Ryan D. Walkup

Amelioration of retinal degeneration and proteolysis in acute ocular hypertensive rats by calpain inhibitor ((1S)-1-((((1S)-1-benzyl-3-cyclopropylamino-2,3-di-oxopropyl)amino)carbonyl)-3-methylbutyl)carbamic acid 5-methoxy-3-oxapentyl ester.

BACKGROUND Our recent study suggested involvement of calpain-induced proteolysis in retinal degeneration and dysfunction in acute ocular hypertensive rats. The purpose of the present study was to determine if an orally available form of calpain inhibitor, ((1S)-1-((((1S)-1-benzyl-3-cyclopropylamino-2,3-di-oxopropyl)amino)carbonyl)-3-methylbutyl)carbamic acid 5-methoxy-3-oxapentyl ester (SNJ-1945), ameliorated retinal degeneration induced by acute hypertension in rats. To help extrapolate the effect of SNJ-1945 from the rat model to the human glaucomatous patient, in vitro inhibition of calpain-induced proteolysis by SNJ-1945 in monkey and human retinal proteins was compared with proteolysis in rat proteins. METHODS Intraocular pressure (IOP) in rats was elevated to 110 mm Hg for 50 min. SNJ-1945 was administrated i.p. or orally before ocular hypertension. Retinal degeneration was evaluated by hematoxylin and eosin (H&E) staining and cell counting. Transcripts for calpains and calpastatin in rat, monkey, and human retinas were measured by quantitative RT-PCR. Calpain activities were determined by casein zymography. Soluble retinal proteins from rat, monkey, and humans were incubated with calcium to activate calpains, with or without SNJ-1945. Proteolysis of calpain substrate alpha-spectrin was analyzed by immunoblotting. RESULTS Elevated IOP caused retinal degeneration and proteolysis of alpha-spectrin. Both i.p. and oral administration of SNJ-1945 inhibited proteolysis of alpha-spectrin and ameliorated retinal degeneration. Transcript levels for calpain 1 and calpastatin were similar in rat, monkey, and human retinas. Calpain 2 transcript levels were higher in rats compared with monkey and human. Appreciable caseinolytic activities due to calpains were observed in monkey and human retinas. Incubation of retinal soluble proteins with calcium led to proteolysis of alpha-spectrin due to calpains in rat, monkey, and human samples. SNJ-1945 similarly inhibited proteolysis in all species. CONCLUSION Our results suggested that orally available calpain inhibitor SNJ-1945 might be a possible candidate drug for testing in preventing progression of glaucomatous retinal degeneration.

Mechanism for carbachol-induced secretion of lacritin in cultured monkey lacrimal acinar cells.

PURPOSE Lacritin protein is highly expressed in the lacrimal gland, secreted into tear fluid, and detected only in primates. The mechanism for lacritin secretion has not been fully investigated, because a system for culturing primate lacrimal acinar cells had not been established. The purposes of the present study were (1) to develop a procedure to culture lacrimal acinar cells from monkey and (2) to determine the mechanism for the secretion of lacritin in the culture system. METHODS Acinar cells from monkey lacrimal gland were cultured and characterized. Lacritin and other proteins were detected by immunohistochemistry, immunocytochemistry, and immunoblot analysis. Secreted proteins were also detected in the medium from stimulated acinar cells. mRNAs were determined by microarray and qPCR. Intracellular calcium levels were measured by calcium-4 assay. RESULTS Acinar cells cultured for 1 day contained adequate amounts of lacritin, lactoferrin, and lipocalin for use in lacritin secretion studies. The cholinergic agonist carbachol (Cch) stimulated the secretion of lacritin and increased intracellular Ca2+. Cch-induced lacritin secretion was inhibited by the store-operated calcium (SOC) channel inhibitor YM58483 and the PKC inhibitors GF109203 and Ro-32-0432. Cch-induced lacritin secretion was not inhibited by MAPKK inhibitor U0126, although p42/p44 MAPK was phosphorylated. Cch also enhanced gene transcription, which was inhibited by U0126, GF109203, and calcium chelators. CONCLUSIONS Successful culture of monkey lacrimal acinar cells showed that, among the prevalent tear proteins, the secretion of lacritin involved the PKC/Ca2+ pathway, not the p42/p44 MAPK pathway. Induction of transcription by Cch involved the independent p42/p44 MAPK and PKC pathways.

Lacritin-Induced Secretion of Tear Proteins From Cultured Monkey Lacrimal Acinar Cells

PURPOSE During inflammation of the ocular surface, increased proinflammatory cytokines depress tear protein secretion, suggesting that a decline in lacrimal cell function contributes to dry eye. Lacritin, a glycoprotein secreted from lacrimal acinar cells, may function as an autocrine factor to stimulate tear protein secretion. The purpose of the present experiment was to investigate lacritin-induced protein secretion in normal and cytokine-pretreated (inflammation model) monkey acinar cells. METHODS Acinar cells from monkey lacrimal glands were cultured with or without tumor necrosis factor alpha (TNF-α) plus interferon gamma (IFN-γ). Protein secretion was induced by lacritin or carbachol (Cch, positive control). Proteins were detected and identified by immunoblotting and immunocytochemistry. Intracellular Ca(2+) was measured with the fluorophore Calcium-4, and cell damage was determined by LDH leakage into the culture medium. RESULTS In cultured monkey acinar cells, lacritin stimulated tear protein secretion in normal cells without elevating intracellular Ca(2+). In contrast, Cch elevated intracellular Ca(2+) and release of tear proteins. This contrast suggested an alternate, calcium-independent mechanism for lacritin-induced protein secretion. TNF-α plus IFN-γ caused LDH leakage from sensitive human corneal epithelial cells, but even higher doses of TNF-α plus IFN-γ did not cause LDH leakage from monkey acinar cells, suggesting a higher tolerance against these cytokines. In cytokine-treated acinar cells, lacritin stimulated protein secretion as much as that in normal cells. In contrast, Cch-induced elevation of Ca(2+) and release of proteins were depressed by cytokines. CONCLUSIONS Lacritin might be a useful biotechnology-based treatment agent against ocular surface diseases where endogenous lacritin is inadequate.

Contribution of Calpain to Cellular Damage in Human Retinal Pigment Epithelial Cells Cultured with Zinc Chelator

Purpose: We previously showed involvement of calpains in neural retina degeneration induced by hypoxia and ischemia-reperfusion. Age-related macular degeneration (AMD) is one of the leading causes for loss of vision. AMD showed degeneration of neural retina due to dysfunction and degeneration of the retinal pigment epithelium (RPE). RPE performs critical functions in neural retina, such as phagocytosis of shed rod outer segments. The purpose of the current study was to determine the contribution of calpain-induced proteolysis to damage in cultured human RPE cells. Zinc chelator TPEN was used to induce cellular damage as zinc deficiency is a suspected risk factor for AMD. Methods: In RPE/choroid preparations from normal and AMD patients, calpain mRNAs were measured by qPCR, and calpain activity was assessed by casein zymography. Third- to fifth-passage cells from human RPE cells were cultured with TPEN. Cell damage was morphologically assessed under the phase-contrast microscope, and TUNEL staining was performed to detect apoptosis. Leakage of lactate dehydrogenese (LDH) into the medium was measured as a marker of RPE cell damage. Activation of calpains and proteolysis of the known calpain substrate α -spectrin were assessed by immunoblotting. To further confirm calpain-induced proteolysis, calpain in homogenized RPE was also activated directly by addition of calcium. Results: RPE/choroid from normal patients expressed mRNAs for calpain 1, calpain 2, and calpastatin moderately, and calpain 2 activity tended to be lower in AMD patients. TPEN caused RPE cell damage with positive TUNEL staining. TPEN also caused leakage of LDH into the medium from RPE cells, and calpain inhibitor SJA6017 inhibited the leakage. Caspase-3 inhibitors z-VAD and z-DEVD also showed inhibitory effects. Immunoblotting for calpain and α -spectrin showed activation of calpain in RPE cells cultured with TPEN. Proteolysis by activated calpain was confirmed by addition of calcium to homogenized RPE. Conclusions: These results suggested that activation of calpain contributed to cellular damage induced by TPEN in cultured human RPE cells.