Calpain II in two in vivo models of sugar cataract

doi:10.1016/0014-4835(90)90151-J

Experimental Eye Research

Volume 51, Issue 4, October 1990, Pages 393-401

https://doi.org/10.1016/0014-4835(90)90151-J Get rights and content

Abstract

Cataracts were produced in rat lenses by either feeding a diet containing 50% galactose or by inducing diabetic condition by intravenous injection of streptozotocin. Proteolysis of crystallins, protease activity of calpain II enzyme (EC 3.4.22.17), and presence of calpain molecule (antigen) were determined at four cataract stages—I, cortical vacuoles, II, vacuoles plus hazy cortex, III, nuclear cataract, and IV, mature cataracts. Calpain activity was normal or moderately elevated at early stages of galactose and diabetic cataracts. Later stages III and IV showed proteolysis of lens crystallins, increased proportion of insoluble proteins, loss of calpain enzyme activity and calpain molecule from the soluble fraction, and reduced amounts of calpain associated with insoluble pellet. In galactose cataract, the largest increase in lens calcium were found when proteolysis was present. These results provide evidence for calpain-induced proteolysis of lens crystallins in two in vivo models of sugar cataracts in rodents.

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Quercetin inhibited epithelial mesenchymal transition in diabetic rats, high-glucose-cultured lens, and SRA01/04 cells through transforming growth factor-β2/phosphoinositide 3-kinase/Akt pathway
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At least 5 min later, the rats were anaesthetised by intraperitoneally injecting 10% chloral hydrate at a dose of 0.35 mL/100 g body weight. The cataract was scored on a scale of I–V according to the description of Azuma et al. (1990). Grade I was a normal clear lens, grade II showed subcapsular opacity, grade III exhibited nuclear cataract, grade IV showed a strong nuclear cataract, and grade V exhibited dense opacity involving the entire lens.
Diabetic cataract (DC), an identified life-threatening secondary complication of diabetes mellitus, has proven to be a dilemma because of its multifactorial caused and progression. An increasing number of studies have shown that in addition to the maillard reaction, enhanced polyol pathway, and oxidative insults, epithelial mesenchymal transition (EMT) is related to the prevalence of DC. Quercetin, a classic flavonoid with multiple pharmacological effects has been reported to possess therapeutic efficacy in the management and treatment of this disease. However, the mechanism underlying its therapeutic efficacy in EMT of lens epithelial cells (SRA01/04) and contribution to resolving DC remains a mystery. Therefore, in this study, we investigated the effects of quercetin on EMT of SRA01/04 and high-glucose (HG)-induced lens opacity accompanied by lens fibrosis induced by type-1 diabetes. Furthermore, we sought to clarify the specific mechanisms underlying these effects. At week 14 after streptozotocin (STZ) intraperitoneal administration, diabetic rats showed lens opacity accompanied with diminished antioxidant function, enhanced polyol pathway activity, and non-enzymatic glycation. Western blotting confirmed EMT in rat SRA01/04 cells with significantly increased α-smooth muscle actin (α-SMA) and decreased E-cadherin expressions. Treatment of the lens with quercetin ameliorated the oxidative stress, inhibited aldose reductase (AR) activation, reduced advanced glycation end product (AGE) production, and finally suppressed EMT in the early stages. Our in vitro results showed that high-glucose activated the transforming growth factor-β2/phosphoinositide 3-kinase/protein kinase B (TGF-β2/PI3K/Akt) signalling and EMT in SRA01/04 cells. Further, induced oxidative stress, activation of aldose reductase, and accumulation of advanced glycation end products were also involved in this process. Quercetin was potent enough to effectively ameliorate the high glucose (HG)-induced EMT of SRA01/04 cells by inhibiting the activation of TGF-β2/PI3K/Akt, enhancing the antioxidant capacity, inhibiting AR activity, and reducing AGE production. From the whole animal to tissues, and finally the cellular level, our results provide considerable evidence of the therapeutic potential of quercetin for DC. This might be due to its inhibition of EMT mediated through inhibition of the TGF-β/PI3K/Akt pathway.
Lens crystallin modifications and cataract in transgenic mice overexpressing acylpeptide hydrolase
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A link between cataracts and increased proteolytic activity or crystallin fragmentation has emerged from many studies of transgenic mice (26–28). Proteolysis of crystallins is known to occur in nonhuman lenses during aging (29–32) and in animal models of cataract (33–36). Extensive studies of calpain, including Lp82 (37–39), and of transgenic mice expressing the HIV protease (40) have confirmed that proteolysis of crystallins is one of the factors in cataract formation.
The accumulation of crystallin fragments in vivo and their subsequent interaction with crystallins are responsible, in part, for protein aggregation in cataracts. Transgenic mice overexpressing acylpeptide hydrolase (APH) specifically in the lens were prepared to test the role of protease in the generation and accumulation of peptides. Cataract development was seen at various postnatal days in the majority of mice expressing active APH (wt-APH). Cataract onset and severity of the cataracts correlated with the APH protein levels. Lens opacity occurred when APH protein levels were >2.6% of the total lens protein and the specific activity, assayed using Ac-Ala-p-nitroanilide substrate, was >1 unit. Transgenic mice carrying inactive APH (mt-APH) did not develop cataract. Cataract development also correlated with N-terminal cleavage of the APH to generate a 57-kDa protein, along with an increased accumulation of low molecular weight (LMW) peptides, similar to those found in aging human and cataract lenses. Nontransgenic mouse lens proteins incubated with purified wt-APH in vitro resulted in a >20% increase in LMW peptides. Crystallin modifications and cleavage were quite dramatic in transgenic mouse lenses with mature cataract. Affected lenses showed capsule rupture at the posterior pole, with expulsion of the lens nucleus and degenerating fiber cells. Our study suggests that the cleaved APH fragment might exert catalytic activity against crystallins, resulting in the accumulation of distinct LMW peptides that promote protein aggregation in lenses expressing wt-APH. The APH transgenic model we developed will enable in vivo testing of the roles of crystallin fragments in protein aggregation.
Magnesium deficiency: Does it have a role to play in cataractogenesis?
2012, Experimental Eye Research
Citation Excerpt :
The increased cellullar calcium thereafter causes activation of the enzyme calpain, which is a calcium-dependent intracellular cystein protease. Several isoforms of calpain are present in the lens, however calpain II appears to be the major form involved in cataractogenesis (Hightower et al., 1987; Azuma et al., 1990; Baruch et al., 2001). Activated calpain II causes proteolysis of the lens proteins, noticeably crystalline.
Magnesium is one of the most important regulatory cation involved in several biological processes. It is important for maintaining the structural and functional integrity of vital ocular tissues such as lens. Presence of high magnesium content especially in the peripheral part of lens as compared to aqueous and vitreous humor has been observed. Magnesium plays significant role as a cofactor for more than 350 enzymes in the body especially those utilizing ATP. Membrane associated ATPase functions that are crucial in regulating the intracellular ionic environment, are magnesium-dependent. Moreover, the enzymes involved in ATP production and hydrolysis are also magnesium-dependent. Magnesium deficiency by interfering with ATPase functions causes increased intracellular calcium and sodium and decreases intracellular potassium concentration. Furthermore, magnesium deficiency is associated with increased oxidative stress secondary to increased expression of inducible nitric oxide synthase and increased production of nitric oxide. Thus the alterations in lenticular redox status and ionic imbalances form the basis of the association of magnesium deficiency with cataract. In this paper we review the mechanisms involved in magnesium homeostasis and the role of magnesium deficiency in the pathogenesis of cataract.
Effect of nifedipine on severe experimental cataract in diabetic rats
2008, Journal of Pharmacological Sciences
We examined the effects of Ca²⁺-channel blockers on sugar cataract formation in streptozotocin (65 mg/kg, i.v.)-induced diabetic rats that were given 5% D-glucose as drinking water. The diabetic rats were treated with an L-type Ca²⁺-channel blocker, nifedipine or verapamil, for 9 weeks from the 3rd day of streptozotocin injection. Using the full lens images of the horizontal plane captured with the new digital camera system that we developed recently, the cataract formation was quantitatively assessed in parallel with the conventional scaling method. In the animal model of diabetes mellitus, the cataracts at the peripheral region of the lens were detected 2 weeks after induction of hyperglycemia and progressed depending on the length of the diabetic period. The majority of them developed mature cataracts after 9 weeks of hyperglycemia. Nifedipine slowed the progression rate of diabetic cataracts without affecting the period of time required for the onset of this disease, whereas verapamil had no significant inhibitory effect on the diabetic cataract. These findings suggest that nifedipine may be considered as a candidate drug to suppress the progression of diabetic cataracts.
The calpains in aging and aging-related diseases
2003, Ageing Research Reviews
Calpains are a family of calcium-dependent cysteine proteases under complex cellular regulation. By making selective limited proteolytic cleavages, they modulate the activity of enzymes, including key signaling molecules, and induce specific cytoskeletal rearrangements, accounting for their roles in cell motility, signal transduction, vesicular trafficking and structural stabilization. Calpain activation has been implicated in various aging phenomena and diseases of late life, including cataract formation, erythrocyte senescence, diabetes mellitus type 2, hypertension, arthritis, and neurodegenerative disorders. The early and pervasive involvement of calpains in Alzheimer’s disease potentially influences the development of β-amyloid and tau disturbances and their consequences for neurodegeneration and neuronal cell loss.
The relationship between osmotic stress and calcium elevation: In vitro and in vivo rat lens models
1998, Experimental Eye Research
Bothin vivoandin vitromodels were employed in the present study to assess the relative contribution of osmotic stress and increasing calcium levels to the development of sugar cataracts. In galactose cataract obtained from galactosemic weanling rats, the concentration of total calcium increased by nearly 10% at the first sign of visible opacification observed on the fourth day post-galactose feeding. After 7 days of galactose feeding, calcium levels continued to rise, to 0.8 mm. During the first 10 days, loss of lens transparency and calcium elevation was gradual and steady, with precipitous changes occurring on days 11 and 12. In groups of rats where galactose feeding was stopped after 7 days, cataract reversal was followed during the next 5 weeks. During the initial first week of recovery, calcium influx and elevation in the lens continued but began to decline steadily thereafter. After 3 weeks of recovery, lens transparency had returned to almost normal. Calcium levels continued to decline and reached normal levels between day 34 and 42, nearly 4 weeks after removal of the galactose diet.
The relationship between osmotic stress and calcium elevation was investigated more directly by culturing normal rat lenses in hypoosmotic medium (280 mOsm) to create osmotic gradients similar to that in galactosemic lenses. The results showed that during the first day of culture (12 hr), osmotically stressed lenses gained 3 mg of water, became opaque and gained excess calcium (7 mmcompared to 0.7 mm). Microscopic vacuoles appeared to accompany the process of opacification and contributed to increased light scattering and the loss of lens transparency.
Additional experiments were designed to further distinguish between the effects of osmotic stress and calcium elevation on the opacification process. Thus, lenses were incubated in control and high-calcium medium (20 mm) at 300 mOsm. Within 12 hr of incubation, calcium elevation progressed to 1.37 mm, nearly doubling the normal value. Although opacification was observed in these lenses, no sign of vacuoles was evident. Collectively, the findings from this study support the premise that an early influx of calcium is brought about by osmotic stress and is responsible for the observed loss in transparency in osmotic (sugar) cataract.

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Calpain II in two in vivo models of sugar cataract

Abstract

Exp. Eye Res.

Curr. Eye Res.

Limited proteolysis of MP26 in lens fiber membranes of galactose-induced cataract in the rat

Curr. Eye Res.

Quantitative microlocalization of diffusible ions in normal and galactose cataractous rat lens by secondary ion mass spectrometry

J. Microsc.

Origin if urea-insoluble protein in selenite cataract. Role of beta-crystallin proteolysis and calpain II

Invest. Ophthalmol. Vis. Sci.

Calcium-activated proteolysis in lens nucleus during selenite cataractogenesis

Invest. Ophthalmol. Vis. Sci.

Role of proteolysis in lens: A review

Lens Eye Toxicol. Res.

Calpain II in human lens

Invest. Ophthalmol. Vis. Sci.

Standard atomic absorption conditions. Ca determination by flame atomization

Regional distribution of calcium in alloxan diabetic rabbit lens

Curr. Eye Res.