Search PubMed⌕ Search

Biomedical subjects

S Lenzen

Publications and source records attributed to S Lenzen.

At least 73 records · Page 4Linked to original sources
PubMed1991

Effects of IP3, spermine, and Mg2+ on regulation of Ca2+ transport by endoplasmic reticulum and mitochondria in permeabilized pancreatic islets.

Inositol 1,4,5-trisphosphate (IP3) increased the free-Ca2+ concentration in the incubation medium of permeabilized ob/ob mouse pancreatic islets. Spermine decreased the free-Ca2+ concentration through stimulation of mitochondrial Ca2+ uptake and attenuated the effect of IP3. Mg2+ antagonized the effects of spermine, thereby increasing the free-Ca2+ concentration and enhancing the effect of IP3 on the free-Ca2+ concentration. Because IP3 releases Ca2+ from endoplasmic reticulum, these results indicate that endoplasmic reticulum and mitochondria can interact in the regulation of the free-Ca2+ concentration in the cytosol of the pancreatic beta-cell.

Animals↗
PubMed1991

Recombinant Q53E- and Q53N--chicken egg white cystatin variants inhibit papain, actinidin and cathepsin B.

A cloned synthetic gene coding for (AEF S1M M29I M89L) chicken egg white cystatin was modified site-specifically at position Q53 by cassette mutagenesis. Two recombinant variants were isolated from a pIN-III-ompA E. coli expression system and purified by Cm-papain affinity chromatography. The mutations at the position 53 were confirmed by amino acid composition and amino acid sequence analysis of the appropriate tryptic peptides. The complexes of both cystatin variants, the Q53E- and Q53N-variant with papain, display Ki values similar to those determined with native chicken cystatin. However, the Ki values of the complexes with actinidin are hundredfold and with cathepsin B three hundredfold higher than with the native chicken cystatin. The different inhibition kinetics of these variants compared to wild type chicken cystatin emphasizes the specificity of single amino acid substitutions for optimal contacts between the binding segments of enzyme and inhibitor.

Amino Acid Sequence↗
PubMed1990

Quantitation of hexadecylphosphocholine by high-performance thin-layer chromatography with densitometry.

A method for the determination of the antineoplastic ether phospholipid hexadecylphosphocholine (HePC) is presented, based on the separation of the lipids by high-performance thin-layer chromatography charring with a cupric sulphate reagent and quantitation by in situ densitometry. The lower limit of determination is ca. 25 pmol. Concentrated hexane-isopropanol extracts of plasma samples can be applied to the plate without further clean-up, making this method useful for clinical drug monitoring. Additional ion-exchange chromatography and removal of the salt contaminants by gel filtration permits the study of endogenous phospholipids together with HePC from the same sample.

Choline↗
PubMed1990

Alloxan and ninhydrin inhibition of hexokinase from pancreatic islets and tumoural insulin-secreting cells.

Alloxan inhibited hexokinase activity in cytoplasmic fractions of transplantable radiation-induced rat islet cell tumours, ob/ob mouse pancreatic islets, rat liver and rat kidney. Half maximal inhibitory concentrations of alloxan were greater than those previously found for half maximal inhibition of pancreatic islet or liver glucokinase. D-glucose, preferentially the alpha-anomer, and D-mannose protected hexokinase activity against alloxan inhibition. 1,4-Dithiothreitol completely protected against and partially reversed the alloxan inhibition of hexokinase. The ability of various dithiols to reverse the inhibition of hexokinase by alloxan was dependent on the spacing between the SH (thiol) groups. Only dithiols with intermediate spacing between the SH groups were effective. Dithiols with two vicinal SH groups such as 1,2-dimercaptoethane and 2,3-dimercaptopropanol (BAL) and dithiols with more widely spaced SH groups such as 1,5-dimercaptopentane were ineffective. Thus a reaction of alloxan with two SH groups in the sugar binding site of the hexokinase with the formation of a disulfide bond may be involved in the reversible inhibition of the enzyme. Ninhydrin also inhibited hexokinase from all four tissues studied. The half maximal inhibitory concentrations of ninhydrin were lower than those of alloxan. Inhibition of hexokinase may be an important factor in the general cytotoxic action of ninhydrin. However, inhibition of pancreatic islet hexokinase is unlikely to be the initial event in the pancreatic B-cell toxic action of alloxan, even if inhibition of hexokinase by high concentrations of alloxan may contribute to the B-cell toxic action.(ABSTRACT TRUNCATED AT 250 WORDS)

Alloxan↗
PubMed1989

Regulation of transmembrane ion transport by reaction products of phospholipase A2. I. Effects of lysophospholipids on mitochondrial Ca2+ transport.

Lysophospholipids inhibited mitochondrial Ca2+ uptake, induced a net Ca2+ efflux, and thereby increased the extramitochondrial Ca2+ concentration. The inhibitory potency decreased in the order lysophosphatidylcholine (LPC) = lysophosphatidylglycerol (LPG) greater than lysophosphatidylinositol (LPI) greater than lysophosphatidylserine (LPS) much greater than lysophosphatidylethanolamine (LPE). This relative order is in inverse relation to the ability of the various phospholipid head-groups to build up intermolecular hydrogen bonds with neighbouring membrane lipids. This indicates that changes in Ca2+ transport induced by lysophospholipids are mediated by the interaction of the lysophospholipids with the mitochondrial membrane bilayer structure. The mitochondrial membrane potential, which is the main driving force for mitochondrial Ca2+ uptake, was affected in the same order by the various lysophospholipids. This reduction of the mitochondrial membrane potential may be the underlying cause for the inhibition of the mitochondrial Ca2+ uniport and the resulting release of Ca2+ from the mitochondria.

Animals↗
PubMed1989

Regulation of transmembrane ion transport by reaction products of phospholipase A2. II. Effects of arachidonic acid and other fatty acids on mitochondrial Ca2+ transport.

The effects of arachidonic acid and other fatty acids on mitochondrial Ca2+ transport were studied. Cis-unsaturated fatty acids generally strongly inhibited mitochondrial Ca2+ uptake, induced a net Ca2+ efflux, and thereby increased the extramitochondrial Ca2+ concentration, whereas trans-unsaturated fatty acids were ineffective. Saturated fatty acids exhibited slight activity at chain lengths from C(10) to C(14) only. The structure-activity relationship and the inability of some of the effective fatty acids such as palmitoleic and myristoleic acid to be metabolized to eicosanoids suggest that Ca2+ release was induced by the fatty acids themselves and resulted from changes in the mitochondrial membrane bilayer structure. There was a correlation between Ca2+-releasing potency and reduction of mitochondrial membrane potential, which is the main driving force for mitochondrial Ca2+ uptake. There were, however, considerable differences compared with the effects of lysophospholipids on the membrane potential. The mechanism of action of fatty acids may be that of a fluidizing effect on the hydrophobic core of the membrane, thereby modulating the activity of integral membrane proteins of the respiratory chain.

Animals↗
PubMed1989

Control of insulin secretion by sulfonylureas, meglitinide and diazoxide in relation to their binding to the sulfonylurea receptor in pancreatic islets.

Sulfonylureas inhibit an ATP-dependent K+ channel in the B-cell plasma membrane and thereby initiate insulin release. Diazoxide opens this channel and inhibits insulin release. In mouse pancreatic islets, we have explored whether other targets for these drugs must be postulated to explain their hypo- or hyperglycaemic properties. At non-saturating drug concentrations the rates of increase in insulin secretion declined in the order tolbutamide = meglitinide greater than glipizide greater than glibenclamide. The same rank order was observed when comparing the rates of disappearance of insulin-releasing and K+ channel-blocking effects. The different kinetics of response depend on the lipid solubility of the drugs, which controls their penetration into the intracellular space. Allowing for the different kinetics, the same maximum secretory rates were caused by saturating concentrations of tolbutamide, meglitinide, glipizide and glibenclamide. A close correlation between insulin-releasing and K+ channel-blocking potencies of these drugs was observed. The relative potencies of tolbutamide, meglitinide, glipizide and glibenclamide corresponded well to their relative affinities for binding to islet-cell membranes, suggesting that the binding site represents the sulfonylurea receptor. The biphasic time-course of dissociation of glibenclamide binding indicates a complex receptor-drug interaction. For diazoxide there was no correlation between affinity of binding to the sulfonylurea receptor and potency of inhibition of insulin secretion. Thus, opening or closing of the ATP-dependent K+ channel by diazoxide or sulfonylureas, respectively, appears to be due to interaction with different binding sites in the B-cell plasma membrane. The free concentrations of tolbutamide, glipizide, glibenclamide and diazoxide which are effective on B-cells are in the range of therapeutic plasma concentrations of the free drugs. It is concluded that the hypo- and hyperglycaemic effects of these drugs result from changing the permeability of the ATP-dependent K+ channel in the B-cell plasma membrane.

ATP-Binding Cassette Transporters↗
PubMed1988

Concentration-dependent effects of tolbutamide, meglitinide, glipizide, glibenclamide and diazoxide on ATP-regulated K+ currents in pancreatic B-cells.

The influence of the hypoglycemic drugs tolbutamide, meglitinide, glipizide and glibenclamide on ATP-dependent K+ currents of mouse pancreatic B-cells was studied using the whole-cell configuration of the patch-clamp technique. In the absence of albumin, tolbutamide blocked the currents half maximally at 4.1 mumol/l. In the presence of 2 mg/ml albumin half maximal inhibition of the currents was observed at 2.1 mumol/l meglitinide, 6.4 nmol/l glipizide and 4.0 nmol/l glibenclamide. The hyperglycemic sulfonamide diazoxide opened ATP-dependent K+ channels. Half maximally effective concentrations of diazoxide were 20 mumol/l with 0.3 mmol/l ATP and 102 mumol/l with 1 mmol/l ATP in the recording pipette. Thus, the action of diazoxide was dependent on the presence of ATP in the recording pipette. The free concentrations of the drugs which influenced ATP-dependent K+ currents were comparable with the free plasma concentrations in humans and the free concentrations which affected insulin secretion in vitro. The results support the view that the target for the actions of sulfonylureas and of diazoxide is the ATP-dependent K+ channel of the pancreatic B-cell or a structure closely related to this channel.

Adenosine Triphosphate↗
PubMed1988

Glucose both inhibits and stimulates insulin secretion from isolated pancreatic islets exposed to maximally effective concentrations of sulfonylureas.

Isolated pancreatic islets from mice were perifused with media containing maximally effective concentrations of glibenclamide (0.1-10 mumol/l) or glipizide (1 mumol/l). In these islets an increase of the glucose concentration from 10 mmol/l to 40 mmol/l or addition of D-glyceraldehyde (20 mmol/l) caused a temporary decrease in insulin release which was followed by a sustained enhancement of release. alpha-Ketoisocaproate (3 or 20 mmol/l) did not inhibit insulin release; at high concentration it was an even stronger secretagogue than D-glucose or D-glyceraldehyde. It is concluded that high energy phosphates couple B-cell fuel metabolism and insulin release by acting both on the ATP-dependent K+ channel and on other targets not yet identified.

Animals↗
PubMed1988

Structural requirements of alloxan and ninhydrin for glucokinase inhibition and of glucose for protection against inhibition.

1. In order to elucidate the mechanism underlying the interactions between glucose and alloxan when competing for the sugar binding site of glucokinase from pancreatic B-cells or liver, the structural requirements of the enzyme for inhibition by alloxan and for protection by glucose were determined. 2. With a half-maximal inhibitory concentration of 5 microM, alloxan was the most potent pyrimidine derivative inhibitor of glucokinase. Uramil was a less potent enzyme inhibitor. A variety of other pyrimidine derivatives and related substances were ineffective. 3. Ninhydrin also inhibited glucokinase with a half-maximal inhibitory concentration of 5 microM. Isatin was a slightly less potent enzyme inhibitor. Several other indoline derivatives were ineffective. 4. Only glucose derivatives with a sufficiently bulky substituent in position C-2, such as the glucokinase substrates glucose and mannose and the inhibitors mannoheptulose, glucosamine, and N-acetylglucosamine, protected glucokinase against inhibition by alloxan by binding to the active site of the enzyme. Glucose epimers which differed in other positions did not protect the enzyme against alloxan inhibition. 5. DTT (dithiothreitol) protected glucokinase against inhibition by alloxan and reversed the inhibition of the enzyme induced by alloxan. Thus the mechanism of glucokinase inhibition by alloxan and other inhibitors, such as uramil and ninhydrin, is an oxidation of functionally essential SH groups of the enzyme, where the most reactive keto group of the inhibitor acts as the hydrogen acceptor. The protective action of glucose and several C-2 epimers demonstrates that these functionally essential SH groups are situated in the sugar binding site of the glucokinase. 6. The present results support our contention, that the pancreatic B-cell glucokinase is the major target mediating the inhibition of insulin secretion by alloxan.

Alloxan↗
PubMed1988

Inhibition of glucokinase by alloxan through interaction with SH groups in the sugar-binding site of the enzyme.

Alloxan is known to inhibit pancreatic B cell and liver glucokinase and glucose protects the enzyme against inhibition. The dithiol 1,4-dithiothreitol (1,4-DTT) protected against and reversed the inhibition of glucokinase by alloxan. An investigation into the structure-activity relationship using a variety of different dithiols demonstrated that the ability of the dithiols to protect against and to reverse the inhibition of glucokinase by alloxan was dependent on the spacing between the SH (thiol) groups of the various dithiols. Only 1,3-dimercaptopropane, 1,4-dimercaptobutane, 1,4-dithioerythritol, and 1,4-DTT, with intermediate spacing between the SH groups, reversed the inhibition of glucokinase induced by alloxan. Dithiols with two vicinal SH groups such as 1,2-dimercaptoethane and 2,3-dimercaptopropanol (BAL) were ineffective in the same way as dithiols with more widely spaced SH groups such as 1,5-dimercaptopentane and 1,6-dimercaptohexane. Except for 1,6-dimercaptohexane, all dithiols also protected glucokinase against the inhibition of alloxan. The monothiol cysteine, but not glutathione, a tripeptide monothiol, also protected glucokinase against alloxan inhibition but both were unable to reverse the inhibition. Like alloxan, other dithiol reagents such as ninhydrin, N-ethylmaleimide, and maleimide inhibited glucokinase. Glucose and 1,4-DTT protected glucokinase against this inhibition. 1,4-DTT partially reversed this inhibition. It is concluded, therefore, that the mechanism of inhibition of glucokinase by alloxan is a reaction of alloxan with two adjacent SH groups in the depth of the sugar-binding site of the glucokinase, with formation of a disulfide bond and concomitant inactivation of the enzyme. Because glucokinase can couple changes in the blood glucose concentration to changes in the glycolytic flux rate and corresponding changes in the rate of insulin secretion, it may function as a glucose signal recognition enzyme in the pancreatic B cell. This mechanism of interaction of alloxan with glucokinase may thereby provide an explanation for the ability of alloxan to inhibit glucose-induced insulin secretion.

Alloxan↗
PubMed1987

Monoamine oxidase in rat and bovine endocrine tissues.

Monoamine oxidase (MAO) was characterized in tissue homogenates from rat pancreatic islets, rat neurohypophysis and adenohypophysis, and rat and bovine adrenal medulla and adrenal cortex. Phenylethylamine was preferentially deaminated by rat pancreatic islet and bovine adrenal medulla MAO and with slight preference by rat neurohypophysis MAO, whereas 5-hydroxytryptamine was preferentially deaminated by MAO from all other endocrine tissues. Tyramine was a good substrate for all tissues. Clorgyline, a selective inhibitor of MAO-A, preferentially inhibited deamination of 5-hydroxytryptamine by all tissue homogenates, whereas deprenyl, a selective inhibitor of MAO-B, preferentially inhibited deamination of phenylethylamine. Km values for 5-hydroxytryptamine and tyramine were higher by one to two decimal powers than for phenylethylamine in homogenates from all endocrine tissues. Km values were significantly lower for 5-hydroxytryptamine and significantly higher for phenylethylamine in rat and bovine adrenal cortex than in adrenal medulla. According to these results, the contributions of MAO-B to total enzyme activity were 70% for rat pancreatic islets, 45% for rat neurohypophysis, 15% for rat adenohypophysis, 20% for rat adrenal medulla, 10% for rat adrenal cortex, 60% for bovine adrenal medulla, and 20% for bovine adrenal cortex. PC 12 cells also contained predominantly MAO-A (90%); however, an increased Km for phenylethylamine and a sensitivity of deamination of this MAO-B substrate to inhibition by clorgyline are indicators of abnormal behavior of MAO in this clonal rat pheochromocytoma cell line.

Adrenal Cortex↗
PubMed1987

Glucokinase in pancreatic B-cells and its inhibition by alloxan.

Characterization of glucokinase in pancreatic B-cells from ob/ob mice and from rat liver revealed identical characteristics. A narrow substrate specificity; high Km values for the two substrates, D-glucose and D-mannose, in the range of 10 and 20 mmol/l, respectively; higher Vmax values for D-glucose than for D-mannose; inhibition of glucokinase activities by D-mannoheptulose and by a specific glucokinase antibody. These characteristics distinguish glucokinase in soluble cytoplasmic fractions of pancreatic B-cells and liver from low Km hexokinases. Alloxan is a pancreatic B-cell cytotoxic agent, which has been widely used as a tool for the elucidation of the mechanisms of insulin secretion, because its inhibitory action on insulin secretion has been presumed to be intimately related to the mechanism of glucose-induced insulin secretion. Alloxan inhibited glucokinase but not hexokinase activity in cytoplasmic fractions of pancreatic B-cells and liver. The half maximal inhibitory concentration of alloxan was 5 mumol/l. Glucokinase activity was protected from alloxan toxicity only by D-glucose and D-mannose; the alpha anomer of D-glucose provided significantly greater protection than the beta anomer. The non-metabolizable sugar 3-O-methyl-D-glucose did not provide protection of glucokinase activity against inhibition by alloxan. Thus, inhibition of pancreatic B-cell glucokinase may contribute to the inhibition of glucose-induced insulin secretion by alloxan. These results support the contention that glucokinase regulates the metabolic flux rate through the glycolytic chain in the pancreatic B-cell and thereby generates the signal for glucose-induced insulin secretion.

Alloxan↗
PubMed1987

Defective regulation of glucokinase in rat pancreatic islet cell tumours.

The role of glucokinase in the regulation of insulin secretion was examined in normal rat pancreatic islets and in chemically- and radiation-induced rat pancreatic B-cell tumours which show an impaired insulin secretory response to glucose. In normal rats glucokinase activity in cytoplasmic fractions of pancreatic islets was decreased with the duration of fasting and increased by refeeding or insulin administration. This observation is consistent with the induction of glucokinase by insulin. Hexokinase activity was only slightly reduced during fasting. Glucokinase activity decreased in cytoplasmic fractions of streptozotocin-nicotinamide-induced rat pancreatic islet cell tumours. Glucokinase activity contributed about 75% to the total glucose phosphorylation capacity in cytoplasmic fractions of normal pancreatic islets and of small (less than 1 mg) streptozotocin-nicotinamide-tumours. This proportion decreased to about 20% in the large streptozotocin-nicotinamide tumours. Glucokinase activity in cytoplasmic fractions of transplantable radiation-induced NEDH (New England Deaconess Hospital) rat B-cell tumours was seven times lower than in normal pancreatic islets and contributed only 15% to the total glucose phosphorylation capacity. In contrast, hexokinase activity of the NEDH tumour B-cells was 2.5 times higher than normal. Decreased glucokinase activity in the chemically- and radiation-induced tumour B-cells appears to result from a loss of the ability of insulin to induce this enzyme and may explain the lack of insulin secretory responsiveness of these tumour B-cells.

Adenoma, Islet Cell↗
PubMed1986

Interactions between spermine and Mg2+ on mitochondrial Ca2+ transport.

The effects of the polyamine spermine on the regulation of Ca2+ transport by subcellular organelles from rat liver, heart, and brain were investigated using ion-sensitive minielectrodes and a 45Ca2+ tracer method. Spermine stimulated Ca2+ uptake by mitochondria but not by microsomes. In the presence of spermine, isolated mitochondria could maintain a free extramitochondrial Ca2+ concentration of 0.3-0.2 microM. Stimulation of the initial rates of Ca2+ uptake and 45Ca2+ cycling of mitochondria by spermine shows that this was accomplished through a decrease of the apparent Km for Ca2+ uptake by the Ca2+ uniporter. The half maximally effective concentration of spermine (50 microM) was in the range of physiological concentrations of this polyamine in the cell. Spermidine was five times less effective. Putrescine was ineffective. The stimulation of mitochondrial Ca2+ uptake by spermine was inhibited by Mg2+ in a concentration-dependent manner. However, the diminished contribution of the mitochondria to the regulation of the free extraorganellar Ca2+ concentration could mostly be compensated for by microsomal Ca2+ uptake. Spermine also reversed ruthenium red-induced Ca2+ efflux from mitochondria. It is concluded that spermine is an activator of the mitochondrial Ca2+ uniporter and Mg2+ an antagonist. By this mechanism, the polyamines can confer to the mitochondria an important role in the regulation of the free cytoplasmic Ca2+ concentration in the cell and of the free Ca2+ concentration in the mitochondrial matrix.

Animals↗