PIP2 hydrolysis underlies agonist-induced inhibition and regulates voltage gating of two-pore domain K+ channels

Two-pore (2-P) domain potassium channels are implicated in the control of the resting membrane potential, hormonal secretion, and the amplitude, frequency and duration of the action potential. These channels are strongly regulated by hormones and neurotransmitters. Little is known, however, about the mechanism underlying their regulation. Here we show that phosphatidylinositol 4,5-bisphosphate (PIP₂) gating underlies several aspects of 2-P channel regulation. Our results demonstrate that all four 2-P channels tested, TASK1, TASK3, TREK1 and TRAAK are activated by PIP₂. We show that mechanical stimulation may promote PIP₂ activation of TRAAK channels. For TREK1, TASK1 and TASK3 channels, PIP₂ hydrolysis underlies inhibition by several agonists. The kinetics of inhibition by the PIP₂ scavenger polylysine, and the inhibition by the phosphatidylinositol 4-kinase inhibitor wortmannin correlated with the level of agonist-induced inhibition. This finding suggests that the strength of channel PIP₂ interactions determines the extent of PLC-induced inhibition. Finally, we show that PIP₂ hydrolysis modulates voltage dependence of TREK1 channels and the unrelated voltage-dependent KCNQ1 channels. Our results suggest that PIP₂ is a common gating molecule for K⁺ channel families despite their distinct structures and physiological properties.     

Lectin-induced increase in clonogenic growth of haematopoietic progenitor cells

Abstract: The galactoside-specific plant lectin, Viscum album agglutinin (VAA-I) increases cellular parameters of natural host defence. It also binds to a variety of haematopoietic cells, including progenitors. We investigated whether VAA-I has a stimulatory effect on haematopoietic progenitor cells. Peripheral blood progenitor cells from 7 healthy volunteers were cultured in a colony assay with VAA-I plus erythropoietin (EPO) and stem cell factor (SCF). At 50 pg/ml VAA-I induced a significant increase in the cytokine-dependent clonogenic growth (52% in median, p<0.05). In another set of experiments purified CD34+ cells were isolated from the bone marrow aspirate of 4 patients with non-metastatic breast cancer using fluorescence-activated cell sorting. Binding to CD34+ cells was demonstrated by using directly fluorescence-conjugated VAA-I. Co-incubation with d -galactose significantly abrogated this effect. CD34+ cells were cultured in the presence of EPO, SCF, interleukin-3, granulocyte/monocyte colony-stimulating factor and granulocyte colony-stimulating factor. VAA-I alone had no measurable effect on the clonogenic growth of the isolated cells. However, at concentrations of 100 and 250 pg/ml VAA-I increased the cytokine-dependent proliferation and differentiation of CD34+ cells by a median of 75 and 85%, respectively. The results show that VAA-I binds to haematopoietic progenitor cells and has a co-stimulatory effect on their proliferation.     

Cellular Distribution of G Protein Goa in Pituitary Lactotrophs: Effects of Dopamine

Membrane-bound GTP-binding (G) proteins mediate signal transduction in a variety of cell systems. The exact mechanisms of G proteins action are still under investigation but they appear to involve effectors located in the plasma membrane as well as in other parts of the cell. With this study, we investigated the cellular and ultrastructural localization of G protein subunits, and particularly of Goa, in normal rat anterior pituitaries and in estrone-induced rat adenomatous lactotrophs. We also evaluated the effects of Goα cellular redistribution in rat adenomatous lactotrophs following short-term exposure to dopamine (DA). Using the Protein A-gold (PAG) methodology, Goα was found to be present in the cysternae of the endoplasmic reticulum of normal pituitary cells and of adenomatous lactotrophs. In the latter, Goα could be co-localized with prolactin (PRL). By immunoblots, using specific antisera, significant amounts of Goα and Gs42α, together with smaller amounts of Giα, Gs47α and Gβ were found to be present in the uncontaminated supernatant fraction of adenomatous lactotrophs. Unexpectedly, exposure of the cells to DA induced a rapid and short-lived decrease in the cytosolic fraction of Goα and Gβ associated with a decrease of PRL release. Since cytosolic Goα can be ADP-ribosylated by pertussis toxin (PT) and is therefore in a heterotrimeric form, our data suggest that the soluble Go protein may play a role during lactotrophs' exposure to an inhibitor of PRL release, perhaps through its relocalization after being internalized with the D₂ receptor or by being used for interaction with intracellular and/or membrane-bound effectors.     

Positive Outcome Expectancies and Smoking Behavior: The Role of Expectancy Accessibility

This study examined the association between smoking outcome expectancy accessibility and smoking behavior. Daily smokers completed a smoking expectancy accessibility task in which they made timed judgments to a series of positive consequences of smoking either after 6 hr deprivation or within 10 min of smoking. Participants then completed a questionnaire battery that contained assessments of smoking behavior and smoking outcome expectancies. Results of hierarchical regression analyses showed that expectancy accessibility was associated with the number of cigarettes smoked per day even when controlling for corresponding questionnaire measures of smoking expectancies. Moreover, smoking expectancy accessibility predicted urge to smoke ratings following exposure to a smoking cue after controlling for the effects of deprivation. Findings suggest that smoking expectancy accessibility may play a central role in smoking behavior and that individual differences in this attribute may be assessed directly through reaction time assessment.     

Iodine-125 Brachytherapy of Brain Stem Tumors

PURPOSE: To report on iodine-125 ((125)I) interstitial irradiation in the treatment of brain stem tumors. PATIENTS AND METHODS: Two patients with brain stem tumors were treated with CT- and image fusion-guided (125)I stereotactic brachytherapy. RESULTS: By March 2003, the patients had been followed up for 47 and 13 months, respectively. In case 1, the tumor volume was 1.98 cm(3) on the control CT, indicating a 65.5% shrinkage as compared to a target volume of 5.73 cm3 at the time of brachytherapy. In case 2, shrinkage was more distinct. After irradiation, the cyst volume was 0.16 cm(3) on the control MRI, indicating a 97.4% shrinkage as compared to a target volume of 6.05 cm(3) at the time of brachytherapy, i. e., the metastasis had virtually disappeared. CONCLUSION: CT- and image fusion-guided (125)I stereotactic brachytherapy can be performed during the biopsy session. The procedure can be well planned dosimetrically and is surgically precise.     

Staph. Epi. Peritonitis in CAPD

Staphylococcus epidermidis is frequently resistant to first-generation cephalosporins. Although I have generally changed the therapy of S. epidermidis CAPD peritonitis to vancomycin when laboratory reports indicate cephalosporin resistance, it frequently appeared that improvement from the cephalosporin was taking place at the time of antibiotic conversion. Should the antibiotics be changed in this setting?