A line of Chinese hamster ovary (CHO) cells called CK1.4 was produced by transfection with the gene for the bovine cardiac Na(+)-Ca2+ exchanger. CK1.4 cells stably expressed substantial exchange activity and exchanger protein as shown by immunoprecipitation. Exchange activity was quantified as 45Ca2+ influx that depended on both increasing intracellular Na+ and lowering the concentration of external Na+. Replacing external Na+ with K+ slightly increased 45Ca2+ uptake by CK1.4 cells with basal Na+ and greatly increased 45Ca2+ uptake by Na(+)-loaded cells. Neither exchange activity nor exchanger protein was detected in the nontransfected parental line. By contrast to CK1.4 cells, replacing external Na+ with K+ decreased 45Ca2+ uptake in the nontransfected cells whether or not they were Na+ loaded. Changes in cytosolic free Ca2+ determined with fura-2 were consistent with the 45Ca2+ uptake data. Analysis of poly(A)(+)-RNA by Northern blot confirmed that CK1.4 cells, but not the parental line, expressed the exchanger. Expression of the exchanger was also observed in aortic myocytes and a renal epithelial cell line (LLC-MK2) but not in other lines of renal epithelial cells (MDCK, LLC-PK1) or human dermal fibroblasts. The cardiac exchanger produced substantial 45Ca2+ efflux from CK1.4 cells in response to hormone-evoked release of stored Ca2+. CK1.4 cells are an attractive model for studies of the regulation of the cardiac exchanger because they stably express sufficient exchanger for biochemical and immunological analysis.

No abstract

Cd2+ provokes an immediate production of inositol trisphosphate and the release of Ca2+ from internal stores in human fibroblasts and some other mammalian cells. Ni2+, Co2+, Fe2+, and Mn2+ evoke the release of stored Ca2+, but are less potent than Cd2+ (apparent K0.5 = 40 nM). Zn2+ and Cu2+ competitively inhibit Ca2+ release evoked by Cd2+ without affecting Ca2+ release by hormones such as bradykinin. Zn2+ has the same apparent Ki value (80-90 nM) towards the five agonist metals, which suggests that the metals interact with the same site. Many other divalent cations neither released stored Ca2+ nor affected Cd(2+)-evoked Ca2+ release. The agonist metals appear to activate phospholipase C via a G protein rather than a tyrosine kinase. The production of reactive oxygen species is probably not involved in Ca2+ release by the metals. Cd2+ and other stimuli that raise cytosolic-free Ca2+ induce cyclic (AMP) production, apparently by activating a calmodulin-dependent adenylyl cyclase. We suggest that an orphan receptor mediates the hormonelike responses to Cd2+ and the other agonist metals. The receptor is referred to as an orphan because its physiological stimulus is unknown. Growth of the fibroblasts in high Zn2+ desensitizes them to the five agonist metals without affecting Ca2+ release by bradykinin or histamine. A several hour incubation in culture medium with normal Zn2+ fully restores responsiveness to the five active metals. Growth in high Zn2+ appears to repress the synthesis of the putative orphan receptor because inhibitors of RNA or protein synthesis, or asparagine-linked glycosylation, prevented the restoration of metal responsiveness.(ABSTRACT TRUNCATED AT 250 WORDS)

No abstract

Cd2+ provokes an immediate production of inositol trisphosphate and the release of Ca2+ from internal stores in human fibroblasts and some other mammalian cells. Ni2+, Co2+, Fe2+, and Mn2+ evoke the release of stored Ca2+, but are less potent than Cd2+ (apparent Ko05 = 40 nM). Zn2+ and Cu2+ competitively inhibit Ca2+ release evoked by Cd2, without affecting Ca2+ release by hormones such as bradykinin. Zn2+ has the same apparent Ki value (80-90 nM) towards the five agonist metals, which suggests that the metals interact with the same site. Many other divalent cations neither released stored Ca2+ nor affected Cd2+-evoked Ca2+ release. The agonist metals appear to activate phospholipase C via a G protein rather than a tyrosine kinase. The production of reactive oxygen species is probably not involved in Ca2+ release by the metals. Cd2+ and other stimuli that raise cytosolic-free Ca2+ induce cyclic (AMP) production, apparently by activating a calmodulin-dependent adenylyl cyclase. We suggest that an orphan receptor mediates the hormonelike responses to Cd2+ and the other agonist metals. The receptor is referred to as an orphan because its physiological stimulus is unknown. Growth of the fibroblasts in high Zn2+ desensitizes them to the five agonist metals without affecting Ca2+ release by bradykinin or histamine. A several hour incubation in culture medium with normal Zn2+ fully restores responsiveness to the five active metals. Growth in high Zn2+ appears to repress the synthesis of the putative orphan receptor because inhibitors of RNA or protein synthesis, or asparagine-linked glycosylation, prevented the restoration of metal responsiveness. Experiments with lectins and neuraminidase support the view that a cell surface sialoprotein mediates Cd2+ responsiveness. Cd2+ evokes rapid changes in 132P] incorporation by certain proteins, as would be expected for the activation of a phospholipase C-coupled receptor. Cd2+ and the other metals that trigger hormonelike messenger production, also induce protooncogenes. These observations have revealed a new target for certain metals which is extraordinary with respect to metal potency and specificity. Additionally, the work reviewed here supports the view that certain metals can promote cell growth, which results in part from the fortuitous induction of hormonelike signals. -Environ Health Perspect 102(Suppl 3): 181-189 (1994).