Biomedical subjects

B Hellman

Publications and source records attributed to B Hellman.

At least 55 records · Page 3Linked to original sources

PubMed1995

Demonstration of chlorobenzene-induced DNA damage in mouse lymphocytes using the single cell gel electrophoresis assay.

The DNA damaging effect of chlorobenzene was investigated in peripheral lymphocytes and bone marrow cells from C57BL/6 female mice using a gel electrophoresis assay for DNA from single cells ('the single cell gel electrophoresis assay') under alkaline conditions. The effect of chlorobenzene was studied both after single and repeated intraperitoneal injections of 750 mg/kg body weight. The cytostatic agent cyclophosphamide (150 mg/kg, i.p.) was used as a reference substance, and vehicle-treated mice as controls. DNA damage was recorded 16 h after the (last) injection, using an automated computerized image analysis system specifically designed for the single cell gel electrophoresis assay. There was evidence of chlorobenzene-induced DNA damage after 3 days of repeated exposure in peripheral lymphocytes, but no indications of such an effect in bone marrow cells. Cyclophosphamide induced significant damage to DNA both in bone marrow cells and lymphocytes, the effect being most pronounced in the latter cells. It is concluded that high-dose exposure to chlorobenzene is associated with genotoxicity to peripheral lymphocytes. However, this solvent is apparently not a major hazard to bone marrow cells, even after repeated high-dose exposure.

Analysis of Variance↗

PubMed1995

Alpha 2-adrenergic stimulation counteracts glucose-induced rise of sodium in pancreatic islets exposed to ouabain.

The effects of alpha 2-adrenergic activation by clonidine on sodium handling were analysed in beta-cell-rich pancreatic mouse islets. In the steady-state situation, clonidine (1 microM) amplified lowering of sodium induced by 20 mM glucose, while the content remained unchanged in 3mM glucose. The loss of sodium in Na(+)-deficient medium was stimulated by glucose but was not affected by clonidine. This agonist also did not influence the ouabain-induced uptake of sodium at 3 mM glucose but partially counteracted additional uptake in response to 20 mM glucose. Although lacking effects of its own, 5 microM yohimbine completely counteracted the action of clonidine. The glucose amplification of the ouabain-induced uptake of sodium was suppressed also by 10 microM of the Ca(2+)-channel blockers methoxyverapamil and diltiazem. Both tolbutamide (100 microM) and dibutyryl cyclic AMP (1 mM) mimicked the action of glucose by promoting clonidine-sensitive uptake of sodium in the presence of ouabain. It is concluded that activation of alpha 2-adrenoceptors has profound effects on the sodium handling of pancreatic beta-cells exposed to glucose and other stimulators of insulin release.

Adrenergic alpha-2 Receptor Agonists↗

PubMed1995

Glucose induces oscillations of cytoplasmic Ca2+, Sr2+ and Ba2+ in pancreatic beta-cells without participation of the thapsigargin-sensitive store.

Individual pancreatic beta -cells were used to study the glucose effects on the handling of Ca2+, Sr2+ and Ba2+. In extracellular medium containing one of these ions, single beta -cells responded to 11 mM glucose with large amplitude oscillations in cytoplasmic Ca2+, Sr2+ or Ba2+ with indistinguishable average frequencies (0.30-0.33/min). The oscillations disappeared after hyperpolarization with 400 microM diazoxide. Under such hyperpolarization, glucose stimulated the sequestration of Ca2+ and Sr2+ but not of repetitively mobilized by consecutive exposures to 100 microM carbachol. A 2-3 min exposure to 100 nM of the intracellular Ca(2+)-ATPase inhibitor thapsigargin also mobilized Ca2+ and Sr2+ and irreversibly abolished subsequent release by carbachol. However, thapsigargin did not prevent the large amplitude oscillations in Ca2+, Sr2+ or Ba2+ under non-hyperpolarizing conditions although the frequency of the Ca2+ oscillations was almost doubled. The results indicate that the slow oscillatory behavior of glucose-stimulated individual beta -cells does not depend on inositol 1,4,5-trisphosphate mediated release of intracellular Ca2+.

Animals↗

PubMed1995

The concepts of tail moment and tail inertia in the single cell gel electrophoresis assay.

Single cell gel electrophoresis under alkaline conditions is a technique used to detect primary DNA damage in individual mammalian cells. Cells embedded in agarose on microscope slides are subjected to lysis, unwinding of DNA and electrophoresis at high pH. After staining with a fluorescent dye, cells with DNA damage display increased migration of genetic material from the cell nucleus. The damage is quantified by measuring the displacement between the genetic material of the nucleus ('comet head') and the resulting 'tail'. The torsional moment of the tail ('tail moment') has been suggested to be an appropriate index of induced DNA damage in considering both the migration of the genetic material as well as the relative amount of DNA in the tail. In the present paper it will be shown that the moment of inertia ('tail inertia'), a not previously described tail parameter, provides a more precise description of the distribution of individual DNA fragments within the tails. The tail inertia was also found to be the most sensitive indicator of the DNA damage induced in peripheral lymphocytes from mice given a single intraperitoneal injection of cyclophosphamide (150 mg/kg b.w.). It is concluded that the tail inertia is an important complement to other tail parameters when looking for damage of DNA with the single cell gel electrophoresis assay.

Animals↗

PubMed1994

Variations in ATP-sensitive K+ channel activity provide evidence for inherent metabolic oscillations in pancreatic beta-cells.

The cell-attached configuration of the patch clamp technique was used for studying slow variations in the activity of the ATP-sensitive K+ channels in pancreatic beta-cells isolated from mouse and man. In 0 or 3 mM glucose, the fraction of time the channels were open exhibited oscillations with frequencies in the 0.25-0.40/min range. This phenomenon is a strong argument for inherent fluctuations in the ATP production of the beta-cells. Variations in metabolism may thus be a major determinant for the characteristic large amplitude oscillations of cytoplasmic Ca2+ with equivalent frequency.

Action Potentials↗

PubMed1994

Sulfonylureas mimic glucose in stimulating the uptake of Na+ in pancreatic islets exposed to ouabain.

The increase of sodium in response to ouabain inhibition of the Na+/K+ pump was measured in beta-cell-rich pancreatic islets from ob/ob mice using integrating flame photometry. D-Glucose promoted the uptake of sodium when added at a concentration of 6 mM or above. The hypoglycemic sulfonylurea compound, tolbutamide, mimicked the action of D-glucose in stimulating the sodium uptake at concentrations of 10 microM or above. There was no stimulation beyond that obtained with 20 mM glucose during exposure to 100 microM tolbutamide. Other test substances also affected sodium uptake in a way reflecting known effects on insulin release. Accordingly, the sodium uptake was stimulated with glibenclamide, glipizide, HB 699 (4-[2-(5-chloro-2-methoxybenzamido)ethyl]benzoic acid) and high K+. Sulphonamides (sulfamethazole, sulfadiazine and sulfadoxine) had practically no effect when added at a concentration of 1 mM. Sodium uptake in response to glucose and tolbutamide was antagonized by 400 microM diazoxide or 4 microM tetrodotoxin. It is concluded that both glucose and hypoglycemic sulfonylureas stimulate Na+ entry into the pancreatic beta-cells, a process presumably involving depolarization.

Animals↗

PubMed1994

Synchronous oscillations of cytoplasmic Ca2+ and insulin release in glucose-stimulated pancreatic islets.

The cytoplasmic Ca2+ concentration ([Ca2+]i) was measured in single pancreatic mouse islets superfused in a system allowing concomitant recordings of insulin release. When glucose was raised from 3 to 11 mM, [Ca2+]i responded by a transient lowering followed by a rise to an average level of 192 +/- 11 nM. In 77% of the islets the rise was associated with the gradual appearance of oscillations, which were either fast (2-7/min), slow (0.3-0.9/min), or a combination of both types. The characteristics of the fast [Ca2+]i oscillations were those expected from a relationship with the electrical burst activity in islets. Accordingly, in most cases the fast oscillations were remarkably regular. The slow [Ca2+]i oscillations had characteristics similar to the large amplitude ones in individual beta-cells. Whereas glucagon and dibutyryl cAMP could transform slow islet oscillations into fast ones, the alpha 2-adrenergic agonist clonidine had the opposite effect. The rapid islet oscillations were also facilitated by elevated concentrations of extracellular Ca2+. Reinforcing the arguments for [Ca2+]i oscillations as responsible for a pulsatile insulin secretion it was possible to demonstrate that the release of the hormone from single islets is synchronized with the slow [Ca2+]i oscillations.

Animals↗

PubMed1994

Glucose-induced oscillations of Ba2+ in pancreatic beta-cells occur without involvement of intracellular mobilization.

Ba2+ was used as a substitute for Ca2+ in analyzing the mechanisms responsible for glucose-induced Ca2+ oscillations in pancreatic beta-cells. The 340/380-nm fluorescence excitation ratio was recorded in individual mouse beta-cells loaded with the indicator fura-2. In 3 mM glucose, Ba2+ entered the cell in a concentration-dependent manner and was partially extruded when the ion was removed from the medium. The extrusion of Ba2+ from the beta-cell was dependent on external Na+, suggesting that Ba2+ can substitute for Ca2+ in Na+/Ca2+ countertransport. When extracellular Ba2+ was kept between 0.3 and 0.5 mM, large amplitude oscillations (0.1-0.4 min-1) were induced by glucose at concentrations above 7 mM. The oscillations were often transformed into a sustained elevation either by increase of the glucose or Ba2+ concentrations or by the additions of glucagon, forskolin, or carbachol. Although Ba2+ could substitute for Ca2+ in the glucose-induced large amplitude oscillations, there were no Ca(2+)-like pronounced spikes superimposed on an elevated cytoplasmic Ba2+ after elevation of cyclic AMP. Neither could Ba2+ substitute for Ca2+ in being incorporated in response to glucose into a pool mobilizable by carbachol. The studies indicate that cations other than Ca2+ can oscillate in response to glucose, and that such oscillations do not require mobilization from internal pools sensitive to inositol 1,4,5-trisphosphate.

Animals↗

PubMed1994

Glucose induces oscillatory Ca2+ signalling and insulin release in human pancreatic beta cells.

Mechanisms of pulsatile insulin release in man were explored by studying the induction of oscillatory Ca2+ signals in individual beta cells and islets isolated from the human pancreas. Evidence was provided for a glucose-induced closure of ATP-regulated K+ channels, resulting in voltage-dependent entry of Ca2+. The observation of step-wise increases of capacitance in response to depolarizing pulses suggests that an enhanced influx of Ca2+ is an effective means of stimulating the secretory activity of the isolated human beta cell. Activation of muscarinic receptors (1-10 mumol/l carbachol) and of purinergic P2 receptors (0.01-1 mumol/l ATP) resulted in repetitive transients followed by sustained elevation of the cytoplasmic Ca2+ concentration ([Ca2+]i). Periodic mobilisation of intracellular calcium was seen also when injecting 100 mumol/l GTP-gamma-S into beta cells hyperpolarized to -70 mV. Individual beta cells responded to glucose and tolbutamide with increases of [Ca2+]i, manifested either as large amplitude oscillations (frequency 0.1-0.5/min) or as a sustained elevation. Glucose regulation was based on sudden transitions between the basal and the two alternative states of raised [Ca2+]i at threshold concentrations of the sugar characteristic for the individual beta cells. The oscillatory characteristics of coupled cells were determined collectively rather than by particular pacemaker cells. In intact pancreatic islets the glucose induction of well-synchronized [Ca2+]i oscillations had its counterpart in 2-5 min pulses of insulin. Each of these pulses could be resolved into regularly occurring short insulin transients. It is concluded that glucose stimulation of insulin release in man is determined by the number of beta cells entering into a state with Ca(2+)-induced secretory pulses.

Adenosine Triphosphate↗

PubMed1994

Supramaximal decrease of sulphonylurea-induced accumulation of sodium in pancreatic islets.

Sodium was measured in beta-cell-rich pancreatic islets of mice under steady state conditions or after 6 min of exposure to 1 mM ouabain. The islet content of sodium increased when 100 microM tolbutamide or 1 microM glipizide were added to an albumin-containing (1 mg ml-1) medium, but remained unaffected at 10-fold higher concentrations. Both sulphonylurea compounds promoted the uptake of Na+ in the presence of ouabain. Whereas tolbutamide was stimulatory at 10 microM or above in a medium containing 10 mg ml-1 albumin, only 0.1 microM was required in the absence of albumin. In the latter situation there was a reduction of the stimulatory action with increase of the tolbutamide concentration from 100 to 1000 microM. The inhibitory component in the sulphonylurea action on the Na+ uptake was particularly impressive with glipizide, maximal stimulation being reached at 10 microM in the presence of 1 mg ml-1 albumin. Diazoxide (400 microM) modified the glipizide action on Na+ uptake, making 1000 microM stimulatory instead of 1 microM. The latter concentration of glipizide became inhibitory after removal of K+. Glipizide stimulated the Na+ uptake both at low and high concentrations in a medium deficient in Ca2+ or when the cotransport of Na+, K+ and Cl- was blocked by 20 microM bumetanide. The observation that the sulphonylurea-induced islet accumulation of sodium is diminished at supramaximal concentrations reinforces existing arguments for additional effects of high concentrations of hypoglycemic sulphonylureas.

Animals↗

PubMed1994

Local changes of medium in studies of individual cells.

A procedure is described for changing the medium surrounding individual cells attached to the bottom of a cell chamber. A small hole at the "apex" of a plastic U-tube allowed application and withdrawal of medium. The medium to be applied was perfused through the U-tube by pressure at one end and suction at the other. To prevent premature delivery of new medium from the U-tube, suction of the outlet dominated resulting in a net withdrawal of medium from the cell chamber. The flow of medium through the hole could be reversed rapidly by arresting the suction with an electromechanical valve. In this way it was possible to obtain 95% replacement of medium within 60 ms. A pressure transient arising from the closure of the valve was damped by the presence of a small air bubble in the system. To secure a precise deposition of medium and minimize the risk of mechanical disturbances to the cell it was essential to be able to inspect the medium changes visually. For this purpose the fluorescent indicator rhodamine B bound to dextran proved satisfactory. Free rhodamine B could not be used because it had biological effects, as was evident from studying ATP-regulated K+ channels in pancreatic beta-cells. When using a purpose-designed syringe pump for perfusing the U-tube, the technique allows well controlled exposure of individual cells to test substances added together with dextran-linked rhodamine B.

Adenosine Triphosphate↗

PubMed1993

Glucose-induced cycles of insulin release can be resolved into distinct periods of secretory activity.

The periodic behaviour of glucose-stimulated insulin release was studied using islets isolated from ob/ob-mice and rats. A single islet was perifused at a rate of 150 microliters/min and insulin measured in samples of the medium taken at intervals as short as 3 s. Exposure to 11 mM glucose resulted in intermittent insulin release, implying that previously observed 2-3 min cycles of secretion could be resolved into regularly occurring fast (< 25 s) transients. A further raise of glucose to 20 mM was associated with increase of the amplitudes of the transients leaving their frequencies unaffected. The discovery of the insulin transients raises the question whether there is a quantal release of the hormone in response to glucose-induced rapid oscillations of cytoplasmic Ca2+ in the pancreatic beta-cells.

Activity Cycles↗

PubMed1993

Dual effects of Na/K pump inhibition on cytoplasmic Ca2+ oscillations in pancreatic beta-cells.

Inhibition of the Na/K pump by ouabain or removal of K+ resulted in gradual increase of intracellular sodium in beta-cell-rich pancreatic islets from ob/ob-mice exposed to 3 mM glucose. In individual beta-cells this action of ouabain was paralleled by closure of ATP-regulated K+ channels and a slow elevation of the cytoplasmic Ca2+ concentration ([Ca2+]i). In most beta-cells an increase of the glucose concentration to 11-20 mM induced large amplitude oscillations of [Ca2+]i with a frequency of 0.2-0.5/min. Ouabain had dual actions on these glucose-induced oscillations in promoting their appearance and at higher concentrations transforming them into a sustained increase of [Ca2+]i. At 100 microM ouabain reduced the frequency of the glucose-induced oscillations but nevertheless raised the time-average [Ca2+]i by increasing the amplitudes and half-widths of the Ca2+ peaks. When high concentrations of ouabain or removal of K+ transformed the oscillations into a sustained increase of [Ca2+]i, the level reached exceeded that obtained in response to rise of glucose alone. By favoring Ca2+ entry and counteracting removal of the cation from the cytoplasm, Na/K pump inhibition perturbs the balance between the processes determining glucose-induced oscillations of [Ca2+]i.

Animals↗

PubMed1993

Regulation of proliferation in JEG-3 cells by a 500-kDa Ca2+ sensor and parathyroid hormone-related protein.

JEG-3 cells are derived from human trophoblasts and demonstrated to express a 500-kDa Ca2+ sensing protein, which elicits biphasic elevations of cytoplasmic Ca2+ concentrations ([Ca2+]i) and mediates Ca2+ regulation of parathyroid hormone-related protein (PTHrP) release from placental cytotrophoblasts. Cytocentrifuged JEG-3 cells were immunostained by monoclonal and polyclonal antiserum toward PTHrP (1-34) and (38-64). Elevation of external Ca2+ from 0.5 to 3.0 mM induced only a sluggish rise in [Ca2+]i and no stimulation of cAMP production despite a more than twofold elevation of PTHrP(1-34) release. Monoclonal antibodies recognizing functionally discrepant epitopes of the Ca2+ sensor protein substantiated uncoupling of this sensor in the Ca(2+)-regulated PTHrP release. Exogenous activation of protein kinase C by a phorbol ester strongly augmented the secretion of PTHrP(1-34), whereby uncoupling of the Ca2+ sensor was partially reversed. This functional differentiation was associated with reduced [3H]thymidine incorporation in JEG-3 cells. Proliferation of these cells was inhibited by 71% upon rise of extracellular Ca2+ from 0.5 to 3.0 mM, and this inhibition was abolished by antibody-mediated interference with the Ca2+ sensor function. PTHrP(1-86) and PTH(1-34) at concentrations up to 10(-7) M decreased proliferation and stimulated the cAMP content of JEG-3 cells. The findings support concomitant Ca2+ sensor and PTH/PTHrP receptor expression in JEG-3 cells, and that Ca2+ inhibits proliferation by actions on the Ca2+ sensor as well as by stimulation of PTHrP release possibly mediating autocrine growth inhibition.

Calcium↗

PubMed1993

Glucose-induced amplitude regulation of pulsatile insulin secretion from individual pancreatic islets.

The insulin secretory response to glucose was studied in single pancreatic islets isolated from ob/ob mice and rats. The perfusate from an individual islet was collected during 18-s periods and analyzed for insulin with an ELISA technique. Increase of the glucose concentration from 3 to > or = 5.5 mM resulted in pulses of insulin release often originating from the basal level and having a frequency of 0.4/min. Glucose regulation of insulin release from the individual islet was manifested by alterations of the amplitudes of the pulses but not of their frequency. It is concluded that the large amplitude oscillations of cytoplasmic Ca2+ known to occur in the pancreatic beta-cells have their counterpart in pulses of insulin release and that glucose stimulation of the secretory activity may be the result of recruitment of more beta-cells into an oscillatory state.

Animals↗

PubMed1993

Blood lead concentrations of Swedish preschool children in a community with high lead levels from mine waste in soil and dust.

The lead concentration in capillary blood was investigated in 49 preschool children (0.7-7.4 years of age) visiting a day-care center in a Swedish community with high lead contamination from mining and milling in soil and dust in populated areas [up to 1400 and 14,000 micrograms.g-1 (6.76 and 67.63 mumol.g-1) of dry weight, respectively]. The blood lead levels were examined twice (in April and in September) in 33 of the children. The lead levels were low on both sampling occasions [arithmetic mean 31 (SD 13, median 30, range 13-79) micrograms.l-1, ie, arithmetic mean 0.15, (SD 0.06, median 0.14, range 0.06-0.38) mumol.l-1]. Whereas children up to four years of age showed significantly increased levels from April to September, a significant decrease was seen in older children. The level of lead in soil at home, gender, smoking habits at home, and estimated level of hand-to-mouth activity did not appear as strong determinants of lead in blood. The results indicate that lead from mine waste in soil and dust fallout does not constitute a significant health hazard for preschool children in Falun.

Child↗

PubMed1992

Cytoplasmic Ca2+ oscillations in pancreatic beta-cells.

In the last 15 years it has been a growing interest in the cyclic variations of circulating insulin [46]. After the suggestion that this phenomenon may be due to oscillations of the beta-cell membrane potential [8,39], it was demonstrated that [Ca2+]i oscillates in the glucose-stimulated beta-cell with a similar frequency to that of pulsatile insulin release. The present review describes four types of [Ca2+]i oscillations in the pancreatic beta-cell. The slow sinusoidal oscillations, referred to as type-a, are those which most closely correspond to pulsatile insulin release. Although not affecting the properties of the type-a oscillations in individual beta-cells, the concentration of glucose is a determinant for their generation and further transformation into a sustained increase. Accordingly, cytoplasmic Ca2+ is regulated by sudden transitions between oscillatory and steady-state levels at threshold concentrations of glucose, which are characteristic for the individual beta-cell. This behaviour explains the observation of a gradual recruitment of previously non-secreting cells with increase of the extracellular glucose concentration [44]. However, it still remains to be elucidated how the sudden transitions between these three states translate into the co-ordinated slow oscillations of [Ca2+]i in the intact islet. Cyclic variations of circulating insulin require a synchronization of the [Ca2+]i cycles also among the islets in the pancreas. It is still an open question by which means the millions of islets communicate mutually to establish a pattern of pulsatile insulin release from the whole pancreas. The discovery that the beta-cell is not only the functional unit for insulin synthesis but also generates the [Ca2+]i oscillations required for pulsatile insulin release has both physiological and clinical implications. The fact that minor damage to the beta-cells prevents the type-a oscillations with maintenance of a glucose response in terms of raised [Ca2+]i reinforces previous arguments [54] that loss of insulin oscillations is an early indicator of type-2 diabetes. Further analyses of the [Ca2+]i oscillations in the beta-cells should include not only the mechanisms for their generation and subsequent propagation within or among the islets but also how modulation of their frequency affects the insulin sensitivity of various target cells. The latter approach may be important in the attempts to maintain normoglycemia under conditions minimizing the vascular effects of insulin supposed to precipitate hypertonia and atherosclerosis [70,71,77].

Animals↗

PubMed1992

Glycine transformation of Ca2+ oscillations into a sustained increase parallels potentiation of insulin release.

Increase of the glucose concentration from 3 to 11 mM resulted in a triphasic release of insulin from perifused ob/ob-mouse beta-cells. A slight inhibition was followed after 2 min by a marked peak and a less pronounced sustained response. At the lower glucose concentration glycine had only marginal effects. However, in the presence of 11 mM glucose, 1-10 mM glycine triggered an immediate and dose-dependent response with an initial peak of insulin release followed by sustained stimulation. In individual beta-cells, rise of the glucose concentration from 3 to 11 mM induced initial lowering of the cytoplasmic Ca2+ concentration ([Ca2+]i) followed by large amplitude oscillations from a level of 50-90 nM to peak values exceeding 300 nM. Already at a concentration of 1 mM, glycine transformed the oscillatory pattern into a sustained level with increase of time-average [Ca2+]i. This elevation became more pronounced in the presence of 10 mM glycine. The effects of glycine on insulin release and [Ca2+]i required extracellular Na+ and were reproduced with the N-methyl analogue sarcosine. It is suggested that glycine potentiation of secretion reflects the elevation of time-average [Ca2+]i both by increased entry and reduced elimination of the cation from the cytoplasm.

Animals↗