Jan-Peter Hildebrandt

The effects of bradykinin on K+ currents in NG108–15 cells treated with U73122, a phospholipase C inhibitor, or neomycin

Bradykinin has multiple effects on differentiated NG108–15 neuroblastoma×glioma cells: it increases Ins(1,4,5)P3 production and intracellular Ca2+ concentration [Ca2+]i, evokes a Ca2+ activated K+ current (IK(Ca)) and inhibits M current (IM). We studied the effect of the aminosteroid U73122 and the antibiotic neomycin, both putative blockers of phospholipase C (PLC), on these four bradykinin effects. Preincubation with 1 or 5 μm U73122 for 15 min partly suppressed Ins(1,4,5)P3 generation and the increase in [Ca2+]i induced by 1 μm bradykinin. U73122 10 μm caused total and irreversible inhibition. The inactive analogue U73343 was without effect. Resting levels of Ins(1,4,5)P3 were not affected. However, resting [Ca2+]i was increased by 10 μm U73122, but not by U73343. Individual cells responded to 10 μm U73122 with a small increase in [Ca2+]i, followed in some cells by a large further rise. Pretreatment of whole‐cell clamped cells with 1 μm U73122 for 30 min reduced the bradykinin‐induced IK(Ca) to a fifth of its normal size. To suppress it totally, a 7–12 min pretreatment with 5 μm U73122 was required. Again, U73343 was without effect. U73122 and U73343 at concentrations of 5–10 μm irreversibly decreased the holding current (Ih) which at a holding potential of −30 or −20 mV mainly flows through open M channels. The decrease was often preceded by a transient increase. M current (IM) measured with 1 s pulses, was also decreased by 5–10 μm U73122 and U73343, but short applications of U73122 could cause a small increase. The bradykinin‐induced inhibition of IM was not affected by U73122. Preincubation with 1 or 3 mm neomycin for 15 min did not affect Ins(1,4,5)P3 generation and the increase in [Ca2+]i induced by bradykinin. Pretreatment with 3 mm neomycin for about 20 min diminished the bradykinin‐induced IK(Ca) to a fifth of its normal size. The four main conclusions drawn from the results are: (a) U73122 suppresses bradykinin‐induced PLC activation and IK(Ca), but not IM inhibition. (b) This indicates that the transient outward current IK(Ca), but not the decrease of IM in response to bradykinin, is mediated by PLC. (c) U73122 itself inhibits IM and mobilizes Ca2+ from intracellular stores. (d) Externally applied neomycin is not an effective inhibitor of PLC‐mediated signalling pathways in NG108–15 cells.

Effects of Staphylococcus aureus-hemolysin A on calcium signalling in immortalized human airway epithelial cells

Part of the innate defence of bronchial epithelia against bacterial colonization is secretion of salt and water which generally depends on coordinated actions of receptor-mediated cAMP- and calcium signalling. The hypothesis that Staphylococcus aureus-virulence factors interfere with endogenous signals in host cells was tested by measuring agonist-mediated changes in [Ca(2+)](i) in S9 cells upon pre-incubation with bacterial secretory products. S9 cells responded to mAChR-activation with calcium release from intracellular stores and capacitative calcium influx. Treatment of cells with culture supernatants of S. aureus (COL) or with recombinant alpha-hemolysin (Hla) resulted in time- and concentration-dependent changes in [Ca(2+)](i). High concentrations of Hla (2000 ng/ml) resulted in elevations in [Ca(2+)](i) elicited by accelerated calcium influx. A general Hla-mediated permeabilization of S9 cell membranes to small molecules, however, did not occur. Lower concentrations of Hla (200 ng/ml) induced a reduction in [Ca(2+)](i)-levels during the sustained plateau phase of receptor-mediated calcium signalling which was abolished by pre-incubation of cells with carboxyeosin, an inhibitor of the plasma membrane calcium-ATPase. This indicates that low concentrations of Hla change calcium signalling by accelerating pump-driven extrusion of Ca(2+) ions. In vivo, such a mechanism may result in attenuation of calcium-mediated cellular defence functions and facilitation of bacterial adherence to the bronchial epithelium.

Small bite, large impact-saliva and salivary molecules in the medicinal leech, Hirudo medicinalis.

Blood-sucking leeches have been used for medical purposes in humans for hundreds of years. Accordingly, one of the most prominent species has been named Hirudo medicinalis by Carl Linne in 1758. Feeding on vertebrate blood poses some serious problems to blood-sucking ectoparasites, as they have to penetrate the body surface of the host and to suppress the normal reactions of the host to such injuries (swelling, pain, inflammation) to remain undetected during the feeding period. Furthermore, the parasites have to take measures to inhibit the normal reactions in host tissues to blood vessel damage, namely hemostasis and blood coagulation (platelet aggregation and activation, activation of thrombin and formation of fibrin clots). During evolution, leeches have acquired the ability to control these processes in their hosts by transferring various bioactive substances to the host. These substances are supposedly produced in unicellular salivary gland cells and injected into the wound at the feeding site through tiny salivary ductule openings in the jaws that the leech uses to slice open the host body surface and to cut blood vessels in the depth of the wound. This review summarizes current knowledge about the salivary gland cells and the biological effects of individual saliva components as well as hints to the potential usefulness of some of these compounds for medical purposes.

Chloride and Potassium Conductances of Mouse Pancreatic Zymogen Granules Are Inversely Regulated by a 80-kDa mdr1a Gene Product

Cl and cation conductances were characterized in zymogen granules (ZG) isolated from the pancreas of wild-type mice (+/+) or mice with a homozygous disruption of the multidrug resistance P-glycoprotein gene mdr1a (-/-). Cl conductance of ZG was assayed in isotonic KCl buffer by measuring osmotic lysis, which was induced by maximal permeabilization of ZG membranes (ZGM) for K with valinomycin due to influx of K through the artificial pathway and of Cl through endogenous channels. To measure cation conductances, ZG (pH 6.0-6.5) were suspended in buffered isotonic monovalent cation acetate solutions (pH 7.0). The pH gradient was converted into an outside-directed H diffusion potential by maximally increasing H conductance of ZGM with carbonyl cyanide m-chlorophenylhydrazone. Osmotic lysis of ZG was induced by H diffusion potential-driven influx of monovalent cations through endogenous channels and nonionic diffusion of the counterion acetate. ZGM Cl conductances were not different in (-/-) and (+/+) mice (2.6 ± 0.3 hversus 3.1 ± 0.2 h (relative rate constant)). The nonhydrolyzable ATP analog adenosine 5′-(β,-methylene)triphosphate (AMP-PCP) (0.5 mM) activated the Cl conductance both in (+/+) and (-/-) mice. However, activation of Cl conductance by AMP-PCP was reduced in (-/-) mice as compared with (+/+) mice (5.0 ± 0.4 hversus 7.6 ± 0.7 h; p < 0.005). In contrast, ZGM K conductance was increased in (-/-) mice as compared with (+/+) mice (14.2 ± 2.0 hversus 8.5 ± 1.2 h; p < 0.03). In the presence of 0.5 mM AMP-PCP, which completely blocks K conductance but leaves a nonselective cation conductance unaffected, there was no difference between (-/-) and (+/+) mice (5.3 ± 0.7 hversus 3.2 ± 0.5 h). In Western blots of ZGM from wild-type mice, a polyclonal MDR1 specific antibody labeled a protein band of ≈80 kDa. In mdr1a-deficient mice, the intensity of this band was reduced to 39 ± 7% of the wild-type signal. This indicates that a mdr1a gene product of ≈80 kDa enhances the AMP-PCP-activated fraction of mouse ZGM Cl conductance and reduces AMP-PCP-sensitive K conductance.

Virulence factors of Staphylococcus aureus induce Erk-MAP kinase activation and c-Fos expression in S9 and 16HBE14o-human airway epithelial cells

Part of the innate defense of bronchial epithelia against bacterial colonization is regulated secretion of salt, water, and mucus as well as defensins and cytokines involving MAP kinase activation and alterations in early gene expression. We tested two different types of immortalized human airway epithelial cells (S9, 16HBE14o-) for activation of Erk-type MAP kinases and for expression of c-Fos on treatment with Staphylococcus aureus culture supernatants from the stationary growth phase [optical density (OD)(540 nm) = 10] or with recombinant S. aureus hemolysins A and B (Hla, Hlb). OD10 supernatants activated Erk-type MAP kinases and c-Fos expression in a concentration-dependent manner. Hla induced Erk-type kinase phosphorylation in S9 but not in 16HBE14o- cells. Hlb induced Erk activation in either cell type. Basal and stimulated levels of Erk-type MAP kinase phosphorylation were sensitive to the Mek1 inhibitor PD-98059, indicating that the bacterial products activated the entire signaling cascade that coregulates IL-8 induction and secretion. While c-Fos expression was enhanced by OD10 supernatants, Hla, and Hlb in S9 cells, 16HBE14o- cells responded to OD10 supernatant and Hlb but not to Hla. In S9 cells, PD-98059 suppressed c-Fos upregulation by OD10 supernatant, Hla, or Hlb, indicating that c-Fos expression requires activation of Erk-type MAP kinases. In 16HBE14o- cells, however, c-Fos expression by OD10 supernatant was sensitive to PD-98059, while that induced by Hlb was not. This indicates that ingredients of OD10 supernatants other than Hla or Hlb are activating Erk-type MAP kinases in 16HBE14o- cells and that other intracellular signaling systems apart from Erk-type MAP kinases contribute to Hlb-mediated regulation of c-Fos. Thus interaction of bacterial factors with airway epithelial cells may be highly cell type specific.

Downregulation of aquaporins 1 and 5 in nasal gland by osmotic stress in ducklings, Anas platyrhynchos: implications for the production of hypertonic fluid

SUMMARY Using primers against highly conserved regions of mammalian and bird aquaporins in RT–PCR experiments, we amplified products derived from duck (Anas platyrhynchos) nasal gland RNA that were identified as homologues of mammalian and chicken aquaporin 1 and aquaporin 5 cDNAs by sequencing. Using digoxigenin-labelled probes derived from these PCR products in northern blot analyses of mRNA isolated from nasal glands of untreated (naïve) or osmotically stressed ducklings (replacement of drinking water with a 1% NaCl solution), we observed a decrease in aquaporin 1 (AQP1) and aquaporin 5 (AQP5) mRNA abundance (by approximately 40%) during saline adaptation in the animals. Western blot analysis of AQP1 and AQP5 expression in the glands revealed that protein abundance decreased in a similar fashion. Immunohistochemical analysis of AQP1 distribution in cryosections of nasal gland indicated that AQP1 is mainly expressed in endothelial cells of the capillaries, but definitely not in the secretory or ductal cells of the gland. AQP5 distribution in the gland, however, seems to be different, since staining was exclusively observed in apical and basolateral plasma membranes of individual epithelial cells of the primary and central ducts, which collect fluid from the secretory tubules. The observations are consistent with the hypothesis that strongly hyperosmotic fluid is produced by the secretory cells at very low (unstimulated gland) or high (activated gland) rates. In the unstimulated gland, secretions may be diluted by aquaporin-mediated transcellular water flux while passing through the ductal system flushing the glandular ducts, thereby potentially preventing ascending infections. In the activated gland, however, downregulation of aquaporins in capillaries and duct cells may prevent dilution of the initially secreted fluid, enabling the animals to excrete large volumes of a highly concentrated salt solution.

Staphylococcus aureus Hemolysin A Disrupts Cell–Matrix Adhesions in Human Airway Epithelial Cells

Treatment of primary or immortalized human airway epithelial cells (16HBE14o-, S9) or alveolar cancer cells (A549) with recombinant hemolysin A (rHla), a major virulence-associated factor of Staphylococcus aureus, induces alterations in cell shape and formation of paracellular gaps in the cell layer. Semiquantitative Western blotting using extracts of freshly isolated airway tissue (nasal epithelium) or 16HBE14o- model cells revealed that phosphorylation levels of focal adhesion kinase (Fak) and paxillin were altered upon treatment of tissue or cells with rHla. Immune fluorescence analyses showed that rHla treatment of 16HBE14o- cells results in losses of vinculin and paxillin from focal contacts and a net reduction in the number of focal contacts. The actin cytoskeleton was strongly remodeled. We concluded that treatment of cells with rHla activates Fak signaling, which accelerates focal contact turnover and prevents newly formed focal contacts (focal complexes) from maturation to focal adhesions. The inability of rHla-treated cells to form stable focal adhesions may be one factor that contributes to gap formation in the cell layer. In vivo, such changes may disturb the defensive barrier function of the airway epithelium and may facilitate lung infections by S. aureus.

S. aureus haemolysin A-induced IL-8 and IL-6 release from human airway epithelial cells is mediated by activation of p38- and Erk-MAP kinases and additional, cell type-specific signalling mechanisms.

Soluble virulence‐associated factors of Staphylococcus aureus like haemolysin A (Hla) induce secretion of chemo/cytokines from airway epithelial cells. To elucidate the potential roles of specific signalling pathways in this response, we treated 16HBE14o‐, S9 or A549 cells with recombinant Hla (rHla). In a dose‐dependent manner, rHla induced secretion of IL‐8 in all three cell types, but IL‐6 release only in 16HBE14o‐ and S9 cells. rHla‐mediated secretion of IL‐8 and IL‐6 was suppressed by pre‐incubation of cells with inhibitors of Erk type or p38 MAP kinases, indicating that activation of these signalling pathways is essential for IL‐8 release in all three cell types and for IL‐6 release in 16HBE14o‐ and S9 cells. The rHla‐mediated phosphorylation and activation of p38 MAP kinase seem to depend on elevations in [Ca2+]i, an early response in rHla‐treated cells. Inhibitors of calmodulin or calcium/calmodulin‐dependent kinase II attenuated rHla‐mediated release of IL‐8 in 16HBE14o‐ and A549 cells and of IL‐6 in 16HBE14o‐ cells. This indicates that rHla may mediate simultaneous activation of calmodulin‐dependent processes as additional prerequisites for chemo/cytokine secretion.However, the inhibitors of calmodulin‐dependent signalling did not affect rHla‐induced p38 MAP kinase phosphorylation, indicating that this pathway works in parallel with p38 MAP kinase.

A Gq-type G protein couples muscarinic receptors to inositol phosphate and calcium signaling in exocrine cells from the avian salt gland

SummaryMuscarinic acetylcholine receptor (mAChR) activation in isolated cells from the nasal salt gland of the domestic duck (Anas platyrhynchos) results in a rapid increase in the rate of phosphatidylinositol hydrolysis and pronounced intracellular calcium signals. Both responses can be elicited by treating these cells with fluoroaluminate (AlF4−) indicating the involvement of a heterotrimeric G protein in the transmembrane signaling process. To characterize this G protein, electrophoretically separated membrane proteins were blotted onto nitrocellulose filters and probed with peptide-antibodies raised against portions of different α-subunits of mammalian G proteins. We could demonstrate the presence of at least four different G proteins in salt gland cell membranes. Two of these proteins (40 and 41 kD) were ADP-ribosylated by pertussis toxin and were recognized by an antiserum against a common sequence in all G protein α-subunits. One protein (46 kD) was a cholera toxin-substrate and was recognized by a Gs-specific antiserum; the other (42 kD) was recognized by Gq-specific antisera and was resistant to ADP-ribosylation. Since the initial inositol phosphate production upon receptor activation with carbachol and the resulting calcium signals were not affected by pertussis toxin-pretreatment of salt gland cells, we conclude that muscarinic receptors are coupled to phospholipase C by a Gq-type G protein.

Staphylococcus aureus alpha-toxin mediates general and cell type-specific changes in metabolite concentrations of immortalized human airway epithelial cells.

Staphylococcus aureus alpha-toxin (Hla) is a potent pore-forming cytotoxin that plays an important role in the pathogenesis of S. aureus infections, including pneumonia. The impact of Hla on the dynamics of the metabolome in eukaryotic host cells has not been investigated comprehensively. Using 1H-NMR, GC-MS and HPLC-MS, we quantified the concentrations of 51 intracellular metabolites and assessed alterations in the amount of 25 extracellular metabolites in the two human bronchial epithelial cell lines S9 and 16HBE14o− under standard culture conditions and after treatment with sub-lethal amounts (2 µg/ml) of recombinant Hla (rHla) in a time-dependent manner. Treatment of cells with rHla caused substantial decreases in the concentrations of intracellular metabolites from different metabolic pathways in both cell lines, including ATP and amino acids. Concomitant increases in the extracellular concentrations were detected for various intracellular compounds, including nucleotides, glutathione disulfide and NAD+. Our results indicate that rHla has a major impact on the metabolome of eukaryotic cells as a consequence of direct rHla-mediated alterations in plasma membrane permeability or indirect effects mediated by cellular signalling. However, cell-specific changes also were observed. Glucose consumption and lactate production rates suggest that the glycolytic activity of S9 cells, but not of 16HBE14o− cells, is increased in response to rHla. This could contribute to the observed higher level of resistance of S9 cells against rHla-induced membrane damage.