Secretin dissipates red aridine orange fluoescence from pancreatic duct epithelium

This study was undertaken to elucidate whether duct cells in the pancreas contain acidic cytoplasmic compartments regulated by secretion. Microdissected pancreatic ducts from pigs were examined by acridibe orange (AO) and 2′, 7′-biscarboxyethyl-5(6)-carboxyfluorescein/tetraacetioxymethyl ester (BCECF/AM) epifluorescence microscopy. Estimated cytoplasmic pH using BCECF fluorescence was 7.43pL0.04 and was not changed by altering CO₂ tension in the incubation mdium. The epithelium of acridine orange incbated peripheral interlobular pancreatic ducts exhibited green and fluorescence was sen in resting pancreatic ducts and was greatly accentuated by raising CO₂ in the incubation medium with chloroqyuine or NH₄Cl or the protonophores carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) or carbonyl cyanide M-chlorophenylhydrazone (CCCP), leaving uniform gren fluoresence. These findings suggest that pancreatic duct cells contain CO₂-dependent acidic compartments which vanishduring seceatin stimulation and which may be cytoplasmic tubulovesicles.     

Target sites for the inhibition of prostacyclin effect in guinea-pig ileum

Summary In the guinea-pig terminal ileum a maximally effective concentration of prostacyclin (PGI₂) (1 ol/l) induced contractions that were partially resistant to tetrodotoxin (TTX) 0.1 mol/l, to low temperature (20°C) and to atropine (30 nmol/l). Half maximum contractions evoked by PGI₂ (20 nmol/l) were abolished by TTX and by low temperature, which did not modify the response to exogenous acetylcholine (ACh), as well as by atropine. Procaine (5–500 ol/l) caused a concentration-dependent inhibition of contractions induced by PGI₂ (20 nmol/l and 1 mol/l) and by equieffective concentrations of ACh (20 nmol/l and 0.4 ol/l, respectively). The order of magnitude for this inhibition was ACh 20 nmol/l = PGI₂ 20 nmol/l > PGI₂1 mol/l > ACh 0.4 mol/l. In preparations exposed to TTX or to low temperature procaine (50 mol/l) did not affect the residual response to PGI₂ (1 mol/l). Quercetin (1 and 5 ol/l) inhibited the effect of PGI₂ and, at higher concentrations, it also caused partial depression of the responses to ACh. Quercetin did not alter TTX-resistant and low temperature-resistant contractions induced by PGI₂ 1 mol/l. Carbonyl cyanide-trifluoromethoxyphenyl hydrazone (FCCP) (0.1–1 ol/l) reduced the effect of PGI₂ and of ACh to approximately the same extent and inhibited the residual response to PGI₂ 1 mol/l in preparations treated with TTX or expressed to low temperature. The present results show that PGI₂, besides acting on cholinergic neurons, also exerts a direct effect on smooth muscle cells and FCCP can be used to block this effect. In contrast procaine and quercetin selectively inhibit the ACh-mediated component of PGI₂ action. Send offprint requests to R. M. Gaion     

Rapid reduction of ATP synthesis and lack of free radical formation by MPP+ in rat brain synaptosomes and mitochondria

MPTP is a neurotoxin thought to damage dopaminergic neurons through free radical formation. MPTP is metabolized in the brain to MPP(+), which is taken up into dopaminergic neurons via the dopamine transporter and assumed to impair mitochondrial function. We used striatal synaptosomes and telencephalic mitochondria to further investigate MPP(+) mechanism of action. For comparison, the respiratory toxins FCCP, a cyanide analog that uncouples mitochondrial ATP production, and rotenone, a NADH dehydrogenase inhibitor, were also tested. FCCP, MPP(+) and rotenone caused a rapid but stable decrease in [3H]dopamine (DA) uptake by striatal synaptosomes. Two free radical scavengers, the salen-manganese complex EUK-134, and the spin trap s-PBN, did not prevent MPP(+)-induced decrease in DA uptake. However, addition of ATP during synaptosome preparation resulted in partial recovery of MPP(+)-induced [3H]DA uptake decrease. Generation of oxygen free radicals by treatment of telencephalic mitochondria with MPP(+), FCCP, or rotenone, was evaluated by measuring DCF fluorescence, while light emission by the luciferin-luciferase complex was used to determine ATP levels. MPP(+), unlike rotenone, did not produce oxygen free radicals, but rather blocked ATP production in mitochondria, as did FCCP and rotenone. Taken together, these results suggest that MPP(+) toxicity, at least during its initial stages, is primarily due to a decrease in ATP synthesis by mitochondria and not to free radical formation.     

Concentration dependent mitochondrial effect of amiodarone

Although, the antiarrhythmic effect of amiodarone is well characterized, its effect on post-ischemic heart and cardiomyocytes, as well as the mechanism of its toxicity on extracardiac tissues is still poorly understood. In this study, we analyzed energy metabolism in situ during ischemia-reperfusion in Langendorff-perfused heart model by measuring the high-energy phosphate metabolites using 31P NMR spectroscopy. The toxicity of amiodarone on cardiomyocytes and cell lines of extracardiac origin, as well as direct effect of the drug on mitochondrial functions in isolated mitochondria was also analyzed. Amiodarone, when was present at low concentrations and predominantly in membrane bound form, protected heart and mitochondrial energy metabolism from ischemia-reperfusion-induced damages in Langendorff-perfused heart model. Toxicity of the drug was significantly higher on hepatocytes and pancreatic cells than on cardiomyocytes. In isolated mitochondria, amiodarone did not induce reactive oxygen species formation, while it affected mitochondrial permeability transition in a concentration dependent way. Up to the concentration of 10 microM, the drug considerably inhibited Ca(2+)-induced permeability transition, while at higher concentrations it induced a cyclosporin A independent permeability transition of its own. At concentrations where it inhibited the Ca(2+)-induced permeability transition (IC(50)=3.9+/-0.8 microM), it did not affect, between 6 and 30 microM it uncoupled, while, at higher concentrations it inhibited the respiratory chain. Thus, the concentration dependent nature of amiodarone's effect on permeability transition together with the different sensitivities of the tissues toward amiodarone can be involved in the beneficial cardiac and the simultaneous toxic extracardiac effects of the drug.     

Antioxidant and pro-oxidant properties of pyrroloquinoline quinone (PQQ): implications for its function in biological systems

Pyrroloquinoline quinone (PQQ) is a novel redox cofactor recently found in human milk. It has been reported to function as an essential nutrient, antioxidant and redox modulator in cell culture experiments and in animal models of human diseases. As mitochondria are particularly susceptible to oxidative damage we studied the antioxidant properties of PQQ in isolated rat liver mitochondria. PQQ was an effective antioxidant protecting mitochondria against oxidative stress-induced lipid peroxidation, protein carbonyl formation and inactivation of the mitochondrial respiratory chain. In contrast, PQQ caused extensive cell death to cells in culture. This surprising effect was inhibited by catalase, and was shown to be due to the generation of hydrogen peroxide during the autoxidation of PQQ in culture medium. We conclude that the reactivities of PQQ are dependent on its environment and that it can act as an antioxidant or a pro-oxidant in different biological systems.     

Enhancement of proteolytic processing of the beta-amyloid precursor protein by hyperforin

We studied the effect of hyperforin, a component of St. John's wort (Hypericum perforatum) extracts, on the processing of the amyloid precursor protein (APP) in rat pheochromocytoma PC12 cells, stably transfected with human wildtype APP. We observed transiently increased release of secretory APP fragments upon hyperforin treatment. Unique features, like a strong reduction of intracellular APP and the time course of soluble APP release, distinguished the effects of hyperforin from those of alkalizing agents and phorbol esters, well known activators of secretory processing of APP. Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), a protonophore, induced an almost identical decrease in intracellular pH in PC12 cells as does hyperforin. Despite this, FCCP induced a less pronounced release of soluble APP fragments and only slightly reduced intracellular APP levels. These results suggest that hyperforin is an activator of secretory processing of APP with a novel mechanism of action not solely dependent on its effects on intracellular pH.     

Age-related changes in neuronal glucose uptake in response to glutamate and beta-amyloid

Energy supplies that may decline with age are crucial for cells to maintain ionic homeostasis and prevent neuron death. We examined baseline glucose transporter expression and rate of glucose uptake in cultured hippocampal neurons from embryonic, middle-age (12-month-old), and old (24-month-old) rats and exposed the neurons to glutamate, beta-amyloid, and mitochondrial inhibitors. Without stress, the rate of glucose uptake was similar in middle-age and old neurons, and the rate of glucose uptake in embryonic neurons was threefold greater than that in middle-age and old neurons. Glucose uptake increased in the presence of mitochondrial inhibitors (FCCP and oligomycin) for embryonic and middle-age neurons. The old neurons failed to increase glucose uptake. In the presence of glutamate, FCCP, and oligomycin, embryonic neurons showed a decrease in glucose uptake and the middle-age and old neurons showed no change in glucose uptake. Middle-age neurons took up significantly more glucose than old neurons when under mitochondrial and glutamate stress. In the presence of beta-amyloid, only embryonic neurons increased glucose uptake; middle-age and old neurons did not. Fluorescence imaging of immunoreactive glut3 in response to beta-amyloid demonstrated a 16-49% increase in glut3 immunoreactivity at the plasma membrane for the three ages. The results suggest that old neurons were not able to upregulate glucose uptake to ensure cell survival. Neuron aging does not indicate a defect in normal glut3 function; rather, our results suggest that mechanisms regulating glucose uptake under stress fail to react in time to ensure cell survival.     

The mechanism and spread of pacemaker activity through myenteric interstitial cells of Cajal in human small intestine

BACKGROUND & AIMS: It has been generally assumed that interstitial cells of Cajal (ICC) in the human gastrointestinal tract have similar functions to those in rodents, but no direct experimental evidence exists to date for this assumption. This is an important question because pathologists have noted decreased numbers of ICC in patients with a variety of motility disorders, and some have speculated that loss of ICC could be responsible for motor dysfunction. Our aims were to determine whether myenteric ICC (ICC-MY) in human jejunum are pacemaker cells and whether these cells actively propagate pacemaker activity. METHODS: The mucosa and submucosa were removed, and strips of longitudinal muscle were peeled away to reveal the ICC-MY network. ICC networks were loaded with the Ca(2+) indicator fluo-4, and pacemaker activity was recorded via high-speed video imaging at 36.5 degrees C +/- 0.5 degrees C. RESULTS: Rhythmic, biphasic Ca(2+) transients (6.03 +/- 0.33 cycles/min) occurred in Kit-positive ICC-MY. These consisted of a rapidly propagating upstroke phase that initiated a sustained plateau phase, which was associated with Ca(2+) spikes in neighboring smooth muscle. Pacemaker activity was dependent on inositol 1,4,5-triphosphate receptor-operated stores and mitochondrial function. The upstroke phase of Ca(2+) transients in ICC-MY appeared to result from Ca(2+) influx through dihydropyridine-resistant Ca(2+) channels, whereas the plateau phase was attributed to Ca(2+) release from inositol 1,4,5-triphosphate receptor-operated Ca(2+) stores. CONCLUSIONS: Each ICC-MY in human jejunum generates spontaneous pacemaker activity that actively propagates through the ICC network. Loss of these cells could severely disrupt the normal function of the human small intestine.     

Effects of carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone on the growth inhibition in human pulmonary adenocarcinoma Calu-6 cells

Carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) is an uncoupler of mitochondrial oxidative phosphorylation in eukaryotic cells. Here, we evaluated the in vitro effects of FCCP on the growth of Calu-6 lung cancer cells. FCCP inhibited the growth of Calu-6 cells with an IC₅₀ of approximately 6.64 ± 1.84 μM at 72 h, as shown by MTT. DNA flow cytometric analysis indicated that FCCP induced G1 phase arrest below 20 μM of FCCP. Treatment with FCCP decreased the level of CDKs and cyclines in relation to G1 phase. In addition, FCCP not only increased the p27 level but also enhanced its binding with CDK4, which was associated with hypophosphorylation of Rb protein. While transfection of p27 siRNA inhibited G1 phase arrest in FCCP-treated cells, it did not enhance Rb phosphorylation. FCCP also efficiently induced apoptosis. The apoptotic process was accompanied with an increase in sub-G1 cells, annexin V staining cells, mitochondria membrane potential (MMP) loss and cleavage of PARP protein. All of the caspase inhibitors (caspase-3, -8, -9 and pan-caspase inhibitor) markedly rescued the Calu-6 cells from FCCP-induced cell death. However, knock down of p27 protein intensified FCCP-induced cell death. Moreover, FCCP induced the depletion of GSH content in Calu-6 cells, which was prevented by all of the caspase inhibitors. In summary, our results demonstrated that FCCP inhibits the growth of Calu-6 cells in vitro. The growth inhibitory effect of FCCP might be mediated by cell cycle arrest and apoptosis via decrease of CDKs and caspase activation, respectively. These findings now provide a better elucidation of the mechanisms involved in FCCP-induced growth inhibition in lung cancer.