Georg Bischof

Possible regulation of capacitative Ca2+ entry into colonic epithelial cells by NO and cGMP.

A possible role of the nitric oxide (NO)/cGMP pathway in the regulation of Ca2+ entry into HT29/B6 human colonic epithelial cells was investigated using digital image processing of Fura-2 fluorescence and immunoblotting for nitric oxide synthase (NOS). We tested the hypothesis that Ca2+ store depletion causes increased NOS activity and [NO], which is stimulatory to Ca2+ entry by increasing guanylate cyclase (GC) and [cGMP]. Cells were incubated in 95 mM K(+)-containing solutions to depolarize the cell membrane potential and thereby exclude effects of NO and CGMP on K+ or Cl- channels, which might affect Ca2+ entry. Sodium nitroprusside (SNP, 0.5 microM and 30 microM), a NO donor, only slightly raised intracellular [Ca2+] ([Ca2+]i) in resting cells, but in 100 microM carbachol-stimulated cells the sustained, elevated Ca2+ plateau (reflecting Ca2+ entry) as well as Ba2+ entry were increased by 0.5 microM SNP, while 5, 10 or 30 microM SNP either had no effect or were inhibitory. Pretreatment of cells with the NOS inhibitor N-nitro-L-arginine (1 mM) reduced carbachol-stimulated Ca2+ entry, and simultaneous treatment with 0.5 microM (but not 30 microM) SNP restored Ca2+ influx. 8-Br-cGMP (1 mM) had little effect on [Ca2+]i or on rates of Ca2+ or Ba2+ influx into resting cells, but there were large effects on cells in which capacitative Ca2+ entry was activated by carbachol or cyclopiazonic acid (10 microM). The GC inhibitor LY83583 (10 microM) reduced carbachol-stimulated Ca2+ entry, and this entry was restored with 8-Br-cGMP. Western blotting revealed that endothelial-type NOS was present in the particulate fraction of cells. The data are consistent with the notion that Ca2+ entry into HT29/B6 cells is regulated by endothelial NOS/NO and GC/cGMP, but effects are most pronounced in store-depleted cells. Thus, NO and cGMP appear to potentiate the action of messengers released from the store during the emptying process, but NO and cGMP have only small effects of their own to open the Ca2+ channel in the plasma membrane. High [SNP] appeared to be inhibitory while low [SNP] was stimulatory, indicating that a precise range of [NO] may be required for effective stimulation of Ca2+ entry.

Effects of extracellular pH on intracellular pH-regulation and growth in a human colon carcinoma cell-line

Mechanisms of intracellular pH (pHi) regulation seem to be involved in cellular growth and cell division. Little is known about how extracellular acidosis, known to occur in central regions of solid tumors, or alkaline conditions affect pHi regulation in colonic tumors. pHi changes in the colonic adenocarcinoma cell-line SW-620 were recorded by spectrofluorimetric monitoring of the pH-sensitive, fluorescent dye BCECF, and proliferative activity was assessed by [3H]thymidine uptake. Resting pHi in Hepes-buffered solution was 7.53 +/- 0.01 (n = 36). Both 1 mM amiloride and Na(+)-free solution inhibited pHi recovery from acidification and decreased pHi in resting cells. In HCO3-/CO2-buffered media resting pH1 was 7.42 +/- 0.01 (n = 36). Recovery from acidification was Na(+)-dependent, CI(-)-independent, and only partially blocked by 1 mM amiloride. In the presence of amiloride and 200 microM H2DIDS pHi recovery was completely inhibited. In Na(+)-free solution pHi decreased from 7.44 +/- 0.04 to 7.29 +/- 0.03 (n = 6) and no alkalinization was observed in CI(-)-free medium. Addition of 5 microM tributyltin bromide (an anion/OH-exchange ionophore) caused pHi to decrease from 7.43 +/- 0.05 to 7.17 +/- 0.08 (n = 5). The effects of pH0 on steady-state pHi, pHi recovery from acidification and proliferative activity after 48 h were investigated by changing buffer [CO2] and [HCO3-]. In general, increases in pH0 between 6.7 and 7.4 increased pHi recovery, steady-state pHi and growth rates. In summary, SW-620 cells have a resting pHi > 7.4 at 25 degrees C, which is higher than other intestinal cells. Acid extrusion in physiological bicarbonate media is accomplished by a pHi-sensitive Na+/H+ exchanger and a pHi-insensitive Na(+)-HCO3-cotransporter, both of which are operational in control cells at the resting pHi. No evidence for activity of a CI-/HCO3- exchanger was found in these cells, which could account for the high pHi observed and may explain why the cells continue to grow in acidic tumor environments.

Does nitric oxide regulate capacitative Ca influx in HEK 293 cells

It has been proposed that capacitative Ca influx into both pancreatic acinar cells and HT-29 colonic cells is regulated by stimulation of nitric oxide synthase (NOS). NO, in turn, controls cGMP levels through effects on guanylate cyclase. We tested this possibility by measuring Ca (and Ba) entry into human embryonic kidney 293 cells and into 293 cells that had been transfected with the neuronal NOS gene (293/NOS). 293 cells had undetectable levels of NOS, while 293/NOS cells exhibited very high levels [Bredt D.S., Ferris C.D., Snyder S.H. Nitric oxide synthase regulatory sites. J Biol Chem 1992; 267: 10976-10981]. Ca (or Ba) entry into single cells was measured as the rate of increase of the Fura-2 fluorescence ratio (digital imaging microscopy) during rapid changes from Ca-free (or Ba-free) to Ca- (or Ba-) containing solutions (using high K to depolarize the membrane potential). cGMP levels (EIA method) were measured to correlate to rates of Ca entry. 100 microM ATP caused release of Ca from internal stores, but no sustained plateau due to Ca entry in either 293 or 293/NOS cells. Cyclopiazonic acid (CPA, which inhibits the Ca pump of the internal store, allowing Ca to leak from the store) caused apparent Ca entry to increase 5-10-fold from similar, low levels in both 293 and 293/NOS cells. CPA-stimulated Ca entry was unaffected by the NOS inhibitor N-nitro-L-arginine (L-NA) in either 293 or 293/NOS cells. In 293 cells [cGMP] was low; ATP and CPA both increased [cGMP] by 2-fold, and the guanylate cyclase inhibitor LY83583 and L-NA decreased [cGMP] by 50-75%. [cGMP] was 20-fold higher in 293/NOS cells than in 293 cells; these [cGMP] were not affected by ATP and CPA, but were effectively decreased by 80-90% by L-NA and by LY83583. Thus, [cGMP] and Ca or Ba entry showed no relationship to each other: Ca entry was small into cells in which [cGMP] was either low (resting 293, CPA + L-NA or CPA + LY83583), intermediate (ATP-treated 293) or high (resting 293/NOS). Similarly, Ca entry was high into cells in which [cGMP] was low (CPA + L-NA- or CPA + LY83583-treated 293), intermediate (CPA-treated 293 and CPA + L-NA-treated 293/NOS) or high (CPA- or ATP-treated 293/NOS). We conclude that, as in most other non-excitable cells, Ca entry into 293 cells is stimulated by loss of Ca from the store but, unlike pancreatic and colonic cells, this capacitative Ca entry does not appear to be regulated by NO and cGMP. Therefore, although capacitative entry across the plasma membrane may be regulated by NO and cGMP in Gl epithelial cells, this regulation does not occur in all cells.

Quality of life after sympathetic surgery at the T4 ganglion for primary hyperhidrosis: Clip application versus diathermic cut

INTRODUCTIONnLimited procedures at the T4 ganglion show low rates of compensatory sweating (CS). The aim of the study was to compare endoscopic sympathetic block (ESB) via clip application with endothoracic sympathicotomy (ETS) via diathermy with special regard on patients quality of life (Qol).nnnPATIENTS AND METHODSnTreatment success, side effects and patient satisfaction were evaluated in a prospectively gathered database of a tertiary-care referral hospital. Two disease-specific Qol questionnaires were used (Keller, Milanez de Campos).nnnRESULTSn406 operations were performed in 205 patients (ESB4 Nxa0=xa0114, ETS4 Nxa0=xa091) with a median follow-up of 12 months. Both procedures improved Qol significantly (Pxa0<xa00.001) and the degree of improvement was equal in both groups. Palmar and axillary HH were ameliorated after both procedures (Pxa0<xa00.001). Accordingly, plantar HH decreased after ESB4 (Pxa0=xa00.002), while remaining unaltered after ETS4. Nineteen patients (9.3%) reported CS and 10 patients (4.9%) judged it as disturbing. Nine of the latter belonged to the ETS4 group compared to one ESB patient (Pxa0=xa00.015). Patients developed higher rates of plantar CS after ETS4 compared to ESB4 (Pxa0=xa00.006). Five patients (2.4%) from both cohorts reported persistence of axillary HH. Recurrence of axillary symptoms was found in 5 ESB4 patients. Satisfaction rates did not differ significantly.nnnCONCLUSIONnPatients Qol and satisfaction rates are similar in both treatment groups for upper limb HH. Outcome and recurrence rates speak in the favor of ETS4, severity of CS and potential reversibility argue for ESB4.

Zur Pathogenese der Antibiotikakolitis: Wirkung von Clostridium difficile Toxin A und B auf die humane Kolonschleimhaut in vitro

ZusammenfassungGrundlagen: Clostridium difficile ist der Erreger der Antibiotika-assoziierten Enterokolitis. In Tierstudien schädigt Clostridium difficile Toxin A, aber nicht Toxin B die Schleimhaut des Gastrointestinaltraktes. Die Wirkung der Toxine auf die humane Kolonschleimhaut ist unbekannt. Deshalb untersuchten wir in dieser Studie die Wirkung von Clostridium difficile Toxin A (TxA) und Toxin B (TxB) auf die humane Kolonschleimhaut in vitro.nMethodik: Schleimhautpräparationen humanen Kolons wurden in Ussing-Kammern eingebracht und 5 h mit Puffer (Kontrollen) oder Puffer, der an der luminalen Seite 5 bis 20 µg/ml TxA bzw. 0,06 bis 5,0 µg/ml TxB enthielt, inkubiert. Potentialdifferenz (PD), Kurzschlußstrom (Isc) wurden gemessen, der Widerstand (R) errechnet. Das Ausmaß der Mukosaschädigung wurde an mit Hämatoxylin und Eosin (HE) gefärbten Schnitten mittels computergesteuerter Morphometrie gemessen. Das filamentöse (F-) Aktin des Epithelzellskelettes wurde mit Rhodamin beladenem Phalloidin auf Gefrierschnitten gefärbt und im Fluoreszenzmikroskop untersucht.nErgebnisse: Während die Kontrollen eine normale Morphologie aufwiesen, zeigte die Histologie eine konzentrationsabhängige Schädigung des Oberflächenepithels der Kolonschleimhaut durch TxA und TxB. Das Epithel der Krypten blieb auch bei höchsten Konzentrationen intakt. Entsprechend waren in der Morphometrie nach 5 h luminaler Inkubation mit 5, 10 und 20 µg/ml TxA jeweils 18,6 ± 3%, 33,3 ± 2% bzw. 54 ± 2% der Mukosa geschädigt. Nach Inkubation mit 0,25, 0,5 und 1,0 µg/ ml TxB waren jeweils 9,8 ± 0,6%, 15,6 ± 0,2% bzw. 28,6 ± 0,3% geschädigt (p <0,05). Elektrophysiologie: Zu Versuchsbeginn betrug R in der Kontrollgruppe 174 ± 13 Ohm x cm2, nach 5 h 177 ± 11 Ohm x cm2. 5 h Inkubation mit 5, 10 bzw. 20 µg/ml TxA führte zu einem R-Abfall von jeweils etwa 58, 82 bzw. 136 Ohm x cm2. 5 h Inkubation mit 0,5, 1,0 bzw. 5 µg/ml TxB verursachte einen R-Abfall von jeweils etwa 85, 94 bzw. 169 Ohm x cm2 (p <0.05). Fluoreszenzmikroskopie: Beide Toxine zerstörten das F-Aktin des Zellskelettes der Epithelzellen.nSchlußfolgerungen: Histologie, Morphometrie und Elektrophysiologie zeigen, daß Clostridium difficile TxA und B das Epithel der humanen Kolonschleimhaut in vitro konzentrationsabhängig schädigen und Toxin B, was die Konzentration betrifft, etwa 10mal wirksamer ist als Toxin A.SummaryBackground: In animal models of Clostridium difficile enteritis, toxin A, but not toxin B, appears to mediate intestinal damage. In this study we investigated the morphologic and electrophysiologic effects of purified C. difficile toxins A and B on human colonic mucosa in vitro.nMethods: Human colonic mucosal sheets were mounted in Ussing chambers and incubated with buffer or luminal buffer containing 5 to 20 µg/ml of toxin A and 0.06 to 10 µg/ml of toxin B for 5 h. Potential difference (PD) and short-circuit current (Isc) were measured, resistance (R) was calculated. Histology and Morphometry were performed on hematoxyline and eosine (HE) stained paraffin embedded sections. Flourescent staining of Factin was performed using rhodamin labelled phalloidin.nResults: Histology: Both toxins caused a dose dependent damage of the surface epithelium, while the epithelium lining the crypts remained intact. Morphometry showed that luminal incubation with 5, 10 and 20 µg/ml of TxA caused mucosal damage of 18.6 ± 3%, 33.3 ± 2% and 54 ± 2% respectively. Inkubation with 0.25, 0.5 and 1.0 µg/ml of TxB caused damage of 9.8 ± 0.6%, 15.6 ± 0.2% and 28.6 ± 0.3% respectively (p <0.05). Electrophysiology: At the beginning of the experiments R was 174 ± 13 Ohm x cm2 in the controlgroup, after 5 h R was 177 ± 11 Ohm x cm2. 5-h incubation of tissues with 5, 10 and 20 µg/ml of TxA caused R to drop by 58, 82 and 136 Ohm x cm2, respectively. 5-h incubation with 0.5, 1.0 bzw. 5 µg/ml of TxB caused R to drop by 85, 94, and 169 Ohm x cm2, respectively (p <0.05). Flourescent microscopy of phalloidin stained sections showed that both toxins caused disruption and condensation of cellular filamentous (F-) actin.nConclusions: Our results demonstrate that the human colon is approximately 10 times more sensitive to the damaging effects of toxin B than toxin A, suggesting that toxin B may be more important that toxin A in the pathogenesis of C. difficile colitis in man.

Endoscopic Sympathetic Surgery

Endoscopic thoracic sympathectomy (ETS) is an effective treatment option for patients suffering from primary hyperhidrosis, facial blushing, and various vascular disorders.

Fundoplikatio — Operation laparoskopisch oder konventionell

SchlußfolgerungenDie laparoskopische Methode ist der konventionellen bezüglich der postoperativen Früh- und Spätergebnisse gleichwertig. Wegen der geringeren postoperativen Schmerzen, der kürzeren Hospitalisation und Rehabilitation und des geringeren immunologischen Operationszeitraumes ist die laparoskopische der konventionellen Technik vorzuziehen. Voraussetzung ist eine korrekte Operationsindikation unter Beachtung der Pathophysiologie der Refluxkrankheiten und eine geübte laparoskopische Operationstechnik.