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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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Ca2+ transport in neurites of NGF-differentiated PC12 cells. (A) Typical time course of Ca2+ clearance in neurites of a PC12 cell (gray line). Due to the noisiness of the traces, we fitted the original data with a double exponential (black line) to calculate Ca2+ transport curves. (B) Ca2+ transport curves for control and 4-blocked experiments in neurites (black lines with circles, and gray lines with triangles, respectively, n = 13). Data from cell somata are shown as well (broken black line is control from Fig. 10, broken gray line is 4-blocked from Fig. 10). (C) Ca2+ transport curves for the BHQ-dependent fluxes in NGF-differentiated cells. The dotted gray line and triangles are from neurites (n = 10), the solid gray line are from cell somata (Fig. 6), and the black line represents the residual Ca2+ transport for neurites (B). (D) Ca2+ transport capacity curves for SERCA. The two different shades of gray lines and squares represent the SERCA data for neurites sorted into two classes (n = 11 each) (N). The black line represents SERCA Ca2+ transport in soma from Fig. 9 (S). (E–G) Ca2+ transport curves for MtU (E), PMCA (F), and NCX (G). Gray line and symbols are from neurites (n = 16, 13, 11, respectively), and the black lines represent capacity data from somata (Fig. 9). (H) The control total Ca2+ transport curve from neurites (black line, Fig. 10 B) is compared with the sum of the individual activities obtained from neurites (gray line).
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fig10: Ca2+ transport in neurites of NGF-differentiated PC12 cells. (A) Typical time course of Ca2+ clearance in neurites of a PC12 cell (gray line). Due to the noisiness of the traces, we fitted the original data with a double exponential (black line) to calculate Ca2+ transport curves. (B) Ca2+ transport curves for control and 4-blocked experiments in neurites (black lines with circles, and gray lines with triangles, respectively, n = 13). Data from cell somata are shown as well (broken black line is control from Fig. 10, broken gray line is 4-blocked from Fig. 10). (C) Ca2+ transport curves for the BHQ-dependent fluxes in NGF-differentiated cells. The dotted gray line and triangles are from neurites (n = 10), the solid gray line are from cell somata (Fig. 6), and the black line represents the residual Ca2+ transport for neurites (B). (D) Ca2+ transport capacity curves for SERCA. The two different shades of gray lines and squares represent the SERCA data for neurites sorted into two classes (n = 11 each) (N). The black line represents SERCA Ca2+ transport in soma from Fig. 9 (S). (E–G) Ca2+ transport curves for MtU (E), PMCA (F), and NCX (G). Gray line and symbols are from neurites (n = 16, 13, 11, respectively), and the black lines represent capacity data from somata (Fig. 9). (H) The control total Ca2+ transport curve from neurites (black line, Fig. 10 B) is compared with the sum of the individual activities obtained from neurites (gray line).

Mentions: We tried to measure Ca2+ transport in the extensive network of neurites that emerged from our cells upon NGF differentiation. The Ca2+ signals from thin neurites were quite noisy compared with those from the much larger somata because the fluorescence was much dimmer. Therefore, before taking the derivatives of these traces, we fitted the Ca2+ clearance phase for each field of neurites with a double exponential function (Fig. 10 A) and used these functions in our subsequent analysis instead of the original data. Even so, the variability in the Ca2+ transport curves was higher than for somata (Fig. 10 B, black lines) and the results must be regarded as somewhat qualitative. The control transport curve for neurites was statistically indistinguishable from the control curve for somata. The 4-blocked protocol showed that the residual Ca2+ transport of neurites and somata also are similar (Fig. 10 B, gray lines). We next compared the additional BHQ-sensitive transport. As shown in Fig. 10 C, BHQ increased Ca2+ clearance in neurites relative to the 4-blocked protocol. This effect is similar to that seen in undifferentiated PC-12 cells, but unlike that recorded from the somata of differentiated cells (Fig. 10 C). This was the first indication of differences in Ca2+ handling in the neurites versus the somata of differentiated PC12 cells.


Calcium transport mechanisms of PC12 cells.

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

Ca2+ transport in neurites of NGF-differentiated PC12 cells. (A) Typical time course of Ca2+ clearance in neurites of a PC12 cell (gray line). Due to the noisiness of the traces, we fitted the original data with a double exponential (black line) to calculate Ca2+ transport curves. (B) Ca2+ transport curves for control and 4-blocked experiments in neurites (black lines with circles, and gray lines with triangles, respectively, n = 13). Data from cell somata are shown as well (broken black line is control from Fig. 10, broken gray line is 4-blocked from Fig. 10). (C) Ca2+ transport curves for the BHQ-dependent fluxes in NGF-differentiated cells. The dotted gray line and triangles are from neurites (n = 10), the solid gray line are from cell somata (Fig. 6), and the black line represents the residual Ca2+ transport for neurites (B). (D) Ca2+ transport capacity curves for SERCA. The two different shades of gray lines and squares represent the SERCA data for neurites sorted into two classes (n = 11 each) (N). The black line represents SERCA Ca2+ transport in soma from Fig. 9 (S). (E–G) Ca2+ transport curves for MtU (E), PMCA (F), and NCX (G). Gray line and symbols are from neurites (n = 16, 13, 11, respectively), and the black lines represent capacity data from somata (Fig. 9). (H) The control total Ca2+ transport curve from neurites (black line, Fig. 10 B) is compared with the sum of the individual activities obtained from neurites (gray line).
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Related In: Results  -  Collection

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fig10: Ca2+ transport in neurites of NGF-differentiated PC12 cells. (A) Typical time course of Ca2+ clearance in neurites of a PC12 cell (gray line). Due to the noisiness of the traces, we fitted the original data with a double exponential (black line) to calculate Ca2+ transport curves. (B) Ca2+ transport curves for control and 4-blocked experiments in neurites (black lines with circles, and gray lines with triangles, respectively, n = 13). Data from cell somata are shown as well (broken black line is control from Fig. 10, broken gray line is 4-blocked from Fig. 10). (C) Ca2+ transport curves for the BHQ-dependent fluxes in NGF-differentiated cells. The dotted gray line and triangles are from neurites (n = 10), the solid gray line are from cell somata (Fig. 6), and the black line represents the residual Ca2+ transport for neurites (B). (D) Ca2+ transport capacity curves for SERCA. The two different shades of gray lines and squares represent the SERCA data for neurites sorted into two classes (n = 11 each) (N). The black line represents SERCA Ca2+ transport in soma from Fig. 9 (S). (E–G) Ca2+ transport curves for MtU (E), PMCA (F), and NCX (G). Gray line and symbols are from neurites (n = 16, 13, 11, respectively), and the black lines represent capacity data from somata (Fig. 9). (H) The control total Ca2+ transport curve from neurites (black line, Fig. 10 B) is compared with the sum of the individual activities obtained from neurites (gray line).
Mentions: We tried to measure Ca2+ transport in the extensive network of neurites that emerged from our cells upon NGF differentiation. The Ca2+ signals from thin neurites were quite noisy compared with those from the much larger somata because the fluorescence was much dimmer. Therefore, before taking the derivatives of these traces, we fitted the Ca2+ clearance phase for each field of neurites with a double exponential function (Fig. 10 A) and used these functions in our subsequent analysis instead of the original data. Even so, the variability in the Ca2+ transport curves was higher than for somata (Fig. 10 B, black lines) and the results must be regarded as somewhat qualitative. The control transport curve for neurites was statistically indistinguishable from the control curve for somata. The 4-blocked protocol showed that the residual Ca2+ transport of neurites and somata also are similar (Fig. 10 B, gray lines). We next compared the additional BHQ-sensitive transport. As shown in Fig. 10 C, BHQ increased Ca2+ clearance in neurites relative to the 4-blocked protocol. This effect is similar to that seen in undifferentiated PC-12 cells, but unlike that recorded from the somata of differentiated cells (Fig. 10 C). This was the first indication of differences in Ca2+ handling in the neurites versus the somata of differentiated PC12 cells.

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