title: Acquired cadmium resistance in metallothionein-I/II(-/-) knockout cells: role of the T-type calcium channel Cacnalpha1G in cadmium uptake.
abstract: Metallothioneins (MTs) are cytoplasmic proteins that sequester certain divalent cations and are considered a primary cellular defense against the toxic transition metal cadmium (Cd(2+)). MT-I/II(-/-) knockout [MT(-/-)] cells are available and serve as an excellent tool to study non-MT-related mechanisms in metal tolerance. In the current study, Cd(2+)-resistant MT(-/-) (CdR) and CdR revertant (CdR-rev) cell lines were developed and characterized to investigate non-MT-mediated cellular protection mechanisms. Resistance to Cd(2+) was approximately 70-fold higher in CdR than the parental MT(-/-) cell line (IC(50) = 20 versus 0.3 microM, respectively) and was stable in the absence of Cd(2+) for 35 days. Accumulation of Cd(2+) by the CdR cell line was reduced by approximately 95% compared with parental cells, primarily because of a decreased Cd(2+) uptake. Cd(2+) uptake by the MT(-/-) parental cell line was independent of sodium, energy, and electrogenic potential. Uptake was saturable (K(m) = 65 nM; V(max) = 4.9 pmol/mg/min) and pH-dependent (maximal at pH 6.5-7). Potent inhibitors of Cd(2+) uptake included Zn(2+) (IC(50) = 7 microM), Mn(2+) (IC(50) = 0.4 microM), and the T-type Ca(2+) channel antagonist mibefradil (IC(50) = 5 microM), whereas other metals (including Fe(2+)) and L-type Ca(2+) channel antagonists had little effect. Immunoblot and real-time reverse transcription-polymerase chain reaction analysis indicated that the Cacnalpha(1G) T-type Ca(2+) channel was expressed at a reduced level in CdR compared with the parental MT(-/-) cell line, suggesting it is important for Cd(2+) uptake. The CdR1-rev cell line was found to have a Cd(2+) uptake and sensitivity level in between that of the CdR1 and MT(-/-) cell lines. Consistent with this was an intermediate expression of Cacnalpha(1G) in the CdR-rev cell line. These data suggest that decreased expression of Cacnalpha(1G) protects cells from Cd(2+) exposure by limiting Cd(2+) uptake.
authors.affiliation: Inorganic Carcinogenesis Section, Laboratory of Comparative Carcinogenesis, National Cancer Institute and National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA.
authors.forename: Elaine M
authors.initials: EM
authors.lastname: Leslie
authors.forename: Jie
authors.initials: J
authors.lastname: Liu
authors.forename: Curtis D
authors.initials: CD
authors.lastname: Klaassen
authors.forename: Michael P
authors.initials: MP
authors.lastname: Waalkes
mesh_terms: D000818:Animals; D051398:Aquaporin 1 / Q000235:genetics / Q000378:metabolism; D002104:Cadmium / Q000494:pharmacology / Q000633:toxicity*; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D002121:Calcium Channel Blockers / Q000494:pharmacology; D020747:Calcium Channels, T-Type / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D027682:Cation Transport Proteins / Q000235:genetics / Q000378:metabolism; D027963:Chloride-Bicarbonate Antiporters / Q000235:genetics / Q000378:metabolism; D004351:Drug Resistance / Q000235:genetics / Q000502:physiology*; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D017136:Ion Transport / Q000187:drug effects / Q000235:genetics; D033862:Iron-Binding Proteins / Q000235:genetics / Q000378:metabolism; D008668:Metallothionein / Q000235:genetics; D008670:Metals / Q000494:pharmacology; D051379:Mice; D018345:Mice, Knockout; D012964:Sodium / Q000378:metabolism / Q000494:pharmacology; D000099236:Solute Carrier Family 11, Member 2
chemical_list: D051398:Aquaporin 1; D002104:Cadmium; D002118:Calcium; D002121:Calcium Channel Blockers; D020747:Calcium Channels, T-Type; D027682:Cation Transport Proteins; D027963:Chloride-Bicarbonate Antiporters; D033862:Iron-Binding Proteins; D008668:Metallothionein; D008670:Metals; D012964:Sodium; D000099236:Solute Carrier Family 11, Member 2; C498197:Cacna1g protein, mouse; C499256:Slc39a8 protein, mouse; C480025:Slc4a9 protein, mouse