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PubMed2002

Histamine release by hydrochloric acid is mediated via reactive oxygen species generation and phospholipase D in RBL-2H3 mast cells.

In order to investigate the underlying mechanism of HCl in oesophagitis, the inflammatory response to HCl was observed in RBL-2H3 mast cells. Rat basophilic leukemia (RBL-2H3) cells were used to measure histamine release, arachidonic acid (AA) release, reactive oxygen species (ROS) and peroxynitrite generation induced by HCl. Exogenous HCl increased the level of histamine release and ROS generation in a dose dependent manner, whereas it decreased the spontaneous release of [3H] AA and the spontaneous production of peroxynitrite. Mepacrine (10 microM), oleyloxyethyl phosphorylcholine (10 microM) and bromoenol lactone (10 microM) did not affect both the level of histamine release and ROS generation induced by HCl. U73122 (1 microM), a specific phospholipase C (PLC) inhibitor did not have any influence on level of histamine release and ROS generation. Propranolol (200 microM), a phospholipase D (PLD) inhibitor, and neomycin (1 mM), a nonspecific PLC and PLD inhibitor, significantly inhibited both histamine release and ROS generation. Diphenyleneiodonium (10 microM), a NADPH oxidase inhibitor, and tiron (5 mM), an intracellular ROS scavenger significantly inhibited the HCl-induced histamine release and ROS generation. These findings suggest that the inflammatory responses to HCl is related to histamine release and ROS generation, and that the ROS generation by HCl may be involved in histamine release via the PLD pathway in RBL-2H3 cells.

Animals↗

PubMed1997

[Rashkind PDA occluder and detachable metal spiral for interventional occlusion of the ductus arteriosus Botalli. Indications, outcome and complications].

The intention of this study was to determine the efficacy and complication rate of the interventional duct occlusion. Furthermore, we wanted to compare the meanwhile well-known Rashkind-PDA-Occluder system (ROS) with the results of the considerably cheaper detachable duct occlude coils (PDA-S). For this purpose, we analyzed the data of 53 consecutive patients who received a total of 28 ROS and 25 PDA-S. The ROS was implanted in the slightly larger ducts (O 3.1 +/- 0.6 mm), the PDA-S in the smaller ones (O 2.2 +/- 0.6 mm). The rate of residual shunting was 20% in the ROS-group (follow-up: 104 to 1099 days, mean 580 +/- 300 days) and 7.7% in the PDA-S-group (follow-up: 90 to 384 days, mean 161 +/- 101 days). One patient after ROS showed a significant residual shunting and received 2.5 years after the first procedure a PDA-S which eliminated the shunt. In a second patient the attempt of implanting two coils in a 4 mm duct resulted in the embolization of the first coil into the right pulmonary artery. The coil could not be retrieved and was left in the pulmonary artery without sequelae. All other patients received only one ROS or one PDA-S. There were no further complications. The complication rate for all interventional duct occlusions therefore was 1.9% (0% for the ROS-group; 4.0% for the PDA-S-group). In comparison with the Gianturco coils that have been introduced since 1975, the new detachable coils can be manipulated much more easily, because they remain fixed at the delivery wire until an ideal position is achieved. Then the coil is set free by unscrewing it from the delivery wire. For small ducts with a diameter up to 3 mm the detachable coil is an effective and cheap alternative which can be handled easily. Because of the smaller delivery systems in comparison to the ROS (4-5 F versus 8 F or 11 F) the PDA-S is also suitable for small children and infants. larger ducts should be closed with the ROS.

Angioplasty, Balloon↗

PubMed2005

Variability analysis of oscillatory airway resistance in children.

Testing pulmonary function in preschool children is problematic since individual cooperation is often insufficient to yield adequate results. This is especially the case when repetitive measurements are carried out, e.g., during provocation tests in order to diagnose bronchial hyperresponsiveness. We therefore sought to determine whether the variability of the oscillatory airway resistance, which can be measured with minimal patient cooperation, might serve as an approach to separate asymptomatic children with asthma from age-matched healthy controls. Monofrequent forced oscillation technique was used to measure the oscillatory resistance (Ros) continuously at a repetition rate of 10 Hz. Forty consecutive values of Ros at the end of expiration (Ros(pe)) and at the end of inspiration (Ros(pi)) were used to calculate variability measures [standard deviation (SD), averaged deviation (LD), root mean square successive differences (RMSSD)] known from heart rate variability analysis to describe the airway diameter variability in 36 asthmatics and 21 healthy controls. Airway diameter variability of Ros(pi) and Ros(pe) as expressed by SD from the mean (P = 0.01 and 0.05, respectively), from LD (P = 0.01 and 0.21, respectively) and from the RMSSD (P = 0.006 and 0.03, respectively) was greater in patients with asthma. Mean values of Ros(pi) and Ros(pe) were not different between groups. Airway diameter variability is higher in asymptomatic children with asthma than in healthy controls. Future research should evaluate whether the measurement of the variability of Ros(pi), which does not require the patient's cooperation, can give similar information as challenge tests with respect to bronchial hyperresponsiveness.

Airway Resistance↗

PubMed2005

Xanthine oxidase, nitric oxide synthase and phospholipase A(2) produce reactive oxygen species via mitochondria.

The formation of reactive oxygen species (ROS) has been suggested to be associated with excitotoxicity but the involvement of cytoplasmic enzymes in ROS formation is not clearly known. In the present study, we examined the role of xanthine oxidase (XO), nitric oxide synthase (NOS) and phospholipase A(2) (PLA(2)) in glutamate-induced oxidative stress in rat cortical slices. Glutamate-induced ROS formation and mitochondrial depolarization were measured in rat cortical slices in presence of allopurinol, L-NAME and 4-bromophenacylbromide, the specific inhibitors of XO, NOS and PLA(2), respectively. Upon stimulation of slices with glutamate, a significant increase in ROS formation and mitochondrial depolarization was observed. However, pretreatment of slices with allopurinol, L-NAME and 4-bromophenacylbromide inhibited the glutamate-induced ROS formation and mitochondrial depolarization. The glutamate-induced ROS formation was dependent on the concentration of these inhibitors and also on the duration of the treatment. Allopurinol was found to be less effective as compared to L-NAME and 4-bromophenacylbromide. The combined treatment of slices with these enzyme inhibitors showed further inhibition in ROS formation and mitochondrial depolarization. The inhibition in ROS formation as well as mitochondrial depolarization by allopurinol, L-NAME and 4-bromophenacylbromide clearly suggests that the activation of XO, NOS and PLA(2) by calcium during glutamate receptor stimulation may release some chemicals which depolarize mitochondria resulting in ROS formation.

Acetophenones↗

PubMed2004

New concepts in reactive oxygen species and cardiovascular reperfusion physiology.

Increasingly complex behavior of free radicals and reactive oxygen species (ROS) are noted within biological systems. Classically free radicals and ROS were considered injurious, however current mechanisms describe both protective and deleterious effects. A burst of ROS has been well described with the first moments of reperfusion and is associated with injury. However ROS can also be protective as signal preconditioning protection and induce stress responses that lead to survival. ROS generation is appreciated to occur during ischemia despite the low oxygen tension, from a likely mitochondria source, and ROS-induced ROS release may amplify its signal. The burst of ROS seen during reperfusion may originate from a different cellular source than during ischemia and is not yet fully identified. ROS and cellular redox conditions regulate a large number of vital pathways (energy metabolism, survival/stress responses, apoptosis, inflammatory response, oxygen sensing, etc). While cellular systems may demonstrate reperfusion injury, whole organ and animal models continue to report contradictory results on reperfusion injury and the role of antioxidants as a therapy. Collectively, these data may offer insight into why clinical trials of antioxidants have had such mixed and mostly negative results. Future antioxidant therapies are likely to be effective but they must become: more specific for site of action, not have deleterious effects on other signaling pathways, be targeted to a specific reactive oxygen species or cellular compartment, and be "time sensitive" so they deliver the correct therapy at precisely the correct time in ischemia and reperfusion.

Animals↗

PubMed2005

Serum withdrawal kills U937 cells by inducing a positive mutual interaction between reactive oxygen species and phosphoinositide 3-kinase.

Reactive oxygen species (ROS) can be generated following cell stimulation and function as intracellular signaling molecules. To determine signaling components involved in ROS induction, human U937 blood cells grown in 10% serum were exposed to serum-free media. It was previously reported that serum withdrawal (SW) killed cells by elevating cellular ROS levels. This study showed that SW activates phosphoinositide 3-kinase (PI3K). PI3K activation was evident after the ROS levels began increasing, and an antioxidant blockade of this increase resulted in PI3K activation suppression. Interestingly, the inhibition of PI3K activity/activation using either its specific inhibitor or dominant-negative mutant attenuated the subsequent additional increase in the ROS levels. These results suggest that SW-induced ROS activate PI3K, which in turn promotes the process leading to ROS accumulation. The present study also revealed that both ROS and PI3K support SW-induced cell death by activating stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK). Overall, it appears that SW triggers a positive mutual interaction between ROS and PI3K, which amplifies signals required for the induction of an SAPK-dependent death pathway.

Catalase↗

PubMed2005

Cells discover fire: employing reactive oxygen species in development and consequences for aging.

The free radical theory of aging states that aging results from the accumulated damage caused by reactive oxygen species (ROS). Herein, we provide a critique of the theory that aims to point out the theory's weaknesses and put forward ideas for how future experiments must adjust to several emerging concepts. In the same way fire is dangerous and nonetheless humans learned how to use it, it now appears that cells evolved mechanisms to control and use ROS. The way ROS are used as signaling molecules in many crucial biological functions suggests ROS are not unwanted by-products of metabolism. We hypothesize that the connection between ROS and cellular processes like growth, proliferation, and apoptosis may explain why long-lived animals appear to have lower levels of ROS production: the longer development of long-lived animals may lead to lower steady state levels of ROS. With age, antioxidant systems become deregulated, just like so many other cellular components, and so oxidative damage occurs. Therefore, the production of ROS is not merely a cause of havoc but rather a complex and critical system whose disruption in disease and aging leads to oxidative damage. Potential roles of ROS in aging are discussed under this model.

Aging↗

PubMed2005

Effect of reactive oxygen species produced by spermatozoa and leukocytes on sperm functions in non-leukocytospermic patients.

OBJECTIVE: To investigate whether there is an impact of different sources of reactive oxygen species (ROS) on sperm functions. DESIGN: Prospective study. SETTING: Patients at the Center for Dermatology and Andrology, Giessen, Germany. PATIENT(S): Semen collected from 63 randomly collected patients attending the IVF unit of the University of Giessen, Germany. INTERVENTION(S): Only patients with nonleukocytospermia were included in this study. MAIN OUTCOME MEASURE(S): Sperm count and motility before and after sperm separation by swim-up, morphology, DNA fragmentation, and extrinsic (by leukocytes) and intrinsic ROS production (by spermatozoa) were evaluated. RESULT(S): Leukocytes correlated significantly with extrinsic ROS production (r = 0.576), but markedly less with intrinsic ROS production (r = 0.296). Sperm count, morphology, and motility in the ejaculate were markedly more affected by extrinsic than by intrinsic ROS. The DNA fragmentation was strongly positively correlated with intrinsic ROS production, whereas this correlation was weaker for extrinsic ROS production. No correlation was found between DNA fragmentation and the number of leukocytes, whereas the correlations with motility in the ejaculate and the motile sperm count after swim-up were highly significant. Moreover, significant differences were observed for extrinsic and intrinsic ROS production between groups of patients having a high (> or = 1 x 10(6)/mL) and a low number (<1 x 10(6)/mL) of leukocytes in the ejaculate. CONCLUSION(S): The origin of ROS seems to have an influence on the site of the damage. Because leukocyte counts <1 x 10(6)/mL caused a significant decrease of motility and DNA integrity, the threshold given by the World Health Organization (WHO) should be re-evaluated.

Adult↗

PubMed2006

Reactive oxygen species as an independent marker of male factor infertility.

OBJECTIVE: To determine the abnormal patterns of reactive oxygen species (ROS) production in male factor infertility (MFI) patients and to define the ROS reference values in such patients. DESIGN: A retrospective study. SETTING: Male infertility clinic at a tertiary healthcare center. PATIENT(S): We examined 132 MFI patients (all normal sperm parameters, n = 24, and all abnormal sperm parameters, n = 38) and 34 healthy donors. INTERVENTION(S): Routine semen analysis, measurement of ROS. MAIN OUTCOME MEASURE(S): Sperm parameters, ROS levels (10(4) cpm/20 x 10(6) sperm). RESULT(S): Normal, healthy donors had significantly higher (P<.0001) sperm concentration, motility, and morphology compared with all MFI patients. Univariate analysis indicated a significant association between MFI and log (ROS + 1) (odds ratio [OR] = 3.84), besides sperm parameters and age. A multivariate model using logistic regression analysis also indicated an independent association of log ROS with MFI (OR = 4.25). The ROS cutoff values of 1.2-1.4 had a sensitivity of 0.70-0.78 with a corresponding specificity of 0.82. However, at a cutoff point of 1.2, the OR was 68.6, which increased with an increase in the cutoff. CONCLUSION(S): High ROS is an independent marker of MFI, irrespective of whether these patients have normal or abnormal semen parameters. We suggest the inclusion of ROS measurement as part of idiopathic infertility evaluation. Treatment with antioxidants may be beneficial in such patients.

Adult↗

PubMed2005

Involvement of UVB-induced reactive oxygen species in TGF-beta biosynthesis and activation in keratinocytes.

TGF-beta produced by keratinocytes in response to UVB (290-320 nm) is a potential mediator for effects of acute and chronic solar radiation on skin. This study was designed to determine whether reactive oxygen species (ROS) mediate UVB-induced TGF-beta biosynthesis in keratinocytes and the subsequent activation of the latent TGF-beta complex. UVB irradiation elevated both total (latent plus active) and active TGF-beta in the keratinocyte supernatants, with a greater increase in the active form. UVB irradiation induced up to a 30% increase in ROS, and the ROS were detected up to 90 min after irradiation. NAC and Trolox, cytoplasmic ROS scavengers, abolished the UVB-induced TGF-beta and intracellular ROS, suggesting that UVB-induced ROS are involved in TGF-beta regulation. Inhibitors of NADPH oxidase activity, DPI and apocynin, decreased UVB-induced ROS. The increase in NADPH oxidase activity was mediated by EGFR activation. UVB-induced ROS also activated latent TGF-beta complex by stimulating MMP-2 and -9 activities. In summary, physiological doses of UVB increase intracellular ROS, which upregulate TGF-beta biosynthesis and activation of TGF-beta through increased activity of MMPs.

ErbB Receptors↗

PubMed2005

Salt stress-induced programmed cell death in tobacco protoplasts is mediated by reactive oxygen species and mitochondrial permeability transition pore status.

The status of mitochondrial permeability transition pore (PTP) and levels of reactive oxygen species (ROS) play key roles in regulating apoptosis in animal cells. To investigate if the PTP and cellular oxidation-reduction state are also involved in salt stress-induced programmed cell death (PCD) in tobacco (Nicotiana tabacum, cultivar BY-2) protoplasts, flow cytometry was used to simultaneously monitor ROS levels, PTP status and PCD. Increased ROS and decreased mitochondrial membrane potential (delta psi(m)) were observed before the appearance of PCD. Pre-treatment with an inhibitor of the PTP opening, cyclosporin A (CsA), effectively retarded the onset of PCD, the delta psi(m) decrease and the ROS content increase. Addition of ascorbic acid (AsA) during the salt stress significantly decreased the percentage of protoplasts undergoing PCD and ROS levels but increased delta psi(m). Hydrogen peroxide effectively induced the appearance of PCD and caused an increase in ROS and a decrease in delta psi(m). Pre-treatment of protoplasts with CsA weakened the effects of H2O2. All these results suggest that the open state of PTP and ROS are necessary elements for salt stress-induced PCD in tobacco protoplasts. The open states of PTP and ROS could promote each other suggesting that ROS could lead to a self-amplifying process. This positive feedback loop may act as an all-or-nothing switch, which is in good accordance with the hypothesis that PTP is an important coordinator and executioner of PCD in both animals and plants.

Apoptosis↗

PubMed2005

Mitochondria-targeted redox probes as tools in the study of oxidative damage and ageing.

Mitochondrial reactive oxygen species (ROS) and oxidative damage are associated with a range of age-related human pathologies. It is also likely that mitochondrial ROS generation is a factor in stress response and signal transduction pathways. However, current methods for measuring and influencing mitochondrial ROS production in vivo often lack the desired specificity. To help elucidate the potential role of mitochondrial ROS production in ageing, we have developed a range of mitochondria-targeted ROS probes that may be useful in vivo. This was achieved by covalently attaching a lipophilic cation to a ROS-reactive moiety causing its membrane potential-dependent accumulation within mitochondria. Mitochondria-targeted molecules developed so far include antioxidants that detoxify mitochondrial ROS, probes that react with mitochondrial ROS, and reagents that specifically label mitochondrial protein thiols. Here, we outline how the formation and consequences of mitochondrial ROS production can be investigated using these probes.

Aging↗

PubMed2005

Unifying mechanism for addiction and toxicity of abused drugs with application to dopamine and glutamate mediators: electron transfer and reactive oxygen species.

There are many unknown aspects concerning the mode of action of abused drugs. Recently, a unifying theme for toxicity and addiction was reported based on electron transfer (ET), reactive oxygen species (ROS), and oxidative stress (OS). The main drugs involved are nicotine, cocaine, alcohol, phencyclidine, ecstasy, amphetamines, morphine-heroin, tetrahydrocannabinol, and therapeutic drugs (benzodiazepines, phenytoin, phenobarbital, aspirin, and acetaminophen). A major source of ROS is ET functionalities, of which the main ones found in abused drug metabolites are quinones and imines (or iminiums). Minor types are the nitroxide metabolite from cocaine, and alpha-dicarbonyl from alcohol. The theoretical approach enjoys support from reports on formation of ET metabolites, generation of ROS, protection by antioxidants (AOs), electrochemical studies, and cell signaling. Dopamine (DA) mediation of drug abuse has been the focus of much attention during the past decades. Recently, a similar role for glutamate (Glu) has come under study. Superficially, from a mechanistic vantage point, these findings might be regarded as in conflict with the ET-ROS-OS scheme. Many investigators believe that each drug or mediator operates by its own distinct mechanism. The present report provides evidence that a commonality in mode of action exists for both abused drugs and the DA-Glu operators. In the case of DA, oxidative metabolism yields o-quinones and semiquinones which can redox cycle with oxygen to provide various ROS. Electrochemical studies support the possibility of ET transformations by these quinones in the biological domain. In relation to cell signaling, DA is involved in formation of cAMP followed by a cascade of other events. A similar scenario exists in the case of Glu, in which an iminocarboxylic acid metabolite is hypothesized to play an ET role. The various phenomena are rationalized within the context of ET-ROS-OS, as was done earlier for abused drugs. Thus, a common mode of action may exist for both categories since all provide metabolites with similar properties. Stimulation of mediator production by abused drugs can occur with subsequent oxidation by ROS, some of which may be supplied by the drugs. In addition to prevention, the difficult topic of addiction mechanism is addressed from the viewpoint of ET and ROS involvement. Low levels of ROS would pertain since high concentrations are related to toxicity. A report that tetrahydrocannabinol does not comprise a substantial risk is in accord with little evidence of OS and absence of ET functionalities in the drug and reported metabolites.

Dopamine↗

PubMed1997

Impaired mitochondrial function, oxidative stress and altered antioxidant enzyme activities following traumatic spinal cord injury.

Glutamate-induced excitotoxicity involving the formation of reactive oxygen species (ROS) has been implicated in neuronal dysfunction and cell loss following ischemic and traumatic injury to the central nervous system (CNS). ROS are formed in mitochondria when energy metabolism is compromised, and are inactivated by the ROS scavengers superoxide dismutase (SOD), catalase, and glutathione (GSH). ROS can impair the function of several cellular components including proteins, nucleic acids, and lipids. In the present study, we measured indicators of mitochondrial metabolic activity, ROS formation, lipid peroxidation, and antioxidant enzyme activities in synaptosomes obtained from rat spinal cord at early times following traumatic injury. Mitochondrial metabolic activity was found to significantly decrease as early as 1 h following injury, and continued to be compromised over the remaining postinjury time points. ROS formation was found to be significantly increased at 4 and 24 h following injury, while lipid peroxidation levels were found to be significantly increased in the injured spinal cord at 1 and 24 h, but not 4 h following injury. SOD enzyme activity was unchanged at all postinjury time points, while catalase activity and GSH levels were significantly increased at 24 h following injury. These findings indicate that impaired mitochondrial function, ROS, and lipid peroxidation occur soon after traumatic spinal cord injury, while the compensatory activation of molecules important for neutralizing ROS occurs at later time points. Therapeutic strategies aimed at facilitating the actions of antioxidant enzymes or inhibiting ROS formation and lipid peroxidation in the CNS may prove beneficial in treating traumatic spinal cord injury, provided such treatments are initiated at early stages following injury.

Animals↗

PubMed1987

Cytochemical localization of Mn2+-dependent pyrimidine 5'-nucleotidase activity in isolated rod outer segments.

A cytochemical method was developed for localization in isolated rod outer segments of manganese-dependent pyrimidine 5'-nucleotidase (MDPNase), an enzyme activity with possible relevance to shedding that we recently reported in photoreceptors and retinal pigment epithelial (RPE) cells in the intact rat retina. The purpose of this study was to eliminate the possibility that the previously observed cytochemical staining of the rods was due to diffusion of reaction product from the RPE cell lysosomes, which were also heavily stained. Rod outer segments (ROS) were isolated on continuous sucrose gradients from retinal homogenates prepared from rats raised in cyclic light (12 hr light:12 hr dark) and killed during the first 2 hr after light onset. ROS-containing bands were removed from the gradients and the isolated rods were fixed in 0.25% glutaraldehyde and pelleted. Chopped sections of the pellets were incubated in cytochemical medium for MDPNase activity and processed for light- and electron-microscopic localization of the enzyme activity. Two patterns of cytochemical staining were seen in ROS isolated from retinas obtained at this time of day. A few of the pellets contained clusters of ROS that were heavily coated along their surfaces and seemingly interconnected by thick strands of highly reactive extracellular material that displayed a punctate pattern of cytochemical staining. This material may have originated from the apical processes of the RPE cells, which were heavily stained in tissue fixed in situ around the time of light onset. The second staining pattern, visible only by electron microscopy, was more commonly observed. In the majority of the isolated ROS profiles, discrete streaks of cytochemical reaction product were seen in association with the internal aspects of the discs, at sites that seemed to correspond to the rims, and to narrow zones within the disc interiors. This distribution of reactive sites closely resembled that observed over most of the length of the ROS in the intact retina fixed at the same time of day. Occasionally, ROS profiles were encountered in which additional reactive sites were localized to the interdisc spaces between the plasma membrane and the rims of the discs. The latter pattern resembled the distribution of reaction product seen during this period over the tips of the ROS fixed in situ. As in the intact retinas, the cytochemical staining of the isolated ROS was inhibited by fluoride ions and strongly stimulated by manganese ions.(ABSTRACT TRUNCATED AT 400 WORDS)

5'-Nucleotidase↗

PubMed1987

Phorbol ester- and light-induced endogenous phosphorylation of rat rod outer-segment proteins.

We have previously described the presence of a C-kinase in bovine retinal rod outer segments (ROS) (Kapoor and Chader, 1984). In this study, we have labeled rat retinas with freshly neutralized radiolabeled sodium phosphate (32P or 33P) by intravitreal injection and compared the phosphorylation patterns of ROS proteins induced by light and specific activators of the C-kinase phosphorylation system. Except for light treatment, all procedures were carried out in complete darkness using an infrared image converter. Incubation of 33P-labeled retinas in light for 5 min resulted in the phosphorylation of rhodopsin, 80-, 65-, 47-, 44-, and 15,000 MW proteins of crude ROS. Incubation of 33P-labeled retinas with 0.5 microM 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in the phosphorylation of several proteins including those at 80-, 65-, 47-, 44-, and 15,000 MW in crude ROS. ROS prepared in complete darkness did not exhibit any phosphorylation of proteins whereas ROS prepared in red light exhibited variable low phosphorylation of 80-, 47-, 44- and 15,000 MW proteins. 1-oleoyl-2-acetylglycerol (OAG) at 500 micrograms ml-1 caused the phosphorylation of the same proteins as observed with TPA. TPA (0.5-500 microM) and OAG (150-500 micrograms ml-1) did not induce rhodopsin phosphorylation. When purified ROS were prepared from 33P-pre-labeled retinas, the complete darkness control did not exhibit phosphorylation of any proteins. TPA, however, induced the phosphorylation of 80- and 65,000 MW proteins and light induced the phosphorylation of 80-, 65,000 MW proteins as well as opsin monomer and dimer. Affinity chromatography of phosphorylated ROS proteins on con A-Sepharose revealed that TPA does not induce rhodopsin phosphorylation whereas light does. Since light and TPA induced the phosphorylation of 80- and 65,000 MW proteins in ROS, it is possible to suggest at least a partial linkage of light- and C-kinase-mediated effects in situ.

Animals↗

PubMed2003

Acetylcholine-induced production of reactive oxygen species in adult rabbit ventricular myocytes is dependent on phosphatidylinositol 3- and Src-kinase activation and mitochondrial K(ATP) channel opening.

Acetylcholine (ACh), like ischemic preconditioning (PC), protects against infarction and is dependent on generation of reactive oxygen species (ROS). To investigate the mechanism by which ACh causes ROS production, isolated adult rabbit cardiomyocytes underwent a timed incubation in reduced MitoTracker Red, which is oxidized to a fluorescent form after exposure to ROS. The mitochondrial ATP-sensitive potassium (mK(ATP)) channel opener diazoxide (50 microM) increased fluorescence by 47 +/- 9% (P = 0.007), indicating that opening of mK(ATP) leads to ROS generation, and that increase was blocked by the mK(ATP) blocker 5-hydroxydecanoate (5HD, 1 mM); 250 microM ACh caused a similar increase in ROS generation (+45 +/- 6% for all experiments, P < 0.001). ACh-induced ROS production was prevented by (1) blockade of muscarinic surface receptors with 100 microM atropine (-6 +/- 2%, P = n.s.) or 250 nM 4-DAMP (+5 +/- 13%, P = n.s.), indicating that ACh's effect was receptor mediated; (2) closing K(ATP) channels with either the non-selective channel closer glibenclamide (50 microM) (-1.2 +/- 17%, P = n.s.) or the selective mK(ATP) closer 5HD (-1.8 +/- 9%, P = n.s.), indicating that increased ROS production involved opening of mK(ATP); (3) blockade of mitochondrial electron transport chain with 200 nM myxothiazol (-4 +/- 9%, P = n.s.), indicating ROS came from the mitochondria; (4) addition of 100 nM wortmannin (-13 +/- 12%, P = n.s.), indicating that phosphatidylinositol 3-(PI3)-kinase was involved; and (5) blockade of Src-kinase with 1 microM PP2 (-2 +/- 5%, P = n.s.), indicating the involvement of an Src-kinase. These results support the hypothesis that occupation of muscarinic surface receptors by ACh causes activation of PI3- and Src-kinases that then open mK(ATP) resulting in mitochondrial ROS generation and triggering of the preconditioned state.

Acetylcholine↗

PubMed1997

Reactive oxygen species generation by seminal cells during cryopreservation.

OBJECTIVES: To test the hypothesis that conventionally used procedures for semen cryopreservation may cause an increase in the production of reactive oxygen species (ROS) by sperm or by seminal leukocytes, which may contribute to poor sperm function following cryopreservation. METHODS: Eighteen semen specimens with normal parameters from healthy male donors 22 to 40 years of age were each divided into two portions. The first portion was combined 1:1 with Test Yolk Buffer-Glycerol Freezing Medium and was frozen by gradual cooling into liquid nitrogen (-196 degrees C). The second portion was washed and the cells were resuspended in Sperm Washing Medium (SWM) and incubated at room temperature to serve as controls. After a period of treatment, frozen samples were thawed and semen cells were washed and resuspended in SWM. ROS generation by semen cells from each treatment group was measured on a luminometer. Sperm motility, sperm viability, and sperm membrane integrity were also measured in both control and freeze-thaw samples. To further assess ROS generation by semen cells during the cooling process, aliquots of washed semen cells and purified polymorphonuclear leukocytes (PMNs) were incubated separately at different temperature conditions (37 degrees C, 22 degrees C, 4 degrees C, and -20 degrees C). ROS activity in each treatment group was measured and compared with each other. RESULTS: In both semen cells and PMNs, ROS activity increased significantly during the cooling process. The highest ROS levels were recorded in both groups when cooled to 4 degrees C. The ROS levels were extremely low in samples cooled to -20 degrees C and in freeze-thaw samples, probably due to marked loss of cell viability. CONCLUSIONS: Gradual reduction of temperature during the process of semen cryopreservation can cause a significant ROS generation by semen cells. ROS is particularly elevated during cooling if the semen sample is contaminated by more than 0.5 x 10(6) leukocytes. Removal of leukocytes from semen samples or treatment with antioxidants prior to cryopreservation may improve sperm viability and function.

Adult↗