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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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Effects of BHQ and GPN on undifferentiated PC12 cells. Ca2+ transport curves (−d[Ca2+]cyt/dt vs. [Ca2+]cyt) are shown. (A) The light gray data points show cells treated with 10 μM BHQ in addition to the 4-blocked protocol (n = 15). The dark gray data points show residual transport in the 4-blocked cells of Fig. 1 with a superimposed smooth curve calculated from the residual transport function in a kinetic model discussed in Appendix. (B) The gray curve shows cells treated with GPN, whereas the black curve shows a truncated version of the control Ca2+ transport curve from Fig. 1.
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fig4: Effects of BHQ and GPN on undifferentiated PC12 cells. Ca2+ transport curves (−d[Ca2+]cyt/dt vs. [Ca2+]cyt) are shown. (A) The light gray data points show cells treated with 10 μM BHQ in addition to the 4-blocked protocol (n = 15). The dark gray data points show residual transport in the 4-blocked cells of Fig. 1 with a superimposed smooth curve calculated from the residual transport function in a kinetic model discussed in Appendix. (B) The gray curve shows cells treated with GPN, whereas the black curve shows a truncated version of the control Ca2+ transport curve from Fig. 1.

Mentions: In addition to the four canonical mechanisms of Ca2+ transport, SPCAs can transport Ca2+ into the Golgi and perhaps into distal secretory compartments. SPCAs are not inhibited by TG but are reported to be inhibited by high concentrations of BHQ (Wuytack et al., 2002). We performed a 5-blocked experiment in which we blocked SPCA with added 10 μM BHQ after blocking the other four mechanisms of Ca2+ transport with the 4-block cocktail. Surprisingly, adding BHQ slightly speeded Ca2+ clearance above 800 nM [Ca2+]cyt (Fig. 4 A). This might occur if SPCAs normally fill some compartment with Ca2+ that releases the Ca2+ when [Ca2+]cyt is high; BHQ would prevent this compartment from being full, so that less Ca2+ is released into the cytoplasm during the clearance phase in the presence of BHQ. The effect is small. In a previous study, we observed that dense core secretory granules release Ca2+ after plasma membrane depolarization in pancreatic β cells and were disrupted by the dipeptide GPN (Duman et al., 2006). Therefore we wanted to see whether the BHQ-sensitive compartment in PC12 cells also was disturbed by pretreatment for 20 min with 50 μM GPN. Below 800 nM [Ca2+]cyt, clearance was completely unaltered by GPN (Fig. 4 B). Unfortunately, we were unable to collect data from high Ca2+ ranges because these cells exhibited lower peak Ca2+ values than control cells did. The experiment was therefore not conclusive.


Calcium transport mechanisms of PC12 cells.

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

Effects of BHQ and GPN on undifferentiated PC12 cells. Ca2+ transport curves (−d[Ca2+]cyt/dt vs. [Ca2+]cyt) are shown. (A) The light gray data points show cells treated with 10 μM BHQ in addition to the 4-blocked protocol (n = 15). The dark gray data points show residual transport in the 4-blocked cells of Fig. 1 with a superimposed smooth curve calculated from the residual transport function in a kinetic model discussed in Appendix. (B) The gray curve shows cells treated with GPN, whereas the black curve shows a truncated version of the control Ca2+ transport curve from Fig. 1.
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Related In: Results  -  Collection

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fig4: Effects of BHQ and GPN on undifferentiated PC12 cells. Ca2+ transport curves (−d[Ca2+]cyt/dt vs. [Ca2+]cyt) are shown. (A) The light gray data points show cells treated with 10 μM BHQ in addition to the 4-blocked protocol (n = 15). The dark gray data points show residual transport in the 4-blocked cells of Fig. 1 with a superimposed smooth curve calculated from the residual transport function in a kinetic model discussed in Appendix. (B) The gray curve shows cells treated with GPN, whereas the black curve shows a truncated version of the control Ca2+ transport curve from Fig. 1.
Mentions: In addition to the four canonical mechanisms of Ca2+ transport, SPCAs can transport Ca2+ into the Golgi and perhaps into distal secretory compartments. SPCAs are not inhibited by TG but are reported to be inhibited by high concentrations of BHQ (Wuytack et al., 2002). We performed a 5-blocked experiment in which we blocked SPCA with added 10 μM BHQ after blocking the other four mechanisms of Ca2+ transport with the 4-block cocktail. Surprisingly, adding BHQ slightly speeded Ca2+ clearance above 800 nM [Ca2+]cyt (Fig. 4 A). This might occur if SPCAs normally fill some compartment with Ca2+ that releases the Ca2+ when [Ca2+]cyt is high; BHQ would prevent this compartment from being full, so that less Ca2+ is released into the cytoplasm during the clearance phase in the presence of BHQ. The effect is small. In a previous study, we observed that dense core secretory granules release Ca2+ after plasma membrane depolarization in pancreatic β cells and were disrupted by the dipeptide GPN (Duman et al., 2006). Therefore we wanted to see whether the BHQ-sensitive compartment in PC12 cells also was disturbed by pretreatment for 20 min with 50 μM GPN. Below 800 nM [Ca2+]cyt, clearance was completely unaltered by GPN (Fig. 4 B). Unfortunately, we were unable to collect data from high Ca2+ ranges because these cells exhibited lower peak Ca2+ values than control cells did. The experiment was therefore not conclusive.

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