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F M Ashcroft

Publications and source records attributed to F M Ashcroft.

At least 91 records · Page 5Linked to original sources
PubMed1996

The Ba2+ block of the ATP-sensitive K+ current of mouse pancreatic beta-cells.

We studied the block of whole-cell ATP-sensitive K+ (KATP) currents in mouse pancreatic beta-cells produced by external Ba2+. Ba2+ produced a time-and voltage-dependent block of KATP currents, both the rate and extent of the block increasing with hyperpolarization. With 5.6 mM [K+]o, the relationship between the steady-state KATP current and [Ba2+]o was fit by the Hill equation with a Kd of 12.5 +/- 2.8 microM at -123 mV and of 0.18 +/- 0.02 mM at -62 mV. The Hill coefficient (n) was close to 1 at all potentials indicating that binding of a single Ba2+ ion is sufficient to block the channel. When [K+]o was raised to 28 mM the Kd was little changed (12.4 +/- 4.1 microM at -123 mV, 0.27 +/- 0.05 mM at -62 mV) and n was unaffected, suggesting that K+ does not interact with the Ba2+ binding site. The kinetics of Ba2+ block were slow, 10 microM Ba2+ blocking the KATP current with a time constant of 20 ms at -123 mV in 28 mM [K+]o. The blocking rate constant was calculated as 1.7 mM-1ms-1 and the unblocking rate as 0.02 ms-1, at -123 mV. The data are discussed in terms of a model in which Ba2+ binds to a site at the external mouth of the channel to inhibit the KATP channel.

Adenosine Triphosphate↗
PubMed1996

Sequence variations in the human Kir6.2 gene, a subunit of the beta-cell ATP-sensitive K-channel: no association with NIDDM in while Caucasian subjects or evidence of abnormal function when expressed in vitro.

The ATP-sensitive K-channel plays a central role in insulin release from pancreatic beta cells. This channel consists of two subunits: a sulphonyl-urea receptor, SUR1, and an inwardly rectifying K-channel subunit, Kir6.2. We screened 135 white Caucasian patients with non-insulin-dependent diabetes mellitus (NIDDM) and 90 non-diabetic subjects for mutations in the Kir6.2 gene by single-stranded conformational polymorphism (SSCP) analysis. We identified one silent mutation (A190A) and four missense mutations (E23K, L270V, I337V and S385C) in normal and diabetic individuals. In a single diabetic subject, we identified a two-amino acid insertion (380KP). We also screened 39 Afro-Caribbean diabetic subjects and identified one additional missense (L355P) and one more silent (S363S) mutation. The E23K and I337V variants were completely linked. The common variants (E23K, 1337V and L270V) were found with similar frequency in diabetic and normal subjects. Diabetic subjects with the variants responded normally to sulphonylurea therapy. When mutant Kir6.2 subunits were coexpressed with SUR1 in Xenopus oocytes, there was no difference in the sensitivity of the whole-cell currents to metabolic inhibition or to the sulphonylurea tolbutamide. We therefore conclude that mutations in Kir6.2 are unlikely to be a major cause of NIDDM.

Amino Acid Sequence↗
PubMed1996

Mechanisms of the glycaemic effects of sulfonylureas.

Sulfonylureas are widely used to treat non-insulin dependent diabetes mellitus. These drugs exert their hypoglycaemic effects by stimulating insulin secretion from the pancreatic beta-cell. Their primary mechanism of action is to close ATP-sensitive K-channels in the beta-cell plasma membrane, and so initiate a chain of events which results in insulin release. Recent studies have shown that the beta-cell ATP-sensitive K-channel is a complex of two proteins: a pore-forming subunit (Kir6.2) and a drug-binding subunit (SUR1) which functions as the receptor for sulfonylureas. This review summarizes recent advances in our understanding of the molecular mechanism of sulfonylurea action, focusing on the relationship between the sulfonylurea receptor and the K-ATP channel. Earlier studies are also re-examined in the light of new findings.

ATP-Binding Cassette Transporters↗
PubMed1995

Cloning and functional expression of the cDNA encoding a novel ATP-sensitive potassium channel subunit expressed in pancreatic beta-cells, brain, heart and skeletal muscle.

A cDNA clone encoding an inwardly-rectifying potassium channel subunit (Kir6.2) was isolated from an insulinoma cDNA library. The mRNA is strongly expressed in brain, skeletal muscle, cardiac muscle and in insulinoma cells, weakly expressed in lung and kidney and not detectable in spleen, liver or testis. Heterologous expression of Kir6.2 in HEK293 cells was only observed when the cDNA was cotransfected with that of the sulphonylurea receptor (SUR). Whole-cell Kir6.2/SUR currents were K(+)-selective, time-independent and showed weak inward rectification. They were blocked by external barium (5 mM), tolbutamide (Kd = 4.5 microM) or quinine (20 microM) and by 5 mM intracellular ATP. The single-channel conductance was 73 pS. Single-channel activity was voltage-independent and was blocked by 1 mM intracellular ATP or 0.5 mM tolbutamide. We conclude that the Kir6.2/SUR channel complex comprises the ATP-sensitive K-channel.

ATP-Binding Cassette Transporters↗
PubMed1995

Effects of divalent cations on exocytosis and endocytosis from single mouse pancreatic beta-cells.

1. The effects of the divalent cations Ca2+, Ba2+ and Sr2+ on exocytosis and endocytosis from single isolated mouse pancreatic beta-cells were investigated by monitoring changes in cell capacitance. 2. The immediate increase in capacitance elicited by a single depolarization from -70 to +20 mV was dependent on the divalent cation species, with Ca2+ (8.2 +/- 1.1 fF pC-1) > Ba2+ (1.0 +/- 0.2 fF pC-1) > Sr2+ (0.7 +/- 0.2 fF pC-1) in perforated-patch recordings. 3. In Ba2+ solutions alone there was subsequently an additional slow increase in capacitance (to 4.3 +/- 1.1 fF pC-1). This second phase of exocytosis was unaffected by preincubation with colcemid (20 microM, 45 min) or cytochalasin D (10 microM, 15 min), suggesting that interaction of secretory granules with microtubules or microfilaments is not involved. 4. An increase in cell capacitance was elicited by depolarization in Ba2+ solutions when intracellular Ca2+ was buffered with 10 mM EGTA. Infusion of the beta-cell with Ba2+ also stimulated exocytosis although the rate was much slower (1.1 +/- 0.2 fF s-1; 8 microM free Ba2+) than for Ca2+ (39 +/- 5 fF s-1; 2 microM free Ca2+). These data indicate that Ba2+ does not evoke secretion by promoting Ca2+ release from internal stores. 5. The lower efficacy of Ba2+ in supporting exocytosis may be related to the fact that this cation does not activate calmodulin-dependent processes and the slow second phase of secretion may result from this ion being removed only slowly from the cytoplasm. 6. Endocytosis was faster in Sr2+ than in Ca2+ or Ba2+ solution, and the speed increased when the external concentration of all three divalent cation species was raised. The ability of Ba2+ to support endocytosis suggests calmodulin-dependent processes are not involved. These data suggest membrane retrieval is regulated differently from exocytosis in beta-cells.

Animals↗
PubMed1995

Characterization and variation of a human inwardly-rectifying-K-channel gene (KCNJ6): a putative ATP-sensitive K-channel subunit.

The ATP-sensitive K-channel plays a central role in insulin release from pancreatic beta-cells. We report here the cloning of the gene (KCNJ6) encoding a putative subunit of a human ATP-sensitive K-channel expressed in brain and beta-cells, and characterisation of its exon-intron structure. Screening of a somatic cell mapping panel and fluorescent in situ hybridization place the gene on chromosome 21 (21q22.1-22.2). Analysis of single-stranded conformational polymorphisms revealed the presence of two silent polymorphisms (Pro-149: CCG-CCA and Asp-328: GAC-GAT) with similar frequencies in normal and non-insulin-dependent diabetic patients.

Adenosine Triphosphate↗
PubMed1995

Metabolic inhibition and low internal ATP activate K-ATP channels in rat dopaminergic substantia nigra neurones.

The patch-clamp technique was used to study whole-cell currents of acutely dissociated rat substantia nigra (SN) neurones. In perforated-patch current-clamp recordings, inhibition of mitochondrial metabolism by rotenone (5 microM) produced a hyperpolarisation and inhibited electrical activity. These effects were reversed by the sulphonylureas tolbutamide (0.5 mM) or glibenclamide (0.5 microM). Under voltage-clamp conditions, rotenone induced a time- and voltage-independent K+ current which was selectively blocked by sulphonylureas. The glibenclamide-sensitive current reversed at -81.7 +/- 2.7 mV (n = 5) and showed marked inward rectification. Intracellular dialysis with 0.3 mM adenosine 5'-triphosphate (ATP), but not 2 mM or 5 mM ATP, in standard whole-cell recordings also resulted in activation of a sulphonylurea-sensitive K+ current with similar properties (reversal potential, -81.9 +/- 2.5 mV, n = 5). The close similarity in the properties of the ATP-sensitive K+ current observed in whole-cell recordings and the K+ current activated by metabolic inhibition in perforated-patch recordings suggest that they both result from activation of the same type of ATP-sensitive K+ channel. Sulphonylureas had no effect on electrical activity or membrane currents in the absence of rotenone in perforated-patch recordings, or in cells dialysed with 5 mM ATP, indicating that in SN neurones these drugs are selective for the ATP-sensitive K+ current.

Action Potentials↗
PubMed1995

A fluorimetric and amperometric study of calcium and secretion in isolated mouse pancreatic beta-cells.

We have examined the temporal relationship between intracellular Ca2+ concentration ([Ca2+]i) and secretion in single intact pancreatic beta-cells. Secretion was detected as the release of 5-hydroxytryptamine from pre-loaded beta-cells, using amperometry, and changes in [Ca2+]i were monitored by microfluorimetry. Stimulation of beta-cells by elevation of the extracellular K+ concentration ([K+]o), acetylcholine or glucose increased [Ca2+]i and, after a delay of 2-7 s, evoked amperometric currents. In the presence of glucose, we observed oscillations in [Ca2+]i which were associated with oscillations in the amplitude and frequency of amperometric currents: however, the temporal correlation was not exact, suggesting that there is a significant latency between the increase in average [Ca2+]i and exocytosis. Both the amplitude and frequency of the amperometric currents elicited by 50 mM KCl declined with successive stimulation, but were restored by agents which elevate intracellular adenosine 3':5':cyclic monophosphate (cAMP). This suggests that beta-cells may possess a readily releasable pool of granules which is replenished by cAMP. The variable amplitude of the amperometric currents is discussed in terms of a model in which several secretory granules fuse simultaneously with the plasma membrane.

Acetylcholine↗
PubMed1995

Effect of L-NMMA, cromakalim, and glibenclamide on cerebral blood flow in hypercapnia and hypoxia.

Sulfonylureas reduce cerebral blood flow (CBF) during hypoxia but not during hypercapnia, whereas blockers of nitric oxide (NO) synthesis reduce hypercapnic CBF. However, the effect of NO blockers on hypoxic CBF is uncertain. CBF was measured in the cortex of 51 enflurane-anesthetized rats by the hydrogen clearance technique during eucapnia, hypercapnia (arterial PCO2 65 Torr), and hypoxia (arterial PO2 40 Torr). CBF increased twofold in both hypercapnia and hypoxia from eucapnia. Intracortical (ic) NG-monomethyl-L-arginine (L-NMMA, 100 microM-5 mM) attenuated both the hypercapnic and hypoxic dilations by 60-70%, and L-arginine (300 mg/kg iv) partially reversed these effects. Glibenclamide (10 microM ic) and L-NMMA gave no further attenuation of the hypoxic dilation than L-NMMA alone. Cromakalim (10 microM, ic) increased CBF in eucapnia, but this was not seen in the presence of glibenclamide. The adenosine antagonist 8-phenyl-theophylline did not attenuate the hypoxic dilation. This suggests that NO synthesis plays a major role in the regulation of CBF in hypercapnia and hypoxia. But the combined effects of glibenclamide and L-NMMA do not further attenuate CBF in hypoxia.

Animals↗
PubMed1995

No evidence for mutations in a putative beta-cell ATP-sensitive K+ channel subunit in MODY, NIDDM, or GDM.

The beta-cell ATP-sensitive K+ (K-ATP) channel has a major role in glucose-induced insulin secretion. Screening the entire coding sequence of the gene for a putative beta-cell K-ATP channel subunit, K-ATP2, with single-strand conformation polymorphism did not show any mutations associated with diabetes in white Caucasian diabetic patients, including five pedigrees with maturity onset diabetes of the young (MODY), 25 patients with noninsulin-dependent diabetes mellitus (NIDDM) selected for marked beta-cell deficiency, 25 selected for mild diabetes presenting before age 50 years with fasting plasma glucose levels < 10 mmol/l, 25 unselected NIDDM patients, and 25 subjects with gestational diabetes mellitus (GDM) and subsequent raised fasting plasma glucose. In five large MODY pedigrees, linkage analysis with simple tandem-repeat polymorphisms (STRPs) near the K-ATP2 gene excluded linkage. In a population association study, no linkage disequilibrium for the STRP was found between 237 unselected white Caucasian NIDDM patients and 104 geographically matched and age-matched white Caucasian nondiabetic subjects. In addition, two silent polymorphisms were found with similar frequency in nondiabetic and diabetic subjects. Mutations in the gene for K-ATP2 are unlikely to be a major cause of MODY, NIDDM, or GDM.

Adenosine Triphosphate↗
PubMed1994

Effects of intracellular pH on ATP-sensitive K+ channels in mouse pancreatic beta-cells.

1. The effects of intracellular pH (pHi) on the ATP-sensitive K+ channel (K+ATP channel) from mouse pancreatic beta-cells were examined in inside-out patches exposed to symmetrical 140 mM K+ solutions. 2. The relationship between channel activity and pHi was described by the Hill equation with half-maximal inhibition (Ki) at pHi 6.25 and a Hill coefficient of 3.7. 3. Following exposure to pHi < 6.8, channel activity did not recover to its original level. Subsequent application of trypsin to the intracellular membrane surface restored channel activity to its initial level or above. 4. At -60 mV the relationship between pHi and the single-channel current amplitude was described by a modified Hill equation with a Hill coefficient of 2.1, half-maximal inhibition at pHi 6.48 and a maximum inhibition of 18.5%. 5. A decrease in pHi reduced the extent of channel inhibition by ATP: Ki was 18 microM at pH 7.2 and 33 microM at pH 6.4. The Hill coefficient was also reduced, being 1.65 at pH 7.2 and 1.17 at pH 6.4. 6. When channel activity was plotted as a function of ATP4- (rather than total ATP) there was no effect of pHi on the relationship. This suggests that ATP4- is the inhibitory ion species and that the effects of reducing pHi are due to the lowered concentration of ATP4-. 7. Changes in external pH had little effect on either single-channel or whole-cell K+ATP currents. 8. The effects of pHi do not support a role for H+ in linking glucose metabolism to K+ATP channel inhibition in pancreatic beta-cells.

Adenosine Triphosphate↗
PubMed1994

Determination of the molecular mass of the native beta-cell sulfonylurea receptor.

In the present study we have determined the molecular mass of the beta-cell sulfonylurea receptor in its native form by two different experimental approaches; gel filtration chromatography and radiation inactivation analysis. We first confirmed that the denatured photolabelled MIN6 beta-cell receptor had a molecular size of 141 +/- 2 kDa (mean +/- S.E., n = 8). Under non-denaturing conditions, using gel filtration chromatography, apparent molecular masses of 166 +/- 1 kDa (mean +/- S.E., n = 3) and 182 +/- 5 kDa (mean +/- S.E., n = 4) were determined for the photoaffinity-labelled and unlabelled sulfonylurea receptor, respectively. We conclude that in the solubilized state the receptor exists as a monomer. Radiation inactivation analysis indicated that the receptor has a target size of 250 +/- 30 kDa (mean +/- S.E., n = 7). This value for the molecular mass is larger than that obtained from SDS-PAGE following photolabelling of the receptor (141 kDa) suggesting that the beta-cell sulfonylurea receptor is composed of more than one subunit in the native membrane.

ATP-Binding Cassette Transporters↗
PubMed1994

Stimulus-secretion coupling in pancreatic beta cells.

Insulin secretion is triggered by a rise in the intracellular Ca2+ concentration that results from the activation of voltage-gated Ca2+ channels in the beta-cell plasma membrane. Multiple types of beta-cell Ca2+ channel have been identified in both electrophysiological and molecular biological studies, but it appears that the L-type Ca2+ channel plays a dominant role in regulating Ca2+ influx. Activity of this channel is potentiated by protein kinases A and C and is inhibited by GTP-binding proteins, which may mediate the effects of potentiators and inhibitors of insulin secretion on Ca2+ influx, respectively. The mechanisms by which elevation of intracellular Ca2+ leads to the release of insulin granules is not fully understood but appears to involve activation of Ca2+/calmodulin-dependent protein kinase. Phosphorylation by either protein kinase A or C, probably at different substrates, potentiates insulin secretion by acting at some late stage in the secretory process. There is also evidence that small GTP-binding proteins are involved in regulating exocytosis in beta cells. The identification and characterisation of the proteins involved in exocytosis in beta cells and clarification of the mechanism(s) of action of Ca2+ is clearly an important goal for the future.

Animals↗
PubMed1994

Effects of internal chloride on ATP-sensitive K-channels in mouse pancreatic beta-cells.

The effect of internal Cl- on the K-ATP channel of pancreatic beta-cells was examined. Reducing Cl- from 140mM to 14mM potentiated channel activity (NPO) approximately 4.5 fold in 60% of patches but was without effect in 40% of patches. The Ki for channel inhibition by ATP was not changed. The inhibitory effect of Cl- interacted with the stimulatory action of MgGDP. In 140mM Cl-, 1mM MgGDP increased NPO in Cl(-)-sensitive patches only: when NPO was potentiated by 14mM Cl-, no further increase was produced by MgGDP. These observations suggest that MgGDP and low Cl- solution may increase channel activity via a similar mechanism.

Adenosine Triphosphate↗
PubMed1994

Differential expression of the inwardly-rectifying K-channel ROMK1 in rat brain.

We have investigated the distribution of the inwardly-rectifying K-channel ROMK1 in rat brain using Northern blotting and in situ hybridization. High levels of expression were found in cortex and hippocampus but not in substantia nigra, ventromedial hypothalamus, cerebellum or striatum. PCR primers generated against ROMK1 amplified a 300 bp cDNA fragment from cortex and hippocampus identical in sequence to ROMK1.

Animals↗
PubMed1994

Block of ATP-sensitive K+ channels in isolated mouse pancreatic beta-cells by 2,3-butanedione monoxime.

1. The patch-clamp technique has been used to examine the action of the chemical phosphatase 2,3-butanedione monoxime (BDM) on ATP-sensitive K+ channels (KATP-channels) from mouse isolated pancreatic beta-cells in the absence of ATP and Mg2+. 2. BDM reversibly inhibited whole-cell KATP-currents with a concentration for half maximal inhibition (K(i)) of 15 +/- 1 mM and a Hill coefficient (n) of 2.5 +/- 0.2 (n = 4). 3. In outside-out patches, external BDM reversibly reduced the activity of single KATP-channels with an affinity similar to that observed in whole-cell recordings (K(i) = 11 +/- 3 mM, n = 2.0 +/- 0.3, n = 7). In inside-out patches, internally applied BDM also reversibly blocked the activity of KATP-channels (K(i) = 31 +/- 2 mM, n = 2.2 +/- 0.4, n = 8). In both excised patch configurations, BDM decreased the mean open life-time and the burst duration, thereby producing a decrease in the channel open probability. The drug had no effect on the short intraburst closed times. 4. BDM had no effect on the single-channel current amplitude. 5. The results suggest that BDM blocks the KATP-channel directly, by mechanisms independent of channel dephosphorylation.

Adenosine Triphosphate↗