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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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Determination of κ in PC12 cells. Time courses of simultaneous fura-2 photometry and standard whole-cell recording used to determine κ in PC12 cells. Cytoplasmic Ca2+ (black line, left axis) and Ca2+ current (gray line, right axis) were monitored before and after depolarization from −80 to 0 mV for 1 s as described in Materials and methods. The black arrow marks the onset of the depolarization, and the gray arrow indicates the baseline for the current measurement. This result is typical of nine cells used for such experiments.
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fig11: Determination of κ in PC12 cells. Time courses of simultaneous fura-2 photometry and standard whole-cell recording used to determine κ in PC12 cells. Cytoplasmic Ca2+ (black line, left axis) and Ca2+ current (gray line, right axis) were monitored before and after depolarization from −80 to 0 mV for 1 s as described in Materials and methods. The black arrow marks the onset of the depolarization, and the gray arrow indicates the baseline for the current measurement. This result is typical of nine cells used for such experiments.

Mentions: Finally, we determined the endogenous Ca2+ binding ratio of PC12 cytoplasm. Only a small fraction of Ca2+ in cells is free; the rest is bound to and buffered by a number of intracellular species. The Ca2+ binding ratio (κ) is the number of bound Ca2+ ions per free Ca2+ ion. To lower the free Ca2+ of a buffered cell by a given amount, the Ca2+ transport machinery of the cell must move 1 + κ times as many Ca2+ ions out of the cytoplasm as it would if there were no buffer. We determined κ for PC12 cells by measuring Ca2+ currents and [Ca2+]cyt simultaneously in cells under the whole-cell configuration. Current measurements give the total amount of Ca2+ that entered the cell (Fig. 11, gray line), and fura-2 measurements give the change in free Ca2+ that occurred as a result (Fig. 11, black line). Using this technique and Eqs. 1 and 2 of Materials and methods, we determined that the endogenous κ in undifferentiated PC12 cells is 268 ± 85 (n = 9).


Calcium transport mechanisms of PC12 cells.

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

Determination of κ in PC12 cells. Time courses of simultaneous fura-2 photometry and standard whole-cell recording used to determine κ in PC12 cells. Cytoplasmic Ca2+ (black line, left axis) and Ca2+ current (gray line, right axis) were monitored before and after depolarization from −80 to 0 mV for 1 s as described in Materials and methods. The black arrow marks the onset of the depolarization, and the gray arrow indicates the baseline for the current measurement. This result is typical of nine cells used for such experiments.
© Copyright Policy
Related In: Results  -  Collection

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

fig11: Determination of κ in PC12 cells. Time courses of simultaneous fura-2 photometry and standard whole-cell recording used to determine κ in PC12 cells. Cytoplasmic Ca2+ (black line, left axis) and Ca2+ current (gray line, right axis) were monitored before and after depolarization from −80 to 0 mV for 1 s as described in Materials and methods. The black arrow marks the onset of the depolarization, and the gray arrow indicates the baseline for the current measurement. This result is typical of nine cells used for such experiments.
Mentions: Finally, we determined the endogenous Ca2+ binding ratio of PC12 cytoplasm. Only a small fraction of Ca2+ in cells is free; the rest is bound to and buffered by a number of intracellular species. The Ca2+ binding ratio (κ) is the number of bound Ca2+ ions per free Ca2+ ion. To lower the free Ca2+ of a buffered cell by a given amount, the Ca2+ transport machinery of the cell must move 1 + κ times as many Ca2+ ions out of the cytoplasm as it would if there were no buffer. We determined κ for PC12 cells by measuring Ca2+ currents and [Ca2+]cyt simultaneously in cells under the whole-cell configuration. Current measurements give the total amount of Ca2+ that entered the cell (Fig. 11, gray line), and fura-2 measurements give the change in free Ca2+ that occurred as a result (Fig. 11, black line). Using this technique and Eqs. 1 and 2 of Materials and methods, we determined that the endogenous κ in undifferentiated PC12 cells is 268 ± 85 (n = 9).

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