Electrophysiological consequences of leukotrienes applied on isolated rat retina

Scotopic vision is the result of a cascade of light-dependent biochemical events in rod outer segments (ROS) involving mainly a cGMP-modulation of sodium current. This modification of ionic currents induces changes of membrane potential which generates electroretinographic (ERG) waves. As (i) ERG disturbances are commonly recorded in hypoxic and inflammatory retinal diseases (ii) leukotrienes (LTs), a very potent mediators of inflammation, disturb ionic exchanges in several artificial or natural membrane systems, we undertook the investigation of the effects of LTs on ERG record in mammalian isolated retina. LTB4, LTC4 and LTD4, all induced a dose-dependent marked reduction of the b wave amplitude of ERG. This effect is correlated with a significant decrease in the survival time of the retina. The analysis of the modification of ERG indicates that LTs exhibit a real toxic effect since b wave is mainly affected while P III wave is unchanged. Comparatively with other nervous cells, this phenomenon may be attributed to an increase in Na+ permeability of ROS. It is suggested that LTs may be involved in the development of inflammatory or ischemic retinal diseases.     

Evaluation of the role of leukotrienes on ovum implantation in mice

Prostaglandins are considered to be one of the important mediators of ovum implantation. Various lipoxygenase products also have been implicated along with PGs for this process. A specific rather than preferential inhibitor of 5-lipoxygenase is used to investigate the role of leukotrienes in the event of implantation and decidualization process in mice. AA861, a selective inhibitor of 5-lipoxygenase is used in different dose levels like 50,100 and 500 μg in (A) intact pregnant mice (on D1-D4 and D2-D4 and D4); (B) delayed mice on (D3-D8); (C) pseudopregnant traumatized mice (on D1-D4). All the experimental animals of group A were killed on D6. Estrogen injected delayed animals of group  were killed 48 h after the induction of implantation. Implantation sites were counted as blue spot and compared with those of control animals. Traumatized animals of group C were killed 24 h after the mechanical traumatization and uterine weights were compared with those of vehicle treated controls. Results show that AA861 could not inhibit ovum implantation in either intact or ovariectomized delayed animals. It also did not show any adverse effect on tubal transport or development of embryos. AA861 did not have any inhibitory role on decidualization of pseudopregnant uteri also. This experiment shows that a selective inhibitor of 5-lipoxygenase enzyme may not impair the implantation in mice indicating a doubt about the involvement of 5-lipoxygenase products in implantation.     

Glycogen metabolism in the rat retina

It has been reported that glycogen levels in retina vary with retinal vascularization. However, the electrical activity of isolated retina depends on glucose supply, suggesting that it does not contain energetic reserves. We determined glycogen levels and pyruvate and lactate production under various conditions in isolated retina. Ex vivo retinas from light- and dark-adapted rats showed values of 44 +/- 0.3 and 19.5 +/- 0.4 nmol glucosyl residues/mg protein, respectively. The glycogen content of retinas from light-adapted animals was reduced by 50% when they were transferred to darkness. Glycogen levels were low in retinas incubated in glucose-free media and increased in the presence of glucose. The highest glycogen values were found in media containing 20 mm of glucose. A rapid increase in lactate production was observed in the presence of glucose. Surprisingly, glycogen levels were the lowest and lactate production was also very low in the presence of 30 mm glucose. Our results suggest that glycogen can be used as an immediate accessible energy reserve in retina. We speculate on the possibility that gluconeogenesis may play a protective role by removal of lactic acid.     

Crucial role of intracellular effectors on glycogenolysis in the isolated rat heart: Potential consequences on the myocardial tolerance to ischemia

The role played by glycogenolysis in the ischemic heart has been recently put into question because it is suspected that a slowing down of this process could be beneficial for the tolerance of the myocardium to ischemia. The role of the intracellular effectors that control the rate of glycogenolysis has therefore regained interest. We aimed to understand the role played by those intracellular effectors which are directly related to the energy balance of the heart. To this end, we review some of the previously published data on this subject and we present new data obtained from P-31 and C-13 NMR spectroscopic measurement on isolated rat heart. Two conditions of ischemia were studied: 15 min global no-flow and 25 min low-flow ischemia. The hearts were isolated either from control animals or from rats pre-treated with isoproterenol (5 mg.kg^–1 b.w. i.p.) 1 h before the perfusion in order to C-13 label glycogen stores. Our main results are as follows: (1) the biochemically determined glycogenolysis rate during the early phase of ischemia (up to 10–15 min) was larger in no-flow ischemia than in low-flow conditions for both groups, (2) direct measurement of the glycogenolysis rate, as determined by C-13 NMR, after labelling of the glycogen pool in the hearts from isoproterenol-treated rats, confirms the estimations from the biochemical data, (3) glycogenolysis was slower in the hearts from pre-treated animals than in control hearts for both conditions of ischemia, (4) the total activity of glycogen phosphorylase (a + b) increased, by 50%, after 5 min no-flow ischemia, whereas it decreased by 42% after the same time of low-flow ischemia. However, the ratio phosphorylase a/a + b was not altered, whatever the conditions, (5) the concentration of inorganic phosphate (Pi) increased sharply during the first minutes of ischemia, to values above 8–10 mM, under all conditions studied. The rate of increase was larger during no-flow ischemia than during low-flow ischemia. The concentration of Pi was thereafter higher in controls than in the hearts from isoproterenol-treated animals.The calculated cytosolic concentration of free 5 AMP increased sharply at the onset of ischemia, reaching in a few minutes values above 30 M in controls and significantly lower values, around 15 M, in the hearts from isoproterenol-treated rats. (6) The hearts from isoproterenol-treated rats displayed a reduced intracellular acidosis, when compared to controls, under both conditions of ischemia.We conclude that the intracellular effectors, mainly free AMP, play an essential role in the control of glycogenolysis via allosteric control of phosphorylase b activity. The alteration in the concentration of free Pi, the substrate of both forms of phosphorylase, can also be considered as determinant in the control of the rate of glycogenolysis.The attenuation of ischemia-induced intracellular acidosis in the hearts from isoproterenol-treated rats could be a consequence of a reduced glycogenolytic rate and is likely to be related to a better resumption of the mechanical function on reperfusion.     

Light exposure stimulates arachidonic acid metabolism in intact rat retina and isolated rod outer segments

This paper presents evidence that short-term exposure to light increases synthesis of hydroxyei-cosatetraenoic acid (HETE) and prostaglandin D₂ (PGD₂) and stimulates the uptake and metabolism of 204 in phospholipids and triacylglycerols in rat retina. There was a time-dependent increase in incorporation of 1-¹⁴C-204 into glycerolipids in both dark-adapted and light-exposed groups. Exposure to light for 15 or 30 min enhanced the acylation of 204 into phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine and triacylglycerols. In the light-exposed groups there was a large increase in the conversion of 204 to leukotriene B₄, two diHETEs, 5-HETE, 15-HETE, and PGD₂. The stimulation of HETE synthesis by light could be due to early stages of light-induced lipid peroxidation in visual cells. To examine this, we studied peroxidation of 204 in isolated rod outer segments (ROS). There was more oxidation of 204 in light-exposed ROS, as compared to ROS incubated in the dark. Vitamin E and nordihydroguaiaretic acid inhibited the light-induced formation of some of these products. The data indicate that photo-oxidation of 204 in ROS is accompanied by enzymatic oxygenation that is stimulated by light. Increased production of HETEs and PGD₂ may be a consequence of the light-induced stimulation of the metabolism of 204 in membrane phospholipids in the retina.     

Electrophysiological properties of isolated rat liver cells

The electrophysiological properties of isolated rat liver cells were studied using the patch clamp method in whole-cell configuration. The membrane potential in isolated hepatocytes was -42 +/- 7 mV (n = 20). The input resistance (Rin) and the time constant (tau m) were 51 +/- 17 M (the range of 34 to 180 M omega) (n = 20) and 4.2 +/- 1.0 msec (the range of 3 to 16.5 ms) (n = 20). Assuming that the specific membrane capacitance is 1 microF/cm2, the membrane resistance and membrane capacitance were 42. +/- 9.0 K omega cm2 and 87 +/- 27 pF. These values indicate that isolated rat hepatocytes are not abnormally permeable or leaky. The current-voltage relationship was linear with no rectification. The depolarizing pulse from the resting potential did not induce fast or slow inward currents even when norepinephrine or high Ca2 (3.6 mM) were applied. This indicates that there is no voltage-sensitive Ca2+ channel in the isolated hepatocytes.     

Secretion of prostaglandins and leukotrienes by endometrial cells in cows with subclinical and clinical endometritis

The aims of this study were (1) to measure the secretion of prostaglandin F_2α (PGF_2α), prostaglandin E₂ (PGE₂), leukotriene B₄ (LTB₄), and leukotriene C₄ (LTC₄) by endometrial cells collected by a cytobrush from healthy cows and cows with subclinical and clinical endometritis in the fourth week postpartum, and (2) to evaluate the relationship between the mediators' levels of secretion and the number of polymorphonuclear neutrophils (PMNs) in the uterine smears of cows with subclinical endometritis. The study included cows without any signs of clinical endometritis (n = 63) and cows with clinical endometritis as a positive control (ENDOM, n = 12). Two different threshold ratios (>5% and >18% of PMNs) were used to categorize the cows without clinical signs as with or without cytologic endometritis (CE). Considering the first or second threshold, the animals with CE were included in group CE POS I or CE POS II, whereas the healthy cows were assigned to group CE NEG I or CE NEG II, respectively.     

Methamphetamine and lipid peroxidation in the rat retina

BACKGROUND: The use of psychoactive drugs during adolescence and early adult life has increased in the last few decades. It is known that developmental exposure to psychostimulants affects the sensory systems, and the retina has been shown to be a target tissue. This work was conducted to evaluate the pattern of lipid peroxidation in the rat retina following prenatal exposure to methamphetamine (MA). METHODS: Pregnant female Wistar rats were given MA (5 mg/kg of body weight/day; SC, in 0.9% saline) from GD 8 to 22. Offspring were sacrificed at postnatal days (PNDs) 7, 14, and 21. The retinas were homogenized, and both the total antioxidant and superoxide dismutase (SOD) activities were measured by enzymatic-colorimetric methods. The lipid peroxidation byproducts (malondialdehyde [MDA] and MDA-like metabolites) were measured by the thiobarbituric acid test. RESULTS: Total antioxidant levels were lower in the MA group at PND 21 in both males and females. The activity of SOD was higher in PND 7 females from the MA group. MDA levels were higher in the MA group at PND 21 in both genders. CONCLUSIONS: These findings suggest that prenatal-induced MA toxicity in the retina may be related to lipid peroxidation processes and oxidative stress.     

Serotonin synthesis and its light-dark variation in the rat retina

Retinal circadian rhythms are driven by an intrinsic oscillator, using chemical signals such as melatonin, secreted by photoreceptor cells. The purpose of the present work was to identify the origin of serotonin, the precursor of melatonin, in the retina of adult rat, where no immunoreactivity for serotonin or tryptophan hydroxylase had ever been detected. To demonstrate local synthesis of serotonin in the rat retina, substrates of tryptophan hydroxylase, the first limiting enzyme in the serotonin pathway, have been used. Tryptophan, in the presence of an inhibitor of aromatic amino acid decarboxylase, enhanced 5-hydroxytryptophan levels, whereas alpha-methyltryptophan, a competitive substrate inhibitor, was hydroxylated into alpha-methyl-5-hydroxytryptophan. Tryptophan hydroxylase substrate concentration was higher in the dark period than in the light period, and formation of hydroxylated compounds was increased. The presence of tryptophan hydroxylase mRNA in the rat retina was confirmed by RT-PCR. Taken together, the results support the local synthesis of serotonin by tryptophan hydroxylation, this metabolic pathway being required more critically when 5-HT is used for melatonin synthesis.     

Apoptosis and necrosis occurring in excitotoxic cell death in isolated chick embryo retina

Excitotoxic studies using isolated chick embryo retina indicated that such an in vitro model provides a valid tool to characterize the effect of different agonists for subtypes of glutamate ionotropic receptors. In retinas maintained for 24 h in a Krebs medium, after a brief exposure (30 min) to glutamate agonists, we compared the effects produced by NMDA and non-NMDA-agonists, such as kainic acid (KA) or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). Delayed retinal damage was assessed by measuring lactate dehydrogenase (LDH) present in the medium after exposure to the previously named agonists. Although at high concentrations, both KA and AMPA produced more relevant release than NMDA, 7-8% of total retinal LDH was released after exposure to a 50 microM concentration of non-NMDA agonists. These values were similar to those obtained after 100 microM NMDA. In this regard, retinal tissue appeared to be less sensitive to excitotoxicity based on the activation of NMDA receptor subtype. All three agents produced histopathological lesions typical for excitotoxic damage. A delayed form of excitotoxicity observed in retina segments was predominated by necrotic features. However, the activation of apoptotic machinery early during the incubation period subsequent to brief exposure to NMDA (100 microM) was also present. The activation of caspase enzymes was studied by a fluorometric protease activity assay as well as by western blot analysis. Caspase-3-like activity reached the highest value within 3 h of incubation after exposure to excitotoxin, then the level of enzyme activity declined to lower values. As confirmed by a time-related appearance of TUNEL-positive nuclei, apoptotic features appeared to be specific for retina response to NMDA. In contrast, the exposure to a 50 microM concentration of KA or AMPA induced necrotic cell damage which was evident through the incubation, leading to a delayed mechanism of excitotoxicity. These observations provide evidence that in the retinal model, with regard to agonist concentrations and subtype of glutamate receptors, the cascade of events leading to excitotoxicity may result in either apoptotic or necrotic neuronal cell damage.     

Metabolism of cysteinyl leukotrienes by the isolated perfused rat kidney.

The metabolism of cysteinyl leukotrienes by the isolated perfused rat kidney was investigated. For this purpose LTC4, LTD4 or LTE4 were studied in separate experiments. The isolated perfused rat kidney metabolized all cysteinyl leukotrienes to the final metabolite N-acetyl-LTE4. In the presence of 5% albumin 50% of LTC4 was metabolized to LTD4 (22%), LTE4 (15%) and N-acetyl-LTE4 (13%) within 60 min. Excretion of radioactivity into urine was less than 1%. In contrast, in the absence of albumin, LTC4 was completely metabolized within 45 min to N-acetyl-LTE4, the sole and final metabolite of LTC4 found in the perfusion medium as well as in urine. After 60 min 19% and 42% of total radioactivity were found in the perfusion medium and in urine, respectively. Isolated glomeruli metabolized LTC4 to LTD4 and to LTE4 but not to N-acetyl-LTE4 at a rate comparable to the rate observed by the isolated perfused kidney in the absence of albumin. In contrast to isolated glomeruli isolated tubuli metabolized LTE4 to N-acetyl-LTE4 at a rate comparable to that observed by the isolated perfused kidney in the absence of albumin. The present study shows that the isolated perfused rat kidney metabolizes cysteinyl leukotrienes to the sole and final metabolite N-acetyl-LTE4. In the presence of albumin metabolism is slowed down and excretion of N-acetyl-LTE4 into urine is prevented.     

Electrophysiological studies with repeated episodes of ischaemia on isolated rat heart

In order to know the beneficial effect of preconditioning electrocardiography recording were used as tool to assess myocardial malfunction and for this perfusion apparatus was setup. Electrophysiological changes for each heart were recorded during perfusion at 1, 2, 3, 5, 10, 20, 30 and 60 min of global ischaemia and also during the equal period of reperfusion. Recordings dembnstrate that the normal rate was about 240 beats/min with an "R" amplitude of 4mV. During the first ischaemic episode of 1min the rate was 180 +/- 15 beats/min (counted as per 'R' wave deflection), at 2 mins it was 60 +/- 6 beats/min, at 3 min the rate was 40 +/- 2 beats/min, at 5 mins of ischaemia it was 90 +/- 6 beats/min, at 10 min 20 +/- 2 beats/min, at 20 min the rate was 60 +/- 4 beats/min, and at 30 mins there were nil beats/min. The recovery during all the periods of reperfusion was restored to between 120 and 180 beats/min in all episodes. Further after a 60 min of ischaemia the heart stopped to elicit any mechanical response. It is concluded that short term ischaemia can induce a resilient effect on the beating of the heart after a few episodes as seen subsequent to 1 and 2 min of ischaemia. Further, preconditioning was beneficial up to 30 min, beyond which the heart showed signs of fatigue and irreversible injury.     

Control of experimental pulmonary tuberculosis depends more on immunostimulatory leukotrienes than on the absence of immunosuppressive prostaglandins

Prostaglandins (PGs) and leukotrienes (LTs) are produced in Mycobacterium tuberculosis (Mtb)-infected lungs and have immune suppressive and protective effects, respectively. Considering that both of these mediators are produced during mycobacterial infection, we investigated the specific and relative biological importance of each in regulating host response in experimental tuberculosis. Administration of celecoxib, which was found to reduce lung levels of PGE₂ and increase LTB₄, enhanced the 60-day survival of Mtb-infected mice in 14%. However administration of MK-886, which reduced levels of LTB₄ but did not enhance PGE₂, reduced 60-day survival from 86% to 43% in Mtb-infected mice, and increased lung bacterial burden. MK-886 plus celecoxib reduced survival to a lesser extent than MK-886 alone. MK-886- and MK-886 plus celecoxib-treated animals exhibited reduced levels of the protective interleukin-12 and gamma-interferon. Our findings indicate that in this model, the protective effect of LTs dominates over the suppressive effect of PGs.     

The effects of glycerol treatment on the isolated rat heart

Summary Isolated rat hearts were perfused with a balanced electrolyte solution containing 1000mM glycerol for 15min and then perfused with normal electrolyte solution for up to 32 min. The perfusion with hypertonic glycerol solution and subsequent washout is termed glycerol treatment. Initially, glycerol removal causes swelling and rupture of the T-system in ventricular myocardial cells which correlates temporally with a period of cardiac arrest. Contractility returns during further glycerol removal and concomitant recovery of the T-system is observed. Atomic absorption spectometry and neutron activation analysis were used to measure ventricular sodium, potassium and calcium ion content. There is no apparent correlation between changes in ion content and cardiac arrest or recovery. The water movements were calculated from wet weight, dry weight and inulin space, and confirmed by morphometric analysis of extracellular and intracellular space. It is suggested that the swelling and rupture of the T-system is due to the rapid water movements that were observed during the onset of glycerol removal. Ultrastructural analysis of glycerol-treated atrium from the same hearts shows damage of mitochondria and of the L-system and intracellular edema. The structural changes are correlated with a loss of atrial contraction. As in ventricular myocardium, resumption of contraction is associated with an almost complete recovery from ultrastructural damage.The studies were supported by the German Research Foundation within the SFB 90 Cardiovasculäres System     

Acetyl- and butyrylcholinesterase in normal and diabetic rat retina

We studied the composition of molecular forms of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) in normal and streptozotocin-induced diabetic rat retinas. Tissues were sequentially extracted with saline (S₁) and saline-detergent buffers (S₂). 50% decrease in the amphiphilic G₄ and G₁ AChE molecular forms was observed in the diabetic retina compared to the controls. Less than 5% of the cholinesterase activity was due to BChE. 60% of the BChE activity in normal retina was brought into solution and evenly distributed between S₁ and S₂. In spite of the low BChE activity in the retina it was possible to detect globular forms (G^A ₁, G^A ₂, G^A ₄, G^H ₄) and a small proportion of an asymmetric form (A₁₂) in the S₁ extract. The G^A ₄ and G^A ₁ forms were found in the S₂ extract. In the diabetic retina the activity of G^A ₄ and G^A ₁ BChE molecular forms was reduced 60% and 40% respectively. Our results indicate that diabetes caused a remarkable decrease in the activity of cholinesterase molecular forms in the retina. These decrease might participate in the alterations observed in the diabetic retina.     

Effects of taurine on the phosphorylation of specific proteins in subcellular fractions of the rat retina

The effects of 20 mM taurine on the phosphorylation of specific proteins in mitochondrial and rod outer segment subcellular fractions of the rat retina were measured. A band of protein with an apparent molecular wieght of 20K was consistently inhibited by taurine. Densitometry measurements performed on gel electrophoresis autoradiograms from the mitochondrial fraction demonstrated a 42.7±8.3% decrease due to taurine (20 mM) in the area corresponding to radioactivity from the 20K phosphoprotein. However, only a 21.2±9.0% decrease was observed due to taurine in the rod outer segment preparation. These data suggest that taurine is exerting its primary effect on the phosphorylation of the 20K molecular weight protein in the mitochondria of the retina. In addition, calmodulin and phorbol ester had no effect on the phosphorylation of the 20K molecular weight protein.     

Differentiation potential of bone marrow mesenchymal stem cells into retina in normal and laser-injured rat eye

Bone marrow mesenchymal stem cells (MSCs) can develop into hematopoietic and mesenchymal lineages but have not been known to participate in the production of retina. Here we report that bone marrow mesenchymal stem cells, after being subretinally transplanted into normal or Nd: YAG laser-injured rat eye, can integrate into RPE layer, photoreceptor layer, bipolar cell layer and ganglion layer. DAPI-labeling detection was used to trace the origin of the repopulating cells. DAPI fluorescence was used to identify retina cells of bone marrow origin 10, 20, 35 and 50 days after transplantation. No formation of rosettes was found but some random cells were found at the end of the observation. MSCs-originated cells spread more widely in the injured retinas than in the normal ones. Immunohistochemical detection showed that though the cells could express neuronal nuclei (NeuN), neuron specific enolase (NSE), glial fibrillary acidic protein (GFAP) and cytokeratin (CK), the proteins expression in the injured transplantation group was abnormal in some region compared with that in the normal transplantation group. Electroretinogram (ERG) showed that ERG-b wave of the injured transplantation group is significantly higher than that of the two laser-injured control groups. These results suggest that a proportion of MSCs can differentiate into retina-like structure in vivo and the differentiation differs in normal and laser-injured retinas.     

Lipid peroxidation and antioxidant enzymatic systems in rat retina as a function of age

In the present study, we have assayed the enzymatic activity of Cu,Zn–SOD, Mn–SOD, GSH–Px, GSH-Red, Cat, and G6PD in rat retina as a function of age. Conjugated diene levels and MDA formation were also determined. The conjugated diene levels in rat retina were found to increase significantly with age, accompanied by a marked decrease in GSH–Px and Cat activities. No agerelated change in MDA levels and in GSH-Red and G6PD activity was found, whereas a significant increase in SOD activity was observed between 1 and 4 months. Decreased GSH–Px and Cat activity is related to increased lipid peroxidation with age.     

Differentiation potential of bone marrow mesenchymal stem cells into retina in normal and laser-injured rat eye

Withinthebonemarrowstromathereexistsasubsetofnonhematopoieticcellsreferredtoasmes-enchymalstemormesenchymalprogenitorcells.Mesenchymalstemcells(MSCs)areapopulationofpluripotentcellswithinthehuman,birdorrodentbonemarrowmicroenvironmentdefinedbytheirability,eitherinvitroorinvivo,todifferentiateintocellsoftheosteogenic,chondrogenic,tendonogenic,adipo-genic,neuralcellsandmyogeniclineages[1].Themethodologiestoisolateandculture-expandMSCsfromhumanbonemarrowforestablishingthecellularortissuediffere…