Biomedical subjects

B Hellman

Publications and source records attributed to B Hellman.

At least 91 records · Page 5Linked to original sources

PubMed1989

Three types of cytoplasmic Ca2+ oscillations in stimulated pancreatic beta-cells.

Oscillations of cytoplasmic Ca2+ (Ca2+i) involved in cell regulation have recently attracted considerable attention. In the pancreatic beta-cells an intermediate concentration of glucose (11 mM) induces large oscillations of Ca2+i with periods of 2 to 6 min. Procedures stimulating insulin secretion further, such as raising glucose to 20-30 mM or adding carbachol, ATP, theophylline, glucagon, or forskolin, often changed these oscillations into a steady increase of Ca2+i. In addition, forskolin and glucagon triggered prominent 9- to 14-s Ca2+i spikes during the intervals of increased Cai2+, whereas carbachol and ATP initiated a series of rapid spikes of decreasing magnitude and increasing duration (6-11 s). All types of oscillations depended on the presence of extracellular Ca2+i, but carbachol and ATP also induced single Cai2+ transients in the absence of the cation. The results demonstrate hitherto unknown oscillations of Ca2+i in the pancreatic beta-cell which are dependent in different ways on Ca2+ entry.

Adenosine Triphosphate↗

PubMed1989

Plasma membrane associated ATP as a regulator of the secretory activity of the pancreatic beta-cell.

beta-Cell-rich pancreatic islets from ob/ob-mice were used for evaluating how ATP associated with the plasma membrane participates in the regulation of insulin release. Increase of Ca2+ initiates insulin release from permeabilized beta-cells only in the presence of Mg-ATP. When bound to the inner part of the plasma membrane ATP depolarizes the beta-cells by closing a glucose-regulated K+-channel. It is possible that ATP in a plasma membrane compartment modulates insulin release also by stimulating ion pumps and exchange processes. ATP can regulate the secretory activity by binding also to the exterior of the beta-cells. The addition of ATP resulted in stimulation of insulin release related to polyphosphoinositide breakdown. It is suggested that the granule fusion with the plasma membrane is followed by release of sufficient amounts of ATP and ADP for activating a P2-purinoceptor. This receptor may consequently be part of a system for amplifying the secretory response to glucose and other agents facilitating the entry of Ca2+.

Adenosine Triphosphate↗

PubMed1989

The effects of glibenclamide and its non-sulfonylurea analogue HB 699 on the sodium content of rat pancreatic islets.

Sodium contents were determined in rat pancreatic islets using integrating flame photometry. Whereas the sodium content decreased in the presence of glucose, it increased when 0.1-100 mumol/l glibenclamide was added to a medium containing 3 mmol/l glucose. The complexity of the glibenclamide action became evident with its reversal after removal of extracellular Ca2+ and the observation that the sulfonylurea counteracted the increase of sodium obtained after removal of K+. The effects of glibenclamide were mimicked by 1 mmol/l of its non-sulfonylurea analogue HB 699 with the exception that the latter compound being without suppressive action on the sodium content in medium deprived of Ca2+. Also exposure to 1 mmol/l sulfadiazine resulted in a Ca2+-dependent increase of sodium. The results suggest a role for sodium in amplifying the secretory response to the increased entry of Ca2+ obtained with the depolarisation of the beta-cells with glibenclamide or HB 699.

Animals↗

PubMed1989

Reversal of glucose-induced inhibition of insulin release by dibutyryl cyclic AMP.

Insulin release in response to glucose was measured after culture of islets from ob/obmice in a medium deficient in Ca2+. When present at a concentration of 6 mmol/l, glucose inhibited, insulin release. After activation of the alpha2-adrenergic receptors by 1 mumol/l clonidine also 20 mmol/l glucose became inhibitory. The inhibitory action of glucose on insulin release disappeared in the presence of 1 mmol/l dibutyryl cyclic AMP. Raising the glucose concentration from 3 to 20 mmol/l resulted in increased cytoplasmic Ca2+ after an initial depression. Whereas clonidine removed the Ca2+ increase, this phase was partly restored after simultaneous addition of dibutyryl cyclic AMP. It is concluded that cyclic AMP plays a major role in controlling the balance between the stimulatory and inhibitory components in the glucose action on insulin release.

Animals↗

PubMed1989

Glucose effects on cytoplasmic Ca2+ of individual pancreatic beta-cells recorded by two procedures for dual-wavelength fluorometry.

Two different dual-wavelength microfluorometric systems were used to measure the cytoplasmatic Ca2+ concentration (Cai2+) of individual pancreatic beta-cells loaded with the indicator fura-2. Fluorescence was excited continuously at 340 nm with frequent manual checks at 360 nm. Alternatively, the 340/380 nm fluorescence excitation ratio was recorded based on automated samples at the two wavelengths every 10 msec. Irrespective of the experimental technique a rise of the glucose concentration from 3 to 20 mM resulted in an initial lowering of Cai2+ followed by a rise with a peak and a subsequent sustained intermediary level. The increase of Cai2+ depended on the presence of extracellular Ca2+. In some cells the pattern differed and the increase of the glucose concentration induced large oscillations of Cai2+. Glucose-induced increase of Cai2+ was associated with small rapid fluctuations, which were detectable only with the fast time-sharing approach. The characteristic Cai2+ response to glucose was maintained by some beta-cells after culture for more than 3 weeks. The results indicate that the glucose sensitivity of individual beta-cells varies, and that many electrically coupled cells within an islet collectively determine the characteristic pattern of rhythmic depolarization.

Animals↗

PubMed1989

Ca2+ oscillations in pancreatic beta-cells exposed to leucine and arginine.

Dual-wavelength microfluorometry with the fura-2 indicator was employed for continuous recordings of cytoplasmic Ca2+ (Ca2+i) in individual pancreatic beta-cells isolated from ob/ob-mice. When added to a medium containing 3 mmol l-1 glucose, both 10 mmol l-1 leucine and 20 mmol l-1 arginine induced rises in Ca2+i with periodic fluctuations. In the case of leucine, this increase was preceded by initial lowering followed by high-amplitude oscillations with a periodicity of 2-6 min. In a glucose-free medium arginine had no effect, and leucine was unable to induce more than a single peak of Ca2+i increase. When present at a concentration of 1 mmol l-1, leucine sometimes induced a couple of high-amplitude oscillations at 3 mmol l-1 glucose but lowered Ca2+i permanently in a glucose-free medium. It is likely that the high-amplitude oscillations of Ca2+i are related to the electrical activity of the beta-cells. Provided that some glucose was present, leucine initiated a similar type of Ca2+i response as obtained during glucose-induced insulin release. The observed leucine effect is therefore compatible with a role of glycolysis in generating high-amplitude Ca2+ oscillations and pulsatile insulin release.

Animals↗

PubMed1989

Leucine induces initial lowering of cytoplasmic Ca2+ in pancreatic beta-cells without concomitant inhibition of insulin release.

The early effects of glucose and leucine on cytoplasmic Ca2+ and insulin release were compared in suspensions of cells prepared by dispersal of the beta-cell-rich pancreatic islets of ob/ob-mice. Adequate temporal resolution was achieved by continuously recording the 340/380 nm fluorescence excitation ratio from cells loaded with the Ca2+ indicator fura-2 and measuring insulin in the perifusate from cells mixed with polyacrylamide beads. Raising the glucose concentration from 3 to 20 mM resulted in concomitant reductions of cytoplasmic Ca2+ and insulin release during the first minute. Whereas 10 mM leucine was as efficient as glucose in inducing temporary lowering of cytoplasmic Ca2+, this amino acid did not depress insulin release. It is concluded that the initial decrease of cytoplasmic Ca2+ is a phenomenon coupled to stimulation of the metabolism. The leucine-induced lowering of Ca2+ may essentially reflect changes in cytoplasmic pools other than in a peripheral one regulating insulin release.

Animals↗

PubMed1988

Sulphonamide modulation of sodium content in rat pancreatic islets.

Sodium was measured in rat pancreatic islet exposed to tolbutamide, glipizide, diazoxide or sulfisomidine. When added to a medium with physiologically balanced cations these sulphonamides induced a significant rise of the islet content of sodium. The insulin-releasing compounds, tolbutamide and glipizide, had effects opposite to those of the hyperglycemic diazoxide in counteracting the increase of sodium obtained with removal of K+. The tolbutamide-induced increase in sodium was reversed to a decrease when Ca2+ was omitted from the incubation medium. The increase of sodium, which was also seen with non-hypoglycemic sulphonamides, is itself not sufficient for initiating insulin release. However, it may well represent an important mechanism contributing to the secretory response initiated by Ca2+ entry into the sulfonylurea-depolarized beta-cell.

Animals↗

PubMed1988

Glucose-induced oscillations of cytoplasmic Ca2+ in the pancreatic beta-cell.

The cytoplasmic calcium concentration (Ca2+i) was measured in individual mouse pancreatic beta-cells loaded with fura-2 by recording the 340/380 nm fluorescence excitation ratio. An increase of the glucose concentration from 3 to 20 mM, caused initial lowering of Ca2+i followed by a rise with a peak preceding constant elevation at an intermediary level. However, at 11 mM glucose there were large Ca2+i oscillations with a frequency of 1 cycle per 2-6 min. The results indicate that both first and second phase secretion depend on elevated Ca2+i, and that many electrically coupled cells collectively determine the pace of rhythmic depolarization.

Animals↗

PubMed1988

Dual effect of glucose on cytoplasmic Ca2+ in single pancreatic beta-cells.

The cytoplasmic Ca2+ concentration (Ca2+i) was measured in single pancreatic beta-cells from ob/ob-mice using the fluorescent indicator fura-2. Raising the glucose concentration from 3 to 20 mM resulted in 25% initial lowering of Ca2+i, followed by 250% rise above the basal level of 49 +/- 3 nM. Tolbutamide (100 microM) was as effective as glucose in increasing Ca2+i, although its action was more rapid and not preceded by any reduction. The results support the concept that stimulated removal of Ca2+ from the cytoplasm is an essential part of the physiological glucose effect on the pancreatic beta-cells.

Animals↗

PubMed1988

Voltage-activated currents in guinea pig pancreatic alpha 2 cells. Evidence for Ca2+-dependent action potentials.

Glucagon-secreting alpha 2 cells were isolated from guinea pig pancreatic islets and used for electrophysiological studies of voltage-activated ionic conductances using the patch-clamp technique. The alpha 2 cells differed from beta cells in producing action potentials in the absence of glucose. The frequency of these potentials increased after addition of 10 mM arginine but remained unaffected in the presence of 5-20 mM glucose. When studying the conductances underlying the action potentials, we identified a delayed rectifying K+ current, an Na+ current, and a Ca2+ current. The K+ current activated above -20 mV and then increased with the applied voltage. The Na+ current developed at potentials above -50 mV and reached a maximal peak amplitude of 550 pA during depolarizing pulses to -15 mV. The Na+ current inactivated rapidly (tau h approximately 0.7 ms at 0 mV). Half-maximal steady state inactivation was attained at -58 mV, and currents could no longer be elicited after conditioning pulses to potentials above -40 mV. The Ca2+ current first became detectable at -50 mV and reached a maximal amplitude of 90 pA (in extracellular [Ca2+] = 2.6 mM) at about -10 mV. Unlike the Na+ current, it inactivated little or not at all. Membrane potential measurements demonstrated that both the Ca2+ and Na+ currents contribute to the generation of the action potential. Whereas there was an absolute requirement of extracellular Ca2+ for action potentials to be elicited at all, suppression of the much larger Na+ current only reduced the upstroke velocity of the spikes. It is suggested that this behavior reflects the participation of a low-threshold Ca2+ conductance in the pacemaking of alpha 2 cells.

Action Potentials↗

PubMed1988

Characterization of the glucose-induced lowering of sodium in mouse pancreatic beta-cells.

The mechanisms for glucose regulation of the sodium content of the pancreatic beta-cells were examined using aggregates of cells prepared from ob/ob-mice of a local colony. Exposure to glucose rapidly resulted in a protracted lowering of the sodium content as estimated with integrating flame photometry. Sodium became decreased after addition of 1 mmol l-1 glucose, and this effect was maximal with 5 mmol l-1 of the sugar. The effects of low glucose concentrations on the sodium content could not be mimicked by the poorly metabolized 3-o-methyl-D-glucose, and it disappeared in the presence of the metabolic inhibitor antimycin A. The significance of the Na/K pump for maintaining low sodium was illustrated by a substantial increase of the element in the presence of ouabain. However, there was no indication that glucose-induced lowering of sodium reflected activation of this pump when measuring the ouabain-sensitive uptake of 86Rb+. Neither bumetanide nor the bromo derivatives of cyclic AMP or cyclic GMP modified the glucose action on the sodium content. In evaluating whether the effect of glucose was mimicked by other inhibitors of the K+ permeability it was observed that 100 mumol l-1 quinine, but not tolbutamide, decreased sodium. It is concluded that the beta-cell is exceptional among excitable cells in responding to its natural physiological stimulant (glucose) by reduction of sodium. Acting in this way glucose facilitates the removal of Ca2+ from the cytoplasm.

3-O-Methylglucose↗

PubMed1988

Diazoxide unmasks glucose inhibition of insulin release by counteracting entry of Ca2+.

The interaction of diazoxide with the effects of glucose on the insulin-releasing mechanism was analyzed in beta-cell-rich pancreatic islets isolated from ob/ob mice. When added at a concentration of 400 microM to a medium containing 1.28 mM Ca2+, diazoxide converted glucose stimulation of insulin release into inhibition. Further addition of 2 mM theophylline restored the insulin secretory response to glucose. The paradoxical glucose inhibition of insulin release was accounted for by a diazoxide interaction with the entry of Ca2+, unmasking a capacity of the sugar to lower cytoplasmic Ca2+ below its resting concentration.

Animals↗

PubMed1988

Influence of glucose on the sodium content of beta-cell-rich pancreatic islets exposed to sulphonamides and amiloride.

Sodium was measured in beta-cell-rich pancreatic islets isolated from ob/ob-mice starved overnight. Exposure to glucose (5 or 20 mmol/l) resulted in about a 30% reduction of the sodium content whether or not the Na+/K+ pump was inhibited by removal of K+. The glucose effect was not potentiated after amiloride depression of Na+/H+ exchange, and it disappeared when combining removal of K+ with the addition of the hyperglycemic sulphonamide diazoxide (400 mumol/l). Tolbutamide (100 mumol/l) counteracted the reduction of sodium obtained with 5 mmol/l glucose both in the presence or absence of extracellular K+. It is concluded that closure of ATP-regulated K+ channels does not necessarily result in a lowering of the sodium content. The pancreatic beta-cells can be regarded as exceptional among the excitable cells in not responding to their natural physiological stimulus (glucose) with increase of sodium.

Adenosine Triphosphate↗

PubMed1988

Opposing effects of glucose and tolbutamide on the sodium content of rat pancreatic islets.

Integrating flame photometry was employed for measuring sodium in rat pancreatic islets incubated in media buffered with HEPES or bicarbonate. The sodium content decreased by nearly 40% when the islets were exposed to 5 mmol/l glucose, no further reduction being seen with additional rise of the concentration to 20 mmol/l. Whereas the depressing effect of glucose was mimicked by 100 mumol/l quinine, increased sodium contents were noted after inhibition of the Na/K pump (removal of extracellular K+ or addition of 1 mmol/l ouabain) or exposure of the islets to 1 mmol/l tolbutamide. Although promoting sodium accumulation in the islet cells, tolbutamide counteracted the increase in sodium obtained on withdrawal of K+ from the incubation medium. It is suggested that tolbutamide in addition to its major effect in promoting the entry of Ca2+ also facilitates insulin release by suppressing the outward transport of this ion.

Animals↗

PubMed1988

Glucose-induced increase of potassium in pancreatic beta-cells associated with reduced mobilization of the ion.

The potassium contents of beta-cell-rich pancreatic islets from ob/ob-mice were measured with an integrating flame photometer. After exposure to 5 mM glucose islet potassium increased by 17 +/- 2%, no additional effect being seen with increase of the sugar to 20 mM. Glucose counteracted the loss of islet potassium obtained on removal of the ion from the incubation medium, halving the initial disappearance rate. Whereas the effect of glucose in suppressing the mobilisation of potassium was mimicked by tolbutamide and quinine, it was antagonized by diazoxide. It is concluded that the glucose interference with the outward transport of K+ is sufficient to raise the beta-cell content of the ion.

Animals↗

PubMed1987

Characterization of the inositol 1,4,5-trisphosphate-induced Ca2+ release in pancreatic beta-cells.

Pancreatic beta-cells isolated from obese-hyperglycaemic mice released intracellular Ca2+ in response to carbamoylcholine, an effect dependent on the presence of glucose. The effective Ca2+ concentration reached was sufficient to evoke a transient release of insulin. When the cells were deficient in Ca2+, the Ca2+ pool sensitive to carbamoylcholine stimulation was equivalent to that released by ionomycin. Unlike intact cells, cells permeabilized by high-voltage discharges failed to generate either inositol 1,4,5-triphosphate (InsP3) or to release Ca2+ after exposure to carbamoylcholine. However, the permeabilized cells released insulin sigmoidally in response to increasing concentrations of Ca2+. Also in the absence of functional mitochondria these cells exhibited a large ATP-dependent buffering of Ca2+, enabling the maintenance of an ambient Ca2+ concentration corresponding to about 150 nM even after several additional pulses of Ca2+. InsP3, maximally effective at 6 microM, promoted a rapid and pronounced release of Ca2+. The InsP3-sensitive Ca2+ pool was rapidly filled and lost its Ca2+ late after ATP depletion. The transient nature of the Ca2+ signal was not overcome by repetitive additions of InsP3. It was possible to restore the response to InsP3 after a delay of approx. 20 min, an effect which had less latency after the addition of Ca2+. These latter findings argue against degradation and/or desensitization as factors responsible for the transiency in InsP3 response. It is suggested that Ca2+ released by InsP3 is taken up by a part of the endoplasmic reticulum (ER) not sensitive to InsP3. On metabolism of InsP3, Ca2+ recycles to the InsP3-sensitive pool, implying that this pool indeed has a very high affinity for the ion. The presence of functional mitochondria did not interfere with the recycling process. The ER in pancreatic beta-cells is of major importance in buffering Ca2+, but InsP3 only modulates Ca2+ transport for a restricted period of time following immediately upon its formation. Thereafter the non-sensitive part of the ER takes over the continuous regulation of Ca2+ cycling.

Animals↗

PubMed1987

The actions of arginine and glucose on glucagon secretion are mediated by opposite effects on cytoplasmic Ca2+.

Cytoplasmic Ca2+ (Ca2+i) was monitored in single guinea-pig pancreatic alpha 2-cells exposed to modulators of glucagon release. The stimulatory amino acid arginine raised Ca2+i from 62 to 160 nM, whereas the inhibitor glucose reduced both the latter concentration and basal Ca2+i by 30%. Epinephrine which potentiates arginine-stimulated secretion by increasing cAMP, does so without affecting Ca2+i. The results indicate that glucagon secretion is positively modulated by Ca2+i. It is suggested that glucose-induced lowering of Ca2+i is a fundamental effect in cells where the sugar is readily metabolized.

Animals↗