Search

To examine the role of calcium-dependent and -independent proteolytic activity in the globulization of isolated fiber cells and glucose-induced lens opacification.Fiber cells from rat lens cortex were isolated, and the [Ca(2+)](i) and protease activity in the isolated fibers were determined by using a calcium binding dye and the protease substrate t-butoxycarbonyl-Leu-Met-7-amino-4-chloromethylcoumarin (BOC-Leu-Met-CMAC). The activity of calpain in the lens cortex homogenate was determined with fluorescein-casein in the presence of Ca(2+) and that of fiber cell globulizing aminopeptidase (FCGAP) with BOC-Leu-Met-CMAC and reduced glutathione (GSH) in the absence of Ca(2+). The lens proteases-calpain and the novel aminopeptidase FCGAP were partially purified by diethylaminoethyl (DEAE) gel column chromatography. Single fiber cells were isolated from rat lens, plated on coverslips, and placed in a temperature-controlled chamber. Their globulization time was determined by the appearance of light-scattering globules in the absence and the presence of protease inhibitors including the aminopeptidase inhibitor bestatin. To investigate the effect of the protease inhibitors E-64 and bestatin on the prevention of hyperglycemic cataract, the rat lenses were cultured in medium 199 in the presence of 5.5 and 50 mM glucose and in the absence and the presence of protease inhibitors. Changes in light transmission by the lenses were determined by digital image analysis.Normal levels of lens fiber cell [Ca(2+)](i), determined by using a cell-permeable dye were approximately 100 nM, and the protease activity determined with BOC-Leu-Met-CMAC was maximum at [Ca(2+)](i) of approximately 500 nM. A large fraction of the FCGAP that cleaves BOC-Leu-Met-CMAC was separated from calpain, which cleaves fluorescein-casein, by diethylaminoethyl (DEAE) gel column chromatography. The FCGAP did not bind to the column, whereas calpain bound to the column and was eluted by approximately 180 mM NaCl. Unlike calpain, the FCGAP did not require calcium for activation and did not cleave fluorescein-casein. However, the Ca(2+)-dependent calpain activated FCGAP, indicating that the latter may exist in pro-protease form. The FCGAP was selectively inhibited by the specific aminopeptidase inhibitor bestatin, indicating that FCGAP could be an aminopeptidase. However, the FCGAP was found to be immunologically distinct from leucine aminopeptidase and calpain. Perfusion of the isolated rat lens fiber cells with Ringer's solution led to their globulization in 30 +/- 3 minutes. Addition of 0.5 mM of the protease inhibitors E-64 and leupeptin increased the globulization time to 60 and 100 minutes, respectively, whereas no globulization of the fiber cells was observed for 4 hours in the presence of 0.05 mM bestatin. In rat lens cultured in medium containing 50 mM glucose, both E-64 and bestatin (0.05 mM each) significantly reduced the extent of opacification, indicating that an aminopeptidase, downstream to a Ca(2+)-dependent protease, may be involved in mediating cataractogenic changes.In addition to calpain, a Ca(2+)-independent novel protease, FCGAP, a novel aminopeptidase, represents a significant fraction of the total proteolytic activity in the lens. Inhibition of FCGAP by bestatin attenuates Ca(2+)-induced globulization of the isolated fiber cells in vitro and hyperglycemia-induced opacification of cultured rat lens.

The role of the Na+/Ca2+exchanger in intracellular Ca2+regulation was investigated in freshly dissociated catfish retinal horizontal cells (HC). Ca2+-permeable glutamate receptors and L-type Ca2+ channels as well as inositol 1,4,5-trisphosphate-sensitive and caffeine-sensitive intracellular Ca2+ stores regulate intracellular Ca2+ in these cells. We used the Ca2+-sensitive dye fluo 3 to measure changes in intracellular Ca2+ concentration ([Ca2+]i) under conditions in which Na+/Ca2+exchange was altered. In addition, the role of the Na+/Ca2+exchanger in the refilling of the caffeine-sensitive Ca2+ store following caffeine-stimulated Ca2+ release was assessed. Brief applications of caffeine (1–10 s) produced rapid and transient changes in [Ca2+]i. Repeated applications of caffeine produced smaller Ca2+ transients until no further Ca2+ was released. Store refilling occurred within 1–2 min and required extracellular Ca2+. Ouabain-induced increases in intracellular Na+ concentration ([Na+]i) increased both basal free [Ca2+]iand caffeine-stimulated Ca2+release. Reduction of external Na+concentration ([Na+]o) further and reversibly increased [Ca2+]iin ouabain-treated HC. This effect was not abolished by the Ca2+ channel blocker nifedipine, suggesting that increases in [Na+]ipromote net extracellular Ca2+influx through a Na+/Ca2+exchanger. Moreover, when [Na+]owas replaced by Li+, caffeine did not stimulate release of Ca2+ from the caffeine-sensitive store after Ca2+ depletion. The Na+/Ca2+exchanger inhibitor 2′,4′-dimethylbenzamil significantly reduced the caffeine-evoked Ca2+response 1 and 2 min after store depletion.

Retinal photoreceptors transmit light stimuli via electrical signals generated by a change in the photocurrent. This voltage-sensitive current is gated by cGMP and allows the entry of both Na+ and Ca2+ into the cell. cGMP levels are controlled by the opposing actions of guanylate cyclase and phosphodiesterase, which are in turn controlled by buffered Ca2+ and photon absorption, respectively. We have constructed a model that reproduces photocurrent responses to light stimuli. In addition to the processes noted above. the actions of the Na+/K+ and Na+/Ca2+-K+ exchangers present in the photoreceptor inner and outer segments are considered. Previously published models have not incorporated all of these processes in a biologically relevant manner. Model light responses are compared to those described in the literature, and the role of Ca2+ buffering in light adaptation is explored.

Pancreatico-pleural fistula is a rare condition in which pancreatic enzymes drain directly in to the pleural cavity, most commonly from an enlarging pseudocyst. We review the literature on the causes, investigations and treatment of pancreatico-pleural fistulae and compare this with our own experience of the case of a 41 year old man with a left sided pancreatico-pleural fistula associated with pancreatic duct obstruction. The fistula could not be demonstrated by USS, CT or ERCP, and after these investigations the patient was managed conservatively. However, deterioration in the patients' condition led to an urgent but not emergency laparotomy and operative pancreatogram. This demonstrated the distally obstructed pancreatic duct, with associated pleural fistula for which aggressive surgical intervention was indicated. The patient subsequently completely recovered.

A controlled trial has been conducted to determine the safety of early drain removal after axillary clearance as part of conservation treatment in early breast cancer. A total of 84 patients was entered into the study of whom 41 had the drain removed after 5 days, irrespective of the volume of fluid draining, and 43 had drains removed when fluid was less than 20 ml per day. Of the standard drainage group, 28% required percutaneous aspiration of lymph because of subsequent accumulation, compared with 49% of the short-term drainage group. Early drain removal was not associated with any increase in wound complications nor in cosmetic outcome, but did enable earlier discharge of patients from hospital.

Increases in the extracellular hydrogen ion concentration ([H + ] o ) but not the intracellular concentration ([H + ] i ) antagonized the inward going membrane currents recorded from isolated cone horizontal cells during application of quisqualate, α-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid, and kainate. The pK determined from a titration curve was 6.5 with a slope greater than 1, indicating protonation of several histidines. The reduction in membrane current was voltage-independent. The affinity of the agonist for the receptor, the single-channel conductance, and the open time were unaffected by [H + ] o· [H + ] o antagonism was not the result of charge neutralization such as screening surface charge. Diethylpyrocarbonate, a histidine-modifying reagent, reduced the agonist-induced current, but disulfide- and sulfhydryl-modifying reagents were ineffective. These results suggest that histidine groups on the external face of the channel protein provide a functional site regulating channel gating.