The standardized Ginkgo biloba extract Egb-761 protects vascular endothelium exposed to oxidized low density lipoproteins.

Dietary antioxidants are frequently proposed as protective agents for the vascular endothelium during the onset of atherosclerosis. This protection may occur at two distinct levels. First, they prevent oxidative modification of atherogenic lipoproteins (LDL). Second, they can provide a cellular protection against oxidized LDL-mediated endothelium dysfunction, although this mechanism remains poorly considered in many instances. To gain insight into the mechanism underlying such cellular protection against oxidized LDL, we examined the impact of a popular traditional medicine, an extract from Ginkgo biloba with well-known antioxidant properties, on two endothelial cells properties: cell adhesion and ionic homeostasis. Cellular lipoperoxides levels were also measured as a marker of cellular oxidative stress. Human umbilical-vein endothelial cells were exposed to native (nat-) or oxidized (ox-) LDL, the latter prepared to be compatible with clinically observed levels of oxidation. Although nat-LDL had little effect, ox-LDL increased endothelial adhesive properties (35%, p<0.01) and lipoperoxidation (45%, p<0.01). Na,K-ATPase activity, a key regulator of ionic homeostasis, was significantly decreased after exposure to nat-LDL (30%, p<0.01) and dramatically depressed after exposure to ox-LDL (65%, p<0.001). The standardized preparation of Ginkgo biloba EGb-761 totally protected adhesive properties and endothelial lipoperoxide levels. Moreover, it limited the decrease in Na,K-ATPase activity induced by ox-LDL to levels similar to nat-LDL. This suggests that EGb-761 protects endothelial adhesive properties and helps prevent the disruption of ionic homeostasis. The EGb-761-mediated inhibition of ox-LDL-induced lipoperoxide levels in endothelial cells appears to be an important mechanism by which Ginkgo biloba extract protects endothelial properties.

Chapter 11.6 Clays and Clay Minerals as Drugs.

This chapter discusses the applications of clays and clay materials as drugs. Clay minerals are efficient against several aggressors that cause major disorders of the gut. These beneficial effects of clay minerals (on the gastrointestinal mucosa) are associated with two mechanisms of action: (1) adsorption of the aggressors or their toxic secretions and (2) modification of the thickness and rheological properties of the adherent mucus, reinforcing the natural defenses of the gastrointestinal mucosa. At the surface of the gut, a mucus gel adheres to the epithelial cells of the mucosa. This adherent mucus is dynamic, being continuously secreted by the calceiform cells and regularly eroded by environmental aggressors present in the gut lumen. The mucus gel is largely composed of glycoprotein polymers, lipids, and proteins linked together by covalent bonds. It acts as a physical barrier protecting the mucosa against penetration by extraneous molecules and mechanical injury. By maintaining a pH gradient and competing with the epithelial surface for microorganisms, the mucus gel also acts as a chemical barrier.

Protective effect of cicletanine on renal function in diabetic spontaneously hypertensive rats.

Hypertension and diabetes are commonly associated and strongly predispose to renal injuries. In general, antihypertensive therapies protect from these damages, but the effect of cicletanine, a new type of antihypertensive drug, is unknown. This study examines the effects of cicletanine on renal failure in spontaneously hypertensive rats with diabetes (SHRD). Diabetes mellitus was induced with streptozotocin in uninephrectomized SHR. Rats received the vehicle, 10 mg or 50 mg/kg per day of cicletanine for 6 weeks. Age-matched untreated Wistar-Kyoto rats were used as controls. Systolic blood pressure (SBP), microalbuminuria and proteinuria were assessed throughout the treatment. At the end of the study, creatinine clearance measurements and histological analysis of kidneys were performed. Cicletanine did not affect SBP but decreased the elevated albuminuria of diabetic SHR in a dose-dependent manner. Similar results were obtained for proteinuria. Treatment with the high dose of cicletanine also normalized the altered creatinine clearance of diabetic SHR. These results indicate that cicletanine has a renal-protective action, probably blood pressure-independent, in a model combining hypertension and diabetes. The mechanism of renal-protection of cicletanine is not clearly established but may be due to the stimulation of arterial prostacyclin synthesis and/or to the reduction of intraglomerular capillary pressure.

Nitric oxide, peroxynitrite and cGMP in atherosclerosis-induced hypertension in rabbits: beneficial effects of cicletanine.

We studied the effect of the furopyridine derivative antihypertensive drug, cicletanine, on blood pressure, vascular nitric oxide (NO) and cyclic guanosine 3':5'-monophosphate (cGMP) content in the aorta and the renal and carotid arteries, aortic superoxide production, and serum nitrotyrosine level in hypertensive/atherosclerotic rabbits. The effect of cicletanine was compared to that of furosemide. Rabbits were fed a normal or a cholesterol-enriched (1.5%) diet over 8 weeks. On the 8th week, the rabbits were treated per os with 2 x 50 mg/kg daily doses of cicletanine, furosemide, or vehicle for 5 days (n = 5-6 in each groups). The cholesterol diet increased mean arterial blood pressure (MABP) from 86 +/- 1 to 94 +/- 2 mm Hg (p < 0.05). Cicletanine decreased MABP in atherosclerotic rabbits to 85 +/- 1 mm Hg (p < 0.05), but it did not affect MABP in normal animals. Furosemide was without effect in both groups. In normal animals, NO content (assessed by electron spin resonance after in vivo spin trapping) in the aorta and the renal and carotid arteries was increased by cicletanine, and the drug increased cGMP in the renal artery as measured by radioimmunoassay. The cholesterol-enriched diet decreased both vascular NO and cGMP and increased aortic superoxide production assessed by lucigenin-enhanced chemiluminescence and serum nitrotyrosine determined by ELISA. In atherosclerotic animals, cicletanine increased NO and cGMP content in the aorta and the renal and carotid arteries and decreased aortic superoxide production and serum nitrotyrosine. Furosemide did not influence these parameters. We conclude that cicletanine lowers blood pressure in hypertensive/atherosclerotic rabbits. The antihypertensive effect of the drug in atherosclerosis may be based on its beneficial effects on the vascular NO-cGMP system and on the formation of reactive oxygen species.

Inhibition of type 4 phosphodiesterase by rolipram and Ginkgo biloba extract (EGb 761) decreases agonist-induced rises in internal calcium in human endothelial cells.

The effects of Gingko biloba extract EGb 761 on 5 isolated, vascular, cyclic nucleotide phosphodiesterase (PDE) isoforms were evaluated. EGb 761 preferentially inhibited PDE4 (IC(50)=25.1 mg/L), the isoform that is mainly present in endothelial cells, in a competitive manner (K:(i)=12.5 mg/L). Because changes in cyclic nucleotide levels may affect intracellular calcium ([Ca(2+)](i)) levels in endothelial cells, we examined the effects of EGb 761 on both resting [Ca(2+)](i) levels and agonist-induced rises in [Ca(2+)](i) in single human umbilical vein endothelial cells (HUVECs) in culture. The effects of EGb 761 were compared with those of rolipram, a selective PDE4 inhibitor that increases cellular cAMP levels, and the cAMP analogue dibutyryl cAMP (db-cAMP). EGb 761 (20 and 100 mg/L), rolipram (50 micromol/L), and db-cAMP (100 micromol/L) significantly inhibited histamine-, ATP-, and thrombin-induced [Ca(2+)](i) increases in HUVECs without modifying resting [Ca(2+)](i) levels. Similar results were obtained by using a Ca(2+)-free bath solution. EGb 761 (100 mg/L), but not rolipram (50 micromol/L) or db-cAMP (100 micromol/L), also inhibited Ca(2+) influx into cells having thapsigargin-depleted internal Ca(2+) stores and bathed in a Ca(2+)-free external solution. Our results are consistent with an inhibition of PDE activity that causes a reduction of agonist-induced increases in [Ca(2+)](i) in HUVECs, mainly by inhibition of Ca(2+) mobilization from internal stores. It thus may be that the cardiovascular effects of EGb 761 involve inhibition of PDE4 activity and subsequent modification of Ca(2+) signaling in endothelial cells.

Relaxation of vascular smooth muscle by cicletanine in aged wistar aorta under stress conditions: importance of nitric oxide.

The vascular mechanism of action of cicletanine, an antihypertensive agent, was studied on isolated Wistar rat aortas (24-months-old) in presence and in absence of endothelium in two different stress conditions, normoxic and hypoxic, in presence of norepinephrine (NE). Under normoxic conditions, in presence of endothelium, cicletanine (10(-9)-10(-5)M) induced a concentration-dependent relaxation, whereas in absence of endothelium, cicletanine (10(-9)-10(-5)M) was ineffective although it relaxed the smooth muscle at higher concentrations (10(-4)M). At pharmacologic concentrations (below or equal 10(-5)M), relaxation induced by cicletanine, in presence of endothelium, was prevented by N(omega)-nitro-L-arginine (L-NNA) (P <.005) and relaxation induced by the highest concentration (10(-4)M) was reversed by BaCl2 (P <.005). Under hypoxic conditions, in presence of NE and endothelium, the aorta displayed an increased developed tension that was significantly (P <.05) attenuated by cicletanine (10(-5)M) and insensitive to indomethacine (10(-7)M). When the two compounds were added together, the relaxation induced by cicletanine was significantly improved (P <.005). These results indicated that cicletanine, under stress conditions, relaxes vascular smooth muscle through an endothelium-dependent action mediated by the nitric oxide (NO) synthase pathway. We proposed that the observed vascular effects could be associated with the counter-regulation mechanisms linked to the antihypertensive action of cicletanine.

Cicletanine reverses vasoconstriction induced by the endogenous sodium pump ligand, marinobufagenin, via a protein kinase C dependent mechanism.

RATIONALE: Cicletanine (CIC), an anti-hypertensive compound with direct vascular and natriuretic actions, is especially effective in salt-sensitive hypertension, in which dysregulation of the sodium pump plays an important pathogenic role, and digitalis-like cardiotonic steroids contribute to increased vascular tone. The purpose of the present study was to investigate whether, and by what mechanisms, cicletanine antagonizes the vasoconstrictor effects of cardiotonic steroids in isolated human arteries. METHODS: The effects of cicletanine on vascular tone were studied in isolated, endothelium-denuded rings of 2nd-3rd-order branches of human mesenteric arteries pre-contracted with bufodienolide marinobufagenin (MBG), an Na/K-ATPase inhibitor, or endothelin-1 (ET-1). Na/K-ATPase activity was measured in sarcolemmal membranes from the mesenteric artery. Activity of rat brain protein kinase C (PKC) was measured using the PepTag phosphorylation assay. RESULTS: MBG and ET-1 both induced sustained vasoconstriction in human mesenteric artery rings, and cicletanine relaxed rings pre-contracted with either MBG (EC50 = 11 +/- 2 micromol/l) or ET-1 (EC50 = 6.4 +/- 1.1 micromol/l). Although 8-Br-cGMP (100 micromol/l) caused complete vasorelaxation of arterial rings pre-contracted with ET-1, it did not affect the MBG-induced vasoconstriction. An activator of PKC, phorbol diacetate (PDA) (50 nmol/l), attenuated CIC-induced vasorelaxation of mesenteric artery rings pre-contracted with MBG (EC50 > 100 micromol/l), but not rings pre-contracted with ET-1 (EC50 = 6.5 +/- 1.2 micromol/l). In mesenteric artery sarcolemma, 100 nmol/l MBG inhibited the Na/K-ATPase by 68 +/- 5% and cicletanine (100 micromol/l) attenuated this Na/K-ATPase inhibition by 85 +/- 6%. In the PepTag PKC assay, cicletanine produced a concentration-dependent inhibition of rat brain PKC activity (IC50 45 +/- 11 micromol/l). In the presence of 50 nmol/l PDA, 100 micromol/l cicletanine did not antagonize the Na/K-ATPase inhibition by MBG, and did not inhibit the PKC from rat brain. CONCLUSIONS: Cicletanine antagonizes vasoconstriction induced by Na/K-ATPase inhibition via a PKC-dependent mechanism that does not involve inhibition of cyclic GMP phosphodiesterase (cGMP-PDE). This mechanism of action may be relevant to the greater potency of cicletanine in salt-sensitive hypertension in which plasma levels of endogenous digitalis-like cardiotonic steroids are elevated. Our findings also suggest that PKC is an important factor for cardiotonic steroid-Na/K-ATPase interactions on the vascular tone, and is therefore a potential target for therapeutic intervention in hypertension.

The protective effect of EGb 761 in isolated ischemic/reperfused rat hearts: a link between cardiac function and nitric oxide production.

This study was conducted to evaluate the effect of Ginkgo biloba extract (EGb 761) on the nitric oxide (NO) production in relation to the recovery of postischemic cardiac function in isolated working rat hearts. Rats were orally treated with various doses (25, 50, 75, and 100 mg/kg/day) of EGb 761 for 10 days. Hearts were isolated in "working mode" and subjected to 30-min ischemia followed by 120 min of reperfusion. EGb 761 inhibited NO production measured by electron spin-resonance spectroscopy (ESR), and improved the recovery of postischemic cardiac function (coronary flow, aortic flow, left ventricular developed pressure and its first derivative) in the ischemic/reperfused myocardium. Thus in rats treated with 25, 50, 75, and 100 mg/kg/day of EGb 761 and in hearts subjected to 30-min ischemia followed by 120 min of reperfusion, aortic flow was increased from its postischemic drug-free control value of 8.0+/-0.4 to 8.6+/-0.4 ml/min (NS), 17.3+/-0.9 ml/min (p<0.05), 21.5+/-1.1 ml/min (p<0.05), and 23.6+/-1.2 ml/min, respectively. The same recovery in postischemic coronary flow, left ventricular developed pressure, and its first derivative also was observed. In the initial phase of reperfusion, NO production measured by ESR was reduced by 85% in the 75 mg/ kg/day of EGb 761-treated group in comparison with the drug-free ischemic/reperfused hearts. Inducible NO synthase (iNOS) messenger RNA (mRNA) measured by reverse transcription-polymerase chain reaction (RT-PCR) also was reduced by 41 and 58% in the groups treated with 75 and 100 mg/kg/day of EGb 761, respectively. Our findings show that EGb 761 directly acts as an NO scavenger and concomitantly inhibits the expression of iNOS mRNA. Thus, EGb 761 may act as a potent inhibitor of NO production under the condition of ischemia/reperfusion, improving the recovery of postischemic cardiac function.

EGb 761 protects liver mitochondria against injury induced by in vitro anoxia/reoxygenation.

The present study investigated the protective effects of Ginkgo biloba extract (EGb 761) on rat liver mitochondrial damage induced by in vitro anoxia/reoxygenation. Anoxia/reoxygenation was known to impair respiratory activities and mitochondrial oxidative phosphorylation efficiency. ADP/O (2.57 +/- 0.11) decreased after anoxia/reoxygenation (1.75 +/- 0.09, p < .01), as well as state 3 and uncoupled respiration (-20%, p < .01), but state 4 respiration increased (p < .01). EGb 761 (50-200 microg/ml) had no effect on mitochondrial functions before anoxia, but had a specific dose-dependent protective effect after anoxia/reoxygenation. When mitochondria were incubated with 200 microg/ml EGb 761, they showed an increase in ADP/O (2.09 +/- 0.14, p < .05) and a decrease in state 4 respiration (-22%) after anoxia/reoxygenation. In EPR spin-trapping measurement, EGb 761 decreased the EPR signal of superoxide anion produced during reoxygenation. In conclusion, EGb 761 specially protects mitochondrial ATP synthesis against anoxia/reoxygenation injury by scavenging the superoxide anion generated by mitochondria.

[Lack of effect of cicletanine and its sulfoconjugated metabolite on the thiazide receptor expressed in Xenopus oocytes]. / Absence d'effet de la ciclétanine et de son métabolite sulfoconjugué sur le récepteur aux thiazides exprimé dans des ovocytes de Xenopus.

UNLABELLED: Although the renal receptor at which cicletanine acts is unknown, cicletanine was assumed to act like thiazide diuretics. Here we tested cicletanine and its natriuretic metabolite, cicletanine-sulfate, for inhibitory activity against the thiazide-sensitive NaCl cotransporter expressed in Xenopus oocytes. The renal thiazide-sensitive NaCl cotransporter was expressed in Xenopus laevis oocytes injected with rat cRNA TSCr (TSCr: thiazide-sensitive cotransporter from rat kidney) and both, racemic (+/-) cicletanine and its sulfoconjugated metabolite were tested for inhibitory activity against oocyte 22Na+ uptake catalyzed by this cotransporter. Polythiazide was used as reference thiazide. Polythiazide fully inhibited NaCl cotransporter function with IC50 approximately 1.2 x 10(-7) M. Conversely, neither cicletanine, nor cicletanine sulfate were able to inhibit such cotransporter, i.e.: a minimum concentration of 10(-4) M of cicletanine was necessary to induce a slight cotransporter inhibition (29.5 +/- 18.2%). Cicletanine sulfate was inactive, even at 10(-4) M. IN CONCLUSION: (i) the natriuretic metabolite of cicletanine (cicletanine sulfate) is unable to inhibit thiazide-sensitive NaCl cotransporter and (ii) inhibition of such cotransporter by cicletanine required concentrations equal or higher than 10(-4) M--concentrations much more higher than urinary therapeutic ones in humans (approximately 10(-6) M). These results clearly demonstrate that cicletanine does not act like thiazide diuretics.

[Mechanism of action of cicletanine on the relationship between endothelium and vascular smooth muscle in the rat thoracic aorta under normoxic and hypoxic conditions]. / Mode d'action du ciclétanine sur la relation endothélium-muscle lisse vasculaire de l'aorte thoracique de rat en conditions normoxiques et hypoxiques.

OBJECTIVE: The vascular mechanism of action of cicletanine, an antihypertensive agent, was studied on isolated Wistar rat aorta in presence and in absence of endothelium both in normoxic and hypoxic conditions. DESIGN AND METHODS: Isolated aorta, from 24 month-old rats, were precontracted with noradrenaline (10(-7) M), in presence and in absence of endothelium and exposed to cumulative cicletanine concentrations in presence and absence of either L-NNA (10(-4) M) or indomethacin (Indo) (10(-7) M). Thereafter, aorta were precontracted by noradrenaline 10(-7) M, and hypoxia was induced by switching gas mixture from 95%O2/5%CO2 to 95%N2/5%CO2 during 10 minutes. Results are expressed as mean +/- sem and statistical analysis were done using one-way analysis of variance. RESULTS: When aorta were precontracted with noradrenaline (10(-7) M), in presence of endothelium, cicletanine (10(-9)-10(-4) M), induced a biphasic concentration-dependent relaxation (EC50 approximately 10(-7) M and 3 x 10(-5) M). In absence of endothelium, the effect of cicletanine was abolished (10(-9) and 10(-5) M). Whereas, at higher concentration (10(-4) M), the magnitude of the relaxation reached 94 +/- 2% and 67 +/- 5% of the initial developed tension in presence and in absence of endothelium respectively. The endothelium-dependent relaxation induced by cicletanine was significantly reduced by Indo (10(-7) M) (p < 0.05) and L-NNA (10(-4) M) (p < 0.005). Addition of 10 mM of BaCl2 significantly reversed the relaxation induced by the higher concentration of cicletanine used (p < 0.005). Under hypoxic conditions, the aorta, in presence of endothelium, displayed an increased developed tension which was significantly attenuated by cicletanine. CONCLUSION: These results indicated that cicletanine relaxes vascular smooth muscle through both, an endothelium-dependent action which was mediated by cyclooxygenase and NOsynthase pathways and an endothelium-independent action that was mediated through K+ channels opening. Under hypoxic conditions, our findings indicate that the effects of cicletanine, appear related to an endothelium protective action associated to NO release.

Functional protection of photoreceptors from light-induced damage by dimethylthiourea and Ginkgo biloba extract.

PURPOSE: To investigate the functional protective effect of a synthetic (dimethylthiourea, DMTU) and a natural antioxidant (Ginkgo biloba extract, EGb 761) against light-induced retinal degeneration. METHODS: Wistar rats were exposed for 24 hours to 1700-lux light after treatment with DMTU or EGb 761. Electroretinograms were recorded before and on day (D)1, D3, D8, D15, D22, and D29 after light exposure. The b-wave amplitude was plotted against log L (ganzfeld luminance), providing the b-wave sensitivity curve. The Naka-Rushton function fitted to the sensitivity curve enabled derivation of the parameters Bmax (saturated amplitude) and K (luminance-inducing Bmax/2). In addition, rats from each group were killed for retinal morphometric analyses. RESULTS: In the untreated group, light exposure caused collapse of the b-wave sensitivity curves. Bmax was reduced by 51% at D1 without subsequent recovery. K increased temporarily, reverting to normal values 8 days later. The outer nuclear layer thicknesses decreased markedly in the superior retina. In the treated groups, light exposure had a weaker effect on sensitivity curves. The values of Bmax were not significantly different from those in the unexposed-untreated group, although K increased temporarily. Retinal morphometry was preserved. CONCLUSIONS: Dimethylthiourea and EGb 761 afford functional protection against light-induced retinal damage.

Cardioprotective effect of enantiomers of cicletanine (BN50417, BN50418) in ischaemic/reperfused isolated working rat hearts: interaction with glibenclamide.

In the present study, interaction of the ATP-sensitive K+-channel blocker glibenclamide with enantiomers of the antihypertensive drug, cicletanine, was studied on ischaemic myocardial function, lactate-dehydrogenase (LDH) release, and early reperfusion-induced ventricular fibrillation (VF). Isolated working rat hearts subjected to 10-min coronary artery occlusion followed by 2-min reperfusion were perfused with 1.5x10(-5)-6.0x10(-5)M D-cicletanine[+] (BN50417) and L-cicletanine[-] (BN50418), respectively. Their interaction with 10(-7) M glibenclamide was also studied. The most effective concentration of BN50418 (3x10(-5) M) increased ischaemic aortic flow (AF) from its non-treated control value of 20.3+/-1.16 to 30.3+/-2.6 ml min-1(P<0.01), decreased left ventricular end-diastolic pressure (LVEDP) from 1.81+/-0.05 to 0.97+/-0.08 kPa (P<0.001), attenuated ischaemia-induced increase in LDH leakage from 164+/-41 to 14.8+/-20 mU min-1g-1 wet wt. (P<0.01) at the 10th-min of coronary occlusion, and reduced VF upon reperfusion. Glibenclamide did not considerably affect cardiac performance, however, it inhibited the anti-ischaemic but not the antiarrhythmic effect of BN50418. BN50417 (3x10(-5) M) tended to improve ischaemic AF to 24.2+/-1.1 ml min-1, and significantly attenuated ischaemia-induced increase in LVEDP to 1.3+/-0.08 kPa (P<0.01), relative increase in LDH release to 29.4+/-44 mU min-1g-1(P<0.05), and alleviated reperfusion-induced VF. Glibenclamide abolished the anti-ischaemic and antiarrhythmic effect of BN50417. The cardioprotective effect of both enantiomers of cicletanine involves a glibenclamide-sensitive mechanism, however, the antiarrhythmic effect of BN50418 is not glibenclamide sensitive. BN50418 is the more potent enantiomer of cicletanine in terms of its cardioprotective effect.

Ginkgo biloba extract (EGb 761) protects Na,K-ATPase activity during cerebral ischemia in mice.

Neuroprotective drugs such as Ginkgo biloba extract (EGb 761) could prevent the ischemia-induced impairment of the Na,K-ATPase activity. In this study, Na,K-ATPase activity and expression, contents in fatty acids and malondialdehyde, an index of lipoperoxidation, were compared in the ipsilateral (ischemic) and the contralateral (unlesioned) cortices after 1 h of unilateral focal cortices cerebral ischemia in the mouse. EGb 761 (110 mg/kg) was administered daily to half of the animals for 10 days before ischemia. Ischemia significantly reduced Na,K-ATPase activity by about 40% and increased malondialdehyde content; EGb 761 pretreatment abolished these effects. The free radical scavenger properties of EGb 761 are a potential mechanism by which Na,K-ATPase injury and lipoperoxidation are prevented.

Effects of EGb761 and superoxide dismutase in an experimental model of retinopathy generated by intravitreal production of superoxide anion radical.

BACKGROUND: A study was carried out to investigate the effect of two antioxidants--Ginkgo biloba extract (EGb761) and superoxide dismutase (SOD)--in an experimental model of vitreoretinopathy obtained by direct production of oxygen free radicals in the vitreous cavity. METHODS: Twenty-eight pigmented rabbits were used. Vitreoretinopathy was induced by intravitreal injection of 50 microliters of a mixture composed of 40 nmol of xanthine and 0.001 IU of xanthine oxidase. Rabbits were randomly distributed into four groups: Group 1 (n = 8) did not receive any treatment and served as a positive control. Groups 2 (n = 8) and 3 (n = 8) received for 1 month EGb761 given orally at a dose of 100 mg/kg/day, respectively 1 day after and 1 week before induction of retinopathy. Group 4 (n = 4) was treated by three intramuscular injections of 15,000 IU/kg of SOD, 24 h before induction and 24 and 48 h thereafter. Clinical evaluations and electroretinograms (ERG) were repeatedly performed until the animals were killed at day 28. Histological examinations and immunohistological procedures were performed to ascertain the origin and characteristics of the cellular proliferation and to compare vitreoretinal structures in the four groups. RESULTS: Intravitreal injection of xanthine-xanthine oxidase produced a strong inflammatory response with vitreous infiltrates and epiretinal membrane formation, inconstantly associated with retinal detachment. ERG showed a decrease of the a-, b- and c-waves beginning within a few hours after injection. Histologic evaluation found an intravitreal and epiretinal infiltration by leukocytes and epithelial-derived cells, dense vitreoretinal membranes and retinal detachments with occasional neovascularization. In the treated groups (groups 2-4), all clinical, electric and histologic data were significantly improved compared to the control group. However, no difference could be found among the three treated groups. CONCLUSION: This study demonstrates the strong pathologic effects of free radical production on the retina and the close relationships between free radicals, inflammatory pathways and vitreoretinal proliferative disorders. It also confirms the pharmacological interest of prevention by antioxidants and free radical scavengers.

Dual effect of cicletanine on the Na+-H+ exchanger in chick embryonic cardiomyocytes.

Na+-H+ exchanger is thought to play an important role in the regulation of the intracellular pH (pHi) and in the cardiac cell injury induced by ischemia and reperfusion. The aim of this study was to assess the effect of cicletanine, an antihypertensive compound in humans, which has been reported to have cardioprotective effect under an ischemia-reperfusion process, on Na+-H+ exchanger activity in ventricular cardiomyocytes isolated from chick embryo. Na+-H+ exchanger activity was assessed by the rate of pHi recovery after an acid load. A dual effect was observed: at low concentration of cicletanine (10(-7) M), Na+-H+ exchanger activity was significantly decreased, whereas at higher concentrations (10(-6)-10(-5) M), a significant stimulation of the exchanger was observed. These results suggest that cicletanine modulates pHi recovery in cardiac cells after cellular acid load.

Vasorelaxant effects of cicletanine and its (+)- and (-)-enantiomers in isolated human pulmonary arteries.

The purpose of the study was to investigate, in isolated human pulmonary artery, the ability of cicletanine and its (-) and (+)-enantiomers to attenuate the endothelin-1 (Et-1) induced vasoconstriction, and to potentiate vasorelaxation (relative to plateau of the effect of Et-1) by sodium nitroprusside (SNP) and human atrial natriuretic peptide (ANP). In pulmonary artery rings, Et-1 induced a concentration-dependent vasoconstriction with median effective concentration (EC50 = 26+/-2.8 nmol/L. Pretreatment of the vessels with 100 micromol/L (+/-)-cicletanine reduced the effect of Et-1 (EC50 = 36+/-3.5 nmol/L; P < .01). (-)-enantiomer displayed greater capacity to antagonize the vasoconstrictor action of Et-1 (EC50 = 47+/-4.2 nmol/L) v (+)-enantiomer (EC50 = 29.9+/-6.5 nmol/L; P < .01). In arterial rings, precontracted with 10 nmol/L Et-1, ANP caused vasorelaxation (EC50 = 9.7+/-1.9 nmol/ L). The relaxant effect of ANP was potentiated by 100 micromol/L of (-)-(EC50 = 4.2+/-0.6 nmol/L; P < .01), but not (+)-cicletanine (EC50 = 7.6+/-0.7 nmol/L). Sodium nitroprusside relaxed pulmonary artery rings precontracted with 10 nmol/L Et-1 (EC50 = 41+/-11 nmol/L). The effect of SNP was potentiated by 10 micromol/L (+/-)-cicletanine (EC50 = 9.0+/-0.7 nmol/L; P < .05). The potentiating effect of 10 micromol/L (+)-cicletanine was weaker (EC50 = 7.9+/-1.8 nmol/L) than that of (-)-enantiomer (EC50 = 3.3+/-0.54 nmol/L; P < .05). The relaxant effect of SNP was not further potentiated by 100 micromol/L (+/-)-cicletanine. The present results demonstrate that, cicletanine antagonizes Et-1 induced vasoconstriction in an isolated human pulmonary artery and potentiates vasorelaxation by two guanylate cyclase activators, ANP and SNP. (-)-Cicletanine displays greater vasorelaxant activity v (+)-enantiomer.

Scanning electron microscopy after cryofracture of normal and ischemic rat retinas.

The authors described the use of scanning electron microscopy after cryofracture to analyze rat retinas after experimental ischemia-reperfusion sequence. In Sprague-Dawley albino rats, intraocular hypertony by cannulation of anterior chamber was performed for 1 h on one eye. The other eye served as control. After 48 h of reperfusion, retinas were dissected. They were frozen and fractured before ultrastructural analysis by a scanning electron microscope. In the ischemic eyes the thickness of the photoreceptors layer was reduced, through internal disorganization resulting in misalignment of rods. There was swelling of the outer segments, a loss of adhesion between segments and superficial necrosis. Scanning electron microscopy after cryofracture permitted an analysis of the external morphology of retinal cells and intercellular components, especially the outer layers.

Inhibition by (-)-cicletanine of the vascular reactivity to angiotensin II and vasopressin in isolated rat vessels.

In pithed rats, the levorotatory (-)-enantiomer of cicletanine reduces the pressor responses to angiotensin II (AII) and also, to a lesser extent, those to arginine-vasopressin (AVP). Here we have attempted to characterize further these inhibitory effects by studies of isolated perfused rat kidney and mesenteric vascular beds. In the isolated rat kidney, (-)-cicletanine behaves as a noncompetitive antagonist of AII- and AVP-receptor stimulation, with Ki values of 9.6 and 208 micromol/L respectively. In the isolated mesenteric vascular bed, (-)-cicletanine antagonized both AII dependent contractions with an inhibitory concentration (IC50) of 54.0 +/- 20.5 micromol/L (n = 6), and AVP dependent contractions with an IC50 of 31.6 +/- 5.0 micromol/L (n = 8). In conclusion, (-)-cicletanine antagonizes AII more effectively in rat kidney than in mesenteric vascular beds. Moreover, in rat kidney vascular beds (-)-cicletanine is more potent in blocking the pressor responses to AII than in blocking those to AVP. A selective blockade of AII induced contractions in kidney vascular beds can be one factor explaining both the greater antagonistic potency of (-)-cicletanine against AII compared with AVP in pithed rats, and the renal protective properties of cicletanine in both hypertensive and aged rats.

A Ginkgo biloba extract (EGb 761) prevents mitochondrial aging by protecting against oxidative stress.

The effect of aging on indices of oxidative damage in rat mitochondria and the protective effect of the Ginkgo biloba extract EGb 761 was investigated. Mitochondrial DNA from brain and liver of old rats exhibited oxidative damage that is significantly higher than that from young rats. Mitochondrial glutathione is also more oxidized in old than in young rats. Peroxide formation in mitochondria from old animals was higher than in those from young ones. According to morphological parameters (size and complexity), there are two populations of mitochondria. One is composed of large, highly complex mitochondria, and the other population is smaller and less complex. Brain and liver from old animals had a higher proportion of the large, highly complex mitochondria than seen in organs from young animals. Treatment with the Ginkgo biloba extract EGb 761 partially prevented these morphological changes as well as the indices of oxidative damage observed in brain and liver mitochondria from old animals.