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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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Four canonical Ca2+ transporters account for most Ca2+ clearance in undifferentiated PC12 cells. (A) Representative time course of [Ca2+]cyt in two single undifferentiated PC12 cells in response to KCl depolarization (black bar). The control cell is shown in black, and the 4-blocked cell (see text) is in gray. Colored lines superimposed on these two traces are simulated time courses of Ca2+ clearance using a kinetic model with four canonical transport mechanisms and residual transport discussed in Appendix. (B) Transport curves of Ca2+ clearance, −d[Ca2+]cyt/dt, calculated from the clearance phases of the cells in A and plotted against [Ca2+]cyt. In 4-blocked cells, transport is greatly slowed. (C) Mean clearance rates for control (black open circles, n = 88) and 4-blocked (gray open circles, n = 62) cells. (D) Representative time course of [Ca2+]cyt in single undifferentiated PC12 cells in response to 3-s (black line) and 30-s (blue line) KCl depolarizations. (E) Ca2+ clearance, plotted as in B, for the two cells shown in D. (F) Mean clearance rates for 3-s depolarized (black, n = 88), 30-s depolarized (blue, n = 12), and TG-treated and 30-s depolarized (green, n = 9) cells, shown as in C.
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fig1: Four canonical Ca2+ transporters account for most Ca2+ clearance in undifferentiated PC12 cells. (A) Representative time course of [Ca2+]cyt in two single undifferentiated PC12 cells in response to KCl depolarization (black bar). The control cell is shown in black, and the 4-blocked cell (see text) is in gray. Colored lines superimposed on these two traces are simulated time courses of Ca2+ clearance using a kinetic model with four canonical transport mechanisms and residual transport discussed in Appendix. (B) Transport curves of Ca2+ clearance, −d[Ca2+]cyt/dt, calculated from the clearance phases of the cells in A and plotted against [Ca2+]cyt. In 4-blocked cells, transport is greatly slowed. (C) Mean clearance rates for control (black open circles, n = 88) and 4-blocked (gray open circles, n = 62) cells. (D) Representative time course of [Ca2+]cyt in single undifferentiated PC12 cells in response to 3-s (black line) and 30-s (blue line) KCl depolarizations. (E) Ca2+ clearance, plotted as in B, for the two cells shown in D. (F) Mean clearance rates for 3-s depolarized (black, n = 88), 30-s depolarized (blue, n = 12), and TG-treated and 30-s depolarized (green, n = 9) cells, shown as in C.

Mentions: Cytoplasmic Ca2+ measurements were done at elevated temperature by ratiometric fura-2 or fura-4F (Invitrogen) photometry. Cells were loaded with the acetoxymethyl (AM) ester of the desired dye dispersed in 10% pluronic and diluted to 10 μM in modified Ringer's solution (in mM: 130 NaCl, 2.5 KCl, 1 MgCl2, 2 CaCl2, 10 glucose, and 10 HEPES, pH 7.3) at room temperature for 20–25 min and transferred into a recording chamber perfused with the modified Ringer's solution using a local perfusion device heated to 37°C. To determine the [fura-2] in PC12 cells, we loaded cells with known concentrations of fura-2 salt using whole-cell patch configuration (see below). By comparing the amount of light collected from resting cells so loaded to similarly sized cells loaded with fura-2-AM, we estimated that the average PC12 cell contains 300 nM fura-2 under our loading conditions. This number was used in our mathematical model and in translating the rate of change of [Ca2+]cyt into absolute fluxes. We used a 3-s or, in Fig. 1 C only, 30-s exposure to high-K+ depolarizing solution to impose a cytoplasmic Ca2+ load. This solution contained in mM: 70 KCl, 67 NaCl, 2 CaCl2, 1 MgCl2, 10 glucose, and 10 HEPES, pH 7.3. In control experiments, a nominally Ca2+-free solution (modified Ringer's without CaCl2) was applied to the cells as they recovered from depolarization. The composition of this clearance solution and of the high K+ solution was sometimes changed as noted. Nominally Ca2+-free Ringer's solution was adequate to prevent store-operated currents activated by thapsigargin from contaminating the measurements (Fig. S1). We recorded only from one cell on each coverslip to ensure that each recorded cell was “naïve” with respect to previous Ca2+ elevations.


Calcium transport mechanisms of PC12 cells.

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

Four canonical Ca2+ transporters account for most Ca2+ clearance in undifferentiated PC12 cells. (A) Representative time course of [Ca2+]cyt in two single undifferentiated PC12 cells in response to KCl depolarization (black bar). The control cell is shown in black, and the 4-blocked cell (see text) is in gray. Colored lines superimposed on these two traces are simulated time courses of Ca2+ clearance using a kinetic model with four canonical transport mechanisms and residual transport discussed in Appendix. (B) Transport curves of Ca2+ clearance, −d[Ca2+]cyt/dt, calculated from the clearance phases of the cells in A and plotted against [Ca2+]cyt. In 4-blocked cells, transport is greatly slowed. (C) Mean clearance rates for control (black open circles, n = 88) and 4-blocked (gray open circles, n = 62) cells. (D) Representative time course of [Ca2+]cyt in single undifferentiated PC12 cells in response to 3-s (black line) and 30-s (blue line) KCl depolarizations. (E) Ca2+ clearance, plotted as in B, for the two cells shown in D. (F) Mean clearance rates for 3-s depolarized (black, n = 88), 30-s depolarized (blue, n = 12), and TG-treated and 30-s depolarized (green, n = 9) cells, shown as in C.
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fig1: Four canonical Ca2+ transporters account for most Ca2+ clearance in undifferentiated PC12 cells. (A) Representative time course of [Ca2+]cyt in two single undifferentiated PC12 cells in response to KCl depolarization (black bar). The control cell is shown in black, and the 4-blocked cell (see text) is in gray. Colored lines superimposed on these two traces are simulated time courses of Ca2+ clearance using a kinetic model with four canonical transport mechanisms and residual transport discussed in Appendix. (B) Transport curves of Ca2+ clearance, −d[Ca2+]cyt/dt, calculated from the clearance phases of the cells in A and plotted against [Ca2+]cyt. In 4-blocked cells, transport is greatly slowed. (C) Mean clearance rates for control (black open circles, n = 88) and 4-blocked (gray open circles, n = 62) cells. (D) Representative time course of [Ca2+]cyt in single undifferentiated PC12 cells in response to 3-s (black line) and 30-s (blue line) KCl depolarizations. (E) Ca2+ clearance, plotted as in B, for the two cells shown in D. (F) Mean clearance rates for 3-s depolarized (black, n = 88), 30-s depolarized (blue, n = 12), and TG-treated and 30-s depolarized (green, n = 9) cells, shown as in C.
Mentions: Cytoplasmic Ca2+ measurements were done at elevated temperature by ratiometric fura-2 or fura-4F (Invitrogen) photometry. Cells were loaded with the acetoxymethyl (AM) ester of the desired dye dispersed in 10% pluronic and diluted to 10 μM in modified Ringer's solution (in mM: 130 NaCl, 2.5 KCl, 1 MgCl2, 2 CaCl2, 10 glucose, and 10 HEPES, pH 7.3) at room temperature for 20–25 min and transferred into a recording chamber perfused with the modified Ringer's solution using a local perfusion device heated to 37°C. To determine the [fura-2] in PC12 cells, we loaded cells with known concentrations of fura-2 salt using whole-cell patch configuration (see below). By comparing the amount of light collected from resting cells so loaded to similarly sized cells loaded with fura-2-AM, we estimated that the average PC12 cell contains 300 nM fura-2 under our loading conditions. This number was used in our mathematical model and in translating the rate of change of [Ca2+]cyt into absolute fluxes. We used a 3-s or, in Fig. 1 C only, 30-s exposure to high-K+ depolarizing solution to impose a cytoplasmic Ca2+ load. This solution contained in mM: 70 KCl, 67 NaCl, 2 CaCl2, 1 MgCl2, 10 glucose, and 10 HEPES, pH 7.3. In control experiments, a nominally Ca2+-free solution (modified Ringer's without CaCl2) was applied to the cells as they recovered from depolarization. The composition of this clearance solution and of the high K+ solution was sometimes changed as noted. Nominally Ca2+-free Ringer's solution was adequate to prevent store-operated currents activated by thapsigargin from contaminating the measurements (Fig. S1). We recorded only from one cell on each coverslip to ensure that each recorded cell was “naïve” with respect to previous Ca2+ elevations.

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