Ocular inflammation and corneal permeability alteration by benzalkonium chloride in rats: a protective effect of a myosin light chain kinase inhibitor.

PURPOSE: The aim of this study was to evaluate the interest of an ophthalmic eyedrop preparation containing a myosin light chain kinase (MLCK) inhibitor, ML-7, in the treatment of ocular surface. The local protective effect on the inflammation and the increase of corneal permeability induced by benzalkonium (BAK) was evaluated. METHODS: An ocular instillation of 10 lL BAK at a concentration of 0.1% in PBS was performed on rats. The eyes were rinsed with sterilized water, 10 minutes after BAK preceded by instillation at T -24, -12, and -0.5 hours of 10 lL ofML-7: 100 µg (10 µL) into a gel form vehicle. All animals were sacrificed 6 hours after BAK instillation. The eyes were isolated for study in a masked manner. The ocular surface inflammation was assessed by measuring the inflammatory cell infiltration by a histologic quantitative analysis and for total ocular myeloperoxidase (MPO) activity. The tight junction permeability was tested. RESULTS: Instillation of 0.1% BAK increased the inflammation of the eye. The quantitative analysis showed an increase in the number of eosinophil and neutrophil polynuclears, and MPO activity. Pretreatment with ML-7 reduced inflammation (P < 0.05). The vehicle alone produced no notable effects. BAK instillation also thickened the fluorescent corneal front on frozen sections, indicating an increase of tight junction permeability. Pretreatment with ML-7 suppressed BAK-induced alterations of paracellular permeability while the vehicle had no visible effects. CONCLUSIONS: Our study indicates that the inhibition of corneal cytoskeleton contraction by an MLCK inhibitor prevents BAK-induced ocular inflammatory response, and that ML-7 may be a new and original preparation in the treatment of ocular surface pathologies.

Myocardial PKC beta2 and the sensitivity of Na/K-ATPase to marinobufagenin are reduced by cicletanine in Dahl hypertension.

Marinobufagenin (MBG), an endogenous ligand of alpha-1 Na/K-ATPase, becomes elevated and contributes to hypertension in NaCl-loaded Dahl-S rats (DS). Protein kinase C (PKC) phosphorylates alpha-1 Na/K-ATPase and increases its MBG sensitivity. Cicletanine, an antihypertensive compound with PKC-inhibitory activity, reverses MBG-induced Na/K-ATPase inhibition and vasoconstriction. We hypothesized that increased PKC levels in sodium-loaded hypertensive DS would sensitize alpha-1 Na/K-ATPase to MBG and that PKC inhibition by cicletanine would produce an opposite effect. We studied the effects of cicletanine on systolic blood pressure, left ventricular PKC isoforms, cardiac alpha-1 Na/K-ATPase levels, and sensitivity to MBG in hypertensive DS. Seven DS received 50 mg x kg(-1) x d(-1) cicletanine, and 7 DS received vehicle during 4 weeks of an 8% NaCl diet. Vehicle-treated rats exhibited an increase in blood pressure, left ventricular mass, MBG excretion (74+/-11 vs 9+/-1 pmol/24 h, P<0.01), myocardial alpha-1 Na/K-ATPase protein, and PKC beta2 and delta. The sensitivity of Na/K-ATPase to MBG was enhanced at the level of high-affinity binding sites (IC50, 0.8 vs 4.4 nmol/L, P<0.01). Cicletanine-treated rats exhibited a 56-mm Hg reduction in blood pressure (P<0.01) and a 30% reduction in left ventricular weight, whereas cardiac alpha-1 Na/K-ATPase protein and MBG levels were unchanged. In cicletanine-treated rats, PKC beta2 was not increased, the sensitivity of Na/K-ATPase to MBG was decreased (IC50=20 micromol/L), and phorbol diacetate-induced alpha-1 Na/K-ATPase phosphorylation was reduced versus vehicle-treated rats. In vitro cicletanine treatment of sarcolemma from vehicle-treated rats also desensitized Na/K-ATPase to MBG, indicating that this effect was not solely attributable to a reduction in blood pressure. Thus, PKC-induced phosphorylation of cardiac alpha-1 Na/K-ATPase is a likely target for cicletanine treatment.

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

Disturbances of Na,K-ATPase activity are implicated in the pathophysiology of cerebral ischemia. Previous experiments have shown that EGb 761 protects NaK-ATPase activity against one hour of cerebral ischemia. In the brain however, the 3 isoenzymes responsible for Na,K-ATPase activity may be differentially affected by various times of ischemia. In the present study, we investigated the effect of a longer period of ischemia, and the protection provided by a pre-treatment with EGb 761 on each of the 3 cerebral NaK-ATPase isoenzymes. In control and EGb 761 pre-treated mice exposed to a 6 hr unilateral occlusion of the middle cerebral artery, Na,K-ATPase activity was decreased by 60% and lipid peroxidation was increased by 40% in the ipsilateral (ischemic) cortex compared to the contralateral one. In parallel, membrane integrity was altered. The alteration of NaK-ATPase activity, as a whole, resulted from a decrease in the activity of the 3 isoenzymes. The two isoenzymes of high ouabain affinity however, had their affinities decreased while the sensitivity of the lowest affinity isoenzyme was increased. Pre-treatment with EGb 761 abolished the differences observed between ipsi- and contralateral cortex, with the exception of the change in ouabain affinity of the low affinity isoenzyme. Ischemia also induced changes in Na,K-ATPase isoenzyme ouabain affinities in the contralateral cortex that where not prevented by EGb 761.

Ginkgo biloba extract EGb 761 protects against mitochondrial aging in the brain and in the liver.

According to the free radical theory of aging, oxygen-derived free radicals causes the age-associated impairment at the cellular and tissue levels. The mitochondrial theory of aging points to mitochondria, and specially mitochondrial DNA, as the major targets of free radical attack upon aging. Thus, oxidative damage to mtDNA accumulate with age in human and rodent tissues and also is inversely related to maximum life span of mammals. Mitochondrial deficits, such as a decrease in mitochondrial membrane potential, occur upon aging due to oxidative damage. The age-related mitochondrial oxidative stress may be prevented by late onset administration of certain antioxidants, such as Ginkgo biloba extract EGb 761. These antioxidants may also delay the physiological impairment associated with aging.