SEN1500, a novel oral amyloid-ß aggregation inhibitor, attenuates brain pathology in a mouse model of Alzheimer's disease.

INTRODUCTION: Amyloid-ß (Aß) aggregation is thought to be a major pathogenic event underlying the neuropathology of Alzheimer's disease (AD). The development of new drugs inhibiting the Aß aggregation process is, therefore, important. SEN1500, an orally bioavailable and CNS-penetrant Aß aggregation inhibitor, has previously been shown to reduce spatial learning and memory deficits in an APP transgenic mouse model. To verify that the pharmacological properties of SEN1500 are not unique to this model, we investigated brain Aß pathology, neuroinflammation, as well as memory in a different mouse model of AD expressing the human amyloid precursor protein with Swedish and London mutations (APPSL). MATERIALS & METHODS: APPSL transgenic mice and non-transgenic littermates were treated with SEN1500 via food pellets from three months of age for four months. At the end of the treatment, animals were tested for memory deficits using the contextual fear conditioning test and brain tissue was analyzed for soluble and insoluble amyloid-ß1-38, -40, -42, ß-amyloid plaques, ß-sheet plaque cores, as well as for astrocytosis and activated microglia. RESULTS: SEN1500 treatment lowered insoluble Aß levels and ß-amyloid plaque load in the brain compared with control-treated APPSL mice. Activated microglia were significantly reduced in the cortex but not the hippocampus of SEN1500-treated APPSL mice. Memory deficits of APPSL mice could not be rescued by SEN1500. DISCUSSION: SEN1500 is not only able to reduce Aß pathology and activated microglia but also to improve learning and memory as previously shown, making SEN1500 a potential candidate for human AD treatment. This Aß aggregation inhibitor could be a promising therapeutic agent for the disease-modifying treatment of AD.

Aß oligomer toxicity inhibitor protects memory in models of synaptic toxicity.

BACKGROUND AND PURPOSE: Amyloid-ß (Aß) aggregation into synaptotoxic, prefibrillar oligomers is a major pathogenic event underlying the neuropathology of Alzheimer's disease (AD). The pharmacological and neuroprotective properties of a novel Aß aggregation inhibitor, SEN1269, were investigated on aggregation and cell viability and in test systems relevant to synaptic function and memory, using both synthetic Aß(1-42) and cell-derived Aß oligomers. EXPERIMENTAL APPROACH: Surface plasmon resonance studies measured binding of SEN1269 to Aß(1-42) . Thioflavin-T fluorescence and MTT assays were used to measure its ability to block Aß(1-42) -induced aggregation and reduction in cell viability. In vitro and in vivo long-term potentiation (LTP) experiments measured the effect of SEN1269 on deficits induced by synthetic Aß(1-42) and cell-derived Aß oligomers. Following i.c.v. administration of the latter, a complex (alternating-lever cyclic ratio) schedule of operant responding measured effects on memory in freely moving rats. KEY RESULTS: SEN1269 demonstrated direct binding to monomeric Aß(1-42) , produced a concentration-related blockade of Aß(1-42) aggregation and protected neuronal cell lines exposed to Aß(1-42) . In vitro, SEN1269 alleviated deficits in hippocampal LTP induced by Aß(1-42) and cell-derived Aß oligomers. In vivo, SEN1269 reduced the deficits in LTP and memory induced by i.c.v. administration of cell-derived Aß oligomers. CONCLUSIONS AND IMPLICATIONS: SEN1269 protected cells exposed to Aß(1-42) , displayed central activity with respect to reducing Aß-induced neurotoxicity and was neuroprotective in electrophysiological and behavioural models of memory relevant to Aß-induced neurodegeneration. It represents a promising lead for designing inhibitors of Aß-mediated synaptic toxicity as potential neuroprotective agents for treating AD.

Exploiting high-throughput ion channel screening technologies in integrated drug discovery.

Ion channels are increasingly being implicated in disease. Although existing drugs that modulate channel function currently represent a key class of pharmaceutical agents, future ion channel drugs could help to treat an even wider variety of diseases. Despite their disease relevance, ion channels remain largely under exploited as drug targets, chiefly resulting from the absence of screening technologies that provide the throughput and quality of data required to support medicinal chemistry. Although some technical challenges still lie ahead, this historic bottleneck in drug discovery is now being bypassed by newer technologies that can be fully integrated into the early stages of drug discovery and will allow the discovery of novel therapeutic agents. Sequencing the human genome has greatly added to the number of potential drug targets but selecting suitable ion channels for drug discovery research should be based on the potential therapeutic relevance of the channel and not just the availability of suitable screens. Currently, ion channel drug discovery is focused on the need to identify compounds that can provide tractable starting points for medicinal chemistry. Advances in laboratory automation have brought significant opportunities to increase screening throughput for ion channel assays but careful assay configuration to model drug-target interactions in a physiological manner remains an essential consideration. Ion channel screening platforms are described in this review to provide some insight into the variety of technologies available for screening, together with some of their inherent advantages and limitations.

Neuropeptides in drug research.

Neuropeptides have been a subject of considerable interest in the pharmaceutical industry over the last 20 years or more. Many drug discovery teams have contributed to our understanding of neuropeptide biology but no significant drugs that act selectively upon neuropeptide receptors have yet emerged from the clinic. There are, however, a plethora of clinically useful drugs that act at other classes of neurotransmitter and neuromodulator receptors, many of them discovered over the last 20 years. Nevertheless, we think that the future for the discovery of novel drugs acting at neuropeptide receptors looks bright for two reasons: (1) there has been a substantial increase in our understanding of the function of neuropeptides; and (2) high-throughput screening (HTS) against neuropeptide receptors has now begun to yield many interesting drug-like molecules, rather than peptides, that have the potential to become clinically useful drugs. The objective of this review is to summarise our current understanding of specific areas of neuropeptide biology and pharmacology in the CNS as well as the PNS. We will also speculate on where we think the new generation of neuropeptide agonists and antagonists could emerge from the clinic.

The pharmacology of apoptosis.

Apoptosis is an area of intense scientific interest, which encompasses the study of and triggers mechanisms involved in mediating the cell biology of programmed cell death. A number of low molecular weight compounds have been used to inhibit or enhance this fundamental cellular process and so apoptosis has now become amenable to pharmacological manipulation. In this review Ross Kinloch, Mark Treherne, Mike Furness and Iradj Hajimohamadreza will focus on the current literature describing the pharmacology of apoptosis, with particular reference to the therapeutic potential that could arise from the development of pro- and anti-apoptotic drugs. The pivotal role of apoptosis in such diverse pathological processes as tumour growth, the immune response and neurodegeneration suggests that an understanding of how apoptosis can be regulated by drugs will become increasingly important to the pharmaceutical industry.

The application of high-throughput screening to novel lead discovery.

The ability to discover new lead compounds for novel therapeutic targets is a pivotal step in drug discovery programmes. High-throughput screening (HTS) utilises a number of platforms for the rapid screening of novel targets to accelerate this process. Key issues in HTS include assay configuration and the ability of a high-throughput screen to predict drug-target interactions accurately. This review highlights a number of issues in the HTS process and describes three key target areas that are likely to be sources of novel, therapeutically important drugs. Particular emphasis is placed on the mechanistic basis of drug-target interactions that are of prime importance in the design of HTS approaches. Critical aspects of information management related to HTS are summarised.

Activation by intracellular ATP of a potassium channel in neurones from rat basomedial hypothalamus.

1. Cell-attached recordings from isolated glucose-sensitive hypothalamic neurones show that on removal of extracellular glucose there is an increased action current frequency concomitant with decreased single-channel activity. Conversely activation of single K+ channels was observed when extracellular glucose was increased. Isolation of membrane patches into the inside-out configuration following cell-attached recording demonstrated the presence of an ATP-activated K+ channel. 2. The ATP-activated K+ channel was characterized by a mean single-channel conductance of 132 pS in symmetrical 140 mM KCl solutions. Single-channel open-state probability (Po) was not calcium dependent, and the presence of calcium did not prevent activation of the channel by ATP. 3. Activation of the channel by ATP was concentration dependent and the Po of the ATP-activated channel was unaffected by membrane voltage, regardless of the degree of activation elicited by ATP. 4. Open and closed time histograms were constructed from inside-out and cell-attached recordings and were consistent with a single open and two closed states. Channel openings were grouped in bursts. Application of ATP, in isolated patches, and glucose, in cell-attached patches, increased the burst duration and number of bursts per second and decreased the slow closed-state time constant. In neither case was there a significant change in the fast closed-state time constant nor the open-state time constant. 5. The non-hydrolysable ATP analogue adenylylimidodiphosphate (AMP(PNP)) and 'Mg2(+)-free' ATP produced little change in the Po of the ATP-activated K+ channel when applied to the intracellular surface of excised patches. These results suggest that activation of this channel is via an enzymic mechanism. 6. ADP, GTP and GDP also activated the channel in a Mg(2+)-dependent manner. ADP and ATP activated the channel in an additive manner and neither GTP nor GDP inhibited channel activity induced by ATP. 7. It is concluded that the ATP-activated K+ channel observed in isolated inside-out patches from hypothalamic neurones is the same as the channel activated by an increase in the concentration of extracellular glucose in cell-attached recordings from glucose-sensitive neurones.

Development of spinal cord in the isolated CNS of a neonatal mammal (the opossum Monodelphis domestica) maintained in longterm culture.

The CNS of the newly born opossum removed in its entirety survives and maintains its electrical excitability in suitable culture media for up to ten days at 25 degrees C. The structure of the developing neonatal spinal cord has been studied in the intact animal and in the cultured CNS. The differentiation and survival of individual cells and subcellular structures were followed at the light and electron microscopic level. The expression of cell markers in neuronal and glial cells was studied immunocytochemically using commercially available antibodies. Both mono- and polyclonal antibodies raised against antigens from several other species cross-reacted with Monodelphis antigens. The spinal cord of preparations removed from three-day-old-animals showed many neuron specific enolase-positive large neurons in the ventral horn as well as vimentin- and glial fibrillary acidic protein-positive radial glial cells and numerous small diameter unmyelinated axons, abundant dendrites and synaptic structures. From post natal day 5 to post natal day 8 continued differentiation of neurons and differentiation of radial glial cells into astrocytes were apparent. Radial glial fibres and astrocytes reacted positively to antibodies against glial fibrillary acidic protein. Myelin had not appeared at 8 days. A comparison of material obtained from postnatal day 3-postnatal day 4 preparations fixed immediately after dissection and from postnatal day 3-postnatal day 4 preparations fixed after 5 days in culture showed growth with continued mitotic activity of the neuroepithelial cells and further neuronal and glial maturation in the spinal cord especially in the more rostral end. In successful experiments in vitro, the preservation of individual cells, organelles, membranes and synapses was similar in the freshly dissected and cultured preparations apart from a distinct loss of the youngest and some of the oldest neurons in the spinal cord. Also the main fibre tracts (dorsal, lateral and ventromedial funiculus) survived. Virtually all preparations that had not been damaged or injured showed these results. Possible reasons for the death or survival of individual neuronal or glial cell populations in these preparations are discussed.

Development of connections by axons growing through injured spinal cord of neonatal opossum in culture.

The ability of neurites to grow through a lesion and form synaptic connections has been analyzed in a developing mammalian spinal cord in vitro. After isolation of the entire central nervous system (CNS) of the newly born South American opossum (Monodelphis domestica) the spinal cord was crushed. Outgrowth through and beyond the lesion was observed in living preparations for 2-5 days by staining axons with carbocyanine dyes. The structure of the acute crush and the growing neurites was examined by light and electron microscopy in tissue fixed immediately after the crush had been made. All axons had been severed and the site was filled with debris and amorphous vesicular structures. By 3 days after injury, numerous labelled neurites had grown into the lesion; by 4 days, many had extended several millimetres beyond it. At this time normal axonal profiles were apparent in electron micrographs of the crush site. Although fewer axons grew across the lesion than had been severed by the crush, the amplitudes of compound action potential volleys conducted across the crush in injured preparations were comparable with those recorded from uninjured spinal cords. Physiological experiments made with raised concentrations of extracellular magnesium in the culture fluid indicated that growing axons had formed synaptic connections. Thus, delayed major peaks of the response were abolished while the small component corresponding to through conduction remained unaffected by magnesium. These experiments demonstrate the development of synaptic interactions by the growing neurites and confirm the far greater powers of repair in neonatal mammals compared to adults. They set the stage for comparing molecular mechanisms involved in development and regeneration of the mammalian CNS.

Growth of axons through a lesion in the intact CNS of fetal rat maintained in long-term culture.

The ability of neurons in the central nervous system (CNS) to grow through a lesion and restore conduction has been analysed in developing spinal cord in vitro. The preparation consists of the entire CNS of embryonic rat, isolated and maintained in culture. Conduction of electrical activity and normal morphological appearance (light microscopical and electron microscopical) were maintained in the spinal cord of such preparations for up to 7 d in culture. A complete transverse crush of the spinal cord abolished all conduction for 2 d. After 3-5 d, clear recovery had occurred: electrical conduction across the crush was comparable with that in uninjured preparations. Furthermore, the spinal cord had largely regained its gross normal appearance at the crush site. Axons stained in vivo by carbocyanine dyes had, by 5 d, grown in profusion through the lesion and several millimetres beyond it. These experiments, like those made in neonatal opossum (Treherne et al. 1992) demonstrate that central neurons of immature mammals, unlike those in adults, can respond to injury by rapid and extensive outgrowth of nerve fibres in the absence of peripheral nerve bridges or antibodies that neutralize inhibitory factors. However, unlike the opossum, in which outgrowth occurred at 24 degrees C, although there was prolonged survival of rat spinal cords at this temperature, outgrowth of axons across the lesion required a temperature of 29 degrees C. With rapid and reliable regeneration in vitro it becomes practicable to assay the effects of molecules that promote or inhibit restoration of functional connections.

Extracellular cations modulate the ATP sensitivity of ATP-K+ channels in rat ventromedial hypothalamic neurons.

ATP-sensitive K+ (ATP-K+) channels underlie the glucose-sensing nature of pancreatic beta-cells by way of their inhibition by intracellular ATP. Recently it has been proposed that ATP-K+ channels have a similar function in certain hypothalamic neurons that become excitable in raised concentrations of extracellular glucose. The aim of this study was to assess the ATP sensitivity of ATP-K+ channels in inside-out membrane patches excised from glucose-sensing neurons that were acutely isolated from the ventromedial nucleus of rat hypothalamus. ATP-K+ channels were less sensitive to ATP in neurons than in other tissues. Moreover, the sensitivity of neuronal ATP-K+ channels to inhibition by intracellular ATP was modulated by extracellular cations. Under physiological ionic gradients (i.e. high extracellular Na+ and low K+), intracellular ATP produced a concentration-dependent inhibition of channel activity, with a half-maximal inhibition (Ki) of 2.32 mM. A non-hydrolysable analogue of ATP, AMP(PNP), was similarly effective. In symmetrical K+ (i.e. no extracellular sodium), channel activity was tenfold more sensitive to ATP (Ki of 0.21 mM). A parallel study on ATP-K+ channels from an insulin-secreting beta-cell line (CRI-G1) showed that, in contrast to the neuronal data, extracellular cations had no effect on the ATP sensitivity of the channel.

Restoration of conduction and growth of axons through injured spinal cord of neonatal opossum in culture.

The ability of neurons in the central nervous system to grow through a lesion and restore conduction has been analyzed in a developing spinal cord. The preparation consists of the entire central nervous system of the newly born opossum (Monodelphis domestica), isolated and maintained in culture. Cell division, cell migration, and reflexes are maintained in such preparations for up to 8 days in culture. In the present experiments, massive lesions were produced by crushing the spinal cord, which abolished all conduction for a day. By 2-3 days after injury, electrical conduction across the crush could be observed. After 4-5 days, clear recovery had occurred: the amplitude of the conducted volley was comparable to that in acute preparations. In such preparations, the spinal cord had largely regained its normal appearance at the crush site. Axons stained by carbocyanine dyes or horseradish peroxidase had, by 4 days, grown in profusion through the lesion and several millimeters beyond it. These experiments demonstrate that neurons in the central nervous system of newly born mammals, unlike those in adults, can respond to injury by rapid and extensive outgrowth in the absence of peripheral nerve bridges or antibodies that neutralize inhibitory factors of myelin. With rapid and reliable regeneration occurring in vitro, it becomes practicable to assay the effects of molecules that promote or inhibit the restoration of functional connections.

The intact central nervous system of the newborn opossum in long-term culture: fine structure and GABA-mediated inhibition of electrical activity.

1. The entire central nervous system (CNS) of the newly born, South American opossum (Monodelphis domestica) was isolated and maintained in basal medium, Eagle's (BME) with 0.2% foetal calf serum and antibiotics. Isolated CNS preparations remained electrically excitable for up to 10 days. The fine structure of the spinal cord was normal after 5 days in culture: axons, synapses, dendrites and glia were virtually unchanged. Signs of degeneration were evident only in dorsal areas of the spinal cord, which had been denervated by removal of the dorsal root ganglia during dissection. 2. Amino acid transmitters such as glycine, glutamate, N-methyl-D-aspartate (NMDA) and gamma-aminobutyric acid (GABA), applied to the bathing fluid, rapidly and reversibly inhibited synaptic transmission in cervical segments of the spinal cord. GABA (10-100 mumol l-1) produced a dose-dependent reduction in the magnitude of ventral root responses evoked by dorsal root stimulation. GABA also inhibited synaptically activated compound action potentials produced by spinal cord stimulation. Dose-response curves for GABA obtained in different preparations were highly reproducible. 3. Both GABAA and GABAB receptors were reversibly activated by selective agonists and inhibited by specific antagonists. The actions of GABA were potentiated by benzodiazepines, competitively antagonised by bicuculline (a selective GABAA antagonist) and mimicked by muscimol (a GABAA agonist). Baclofen (a specific GABAB agonist) also inhibited electrical activity and was competitively antagonised by the GABAB antagonist, CGP 35348. 4. After 5 days of culture in BME or minimal essential medium (MEM), GABA dose-response curves were unchanged from those observed immediately after removal of the CNS. The inhibitory potency of baclofen was also unaffected by culture in BME. By contrast, after 5 days of culture in MEM, baclofen no longer inhibited electrical activity. This difference between BME and MEM could be attributed to the higher content of L-histidine in MEM. Thus, addition of 150 mumol l-1 L-histidine to BME produced similar results to culture in MEM: the inhibitory action of baclofen was virtually abolished after 3-5 days. L-Histidine had no effect on freshly dissected preparations. Chronic application of L-histidine did not affect glycine or glutamate responses after 5 days. Addition of D-histidine or other amino acids, such as arginine, to BME did not abolish the responses to baclofen. 5. These results show that the isolated CNS of the newborn opossum survives well in long-term culture and that it provides a useful preparation to study receptor development and plasticity of an intact mammalian CNS in vitro.(ABSTRACT TRUNCATED AT 400 WORDS)

Regulation of GABAB receptors by histamine and neuronal activity in the isolated spinal cord of neonatal opossum in culture.

The aim of these experiments has been to analyse the properties of receptors for the transmitter gamma-aminobutyric acid (GABA) in developing mammalian nervous system. Changes in responses of GABAB receptors have been measured after alterations of the chemical environment and the level of electrical activity. We have previously shown that when the central nervous system (CNS) of the new-born opossum, Monodelphis domestica, is cultured for three to five days in the presence of histidine, inhibition by baclofen, a GABAB agonist, disappears (Stewart et al. 1991). We have now investigated whether histidine acts indirectly by way of conversion to histamine. As with histidine, culture with 150 microM histamine for five days virtually abolished the inhibition by baclofen. The effects of histidine, as well as histamine, were blocked by mepyramine, a histamine H1-receptor antagonist, and by ranitidine, an H2-antagonist. Tetrodotoxin (TTX), which blocks all electrical activity, protected preparations from the action of histidine but not histamine. Our results suggest that histidine is converted to histamine, which reduces the efficacy of GABAB agonists. We conclude that, in the developing mammalian CNS, transmitter levels and electrical activity can selectively influence the properties of receptors.

Inhibition by cations of antagonist binding to histamine H1-receptors: differential effect of sodium ions on the binding of two radioligands.

1. Measurements have been made of the inhibition by mono- and divalent cations of the binding of [3H]-(+)-N-methyl-4-methyldiphenhydramine ([3H]-QMDP) to histamine H1-receptors in homogenates of guinea-pig cerebellum. 2. The binding of [3H]-QMDP was inhibited by monovalent cations with an order of potency Li+ = Na+ greater than K+ greater than Cs+ = Rb+. The IC50 for Li+ was 39 mM, but that for K+ was 132 mM. Hill coefficients for inhibition curves for Li+ and Na+ were less than 1. 3. Divalent cations also inhibited the binding of [3H]-QMDP. The most potent cations examined were Hg2+, Cd2+ and Zn2+, with IC50 values of 5, 17 and 41 microM, respectively. Ca2+ and Mg2+ were relatively weak inhibitors (IC50 12 and 34 mM, respectively). The potency of Ni2+, Co2+ and Mn2+ was intermediate between these groups. Hill coefficients for inhibition curves for Hg2+, Cd2+ and Zn2+ were greater than 1, but Hill coefficients for the other cations were less than 1. 4. Both mono- and divalent cations also inhibited the binding of [3H]-mepyramine. The divalent cations were approximately equipotent in inhibiting the binding of [3H]-QMDP and [3H]-mepyramine. The same was true for Li+. However, Na+ was markedly more effective against [3H]-QMDP binding than against the binding of [3H]-mepyramine. 5. The effect of 40 mM Li+ on the parameters of binding of [3H]-mepyramine was to increase the best-fit value of the concentration giving half-maximal binding EC50, by approximately 2 fold without having any significant effect on the maximum amount of binding. Cd2+ (15 microM) caused both an increase in EC 0 and a decrease in Bmax (32 +/- 4% inhibition). Na+, 100 mm, had no significant effect on either EC50 or Bmax for [3H]-mepyramine binding.

The regional distribution of sulphonylurea binding sites in rat brain.

Sulphonylureas such as glibenclamide, which are used in the treatment of Type-2 diabetes, are inhibitors of ATP-sensitive potassium channels. These channels link cellular metabolism to membrane electrical activity and it is likely that they are closely associated with glibenclamide binding sites. Quantitative autoradiography was used to localize high-affinity [3H]glibenclamide binding sites in coronal sections of rat brain. The relative density of binding sites was found to correlate well with the relative capacity of sites determined in homogenate assays. There was no evidence of any variation of affinity between brain regions. The highest levels of binding were found in the substantia nigra with high levels in the globus pallidus, cerebral cortex, hippocampus and caudate-putamen, intermediate levels in the cerebellum, and low levels in the hypothalamus and pons. The density of [3H]glibenclamide binding sites was low in glucose-responsive brain regions, known to contain ATP-sensitive potassium channels that are inhibited by sulphonylureas. However, higher densities were associated with brain regions (often limbic structures) active during temporal lobe epilepsy. In at least two of these structures, the CA3 region of the hippocampus and the substantia nigra, it is probable that these sites are coupled to ATP-sensitive potassium channels. These results are discussed with reference to the reported actions of ATP-sensitive potassium channels on CNS function.

Inhibition by cations of antagonist binding to histamine H1-receptors.

The binding of the quaternary radioligand [3H] QMDP to the histamine H1-receptor was inhibited by a series of mono- and di-valent cations. The order of potency was Hg2+ greater than Cd2+ greater than Zn2+ greater than Ni2+ greater than Co2+ greater than Mn2+ greater than Ca2+ greater than Mg2+ greater than Li+ = Na+ greater than K+ greater than Cs+. The binding of [3H] mepyramine, a tertiary amine, was inhibited by the divalent cations to a similar extent as the binding of [3H] QMDP. Li+ also had a similar potency against the two ligands, but Na+ was a much more potent inhibitor of the binding of [3H] QMDP than that of [3H] mepyramine.

Calcium-activated potassium channels in rat dissociated ventromedial hypothalamic neurons.

Abstract A potassium-selective channel, characterized by a single channel conductance of 160 pS was demonstrated to be present in rat freshly dispersed ventromedial hypothalamic nucleus neurons. The single channel activity was shown to be dependent, using inside-out membrane patches, upon the presence of intracellular calcium ions, with maximal sensitivity between 10(-6) and 10(-6) M[Ca(2+)], and to be modulated by membrane voltage, depolarization causing an increase in open-state probability in the presence of an activating concentration of calcium. Therefore these properties place this channel into the category of a large conductance (maxi-K(+)) calcium-activated potassium (Ca(2+)-K(+)) channel. This channel is active in cell-attached recordings from glucoreceptive cells when depolarized by glucose or tolbutamide with openings often associated with action current repolarization. These openings were shown to be abolished in the presence of extracellular Cd(2+) and La(3+) ions, which block calcium channels, suggesting that extracellular calcium entry upon cell depolarization is responsible for their activation. On a few occasions, a larger conductance (250 pS) Ca(2+)-K(+) channel was observed in inside-out membrane patches isolated from ventromedial hypothalamic nucleus neurons. In contrast to the 160 pS channel, the presence of intracellularly-applied ATP caused a concentration-dependent, reversible inhibition of its open-state probability.

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.