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  • 1
    ISSN: 1432-2013
    Keywords: Key words Ca2+ influx ; Fura-2 ; cAMP ; Forskolin ; Carbachol ; HT29 ; Second messenger ; Patch-clamp technique
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract In HT29 colonic epithelial cells agonists such as carbachol (CCH) or ATP increase cytosolic Ca2+ activity ([Ca2+]i) in a biphasic manner. The first phase is caused by inositol 1,4,5-trisphophate-(Ins P 3-) mediated Ca2+ release from their respective stores and the second plateau phase is mainly due to stimulated transmembraneous Ca2+ influx. The present study was undertaken to examine the effect of increased adenosine 3′,5′-cyclic monophasphate (cAMP) (forskolin 10 μmol/l = FOR) on the Ca2+ transient in the presence of CCH (100 μmol/l). In unpaired experiments it was found that FOR induced a depolarization and reduced cytosolic Ca2+ ([Ca2+]i, measured as the fura-2 fluorescence ratio 340/380 nm) significantly. Dideoxyforskolin had no such effect. The effect of FOR was abolished when the cells were depolarized by a high-K+ solution. In further paired experiments utilizing video imaging in conjunction with whole-cell patch-clamp, [Ca2+]i was monitored separately for the patch-clamped cell and three to seven neighbouring cells. In the presence of CCH, FOR reduced [Ca2+]i uniformly from a fluorescence ratio (345/380) of 2.9 ± 0.12 to 1.8 ± 0.07 in the patch-clamped cell and its neighbours (n = 48) and depolarized the membrane voltage (V m) of the patch-clamped cells significantly and reversibly from −54 ± 7.4 to −27 ± 5.9 mV (n = 6). In additional experiments V m was depolarized by 15–54 mV by various increments in the bath K+ concentration. This led to corresponding reductions in [Ca2+]i. Irrespective of the cause of depolarization (high K+ or FOR) there was a significant correlation between the change in V m and change in [Ca2+]i. These data indicate that the cAMP-mediated attenuation of Ca2+ influx is caused by the depolarization produced by this second messenger.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1432-2013
    Keywords: Key words BCECF ; Fura-2 ; pHi ; [Ca2+]i ; HT29 ; Carbachol ; Neurotensin ; ATP ; InsP3 ; Cell volume ; Calcein
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract  In this study we examined the influence of intracellular pH (pHi) on agonist-induced changes of intracellular Ca2+ activity ([Ca2+]i) in HT29 cells. pHi and [Ca2+]i were measured microspectrofluorimetrically using BCECF and fura-2, respectively. Buffers containing trimethylamine (TriMA), NH3/NH4 + and acetate were used to clamp pHi to defined values. The magnitudes of the peak and plateau of [Ca2+]i transients induced by carbachol (CCH, 10–6 mol/l) were greatly enhanced by an acidic pHi and nearly abolished by an alkaline pHi. The relationship between pHi and the [Ca2+]i peak was nearly linear from pHi 7.0 to 7.8. This effect of pHi was also observed at higher CCH concentrations (10–4 and 10–5 mol/l), at which the inhibitory effect of an alkaline pHi was more pronounced than the stimulatory effect of an acidic pHi. An acidic pHi shifted the CCH concentration/response curve to the left, whereas an alkaline pHi led to a rightward shift. The influence of pHi on [Ca2+]i transients induced by neurotensin (10–8 mol/l) or ATP (5 × 10–7 mol/l) was similar to its influence on those induced by CCH, but generally not as pronounced. Measurements of cellular inositol 1,4,5-trisphosphate (InsP 3) showed no changes in response to acidification with acetate (20 mmol/l) or alkalinization with TriMA (20 mmol/l). The InsP 3 increase induced by CCH was unaltered at an acidic pHi, but was augmented at an alkaline pHi. Confocal measurements of cell volume showed no significant changes induced by TriMA or acetate. Slow-whole-cell patch-clamp experiments showed no additional effect of CCH on the membrane voltage (V m) measured after TriMA or acetate application. We conclude that pHi is a physiological modulator of hormonal effects in HT29 cells, as the [Ca2+]i responses to agonists were significantly changed at already slightly altered pHi. The measurements of InsP 3, cell volume and V m show that pHi must act distally to the InsP 3 production, and not via changes of cell volume or V m.
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  • 3
    ISSN: 1432-2013
    Keywords: [Ca2+]i export ; Thapsigargin ; fura-2 ; HT29 ; CFPAC-1 ; ATP ; Carbachol ; Neurotensin
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract There is increasing evidence that some agonists not only induce intracellular Ca2+ increases, due to store release and transmembranous influx, but also that they stimulate Ca2+ efflux. We have investigated the agonist-stimulated response on the intracellular Ca2+ activity ([Ca2+]i) in the presence of thapsigargin (10−8 mol/l, TG) in HT29 and CFPAC-1 cells. For CFPAC-1 the agonists ATP (10−7–10−3 mol/l, n=9), carbachol (10−6–10−3 mol/l, n=5) and neurotensin (10−10–10−7 mol/l, n=6) all induced a concentration-dependent decrease in [Ca2+]i in the presence of TG. Similar results were obtained with HT29 cells. This decrease of [Ca2+]i could be caused by a reduced Ca2+ influx, either due to a reduced driving force for Ca2+ in the presence of depolarizing agonists or due to agonist-regulated decrease in Ca2+ permeability. Using the fura-2 Mn2+ quenching technique we demonstrated that ATP did not slow the TG-induced Mn2+ quench. This indicates that the agonist-induced [Ca2+]i decrease in the presence of TG was not due to a reduced influx of Ca2+ into the cell, but rather due to stimulation of Ca2+ export. We used the cell attached nystatin patch clamp technique in CFPAC-1 cells to examine whether, in the presence of TG, the above agonists still led to the previously described electrical changes. The cells had a mean membrane voltage of −49±3.6 mV (n=9). Within the first 3 min ATP was still able to induce a depolarization which could be attributed to an increase in Cl− conductance. This was expected, since at this time after TG stimulation all Ca2+ agonists still liberated some [Ca2+]i. When TG incubation was prolonged, agonist application led to strongly attenuated or to no electrical responses. Therefore, the agonist-stimulated [Ca2+]i decrease cannot be explained by the reduction of the driving force for Ca2+ into the cell. In the same cells hypotonic swelling (160 mosmol/l, n=15) still induced a further [Ca2+]i increase in the presence of TG and concomitantly induced Cl− and K+ conductances. We conclude that the agonist-induced decrease of [Ca2+]i in the presence of TG probably unmasks a stimulation of [Ca2+]i export.
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  • 4
    Electronic Resource
    Electronic Resource
    Springer
    Langenbeck's archives of surgery 382 (1997), S. S5 
    ISSN: 1435-2451
    Keywords: Key words Peritonitis ; Candidiasis ; Mycosis ; Fungal infection ; Antifungal therapy ; Schlüsselwörter Peritonitis ; Candidiasis ; Mycosis ; Fungal-Infektion ; Antifungal-Therapie
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Description / Table of Contents: Zusammenfassung Obwohl 20% der Bevölkerung eine Pilzkolonisation des Gastrointestinaltrakts aufweisen, spielen Mykosen in der Initialphase der sekundären Peritonitis eine untergeordnete Rolle. Das Risiko für eine Pilzinfektion steigt nach ausgedehnten operativen Eingriffen, bei Breitspektrumantibiose, parenteraler Ernährung, Katheterismus, Immunsuppression etc. deutlich an. Innerhalb der letzten Jahre nahmen bei nosokomialen Infektionen Mykosen (überwiegend Candida spp.) deutlich zu. Intraabdominale Infektionen bei CAPD-Patienten werden in ca. 5% der Fälle durch Pilze verursacht. Bei Peritonitiden aufgrund Anastomoseninsuffizienz steigt die Inzidenz der Mykosen deutlich an, wobei die Letalität bis zu 80% beträgt. Im Verlauf der schweren Pankreatitis tritt bei bis zu 5% der Nekroseinfektionen eine invasive Mykose auf. Die Klinik der invasiven Pilzinfektion gleicht dem septischen Syndrom und ist in diesem Stadium mit einer Häufigkeit von bis zu 50% mit Fungämien vergesellschaftet. Da die meisten fakultativ pathogenen Pilze Teil der physiologischen Flora sind, ist die Interpretation kultureller Nachweise schwierig. Zur Diagnose einer invasiven Mykose können histopathologische Methoden sowie serologische Candidaantigen- und -antikörpernachweis hilfreich sein. Therapeutisch stehen mit Amphotericin B, Flucytosin und Fluconazol 3 hochwirksame Substanzen für die i.v.-Applikation zur Verfügung. Amphotericin B wird in einer Dosierung bis zu 1 mg/kg und Tag, in der liposomalen Galenik bis 3 mg/kg und Tag verabreicht. Flucytosin (0,15–0,2 g/kg und Tag) ist gut liquorgängig und hat in der Kombination mit Amphotericin B eine synergistische Wirkung. Fluconazol stellt bei empfindlichen Pilzen (Ausnahmen C. glabrata und C. krusei) in einer Dosierung von 200–800 mg/Tag eine ähnlich wirksame und nebenwirkungsärmere Alternative dar.
    Notes: Abstract Although there is a 20% yeast colonization in the gastrointestinal tract of the population, fungal infections appear only rarely in secondary peritonitis. The risk of severe mycosis increases after a major operation and when a patient is taking broad-spectrum antibiotics, is on total parenteral nutrition, is catheterized, and/or is immune-suppressed. In the past years the incidence of nosocomial fungal infections (usually Candida spp.) has risen significantly. Five percent of CAPD-related peritonitis is caused by fungi. In enteral anastomosis breakdown, invasive mycosis occurs more often, with an accompanying lethality of up to 80%. In severe pancreatitis, up to 5% of peripancreatic necrosis is infected with fungi. The clinical course of severe mycosis, like the septic syndrome, is associated with fungemia in up to 50% of cases. As most of the facultative pathogenic fungi are part of the physiological flora, it is difficult to interpret mycological cultures. In order to diagnose invasive fungal infections, histopathological techniques and serologic tests for antigens and antibodies are available. Three antifungal agents (amphotericin B, flucytosine, fluconazole) are available for intravenous administration. Amphotericin B is given at doses of up to 1 mg/kg per day, in liposomal galenism up to 3 mg/kg per day. Combining amphotericin B with flucytosine (150–200 mg/kg per day) a synergistic effect is reached. Fluconazole at a dosage of 200–800 mg per day represents an alternative with similar antifungal activity and lower side effects.
    Type of Medium: Electronic Resource
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  • 5
    Electronic Resource
    Electronic Resource
    Springer
    Langenbeck's archives of surgery 382 (1996), S. S5 
    ISSN: 1435-2451
    Keywords: Peritonitis ; Candidiasis ; Mycosis ; Fungal infection ; antifungal therapy ; Peritonitis ; Candidiasis ; Mycosis ; Fungal-Infektion ; Antifungal-Therapie
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Description / Table of Contents: Zusammenfassung Obwohl 20% der Bevölkerung eine Pilzkolonisation des Gastrointestinaltrakts aufweisen, spielen Mykosen in der Initialphase der sekund=:aren Peritonitis eine untergeordnete Rolle. Das Risiko für eine Pilzinfektion steigt nach ausgedehnten operativen Eingriffen, bei Breitspektrumantibiose, parenteraler Ernährung, Katheterismus, Immunsuppression etc. deutlich an. Innerhalb der letzten Jahre nahmen bei nosokomialen Infektionen Mykosen (überwiegendCandida spp.) deutlich zu. Intraabdominale Infektionen bei CAPD-Patienten werden in ca. 5% der Fälle durch Pilze verursacht. Bei Peritonitiden aufgrund Anastomoseninsuffizienz steigt die Inzidenz der Mykosen deutlich an, wobei die Letalität bis zu 80% beträgt. Im Verlauf der schweren Pankreatitis tritt bei bis zu 5% der Nekroseinfektionen eine invasive Mykose auf. Die Klinik der invasiven Pilzinfektion gleicht dem septischen Syndrom und ist in diesem Stadium mit einer Häufigkeit von bis zu 50% mit Fungämien vergesellschaftet. Da die meisten fakultativ pathogenen Pilze Teil der physiologischen Flora sind, ist die Interpretation kultureller Nachweise schwierig. Zur Diagnose einer invasiven Mykose können histopathologische Methoden sowie serologische Candidaantigen- und-antikörpernachweis hilfreich sein. Therapeutisch stehen mit Amphotericin B, Flucytosin und Fluconazol 3 hochwirksame Substanzen für die i.v.-Applikation zur Verfügung. amphotericin B wird in einer Dosierung bis zu 1 mg/kg und Tag, in der liposomalen Galenik bis 3 mg/kg und Tag verabreicht. Flucytosin (0,15–0,2 g/kg und Tag) ist gut liquorgängig und hat in der Kombination mit Amphotericin B eine synergistische Wirkung. Fluconazol stellt bei empfindlichen Pilzen (AusnahmenC. glabrata undC. krusei) in einer Dosierung von 200–800 mg/Tag eine ähnlich wirksame und nebenwirkungsärmere alternative dar.
    Notes: Abstract Although there is a 20% yeast colonization in the gastrointestinal tract of the population, fungal infections appear only rarely in secondary peritonitis. The risk of severe mycosis increases after a major operation and when a patient is taking broad-spectrum antibiotics, is on total parenteral nutrition, is catheterized, and/or is immune-suppressed. In the past years the incidence of nosocomial fungal infections (usuallyCandida spp.) has risen significantly. Five percent of CAPD-related peritonitis is caused by fungi. In enteral anastomosis breakdown, invasive mycosis occurs more often, with an accompanying lethality of up to 80%. In severe pancreatitis, up to 5% of peripancreatic necrosis in infected with fungi. The clinical course of severe mycosis, like the septic syndrome, is associated with fungemia in up to 50% of cases. As most of the facultative pathogenic fungi are part of the physiological flora, it is difficult to interpret mycological cultures. In order to diagnose invasive fungal infections, histopathological techniques and serologic tests for antigens and antibodies are available. Three antifungal agents (amphotericin B, flucytosine, fluconazole) are available for intravenous administration. Amphotericin B is given at doses of up to 1 mg/kg per day, in liposomal galenism up to 3 mg/kg per day. Combining amphotericin B with flucytosine (150–200 mg/kg per day) a synergistic effect is reached. Fluconazole at a dosage of 200–800 mg per day represents an alternative with similar antifungal activity and lower side effects.
    Type of Medium: Electronic Resource
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