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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.

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Comparison of Ca2+ transport capacities between undifferentiated and NGF-differentiated PC12 cells. Experimental Ca2+ transport curves (−d[Ca2+]cyt/dt vs. [Ca2+]cyt) are shown as symbols. Black symbols are from undifferentiated cells and red symbols are from NGF-differentiated cells. Capacity data from undifferentiated cells are reproduced from Fig. 2. The capacity data for MtU and residual Ca2+ transport in differentiated cells are reproduced from Figs. 8 and 7, respectively. Smooth black and red curves represent fitted functions from the mathematical model in Appendix using the same color code. (A) Capacities and fits of SERCA. The NGF-differentiated points result from 2-blocked experiments where PMCA and NCX were inhibited, corrected for MtU and residual Ca2+ transport (n = 11). (B) Capacities and fits of MtU. (C) Capacities and fits of PMCA. The NGF-differentiated curve is the result of 2-blocked experiments in which SERCA and NCX were inhibited, corrected for MtU and residual Ca2+ transport (n = 16). (D) Capacities and fits of NCX. The NGF-differentiated curve points result from 2-blocked experiments in which SERCA and PMCA were inhibited, corrected for MtU and residual Ca2+ transport (n = 12). (E) Capacities and fits of the residual Ca2+ transport. (F) The NGF-differentiated control values (black) are shown with the sum of the five capacity curves from NGF-differentiated cells (blue) from A–E and the sum of the fitted functions (red).
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fig9: Comparison of Ca2+ transport capacities between undifferentiated and NGF-differentiated PC12 cells. Experimental Ca2+ transport curves (−d[Ca2+]cyt/dt vs. [Ca2+]cyt) are shown as symbols. Black symbols are from undifferentiated cells and red symbols are from NGF-differentiated cells. Capacity data from undifferentiated cells are reproduced from Fig. 2. The capacity data for MtU and residual Ca2+ transport in differentiated cells are reproduced from Figs. 8 and 7, respectively. Smooth black and red curves represent fitted functions from the mathematical model in Appendix using the same color code. (A) Capacities and fits of SERCA. The NGF-differentiated points result from 2-blocked experiments where PMCA and NCX were inhibited, corrected for MtU and residual Ca2+ transport (n = 11). (B) Capacities and fits of MtU. (C) Capacities and fits of PMCA. The NGF-differentiated curve is the result of 2-blocked experiments in which SERCA and NCX were inhibited, corrected for MtU and residual Ca2+ transport (n = 16). (D) Capacities and fits of NCX. The NGF-differentiated curve points result from 2-blocked experiments in which SERCA and PMCA were inhibited, corrected for MtU and residual Ca2+ transport (n = 12). (E) Capacities and fits of the residual Ca2+ transport. (F) The NGF-differentiated control values (black) are shown with the sum of the five capacity curves from NGF-differentiated cells (blue) from A–E and the sum of the fitted functions (red).

Mentions: Throughout the manuscript, curves that were obtained directly from experimental data are shown with open symbols and solid lines; the lines may connect the symbols or may represent output of a model. Points from mathematical operations performed on experimental results are shown with closed symbols and dotted lines. With the exception of the control Ca2+ transport curves, curves that have appeared in previous figures are shown as lines and error bars without symbols. In Fig. 9, we bypassed some of our rules and switched to closed symbols in panels A, B, C, and D for clarity.


Calcium transport mechanisms of PC12 cells.

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

Comparison of Ca2+ transport capacities between undifferentiated and NGF-differentiated PC12 cells. Experimental Ca2+ transport curves (−d[Ca2+]cyt/dt vs. [Ca2+]cyt) are shown as symbols. Black symbols are from undifferentiated cells and red symbols are from NGF-differentiated cells. Capacity data from undifferentiated cells are reproduced from Fig. 2. The capacity data for MtU and residual Ca2+ transport in differentiated cells are reproduced from Figs. 8 and 7, respectively. Smooth black and red curves represent fitted functions from the mathematical model in Appendix using the same color code. (A) Capacities and fits of SERCA. The NGF-differentiated points result from 2-blocked experiments where PMCA and NCX were inhibited, corrected for MtU and residual Ca2+ transport (n = 11). (B) Capacities and fits of MtU. (C) Capacities and fits of PMCA. The NGF-differentiated curve is the result of 2-blocked experiments in which SERCA and NCX were inhibited, corrected for MtU and residual Ca2+ transport (n = 16). (D) Capacities and fits of NCX. The NGF-differentiated curve points result from 2-blocked experiments in which SERCA and PMCA were inhibited, corrected for MtU and residual Ca2+ transport (n = 12). (E) Capacities and fits of the residual Ca2+ transport. (F) The NGF-differentiated control values (black) are shown with the sum of the five capacity curves from NGF-differentiated cells (blue) from A–E and the sum of the fitted functions (red).
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Related In: Results  -  Collection

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fig9: Comparison of Ca2+ transport capacities between undifferentiated and NGF-differentiated PC12 cells. Experimental Ca2+ transport curves (−d[Ca2+]cyt/dt vs. [Ca2+]cyt) are shown as symbols. Black symbols are from undifferentiated cells and red symbols are from NGF-differentiated cells. Capacity data from undifferentiated cells are reproduced from Fig. 2. The capacity data for MtU and residual Ca2+ transport in differentiated cells are reproduced from Figs. 8 and 7, respectively. Smooth black and red curves represent fitted functions from the mathematical model in Appendix using the same color code. (A) Capacities and fits of SERCA. The NGF-differentiated points result from 2-blocked experiments where PMCA and NCX were inhibited, corrected for MtU and residual Ca2+ transport (n = 11). (B) Capacities and fits of MtU. (C) Capacities and fits of PMCA. The NGF-differentiated curve is the result of 2-blocked experiments in which SERCA and NCX were inhibited, corrected for MtU and residual Ca2+ transport (n = 16). (D) Capacities and fits of NCX. The NGF-differentiated curve points result from 2-blocked experiments in which SERCA and PMCA were inhibited, corrected for MtU and residual Ca2+ transport (n = 12). (E) Capacities and fits of the residual Ca2+ transport. (F) The NGF-differentiated control values (black) are shown with the sum of the five capacity curves from NGF-differentiated cells (blue) from A–E and the sum of the fitted functions (red).
Mentions: Throughout the manuscript, curves that were obtained directly from experimental data are shown with open symbols and solid lines; the lines may connect the symbols or may represent output of a model. Points from mathematical operations performed on experimental results are shown with closed symbols and dotted lines. With the exception of the control Ca2+ transport curves, curves that have appeared in previous figures are shown as lines and error bars without symbols. In Fig. 9, we bypassed some of our rules and switched to closed symbols in panels A, B, C, and D for clarity.

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