Thapsigargin-sensitive cationic current leads to membrane depolarization, calcium entry, and insulin secretion in rat pancreatic beta-cells

Glucose-induced insulin secretion by pancreatic beta-cells depends on membrane depolarization and [Ca2+]i increase. We correlated voltage- and current-clamp recordings, [Ca2+]i measurements, and insulin reverse hemolytic plaque assay to analyze the activity of a thapsigargin-sensitive cationic channel that can be important for membrane depolarization in single rat pancreatic beta-cells. We demonstrate the presence of a thapsigargin-sensitive cationic current, which is mainly carried by Na+. Moreover, in basal glucose concentration (5.6 mM), thapsigargin depolarizes the plasma membrane, producing electrical activity and increasing [Ca2+]i. The latter is prevented by nifedipine, indicating that Ca2+ enters the cell through L-type Ca2+ channels, which are activated by membrane depolarization. Thapsigargin also increased insulin secretion by increasing the percentage of cells secreting insulin and amplifying hormone secretion by individual beta-cells. Nifedipine blocked the increase completely in 5.6 mM glucose and partially in 15.6 mM glucose. We conclude that thapsigargin potentiates a cationic current that depolarizes the cell membrane. This, in turn, increases Ca2+ entry through L-type Ca2+ channels promoting insulin secretion.     

SITS Blockade Induces Multiple Subconductance States in a Large Conductance Chloride Channel

The effect of the chloride channel blocker 4-acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid (SITS) on the gating and amplitude of an endothelial chloride channel was explored using the outside-out configuration of the patch-clamp technique. Under control conditions the channel displayed two main gating modes: shut and fully open. Transitions to equally spaced subconductance states were rarely observed (less than 10 events/minute). At low concentrations (<45 μm), SITS increased the number of transitions to the three subconductance states in a concentration-dependent manner, while reducing the number of transitions to the fully open state. This effect was maintained after removing SITS from the bath solution, suggesting that the modifications in the channel induced by SITS were irreversible. All four conducting states had similar current-voltage relationships. At higher concentrations (>45 μm), SITS reduced the amplitude of all conducting states (three subconductances and fully open). This effect was fully reversible upon SITS removal from the bath solution. A half-inhibitory concentration (IC₅₀) of 55.6 ± 2.7 μm (+60 mV) and 66.7 ± 2.2 (−60 mV) was obtained from the fitting to a Langmuir function. All these results are compatible with the existence of two SITS binding sites in the chloride channel: one of high affinity responsible for the increment in the number of transitions to subconductance states, and one low affinity binding site involved in the reduction of the amplitude of all conducting states. Received: 29 October 1998/Revised: 16 February 1999     

Variation of Transhexadecenoic Acid Content in Two Triazine Resistant Mutants of Chenopodium album and Their Susceptible Progenitor

Two atrazine resistant nutants of Chenopodium album L. and their susceptible progenitor were analyzed for lipid composition. In the phosphatidyldiacylglycerol the Δ3-trans-hexadecenoic acid (C16:1 trans) percentage was higher in the two resistant phenotypes. However, this difference appears later in the development of the leaves and is not clearly observed in young leaves and seedlings. Thus, the increase of the C16:1 trans during the leaf development of the resistant phenotypes is probably a secondary effect of the psbA mutation that arises in compensation for some photosynthesis deficiency. The significance of the lipid differences shown between the two resistant mutants is discussed in terms of whether they are responsible of the two different levels of herbicide resistance observed in the field.     

Interaction of lipid peroxidation products with nuclear macromolecules

The interaction of lipid peroxidation products with nuclear macromolecules was investigated in rat liver nuclei labelled with [3H]arachidonic acid. Lipid peroxidation reactions were driven both non-enzymatically and enzymatically by the addition of ascorbate-Fe2+ or NADPH-ADP-Fe3+, respectively, to the incubation mixtures. The extent of peroxidation was evaluated by the formation of thiobarbituric acid chromophore and of radioactive hydrophilic peroxidation products. The results obtained show that: (1) nuclear membrane lipid peroxidation products formed during incubation interact with DNA and total nuclear proteins; (2) non-enzymatic lipid peroxidation processes induced a 40% larger association of peroxidation products to DNA compared to processes driven enzymatically, whereas the corresponding interaction with total nuclear proteins was similar in both peroxidation systems; (3) the radioactivity associated with histones decreased during incubation in the presence of ascorbate-Fe2+ or NADPH-ADP-Fe3+, and increased in control samples (no additions); (4) inhibition of lipid peroxidation by the iron chelator Desferrioxamine B prevented the association of peroxidation products to nuclear macromolecules; (5) the levels of radioactivity found in DNA after 180 min of incubation would represent the formation of 0.6-1.0 adducts per 10(6) DNA bases. The results obtained provide evidence for an interaction between lipid peroxidation products and chromatin in the interior of the cell nucleus.     

46,XX,-12,+der(12),rcp(3;12)(p25.1;p13.31)pat karyotype in a girl. Probable subregional assignment of glyceraldehyde-3-phosphate dehydrogenase locus to 12p13.1----p13.31 by exclusion mapping

A female infant with partial trisomy 3p and a terminal deletion 12p, due to a paternal (3;12)(p25.1;p13.31) translocation is described. Normal glyceraldehyde-3-phosphate (GAPD) activity in the proposita tentatively excludes GAPD locus from the deleted segment. Therefore, the region for this locus is reduced to 12p13.1----p13.31.     

Red blood cell sorbitol dehydrogenase deficiency in a family with cataracts

Summary Sorbitol dehydrogenase (SORD) was quantitatively assayed in a family in which four out of five brothers and their father had bilateral cataracts. Three sibs (two of them with cataracts) and both their father and paternal grandfather had SORD activity of about 25% of the reference values; of the other two affected sibs one had about 50% and the other had 75%; the mother and two paternal uncles had about 75%. These results do not define a clear cataract-SORD deficiency etiopathogenic relationship, nevertheless, they strongly suggest activity polymorphism in human red cell SORD, which would be highly relevant not only to the study of cataracts but of other major complications in diabetes.     

Mechanisms controlling choline transport and acetylcholine synthesis in motor nerve terminals during electrical stimulation

Electrical stimulation of the chick ciliary nerve leads to a frequency-dependent increase in the Na+-dependent high affinity uptake of [3H]choline (SDHACU) and its conversion to acetylcholine (ACh) in the nerve terminals innervating the iris muscle. The forces that drive this choline (Ch) uptake across the presynaptic membrane were evaluated. Depolarization with increased [K+] out or veratridine decreases Ch accumulation. In addition to the electrical driving force, energy is provided by the Na+ gradient. Inhibition of the Na,K-ATPase decreased the Ch taken up. Thus, changes in the rate of Ch transport are dependent on the electrochemical gradients for both Ch and Na+. Ch uptake and ACh synthesis were increased after a conditioning preincubation with high [K+] out or veratridine. As is the case for electrical stimulation, this acceleration of Ch uptake and ACh synthesis was strongly dependent on the presence of Ca++ in the incubation medium. Na+ influx through a TTX-sensitive channel also contributed to this acceleration. Inasmuch as membrane depolarization reduces the initial velocity of Ch uptake and ACh synthesis, their increases during electrical stimulation therefore cannot be the direct effect of the depolarization phase of the action potential. Instead they are the result of the ionic fluxes accompanying the presynaptic spike. It is concluded that stimulation of Ch uptake and ACh synthesis by nerve activity depends first, on the ACh release elicited by Ca++ influx after depolarization and second, on the activation of the Na,K-ATPase due to Na+ entry. Furthermore, it is suggested that the release of ACh after stimulation drives translocation of cytoplasmic ACh into a protected compartment (probably vesicular). This recompartmentation of intraterminal ACh stimulates ACh synthesis by mass action, allowing further accumulation of Ch.     

A simple rapid fluorescent assay for adenosine deaminase activity.

A simple, rapid (2 hours), fluorescent test for the activity of blood adenosine deaminase (ADA) is described. The test which can be performed on both heparinized and dried blood, is based on the conversion of adenosine to inosine and ammonium in the presence of ADA. The enzyme activity is visually estimated by the oxidation of NADH (fluorescent) to NAD+ (non-fluorescent) in a coupled reaction with glutamate dehydrogenase. The disappearance of fluorescence indicates ADA activity in the sample. The advantages are discussed of the use of this test for the study of the autosomal recessive severe combined immunodeficiency.     

Progesterone levels and relationship with the diagnosis of a corpus luteum by rectal palpation during the estrous cycle in Zebu cows

To assess the accuracy of rectal palpation for detecting functional luteal tissue during the estrous cycle in Zebu cattle, 20 mature non-lactating Indobrazil cows were palpated twice weekly for 7 1/2 weeks. Blood samples were drawn for progesterone analyses at each palpation. Circulating serum progesterone levels were below 0.5 ng/ml from days 0-4 (Day 0 = day of estrus); they increased thereafter, reaching maximum levels of 3.1 ng/ml on days 9 and 10. Values declined sharply to less than 0.5 ng/ml on day 18. Regardless of the stage of the estrous cycle in 71.3% of the cases (117 out of a total of 164 observations) the circulating progesterone levels corresponded to the results of rectal examination. The criteria to assess this relationship were that the presence of CL as determined by rectal palpation would be accompanied by levels of progesterone higher than 0.5 ng/ml, whereas absence of CL would be accompanied by levels less than 0.5 ng/ml. The correlation was significantly higher (P<0.05) on days 5-17 (77.9%) than on days 0-4 (57.5%) and 18-20 (65%). To assess the correlation of both rectal examination and progesterone levels with the stage of the estrous cycle, we expected that on days 0-4 and 18-20 no palpable CL and progesterone levels less than 0.5 ng/ml would occur, whereas on days 5-17 palpable CL and progesterone levels higher than 0.5 ng/ml would be found. On this basis, a correlation of 45% (18 out of 40 observations) between expected and observed values was found on days 0-4, 76% (79 out of 104) on days 5-17 and 60% (12 out of 20) on days 18-20 of the estrous cycle. Of the total of 55 observations which fell outside the expected values, 71% was due to a wrong diagnosis of CL; 14.5% was due to progesterone levels higher or lower than the expected values, and 14.5% to both laboratory and rectal palpation findings.