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Biomedical subjects

B Hellman

Publications and source records attributed to B Hellman.

At least 145 records · Page 8Linked to original sources
PubMed1984

Glibenclamide is exceptional among hypoglycaemic sulphonylureas in accumulating progressively in beta-cell-rich pancreatic islets.

Six hypoglycaemic sulphonylurea compounds were compared with regard to their ability to bind to beta-cell-rich pancreatic islets microdissected from ob/ob-mice. Glibenclamide differed from carbutamide, tolbutamide, chlorpropamide, glibornuride and glipizide in not being rapidly bound to an equilibrium, but accumulating progressively in amounts far exceeding the water space. An inhibitor of the anion channels in the beta-cell membrane, 4-acetamido-4'-isothiocyanate-stilbene-2,2'-disulphonic acid (SITS), suppressed the islet uptake of glibenclamide and to some extent also that of carbutamide and glibornuride. The unusual uptake characteristics of glibenclamide had their counterpart in a retardation of its maximal action in promoting the entry of Ca2+ into the beta-cells.

Animals↗
PubMed1983

The interaction between manganese and calcium fluxes in pancreatic beta-cells.

Electrothermal atomic-absorption spectroscopy was employed for measuring manganese in beta-cell-rich pancreatic islets isolated from ob/ob mice. The efflux from preloaded islets was estimated from the amounts remaining after 30 min of subsequent test incubations in the absence of Mn2+. An increase in the extracellular Mg2+ concentration promoted the Mn2+ efflux and removal of Na+ from a Ca2+-deficient medium had the opposite effect. Addition of 25 mM-K+ failed to affect Mn2+ outflow as did 3-isobutyl-1-methylxanthine and dibutyryl cyclic AMP. Whereas tolbutamide caused retention of manganese, the ionophore Br-X537A promoted an efflux. D-Glucose was equally potent in retaining the islet manganese when the external Ca2+ concentration ranged from 15 microM to 6.30 mM. Subcellular-fractionation experiments indicated a glucose-stimulated incorporation of manganese into all fractions except the microsomes. The effect was most pronounced in the mitochondrial fraction, being as high as 164%. The glucose-induced uptake of intracellular 45Ca was abolished in the presence of 0.25 mM-Mn2+. When added to medium containing 2.5 mM-Mn2+, glucose even tended to decrease 45Ca2+ uptake. The inhibitory effect of Mn2+ was apparent also from a diminished uptake of 45Ca into all subcellular fractions. The efflux of 45Ca2+ was markedly influenced by Mn2+ as manifested in a prominent stimulation followed by inhibition. In addition to demonstrating marked interactions between fluxes of Mn2+ and Ca2+, the present studies support the view that the glucose inhibition of the efflux of bivalent cations from pancreatic beta-cells is accounted for by their accumulation in the mitochondria.

1-Methyl-3-isobutylxanthine↗
PubMed1983

Depolarization-independent net uptake of calcium into clonal insulin-releasing cells exposed to glucose.

Insulin release, net fluxes of Ca2+, and glucose metabolism were studied in a clonal cell line (RINm5F) established from a transplantable rat islet tumor. The insulin content amounted to only 0.03% of that of the total protein and decreased even further with subsequent passages. The insulin secretion was as high as 10 to 20% of the total hormone content per hour. Insulin release was stimulated by K+ depolarization but not by exposure to glucose. In contrast to this secretory pattern, glucose but not K+ stimulated the net uptake of Ca2+ at micromolar concentrations of the ion. The glucose effect was not mimicked by 20 mM 3-O-methylglucose. It was as pronounced at 1 mM as at 20 mM of the sugar and corresponded to an uptake of 119 fmol cm-2 s-1. Glucose metabolism was typical for tumor cells with a high glycolytic flux and an oxidation-to-utilization ratio as low as 0.05-0.15. Maximal oxidative degradation was attained already at 1 mM. This concentration was also equivalent to the Km for glucose utilization, indicating a substantial left-hand shift of the normal dose-response curve. It is suggested that glucose induces a depolarization-independent net uptake of Ca2+ by favouring intracellular buffering of the cation.

Adenoma, Islet Cell↗
PubMed1983

Reduction of the cytosolic calcium activity in clonal insulin-releasing cells exposed to glucose.

The cytosolic Ca2+ activity of insulin-releasing clonal cells (RINm5F) was studied with the intracellular fluorescent indicator quin-2. When the extracellular Ca2+ concentration was 1 mM, the basal cytosolic Ca2+ activity was 101 +/- 5 nM. Depolarization with 25 mM K+ increased this Ca2+ activity to at least 318 nM, an effect completely reversed by the voltage-dependent channel blocker D-600. In the presence of K+ alone these channels appeared to have a half-life of 6.7 +/- 0.8 min. In contrast to the action of K+, exposure of the RINm5F cells to 4 mM glucose resulted in a reduction of the cytosolic Ca2+ activity. This effect was observed during K+ depolarization but was more pronounced under basal conditions when it amounted to 20%. The data provide the first direct evidence that glucose can decrease the cytosolic Ca2+ activity in beta-cells. Unlike the case in normal beta-cells the glucose effect on the voltage-dependent Ca2+ channels in the RINm5F cells is apparently not sufficient to overcome the intracellular buffering of Ca2+. A defective depolarization is therefore a probable cause of the failing insulin secretion of RINm5F cells exposed to glucose.

Adenoma, Islet Cell↗
PubMed1983

Interactions between magnesium and calcium in beta-cell-rich pancreatic islets.

Calcium-magnesium interactions, total amounts of intracellular magnesium, and insulin release were studied in beta-cell-rich pancreatic islets from ob/ob mice. Mg2+ inhibited the uptake of intracellular 45Ca and insulin release induced by glucose or high concentrations of potassium. Omission of Mg2+ from a Ca2+-deficient medium resulted in an increased efflux of 45Ca, whereas the characteristic glucose inhibition of the efflux was diminished. After addition of Mg2+ to a Mg2+-depleted medium, the glucose-stimulated 45Ca efflux was markedly reduced. Mg2+ inhibited the basal efflux of 45Ca, and this effect was preceded by a transient stimulation. Ca2+ but not Mg2+ stimulated 45Ca efflux in a medium depleted of Ca2+, Mg2+, and Na+. The data indicate that Mg2+ interferes with Ca2+ entry through voltage-dependent Ca2+ channels. Mg2+ may also inhibit the outward transport of Ca2+ from the cells at a site different from the Na+-Ca2+ countertransport mechanism. The total amount of intracellular magnesium remained unaffected by glucose and was not changed unless the ionic composition of the mediums were changed grossly. Under physiological conditions it is therefore unlikely that fluctuations in the intracellular Mg2+ concentration are part of the mechanism by which the functionally important Ca2+ is regulated.

Animals↗
PubMed1982

Glucose inhibits insulin release induced by Na+ mobilization of intracellular calcium.

45Ca2+ incorporated in response to glucose was selectively mobilized from the beta-cell-rich pancreatic islets of ob/ob-mice after raising the intracellular Na+ by removal of K+ or addition of ouabain or veratridine. Also studies of insulin release indicated opposite effects of glucose and Na+ on the intracellular sequestration of calcium. The fact that glucose inhibits insulin release induced by raised intracellular Na+ indicates that this sugar can lower the cytoplasmic [Ca2+]. The concept of a dual action of glucose on the cytoplasmic [Ca2+]. The concept of a dual action of glucose on the cytoplasmic [Ca2+] might well explain previous observations of an inhibitory component in the glucose action on the 45Ca2+ efflux.

Animals↗
PubMed1982

Stimulation of the insulin secretory mechanism following barium accumulation in pancreatic beta-cells.

Electrothermal atomic absorption spectroscopy was employed for measuring barium in beta-cell-rich pancreatic islets microdissected from ob/ob-mice. Both the uptake and efflux of barium displayed two distinct phases. There was a 4-fold accumulation of barium into intracellular stores when its extracellular concentration was 0.26 mM. Unlike divalent cations with more extensive intracellular accumulation, the washout of Ba2+ was not inhibited by D-glucose. Ba2+ served as a substitute for Ca2+ both in maintaining the glucose metabolism after removal of extracellular Ca2+ and making it possible for glucose to stimulate insulin release. Furthermore, Ba2+ elicited insulin release in the absence of glucose and other secretagogues. The latter effect was reversible and was markedly potentiated under conditions known to increase the beta-cell content of cyclic AMP. It is likely that the observed actions of Ba2+ are mediated by Ca2+, since Ca2+ -dependent regulatory proteins, such as calmodulin, apparently cannot bind Ba2+ specifically.

Animals↗
PubMed1982

Manganese accumulation in pancreatic beta-cells and its stimulation by glucose.

Electrothermal atomic-absorption spectroscopy was employed for measuring manganese in beta-cell-rich pancreatic islets microdissected from ob/ob mice. The islet content of endogenous manganese was 80 mumol/kg dry wt., which is about half as much as found in the exocrine pancreas. The initial uptake was characterized by two components, with approximate Km values of 35 microM and 3.7 microM respectively. After 60 min of incubation with 0.25 mM-Mn2+, the intracellular concentration of manganese corresponded to an almost 25-fold accumulation compared with that of the extracellular medium. When exposed to 20 mM-D-glucose, the islets retained more manganese, owing to suppression of its mobilization. The glucose inhibition of efflux was prompt and reversible, as indicated from direct recordings of manganese in a perifusion medium. D-Glucose was an equally potent inhibitor of efflux in the presence of 15 microM- and 1.28 mM-Ca2+. The inhibitory action disappeared when metabolism was suppressed by adding 0.1 mM-N-ethylmaleimide or by lowering the temperature from 37 degrees C to 2 degrees C. At a concentration of 0.25 mM, Mn2+ abolished the insulin-releasing action of D-glucose, exerting only moderate suppression of its metabolism. The addition of Mn2+ resulted in inhibition of basal insulin release in the presence of 1.28 mM-Ca2+, but not in a Ca2+-deficient medium. The studies indicate that the previously observed phenomenon of glucose inhibition of 45Ca efflux has a counterpart in the suppression of manganese mobilization from the pancreatic islets. With the demonstration of a pronounced glucose inhibition of manganese efflux, it is evident that Mn2+ may represent a useful tool for exploring the mechanism of glucose-induced retention of calcium in the pancreatic beta-cells.

Animals↗
PubMed1982

Amounts and distribution of intracellular magnesium and calcium in pancreatic beta-cells.

beta-Cell-rich pancreatic islets were incubated for 60-120 min in the presence of 1 mM or 20 mM glucose and analysed with regard to their contents of magnesium and calcium and how these elements were distributed among subcellular fractions. The islets contained 42 mmol magnesium per kg protein with as much as 70 mmol per kg protein in the microsomal fraction. Both the total amount and intracellular distribution of magnesium remained unaffected after raising the glucose concentration of the incubation medium. The islet content of calcium was twice as high as that of magnesium, the mitochondria and secretory granules accounting for most of the calcium in the sedimentable fractions. In both organelles a substantial fraction of calcium was exchangeable as indicated from the incorporation of 45Ca during 90 min of incubation of the islets. When raising the glucose concentration to 20 mM the percentage exchange of calcium increased from 10 to 27 in the mitochondria and from 13 to 28 in the secretory granules. The glucose stimulation of 45Ca uptake was not associated with a statistically significant increase in the total amounts of calcium. However, in addition to stimulating calcium/calcium exchange, it cannot be excluded that glucose also induces a net accumulation of intracellular calcium in the beta-cells.

Animals↗
PubMed1982

Evidence for a slowly exchangeable pool of calcium in the pancreatic beta cell plasma membrane.

1. Exposure to media deprived of Ca2+ resulted in prompt and transient stimulation of 45Ca efflux from beta cell-rich pancreatic islets microdissected from ob/ob-mice and to some extent also from the isolated neurohypophysis. 2. Particular high efflux rates were reached when the Ca2+-deficient medium contained EGTA, but there was no effect of the chelator on the total amount of radioactivity mobilized from the islets. 3. The removal of extracellular Ca2+ was less effective in promoting the 45Ca efflux in the absence of Na+ and no stimulatory response was seen in the presence of 1 mM-La3+. 4. The 45Ca washout was stimulated whether or not the media used for the loading or subsequent perifusion of the islets were supplemented with 20 mM-D-glucose. However, there was no response to a second exposure to a Ca2+-deficient medium even subsequent to redistribution of intracellular calcium induced by temporary lowering of the temperature. 5. It is suggested that the islet 45Ca released by the removal of extracellular Ca2+ originates from a distinct plasma membrane pool which is exchanged slowly compared to most of the calcium at the beta cell periphery.

Animals↗
PubMed1982

Calcium, magnesium, and zinc contents in organelles of prostatic origin in human seminal plasma.

Human seminal plasma contains organelles (granules and vesicles) of prostatic origin. A Mg2+- and Ca2+-dependent ATPase activity, associated with the membranes of the organelles, was determined and related to the contents of calcium, magnesium and zinc in the granules. The samples were obtained from total ejaculates of 42 men. Fourteen had normal spermiograms. Sperm-free, post-vasectomy ejaculates were obtained in 12 cases. In 16 other men, 9 had sperm concentrations less than 40 X 10(6)/ml and 7 had asthenospermia and/or teratazoospermia. No statistically significant intergroup differences were found when the organelle contents of the metals were determined with flameless atomic absorption spectroscopy. In fractionated ejaculate specimens, the distribution of the metals was correlated to the organelles rather than to the amorphous substance also present in ejaculate. In comparison with the surrounding seminal plasma, an unambiguous enrichment of metals was obtained in the organelles. It is suggested that the organelles exert a regulatory function on spermatozoa by modulating in their microenvironment the concentration of divalent cations necessary for the spermatozoan motility.

Adenosine Triphosphatases↗
PubMed1982

The mechanism of sulfonylurea stimulation of insulin release.

The mechanisms for sulfonylurea stimulation of insulin release were explored by studying how these compounds interacted with beta-cell-rich pancreatic islets isolated from ob/ob-mice. Although sulfonylureas from the "second generation" were taken up to a greater extent, there was no direct correlation between the binding to the islets and the stimulation of insulin release. Drugs, which are known to augment the hypoglycemic action of the sulfonylureas, displaced these compounds from serum albumin to the islets. Sulfonylurea binding to the beta-cells is supposed to result from a hydrophobic interaction of the drug with the beta-cell surface counteracted by electrostatic repulsion from fixed negative charges at the cell surface. Like glucose, the sulfonylureas stimulate insulin release by promoting the Ca2+ influx into the beta-cells. The enhanced Ca2+ influx cannot be accounted for by Ca2+-ionophoretic activity but is secondary to a depolarisation of the beta-cells by a mechanism which may involve a reaction with thiol groups in the plasma membrane.

Animals↗
PubMed1982

The insulin-releasing activity of the tropical plant momordica charantia.

An aqueous extract from the unripe fruits of the tropical plant Momordica charantia was found to be a potent stimulator of insulin release from beta-cell-rich pancreatic islets isolated from obese-hyperglycemic mice. The stimulation of insulin release was partially reversible. It differed from that of D-glucose and other commonly employed insulin secretagogues in not being suppressed by L-epinephrine and in even being potentiated by the removal of Ca2+. This anomalous behaviour was not associated with general effects on the metabolism of the beta-cells as indicated by an unaltered oxidation of D-glucose. Studies of 45Ca fluxes suggest that the insulin-releasing action is the result of perturbations of membrane functions. In support for the idea of direct effects on membrane lipids, the action of the extract was found to mimic that of saponin in inhibiting the Ca2+/H+ exchange mediated by the ionophore A23187 in isolated chromaffin granules and release Ca2+ from preloaded liposomes.

Animals↗
PubMed1982

Lack of Ca2+ ionophoretic activity of hypoglycemic sulfonylureas in excitable cells and isolated secretory granules.

Beta-Cell-rich pancreatic islets, neurohypophyses, and adrenal medullae were used for exploring whether hypoglycemic sulfonylureas exhibit Ca2+ ionophoretic properties. Exposure of these excitable organs to depolarizing concentrations of K+ resulted in stimulation both of 45Ca uptake and efflux. Although tolbutamide does not bind preferentially to pancreatic islets, this sulfonylurea was specific in stimulating the fluxes of 45Ca in these endocrine specimens. A chromaffin granule preparation was used for studies of both the net transport of Ca2+ with the metallochromic indicator arsenazo III and the proton concentration gradient (delta pH) with the fluorescent probe 9-aminoacridine. Even at high concentrations, tolbutamide and glibenclamide did not mediate Ca2+ -H+ exchange diffusion whether or not the granules were made permeable to protons by the addition of the protonophore carbonyl cyanide rho-trifluoromethoxyphenylhydrazone, nor did the sulfonylureas affect Ca2+ -H+ exchange diffusion induced by the addition of A-23187. The data indicate that the Ca2+ fluxes associated with sulfonylurea-stimulated insulin secretion do not result from the Ca2+ -ionophoretic properties of the drugs but rather reflect depolarization of the beta-cells.

Animals↗
PubMed1981

Influence of external calcium ions on labelled calcium efflux from pancreatic beta-cells and insulin granules in mice.

Addition of Ca2+ to a glucose-free perifusion medium stimulated the efflux of 45Ca from prelabelled pancreatic islets isolated from ob/ob-mice. This effect differed from that of glucose in being transient, markedly stimulated by previous exposure to a Ca2+-deficient medium and resulting in mobilization also of substantial amounts of 45Ca incorporated in the absence of glucose. The glucose action on 45Ca efflux reflected the balance between inhibitory and stimulatory components which differed with respect to their chronological order and sensitivity to glucose. The magnitude of the stimulatory phase was related linearly to the extracellular concentration of Ca2+ up to 2.40 mM. The efflux of 45Ca from isolated secretory granules was stimulated by Mg-ATP. The latter made the 45Ca efflux from the granules sensitive to Ca2+; significant stimulation being seen when increasing the medium Ca2+ from 0.1 to 10 microM. The results support the concept of an efficient Ca/Ca exchange mechanism in the depolarized beta-cells, emphasizing a role for the secretory granules in this process.

Animals↗
PubMed1981

Thulium binding to the pancreatic beta-cell membrane.

Radioactive thulium (171Tm) was used to probe cation-binding sites in the plasma membrane of beta-cell-rich pancreatic islets microdissected from noninbred ob/ob mice. Temporal studies revealed that 171Tm uptake was rapid, reaching isotopic equilibrium by 60 min. Analysis of the concentration dependence of 171Tm uptake revealed at least two components. At low 171Tm concentrations (0.003--0.18 micrometer), there was a saturable low capacity component, capable of accommodating less than 20 mumol/kg dry weight. At higher 171Tm concentrations (1.0--500 micrometers), a nonsaturable high capacity component capable of binding more than 100 mmol/kg dry weight was observed. 171Tm taken up at 0.18 micrometer exhibited a high degree of mobility. The uptake of 171Tm at this concentration was increased by incubation with chlorpromazine at concentrations known to increase the permeability of the beta-cell plasma membrane. At 0.18 micrometer, exposure to 20 mM D-glucose reduced 171Tm uptake compared with that in islets incubated in the absence of sugar or in the presence of sugars which lack stimulatory effects on insulin release. Such an effect was not observed at 125 micrometers, and none of the sugars influenced the 171Tm uptake of the exocrine pancreas. These data raise the possibility that cation-binding sites in the beta-cell plasma membrane are of physiological significance in the regulation of insulin secretion.

Animals↗