hypersensitivity. Glycine, N, N-dimethyl-, 2-[[2-[[(2-benzofuranylmethoxy)carbonyl]amino]-3-(1H-indol-3-yl)-2 -me thyl-1-oxopropyl] amino]-2-phenylethylester, bisulfate, [R-(R*,R*)] (PD 156982) is a selective NK1 receptor antagonist with nanomolar affinity for the human (IC50 = 1.4 nM) and guinea pig (IC50 = 9.6 nM) NK1 receptors. However, it has approximately two orders of magnitude lower affinity for the rodent NK1 receptor (IC50 = 820 nM). In electrophysiological studies, PD 156982 inhibited NK1 receptor-mediated responses in the guinea pig locus ceruleus, in a competitive manner, with an equilibrium constant of 13.9 nM. The intracerebroventricular (10-100 microg/animal) but not systemic administration of PD 156982 (1-100 mg/kg, s.c.) blocked the [Sar9, Met(O2)11] substance P-induced gerbil foot tapping response. This indicates that PD 156982 is unable to penetrate into the central nervous system. However, PD 156982 (10-100 mg/kg, s.c.) blocked the mechanical hypersensitivity induced by administration of substance P into the plantar surface of a rat paw. This suggests that PD 156982 can effectively antagonize peripheral NK1 receptors in vivo. The chemically related compound carbamic acid, [1-(1H-indol-3-ylmethyl)-1-methyl-2-oxo-2-[(1-phenylethyl)amino]et hyl ]-, 2-benzofuranylmethyl ester, [R-(R*,S*)] (CI-1021) is also a selective NK1 receptor antagonist but can penetrate into the central nervous system. PD 156982 (10-100 mg/kg, s.c.) failed to block streptozocin (75 mg/kg, i.p.) induced mechanical hypersensitivity. In contrast, CI-1021 dose-dependently (3-100 mg/kg, s.c.) blocked this hypersensitivity state with a minimum effective dose of 10 mg/kg. At these doses CI-1021 also antagonized mechanical hypersensitivity mediated by central NK1 but not NK2 receptors in the rat. It is suggested that the central NK1 receptor may play an important role in diabetes-induced hypersensitivity.

A summary of mechanistic hypotheses of gabapentin pharmacology.

Although the cellular mechanisms of pharmacological actions of gabapentin (Neurontin) remain incompletely described, several hypotheses have been proposed. It is possible that different mechanisms account for anticonvulsant, antinociceptive, anxiolytic and neuroprotective activity in animal models. Gabapentin is an amino acid, with a mechanism that differs from those of other anticonvulsant drugs such as phenytoin, carbamazepine or valproate. Radiotracer studies with [14C]gabapentin suggest that gabapentin is rapidly accessible to brain cell cytosol. Several hypotheses of cellular mechanisms have been proposed to explain the pharmacology of gabapentin: 1. Gabapentin crosses several membrane barriers in the body via a specific amino acid transporter (system L) and competes with leucine, isoleucine, valine and phenylalanine for transport. 2. Gabapentin increases the concentration and probably the rate of synthesis of GABA in brain, which may enhance non-vesicular GABA release during seizures. 3. Gabapentin binds with high affinity to a novel binding site in brain tissues that is associated with an auxiliary subunit of voltage-sensitive Ca2+ channels. Recent electrophysiology results suggest that gabapentin may modulate certain types of Ca2+ current. 4. Gabapentin reduces the release of several monoamine neurotransmitters. 5. Electrophysiology suggests that gabapentin inhibits voltage-activated Na+ channels, but other results contradict these findings. 6. Gabapentin increases serotonin concentrations in human whole blood, which may be relevant to neurobehavioral actions. 7. Gabapentin prevents neuronal death in several models including those designed to mimic amyotrophic lateral sclerosis (ALS). This may occur by inhibition of glutamate synthesis by branched-chain amino acid aminotransferase (BCAA-t).

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.

Characterization of the inward current induced by metabotropic glutamate receptor stimulation in rat ventromedial hypothalamic neurones.

1. Whole-cell patch clamp recordings were made from rat ventromedial hypothalamic neurones in slices of brain tissue in vitro. Bath application of 50 microM (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) depolarized all neurones tested by activation of an inward current of approximately 55 pA at -60 mV. 2. The inward current elicited by 1S,3R-ACPD was unaffected by K+ channel blockade with external Cs+, Ba2+ or TEA. However, the current was significantly reduced by replacement of the external NaCl with either Tris-HCl or LiCl. 3. The 1S,3R-ACPD-induced current was reduced by the heavy metal ions Ni2+ or La3+ and also by the Na(+)-Ca2+ exchange current inhibitor 3',4'-dichlorobenzamil. 4. The effects of 1S,3R-ACPD were mimicked by the group I metabotropic agonist 3,5-dihydroxyphenylglycine (DHPG) but not by the group III selective agonist, L-2-amino-4-phosphonobutanoate (L-AP4). Furthermore, the effects of 1S,3R-ACPD were inhibited by the metabotropic antagonists alpha-methyl-4-carboxyphenylglycine (MCPG) and 1-aminoindan-1,5-dicarboxylic acid (AIDA) but not by the presynaptic metabotropic receptor antagonists alpha-methyl-4-phosphonophenylglycine (MPPG) or alpha-methyl-4-tetrazolylphenylglycine (MTPG). 5. Photorelease of caged GDP beta S inside neurones irreversibly blocked the 1S,3R-ACPD-induced current whilst photolysis of caged GTP gamma S inside neurones irreversibly potentiated this current. 6. The PLC inhibitor U-73,122 significantly reduced the size of the inward current induced by 1S,3R-ACPD. This effect was not mimicked by the inactive analogue U-73,343. 7. Flash photolysis of the caged calcium chelator diazo-2 inside neurones diminished the response to 1S,3R-ACPD. 8. It is concluded that group I metabotropic glutamate receptors depolarize neurones in the VMH by activation of a Na(+)-Ca2+ exchange current through a G-protein coupled increase in intracellular Ca2+.

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.

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.

Potassium channel dysfunction in hypothalamic glucose-receptive neurones of obese Zucker rats.

1. We have shown, using intracellular and cell-attached recordings, that glucose-receptive (GR) neurones of obese Zucker rats exhibit abnormal electrophysiological responses to changes in extracellular glucose concentration, whereas GR neurones of lean Zucker and control rats respond normally. 2. In inside-out recordings from obese rat GR neurones it was shown that the 150 pS ATP-sensitive K+ (KATP) and the 160 pS calcium-activated K+ (KCa) channels were absent, whereas both were present in GR neurones of lean Zucker and control rats. 3. The potassium channel most frequently observed in inside-out patches from obese GR neurones was characterized by a conductance of 213 pS, was activated by raising internal calcium and inhibited by application of internal ATP. This channel (which we have termed Kfa) was not observed in lean or control rat GR neurones. 4. Tolbutamide (100 microM) was found to induce no effect or to elicit a small depolarization of obese rat GR neurones in the absence of glucose, in contrast to its clear excitatory actions on control or lean Zucker GR neurones. 5. Intracellular, cell-attached and inside-out recordings from obese rat non-GR neurones showed that there was no alteration in their membrane properties or firing characteristics or in the characteristics of the large-conductance calcium-activated K+ channel (KCa) present in these neurones as compared with lean and control rats. 6. It is concluded that the Kfa channel is specific to GR neurones of obese Zucker rats and that the presence of this channel coupled with the absence of KATP and KCa channels results in the abnormal glucose-sensing response of these neurones.

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.

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.

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.

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.

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.

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)

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.