title: Calcium homeostasis of isolated single cortical fibers of rat lens.
abstract: OBJECTIVE
  To investigate the calcium homeostasis in single fiber cells isolated from rat ocular lens cortex and to quantify the changes in the concentration of free intracellular calcium [Ca2+]i during the process of disintegrative globulization.
  
  
  METHODS
  Individual fiber cells from the cortex of the adult rat lens were isolated by treatment with trypsin in ion-free buffered sucrose. The isolated fiber cells were loaded with the acetoxymethyl esters of Fluo-3 or Calcium Green-2, or with Fluo-3 and Fura Red, and changes in [Ca2+]i of single cortical fibers were measured using a microfluorometer. The time course of increase of [Ca2+]i in fiber cells exposed to Ringer's solution was measured, and the effects on the increase of [Ca2+]i of calcium channel blocker, verapamil, Na-Ca exchange inhibitors Ni2+ and Zn2+, and protease inhibitor, leupeptin, Na+-free and K+-free media and Ca2+-containing isotonic sucrose solution, were investigated.
  
  
  RESULTS
  In Hepes sucrose solution (containing approximately 1.5 microM Ca2+), the isolated fiber cells maintained stable values of [Ca2+]i at 99.6+/-10 nM (n = 32). Exposure of the isolated fibers to Ringer's solution (containing 2 mM Ca2+) led to a monoexponential increase of [Ca2+]i at a rate of 0.12 min(-1). This increase in [Ca2+]i was accompanied by disintegration of the isolated fibers into discrete but resealed globules. Changes in [Ca2+]i, monitored by using a two-dye ratiometric method using Fura Red and fluo-3, showed a progressive increase in [Ca2+]i in fibers exposed to Ringer's solution, preceding globulization. The [Ca2+]i in the globules in Ringer's solution, determined using Calcium Green-2, was 3.6+/-0.7 microM (n = 23). Compared with that in fibers in Ringer's solution, the rate of increase of [Ca2+]i in fibers was much slower in the presence of 50 microM verapamil (0.047 min[-1]), in Na+-free (0.086 min[-1]) and in K+-free (0.062 min[-1]) Ringer's solution, or when the fibers were suspended in Hepes-sucrose solution, containing 2 mM Ca2+ (0.046 min[-1]). After 30 minutes, the [Ca2+]i of fiber cells exposed to Ringer's solution, containing 2 mM Ni2+ (574.7+/-29 nM; n = 7) or Zn2+ (402.6+/-77 nM; n = 7) was significantly lower (P < 0.001) compared with that in fiber cells exposed to Ringer's solution alone (1995+/-461 nM, n = 10). In Ringer's solution, leupeptin delayed globulization without significantly affecting the increase in [Ca2+]i. The [Ca2+]i of fiber cells isolated from outer and inner cortex and suspended in Hepes-sucrose was comparable; however, after 15 minutes of exposure to Ringer's solution, [Ca2+]i in fibers from the outer cortex was approximately three times higher than [Ca2+]i in those from the inner cortex.
  
  
  CONCLUSIONS
  Exposure to high (millimolar) concentrations of calcium in the external medium leads to an increase in [Ca2+]i of isolated individual fiber cells, which precedes disintegrative globulization. The protective effects of Na+-free and K+-free solutions on globulization appear to be due to a lower rate of increase of [Ca2+]i. Part of the calcium influx may be mediated by L-type calcium channels and by Na-Ca exchange, operating in reverse. Proteolytic inhibitors do not affect the increase in [Ca2+]i but delay globulization by inhibiting calcium-mediated proteolysis. The isolated fiber cells and the disintegrated globules maintain a 100- to 300-fold gradient of calcium across their plasma membranes.
authors.affiliation: Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston 77555-0647, USA.
authors.forename: S K
authors.initials: SK
authors.lastname: Srivastava
authors.forename: L F
authors.initials: LF
authors.lastname: Wang
authors.forename: N H
authors.initials: NH
authors.lastname: Ansari
authors.forename: A
authors.initials: A
authors.lastname: Bhatnagar
mesh_terms: D000814:Aniline Compounds / Q000378:metabolism; D000818:Animals; D001572:Benzofurans / Q000378:metabolism; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002121:Calcium Channel Blockers / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000378:metabolism; D006706:Homeostasis; D007093:Imidazoles / Q000378:metabolism; D007904:Lens Cortex, Crystalline / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D007976:Leupeptins / Q000494:pharmacology; D009532:Nickel / Q000494:pharmacology; D009930:Organic Chemicals; D051381:Rats; D017207:Rats, Sprague-Dawley; D013050:Spectrometry, Fluorescence; D014700:Verapamil / Q000494:pharmacology; D014966:Xanthenes / Q000378:metabolism; D015032:Zinc / Q000494:pharmacology
chemical_list: D000814:Aniline Compounds; D001572:Benzofurans; D002121:Calcium Channel Blockers; D005456:Fluorescent Dyes; D007093:Imidazoles; D007976:Leupeptins; D009930:Organic Chemicals; D014966:Xanthenes; C071336:calcium green; C082877:fura red; C059715:Fluo-3; D009532:Nickel; D014700:Verapamil; D015032:Zinc; C032854:leupeptin; D002118:Calcium