Carbonyl Cyanide 3-chlorophenylhydrazone Research Articles

Acetylation of H4K12 in porcine oocytes during in vitro aging: potential role of ooplasmic reactive oxygen species

Deterioration in the quality of mammalian mature oocytes during metaphase-II (M-II) arrest is called “oocyte aging”. Although histone acetylation may affect the progression of aging in murine oocytes, the mechanism is unknown. The objective was to determine the role of ooplasmic reactive oxygen species (ROS) in acetylation of histone H4 at lysine 12 (acH4K12) in porcine aged oocytes in vitro. Based on immunostaining with a specific antibody, acetylation of H4K12 in porcine oocytes increased during in vitro aging, which coincided with changing patterns of ooplasmic ROS content. Furthermore, both hydrogen peroxide (H 2O 2), and the mitochondrial membrane potential disrupter, carbonyl cyanide 3-chlorophenylhydrazone (CCCP), which can moderately elevate oocyte ROS content, significantly increased acetylation levels of H4K12 in porcine oocytes. It was noteworthy that acetylation in the CCCP group was decreased when ROS was counteracted by cysteine, a common antioxidant. In addition, the intracellular mRNA abundance of acetyltransferase gene HAT1 in aged and H 2O 2 treated oocytes was higher than in M-II phase oocytes, suggesting that HAT1 was involved in this reaction. After parthenogenetic activation, a lower proportion of oocytes developed to the blastocyst stage after CCCP or H 2O 2 treatment when compared with M-II phase oocytes (20 and 0% for CCCP and H 2O 2 groups, respectively, versus 42% for the M-II group, P < 0.05). In conclusion, elevated levels of H4K12 acetylation were attributed to increased ooplasmic ROS content during porcine oocyte aging in vitro.

Ca 2+ homeostasis, Ca 2+ signalling and somatodendritic vasopressin release in adult rat supraoptic nucleus neurones

Multiple mechanisms that maintain Ca 2+ homeostasis and provide for Ca 2+ signalling operate in the somatas and neurohypophysial nerve terminals of supraoptic nucleus (SON) neurones. Here, we examined the Ca 2+ clearance mechanisms of SON neurones from adult rats by monitoring the effects of the selective inhibition of different Ca 2+ homeostatic molecules on cytosolic Ca 2+ ([Ca 2+] i) transients in isolated SON neurones. In addition, we measured somatodendritic vasopressin (AVP) release from intact SON tissue in an attempt to correlate it with [Ca 2+] i dynamics. When bathing the cells in a Na +-free extracellular solution, thapsigargin, cyclopiazonic acid (CPA), carbonyl cyanide 3-chlorophenylhydrazone (CCCP), and the inhibitor of plasma membrane Ca 2+-ATPase (PMCA), La 3+, all significantly slowed down the recovery of depolarisation (50 mM KCl)-induced [Ca 2+] i transients. The release of AVP was stimulated by 50 mM KCl, and the decline in the peptide release was slowed by Ca 2+ transport inhibitors. In contrast to previous reports, our results show that in the fully mature adult rats: (i) all four Ca 2+ homeostatic pathways, the Na +/Ca 2+ exchanger, the endoplasmic reticulum Ca 2+ pump, the plasmalemmal Ca 2+ pump and mitochondria, are complementary in actively clearing Ca 2+ from SON neurones; (ii) somatodendritic AVP release closely correlates with intracellular [Ca 2+] i dynamics; (iii) there is (are) Ca 2+ clearance mechanism(s) distinct from the four outlined above; and (iv) Ca 2+ homeostatic systems in the somatas of SON neurones differ from those expressed in their terminals.

Role of perinuclear mitochondria in the spatiotemporal dynamics of spontaneous Ca2+ waves in interstitial cells of Cajal‐like cells of the rabbit urethra

Although spontaneous Ca(2+) waves in interstitial cells of Cajal (ICC)-like cells (ICC-LCs) primarily arise from endoplasmic reticulum (ER) Ca(2+) release, the interactions among mitochondrial Ca(2+) buffering, cellular energetics and ER Ca(2+) release in determining the spatiotemporal dynamics of intracellular Ca(2+) remain to be elucidated. Spontaneous Ca(2+) transients in freshly isolated ICC-LCs of the rabbit urethra were visualized using fluo-4 Ca(2+) imaging, while the intracellular distribution of mitochondria was viewed with MitoTracker Red. Spontaneous Ca(2+) waves invariably originated from the perinuclear region where clusters of mitochondria surround the nucleus. Perinuclear Ca(2+) dynamics were characterized by a gradual rise in basal Ca(2+) that preceded each regenerative Ca(2+) transient. Caffeine evoked oscillatory Ca(2+) waves originating from anywhere within ICC-LCs. Ryanodine or cyclopiazonic acid prevented Ca(2+) wave generation with a rise in basal Ca(2+), and subsequent caffeine evoked a single rudimentary Ca(2+) transient. Inhibition of glycolysis with 2-deoxy-glucose or carbonyl cyanide 3-chlorophenylhydrazone, a mitochondrial protonophore, increased basal Ca(2+) and abolished Ca(2+) waves. However, caffeine still induced oscillatory Ca(2+) transients. Mitochondrial Ca(2+) uptake inhibition with RU360 attenuated Ca(2+) wave amplitudes, while mitochondrial Ca(2+) efflux inhibition with CGP37157 suppressed the initial Ca(2+) rise to reduce Ca(2+) wave frequency. Perinuclear mitochondria in ICC-LCs play a dominant role in the spatial regulation of Ca(2+) wave generation and may regulate ER Ca(2+) release frequency by buffering Ca(2+) within microdomains between both organelles. Glycolysis inhibition reduced mitochondrial Ca(2+) buffering without critically disrupting ER function. Perinuclear mitochondria may function as sensors of intracellular metabolites.

Characterization of Ca2+ Signalling in Postnatal Mouse Retinal Ganglion Cells: Involvement of OPA1 in Ca2+ Clearance

Purpose: The regulation of Ca2+ entry and removal is a fine-tuned process which remains not well understood in mouse retinal ganglion cells (RGCs). The latter are known to be sensitive to dysfunctions of mitochondria, organelles playing a pivotal role in Ca2+ reuptake.Methods: We first described the Ca2+ signals of RGCs in response to varied drugs with Fura-2 imaging, and secondly tested the role of optic atrophy 1 or OPA1, the gene responsible for Autosomal Dominant Optic Atrophy, on mitochondrial ability to capture intracellular Ca2+ in cells transfected with the OPA1 small interfering ribonucleic acids (siRNAs).Results: In control RGCs, K+-evoked [Ca2+]i increase was blocked by the Ca2+ channel antagonists (Ni2++ Cd2+) and GABAA receptor agonist muscimol-induced [Ca2+]i responses were attenuated by the GABAA receptor antagonists, picrotoxin and gabazine. We also prove the presence of NMDA and AMPA/Kainate (glutamate receptor agonists) responsive receptors in this model. Application of cyclopiazonic acid, an inhibitor of Ca2+-ATPase pumps of the intracellular Ca2+ stores, induced an increase in [Ca2+]i while ryanodine or caffeine had no effect on resting [Ca2+]i. Spontaneous Ca2+ oscillations in contacting neurons highlighted the importance of cross-talks between RGCs during maturation. The mitochondrial respiration uncoupler, carbonyl cyanide 3-chlorophenylhydrazone (CCCP), induced robust raises of intracellular Ca2+ after K+ application, with a more pronounced effect in cells silenced for OPA1, which could lead to cell death.Conclusions: Our results indicate an important role of OPA1 in mitochondrial dependent Ca2+ homeostasis and cell survival in RGCs, suggesting a possible patho-physiological mechanism involved in inherited optic neuropathies.

Fluconazole Transport into Candida albicans Secretory Vesicles by the Membrane Proteins Cdr1p, Cdr2p, and Mdr1p

A major cause of azole resistance in Candida albicans is overexpression of CDR1, CDR2, and/or MDR1, which encode plasma membrane efflux pumps. To analyze the catalytic properties of these pumps, we used ACT1- and GAL1-regulated expression plasmids to overexpress CDR1, CDR2, or MDR1 in a C. albicans cdr1 cdr2 mdr1-null mutant. When the genes of interest were expressed, the resulting transformants were more resistant to multiple azole antifungals, and accumulated less [(3)H]fluconazole intracellularly, than empty-vector controls. Next, we used a GAL1-regulated dominant negative sec4 allele to cause cytoplasmic accumulation of post-Golgi secretory vesicles (PGVs), and we found that PGVs isolated from CDR1-, CDR2-, or MDR1-overexpressing cells accumulated much more [(3)H]fluconazole than did PGVs from empty-vector controls. The K(m)s (expressed in micromolar concentrations) and V(max)s (expressed in picomoles per milligram of protein per minute), respectively, for [(3)H]fluconazole transport were 0.8 and 0.91 for Cdr1p, 4.3 and 0.52 for Cdr2p, and 3.5 and 0.59 for Mdr1p. [(3)H]fluconazole transport by Cdr1p and Cdr2p required ATP and was unaffected by carbonyl cyanide 3-chlorophenylhydrazone (CCCP), whereas [(3)H]fluconazole transport by Mdr1p did not require ATP and was inhibited by CCCP. [(3)H]fluconazole uptake by all 3 pumps was inhibited by all other azoles tested, with 50% inhibitory concentrations (IC(50)s; expressed as proportions of the [(3)H]fluconazole concentration) of 0.2 to 5.6 for Cdr1p, 0.3 to 3.1 for Cdr2p, and 0.3 to 3.1 for Mdr1p. The methods used in this study may also be useful for studying other plasma membrane transporters in C. albicans and other medically important fungi.

In Vitro and in Vivo Enhanced Generation of Human A9 Dopamine Neurons from Neural Stem Cells by Bcl-XL

Human neural stem cells derived from the ventral mesencephalon (VM) are powerful research tools and candidates for cell therapies in Parkinson disease. Previous studies with VM dopaminergic neuron (DAn) precursors indicated poor growth potential and unstable phenotypical properties. Using the model cell line hVM1 (human ventral mesencephalic neural stem cell line 1; a new human fetal VM stem cell line), we have found that Bcl-X(L) enhances the generation of DAn from VM human neural stem cells. Mechanistically, Bcl-X(L) not only exerts the expected antiapoptotic effect but also induces proneural (NGN2 and NEUROD1) and dopamine-related transcription factors, resulting in a high yield of DAn with the correct phenotype of substantia nigra pars compacta (SNpc). The expression of key genes directly involved in VM/SNpc dopaminergic patterning, differentiation, and maturation (EN1, LMX1B, PITX3, NURR1, VMAT2, GIRK2, and dopamine transporter) is thus enhanced by Bcl-X(L). These effects on neurogenesis occur in parallel to a decrease in glia generation. These in vitro Bcl-X(L) effects are paralleled in vivo, after transplantation in hemiparkinsonian rats, where hVM1-Bcl-X(L) cells survive, integrate, and differentiate into DAn, alleviating behavioral motor asymmetry. Bcl-X(L) then allows for human fetal VM stem cells to stably generate mature SNpc DAn both in vitro and in vivo and is thus proposed as a helpful factor for the development of cell therapies for neurodegenerative conditions, Parkinson disease in particular.

CwrA, a gene that specifically responds to cell wall damage in Staphylococcus aureus

Transcriptional profiling data accumulated in recent years for the clinically relevant pathogen Staphylococcus aureus have established a cell wall stress stimulon, which comprises a coordinately regulated set of genes that are upregulated in response to blockage of cell wall biogenesis. In particular, the expression of cwrA (SA2343, N315 notation), which encodes a putative 63 amino acid polypeptide of unknown biological function, increases over 100-fold in response to cell wall inhibition. Herein, we seek to understand the biological role that this gene plays in S. aureus. cwrA was found to be robustly induced by all cell wall-targeting antibiotics tested - vancomycin, oxacillin, penicillin G, phosphomycin, imipenem, hymeglusin and bacitracin - but not by antibiotics with other mechanisms of action, including ciprofloxacin, erythromycin, chloramphenicol, triclosan, rifampicin, novobiocin and carbonyl cyanide 3-chlorophenylhydrazone. Although a DeltacwrA S. aureus strain had no appreciable shift in MICs for cell wall-targeting antibiotics, the knockout was shown to have reduced cell wall integrity in a variety of other assays. Additionally, the gene was shown to be important for virulence in a mouse sepsis model of infection.

Mutant prevention concentrations of fluoroquinolones against Campylobacter jejuni isolated from chicken

The mutant prevention concentration (MPC) and mutant selection window (MSW) concepts have been used to evaluate antibiotic concentration ranges that prevent the emergence of antibiotic resistant mutants. Campylobacter jejuni is highly mutable to fluoroquinolone (FQ) antibiotics, but it is unknown if the MPC concept can be used to prevent mutant emergence. In this study, the MPCs of three FQs including enrofloxacin, norfloxacin and ciprofloxacin were determined using 13 C. jejuni isolates. Also, first- and second-step FQ-resistant mutants were selected and the mutations in gyrA and gyrB as well as the contribution of efflux pump to FQ resistance were investigated. The MICs of all selected mutants were determined in the presence or absence of the efflux pump inhibitors carbonyl cyanide 3-chlorophenylhydrazone (CCCP) and reserpine. Our results revealed that the three tested FQs had different MPC ranges and the MPC order was norfloxacin > ciprofloxacin > enrofloxacin, suggesting a better in vitro efficacy of enrofloxacin over ciprofloxacin and norfloxacin in reducing the emergence of C. jejuni mutants. The results also confirmed the single-step mechanism of acquired FQs resistance in C. jejuni mutants. Both point mutations (Thr-86-Ile and Asp-90-Asn) in the gyrA gene and the function of efflux pumps contributed to the acquired resistance to ciprofloxacin and norfloxacin, while gyrA mutations (Thr-86-Ile and Asp-90-Asn) were the main mechanism for enrofloxacin resistance. These findings provide new insights into the development and mechanisms of FQ resistance in Campylobacter.

Methylglyoxal augments intracellular oxidative stress in human aortic endothelial cells

Methylglyoxal (MGO) is a non-enzymatic metabolite in the glycolytic pathway and its concentration in blood and tissues is elevated in diabetes and renal failure. MGO induces tissue injuries via ROS; however, the mechanism remains to be clarified. The present study examined the harmful actions of MGO. Human aortic endothelial cells were assessed under real-time fluorescent microscopy with continuous superfusion. Increases in intracellular ROS were measured with fluorescent indicator, 5-(and-6)-chloromethyl-2’,7’-dichlorodihydrofluorescein diacetate acetyl ester (DCFH-DA). The addition of MGO rapidly increased the ROS in a dose-dependent manner. The increment of DCF was entirely abolished by pre-treatment with superoxide anion scavenger and membrane-permeable catalase, indicating that MGO induces superoxide production. The increment was completely inhibited by 2-thenoyltrifluoroacetone or carbonyl cyanide 3-chlorophenylhydrazone and partially inhibited by N-methyl-L-arginine. These data suggest that MGO stimulates superoxide production from mitochondria and partially stimulates nitric oxide synthase in human endothelial cells.

NBDT (3‐(N‐(7‐nitrobenz‐2‐oxa‐1,3‐diazol‐4‐yl)amino)‐3‐toluene)—A novel fluorescent dye for studying mechanisms of toluene uptake into vital bacteria

Uptake of small hydrophobic substances such as toluene into bacteria is widely assumed to occur by passive diffusion. Some toluene degrading bacteria, however, are described to contain uptake systems which may be involved in the transport of this compound. In this study, a fluorescently labeled toluene analogue dye (3-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-3-toluene; NBDT), flow cytometry, and shot gun proteome analysis were used to follow toluene uptake into bacteria in more detail. The new dye has excitation peaks at 444 and 475 nm and an emission peak at 537 nm. The toluene-degraders P. putida mt-2 and P. putida F1 as well as P. putida KT2440 and E. coli K12 as negative controls were included. To enable quantification of NBDT uptake, carbonyl cyanide 3-chlorophenylhydrazone (CCCP) was added to inactivate NBDT efflux pumps. The porin inhibitor cadaverine was added to study the porin-mediated influx of toluene. Cadaverine reduced NBDT uptake by toluene-grown P. putida mt-2 and F1 by 25% and 42%, respectively, thus revealing an involvement and possibly a regulatory function of porins in the uptake of the toxic substrate toluene. Shot gun proteome measurements gave evidence for the presence of toluene transporting porins in P. putida mt-2 grown on toluene but not when grown on glucose.

Plasma membrane calcium ATPase 4 and the remodeling of calcium homeostasis in human colon cancer cells

A remodeling of calcium homeostasis has been identified as a characterizing feature of some cancers. Possible consequences of this include alterations in many pivotal physiological responses including apoptosis, proliferation and gene transcription. An alteration in calcium homeostasis can occur via changes in the expression of proteins that transport calcium and examples of cancers where this is seen includes the prostate and breast. A specific isoform of the calcium efflux pump, plasma membrane Ca(2+)-ATPase (PMCA) 4, is significantly upregulated during differentiation of the HT-29 colon cancer cell line suggesting that it may also be altered in colon cancer. We now report that differentiated HT-29 colon cancer cells have pronounced plasma membrane PMCA4 localization, consistent with augmented calcium efflux. Assessment of PMCA4 transcription in human colon cancer samples suggests that PMCA4 is significantly (P < 0.000001) downregulated early in the progression of some colon cancers as these cells become less differentiated. Inhibition of PMCA4 using small interfering RNA did not induce cell death or augment sensitivity to the mitochondrial uncoupler carbonyl cyanide 3-chlorophenylhydrazone (CCCP) or tumor necrosis factor-related apoptosis-inducing ligand. Reversing the colon cancer remodeling of PMCA4 by overexpression reduced cellular proliferation (P < 0.01) and downregulated transcription of the calcium sensitive early response gene FOS. Our studies suggest that the remodeling of the calcium signal in colon cancer is associated with compromised calcium efflux at a level that promotes proliferative pathways while avoiding increased sensitivity to apoptotic stimuli.

Repression of induced apoptosis in the 2-cell bovine embryo involves DNA methylation and histone deacetylation

Apoptosis in the bovine embryo cannot be induced by activators of the extrinsic apoptosis pathway until the 8–16-cell stage. Depolarization of mitochondria with the decoupling agent carbonyl cyanide 3-chlorophenylhydrazone (CCCP) can activate caspase-3 in 2-cell embryos but DNA fragmentation does not occur. Here we hypothesized that the repression of apoptosis is caused by methylation of DNA and deacetylation of histones. To test this hypothesis, we evaluated whether reducing DNA methylation by 5-aza-2′-deoxycytidine (AZA) or inhibition of histone deacetylation by trichostatin-A (TSA) would make 2-cell embryos susceptible to DNA fragmentation caused by CCCP. The percent of blastomeres positive for TUNEL was affected by a treatment × CCCP interaction ( P < 0.0001). CCCP did not cause a large increase in the percent of cells positive for TUNEL in embryos treated with vehicle but did increase the percent of cells that were TUNEL positive if embryos were pretreated with AZA or TSA. Immunostaining using an antibody against 5-methyl-cytosine antibody revealed that AZA and TSA reduced DNA methylation. In conclusion, disruption of DNA methylation and histone deacetylation removes the block to apoptosis in bovine 2-cell embryos.

Repression of the Intrinsic Pathway to Apoptosis in the Two-Cell Bovine Embryo Involves Histone De-Acetylation and DNA Methylation.

Apoptosis in the bovine embryo cannot be induced by activators of the intrinsic or extrinsic apoptosis pathways until the 8-16 cell stage. Depolarization of mitochondria with the decoupling agent carbonyl cyanide 3-chlorophenylhydrazone (CCCP) can activate caspase-3 in two-cell embryos but DNA fragmentation does not occur. Here we hypothesized that the repression of apoptosis is caused by hyperacetylation of histones and methylation of DNA. To test this hypothesis, we evaluated whether reducing DNA methylation or histone deacetylation would make two-cell embryos susceptible to DNA fragmentation caused by CCCP. Bovine oocytes were maturated and fertilized in vitro for 18 h. Putative zygotes were then treated for 12 h with vehicle, 100 μM 5-aza 2′ deoxycytidine (AZA), to cause DNA demethylation, or 100 nM Trichostatin A (TSA), to inhibit histone deacethylase. Embryos were then treated + 100 μM CCCP. Fragmentation of DNA was determined at 24 h after CCCP using the TUNEL reaction. The experiment was replicated 6 times using a total of 458 embryos. The percent of blastomeres positive for TUNEL (% TUNEL positive nuclei) was affected by a treatment x CCCP interaction (P<0.0001). CCCP did not cause a large increase in % TUNEL positive nuclei in control embryos (2.0 +3.4% vs 7.7 + 5.5%) but caused a large increase in % TUNEL positive nuclei for embryos treated with AZA (5.4 + 2.9% vs 42.3 + 3.2%). Results for TSA indicated an increase in %TUNEL positive nuclei in embryos without CCCP and only a slight increase with CCCP treatment (17.1 + 2.8% vs 24.9 + 4.2%). Further analysis of TSA treated embryos focused on the subset of embryos that were < 8 cells (i.e., those that are ordinarily not susceptible to apoptosis). In this subset, which included 68% of the TSA-treated embryos, there was a clear increase in % TUNEL positive nuclei after CCCP treatment (10.0 + 4.2% vs 24 + 4.5%, P<0.025). In conclusion, disruption of DNA methylation and histone deacetylation can remove the block to apoptosis in bovine two-cell embryos. Moreover, inhibition of histone acetylation leads to some spontaneous apoptosis in the absence of a proapoptotic signal. It is likely that interactions between methylated DNA and histones masks DNA at early stages of development and prevents access of caspase-activated DNAses to DNA. Support: USDA CSREES Grant # 2007-35203-18070. (poster)

Role of Ca2+ in injury-induced changes in sodium current in rat skeletal muscle

Characteristics of voltage-dependent sodium current recorded from adult rat muscle fibers in loose patch mode were rapidly altered following nearby impalement with a microelectrode. Hyperpolarized shifts in the voltage dependence of activation and fast inactivation occurred within minutes. In addition, the amplitude of the maximal sodium current decreased within 30 min of impalement. Impalement triggered a sustained elevation of intracellular Ca(2+). However, buffering Ca(2+) by loading fibers with AM-BAPTA did not affect the hyperpolarized shifts in activation and inactivation, although it did prevent the reduction in current amplitude. Surprisingly, the rise in intracellular Ca(2+) occurred even in the absence of extracellular Ca(2+). This result indicated that the injury-induced Ca(2+) increase came from an intracellular source, but it was not blocked by an inhibitor of release from the sarcoplasmic reticulum, which suggested involvement of mitochondria. Ca(2+) release from mitochondria triggered by carbonyl cyanide 3-chlorophenylhydrazone was sufficient to cause a reduction in sodium current amplitude but had little effect of the voltage dependence of activation and fast inactivation. Our data suggest the effects of muscle injury can be separated into a Ca(2+)-dependent reduction in amplitude and a largely Ca(2+)-independent shift in activation and fast inactivation. Together, the impalement-induced changes in sodium current reduce the number of sodium channels available to open at the resting potential and may limit further depolarization and thus promote survival of muscle fibers following injury.

The effect of substrate, ADP and uncoupler on the respiration of tomato pollen during incubation in vitro at moderately high temperature

Pollen of tomato cv. Supermarmande was collected from greenhouse-grown plants at various intervals throughout the year and arbitrarily classified as of high, medium or low respiratory activity on the basis of CO(2) production during 8 h incubation in vitro at 30 degrees C, a temperature that is considered to be moderately high for tomato fruit set. After an initial burst of respiration during the first stage of hydration at 30 degrees C (>1 h), the respiration rate of pollen of all three categories declined, the decrease being greater in the lots with a low or medium respiratory activity than in the high category. During hydration (10 min after the start of incubation), the addition of succinate or reduced beta-nicotinamide adenine dinucleotide (NADH) to the substrate increased the respiratory rate of slowly-respiring pollen more than that of fast-respiring pollen, but carbonyl cyanide 3-chlorophenylhydrazone (CCCP) and adenosine 5'-diphosphate (ADP) had less effect. After 1-4 h incubation, the respiration rate of the slow- or medium-respiring pollen lots had decreased, but was stimulated by succinate or NADH, and to a lesser degree by ADP. By 7 h, the respiration rate of all pollen lots had declined and was stimulated less by substrate, ADP or CCCP. The oxidation of NADH by tomato pollen contrasts with the failure of other pollen species to utilize this substrate; moreover, a synergistic effect of NADH and succinate was consistently observed. We conclude that the decline in respiration during incubation for up to 4 h at 30 degrees C may reflect a lack of respiratory substrate. After 7 h, however, the decreased response to substrate indicates a loss of mitochondrial integrity or an accumulation of metabolic inhibitors. It is concluded that at 30 degrees C (a moderately high temperature for tomato pollen), the initially high rate of respiration leads to exhaustion of the endogenous respiratory substrates (particularly in pollen with low to medium respiratory activity), but subsequently to ageing and a loss of mitochondrial activity.

W1551 Extracellular ATP Synthase Generates Extracellular ATP, Regulating Bicarbonate Secretion in Rat Duodenum

The pH of the alkaline microclimate overlying the duodenal enterocyte brush border is regulated by an ecto-purinergic signaling system consisting of intestinal alkaline phosphatase (IAP), extracellular ATP and P2Y receptors. This system regulates the rate of duodenal bicarbonate secretion (DBS). IAP inhibition increases DBS and non-lytic ATP release into the lumen, the mechanism of which has not been clarified. Ecto-F1F0-ATP synthase has been localized to the plasma membrane of several non-epithelial cell types. We hypothesized that ecto-ATP synthase generates extracellular ATP, regulating DBS as part of the ectopurinergic signaling system. We measured DBS with flow-through pH and CO2 electrodes with the perfusate ATP content measured by luciferin-luciferase bioassay. We tested the effect of perfusion of the competitive AP inhibitor glycerol phosphate (GP, 10 mM) with or without the addition of ATP synthase inhibitors oligomycin (Om, 5 μg/ml), piceatannol (Pic, 20 μM) or resveratorol (Res, 20 μM), or the mitochondrial uncoupler carbonyl cyanide 3-chlorophenylhydrazone (CCCP, 1 μM) on DBS and ATP output. GP increased DBS and ATP output. Pic or Res inhibited GP-induced augmented DBS and ATP output. GP, or Pic or Res with GP had no effect on LDH release, confirming the absence of lytic cellular injury. Om or CCCP had a lesser effect on GP-induced DBS, but inhibited GP-induced ATP output accompanied by increased LDH release, consistent with cellular injury. Furthermore, α and β subunits of F1 complex of ATP synthase were immunolocalized to the brush border membranes (α β) in the lamina propria mucosa. Furthermore, phosphate buffer saline (pH 7.0, 10 mM) increased ATP output, suggesting that excess supply of inorganic phosphate (Pi) may enhance ATP synthesis and/or inhibit ATP degradation. Luminal ATP release unmasked by IAP inhibition is dependent on extracellular ATP synthesis, consistent with the presence of ecto-ATP synthase on the brush border. Extracellular ATP synthesis and ATP degradation by IAPmay coordinately regulate ATP-P2Y signaling for DBS as part of a novel ecto-purinergic signaling system.

W1553 Glutamate Reduces Helicobacter pylori-Induced Gastric Atrophy in Rodents - Possible Involvement of Chief Cell Protection via Glutamate Metabolism-

The pH of the alkaline microclimate overlying the duodenal enterocyte brush border is regulated by an ecto-purinergic signaling system consisting of intestinal alkaline phosphatase (IAP), extracellular ATP and P2Y receptors. This system regulates the rate of duodenal bicarbonate secretion (DBS). IAP inhibition increases DBS and non-lytic ATP release into the lumen, the mechanism of which has not been clarified. Ecto-F1F0-ATP synthase has been localized to the plasma membrane of several non-epithelial cell types. We hypothesized that ecto-ATP synthase generates extracellular ATP, regulating DBS as part of the ectopurinergic signaling system. We measured DBS with flow-through pH and CO2 electrodes with the perfusate ATP content measured by luciferin-luciferase bioassay. We tested the effect of perfusion of the competitive AP inhibitor glycerol phosphate (GP, 10 mM) with or without the addition of ATP synthase inhibitors oligomycin (Om, 5 μg/ml), piceatannol (Pic, 20 μM) or resveratorol (Res, 20 μM), or the mitochondrial uncoupler carbonyl cyanide 3-chlorophenylhydrazone (CCCP, 1 μM) on DBS and ATP output. GP increased DBS and ATP output. Pic or Res inhibited GP-induced augmented DBS and ATP output. GP, or Pic or Res with GP had no effect on LDH release, confirming the absence of lytic cellular injury. Om or CCCP had a lesser effect on GP-induced DBS, but inhibited GP-induced ATP output accompanied by increased LDH release, consistent with cellular injury. Furthermore, α and β subunits of F1 complex of ATP synthase were immunolocalized to the brush border membranes (α β) in the lamina propria mucosa. Furthermore, phosphate buffer saline (pH 7.0, 10 mM) increased ATP output, suggesting that excess supply of inorganic phosphate (Pi) may enhance ATP synthesis and/or inhibit ATP degradation. Luminal ATP release unmasked by IAP inhibition is dependent on extracellular ATP synthesis, consistent with the presence of ecto-ATP synthase on the brush border. Extracellular ATP synthesis and ATP degradation by IAPmay coordinately regulate ATP-P2Y signaling for DBS as part of a novel ecto-purinergic signaling system.

Polymorphic ROS scavenging revealed by CCCP in a lizard

Ingestion of antioxidants has been argued to scavenge circulating reactive molecules (e.g., free radicals), play a part in mate choice (by mediating access to this important resource), and perhaps increase life span. However, recent work has come to question these relationships. We have shown elsewhere in the polychromatic lizard, Ctenophorus pictus, that diet supplementation of carotenoids as antioxidants does not depress circulating natural reactive oxygen species (ROS) levels and leads to no corresponding improvement of color traits. However, a much stronger test would be to experimentally manipulate the ROS levels themselves and assess carotenoid-induced ROS depression. Here, we achieve this by using carbonyl cyanide 3-chlorophenylhydrazone, which elevates superoxide (SO) formation approximately threefold at 10 microM in this model system. We then look for depressing effects on ROS of the carotenoids in order to assess whether 'super-production' of SO makes carotenoid effects on elevated ROS levels detectable. The rationale for this treatment was that if not even such elevated levels of SO are reduced by carotenoid supplementation, the putative link carotenoids, ROS depression, and mate quality (in terms of antioxidant capacity) is highly questionable. We conclude that there is no significant effect of carotenoids on mean SO levels even at the induced ROS levels. However, our results showed a significant interaction effect between carotenoid treatment and male color, with red males having higher ROS levels than yellow males. We suggest that this may be because different pigments are differently involved in the generation of the integumental colors in the two morphs with concomitant effects on ROS depletion depending on carotenoid uptake or allocation to coloration and antioxidation.

α 1-adrenoceptor modulation of spontaneous electrical waveforms in the guinea-pig prostate

The aim of this study was to investigate the effects of phenylephrine on the spontaneous slow wave and pacemaker activity in the guinea-pig prostate. Membrane potential recordings were made using intracellular microelectrodes. Guinea-pig prostatic cells either displayed ‘slow wave’ activity or ‘pacemaker’ potentials. In the presence of nifedipine, none of the parameters measured were significantly different between both waveforms. Phenylephrine (1 µM) or histamine (5 µM) increased the frequency of slow waves and pacemaker potentials in the absence or presence of nifedipine (1 µM). In the presence of nifedipine (1 µM), the addition of either cyclopiazonic acid (CPA, 10 µM) or carbonyl cyanide 3-chlorophenylhydrazone (CCCP, 1–10 µM) caused an initial transient increase in frequency of the spontaneous electrical activity that was associated with a membrane depolarisation of 3–7 mV before abolishing activity. Following the cessation of electrical activity, phenylephrine (1 µM) was unable to restore the spontaneous electrical events. Although niflumic acid (10–100 µM) (in the presence of 1 µM nifedipine) similarly abolished all activity, electrical activity was promptly initiated upon the addition of phenylephrine (1 µM). These results demonstrate that in the presence of nifedipine, there are no differences between the slow waves and pacemaker potentials suggesting that both activities arise from cells that are well coupled. Both phenylephrine and histamine increased the frequency of pacemaker activity which is likely to; at least, partly explain the increase in slow wave discharge also evoked by these agents. In the presence of nifedipine, the effects observed upon α 1-adrenoceptor activation were dependent on Ca 2+ cycling by both the intracellular Ca 2+ stores and mitochondria.

Can programmed cell death be induced in post-ejaculatory bull and stallion spermatozoa?

Apoptosis is common during spermatogenesis. Here, it was tested whether apoptosis could be induced in sperm after ejaculation. There were several lines of evidence to indicate that sperm are resistant to induction of apoptosis. First, incubation of bull sperm at temperatures characteristic of normothermia (38.5 °C) or heat shock (40 and 41 °C) for 4 h did not increase the proportion of sperm positive for the TUNEL reaction. There was also no reduction in mitochondrial polarity caused by exposure to 40 or 41 °C. Incubation at 38.5 °C (least-squares mean ± SEM = 4.0 ± 1.4%), 40 °C (6.2 ± 1.4%), and 41 °C (7.0 ± 1.4%) for 24 h did increase the proportion of sperm that were TUNEL positive slightly as compared to non-incubated control sperm (1.0 ± 1.4%). However, the increase in TUNEL labeling was not affected by incubation temperature and occurred even in the presence of the group II caspase inhibitor, z-DEVD-fmk. In addition, exposure of bull sperm to carbonyl cyanide 3-chlorophenylhydrazone (CCCP), which depolarizes mitochondrial membranes, did not increase TUNEL labeling. Stallion sperm were also resistant to increased TUNEL labeling in response to incubation at 41 °C for 4 h or exposure to CCCP. Western blotting was performed to determine whether failure of induction of apoptosis was due to aberrant caspase activation. Procaspase-9 was detected in bull sperm, but cleavage to caspase-9 was not induced by short-term aging at 38.5, 40, or 41 °C, or exposure to CCCP. Procaspase-3 was not detected in bull spermatozoa. In conclusion, post-ejaculatory bull and stallion sperm were resistant to induction of apoptosis; this resistance, at least in bulls, was due to refractoriness of mitochondria to heat shock-induced depolarization, lack of activation of procaspase-9, and an absence of procaspase-3.