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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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Most Ca2+ transporters work at or near capacity in undifferentiated PC12 cells. In each panel of the left column (A, C, E, and G), Ca2+ transport curves from various 1-blocked experiments are shown in gray, and control Ca2+ transport curves from Fig. 1 (−d[Ca2+]cyt/dt vs. [Ca2+]cyt) are shown in black. In each panel in the right column (B, D, F, and H), the activity of a given transporter, calculated as the difference between the control Ca2+ transport curve and the 1-blocked Ca2+ transport curve to the left, is shown in gray. The capacity trace obtained from the 3-blocked experiments of Fig. 2 is shown in black. (A and B) SERCA was inhibited with 1 μM TG (n = 12). (C and D) MtU was inhibited by treatment with 2 μM CCCP (n = 16). (E and F) PMCA was inhibited by raising extracellular pH to 9.0 (n = 16). (G and H) NCX was inhibited by replacing extracellular Na+ with Li+ (n = 16).
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fig3: Most Ca2+ transporters work at or near capacity in undifferentiated PC12 cells. In each panel of the left column (A, C, E, and G), Ca2+ transport curves from various 1-blocked experiments are shown in gray, and control Ca2+ transport curves from Fig. 1 (−d[Ca2+]cyt/dt vs. [Ca2+]cyt) are shown in black. In each panel in the right column (B, D, F, and H), the activity of a given transporter, calculated as the difference between the control Ca2+ transport curve and the 1-blocked Ca2+ transport curve to the left, is shown in gray. The capacity trace obtained from the 3-blocked experiments of Fig. 2 is shown in black. (A and B) SERCA was inhibited with 1 μM TG (n = 12). (C and D) MtU was inhibited by treatment with 2 μM CCCP (n = 16). (E and F) PMCA was inhibited by raising extracellular pH to 9.0 (n = 16). (G and H) NCX was inhibited by replacing extracellular Na+ with Li+ (n = 16).

Mentions: If the four clearance mechanisms operated independently, any combination of them should be additive. To verify additivity and to cross-check the conclusions of our 3-blocked measurements, we performed a series of 1-blocked experiments in which only one transporter was inhibited at a time. The “activity” of that transporter was determined by subtracting the 1-blocked curve from the control curve. We refer to the subtracted curves as “activity curves” (Fig. 3). An activity curve differs from a capacity curve in that the former is intended to show what a given transporter accomplishes while working in concert with other Ca2+ transporters, whereas the latter shows the results of that transporter working alone. The panels on the left show the 1-blocked transport curves in gray and the control curves in black. The panels on the right show the activity curves for each mechanism, i.e., the differences between the two curves in the left panel, in gray, compared with the capacity curves obtained from the 3-blocked experiments (Fig. 2) in black. The activity curves are quite noisy as each point represents a small difference between two large numbers. For SERCA pumps, the capacity and activity curves are not obviously different, transport remaining small until [Ca2+]cyt rises above 1000 nM (Fig. 3, A and B). Likewise, for the MtU, the curves seem similar and small at all but high (≥800 nM) values of [Ca2+]cyt (Fig. 3, C and D). For PMCA, on the other hand, the calculated activity falls well below the capacity for five consecutive data points below 800 nM [Ca2+]cyt (Fig. 3, E and F). Under these 1-blocked conditions, the NCX was the most robust transporter of Ca2+, and it operated at capacity until ∼1000 nM Ca2+; where it fell below the capacity curve (Fig. 3, G and H). These calculations indicate that the canonical Ca2+ transporters transport at or a little below capacity over the tested range of [Ca2+]cyt, explaining why the sum of individual transport capacities slightly exceeds the observed total transport at higher values of [Ca2+]cyt (Fig. 2 C). The deviation from additivity for the NCX, for example, might imply that when the other transporters are operating, the NCX experiences a lower local [Ca2+]cyt than the average value that fura-2 dye reports (see Discussion).


Calcium transport mechanisms of PC12 cells.

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

Most Ca2+ transporters work at or near capacity in undifferentiated PC12 cells. In each panel of the left column (A, C, E, and G), Ca2+ transport curves from various 1-blocked experiments are shown in gray, and control Ca2+ transport curves from Fig. 1 (−d[Ca2+]cyt/dt vs. [Ca2+]cyt) are shown in black. In each panel in the right column (B, D, F, and H), the activity of a given transporter, calculated as the difference between the control Ca2+ transport curve and the 1-blocked Ca2+ transport curve to the left, is shown in gray. The capacity trace obtained from the 3-blocked experiments of Fig. 2 is shown in black. (A and B) SERCA was inhibited with 1 μM TG (n = 12). (C and D) MtU was inhibited by treatment with 2 μM CCCP (n = 16). (E and F) PMCA was inhibited by raising extracellular pH to 9.0 (n = 16). (G and H) NCX was inhibited by replacing extracellular Na+ with Li+ (n = 16).
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fig3: Most Ca2+ transporters work at or near capacity in undifferentiated PC12 cells. In each panel of the left column (A, C, E, and G), Ca2+ transport curves from various 1-blocked experiments are shown in gray, and control Ca2+ transport curves from Fig. 1 (−d[Ca2+]cyt/dt vs. [Ca2+]cyt) are shown in black. In each panel in the right column (B, D, F, and H), the activity of a given transporter, calculated as the difference between the control Ca2+ transport curve and the 1-blocked Ca2+ transport curve to the left, is shown in gray. The capacity trace obtained from the 3-blocked experiments of Fig. 2 is shown in black. (A and B) SERCA was inhibited with 1 μM TG (n = 12). (C and D) MtU was inhibited by treatment with 2 μM CCCP (n = 16). (E and F) PMCA was inhibited by raising extracellular pH to 9.0 (n = 16). (G and H) NCX was inhibited by replacing extracellular Na+ with Li+ (n = 16).
Mentions: If the four clearance mechanisms operated independently, any combination of them should be additive. To verify additivity and to cross-check the conclusions of our 3-blocked measurements, we performed a series of 1-blocked experiments in which only one transporter was inhibited at a time. The “activity” of that transporter was determined by subtracting the 1-blocked curve from the control curve. We refer to the subtracted curves as “activity curves” (Fig. 3). An activity curve differs from a capacity curve in that the former is intended to show what a given transporter accomplishes while working in concert with other Ca2+ transporters, whereas the latter shows the results of that transporter working alone. The panels on the left show the 1-blocked transport curves in gray and the control curves in black. The panels on the right show the activity curves for each mechanism, i.e., the differences between the two curves in the left panel, in gray, compared with the capacity curves obtained from the 3-blocked experiments (Fig. 2) in black. The activity curves are quite noisy as each point represents a small difference between two large numbers. For SERCA pumps, the capacity and activity curves are not obviously different, transport remaining small until [Ca2+]cyt rises above 1000 nM (Fig. 3, A and B). Likewise, for the MtU, the curves seem similar and small at all but high (≥800 nM) values of [Ca2+]cyt (Fig. 3, C and D). For PMCA, on the other hand, the calculated activity falls well below the capacity for five consecutive data points below 800 nM [Ca2+]cyt (Fig. 3, E and F). Under these 1-blocked conditions, the NCX was the most robust transporter of Ca2+, and it operated at capacity until ∼1000 nM Ca2+; where it fell below the capacity curve (Fig. 3, G and H). These calculations indicate that the canonical Ca2+ transporters transport at or a little below capacity over the tested range of [Ca2+]cyt, explaining why the sum of individual transport capacities slightly exceeds the observed total transport at higher values of [Ca2+]cyt (Fig. 2 C). The deviation from additivity for the NCX, for example, might imply that when the other transporters are operating, the NCX experiences a lower local [Ca2+]cyt than the average value that fura-2 dye reports (see Discussion).

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