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Biomedical subjects

F M Ashcroft

Publications and source records attributed to F M Ashcroft.

At least 109 records · Page 6Linked to original sources
PubMed1993

Substrate-dependent changes in mitochondrial function, intracellular free calcium concentration and membrane channels in pancreatic beta-cells.

Microfluorimetric and patch-clamp techniques have been combined to determine the relationship between changes in mitochondrial metabolism, the activity of KATP channels and changes in intracellular free calcium concentration ([Ca2+]i) in isolated pancreatic beta-cells in response to glucose, ketoisocaproic acid (KIC) and the electron donor couple tetramethyl p-phenylenediamine (TMPD) and ascorbate. Exposure of cells to 20 mM glucose raised NAD(P)H autofluorescence after a delay of 28 +/- 1 s (mean +/- S.E.M., n = 30). The mitochondrial inner membrane potential, delta psi m (monitored using rhodamine 123 fluorescence), hyperpolarized with a latency of 49 +/- 6 s (n = 17), and the [Ca2+]i rose after 129 +/- 13 s (n = 5). The amplitudes of the metabolic changes were graded appropriately with glucose concentration over the range 2.5-20 mM. All variables responded to KIC with shorter latencies: NAD(P)H autofluorescence rose after a delay of 20 +/- 3 s (n = 5) and rhodamine 123 changed after 21 +/- 3 s (n = 6). The electron donor couple, TMPD with ascorbate, rapidly hyperpolarized delta psi m and raised [Ca2+]i. When [Ca2+]i was raised by sustained exposure to 20 mM glucose, TMPD had no further effect. TMPD also decreased whole-cell KATP currents and depolarized the cell membrane, measured with the perforated patch configuration. These data are consistent with a central role for mitochondrial oxidative phosphorylation in coupling changes in glucose concentration with the secretion of insulin.

Animals↗
PubMed1993

Calcium-independent potentiation of insulin release by cyclic AMP in single beta-cells.

How does cyclic AMP potentiate insulin secretion from pancreatic islet beta-cells? This question is fundamental to understanding how hormones such as glucagon, which elevates cAMP, stimulate insulin secretion and so contribute to the normal secretory response of the islet. It is well established that a rise in the cytoplasmic Ca2+ concentration ([Ca2+]i) is essential for insulin secretion and therefore cAMP has been proposed to act by elevating [Ca2+]i. But studies on permeabilized beta-cells indicate that cAMP increases insulin release even when [Ca2+]i is held constant. We have used microfluorimetry and the patch-clamp technique to measure changes simultaneously in Ca2+ currents, [Ca2+]i and exocytosis in a single beta-cell in response to cAMP. We show here that cAMP, through activation of protein kinase A, increases Ca(2+)-influx through voltage-dependent L-type Ca2+ channels, thereby elevating [Ca2+]i and accelerating exocytosis. More importantly, cAMP also promotes insulin release by a direct interaction with the secretory machinery, which accounts for as much as 80% of its effect.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1993

The effect of tolbutamide on cerebral blood flow during hypoxia and hypercapnia in the anaesthetized rat.

The increase in blood flow in the cerebral cortex of the anaesthetized rat during hypoxia and hypercapnia was investigated. Cerebral blood flow (CBF) was measured using the hydrogen clearance method with acutely implanted platinum electrodes. Hypoxia (PaO2 35.3 +/- 2.4 Torr) and hypercapnia (PaCO2 68.1 +/- 5.1 Torr) increased basal CBF from 76.3 +/- 9.0 ml/100g/min to 168.1 +/- 20.1 ml/100g/min and 162.4 +/- 31.9 ml/100g/min respectively. The sulphonylurea tolbutamide (1mM in 1%DMSO) had no significant effect on CBF in hyperoxia or in hypercapnia. However, it attenuated the increase of CBF during hypoxia by 66 +/- 11% (p < 0.01). This suggests that opening of tolbutamide-sensitive potassium channels may be involved in the process of hypoxic vasodilation in the rat cerebral cortex.

Anesthesia↗
PubMed1993

Two types of potassium channel regulated by ATP in pancreatic B cells isolated from a type-2 diabetic human.

Two types of K channel regulated by ATP were observed in pancreatic beta cells from a type-2 diabetic man. One type had a conductance of 67 pS at -70 mV in symmetrical 140 mM KCl and was inhibited by intracellular ATP with a half-maximal concentration of 40 microM. ATP inhibition was antagonised by ADP. Tolbutamide inhibited the whole-cell K currents half-maximally at 25 microM. This channel has properties similar to those found for the ATP-sensitive K channel in rodent and normal human beta cells. The second channel type observed was an ATP-activated K channel. It had a conductance of 37 pS at -70 mV in symmetrical 140 mM KCl and was activated half-maximally by 9 microM intracellular ATP. This channel was unaffected by 1 mM tolbutamide. In cell-attached patches, one beta cell out of four tested responded to 20 mM glucose with depolarization. The role of the ATP-activated K channel with respect to the (patho)physiology of the beta cell is uncertain.

Adenosine Diphosphate↗
PubMed1993

Modification of K-ATP channels in pancreatic beta-cells by trypsin.

The inside-out configuration of the patch-clamp method was used to study the effects of trypsin on the activity of ATP-sensitive potassium (K-ATP) channels from isolated mouse pancreatic beta-cells. Trypsin (20 micrograms/ml) irreversibly enhanced channel activity around twofold by reducing the interburst intervals without altering the burst kinetics. No effect on the single channel conductance or the inward rectification produced by internal Mg2+ was observed: however, the protease did reduce the inhibitory effect of Mg2+ on channel activity. Trypsin both prevented rundown of K-ATP channel activity and reactivated the channels after complete rundown. These effects of trypsin were absent in the presence of trypsin inhibitor. The protease also reduced the inhibitory effect of ATP on channel activity, increasing the dissociation constant from 7 to 49 microM. Trypsin removed the activating effect of ADP (0.1 mmol/l) on channel activity and reduced the inhibitory effect of tolbutamide (0.5 mmol/l). Carboxypeptidase A did not activate K-ATP channels in excised patches, although it was able to slightly reactivate channels after complete rundown, whereas chymotrypsin increased K-ATP channel activity but it did not produce reactivation. The effects of papain were similar to those of trypsin.

Adenosine Diphosphate↗
PubMed1993

Transporters, channels and human disease.

The past year has seen significant advances in our understanding of the molecular biology of ion channels and transporters and their role in human disease. The star of the show has to be the cystic fibrosis chloride channel about which an enormous amount of information has been accumulated, and the functional effects of some of the mutations found in cystic fibrosis patients have been characterized.

Biological Transport↗
PubMed1993

Exocytosis elicited by action potentials and voltage-clamp calcium currents in individual mouse pancreatic B-cells.

1. Measurements of membrane capacitance, as an indicator of exocytosis, and intracellular Ca2+ concentration ([Ca2+]i) were used to determine the Ca2+ dependence of secretion in single pancreatic B-cells. 2. Exocytosis was dependent on a rise in [Ca2+]i and could be evoked by activation of voltage-dependent Ca2+ currents. The threshold for depolarization-induced release was 0.5 microM [Ca2+]i. Once the [Ca2+]i threshold was exceeded, exocytosis was rapidly (< 50 ms) initiated. When individual pulses were applied, exocytosis stopped immediately upon repolarization and the Ca2+ channels closed, although [Ca2+]i remained elevated for several seconds. 3. During repetitive stimulation (1 Hz), when [Ca2+]i attained micromolar levels, exocytosis also took place during the interpulse intervals albeit at a slower rate than during the depolarizations. 4. Exocytosis could be initiated by simulated action potentials. Whereas a single action potential only produced a small capacitance increase, and in some cells even failed to stimulate release, larger and more consistent responses were obtained with > or = four action potentials. 5. Comparison of the rates of exocytosis measured in response to depolarization, mobilization of Ca2+ from intracellular stores or infusion of Ca2+ through the patch pipette suggests that [Ca2+]i at the secretory sites attains a concentration of several micromolar. This is much higher than the average [Ca2+]i detected by microfluorimetry suggesting the existence of steep spatial gradients of [Ca2+]i within the B-cell. 6. Inclusion of inhibitors of Ca2+/calmodulin-dependent protein kinase II in the intracellular solution reduced the depolarization-induced exocytotic responses suggesting this enzyme may be involved in the coupling between elevation of [Ca2+]i to stimulation of the secretory machinery. 7. The size of the unitary exocytotic event was 2 fF, corresponding to a secretory granule diameter of 250 nm. 8. Over short periods, exocytosis may be extremely fast (1 pF/s or 500 granules/s), which is much higher than the rate of endocytosis (18 fF/s or 9 granules/s). Since the latter is in better agreement with the maximum rate of insulin secretion from islets (approximately 2 granules/s), we suggest that membrane retrieval may set an upper limit on the rate of exocytosis during extended periods of secretion.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1992

Ca(2+)-activated K+ channels from an insulin-secreting cell line incorporated into planar lipid bilayers.

This study evaluates the use of the planar lipid bilayer as a functional assay of Ca(2+)-activated K+ channel activity for use in purification of the channel protein. Ca(2+)-activated K+ channels from the plasma membrane of an insulin-secreting hamster Beta-cell line (HIT T15) were incorporated into planar lipid bilayers. The single channel conductance was 233 picoSiemens (pS) in symmetrical 140 mmol/l KCl and the channel was strongly K(+)-selective (PCl/PK = 0.046; PNa/PK = 0.027). Channels incorporated into the bilayer with two orientations. In 65% of cases, the probability of the channel being open was increased by raising calcium on the cis side of the bilayer (to which the membrane vesicles were added) or by making the cis side potential more positive. At a membrane potential of + 20 mV, which is close to the peak of the Beta-cell action potential, channel activity was half-maximal at a Ca2+ concentration of about 15 mumol/l. Charybdotoxin greatly reduced the probability of the channel being open when added to the side opposite to that at which Ca2+ activated the channel. These results resemble those found for Ca(2+)-activated K+ channels in native Beta cell membranes and indicate that the channel properties are not significantly altered by incorporation in a planar lipid bilayer.

Animals↗
PubMed1991

Separate processes mediate nucleotide-induced inhibition and stimulation of the ATP-regulated K(+)-channels in mouse pancreatic beta-cells.

The mechanisms by which nucleotides stimulate the activity of the ATP-regulated K(+)-channel (KATP-channel) were investigated using inside-out patches from mouse pancreatic beta-cells. ATP produces a concentration-dependent inhibition of channel activity with a Ki of 18 microns. The inhibitory action of ATP was counteracted by ADP (0.1 mM) and GDP (0.2 mM) but not GTP (1 mM). Stimulation of channel activity was also observed when ADP, GDP and GTP were applied in the absence of ATP. The ability of ADP and GDP to reactivate KATP-channels blocked by ATP declined with time following patch excision and after 30-60 min these nucleotides were without effect. During the same time period the ability of ADP and GTP to stimulate the channel in the absence of ATP was lost. In fact, ADP now blocked channel activity with 50% inhibition being observed at approximately 0.1 mM. By contrast, GDP remained a stimulator in the absence of ATP even when its ability to evoke channel activity in the presence of ATP was lost. These observations show that nucleotide-induced activation of the KATP-channel does not involve competition with ATP for a common inhibitory site but involves other processes. The data are consistent with the idea that nucleotides modulate KATP-channel activity by a number of different mechanisms that may include both regulation of cytosolic constituents and direct interaction with the channel and associated control proteins.

Adenosine Diphosphate↗
PubMed1991

Identification and electrophysiology of isolated pars compacta neurons from guinea-pig substantia nigra.

Single neurons from the substantia nigra of the adult guinea-pig were dissociated. Two morphological types of neurons were identified: (i) large multipolar neurons (20-40 microns greatest diameter) and (ii) small fusiform neurons (less than 20 microns diameter). Neurons of both types showed catecholamine fluorescence and were retrogradely labelled by rhodamine-conjugated microspheres injected into the striatum. Two types of neuron (Type A and Type B) were also identified electrophysiologically in perforated patch recordings of membrane potential and whole-cell current. Type A neurons had a low spontaneous firing frequency (5 Hz) broad action potentials (4 ms) and a threshold of approximately -45 mV. They were inhibited by the dopamine D2 receptor agonist, quinpirole. Type B neurons were characterized by a faster firing frequency (25 Hz), shorter action potential duration (1.5 ms) and a threshold of approximately -50 mV. These cells were unaffected by quinpirole. Large multipolar neurons were usually found to be of the A type and small fusiform, neurons of the B type. The properties of the large multipolar, Type A neurons suggest that they form part of the nigrostriatal, dopaminergic population of the pars compacta.

Action Potentials↗
PubMed1991

Amantadine and sparteine inhibit ATP-regulated K-currents in the insulin-secreting beta-cell line, HIT-T15.

1. The effects of pharmacological agents that potentiate insulin release were studied on ATP-regulated K-currents (K-ATP currents) in the insulin-secreting beta-cell line HIT-T15 by use of patch-clamp methods. 2. The tricyclic drug, 1-adamantanamine (amantadine), reversibly inhibited both whole-cell currents (with a Ki of 120 microM) and single channel currents in inside-out patches. This effect was principally due to an increase in a long closed state which reduced the channel open probability. The related compound, 1-adamantanol, in which the amino group is substituted by a hydroxyl one, did not inhibit K-ATP currents substantially. 3. The alkaloid, sparteine, reversibly inhibited both whole-cell K-ATP currents (Ki = 171 microM) and single channel currents in inside-out patches. 4. The results suggest that sparteine and amantadine can block the K-ATP channel from either side of the membrane and support the idea that at least part of the stimulatory effect of these agents on insulin secretion results from inhibition of this channel.

Adenosine Triphosphate↗
PubMed1991

Voltage-activated calcium and potassium currents in human pancreatic beta-cells.

1. The whole-cell configuration of the patch clamp technique was used to study inward and delayed outward currents in beta-cells isolated from human pancreatic islets. 2. The delayed outward current activated at about -20 mV and increased linearly with further depolarization. The instantaneous current-voltage (I-V) relation, measured by tail current analysis, reversed at -70 mV. This is close to the K+ equilibrium potential and suggests the outward current is carried primarily by potassium ions. In support of this idea, outward currents were abolished when internal K+ was replaced by the impermeant cation N-methyl-D-glucamine (NMG). 3. The voltage dependence of K+ current activation could be fitted by a sigmoidal function with a mid-point at +1 mV. K+ currents showed voltage-dependent inactivation which was half-maximal at -25 mV. 4. Inward currents were studied after outward currents were suppressed by replacing internal potassium with NMG. In 5 mM [Ca2+]o, the inward current activated between -50 and -40 mV, had a peak amplitude at -10 mV and reversed at potentials positive to +60 mV. The voltage dependence of inward current activation was sigmoidal with half-maximal activation at -10 mV in 5 mM [Ca2+]o and at -22 mV in 5 mM [Ba2+]o. 5. Inward currents were unaffected by tetrodotoxin (TTX), but could be blocked by cadmium ions. Barium was also capable of carrying inward current. This pharmacology is consistent with inward currents flowing through Ca2+ channels. 6. The inactivation of the inward current was dependent on calcium entry. In two-pulse experiments, the voltage dependence of inactivation was U-shaped, and resembled that of the calcium current. Barium currents showed little inactivation. 7. In two-pulse experiments the degree of inward current inactivation during the test pulse was related to the amount of calcium entry during the first pulse. Calcium entering at more positive potentials was less effective at producing inactivation. 8. Calcium and barium currents also showed a slow, voltage-dependent inactivation when the holding potential was changed between -100 and -40 mV. This inactivation developed with a time course of seconds. 9. The Ca2+ and K+ currents described here are similar to those reported for rodent beta-cells and indicate the rodent beta-cell provides a good model for that of man.

Action Potentials↗
PubMed1990

Expression of voltage-gated K+ channels in insulin-producing cells. Analysis by polymerase chain reaction.

We have used the polymerase chain reaction (PCR) with primers against the S5 and S6 regions of voltage-gated K+ channels to identify 8 different specific amplification products using poly(A)+ RNA isolated from islets of Langerhans from obese hyperglycemic (ob/ob) mice and from the two insulin-producing cell lines HIT T15 and RINm5F. Sequence analysis suggests that they derive from mRNAs coding for a family of voltage-gated K+ channels; 5 of these have been recently identified in mammalian brain and 3 are novel. These hybridize in classes to different mRNAs which distribute differently to a number of tissues and cell lines including insulin-producing cells.

Animals↗
PubMed1990

Simultaneous recordings of glucose dependent electrical activity and ATP-regulated K(+)-currents in isolated mouse pancreatic beta-cells.

Membrane potential and membrane currents were recorded from single mouse pancreatic beta-cells using the perforated patch whole-cell recording technique at 30 degrees C. Single beta-cells maintained in primary tissue culture exhibited glucose-dependent electrical activity similar to that reported for freshly isolated intact islets. The resting input conductance (5.1 +/- 0.9 nS) was determined by ATP-regulated K+ (KATP) channels as it was blocked by 1 mM tolbutamide. 8 mM glucose decreased the input conductance by 80%. The input conductance at -70 mV was of a similar value during the plateau phase and during the silent phase of electrical activity in 8 mM glucose. This suggests that oscillations of KATP channel activity do not underlie the slow waves.

Adenosine Triphosphate↗