Is Ca2+ -Calmodulin-Dependent Protein Phosphorylation in Rat Brain Modulated by Carboxylmethylation?

Calmodulin stimulation of protein kinase activity in calmodulin-depleted preparations of rat brain cytosol or synaptosomal membranes was attenuated by prior carboxylmethylation of the enzyme source with purified protein-O-carboxylmethyltransferase. Similarly, calmodulin stimulation of highly purified Ca2+-calmodulin-dependent protein kinase was reduced if the kinase was exposed to methylating conditions prior to addition of calmodulin. Biochemical and acidic sodium dodecyl sulfate-gel electrophoretic analyses indicated that all sources of protein kinase activity were substrates for methylation. The specific activity of methyl group incorporation into protein kinase increased with increasing purity of the preparation, reaching values of 1.72 pmol CH3/micrograms protein or 0.15-1.12 mol CH3/mol of holoenzyme. Analysis of ATP binding in cytosol with the use of the photoaffinity probe [32P]8-azido-ATP indicated that carboxylmethylation reduced ATP binding. These results suggest that carboxylmethylation of Ca2+-calmodulin protein kinase may modulate the activity of this enzyme in rat brain.     

Glycation of lens MIP26 affects the permeability in reconstituted liposomes.

We studied the role of glycation of lens putative gap junctional protein, MIP26, on the permeability as well as on calmodulin mediated gating activity in reconstituted liposomes. Calf lens membranes were incubated with 0-100 mM glucose for 3 days and MIP26 was isolated. There was a glucose concentration dependent increase in the glycation of MIP26 which reached to 2.48 moles/mole of protein with 100 mM glucose. Gel electrophoresis showed that there was no degradation of MIP26 to MIP22 during incubation. Channel permeability was determined by reconstituting MIP26 into asolectin liposomes. There was a MIP26 glycation dependent decrease in the permeability to sucrose. Furthermore, proteoliposomes containing nonglycated MIP26 showed complete uncoupling of the channels with calmodulin whereas the channels containing glycated MIP26 were only partially uncoupled. These results suggest that glycation of MIP26 does interfere with the gating activity in reconstituted liposomes.     

The stimulus-secretion coupling of glucose-induced insulin release. LIII. Calcium-dependency of the cyclic AMP response to nutrient secretagogues

Above a threshold value in excess of 5.6 mM, D-glucose increases the amount of cyclic AMP measured by radioimmunoassay in pancreatic rat islets and their surrounding incubation medium. As judged from the cyclic AMP content of islets exposed to isobutylmethylxanthine (1.0 mM), the glucose-induced increment in the rate of cyclic AMP generation represents a rapid and sustained phenomenon. The stimulant action of glucose on cyclic AMP accumulation is mimicked by L-leucine, and L-glutamine, these amino acids acting synergistically of one another. Trifluoperazine slightly decreases but fails to abolish the effect of glucose. In the absence of extracellular Ca2+, however, the cyclic AMP response to D-glucose, L-leucine and/or L-glutamine is severely impaired. These findings are compatible with the view that an increase in the generation rate of cyclic AMP participates in the process of nutrient-stimulated insulin release. This increase could be secondary to the nutrient-induced accumulation of Ca2+ in the islet cells leading to activation of adenylate cyclase by calmodulin.     

Effect of thioloxidant diazene dicarboxylic acid bis-(N'-methylpiperazide) (DIP) on 45Ca2+ net uptake into rat pancreatic islets

The effect of DIP (an oxidant of glutathione) on 45Ca2+ net uptake induced by a variety of stimulators of insulin secretion was studied in rat pancreatic islets. In addition the effect of exogenous glutathione (GSH) on 45Ca2+ net uptake in response to glucose was tested. DIP (0.1 mM) inhibited the increase of 45Ca2+ net uptake in the presence of glucose (16.7 mM) and glyceraldehyde (10 mM). A similar inhibitory effect could be demonstrated, when 45Ca2+ net uptake was enhanced by tolbutamide (100 micrograms/ml), glibenclamide (0.5 micrograms/ml), b-BCH (20 mM), 2-ketoisocaproate (20 mM), arginine (20 mM) in the presence of 3 mM glucose or by high extracellular potassium (20 mM). The increase of 45Ca2+ net uptake stimulated by leucine (20 mM) plus glucose (3 mM) was further augmented by DIP. Exogenous GSH did not affect 45Ca2+ net uptake in the presence of (5.6-16.7 mM) glucose. It is suggested that 45Ca2+ net uptake of pancreatic islets depends on the redox state of islet thiols regardless of whether uptake is promoted via inhibition of potassium efflux (nutrients, sulfonylureas) or by high potassium and arginine. The voltage sensitive calcium-channel is the site of action of critical thiols. It is possible that these thiols are localized at the inner side of the plasma membrane.     

Effect of tetracaine and lidocaine on insulin release in isolated mouse pancreatic islets

The effect of tetracaine and lidocaine on insulin secretion and glucose oxidation by islets of ob/ob-mice was measured. Tetracaine, at a concentration of 1 microM to 0.1 mM, did not markedly influence the basal (3 mM glucose) insulin secretion, whereas 0.5-3.5 mM induced a marked increase. At 7 mM glucose, there was a dose-dependent increase with 0.1-2.5 mM tetracaine. Insulin release induced by 20 mM glucose was potentiated by 0.1 mM and 0.5 mM tetracaine, but this effect disappeared at 1 mM tetracaine. The stimulatory effect of 0.5-1 mM tetracaine on basal insulin release was blocked by the secretory inhibitors, adrenaline (1 microM), clonidine (1 microM) and by Ca2+-deficiency, but the stimulation by 3.5 mM tetracaine was not reduced by 1 microM clonidine or Ca2+ deficiency. Atropine (10 microM) did not affect the stimulation by 0.5 mM tetracaine at 3 mM glucose or by 0.25 mM tetracaine at 20 mM glucose. Tetracaine, at 0.1 mM, potentiated the secretory stimulation of 20 mM L-leucine, 20 mM D-mannose, or 1 microM glibenclamide. Mannoheptulose, 10 mM, abolished the combined effects of 0.1 mM tetracaine and 10 mM glucose. Lidocaine, 1-5 mM, stimulated basal insulin release, but 1 microM-1 mM of the drug did not affect glucose-induced (20 mM glucose) insulin release and 5 mM lidocaine inhibited glucose stimulation. The oxidation of 10 mM D-[U-14C]glucose was slightly enhanced by 0.1 and 1 mM tetracaine. The results indicate that tetracaine and lidocaine, at certain concentrations, can induce insulin release and that tetracaine potentiates secretion induced by other secretagogues. It is concluded that these effects may be associated with beta-cell functions related to the adrenergic receptors but probably not to cholinergic receptors.     

Successful cultivation of isolated islets of Langerhans without attachment: relationship between Glucose- and theophylline-induced insulin release and insulin content in rats islets after cultivation.

The effect of various inhibitors of insulin secretion such as mannoheptulose (20 mM), atropine (1 mM), diphenylhydantoin (20 microng/ml), high concentration of Mg++ (5.3 mM) in the presence of 20 mM glucose (control) on insulin content and secretion from collagenase-isolated rat pancreatic islets was studied in vitro by cultivation of islets up to 5 or 9 days in glass Petri dishes without attachment. In a following short-term incubation for 60 min the glucose-induced insulin release without and with theophylline (5 mM) was investigated. Islets cultivated at 5 mM glucose and at 20 mM glucose with the inhibitors mannoheptulose or atropine lost the responsiveness to glucose and theophylline whereas such islets cultivated at 20 mM glucose alone or with diphenylhydantoin (DPH) or 5.3 mg Mg++ showed a stimulation of insulin secretion by glucose and theophylline. Compared, however, with freshly isolated islets all cultivated islets were restricted in their maximal glucose response and this defect was not evoked alone by quantitative changes in islet insulin content. Nevertheless, culture conditions which facilitate a net increase of insulin (content and release) during cultivation influenced also positively the glucose-induced insulin release without and with 5 mM theophylline in the following short-term experiments.     

Glycation of calmodulin: chemistry and structural and functional consequences

In the presence of Ca2+ and glucose, calmodulin incorporates 2.5 mol of glucose/mol of protein. In the absence of Ca2+, only 1.5 mol of glucose is incorporated per mole of calmodulin. Glycation of calmodulin is associated with variable reductions in its capacity to activate three Ca2+/calmodulin-dependent brain target enzyme systems, including adenylyl cyclase, phosphodiesterase, and protein kinase. In addition, glycated calmodulin exhibits a 54% reduction in its Ca2+ binding capacity. Isolated CNBr cleavage fragments of glycated calmodulin suggest that glycation follows a nonspecific pattern in that each of seven available lysines is susceptible to modification. A limit observed on the extent of glycation appears related to the accompanying increase in negative charge on the protein. Glycation results in minimal structural rearrangements in calmodulin, and the Ca2+-induced increase in alpha-helix content and radius of gyration is the same for glycated and unmodified calmodulin. Since glycated calmodulin's Ca2+ binding capacity is reduced, this implies that the Ca2+-induced conformational changes in calmodulin do not require all four Ca2+ binding sites to be occupied. Examination of the lysine positions in calmodulin suggests that Ca2+ binding to domains II and IV is sufficient to induce these changes. The functional consequences of calmodulin glycation therefore cannot be attributed to inhibition of these conformational changes. An alternative explanation is that the inhibition arises from interference at the target enzyme binding site by bound glucose. While glycation shows minimal structural effects, a large pH dependence is observed for the alpha-helix content of unmodified calmodulin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glucose-sensitive conductances in rat pancreatic beta-cells: contribution to electrical activity

The perforated patch technique was used to assess the relative contribution of K(ATP) channel activity, assessed from input conductance (G(input)), and volume-sensitive anion channel activity to the induction of electrical activity in single isolated rat pancreatic beta-cells by glucose, 2-ketoisocaproate and tolbutamide. In cells equilibrated in the absence of glucose, the membrane potential was -71 mV and G(input) 3.66 nS. Addition of 8 mM glucose resulted in depolarisation, electrical activity and a reduction in G(input), reflecting an inhibition of K(ATP) channels. Cells equilibrated in 4 mM glucose had a membrane potential of -59 mV and a G(input) of 0.88 nS. In this case, a rise in glucose concentration to 8-20 mM again resulted in depolarisation and electrical activity, but caused a small increase in G(input). 2-Ketoisocaproate also evoked electrical activity and an increase in G(input), whereas electrical activity elicited by addition of tolbutamide was accompanied by reduced G(input). Increasing the concentration of glucose from 4 to 8-20 mM generated a noisy inward current at -70 mV, reflecting activation of the volume-sensitive anion channel. The mean amplitude of this current was glucose-dependent within the range 4-20 mM. Addition of 2-ketoisocaproate or a 15% hypotonic solution elicited similar increases in inward current. In contrast, addition of tolbutamide failed to induce the inward current. It is concluded that K(ATP) channel activity is most sensitive to glucose within the range 0-4 mM. At higher glucose concentrations effective in generating electrical activity, activation of the volume-sensitive anion channel could contribute towards the nutrient-induced increase in G(input).     

Incorporation of (U-14C)-glucose into glycogen in normal rat pancreatic islets

The incorporation of glucose into glycogen was determined in pancreatic islets isolated from normal rats and incubated with glucose (5 or 20 mM) and compounds known to affect glycogen metabolism in other tissues. Incubation of pancreatic islets with glucose (20 mM) induced a marked increase in radioactive glycogen. Exposure to epinephrine in the presence of glucose (20 mM) slightly increased incorporation of glucose into glycogen. In contrast the incorporation of glucose into glycogen was not affected when isolated islets were exposed to glucagon or insulin, whereas anti-insulin serum in the incubation medium decreased radioactive glycogen formation.     

Calmodulin activates bovine-cardiac myosin light-chain kinase by increasing the affinity for myosin light-chain 2

The basic mechanism by which calmodulin activates bovine-cardiac muscle myosin light-chain kinase was investigated using highly purified preparations of mixed bovine-cardiac myosin light chains or isolated myosin light chain 2. The apparent contamination of these substrate proteins by calmodulin, as detected by activation of calmodulin-sensitive phosphodiesterase, was less than 4 parts/million and was undetectable by antibodies against calmodulin. The apparent KA for calmodulin was 2 nM and 20 nM in the presence of isolated myosin light-chain 2 and mixed myosin light chains, respectively. Purified bovine cardiac troponin C activated myosin light-chain kinase by about 10% at a concentration of 2 microM. Mixed myosin light chains were phosphorylated in the absence and presence of calmodulin and in the presence of calcium with a V of 11.1 and 11.0 mumol phosphate transferred min-1 (mg enzyme)-1, respectively. The apparent Km values for mixed myosin light chains were 8.0 and 0.35 mg/ml in the absence and presence of calmodulin, respectively. Similarly calmodulin lowered the Km value for isolated myosin light-chain 2 over 20-fold and increased the V value only about 1.5-fold. Activity observed in the absence of calmodulin was dependent on the presence of calcium and was suppressed by chelating free calcium either before or during a phosphorylation reaction. The apparent KA for calcium was 1.2 microM and 0.4 microM in the absence and presence of calmodulin. Activity in the absence of calmodulin was inhibited at very high concentrations of the 'specific' calmodulin antagonists W-7, trifluoperazine and R24571 with apparent IC50 values of 0.3 mM, 0.2 mM and 0.02 mM. Antibiotics raised against calmodulin suppressed completely the kinase activity in the presence of calmodulin but had no effect on the activity measured in its absence. These results suggest that calmodulin stimulates the activity of bovine-cardiac myosin light-chain kinase by increasing over 20-fold the affinity for its substrate myosin light-chain 2.     

Acute and chronic effects of glucose and carbachol on insulin secretion and phospholipase C activation: studies with diazoxide and atropine

The acute and chronic effects of 20 mM glucose and 10 microM carbachol on beta-cell responses were investigated. Acute exposure of rat islets to 20 mM glucose increased glucose usage rates and resulted in a large insulin-secretory response during a dynamic perifusion. The secretory, but not the metabolic, effect of 20 mM glucose was abolished by simultaneous exposure to 100 microM diazoxide. Glucose (20 mM) significantly increased inositol phosphate (IP) accumulation, an index of phospholipase C (PLC) activation, from [(3)H]inositol-prelabeled islets. Diazoxide, but not atropine, abolished this effect as well. Unlike 20 mM glucose, 10 microM carbachol (in the presence of 5 mM glucose) increased IP accumulation but had no effect on insulin secretion or glucose (5 mM) metabolism. The IP effect was abolished by 50 microM atropine but not by diazoxide. Chronic 3-h exposure of islets to 20 mM glucose or 10 microM carbachol profoundly reduced both the insulin-secretory and PLC responses to a subsequent 20 mM glucose stimulus. The adverse effects of chronic glucose exposure were abolished by diazoxide but not by atropine. In contrast, the adverse effects of carbachol were abolished by atropine but not by diazoxide. Prior 3 h of exposure to 20 mM glucose or carbachol had no inhibitory effect on glucose metabolism. Significant secretory responses could be evoked from 20 mM glucose- or carbachol-pretreated islets by the inclusion of forskolin. These findings support the concept that an early event in the evolution of beta-cell desensitization is the impaired activation of islet PLC.     

Heme oxygenase-1 is an important modulator in limiting glucose-induced apoptosis in human umbilical vein endothelial cells

Hyperglycaemia is associated with oxidative stress. The inducible isoform of heme oxygenase (HO-1) is an effective system to counteract oxidative stress, yet it is unclear how hyperglycaemia affects HO-1. In this study, we explored: 1) the HO-1 protein content and HO activity in human umbilical vein endothelial cells (HUVECs) exposed to different glucose concentrations, and 2) the mechanisms which account for the high glucose-induced effects on HO-1. We evaluated HO-1 protein expression, HO activity, apoptosis and reactive oxygen species (ROS) in HUVECs treated for 48 h with 5.5, 10 and 20 mM glucose. A dose-dependent production of reactive oxygen species was observed. At 10 mM glucose, an increase of HO-1 protein expression and HO activity was observed, whereas at 20 mM, there was no change in protein content and activity relative to at 5.5 mM glucose. HO-1 protein expression in HUVECs exposed to 20 mM of glucose was increased in the presence of 20 U/ml superoxide dismutase (SOD). HO-1 gene silencing augments ROS production both at 5.5 and 10 mM glucose, leading to an increased apoptosis. We conclude that, in endothelial cells, the regulation of HO-1 by glucose is dependent upon levels of glucose itself. Lack of homeostatic HO-1 upregulation fails to protect from oxidative damage and results in a higher rate of apoptotic cell death.     

Insulin secretion: Combined tolbutamide, forskolin and TPA mimic action of glucose

We have proposed that the two phases of glucose-induced insulin secretion are regulated by two distinct branches of the calcium messenger system: the initial phase by a calmodulin branch, and the sustained phase by a C-kinase branch. To provide further support for this concept, we examined the separate and combined effects of tolbutamide, TPA, and forskolin upon insulin secretion from rat islets perifused in the absence of added fuels. Addition of 200 μM tolbutamide to the perifusate induces only a first phase of insulin secretion, addition of 200 nM TPA only a second phase, and addition of 10 μM forskolin only a small elevation in the basal rate of secretion. The combination of tolbutamide and TPA induces a biphasic secretory response qualitatively and quantitatively similar to that evoked by an increase in glucose concentration from 2.75 to 7 mM. The combination of TPA, tolbutamide, and forskolin evokes a biphasic pattern of insulin secretion qualitatively and quantitatively similar to that evoked by an increase in glucose concentration from 2.75 to 10 mM.     

The role of free calmodulin in the regulation of calcium-pump activity in erythrocytes

The activity of Ca-pump in inside-out oriented vesicles obtained from erythrocyte membranes after their 30 min treatment with EGTA at 20 degrees C (membranes A) and 37 degrees C (membranes B) was investigated. It was shown that in membranes A placed into an incubation medium containing 0.1 mM EGTA (pH 7.4) the overall effect of exogenous calmodulin is due to the increase in the maximal activity of the enzyme, its affinity for Ca2+ being unaffected thereby. In membranes B placed into the same medium (pH 6.75) the activation of the Ca-pump by calmodulin is due to the increased affinity for Ca2+ at a constant maximal activity of the enzyme. The dependencies of the value of the calmodulin-stimulated component of membranes A and the Ca2+-binding capacity of calmodulin measured by the intensity of N-phenyl-1-naphthylamine fluorescence on the concentration of free Ca2+ are coincident. In the case of membranes B, the stimulation of Ca-pump by calmodulin occurs at much lower Ca2+ concentrations than the Ca2+ binding-induced conformational shifts in calmodulin. The experimental results suggest that the affinity of the Ca-pump for Ca2+ may affect calmodulin existing in a Ca2+-independent state. The hydrophobic interactions between the Ca-calmodulin complex and the Ca-ATPase molecule are apparently essential for the regulation of the maximal enzyme activity.     

Considerations on the lactate consumption by CHO cells in the presence of galactose

A CHO cell line producing t-PA was cultured using glutamate and glucose or galactose to decrease the formation of metabolic end-products and therefore improving the process. In batch cultures using glutamate (6 mM) with glucose at two different levels (5 and 20 mM) or with glucose and galactose (5 and 20 mM, respectively) a remarkable difference in cell culture parameters was evidenced. For 20 mM glucose, a usual cell pattern was observed with lactate built-up in the medium. For 5 mM glucose, cell growth was arrested due to glucose depletion and only a limited use of the excreted lactate could be observed, not supporting cell growth sufficiently. However, when glucose 5 mM and galactose 20 mM were used together, cells consumed the glucose first and, interestingly, in a second phase they continued growing on galactose with the simultaneous consumption of the endogenous lactate. Under these conditions, cell growth was even improved with respect to growth on 20 mM glucose, used as a control. This metabolic behavior is further investigated by using metabolic flux analysis, suggesting that the lactate produced is not used in the oxidative metabolism through the TCA cycle. Metabolic fate of the lactate consumed is discussed.     

Carbohydrate-dependent induction of fatty acid synthase in primary cultures of rat hepatocytes

The glucose and insulin-dependent long-term regulation of fatty acid synthase was studied in primary cultures of hepatocytes from adult female rats. Under basic culture conditions, i.e. 5.5 mM glucose and 0.5 nM insulin, the enzyme activity was continuously decreased over 6 days. In the presence of 100 nM insulin this decrease was reduced but it was not prevented. Enhancement of glucose to 20 mM was followed by an increase of the enzyme activity; after 5 days of treatment the activity was three times higher than under basic culture conditions. The simultaneous presence of 20 mM glucose and of 100 nM insulin resulted in a much more pronounced increase of the activity; after 5 days of treatment the activity was eight times higher than under basic culture conditions. The enhancement was prevented by inhibition of glycolysis. This may indicate that the increase of fatty acid synthase was mediated by a metabolite of glucose rather than by glucose itself. The coordinate regulation of fatty acid synthase and of other lipogenic enzymes was specific as demonstrated by comparison with the activity of lactate dehydrogenase and with synthesis and degradation of cytosolic proteins. The enhancement of the enzyme protein, demonstrated by rocket immunoelectrophoresis, was due to an increase in the rate of enzyme synthesis by 600% as well as to a prolongation of the apparent half-life of the enzyme by 50% (45 h).     

Effects of sugars on leucine and lysine uptake by intestinal cells from rats fed sucrose and stock diets.

The effect of various dietary sugars on the uptake of 1 mM leucine and 1 mM lysine by intestinal cells isolated from stock-fed and sucrose-fed rats was determined. Leucine uptake was activated by 10 mM fructose and inhibited by 10 mM glucose or 20 mM sucrose on both diets. The major dietary effect noted was a significant increase in the inhibition of leucine by glucose in the sucrose-fed rats. The uptake of lysine was minimally affected by the sugars irrespective of the diet fed. These results demonstrate an important dichotomy in the properties of glucose and fructose transport in the intestine and suggest that dietary fructose may increase the transport of certain amino acids.     

Characterization of Free Radical Defense System in High Glucose Cultured HeLa-tat Cells: Consequences for Glucose-induced Cytotoxicity

HeLa cell line stably transfected with the tat gene from human immunodeficiency virus type 1 has a decreased antioxidant potential. In this work, we used this model to investigate the effect of a high glucose level (20 mM) on the glucose induced cytotoxicity and on the antioxidant system. In comparison to cell culture under control medium, HeLa-wild cell cultured under 20 mM glucose did not exhibit necrosis or apoptosis, contrary to HeLa-tat cell presenting a significant increase in necrotic or apoptotic state. Moreover after 48 h culture under high glucose level the HeLa-tat proliferation rate was not higher than the one of HeLa-wild cells. In HeLa-wild cell high glucose level resulted in an induction of glutathione reductase activity in opposition to HeLa-tat cells where no change was observed. High glucose level resulted in 20% increase in GSSG/GSH ratio in HeLa-wild cells and 38% increase in HeLa-tat cells. Moreover, high glucose level resulted in a dramatic cytosolic thiol decrease and an important lipid peroxidation in HeLa-tat cells. No significant change of these two parameters was observed in HeLa-wild cells. In both cell lines, high glucose resulted in an increase of total SOD activity, as a consequence of the increase in Cu,Zn-SOD activity. High glucose did not result in an increase of Mn-SOD activity in both cell lines. As a consequence of tat tranfection Mn-SOD activity was 50% lower in HeLa-tat cells in comparison to HeLa-wild cells. This work emphasizes the importance of the antioxidant system in the glucose induced cytotoxicity.     

Characterization of free radical defense system in high glucose cultured HeLa-tat cells: consequences for glucose-induced cytotoxicity

HeLa cell line stably transfected with the tat gene from human immunodeficiency virus type 1 has a decreased antioxidant potential. In this work, we used this model to investigate the effect of a high glucose level (20 mM) on the glucose induced cytotoxicity and on the antioxidant system. In comparison to cell culture under control medium, HeLa-wild cell cultured under 20 mM glucose did not exhibit necrosis or apoptosis, contrary to HeLa-tat cell presenting a significant increase in necrotic or apoptotic state. Moreover after 48 h culture under high glucose level the HeLa-tat proliferation rate was not higher than the one of HeLa-wild cells. In HeLa-wild cell high glucose level resulted in an induction of glutathione reductase activity in opposition to HeLa-tat cells where no change was observed. High glucose level resulted in 20% increase in GSSG/GSH ratio in HeLa-wild cells and 38% increase in HeLa-tat cells. Moreover, high glucose level resulted in a dramatic cytosolic thiol decrease and an important lipid peroxidation in HeLa-tat cells. No significant change of these two parameters was observed in HeLa-wild cells. In both cell lines, high glucose resulted in an increase of total SOD activity, as a consequence of the increase in Cu,Zn-SOD activity. High glucose did not result in an increase of Mn-SOD activity in both cell lines. As a consequence of tat tranfection Mn-SOD activity was 50% lower in HeLa-tat cells in comparison to HeLa-wild cells. This work emphasizes the importance of the antioxidant system in the glucose induced cytotoxicity.     

Inhibition of ATP-regulated K+ channels precedes depolarization-induced increase in cytoplasmic free Ca2+ concentration in pancreatic beta-cells

The effects of glucose, diazoxide, K+, and tolbutamide on the activity of K+ channels, membrane potential, and cytoplasmic free Ca2+ concentration were investigated in beta-cells from the Uppsala colony of obese hyperglycemic mice. With [K+]e = [K+]i = 146 mM, it was demonstrated that the dominating channel at the resting potential is a K+ channel with a single-channel conductance of about 65 picosiemens and a reversal potential of about +70 mV (pipette potential). This channel is characterized by complex kinetics with openings grouped in bursts. The channel was completely inhibited by 20 mM glucose in intact cells or by intracellularly applied Mg-ATP (1 mM). The number of active channels was markedly reduced already by 5 mM glucose. However, the single channel current of the channels remaining active was unaffected, indicating no major depolarization. To evoke a substantial depolarization of the membrane and thereby action potentials, a total block in channel activity was necessary. This could be achieved either by increasing the concentration of glucose to 20 mM or by combining 5 mM glucose with 100 microM tolbutamide. In both cases, the effect was counteracted by the hyperglycemic sulfonamide diazoxide. The effects on single channel activity were paralleled by changes in membrane potential and cytoplasmic free Ca2+ concentration, also when the latter measurements were performed at room temperature. The transient increase in the number of active channels and the resulting hyperpolarization observed after raising the glucose concentration to 20 mM probably reflected a drop in cytoplasmic ATP concentration. It is suggested that ATP works as a key regulator of the beta-cell membrane potential and thereby the opening of voltage-activated Ca2+ channels.