Role of ascorbic acid in the modulation of inhibition of platelet aggregation by polymorphonuclear leukocytes

OBJECTIVES: We investigated the modulatory effect of ascorbate on the inhibition of platelet aggregation response by polymorphonuclear leukocytes (PMNs) and characterized the mechanism of the inhibitory response. BACKGROUND: PMNs have been reported to play a significant role in vascular homeostasis by releasing various factors including short-lived reactive oxygen species (ROS) and nitric oxide (NO). NO prevents the activation of circulating platelets and plays a significant role in hemostasis. In addition, PMNs also have the capacity to store very high concentrations of ascorbate. The physiological implications of storing such high concentrations of an antioxidant by a cell-releasing free radicals is unknown, viz. a viz. hemostatic regulation. METHODS: ADP-induced aggregation in human, monkey and rat platelet-rich plasma (PRP) was monitored in the presence of PMNs treated with varying concentrations of ascorbate/dehydroascorbate. NO generation from rat and human PMNs treated with ascorbate was monitored on a FACS Calibur flow cytometer and intraplatelet cyclic guanosine 3',5'-monophosphate (cGMP) levels was also measured. RESULTS: PMNs induced a cell number and time-dependent inhibition of ADP-induced aggregation. The PMNs dependent inhibition was enhanced significantly at 30 min by ascorbate (300 microM). Ascorbate seemed to exert its effects through its oxidized product, dehydroascorbate, as the effects was prevented in the presence of D-glucose (10 mM). Dehydroascorbate elicited significant potentiation of the PMNs induced inhibitory responses and these effects were mediated by the release of NO and subsequent activation of platelet guanylyl cyclase. Flow cytometry experiments with human and rat PMNs confirmed the release of NO and the elevated platelet cGMP levels confirmed NO-mediated activation of guanylyl cyclase. CONCLUSIONS: Ascorbate in circulation seems to prevent the activation of platelets by enhancing the release of antiaggregatory NO, from neighbouring or cohabitant PMNs. The ascorbate effect is mediated through its conversion to dehydroascorbate, subsequently, gets taken up by the cell and converted back to ascorbate. Intracellular ascorbate potentiates the release of NO from the PMNs and subsequently activates guanylyl cyclase in the platelets.     

Inhibition of platelet aggregation by rat globin

This study was undertaken to identify and characterize the anti-aggregatory protein factor present in rat polymorphonuclear leukocytes (PMNs) supernatant. Since the purified protein exhibited sequence homology to beta globin, globin was also isolated from rat blood by acid-acetone precipitation and was purified on Superdex-75 column in FPLC. Elution of rat globin on the gel filtration column yielded two peaks of approximately 60 and 30 kDa as observed in the PMNs supernatant. Purity of globin and eluted fractions was further evaluated by SDS-PAGE. Platelet aggregation induced by agonists viz. adenosine-5'-diphosphate (ADP; 2-5 microM), arachidonic acid (AA; 10 microM), A23187 (2.50 microg/ml) was inhibited by globin and the purified fractions. ADP-induced rise in intracellular calcium levels and expression of CD62 on the platelets were reduced by both globin and active fraction of PMNs supernatant. Results obtained suggest that globin or globin-related protein present in the PMNs supernatant inhibits platelet aggregation response.     

Risk factors for the development of second primary tumors among men after laryngeal and hypopharyngeal carcinoma

BACKGROUND: Second primary tumors (SPT) constitute a major threat to the survival of patients with laryngeal carcinoma. However, to the authors' knowledge little is known regarding the risk factors for developing SPTs or about the strategy to be followed to avoid them. METHODS: Eight hundred seventy-six male patients with laryngeal/hypopharyngeal carcinoma enrolled in a population-based, case-control study in 5 centers from South Europe during 1979-1982 were followed up to ascertain the occurrence of SPTs. Standardized incidence ratios were calculated to estimate the risk of SPT occurring in the cohort. Cox proportional hazard models were fitted to estimate the hazard ratio for development of SPTs in relation to use of tobacco smoking, alcohol consumption, and dietary habits before the first primary tumor. RESULTS: One hundred forty-five patients developed an SPT with an annual average rate of 2.1%. An excess risk of developing an SPT of the tongue, mouth, esophagus, and lung was observed. No elevated risks of SPTs were observed in other organs. Alcohol consumption strongly increased the risk of developing an SPT of the upper aerodigestive tract (UADT). Heavy cumulative cigarette smoking increased the risk of developing a lung SPT. A protective effect of high intake of citrus fruit was noticed for SPT in the lung, whereas high butter intake was associated with an increased risk for SPT of the UADT. CONCLUSIONS: Continuous medical surveillance was essential in the UADT and lung to reduce the risk from an SPT after initial laryngeal/hypopahryngeal carcinoma. Alcohol consumption before the first primary tumor was a risk factor for SPT of the UADT. The study suggested a protective role of citrus fruits in the development of a lung SPT.     

AMPA receptor regulation mechanisms: future target for safer neuroprotective drugs

The post-synaptic AMPA receptors play an important role in mediating fast excitatory transmission in the mammalian brain. Over-activated AMPA receptors induce excitotoxicity, implicated in a number of Chronic neurodegenerative disorders such as Parkinson's disease, Huntington's disease, and AIDS encephalitis. AMPA receptor antagonists offer protection against neurodegeneration in the experimental models even if they are given 24 h after the injury. Because AMPA receptors seem to be involved in the neurodegenerative diseases, modulating the activity of the AMPA receptors could be an attractive approach to reduce or prevent excitotoxicity. Studies conducted recently have exhibited a number of new mechanisms for AMPA receptor regulation. Modulations of these were found to have protective implications. AMPA receptor depolarization and desensitization are protective to the neurons. Receptor desensitization depends on the receptor subunit composition. The R/G editing site and the flip/flop cassettes in AMPA receptor subunits contribute to a great extent in receptor desensitization and recovery rates. Molecules that could quicken receptor desensitization or delay recovery could be of use. AMPA receptors limit neuronal entry of Ca2+ ions by regulating Ca2+-permeability. Ca2+-permeable receptor channels are made up of GluR1, GluR3, or GluR4 subunits, whereas presence of the GluR2 subunit restricts Ca2+ entry and renders the receptor Ca2+-impermeable. GluR2 levels, however, experience a fall after neuronal insult rendering the AMPA receptors Ca2+-permeable, thus factors that could interfere with this event might prove to be very beneficial against excitotoxicity. AMPA receptor clusters are stabilized by PSD-95, which requires palmitoylation at two sites. Targeting palmitoylation of the PSD-95 can also be a useful approach to disperse AMPA clusters at the synapse. In the perisynaptic region, mGluRs are present a little away from the synapse and are among the glutamate transporters, which require high-frequency firing for activation. On activation they might enhance the activity of NMDA receptors at the synapse to increase the levels of AMPA receptors. AMPA receptors surfaced at this juncture can contribute to heavy Ca2+ influx. Thus, blocking this pathway could be of considerable importance in preventing the excitotoxicity. A number of proteins such as the GRIP, PICK, and NSF also modulate the functions of AMPA receptors. Polyamines also block Ca2+ permeable AMPA receptors and thus are protective. NO and cGMP also play an important role in negatively regulating AMPA receptors and thus could offer protection. Modulation of AMPA receptor by different mechanisms has been discussed in the present review to implicate importance of these targets/pathways for safer and future neuroprotective drugs.     

Studies on the immunomodulatory effects of Boerhaavia diffusa alkaloidal fraction.

The alkaloidal fraction of Boerhaavia diffusa was studied for its effect on cellular and humoral functions in mice. Oral administration of the fraction (25-100 mg/kg) significantly inhibited SRBC-induced delayed hypersensitivity reactions in mice. However, the inhibition was observed only during post-immunisation drug treatment, while no effect during pre-immunisation drug treatment was observed. A significant dose-related increase in antibody titre was observed during pre- and post-immunisation treatment. The alkaloidal fraction failed to show any blastogenic responsiveness of murine splenocytes to Concanvalin A (Con A) and lipopolysaccharide (LPS). Similarly, it did not display any mitogenic activity. Thus, the present study has shown the in vivo immunostimulatory activity of B. diffusa alkaloidal fraction without an in vitro effect.     

Role of endogenous reactive oxygen derived species and cyclooxygenase mediators in 5-hydroxytryptamine-induced contractions in rat aorta: relationship to nitric oxide.

Endogenous reactive oxygen species (superoxide anion, hydroxyl radical and hydrogen peroxide), endothelium-derived nitric oxide and cyclooxygenase mediators are involved in the regulation of vascular smooth muscle tone. An imbalance of these mediators can have profound implications in various cardiovascular disorders. Involvement of endogenous reactive oxygen species, endothelium-derived nitric oxide (NO) and cyclooxygenase mediators in 5-hydroxytryptamine- (5-HT-) induced contractions of endothelium intact rat aortic rings have been investigated in the present study. The contribution of each of the endogenous reactive oxygen species in mediating 5-HT-induced contractions was studied by pretreating the rings with their respective scavengers. Pretreatment of the rings with superoxide dismutase (superoxide radical scavenger), catalase (H (2)O (2)inactivator), mannitol (extracellular OH. scavenger), or thiourea (intracellular OH. radical scavenger) significantly depressed the 5-HT-induced contractions in the aortic rings. The responses to 5-HT in the presence of SOD or catalase were augmented byL -NAME pretreatment. Though aminotriazole partially inhibited the catalase activity, it inhibited 5-HT-induced contractions significantly. The results obtained thus suggest that endogenous generation of ROS (O(2).(-), H (2)O (2)and OH.) modulates 5-HT-induced rat aortic ring contractions. In addition, H (2)O (2)generated in the endothelium seems to regulate the vascular response and also act as a mediator to release other vasoactive substances. Basal production of NO by the endothelium seems to affect the vascular response due to its interaction with ROS mediators.