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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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NGF-differentiated PC12 cells have greater Ca2+ rises in response to depolarization than undifferentiated cells. Peak [Ca2+]cyt for PC12 cells loaded with fura-4F and depolarized with 70 mM KCl are shown for both undifferentiated (No NGF, n = 20) and differentiated (NGF, n = 13) cells. Open gray circles represent individual cells, and closed black circles are means.
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fig5: NGF-differentiated PC12 cells have greater Ca2+ rises in response to depolarization than undifferentiated cells. Peak [Ca2+]cyt for PC12 cells loaded with fura-4F and depolarized with 70 mM KCl are shown for both undifferentiated (No NGF, n = 20) and differentiated (NGF, n = 13) cells. Open gray circles represent individual cells, and closed black circles are means.

Mentions: The error bars and statistics given for means represent SEM. To combine data from multiple cells, we binned data points by the values of [Ca2+]cyt. The bins were 100 nM wide from 0 to 1000 nM, and 200 nM wide above 1000 nM. We then averaged the binned data from a pool of cells—[Ca2+]cyt for the abcissae and d[Ca2+]cyt/dt for the ordinates—and report the average value in the figures. Note that due to a large spread in peak Ca2+ values (Fig. 5), not every cell that we measured contributed to every bin. All of these mean Ca2+ transport curves (d[Ca2+]cyt/dt vs. [Ca2+]cyt) are shown ± SEM of both x and y values. At times, we performed mathematical operations on Ca2+ transport curves. Most frequently, this involved addition and subtraction operations. To add or subtract curves, we averaged the concentrations and performed the desired operation on the transport rates for each bin. SEMs combined according to the standard formula for addition (Goldstein, 1964).


Calcium transport mechanisms of PC12 cells.

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

NGF-differentiated PC12 cells have greater Ca2+ rises in response to depolarization than undifferentiated cells. Peak [Ca2+]cyt for PC12 cells loaded with fura-4F and depolarized with 70 mM KCl are shown for both undifferentiated (No NGF, n = 20) and differentiated (NGF, n = 13) cells. Open gray circles represent individual cells, and closed black circles are means.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: NGF-differentiated PC12 cells have greater Ca2+ rises in response to depolarization than undifferentiated cells. Peak [Ca2+]cyt for PC12 cells loaded with fura-4F and depolarized with 70 mM KCl are shown for both undifferentiated (No NGF, n = 20) and differentiated (NGF, n = 13) cells. Open gray circles represent individual cells, and closed black circles are means.
Mentions: The error bars and statistics given for means represent SEM. To combine data from multiple cells, we binned data points by the values of [Ca2+]cyt. The bins were 100 nM wide from 0 to 1000 nM, and 200 nM wide above 1000 nM. We then averaged the binned data from a pool of cells—[Ca2+]cyt for the abcissae and d[Ca2+]cyt/dt for the ordinates—and report the average value in the figures. Note that due to a large spread in peak Ca2+ values (Fig. 5), not every cell that we measured contributed to every bin. All of these mean Ca2+ transport curves (d[Ca2+]cyt/dt vs. [Ca2+]cyt) are shown ± SEM of both x and y values. At times, we performed mathematical operations on Ca2+ transport curves. Most frequently, this involved addition and subtraction operations. To add or subtract curves, we averaged the concentrations and performed the desired operation on the transport rates for each bin. SEMs combined according to the standard formula for addition (Goldstein, 1964).

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