Effect of the plant product croton oil on the in vivo incorporation of 3H-thymidine. Evidence for a pronounced inhibition in the thymus.
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Animals↗
Biomedical subjects
B Hellman
Publications and source records attributed to B Hellman.
Modifying effects of a single injection of phenobarbital on the inhibitory action of benzo(a)pyrene on 3H-thymidine incorporation into various organs of the mouse.
The effects of the procarcinogen benzo(a)pyrene and the enzyme inducer phenobarbital on the DNA turnover in various organs of male C57BL mice were evaluated by measuring the incorporation of [6-3H]thymidine. When injected intraperitoneally 48 h before sacrifice, benzo(a)pyrene (28.8 mg/kg body weight) inhibited the incorporation of 3H-thymidine into the spleen, thymus, testis, and small intestine. A corresponding analysis with phenobarbital sodium (75 mg/kg b.w.) revealed reduced incorporation of 3H-thymidine into the pancreas (after 24 h) and bone marrow (after 72 h). The effects of combining benzo(a)pyrene and phenobarbital was examined by injecting the latter agent either 24 h before or after the polycyclic hydrocarbon. A previous injection of phenobarbital resulted in a potentiation of the inhibitory action of benzo(a)pyrene in the spleen. However, when phenobarbital was given after benzo(a)pyrene, there was a five-fold increase of the 3H-thymidine incorporation into the liver in comparison to the controls given vehicle. The demonstration that a single injection of phenobarbital has modifying effects on the 3H-thymidine incorporation both when administered alone and in combination with benzo(a)pyrene indicates that enzyme inducers may influence the outcome in genotoxicity tests.
Animals↗
Beta-cell cytoplasmic Ca2+ balance as a determinant for glucose-stimulated insulin release.
The introduction of new techniques and the access to clonal lines of insulin-secreting cells have enabled re-evaluation of glucose effects on Ca2+ movements in pancreatic beta cells. It became evident that glucose, in addition to stimulating the entry of Ca2+, also promotes active sequestration of the ion in intracellular stores and its extrusion from the beta cells. The balance between these processes will determine the activity of Ca2+ in the cytoplasm and consequently the rate of insulin release. With the demonstration that glucose can not only increase but also lower cytoplasmic Ca2+, it follows that exposure to the sugar under certain conditions results in a paradoxical inhibition of insulin release. In diabetic patients this may be manifest as prompt reduction of circulating concentrations of insulin and C-peptide after an intravenous injection of glucose. The concept of the dual action of glucose might aid in explaining a number of poorly understood phenomena, such as the induction of rhythmic oscillations of the membrane potential of beta cells and the fact that their secretory response is improved by prolonged exposure to glucose and after priming with the sugar.
Animals↗
Tissue-specific inhibition of [3H]thymidine incorporation into DNA by carcinogenic N-nitrosamines.
The N-nitrosamines N-nitrosodimethylamine (DMN), N'-nitrosonornicotine (NNN) and 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) were injected intraperitoneally 24 h before sacrifice in F344 rats and C57BL mice in doses of 297 mumoles/kg b.w. and 148 mumoles/kg b.w., respectively. 2 h before sacrifice, the animals were given an intraperitoneal injection of [3H]thymidine. The results showed that the examined N-nitrosamines inhibited the incorporation of [3H]thymidine into DNA in a few tissues of the rats and the mice. The results indicated that the N-nitrosamines exerted a tissue-specific inhibition of the [3H]thymidine incorporation in the tissues reported to be involved in the biotransformation of these substances. The observed inhibitory effects on the incorporation of [3H]thymidine by DMN, NNN and NNK were also correlated to a considerable extent to the reported sites of carcinogenicity. The present study indicates that measurements of [3H]thymidine incorporation into DNA in various tissues of experimental animals is a useful short-term bioassay to evaluate the potential tissue-specific carcinogenicity of the N-nitrosamines. The method may also be useful as a complement to other short-term in vivo tests in the screening of potential genotoxicity of several other chemicals.
Animals↗
Effects of altered Ca2+ and Mg2+ concentrations on proliferation and functional differentiation of the clonal insulin-producing cells RINm5F.
Effects of calcium and magnesium on proliferation and functional differentiation were investigated in the clonal insulin-producing rat cell line RINm5F. Variations of the extracellular concentrations of Ca2+ and Mg2+ had only minor effects on the cellular contents of these elements. Even when the medium was depleted of extracellular Ca2+ by the addition of EGTA, the Ca/Mg ratio corresponded to approximately 0.1. In a Mg2+-deficient medium a lowering of the extracellular calcium to 0.13 mM reduced the amount of insulin in the media but increased that in the cells. Irrespective of the concentration of Mg2+, depletion of Ca2+ was associated with low media levels of insulin and a reduced proliferation rate. In addition, the cells became smaller in size and showed an increased ability to exclude trypan blue. a rise of the magnesium concentration to 6.30 mM was accompanied by a decrease of the cellular content of insulin despite reduced amounts of hormone in the medium. This finding might be due to suppression of insulin biosynthesis, suggesting that the RINm5F cells have a markedly different metabolism and/or sensitivity to extracellular magnesium than non-tumorigenic beta-cells.
Adenoma, Islet Cell↗
Effects of zinc during culture of an insulin-producing rat cell line (RINm5F).
The effects of various Zn2+ concentrations on cell proliferation, insulin secretion and contents of insulin and zinc were studied in a clonal cell line (RINm5F) established from a transplantable rat islet tumor. The RINm5F cells were equally effective in proliferating and releasing insulin at zinc concentrations ranging from 0.013 to 0.073 mM. The percentage of cells able to exclude trypan blue was significantly less in cultures with 0.073 mM Zn in the medium. Increasing the extracellular concentrations of Zn2+ to 0.044 and 0.073 mM, respectively, resulted in a 40-60% reduction in the cellular content of insulin. There was a significant increase (73%) in the cellular content of zinc only when increasing the extracellular concentration of the element to 0.073 mM. The addition of 0.2 mM EGTA to a zinc-deficient medium had no effect on proliferation, insulin release or content of insulin and zinc, indicating the presence of a stable endogenous pool of zinc maintaining the function of the RINm5F cells for at least 5 days in culture.
Adenoma, Islet Cell↗
Proton microprobe analysis of pancreatic beta-cells.
Freeze-dried pancreas sections from obese-hyperglycemic mice were subjected to proton bombardment and the elemental contents in the beta-cells and the exocrine part were obtained from the characteristic X-rays emitted. Quantitative data were provided for 18 different elements. The mole ratio between K and Na exceeded 10, implying that neither the sample preparation nor the irradiation had induced significant diffusive changes. With the demonstration of this high K/Na ratio it seems likely that also the beta-cells are equipped with an efficient Na+/K+ pump. The beta-cells contained about 70 mmoles Cl per litre cell water. Observed amounts of Ca and Mg were equivalent to those previously recorded by electrothermal atomic absorption spectroscopy. The significant role of Zn for the storage of insulin was emphasized by the demonstration of 3 times as much of this element in the beta-cells as compared with the exocrine pancreas. In addition, the sensitivity of the proton microprobe enabled measurements of various trace elements such as Rb, Cr, Cu, Al and Pb not previously demonstrated in the pancreatic beta-cells.
Animals↗
The dual action of glucose on the cytosolic Ca2+ activity in pancreatic beta-cells. Demonstration of an inhibitory effect of glucose on insulin release in the mouse and man.
The cytosolic Ca2+ activity was measured with the fluorescent indicator quin-2 in pancreatic beta-cells obtained from obese-hyperglycemic mice. When present at a concentration of 20 mmol/l in a medium physiologically balanced in cations, glucose induced a rise of cytosolic Ca2+ after a delay of 1--3 min. At lower concentrations of extracellular Ca2+ this effect was not only prevented but the sugar promptly reduced the cytosolic Ca2+ activity. The dual effect of glucose on cytosolic Ca2+ had its counterpart in the release of insulin. Whereas 20 mmol/l of glucose stimulated the release of insulin from mouse islets previously stored in a Ca2+-deficient medium, the sugar was clearly inhibitory when present at a concentration of 6 mmol/l. Intravenous glucose tolerance tests revealed a temporary glucose depression of the serum concentrations of insulin and C-peptide in several patients with diabetes. In a mentally retarded girl with hyperinsulinemia associated with acanthosis nigricans the glucose suppression of circulating insulin was prolonged and sufficiently pronounced to suggest an almost complete inhibition of the secretory activity of the pancreatic B-cells.
Aminoquinolines↗
Effects of glucose deprivation and altered Ca2+ concentrations on clonal insulin-producing cells (RINm5F).
The effects of various Ca2+ concentrations and glucose deprivation on cell proliferation, insulin secretion and contents of insulin, calcium and magnesium were studied in a clonal cell line (RINm5F) established from a transplantable rat islet tumor. The RINm5F cells were equally effective in proliferating and releasing insulin at Ca2+ concentrations ranging from 0.16 to 4.4 mM. In contradistinction to normal beta-cells, the amounts of calcium in the RINm5F cells remained relatively constant after lowering the extracellular concentration of Ca2+ and inhibiting the entrance of the ion with D-600. However, culture at 4.4 mM Ca2+ resulted in substantial incorporation of cellular calcium and a simultaneous reduction of the insulin content. Cells deprived of glucose had a significantly lower dry weight, were less able to exclude trypan blue and contained and released less insulin. Since protein biosynthesis is strictly dependent upon an adequate intracellular concentration of Mg2+, these long term effects of glucose removal might be related to the concomitant decrease in the cellular content of magnesium.
Animals↗
Glucose inhibits insulin release when not promoting the entry of calcium into the beta-cells.
The basal Ca2+ permeability of islets from ob/ob-mice was raised by culture in a Ca2+-deficient medium. The resulting secretory activity upon transfer to a higher Ca2+ concentration was significantly inhibited by 6 mM glucose although higher concentrations of the sugar further stimulated insulin release. In the presence theophylline, the inhibitory effect of 6 mM glucose was altered into a stimulatory one. After blocking the voltage-dependent channels for Ca2+ with D-600, 20 mM glucose did not enhance but significantly inhibited insulin release. The demonstration of a paradoxical glucose inhibition of insulin release is in accordance with recent reports that the sugar not only increases but also can lower the cytoplasmic Ca2+ activity in the pancreatic beta-cells.
Animals↗
Interaction of sulfonylurea with the pancreatic B-cell.
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4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
Dual effects of glucose on the cytosolic Ca2+ activity of mouse pancreatic beta-cells.
The cytosolic Ca2+ activity of mouse pancreatic beta-cells was studied with the intracellular fluorescent indicator quin2 . When the extracellular Ca2+ concentration was 1.20 mM, the basal cytosolic Ca2+ activity was 162 +/- 9 nM. Stimulation with 20 mM glucose increased this Ca2+ activity by 40%. In the presence of only 0.20 mM Ca2+ or after the addition of the voltage-dependent Ca2+ -channel blocker D-600, glucose had an opposite and more prompt effect in reducing cytosolic Ca2+ by about 15%. It is concluded that an early result of glucose exposure is a lowering of the cytosolic Ca2+ activity and that this effect tends to be masked by a subsequent increase of the Ca2+ activity due to influx of Ca2+ through the voltage-dependent Ca2+ channels.
Animals↗
Glucose inhibits 45Ca efflux from pancreatic beta-cells also in the absence of Na+-Ca2+ countertransport.
During perifusion with medium deprived of Ca2+, addition of glucose or omission of Na+ resulted in prompt and quantitatively similar inhibitions of 45Ca efflux from beta-cell rich pancreatic islets microdissected from ob/ob mice. Glucose had no additional inhibitory effect when Na+ was isoosmotically replaced by sucrose or choline+. When K+ was used as a substitute for Na+, the inhibitory effect of Na+ removal on 45Ca efflux became additive to that of glucose. The observation that glucose can be equally effective in inhibiting 45Ca efflux in the presence or absence of Na+ is difficult to reconcile with the postulate that the Na+-Ca2+ countertransport mechanism is a primary site of action for glucose.
Animals↗
Effect of hypoglycemic sulfonylureas on Ca2+ fluxes across lipid bilayers.
Black lipid membranes and liposomes loaded with Ca2+ or 5,6-carboxyfluorescein were used for exploring the mechanism of action of insulin-releasing sulfonylureas. Unlike the Ca2+/H+ exchanging ionophore A-23187, tolbutamide did not stimulate the net efflux of Ca2+ from the liposomes. Glibenclamide caused a sustained release of Ca2+, but this effect could be attributed to labilization of the liposomal membrane as indicated by a quantitatively similar loss of the stability marker 5,6-carboxyfluorescein. Unlike the neutral ionophore nonactin or the channel forming quasi-ionophore gramicidin A, the sulfonylureas did not alter the conductance of black lipid membranes in medium containing Na+, K+, Ca2+, Mg2+, and Cl-. It is concluded that the sulfonylureas tested lack ionophore properties but that glibenclamide can labilize membranes.
Biological Transport↗
The in vivo uptake of tritiated thymidine as a potential short-term test of toxic effects of polycyclic aromatic hydrocarbons in different organs.
A rapid in vivo test for toxicity is described where the test substance is allowed to distribute and metabolize in the intact mouse. Quantitative data are provided on the effect of 3 different polycyclic aromatic hydrocarbons on the DNA turnover in various organs, measured as the incorporation of tritiated thymidine. In accordance with previously reported carcinogenic potencies, 7,12-dimethylbenz[alpha]anthracene (DMBA) was more potent in inhibiting the thymidine incorporation than benzo[alpha]pyrene (B[alpha]P). The inhibitory effect was most pronounced in spleen, lung, pancreas, small intestine and kidney, resulting in a decrease of incorporated activity with up to 80%. There was no evidence for the existence of specific target organs for DMBAs effects on thymidine incorporation as indicated by an inhibitory action in all organs studied. A decreased thymidine incorporation after administration of DMBA could also be demonstrated with whole-body autoradiography. The inhibitory effect of B[alpha]P was most pronounced in thymus, spleen, small intestine and testis, the average decrease of incorporated activity being more than 40% after 48 h. Contrary to the wide action of the above mentioned polycyclic aromatic hydrocarbons, an equimolar dose of anthracene lacked significant effects on the various organs.
9,10-Dimethyl-1,2-benzanthracene↗
Evidence for glucose stimulation of intracellular buffering of calcium in the pancreatic beta-cell.
Glucose-induced movements of Ca2+ in pancreatic beta-cells were analysed using islets isolated from ob/ob mice and insulin-releasing cells from a clonal cell line (RINm5F). Addition of glucose to a perifusion medium resulted in an inhibited efflux of 45Ca from the islets both when the extracellular Ca2+ concentration was lower or higher than that in the cytosol. Glucose inhibition of 45Ca efflux was seen also after altering the Na+ gradient across the plasma membrane provided that the cytosolic K+ activity was maintained. The glucose suppression of 45Ca efflux corresponded to a stimulated net uptake of Ca2+ in the RINm5F cells. Also in this case the glucose effect was maintained when Na+ was replaced with K+ and suppressed after substitution with choline. During perifusion of islets with a Ca2+-deficient medium the removal of glucose resulted in a temporary stimulation of insulin release. The results suggest that glucose, in addition to stimulating the entry of Ca2+, also promotes active sequestration of the ion in intracellular stores. The balance between these processes will determine the activity of cytosolic Ca2+ and consequently the rate of insulin release.
Animals↗
Mobilization of 45Ca from insulin-producing RINm5F cells attached to microcarriers.
A technique was designed allowing studies of 45Ca efflux from insulin-releasing cells of a clonal cell line (RINm5F) attached to fibronectin-coated plastic beads. Supporting the existence of Ca2+-Ca2+ and Na+-Ca2+ exchange mechanisms, the efflux of 45Ca in a Ca2+-deficient perifusion medium was increased after introduction of Ca2+ and decreased after removal of Na+. The washout of radioactivity was similar to that from pancreatic islets in also being inhibited by glucose in the presence of low concentrations of extracellular Ca2+. During perifusion with a medium containing 2.56 mM Ca2+, increasing the K+ concentration resulted in an increased 45Ca efflux, a process counteracted by D-600. Also, exposure to tolbutamide resulted in a distinct stimulatory peak. Glucose was a poor stimulator of 45Ca efflux, suggesting that this sugar lacks the ability to depolarize the RINm5F cells sufficiently to induce normal opening of the voltage-dependent channels in the plasma membrane. Such a defect might well explain why the RINm5F cells do not respond to glucose with stimulation of insulin release.
Animals↗
Differentiation between the short and long term effects of glucose on the intracellular calcium content of the pancreatic beta-cell.
The problem of how glucose affects the intracellular (La3+-nondisplaceable) calcium content of pancreatic beta-cells was approached by combining measurements of 45Ca in ob/ob-mouse islets loaded to isotopic equilibrium with determinations of calcium using electrothermal atomic absorption spectroscopy. Whereas short term changes of the glucose concentration induced marked alterations of insulin release, the islet content of intracellular 45Ca was remarkably stable. The chronic actions of glucose differed from the acute ones in being readily demonstrable and sometimes resulting even in a suppression of the calcium content. Thus, after 7 days of culture in 20 mM glucose, the amount of intracellular calcium was actually lower than when the islets were cultured at 5.5 mM glucose. The long term effect of glucose in suppressing the islet content of intracellular calcium was associated with degranulation and loss of immunoreactive insulin, indicated both from staining of the beta-cells and measurements of the extracted hormone by RIA. The previously unknown ability of glucose to suppress the islet content of intracellular calcium may consequently result from mobilization of the secretory granules.
Animals↗