Translocon closure to Ca2+ leak in proliferating vascular smooth muscle cells

Abstract

Vascular smooth muscle cells have a proliferative phenotype that is important in vascular development, adaptation, and disease. Intracellular calcium handling is thought to play pivotal roles in determining the properties of these cells, and thus previously unrecognized mechanisms for transmembrane calcium movement are of potential interest. An unsolved question is the mechanism of constitutive (passive) calcium leak from the intracellular stores. Studies of other cell types have suggested that the translocon is a calcium leak pathway. Here we investigated the contribution of the translocon in proliferating vascular smooth muscle cells. Calcium leak into the cytoplasm was measured using fura-2, and protein synthesis was measured using radioactive methionine. Puromycin, emetine, and anisomycin are chemicals that inhibit protein synthesis, acting via the translocon; all three agents strongly inhibited protein synthesis in the smooth muscle cells within 1 h. Puromycin, which opens the translocon, evoked a transient increase in cytoplasmic calcium that was similar in amplitude to the calcium rise evoked by thapsigargin. The puromycin effect was abolished by thapsigargin. The treatment of cells for 1 h with emetine or anisomycin, which close the translocon, inhibited the calcium release evoked by puromycin but not the calcium release evoked by extracellular ATP, endothelin-1, or the calcium ionophore ionomycin. Thapsigargin-evoked calcium rises were slightly suppressed by emetine but unaffected by puromycin or anisomycin. The data suggest that the translocon has the capacity to act as a calcium leak pathway in the ribosomal endoplasmic reticulum but that it is normally closed and lacks relevance to physiological calcium leak mechanisms.

Fig. 1.

Chemical inhibition of protein synthesis. Radioactive methionine incorporation in vascular smooth muscle cells (VSMCs) treated for 1 h with vehicle control, puromycin (200 μM), anisomycin (200 μM), or emetine (40 μM) (n = 3 independent experiments; N = 8 wells; P < 0.05 for each inhibitor relative to control). cpm, Counts per minute.

Fig. 2.

Puromycin-evoked Ca²⁺ release. All graphs show measurement of intracellular Ca²⁺ (Ca ) from VSMCs in the absence of extracellular Ca²⁺ (0 Ca²⁺). Data in A and B are each from single paired-independent experiments. A: effect of extracellular application of puromycin (200 μM; a) or vehicle control (b). B: effect of extracellular application of puromycin (200 μM) after pretreatment with vehicle control (a) or 1 μM thapsigargin (b) for 0.5 h at room temperature. ΔF, change in fluorescence; T, time.

Fig. 3.

Inhibition of puromycin-evoked Ca²⁺ release by translocon inhibitors. All graphs show measurement of intracellular Ca²⁺ from VSMCs in the absence of extracellular Ca²⁺ (0 Ca²⁺). Data in A and B are each from single paired-independent experiments. A: effect of puromycin (200 μM) after pretreatment with vehicle control (a) or 40 μM emetine (b) for 1 h. B: effect of puromycin (200 μM) after pretreatment with vehicle control (a) or 400 μM anisomycin (b) for 1 h.

Fig. 4.

Lack of effect of translocon inhibitors on constitutive Ca²⁺ leak. All graphs show measurement of intracellular Ca²⁺ from VSMCs in the absence of extracellular Ca²⁺ (0 Ca²⁺). Data in A and B are each from single paired-independent experiments. A: effect of thapsigargin (3 μM) after pretreatment with vehicle control (a) or 400 μM anisomycin (b) for 1 h. B: effect of ionomycin (5 μM) after pretreatment with vehicle control (a) or 40 μM emetine (b) for 1 h.

Fig. 5.

Lack of effect of translocon inhibitors on Ca²⁺ release evoked by physiological agonists. All graphs show measurement of intracellular Ca²⁺ from VSMCs in the absence of extracellular Ca²⁺ (0 Ca²⁺). Data in A and B are each from single paired-independent experiments. A: effect of endothelin-1 (100 nM) after pretreatment with vehicle control (a) or 40 μM emetine (b) for 1 h. B: effect of ATP (100 μM) after pretreatment with vehicle control (a) or 400 μM anisomycin (b) for 1 h.