The new cardiotonic agent sulmazole is an A1 adenosine receptor antagonist and functionally blocks the inhibitory regulator, Gi

Although many of the new cardiotonic agents are known to increase cAMP and to inhibit with variable potency a low Km cAMP phosphodiesterase, there is still debate as to the mechanism(s) by which these agents act. In a rat adipocyte membrane model we demonstrate that only approximately 50% of the effect of the new cardiotonic agent sulmazole on cAMP accumulation can be attributed to phosphodiesterase inhibition and that the remaining production of cAMP involves stimulation of adenylate cyclase activity. Two distinct pathways for stimulation of adenylate cyclase are herein reported. Sulmazole, UD-CG 212 CL, enoximone, piroximone, amrinone, and milrinone are all shown to be competitive antagonists of inhibitory A1 adenosine receptors, with EC50 values of 11-909 microM. Elimination of the effects of endogenous adenosine with adenosine deaminase reveals a third distinct mechanism for activation of adenylate cyclase. This mechanism appears to involve Gi, the inhibitory guanine nucleotide-regulatory protein, in that sulmazole attenuates the capacity of GTP to inhibit adenylate cyclase activity, and covalent modification of Gi by pertussis toxin treatment abolishes the capacity of sulmazole to mediate stimulation. Thus, functional blockade of Gi activity is the likely mode of action. Restoration of sulmazole's stimulatory effect on adenylate cyclase activity in pertussis toxin-treated membranes can be accomplished by reconstituting purified preparations of either Gi or mixtures of Gi/Go into treated adipocyte membranes. Of note, this stimulatory effect is completely reversed by inhibitory receptor agonists. Thus, the new cardiotonic agent sulmazole mediates increases in cAMP accumulation by mechanisms other than phosphodiesterase inhibition, including A1 adenosine receptor antagonism and inhibition of Gi function.     

II. Expression of TNF-α and TNFR p55 in Cultured Amniochorion

PROBLEM: Preterm labor and PROM are major complications of pregnancy. We have reported the possible role of amniochorionic membrane as it relates to the production of cytokines and the early onset of labor. Amniochorion is capable of responding to an infectious process with the production of IL-6 and IL-1β. Here we examine the expression of TNF-α and TNFR in amniochorion. METHOD: Amniochorionic membranes were collected and maintained in an organ explant system. Samples were frozen and/or fixed for RT-PCR, in situ hybridization, and immunocytochemistry. RESULTS: RT-PCR demonstrated mRNA for TNF-α and in situ hybridization localized mRNA in chorion and amnion. Immunocytochemistry demonstrated TNF-α peptide in amnion but not in chorion. Immunocytochemical localization of TNFR indicates presence of that peptide in both amnion and chorion. CONCLUSIONS: We conclude that the fetal membranes are sources of TNF-α and TNFR, supporting our previous work indicating that fetal membranes are active participants in the response to intraamniotic infection.     

Effect of fish oil pretreatment on isoproterenol-induced changes in myocardial membrane phospholipids

OBJECTIVE: In the present study, the protective effect of fish oil treatment on the fatty acid composition in isoproterenol (IPH)-induced myocardial infarction was studied in male albino Wistar rats. METHODS: Rats were injected for 2 consecutive days with IPH (60 mg/kg body weight) at 24-h intervals to induce myocardial infarction. Fish oil was administered orally at a dose of 0.05 mL/d for 45 d, after which serum and heart tissue were assayed for lipid profile, lipoprotein changes, and myocardial membrane phospholipid fatty acid composition. RESULTS: Biochemical assessment of myocardial infarction was done by measuring the activities of creatinine kinase and lactate dehydrogenase, which were significantly elevated in the rats administered with IPH. Further, the administration of IPH modified the fatty acid composition and analysis of fatty acids showed there was an increase in the omega-3/omega-6 ratio in phospholipid pool. In addition, increased levels of total cholesterol, free cholesterol, ester cholesterol, phospholipids, triacylglycerols and free fatty acid was observed in serum and heart tissue of IPH-induced rats. The fish oil treatment for a period of 45 d decreased the levels of cardiac markers (creatinine kinase and lactate dehydrogenase) and reversed the biochemical lesions induced by IPH. CONCLUSION: Our study suggests that fish oil treatment has a hypolipidemic effect and has potential use in the treatment of myocardial infarction.     

Modulatory effect of fish oil on the myocardial antioxidant defense system in isoproterenol-induced myocardial infarction

The effect of fish oil treatment on the activities of antioxidants such as superoxide dismutase, catalase, glutathione peroxidase, glutathione-s-transferase, and glutathione, as well as the level of the lipid peroxidation marker thiobarbituric reactive substance was studied in isoproterenol-induced myocardial infarction (MI). To confirm the induction of MI by isoproterenol, we studied the activities of cardiac marker enzymes like creatinine kinase and lactate dehydrogenase and the level of troponin. The biochemical lesions due to the activation of lipid peroxidation and decrease in antioxidant status are significantly implicated in experimental isoproterenol-induced MI. The results indicate that the protective effect of fish oil is achieved by decreasing the peroxide concentration and normalizing antioxidant defense enzymes.     

Brief daily periods of unrestricted vision can prevent form-deprivation amblyopia

PURPOSE: To characterize how the mechanisms that produce unilateral form-deprivation amblyopia integrate the effects of normal and abnormal vision over time, the effects of brief daily periods of unrestricted vision on the spatial vision losses produced by monocular form deprivation were investigated in infant monkeys. METHODS: Beginning at 3 weeks of age, unilateral form deprivation was initiated in 18 infant monkeys by securing a diffuser spectacle lens in front of one eye and a clear plano lens in front of the fellow eye. During the treatment period (18 weeks), three infants wore the diffusers continuously. For the other experimental infants, the diffusers were removed daily and replaced with clear, zero-powered lenses for 1 (n=5), 2 (n=6), or 4 (n=4) hours. Four infants reared with binocular zero-powered lenses and four normally reared monkeys provided control data. RESULTS: The degree of amblyopia varied significantly with the daily duration of unrestricted vision. Continuous form deprivation caused severe amblyopia. However, 1 hour of unrestricted vision reduced the degree of amblyopia by 65%, 2 hours reduced the deficits by 90%, and 4 hours preserved near-normal spatial contrast sensitivity. CONCLUSIONS: The severely amblyogenic effects of form deprivation in infant primates are substantially reduced by relatively short daily periods of unrestricted vision. The manner in which the mechanisms responsible for amblyopia integrate the effects of normal and abnormal vision over time promotes normal visual development and has important implications for the management of human infants with conditions that potentially cause amblyopia.     

Ameliorative effect of vitamins (alpha-tocopherol and ascorbic acid) on PCB (Aroclor 1254) induced oxidative stress in rat epididymal sperm

The aim of this study was to investigate the possible protective role of vitamins on PCB (Aroclor 1254)-induced spermiotoxicity using qualitative, quantitative and biochemical approaches. Adult male albino rats of Wistar strain were randomly divided into four groups, each group consists of six animals. The control group received corn oil, the second group of rats were administered Aroclor 1254 at a dose of 2 mg/kg bw/day intraperitoneally for 30 days. The third group of rats were treated with Aroclor 1254 along with alpha-tocopherol (50 mg/kg of bw/day) for 30 days, while the fourth group of rats were treated with Aroclor 1254 along with ascorbic acid (100 mg/kg bw/day) orally for 30 days. Twenty-four hours after the last treatment, control and experimental animals were killed by decapitation. Sperm was collected from the cauda epididymal region and its count and motility were detected. Sperm was sonicated and used for the estimation of reactive oxygen species (ROS) [hydroxyl radical (HO(*)) and hydrogen peroxide (H(2)O(2))], non-enzymic antioxidants [alpha-tocopherol, ascorbic acid and reduced glutathione (GSH)], activity of enzymic antioxidants [superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) glutathione reductase (GR) and glutathione-S-transferase (GST)] and lipid peroxidation (LPO). The result of this experiment shows that PCB significantly decreases the level of alpha-tocopherol, ascorbic acid and GSH and the activities of SOD, CAT, GPx, GR and GST with elevated levels of ROS and LPO. In addition, decreased epididymal sperm motility and count were observed. Simultaneous supplementation with alpha-tocopherol and ascorbic acid restored these parameters to that of normal range. In conclusion, alpha-tocopherol and ascorbic acid exhibited protective effect on sperm by inhibiting PCB-induced ROS generation.     

Differences in the placental membrane cytokine response: a possible explanation for the racial disparity in preterm birth

PROBLEM: The prematurity rate is higher in African-Americans (AA) compared with Caucasians (C). As spontaneous preterm labor has been hypothesized to be a host inflammatory response disease racial differences in human placental membrane inflammatory cytokine and prostaglandin pathway gene expression patterns between AA and C were examined in this report. METHOD OF STUDY: Placental membranes (amniochorion) collected from AA and C women from cesareans at term were maintained in an organ explant system and stimulated with endotoxin (lipopolysaccharide, LPS). Microarray analysis and enzyme-linked immunosorbent assay was performed on mRNAs and culture media from AA- and C-derived membranes to document any differences in mRNA expression and protein production of IL-1, IL-6, IL-8, IL-10 and expression of cyclooxygenase 1 (COX-1), COX-2 and 15-hydroxyprostaglandin dehydrogenase (PGDH). RESULTS: Increased mRNA expression of IL-1, IL-8 and COX-2 in AA and IL-6, IL-10, COX-1 and PGDH in C were documented after LPS stimulation. Concentration of IL-1 was significantly higher in media derived from AA whereas IL-6 and IL-10 concentrations were higher in C with no differences observed in IL-8 after LPS stimulation compared with respective unstimulated controls. CONCLUSION: These data document ethnic diversity in placental membrane immune response.     

Activation of the adenosine A3 receptor in RAW 264.7 cells inhibits lipopolysaccharide-stimulated tumor necrosis factor-alpha release by reducing calcium-dependent activation of nuclear factor-kappaB and extracellular signal-regulated kinase 1/2

Bacterial lipopolysaccharide (LPS) activates the immune system and promotes inflammation via Toll-like receptor (TLR) 4, which regulates the synthesis and release of tumor necrosis factor (TNF)-alpha and other inflammatory cytokines. Previous studies have shown that the nucleoside adenosine suppresses LPS-stimulated TNF-alpha release in human UB939 macrophages by activating an adenosine A(3) receptor (A(3)AR) subtype on these cells. In this study, we examined the mechanism(s) underlying A(3)AR-dependent inhibition of TNF-alpha release in a mouse (RAW 264.7) cell line. Treatment of RAW 264.7 cells with LPS (3 mug/ml) increased TNF-alpha release, which was reduced in a dose-dependent manner by adenosine analogs N(6)-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (IB-MECA) and R-phenylisopropyladenosine and reversed by selective A(3)AR blockade. The increase in TNF-alpha release was preceded by an increase in intracellular Ca(2+) levels. Inhibition of intracellular Ca(2+) release by IB-MECA, a selective agonist of the A(3)AR, or with BAPTA-AM, an intracellular Ca(2+) chelator, reduced LPS-stimulated TNF-alpha release. Activation of the A(3)AR or inhibition of intracellular Ca(2+) release also reduced LPS-stimulated nuclear factor-kappaB (NF-kappaB) activation and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation. Similar inhibition by A(3)AR was observed for LPS-stimulated inducible nitric-oxide synthase. These data support the contention that inhibition of LPS-stimulated release of inflammatory molecules, such as TNF-alpha and NO via the A(3)AR, involves suppression of intracellular Ca(2+)signaling, leading to suppression of NF-kappaB and ERK1/2 pathways.