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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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Capacities and activities of Ca2+ transporters in NGF-differentiated PC12 cells. Results are shown as Ca2+ transport curves (−d[Ca2+]cyt/dt vs. [Ca2+]cyt). In the left column (A, C, E, and G), data from 1-blocked experiments for each of the four canonical Ca2+ transport mechanisms (gray lines and symbols) are compared with control NGF-differentiated cells (black lines and circles) from Fig. 6. In the right column (B, D, F, and H), the capacity of each transporter, obtained from 3-blocked experiments and corrected for residual Ca2+ transport, is shown in black. The activity of each Ca2+ transporter type, calculated as the difference between the two traces in the lefthand panel, is shown in gray: (A and B) SERCA (n = 33 for 1-blocked experiments, n = 17 for 3-blocked experiments); (C and D) MtU (n = 21 for 1-blocked experiments, n = 17 for 3-blocked experiments); (E and F) PMCA (n = 17 for 1-blocked experiments, n = 16 for 3-blocked experiments). (G and H) NCX (n = 19 for 1-blocked experiments, n = 16 for 3-blocked experiments).
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fig7: Capacities and activities of Ca2+ transporters in NGF-differentiated PC12 cells. Results are shown as Ca2+ transport curves (−d[Ca2+]cyt/dt vs. [Ca2+]cyt). In the left column (A, C, E, and G), data from 1-blocked experiments for each of the four canonical Ca2+ transport mechanisms (gray lines and symbols) are compared with control NGF-differentiated cells (black lines and circles) from Fig. 6. In the right column (B, D, F, and H), the capacity of each transporter, obtained from 3-blocked experiments and corrected for residual Ca2+ transport, is shown in black. The activity of each Ca2+ transporter type, calculated as the difference between the two traces in the lefthand panel, is shown in gray: (A and B) SERCA (n = 33 for 1-blocked experiments, n = 17 for 3-blocked experiments); (C and D) MtU (n = 21 for 1-blocked experiments, n = 17 for 3-blocked experiments); (E and F) PMCA (n = 17 for 1-blocked experiments, n = 16 for 3-blocked experiments). (G and H) NCX (n = 19 for 1-blocked experiments, n = 16 for 3-blocked experiments).

Mentions: We determined the capacity of the four canonical transporters in differentiated cells by performing 3-blocked experiments, and we determined their activities when other transporters were active using 1-blocked experiments. As shown in Fig. 7, the capacity curves nearly overlaid the noisier activity curves for SERCA, PMCA, and NCX in differentiated cells. However, the component of transport attributed to MtU is clearly discrepant between the two measures. Application of CCCP at the moment of the KCl treatment diminished subsequent Ca2+ transport by almost 50% at [Ca2+]cyt values above 1000 nM (Fig. 7 C). At face value, this result contrasted with our 3-blocked experiments, which suggested that the mitochondria might even release a little Ca2+ into the cytoplasm at these high values of [Ca2+]cyt and transported little net Ca2+ below 1000 nM [Ca2+]cyt (Fig. 7 D). Clearly in differentiated cells the 1-blocked (CCCP) experiments are not correctly extracting the mitochondrial component of flux seen with the three blocked experiments (high pH, TG, Li+).


Calcium transport mechanisms of PC12 cells.

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

Capacities and activities of Ca2+ transporters in NGF-differentiated PC12 cells. Results are shown as Ca2+ transport curves (−d[Ca2+]cyt/dt vs. [Ca2+]cyt). In the left column (A, C, E, and G), data from 1-blocked experiments for each of the four canonical Ca2+ transport mechanisms (gray lines and symbols) are compared with control NGF-differentiated cells (black lines and circles) from Fig. 6. In the right column (B, D, F, and H), the capacity of each transporter, obtained from 3-blocked experiments and corrected for residual Ca2+ transport, is shown in black. The activity of each Ca2+ transporter type, calculated as the difference between the two traces in the lefthand panel, is shown in gray: (A and B) SERCA (n = 33 for 1-blocked experiments, n = 17 for 3-blocked experiments); (C and D) MtU (n = 21 for 1-blocked experiments, n = 17 for 3-blocked experiments); (E and F) PMCA (n = 17 for 1-blocked experiments, n = 16 for 3-blocked experiments). (G and H) NCX (n = 19 for 1-blocked experiments, n = 16 for 3-blocked experiments).
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fig7: Capacities and activities of Ca2+ transporters in NGF-differentiated PC12 cells. Results are shown as Ca2+ transport curves (−d[Ca2+]cyt/dt vs. [Ca2+]cyt). In the left column (A, C, E, and G), data from 1-blocked experiments for each of the four canonical Ca2+ transport mechanisms (gray lines and symbols) are compared with control NGF-differentiated cells (black lines and circles) from Fig. 6. In the right column (B, D, F, and H), the capacity of each transporter, obtained from 3-blocked experiments and corrected for residual Ca2+ transport, is shown in black. The activity of each Ca2+ transporter type, calculated as the difference between the two traces in the lefthand panel, is shown in gray: (A and B) SERCA (n = 33 for 1-blocked experiments, n = 17 for 3-blocked experiments); (C and D) MtU (n = 21 for 1-blocked experiments, n = 17 for 3-blocked experiments); (E and F) PMCA (n = 17 for 1-blocked experiments, n = 16 for 3-blocked experiments). (G and H) NCX (n = 19 for 1-blocked experiments, n = 16 for 3-blocked experiments).
Mentions: We determined the capacity of the four canonical transporters in differentiated cells by performing 3-blocked experiments, and we determined their activities when other transporters were active using 1-blocked experiments. As shown in Fig. 7, the capacity curves nearly overlaid the noisier activity curves for SERCA, PMCA, and NCX in differentiated cells. However, the component of transport attributed to MtU is clearly discrepant between the two measures. Application of CCCP at the moment of the KCl treatment diminished subsequent Ca2+ transport by almost 50% at [Ca2+]cyt values above 1000 nM (Fig. 7 C). At face value, this result contrasted with our 3-blocked experiments, which suggested that the mitochondria might even release a little Ca2+ into the cytoplasm at these high values of [Ca2+]cyt and transported little net Ca2+ below 1000 nM [Ca2+]cyt (Fig. 7 D). Clearly in differentiated cells the 1-blocked (CCCP) experiments are not correctly extracting the mitochondrial component of flux seen with the three blocked experiments (high pH, TG, Li+).

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