Forskolin-induced block of delayed rectifying K+ channels in pancreaticβ-cells is not mediated by cAMP

K⁺ channels in the membrane of murine pancreatic -cells were studied using the patch-clamp technique. The delayed outward current was activated in whole-cell experiments by depolarizing voltage pulses to potentials between –30 mV and 0 mV. Forskolin blocked the current rapidly (<5 s) and reversibly with 50% inhibition at 13 M. The inhibition did not depend on a stimulation of the adenylate cyclase since it occurred even in presence of 1 mM cAMP in the pipette solution which replaced the cytoplasm. Membrane permeant cAMP analogues and phosphodiesterase inhibitors did not influence the delayed outward current. In experiments on outside-out patches forskolin (100 M) shortened the openings of a channel of about 10 pS conductance at 0 mV and a time course of activation and inactivation similar to the whole-cell current. Another smaller, slowly activating channel and the Ca²⁺- and ATP-dependent K⁺ channels were influenced only weakly or not at all. It is therefore concluded that the 10-pS channel generates most of the delayed outward K⁺ current in murine pancreatic -cells. The Ca²⁺-independent part of the delayed outward current in bovine adrenal chromaffin cells was also blocked by forskolin (100 M).     

The nonselective cation channel in the basolateral membrane of rat exocrine pancreas

Nonselective Ca²⁺-sensitive cation channels in the basolateral membrane of isolated cells of the rat exocrine pancreas were investigated with the patch clamp technique. With 1.3 mmol/l Ca²⁺ on the cytosolic side, the mean openstate probabilityP _o of one channel was about 0.5. In insideout oriented cell-excised membrane patches the substances diphenylamine-2-carboxylic acid (DPC), 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) and 3,5-dichlorodiphenylamine-2-carboxylic acid (DCDPC) were applied to the cytosolic side. These compounds inhibited the nonselective cation channels by increasing the mean channel closed time (slow block). 100 mol/l of NPPB or DPC decreasedP _o from 0.5 (control conditions) to 0.2 and 0.04, respectively, whereas 100 mol/l of DCDPC blocked the channel completely. All effects were reversible. 1 mmol/l quinine also reducedP _o, but in contrast to the abov mentioned substances, it induced fast flickering. Ba²⁺ (70 mmol/l) and tetraethylammonium (TEA⁺; 20 mmol/l) had no effects. We investigated also the stilbene disulfonates 4-acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid (SITS), 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS) and 4,4-dinitro-2,2-stilbenedisulfonate (DNDS). 10 mol/l SITS applied to the cytosolic side increasedP _o from 0.5 to 0.7 and with 100 mol/l SITS the channels remained nearly permanently in its open state (P _o1). A similar activation of the channels was also observed with DIDS and DNDS. These effects were poorly reversible. The stilbene disulfonates acted by increasing the channel mean open time. When the channel was inactivated by decreasing bath Ca²⁺ concentration to 0.1 mol/l, addition of 100 mol/l of SITS had no effect. Similarly, reducing bath Ca²⁺ concentration from 1.3 mmol/l in presence of 100 mol/l SITS (channels are maximally activated) to 0.1 mol/l, inactivated the channels completely. These results demonstrate, that SITS can only activate the channels in the presence of Ca²⁺. SITS had no effects, when applied to the extracellular side in outside out patches. In summary, the substances DPC, NPPB and DCDPC inhibit nonselective cation channels, where DCDPC has the most potent and NPPB the smallest effect; whereas SITS, DIDS and DNDS activate the channel when applied from the cytosolic side in the presence of Ca²⁺ ions.     

The role ofγδ T cells in the normal and disordered immune system

Summary A small population of T cells does not express the conventional T cell receptor characterized by the and polypeptide chains (TCR) but instead, two polypeptides termed and (TCR). This alternative receptor is able to recognize antigen. It appears early in T cell ontogeny, but its role in the thymus prior to the availability of TCR remains unclear. In selected sites such as skin or gut TCR predominates in mice which might suggest a role of T cells in the first line of defense against infection, T cells secrete lymphokines and display cytotoxic activity. However, their activation requirements may differ from what is known for T cells since MHC-nonrestricted and also CD4 and CD8 negative T cells have been described. Preferential activation by mycobacterial antigens possibly indicates a special repertoire of the T cells. In various diseases slightly increased numbers of T cells were found, but these preliminary studies have not yet provided evidence for a major pathogenetic role of T cells.List of abbreviations C constant region (immunoglobulin or TCR gene segment) - CD4 cluster of differentiation 4 (mainly on helper cells) - CD8 cluster of differentiation 8 (mainly on cytotoxic cells) - D diversity region (immunoglobulin or TCR gene segment) - DNA desoxyribonucleic acid - IL2 interleukin 2 - J joining region (immunoglobulin or TCR gene segment) - kD kiloDalton - MHC major histocompatibility complex - NK natural killer (cells) - RA rheumatoid arthritis - TCR T cell receptor - V variable region (immunoglobulin or TCR gene segment)     

Flow Cytometric Analysis of In Vitro Proinflammatory Cytokine Secretion in Peripheral Blood from Multiple Sclerosis Patients

The cytokines, interferon- (IFN-), tumor necrosis factor- (TNF-rpar;, and interleukin-2 (IL-2) are important endogenous proinflammatory proteins and have been linked to disease activity in multiple sclerosis. In this study, we use flow cytometric methodology to compare the secretion of IFN-, IL-2, and TNF- from peripheral blood-derived T cells of multiple sclerosis patients to the secretion in healthy controls. The percentages of IFN-, IL-2, and TNF- secreting cells are not significantly different between multiple sclerosis patients and controls. However, the TNF- secreting CDS cell percentage is correlated with the IFN- and IL-2 secreting CD3 cell percentages in multiple sclerosis patients. In the controls, only the TNF- secreting CD3 cell percentage is correlated with IFN-. These findings show that correlated secretion of cytokines occurs in multiple sclerosis and suggest that concerted intercytokine interactions may play an important role in the disease.     

The isolated frog skin epithelium: Presence of α and β adrenergic receptors regulating active sodium transport and water permeability

Summary The effects of stimulation of and adrenergic receptors on short circuit current (S.C.C.), Na⁺ and Cl^– fluxes and osmotic water permeability were studied on isolated frog skin epithelial layers separated from the dermis.Low norepinephrine doses (final concentrations in the incubation medium ranging from 5×10^–9 to 10^–8 M) produced increased water permeability and S.C.C. The latter was entirely accounted for by an increase in the active Na⁺ influx. Na⁺ outflux and Cl^– fluxes were not modified. Both these effects disappeared after treatment with the blocking agent, Propranolol. Higher norepinephrine doses (final concentrations: 10^–7 to 10^–6 M) produced: 1. an increase in water permeability lower than that produced by low doses, the highest doses failing to increase water permeability, and 2. a triphasic change in S.C.C.: after an initial increase, S.C.C. dropped to its resting value and then rose again to a sustained value. Na⁺ and Cl^– flux measurements showed that the variation in S.C.C. reflected variations in active Na⁺ transport. When the same high norepinephrine doses were applied after treatment with the blocking agent Phentolamine, the effects observed were identical to those obtained with low doses.On blocked preparations, large doses of norepinephrine inhibited the water permeability and sodium transport increases induced by theophylline or oxytocin but did not modify those induced by 35-cyclic AMP. The inhibition was suppressed after blocking receptors.From the foregoing, it was concluded that both and adrenergic receptors are present in frog skin epithelial cells and are involved in the regulation of water and sodium permeability.It is suggested that the inhibitory effect of stimulation resulted from the inhibition of cyclic-AMP generating system, the activity of which is under the positive control effect of oxytocin and stimulation.     

Intracellular pH regulation of human colonic crypt cells

In order to investigate the regulation of intracellular pH (pH_i) in freshly isolated human colonocytes, we have used a newly developed technique for the rapid isolation and covalent attachment of these cells to glass surfaces and microspectrofluorimetric measurement of the pH-sensitive fluorescence of 2,7-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF)-loaded specimens in a perfusion chamber (37°C). In N-2-hydroxyethylpiperazine — N — 2 — ethanesulphonic — acid — (HEPES)-buffered Ringer solution (HBS) a baseline pH_i of 7.35±0.03 (mean ± SD; n=42) was found for human colonocytes and in HBS, NH₄Cl-prepulse-induced intracellular acidification in colonocytes is reversed rapidly by the ubiquitous amiloride-sensitive (1 mmol/l) Na⁺/H⁺ exchanger. Switching from HBS to HCO ₃ ^– buffered solution (BBS) led to a transient intracellular acidification (7.29±0.09), followed by a recovery to a final resting pH_i of 7.43±0.03. One-third of the acid extrusion in BBS is amiloridesensitive; the remaining two-thirds are caused by the dihydroderivative of 4,4-diisothiocyanatostilbene-2,2-disulphonic acid (H₂DIDS)-sensitive HCO ₃ ^– -dependent mechanisms. The functional activity of an acid-extruding Na⁺/HCO ₃ ^– cotransporter in human colonocytes was observed in response to the reintroduction of Na⁺ into amiloride-containing Na⁺/Cl^–-free BBS. In addition, the mechanism leading to alkalinization (7.56±0.05) in Cl^–-free BBS was identified as Na⁺-dependent Cl^–/HCO ₃ ^– exchange, by its H₂DIDS sensitivity and the specific requirement for Cl^– and Na⁺. The intrinsic buffering capacity ( _i) of the human colonocytes was calculated from pH changes induced by sequential NH₄Cl-loading steps during blockage of acid/base transporters. With _i=80 mmol · l^–1 · pH unit^–1 for the pH interval ranging from 6.9 to 7.1 (n=8) the colonocytes exhibited a relatively high intrinsic buffering in comparison with other cell types. In conclusion, the freshly isolated human colonocytes express a Na⁺/H⁺ exchanger, a Na⁺/HCO ₃ ^– cotransporter and a Na⁺-dependent Cl^–/HCO ₃ ^– exchanger, all of them likely to be involved in the regulation of pH homeostasis in vivo in the presence of widely varying extracellular conditions. Maintenance of a stable pH_i of human colonocytes seems to be facilitated by a comparatively high _i at physiological pH values.     

Increase in TCRγδ T lymphocytes in synovia from rheumatoid arthritis patients with active synovitis

To determine the relative presence of TCR+ and TCR+ T cells in synovial tissue from patients with various types of inflammatory synovitis and in tissues from patients with a number of chronic T cell-mediated conditions, we stained frozen tissue sections with monoclonal antibodies in indirect immunofluorescence assays. In tissues obtained from patients with chronic T cell-mediated granulomatous conditions (Wegener's granulomatosis, lymphomatoid granulomatosis, granuloma annulare, Langerhan's cells granulomatosis, pigmented villonodular synovitis, Takayasu's arteritis, and talc granulomatosis), the T cells present were predominantly TCR+, without an increased presence of TCR+ cells. In contrast, 6 of 14 (43%) synovia from patients with rheumatoid arthritis (RA) showed increased TCR+ T cells (3–10 cells/hpf). The RA synovia with increased TCR+ cells present had an increased tissue inflammation score compared to RA synovia with few TCR+ cells [18.6±5.8 versus 11.6±4.2 (mean±SE),P<0.05]. In contrast, synovia from patients with osteoarthritis, systemic lupus erythematosus, and trauma did not show an increased presence of TCR+ T cells. Thus, in cellular inflammatory infiltrates the presence of increased TCR cells is not a component of noninfectious granulomatous inflammation but is found in approximately 40% of RA synovia with high levels of inflammation.     

Spontaneous Mucosal Lymphocyte Cytokine Profiles in Children with Autism and Gastrointestinal Symptoms: Mucosal Immune Activation and Reduced Counter Regulatory Interleukin-10

A lymphocytic enterocolitis has been reported in a cohort of children with autistic spectrum disorder (ASD) and gastrointestinal (GI) symptoms. This study tested the hypothesis that dysregulated intestinal mucosal immunity with enhanced pro-inflammatory cytokine production is present in these ASD children. Comparison was made with developmentally normal children with, and without, mucosal inflammation. Duodenal and colonic biopsies were obtained from 21 ASD children, and 65 developmentally normal paediatric controls, of which 38 had signs of histological inflammation. Detection of CD3⁺ lymphocyte staining for spontaneous intracellular TNF, IL-2, IL-4, IFN, and IL-10, was performed by multicolor flow cytometry. Duodenal and colonic mucosal CD3⁺ lymphocyte counts were elevated in ASD children compared with noninflamed controls (p<0.03). In the duodenum, the proportion of lamina propria (LP) and epithelial CD3⁺TNF⁺ cells in ASD children was significantly greater compared with noninflamed controls (p<0.002) but not coeliac disease controls. In addition, LP and epithelial CD3⁺IL-2⁺ and CD3⁺IFN⁺, and epithelial CD3⁺IL-4⁺ cells were more numerous in ASD children than in noninflamed controls (p<0.04). In contrast, CD3⁺IL-10⁺ cells were fewer in ASD children than in noninflamed controls (p<0.05). In the colon, LP CD3⁺TNF⁺ and CD3⁺IFN⁺ were more frequent in ASD children than in noninflamed controls (p<0.01). In contrast with Crohns disease and non-Crohns colitis, LP and epithelial CD3⁺IL-10⁺ cells were fewer in ASD children than in nondisease controls (p<0.01). There was a significantly greater proportion of CD3⁺TNF⁺ cells in colonic mucosa in those ASD children who had no dietary exclusion compared with those on a gluten and/or casein free diet (p<0.05). There is a consistent profile of CD3⁺ lymphocyte cytokines in the small and large intestinal mucosa of these ASD children, involving increased pro-inflammatory and decreased regulatory activities. The data provide further evidence of a diffuse mucosal immunopathology in some ASD children and the potential for benefit of dietary and immunomodulatory therapies.     

Energetics of anaerobic glycolysis in dog gastrocnemius

Thermally isolated gastrocnemii were stimulated to exhaustion, by rhythmic isotonic (70 N) tetanic contractions, during complete occlusion of blood flow. Enthalpy change (h=work + heat) and work output (w) (kJ/kg) were obtained from records of deep muscle temperature and shortening. The lactate produced (LA, mol/kg) was measured in the outflow after reestablishement of blood flow. The following relationships were obtained:h=76 LA+1.2, andw=19.8 LA+0.30. As the energy liberated at exhaustion by alactic energy sources (P and O₂ stores) is constant, h/ LA=76 (±10.5, S.E.) kJ/mol is the enthalpy change for lactate formation ( ). The neutralization heat was estimated on muscle homogenates at 12 kJ/mol, leaving 64 kJ/mol for of LA formation proper. The mechanical efficiencies of work related to LA formation (E _LA) and of that not related to LA formation (E _nonLA) were practically identical (0.25). From these values and from , the enthalpy change of P splitting was estimated in the range 52–62 kJ/mol, depending on the value of the ratio P/ LA assumed in the calculation.     

Renal tubular reabsorption of taurine,γ-aminobutyric acid (GABA) andβ-alanine studied by continuous microperfusion

Summary Renal tubular reabsorption of taurine, -aminobutyric acid (GABA), and -alanine was studied in vivo et situ by continuous microperfusion of single proximal tubules of the rat. In each case, reabsorption was much slower than that for other amino acids that have been studied. With a concentration of 0.1 mmol/l in initial perfusate, about 60% of initial load was reabsorbed over perfusion distance of 3 mm. Taurine reabsorption saturated with only 2.17 mmol/l in initial perfusate. Assuming simple two-parameter kinetics, upper limits for K _m of 0.54 mmol/l and forV _max of 0.59 pmol·cm^–1·s^–1 for tubular reabsorption of taurine were estimated. High (20 mmol/l) concentrations of taurine or -alanine in perfusate completely inhibited GABA reabsorption, butl-phenylalanine (20 mmol/l) had no significant effect. The results indicate that the three amino acids are reabsorbed slowly from the proximal tubule by what may be a common transport system. This system appears to have a high affinity but low capacity and to be different from other known renal tubular transport systems for amino acids.Supported by National Science Foundation Research Grant No. PCM 75-09918 and by grant No. 1740 of the Austrian Fonds zur Förderung der Wissenschaftlichen Forschung.     

Coexpression of Fcγ receptor IIIA and interleukin-2 receptor β chain by a subset of human CD3+/CD8+/CD11b+ lymphocytes

In this study we identify and characterize a subset of human peripheral blood T cells, present in all individuals, that has features previously described for T cells either separately or in special circumstances. These cells are found in purified suspensions of resting peripheral blood lymphocytes within the CD8⁺ T lymphocytes, express T cell receptor (TCR), and can be identified and isolated because of high-density expression of surface CD11b (TCR⁺/CD3⁺/CD8⁺/CD11b⁺ cells). They coexpress constitutively the IL-2 receptor chain, FcRIIIA, and CD56. Although they do not mediate spontaneous cytotoxicity, CD3⁺/CD8⁺/CD11b⁺ cells have cytotoxic potential, demonstrated in redirected cytotoxicity assays with P815 target cells in the presence of anti-FcRIII (CD16) or anti-CD3 monoclonal antibodies. Stimulation of CD3⁺/CD8⁺/CD11b⁺ cells with rIL-2 induces proliferation, cytotoxicity against NK-sensitive and NK-resistant target cells, and expression of surface activation antigens, including IL-2 receptor chain (CD25). CD3⁺/CD8⁺/CD16⁺/CD56⁺ cell clones with cytotoxic functions including those mediated by engagement of surface CD16 were obtained by limiting-dilution cloning of purified CD3⁺/CD8⁺/CD11b⁺ cells in the presence of rIL-2 and autologous feeder cells. Our data support the hypothesis that the CD3⁺/CD8⁺/CD11b⁺/CD16⁺ cells represent a discrete peripheral blood lymphocyte subset that could be the physiological counterpart of that expanded in several pathological conditions and in large granular lymphocyte lymphocytosis.     

Effects of antisense oligodeoxynucleotide hybridization on in vitro translation of potato virus Y RNA

Potato virus Y (PVY), a potyvirus, has an RNA genome containing 9704 nucleotides of which 185 belong to the 5 nontranslated region (NTR). Contrary to most eukaryotic mRNAs that have a cap structure, the potyvirus RNA has a genome-linked protein (VPg). In order to understand the mechanisms of PVY RNA translation initiation, hybrid-arrest translation was used to localize sequences involved in binding of proteins and/or ribosomes. The 5 NTR was fused to the -glucuronidase (GUS) reporter gene. Six antisense oligodeoxynucleotides were used for hybridization, and the efficiency of the in vitro translation of the hybridized mRNA was modified to different levels depending upon the position of the oligodeoxynucleotide used. The highest inhibition was obtained with an oligodeoxynucleotide hybridized to the 5 end. In addition, translation of GUS mRNA containing the PVY 5 NTR was greatly enhanced when this mRNA was capped. These results differ from those obtained with the tobacco etch virus (TEV) and three picornaviruses, but are similar to those obtained with capped mRNA.     

Ion-induced release of LHRH,TRH and α-MSH from hypothalamic synaptosomes and its inhibition by vinblastine

Homogenates of rat hypothalamic tissue were fractionated by means of discontinuous sucrose density gradient centrifugation. Immunoreactive luteinizing hormone releasing hormone (LHRH), thyrotropin releasing hormone (TRH), and -melanocyte stimulating hormone (-MSH) were found to be concentrated in the synaptosome-enriched fraction. This fraction was suspended in 0.32 M sucrose and the release of the three peptides was investigated. After incubation, the synaptosomes were re-isolated by ultrafiltration, and the concentration of each peptide in the ultrafiltrate was determined by radioimmunoassay. When the synaptosomal fraction was incubated at 30° C in 0.32 M sucrose containing either 60 mM K⁺-2 mM Ca²⁺ or 140 mM Na⁺ alone a release of LHRH, TRH, and -MSH occurred. Of the total content 30–50% of LHRH but only about 10% of TRH and -MSH was releasable. When the synaptosome preparation was preincubated for 30 min at 30°C with 10^–4 M vinblastine. K⁺- as well as Na⁺-induced release of LHRH, THR, and -MSH was inhibited, and the stimulatory effect of each cation was almost totally blocked by preincubation with 5×10^–4 and 10^–3 M vinblastine. The inhibitory action of vinblastine (5×10^–4 M) did not affect the oxidation of glucose to CO₂ by the synaptosomes. The results of the present investigation demonstrate that synaptosome-enriched fractions of hypothalamic origin are more stable with respect to LHRH, TRH, and -MSH release during incubation in isotonic sucrose than they are in ionic solutions, and that the peptides are released by a vinblastine-sensitive mechanism.Supported by grants from the National Institute of Arthritis, Metabolism, and Digestive Diseases (AMO 1237), the National Institute of Child Health and Human Development (HDO8672), and the National Institutes of Health contract (5-P17-HL1487-06)Supported by Grant No. 512-6951 from the Danish Medical Research Council     

Expression of estrogen receptors-α and -β in primary breast neoplasms and tumors exposed to neoadjuvant hormonal therapy

We studied the content and expression of mRNA for estrogen receptors receptors- and - in breast tumors before and after 3-month neoadjuvant hormone therapy with antiestrogen tamoxifen and/or aromatase inhibitors. Expression of estrogen receptors- and - was most often detected in ER⁺PR⁺ tumors and most significantly decreased in these neoplasms after exemestane therapy. Immunocytochemical and radioligand assays showed that tamoxifen and anastrozole have little effect on the number of estrogen receptors- The number of progesterone receptors in tumors decreased by the end of anastrozole therapy. Estrogen receptors- were immunocytochemically revealed in 50% primary breast tumors. Anastrozole slightly decreased, while tamoxifen increased the incidence of these receptors. Interruption of signaling through estrogen receptors and suppression of estrogen biosynthesis had different effects on the receptor status of neoplasms and distribution of estrogen receptors- and -.Translated from Byulleten Eksperimentalnoi Biologii i Meditsiny, Vol. 138, No. 11, pp. 559–562, November, 2004     

alpha1-acid glycoprotein possesses in vitro pro- and antiinflammatory activities

We studied the effects of ₁-acid glycoprotein on tumor necrosis factor- (TNF-) and interleukin-10 (IL-10) production and lymphocyte response to phytohemagglutinin in cultured peripheral blood mononuclear leukocytes from 6 healthy donors. We observed 2 opposite responses to ₁-acid glycoprotein: first, stimulation of TNF- and IL-10 production and inhibition of lymphocyte proliferation, and second, suppression of cytokine production and stimulation of lymphocyte proliferation. In cell cultures isolated from 4 of 6 donors, the TNF-/IL-10 ratio remained unchanged after addition of native ₁-acid glycoprotein, but some fractions isolated by chromatography on concanavalin A-Sepharose changed this parameter. These changes were most pronounced after treatment with fraction C enriched with molecules with incomplete (biantennary) carbohydrate chains. The mechanisms of ₁-acid glycoprotein-induced effects on peripheral blood mononuclear leukocytes are discussed.     

Modulation of hematopoietic colony formation of stem cells in peripheral blood by anti-TGF-β in patients with severe immunosuppression

Summary The influence of transforming growth factor- (TGF-) on hematopoiesis has been evaluated by adding blocking antibodies against TGF- to colony forming assays (CFU-c). When optimum concentrations of recombinant growth factors, granulocyte-macrophage colony stimulating factor (GM-CSF), and interleukin-3 (IL-3) were added to stem cells from the peripheral blood of healthy individuals and certain patients with tumors or HIV infection, the anti-TGF- capable of blocking 5 ng/ml of active TGF- had no significant influence on erythroid or myeloid colony formation. However, in certain immunosuppressed individuals, anti-TGF- resulted in a significant decrease of erythroid colony formation and slight suppression of myeloid colony formation. The significant inhibition of hematopoiesis by plasma of HIV patients could be due to the presence of active forms of TGF-. The results of the blocking experiments are consistent with the concept that TGF- in low concentrations is essential for erythropoiesis and myelopoiesis but that higher levels of TGF- primarily inhibit erythropoiesis in vitro. TGF- serves as a coordinating factor when efficient recruitment of granulocytes and monocytes is more essential than erythropoiesis and stem cell growth.Abbreviations BFU-E burst forming unit-erythroid - CFC colony forming cells - CFU-GEMM colony forming unit-granulocyte/erythroid/macrophage/megacaryocyte - CFU-GM colony forming unit-granulocyte/macrophage - EPO erythropoietin - GM-CSF granulocyte/macrophage-colony stimulating factor - HIV human immunodeficiency virus - IL-1 interleukin-1 - IL-3 interleukin-3 - IMDM Iscove's Modified Dulbecco's medium - PBS phosphate buffered saline - TGF- transforming growth factor- - TNF- tumor necrosis factor-     

Dual effects of ATP on K+ currents of mouse pancreatic β-cells

K⁺ currents through ATP-dependent channels were recorded from inside-out patches of -cell membrane as previously described (Rorsman and Trube 1985). Channels were opened by removing ATP from the intracellular side of the membrane. The open probability and/or the number of active channels declined spontaneously (run-down) when ATP was absent for periods longer than about 30 s. Channels subject to the run-down could be activated again after applying a blocking concentration (>0.1 mM) of ATP in presence of 1 mM MgCl₂ for at least 2 min. ATP in absence of Mg and the ATP-analogues AMP-PNP, AMP-PCP and ATPS were ineffective in reactivating the channels. This suggests that phosphorylation of the channels or associated proteins of hydrolysis of ATP may be necessary for keeping the channels available. In contrast to the differential effects on the run-down, ATP in presence and absence of Mg and the ATP analogues were similarly effective in blocking the channels at concentrations above 0.1 mM. Using an experimental protocol avoiding the run-down ghe dose-inhibition curve for ATP was found to reach 50% at 18 M.     

Comparison of two models of bleomycin-induced lung fibrosis in mouse on the level of leucocytes and T cell subpopulations in bronchoalveolar lavage

Bleomycin produces pulmonary fibrosis in mice when given as a single intratracheal instillation or as multiple subcutaneous injections. Both models are associated with a significant deposition of collagen in the lungs but differ in the location of the initial lesions. Intratracheal instillation of bleomycin directs lesions to peribronchial or peribronchiolar sites, whereas subcutaneous injection produces lesions in subpleural and perivascular locations. It was therefore of interest to analyse the bronchoalveolar lavage (BAL) fluid for differences in the cellular composition, especially of intraepithelial T lymphocytes characterised by the expression of the integrin E7.Intracheal instillation of bleomycin induced a 5 to 6 times higher number of neutrophils and lymphocytes in BAL fluid than the subcutaneous model. Furthermore, intratracheal instillation of bleomycin induced the infiltration of eosinophilic neutrophils, a peculiar subtype of neutrophils often found in rodents, which were not found in BAL after subcutaneous injection of bleomycin. In both models of bleomycin injection, however, CD4⁺, CD8⁺, NK, and T lymphocytes were detected with dominance of the CD4⁺ T cell population. Analysis of the expression of the integrin E7 revealed similar numbers of E7⁺ cells in the CD4⁺ and T cell populations in both models but significantly lower numbers of E7⁺ T cells were found in the BAL fluid within the CD8⁺ T cell population after subcutaneous injection of bleomycin compared to intratracheal instillation.These results show that a difference in route of bleomycin administration not only causes changes in the localisation of the lesions but that this variation may be reflected in alterations within the BAL leucocyte population especially within the intraepithelial T lymphocytes.     

Aldosterone does not stimulate the Na:K pump in isolated turtle colon

The study of the mechanisms by which mineralocorticoids stimulate sodium absorption across distal epithelia has focused on three possible sites of action: apical sodium permeability, the basolateral NaK pump, and the production of high-energy substrates. Recently we developed a method for direct measurement of the current generated by the basolateral NaK pump of the turtle colon [15]. In the presence of mucosal amphotericin-B and serosal barium the short-circuit current across the colon can be equated with the current produced by active electrogenic exchange of sodium for potassium across the basolateral membrane. This pump current is a measure of the transport capacity of the epithelial NaK pump that is uncomplicated by changes in apical membrane sodium permeability. Pump currents, thus defined, were compared in control tissues and tissues treated with aldosterone in vitro. After 9h Na absorption was increased 4-fold in the aldosterone-treated tissues but the values of the pump current were identical in the two groups. This result indicates that acute stimulation of sodium absorption by aldosterone does not occur by stimulating the NaK pump directly.     

Synoviocytes in chronic synovitis in situ and cytokine stimulated synovial cells in vitro neo-express α1, α3 and α5 chains of β1 integrins

The expression of the 1 integrins was examined immunohistochemically in synoviocytes from normal synovial membrane and from chronic synovitis of different aetiology and intensity. Normal synoviocytes were 61-positive but lacked 1 through 5. In mild inflammation type A synoviocytes neo-expressed 1, 3, and 5 chains. In severe inflammation both type A and B synoviocytes expressed 3, 4, 5, and 6 chains. The effects of inflammatory cytokines, as single agents or in combination, on the 1 integrin expression in cultured normal synoviocytes was determined by immunocytochemistry and flow cytometry. The 1 chain, while absent in unstimulated synoviocytes, was induced by interleukin-1 (IL-1), tumour necrosis factor- (TNF-), and interferon- (INF-). This effect was enhanced by combining IL-1 and TNF-. Expression of the 3 chain was up-regulated by IL-1 and, more intensely, by IFN-. Transforming growth factor (TGF-) inhibited the up-regulating effect of IL-1 and antagonized the effect of IFN- on 3 chain expression. Expression of the 5 chain was up-regulated significantly by co-stimulation through IL-1 together with TGF- or TNF-. Thus, the 1 integrin profile of cytokine activated synoviocytes in vitro resembled that of synoviocytes in synovitis in situ. These data suggest that IL-1, TNF-, IFN-, and TGF- are likely to be among the effectors regulating 1 integrin expression in synoviocytes in vivo.