Limits...
Calcium transport mechanisms of PC12 cells.

Duman JG, Chen L, Hille B - J. Gen. Physiol. (2008)

Bottom Line: Our results indicate that Ca2+ transport in undifferentiated PC12 cells is quite unlike transport in adrenal chromaffin cells, for which they often are considered models.Transport in both cell states more closely resembles that of sympathetic neurons, for which differentiated PC12 cells often are considered models.Comparison with other cell types shows that different cells emphasize different Ca2+ transport mechanisms.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics University of Washington School of Medicine, Seattle, WA 98195, USA.

ABSTRACT
Many studies of Ca2+ signaling use PC12 cells, yet the balance of Ca2+ clearance mechanisms in these cells is unknown. We used pharmacological inhibition of Ca2+ transporters to characterize Ca2+ clearance after depolarizations in both undifferentiated and nerve growth factor-differentiated PC12 cells. Sarco-endoplasmic reticulum Ca2+ ATPase (SERCA), plasma membrane Ca2+ ATPase (PMCA), and Na+/Ca2+ exchanger (NCX) account for almost all Ca2+ clearance in both cell states, with NCX and PMCA making the greatest contributions. Any contribution of mitochondrial uniporters is small. The ATP pool in differentiated cells was much more labile than that of undifferentiated cells in the presence of agents that dissipated mitochondrial proton gradients. Differentiated PC12 cells have a small component of Ca2+ clearance possessing pharmacological characteristics consistent with secretory pathway Ca2+ ATPase (SPCA), potentially residing on Golgi and/or secretory granules. Undifferentiated and differentiated cells are similar in overall Ca2+ transport and in the small transport due to SERCA, but they differ in the fraction of transport by PMCA and NCX. Transport in neurites of differentiated PC12 cells was qualitatively similar to that in the somata, except that the ER stores in neurites sometimes released Ca2+ instead of clearing it after depolarization. We formulated a mathematical model to simulate the observed Ca2+ clearance and to describe the differences between these undifferentiated and NGF-differentiated states quantitatively. The model required a value for the endogenous Ca2+ binding ratio of PC12 cell cytoplasm, which we measured to be 268 +/- 85. Our results indicate that Ca2+ transport in undifferentiated PC12 cells is quite unlike transport in adrenal chromaffin cells, for which they often are considered models. Transport in both cell states more closely resembles that of sympathetic neurons, for which differentiated PC12 cells often are considered models. Comparison with other cell types shows that different cells emphasize different Ca2+ transport mechanisms.

Show MeSH

Related in: MedlinePlus

CCCP inhibits primary active transporters in NGF-differentiated PC12 cells. Results are shown as Ca2+ transport curves (−d[Ca2+]cyt/dt vs. [Ca2+]cyt). (A) Data from control NGF-differentiated cells (from Fig. 7) are shown as black lines and circles, and data from 2 μM CCCP-treated cells (Fig. 7) are shown as a gray line. CCCP-treated cells were also concomitantly treated with 5 μM oligomycin (gray lines and triangles, n = 28). We obtained the same results when cells were treated with oligomycin for 30 s before application of CCCP as with continuing application of oligomycin (data not shown). (B) Residual Ca2+ transport in NGF-differentiated cells (gray line, from Fig. 6) and NGF-differentiated cells treated with 5 μM oligomycin (black line and triangles, n = 16). (C–E) Ca2+ transport curves for 2-blocked experiments (black lines and symbols) and estimated results of these experiments based on transporter capacities shown in Fig. 7 (gray lines and symbols). Residual Ca2+ transport (from Fig. 6) is shown as a gray line in E. In C, cells were treated with CCCP and TG (n = 15) and compared with the sum of the transport curves for NCX, PMCA, and residual Ca2+ transport. In D, cells were treated with CCCP and pH 9.0 (n = 16) and compared with the sum of the transport curves for NCX, SERCA, and residual Ca2+ transport. In E, cells were treated with CCCP and Li+ (n = 20) and compared with the sum of the transport curves for SERCA, PMCA, and residual Ca2+ transport.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2279173&req=5

fig8: CCCP inhibits primary active transporters in NGF-differentiated PC12 cells. Results are shown as Ca2+ transport curves (−d[Ca2+]cyt/dt vs. [Ca2+]cyt). (A) Data from control NGF-differentiated cells (from Fig. 7) are shown as black lines and circles, and data from 2 μM CCCP-treated cells (Fig. 7) are shown as a gray line. CCCP-treated cells were also concomitantly treated with 5 μM oligomycin (gray lines and triangles, n = 28). We obtained the same results when cells were treated with oligomycin for 30 s before application of CCCP as with continuing application of oligomycin (data not shown). (B) Residual Ca2+ transport in NGF-differentiated cells (gray line, from Fig. 6) and NGF-differentiated cells treated with 5 μM oligomycin (black line and triangles, n = 16). (C–E) Ca2+ transport curves for 2-blocked experiments (black lines and symbols) and estimated results of these experiments based on transporter capacities shown in Fig. 7 (gray lines and symbols). Residual Ca2+ transport (from Fig. 6) is shown as a gray line in E. In C, cells were treated with CCCP and TG (n = 15) and compared with the sum of the transport curves for NCX, PMCA, and residual Ca2+ transport. In D, cells were treated with CCCP and pH 9.0 (n = 16) and compared with the sum of the transport curves for NCX, SERCA, and residual Ca2+ transport. In E, cells were treated with CCCP and Li+ (n = 20) and compared with the sum of the transport curves for SERCA, PMCA, and residual Ca2+ transport.

Mentions: Why does CCCP block so much clearance activity in the 1-blocked experiments? We suggest that even a short CCCP treatment stops not only the MtU but also the two ATP-requiring transporters. CCCP collapses the mitochondrial proton gradient, stopping formation of new ATP by mitochondria and allowing cellular ATP to deplete if glycolysis cannot keep up with ATP demand. Treatment with oligomycin can be used to keep the F1F0 ATPase from breaking down ATP once the proton gradient is gone (Babcock et al., 1997). However, addition of 5 μM oligomycin, either coincident with CCCP or as a 30-s preincubation before CCCP was added, only weakly restored some missing Ca2+ transport (Fig. 8 A). Oligomycin had little effect on residual Ca2+ transport in 4-blocked experiments (Fig. 8 B). To determine which non-MtU transporters were affected by CCCP, we performed a series of 2-blocked experiments, treating the cells with both CCCP and one other inhibitor and allowing the other two mechanisms to function. We compared these results to curves calculated from the 3-blocked experiments in Fig. 7. For example, in Fig. 8 C, we compare a 2-blocked experiment in which MtU and SERCA are inhibited (black line) with the calculated sum of the capacities of NCX, PMCA, and the residual Ca2+ transport (gray line). The two curves are similar, suggesting that the fluxes allowed to operate in this experiment are not strongly affected by CCCP. In Fig. 8 D, we inhibited both MtU and PMCA (black line) and compared the transport curve to the sum of the capacities of SERCA, NCX, and the residual Ca2+ transport. Once again, the curves are quite similar with CCCP being only slightly lower. However, when we inhibited NCX in addition to CCCP treatment, we obtained the unambiguous result that the remaining transport was far below that expected for the sum of SERCA, PMCA, and the residual Ca2+ transport (Fig. 8 E). In fact, the observed transport was virtually identical to residual Ca2+ transport. These results indicate that both SERCA and PMCA are inhibited by CCCP treatment, as might be expected since both require ATP, and that when only one of these two transporters is working, a differentiated cell can supply almost enough ATP to keep up, but when both are operating it cannot.


Calcium transport mechanisms of PC12 cells.

Duman JG, Chen L, Hille B - J. Gen. Physiol. (2008)

CCCP inhibits primary active transporters in NGF-differentiated PC12 cells. Results are shown as Ca2+ transport curves (−d[Ca2+]cyt/dt vs. [Ca2+]cyt). (A) Data from control NGF-differentiated cells (from Fig. 7) are shown as black lines and circles, and data from 2 μM CCCP-treated cells (Fig. 7) are shown as a gray line. CCCP-treated cells were also concomitantly treated with 5 μM oligomycin (gray lines and triangles, n = 28). We obtained the same results when cells were treated with oligomycin for 30 s before application of CCCP as with continuing application of oligomycin (data not shown). (B) Residual Ca2+ transport in NGF-differentiated cells (gray line, from Fig. 6) and NGF-differentiated cells treated with 5 μM oligomycin (black line and triangles, n = 16). (C–E) Ca2+ transport curves for 2-blocked experiments (black lines and symbols) and estimated results of these experiments based on transporter capacities shown in Fig. 7 (gray lines and symbols). Residual Ca2+ transport (from Fig. 6) is shown as a gray line in E. In C, cells were treated with CCCP and TG (n = 15) and compared with the sum of the transport curves for NCX, PMCA, and residual Ca2+ transport. In D, cells were treated with CCCP and pH 9.0 (n = 16) and compared with the sum of the transport curves for NCX, SERCA, and residual Ca2+ transport. In E, cells were treated with CCCP and Li+ (n = 20) and compared with the sum of the transport curves for SERCA, PMCA, and residual Ca2+ transport.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2279173&req=5

fig8: CCCP inhibits primary active transporters in NGF-differentiated PC12 cells. Results are shown as Ca2+ transport curves (−d[Ca2+]cyt/dt vs. [Ca2+]cyt). (A) Data from control NGF-differentiated cells (from Fig. 7) are shown as black lines and circles, and data from 2 μM CCCP-treated cells (Fig. 7) are shown as a gray line. CCCP-treated cells were also concomitantly treated with 5 μM oligomycin (gray lines and triangles, n = 28). We obtained the same results when cells were treated with oligomycin for 30 s before application of CCCP as with continuing application of oligomycin (data not shown). (B) Residual Ca2+ transport in NGF-differentiated cells (gray line, from Fig. 6) and NGF-differentiated cells treated with 5 μM oligomycin (black line and triangles, n = 16). (C–E) Ca2+ transport curves for 2-blocked experiments (black lines and symbols) and estimated results of these experiments based on transporter capacities shown in Fig. 7 (gray lines and symbols). Residual Ca2+ transport (from Fig. 6) is shown as a gray line in E. In C, cells were treated with CCCP and TG (n = 15) and compared with the sum of the transport curves for NCX, PMCA, and residual Ca2+ transport. In D, cells were treated with CCCP and pH 9.0 (n = 16) and compared with the sum of the transport curves for NCX, SERCA, and residual Ca2+ transport. In E, cells were treated with CCCP and Li+ (n = 20) and compared with the sum of the transport curves for SERCA, PMCA, and residual Ca2+ transport.
Mentions: Why does CCCP block so much clearance activity in the 1-blocked experiments? We suggest that even a short CCCP treatment stops not only the MtU but also the two ATP-requiring transporters. CCCP collapses the mitochondrial proton gradient, stopping formation of new ATP by mitochondria and allowing cellular ATP to deplete if glycolysis cannot keep up with ATP demand. Treatment with oligomycin can be used to keep the F1F0 ATPase from breaking down ATP once the proton gradient is gone (Babcock et al., 1997). However, addition of 5 μM oligomycin, either coincident with CCCP or as a 30-s preincubation before CCCP was added, only weakly restored some missing Ca2+ transport (Fig. 8 A). Oligomycin had little effect on residual Ca2+ transport in 4-blocked experiments (Fig. 8 B). To determine which non-MtU transporters were affected by CCCP, we performed a series of 2-blocked experiments, treating the cells with both CCCP and one other inhibitor and allowing the other two mechanisms to function. We compared these results to curves calculated from the 3-blocked experiments in Fig. 7. For example, in Fig. 8 C, we compare a 2-blocked experiment in which MtU and SERCA are inhibited (black line) with the calculated sum of the capacities of NCX, PMCA, and the residual Ca2+ transport (gray line). The two curves are similar, suggesting that the fluxes allowed to operate in this experiment are not strongly affected by CCCP. In Fig. 8 D, we inhibited both MtU and PMCA (black line) and compared the transport curve to the sum of the capacities of SERCA, NCX, and the residual Ca2+ transport. Once again, the curves are quite similar with CCCP being only slightly lower. However, when we inhibited NCX in addition to CCCP treatment, we obtained the unambiguous result that the remaining transport was far below that expected for the sum of SERCA, PMCA, and the residual Ca2+ transport (Fig. 8 E). In fact, the observed transport was virtually identical to residual Ca2+ transport. These results indicate that both SERCA and PMCA are inhibited by CCCP treatment, as might be expected since both require ATP, and that when only one of these two transporters is working, a differentiated cell can supply almost enough ATP to keep up, but when both are operating it cannot.

Bottom Line: Our results indicate that Ca2+ transport in undifferentiated PC12 cells is quite unlike transport in adrenal chromaffin cells, for which they often are considered models.Transport in both cell states more closely resembles that of sympathetic neurons, for which differentiated PC12 cells often are considered models.Comparison with other cell types shows that different cells emphasize different Ca2+ transport mechanisms.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics University of Washington School of Medicine, Seattle, WA 98195, USA.

ABSTRACT
Many studies of Ca2+ signaling use PC12 cells, yet the balance of Ca2+ clearance mechanisms in these cells is unknown. We used pharmacological inhibition of Ca2+ transporters to characterize Ca2+ clearance after depolarizations in both undifferentiated and nerve growth factor-differentiated PC12 cells. Sarco-endoplasmic reticulum Ca2+ ATPase (SERCA), plasma membrane Ca2+ ATPase (PMCA), and Na+/Ca2+ exchanger (NCX) account for almost all Ca2+ clearance in both cell states, with NCX and PMCA making the greatest contributions. Any contribution of mitochondrial uniporters is small. The ATP pool in differentiated cells was much more labile than that of undifferentiated cells in the presence of agents that dissipated mitochondrial proton gradients. Differentiated PC12 cells have a small component of Ca2+ clearance possessing pharmacological characteristics consistent with secretory pathway Ca2+ ATPase (SPCA), potentially residing on Golgi and/or secretory granules. Undifferentiated and differentiated cells are similar in overall Ca2+ transport and in the small transport due to SERCA, but they differ in the fraction of transport by PMCA and NCX. Transport in neurites of differentiated PC12 cells was qualitatively similar to that in the somata, except that the ER stores in neurites sometimes released Ca2+ instead of clearing it after depolarization. We formulated a mathematical model to simulate the observed Ca2+ clearance and to describe the differences between these undifferentiated and NGF-differentiated states quantitatively. The model required a value for the endogenous Ca2+ binding ratio of PC12 cell cytoplasm, which we measured to be 268 +/- 85. Our results indicate that Ca2+ transport in undifferentiated PC12 cells is quite unlike transport in adrenal chromaffin cells, for which they often are considered models. Transport in both cell states more closely resembles that of sympathetic neurons, for which differentiated PC12 cells often are considered models. Comparison with other cell types shows that different cells emphasize different Ca2+ transport mechanisms.

Show MeSH
Related in: MedlinePlus