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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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Plasma membrane Ca2+ transporters have higher capacities than intracellular Ca2+ transporters in undifferentiated PC12 cells. Clearance rate, −d[Ca2+]cyt/dt, is plotted against [Ca2+]cyt for cells treated with 3-blocked protocols that allow only one transporter to work at a time. All gray data points are corrected for residual Ca2+ transport after 4-blocked experiments. Black curves represent data from control cells, as shown in Fig. 1 (n = 88). (A) Capacities of the intracellular transporters, SERCA (squares, n = 15) and MtU (triangles, n = 24). (B) Capacities of the plasma membrane transporters, PMCA (inverted triangles, n = 16) and NCX (diamonds, n = 16). (C) The sum of the four capacity (gray) curves in A and B plus the residual Ca2+ transport from Fig. 1 is compared with the control curve. The smooth curves superimposed on the gray data points of A and B and on the black points of C are calculated from the individual functions of a kinetic model with four canonical transport mechanisms and residual transport discussed in Appendix.
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fig2: Plasma membrane Ca2+ transporters have higher capacities than intracellular Ca2+ transporters in undifferentiated PC12 cells. Clearance rate, −d[Ca2+]cyt/dt, is plotted against [Ca2+]cyt for cells treated with 3-blocked protocols that allow only one transporter to work at a time. All gray data points are corrected for residual Ca2+ transport after 4-blocked experiments. Black curves represent data from control cells, as shown in Fig. 1 (n = 88). (A) Capacities of the intracellular transporters, SERCA (squares, n = 15) and MtU (triangles, n = 24). (B) Capacities of the plasma membrane transporters, PMCA (inverted triangles, n = 16) and NCX (diamonds, n = 16). (C) The sum of the four capacity (gray) curves in A and B plus the residual Ca2+ transport from Fig. 1 is compared with the control curve. The smooth curves superimposed on the gray data points of A and B and on the black points of C are calculated from the individual functions of a kinetic model with four canonical transport mechanisms and residual transport discussed in Appendix.

Mentions: We then assessed the capacity of each of these classes of transporters by performing 3-blocked experiments in which we blocked three classes of transporters and examined the ability of the unblocked transporter to clear Ca2+. The results of these experiments are shown in Fig. 2. In each panel, the control curve represents transport in the absence of inhibitors, and the remaining curves in Fig. 2 (A and B) show the transport when only one of the classical mechanisms is operating alone; these curves have been corrected for the small residual Ca2+ transport that remains in the 4-blocked experiments (Fig. 1 C). We call the results of these experiments “capacity curves” because they show what one transport mechanism could accomplish essentially alone. Fig. 2 A shows the capacity of the two intracellular Ca2+ transporters. Compared with overall transport, the SERCA makes little contribution except above 800 nM [Ca2+]cyt. The MtU component is practically negligible, being comparable to the residual transport component of 4-blocked experiments (Fig. 1). Fig. 2 B shows the capacities of the two plasma-membrane transporters. The PMCA and NCX each account for almost half of the total Ca2+ transport up to 900 nM [Ca2+]cyt. In accordance with work on other cells, at higher concentrations, the PMCA appears to saturate, whereas the NCX rate continues to rise and accounts for ∼70% of the total Ca2+ transport. Fig. 2 C shows the sum of the capacity curves for these four transporters plus the residual Ca2+ transport compared with the observed transport in the control. Together, these data show that the Ca2+ transporting capacity of PC12 cells is dominated by the two plasma membrane transporters and that, up to 1000 nM [Ca2+], the transporters are operating near capacity.


Calcium transport mechanisms of PC12 cells.

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

Plasma membrane Ca2+ transporters have higher capacities than intracellular Ca2+ transporters in undifferentiated PC12 cells. Clearance rate, −d[Ca2+]cyt/dt, is plotted against [Ca2+]cyt for cells treated with 3-blocked protocols that allow only one transporter to work at a time. All gray data points are corrected for residual Ca2+ transport after 4-blocked experiments. Black curves represent data from control cells, as shown in Fig. 1 (n = 88). (A) Capacities of the intracellular transporters, SERCA (squares, n = 15) and MtU (triangles, n = 24). (B) Capacities of the plasma membrane transporters, PMCA (inverted triangles, n = 16) and NCX (diamonds, n = 16). (C) The sum of the four capacity (gray) curves in A and B plus the residual Ca2+ transport from Fig. 1 is compared with the control curve. The smooth curves superimposed on the gray data points of A and B and on the black points of C are calculated from the individual functions of a kinetic model with four canonical transport mechanisms and residual transport discussed in Appendix.
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Related In: Results  -  Collection

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fig2: Plasma membrane Ca2+ transporters have higher capacities than intracellular Ca2+ transporters in undifferentiated PC12 cells. Clearance rate, −d[Ca2+]cyt/dt, is plotted against [Ca2+]cyt for cells treated with 3-blocked protocols that allow only one transporter to work at a time. All gray data points are corrected for residual Ca2+ transport after 4-blocked experiments. Black curves represent data from control cells, as shown in Fig. 1 (n = 88). (A) Capacities of the intracellular transporters, SERCA (squares, n = 15) and MtU (triangles, n = 24). (B) Capacities of the plasma membrane transporters, PMCA (inverted triangles, n = 16) and NCX (diamonds, n = 16). (C) The sum of the four capacity (gray) curves in A and B plus the residual Ca2+ transport from Fig. 1 is compared with the control curve. The smooth curves superimposed on the gray data points of A and B and on the black points of C are calculated from the individual functions of a kinetic model with four canonical transport mechanisms and residual transport discussed in Appendix.
Mentions: We then assessed the capacity of each of these classes of transporters by performing 3-blocked experiments in which we blocked three classes of transporters and examined the ability of the unblocked transporter to clear Ca2+. The results of these experiments are shown in Fig. 2. In each panel, the control curve represents transport in the absence of inhibitors, and the remaining curves in Fig. 2 (A and B) show the transport when only one of the classical mechanisms is operating alone; these curves have been corrected for the small residual Ca2+ transport that remains in the 4-blocked experiments (Fig. 1 C). We call the results of these experiments “capacity curves” because they show what one transport mechanism could accomplish essentially alone. Fig. 2 A shows the capacity of the two intracellular Ca2+ transporters. Compared with overall transport, the SERCA makes little contribution except above 800 nM [Ca2+]cyt. The MtU component is practically negligible, being comparable to the residual transport component of 4-blocked experiments (Fig. 1). Fig. 2 B shows the capacities of the two plasma-membrane transporters. The PMCA and NCX each account for almost half of the total Ca2+ transport up to 900 nM [Ca2+]cyt. In accordance with work on other cells, at higher concentrations, the PMCA appears to saturate, whereas the NCX rate continues to rise and accounts for ∼70% of the total Ca2+ transport. Fig. 2 C shows the sum of the capacity curves for these four transporters plus the residual Ca2+ transport compared with the observed transport in the control. Together, these data show that the Ca2+ transporting capacity of PC12 cells is dominated by the two plasma membrane transporters and that, up to 1000 nM [Ca2+], the transporters are operating near capacity.

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