title: Novel approach for generation of low calcium reagents for investigations of heavy metal effects on calcium signaling.
abstract: BACKGROUND
  Lead exposure can cause learning disabilities, memory loss and severe damage to the nervous system. However, the exact mechanism by which lead causes learning disabilities is not fully understood. The effects of lead on calcium-regulated signaling pathways are difficult to study biochemically; with the traditional method of controlling the free calcium concentration with EGTA, the exact concentrations of free lead and calcium ions in solution are interdependent and prone to error because EGTA also buffers lead.
  
  
  RESULTS
  In our approach, we first reduced the free calcium concentration in the solution using calcium-binding resins before adding lead to buffers. The solution was sequentially treated with Chelex-100 ion exchange resin, followed by immobilized BAPTA resin. The final concentration of free calcium in the solution was measured with Fluo-3 indicator. Our protocol successfully produced buffers with free calcium levels below 15 nM, which is substantially below threshold for activation of calcium-dependent enzymes in signaling pathways (which is typically a few hundred nanomolar calcium, when determined in vitro).
  
  
  CONCLUSIONS
  This method provides an improved approach to study the effect of heavy metals on calcium-stimulated signaling pathways.
authors.affiliation: Department of Pharmacology, University of Maryland School of Medicine, 655 West Baltimore Street, Baltimore, MD 21201-1559, USA.
authors.forename: Katelyn Y
authors.initials: KY
authors.lastname: Niu
authors.forename: Nathaniel C
authors.initials: NC
authors.lastname: Noyes
authors.forename: Thomas W
authors.initials: TW
authors.lastname: Abrams
mesh_terms: D000818:Animals; D002021:Buffers; D002118:Calcium / Q000737:chemistry*; D020013:Calcium Signaling / Q000187:drug effects*; D004533:Egtazic Acid / Q000737:chemistry; D007854:Lead / Q000737:chemistry / Q000633:toxicity; D008297:Male; D019216:Metals, Heavy / Q000009:adverse effects* / Q000737:chemistry*; D051381:Rats; D012996:Solutions / Q000737:chemistry
chemical_list: D002021:Buffers; D019216:Metals, Heavy; D012996:Solutions; D007854:Lead; D004533:Egtazic Acid; D002118:Calcium