9-Aminoacridine- and tetraethylammonium-induced reduction of the potassium permeability in pancreatic B-cells. Effects on insulin release and electrical properties.

The effects of 9-aminoacridine and tetraethylammonium on insulin release and rubidium efflux from perifused rat islets were investigated and correlated with their effects on the electrical properties of mouse B cells studied with microelectrode techniques. 9-Aminoacridine (0.05--1 mmol/l) and tetraethylammonium (2--40 mmol/l) produced a dose-dependent, reversible potentiation of glucose-stimulated insulin release. This effect was rapid, affected both phases of secretion and was maximum in the presence of 6 mmol/l glucose, but no longer significant at 20 mmol/l glucose. It was unaltered by atropine or propanolol, and abolished by mannoheptulose or omission of extracellular calcium. 9-Aminoacridine, but not tetraethylammonium, also induced insulin release in the absence of glucose stimulation. Neither drug modified glucose metabolism in islet cells and only 9-aminoacridine increased 45Ca2+ uptake. In the presence of 0, 3 or 6 mmol/l glucose, but no longer at 20 mmol/l glucose, 9-aminoacridine and tetraethylammonium reduced the rate of 86Rb+ efflux from the islets. Both drugs also slightly reduced 86Rb+ uptake by islet cells. In the presence of 11 mmol/l glucose, 9-aminoacridine reduced the amplitude and the duration of the polarization phases between the bursts of electrical activity; concomitantly these periods of spike activity were markedly prolonged. At lower glucose concentrations (3 or 7 mmol/l), 9-aminoacridine progressively depolarized B cells and induced electrical activity in otherwise silent cells. Tetraethylammonium also suppressed the repolarization phases between the bursts of spikes in the presence of a stimulating concentration of glucose. At low glucose, tetraethylammonium produced only a limited and not maintained depolarization. These results show that a reduction of the potassium permeability in pancreatic B cells potentiates the insulin-releasing effect of glucose and may even stimulate secretion. They also suggest that the initial depolarizing effect of glucose is due to a reduction of the potassium permeability, whereas the repolarization at the end of each burst of electrical activity is mediated, at least in part, by an increase in the potassium permeability of B cells.

Pancreatic acinar cell function: measurement of intracellular ions and pH and their relation to secretion.

1. Isolated mouse pancreatic acini were used to investigate the effect of secretagogues on acinar cellular electrolytes and cell pH. The effect of changes in the acid-base status of the incubation medium on acinar cellular electrolytes, cell pH and amylase release were also studied. 2. Carbachol at concentrations of 10(-6) or 10(-5) M was without any effect on the intracellular concentrations of total Na+, Na+ exchangeable with 22Na+, K+ and Cl-, and did not influence cell pH as determined by the DMO method. 3. Changes in pHe achieved by varying the HCO3- concentrations at constant CO2, varying the CO2 concentration at constant HCO3- or by titration of a HCO3-/CO2 free HEPES buffered medium did not influence intracellular electrolyte values. 4. pHi changed linearly with pHe by about 1.2 units/pHe unit change over the pHe range of 7.7--6.5. pHi, however, did not change in response to a change in the CO2 tension when the HCO3- concentration was adjusted to keep pHe at 7.4. 5. Lowering pHe below 7.1 inhibited carbachol and CCK8-stimulated amylase release. By contrast a decrease of pHe to 6.8 was without significant effect on basal and secretagogue increased 45Ca2+ efflux from pancreatic acini. 6. In conclusion the pH sensitivity of amylase release by acinar cells is probably related to changes in pHi. Since Ca2+ mobilization is not affected, the pH sensitive step is probably in the mechanism by which Ca2+ activates the release of zymogen granules contents by exocytosis.

Ionic mechanisms of the glucose-induced membrane potential changes in B-cells.

The ionic basis of the glucose-induced membrane potential changes in pancreatic B-cells was investigated. The results suggest that the initial depolarization of the membrane in response to a stimulation with glucose is due to a decrease of the K permeability. This depolarization seems to open a voltage-dependent Ca-channel and thereby an additional depolarization, the depolarization phase of the slow waves, is initiated. Insulin release is then triggered by the entering Ca ions. The fast spike activity may be a consequence of the exocytotic process. The polarization phase of the slow waves seems to be caused by the activity of an electrogenic Na-K-pump and a calcium-dependent increase of the K permeability. The activity of the Na-pump is considered to be regulated by the intracellular Na concentration.

Localization of ATP-dependent calcium transport activity in mouse pancreatic microsomes.

Electron-dense deposits representing calcium oxalate crystals which result from ATP-dependent calcium uptake have been localized within vesicles of of a heavy microsomal fraction prepared from mouse pancreatic acini. In the absence of either ATP or oxalate, no electron-dense deposits could be observed. By subfractionation of microsomes on discontinuous sucrose gradients, it could be shown that the highest energy-dependent calcium transport activity was associated with the rough endoplasmic reticulum. In rough microsomes, the 45Ca2+-uptake measured was 7 times greater than that of smooth microsomes in the presence of ATP and oxalate and about 3 times greater in he presence of ATP alone. When ribosomes were released from the rough endoplasmic reticulum vesicles by treatment with KCl in the presence of puromycin, the stripped microsomes showed a 40% increase in the specific 45Ca2+-uptake activity measured in he presence of ATP and oxalate and an increase of 80 to 90% in the presence of ATP alone. From these results it can be concluded that the calcium transport activity of microsomes prepared from mouse pancreatic acini is located predominantly in the rough endoplasmic reticulum membrane.

Real-time confocal laser scan microscope for examination and diagnosis of the eye in vivo.

A confocal laser scan microscope is designed for the diagnosis and the examination of the anterior segment of the human eye in vivo. Any contact of the eye with the instrument optics or an immersion fluid is avoided to preclude the risk of infection or injury. Normal eyes of nine volunteers are observed and investigated. Nerve fibers and keratocytes in the stroma and the endothelium of the cornea, the capsule, and the suture of the lens, and threadlike structures in the vitreous can be observed. Cellular details in all tissues investigated can be resolved.

Lipoteichoic acid inhibits interleukin-2 (IL-2) function by direct binding to IL-2.

Lipoteichoic acid (LTA) is associated with the cell envelope of most gram-positive bacteria. Although previously thought to act mainly as a virulence factor by virtue of its adhesive nature, evidence is now provided that LTA can also suppress the function of interleukin-2 (IL-2), an autocrine growth factor for T cells. LTA from four separate bacterial strains lowered the levels of detectable IL-2 during a peripheral blood mononuclear cell response to the antigen tetanus toxoid (TT). T-cell proliferation in response to TT was similarly inhibited by LTA. In contrast, levels of detectable gamma interferon increased. In addition, LTA inhibited IL-2 detection by enzyme-linked immunosorbent assay (ELISA) and blocked the proliferative response of an IL-2-dependent T-cell line to soluble IL-2. Further studies using ELISA demonstrated that LTA blocks IL-2 detection and function by binding directly to IL-2. Flow cytometric analysis revealed that IL-2 binding to T cells is inhibited in the presence of purified LTA but not LTA plus anti-LTA monoclonal antibody. In summary, these studies demonstrate a novel effect of LTA on the immune response through direct binding to IL-2 and inhibition of IL-2 function. Importantly, gram-positive organisms from which LTA is obtained not only play an important role in the pathology of diseases such as bacterial endocarditis, septic shock, acute respiratory distress syndrome, and multiple organ failure but also comprise a significant portion of commensal populations within the human host. Inhibition of IL-2 function by LTA may represent yet another mechanism by which gram-positive bacteria dampen the host immune response and facilitate survival. Thus, LTA provides a potential target for therapeutic intervention when gram-positive organisms are involved.